Stable Isotopes in Pharmaceutical Research, Volume 26 (Pharmacochemistry Library)

PREFACE

Stable isotope techniques offer advantages in safety, sensitivity, specificity and economy for many types of pharmaceutical investigations when compared with conventional techniques. Nevertheless, pharmaceutical researchers have been slow to embrace stable isotope techniques. This book was written in the hope that putting together in one place comprehensive reviews of the many applications of stable isotopes, and the background material necessary to understand the applications, will lead to increased usage of stable isotopes in pharmaceutical research. The chapters in this book were contributed by a very talented and diverse group of investigators. The editor wishes to thank these investigators for their excellent contributions. THOMAS R. BROWNE, M.D.

CHAPTER 1

STABLE ISOTOPES: ORIGINS AND SAFETY

PETER D. KLEIN and E. ROSELAND KLEIN Meretek Diagnostics, Inc., Medical Towers Building, 1709 Dryden Road, Suite 1513, Houston, Texas

1. THE DISCOVERY OF STABLE ISOTOPES Approximately 80 years ago, Fredrick Soddy was studying the chemistry of mesothorium and, after a review of the known methods of preparation, concluded that thorium X, mesothorium, and radium formed a chemically inseparable trio. He questioned, " . . . whether some of the common elements may not in reality be a mixture of chemically nonseparable elements in constant proportions differing step-wise by whole units in atomic weight, which would account for the lack of regular relationships between the numerical values of the atomic weights" (1). To this property of multiple radioactive preparations, he gave the name "isotope" to signify their occupancy of the same place in the table of atomic numbers. Three years later, J.J. Thomson examined the positive rays of atmospheric gases and found evidence for two forms of neon at masses 20 and 22. The value accepted at that time for the atomic weight of neon was 20.2, and the inability of Thomson's equipment to achieve unequivocal resolution of mass 20.0 from 20.2 left his postulate, that neon was a mixture of two isotopes, unproved. It was not until after World War I that Aston reported, in a one-paragraph letter to Nature, the finding of two isotopes of neon with masses at 20.0 and 22.0 (determined with a precision of 0.1) (2); within the next year, his manuscript was published (3). He then designed and constructed a new mass spectrometer and embarked on the espousal of the whole number rule. Aston (4) reported that each of the isotopes of lithium, boron, sulfur, chlorine, argon, krypton, tin, xenon, and mercury had a mass number that differed by less than 0.07 from a whole number. This report contained the first account

of an element with a stable isotope (S) relevant to biologic studies. Perhaps even more memorable was Aston's description of his mass spectrometer, "It behaves at times in the most capricious and unaccountable manner.., when by good fortune, all is well, the arrangement is capable of good performances. Thus after a favorable setting of the apparatus, six elements were successfully analyzed in as many working days. On the other hand, after dismantling became imperative and it had to be cleaned and rebuilt, exactly as before as far as any one could tell, no results of any value were obtained during weeks of work" (5). There followed an extensive search among the elements of the atomic table for the existence of other isotopes. Giauque and Johnstone (6), who had examined the atmospheric bands of oxygen, reported in 1929 the finding of an oxygen isotope with an atomic mass of 18; this discovery was followed by the report of a second isotope with a mass of 17 (7). During the same year, studies of carbon arc spectra obtained in a vacuum oven by King and Birge (8, 9) showed the existence of a 13C species. Within that year, Naude measured the absorption spectrum of nitrous oxide in the ultraviolet region and confirmed not only the existence of the oxygen isotopes, but also the presence of an isotope of nitrogen with a mass of 15 (10, 11). There remained a discrepancy between the atomic weight of hydrogen as determined by chemical means (1.00777) and that determined by mass spectroscopy (1.00756). Birge and Menzel (12) postulated that an isotope of hydrogen with an abundance of 1 part in 4,500 parts hydrogen would account for this difference. On the basis of this possibility, Urey et al. (13) were inspired in 1932 to make a limited number of assumptions about the spectral properties of such an isotope and sought evidence for its existence in hydrogen spectra. A line that corresponded to the putative isotope was found only after over-exposure of the principal lines by some 4,000-fold. Convincing proof was obtained when they evaporated 6 L liquid hydrogen and collected the last 2-3 mL as gas; the intensity of the isotopic spectra was increased 5fold, and the final significant gap in the atomic table was closed.

2. THE ENRICHMENT OF STABLE ISOTOPES Little or no attempt was made to obtain enriched fractions of stable isotopes until after the discovery of deuterium. In 1933, Lewis (of the legendary Lewis and Randall, authors of the classic treatise, Thermodynamics) reported that he had achieved an enrichment of heavy water to 35 percent through a series of electrolytic dissociations. In his letter to the Journal of the American

Chemical Society, he concluded, "The separation of any isotope in sufficient quantity to permit investigation.., suggests a wide range of interesting experiments but the isotope of hydrogen is, above all others, interesting to chemists. I believe that it will be so different from common hydrogen that it will be regarded almost as a new element. If this is true the organic chemistry of compounds containing the heavy isotope of hydrogen will be a fascinating study" (14). Later that year, Lewis and MacDonald (15) reported that they had produced 0.3 mL of pure D20 and described its physical properties including density, freezing and boiling points. They concluded, "In the various ways in which water is said to be an abnormal liquid, H2H20 seems to be more abnormal, but the differences between the two become smaller with rising temperature". For many years, deuterium production was based on the electrolysis of water with recombination of the hydrogen and oxygen through combustion. Beginning with the solution from commercial electrolysis cells with an enrichment of 0.005 percent, Urey (16) found that through five stages the enrichments were 1, 2.2, 6.5, 16-20 and 40 percent, respectively. He quoted a price of $15 to $20 per gram for the enriched samples, which in those days prohibited studies that required large volumes at high enrichments. As we shall see, many of the earliest studies were undertaken with enrichments of 2-5 percent. Not until the H2S:H20 dual exchange system was developed in the late 1950s did the price of deuterium drop to $0.20 per gram, thus enabling large-scale studies to be undertaken (17). Urey and coworkers (18) developed fundamental calculations underlying the enrichment process through exchange equilibria in 1935, and then reported in quick succession on ~5N enrichment (19), ~80 enrichment (20, 21) and ~3C preparation (22). Six years later, Nier and Bardeen (23) reported that they had achieved an enrichment of ~3C-methane to 11 percent using a thermal diffusion process set up in the stairwell of their building. Through Nier's design of a gas isotope ratio mass spectrometer (24) and his collaborations with Wood, ~3C was to enable new insights into carbon fixation and the Krebs cycle.

3. THE APPLICATION OF STABLE ISOTOPES AS TRACERS

The development of biochemistry in the late 1930s can be experienced by reading the review articles of Schoenheimer (25), Schoenheimer and Rittenberg (26), and the 1948 recollections of Clarke (27). It was a time of discovery, new concepts and rapid growth. As recounted by Clarke, Urey prepared a quantity of enriched heavy water after which he was awarded a grant from

the Rockefeller Foundation to develop applications for biologic processes. A portion of the grant was designated for the salary of a specialist to be trained to work with heavy water. That person was David Rittenberg, who was commissioned to find interesting biologic applications. He soon encountered Rudolph Schoenheimer, and the two established a formidable alliance. Their work began with a simple synthesis: they hydrogenated linseed oil to produce a deuterium-containing fat to administer to mice. On the basis of the limited amount of food fed to the animals, they expected the deuterium to be liberated promptly by oxidation of the fat to C02 and water. (Their analysis of deuterium-containing fluids was based on a densitometric method and could detect 0.001 percent enrichment.) They were totally surprised by the outcome of the study: first, the total amount of deuterium did not appear in the urine, and second, one-half of the label was recovered from fat depots. In a series of succinct tables, they reported the principles of fatty acid synthesis, storage, oxidation, desaturation, saturation and chain lengthening and shortening. They predicted early in their work that cholesterol was assembled from many units that were two carbons in length. Soon thereafter, the conversion of phenylalanine to tyrosine was demonstrated in the rat, even in the presence of large amounts of dietary tyrosine. With the availability of ~SN in the late 1930s, Schoenheimer et al. (28) studied amino acid and protein metabolism and rapidly demonstrated the incorporation of dietary ammonium citrate into amino acids of body proteins, with the exception of lysine. Moreover, there was interconversion between nitrogen-containing amino acids, such as glycine and the protein amino acids, because the latter were found to contain ~SN when hydrolyzed. When they studied the metabolism of leucine, containing a deuterium-labeled chain and an ~SN amino group, they discovered that leucine isolated from protein had lost 35 percent of its ~SN and that this nitrogen was now found in many other amino acids. They commented on the distinction drawn between exogenous and endogenous nitrogen metabolism, "It is scarcely possible to reconcile our finding with any theory which requires a distinction between these two types of nitrogen. It has been shown that nitrogenous groupings of tissue protein are constantly involved in chemical reactions; peptide linkages open, the amino acids liberated mix with others of the same species of whatever source, diet or tissue. This mixture.., while in the free state takes part in a variety of chemical reactions: some reenter directly into vacant positions left open by the rupture of peptide linkages, other transfer their nitrogen to deaminated molecules to form new amino acids...". The use of ~3C in biologic studies appears to have proceeded at a more measured pace than that of deuterium. The incorporation of ~3C into succinic,

pyruvic and formic acids by bacterial fixation was demonstrated by Nier and coworkers in 1941 (29) and led the way for subsequent demonstrations of the fixation of CO2 during photosynthesis. Olsen et al. (30) also spent considerable effort to synthesize ~3C-glycine, measure its rate of catabolism in the rat, and show the conversion of glycine to glycogen. An examination of Nier's bibliography (31) shows work with a consistent thread of biochemical and clinical applications. Some years ago he described how his entry into stable isotope studies was fortuitous; he and his coworkers just happened to have a ~3C-methane column operating in the stairwell, and they built the first ~3C mass spectrometer. The Iongtime collaboration between Nier and Wood was the result of a chance encounter when Wood visited his brother who was studying medicine at the Mayo Clinic. The eventual exploitation of the stable isotope ~80 to measure CO2 production in free-living subjects, and hence their energy expenditure, was brought about by the collaboration of Nier with Lifson from the Department of Physiology at the University of Minnesota (32). Together they showed that the oxygen of respiratory carbon dioxide was in isotopic equilibrium with the oxygen of body water and that the ~80 content of CO2 could be predicted from the rate of oxygen consumption and the weight of total body water.

4. THE BIOLOGIC CONSEQUENCES OF ENRICHED STABLE ISOTOPE USAGE

The biologic effects of stable isotopic substitution in enzymatic, cellular or physiologic processes can be subdivided into two categories: those that involve deuterium and those that involve all other elements found in an organism. Because of the large mass difference between deuterium and hydrogen, there is a corresponding effect on the chemical-bond reactivity. This same difference helps in the concentration and enrichment of deuterium in virtually limitless quantities. These quantities have enabled extensive investigation of the enrichment level required for toxicologic manifestations. However, the mass differences for isotopes of higher elements are much smaller; their physical properties are more similar, and thus, the enrichment of such isotopes becomes much more expensive. Moreover, the quantities necessary to investigate high levels of exposure become cost-prohibitive. Therefore, toxicity studies of the stable isotopes of biologic interest were undertaken in inverse order of their discovery (~80, discovered in 1929, toxicity determined in 1975; ~3C, discovered in 1929, toxicity determined in 1973; 2H, discovered in 1932 and toxicity determined in 1933). Thus, it was within the same year that deuterium was discovered, that the first studies of its biologic effects started. The zeal with which Lewis pursued this problem is evident in

his letter to the Journal of the American Chemical Society: "Even before I had succeeded in concentrating the isotope of hydrogen, I predicted that H=H=O would not support life and would be lethal to higher organisms. As soon as heavy water became available, experiments to test this idea were begun but it was necessary to choose an experiment which would require the minimum of biological techniques and also very small quantities of water" (33). Lewis chose to use tobacco seeds, germinated in sealed glass capillaries with 0.02 mL ordinary water or pure D20. Seeds in pure D20 did not sprout, but those in 50-percent D20 sprouted in normal time, thus giving the first LDso data for deuterium. Lewis concluded, "1 have long desired to determine the proportions of isotopes in living matter, in order to see whether the extraordinary selective power of living organisms, which is exemplified by their behavior toward optical isomers might lead to a segregation of isotopes in some of the substances that are necessary to growth. The marked biochemical differences between the two isotopes of hydrogen lends a further incentive to this search" (33). Taylor et al. (34) compared the toxicity of 92 percent and 30 percent heavy water in tadpoles, aquarium fish, flat worms and paramecia. All species succumbed in the highest concentration of deuterium within one to three hours, but survived in the 30-percent concentration. Lewis conducted the first mammalian study in which a mouse received 0.66 g pure D20 and showed evidence of thirst, but survived (35). Between 1934 and 1939, 216 papers were published on the biologic effects of deuterium. Most of the papers appeared within the first three years; the last seven were published in 1939 (36). Thus, after the classic studies were completed, it was not until the price of deuterium dropped from $20 to $0.20 per gram that interest was rekindled (see Table 1). Two papers of major importance that summarized the new work were by Katz, a review of chemical and biologic studies (37), and Thomson, a definitive monograph on the biologic effects of deuterium that discusses every consequent aspect of acute, chronic and low-level exposure to deuterium (36). The depth of study is much more attenuated for ~3C and is represented by several publications that resulted from work at the Los Alamos Scientific Laboratory. No evidence of toxicity was found at the highest enrichments attained (60 percent) in two mice (38), nor in mouse embryos cultured in media containing uniformly labeled ~3C-glucose as the sole energy source (39). More recently, Berthold et al. (40) fed a laying hen a ration which contained 50 percent Spirulina platensis grown in an atmosphere of pure ~3CO2. Over the course of 30 days, between 20 and 70 percent of all carcass amino

TABLE 1. Natural Stable Isotopic Content of the Human Body, Daily Consumption of Stable Isotopes, and Quantities of Stable Isotopes Used in Conventional Tracer Studies

Parameter

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acids were replaced with labeled forms. The hen laid 28 eggs during this time, and an analysis of the amino acids in the egg albumen showed two different and distinctive patterns (see Figure 1). Nonessential amino acids, exemplified by glutamine/glutamate, were catabolized and underwent extensive resynthesis. However, essential amino acids, such as phenylalanine, were incorporated intact and appeared as fully labeled isotopomers in the protein. This unexpected consequence of feeding fully ~3C-labeled nutrients has proved to be a valuable tool in identifying essential dietary components. To date, there has been no report of an investigation of whole organism response to 15N-enrichment levels. However, the physical chemistry of 180, has been well reported by Staschewski (41). Only because of the substantial resources of the Stable Isotope Department of the Weizmann Institute, however, was it possible for Samuel and colleagues (42-44) to raise three generations of mice in an atmosphere of 90 percent 1802 and to provide their drinking water as 90 percent H2180. No physiologic or biochemical effects were noted, and the mice reproduced normally through each generation without an increase in infant mortality. There is a large margin of safety in the use of stable isotopes in human studies. The enrichment of total body water with deuterium may be as high as 1 to 2 percent. In the cases of 13C, lSN or 180, the cost of substantial tissue isotope replacement is so prohibitive that research or diagnostic applications are unlikely to be undertaken. Nevertheless, it is useful to have an appreciation of the magnitude of an effect achieved with the introduction of a heavy isotope such as 13C. Biological processes which transform 13C-containing molecules, such as photosynthesis, discriminate slightly against the heavier (and less reactive) 13C in C02. The level of this discrimination can only be detected using the high precision of gas isotope ratio mass spectrometer

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measurements. For example, there are 11,117 molecules of ~3C02 in every 1,000,000 molecules of atmospheric C02. In the process of starch formation in a leaf, between 121 and 284 ~3C02 molecules are left behind for every 1,000,000 molecules of C02 used. The result is a product 1.09-2.55 percent lighter in ~3C than the starting material. Thus, biological systems tend to reject rather than retain the heavier form of isotope, but overall will reflect the isotopic composition of the diet. For example, Boutton et al. (45) studied cows whose diets were either switched from an alfalfa (with an isotopic abundance of -24%o) to a corn (-11%o vs. PDB) or vice versa. The milk produced by these cows quickly reflected the altered isotopic intake and reached equilibrium within 3-4 days. For these reasons, and the more important feature - absence of radiation - the use of these tracers in protected populations has been strongly favored by human investigation review committees. However, this perspective must be provided in an accurate manner to the subject or guardian from whom informed consent is required. The information in Table 1 may be used for this purpose. Listed is the natural abundance for each isotope, calculated as mg/kg of body weight, compared with normal daily intake, and with the amount used in most foreseeable studies. These values demonstrate the absence of any perturbation in body composition when stable isotopes are used. Stable isotopes offer a number of specific advantages to clinical pharmacologists, because they are ideally suited to answer the recurrent types of questions posed in studies. These applications have been the subject of several excellent reviews by Browne (46, 47) who has classified them according to the following types: use in isotope dilution techniques to measure the concentrations of drugs in biological fluids; determination of absorption, bioavailability, and distribution; biotransformation and excretion, including metabolite identification, mechanisms of drug metabolism, and quantitation of drug transformation and elimination. To these applications can be added the determination of compliance and the assessment of unwanted side effects of drugs. Examples of stable isotope applications in pediatric pharmacology are reviewed by Pons in this volume (48). When the applications of stable isotopes are reviewed today, their status remains true to the vision of Rudolph Schoenheimer when almost 60 years ago, he wrote, "The chemical constituents of the living body represent links in a chain of continuous reactions in which apparently all organic substances, even those of the storage material, are involved. It is with this aspect of the dynamic processes of life that the biochemist is especially concerned. The isotopes of those elements which are present in natural organic compounds,

10 presented to the biochemist by the physical chemist will certainly furnish a better insight into the details of this intricate m e c h a n i s m " (25).

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.

F. Soddy, J. Chem. Soc., 90 (1911) 72. F.W. Aston, Nature, 104 (1919) 334. F.W. Aston, Philos. Mag., 39 (1920) 449. F.W. Aston, Proc. Roy. Soc. Lond., Al15 (1927) 487. F.W. Aston, Mass Spectra and Isotopes, 2nd edn (Edward Arnold, London, 1924) p. 99. W.F. Giauque and H.L. Johnstone, Nature, 123 (1929) 318; J. Am. Chem. Soc., 51 (1929) 1436. W.F. Giauque and H.L. Johnstone, Nature, 123 (1929) 813; J. Am. Chem. Soc., 51 (1929) 3528. A.S. King and R.T. Birge, Nature, 124 (1929) 127; Phys. Rev., 34 (1929) 376. R.T. Birge, Nature, 124 (1929) 182; Phys. Rev., 34 (1929) 379. S.M.Naud~, Phys. Rev., 34 (1929) 1498. S.M. Naude, Phys. Rev., 36 (1930) 333. R.T. Birge and D.H. Menzel, Phys. Rev., 37 (1931) 1669. H.C. Urey, F.G. Brickwedde and G.M. Murphy, Phys. Rev., 40 (1932) 1. G.N. Lewis, J. Am. Chem. Soc., 55 (1933) 1297. G.N. Lewis and R.T. MacDonald, Chem. Phys., 1 (1933) 341. H.C. Urey, Science, 78 (1933) 566. G. Weiss, Chem. Eng. Tech., 30 (1958) 433. H.C. Urey and L.J. Greiff, J. Am. Chem. Soc., 57 (1935) 321. H.C. Urey and A.H. Aten Jr, Phys. Rev., 50 (1936) 575. J.R. Huffman and H.C. Urey, Ind. Eng. Chem., 29 (1937) 531. H.C. Urey, G.B. Pegram and J.R. Huffman, Phys. Rev., 4 (1936) 623. H.C. Urey, A.W.H. Aten Jr and A.S. Keston, Phys. Rev., 4 (1936) 622. A.O. Nier and J. Bardeen, J. Chem. Phys., 9 (1941) 690. A.O. Nier, Rev. Sci. Inst., 11 (1940) 212. R. Schoenheimer, Harvey Lectures XXXII (Academic Press, Amsterdam, 1936-37) p. 122. R. Schoenheimer and D. Rittenberg, Physiol. Rev., 20 (1940) 218. H.T. Clarke, A Symposium on the Use of Isotopes in Biology and Medicine (The University of Wisconsin Press, Madison Wl, 1948) p. 3. R. Schoenheimer, S. Ratner and D. Rittenberg, J. Biol. Chem., 130 (1939) 703. H.G. Wood, C.H. Werkman and A. Hemingway et al., J. Biol. Chem., 139 (1941) 365. N.S. Olsen, A. Hemingway and A.O. Nier, J. Biol. Chem., 148 (1943) 611. T.T. Scolman, W.H. Johnson and O.C. Alfred et al., Int. J. Mass. Spectrom. Ion. Phys., 8 (1972) 241. N. Lifson, G.B. Gordon, M.B. Visscher and A.O. Nier, J. Biol. Chem., 180 (1949) 803. G.N. Lewis, J. Am. Chem. Soc., 4 (1933) 3503. H.S. Taylor, W.W. Swingle and H. Eyring et al., J. Chem. Phys., 1 (1933) 751. G.N. Lewis, Science, 79 (1934) 151.

11 36. J. F. Thomson, Biological Effects of Deuterium, (Macmillan, New York, 1963) p. 133. 37. J.J. Katz, Am. Sci., 47 (1960) 544. 38. C.T. Gregg, J.Y. Hutson and J.R. Prine et al., Life Science, 13 (1973) 775. 39. C.T. Gregg, D. Ott and L. Deaver et al., Proceedings of the Second International Conference on Stable Isotopes, E.R. Klein and P.D. Klein (eds) (US Department of Commerce, Springfield, VA, CONF-751027 NTIS, 1975) p. 64. 40. H.K. Berthold, D.L. Hachey and P.J. Reeds et al., Proc. Nat. Acad. Sci., 88 (1991) 8091. 41. D. Staschewski, Agnew Chem., 13 (1974) 367. 42. D. Samuel, Proceedings of the Second International Conference on Stable Isotopes, E.R. Klein and P.D. Klein (eds) (US Department of Commerce, Springfield, VA, CONF-751027 NTIS 1975) p. 196. 43. D. Samuel, D. Wolf and A. Meshorer et al., Proceedings of the Second International Conference on Stable Isotopes, E.R. Klein and P.D. Klein (eds) (US Department of Commerce, Springfield, VA, CONF-751027 NTIS 1975) 203. 44. D. Wolf, H. Cohen and A. Meshorer et al., Stable Isotopes: Proceedings of the Third International Conference, E.R. Klein and P.D. Klein (eds) (Academic Press, New York, 1979) p. 360. 45. T.W. Boutton, H.F. Tyrell and B.W. Patterson et al., J. Anim. Sci., 66 (1988) 2636. 46. T. R. Browne, J. Clin. Pharmacol., 26 (1986)485. 47. T. R. Browne, Clin. Pharmacokinet., 18 (1990) 423. 48. G. Pons and E. Rey, Stable Isotopes in Pharmaceutical Research, T.R. Browne (ed) (Elsevier Science Amsterdam, 1996) p. 347.

13

CHAPTER 2

ISOTOPE EFFECT: IMPLICATIONS FOR PHARMACEUTICAL INVESTIGATIONS

THOMAS R. BROWNE Departments of Neurology and Pharmacology, Boston University School of Medicine; Neurology Service, Boston Department of Veterans Affairs Medical Center

1. INTRODUCTION

Any bond involving a heavy isotope and another atom will be stronger than the same bond between the corresponding light isotope and that atom. The greater mass of the heavy isotope results in greater force bonding it to the other atom and a greater energy of activation to break the bond (see Figure 1). In any reaction in which the breaking of this bond is the rate-limiting step, the reaction will proceed slower for the molecule with the heavy isotope due to this "kinetic isotope effect". In addition to kinetic isotope effects there are other ("secondary")isotope effects due to differences in bond length, bond angle, etc., of bonds involving heavy and light isotopes.

2. KINETIC ISOTOPE EFFECT

The physical chemistry of kinetic isotope effect on simple chemical reactions is reviewed in references 1-6. Kinetic isotope effects are modified during enzymatically mediated reactions following a set of rules reviewed in Chapter 15. This section reviews those aspects of the huge theoretical and empirical literature on kinetic isotope effect relevant to the design and performance of pharmaceutical investigations. Kinetic isotope effect is proportional to the mass difference between different isotopes of the same atom. Substitution of deuterium for hydrogen (100 percent mass difference) results in larger isotope effects than substitution of 13C for 12C, or lSN for ~4N (

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27 to be vaporized and thermally stable, this technique is being replaced by the newer thermospray, electrospray and API interfaces.

2.3.3. Thermospray ( TS) interface The TS interface has been effective for solving practical everyday bio-analytical problems requiring LC-MS (5, 8). Because of the potential for thermal degradation of heat-labile bio-analytes in the flash heater tube, TS can be a problem, and the likely reason for its replacement with electrospray and atmospheric pressure ion spray interfaces. This interface is both an inlet and ionizing interface that create ions via electrolyte-mediated chemical ionization during the course of solvent evaporation without an external ionization source. The TS interface allows the direct introduction of up to 2 mL/min of the eluant from the LC column into the MS. The function of this interface is to flash vaporize the LC eluant which assists in nebulizing the remaining liquid into a mist of fine droplets. It is during the rapid desolvation of the mist that the analyte molecules in the concentrated droplets become ionized due to the presence of a volatile buffer (i.e. ammonium acetate) in the LC eluant. The solvent vapor and other vaporized or nonvaporized nonionic substances in the eluant are drawn into the vacuum pump. This interface is applicable to polar nonvolatile analyte molecules that are thermally stable in aqueous/mixed aqueous LC mobile phases, with sensitivity in the low picogram range. Nonvolatile buffers (i.e. phosphates) and salts are not compatible with this interface. Although atmospheric pressure ionization interfaces are competing with thermospray interfaces, the latter have the advantage of larger flow volumes.

2.3.4. Continuous-flow or dynamic fast-atom bombardment interface ( CF-FAB) The CF-FAB interface has been a popular type of interface for the analysis of nonvolatile thermally labile ionic bio-analytes, such as peptide, proteins, oligonucleotides and oligosaccharides (9). With this interface, the LC eluant (typically 5-15 ~L/min) is mixed with a FAB matrix nonvolatile solvent, usually glycerin either in the mobile phase or added post-column, and forced through a narrow bore silica capillary onto the FAB target. The volatile solvents evaporate leaving a thin uniform film of glycerol on the surface of the FAB target which is then subject to bombardment from the FAB source. The ions are generated by bombardment of the continuously renewed liquid film with a

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m/z Figure 3. Background-subtracted continuous flow FAB spectrum of the peptide FTVWlEGVMR (MW 1237). Masses of amino acid sequence related fragment ion are also indicated (19). high energy beam of atoms. The advantage of CF-FAB over the conventional static FAB is less background signal from the matrix solvent (Figure 3).

2.3.5. Particle-beam (PB) interface The PB interface consists of a nebulizer, a desolvation chamber and a momentum separator that are connected to the ion source (5, 8, 10). The principle is analogous to the jet separator used with GC-MS to separate the solvents in the mobile phase from the analyte. With the PB interface, the liquid eluant is first converted into a mist of fine droplets which is desolvated as it is swept into the momentum separator. The separator increases the momentum of the more heavier analyte particles across a narrow gap into a heated capillary tube (the skimmer) for transport to the ion source, causing the lighter solvent molecules to diffuse away from the heavier analyte particles into the vacuum pumps. As a result of this process, a particle beam of analyte molecules that is nearly devoid of solvent enters the ion source. As the particle beam collides with the heated target in the ion source, the analyte molecules are flash vaporized prior to ionization by either El or CI. Because of the need for thermal vaporization, PB has been used mostly for volatile analytes and with LC eluants that are comprised largely of organic solvents.

29 3. THE ION SOURCE

3.1. Electron Ionization (El) El has been the most widely used ionization technique in mass spectrometry because of its extensive ion fragmentation for structural analysis of analytes and its reproducibility. Organic molecules that have been vaporized at high temperatures under a low vacuum (about 10 -s Torr) enter the ion source chamber and are bombarded by an electron beam. This causes molecules to lose an electron and generate a positively charged radical-cation (M-i-), the molecular ion, whose mass is the molecular weight of the original molecule. The low vacuum of the ion source minimizes collisions between the ions and neutral molecules preventing ion-molecule reactions. The highly energized molecular ion decomposes unimolecularly in a predictable fashion into fragment ions according to the strength of the different covalent bonds of which the analyte is composed (Eqs. 1 and 2). The fragment ions will continue to fragment until stable fragment ions are formed. The radical character of the molecular ion contributes to rearrangement reactions and the formation of unusual fragment ions. The fragment ions are characteristic of the original molecule and can be used to reconstruct the original molecule. Collectively, the combination of the molecular ion and the fragment ions constitute the mass spectrum of the analyte and can be used as a fingerprint for comparison with mass spectral libraries (Figure 4). El-MS is usually used only to detect positive ions. The negative ions and neutral fragments are pumped away. M + e--,

M-i- + 2 e -

M-i- -~ A § + B'

(1) (2)

--> CM-i- + D

3.2. Chemical Ionization (CI) Unlike the El mass spectrum with its molecular ion and abundance of fragment ions, the CI mass spectrum contains a quasi-molecular ion ([M + H] § and a low abundance of fragment ions (Figure 4). In some cases the molecular ion may attach itself to the reagent gas (e.g. [M + NH4] + with ammonia as the reagent gas). Consequently, the CI mass spectrum is more likely to give molecular weight information, but lacks the detail of the fragment ions that makes the El spectrum so useful for determining the structure of an analyte. Therefore, CI and El can complement each other in structural analysis. CI is

30

B

A

le~_.

112 (M~'H)*

82

O C C~ "O C: :3 ,,,ID

/CH2CH2NH2 H

mamm

dime

m

m i r a

rr,

le...

J[ I

"

.~0

/ M"" "

'"

"

~

-

-

.

I.

9

.

.

I

.

.

. .

.

.

.

.

.

I~!)

.

,

i,,=

"

.

I

.

,

,

100

MIZ Figure 4. (a) Electron ionization, and (b) chemical ionization spectra of histamine (MW

111) (8). commonly referred to as a soft or mild ionization technique. For CI to occur, the analyte molecule must be in the gas state, at a lower vacuum than for El (about 10 -1 to 10 -2 Torr), and the presence of a reagent gas. The reagent gas (e.g. methane, ammonia or in some cases solvent molecules) is in large excess in the ion source and undergoes El to produce reactant ions that can abstract a proton from the vaporized analyte to generate a quasi-molecular ion ([M + H] § (Eq. 5). Because the proton transfer reaction is a mild ionization process and occurs with lower energy than El, the quasi-molecular ion ([M + H] § is not highly energized and therefore, fragment ions are either of low abundance or absent altogether. The extent of fragmentation of the quasimolecular ion depends upon its internal energy gained from the ability of the vaporized molecule to abstract the proton from the reagent gas. For example, more fragmentation of the quasi-molecular ion occurs with methane as the reagent gas than with ammonia.

CH4 + e---, CH; + 2e-

(3)

31

CH~- + CH4---, CH~- + CH; CH~- + M--, [M + H] § + CH4

(4) (6)

Negative ions as well as positive ions are formed in the El and CI ion sources, but are not observed, because the electrical fields used, are for accelerating and detecting positive ions (5). By changing the polarity of the fields, the negative ions can be observed. Under El conditions, the high energy of the electron causes the negative ions formed to decompose into low mass ions that are of little value for obtaining molecular weight and structural information. However, if the electron is de-energized through numerous collisions with neutral molecules (Eq. 6), the electron can be more efficiently captured by those molecules that can stabilize an excessive negative charge to generate the negative molecular anion ([M_]). Those functional groups that have a high affinity for capturing electrons and stabilizing the negative charge are the halogens, oxygen, aromatic nitro groups and highly conjugated aromatic systems. This process is more successful with CI and is therefore referred to M + e---, M_

(6)

as negative chemical ionization (NCI). Depending upon its internal energy, the negative molecular ion can decompose into fragment ions, similar to that of a positive CI spectrum. Depending upon the type of electron capturing functional group(s), NCI can reach sensitivity below the femtomole level. Therefore, derivatization of the analyte with fluorinated reagents is a common technique for enhancing the sensitivity for trace analysis. Mass spectra recorded in the negative-ion mode are often complementary to the results from positive-ion spectra and is less subject to interferences from impurities that do not produce stable negative ions. For CI, FAB and APCI, negative-ion spectra have become routine and highly useful. Most instruments currently manufactured have negative-ion capability as an option.

3.3. Fast-Atom Bombardment (FAB) Ionization Both El and CI require sample vaporization prior to ionization. With highly polar and ionic nonvolatile biomolecules vaporization prior to ionization may lead to their thermal degradation without the production of a useful mass spectrum. Therefore, the need to volatilize these relatively nonvolatile and thermally unstable compounds without the analyte having to be volatilized

32 in an inlet system prior to ionization, led to the development of desorption ionization techniques (8). Desorption ionization relies upon the localized rapid vaporization of the analyte from a high energy source to produce gaseous ions that are expelled (desorbed) from the surface of the target into the mass analyzer. The gaseous ions are formed directly by focusing a high energy beam of xenon or argon atoms (fast atom bombardment, FAB) at the analyte that is attached to a solid target surface or in a liquid matrix. These gaseous ions expel (desorb) themselves from the solid surface of the beam target or liquid matrix forming a blanket of gaseous ions at the surface interface. These ions are swept into the mass analyzer generating a mass spectrum, similar to that from CI. FAB depends upon ion-molecule interactions for the generation of a quasi-molecular ion and a low abundance of fragment ions with unimolecular ion fragmentation occurring at the weaker covalent bonds. Therefore, FAB shares many characteristics with CI-MS. If an electrolyte is present, an adduct-molecular ion from the electrolyte cation and molecular ion is observed in the spectrum, e.g. [M + NH4]+ from ammonium acetate. Thus, with FAB techniques, the molecular weights of biomolecules greater than 10,000 Daltons have been desorbed. With CF-FAB, the analyte is mixed with glycerin and subjected to a beam of high energy xenon atoms which ionizes the analyte, causing the analyte ions to be expelled from the surface of the glycerin. The nonvolatile glycerin not only provides a matrix for the analyte but also helps to dissipate the thermal energy of the xenon atoms protecting the analyte from thermal degradation. The glycerin is also ionized by the beam of atoms, contributing to the formation of the quasi-molecular ion via solvent-mediated chemical ionization of the analyte from proton transfer reactions between the analyte and the ionized glycerin. The attachment of ionized glycerin molecules to molecular and fragment ions contributes to the complexity of the mass spectrum. The coupling of LC with FAB has extended mass spectrometry to the analysis of peptides, proteins and other thermolabile and ionic substances. The appearance of fragment ions from peptides provides information to confirm the amino acid sequence of the peptide. Matrix-assisted laser desorption ionization (MALDI) is similar to FAB except that the gaseous ions are produced by the laser desorption ionization process. This technique has become a popular method for the mass analysis of peptides to confirm its amino acid sequence. A pulsed high energy laser beam irradiates the solid analyte on the laser target surface producing molecular ions from thermally labile and relatively nonvolatile biomolecules without the problem of the formation of adduct ions from the liquid matrix or from volatile electrolytes common to FAB techniques. The solid analyte is dispersed in a

33 radiation-absorbing matrix (i.e. a fine metal powder) in order to limit thermal degradation to the analyte. Unlike the other desorption ionization techniques, MALDI is not a 'soft' ionization technique as with CF-FAB and does not rely upon ion-transfer reactions for the generation of the ions in the mass spectrum. The mechanism of ion generation remains a mystery. This type of desorption ionization is tolerant of nonvolatile buffers, salts and other substances. MALDI is coupled with the time-of-flight mass analyzer for the production of the mass spectrum.

3.4. Thermospray Ionization (TSI) TSI was introduced for converting ions in solution into gaseous ions for the mass analysis of ionic analytes (10). This method is similar to the direct introduction of a liquid in vacuo into the heated inlet chamber for desolvation prior to the sample entering the ion source. A solution of the analyte containing ammonium acetate (a volatile electrolyte)is forced through a heated capillary tube and is nebulized into a mist of fine droplets. A heated filament or electrical discharge may be used to facilitate ionization of the analyte and solvent molecules in the mist in the absence of a volatile electrolyte. As the droplets decrease in size from evaporation, the volatile electrolyte ammonium acetate yields gaseous ammonium and acetate ions which ionize the analyte molecules through an electrolyte-mediated chemical ionization (charge exchange) mechanism (6). This causes the droplets to develop an excessive charge and the eventual expulsion (desorption) of the ionized analyte molecule from the surface of the droplet into the mass analyzer. TSI is a mild ionization process and only ions indicative of molecular weight are present and structurally informative fragment ions are minimal (Figure 5). Consequently, the TSI mass spectra are similar to that from CI and lack the detail that make El spectra so useful for identification purposes. As a result of the presence of volatile electrolytes, an electrolyte adduct-molecular ion (for example [M + NH4] § with ammonium acetate) as well as the quasi-molecular ion ([M + H] § are found in the mass spectrum. Because of the large volume of vaporized solvent and water, extra large vacuum pumps are needed to draw off the solvent and water in order to maintain the vacuum in the inlet and mass analyzer.

3.5. Atmospheric Pressure Ionization (API) The capability of ionizing nonvolatile polar and ionic molecules at atmospheric pressure without high temperatures, has extended the applicability

34 le~L

ee._ 4) 0 C m

1TT

M

j coax H o ~ q

I

, ,

194

J """:! .......

"0

:

\,

!tl4

C:




e.--

m 4)

, . , . .

353

m.m ~

le_

m/z Figure 5. Thermospray spectrum of 4-methylumbelliferyl glucuronide (MW 352) (20). of MS to the analysis of drug-conjugate metabolites, peptides, proteins, oligonucleotides and other biomolecules. API is a soft ionization technique for the nonthermal ionization of the molecule at atmospheric pressures by spraying the analyte solution into an electrical field (5, 11). The techniques are called electrospray ionization, ionspray ionization, or atmospheric pressure chemical ionization, which can be coupled to magnetic sector, quadrupole mass filter analyzers, ion trap mass analyzers or time-of-flight mass analyzers.

3.6. Electrospray (ES) Ionization ES ionization depends upon the production of gaseous ions from ions in solution for a broad range of molecules, making it widely used for interfacing of LC to MS. ES is a soft ionization process, but unlike TSI, the process is without any thermal input in the ionization process (5, 11, 12). In ES, the analyte solution emerges from the capillary as a mist of fine droplets into a strong electrical field. The nebulization of the eluant into fine droplets is

35 assisted by a secondary or make-up gas flow (pneumatic nebulization). As the charged droplets decrease in size because of solvent evaporation, the charged analyte ions are expelled from the surface of the droplet. Uncharged nonvolatile material is swept away from the inlet to the mass analyzer by the secondary gas flow. Up to this point, the process is occurring at atmospheric pressure. The charged analyte ions are then drawn through a small orifice into the low pressure of the mass analyzer. As a result of the soft ionization process, fragmentation is usually absent and only molecular weight information is available. Peptide, proteins and oligonucleotides form multiply charged ions that give rise to clusters of peaks carrying slightly different numbers of charges. The multiply charged ions are produced primarily as a result of proton attachment to available basic sites in the molecules. MS separates ions according to their m/z ratio rather than mass. For example, an ion with a mass of 10,000 Daltons with 10 charges will be recorded at 'mass' 1000, reducing the mass range required from the analyzer. The addition of different volatile mobile phase buffers may facilitate structural recognition of fragments through changes in cluster ion formation. Because ES is readily coupled to LC-MS and CE-MS, these techniques are newer methods for obtaining molecule weight information for biomolecules (4). ES and MALDI are complementary techniques for the mass analysis of biomolecules. However, the quality of information from ES exceeds that from MALDI with better resolution and sensitivity. The application of ES in LC-ES-MS requires flow rates less than 10 i~L/min which could require the use of a post-column flow splitter with traditional or minibore columns >2 mm i.d. However, direct coupling with complete transfer of the injected volume into the MS can be achieved with microbore/nanobore LC (50 ~L/min and columns >2 mm i.d., a post-column flow splitter may be required. IS spectra are usually devoid of structural information since they mostly contain quasi-molecular ion [M + H] § and cluster ions. IP is more practical than ES because of the higher flow rates and no splitting or low post-column flow-split ratios.

36

3.8. Atmospheric Pressure Chemical Ionization (APCl) APCI is the chemical ionization of analytes in an ion source operated at atmospheric pressure (5, 11). For atmospheric pressure chemical ionization to occur, a heated nebulizer converts the liquid eluant from the column into a mist of fine droplets and vaporized solvent molecules. The mist is swept into the vicinity of the ion source and ionized by a corona discharge, generating positive and negative ions. The ionization of the analyte molecules is achieved by mechanisms similar to those with conventional CI, except that the protonated solvent and water molecules serve as the reagent gas (solventmediated ionization). APCI can operate in either the positive- or negative-ion mode. In the positive-ion mode, the formation of positive ions occurs from proton transfer with the solvent ions, adduct formation or charge-exchange reactions between the buffer and the analyte. Whereas in the negative mode, the negative ions are formed from proton abstraction, electron capture, or anion attachment reactions. APCI is run with conventional bore HPLC columns at nominal flow rates of 2 mL/min and the use of volatile or nonvolatile buffers are permitted. This technique appears to be more sensitive than TS.

4. THE MASS ANALYZER

See Table 2.

4.1. Magnetic Sector Analyzer The magnetic field of the magnetic sector analyzer is scanned causing the positively charged ions to accelerate and follow different circular paths according to their m/z ratios. For any one magnetic field strength, those ions with a given m/z ratio and equal energy will follow the path with the correct radius and arrive at the detector, whereas, those ions with an incorrect radius will be deflected to the sides of the analyzer and not arrive at the detector. By scanning the magnetic field, a complete mass spectrum can be obtained. The magnetic sector analyzer can be either a unit resolution single focusing or high resolution double focusing analyzer. The latter utilizes a combination of electromagnetic and magnetic fields to obtain m/z ratios with masses calculated to 3 or 4 decimal places. The mass analyzer is maintained at an internal pressure of about 10 -5 Torr, which keeps the ion from colliding with itself or with other ions or molecules in the system.

37 TABLE 2. Experimental Options in Mass Spectrometry

Ionization techniques electron ionization chemical ionization negative chemical ionization fast atom bombardment matrix assisted laser desorption thermospray ionization electrospray/ionspray ionization atmospheric pressure chemical ionization Mass analyzers magnetic sector scanning quadrupole mass filter ion trap time-of-flight MS/MS (collision activated decomposition) single- and multiple-ion scanning

4.2. Quadrupole Mass Filter Analyzer The quadrupole mass filter is composed of four circular rods that serve as electrodes using a combination of dc voltages and oscillating radio frequencies to filter ions within a limited range of m/z ratios. For any one dc voltage and radio frequency, only those ions with a given m/z ratio will follow a stable path and arrive at the detector, whereas, the other ions will develop an unstable path and collide with the rods without arriving at the detector. When the voltages to the rods are scanned at a fixed radio frequency, the m/z range of ions is varied allowing all the ions to traverse the length of the analyzer to the detector, thus, an entire mass spectrum can be recorded. The quadrupole MS is more rugged, less expensive with faster scan times than the magnetic sector analyzer, except that it resolves ions that differ in mass by one unit. Its range has been extended to 3000-4000 m/z.

4.3. Ion-Trap Mass Analyzer The quadrupole ion-trap mass analyzer utilizers similar principles to the quadrupole mass filter except that ions from the ion source are trapped for several seconds within a circular electromagnetic field before being expelled from the trap to the detector (13). The trap consists of a central ring electrode and

38 a pair of end-cap electrodes that control the entrance and exit of the ions. The ions from the ion source enter the ion trap through the upper end cap, and circulate in a stable orbit within the cavity of the ring electrode. Increasing the voltage to the ring electrode causes the orbits of the more heavier ions to become stabilized, while those for the lighter ions become destabilized, and are expelled from the ring electrode cavity through electrostatically controlled slits in the lower end cap into the detector. The ion trap is a powerful mass analyzer for the storage of ions over a wide mass range with excellent detection limits. This analyzer has been used as both an ion source and ion trap with El, CI, MALDI, and as an ion trap from external ion sources, API, ES and TS. The ion trap achieves its high sensitivity through ion storage and integration of the ion signal over an extended period of time to allow the detection of relatively strong signals from a weak ion beam. Thus, ion traps are especially useful for the trace analysis of analytes. Ion-trap spectrometers are more compact and less costly than the quadrupole instruments.

4.4. Time-of-Flight Mass Analyzer The time-of-flight mass spectrometers are relatively simple, inexpensive instruments that can analyze ions of very large and broad mass ranges with high sensitivity, reasonable resolution and mass accuracy (14, 15). This mass analyzer has become a widely used tool for the structural analysis of biological macromolecules such as proteins, carbohydrates and oligonucleotides. The time-of-flight mass analyzer utilizes the principle of the time it takes for an ion to cover a fixed distance from the ion source to the detector along a tube free of any electrical or magnetic control. After an initial acceleration from the ion source, the burst of ions will drift along the field-free tube according to their m/z ratios, and arrive at the detector at different time intervals. The velocity of an ion is inversely proportional to the square root of its mass. Therefore, small ions will travel much faster than heavy ions. This analyzer is more suited to desorption ionization methods, such as the pulsed matrixassisted laser desorption ionization method (MALDI), than from ion sources that generate ions continuously (e.g. El, CI, TS, ES).

5. THE DETECTOR, SIGNAL PROCESSOR/DATA SYSTEM, AND VACUUM SYSTEM See above.

39

5.1. Tandem M S - M S MS/MS instrumentation has found increasing use in LC-MS in conjunction with the soft ionization techniques used with TSI, FAB and API because their mass spectra usually show the quasi-molecular ion and are devoid of much fragmentation, therefore, minimal structural information. However, tandem MS affords fragmentation spectra for selected individual ions that allow full characterization for the identification of the analyte in question with improved sensitivity as compared with conventional MS (16). Most MS/MS systems consist of two mass analyzers arranged in tandem separated by a collision cell (16). A soft ionization source of the first MS produces predominately singly charged ions that selectively transmits an analyte ion of a specified mass (m/z) into the collision cell, where these fastmoving ions collide with neutral gas molecules, such as helium or argon. The collision cell induces fragmentation of the selected ion into numerous daughter fragment ions, which are scanned by the second analyzer generating a mass spectrum of the daughter ions (Figure 6). Thus, with MS/MS, a pure

(a)

145

100OCONHCH a

173

[185 k

100 (b)

-~ ~ . @

77

ft.

115

91

Precursor ion

39

29 ] ,

s,,

65 I ,

145 !

1

lOO nl/z

Figure 6. A MS/MS spectrum of carbaryl" (a) the methane CI mass spectrum, and (b) the argon collision daughter fragment ions of the m/z ion at 145.

40 ionic species can be individually separated from other interfering ions such as background ions in the mobile phase or from other unresolved components of the sample, and be structurally identified. Little sample cleanup is required. If the quasi-molecular ion is selected for transmission to the second analyzer, none of the background or other ions will reach the collision cell, unless these ions have a similar m/z ratio. In other words, you do not need to have a completely resolved chromatogram, and only minimal sample cleanup is required if using LC-MS-MS. Tandem MS is a technique that will find wider application in the solution of bio-analytical problems. Figure 6 depicts the results from a LC-CI-MS/MS analysis of the pesticide, carbaryl (17). In Figure 6a, the methane CI-spectrum produced the major fragment at m/z 145, corresponding to protonated a-naphthol. The product ion spectrum (Figure 6b) yielded a large number of fragment ions that confirmed, without question, the aromaticity of the pesticide and the structural confirmation of the pesticide as carbaryl. Tandem MS-MS is reviewed in greater detail in Chapter 5.

6. DATA ANALYSIS

6.1. Quantitative Analysis The basis for obtaining quantitative data is the accurate measurement of the abundance of a selected mass ion. Because of the lack of reproducibility of the ion source for the generation of the selected ion, quantitation is best achieved using an internal standard. The best accuracy is obtained using the analyte that has been isotopically labeled so that there is at least a separation of 3 m/z between the unlabeled and the labeled analyte in the mass spectrum. Deuterium is an excellent source for isotopically labeling the analyte. Also, the isotopic purity of the labeled analyte has to be included in the quantitation of the analyte. To enhance the sensitivity for the measurement of the ions, the data acquisition system can record only the ion current of selected ions characteristic of the analyte (selected ion monitoring, SIM) rather than measuring the ion current for the entire spectrum.

6.2. The Mass Spectrum The mass spectrum of benzoic acid is shown in Figure 7 (2). The x-axis is the m/z ratio. The peaks in the spectrum are normalized to the height of the most abundant or base peak (100 percent), the y-axis. The singly charged molecular (or parent) ion, if present, is typically the highest mass peak in the spectrum,

41

~

105

base peak

122

,o, I

I

C I~-OH / I 5

Mr-122

77

molecular ion 77

~LL,L ..... 11

9. , . , . , - , - . i r , . ,

, ,,'~i .,.~ r , , ' , , i 9 Ioo

MIZ Figure 7. The El mass spectrum of benzoic acid.

unless cluster ions, adduct ions or impurities are present. The lower mass ions (fragment or daughter), arise by decomposition of the molecular ion, either directly or by multi-step pathways (18). Especially for El, the normal routes of fragmentation are well understood and predictable, so that the spectrum of the unknown can be interpreted to reveal the molecular structure. The patterns are quite reproducible, and extensive mass spectral libraries are available for computer or manual search. By analyzing the fragment ions of the analyte, the whole molecule can be reconstructed. Even without a complete reconstruction of the analyte, a surprising amount of information about the structure of the analyte can be obtained. The abundance of any ion is its ability to stabilize a charge, without further bond cleavage from the excess internal energy remaining in the ion. Individual laboratories often assemble their own libraries appropriate for particular projects. Ions of low abundance are associated with each of the larger peaks. These correspond to the natural abundance of isotopes such as 13C, 2H, lSN, 180,

42 34S, 3701 and 8~Br. Because the MS can easily differentiate isotopes, the technique lends itself to studies that utilize the incorporation of stable isotopes into compounds of interest for purposes of quantitation, or for investigations of reaction mechanism or metabolic disposition. When the natural abundance of a stable isotope is high, the distribution pattern for the various ion clusters can be used to calculate the number of those atoms in each cluster. Figure 8 shows the El mass spectrum of pentachlorophenol (2). In the inset are the calculated distributions for peak abundances when 1-5 chlorine atoms are present. One can easily judge how many chlorine atoms are included in the molecular ion and in each of the major fragments.

,1~176176 ..

--

X +2

X

.--L

.

+4

X +4

.I--

-.

t--

-"

X +4 + 8

."

.

.

X +4 + 8

OH CI ~ ~ ' ~

CI

CI ~

CI

Y

9

!~

\"r"

CI

"-I-"I "t ~'I"-I"

1oo

200

l-,-r

I',I-'I

300

MIZ Figure 8. The El mass spectrum of pentachlorophenol. [Inset] the calculated distribution of the isotopic abundances for 1-5 chlorine atoms.

43 As the molecular weight of the analyte increases, the contribution of even the less-abundant stable isotopes becomes significant. For analytes with large molecular weights, such as polypeptides, the contribution of the less-abundant isotopes can become very significant when many atoms are present in the molecule. These factors must be given careful consideration when an analysis is planned so that the determination of the number and location of the labeled atoms will be experimentally feasible. Incorporation of a sufficient number of heavy atoms to shift the labeled peak away from the normal abundance cluster is an obvious advantage. Derivatization of the sample may be necessary in order to achieve volatility or, to minimize or direct, mass spectral fragmentation. Selection of a derivative should take into account both the chromatographic properties of the products and the distribution of ion current in their mass spectra. The mass spectrum of the derivative of choice will have a molecular-weight related ion with good abundance and structurally significant high-mass fragments (which retain the label, if one is used). This assures the uniqueness of the analysis and minimizes interference from low-mass background ions. Figure 9 shows the trimethylsilyl (TMS) and methyl esters of phenyl acetic acid (2). In this case, the methyl ester is clearly preferable; the spectrum of the TMS ester has only a very low abundance molecular-ion, and the base peak corresponds to the TMS group. The abundance of individual ions in the spectra of compounds of interest can be plotted as a function of time, producing a signal that is relatively free

I

I

159

i . ~ . ~ , C-~ 9,1 o 173

9o =.

8 i I I

9,

73 M +"

119

208

M-+" 150

=.

M+, M+

.,.,,

,,,!,i

j"

i,..~L,.,,.,.,,.I.

L L2

j~

' ,-,-,-.,

..,.,_,.,.l.,:,:.i.

9 d

,

.i-,-,-,,--r-,.,.,-i

MIZ

Figure 9. El mass spectra of phenylacetic acid derivatives" (a) the trimethylsilyl ester, and (b) the methyl ester.

44 of interference. This type of trace (a mass chromatogram) can be generated from sets of complete spectra in order to locate components with specific molecular weights or a common structural feature in a complex mixture or to enhance chromatographic resolution. Specific ion signals can be recorded with very high sensitivity by selectively detecting one or a few ions during the entire experiment (single- or multiple-ion monitoring). The former approach is useful for qualitative analysis or for profiling of complex mixtures; the latter approach provides good quantitative results. Exact mass measurements made at high resolution have sufficient accuracy for determination of elemental composition of molecular and fragment ions. This information helps in the elucidation of structure of unknowns and also in the characterization of new compounds that cannot be isolated in quantities necessary for elemental analysis. In those cases in which the El mass spectrum does not include a molecular ion, the elemental composition of the compound may still be determined by employing one of the several soft ionization techniques to assign a composition to the (M + H) § or M+ ion or other appropriate molecular-weight related species. High-resolution measurements may also be necessary when impurities that produce ions isobaric with those of interest are present. Thus, the analyst today has the choice of a wide range of mass spectral capabilities. These are summarized in Table 2. The selection of the method to be employed will be dependent on the information sought, the type of analyte (its volatility or nonvolatility, thermostability or thermolability) to be analyzed, and the level of sophistication (of both operator and instrument). This is clearly the era of biological mass spectrometry, the study of macromolecular science in the overall context of human health and disease (4). The technical advances in creating ions efficiently and reproducibly from nonvolatile polar and labile biopolymeric substances has redefined bio-analytical chemistry. This dynamic field has much to contribute toward the solution of pharmacologic and biochemical problems.

REFERENCES 1. R.J. Anderegg, Mass Spectrometry: An Introduction, in Biomedical Applications of Mass Spectrometry, Vol. 34, Methods of Biochemical Analysis (John Wiley, New York, 1990), pp. 1-89. 2. C.E. Costello, Fundamentals of Present Day Mass Spectrometry, J. Clin. Pharmacol., 26 (1986) 390. 3. W.H. McFadden, Techniques of Combined GC-MS (John Wiley, New York, 1973). 4. A.L. Burlingame, R.K. Boyd and S.J. Gaskell, Mass Spectrometry, Anal. Chem., 66 (1994) 634R.

45 5. W.M.A. Niessen and A.P. Tinke, Liquid Chromatography-Mass Spectrometry: General Principles and Instrumentation, J. Chromatogr. A., 703 (1995) 37. 6. W.M.A. Niessen and J. van der Greef, Liquid Chromatography-Mass Spectrometry (Marcel Dekker, New York, 1992). 7. E. Gelpi, Biomedical and Biochemical Applications of Liquid ChromatographyMass Spectrometry, J. Chromatogr. A., 703 (1995) 59. 8. J.R. Chapman, Mass Spectrometer as an LC Detection Technique, in: A practical Guide to HPLC Detection, D. Parriott (ed) (Academic Press, New York, 1993). 9. R.M. Caprioli (ed), Continuous-Flow Fast Atom Bombardment Mass Spectrometry (John Wiley, New York, 1990). 10. A.L. Yergey, C.G. Edwards, I.A.S. Lewis and M.L. Vestal, Liquid Chromatography/Mass Spectrometry: Techniques and Applications (Plenum Press, New York, 1990). 11. E.C. Huang, T. Wachs, J.J. Conboy and J.D. Henion, Atmospheric Pressure Ionization Mass Spectrometry: Detection of the Separation Sciences, Anal. Chem., 62 (1990) 713A. 12. P. Kebarle and L. Tang, From Ions in Solution to Ions in the Gas Phase: The Mechanism of Electrospray Mass Spectrometry, Anal. Chem., 65 (1993) 972A. 13. S.A. McLuckey, G.J. van Berkel, D.E. Goeringer and G.L. Glish, Ion Trap Mass Spectrometry of Externally Generated Ions, Anal. Chem., 66 (1994) 689A. 14. R.J. Cotter, Time-of-Flight Mass Spectrometry for the Structural Analysis of Biological Molecules, Anal. Chem., 64 (1994) 1027A. 15. M.G. Qian and D.M. Lubman, A Marriage Made in MS, Anal. Chem., 67 (1995) 234A. 16. F.W. McLafferty (ed), Tandem Mass Spectrometry (John Wiley, New York, 1983). 17. T. Cairns and R.A. Baldwin, Pesticide Analysis in Food by MS, Anal. Chem., 67 (1995) 552A. 18. F.W. McLafferty, Interpretation of Mass Spectra, 3rd edn (University Science Books, Mill Valley, California, 1980). 19. D.J. Bell, M.D. Brightweil, W.A. Neville and A. West, Rapid Commun. Mass Spectrom., 4 (1990) 88. 20. D.J. Liberato, C.C. Fenselau, M.L. Vestal and A.L. Yergey, Anal. Chem., 55 (1983) 1741.

47

CHAPTER 4

MASS SPECTROMETRY: LIQUID C H R O M A T O G R A P H Y - MASS SPECTROMETRY

JAMES E. EVANS E. K. Shriver Center, Inc., 200 Trapelo Rd., Waltham, MA 02254

1. INTRODUCTION 1.1. Background

For nearly thirty years, mass spectrometry (MS) has been the premier technique for the detection, structure determination and quantitation of compounds labeled with stable isotopes. High-performance liquid chromatography (HPLC) developed over this same period into the preferred technique for separation and measurement of compounds in biological research. Analysis by HPLC requires much less sample isolation and derivatization and is applicable to a much wider range of compounds than the previously well-established gas chromatography. The on-line combined technique of LC-MS provides the advantages of both and is widely recognized as the most powerful tool available for analysis with high sensitivity and specificity of low concentrations of drugs and their metabolites in biological matrices. The ability of LC-MS to distinguish and measure compounds labeled with stable isotopes with the same analytical prowess makes it the obvious choice when tracer studies with pharmaceuticals are considered. Yet, only a few examples of the application of stable isotope-labeled pharmaceuticals with LC-MS analysis are found in the literature. Reasons for this may include what seems to be the small, albeit increasing, role stable isotopes have in studies of drug disposition and the fact that easy to use high-performance LC-MS instrumentation has only become available over the last five years. LC-MS instrumentation has now developed to the point that it can conveniently solve the difficult bioanalytical problems encountered in such studies. This chapter

48 will describe the operation and capabilities of LC-MS instrumentation and illustrate how it can be best used for stable isotope tracer studies of drug disposition. Since the early 1970s, intense research activity resulted in the development of highly successful approaches for interfacing the rapidly developing technique of HPLC directly to MS. The primary impetus for this was to extend the demonstrated benefits of combined GC-MS to the majority of compounds that are too involatile or thermally labile to pass through a GC column even after derivatization. During this developmental period more than 25 LC-MS interfaces were reported in the literature and about ten of these have been offered commercially (1). Today only five interfaces have survived the competition and are commercially available. These are continuous flow fast atom bombardment (CF/FAB), particle beam (PB), thermospray (TS), electrospray (ES), ionspray (IS) and atmospheric pressure chemical ionization (APCI). LC-MS now makes possible the convenient analysis of a very broad range of compounds in complex matrices often with very high sensitivity and precision. These compounds include polar, involatile, thermally liable and/or high molecular weight analytes such as peptides, proteins, oligosaccharides, polynucleotides, etc. Mass spectrometric analyses of many of these were unimaginable twenty-five years ago. 1.2. L C - M S Interfaces

LC-MS interfaces are conveniently divided into two groups: those that deliver the LC analytes to a conventional ion source for subsequent ionization and those that ionize the analytes and transmit the ions to the mass spectrometer. These are referred to respectively as transport and ionization type interfaces. Figure 1 provides an indication of the amount of developmental and applications effort that is going into each LC-MS interfacing technique. The ionization type interfaces, notably the API interfaces, are currently attracting the most interest and developmental effort. Transport interfaces deliver analytes from the HPLC eluate to a conventional MS ion source where they are ionized and subsequently mass analyzed. They include the direct liquid introduction (DLI), moving belt (MB) interface, PB and CF/FAB interfaces. DLI and MB were the first successful techniques developed for LC-MS interfacing and were available commercially in the late 1970s. The DLI interface, conceptually and physically the simplest of all LC-MS interfaces, functions by spraying the chromatography eluate at a few I~l/min directly into a conventional chemical ionization (CI) ion source. There both the sample and solvent are thermally vaporized and the sample components

49 400 350

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I-IAPCI i E S I

300 t~ o 250 O. o 200 t_ O

E 150

:3 Z

100 50

1991

1992

1993 1994 1995 Meeting Year Figure 1. Graph of number of presentations concerning different LC-MS interfacing methods presented at the annual American Society of Mass Spectrometry Conferences from 1991 through 1995.

ionized by CI using the vaporized solvent as the reagent gas. Two transport interfaces, MB and PB, deliver solute nearly free of LC solvent to the ion source and have the advantage of allowing conventional electron impact (El) or CI mass spectra to be acquired. PB, MB and DLI interfaces have the limitation that analytes must have sufficient volatility and stability to be thermally vaporized before ionization can occur. While this condition limits the analysis to such compounds, it is a much less severe limitation than is encountered by GC-MS analyses. Indeed compounds that were traditionally considered quite nonvolatile or thermally labile have been successfully analyzed by M B LC-MS with chemical ionization (2, 3) and can provide spectra that are similar to those obtained by direct chemical ionization (DCI). Vaporization of highly nonvolatile or thermally labile biopolymers remains a problem for these interfaces. There is no such requirement for direct volatilization with CF/FAB and its range of application extends to biopolymers with molecular weights

50 up to about 5,000. The transport interfaces discussed in more detail below are currently used for a variety of analyses but are losing popularity in favor of the newer atmospheric pressure ionization interfaces (API). Because the DLI and MB interfaces are little used and not presently commercially available they will not be discussed in detail below. While the popularity of transport interfaces as a group has decreased enormously in favor of the API interfaces, the MB, PB and CF/FAB techniques still retain areas of application in which they are quite useful. LC-MS interfaces that serve as ionization sources include the thermospray interface (TSP) and three API interfaces: ES, IS and APCI. TSP achieved a very high level of popularity and was the dominate LC-MS interface from the mid1980s until recently, especially for drug analysis and development studies. Since the announcement of ES by Fenn and coworkers (4) it, and subsequently APCI and IS, have become the dominate LC-MS interfaces. TSP is now being replaced by those techniques that offer higher sensitivity and are easier to use with a much wider range of compounds. The atmospheric pressure ionization interfaces are having a tremendous impact on the analysis of analytes with a very wide range of properties and are much more universal for analysis of compound types than any of the earlier techniques. Currently a large portion of instrumentation development in LC-MS is directed toward further development of API interfaces.

1.3. Objectives of this Review This chapter will present a practical overview of LC-MS techniques, providing guidelines that will indicate the potential and limitations of each technique for analysis of various sample/compound types and the compatibility of each interface with various types of separation systems. Features of each interface that make it particularly useful or less applicable for stable isotope tracer studies will be noted. The capabilities that are desirable for stable isotope tracer experiments generally include high sensitivity, accuracy, reproducibility and, of course, a capacity of good mass discrimination. Rather than presenting an all inclusive overview of interfacing techniques and the history of their development, this chapter will be limited to those that are available commercially. Because only very limited use of LC-MS has been made for studies with stable isotope tracers, examples used here will usually be drawn from studies in which they were not utilized but that will illustrate the potential of the technique for stable isotope tracer studies. A number of useful recent reviews of LC-MS development and applications have appeared that can provide the reader with further information concern-

51 ing these techniques. One by Burlingame (5) is highly recommended and appears biannually in Analytical Chemistry, and covering the major developments in mass spectrometry over the preceding two-year period. Because LC-MS interfacing has been one of the most active areas of development in mass spectrometry, much of this review concerns LC-MS. A good recent review of the general principles and instrumentation is by Niessen and Tinke (6). Several reviews of the application of LC-MS in pharmaceutical or biomedical research are worth note (7-9). Other reviews related to specific interfaces will be noted in the discussion of each technique.

2. LC-MS INTERFACING METHODS 2.1. Transport Interfaces 2.1.1. Particle beam L C-MS The PB interface is now well established and popular as a user-friendly and versatile LC-MS interface (Figure 2). The jet separator developed by Ryhage (10) for interfacing packed column GC-MS served as a model for development of the PB interface. Takeuchi et al. (1 1) reported the use of a device similar to a GC-MS glass jet separator to interface micro column HPLC to MS with limited success. Willoughby and Browner (12) were then largely responsible Two Stage Momentum Separator

He in

Nebulizer ----

LC Solvent

Heated Desolvation Chambel

/'

~

Ion Source i lli

~1

I'

Vacuum Pumps

Figure 2.

Schematic representation of a particle beam LC-MS interface.

52 for developing an understanding of many of the critical factors for achieving high enrichment and recovery of solutes from liquid flow streams. The insight gained from their work led to today's commercial PB interfaces. Most mass spectrometer manufacturers now offer PB interfaces for their instruments instead of the once commonly offered MB interfaces. Because PB delivers the concentrated LC analytes nearly free from solvent (enrichment of about 10s) to the ion source, it is highly versatile and has been used with most types of mass spectrometers and with a wide array of ionization sources including El, CI, FAB, laser desorption, high temperature surface ionization and chemical reaction interface mass spectrometry (CRIMS, discussed in Chapter 6 of this volume). The PB LC-MS interface is the only one currently commercially available that allows the acquisition of El and CI mass spectra that are identical to those obtained by conventional GC-MS or direct probe inlets. These El spectra are useful for structure elucidation based on established rules and can be searched against library reference spectra. PB also has few limitations for analysis of different compound types and chromatographic solvents beyond those of the ionization source used. Ease of use, one of the advantages of this technique, results from its simple design and operation. It is the most popular of the transport-type interfaces and the only one that is currently undergoing active development. A recent detailed review of the development, operation and use of this interface by Creaser and Stygall (13) is highly recommended. Figure 2 is a schematic representation of the PB interface that will serve to illustrate its operation. It functions solely by aerodynamic means, enriching solute from an LC flow-stream and transporting it directly to a conventional MS ion source. Several important and distinct steps are involved in this process. The first is the conversion of the LC solvent into an aerosol of small uniform droplets by nebulization in a stream of helium. This aerosol then passes through a desolvation chamber that is maintained slightly above ambient temperature, where much of the solvent evaporates and residues from the dissolved analytes form particles. The resulting suspension of solute particles in solvent vapor and helium nebulizing gas is collimated by passing through a small orifice into the first pumped chamber (about 103 Pa) of the 2-stage momentum separator where much of the gasses are pumped away. The beam of more massive particles continues on through an in-line skimmer into the second chamber (about 100 Pa) where the process is repeated. Finally, the collimated particle beam continues its line-of-site path through a transfer tube into the ion source of the MS. The design and operation of the nebulizer and the diameter of the sampling orifice, the temperature of the desolvation chamber and the spacing of the skimmers in the momentum separator are

53 critical for high transmission of solute containing little solvent to the mass spectrometer ion source (12). For highest transmission of analyte the particles should retain some solvent to increase their mass. Huang and coworkers (14, 15) investigated the interaction between mobile phase composition, flow rate, helium nebulizer gas pressure and desolvation chamber temperature on the optimization of two commercial PB interfaces from Hewlett-Packard and Extrel. They found that these interfaces behaved quite differently with respect to those variables and that adjustment of nebulizer flow and desolvation chamber temperature for different solvent compositions was critical to obtain maximum sensitivity. One commercial PB interface that departs significantly in design from others is the Vestec Universal Interface (16). It incorporates a heated thermospray vaporizer in place of the pneumatic nebulizer for more complete vaporization of the solvent and a membrane separator that removes much of the solvent vapor prior to the momentum separator. Helium is admitted separately to serve as a transport gas to carry the aerosol through the membrane separator and the momentum separator. This device has been reported to achieve a much more complete removal of the solvent than other PB designs, and to be the only PB interface suitable for use with LC-CRIMS where the usual levels of residual solvent would swamp the microwave reaction interface (17). Problems with low transfer efficiencies (18), nonlinear response and chromatographic band-broadening have been widely reported with L C - P B - M S interfaces. While these problems are of considerable consequence they do not preclude accurate quantitative analysis when the proper measures are taken. They do indicate that a better understanding of the processes of nebulization, desolvation and momentum separation needs to be gained for future design modifications if PB is to gain wider popularity. As discussed below, a number of reports have demonstrated that accurate and reliable data can be obtained with L C - P B - M S without difficulty. Nonlinear calibration curves have been of major concern in the use of PB for quantitative analysis. Response generally varies exponentially with analyte concentration for reasons that are not presently well understood. This results in poor detection limits and a limited dynamic range. Factors, including particle size and particle charging, have been investigated as the source of this problem without clearly identifying its basis. Investigations have demonstrated that some measures can be taken to considerably improve both response and linearity. One measure is to add a carrier compound (often ammonium acetate) to the chromatographic solvent. Apffel and Perry (18) investigated this carrier effect with a number of analytes and additives and found that the effect was more pronounced with some combinations than

54

others. In general, this effect is substantial and can produce, in many cases, considerable signal enhancement and linear calibration curves. This ammonium acetate carrier effect is not observed with the Vestec Universal Interface, presumably because the membrane separator removes most of the ammonium acetate before the momentum separator. The other effective approach to correction of this problem has been the use of co-eluting stable isotope-labeled internal standards. This also results in linear calibration curves and in most cases considerable signal enhancement. A good discussion of the problems with nonlinear calibration curves and poor analyte yield is presented by Creaser and Stygall (13). The PB interface works well for the analysis of compounds in a midrange of volatility and with considerable thermal stability. A problem occurs with less massive, or more volatile, analytes because they are vaporized and pumped away with the helium and solvent vapor and not transmitted as particles to the mass spectrometer. Another limitation arises with compounds of low volatility or low thermal stability. When these particles arrive in the ion source they must be thermally vaporized before ionization by gas phase techniques such as CI and El. This vaporization occurs by collision with the heated wall of the ion source and provides a major limitation to the PB analysis of these compounds. This, together with the limit on higher volatility compounds, results in a volatility range for analytes that is somewhat restricted. Richardson and Browner (19) demonstrated that much higher than usual ion source temperatures or laser vaporization leads to much higher sensitivity with much less thermal decomposition when thermally labile/low volatility compounds are analyzed. Ionization methods that do not require thermal vaporization have been used in combination with the PB interface and have shown promise for the analysis of compounds with low volatility, poor thermal stability or higher molecular weight. FAB using a PB interface has been demonstrated (20, 21) and provided good spectral quality and high sensitivity without the flow rate limitation or solvent load experienced with continuous flow FAB. PB-massive particle impact ionization-MS (22) is a somewhat similar technique to FAB and uses massive glycerol cluster impact to ionize the sample in place of FAB. This approach shows promise for the analysis of compounds beyond the molecular weight range of FAB because it produces multiply charged ions similar to the API methods such as ES. Early reports of attempts to perform matrix-assisted laser desorption/ionization (MALDI) directly on matrix containing particles in the ion source were presented and shown to be promising (19, 23). PB-hyperthermal surface ionization-MS that produces ions by collision of a supersonic molecular beam of analyte particles with a platinum, tungsten

55 or rhenium surface has been developed using a modified PB interface (24). The developers reported ionization efficiencies ten times higher than normal El ionization for polycyclic aromatic hydrocarbons using this technique. PBchemical reaction interface-MS (LC-PB-CRIMS) has been reported for the selective detection and measurement of stable isotopes of carbon, hydrogen, nitrogen, oxygen, sulfur, phosphorous, selenium, bromine and chlorine in organic compounds (17). The LC-PB-CRIMS technique has shown considerable promise and is covered in Chapter 6 of this volume. Furthermore, developments of ionization techniques that take advantage of PB's ability to deliver highly enriched analyte from an LC flow stream are expected. Several recent applications demonstrate quantitation of pharmaceuticals in biological matrices using LC-PB-MS. These are selected to demonstrate that nonlinearity and other problems do not preclude high quality quantitative analyses with good sensitivity. Many of these applications used electron capture negative ion chemical ionization (NCI) to achieve high sensitivity. Girault et al. (25) reported a LC-PB-NCI/MS assay for the measurement of BN50727 (a platelet activating factor antagonist) in human plasma and urine. They used a simple solid-liquid extraction procedure after addition of a chemical analog internal standard prior to injection. A quantification limit of 1 ng/ml and very good reproducibility were reported. The procedure was used to obtain preliminary pharmacokinetic data. The same group also developed a similar method for another platelet activating factor antagonist with similar success (26). The measurement of the antibiotic tylosin in bovine muscle by LC-PB-NCI/MS was reported to be useful as a confirmatory technique for residues in animal products (27). Similar assays for spiramycin (28) and chloramphenicol (29) residues have been reported by the same group. Celma (30) reported a method for measurement of an aza alkyl lysophospholipid in rat plasma after a single liquid-liquid extraction using a polymeric reversed phase column and LC-PB-EI/MS. He was able to make measurements in the range from 25 ng/ml to 5 i~g/ml with good accuracy and precision and applied the procedure in pharmacokinetic studies. The immunosuppressant FK506 and its metabolites were determined in blood and urine by Christians et al. (31) using LC-PB-NCI/MS. They achieved a 25 pg limit of detection for standard solutions and a limit of quantification of 250 pg for blood (CV 11.3 percent at 5 ng). An excellent example of the use of LC-PB-MS for stable isotope tracer metabolism studies has appeared from Markey's group (32). They reported quantitative studies on the conversion of [13C6]-L-tryptophan to [13C6]-L-kynurenine in interferon-~/stimulated human monocytes and poke weed mitogen stimulated gerbil lung and brain slices. The effect of whole body stimulation

56 with interferon--y on plasma levels of endogenous of L-kynurenine was assessed. After addition of 180- or 2H-labeled internal standards to biological samples, they used a simple combined extractive derivatization to form electron capturing pentafluorobenzyl derivatives of tryptophan and kynurenine. These were separated on either a 1.1 or a 2.1 mm i.d. normal phase silica column using gradient elution with toluene-ethyl acetate mixtures at 0.4ml/min flow rate. LC-PB-NCI/MS was performed to obtain standard curves that were linear over the range of 1-250 ng/sample with limits of detection from 10-50pg injected. Excellent recovery and reproducibility values were reported. Recently this group reported improvement of the derivatization procedure and inclusion of a number of additional tryptophan metabolites in the analysis (33). 2.1.2. Continuous flow FAB

The introduction of fast atom bombardment mass spectrometry (FAB-MS) by Barber et al. (34) caused a revolution in biochemistry and mass spectrometry. FAB-MS is also used here to refer to the similar technique of liquid secondary ion emission-MS (liquid-SIMS or LSIMS). It made possible for the first time the MS analysis of many compounds that were polar, thermally labile and/or of higher molecular weight. Before FAB-MS, it was necessary to convert such compounds to volatile and thermally stable derivatives, if possible, for analysis by gas phase ionization techniques or to use field desorption ionization, which was only marginally successful with a select group of compounds. Because FAB produces gas phase ions by bombardment with high energy atoms of the surface of a solution of analytes in a low volatility matrix, it eliminates the need for thermal vaporization. FAB ionization causes little fragmentation and produces primarly pseudomolecular ions even with very labile compounds. This made possible the analysis of previously unapproachable compounds. These included a large variety of biological compounds such as peptides, oligosaccharides, drug conjugates, etc. This capability generated a tremendous interest among biologists and resulted in intense research efforts to further develop FAB-MS, and subsequently many other new MS methods, for the analysis of such compounds. One of the main disadvantages of FAB is the intense matrix ions that obscure portions of the spectra, especially at low mass. Another disadvantage that makes mixture analysis difficult is ion suppression that occurs when better responding components of mixtures suppress ion production from other components. A second phase of this revolution began with attempts to combine FAB

57 Frit

Fused Silica Capillary

/

LC i n _ _ ~ , ~ 4 ~ l ~ .

.~

Wick// Fast Atom

Ii AnMiSzer

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Beam//" ""'"'""

Figure 3. Schematic representation of a continuous flow fast atom bombardment probe.

with a separation technique (HPLC). The first LC-FAB-MS techniques used a moving belt transport interface (35). The fast atom beam was focused directly onto the belt where the residues were ionized. Addition of matrix to the belt was difficult and often had little or negative effect on the resulting spectra. Moving belt FAB was difficult to use and did not provide the necessary sensitivity or the soft ionization of probe FAB to make it a method that was useful for practical analyses. Continuous flow FAB (CF/FAB, also termed frit FAB or dynamic FAB) was introduced by Ito et al. (36) as a much simpler and more successful approach to on-line LC-FAB-MS. Figure 3 illustrates a contemporary device that is not very different from the one they used. Mobile phase containing a few percent glycerol (to serve as the FAB matrix) flows through a capillary tube onto a stainless steel frit at the tip of the FAB probe. The volatile components of the solvent evaporate leaving the analyte dissolved in the remaining glycerol matrix on the surface of the frit. There it is the target for the fast atom beam. Ito et al. (36) used nano-scale chromatography with a packed capillary column that ran through the FAB probe and terminated at the frit. Today's CF/FAB interfaces differ from this original design only by having a heated probe tip to avoid evaporative cooling and freezing of solvent and the addition of a wick to absorb excess solvent and matrix. Caprioli et al. (37) reported a modified design that allowed direct introduction of samples either from an external HPLC stream or reaction vessel or by direct injection. This design differed significantly from Ito's in that the flow from an unpacked capillary went directly onto a copper target at the probe tip and used no frit. With the capillary serving as a transfer line, analysis of flow streams from a variety of sources including HPLC columns, capillary electrophoresis, direct injections of samples, on-line monitoring of incubations and in vivo microdialysis were

58 reported. A book edited by Caprioli (38) summarizes many aspects of the development and application of CF/FAB. CF/FAB is now a well-established technique that has proved useful for analysis of a wide variety of compounds. It provides high sensitivity and, consequently, selectivity for the analysis of compounds that have amphiphilic character and/or a strong ionic functional group. Other compounds that do not have this character are also successfully analyzed but usually with lower sensitivity. The design of CF/FAB interfaces seems to be quite stable and not to have undergone much development in the last five years. Most commercial interfaces available today are of the frit-FAB design and use a 50 i~m i.d. fused silica capillary to transfer the analyte-containing solvent to the probe. The flow rate is limited to about 5-10 i~l/min by the vacuum requirements of the mass spectrometer. Because of this it is necessary to split the flow from HPLC columns with a larger i.d. than about 0.5 mm. The requirement for matrix in the solvent has been met either by addition pre-column to the LC mobile phase or post column via a coaxial delivery system that mixes the matrix with the mobile phase at the probe tip. Reports have appeared to demonstrate that addition of a viscous matrix such as glycerol to the solvents used for chromatography has a detrimental effect on chromatographic efficiency (39, 40). This effect is of considerable consequence at levels above about 5 percent matrix in the chromatographic mobile phase. It is now normal practice to include from 1 to 5 percent matrix in the chromatographic mobile phase. This level of matrix is sufficient to provide optimal FAB results but does not impair chromatographic efficiency to a significant degree. Cesium and massive cluster ion sources have been used in place of FAB and provide improved sensitivity because these beams can be focused to provide high intensity on the target. The massive cluster ion source offers the additional advantage of producing multiply charged ions from higher molecular weight compounds (similar to electrospray) allowing them to be detected by analyzers that have an upper m/z limit of less than 2,000. CF/FAB has very significant advantages over static FAB in addition to the ability to couple on-line to LC. These include increased sensitivity, the ability to effectively subtract background from spectra and much less ion suppression of compounds in mixtures. The increase in sensitivity is often on the order of a 100-fold or more. It results from both reduced ion suppression and a much reduced matrix background. The ability to reliably subtract background allows one to obtain clean spectra of compounds even in mass ranges that are dominated by matrix ions. Reduced ion suppression improves the capabilities for performing quantitative analysis.

59 The quantitative capabilities for CF/FAB have proved to be quite good. Detection limits are often in the low picomole range with linear ranges greater than two orders of magnitude. These capabilities plus the ability to use a wide variety of mobile phase compositions (from 100 percent organic to 100 percent aqueous) make LC-CF/FAB-MS a very useful technique for analysis of many compounds. Compounds that have a polar ionic group and a fairly hydrophobic moiety produce the highest sensitivity. Examples of these include: bile acids; drug conjugates with glucuronic acid, glutathione and sulfates; and other compounds that can be classified as ionic surfactants. Other compounds such as peptides, oligosaccharides, phospholipids, steroids, etc., have also been analyzed very successfully by these techniques but usually with somewhat less sensitivity. A number of reports demonstrating the qualitative and quantitative capabilities of LC-CF/FAB-MS analysis systems have appeared. These were reviewed through 1991 by Caprioli and Suter (41) and applications in forensic analysis were recently reviewed by Sato et al. (42). Some recent examples of quantitation using this technique illustrate its capabilities for metabolite identification and measurement. An elegant automated system for analysis of diethylstilbesterol (DES) isomers in urine was described by Davoli et al. (43). Samples with added stable isotope-labeled DES internal standards were extracted on an immunoaffinity column. The eluate was concentrated on a C-18 cartridge, chromatographed on a 3 mm i.d. C-18 HPLC column with 0.8 percent glycerol matrix included in the mobile phase. The HPLC eluate (200 l~l/min) was split with about 1 percent going to CF/FAB-MS where spectra were acquired from m/z 40-400. Selected ion plot peak areas were used for measurements. The total process was under computer control with a cycle time of 28 min. They reported detection limits of 2 ng/ml of sample for both the cis and trans isomers of DES with 4.6 and 4.8 percent standard errors respectively. Evans et al. (44) reported a method for separation and measurement of urinary bile acids using gradient elution, micro-HPLC-negative ion CF/FAB-MS. They reported detection limits in the pg injected range for a number of bile acids with 1:10 post column splitting. The simultaneous structure-activity determination of disulfiram photolysis products by micro-HPLC-CF/FAB combined with an aldehyde dehydrogenase inhibition assay has been reported (45). The flow from the column was split with 5 percent going to the MS and 95 percent to the inhibition assay. Using this technique Evans et al. were able to closely couple the mass spectral identification to the inhibitory activity of the products. These reports along with many others illustrate the potential of LC-CF/FAB for drug disposition studies with stable isotope-labeled corn-

60 pounds. This potential lies in its capabilities for separation, structure identification and sensitive detection and measurement of a wide variety of compounds, with its best applications being with polar conjugates and peptides.

2.2. Ion Source Interfaces 2.2.1. Thermospray The TSP LC-MS interface was until recently the most used of all LC-MS interfacing techniques and has probably solved more practical analytical problems than any other. This is especially true in the pharmaceutical industry where it has played a very important role in studies of drug disposition. It is now being displaced by the newer API techniques. This trend is expected to continue because most of the compounds TSP analyzed best are now better analyzed, with greater ease, by the API techniques. For this reason this discussion of TSP will be brief. TSP was invented and developed largely through the efforts of Vestal and coworkers (46). Reviews of TSP that deal in considerable detail with its development and application have appeared (47-49). A schematic representation of a TSP interface is shown in Figure 4. It operates by rapidly heating the LC solvent (to 200-300 ~ in the heated entrance tube) forming an aerosol that sprays directly into the heated source. There under reduced pressure, the droplets rapidly desolvate and ionization takes place by solvent-mediated chemical ionization processes. The solvent must contain ammonium acetate or another volatile buffer as a source of ions for true thermospray ionization to occur. For some difficult analytes ionization can be promoted by the use of an external energy source such as an electron beam or discharge source

Sampling cone

MS

Vapirizer LC in-*.

.. '

J

m

; Vacuum Pump

:

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Figure 4. Schematic representation of a thermospray LC-MS interface.

61

to promote ionization of the solvent and subsequently the sample. This mode of operation is often termed assisted thermospray. Controversy is still abundant over the details of TSP ionization. The interface operates well at 1-2 ml/min flow rates that are compatible with normal 4.6 mm HPLC columns and with reverse phase solvents that contain 20-80 percent water, as long as nonvolatile components are absent. This is a major convenience since it allows operation with many already established reversed phase HPLC separations without modification. TSP has achieved considerable success with polar compounds that are of relatively low molecular weight and of appreciable volatility. The initial heating process is quite severe and causes the thermal decomposition of many labile analytes, limiting its usefulness for these. Otherwise, it is a soft ionization technique in that little fragmentation is present and ammonium adducts of the molecular ions are usually strong ions in the positive ion mode. Operation requires the fine optimization of the vaporizer and source body temperatures, both of which are very sensitive to changes in solvent flow rate or composition (gradient operation is very difficult) often resulting in unstable operation over time. This optimization is compound dependent and differs even for compounds that are relatively similar, making analysis for unknowns or mixtures uncertain. Its quantitative capabilities can be good in cases where stable isotopomer or closely related internal standards are used. In an exemplary report, LC-TSP-MS was used for stable isotope tracer studies to determine the steady state pharmacokinetics of carbamazepine and its epoxide in patient blood samples (50). Moor et al. used [lSN, 13C]carbamazepine and [~SN, ~3C]-carbamazepine epoxide as tracer compounds with their d4 isotopomers as internal standards. Following a single extraction, samples were analyzed by reversed phase HPLC-TSP-MS. The good sensitivity and reproducibility for these compounds allowed for the precise determination of each of these analytes in 0.25 ml pediatric blood samples. The reader is referred to the reviews mentioned above and those by Baillie (7), Blair (8) and Burlingame et al. (5) for further examples of the application of LC-TSP-MS. It will probably continue to be used for some time to carry out established assays but will most likely be replaced by the new API techniques in the future. 2.2.2. Atmospheric pressure ionization (API)

The API techniques are the most used and intensely developed of all LC-MS techniques today. The announcement of electrospray (ES) ionization by Fenn and coworkers (4) began the revolution that has resulted in the further devel-

62 opment of ES, and the derived technique IS, into very robust analytical tools for analysis of a very wide range of analytes. What was most exciting in their early reports (for a review of these see Ref. 51) was the demonstration that ES could generate multiply-charged ions with very high efficiency from intact proteins having molecular weights up to 40,000 and that the high charge states on these ions allowed them to be analyzed using relatively inexpensive quadrupole instruments with m/z limits of less than 2,000. The third API technique, APCI, was first reported by Horning et al. (52); (reviewed by Carroll et al. (53)) as both a GC-MS and LC-MS interface but its potential was not fully appreciated until recently. These three API ion source/LC interfaces have much in common in design. They are now the most used and rapidly developing techniques for LC/MS today. This popularity seems to be especially high in studies of drug disposition (8). The abilities of ES and IS for the analysis high molecular weight biopolymers (e.g. peptides, proteins, oligosaccharides, polynucleotides, etc.) as well as small molecules opened up a whole new world for mass spectrometry in biology. The impact of these developments is now becoming apparent and new areas of application are rapidly developing. 2.2.2.1. Electrospray and ionspray In ES ionization, nebulization of the liquid flow stream occurs solely by electrostatic means in a strong electrostatic field. Multiply-charged analyte ions then result from residual charge left on solute molecules after evaporation of the solvent. Figure 5 is a schematic representation that will serve to illustrate the operation of all three API interfaces. Many variations on the design shown here are discussed in the reviews mentioned above. In ES, ion production

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63 occurs in two steps. In the first step, solvent flows in through a stainless steel tube (1-5 i~l/min) with its outlet at atmospheric pressure and at several thousand volts potential relative to the surrounding chamber (negative potential for positive ion production, positive potential for negative ion production). An aerosol of highly charged droplets of the analyte-containing solvent is generated by a charge dispersal mechanism. The next stage is evaporation of solvent from the droplets by collision with a dry bath gas that is flowing counter to the direction of ion travel. This serves to exclude large droplets from the mass spectrometer and aid in desolvation of ions as they are drawn electrostatically toward the ion exit. As the neutral solvent evaporates, the concentration of charge on the droplets increases to the point that Raleigh fission occurs, eventually resulting in multiply-charged analyte ions. These are then electrically drawn through pressure reduction stages into the mass analyzer. Recent reviews of the ES ionization process and its applications have appeared (51, 54) and are recommended for a detailed discussion of the processes involved. Not all types of compounds provide a good response with ES and IS ionization (55). Compounds that respond best are those that are ionic in solution. Those that can be ionized through Brensted or Lewis acid/base chemistry also respond well providing (M + H) § or other cation adducts or (M - H)- ions. A sizable majority of the compounds of interest to biologists and pharmacologists are good responders. Neutral, nonpolar compounds give very low ion yields and are not usually detected by ES-MS with useable sensitivity. Van Berkel and associates have devised several chemical derivatization strategies for such compounds to generate "ES-active" forms by derivatization for a variety of functional groups before analysis (56) and an on-line post-separation derivatization for aromatic and highly conjugated compounds (57). These and other efforts have successfully extended the ES advantages of high sensitivity and in some cases selectivity to a broader range of compounds. ES ionization imposes limitations on flow rate, ionic strength and dielectric constant that put restraints on its use as an LC-MS detector. Electrolyte concentrations higher than 10-4N result in poor ion production, unstable operation and limit the flow rate to very low values. Solvents with low dielectric constants (e.g. methanol) give the highest ion yields, while high dielectric constant solvents (e.g. water) require higher ES voltages and result in lower ion production. The problems caused by highly aqueous solvent systems have been overcome to some extent by mixing methanol or acetonitrile with the sample stream (usually at the probe tip) to achieve better ion production and better stability. Still the use of ES as a HPLC detector is difficult and only

64

a few examples of such have appeared. Capillary electrophoresis and capillary isotachophoresis are techniques that give very high separating power and operate at very low flow rates (0-100 nl/min). They are well suited to interfacing with ES-MS by addition of a post capillary make-up solvent. Many examples have appeared demonstrating the utility of these in a wide variety of analyses including peptides and proteins for which they are most popular (58). The ionspray (IS)interface was developed as a solution to the problems of interfacing HPLC to ES. The significant modification is that, instead of nebulization solely by electrostatic means, a pneumatic nebulizer is used to assist in aerosol formation. This overcomes many of the solvent composition limitations of ES and allows flow rates of 200 i~l/min or higher. Other thermal and ultrasonic assisted ES nebulization devices have been used with similar results. While much higher flow rates are allowed with IS, allowing it to be used with conventional HPLC systems, the sensitivity does not increase as a result of a higher rate of sample delivery. Response in ES and IS depends upon analyte concentration and not its rate of sampling. Another advantage of IS is that gradient elution, which is generally not possible with ES, is usually not difficult. The analysis of large biomolecules by ES and IS coupled to MS or MS/MS has been the major impetus behind the development of these interfaces. These efforts have generated an extensive literature that was recently reviewed by Loo et al. (54). Molecules with molecular weights extending to over 100,000 yield mass spectra consisting of a series of multiply-charged (M + nil) n+ ions (n approximates Mr/I,000) differing by one charge. These fall in a m/z range that can be analyzed using mass spectrometers with mass ranges of m/z 2,000 or less. The spectra, consisting of ions of differing charge states, can be easily deconvoluted by MS data systems to spectra showing only the singly charged species. Molecular weights can be determined with an accuracy of about 0.01 percent, making ES and IS the most accurate way to determine molecular weights of large molecules. Structural information for biopolymers can be obtained by collisional activation dissociation (CAD) MS/MS of a selected multiply-charged molecular ion. The resulting product ions also occur in a series of charge states and are highly useful for determining the sequence of biopolymers. For higher molecular weight compounds, the resulting MS/MS spectra are very complex, containing multiple fragment ions, all with multiple-charge states, and assignment of charge state can be impossible. The use of higher resolution instruments, such as magnetic sector or FTICR mass spectrometers, can allow the measurement of one m/z unit separation of 13C isotope ions to determine charge state of individual ions

65 and their atomic mass, allowing better mixture resolution. MS/MS analyses have been used to obtain sequence information on proteins in excess of 65,000 molecular weight. Analysis of small molecules by ES and IS is highly effective for many compound classes. Generally for compounds of less than molecular weight 1,000, little multiple charging is seen unless multiple anionic or cationic functional groups are present. Very little fragmentation is seen even for very labile compounds when the temperature and flow rate of the bath gas are proper (51). Even though the current interest in ES/IS is due to its capabilities for analysis of large molecules, a major area for application of LC-ES/IS-MS is the analysis of small molecules. API techniques seem likely to replace others for most analyses of small molecules by LC-MS. The number of reports of applications of ES and IS LC-MS to pharmaceutical analysis has become quite large and are appearing at an increasing rate. This is an indication of the acceptance of these techniques as near universal interfaces for LC-MS. Only a few examples from this large literature can be mentioned here. The reader is referred to the recent review by Loo et al. (54) for examples of its use for analysis of large molecules and to the review of mass spectrometry in studies of drug disposition and pharmacokinetics by Blair (8) for other examples of its use in analysis of small molecules. In one study, Moseley and Unger (59) evaluated the combination of packed capillary chromatography with ES-MS for the characterization of protein mixtures in the development of pharmaceuticals. They concluded that it performs very favorably compared to SDS/PAGE gel electrophoresis that is traditionally used for this purpose, by providing good chromatographic separation with sensitive detection and accurate molecular weight determination. An excellent example of the measurement capabilities of LC-IS-MS was reported by Murphy et al. (60). They demonstrated the rapid (2.5 min), sensitive (0.075-5.0 ng/ml), and accurate LC-IS-MS/MS determination of xanomeline (a muscarinic receptor agonist) in human plasma. This method was used clinically for determination of the pharmacokinetics of this compound. Another example of quantitation of small molecules by LC-ES-MS was reported by Pacifici et al. (61). They measured levels of morphine and its 3- and 6-glucuronides in serum, achieving detection limits of 10, 100 and 50 ng/ml respectively, and applied the assay in pharmacokinetic studies. 2.2.2.2. A t m o s p h e r i c pressure chemical ionization

An APCI interface physically resembles an ES/IS interface and APCI operation is a possible operating mode with many ES/IS interfaces, but the ionization process appears to be distinctly different (62). The consequential differences

66 between ES/IS and APCI are that a corona discharge needle is placed in the source and high voltages are not used to form an aerosol or charge the droplets in APCI. Unlike ES/IS, APCI is a gas phase process and requires thermal volatilization of the analytes before ionization. The mechanisms involved are the same as in a standard CI ion source: ion molecule reactions, charge transfer and electron capture. APCI uses the chromatographic solvent vapor as the reagent gas. In operation, the solvent is nebulized via a heated pneumatic nebulizer and the aerosol is carried through a heated tube, where the solvent and analytes are evaporated, then to an ionization region where a corona discharge needle generates solvent reagent ions for APCI. As in ES and IS, the neutral volatile solvent is swept away by a counter-current flow of drying gas while the analyte ions are drawn into the mass spectrometer by electrostatic forces. A wide range of polarities of solvents that may also contain nonvolatile buffers can be used with flow rates up to 2 ml/min, making APCI one of the least restrictive on the HPLC system of all LC-MS interfaces. APCI as a gas phase ionization technique is complementary to the solution ionization techniques ES/IS. It is useful for analysis of lower molecular weight compounds that have sufficient thermal stability and volatility. It does not produce the multiply-charged ions seen with ES/IS but gives spectra that are very similar to those obtained with normal CI. As such it has found much use for the LC-MS analysis of pharmaceuticals. Its ease of use, and the lack of restrictions it places on HPLC systems, have led to its broad acceptance as a replacement for the once pervasive TSP interface. As in TSP, the major limitation of APCI for analysis of polar, higher molecular weight compounds derives from the requirement that analytes be thermally vaporized for ionization. A major strength is that it gives a more constant ion yield with diverse types of compounds than the solution ionization techniques. This is because APCI does not require charged analytes in solution to achieve high sensitivity. Its general applicability to quantitative analysis of small molecules makes it a natural choice for many of the analyses performed with pharmaceuticals. Numerous examples can be found of LC-APCI-MS applied to pharmaceutical research. These have been reviewed recently by Bruins (63) and Gelpi (9). One study of the relative performance of APCI, ES and TSP for use in studies of drug metabolism paid particular attention to compound polarities and the resulting sensitivity achieved by each technique (64). They found that the best sensitivities for nonpolar compounds were obtained with APCI but, as expected, it was not as useful for hydrophilic compounds such as glutathione conjugates. TSP was reported to be useable for all compounds studied as was ES. However, ES gave 10-100 times more sensitivity for hydrophilic analytes. The determination of pilocarpine (used to treat glaucoma) in aque-

67 ous humour by LC-APCI-MS was reported by Matsuura et al. (65). They obtained linear calibration curves from 2 ng to 10 ~g/ml of sample and excellent intra- and inter-day precision without the use of internal standards. This provides an example of the high stability (and sensitivity) that can be expected with LC-APCI-MS. A elegant LC-APCI-MS/MS multiple reaction monitoring assay for MK-434 (a 5(x-reductase inhibitor) and its two principal metabolites in plasma was reported (66). Using less than 5-min runs they were able to measure concentrations from 0.5-50 ng/ml with high precision, accuracy and specificity. Fraser et al. (67) compared LC-APCI-MS/MS to GC-MS for the analysis of an inhibitor of acylcoenzyme A cholesterol acyltransferase in plasma. The LC-MS/MS method had the advantage of not requiring the extensive sample clean-up and derivatization of the GC-MS procedure and provided increased sensitivity, selectivity and speed. Many other examples of the usefulness of LC-APCI-MS can be found that are models for development of new assays.

3. CONCLUSIONS AND FUTURE DIRECTIONS

The development of methods for interfacing HPLC directly to MS has proceeded with vigor for the last twenty-five years or so resulting in the mature techniques discussed here. LC-MS now finds routine use and has become an extremely important technique in a number of application areas, including many phases of pharmaceutical development. It is expected that growth in reliance on LC-MS will increase with the increased availability of modern interfaces in laboratories. It remains to be seen what other LC-MS interfacing techniques may be introduced or what improvements may be made to the present ones. The API interfaces exceed the capabilities envisioned by their inventors and they can be expected to be the most useful for solving problems in biology for some time to come. They appear to have achieved a high degree of development, which is not to say that further improvements are unimaginable or unlikely to occur. It is expected that certain apparent limitations will be addressed and improved devices developed. For instance, the API techniques provide high sensitivity by producing nearly total ionization of analytes, but today's interfaces can only direct a small percentage of those ions into the mass spectrometer. Efforts to improve ion transmission are likely to result in an enormous improvement in sensitivity. Similarly, the PB interface only obtains an efficiency of a few percent for transmission of solute to the mass spectrometer ion source. Further developments can be expected to increase this efficiency.

68 The further development of highly popular MALDI techniques for use with LC-MS, possibly with PB or continuous flow probes, seems a likely area of future accomplishment. While other developments will surely occur, it seems unlikely that, with the major goals met or wildly exceeded, there will be the same level of innovation in the next twenty-five years as the first. Much of the developmental work that used to be done in academic laboratories has now moved into those of the instrument manufacturers where further product development can be expected to be more incremental and less innovative. The increasing domination of the API techniques is evident from the reports presented at the annual meeting of the American Society of Mass Spectrometry (ASMS). Figure 1 is an update of a similar graph by Niessen and Tinke (6) and shows the number of papers reporting on the use or development of each LC-MS interfacing technique at each annual meeting. Clearly, ES/IS have come to dominate. At the 1995 conference, of the 464 reports concerning LC-MS interfacing techniques, 360 (84 percent) of them concerned ES/IS. Most of these reports concerned applications of API techniques to biological problems and did not deal with further development of the devices, indicating general satisfaction with their present capabilities. It should be noted that many of these reports concern direct injection of samples into a flow stream and not actual LC-MS experiments. This is particularly true for ES which is often used for characterization of isolated macromolecules. Reports at this conference generally deal with methods in their developmental or early application phases and may not reflect overall use in routine analysis, which probably relies more on well-established methods. Indeed some LC-MS interfacing techniques that are not or are poorly represented here may still be the best available in particular situations. This author feels this way about the now little used moving-belt interface for complex lipid analysis (2, 3, 68, 69). No ASMS papers dealt with MB-MS in 1995. A variety of liquid mobile phase separation techniques have been directly interfaced to mass spectrometry including: capillary liquid chromatography (70), capillary electrophoresis and isotachophoresis (58), normal column HPLC, (pseudo)electrochromatography (71), ion chromatography (72) and thin layer chromatography (TLC-MS) (73). Many of these have solvent composition or flow rate requirements that make them especially suited to use with one or more of the LC-MS interfaces mentioned above. Table 1 presents a generalization of the operating parameters that apply to each interface and limit its applicability as a interface for chromatography-MS. Apparently almost any conceivable liquid-based separation technique has been interfaced to MS using one of these interfaces, without serious operational compromise for either the separation or mass spectrometry systems. The routine use of corn-

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70 bined liquid separation-MS techniques is now practical and convenient for most bioorganic compounds. In the future we can expect the use of these techniques to vastly expand as the availability of lower cost, easy-to-use instrumentation increases.

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73

CHAPTER 5

STABLE ISOTOPES IN PHARMACEUTICAL RESEARCH: TANDEM MASS SPECTROMETRY

CECILIA BASIC ~, SUSAN F. SILVERTON 2 and RICHARD A. YOST 3 1Department of Chemistry, Villanova University, Villanova, PA 19085-1699; 2Department of Oral Medicine, School of Dental Medicine, 525 Levy Research Building, 4010 Locust St., University of Pennsylvania, Philadelphia, PA 19104-6002; 3Department of Chemistry, University of Florida, Gainesville, FL 32611-7200

1. INTRODUCTION

Tandem mass spectrometry (MS/MS) is a powerful analytical tool which not only allows the quantitative analyses of complex biological mixtures but also provides a high degree of molecular specificity. In contrast to the technologies of high-performance liquid chromatography (HPLC), gas chromatography/ mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) which have been adapted relatively easily into pharmaceutical research, MS/MS has been under utilized. Part of this under utilization is due to the instrument cost, and part to the inherent individuality of each new application which requires appreciable research investment. Where, then, has MS/MS been used successfully, and in particular, where has its use in concert with stable isotopes significantly advanced the field of pharmaceutical research? To address this question, a series of illustrative examples in which stable isotopes have been used in conjunction with MS/MS methods will be presented. Prior to this, however, a general introduction to MS/MS will be provided, including descriptions of the more common types of MS/MS instruments, summaries of their relative performance capabilities, descriptions of the types of tandem scan methods available, and the qualitative and quantitative information these scan methods can provide. The chapter is designed so that readers who are more familiar with MS/MS may proceed directly to the

74 series of illustrative examples, while others may benefit from the material presented in the first part of the chapter. It should be stressed that the overview of MS/MS is by no means exhaustive, nor is the presentation of the stable isotope applications. Rather, the chapter is designed to familiarize the reader with the instruments and terminology encountered in MS/MS and to highlight the power of MS/MS when used in conjunction with stable isotopes. Readers are encouraged to seek more detailed information in the references provided or in the additional reading list.

2. TANDEM MASS S P E C T R O M E T R Y - AN-OVERVIEW

Two types of scan methods can be employed in conventional MS analyses: normal (full-scan) MS and selected-ion monitoring (SIM). In full-scan MS, the mass-to-charge (m/z) ratios of either all the ions (or a range of ions formed in the ion source) are recorded. For relatively pure compounds undergoing sufficient fragmentation in the source, a normal mass spectrum can provide molecular weight (MW) information as well as ion structural information vis-a-vis the m/z ratios of the fragment ions. In SIM, only ions of a single m/z ratio (or a selected few m/z ratios) are transmitted and detected, providing improved sensitivity due to the increased signal-to-noise (S/N) ratio obtained by dwelling only on the ion signal of interest rather than on the background ions. SIM is of value in the quantitation of targeted compounds (1). The ability to obtain unambiguous MW and ion structural information from normal mass spectra becomes limited in the analysis of impure compounds or complex mixtures, since the normal mass spectrum of a mixture cannot definitively establish which ions are molecular ions (e.g. M § or M-) nor can it establish any links between a given molecular ion and its associated fragment ions. While SIM can serve to select a specific targeted ion for analysis, it does not provide any structural information. Normal MS also has limitations in analyses using "softer" ionization methods, such as fast atom bombardment (FAB; see Appendix). Many softer ionization methods used in biomolecular analyses result in the formation of a single ion characteristic of the compound of interest, most commonly the protonated MH § or deprotonated [ M - H ] - molecular ion. Normal FAB/MS can provide accurate MW information, but little or no structural information due to the lack of fragment ions in the mass spectrum. MS/MS analyses involve the initial separation, or mass-selection, of an ion of interest from the ensemble of ions formed in the source, the dissociation of this precursor (or parent) ion in a collision region of the mass spectrometer,

75 and the subsequent mass-analysis of the resulting product (or daughter) ions. Dissociation is usually achieved via collision-induced dissociation (CID, or collisionally-activated dissociation, CAD) with a neutral target gas. The two stages of mass-analysis, separated by a dissociation step, allow the ability to establish relationships between a given ion and its associated fragment ions and provides enhanced fragmentation to gain detailed ion structural information. Thus, MS/MS provides a greater degree of selectivity and molecular specificity than that found in conventional MS methods (2).

2.1. Tandem Mass Spectrometers Tandem mass spectrometers can be broadly divided into two main types: (i) those in which the mass-selection, dissociation, and mass-analysis steps are performed in different spatial regions of the mass spectrometer (tandemin-space); and (ii) those in which the steps are performed within the same region of the mass spectrometer but at sequentially controlled times (tandemin-time). Tandem-in-space instruments require at least two coupled mass analyzers separated by a collision region (Figure 1), while tandem-in-time methods are performed with a single mass-analyzing device which can trap and store ions. Both ion cyclotron resonance (ICR) and quadrupole ion-trap mass spectrometers (QITMS) are tandem-in-time instruments. While these

ion source

first mass analyzer

collision region

second mass analyzer detector

'~e

.O.o o 9

+~

I

ill

+o

'

+.

+o

~176 I . . . . . .

+,

ill

II

ii i

i

..,.

II

~

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I-4'e §

+.%+ i

+ o ~o §

o§ o+

I-data

system Q B E BE EB

He, Ar, Xe, N2

Q B E BE EB

Figure 1. Schematic of a tandem-in-space mass spectrometer illustrating mass-selection, collision-induced dissociation (CID) and subsequent mass-analysis of fragment ions. (Q, quadrupole mass filter; B, magnetic sector and E, electric sector.)

76 ion-trapping mass spectrometers have been successfully used to analyze biological molecules (3, 4), they have not seen widespread use in routine MS/MS analyses and as such will not be discussed further. Interested readers are referred to several reviews of both ICR (3, 5, 6) and QITMS (4, 7, 8) instruments. Three tandem-in-space mass spectrometers have found widespread use in pharmacological studies: triple quadrupole (QlqQ2), double-focussing sector (BE and EB), and hybrid sector (BEqQ and EBqQ) mass spectrometers. The performance capabilities of these instruments are dictated in part by the operating principles of the different mass analyzers, i.e. the way in which quadrupole mass filters (Q) (9-11), and magnetic (B) and electric (E) sectors (9, 12, 13) separate ions of differing m/z ratios, and by differences in the design and operation of rf-only quadrupole collision cells (q) versus the collision cells used in sector mass spectrometers. The performance characteristics of the different tandem mass spectrometers can be assessed based on (9): (i) the maximum m/z ratio which can be measured; (ii) the resolving power of the instrument and the concomitant ability to perform accurate mass measurements; (iii) the ability to rapidly switch from one MS/MS scan mode to another; (iv) the ability to perform high- versus low-energy CID; (v) the ease with which the MS can be interfaced to alternate ionization methods and auxiliary GC and LC equipment; and (vi) the cost of the instrument. The performance capabilities of Q~qQ2, BE and EB, and hybrid mass spectrometers are summarized in Table 1 (3) and discussed in greater detail below.

2.1.1. Triple quadrupole mass spectrometers Triple quadrupole mass spectrometers (QlqQ2, Figure 2) make use of quadrupole mass filters (Q) to perform the first and second stages of mass-analysis and an rf-only quadrupole collision cell (q). (Most commercial instruments employ hexapole or octopole collision cells.) Mass-analysis in quadrupole mass filters is based on the inherent stability, or instability, of an ion's trajectory as it traverses the length of the two-dimensional quadrupolar field formed by applying alternating radio-frequency (rf) and direct current (dc) potentials to a set of four parallel, hyperbolic (or circular) rods. Ions entering the mass filter must have kinetic energies of less than 100 eV in order to experience enough cycles of the rf to establish stability/instability and thus provide adequate mass resolution. It is this feature that dictates the use of low-energy CID on quadrupole-based tandem instruments. MS/MS analyses using lowenergy CID on a Q~qQ2 instrument was first demonstrated by Yost and Enke (14).

77 TABLE 1. Performance Characteristics of Common Tandem-in-space Mass Spectrometers a'b

Mass analyzer

Maximum m/z ( D a )

Resolving power

Rapid scanning?

High- vs. low-energy CID

Higherorder MS/MS?

Triple quadrupole Double-focussing sector, BE or EB Hybrid sector BE or EB Q Multiple sector

_m .e 40

[M + H20] +

20

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0

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9

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300

250

m/z 100-

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"== e0: 0 ,4..,;

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-81

MH + 213

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50

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77

104 120

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150

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m/z Figure 7. M S spectra of pure z o n i s a m i d e : (a) n o r m a l p o s i t i v e CI mass s p e c t r u m ; and (b) p r o d u c t ion s p e c t r u m o f the M H + ion (m/z 213) of z o n i s a m i d e (34).

a.

100-

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extract

91

259

229

80.

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, 300

m/z

Figure 8. MS/MS spectra of urine extract" (a) neutral loss spectrum (81 u); and (b) precursor ion scan in which Q2 was set to transmit the product ion at m/z 132 (34).

92 O

!!

CH~--~-NH 2

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zonisamide or

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- 81

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D

Figure 9. Metabolites of the antiepileptic drug zonisamide identified using the MS/MS analysis scheme in Figure 6 (34).

It can be seen that metabolites A and B retain the intact, unmetabolized sulfonamide portion of the parent drug and thus undergo facile neutral loss of 81 while metabolites C and D retain the intact, unmetabolized ring structures of the parent drug and thus form a prominent m/z 132 fragment ion. The neutral loss and precursor ion scans thus reflect different sites of metabolism and, therefore, provide complimentary metabolite information. It is interesting to note that it was possible to distinguish between the two isomeric monohydroxy metabolites A and C because metabolism occurred on different parts of the parent drug. No structures were assigned to the ions at m/z 215 and 299 because no plausible metabolite structures could be deduced from their product ion spectra (34). This study illustrates how the molecular specificity inherent in MS/MS

93 methods can lead to the assignment of metabolite structures from a biological extract without isotopic labeling. MS/MS analyses in conjunction with stable isotopes leads to an even greater degree of molecular specificity; the remainder of this chapter is dedicated to presenting a series of illustrative examples of MS/MS analyses using stable isotopes.

3. M S / M S ANALYSES USING STABLE ISOTOPES

Given the wide flexibility of MS/MS instruments and techniques, we are limited to describing only a few selected examples of MS/MS utilizing stable isotopes representing several different approaches for quantitation, identification, and characterization of compounds of pharmacological interest. The examples fall into two main categories: (i) quantitation using stable isotope dilution MS/MS; and (ii) the use of stable isotope tracers in MS/MS analyses for identification and structure elucidation. The primary purpose of the following sample studies is to illustrate the range of MS/MS applications in pharmacological research. As such, the summaries are focussed on the MS/MS methods employed and the contributions of the MS results to the overall conclusions of the study. Details of the MS methods have been presented in italics with specific terms or MS/MS methodologies described in the first part of the Chapter. Since many of the studies use fast atom bombardment (FAB), a summary of this ionization method has been presented in the Appendix. A list of additional reading including other MS/MS stable isotope applications has also been provided.

3.1. Quantitation Using Isotope Dilution MS~MS The selectivity of two stages of mass-analysis in MS/MS is of particular value in quantitating trace components in complex biological samples. Alternating MS/MS scans (product, precursor, neutral loss or SRM, Figure 5) permit monitoring of the analyte and its isotopically labeled internal standard for quantitation. In isotope dilution MS/MS, quantitation is achieved by measuring the ion intensities (or areas) corresponding to the unlabeled compound and that of a known amount of labeled internal standard. The ratios can be determined from the product, precursor, neutral loss, or SRM spectra and then compared to an appropriately constructed standard curve. The use of ratio measurements increases the precision of quantitation in that it accounts for changes in the ion intensities due to changes in the extraction efficiency, variability in injection volumes, adsorption losses, and any chemi-

94 cal interferences. It also allows quantitation of the targeted compound independent of volume. The following examples illustrate the use of isotope dilution MS/MS to quantitate targeted compounds in a series of biological matrices. Results of the quantitative studies were used for the diagnosis and rapid screening of disease states (36-38), for tracking targeted compounds in tissue (39-42), and for pharmacokinetics studies (43). 3.1.1. Carnitines in urine and plasma: diagnosis of carnitine deficiency

L-Carnitine (3-hydroxy-4-N-trimethylammonium butyrate) serves as a carrier of acyl groups across the mitochondrial inner membrane and is essential for the transport and metabolism of fatty acids (36, 37). Genetic disorders of fatty acid or amino acid catabolism may result in accumulation of abnormal, disease-specific, carnitines with the concomitant depletion of carnitine stores. Since carnitine is used in vivo as a scavenger of accumulated toxic metabolites, the depletion of carnitine can lead to sudden death (37). As such, a rapid and reliable method for the routine assay of carnitines in biological fluids was required for early diagnosis of carnitine deficiency (36). To this end, total and free carnitine in urine and free and short-chain carnitines in plasma were assayed using isotope dilution in combination MS/MS. The carnitines were butylated prior to MS/MS analysis to increase the intensities of the FAB ionization signals. The results were then compared to those obtained using the standard method of radioenzymatic assay (REA) (36). A Q 7qQ2 mass spectrometer and FAB ionization were employed. Product ion scans of the MH § ions of butylated synthetic carnitine (m/z 218) and the butylated internal standard, D3-carnitine (m/z 221), were performed using Ar collision gas at a pressure of 0.2 to 0.5 • 10-6 Torr and an E/ab of 15 e V. The MH + product ion spectra revealed a common fragment ion at m/z 103. The butylated carnitine levels in the urine and plasma extracts were therefore determined from the ratios of the nondeuterated and deuterated MH § ions in the precursor ion spectra obtained by scanning the Q7 from m/z 200 to 240 and setting Q2 to detect the ion at m/z 103.

Figure 10 shows the m/z 103 precursor ion spectra of butylated carnitine and butylated carnitine plus the isotopically labeled internal standard in a urine sample. Calibration curves for carnitine in urine constructed from the precursor ion spectra showed excellent linearity over the ranges 0-1500 nmol/ml

95 1oo

1oo

b.

o~ 215

220 m/z

225

215

220

225

m/z

Figure 10. Precursor ion spectra of (a) butylated carnitine (m/z 218), and (b) butylated carnitine plus the butylated isotopically labeled internal standard D3-carnitine (m/z 221). The product ion at m/z 103 was monitored (36). Adapted with permission from Elsevier Science.

(R = 0.999), and 0-20 nmol/ml (R = 0.998). The calibration range for free carnitine in plasma using MS/MS was 0-200 nmol/ml. Similar coefficients of variation (

786

/I\ 000

5O

641

600 100

700 C,

9

931

e

784

800

784

o/ o o -Pro -.,

.

.

.

.

687

.

6OO

7O0

1

800

m/z Figure 16. Low-energy CID spectra of (a) the MH § ion of angiotensin III (m/z 931); (b) the MH § ion of 1802-1abeled analogue (m/z 935); and (c) MS/MS/MS spectrum resulting from mass-selection and high-energy CID of the MH § ion of AIII (m/z 931) followed by selection and subsequent low-energy CID of the m/z 784 fragment ion (50). Reproduced with permission from John Wiley.

protonated hexapeptide Arg-VaI-Tyr-Ile-His-Pro. The MS/MS/MS product ion spectrum of m/z 784 (Figure 16c) shows only one significant fragment ion at m/z 687, corresponding to the loss of the terminal amino acid, Pro. Thus, the first-generation product ion at m/z 784 fragments in a similar manner to that of the MH § ion. The formation of these "internal" fragment ions is attributed,

112 therefore, to the formation of an intermediate zwitterion due to interaction between the terminal carboxy oxygens and the oxygen of the first peptide bond (50). The MS/MS/MS scanning capability of the hybrid mass spectrometer thus allowed the facile elucidation of the mechanism of formation of these "internal" peptide fragment ions.

4. S U M M A R Y

MS/MS is a versatile method for quantifying and characterizing compounds of pharmacological interest. When used in conjunction with stable isotope methods, an even greater degree of molecular specificity is attained. Advantages of these combined methodologies include their versatility (analyses range from quantitation, to pharmacokinetics, to structure elucidation), the small requisite sample sizes (e.g. 11 i~1 dried blood spots), the speed of the analyses (min vs. hr), and the inherent selectivity which allows for the detection of trace components in complex biological matrices. Thus, MS/MS offers an attractive compliment to more conventional pharmacological methods of analysis.

5. APPENDIX

5.1. Fast Atom Bombardment Ionization

Fast atom bombardment (FAB) is a method of producing intact molecular ions of compounds present in the liquid phase (52). FAB is performed by mixing the sample of interest in a liquid matrix, depositing it on a sample probe and then bombarding the sample under vacuum with a beam of atoms of high kinetic energy (8-10 keV). Common incident beams include Ar or Xe. If the sample is bombarded with a beam of high-energy ions (e.g. Cs § ions), then the method is referred to as liquid secondary ion mass spectrometry, or LSIMS. Typically, 1 ~1 of the sample solution is combined with 3-5 i~1 of the matrix and 2 i~1 of this solution is subjected to bombardment. Positive ion FAB typically leads to the formation of MH § or multiply protonated [M + nil] n§ ions. Negative-ion FAB leads to the formation of [M - H]- ions. Often, the molecular ion is the only ion in the spectrum indicative of the compound of interest. Thus, FAB is a facile method for determining the MW of biological compounds. The judicious choice of a matrix is critical in obtaining good quality FAB

113 spectra. It is not unheard of for a sample to produce a strong signal with one matrix and no signal at all with another. Classical matrices include gylcerol, thioglycerol and nitrobenzyl alcohol. The liquid matrix serves two main purposes: (i) it keeps the sample in the liquid phase as it is being introduced into the vacuum chamber; and (ii) it reduces damage to the sample from the bombarding beam by constantly diffusing fresh sample to the surface. Detailed discussions of FAB matrices have been presented (53, 54). The principal drawback of FAB analyses is the often high chemical-noise background formed by the clustering and fragmentation of the sample matrix. The sample also has limited lifetime upon continued bombardment. Also, FAB analyses can suffer from suppressed sample ionization due to the migration of the sample away from the liquid/vacuum interface. Continuous flow FAB sources (CF-FAB) were developed to overcome the limitations of conventional FAB sources by providing a continuous flow of sample matrix into the ion source via a sample introduction probe. The continuous introduction of liquid allows the use of much less viscous and more volatile sample carriers such as 5 percent glycerol in water, methanol, or acetonitrile. Typically, 0.5 to 1 ~1 of sample is injected into the flowing solvent stream and introduced to the bombarding atom, or ion beam, in the ion source. CF-FAB analyses has decreased ion suppression effects, deceased background noise due to matrix ions and thus overall improved limits of detection. A more detailed discussion of CF-FAB sources and methods is available (55).

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115 45. Z-M. Yuan, P.B. Smith, R.B. Brundrett, M. Colvin and C. Fenselau, Drug Metab. Dispos., 19 (1991) 625. 46. D-H. Chin, C-H. Zeng, C.E. Costello and I.H. Goldberg, Biochem., 27 (1988) 8106. 47. O.D. Hensens, R.S. Dewey, T.M. Liesch, M.A. Napier, R.A. Reamer, J.L. Smith and G. Albers-Schonberg, Biochem. Biophys. Res. Comm., 113 (1983) 538. 48. P. Wheelan, J.A. Zirroli and R.C. Murphy, Biol. Mass Spectrom., 22 (1993) 465. 49. M.J. Contado and J. Adams, Anal. Chim. Acta, 246 (1991) 187; J. Adams and M.L. Gross, Anal. Chem. 59 (1987) 1576; K.B. Tomer, N.J. Jensen and M.L. Gross Anal. Chem., 58 (1986) 2429. 50. G.C. Thorne and S.J. Gaskell, Rapid Commun. Mass Spectrom., 3 (1989) 227. 51. P. Roepstorff and J. Fohlman, Biomed. Environ. Mass Spectrom., 11 (1984) 601. 52. M. Barber, R.S. Bordoli, G.J. Elliott, R.D. Sedgwick and A.A. Tyler, Anal. Chem., 54 (1982) A645. 53. E. DePauw, A. Agnello and F. Derwa, Mass Spectrom. Rev., 10 (1991) 283. 54. E. DePauw, Meth. Enzym., 193 (1990) 201. 55. R.M. Caprioli and W.T. Moore, Meth. Enzym., 193 (1990) 21.

6.1. Additional Reading D.J. Ashworth, W.M. Baird, C. Chang, J.D. Ciupek, K.L. Busch and R.G. Cooks, Chemical Modification of Nucleic Acids. Methylation of Calf Thymus DNA Investigated by Mass Spectrometry and Liquid Chromatography, Biomed. Environ. Mass Spectrom., 12 (1985) 309. M.J. Bennett, P.M. Coates, D.E. Hale, D.S. Millington, N. Pollitt, P. Rinaldo, C.R. Roe and K. Tenaka, Analysis of Abnormal Urinary Metabolites in the Newborn Period in Medium-Chain AcyI-CoA Dehydrogenase Deficiency, J. Inherit. Metab. Dis., 13 (1990) 707. C. Chang, D.J. Ashworth, I. Isern-Flecha, X-Y. Jiang and R.G. Cooks, Modification of Calf Thymus DNA by Methyl Methanesulfonate. Quantitative Determination of 7Methyldeoxyguanosine by Mass Spectrometry, Chem. Biol. Interact., 57 (1986) 295. R.B. Cole, C.R. Guenat and S.J. Gaskell, Effect of Experimental Conditions on the Daughter Ion Spectra Derived from Tandem Mass Spectrometry of Steroid Glucuronidides, Anal. Chem., 59 (1987) 1139. A. Dell, Mass Spectroscopy of Carbohydrates, Adv. Carbohydr. Chem. Biochem., 45 (1987) 19. C. Dass, J.J. Kusmierz and D.M. Desiderio, Mass Spectrometric Quantification of Endogenous/3-endorphin, Biol. Mass Spectrom., 20 (1991) 130. M. Dawson, M.D. Smith and C.M. McGee, Gas Chromatography/Negative Ion Chemical Ionization/Tandem Mass Spectrometric Quantification of Indomethacin in Plasma and Synovial Fluid, Biomed. Environ. Mass Spectrom., 19 (1990) 453. D.M. Desiderio and M. Kai, Field Desorption Mass Spectral Meaurement of Enkephalins in Canine Brain with 180 Peptide Standards, Int. J. Mass Spectrom. Ion Phys., 48 (1988) 261. D.M. Desiderio and I. Katakuse, Fast Atom Bombardment-Collision Activated Dissociation-Linked Field Scanning Mass Spectrometry of the Neuropeptide Substance P, Anal. Biochem., 129 (1983) 425. D.M. Desiderio, I. Katakuse and M. Kai, Measurement of Leucine Enkephalin in Caudate Nucleus Tissue with Fast Atom Bombardment-Collision Activated DissociationLinked Field Scanning Mass Spectrometry, Biomed. Mass Spectrom., 10 (1983) 426. B. Domon and C.E. Costello, Structure Elucidation of Glycosphingolipids and

116 Gangliosides using High-Performance Tandem Mass Spectrometry, Biochem., 27 (1988) 1534. B. Domon, J.E. Vath and C.E. Costello, Analysis of Derivatized Ceramides and Neutral Glycosphingolipids by High-Performance Tandem Mass Spectrometry, Anal. Biochem., 184 (1990) 151. C. Fenselau and P.B.Smith, High-Performance Tandem Mass Spectrometry in Metabolism Studies, Xenobio., 22 (1992) 1207. A. Ferretti, V.P. Flanagan and E.J. Maida, GC/MS/MS Quantification of 11-Dehydrothromboxane B2 in Human Urine, Prostaglandins Leukot. Essent. Fatty Acids, 46 (1992) 271. G. Fridland, Measurement of Opiod Peptides with Combinations of Reversed Phase High- Performance Liquid Chromatography, Radioimmunoassay, Radioreceptorassay and Mass Spectrometry, Life Science, 41 (1987) 809. G. Fritz, D. Fetterolf, R.A. Yost and B.D. Anderson, Oxygen Fixation into Hydroxyproline in Etiolated Maize Seedlings: Verification by Tandem Mass Spectrometry, Plant Physiol., 73 (1983) 860. S.J. Gaskell, C. Guenat, D.S. Millington, D.A. Maltby and C.R. Roe, Differentiation of Isomeric Acylcarnitines Using Tandem Mass Spectrometry, Anal. Chem., 58 (1986) 2801. P.E. Haroldsen and S.J. Gaskell, Quantitative Analysis of Platelet Activating Factor using Fast Atom Bombardment/Tandem Mass Spectrometry, Biomed. Environ. Mass Spectrom., 18 (1989) 439. P.E. Haroldsen, M.H. Reilly, H. Hughes and S.J. Gaskell, Characterization of Glutathione Conjugates by Fast Atom Bombardment/Tandem Mass Spectrometry, Biomed. Environ. Mass Spectrom., 15 (1988) 615. K.E. Ibrahim, J.M. Midgley, M.W. Crouch, M.B. Budd, R.A. Yost and C.W. Williams, Quantitative Measurement of Octopamines and Synephrines in Urine using Capillary Column Gas Chromatography Negative Ion Chemical Ionization Tandem Mass Spectrometry, Anal. Chem., 56 (1984) 1695. I. Isern-Flecha, X-Y. Jiang, R.G. Cooks, W. Pfleiderer, W-G. Chae and C. Chang, Characterization of an Alkylated Dinucleotide by Desorption Chemical Ionization and Tandem Mass Spectrometry, Biomed. Environ. Mass Spectrom., 14 (1987) 17. B.L. Kleintop, R.A. Yost and C.R. Abolin, Alternating RF/DC Isolation for Quantitation with Co-Eluting Internal Standards in Gas Chromatography/Ion Trap Mass Spectrometry, J. Am. Soc. Mass Spectrom., 3 (1992) 85. D.S. Millington, N. Kodo, D.L. Norwood and C.R. Roe, Tandem Mass Spectrometry: A New Method for Acyl Carnitine Profiling with Potential for Neonatal Screening for Inborn Errors of Metabolism, J. Inherit. Dis., 13 (1990) 321. D.S. Millington, D.A. Maltby and C.R. Roe, Rapid Detection of Argininosuccinic Aciduria and Citrullinuria by Fast Atom Bombardment and Tandem Mass Spectrometry, Clin. Chim. Acta, 155 (1986) 173. D.S. Millington, C.R. Roe and D.A. Maltby, Application of High-Resolution Fast Atom Bombardment and Constant B/E Ratio Linked Scanning to the Identification and Analysis of Acylcarnitines in Metabolic Disease, Biomed. Mass Spectrom., 11 (1984) 236. R.C. Murphy and K.L. Clay, Preparation of 180 Derivatives of Eicosaniods for GC-MS Quantitative Analysis, Meth. Enzym., 86 (1982) 547. J.O. Naim, D.M. Desiderio, J. Trimble and J.R. Hinshaw, The Identification of Serum Tuftsin by Reverse-Phase High-Performance Liquid Chromatography and Mass Spectrometry, Anal. Biochem., 164 (1987) 221. M.J. Raftery, U. Justensen, H. Jaeschke and S.J.Gaskell, Mass Spectrometric Quantifi-

117 cation of Cystein-Containing Leukotrienes in Rat Bile Using 13C-Labeled Internal Standards, Biol. Mass Spectrom., 21 (1992) 509. M.J. Raftery, G.C. Thorne, R.S. Orkiszewski and S.J. Gaskell, Preparation and Tandem Mass Spectrometric Analysis of Deuterium-Labeled Cysteine-Containing Leukotrienes, Biomed. Environ. Mass Spectrom., 29 (1990) 465. C.R. Roe, D.S. Millington, D.A. Maltby, T.P. Bohan, S.G. Kahler and R.A. Chalmers, Diagnostic and Theraputic Implications of Medium-Chain Acylcarnitines in the Medium-Chain AcyI-CoA Dehydrogenase Deficiency, Pediatr. Res., 19 (1985) 459. C.R. Roe, D.S. Millington, D.L. Norwood, N. Kodo, H. Sprecher, B.S. Mohammed, M. Nada, H. Schultz and R. McVie, 2, 4-DienoyI-Coenzyme A Reductase Deficiency: A Possible New Disorder of Fatty Acid Oxidation, J. Clin. Invest., 85 (1990) 1703. C.R. Roe, D.S. Millington, S.G. Kahler, N. Kodo and D.L. Norwood, Carnitine Homeostasis in the Organic Acidurias Fatty Acid Oxidation: Clinical Biochemical and Molecular Aspects, K. Tanaka and P.M. Coates (eds) (Alan R. Liss, New York, 1990) pp. 383-402. C.R. Roe, N. Terada and D.S. Millington, Automated Analysis for Free and Short-Chain Acylcarnitine in Plasma with a Centrifugal Analyzer, Clin. Chem., 38 (1992) 2215. M.H. Shaffer, B.E. Noyes, C.A. Slaughter, G.C. Thorne and S.J. Gaskell, The Fruitfly Drosophila Melanogaster Contains a Novel Charged Adipkinetic-Hormone-Family Peptide, Biochem. J., 269 (1990) 215. B.N. Singh, C.E. Costello and D.H. Beach, Structures of Glycophosphosphingolipids of Tritichomonas Foetus: A Novel Glycophosphosphingolipid, Arch. Biochem. Biophys., 286 (1991) 409. B.N. Singh, C.E. Costello, D.H. Beach and G.G. Holtz, DI-O-Alkylglycerol, Mono-OAlkylglycerol and Ceramide Inositol Phosphates of Leishmania Mexicana Promastigotes, Biochem. Biophys. Res. Comm., 157 (1988) 1239. F.S. Tanzer, E. Tolun, G.H. Fridland, C. Dass, J. Killmar, P.W. Tinsley and D.M. Desiderio, Methionine-Enkephalin Peptides in Human Teeth, Peptide Prot. Res., 32 (1988) 117. D. Tsikas, Analysis of Cysteinyl Leukotrienes and Leukotriene by Gas Chromatograp h y - (Tandem) Mass Spectrometry, Eicosanoids, 5 (1992) 57. J.L.K. Van Hove, W. Zhang, S.G. Kahler, C.R. Roe, T-S. Chen, N. Ternada, D.H. Chace, A.K. lafolla, J-H. Ding and D.S. Millington, Medium-Chain AcyI-CoA Dehydrogenase (MCAD) Deficiency: Diagnosis by Acylcarnitine Analysis in Blood, Am. J. Hum. Genet., 52 (1993) 958. J.Y. Wescott, K.R. Stenmark and R.C. Murphy, Analysis of Leukotriene B4 in Human Lung Lavage by HPLC and Mass Spectrometry, Prostaglandins, 31 (1986) 227. B.K. Wong, T.F. Woolf, T. Chang and L.R. Whitfield, Metabolic Disposition of Trimetrexate, A Nonclassical Dihydrofolate Reductase Inhibitor in Rat and Dog, Drug Metab. Dispos. Biol. Fate Chem., 18 (1990) 980. Z. Yamaizumi, H. Kasai, S. Nishimura, C.G. Edmonds and J.A. McCIosky, Stable Isotope Dilution Quantification of Mutagens in Cooked Foods by Combined Liquid Chromatography-Thermospray Mass Spectrometry, Mutation Res., 173 (1986) 1. J.A. Zirolli, K.L. Clay and R.C. Murphy, Tandem Mass Spectrometry of Negative Ions from Choline Phospholipid Molecular Species Related to Platelet Activating Factor, Lipids, 26 (1991 ) 1112.

119

CHAPTER 6

MASS SPECTROMETRY: ISOTOPE RATIO MASS SPECTROMETRY

THOMAS R. BROWNE ~, GEORGE K. SZABO ~ and ALFRED AJAMI 2 1Departments of Neurology and Pharmacology, Boston University School of Medicine; Neurology Service, Boston Department of Veterans Affairs Medical Center; 2Tracer Technologies, Inc.

Isotope ratio mass spectrometry (IRMS) determines the ratio of isotopes of a given element (e.g. H: 2H, 12C: 13C, ~4N: ~SN) present in a specimen. Typically, the total amount of each isotope also is determined. The amount of element derived from labeled drug in a specimen can be computed from the excess (over normal abundance) in labeled element isotope ratio and the total amount of element. The instruments that are traditionally associated with IRMS use multi-collector mass spectrometers that have the precision necessary to measure very small differences in isotope ratios that arise in nature. Thus, IRMS offers the potential of detecting and quantitating added label in any whole biological matrix. In pharmacologic studies, this procedure offers simplicity and flexibility similar to radioactive tracer procedures without the administrative and safely concerns of radioactive studies. Historically, whole biologcal matrix samples were combusted to elemental gases off-line, and manually collected and transfered to a dual inlet IRMS system. Two variant types of IRMS have been applied to pharmacologic research: continuous flow-isotope ratio mass spectrometry (CF-IRMS) and chemical reaction interface mass spectrometry (CRIMS). CF-IRMS uses a multi-collector mass spectrometer to measure isotope ratios of combusted gas species in a continuous flow of inert gas, while CRIMS uses traditional selected ion monitoring mass spectrometry (SIM-MS) measuring isotope ratios of small microwaveinduced plasma ion species. Neither of these techneques are classic IRMS, however, both are based on the concept of isotope ratio monitoring of tracer excess above natural abundance in the biomatrix. Both methods have been applied to mass balance/metabolite identification studies (see Chapter 11).

120 Both methods report promising preliminary results, but neither method is fully validated (see Chapter 11).

1. CONTINUOUS FLOW-ISOTOPE RATIO MASS SPECTROMETRY (CF-IRMS) 1.1. Technique

In CF-IRMS (Figure 1) organic sample is introduced into the high temperature combustion chamber of an elemental analyzer and flash combusted with a pulse of oxygen. The oxidized combustion gases (C02, N2, NOx and H20) are carried by a continuous stream of helium (hence, "continuous flow") through clean-up phases to a gas chromatography-isotope ratio mass spectrometry (GC-IRMS) device (eliminating the problematic step of transferring by hand combustion gas species to IRMS). NOx is reduced to N2 in a reduction tube (Cu); H20 is removed in an H20 trap; and C02 is removed (for N2 determina-

Samples in ------r Autosampler He -i02

Switchable H20.trap CO2 trap I

i

pulse

Combustion tube 11020~C)

Reduction tube (6OO~

Control valve

.! _J processing To atmosphere Results out Figure 1. Schematic diagram of CF-IRMS instrument. From Browne et al. (3) with permission.

121 tions) in a C02 trap. N2 and C02 are separated on a GC column and carried to an ion source where an ion beam is generated for each combustion gas. In the magnetic sector of the MS, the beam is separated into multiple beams depending on the mass-to-charge ratio of the various isotopically labeled ions. The beam for N2 is separated into three ion beams in the magnetic sector, and ion currents are measured at preset masses (m/z 28, 29, 30) in Faraday cup collectors. The intensity of ions at these m/z values are used to calculate 14N/14N, 15N/14N and 1SN/15N ratios. The mass spectrometer data system also calculates total nitrogen. In a biological sample containing a 15N tracer, an increase in masses 29 and 30 above natural abundance (atom percent) will correlate with the concentration of enriched tracer as atom percent excess (APE). Similar considerations apply for 13C (1) except that the mass spectrometer is tuned to measure m/z 44([12C02]), 45([13C 02] + [12C 170 160]) and 46([12C 180 160]), 1.2. H i s t o r y

IRMS with 15N and 13C tracers has been used extensively in the past for environmental, metabolic and nutritional studies but has been used little for pharmacokinetic studies (1-8). Hachey et al. (1) provided an excellent review of technical considerations of IRMS for nutrition and biomedical research. Benedetti and Pataky (4) measured the elimination of lSN-labeled urea in rats using an early spectroscopic analytical method. In 1974, Von Unruh et al. (9) and in 1976 Sano et al. (10) each demonstrated mass balance and metabolite identification techniques with 13C, labeled aspirin in human urine using combustion/mass spectrometric isotope ratio measurements. In 1985, Nakagawa et al. (11) performed a successful IRMS mass balance study of antipyrine in rats using 15N and 13C labeling. These studies did not lead immediately to routine animal or human applications, presumably because of the problems with early IRMS methods discussed below. Goodman and Brenna (12) described a high precision (at natural abundance) gas chromatographic combustion-isotope ratio mass spectrometric (GCC-IRMS) system capable of performing metabolite identification and mass balance for pharmaceuticals. Similar to the GC-pyrolizer-MS system used earlier by Sano et al. (10), the GCCIRMS system relies on isotope ratio measurements from combusted GC peaks. However, this is a problem in metabolite studies where thermolabile polar conjugates are present. Inadequate derivitization of nonvolatile drugs and metabolites would also be a problem with methods that used GC as the preliminary separation technique. These issues would also prove to be limitations for early GC-CRIMS methods. In 1993, Browne and coworkers (3,

122 13-16) were the first to provide evidence that stable isotope labeling and newly developed, commercially available CF-IRMS instruments could be used for whole matrix human mass balance studies and detection of labeled liquid chromatographic (LC) peaks for metabolite identification studies. However, it should be noted that LC peaks were individually collected and processed offline as discrete samples for CF-IRMS analysis. The optimal instrument would directly interface an HPLC to a continuous flow combustion isotope ratio mass analyser. Classic IRMS instrumentation was problematic because: (1) each instrument was unique and "made by hand"; (2) complete liberation of all atoms of a given molecule by manual oxidation techniques was difficult; (3) transfer by hand of N2 and CO2 gases from Dumas combustion techniques to IRMS was problematic; (4) each specimen was run manually (lack of automation); and (5) factors 1-4 made IRMS difficult and inconsistent. Recently, refined commercially-available instruments using a helium carrier gas to carry combustion products to the IRMS have become available from three sources (Europa Scientific, Ltd.; Finnegan MAT; and Micromass, Inc.). The authors purchased a Europa (Europa Scientific, Inc., Franklin, Ohio, USA) ANCL-SL (elemental analyzer) 20/20 (mass analyzer) CF-IRMS and found the instrument performed up to specifications and with very high precision as delivered (see Chapter 11 ).

1.3. Assumptions, Validation, Applications, Advantages and Disadvantages These topics are covered for pharmacologic applications of CF-IRMS in Chapter 11.

2. CHEMICAL REACTION INTERFACE MASS SPECTROMETRY (CRIMS)

2.1. Technique In CRIMS (Figure 2), the effluent molecules from a gas chromatograph (GC) or HPLC column (after solvent removal) are converted to low molecular weight ionic species, using a reactant gas in a microwave induced helium plasma with sufficient energy to break all chemical bonds (17-26). In the presence of an abundance of the reactant gas, the atoms of each analyte recombine into a common set of simple molecular species. The labeled and unlabeled species of small molecules are then detected and quantitated by selected ion monitoring (SIM) with either a magnetic sector or quadrupole mass spectrometer or

123

Reactant Gas !

i Micr~ Power

.........

I

Liquid Chromatograph I

I

Universal _ ~ Interface

I

Chemical Reaction Interface ii

LC S~ ! Removal I

Ill II

Mass Spectrometer

IIII

I

I'

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Figure 2. Schematic diagram of HPLC-CRIMS instrument.

an isotope ratio mass spectrometer (19). For example, in deuterated drug studies, H2 is used as a reductive reactant gas. The resulting CRIMS products are: HD (from deuterium labeling), CH4, HCN, H2S, H20 and C2H2. However, an MS with sufficient resolving power is required to measure HD at m/z 3.022 (18, 20). Strongly oxidizing reactant gases such as S02 are more useful for ~3C and ~5N isotopic detection and SIM can be performed at m/z 45 (~3C02) and m/z 31 (15N0) on a conventional quadrapole MS. Element selective detection (e.g. CL, Br, S, P, Se) is also possible with CRIMS methods for drug and metabolite molecules that contain these elements. Because of the inability of GC to analyse underivatized nonvolatile/thermolabile compounds (e.g. drug conjugates), only HPLC-CRIMS is satisfactory as a general method for mass balance/metabolite identification studies of new drugs with unknown metabolites.

2.2. History Recently, Abramson (20) provided an extensive review of the developments in CRIMS technology. CRIMS was introduced in 1982 (21) for use in conjunction with GC. In the following years a number of studies were performed using GC-CRIMS and deuterium, 13C and 15N labeling (22-24). The first attempt to combine HPLC with CRIMS utilized a moving belt interface (25). Later, a particle beam LC-MS interface (the Vestec Universal Interface) was used to join HPLC with CRIMS (26). This is the current state-of-the-art HPLC-CRIMS instrument which has been successfully applied to separation and detection

124 of deuterium, 13C and 15N labeled drugs (17-18, 26). More recently, postcolumn modifications to the HPLC interface have improved sensitivity such that LC-CRIMS had superior metabolite identification over on-line radiometric ~4C detection of a doubly labeled (~4C ~5N) anxiolytic drug (buspirone) in bile and urine (27-29). The principle need for post-column modifications of the particle beam LC-MS interface arises from the fact that HPLC gradient elution techniques are necessary to resolve polar metabolites. Unfortunately, aqueous mobile phase containing polar metabolites tends not to form uniform finely nebulized particles that readily desolvate in the universal interface. Post-column addition of an organic modifier (e.g. methanol) to the LC eluent stream reduces the variability of CRIMS response and improves instrument sensitivity for quantitation (27). This modification is critical for LC-CRIMS mass balance studies since accurate mass balance quantitation by this technique requires the measurement of the sum of all the possible labled peaks in a given sample. This is not a trivial point since each sample analyzed by LCCRIMS will have labeled peaks that cover a wide dynamic range of isotope ratio measurments. Thus, a drug that forms many minor metabolites may give total mass balance quantitation errors simply due to a lack of sensitivity of individual isotope ratio measurments for minor metabolites. As these technical details are obviated, LC-CRIMS promises to be an exciting and powerful technique for pharmacologic tracer studies.

2.3. Assumptions, Validation, Applications, Advantages and Disadvantages These topics are covered for pharmacologic applications of CRIMS in Chapter 11.

ACKNOWLEDGMENTS Supported by the United States Department of Veterans Affairs. We wish to thank Fred P. Abramson, Ph.D. for a critical review of this paper.

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125 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

A.L. Burlingame, T.A. Baillie and R.R. Russell, Anal. Chem., 64 (1992) 467R. E.B. Fenn, P.J. Garlick and W.A. McNurlan et al., Clin. Sci., 61 (1981) 217. R. Guilluy, F. Billion-Fey and J.L. Brazier, J. Chromatogr., 562 (1981) 341. C.H. Suelters and J.T. Watson, Biomedical Applications of Mass Spectrometry (Wiley, Chichester, 1990), p. 1. G.E. Von Unruh, D.J. Hauber and D.A. Schoeller et al., Biomed. Mass Spectrom., 1 (1974)345. M. Sano, Y. Yotsui and H. Abe et al., Biomed. Mass Spectrom., 3 (1976) 1. A. Nakagawa, A. Kitagawn and M. Asami et al., Biomed. Mass Spectrom., 12 (1985) 502. K.J. Goodman and J.T. Brenna, Anal. Chem., 64 (1992) 1088. G.K. Szabo, T.R. Browne and A. Ajami, in 5th International Symposium on the Synthesis and Applications of Isotopes and Isotopically Labeled Compounds, J. Allen and R. Voges (eds), (Wiley, Chichester, 1995), p. 443. T.R. Browne, G.K. Szabo and A. Ajami, in 5th International Symposium on the Synthesis and Applications of Isotopes and Isotopically Labeled Compounds, J. Allen and R. Voges (eds), (Wiley, Chichester, 1995), p. 435. T.R. Browne, G.K. Szabo and A. Ajami, J. Clin. Pharmacol., 35 (1995) 935. T.R. Browne, G.K. Szabo and A. Ajami, J. Clin. Pharmacol., 35 (1995) 935. Y. Teffera, F.P. Abramson and M. McLean et al., J. Chromatogr., 620 (1993) 89. Y. Teffera and F.P. Abramson, Biomed. Mass Spectrom., 23 (1994) 776. Y. Teffera and F.P. Abramson, Abstracts, 42nd ASMS Conference on Mass Spectrometry (1994) p. 863. F.P. Abramson, Mass Spectrom. Rev., 13 (1994) 341. S.P. Markey and F.P. Abramson, Anal. Chem., 54 (1982) 2375. D.H. Chace and F.P. Abramson, in Synthesis and Applications of Istopically Labeled Compounds, T.A. Baillie and J.R. Jones (eds), (Elsevier, Amsterdam, 1989), p. 253. D.H. Chace and F.P. Abramson, Anal. Chem., 61 (1989) 2724. D.H. Chace and F.P. Abramson, J. Chromatogr., 527 (1990) 1. M. Moini and F.P. Abramson, Biomed. Mass Spectrom., 20 (1991) 308. F.P. Abramson, M. McLean and M. Vestal, in Synthesis and Applications of Istopically Labeled Compounds, E. Buncel and G.W. Kabola (eds), (Elsevier, Amsterdam, 1992), p. 113. F.Y. Hsieh, C.A. Goldthwaite and B. Nobes, Abstracts, 42nd ASMS Conference on Mass Spectrometry (1994) p. 615. C.A. Goldthwaite, F.Y. Hsieh and B. Nobes, Abstracts, 42nd ASMS Conference on Mass Spectrometry (1994) p. 618. F.Y. Hsieh, C.A. Goldthwaite and B. Nobes, Abstracts, 43rd ASMS Conference on Mass Spectrometry (1995) p. 566.

127

CHAPTER 7

ALTERNATIVES TO MASS SPECTROMETRY FOR QUANTITATION OF STABLE ISOTOPES: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH CONVENTIONAL ULTRAVIOLET DETECTION

GEORGE K. SZABO, ROBERT J. PERCHALSKI*, DAVID G. BROWNE and THOMAS R. BROWNE Neuropharmacology Laboratory, Department of Neurology, Boston University School of Medicine; Research Service, Boston Department of Veterans Affairs Medical Center; *Consultant, PO Box 12906 Gainesville, FL 32606

1. I N T R O D U C T I O N 1.1. Rationale for an Alternative

The rationale for selective quantitation of stable isotope labeled (SIL) compounds in pharmaceutical research has been discussed extensively in other chapters in this book as well as in the scientific literature at large (1-7). Historically, mass spectrometry (MS) in all its forms has been the mainstay of analytical methodology for both qualitative and quantitative studies using stable isotopes. So what is the rationale for seeking an alternative to mass spectrometry? The mass spectrometer traditionally fulfils the role of a highly selective and sensitive detector placed at the tail end of some chromatographic (separation) process. Mass-selective detection, however, involves ion formation, mass filtration and ion detection, all of which must be carefully controlled by an elaborate support system of vacuum pumps, magnets or radio-frequency controllers and a sophisticated data acquisition and storage system. Therefore, most mass spectrometric detectors are more complex and

128

expensive than any of the separation techniques and instrumentation that precede the mass analyzer. Such complexity and expense for purchase, operation and continuing maintenance has kept mass spectrometry out of the mainstream as a tool for general quantitative analysis. Although mass spectrometry, and even tandem mass spectrometry is widely used in large pharmaceutical companies, there are many investigators in small pharmaceutical firms, universities and patient care facilities who do not have access to this technology. Over a decade ago, Hoffman and Porter (8) recognized the advantage of eliminating the costly mass spectrometer for certain stable isotope applications. They developed a gas chromatographic (GC) method that resolved the antiepileptic drug (AED) valproic acid (VPA) from deuterated [2H] VPA analogues. Within six years of this application, our group had published a liquid chromatographic (LC) method that resolved two other antiepileptic drugs (phenytoin and carbamazepine) from their respective deuterated analogues (9). The critical point in this methodological approach was that mass discrimination was shifted from a detection process to a chromatographic process. Traditionally for SlL tracer studies, mass discrimination came at the end of some cleanup chromatographic process that would isolate the tracer and parent drug from potential interferences endogenous to the matrix. The purpose in GC-MS, or LC-MS was to produce an isographic peak. This peak contained tracer, parent drug and possibly an internal standard. The peak components were then fragmented to give ionic species for mass spectral identification and quantitation. An alternative to this process is to use a chromatographic isotopic separation of the tracer and its parent compound and measure the separated peaks by some simple general or selective detector against some internal standard. Depending on the intended tracer application (mass balance, drug interaction, bioequivalence, etc.), the alternative chromatograghic method has advantages of simplicity, lower cost, and reproducibility over the mass spectrometric methods. The greatest disadvantages are that deuterium is the sole source of heavy isotope, a high degree of isotope substitution is required and isotopic separation of the deuterated analogues cannot be guaranteed. The advantages and disadvantages of each method of mass discrimination and quantitation will be compared and contrasted in detail throughout this chapter. The purpose of this chapter is to review the work that has been done with chromatographic separation of stable isotope-labeled drugs for pharmacological studies.

129 1.2. Isotope Effect

The primary feature of this alternative method to mass spectrometric analysis is its reliance on a beneficial chromatographic deuterium isotope effect. Twenty years ago Blake et al. (10) wrote a comprehensive review of "Studies with Deuterated Drugs" in which adverse deuterium isotope effects were prominently featured. Van Langenhove (11) has elegantly described the consequences of adverse in vivo isotope effects for pharmacologic studies and cautions investigators on the choice and placement of stable isotopes in drug molecules. Chapter 2 in this text is devoted to a full discussion of in vivo isotope effects. A distinction should be made between the adverse in vivo kinetic (physicochemical and biochemical) deuterium isotope effects, and the chromatographic deuterium isotope effect that alternative chromatographic methods depend on for mass discrimination. An adverse in vivo isotope effect changes the rate at which a SIL compound is absorbed, metabolized, or eliminated, such that the SIL compound's behavior in the body is significantly different from that of it's unlabeled analogue. A chromatographic isotope effect results in a physicochemical difference in the rate of migration between a SIL compound and it's unlabeled analogue during the chromatographic process. The primary considerations for isotopic separation are the placement and number of deuterium atoms in a SIL molecule necessary to achieve baseline resolution. For SIL tracer studies, chromatographic conditions must be produced and maintained so that a sensitive, selective and cost effective quantitative assay is achievable. Some drug molecules lend themselves to creative deuteration allowing chromatographic isotopic separation and quantitation without mass spectrometry and without in vivo isotope effects (8, 9, 12, 14). Historically, alternative methods to mass spectrometry were not created so much by design; rather, they evolved from an SIL tracer study designed for mass spectrometric analysis. Von Unruh (13) used a tetradeutero valproic acid (VPA) isotopomer for a study of elimination kinetics in patients with epilepsy on maintenance doses of VPA. This GC-MS study was conducted on a large bore packed GC column that probably did not chromatographically resolve the isotopomers. Later, Hoffman and Porter (8) and Durden and Boulton (14) found that highly efficient capillary glass GC columns could resolve valproic acid from its deuterated analogues. Heavily deuterated isotopomers containing five or more deuterium atoms had been extensively used in GCMS assays as analytical internal standards because they were relatively inexpensive and easy to synthesize with high isotopic purity. Concern over primary and secondary isotope effects, however, often caused these compounds

130 to be passed over as tracers in SIL tracer studies in favor of more costly ~3C and ~SN analogues. Hoffman and Porter (8) were the first to demonstrate the use and potential benefits of a deuterated analogue as the tracer in a pharmacokinetic study using GC assay without MS. In our laboratory, decadeuterophenytoin ([2H~o]-PHT, Figure 1) was the mainstay internal standard for our early GC-MS work (15). Decadeuterocarbamazepine ([2Hlo]-CBZ, Figure 1) was first synthesized for us with the intended use as a GC-MS internal standard. We had noticed in the literature that other investigators had demonstrated bioequivalence of heavily deuterated isotopomers for SIL tracer studies (8, 16, 17). There were also reports of isotopic separation of deuterated compounds from their undeuterated analogues using liquid chromatography (LC) (18-22). We postulated that an LC assay with simple ultraviolet detection could be developed for these decadeuterated compounds, and were able to achieve base-line resolution of both deuterated isotopomers from their unlabeled analogues with a single isocratic LC method (5).

2. AN ALTERNATIVE METHOD

In a semi-preparative reversed phase LC method that was used for cleanup prior to GC-MS analysis, we suspected that isotopic substitution caused a decrease in the retention time of [2Hlo]-PHT relative to phenytoin (PHT) and the 13C, ~SN2-PHT analogue. Under these conditions (described below), [2H~o]PHT, ~3C, ~SN2-PHT and PHT co-eluted. If the fraction collector missed the front portion of the peak even by a few seconds, however, subsequent GCMS ion intensity readings for the deuterated internal standard were lower than when the fraction collection window started well before the front of the peak. From this observation we suspected that some isotopic separation was taking place. The mobile phase for this LC procedure was strongly organic (>50 percent) and the column packing was 10 i~m particle C18 reversed phase. Phenytoin and carbamazepine, being very nonpolar compounds, eluted late at 16.1 and 18.6 min, respectively (23). Simply lengthening the elution time of this assay by decreasing flow rate, or decreasing organic modifier, did not give base line resolution of the [2H~o] isotopomers from their parent analogues. We then tried a reversed phase C~8 column with smaller particles (5 i~m or less) and with large C~8 chain density (l~mol/m 2) to gain resolving power, and addition of a small amount of tetrahydrofuran (THF) to the mobile phase to gain more control over the carbamazepine analogues. With the addition of 0.5 percent V/V THF to a 25 percent aqueous acetonitrile mobile

131 Carbamazepine Internal Standard

Parent Drug

Isotopic Analogue

c,'=o

NH2

D

(~=O D i

NHz

NHz

Metabolites

,-, DOD

i

D

c=o

D

~=o o

NH2

I~IH2 Phenytoin

Parent Drug

Isotopic Analogue D D

H

D

Metabolites

D

o D

H

.o~

D D

~..

.O~yo ~.. Ethotoin

Parent Drug H

"~o

Isotopic Analogue D

~

"~o

N H =" ~'---N\ /D 9 O/'~- ~C/, /H O D/C, / D H /C\ D/C\D H H Figure 1. Chemical structures and isotopic labeling of drug standards and metabolites described in our studies. Deuterium [2H] substitution for protium [H] on a molecule is presented by [D].

132 CBZ

PHT-dIo

A 6

. . . . 1o TIME

2o

ao

4"o

So

MIN.

Figure 2(A). Isocratic isothermal (room temperature) chromatographic separation of carbamazepine (CBZ), phenytoin (PHT) and their respective decadeuterated isotopomers (CBZ-dlo)and (PHT-dlo).

phase, [2Hlo]-CBZ was not only resolved from CBZ and [2Hlo]-PHT from PHT but the CBZ/deutero-analogue pair eluted earlier than the PHT/deutero-anaIogue pair (see Figure 2A), the reverse of the order without THF. At ambient temperature the run time was almost 50 rain long. Increasing the column and pre-column temperature to 55~ reduced the run time to 30 rain, and improved peak shape and resolution by increasing rates of mass transfer in the stationary and mobile phases (24). Subsequent modifications to the mobile phase (Figure 2B) allowed the addition of an internal standard (10,11-dihydrocarbamazepine) which eluted between the isotopomer/analogue pairs (Figure 2B) (9). It should be noted that not all high efficiency small particle C~8-columns gave such robust resolution. The BioAnalytical Systems Phase II ODS 5 (25 cm x 4.6 mm i.d.) stainless steel column used in this assay gave the best results at the time this work was done; however, recent advances in solid support treatment and stationary phase bonding chemistry should allow even more efficient and reproducible separations. After chromatograghic optimization, a rigorous multi-sample validation study was conducted to demonstrate assay precision and accuracy suitable for SIL tracer studies (25). Investigators should choose reversed phase columns for such applications first by a column's proven ability to resolve a drug from its metabolites and possible

133 10.11

CsZ 0.S.)

a-=N o

B

i 10

2"0

30

RUN TIME IN MINUTES Figure 2(B). Isocratic, isothermal (55~ chromatographic separation of the above compounds plus the 10,11-dihydrocarbamazepine (10,11-dihydro-CBZ) structural analogue internal standard (IS) described in Szabo eta/. (9).

interferences (endogenous species and other drugs). Standard LC method development techniques can be applied to this task. If isotopic separation of deuterated isotopomers from their parent analogue is shown to be possible, a number of replicate columns and variations on chromatographic conditions should be tested before an assay is deemed suitable for tracer studies.

134 3. ISOTOPIC SEPARATION

3.1. Synthetic Considerations

Although isotopic separation is by definition an isotope effect, the rule of thumb for synthesis of a tracer compound still should be to construct the molecule to minimize in vivo isotope effects. Deuteriums should not be placed in metabolically active sites where they might be exchanged or lost altogether during biotransformation. Sometimes the synthesis itself will determine the placement of deuterons, and thus the scope of the tracer application may have to be modified to accomodate the compound. For instance, [2H~o]-CBZ was synthesized by a metal catalyzed deuterium substitution reaction, in which all protiated ring carbons, including the bridgehead carbons at the 10 and 11 positions, were deuterated. The major metabolite of carbamazepine is carbamazepine 10,11-epoxide (CBZ-E). This compound is found in blood and has anti-epileptic properties (26). [2H~o]-CBZ will form decadeutero 10,11epoxide ([2H~o]-CBZ-E, Figure 1); yet there is no way of predicting if a possible in vivo kinetic isotope effect during epoxidation of the deuterated bridgehead (10,11)-carbons is significant enough to exclude [2H~o]-CBZ in an isotope tracer study. The point here is that the substitution reaction of the entire ring system involves fewer synthetic steps than building [2H8]-CBZ carbon by carbon with protiated 10,11-carbons and deuterated benzene rings. Thus, synthesizing [2H~o]-CBZ is simpler and less costly than [2H8]-CBZ. The decadeutero phenytoin was synthesized by the incorporation of a [2H6] benzene precursor (a common NMR solvent) into the final phenytoin molecule. This relatively simple and inexpensive synthetic pathway provided a tracer with sufficient deuteration for chromatographic separation. This tracer compound has a vulnerable deuterium in the active site of the major metabolic pathway. Para-hydroxylation of the phenyl ring is the metabolic pathway that turns the decadeutero phenytoin into a nonadeutero parahydroxyphenytoin ([2Hg]-p-HPHT) metabolite (Figure 1). Caution should always be exercised when isotopic labeling occurs at metabolically active sites; however, Browne et al. (27), Mamada et al. (16) and Hoffman and Porter (8) each have demonstrated pharmacokinetic equivalence of heavily deuterated carbamazepine, phenytoin and valproic acid tracer compounds with their corresponding unlabeled analogues. Although the last two methods were GC and not LC methods, the critical feature in these methods was the elimination of mass spectrometry for tracer quantitation.

135

3.2. Chromatographic Considerations For some time, researchers have investigated the physicochemical effects of substituting deuterium for hydrogen. Results of these studies, some of which are chromatographic, indicate several effects which are linked, including: 1. carbon-deuterium bonds are shorter than carbon-hydrogen bonds (28); 2. molar volume decreases when deuterium is substituted for hydrogen (29, 30); 3. carbons bonded to deuterium have lower polarizability than carbons bonded to hydrogen (29, 31); 4. deuterated compounds have lower lipophilicity than their protiated analogues and behave accordingly in reversed phase (elute earlier) and normal phase (elute later) chromatographic systems (22, 32, 33, 34). The magnitude of the beneficial isotope effect which allows chromatographic resolution of isotopomers depends on the number and location of deuterium atoms in the molecule (35). The general resolution equation can be the best guide for optimizing the separation (36). This equation:

Rs =

(4)

(~x- 1)k/-~

[']

1 +k'

indicates that resolution between closely spaced components (Rs) is dependent on column efficiency (N, a measure of band broadening), selectivity of the mobile and stationary phase combination for the species to be separated (e, a measure of band separation), and retention as measured by the capacity factor (k', retention factor). Column efficiency (N) is dependent on the quality of the column hardware, packing material and the column packing process; however, it is also dependent on the functional groups present in the solutes (isotopomers). Assuming that hardware is optimized (minimum dead volume), selectivity (a) depends on the stationary phase and mobile phase interactions with each other and with the analyte. Successful separation of isotopomers is dependent on small differences in their affinity for the stationary phase. Therefore, the stationary phase should be chosen to give narrow symmetrical peaks for the solutes with moderate to long retention times. The time required for the separation (as measured by k') is the least important of the three parameters in this case. If selectivity is adequate, retention time

136 can be adjusted easily by changing the mobile phase or increasing column temperature to achieve the separation in a reasonable time. Chromatography is always a trade-off between resolution and speed. In order to gauge resolution, peak retention time (k' as a unit of time t) is a generally acceptable means to measure resolution. For any particular separation on the same column, the above equation can be redefined as follows (35): t~ (1/2)(twl + tw2) t2-

a s --

where relative peak separation (Rs) is calculated from the difference of retention times of adjacent peaks at tl and t2, divided by the average peak base widths of twl and tw2. This concept is graphically demonstrated in Figure 3B. Generally, the above principles will provide a system that will separate isotopomers; however, each pair of analytes will probably require independent optimization. For example, in the system described above for separation of isotopomers of phenytoin and carbamazepine, deuterated ethotoin (2HloEHN, mol. wt. 214.2, %2H = 4.7 percent) is not separated from ethotoin (EHN, mol. wt. 204.2) at k ' = 5.4. As shown in Figure 3A, the Rs value (using the second equation) was only 0.6 for both ethotoins. At the same retention time, however, deuterated carbamazepine epoxide (2H~o-CBZE, mol. wt. 262.3, %2H = 3.8 percent) is completely separated from carbamazepine epoxide (CBZE, tool. wt. 252.3). In this case with the ethotoins, it was necessary to reduce the acetonitrile concentration in the mobile phase from 16 to 6 percent to achieve baseline separation of the EHN isotopomers at k ' = 12.7. THF concentration remained at 4 percent. As shown in Figure 3B the peaks are resolved to an Rs value of 1.1. Selecting a reversed phase column can be a confusing experience. Since the introduction of bonded phase liquid chromatography in the late 1960s (37), people have recognized that all bonded phases of a particular type (i.e., C8, C~8, nitrile) are not equivalent. In fact, one of the major concerns with using bonded phase columns for analyses, particularly in regulated environments, such as pharmaceutical development and environmental monitoring, has been nonreproducibility. This non-reproducibility has not just been between nominally equivalent phases from different manufacturers, but even between lots of material from the same manufacturer. Fortunately, the cooperative efforts of manufacturers of chromatography stationary phases and columns, and academic and industrial researchers over the past 20 years, have resulted in significant improvements in lot-to-lot reproducibility of bonded phases, increased column efficiencies and much greater understand-

137

A

Rs= 0 . 6

iuu

t2

I~1 i

tl

I

1 i

Rs = 1.1

tw 1

t---~ Figure 3. Chromatograms showing relative separation (resolution Rs) of ethotoin (t2) and decadeuterated ethotoin (tl). (A) Using condition described in Szabo et al. (9), and (B) Modifying the above conditions by reducing the acetonitrile content of the mobile phase from 16 to 6 percent.

138 ing of solute interactions with the mobile and stationary phases. Much of this information is available in a special issue of the Journal of Chromatography (38). Cox (39) published a very informative review of the effects of silica structure on reversed phase separations. He concluded that traces of heavy metals in the silica cause most of the problems with reversed phase separations. Olsen and Sullivan (40) used principal components and cluster analysis to categorize 17 different C18 reversed phase columns. Columns were grouped according to separations and peak shapes obtained for selected test mixtures which characterize the stationary phase according to hydrophobic and free silanol interactions, trace metal activity and shape selectivity. This type of information can make column selection much less confusing and increase the ruggedness of HPLC methods. Carr et al. (41) present evidence that shows that retention of nonpolar solutes in reversed phase chromatography is primarily due to attractive interactions between the solute and the stationary phase rather than to repulsive interactions between the solute and mobile phase (solvophobic interactions). Tchapla et al. (42), and Dorsey and Cooper (43), have reviewed retention mechanisms for bonded phase chromatography. Although there is some controversy surrounding this subject, Tchapla et al. (42) concluded that different models for retention apply under different operating conditions, and that partition, adsorption and solvophobic theories are all valid in certain circumstances. 3.3. Detection and Quantitation

The choice of LC detection method to use once base-line isotopic separation is achieved, depends on the drug under investigation. The anti-epileptics we studied are molecules with strong chromophores that readily absorb ultraviolet (UV) light, so that any of the fixed and variable wavelength, or even photodiode array UV/VIS detectors, would be appropriate. The lower limit of quantitation (LLQ) (25) for UV detection generally ranges from the high nanogram to low microgram per milliliter of analyte concentration. Drugs that are therapeutically in the sub-~g/mL range could be quantitated by electrochemical or fluorescence detectors; however, these more selective detection methods might require use of post-column derivatization techniques to enhance drug detection sensitivity. Pre- or post-column derivatization always adds an additional level of technical complexity and a potential source of error to a quantitative assay. The point is that the LC alternative method should not become more technically labor intensive than the mass spectrometry it seeks to avoid.

139 4. CONCLUSION

The LC method we developed (5, 9) was designed for drug interaction studies. Phenytoin and carbamazepine are the two most commonly prescribed antiepileptic drugs against which a new AED entity would be compared. Drug interaction studies generally require quantitative analysis of multiple serialblood samples from multiple subjects given SIL tracers on multiple occasions. For such a volume of samples, a stable, reproducible LC method is a costeffective alternative to mass spectrometry. Bio-equivalence of the heavily deuterated tracers has been demonstrated. Moderately nonpolar and apolar deuterated drugs are well suited for iosotopic separation by chromatographic methods. The polar neurotransmitter dopamine in a heavily deuterated form has been resolved from its unlabeled analogue on a reversed phase C18 column using ion pair chromatography (35). Thus, reversed phase LC separation of polar drugs and heavily deuterated analogues seems possible as long as the hydrophobic and lipophilic interactions of column and compound are optimized. Similar applications for other drug classes are possible if investigators take the necessary steps to show pharmacokinetic equivalence of labeled and unlabled forms, and pay careful attention to the number and location of deuterium atoms on the labeled analogue.

ACKNOWLEDGEMENT

The authors wish to acknowledge the support of the United States Department of Veterans Affairs.

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1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

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141

CHAPTER 8

ALTERNATIVES TO MASS SPECTROMETRY FOR QUANTITATING STABLE ISOTOPES: APPLICATION OF NUCLEAR MAGNETIC RESONANCE IN BRAIN METABOLIC RESEARCH

K.L. BEHAR Department of Neurology, Yale University School of Medicine, New Haven, Connecticut

1. I N T R O D U C T I O N

The explosive growth in the application of NMR spectroscopy to the study of brain metabolism and function is a testament to the versatility of this noninvasive and clinically applicable method. NMR-active isotopes exist for the majority of the biologically important nuclei but only a few of these (e.g. 1H, 31p, ~3C, ~SN, ~gF) are widely used in brain studies. A steady progression of technical developments has led to studies that span from the imaging of anatomy, diffusion, and function to the spectroscopic imaging of metabolite concentrations and metabolic fluxes (1). ~H is the most sensitive and abundant of the stable NMR-active nuclei. Water is the most concentrated of brain constituents and measurement of its spatial distribution in the brain forms the basis of magnetic resonance imaging (MRI). Detection of the low molecular weight metabolites (e.g. glucose, lactate, glutamate) requires that water and peripheral tissue lipid resonances be suppressed with great efficiency (>104-10S-fold). The list of compounds that have been reported in ~H spectra of brain now exceed twenty (2, 3). Neurochemical signatures of cell-type and density and/or disease processes have been identified in the 1H NMR spectrum and combined with imaging to create intensity maps of the resonances of these compounds (1). The relatively high concentrations (>O.5mM) of phosphorylated com-

142 pounds of key importance in cellular oxidative reactions, e.g. adenosine triphosphate (ATP), phosphocreatine (PCr), and inorganic phosphate (P~), have lead to the widespread use of 3~p NMR in the study of cerebral energetics in vivo (1, 4, 5). Although 31p is intrinsically less NMR sensitive than 1H (6.6 percent), the high natural abundance of the 3~p isotope in nature (100 percent) requires no specific enrichment for its detection. A unique capability of 3~p NMR is that intracellular pH can be determined simultaneously with the measurement of phosphorylated compounds (6). Phosphodiesters and monoesters detected in 3~p NMR spectra are associated with cellular membrane metabolism and these substances are altered in some disease states. A powerful application of NMR lies in the investigation of brain glucose metabolism and the quantitative determination of pathway fluxes (7, 8). The basic mechanisms regulating the transport and conversion of glucose to cellular energy can be studied using isotopic labeling techniques (e.g. ~3C, ~SN, ~gF). The use of ~3C and ~H NMR in conjunction with labeled isotopes of glucose and other substrates in vivo and in vitro have provided new information on the interactions between neurons and glia (4, 9). Measurements of the inhibitory neurotransmitter 7-aminobutyric acid (GABA) during anti-epileptic therapy in animals and humans highlights the role that NMR can play in the discovery and testing of new pharmaceutical agents (10). This chapter does not represent a comprehensive review of the literature; instead, the author has sought to show some examples of how NMR can be used to investigate cerebral metabolism and monitor both the progression of disease and treatment. Consequently, many important studies were not included and the reader is encouraged to consult the excellent reviews (1, 4 9, 11) and references within the text for detailed information concerning each application.

1.1. Some Basic Principles of NMR 1.1.1. Nuclear spin and the NMR phenomenon The basis for the NMR phenomenon arises from an intrinsic property of nuclei having odd numbers of protons or neutrons and unpaired nuclear spins. Such nuclei possess a net spin angular momentum. Because nuclei are charged particles, nuclei with spin possess a magnetic moment. In the presence of an applied static magnetic field (Bo), the nuclear moments orient themselves in discrete states given by the spin quantum number,/, w h e r e / = _+1/2 or ->1.

143 Nuclei w i t h / = 0 are nonmagnetic (e.g. ~2C) and do not give rise to an NMR signal. For a spin-l/2 nucleus, the orientation of the magnetic moment lies either with or against the field. Most of the nuclei studied in biological systems are spin-l/2 (e.g. ~H, ~3C, 3~p, ~9F) and in the presence of a magnetic field can be described by transitions between two energy levels, although spin-3/2 nuclei may be encountered (e.g. Na § Li § leading to more energy levels and more complex relaxation behavior. The proportion of spins aligned (lower energy) or opposed (higher energy) with the field can be described statistically and is given by the Boltzman distribution. At ambient temperature, there exists only an extremely small excess population of spins aligned with the Bo field (i.e. low energy state). At thermal equilibrium this small spin population difference gives rise to a "net magnetization" and it is this property, when suitably prepared, that is measured in the NMR experiment. The equilibrium net magnetization is proportional to the strength of the applied static magnetic field and the total number of nuclear spins in the sample. The intensity of an NMR signal relative to noise (i.e. detection sensitivity) is proportional to this population difference, increasing with both field strength and sample concentration. The interaction between the applied magnetic field and each nuclear moment induces the latter to precess about the direction of the applied field. Because the net magnetization is given by the vector sum of the individually precessing moments, it can also be viewed in terms of a vector precessing about the direction of the static field. The precession frequency is proportional to the applied field and is given by the Larmor equation:

v = 712]-[ x Bo

where v is the Larmor frequency, 7 is the gyromagnetic ratio and is a constant that depends on the intrinsic properties of the nucleus, and Bo is the external magnetic field strength. The frequencies encountered in the majority of clinical and high resolution NMR applications are in the MHz range (~--2-500 MHz) for magnet strengths between 1 and 12 Tesla. The highest field large bore clinical system currently available operates at 4 Tesla, although higher field systems are currently being considered. For a given field strength each NMRactive nucleus (e.g. ~H, ~3C, etc) will be resonant at a characteristic frequency as defined in the Larmor equation. A more detailed discussion of NMR theory and its application may be found in (1) and the many excellent references cited therein.

144

1.1.2. Relaxation: the concept of T1 and T2 Detection of an NMR signal involves a perturbation of the magnetization from equilibrium. A radio-frequency (rf) pulse oscillating at the resonance frequency defined by the Larmor condition is used to perturb the magnetization from equilibrium. The perturbed nuclear spins return to thermal equilibrium, through the process of relaxation with a rate defined by the spinlattice relaxation time, TI. The T1 (spin-lattice) relaxation time refers to the longitudinal (along z-axis) vector component of the magnetization (Mz) as it returns to its equilibrium value (Mo). The intensity of an NMR signal is directly proportional to Mo prior to the rf perturbing pulse. As the magnetization is tipped away from equilibrium along the z-axis and into the xy-plane, randomizing interactions between the spins and their environment causes a loss of phase coherence and magnitude of transverse magnetization, Mxy. The decay of the transverse magnetization is defined as T2 (spin-spin) relaxation. Only transverse magnetization is detected directly with the receiver coil and it is this signal that is represented by the free-induction-decay (FID). Spatial inhomogeneity in the static magnetic field also contributes to T2; differences in precession rates between spatially separated nuclear spins results in a more rapid loss of phase coherence and decay of Mxy than if the field were the same everywhere. This decay is characterized by an apparent T2 relaxation time (Tt) where Tt < T2. Because the resonance linewidth is equal to 1/(]-[ x T2), field inhomogeneity leads to shorter values of Tt and broadening of the resonance width. Overlap between closely spaced resonances is a problem regularly encountered in NMR spectroscopy in vivo. The process of "shimming" involves the adjustment of a secondary set of gradient coils to smooth out field irregularities across the sample.

1.1.3. Detection of the NMR signal The tiny oscillating magnetic field generated by the sample can be detected with a suitable antenna (i.e. loop of wire) oriented perpendicular to the direction of the static field. Transmission and reception of rf is often obtained from a single antenna coil although different coils may be used for this purpose, such as for applications involving NMR of several different nuclei from the same sample or subject. The small currents created by the oscillating field detected by the coil is amplified, digitized, and sent to a computer for further processing. Standard processing techniques involve a Fourier transformation of the digitized free induction decay to give the frequency spectrum.

145 1.1.4. Chemical shift

The resonance frequency of a given magnetic nucleus (e.g. a hydrogen atom in glucose) is determined by its electronic and chemical environment and the given magnetic field strength. The electrons within molecules shield their nuclei from the external magnetic field resulting in slight differences in their resonance frequencies. This nuclear shielding is proportional to the applied field strength. The ratio of the shielding to field strength is referred to as the "chemical shift". The chemical shift is expressed as a fraction of the applied field strength in parts-per-million (ppm) from a suitable reference and is an identifying characteristic of the NMR spectrum of a particular molecule. 1.1.5. Spin coupling

The interaction between the magnetic fields of neighboring magnetic nuclei (e.g. 1H-13C) within a molecule induces additional splitting of the energy levels for each nucleus giving rise to the phenomenon of spin-spin or J coupling. The multiplicity of resonances observed for covalently bound methylene protons of amino acids in the 1H NMR spectrum or the heteronuclear ~3C-~H splitting observed in the ~H or ~3C spectrum of a 13C labeled metabolite are examples of this type of interaction. The coupling constant, J, is given in Hz and is independent of field strength. Although a detailed discussion of spin coupling is outside the scope of this review, it is important to note that spinspin or J-coupling is the basis of many of the spectral editing techniques currently used to select and simplify the in vivo NMR spectrum (e.g. lactate and GABA) from the large number of overlapping resonances of other compounds.

2. 1H NMR IN THE NEUROCHEMICAL DETECTION OF BRAIN PATHOLOGY 2. I. N-acetylaspartate as a NeuronaI-Axonal Marker

N-acetylaspartate (NAA)is highly concentrated in animal and human brain (5-8 mM) and is the most prominent metabolite resonance in the ~H NMR spectrum. Immunohistochemical and cell culture techniques have shown that NAA is confined mainly to neurons (11, 12). While NAA is not detected in 1H spectra of astroglial cultures (13) and tumors of glial origin (14-16), significant levels have been found in some immature oligodendroglia, where it may have a role in the synthesis of myelin (13). Decreased levels of N-

146 acetylaspartate, and by inference reduced neuronal density, has been observed in the necrotic regions of cerebral infarcts (17, 18), epileptic foci (19), and in patients with acquired human immunodeficiency syndrome (20), Creutzfeldt-Jakob Disease (21), and Alzheimer's dementia (22). However, decreased NAA levels may not always be related to neuronal death, as reversible changes have been observed in white matter lesions in patients with multiple sclerosis (23, 24), which may be related to changes in synthesis and/or degradation of NAA. Although the function of NAA (and myo-inositol, see below) in the brain remains obscure, heightened interest for their use as cell-type specific markers has resulted in a renewed interest in the metabolic pathways involved in their synthesis and degradation. 2.2. Myo-inositol as a Glial Marker

Myo-inositol has been identified as a possible glial marker based on the finding that high levels are present in cultured astrocytes but not neurons (25). The role of myo-inositol in cerebral metabolism is unclear but some evidence indicates it functions as an osmolyte. Increased levels of myoinositol have been observed in ~H NMR spectra of the cerebri of patients with Alzheimer's (22) and Creutzfeldt-Jakob dementia (21), whereas reduced levels are observed in hepatic encephalopathy (26). These conditions are known to affect glial cells. As with all metabolite markers, changes in levels may reflect either altered synthesis or degradation within a given cell or a change in cell number. The coordination of studies linking clinical disease states and cellular changes with appropriate animal and in vitro models are needed to elucidate the function of myo-inositol. 2.3. Macromolecules: Lipid and Protein as Markers of Brain Disease

Low molecular weight metabolites represent only a part of the 1H NMR spectrum and techniques have been developed to reveal the broad (shorter T2), but informative, background of underlying macromolecules (27). In normal nondiseased brain tissue, the majority of macromolecule resonances arise from cytosolic proteins (27, 28) with few if any resonances identifiable as lipids. During brain injury, such as stroke, tumors, and active multiple sclerosis lesions, loss of cellular membrane integrity, myelin breakdown, and possibly macrophage infiltration can lead to marked increases in lipid signals (18, 23, 24, 29). The ability to follow metabolic changes during the inflammatory process noninvasively should aid in the monitoring of new therapies targeted at reducing the inflammation associated with brain injury.

147 3. 31p NMR MEASURES OF BRAIN ENERGY METABOLISM

3.1. High Energy Phosphate Metabolism, pH and Mg 2§ The concentrations of the high energy phosphates, phosphocreatine (PCr) and adenosine triphosphate (ATP), are substantial in brain (5 and 3 ~mol/g, respectively), and are readily detected using 31p NMR. Signal-averaging times of 1-5 min to acquire a 0.5-1 cc3 volume of animal brain and 30-60 min for a -100 cc3 volume of human brain are typical using surface coil techniques. 3~p NMR is particularly useful in the determination of intracerebral pH based on the pH dependence of the chemical shift of inorganic phosphate (6). Free intracellular [Mg 2§ can be estimated from the chemical shift of ATP (30). Based on the equilibrium reaction maintained by creatine kinase (see below), both [ADP] and [AMP] concentrations can be calculated from the measured changes in [PCr], [ATP] and [H § in the 31p NMR spectrum (31). Changes in PCr, ATP, P~ and pHi in the 3~p NMR spectrum are sensitive indicators of tissue oxygenation. Cerebral hypoxia and ischemia leads to rapid depletion of high energy phosphates, elevation of P~, and cellular acidosis in animals (32) and in patients with stroke (33). Phosphomonoesters (PME) and phosphodiesters (PDE) in 3~p NMR spectra, which are linked to cellular phospholipid synthesis and catabolism, are altered in some disease states such as AIDS (20) and Alzheimer's dementia (34). As for other NMR-active nuclei detected in vivo, phosphorylated substances must be relatively mobile on the NMR time-scale to give narrow peaks in the 3~p NMR spectrum; phosphorylated proteins, which are low in concentration and restricted in motion, cannot generally be observed in vivo.

3.2. Creatine Kinase Flux Measured In Vivo Using Magnetization Transfer Cellular levels of phosphocreatine and ATP in brain tissue are enzymatically linked through the creatine kinase (CK) reaction. It has been possible under certain conditions to extract information on high energy phosphate turnover by measurement of the unidirectional fluxes for phosphoryl group transfers catalyzed by CK using magnetization transfer techniques (35). In the saturation transfer method, a low power rf field is applied to saturate either of the phosphate resonances of PCr or the ?,-phosphate of ATP, which is the phosphate group undergoing chemical exchange with creatine. Transfer of this "magnetization label" to creatine by the CK reaction results in a reduction in the intensity of PCr. By increasing the length of time between the end of the

148 saturating rf and the acquisition of the 31p spectrum in successive experiments, a time course of the recovery of PCr to its equilibrium state (or ATP7 if PCr is saturated) is obtained and used to derive the pseudo-first-order unidirectional rate constants for the forward reaction (PCr~ ATP) and the reverse reaction (ATP-~ PCR). In principal, both forward and reverse rate constants and fluxes can be determined. However, the reverse rate constant is more difficult to measure accurately (36) so that many investigators report only the forward rate constant. Results of a saturation transfer study of rat brain showed that phosphoryl group exchange between PCr and ATP is several-fold faster (>5 times) than the rate of ATP utilization (37). Evidence that CK is operating at, or near, equilibrium has been reported both in rabbit brain in vivo (36) and in superfused brain slices (38). The fast rates of exchange between PCr and ATP relative to the rate of ATP hydrolysis probably accounts for the lack of ATP changes during acute hypoxia or seizures (31, 39). In human grey matter the CK forward flux is 2-fold greater than in white matter (40), suggesting that CK activity is greater in neuron-rich areas. An activity dependent relationship in both the forward rate constant and flux of CK has been reported during thiopental anesthesia and seizures in rats (41). Changes in CK kinetics are also seen during development where a 4-fold increase is observed in the CK forward reaction rate over a narrow time period between 12-17 days after birth. This time period parallels the increase in the activity of the mitochondrial CK iso-enzyme when the latter is expressed as a percentage of total brain CK activity (42). The timing for the increase both in CK flux and mitochondrial CK expression is also coincident with development-dependent changes observed in the time course of PCr and ATP depletion during prolonged hypoxia. These studies suggest that ATP utilization and synthesis are closely associated with the maturational increase in CK flux.

4. NMR MEASURES OF CARBOHYDRATE METABOLISM

Glucose is the major fuel of the mature brain accounting for >90 percent of oxygen consumed in the well nourished state. During development, substrates other than glucose contribute appreciably; lactate and ketones (/3hydroxybutyrate and acetoacetate) may be oxidized to a greater extent than glucose, a process that appears to be related both to neuronal and glial development and the concentrations of these fuels in the blood. The availability of ~3C labeled isotopes of glucose, lactate and /3-hydroxybutyrate

149 provides the opportunity to explore many facets of cerebral energy metabolism. NMR spectroscopy permits measurement not only of the properties of key steps in the metabolism of glucose, such as the affinity and capacity of glucose transport, but the rates of the major pathways of energy metabolism. The determination of the TCA cycle flux allows the determination of several other fluxes, which include: glucose utilization, a-ketoglutarate/glutamate exchange, glutamine synthesis, and GABA synthesis. These pathways are of fundamental importance in brain energy metabolism, are altered during disease, and are uniquely suited to measurement with NMR methods.

4.1. Glucose Transport The concentration of intracellular glucose in the brain (G;) is determined by the kinetic parameters of the glucose transport proteins that reside in the microvasculature of the blood-brain-barrier (Kt, Tmax), the blood glucose concentration (Go), and the rate of glucose metabolism by the brain (CMRg~u). Glucose levels have been measured in rat (43) and human brain using 13C NMR (44, 45) and ~H NMR methods (46-48). At steady state, when blood and brain levels of glucose are constant, the unidirectional inflow and outflow velocities can be described in terms of the Michaelis-Menten formulation according to the following equation:

/max"~_

dG* = ('Go x Tmax~_ ( G ; x dt \ i G o +~)t) J (G~-+ Kt) /

CMRg,u = 0.

When this equation is fitted to sets of paired measurements of blood and brain glucose and iterated with the parameters Kt and the ratio of Tmax-tOCMRg~u, the values of Kt and Tmax of glucose transport are obtained. Both rat and human brain glucose transport kinetic parameters have been determined in this way using 13C NMR detection of [1-13C]-Iabeled glucose (43, 44). The observation that Tmax/CMRg~u > 1 in both rat and human brain in vivo shows that under normal conditions, transport is not limiting the utilization of glucose. Brain diseases associated with decreased transporter density and Tmax, e.g. Alzheimer's Disease, would be expected to narrow this margin. Another approach is to measure the change in the glucose signal in the brain following a rapid step-up in blood glucose concentration. The glucose infused may be either labeled or unlabeled, the choice determining the parti-

150 cular detection technique. ~H NMR detection of glucose in difference spectra of human occipital cortex (46) allows spectra to be acquired in as little as 3 min (48, 50). The rapid time resolution possible with ~H NMR allows dynamic changes in individual subjects to be assessed from the rise of the brain glucose signal (48). Intersubject variability can also be assessed directly. Because the transport kinetic parameters can be determined for individual subjects, the step-up method should be of particular value in clinical investigations of suspected glucose transport alterations and measurements of glucose utilization in paired stimulus-response functional studies. In the absence of changes in the transport parameters, changes in glucose levels during cortical activation can be related to changes in glucose utilization. Glucose levels have been observed to decrease in visual cortex during photic stimulation in humans which is consistent with an increased rate of glycolysis (7, 8, 49, 50).

4.2. Glucose Utilization 4.2.1. Glycolysis and lactic acid

The end product of the anaerobic utilization of glucose in mammalian brain is lactic acid. Under basal aerobic conditions, brain lactate levels measured in vivo using 1H NMR editing techniques are about 1 mM. Although hypoxia and ischemia can lead to high concentrations of lactate in vivo (17, 18, 32, 51), increased levels can be produced when the rate of glycolysis exceeds oxidation and lactate removal by the blood (e.g. seizures or hypocapnia), a subject that has fueled recent controversy in studies of functional activation (7, 8). Under hypoxic or ischemic conditions, glucose utilization through the glycolytic pathway can be determined from the change in the lactate concentration verses time in a series of sequentially acquired 1H NMR spectra. Lactate can be measured either without isotopic labeling (e.g. difference spectroscopy (51) or editing (52)) or after the enrichment of the precursor glucose pool (53) and both approaches have their specific advantages. Because the heteronuclear ~3C-edited ~H difference technique (53) permits both labeled and unlabeled species to be measured from a single set of acquired spectra, the fractional enrichment of lactate-C3 reflects the sum of the pathways contributing carbon atoms (both ~3C and ~2C) to the C3 position of lactate. During ischemia, the brain is essentially a closed system such that inflow of glucose and outflow of products is halted and oxidative pathways are inhibited. Under conditions of constant glucose labeling in the blood, the contribution of unlabeled endogenous glycogen to pyruvate (and lactate) production during ische-

151 mia resulted in the dilution of labeled lactate from which the concentration of glycogen could be estimated (53). Because glycogen is known to be localized in glia, its hydrolysis is a measure of glial metabolism; in principal similar measurements could be used to determine the contribution of glial glycogen metabolism during seizures. ~3C-labeling of the large lactate pool observed in human stroke (54) has shown that the lactate observed in ~H NMR spectra is metabolically active and in communication with blood glucose. Lactic acid may also be produced actively from leukocytes within weeks of cerebral infarction (18) and may be suspected when macrophage-associated lipid resonances are present. The ability to monitor these changes noninvasively provides new opportunities to assess the efficacy of different therapeutic approaches.

4.2.2. Hexose monophosphate shunt The hexose monophosphate shunt (HMP) or alternatively, the pentose phosphate cycle, diverts glucose carbon from the glycolytic pathway for the production of pentoses for nucleic acid synthesis and NADPH for synthesis of lipids and maintenance of reduced glutathione. In the adult brain, HMP activity accounts for only 3-5 percent of glucose metabolism but is increased during oxidative stress as shown in studies of rat brain using 13C NMR (55, 56). Discrimination between differently labeled glucose isotopes in the HMP pathway occurs at the level of 6-phosphogluconate dehydrogenase, which catalyzes the decarboxylation of C1 of 6-phosphogluconate produced from glucose-6-phosphate. For glucose labeled at C1, flux through the HMP pathway will result in the loss of label as 13C02 and in a lower enrichment of lactate C3. The measurement of HMP flux has been accomplished by monitoring label incorporation into lactic acid after the addition of equal mixtures of [1~3C] and [6-~3C]glucose to transformed glial cells in culture (57); an approach that corrects for the significant recycling of trioses that can lead to overestimation of HMP flux when using [1-~3C]glucose alone. HMP flux can also be determined from the relative distribution of ~3C in the C4 and C5 of glutamate at steady state using [2-13C]glucose (58).

4.2.3. Fluoro-deoxyglucose and alternate pathways of glucose metabolism Fluoro-deoxyglucose (FDG) competes with glucose for transport into brain tissue and is readily phosphorylated by hexokinase to fluoro-deoxyglucose6-phosphate (FDG-6-P). When FDG is administered in the blood in tracer

152 quantities, the fluorinated tag is trapped in tissue predominately as FDG-6-P, since the latter is not a substrate of phosphohexose isomerase (glycolytic pathway) and is only slowly metabolized by enzymes of the HMP pathway. However, under conditions of high loading doses (>400 mg/kg) of FDG, the product FDG-6-P may rise substantially in brain (>1 ~mol/g) as shown in rats using both 31p NMR (59) or ~gF NMR (60, 61). FDG-6-P can be metabolized further, albeit slowly, by the enzymes of the HMP and aldose reductase sorbitol (ARS) or polyol pathways. Administration of the [2-~9F] and [3-~9F] analogues of DG to rats leads to the appearance in brain tissue of the corresponding [2-~9F] and [3-19F] analogues of fluoro-deoxy-6-phosphogluconate, fluoro-deoxyfructose, and fluoro-deoxysorbital, metabolites of the HMP and ARS pathways (60, 61). Pre-treatment of rats with sorbinil, an inhibitor of aldose reductase, at pharmacologic doses reduced the flux of FDG through the ARS pathway (61, 62). ~gF NMR employing high loading doses of 2- and 3-fluorinated analogues of FDG in conjunction with analysis of fluorinated metabolites in extracts may be useful probes to investigate the regulation of the HMP and ARS pathways in the brain.

4.3. TCA Cycle Flux and Oxygen Consumption (CMR02) The metabolism of glucose in the brain can be traced in great detail through the use of 13C-labeled isotopes (7-9). The position of the isotopic label in the glucose molecule determines the specific labeling patterns observed in the various metabolites of glucose (Figure 1). For example, if the glucose molecule is labeled at C1, then pyruvate produced from glucose by the glycolytic pathway will be labeled at C3. Rapid chemical exchange between pyruvate and lactate catalyzed by lactate dehydrogenase will result in the labeling of lactate at C3. Oxidation of pyruvate in the TCA cycle leads to labeling of eketoglutarate C4, and through rapid isotopic exchange with glutamate, labelling of glutamate C4. The rate of isotopic exchange between a-ketoglutarate and glutamate is >60 times faster than the TCA cycle flux in rat and human brain (63, 64, 91). The fast exchange rate together with the high concentration of glutamate in brain tissue (about 10-12mM) makes glutamate a highly efficient ~3C-label trap. Glutamine and GABA synthesized from glutamate will be labeled initially at C4 and C2, respectively. Movement of the ~3C label around the TCA cycle results in the labeling of other carbon positions in glutamate and its products. The rate at which these other positions are labeled (e.g. C3) relative to C4 is a function of the rate of a-ketoglutarate/glutamate exchange and the rate of TCA cycling. The isotopic labeling of glutamate can

153

LABELING

OF BRAIN METABOLITES

FROM

13C G L U C O S E

CCCCCC Glucose

ccc ~

' ,I, ~cc ccc

Lactate

i,3,yco,~,, I

J/ pyruvate

o

CCCC o

cccc ~

ccccc ~__ @

J

ket~lu~r~e

Glutamate

1

succlnate oo 9

eoo

CCCCC

CCCC

Glutamlne

GABA

1

1

Figure 1. Labeling of brain metabolites from [1-13C]glucose. Glutamate efficiently traps 13C, first at C4 (filled circle) and then at other carbon positions due to movement through the TCA cycle (open circles). Carbon numbering is left-to-right as indicated for each molecule.

be detected either with 1H NMR using 13C-editing (65) or direct 13C NMR (91). When the labeled glucose is raised rapidly in blood and held constant, typically 50-60 percent enrichment, glutamate is labeled rapidly at C4 and more slowly at C3 as shown in human brain (Figure 2). Analysis of the time courses of the ~3C enriched spectral peaks using mathematical modelling permits both a-ketoglutarate/glutamate exchange and TCA cycle flux to be determined (63, 64). The glutamate measured with NMR may be associated with the "large glutamate pool" of neurons (65). The TCA cycle rate is coupled to oxygen consumption and can be used as an indirect but sensitive measure of CMRO2 (7). Measurement of the carbon-carbon J-couplings of ~3C-labeled metabolites in conjunction with isotopomer analysis (66-68) can give specific information about metabolism occurring within neurons and astrocytes (see below).

154

Time course of glutamate and other amino acids labelled from [1J3C]glucose in ~3C NMR spectra of human occipital cortex GluC4I 60 min "~

~ GlnC4 GIuC3

5

(ppm)

25

30 min

GluCA

....

c

;4'

'

0 min J0

i

i,

1

30 (ppm)

Figure 2. Time course of 13C labeling of glutamate, glutamine and aspartate in 13C NMR spectra of human occipital cortex during an intravenous infusion of [1-~3C]glucose. Each spectrum was acquired in 4-min blocks. (Reproduced from Gruetter et al., 1994, Ref. 91.)

5. METABOLIC COMPARTMENTATION" NMR MEASUREMENTS OF NEURONAL AND GLIAL METABOLISM Functional nervous tissue represents a bewildering degree of complexity where compartmentation exists at the anatomic, cellular and subcellular level. A major challenge of contemporary NMR spectroscopy as applied to the brain

155 is the development of methods and strategies to assess the contribution of major cell populations (e.g. neurons and glia) to the signals that are measured. Methodological developments include techniques to identify, select, and quantitate resonance intensities and isotopic enrichment (e.g. homonuclear and heteronuclear editing (10, 52, 53, 65)) while isotopic labeling strategies take advantage of known metabolic pathways and cell-specific enzymatic reactions to provide detailed information about neuronal and glial metabolism (67-69). The metabolism of glutamate and GABA associated with nerve terminals has been linked to a substrate cycle between neurons and astrocytes involving glutamate, GABA, and glutamine, generally termed the "glutamine cycle" (70). The efficient functioning of the glutamine cycle is made possible by the physical segregation of certain enzymes between neurons and glia. For example, glutamine synthetase and pyruvate carboxylase are astroglial enzymes. GABA synthesis catalyzed by glutamic acid decarboxylase is confined to neurons. Glutaminase catalyzes the production of glutamate from glutamine and is enriched in neurons. NMR measurements of the fluxes associated with these enzymatic reactions permit investigation of neuron and glia specific metabolic pathways. The synthesis of GABA in neurons and glutamine in astroglia are important examples. These reactions involve rearrangements of both carbon and nitrogen groups and provide opportunities for NMR investigations employing ~3C and 15N isotopes in vivo and in vitro as described below. 5. I. Information Contained in Isotopomer Distributions

A powerful method to determine the contributions of competing metabolic pathways occurring in vivo is based on the analysis of homonuclear 13C-13C couplings in 13C NMR spectra (66). Analysis of the mutiplet structure of ~3C NMR spectra of metabolites isolated from extracts of whole tissue, brain slices, or cell cultures following the use of doubly or universal labeled isotopes (e.g. [1,2-~3C]glucose, [U-~3C glucose], [1,2-~3C]acetate, [U-13C]glutamate) has been used to assess metabolism of neurons and glia in vitro and in vivo. For the case of two adjacent ~3C atoms in a molecule, spin coupling between the nuclei results in the splitting of each resonance into a doublet with a characteristic coupling constant, J (Hz). Singlet resonances arise from the 1.1 percent natural abundance of ~3C and reflect the contribution of unlabeled pathways to the carbon atom at that position in the molecule. Since the probability of observing a doublet in the ~3C spectrum from natural abundance ~3C is low, only 0.0001 percent, detection of a doublet indicates that the doubly

156 labeled carbon skeleton is incorporated intact; other pathways incorporating unlabeled carbon atoms will lead to isotopic dilution of adjacent carbons and the appearance of singlets. Therefore, the proportion of doublets to singlets at steady state represents the ratio of fluxes from labeled and unlabeled pathways. Differences in the isotopomer distributions between the TCA cyclelinked amino acids, glutamate, glutamine, and GABA is consistent with physically separate TCA cycles and the "compartmentation" of their metabolism (67-69). The distribution of ~3C labeling among the five carbons atoms of glutamate gives specific information on the flows of carbon into the cycle. Amino acids synthesized from glutamate, e.g. GABA and glutamine, will reflect the isotopic distribution of the glutamate and TCA cycle involved in their synthesis. Analysis of the isotopomer distributions by appropriate steady state mathematical models (66, 71, 72) has allowed the relative contributions of these pathways in neurons and glia to be assessed (68, 72). 5.2. Differentiation of Neuronal and Glial Metabolism from Isotopic Labeling

The distribution of isotopic labeling of glutamate, glutamine, and GABA in NMR spectra obtained from extracts of animal brain tissue in vivo (67, 68, 73) and in vitro (69, 74) following application of 13C-labeled substrates has been interpreted in terms of neuronal and glial compartmentation and the "glutamine cycle". Particularly advantageous for isotopic studies is the observation that neurons and glia can be differentiated by their preference for different substrates. Whereas neurons are almost solely dependent on glucose, glial cells are capable of metabolizing acetate carbon (9). The metabolism of [113C] or [1,2-~3C] acetate in rats leads to different isotopomer distributions of glutamate and glutamine in ~3C spectra of brain extracts (67). In superfused brain slice preparations glutamine is more highly labeled than glutamate from [2-~3C]acetate but not [1-~3C]glucose (69). Glutamine labeling from acetate occurs readily in cultured glial cells and co-cultures of glia and neurons but not in neuronal cultures ( 7 5 ) - a finding that is consistent with the known localization of glutamine synthesis in glia. Because glutamate serves as the direct precursor of glutamine, unequal 13C isotopic distributions between glutamate and glutamine must arise from physically separate glutamate pools. Together these findings are consistent with separate TCA cycles and separate pools of glutamate in neurons and glia. In vitro findings have provided an important framework for the interpretation of labeling patterns observed in vivo and will continue to be important to the development and refinement of realistic multicompartment metabolic models for the analysis of in vivo data in animal and human brain (63, 64, 66, 71, 72).

157 5.3. Glutamine Synthesis

Glutamine synthesis from [1-13C]glucose has been measured from the time course of glutamine labeling in13C NMR spectra obtained from extracts of rat brain (73), rat brain in vivo (76), and human occipital cortex (91). Metabolic modeling analysis of the glutamine and glutamate enrichment time courses in conscious human brain and rat brain in vivo have indicated that glutamine synthesis is more rapid than generally thought and may reflect, in part, the rapid exchange of labeled glutamate and GABA between neurons and glia (64, 76). This finding is consistent with a study of brain slices (77), where glutamine labeling from [13C]glucose was found only following KCI depolarization, suggesting that glial glutamine synthesis from glucose is enhanced in response to increased activity. The concentration of blood and brain ammonia has a major role in the regulation of brain glutamine and the glutamine cycle. Brain glutamine levels are increased in hyperammonemia as shown in experimental animal models (78-80) and human patients with hepatic encephalophy (26). Elevated glutamine has also been observed in some epilepsy patients on anti-epileptic medications (81) as discussed below. The time course of [5-15N] glutamine labeling in 15N NMR spectra following administration of ~SN-labeled ammonium chloride has permitted the rate of glutamine synthesis to be measured in the hyperammonemic rat brain in vivo (80). The disappearance of 15N-labeled glutamine in 15N-NMR spectra following the administration of an inhibitor of glutamine synthesis has been used to estimate the rate of phosphate-dependent glutaminase (82). The reaction catalyzed by glutaminase is believed to be crucial for the neuronal replenishment of transmitter glutamate and GABA. 5.4. Anaplerotic Flux and the Contribution of Gila to Glucose Metabolism

The major anaplerotic enzyme in brain tissue, pyruvate carboxylase (PC), is highly enriched in glial cells and is the main pathway capable of replenishing the carbon skeletons of TCA cycle intermediates lost from glia during the dynamic process of neurotransmitter cycling and ammonia detoxification. The proportion of glucose metabolized through PC represents about 10 percent of total glucose metabolism (83). Isotopic labeling of brain glutamate and glutamine from glucose or acetate can be used to measure PC activity. Pyruvate formed from glucose through the glycolytic pathway may undergo direct decarboxylation and acetyI-CoA formation via pyruvate dehydrogenase (PDH) or carboxylation to oxaloacetate via pyruvate carboxylase (PC). Because

158 c~-ketoglutarate C4 (and glutamate C4 by exchange) originates from the methyl group of acetyI-CoA, labeling at that position and that of glutamine C4 will be derived only from C3-1abeled pyruvate. In contrast, carboxylation of pyruvate labeled at C3 will lead to an additional flow of label into oxaloacetate C2 and/or C3 (depending on the state of equilibration with fumarase) leading to more label at glutamate C2 (and glutamine C2) than would be expected based on flux through PDH alone. Values of 38 percent of total glial metabolism (72) and 60 percent (84) of PDH flux have been calculated for the contribution of the PC pathway using this approach. If the flux through PC accounts for 10 percent of total glucose metabolism, then glia could account for ~--27 percent of brain glucose metabolism (84). 5.5. Detection of Neurotransmitters In Vivo: The Unique Case of GABA

The concentration of substances that can be detected in vivo is limited generally to a few hundred micromolar which exceeds by 7-fold the concentrations of neurotransmitters such as acetylcholine, dopamine, or serotonin. However, the amino acids glutamate and GABA, which exist at millimolar levels, are readily detected and quantitated with specialized techniques. For example, GABA (0.9-1.0 mM) can be measured in 1H spectra of single brain volumes of about 8 mL in 501~M; a limit that depends on the number of protons on the groups being detected (e.g.--CH, INCH2,--CH3, etc.), multiplicity due to spin-coupling, and the presence of other overlapping resonances from nondrug molecules. The drug may be detected in the biofluid with little or no sample preparation; for nonvolatile substances, simple lyophilization and dissolution into D20 will often suffice. In some cases, quantitation and selectivity may be improved by prior chromatography using solid phase extraction (92) or HPLC (93; and references therein). Several drugs have been detected in plasma and urine, including oxypentifylline, ibuprofen (93), acetaminophen (92, 94), naproxen (92), penicillins (95) and metronidazole (96). For example, numerous conjugates of paracetamol (e.g. cysteinyl, Nacetylcysteinyl, glucuronide and sulfur conjugated paracetamols) appear in urine within a few hours of a subject ingesting the compound (97). Increased levels of these conjugates and specific changes in their ratios have been reported in cases of paracetamol (acetaminophen) overdose and may be related to the hepatotoxicity of this drug (98). New metabolites of drug metabolism have been described, such as the appearance of diketopiperazine in the ~H spectrum of urine after administration of ampicillin (95). Specific labeling of drugs with ~3C and ~gF (see below) can provide much additional information about drug metabolism. In addition, studies of the metabolism of drugs in cell suspensions, such as that of acetaminophen in hepatocytes can lead to detailed information about cell and organ specific metabolism of the drug (94). Similar techniques should be as readily adaptable to in vivo and in vitro investigations of drug metabolism in the CNS.

7.2. ~gF NMR Detection of Fluorinated Drugs and Ion Sensitive Ligands The 19F nucleus possesses a high NMR sensitivity relative to 1H (83 percent) and is the predominant isotope of fluorine with a natural abundance of 100 percent. Fluorine is not a natural constituent of biological tissue and presents

161 no background spectrum. Therefore, 19F NMR is ideally suited for pharmacokinetic investigations of drugs and their metabolites. 7.2.1. Neuroleptics

Several of the neuroleptics used in the treatment of psychiatric disorders contain atoms of fluorine and can be measured using 19F NMR. 19F NMR detection of fluphenazine (99, 100), trifluoperazine (100, 101), fluvoxamine (102), and fluoxetine + norfluoxetine (103, 104) have been reported in the brains of animals and human patients following administration of these compounds. Renshaw et al. (103) reported in a small number of patients administered fluoxetine, that the parent compound and its active metabolite norfluoxetine, which is not resolved from fluoxetine in the ~9F spectrum, accumulates more slowly and to a greater extent in brain (brain-to-plasma ratio, 2.6), long after steady state levels were reached in the plasma. Studies of autopsy brain samples of patients treated with fluoxetin confirmed the presence-of norfluoxetin (104). The tri-fluorinated serotonin selective reuptake inhibitor fluvoxamine (102) required days to weeks for the drug to attain steady state levels in the brains of patients under treatment. The T1 spin-lattice relaxation times reported for these lipophilic drugs in human brain 19F NMR spectra appear to be highly variable, possibly reflecting the known binding of these compounds to membranes and proteins in plasma and tissue. Reported differences in the chemical shifts of these resonances between human brain spectra and solutions of the pure compound have been ascribed to binding of the drug to its receptor; however, sample temperatures were not reported. Because 19F shifts are very temperature sensitive, ~9F spectra of pure solutions of the drug should be obtained at 37 ~ in order to match physiological conditions. Some of the problems encountered in the detection of these compounds appear to be related to poor field homogeneity, low signal-to-noise ratios and/or lack of spatial localization. Uncertainties in spectral resonance assignments, quantitation, and relaxation times in ~9F NMR spectra of brain could be readily assessed in animals administered the drug in question. 7.2.2. Anesthetics

Accumulation of the fluorinated inhalation anesthetics halothane, methoxyflurane, and isoflurane was first reported in rabbit brain using ~9F NMR (105). Significant levels (20 percent of maximum signal) of these fluorinated compounds were detected in their brains up to 4 days following termination of

162 the anesthetic, a result indicating that the half-life for their clearance was longer than originally thought. Subsequent studies of halothane and other related anesthetics (e.g. enflurane) have focused on the kinetics of accumulation and clearance and the formation of metabolites from the parent compound. A study of halothane distribution in rabbit brain (106) indicated that elimination is biexponential; the appearance of multiple halothane peaks was interpreted as representing the distribution of halothane between distinct chemical environments within the brain. A long-lived water soluble metabolite of halothane detected in the in vivo ~gF spectrum was tentatively assigned to trifluoroacetate, a compound that could have specific effects on glial metabolism. The studies show clearly that the elimination kinetics of inhalation anesthetics such as halothane from the brain are not perfusion-limited as previously believed.

7.2.3. Chemotherapeutic drugs ~gF NMR is an important tool in the pharmacokinetics evaluation of the anticancer fluorinated pyrimidines, which include 5-fluorouracil, 5-fluorouridine and 5-fluoro-deoxyuridine (107). The rates of activation and clearance of the fluoropyrimidines to the fluorinated nucleosides and nucleotides are important both in the chemotherapeutic effectiveness and toxicity of these agents. The major catabolic pathway of 5-fluorouracil (5-FU) to ~-fluoro-/3-alanine (FBAL) has been shown in the livers of both mice (107) and humans (108) in vivo and in the plasma and urine of cancer patients treated with 5-FU (109). Trapping of 5-FU into the tumors may be an important factor in therapeutic outcome (110). The conversion of 5-FU to the toxic fluoro-nucleosides, deoxynucleosides and deoxynucleotides (single unresolved peak in 19F NMR spectrum in vivo) in the anabolic pathway are observed in tumors but not in liver (107).

7.2.4. ~gF Indicators of ion concentrations ~gF NMR spectroscopy has been used in conjunction with ion sensitive fluorinated ligands for the measurement of Ca2§ (fluoro-BAPTA (111, 112)), Na § (FCryp-1 (113)), pH (fluoromethyl alanines (114)), and oxygen tension (perfluorocarbons (115, 116)). A review of the many applications of 19F indicators in vivo may be found in (117). For the polar calcium and pH indicators, intracellular loading is achieved by use of their membrane permeant acetoxymethyl ester derivatives. Nonspecific esterases cleave the ester linkages and effectively trap the indicator within the cell. ~gF NMR measurements of intra-

163 cellular Ca2+ in superfused brain slices using the Ca2+ indicator fluoro-BAPTA showed increased [Ca;2 + ] under conditions known to raise [Ca,-2+] levels (112, 118). 19F NMR measurements of [Ca;2 + ] using fluoro-BAPTA combined with 31p NMR of high energy phosphates has shown that a disturbance of energy metabolism in response to the excitotoxic amino acid NMDA occurs independently of the NMDA-mediated rise in [Ca,.2+] (119). Deutsch and Taylor (114, 120) have described a number of fluorinated amino acid and aniline derivatives for use as intracellular pH indicators. The di- and tri-fluoromethyl alanines have pKa's in both the acid and neutral pH range (114) and provide potentially more accurate values of pH/than 3~p NMR under conditions of moderate to extreme acidosis (e.g. ischemia) where the P; chemical shift is relatively insensitive to changes in pH. ~gF NMR measurements of intracerebral pH in rat brain following intraventricular loading of the fluoromethyl alanines (121) yielded pH values slightly more acid than pH values obtained with 3~p NMR suggesting the possibility of their selective accumulation into a compartment with lower pH (neurons?).

7.3. Detection and Quantitation of Lithium

Lithium is used in the treatment of bipolar affective disorders. Detailed information on the biodistribution of lithium in the brain and body tissues in vivo has been limited by the absence of appropriate measurement techniques. The major isotope of lithium, 7Li, has a relatively high intrinsic NMR sensitivity (27 percent of 1H) and a high natural abundance (92.6 percent). Measurements of lithium levels in 7Li NMR spectra of animal and human brain (122-125) and pharmacokinetics following single intraperitoneal doses (126) have been reported in rats. Lithium accumulation in the brain progresses more slowly than in serum or muscle with a time course of hours to days; elimination from the brain is also slower with a half-time of 48 h (124). Consistent with the quadrupolar relaxation mechanism of this spin-3/2 nucleus and its binding to macromolecules in the tissue, both T1 and T2 appear biexponential and can be described by more than a single rate constant (123). The different relaxation times have been used to calculate a correlation time of 3.6 x 10 -8 s between free and bound lithium in the immature dog brain (123). The relatively long T~ relaxation time of 7Li in brain tissue (~3-7 s) has limited the practical application of 7Li spectroscopic imaging to relatively low spatial resolution.

164

ACKNOWLEDGEMENT The author wishes to acknowledge the support of grant HD32573 from the National Institutes of Health.

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169

CHAPTER 9

ALTERNATIVE TO MASS SPECTROMETRY FOR QUANTITATING STABLE ISOTOPES: ATOMIC EMISSION DETECTION

J.L. BRAZIER Faculty of Pharmacy, Universit& de Montreal, CP 6128 Succursale Centre-Ville, Montreal, H3C 3JC, Quebec, Canada

1. I N T R O D U C T I O N

Atomic emission detection is becoming an increasingly popular and powerful methodology for gas chromatographic analysis. This mode of detection allows multielement measurements and combines the temporal selectivity and high resolution of capillary gas chromatography with the specificity of the spectral resolution given by atomic emission. Gas chromatography-atomic emission detection (GC-AED) is performed by passing eluates from GC directly into a plasma induced by microwave where elements are atomized, excited and then returned to their ground state. This results in emission of photons with a wavelength characteristic of the emitting atom. Such a system allows the qualitative and quantitative determinations of a wide range of isotopes and elements and it may be feasible to attain elemental and/or isotopic composition via the calculation of inter-element ratios. Interest in methodologies using stable isotopically labeled (SIL) molecules continuously increases with the development of analytical methods like gas chromatography coupled to mass spectrometry, isotope ratio mass spectrometry or atomic emission spectroscopy. SIL molecules can be used as ideal internal standard for quantitative analysis (see Chapter 12). They are used much more as nonradioactive tracers in various fields of biology such as biochemistry, clinical pharmacology and clinical pharmacokinetics (see Chapters 15-21). They are also powerful tools for medical diagnosis (see Chapter 20). Among the main stable isotopes, 13C is widely used because it

170 does not give rise to significant biological isotope effects, while deuterium can induce kinetic isotope effects or metabolic switching (see Chapter 2). lSN can be used in the same manner as 13C for the labeling of organic molecules with nitrogen containing chemical groups.

2. STABLE ISOTOPES AND ATOMIC EMISSION

The past decades have seen a keen and growing interest in the applications of stable isotopically labeled molecules to chemical and biological problems. Due to a quantitative and specific labeling, SIL molecules can be used as perfect tracers in the studies of mechanisms, metabolic pathways and many other fields in pharmacology, biochemistry, diagnosis methodologies and functional investigations. The absence of radioactivity of stable isotopes has opened very wide areas of applications for investigations in humans because of the safety of these tracers. From the analytical point of view, the main driving forces for this interest are: - the application of magnetic nuclear resonance techniques to many complex chemical or biological molecules, facilitated by the judicious use of stable isotope labeling on various molecular sites. - the development of gas chromatographic mass spectrometric techniques, either in the fields of organic mass spectrometry or isotopic mass spectrometry. More recently, the development of continuous flow gas chromatography-isotope ratio mass spectrometry (CF-GC-IRMS) has given a new dimension to stable isotopic labeling in many biological applications. The areas covered by these applications go from authentication of natural compounds to noninvasive methodologies for medical diagnosis (1) and forensic sciences (2) (see Chapters 8-20). - the availability of deuterated compounds, together with the increasing availability of ~3C, ~SN, ~80, and even depleted ~2C and ~4N molecules in high isotopic purity. In the early years of SIL work, the price of analytical instrumentation and isotopes prevented the rapid development and wide use of SIL molecules, despite their great interest, The development of new generations of analytical instrumentation and electronics and new analytical techniques, together with the publication of numerous and ever-increasing number of papers on the various applications

171 of stables isotopes, progressively lowered the costs of these methodologies and opened much wider fields for their applications. Despite the many uses applicable to SIL, certain inherent limitations still prevail. One is the isotopic effect. From the physical and physicochemical point of view, isotopic effects create differences in physical and physicochemical properties which can be used in various areas especially for isotope detection. But these isotope effects have to be avoided when using SIL molecules as tracers. The marked biological effects of deuterium labeling are not observed when ~3C is substituted for ~2C, or ~SN for ~4N. The source of this differential effect is likely to be the much larger kinetic isotope effect associated with deuterium as compared to the stable isotopes of carbon and nitrogen. It is obvious that the heavy isotopes of carbon and nitrogen may be expected to have qualitatively effects similar to those induced by deuterium, but the magnitude of these effects is generally small enough to be within the range of the normal biological control systems (see Chapter 2 for a detailed review of isotope effect). Consequently, this chapter treats the detection of molecules labeled with 2H, ~3C and ~SN, and used as markers, or tracers, in biological investigations and trials. In the domain of atomic spectroscopy, let us remember that the first direct observation of deuterium was made in 1931 by Urey et al. (3), who observed weak satellites of four of the Balmer lines of hydrogen which were shifted to a shorter wavelength by an amount ranging from 1.9~ for He at 6536~ to 1.12 ~ for H$ at 4102 ~. Within experimental error, the shifts were in exact agreement with the prediction of quantum mechanics for the effect of mass " 2 " nucleus on the reduced mass of the atom. For multielectron atoms, isotope effects are manifest not only in the changes in hyperfine structure rising from nucleus spin changes, but also in small shifts in the energy of electrons which may be attributed to changes in the nuclear dimensions. Using such isotopic shifts, spectroscopic methods have been used for the analysis and determination of the isotopic composition of hydrogen by Veinbert and Zaidel (4), carbon by Zaidel and Ostrovskaya (5), and nitrogen by Zaidel and Ostrovskaya (6). The development of theoretical and analytical atomic spectroscopy has assumed an increasingly important place in element detection and, more recently, in isotope detection. The development and current status of atomic emission spectroscopy (AES), and the concept and implementation of chromatographic detection, led to the coupling between a chromatographic separation and atomic emission spectroscopy or detection (GC-AES, GC-AED).

172 These analytical devices allow for an element selective detection. Moreover, the objective of element selective chromatographic detection is to obtain quantitative and qualitative information on eluates, generally in the presence of interfering background matrix, by virtue of their elemental composition. Element selective detectors have been developed for gas chromatography: (1) Alkali Flame Thermoionic Detector (AFID, NPD) for nitrogen and phosphorus; (2) Flame Photometric Detectors (FPD) for sulfur and phosphorus; and (3) Hall detector for halogens, nitrogen and sulfur. Despite their wide use, none of these detectors is able to detect several elements simultaneously and the number of elements that can be monitored is restricted. So the development of detectors using atomic spectroscopy interfaced with chromatographic separation is a very important improvement in analytical chemistry because atomic spectroscopy may be recognized as the most fundamental analytical technique for elemental determination.

3. PHYSICAL BASIS OF ISOTOPE ATOMIC EMISSION DETECTION

3.1. Carbon Isotopes In 1956, Ferguson and Broida (7) reported stable carbon isotope analysis by optical spectroscopy. The reported work concerned the use of the C2 radiation from a flame for the measurement of relative concentrations of carbon isotopes 12C and 13C. Acetylene samples with various ~3C contents were burned and the emission spectra of the flames were recorded. The relative intensity of the 1,0 bandheads of the 3]-[-3R system due to the isotopic C2 radicals ~2C-~2C, ~2C-13C, and ~3C-~3C in the 4735-4755 ~ region were measured. Plots of the observed ~2C-~3C/12C-~2C bandhead intensity vs. ~3C acetylene contents were studied. With samples containing less than 15 atom percent carbon 13, the ~3C-~2C and 12C-~3C intensities were very nearly proportional to the expected isotopic C2 concentrations calculated by assuming a random distribution of the isotopes in C2. It was estimated that the measured intensity ratio alone could be used directly to determine the 13C abundance to the nearest 1 percent in this range. When oxygen is introduced in the discharge tube, intense CO bands appear in the spectrum. Among the CO bands, the 12CO band at 4123~ is the most convenient one for spectral analysis when the ~3C content is low, since the head of the corresponding band of the ~3CO molecule is displaced 8.2 toward the red from the ~2CO band and is free from its superimposed rotational structure. The problem of the differential detection of both ~2C and

173

0.8nm

4 ~ A

.L

'1;0

3411

342

344

346

nm

Figure 1. (0--> 3) and (1--> 4) emission bands of the fourth positive system of 12CO and 13CO.

13C from 13C-labeled molecules is much more difficult because the molecular bands corresponding to ~3CO and ~2C, respectively, are quite superimposed, and particular recipes are necessary to estimate the specific proportion of each carbon isotope. Figure 1 shows the 0-3 and 1-4 bands of the fourth positive system of CO. Figure 2 shows the three-dimensional display of the snapshot of 12C and ~3C bands recorded during the elution of the chromatographic peak of caffeine labeled with 13C on the methyl groups, in the wavelength range (338-346nm). It can be seen that the emission spectra are overlapping, but the respective signal of both carbon isotopes can be automatically extracted from the whole analytical response using the recipe developed by Quimby et al. (8). These results have been obtained on a GC AED system (HP5921), the spectrum being recorded by a diode array detector. The Quimby's recipe is the software algorithm used to make an element selective chromatogram. It is conceived to detect the raw atomic emission from the element and the interferent. A particular recipe was adjusted to reject compounds with the ~3C natural abundance (1.1 percent). With such a recipe, a selectivity of 2,500 was achieved for ~3C-labeled compounds over unlabeled compounds, otherwise the selectivity for spiked compounds could not exceed 100.

174

~2C0 ~SCO

~2C0

16.2

16:71'1"1 340

min

342

t 344

f 346

Fim

Figure 2. GC-AED: three-dimensional display of the snapshots of 12COand 13COemission bands recorded during the elution of a chromatographic peak corresponding to [~3C3] caffeine between Rt = 16.2 and 16.7 min (A = 338-346 nm).

Remember that selectivity corresponds to the multiplying factor of the 12C amount, necessary to produce the same analytical response as that obtained for the measured element. The band spectra of CO near 171 nanometers can be used to detect 13C. The spectra in second order (A13C = 341.712 nm and A~2C = 342.574 nm) are preferred. The CO molecular bands are emitted inside the plasma cavity using 02 and H2 as reactant gas. The wavelength difference of the first-order bands (A13C 170.86 nm and A~2C = 171.28 nm) is ~A = 0.43 nm, and is too narrow for a selective detection of both isotopes. As CO has to be produced inside the plasma, the flow rates of the plasma gas and reactant gases have to be optimized to obtain the maximum residence time of the element inside the plasma for a best yield of the reaction, and to obtain the highest sensitivity of ~3C detection. Such an optimization carried out for the detection of ~3C using an experimental design has been published by Leclerc et al. (9).

3.2. Nitrogen Isotopes As for carbon isotopes, an alternative method of determining 14N/15N ratios has been developed by molecular emission spectroscopy, for bands in the

175 electronic vibrational spectrum of the nitrogen molecule which show an isotope shift (10, 11). The advantage of this optical method is that very small samples can be measured. As little as 0.2 ~g of total nitrogen was sufficient for Goleb and Middleboe (12) to obtain 14N/15N ratios. In the many optical emission spectroscopies described in the literature for the 14N/1SN ratio determination, isotopically shifted bandheads of a single transition corresponding to the electronic transition system C (3]-[u) ~-> B (3]-[g) of the 2-~ 0 transition have been used. In this system, the I"N-I"N bandhead has a wavelength of 297.7 nm, the 14N-1SN bandhead 293.3 nm and the lSNlSN bandhead 298.9 nm. The possible use of the 3--, 0 bandhead is weak and can suffer interferences from coincident OH bands in the 281-283 nm region. The large isotope shifts of the (3-0) and (4-1) bandheads produce a pattern of overlapping bands. However, Burridge and Hewitt (13) demonstrated that the simultaneous use of bandheads from two transitions (3--> 0) and (4-> 0) can provide an adequate basis for the optical emission spectroscopy determination of 14N/1SN ratios. A14N2 (3.0) = 281.98 nm, A14N 15N(4.1 ) = 282.11 nm A14N-1SN (3.0)= 282.71 nm and AlSN2 (4.1)= 282.78 nm The detection of 14N and lSN can be carried out using the emission of the diatomic molecule CN in the 420 nm region. Figure 3 shows the 3D emission spectrum obtained from caffeine labeled with lSN at the 1 and 3 positions by gas chromatography coupled to the atomic emission detector HP 5921 equipped with a diode array detector. The spectrum shows two bands corresponding to the B2]-[-A2]-[ system. The Arnax are 420.1677 nm for 15N and 421.4646 nm for 14N, respectively. The wavelength difference 8,~ is only 1.2969 nm. As for carbon isotopes, a careful optimization of the plasma gas (He) and reactant gases (CH4, 02, N2) flow rates has to be carried out for an optimal formation of CN inside the plasma and excellent sensitivity and limit of detection. Recently, a new plasma discharge source for assaying nitrogen isotope abundance has been designed by Hoult and Preston (14).

3.3. Isotopes of Oxygen and Hydrogen The detection of oxygen isotopes 180 and 160 is difficult. It can be performed from the emission of the CO band using ethane, or methane, as reagent gases. If the 180 chromatograms are too long or recorded too often, the diode array can be damaged because the emission line of nitrogen near 175 nm is

176

C15N

7

8. min

423 nm

Figure 3. GC-AED: three-dimensional display of the snapshots of C14N and C15N recorded during the elution of a chromatographic peak corresponding to 1.3-[15N2], 2-[13C] caffeine.

so intense that the photodiode array becomes blind rapidly. The CO emission band detected at 171 nm for 180 determination corresponds to that used for the 13C determination as 13CO. Oxygen can also be detected when reacted with hydrogen as an OH radical, the Amax of this band is located near 307 nm. At last hydrogen isotopes can also be measured using atomic emission detection. The emission lines recorded are 656,039 nm for 2H and 656,302 nm for 1H, respectively. It can be seen that for increasing the resolution molecular bands are generally used for isotope determination using atomic emission, especially when it is coupled to gas chromatography. Table 1 gives the main characteristics of these determinations. Table 2

TABLE 1. Molecular Bands Used for Isotope AE Detection with ~maxand Reagent Gases Used for Producing the Chemical Emitting Species Inside the Plasma

Isotope

Molecular band

Wavelength (nm)

Reagentgas

12C, 13C, 14C

CO OH CN CO

171 307 419 171

Oxygen Oxygen Ethane, methane Ethane, methane

2H, 1H 14N, ~SN 160, 180

177 TABLE 2. Selectivity vs. Carbon for the Determination of Stable Isotopes by GC-AED

Isotope

Selectivity vs. carbon

13C 2H* 1SN 180

2400 2200 2500 2900

*The selectivity of 2H VS. 1H at 656.1 nm is 300.

TABLE 3. Atomic Weight and Terrestrial Natural Abundance of Various Stable Isotopes

Element

Atomic weight

Terrestrial natural isotopic abundance (percent)

1H 2H 12C 13C 14N 15N 160 170 180

1.0078 2.0141 12.000 13.003 14.003 15.004 14.994 16.999 17.999

99.985 0.015 98.85 1.11 99.63 0.36 99.759 0.037 0.204

shows the values of selectivity vs. carbon for the main stable isotopes determined by GC-AED using the GC AED HP 5921. This table shows that the selectivity of stable isotope detection by atomic emission is excellent, but not sufficient to measure variations in the natural abundance range of stable isotopes found in different biological materials (Table 3).

4. C H R O M A T O G R A P H Y COUPLED WITH A T O M I C SPECTROSCOPY DETECTION

Different types of atomic spectroscopy have been interfaced with chromatographic systems. Among these are atomic absorption (AAS), flame emission (FES), atomic fluorescence (AFS) and atomic emission (AES) spectroscopies.

178 The capability of AES for simultaneous multielement measurement, while maintaining a wide dynamic measurement range and good selectivities and sensitivities over background elements, has led to atomic plasmas becoming widely used during the last decade (15). The main advantages of chromatography AES are: - monitoring the elemental composition of eluates directly with high elemental sensitivity. - monitoring of particular molecular functionality by means of interelement ratio measurement or by using specific derivatization reagents containing elemental labels. toleration of nonideal chromatographic separation. Elemental detection is very specific and its inherent selectivity enables quantitation of compounds with poor chromatographic resolution when working on complex matrixes like biological samples. - simultaneous detection of various elements, computation of interelement ratios, determination of empirical and molecular formula from a chromatographic peak. - detection and quantitation of some isotopes, especially those of elements constituting organic molecules: ~3C, 2H, ~5N and ~80. -

4.1. Plasma Sources The main atomic plasma emission detectors that have been developed for GC coupling can be summarized as follows: ICP: Inductibility Coupled Plasma In such a plasma the discharge results from interaction of a radiofrequency fields with argon flowing through a quartz tube set within a copper coil. The ICP discharge is well suited for liquid chromatographic detection since it is usually configured for a liquid inlet stream (spray chamber, nebulizer). DCP: Direct-Current Plasma Direct-current excitation sources for atomic emission involve a low voltage (10-500), high current (1-35 A) discharge. The direct-current plasma jet stabilized by flowing inert gas (argon) can be coupled with liquid chromatography. The argon based ICP and DCP are interfaced with HPLC or supercritical fluid chromatography for specific elemental determinations.

179 MIP: Microwave-Induced Plasma MIP is the most used plasma source for gas chromatographic (GC) applications. A helium plasma is maintained within a resonant cavity which allows focusing the power from a microwave source (2.46 GHz) into a discharge cell or discharge tube (quartz). Although the plasma temperature is lower in helium MIP than in ICP or DCP, the chemistry of helium discharges produces high spectral emission intensities. The first presentation of the use of a microwave-induced plasma as a detector for GC was performed in 1965 by McCormack et al. (16) using an argon plasma working at atmospheric pressure. Bache and Lisk (17) studied a plasma of helium at low pressure, the device was later improved by Van Delhen et al. (18). There was a very large increase in the applications of GC coupled to microwave plasma (19) after the development of the Beenakker cavity (20, 21). More recently, Yieru et al. (22) showed that the chemical structure, as well as the microwave energy, could influence the analytical response. SWP: Surface Wave Plasma SWP is an alternative to the electromagnetic resonant cavity for the transmission of microwave energy to the plasma SWP using a "Surfatron" power launching device (23). GC-MIP Coupling In a GC-AED system, the separation of sample components takes place on a capillary column within a standard gas chromatograph oven. The column is extended out of the oven through a heated interface and directly connected to the detector. Thus, the entire column effluent enters the microwave-induced plasma. Make-up and reagent gases are automatically added according to the element to be recorded in order to generate chosen chemical species inside the plasma. The cavity can be a Beenakker type resonant cavity which enables the microwave energy to be focused in the centre of a quartz discharge tube. The capillary column ends just before the beginning of the plasma. The discharge tube has to be cooled by a water circulation in order to avoid or minimize erosion and interactions of sample components with the silica tube walls. The atomic emission exits the discharge tube and is transmitted into the spectrometer. The exit chamber and window are flushed by pure helium. In the spectrometer, the light is diffracted by a holographic grating which projects it onto a flat focal plane. An adjustable photodiode array covering 50 nm can move along this focal plane to provide a good resolution of the monitored emission lines and allows one to obtain elemental chromatograms and atomic spectra. The signal recorded by each of the 211 photodiodes can

180 be treated for background and interference subtraction, especially for isotope determination (natural isotope abundance). This type of analytical instrument and detector allows one to acquire two kinds of analytical information.

4.2. Elemental Chromatograms By recording all along the chromatographic run the various emission lines corresponding to particular compounds, it is possible to obtain the chromatograms corresponding to each of the elements or isotopes monitored. Figure 4 shows the elemental chromatogram of a mixture of xanthines containing caffeine labeled with deuterium. The elements monitored were carbon at 193.03 nm, hydrogen at 656.30 nm and deuterium at 656.04 nm. The chromatogram of deuterium clearly points out the presence of deuterium-labeled caffeine with a retention time of 6.5 min. The qualitative response on the presence of such or such element can be completed by the quantitative measurement of each elemental response. So it is possible to quantify every element and to calculate interelement ratios which are very useful for structure or empirical formula determinations. These values can afford additional information to mass spectrometric analysis, especially in the presence of labeling stable isotopes.

3000

C i93.03 nmJ

2000

t JD656"~nmJ'/

1000.

o-

.....

,~

~

Ill

7

~

~

1"0 min

I

Figure 4. Elemental chromatogram of a mixture of xanthines containing deuterated caffeine. Elements monitored: C at 193.03 nm, 1H at 656.30 nm and 2H at 656.04 nm.

181 4.3. Snapshot

Snapshots are three-dimensional displays (A, retention time, intensity) recovered within a wavelength range during the elution of a chromatographic peek. These 3D spectra which are specific for an element or isotope certify the presence of this particular atom in the analyzed molecule (Figures 2 and 3).

5. LIMIT OF DETECTION OF ISOTOPES DETECTION 5.1. Limit of Detection of 7SC at 341.712nm

The limit of detection of isotopes can be expressed according to several methods. We will express the limit of detection according to the IUPAC method (24), Oppenheimer's method (25) and that of Quimby and Sullivan (26). IUPAC M e t h o d The limit of detection is defined as the concentration (CL) obtained from the smallest measurement (XL) which can be detected with a sufficient confidence level during a given analytical procedure. First, the standard deviation of the noise measurement Sb is determined from a blank sample (Sb) in the interval of retention times where the studied compound will be eluted. According to the International Union of Pure and Applied Chemistry (IUPAC), the following equation can be used: XL = XB + KSB

where K is a numeric factor chosen according to the expected confidence level. If K = 3, the confidence level is 99.86 percent, there is a 0.14 percent risk in obtaining a signal which is only a fluctuation of the noise. The CL concentration is a function of XL according to, CL = (KSB)Im

CL = (3SB)/m

This method, used for the calculation of the limit of detection, generally gives values which are artificially low. It is why IUPAC recommended the use of K = 3 (CLUE=31). Indicative comparisons between analytical methods and/or instruments can be carried out using CL values calculated according to the IUPAC method.

182 Oppenheimer" s M e t h o d Oppenheimer defines the limit of detection X~ using the regression lines and their confidence intervals. These limits are expressed in terms of concentrations with an e and/3 risk. This method is restricting but it is statistically the more relevant and very close to the analytical factuality. The limit of detection Y1 for a signal X~, form a regression line y = aX = b is given by, Y~ = b + (t(~_,) + t(1-f3))Sr[1 + 1/n + {E(x)2/S(X)})] ~

where b = estimated intercept; t(l_,) and t(l_~)= the values of the student's test for = a =/3 = 0.05; n = number of replicates; Sr = residual standard deviation; E(X) 2 = X mean square; and S(X) standard deviation of X values. Calculation of the Residual Standard Deviation The residual standard deviation is calculated using the following equation: Sr

=

{,~-~(Yest

-

Ymes)2/( n -

2)} ~

where Yest = estimated Yvalue using the regression line; Ymes = measured Y value; and n = number of data. M e t h o d of Quimby and Sullivan According to Quimby and Sullivan, the limit of detection for a given element is the amount of the element necessary to give a peak twice the height of the noise (X2) divided by the width at middle height of the peak (Wo.5) expressed in seconds: LD = (2 * X2)lWo.s

This way of expressing results (pg/s) is difficult to compare with the limit of detection expressed as analytical concentrations; nevertheless, it can be use to compare the sensitivities of various analytical methods or instruments. After applying a four-factor experimental design corresponding to the various flow rates of plasma gas and reagents gas, Leclerc et al. (9) determined the limit of detection of ~3C from an anti-inflamatory drug, Fenoprofen, transformed into its ~3CH3 methyl ester. Under their experimental conditions, and after injection of 1 i~1 of the analytical sample, the limits of detection were: 1 pg/i~l according to the IUPAC method, 12 pg/l~l according to Oppeinheimer and 0.1 pg/s according to Quimby and Sullivan. The detec-

183 TABLE 4. Limits of Detection Measured from Trideuteromethylcaffeine by GC-AED (C, N, 1H, 2H) and Expressed According to Quimby, Oppenheimer and IUPAC Methods Method

C

N

1H

2H

Quimby (pg/s)

1.07

8.30

0.40

6.50

IUPAC (pg) (pg/s)

1.04 0.20

13.80 4.60

1.70 0.30

6.50 1.10

52.00 8.70

287.00 47.80

13.00 2.10

66.00 11.00

Oppenheimer (pg) (pg/s)

tion of 12CO and 13CO was performed by recording the emission lines at 341.712 and 342.574 nm, respectively.

5.2. Limit of Detection of Deuterium The same methods of determination of the limit of detection were carried out on deuterium using the molecule of caffeine labeled with 9 deuterium atoms on its three methyl groups, on positions: N1, N3 and N7, as analyte. According to the three methods previously described, the results are presented in Table 4 for carbon measured at 193.031 nm, nitrogen at 174.261 nm, 1H at 656.302 nm and 2H at 656.039 nm (27).

5.3. Limit of Detection of Nitrogen 75N The limit of detection of lSN was determined after optimization of four factors affecting sensitivity: helium flow rate (plasma gas), methane pressure, as well as oxygen and hydrogen pressures (reagent gas). Emission lines of ~SN and I"N were detected at 420.17 and 421.46 nm, respectively. The results for the limit of detection are shown in Table 5. The molecule used for these optimization and limit of detection determinations was caffeine labeled with two atoms of lSN at positions 1 and 3. According to Quimby and Sullivan, the minimum detectable limits for deuerium and 180 are 16.0 and 140 pg/s, respectively.

184 TABLE 5. Limit of Detection of lSN Form [~5N2(1-3)]Caffeine

Method

lSN

IUPAC (pg) (pg/s)

4.6 1.9

Quimby (pg) (pg/s)

5.52 2.3

Oppenheimer (pg) (pg/s)

100 36

6. LINEARITY

Analytical parameters, such as selectivity and limit of detection or minimum detectable level, are good when using GC coupled with atomic emission detection. Linearity and linear dynamic range are also very good. The values of the extent of the linear dynamic range for several elements are shown in Table 6. During a linearity study of deuterium performed by Bannier et al. (27), the possible variation of the response according to the number of deuterium atoms substituting for hydrogen atoms in a caffeine molecules has been studied for the same overall deuterium concentration. Three caffeine isotopomers were used [7C2H3] caffeine (7d3 caf), 1,3 [C2H312 caffeine (1,3 d6 caf) and

TABLE 6. Atomic Emission Line, Wavelength of Emission; Minimum Detectable Level and Linear Dynamic Range of Several Common Elements Measured by GC-AED

Element

Wavelength (nm)

Minimum detectable level (pg/s)

Linear dynamic range (K)

C 1H

193.1 486.1 656.1 777.2 174.2 180.7

0.2 1.0 2.0 50.0 15.0 1.0

10 9 10 3 10 10

2H O N S

185 12000

12C

10000

9

J

9

8000

6000

4000 -"

.

.

.

.

.

. _ _

. _ _ . _ _

. _ _

2000

0 0

5

10

15

20

25

NANOGRAMS OF 12C AS CAFFEINE Figure 5. Mixture of 13C labeled (constant amount = 6.9ng of 13C) and varying amounts of unlabeled caffeine measured by GC AED using ~2C and 13C signals.

1,3,7 [C2H313 caffeine (d9 caf). An effect of the number of deuterium atoms per molecule would make the slope of the regression lines, calculated between the deuterium signal and the deuterium amount, exhibit a change for each isotopomer. The parallelism of the lines was checked and showed that there is no influence of the number of deuterium atoms per molecule of caffeine on the deuterium quantitative determination. For the same deuterium concentration, the detection is the same whatever the number of deuterium atoms in the isotopomer. Figure 5 shows the responses of 12C and lSC in a mixture containing a constant amount of caffeine labeled with 13C (corresponding to 6.9 ng of ~3C) and increasing amounts of unlabeled caffeine (up to 25 ng). This figure clearly shows that AED responses are independent of the other isotope when monitoring two isotopes of the same element. The same results are shown on Figure 6 with a mixture containing caffeine labeled with lSN (constant amount of 7.8 ng ~SN) and increasing amounts of the unlabeled molecule. This result leads to the same conclusion than that obtained with carbon isotopes. Dual isotope measurements by gas chromatography coupled with atomic

186 5000 4500

14N

4000 3500 3000 2500 2000 -'-'-'"11

. . . .

1500

B"

O/

""

-1 . . . . 7,8

,, -

-

-

--,-

NANOGRAMS

-

OF

--~

15[~

. . . .

r

1000 500

0

5

10

NANOGRAMS

15

20

OF

14N A S

25

30

35

CAFFEINE

Figure 6. Mixture of lSN labeled (constant amount=7.8ng of lSN) and varying amounts of unlabeled caffeine measured by GC-AED using ~4N and ~SN signals.

emission detection may enhance results for quantitative analysis. Adding a known amount of an isotopically labeled form of a target analyte in each sample can compensate for irreproducibilities or uncertainties associated with sample pretreatment, as well as fluctuations in AED parameters (28).

7. SOME APPLICATIONS OF GC-AED ISOTOPE DETECTION

7.1. Determination of the Number of Isotopes Incorporated into a Molecule (27) The various isotopomers used in the reported study were: 1-mono deuteromethyl caffeine, 3-mono deuteromethyl caffeine, 7-mono deuteromethyl caffeine: (84 ng/l~l) and 1.3-dideuteromethyl caffeine, 1,7-dideuteromethyl caffeine, 3,7 dideuteromethyl caffeine (82 ng/~l). The analytical signals corresponding to 1H, 2H and 12C were recorded and the areas corresponding to elution peaks were measured. In order to check the influence of the number of the deuterium atoms and of the location of these atoms on the analytical response, the 2H/~2C ratios were calculated as follows:

187 2H/12C = (area of the 2H peak/number of 2H atoms)/(area of the ~2C

peak/number of ~2C atoms) There are three deuterium atoms in the monodeuteromethyl isotopomers of caffeine and six in the various dideuteromethyl isotopomers. All the caffeine molecules contain eight carbon atoms. The 2H/12C values calculated from isotopomers containing three atoms of deuterium (d3 caffeine) were respectively: 0.011 (1-d3 caffeine), 0.010 (7-d3 caffeine), 0.011 (3-d3 caffeine). The same ratios calculated from the d6 isotopomers gave the following values: 0.012 (1.3 d6 caffeine), 0.013 (1.7 d6 caffeine) and 0.012 (3.7 d6 caffeine). The mean value of the 2H/12C ratio from all the d3 isotopomers (0.011 _+ 0.00062) was not significantly different (p < 0.01) from that obtained from the d6 isotopomers (0.012 _+ 0.00048). In order to calculate the number of deuterium atoms present in every other isotopomer, the factor of proportionality between the responses of deuterium and carbon, KCDw a s calculated from 1-d3 caffeine taken as standard

Kco = [(Number of 2H atoms) x (area of C)]/[(Number of C atoms) x (area of 2H)] The number of deuterium atoms in each isotopomer was calculated as follows: Number of 2H atoms = KCD • number of C x (area of 2H/area of C) where Kco is the factor of proportionality between C and 2H; Number of C is the number of carbon atoms in the standard (8 in 1-d3 caffeine); Area of C and 2H are the areas of the corresponding peaks of the measured isotopomer. The results are shown in Table 7. It can be seen from these results that there is an unequivocal determination of the number of deuterium atoms in the various deuterated isotopomers. Moreover, these results show that there is no influence of the location of the labeling on the determination of the number of labeling isotopes.

7.2. Studies of Metabolic Pathways Pharmaceutical and biomedical analysis needs much more efficient separative techniques like capillary gas chromatography coupled with specific detectors. The interest of coupling such specific modes of detection with highly resolu-

188 TABLE 7. Number of Deuterium Atoms Measured in Each Caffeine Isotopomer

Caffeine isotopomer

1-d3

KCD

87.47

7-d3

3-d3

1,3-d6

1,7-d6

3,7-d6

Number of 2H calculated

3.00

2.72

2.85

6.00

6.16

6.05

Number of 2H theoretical

3

3

3

6

6

6

tive techniques increases with the development of various kinds of chromatographic systems coupled with organic mass spectrometry (MS), isotope ratio mass spectrometry (IRMS) and atomic emission detection (AED). Such a detector can be used for the determination of drug metabolites after administration of a parent drug labeled with the stable isotope 13C. The use of stable isotopically labeled (SIL) drugs for the studies of metabolic pathways in humans increases from year to year. These SIL molecules act as safe and nonradioactive tracers. They can be used according to the "ion cluster" technique where a mixture of unlabeled and SIL parent drug is administered to a subject. Thus, all the metabolites which are formed from this mixture are also labeled with the same isotopic content as the parent drug. They can be detected easily from the total ion current by the presence of ion clusters corresponding to unlabeled and labeled ions. Also the SIL parent drug can be administered alone and its labeled metabolites specifically detected by atomic emission detection. For example, the study of the urinary metabolites of caffeine (1,3,7-trimethylxanthine) labeled with three atoms of 13C on the three methyl groups located at the N1, N3 and N7 positions was reported by Boukraa et al. (29). Caffeine can be used as a metabolic probe for exploring oxidative metabolic pathways (cyt P450 IIA2) and the numerous factors that can potentially alter these enzymatic activities. Demethylation mediated by microsomial mono-oxygenases and oxidative reactions on the C8 position, lead to the production of di- and monomethylxanthines (MX), tri-, di- and monomethyl uric acids (MU) as well as ring-opened uracil related metabolites. With such an extensive metabolism, more than 85 percent of the dose of caffeine administered to a human is eliminated as urinary metabolites. If caffeine is labeled with ~3C on the three methyl groups, all the methylated

189 metabolites which are eliminated are also labeled and can be detected from the biological fluids by the specific signal of 13C. For this trial a GC AED System (Hewlett Packard HP 5921) was used. This system consists of a HP 5890 Series II standard gas chromatograph equipped with a HP 7673A auto-sampler and a HP 5895 A Chemstation. The choice of the plasma gas (Helium 99.9999 d. percent) and reagent gas (H2 and 02 under a pressure of 1 bar) and their flow rates were adjusted in order to optimize to the yield of atomic emission of the selected elements. The emission lines monitored were: 348.424nm for nitrogen, 342.574nm for 12C and 341.712 nm for ~3C. The overall flow rate in the detector was 32 ml/min. Caffeine metabolites were extracted from urine and transformed into their pentylated derivatives on the residual free NH groups before injection into the chromatographic system. Figure 7 shows the chromatograms obtained by gas chromatographyatomic emission detection from an aqueous solution containing caffeine and its metabolites at a concentration of 20 i~g/ml for methylxanthines (MX) and 30 i~g/ml for methyluric acids (MU). An aliquot of this solution was submitted to the whole analytical procedure described above. Figures 7a, 7b and 7c, respectively, show the elemental chromatograms monitored at 342.574 nm for ~2C (a), 341.712 nm for ~3C (b) and 348.424 nm for N. As these wavelengths are inside the 50-nm interval covered by the photodiode array, the three emission lines were recorded simultaneously during the same chromatographic run. When these three emission lines are used for detection, the compounds of interest which contain both stable isotopes of carbon (~2C and 13C) and N can be detected perfectly well. It can be observed that the chromatograms corresponding to the detection of ~2C and ~3C are almost identical because these compounds display an isotopic abundance which is the natural ~3C abundance (#1.1 percent). These results show that the AED is able to detect ~3C from organic compounds with an isotope enrichment corresponding to the terrestrial natural abundance of this isotope (1.01-1.15 percent). A blank urine from a subject who was not a caffeine consumer was spiked with a solution containing caffeine labeled with 13C and twelve unlabeled metabolites as well as IBMX (internal standard) in order to obtain a final concentration of 20 ~g/ml for MX and 30 i~g/ml for MU. An aliquot sample was processed according the described procedure. Figure 8 shows the elemental chromatograms from the extracted urine where the following elements or isotopes are recorded: ~2C (a), ~3C (b) and N (c). As every compound present in urine contains both carbon isotopes, the

190 i

12C 9 342.574 n m 1200-.

3 3'

8

4

IOO0"

r~

67

9

2

800-

~ 400-. 2000:

11

I

600-..

"

13

X,o ! "

I-8

"

"

-2.0-

"

-

- 52

-

- 2 ~

"

~

"

~

t "

-~-

- - ~

"

3 ~

-

min

i

i30"

,

341.712 n m

33' 8 4

~0-

9

26o-

2

I

.

"

1

11

13

o

.....

18

"

- 2"0

. . . .

52-

-

-2~1

"

"

"

2'6"."

" 2~

30

.....

-

"

-

32

3"4 . . . . . . . .

. . . .

min

N ' 348,424 nm ~ ' 5001

5O0~00:

3001

I00" O"

.

.

18

.

,

.

.

.

.

20

.

.

.

.

.

.

22

. 2"4"

"

"

~b

.....

28" ,

"

"3"0-

"

-3~2"

"

"

,~4 . . . .

m i n ,

Figure 7. GC-AED" elemental chromatograms of 12CO at 342.574nm (7a), 13CO at 341.712 nm (7b), N at 348.424 nm (7c). Caffeine (1) and its metabolites (2) 1.3 MX, (3) 1.7 MX, (3')3.7 MX, (4)IBMX, (5) TMU, (6)3 MX, (7) 1 MX, (8) 7 MX, (9) 1.3 MU, (10) 1.7 MU, (11)3.7 MU, (12) 1 MU, (13)3 MU, (14) 7 MU.

191

12C : 343 n m

3 000

13C : 342 n m

2 000. i

1 000'

lO

15

"

2b

2~5 min. ;

Figure 8. Elemental chromatograms for 12C (a), 13C (b) and N (c) from an urine extract containing 3-[~3C] caffeine and its metabolites.

corresponding elemental chromatograms are quite nonspecific and unable to detect the presence of caffeine and its various metabolites. Numerous molecules excreted by urine also contain nitrogen and the N chromatogram is no more specific for caffeine detection. The very large peak on the chromatogram corresponds to hippuric acid whose concentration in urine is very high. A specific detection has been performed by GC-AED. The 13C chromatogram in Figure 8b corresponds to the analytical response given by the natural abundance of this carbon isotope. All the compounds which appear on this chromatogram have the same 13C enrichment (#1.1 percent) caffeine excepted which is artificially enriched on the three methyl groups. This ~3C elemental chromatogram can be processed in order to subtract the natural abundance of ~SC. The signal used for this subtraction is taken from a portion of the chromatogram free of peaks corresponding to labeled molecules (1013 min). The use of this "Suppress" function results in the total removal of the natural ~3C response. Hence, only the compounds enriched with ~3C over the natural abundance are specifically detected. The same method was used to find the various metabolites of caffeine from the urine of a subject who absorbed an oral dose of 50 mg of caffeine labeled with three atoms of ~3C. The ~3C enrichment of each methyl group was 99 percent, so the total amount of ~3C administered as caffeine was 9.8mg. Figure 9a shows the isotopic 13C chromatogram from the urine collected before caffeine intake spiked with the internal standard ~3C isobutyl methyl xanthine and processed to subtract the natural ~3C contribution. No significant

192

I

1.4E4-" 1.2E4-

13

4

CO : 341.712 nm

1000080006000-

5 6

4000.

_~

I [iii i i I lb

b

...... ........o "

--

.... ---

~Omin

Figure 9. (a) 13C chromatogram of a blank urine spiked with [13C] isobutylmethylxanthine (internal standard) obtained after subtraction of the ~3C natural abundance ("Suppress" command). (b) ~3Cchromatogram of a urine extract after ~3C natural abundance subtraction. Urine collected after 3-[~3CH3] caffeine administration. (1) 3-[13C] TMX; (2) 3-[~3C]theophylline; (3) 3-[13C]theobromine; (4) 3-[~3C]trimethyluric acid; (5) 3-[~3CH3] xanthine; (6) 3-[~3CH3] dimethyluric acid.

peak can be observed on the chromatogram because all the molecules extracted from urine and eluted from the chromatographic column have the same 13C content which is the natural one. Figure 10 shows the subtracted 13C chromatograms of urine samples collected at various times after labeled caffeine intake. The only peaks which can be observed on this subtracted chromatogram are those of caffeine and its urinary metabolites, because these molecules are enriched in ~3C over the natural abundance and are not erased by the natural ~3C subtraction. Figure 11 shows the chromatogram of an urine extract corresponding to a subject who absorbed orally 100 mg of 3-~3CH3 labeled caffeine. After subtracting the natural abundance, only the metabolites containing a ~3C atom on the 3N position can be detected. This example clearly shows that atomic emission detection, coupled with gas chromatography, is a powerful tool for the screening of compounds from complex matrices and mixtures using the selective detection of an element

193

3C0 9 3 4 1 . 7 1 2 n m 2

4

1 400 1 200

I

3

5 7

8

1112

I 000 i""-

d

800 600' 400" 200" 5

10 "

15

20

25

min

Figure 10. 13Cchromatogram of urine extracts after 13C natural abundance subtraction. Urine collected after 1,3,7-[13 (CH3)3] caffeine absorption. (a) to-2H; (b) t2-6H; (c) t612H; (d) t~2-24H. (1) 1,3,7-13 C]3 TMX; (2) 7 [~3C] IBMX; (3) 1,3-~3C]2 MX; (4) 1,7-~3C]2 MX and 3,7[13C]2 MX; (5) 3-~3C] MX; (6) 1-~3C]MX; (7) 7-~3C] MX; (8) 1,7-~3C]2MU; (9) 1,3-[13C]2 MU; (10) 3,7-[~3C]2 MU; (11) 3-[~3C] MU; (12) 1-[~3C] MU; (13) 7-[~3C] MU.

or isotope. Despite the very small wavelength difference between the emission lines of 12C0~ and 13C0~ radicals (0.85 nm for the second order), the algorithm proposed by Quimby allows the extraction of both isotopic signals and the perfect recording of the specific ~3C chromatogram. These secondorder emission lines are preferred to the first-order one (at 171.3 and 170.8 nm, respectively) which are more intense but too close one from each other to allow a good extraction of the ~3C response at the ~3C natural abundance level. The processing of the ~3C chromatogram, and the judicious use of the "Suppress" function, allows the subtraction of the natural contribution of ~3C and a specific detection of the only molecules whose ~3C enrichment is over the natural one. Hence, gas chromatography coupled with atomic emission detection is a very appropriate tool for metabolic studies where the parent drug is labeled with stable isotopes (like 13C). If the labeled site is chosen correctly so as not to be removed during the metabolic process, the label remains in all the metabolites formed from the parent drug. A nonspecific extraction of the biologic fluid to be studied, followed by a chromato-

194

~3C0" 341.712 nm

40.

20.

2 ,.,, i13

10

4 5

15

6

20 min

Figure 11. 13C chromatogram of a urine extract after 13C natural abundance subtraction. Urine collected after 3-[~3CH3] caffeine administration. (1) 3-[~3C]TMX; (2) 3-[13C]theophylline; (3) 3-[13C]theobromine; (4) 3-[13C]trimethyluric acid; (5) 3-[~3CH3] xanthine; (6) 3-[~3CH3] dimethyluric acid.

graphic analysis with 13C atomic emission detection with the subtraction of the 13C natural abundance, allows one to easily point out the various ~3C enriched compounds corresponding to the drug metabolites from the whole chromatogram. Hence, a specific profile of metabolites can be obtained. Another alternative to detect metabolite is GC-MS and the ion-cluster technique with coadministration of a mixture of both unlabeled and labeled parent drugs. When using such a method, ion-clusters have to be detected for each unknown metabolite. An algorithm can be used to systematically seek these cluster all along the total ion current. This detection can be very difficult if the compounds are at a very low level and their corresponding ion-cluster within the noise intensity. An alternative is to use the selected ion monitoring mode (as shown above).The disadvantage of this method is the necessity to know the characteristic fragment-ions of the metabolites and generally neither the metabolites nor their characteristic ions are known before the study of metabolic pathways. Consequently, the major advantages of GC-AED for the screening of the metabolites of labeled drugs relies on the possibility to: (i) monitor various elements or isotopes, among them the tracer isotope ~3C, during the same chromatographic run,

195 (ii) use and compare the elemental responses for interelement ratios calculations, (iii) use the subtraction of the ~3C natural abundance from the whole ~3C chromatogram in order to easily point out only the labeled compounds.

7.3. Bioavailability Studies A study was designed in our laboratory (LEACM - unpublished results) by Besacier and Croin to compare the performance of GC-MS and GC- AED in bioavailability simulations. The study design was that an i.v. dose of unlabeled caffeine was administered simultaneously with an oral dose of either 3(13CH3) caffeine or 1,3,7 (13CH3)3 caffeine. Plasma samples were spiked with these caffeine isotopomers in order to simulate pharmacokinetic curves corresponding to a theoretical bioavailability of the oral dose of 70 percent vs. the i.v. dose. Plasma samples were then assayed by GC-MS (HP 5972) and GC-AED (HP 5921) using ~3CH3 isobutylmethylxanthine (IBMX) as internal standard. When 3(~3CH3) caffeine is used with caffeine and their concentrations measured by GC-MS, there is an important overlapping of the molecular ionclusters of both isotopomers which are used for the quantification. When the mixture of the extracted unlabeled caffeine and labeled caffeine is analyzed by AED, the two isotopomers are coeluted from the chromatographic column and it is not possible to detect the proportion of the analate due to the labeled isotopomer. The response corresponding to the ~3C signal is the sum of the ~3C natural abundance of the unlabeled molecule and of the 13C content of the labeled one. The problem is the same with the ~2C response. Two methods can be used to selectively obtain the signal of ~2C from the unlabeled molecule and of ~3C from the labeled one. The first is the use of the recipe allowing one to subtract the ~3C natural abundance, the second is the resolution of a series of equations derived from standard curves. The use of the 13C subtraction has been discussed above, and so we will develop the mathematical method. S(~2C) and S(~3C) are the areas of unlabeled and labeled molecules, respectively, measured from the ~2C and ~3C chromatograms of the mixture. Se(12C) and Se(~3C) are the areas corresponding to the internal standard measured on the same chromatograms. [M] and [M*] are the respective concentrations of unlabeled and labeled molecules and [Se] the internal standard concentration. We can develop the

196 system of four equations: S(12C)/Se(~2C) = (A1 x [M]/[Se] + B1)+ (A2 x [M*]/[Se] + B2)

(1)

S(120)/Se(13C)- (A3 x [M]/[Se] + B3)+ (A4 x [M*]/[Se] + B4)

(2)

S(13C)/Se(120) = (A5 x [M]/[Se] + B5)+ (A6 x [M*]/[Se] + B6)

(3)

S(13C)/Se(13C) = (A7 x [M]/[Se] + B7)+ (A8 x [M*]/[Se] + B8)

(4)

The A1, B1, A3, B3, A5, B5, A7 and B7 coefficients are determined from the standard curves of the unlabeled molecules. The A2, B2, A4, B4, A6, B6, A8 and B8 coefficients are determined from the standard curves of the labeled molecule. Combining Eqs. (1) to (4), [M] and [M*] can then be calculated according four different ways. For example, combining Eqs. (1) and (3): [M*]/[Se] = [(S~3C)/Se(~2C)- B5 - B 6 - A5) x (S(~2C)/Se(~2C) - B1 - B 2 ) / A 1 ] / ( - A 6 - A2 x A5/A1) and [M]/[Se] = [(S(~2C)/Se(12C)- B1 - B2 - A2) x ([M*]/[Se])]/A1 In the bioavailability study simulation the following standard curves were calculated for caffeine concentrations ranging from 0 to 50 ng/l~l: S(~2C)/Se(~2C) = S(~2C)/Se(~3C) = S(~3C)/Se(~2C) = S(~3C)/Se(~3C) =

f([Caf]/[IBMX]) f([Caf]/[IBMX]) f([Caf]/[IBMX]) f([Caf]/[IBMX])

Table 8 shows the parameters of the standard curves corresponding to unlabeled and tri- ~3C-labeled caffeine molecules. The concentrations were calculated from the following derived equations: S(12C)/Se(12C) = (A1 x [M]/[Se] + B1)+ (A2 x [M*]/[Se] + B2)

(1)

S(12C)/Se(12C) = (A5 x [M]/[Se] + B5)+ (A6 x [M*]/[Se] + B6)

(4)

The correlations between measured concentrations and target values were

197 TABLE 8. Regression Lines Parameters of a Mixture of Labeled and Unlabeled Caffeine Determined by GC-AED

S(12C)/Se(12C)

Unlabeled caffeine

Labeled caffeine

Slope Intercept Coef of regression S(12C)/Se(13C)

0.84 _+0.02 0.06 _+0.03 99.49%

0.549 _+0.009 0.03 _+0.01 99.77%

Slope Intercept Coef of regression S(13C)/Se(12C)

3.56 +_0.09 0.2 -+ 0.1 99.52%

2.27 _+0.04 0.13 -+ 0.017 99.73%

Slope Intercept Coef of regression S(13C)/Se(13C)

0.115 _+0.002 0.007 _+0.003 99.78%

0.409 _+0.008 0.02 _+0.01 99.73%

Slope Intercept Coef of regression

0.486 _ 0.008 0.027 _+0.01 99.80%

1.69 _+0.03 0.09 _ 0.05 99.71%

excellent on the i.v. and oral pharmacokinetic curves. r = 0.9997 for the ~3C labeled caffeine concentrations (oral) r = 0.9971 for the unlabeled caffeine concentration (i.v.) The derived kinetic parameters were in good agreement. Two simulations were performed for the absolute bioavailability study. Unlabeled caffeine was administered by i.v. route and either 3-13CH3 caffeine or 1,3,7 (13CH3) caffeine were simultaneously administered by oral route. Table 9 shows the target pharmacokinetic parameters and the measured values derived from both labeled caffeine plasma levels determined by 13C atomic emission. For relative bioavailability, the two 13C-labeled caffeine isotopomers were administered orally in a two-phase experimental design. Each isotopomer was administered with unlabeled caffeine. As in Table 9, Table 10 shows the pharmacokinetic parameters derived from plasma curves. It can be seen from this example that molecules labeled with ~3C can be used for biopharmaceut-

198 TABLE 9. Comparison of Pharmacokinetic Parameters Obtained from Plasma Level of 313CH3 Caffeine, and 1,3,7 ~3(CH3)3 Caffeine Administered Orally, with the Target Values of the Model. tm.x = Time for Plasma Peak, Cm.x = Maximum Plasma Level, AUC = Area Concentration-time Curves, F = Factor of Bioavailability

Target values

tmax (h -1)

2.54

2.00

2.50

Cmax (mg/I)

3.13

3.14

3.11

34.46

30.18

32.10

AUC(0-inf)(mg.h.1-1) Difference (%) F (%)

~

(~= 12.4

70

3-~3CH3 caffei ne

1,3,7 (13CH3)3 caffeine

Parameter

~" (~= 6.84 61.26

~/ 69.35

TABLE 10. Comparison of Pharmacokinetic Parameters Obtained from Plasma Level of 3(13CH3) Caffeine, and 1,3,7 13(CH3)3 Caffeine Administered Orally, With the Target Values of the Model. tm.x = Time for Plasma Peak, Cm.x = Maximum Plasma Level, AUC = Area Concentration-time Curves, F = Factor of Bioavailability.

Parameter

3-(13CH3)

Target values

caffeine

1,3,7 (13CH3)3 caffeine

tmax (h)

2.54

2.50

2.50

Crnax (mg/I)

3.13

3.18

2.89

AUC(0-inf)(mg.h.1-1)

34.46

35.22

32.80

Difference (%)

~ '~

F (%)

70

(~= 2.19

~/ ~ = 4.83 71.53

~/ 66.62

ical studies using an experimental design with co-administration of unlabeled and labeled molecules and that AED is quite functional for the determination of both isotopomers. And that even with only one labeling isotope the experimental results are in very good agreement with the target values calculated by the theoretical pharmacokinetic model.

199 8. GC-AED COMPARED TO OTHER ANALYTICAL INSTRUMENTS FOR ISOTOPE MEASUREMENT

When stable isotopes are used several analytical methods are now available to measure their concentration, or to measure isotopes ratios between labeled and unlabeled compounds. Numerous mass spectrometric methods have been utilized in their detection. The most common of these methods has been the twin ion or ion-cluster method which allows both detection of metabolites using the ion cluster and structural identification by their mass spectrum. This technique is structure-dependent, and labeled compounds or metabolites in low amounts may not be detected due to interference with overlapping mass spectra of other compounds. Structure-independent detection may be an advantage when the structure of the investigated compounds are not known. Several structure-independent detectors have been developed. Among them, three kinds of analytical instruments can be mentioned for isotope determination:

- the first correspond to chromatography atomic emission spectroscopy coupling and especially GC-AED. - the second is CRIMS (chemical reaction interface mass spectrometry). Markey and Abramson (30) developed the chemical reaction interface. It is a microwave powered device that completely decomposes a complex molecule to its elements in the presence of helium. The addition of reagent gas forms stable products such as C02, S02 and H20 which reflect the elemental composition of the original analyte and are detected by a conventional quadrupole mass spectrometer. CRIMS is able to selectively detect compounds labeled with 13C, lSN and 2H from a biological matrix (31). HPLC can also be interfaced with such CRIMS (32). Detection limits are in the low to high ng/ml concentration range, and quantitative precision is in the 3-6 percent range (33). See Chapters 8 and 13 for more details on CRIMS. - t h e third is CF-GC-IRMS (continuous flow gas chromatography mass spectrometry). This isotope ratio mass spectrometer is able to measure isotopes ratios of carbon and nitrogen from very small samples with high precision and accuracy. For ~3C/~2C ratio measurements, organic carbon has to be transformed into C02, and for ~SN/~4N ratio measurement organic nitrogen into N2. Special devices have been developed for the determination of ~3C/~2C ratio from C02 contained in gaseous samples produced by micro-C02 generators.

200 Deruaz et al. (34) compared the limit of isotope enrichment detectable by GC-MS, GC AED and GC IRMS using ~3C-labeled progesterone as analyte. Progesterone (4 p r e g n e n e - 3.20 dione) was labeled with two atoms of ~3C at positions 3 and 4. It was chromatographed as its 2-enol pentafluoropropionic ester. The limit of isotope enrichment was the smallest value of molar enrichment significantly different from the response given by the unlabeled compound. For 10 ng of progesterone injected into the GC MS system (HP 5970A), the limit of isotope enrichment was 0.6 percent (over the natural ~3C abundance). The ~3C enrichment which can be determined routinely for progesterone by GC MS is about 2 percent (measurement on the molecular and base peak at m/2 = 462). When using GC AED, and the same chromatographic conditions, the limit of isotope enrichment was 1.8 percent for 10 ng of progesterone injected (HP 5921A). GC IRMS is specifically dedicated for the precise and accurate determination of small isotope enrichment. The smallest amount of CO2 necessary for a carbon isotope ratio is 10 nmol. That amount corresponds to 524 ng of unlabeled progesterone. The limit of isotope enrichment obtained by GC IRMS was 4.810 -3 percent for 2 nmol of progesterone injected (628 ng) (VG ISOCHROM II). See Chapters 6 and 21 for more details on CF-GC-IRMS. It can be observed from these results that GC AED and GC MS can measure carbon isotope enrichments in the same range of values (---2 percent) with small samples. GC IRMS is a complementary method which allows the determination of very small enrichments but needs larger amounts of analytical samples. In thesame way, the comparison between a quadrupole mass spectrometer (RIAL QM 130-CS) and an emission spectrometer (JASCO NIA 1) was reported on the determination of ~SN abundance in low enrichment nitrogenous materials (0.36-0.99 percent) by Cervelli et al. (35).

9. CONCLUSION

Current AED instrumental development allows one to attain high levels of sensitivity and specificity in elemental detection. With chemical, instrumental and algorithmic techniques, it is possible to lessen the interferences which limited the applications of AES coupled to chromatography. Hence, technique is now found to be practical and broadly useful in the analytical laboratory and in the areas of analysis of complex mixtures like biological samples, in biochemistry and pharmacology. These analytical performances allow use of a new kind of safe tracers,

201 stables isotopes, which can be easily detected and measured using gas c h r o m a t o g r a p h y coupled to atomic emission spectroscopy.

ACKNOWLEDGEMENTS The author thanks Dr D. Deruaz w h o has made productive this area of research in the LEACM, and M. Buisson for her skilful typographic assistance.

REFERENCES 1. R. Guilluy, F. Billion-Rey, C. Pachiaudi, S. Normand, J.P. Riou, E.J. Jumeau and J.L. Brazier, Anal. Chim. Acta, 259 (1992) 193. 2. J.L. Brazier, in Forensic Application of Mass Spectometry, in J. Yinon (ed) CRC Series Modern Mass Spectrometry (CRC Press, Boca Raton, FL, 1995), p. 259. 3. H.C. Urey, F. G. Brickwedde and G.H. Murphy, Phys. Rev. (1931) 722. 4. G.V. Veinbert, A.N. Zaidel and A. Petrov, Optr. I Spektr., 2 (1950) 972. 5. A.N. Zaidel and G.V. Ostrovskaya, Optics and Spectroscopy, 9 (1960) 78. 6. A.N. Zaidel and G.V. Ostrovskaya, Optr. I Spektr., 1 (1960) 137. 7. R.E. Ferguson and H.P. Broida, Anal. Chem., 28 (1956) 1436. 8. B.D. Quimby, P.C. Dryden and J.J. Sullivan, Anal. Chem., 62 (1990) 2509. 9. F. Leclerc, D. Deruaz, A. Bannier and J.L. Brazier, Anal. Lett., 27 (1994) 1325. 10. M. Hoch and H.R. Weisser, Helv. Chim. Acta, 23 (1950) 2128. 11. H.P. Broida and M.W. Chapman, Anal. Chem., 30 (1958) 2049. 12. J.A. Goleb and V. Middleboe, Anal. Chim. Acta, 43 (1968) 221. 13. J.C. Burridge and I.J. Hewitt, Anal. Chim. Acta, 153 (1983) 347. 14. D.I. Hoult and C.M. Preston, Rev. Sci. Instrum., 63 (1992) 1927. 15. P.C. Uden, Atomic Spectral Chromatographic Detection in Element Specific Detection by Atomic Emission Spectroscopy, in P.C. Uden (ed), ACS Symposium series 479 (1992), p. 1. 16. J.A. McCormack, S.C. Tong and W.D. Cooke, Anal. Chem., 37 (1965) 1470. 17. D.H Chose and F.P. Abramson, Anal. Chem., 61 (1989) 2724. 18. J.P. Van Delhen, P.A. De Lezenne Coulander and L. De Galan, Spectrochim. Acta, 33B (1978) 545. 19. D. Deruaz and J.M. Mermet, Analusis, 14 (1986) 107. 20. C.I.M. Beenakker, Spectrochim. Acta, 31B (1976) 483. 21. C.I.M. Beenakker, Spectrochim. Acta, 32B (1977) 173. 22. H. Yieru, O. Qingyu and Y. Weile, J. Chromatogr. Sci., 28 (1990) 584. 23. J. Hubert, M. Moisan and A. Ricard, Spectrochim. Acta, 34B (1979) 1. 24. G.L. Long and J.D. Winefordner, Anal. Chem., 55 (1983) 712. 25. R L. Oppenheimer, T. Capizzi, R. Weppelman and H. Mehta, Anal. Chem., 55 (1983) 638. 26. B.D. Quimby and J.J. Sullivan, Anal. Chem., 62 (1990) 1027. 27. A. Bannier, D. Deruaz, C.Weber and J.L. Brazier, Anal. Lett., 25 (1992) 1073. 28. T.C. Laurence and R.L. Terry, J. Chromatogr., 586 (1991) 309. 29. M.S. Boukraa, D. Deruaz, A. Bannier, M. Desage and J.L. Brazier, J. Pharm. Biomed. Anal., 12 (1994) 185.

202 30. S.P. Markey and F.P. Abramson, Anal Chem., 54 (1982) 2375. 31. D.H. Chace and F.P. Abramson, Anal. Chem., 61 (1989) 2724. 32. F.P. Abramson, M. McLean and M. Ustal, in Synthesis and Applications of Isotopically Labeled Compounds, E. Buncel and G.C. Kabalka (eds) (1991), p. 133. 33. D.H. Chace and F.P. Abramson, Biomed. Environ. Mass Spectrom., 19 (1990) 117. 34. D. Deruaz, B. Deruaz, A. Bannier, M. Desage and J.L. Brazier, Analusis, 22 (1994) 241. 35. S. Cervelli, F. Di Govanni and S. Ferrari, Rapid Comm. Mass Spectrom., 5 (1991) 48.

203

CHAPTER 10

THE USE OF ISOTOPES FOR PROBING LIGAND-PROTEIN INTERACTIONS AND LIGAND STRUCTURE: THE RHODOPSIN G-PROTEIN COUPLED MEMBRANE RECEPTOR PARADIGM

PETER J.E. VERDEGEM, JOHAN LUGTENBURG and HUUB J.M. DE GROOT Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands

1. INTRODUCTION

Rhodopsins are the light-collecting and transducting molecules in visual perception for three phyla of animals. Vertebrate, mollusks and anthropods. The most studied is bovine rhodopsin, which is the 40kDa G-protein coupled photoreceptor in the rod outer segments of the bovine retina that initiates the visual signal transduction cascade (1). It is an intrinsic membrane protein with 348 amino acids, which is folded into seven hydrophobic e-helices flanked by hydrophilic loops (Figure 1) (2, 3). The light-absorbing ligand in the active site of rhodopsin is called the chromophore. It is an 11-cis-retinylidene group covalently bound with a Schiff base link to lysine-296 of the polypeptide chain. The chromophore in rhodopsin is positively charged since the Schiff base is protonated. The charge is stabilized by a negative counterion in the protein. Glu 113 is thought to represent the primary counterion in rhodopsin, although it is likely part of a hydrogen-bonded network involving one or more water molecules, similar to the complex counterion in the somewhat related bacteriorhodopsin protein (4, 5). Capture of a photon by rhodopsin initiates the photochemical isomerization of the 11-cis chromophore to a distorted alI-E configuration within ~--200fs, leading to the primary photointermediate bathorhodopsin (6). The groundstate energy of bathorhodopsin is 145 kJ/mol higher than for rhodopsin and

204

205

Rhodopsin light Bathorhodopsin

l l l l

Lumirhodopsin

Metarhodopsin I

Metarhodopsin II

opsin + a/l-E-retinal Figure 2. The photosequence of rhodopsin.

--~60 percent of the photon energy of ---2 eV is temporarily stored in the primary photoproduct. Subsequent dark-reactions lead to the other intermediates in the sequence, lumirhodopsin, metarhodopsin I and II, and finally opsin and free all-E-retinal (Figure 2). Thus, a cis-trans photoisomerization is sufficient to convert the chromophore from an excellent antagonist into an optimal agonist for the receptor. The apoprotein is selective with respect to the binding of the 11-cis isomer. After the photosequence has ended, enzymatic isomerization of the all-Eretinal to the 11-cis form is necessary before it can bind again with the opsin to form the light active rhodopsin. The metarhodopsin I to II transition involves the discharge of the Schiff base through deprotonation and leads to the activation of the G-protein transducin and visual signal transduction (7).

Figure 1. The structure of the rhodopsin G-protein coupled visual photoreceptor, with the seven trans membrane e-helices and the 11-cis-retinylidene chromophore. Color picture courtesy of Prof. S.O. Smith.

206 In this chapter some recent applications of enrichment with stable 13C isotopes and Magic Angle Spinning (MAS) NMR studies of rhodopsin are presented. It will be demonstrated that comprehensive information about the electronic and spatial structure of the ligand can be obtained when bound to the membrane with isotope labeling and MAS NMR. First, we present a short overview of the labeling and MAS NMR studies aiming at a characterization of ligand-protein interactions for rhodopsin via the determination of chemical shifts. These studies have been performed in collaboration with several other research groups. To complement the NMR shift investigations, we discuss experimental data for the spatial structure of the ligand in the binding site in rhodopsin. To illustrate the capabilities of MAS NMR for studying photoproducts, shift data on the ligand-protein interactions in bathorhodopsin, structural data for the pre-discharge metarhodopsin I, and finally, shift data for the discharged metarhodopsin II photointermediate, will be reviewed. This includes a brief presentation of some of the newest structural results that were obtained recently in the last year in our own laboratories and will be published extensively elsewhere. The rhodopsin can be considered a paradigm for other G-protein coupled receptors. With hundreds of potential drug targets within companies worldwide, the importance of tools for the study of ligand structure and ligand-protein interactions can hardly be overestimated.

2. EXPERIMENTAL METHODS

MAS NMR, in conjunction with selective isotope enrichment, is the method of choice for NMR investigations of membrane protein receptors when in the membrane in their natural environment. With technologies for membrane protein expression continuously improving, it can be anticipated that many membrane receptors will become available in sufficient quantity for NMR spectroscopy in the near future. In this review, the major focus will be on the characterization of ligand-protein interactions at the atomic level, and how to examine specific details of the ligand structure with pairs of isotopes, incorporated by total synthesis at strategic positions in the ligand.

2.1. Magic Angle Spinning NMR MAS NMR is a technique for obtaining high-resolution NMR data from solids. In a MAS NMR experiment, the chemical shift anisotropy broadening of the NMR response in the solid state is suppressed by macroscopic sample rotation around an axis at the magic angle /3,7,= 54o44' with respect to the

207 applied magnetic field. A detailed treatment of the MAS averaging of the NMR response of a solid can be found elsewhere (8). Briefly, during MAS individual molecules are subject to physical sample rotation and the chemical shift of every molecule varies periodically in time. Since many different chemical shift trajectories will be possible, the macroscopic nuclear magnetization collapses in a very short time, but refocuses after every completed rotor cycle. In the frequency domain MAS generates an infinite number of sidebands at integral multiples of the spinning speed O~r,with respect to the isotropic shift (r~, which is the average chemical shift experienced by every molecule in the rotating sample.

2.2. The Rotational Resonance MAS NMR Technique Rotational resonance is a high resolution solid state NMR technique that 1 allows the measurement of internuclear distances between / = ~ nuclei through the interference of the MAS with the homonuclear dipole interactions within the pair of spins (9, 10). Rotational resonance occurs when (.Or matches the difference in resonance frequency A~O~s= ~o~- ~Os of the two spins. In an established approach to measure distances between a pair of ~3C atoms, one of the spins is selectively inverted and rotor-driven exchange of magnetization is followed in time by collecting a series of 1D datasets (11). At the n = 1 rotational resonance condition, the line shapes change and a broadening, or in favorable cases, a splitting of the line shape A(o~ can be observed. Recently, the relationship between A~o~ and the scaled dipolar interaction b~s/27r~/8 was investigated and calibrated experimentally with a series of four doubly-labeled retinal model compounds (12). Here,

b,s = t -j['~tOt ,)/2~ r3s

(1)

is the dipolar coupling constant. Analysis of the A(o~, by taking the second derivative of the rotational resonance spectrum and subsequent fitting of the shape with a pair of second derivative Lorentzians, provides a way to measure internuclear distances accurately (12). A linear relationship

bls/27r~/8 = al(A~ol/2~T) + ao

(2)

was found with a~ = 1.15 and ao a small offset depending on the total line

208 width (12). Using Eqs. (1) and (2), dipolar couplings b~s and internuclear distances r~s can be calculated from the experimentally determined A~o~.

3. MAS NMR INVESTIGATIONS OF RHODOPSIN

3.1. Ligand-Protein Interactions in Bovine Rhodopsin

The NMR response can be used to probe, with atomic resolution, the chemical environment of ligands bound to membrane receptors. The technique complements other spectroscopic approaches, in particular resonance Raman spectroscopy with isotope labeling to assign vibrations. By labeling with isotopes in the ligand, an NMR assay of the ligand-protein interactions can be constructed, for instance, by comparing the chemical shifts for the retinylidene in the protein with the corresponding shifts for a protonated Schiff base model in solution. This is illustrated in Figure 3, which shows the 11-cisretinylidene protonated Schiff base, like the antagonist-type ligand in rhodopsin. To construct a shift assay for the chromophore in situ, retinals labeled along the polyene chain were first synthesized (13) and reconstituted with bovine opsin (14). Subsequently, NMR experiments were performed to determine the ~3C shifts of the isotope labels in the protein and the signals for protonated Schiff bases in solution (15). The shift differences are reliable indicators of ligand-protein interactions. In Figure 3 they are encoded with colors, going from blue via green to yellow and red for increasing interaction strength. The result is a genuine assay of ligand-protein interactions with atomic resolution, almost like a "snapshot" of the ligand when bound to the receptor, in its natural lipid environment. Chromophore-protein interactions are observed for almost the entire length of the conjugated chain. The strongest interactions, indicated by the red and yellow spots, are detected for the cis region of the chromophore. This is of interest, since it is exactly the section of the chromophore involved in the isomerization to the agonist form of the ligand. It can be concluded that for rhodopsin the shift assay works particularly well, since the region of the chromophore that is most critical for the function, is immediately transpiring from the representation of the NMR data. Using the shift differences and by correlating the NMR data with other experimental results from e.g. site-directed mutagenesis studies, Han et al. (16) were able to identify the location of the counterion to the Schiff base. Using this counterion as an anchor, they were also able to fit the chromophore into the electron diffraction map that was recently obtained for rhodopsin (2),

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209

210 thereby providing the first comprehensive low-resolution structure for a Gprotein coupled membrane receptor shown in Figure 1 (3). In principle, similar assays can also be made based on other NMR characteristics. For instance, the examination of the variations in relaxation properties can help to detect steric nonbonding ligand-protein and intra-ligand interactions. The measurements presented in Figure 3 were performed using selectively labeled compounds, which is a laborious procedure, since every labeled position needs a separate organic synthesis. However, recent advancements in 2D MAS NMR dipolar correlation spectroscopy allows the use of uniformly labeled ligands to obtain a complete picture in one set of labeling and reconstitution experiments, with improved accuracy (17, 18). Such experiments will benefit greatly from novel ultra-high field MAS NMR equipment currently under development with the NMR industries.

3.2. Probing the Spatial Ligand Structure in Bovine Rhodopsin With NMR and isotope labeling we can examine the structure of the isomerization region by measuring critical intra-ligand internuclear distances. In Figure 4A, the chemical structure of the 11-cis-retinylidene chromophore in rhodopsin is shown. It is thought that nonbonding interactions between the C10-H and the C20-H3 provoke an out-of-plane deformation in the is0merization region of the chromophore which is of prime importance for the speed and efficiency of the isomerization. The presence of an out-of-plane deformation can be verified with 1D MAS NMR rotational resonance spectroscopy (19). The particular focus will be on the r~o,2o, between the vinylic C10 and the C20 methyl group, which were labeled for this purpose. The doubly labeled retinal was reconstituted into freshly prepared bovine opsin maintained in its natural lipid environment. In Figure 5, the off rotational resonance MAS spectrum of the reconstituted [10,20-~3C2]-rhodopsin is shown. The narrow label resonances are indicated with a filled circle for the vinylic signal with (~; = 127.1 ppm, and with a square for the methyl response with ~; = 15.9 ppm. To determine the n = 1 rotational resonance condition &~s from the NMR response, both label resonances were analyzed with Lorentzians and, from the center frequencies ~O~oand (02o, the rotational resonance condition was calculated as A~O~s= 12.592 kHz. The vinylic part of the rotational resonance spectrum of [10,20-~3C2]-rhodop sin, collected with OJr= A~O~S,is depicted in Figure 6A, showing in the upper trace the C10 signal superimposed on the broad resonance from the unsaturated lipids in the natural membrane. At rotational resonance, a small splitting of the label response due to rotational resonance dipolar recoupling can be

211 16

17

, , o, i l . :

A 2

~

12

13

3 4

18

,5 L%NtH LLys296 2O | FI

Lys296

Figure 4. Chemical structures of 11-cis retinylidene (A) and alI-E retinylidene in rhodopsin. In scheme A the IUPAC numbering for the entire chromophore group is indicated, while in scheme B the two positions that were labeled with isotopes to measure the out-of-plane distortion in the rhodopsin and the metarhodopsin-I intermediate are emphasized with their IUPAC numbers.

observed. To analyze the data, we first apply a 40 Hz apodization to suppress background noise, and fit the second derivative spectrum with a pair of second derivative Lorentzians, yielding A~o1/2~r=75_+4Hz. The second derivative of the signal and the computer analysis are shown in the lower traces in Figure 6. In the second derivative, the splitting is enhanced which facilitates the analysis. Calculation of the scaled dipolar coupling constant using Eq. (2) gives Ib,sl/2~/8 - 98 _+ 4 Hz, corresponding with an internuclear distance rlo,2o=0.302-+0.01 nm, significantly longer than the rlo,2o = 0.295 nm expected for a predominantly planar chromophore. The rlo,2o distance measurement provides direct and unambiguous experimental evidence that the chromophore is indeed the 11-cis form and that a considerable outof-plane distortion is present in the isomerization region of the chromophore of rhodopsin, probably due to nonbonding interactions between C10-H and C13-methyl. Simple molecular modeling, using the crystal structure of 11-cis12-s-cis-retinal (20) and our internuclear distances, provides an estimate of the angle between the C7-C10 and C13-C15 planes of the chromophore of

212

I

I

I

I

I

I

250

200

150

l O0

50

0

Chemical shift (ppm)

Figure 5. 100.6 MHz 13C-CP/MAS spectrum of [10,20-13C2]-rhodopsin collected with ~r/2~T = 7.000 kHz. The markers indicate the centerbands of the C10 (O) and C20 (11) label resonances.

---42~ in agreement with ab-initio molecular dynamics results (21). The distance between C11 and C20 has also been determined, r11,2o = 0.294 nm. This measurement confirms the previous conclusions with respect to the out-ofplane deformation of the chromophore (19). Using a limited set of precise distance constraints, Bifone et al. (22) were able to model the structure of the isomerization region. In particular, they found a distribution of the ---42~ torsion over the Clo-C~-C~2-C~3 moiety, which has been confirmed recently with a novel MAS NMR method for measuring torsional angles (23).

3.3. Probing the Electronic and Spatial Structure of Intermediates An accurate determination of the electronic and spatial structure of the ligand after activation will be essential also for understanding the molecular mechanisms behind the initial stages of visual signal transduction. The retinylidene ligand in rhodopsin and its photoproducts are brightly coloured, which makes them accessible to an extensive array of optical techniques. Recent ultrafast time-resolved optical absorption experiments, and resonance Raman experiments, have provided conclusive evidence that both the speed and the effi-

213

'

140

I

'

I

135

'

130

I

'

125

I

'

120

I

'

115

-

110

Metarhodopsin-I

,

'

140

I

135

,

"

I

130

'

I

125

'

I

120

'

I

115

'

llO

Chemical shift (ppm) Figure 6. Vinylic region of 100.6 MHz 13C n = 1 rotational resonance CP/MAS spectra of [10,20-13C2]-rhodopsin and -metarhodopsin-I. The lower traces represent the second derivatives of the NMR response with the computer, analyses indicated by the dashed lines.

214 ciency of the isomerization strongly depend on a precise tuning of the structure in the central part of the ligand, well in line with the MAS NMR shift assay discussed in the previous section (24, 25). In addition, the isomerization is ultrafast and is completed in less than ---200 fs (26). Even a small ligand cannot undergo major structural changes on such a short timescale. The observation that the photochemical isomerization is ultrafast thus implies that the bathorhodopsin photoproduct constitutes a formal alI-E chromophore in globally the binding pocket optimized to accommodate the 11-cis form. This inference is supported by the ab initio molecular dynamics simulations, which provided compelling evidence that the conformation of the ligand in the rhodopsin inactive ground state, together with its stabilization by the counterion or complex, largely determines the structure, the amount of strain, and the charge delocalization of the chromophore in the active bathorhodopsin state (27). An accurate determination of the electronic structure of the chromophore in bathorhodopsin along the polyene chain, and in the vicinity of C11C12 double bond, is then also essential to understand the mechanisms of isomerization and energy storage in the primary photointermediate, which can be considered as the agonist form of the ligand. The photointermediates in the rhodopsin photosequence can be trapped at low temperature and accessed with MAS NMR once isotopes have been incorporated. To achieve this, either reconstituted rhodopsin or the 9-cis isorhodopsin receptor is activated by illumination with an intense light source, with the sample packed in the NMR rotor and cooled to liquid nitrogen temperature. In this procedure, the bathorhodopsin primary photointermediate is formed with good efficiency. Subsequently, the NMR rotor can be inserted in a pre-cooled probe. By raising the temperature in steps, the various photointermediates can be trapped and their electronic and spatial structure examined with MAS NMR. Again the NMR complements the vibrational at studies where structural information about the chromophone can be obtained from resonance Raman spectroscopy and isotope labeling to assign the normal modes. MAS NMR spectra were obtained for the trapped bathorhodopsin photointermediate, starting from isorhodopsin samples reconstituted with retinals labeled at positions 8, 10, 11, 12, 13, 14 or 15 (28). A shift assay of the ligandprotein interactions in the isomerization region of the primary photointermediate was constructed by comparison with the shifts from alI-E retinal protonated Schiff bases. The largest difference amounts to 6.2 ppm and is observed for the position 13 in the bathorhodopsin photoproduct. Small differences in chemical shift between bathorhodopsin and the alI-E protonated Schiff base chloride salt are also observed at positions 10, 11 and 12. The effects are almost equal in magnitude to the shifts observed in rhodopsin (Figure 3).

215 Consequently, the energy stored in the primary photoproduct does not give rise to any substantial change of the average electron density at the labeled positions. The data indicate that the electronic and structural properties of the protein environment are similar to those in rhodopsin and isorhodopsin. In particular, the counterion, which is thought to be located near C13, appears not to change its position significantly with respect to the chromophore upon isomerization. The NMR provides strong evidence that the light energy is rapidly converted into strain, and is not primarily stored in the form of charge separation between chromophore and protein. Starting from the rhodopsin structure and our NMR charge and distance constraints, the bathorhodopsin structure and energy storage function were modelled with ab-initio molecular dynamics methods (27). It appears that the energy storage is mediated by a charge defect or soliton, which is already present in the ground state (22). After excitation the molecule is bistable, in the sense that the defect can easily move between the two extremes of the polyene chain. Fully in line with the MAS NMR shift data for the bathorhodopsin intermediate, it is the interaction with the counterion that locks the defect at the Schiff base end of the chromophore. This yields a distribution of electronic change similar to rhodopsin and gives rise to a strained molecule capable of accumulating the energy for triggering the subsequent steps in the sequence. In this respect, it is also of interest that discharge of the Schiff base probably requires the motion of another solitonic defect at the same energy scale, e.g. a proton in a hydrogen bonded network, from the Schiff base nitrogen into the protein environment. It is fair to state that these novel functional descriptions have emerged from the interpretation of the data from NMR and isotope labeling studies (5, 22). Very recently, we have been able to obtain genuine structural data for various intermediates in the rhodopsin sequence. This is illustrated in Figure 6, which shows the vinylic region of a dataset collected after illumination and accumulation of the [10,20-~3C2] metarhodopsin I intermediate together with its second derivative trace. The response of the [10,20-~3C2] yields two contributions to the second derivative spectrum, with cr/= 130.6 ppm from the metarhodopsin I intermediate and with cr; = 127.1 ppm from the rhodopsin that could not be converted in the illumination procedure. Although the data for Figure 6 were collected at the n = I rotational resonance condition for the [10,20-~3C2] in the metarhodopsin I, there is no resolved splitting of the second derivative signal like in Figure 6 for the rhodopsin. This shows that the C10C20 distance is longer in metarhodopsin I than for rhodopsin, which is due to the photoisomerization to the alI-E form (Figure 4B). The distance in the photointermediate can be estimated by measuring the excess broadening of

216 the n = 1 rotational resonance in the second derivative response. From Figure 6 an rlo,2o > 0.4 nm can be estimated, which indicates that the chromophore is almost planar in the metarhodopsin I intermediate. This would imply a considerable structural change of the ligand of ~--0.5 nm already before the discharge of the chromophore and the activation of transducin by the deprotonated metarhodopsin II form occurs. The actual discharge of the ligand has been investigated with ~3C MAS NMR and retinal labeled at C14 or C15. To achieve this, MAS !3C NMR spectra were collected from 13C-labeled rhodopsin reconstituted into 1,2dipalmitoleoylphosphaditylcholine bilayers to increase the amount of meta II trapped at low temperature (29). Both the C13 and the C15 shifts are characteristic of an unprotonated Schiff base, providing unambiguous evidence that the ligand is discharged in the meta I to meta II transition.

4: CONCLUSIONS

NMR in conjunction with isotope enrichment can provide assays of ligandprotein interactions to atomic resolution for ligands when bound to the receptor. In the future, such NMR assays will be useful for the identification and characterization of pharmacophores, conformational and configurational changes of the ligand induced by binding, charging or discharging, nonbonding interactions and novel functional mechanisms for triggering signal transduction. 1-Dimensional rotational resonance and other, more sophisticated MAS NMR techniques, in conjunction with specific isotope enrichment, are powerful methods for obtaining accurate structural information of biological systems. It is illustrated how the ligand conformation in the rhodopsin Gprotein coupled receptor can be examined with MAS NMR. Our newest data measure the out-of-plane deformation in the isomerization region, which is essential for the molecular mechanism of function, since it determines the speed and efficiency of the first step in the visual signal transduction process (25). After capture of a photon and isomerization to an alI-E conformation in bathorhodopsin, the chromophore adapts a relaxed alI-E structure already in the pre-discharge metarhodopsin.

217

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

L. Tang, T.G. Ebrey and S. Subramaniam, Isr. J. Chem., 35 (1995) 193. J. Baldwin, EMBO J., 12 (1993) 1693. M. Han and S.O. Smith, Biochemistry, 34 (1995) 1425. H.J.M. De Groot, G.S. Harbison, J. Herzfeld and R.G. Griffin, Biochemistry, 28 (1989) 3346. H.J.M. De Groot, S.O. Smith, J. Courtin, E.M.M. van den Berg, C. Winkel, J. Lugtenburg, R.G. Griffin and J. Herzfeld, Biochem., 29 (1990) 6873. R.W. Schoenlein, L.A. Peteanu, R.A. Mathies and C.V. Shank, Science, 254 (1991) 412. K.P. Hofmann, O.P. Ernst, S. J~ger, Isr. J. Chem., 35 (1995) 339. M. Mehring, Principles of High Resolution NMR in Solids (Springer-Verlag, Berlin, 1983). M.H. Levitt, D.P. Raleigh, F. Creuzet and R.G. Griffin, J. Chem. Phys., 92 (1990) 6347. F. Creuzet, A. McDermott, R. Gebhard, K. van der Hoef, M.B. Spijker-Assink, J. Herzfeld, J. Lugtenburg, M.H. Levitt and R.G. Griffin, Science, 251 (1991) 783. D.P. Raleigh, M.H. Levitt and R.G. Griffin, Chem. Phys. Lett, 146 (1988) 71. P.J.E. Verdegem, M. Helmle, J. Lugtenburg and H.J.M. De Groot, J. Amer. Chem. Soc., 119 (1997) 169. M. Groesbeek and J. Lugtenburg, Photochem. Photobiol., 56 (1992) 903. W.J. De Grip, F.J.M. Daemen and S.L. Bonting, Methods in Enzymol., 67 (1980) 301. S.O. Smith, I. Palings, M.E. Miley, J. Courtin, H.J.M. De Groot, J. Lugtenburg, R.A. Mathies and R.G. Griffin, Biochem., 30 (1991) 1991. M. Han, B.S. De Decker and S.O. Smith, Biophys. J., 65 (1993) 899. T.S. Balaban, A.R. Holzwarth, K. Schaffner, G.-J. Boender and H.J.M. De Groot, Biochem., 34 (1995) 15259. T. Egorova-Zachernyuk, B. Van Rossum, G.-J. Boender, E. Franken, J. Ashurst, J. Raap, P. Gast, A. Hoff, H. Oschkinat and H. De Groot, Biochem., 36 (1997) 7513. P.J.E. Verdegem, P.H.M. Bovee-Geurts, W.J. De Grip, J. Lugtenburg and H.J.M. De Groot, submitted. R.D. Gilardi, I.L. Karle and J. Karle, Acta Cryst., B28 (1972) 2605. A. Bifone, H.J.M. De Groot and F. Buda, Chem. Phys. Lett., 248 (1996) 165. A. Bifone, H.J.M. De Groot and F. Buda, J. Phys. Chem., 101B (1997) 2954. X. Feng, P.J.E. Verdegem, Y.K. Lee, D. Sandstr6m, M. Eden, P.H.M. Bovee-Geurts, W.J. De Grip, J. Lugtenburg, H.J.M. De Groot and M. H. Levitt, J. Amer. Chem., in press. Q. Wang, G.G. Kochendoerfer, R.W. Schoenlein, P.J.E. Verdegem, J. Lugtenburg, R.A. Mathies and C.V. Shank, J. Phys. Chem., 100 (1996) 17388. G.G. Kochendoerfer, P.J.E. Verdegem, I. van der Hoef, J. Lugtenburg and R.A. Mathies, Biochem., 35 (1996) 16230. Q. Wang, R.W. Schoenlein, L.A. Peteanu, R.A. Mathies and C.V. Shank, Science, 266 (1996) 422. F. Buda, H.J.M. De Groot and A. Bifone, Phys Rev. Lett., 77 (1996) 4474. S.O. Smith, J. Courtin, H.J.M. De Groot, R. Gebhard and J. Lugtenburg, Biochem., 30 ( 1991 ) 7409. S.O. Smith, H.J.M. De Groot, R. Gebhard and J. Lugtenburg, Photochem. Photobiol., 56 (1992) 1035.

219

CHAPTER 11

MASS BALANCE

THOMAS R. BROWNE, 1 GEORGE K. SZABO ~ and ALFRED AJAMI 2 1Departments of Neurology and Pharmacology, Boston University School of Medicine, Neurology Service, Boston Department of Veterans Affairs Medical Center; 2MassTrace, Woburn, MA

1. INTRODUCTION

Regulatory agencies in many countries require a human mass balance/metabolite identification (MB/MI) study as part of the testing of a new drug. Historically, MB/MI studies have been performed by: (1) administering radioactive (14C) labeled drug; (2) measurement of radioactivity in urine and feces (to measure mass balance); (3) chromatography of urine to divide dissolved material into peaks; (4) detection and quantitation of peaks containing drug or metabolite by measurement of radioactivity; and (5) identification of structure of drug or metabolite in "hot" chromatography peaks by various mass spectrometry techniques. Preliminary data on the pharmacokinetics of the drug can be obtained from radioactivity versus time relationships in serum, blood and urine. The analytic aspects of this methodology are simple and have served pharmaceutical research well for many years. Recently, alternatives to traditional radioactive labeling techniques for performing MB/MI studies have been sought for several reasons: (1) regulations for storage and disposal of radioactive specimens have become more restrictive; (2) institutional review boards are increasingly reluctant to approve any work exposing humans to radioactivity; (3) it has been nearly impossible to perform radioactive tracer studies in children, even though drug metabolism in children often differs significantly from adults; (4) synthesis of radioactive analogues of some compounds can be difficult; and (5) the sponsor may be assuming long-term liability risk if the subjects or employees later develop cancer or other diseases. These considerations may increase the cost and delay performance of MB/MI studies. Human MB/MI data should be obtained as early in drug development as possible to determine the presence and

220 extent of potentially active or toxic metabolites and to guide future pharmacokinetic studies (e.g. collection of patient urine samples for drug analysis may, or may not, be useful, depending on results of MB/MI studies). This chapter reports on work to develop simple general methods for performing an MB/MI study on any new drug using stable isotope labeling (SIL) and detection as an alternative to radioactive labeling and detection. Two SIL techniques show promise of achieving this objective of a simple general MB/MI methodology: (1) continuous flow-isotope ratio mass spectrometry and (2) high-performance liquid chromatography combined with chemical reaction interface mass spectrometry. There are many examples of combining SIL and MS for identification of specific metabolites of specific drugs which are covered in Chapters 3-5, and 12.

2. CONTINUOUS FLOW-ISOTOPE RATIO MASS SPECTROMETRY (CF-IRMS) 2.1. Technique and History These topics are covered in Chapter 6.

2.2. Assumptions in Using CF-IRMS for MB/MI Studies Use of CF-IRMS methods for performing human MB/MI studies assumes: (1) simple, reliable CF-IRMS instruments are commercially available; and (2) the commercially-available CF-IRMS instruments possess the necessary sensitivity, precision and accuracy to determine label in urine, feces, serum and blood and to detect and quantify labeled drug and metabolites in HPLC peaks collected from urine, feces and serum.

2.2.1. Reliable instrumentation Early IRMS instrumentation was problematic because: (1) each instrument was unique and "made by hand"; (2) complete liberation of all atoms of a given molecule by oxidation was difficult; (3) transfer by hand of N2 and C02 gases from elemental analyzer to IRMS was problematic; (4) each specimen was run by hand (lack of automation); and (5) factors 1-4 made IRMS difficult and inconsistent (1, 2). Recently, refined commercially-available instruments using a helium carrier gas to carry combustion products to the IRMS have become available from tl~ree sources (Europa Scientific, Ltd., Finnegan MAT and VG Instruments). The authors purchased a Europa (Europa Scientific, Inc.,

221 Franklin, Ohio, USA) ANCL-SL (elemental analyzer) 20/20 (mass analyzer) CFIRMS and found the instrument performed up to specifications and with very high precision as delivered (see below).

2.2.2. Adequate sensitivity: Theoretical computations It is possible to compute the lowest quantity of drug quantifiable with a precision (coefficient of variation, CV) of 5 percent or less for a given drug using the maximal resolution and minimal total sample size of a CF-IRMS instrument, the carbon or nitrogen content of a biological specimen, and the molecular weight and 15N or 13C content of the tracer drug. The equation for this computation is as follows: LQ = MR,- x M(c,n) x Tmw x N

(1)

where MR/is a mass spectrometer's instrument resolution taken as the mole ratio of 15N/14N focused on masses 29/28 or 13C/~2Cfocused on masses 45/44 at natural abundance which can be measured with 5 percent or better precision (data taken from manufacturer's or literature values); M(c,n)is the moles of natural abundance isotopolog(s) per unit volume (time) in the biological matrix that is to be spiked with 15N or 13C tracer (data taken from published elemental composition of various biological matrices); Tmw is the molecular weight of a tracer drug (assumed to be 200 g/mol in this paper); N is the number of labeled atoms per mole of tracer drug. The results of Eq. (1) applied to a typical CF-IRMS instrument are shown in Table 1. Note the following: (1) procedures which reduce background carbon or nitrogen (deproteinization, extraction, chromatography) increase sensitivity; (2) sensitivity for a given molecule increases directly with the number of atoms labeled with an additional neutron; and (3) the theoretical sensitivity of CF-IRMS appears sufficient to perform MB/MI studies on drugs of medium or low (but not high) potency using one 15N or two 13C labels.

2.2.3. Adequate sensitivity, precision and accuracy: Empirical studies In 1993, we presented preliminary evidence that an early commercial CFIRMS instrument (Europa Roboprep CN/Tracer Mass) may possess sufficient sensitivity, precision and accuracy to quantitate some drugs with one 13C or two 15N labels (2, 3). Stable isotope labeling in therapeutic and subtherapeutic quantities of 15N2 13C-phenobarbital were quantitated in urine and in HPLC peaks from urine. Standard curves were reproducible and linear (r2> 0.985)

222 TABLE 1. Lowest Quantity of Drug Quantifiable with a Precision (CV) of 5% or less Using CF-IRMS 1

Desired value

lSN1 label

~3C~ label

~3C2 label

A. Total label Blood (whole)

0.1 ~g/mL

42.2 i~g/mL

>42.2

>42.2

Blood (deproteinized)

0.1 ~g/mL

0.006 ~g/mL

Serum (whole) 0.1

0.1 i~g/mL

1.4 i~g/mL

Serum (deproteinized)

0.1 i~g/mL

0.006 i~g/mL

0.006 ~g/mL

0.003~g/mL

Urine (whole)

1 i~g/mL

1.0 i~g/mL

1.4 i~g/mL

0.7 ~g/mL

Feces (whole)

1 mg/24 hr

0.3 mg/24 hr

2.3 mg/24 hr

1.2 mg/24 hr

Feces (extracted)

1 mg/24 hr

0.05 mg/24 hr

0.7 mg/24 hr

0.4 mg/24 hr

0.006 i~g/mL

0.003~g/mL

0.006 ~g/mL >1.4 i~g/mL

0.003~g/mL >1.4 ~g/mL

B. Labeled drug or metabolite in an HPLC peak drug Serum or urine

0.02 ~g/mL

0.006 ~g/mL

1Europa ANCL-SL 20/20 instrument.

over the range of 3-100 i~g/ml for whole urine (15N2 or 13C labeling) and 0.1-8.0 ~g/ml for HPLC peaks derived from urine (lSN2 labeling). The lower limit of quantitation values for urine drug concentration were 0.46-2.62 ~g/ml in whole urine and 0.10-0.701~g/ml in HPLC peaks. Validation samples quantitated with these standard curves yielded close to expected values. We have been working since then on further empirical verification of CFIRMS analytic determinations of stable isotope-labeled drug concentration in biological matrices using a newer CF-IRMS instrument (Europa ANCL-SL 20/20). We calculated the new instrument should be more sensitive than the older instrument (4). Several interim reports of our (not yet completed) work to confirm these calculations are available. We first studied ~SN~ labeled drug in human urine (5, 6). Standard curves of atom percent excess of ~SN (above natural abundance) times total nitrogen values versus drug (~SN-acetaminophen) concentration were regressed over a concentration range (in whole urine) of 1.0 to 200.0 ~g/ml (Figure 1). Weighted (1/X 2) and unweighted least squares linear regression analysis techniques gave coefficient of determination (r 2) and lower limit of quantitation (LLQ)

X I,--I

r'Y'

I-
" rr
45 days) regimens of phenytoin monotherapy were studied with intravenous tracer doses of 150 mg of 13ClSN2-1abeled sodium phenytoin. Labeled and unlabeled phenytoin plasma concentrations were followed for 48 hr after each infusion. 3.2.2. First method

Elimination phase plasma concentration versus time relationships of labeled phenytoin were inspected visually and tested statistically for semilog linearity using procedures described previously. 3.2.3. Second method

Tracer dose clearance and steady state clearance computed with Eq. (2) were determined for each patient as described previously. Differences between tracer dose clearance and steady state phenytoin clearance values were tested by the Student's paired t-test (i.e. probability of a type I or alpha error was computed). The probability of failing to detect a truly significant difference of 20 percent or greater in clearance values was determined by the method of Glenberg (i.e. the probability of a type II or beta error was computed) (21). 3.2.4. Results of first method

The elimination phase plasma concentration versus time relationships for ~3C~SN2-phenytoin after each infusion appeared linear on semilog plots. The

269 TABLE 2. First Method: Correlation Coefficients tration Versus Time Relationships

Patient number

r2

(p)

1 2 3 4 5 6 7 8

0.994 0.999 0.998 0.977 0.994 0.998 0.990 0.981

(< 0.001 ) ( 1.0

at the nondeuterated site from a 50:50 mixture of normal and deuterated substrate is measured to determine the isotope effect. In the noncompetitive experiment, the isotope effect is determined from the ratio of products formed at the nondeuterated site when normal and deuterated substrates are independently incubated with enzyme. The value of the isotope effects to be expected for each combination of experimental design and mechanism are given in Table 1. A value of 1.0 for the product formation rate ratio between normal and deuterated substrates, VH/VO, in the competitive experiment, but 1.0 for the ratio of V/Ks between normal and deuterated substrates, (Vrnax/Krn)H/(Vmax/Krn)D in the noncompetitive experiment is diagnostic for a dissociative mechanism. Values of 1.0 in both kinds of experiment are diagnostic for a parallel path mechanism while values of l:z: < n,,, l-m nr, W

_j

1

0

--1

0

I

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I

100

200 TIME (MIN)

300

400

Figure 3. The graph represents mean serum levodopa levels obtained over a 6-hr period following an intravenous bolus infusion of 150 mg of stable isotope labeled levodopa at time 0. Each point is the average of nine patients. Error bars indicate standard deviation.

328

SERUM HVA LEVELS AFTER INFUSION OF STABLE ISOTOPE LABELED LEVODOPA 400 Filled circle - +6 HVA Open circle +0 HVA 3OO

S" z~

200

-r" 100

0

, I

0

100

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300

400

Figure 4, The graph represents mean labeled (filled circles) and unlabeled (open circles) serum concentrations of homovanillic acid over a 6-hr period following an intravenous infusion of 150 mg of stable isotope labeled levodopa. Each point is the average of nine patients. Error bars indicate standard deviation.

4.2. Systemic Homovanillic Acid Levels It can be seen in Figure 4 that large amounts of the infused stable isotope labeled levodopa were decarboxylated in the periphery to homovanillic acid despite pretreatment with 50 mg of carbidopa (twice the carbidopa used in a standard oral carbidopa/levodopa tablet). Concentrations of the labeled homovanillic acid rose quickly and peaked at 60 min to a mean of 162 ng/ml accounting for approximately 85 percent of the total blood homovanillic acid. Unlabeled homovanillic acid serum concentrations, on the other hand, remained fairly stable and low throughout the 6-hr period with no suggestion of increased production.

4.3. Cerebrospinal Fluid Homovanillic Acid Levels We found that over 50 percent of homovanillic acid in lumbar cerebrospinal fluid was labeled 6 hr after a single bolus infusion of 150 mg of stable isotope

329

% LABELED HVA IN LUMBAR CSF 6 HOURS AFTER BOLUS INFUSION OF 150 MG LABELED LEVODOPA 70 60 < > I a

50

LU

4O


. z uJ 2: (1,,

1

-..~

o x

-

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-

98

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-

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I

10

20

,,

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,

30

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40

I 50

TIME AFTER PHENYTOIN INFUSSION ( hours ) Figure 1. Serum concentration versus time relationships for 150 mg 13C15N2-phenytoin i.v. tracer doses for patient on chronic phenytoin therapy before (Week O) and 4 and 12 weeks after adding chronic phenobarbital therapy. From Browne et al. (1) with permission.

1.2. Assumptions SIL tracer m e t h o d s a s s u m e a b s e n c e of m e t a b o l i c i s o t o p e effect and a b s e n c e of " d e e p pool e f f e c t " on m e a s u r e d p h a r m a c o k i n e t i c p a r a m e t e r s , The a b s e n c e of t h e s e effects can be d e m o n s t r a t e d using s t r a i g h t f o r w a r d t e c h n i q u e s described in C h a p t e r s 2, 14 and 16 and Refs, 4, 5 and 6, Thus, SIL t r a c e r s t u d i e s are based u p o n t w o a s s u m p t i o n s w h i c h can be verified,

339 10 X . . . . . .

0---

WEEK

0

X

WEEK

4

--0

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1

X

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.7

-

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-

X

L

0

10

20

30

40

50

TIME AFTER PHENYTOIN INFUSION ( hours ) Figure 2. Serum concentration versus time relationships for 150 mg 13ClSN2-phenytoin i.v. tracer doses for patient on chronic phenytoin therapy before (Week 0) and 4 and 12 weeks after adding chronic carbamazepine therapy. From Browne et al. (2) with permission.

340 TABLE 1.

Phenytoin Pharmacokinetic Values Before (Week 0) and 4 and 12 Weeks After Adding Phenobarbtal in Six Subjects Before

Week 4

Week 12

Significance a

Mean steady state plasma concentration (l~g/mL)

12.2 b _+ 5.4

13.2 _+ 5.7

15.0 +_ 5.6

0.51

Clearance (mL/min/kg)

0.29 +_ 0.18

0.31 _+ 0.19

0.29 _+ 0.20

0.43 c

Elimination half-life (hr) (L/kg)

34.3 _+ 16.4

29.6 _+ 12.5

32.5 _+ 17.2

0.53

Volume of distribution

0.69 -+ 0.06

0.64 _+ 0.09

0.60 +_ 0.05

0.10

aBy analysis of variance. bMean _ SD. Probability of missing a truly significant change of 20 percent or greater (Type II or beta error)

asmple

COLUMN

2

6its CHROflHI~RflPH Figure 2. Continuous flow isotope ratio mass spectrometer equipped with a TCD gas chromatograph for the separation of gas mixtures and the dual inlet dynamic interface.

383 displays the scheme of the dual inlet dynamic interface which allows the online preparation of the CO= to be isotopically analyzed. Obviously, water must be prevented from entering the mass spectrometer because the partial protonation of CO2 induces the formation of CO2H§ ions, thus interfering with the isotopic measurement. After the changing over valve, CO= enters the ion source and ions at m/z 44, 45 and 46 are continuously recorded (8). For the measurement of the isotopes of carbon, various devices or analytical systems allowing the preparation and purification of the CO=, to be isotopically analyzed, can be connected to the sample line of the dual dynamic interface.These devices can be: (i) a gas chromatograph and a combustion oven; (ii) an elemental analyser; or (iii) a gas chromatograph for the separation of gaseous mixtures containing CO=. When CO2 is present in a gaseous mixture it is possible to directly couple a gas chromatograph to the dynamic interface. The components of the mixture of gases can be separated on a column packed with Hayesep Q Porapack and detected using a thermal conductivity detector. A switching valve allows only CO2 to enter the isotope ratio mass spectrometer for isotopic measurement. Amounts of CO2 as small as 10 nanomoles are sufficient to ensure an accurate and precise determination of the 13C/12C ratio (9, 10). Various kinds of samples can be of interest in the area of drug metabolism. Among them are breath samples from small animals or humans, collected after administration of 13C-labeled substrates, atmospheres of in vitro, tissues, cells or culture media. It is also possible to associate CO2 micro-generators that can generate micro-volumes of CO2 using either chemical or enzymatic reaction from substrates to be analyzed for their isotopic content. The resulting CO2 produced by the reaction is purified on line by gas chromatography and immediately measured for its 13C content. The statistical combination of the isotopes of carbon (12C, 13C) and oxygen (160, 170, 180) to generate the CO2 molecules gives rise to the formation of various isotopomers whose molecular weights are 44, 45 and 46, respectively. In order to obtain a high precision and accuracy, reference gases of known isotopic composition are used and the dual inlet system allows an alternative admission of both sample gas and reference gas into the ionization source via a gas-switching valve. The measurement of the various ion beams allows the calculation of the 13C enrichment of the sample: ~13C (%o). The 13C abundance is generally expressed as (~13C (%o) according to the following relation:

813C (%o)= ([(13C/12C)sample/(13C/l=C)PDB]- 1)x 1000

384 This delta 813C (%o) value measures the variations in part per thousand of the carbon isotope ratio from the standard. For carbon, PDB was selected as the international reference. PDB is the Pee Dee Belemnitella (a fossil from the Pee Dee geological formation of South Carolina). The ~3C/~2C ratio from the calcium carbonate of this fossil is 0.011237. So compared to PDB, most of the natural compounds display a negative delta value, which is also true for CO2 values. For isotopic CO2 measurements, micro-breath samples are injected into the chromatographic column. A switching valve placed beyond the thermoconductibility detector (TCD) is activated between t~ and t2 (Figure 3), so that the CO2 separated form the breath sample is entirely transferred to the sample transfer line. Pulses of reference CO2 are admitted into the reference transfer line at preprogrammed times so that the peak of CO2 to be measured is framed by pulses of reference gas.

3. 13C0z AND DRUG METABOLISM 3.1. Action on Cyt P450-Mediated Drug Metabolism Enzymes The liver is the main organ involved in metabolism, especially in the transformations of endogenous as well as exogenous substances. The diversity and complexity of liver functions make its examination very difficult. Many mechanisms of biotransformation are based on oxidative reactions carried out by the cytochrome P450-dependent monooxygenase system. The reactivity of these enzymes can be modified by many chemical compounds, xenobiotics and drugs which react as inducers or inhibitors. Breath tests using ~3C-labeled substrates are used for diagnosis purpose and allow measurement of the metabolic activity of these mono-oxygenase systems in order to explore the liver function. As these enzymes can be induced or inhibited by drugs or chemicals, breath or micro-breath tests can be used to measure the changes in enzyme activities which can modify drug metabolism. During preclinical studies, 13CO2-breath tests can be used on small animals in order to detect inducing or inhibitory activities of drugs or metabolites as well as xenobiotics. Breath tests can also be utilized to measure modifications of the liver oxydative metabolism induced by drugs, or to monitor a potential hepatotoxicity.

385

TCD 51GRILL 0.4. N2+O

mV

CO 2

a

TCB Chrom~ogrm o_

tl~ i

BI]flmqq 51GIlflL

~t2 " ' H I ~ f l R T C U T " T I m E

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Figure 3. Procedure and time sequences for a typical GC-IRMS run. (a) TCD trace for

column switching and selective transfer of purified C02 to the IRMS source. (b) Record of the traces corresponding to ions at m/z = 44, 45 and 46 from the sample and the reference C02.

3.1.1. Induction

Enzyme induction by drugs and other xenobiotic chemicals, was discovered initially by virtue of the profound effects that induction can have on many pharmacologic responses as reported by Conney (11). Over the past decades,

386

many more situations have been uncovered in which induction of drugmetabolizing enzymes has been shown to significantly alter pharmacologic or toxic responses in vivo in laboratory animals and humans (12). It is of importance to rapidly detect inducing activities because there are two primary practical concerns related to P450 induction. First, induction may alter the. efficacy of therapeutic agents. Second, induction may create an undesirable umbalance between rates of "toxification" or "detoxification" in organisms exposed to drug or environmental chemicals. Some drugs that act as cytochrome P450 inducers are also substrates for the enzymes that they induce, they may stimulate their own metabolism, enhance their clearance and, thereby, reduce the duration or intensity of their pharmacodynamic effect. Many xenobiotic chemicals, drugs and/or metabolites can stimulate the metabolism and clearance of unrelated compounds. In the cases of drugs with low therapeutic index, induction may severely compromise efficacy. Since liver is the major site of xenobiotic metabolism, it is also frequently the prime target for toxicity from reactive metabolites. Studies of P450 induction in humans are intrinsically more difficult than in laboratory animals, however, the continued development of various technical approaches has led to considerable progress in this area. In vivo probes, among them micro-breath tests using 13C02, can be used in laboratory animals as well as in humans. The rapid screening of an inductive activity can be carried out on small animals as described by Guilluy et al. (9). Rats or mice are housed in tight all-glass metabolic cages swept by a continuous flow of air in order to ensure physiologic breathing conditions. Expired gases can be collected, either automatically or manually, in glass vacutainer at various times after 13C-labeled substrate administration. Small samples of breath air are collected because only 250 ~1 are needed for the measurement of the 13C/~2C ratio of the expired C02, by CFIRMS. Various substrates can be used to study the activity of phenobarbitalinducible P450 or other isoforms of cytochrome P450. Moreover, when using caffeine as metabolic probe the various N-CH3 groups at positions 1, 3 and 7 can be labeled separately allowing one to obtain informations on various isoforms of cytochrome P450. Figure 4 shows the variation of the ~3C enrichment of the C02 expired by a rat after administration of 1,3,7 [(13CH3)3]-caffeine as substrate in a twosequence trial. The first sequence corresponds to the measurement of the basal metabolism of the rat. Sequence II was performed after cytochrome P450 induction carried out by a single i.p. (30 mg/kg) of 3-methyl-cholanthrene, 24hr before the second administration of labeled caffeine. It can be observed that immediately after [~3C]-caffeine administration, the 13C02

387

APE/O /-J~: 12 /

I CBBASAL

t

it~3MC

10 8

-2

J

Minutes 0

5

10

15

20

30

40

60

80

100 120 150 180

Figure 4. Kinetics of the expired 13002 by a rat after 1,3,7 [13C3]-caffeine administration. Basal kinetics and kinetic after one injection of 3 methylcholantrene (3 MC).

enrichment rises very rapidly up to +30.74 i~/ooPDB within 20 min. The maximum (~130po B difference versus basal value was +53.88(~%o, and was reached after only 30 min. This large change in C02 excretion clearly indicates the inductive effect of 3-methylcholanthrene on caffeine N-demethylase activity. Figure 5 shows a typical profile of the 13002 enrichment ((~130PDB versus time) from a rat after administration of [13C2]-aminopyrine (sequence I), and of the same substrate after a 6-day administration of phenobarbital (sequence II). In sequence II, immediately after [13C2]-aminopyrine injection, the 13002 enrichment of the expired air increases from -23.70(~%o (basal value) to --4.96(~130pDa, 20 min after administration. The maximum enrichment difference between sequence II (induced) and sequence I (noninduced)is +21.12 (~%o, 10 min after [13C2]-aminopyrine administration. Such an increase in the rate of excretion of 13002 derived from N-demethylation of isotopically labeled aminopyrine clearly indicates the inductive effect of phenobarbital. 3. 1.2. Inhibition

The activity of cytochrome P450 may be decreased by four main mechanisms. Thus, (i) a drug may compete with other substrates for reversibly binding to cytochrome P450; (ii) a reactive metabolite may covalently bind to a nitrogen

388

10

~v

i

C]BASAL

,,,

AN

5 ~ //1

_ m

m

m

~

mPHENO a

/ l

l

l

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lV -5 -10 -15 -20

m"

-25

Minutes

-30

-10

-5

3

10

16

22

30

45

60

75

90

120 150 180

Figure 5. Kinetics of 13CO2expired by a rat after [13C2]-aminopyrine before and after 6 days of treatment with phenobarbital (Pheno).

atom of the heme and destroy cytochrome P450 (13); (iii) reactive metabolites may also covalently bind to the apoprotein itself, with the formation of a hypoactive cytochrome P450; and (iv) either the drug itself, or one of its metabolites, may form complexes with the iron of cytochrome P450. Cimetidine, an H2-receptor antagonist, has the additional pharmacologic property of impairing hepatic microsomal drug oxidizing capacity (14). When co-administered with a number of drugs that are biotransformed by oxidative processes, cimetidine impairs metabolic clearance, leading to modified pharmacokinetic parameters: increased elimination half-life, increased area under plasma concentration versus time curve, and higher steady-state concentration during multiple dosing (15). This inhibition of drug oxidizing capacity is not an invariable component of the action of H2 antagonists, since ranitidine does not share this inhibition property. As a matter of fact, ranitidine does not impair the metabolism of several drugs including antipyrine, warfarin, propranolol, theophylline or phenytoin (16). Bell et al. (17), and Gerber et al. (18), have demonstrated that ranitidine does not inhibit the liver cytochrome P450 mixed function oxidase system because contrarily to cimetidine, ranitidine does not possess the imidazole ring thought to be the site of binding to cytochrome P450. Various studies of the pharmacokinetics of drugs associated with cimetidine and ranitidine have demonstrated inhibitory effect of cimetidine, whereas ranitidine had no effect.

389

APE/O C3BASAL

20

mCIMET IIDIFF

15 10

-5

Minutes

-10 0

5

10

15

20

30

40

50

60

70

80

Figure 6. Kinetics of 13CO2expired by a rat after [13C2]-aminopyrine in normal conditions (Basal) and 45 min after receiving a dose of cimetidine (Cimet). The difference between the two sequences corresponds to the (Diff) bars.

Micro-breath tests, using 13C-labeled aminopyrine and carried out on small laboratory animals, can provide fast and comprehensive information on these kinds of modifications of cytochrome P450. Figure 6 shows the kinetics of 13CO2 exhalation after [~3C]-aminopyrine intake in the basal conditions and the modified kinetics, when the same rat received cimetidine, 45 rain before aminopyrine administration. The figure also shows the difference between the two sequences expressed in atom percent excess versus the basal kinetics (APE/o). Figure 7 shows the results of the same trial carried out with ranitidine. The different behaviors of these two H= antagonists on the oxidizing activity is clearly shown using the rapid screening with ~3C-breath test on small animal. In 1991, Brady et al. (19) showed that hepatotoxicity of N-nitrosodimethylamine or CCI4 in rats was blocked by pretreatment with disulfiram. The authors demonstrated that cytochrome P450 II E1 was inhibited and inactived by disulfiram and/or its metabolites and that this mechanism could account for many of the reported inhibitory actions of disulfiram against chemically induced toxicity and carcinogenesis. P450 II El-dependent activities are decreased by treating animals with organic sulphur compounds such as diallyl sulfide, phenethyl isothiocyanide and dimethylsulfide. Disulfiram, a drug used

390

APE/O C3BASAL

25

mRANITIDINE INDIFFERENCE

20 15 10

Minutes

-5

0

5

10

15

20

30

40

50

60

70

80

Figure 7. Kinetics of 13C02 expired by a rat after [13C2]-aminopyrine in normal conditions (Basal) and 45 rain after ranitidine intake, There is no significant difference between the two sequences,

in avoidance therapy for alcohol abuse, and its metabolites, diethydihiocarbamate (DDTC) and CS2, exhibit cancer protective properties that resemble those described for diallyl sulfide. DDTC is rapidly formed from disulfiram in vivo. Disulfiram and its metabolites were tested by Brazier et al. (20) for their inhibitory effect using micro-[13C]-aminopyrine breath test (ABT) and [13C]caffeine breath test (CBT). With this technique, it is possible to design studies, in vivo, where the inhibitory substances to be tested can be administered at various time before administration of the ~3C-labeled probes in order to study where inhibition takes place and its duration. Figure 8 shows the inhibitory effect of a 164-mg/kg dose of DDTC administered 5 min only, before ~3C aminopyrine administration. A significant inhibitory effect of DDTC can be observed from this ABT result. As caffeine metabolism in rats gives rise to the production of ~3CO2 for a long time but with small ~3C enrichment, in normal conditions, CBT is not an ideal model to study cytochrome P450 inhibition. If rats are pretreated with 3MC, cytochrome P450 allows the metabolism to generate ~3CO2 giving a higher ~3C enrichment of the expired gas. Figure 9 shows the effect of a 164-mg/kg dose of DDTC administered only 5 minutes before [~3C]-caffeine intake in a rat pretreated by 3MC. The inhibi-

391

APE,'~,~DTC'164 mg/kg - 5 min before AB 40"

. . . . . . . . . . . . . . . . . . . . . .

E3BASAL mDTC Imlnhib

30 20 ~. 10

-10 -20 ~

Minutes 0

5

10

15

20

30

40

60

80

100 120 150 180

Figure 8. Kinetics of 13C02 exhaled by rats after [~3C2]-aminopyrine in normal conditions (Basal) and 5 rain after administration of a dose of 164 mg/kg of diethyldithiocarbamate. The (Inhib) bars shows the difference between the two sequences and the inhibition process.

APE/O

,o/

C3 Caf-3 MC mDDTC iDiff

20 J' ~

DDTCi!~-mg/kg" 5 min before CBT -40

,,//

...................................................................................

0

5

10

15

20

30

40

60

80

100 120 150 180min

Figure 9. Kinetics of ~3C2 expired by rats receiving [13C3] labeled caffeine after 3MC induction (Caf-3MC), by the same rats, 5 rain after a Na-diethylditihocarbamate administration of 164 mg/kg. The (Diff) bars clearly show the cytochrome P450 inhibition.

392

/CS2 30

........................................... mDIFF !

/

20 ~ 10 ~

-10

.,

1

-20 -30

3CS2: .4_ 5

10

mmol/Kg 2.5 H before ABT 15

20

30

40

60

80

100 120 150 180rain

Figure 10. Kinetics of 13CO2expired by rats after [13C2]-aminopyrine administration in normal conditions (Basal) and 2.5 hr after CS2 administration (3.4 mmol/kg). The (Diff) bars show the inhibitory effects of CS2 on cytochrome P450-dependent monooxygenases.

tory effect is extremely rapid and intense and the 13CO2 level is quite identical to that observed in a noninduced rat. These two examples clearly show that both ABT and CBT on small animals allow one to detect easily the inhibitory effect of DDTC on cytochrome P450. The same results can be obtained with the degradation product of DDTC, CS2. Figure 10 show its inhibitory effect on the ABT. It can be observed that 2.5 hr after CS2 administration, inhibition still persists in rats. Figure 11 presents the effect of a CS2 dose administered 20 min after [13C]caffeine intake in 3MC-pretreated rats. The ~3C enrichment is maximum in these rats. When CS2 is administered, the ~3C enrichment immediately falls down and reaches the basal level within 1 hr, whereas in rats not receiving CS2 this ~3C enrichment level is not reached after 3 hr. These results were obtained in vivo with each animal being its own standard, and without killing animals before enzyme activity measurement. These results are in total agreement with those previously obtained, in vitro, on microsomes. The inhibition of cytochrome P450 by disulfiram, DDTC and CS2 can be reflected well by the results of ABT and CBT which can be extended to many other drugs or xenobiotics.

393 CS2

APE/O/ mCS2 ................................ C3Induct 3MC

30 20 10

0

5

10

15

20

30

40

60

80

100 120 150 180

min

Figure 11. Kinetics of 13C02exhaled by rats receiving [13C3]-Iabeled caffeine after 3MC induction (induc- 3MC). Effect of 3.4 mmol/kg of CS2 administration, 20 min after labeled caffeine intake in rats induced by 3MC.

The previous examples on induction and inhibition of cytochrome P450 dependent drug metabolizing activities show that these noninvasive methods are reliable and have a number of methodological advantages. They do not raise the ethical problems caused by the sacrifice of animals (the administration of the test-labeled compounds is not considered as an invasive procedure). They permit the recurring use of the same animal, and the use of each animal as its own control; this drastically reducing the number of animals. Finally, they reflects the overall rate of metabolism.

3.2. Hepatotoxicity Breath tests can be used to evaluate in vivo the hepatotoxicity of drugs or chemicals. Mion et al. (21) conducted a study to evaluate in vivo the hepatotoxic effects of CCI4 administration to rats using breath tests. Carbon tetrachloride (CCI4) hepatotoxicity is mainly due to the formation of reactive metabolites through the cytochrome P450 system. In vitro incubation of liver microsomes in the presence of CCI4 produces a "suicidal" destruction of CCI4 bioactivating mechanism as a result of cytochrome P450 inhibition. In vivo,

394 a small dose of CCI4 decreases the toxicity of a second lethal CCI4dose: the first dose inhibits cytochrome P450, leading to a decreased bioactivation of the second dose. Mion's study was designed to evaluate in vivo the CCI4 autoprotection phenomenon and its temporal relationships with cytochrome P450 inhibition, based on 13C-breath tests: these noninvasive tests allow repeated enzymatic measurements on the same animal, each rat being its own control. Cytochrome P450 activities were measured by the [~3C]-aminopyrine breath test. The metabolic liver function was assessed by the [~3C]-galactose breath test. This test explores the hepatic clearance of galactose, of which the rate limiting step is the galactose kinase. This enzymatic process is not involved in CCI4 bioactivation, and a decrease of the test should reflect the CCI4-induced decrease of liver function. Some transaminase activities were measured in order to assess the severity of CCI4-induced liver damage. The effects of a single intra-gastric administration of CCI4 are an almost complete inhibition of N-demethylation on day 1, as assessed by the ABT (Aminopyrine Breath-Test). A slight, but significant, recovery was detectable on day 3. Seven days later, results of the ABT were identical to day 0. The galactose breath test (GBT) values also decreased significantly on day 3 and were not significantly different from day 0, or day 7 (Figure 12). A second CCI4 administration was performed either 3 or 7 days after the first one, to evaluate in vivo the duration of the CCI4 "autoprotection" duration. When CCI4 was given on day 3, the cytolytic effect was much less pronounced and the GBT values were not decreased. On the contrary, when administrated 7 days after the first CCI4 intake, the decrease of the GBT value was identical to the one induced by the first dose (Figure 13). These results clearly indicate an inhibition of aminopyrine N-demethylase lasting at least 3 days after CCI4 administration and a complete recovery 7 days later. These results are in accordance with in vitro microsomal studies carried out by Glend (22). This example demonstrate the facility and usefulness of noninvasive 13C-breath test to study in vivo, the evaluation of enzymatic activities. It is well known that methotrexate (MTX) exhibits hepatotoxicity during treatment (23). In order to assess the damage induced by this hepatotoxicity, liver biopsies regularly taken during MTX therapy have been proposed by Philips et al. (24). Guitton et al. (25) tried to use the ABT to detect the effects of MTX on P450-dependent mono-oxygenases during treatment and developed an animal model. In this study, three groups of rats were used: a control group, a group receiving a single dose 70 mg/kg of MTX by intraperitoneal route and a third group treated orally with 1 mg/kg of MTX every 2 days. ABT were performed throughout the study to monitor the metabolic activity. At

395 CCI4

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421 have been reported which generate information similar to that obtained with 14C-labeled drug and scintillation counting (see Chapters 6 and 11). The SIL/IRMS methods appear feasible for studies of low and medium (but not high) potency drugs. The overall cost of a mass balance/metabolite identification study performed with SlL- or 14C-labeling is approximately equal (see Table 2). However the SlL/IRMS method offers significant advantages: (1) the SlL/IRMS methods eliminates the five problems associated with ~4C-labeled studies listed above; and (2) the SIL/IRMS study can be combined with the singledose volunteer study, with a modest reduction in the overall cost of the two studies (see below).

4. SINGLE-DOSE VOLUNTEER AND SINGLE-DOSE PATIENT STUDIES

Single-dose studies typically collect information on the pharmacokinetics, safety and dose ranging of a new drug after administration of a single dose. A modest number of volunteers (e.g. 20) are studied to obtain information on dose ranging. Single-dose studies can be performed using SIL drug and MS detection (see Chapters 13, 16 and 18). However, this methodology usually is more expensive than standard methods (e.g. unlabeled drug, high performance liquid chromatography (HPLC) with ultra violet (UV) detection) because of the added costs of isotope synthesis and MS analytic methodology (Table 2). There are instances where quantitation of SIL-labeled drug with MS is more practical than quantitation of nonlabeled drug with conventional techniques, especially when inexpensive "table-top" mass spectrometers can be employed. Examples include high potency (i.e. low plasma concentration) drugs and drugs with low response to ultraviolet detection.

5. COMBINED MASS BALANCE/METABOLITE IDENTIFICATION AND SINGLE-DOSE VOLUNTEER STUDIES

Both mass balance/metabolite identification and single-dose volunteer studies involve administering a single dose of drug to healthy volunteers, collecting blood and urine for pharmacokinetic studies, and collecting safety data. The blood and urine specimens collected in a mass balance/metabolite identification study performed with ~4C-labeled drug cannot be used for later pharmacokinetic analysis because of problems with radioactive decay of sample, storage of radioactive specimens and radioactive contamination of analytic

422 instruments (e.g. HPLC) used to measure the concentration of drug in blood and urine for pharmacokinetic calculations. SIL-labeled drug is stable indefinitely in blood and urine samples and contains no radioactivity. Studies performed with single doses of SlL-labeled drug can have the SIL label "counted" by IRMS studies for mass balance determinations, and the blood and urine can then be stored for later analysis by HPLC or other methods for pharmacokinetic computations. Thus, the same subject and specimens can be used for both studies. Such a combination would result in a modest reduction in cost, time and number of volunteers (see Tables 1 and 2).

6. MULTIPLE-DOSE VOLUNTEER STUDY

Such studies are performed to determine the type of pharmacokinetic properties (linear, concentration-, or time-dependent) and the pharmacokinetic values (clearance, half-life dependent, volume of distribution) a new drug has during chronic administration and to obtain information on safety during long term administration. The standard method for performing such a study would be: (1) administer a single dose of drug and determine single-dose pharmacokinetic parameters from plasma concentration versus time relationships; (2) administer the drug chronically for a period of time; (3) stop the drug; (4) redetermine pharmacokinetic parameters from plasma concentration versus time relationships during the terminal "wash out" of drug; and (5) compare single dose and terminal pharmacokinetic values. A more satisfactory SIL method has been reported in which steps 3 to 5 are varied as follows: (3) administer a SlL-tracer dose of drug while the volunteer continues to take the drug; (4) determine tracer dose pharmacokinetic properties from tracer dose plasma and urine concentration versus time relationships; and (5) discontinue drug administration when tracer study is complete (see Chapter 16). This SIL technique will accurately determine the type of pharmacokinetic properties and the pharmacokinetic values (including Kr~ and Vva• for drugs with concentration-dependent pharmacokinetic properties). This SIL technique is superior to studying terminal plasma concentration versus time relationship because the SlL method measures true steady state pharmacokinetic values. Measurement of plasma concentration versus time relationships after stopping drug (i.e. when plasma concentration is constantly falling) does not permit direct determination of pharmacokinetic values for drugs with concentration-dependent pharmacokinetics. There are no economic benefits to utilizing SIL techniques to perform only

423 a multiple-dose volunteer study. Indeed, the SIL method is more costly because of the added costs of isotope synthesis and MS analysis (see Tables 1 and 2). The advantages of the SIL method are: (1) detailed and accurate information on changes in pharmacokinetic parameters during chronic administration for and drugs with concentration-dependent or time-dependent pharmacokinetics at steady state plasma concentration; and (2) improved statistical power to exclude concentration-dependent or time-dependent changes in drugs without such changes (see Chapter 16). Moreover, economic benefits can be obtained by combining a SIL multiple-dose volunteer study with other SIL studies (see below).

7. ABSOLUTE OR RELATIVE BIOAVAILABILITY/BIOEQUIVALENCE STUDIES

The classic technique for performing bioavailability/bioequivalence studies is a cross-over technique in which volunteers receive a dose of drug using the marketed preparation during one test session and a reference dose of drug as an i.v. infusion (absolute bioavailability), or an oral solution (relative bioavailability) during a second test session. Plasma concentration versus time relationships after each test dose of drug are used to compute area under the plasma concentration versus time curve (AUC) values. The ratio of AUC for marketed drug/AUC for i.v. solution (or oral solution) is a measure of absolute (or relative) bioavailability. Typically, this method requires study of approximately 24 volunteers in order to obtain adequate statistical power to demonstrate equivalence (i.e. adequate statistical power to detect a truly significant difference of 0.20 with a power of at least 0.80). The principal determinant of the number of subjects need is intraindividual variability in absorption and elimination between test sessions. Such variability produces random variation in AUC values, reduces statistical power, and increases the number of subjects required. An attractive alternative SIL methodology is available for performing bioavailability/bioequivalence studies. The marketed drug preparation is administered simultaneously with SIL reference preparation (i.v. solution or oral solution), and AUC values for each formulation are determined from plasma concentration versus time relationships for labeled and unlabeled drugs. This technique eliminates intraindividual variability and greatly improves statistical power. For a typical drug, 24 subjects are required to demonstrate equivalence of two preparations with adequate statistical power with the cross-over technique and only eight or less subjects with the SIL technique. Furthermore,

424 the subjects in an SIL study receive the test drug only once, reducing risk, hospital days and analytic specimens. See Wolen (4) and Chapter 13 for details on the methodologic and statistical aspects of SlL bioavailability/bioequivalence studies. The above considerations apply to "typical" drugs. Drugs with first pass metabolism have greater intraindividual variability in the AUC values obtained with the same formulation administered at different times than drugs without first pass metabolism. This increases the number of subjects necessary to obtain adequate statistical power. SlL techniques obviate these differences by simultaneous administration of both formulations and dramatically reduce the number of subjects needed to show equivalence with adequate statistical power. Drugs with saturatable first pass metabolism, nonlinear pharmacokinetics, prodrugs, and enantomers all present special problems for bioavailability/bioequivalence studies which can be solved using SIL methods (see Chapter 13).

8. COMBINED MULTIPLE-DOSE VOLUNTEER AND ABSOLUTE BIOAVAILABLITY STUDIES

Both of these types of study involve administering test drug to volunteers, collecting blood and urine for pharmacokinetic studies, and making safety observations (see Tables 1 and 2). By administering an SIL reference dose of drug (i.v. or oral solution) with the first chronic dose of drug (marketed drug preparation), it would be possible to determine simultaneously the absolute or relative bioavailability of the drug as well as the drug's single-dose pharmacokinetic parameters. This combined procedure would result in a dramatic reduction in subjects, hospital days, analytic specimens and cost (see Tables 1 and 2).

9. MULTIPLE-DOSE PATIENT STUDY

The objectives of this study are to obtain data on" (1) pharmacokinetic properties of the new drug during chronic administration (i.e. determine if a new drug has linear, concentration-dependent, or time-dependent pharmacokinetic properties); (2) drug interactions of new drug with medications with which the new drug will be co-administered (e.g. interactions of a new antiepileptic

425 drug with phenytoin or carbamazepine); and (3) preliminary data on safety and efficacy of the new drug. The standard methodology for obtaining multiple-dose pharmacokinetic data in patients is similar to the methodology employed in multiple-dose volunteer studies (see above). Usually 12 to 24 subjects are studied. Drug interactions of a new drug with an old drug usually are determined by measuring trough plasma concentration of old drug before and after adding new drug. Drug interactions of old drug with new drug usually are determined by administering a single dose of new drug to patients with a therapeutic plasma concentration of old drug and determining pharmacokinetic parameters of the new drug from plasma concentration versus time relationships. Pharmacokinetic parameters of new drug in the presence of old drug are compared with literature values of pharmacokinetic parameters of the new drug when taken alone. These two methods of studying drug interactions are highly suspect because they are dependent on multiple assumptions which often are untrue, almost never verified, and introduce variance into results. These multiple assumptions and their consequences have been reviewed in detail elsewhere (5). Drug interaction studies utilizing older methods typically involve 12 to 24 patients. Because of the multiple sources of variance in traditional drug interaction study methods, such methods lack statistical power. In particular, it is not uncommon for a study of 12, or even 24, subjects to lack adequate statistical power to prove the absence of difference in pharmacokinetic parameters of one drug in the presence of the other (i.e. power to detect a truly significant difference of 0.20 with a power of 0.80). Thus, the traditional methods employed on a multiple-dose patient study require 24 or more subjects and may yield suspect results. SIL tracer methods exist for determining presence or absence of pharmacokinetic changes during chronic administration (i.e. determining if a drug has linear, concentration-dependent, or time-dependent pharmacokinetic properties), including differential effects for enantiomers (see Chapter 16) and for determining presence or absence of pharmacokinetic drug interactions (see Chapter 18). These consist of administering a SIL-tracer dose of new drug before and during chronic administration and administering a tracer dose of old drug before and during the chronic administration of new drugs. These methods eliminate intraindividual variability and many other sources of variability (see Browne, et al. (5) for detailed discussion). There is increased statistical power because of reduced variance. Indeed the presence or absence of pharmacokinetic changes of new drug during chronic administration or of old drug after adding new drug can often be demonstrated using eight or

426 fewer subjects with 0.80 power to detect a truly significant difference of 0.20 (6, 7). Thus, two separate SlL studies to determine new drug pharmacokinetic properties during chronic administration and drug interactions between a new drug and an older drug would usually require 16 subjects. The cost of two SIL studies with eight subjects each would be approximately the same as studying 24 subjects with the traditional techniques (see Tables 1 and 2). The savings in subjects with SIL techniques would be offset by costs for SlL drug and MS determinations. In Chapter 7 it is suggested that deuterated analogs of many drugs can be separated from non-labeled drug by HPLC and quantitated by UV detection, an inexpensive and readily available technique. New antiepileptic drugs must be tested for drug interactions with the standard older antiepileptic drugs, carbamazepine and phenytoin. We have shown that deuterated analogs of carbamazepine and phenytoin can be synthesized, separated from nonlabeled analogs by HPLC, and quantitated by HPLC/UV (8, 9). It has been shown the deuterated analogs of carbamazepine and phenytoin do not exhibit a metabolic isotope effect in man (8, 10). This method was developed as a standard, inexpensive method of performing drug interaction studies of the effect of new antiepileptic drugs on older standard drugs. Other such deuterium/HPLC/UV techniques probably will be developed to study the interactions of new drugs with currently administered older drugs. It is possible to combine SlL studies of new drug pharmacokinetics during chronic administration and pharmacokinetic drug interactions of new and old drugs into one study utilizing eight subjects. Simultaneous administration of SlL-tracer doses of new and old drugs would be performed before and during chronic administration of new drugs to subjects continuously taking old drugs. Pharmacokinetic values obtained before and during chronic administration of new drug would determine if the new drug has time-dependent pharmacokinetic changes. Pharmacokinetic values of new drug obtained at low and therapeutic plasma concentration of new drug would determine if the new drug has concentration-dependent pharmacokinetic properties. Pharmacokinetic values of new drug in the presence of old drug would provide information on drug interactions of old drug with new drug when compared with pharmacokinetic values of new drug administered alone. Pharmacokinetic values of old drug obtained before and during chronic administration of new drug would determine presence or absence of drug interaction of new drug with old drug. The reduction in subjects from 24 subjects with traditional methods to eight subjects with combined SIL methods would reduce total costs for the study by more than 50 percent (see Tables 1 and 2).

427 10. MAXIMALLY EFFICIENT UTILIZATION OF SIL METHODS

Let us now investigate the possibilities of making maximal and optimal use of SIL and combined SlL techniques throughout a Phase I drug development program. Note first that the fixed development costs associated with utilization of SIL techniques (isotope synthesis and formulation, MS method development, regulatory approval) would be spread over several studies. The logic of this optimized SIL Phase I program can be followed by referring to Table 1. The six volunteers used in the SIL mass balance/metabolite identification study will yield excellent single-dose volunteer data. This will reduce the number of subjects in studies #1 and #2 of Table 1 from 26 to 20 and will reduce the cost by $80,000. SIL methods reduce the cost of the absolute bioavailability study alone from $960,000 to $222,000, a savings of $738,000. Combining the absolute bioavailability study with the multiple-dose volunteer study will reduce the total number of subjects required from 44 to 20 and will reduce the total cost by $860,000. Using traditional methods, obtaining information on multiple-dose patient pharmacokinetics and multiple-dose patient drug interactions would require at least 24 subjects compared with eight subjects using the combined SIL study method described above. Thus, the stable isotope method reduces multiple-dose patient subjects by 16 and costs by $1,000,000 (see Table 1). The combined savings over Phase I from combining studies and using stable isotope methods is 46 subjects and almost $2,000,000 (see Table 1). The time savings realized by eliminating 46 early Phase I study patients will vary from 12 to 24 months depending on company logistics. It needs to be emphasized that the data obtained using the proposed stable isotope combination methods are not "short cut" or second rate. In fact, the data obtained is equal or superior to data obtained by standard methods for several reasons. First, the bioavailability, pharmacokinetic, and drug interaction data obtained with the proposed stable isotope methods have been shown to have statistical power which is adequate for FDA purposes and is often superior to standard methods (see Chapters 11, 13, 16 and 18). This assures that the more long-term and expensive late Phase I and Phase II studies (safety, dose ranging, efficacy) will be carried out optimally. Second, the early availability of mass balance/metabolite identification data can prevent major problems discussed above. Third, the number of subjects required for safety and dose ranging studies (Studies 2, 3 and 5 in Table 1) is not reduced. To date, the combined SIL methods described in this paper have not been

428 utilized in drug development. However, the combined SlL methods represent a straightforward combination of published single study methods. We currently are performing a demonstration project to demonstrate the SlL combination studies described in this chapter. We hope others will utilize these combined SlL methods in the near future.

ACKNOWLEDGMENT Supported by the United States Department of Veterans Affairs.

REFERENCES 1. T.R. Browne, Clin. Pharmacokinet., 18 (1990) 423. 2. R. L. Wolen and W.A. Garland, in Synthesis and Applications of Isotopically Labeled Compounds, T.A. Baille and J.R. Jones (eds) (Elsevier, Amsterdam, 1989) p. 147. 3. T.R. Browne, in New Antiepileptic Drug Development: Preclinical and Clinical Aspects, J.A. French, M.A. Dichter and I.E. Leppik (eds) (Elsevier, Amsterdam, 1993) p. 31. 4. R.L. Wolen, J. Clin. Pharmacol., 26 (1986) 419. 5. T.R. Browne et al., in Antiepileptic Drug Interactions, W.H. Pitlick (ed) (Demos, New York, 1989) p. 1. 6. T.R. Browne et al., Neurology, 38 (1988) 639. 7. T.R. Browne et al., Neurology, 38 (1988) 1146. 8. G.K. Szabo et al., J. Chromotogr., 535 (1990) 271. 9. G.K. Szabo et al., J. Clin. Pharmacol., 34 (1994) 242. 10. T. R.Browne et al., Neurology, 44 (1994) 2410.

429

SUBJECT INDEX

Absolute bioavailability 246, 248, 250, 354, 417, 418, 420, 423, 427 Absorption 243, 309, 341,369, 419, 423 Absorption half-life 337 ABT 392 Accelerating voltage 86 Acceptance specifications 414 Accumulation 160 Accuracy 220, 221,226, 228 Acetaminophen 160, 223, 224, 225, 226, 227, 228, 231,231,374, 405, 407 Acetic anhydride-d6 234 Acetoacetate 148 Acetylcholine 158 AcetyI-CoA 158 Acetylcysteinyl 160 Acid hydrolysis 408 Adenosine triphosphate (ATP) 142, 147 Administration technique 261 ADP 147 Adsorption losses 93 Advantages of SlL tracer methods 312, 344 Advantages of the CF-IRMS method 228 AED 139, 195, 198, 200 AED parameters 186 AES 200 Albumen 8 Alcoholic liver disease 375 Aldehyde dehydrogenase inhibition 59 Aliphatic 14 Aliphatic hydroxylation 289 Alkali flame thermoionic detector 172 alI-E retinylidene 211 Alzheimer's disease 146, 149 Amine radical cation 290

Amino acid catabolism 94 Amino acid residues 281 Amino acid sequence 32 Amino acid 28, 97, 95,111 ~/-aminobutyric acid 142 Aminopyrine 349, 362, 373, 387, 391, 397, 401 Aminopyrine breath test 375, 390, 394 Aminopyrine N-demethylation 374 Ammonia 29, 30, 224, 407 Ammonium acetate 27, 33, 54 Ammonium carbonate 224, 225, 225 Ammonium hippurate 407 AMP 147 Amphiphilic 58 Analyte thermal stability required 69 Analyte 28, 36 Analyte-containing solvent 58, 63 Anaplerotic Flux 157 Anesthetics 161 Aniline 279 Anionic cationic functional groups 65 Anisotropy 206 Anthropods 203 Anticonvulsant drugs 99 Antiepileptic drug 128, 157, 158, 430 Antiepileptic properties 134 Anti-inflamatory drug 182 Antimicrobial agents 363 Antineoplastic agent 100 Antipyrine 407 Antipyrine clearance 373 Antitumor reactions of neocarzinostatin 104 APCI 26, 31, 50, 65, 66 APE 227 APEXTC 226 APEXTN 223, 225, 227 API 27, 38, 39, 65

430 Approval of a drug or device for marketing 415 Ar collision gas 94, 96 Arachidonic acid 108, 110 Area under the plasma concentration versus time curve (AUC) 198, 247, 264, 298, 310, 423 Argon 1, 32 Argon plasma 179 Aromatic 63 Aromaticity 40 Aromatic nitro groups 31 Array detector 21 Arteriovenous difference in drug plasma concentration 270 Aspartate 154 Assessment of exocrine pancreatic function 369 Assessment of fat malabsorption 371 Assessment of organ functions 367 Assessment of pancreatic insufficiency 369 Assessment of steatorrhea 371 Association of official analytical chemists 409 Assumptions in using CF-IRMS for MB/MI studies 220 Assumptions of SlL tracer methods 310 Atmospheric bands 2 Atmospheric pressure chemical ionization (APCI) 34, 36, 37, 48, 65 Atmospheric pressure interfaces 23, 25 Atmospheric pressure ion spray interfaces 27 Atmospheric pressure ionization (API) 20, 33, 61,239 Atmospheric pressure ionization interfaces (API) 50 Atmospheric pressure ionization-mass spectrometry (API/MS) 233 Atom percent excess (APE) 121 Atomic absorption (AAS) 177 Atomic emission line 184 Atomic emission spectroscopy (AES) 169, 170, 171,176, 179, 184, 189, 193, 201

Atomic fluorescence (AFS) 177 Atomic mass 2, 65 Atomic numbers 1 Atomic spectroscopy detection 177 Atomic table 2 Atomic weight 177 ATP hydrolysis 148 AUC 198, 424 Auto-induction 352 Aziridinium ion 101 B/E linked-scan 84, 85, 97, 103 Bacterial fixation 5 Bacterial overgrowth 365 Bacteriorhodopsin protein 203 Basal 390 Basal kinetics 387 Bathorhodopsin 203, 205 BE 76, 82, 87 BEB 79 BEEB 79, 80, 85 Beenakker type resonant cavity 179 Bell-shaped curve 289 Benoxaprofen 248 Benzoic acid 241,400, 41 Benzoxylcarbonyl methyl esters 100 Benzylic hydroxylation 235, 290 Benzylic radical 289 1-benzymidazole 397 BEqQ mass spectrometers 82 BEqQ 76, 85, 86 Bile acids 59 Bile 100 Bio-analytes 23 Bio-analytical chemistry 44 Bio-analytical problems 40 BioAnalytical systems 132 Bioavailability 9, 309, 318, 319, 354, 424 Bioavailability studies 195, 196, 245 Bioavailability/bioequivalency studies 250, 252, 257 Bioequivalence 243, 250, 252, 257, 354 Biofluid 160 Biologic fluid 193 Biological compounds 100, 112

431 Biological effects 171 Biological fluids 189 Biological macromolecules 38 Biological mass spectrometry 44 Biological materials 177 Biological matrices 100, 199, 229 Biological molecules 170 Biological polymers 100 Biological samples 108, 178 Biomedical analysis 187 Biomedical mass spectrometry 19 Biomolecules 32, 35, 64 Biopolymeric substances 44 Biopolymers 49, 62, 64, 81 Biotransformation 9, 275, 297, 315, 348, 353, 384 Blood 96, 220, 222 Blood (deproteinized) 222 Blood glucose concentration 149 Blood samples 99 Blood-brain barrier 315, 324, 332 Blood-cerebrospinal fluid barrier 324 Blood-cerebrospinal fluid transfer 329 BN50727 55 Boltzman distribution 143 Bolus infusion methodology 327 Bond angle 15 Bond length 15 Boron 1 Bovine retina 203 Bovine rhodopsin 203 Brain energy metabolism 147 Brain metabolites 153 Brain pathology 145 Brain slices 157 Brain tissue 97 Brainstem 98 Branched reaction pathway 286 Breath test 307, 361,363, 379, 380, 401,416 Bromine 55 BSA 234 BSP clearance 374 BSTFA 234 BSTFA-d18 234 Buspirone 124 Butylated carnitine levels 94

[13C 02] 121 CmD 14 D20 3, 6 12C 13, 319 12CO 173, 183 13C 2, 4, 6, 7, 9, 13, 15, 17, 41,104, 119, 121,123, 124, 130, 141,143, 170, 171,172, 174, 176, 178, 182, 188, 189, 191,194, 195, 199, 319 13CO 183,190 13CO2 6, 9, 123, 173, 176, 363 14C 419, 421 14N 13 14N/14N 121 15N 4, 7, 13, 15, 17, 41,119, 121,123, 130, 141, 170, 178, 183, 185, 199, 319 15N/14N 121 15N/15N 121 180 5, 7, 41, 170, 178 19F 141, 143 31p 141, 142, 143 34S 42 35CI 104 37CI 42, 104 81Br 42 1H 141, 143 2H 119, 178, 199 2H 7, 41 12C-13C intensities 172 13C atomic emission detection 194 13C bands 173 13C chromatogram 191, 192, 193 13C glucose 153 13C hiolein 373 13C isobutyl methyl xanthine 191,192 13C isotope 64, 206 13C isotopomer-based NMR method 159 13C labeled metabolite 145 13C labeling 7, 148, 151,173, 180, 185, 236, 380 13C MAS NMR 216 ~3C mass spectrometer 5 ~3C NMR spectra 155, 159 13C NMR 149, 151, 153 13C octanoic acid 367 ~3C signal 195

432 ~3C spectra 156 13C spectrum 145 ~3C subtraction 195 13C triglycerides 369 13C xylose 366 13C 150, 154, 155, 159, 169, 185 ~3C/12C isotope ratio 401 13C/12C ratio 199, 383 13C-CO2 enrichment 349 ~3C2-acetaminophen 226 ~3C6-acetaminophen 228 ~3C6-1evodopa 229 13C-Aminopyrine Breath Test 374 ~3Carbon 236 13C-breath tests 361 ~3C-containing molecules 7 13C-edited 150 ~3C-editing 153 ~3C-glycine 5 ~3CH3 isobutylmethylxanthine 195 ~3CH3 methyl ester 182 ~3C-labeled acetate 367 ~3C-labeled bicarbonate 367 13C-labeled caffeine molecules 196, 197 13C-labeled compounds 173 ~3C-labeled hiolein 372 13C-labeled isotopes 152 13C-labeled metabolites 153 13C-labeled mixed triglyceride breath test 369 13C-labeled polyunsaturated fatty acids 159 ~3C-labeled progesterone 200 ~3C-methane 5 13C-NAPA 246 13C-NMR 240, 241 13CO2 excretion 370, 372 ~3CO2 recovery rate 373 13CO2 151,384, 398 ~3C-octanoate 369 ~3C-octanoic acid breath test 367, 3 6 8 , 369 ~3C-octanoic breath test 365, 367, 368, ~3C-urea 363 ~3C-xylose breath test 366

14C bile acid test 366 14C trioctanoin 371 14C tripalmitin 371 ~"C-breath tests 362 14C-labeled triglycerides 371 35CI 103 l"C-xylose breath test 366 2-[~3C] caffeine 176 C/D/N Isotopes Inc. 406 C13H120~ 79 C15H~-o 79 C~e reversed phase columns 138 C-18 cartridge 59 C3-1abeled pyruvate 158 Ca=+ 162 [1,2,3-~3C3]acrylic acid 241 [1,2-13C]acetate 155 1,2,3[carboxyl ~3C] octanoyl glycerol 370 1,3,7-~3C trimethyl-xanthine 349 Caffeine 175, 180, 183, 184, 185, 186, 187, 188, 189, 190, 191,192, 195, 197 198, 348, 349, 401,307 Caffeine breath test (CBT) 390 Caffeine isotopomer 184, 188 Caffeine metabolites 189 Calcium 266 Calculation of the residual standard deviation 182 Cambridge Isotope Laboratories 321, 406 Capacity factor 135 Capillary column 179 Capillary electrophoresis 20, 57, 64, 68 Capillary gas chromatography 169, 187 Capillary glass GC columns 129 Capillary isotachophoresis 64 Capillary liquid chromatography 68 Capillary tube 25 Capillary 25, 28 Carbamazepine 98, 128, 130, 131, 132, 134, 136, 139, 262, 308, 339, 340, 345, 352, 407, 425, 426 Carbamazepine clearance 307 Carbamazepine-epoxide 98

433 Carbaryl 40 Carbidopa 317, 318, 334 Carbohydrate metabolism 148 Carbohydrates 38 Carbon dioxide 381,399 Carbon isotope 172, 175, 185, 189, 191 Carbon radical 287 Carbon 407, 55 Carbon-deuterium bonds 135 Carbon-based radical 290 Carbon-hydrogen bond cleavage reaction 275, 287 Carbon-hydrogen bond oxidation 285 Carboxy terminal group 109 Carboxyl oxygen 110 Carboxyl-labeled substrates 373 Cardiac arrhythmia 316, 317, 334 Carnitine 95 Carnitine deficiency 94 Carnitines in plasma 94 Carnitines in urine 94 Catalog cost for stable isotope-labeled analogues of common drugs 407 Catalytic hydrogenation 244 Cationic functional groups 65 CBT 392 CBZ 99 CBZ/deutero-analogue 132 CE-MS 35 Cellular Ca2+ 163 Cellular phospholipid synthesis 147 Center for devices and radiological health 415 Center for drug evaluation and research 415 Central dopamine metabolism 320 Central levodopa metabolism 319 Central levodopa/dopamine metabolism 319 Central nervous system 315, 317 Cerebellum 98 Cerebral lipid metabolism 159 Cerebral metabolism 146 Cerebrospinal fluid 262, 263, 324 Cerebrospinal fluid homovanillic acid levels 328

Certificate of analysis 414 Cesium 58 CF/FAB 26, 33, 49, 50, 58 CF/FAB interfaces 48, 57 CF-GC-IRMS (continuous flow gas chromatography mass spectrometry) 199 CF-IRMS 122, 220, 221,222, 228, 229, 230, 232, 386, 398, 399, 400 CFR 412 [1-13C]glucose 151, 153, 154, 156, 157 [2-13C]glucose 151 [6-~3C]glucose 151 [1,2-13C]glucose 155 Charge delocalization 214 Charge dispersal mechanism 63 Charge transfer 66 Charged analyte ions 35 Charged analyte molecule or parent ion 20, 21 Charged ions 58 Charged molecules 19 Charged radical-cation 29 Charge-exchange 26 Chemical and isotopic purity 409 Chemical Ionization (CI) 20, 25, 29, 37 Chemical ionization processes 60 Chemical purity 409 Chemical reaction interface mass spectrometry (CRIMS) 52, 119, 122, 220, 230 Chemical shift 145, 147, 207, 240 Chemotherapeutic drugs 162 3-13CH3 labeled caffeine 192 Chloramphenicol 55 Chlorine 1, 55 Cholestatic liver disease 374 Cholesterol 4 Cholesteryl octanoate 362 Cholesteryl octanoate breath test 371 Cholesteryl-[1-~3C] octanoate 370 Chromatographic efficiency 58 Chromatographic isotopic separation 128 Chromatography 177, 178, 200, 221 Chromophore 203, 208, 211,215 Ch ro mopho re-protei n interactions 208

434 Chronic active hepatitis 374 Chronic alcoholism 97 11-cis retinylidene 211 CI mass spectra 49 CI 31, 38, 52, 54 Cibenzoline 309 CID 76, 82, 86, 87, 88, 89, 98, 99 Cimetidine 388 Cirrhosis 374 CK activity 148 CK expression 148 CK flux 148 CK iso-enzyme 148 CK kinetics 148 CL 297, 298, 311,342, 343, 345 [1-13C]-Iabeled glucose 149 Clearance via production 313, 341 Clearance 264, 267, 268, 269, 270, 271,272, 273, 297, 298, 300, 301, 305, 306, 308, 310, 337, 340, 345, 422 Clinical gastroenterology 373 Clinical studies 323 [13C6]-L-kynurenine 55 Clobazam 234 Cluster ion formation 35 [~3C6]-L-tryptophan 55 Cmax (mg/I) 198 CO2 breath test (CBT) 348, 349, 350, 351 CO2 excretion rate 351 CO2 380 Coaxial delivery system 58 Code of federal regulations 412 Coefficient of regression 197 Coefficient of variation 221 Coefficients 196 Collision region 75 Collision 63 Collisional activation dissociation (CAD) 64, 75 Collision-induced dissociation 81 Collision-induced dissociation (CID) 75 Collisions 22 Colors 208 Column efficiency (N) 135 Combined mass balance/metabolite

identification and single-dose volunteer studies 421 Combined multiple-dose volunteer 424 Comparative isotope ratio measurements 382 Competitive or non-competitive inhibition 306 Complementary techniques 35 Complex mixtures 22 Compliance 312, 313, 341,343, 344, 345, 355 Concentration 343 Concentration-dependent (nonlinear) pharmacokinetics 297, 298, 299, 301,303, 305, 311,312, 344, 422, 423, 426 Conjugated aromatic systems 31 Conjugated compounds 63 Container closure system 414 Continuous flow fast atom bombardment (CF/FAB) 48, 56, 57 Continuous flow fast atom bombardment interface (CFFAB) 23, 24 Continuous flow gas chromatographyisotope ratio mass spectrometry (CFGC-IRMS) 170 Continuous flow-isotope ratio mass spectrometry (CF-IRMS) 119, 120, 220, 379, 381,382 Continuous-flow 27 Cortex 98 Cortisol 231 Cost of an MB/MI study 229 Cost 406, 407, 422, 426 Coupling constant 211 Covalent bonds 29 CP/MAS spectra 213 [1,2,313C3]propionic acid 241 Creatine kinase 147 Creutzfeldt-Jakob disease 146 [10,20-13C2]-rhodopsin 212 CRIMS (chemical reaction interface mass spectrometry) 122, 199, 232 CRIMS 122, 232 Cross-over study designs 245, 246, 251,254, 257

435 Cross-over technique 423 CSF 160, 263 Custom synthesis 406, 409 CVP 304, 305 Cyclic aziridinium intermediate 101, 103, 104 Cyclophosphamide 101, 103, 104 CYP 1A2 349, 350 CYP2B1 286, 292 CYP2D6 294 Cystic fibrosis patients 356 Cytochrome P450 275, 276, 277, 287, 288, 289, 290, 291,292, 380, 384, 388, 389, 393, 394, 397 Cytochrome P4502B1 287 Cytosolic proteins 146 D2-imipramine 247 D2-nicotine 249 D20 160 D3-methadone 246 D2-1abeled analogues 108 D2-1abeled fragment 104 D20 105 D3-carnitine 94 D3-GSH-NMF 100, 101, 102 D3-NMF 100, 101, 102 D4-5-hydroxytrypta mine 98 D4-CBZ 99 D4-1abeled analogue 98, 104 D4-1abeled indolethylamine 97 D4-tryptamine 98 D4-Tyr 96 Do-ME 96, 97 D6-5-HMTLN 98 D8-HTLN 98 Dg-TLN 98 Daltons (Da) 77 Data acquisition 40 Data analysis 40 Daughter fragment ions 39 N-dealkylation 289, 291 O-dealkylation reactions 290 Debrisoquine 351 Decadeuterated compounds 130 Decadeuterated ethotoin 137 Decadeutero phenytoin 134

Decadeuterocarbamazepine 130 Decadeuterophenytoin 130 Decarboxylation 348 Deep peripheral compartment 264 Deep pool effect 261,264, 265, 266, 267, 268, 269, 270, 272, 272, 273, 310, 338, 344 Demethylation of caffeine 351 Demethylation 188, 348, 349, 350, 408 N-demethylation 279, 289, 349, 350, 401 Demethylsufoxide 401 3D emission spectrum 175 Deproteinization 221 Deprotonated [M + H] + molecular ion 74 Deprotonation 290 Derivatives 22 Derivatization reagents 178 Desorption ionization method 38 Detection limits 53 Detection of neurotransmitters in vivo 158 Detection of the NMR signal 144 Determination of absorption 9 Determination of stable isotopes 177 Detoxification 386, 397 Deuterated caffeine 180 Deuterated compounds 106, 128, 134, 135, 139, 170, 426 Deuterated ethotoin 136 Deuterated internal standard 130 Deuterated isotopomers 133, 187 Deuterium [2H] substitution 131 Deuterium atoms 185, 187 Deuterium concentration 184 Deuterium ion cluster techniques 238 Deuterium isotope effect 275, 276, 277, 278, 280, 286, 290, 294, 295 Deuterium labeling 4, 15, 16, 123, 180, 244, 347 Deuterium substitution reaction 134 Deuterium 2, 3, 4, 5, 14, 15, 17, 40, 129, 134, 170, 171,180, 184, 185, 188, 234, 235 Dextrose injection 323 Diagnostic breath tests 362

436 Diarrhea 366 1,2-di b ro m e-3-ch Io ropropa ne (DBCP) 239 2,3-didehydrosparteine 294, 295 5,6-didehydrosparteine 294 Dideuteromethyl isotopomers 187 Dietary ammonium citrate 4 Diethydihiocarbamate (DDTC) 390, 391 Diethylstilbesterol (DES)isomers 59 Di-GSH-derivative 101 10,11-dihydrocarbamazepine 132, 133 Dihydroxyphenylacetic acid 319 Dimethylanilines 289, 291 Dimethylsulfide 389 Dipole moment 15 Direct chemical ionization (DCI) 49 Direct current (dc) 76 Direct insertion probe 19 Direct liquid introduction interface (DLI) 23, 25, 26, 48, 49 Direct-current plasma 178 Disadvantages of HPLC-CRIMS 231 Disadvantages of SlL tracer methods 313, 345 Disadvantages of the CF-IRMS methods 230 Dissociative mechanism 293, 294 Distribution 9, 273 Disulfiram 59, 389 Divisions of FDA Center for Drug Evaluation and Research 413, 415 D-labeling method 106 DLI interfaces 23, 25, 26, 48, 49 1D MAS NMR 210 2D MAS NMR dipolar correlation spectroscopy 210 DMSO 397 DNA 105, 106, 107 DNA damage 104 Dopa-decarboxylase activity 317 Dopa-decarboxylase inhibitor 315 Dopa-decarboxylase 317, 322 Dopamine 158, 317, 322, 334 Dose ranging 418, 421,427 Dose-dependent pharmacokinetic changes 353

Dosing interval 303 Dosing rate 303, 304, 305, 343 Double-focussing 77 Double-focussing mass spectrometers 78 Double-focussing sector mass spectrometers 78 Drug conjugates 56 Drug disposition 65 Drug distribution 261 Drug interaction studies 337, 417 Drug interactions 261,297, 298, 309, 337, 344, 353, 418, 424, 425, 426, 427 Drug master file (DMF) 414 Drug metabolic pathways 348 Drug metabolism 20, 352, 384 Drug metabolites 89 Drug monitoring 20 Drug protein binding 341 Drug-conjugate metabolites 34 Drug-conjugates 24 Drug-free bile sample 101 Drugs in biological fluids 9, 160 Drug-tissue entry rate constant 262, 264 Dual inlet dynamic interface 382 Dual inlet IRMS system 119 Dual selective detection of lSN label in N2 gas and subsequent 13C label in CO2 gas 225 Dumas combustion techniques 122 D-xylose 362 Dynamic fast-atom bombardment interface (CF-FAB) 27 Dynamic interfaces 382 Dynamic measurement AES 178 Dynamic processes 9 Elab 96 EB 76, 82, 85, 87 EB double-focussing 96 EBE 79 EBEB 79, 80, 85 EBEB mass spectrometer 103 EBqQ 76, 80 EBqQ hybrid mass spectrometer Economic evaluation 417

100

437 Economic savings 313, 345 Effect of drugs 355 Efficacy 418, 425, 427 El 24, 25, 38, 52, 54 El mass spectrum 42 Electric field strengths 84 Electric sector 75, 76, 78, 80, 87 Electrospray (ES) 48 Electrolysis 3 Electrolyte-mediated chemical ionization 33 Electrolytic dissociations 2 Electron capture (EC) 66, 97 Electron donation 15 Electron impact (El) 49 Electron ionization 20, 29, 340, 37 Electron multiplier 21 Electron spin resonance (ESR) 400 Electron transfer mechanism 290, 291 Electronegativity 292 Electronic and chemical environment 145 Electronic effects 289 Electronic transition system 175 Electronic vibrational spectrum 175 Electrospray and ionspray 62 Electrospray (ES) 23, 25, 26, 27 Electrospray (ES)ionization 34 Electrospray/ionspray ionization 37 Electrospray/ionspray LC-MS interface 62 Elemental analysis 44 Elemental chromatogram 180, 191 Elemental labels 178 Elimination 9, 344 Elimination half-life 264, 297, 298, 299, 300, 301,305, 309, 310, 337, 340, 343 Elimination rate constant 299, 300, 301,303 Emission spectrometer 200 Enantiomer 255, 256, 308, 341,351, 424, 425 Enantiomeric interconversion 255 Enantiomeric purity 409 Enantiomeric racemization 255 Enantioselectivity of drugs 351,354

Enantiospecific syntheses 408 Endogenous 276, 384 Endogenous levels 97 Endoscopy 363 Energy of activation 13 Energy-to-charge ratio 84 Enflurane 162 Enkephalins 96 Enrichment 2 Entry half-life 262, 264 Enzymatic maturation 351 Enzyme binding kinetics 15 Enzyme induction 272, 298, 385 Enzyme structure 288 Enzyme substrate complex (ES) 293 Enzyme urease 363 Enzymeoxene 293 Enzyme-substrate complex 283 EOS 293 Epidemiological studies 365 Equilibrium 9, 144 ES ionization 63 ES 38, 50 ES/IS 65, 66 ES-MS 63, 64 ES-active 63 ESD 279, 281,282, 283 ESH 279, 281,282, 283 Essential amino acids 7, 8 Ethane 176 Ethotoin 131, 137, 407 EURISO-TOP 406 Excretion 9, 160, 275, 297, 315 Exocrine insufficiency 370 Exocrine pancreatic function 372 Exogenous 276 Exogenous pancreatic enzymes 356 Expired labeled C02 349 Extraction 221 Extraction efficiency 93 19F NMR measurements 162 19F NMR spectroscopy 162 ~9FNMR 152,161,161,163 ~9F nucleus 160 ~9F spectrum 161, 162 ~9F indicators 162

438 19F NMR detection of fluorinated drugs 160 2-19F 152 FAB 28, 31, 39, 52, 57, 96 FAB ionization 94, 108 FAB ionization source 97, 100, 104 FAB mass spectra 102 FAB probe tip 95 FAB/MS spectrum 101, 106 FAB/MS 74, 104, 105, 107 FAB/MS/MS 104 Facilities data 412 Factor of bioavailability 198 Faraday cup collectors 121,382 Fast atom bombardment (FAB) 20, 25, 31, 32, 37, 74, 93, 112 Fast atom bombardment ionization 112 fast atom bombardment mass spectrometry (FAB-MS) 56, 239 Fat malabsorption 356, 362, 369 Fatty acid catabolism 94 Fatty acid metabolism 159 FDA Center for Drug Evaluation and Research 412, 413, 415 FDA Division 413 FDAPhasel 229,418 FDA 229, 405, 409, 412, 413, 414, 415, 416, 417 FDG-6-P 152 FDL 412 Feces 219,220 Feces (extracted) 222 Feces (whole) 222 Fenoprofen 182 [FeO]3+-substrate complex 288 [18F] fluorodopa 319 FFR1 85, 86, 87 FFR2 85, 86 First field-free region (FFR1) 78 First pass metabolism 424 First quadrupole mass filters 77 First-order 193 FK506 55 Flame emission (FES) 177 Flame photometric detectors 172 Fluorinated dopamine 319

Fluoro-BAPTA 162, 163 Fluoro-deoxy-6phosphogluconate 152 Fluoro-deoxyfructose 152 Fluoro-deoxyglucose 151 FIuo ro-d eo xy g Iu cose-6- p h osph ate (FDG-6-P) 151 Fluoro-deoxysorbital 152 5-fluoro-deoxyuridine 162 5-fluorouracil 162 Fluoromethyl alanines 162 Fluorometric assays 96 e-fluoro-/3-alanine 162 Fluoxetine + norfluoxetine 161 Fluphenazine 161 Fluvoxamine 161 Food and Drug Administration (FDA) 411 Food Chemicals Codex 409 Food Drug and Cosmetic Act of 1938 415 Forensic analysis 59 Form FDA1571 412 Form(s) FDA 1572 412 Formaldehyde 349 Formic acid 5, 349 Formyltetrahyd rofolate 349 Fourier transform analysis 144, 240 Four-sector mass spectrometers 79 Fraction absorbed 312, 313, 337, 343, 344, 345 Fragment 64, 110, 433 Fragment ion 28, 106, 22, 29, 30, 33, 41, 194 Fragment ions (F+) 81 Fragment or daughter ions 21 Fragmentation of angiotensin III 110 Fragmentation 69 Free-induction-decay 144 Frequency domain MAS 207 Frequency spectrum 144 Full-scan MS 82 GABA 155, 156 GABA metabolism 158 GABA spectra 158 GABA synthesis 149

439 GABA-T inhibition 158 GABA-T inhibitor 159 GABA-transaminase 158 Galactose breath test 394 Galactose elimination 374 Gas chromatograph (GC) 122 Gas chromatographic combustionisotope ratio mass spectrometric (GCC-IRMS) 121 Gas chromatographic-mass spectrometry (GC-MS) 73, 170, 237, 319, 326, 337 Gas chromatography 175, 185, 383 Gas chromatography-atomic emission detection (GC-AED) 169, 189 Gas chromatography-isotope ratio mass spectrometry (GC-IRMS) 120 Gas or liquid chromatograph 19 Gastric emptying 371 Gastric lipase 370 Gastrointestinal disorders 361,363 Gastrointestinal infections 363 Gastrointestinal or liver dysfunction 362 Gastrointestinal tract 355, 373 GCAED 185,189,200 (GC-AES GC-AED) 171 GC-IRMS 200, 385, 398 GC/MS 97, 234, 250 GC-MS 20, 25, 28, 48, 51, 62, 67, 97, 234, 241,250, 341,344, 345 GC-MS Inlet 24 GC/MS/MS 97, 98 GC/MS-SlM quantitation 98 GC-pyrolizer-MS system 121 GC-AED 174, 176, 177, 179, 184, 186, 191,194, 195, 197, 199 GC-AED isotope detection 186 GC-MIP coupling 179 GC-MS 194, 195, 200, 238 Gentamicin 264, 266 Glucose 141,157 [13C]glucose 157 Glucose metabolism 151,157, 158 Glucose oxidation 398 Glucose signal 149 Glucose transport 149

Glucose utilization 150 Glucuronic acid 59 Glucuronide 160, 238 3-glucuronides 65 6-glucuronides 65 Glutamate 141,152, 154, 156, 157 Glutamate enrichment time courses 157 Glutamine cycle 155 Glutamine synthesis 149, 156, 157 Glutamine synthetase 155 Glutamine 7, 154, 155, 156, 156, 157 Glutathione 151, 59 Glutathione conjugates 66, 100 Glutathione conjugation 101 Glutathione (GSH) 100 Glycerin 32 Glycerol 58, 369 1,2-glyceryl dinitrate 253, 254 1,3-glyceryl dinitrate 253, 254 Glycine 4, 5 Glycogen 5,150, 151 Glycolysis 150 Gopher systems 412 G-protein transducin 206 G-protein 203 Gram-negative bacterium 363 Growth and development 307 Growth hormone therapy 351 Growth hormone-deficient children 351 GSH 105 GSH conjugation 104 GSH-NMF 100, 101, 102 GSH-cyclophosphamide 104 Guidelines for ordering stable isotopelabeled drugs 408 Gylcerol 113 2H-labeled internal standards 56 1H NMR detection 150, 160 1H NMR spectrum 141, 145, 146 1H NMR 153 2Hp2C ratios 186 2H/~2C values 187 Halogens 31 [2Hlo]-CBZ 132

440 Heat labile biomolecules 23 Heated inlet chamber 33 Helicobacter pylori 355, 363, 411, 416 Helium carrier gas 122 Helium 52, 53, 54, 179, 220 Heme-oxene-substrate complex (EOS) 293 Hepatic antipyrine metabolism 373 Hepatic encephalopathy 146 Hepatic functional impairment 374 Hepatic metabolism 315, 371,373 Hepatocytes 235 Hepatotoxicity 384, 389, 393 Heptafluorobutyryl derivatives (HFBTEN) 97 Heteronuclear 13C-1H 145 Hexapole collision cell 76, 77 Hexokinase 151 Hexose monophosphate shunt (HMPS) 151,398 High-energy CID 78 High molecular weight biomolecules 20 High molecular weight biosubstances 24 High performance liquid chromatography (HPLC) 47, 220, 236, 355, 421 High temperature surface ionization 52 High-energy CID 80, 81, 85, 111 High-resolution capabilities of EB and BE mass spectrometers 79 Hiolein 362, 371 Hippocampus 98 Hippuric acid 241 Histamine 30 Histology 365 Histopathology 364 HMPS 399 5-HMTLN 98 Homogenization process 397 Homovanillic acid 315, 318, 319, 320, 321,324, 325, 326, 328, 329, 332, 333 HPLC columns 57, 58 HPLC flow 69 HPLC methods 138

HPLC technology 23 HPLC 24, 36, 51, 57, 64, 69, 100, 103, 160, 178, 222, 230, 232, 422, 426 HPLC-TSP-MS 61 HPLC-CRIMS 230, 231,232 HPLC-CRIMS aplications to mass balance studies 231 HPLC-CRIMS instrument 123 HPLC-CRIMS: advantages 231 5-HTLN 98 Human pituitary 96 Human plasma 65 Hybrid EBqQ 82 Hybrid instruments 87 Hybrid mass spectrometers 76, 81, 85 Hybrid sector 76, 77 Hybrid sector mass spectrometers 80 Hydoxylation 234 Hydrochloric acid 325 Hydrogen 55 Hydrogen atom abstracting species tert-butoxy radical 291 Hydrogen atom abstraction 288 Hydrogen atom abstraction mechanisms 291 Hydrogen atom radical recombination mechanism 288 Hydrogenated linseed oil 4 Hydrolization 372 Hydrophilic compounds 66 Hydrophilic loops 203 Hydrophobic c~-helices 203 Hydrophobic moiety 59 /3-hydroxybutyrate 148 6-hydroxydopamine 317 e-hydroxy fragmentation 108, 109 Hydroxy functional group 107 Hydroxyl radicals (OH~ 290, 400 Hydroxylation 279, 287, 400 o~-hydroxylation 285, 286 5-hydroxymethyltryptoline (5HMTLN) 97 5-hydroxytryptoline (5-HTLN) 97 Hyperactivity 320 Hyperlipidemia 372 Hypothalamus 98 Ibuprophen

160, 309

441 ICON Services Inc. 406 ICR 76 Identification purposes 33 Identity testing 414 Immunoaffinity column 59 Increased column efficiencies 136 IND 411,412, 413, 414, 415 Indoleamines 97 Indolethylamine internal controls 98 Indolethylamines 97 Inductibility coupled plasma 178 Induction 385 Induction (inhibition) 341 Inert target gas 81 Inflammation 146 Inflammatory reaction 395 Inhibition 387 Initial electron transfer mechanism 290 Initial hydrogen atom abstraction mechanism 290 Injection volumes 93 Inlet system 19 Institutional review board (IRB) 219, 412, 419 Inter-element ratios 169 Interface 53, 67 Interferon-~/ 55, 56 Intermolecular deuterium isotope effect 293 Intermolecular isotope effect 292 Internal standard 22, 40, 54, 98, 132, 195 Intersubject variability 150 Intra-ligand interactions 210 Intra-ligand internuclear distances 210 Intramolecular competition 277 Intramolecular competitive design 277 Intramolecular deuterium isotope effect 280, 282, 283, 285 Intramolecular isotope effect 277, 282, 291 Intrinsic isotope effect 275, 284 Intrinsic primary isotope effect 291, 292

Invasive tests 363, 365 Investigational new drug application 411 Investigator data 412 in vitro models 146 in vivo 250, 315, 316, 318, 334, 347, 375 in vivo isotope effects 129, 134 in vivo microdialysis 57 in vivo NMR spectrum 145 Ion accelerating voltage 79 Ion chromatography 68 Ion cluster 233 Ion cyclotron resonance (ICR) 75 Ion fragments 19 Ion molecule reactions 66 Ion sensitive ligands 160 Ion source 20, 28, 38 Ion source interfaces 60 Ion trap mass analyzer 21 Ion trap 37 Ion-cluster 194 Ionic and polar 19 Ionic substances 32 Ionic surfactants 59 Ionization mass spectrometry (APIMS) 238 Ionization modes 69 Ionization process 34 Ionization techniques 32 Ionization-MS 54 Ion-molecule interactions 32 Ionspray (IS) 26, 48 Ionspray (IS)interface 64 Ionspray (IS) ionization 34, 35 Ionspray API-MS detection 237 Ion-trap mass analyzer 37 Ion-trapping mass spectrometers 38, 76 IRMS 121, 122, 421,422 Irreversible inhibitors 159 IS 50 IS ionization 63 Isolation valve 382 Isomeric purity 409 Isotachophoresis 68 Isotec Inc. 406

442 Isotope atomic emission 172, 176 Isotope cluster technique 233, 348 Isotope dilution MS/MS 93, 94, 95, 97 Isotope effect 129, 134, 224, 225, 270, 275, 277, 280, 288, 290, 293, 310, 320, 332, 347, 352, 357, 379, 406, 408 Isotope enrichment 200 Isotope labeling 206, 215 Isotope labeling (SIL) 220 Isotope measurement 199 Isotope peak shift technique 234 Isotope ratio mass spectrometry (IRMS) 119, 123, 169, 188, 236, 238, 361,362, 419 Isotope synthesis 427 Isotope tracers 100 Isotopes detection 181 Isotopes of oxygen and hydrogen 175 Isotopes ratios 199 Isotopic dilution 156 Isotopic effect 171 Isotopic enrichment 155 Isotopic labeling 15, 16, 64, 93, 100, 150, 156 Isotopic purity 129, 409 Isotopic separation 129, 130, 133, 134, 138 Isotopic shifts 171 Isotopically labeled internal standard 94 Isotopically labeled internal standard D3-carnitine 95 Isotopically labeled species 98 Isotopomer distributions 155, 156 Isotopomer/analogue pairs 132 Isotopomers 129, 135, 136, 185, 186, 399 IUPAC (pg) 183, 184 IUPAC method 181 J-coupling 145 Jet separator 28

Kco 187 e-ketoanalogues 375 Keto-enol tautomerism 236 (x-ketoglutarate 152, 153, 157

(x-ketog Iuta rate/g Iuta m ate exchange 149 Ketoisocaproic acid 362 KICA breath test 376 Kinetic energies 81 Kinetic energy filter 87 Kinetic isotope effect 13, 14, 16, 17 Kinetic-energy-to-charge ratios 78 Kinetics of drugs 354 Kinetics of the expired 13CO2 387 Kr. 298, 299, 303, 304, 422 K,-nNmax 305 k! retention factor 135 Krypton 1 Kynurenine 56 Labeled caffeine 195, 197 Labeled compounds 195 Labeled drugs 15 Labeled homovanillic 330 Labeled isotopomer 195 Labeled lactate 151 Labeled levodapa 330 Labeled parent drugs 194 Labeling isotopes 187 Labile compounds 56 Lactate dehydrogenase 152 Lactate 141, 150 Lactic acid 150 Larmor condition 144 Larmor equation 143 Larvae 397 Laser desorption 52 Last-in first-out phenomenon 271, 272 LC technology 24 LC-APCI-MS 66, 67 LC-CF/FAB-MS 59 LC-CI-MS/MS 40 LC-CRIMS 53 LC-ES/IS-MS 65 LC-ES-MS 35 LC-FAB-MS 57 LC-MS 20, 23, 24, 35, 39, 47, 48, 51, 65, 243 LC-MS detector 63 LC-MS inlets 25

443 LC-MS interface 26, 48, 49, 51, 52, 62, 66, 69 LC-MS-MS 40, 243 LC-PB-CRIMS 55 LC-PB-EI/MS 55 LC-PB-MS 53 LC-PB-NCI/MS 55, 56 LC-TSP-MS 61 L-Carnitine 94 LC-CRIMS 124 LC-MS with atmospheric pressure ionization interface 239 LC-MS with fast atom bombardment interface 239 LC-MS with thermospray interface 238 L-dopa 407 Least square linear regression analysis 225 Less-abundant isotopes 43 Leucine 4, 356 Levodopa central and peripheral metabolism 318 Levodopa-induced clinical response 319 Levodopa metabolism studies 333 Levodopa 226, 315, 316, 322, 323, 325, 332 Liability risk 419 Ligand-protein interactions 203, 206, 208, 210 Limit of detection of 13C 181 Limit of detection of deuterium 183 Limit of detection of nitrogen lSN 183 Limit of detection 181, 184 Limited dynamic range 53 Limits of detection 183 Linear calibration curves 54 Linear dynamic range 184 Linear pharmacokinetics 267, 271, 272, 298, 299, 301 Linear regression analysis 222 Linked-scan 87 Linoleic acid 108 Linolenic acid 108 Lipid resonances 151 Lipophilic interactions 139

Lipophilicity 15 Liquid chromatography/mass spectrometry (LC/MS) 73, 238 Liquid inlet stream 178 Liquid matrix 32 Liquid secondary ion emissionMS 56, 96 Liquid/vacuum interface 113 7Li spectroscopic imaging 163 Lithium 1 Liver biopsy 263 Liver function breath tests 373, 375 L-kynurenine 56 LLQ 224 Long-chain fatty acids 372 Low or high molecular-weight compounds 19 Low thermal stability 54 Low volatility compounds 54 Low-energy CID 76, 80, 81, 85, 110 Lower limit of quantitation (LLQ) 222, 230 Low-resolution structure 210 Lumbar puncture 263, 264, 320, 330 Luminal lipase 369 Lumirhodopsin 205 Lysine 4 m/z ratio 21, 22, 35, 36, 37, 38, 40, 64, 76, 77, 79, 84, 86, 88 Magic Angle Spinning (MAS) NMR 206 Magnetic moment 142 Magnetic nuclear resonance 170 Magnetic sector analyzer 21, 36 Magnetic sector scanning 37 Magnetic sector 75, 78, 79, 80, 122 Magnetization transfer 147 Magnitude of kH/ko 287 Magnitude of transverse magnetization 144 Major fragments 42 Make-up gas flow 35 Malabsorption 370, 372 MALDI, 38 Mannitol 401 Manufacture 414

444 Maprotiline 247 Margin of safety 7 MAS NMR investigations of rhodopsin 208 MAS NMR 206, 212, 214, 215, 216 Mass analyzer 21, 32, 33, 34, 35, 36, 37, 38, 39, 75 Mass balance 124, 219, 232, 422 Mass balance/metabolite identification study 219, 417, 418, 421,427 Mass isotopomer patterns 8 Mass measurement 44, 104 Mass number 1 Mass-selection 74 Mass shifts 106 Mass spectra 43 Mass spectral identification and quantitation 128 Mass spectrometer (MS) 1, 19, 54, 58, 63, 64, 74, 75, 81, 86, 87, 221 Mass spectrometric detectors 127 Mass spectrometric fragmentation pattern 15 Mass spectrometry (MS) 20, 22, 29, 47, 65, 127, 128, 134, 139, 188, 219, 233, 352, 419 Mass spectrum 22, 29, 31, 36, 40, 41, 90, 348 Mass to charge ratio 19 Mass Trace 406 Mass-analysis 75 Mass-analyzed-ion-kinetic-energy spectrometry 85 Mass-analyzer 84s 87 Mass-analyzing 110 Mass-selected 86 Mass-selection 84 Mass-selective detection 127 Mass-to-charge (m/z) ratio 21, 74, 121 Matrix ions 58 Matrix-assisted laser desorption ionization (MALDI) 20, 32, 37, 54 Maturation of N-demethylation of caffeine 350 Maximum plasma concentration 198, 309 Maximum residence time 174

Maximum velocity 298 MB 49 MB interfaces 52 MB/MI 219, 220, 221,228, 229, 232 MB/MI study (14C) 420 ME 97 Mean steady state plasma drug concentration 343 Measurement of distribution half-life and volume of distribution 261 Measurement of drug plasma concentration 342 Measurement of drug plasma concentration method 345 Measurement of gastric emptying 367, 368 Measurement of rate of entry of drug into tissues 261 Mechanisms of drug metabolism 9 Medical imaging surgical and dental drug products division of the center for drug evaluation 415 Medical Library Electronic Reference Network (LERN) 412 Mefloquine 248 Membrane integrity 146 Membrane protein receptors 206 Membrane receptors 206 Mercury 1 Metabolic compartmentation 154 Metabolic disposition 42 Metabolic isotope effect 266, 310, 338, 426 Metabolic pathway 15, 134, 146, 187, 194, 303, 304 Metabolic process 193 Metabolism-free radicals 398 Metabolite 92, 132, 188, 190, 191,194 Metabolite detection 232 Metabolite formation 305 Metabolite identification 9, 59, 231, 235 Metal activity 138 Metaproterenol 248 Metarhodopsin I 205, 213 Metarhodopsin II 205 Metastable ion 82

445 Metastable ion decomposition 81 Methadone 256 Methane 30, 39, 176 Methanol 401 Methionine-dependent folate pathway 380 Method of Quimby and Sullivan 182 Methods to test for deep pool effect 266 Methotrexate (MTX) 394 Methoxsalen 248 3-methylcholanthrene 387 Methyl ester 43 Methylene chloride 325 a-methyltestosterone 248 Methyltryptoline (MTLN) 97 4-methylumbelliferyl glucuronide 34 Methyluric acids 189 Methylxanthine 189, 348 Metronidazole 160 Mg 2§ 147 MH § ion 90, 97, 99, 100, 101,102, 103, 104, 106, 107, 110, 111 MH + ions of butylated Phe and DsPhe 96 (M+H) + 63, 109 [M + H] § carboxylate anions 108 [M-HF] § ions 98 MHz range 143 Michaelis constant 298 Michaelis-Menten formulation 149 Microbore/nanobore LC 35 Micro-breath test 389, 397 Micro-HPLC-negative ion 59 Microsomal enzymes 375 Microsomial mono-oxygenases 188 Microwave-induced plasma 179, 230 MIKES 85 MIKES spectra 85 Mild ionization technique 30 Mitochondria 375 Mitochondrial/3-oxidation of fatty acids 159 Mitochondrial dysfunction 375 Mitochondrial inner membrane 94 Mixed triglyceride (1,3 distearyl 2113C] octanoyl glycerol) 370

Mixed triglyceride or cholesteryl octanoate breath test 370 MK-434 67 Mobile phase 28, 53 Molar volume 15 Molecular emission spectroscopy 174 Molecular ion 29 Molecular modeling 211 Molecular structure 41 Mollusks 203 Momentum separator 28, 52, 53, 54 Momentum-to-charge 78 Monoamine oxidase 322 Monodeuteromethyl isotopomers 187 Monohydroxy metabolites 92 Monohydroxy unsaturated fatty acids 108 Monoxide 407 Morphine 65 Moving belt 26 Moving belt (MB)interface 48 Moving-belt interface 25 MPTP 317 MS 20, 101,220, 426, 427 MS/MS 39, 65, 74, 77, 78, 79, 89, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 104, 108 MS/MS (collision activated decomposition) 37 MS/MS analyses 81, 82, 93 MS/MS analysis scheme for identifying drug metabolites 88 MS/MS instruments 88 MS/MS Phe/Tyr ratios 96 MS/MS scan methods 82 MS/MS scan modes 83 MS/MS scans 93 MS/MS spectrum 39 MS/MS/MS analyses 86 MS/MS/MS 110, 111, 112 MTLN 98 Mucosal absorption 371 Multi-collector mass spectrometer 119 Multicompartment metabolic models 156 Multiple ionization modes 69

446

Multiple sector 77 Multiple sector mass spectrometers 85 Multiple-charge 64 Multiple-dose patient study 417, 418, 420, 427 Multiple-dose volunteer study 417, 418, 420, 422, 427 Multiply-charged 64 Multiply-charged ions 69 Multiply-charged molecular ion 64 Multiply-labeled octanoate 159 Multi-step pathways 41 Myelin breakdown 146 Myo-inositol as a Glial Marker 146 14N/15N ratios 174, 175, 199 lSN NMR spectra 157 15N 13C2-acetaminophen 226 lSN 155 lSN2-PHT 130 15N-acetaminophen 222, 224, 225 lSN-enrichment 7 lSNitrogen 236 lSN-labeled 157 15N-nitroglycerin 251,253 15NO 123 N,N-diethyl-13C-benzamide 241 N7 methylation of theophylline 348 NaBD4 106 NaBH4/H20/CH3OH 106, 107 N-acetylaspartate 145 N-acetylprocainamide (NAPA) 246 NADPH 151 Nano-scale chromatography 57 Naproxen 160 National Institute of Standards and Technology (NIST) 409 Natural abundance 227 [lSN 13C]-carbamazepine 61 NCS-chrom 104, 105, 106, 108 NCS-chrom A 107 N-demethylation of caffeine 349 Nebulizer 28, 53 Necroinflammatory activity 374 Negative charge 31 Negative chemical ionization (NCI) 31

Negative ion chemical ionization (NCl) 55 Negative molecular anion 31 Negatively charged alkene 109 Neon 1 Net magnetization 143 Net spin angular momentum 142 Neurochemical detection 145 Neuroleptics 161 Neuronal and glial metabolism 156 Neuronal density 146 Neuronal-axonal marker 145 Neutral aldehyde fragment 109 Neutral loss 88, 92, 93 Neutral loss scan 82, 83, 87, 100 Neutral loss spectrum 88, 91 Neutral loss-precursor scans 87 Neutral particles 22 Neutrals (Ni) 81 [5-1SN] glutamine 157 Nicotine 248, 249 NIH shift 236 Nimodipine 309 Nitrobenzyl alcohol 113 Nitrodisc | 252, 253 Nitro-Dur II| 252, 253 Nitrogen isotope 174, 175 Nitroglycerin 250, 252, 253, 254 N-methylformamide 100 N-methylformamide (NMF) 239 N-methyI-N-(trideuteromethyl) aniline 279 NMF 102 NMF conjugates 100 NMR 143, 144, 206 NMR analysis 240 NMR measures 148 NMR phenomenon 142 NMR shifts 209 NMR spectra 154 NMR spectroscopy 141,144, 149, 154, 240 NMR-active isotopes 141 NMR-active nuclei 141 N,N-bis(dideuteriomethyl) aniline 279, 280 N,N-bis(dideuteriomethyl) aniline analogs 280

447 n-octanes 284 Nonalcoholic liver disease 376 Noncompliance 337 Nondissociative 294 Nondissociative kinetic mechanism 294 Nondissociative mechanism 293 Nonessential amino acid 8 Noninvasive methods 379 Noninvasive tests 363, 370, 394 Nonlinear and time-dependent pharmacokinetics 313 Nonlinear calibration curves 53, 54 Nonlinear pharmacokinetics 267, 270, 271,272, 343, 424 Non-linear response 69 Nonpolar compounds 19 Nonradioactive tracers 188 Nonrenal clearance 309 Nonreproducibility 136 Nonvolatile 23, 26, 44, 49 Nonvolatile buffers 27 Nonvolatile polar and ionic molecules 33 Nonvolatile/thermolabile compounds 123 Normal column HPLC 68 Normal (full-scan) MS 74 Normal phase 26 Nuclear spin 142 160 isotope 319 180 chromatograms 175 1802-labeled analogue 111 180-labeled internal standards 56 O-acetyl carboxypentafluorobenzyl ester 326 Occipital cortex 154 Octane-1,2,3-2H7 285 Octane-l-2H 3 285 Octanoic acid 362, 367 1-octanol 286 2-octanol 286 Octapole collision cell 76, 77 O-demethylation 278 Oleic acid 108 Oligonucleotides 24, 27, 34, 35, 38

Oligosaccharides 24, 27, 48, 56, 59, 62 3-O-methyldopa 319 Opioid peptides 96 Oppenheimer (pg) 183, 184 Oppenheimer's method 181, 182 Opsin + all-E-retinal 205 ORA gold disc databases 412 Oral route 197 Ordering from catalogs 405, 406 Organic molecules 170, 178 Orifice 52 Out-of-plane deformation 210 Oxene 293 Oxene-heme complex 288 Oxidation 369 Oxidative pathways 150 Oxidative stress 399 Oxygen consumption 152 Oxygen isotope 2 Oxygen 31, 55 Oxypentifylline 160 31p NMR 142, 147, 152, 163 31p spectrum 148 P450 catalyzed oxidative reactions 289 P4502B1 286, 288 P4502B4 288 P450b 286 P451A1 288 Palmitic acid 362, 372 Palmitic acid breath tests 371 Pancreatic carboxyl ester lipase 370 Pancreatic function tests 371 Pancreatic insufficiency 370 Pancreatic lipase 370 Pancreatic resection 370 Paracetamol 160 Para-ditrideuteromethoxybenzene 278 Para-hydroxylation 134 Parallel pathway mechanism 293, 294 Para-methoxyanisole 278 Para-trideuteromethoxyanisole 278 Parent drug 188 Parkinson disease 315, 316, 317, 318, 319, 320, 321,322, 331,333, 334

448 Particle beam (PB) 26, 48 Particle beam LC-MS 51 Particle beam LC-MS interface 51 Particle charging 53 Particle size 53 Particle-beam (PB)interface 28 Particles 52 Pattern of fragment ions 21 PB 49 PB interface 28, 48, 52, 53, 54, 55 PB-massive particle 54 Peak retention time (k! as a unit of time t) 136 Pediatric pharmacology 347, 351 Pediatrics 307 Pee Dee Belemnitella 384 Penicillins 160 Pentachlorophenol 42 Pentafluorobenzyl derivatives 56 Pentapeptide methionine enkephalin (ME) 96 Pentapeptides 97 Pentose phosphate cycle 151 Pentylfluroroaryl 238 Peptide 27, 28, 32, 35, 56, 59, 62, 64 Perferryloxy heme oxidizing species 276 Peripheral decarboxylation ,317 Peripheral dopa-decarboxylase 315 Peripheral levodopa metabolism 315, 316, 317, 318, 321,334 Peripheral levodopa toxicity 318 Pesticide 40 PET scanning 319 Pharmaceuticals 66 Pharmacokinetic applications 244 Pharmacokinetic parameters 198 Pharmacokinetic properties (linear, concentration-, or timedependent) 422 Pharmacokinetic studies 22, 351,353 Pharmacokinetics 20, 65, 98, 219 Pharmacologic 316 Phasel 419,427 Phase I human testing 417 Phase II studies 427 Phenacetin 405, 407

Phenethyl isothiocyanide 389 Phenobarbital 221,261,262, 263, 299, 300, 338, 340, 345, 387, 405, 407 Phenylacetic acid derivatives 43 Phenylalanine 4, 7, 95 Phenylbutazone 341,342 Phenylketonuria (PKU) 95, 96 Phenytoin 128, 130, 131,132, 134, 136, 139, 248, 261,262, 268, 269, 270, 271,301,302, 304, 305, 307, 312, 338, 339, 340, 345, 407, 425, 426 Phenytoin dihydrodiol 340 Phorbol myristate acetate 399 Phosphatase 256 Phosphate 27, 256 Phosphate metabolism 147 Phosphocreatine (PCr) 142, 147 Phosphodiesters 147 Phosphohexose isomerase 152 Phospholipids 59 Phosphomonoesters 147 Phosphoramide 101, 103, 104 Phosphorous 55 Phosphorylated compounds 142 Photochemical isomerization 214 Photodiode array 176, 179, 189 Photointermediates 214 Photoisomerization 205, 215 Photon 203 Photoreceptor 203 Photosynthesis 5 p-HPPH 302, 305 PHT/deutero-analogue pair 132 Pilocarpine 66 Pilot bioavailability/bioequivalency study 250 PKU 96 Placental transfer of drugs 353 Plasma 98, 99, 160, 175, 176 Plasma clearnace 309 Plasma levels 197 Plasma sources 178 Platinum 54 Pneumatic nebulization 35 Pneumatic nebulizer 53, 64 Polar and ionic 23 Polarity 15

449 Polycyclic aromatic hydrocarbons 55 Polymorphonuclear leukocytes (PMNL) 398 Polynucleotides 48, 62 Polypeptides 43 Polytherapy 98 Positive- or negative-ion 36 Positively charged ions 36 Post capillary 64 Post column 58 Post-column derivatization 138 Post-column flow-split ratios 35 Post-column jet separator 24 Post-mortem pituitaries 96 Post-natal age 350 Post-separation derivatization 63 Potassium cyanide 407 Potential toxicity 160 p-pydroxy-phenylphenylhydantoin 340 Precision 220, 221,228 Pre-column 58 Precursor 93 Precursor ion scan 82, 83, 86, 88, 91, 92 Precursor (or parent) ion 74 Prednisone 357 Primary isotope effect 286 Principal lines 2 Probe 57, 58 Procainamide 264, 266 Procurement of stable isotope-labeled pharmaceuticals 405 Pro-drug formulations 256, 424 Product inhibition 303, 306, 307 Product ion scans 82, 83, 84 Product ion spectrum 84 Product (or daughter)ions 75 Progesterone 200 Propoxyphene 256 Propranolol 256 Protein binding 15, 337 Protein metabolism 356 Proteins 24, 27, 32, 35, 38, 62, 64, 146 Protocol 412 Proton exchange 236 Protonated hexapeptide 111

Protonated MH + 74 Protonated Schiff base 208 (Pseudo)electroch romatog raphy 68 Pseudo-first-order unidirectional rate constants 148 Pseudomolecular ions 56 Pseudoracemate technique 235, 255, 256, 257, 308, 309, 341,342 Psychiatric disorders 96, 161 Pulmonary disease 375 Pulmonary excretion 371 Pyruvate carboxylase 155, 157 Pyruvate dehydrogenase 157 Pyruvate 5, 150, 152, 157 Q/E linked-scan 85 Q1 94, 96 QlqQ2 86, 88 QlqQ2 mass spectrometer 94, 96, 97, 99 Q2 89, 94, 96 QITMS 76 Quadrupole 38, 62, 77 Quadrupole ion-trap mass spectrometers (QITMS) 37, 75 Quadrupole mass filter 37, 75, 76, 79, 80, 84, 87 Quadrupole mass filter analyzer 21, 34, 37 Quadrupole mass spectrometer 122 Qualitative and quantitative determinations 169 Quantify tryptoline (TLN) 97 Quantitation of drug transformation 9 Quantitative analysis 22, 40, 53, 58, 66 Quantitative capabilities 61 Quantitative capabilities for CF/FAB 59 Quantitative MS/MS methods 99 Quantitative statement of composition 414 Quantum mechanics 171 Quasi-molecular ion 29, 30, 32, 33, 39, 40 Quasi-molecular ion [M + H] + 35 Quick urease testing 364

450 Quimby (pg/s)

183, 184

R-(+)-D6-propranolol 256 R-(+)-propranolol 255 Racemates 308 Radiation 231 Radiation-absorbing matrix 33 Radio frequency 37 Radioactive 219, 228, 229, 344, 419, 421,422 Radioactive (14C) 219 Radioactive flow detectors 236 Radioactive isotopes 236, 243, 356 Radioactive label 88, 229 Radioactive labeled levodopa 318 Radioactive tracer studies 341,342, 343 Radioenzymatic assay (REA) 94 Radio-frequency controllers 127 Radio-frequency (rf) 76, 144 Radioimmunoassay (RIA) 96 Radionucleotides 319 Radioreceptor assay (RRA) 96 Raman spectror 214 Ranitidine 388, 390 Rapid urease test (CLOtestTM) 365 Rate-limiting step 13, 14, 361,370 REA 95 Reaction mechanism 42 Reactive metabolites 388 5(x-reductase inhibitor 67 Regression lines parameters 197 Regulatory approval 417, 427 Regulatory aspects 411 Relative bioavailability 197, 245, 246, 247, 354, 423, 424 Relative peak separation 136 Relaxation 144, 210 Reliable instrumentation 220 Renal biopsy 264 Renal clearance 309 Renal impairment 309 Reproducible LC method 139 Reservoir inlet 19 Residual solvents 414 Resolution 104, 221 Resolution equation 135

Resonance Raman experiments 212 Respiratory carbon dioxide 5 Retention mechanisms 138 Retention time 135 Retinals 208 Reversed phase 26 Reversed phase chromatography 138 Reversed phase column 136 Reversed phase separations 138 Revision of investigational new drug application regulations (1987) 415 Revision of new drug application regulations (1985) 415 Rf-only quadrupole 77 Rf-only quadrupole collision cells 76, 80, 82 Rhodopsin 203, 205, 206, 211,212, 215 RIA 97 Rotational resonance 207 Rotational resonance dipolar recoupling 211 Routes of metabolism 337, 345 RRA 97 (R)-1,1,1 -tride ute ro-2phenylpropane 282

Safety 312, 344, 418, 421,424, 425, 427 Safety of stable isotope labeling 307, 310 Saturatable first pass metabolism 424 Scan rate 22 Scavengers 400 Schiff base 203, 206, 214, 215, 216 Scintillation counting 421 SDS/PAGE gel electrophoresis 65 Secondary isotope 13, 15 Second field-free region (FFR2) 78 Second linear pathway 306, 307 Second quadrupole mass filters 77 Secondary isotope effects 129, 286 Second-generation product ions 110 Selected ion monitoring (SIM) 74, 122 Selected ion monitoring mass spectrometry (SIM-MS) 119

451 Selected-reaction monitoring (SRM) 82, 83, 88 Selectivity (a) 135 Selectivity 184 Selenium 55 Sensitivity 74, 220, 221,228 Sensitivity and specificity of diagnostic tests 365 Sequence-specific fragment 110 Serology 363 Serotonin 158, 161 Serum 65, 220, 226, 324 Serum (deproteinized) 222 Serum (whole) 222 Serum concentration versus time relationships 16 Serum whole matrix 228, 229 Serum whole matrix diluted 228, 229 Shape selectivity 138 Shelf-life 414 Signal processor/data system 21, 22 Signal-averaging times 147 Signal-to-noise (S/N) ratio 74, 98 SIL labeling 220, 229, 415 SIL molecules 170 SIL tracer dose 299 Silica capillary 58 Single- and multiple-ion scanning 37 Single dose of drug 2 method 343 Single dose volunteer safety/pharmacokinetic study 229 Single labeled analogue 22 Single-dose experiment 352 Single-dose kinetics 355 Single-dose patient 417, 418 Single-dose volunteer and single-dose patient studies 418, 420, 421,427 Site-directed mutagenesis studies 208 Skin metabolism 248 Snapshot 181 Sodium acetate 407 Solid phase extraction 160 Solid-liquid extraction 55 Solids 19 Soliton 215 Solubilization 371 Solute 138

Solvent-mediated chemical ionization 25 Solvent-mediated CI, 26 [34S]omeprazaole 240 [34S]omeprazole 237 Sorbinil 152 Source temperature 22 Sources for stable isotope-labeled drugs 406 Spatial ligand structure 210 Spectral characteristics 69 Spin coupling 145 Spin-lattice relaxation time 161,144 Spiramycin 55 SRM 88, 93 SRM mass spectra 97 Stability protocol 414 Stable fragment ions 29 Stable isotope 1, 2, 7, 9, 14, 22, 42, 43, 47, 48, 50, 55, 59, 73, 74, 93, 98, 99, 100, 101, 112, 127, 128, 129, 170, 171,177, 180, 189, 193,201,229, 255, 257, 316, 319, 320, 379 Stable isotope administration technique 353 Stable isotope breath tests 363 Stable isotope labeled (SIL) tracer techniques 3, 93, 261,263, 265, 297, 337, 353 Stable isotope labeled derivatizing agent 234 Stable isotope labeled levodopa 321, 324, 330, 330 Stable isotope labeling 47, 347, 351, 356 Stable isotope methodology 5, 232, 233, 246 Stable isotope synthesis 257 Stable isotope technology 316, 333 Stable isotope utilization 244 Stable isotope-labeled DES internal standards 59 Stable isotopically labeled (SIL) 5, 169, 188, 379 Stable or labile compounds 19 Staggered stable isotope administration technique 262, 263, 264

452 Standard curve 93 Standard deviation 181 Standard fluorometric methods 95 Start-up costs 417 Stationary phase 138 Statistical power 345, 423, 424, 425 Steady state clearance 267, 268, 269, 270 Stearate 369 Stereoselective hydroxylation 281 Stereoselective metabolism 351 Stereoselective pharmacokinetic studies 255 Stereoselectivity 281 Steroids 59 Stopping drug administration 343 Storage and disposal of radioactive specimens 419 Storage conditions 414 Striatum 98, 317 (S)- 1,1,1-t rid eute ro-2phenylpropane 281,282 Structural analysis 29 Structure elucidation 31, 52, 100 Structure-independent detection 199 Study costs 420 Substrate saturation 305 Succinic 4 Sulfate 59, 238 Sulfonamide 88, 92 3"Sulfur 237 Sulfur 1, 55 Sulfur conjugated paracetamols 160 Superoxide ions 398 Suprofen 309 Surface wave plasma 179 Synchronously scan 87 Synthesis and formulation of SII drug 134, 417 Systemic homovanillic acid levels 328 Systemic levodopa levels 327 2S-[2H]-5,6-didehydrosparteine 295 2S-[2H]-sparteine 295

tl/2 297, 311,342, 343, 304, 312 tmax (h-l) 198 T2 relaxation time 144

Tamoxifen-ds 234 Tamoxifen-do 234 Tandem-in-space mass spectrometers 75, 76, 77 Tandem-in-time 75 Tandem-in-time ion-trapping mass spectrometers 86 Tandem mass spectra 81 Tandem mass spectrometry (MS/MS) 39, 40, 73, 74, 75, 128 Tandem scan methods 73 Target pharmacokinetic parameters 197 TCA cycle 152, 156, 157 TCA cycle flux 149, 152, 153 TCD 385 Telazol 324 Teratogenicity 357 Terminal carboxy oxygens 112 Terodiline 248 Terrestrial natural abundance of various stable isotopes 177 Tetradeuterium-labeled CBZ 352 Tetradeutero 129 Tetrahydrofuran 130 Theophylline 248, 348, 353, 407 Theoretical computations 221 Thermal conductivity detector 383 Thermal spray TS, 20 Thermal stability 54 Thermally unstable compounds 31 Thermoconductibility detector (TCD) 384 Thermolabile analytes 23 Thermolabile substances 32 Thermolability 44 Thermospray (TS) 25, 26, 27, 48, 60, 61 Thermospray interface (TS) 23, 27, 50 Thermospray ionization (TSI) 33, 37 Thermospray mass spectrometry 239 Thermospray spectrum 34 Thermospray vaporizer 53 Thermostability 44 THF 136 Thin layer chromatography 68 Thioglycerol 113

453 Three-dimensional displays 181 Three-sector mass spectrometers 79 Thymidine 108 Thyroid disease 372 Time (t) to reach a given serum concentration 304 Time for plasma peak 198 Time to maximum plasma concentration 309 Time to reach steady state plasma concentration 303, 305 Time-dependent pharmacokinetic properties 297, 298, 299, 301,312, 313, 344, 352, 423, 426 Time-of-flight 37 Time-of-flight mass spectrometers 38 Tin 1 Tissue metabolism 315 TLNs 97, 98 TMS derivatives 235 Tobramycin 264, 266 Torsional angles 212 Toxicity studies 5 Toxification 386 Tracer dose 9, 264, 266, 267, 268, 269, 270, 271,272, 273, 297, 299, 300, 301,303, 337, 338, 339, 341, 422, 425 Tracer dose area under the plasma concentration versus time curve (AUC) 337 Tracer dose elimination rate constant (k) 298 Transchain amino acids 375 Transdermal delivery systems 248, 251 Transdermal nicotine systems 249 Transderm-Nitro 10| 250 Transderm-Nitro | 252, 253 Transfer time 385 Transmission 144 Transport interfaces 51 Transport kinetic parameters 150 Trazodone 248 Trideuteromethylcaffei ne 183 Trifluoperazine 161 Trifluoroacetamido 238

Triglyceride breath tests 371 Triglycerides 372 Trimethylsilyl (TMS) 43, 238 Trimethylsilyl ester 43 Trioctanoin 362, 370, 371 Triolein 362, 371 Tripalmitin 372 Triple quadrupole mass spectrometer 76, 77 Tryptolines 97, 98 Tryptophan 56 TS 36, 38 TSI 33, 39 TSP interface 66 TSP LC-MS interface 60 TSP 50 Tungsten 54 Turnover of amino-acids 356 Two double-focussing instruments 80 Tylosin 55 Tyr-Gly-Gly-Phe-Met 96 Tyrosine (Tyr) 4, 95, 96 Tyrosi ne-g lyci n e-g lyci ne-phe nyla la ninemethionine 96 [U-13C] glucose 155 [U-13C]glutamate 155 Ultra-high field MAS NMR 210 Ultraviolet detection 421 Uncharged nonvolatile material 35 Uncompetitive inhibition 306 Unimolecular 82 United States Food and Drug Administration 417 Unlabeled caffeine 185, 195, 197 Unlabeled compound 200 Unlabeled internal standard 98 Unsaturated fatty acids 110 Uracil 188 Urea breath test 363 Urea 224, 407, 411,416 Urease 224 Uric acids 188 Urinary bile 59 Urinary excretion 188, 313, 337, 340, 341,345 Urine (diluted) 224, 225

454 Urine (urease treated) 224, 225 Urine and plasma extracts 94 Urine diluted with water 224 Urine whole matrix 226 Urine 100, 160, 189, 191,192, 219, 220, 222, 223, 224, 225 US Pharmacopoeia 409 USP 409 UV detection 426

Vinylic fragmentation 109 Visual signal transduction 206 Volatile analytes 28 Volatile buffer 27 Volatile electrolyte 33 Volatility 26, 44 Voltages 22 Volume of distribution (Vd) 262, 271, 272, 297, 300, 301,309, 311,337, 340, 341,344, 422

Vmax 298, 299, 303, 304, 422 1Nmax 307 Vacuum pumps 25 Vacuum system 22 Validation of analytic method 417 Valproic acid 99, 128, 134 Van der Walls forces 15 Vaporized analyte molecules 25 Vd 298, 311,312, 343, 344 Verapamil 246, 256 Vertebrate 203 Vestec universal interface 54 ~/-vinyl 159

Warfarin 309, 341,342 Washout of drug 266, 422 Wavelength 176 Weighted (1/X2) least square linear regression 224, 225, 226, 228, 229 Whole urine 222 Xanomeline 65 Xanthines 180 Xenon 1, 32 Xylose breath test Zonisamide

365, 366

88, 89, 90