CHROMATOGRAPHY OF STEROIDS, Volume 8 (Journal of Chromatography Library)

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Journal of Chromatography Lbrary

-

Volume 8

CHROMATOGRAPHY OF STEROIDS

JOURNAL OF CHROMATOGRAPHY LIBRARY Volume 1 Chromatography of Antibiotics by G. H. Wagman and M. J. Weinstein Volume 2 Extraction Chromatography edited by T. Braun and G. Ghersini Volume 3 Liquid Column Chromatography. A Survey of Modern Techniques and Applications edited by Z . Deyl, K. Macek and J. Janak Volume 4 Detectors in Gas Chromatography by J. SevEik Volume 5 Instrumental Liquid Chromatography. A Practical Manual on High-Performance Liquid Chromatographic Methods by N. A. Parris Volume 6 Isotachophoresis. Theory, Instrumentation and Applications by F. M. Everaerts, J. L. Beckers and Th. P. E. M. Verheggen Volume 7 Chemical Derivatization in Liquid Chromatography by J. F. Lawrence and R. W. Frei Volume 8 Chromatography of Steroids by E. Heftmann

Journal of Chromatography Library - Volume 8

CHROMATOGRAPHY OF STEROIDS Erich Heftmann Western Regional Research Center, United States Department of Agriculture, Berkeley, Calif.

ELSEVIER SCIENTIFIC PUBLISHING COMPANY AMSTERDAM

-

OXFORD - NEW YORK

1976

ELSEVIER SCIENTIFIC PUBLISHING COMPANY 335 Jan van Galenstraat P.O. Box 211, Amsterdam, The Netherlands

Distributors for the United States and Canada: ELSEVIER /NORTH-HOLLAND INC. 52, Vanderbilt Avenue New York, N.Y. 10017

L i b r a r y of Congress Cataloging in Publicalion D a t a

Heftrnann, Erich. Chromatography of s t e r o i d s .

(Journal of chromatography l i b r a r y ; V. 8) Bibliography: p. I n c l u d e s index. 1. Steroids--Analysis. 2. Chromatographic a n a l y s i s . I. T i t l e . 11. S e r i e s . ~ ~ 4 2 6 . ~ 4 547’ -731 76-24897

ISBN: 0-444-41441-x Copyright 0 1976 by Elsevier Scientific Publishing Company, Amsterdam All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the publisher, Elsevier Scientific Publishing Company, Jan van Galenstraat 335, Amsterdam

Printed in The Netherlands

TO BRIGITTE

This Page Intentionally Left Blank

Contents Preface

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

IX

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

X

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

1

Commercial Products 1. Introduction

2. Liquid column chromatography . . . . . . . . . . . . . . . . . . 2.1. Sorbents 2.2. Instrumentation . . . . . . . . . . 2.3. High-pressure liquid column chromatography .

. . . . . . . .

. . . . . . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . . . .

3 3 8 10

3 . Paper and thin-layer chromatography . . 3.1. Paper chromatography . . . . . 3.2. Thin-layer chromatography . . . . 3.3. Sorbents . . . . . . . . . 3.4. Layers . . . . . . . . . . 3.5. Development . . . . . . . . 3.6. Detection . . . . . . . . . 3.7. Quantitative and radiochemical methods 3.8. Steroid derivatives . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

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13 13 14 14 16 17 18 24 25

4 . Gas chromatography . . . . . . . . . . . 4.1. Introduction . . . . . . . . . . . . 4.2. Steroid derivatives . . . . . . . . . . . 4.3. Packed columns . . . . . . . . . . . 4.4. Coated capillaries . . . . . . . . . . . 4.5. Instrumentation . . . . . . . . . . . 4.6. Gas chromatography-mass spectrometry combination 4.7. Quantitative and radiochemical methods . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. .

29 29 29 33 37 41 41 43

5 . Relationsbetweenstructureandchromatographicmobility . . . . . 5.1. RM values in liquid chromatography 5.2. RM values in gas chromatography . . . . . . 5.3. Group retention factors . . . . . . . . . 5.4. Steroid numbers . . . . . . . . . . . . 5.5. Other indices . . . . . . . . . . . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . . . . . .

45 45 46 47 48 53

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

55 55 57 61

6 . Sterols . . . . . . . . . 6.1. Liquid column chromatography . 6.2. Thin-layer chromatography . . 6.3. Gas chromatography . . . .

7. Bile acids and alcohols . . . . 7.1. Liquid column chromatography 7.2. Thin-layer chromatography . 7.3. Gas chromatography . . .

. . . . . . . . .

. . . . . . . . .

. . . . . .

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

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

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

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

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

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

VII

71 71 71 74

VIII

CONTENTS

8. Estrogens . . . . . . . . 8.1. Liquid column chromatograpRy 8.2. Thin-layer chromatography . . 8.3. Gas chromatography . . . .

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

9 . Androstane derivatives . . . . 9.1. Liquid column chromatography 9.2. Thin-layer chromatography . . 9.3. Gas chromatography . . . .

. . . .

10. Pregnane derivatives . . . . 10.1. Thin-layer chromatography 10.2. Gas chromatography . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . . . .

79 79 81 83

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

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

.

87 87 87 91

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

11. Corticosteroids . . . . . . . 11.1. Liquid column chromatography 11.2. Thin-layer chromatography . 11.3. Gas chromatography . . .

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

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

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

12, Miscellaneous steroid hormones . . . . . . . . 12.1. Introduction . . . . . . . . . . . . . 12.2. Hormones in urine . . . . . . . . . . . 12.3. Hormones in other biological specimens . . . . 12.4. Hormones in pharmaceuticals . . . . . . . 13. VitaminsD

. . . . .

. . . . . . . . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . . . .

93 93 96 99 99 101 103 107 107 107 109 109

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

113

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

115

14. Molting hormones

15. Steroid sapogenins and alkaloids

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

117

16. Cardenolides and bufadienolides

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

121

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

125

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

127

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

193

List of Abbreviations References Subject index

Chromatography has become one of the most important methods in steroid research. Yet, since the appearance of Neher's booksteroid Chromatography in 1964 [769], no comprehensive review of this subject has been published. Having accumulated over 2000 reprints on the chromatography of steroids since 1964, I can well understand the reluctance of my colleagues to write such a review, but I do feel a need to organize this information for my own benefit as well as theirs. In citing the literature, I obviously had to be selective in order to keep this monograph within a reasonable size. Even after eliminating all the material presented by Neher, I found myself with more information than I could possibly use. I could have solved my problem by referring the reader to the numerous review articles, chapters, and books on individual aspects of steroid chromatography. However, I feel that the reader is entitled to enough detail so that he can at least decide which articles to look up and, preferably, so that he can repeat experiments without referring to the original papers. 1 therefore decided to omit that part of the literature which I found less original and more difficult to obtain. I am fully aware of the risk of incurring the wrath of some colleagues who may feel slighted by such omissions, but I hope that more unbiased readers will appreciate my effort at simplifying their literature search. The literature citations have been handled by the methods currently used by Chemical Abstracts and other publications of the American Chemical Society. For the more common steroids, I have used the trivial names. The systematic names (IUPAC-IUB 1967 revised tentative rules) [SO71 are shown in the subject index. It is assumed that the readers are familiar with the general terminology, theory, and techniques of chromatography. These aspects are only covered as they relate to steroids. Readers requiring further information on chromatography are referred to my book Chromatography [422]. Some background material on steroids and a relatively recent guide to the steroid literature will be found in my book Steroid Biochemistry [420]. Extensive bibliographies on chromatography are being published regularly in the Journal of Chromatography and other analytical journals, biennially in Analytical Chemistry, in several Elsevier books [233,234,681,682], as well as by various manufacturers of chromatographic equipment and supplies. Many other aspects of steroid analysis are covered in my recent bookModern Methods of Steroid Analysis [421]. Most of this book was written while I was at the Federal Institute for Lipid Research of the German Federal Republic in Munster under the terms of a U S . Senior Scientist Award by the Humboldt Foundation. I am deeply grateful to the Director of the H.P. Kaufmann Institute, Professor H.K. Mangold, and its staff as well as to the staff of the Humboldt Foundation for their most generous support and cordial welcome. ERICH HEFTMANN

Minster, October 1975

IX

Commercial Products

x

Trade designation

Chemical nature

Source

Adsorbosil-CABN Alpha-8-Metricel Amberlite IRC-50 Amberlite XAD-2 Amberlyst XN-1006 Amberlyst A-6 AN-600 Anakrom ABS ANH BDSA BSA BSTFA Celite Cellex E Centri-Chrom Chromosorb CMDMCS CMDMTMDS

AgN03 on SiOz membrane filter polyacrylic acid macroporous styrene-divinylbenzene resin anion-exchange resin anion-exchange resin 50% cyanoethyl methyl silicone silanized diatomaceous earth cyanoethyl silicone bis (dime thylsilyl) acetamide N, 0-bis (trimethylsilyl) ace tamide N, 0-bis (t rimethylsilyl) trifluoroacetamide diatomaceous earth ECTEOLA-cellulose centrifugally accelerated LC system styrene-divinylbenzene copolymer chloromethyldimethylchlorosilane 1,3-bis(chloromethyldimethyl)-1,1,3,3-tetramethyldisilazane cyanoethyl methyl silicone porous silica permanently bonded octadecylsilane Carbowax 2OM-terephthalic acid silicone sym-dichlorotetrafluoroacetone diethylene glycol succinate

Applied Science Laboratories, Stage College, Pa., U.S.A. Gelman Instruments, Ann Arbor, Mich., U.S.A. Rohm & Haas, Philadelphia, Pa., U.S.A. Rohm & Haas, Philadelphia, Pa., U.S.A. Rohm & Haas, Philadelphia, Pa., U.S.A. Rohm & Haas, Philadelphia, Pa., U.S.A. Analabs, North Haven, Conn., U.S.A. Analabs, North Haven, Conn., U.S.A. DuPont de Nemours, Wilmington, Del., U.S.A. Supelco, Bellefonte, Pa., U.S.A. Supelco, Bellefonte, Pa., U.S.A. Supelco, Bellefonte, Pa., U.S.A. Alltech Associates, Arlington Heights, Ill., U.S.A. Bio-Rad, Richmond, Calif., U.S.A. Ivan Sorvall, Norwalk, Conn., U.S.A. Analabs, North Haven, Conn., U.S.A. Pierce Chemical Co., Rockford, Ill., U.S.A.

CNSi Corasil Corasil CIS CTpA DC-200 DCTFA DEGS

Pierce Chemical Co., Rockford, Ill., U.S.A. General Electric, Schenectady, N.Y., U.S.A. Waters Associates, Milford, Mass., U.S.A. Waters Associates, Milford, Mass., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. Regis Chemical Co., Morton Grove, Ill., U.S.A. Applied Chemical Corp., Morristown, N.J., U.S.A. Regis Chemical Co., Morton Grove, Ill., U.S.A.

Dexsil-300 Diatoport S DMMCS Epon Resin 1001 F-50, etc. Factice 31-B Florisil Gas-Chrom Gas Quat L GE-F-50 HCP HFBA HI-EFF-8BP Hyamine hydroxide ITLC JXR KGn, Kalignost L45 LA-1 Lipidex Michrome No. 64 Micropak Si 60 MSTFA NGS NPGA o v - I , ov-101 OV-17,0V-25 OV-225

polycarboranesiloxane Analabs, North Haven, Conn., U.S.A. diatomaceous earth F & M Scientific Corp., Avondale, Pa., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. dime thylmonochlorosilane Alltech Associates, Arlington Heights, Ill., U.S.A. bisphenol-epichlorohy drin polymer see Versilube polymer from soybean oil Carter-Bell Manuf. Co., Springfield, N.J., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. magnesium silicate diatomaceous earth Applied Science Laboratories, State College, Pa., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. trioctadecylmethylammonium bromide Applied Science Laboratories, State College, Pa., U.S.A. see Versilube DuPont de Nemours, Wilmington, Del., U.S.A. ethylene-propylene copolymer PCR Inc., Gainesville, Fla., U.S.A. heptafluorobutyric anhydride Applied Science Laboratories, State College, Pa., U.S.A. cyclohexanedimethanol succinate diisobutylcresoxyethoxyethyl dimethyl J.T. Baker,Phillipsburg,N.J.,U.S.A. benzylammonium hydroxide Gelman Instruments, Ann Arbor, Mich., U.S.A. (Instant Thin-Laye r Chromatography) adsorbent-impregnated glass-fiber paper Supelco, Bellefonte, Pa., U.S.A. dime thylpolysiloxane sodium tetraphenylborate Eastman, Rochester, N.Y., U.S.A. General Electric, Schenectady, N.Y. methyl silicone Rohm & Haas, Philadelphia, Pa., U.S.A. n-dodecenal(trialkylmethy1)amine Pharmacia, Uppsala, Sweden hydroxyalkoxypropyl Sephadex E. Gurr Ltd., London SW 14, Great Britain primuline Varian Associates, Walnut Creek, Calif., U.S.A. silica gel N-methyl-N-trimethylsilyltrifluoroacetamide Supelco, Bellefonte, Pa., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. neopentyl glycol succinate Applied Science Laboratories, State College, Pa., U.S.A. neopentyl glycol adipate Applied Science Laboratories, State College, Pa., U.S.A. methyl silicones Applied Science Laboratories, State College, Pa., U.S.A. phenyl methyl silicones Applied Science Laboratories, State College, Pa., U.S.A. cyanopropyl phenyl methyl silicone Continued on p . XII

h icr

e

F

V

icr

0

E

Commercial products (continued) Trade designation

Chemical nature

Source

Permaphase ETH Permaphase ODS PhSi Plaskon CTFE-2300 PMPE Polygram Sil G Polyimide Poragel PN Porasil A PZ- 176 QF-1 Regisil SCX SE-30 SE-30 “ultraphase” SE-52 Sephadex Sephadex LH-20 SI-100 silanox 101 SILAR-5CP Silica Gel 1B-F Silica Gel F Silica Gel G Silica Gel HS SP-400 SP-525 SP-1000

ether-bonded controlled-porous surface beads octadecylsilane bonded to Zipax see XE-61 trifluoroethylene polymer polymetaphenoxylene silica gel on polyester sheets

DuPont de Nemours, Wilmington, Del., U.S.A. DuPont de Nemours, Wilmington, Del., U.S.A.

polystyrene gel porous silica polyphenyl ether sulfone fluoroalkyl polysiloxane BSTFA+TMCS (99: 1) strong cation exchanger methylpoly siloxane “improved” methyl silicone methyl phenyl silicone cross-linked dextran hydroxypropyl ether of dextran silica silica cyanoalkyl phenyl silicone flexible precoated TLC sheet silica plus fluorophor silica plus gypsum silanized silica chlorophenyl silicone aromatic hydrocarbon modified Carbowax 20M

Allied Chemical Corp., Morristown, N.J., U.S.A. Varian Associates, Wainut Creek, Calif., U.S.A. Macherey, Nagel & Co., Duren, G.F.R. Pennzoil United, Shreveport, La., U.S.A. Waters Associates, Milford, Mass., U.S.A. Waters Associates, Milford, Mass., U.S.A. Pennzoil United, Shreveport, La., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. Regis Chemical Co., Morton Grove, Ill., U.S.A. DuPont de Nemours, Wilmington, Del., U.S.A. Analabs, North Haven, Conn., U.S.A. Phase Separation, Queensferry, Great Britain General Electric, Schenectady, N.Y., U.S.A. Pharmacia, Uppsala, Sweden Pharmacia, Uppsala, Sweden Merck, Darmstadt, G.F.R. Cabot, Boston, Mass., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. Baker, Phillipsburg, N.J., U.S.A. Merck, Darmstadt, G.F.R. Merck, Darmstadt, G.F.R. Merck, Darmstadt, G.F.R. Supelco, Bellefonte, Pa., U.S.A. Supelco, Bellefonte, Pa., U.S.A. Supelco, Bellefonte, Pa., U.S.A.

SP-240 1 Spherosil XOA-400 Supelcoport Sylon-CT TCTFA TMCBA TMDS TMSDEA Versilube Vydac XE-60 XE-6 1

Z Zipax Zorbax SIL

trifluoropropyl methyl silicone silica GC support silanizing solution 1,1,3-trichlorotrifluoroacetone tetramethylcyclobutanedioladipate tetramethyldisilazane trimethylsilyldiethylamine methyl chlorophenyl silicone porous silica layer on solid core cyanoethyl silicone phenyl methyl silicone ethylene glycol, succinic acid, and methyl siloxane copolymer porous-layer support porous silica microspheres

Supelco, Bellefonte, Pa., U.S.A. Pechiney, Saint Gobain, France Supelco, Bellefonte, Pa., U.S.A. Supelco, Bellefonte, Pa., U.S.A. Allied Chemical Corp., Morristown, N.J., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. Supelco, Bellefonte, Pa., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. Applied Science Laboratories, State College, Pa., U.S.A. General Electric, Schenectady, N.Y., U.S.A. General Electric, Schenectady, N.Y., U.S.A. DuPont de Nemours, Wilmington, Del., U.S.A. DuPont de Nemours, Wilmington, Del., U.S.A.


Chapter 1

Introduction The steroids constitute a very large group of natural and synthetic compounds with a broad range of biological activities. Because they are of considerable importance in medicine, there has been a great deal of interest in various aspects of steroid chemistry, including their analysis. In biological extracts, steroids usually occur in low concentration and invariably in association with numerous structurally related compounds. The latter may be present in considerably higher concentrations, but may not have comparable biological activities. Thus, it is desirable that analytical methods for steroids be both specific and sensitive [ 1 121. As in the analysis of other groups of products, there is a strong trend toward instrumentation and automation in steroid analysis. Because most physical methods of analysis are not sufficiently selective, the analytical samples must be purified and, preferably, fractionated. The most efficient fractionation method available to the steroid chemist is chromatography. This is one reason for the strong association between steroid chemistry and chromatography. The other reason is that scientists who develop instrumental methods of analysis, including chromatographic methods, look toward the steroid analyst as a relatively well-endowed user with many challenging problems. One of these problems is that the associated steroids in an analytical sample are often analogous or isomeric compounds with very similar physical and chemical properties. Another problem is that steroids may belong to several solubility classes, ranging from rather hydrophilic to very lipophilic compounds. Some of these compounds are highly reactive and even unstable, whereas others are extremely sluggish and may even be devoid of analytically useful functional groups. So far, no single chromatographic method has been able to overcome all of these problems, and the analyst must have the ability to select the technique appropriate to the goal [770]. Thus, in addition to the obvious constraints of his knowledge and skill, available time and facilities, the analyst is limited by the chemical nature and physical condition of the steroids in samples of biological or synthetic origin. A few generalizations about the selection of techniques are in order, although many exceptions will be found in the examples of chromatographic analyses presented in the following pages. As a rule, such methods as ion exchange and electrophoresis are only suitable for ionic or ionogenic substances and are therefore largely inapplicable to neutral steroids. Generally speaking, partition chromatography will be more successful in separating the homologs of the more hydrophilic steroids, whereas adsorption chromatography is more apt t o resolve mixtures of analogous or isomeric steroids having a more lipophilic character. For crude or bulky sam.ples, old-fashioned column chromatography is still the method of choice, although it is not as efficient with respect to resolution, labor, and time as other chromatographic techniques. Qualitative analysis is most efficiently performed by thin-layer chromatography (TLC), because a number of samples and reference compounds can be tested simultaneously. For quantitative analysis and for the best resolution, gas chromatography (GC) is preferred, but high-pressure liquid chromatography (HPLC) has several potential

1

2

INTRODUCTION

advantages over GC. It provides larger capacity and greater choice of parameters and it usually requires no derivatization, while offering advantages of speed, convenience, and sensitivity that rival GC.

Chapter 2

Liquid column chromatography 2.1. SORBENTS

As the specific examples of liquid column chromatography (LC) in this volume show, silica is by far the most useful sorbent for steroids. Some of the important work on the relation between the structure of silica gel and the chromatographic behavior of steroids goes back to 1961, but it must be mentioned here because it was not covered in Neher’s book. Klein [566] has systematically studied the effect of surface area, pore volume, and average pore diameter of different types of silica on the resolution of sterol acetates by LC. Only when the pore diameter is large relative to the size of the sterol molecule can the molecule be attracted by a flat surface. Thus, 24-dehydrosterols, which have a smaller cross-section than sterols with a nuclear double bond, are more strongly adsorbed than the latter on silica gels with small pore diameters. Activation of silica gel by heat increases the range of surface energies and results in broadening or trailing of chromatographic zones [ 5671. Deactivation, which is best accomplished by exposure to an atmosphere of controlled humidity, will improve the performance of a silica gel only if it is adjusted to the surface strilcture of that particular silica. The water content of silica depends on the water content of the solvent [69]. By gradually increasing it, one can change the chromatographic system continuously from an adsorption to a partition system. It is even possible to achieve some sort of gradient elution effect by using a wet nonpolar solvent as eluent for a dry silica column. An example of the use of silica columns is the preliminary isolation of steroids from a crude lipid extract [429]. Up to 1 g of mixture, dissolved in pentane-diethyl ether (4: 1) can be fractionated on a column, 1 cm in internal diameter (I.D.), which has been packed with 7 g silicic acid (activity grade IIB), slurried in pentane-diethyl ether. Neutral lipids, sterols, and steryl esters are eluted by 150 ml pentane-diethyl ether (4: l), other steroids by 150 ml acetone-chloroform (2: l), and more polar lipids by 150 ml methanol. Another example is the use of Silica Gel G with a water content of 10%in a 5-cm X 1.8-cm column [210]. About 10-1 5 mg of lipid mixture can be fractionated on such a column by elution with a series of solvents. Thus, 60 ml petroleum ether (b.r. 30-754 elutes the hydrocarbons, 50 m l 6 % diethyl ether in petroleum ether the cholesteryl esters, and 160 ml of 10%ethyl acetate in petroleum ether the triglycerides, followed, in the last 60 ml, by cholesterol. The activity and thus the chromatographic properties of alumina can be reversibly modified in situ by passing organic solvents with different water content through a column of alumina [283]. Useful separations can be achieved on alumina thus deactivated. Alumina, although quite selective, is now discredited by the various alterations the more active grades have been reported to produce, and it is rarely used nowadays. However, an interesting method of labeling steroids by alumina chromatography should be mentioned [569]. By taking advantage of the enolization of ketosteroids on basic

3

TABLE 2.1

P

ELUTION VOLUMES OF STEROIDS ON DIFFERENT GELS IN DIFFERENT SOLVENTS EXPRESSED AS PER CENT OF TOTAL BED VOLUME 12781 Compound

LH-20

G-25-36

Dichloromethane

Isopropanol

Propanol

Methanol

45.5 45.0 61.5 59.0 73.5 44.0 151.5 42.0 43.5 48.5 48.0 51.0 50.0 71.0 239.5 -

69.0 72.5 69.0 74.5 70.5 77.5 72.5 90.0 77.5 68.0 70.5 74.5 88.0 78.0 84.5 81.5 115.5 113.5

70.0 73.5 70.0 75.0 72.0 72.0 85.0 75.0 69.0 71.5 71.0 86.5 78.5 83.0 81.5 114.0 109.5

76.0 76.0 -

Dichloromethane

Propanol

G-15-38

G-2540

Methanol

Dichloromethane

Dichloromethane

98.0 91.0 93.0 84.5 94.0 82.5 100.5 102.5

47.0 45.0 54.0 59.0 44.0 74.5 45 .O 43.0 5 2.0 54.5 52.5 63.5 138.0 -

57.0 55 .O 64.0

~

5aCholestane 5aCholestan-3-0ne 5a€holestan-3p-o1 5pCholestan-3-one 5p€holestan-3p-ol 5 @€holestane-3,12-dione 5pCholestane-3a, 12adiol 5p€holestane-3,7,12-trione Sp€holestane-3a,7a, 12a-trio1 30-Methoxyd-cholestene 3p-AcetoxyJ-cholestene 5a-Pregnane Progesterone 5a-Androstane 5a-Androstan-17-0ne 5a-Androstan-l7p-01 Estrone Estradiol

-

73.5 73.5 76.0 76.0 77.5 83.5 83.5 99.0 98.0

54.5 53.0

67.0

-

64.5

-

80.0 52.0 132.5 53.0

-

59.5 56.5 62.5 59.5 77.0 202.5

-

75.5 79.0 71.0 -

74.0 82.0 72.5 100.5 74.0 75.5 82.5 92.5 83.5 90.5 80.5 133.0 113.0

-

93.0 -

100.5 95.0 92.0 91.0

55.0 -

68.5 53.0 87.0 55 .O 54.0 62.5 57.0 -

-

-

SORBENTS

5

alumina, HTO on the column can be made to exchange with H in such ketosteroids as 5a-cholest-7-en-3-one,and sizeable quantities of tritiated ketosteroids of high purity (5 -10 mCi/mmole) can be prepared by sirnple chromatographic development. This procedure can also be utilized for analytical purposes [568]. A mixture of ketosteroids passing through a column of tritiated alumina becomes self-labeled, and the individual compounds are readily detected and measured in submicrogram quantities by monitoring the column effluent. Ion-exchange resins are useful sorbents for partition chromatography of nonionic compounds. Seki and Matsumoto [954] have pioneered in the application of partially esterified cation exchangers to steroids (cf. p.99). Columns of neutral resins, such as polystyrene with 2% divinylbenzene cross-linkages, can be developed with benzene to fractionate mixtures of lipids, including sterols and steryl esters [ 10561. Nystrom and Sjijvall [800] began to use lipophilic dextran preparations as supports for the stationary phase in reversed-phase partition chromatography of lipids in 1964. Methylated Sephadex G-25 was mixed with lipid solvents in which the sorbent does not float, e.g., chloroform-methanol (1 : l ) , and the slurry was poured into a chromatographic tube. The same solvent mixture eluted cholesterol, some of its esters, and various bile acids from this column, in generally decreasing order of polarity, but other solvent mixtures and other lipids gave other elution orders [801,802]. The effects noted were clearly not due to reversed-phase partition alone, but gel permeation, ordinary partition, and perhaps also adsorption may have contributed to them, depending on the degree of cross-linking and methylation of the dextran preparation and the chloroform/methanol ratio of the eluent. The theoretical basis of steroid chromatography on lipophilic Sephadex gels was studied in more detail by Eneroth and Nystrom [278]. Table 2.1 shows the per cent total bed volume (PTV), i.e. the milliliters of each solvent which would have been required to elute the compounds shown from a Sephadex column, if its total volume had been 100 ml. Sephadex LH-20 is a cross-linked dextran with hydroxypropyl ether groups, and Sephadex G-I5 and G-25 are dextran gels which exclude polysaccharides above a molecular weight of 1500 and 5000, respectively. The second number in the Sephadex G series denotes the per cent methoxyl content of the dextran. Generally, the PTV value of steroids without hydroxyl groups increases with the polarity of the solvent and with the methoxyl content of the sorbent, whereas the reverse is true of steroids carrying hydroxyl groups. The elution order with nonpolar solvents, such as dichloromethane, follows the general order of increasing polarity of the samples, whereas polar solvents usually elute steroids in decreasing order of polarity. For any given sample and solvent combination, the more porous gel gives the higher PTV value. Thus, combinations of a relatively nonpolar gel with a relatively polar solvent exhibit reversed-phase partition behavior, whereas ordinary partition chromatography may be at work in combinations of relatively polar gels with relatively nonpolar solvents. Ellingboe et al. [268] have prepared a number of long-chain alkyl ethers of Sephadex and tested them as column packing materials for partition and reversed-phase partition chromatography of sterols, bile acids, and steroid hormones. The reversed-phase systems, such as Sephadex G-25 with a hydroxyalkyl (CIs-Cls) group content of 71% by weight, eluted with methanol-heptane (19: l), separated C2,, CZs, and C29 sterols; Sephadex with

6

LIQUID COLUMN CHROMATOGRAPHY

TABLE 2.2 PER CENT TOTAL BED VOLUME OF STEROIDS ON A HYDROXYCYCLOHEXYL SEPHADEX COLUMN ELUTED WITH BENZENE m i Steroid

PTV**

OH* ~

5aCholestan-3-one 4Cholesten-3-one SCholesten-3-one 5aCholestan-3p-01 (cholestanol) SaCholestan-3a-01 (epicholestanol) 5pCholestan-30-01 (epicoprostanol) 5pCholestan-3p-01 (coprostanol) 5Cholesten-3p-ol (cholesterol) SCholesten-3a-01 (epicholesterol) 3p-Hydroxydaandrostan-17-0ne (epiandrosterone) 3a-Hydroxy-5a-androstan-1"-one (androsterone) 3a-HydroxyJp-androstan-17-one (etiocholanolone) 3p-Hydroxy-5p-androstan-1 "-one 3a-Hydroxy-5p-pregnan-20-one 3p-Hydroxy-5p-pregnan-2O-one 5Cholestene-3p,7pdiol S€holestene-3p, 7adiol 5pCholan-7p-01 Sp€holan-7c~-ol 11a-Hydroxypregn-4ene-3,2Odione 1lp-Hydroxypregn-4ene-3,20-dione 5a-Androstan-l7p-01 Sa-Androstan-l7a-01 5-Pregnene-3p,2Opdiol 5-Pregnene-3@,20a-diol 4-Pregnene-3,20-dione (progesterone) 3p-Hydroxy-5-androsten-17-0ne(dehydroepiandrosterone) 3p-Hydroxy-5p-pregnan-20-one 3p-Hydroxy-5-pregnen-20-one(pregnenolone) 5a-Pregnane-3p, 20pdiol 5-Pregnene-3p,2Opdiol -

e a

e a

e a

e a

e a

e a

e a

e a

e a rL-e

ILa more hindered less hindered

55.8 56.6 55.3 88.4 82.9 87.0 87.0 94.4 70.1 114 104 107 104 99.5 99.5 170 162 67.0 67.0 115 108 103 95.2 170 183 62.9 . 108 97.2 105 193 21 1

*Conformation: a = axial; e = equatorial. **Per ceilt total bed volume,

55% hydroxyalkyl (C1l-Cl4) group content, eluted with methanol-water-l,2-dichloroethane (7:3: l), separated bile acids; and a partition column of Sephadex with 58% hydroxyalkyl (CI5-Cl8) group content, eluted with heptane-chloroform (4:l), separated various pregnane derivatives. Using the lipophilic dextran with 50% hydroxyalkyl groups in columns eluted with benzene or benzene-isopropanol(3: l), Brooks and Keates [ 1181 further investigated the chromatographic behavior of many steroids. After experimenting with various other lipophilic dextran gels [20], Anderson et al. [23] observed a considerable enhancement in selectivity when dextran gels were substituted with hydroxycyclohexyl residues. Elution with benzene gave the PTV values shown in Table 2.2. Pairs of steroids with epimeric hydroxyl groups were generally

I

SORBENTS

resolved, unless they had Rings A and B cis-fused (50-steroids). Moreover, this chromatographic system separated 5-unsaturated steroids from their saturated Sar-analogs. In both 5a- and As-steroids, the equatorial alcohols were more retarded than their axial epimers. This is illustrated in Fig. 2.1. The mechanism underlying this separation appears to be adsorption of the steroids t o the gel, more specifically, hydrogen bonding to its ether linkages.

I

A

hours

8 0

60

80

I

lw

1

SEV

Fig. 2.1. Separation of epimeric 3hydroxysteroids on a hydroxycyclohexyl Sephadex column (1 m X 3 mm I.D.), eluted with benzene. A = epicholesterol; B = epicholestanol; C = cholestanol; D = cholesterol; E = androsterone; F = epiandrosterone. SEV = standard elution volume. (Reproduced from J. Chrornarogr., 82 (1973) 340, with permission; [23].)

The use of LH-20 columns is recommended for clinical analyses, where groups of steroids must be isolated from blood or urine samples (cf. Chapter 12, Sections 2 and 3), particularly for purposes of radioimmunoassay or competitive protein binding assay [ 100, 142,754,821,9551. They have also been found useful in biosynthetic studies of plants for the convenient separation of carotenoids from sterols [ 10291. Various other aspects of chromatography on lipophilic Sephadex have been reviewed [978]. For instance, it is used in recycling and capillary column chromatography with automatic detection systems [804]. Marker dyes facilitate the location of steroid fractions in routine applications [248]. Lipophilic Sephadex also exhibits cation-exchange properties when electrolytes are present in the solvents or samples [981]. This effect can be exploited for the isolation of conjugated steroids from biological sources. For instance, from a column of methylated Sephadex, eluted with a 1:1 mixture of chloroform and a 0.02 M methanolic solution of some salt, free steroids emerge before steroid monosulfates, which are followed by disulfates. The mixed steroid sulfates in cu. 5 ml plasma can be separated on a 4-g column of Sephadex LH-20 by elution of the monosulfates with 30-60 ml chloroform-0.01 M NaCl in methanol (1 : 1) and then of the disulfates with 65- 115 ml methanol [ 5 141. The separation of individual steroid sulfates can be accomplished by liquid-liquid partition chromatography on Celite columns [138]. The use of polyamide columns for the isolation of steroid conjugates has also been reported [807].

8


2.2. INSTRUMENTATION As in other fields of application, there has been a continuous trend toward automation in steroid analysis by liquid column chromatography. This section deals with instrumentation applied to gravity flow; high-pressure chromatography is treated in Section 2.3. One of the earliest solvent pumping systems for gradient elution in the Steroid Analyzer [19] consisted of a metal cam, shaped like a plot of solvent ratio us. time. The position of the arm of a linear potentiometer, following this cam, then regulated the rate of two reciprocating piston pumps, which delivered two solvents into a mixing chamber. This allowed elution programs to be designed for any particular fractionation [3 181. Fig. 2.2 illustrates this for the programmed gradient elution of corticosteroids. The solvent gradient system was subsequently modernized by substituting the more flexible punched tape for the metal cam to control the pumps [521] and an improved mixing chamber with minimum hold-up [522]. The Steroid Analyzer is suitable not only for multiple quantitative assays on collected fractions, but also for the isolation of minor components from large amounts of contaminants [520]. Another gradient system designed for LC of lipids produces concave gradients by the controlled addition of one solvent to the other in a mixing chamber with a cam-operated positive-displacement pump [772]. Equations permitting the calculation of any elution gradient produced in this manner have been derived by Cavina et al. [ 1581. Castellana and Kelly [ 1461 have calculated the shape of elution gradients that can be generated without the use of pumps by connecting two vessels of a certain shape, containing solvents of different densities. The application of such gradients to steroid chromatography was demonstrated.

Fig. 2.2. Programmed gradient elution of adrenocortical hormones. The tube number is plotted against elution composition, in % dichloromethane (DCM) in light petroleum ether, in the top portion and against UV absorbance of the fractions in the bottom portion. Q = Deoxycorticosterone; A = dehydrocorticosterone; S = deoxycortisol; B = corticosterone; E = cortisone; ALDO = aldosterone; F = cortisol. (Reproduced from Anal. Chem., 35 (1963) 2020, with permission; [318].)

INSTRUMENTATION

9

Vestergaard [ 11 16-1 1181 has made many contributions to the automation of steroid analysis by liquid column chromatography. He can now elute 25 columns simultaneously [ 1 1211 by pumping a solvent mixture with nitrogen at low pressure from a mixing bottle. The polarity of the eluent is gradually increased by metered addition of a more polar solvent to the mixing bottle. The chromatographic tubes are 6-ft.-long PTFE capillaries [ 1 1231. They are packed by either pumping a slurry from a wider tube into them or, preferably, by allowing the dry sorbent to enter the capillaries from funnels which are attached to a shaking machine [927]. Eluate fractions are analyzed with the aid of a computerized multichannel high-capacity read-out system [ 11 19,11201 . It has a capacity of 100 chromatograms per day when used as a three-channel instrument, but it can be expanded to 400 chromatograms per day by the use of twelve channels. In isocratic elution, a recording refractometer can be used for monitoring the column effluent. For RI determinations with changing eluent composition, the solvents must be evaporated and the residues must be redissolved in a single solvent. This is accomplished automatically in a device which collects the effluent on a moving Mylar or PTFE tape [ 11541. The tape passes from the column outlet first through an evaporating chamber and then past a solvent outlet, where the residue is redissolved, to an aspirator, which draws the solution into a recording refractometer. A universal detector for LC with great potential is the flame-ionization detector (FID). It requires a device for transferring an aliquot of the column effluent to the detector after the removal of the eluent. In the design of Haahti and Nikkari [382] the transfer is effected by an endless gold chain, which passes through a stream of hot air, where the solvent is evaporated, and then through the flame of a hydrogen FID. The combustion of the residue on the chain gives rise to an ionization current, which is amplified to give a detector response. Karmen’s [533] design differs from the one just described in the construction of the FID. Here, the chain passes through a T-tube, in which the volatile components of the residue are evaporated in a stream of hot nitrogen, and the nonvolatile components are then pyrolyzed. The resulting vapors are aspirated into the hydrogen-nitrogen line of the FID to ensure a quantitative transfer. The Barber-Colman Model 5400 Liquid Chromatography Detector, based on the design of Haahti and Nikkari [382], has been used in forerunners of HPLC, such as the microcolumn system of Stouffer and Oakes [ 10141. That system includes a miniature gradient mixer and permits rapid analysis of lipid mixtures in submicrogram quantities. Cavina et al. [ 1581 have made extensive use of the Barber-Colman FID. Combining elution with a concave gradient (see p. 8) of diethyl ether in petroleum ether (b.r. 65-75?, conventional silicic acid column chromatography, stream splitting, continuous detection, and gravimetric analysis of the eluate, they were able to fractionate milligram amounts of neutral lipids into classes, including sterols and steryl esters. In the Pye System 2 Liquid Chromatograph for HPLC, the effluent coats a steel wire, which passes through an evaporator oven and then through a pyrolyzer oven [ 1 1681. A stream of inert gas transports the vapors from the pyrolyzer into a hydrogen FID. The liquid spectroradiochromatograph is an apparatus for the automatic determination and radioassay of steroids and other nonsaponifiable lipids in the effluent from a chromatographic column [221]. In addition to an automatic gradient elution device, the machine features a special sample injector and three monitors: a recording spectrophoto-

10


meter, a scintillation counter (both equipped with flow cells), and an argon ionization detector. The response of all three monitors is recorded, and the effluent is collected in a fraction collector. A device for forcing eluents through very fine silica gel consists of a centrifuge head with assemblies of short chromatographic columns and loading funnels in radial array [889, 8901. After these assemblies have been centrifuged, the adsorbent may be extruded and stained. Steroid mixtures applied to columns of microparticulate silica, ranging in particle size from 0.02-0.7 pm [ 11831, were sharply separated. The commercial instrument (Centri-Chrom) also permits the collection of eluents in attached cups, but steroids may also be eluted from sections of the extruded column.

2.3. HIGH-PRESSURE LIQUID COLUMN CHROMATOGRAPHY Siggia and Dishman [971] were among the first to apply HPLC to steroid analysis. Best results were obtained by dry-packing of glass tubes, 50 cm X 2 mm I.D., with a trifluoroethylene polymer (Plaskon CTFE-2300, OAc > TMS > TFA, whereas on SE-30 the order is OAc > TMS 2 OH > TFA, and on XE-60 it is OAc > OH > TMS. Thus, the polarities of the columns are in decreasing order: HI-EFF-8B = NGS > XE-60 > SE-30. Further studies on the retention behavior of androstane derivatives have been published by Hiscoe et al. [443]. Other derivatives useful for GC of androstane derivatives are the n-butyrates [916], formates [690], chloroacetates [826], CMDMS ethers [ 1053],O-methoximes [294], and 0-butyl- and 0-pentyloximes [45]. The extent of preliminary purification depends on the biological source and concentration of the CI9 steroids [705]. For the quantitative estimation of the major urinary 17ketosteroids, no purification was used [906] or the urine extract was fractionated by TLC in ethyl acetate-benzene (2:3) [68]. The dehydroepiandrosterone zone which is eluted also contains androsterone and etiocholanolone. Before GC analysis of 5a-androstane-3a,l7pdiol, the urine extract was purified by alumina column chromatography as well as silica gel TLC [72]. Combinations of TLC and GC were used to resolve the four epimers of 5aandrostane-3,17-diol [74] and other urinary androstanediols [73,761]. The determination of androsterone and dehydroepiandrosterone sulfates in serum by GC was preceded by chromatography of an acetone-ethanol extract on methylated Sephadex G-25, solvolysis, and another column chromatogram on silicic acid [980]. Similarly, dehydroepiandrosterone

92

ANDROSTANE DERIVATIVES

sulfate was isolated first by chromatography on the anion-exchange resin Amberlyst XN1006 19841. The analysis of testosterone in urine by GC involved gradient elution from an A1203 column [920] or two A1203 chromatograms, one of the free steroid and one of its TMS ether [1122]. In another approach, the acetate was purified by gradient elution from a disposable SiOz column [ 1 1491. Purification by TLC can be simple if the urine extract has gone through a Girard separation [338]. Otherwise, two developments by horizontal TLC [673], or TLC of testosterone followed by TLC of its acetate [493,999], may be required. Demish and Staib [229] found that the TMS ethers of testosterone and its hydroxylated metabolites are adequately purified by TLC with cyclohexane-ethyl acetate (4: 1). The testosterone concentration in plasma is so low that it can only be determined by the ECD. Labeled testosterone may be added to trace the steroid in the purification process and to correct for manipulative losses. The plasma extract is usually subjected to TLC, the eluted testosterone is converted to an electron-capturing derivative, and then the derivative is purified by TLC [ 1951, but two alumina columns may be similarly employed [ 10521. Suitable derivatives are: the heptafluorobutyrate [617], the monochioroacetate [ 1261, the hexadeca- and eicosafluoroundecanoate [563], the iodomethyldimethylsilyl ether [ 10521, and the 0-methyldichlorotetrafluoroketal[753]. The bromomethyldimethylsilyl ether of testosterone is suitable for the estimation of testosterone by GC-MS, because it is formed quantitatively and shows a relatively intense molecular ion [180]. When 'H-labeled testosterone is added to the plasma as an internal standard, no elaborate purification is necessary, but a simple extraction, followed by derivatization, permits the accurate determination of as little as 0.8 pg testosteroneflO0 ml plasma by multiple peak monitoring. Dehydroepiandrosterone in plasma can be determined by the ECD if it is oxidized t o 4-androstene-3,6,17-trione, which is electron-capturing [525].

Chapter 10

Pregnane derivatives This chapter deals with the chromatography of CZl steroids, with the exception of the corticosteroids, which are treated in Chapter 1 1, but it includes the Czl alkamines. The pregnane derivatives are relatively stable and nonpolar compounds, substituted a C-3 and often at C-20 as well. They are metabolically related t o progesterone, which contains an a$-unsaturated carbonyl group, like most steroid hormones. The chief urinary metabolite, 5/3-pregnane-3a,20cw-diol,is excreted as the glucosiduronate. There have been very few publications on LC in the past ten years. A method for the determination of urinary pregnanediol was based on the centrifugal microparticulate-bed chromatography described on p. 10 [926]. Gel chromatography on Sephadex LH-20 was used in the isolation of Sa-pregnane-3a,ZOa,21-triol [979] and hydroxyalkoxypropyl Sephadex in the protein-binding assay of 17-hydroxyprogesterone [456]. King er al. [554] have developed a convenient assay method for progesterone in oil, based on HPLC. The progesterone is extracted with 85% ethanol, and the extract is injected into a 1-m X 2.1-mm I.D.column, packed with spherical glass microbeads, 30 pm in diameter, which have been coated with 1% octadecylsilane. Reversed-phase partition with an 18% aq. isopropanol solution at 42-44°C separates progesterone from the other ingredients in the preparation.

10.1. THIN-LAYER CHROMATOGRAPHY The definitive work on TLC of 21-deoxypregnane derivatives was done by Lisboa [652,656,658]. Table 10.1 excerpts some of the ~ R values F from that work. In many cases where resolution was not possible by a single development, it was accomplished by the simple expedient of multiple development. For unsaturated steroids, argentation TLC frequently proved to be effective. The order of mobilities was generally for oxygen functions at C-20: 0 > /3 > a , and for oxygen functions at C-3: 3/3(50) > 3a(5a) > 3/3(5a) > 3a(5P), i.e., as in other series,'the steroids with axial hydroxyl groups were more mobile than those with equatorial ones. In addition to the nonspecific detection methods, Lisboa used the 2,4-dinitrophenylhydrazine reagent for ketonic steroids, the UV absorption on Silica Gel GFZ54 plates for 4-en-3-ones, and the nitroprusside reaction for the methylketones. A highly specific brickred color is produced when 1 1/3-hydroxyprogesterone on Silica Gel G is sprayed with conc. HZSO4and heated at 70-72°C for 0.5-1.0 min [1073]. TLC is used extensively for the quantitative estimation of 5P-pregnane-3a,20a-dioland its congeners in the urine. This usually involves enzymatic or acid hydrolysis and extraction of the urine, followed by TLC with, e.g., chloroform-acetone (9: 1). The steroid may be located with water or iodine vapor, eluted with chloroform-methanol (1 : l), and determined colorimetrically [309,806,1022]. Alternatively, the H,S04 reaction can be performed directly on the scraped-off silica gel [49,453,810,844,933,1048].The direct densitometric procedures are more convenient [79,536]. TLC was also used in the

93

94

PREGNANE DERIVATIVES

TABLE 10.1 ~ R F V A L U E SOF PREGNANE DERIVATIVES IN TLC ON SILICA GEL (Data from Refs. 652,656,658)

Substituents*

Solvent system*

3

4

5

6

a a

-

a

-

-

-

a

-

-

-

P P

-

a a

-

-

-

a a

-

p p

-

P P P

-

P P

-

a

-

a P

P P

-

a

-

a

a

-

a

-

a

-

a a

-

p p

a -

P

-

P

-

-

-

-

a

P P P P P 0

-

-

p

p

P

-

-

a a

a P

a

-

-

-

-

0

-

-

a a

-

a

-

a

-

P

-

P

-

-

a

-

p

-

-

a

-

a

-

a P a

a

-

p

a

-

-

6 P 0

-

O

A

-

-

0 O 0 a

-

a P 0

P

-

a

a

-

P 0

-

o

-

p

-

o

0

-

0

0

-

p a

-

0 o

a

-

o

O

A

-

O

A

-

O

A

-

-

0

P

-

-

P

a a

P a

0 P

P P P &

a a

-

a

P

a

P

-

a

&

a -

P

-

-

-

-

-

-

0

a a

-

0

-

-

-

0 -

a

a

p P

-

-

-

-

-

P

-

-

-

-

-

-

-

a a

p

-

-

a

a

-

-

a

a

1 1 1 6 1 7 2 0 1

-

-

-

-

a a

(Y

a a P 0 0 0 0 0 0 0

0

a a a a -

a 0 0 0 0 0 0 0 0 0 0 0

-

P

-

a

P

59 52 51

49 54 56 59 30 27 24 21 18 15 26 34 32 39 34 36 9 I 57 64 55 59

56 52 26 23 46 50 24 62 66 64 66 42 48 49 45 56 53 53 56 50 44

2

3

4

5

6

7

8

9

32 34 27 31 22 25 34 37 -

55 55 50

31 34 33 35 28 31 37 40 11 13 9 7

68 70 65 70 65 68 70 I0 47 47 39 38 32 30

59 52 56 50 56 59 61 34 34 26 25 18 16 30 39 37 45 41 45 3 4 65 63 25 22 52 58 63 68 70 66

58

35 34 30 33 33 36 40 41 14 13 14 11 10 8 20 26 23 29 25 27

-

-

5 11 8 16 10 13 38 42 33 36 39 29

-

4 15

25

-

38 53 50 38 10 14 8 12 26 22 27 27 24 16

-

52 50 52 57 30 30 26 24 20 16 23 35 32 41 33 38 4 5 -

71 62 61 33 29 42 52 25 75 75 73 71 54 57 44 37 76 13 14 63 60 51

5

4 7 15 12 19 14 18 50 60 45 52 49 45 12 9 20 33 67 70 69 57 38 40 19 26 69 66 66 55

52 27

45 55

51 61 58 13 10 76 71 74 -

38 37 64 68 72 78 76 75 54 59 62 57 68 65 65 70 67 61

40

47 49 41 56 54

56 53 56 52

54 54 57 40 39 41 37 36 32 39 47 44 49 45 17 19 62 65 58 59 61 61 46 43 57 61 68 70 70 54 -

58 -

49 47 67 68 67 56 54

50

55

55

-

-

-

-

-

-

-

47 51 42 47 41 44 18 15 30 36

-

-

-

56 63 63 47 31 33 19 24 53 49 51 38 36 32

42 48 -

-

-

-

-

20 18 -

THIN-LAYER CHROMATOGRAPHY

95

TABLE 10.1 (continued)

_-

-.

Substituents* 3 4 5

6 a

1 -

1

1 -

-

0

-

O O O

A A A

-

O

A

-

-

O

A

-

-

O O O O O

A A A A A

-

0

-

0

O

A

-

O

A

-

O @ 0 P

A -

A A A

-

@

-

A

6 p 6

-

@ P

-

& a

-

@ P P @

-

6

-

-

-

6 -

-

a

A

a

-

-

-

OL

-

-

a a a

-

P 0 -

-

-

A A A

-

A A

-

A A A A

-

A A

-

A

-

-

a

0 p

a

-

A a

-

-

-

a

a a a

-

-

a

-

a a

-

-

a a a

-

-

-

-

0

a

-

-

-

-

-

-

-

-

Solvent system* 1 1 2 0 1 2 3 4 P 39 10 48 19 65 P 34 5 38 11 0 41 12 51 27 a 36 9 48 23 0 59 38 12 68 14 0 49 15 55 31 61 0 46 14 51 40 60 0 31 6 43 24 43 0 29 6 47 35 44 0 56 21 64 52 12 0 43 11 40 20 62 0 26 6 5 6 3 5 44 16 I0 60 62 0 0 54 31 58 48 12 53 31 57 49 71 0 0 35 9 41 21 52 0 51 28 50 33 70 a 44 13 37 13 66 0 34 I 31 11 51 0 35 12 47 26 52 a 50 21 48 33 66 p 53 31 50 36 69 a 30 2 32 11 42 21 2 2 2 8 4 1 a 30 14 38 15 46 25 8 26 10 44 a 38 11 36 14 57 p 41 16 40 22 61 a 29 5 21 6 51

5 44

-*

42 31 46 34

-

-

-

38 58 49 33 44 26 21 27 26 40 43 31

6

7 51 -

-

65 55 54 45 52 58 50 60 56 55 41 53 46 40 49 50 42 48 43 36 44 41 36

8 27 22 32 28 55 30 32 20 25 40 22 29 44 42 42 26 35 31 23 25 33 34 19 16 22 14 23 30 19

*See the footnote and the legend to Table 9.1 (pp. 88 and 89).

specific analysis of urinary pregnanediol by the double isotope derivative dilution technique [265]. In the determination of progesterone and its congeners in plasma similar approaches have been used. For the elution from silica gel, methanol should be used [ 10041. Hobzovi and Novak [448] have chromatographed the 2,4-dinitrophenylhydrazoneof progesterone, which is easily assayed by spectrophotometry. For direct scanning, Keller et al. [548] and Weiss [ 11531 determined the absorption at 254 nm of progesterone on fluorescent silica gel, whereas Egg [264] measured the fluorescence produced when progesterone on an A1203FZs4 plate Type T is heated at 150°C for 20 min. TLC was likewise used for the purification of progesterone in double isotope derivative methods [895,1158]. The separation of CZl steroidal bases by TLC has been investigated by Libler and t e r n 9 [623,624]. Using benzene or diethyl ether, previously saturated with NH40H, in chambers with a NH3-saturated dtmosphere, they systematically studied the effect of substituents on chromatographic mobility. Because the axial substituents were less polar

9 -

-

40 14 14 I 8 22 7

-

21 -

-

-

96

PREGNANE DERIVATIVES

than the equatorial ones, they observed that the order of mobility for alkaloids substituted with a -N(CH3)2 group at C-3 is: 3a,5a > 3a,As > 30,50 > 3a,5p > 3/3,A5 > 30,5a, and for those with a -N(CH3)2 group at C-20 the 0-substituted steroids move faster than the a-substituted ones. Bennett and Heftmann [64,65] used 0.1 N NaOH instead of water in the preparation of the silica gel plates and chromatographed Holarrhena alkaloids with various water-saturated solvent systems.

10.2. GAS CHROMATOGRAPHY

Earlier experiments having shown that progesterone [337] and other pregnane derivatives [773] can be chromatographed on SE-30 and other packed columns, investigators began to apply GC to the analysis of pregnanediol in urine [204,515,840] and bile [6]. The free steroids were used, as well as their acetates [ 11731, TMS [ 15 I ] , and MO [ 101 derivatives. For the analysis of pregnanediol in urine, preliminary purification by preparative TLC, e.g., with chloroform-acetone (9: 1) [63 1] was recommended, although other methods, e.g., precipitation by 70% (NH4)?S04 [ 1811, have been proposed. Nowadays, however, urine extracts are usually not laboriously purified. Pregnanediol diacetate is analyzed on a 6-ft. column of 3% SE-30 at 250°C [ 182,3621, or 1% SE-30 at 270°C [856], on a 5-ft. column of 2% OV-101 at 230-250°C [736], or 3%OV-1 at 240°C [913]. Free pregnanediol can be determined on a column of 0.5% NPGA at 235°C [717] or 3.8% SE-30 with programmed temperature [27]. For routine analyses of pregnanediol, one of the available automatic sample injection systems may be used to increase the processing rate [27,856,913]. Occasionally, other progesterone metabolites may be sought in the urine. For instance, in the presence Metcalf [7 181 has analyzed pregnanolone (3a-hydroxy-50-pregnan-20-one) of pregnanediol on a 2% XE-60 column, but found that pregnanetriol (50-pregnane3a,l7a,20a-triol) interferes. A method for the simultaneous determination of pregnanediol and pregnanetriol by GC was perfected by Rahman [874]. Pregnanediol, pregnanetriol, pregnanetriolone (3a, 17a,20a-trihydroxy-S/3-pregnan-l1 -one), and pregnanetetrol(50pregnane-3ql lp,l7a,2Oa-tetrol), when chromatographed in the form of TMS ethers on 1.5% SE-30 at 219'C gave retention times relative to cholestane of 0.96, 1.45, 1.93, and 2.38, respectively [555]. For an additional estimation of urinary S-pregnene3/3,17a,2ktriol, which gives an R T of 1.65 under these circumstances, that steroid is isolated by preliminary chromatography through an alumina column 15561. Pregnanediol, pregnanetriol, and pregnanetriolone have also been analyzed as the TMS ethers without preliminary chromatography [213] or after fractionation on alumina thin-layer plates [877]. Another approach to the simultaneous analysis of pregnanetriol and pregnanetriolone is to convert them to the acetates after alumina column chromatography and to separate the acetates by GC on a 1.2-m X 3-mm I.D. column of 3.8% SE-30 at 230-238'C [632]. The low concentration of progesterone in plasma makes it necessary to account for losses in the isolation procedure. This may be accomplished by adding radioactive progesterone as an internal standard at the beginning of the isolation [ 196, 11821. Preliminary purification by TLC [675], which is almost invariably needed, utilizes the W ab-

GAS CHROMATOGRAPHY

91

sorption of progesterone 15481 or the radioactivity of the tracer [ 11821 for detection. Use of solid injection into a 1% XE-60 column with a FID was recommended [ 118.51. The ECD may be used if progesterone has been reduced to 20P-hydroxy-4-pregnen-3-one by 200-hydroxysteroid dehydrogenase and the chloroacetate has been prepared [73 11. A more convenient electron-capturing derivative is the HFB 3-en01 ester of progesterone, which is prepared by heating the sample with 10 1.11 abs. tetrahydrofuran, 25 1.11 benzene, and 25 p1 HFBA at 65°C for 30 min [304]. Pregnenolone (3fl-hydroxy-5-pregnen-20-one) in biological samples can be determined in the nanogram range if the sample has been purified by TLC and the steroid enzymatically reduced (see above) [868]. The 3,20-dichloroacetate is then assayed by an ECD. For the analysis of 16a-hydroxyprogesterone in amniotic fluid, the purified steroid was converted to the TMS ether, which was chromatographed on a 6-ft. column of 3% QF-1 or OV-1 [325]. The determinations of progesterone in milk by GC and by radioimmunoassay give essentially the same results [798].


Chapter I I

Corticosteroids The adrenocortical hormones are characterized by a A4-3-keto group and an a-ketol side chain. Both features facilitate their detection by TLC and HPLC, but they are rather unstable in GC. The individual hormones differ from each other by oxygen substituents, usually at C-11, (2-17, or C-18. The urinary metabolites are usually saturated compounds, many of which retain the hydroxyl group at C-2 1. They are largely conjugated with glucuronic or sulfuric acid at (2-3. There has been much interest in the analysis of both natural and synthetic corticosteroids, but no comprehensive reviews have been written on this subject.

11.1. LIQUID COLUMN CHROMATOGRAPHY

Silica gel columns are useful, not only for the preliminary fractionation of corticosteroids [786], but also for the automatic determination of individual 17-ketosteroids and adrenocortical hormones [520,626,627]. For that analysis, a column of silicic acid with 60% water content is eluted with a linear gradient of increasing dichloromethane concentration in petroleum ether (cf. also Fig. 2.2). Apparatus has been designed for the simultaneous development of twelve capillary columns filled with silica gel, having ca. 20% water content, by a gradient of acetone in chloroform [1124] (see p. 9). An elegant machine for this type of work was devised by Cavina et al. [ 140,1551. It provides a suitable gradient of methanol in chloroform with solvent delivery at 500-600 p.s.i. to a column, 560 mm X 6 mm, containing silicic acid, 325 mesh, of known water content. The corticosteroids are measured by a UV detector as well as by a FID. Celite has been used for partition columns in corticosteroid chromatography. For instance, the double isotope analysis of aldosterone involves three Celite columns [545]. It should be noted that under these conditions there is some isotope fractionation, the tritiated aldosterone separating from unlabeled aldosterone [ 1631. From the partition coefficients, Weber et al. [ 1 1501 predicted the elution curves of various synthetic corticosteroids on a Celite column. Partially esterified Amberlite IRC-50 was used in the separation of 17-hydroxycorticosteroids and 17-ketosteroids by column chromatography [954]. Subsequently, Seki [953] found that Sephadex LH-20 columns also produce partition chromatograms of 17-hydroxycorticosteroids. Such columns have been used to separate labeled corticosteroids in biosynthetic research [960]. Sephadex LH-20 has also found use in the purification of aldosterone [219,1140] and deoxycorticosterone [ 1491 for radioimmunoassays. A marker dye facilitates the collection of the aldosterone fraction [494]. An automated procedure has been devised for the isolation of corticosterone, 1 1-deoxycortisol, aldosterone, cortisone, and cortisol by chromatography through 15-g Sephadex LH-20 columns of 11 mm I.D. [974]. A battery of six columns is simultaneously eluted with metered quantities of dichloromethane-methanol(49: l), and the eluate fractions are individually collected.

99

CORTICOSTEROIDS

100

$ 0 (u

*0 C

P

P 0, R 0 3 > c

8 2

I

I

’pRetention time (min)

Fig. 11.1. HPLC of a steroid mixture on a column of Spherosil XOA-400 with the organic phase of a 948:35: 17 mixture of dichloromethane, ethanol, and water. (Reproduced from Z . Klin. Chern. Klin. Biochem., 12 (1974) 194, with permission; [433].)

Because corticosteroids absorb UV light and have suitable solubility properties, they are ideal subjects for HPLC. Interest in this application is especially great, because GC is not easily adapted to corticosteroids. The first experiments with silica and bonded layers of octadecyltrichlorosilane or cyanoethylsilicone as column packing materials gave moderately good resolution of adrenocortical hormones [822,1071,1170]. Using Zorbax SIL (porous siiica microspheres) as adsorbent and dichloromethane-methanol-water (48: 1:1) as eluent, adequate separation of cortisone, prednisone, cortisol, and prednisolone (in order of elution) could be accomplished [835], and the procedure was found applicable to the quantitative analysis of plasma 110741. Hesse et al. 14331 have devised a convenient routine method for the determination of nanogram quantities of corticosterone and was packed with cortisol in plasma, based on HPLC. The column, 300 mm X 2 mm I.D., Spherosil XOA-400,4-8 pm, and eluted with the organic phase of a 948:35: 17 mixture of dichloromethane, ethanol, and water at a rate of 1 ml/min. A model chromatogram is shown in Fig. 1 1 . l . A “preparative” application of HPLC was recently announced by Pei el aE. 18421. A Vydac column, 410 mm X 15.8 mm I.D., separated 0.87 mg of a five-component corticosteroid mixture in 30 min by elution with heptane-chloroform-methanol(79: 19:2) at 100 p.s.i. and 15 ml/min. It is fair to say that these accomplishments will be considerably improved upon by the time this book has been published.

THIN-LAYER CHROMATOGRAPHY

101

11.2. THIN-LAYER CHROMATOGRAPHY There have been some attempts to use polyamide layers for TLC of corticosteroids [321,480], and an evaluation of the applicability of silica layers on polyester sheets has been published [869]. However, the bulk of the TLC methods presently used is based on conventional gypsum-bound silica on glass plates. Table 1 1.1 summarizes the results observed on Silica Gel G plates by Lisboa [655,658]. Table 1 1.2 gives additional ~ R F values, obtained with Polygram Sil G sheets by Smith and Hall [993]. The separation of Compound S (1 7a,2 l-dihydroxy-4-pregnene-3,2O-dione) from Comis difficult, but succeeds with a 4: 1 pound B (1 1/3,2l-dihydroxy-4-pregnene-3,20-dione) mixture of diisopropyl ether-acetone [640]. Compound B separates from aldosterone and 18-hydroxycorticosterone with chloroform-acetone (3: 2) ( ~ R values: F 5 6 , 3 3 , and 18, respectively) [336]. Two-dimensional systems for difficult separations have been described [70,870]. Thus, aldosterone can be very neatly separated from other corticosteroids by two-dimensional TLC with ethyl acetate-95% ethanol (7:3) or with chloroform-95% ethanol (49: 1) 19701. Spraying of the chromatoplates with a concentrated solution of ascorbic acid in ethanol will inhibit air oxidation of corticosteroids during radioautography [324]. Because TLC is inadequate and ion exchange is too cumbersome for the separation of conjugated corticosteroids, Kornel et al. [ 5921 have recently devised PC procedures based on continuous development of Whatman No. 3MM filter paper strips, which are in some instances treated with boric acid. The solvent systems are mixtures of petroleum ether, isopropanol, and water, and the method permits closely related conjugates of corticosteroids and of androstane derivatives to be separated. Lisboa [648] has described no less than 34 reactions suitable for the detection of corticosteroids and related compounds on thin-layer chromatograms. In addition to general tests, such as treatment with 50% H2S04, which may nevertheless produce very sensitive and specific responses [235], there are quite a few which are due to characteristic features of corticosteroids, such as the a$-unsaturated carbonyl group or the a-ketol side chain. The former is responsible for the W absorption and for the reactions with tetranitromethane and with Os04 and the latter for the reduction of phosphomolybdic or arsenomolybdic acid and of various tetrazolium salts. The sensitivity of the tetrazolium test is greatly increased when Tetrazolium Blue is incorporated in the silica layer during preparation [ 11 131. Most of the numerous methods for the determination of urinary corticosteroids by TLC are based on enzymatic hydrolysis, extraction, isolation from a thin-layer chromatogram on the basis of W absorption or Tetrazolium Blue reduction, and elution with ethanol or chloroform-acetone (1: 1). The eluate may be rechromatographed and the individual zones separately eluted and analyzed with Tetrazolium Blue [ 162,7841, H2S04 [ 10281, or Porter -Silber reagent [97,482]. An exacting technique and apparatus permits the accurate determination of corticosteroids in nanogram quantities [308]. The steroids are detected in W light on layers of Macherey-Nagel HRzS4 silica gel and are then eluted for fluorimetric analysis with minimal interference. An interesting approach to the estimation of corticosteroids is the reaction of 14C-labeledTetrazolium Blue with the zones on a paper chromatogram, which are then

TABLE 11.1 hRpVALUES FOR CORTICOSTEROIDS IN TLC ON SILICA GEL G (Data from Refs. 655 and 658) Substituents*

Solvent system*

3

4

6

O

A

-

O

A

-

O O O O O

A A A A A

-

O O O O O

A A A

-

A

D

A

O

A

O

A

O O O

A A A

-

O O O O O

A A A A A

11 0

16

17

18

-

-

-

-

-

-

-

a

a

0 O O D -

-

a

-

6

-

-

21

1

2

3

4

5

6

7

8

9

0 0 0 0 0 0 0 0 P

OH OH OH

40 22 13 14 38 31 16 16 9 9 12 15 3 28 16 18 21 21 17 -

13 9 -

62 31 20 22 41 38 30 26 16 17 14 21 4 18 11 16 29 14 49 34 16 24

51 15 7 9 22

55 37 22 26 57 42 30 26 20 21 26 28 9 44 28 23 44 39 37 47 31 35

46

55 38 26 31 49 42 31 32 25 25 24 30 12 37 25 26 42 43 28

38 16 9 11 23 22 16 16 13 12 6 10 -

20 5 8 -

a

-

OH 0

a

-

a

-

a

-

-

a

-

0 P

a a

-

P

a

-

0 0 0 0 0 0 0

a

-

0

a

-

0

a

-

a

-

-

a

-

-

P -

p O O O O

a a

a a

-

-

-

a

20

-

-

19

OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH

*See the footnote and the legend to Table 9.1 (pp. 88 and 89).

4 5 -

-

9 39 24 15 13 -

8

26 14

28 21 23 29 20

-

-

23 17 -

-

8 5 -

cl 0 ;a 4

ii

3m F

s

GAS CHROMATOGRAPHY

103

eluted and counted [ 1 1 141. The double isotope analysis of aldosterone involves both TLC and PC [ 1101. A rather complex procedure is evidently necessary for the specific determination of the very low quantities of aldosterone in blood. The direct densitometric evaluation of corticosteroids on thin-layer chromatograms requires staining with Tetrazolium Blue if the instrument is a spectrophotometer [400]. With a fluorometer, the quenching of the background fluorescence can be read directly [ 10831. The fluorometer can also be used for corticoid metabolites lacking the a,p-unsaturated carbonyl group by scanning a layer containing a fluorescence indicator before and after it has been treated with Tetrazolium Blue [ I ] . The analysis of aldosterone in urine requires rather efficient TLC procedures. Ripa [896] purified the urine extract by chromatography on a silica gel column and applied the eluate to a thin layer of kieselguhr. Two-dimensional chromatography with chloroform-ethanol (9: 1) and diethyl ether-benzene-acetone (5:3:2) was effective in isolating aldosterme. In the method of Gerdes and Staib [351], the aldosterone zone from TLC on silica gel with chloroform-ethanol (9: 1) is eluted, the eluate acetylated, the product chromatographed with cyclohexane-chloroform-acetic acid (4:5: l), again eluted, and chromatographed a third time with chloroform-ethanol (9: 1). Cavina et al. [ 1531 use a silica column, followed by TLC with chloroform-methanol-water (225 :25:2), but then resort to two-dimensional TLC with ethyl acetate-methanol-water (85: 15: 1) in the first dimension and continuous development for 4%h with benzene-acetone-acetic acid (140:60: 1) in the second. Other examples of the application of TLC to the analysis of specific adrenocortical steroids are the determination of 6P-hydroxycortisol [7 11 and of corticosterone [915], TLC is now being replaced by radioimmunoassay methods for hormone analysis, but continues to be useful in the preliminary purification of biological extracts. Material interfering with the radioimmunoassay of urinary aldosterone, which was not eliminated by two silica gel chromatograms, with dichloromethane-96% acetic acid-methanol (1 8:2: 1) and with chloroform-95% methanol (23:2), was finally removed by TLC on a cellulose layer with 95% ethanol [937]. PC is now eclipsed by TLC, but continues to find use in the analysis of conjugated hormones. Aldosterone 18-glucosiduronate, tetrahydroaldosterone glucosiduronate, and aldosterone sulfate have been isolated from urine by PC [374].

11.3. GAS CHROMATOGRAPHY The analysis of corticosteroids by GC has recently been reviewed by Menini [712]. Early attempts to exploit the thermal decomposition of corticosteroids during GC for analytical purposes having failed [368], Tsuda et al. [ 10801 experimented with H I 0 4 and NaBi03 oxidation prior to injection. The 17-ketosteroids produced upon injection of the tetrahydro derivatives can be utilized for quantitative analysis [670], but in this case it is also preferable to carry out a NaBi03 oxidation before GC [ 10001. Complete schemes for the determination of corticoids in biological fluids, based on either NaBiOJ 1411 or HI04 [564] oxidation or both [53], have been presented. The procedure of Craig and Chamberlain [207] involves NaBH4 reduction, followed by HI04 oxidation, and finally acetylation.

c

P

TABLE 11.2

hRF VALUES FOR NATURAL CORTICOSTEROIDS IN TLC ON POLYGRAM SIL G [993] Solvent systems: 1 = 1,2-dichloroethane-ethanol(7: 1); 2 = 1,2dichloroethane-rert.-butylalcohol (17:3);3 = 1,2-dichloroethane-acetone (67:33);4 = 1,2-dichloroethanedioxane (3:l);5 = 1,2dichloroethane-acetic acid (31:9);6 = 1,2dichloroethane-pyridine;I = propyl acetate; 8 = isobutyl methyl ketone; 9 = nitromethane-ethanol (37:3);10 = anisole-ethanol (9:l);1 1 = diisopropyl ether-ethanol (9:l);12 = diisopropyl ether-tert.-butyl alcohol (4:l); 13 = diisopropyl ether-acetone (7:3);14 = diisopropyl ether-dioxane (4:1); 15 = diisopropyl ether-acetic acid (3:1); 16 = diisopropyl ether-pyridine (4:l). Substituents*

3

4

5

O

A

-

a

-

a O a a O a a

A A A

p a p

O

a f

0 a O a

a

O 0 O

f

Solvent system

f

p

6 f

17

18

-

a a a

-

-

-

-

-

0

-

0

a

-

o

-

-

P

0

-

p

A

a -

-

-

p

A -

-

A

-

p

A A

-

-

11

p (Y

p

0 o p p p p

-

-

D

20

0 0 0 O O O O O O 0 0 0 0 0 0 (Y 0 a 0 a 0 a! 0 0 0 0 O O O

*Substituents, see Table 9.1 (pp. 88 and 89).

21

1

2

3

4

5

6

7

8

9

1 0 1 1 1 2 1 3 1 4 1 5 1 6

OH OH

66 44 50 42 26 30 55 28 33 38 26 27 36 21 26 18 19 23 14 27

61 45 55 43 25 33 50 24 30 34 22 25 34 17 22 12 15 20 10 23

66 50 59 52 28 37 51 25 31 40 24 29 41 19 27 14 17 21 13 21

82 70 75 64 41 51 72 45 53 51 41 46 50 25 34 18 23 28 13 36

66 61 65 50 41 44 54 41 44 45 37 40 33 22 24 16 19 15 6 30

69 51 59 55 32 40 59 30 38 43 26 31 48 24 33 18 20 30 19 34

53 45 57 54 43 55 32 18 24 34 26 32 41 21 39 30 36 26 26 18

53 42 59 54 40 56 35 16 24 34 25 32 41 22 37 26 34 25 24 12

58 45 51 48 29 41 54 35 39 43 28 30 45 27 33 20 22 27 21 25

60 47 54 46 30 37 49 31 36 41 31 34 36 21 31 20 24 27 19 29

OH H H H OH OH

OH OH OH OH OH

OH OH OH OH

OH OH H

46 49 54 47 46 53 17 16 20 29 27 32 33 30 34 30 34 21 22 14

53 61 70 62 64 72 23 19 29 39 40 48 44 43 49 50 56 33 35 21

55 51 61 48 42 52 33 20 28 36 29 38 34 24 33 26 31 22 18 20

42 37 49 35 27 38 20 12 18 22 18 27 20 12 17 14 19 14 10 16

37 48 57 35 42 50 15 17 20 20 26 29 18 21 20 24 27 10 9 9

58 55 62 47 43 47 36 30 34 39 33 39 31 25 29 24 26 21 14 31

8

2

; 4

E! U

CA

GAS CHROMATOGRAPHY

105

Many other derivatives of corticosteroids have been proposed for use in GC. Steroids with a dihydroxyacetone side chain react with formaldehyde to produce thermostable bismethylenedioxy derivatives [560]. Acetylation of the side chain also increases the thermal stability of corticosteroids [ 11 11. The acetates and chloroacetates may be analyzed with the ECD [878]. Rosenfeld [903] prepared the TMS ethers of cortisol metabolites and found them suitable for GC on 3% QF-1 at 220°C and on 3% SE-30 at 235°C. The TMS ethers of corticosteroid methyloximes are quite stable on 1% SE-30 at 220°C 14.591, although the cortisone and corticosterone derivatives are not separated [411]. To prevent sample losses due to acidic materials in the column, the first 3 cm should be packed with 10%SE-30 and the sample should contain an excess of TMSI [ 10501. Instead of using a carbonyl reagent for corticosteroids with ketol or dihydroxyacetone side chains, Chambaz et al. [ 166,1691 have made use of the enolization of the keto group in the presence of nucleophilic agents to produce the TMS-enol-TMS derivatives. The reaction was carried out overnight in a closed vessel at room temperature. First, 0.2 ml of a solution of 50 mg potassium acetate per milliliter methanol was evaporated in the vessel, then 50 yl of a solution of 1 mg steroid per milliliter ethanol was added and likewise evaporated. Finally, 200 yl BSTFA was added. Upon completion of the reaction, 2-5 pl of this solution were directly injected into the gas chromatograph. The derivatives, which formed in quantitative yield, were shown to be the TMS ethers of the 20-enols. The 3-en01 ether group was hydrolyzed when the reaction mixture was evaporated t o dryness. Single peaks for each of the derivatives were obtained on a 12-ft. 1% OV-101 column at programmed temperatures. Bailey [42] has prepared the 20,2 1-acetonides of 20,2 1-dihydroxycorticoids and found them useful for the separation of the 20-dihydrometabolites of cortisone and cortisol. Kelly [550,551] described the preparation and properties of siliconides formed by the action of dimethyldiacetoxysilane and triethylamine on steroids with a dihydroxyacetone side chain. Brooks and Harvey [ 1 151 have compared the GC properties of various cyclic boronates. The methylboronate of 4 y g of Compound S was formed in 85% yield in 15 min when it was kept at room temperature in a closed vessel with 4 molar equivalents of methylboronic acid in 6 yl of ethyl acetate. Comparison of various corticosteroid derivatives by GC-MS showed that boronates could be estimated in the subnanogram range [46]. The sensitivity, specificity, and versatility of GC is nowhere better illustrated than in the determination of aldosterone. Relatively simple but less sensitive methods are based on the use of a FID. For instance, by isolating aldosterone from urine with an Amberlite XAD-2 column and purifying the eluate by TLC, a concentrate is obtained which may be oxidized with HI04.The aldosterone ylactone is isolated by TLC and is determined by GC with a sensitivity of about 1 yg/l, 10%precision, and 95-99% accuracy [608,638]. Similar results were reported with a method in which the preliminary purification involved in succession, PC, acetylation, PC, and TLC [ 1 1651. Greater sensitivity is achieved with the ECD. Fabre et al. [292] have estimated the concentration of tetrahydroaldosterone in a 10-ml sample of blood by preliminary purification with TLC, followed by HI04 oxidation to the ylactone, and conversion of the product to the 30-chlorodifluoroacetyltetrahydroaldosterone ylactone, which was accurately determined with the ECD. Aldosterone was assayed by the same procedure after enzymatic reduction to the tetrahydro derivative.

106

CORTICOSTEROIDS

Other electron-capturing derivatives of aldosterone that have been tested include the HFB [698] and MO-HFB [465] esters, The most specific assays are based on GC-MS. Amounts of aldosterone down to 100 pg can be measured in 10 ml of plasma by a method involving chromatography on Amberlite XAD-2, preparation of the acetal and its purification by TLC, preparation of the HFA derivative of the acetal, and GC-MS [969]. The amount is estimated by addition of 4-['4C]-aldosterone to the plasma and measuring the 12C/'4C ratio by MS. Using mass fragmentography, highly specific and sensitive analyses can be performed with a minimum of purification. The 18-hydroxycorticosteroids [865,866] and the hydrogenated metabolites of corticosterone [ 1011 in animal tissues as well as the cortisol level in the plasma [85] have been determined in that manner. Obviously, only the cost of the instruments stands in the way of widespread adoption of this technique.

Chapter 12

Miscellaneous steroid hormones 12.1. INTRODUCTION Individual groups of natural steroid hormones are discussed in Chapters 8- 11 and 14, but Chapter 12 deals with the chromatographic analysis of biological samples for steroid hormones belonging to different classes and with the chromatography of synthetic steroid hormones. Surveys of these methods are scattered in various textbooks and symposium volumes on clinical and pharmaceutical analysis, chromatography, and MS. Among recent review articles in readily available journals, special mention should be made of those dealing with the applications of GC [ 108,168,730,850,968,1027]. General schemes for the work-up of complex biological mixtures in preparation for GC have been published [562,67 1,8391. They consist of various combinations of solvent partition, LC, PC, TLC, and derivatization. Global hormone analysis has been proposed by two developments of a thin-layer plate with benzene-ethanol (19: 1) [961] or by elution of 7 g Sephadex LH-20 in a chromatographic tube, 2 cm I.D., with 50 ml of a 100:100: 1 :1 mixture of heptane-chloroform-methanol-water (androgens + progesterone), followed by 60 ml of a 19: 1 mixture of dichloromethane-methanol (estrogens) [416]. TLC has been useful in the purification of various steroid derivatives, synthesized for conjugation to protein in radioimmunoassays [50]. R F values have been reported for the hemisuccinates, hydrazones with p-hydrazinobenzoic acid, and other derivatives of steroids.

12.2. HORMONES IN URINE The determination of steroid hormones in urine by GC has been reviewed by Gleispach [358]. As is evident from Chapters 8-1 1, chromatography is extensively used in the extraction of steroid hormones from urine. TLC [561,853], LC on A1203 [216,938], and combinations of both [526] have been standard procedures for the isolation of 17-ketosteroids,pregnanediol, and other relatively stable urinary metabolites prior to GC. The method of isolating steroid conjugates with the aid of a neutral resin, Amberlite XAD-2, is due to Bradlow [103]. A chromatographic tube, 35 cm X 7 cm, is filled with a slurry of 1 kg of the resin in water and back-flushed to remove fines. When 2 1 of urine are percolated through this column, all the steroid conjugates are retained, and impurities may be washed out with 4 1 of water. The conjugates are then quantitatively recovered by elution with 5 1 of methanol. Acidic impurities remaining after hydrolysis of the conjugates may be removed by passage through Amberlyst A-26 [959] . Steroid N-acetylglucosaminides can be isolated on Amberlite XAD-2 prior to GC analysis [762]. Group separations prior to GC by means of Sephadex LH-20 have been described by Setchell and Shackleton [9571 and by B6gue et al. [56]. Janne [511,512] has used Sephadex LH-20 to separate mono-, di-, and trisulfates of C19 and CZl steroids, which were subsequently hydrolyzed. In the work of Anderson et al. [2 1,221 the steroid conjugates were subjected to differential

107

MISCELLANEOUS STEROID HORMONES

108

% 0

10 Q L

b"

c

aJ

c 13 a,

10

15

20

25

30

35

Retention time (min)

Fig. 12.1. High-resolution gas chromatogram of MO-TMS derivatives of the urinary steroids of a healthy man. 1 = Androsterone; 2 = etiocholanolone; 5 = 11-ketoetiocholanolone; 7 = 11-hydroxyandrosterone; 9 = allopregnanediol; 10 = pregnanediol; 14 = pregnanetriol; 17 = cholesterol; 18 = tetrahydrodehydrocorticosterone; 19 = tetrahydrocorticosterone; 20 = allotetrahydrocorticosterone; 21 = tetrahydrocortisone; 22 = tetrahydrocortisol; 23 = allotetrahydrocortisol; 24 = cortolone; 25 = p-cortolone. (Reproduced from Z. Klin. Chem. Klin. Biochem., 9 (1971) 45, with permission; [ 11311.)

hydrolysis and then fractionated on lipophilic Sephadex. There are many reports on GC of underivatized urinary steroids [680,923], but most investigators prefer to use the TMS ethers or MO-TMS [342] derivatives. The BO and BO-TMS derivatives are especially advantageous, because they are better resolved in GC and give a characteristic MS [232]. The MO-HFB derivatives, which exhibit very good thermal stability, are especially suitable for the analysis of nanogram quantities by the ECD [708]. Ros [YO21 preferred the enol-TMS derivatives to the double derivatives. With the advent of high-resolution capillary GC and greater availability of the GC-MS combination, there has been much interest in urinary steroid patterns or profiles. Vollmin [1129-11311 has presented some impressive examples of the power of this new tool, one of which is shown in Fig. 12.1. The technique is described in Chapter 4. Similar metabolic profiles have been obtained by Luyten and Rutten [679], by German and Horning [353] and by Horning et al. [463], who used the MO-TMS as well as the BO-TMS derivatives. For the separation of the dihydroxysteroids in urine, the TMS, TFA, and HFB derivatives were used [75]. In view of the speed and the accuracy of the analysis, Bailey et al. [43] have recommended an automatic version with solid sampler for routine application. GC has also been applied to the analysis of synthetic steroids and their metabolites in urine. Following the administration of spironolactone, the aldadiene levels in urine and plasma were determined by GC [ 1711. The urinary metabolites of Nilevar [123], 16achloroestrone methyl ether [763], dimethisterone and norethisterone [ 10121, and methandienone [242,306] have been identified by GC.

HORMONES IN OTHER BIOLOGICAL SPECIMENS

109

12.3. HORMONES IN OTHER BIOLOGICAL SPECIMENS For the determination of steroid hormones in blood the purification methods are similar to the ones described for urine, but electron-capturing derivatives are usually preferred because the concentrations are quite low [732]. Sjovall and Vihko [982,983] separated the steroid monosulfates from disulfates in blood on Sephadex LH-20 and determined the steroids separately by GC-MS. In preparation for radioimmunoassays, a column of 2.5 g of Lipidex separated the following steroids in an extract of 1-2 ml of (19: 1): progesterone plasma by elution with petroleum ether (b.r. 66-68")-chloroform in 10-20 ml, testosterone in 48-72 ml, and 17-hydroxyprogesterone in 72-105 ml [513]. Similarly, a combination of chromatography on Amberlite XAD-2, Lipidex, and Sephadex LH-20 was used for the GC-MS analysis of neutral steroids in plasma by Axelson and Sjovall [38]. A micro column of 2 ml Celite-ethylene glycol ( 2 :1, w/v) also proved effective in separating estrogens, androgens, and progesterone in connection with competitive protein-binding assays and radioimmunoassays [928]. With single-ion monitoring the GC-MS analysis has become so specific that no elaborate purification of the blood extract is necessary. Testosterone and progesterone were determined in 1 ml of plasma by adding 4-methyl-19-nortestosterone as an internal standard for testosterone and 4-ethylandrostenedione as an internal standard for progesterone [223]. After simple extraction, the dienol-HFB derivatives were prepared and peak heights were determined by single-ion detection. The results agreed well with those of radioimmunoassays [224]. Selective ion detection has been used to determine norethisterone [ 10131 and mass fragmentography t o determine megestrol acetate [ 1 11 in plasma by a GC-MScomputer system. For the analysis of adrenal and sex hormones in cow's milk and tissues by TLC, Duthie et al. [250] have devised new solvent systems. A scheme for the analysis of human ovarian tissue, based on TLC before and after acetylation and on GC, was published by Poteczin el ai. [864]. Axelson et al. [37] have determined neutral tissue steroids by a procedure involving chromatography on Lipidex, preparation of MO derivatives, chromatography on Arnberlyst A-26, trimethylsilylation, and computerized GC-MS. Lipidex chromatography, TLC, and GC-MS were also involved in-the identification of metabolites in placental rnicrosomes [ 1071. High-resolution capillary GC was successfully applied to the trace analysis of steroids in tissues [226,721]. GC has also found use in the determination of some synthetic glucocorticoids in rat muscle [973]. The CI9O2 and CZ1O2steroid mono- and disulfates [622] and glucosiduronates [621] were analyzed by separating them first on Sephadex LH-20 and then hydrolyzing them prior to GC. Other applications of GC include the analysis of amniotic fluid [929] and feces [286,287].

12.4. HORMONES IN PHARMACEUTICALS The structure of the 17a-ethynyl steroids suggests, of course, the use of argentation chromatography. They have, in fact, been chromatographed on a silver-impregnated

110


Florisil column [6 161 and on a silver-sulfoethyl cellulose column [845]. A reversed-phase partition column, with heptane held stationary on silanized Celite and with ethanol as the mobile phase, was used for the analysis of testosterone propionate in oil injectables [989]. For the automatic analysis of steroid pharmaceuticals, Cavina etal. [ 156,1571 have used elution of a silicic acid column with a gradient of diethyl ether in petroleum ether (b.r. 65-75") and continuous monitoring of the eluate (see p. 9). Many analysts are beginning to use HPLC for pharmaceuticals. Corticosteroid creams and ointments have been assayed by eluting a silicic acid column with dichloromethane95% ethanol (19: 1) [339], a column of fl,fl'-oxydipropionitrile on Zipax with a hexaneethyl acetate-acetonitrile (474:25: 1) mixture [733], or a column of silanized Porasil with a 2,2,4-trimethylpentane-isopropanol-acetonitrile (1 38:37:25) mixture [628]. Permaphase ODS columns have been used for a variety of synthetic steroid hormones [44], including the synthetic estrogens [901]. Other column materials included Permaphase ETH [ 135,9011 and a spherical porous copolymer of styrene-divinylbenzene [333]. For TLC of pharmaceutical preparations, some use of polyamide [320] and polyvinylpyrrolidone [217,218] plates has been reported, and continuous [154] as well as twodimensional [ 159,10331 development was occasionally used. Silver nitrate-impregnated silica gel has proved selective for unsaturated steroids [ 10091. In the case of the 1 7 ~ ethynyl estrogens, argentation TLC has been advocated I2841 but found to result in some TABLE 12.1

~ R VALUES F FOR ESTROGEN AND PROGESTERONE ANALOGS Solvent systems: 1 = heptane-acetone (4:l) [60]; 2 = cyclohexane-ethyl acetate-acetone (15: 15:2) [60]; 3 = benzene-methanol (19:l) (972];4 = benzene-acetone (4:l) [972]; 5 = chloroformmethanol (9: 1) [972] ; 6 = dichloromethane-methanol-water (300: 18:l) [972]. Compound

Progesterone 17-Hydroxyprogesterone acetate Medroxyprogesterone acetate Norethindrone Norgestrel Allylestrenol Lynestrenol Ethylestrenol Norethynodrel Vinylestrenolone Ethynodiol diacetate Estrone Estradiol Estradiol benzoate Ethynylestradiol Mestranol Chlormadinone acetate Dimet histerone Megestrol acetate

Solvent system -1 2 3 28 19 22 15 16 57 42 53 24 29 40 18 12 22 13 32 -

32 26 30 22 26 66 60 59 38 39 50 33 22 32 26 48 -

-

29 37 -

61 42 -

77 23 57 55 40 52

4 37 45 63 -

51 68 -

5

6

-

-

-

-

57 59

48 53

65

69 57 84 39 68 74 57 70

-

63

-

76

-

-

-

-

43 56 47 46 43

46 65 72 61 71

-

HORMONES IN PHARMACEUTICALS

111

TABLE 12.2 h R F VALUES FOR SYNTHETIC CORTICOSTEROIDS Solvent systems: 1 = 1,2-dichloroethane-methanol-water (475:25: l ) , relative to hydrocortisone acetate = 100 [ 1921 ; 2 = 1,2-dichloroethane-2-methoxyethyl acetate-water (80:20: l), relative to hydrocortisone acetate = 100 [ 192); 3 = cyclohexane-ethyl acetate-water (25:75:1), relative to hydrocortisone acetate = 100 [192]; 4 = heptane-chloroform-acetic acid (1:1:2) [1127]; 5 = chloroform-acetic acid (9:l) [ 11271 ; 6 = cyclohexane-ethyl acetate (3:17) [ 11271 ; 7 = 1,2-dichloroethane-methyl acetate-water (2:1:1), relative to cortisone acetate = 100 [ 3921; 8 = 1,2-dichloroethane-dioxane-water (2: l : l ) , relative to hydrocortisone = 100 [ 3921 ; 9 = dichloromethane-diethyl ether-methanol-water (385:75:40:6) [59] ;dichloromethane-dioxane-water (2:l: 1) [59]. Compound

Solvent system 1

2

3

4

5

6

7

8

9

10 -

Deoxycorticosterone acetate Deoxycor ticosterone Cortisone acetate Prednisone acetate Dexamethasone acetate Fludrocortisone acetate 6a-Methylhydrocortisne 21-acetate Hydrocortisone acetate Methylprednisolone acetate Prednisolone acetate Triamcinolone acetonide Cortisone F’rednisone 6a-Methylhydrocortisone Dexamethasone Hydrocortisone Fludrocortisone Betamethasone Methylprednisolone Prednisolone 16a-Hydroxyfludrocortisone Triamcinolone

244 176 125 116 111 103 100 100 92 78 59 51 43 27 27 24 24 22 22 19 14

8

251 196 145 124 114 114 110 100 90 82 73 63 51 31 35 29 33 35 24 22 18 14

142 98 102 89 118 116 108 100 100 92 74 53 44 50 68 45 63 69 45 39 31 27

-

82 -

-

58 76 -

68 88 64 50 32

192 100 79 79 87 66 53 50 19 19 -

26

-

59

188 -

87 82 77 79

-

-

-

75 73 70 47 62 35 50 38 44 42 37

-

139 137 121 105 100 100 100 89 83 -

77 70 65 66 64 60 55 42 50 43 40 44 36 35 25 -

decomposition [39]. A spray of 0.1% AgN03 in 0.5 M NH,OH, followed by a spray of 0.02% eosin or sodium fluoresceinate in ethanol is a useful detection reagent for these compounds [897]. The RF values of a number of substituted estrone and estradiol analogs have been reported [ 10351. Table 12.1 gives hRF values for some estrogen and progesterone analogs. TLC has been used to identify 21-aminocorticosteroids [361], prednisone and prednisolone in oily solutions [774], impurities in prednisolone preparations [787], and the etiocholenic acids produced by the oxidation of corticosteroids [251]. Table 12.2 lists the hRF values of a few synthetic corticosteroids in TLC on silica gel. Some natural corticosteroids are included for reference. Rosetti [905] has studied the effect of multiple development with water-saturated diethyl ether of silica plates with different water content on the resolution of synthetic corticosteroids. Two or three developments of plates kept at 20% humidity gave the best results.

112


Structure-mobility correlations have been attempted for many synthetic steroids [243, 404,4051, including androgen [834] and progesterone [867] analogs. For quantitative analysis, not only elution and colorimetry [148] or IR spectrometry [76] have been used, but also direct densitometry [966]. GC has not been applied extensively to pharmaceutical analysis. Schulz [946] has compared GC with TLC in the assay of 17a-ethynylestradiol3-methylether. Both methods were equally accurate, but GC was significantly faster. A method for the analysis of ethynylestradiol by GC was published by Talmage et al. [ 10391. Adhikary and Harkness [4] have explored the carbon skeleton chromatography of synthetic steroid hormones. In this method, the steroids are subjected to high-temperature catalytic reduction prior to GC. Synthetic steroids can be isolated from oil solutions in an adequate state of purity for GC by a simple partition between hexane and 85% ethanol [ 1611. TLC was used to isolate estradiol monoesters from oil solutions prior to analysis by GC [ 160,7371. The GC-MS combination has been applied to fluorinated corticosteroids [ 1201.

Chapter 13

Vitamins D The chromatography of the vitamins D p r e s a t s major problems in addition t 6 the difficulties of separating a group of sterols differing in only minor structural details of the side chain. They are unstable to light, air, heat, and strong adsorbents. On the other hand, they are easily detected because they absorb UV light. Many aspects of the chromatography of vitamins D have been reviewed [227,540,758]. Norman and DeLuca [791] have devised a preparative method for separating the products of W irradiation of ergosterol and 7-dehydrocholesterol. By eluting a column, 58 cm X 1.5 cm, of 24 g silicic acid with 10% diethyl ether in petroleum ether (b.r. 6570'), they were able to separate at least 25 mg of an irradiation mixture into vitamin D2, tachysterol, , and ergosterol. Subsequently, Dollwet and Norman [240] accomplished the resolution of vitamins D2 and D3 by reversed-phase partition chromatography. When a column, 214 cm X 0.8 cm I.D., of Factice was developed with acetone-water (19: l), separate bands of vitamin D2, vitamin D3, ergosterol, and 7-dehydrocholesterol emerged, in that order. By using 29 g of silica gel containing 12% of alumina in a 60-cm X 1-cm column, Mermet-Bouvier [7 161 was able to fractionate the photochemical isomers of ergosterol with diethyl ether to yield, in the order of elution: toxisterol, A, previtamin D2, lumistero12, tachystero12, and ergosterol. To isolate metabolites of vitamin D3, Holick and DeLuca [455] have used a 600-mm X 1 1-mm column of 20 g Sephadex LH-20, which was eluted with chloroform-petroleum ether (b.r. 65-70') mixtures, a 13:7 mixture being particularly useful. As one would expect, HPLC is eminently suitable for the fractionation of fat-soluble vitamins [ 11601. Applications to the vitamin D field are just beginning to appear. Krol et al. [601] have shown that the separation of calciferol from precalciferol by HPLC on a Vydac column takes 1.2 min, whereas an equivalent analysis on a Lipidex column takes more than 1 h. Use of a 500-mm X 2.1-mm I.D. column of Permaphase ODS with a linear elution gradient of methanol in water allowed Matthews e t al. [704] to separate a number of hydroxylated cholecalciferol metabolites in a matter of minutes (see Fig. 13.1). Solvent systems for TLC of vitamins D and related sterols include: petroleum ether (b.r. 65-7O0)-acetone (9:l) for the separation of vitamin D2 from ergosterol [791], hexane-acetone-water (45 :5:1) for the separation of vitamin D3 from 7-dehydrocholesterol [833], hexane-ethyl methyl ketone (5: 1) for the isomers of vitamin D2 [586], carbon tetrachloride-acetone (10: l ) , or benzene-acetone (1 0: 1) for the separation of the photochemical isomers of ergosterol on either silica or alumina plates [714], and petroleum ether-benzene (1 :1) or hexane-chloroform (1 : 1) for ergocalciferol and related sterols on alumina plates [860]. The incorporation of BHT and squalane in the solvents inhibits air oxidation [403]. Chen [ 1841 has studied the use of various fluorescent dyes for the detection of vitamin D on thin-layer plates. An SbC13 spray produces a variety of colors with vitamin D analogs [860], A recurrent problem in the analysis of biological extracts for vitamin D is the removal of cholesterol. This may be accomplished by TLC with 1,2-dichloroethane-isobutylmethyl

113

VITAMINS D

114

L

I

5

10

I

I

I

15

20

25

Time in minutes

Fig. 13.1. HPLC of hydroxylated cholecalciferol (HCC) derivatives on Permaphase ODs. A linear solvent gradient was used from methanol-water 3:7 to 8: 2 at the rate of 2%/min. (Reproduced from FEBS Lett., 4 8 (1974) 123, with permission; [704].)

ketone (9:1) [855] or dichloromethane [312], or by TLC on 5% (w/w) AgN03-Silica Gel G with chloroform-acetone (9:1) [985,986]. The quantitative estimation of vitamin D may be based on visual comparison of zones on thin-layer plates after they have been sprayed with 50% H2S04 [418] or SbC13 in chloroform [ 1015,11431 or on elution of the zones and either W spectrophotometry [ 10851 or spectrophotometry after the Nield reaction [403]. A method for the specific determination of nanogram quantities of vitamin D3 is based on isotope dilution [139]. By adding the sample to a mixture of I4Clabeled vitamin D3 and tetracyanoethylene, the amount of radioactive product, which is estimated by scanning a thin-layer chromatogram, is decreased, depending on the amount of vitamin D3 in the sample. The vitamins D undergo thermal rearrangement to the pyro and isopyro compounds during GC, but the peak areas of these products can nevertheless be used for quantitative estimation [36,757,1115].The vitamins have been chromatographed as such or in the form of their TMS or TFA derivatives on 3.4%butanediol succinate, SE-30, SE-52, or mixed SE-52 t cyanoethyl methylpolysiloxane columns. Recent experiments by Fisher et aZ. [311] indicate that it is best to silylate the sample by incubation at 4°C in the dark with 1 volume of pyridine and 0.1 volume of MSTFA and t o separate the products by GC on 4% OV-225.Even better resolution was obtained by GC on 4%Dexsil300 [260]. Another approach has been the conversion of the vitamins to isovitamins with SbC13 [755] and purification of the products by silicic acid column chromatography [285]. Isovitamins D, and D3 were separated by GC on a 3% OV-1 column [756]. To make this method more sensitive, Wilson et al. [ 1 1611 esterified the isotachysterols with HFBA at O°C, thus enabling as little as 10 ng of the vitamins to be determined with a 63Ni-ECD.Sheppard el d . [964] preferred acetyl chloride to SbC13 as a reagent for complete isomerization of the vitamins D. The isotachysterols were then chromatographed on 3% JXR [963]. The separation of eight photochemical isomers, produced by prolonged irradiation of ergosterol, has been accomplished without derivatization 17 151. From a Pyrex column, 10 ft. X 0.08 in. I.D., packed with 3%XE-60 on Gas-Chrom Q, at 225"C, three toxisterols, pyrocalciferol, lumisterol, isopyrocalciferol, ergosterol, and tachysterol were eluted, in that order, and the eluate fractions were analyzed by W spectrometry.

Chapter 14

Molting hormones This is a short chapter, because molting hormones have been rather recently discovered. The reason for this is that they are sterols of unusually hydrophilic character, which were apparently discarded in earlier work on sterol isolation. The molting hormones owe their hydrophilic character to the numerous hydroxyl groups, both in the nucleus and in the side chain. Like most steroid hormones, they contain an a,@nsaturated carbonyl group, a useful feature for W analysis. Several dozen representatives of this subgroup of sterols are known, differing in both the number of carbon atoms and the number and distribution of hydroxyl groups. Hori [458] used a 150-cm Amberlite XAD-2 column for the automatic analysis of molting hormones by linear gradient elution with from 20 to 70% ethanol in water. The elution, monitored by a UV detector, took 12 h and yielded the following sequence: ecdysterone, inokosterone, cyasterone, pterosterone, ponasterone A, i.e., the hormones emerged in decreasing order of polarity. An Amberlite XAD-2 column was also used by Schooley et al. [941] for the isolation of insect-molting hormones from a plant extract. A 500-g column retained the hormones in 4 1 of plant extract in 20% aq. methanol solution. After it had been washed with 3 1 of 33% methanol, the column was eluted with 3 1 of 70% methanol to recover 99% of the ponasterones A and B. HPLC with Poragel PN and a linear gradient of methanol in water was used for hormone analysis [940]. Nigg et al. [782] used a 3-m X 2-mm I.D. column, packed with Corasil II,37-50 pm, and various mixtures of chloroform and 95% ethanol to separate up to nine of the hormones. The molting hormones are separable by TLC [924], but, being hydrophilic, they can be very well analyzed by PC [357]. The solvent systems for PC were prepared by equilibrating toluene or benzene with 40-50% aq. 2-butanol or isopropanol. A number of color reactions, suitable for TLC of molting hormones, have been described [45 11. They may be assayed on the thin-layer plate by a densitometric method [924]. For GC, the insect-molting hormones are silylated by heating 1 mg of the steroid with 0.5 ml BSA and 0.1 ml pyridine at 80°C for 1 min [541]. Ikekawa et al. [501] and Miyazaki et al. [725] have prepared the HFB derivatives by an exchange reaction, heating 0.5 mg of steroid, 20 pl TMSI, 20 pl HFBI, and 2 p1 HFBA at 50°C for 2 h. The TMS ethers were separated on 1.5% OV-101 at 275°C and the HFB esters on 1% OV-101 at 260°C. Picogram amounts of the HFB-TMS derivatives could be determined by an ECD, and the TMS derivatives were suitable for mass fragmentography. Morgan and Woodbridge [740] have chromatographed the MO-TMS derivatives of molting hormones, but subsequently Poole et al. [862] found that their TMS ethers can be detected at the picogram level by the ECD, probably owing to the 7-en-6-onegroup. The results of GC analysis agree well with those obtained by radioimmunoassay 1981.

115


Chapter 15

Steroid sapogenins and alkaloids The C27 sapogenins and alkaloids occur in plants in the form of their glycosides, the saponins and glycoalkaloids. Because of their hydrophilic nature, these glycosides are sometimes still separated by PC. The sapogenins are relatively stable and inert compounds, having a hydroxyl group at C-3 and differing from their analogs only in the number and location of additional oxygen functions and in the junction of Rings A and B. The alkaloids are too weakly ionized to be effectively separated by ion-exchange and electrophoretic methods and are treated together in this chapter because they are isolated and chromatographed along with the sapogenins. The isolation of sapogenins and alkaloids from plant material almost invariably involves hydrolysis, extraction, and column chromatography. Suitable column materials are A1203 [830], Si02 [413], and ion exchangers [24-261. HPLC has also been applied to the steroidal alkaloids 14891. The chromatographic column, up t o 16 ft. long and 3/8 in. O.D., was packed with Porasil A, 37-75 pm, and eluted with a concave gradient of 97% aq. acetone in a 2: 1 mixture of acetone-hexane. Analysis of the effluent by TLC showed the elution sequence: tomatidine, solanidine, solasodine, rubijervine, veratramine, jervine. TLC of saponins has recently been reviewed by Hiller and Woitke [438]. Table 15.1 shows the hRF values observed with various solvent systems. Additional data were published by Blunden and Hardman [90] and by Elmunajjed e t al. [270]. The separation of Sol- and 5-unsaturated sapogenins is generally poor, unless AgN03-impregnated layers are used [297], Ronsch and Schreiber [898] and Schreiber et al. [942] had observed earlier that silica and alumina layers, impregnated with 7-1 5% AgN03, can accomplish the separation of steroidal sapogenins and alkaloids belonging to the As- and Sol-series. Subsequently, Rozumek [909] has determined that the optimum resolution of such pairs is obtained with a AgN03 content of 4.2-6.3%. Continuous development improves the separation. Another possible solution for this problem is the addition of bromine to the solvent system [298]. Table 15.2 lists the hRF values observed by Hunter et al. [490] for a series of steroidal alkaloids. Additional data for TLC of Veratrum alkaloids may be found in Zeitler’s article [ 1 1901. The quantitative analysis of individual sapogenins and alkaloids by PC or TLC requires detection, usually with iodine [2], and elution [ 13,380,668,83 11. Densitometric estimation involves a staining process, a SbC13 spray being the preferred method [91,92, 105, 6663. Boll [95] has separated glycoalkaloids on Silica Gel G plates with the organic phase of either of two solvent systems: ethyl acetate-water-pyridine (3:3: 1) or chloroform-water1% NH40H (2:2: 1). Another suitable solvent system, 95% ethanol-acetic acid (3: I), was described by Paquin and Lepage [832]. For the fractionation of steroid saponins, a mixture of 2.5 g cellulose powder, pretreated with 2% HCI, and 0.3 g gypsum was slurried in 1 1 ml water. The slurry was used to make thin-layer plates, which were developed with butanol, saturated with 5% acetic acid [688]. Kawasaki and Miyahara [544] used silica gel plates for the saponins and their acetates, whereas Paseshnichenko and Borikhina [837] have

117

STEROID SAPOGENINS AND ALKALOIDS

118 TABLE 15.1

hRF VALUES OF SAPOGENINS IN TLC Solvent systems 1-7 were used on silica gel layers, 8-12 on alumina-sintered glass plates, 13-18 on silica-sintered glass plates, 19-22 on AgN0,-impregnated alumina, and 23-26 on AgN0,-impregnated silica gel. 1 = Chloroform-ethanol (19: 1) [ 10371 ; 2 = chloroform-acetone (9: 1) [ 10371 ; 3 = benzeneacetone(l7:3) [1037];4 = benzene-methanol(23:2) [1037];5 = hexane-ethyl acetate (1:l) [1037]; 6 = hexane-acetone (4:l) [1037]; 7 = benzene-ethanol (17:3) [703]; 8 = benzene-ethanol (17:l) [818]; 9 = benzene-ethyl acetate (4:l) [818]; 10 = chloroform-acetone (9: 1) [818]; 11 = chloroform-ethanol (9:l) [818]; 12 = chloroform-methanol (9:l) (8181; 13 = benzene-ethanol (17:l) [814]; 14 =benzeneethyl acetate (4:l) [814] ; 15 = benzene-ethyl acetate (2:l) [814]; 16 = chloroform-ethyl acetate (4: 1) [814]; 17 = chloroform-acetone (9:l) [814]; 18 = chloroform-acetone (4:l) [814]; 19 = hexaneethyl acetate (20: 1) [ 2971 ;20 = cyclohexane-ethyl acetate (10: 1) [ 2971 ; 21 = hexane-ethyl acetate (5:l) [ 2971; 22 = cyclohexane-chloroform-ethyl acetate (20:4:3) [297] ; 23 = hexane-ethyl acetate (5: 1) [ 2971; 24 = cyclohexane-chloroform-ethyl acetate (20:4:3) [ 2971; 25 = toluene-chloroform (10:3) [297]; 26 = toluene-chloroform-ethyl acetate (20:4:3) [297]. Compound*

Sarsasapogenin Smilagenin Diosgenin Tigogenin Pennogenin Gentrogenin Hecogenin Convallamarogenin Isorhodeasapogenin Rhodeasapogenin Y onogenin Gitogenin To korogenin Metagenin Kitigenin Kogagenin Chiapagenin Isochiapagenin Chlorogenin p-Chlorogenin Digitogenin Neogitogenin Neotigogenin Neoyonogenin

Solvent system 1

2

65

39

-

-

59 59 53 49 46 39 39 37 21 19 9 6 4 3

35 35 26 24 22 21 18 18 11 7 2 1 1 1

-

-

-

-

-

-

-

-

-

-

3

4

46 51 41 42 39 39 30 42 25 32 22 37 24 30 25 38 25 32 11 17 11 16 2 1 2 1 9 1 1 0 1 8 -

5

-

-

51 46 46 35 26 21 28 28 28 13 9 3 2 0 0 -

-

-

-

-

-

-

-

-

-

-

-

6

7

8

42 67 - 83 34 57 73 29 53 77 22 50 75 16 42 80 16 50 76 22 42 77 22 47 22 47 9 30 34 11 35 28 4 2 7 6 1 2 3 1 2 0 0 23 2 - 69 69 46 - 60 - 18 26 - 76 - 38

9

1 0 1 1 1 2

-

80 61 67 56 70 59 50

75 60 67 55 62 58 42 -

9 6 0 0 41 41 7 15 0 6 66 8

-

19 40 41 18 20 21 20 1 1

0

-

0 11 10 1 3 0 1 41 1

-

6 5 0

0 39 40 7 13 0 5 65 7

85 85 83 81 82 81 77 54 56 30 16 69 68 48 56 48 58 81 55

*Compounds chromatographed as genins, except in Solvent systems 19-26, where the acetates were chromatographed.

119

STEROlD SAPOGENINS AND ALKALOIDS

13

14

15

16

17

18

71 71 63 63 53 46 46 44

58 61

66 67 64 59 52 45 40 39 15 10 3

65 69 65 60 52 47 44 37 12 9 1

70 73 68 65 58 55 51 47 20 16 4 0 38 38 10 15 32 16 66 19

80 87 75 77 75 71 69 67 -

-

25 18 16 2 31 31 12 16 3 18 63 26

55 50 36 29 24 24 6 3 2

-

-

-

0 15 15 2 5 0 5 48 7

0 32 32 6 10 0 10 62 15

-

0 30 29

6 10 9 60 12

-

40 41 17 '8 60 60 35 43 42 41 72 41

19

20

21

22

23

24

25

26

120

STEROID SAPOGENINS AND ALKALOIDS

TABLE 15.2

hRF VALUES OF STEROIDAL ALKALOIDS IN TLC ON SILICA GEL [490] Solvent systems: 1 = hexane-ethyl acetate (1:l); 2 = hexane-ethanol (1:l); 3 = dichloromethanemethanol (23:2); 4 = dichloromethane-methanol(9: 1); 5 = dichloromethane-acetone (4: 1); 6 = hexane-acetone (1: 1); 7 = dichloromethane-methanol-acetic acid (85: 13:2); 8 = dichloromethanemethanol-NH,OH (100: 100:1). Compound

Tomatidine Tomatidenol Soladulcidine Solasodine Veramine 9a-Hydroxytomatidine 7wHydroxytomatidine 7a, 1la-Dihydroxytomatidine 9 q 1 1a-Dihydroxytomatidine Demissidine Solanidine S p-Solanidan-3-one

4-Solaniden-3-one Rubijervine Isorubijervine Veralobine Verarine Cyclopamine Jervine Veratramine Veramarine Solanocapsine Verazine Tomatillidine Etioline Veralkamine

Solvent system 1

2

3

4

5

6

7

47 47 26 27 10 8 4 0 0 58 59 75 63 15 19 11 3 3 0 5 3 0 69 69 1 3

90 92 87 85 75 86 88 69 69 85 83 85 83 73 81 75 60 46 35 16 29 2 92 91 47 52

49 46 40 41 21 21 20 5 5 21 42 61 48 17 21 43 22 25 26 28 12 0 59 61 12 19

66 66 53 59 35 31 35 6 6 33 61 50 50 13 33 43 17 21 19 17 8 0 64 69 14 14

37 40 23 23 9 7 5 0 0 19 15 43 39

87 89 85 82 60 60 53 10 10 87 95 0 0 56 60 64 19 17 14 36 11 0 84 84 9 22

73 94 73 96 71 91 70 96 68 93 45 91 37 93 10 93 10 93 57 97 81 89 91 100 71 89 55 82 43 91 55 100 64 13 51 81 53 83 31 91 27 60 5 1 2 92 97 92 97 47 77 60 73

I 11 17 3 5 3 11 5 0 83 82 3 5

8

preferred alumina plates. There has been very little research activity in the application of GC to steroid sapogenins and alkaloids. Cooke [ 1981 and Rozanski [908] have shown that a crude Dioscoreu extract can be assayed for diosgenin very simply by injecting it in a column of 2.5% SE-30. Cowley et ul. [203] have prepared the TMS ethers of sapogenins by heating them in 1 ml tetrahydrofuran with 0.4 ml HMDS, 0.1 ml TMCS, and 0.05 ml pyridine. Good separation and quantitative data were obtained by chromatographing the TMS ethers on a 9-ft. X 1/8-in. column of 1.2% SE-30 at 26OoC. A method for the analysis of permethylated derivatives of glycoalkaloids by GC on OV-1 or Dexsil300 columns has recently been published [428].

Chapter 16

Cardenolides and bufadienolides The cardenolides and the homologous bufadienolides do not occur together, but they are discussed together because the same chromatographic methods are applicable to both groups of steroids. In plants, they occur in the form of glycosides, which are quite hydrophilic and therefore still frequently analyzed by PC. The genins, which contain at least two hydroxyl groups, at C-3 and at C-14, and an unsaturated lactone in the side chain, are also rather polar. The lactone ring may be exploited for detection, but it makes the molecule relatively unstable. The older preparative methods for isolating cardiac glycosides from plants with silicic acid columns [96,202,528] have been modified by automation [589] and by use of socalled dry columns [590], containing finer silica particles. A column of Sephadex (3-200, eluted with 30% methanol, was used for the isolation of heart glycosides from animal tissues [370]. El-Olemy and Stohs [272] found that a Sephadex LH-20 column, eluted with 95% ethanol, separated cardiac glycosides from aglycones. The first application of HPLC to heart glycosides was reported by Evans [288]. An SCX ion-exchange column, 3 ft. X 1/4 in. O.D., eluted with 4%amyl alcohol at 45"C, produced in consecutive fractions: digitoxigenin, its monodigitoxoside, its bisdigitoxoside, digitoxin, and lanatoside A. Only a few examples of the use of PC for the quantitative analysis of cardiac glycosides can be given. Lanatoside C was isolated by continuous PC with benzene-ethyl acetateformamide (30:69: 1) on formamide-impregnated paper, which was subsequently treated with xanthydrol reagent [267]. Comparing the use of this solvent system on filter paper and on talc layers, it was found that the former is more suitable for quantitative analysis [ 11961. Instead of xanthydrol, dixanthylurea [85 11 or 2,2',4,4'-tetranitrobiphenyl [872] may be used as color reagents. A convenient method of direct densitometry for paper chromatograms has been described [645]. TLC of cardiac glycosides has recently been reviewed [792]. Reviews on cardenolides [811] and bufadienolides [824] are available in Japanese. Earlier methods of TLC were adaptations of PC, and in many cases employed partition systems. Cellulose powder [871], talc [1194,1195], kieselguhr [857,995,1142], and silica gel [lo41 were impregnated with formamide, and thin-layer chromatograms were developed with partially miscible solvents. Impregnation of silica gel with borax was found to retard specifically glycosides with cis vicinal hydroxyl groups [880]. Current TLC methods are based on the adsorption on silica or alumina [349,607,818, 10241. Table 16.1 summarizes a few of the observed mobilities on silica gel. Additional solvent systems have been described for the separation of cardenolides from plant sterols [94], for cardenolide acetates [873], and for the bufadienolides [504,587]. Special solvent mixtures have been recommended for the analysis of cardiac glycosides in Digitalis [136,295,323,629,684],Strophanthus[201,449],Convallaria I6091 ,Apocynum [352], Eiysimum [888], and Acokanthera [62] species. Repeated and successive developments [ 1441 as well as continuous development [434] improve the resolution. The relationship between structure and mobility in TLC has been studied by Nover et al. [793] and by

121

+

TABLE 16.1

N N

~ R VALUES F OF CARDENOLIDES AND THEIR GLYCOSIDES IN TLC ON SILICA GEL Solvent systems: 1 = chloroform-acetone (13:7) [ 11931; 2 = diethyl ether-methanol (9:l) [1193]; 3 = chloroform-isopropanol(9:l) [ 11931;4 = chloroform-ethanol (9:l) [ 11931; 5 = toluene-methanol (4:l) [ 1193);6 = cyclohexane-acetone-acetic acid (49:49:2) [ 11931; 7 = ethyl acetatehexane-acetic acid (8:l:l) [ 11931; 8 = cyclohexane-acetone-acetic acid (65:33:2) [ 1281; 9 = hexane-ethyl acetate-ethanol (3:15:2) + 2.2% water [ 7931 ; 10 = ethyl acetate-pyridine (9: 1) + 2.7% water [ 7931; 11 = ethyl methyl ketone + 5.4% water [ 7931; 12 = toluene-ethyl acetate-propanol (10:33:7) + 2.2% water [793]; 13 = ethyl acefate-chloroform-acetic acid (18:l:l) [144]; 14 = two developments with System 13 [144]; 15 = cyclohexane-acetone-acetic acid (65:33:2) followed by a second development with (49:49:2) [ 1441; 16 = cyclohexane-acetone-acetic acid (65:33:2) followed by a second development with ethyl acetate-chloroform (9: 1) [ 1441; 17 = two developments with chloroform-isopropanol-acetone (16:1:3) [144]. Solvent system Compound ~~

~

1

2

~

-

~

3

4

_

5

_

6

_

7

_

8

9

10

11

12

13

14

15

16

17

43 26 22 19 16 12 10 9 20 12 9 41 18

48 35 34 26 24 23 23 31 25 24 41 29

38

79 73 67 60 51 46 41 36 -

79 60 42 30 38 30 19 12

78 56 45 36 40 29 22 18 -

-

-

-

-

-

-

34

29

-

-

25 23 39

68 64 51 47 50 47 38 36 55 50 38 66 48 32 41 46 33 -

93 85 73 57 71 63 42 28

12 -

62 56 47 39 42 41 32 26 45 46 29 62 41 27 38 38 27 -

14 70 60 57 68 59 51

21

53 44 35 26 24 9 30 12 33 15 22 23 12 35

71 62

33 27 28 24 19 17 30 25 18 40 32 18 26 26

45 33 21 15 23 19 11 8 23 19 8 44 23 16 12 23

68 70 59 53 55 55 44 42 55 57 44 72

-

38 30 28 22 20 19 20 29 26 24 36 33 26 29 24 33

-

~

.

~

~

~

Digitoxigenin Digitoxigenin monodigitoxoside Digitoxigenin bisdigitoxoside Digitoxin Digoxigenin Digoxigenin monodigitoxoside Digoxigenin bisdigitoxoside Digoxin Git oxigenin Gitoxigenin monodigitoxoside Gitoxin Gitaloxigenin Periplogenin Strophanthidol Strophanthidin Sarmentogenin Diginatigenin Acovenosigenin

35 19 15 9 16 11 7 4 18 9 4 32 16 12 24

43 34 30 24 26 23 17 14 28 22 14 42 29 23 31 ~

-

30

-

-

-

55

43 51 53 36 -

51

66 65 49 76 62 48 67 62 55

-

44 31 43 36 22 15

-

-

-

-

-

-

-

-

-

-

-

CARDENOLIDES AND BUFADIENOLIDES

123

Ziillich et al. [ 1 1931. For the detection of cardiac glycosides, sulfuric acid or aldehyde reagents, such as anisaldehyde, in sulfuric acid are generally used. Fluorescence may be produced by a perchloric acid spray [523] or modified Jensen reagent [519]. It is prepared by adding 2.1 ~ 1 3 0 % HzOz to a mixture of 20 mg ascorbic acid in 19 ml methanol and 30 ml conc. HCI and permits the detection of as little as 0.01 pg of the steroids. For quantitative analysis, visual comparison of the fluorescence can be used, which is produced by a spray reagent consisting of 60 ml acetic acid, 5 ml HzSO4, and 1 ml9% FeC13 [246]. Zones detected by their fluorescence or by other nondestructive methods can be eluted and determined colorimetrically [81,200,348,435] or fluorimetrically I5191 The UV absorption of bufadienolides at 355 nm 2 h after the addition of 1 N methanolic KOH can also be used for estimation [1007]. Kartnig and Danhofer [537] assayed Strophanthus glycosides by TLC on MgO by scraping off the adsorbent and heating it with 0.2 ml conc. HCl + 3 ml HzSO4 at 60°C for 15 min. The absorption at the maximum around 400 nm was then determined. The dersitometric estimation of cardenolides [289, 7 131 and bufadienolides [588] has also been described. The fluorescence, produced by the Jensen reagent [238] or by HCl vapors [326,327] can be measured by a scanning fluorimeter. Jelliffe and Blankenhorn [5 181 introduced GC into the analysis of cardenolides. which were separated in the form of their TMS ethers. The application of the GC-MS combination to these derivatives was later studied by Maume etal. [709] and several modifications were published [ 1162,1163j. Tan [ 10401 has proposed the TMS ethers of the 14-anhydrocardenolides as derivatives, and Watson and K,ilman [ 11471 and Watson et al. [ 1 1481 have been able to measure nanogram quantities of Ihe HFB esters with the ECD. The HFB derivatives are also suitable for the identification of cardenolides by GC-MS [ 1 1461.


List of abbreviations Proportions of solvents are v/v, unless otherwise stated.

A a

abs . AFID aq. atm BDSA BHT BN BO b.r. BSA BST F A BTPPC ca . cm CMDMS conc. Dns DEAE DMH DMS e ECD ECTEOLA FID ft. g GC GC-MS HFB HFBA HFBl HM DS HPLC h hRF I.D. in.

IR

Angstrom, 10-l0m axial absolute alkali flame-ionization detector aqueous atmosphere bis(dimethylsily1) acetamide butylated hydroxytoluene Brenner-Niederwieser benzyl oxime boiling range N, 0-bis(trimethylsily1) acetamide N, 0-bis (t r imethylsilyl) t rifluoroacet amide benzyltriphenylphosphonium chloride about centimeter, 10-'m chloromethyldimethylsilyl concentrated 1-dimet hylaminonapht halene-5 -sulfonyl die thylaminoethyl N, N-dimethylhydrazone dime thylsilyl equatorial electron-capture detector epichlorohydrin of triethanolamine flame-ionization detector feet gram gas chromatography (ic) gas chromatography-mass spectrometry heptafluorobutyrate heptafluorobutyric anhydride hexafluorobutyrylimidazole hexamethyldisilazane high-pressure liquid chromatography hour R F X 100 internal diameter inches infrared

125

LIST OF ABBREVIATIONS

126

1 LC

M m mCi ME mg Pg min ml P1 mm Pm mmole MO MS MSTFA N ng O.D. PC PFBO Pg PIPSYL p.s.i. PTFE PTV

RI rPm satd. sec TFA TLC TMCS TMS TMSDEA TMSI

uv vlv wlv

WIW

liter liquid column chromatography molar meter millicurie, 1O - 3 ~ i methyl ether milligram, 1O-3g microgram, 10-6g minutes milliliter, 10-~1 microliter, millimeter, I r 3 m micrometer, 1W6m millimole methyl oxime, methoxime mass spectrometry

N-methyl-N-trimethylsilyltrifluoroacetamide normal nanogram, w 9 g outside diameter paper chromatography pentafluorobenzyl oxime picogram, lO-''g p-iodobenzenesulfonyl pounds per square inch poly(tetrafluoroethy1ene) per cent total bed volume refractive index rotations per minute saturated seconds trifluoroacetate thin-layer chromatography trimethylchlorosilane trime thylsilyl trime thylsilyldiethylamine trime thylsilylimidazole ultraviolet volume/volume weight/volume weightlweight

References 1 R. Abraham, K.-F. Gutte and E. Hild, Fractionation of urinary corticosteroids by thinlayer chromatography and direct photometric evaluation of chromatograms, Clin. Chim. Acfa, 27 (1970) 395-400 (Ger.). 2 G. Adam and K. Schreiber,Solanum alkaloids. 26. Preparative separation of stereoisomeric iminocholestanes and other steroids by means of thin-layer chromatography and using iodine as indifferent tracer,Z. Chem., 3 (1963) 100-102 (Ger.). 3 G. Adessi, D. Eichenberger, T.Q. Nhua and M.-F. Jayle, Determination by gas chromatography of the seven principal urinary estrogens at the end of gestation, Clin. Chim. Acfa, 55 (1974) 323-331 (Fr.). 4 P.M. Adhikary and R.A. Harkness, Production of the parent hydrocarbons from steroid drugs and their separation by gas chromatography, J. Chromafogr., 42 (1 969) 29-38. 5 H. Adlercreutz, Studies on the hydrolysis of gel-filtered urinary oestrogens, Acfa Endocrinol., 57 (1968) 49-68. 6 H. Adlercreutz and T. Luukkainen, Gas-chromatographic identification of pregnanediol and some of its isomers in bile of pregnant women,Ann Med. Exp. Biol. Fenn., 42 (1964) 161-167. 7 H. Adlercreutz and T. Luukkainen, Gas-chromatographic studies on estrogens in the bile of pregnant women, Biochim. Biophys. Acfa, 97 (1965) 134-141. 8 H. Adlercreutz and T. Luukkainen, Identification and determination of oestrogens in various biological materials in pregnancy, Ann. Clin. Res., 2 (1970) 365-380. 9 H. Adlercreutz and T. Luukkainen, Methods for the gas chromatography and mass spectrometry of new estrogens, 2. Klin. Chem. Klin. Biochem., 9 (1971) 421-426 (Ger.). 10 H. Adlercreutz, T. Luukkainen and W. Taylor, Gas-chromatographic and mass-spectrometric investigations of sodium pregnanediol glucuronidate, Eur. J. Steroids, 1 (1 966) 117-133. 11 H. Adlercreutz, U. Nieminen and H.S. Ervast, A mass-fragmentographic method for the determiration of megestrol acetate in plasma and its application t o studies on the plasma levels after administration of the progestin to patients with carcinoma corporis uteri, J. Steroid Biochem., 5 (1974) 619-626. 12 H. Adlercreutz, A. Salokangas and T. Luukkainen, Measurement of estrogens in biological materia1,Mern. SOC. Endocrinol., 16 (1967) 89-1 13. 13 A. Akahori, K. Murata, F. Yasuda, S . Nagase, M. Togami and T. Okanishi, Colorimetric determination of steroidal sapogenins, Shionogi Kenkyusho Nempo (Ann. Rep. ShionogiRes. Lab.), 16 (1966) 74-77 (Jap.). 14 G. Alexander and G.A.F.M. Rutten, Preparation of polar phase coated open tubular columns for steroid analysis, Chromafographia, 6 (1973) 231-233. 15 G. Alexander and G.A.F.M. Rutten, Surface characteristics of treated glasses for the preparation of glass capillary columns in gas-liquid chromatography, J. Chromafogr., 99 (1974) 81-101.

127

128

REFERENCES

16 J.P. Allais and M. Barbier, Preparation of pure p-sitosterol by thin-layer chromatography on siIver nitrate impregnated alumina, Qual. Plant. Mater. Veg., 16 (1968) 215--222 (Fr.). 17 E. Alsat, C. Corpechot, C , Ego, E. Richard and L. CCdard, Ultrasensitive assay method for estriol by gas chromatography with electron capture. Formation of the 16,17-diheptafluorobutyrate of 3-methylestriol,J. Chromatogr., 44 (1969) 383- 385 (Fr.). 18 M.W. Anders and G.J. Mannering, New peak-shift technique for gas-liquid chromatography. Preparation of derivatives on the column,Anal. Chem., 34 (1962) 730 733. 19 F.O. Anderson, L.R. Crisp, G.C. Riggle, G.G. Vurek, E. Heftmann, D.F. Johnson, D. Franqois and T.D. Perrine, Steroid analyzer,Anal. Chem., 33 (1961) 1606- 1610, 20 R.A. Anderson, C.J.W. Brooks and B.A. Knights, Hydroxyalicyclic derivatives of Sephadex LH-20 for lipophilic gel chromatography, J . Chromatogr., 83 (1 973) 39-44. 21 R.A. Anderson, E.M. Chambaz, G. Defaye, C. Madani, T.A. Baillie and C.J.W. Brooks, Steroids in the human newborn. Lipophilic gel separation and gas-phase analysis, J. Chromatogr. Sci., 12 (1974) 636-641. 22 R.A. Anderson, G. Defaye, C. Madani, E.M. Chambaz and C.J.W. Brooks, Lipophilic gel and gas-phase analysis of steroid hormones. Application to the newborn, J. Chromatogr., 99 (1974) 485-494. 23 R.A. Anderson, B.A. Knights and C.J.W. Brooks, Preparation and evaluation of a hydroxycyclohexyl derivative of Sephadex LH-20, J. Chromafog., 8 2 (1973) 337-342. 24 L.G. Andreeva, Ion-exchange sorption of glycoalkaloids of Solanum laciniatum, Khim.Farm. Zh., 8 (1974) 52-56 (Russ.). 25 L.G. Andreeva, L.G. Demina and M.A. Romanchuk, Use of ion exchangers for separating Solanum laciniatum glycoalkaloids, Khim.-Farm. Zh., 3 (1969) 42-46 (Russ.). 26 L.G. Andreeva and M.A. Romanchuk, Ion-exchange method for determining solasodine in Solanum laciniatum, Khim.-Farm. Zh., 4 (1970) 43-45 (Russ.). 27 A P . Anker and E.J. van Kampen, Quick and accurate pregnanediol determination in urine during pregnancy by semi-automated gas-liquid chromatography, Clin. Chim. Acta,42(1972) 335-341. 28 W.L. Anthony and W.T. Beher, Color detection of bile acids using thin-layer chromatography, J. Chromatogr., 13 (1964) 567-570. 29 C. Apter, R. Chayen, S. Could and A. Harell, Determination of individual urinary 17-ketosteroidsas the DANSYL-hydrazones, Clin. Chim. Acta, 42 (1972) 115-1 18. 30 L. Aringer, P. Eneroth and J.A. Gustafsson, Trimethylbromosilane-catalyzed trimethylsilylation of slow-reacting hydroxy- and oxosteroids in gas chromatographic--mass spectrometric analysis, Steroids, 17 (1971) 377-398. 31 J.E. Arnold and H.M. Fales, Comparison of column and connector materials in the gas chromatography of sensitive, high-molecular-weight compounds, J. Gas Chromatogr., 3 (1965) 131-133. 32 J . Attal and K.B. Eik-Nes, Measurement of free 170-estradiol in blood plasma by gasliquid chromatography using an electron-capture detector, Anal. Biochem., 26 (1968) 398-41 1. 33 J . Attal, S.M. Hendeles and K.B. Eik-Nes, Determination of free estrone in blood plasma by gas-phase chromatography with electron-capture detection, Anal. Eiochem., 20 (1967) 394-410. 34 J . Attal, S.M. Hendeles, J.A. Engels and K.B. Eik-Nes, Elution of steroids after thin-

REFERENCES

129

layer chromatography,J. Chromatogr., 2 7 (1967) 167-1 71. 35 J . Avigan, D.S. Goodman and D. Steinberg, Thin-layer chromatography of sterols and steroids,J. LipidRes., 4 (1963) 100-101. 36 L.V. Avioli and S.W. Lee, Detection of nanogram quantities of vitamin D by gas-liquid chromatography,Anal. Biochem., 16 (1966) 193-199. 37 M. Axelson, G. Schumacher and J. Sjovall, Analysis of tissue steroids by liquid-gel chromatography and computerized gas chromatography-mass spectrometry, J. Chromatogr. Sci., 12 (1974) 535-540. 38 M. Axelson and J . Sjovall, Separation and computerized gas chromatography-mass spectrometry of unconjugated neutral steroids in plasma, J. Steroid Biochem., 5 (1974) 733-738. 39 M.T.A. Aziz and K.1.H. Williams, Decomposition of 17cu-ethynylestradiol and its ethers on silver nitrate-impregnated silica gel thin-layer plates, Steroids, 12 (1968) 167-170. 40 P. Back, J . Sjovall and K. Sjovall, Monohydroxy bile acids in plasma in intrahepatic cholestasis of pregnancy. Identification by computerized gas chromatography-mass spectrometry,Med. Biol., 52 (1974) 31-38. 41 E. Bailey, The estimation of cortisol, prednisolone, and some of their unconjugated metabolites in biological fluids using gas-liquid chromatography, Mem. SOC.Endocrinol., 16 (1967) 183-197. 42 E. Bailey, The gas-liquid chromatographic analysis of some 20,21 -dihydroxycorticosteroids as 20,21-acetonides, Steroids, 10 (1967) 527-545. 43 E. Bailey, M . Fenoughty and J.R. Chapman, Evaluation of a gas-liquid chromatographic method for the determination of urinary steroids using high-resolution open-tubular glass capillary columns,J. Chromatogr., 96 (1974) 33-46. 44 F. Bailey and P.N. Brittain, High-efficiency liquid chromatography in pharmaceutical analysis, J. Chromatogr., 83 (1973) 43 1-437. 45 T.A. Baillie, C.J.W. Brooks and E.C. Horning, 0-Butyloximes and 0-pentyloximes as derivatives for the study of ketosteroids by gas chromatography, Anal. Lett., 5 (1972) 351 -361. 46 T.A. Baillie, C.J.W. Brooks and B.S. Middleditch, Comparison of corticosteroid derivatives by gas chromatography-mass spectrometry,Anal. Chem., 44 (1972) 30-37. 47 D.R. Baker, R.A. Henry, R.C. Williams and D.R. Hudson, Preparative columns in highspeed liquid chromatography,J. Chromatogr., 83 (1973) 233-243. 48 J.A. Ballantine, J.C. Roberts and R.J. Morris, Sterols of the cockle Cerastoderma edule. Evaluation of thermostable liquid phases for the gas-liquid chromatographic-mass spectrometric analysis of the trimethylsilyl ethers of marine sterols, J. Chromatogr., 103 (1975) 289-304. 49 H.O. Bang, A simplified method for the quantitative determination of pregnanediol in urine, J. Chromatogr., 14 (1964) 520-523. 50 U. Barbieri, A. Massaglia, M. Zannino and U. Rosa, Thin-layer chromatography of steroid derivatives for radioimmunoassay, J. Chromatogr., 69 (1 972) 15 1- 155. 51 J.D. Baty and A.P. Wade, Analysis of steroids in biological fluids by computer-aided gas-liquid chromatography-mass spectrometry, Anal. Biochem ., 57 (1974) 27-37. 52 M.A. Baydarovtseva, B.A. Rudenko, V.F. Kucherov and M.I. Kuleshova, Chromatography of sterols, alkaloids and other drugs using steam as the mobile phase, J. Chro-

130

REFERENCES

matogr., 104 (1975) 277-281. 53 R.N. Beale, D. Croft, R.F. Taylor and D. Powell, Gas-chromatographic quantitation of steroids in health and disease. 4. Determination of conjugated cortisol metabolites in urine,Steroids, 18 (1971) 641-650. 54 F. Begemann, Simple method for the isolation of free and conjugated bile acids from serum,Z. Klin. Chem. Klin. Biochem., 10 (1972) 29-32 (Ger.). 55 F. Begemann, Identification of lipids and bile acids, separated by thin-layer chromatography, by observing the fluorescence with 2,2’-phenylene-bis(4-methyl-5-phenyloxazole), J. Chromatogr., 78 (1973) 434-435 (Ger.). 56 R.J. BBgue, J. DesgrBs, P. Padieu and J.A. Gustafsson, Method of analysis of urinary steroids of human pregnancy by gas-liquid chromatography and gas chromatographymass spectrometry of Sephadex LH-20 chromatographic fractions, J. Chromatogr. Sci., 12 (1974) 763-766. 57 I. BeliE and H. SoEiE, Specificity of the chromogenic reaction of steroids with sulfuric acid, Chrumatugraphia,4 (1 97 1) 266. 58 C.G. Beling, Gel filtration of conjugated urinary estrogens and its application in clinical assays, Acta Endocrinul., Suppl., 79 (1963) 98 pp. 59 G. Bellomonte, G. Carelli, G. Verzura, G. Cavina and E. Cingolani, Use of thin-layer chromatography in the identification and the control of purity of corticosteroids, Farmaco (Pavia),Ed. Prat., 25 (1970) 446-456 (Ital.). 60 G. Bellomonte, G. Moretti, G. Verzura, G. Cavina and E. Cingolani, Thin-layer chromatographic separation and identification of progestational and estrogenic steroids, Farmaco (Pavia),Ed. Prat., 25 (1970) 744-752 (Ital.). 61 G. Bellomonte, G. Moretti, G. Verzura, G. Cavina and E. Cingolani, Separation and identification of androgenic and anabolic steroids by thin-layer chromatography, Ann. Chim. (Rome),61 (1971) 756-764 (Ital.). 62 K. Benmerabet, L. Abed and F . Pieri, Thin-layer chromatography of the cardenolides of Acokanthera spectabilis,Plant. Med. Phytother., 8 (1974) 89-95 (Fr.). 63 R.D. Bennett and E. Heftmann, Devices for continuous development and sample application in preparative thin-layer chromatography, J. Chromatogr., 12 (1 963) 245248. 64 R.D. Bennett and E. Heftmann, Biosynthesis of Holarrhena alkaloids from cholesterol, Arch. Biochem. Biuphys., 112 (1 965) 6 16-620. 65 R.D. Bennett and E. Heftmann, Biosynthesis of Holarrhena alkaloids from pregnenolone and progesterone, Phytochemistry, 4 (1965) 873-879. 66 R.D. Bennett and E. Heftmann, Separation of closely related steroids by an improved technique for continuous development of thin-layer chromatograms, J. Chromatogr., 21 (1966) 488-490. 67 E.D. Bergmann, R. Ikan and S. Harel, Thin-layer chromatography of 0-sitosteryl esters, J. Chromatogr., 15 (1964) 204-206. 68 C.R. Berrett and C. McNeil, Quantitation of major 17-ketosteroid fractions by gasliquid chromatography, Clin. Chem., 12 (1966) 399-405. 69 L.V. Berry and H. Engelhardt, Influence of the water content of the solvent on chromatographic separations, J. Chromatogr., 95 (1974) 27-38. 70 G. Bertellini, Assay of a-ketolic steroids in natural extracts by two-way thin-layer

REFERENCES

131

chromatography, Boll. Chim. Famt., 105 (1966) 836-842. 71 K. Berthold and H. Staudinger, Quantitative determination of 60-hydroxycortisol by use of thin-layer chromatography with fluorescent indicator, Z. Klin. Chem., 4 (1966) 130-133 (Ger.). 72 F.L. Berthou, L.G. Bardou and H.H. Floch, Measurement of 5a-androstane-3a,l7/3-diol and SP-androstane-3cu,170-diol in the urine of healthy men and women, J. Stemid Biochem., 2 (1971) 141-153. 73 F.L. Berthou, L.G. Bardou and H.H. Floch, Separation of urinary androstanediol and pregnanediol isomers by a combined gas-liquid chromatography -thin-layer chromatography method, J. Chromatogr.,93 (1974) 149-165. 74 F.L. Berthou, R.F. Morfin, D. Picart and L.G. Bardou, Analysis of 5a-androstanediol monoacetates by thin-layer and gas-liquid chromatography and mass spectrometry, J. Chromatogr., 88 (1974) 271-279. 75 F.L. Berthou, D. Picart, L.G. Bardou and H.H. Floch, Separation and identification of human urinary dihydroxysteroids by high-resolution glass capillary columns, J. Chromatogr. Sci., 12 (1974) 662-667. 76 K. Beyermann and E. Roder, Combination of thin-layer chromatography and infrared spectrographic determination of microgram amounts, as illustrated by the analysis of oral contraceptives, Z. Anal. Chem., 230 (1967) 347-355 (Ger.). 77 B.R. Bhavnani, A simple staining technique for the detection of steroids on silica gel thin-layer glass plates, J. Chrornatogr.,65 (1972) 454-455. 78 G.L. Biagi, A.M. Barbaro and M.C. Guerra, Partition data of chemotherapeutic and steroid agents determined by reversed-phase thin-layer chromatography, Advan. Chem. Ser., 114 (1972) 61-79. 79 P. Bianchini, R. Casetta and B. Osima, Determination of urinary pregnanediol and pregnanetriol by direct photoreflectometry of thin-layer chromatograms, Biochim. Biol. Sper., 5 (1966) 325-331 (Ital.). 80 T. BiCan-FiJter, Quantitative separation and estimation of steroids by thin-layer chromatography. 1. Determination of progesterone and testosterone propionate in oil solutions, J. Chromatogr., 22 (1966) 465-468. 81 T. BiCan-FiSter and J. MerkaS, Colorimetric assay of digitoxin and gitoxin in mixtures after thin-layer chromatographic separation, J. Chromatogr.,41 (1969) 91-95. 82 D.C. Bicknell and D.B. Gower, The separation of C19-16-unsaturated steroids from Czland other C19-steroids by two-dimensional thin-layer chromatography, J. Chromatogr., 61 (1971) 358-360. 83 D.C. Bicknell and D.B. Gower, Separation of some 16-androstenes on hydroxyalkoxypropylsephadex (Lipidex), J. Chromatogr., 110 (1975) 210-212. 84 R.H. Bishara and I.M. Jakovljevic, Separation of some estrogens by thin-layer chromatography,f. Chromatogr.,41 (1969) 136-138. 85 1. Bjorkhem, R. Blomstrand, 0. Lantto, A. Lof and L. Svensson, Plasma cortisol determination by mass fragmentography, Clin. Chim. ACfQ,56 (1974) 241-248. 86 I. Bjorkhem, R. Blomstrand and L. Svensson, Serum cholesterol determination by mass fragmentography, Clin. Chim. Acra, 54 (1974) 185-193. 87 I. Bjorkhem and H. Danielsson, Assay of liver microsomal cholesterol 7a-hydroxylase using deuterated carrier and gas chromatography-mass spectrometry, Anal. Biochem.,

132

REFERENCES

59 (1974) 508-516. 88 S. Bleecken, G. Kaufmann and K. Kummer, Quantitative determination of tritium-

labeled compounds on thin-layer chromatograms, J. Chromatogr., 19 (1965) 105-1 13 (Ger.). 89 G.A. Blondin, B.D. Kulkarni, J.P. John, R.T. Van Aller, P.T. Russell and W.R.Nes, Identification of steroidal 5,8-peroxides by gas-liquid chromatography, Anal. Chem., 39 (1967) 36-40. 90 G. Blunden and R. Hardman, Thin-layer chromatography of Dioscorea sapogenins, J. Chromatogr., 15 (1964) 273-276. 91 G. Blunden and R. Hardman, Quantitative estimation of monohydroxy saturated steroidal sapogenins in plant materials by densitometric thin-layer chromatography, J. Chromatogr., 34 (1968) 507-5 14. 92 G. Blunden, R. Hardman and J.C. Morrison, Quantitative estimation of diosgenin in Dioscorea tubers by densitometric thin-layer chromatography, J. Pharm. Sci., 56 (1967) 948-950. 9 3 L.E. Bottiger and B.P. Lisboa, Fractionation and determination of 17-ketosteroids by means of column and thin-layer chromatography, 2. Klin. Chem. Klin. Biochem., 5 (1967) 176-182. 94 M.L. Boisio, Evaluation of several solvent systems in the thin-layer chromatographic separation of cardiotonic aglycones and glycosides from cholesterol, phytosterols, and their esters, J. Chromatogr., 73 (1972) 279-286. 95 P.M. Boll, Alkaloidal glycosides from Solanum dulcamara. 2. Three new alkaloidal glycosides and a reassessment of soladulcamaridine, Acfa Chem. Scand., 16 (1962) 1819-1 830. 96 E. Bombardelli, Quantitative and qualitative determination of proscillaridine A, Fifoterapia, 36 (1965) 72-76 (Ital.). 97 R. Bonaly, C. Merlin and H. Colonval, Fractionation of urinary 17-hydroxycorticosteroids by thin-layer chromatography, Phurm. Biol., 5 (1968) 591-595 (Fr.). 9 8 D.W. Borst and J.D. O’Connor, Trace analysis of ecdysones by gas-liquid chromatography, radioimmunoassay and bioassay, Steroids, 24 (1974) 637-656. 99 M. Boucard, V. Massa, J.J. Serrano, P. Susplugas and D. Sincholle, Chromatography of bile. 1. Direct fluorimetry of biliary acid chromatograms, Trav. SOC.Pharm. Montpellier, 34 (1974) 19-25 (Fr.). 100 A.R. Bourne, A convenient system for the separation of some steroids on Sephadex LH-20, J. Chromatogr., 9 2 (1974) 465-466. 101 P. Bournot, B.F. Maume and P. Padieu, Sex-linked specificity of the hepatic metabolism of steroids in rats, Mass fragmentography as a method for the assay of hydrogenated metabolites of corticosterone in the liver, Biomed. Muss Spectrom., 1 (1974) 29-39. 102 G.S. Boyd and H.R.B. Hutton, Detection of steroids on chromatoplates using a nondestructive method, Biochim. Biophys. Acfa,69 (1963) 419-420. 103 H.L. Bradlow, Extraction of steroid conjugates with a neutral resin, Steroids, 11 (1968) 265-272. 104 P. Braeckman, R. van Severen and F. Haetinck, Control of Digitalis glycosides and of the stability of tincture of Digitalis by thin-layer chromatography, Pharm. Tijdschr.

REFERENCES

133

Belg., 40 (1963) 129-146 (Dutch). 105 K.R. Brain and R. Hardman, An improved method of densitometric thin-layer chromatography as applied to the determination of sapogenin in Dioscorea tubers, J. Chromafogr.,38 (1968) 355-363. 106 W.E. Braselton, Jr., J.C. Orr and L.L. Engel, Twin-ion technique for detection'of metabolites by gas chromatography-mass spectrometry. Intermediates in estrogen biosynthesis, Anal. Biochem ., 53 (1 973) 64-85. 107 W.E. Braselton, Jr., J.C. Orr and L.L. Engel, Identification by gas chromatographymass spectrometry of intermediates in the aromatization of modified C19-steroids by human placental microsomes, Steroids, 24 (1974) 41 1-433. 108 H. Breuer, Modern methods for the determination of steroid hormones, Z. Anal. Chem., 21 2 (1965) 253-267 (Ger.). 109 T. Briggs and S.R. Lipsky, The formation of partial trimethylsilyl ethers of bile acid methyl esters, and their analysis by gas chromatography, Biochim. Biophys. Acfa, 97 (1965) 579-588. 110 A.H. Brodie, N. Shimizu, S.A.S. Tait, J.F. Tait and S. Baniukiewicz, A method for the measurement of aldosterone in peripheral plasma, using [3H]-acetic anhydride, J. Clin. Endocrinol. Metab., 27(1967) 997-1011. 1 11 C.J.W. Brooks, Studies of acetylated corticosteroids and related 20-oxopregnane derivatives by gas-liquid chromatography, Anal. Chem., 37 (1965) 636-641. 112 C.J.W. Brooks, R.V. Brooks, K. Fotherby, J.K. Grant, A. Klopper and W. Klyne, The identification of steroids, J. Endocrinol., 47 (1970) 263-272. 113 C.J.W. Brooks, E. Chambaz and E.C. Horning, Thin-layer and column chromatographic group separations of steroids as trimethylsilyl ethers. Isolation for gas-liquid chromatographic analysis of pregnanediol and estriol in pregnancy urine, Anal. Biochem., 19 (1967) 234-242. 1 14 C.J.W. Brooks and L. Hanaineh, Correlation of gas-liquid chromatographic behavior and structure of steroids,Biochem. J., 87 (1963) 151-161. 115 C.J.W. Brooks and D.J. Harvey, Comparative gas chromatographic studies of corticosteroid boronates, J. Chromafogr., 54 (1971) 193-204. 116 C.J.W. Brooks, W. Henderson and G. Steel, The use of trimethylsilyl ethers in the characterization of natural sterols and steroid diols by gas chromatography-mass spectrometry, Biochim. Biophys. Acfa,296 (1973) 431-445. 117 C.J.W. Brooks, E.C. Horning and J.S. Young, Characterization of sterols by gas chromatography-mass spectrometry of the trimethylsilyl ethers, Lipids, 3 (1968) 39 1-402. 118 C.J.W. Brooks and R.A.B. Keates, Gel filtration in lipophilic solvents using hydroxyalkoxypropyl derivatives of Sephadex, J. Chrornafogr.,44 (1969) 509-521. 119 C.J.W. Brooks, B.A. Knights, W. Sucrow and B. Raduchel, The characterization of 24-ethylidene-sterols, Steroids, 20 (1972) 487-497. 120 C.J.W. Brooks and A.M. Lawson, Studies of fluorinated corticosteroids based on gas chromatography-mass spectrometry, Excerpfu Med. Inf. Congr. Ser., 219 (1971) 238-247. 121 C.J.W. Brooks and B.S. Middleditch, Uses of chloromethyldimethylsilyl ethers as derivatives for combined gas chromatography-mass spectrometry of steroids, Anal.

134

REFERENCES

Lett., 5 (1972) 611-618. 122 C.J.W. Brooks and B.S. Middleditch, Mass spectrometry in steroid analysis, in E. Heftmann (Editor), Modern Methods of Steroid Analysis, Academic Press, New York, 1973, pp. 139-198. 123 C.J.W. Brooks, A.R. Thawley, P. Rocher, B.S. Middleditch, G.M. Anthony and N.G. Stilwell, Characterization of steroidal drug metabolites by combined gas chromatography-mass spectrometry, J. Chromatogr. Sci., 9 (1971) 35-43. 124 C.J.W. Brooks and J. Watson, Quantitative thin-layer chromatography of trimethylsilyl ethers of hydroxylic steroids, J. Chromatogr., 31 (1967) 396-404. 125 S.C. Brooks and V.C. Godefroi, Quantitative collection of micro amounts of steroids from gas-liquid chromatography, Anal. Biochem., 7 (1964) 135-146. 126 A.C. Brownie, H.J. van der Molen, E.E. Nishizawa and K.B. Eik-Nes, Determination of testosterone in human peripheral blood using gas-liquid chromatography with electron-capture detection, J. Clin. Endocrinol. Merab., 24 (1964) 1091-1 102. 127 A. Bruusgaard, Quantitative determination of the major 3-hydroxy bile acids in biological material after thin-layer chromatographic separation, Clin. Chim. Acta, 28 (1970) 495-504. 128 W.H. Bulger, R.E. Talcott and S.J. Stohs, Separation of the hydroxylation products of digitoxigenin by thin-layer chromatography, J. Chromatogr., 70 (1972) 187-1 89. 129 I.E. Bush, The Chromatography of Steroids, International Series of Monographs on Pure and Applied Biology, Biochemical Division, Vol. 2, Pergamon, New York, 1961. 130 I.E. Bush, Quantitative estimation of substances on paper chromatograms. 1. Machine for the treatment of paper strips with chemical reagents, J. Chromatogr., 23 (1966) 94-1 19. 131 I.E. Bush, Automation of steroid analysis, Science, 154 (1966) 77-83. 132 I.E. Bush, Automation of the analysis of urinary steroids using quantitative paper chromatography and a small laboratory digital computer, Clin. Chem., 14 (1968) 491-5 12. 133 E. Butruk, J. Vaedtke and B. Samocuik, Thin-layer partition chromatography for urinary metabolites of cortisol, J. Chromatogr.,32 (1968) 31 1-314. 134 A.G. Butterfield, B.A. Lodge and N.J. Pound, High-speed liquid chromatographic separation of equine estrogens, J. Chromatogr. Sci., 11 (1973) 401-405. 135 A.G. Butterfield, B.A. Lodge, N.J. Pound and R.W. Sears, Combined assay, identification, and foreign related steroids test for methandrostenolone by high-speed liquid chromatography, J. Pharm. Sci., 64 (1975) 441-443. 136 L.M.G. Cabrera and D.P.N. Tsao, Separation ofDigitalis glycosides by thin-layer chromatography, J. Philipp. Pharm. Ass., 56 (1970) 5-8. 137 G. Cagnazzo, A. Ros and G. Bignardi, Gas-chromatographic determination of urinary estrogens during pregnancy and normal menstrual cycle by dry sampling, J. Chromatogr., 19 (1965) 185-189. 138 H.I. Calvin, K.D. Roberts, C. Weiss, L. Bandi, J.J. Cos and S. Lieberman, Column liquid-liquid partition chromatography of steroidal sulfates, Anal. Biochem., 15 (1966) 426-436. 139 T.H. Campion and S . Dilley, Measurement of nanogram quantities of vitamin D3, Anal. Lett., 6 (1973) 139-145. '

REFERENCES

135

140 A. Cantafora, G. Cavina, G. Moretti and B. Gallinella, Analysis of corticosteroids by gradient elution liquid chromatography. Application to the analysis of cortical extracts for pharmaceutical use, Furmaco (Pavia),Ed. Bat., 29 (1974) 35 1-365. 141 D.I. Cargill, The separation of cholesterol from related stanols and stanones by thinlayer chromatography, Analyst (London),87 (1 962) 865-869. 142 B.R. Carr, G. Mikhail and G.L. Flickinger, Column chromatography of steroids on Sephadex LH-20, J. Clin. Endocrinol. Metab., 33 (1971) 358-359. 143 H. Carstensen, Chromogenic steroid reactions induced by silica in dilute sulfuric acid, useful for detection of steroids on thin-layer chomatoplates without heating, and a note on the specificity of the so-called Oertel, Allen, and Jensen reactions, Eur. J. Steroids, 1 (1966) 233-286. 144 M.L. Carvalhas and M.A. Figueira, Comparative study of thin-layer chromatographic techniques for separation of digoxin, digitoxin and their main metabolites, J. Ciiromatogr., 86 (1 973) 254-260. 145 O.W. Cass, A.E. Cowen, A.F. Hofmann and S.B. Coffin, Thin-layer chromatographic separation of sulfated and nonsulfated lithocholic acids and their glycine and taurine conjugates, J. Lipid Res., 16 (1975) 159-160. 146 F.S. Castellana and W.G. Kelly, Elution gradients generated under hydrostatic equilibrium employing various devices and solvents of different density, J. Chromatogr. Sci., 11 (1973) 429-434. 147 T. Castellani, P. Kjelgaard-Nielsen and J. Wolff-Jensen, Relationship between densitometric peak area and concentration of lipid in the quantitative analysis of a lipid mixture separated by thin-layer chromatography,J. Chromatogr., 104 (1975) 123-133. 148 E. CastrBn, Determination of hormones in geriatric vitamin preparations by thin-layer chromatography, Farm. Aikak., 74 (1965) 189-194. 149 A. Castro, D. Bartos, B. Jelen and M. Kutas, Radioimmunoassay for deoxycorticosterone in human peripheral plasma using Sephadex LH-20 chromatography, Steroids, 22 (1973) 851-867. 150 D.M. Cathro, J. Cameron and K. Birchall, The application of paper chromatography to the study of steroid structure,J. Chromatogr., 17 (1965) 362-372. 151 A . Cavalleri and A. Salvadeo, Gas-chromatographic determination of urinary pregnanediol, Biochim. Biol. Sper., 5 (1966) 332-338 (Ital.). 152 A. Cavalleri, A. Salvadeo and A. Favino, Gas-chromatographic analysis of urinary 1 I-deoxy-l7-ketosteroids as trimethylsilyl ethers, Folia Endocrinol., 17 (1 964) 646657 (Ital.). 153 G. Cavina, G. Giocoli and D. Sardini, Thin-layer chromatographic separation of urinary aldosterone for determination with the Tetrazolium Blue reaction, Steroids, 14(1969) 315-325. 154 G. Cavina and G. Moretti, Quantitative separation of steroids in oily solutions by means of thin-layer chromatography with continuous elution, J. Chromatogr., 22 (1966) 41-51. 155 G. Cavina, G. Moretti and A. Cantafora, Analysis of corticosteroids in mixtures by gradient elution liquid chromatography, J. Chromatogr., 80 (1973) 89-100. 156 G. Cavina, G. Moretti, A. Mollica and R. Antonini, Quantitative separation of steroids in oil solutions by column chromatography with continuous monitoring of the eluate

136

REFERENCES

using a flame-ionization detector, Farmaco (Pavia),Ed. Prat., 26 (1971) 275-287. 157 G. Cavina, G. Moretti, A . Mollica and R. Antonini, Analysis of steroid mixtures by column chromatography with continuous monitoring of the eluate by a flameionization detector, 1. Chromatogr., 60 (1971) 179-184. 158 G. Cavina, G. Moretti, A. Mollica, L. Moretta and P. Siniscalchi, Analytical and preparative column chromatography of neutral lipids with continuous monitoring of the eluate by flame ionization detector, J. Chromatogr., 44 (1969) 493-508. 159 G. Cavina, G. Moretti and M. Petrella, A solvent system for the separation of steroids with estrogenic and progestational activity by two-dimensional thin-layer chromatography, J. Chromatogr., 103 (1975) 368-371. 160 G. Cavina, G. Moretti and J. Sardi de Valverde, Quantitative separation of estrogens from oil solutions by thin-layer chromatography and their gas-chromatographic and colorimetric determination. Ann. Inst. Super. Sanita, 4, Parts 1-2 (1968) 75-89 (Ital.), 161 G . Cavina, G.Moretti and P. Siniscalchi, Separation and determination of steroids in oil solution. 4. Gas-liquid chromatographic analysis of anabolic, androgenic, estrogenic, and progestational steroids,J. Chromatogr., 47 (1970) 186-194. 162 G. Cavina and C. Vicari, Determination of corticosteroids in human urine extracts by thin-layer chromatography, Boll. SOC.Ital. Biol. Sper., 39 (1963) 1953-1956 (I tal.). 163 V. Cejka and E.M. Venneman, Difference in chromatographic behavior of aldosterone and 1,2-[3H]-aldosterone,Clin. Chim. Acta, 11 (1965) 188-190. 164 J.R.G. Challis and R.B. Heap, Steroid mono- and diheptafluorobutyrates. Preparation, purification, and estimation, J. Chromatogr., 50 (1970) 228-238. 165 B.E. Cham, J.J. Hurwood, B.R. Knowles and L.W.Powell, Rapid, sensitive method for the separation of free cholesterol from ester cholesterol, Clin. Chim. Acta, 49 (1973) 109-113. 166 E.M. Chambaz, G. Defaye and C. Madani, Trimethylsilyl ether-enol-trimethylsilyl ether. New type of derivative for the gas-phase study of hormonal steroids, Anal. Chem., 45 (1973) 1090-1098. 167 E.M. Chambaz and E.C. Horning, Conversion of steroids to trimethylsilyl derivatives for gas-phase analytical studies. Reactions of silylating agents, Anal. Biochem., 30 (1969) 7-24. 168 E.M. Chambaz and C. Madani, Gas chromatography in analysis of hormonal steroids. Possible application in their physiopathological investigation, Rev. Fr. Gynecol. Obstet., 65 (1970) 263-271 (Fr.). 169 E.M. Chambaz, C. Madani and A. Ros, Trimethylsilyl-enol-trimethylsilyl.New type of derivative for the gas-phase study of dihydroxyacetone side-chain saturated corticosteroid metabolites, J. Steroid Biochem., 3 (1972) 741 -747. 170 J. Chamberlain, Characterization of 3-0x0-A4-steroids by gas chromatography of enol heptafluorobutyrates, J. Chromatogr., 28 (1967) 404-405. 171 J. Chamberlain, Gas-chromatographic determination of levels of aldadiene in human plasma and urine following therapeutic doses of spironolactone, J. Chromatogr., 55 (1971) 249-253. 172 J. Chamberlain, B.A. Knights and G.H. Thomas, Analysis of steroid metabolites by

REFERENCES

137

gas chromatography, J. Endocrinol., 26 (1963)367-387. 173 J . Chamberlain, B.A. Knights and G.H. Thomas, A system of analysis by gas chromatography of 17a-and 17P-pregnane-3,20-diols and their identification as metabolites of progesterone in man, monkey, rabbit, and guinea pig, J. Endocrinol., 28 (1964)235-246. 174 J. Chamberlain and G.H. Thomas, Characterization of steroid ketones by gas chromatography, Biockem. J . , 86 (1963)3 ~ . 175 J. Chamberlain and G.H. Thomas, RM values in adsorption chromatography, J. Ckromatogr., 1 1 (1963)408-409. 176 J . Chamberlain and G.H. Thomas, Characterization of 20-oxosteroids by gas chromatography,AnaZ. Biockem., 8 (1964)104-1 15. 177 R. Chambers, A. Dos Reis Valle and K. Fotherby, Method for the estimation of 1 l-deoxy-l7-ketosteroids in urine, Clin. Ckim. A m , 17 (1967) 135-137. 178 E.Chang, Partition thin-layer chromatography of steroids of the CIp series, Steroids, 4 (1964)237-247. 179 J.R. Chapman and E. Bailey, Use of chloromethylsilyl ether derivatives for the determination of hydroxylated steroids by gas chromatography-mass spectrometry, Anal. Chem., 45 (1973)1636-1641. 180 J.R. Chapman and E. Bailey, Determination of plasma testosterone by combined gas chromatography-mass spectrometry, J. Ckromatogr., 89 (1974)215-224. 181 S.C. Chattoraj, A. Scommegna and L. Rambow, A gas-chromatographic technique for the simultaneous determination of urinary pregnanediol and pregnanetriol after ammonium sulfate precipitation, Steroids, 9 (1967)327-346. 182 S.C. Chattoraj and H.H. Wotiz, A routine method for the gas-chromatographic determination of pregnanediol, Fertil. Steril., 18 (1967)342-352. 183 L. Chen and H. Hsiu, Polyamide chromatography of estrogens and related compounds. 3. Chromatographic behavior of estrogens on poly(ethy1ene terephthalate)-supported and glass-supported polyamide thin layers, Bull. Taipei Med. Coll., (1973)125-132. 184 P.S. Chen, Jr., Fluorescent dyes and thin-layer chromatography applied to detection of vitamin D and related sterols in tuna liver oil,AnaZ. Chem., 37 (1965)301-302. 185 K.Chmel, P. Pihera and V. Schwarz, Chromatography on Silpearl UVz54 silica gel. 1. Chromatography of steroidal compounds, Chem. Listy ,67(1973)649-653 (Czech). 186 S.J. Clark and H.H. Wotiz, Separation and detection of nanogram amounts of steroids, Steroids, 2 (1963)535-540. 187 S.J. Clark and H.H. Wotiz, Gas chromatography of steroid hormones, in E. Heftmann (Editor),Modern Methods of Steroid Analysis, Academic Press, New York, 1973, pp. 71-102. 188 J.-R. Claude, Separation of cholesterol, desmosterol, and 5-dihydrocholesterol by thin-layer chromatography after propionylation, J. Ckromatogr., 17 (1965)596-599 (Fr.). 189 J.-R. Claude, Separation and identification of sterols with substituted A and B rings by thin-layer and gas chromatography, J. Ckromatogr., 23 (1966)267-273 (Fr.). 190 J.-R. Claude, Thin-layer chromatography of sterols. Application t o the study of biological and pharmacological problems, Bull. SOC.Ckim. Fr., (1973)114-1 17 (Fr.). 191 J.-R. Claude and J.-L. Beaumont, Method of systematic study of serum sterols by

138

REFERENCES

thin-layer chromatography,*Ann. Biol. Clin. (Paris), 22 (1964) 815-833 (Fr.). 192 C.J. Clifford, J.V. Wilkinson and J.S. Wragg, Thin-layer chromatography in a limit test for related foreign steroids,J. Pharm. Pharmacol., Suppl., 16 (1964) 11T-16T. 193 S . Cohen and E. Oran, Conjugated estrogens. 2. A new method for isolating estriol monoglucosiduronide from normal human pregnancy urine, Can. J. Biochem ., 50 ( 1972) 1245- 1248. 194 G.L. Cohn and E. Pancake, A standardized technique for the separation of steroids by thin-layer chromatography, Nature (London), 201 (1964) 75-76. 195 W.P. Collins, J.M. Sisterson, E.N. Koullapis, M.D. Mansfield and I.F. Sommerville, Evaluation of gas-liquid chromatography for the determination of plasma testosterone using nickel-63 electron-capture detection, J. Chromatogr., 37 (1968) 33-45. 196 W.P. Collins and I.F. Sommerville, Quantitative determination of progesterone in human plasma by thin-layer and gas-liquid radiochromatography, Nature (London), 203 (1964) 836-839. 197 B.A. Cooke, Determination of specific activities of labeled steroids using gas-liquid chromatography and a fraction collector, Anal. Biochem., 32 (1969) 198-203. 198 B.K. Cooke, Determination of diosgenin in Dioscorea deltoidea and Dioscorea sylvatica by using gas-liquid chromatography, Analyst (London), 95 (1970) 95--97. 199 J.W. Copius Peereboom and H.W. Beekes, The analysis of mixtures of animal and vegetable fats. 5. Separation of sterol acetates by thin-layer chromatography in reversed-phase systems and on Silica Gel G-silver nitrate layers, J. Chromatogr., 17 (1965) 99-113. 200 G.L. Corona and M. Raiteri, Separation and quantitative determination of k-strophanthin glycosides by thin-layer chromatography, J. Chromatogr., 19 (1965) 435-437. 201 G.L. Corona, M. Raiteri and G . Tieghi, Identification of the components of k-strophanthin by thin-layer chromatography, Farmaco (Pavia), Ed. Prat., 19 (1964) 574-579 (Ital.). 202 G.L. Corona, R. Rondanelli and A. Mascherpa, Fractionation of Digitalis glycosides by column chromatography, Farmaco (Pavia), Ed. Prat., 18 (1963) 549-557 (Ital.). 203 P.S. Cowley, F.J. Evans and R.F.A. Ginman, A gas-liquid chromatographic determination of the ratio of gitogenin and digitogenin in mixtures, J. Chromatogr., 54 (1971) 185-191. 204 R.I. Cox, Gas chromatography in the analysis of urinary pregnanediol, J. Chromatogr., 12 (1963) 242-245. 205 R.I. Cox and A.R. Bedford, The use of double derivatives in the gas chromatography of urinary estrogens, Steroids, 3 (1964) 663-669. 206 J. Coyotupa, K. Kinoshita, R.-Y. Ho, C. Chan, W. Paul, M . Foote and S . Kushinsky, Variable decomposition by environmental contaminants in air of estrogens on glass plates coated with silica gel for thin-layer chromatography, Anal. Biochem., 34 (1970) 71-73. 207 A. Craig and J . Chamberlain, Group determination of urinary adrenal steroids by gas chromatography, Ann. Clin. Biochem., 9 (1972) 88-90. 208 B.G. Creech, Separation and determination of ketosteroids, pregnanediol, and pregnanetriol on one column,J. Gas Chromatogr., 2 (1964) 194-195. 209 0. CrCpy, 0. Judas and B. Lachkse, Detection of conjugated steroids separated by

REFERENCES

139

thin-layer chromatography, J. Chromatogr., 16 (1964) 340-344 (Fr.). 210 Q.E. Crider, P. Alaupovic, J. Hillsberry, C. Yen and R.H. Bradford, Separation of lipids by Silica Gel C column chromatography, J. Lipid Res., 5 (1964) 479-481. 21 1 L.E. Crocker and B.A. Lodge, Thin-layer chromatographic separation of equine estrogens on Silica Gel H-silver nitrate plates, J. Chromatogr., 62 (1971) 158-160. 21 2 L.E. Crocker and B.A. Lodge, Thin-layer chromatographic separation of conjugated estrogens on Silica Gel G-silver nitrate plates, J. Chromatogr., 69 (1972) 419-420. 213 H.C. Curtius, Gas-chromatographic determination of pregnanediol, pregnanetriol, and pregnanetriolone in urine, 2. Klin. Chem., 4 (1966) 114-1 19 (Ger.). 214 H.C. Curtius, Gas chromatography of steroids, in H.C. Curtius (Editor), Clinical Biochemistry. Vol. 1.Principles and Methods, Walter de Gruyter, Berlin, 1974, pp. 700-753. 215 H.C. Curtius and M. Muller, Combination of gas chromatography and thin-layer chromatography for the investigation of steroids, J. Chromatogr., 32 (1968) 222229 (Cer.). 216 H.C. Curtius, M. Muller and B. Manella, Gas-liquid chromatography of 17-ketosteroids and progesterone metabolites of urine. Comparison of different methods of hydrolysis, J. Chromatogr., 30 (1967) 410-427. 217 J.M.Dalmau, J.M. PIP Delfina and A. del Pozo Ojeda, Identification of 9wfluorinated homologues of hydrocortisone and prednisolone by thin-layer chromatography on insoluble polyvinylpyrrolidone, J. Chrornatogr., 48 (1970) 118-1 22. 21 8 J.M. Dalmau, J.M. P1L-Delfina and A. del Pozo Ojeda, Separation and determination of the synthetic epimeric corticosteroids dexamethasone and betamethasone in mixtures by thin-layer chromatography on insoluble polyvinylpyrrolidone, J. Chromatogr., 78 (1973) 165-171. 219 J.Y. Daniel, H. Mion and D. Soulas, Radioimmunoassay of plasma aldosterone by chromatography on Sephadex LH-20, Clin. Chim. Acta, 55 (1974) 235-243. 220 D. Darlington, B.A. Knights and G.H. Thomas, Some comments on the calculation of relative retention times of steroids,J. Gas Chromatogr., 1 (1963) 21-23. 221 B.H. Davies and E.I. Mercer, The liquid spectroradiochromatograph - an automated method for the simultaneous separation, identification, purification, estimation, and radioassay of the components of a mixture of unsaponifiable lipids, J. Chromatogr., 46 (1970) 161-172. 222 G. De Angelis, Chromatography on anodically oxidized aluminum. 2. Ascendent chromatography and separation of the main estrogens and ketosteroids, Sci. Tee., 4 (1960) 75-89. 223 L. Dehennin, A. Reiffsteck and R. Scholler, Quantitative method for the estimation of testosterone and progesterone in human plasma, using the gas chromatographmass spectrometer combination with single-ion monitoring, J. Steroid Biochem., 5 (1974) 81-86. 224 L. Dehennin, A. Reiffsteck and R. Scholler, Simultaneous estimation of testosterone, progesterone, and androstenedione by gas chromatography-mass spectrometry with a single-ion detection. Correlation with radioimmunoassay. J. Steroid Biochem., 5 (1974) 767-768. 225 L. Dehennin and R. Scholler, A new derivative for the gas-liquid chromatography

140

226

227

228 229

230

231

232

233 234 235 236 237

238

239 240 241 242 243

REFERENCES

with electron-capture detection of steroidal secondary alcohols, J. Chromatogr., 1 11 (1975) 238-241. M. Delaforge, B.F. Maume, P. Bournot, M. Prost and P. Padieu, Use of gas-liquid chromatography with glass capillary columns for steroid trace analysis in tissues, J. Chromatogr. Sci., 12 (1974) 545-549. H.F. DeLuca, M.H. Zile and P.F. Neville, Chromatography of vitamins A and D, in G.V. Marinetti (Editor), Lipid Chromatographic Analysis, Vol. 2, Marcel Dekker, New York, 1969, pp. 345-457. K. Demisch, Rapid thin-layer chromatographic method for the separation of the 17-ketosteroids in urine, J. Chromatogr., 36 (1968) 246-249 (Ger.). K. Demisch and W. Staib, An improved method for isolation and identification of hydroxylated testosterone metabolites as trimethylsilyl ethers from biological material, Eur. J. Biochem., 9 (1969) 378-382. B. Desfosses, R. Condom and R. Emiliozzi, Vapor-phase chromatographic behavior of 0-D-glucopyranuronosides (tri-0-acetylated methyl esters of hydroxy steroids), J. Chromatogr., 38 (1968) 290-292 (Fr.). P.G. Devaux, M.G. Horning, R. Hill and E.C. Horning, 0-Benzyloximes: Derivatives for the study of ketosteroids by gas chromatography. Application to urinary steroids of the newborn human,Anaf. Biochem., 41 (1971) 70-82. P.G. Devaux, M.G. Horning and E.C. Horning, Benzyloxime derivatives of steroids. New metabolic profile procedure for human urinary steroids, Anal. Lett., 4 (1 97 1) 151-160. Z. Deyl, K. Macek and J. Janik, Liquid Column Chromatography, Elsevier, Amsterdam, 1975. Z. Deyl, J . Rosmus, M. Juficovi and J. Kopeckq, Bibliography of Column Chromatography, 1967-1970, Elsevier, Amsterdam, 1973. I.M. Diab and A.W. Gomoll, Color characterization of adrenal corticoids on thinlayer chromatograms, Steroids, 8 (1966) 109-1 10. N.W. DiTullio, C.S. Jacobs, Jr. and W.L. Holmes, Thin-layer chromatography and identification of free sterols, J. Chromatogr., 20 (1965) 354-357. B. Doboszynska and E. Burakowska, Application of thin-layer chromatography for the quantitative determination of 7-dehydrocholesterol,Bull. Acad. Pol. Sci., Ser. Sci. Biof., 19 (1971) 553-561. E. Doelker, I. KapCtanidis and A. Mirimanoff, Application of remission fluorimetry to the direct estimation of Digitalis preparations on chromatoplates, Pharm. Acta Helv., 44 (1969) 647-651 (Fr.). P. Doerr, Thin-layer chromatography and elution of picogram amounts of estradiol, J. Chromatogr., 59 (1971) 452-456. H.H.A. Dollwet and A.W. Norman, Chromatographic separation of vitamins D2 and D3 and related compounds,Anaf. Biochem., 25 (1968) 297-306. R.J. Dolphin, The analysis of estrogenic steroids in urine by high-speed liquid chromatography, J. Chromatogr., 83 (1973) 421-429. M. Donike, Problem of the detection of anabolic steroids. Gas-chromatographic and mass-specific possibilities, Sportarzt Sportmed., 26 (1975) 1-6 (Ger.). N J . Doorenbos and R.K. Sharma, Steroids. 29. Separation and characterization of

REFERENCES

244

245 246

247

248 249 250 251 252 253 254

255

256

257 258 259

260

261

141

various classes of steroids by thin-layer chromatography, J. Chromatogr., 29 (1967) 393-395. J. Dougan and L. Tan, Detection and quantitative measurement of fecal water pollution using a solid-injection gas-chromatographic technique and fecal steroids as a chemical index,J. Chromatogr., 86 (1973) 107-1 16. D.R. Douglas and H.S. Black, Inexpensive method for collection of radioactive compounds from gas-chromatographic effluents, Chromatographia,6 (1973) 229-230. M.L. DOW,R.D. Kirchhoefer and J.F. Brower, Rapid identification and estimation of gitoxin in digitoxin and digoxin tablets by thin-layer chromatography, J. Pharm. Sci., 60 (1971) 298-299. F. Dray and I. Weliky, Identification of 0-methyloximes of ketosteroids by gas chromatography, thin-layer chromatography, mass spectra, and kinetic studies, Anal. Biochem., 34 (1970) 387-402. P.A. Drewes and A.J. Kowalski, Some marker dyes for locating steroids eluted from Sephadex LH-20 columns, Clin. Chem., 20 (1974) 1451-1453. B. Duperray, Analysis of a mixture of bile acids,MethodesPhys. Anal., 4 (1968) 91-92 (Fr.). A.H. Duthie, K.R. Simmons and B.R. Urey, New thin-layer chromatographic solvent systems for separating steroid hormones, J. Chromatogr., 44 (1969) 193-194. J. Duvivier, Thin-layer chromatography of some etiocholenic acids produced by oxidation of corticosteroids, J. Chromutogr., 19 (1965) 352-357 (Fr.). R. Dvir and R. Chayen, A chromatographic and fluorimetric method for the determination of oestriol in pregnancy urine,J. Chromatogr.,45 (1969) 76-81. R. Dvir and R. Chayen, Thin-layer chromatography of DANSYL oestrogens, J. Chromatogr., 52 (1970) 505-506. W.G. Dyer, J.P. Gould, N.A. Maistrellis, T.C. Peng and P. Ofner, Thin-layer chromatography of some C19-steroids. Use of a simple standardized procedure, Steroids, 1 (1963) 271-285. C. Eaborn, C.A. Holder, D.R.M. Walton and B.S. Thomas, Gas-chromatographic assay at nanogram,levels. (Halomethyl) dimethylsilyl steroid ethers, J. Chem. SOC.,(1969) 2502-2503. M.A. Eastwood and D.Hamilton, A sensitive nondestructive method for the demonstration of bile acids on thin-layer chromatography, Biochem. J., 105 (1967) 37c. M.A. Eastwood, D. Hamilton and L. Mowbray, A method for the estimation of bile acid conjugates and bile acids in biological fluids,J. Chromatogr., 65 (1972) 407-41 1. W.R. Eberlein, A stain of 30-hydroxy As-steroids and sterols, J. Clin. Endocrinol. Metab., 25 (1965) 288-289. W.R. Eberlein, Measurement of low levels of estrone and 170-estradiol in urine, employing ion-exchange, thin-layer and gas-liquid chromatography, Steroids, 14 (1969) 553-573. D.O. Edlund and F.A. Filippini, Improvements and modification of the gas-liquid chromatographic determination of vitamin D2 in multiple vitamin tablets and raw materials,J. Ass. Offic. Anal. Chem., 56 (1973) 1374-1377. R.W.H. Edwards and D.J.H. Trafford, The use of tert.-butyl chromate for the location

142

REFERENCES

of steroids on paper chromatograms, J. Chromatogr.,21 (1966) 275-279. 262 W. Eechaute and G. Demeester, Fluorimetric determination of total estrogens after gel filtration of enzymatically hydrolyzed urine, J. Clin. Endocrinol. Metab., 25 (1 965) 480-490. 263 W. Eechaute, G. Demeester and I. Leusen, Determination of estrone, estradiol-l7/3, and estriol after gel filtration of enzymically hydrolyzed urine, Clin. Chim. Acta, 12 (1965) 359-360. 264 D. Egg, Determination of progesterone in plasma by direct evaluation of thin-layer chromatograms,J. Chromatogr.,86 (1973) 151-157 (Ger.). 265 E.N. Ehrlich and M.L. Laves, A method for measuring urinary pregnanediol by a double isotope derivative dilution technique utilizing thin-layer chromatography, J. Lab. Clin. Med., 65 (1965) 869--882. 266 K.B. Eik-Nes and E.C. Horning, Gas Phase Chromatography of Steroids. Monographs on Endocrinology, Vol. 2, Springer, Berlin, 1968. 267 A. Elbanowska and F. Kaczmarek, The quantitative determination of the cardenolide complex and lanatoside C in the leaves of Digitalis lanata,Herba Pol., 12 (1966) 173-177 (Pol.). 268 J . Ellingboe, E. Nystrom and J. Sjovall, Liquid-gel chromatography on lipophilichydrophobic Sephadex derivatives, J. Lipid Res., 11 (1970) 266-273. 269 W.H. Elliott, L.B. Walsh, M.M. Mui, M.A. Thorne and C.M. Siegfried, Bile acids. 28. Gas chromatography of new bile acids and their derivatives,J. Chromatogr.,44 (1969) 452-464. 270 D.T. Elmunajjed, M.B.E. Fayez and A.S. Radwan, Steroid sapogenins. 9. Thin-layer chromatography, Phytochemisiry, 4 (1965) 587-592. 271 M.M. El-Olemy and S.J. Stohs, Chromatography on lipophilic dextran gels for fractionation of low-molecular-weight compounds. 1. Steroid digitonides, J. Pharm. Sci., 64 (1975) 161-162. 272 M.M. El-Olemy and S.J. Stohs, Chromatography on lipophilic dextran gels for fractionation of low-molecular-weight compounds. 2. Separation of cardiac glycosides from cardiac aglycones,J. Phurm. Sci., 64 (1975) 163-164. 273 P. Eneroth, Thin-layer chromatography of bile acids, J. Lipid Res., 4 (1963) 11-16. 274 P. Eneroth, Thin-layer chromatography of bile alcohols and bile acids, in G.V. Marinetti (Editor), Lipid ChromatographicAnalysis, Vol. 2, Marcel Dekker, New York, 1969, pp. 149-186. 275 P. Eneroth, B. Gordon, R. Ryhage and J. Sjovall, Identification of mono- and dihydroxy bile acids in human feces by gas-liquid chromatography and mass spectrornetry,J. LipidRes., 7 (1966) 511-523. 276 P. Eneroth, B. Gordon and J. Sjovall, Characterization of trisubstituted cholanoic acids in human feces,J. Lipid Res., 7 (1966) 524-530. 277 P. Eneroth, K. Hellstrom and J. Sjovall, A method for quantitative determination of bile acids in human feces, Acta Chem. Scand., 22 (1968) 1729-1744. 278 P. Eneroth and E. Nystrom, A study of liquid-gel partition of steroids and steroid derivatives on lipophilic Sephadex gels, Biochim. Biophys. Acta, 144 (1 967) 149- 161. 279 P. Eneroth and J . Sjovall, Methods of analysis in the biochemistry of bile acids, Methods Enzymol., 15 (1969) 237-257.

REFERENCES

143

280 P. Eneroth and J. Sjovall, Extraction, purification, and chromatographic analysis of bile acids in biological materials, in P.P. Nair (Editor), Bile Acids, Vol. 1, Plenum Press, New York, 1971, pp. 121-171. 281 L.L. Engel, A.M. Neville, J.C. Orr and P.R. Raggatt, Quantitative gas chromatography of steroid methoxime-trimethylsilylethers, Steroids, 16 (1970) 377-386. 282 H. Engelhardt, J. Asshauer, U. Neue and N. Weigand, Separations on heavily loaded small-particle columns in high-speed liquid chromatography, Anal. Chem ., 46 (1 974) 336-340. 283 H. Engelhardt and H. Wiedemann, High-speed liquid chromatography on alumina of different activity levels,Anal. Chem., 45 (1973) 1641-1646. 284 A. Ercoli, R. Vitali and R. Gardi, Adsorbents for detection, isolation, and evaluation of ethynyl steroids, Steroids, 3 (1964) 479-485. 285 P. Erdody and T.K. Murray, Improved method for determination of vitamins Dz and D3 in pharmaceuticals by gas chromatography, J. Ass. Offic. Anal. Chem., 53 (1970) 189- 190. 286 H. Eriksson and J.-A. Gustafsson, Excretion of steroid hormones in adults. Steroids in faeces from adults,Eur. J. Biochem., 18 (1971) 146-150. 287 H. Eriksson, J . 4 . Gustafsson and J. Sjovall, Excretion of steroid hormones in adults. CI9 and Czl steroids in feces from pregnant women,Eur. J. Biochem., 12 (1970) 520-526. 288 F.J. Evans, High-pressure liquid chromatography of digitoxigenin and its glycosides, J. Chromatogr., 88 (1974) 41 1-412. 289 F.J. Evans, P.A. Flemons, C.F. Duignan and P.S. Cowley, A new thin-layer densitometric technique for the assay of cardenolides from Digitalis purpurea, J. Chromatogr., 88 (1 974) 34 1-346. 290 E. Evrard and G. Janssen, Gas-liquid chromatographic determination of human fecal bile acids, J. Lipid Res., 9 (1968) 226-236. 29 1 D. Exley, The ultramicro detection of steroids using gas-liquid chromatography with electron-capture detection, Mem. SOC.Endocrinol., 16 (1967) 117-1 28. 292 L.F. Fabre, Jr., D.C. Fenimore, R.W. Farmer, H.W. Davis and G. Farrell, Determination of aldosterone and tetrahydroaldosterone in blood by electron-capture gas chromatography, J. Chromatogr. Sci., 7 (1969) 632-638. 293 G. Faglia, A. Liuzzi and G. Norbiato, Technical review of a thin-layer chromatographic method for the determination of urinary testosterone, Biochim. Biol. Sper., 5 (1966) 291-296 (Ital.). 294 H.M. Fales and T. Luukkainen, 0-Methyloximes as carbonyl derivatives in gas chromatography, mass spectrometry, and nuclear magnetic resonance, Anal. Chem., 37 (1965) 955-957. 295 L. Fauconnet and M. Waldesbuhl, Analysis of the cardenolides of Digitalis by thinlayer chromatography, Pharm. Acta Helv., 38 (1963) 423-429 (Fr.). 296 0. Fausa and B.A. SkAlhegg, Quantitative determination of bile acids and their conjugates using thin-layer chromatography and a purified 3a-hydroxy steroid dehydrogenase, Scand. J. Gastroenterol., 9 (1974) 249-254. 297 M.B.E. Fayez and A S . Radwan, Steroid sapogenins. 12. Thin-layer chromatography of steroid sapogenins as acetates, 2. Anal. Chem., 227 (1967) 30-34.

144

REFERENCES

298 M.B.E. Fayez and A.A. Saleh, Constituents of local plants. 15. Thin-layer chromatographic method for the detection and determination of steroidal alkaloid glycosides and aglycones in plant tissue, 2. Anal. Chem., 246 (1969) 380-383. 299 T. FehCr, Structural analysis of CI9-steroids by means of chromatography, J. Chromatogr., 19 (1965) 551-555. 300 T. FehCr, Application of thin-layer chromatography to purification, separation, and quantitative determination of steroid metabolites, Mikrochim. Zchnoanal. A cta, (1965) 105-116. 301 T. Feher, Use of gas-chromatographic methods in the analysis of steroids, Magv. Kern Lapja, 29 (1974) 202-209 (Hung.). 302 T. FehCr and L. Bodrogi, Thin-layer chromatography of steroids. 1. Analysis of biologically important androstanes, Magy. Kern. Fob., 77 (1971) 360-365 (Hung.). 303 T. FehCr and L. Bodrogi, Correlation between the chemical structure and retention behavior of C19-steroids,J. Chromatogr.,71 (1972) 17-21. 304 T. FehCr, K.G. FehCr and L. Bodrogi, A simple gas-liquid chromatographic method with electron-capture detection for the determination of progesterone in the blood of humans and domestic animals,J. Chromatogr., 111 (1975) 125-132. 305 T. FehCr and M.H. Kazik, Thin-layer chromatography of steroids. 2. Analysis of biologically important bile acids, Magy. Kem. Fob., 78 (1972) 186-190 (Hung.). 306 T. FehCr and E. Sirfy, Gas-chromatographic method for the determination of nerobol in human urine,Acta Pharm. Hung., 44 (1974) 168-172 (Hung.). 307 G.L. Feldman and J.F.R. Kuck, Jr., Quantitative gas chromatography of sterols in the free form,Lipids, 1 (1966) 158-159. 308 J.D. Few and T.J. Forward, The quantitative thin-layer chromatography of corticosteroids, J. Chromatogr., 36 (1968) 63-73. 309 P. Fischer, Detection of pregnancy and the quantitative determination of pregnanediol by thin-layer chromatography, Schweiz. Apoth.-Ztg., 103 (1965) 56-59, 137140, 182-186 (Ger.). 3 10 W. Fischer-Rasmussen, Gas-liquid chromatographic method for the measurement of estriol in human plasma during the second half of pregnancy, J. heroid Biochem., l(1970) 127-137. 31 1 A.L. Fisher, A.M. Parfitt and H.M. Lloyd, Gas-liquid chromatography of vitamin D as trimethylsilyl derivatives,J. Chromatogr., 65 (1972) 493-499. 312 A.L. Fisher, A.M. Parfitt and H.M. Lloyd, Removal of cholesterol from serum extracts of vitamin D by thin-layer chromatography, J. Chromatogr., 65 (1972) 571-576. 3 13 W.H. Fishman, F. Harris and S . Green, Two-dimensional thin-layer chromatography of estradiol and estriol glucosiduronic acids extracted from buffered solution, Steroids, 5 (1965) 375-383. 3 14 F.A. Fitzpatrick and S. Siggia, High-resolution liquid chromatography of derivatized nonultraviolet-absorbing hydroxysteroids, Anal. Chem., 45 (1973) 2310-2314. 31 5 F.A. Fitzpatrick, S . Siggia and J. Dingman, High-speed liquid chromatography of derivatized urinary 17-ketosteroids,Anal. Chem., 44 (1972) 221 1-2216. 316 G.L. Flickinger and J.C. Touchstone, Separation of free estrogens and their monosulfate esters by thin-layer chromatography, J. Chromatogr., 36 (1968) 250-252. 3 17 J.T. France, N.L. McNiven and R.I. Dorfman, Determination of microquantities of

REFERENCES

318 3 19 320

321

322

323

324 325

326

327

328 329 330 33 1 332 333

334

145

androsterone, etiocholanolone, and dehydroepiandrosterone by gas-liquid chromatography, Acta Endocrinol., Suppl., 90 (1964) 71-80. D. Franqois, D.F. Johnson and E. Heftmann, Programmed gradient elution chromatography with the steroid analyzer,Anal. Chem., 35 (1963) 2019-2022. . I.D. Frantz, Jr., Chromatography of unesterified sterols on silicic acid-Super-Cel, J. LipidRes., 4 (1963) 176-178. U. Freimuth, M. Buchner, B. Zawta, W. Hub1 and D. Keibel, Polyamide thinlayer chromatography of steroids and indole derivatives, Deut. Gesundheitswes., 21 (1966) 2039-2041 (Ger.). U. Freimuth, B. Zawta and M. Buchner, Separation of corticosteroids by means of thin-layer chromatography on polyamide layers, Acta Biol. Med. Ger., 13 (1 964) 624-628 (Ger.). U. Freimuth, B. Zawta and M. Buchner, New chromatographic system for the separation of structurally isomeric free bile acids, J. Chromutogr., 30 (1967) 6076 10 (Ger.). J.A. Fresen, Identification and purity control of digitoxin (digitoxoside) and digoxin with the aid of thin-layer chromatography, Pharm. Weekbl., 100 (1965) 1509-1513 (Dutch). S. FrgaEiC and Z. Kniewald, Ascorbic acid as an antioxidant in thin-layer chromatography of corticosteroids, J. Chromatogr., 94 (1974) 291 -293. E. Friedrich, L. Siekmann and A.E. Schindler, Identification and quantitation of 16a-hydroxyprogesterone in human amniotic fluid, Clin. Chim. Acta., 56 (1974) 127- 130. J .M.G.J. Frijns, Chromatographic isolation and direct spectrofluorometric determination from the chromatogram of drugs in microgram doses. 1. Injection solutions containing digoxin, digitoxin, and acetyldigitoxin, Pharm. Weekbl., 105 (1970) 209-226 (Dutch). J.M.G.J. Frijns, Chromatographic isolation and direct spectrofluorimetric determination from the chromatogram of drugs in microgram doses. 2. Injection solutions containing deslanoside and ouabain, Pharm. Weekbl., 105 (1970) 709-719 (Dutch). B. Frosch, The quantitative determination of conjugated bile acids in serum after thinlayer chromatographic separation, Arzneim.-Forsch., 15 (1965) 178-184 (Ger.). B. Frosch and H. Wagener, Thin-layer chromatographic separation of bile acids, Z . Klin. Chem., 1 (1963) 187-188 (Ger.). B. Frosch and H. Wagener, The quantitative determination of bile acids by thin-layer chromatography, Klin. Wochenschr., 42 (1964) 192-196 (Ger.). B. Frosch and H. Wagener, The quantitative determination of bile acids from human bile by thin-layer chromatography, Klin. Wochenschr., 42 (1964) 901-908 (Ger.). W. Furst, Oxidation products of ergosterol. I. Chromatographic separation of new oxidation products of ergosterol, Arch. Pharm., 298 (1965) 795-804 (Ger.). K. Fujita, Y. Arikawa and S. Ganno, High-speedliquid chromatograpjy. 2. Highspeed liquid chromatography using a spherical porous copolymer of styrene-divinylbenzene, Nippon Kagaku Kaishi, (1975) 463-468 (Jap.). R. Fumagalli, Gas chromatography of cholesterol and sterol precursors, in G.V. Marinetti (Editor), Lipid Chromatographic Analysis, Vol. 2, Marcel Dekker, New York, 1969,

146

REFERENCES

pp. 187-213. 335 R. Fumagalli, P. Capella and W.J.A. VandenHeuvel, Gas-chromatographic determination of cholesterol-desmosterol ratios, Anal. Biochem ., 10 (1965) 377-386. 336 T. Furukawa and T. Nozu, Separation of aldosterone, 18-hydroxycorticosterone and corticosterone by thin-layer chromatography, Nippon Yakubutsugaku Zasshi, 66 (1970) 651-658 (Jap.). 337 W. Futterweit, N.L. McNiven and R.I. Dorfman, Gas-chromatographic identification of progesterone in human pregnancy plasma, Biochim. Biophys. Acta, 71 (1963) 474 -476. 338 W. Futterweit, N.L. McNiven, L. Narcus, C . Lantos, M . Drosdowsky and R.I. Dorfman, Gas-chromatographic determination of testosterone in human urine, Steroids, 1 (1963) 628-642. 339 E. Gaetani and C.F. Laureri, Liquid-solid chromatography at high pressure. Separation and assay of corticosteroids, Farmaco (Pavia),Ed. Prat., 29 (1974) 110-1 18 (I tal .). 340 G. Galli and E.G. Paoletti, Separation of cholesterol-desmosterol acetates by t h n layer and column chromatography on Silica Gel G-silver nitrate, Lipids, 2 (1967) 72-75. 341 G. Galli and E.G. Paoletti, Quantitative separation of C2,-sterol precursors of cholesterol, Lipids, 2 (1967) 84-85. 342 W.L. Gardiner and E.C. Horning, Gas-liquid chromatographic separation of C19 and CZl human urinary steroids by a new procedure, Biochim. Biophys. Acta, 1 15 (1966) 524-526. 343 M. Gassiot, E. Fernandez, G. Firpo, R. Carbo and M. Martin, Empirical quantum chemical approach to structure-gas chromatographic retention index relationships. 1. Sterol acetates,J. Chromatogr., 108 (1975) 337-344. 344 M. Gassiot-Matas and E. Julia-Danes, Pyrolysis gas chromatography of some sterols, Chromatographia, 5 (1972) 493-501. 345 M. Gassiot-Matas, M. Sanchez-Dalmau and R. Palli-Cama, Analysis of sterols by gas chromatography, Afinidad, 29 (1972) 1153-1 163 (Span.). 346 H.P. Gelbke and R. Knuppen, A new method for preventing oxidative decomposition of catechol estrogens during chromatography, J. Chromatogr., 71 (1972) 465-47 1. 347 HI'. Gelbke and R . Knuppen, Identification and quantitative determination of 2-hydroxyestriol in human late-pregnancy urine, J. Steroid Biochem., 5 (1 974) 1-7. 348 G.L. Genkina and N.K. Abubakirov, Separate determination of basic groups of glycosides in Strophanthus kombe' preparations, Khim.-Farm. Zh., 1 (1967) 47-49 (Russ.) . 349 G.L. Genkina, A.Kh. Sharipov and N.K. Abubakirov, Thin-layer chromatography and determination of k-strophanthin-0 and its glycosides, Med. Prom. SSSR , 18 (1 964) 40-44 (Russ.). 350 G. Gerali, G. Lugaro and L. Ferrari, Steroids. 4. Chromatography of some androstene derivatives, Farmaco (Pavia),Ed. Sci., 20 (1965) 148-157 (Ital.). 35 1 H. Gerdes and W. Staib, Thin-layer chromatographic separation and fluorometric determination of aldosterone in human urine. Klin. Wochenschr., 43 (1965) 789-793 (Ger .). 352 H. Gerlach, W. Grundmann and R. Giessner, The cardioactive glycosides from

REFERENCES

147

Apocynum cannabinum roots. 1. Isolation and separation of the glycosides, Pharmazie, 20 (1965) 450-455 (Ger.). 353 A.L. German and E.C. Horning, Thermostable open-tube capillary columns for the high-resolution gas chromatography of human urinary steroids, J. Chromatogr. Sci., 11 (1973) 76-82. 354 A.L. German, C.D. Pfaffenberger, J.-P. Thenot, M.G. Horning and E.C. Horning, Anal. Chem., 45 (1973) 930-935. 355 Y. Ghoos, G. Vantrappen and D. Mayer, The separation of bile acids from neutral lipids by thin-layer chromatography, J. Chromatogr., 76 (1 973) 425-43 1. 356 G.F. Gibbons, K.A. Mitropoulos and K. Ramananda, Method for the rapid qualitative and quantitative analysis of 4,4-dimethyl sterols, J. Lipid Res., 14 (1973) 589-592. 357 M.W. Gilgan and T.E. Farquharson, Paper-chromatographic separation of a-ecdysone, ecdysterone, inokosterone, makisterone A, and ponasterone A, Steroids, 22 (1973) 365-372. 358 H. Gleispach, Determination of steroid hormones in urine by means of gas chromatography,Rev. Diagn. Biol., 20 (1971) 39-1 16. 359 H. Gleispach, The use of different silylating agents for structure analyses of steroids, J. Chromatogr., 91 (1974) 407-412. 360 G. Condos, B. Matkovics and 0. Kovacs, Thin-layer chromatography of steroid oximes. l.,Microchem. J., 8 (1964) 415-423. 361 S. Gorog and G . Hajbs, Analysis of steroids. 13. Detection of 21-aminocorticosteroids on thin-layer chromatograms, J. Chromatogr.,43 (1969) 541-542. 362 J.W. Goldzieher, C. Matthijssen, C. Gual, B.A. Vela and A. De La Pena, A simplified gas-chromatographic method for large numbers of urinary pregnanediol determinations, Amer. J. Obstet. Gynecol., 98 (1967) 759-766. 363 S.K. Goswami and C.F. Frey, Spray detection of bile acids on thin-layer chromatograms,J. Chromatogr., 47 (1970) 126-127. 364 S.K. Goswami and C.F. Frey, Manganous chloride spray reagent for cholesterol and bile acids on thin-layer chromatograms, J. Chromatogr., 53 (1970) 389-390. 365 S.K. Goswami and C.F. Frey, A novel method for the separation and identification of bile acids and phospholipids on thin-layer chromatograms, J. Chromatogr., 89 (1974) 87-91. 366 S.K. Goswami and C.F. Frey, Separation of bile acids from neutral lipids on thmlayer chromatograms,J. Chromatogr., 100 (1974) 200-201. 367 H. Gottfried, The gas-chromatographic separation of submicrogram amounts of sex steroids, Steroids, 4 (1964) 387-393. 368 H. Gottfried, Thermal decomposition of corticosteroids during gas chromatography, Steroids, 5 (1965) 385-397. 369 D.B. Cower, Chromatographic separation of C ,,-16-dehydrosteroids,J. Chromatogr., 14 (1964) 424-431. 370 K. Grade, W. Forster and S. Schulzeck, An improved method for the isolation of heart glycosides from tissues by using Sephadex (3-200, Biochem. Pharmacol., 16 (1967) 1299- 1303. 37 1 J.K. Grant (Editor), The Gas-Liquid Chromatography of Steroids, University Press, Cambridge, 1967, 294 pp.

148

REFERENCES

372 R.A. Greenstreet, Collection of steroids eluted from a gas chromatography column, J. Chromatogr.,33 (1968) 530-533. 373 J.A. Gregg, New solvent systems for thin-layer chromatography of bile acids, J. Lipid Res., 7 (1966) 579-581. 374 J.H. Grose, W. Nowaczynski, 0. Kuchel and J. Genest, Isolation of aldosterone urinary metabolites, glucuronides, and sulfate, J. Steroid Biochem ., 4 (1 973) 55 1566. 375 F.K. Grutte and H. Gartner, Thin-layer chromatographic separation of bile acids, particularly free dihydroxycholanic acids, J. Chromatogr., 41 (1969) 132-1 35 (Ger.). 376 S.M. Grundy, E.H. Ahrens, Jr. and T.A. Miettinen, Quantitative isolation and gasliquid chromatographic analysis of total fecal bile acids, J. Lipid Res., 6 (1965) 397410. 377 C. Grunwald, Analysis of free phytosterols by gas chromatography using liquid phase OV-101,J. Chromatogr.,44 (1969) 173-175. 378 C. Grunwald, Quantitative analysis of free phytosterols by gas chromatography using stationary phase OV-101,Anal. Biochem., 34 (1970) 16-23. 379 D. Gupta, E. Breitmaier, G. Jung, G. von Lucadou, H. Pauschmann and W. Voelter, Comparison of trimethylsilyltrifluoroacetyland heptafluorobutyrate derivatives of steroids by gas chromatography, Chromatographia, (1971) 572-575 (Ger.). 380 A.R. Guseva, V.A. Paseshnichenko, M.G. Borikhina and R.K. Moiseev, Assay of steroid glycoalkaloids in Solanum laciniatum,Biokhimiya, 30 (1965) 260-264 (Russ.). 381 E. Haahti and T. Nikkari, Separation and isolation of waxes and sterol esters of skin surface fat with thin-layer chromatography, Acta Chem. Scand., 17 (1963) 536-537. 382 E. Haahti and T. Nikkari, Continuous detection of fractions of effluents of silicic acid chromatography, Acta Chem. Scand., 17 (1963) 2565-2568. 383 E. Haahti, T. Nikkari and K. Juva, Fractionation of serum and skin sterols and skin waxes with chromatography on silica gel impregnated with silver nitrate, Acta Chem. Scand., 17 (1963) 538-540. 384 R. Hahnel, Use of Sephadex ion exchanger for the separation of-conjugated urinary estrogens, Anal. Biochem., 10 (1965) 184-192. 385 R. Hahnel and M. Ghazali bin Abdul Rahman, Improved gradient elution for the separation of urinary steroid conjugates on DEAE-Sephadex columns, Clin. Chim. Acta, 13 (1966) 797-799. 386 R. Hahnel and M. Ghazali bin Abdul Rahman, Separation of urinary estrogen sulfates from estrogen glucosiduronates on DEAE-Sephadex columns, Biochem. J., 105 (1 967) 1047-1053. 387 R. Hahnel and N. bin Muslim, Tables for the identification of steroid conjugates, Chromatogr. Rev., 11 (1969) 215-364. 388 H. Hafez-Zedan and R. Plourde, “Spore plate method” for transformation of steroids by fungal spores entrapped in Silica Gel G,AppZ. Microbiol., 21 (1971) 815-819. 389 D.D. Hagerman and J.M. Spencer, Thin-layer chromatography of some 30-hydroxyA5-steroids,Steroids,4 (1964) 547-556. 390 J. Hakl, Steroids. 1. Application of thin-layer chromatography to the separation of 2,4-dinitrophenylhydrazonesof the physiologically important 17-ketosteroids, J. Chromatogr., 61 (1971) 183-186.

REFERENCES

149

391 J. Hakl, Steroids. 2. The use of multiple development in the correlation of the chemical constitutions and the chromatographic migrations of some 17-ketosteroids and their 2,4-dinitrophenylhydrazones,J. Chrornatogr., 71 (1972) 3 19-328. 392 A. Hall, Thin-layer chromatography of corticosteroids, J. Pharm. Pharmacol., Suppl., 16 (1964) 9T-10T. 393 H. Halpaap, Preparative thin-layer chromatography, Chern.-Ing.-Tech., 35 (1963) 488-493 (Ger.). 394 H. Halpaap, Preparative thin-layer chromatography. 2. Fundamentals of preparative layer chromatography, Chern.-Ztg., 89 (1965) 835-849 (Ger.). 395 H. Halpaap, Standardization of thin-layer chromatography by use of ready-made plates, J. Chromatogr., 33 (1968) 144-163 (Ger.). 396 H. Halpaap and H. Bausch, Extensions of the thin-layer chromatographic technique by ready-made preparations in the form of rolls, J. Chromatogr., 48 (1970) 144-160 (Ger.). 397 H. Halpaap and K. Klatyk, Chromatographic properties of silica gels of different pore structure, J. Chrornatogr., 33 (1968) 80-89 (Ger.). 398 R.J. Hamilton, W.J.A. VandenHeuvel and E.C. Horning, An extension of the steroid number concept to relations between the structure of steroids and their gas-chromatographic retention times observed with selective phases, Biochirn. Biophys. Acta, 70 (1963) 679-687. 399 B.L. Hamman and M.M. Martin, Separation of six urinary 17-ketosteroids by twodimensional thin-layer chromatography. Control values and response to ACTH, J. Clin. Endocrinol. Metab., 24 (1964) 1195-1205. 400 B.L. Hamman and M.M. Martin, Direct spectrophotometric quantitation of steroid chromatograms. 1. The measurement of corticosteroids, Anal. Biochem., 20 (1967) 423-43 1. 401 B.L. Hamman and M.M. Martin, Steroid reactions on thin-layer films, Steroids, 10 (1967) 169-183. 402 B.L. Hainman and M.M. Martin, Direct spectrophotometric quantitation of 17-ketosteroids on thin-layer chromatograms, J. Lab. Clin. Med., 71 (1968) 1028-1033. 403 K.H. Hanewald, F.J. Mulder and K.J. Keuning, Analysis of fat-soluble vitamins. 9. Thin-layer chromatographic assay of vitamin D in high-potency preparations, J. Pharm. Sci., 57 (1968) 1308-1312. 404 S. Hara and K. Mibe, Systematic analysis of steroids. 7. Thin-layer chromatography of steroidal pharmaceuticals, Chern. Phamz. Bull. (Tokyo), 15 (1967) 1036-1040. 405 S. Hara and K. Mibe, Thin-layer chromatography of steroidal pharmaceuticals, Anal. Chem., 40 (1968) 1605-1608. 406 S. Hara and M. Miyaki, Systematic analysis of steroids. 6. Relation between steroid structure and mobility in adsorption liquid-phase chromatography, Chem. Pharm. Bull. (Tokyo), 15 (1967) 1032-1035. 407 S . Hara and M. Takeuchi, Systematic analysis of bile acids and their derivatives by thin-layer chromatography, J. Chrornatogr., 11 (1963) 565-567. 408 S . Hara and M. Takeuchi, Systematic analysis of steroid hormones by thin-layer chromatography, Endocrinol. Jpn., 10 (1 964) 202-207. 409 S. Hara, M. Takeuchi, M. Tachibana and G. Chihara, Systematic analysis of steroids.

150

REFERENCES

4. Thin-layer chromatography and densitometry of bile components, Chem. Pharm. Bull. (Tokyo), 12 (1964) 483-488. 410 S. Hara, H. Tanaka and M. Takeuchi, Systematic analysis of steroids. 5. Densitometric analysis of thin-layer chromatograms, Chem. Pharm. Bull. (Tokyo), 12 (1964) 626 630. 41 1 S . Hara, T. Watabe and Y. Ike, Direct analysis of corticosteroids by gas chromatography as trimethylsilyl ethers of methyloximes, Chem. Pharm. Bull. (Tokyo), 14 (1966) 1311-1314. 412 S. Hara, T. Watabe, Y. Ike and N. Ikekawa, Systematic analysis of steroids. 8. Molecular structure of C19 and C18 steroid derivatives and retention times in gas chromatography, Chem. Pharm. Bull. (Tokyo), 15 (1967) 1041-1044. 413 R. Hardman and T.M. Jefferies, Combined column chromatographic and infrared spectrophotometric determination of diosgenin and yamogenin in Fenugreek seed, Analyst (London),97 (1972) 437-441. 414 I.S. Hartman and H.H. Wotiz, A method for the simultaneous separation of C1902 and CI9O3 17-ketosteroids and progesterone metabolites by gas chromatography, Steroids, 1 (1963) 33-38. 41 5 I.S. Hartman and H.H. Wotiz, Steroid metabolism. 19. The relation of gas-chromatographic retention time to the structure of steroids and derivatives, Biochim. Biophys. Acta, 90 (1964) 334-348. 416 H. Hauser and H.O. Gunther, Group separation of steroid hormones and stilbenes from biological material,J. Chromatogr., 95 (1974) 232-234 (Ger.). 417 J.C. Hauton and C. Greusard, Thin-layer adsorption chromatography. Role of the “mobile phase”. The mechanism of separation., Bull. SOC.Pharm. Marseille, 15 (1966) 31-49 (Fr.). 418 L.T. Heaysman and E.R. Sawyer, The determination of vitamin D in pharmaceutical preparations by thin-layer chromatography, Analyst (London),89 (1964) 529-533. 419 E. Heftmann, Thin-layer chromatography of steroids, Chromatogr. Rev., 7 (1965) 179-195. 420 E. Heftmann, Steroid Biochemistry, Academic Press, New York, 1970. 421 E. Heftmann (Editor), Modern Methods ofsteroid Analysis, Academic Press, New York, 1973. 422 E. Heftmann (Editor), Chromatography, Van Nostrand-Reinhold, New York, 3rd Ed., 1975. 423 E. Heftmann, S.-T. KO and R.D. Bennett, Response of steroids to sulfuric acid in thin-layer chromatography, J. Chromatogr., 2 1 (1 966) 490-494. 424 J. Heikkila and H. Adlercreutz, Determination of urinary 150-hydroxyestriol and estriol. Preliminary results of 1Sa-hydroxyestriol determinations in pregnancy urine, J. Steroid Biochem ., 1 (1 970) 243-253. 425 R.J. Heitzman and G.H. Thomas, Evaluation by gas chromatography of the urinary steroids of the pregnant dairy cow, J. Endocrinol., 33 (1965) 455-467. 426 0. Helmich and J. Hradec, Quantitative determination of cholesteryl 14-methylhexadecanoate by radio-gas chromatography, J. Chromatogr., 91 (1974) 505-5 12. 427 R.A. Henry, J.A. Schmit and J.F. Dieckman, Analysis of steroids and derivatized . steroids by high-speed liquid chromatography, J. Chromatogr. Sci., 9 (1971) 513-520.

REFERENCES

151

428 S.F. Herb, T.J. Fitzpatrick and S.F. Osman, Separation of potato glycoalkaloids by gas chromatography, 1.Agr. Food Chem., 23 (1975) 520-523. 429 R. Hernandez, Jr. and L.R. Axelrod, Chromatographic separation of the steroids from total lipid extracts,Anal. Chem., 35 (1963) 80-83. 430 F. Hertelendy and R.H. Common, Thin-layer chromatography of the four estriol epimers and their methyl ethers,Steroids, 2 (1963) 135-142. 43 1 J.E. Herz and E. Gonzllez, Characterization of cholestanols and cholestanones by thin-layer and gas chromatography. Steroid boranes. 2., J. Chromatogr., 3 4 (1968) 251 -253. 432 C.Hesse and W. Hovermann, Three-component two-phase system for high-speed liquid chromatography, Chromatographia, 6 (1973) 345-348 (Ger.). 433 C. Hesse, K. Pietrizik and D. Hotzel, Specific determination of corticosterone and cortisol in the nanogram range,Z. Klin. Chem. Klin. Biochem., 12 (1974) 193-197 (Ger.). 434 D.Heusser, Evaluation of various medicinal plants or their preparations with the help of thin-layer chromatography, Planta Med., 12 (1 964) 237-245 (Ger.). 435 D. Heusser, Quantitative determinations by means of thin-layer chromatography, Pharm. Tijdschr. Belg., 42 (1965) 263-270 (Ger.). 436 W.Heyns, A. Hendrikx and P. de Moor, Gas-chromatographic determination of 11-deoxy-17-ketosteroids in urine, Acta Clin. Belg., 19 (1964) 346-359 (Fle.). 437 J. Hilgenfeldt, I. Franzen, M. Goetze and V. Klingmiiller, Standardization and interpretation of thin-layer chromatograms of 17-ketosteroids in urine with the aid of a Zeiss chromatogram spectrophotometer, Aerztl. Lab., 19 (1973) 339-345 (Ger.). 438 K. Hiller and H.-D. Woitke, Saponins, in K. Macek (Editor), Pharmaceutical Applications of Thin-Layer and Paper Chromatography, Elsevier, Amsterdam, 1972, pp. 393408. 439 M. Hiroi, Gas chromatography of estrogen. 1. The optimal conditions, Horumon to Rinsho, 15 (1967) 183-189 (Jap.). 440 M. Hiroi, Gas chromatography of estrogens. 2. Trifluoroacetylation and heptafluorobutyration of estrogens, Horumon to Rinsho, 15 (1967) 340-344 (Jap.). 441 M. Hiroi, Gas chromatography of estrogens. 3. Determination of blood estrogen by heptafluorobutyration and thin-layer chromatography, Horumon to Rinsho, 16 (1968) 3 19-323 (Jap.). 442 J. Hirsch, Factice chromatography. An automatically monitored, liquid-gel system for the separation of nonpolar lipids,J. Lipid Res., 4 (1963) 1-10. 443 A. Hiscoe, D.W. Mathieson and R.H. Perrett, Gas-liquid chromatography of ketoand hydroxyandrostanes, 1.Chromatogr., 81 (1973) 144-149. 444 K.Ho, S.K. Peng and C.B. Taylor, Identification and quantitation of dietary and fecal neutral sterols by mass spectrometry, Atherosclerosis, 15 (1972) 249-264. 445 R.Hobkirk and S. Davidson, Behaviour of dehydroisoandrosterone, testosterone, and their conjugates on DEAE-Sephadex, 1.Chromatogr., 54 (1971) 4 3 1-432. 446 R. Hobkirk, P. Musey and M. Nilsen, Chromatographic separation of estrone and 170estradiol conjugates on DEAE-Sephadex, Steroids, 14 (1969) 191-206. 447 R.Hobkirk and M. Nilsen, Separation of monoglucosiduronate conjugates of estrone and 17P-estradiol by DEAE-Sephadex chromatography, Anal. Biochem., 37 (1970)

152

REFERENCES

337-344. 448 J. Hobzova and M. Novik,Separation of progesterone and its determination in human plasma during the menstrual cycle, Endokrinologie, 52 (1968) 366-372 (Ger.). 449 L. Horhammer, H. Wagner and H. Konig, New methods in pharmacognosy. 6. Chromatography of the constituents of Strophanthus seeds and their pharmaceutical preparations, Deut. Apoth.-Ztg., 103 (1963) 502-504 (Ger.). 450 B. Hoffmann, A modified detection reaction of estrogens on paper and thin-layer chromatograms, J. Chromatogr., 34 (1968) 269 (Ger.). 451 H. Hoffmeister and C. Rufer, Chemistry of ecdysone. 4. Color reactions of saturated and unsaturated steroidal ketones, Chem. Ber., 98 (1965) 2376-2382 (Ger.). 452 A.F. Hofmann, Thin-layer chromatography of bile acids and their derivatives, in A.T. James and L.J. Morris (Editors), New Biochemical Separations,Van Nostrand, New York, 1964, pp. 261-282. 453 M. Hofmann, Simple method for thin-layer chromatographic determination of pregnanediol in the urine, Deuf. Gesundheitswes.,29 (1974) 185-188 (Ger.). 454 J.L. Hojnacki and S.C. Smith, Separation of six lipid classes on one thin-layer chromatogram, J. Chromatogr., 90 (1974) 365-367. 455 M.F. Holick and H.F. DeLuca, New chromatographic system for vitamin D, and its metabolites. Resolution of a new vitamin D3 metabolite, J. Lipid Res., 12 (1971) 460-465. 456 T.H. Holmdahl and J. Sjovall, Liquid-gel chromatography on hydrophobic Sephadex and competitive protein binding of 17a-hydroxyprogesterone in plasma, Steroids, 18 (1971) 69-76. 457 C.G. Honegger, Application of activity gradients in thin-layer chromatography, Helv. Chim. Acta, 47 (1964) 2384-2387 (Ger.). 458 M. Hori, Automatic column-chromatographic method for insect-molting steroids, Steroids, 14 (1969) 33-46. 459 E.C. Horning, C.J.W. Brooks, L. Johnson and W.L. Gardiner, Separation, identification, and estimation of human steroid hormones and their metabolites. Applications to adrenocortical steroids, Separ. Sci., 1 (1966) 555-573. 460 E.C. Horning, C.J.W. Brooks and W.J.A. VandenHeuvel, Gas-phase analytical methods for the study of steroids, Advan. Lipid Res., 6 (1968) 273-392. 461 E.C. Horning, D.I. Carroll, I. Dzidic, K.D. Haegele, M.G. Horning and R.N. Stillwell, Liquid chromatograph-mass spectrometer-computer analytical system, J. Chrumatogr., 99 (1974) 13-21. 462 E.C. Horning, M.G. Horning, N. Ikekawa, E.M. Chambaz, P.I. Jaakonmaki and C.J.W. Brooks, Studies of analytical separations of human steroids and steroid glucuronides, J. Gas Chromatogr., 5 (1967) 283-289. 463 E.C. Horning, M.G. Horning, J. Szafranek, P. van Hout, A.L. German, J.P. Thenot and C.D. Pfaffenberger, Gas-phase analytical methods for the study of human metabolites. Metabolic profiles obtained by open-tubular capillary chromatography, J. Chromatogr., 91 (1974) 367-378. 464 E.C. Horning, T. Luukkainen, E. Haahti, B.G. Creech and W.J.A. VandenHeuvel, Studies of human steroidal hormones by gas-chromatographic techniques, Recent bog. Hormone Res., 19 (1963) 57-106.

REFERENCES

153

465 E.C. Horning and B.F. Maume, Derivatives of aldosterone for gas-phase analysis, J. Chromatogr. Sci.,7 (1969)411-418. 466 E.C. Horning and W.J.A. VandenHeuvel, Qualitative and quantitative aspects of the separation of steroids, in J.C. Giddings and R.A. Keller (Editors), Advances in Chromatography, Vol. 1, Marcel Dekker, New York, 1965, pp. 153-198. 467 E.C. Horning and W.J.A. VandenHeuvel, Gas-chromatographic separations of steroids and related substances, Riv. Ztal. Sostance Grasse, 4 2 (1965) 418-429 (Ital.). 468 E.C. Horning, W.J.A. VandenHeuvel and B.G. Creech, Separation and determination of steroids by gas chromatography,Methods Biochem. Anal., 11 (1963) 69-147. 469 M.G. Horning, A.M. Moss and E.C. Horning, Formation and gas-liquid chromatographic behavior of isomeric steroid ketone methoxime derivatives, Anal. Biochem., 22 (1 968) 284-294. 470 H.-J. Horst, Chromatographic separation of estrone, estradiol-l7p, and estriol on Sephadex G-10, J. Chromatogr., 58 (1971) 227-234 (Ger.). 47 1 H.-J. Horst, Adsorption and purification of free estrogenic steroids with Sephadex G-25-Fine,Z. Veteriniirmed., ReiheA, 18 (1971) 93-103 (Ger.). 472 H.-J. Horst, E. Grunert and M. Stoye, Separation of estradiol and estriol epimers, J. Chromatogr., 69 (1972) 395-398 (Ger.). 473 C. Horvath, Quantitative determination of cholesterol in autooxidation mixtures by thin-layer chromatography, J. Chromatogr., 22 (1966) 52-59. 474 H.-C. Hsiu, Migration rates of estrogens and elution capacity of solvents on polyamide thin-layer chromatography, Bunseki Kagaku (Jap. Anal.), 20 (1971) 1026-1031 (Jap.1. 475 H.-C. Hsiu, Polyamide thin-layer chromatography of estrogen-related compounds and influence of chemical structure on migration rates, Bunseki Kagaku (Jap. Anal.), 23 (1 974) 1226- 123 1 (Jap.). 476 C.T.L. Huang and B.L. Nichols, New solvent systems for the separation of free and conjugated bile acids, J. Chromatogr., 101 (1974) 235-239. 477 C.T.L. Huang and B.L. Nichols, New solvent systems for the separation of free and conjugated bile acids. 2. Separation of free bile acids as a group, J. Chromatogr., 109(1975)427-431. . 478 J.F.K. Huber, J.A.R.J. Hulsman and C.A.M. Meijers, Qualitative analysis of trace amounts of estrogenic steroids in pregnancy urine by column liquid-liquid chromatography with ultraviolet detection, J. Chromatogr., 62 (1971) 79-91. 479 J.F.K. Huber, C.A.M. Meijers and J.A.R.J. Hulsman, Prediction of partition coefficients in liquid-liquid systems and its experimental verification for steroids by static and chromatographic measurements,Anal. Chem., 44 (1971) 111-116. 480 W. Hubl, Qualitative fractionation of corticosteroids by means of polyamide thinlayer chromatography and subsequent documentation, 2. Chem., 6 (1966) 225-226 (Ger.). 481 W. Hubl and K. Schollberg, Fluorimetric determination of testosterone, epitestosterone, and androst-4-ene-3,17-dione after thin-layer chromatographic isolation from urine, Acta Endocrinol., 58 (1968) 353-363 (Ger.). 482 B. Hubschman, A. Racadot and M. Linquette, Fractionation of urinary 17-hydroxycorticosteroids by thin-layer chromatography, Lille Med., 12 (1967) 548-555 (Fr.).

154

REFERENCES

483 H. Huck, The product of break-through time and peak height as a quantitative measure in the determination of steroids by gas chromatography, J. Chromatogr., 62 (1971) 47-52 (Ger.). 484 H. Huck, The aluminum oxide fluorescence of A4-3-ketosteroids, Chromatographia, 6 (1973) 46-49 (Ger.). 485 H. Huck, Fluorimetric determination of testosterone on A1203 by thin-layer chromatographic separation of the trimethylsilyl ethers, J. Chromatogr., 1 10 (1975) 125-131 (Ger.). 486 W. Hiibner and W. Staib, Fluorimetric determination of testosterone after thin-layer chromatographic isolation from human urine, Klin. Wochenschr.,45 (1967) 674-678 (Ger.). 487 D.H. Hunneman, Use of dimethylsilyl ethers in the gas chromatographic-mass spectrometric analysis of steroids, in A. Frigerio and N. Castagnoli (Editors), Mass Spectrometry in Biochemistry and Medicine, Raven Press, New York, 1974, pp. 131138. 488 S.M.V. Hunt, Apparatus for the elution of steroids from thin-layer chromatograms, Lab. Pract., 16 (1967) 601. 489 I.R. Hunter, M.K. Walden, J.R. Wagner and E. Heftmann, High-pressure liquid chromatography of steroidal alkaloids, J. Chromatogr., 119 (1976) 223-226. 490 I.R. Hunter, M.K. Walden, J.R. Wagner and E. Heftmann, Thin-layer chromatography of steroidal alkaloids,J. Chromatogr., 118 (1976) 259-262. 491 A.R. Hurwitz, H.J. Burke and R.A. Marra, Separation of some estrogen sulfates from their oxidation products,J. Pharm. Sci., 56 (1967) 1509-1512. 492 P.M. Hyde and W.H. Elliott, Separation of 0-sitosterol and campesterol on hydrophobic hydroxyalkyl Sephadex LH-20, J. Chromafogr.,67 (1972) 170-1 72. 493 H. Ibayashi, M. Nakamura, S . Murakawa, T. Uchikawa, T. Tanioka and K. Nakao, Determination of urinary testosterone using thin-layer chromatography and gas chromatography, Steroids, 3 (1964) 559-568. 494 S. Ichikawa, K. Murata and R. Shigiya, Application of a marker dye on Sephadex LH-20 column chromatography in radioimmunoassay of plasma aldosterone, Endocrinol. Jpn., 21 (1974) 361-365. 495 D.R. Idler and D.A. Horne, Recovery of micro quantities of steroids from silica gel followingrepeated thin-layer chromatography, Steroids, 1 1 (1968) 909-914. 496 D.R. Idler, N.R. Kimball and B. Truscott, Destruction of micro quantities of steroids on silica gel, as shown by repeated thin-layer chromatography, Steroids, 8 (1966) 865-876. 497 D.R. Idler and L.M. Safe, Separation of steryl acetates by silver-ion chromatography, Steroids, 19 (1972) 315-323. 498 R. Ikan and M. Cudzinovski, Separation of sterols and corresponding stanols on tlun layers of silica impregnated with silver nitrate,J. Chromatogr., 18 (1965) 422-423. 499 R. Ikan, S. Harel, J. Kashman and E.D. Bergmann, The separation of sterols and corresponding stanols by thin-layer chromatography, J. Chromatogr., 14 (1964) 504-506. 500 R. Ikan and E. Rapaport, Test tube and glass rod thin-layer chromatography, J. Chem. EdUC., 44 (1967) 297-298.

REFERENCES

155

501 N. Ikekawa, F. Hattori, J. Rubio-Lightbourn, H. Miyazaki, M. Ishibashi and C. Mori, Gas-chromatographic separation of phytoecdysones, J. Chromarogr.Sci., 10 (1 972) 233-242. 502 N. Ikekawa, R. Watanuki, K. Tsuda and K. Sakai, Correlation between molecular structure of sterols and retention time in gas chromatography, Anal. Chem., 40 (1968) 1139-1141. 503 M. Inaba, T. Nakao and K. Kamata, Double isotope derivative assay of 170-estradiol and estriol. Preliminary study on pipsylation of estrogen and acetylation of pipsylated estrogen, Endocrinol. Jpn., 14 (1967) 357-362. 504 M. Ishikawa and T. Miyasaka, Bufodienolides. 1. Thin-layer chromatography of toad poison, Shika Zairyo Kenkyusho Hokoku, 2 (1962) 397-400 (Jap.). 505 T.T. Ishikawa, J. MacGee, J.A. Morrison and C.J. Glueck, Quantitative analysis of cholesterol in 5 t o 20 pl of plasma, J. Lipid Res., 15 (1974) 286-291. 506 T. Itoh, T. Tamura, T. Iida and T. Matsumoto, Gas-chromatographic differentiation of 4-demethyl, 4-monomethyl, and 4,4-dimethyl sterols, Steroids, 23 (1974) 687-698. 507 IUPAC-IUB Commission on Biochemical Nomenclature, Revised tentative rules for nomenclature of steroids,Steroids, 13 (1969) 277-310; Biochemistry, 8 (1969) 2227-2242. 508 P.I. Jaakomaki, K.A. Yarger and E.C. Horning, Gas-liquid chromatographic separation of human urinary steroid glucuronides, Biochim. Biophys. Acta, 137 (1967) 216-219. 509 G.M. Jacobsohn, Quantitation of estrone, estradiol, and estriol on thin-layer chromatograms by a photogrammetric procedure, Anal. Chem., 3 6 (1964) 275-279. 510 G.M. Jacobsohn, Photogrammetry of estrogens on thin-layer chromatograms by a one-step photographic procedure, Anal. Chem., 36 (1964) 2030-2032. 51 1 0. Janne, Quantitative determination of neutral steroid mono- and disulfates in human urine, Clin. Chim. Acta, 29 (1970) 529-540. 51 2 0. Janne, Identification and quantitation of CI9 and Czl steroid trio1 sulfates in adult human urine, J. Steroid Biochem., 2 (1971) 33-41. 513 0. Janne, D. Apter and R. Vihko, Assay of testosterone, progesterone and 17ahydroxyprogesterone in human plasma by radioimmunoassay after separation on hydroxyalkoxypropyl Sephadex, J. Steroid Biochem., 5 (1974) 155-162. 514 0. Janne, R. Vihko, J. Sjovall and K. Sjovall, Determination of steroid mono- and disulfates in human plasma, Clin. Chim. Acra, 23 (1969) 405-412. 515 A.P. Jansen, Determination of pregnanediol in urinary extracts by gas-liquid chromatography, Clin. Chim. Acta, 8 (1963) 785-787. 5 16 J. Jeffery, A chromatographic staining technique useful for 3P-hydroxy-5-ene steroids and certain steroid drugs, J. Chromarogr., 59 (1971) 216-219. 5 17 J. Jeffery, A simple staining technique useful for distinguishing 3-hydroxy-4-ene from 3-hydroxy-5-enesteroids on silica gel thin layers, and for other purposes, J. Chromatogr., 67 (1972) 188-190. 518 R.W. Jelliffe and D.H. Blankenhorn, Gas chromatography of digitoxigenin and digoxigenin, J. Chromatogr., 12 (1963) 268-270. 519 R.W. Jelliffe, C.J. Page, E.J. Escamilla, S.L. Morgan and R.G. Stephinson, An ultramicro fluorescent spray reagent for detection and quantification of cardiotonic steroids on thin-layer chromatograms, J. Chromarogr.,27 (1 967) 172-1 79.

156

REFERENCES

520 D.F. Johnson, Gradient elution and thin-layer chromatography in the analysis of corticosteroids and 17-ketosteroids, in E. Heftmann (Editor),Modern Methods of Steroid Analysis, Academic Press, New York, 1973, pp. 55-70. 521 D.F. Johnson, D. Franqois, G.C. Riggle and C.J. Ramsden, Design and application of an automated punched-tape system in programmed gradient elution chromatography, Ann. N. Y. Acad. Sci , 130 (1965) 792-800. 522 D.F. Johnson, N.S. Lamontagne, G.C. Riggle and F.O. Anderson, Tape-controlled gradient elution chromatography system for steroid analysis,Anal. Chem., 43 (1971) 1712-1 71 5. 523 E.J. Johnston and A.L. Jacobs, Thin-layer chromatography of cardiac glycosides, J. Pharm. Sci., 55 (1966) 531. 524 D. Jones, D.E. Bowyer, G.A. Gresham and A.N. Howard, Improved spray reagent for detecting lipids on thin-layer chromatograms, J. Chromarogr.,23 (1966) 172- 174. 525 F.H. de Jong and H.J. van der Molen, Determination of dehydroepiandrosterone and dehydroepiandrosterone sulfate in human plasma using electron-capture detection of 4-androstene-3,6,17-trione after gas-liquid chromatography, J. Endocrinol., 53 (1 972) 46 1-474. 526 R.A. Jungmann, E. Calvary and J.S. Schweppe, Quantitative analysis of urinary 1 1-deoxy-17-ketosteroids and pregnanediol by gas-liquid chromatography, J. Clin. Endocrinol. Metab., 27 (1967) 355-364. 527 F.J. Kabot and L.S. Ettre, Instrumental aspects of steroid analysis by gas chromatography, J. Gas Chromatogr., 2 (1964) 21-33. 528 F. Kaiser, Chromatographic analysis of a cardiac glycoside from Digitalis species, Arch. Pharm., 299 (1964) 263-274 (Ger.). 529 S. Kamada, H . Tanaka, N. Kawanobe, S. Kunitomi, A. Inohara, A. Akimoto, H. Asagiri and I. Shibata, Modified method for isolation of steroids by chromatography on impregnated thin layers, Horumon To Rinsho, 17 (1969) 542-546 (Jap.). 530 R. Kammereck, W.-H. Lee, A. Paliokas and G.J. Schroepfer, Jr., Thin-layer chromatography of sterols on neutral alumina impregnated with silver nitrate, J. Lipid Res., 8 (1967) 282-284. 531 T. Kanno, K. Tominaga, T. Fujii and F. Funatani, Analysis of serum bile acids by gas-liquid chromatography equipped with 63Ni electron-capture detector, J. Chromarogr. Sci., 9 (1971) 53-58. 532 B.L. Karger and L.V. Berry, Rapid liquid-chromatographic separation of steroids on columns heavily loaded with stationary phase, Clin. Chem., 17 (1971) 757-764. 533 A. Karmen, Flame-ionization detector for liquid-liquid chromatography, Anal. Chem., 38 (1966) 286-290. 534 A. Karmen, I . McCaffrey and B. Kliman, Derivative ratio analysis. A new method for measurement of steroids and other compounds with specific functional groups using radioassay by gas-liquid chromatography, Anal. Biochem., 6 (1963) 3 1-38. 535 A. Karmen and J.L. Marsh, Clinical analyses of steroid hormones by gas chromatography, in G.V. Marinetti (Editor), Lipid ChromatographicAnalysis, Marcel Dekker, New York, 1969, pp. 523-561. 536 N.S. Kartavova, Determination of pregnanediol in urine by micro thin-layer chromatography, Lab. Delo, (1973) 598-600 (Russ.).

REFERENCES

157

537 T. Kartnig and R. Danhofer, Identification and quantitative analysis of Smophanthus glycosides, J. Chromatogr., 52 (1970) 3 13- 320 (Ger.). 538 T. Kartnig and G. Mikula, Identification and determination of sterols, J. Chromatogr., 53 (1970) 537-543 (Ger.). 539 M. Katayama, C.T. Hou, N.C. Chen. T. Hirota, T. Kiribuchi and S. Funahashi, A method for the fractional determination of soybean sterols in four classes by Florisil chromatography,Agr. Bid. Chem. (Tokyo), 38 (1974) 1661-1667. 540 G. Katsui, Vitamins, in K. Macek (Editor), Pharmaceutical Applications of Thin-Layer and Paper Chromatography, Elsevier, Amsterdam, 1972, pp. 469-502. 541 M. Katz and Y. Lensky, Gas-chromatographic analysis of ecdysone, Experientia, 26 (1970) 1043. 542 H.P. Kaufmann and C.V. Viswanathan, Thin-layer chromatography of fats. 10. The analysis of the metabolism of lipids, Fette, Seifen, Anstrichm., 6 5 (1963) 538-543 (Ger .). 543 H.P. Kaufmann and C.V. Viswanathan, The chromatography of cholesterol and its esters, Fette, Seifen, Anstrichm., 6 5 (1963) 8 3 9 4 4 5 (Ger.). 544 T. Kawasaki and K. Miyahara, Thin-layer chromatography of steroid saponins and their derivatives, Chem. Pharm. Bull. (Tokyo), 11 (1963) 1546-1550. 545 R. Kawate, B. Pettitt and V.W. Cole, Determination of the secretion rate of aldosterone by use of double isotopes and column chromatography, Texas Rep. Biol. Med., 24 (1 966) 90- 106. 546 H.L. Kay, Apparatus for dynamic thin-layer chromatography, Nature (London), 209 (1966) 1237-1 238. 547 T. Kazuno and T. Hoshita, Stero-bile acids and bile alcohols. 57. Chromatography of bile alcohols,Steroids, 3 (1964) 55-65. 548 M. Keller and A . Uettwiller, Quantitative thin-layer and gas-chromatographic determination of plasma progesterone, Gynaecologia, 165 (1968) 385-394 (Ger.). 549 T.F. Kellogg, Improved spray reagent for detection of bile acids on thin-layer chromatoplates, J. Lipid Res., 11 (1970) 498-499. 550 R.W. Kelly, Dimethyldiacetoxysilane as a silylating reagent. A new technique for forming siliconides of corticosteroids, J. Chromatogr., 4 3 (1969) 229-232. 551 R.W. Kelly, Gas chromatography of some new silyl derivatives of cortisone, cortisol, and related compounds, Steroids, 13 (1969) 507-5 18. 552 R.W. Kelly, The measurement by gas chromatography-mass spectrometry of oestra1,3,5-triene-3,15a,l6a,l7P-tetrol (oestetrol) in pregnancy urine, J. Chromatogr., 54 (1971) 345-355. 553 J.R. Kent and A.B. Rawitch, Thin-layer chromatography of ketosteroid dinitrophenylhydrazones, J. Chromatogr., 20 (1965) 614-615. 554 R.H. King, L.T. Grady and J.T. Reamer, Progesterone injection assay by liquid chromatography, J. Pharm. Sci., 6 3 (1974) 1591-1596. 555 K. Kinoshita and K. Isurugi, Gas-chromatographic determination of pregnanediol, pregnanetriol, pregnanetriolone, and pregnanetetrol, Nippon Naibumpi Gakkai Zasshi (Folia Endocrinol Jpn.; Jap. J. Endocrinol.), 40 (1964) 978-981 (Jap.). 556 K. Kinoshita, K. Isurugi, Y. Matsumoto and H . Takayasu, Gas-chromatographic estimation of urinary A5-pregnene-3P,17a,2001-triol, Steroids, 11 (1968) 1-1 1.

158

REFERENCES

557 T. Kiribuchi, T. Mizunaga and S. Funahashi, Separation of soybean sterols by Florisil chromatography, and characterization of acylated steryl glucoside, Agr. Biol. Chem. (Tokyo), 30 (1966) 770- 778. 558 D.N. Kirk and P.M. Shaw, Gas chromatography of steroids. Use of steroid derivatives as stationary phases,J. Chem. Soc., C, (1971) 3979-3982. 559 M.A. Kirschner, Electron-capture techniques for steroid analysis, Methods Enzymol., 36,Part A (1975) 58 67. 560 M.A. Kirschner and H.M. Fales, Gas-chromatographic analysis of 17-hydroxycorticosteroids by means of their bismethylenedioxy derivatives,Anal. Chem., 34 (1962) 1548 1551. 561 M.A. Kirschner and M.B. Lipsett, The analysis of urinary steroids using gas-liquid chromatography, Steroids, 3 (1964) 277-294. 562 M.A. Kirschner, M.B. Lipsett and D.R. Collins, Analysis of steroids in urine and blood using gas-liquid chromatography, J. Gas Chromatogr., 2 (1964) 360-364. 563 M.A. Kirschner and J.P. Taylor, New derivatives for electron-capture gas chromatography of steroids. A simplified procedure for measuring plasma testosterone, Anal. . Biochem., 30 (1969) 346-357. 564 G.W. Kittinger, Quantitative gas chromatography of adrenal cortical steroids, Steroids, 11 (1968)47-71. 565 K.A. Klause and M.T.R. Subbiah, Improved resolution of cholestanol and cholesterol by gas-liquid chromatography. Application to pigeon testicular sterols, J. Chromatogr., 103 (1 975) 170-1 72. 566 P.D. Klein, Silica gel structure and the chromatographic process. Effect of pore diameter on the adsorption and differential migration of sterol acetates, Anal. Chem., 33 (1961) 1737-1741. 567 P.D. Klein, Silica gel structure and the chromatographic process. Surface energy and activation procedures, Anal. Chem., 34 (1962) 733-736. 568 P.D. Klein and E.H. Erenrich, Tritiated alumina as a reagent for self-labeling chromatographic analyses,Anal. Chem., 38 (1966) 480-484. 569 P.D. Klein and J.C. Knight, The exchange labeling of 0x0 steroids with tritium by adsorption chromatography on basic alumina,J. Amer. Chem. Soc., 87 (1965) 2657-2661. 570 P.D. Klein, J.C. Knight and P.A. Szczepanik, The behavior of sterols on silica surfaces and at other interfaces, J. Amer. Oil Chem. SOC.,43 (1966) 275-280. 571 P.D. Klein, D.W. Simborg and P.A. Szczepanik, Detection and computation of isotope fractionation in the adsorption chromatography of dual-labeled compounds, Pure Appl. Chem., 8 (1964) 357-370. 572 P.D. Klein and P.A. Szczepanik, The differential migration of sterol acetates on silica gels and its application to the fractionation of sterol mixtures, J. Lipid Res., 3 (1 962) 460-466. 573 P. Knapstein and J.C. Touchstone, Determination of the 2,4-dinitrophenylhydrazones of urine ketosteroids by thin-layer densitometry, J. Chromatogr., 37 (1968) 83-88. 574 P. Knapstein, J.C. Touchstone, P. Menzel and G.W. Oertel, Rapid quantitation of A4-3-ketosteroids by thin-layer densitometry, J. Chromatogr., 44 (1969) 190-1 92. 575 P. Knapstein, L. Treiber and J.C. Touchstone, Rapid quantitation of 2,4-dinitrophenyl-

REFERENCES

159

hydrazones of plasma ketosteroids by thin-layer densitometry, Steroids, 11 (1968) 91 5-924. 576 B.A. Knights, Gas-chromatographic analysis of plant sterols. 1 . Characterization of sterol double bonds using the ARM function, J. Gas Chrumatugr., 2 (1964) 160 162. 577 B.A. Knights, Gas chromatography of plant sterols. 2. Use of a poly(vinylpyrro1idinone)coated support, J. Gas Chromatogr., 2 (1964) 338-339. 578 B.A. Knights, Identification of plant sterols using combined gas-liquid chromatography mass spectrometry, J. Gas Chromatugr., 5 (1967) 273-282. 579 B.A. Knights, The analysis of plant sterols in feces, Mem. Soc. Endocrinol., 16 (1967) 21 1-221. 580 B.A. Knights, Gas-liquid chromatography of sterol glycosides, Anal. Lett., 6 (1973) 495-503. 58 1 B.A. Knights, Qualitative and quantitative analysis of plant sterols by gas-liquid chromatography, in E. Heftmann (Editor), Modern Methods of Steroid Analysis, Academic Press, 1973, pp. 103-138. 582 B.A. Knights and G.H. Thomas, ARM^ values in the gas chromatography of steroids, Chem. Ind. (London),( 1 963) 43-44. 583 B.A. Knights and G.H. Thomas, Gas chromatography of steroids. ARM^ values for hydroxy and acyloxy groups,J. Chem. Soc., (1963) 3477-3480. 584 F. Knuchel and H. Ochs, Quantitative determination of cholesterol ester fractions by thin-layer chromatography, Aerztl. Lab., 20 (1974) 120-125 (Ger.). 585 R. Knuppen, M.L. Rao and H. Breuer, Determination of 25 estrogens in urine by combined paper, thin-layer, and gas chromatography, 2. Anal. Chem., 243 (1968) 263-272 (Ger.). 586 T. Kobayashi, Chemical isomerization of vitamin D. 4. Identification of vitamin D2 and its related compounds by thin-layer chromatographic separation, Bitamin, 34 (1 966) 482-484 (Jap.). 587 M. Komatsu, Y. Kamano and M. Suzuki, Thin-layer chromatography of bufo steroids on silica gel, Bunseki Kagaku (Jap. Anal.), 14 (1965) 1049-1054 (Jap.). 588 M. Komatsu and S. Okano, Thin-layer chromatography of steroids in toad extracts. 2. Quantitative determination of bufo steroids by densitometry of thin-layer chromatograms, Bunseki Kagaku (Jap. Anal.), 15 (1966) 11 15-1 119 (Jap.). 589 M. Komatsu and T. Okano, Separation and qualitative determination of resibufogenin and bufalin on an automatic liquid chromatograph with thermal detection, Bunseki Kagaku (Jap. Anal.), 16 (1967) 118-122 (Jap.). 590 M. Komatsu and T. Okano, Separation of bufo steroids by dry elution column chromatography on silica gel, Yakugaku Zasshi (J. Pharm. Soc. Jap.), 87 (1967) 712718 (Jap.). 591 V.P. Komissarenko and V.N. Demchenko, Determination of the eight most important 1 7-ketosteroid fractions using two-dimensional thin-layer chromatography in human urine, Lab. D e b , (1974) 73-76 (Russ.). 592 L. Kornel, S. Miyabo and Z. Saito, New paper-chromatographic systems for the separation of conjugated corticosteroids, J. Chromatogr., 11 1 (1975) 200-205. 593 J. Korolczuk and I. KwaSniewska, Cupric sulphate as a charring agent in thin-layer chromatography of lipids, J. Chromatogr., 88 (1 974) 428--429.

160

REFERENCES

594 B.P. Korzun, L. Dorfman and S.M. Brody, Separation of some alkaloids, steroids, and synthetic compounds by thin-layer chromatography, Anal. Chern., 35 (1963) 950-952. 595 K.T. Koshy, D.G. Kaiser and A.L. Vanderslik, 0-(2,3,4,5,6-Pentafluorobenzyl)hydroxylamine hydrochloride as a sensitive derivatizing agent for the electroncapture gas-liquid chromatographic analysis of ketosteroids, J. Chromatogr. Sci., 13 (1975)97-104. 596 F.W. Koss and D. Jerchel, Use of aluminum foil as carrier for descending thin-layer chromatography, Natunvissenschuften, 5 1 (1964)382-383 (Ger.). 597 B.A. Kottke, J. Wollenweber and C.A. Owen, Jr., Quantitative thin-layer chromatography of free and conjugated cholic acid in human bile and duodenal contents, J. Chromatogr., 21 (1966)439-447. 598 D. Kritchevsky, D.S. Martak and G.H. Rothblat, Detection of bile acids in thin-layer chromatography, Anal. Biochem., 5 (1963)388-392. 599 D. Kritchevsky and S.A. Tepper, Detection of steroids in thin-layer chromatography, J. Chromatogr., 37 (1968)361-362. 600 G.J. Krol, G.R. Boyden, R.H. Moody, J.C. Comeau and B.T. Kho, Thin-layer separation and detection of free estrogens, J. Chromatogr., 61 (1971)187 -192. 601 G.J. Krol, C.A. Mannan, F.Q. Gemmill, Jr., G.E. Hicks and B.T. Kho, High-efficiency liquid chromatographic separation of vitamin D from precalciferol, J. Chromatogr., 74 (1972)43-49. 602 G.J. Krol, R.P. Masserano, J.F. Carney and B.T. Kho, Quantitative separation of free estrogens by liquid partition chromatography, J. Pharm. Sci.,59 (1970)1483 - 1487. 603 H.S. Kroman and S.R. Bender, An improved method for collecting gas-chromatographically separated estrogens, J. Chromatogr., 13 (1964)242-243. 604 H.S. Kroman, S.R. Bender and R.L. Capizzi, Gas-chromatographic separation and quantitation of estrogens from normal human plasma, Clin. Chim. Acta, 9 (1964) 73-78. 605 H.S. Kroman, M.O. King and S.R. Bender, A method for the gas-chromatographic separation of estrogens employing a solid injection system, J. Chromatogr., 15 (1964) 92-94. 606 H.S. Kroman, M.L. Moskovitz and S.R. Bender, Method for the gas-chromatographic separation of the methoxy derivative of estrogens. J. Gas Chromatogr., 2 (1964)40. 607 V.Krupa, M. Rochova and L. Zathurecky, Separation of some strophanthidin cardenolides by thin-layer chromatography on aluminum oxide, Cesk. Farm., 16 (1967)184-186 (Slo.). 608 W.R. Kulpmann, L. Siekmann and H. Breuer, Improved method for the gas-liquid chromatographic determination of aldosterone in urine, J. Steroid Biochem., 4 (1973) 649-657. 609 E. Kukkonen, Improvement in the investigation of the glycosides of Convallaria majalis, Farm. Aikak., 73 (1964)333-338 (Swed.). 610 A. Kuksis, Gas-chromatographic fractionation of natural steryl ester mixtures, Can. J. Biochem., 42 (1964)407-417. 61 1 A.Kuksis, Gas-liquid chromatography of bile acids, J. Amer. Oil Chem. Soc., 42 (1 965)276-282.

REFERENCES

161

612 A. Kuksis, Newer developments in determination of bile acids and steroids by gas chromatography, Methods Biochem. Anal., 14 (1966) 325-454. 613 A. Kuksis, Gas chromatography of bile acids, in G.V. Marinetti (Editor), Lipid ChromatographicAnalysis, Vol. 2, Marcel Dekker, New York, 1969, pp. 215 312. 614 A. Kuksis, Ionexchange chromatography of bile acids, in P.P. Nair and D. Kritchevsky (Editors), The Bile Acids. Chemistry, Physiology, and Metabolism, Vol. 1. Chemistry, Plenum Press, New York, 1971, Chap. 6 , pp. 173-208. 615 A. Kuksis, Progress in the analysis of lipids. 12. Gas chromatography. 4. Gas chromatography of sterols, steryl esters, and steroid glycosides, Fette, Seifen, Anstrichm., 75 (1973) 420-433. 616 B.D. Kulkarni and J.W. Goldzieher, Isolation of 17aethynyl steroids by column chromatography on silver-impregnated Florid, Steroids, 13 (1969) 467-475. 617 G.L. Kumari, W.P. Collins and I.F. Sommerville, Further studies on the gas-liquid chromatographic determination of CI9-steroids in human plasma using 63Ni electroncapture detection, J. Chromatogr.,41 (1969) 22-36. 618 S. Kunitomi, New analytical method for urinary testosterone, Okayama Zgakkai Zasshi (J. Ohzyama Med. SOC.),84 (1972) 475-486 (Jap.). 619 S. Kushinsky, Stability of estrogens to oxygen during exposure on silica ge1,J. Chromatogr., 71 (1972) 161-164. 620 S. Kushinsky, J. Coyotupa, K. Honda, M. Hiroi, K. Kinoshita, M. Foote, C. Chan, R.Y. Ho, W. Paul and W.J. Dignam, Gas-chromatographic determination of estrone, estradiol, and estriol in nonpregnant plasma, Mikrochim. Acta, (1 970) 491-503. 621 T. Laatikainen and R. Vihko, Identification of CI9O2 and CzlOz steroids in the glucuronide fraction of human bile, Eur. J. Biochem., 10 (1969) 165-171. 622 T. Laatikainen and R. Vihko, Quantitation of CI9O2 and CzlOz steroid mono- and disulfates in human bile,Steroids, 14 (1969) 119-131. 623 L. Labler and V. Cerny, Steroids. 84. Thin-layer chromatography of some steroidal bases and Holarrhena alkaloids, Collect. Czech. Chem. Commun., 28 (1963) 29322940. 624 L. Libler and V. Cerny, Thin-layer chromatography of steroidal bases and Holarrhena alkaloids, in G.B. Marini-Bettblo (Editor), Thin-LayerChromatography,Elsevier, Amsterdam, 1964, pp. 144-148. 625 A.G. Lacko, H.L. Rutenberg and L.A. Soloff, Recovery of labeled cholesterol and cholesterol esters from thin-layer chromatograms, Clin. Chim. Acta, 39 (1 972) 506510. 626 N.S. Lamontagne and D.F. Johnson, Chromatography of adrenocortical steroids on silicic acid columns, J. Chromatogr., 53 (1970) 225-232. 627 N.S. Lamontagne and D.F. Johnson, Determination of 17-ketosteroids and adrenocortical steroids by gradient elution chromatography on a silicic acid column, Steroids, 17 (1971) 365-375. 628 W.C. Landgraf and E.C. Jennings, Steroid determination from complex mixtures by high-pressure chromatographic techniques, J. Pharm. Sci., 62 (1 973) 278-281. 629 A. Latomaki, Thin-layer chromatographic investigations of Digitalis purpurea, Farm. Aikak., 74 (1965) 22-29 (Swed.). 630 H.L. Lau, Factors and artifacts in the formation of the trimethylsilyl ethers of

162

REFERENCES

steroids,J. Gas Chromatogr.,4 (1966) 136-139. 631 H.L. Lau and G.S. Jones, The value of preparative thin-layer chromatography for the routine determination of pregnanediol, Amer. J. Obstet. Gynecol., 90 (1964) 132-1 35. 632 D.M. Lawrence, Gas-chromatographic estimation of urinary pregnane-3a,l7,2Oatrio1 and 1l-ketopregnane-3a,l7,20a-triol,J. Chromatogr.,36 (1968) 344---347. 633 C. Le Borgne de Kaouel, J. Duron, C. Aubert and P. Juret, Thin-layer chromatographic study of sterols in ultraviolet light, J. Chromatogr., 27 (1967) 282-286 (Fr.). 634 P.C. Lee and P.J. Wood, Gas-liquid chromatographic determination of estriol and estetrol in late pregnancy urine, Clin. Chim. Acta, 3 0 (1970) 221-224. 635 T.M. Lees, M.J. Lynch and F.R. Mosher, Continuous-flow thin-layer chromatography, J. Chromatogr., 18 (1965) 595-598. 636 G. Lehnert, W. Mucke and H. Valentin, A method for the analysis of 1 l-deoxy-17ketosteroids in urine by gas chromatography and its application to man, Endokrinologie, 46 (1964) 241-246 (Ger.). 637 G. Lehnert, H. Valentin, W. Mucke and H. Deutzmann, Determination of androsterone, etiocholanolone, and dehydroepiandrosterone in the urine by gas chromatography, Aerztl. Lab., 11 (1965) 143-148 (Ger.). 638 F.Y. Leung and J. Griffiths, Chromatographic method for aldosterone determination in human urine, Clin. Chim. Acta, 37 (1972) 423-432. 639 E. Levin and C. Head, Quantitative analysis of tissue neutral lipids by thin-layer chromatography, Anal. Biochem., 10 (1965) 23-31. 640 S.S. Levin, J.C. Touchstone and T. Murawec, Improved solvent system for thin-layer chromatography of adrenal steroids, J. Chromatogr., 42 (1969) 129-130. 641 K.B. Lie and J.F. Nyc, The chromatography of lipids in test tubes coated with a thin layer of silicic acid,J. Chromatogr., 8 (1962) 75-81. 642 J.E. van Lier and L.L. Smith, Crystalline sterols obtained by gas chromatography, J. Chromatogr., 36 (1968) 7-17. 643 J.E. van Lier and L.L. Smith, Chromatography of some cholesteiol autoxidation products on Sephadex LH-20,J. Chromatogr., 41 (1969) 37-42. 644 J.E. van Lier and L.L. Smith, Sterol metabolism. 13. Chromatographic resolution of the epimeric 24-hydroxycholesterols, J. Chromatogr., 49 (1970) 555-557. 645 P.A. Linley, The evaluation of Digitalis purpurea by direct densitometry from paper chromatograms, Plantahfed., 23 (1973) 272-280. 646 M.B. Lipsett (Editor), Gas Chromatography of Steroids in Biological Fluids, Plenum Press, New York, 1965. 647 B.P. Lisboa, Application of thin-layer chromatography to the steroids of the androstane series,J. Chromatogr., 13 (1964) 391-407. 648 B.P. Lisboa, Characterization of A 4 - 3 - ~ ~ ~ - C 2 1 - ~on t ethin-layer r ~ i d ~ chromatograms by in situ color reactions,J. Chrornatogr., 16 (1964) 136-151. 649 B.P. Lisboa, Thin-layer chromatography of A4-3-oxosteroids of the androstane series, J. Chromatogr., 19 (1965) 81-104. 650 B.P. Lisboa, Separation and characterization of A5-3-hydroxyC&eroids by thinlayer chromatography,J. Chromatogr., 19 (1965) 333-351. 651 B.P. Lisboa, Thin-layer chromatography of steroids, J. Pharm. Belg., 20 (1965) 435-

REFERENCES

163

449 (Fr.). 652 B.P. Lisboa, Application of thin-layer chromatography to the separation of saturated 21-deoxypregnane steroids, Steroids, 6 (1965) 605-625. 653 B.P. Lisboa, Thin-layer chromatography of newer estrogens on Kieselgel G, Clin. Chim. Acta, 13 (1966) 179-199. 654 B.P. Lisboa, Formation and separation of Girard hydrazones on thin-layer chromatography by elatographic techniques, J. Chromatogr., 24 (1966) 4755477, 655 B.P. Lisboa, Separation and characterization of formaldehydogenic A4-3-oxo-CZlsteroids by means of thin-layer chromatography, Steroids, 7 (1966) 41-65. 656 B.P. Lisboa, Separation of 21-deoxy-A4-3-0x0-steroids of the pregnane series by thinlayer chromatographic procedures, Steroids, 8 ( 1966) 3 19-344. 657 B.P. Lisboa, Thin-layer chromatography. Application to C19 steroids, Pharm. Biol., 5 (1968) 463-469 (Fr.). 658 B.P. Lisboa, Application of ascending thin-layer chromatography to As-3-hydroxysteroids of the pregnane series,J. Chromatogr., 39 (1969) 173-181. 659 B.P. Lisboa, Chromatography of sterols and steroids, in G.V. Marinetti (Editor), Lipid ChromatographicAnalysis, Vol. 2 , Marcel Dekker, New York, 1969, pp. 57-147. 660 B.P. Lisboa, Thin-layer chromatography of steroids, sterols, and related compounds, Methods Enzymol., 15 (1969) 3-158. 661 B.P. Lisboa, Steroids, in K. Macek (Editor), Pharmaceutical Applications of ThinLayer and Paper Chromatography, Elsevier, Amsterdam, 1972, pp. 275-348. 662 B.P. Lisboa and E. Diczfalusy, Color reactions for the in situ characterization of steroid estrogens on thin-layer chromatograms, Acta Endocrinol., 43 (1963) 545-560. 663 B.P. Lisboa and R.F. Palmer, Application of thin-layer chromatographic techniques to the separation of A'6C19-steroids and related compounds, Anal. Biochem., 20 (1967) 77-85. 664 B.P. Lisboa, A.I. Palomino and G. Zucconi, New aspects of functional chromatography of steroids on thin layers, Symp. Chromatogr. Electrophor., 5me, Bruxelles, 1969, Presses Acad. Eur., Brussels, 1969, pp. 394-404 (Fr.). 665 B.P. Lisboa and M. Strassner, Gel chromatography of steroid estrogens on Sephadex LH-20,J. Chromatogr., 11 1 (1975) 159-164. 666 C.B. Lockwood, K.R. Brain and T.D. Turner, Simultaneous estimation of steroidal sapogenin in plant extracts by densitometric thin-layer chromatography, J. Chromatogr., 95 (1974) 250-253. 667 K. Lotscher and H. Kern, Liquid-chromatogiaphic separation of steroids, Chimiu, 27 (1 973) 348-349. 668 C. Lopez S h c h e z , J.C. Medina Acevedo and R. Ramirez Soto, Spectrophotometric determination of diosgenin in Dioscorea composita following thin-layer chromatography, Analyst (London),97 (1972) 973-976. 669 R. Lowry, Ferric chloride spray detector for cholesterol and cholesteryl esters on thin-layer chromatograms, J. Lipid Res., 9 (1968) 397. 670 J.A. Luetscher and R.G. Could, Gas-liquid chromatography of the tetrahydro derivatives of cortisol isolated from urine, J. Chromatogr., 13 (1964) 350-353. 671 M. Luisi, A. Carnicelli, G. Gambassi and G.F. Menchini, Determination of blood and urinary estrogens and progesterone by gas chromatography, Biochim. Biol, Sper.,

164

REFERENCES

5 (1966) 173-185 (Ital.). 672 M. Luisi, A. Carnicelli, V. Marescotti, M. Grieco and G . Menchini, Separation of some 17-ketosteroids by means of horizontal thin-layer chromatography, J. Chromatogr., 35 (1968) 430-433. 673 M. Luisi, C. Fassorra, C. Levanti and F. Franchi, Determination of testosterone in human urine by means of horizontal thin-layer and gas-liquid chromatography, J. Chrornatogr., 58 (1971) 213-226. 674 M. Luisi, F. Franchi, G.F. Menchini, D. Barletta, C. Fassorra, F. Ciardella and G. Gagliardi, Radioimmunoassay for plasma testosterone, Steroids Lipids Res., 4 (1973) 213-223. 675 M. Luisi, G. Gambassi, V. Marescotti, C. Savi and F. Polvani, Gas-chromatographic method for the quantitative determination of progesterone in human plasma, J. Chromatogr., 18 (1965) 278-284. 676 M. Luisi, F. Panicucci, E. Gagliardo and V. Marescotti, Steroid metabolism. 1. Gas chromatographic analysis of mixtures of pure estrogens, Folia Endocrinol., 16 (1963) 352-357 (Ital.). 677 M. Luisi, C. Savi and V. Marescotti, The separation of estrogens by horizontal thinlayer chromatography, J. Chromatogr., 15 (1964) 428-429. 678 T. Luukkainen and H. Adlercreutz, Gas chromatography of methylated estrogens and application of the method t o the analysis of human late pregnancy bile, Biochim. Biophys. Acfa, 70 (1963) 700-703. 679 J.A. Luyten and G.A.F.M. Rutten, Analysis of steroids by high-resolution gas-liquid chromatography. 2. Application to urinary samples, J. Chromatogr., 91 (1974) 393406. 680 J.M. Macarulla, J.A. G6mez-Capilla and C. Osorio, Direct analysis of steroids in urine by gas-liquid chromatography, Rev. Esp. Fisiol., 30 (1974) 67-70 (Span.). 681 K. Macek, I.M. Hais, J. Kopeck? and J. GaspariE (Editors), Bibliography ofpaper and Thin-Layer Chromatography, 1961-1965, Elsevier, Amsterdam, 1968. 682 K. Macek, I.M. Hais, J. Kopeck?, J. GaspariE, V. Ribek and J. ChuriEek, Bibliography of Paper and Thin-Layer Chromatography, 1966-1 969, Elsevier, Amsterdam, 1972. 683 K.M. McErlane, Gas-liquid chromatographic separation of equine estrogens, J. Chromafogr. Sci.,12 (1974) 97-100. 684 F. Machovicovi, Separation of certain cardenolides by thin-layer chromatography, Farm Obzor, 35 (1966) 450-453 (Czech.). 685 K.W. McKerns and E. Nordstrand, Simple method for tissue extraction and gaschromatographic separation of estrogens, Biochim. Biophys. Actu, 82 (1964) 198200. 686 L. McLaren, M.N. Myers and J.C. Giddings, Dense gas chromatography of nonvolatile substances of high molecular weight,Science, 159 (1968) 197-199. 687 H.M. McNair, C.R. Dobbs, L.H. Aung, D.C. Damoth and A.J. Luchte, Analysis of steroid hormones by a new direct gas chromatograph-mass spectrometer interface, Dyn. Mass Spectrom., 3 (1972) 301-307. 688 O.S. Madaeva and V.K. Ryzhkova, Application of thin-layer chromatography to the determination of steroid saponins and other polar compounds, Med. Prom. SSSR, 17 (1963) 44-45 (Russ.).

REFERENCES

165

689 S. Maglione, A. Bellastella, R. Gasbarro, A. Ghionni and G. Iacono, New gas-chromatographic method for the determination of urinary estrogens, Biochim. Appl., 15, Suppl. (1968) 542-550 (Ital.). 690 H.L.J. Makin, Gas liquid chromatography of steroid formates. An application in congenital adrenal hyperplasia, J. Endocrinol., 47 (1970) 55-64. 691 I. Makino, K. Shinozaki, S. Nakagawa and K. Mashimo, Measurement of sulfated and nonsulfated bile acids in human serum and urine,J. Lipid Res., 15 (1974) 132-138. 692 I. Makino and J. Sjovall, A versatile method for the analysis of bile acids in plasma, Anal. Lett., 5 (1972) 341-349. 693 M. Makita and W.W. Wells, Quantitative analysis of fecal bile acids by gas-liquid chromatography, Anal. Biochem., 5 (1963) 523-530. 694 M. Malaiyandi, J.P. Barrette and M. Lanouette, Vanadium pentoxide as a chromogenic spray reagent for the qualitative analysis of some organic compounds on thin-layer plates,J. Chromatogr., 101 (1974) 155-162. 695 B.J. Marcus and T.S. Ma, Organic synthesis on the microgram scale. 3. Separation and purification of steroids, Mikrochim. Acta, (1968) 436-441. 696 V.S. Marino, Porous glass as an adsorbent in thin-layer chromatography, J. Chromatogr., 46 (1970) 125-129. 697 R. Masaracchia and A. Gawienowski, Quantitative elution of microgram quantities of steroids from silica gel layers,Steroids, 11 (1968) 717-718. 698 P.A. Mason and R. Fraser, Estimation of aldosterone, 1 1-deoxycorticosterone, 18-hydroxy-l l -deoxycorticosterone, corticosterone, cortisol, and 1 1-deoxycortisol in human plasma by gas-liquid chromatography with electron-capture detection, J. Endocrinol., 64 (1 975) 277-288. 699 T. Masui and E. Staple, The separation of the stereoisomers of bile steroids, 5pcholestane-3a,7a,l2a,24a-tetrol and SP-cholestane-3a, 7a,12a,24P-tetrol, by thinlayer chromatography, Steroids, 9 (1967) 443-450. 700 R.G. Mathews, R.D. Schwartz, C.D. Pfaffenberger, S.-N. Lin and E.C. Horning, Polyphenyl ether sulfones. Thermally stable polar phases for gas chromatography, J. Chromatogr.,99 (1974) 51-61. 701 B. Matkovics and G. Condos, Thin-layer chromatography of isomeric steroid oximes and homo-aza-lactams. 3. Thin-layer chromatography of estra-l,3,5 (lO)-trien3-0117-one and its derivatives, Microchem. J., 13 (1968) 171-173. 702 B. Matkovics, Z . Tegyey and G. Condos, Thin-layer chromatography of isomeric steroid oximes and lactams. 2. Microchem. J., 11 (1966) 548-551. 703 N. Matsumoto, Systematic analysis of steroids. 2. Analysis of steroid sapogenins by thin-layer chromatography, Chem. Pharrn. Bull. (Tokyo), 11 (1963) 1189-1 192. 704 E.W. Matthews, P.G. Byfield, K.W. Colston, I.M. Evans, L.S. Galante and I. MacIntyre, Separation of hydroxylated derivatives of vitamin D3by high-speed liquid chromatography, FEBS Lett., 4 8 (1974) 122-125. 705 C. Matthijssen, J.W. Goldzieher, C. Franco and E.L. Wright, Analysis of total, grouped, and individual urinary 17-ketosteroids. Critical evaluation, Acta Endocrinol., 68 (1971) 311-333. 706 B.F. Maume, P. Bournot, J.C. Lhuguenot, C. Baron, F. Barbier, G. Maume, M. Prost and P. Padieu, Mass fragmentographic analysis of steroids, catecholamines and amino

166

REFERENCES

acids in biological materials, Anal. Chem., 4 5 (1973) 1073-1082. 707 B.F. Maume and J.A. Luyten, Evaluation of gas-chromatographic-mass spectrometric and mass fragmentographic performance in steroid analysis with glass capillary columns, J. Chromatogr. Sci., 11 (1973) 607-610. 708 B.F. Maume, G.M. Maume, J. Durand and P. Padieu, A new type of micro-analysis of steroid hormones in biological materials by gas chromatography with flame-ionization and electron-capture detection. 1. The urinary profile, J. Chromatogr., 58 (1971) 277-282. 709 B.F. Maume, W.E. Wilson and E.C. Horning, Gas-chromatographic and mass-spectrometric study of trimethylsilyl ethers of cardiac aglycones,Anal. Lett., 1 (1968) 401415. 710 R.A. Mead, G.C. Haltmeyer and K.B. Eik-Nes, Determination of free estradiol in peripheral plasma by gas-phase chromatography, J. Chromatogr. Sci., 7 (1969) 554560. 71 1 J.W.A. Meijer, Gas chromatography of steroids, Planta Med., Suppl., (1967) 48-59. 712 E. Menini, Determination of adrenal steroids by gas chromatography and its application to clinical research,Ann. Clin. Res., 2 (1970) 163-178. 713 E. Menziani, P. Sancin and G. Pertusato, Determination of ouabain in Acokanthera abyssinica seeds, Boll. Chim. Farm., 103 (1964) 825-828 (Ital.). 714 R. Mermet-Bouvier, The separation of the photochemical isomers of ergosterol by thin-layer chromatography, J. Chromatogr., 59 (197 1) 226-230. 71 5 R. Mermet-Bouvier, Gas chromatography of the photochemical isomers of ergosterol, J. Chromatogr. Sci., 10 (1972) 733-736. 716 R. Mermet-Bouvier, Analysis of the photochemical isomers of ergosterol by column chromatography, Anal. Chem., 45 (1973) 584--586. 71 7 M.G. Metcalf, Gas-chromatographic assay of urinary pregnanediol, Anal. Biochem., 25 (1968) 510-522. 718 M.G. Metcalf, Rapid gas-chromatographic assay for progesterone metabolites in urine, Clin. Biochem., 6 (1973) 307-320. 719 T.A. Miettinen, E.H. Ahrens, Jr. and S.M. Grundy, Quantitative isolation and gasliquid chromatographic analysis of total dietary and fecal neutral steroids, J. Lipid Res., 6 (1965)411-424. 720 S.A. Miklosi and P.J. McCosker, Gel filtration of estrogen conjugates in bovine urine, J. Endocrinol., 39 (1967) 361- 368. 721 D. Millington, D.A. Jenner, T. Jones and K. Griffiths, Endogenous steroid concentration in human breast tumors determined by high-resolution mass fragmentography, Biochem. J . , 139 (1974) 473-475. 722 Y.W. Mirhom, Deleterious effect of iodine when used as a detecting reagent for estrogens on chromatoplates before their estimation, Clin. Chim. Acra, 30 (1970) 347-349. 723 M.A. Mitchell, Rapid method for the estimation of estriol in pregnancy by gasliquid chromatography, Can. J. Med. Technol., 34 (1972) 106-121. 724 H. Miyazaki, M. Ishibashi, M. Inoue, M. Itoh and T. Kubodera, Simultaneous qualitative and quantitative analyses of bile acids by mass chromatography, J. Chromatogr., 99 (1974) 553-565.

REFERENCES

167

725 H. Miyazaki, M. Ishibashi, C. Mori and N. Ikekawa, Gas-phase microanalysis of zooecdysones, A d . Chem., 45 (1973) 1164 1168. 726 W. Mlekusch, W. Truppe and B. Paletta, Direct fluorimetric determination of serum lipids on thin-layer chromatograms, Clin. Chim. Actu, 49 (1973) 73-77 (Ger.). 727 W. Mlekusch, W. Truppe and B. Paletta, Fluorometric determination of cholesterol in thin-layer chromatograms, J. Chromutogr., 78 (1973) 438-441 (Ger.). 728 W.Mlekusch, W. Truppe and B. Paletta, Application of the sulfophosphovanillin reaction to lipids separated by thin-layer chromatography, J. Chromutogr., 93 (1974) 183-187 (Ger.). 729 H.J. van der Molen, Electron-capture detection of steroids isolated from biological samples, Ann. Clin. Res., 2 (1970) 279-288. 730 H.J. van der Molen, F.H. de Jong and B.A. Cooke, Steroid analysis of the clinical chemistry laboratory with special reference to the role of gas-liquid chromatography, Clin. Chim. Acta, 34 (1971) 169-185. 731 H.J. van der Molen and D. Groen, Determination of progesterone in human peripheral blood using gas-liquid chromatography with electron-capture detection, J. Clin. Endocrinol. Metub., 25 (1965) 1625-1639. 732 H.J. van der Molen and D. Groen, Determination of submicrogram amounts of steroids in blood using electron-capture and flame-ionization detection following gasliquid chromatography, Mem. SOC.Endocrinol., 16 (1967) 155-1 77. 733 J.A. Mollica and R.F. Strusz, Analysis of corticosteroid creams and ointments by high-pressure liquid chromatography, J. Phumz. Sci., 6 1 (1 972) 444-447. 734 R.E. Monroe, Paper-chromatographic separation of sterol mixtures by a reversedphase system,J. Chromatogr., 62 (1971) 161- 164. 735 R. Montalvo and O.H. Wheeler, Gas chromatography of estrone and related compounds, Can. J. Chem., 44 (1966) 100-103. 736 J.W. Moore and W. Cooper, Rapid method for the analysis of pregnanediol in nonpregnancy urine, Clin. Chim. Acta, 56 (1974) 215-220. 737 G. Moretti, G. Cavina and J. Sardi de Valverde, Gas-chromatographic and colorimetric determination of estradjol monoesters in oil solutions after separation by thin-layer chromatography, J. Chromatogr., 40 (1969) 410-416. 738 E.D. Morgan and C.F. Poole, Preparation and assessment of fluorocarbonsilyl ethers as gas chromatography derivatives for steroids, J. Chromutogr., 89 (1974) 225-230. 739 E.D. Morgan and C.F. Poole, Formation of pentafluorophenyldimethylsilyl ethers and their use in the gas-chromatographic analysis of sterols, J. Chromutogr., 104 (1975) 351-358. 740 E.D. Morgan and A.P. Woodbridge, Insect-molting hormones (ecdysones). Identification as derivatives by gas chromatography, J. Chem. SOC.,(1971) 475-476. 741 1. Mori, T. Ikeda and H . 4 . Lee, Separation of urinary 17-ketosteroids by partition thin-layer chromatography, Actu Med. Univ. Kagoshima, 9 (1967) 253-258. 742 Y. Mori and T. Sato, Retention indices of steroids and their group increment with temperature, J. Chromatogr., 76 (1973) 133-139. 743 K. Morimoto, Bile acids and sterols. 60. Synthesis of trihydroxy-24-ethylcoprostanic acid and chromatography of steroidal bile acids,J. Biochem. (Tokyo),55 (1964) 4 10-4 14.

168

REFERENCES

744 C.E. Morreal and T.L. Dao, Protection of estrogenic hormones by ascorbic acid during chromatography, Steroids, 25 (1975) 421 -426. 745 C.E. Morreal, T.L. Dao and P.A. Lonergan, Improved method for the detection of estrone, estradiol, and estriol in low-titer urine, Steroids, 20 (1972) 383- 397. 746 L.J. Morris, Fractionation of cholesterol esters by thin-layer chromatography, J. Lipid Res., 4 (1963) 357-359. 747 J. Morvay, Gas chromatography of estrogens in biological fluids, Acta Med. (Budapest), 29(1973) 145-153. 748 G.E. Mott, R.W. Moore and R. Reiser, Gas-liquid chromatography of hyocholic acid, J. LipidRes., 12(1971) 117-119. 749 J.R. Mueller, J.A. Robinette and M.E. Yannone, An evaluation of “OV” liquid phases for the gas-liquid chromatographic separation of biologically active steroids, J. Steroid Biochem., 3 (1972) 735 - 740. 750 K.D. Mukherjee, A new technique for scanning tubular thin-layer chromatograms, J. Chromatogr., 96 (1974) 242-244. 751 K. Mukherjee, H. Spaans and E. Haahti, New detector systems for thin-layer chromato. graphy, J. Chromatogr., 61 (1971) 317-321. 752 K.D. Mukherjee, H. Spaans and E. Haahti, New methods for detection and quantification in thin-layer chromatography. A study of pyrolysis and combustion systems, J. Chromatogr. Sci., 10 (1972) 193-200. 753 I.A. Muni, C.H. Altshuler, J.C. Neicheril and J.R. Voegeli, Estimation of plasma testosterone and epitestosterone by electron-capture gas-liquid chromatography, Anal. Lett., 5 (1972) 785-799. 754 B.E.P. Murphy, Sepbadex column chromatography as an adjunct to competitive protein binding assays of steroids, Nature (London),New Biol., 232 (1971) 21-24. 755 T.K. Murray, K.C. Day and E. Kodicek, The differentiation of vitamins D2and D3 by gas-liquid chromatography, Biochem. J., 98 (1966) 293-296. 756 T.K. Murray, P. Erdody and T. Panalaks, Determination of vitamins D2 and D3 in pharmaceuticals by gas-liquid chromatography, J. Ass. Offi. Anal. Chem., 51 (1968) 839-842. 757 P.P. Nair, C. Bucana, S. de Leon and D.A. Turner, Gas-chromatographic studies of vitamins D2 and D3,Anal. Chem., 37 (1965) 631-636. 758 P.P. Nair and S . de Leon, Current research on gas-liquid chromatography of the vitamin D group, B o g . Biochem. Pharmacol., 3 (1967) 498-505. 759 P.P. Nair and A . Pinelli, Thin-layer chromatographic separation of steroids and their localization by diazo dyes, J. Chromatogr., 47 (1970) 513-518. 760 P.P. Nair, I.J. Sarlos, D. Solomon and D.A. Turner, Simultaneous separation of 17-ketosteroids and estrogens by biphase gas chromatography, Anal. Biochem., 7 (1 964) 96- 102. 761 T. Nambara and Y.H. Bae, Analytical chemical studies on steroids. 54. Studies on metabolism of 3-deoxysteroids. 9. Determination of urinary metabolites of androst5-en-17-one by gas chromatography, J. Chromatogr., 64 (1972) 239-245. 762 T. Nambara, Y.H. Bae, T. Anjyo and S. Coya, Analytical chemical studies on steroids. 52. Studies on steroid conjugates. 7. Gas chromatography of steroid N-acetylglucosaminides, J. Chromatogr., 62 (1971) 369-372.

REFERENCES

169

763 T. Nambara, Y.N. Bae and M. Nokubo, Analytical chemical studies on steroids. 50. Determination of urinary metabolites of l6a-chloroestrone methyl ether by gas chromatography, J. Chromatogr., 60 (1971) 418-421. 764 T. Nambara, R. Imai and S . Sakurai, Colorimetric determination of cholestan-3a-01 in the presence of cholestan-3P-01 and cholesterol, Yakugaku Zasshi (J. Phurm: SOC. Jap.), 84 (1964) 680-684 (Jap.). 765 T. Nambara and T. Iwata, Analytical chemical studies on steroids. 63. Steroid numbers of androstanones and their oxime derivatives on gas chromatography, Chem. Pharm. Bull. (Tokyo), 21 (1973) 899-902. 766 T. Nambara, T. Iwata and S . Honma, Analytical chemical studies on steroids. 37. Gas chromatography of 2,3-oxygenated estratrienes, J. Chromatogr., 50 (1970) 400-404. 767 T. Nambara, T. Kudo, H. Hosoda, K. Yamanouchi and S. Goya, Analytical chemical studies on steroids. 16. Steroid number contribution by C/D-ring fusion, J. Chromatogr., 31 (1967) 535--538. 768 H. Nau, Sensitive detection of estrogens with an automatic conductivity detector for thin-layer chromatography, J. Chromatogr., 53 (1970) 391- 394 (Ger.). 769 R. Neher, Steroid Chromatography, Elsevier, Amsterdam, 1964. 770 R. Neher, Steroid separation and analysis. The technique appropriate to the goal, in J.C. Giddings and R.A. Keller (Editors), Advances in Chromatography, Vol. 4, Marcel Dekker, New York, 1967, pp. 47-59. 771 R. Neher, Thin-layer chromatography of steroids and related compounds, in E. Stahl (Editor), Thin-Layer Chromatography. A Laboratory Handbook, Springer, Berlin, 2nd Ed., 1967, pp. 302-350 (Ger.). 772 G.J. Nelson, Automated gradient-elution column chromatography, J. Amer. Oil Chem. SOC.,44 (1967) 86-94. 773 J.P. Nelson, Gas chromatography of selected pregnenes and pregnanes, J. Gas Chromatogr., 1 (1963) 27-29. 774 H. Neuninger, Rapid thin-layer chromatographic detection of prednisone and prednisolone in oily solution, Sci. Pharm., 33 (1965) 230-232 (Ger.). 775 F. Neuwald and K.E. Fetting, Thin-layer chromatographic detection of cholesterol oxidation products, Pharm. Ztg.-Nachr., 108 (1963) 1490-1491 (Ger.). 776 B.Kh. Nguen and A.A. Chemerisskaya, Chromatography of 3-oxosteroids in a thin sorbent layer. 2. Use of acid aluminum oxide,Zh. Anal. Khim., 21 (1966) 13751379 (Russ.). 777 E. Nice and K. Williams, Simultaneous estimation of pregnenolone, 17-hydroxypregnenolone, and dehydroepiandrosterone by gas-liquid radiochromatography, Anal. Biochem., 59 (1974) 399-406. 778 S.Z. Nicosia, G . Galli, A. Fiecchi and A. Ros, Base-catalyzed silylation. A quantitative procedure for the gas chromatographic-mass spectrometric analysis of neutral steroids, J. Steroid Biochem ., 4 (1 973) 4 17-425. 779 A. Niederwieser and C.G. Honegger, Gradient elution in thin-layer chromatography, Helv. Chim. Acta, 48 (1965) 893-909 (Ger.). 780 W. Nienstedt, Studies on the anomalous behaviour of steroids in adsorption chromatography, Acta Endocrinol., Suppl., 114 (1967) 1-53. 781 W. Nienstedt, Atypical thin-layer chromatography in the characterization of Czl-

170

REFERENCES

steroids, Eur. J. Steroids, 2 (1 967) 473-48 1. 782 H.N. Nigg, M.J. Thompson, J.N. Kaplanis, J.A. Svoboda and W.E. Robbins, Highpressure liquid solid chromatography of the ecdysones - insect-molting hormones, Steroids. 23 (1974) 507-516. 783 0. Nishikaze, Thin-layer chromatography. 2. Steroid hormones, Taisha, 2 (1965) 777-784 (Jap.). 784 0. Nishikaze, R. Abraham and H. Staudinger, Thin-layer chromatographic fractionation and determination of urinary corticosteroids, J. Biochem. (Tokyo), 54 (1 963) 427-431 (Cer.). 785 R. Noiret, Complete lipid removal from plasma extracts containing 17-ketosteroids (dehydroepiandrosterone, androsterone, etiocholanolone), J. Chromatogr., 34 (1968) 415 418 (Fr.). 786 R. Noiret and R. Devis, New method of fractionating steroid a-ketols (a-ketol side chain) on a silica gel column,Ann. Biol. Clin. (Paris),21 (1963) 373-381 (Fr.). 787 M. Nonclercq, S. Laboureur and G. Atassi, Detection of steroid impurities in prednisolone by thin-layer chromatography, J. Pharm. Belg., 25 (1970) 309-31 6 (Fr.). 788 H.E. Nordby and S. Nagy, An evaluation of recent gas-liquid chromatographic liquid phases for resolution of acety!ated plant sterols, J. Chromatogr., 75 (1973) 187-193. 789 H.E. Nordby and S. Nagy, Preparative argentation column chromatography for separation of sterols from orange vesicles, J. Chromatogr., 79 (1973) 147- 156. 790 A. Norman, Bile acids and steroids. 148. Application of gel filtration of bile acids to studies of lipid complexes in bile, Proc. SOC.Exp. Biol. Med., 116 (1964) 902- 905. 791 A.W. Norman and H.F. DeLuca, Chromatographic separation of mixtures of vitamin D2, ergosterol, and tachysterol,,Anal. Chem., 35 (1963) 1247-1250. 792 L. Nover, Cardiac glycosides and their genins, in K. Macek (Editor), Pharmaceutical Applications of Thin-Layer and Paper Chromatography, Elsevier, Amsterdam, 1972, pp. 349-392. 793 L. Nover, C. Juttner, S. Noack, G. Baumgarten and M. Luckner, Relation between chemical structure and chromatographic behavior of heart glycosides. 5. Thin-layer chromatographic investigations of heart glycosides and their aglycones, J. Chromatogr., 39 (1969) 419-449 (Ger.). 794 M. Novotny and R. Farlow, A simple method for concentrating dilute high-boiling samples for capillary gas chromatography, J. Chromatogr., 103 (1975) 1-6. 795 M. Novotnq and J. Janak, Analysis of steroid compounds by gas chromatography, Chem. Listy, 66 (1972) 693-726 (Czech). 796 M. Novotny and A. Zlatkis, High-resolution chromatographic separation of steroids with open-tubular glass columns, J. Chromatogr. Sci., 8 (1970) 346-350. 797 M. Novotny and A. Zlatkis, Capillary column chromatography of steroids. Evaluation of stationary phases, J. Chromatogr., 56 (1971) 353-356. 798 L.C. Nuti, B.C. Wentworth, H.J. Karavolas, W.J. Tyler and O.J. Ginther, Comparison of radioimmunoassay and gas-liquid chromatographic analyses of progesterone concentrations in cow’s milk, Proc. SOC.Exp. Biol. Med., 149 (1975) 877-880. 799 E. Nystrom, Preparation of methylated Sephadex and lipophilic Sephadex ion exchangers,Ark. Kern., 29 (1968) 99-106. 800 E. Nystrom and J. Sjovall, On the use of methylated Sephadex in gel filtration and

REFERENCES

171

reversed-phase partition chromatography of lipid-soluble substances, Biochem. J., 92 (1964) l o p . 801 E. Nystrom and J. Sjovall, Separation of lipids on methylated Sephadex, Anal, Biochem., 12 (1965) 235-248. 802 E. Nystrom and J. Sjovall, Methylated Sephadex as a support in reversed-phase partition chromatography, J. Chromatogr., 17 (1965) 574-576. 803 E. Nystrom and J. Sjovall, Thin-layer chromatography of bile acids on lipophilic Sephadex, Acta Chem. Scand., 21 (1967) 1974-1976. 804 E. Nystrom and J. Sjovall, Recycling and capillary column chromatography of steroids on lipophilic Sephadex,Ark. Kem., 29 (1968) 107-1 15. 805 T.A. Oboot, Determination of testosterone in peripheral blood plasma using thinlayer chromatography and fluorimetry in the red spectral region, Lab. D e b , (1974) 351-353 (Russ.). 806 G.W. Oertel and K. Groot, Estimation of pregnanediol and pregnanetriol in urine, Clin. Chim. Acta, 11 (1965) 512 518. 807 G.W. Oertel, P. Menzel and D. Wenzel, Steroid conjugates in plasma. 21. Isolation of steroid sulfatides from biological material, J. Steroid Biochem., 1 (1969) 17 23 (Ger.). 808 G.W. Oertel and L.P. Penzes, Determination of estrogens by densitometry of their ABS or DANSYL derivatives, Z . Anal. Chem., 252 (1970) 306-309 (Ger.). 809 G.W. Oertel, M.C. Tornero and K. Groot, Thin-layer chromatography of steroid conjugates,J. Chromatogr., 14 (1964) 509-51 1. 810 M. Ohta, M. Obara and S. Purshottam, A simple method for the determination of urinary pregnanediol and pregnanetriol by thin-layer chromatography, Endocrinol. Jpn., 13 (1966) 14-22. 81 1 M. Okada, Thin-layer chromatography of cardiotonic glucosides, Kagaku No Ryoiki, Zokan (J. Jap. Chem., Suppl.), 64 (1964) 103-1 13 (Jap.). 81 2 T. Okishio and P.P. Nair, An improved column for gas-liquid chromatography of substituted cholanic acids, Anal. Biochem., 15 (1966) 360-363. 813 T. Okishio, P.P. Nair and M. Gordon, Bile acids. The microquantitative separation of cellular bile acids by gas,liquid chromatography, Biochem. J . , 102 (1967) 654-659. 814 T. Okumura, Thin-layer chromatography on precoated adsorbents fixed with fused glass. 10. Thin-layer chromatography of steroidal sapogenins on silica gels and alumina sintered plates, Bunseki Kagaku (Jap. Anal.), 23 (1974) 893-900 (Jap.). 8 15 T. Okumura, Sintered thin-layer chromatography. 12. Thin-layer chromatography on porous metal oxide-sintered plates, Yakugaku Zasshi (J. Pharm. SOC.Jap.), 95 (1975) 304-3 11 (Jap.). 816 T. Okumura and T. Kadono, Thin-layer chromatography on precoated adsorbents fixed with fused glass. 8. Thin-layer chromatography on silica gel and alumina sintered sticks, Bunseki Kagaku (Jap. Anal.), 22 (1973) 980-987 (Jap.). 8 17 T. Okumura and T. Kadono, Thin-layer chromatography on fluorescent sintered plates, Bunseki Kagaku (Jap. Anal.), 22 (1973) 1602-1609 (Jap.). 818 T. Okumura and T. Kddono, Thin-layer chromatography on alumina-sintered glass plates,J. Chromatogr., 86 (1973) 57-64. 819 T. Okumura, T. Kadono and A. Iso’o, Sintered thin-layer chromatography with flameionization detector scanning, J. Chromatogr., 108 (1975) 329-336.

172

REFERENCES

820 T. Okuniura, T. Kadono and M. Nakatani, Thin-layer chromatography on silica gelsintered plates, J. Chromatogr., 74 (1972) 73-84. 821 J . Olivo, J. Vittek, A.L. Southren, G.G. Gordon and F. Rafii, Rapid method for the measurement of androgen kinetics and conversion to estrogens using Sephadex LH-20 column chromatography, J. Clin. Endocrinol. Metab ., 36 (1973) 153-1 59. 822 M .C. Olson, Analysis of adrenocortical steroids in pharmaceutical preparations by high-pressure liquid-liquid chromatography, J. Phamz. Sci., 62 (1973) 2001-2007. 823 R.R. O’Moore and I.W. Percy-Robb, Analysis of bile acids and their conjugates in jejunal juice by thin-layer chromatography and direct densitometry, Clin. Chim. Acta, 43 (1973) 39-47. 824 T. Omoto, Thin-layer chromatography of toad toxin, Kagaku No Ryoiki, Zokan (J. Jap. Chem., Suppl.), 64 (1964) 115-122 (Jap.). 825 G.F. Orlandi, B. Ferramosca, P. Bernardi, G. Abate and G. Del Giudice, Fractionation of urinary neutral 17-ketosteroids by thin-layer chromatography on Alumina G, Biochim. Biol. Sper., 5 (1966) 281-285 (Ital.). 826 M . Palem, A. Maquinay, M. Margoulies and P. Coninx, Determination of testosterone as the chloroacetate by gas chromatography,Rev. Fr. Er. Clin. Biol., 14 (1969) 96-98 (Fr.). 827 A.M. Paliokas, W.-H. Lee and G.J. Schroepfer, Jr., Improved separation of sterols by column chromatography,J. Lipid Res., 9 (1968) 143-145. 828 F. Panicucci, C. Savi, F. Coli and G. Gambassi, The Zimmerman-Peterson-Pierce reaction for the quantitative determination of single androgens separated by thinlayer chromatography, Boll. SOC.Ital. Biol. Sper., 40 (1964) 1042-1044 (Ital.). 829 F. Panicucci, C. Savi, F. Coli and M. Luisi, Determination of pure androgens by thinlayer chromatography and colorimetry, FoIia Endocrinol., 17 (1964) 237-244 (Ital.). 830 V.V. Panina and O.S. Madaeva, Quantitative determination of the content of diosgenin in samples of Solanum laciniatum from different zones of growth in the U.S.S.R., Khim.-Farm. Zh., 1 (1967) 37-38 (Russ.). 831 V.V. Panina and T.B. Pimenova, Method of quantitative determination of diosgenin in solasodine samples by paper chromatography, Med. Prom. SSSR, 20 (1 966) 47-48 (Russ.). 832 R. Paquin and M. Lepage, Separation of solanine, chaconine, and solanidine by thinlayer chromatography, J. Chromatogr., 12 (1963) 57-62 (Fr.). 833 C.K. Parekh and R.H. Wasserman, Preparation of [3H] -vitamin D3 by using column and thin-layer chromatography, J. Chromatogr., 17 (1965) 261-266. 834 C. Parini, G. Gerali, G. Sportoletti and G. Maroni, Steroids. 14. Chromatographic study of new androstene derivatives, Farmaco (Pavia),Ed. Prat., 23 (1968) 645-655 (Ital .). 835 N.A. Parris, High-performance liquid-liquid partition chromatography on porous silica microspheres, J. Chromatogr.Sci., 12 (1974) 753-757. 836 A. Pascaud, M. Pascaud and M. Lievremont, Column fractionation of cholesterol esters, Bull. SOC.Chim. Biol., 48 (1966) 192-193 (Fr.). 837 V. Paseshnichenko and M.G. Borikhina, Separation of water-soluble and waterinsoluble saponins from Dioscorea deltoidea rhizomes, Prikl. Biokhim. Mikrobiol., 10 (1974) 467-471 (Russ.).

REFERENCES

173

838 G.W. Patterson, Relation between structure and retention time of sterols in gas chromatography,AnaZ. Chem., 43 (1971) 1165-1170. 839 A. Patti, P. Bonanno, T.F. Frawley and A.A. Stein, Application of gas-phase chromatography to the separation and identification of steroids in biological fluids. 1. Pregnanediol. 2. 17-Ketosteroids. 3. Estrogens. 4. 17-Hydroxycorticosteroids,Acta Endocrinol., Suppl., 77 (1963) 3-34. 840 A. Patti, P. Bonanno, T.F. Frawley and A.A. Stein, Gas-phase chromatography in separation and identification of pregnanediol in urine and blood of pregnant women, Obstet. Gynecol., 21 (1963) 302- 307. 841 A. Patti and A.A. Stein, Steroid Analysis by Gas--Liquid Chromatography,Thomas, Springfield, 1964. 842 P. Pei, S. Ramachandran and R.S. Henly, Studies on preparative high-speed liquid-solid chromatography, Amer. Lab., 7 (1975) 37-40. 843 J.J. Peifer, Analysis by thin-layer chromatography using microchromatoplates, Mikrochim. Acta, (1962) 529-540. 844 A. Pekkarinen, A routine method for the assay of pregnanediol by thin-layer chromatography during pregnancy, Trans. Meet. Int. Study Group Steroid Hormones, 2nd, Rome, 1965,(1966) 223-227. 845 E.D. Pellizzari, J. Liu, M.E. Twine and C.E. Cook, Novel silver-sulfoethyl cellulose column for purification of ethynyl steroids from biological fluids, Anal. Biochem., 56 (1 973) 178-190. 846 L. Penzes, P. Menzel and G.W. Oertel, Thin-layer chromatography of C19-steroid 2,4-dinitrophenylhydrazoneson polyamide, J. Chromatogr.,44 (1969) 189-190. 847 L.P. Penzes and G.W. Oertel, Determination of steroids by densitometry of derivatives. 1. Assay of estrogens as azobenzene-4-sulfonates,J. Chromatogr., 5 1 (1970) 322--324. 848 L.P. Penzes and G.W. Oertel, Determination of steroids by densitometry of derivatives. 2. Direct fluorometry of DANSYL estrogens, J. Chromatogr., 51 (1970) 325-327. 849 L.P. Penzes and G.W. Oertel, Determination of steroids by densitometry of derivatives. 3. Micro-assay of estrogens as DANSYL derivatives,J. Chromatogr., 74 (1972) 359365. 850 K. Petrak, Quantitative estimation of nanogram and subnanogram amounts of steroids in blood and urine, CRCCrit. Rev. Anal. Chem., 3 (1974) 421-453. 851 K. Pfordte and W. Forster, Quantitative determination of cardenolides and their metabolites in biological materia1,Z. Med. Labortech., 11 (1970) 272-282 (Ger.). 852 C.G. Pierrepoint, Separation of neutral sulfates by thin-layer chromatography, Anal. Biochem., 18 (1967) 181-185. 853 A. Pinelli and M.L. Formento, A precise and sensitive method for the analysis of steroids in small urine samples by thin-layer chromatography and gas-liquid chromatography, J. Chromatogr., 68 (1972) 67-75. 854 A. Pinelli and P.P. Nair, Gas-liquid chromatographic separation of steroids and their derivatives on a dual-component column of high thermal stability, J. Chromatogr., 43 (1969) 223-228. 855 A . Pinelli, F. Witzke and P.P. Nair, Separation of vitamin D from cholesterol by thinlayer chromatography, J. Chromatogr., 42 (1969) 271-274. 856 D.A. Podmore, Routine determination of urinary pregnanediol using a gas chromato-

174

REFERENCES

graph with automatic sample application,J. Clin. Pathol., 19 (1966) 619-621. 857 H. Potter and H. Barisch, Thin-layer chromatographic determination of Digitalis glycosides,Pharmazie, 27 (1972) 315-318 (Ger.). 858 K. Pollow, R. Sinnecker and B. Pollow, Thin-layer chromatography of picogram amounts of estradiol, J. Chrornatogr., 90 (1974) 402-404 (Ger.). 859 F. Polvani, M. Surace and M. Luisi (Editors), Gas-Chromatographic Detemination of Hormonal Steroids, Academic Press, New York, 1968. 860 G. Ponchon and F.X. Fellers, Thin-layer chromatography of vitamin D and related sterols,J. Chromatogr., 35 (1968) 53-65. 861 C.F. Poole and E.D. Morgan, Anomalies in the gas-liquid chromatography of cholesterol heptafluorobutyrate, J. Chromatogr., 90 (1 974) 380-381. 862 C.F. Poole, E.D. Morgan and P.M. Bebbington, Analysis of ecdysones by gas chromatography using electron-capture detection, J. Chrornatogr., 104 (1975) 172175. 863 L. Porgesova and E. Porges, New spray reagents for thin-layer chromatography of some steroids of animal origin, J. Chromatogr., 24 (1966) 478-479 (Ger.). 864 E. Poteczin, T. FehCr, C. Kiss and G. Gyiiry Gas-chromatographic determination of unconjugated dehydroepiandrosterone, androstenedione, testosterone, pregnenolone, and progesterone in human ovarian tissue, Endokrinologie, 6 4 (1975) 15 1-158. 865 M. Prost, P. Bournot and B.F. Maume, Adrenal and liver metabolites of 18-hydroxy11-deoxycorticosteronein the rat. Identification of reduced compounds (1 8-OH-THDOC),Steroids, 25 (1975) 177-188. 866 M. Prost and B.F. Maume, Steroidal hormones of the rat adrenal gland. Analysis of 18-hydroxycorticosteroidsby gas-liquid chromatography coupled with mass spectrometry and mass fragmentography,J. Steroid Biochem., 5 (1974) 133-144 (Fr.: 867 A. Puech, G. Kister and J. Chanal, Structure-mobility correlations in thin-layer chromatography. Applications to some progestational agents, J. Chromatogr., 108 (1975) 345-353 (Fr.). 868 W.P. Pulsinelli, R.K. Tcholakian and K.B. Eik-Nes, Method for the determination of pregnenolone in biological samples using gas-phase chromatography, Anal. Biochem ., 49 (1972) 559-568. 869 R.O. Quesenberry, E.M. Donaldson and F. Ungar, Descending and ascending chromatography of steroids using thin-layer chromatography sheets, Steroids, 6 (1 965) 167- 175. 870 R.O. Quesenberry and F. Ungar, Thin-layer chromatographic systems for adrenal corticosteroids,Anal. Biochem., 8 (1964) 192-199. 871 G. Rabitzsch, Separation of the cardiac glycosides digitoxin and digoxin from their 20,22-dihydro derivatives by multiple thin-layer chromatography on cellulose films, J. Chrornatogr., 35 (1968) 122-125 (Ger.). 872 G. Rabitzsch and S. Jiingling, Butenolide-ring specific paper and thin-layer chromatography of cardenolide glycosides (digitoxin) with the use of 2,2',4,4'-tetranitrobipheny1,J. Chromatogr., 41 (1969) 96-104 (Ger.). 873 G. Rabitzsch and S . Jungling, Thin-layer chromatography of cardenolide derivatives and their acetates,J. Chromatogr., 42 (1969) 146-148 (Ger.). 874 M. Rahman, A simultaneous determination of pregnanetriol and pregnanediol by

REFERENCES

175

gas-liquid chromatography, Clin. Chim. Acta, 51 (1974) 233-240. 875 K.M. Rajkowski and G.D. Broadhead, The monochloroacetylation of oestrogens prior to gas-liquid chromatography with electron-capture detection, J. Chromatogr.,69 (1972) 373 376. 876 K.M. Rajkowski and G.D. Broadhead, Thin-layer chromatography of oestrogens, their derivatives and cholesterol, J. Chromatogr., 89 (1974) 374-379. 877 P.B. Raman, R. Avramov, N.L. McNiven and R.I. Dorfman, A method for the determination of pregnanediol, pregnanetriol, and pregnanetriolone by gas chromatography,Steroids, 6 (1965) 177-193. 878 J.P. Rapp and K.B. Eik-Nes, Gas chromatography with electron-capture detection of some corticosteroid derivatives, J. Gas Chromatogr.,3 (1965) 235-237. 879 V. Ratanosopa, A.E. Schindler, T.Y. Lee and W.L. Herrmann, Measurement of estriol in plasma by gas-liquid chromatography, Amer. J. Obstet. Gynecol., 99 (1967) 295-302. 880 J. Reichelt and J. Pitra, New experience with the application of thin-layer chromatography, Cesk. Farm., 12 (1963) 416 417 (Czech). 881 R. Reimendal and J. Sjovall, Analysis of steroids by off-line computerized gas chromatography-mass spectrometry, Anal. Chem., 44 (1972) 21- 29. 882 R. Reimendal and J. Sjovall, Computer evaluation of gas-chromatographic-massspectrometric analyses of steroids from biological materials, Anal. Chem., 45 (1973) 1083- 1089. 883 P.M. Reisert and D. Schumacher, Separation of CI9 steroids of urine by thin-layer chromatography, Experientia, 19 (1963) 84-86 (Ger.). 884 A.L. Remizov, M.V. Pavlova and L.M. Bershtein, Examination of steroid estrogens as products of their azo-condensation by thin-layer chromatography on silica gel, Probl. Endokrinol., 13 (1967) 105-108 (Russ.). 885 V. Remmers, H. Schmitt, H.G. Solbach, W. Staib and H. Zimmermann, Method for the separation of testosterone and epitestosterone by thin-layer chromatography on silica ge1,J. Chromatogr., 32 (1968) 760-761 (Ger.). 886 R. Repole, R. Gasbarro, S. Maglione and P. Rossi, Direct identification with fluorescein in thin-layer chromatography and determination of pure estrogens, Biochim. Appl., 12 (1965) 176-181 (Ital.). 887 R. Repole, R. Gasbarro, S. Maglione and P. Rossi, Detection of androgens by the use of sodium fluorescein on thin-layer chromatography plates, Rass. Med. Sper., 12 (1965) 133-138 (Ital.). 888 G. Reuter and P. Heller, Thin-layer chromatographic studies of the cardiac-active glycosides of Erysimum crepidifolium, Pharm. Zentralhalle, 105 (1 966) 597-598 (Ger .). 889 E. Ribi, C.J. Filz, G. Goode, S.M. Strain, K. Yamamoto, S.C. Harris and J.H. Simmons, Chromatographic separation of steroid hormones by centrifugation through columns of microparticulate silica, J. Chromatogr. Sci., 8 (1970) 577-580. 890 E. Ribi, C. Filz, K. Ribi, G. Goode, W. Brown, M. Niwa and R. Smith, Chromatography of microbial lipids by centrifugation through microparticulate gel, J. Bacteriol., 102 (1 970) 250-260. 891 G.S. Richardson, I. Weliky, W. Batchelder, M. Griffith and L.L. Engel, Radioautography

176

REFERENCES

of 14C-and 3H-labeled steroids on thin-layer chromatograms, J. Chromatogr., 12 (1963) 115-118. 892 E. Richter, Detection of sterols on silica gel layers with perchloric acid-naphthoquinone,J. Chromatogr., 18 (1965) 164-167 (Ger.). 893 J. Riess, Identification and chromatographic separation of organic phosphates via their methyl esters,J. Chromatogr., 19 (1965) 527-530 (Fr.). 894 F.L. Rigby, H.J. Karavolas, D.W. Nogard and R.C. Wolf, Preparation of steroid heptafluorobutyrates for gas-liquid chromatography utilizing vapor-phase derivatization, Steroids, 16 (1970) 703-706. 895 A. Riondel, J.F. Tait, S.A.S. Tait, M. Gut and B. Little, Estimation of progesterone in human peripheral blood using [3sS]-thiosemicarbazide, J. Clin. Endocrinol. Metab., 25 (1965) 229-242. 896 R. Ripa, A useful modification of the determination of urinary aldosterone by means of thin-layer chromatography, Minerva Med., 55 (1964) 3863-3865 (Ital.). 897 M. Rizk, J.J. Vallon and A. Badinand, Micromethods of characterization and determination of acetylenic steroids by formation of silver acetylide, Anal. Chim. Actu, 70(1974) 457-461 (Fr.). 898 H. Ronsch and K. Schreiber, Solunum alkaloids. 83. Analytical and preparative separation of 5a-saturated from As-unsaturated steroidal alkaloids and sapogenins by thin-layer chromatography on silver nitrate-containing adsorbent layers, J. Chromatogr., 30(1967) 149-154 (Ger.). 899 R. Roman, C.H. Yates, J.F. Millar and W.J.A. VandenHeuvel, Identification of estrogens isolated from pregnant mare's urine, Can. J. Pharm. Sci., 10 (1975) 8-1 1. 900 R. Roman, C.H. Yates, J.F. Millar and W.J.A. VandenHeuvel, Gas-chromatographic analysis of esterified estrogens, Can. J. Pharm. Sci., 10 (1975) 12-16. 901 R.W. Roos, Identification and determination of synthetic estrogens in pharmaceuticals by high-speed, reversed-phase partition chromatography, J. Pharm. Sci., 6 3 (1 974) 594- 599. 902 A. Ros, Simultaneous gas-chromatographic determination of the principal neutral urinary steroids using a glass capillary column. Method and clinical applications, MinervaMed., 65 (1974) 2568-2588 (Ital.). 903 R.S. Rosenfeld, Gas chromatography of some Czl metabolites of cortisol, Steroids, 4 (1964) 147-157. 904 R.S. Rosenfeld, Analysis of sterol extracts for cholestano1,Anal. Biochem., 12 (1965) 483-487. 905 V. Rossetti, Separation of cortisone-like synthetic steroids by silica gel thin-layer chromatography, Biochim. Appl., 12 (1965) 113-123 (Ital.). 906 G.D. Roversi and A. Ferrari, A new method for the gas-chromatographic analysis of the urinary 1 l-deoxy-17-ketosteroids, J. Chromatogr., 24 (1966) 407-41 1. 907 A. Rozanski, Gas-chromatographic determination of campesterol, 0-sitosterol and stigmastero1,Anal. Chem., 38 (1966) 36-40. 908 A. Rozanski, Simplified method of extraction of diosgenin from Dioscorea tubers and its determination by gas-liquid chromatography, Analyst (London), 97 (1 972) 968-972. 909 K.E. Rozumek, Separation of tomatidenol, solasodine, and soladulcidine and of

REFERENCES

910 91 1 912 91 3 914

915

916

917 91 8 919 920 921

922 923

924 925

926 927 928

1I 7

yamogenin, diosgenin, and tigogenin by thin-layer chromatography, J. Chromatogr., 40 (1969) 97-102 (Ger.). M.W. Ruchelman, Zone extractor for thin-layer chromatography, J. Chem. Educ., 44 (1967) 110. M.W. Ruchelman and V.W. Cole, Analysis of urinary 17-ketosteroids by gas-liquid chromatography, Clin. Chem., 12 (1966) 771-788. M.W. Ruchelman and P. Haines, Solubility studies of estradiol in organic solvents using gas-liquid chromatography, J. Gas Chromatogr., 5 (1967) 290-296. M.C. Ruiz Gonzalez, Semiautomatic determination of urinary pregnanediol through gas-liquid chromatography, Rev. Clin. Esp., 134 (1974) 17--22 (Span.). G.A.F.M. Rutten and J.A. Luyten, Analysis of steroids by high-resolution gas-liquid chromatography. 1. Preparation of apolar columns, J. Chromatogr., 74 (1 972) 177193. R. Sable-Amplis, R. Agid and D. Abadie, Determination of small amounts of corticosterone in high-level lipid plasma by means of thin-layer chromatography, J. Chromatogr., 94 (1974) 287-290. P.A. Sadler and A.E. Kellie, The gas--liquid chromatography of carboxylic acid esters of the urinary 1 1-deoxy-17-0x0 steroids. Determination as n-butyrates, Biochem. J., 103 (1967) 768-772. L.A.R. Sallam, A.M.H. El-Refai and I.A. El-Kadi, Thin-layer chromatography of some CZ1,CI9,and C18 steroids,J. Gen. Appl. Microbiol., 15 (1969) 309-315. A. Salvadeo and A. Cavalleri, Gas-chromatographic analysis of urinary 1l-deoxy-17ketosteroids, Boll. SOC.Ital. Biol. Sper., 4 0 (1964) 1047-1050 (Ital.). P. Samuel, M. Urivetzky and G. Kaley, Separation and radioassay of fecal cholesterol and coprosterol using thin-layer chromatography,J. Chromatogr., 14 (1964) 508-509. D.H. Sandberg, N. Ahmad, W.W. Cleveland and K. Savard, Measurement of urinary testosterone by gas-liquid chromatography, Steroids, 4 (1964) 557-568. D.H. Sandberg, N. Ahmad, M. Zachmann and W.W. Cleveland, Measurement of plasma 1 1-deoxy-l7-ketosteroid sulfates by gas-liquid chromatography, Steroids, 6 (1965) 777-791. D.H. Sandberg, J. Sjovall, K. Sjovall and D.A. Turner, Measurement of human serum bile acids by gas-liquid chromatography, J. Lipid Res., 6 (1965) 182-192. A. Sanghvi, C. Wight, A. Serenko and R. Balachandran, Gas-liquid chromatography of underivatized steroids. Simultaneous determination of urinary 17-ketosteroids, pregnanediol, pregnanetriol, and pregnanetriolone, Clin. Chim. Acta, 56 (1 974) 49-57. D. Sardini and J. Krepinsky, Densitometric determination of ecdysones, Farmaco (Pavia),Ed. h a t . , 29 (1974) 723-731 (Ital.). G.A. Sarfaty and H.M. Fales, Methyl haloacetone ketals as steroidal alcohol derivatives in gas-liquid chromatography and combined gas-liquid chromatography-mass spectrometry,Anal. Chem., 42 (1970) 288-291. S.L. Sarkar, K.V. Jogi and A. Mohimen, A new rapid method for estimation of urinary pregnanediol, J. Chromatogr. Sci., 12 (1974) 206-208. J.F. Sayegh and P. Vestergaard, Dry filling of capillary columns for liquid chromatography,J. Chromatogr., 31 (1967) 213-216. H.S. Schiller, E. Mahler, M.A. Brammall and P.H. Anderson, A simplified method for

178

REFERENCES

the separation of steroid hormones and its application to radio ligand assays, Anal. Lett., 7 (1974)473-481. 929 A.E. Schindler, Steroids in amniotic fluid, Fortschr. Geburtsh. Gynaekol., 46 (1972) 1-89 (Ger.). 930 A.E. Schindler and W.L. Herrmann, Determination of steroids by gas-liquid chromatography. 1. Measurement of estriol in pregnancy urine, Gynaecologia, 161 (1966) 446--460(Ger.). 93 1 W. Schink and H. Struck, Two-dimensional separation of steroids by thin-layer chromatography,Med. Welt, (1964) 1525-1526 (Ger.). 932 W. Schlemmer, Quantitative thin-layer chromatography. Assay of drug mixtures by scanning of remission peaks,J. Chromatogr., 63 (1971) 121-129. 933 H.P.G. Schneider and Z. Szereday, A quantitative thin-layer chromatographic method for the determination of pregnanediol, Klin. Wochenschr.,43 (1965) 747-748 (Ger.). 934 J.J. Schneider, Thin-layer and paper chromatography of steroidal 0-D-glucopyranosides, 0-D-glucopyranosiduronic acids, and derivatives, J. Chromatogr., 54 (1971) 97 102. 935 J.J. Schneider and D.K. Fukushima, Thin-layer chromatography of anomeric steroid tri-0-acetyl-D-glucopyranosiduronic methyl esters, D-glucopyranoside tetraacetates, J. Chromatogr., 48 (1 970) and 2’-acetamido-2’-deoxy-tri-O-acetyl-D-glucopyranosides, 5 09- 5 14. 936 J.J. Schneider and M.L. Lewbart, Paper-chromatographic evaluation of the contribution to polarity of hydroxyl and other groups at the C-17,C-20,and C-21 positions of the pregnane side chain, J. Chromatogr., 35 (1968) 287-292. 937 E. Schnurr, An interference factor in the radioimmunoassay of urinary aldosterone, J. Chromatogr., 84 (1973) 165-166 (Ger.). 938 R. Scholler and L. Dehennin, Steroidal hormone analysis by gas chromatography, Bull. SOC.Chim. Fr., (1967)3942-3951 (Fr.). 939 R. Scholler and M.-F. Jayle (Editors), Gas-Phase Chromatography of Hormonal Steroids, Dunod, Paris, 1968;Gordon and Breach, New York, 1968. 940 D.A. Schooley and K. Nakanishi, Application of high-pressure liquid chromatography t o the separation of insect-molting hormones, in E. Heftmann (Editor), Modern Methods of Steroid Analysis, Academic Press, New York, 1973,pp. 37-54. 941 D.A. Schooley, G. Weiss and K. Nakanishi, Simple and general extraction procedure for phytoecdysones based on reversed-phase adsorption chromatography, Steroids, 19 (1972) 377-383. 942 K. Schreiber, 0.Aurich and G. Osske, Solanum alkaloids. 18.Thin-layer chromatography of Solanum steroid alkaloids and steroid sapogenins, J. Chromatogr., 12 (1963) 63-69 (Ger.). 943 I. Schroeder, G. Lopez-Sanchez, J.C.Medina-Acevedo and M. del Carmen Espinosa, Quantitative determination of conjugated or esterified estrogens in tablets by thinlayer chromatography, J. Chromatogr. Sci., 13 (1975)37-40. 944 I. Schroeder, J.C. Medina-Acevedo and G . Lopez-Sanchez, Application of vapor-phase chromatography to the quantitative determination of conjugated estrogens in drugs, J. Chromatogr. Sci., 10 (1972) 183-186. 945 H.-R. Schulten and H.D. Beckey, Potentiality of the coupling of column liquid chromatography and field desorption mass spectrometry, J. Chromatogr., 83 (1973) 31 5--

179

REFERENCES

320. 946 E.P. Schulz, Gas-chromatographic assay of 17a-ethynylestradiol3-methylether in oral progestational agents. Comparison with thin-layer chromatographic assay, J. Pharm. Sci., 54 (1965) 144 147. 947 V. Schwarz and J. Protiva, Steroid derivatives. 45. Quantitative determination of steroids by thin-layer chromatography, Cesk. Farm., 16 (1967) 126-1 28 (Cz'ech). 948 R.M. Scott and J. Pietrzak, The detection of steroids by silicotungstic acid on Gelman sheets, J. Chromatogr.,44 (1969) 633-634. 949 R. Segura and A.M. Gotto, Jr., A new fluorimetric procedure for the detection and quantitation of organic compounds in thin-layer chromatography, J. Chromatogr., 99 (1974) 643-657. 950 R. Segura, J. Or6 and A. Zlatkis, Resolution of steroid glucuronides by thin-layer chromatography on polyamide, J. Chromatogr. Sci.,8 (1970) 449-451. 951 A. Seher and E. Homberg, Investigation of steroid mixtures by thin-layer chromatography, Fette, Seifen, Anstrichm., 70 (1968) 481-485 (Ger.). 952 A. Seher and E. Homberg, Sterol mixtures. 2. Qualitative analysis by thin-layer chromatography, Fette, Seifen, Anstrichm., 73 (1971) 557-560 (Ger.). 953 T. Seki, Chromatographic separation of 17-hydroxycorticosteroidson Sephadex LH20, J. Chromatogr.,29 (1967) 246-247. 954 T. Seki and K. Matsumoto, Chromatographic separation of 17-hydroxycorticosteroids and 17-ketosteroids,J. Chromatogr., 27 (1967) 423-430. 955 T. Seki and T. Sugase, Chromatographic separation of 17-ketosteroids and 17-hydroxycorticosteroids on Sephadex LH-20, 1. Chromatogr.,42 (1969) 503-508. 956 G. Semenuk and W.T. Beher, Quantitative determination of bile acids by direct densitometry of thin-layer chromatograms, J. Chromatogr., 21 (1966) 27-3 1. 957 K.D.R. Setchell and C.H.L. Shackleton, Group separation of plasma and urinary steroids by column chromatography on Sephadex LH-20, Clin. Chim. A m , 47 (1973) 381-388. 958 J. Seth, The selective detection of some steroid acetates using the electron-capture detector, J. Chromatogr., 38 (1968) 139-141. 959 C.H.L. Shackleton, J. Sjovall and 0. Wish, A simple method for the extraction of steroids from urine, Clin. Chim. Acta, 27 (1970) 354-356. 960 B.H. Shapiro and F.G. Peron, Separation of rat corticosteroids on Sephadex LH-20, J. Chromatogr.,65 (1972) 568-570. 96 1 D.P. Sharma and T.A. Venkitasubramanian, Separation of adreno-ovarian steroids by thin-layer chromatography, Anal. Biochem., 49 (1972) 9-14. 962 I.S. Shepherd, L.F. Ross and I.D. Morton, The detection of steroids on Silica Gel G layers, Chem. Ind. (London),(1966) 1706-1 707. 963 A.J. Sheppard and W.D. Hubbard, Gas chromatography of vitamins D2 and D3, Methods Enzymol., 18C (1971) 733-738. 964 A.J. Sheppard, D.E. LaCroix and A.R. Prosser, Separation of vitamins D2 and D3 as isotachysterols Dz and D3 by gas-liquid chromatography, J. Ass. Offic. Anal. Chem., 51 (1968) 834-838. 965 A.P. Shroff and J.K. Garner, A comparison of molybdenum thin-layer chromatographic spray reagents for steroids using spectrodensitometry, J. Chromatogr. Sci., 10 (1972) ~

180

REFERENCES

504-505. 966 A.P. Shroff and C.J. Shaw, In situ quantitation of norethindrone and mestranol by spectrodensitometry of thin-layer chromatograms, J. Chromatogr. Sci., 10 (1 972) 509-512. 967 C.M. Siegfried and W.H. Elliott, Separation of bile acids of rat bile by thin-layer chromatography, J. Lipid Res., 9 (1968) 394-395. 968 L. Siekmann, Mass fragmentography of steroid hormones,J. Steroid Biochem., 5 (1974) 727-732. 969 L. Siekmann, B. Spiegelhalder and H. Breuer, Determination of aldosterone in human plasma by combined gas chromatography-mass spectrometry, Z. Anal. Chem., 261 (1972) 377-381 (Ger.). 970 M.A. Siena, A. Brunelli and 0. Tofanetti, Separation of aldosterone on kieselgel thin layers using different solvent systems, Ann. Univ. Ferrara, Sez. 1 1 ,4 (1 972) 9- 16 (Ital.). 971 S. Siggia and R.A. Dishman, Analysis of steroid hormones using high-resolution liquid chromatography, Anal. Chem., 42 (1970) 1223-1229. 972 M.B. Simard and B.A. Lodge, Thin-layer chromatographic identification of estrogens and progestogens in oral contraceptives, J. Chromatogr., 5 1 (1970) 5 17-524. 973 P.M. Simpson, A method for the estimation of some synthetic glucocorticosteroids in rat muscle,J. Chrornatogr.,77 (1973) 161-174. 974 W.G. Sippell, P. Lehmann and G. Hollmann, Automation of multiple Sephadex LH-20 column chromatography for the simultaneous separation of plasma corticosteroids, J. Chromatogr., 108 (1975) 305-312. 975 J. Sjovall, Gas chromatography of bile acids, in H.A. Szymanski (Editor), Biomedical Applications of Gas Chromatography, Plenum Press, New York, 1964, pp. 151-167. 976 J. Sjovall, Separation and determination of bile acids, Methods Biochem. Anal., 12 (1964) 97-141. 977 J. Sjovall, Gas-liquid chromatography of bile acids, in A.T. James and L.J. Morris (Editors), New Biochemical Separations, Van Nostrand, New York, 1964, pp. 65-79. 978 J. Sjovall, E. Nystrom and E. Haahti, Liquid chromatography on lipophilic Sephadex. Column and detection techniques, Advan. Chromatogr., 6 (1968) 119-1 70. 979 J. Sjovall and K. Sjovall, Identification of 5a-pregnane-3a,20a,2 1-trio1 in human pregnancy plasma, Steroids, 12 (1968) 359-366. 980 J. Sjovall and R. Vihko, Determination of androsterone and dehydroepiandrosterone sulfates in human serum by gas-liquid chromatography, Steroids, 6 (1965) 597-604. 981 J. Sjovall and R. Vihko, Chromatography of conjugated steroids on lipophilic Sephadex, Acta Chem. Scand., 20 (1966) 1419-1421. 30,2001-di982 J. Sjovall and R. Vihko, Identification of 3/3,170-dihydroxyandrost-5-ene, hydroxypregn-Sene and epiandrosterone in human peripheral blood, Steroids, 7 (1966) 447-458. 983 J. Sjovall and R. Vihko, Analysis of solvolyzable steroids in human plasma by cornbined gas chromatography-mass spectrometry, Acta Endocrinol., 57 (1968) 247-260. 984 K. Sjovall, J. Sjovall, K. Maddock and E.C. Horning, Estimation of dehydroepiandrosterone sulfate in human serum by gas-liquid chromatography, Anal. Chem., 14 (1966) 337-346.

REFERENCES

181

985 D. Sklan and P. Budowski, Simple separation of vitamin D from sterols and retinol by argentation thin-layer chromatography,AnaZ. Chem., 45 (1973) 200-201. 986 D. Sklan, P. Budowski and M. Katz, Determination of 25-hydroxycholecalciferol by combined thin-layer and gas chromatography, Anal, Biochem., 56 (1 973) 606609. 987 J. Slemrova, Significance of bacterial steroid degradation for the etiology o'f large bowel cancer. 3. Possibilities for the determination of bile acids in biological materials, Zentralbl. Bakteriol. Parasitenkd., Infektionskr. Hyg., Abt. 1, Orig., Reihe B , 159 (1974) 539-545 (Ger.). 988 A.G. Smith and C.J.W. Brooks, Application of cholesterol oxidase in the analysis of steroids,J. Chromatogr., 101 (1974) 373-378. 989 E. Smith, Application of reversed-phase partition chromatography to the analysis of testosterone propionate in oil injectables, J. Pham. Sci., 56 (1967) 630-634. 990 L.L. Smith and F.L. Hill, Detection of sterol hydroperoxides on thin-layer chromatoplates by means of the Wurster dyes,J. Chromatogr., 66 (1972) 101-109. 991 L.L. Smith, W.S. Matthews, J.C. Price, R.C. Bachmann and B. Reynolds, Thin-layer chromatographic examination of cholesterol autoxidation, J. Chromatogr., 27 (1967) 187-205. 992 L.L. Smith and J.C. Price, Detection of 7-ketocholesterol in oxidized sterol preparations, J. Chromatogr., 26 (1967) 509-5 11. 993 P. Smith and C.J. Hall, Solvents for the adsorption chromatography of adrenocortical steroids, J. Chromatogr., 101 (1974) 202-205. 994 H. SoEiE and I. BeliE, Separation and identification of steroids produced by fermentative oxidation of progesterone, Z. Anal. Chem., 243 (1968) 29 1-294 (Ger.). 995 D. Sonanini, Thin-layer chromatography of Digitalis cardenolides, Pharm. Acta Helv., 39 (1964) 673-678 (Ger.). 996 D. Sonanini and L. Anker, Detection of steroids (androgens, estrogens, gestagens, anabolic agents) by partition thin-layer chromatography, Pham. Acta Helv., 4 2 (1 967) 54-64 (Ger.). 997 D. Sonanini, R. Hofstetter, L. Anker and H. Muhlemann, Detection of pharmacologically important glucocorticoids by partition thin-layer chromatography, Pharm. Acta Helv., 4 0 (1965) 302-307 (Ger.). 998 N.J. de Souza and W.R. Nes, Improved separation of sterols by reversed-phase thinlayer chromatography, J. Lipid Res., 1 0 (1969) 240-243. 999 M. Sparagana, Quantitative gas-chromatographic analysis of urinary testosterone and epitestosterone, Steroids, 5 (1965) 773-789. 1000 M. Sparagana, Determination of urinary tetrahydrocortisone, tetrahydrocortisol, and 3a-allotetrahydrocortisol by gas chromatography, Steroids, 6 (1965) 583-596. 1001 M. Sparagana, Infrared microspectrophotometry of urinary steroids separated by gas chromatography, Steroids, 8 (1966) 219-232. 1002 M. Sparagana, E.H. Keutmann and W.B. Mason, Quantitative determination of individual C 1902 and CI9O3 urinary 17-ketosteroids by gas chromatography, Anal. Chem., 35 (1963) 1231-1238. 1003 R. Stainier, Application of thin-layer chromatography for the separation of certain ketol steroids, J. Pharm. Belg., 20 (1965) 89-1 10 (Fr.).

182

REFERENCES

1004 D.A. Stansfield, The elution of progesterone from silica gel, Biochem. Biophys. Res. Commun., 16 (1964) 398-399. 1005 L. Stlrka, R F values of some estrogens and 3fl-hydroxy-A5-steroidsin thin-layer chromatography without binder, J. Chromatogr., 17 (1965) 599-602. 1006 W.R. Starnes, A.H. Rhodes and R.H. Lindsay, Thin-layer chromatography of 17ketosteroid 2,4-dinitrophenylhydrazones,J. Clin. Endocrinol. Metab., 26 (1966) 1245-1 250. 1007 W. Steidle, Scilla glycosides. 2. Quantitative estimation of hexadienolides in squill extracts,Ann., 662 (1963) 126-132 (Ger.). 1008 P.J. Stevens, Thin-layer chromatography of steroids. Specificity of two location reagents, J. Chromatogr., 14 (1964) 269-273. 1009 P.J. Stevens, Thin-layer chromatography of synthetic steroids on silica gel impregnated with silver nitrate, J. Chromatogr., 36 (1968) 253-258. 1010 P.J. Stevens and A.B. Turner, The use of iodine as a nondestructive location reagent for steroids in thin-layer chromatography, J. Chromatogr., 43 (1969) 282-286. 1011 A. Stiehl, J. Wollenweber and H. Wagener, Separation of free bile acids by thin-layer chromatography with continuous development and their isolation with a modified pilot chromatogram, J. Chromatogr., 43 (1969) 278-281 (Ger.). 1012 W.G. Stillwell, E.C. Horning, M.G. Horning, R.N. Stillwell and A. Zlatkis, Characterization of metabolites of steroid contraceptives by gas chromatography and mass spectrometry, J. Steroid Biochem., 3 (1972) 699-706. 1013 W.G. Stillwell, R.N. Stillwell and E.C. Horning, Analysis of nanogram quantities of norethisterone in plasma using a gas chromatograph-mass spectrometer-computer selective ion detection procedure, Steroids Lipids Res., 5 (1974) 79-90. 1014 J.E. Stouffer and P.L. Oakes, Microcolumn liquid analysis of lipids with flameionization detection, J. Amer. Oil Chem. SOC.,44 (1967) 77-79. 1015 R. Strohecker, Determination of ergosterol in ergocalciferol (vitamin Dz),Deut. Apoth.-Zt., 106 (1966) 1171-1172(Ger.). 1016 H. Struck, H. Karg and H. Jork, Thin-layer chromatographic determination of testosterone and androst-4-ene-3,17-dione from bovine fetal testicular tissue, J. Chromatogr., 36 (1968) 74-83 (Ger.). 1017 R. Stupnicki and M. Marchut, Determination of 17-ketosteroids in urine, Endokrynol. Pol., 17 (1966) 245-253; ibid., Engl. Transl., 137-145. 1018 M.R. Subbaram, Separation of saturated and unsaturated fatty acid esters of cholesterol by gas-liquid chromatography, J. Chromatogr., 15 (1964) 78-80. 1019 M.T. Subbiah, Analysis of sterols. Application to sterol balance studies, Amer. J. Clin. Nutr., 25 (1972) 780-788. 1020 M.T. Subbiah, Measuring bile acids in bile and feces, Ann. Clin. Lab. Sci., 3 (1973) 362-368. 1021 M.T. Subbiah and A . Kuksis, Alkaline solvent systems for thin-layer chromatography of bile acids, J. Lipid Res., 9 (1968) 288-290. 1022 S. Sulimovici, B. Lunenfeld and M.C. Shelesnyak, Practical method for estimation of urinary pregnanediol and allopregnanediol using thin-layer chromatography, Acta Endocrinol., 49 (1965) 97-106. 1.023 S. Sulimovici, B. Lunenfeld and M.C. Shelesnyak, The separation and estimation of

REFERENCES

183

androsterone, etiocholanolone, and dehydroepiandrosterone by use of thin-layer chromatography, Acta Endocrinol., 5 1 (1966) 447-460. 1024 N.-C. Sun and H.-Y. Lang, Application of thin-layer chromatography in the study of natural products. 3. Identification of cardiac glycosides, Yao Hsueh Hsueh Pao, 11 (1964) 101-107 (Chin.). 1025 G.S. Sundaram, H. Singh and H.S. Sodhi, Thin-layer chromatographic separation of chenodeoxycholic and deoxycholic acids, Clin. Chim. Acta, 34 (1971) 425-429. 1026 G.S. Sundaram and H.S. Sodhi, Color detection of bile acids in thin-layer chromatography, J. Chromatogr., 61 (1971) 370 --372. 1027 M. Surace and F. Polvani, Gas chromatography of steroid hormones in clinical investigations, Ann. Obstet. Ginecol., 90 (1968) 35 1-450 (Ital.). 1028 H. Suzuki, Thin-layer chromatography of steroids. Application of thin-layer chromatography to the separation and quantitative analysis of individual free corticoids and the tetrahydro compounds in urinary extracts and adrenal vein blood of dogs, Nippon Naibumpi Gakkai Zasshi (Folia Endocrinol. Jpn.; Jap. J. Endocrinol.), 40 (1965) 1358-1363 (Jap.). 1029 T. Suzuki and K. Hasegawa, Separation of carotenoids, steroids, and the related substances on lipophilic Sephadex,Agr. Biol. Chem., 38 (1974) 871-872. 1030 C.C. Sweeley and T.-C.L. Chang, Gas chromatography of steroids. Relation of structure to molar response in an argon ionization detector, Anal. Chem., 33 (1961) 1860-1863. 1031 L. Swell, Simultaneous determination of mass and radioactivity of labeled sterols and steroids by gas-liquid radiochromatography, Anal. Biochem., 16 (1966) 70-83. 1032 L. Swell, Gas-liquid radiochromatography of intact labeled cholesterol esters, Proc. Soc. Exp. Biol. Med., 121 (1966) 1290-1294. 1033 I. Szkkics and M. Klembala, Thin-layer chromatographic separation of oral contraceptives and some sex steroids,Z. Klin. Chem. Klin. Biochem., 8 (1970) 131-133 (Ger.). 1034 Z. Szereday and L. Sachs, New method of determination of testosterone in urine, Expenentia, 21 (1965) 166 (Ger.). 1035 D.G. Szonyi, K. Kovics and B. Matkovics, Thin-layer chromatography of steroids. 7. Comparative thin-layer chromatographic properties of hydroxyestrones, hydroxyestradiols, and their synthetic intermediates,Microchem.J., 18 (1973) 21 5-219. 1036 J.M. Takics, E. Kocsi, E. Garamvolgyi, E. Eckhart, T. Lombosi, Sz. Nyiredy, Jr., I. BorbCly and G. Krasznai, Contribution to the theory of the retention index system. 6. Calculation of the retention indices of compounds containing halogen atoms or hydroxyl groups, amines, ketones, esters, heterocyclic compounds, adamantanes, silanes and steroids on apolar and polar stationary phases in gas-liquid chromatography,J. Chromatogr., 81 (1973) 1-8. 1037 K. Takeda, S. Hara, A. Wada and N. Matsumoto, A systematic simultaneous analysis of steroid sapogenins by thin-layer chromatography, J. Chromatogr., 11 (1963) 562-564. 1038 M. Takeuchi, Analysis of steroids. 1. Analysis of steroid hormones by thin-layer chromatography, Chem. Pharm. Bull. (Tokyo), 11 (1963) 1183-1 188. 1039 J.M. Talmage, M.H. Penner and M. Geller, Quantitative gas-liquid chromatographic

184

REFERENCES

determination of ethynylestradiol, J. Pharm. Sci., 54 (1965) 1194-1 196. 1040 L. Tan, An improved method for the gas-chromatographic identification of Digitalis cardenolides, J. Chromafogr., 45 (1969) 68-75. 1041 L. Tan, M. Clemence and J. Gass, Gas-chromatographic analysis of fecal pollution sterols on a single combined packed column, J. Chromatogr., 53 (1 970) 209-21 5. 1042 S. Tanaka, M. Satake and K. Sakai, Rapid gas-chromatographic determination of urinary estriol in the end stage of pregnancy,Horumon f oRinsho, 19 (1971) 955999 (Jap.). 1043 E.B. Tapscott and G.L. Dohm, Use of sulfo-phospho-vanillin to quantitate unsaturated neutral lipids in thin-layer chromatography, J. Chromafogr., 107 (1975) 420-422. 1044 G.E. Tarr, Simple method of gradient elution in thin-layer chromatography of lipids, J. Chromafogr., 52 (1970) 357-361. 1045 T. Taylor, Group separation of A4-3-oxosteroids and A5-3P-hydroxysteroids by chromatography on digitonin-silica thin layers, Anal. Biochem., 41 (1971) 435-445. 1046 T. Taylor, Simultaneous separation of common mammalian A4-3-oxosteroids and oestrogens using two-dimensional thin-layer chromatography, J. Chrornafogr., 66 (1972) 177-178. 1047 R.J. Teichman, G.H. Takei and J.M. Cummins, Detection of fatty acids, fatty aldehydes, phospholipids, glycolipids, and cholesterol on thin-layer chromatograms stained with Malachite Green, J. Chromafogr., 88 (1974) 425-427. 1048 H. Temp1 and G. Geyer, Thin-layer chromatographic determination of pregnanetriol and pregnanetriolone in urine, Clin. Chim. Acta, 16 (1967) 179-180. 1049 J.I. Teng, M.J. Kulig and L.L. Smith, Gas-chromatographic differentiation among cholesterol hydroperoxides, J. Chromafogr., 75 (1973) 108-1 13. 1050 J.P. Thenot and E.C. Horning, Gas-chromatographic behavior of 3-ketosteroid methoximes. Application of gas-chromatographic studies of adrenocortical steroid hormone methoxime-trimethylsilyl derivatives, Anal. Leff., 5 (1972) 801-814. 1051 J.H.H. Thijssen and W. Veeman, A gas-chromatographic method for the measurement of small amounts of estrogens in urine, Steroids, 11 (1968) 369-387. 1052 B.S. Thomas, Measurement of plasma testosterone as the iodomethyldimethylsilyl ether by gas-liquid chromatography, J. Chromatogr., 56 (1 971) 37-50. 1053 B.S. Thomas and D.R.M. Walton, The use of haloalkylsilyl ether steroid derivatives for analysis by gas-liquid chromatography, Mem. SOC.Endocrinol., 16 (1967) 199-209. 1054 G.H. Thomas, The R M approach to the structural analysis of steroid metabolites, Mem. SOC.Endocrinol., 16 (1967) 129-139. 1055 M.J. Tikkanen and H . Adlercreutz, Separation of estriol conjugates on Sephadex, Acta Chem. Scand., 24 (1970) 3755-3757. 1056 C.L. Tipton, J.W. Paulis and M.D. Pierson, Gel filtration of lipid mixtures, J. Chromatogr., 14 (1964) 486-489. 1057 I. T6th and I. Faredin, Separation and identification of free and esterified 17-ketosteroids,AcfaMed. (Budapest), 29 (1973) 141-144. 1058 J.C. Touchstone, A.K. Balin and P. Knapstein, Rapid quantitation of estrogens by diazotization and direct thin-layer densitometry, Steroids, 13 (1969) 199-21 1. 1059 J.C. Touchstone, A.K. Balin, T. Murawec and M. Kasparow, Quantitative densito-

REFERENCES

185

metry on thin-layer plates of silica gel impregnated with phosphomolybdic acid, J. Chromatogr. Sci., 8 (1970) 443-445. 1060 J.C. Touchstone, M. Breckwoldt and T. Murawec, Chromatographic properties of the epimeric estriols,J. Chromatogr., 59 (1971) 121-125. 1061 J.C. Touchstone, S.S. Levin and T. Murawec, Quantitative aspects of spectrodensitometry of thin-layer chromatograms, Anal. Chem., 43 (1971) 8584363. 1062 J.C. Touchstone and T. Murawec, Mixed-phase packings of OV substrates for gas chromatography of steroids, J. Chromatogr.Sci., 9 (1971) 446 -447. 1063 J.C. Touchstone and T. Murawec, Methods for determination of steroids by thinlayer chromatography, in J.C. Touchstone (Editor), Quantitative Thin-Layer Chromatography,Wiley, New York, 1973, pp. 131-153. 1064 J.C. Touchstone, T. Murawec and 0. Brual, Improved solvent systems for thin-layer chromatography of estrogens, J. Chromatogr., 37 (1968) 359-360. 1065 J.C. Touchstone, T. Murawec, 0. Brual and M. Breckwoldt, Urinary estrogens. 1. Improved method for quantitation of urinary estrogens, Steroids, 17 (1971) 285304. 1066 J.C. Touchstone, T. Murawec, M. Kasparow and A.K. Balin, Quantitation of estriol in pregnancy urine by diazotic coupling and direct thin-layer densitometry, J. Chromatogr. Sci., 8 (1970) 81-83. 1067 J.C. Touchstone, T. Murawec, M. Kasparow and W. Wortmann, The use of silica gel modified with ammonium bisulfate in thin-layer chromatography, J. Chromatogr., 66 (1972) 172-174. 1068 J.C. Touchstone, T. Murawec and A. Nikolski, Retention slopes of steroids on gas chromatography in a series of combination columns, J. Chromatogr. Sci., 8 (1970) 221-223. 1069 J.C. Touchstone, A. Nikolski and T. Murawec, Combination columns in gas chromatography of estrogens and other steroids, Steroids, 3 (1964) 569-577. 1070 J.C. Touchstone, A. Nikolski and T. Murawec, Improved combination column for gas chromatography of estriol, Steroids, 5 (1965) 423-427. 1071 J.C. Touchstone and W. Wortmann, High-pressure liquid chromatography of corticosteroids, J. Chromatogr., 76 (1973) 244-247. 1072 J.C. Touchstone, C.-H. Wu, A. Nikolski and T. Murawec, Retention behavior of steroids in gas chromatography with a series of combination columns, J. Chromatogr., 29 (1967) 235-238. 1073 H. Traikov and M.K. Birmingham, Tests for 1 lp-hydroxyprogesterone and other CZl steroids, Can. J. Biochem., 44 (1966) 291-292. 1074 F.K. Trefz, D.J. Byrd and W. Kochen, Quantitative determination of cortisol in human plasma by high-pressure liquid chromatography, J. Chromatogr., 107 (1975) 181-189. 1075 L. Treiber and G.W. Oertel, Estimation of ketosteroids with 2,4-dinitrophenylhydrazine. Determination of dehydroepiandrosterone, androsterone, and etiocholanolone in urine, Clin. Chim. Actu, 17 (1967) 81-86. 1076 L. Treiber, W. Rindt and G.W. Oertel, A spot test for ketosteroids on thin-layer chromatograms, J. Chromatogr., 24 (1966) 480. 1077 A. Truswell and W.D. Mitchell, Separation of cholesterol from its companions,

186

REFERENCES

cholestanol and 7-cholestenol, by thin-layer chromatography, J. LipidRes., 6 (1965) 438-441. 1078 R. Tschesche, G. Biernoth and G. Wulff, Pyrene derivatives as fluorescent indicators for thin-layer chromatography, J. Chromafogr., 12 (1963) 342-346 (Ger.). 1079 R. Tschesche, G. Wulff and K.H. Richert, Thin-layer chromatography of steroids, in A.T. James and L.J. Morris (Editors),New Biochemical Separations, Van Nostrand, New York, 1964, pp. 197 245. 1080 K. Tsuda, N. Ikekawa, Y. Sat0 and R. Watanuki, Gas-chromatographicanalysis of oxidation products of adrenal corticosteroids,Anal. Biochem., 16 (1966) 183-1 88. 1081 K. Tsuda, Y. Sato, N. Ikekawa, S. Tanaka, H. Higashikuze and R. Osawa, Bile acids and bile alcohols. 2. Separation of bile acids by gas- liquid chromatography, Chem. Pharm. Bull. (Tokyo), 13 (1965) 720-723. 1082 Y.-C. Tung and K.-T. Wang, Polyamide-layer chromatography of estrogens, Nature (London), 208 (1965) 581-582. 1083 A. Uettwiller and M. Keller, A direct fluorometric quantitative routine thin-layer chromatographic method for determination of free cortisol, cortisone, corticosterone, and Compound S in plasma, J. Chromafogr., 35 (1968) 526-530 (Ger.). 1084 T. Usui, Thin-layer chromatography of bile acids with special reference to separation of keto bile acids, J. Biochem. (Tokyo), 54 (1963) 283-286. 1085 J . Vachek, B. KakiE and M. SarSCnovi, Determination of cholecalciferol (vitamin D3) in aqueous solutions on a thin layer, Cesk. Farm., 16 (1967) 229-231 (Czech). 1086 J. Vaedte, A. Gajewska and A. Czarnocka, Shortening of development time in thinlayer adsorption chromatography. Application to the separation of steroids, J. Chromatogr., 12 (1963) 208-211. 1087 G.V. Vahouny, C.R. Borja and S. Weening, Radioactive and analytical determinations of free and esterified cholesterol following micro thin-layer silicic acid chromatography,Anal. Biochem., 6 (1963) 555-559. 1088 L.B.S. Valle and R.M. de Oliveira-Filho, Application of a histochemical reaction to thin-layer chromatography of steroids,J. Chromatogr., 105 (1975) 201 -205. 1089 H. van Baelen, W. Heyns and P. de Moor, The separation of estrogens on Sephadex, J. Chromatogr., 30 (1967) 226-227. 1090 H. van Baelen, W. Heyns and P. de Moor, Measurement of urinary estrogens using adsorption on Sephadex,J. Clin. Endocrinol. Metab., 27 (1967) 1056-1058. 1091 G.P. van Berge Henegouwen, A. Ruben and K.H. Brandt, Quantitative analysis of bile acids in serum and bile, using gas-liquid chromatography, Clin. Chim. Acta, 54 (1974) 249-261. 1092 F.A. Vandenheuvel, Equipment and techniques for the quantitative recovery of steroids from thin-layer chromatography plates, J. Lab. Clin. Med., 69 (1967) 343350. 1093 F.A. Vandenheuvel, Precise location of steroids in thin-layer chromatoplates with marker dyes,J. Chromatogr., 38 (1968) 373-381. 1094 F.A. Vandenheuvel, Gas-liquid chromatographic studies of reactions and structural relationships of steroids. 1. Positions 3, 11, and 17 in the androstane series, J. Chromatogr., 96 (1974) 47-78. 1095 F.A. Vandenheuvel, Gas-liquid chromatographic studies of reactions and structural

REFERENCES

187

relationships of steroids. 2. Positions 3, 1 1, and 20 in the pregnane series, J. Chromatogr., 103 (1974) 113-134. 1096 F.A. Vandenheuvel, Gas-liquid chromatographic studies of reactions and structural relationships in steroids. 3. 1 la-Hydroxysteroids of the androstane and pregnane series,J. Chromatogr., 105 (1975) 359-375. 1097 F.A. Vandenheuvel and A.S. Court, Reference high-efficiency nonpolar packed columns for the gas-liquid chromatography of nanogram amounts of steroids. 1. Retention time data, J. Chromatogr., 38 (1968) 439-459. 1098 F.A. Vandenheuvel and A.S. Court, Reference high-efficiency nonpolar packed columns for the gas-liquid chromatography of nanogram amounts of steroids. 2 . Identification of steroids, J. Chromatogr., 39 (1969) 1-16. 1099 F.A. Vandenheuvel, G.J. Hinderks, J.C. Nixon and W.G. Layng, Precise ultramicro determination of steroid hormones by combined thin-layer chromatographic and gas-liquid chromatographic analysis, J. Amer. Oil Chem. SOC.,4 2 (1965) 283-290. 1100 W.J.A. VandenHeuvel, The gas-liquid chromatographic behavior of sterol sulfonates. Effect of structure on the nature of their elimination reaction, J. Chromatogr., 26 (1967) 396-403. 1101 W.J .A. VandenHeuvel, The gas-liquid chromatography of dimethylsilyl, trimethylsilyl and chloromethyldimethylsilyl ethers of steroids. Mechanism of silyl ether formation and effect of trimethylsilylation upon detector response, J. Chromatogr., 27 (1 967) 85-95. 1 102 W.J .A. VandenHeuvel, Gas-liquid chromatography of steroid glucuronosides, J. Chromatogr., 28 (1967) 406-408. 1 103 W.J.A. VandenHeuvel, Separation techniques for the gas-liquid chromatography of steroids, in R. Scholler and M.-F. Jayle (Editors), Gas-Phase Chromatography of Hormonal Steroids, Dunod, Paris, 1968, pp. 3-20. 1 104 W.J.A. VandenHeuvel, Further studies on the gas-liquid chromatographic behavior of sulfonate esters, J. Chromatogr., 43 (1969) 21 5-222. 1105 W.J.A. VandenHeuvel and K.L.K. Braly, The gas-liquid chromatographic behavior of methanesulfonates and mixed silyl ethers of bile acids, J. Chromatogr., 31 (1967) 9-19. 1106 W.J.A. VandenHeuvel and E.C. Horning, A study of retention-time relationships in gas chromatography in terms of the structure of steroids, Biochim. Biophys. Acta, 64 (1962) 416-429. 1107 W.J.A. VandenHeuvel and E.C. Horning, Gas-chromatographic characterization of steroid ketones as N,N-dimethylhydrazones, Biochim. Biophys. Acta, 74 (1 963) 560-563. 1108 W.J.A. VandenHeuvel and E.C. Horning, Analysis of steroids by gas-phase chromatography, Bull. SOC.Chim. Biol., 47 (1965) 945-977 (Fr.). 1109 W.J.A. VandenHeuvel and E.C. Horning, Conditions for the separation of steroids by gas-liquid chromatography,Mem. SOC.Endocrinol., 16 (1967) 39-68. 1110 W.J.A. VandenHeuvel, J.L. Smith, G. Albers-Schonberg, B. Plazonnet and P. BClanger, Derivatization and gas chromatography in the mass spectrometry of steroids, in E. Heftmann (Editor), Modern Methods of Steroid Analysis, Academic Press, New York, 1973, pp. 199-219.

188

REFERENCES

1111 P. van Hout, J. Szafranek, C.D. Pfaffenberger and E.C. Horning, Thermostable polarphase open-tubular glass capillary columns, J. Chromatogr., 99 (1974) 103 110. 1112 H.H. Varon, H.A. Darnold, M. Murphy and J. Forsythe, Comparison of methods of detection for free estrogens and estrogen acetates on thin-layer chromatograms, Steroids, 9 (1967) 507-516. 11 13 P. Vecsei, V. KemCny and A. GorgCnyi, Separation of corticosteroids by thin-layer chromatography on silica gel plates containing Blue Tetrazolium, J. Chromatogr., 14 (1964) 506-507. 1114 P. Vecsei, D. Lommer, H.G. Steinacker and H.P. Wolff, Quantitative estimation of corticosteroids by means of Blue [14C]-Tetrazolium, J. Chromatogr., 26 (1967) 533-549. 1115 J. Vessman and G. AhlCn, Quantitative gas chromatography of vitamin D2,Acta Pharm. Suec., 1 (1964) 209-218. 1116 P. Vestergaard, Automation in steroid-analytical procedures, Advan. Biosci., 2 (1968) 18-40. 1117 P. Vestergaard, Simultaneous multicolumn liquid-liquid and liquid-solid chromatography with a computerized read-out system, Clin. Chem., 16 (1970) 651-656. 1118 P. Vestergaard, Liquid column chromatography of hormonal steroids, in E. Heftmann (Editor), Modern Methods of Steroid Analysis, Academic Press, New York, 1973, pp. 1-35. 1119 P. Vestergaard, A. Bachman, T. Piti and M. Kohn, Multi-column liquid chromatography. 4. Computerized on-line spectrophotometric quantitation system for multicolumn liquid chromatography,J. Chromatogr., 111 (1975) 75-92. 1120 P. Vestergaard, L. Hemmingsen and P.W. Hansen, High-capacity multi-channel computerized read-out system for multi-column chromatography, J. Chromatogr., 40 (1969) 16-30. 1121 P. Vestergaard and E. Jacobsen, A system for simultaneous 25-column, capillary column liquid-solid chromatography, J. Chrornatogr., 50 (1970) 239-250. 1122 P. Vestergaard, E. Raabo and S. Vedse, Determination of urinary testosterone in men, women, and children, Clin. Chim. Acta, 14 (1966) 540-542. 1123 P. Vestergaard and J.F. Sayegh, Capillary Teflon columns for adsorption and partition chromatography, J. Chromatogr., 24 (1 966) 422-426. 1124 P. Vestergaard and J.F. Sayegh, Estimation of individual urinary corticosteroids by simultaneous multicolumn capillary column liquid-liquid chromatography, Advan. Automat. Anal., Technicon Int. Congr., 1969,l (1970) 327-332. 1125 D.W. Vidrine and H.J. Nicholas, Two-dimensional thin-layer chromatography of lipophilic compounds. Characterization of a mixed stationary phase permitting both adsorption thin-layer chromatography and reversed-phase partition thin-layer chromatography on one plate. J. Chromatogr., 89 (1974) 92-95. 1126 V. Vlasinich and J.B. Jones, Detection of steroids in thin-layer chromatography with p-toluenesulfonic acid, Steroids, 3 (1964) 707-710. 1127 W. Vlassak and G. Willems, Rapid separation of a-ketolsteroids by thin-layer chromatography,J. Pharm. Belg., 19 (1964) 195-199 (Fr.), 1128 J.A. Vollrnin, Gas-chromatographc separation of steroids on glass capillary columns, Chromatographia, 3 (1970) 233-237.

REFERENCES

189

1 129 J.A. Vollmin, Separation and identification of urinary steroids on glass capillary columns in a combination of gas chromatograph and mass spectrometer, Chromatographia, 3 (1970) 238-241. 1 130 J.A. Vollmin, High-resolution gas chromatography of urinary steroids o n glass capillary columns, Clin. Chim. Acra, 34 (1971) 207-214. 1131 J.A. Vollmin and H.C. Curtius, High-resolution gas chromatography of steroids, Z. Klin. Chem. Klin. Biochem., 9 (1971) 43-46 (Ger.). 1132 W. Volter, G. Jung, E. Breitmaier, G. Boucon, E. Bayer and D. Gupta, Comparison of the trifluoroacetyl and trimethylsilyl ether derivatives for gas chromatography of steroids,Anal. Chim. Acta, 53 (1971) 185-188. 1133 W. Vogt, I. Fischer and M. Knedel, Estrone dimethylphosphinic esters, a new type of derivatives for the sensitive gas-chromatographic determination of steroids, 2. Anal. Chem., 267 (1973) 28-30 (Ger.). 1134 W. Vogt, K. Jacob and M. Knedel, Highly sensitive and selective gas-chromatographic determination of monohydroxysteroids as phosphinic esters with the alkali-flame detector, J. Chromatogr. Sci., 12 (1974) 658-661. 1135 B. de Vries, Quantitative separations of lipid materials by column chromatography on SiOz impregnated with AgN03, Chem. Znd. (London), (1962) 1049 1050. 1136 H.E. Vroman and G.L. Baker, Limits of detection of some lipids in thin-layer chromatography,J. Chromatogr., 18 (1965) 190-191. 1137 H.E. Vroman and C.F. Cohen, Separation of sterol acetates by column and thinlayer argentation chromatography, J. Lipid Res., 8 (1967) 150-1 52. 1 138 H. Wagener and B. Frosch, Two-dimensional thin-layer chromatographic separation of cholic acids, Klin. Wochenschr., 41 (1963) 1094-1095 (Cer.). 1139 S. van der Wal and J.F.K. Huber, High-pressure liquid chromatography with ionexchange celluloses and its application to the separation of estrogen glucuronides, J. Chromatogr., 102 (1974) 353-374. 1140 W. Waldhausl, H. Haydl and H. Frischauf, Determination of aldosterone by Sephadex LH-20 chromatography and radioimmunoassay, Steroids, 20 ( 1972) 727736. 1141 B.L. Walker, A novel charring technique for detection of lipids on thin-layer chromatograms,J. Chromatogr., 56 (1971) 320-323. 1142 M.-C. Wang and T.-H. Chou, Analysis of Digitalis leaves. 1. Thin-layer chromatography of Digitalis glycosides, Yao Hsueh Hsueh Pao, 12 (1965) 720-726 (Chin.). 1143 J. Washuttl and M. Gemeiner, A semi-quantitative determination of vitamin D in horse and swine blood serum, Mikrochim. Acta, (1974) 73-79 (Ger.). 1144 D.J. Watson and D. Bartosik, Thin-layer partition chromatography of steroids using volatile stationary phases,J. Chromatogr., 54 (1971) 91-95. 1 145 D.J. Watson and E.B. Romanoff, Resolution of steroid 2,4-dinitrophenylhydrazone isomers by thin-layer chromatography, J. Chromatogr., 22 (1966) 192-194. 1146 E. Watson, D.R. Clark and S.M. Kalman, Identification by gas chromatographymass spectroscopy of dihydrodigoxin, a metabolite of digoxin in man, J. Pharm. Exp. Therap ., 184 (1973) 424-43 1. 1147 E. Watson and S.M. Kalman, Assay of digoxin in plasma by gas chromatography, J. Chromatogr., 56 (1971) 209-218.

190

REFERENCES

1148 E. Watson, P. Tramell and S.M. Kalman, Identification of submicrogram amounts of digoxin, digitoxin, and their metabolic products. Isolation by chromatography and preparation of derivatives for assay by electron-capture detector, J. Chromatogr., 69 (1972) 157-163. 1 149 J.T. Watson, A simplified determination of urinary testosterone utilizing column and gas-liquid chromatography, J. Chromatogr., 43 (1969) 339--349. 1150 D.J. Weber, T.R. Ennals and H. Mitchner, Versatile system for partition chromatography of corticosteroids and prediction of their elution curves, J. Pharm. Sci., 6 1 (1972) 689-694. 1151 P.A.M. Weiss, Quantitative determination of estriol from urine of pregnant women by chromatogram spectrophotometry, Geburtsh. Frauenheilk., 30 (1 970) 6 2 3 4 2 9 (Ger .). 1152 P.A.M. Weiss, Simple quantitative determination of estrone, estradiol, and estriol by thin-layer chromatography and chromatogram spectrophotometry and its practical application for the determination of estriol in the urine of pregnant women, Endokrinologie, 59 (1972) 273-278 (Ger.). 1153 P.A.M. Weiss, Determination of progesterone in the plasma of pregnant women by means of chromatogram spectrophotometry, Endokrinologie, 61 (1973) 1 8 (Ger.). 1154 N.T. Werthessen, J.R. Beall and A.T. James, Semi-automatic chromatographic determination of neutral lipids, J. Chromatogr., 46 (1970) 149-160. 1155 K.J. Whittle, P.J. Dunply and J.F. Rennock, Detection of lipids on reversed-phase chromatograms with fluorescein, Chem. Znd. (London),(1966) 1303. 1156 W.H.J.M. Wientjens, R.A. de Zeeuw and J. Wijsbeek, Separation of sterols by vaporprogrammed thin-layer chromatography, J. Lipid Res., 11 (1970) 376-377. 1157 P. Wierzchowski, Z. Wierzchowska, W. Poludnikiewicz and L. Konarska, Analysis of urine 17-ketosteroidsby column chromatography on silica gel, Biochem. Med., 5 (1971) 399-403. 1158 W.G. Wiest, A double isotope derivative assay for progesterone and 20a-hydroxypregn-4-en-3-one, Steroids, 10 (1967) 257-278. 1159 J.H. Williams, M. Kuchmak and R.F. Witter, Evaluation of the purity of cholesterol primary standards, Clin. Chem., 16 (1970) 423-426. 1160 R.C. Williams, J.A. Schmit and R.A. Henry, Quantitative analysis of the fat-soluble vitamins by high-speed liquid chromatography, J. Chromatogr. Sci., 10 (1 972) 494501. 1161 P.W. Wilson, D.E.M. Lawson and E. Kodicek, Gas-liquid chromatography of ergocalciferol and cholecalciferol in nanogram quantities, J. Chromatogr., 39 (1969) 75-77. 1162 W.E. Wilson, S.A. Johnson, W.H. Perkins and J.E. Ripley, Gas-chromatographic analysis of cardiac glycosides and related compounds, Anal. Chem., 39 (1967) 40-44. 1163 W.E. Wilson and J.E. Ripley, Role of induction forces interactions in effecting gaschromatographic separation of cardiac glycoside trimethylsilyl ethers, Anal. Chem., 41 (1969) 810-815. 1164 J.V. Wisniewski and S.F. Spencer, New liquid phase for the analysis of steroids, J. Gas Chromatogr., 2 (1 964) 34-35.

REFERENCES

191

1165 0. Worsdorfer, G. Diedrichsen and D. Lommer, Gas-chromatographic method for the estimation of secretion and excretion rates of aldosterone, 2.Klin. Chem. Klin, Biochem., lO(1972) 555-561 (Ger.). 1166 L. Wolfman and B.A. Sachs, Separation of cholesterol and desmosterol by thinlayer chromatography, J. Lipid Res., 5 (1964) 127--128. 1167 J. Wollenweber, B.A. Kottke and C.A. Owen, Jr., Quantitative thin-layer chromatography of chenodeoxycholic acid and deoxycholic acid in human duodenal contents, J. Chromatogr., 24 (1966) 99-105. 1168 H.G.J. Worth and M. MacLeod, Use of a liquid chromatograph in lipid class separation, J. Chromatogr., 40 (1969) 31-38. 1169 B. Wortmann, W. Wortmann and J.C. Touchstone, The use of polyamide for thinlayer chromatographic separation of steroids, J. Chromatogr., 70 (1972) 199-201. 1170 W. Wortmann, C. Schnabel and J.C. Touchstone, Quantitative determination of corticosteroids from plasma by high-pressure liquid chromatography, J. Chromatogr., 84 (1 973) 396-401. 1171 W. Wortmann and J.C. Touchstone, Techniques for determination of specific activity for isotopic materials by thin-layer chromatography, in J.C. Touchstone (Editor), Quantitative Thin-Layer Chromatography,Wiley, New York, 1973, pp. 23-44. 1172 W. Wortmann, B. Wortmann, C. Schnabel and J.C. Touchstone, Rapid determination of estriol of pregnancy (urine) by spectrodensitometry of thin-layer chromatograms, J. Chromatogr. Sci., 12 (1974) 377-379. 1173 H.H. Wotiz, Steroid metabolism. 15. Rapid determination of urinary pregnanediol by gas chromatography, Biochim. Biophys. Acta, 69 (1963) 415-416. 1174 H.H. Wotiz, Steroid metabolism. 14. Modification of the analysis of urinary estrogens by gas chromatography, Biochim. Biophys. Acta, 74 (1963) 122-126. 1175 H.H. Wotiz and S.C. Chattoraj, Determination of estrogens in low- and high-titer urines using thin-layer and gas-liquid chromatography, Anal. Chem., 36 (1964) 1466-1 472. 1176 H.H. Wotiz and S.C. Chattoraj, The role of gas-liquid chromatography in steroid hormone analysis, J. Chromatogr. Sci., 11 (1973) 167-1 74. 1177 H.H. Wotiz and S.J. Clark, Gas ChromatographicAnalysis of Steroid Hormones, Plenum Press, New Yo-rk, 1966. 1178 H.H. Wotiz and S.J. Clark, Developments in the analysis of steroids by gas chromatography,Methods Biochem. Anal., 18 (1970) 339-372. 1 1 79 R.S. Wright, Resolution of some pairs of closely relaled steroids by thin-layer chromatography, J. Chromafogr.,37 (1968) 363-364. 1180 R.S. Wright, Reagent for the nondestructive location of steroids and some other lipophilic materials on silica gel thin-layer chromatograms, J. Chromatogr., 59 (1971) 220-221. 1181 F.S. Wusteman, K.S. Dodgson, A.G. Lloyd, F.A. Rose and N. Tudball, Thin-layer chromatography in the study of ester sulfates,J. Chromatogr., 16 (1964) 334-339. 1182 H. Wyman and 1.F. Sommerville, Description and evaluation of a simple technique for the determination of plasma progesterone by thin-layer and gas-liquid chromatography, Steroids, 12 (1968) 63-86. 1183 C.R. Wynne-Roberts and D.L. Davis, Evaluation of centrifugal chromatography. 1.

192

REFERENCES

Separation of steroid hormones on silica gel, J. Chromatogr. Sci., 11 (1973) 406410. 1184 A. Yamamoto and G. Rouser, Quantitative analysis of bile lipids by dextran gel column chromatography and thin-layer chromatography, Biochim. Biol. Sper., 6 (1967) 135-145. 1185 M.E. Yannone, D.B. McComas and A. Goldfien, The assay of plasma progesterone, J. Gas Chromatogr., 2 (1964) 30-33. 1186 M.E. Yannone, J.R. Mueller and R.H. Osborn, Improved method of steroid analysis by gas-liquid chromatography, Chromatographia, (1970) 13-16. 1187 K. Yasuda, Gas-chromatographic analysis of steroid hormones. 1. Problems in quantitative aspects of gas chromatography,NagoyaJ. Med. Sci., 29 (1967) 191230. 1188 K. Yasuda, Gas-chromatographic analysis of steroid hormones. 2. Gas-chromatographic behavior of trimethylsilyl ethers,Nagoya J. Med. Sci., 30 (1967) 269-307. 1189 1.1. Zaretskaya, L.M. Kogan, O.B.Tikhomirova and I.V. Torgov, Thin-layer chro-

matography of steroids on microplates coated with silica gel, Khim. Mr. Soedin., 2 (1966) 321-325 (Russ.); Chem. Natl. Prod., (1966) 261-264 (Engl. Transl.). 1190 H.-J. Zeitler, Separation of Veratrum alkaloids by thin-layer chromatography, J. Chromatogr., 18 (1965) 180-183 (Ger.). 1191 T. Ziminski and E. Borowski, New spray reagent replacing sulfuric acid in thinlayer chromatography, J. Chromatogr., 23 (1966) 480-482. 1192 A. Zmigrod and H.R. Lindner, Gas-chromatographic separation of ketosteroids as ethylene thioketal derivatives,Steroids, 8 (1966) 119-132. 1193 G . Zullich, W. Braun and B.P. Lisboa, Thin-layer chromatography for the separation of digitoxin, digitoxigenin and related compounds, J. Chromatogr., 103 (1975) 396-401. 1194 J. Zurkowska, M. %ukaszewski and A. Ozarowski, Cardenolide glycosides. 3.

Qualitative and quantitative thin-layer chromatography of glycosides from Digitalis purpurea L.,Acta Pol. Pharm., 20 (1963) 99-104. 1195 J. Zurkowska and A. Oiarowski, Quantitative thin-layer chromatography of a mixture of lanatosides A, B, C, and D on talc,PZantaMed., 12 (1964) 222-227 (Ger.). 1196 J. Zurkowska and A. Ozarowski, Cardenolide glycosides. 13. Chromatographic separation of helveticoside, corchoroside, and strophanthidin, Acra Pol. Pharm., 23 (1966) 59-62.

Subject index

A Acetates 29, 67 Acovenosigenin 122 Adrenocortical hormones, see Corticosteroids Adrenosterone 21,49, 88 Aldadiene 108 Aldosterone 8,99-106 Alkaloids, C,, 23, 95,96 _ _ _ , C,, 23,117,120 Alkamines, see Alkaloids Allopregnanediol 38,52,94,108 Allopregnanolone 13, 21, 35,48,49,52,94 Allotetrahydrocorticosterone 104, 108 Allotetrahydrocortisol 104, 108 Allylestrenol 110 Alumina 3, 15 Androgens, see Androstane derivatives 1,4-Androstadiene-3,17-dione 20, 34, 88 5J6-Androstadien-3p-01 89 1,4-Androstadien-3-one,17phydroxy- 20, 88 _ _ _ , 17p-hydroxy-l7-methyl-, see Methandienolne 4,6-Androstaddien-3-one, 17p-hydroxy- 88 Androstane derivatives 87-92, 107, 109 _ _ _ blood 91, 92 _ _ _ GC 53,90-92 -_- HPLC 10,87 _ _ _ LC 87 _ _ - TLC 20,21,87-91 __- , urinary 89,91, 92 So-Androstane 4, 50, 90 5p-Androstane 90 Sa-Androstane-3p,l6pdiol 20, 49 5a-Androstane-3a,l 7pdi01, see Dihydroan droster o ne 5a-Androstane-3p,17p-diol 20, 49,52, 88, 90 5p-Androstane-3a,l7pdiol 88,90 5p-Androstane-3p,l7pdiol 88,90 Sa-Androstane-3,16-dione 20, 49 Sa-Androstane-3,17-dione 20,48,49, 52, 88, 90 _ _ _ , llp-hydroxy- 88 5p-Androstane-3,17-dione 20,48,88,90 5a-Androstane-ll,17-dione,3a-hydroxy; see 11-Ketoandrosterone 5PAndrostane-ll,l’I-dione, 3a-hydroxy-, see 11-Ketoetiocholanolone

193

Sa-Androstane-3a,l lp,l7p-triol 88 Sp-Androstane-for,llp,l7p-triol 88 Sa-Androstane-3,11,17-trione21 5P-Androstane-3,11,17-trione 88 5a-Androstan-301-01 20 5a-Androstan-30-01 20, 90 Sp-Androstandp-01 90 Sa-Androstan-1701-01 6 5a-Androstan-17p-01 4, 6, 20, 90 5pAndrostan-170-01 90 Androstanolone 35,88,90 5a-Androstan-3-one 90 _ _ _ , 17a-hydroxy- 90 _ _ _ , 170-hydroxy-,see Androstanolone Sp-Androstan-3-one 90 - _ _ , 17a-hydroxy- 90 _ _ - , 17p-hydroxy- 20, 88, 90 Sa-Androstan-16-one, 3p-hydroxy- 20 5a-Androstan-17-one 4, 20,49,52, 90 _ _ _ , 3tuJ lpdihydroxy-, see 11-Hydroxyandrosterone --- , 3p,llpdihydroxy- 35,89 _ _ - , 3a-hydroxy-, see Androsterone _-- , 3P-hydroxy; see Epiandrosterone $3-Androstan-1Tone 90 _ _ _ , 3a,l lp-dihydroxy-, see 1lp-Hydroxyetiocholanolone _ _ _ , 3a-hydroxy-, see Etiocholanolone -_- , 3Phydroxy- 6, 13, 88,90 1,4,6-Androstatriene-3,17-dione88 1,4,6-Androstatrien-3-one, 17p-hydroxy- 88 4-Androstene-3p,l7p-diol20,88 5-Androstene-3p,16pdiol 20 5-Androstene-3p,l7adiol 20, 35, 88 5-Androstene-3p,17p-diol 20, 35,49, 52, 88 _ _ _ , 17a-methyl-, see Methandriol l-Androstene-3J7-dione 20, 88 4-Androstene-3,16-dione 20, 49 4-Androstene-3,17-dione 20, 34,48,49, 52, 88 __- , 6tu-hydroxy- 88 _-- , 6p-hydroxy- 88 -_- , lla-hydroxy- 49,88 -__ , 1lp-hydroxy- 21,49,52,88 _ _ _ , 16a-hydroxy- 88 5-Androstene-l1,17-dione,30-hydroxy- 89 5-Androstene-3p,1lp,l7p-triol 88 5-Androstene-3p,l6a,l7a-triol35

194

4-Androslene-3,11,17-trione, see Adrenosterone 5a-Androst-l6en-3a-01 89 5a-Androst-16en-30-01 89 5p-Androst-16en-3a-ol 89 Androstenolone, see Dehydroepiandrosterone 4-Androsten-3-one, 6pJ7pdihydroxy- 89 _-- , lla,l7a-dihydroxy- 21 _ _ _ , lla,l7pdihydroxy- 89 ---, llp,l7p-dihydroxy- 89 _ _ _ , 16a,l7pdihydroxy- 89 -_- , 17aethynyl-l7-hydroxy-6a,2 1-dimethyl-, see Dimethisterone _ _ _ , 17a-hydroxy-, see Epitestosterone , 170-hydroxy-, see Testosterone 4-Androsten-17-one, 3a-hydroxy- 88 _ _ _ , 3P-hydroxy- 88 5-Androsten-ll-one, 3p,l7pdihydroxy- 89 5-Androsten-16-one, 3p,l7p-dihydroxy- 2 1 5-Androsten-17-one, 3p,l ladihydroxy- 89 _ _ _ , 3p,l lpdihydroxy- 89 _ _._ , 3p,16cudihydroxy-, see 16-Hydroxydehydroepiandrosterone _-- , 3a-hydroxy- 8 8 _-- , 3P-hydroxy-, see Dehydroepiandrosterone Sa-Androst-len-3-one, 170-hydroxy- 20, 88 Androsterone 6, 7, 13, 20, 23, 35, 38,49,S2, 88-91,108 --- glucosiduronate 25, 26 _ _ _ sulfate 25, 26 Argentation chromatography 15, 55,56, 5 9 , 6 0 Avenasterol, see 28-lsofucosterol

B Benzoates 11 Benzyloximes 33 Betamethasone 111 BiIe acids I 1 - 77 _ _ _ , fecal 75 _ _ _ GC 74-71 _ _ _ LC 5 , 6 , 7 1 _ _ _ methyl ethers 74, 75 _ _ - TLC 22,71-74 Bile alcohols 7 1, 73 Botogenin, see Gentrogenin Brassicasterol 59,63, 6 4 , 6 6 Bufadienolides 121, 123 Bufogenins, see Bufadienolides

C Campestadienol 63

SUBJECT INDEX Campestanol 6 3 , 6 4 Campesterol 5 6 , 5 8 , 5 9 , 6 3 , 6 4 , 6 6 , 67 5cu,l4pCard-20(22)~nolide,3@,14-dihydroxy-, see Uzarigenin 5P,14pCard-20(22)enolide, 3p,14-dihydroxy-, see Digitoxigenin _ _ _ , 16p-formoyloxy-3p,l4-dihydroxy-,see Gitaloxigenin

_ _ _ , 3p,5,14,194etrahydroxy-,see Strophanthidol

__- , lp,3p, 14-trihydroxy-, see Acovenosigenin _ _ _ , 3p,5,14-trihydroxy-, see Periplogenin

_ _ _ , 3p,l la,Irl-trihydroxy-, see Sarmentogenin _ _ _ , 3@,12p,l4-trihydroxy-,see Digoxigenin

_-- , 3!3,14,16f3-trihydroxy-,see Gitoxigenin _ _ _ , 3p,5,14-trihydroxy-19-oxo-, see Strophanthidin Cardenolides 22, 121-123 Cardiac genins, see Cardenolides, Bufadienolides _ _ _ glycosides 121-123 CentriChrom 10 Chalinasterol, see 24-Methylenecholesterol Chenodeoxycholic acid 22,12, 76, 77 Chiapagenin 118 Chlormadinone acetate 110 16aChloroestrone methyl ether 108 Chlorogenin 118 Chloromethyldimethylsilyl ethers 32 SwCholanic acid 77 5PCholanic acid 22, 76, 77 SaCholan-24-0ic acid, see 5aCholanic acid __- , 3a,ladihydroxy- 77 _-- , 3p,7adihydroxy- 77 __- , 3a,12adihydroxy- 77 _ _ _ , 3P,12~~-dihydro~y71 _ _ _ , 7a,l2adihydroxy- 77 _ _ _ , 3a,12adihydroxy-7-0~0- 7 1 _ _ _ , 3p,12adihydroxy-7-0~0- 77 _ _ _ , 7e,I 2a-dihydroxy-3-0x0- 77 _ _ _ , 3,7-dioxo- 77 _-- , 3,12-dioxo- 77 _-- , 3a-hydroxy- 77 _ _ _ , 3p-hydroxy- 77 _ _ _ , 7a-hydroxy- 77 _ _ _ , 7p-hydroxy- 77 _-- , 12a-hydroxy- 77 _ _ _ , 12a-hydroxy-3,7-dioxo- 77 _-- , 3a-hydroxy-7-0x0- 77 _-- 7 ~ ~ - h y d r O ~ y - 3 - 077 ~0_-- , 12a-hydroxy-3-0x0- 77 _-- , 12a-hydroxy-7~x0- 77 _-- , 50x0- 77 _ _ _ , 3a,6@,7p-trihydroxy- 77 _ _ _ , 3a,7a,12or-trihydroxy- 77 9

SUBJECT INDEX

195

-_--_

, 3p,7a,l2a-trihydroxy- 77 , 3,7,12-trioxo- 17 SpCholan-24-oic acid, see 5pCholanic acid

--_ _-_

-__

, 3a,6a-dihydroxy-, see Hyodeoxycholic acid --_ , 3a,6p-dihydroxy- 76, 77 --- , 3a,7a-dihydroxy-, see Chenodeoxycholic acid -__ , 3a,7pdihydroxy- 72,76, 77 --_ , 3P,7wdihydroxy- 7 1 --_ , 301,12a-dihydroxy-, see Deoxycholic acid - _ _ , 3p,l2a-dihydroxy- 1 7 --_ , 7a,l2a-dihydroxy- 77 --_ , 3a,7a-dihydroxy-12-oxo- 76, 77 --_ , 3a,l2adihydroxy-7-oxo- 72, 76, 77 _ _ _ , 7a,l2a-dihydroxy-3-0~0- 77 --_ , 3,7-dioxo- 76,17 - - _ , 3,12-dioxo- 76, 77 --_ , 3a-hydroxy-, see Lithocholic acid --_ , 3p-hydroxy-, see lsolithocholic acid --_ , 7a-hydroxy- 77 _ _ _ , 7p-hydroxy- 77 _ _ _ , 12a-hydroxy- 76, 7 1 --_ , 120-hydroxy- 71 --_ , 3a-hydroxy-7,12-dioxo- 72,76, 77 --_ , 12a-hydroxy-3,7-dioxo- 77 --_ , 3a-hydroxy-7-0x0- 16, 77 --_ , 3a-hydroxy-12-0x0- 72, 76, 77 _-- , 7whydroxy-3-oxo- 17 - _ _ , 12~-hydroxy-3-0~0-76, 77 _-_ , 12a-hydroxy-7-0x0- 77 __- , 3-0x0- 76, 77 _-_ , 3a,6a,7a-trihydroxy-, see Hyocholic acid -__ , 3a,6&,7@-trihydroxy- 76, 77 _-_ , 3a,6(3,7a-trihydroxy- 76, 77 _ _ _ , 3a,6p,7p-trihydroxy- 76, 77 --_ , 3a,7a,12a-trihydroxy-, see Cholic acid --_ , 3a,7&12a-trihydroxy- 77 --_ , 3p,70r,l2~~-trihydroxy-77 --_ , 3,7,12-trioxo-, see Dehydrocholic acid 5p-Cholan-7a-ol 6 5pCholan-70-01 6 Cholecalciferol 113, 114 Cholecalciferols, hydroxy- 114 3,SCholestadiene 22, 57, 6 2 Sa-ChoIesta-7,22-dien-3p-ol 6 3 _ _ _ , (24R)-24-ethyl-, see d p i n a s te r o l --_ , (24S)-24-ethyl-, see Chondrillasterol 5a€holesta-7,24-dien-3p-o1 56 Sor€hoIesta-8,14-dien-3p-ol 64 5aCholesta-8,24-dien-3p-ol,see Zymosterol 5,7Cholestadien-3p-o1, see 7-Dehyd~ocholesterol ---, (24R)-24-methyl-, see Campestadienol 5,22-Cholestadien-3p-ol, see 22-D~hydrocholesterol

5,24Cholestadien-3p-ol, see Desmosterol 5,25Cholestadien-3p-ol, see 25-Dehydrocholesterol 1,4Cholestadien-3-one 22, 5 7 ,6 2 3,5Cholestadien-7-one 5 7 ,6 2 4,6Cholestadien-3-one 22,57 5aCholestane 4 , 5 0 , 6 8 5pCholestane-3a,l2a-diol 4 Sp€holestane-3,12-dione 4 5~Cholestane-3or,7a,l2a,24a-tetro:73 Sp€holestane-3a,7a,l2a,24p-tetrol 73 5pCholestane-3a,7a,l2a,26-tetrol 73 5a-Cholestane-3p,5a,6p-triol 57, 62 5p-Cholestane-3a,7a,l2a-triol4 , 7 3 Sp-Cholestane-3,7,12-trione 4 Cholestanol 4 ,6 , 7, 16, 22, 34,40, 49,52, 55, 57,59-64,66 5aCholestan-3a-ol, see Epicholestanol 5a-Cholestan-30-01, see Cholestanol _-_ , 5a,6a-epoxy- 22 _ _ _ , (24R)-24-mcthyl-, see Campestanol --- , (24S)-24-methyl-, see Ergostanol SpCholestan-3a-ol, see Epicoprostanol 5pCholestan-3p-ol, see Coprostanol 50-Cholestan-70-01 59 Cholestanone 4 ,6 , 22,49, 52 Sa-Cholestan-3-one, see Cholestanone 5pCholestan-3-one, see Coprostanone 5aCholestan-6-one, 3p,Sadihydroxy- 5 7 , 6 2 _-- , 30-hydroxy- 22, 5 7 ,6 2 5,7,22Cholestatrien-3p-ol 64 _-- , (24S)-24-methyl-, see Ergosterol 5,7,22(9p,lOa)€holestatrien-3p-ol, (24R)-24methyl-, see Lumisterol, 5,22,24Cholestatrien-3p-ol 64 SCholestene, 3p-methoxy-, see Cholesterol methyl ether 5Cholestene-3p,4pdiol 22, 57,62 5Cholestene-3P,7adiol 6, 5 7 ,6 2 5-Cholestene-3p,7p-diol 6 , 5 7 ,6 2 5Cholestene-3p,24a-diol, see 24a-Hydroxycholesterol 5-Cholestene-3p,24pdiol,see 240-Hydroxycholesterol 5aCholest-8ene-3p,6adiol, see Peniocerol _-_ , 14a-methyl-, see Macdougallin 4Cholestene-3,6-dione 2 2 ,5 7 ,6 2 SChoIestene-16,22-dione, 3p,26-dihydroxy-, see Kryptogenin 4Cholesten-3p-ol 59 5Cholesten-3a-ol, see Epicholesterol

, (24R)-24-ethyl-, see Stigmasterol , (24S)-24-ethyl-, see Poriferasterol _ - _ , (24S)-24-methyl-, see Brassicasterol

196 SCholesten-3p-ol, see Cholesterol -__ , (24R)-24ethyl-, see Sitosterol _-- , (24S)-24ethyl-, see Clionasterol _-- , (E)24(28)ethylidene-, see Fucosterol _-- , (Z)24(28)-ethylidene-, see 28-lsofucosterol ---, (24R)-24-methyl-, see Campesterol __- , 24(28)-methylene-, see Methylenecholesterol (22R ,23R,24R)-5Cholesten-3&01, 22,23methylene-23,24-dimethyl-, see Gorgosterol ---, 22,23-methylene-24-methyl-, see 23-Demethylgorgosterol 5-Cholesten-3p-ol, 7-0x0-, see 7-Ketocholesterol 5aCholest-7en-3p-01, see Lathosterol _-- , (Z)24(28)-ethylidene-, see Citrostadienol --_ , 4a-methyl-, see Lophenol _-- , 14amethyl- 64 SaCholest-7-en-3p-ol, (24S)-24ethyl-, see 7-Chondrllasten-30-01 50Cholest-7en-3po1, see 7Coprostenol Sa-Cholest-8en-30-01 56,64 __- , l4a-methyl- 64 5aCholest-8(14)-en-3pol 63 S~Cholest-d(l4)-en-3p-ol,see 8(14)-Coprostenol 5or-Cholest-14en-3p-ol 63 4-Cholesten-3-one 6, 22,49, 52, 68 5-Cholesten-3-one 6, 22, 57,62 5Cholesten-7-one, 30-hydroxy- 22, 57, 62 hexahydroxy-, see Pterosterone

-_- , 2p,3&14,20,22,25-hexahydroxy-,see Ecdysterone

_-- , 2&30,14,20,22,26-hexahydroxy-,see Inokosterone

_-- , 2a,3a,l4,20,22-pentahydroxy-,see Ponasterone B

-__ , 2p,3P,14,20,22-pentahydroxy-,see Ponasterone A Cholesterol 3,5-7, 13,22, 23,34, 37,49, 52, 55-64,66,68,108,113 -_- blood 61,68,69 -__ methyl ether 4 _-_ oxidase 29 Cholesteryl esters 3-5, 56-58,61 Cholic acid 22, 72, 76, 77 7-ChondrilIasten-3po1 65 Chondrillasterol 63,65 Citrostadienol 65 Clionasterol 65 Coated capillaries 37-41, 108 Compound A, see Dehydrocorticosterone Compound B, see Corticosterone Compound E, see Cortisone Compound I:, see Cortisol

SUBJECT INDEX Compound S, see Deoxycortisol Conjugates 101, 107 Continuous development 17 Convallamarogenin 118 Coprostanol 4,6, 22,40,49,57,59,62-64, 68 Coprostanone 4, 22,68 7Coprostenol 64 8(14)-Coprostenol 64 Cortexolone, see Deoxycortisol Cortexone, see Deoxycorticosterone Corticosteroids 8, 10, 11, 99-106 --- blood 100, 103, 105 _-- GC 103,105,106 _ _ _ HPLC 100,110 --- LC 99,100 _-- pc 101 --- TLC 21,22,101-104, 111 _-- , urinary 103, 105 Corticosterone 8, 21, 99-102, 104 Cortisol 8, 21, 22, 99, 100, 102, 104, 111 Cortisone 8,21, 99, 100, 102, 104, 111 (a)Cortol 22 (or)Cortolone 21, 108 PCortolone 21, 108 Crustecdysone, see Ecdysterone Cyasterone 115 Cycloartanol 65 Cycloartenol 65 3,5Cyclocholestan-6-one 22 9p,19-Cyclo-5a-cholestan-3p-ol, 14a-methyl-, see Pollinastanol Cycloeucalenol 65 9p, 19Cycl0-5crlanostan-3p-ol, see Cycloartanol _-- , 24-methyl-, see 24-Methylcycloartanol _-- , 24-methylene-, see 24-Methylenecycloartanol see Cyclo9p, 19-Cyclo-5orlanost-24-en-3~-ol, artenol 9p,19-Cyclo-5c~-lanost-25en-30-01,(24s)-24methyl-, see Cyclolaudenol Cyclolaudenol 65 90, 19Cyclo-31-nor-5a-lanostan-30-1, 24-methylene, see Cycloeucalenol Cyclopamine, see 1 1-Deoxojervine

D 7-Dehydrocholesterol 22, 34,56,57,59-62, 64,113 22-Dehydrocholestero1 59,63,64 24-Dehydrocholesterol, see Desmosterol 25-Dehydrocholesterol 59,64,66

SUBJECT INDEX Dehydrocholic acid 72,76,77 11-Dehydrocorticosterone 8,21, 102, 104 Dehydroepiandrosterone 6, 13,20, 35, 38, 49,52,88-92 _ _ _ glucosiduronate 25, 26 _ _ _ sulfate 25, 26, 91 23-Demethylgorgosterol 65 Demissidine 49,120 Densitometry 24 11-Deoxojervine 120 Deoxycholic acid 22, 72,76, 77 Deoxycorticosterone 8,21,99, 102, 105, 111 Deoxycortisol 8,21,99, 101, 102, 105 24-Deoxyscymnol 73 Derivative ratio analysis 44 Derivatives 25-27, 29-33,43 Desmosterol 22,49,56,59-64,66 Desoxy-, see DeoxyDexamethasone 111 Digilanides, see Lanatosides Diginatigenin 122 Digitogenin 118 Digitoxigenin 22,121, 122 Digitoxin 121, 122 Digoxigenin 22, 122 Digoxin 122 Dihydroandrosterone 20, 35,49,52, 88, 90, 91 17a-Dihydroequilenin 20 17p-Dihydroequilenin 80 17p-Dihydroequilin 80,82 Dihydrotestosterone, see Androstanolone 24-Dihydrolanosterol 22,64 24-Dihydroobtusifoliol 64 7a,l la-Dihydroxytomatidine 120 9a,l la-Dihydroxytomatidine 120 Dimethisterone 108, 110 Dimethylhydrazones 33 Dimethylsilyl ethers 32 2,4-Dinitrophenylhydrazones 11, 26,89 Diosgenin 23, 118

E Ecdysones, see Molting hormones Ecdysterone 115 Elatography 26 Electron-capture detector 4 1 Epiandiosterone 6,7, 13,35,49,52, 88, 90 Epicholestanol 6 , 7 , 4 0 , 4 9 , 5 2 , 5 7 , 6 1 , 6 2 , 6 4 Epicholesterol 6 , 7 Epicoprostanol 6,22,40,59 16-Epiestriol 20, 80,82,85

197 17-Epiestriol 82, 85 16,17-Epiestriol 80,82, 85 Epipregnanolone 6,13,35,94 Epitestosterone 35, 38,87,88 Equilenin 20,49,80-82 Equilin 20,80-82,86 Ergocalciferol 113, 114 5,7-Ergostadien-3p-o1 64 Sa-Ergosta-7,22-dien-3p-o16 3,64 _-_ , 14a-methyl- 64 5a-Ergosta-7,24(28)dien-3p-o1 64 _ _ _ , 4a-methyl-, see 24-Methylenelophenol 5a-Ergosta-8,14dien-3p-o1 64 _ _ _ , 4a-methyl- 65 5or-Ergosta-8,22-dien-3p-ol,l4a-methyl- 64 5a-Ergosta-8,24(28)dien-3&01 64 _ _ _ , 4a,14adlimethyl-, see Obtusifoliol _ _ _ , l4a-methyl- 64 5,22-Ergostadien-3&01,see Brassicasterol 5,24-Ergostadien-3p-o1 63 5,24(28)-Ergostadien-3P-o1,see 24-Methylenecholesterol Ergostanol 66 5,7,(E)22-Ergostatrien-3p~l, see Ergosterol 5a-Ergosta-7,9( 11),22-trien-3p-o1 64 5a-Ergosta-7,14,22-trien-3p-o1 64 9p,l Oa-Ergostad ,7,22-trien-3p-ol, see Lumisterol, 5-Ergosten-30-01 64 5a-Ergost-7en-3p-ol 64 -__ , 14a-methyl- 65 5a-Ergost-8en-3p-ol 64 _ _ _ , 4a,l4adlimethyl-, see 24-Dihydroobtusifoliol --_ , 4a-methyl- 65 _ _ _ , 14a-methyl- 64 5a-Ergost-8(14)en-3p-ol 63,64 Ergosterol 22,34,58,63,64,66, 113, 114 Ergosteryl esters 58 Estetrol 86 Estradiol, see Estradiol-170 _ _ _ , benzoate 110 ___ , glucosiduronates 79 ___ , sulfates 79 Estradiol-l7a 20, 79,80,82,85 Estradiol-17P 4, 20, 35,49,52, 79, 80,82-85, 110 1,3,5( 10),6,8-Estrapentaene-3,1Ila-diol, see 17a-Dihydroequilenin 1,3,5( 10),6,8-Estrapentaene-3,17p-diol, see 17p-Dihydroequilenin 1,3,5(10),6,8-Estrapentaen-l7-0ne,3-hydroxy-, see Equilenin 1,3,5(10) ,6-Estratetraene-3,17pdiol 20

198

SUBJECT INDEX

1,3,5(10),7-Estratetraene-3,17pdiol,see 17pDihydroequilenin 1,3,5(1 0),7-Estratetraen-l7-one, 3-hydroxy-, see Equilin 3-methoxy1,3,5(1 O)-Estratriene-2,17pdiol, 84 1,3,5( lO)-Estratriene-3,16adiol 20 1,3,5(10)-Estratriene-3,17adiol,see Estradiol-l7a 1,3,5(1 O)-Estratriene-3,17pdiol, see Estradiol-170 _ _ _ , 17crethyny1, see Ethynylestradiol -_- , 2-methoxy-, see 2-Methoxyestradiol 1,3,5 (lO)-Estratriene-4.17p-diol,3-methoxy- 84 1,3,5( 1O)-Estratriene-l6a,l7adiol, 3-methoxy82 1,3,5(10)-Estratriene-l6a,17~diol, 3-methoxy82 1,3,5(1 O)-Estratriene-l6p,17pdiol, 3-methoxy82 1,3,5(1 O)-Estratriene-6,17-dione,3-hydroxy82,85 1,3,5(10)-Estratriene-ll,17-dione,3-hydroxy82 1,3,5(1O)-Estratriene-16,17-dione,3-hydroxy82,85 1,3,5 (1O)-Estratriene-2,3,16a,l7p-tetrol,see 2-Hydroxyestriol 1,3,5(10)-Estratriene-3,6cr,16ar,l7p-tetrol 82

1,3,5(10)-Estratriene-3,15a,16a,17cu-tetroI,see Estetrol 1,3,5( lO)-Estratriene-2,3,17p-triol 82, 84 1,3,5(10)-Estratriene-3,4,17p-triol 84 1,3,5(1O)-Estratriene-3,6a,17p-triol 82, 85 1,3,5(1 O)-Estratriene-3,6p,17p-triol 82 1,3,5 (1O)-Estratriene-3,11&17p-trio1 82 1,3,5(10)-Estratriene-3,16a,l7~-triol, see 17-Epiestriol _ _ _ , 2-methoxy- 85 1,3,5 (1O)-Estratriene-3,160~,170-triol, see Estriol _ _ _ , 2-methoxy-, see 2-Methoxyestriol 1,3,5( lO)-Estratriene-3,16p, 17a-triol, see 16J7-Epiestriol

1,3,5(10)-Estratriene-3,16p,l7ptriol, see 16-Epiestriol

1,3,5(1O)-Estratrien-3-01 20 1,3,5(10)-Estratrien-17p-01, 2,3-dimethoxy- 84 --_ , 17aethynyl-3-methoxy-, see Mestranol

_ _ _ , 3-methoxy-

82 1,3,5( 10)-Estratriend-one, 3,17pdihydroxy85 _ _ _ , 3,16~,17p-trihydroxy- 82 1,3,5(10)-Estratrien-16-one,3,17p-&hydroxy. 20,35,82,85 1,3,5(10)-Estratrien-17-one,3,4-dihydroxy- 84

__- , 3,6adihydroxy-

82

_-- , 3,6p-dihydroxy- 82 --- , 3,110-dihydroxy- 82

_-_ _--

, 3,16adihydroxy- 82,85 , 2,3-dimethoxy- 84 --- , 3-hydroxy-, see Estrone ---, 2-methoxy-3-hydroxy-, see 2-Methoxyest r one --- , 3-methoxy-2-hydroxy- 84 4-Estrenedp,l7pdiol, 17aethynyl-3p,17pdiacetoxy-, see Ethynodiol diacetate 4-Estrene-3,17-dione 20,49 4-Estren-170-01, 17-allyl, see Allylestrenol _-- , 17-ethyl-, see Ethylestrenol --- , 17-ethynyl-, see Lynestrenol 4-Estren-3-one, 17a-ethy1-17-hydroxy, see Nilevar --- , 17aethynyl-l7-hydroxy-, see Norethisterone --- , D-1 7aethynyl-17-hydroxy-18-methyl-, see D-Norgestrel _-- , 17p-hydroxy-, see Nandrolone _-- , 17a-vinyl-l7-hydroxy-, see Vinylestrenolone 5( lO)-Estren-3-one, 17~ethynyl-l7-hydroxy-, see Norethynodrel _-- , 17p-hydroxy- 20 Estriol 15,20, 23,35,38, 79,80, 82, 84, 85 _-- glucosiduronates 79,80 _-plasma 86 --- 3-sulfate 80 _-- 3-sulfate 16-glucosiduronate 80 _-- , urinary 86 Estrogen glucosiduronates 79,81 _-- sulfates 79, 81 Estrogens 10, 11, 79-8ti, 107, 109 _-- GC 83-86 _-- gel chromatography 79, 80 _-- HPLC 79,80,110 _-- LC 79,80 _-- plasma 84 _-- TLC 20,81-83 _-- , urinary 83 Estrone 4,20,35,49,52,79-86,110 --- glucosiduronate 79 _-- sulfate 25,26,79 Ethylestrenol 110 Ethynodiol diacetate 110 Ethynylestradiol 110, 112 Etiocholanolone 6, 13,23,35,38,48, 88-91, I08 --- glucosiduronate 25, 26 Etioline 120

SUBJECT INDEX

F Factice 56 Flame-ionization detector 9, 41 Flophemesyl ethers, see Pentafluorophenyldimethylsilyl ethers Fludrocortisone 111 Fragment ion chromatogram 43 Fucosterol 59,63,65,66

G Gel chromatography 4-7 Gentrogenin 23, 118 Gitaloxigenin 122 Gitogenin 118 Gitoxigenin 22, 122 Gitoxin 122 Glass-fiber paper 14 Glucosides 13, 25 Glucosiduronates 13, 16, 19, 25, 32, 109 Glycoalkaloids 117, 120 Gorgosterol 65 Gradient elution 8, 9, 99 Group retention factors 47,48

199

18-Hydroxysolanidine, see Isorubijervine 7a-Hydroxytomatidine 120 9or-Hydroxytomatidine 120 Hyocholic acid 75-77 Hyodeoxycholic acid 22, 72, 76,77

I Inokosterone 115 Isochiapagenin 118 Isocholesterol 22 28-Isofucosterol 65,66 Isolithocholic acid 76, 77 Isopyrocalciferols 114 Isorhodeasapogenin 118 Isorubijervine 120 Isotachysterols 114 Isotope effects 55, 99

J Jervine 117, 120

K

H Hecogenin 23, 118 Heptafluorobutyrates 29 High-pressure liquid chromatography 10, 11 Holamine 23 Holaphyllamine 23 Holaphylline 23 Hydrocortisone, see Cortisol 1I-Hydroxyandrosterone 38, 89, 108 24a-Hydroxycholesterol 59 240-Hydroxycholesterol 59 18-Hydroxycorticosterone 101 6~-Hydroxycortisol 103, 104 16-Hydroxydehydroepiandrosterone 38,89 2-Hydroxyestriol 86 1 lp-Hydroxyetiocholanolone 35, 38, 89 160-Hydroxyfludrocortisone 111 16a-Hydroxypregnenolone 38,95 17-Hydroxypregnenolone 25,26,95 1 lp-Hydroxyprogesterone 6,93, 95 16a-Hydroxyprogesterone 95,97 17-Hydroxyprogesterone 21, 35, 93, 95, 109 _ _ _ acetate 110 21-Hydroxyprogesterone, see Deoxycorticosterone 12ar-Hydroxysolanidine, see Rubijervine

Ketals 32 11-Ketoandrosterone 23, 35, 38, 88 7-Ketocholesterol 60 11-Ketoetiocholanolone 35, 38, 88, 108 17-Ketosteroids 87,89, 91,99, 107 Kitigenin 118 Kogagenin 118 Kryptogenin 23

L Lanatosides 121 8,24-Lanostadien-30-01, see Lanosterol 8-Lanosten-30-01,see 24-Dihydrolanosterol Lanosterol 22, 58,65 Lathosterol 22,49,56,57,62-64 Lithocholic acid 22, 72, 76, 77 Lophenol 64 Lumisterol, 34,58, 113, 114 Lynestrenol 110

M Macdougallin 65 Mass chromatography 75

200 Mass fragmentography 42,69 Mass spectrometry 11,42,43 Medrol, see 6a-Methylprednisolone Medroxyprogesterone acetate 110 Megestrol 109, 110 Mestranol 110 Me tagenin 118 Methandienone 108 Methandriol 49 Methostenol, see Lophenol Methoximes, see Methyloximes 2-Methoxyestradiol 82, 85 2-Methoxyestriol 82 2-Methoxyestrone 82 6a-Methylcortisone 111 24-Methylcycloartanol 65 Methylene units 53 24-Methylenecholesterol 59,63,64 24-Methylenecycloartanol 65 24-Methylenelophenol 65 24-Methylenepollinastanol 64 6a-Methylhydrocortisone 111 Methyloximes 33 24-Methylpollinastanol 65 6a-Methylprednisolone 111 Methylsterols 63,67 Molting hormones 115

N Nandrolone 20 Neogitogenin 118 Neotigogenin 13,23, 118 Neoyonogenin 118 Nilevar metabolites 108 p-Nitrobenzoates 11 19-Nor-4-androstene-3,17dione, see 4-Estrene-3, 1'I-dione 19-Nor-4-androsten-3-one, 17aethyl-17-hydroxy-, see Nilevar 24-Nor-5,(E)22-cholestadien-3p-ol 5 9 31-Norcycloartanol 64 Norethindrone, see Norethisterone Norethisterone 108-1 10 Norethynodrel 110 D-Norgestrel 110 19-Nortestosterone, 17aethyl-, see Nilevar

0 Obtusifoliol 64 Ostreasterol, see 24-Methylenecholesterol

SUBJECT INDEX

P Paper chromatography 13, 14 Peak-shift technique 29 Peniocerol 65 Pennogenin 118 Pentduorobenzyloximes 33 Pentafluorophenyldimethylsilyl ethers 32 Periplogenin 122 Phosphinic esters 31 Pollinastanol 64 Polyamide 16 Ponasterone A 115 Ponasterone B 115 Poriferasterol 64 Prednisolone 100, 111 Prednisone 22, 100, 111 1,4-Pregnadiene-3,20dione, gar-fluoro-1lp, 16a,17,21tetrahydroxy-, see Triamcinolone _-_ , ~a-fluoro-~lp,l7,21-trihydroxy-16amethyl-, see Dexamethasone _ _ _ , 9a-fluoro-llp, 17,2 1-trihydr oxy- 166methyl-, see Betamethasone ___ , 6a-methyl-1 lp,l7,21-trihydroxy-, see 6a-Meth ylprednisolone ---, 1lp,l7,21-trihydroxy-, see Prednisolone 4,6-Pregnadiene-3,20dione,17-acetoxyd-chloro-, see Chlormadinone acetate ___ , 6-methyl-1 7-hydroxy-, see Megestrol 4,16-Pregnadiene-3,2O-dione 21,95 1,4-Pregnadiene-3,11,20-trione, 17,21-dihydroxy-, see Prednisone 5,16-Pregnadien-20-one, 3p-hydroxy- 21, 95 5a-Pregnane 4,48 Pregnane derivatives 6,93-112 --- blood 95,96 ___ GC 96,97 ___ HPLC 93 __- TLC 21,22,93-96 _-_ , urinary 93,95,96 Pregnanediol 21,23,35,38,93-96,107, 108 _ _ _ glucosiduronate 79, 93 5or-Pregnane-3or,20adiol,see Allopregnanediol 5a-Pregnane-3a,20p-diol 94 5 a-Pregnane-3@,2 Oa-diol 4 9, 94 Sa-Pregnane-3p,2Op-diol 6, 21, 35,49, 52, 94 Sp-Pregnane-3a,2Oa-diol, see Pregnanediol Sp-Pregnane-3a,20pdiol 94 Sp-Pregnane-3p,2Oadiol 21,94 Sp-Pregnane-3p,ZOpdiol 21, 94 Pregnanedione 21,48,49, 52,94 5a-Pregnane-3,20dione 21,48,49, 52, 94 Sp-Pregnane-3,20-dione,see Pregnanedione _ _ _ , 17,21-dihydroxy- 21

SUBJECT INDEX

--_ , 120-hydroxy-

201

21

_ _ _ , 17-hydroxy- 94 _ _ _ , 21-hydroxy- 49

Sa-Pregnane-l1,20-dione, 3a,2 l-dihydroxy104 _ _ _ , 3p,l7-'-dihydroxy- 94 _-- , 3p-hydroxy- 49,94 Sp-Pregnane-l1,20-dione,3ql7-dihydroxy- 94 _ _ _ , 3a,2 1-dihydroxy-, see Tetrahydrodehydrocorticosterone __- , 3 ~ h y d r o x y - 94 _ _ _ , 3a,l7,2l-trihydroxy-, see Tetrahydrocortisone Sp-Pregnane-3~,1lp,l7,20a,21-pentol, see aCortol Pregnanetetrol 96 Sa-Pregnane-3p,6a,l6p,20a-tetrol94 Sa-Pregnane-3p,6p,16p,2Oa-tetrol94 Sp-Pregnane-3a,l lp,l7,20a-tetrol, see Pregnanetetrol Pregnanetriol 23,35,38,94,96 Sa-Pregnane-3a,6a,2Oa-triol94 Sa-Pregnane-3or,6a,2Op-triol 94 Sa-Pregnane-3p,6p,2O@-triol 94 Sp-Pregnane-3a,6a,2Ocr-triol 94 Sp-Pregnane-3a,6a,2Op-triol 94 Sp-Pregnane-3~~,6p,2Op-triol94 Sa-Pregnane-3p,17,2Oa-triol94 Sa-Pregnane-3p,l7,2Op-triol 94 Sp-Pregnane-3a,17,20a-triol, see Pregnanetriol Sp-Pregnane-3a,l7,20p-triol 94 Sp-Pregnane-3p,17,2Ocu-triol 94 Sp-Pregnane-3p,l7,20p-triol 94 Sa-Pregnane-3a,2Oa,21-triol93 Pregnanetriolone 38,94,96 Sp-Pregnane-3,6,20-trione94 Sa-Pregnane-3,11,20-trione49, 52, 94 Sp-Pregnane-3,11,20-trione 21, 94 Sa-Pregnan-3p-01 21 50-Pregnan-3a-01 21 Pregnanolone 6, 13, 21, 38,94, 96 Sa-Pregnan-3-one, 20a-hydroxy- 94 _ _ _ , 20p-hydroxy- 21,49,52,94 50-Pregnan-11-one, 3or,l7,20a-tetrahydroxy-, see aCortolone __- , 3a,l7,20p,21-tetrahydroxy-,see pCortolone _-- , 301,17,2Oa-trihydroxy-,see Pregnanetriolone Sa-Pregnan-20-one, 3a,6a-dihydroxy- 94 _-_ , 3p,l6wdihydroxy- 21 _-_ , 3or,21-dihydroxy- 104 _-_ , 3a-hydroxy- 13,94 _-_ , 3p-hydroxy-, see Allopregnanolone

_-- , 3a,l lp,l7,21-tetrahydroxy-,

see Allotetrahydrocortisol _ _ _ , 3a,l lp,21-trihydroxy-, see Allotetrahydrocorticosterone _ _ _ , 3a,17,21-trihydroxy- 104 Sp-Pregnan-20-one, 3or,6a-dihydroxy- 94 - _ _ , 3@,16adihydroxy- 21 _ _ _ , 3a,l7-dihydroxy- 94 _ _ _ , 3p,l7-dihydroxy- 21, 94 _-- , 3a-hydroxy-, see Epipregnanolone _ _ _ , 3p-hydroxy-, see Pregnanolone _ _ _ , 3a,1 lp,l7,21-tetrahydroxy-, see Tetrahydrocortisol _-_ , 3a,Sp,21-trihydroxy- 104 _-_ , 3a,l lp,21-trihydroxy- 104 _-- , 3a,l7,21-trihydroxy- 104 4-Pregnen-l8-al, 1lp,2 l-dihydroxy-3,20-dioxo-, see Aldosterone Pregnenediol 6,35, 38,95 4-Pregnene-3p,20pdiol 21 S-Pregnene-3p,2Oor-diol,see Pregnenediol S-Pregnene-3p,20p-diol 6, 21, 95 l-Pregnene-3,20-dione, 60-hydroxy- 21 4-Pregnene-3,1l-dione, 17,20or,2l-trihydroxy102 _ _ _ , 17,20p,2l-trihydroxy- 102 4-Pregnene-3,20-dione, see Progesterone _ _ _ , 17-acetoxy-6a-methyl-, see Medroxyprogesterone acetate _-- , 11p,17-dihydroxy- 95 _ _ _ , lla,21-dihydroxy- 102 _ _ _ , 1 lp,21-dihydroxy-, see Corticosterone _ _ _ , 16aJ7-dihydroxy- 95 _-- , 16a,21-dihydroxy- 102 _ _ _ , 17,21-dihydroxy-, see Deoxycortisol _ _ - , 18,21-dihydroxy- 102 _-_ , 19,21-dihydroxy- 102 _-- , 9a-fluoro- 1lp,l60r, 17,2 1-tetr ahydroxy-, see 16a-Hydroxyfludrocortisone _-_ , 9a-fluoro-1 lp,17,2l-trihydroxy-, see Fludrocortisone _ _ _ , 6p-hydroxy- 95 __- , lla-hydroxy- 6, 21,95 _ _ _ , 1lp-hydroxy-, see 1lp-Hydroxyprogesterone _ _ _ , lSa-hydrOXy- 21 _ _ _ , 16a-hydroxy-, see 16a-Hydroxyprogesterone _-_ , 17-hydroxy-, see 17-Hydroxyprogesterone _ _ _ , 21-hydroxy-, see Deoxycorticosterone _ _ _ , 6a-methyl-1 lp,l7,21-trihydroxy-, see 6or-Methylhydrocortisone _ _ _ , 6p,l lp,l7,21-tetrahydroxy-, see 6pHydroxycortisol

202

SUBJECT INDEX

_ _ _ , 1lp,16a,l7,21-tetrahydroxy- 102 _ _ _ , 6p,llp,21-trihydroxy- 102, 104 _ _ _ , lla,l7,21-trihydroxy- 102

_ _ _ , 1lp,17,2l-trihydroxy-, see Cortisol _ _ _ , 1lp,l8,21-trihydroxy-, see 18-Hydroxycorticosterone _ _ _ , 16a,l7,21-trihydroxy- 102 _ _ _ , 17,19,21-trihydroxy- 102 5-Pregnene-3pJ lp,l7a,20a-tetrol 95 Pregnenetriol 38, 95,96 5-Pregnene-3a,l6a,2Oa-triol95 5-Pregnene-3a,l6a,2Op-triol95 5-Pregnene-3p,16a,20a-triol95 S-Pregnene-3p,16a,2Op-triol95 5-Pregnene-3pJ 7,20or-triol, see Pregnenetriol 5-Pregnene-30,17,ZOp-triol 95 4-Pregnene-3,11,20-trione21,95 __- , 17,21-dihydroxy-, see Cortisone _-- , 21-hydroxy-, see 11-Dehydrocorticosterone _-- , 6a-methyl-17,21-dihydroxy-,see 6 w Methylcortisone _-- , 6p,17,21-trihydroxy- 102 _-- , 16a,17,21-trihydroxy- 102 Pregnenolone 6, 13, 21, 35,95,97 _ _ _ sulfate 25, 26 4-Pregnen-3-one, 6a,20pdihydroxy- 95 _ _ _ , 6&20p-dihydroxy- 94 _ _ - , 1 lp,20pdihydroxy- 95 _ _ _ , 17,20a-dihydroxy- 95 --_ , 17,20p-dihydroxy- 95 _ _ _ 20a-hydroxy- 21, 35,94 _-- , 20p-hydroxy- 21,35,94,96 _ _ _ , lla,17,20p,21-tetrahydroxy-102 _ _ _ , 17,20p,21-trihydroxy- 102 5-Pregnen-ll-one, 3p,l7,20a-trihydroxy- 95 5-Pregnen-20-one, 3a-amino, see Holamine _-- , 3p-amin0, see Holaphyllamine _ _ _ , 30,16adihydroxy-, see 16a-Hydroxypregnenolone _-- , 3p,17-dihydroxy-, see 17-Hydroxypregnenolone _ - _ , 3p-hydroxy-, see Pregnenolone _-_ , 3p-methylamino-, see Holaphylline _-- , 3p,l lp,l7-trihydroxy- 95 _ _ _ , 3p,16a,17-trihydroxy- 95 5a-Pregn-2-en-2 0-0ne 4 8 _-- , 16a-methyl- 48 5p-Pregn-16-en-20-one, 30-hydroxy- 21 Preparative GC 41 _ _ _ HPLC 11 --_ TLC 16 . Previtamins D 113 Progesterone 4 , 6 , 10, 21, 23, 34,49, 93-97, 100,107,109, 110

.

Pterosterone 115 Pyrocalciferols 114

R Radiochromatography 25 Regisil 32 Retention constants 53 Rhodeasapogenin 118 RM Values, GC 46,47 --_ , LC 45 Rubijervine 117, 120

S Sapogenins 23, 117-120 Saponins 117 Saringosterol 65 Sarmentogenin 122 Sarsasapogenin 23,118 Scymnol 73 9,lO-Secod ,7,10(19)-cholestatrien-3p-o1, see Cholecalciferol 9,1O-Seco-5,7,lo( 19),22-ergostatetraene-3p-01, see Ergocalciferol Self-labeling 3 , 4 Silica 3, 14 Sintered-glass plates 15 Sitosterol 22, 34,56,58,59,61,63,65-67 Sitosteryl esters 58,59 Smilagenin 23, 118 Soladulcidine 120 5a-Solanidan-3p-ol, see Demissidine 5p-Solanidan-3-one 120 4-Solaniden-3-one 120 Solanidine 23, 117, 120 Solanocapsine 120 5a-Solasodan-3p-ol, see Soladulcidine Solasodine 23, 117, 120 Sorbents 3-7, 14-16 Spectroradiochromatograph 9 a-Spinasterol 63,65 (25R)-5p-Spirostane-lp,3p-dioI, see Isorhodeasapogenin (25S)-5p-Spirostane-lp,3pdiol, see Rhodeasapogenin (25R)-5a-Spirostane-2a,3p-diol, see Gitogenin (25S)-5a-Spirostane-2a,3p-diol, see Neogitogenin (25R)-5a-Spirostane-3p,6a-diol, see Chlorogenin (25R)-5p-Spirostane-lp,2p,3rr,5-tetrol, see Kogagenin (25R )-Sp-Spirostane-lp,2p,3a-triol, see Tokorogenin

203

SUBJECT INDEX

(25R)-5p-Spirostane-2p,3p,lla-triol, see Metagenin (25R)-5a-Spirostane-2a,3p,l5p-triol, see Digitogenin (25R)-5a-Spirostan-3p-ol, see Tigogenin (25R)-5p-Spirostan-3p-oI,see Smilagenin (25S)-5a-Spirostan-3p-ol, see Neotigogenin (25S)-Sp-Spirostan-3p-o1, see Sarsasapogenin (25R)-Sa-Spirostan-l2-one, 3p-hydroxy-, see Hecogenin (25R)-5-Spirostene-2p,3a-diol, see Yonogenin (25S)-5-Spirostene-3p,12p-diol, see Chiapagenin (25R)-5-Spirosten-3p-ol, see Diosgenin (25S)-5-Spirosten-3p-o1,see Yamogenin (25R)-5-Spirosten-l2-one, 3p-hydroxy-, see Gentrogenin Spore plate 26 Stanolone, see Androstanolone Stationary phases 33-37 Steroid Analyzer 8 Steroid numbers 48-53 Sterols 55-69 _-- , fecal 68 GC 61-69 _ _ _ LC 3-5,55-57 _ _ _ , plant 68 _ - _ TLC 22,57-61 Steryl esters 3,5, 56,58, 67 _-- glycosides 56,67 Sa-Stigmasta-7,22-dien-3p-o1, see a-spinasterol 5a-Stigmasta-7,24(28)-dien-3p-o1, 28-iso- 65 _ - ,~4a-methyl-, see Citrostadienol 5a-Stigmasta-7,25-dien-3p-o1 63, 65 5a-Stigmasta-8,14-dien-3p-ol 65 _ _ _ , 4a-methyl- 65 5,7-Stigmastadien-3pol 63, 65 5,(E)24(28)-Stigmastadien-3p-ol, see Fucosterol 5,(Z)24(28)-Stigmastadien-3@-01,See 28-lsofucosterol 5,25-Stigmastadien-3p-ol 63, 65 Stigmastanol 63, 65,66 5,7,22-Stigmastatrien-3p-ol 65 Sa-Stigmasta-7,22,25-trien-3p-o1 63, 65 5a-Stigrnasta-8,14,24(28)-trien-3p-o1 65 5a-Stigmast-7-en-3p-01 63, 65 5a-Stigmast-8-en-30-01 65 -_- , 4a-methyl- 65 ~ _ , 14a-methyl_ 65 5a-Stigmast-8(14)en-3@-01 65 Sa-Stigrnast-22-en-3p-ol 22 Stigmasterol 22,40, 58, 59, 61, 63, 64, 66,67 Stigmasteryl esters 58 Strophanthidin 22, 122 Strophanthidol 122 Sulfates 7, 25, 109 Sulfuric acid test 18, 20-23, 93

T Tachysterols 113 Testosterone 20, 35, 38,49,52,19, 87, 88, 91,92,100,109 - _ _ propionate 110 Tetrahydrocortisol 104, 108 Tetrahydrocortisone 104, 108 Tetrahydrocorticosterone 104, 108 Tigogenin 23,49, 118 Tokorogenin 118 5a-Tomatidan-30-01, see Tomatidine 5-Tomatiden-3p-1 23, 120 Tomatidine 23, 117, 120 Tomatillidine 120 Toxisterols 114 Triamcinolone 11 1 Trimethylsilyl ethers 26, 27, 30-32 Tubular TLC 16, 24, 25 Twin ion technique 44

U Urinary steroid profiles 108 Uzarigenin 22

V Vapor-phase acylation 85 Vapor-programmed TLC 17 Veralkamine 120 Veralobine 120 Veramarine 120 Veramine 120 Verarine 120 Veratramine 120 Verazine 120 Vinylestrenolone 110 Vitamins D 113, 114

Y Yamogenin 13,23 Yonogenin 118

1

Zone extraction 24 Zymosterol 56,64


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