Journal Separation Science

separation science

driving analytical chemistry forwards

Mercury and arsenic speciation analysis by IC-ICP/MS

Comparing TLC methods for constructing medicinal plant fingerprints Determining carcinogenic aromatic amines in cigarette smoke by LC-MS/MS

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separation science

driving analytical chemistry forwards

separation science

driving analytical chemistry forwards

Mercury and arsenic speciation analysis by IC-ICP/MS

contents

Comparing TLC methods for constructing medicinal plant fingerprints Determining carcinogenic aromatic amines in cigarette smoke by LC-MS/MS

Volume 1 / Issue 6 May 2009

www.sepscience.com

Rr

Volume 1 / Issue 6 May 2009

features

06

research round-up 06 A CTAB-based method for the proteomic analysis of wine spoilage microrganisms

08 Rapid and sensitive determination of

16 Mercury and arsenic speciation analysis by IC-ICP/MS Laura Reyes, H.M. Skip Kingston, Andrea Wille, Jürgen Knöll and Alfred Steinbach

six carcinogenic aromatic amines in cigarette smoke by LC-MS/MS

09 HPLC assays for desmethoxyyangonin,

methysticin, kavain and their microsomal metabolites

10 Thermodynamic study of salbutamol interaction with an immobilized β2-adrenoceptor by HPLC

12 Pharmaceutical quality control by

22 A comparison of TLC methods for constructing medicinal plant fingerprints Łukasz Cieśla and Monika Waksmundzka-Hajnos

micellar electrokinetic chromatography

13 Reversed-phase chiral LC–MS/MS

quantitation of reboxetine enantiomers in rat plasma and brain

14 Preparation and ion chromatographic

properties of a new core-shell chromatographic support Al2O3/SiO2-10

15 Detection of Salmonella enteriditis from egg using immunomagnetic beads

regulars

An

Tu

28

application notes High Speed, Ultra-High Sensitivity, and Robustness Needed for the Quantitation of Pharmaceuticals in Blood Plasma, Agilent. Hormones Norgestrel and Ethynyl Estradiol on Allure C18, Restek. Artemisinin in Artemisia Annua Leaf, Camag. Automating Calculations for Rapid Seed Oil Quality Control and Authenticity, Waters.

30

technology update An overview of recent technology advances in separation science and instrumentation.

Separation Science is published by Eclipse Business Media Ltd, 70 Hospital street, Nantwich, Cheshire, CW5 5RP, UK. Copyright 2009 Eclipse Business Media Ltd. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical including by photocopying, recording or information storage and retrieval without permission from the publisher, Eclipse Business Media Ltd. Applications for the copyright owner’s permission to reproduce any part of this publication should be forwarded in writing to Permissions Dept, Separation Science, Eclipse Business Media Ltd, 70 Hospital street, Nantwich, Cheshire, CW5 5RP, UK. Separation Science does not verify any claims or other information appearing in any of the advertisements contained in the publication, and cannot take any responsibility for any losses or other damages incurred by readers in reliance on such content.

separation science

scientific advisory council

driving analytical chemistry forwards

Peter Myers – Chief Scientific Officer [email protected] David Barrow University of Cardiff, UK

Melissa Hanna-Brown Pfizer, UK

technical articles on chromatography and related technologies?

Zongwei Cai Hong Kong Baptist University

Tuulia Hyötyläinen University of Helsinki, Finland

updates on recent research studies?

Yi Chen Chinese Academy of Sciences, Beijing, China

Gongke Li Sun Yat-Sen University, Guangzhou, China

Gert Desmet Vrije Universiteit Brussel, Belgium

Yong-Chien Ling National Tsing Hua University, Taiwan

practical advice on routine analysis? applications of new technology? information on commercial product developments? market trends and opinions?

c n e i c s n ratio

C. Bor Fuh National Chi Nan University, Taiwan Klara Valko, GSK, UK Y.S. Fung Hong Kong University Jean-Luc Veuthey University of Geneva, Switzerland Xindu Geng Northwest University, Xi’an, China Claudio Villani Universita’ degli Studi di Roma “La Luigi Mondello Sapienza”, Italy University of Messina, Italy Cheing- Tong Yan Paul Haddad Center of Environmental Safety and University of Tasmania, Australia Hygene, Taiwan Hian Kee Lee National University of Singapore, Singapore

Edward Browne GSK, Singapore

contactsards try forw is m e h c l a c ti ly a n a g drivin Dean Graimes Publishing Director +44 208 398 0209

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Day One:

Day Three:

Bioscience

Pat Sandra

Advances in Separation Sciences Deriven by the Metabolomics and Proteomics Quest for Biomarkers

Alastair Lewis

Y.S. Fung

Hian-Kee Lee

Eric Chun Yong Chan

Siu Kwan Sze

Enviro

Food

Trace Pollutant Detection in Challenging Environments Solvent-Minimized Sample Preparation for Separation Science An Advanced Proteomic Approach to the Discovery of Microbial Enzymes

Manfred Raida

Multidimensional Gel-free Protein Separation Approaches for In-depth Analysis of Complex Proteomes

Gongke Li

Yi Chen

Paul Haddad

New Approaches to Online Anti-salt Stacking for Direct Capillary Electrophoresis of Biosamples

Molecularly Imprinted Polymers for Trace Analysis of Complicated Samples

Terrorist Explosives by Analysis of Inorganic Residues

Andrew Jenner

Philip Marriott

GC-MS Analysis of Lipid Oxidation and Cholesterol Metabolism

Headspace Analysis of Plant Materials by Using Comprehensive Two-Dimensional Gas Chromatography: Selected Examples

Thomas Walczyk

Element Separation at the Microscale for High-Precision Isotopic Analysis of Biological Samples

Jessie Tong

Multidimensional Gas Chromatographic Analyses of Flavours and Fragrances

Bahruddin Saad

Day Two:

Pharma

TCM

Determination of Biogenic Amines in Food: Conventional and Nonconventional Approaches

Robert Shellie Gert Desmet

One- and Two- dimensional GC-MS for Hop Metabolics

Current and Future Approaches to Speed Up HPLC Separations

Phil Nethercote

The applictaion of Quality by Design Principles to Analytical Method Development, Validation and Transfer.

Sanjay Garg

The Role of Analytical Science and Techniques in Early Phase Drug Discovery and Registration for Clinical Studies

Anne Goh

Online Solid Phase Extraction-LC-MS in DMPK Applications

Edward Browne

Biomarker Analysis for Preclinical Pharmaceutical R&D

Shawn Stanley

Using Narrow Bore Monolithic LC Columns to Enhance Analytical Sensitivity in Equine Doping Control

Ping Li

HPLC and Hyphenated Techniques for Analysing Ingedients in Herbal Medicines

Yizeng Liang

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Rr Research round-up

Key

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Product information

A CTAB-based method for the proteomic analysis of wine spoilage micro-organisms Italy According to a paper in the Journal of Chromatography B [877 (10), 887-891 (2009)] a protocol based on the use of the cationic detergent cetyl-trimethylammonium bromide (CTAB) and its salt-dependent solubility was developed.

Comment

Dr Alessandra Bossi from the Department of Science and Technology at the University of Verona in Italy explained that the contamination of nucleic acid and polysaccharides is a real problem when mapping the proteome of microrganisms by 2D electrophoresis. “Polysaccharides are complex proteins, giving rise to high molecular weight aggregates that do not migrate in gel; plasmids and nucleic acid could also bind to proteins and, due to their external skeleton exposing phosphoric groups, such complexes result in the apparent acidification of the proteins and in their defocusing. Both polysaccharides and nucleic acid cause severe streaks and smears on the gels and pose problems in terms of resolution of the maps. Thus, a protocol for an efficient removal was really needed,” Dr Bossi said. The team proposed a solution to the problem of efficient removal of nucleic acid and polysaccharide residues from the total protein extract of a microrganism prior to proteomic analysis by setting up a protocol based on the use of the cationic detergent cetyl-trimethylammonium bromide (CTAB) and its peculiar solubility properties, which are dependent on the concentration of NaCl in the solution. “CTAB is routinely exploited in molecular biology and genetic for nucleic acid purification and we propose it as treatment before performing two-dimensional electrophoresis of the total protein extracts. Results showed a far superiority of the CTAB protocol, respect the available and already described procedures. More than 500 protein spots were counted on minigels (7 cm length) after the CTAB treatment, which is almost a hundred more than what obtained with other methods, tested for comparison,” Bossi added. She believes the technique proved to be simple and very efficient. “The protocol was applied to several microrganism and to yeasts and the results were always remarkable. The protocol is nowadays employed for mapping other microroganisms, such as Prevotella species and to the treatment of cells from superior plants. We think that this technique is beneficial for the broad community that devotes its scientific interests to proteome research,” she concluded.

6

research round-up

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TOSOH BIOSCIENCE

Rapid and sensitive determination of six carcinogenic aromatic amines in cigarette smoke by LC-MS/MS India Aromatic amines are one of the sources of

of six aromatic amines (aniline, benzidine,

carcinogenicity in cigarette and tobacco smoke, and

o-toluidine, 1-naphthylamine, 2-naphthylamine and

accurate quantification of these chemicals is needed to

4-aminobiphenyl) using gradient LC-ESI-MS/MS and

assess public health risk. As documented in the Journal

determined them from cigarette smoke. All aromatic

of Chromatography A [1216 (15), 3059-3063 (2009)], a

amines were well resolved in 8 minutes using these

validated rapid, sensitive and analyte-specific liquid

gradient conditions. “We also studied the degradation

chromatography/electrospray ionization tandem mass

of benzidine present in cigarette smoke extract. The

spectrometric (LC/MS/MS) method has been developed

separation and detection technique is a robust one and

for the simultaneous determination of six aromatic

will be useful for chromatographers for determination

amines in mainstream cigarette smoke using research

of these chemicals from other sources when present at

reference cigarette 2R4F.

trace levels. We also introduced a recovery calculation

Dr Bidhan Ray from the Department of Chemistry

technique using the standard addition method, without

at Jadavpur University in India, explained that these

using isotopic-labelled standards,” Ray explained.

aromatic amines possess different adverse effect

In this work liquid-liquid extraction was used for the

on human health and are also considered as potent

extraction of aromatic amines from cigarette smoke

genotoxic compounds. “Accurate quantification of trace

followed by liquid chromatographic separation and

levels of these aromatic amines is a challenging task. Our

tandem mass spectrometric detection. “Determination

main aim was to develop a rapid and compound-specific

of all other toxic volatiles from cigarette smoke has been

determination method for aromatic amines, so that

performed and will be published soon. At present we are

the technique can be used for quantification of other

studying the biological effects of the chemicals present

chemicals when present at trace level,” Dr Ray added.

in cigarette smoke on humans in an attempt to uncover

The team developed and optimized the separation

ways of reducing or eliminating them,” he concluded.

8

research round-up

www.sepscience.com

HPLC assays for desmethoxyyangonin, methysticin, kavain and their microsomal metabolites Australia Three novel, simple and reproducible high-performance liquid chromatography quantitative assays with UV detection were developed and validated for three major kavalactones – desmethoxyyangonin, methysticin and kavain – in rat liver microsomes using diazepam as an internal standard; liquid-liquid extraction was used for sample preparation and analysis was performed on a Shimadzu 10A high-performance liquid chromatography system. The study was published in Biomedical Chromatography [23 (1),81-91 (2009)] and authored by Professor Iqbal Ramzan, Dean of the Faculty of Pharmacy at the University of Sydney, Australia, who explained that kava had interested him since childhood. With a background in Pharmacokinetics, Professor Ramzan wanted to contribute to the knowledge about kava, which entailed developing an analytical technique to measure its active constituents. “We are able to assay kava, some of its metabolites to determine the structure of the metabolites,” he said. His fundamental work is designed to see why kava is hepatotoxic. “Using this assay we can see if the heptotoxicity is due to kava itself, its metabolites or both. Also it will allow us to see which specific kava component or kavalactone is the offending species in terms of liver damage,” he added.

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research round-up

9

Thermodynamic study of salbutamol interaction with an immobilized β2-adrenoceptor by HPLC China Investigation of the interaction between drugs and receptors is very important in revealing the biologic basis and mechanism of the drug, and designing new bioactive compounds. A study published in the Journal of Chromatography B [877 (10), 911-918 (2009)], purified the β2-adrenoceptor (β2-AR) from rabbit lung tissues and immobilized it on a surface of macro-pore silica gel through covalent bonds to prepare the stationary phase. Dr Xinfeng Zhao from the Department of Pharmacology at the Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education at Xi’an Jiaotong University in China explained that the technology for screening bioactive compounds of innovative and efficient drugs is a hot topic in pharmaceutical fields. “Traditional Chinese medicine (TCM) and its complex prescription compounds have a complete theoretical system and long clinical history, and have unique advantages in the treatment of cardiovascular diseases. In recent years, TCM have become a major source to develop innovative new drugs. However, they also have the disadvantages of complicated composition and flexible application. Therefore, screening of their bioactive ingredients has become the bottleneck of the entire research process,” Dr Zhao said. Zhao added: “It is well known that drugs have pharmaceutical activities by recognizing and then binding to membrane receptors, so as to activate some cellular signal transmission cascade.” Based on this theory, if these receptor could be bonded onto the surface of the stationary phase, a fast, effective and stable affinity chromatographic method could be established for the screening of active compounds in TCM by taking advantage of the receptor’s unique recognition to drugs and the high separating capacity of liquid chromatography. “The results showed that the association constant of terbutaline with β2-AR in the biochromatographic column was 9.37 × 10-6 M, and the binding sites were 9.76 × 104 M. Salbutamol has two binding sites with β2-AR in the column, for which the association constants were 1.11 × 104 M and 1.34 × 103 M. All the above results demonstrated that the receptor biochromatographic model was a new technology for online investigation of the interactions between receptor and drug. More importantly, our results showed that thermodynamic theory could be used for investigating the binding mechanism of the drug and receptor,” he said. The team believes the results indicate that receptor biochromatography had the properties of recognizing, combing and separating its ligands. “It could be used for screening bioactive compounds in TCM [Chinese Science Bulletin, 2008, 53(6): 842-847]. In our future work, we will combine different receptor columns online by column switching technology to screen bioactive compounds in complex samples such as TCM,” he concluded.

10

research round-up

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Pharmaceutical quality control by micellar electrokinetic chromatography Greece According to the Journal of Pharmaceutical and Biomedical Analysis [49 (2), 201-206 (2009)], a micellar electrokinetic chromatography (MEKC) method has been developed and validated for the determination of nimesulide related compounds in pharmaceutical formulations. “This paper was part of our continuous research cooperation with the Greek pharmaceutical industry aiming to develop advantageous analytical methods for the quality control of formulations during development and production stages. We are mainly focused on the development of reliable and high-throughput methods for the analysis of samples produced from assay, purity, dissolution and in-process tests. On this basis, we developed, validated and applied the first micellar electrokinetic chromatography (MEKC) method for the separation and quantitation of the process and degradation impurities of the non-steroidal anti-inflammatory drug Nimesulide,” explained Dr Paraskevas Tzanavaras from the Laboratory of Analytical Chemistry at the Aristotelian University of Thessaloniki, Greece. After careful investigation of the key variables that affected the behaviour of the target compounds under MEKC conditions, the team was able to separate and quantify the active ingredient and its six related substances within 20 minutes. “The validation of the method in terms of the most critical parameters according to international guidelines (specificity, linearity, accuracy and precision) proved its potential usefulness and efficacy for its intended purposes,” said Dr Tzanavaras. Tzanavaras believes the results confirmed the potential of capillary electrophoretic techniques to the quality control of pharmaceutical formulations. “If properly developed and validated, these techniques offer adequate analytical figures of merit, comparable reliability and performance to traditional and well-established HPLC protocols and dramatic reduction in organic waste generation. We intend to continue our research activities towards this direction,” Tzanavaras concluded.

12

research round-up

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Reversed-phase chiral LC–MS/MS quantitation of reboxetine enantiomers in rat plasma and brain UK A sensitive liquid chromatography–mass spectrometry (LC–MS) method has been developed for stereoselective determination of reboxetine in rat plasma and brain homogenate (LLOQ, 50 pg/mL). Dr Paul Turnpenny from the Discovery Bioanalytical Group at Pfizer Global Research and Development in Sandwich, UK, explained the research was performed to characterize CNS and systemic exposure levels for the potent pharmaceutical agent reboxetine administered in specific receptor occupancy investigations. Stereo-selectivity was required because the two enantiomers are known to exhibit differences in their receptor potency. “Using two different biological matrices and working with low analyte concentrations, the primary focus of the work was to optimize the sensitivity and robustness of the assay. MS detection, as opposed to UV/fluorescence detection in previously described methods, was anticipated to offer the best chance of this,” Dr Turnpenny said. Published in the Journal of Pharmaceutical and Biomedical Analysis [49 (1), 133-139 (2009)] the study’s most significant outcomes include the chiral recognition of the stationary phase α1 acid glycoprotein which enabled good separation to be achieved, although the interaction was extremely sensitive to mobile phase pH and needed adequate buffering (12.5 mM ammonium carbonate). The study also revealed a pH below 6.6 caused co-elution of the enantiomers and above pH 7 excessive analyte retention and peak tailing. The pKa of reboxetine is 8.3, therefore at the assay pH of 6.7 (1.6 units lower) the analyte was still predominantly ionized and only a low percentage of organic modifier (15%) was needed to elute. “I found that the composition of flow entering the MS source was hugely influential upon the detection sensitivity. By introducing a secondary postcolumn organic modifier to aid the desolvation and ionization process, the sensitivity was improved up to 10-20 fold and gave a much smoother response. I also found that column washing was absolutely essential to maintain good chromatography. A small amount of acetic acid (0.05%) helped remove protein debris and enabled >80 sequential injections to be performed with no problem and upwards of 300 in total on the same column,” he added. He believes that for complicated multi-analyte or chiral separations in which the eluting mobile phase contains a low percentage of organic solvent or incompatible pH to MS detection, postcolumn adjustment of the flow can significantly improve both the sensitivity and consistency of the response and is relatively easy to setup. “The stereospecific effects of reboxetine, currently administered in the racemic form, are still of considerable interest to my company and it could be the analytical technique will be utilized in further research to establish an improvement in its clinical use,” he concluded.

separation science — volume 1 issue 6

research round-up

13

Preparation and ion chromatographic properties of a new core-shell chromatographic support Al2O3/SiO2-10 China A new stationary phase Al2O3/SiO2-10 was prepared and characterized by XPS, XRD, SEM and surface analysis and documented in the Journal of Chromatography A [1216 (15), 3054-3058 (2009)]. Main author, Dr Shengxiang Jiang from the Key Laboratory for Natural Medicine of Gansu Province at the Chinese Academy of Sciences in Lanzhou, China, explained, “Some inorganic oxide supports, such as zirconia, titania and alumina have been found to offer some special chromatographic properties. Of the above-mentioned oxides, alumina is the most extensively studied material and it has many desirable ion-exchange properties; however, until now it has not been broadly used.” He believes the main reason is that these alumina supports provide low ion exchange capacities, low resistance to strong acidic and basic conditions, and often result in poorly defined peak shapes and zones in chromatographic applications. “To improve the chromatographic characteristics of alumina-based supports, we employed a layer-bylayer self-assembly technique to develop a new core-shell chromatographic support Al2O3/SiO2-10,” Jiang added. The team found that the core-shell chromatographic support Al2O3/SiO2-10 had a large surface area, and very uniform diameter and surface. “Compared with other alumina supports, this core-shell support had excellent column efficiency, well-defined chromatographic peaks and favourable retention times for the separation of inorganic anions,” he explained. The core-shell chromatographic support appeared to combine the advantages of both core and shell materials, and provided better chromatographic properties. “In my opinion, further research into these core-shell chromatographic materials may result in many excellent supports for the HPLC separation, including fast or multiaction chromatographic supports,” he said. In the future, Jiang plans to choose new shell materials to make further advances in core-shell chromatographic supports; and develop functionalized core-shell chromatographic supports after chemical modifications.

14

research round-up

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Detection of Salmonella enteriditis from egg using immunomagnetic beads USA The types of chemical linkages used to bind antibodies

As far as which bead/antibody interaction provided

to magnetic beads to form immunomagnetic beads

better detection, those results varied among the three

(IMB) were compared in the capture and detection

S. enteriditis strains tested. We concluded that antigenic

of Salmonella enteriditis from egg white, egg yolk

differences among those strains contributed to different

and whole egg in a paper in Food Analytical Methods

degrees of interaction with the immunomagnetic

[2 (1), 14-22 (2009)]. According to Sue Reed from the

beads,” she said. Second, they found that the presence

Eastern Regional Research Center at the United States

of egg yolk resulted in lower signal intensities for all

Department of Agriculture, USA, there are many different

types of beads. “This suggested that egg yolk may have

types of immunomagnetic beads on the market today.

an adverse effect on the immunological interactions

“We were interested to study what differences may

between the bacteria and the immunomagnetic beads,”

exist among different beads with regard to the linking

she added.

chemistry by which antibodies are attached. We chose

Reed believed that based on the results of this work,

two common types of beads having streptavidin/

they have realized that the capture of a target organism

biotin interactions and covalent attachment through

by immunomagnetic beads may vary depending on

carboxyl groups. In addition, we included a commercially

the strain being studied. “In subsequent work, we have

prepared immunomagnetic bead for comparison. We

continued to study the differences in detection among

also chose to use Salmonella enteriditis infected eggs

immunomagnetic beads utilizing different antibody

as a real-world model with which to study the capture

conjugating chemistries. We have expanded our research

abilities of these immunomagnetic beads,” Reed

to include tosylactivated beads, as well as different sizes

explained.

of these beads. Perhaps a different target organism and/

The team’s findings were twofold. Both the

or food matrix will yield more definitive findings,” she

streptavidin-coated and carboxylated beads yielded

concluded.

higher relative signals than the commercially prepared bead. “That is attributed to a much higher background signal associated with the commercially prepared beads.

separation science — volume 1 issue 6

research round-up

15

16

feature article — Speciation analysis with IC-ICP/MS

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Mercury and arsenic speciation analysis by IC-ICP/MS Laura Reyes,1 H.M. Skip Kingston,1 Andrea Wille,2 Jürgen Knöll3 and Alfred Steinbach2 Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, Pennsylvania, USA Metrohm International Headquarters, Herisau, Switzerland 3 Philipps Universität Marburg, Germany

1

2

By means of IC-ICP/MS, different valence states of arsenic and mercury in the form of inorganic and organic species can be sensitively and unambiguously identified in one single run. Owing to the absence of interconversions during sample preparation, the determination of arsenic species in biological and environmental matrices is straightforward and can be performed by traditional speciation analysis down to the sub-ppb level. In contrast, species transformations of mercury during sample preparation require the use of specific isotope dilution mass spectrometry (SIDMS). EPA Method 6800 was applied to evaluate and efficiently compensate for potential errors during measurement.

Introduction

conductivity detection.

Arsenic

Ion chromatography (IC) with

Especially in view of the zero

Arsenic is ubiquitously found in a

conductivity detection has been

tolerance policy concerning

high number of minerals and its

successfully used to determine

chromium, arsenic and selenium

use as a weed killer and rat poison

anionic and cationic substances as

compounds in drinking water

illustrates its high toxicity. Inorganic

well as polar compounds such as

and mercury in food samples,

arsenical derivatives are considered

amines or organic acids. However,

ICP/MS detectors have gained

to be carcinogenic and possibly

in environmental samples a higher

increasing importance. IC-ICP/MS

teratogenic. Therefore, the EPA

sensitivity and selectivity are

can distinguish between different

proposes a maximum allowable

required to test for potentially toxic

oxidation states and chemical forms

drinking water concentration of

substances with low maximum

of a given element. This approach

10 ppb. In environmental and

contaminant levels (MCL).

is called speciation analysis. From

biological samples, more than 20

The coupling of IC with multi-

the toxicological point of view,

arsenic species have been identified.

dimensional detectors such as

individual concentrations of

Depending on their binding

electrospray ionization mass

element-containing species are far

characteristics, they have different

spectrometers (ESI-MS) or

more significant than total element

toxicities and chemical behaviours.

inductively coupled plasma

concentrations as different valence

Based on structural data, IC-ICP/MS

mass spectrometers (ICP/MS)

states of an element often have

allows separation and unambiguous

solves even complex separation

completely different properties.

identification of different arsenic

problems, simultaneously achieving

For example, while chromium(III)

species in inorganic and organic

high sensitivity and selectivity.

is an essential trace element

forms.

Additionally, these hyphenated

for mammals as it is involved in

techniques allow unambiguous

glucose metabolization, all forms of

Mercury

peak identification and are less

hexavalent chromium are regarded

Mercury is found in several forms,

prone to matrix interferences than

as highly toxic and carcinogenic.

particularly as elemental (Hg0),

Figure 1

separation science — volume 1 issue 6

feature article — Speciation analysis with IC-ICP/MS

17

inorganic (Hg2+), or alkylated

death, even in small quantities.

dose for methylmercury (Rf ) of

mercury (CH3Hg ). Of the most

According to the US Food and Drug

0.1 µg/kg of body weight per day,

common mercury species found in

Administration (FDA), the major

while the World Health Organization

the environment, methylmercury is

exposure pathway to methylmercury

(WHO) has set a tolerable dose of

considered the most toxic species.

in humans and wildlife is through

1.6 µg/kg of body weight per week [1].

It is classified as a neurotoxin that

consumption of contaminated fish.

However, mercury species are

rapidly bioaccumulates and can

The US Environmental Protection

prone to interconversion. Mercury

cause major health problems or

Agency (EPA) stipulates a reference

shows a pronounced transformation

+

from inorganic mercury (Hg2+) to the

Table 1

biologically active and highly toxic

Instrumental operation conditions for the determination of the arsenic species

methylmercury (methylation) and

via IC-ICP/MS

vice versa (demethylation). Similarly,

Column

Metrosep A Supp 15 – 150

the extraction techniques used for

Injection volume

10 µL

separation and preconcentration

Column temperature

Ambient

tend to alter the original distribution

Eluent

8 mmol/L NH4NO3, pH = 8.3

of mercury species, which affects the

Elution

Isocratic

legal defensibility of the data.

Flow rate

0.7 mL/min

Speciated isotope dilution mass

ICP/MS

Without reaction or collision mode

spectrometry (SIDMS) has been

m/z

75

developed to correct for species conversions. According to US EPA

Table 2

Method 6800 [2] each species is

Instrumental operation conditions for the determination of mercury species

labelled with a different isotope-

via SIDMS IC–ICP/MS.

enriched spike in the corresponding

RF power

1475 W

form. By measuring the isotope

Plasma gas flow

Ar, 115 L/min

ratio of both the unspiked and

Auxiliary gas flow

Ar, 1 L/min

spiked sample and knowing the

Nebulizer

Quartz, concentric

isotopic ratio of the addition,

Spray chamber

Quartz

interconversions between the

Sample and skimmer cones

Ni, 1.1 and 0.8 mm, respectively

species become traceable and can be corrected.

Measurement parameters of ICP/MS

This article deals with the

Monitoring isotopes

199

Acquisition mode

Time-resolved analysis

determination of organic and

Integration time per mass

0.20 s

inorganic arsenic and mercury

Replicates

1

compounds by means of IC-ICP/MS.

Total analysis time

300 s

Arsenic species (monoisotopic) are

Hg,

Hg,

200

201

Hg and

202

Hg

not prone to interconversion and

Separation conditions Column

DVB-C18 column, 150 × 4.6 mm, 2 µm

are thus determined by traditional

Injection volume

100 µL

speciation analysis. Several

Column temperature

Ambient

established extraction techniques

Eluent

50 mmol/L pyridine,

used for mercury speciation in

5% (v/v) methanol, pH 3,

biological samples are evaluated by

0.5% (w/v) cysteine,

applying both internal SIDMS and

Elution

Isocratic

external calibration.

Flow rate

1 mL/min

18

feature article — Speciation analysis with IC-ICP/MS

www.sepscience.com

Experimental

or analysis procedure.

enzymatic digestion with protease

External calibration: Separation

Extraction methods: The extraction

XIV. The methods are summarized

of mercury species was automated

methods to be evaluated were

in Table 3 [3]. The reference material

using the 858 Professional Sample

based on literature-referenced

used for the comparison of sample

Processor and the 850 Professional

methods such as alkaline extraction

preparation methods was Tuna Fish

IC (both Metrohm AG, Herisau,

with potassium hydroxide (KOH)

Tissue Certified Reference Material

Switzerland) coupled to an ICP/

or tetramethyl ammonium

(ERM-CE464) supplied by IRMM

MS model HP 4500 (Agilent

hydroxide (TMAH) solution; acid

(Geel, Belgium), which is certified for

Technologies, Palo Alto, California,

leaching with hydrochloric acid

total mercury and methylmercury

USA and Yokogawa Analytical

(HCl), nitric acid (HNO3) or glacial

content.

System Inc., Tokyo, Japan). For the

acetic acid (CH3COOH); extraction

Reagents, standard solutions and

determination of arsenic species,

with l-cysteine hydrochloride and

eluents: All reagents used in this

the IC system was coupled to an Agilent 7500ce ICP/MS. The

Figure 1

conditions used during the study are detailed in Tables 1 and 2. Each sample was analysed three times. Quantitation was based on peak areas by external calibration using the arsenic isotope m/z 75 and the most abundant mercury isotope m/z 202. Quantitation using the mercury isotopes m/z 199, 200 and 201 yielded similar results. For the determination of total mercury concentrations, the digested and extracted solutions were analysed by ICP/MS. SIDMS: To correct for mutual

Figure 1: Separation and detection of arsenite, dimethylarsenate, monomethylarsenate and arsenate.

Figure 2

interconversion, Hg2+ and CH3Hg+ compounds were quantified by EPA 6800 protocol specifications, spiking the sample before the extraction with 199Hg2+ and CH3200Hg+ and application of SIDMS equations [2]. The isotopic species reagents and calculation software in a SPC-M Mercury Speciation Kit was provided by Applied Isotope Technologies (AIT, Sunnyvale, California, USA). This double-spike approach allowed tracking of any artifact stemming from methylation/demethylation reactions that might have occurred during the sample preparation and/ separation science — volume 1 issue 6

Figure 2: IC-ICP/MS chromatogram for 10 µg/L Hg2+ and CH3Hg+. Chromatograms obtained at different masses were shifted for clarity. Instrumental operation conditions are given in Table 2.

feature article — Speciation analysis with IC-ICP/MS

19

work were of the highest purity

prepared with deionized water with

species. However, ASB interference

grade (puriss. p.a.). Analytical

a specific resistance higher than

can be overcome by changing the

reagent grade HNO3, HCl, TMAH,

18 MΩ•cm.

chromatographic parameters. Mercury: Figure 2 shows the

potassium hydroxide, optimagrade methanol and HPLC-grade

Results and Discussion

chromatogram of the separation

glacial acetic acid were purchased

Arsenic: IC-ICP/MS allows the

of the divalent mercury ion from

from Fisher Scientific (Pittsburgh,

separation and unambiguous

methylmercury [3] on a polymer-

Pennsylvania, USA). Reagent grade

identification of different arsenic

based C18 reversed-phase column.

l-cysteine, l-cysteine

species in inorganic and organic

Separation was achieved in less than

hydrate, ammonium phosphate

forms. Figure 1 displays the peaks

five minutes and the retention times

dibasic, pyridine and protease XIV

of a 10 µg/L standard solution

were 1.87 ± 0.02 and 2.98 ± 0.03

were obtained from Sigma Aldrich

containing monomethylarsenate,

minutes. Linear calibration curves for

(St. Louis, Missouri, USA). The arsenic

dimethylarsenate, arsenite, arsenate

Hg2+ and CH3Hg+ were obtained in

standard solutions were purchased

and arsenobetaine (ASB). Under the

the range from 1–20 µg/L. Detection

from Fluka (Sigma Aldrich, Buchs,

given conditions (Table 1), ASB is not

limits were 0.46 ± 0.02 and 0.78

Switzerland). All solutions were

separated from the trivalent arsenic

± 0.08 µg/L for Hg2+ and CH3Hg+,

hydrochloride

Table 3

Concentrations of mercury species (in mg/kg Hg) determined in Tuna Fish Tissue Reference Material (ERM-CE464) by external calibration and by EPA Method 6800 (SIDMS). The values are means of ± 95% confidence limits (n = 3). The percentage recoveries of total Hg and CH3Hg+ are indicated in parentheses. Extraction procedure

External calibration Hg

2+

0.12b Extraction technique, extraction reagent

a

EPA Method 6800 (SIDMS)

CH3Hg

Sum of species

Hg

5.12 ± 0.16a

5.24 ± 0.10a

0.12b

+

Temp. Time [°C] [min]

2+

mg/kg Hg

CH3Hg+

Sum of species

5.12 ± 0.16a

5.24 ± 0.10a

mg/kg Hg

A

Sonication/water bath, 25% (w/v) KOH in methanol

70

180

0.06 ± 0.02b

5.05 ± 0.13 (99 ± 3)

5.11 ± 0.13 (98 ± 3)

0.07 ± 0.02b

5.22 ± 0.31 (102 ± 6)

5.29 ± 0.31 (101± 6)

B

Sonication/water bath, 25% (w/v) TMAH in methanol

70

180

0.12 ± 0.03b

5.05 ± 0.18 (99 ± 4)

5.17 ± 0.18 (99 ± 3)

0.07 ± 0.03b

5.20 ± 0.18 (102 ± 4)

5.27 ±0.18 (101 ± 6)

C

Microwave, 5% (w/v) TMAH in methanol

180

20

0.18 ± 0.05b

4.88 ± 0.17 (95 ± 3)

5.06 ± 0.18 (97 ± 3)

0.30 ± 0.07b

5.18 ± 0.13 (101 ± 3)

5.48 ± 0.15 (105 ± 3)

D

Sonication bath, 5 mol/L HCl

25

5

0.07 ± 0.02b

4.29 ± 0.39 (84 ± 8)

4.36 ± 0.39 (83 ± 7)

0.13 ± 0.05b

5.11 ± 0.38 (100 ± 7

5.24 ± 0.38 (100 ± 7)

E

Microwave, 4 mol/L HNO3 (EPA 3200)

180

20

0.06 ± 0.04b

3.94 ± 0.12 (77 ± 2)

4.00 ± 0.13 (76 ± 2)

0.11 ± 0.07b

5.60 ± 0.33 (109 ± 6)

5.71 ± 0.34 (109 ± 6)

F

Microwave, glacial CH3COOH

165

10

0.35 ± 0.08b

3.29 ± 0.14 (64 ± 3)

3.64 ± 0.16 (69 ± 3)

0.27 ± 0.12b

5.12 ± 0.19 (100 ± 4)

5.39 ± 0.22 (103 ± 4)

G

Water bath, 1% L-cysteine hydrochloride

60

120

0.45 ± 0.10b

4.87 ± 0.20 (95 ± 4)

5.32 ± 0.22 (102 ± 4)

1.05 ± 0.14b

5.08 ± 0.25 (99 ± 5)

6.13 v 0.29 (117 ± 5)

H

Hybdridization oven, Enzymatic digestion with protease XIV

37

120

0.16 ± 0.07b

4.42 ± 0.14 (86 ± 3)

4.58 ± 0.16 (87 ± 3)

0.07 ± 0.02b

5.09 ± 0.24 (99 ± 5)

5.29 ± 0.31 (100 ± 5)

certified methyl mercury and total mercury content in Tuna Fish Tissue Certified Reference Material (ERM-CE464) supplied by IRMM (Geel, Belgium) inorganic mercury concentration was calculated as the difference between certified total mercury and methylmercury concentrations.

b

20

feature article — Speciation analysis with IC-ICP/MS

www.sepscience.com

Table 4

respectively.

Extraction procedure

Mean degree of transformation [%]

Table 3 shows the accuracy of the

Methylation

Demethylation

extraction procedures tested by

A

5±3

6±1

both external calibration analysis

B

6±2

4±1

and SIDMS using the Tuna Fish

C

3±2

6±2

Tissue Certified Reference Material

D

5±3

3±1

(ERM-CE464). For seven of the eight

E

18 ± 4

0.8 ± 0.6

extraction procedures evaluated, the

F

4±2

27 ± 5

CH3Hg values calculated by using

G

4±2

4±1

SIDMS were in good agreement with

H

4±2

1.4 ± 0.5

+

the certified reference value. Only for extraction procedure E, a too high

extraction with glacial acetic acid

occur after spiking.

methylmercury content was found.

(procedure F) and extraction with 4

Because of the unique features

Hence, transformations and losses

mol/L HNO3 (procedure E) was used.

and undisputed benefits of EPA

of Hg and CH3Hg can be directly

Owing to the relatively low ratio of

Method 6800, it is expected that

linked to pretreatment steps.

Hg2+ to CH3Hg+, the high methylation

utilization of SIDMS will increase and

Results obtained by external

rate of 18% in procedure E did not

that this valuable tool for optimizing

calibration: Based upon the

cause a significant error regarding

and validating sample preparation

data shown in Tables 3 and 4,

CH3Hg concentration. In contrast, a

procedures for trace-metal

the concentrations found for

pronounced demethylation rate has

speciation, involving extraction,

methylmercury in the alkaline

a considerable effect if, as in the case

separation and detection, will gain

extraction procedures using either

of procedure F, high CH3Hg+ to Hg2+

much wider acceptance by analytical

the ultrasonic-assisted system

ratios are prevailing. Apparently, the

chemists.

(procedures A and B) or the

elevated Hg concentration of

microwave device (procedure C) were

0.27 mg/kg stems from the

Acknowledgments

in good agreement with the certified

pronounced demethylation rate.

We thank Milestone Inc. for the

values at the 95% confidence level.

The SIDMS protocol is an invaluable

financial support to Laura H. Reyes

These procedures yielded similar

tool for overcoming nonquantitative

and for instrumental support. We

methylation and demethylation rates

recoveries and species

also thank Agilent Technologies,

(~ 6%). Although procedure G was

transformations observed during the

Applied Isotope Technologies, Inc.,

suitable for CH3Hg determination,

evaluation of extraction procedures.

Duquesne University and Metrohm

2+

+

+

2+

+

USA, Inc., for instrumental and

inorganic mercury contamination – 0.45 compared with 0.12 mg/kg

Conclusions

Hg2+ – in the extracting reagent was

Ion chromatography coupled to

observed.

an ICP/MS is a powerful tool to

References

Extraction procedures D, E, F and

determine different organic and

[1] C.W. Levenson and D.M. Axelrad, Nutr. Rev.,

H suffered from too low CH3Hg

inorganic species unambiguously

64 (2006), 139-45.

recoveries (64–86%). Despite

in one single run. In the absence

[2] US EPA Method 6800, elemental

frequent use of acid leaching for

of interconversions, traditional

and speciated isotope-dilution mass

the extraction of mercury species

speciation analysis provides accurate

spectrometry, test methods for evaluating

from tuna fish samples, the lowest

results down to the sub-ppb level.

solid waste, physical/chemical methods SW

concentration of methylmercury

Species interconversions, however,

846, Update IVA. US Government Printing

on the ERM-CE464 and the highest

require correction. SIDMS according

Office (GPO), 2007, Washington, DC.

mercury species transformation

to EPA Method 6800 is capable of

[3] L. Reyes et al., Anal. Bioanal. Chem., 390

occurred when microwave-assisted

tracing any interconversions that

(2008), 2123-2132.

+

separation science — volume 1 issue 6

material support.

feature article — Speciation analysis with IC-ICP/MS

21

A comparison of TLC methods for constructing medicinal plant fingerprints Łukasz Cieśla and Monika Waksmundzka-Hajnos Department of Inorganic Chemistry, Medical University of Lublin, Poland.

Chromatographic fingerprinting is one of the most important quality control methods in the analysis of medicinal plants. It has been widely introduced and accepted by WHO, FDA, EMEA, German Commission E, British Herbal Medicine Association and many others. HPTLC with digital scanning is becoming increasingly popular in the development of chromatographic fingerprints. In case of very complex samples, as well as those of closely related plant species or varieties, the use of multidimensional and multimodal thin-layer chromatography may become a common approach. This paper describes two modes of TLC applied for fingerprint construction: one- and multidimensional modes are described, advantages and disadvantages of the both methods are highlighted, and the techniques compared with HPLC. 22

feature article — TLC fingerprinting

www.sepscience.com

Recent decades have brought a

has gained recent attention,

validated and well described [6].

great interest in herbal medicines

especially when a lack of authentic

In TLC detection can be repeated

and botanicals, which take a more

standard substances for active

several times, with and without

balanced and holistic approach

component identification exist [4].

derivatization. In addition, there

when compared with synthetic

A chromatographic fingerprint is

is no need for complicated clean-

drugs. Dietary supplements,

a chromatogram that represents

up procedures, which are often

nutraceuticals, traditional Chinese

the chemical characteristics of

necessary in HPLC to avoid column

medicines, Ayurvedic drugs are

herbal medicine [5]. Although HPLC

contamination. Sample preparation

only a few of the great diversity of

dominates the chromatographic

does not need to be modified even

phytopharmaceuticals present on

fingerprint literature, HPTLC

if one wants to focus on different

the market.

has several unique features that

substance classes present in the

Botanicals are often considered

can offer advantages over other

extract. Correct chromatographic

to be safe, but at the same time

chromatographic techniques

systems must be chosen in

not very effective; however,

used for fingerprint construction.

order to focus on the desired

nothing could be further from

However, we would not like to

constituents group. However, in

the truth. Many herbal medicines

detract from these other techniques,

case of quantitative measurements,

can cause severe side effects,

but rather underline that HPTLC

during chromatographic fingerprint

when overdosed or not properly

can provide reliable results as far

development, HPLC is a preferable

prepared. For example a popular

as fingerprint development is

technique because of its higher

herbal drug, Stephania tetrandra,

concerned. Identification is believed

separation power [3].

is often adulterated with plants

to be one of the leading applications

All aforementioned advantages

containing hepato- and nephrotoxic

of HPTLC, as the technique offers

become even more important in

aristolochic acids [1] (see also Figure

a very visual representation of

the analysis of very complex natural

1 for S. tetrandra fingerprint and

chromatographic results [3]. The

mixtures, containing structural

reference [2]). A proper amount of

major advantage of this technique is

analogues or substances spanning a

active constituents present in the

its ability to compare many samples

wide polarity range.

herb is responsible for its curing

side by side (see Figure 1). In Figure 1

or health-promoting effects. It is,

all samples on the plate are analysed

One-dimensional HPTLC

therefore, important to confirm

under the same conditions, which

fingerprints

not only the identity, but also the

is impossible to perform using the

In traditional one-dimensional thin-

amount or ratios of the active/

sequential mode of HPLC [3]. This

layer chromatographic fingerprints,

marker compounds. The need for

advantage, in turn, leads to another

the analysed sample is compared

quality assurance tools to ensure

– rapid results, as many samples are

with the botanically authenticated

the identity, purity and quality

analysed at the same time, leadings

raw material or properly chosen

of botanical materials has risen

to solvent savings.

chemical reference standards on

dramatically. Botanical drugs are

Many factors can be changed

the same chromatoplate (Figure

usually complex mixtures, and it is

during the chromatographic process,

1) [7]. All the analysed samples

difficult to analyse them with the

which influence the resolution of

are compared with regard to the

use of techniques developed for

analysed compounds. This can also

number, sequence, colour and

synthetic drugs [3].

be considered as a drawback, as it

intensity of the separated zones.

is more difficult to obtain identical

However in very complex samples

HPTLC for fingerprint construction

conditions during development

coeluting bands may appear.

Among a variety of quality control

of each plate. For reproducible

What is more, plant extracts used

methods for herbal materials,

results, all the steps involved in

for fingerprint construction,

chromatographic fingerprinting

method development should be

very often contain constituents

separation science — volume 1 issue 6

feature article — TLC fingerprinting

23

Figure 1

chromatogram is divided into several sections, where characteristic zones are marked and briefly described [7]. However, such an approach does not provide enough information, for example, with closely related plant species, as their chemical content is very similar [8]. In such instances it is of great importance 1

2

3

4

5

6

7

8

9

10 11 12 13 14

15

to gain as much information, from the chromatogram, as is possible. Chemotaxonomic studies are easier to perform after the application of methods enabling the resolution of all, or almost all, constituents present in the sample. Even the smallest differences in content may contribute to uncovering

16

17 18

19 20 21 22

23 24 25 26

27 28 29

chemotaxonomical relationships between closely related plant species [8].

Figure 1: One-dimensional HPTLC fingerprint of different Chinese plants with respect to aristolochic acids. Chromatographic conditions: SiO2, eluent: toluene/ethyl acetate/water/formic acid (20:10:1:1, v/v/v/v), derivatization agent: Tin(II) chloride reagent, observation under UV 366 nm. 1 = Aristolochic acids mixture, 2 = Aristolochic acid A, 3 = Aristolochic acid B, 4 = Aristolochic acid C, 5-8 = Aristolochia fangji, 9-11 = Stephania tetrandra, 12-15 = Aristolochia manshuriensis, 16 = Aristolochic acids mixture, 17-18 = Clematis armandii (Mutong), 19-20 = Clematis chinensis, 21 = Aristolochia tubiflora, 22 = Akebia trifoliata, 23 = Aristolochia mollissima, 24-26 = Asarum heterotropoides, 27 = Asarum sieboldi, 28-29 = Saussurea costus (Aucklandia lappa) [2].

Multidimensional HPTLC fingerprints As already stated, with very complex

spanning a wide polarity range

phenolic acids, flavonoids, etc.).

samples traditional one-dimensional

[8]. It is very difficult to find one

Sometimes ubiquitous constituents,

fingerprints may appear insufficient,

chromatographic system that would

such as amino acids or sterols can

as it is almost impossible to develop

enable satisfactory resolution of the

also provide plant-specific profiles

appropriate analytical methods to

majority of constituents present in

[3]. When nothing or very little

represent all chemical characteristics

this type of sample. In such instances

is known about the sample, one

of constituents in a chromatogram

some constituents are usually eluted

fingerprint is usually insufficient,

[5]. Several solutions have been

with the mobile phase front, or

and generating multiple fingerprints

proposed to this problem: (i) a

remain at the start, and thus are

is recommended [5]. Traditionally,

combination of analytical methods

lost from the fingerprint. However,

in a one-dimensional fingerprint,

with different separation principles,

the separation of all sample

the sequence of zones is usually

as has been demonstrated with

constituents is not always necessary,

presented relative to a point of

Andrographis paniculata analysis

and is very often not needed for

reference (e.g., mobile phase

[10], (ii) development of “multiple

distinguishing plant species [3]. For

front, markers) [9]. The addition

chromatographic fingerprints”,

a one-dimensional fingerprint it is a

of reference substances into the

used, for example, for distinguishing

good approach to focus only on the

extract, such as dyes, should

between Radix Puerariae Lobatae

constituents that are characteristic

rather be avoided. For proper

and Radix Puerariae Thomsonii [4] or

for the analysed plant species,

chromatogram description the

for developing binary fingerprints

rather than include substances

European Pharmacopoeia proposes

of Danshen Dropping Pill (DSDP) [5],

common to many plants (e.g., some

the use of a table, in which the

(iii) application of multidimensional

24

feature article — TLC fingerprinting

www.sepscience.com

and/or multimodal thin-layer

is a must to obtain reproducible

they cannot be removed from the

chromatography methods; for

and reliable results. It should

plate and may cause difficulties

example, HPTLC fingerprints of

always be checked as to whether

in obtaining reproducible results

furanocoumarins for distinguishing

multiple dimensions lead to analyte

[13]. It is also not advisable to apply

different Heracleum spp [8].

decomposition, because this can

aqueous eluents, because water

In the analysis of botanicals and

be the main reason for obtaining

extends the drying step between

herbal preparations the following

unreliable results [11].

each development and if not quantitatively removed may lead to

modes of multidimensional planar chromatography have been

Repeated developments in one

commonly applied: comprehensive

direction

2D planar chromatography realized

Multiple development techniques

Two-dimensional thin-layer

on mono- and bilayers, coupled-

in one direction have occasionally

chromatography

layer chromatography, combinations

been applied in fingerprint

A greater number of fully resolved

of MD-PC (multidimensional planar

construction [8]. The most popular

spots can be obtained using

chromatography) techniques and

is the unidimensional multiple

two-dimensional thin-layer

hyphenated methods [11].

development (UMD) technique [13],

chromatography [16]. In this

This way of constructing

and so will be discussed here. UMD

technique the analysed sample is

chromatographic fingerprints

has been applied, for example, to

first developed in one direction, and

has several interesting features;

fingerprint development of closely

after drying is subjected to another

for example, all separated

related species of the Heracleum

development, perpendicular to the

constituents can be detected on

genus or in cases of Chelidonium

first one. If systems, orthogonal

one chromatoplate and as such are

majus and Fumaria officinalis alkaloid

in properties, are applied a great

easier to compare. With multiple

fractionation, or the analysis of

spot capacity may be obtained

chromatographic fingerprints

coumarins [14]. This method consists

[11]. This method is very useful for

information contained in two or

of the repeated development

closely related plant species, which

more chromatograms should be

of the same plate, with a mobile

are characterized by comparable

combined before employing the lot-

phase of constant composition, for

chemical profiles [8]. Usually, small

to-lot consistency testing methods;

the same distance [13]. After each

differences between the species,

for example, data fusion-based

development the plate is carefully

varieties and forms are difficult

methods have been employed

dried. In this technique spots are

to notice using one-dimensional

for this purpose [5]. In addition,

better resolved because of a spot-

fingerprints, because of spot co-

multidimensional and multimodal

reconcentration mechanism, as

elution. If orthogonal systems are

chromatography enable the analysis

repeated development leads to

applied, in perpendicular directions,

of highly complex samples, as many

narrowing of spots, similar to the

the spots are spread over the whole

parameters can be used during

effect of a preconcentrating zone

plate, and any differences in the

each development step to affect

[15].

amount and spot location are easily

the outcome of the separation.

This method should not be used

observed.

For good resolution and high spot

with chemically labile constituents;

Planar chromatography allows

capacity values, systems orthogonal

for example, carotenoids as they may

two-dimensional separations using

in properties should be used [12].

undergo chemical changes during

the same stationary phase with

However obtaining reliable multiple

the chromatographic process. The

different eluent systems or by using

chromatographic fingerprints

application of nonvolatile mobile

a stationary phase gradient [11].

with the use of multidimensional/

phase additives, such as acetic acid,

For example, graft TLC with silica

multimodal chromatography is not

DMSO (dimethylsulfoxide) and

and RP-18W plates proved to be the

simple or easy. Proper validation

buffers, should also be avoided as

method of the greatest selectivity

separation science — volume 1 issue 6

ghost peak formation.

feature article — TLC fingerprinting

25

differences for the separation of

of the plate in the second direction

Conclusion

furanocoumarins, when compared

(see Figure 2) [8].

Despite many advantages,

with one-dimensional and

One-dimensional separation of

multiple development thin-layer

comprehensive two-dimensional

structural analogues is a difficult

chromatography techniques are not

thin-layer chromatography on

task, as is observed, for example,

always a good analytical choice, and

one adsorbent (CN-silica) [15, 17].

in coumarin analysis [8]. Coumarin

it should always considered whether

Good results are also obtained

fractions from Apiaceae plant

the application of such a technique is

when different multidimensional

material are poorly resolved after

actually needed. First, these methods

and/or multimodal techniques

one-dimensional development;

are usually time-consuming, and

are combined. For example UMD

that is, the separated peaks

if their application offers no great

may be applied hand in hand with

observed on the densitogram do

improvement their use is simply

two-dimensional developments

not belong to pure compounds,

not warranted. Sometimes, changes

[8]. The application of multiple

as proved by checking the UV

in experimental setup are a better

development improves resolution

spectra at each peak’s centre

idea, as has been proven for the

of partly resolved spots in the

and flanks. Comprehensive two-

separation of fatty oils according

orthogonal direction. The use of

dimensional separation results in

to the European Pharmacopoeia

UMD in the first direction allows the

better separation of the analysed

[3], for which changing the

developing distance in the second

compounds, although some of

application mode resulted in better

dimension to be reduced, leading to

the coumarins still are poorly

separation compared with double

shorter analysis time. It is because

resolved. The application of graft

development. The use of HPTLC

of satisfactory spot resolution from

TLC enables the best separation of

plates instead of TLC ones may

the first dimension development.

these compounds. Coumarins are

also bring satisfactory results. With

In addition, UMD together with

first separated on silica gel according

multiple development techniques

methanol, for transferring spots

to differences in their polarity, and

there is always the threat of artifact

from the first to another adsorbent,

then orthogonally via differences in

formation, which may be the result

results in narrowing of starting

structure and dimension of nonpolar

of chemisorption or decomposition

bands and symmetrical, well

molecule fragments, and structural

of some constituents during the

separated spots after development

analogues are resolved (Figure 2).

chromatography process. To check

Figure 2

All @ 300 nm

700.0 600.0 500.0

700.0

400.0

(AU)

300.0

500.0

200.0

400.0

100.0

300.0 2D

200.0 100.0 0.0

5.0

0.0 100.0 (mm) 80.0 70.0 60.0 10.0 50.0 15.0 20.0 40.0 25.0 30.0 30.0 20.0 35.0 40.0 10.0 (mm) 0.0 50.0

Figure 2: Two-dimensional chromatographic fingerprint of Heracleum sibiricum fruit sample adultered with fruit of unknown Apiaceae species. The sample was first triple developed on silica with the use of eluent: 35% (v/v) AcOEt + n-heptane, after the transfer with MeOH it was redeveloped on RP-18W plate with a mixture: 50% (v/v) MeOH + H2O.Spots of adulterant are marked with arrows.

26

feature article — TLC fingerprinting

www.sepscience.com

whether the spots, that appear after

[4] S.B. Chen et al., J. Chromatogr. A, 1121

the second or further development,

(2006) 114.

are not artifacts the sample should

[5] X.H. Fan et al., Anal. Chim. Acta, 555 (2006)

be developed in two-dimensional

217.

mode, with application of the same

[6] A. Schibli and E. Reich, J. Planar

eluent in both directions [11]. All

Chromatogr., 18 (2005) 34.

spots should lie on the diagonal,

[7] E. Reich et al., J. Planar Chromatogr., 15

with artifacts located to the side of

(2002) 244.

the line. An example of this is the

[8] Ł. Cieśla et al. J. Chromatogr. A, 1207 (2008)

double development of Angelica

160.

sinensis sample, according to the

[9] C. Fang et al., J. Planar Chromatogr., 19

European Pharmacopoeia method

(2006) 348.

[3]. It must always be proved that

[10] A. Srivastava et al., Phytochem. Anal., 15

additional zones after repeated

(2004) 280.

developments are not the result of

[11] Ł. Cieśla and M. Waksmundzka-Hajnos, J.

artifacts [3]. With two-dimensional

Chromatogr. A, 1216 (2009) 1035.

separations another problem is the

[12] M. Daszykowski et al., Acta Chromatogr.,

limitation of one sample per plate,

20

and some difficulties may occur in

(2008) 283.

the identification of zones because

[13] C.F. Poole and S.K. Poole, J. Chromatogr.

the standards must be separated on

A, 703 (1995) 573.

a separate plate.

[14] M. Waksmundzka-Hajnos and Ł. Cieśla,

Before developing a

Medicinal Plants in Poland and in the World, 17

chromatographic fingerprint, the

(2009) 24.

most appropriate method should

[15] Ł. Cieśla et al., J. Planar Chromatogr., 21

be carefully considered: HPLC,

(2008) 237.

one-dimensional TLC or one of the

[16] C.F. Poole, J. Chromatogr. A, 1000 (2003)

several modes of multidimensional

963.

TLC. The amount of samples to

[17] Ł. Cieśla et al., J. Planar Chromatogr., 21

be analysed, their complexity,

(2008) 447.

chemotaxonomical relationships between the analysed plants, as well as the aim of analysis (e.g., if there is a need for quantitative results) should be taken into account. References [1] A. Blatter and E. Reich, J. Planar Chromatogr., 17 (2004) 355. [2] www.camag.com/laboratory/methods/ identification.html, accessed on 15th April, 2009. [3] E. Reich and A. Schibli, High-Performance Thin-Layer Chromatography for the Analysis of Medicinal Plants. Thieme, New York, 2008.

separation science — volume 1 issue 6

feature article — TLC fingerprinting

27

An Application notes

Artemisinin in Artemisia Annua Leaf Company: Camag Summary: Artemisinin is an antimalarial substance obtained from the dried leaves of Artemisia annua. This application note from Camag outlines an HPTLC method for the quantification of artemisinin performed on silica gel 60 with cyclohexane, ethyl acetate, acetic acid (20:10:1). Derivatization with modified anisaldehyde reagent allows specific densitometric evaluation of fluoresecence at 520 nm with a 540 nm cut-off filter. Click here to view the application

Automating Calculations for Rapid Seed Oil Quality Control and Authenticity Company: Waters Seed oils are important components of foods, cosmetics and personal-care products. They are mainly extracted from 22 oil crops around the world. Production processing, storage, transportation and distribution are all critical to the quality of seed oils, and cross contamination can occur accidentally or intentionally. This technical note describes a streamlined system solution for seed oil quality control and authentication using UPLC with and Empower 2 software custom field calculation function to automatically determine and report if a seed oil sample passes or fails user-set QC criteria. Click here to view the application

28

application notes

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High Speed, Ultra-High Sensitivity, and Robustness Needed for the Quantitation of Pharmaceuticals in Blood Plasma Company: Agilent

High Speed, Ultra-High Sensitivity, and Robustness Needed for the Quantitation of Pharmaceuticals in Blood Plasma

Summary: This application note demonstrates the performance of an Agilent

Application Note Drug discovery and development: Drug metabolism and pharmacokinetics (DMPK)

Authors

1200 Series Rapid Resolution LC (RRLC) system coupled to an Agilent 6460A Triple

0.9

R 2 = 0.9999

0.8

Stephan Buckenmaier

0.7 Relative Responses

Application Scientist Agilent Technologies, Inc. Hewlett-Packard-Straβe 8 76337 Waldbronn Germany

0.6 0.5 0.4 0.3 0.2 0.1

Quadrupole (QQQ) mass spectrometer for the bioanalysis of human plasma.

0 0

100

200

300

400

500

600

Concentration [pg/mL]

Abstract This Application Note demonstrates the performance of an Agilent 1200 Series Rapid Resolution LC (RRLC) system coupled to an Agilent 6460A Triple Quadrupole (QQQ) mass spectrometer for the bioanalysis of human plasma.

High throughput analysis is shown for a test pharmaceutical and an internal

High throughput analysis is shown for a test pharmaceutical and an internal standard with both drugs eluting well within a 1 minute chromatographic window. Ultra-high sensitivity is demonstrated by linearity across a concentration range typically covered in human microdosing pharmacokinetic studies (1–500 pg/mL plasma). Robustness and reproducibility are shown for this concentration range over a 5-day period during which the LC/MS system was exposed to more than 1000 plasma samples.

standard with both drugs eluting well within a 1 minute chromatographic window. Ultra-high sensitivity is demonstrated by linearity across a concentration range

typically covered in human microdosing pharmacokinetic studies (1–500 pg/mL plasma). Robustness and reproducibility are shown for this concentration range over a 5-day period during which the LC/MS system was exposed to more than 1000 plasma samples. Click here to view the application

Hormones Norgestrel and Ethynyl Estradiol on Allure C18 Company: Restek

Hormones Norgestrel and Ethynyl Estradiol on Allure™ C18 Peak List: 1. uracil (marker) 2. ethynyl estradiol 3. norgestrel

1

Sample: Inj.: Conc.: Solvent:

5µL 100µg/mL mobile phase

Column: Cat. #: Dimensions: Particle size: Pore size:

Allure™ C18 9164565 150 x 4.6mm 5µm 60Å

Conditions: Mobile phase: Flow: Temp.: Det.:

Summary: Restek offers and application sheet showing the separation of the hormones norgestrel and ethynyl estradiol

water:methanol (25:75, v/v) 1.0mL/min. 25°C UV @ 220nm 2

on a 150 x 4.6 mm, 5 µm (60 Å pore size) Allure C18 column,

3

using a water-methanol mobile pahse and UV detection at 0

1

2

3

4

5

min.

LC_0123

Restek Corporation 110 Benner Circle Bellefonte, PA 16823 814-353-1300 • 800-356-1688 • Fax: 814-353-1309 • www.restek.com

220 nm. Click here to view the application

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Tu Technology update

Characterizing glycans from glycoproteins Manufacturer: Waters Manufacturer’s description: Waters Corporation has introduced the UltraPerformance LC (UPLC) analytical solution for the characterization of 2-AB labelled glycans from glycoproteins. The UPLC solution combines ACQUITY UPLC BEH Glycan columns with the ACQUITY UPLC system equipped with fluorescence detection. Current FDA regulations require that firms developing and manufacturing therapeutic proteins are able to accurately characterize the

Key

glycans attached to those proteins to ensure the efficacy and safety of a biopharmaceutical product.

Email the company

According to Waters, initial customer feedback has shown the UPLC solution to be up to three times faster than traditional HPLC for the analysis of 2-aminobenzamide (2AB)-labelled

Product information

glycans from human IgG. In addition, to ensure confidence in long-term use in customervalidated methods all ACQUITY UPLC BEH Glycan columns will be

Applications

quality-control tested with relevant labelled glycan standards for consistent batch-to-batch column performance.

Additional information

Expanding the capabilities of UPLC technology Waters ACQUITY UPLC BEH Glycan columns provide superior resolution for a wide range of glycans and represent the fifth applicationtested UPLC column and eleventh UPLC chemistry added to the ACQUITY UPLC column family, claims Waters. ACQUITY UPLC BEH Glycan columns are produced with the Waters Designed for Six Sigma (DFSS) Phase II ACQUITY UPLC column manufacturing process. According to the company, this process produces superior long-term performance by maximizing efficiency while minimizing column-to-column variability. As part of a Waters ACQUITY UPLC total system solution, this new glycan UPLC column is ideally suited to help scientists get more accurate and precise quantitative results than they can get with traditional HPLC columns and systems.

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Trace Pollutant Detection in Challenging Environments Solvent-Minimized Sample Preparation for Separation Science An Advanced Proteomic Approach to the Discovery of Microbial Enzymes

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Multidimensional Gel-free Protein Separation Approaches for In-depth Analysis of Complex Proteomes

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New Approaches to Online Anti-salt Stacking for Direct Capillary Electrophoresis of Biosamples

Molecularly Imprinted Polymers for Trace Analysis of Complicated Samples

Terrorist Explosives by Analysis of Inorganic Residues

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GC-MS Analysis of Lipid Oxidation and Cholesterol Metabolism

Headspace Analysis of Plant Materials by Using Comprehensive Two-Dimensional Gas Chromatography: Selected Examples

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Determination of Biogenic Amines in Food: Conventional and Nonconventional Approaches

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One- and Two- dimensional GC-MS for Hop Metabolics

Current and Future Approaches to Speed Up HPLC Separations

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The applictaion of Quality by Design Principles to Analytical Method Development, Validation and Transfer.

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The Role of Analytical Science and Techniques in Early Phase Drug Discovery and Registration for Clinical Studies

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Online Solid Phase Extraction-LC-MS in DMPK Applications

Edward Browne

Biomarker Analysis for Preclinical Pharmaceutical R&D

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Using Narrow Bore Monolithic LC Columns to Enhance Analytical Sensitivity in Equine Doping Control

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HPLC and Hyphenated Techniques for Analysing Ingedients in Herbal Medicines

Yizeng Liang

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ICS-2100 Integrated Reagent-Free IC System Manufacturer: Dionex Manufacturer’s description: Reagent-Free IC systems with Eluent Generation (RFIC-EG systems) have been designed to deliver precise, reproducible results. According to the company, automated eluent generation minimizes the time, labour, costs and errors of manually prepared eluents. The systems are now enhanced by RFIC-ESP: electrolytic sample preparation. RFIC-ESP automates sample preparation so that manual preinjection processing such as matrix elimination and neutralization are removed. The ICS-2100 ensures you have what you need to run your samples, claims Dionex. The eluent organizer tray accommodates 1, 2 or 4 L eluent bottles, and the injection port is ergonomically designed for manual sampling. The eluent valve provides positive shutoff of eluent flow prior to the pump for easy servicing and an easy-access door opens to all chromatography components. Leak detection and management allow fast response to system leaks. The ICS-2100 employs TTL controls for external pump, injection valve, range selection, and signal offset for stand-alone operation. Key features of the ICS-2100 include: • Reagent-Free IC, RFIC-EG (eluent generation), RFIC-ESP, integrated auxiliary sample preparation valve (optional) • LCD front-panel control • Dual-piston pump • Column heater • Electrolytic suppression • Digital conductivity detection • Vacuum degas (option) • USB connectivity, plug-n-play • Optical leak detector • Electronic logbook and trending through virtual channels 32

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LCMS-2020 Manufacturer: Shimadzu Manufacturer’s description: Shimadzu has introduced a new compact LCMS-2020 single quadrupole mass spectrometer, which features the world’s fastest scanning capabilities, according to the company. Utilizing patentpending ultrafast (UF) technology, the LCMS-2020 has fast measurement speed and high sensitivity. This provides more accurate detection of trace impurities in pharmaceuticals, environmental pollutants and other contaminants. The new UFscanning technology achieves mass spectrum measurement speeds of 15,000 u/sec without sacrificing sensitivity or resolution, thus obtaining the best chromatography for the fastest LC conditions, Shimadzu states. The UFswitching technology achieves 15 ms polarity switching, enabling accurate data from the fastest chromatographic peaks without any loss of peak height. Shimadzu redesigned the ion transfer section to provide high sensitivity compared with other quadrupole analyzers for commonly measured substances. Users can now inject less and keep the analyser cleaner for longer. It also has improved high mass operation with sensitivity increased for masses above 1,000. In addition to better performance, the LCMS-2020 allows easier maintenance, permitting users to replace the ionization unit and inlet capillary to the MS from the LC, without breaking the vacuum. LCMS-2020 control and data processing are handled by an updated version of LCMSsolution software. The new software easily displays multiple sets of LC or MS data, allowing overlay and analysis of multiple data results for easy searching and comparison. The new software controls the LCMS-2020 and is fully integrated with Shimadzu’s Prominence series of ultrafast and nano HPLCs.

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Improved sensitivity with 7100 CE system Manufacturer: Agilent Manufacturer’s description: Agilent Technologies has introduced the 7100 Capillary Electrophoresis (CE) system, which it claims providing at least 10 times more sensitivity than any other commercial CE instrument. “We consider electrophoresis to be one of our core technologies and are seeing strong interest in CE in a number of areas, such as new biological drug QA/QC, environmental analysis, food safety and life sciences,” said Nitin Sood, General Manager of Agilent’s Electrophoresis business. “Used in standalone mode, as the separations component of a CE/MS or as a complementary, orthogonal technology to LC, the 7100 CE system brings unprecedented HPLC-like sensitivity to a wide range of analytical challenges.” CE is also attracting attention because the technique uses very small amounts of solvent. The 7100 system also requires 25 per cent less bench space, weighs 30 per cent less than its predecessor and uses less power. The sensitivity improvement is the result of a new detector used in combination with proprietary extended light path capillaries or a high-sensitivity cell. The 7100 offers a wide selection of detectors for flexibility and sensitivity, and the instrument is reverse-compatible to the company’s previous CE platform, so existing methods can continue to be used. The instrument performs the full range of CE separation techniques, including capillary electrochromatography for fast separation of closely related compounds. Its standard replenishment system provides high-throughput for unattended operation, and has been improved to use less buffer for the replenishment function. The Agilent 7100 CE system was designed to enhance productivity, reliability and ease 34

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of use. The rugged internal pressure system and improved capillary cooler supports higher currents and/or larger capillary diameters to increase throughput and also to enable a wider range of applications. In addition, the system comes with Chemstation software with an easyto-use graphical user interface and an improved method setup that minimizes start-up and training time. The system’s modular architecture allows fast, easy access to electrodes, prepunchers, electronics and tubing to facilitate routine maintenance and servicing. The quick-change, self-aligning capillary cartridge can be changed out in seconds. It is compatible with all commercially available capillaries. The 7100 CE provides plug-and-play connectivity to Agilent mass spectrometers (MS), combining the short analysis time and high separation efficiency of CE with the molecular weight and structural information of MS. These include the single quadrupole, time-of-flight, ion trap, triple quadrupole, ICP and quadrupole time-of-flight MS systems.

Data reprocessing software for GC/MS and LC/MS Manufacturer: Markes International Manufacturer’s description: ClearView software uses sophisticated Dynamic Background Compensation (DBC) algorithms to distinguish between chromatographic peaks and background/baseline anomalies. It reprocesses stored GC/MS and LC/MS data files, eliminating background ions from the total ion chromatogram (TIC) and improving both spectral purity and peak integration, according to Markes. The software is fast, intuitive and works with standard GC/MS and LC/MS data file formats from vendors such as Agilent Technologies, PerkinElmer, Shimadzu, Thermo Scientific and Varian. Files can be processed individually or as a batch for optimum efficiency. The company states that processing takes about three seconds per file and none of the original GC/MS or LC/MS data/information is lost. ClearView is compatible with all standard GC/MS file formats, offering significant tangible benefits and is a must have for anyone involved in trace analysis, reports Markes. Key features include: • Aids automatic identification of trace components • Eliminates noise and background • Improves the repeatability of integration/quantitation • Effective for SIM, SCAN and SIM/SCAN data ClearView is now available as a 12-month or convenient time-unlimited license. ClearView data reprocessing software is available free on a 30-day trial period. Click here to download 30-day free trial.

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