journal separation science

Volume 1 / Issue 7

June 2009www.sepscience.com

Getting the most out of your � ash chromatography

Understanding ionization techniques in mass spectrometry

Analysing biomarkers of oxidative stress

separationdriving analytical chemistry forwardsscience

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contentsVolume 1 / Issue 7

June 2009www.sepscience.com

Getting the most out of your flash chromatography





Michal Holcapek

16

feature


Volume 1 / Issue 7June 2009

research round-up

Improving performance of simulated moving bed chromatography by fractionation and feed-back of outlet streams

Comparing sample injection techniques for the CZE analysis of water-soluble vitamins

Fluorescence HPLC assay for the monitoring of L-asparaginase activity and L-asparagine depletion in children su� ering from acute lymphoblastic leukaemia

Improving detection of biomarkers for congestive heart failure with nanoLC-FT-ICR-MS


Amino acid a� nity chromatography shows promise in plasmid DNA studies

Chemotaxonomic studies to quantify iso� avones in clover

Rr

regulars

06

06

08

10

11

12

An

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application notesForensic Toxicology Analysis of Non-derivatized Drugs in Urine by Automated Solid Phase Microextraction (SPME) GCxGC-TOFMS, Leco

On-Line Two-Dimensional Separation of Intact Proteins, Dionex

Multiplexed Quantitative Peptide Assays for Protein Biomarkers of Cardiovascular Disease in Human Plasma, Applied Biosystems

HPLC and UHPLC Methods for Melamine, Grace Davison Discovery Sciences.

Carbohydrates, Organic Acids and Alcohols in Wine, Bischo�

Analysis of Pesticide Residues in Apple using Agilent SampliQ QuEChERS European Standard EN Kits by LC/MS/MS Detection, Agilent

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

34

38

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.

12

16

Mr meeting reportA Meeting to mark the o� cial retirement of Dr CK Lim.

24

Cd chrom doctorTechniques for optimizing normal- and reversed-phase � ash puri� cation

28

www.sepscience.com

scienti� c advisory

councilPeter Myers

– Chief Scienti� c O� cer

[email protected]

David Barrow

University of Cardi� , UK

Zongwei Cai

Hong Kong Baptist University

Yi Chen

Chinese Academy of Sciences,

Beijing, China

Gert Desmet

Vrije Universiteit Brussel, Belgium

C. Bor Fuh

National Chi Nan University, Taiwan

Y.S. Fung

Hong Kong University

Xindu Geng

Northwest University, Xi’an, China

Luigi Mondello

University of Messina, Italy

Paul Haddad

University of Tasmania, Australia

Hian Kee Lee

National University of Singapore,

Singapore

Melissa Hanna-Brown

P� zer, UK

Tuulia Hyötyläinen

University of Helsinki, Finland

Gongke Li

Sun Yat-Sen University, Guangzhou,

China

Yong-Chien Ling

National Tsing Hua University,

Taiwan

Klara Valko,

GSK, UK

Jean-Luc Veuthey

University of Geneva, Switzerland

Claudio Villani

Universita’ degli Studi di Roma “La

Sapienza”, Italy

Cheing- Tong Yan

Center of Environmental Safety and

Hygene, Taiwan

Edward Browne

GSK, Singapore

contactsDean Graimes

Publishing Director

+44 208 398 0209

[email protected]

Stephanie Painter

Associate Publisher

+44 1634 855 296

[email protected]

Kevin McGeehan

Associate Publisher

+44 208 398 1750

Karen High� eld

Financial Controller

Bo Zhang

Technical Editor

David Hills

Scienti� c Director

+44 1270 629496

[email protected]

Marita Kritzinger

Assistant Editor

+44 1270 629496

[email protected]

Professor Peter Myers

Chief Scienti� c O� cer

+44 151 601 2020

[email protected]

Will O’Keefe

Graphic Designer

[email protected]

rationdriving analytical chemistry forwardsdriving analytical chemistry forwardsscienccationdriving analytical chemistry forwardsscienc


technical articles on chromatography and related technologies?

updates on recent research studies?

practical advice on routine analysis?

applications of new technology?

information on commercial productdevelopments?

market trends and opinions?

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FoodEnviro

Day One:

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

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

Y.S. Fung

Eric Chun Yong Chan

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

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

Emily HilderOrganic Polymer Monoliths as Temperature Stable Stationary Phases for High Temperature Separations of Intact Proteins and Peptides

Andrew JennerGC-MS Analysis of Lipid Oxidation and Cholesterol Metabolism

C. Bor FuhImmunoassays Using Functional Magnetic Nanopaticles for Biochemical Analysis

Gurmil GendehOptimization Strategies in LC-MS/MS Front-End Separations to Achieve High Throughput, High Resolution and High Selectivity

Zhan ZhaoqiTools for Structural Elucidation & Metabolomics using IT-TOF Technology

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Day Two:

Gert DesmetCurrent and Future Approaches to Speed Up HPLC Separations

John DolanPractical Tools for the Selection of Equivalent and Orthogonal HPLC Columns

Ronald E. MajorsHPLC Column Technology: Smaller and Faster

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

Sanjay GargThe Role of Analytical Science and Techniques in Early Phase Drug Discovery and Registration for Clinical Studies.

Vinayak NadigerPharmaceutical Analysis in Regulated Environments: Perspectives, Challenges and Insights

Phil DeLandThe Next Frontier: Doubling UHPLC Throughput for Pharmaceutical Applications

Edward BrowneBiomarker Analysis for Preclinical Pharmaceutical R&D

Anne GohOnline Solid Phase Extraction-LC-MS in DMPK Applications

Ping LiHPLC and Hyphenated Techniques for Analysing Ingedients in Herbal Medicines

Yizeng LiangSeparation Science for the Quality Control of Traditional Chinese Medicine

Tung-Hu TsaiMethods and Strategy of Microdialysis for Pharmacokinetics in Herbal Medicine

Day Three:

Alastair LewisTrace Pollutant Detection in Challenging Environments

Hian-Kee LeeSolvent-Minimized Sample Preparation for Separation Science

Siu Kwan SzeAn Advanced Proteomic Approach to the Discovery of Microbial Enzymes for

Gongke LiMolecularly Imprinted Polymers for Trace Analysis of Complicated Samples

Paul Haddad

Terrorist Explosives by Analysis of Inorganic Residues

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

Bahruddin SaadDetermination of Biogenic Amines in Food: Conventional and Nonconventional Approaches

Peter Gorst-AllmanComprehensive Two-Dimensional Gas Chromatography Time of Flight Mass Spectrometry (GCxGC-TOFMS) for Environmental Forensic Investigations in Developing Countries

Robert ShellieOne- and Two- dimensional GC-MS for Hop Metabolics

Jessie TongMultidimensional Gas Chromatographic Analyses of Flavours and Fragrances

Mike KindermanNew Developments for the Analysis of Inorganic and Organic Contaminants

Hui-Lai ChinPesticides Analysis by GPC-GCMS

Yuki HashiMelamine Analysis – New Approaches

Pharma TCM

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

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RrResearchround-up

Germany

Professor Andreas Seidel-Morgenstern from

the Max Planck Institute for Dynamics of

Complex Technical Systems in Magdeburg,

Germany, has several years experience of

working with simulated moving bed (SMB)

chromatography, based on exploiting the

elegant principle of simulated moving

countercurrent movement between

stationary and mobile phases. Having seen

the potential for further improvement, a

study was conducted on a novel concept

that combines non-permanent product

withdrawal at one or both outlet ports

(leading periodically to a ‘product’ and a

‘non-product’ fraction), with an internal

recycle and re-feeding of the ‘non-product’

fraction in alternation to the original feed

mixture.

“After studying the possibilities to

systematically vary feed concentrations,

we studied the concept of partial product

withdrawal and optimized feedback. The

idea was inspired from reaction engineering

concepts based on recycling not sufficiently

converted reactants,” Professor Seidel-

Morgenstern said about the research,

published in the Journal of Chromatography A

[1207 (1-2), 55-71 (2008)].

Using simulation studies for linear and

non-linear isotherms, it was shown that in

terms of process performance and product

recovery, this fractionation and feedback

approach (FF-SMB) is superior to both the

conventional SMB process as well as to a

previously reported fractionation and discard

strategy. “The key finding is the fact that the

productivity of SMB chromatography can

be increased significantly, if the product

streams are collected at the outlets only

within certain fractions of the shift times, and

if the not sufficiently separated fractions are

recycled. This concept allows the processing

altogether of more feed and, thus, allows

higher productivities to be achieved,” he

added.

The results will be tested in the near future

for various applications. “If the theoretical

predictions can be confirmed, conventional

processes might be modified in order to

apply the new FF-SMB concept. For this, just

small modifications have to be made in the

hardware,” he concluded.

Improving performance of simulated moving bed chromatography

by fractionation and feed-back of outlet streams

6 research round-up www.sepscience.com

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Comparing sample injection techniques for the CZE analysis of water-soluble vitamins

ChinaA new online concentration method, namely pressure-

assisted field-enhanced sample injection (PA-FESI), was

developed and compared with FESI for the analysis of

water-soluble vitamins by CZE with UV detection and

described in the Journal of Separation Science [32 (7),

1011-1017 (2009)].

Main author, Dr Li Jia from the Key Laboratory of

Laser Life Science & Institute of Laser Life Science at the

South China Normal University in Guangzhou, China,

explained that the relatively low detection sensitivity

with conventional absorbance detectors in CE limits its

application in the analysis of low levels of components

in real-world samples. “In order to enhance the detection

sensitivity in CE, on-line sample concentration technique

represents an effective and versatile way,” Dr Jia added.

As part of the study, conventional sample stacking,

field-enhanced sample stacking (FESI), and two pressure-

assisted-FESI (PA-FESI) methods with and without a water

plug were developed as on-line sample concentration

methods to stack trace amounts of water-soluble

vitamins in CZE and compared. “In comparison with FESI,

PA-FESI with a water plug can achieve an increase in the

injection amounts of analytes, resulting in improvements

of concentration efficiency. FESI and PA-FESI were

suitable for the stacking of the analytes with high

electrophoretic mobilities,” Jia said. Conventional sample

stacking was a universal on-line concentration method

although the concentration efficiency for the vitamins

was less than 10. Using the PA-FESI with a water plug

method, the LODs of the vitamins were in the range from

0.1 to 0.2 ng/mL.

“The developed CZE method in combination with

PA-FESI with a water plug technique was applied to the

analysis of water-soluble vitamins in corns. The successful

evaluation of trace amounts of some water-soluble

vitamins contents (NA and FA) in corns proved that the

developed PA-FESI with a water plug method in CZE can

be applied to complex real-world samples,” he concluded.


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Fluorescence HPLC assay for the monitoring of l-asparaginase activity and l-asparagine depletion in children suffering from acute lymphoblastic leukaemia

Australia

A novel assay for the determination of l-asparaginase activity in

human plasma is described in Biomedical Chromatography [32(2)

152-159] that is based on the HPLC quantitation of l-aspartic acid

produced during enzyme incubation.

Lead author, Dr Christa Nath from the Department of Biochemistry

at the Children’s Hospital in Westmead, Australia, explained that as

originally planned in the study protocol, l-asparaginase was to be

administered every 3 days as per the BFM-95 protocol in Germany.

“However, logistical difficulties arose when drugs needed to be

administered on the weekend. At our institution we administer

l-asparaginase every 3 or 4 days, the latter to avoid administration

on weekends. We wanted to develop the assay to measure

l-asparaginase and l-asparagine to compare l-asparaginase activity

and l-asparagine depletion at 3 versus 4 days after the dose. We

wished to confirm whether there was still therapeutic l-asparaginase

activity and depleted l-asparagine at 4 days after the dose,” Dr Nath

said.

For her, the most significant outcomes of the study were that

while l-asparaginase activity at 96 h postdose was less than the

activity at 72 postdose, there was no difference in l-asparagine levels

suggesting therapeutic equivalence. “We are currently collecting data

to perform a population pharmacokinetic and pharmacodynamic

analysis of l-asparaginase. This analysis will help us to better

understand the factors that affect the decline of l-asparaginase

activity with time, how and why individuals differ, and how these

differences affect l-asparagine concentrations,” Nath added.

This method may also be used to identify those patients with

reduced enzyme activity and reduced clinical efficacy, possibly as a

result of immunological reactions, and who may be candidates for

PEG-asparaginase. It also has an application for patients receiving

PEG-asparaginase. “By measuring l-asparagine levels after the

PEG-asparaginase dose, it is possible to follow the recovery of

l-asparagine levels, which will provide some indication of whether or

not another dose is required, because this information is not clear in

current dosing guidelines,” she concluded.


11research round-upseparation science — volume 1 issue 7

Improving detection of biomarkers for congestive heart failure with nanoLC-FT-ICR-MS

USA

A paper in the Journal of Chromatography B [877 (10), 948-954 (2009)] demonstrates methods that augment the

detection of Brain (B-type) Natriuretic Peptide (BNP-32) by LC-MS. BNP-32 is a biomarker for congestive heart failure

and is measured using ELISA based assays in order to rapidly diagnose and monitor disease progression. The lack of

molecular specifi city aff orded by these assays has recently come into question as emerging studies indicate there are

potentially multiple heterogeneous forms of BNP in circulation with immunoreactive capabilities.

The study was led by Dr David Muddiman from the Department of Chemistry at North Carolina State University in

Raleigh, USA, who explained that FT-ICR mass spectrometry can aff ord unparalleled molecular specifi city, but alone

lacks the sensitivity essential for detection of low abundant biomarkers. “To increase the sensitivity and provide low

limits of detection, it is vital to develop and optimize front-end technologies, in particular, online separations. In our

most recent publication, we detail a comparison of two online sample trapping/clean-up confi gurations and a variety

of packing materials to facilitate detection of BNP-32, a clinically relevant cardiac biomarker, using nanoLC-FT-ICR-MS,”

Dr Muddiman said.

Using BNP-32 tryptic peptides, the team’s eff orts revealed that the continuous, vented column confi guration off ered

superior chromatographic performance relative to the discontinuous confi guration. “The former confi guration

utilizes a continuous bed of stationary phase segmented by a venting tee to direct fl ow during loading and elution.

Using packing material in this manner eliminates dead volume during the gradient elution, and consequently limits

diff usional peak broadening. A novel aspect of this work was the inclusion of a dummy column within the vented

confi guration to provide more rapid pressure stabilization and enhance the chromatographic performance for

tryptic peptides. Collectively, these results provide a benchmark for separation and digestion conditions of BNP-32,”

Muddiman added.

He continues that these fi ndings have resulted in the adoption of the continuous, vented column confi guration

within all their research endeavours utilizing online separations. “To date, this confi guration has been applied to

separations of cell lysates, plasma protein digests, and glycans cleaved from glycoproteins in plasma. For future

studies involving analysis of BNP-32 our fi ndings will serve to facilitate detection and identifi cation of the cardiac

biomarker and its alternative forms,” he concluded.

Amino acid affi nity chromatography shows promise in plasmid DNA studies

Portugal

In a study published in Biomedical Chromatography [23 (2), 160-165 (2009)], focused on the development and

establishment of new chromatographic strategies to purify pDNA, it was possible to implement a new affi nity

chromatography approach, named as amino acids-DNA affi nity chromatography, to purify pDNA. Two diff erent

strategies based on increased sodium chloride (225-250 mm) or arginine (20-70 mm) stepwise gradients are described

to purify supercoiled (sc) isoforms. According to main author, Dr João Queiroz from the Centro de Investigação em

Ciências da Saúde at Universidade da Beira Interior in Covilhã, Portugal, this proves that well-defi ned binding/elution

conditions are crucial to enhance purifi cation performance, resulting in an improvement of the fi nal plasmid yields

and transfection effi ciency.

“In the past few years the main fi eld of our research has been the development of new chromatographic strategies

to effi ciently purify plasmids,” Dr Queiroz said. Gene therapy and DNA vaccination have emerged as two potentially

revolutionary DNA-based therapies. New products have to be developed and the improvement of biotechnological

processes will represent a great advantage in implementing these platforms in the biopharmaceutical industries.

Analysing biomarkers of oxidative stressUSAA robust and rapid high-pressure liquid

chromatography–electrochemical detection (HPLC–ECD)

method was developed and validated for the accurate

determination of ascorbic acid and uric acid in human

plasma. The study was led by Dr Xingnan Li and Professor

Adrian Franke, Director of the Analytical Laboratory

Shared Resource at the Natural Products & Cancer

Biology Program in the Cancer Research Center of Hawaii,

USA, and documented in the Journal of Chromatography B

[877 (10), 853-856 (2009)].

“Ascorbic acid (AA) and dehydroascorbic acid (DHAA)

are important biomarkers for evaluating oxidative stress.

During our ongoing research on oxidative stress factors

and their diagnostic value in disease risk assessment, we

intended to establish a fast, accurate and robust assay for

AA and DHAA analyses,” Professor Franke explained.

The team established an optimized, validated, robust

and rapid HPLC assay with electrochemical detection

(ECD), specifi cally by coulometry, using homogentisic

acid as a stable internal standard for the simultaneous

determination of AA and UA. “DHAA is not responsive

by ECD and is calculated by subtracting native AA from

total AA. The latter is determined after reducing native

DHAA to AA using dithiothreitol. This method was

further improved by applying ultra-HPLC techniques

using 1.9 μm Hypersil Gold C18 columns which

increased sensitivity, lowered detection limit and solvent

consumption twofold, and reduced analysis times over 4

times to 2.5 min. Both methods showed good precision

and accuracy,” Franke said.

After comparing various methods recommended for

clinical and translational research the team presented an

optimized, validated and robust HPLC–ECD method for

the quantifi cation of serum ascorbic, dehydroascorbic,

and uric acid using homogentisic acid as internal

standard. “We also reported on a highly effi cient and

robust UHPLC method for fast turn-around ascorbate

and urate analyses. Our results provide fast, accurate,

and robust method for AA, DHAA and UA analyses from

serum or plasma for clinical and other research,” he

concluded.

Analysing biomarkers of


“The clinical application of plasmid DNA (pDNA) obliges to its recovery and manipulation

with a high level of purity and biological activity. However, because of the biosynthesis of

pDNA in recombinant organisms such as Escherichia coli, the primary pDNA recovery occurs

in a highly impure extract,” he explained. The original idea on the basis of his research was the

application of amino acids to exploit an affinity-based and specific interaction with the pDNA

in order to efficiently purify this biomolecule from all the impurities. This idea was supported

by the natural occurrence of interactions between proteins and nucleic acids molecules, at

biological level.

“Histidine and arginine have been used as amino acid ligands and their ability to isolate

the active sc pDNA proved the presence of specific interactions of pDNA with both amino

acid-based matrices,” he said. The potential of histidine affinity chromatography to efficiently

separate sc pDNA from host impurities was further demonstrated in a study which showed

that only sc pDNA interacted with the histidine ligand, whereas the other isoforms of pDNA

and contaminants did not.

“The pDNA obtained with this histidine affinity approach was of high quality and

contamination with gDNA and endotoxins was within acceptable levels, while neither

RNA nor proteins were detected. Transfection experiments using this purified pDNA also

confirmed that it could efficiently drive gene expression in eukaryotic cells,” he added.

According to Queiroz, the relatively low yield and the high salt concentrations required for

pDNA elution using this strategy highlighted the need for further improvements. “The studies

that employed arginine matrices to purify pDNA revealed the presence of specific interactions

with plasmid molecules, and importantly a significant recognition of the sc isoform. The

multiple interactions that arginine-based matrix was able to promote, allowed the differential

recognition of the biomolecules present in E. coli lysates, representing an important insight to

pDNA purification process. Using this simplified sc pDNA purification process, the majority of

the contaminants were removed, a 79% yield was achieved, sc pDNA was purified under mild

conditions, and showed to be extremely efficient (62%) on cells transfection. Hence,

arginine-chromatography proved to be an interesting option as a late-stage plasmid

purification step,” he explained.

In his view, amino acids-based affinity chromatography represents a particularly promising

approach, because it combines the selectivity of a biological interaction with the simplicity

of a single small molecule as chromatographic ligand. However, the low capacity of available

supports for pDNA still remains to be solved, as well as the low diffusivity of pDNA samples

resulting from their molecular weight. This will require further efforts in order to design more

suitable ligands and supports, in a similar way to the developments already seen for protein

purification matrices.

“Hopefully, some of the findings concerning the chromatographic interactions between

pDNA and amino acids can be applied in future bioseparation methods both for preparative

and analytical purposes. For the moment, it will be intended to apply this purification

strategy in the purification of a specific plasmid with genetic information for p53 expression.

In this particular gene-based treatment, it is expected to recover a delivery system capable

of inserting the p53 gene into tumour cells, thereby inducing cell death. Other research

groups are investing in this field and several studies are in progress. It is considered of crucial

relevance to proceed with this research with the global aim to find an efficient process to

obtain a highly pure pDNA product, efficient on cancer therapies,” he explained.


Chemotaxonomic studies to quantify isoflavones in clover

Poland

A recent paper in the Journal of Separation Science [32 (7), 965-972 (2009)] detailed chemotaxonomic studies

undertaken to establish the qualitative profile and actual amounts of the pharmacologically active isoflavone

aglycones genistein, daidzein, formononetin and biochanin A in aerial parts of thirteen Trifolium L. (clover) species,

native to Poland. A newly elaborated micropreparative technique, developed by Dr Grazyna Zgórka from the

Department of Pharmacognosy with Medicinal Plant Unit at the Medical University of Lublin in Poland,— SPE on

BakerBond octadecyl, cyclohexyl, and phenyl cartridges — was used in combination with ultrasound-assisted

extraction for isolation of isoflavone aglycones from hydrolysed samples.

“For many years I have been working on phytoestrogens, that nowadays attract considerable scientific research

because of health benefits related to menopausal problems and some biodegenerative diseases such as breast and

prostate cancer, osteoporosis, as well as cardiovascular disease, combined with atheromatosis,” Dr Zgórka explained.

Trifolium L. (clover) is an important genus of the Leguminosae (Fabaceae) family containing phytoestrogenic

isoflavone constituents. Isoflavone glycosides and malonylglycosides are metabolized in the mammalian and

human small intestine, releasing pharmacologically active aglycones. Effective extraction techniques, followed by

appropriate hydrolytic procedures are needed to determine the real, total concentration of non-glycosidic isoflavone

phytoestrogens in plant samples examined. “A knowledge on verified, factual amounts of isoflavone aglycones is

undoubtedly of special importance in the establishment of the proper dosage of clover extracts, while performing

in vivo experiments or clinical studies, and for further accurate evaluation of pharmacological activity of these

constituents,” Zgórka added.

The key findings of her work comprised the elaboration of a new micropreparative technique — solid-phase

extraction (SPE) on BakerBond octadecyl, cyclohexyl and phenyl cartridges, in combination with

ultrasound-assisted extraction (UAE), for isolation of isoflavone aglycones from clover samples. “I

documented very high recoveries (> 96%) for four isoflavones using these

micropreparative techniques. To obtain the most reliable qualitative

and quantitative results, I employed photodiode-array (PDA) and

fluorescence (FL) detection, coupled with reversed-phase LC. Using

optimized preparative and detection techniques, I aimed to establish

the clear chemotaxonomic differences, combined with flower colour

variability, within thirteen clover species. Clovers with pink or purple-red

flower corolla possessed the highest concentration levels of isoflavones

(e.g., 3300 µg/g dry wt in Trifolium medium), while in three yellow

flowering clovers (T. aureum, T. dubium and T. campestre) these compounds

were not detected at all,” she added.

Dr Zgórka believes the elaboration of effective extraction techniques (UAE

and SPE), followed by acidic hydrolysis and RP-LC coupled with simultaneous

DAD and FL detection, may be important for both phytochemical

studies, concerning accurate and precise determination of

isoflavone phytoestrogenic compounds in plant material, and

chemotaxonomical investigation of some species belonging

to the Leguminosae (Fabaceae) family. “The method may be

useful in the selection process of new plant sources rich in

active isoflavone bioconstituents,” she concluded.


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18 feature article — Ionization techniques in MS www.sepscience.com

Understanding ionization techniques in mass spectrometryMichal Holcapek, University of Pardubice, Czech Republic

Ionization is the process whereby neutral molecules are converted

into charged species (i.e., ions). The conventional electron ionization

technique can only be used for low-to-moderate molecular weight volatile

compounds, while soft ionization techniques are suitable for the ionization

of a wide range of molecules, regardless of their polarity and molecular

weights, and are capable of creating both positively or negatively

charged ions. Soft ionization techniques, especially atmospheric pressure

ionization techniques (e.g., ESI, APCI and APPI), are well suited for HPLC/

MS coupling. This article on LC/MS describes electron ionization (EI),

chemical ionization (CI) and electrospray ionization (ESI).

19feature article — Ionization techniques in MSseparation science — volume 1 issue 7

identifi cation of the peaks

eluting from the GC column, a

process simplifi ed by the

availability of extensive EI

spectral libraries.

In EI a rhenium or tungsten

fi lament heated to high temperature

produces thermal electrons, which

are accelerated towards an anode by

establishing a potential diff erence.

Large libraries of EI mass spectra

are used for fast identifi cation of

unknown analytes. The high energy

electrons interact with neutral

molecules in the ion source, violently

ejecting valence electrons, a process

represented as:

M + e- M+. + 2e-

and is applicable only for volatile

compounds because it is a gas-phase

ionization technique. EI is considered

a “hard” ionization technique

because the excess internal energy

deposited in the analytes during

the ionization process may lead to

extensive fragmentation and even

to the total absence of the molecular

ion M+. for ~10% of volatile organic

compounds.

EI is mostly coupled to gas

chromatography. This pair is

particularly well suited for several

reasons:

• GCandEIoperateinsimilar

concentration and polarity

ranges,

• EIfragmentationallows

An enormous number of compounds

can be found in nature or are

prepared synthetically. They can

diff er in molecular weights, polarity,

volatility, thermal stability and many

other physicochemical properties.

Because of this variety of properties,

there is no perfect analyser, and

thus mass spectrometers employ

a number of diff erent ionization

techniques. In fact, there are

commercial instruments available

with interchangeable ionizers so

that the ionization technique can be

tailored to the analyte.

Electron ionization (EI)Electron ionization (EI) was the fi rst

ionization technique used in MS,


Chemical ionization (CI)Chemical ionization (CI) is the soft

ionization technique used in

GC/MS. It is a modifi cation of EI,

whereby high energy electrons are

fi rst used to ionize a reacting gas,

which goes on to ionize the analyte

molecules. Fragment ions intensities

are still higher compared with

API techniques used for HPLC/MS

coupling nowadays.

• Thereactiongasisintroducedin

the ionization chamber at a

pressure of approximately

50-100 Pa (~3 x 10-3 to 7 x 10-3

atm).

• Becauseoftheexcessofthis

reactant gas in the source, the

accelerated electrons collide fi rst

with the reaction gas.

• Theresultingreactiongasions

then react with the remaining

neutral molecules of reaction gas

by a series of ion-molecule

reactions.

• Thesecomplexreactionswill

fi nally achieve a relatively

steady-state composition of

reaction ions (e.g., CH5, C2H5 and

C3H5 for methane) which can

fi nally ionize the analyte by ion-

In principle, EI has rather high

vacuum requirements (about

10-5 Pa) to protect the heated

fi lament, which would burn at

elevated pressures, and to avoid

unwanted ion-molecule reactions

within the ion source. The molecular

and fragment ions formed in the ion

source are forwarded to the mass

analysed region by the repeller

electrode and a system of other

focusing and acceleration electrodes

referred to as ion optics.

Particle beam interface

HPLC/MS is mostly coupled to

atmospheric pressure ionization

techniques. Occasionally, however,

EI is used with HPLC separations

of moderately polar molecules in

the 200-800 Da range (e.g., certain

drugs and pesticides) in order to take

advantage of EI spectral libraries.

Such a measurement employs a

particle beam interface to produce

the gas-phase molecules required

for EI.

A particle beam interface transfers

molecules to a gas-phase (Figure 2).

The HPLC effl uent is:

• Nebulizedbyastreamofhelium

(1 and 2)

• Furtherevaporatedinan

evaporation chamber heated to

about 50-80 °C

• Separatedinatwostage

molecular jet separator (4).

• Thelightermobilephaseand

helium nebulizing gas are

selectively pumped off (3)

by vacuum pumps while heavier

analyte molecules continue to

the EI ion source (5).

Figure 2

Figure 2: Graphical reprsentation of a particle beam interface.

© CHROMEDIA

4

5

32

1

Figure 1

© CHROMEDIA

Ions

Ionisation chamber

Anode trap

Heated filament

Electronbeam

Repellerplate

Sample

MoleculesTo

analyser

Figure 1: The ionization principle of the EI technique.


molecule reactions.

CI is used in either polarity mode

and generates mainly even-electron

ions, for example [M+H]+ in the

positive-ion mode or [M-H]- in

the negative-ion mode plus some

fragment ions. Understanding this

ionization mechanism is useful for

the explanation of APCI and other

modern soft ionization techniques.

Electrospray ionization (ESI)ESI is the softest ionization technique

and is well suited for biomolecules,

organometallic compounds, non-

covalent complexes, gas phase

reactivity studies, etc.

The introduction of electrospray

has proven to be a great tool in

biochemistry, allowing the mass

spectrometric characterization and

sequencing of peptides, proteins

and other biopolymers of great

importance to human life and

medicine. In an ESI source:

• Thecolumneffluentisdirected

through a stainless steel capillary.

• Ahighvoltageisappliedtothe

© CHROMEDIA

Ions

Solvent spray

Heated nitrogen drying gas

Dielectric capillary entrance

Nebulizer gas

Figure 4

Fig 4: A set up with Nitrogen as ‘drying gas’ .

Figure 3

© CHROMEDIA

Mobile phase

Capillary- 3 kV

Spray of fine droplets

Taylor cone

Ion evaporation

Desolvation gas

Solvent evaporated from droplets Coulombexplosion

Fig 3: The desolvation gas speeds up the evaporation and explosions.

capillary (ca. 3-5 kV), which is

kept in a coaxial flow of nitrogen

nebulizing gas (Figures 3 and 4).

• Creatingafineaerosolofvery

small droplets, each of which

carries many excess charges at its

surface.

• Thedropletsizeisfurther

diminished in the ion source

region with counter-flow of

heated drying gas due to solvent

evaporation from the droplet

surface (Figure 5).

• Whenthechargedensityatthe

droplet surface reaches a critical

value (the Rayleigh limit), a so

called Coulombic explosion

occurs and several even smaller

droplets are formed, each

carrying some fraction of the

original droplet’s surface charge.

• Theprocessofsolvent

evaporation, droplet contraction

and Coulombic explosions is

repeated until the molecular

adducts are released from the

final droplet.

If a positive voltage is applied to

the capillary, then the droplets will

carry positive charges and finally

positive ions are formed, such as

[M+H]+and[M+Na]+ adducts.

In the negative-ion mode, the base

peak is typically the [M-H]- ion.

Because many mass analysers (and

ion detectors) have an effective

upper m/z limit, ESI is notable for

the formation of multiply charged

ions, which significantly extends

the mass range up to and beyond

100,000 Daltons. This is of obvious

importance to measurement of

biopolymers.

An example: Let´s consider the

simple example of a protein with


amolecularweightMW=60,000

Da. The molecular adduct with for

example60protonswillprovide

a signal at m/z=(60,000+60)/60

=1001.Inpractice,weobservea

distribution of multiply protonated

molecules, each with a diff erent

number of charges, as shown in the

figure.Wecaneasilydeterminethe

MWforunknownproteinsusing

simple algebra:

1. First, we assume that the ion

at m/z 1049.8 carries an unknown

number of charges x, which is

related to its m/z by the equation:

1049.8=(MW+x)/x.

2. Likewise, the neighboring ion

at m/z 991.5 carries an unknown

number (x+1) of charges, giving

the relationship:

991.5=(MW+x+1)/(x+1).

The solution of these two

equations with two unknowns will

yield both the charge numbers of

the ions and the molecular weight of

the protein.

This article was written by Michal

Holčapek, Professor of Analytical

Chemistry, University of Pardubice,

Czech Republic.

Publication of this article was made

possible through collaboration with

Chromedia.

Figure 6

Rela

tive

inte

nsity

(%)

800 1000 1200 1400 1600m/z

100

80

60

40

20892.4

939.2

991.51049.8

1115.5

1189.6

1274

1372.5 1486.6

20+

19+

18+17+

16+

15+

14+

13+12+

Figure 6: ESI spectrum of multiply charged protein (Lysozyme).

Figure 5

© C

HRO

MED

IA

Evaporation Analyte ion ejected

Fig 5: In ESI the droplet size decreases and charged + ions are formed.

Special Subscription Off er“We are a worldwide community

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Recommended Further Reading

Click titles for more information

The Mass Spectrometry Topic Circle

Analysis of PAH’s in foods

LCMS of pesticides

LCMS of lipids

Chromedia is pleased to off er its members a FREE

download of GCXGC:

GCXGC, a 144 page PDF by multidimensional GC expert

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The Mass Spectrometry Topic Circle

Analysis of PAH’s in foods

LCMS of pesticides

LCMS of lipids

RECENRECENF

4–6 November

09.e

u TOPICS focused oBioanalytical TecApplications in are

Residues and conAuthenticity, trac

afa2

00

Authenticity, tracFlavours and odouProcessing and paMycotoxins, mariAllergensGenetically modifNanoparticlesN l f d t

ww

.ra Novel foods, nutr

QA/QC and chem

OTHER EVENTS• A workshop focused

research within the 7programmes in collab

ww p g

Centre AS CR• Vendor seminars intr

advanced food contr• A Seminar on issues a

food: US perspective

SPECIAL OPPORA i f • An opportunity for yothe main program

• IAEAC and RAFA 200• The best poster pres

poster award

DEADLINES: August 1, 2009 Rg , 009July 31, 2009 AAugust 31, 2009 AOctober 15, 2009 LaDecember 6, 2009 M

sp

Symposium chair PSymposium co-chair PSymposium co chair P

4th International Symposium on

T ADVANCES IN T ADVANCES IN FOOD ANALYSISr, 2009 Prague, Czech Republic

on Recent Advances in Analytical & hnologies and Emerging Food-Related eas:

taminantsceability, fraudceability, fraudursackaging contaminants ne and plant toxins

fied organisms (GMO’s)

iti l l t i f dritional supplements, organic foodsmometrics in food analysis

S: on opportunities for young scientists to take part in

7th Framework Programme and other related EC boration with Joint Research Centre and Technology J gy

roducing recent instrumentation and strategies for oland approaches to address chemical contaminants in

e

RTUNITIES FOR YOUNG SCIENTISTS: i i l ib i i hi oung scientists to present an oral contribution within

09 Student Travel Grants sentation honoured with the IAEAC Roland W. Frei

Reduced registration fee deadlinegAbstract submission deadline (oral presentation)Abstract submission deadline (poster presentation)ast minute poster deadline

Manuscript’s submission deadline for publication in pecial issue of Analytica Chimica Acta

rof. Dr. Jana Hajslova (ICT, Prague)rof. Dr. Michel Nielen (RIKILT, Wageningen)rof. Dr. Michel Nielen (RIKILT, Wageningen)

MrMeeting report

Applications in Clinical Biochemistry 2009 A Meeting to Mark the Official Retirement of Dr CK Lim

Chang Kee Lim, latterly of Birkbeck College, University

of London, has been a leader in the application of

separation science in biochemistry for many years. A

capacity audience at Guy’s Hospital, London met to pay

tribute to him and his achievements, and to discuss

current trends in the use of separation science in clinical

chemistry. Sponsorship from ABS, Agilent, ESA, Jasco,

Jaytee Biosciences, Thermo Scientific and Waters is

gratefully acknowledged.

In opening the meeting, David Perrett (Queen Mary,

University of London) recounted some milestones in

Dr Lim’s career. A BSc in Chemistry and Zoology was

followed by a PhD from Westfield College, University of

London. In 1973 Chang Kee went to work with Professor

Charles H. Gray at King’s College Hospital Medical School.

There he began to work on porphyrin metabolism, which

he continued at the Clinical Research Centre (CRC),

A joint meeting of the Royal Society of Chemistry, Separation Science Group and the

Association for Clinical Biochemistry Southern Region

Northwick Park, 1976-1991. With the closure of the CRC,

Chang Kee moved to the MRC Toxicology Unit, firstly at

Carshalton and then in Leicester.

In 2000 Chang Kee moved back to London, still under

the auspices of the MRC, to establish a BioAnalytical

Science Unit at Birkbeck College. Although retired,

Chang Kee continues to edit the journal Biomedical

Chromatography that he helped establish in 1986. Prof.

Perrett also recalled the meeting ‘High Pressure Liquid

Chromatography in Clinical Chemistry’ Chang Kee

organized at King’s College Hospital in 1975, and the

ensuing book ‘HPLC of Small Molecules’ (IRL Press, 1986)

that was a classic in its day.

Porphyrins 40 Years OnThe session Chair, Dennis Wright (Northwick Park

Hospital) recounted the valediction ‘look at the

chemistry’ that guided him in his early PhD studies on

porphyrins with Chang Kee. Porphyrins have been Chang

Kee’s abiding interest, and in the opening lecture he

emphasised the contribution of HPLC and of LC-MS/MS

to the study of porphyrins and the porphyrias.

Two topics stood out. First, it had been claimed in

a paper published in the Proceedings of the National

Academy of Sciences, USA, in 2007 that a porphomethene

inhibitor of uroporphyrinogen decarboxylase, namely

uroporphomethene (m/z 835), was the cause of

porphyria cutanea tarda. However, not only was the LC

retention of ‘uroporphomethene’ incompatible with

what would be expected of a porphomethene, but also

fragmentation characteristic of polyethylene glycol (m/z

835 with product ions at 791, 747, 703 and 659; that is

to say, successive loss of OCH2CH2) was present. This was

wrongly interpreted as loss of CO2 from the porphyrin Chang Kee Lim and wife, Soo.

24 meeting report www.sepscience.com

carboxyl groups.

On a related theme, 2-vinyl 4,6,7-tripropionic acid

porphyrin (harderoporphyrin) had been reported

in the Harderian glands of rodents. However, work

using a neutral extraction procedure and LC-MS/MS

showed that ‘harderoporphyrin’ was protoporphyrin

monoxyloside, protoporphyrin itself and protoporphyrin

monoglucoside being minor components of the gland.

Again, understanding the underlying chemistry of the

analytical procedure was vital to a proper understanding

of the natural products under study.

On a lighter note Chang Kee outlined work undertaken

early in his ‘retirement’ in conjunction with the Centre

for Ornithology, Birmingham and the Natural History

Museum on the origin, evolution and functions of

eggshell colours and patterning. Porphyrins again

– it seems that the colouration of sparrowhawk and

emu eggshells, for example, is due to the presence of

biliverdin and protoporphyin in different proportions.

Vitamin D, Purines, and Drugs of Abuse

Sandra Rainbow (Northwick Park Hospital) pointed

out that vitamin D research was very active, with some

1,000 papers being published in 2007 alone. LC-MS/MS

was rapidly becoming the method of choice for clinical

25-OH-D2 and 25-OH-D3 analysis. Derivatization was

not required and there were advantages of selectivity

over immunoassays. However, an internationally agreed

reference material was lacking and measurement of

1,25-dihydroxyvitamin D remained challenging. Sandra

also drew attention to work using chiral LC-MS/MS that

demonstrated the presence of both 25-OH-D3 and of

epi-25-OH-D3 in patient samples.

Tony Marinaki (GSTS Pathology) outlined work in

screening for inborn errors of purine and pyrimidine

metabolism. For purine analysis a 250 x 3 mm i.d. Waters

Spherisorb ODS1 column with gradient elution gave

a 33 min analysis time. Use of a bridged ethyl hybrid

(BEH) C18 column (Waters Acquity) (1.7 µm aps) gave

improved speed of analysis, resolution, and sensitivity,

but with a higher column back pressure. However, more

recent work had suggested that Supelco Ascentis Express

Halo columns (2.7 µm particles) could give analogous or

better results at lower back pressures.

Finally, Richard Evers (King’s College Hospital) discussed

problems and pitfalls in clinical drugs of abuse screening.

LC-MS/MS offered lowered consumables costs and the

ability to identify unequivocally a range of underivatized

analytes. However, sequential analysis limited

sample throughput, the potential for ion suppression

necessitated great care in method validation, and sample

hydrolysis was still needed in the event that sensitivity

towards morphine and buprenorphine glucuronides was

poor or non-existent. The potential for use of ion trap and

accurate mass technology in overcoming these problems

was just beginning to be developed.

From Tswett to MetabonomicsAfter lunch, David Perrett began by paying tribute

to another pioneer in the application of analytical

methods to clinical problems, Professor Charles Enrique

Dent (1911–1976). Using the then new technique

of 2-dimensional paper chromatography, David

felt that Dent had probably discovered/developed

major insights into more diseases (including Fanconi

syndrome, Hartnup disease, argininosuccinic aciduria,

homocystinuria, cystinuria, and xanthinuria) than anyone

else. David then discussed his own recent research

using capillary electrophoresis with low-wavelength UV

detection in which some 80 compounds can be detected

in urine in 10 minutes. He also took the prize for the

oldest sample discussed during the day with a slide of

the CE-DAD analysis of organic compounds in an extract

of an Egyptian mummy (site of sampling not stated)!

25meeting reportseparation science — volume 1 issue 7

David’s talk neatly picked up the theme initiated

by Chang Kee Lim earlier in the day – it is not simply

‘hypothesis driven’ research (with which funding

bodies seem sometimes to be obsessed) that advances

knowledge, but more often than not it is either the

application of new techniques to old problems, or

the critical appraisal of existing data that leads to key

developments.

Having discussed aspects of ICP-MS operation,

including GC- and LC-ICP-MS, Andrew Taylor (Royal

Surrey County Hospital) gave a brief overview of

metallomics (chemical speciation, dynamics and kinetics

of trace elements in biological systems). Applications

discussed included isotope measurement to help identify

sources of lead exposure, the use of 65Cu administration

as an additional diagnostic test in Wilson’s disease,

diff erentiation of arsenic species in urine, and recent

work aimed at unravelling the complexities of selenium

metabolism. An interesting aspect was the use of

selenium isotope patterns to help identify selenium-

containing species in complex chromatograms. Potential

pitfalls such as ‘polyatomic’ (e.g., 40Ar2+ on 80Se+) and

‘isobaric’ (e.g., 64Ni+ on 64Zn+) interferences were given

due prominence. A clinical problem of gadolinium used

as contrast medium was discussed – 156Gd2+ has the same

m/z as 78Se+.

Edwin Carr (St Helier Hospital, Carshalton) next

discussed catecholamine measurement using HPLC with

electrochemical detection (ED). Nowadays the use of ED

in clinical chemistry is virtually confi ned to diagnosis of

phaeochromocytoma, with urine being the specimen of

choice unless the patient has renal failure. There is almost

universal acceptance that, at present, metanephrines

analysis is superior to other means of diagnosing this

condition, yet the evidence for this is still ignored by

many.

The fi nal talk returned to the ‘omics’ theme with Paul

Thomas (University of Loughborough) discussing volatile

profi ling using GC-MS and other techniques. After

describing investigations into breath volatiles in, for

example, chronic obstructive pulmonary disease, Paul

outlined work to develop a hand-held ‘sniff er’ device

based on ion mobility MS to seek out people trapped as

a result of an earthquake, for example. It seems that not

only do specially trained sniff er dogs have a very short

working period (20 min or so) before needing prolonged

rest, but also suff er high mortality (up to 70 % was

quoted), hence there is a real need for such a device.

All-in-all the day was a fi tting tribute to Chang Kee Lim

and his contribution to the application of separation

science in biochemistry.

This report was compiled and written by

Bob Flanagan

26 meeting report www.sepscience.com

Register Now for your 20% Early Bird Discount Conference Highlights

Singapore


FoodEnviro

Day One:

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

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

Y.S. Fung

Eric Chun Yong Chan

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

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

Emily HilderOrganic Polymer Monoliths as Temperature Stable Stationary Phases for High Temperature Separations of Intact Proteins and Peptides

Andrew JennerGC-MS Analysis of Lipid Oxidation and Cholesterol Metabolism

C. Bor FuhImmunoassays Using Functional Magnetic Nanopaticles for Biochemical Analysis

Gurmil GendehOptimization Strategies in LC-MS/MS Front-End Separations to Achieve High Throughput, High Resolution and High Selectivity

Zhan ZhaoqiTools for Structural Elucidation & Metabolomics using IT-TOF Technology

Bioscience

sponsors:

For all delegate enquiries email [email protected]

26–28 AugustBiopolis Science Park, Singapore

Day Two:

Gert DesmetCurrent and Future Approaches to Speed Up HPLC Separations

John DolanPractical Tools for the Selection of Equivalent and Orthogonal HPLC Columns

Ronald E. MajorsHPLC Column Technology: Smaller and Faster

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

Sanjay GargThe Role of Analytical Science and Techniques in Early Phase Drug Discovery and Registration for Clinical Studies.

Vinayak NadigerPharmaceutical Analysis in Regulated Environments: Perspectives, Challenges and Insights

Phil DeLandThe Next Frontier: Doubling UHPLC Throughput for Pharmaceutical Applications

Edward BrowneBiomarker Analysis for Preclinical Pharmaceutical R&D

Anne GohOnline Solid Phase Extraction-LC-MS in DMPK Applications

Ping LiHPLC and Hyphenated Techniques for Analysing Ingedients in Herbal Medicines

Yizeng LiangSeparation Science for the Quality Control of Traditional Chinese Medicine

Tung-Hu TsaiMethods and Strategy of Microdialysis for Pharmacokinetics in Herbal Medicine

Day Three:

Alastair LewisTrace Pollutant Detection in Challenging Environments

Hian-Kee LeeSolvent-Minimized Sample Preparation for Separation Science

Siu Kwan SzeAn Advanced Proteomic Approach to the Discovery of Microbial Enzymes for

Gongke LiMolecularly Imprinted Polymers for Trace Analysis of Complicated Samples

Paul Haddad

Terrorist Explosives by Analysis of Inorganic Residues

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

Bahruddin SaadDetermination of Biogenic Amines in Food: Conventional and Nonconventional Approaches

Peter Gorst-AllmanComprehensive Two-Dimensional Gas Chromatography Time of Flight Mass Spectrometry (GCxGC-TOFMS) for Environmental Forensic Investigations in Developing Countries

Robert ShellieOne- and Two- dimensional GC-MS for Hop Metabolics

Jessie TongMultidimensional Gas Chromatographic Analyses of Flavours and Fragrances

Mike KindermanNew Developments for the Analysis of Inorganic and Organic Contaminants

Hui-Lai ChinPesticides Analysis by GPC-GCMS

Yuki HashiMelamine Analysis – New Approaches

Pharma TCM

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

CdThe Chrom

Doctor

Techniques for optimizing normal- and reversed-phase flash purification

Traditional flash purification techniques can

prove challenging and time-consuming

for chemists, and many organic synthesis

laboratories are now looking to new

methods to achieve better separations.

This article will examine the parameters

for achieving effective results from flash

chromatography and will discuss method

development examples to illustrate how to

get the most out of flash chromatography.

Setting purification goalsTo effectively use flash chromatography

chemists need to define purification goals.

1. Purity (how pure do I need my product?)

2. Yield (how much product do I need to

isolate?)

3. Speed/throughput (how fast do I need

the pure product?)

Understanding these goals will help with

creating an efficient purification method.

Purification modesFlash chromatography, like other liquid

chromatography techniques can be

performed in several different modes, the

primary modes being:

1. Normal-phase (polar stationary phase,

non-polar mobile phase)

2. Reversed-phase (non-polar stationary

phase, polar mobile phase)

Flash chromatography is a technique used by organic and natural product chemists to quickly purify chemical mixtures. In basic terms, the chemical mixture is passed through a purification cartridge containing a stationary phase (adsorbent) using solvents (mobile phase) to carry them through the cartridge. Compounds within the reaction mixture will interact with the stationary phase and separate.

Elution modesWithin the purification modes there are also

elution modes, which are selected based on

sample complexity. Simple separations can

typically be performed using isocratic elution

while more complex mixtures might require

gradient elution.

In isocratic elution, the mobile phase

strength and selectivity is kept constant

throughout the purification. With gradient

elution the solvent strength increases linearly

or in programmed steps throughout the

purification. Typically, modern flash systems

allow several solvents to be programmed

into a gradient providing a selectivity change

as well as an increase in solvent strength

to improve compound solubility or reduce

compound-silica interactions.

Method developmentFlash purification method development can

be very quick but several criteria must be

evaluated:

1. Solubility – in which solvents is the

sample mixture soluble? This will help

decide on the purification mode. If

soluble in non-polar solvents, normal-

phase is usually the best option, if

soluble in alcohols or water, reversed-

phase is typically best.

2. Chemistry – the target product’s

28 chrom doctor www.sepscience.com

functional groups can play an important

role in method development. For

example, an organic soluble

N-heterocycle may be

chromatographically problematic on

silica requiring a mobile phase of DCM/

MeOH/NH3 in normal-phase. However,

an amine-functionalized silica or

basic alumina will likely provide a better

separation using simpler, more

eco-friendly solvents such as heptanes/

EtOAc.

Once solubility and compound chemistry

have helped decide the chromatographic

mode, chromatographic method

development can begin.

Normal-phase method development: Thin

layer chromatography (TLC) is always a good

method development tool for normal-phase

chromatography. With TLC, it is very easy to

scout various solvent mixtures in parallel to

find the optimal separation conditions.

Compound retention on TLC, measured as Rf

(retention factor), is inversely proportional to

the number of column volumes (CV) required

to isocratically elute the same compound

with the same solvent on a flash cartridge, so

CV = 1/Rf. Retention is influenced primarily

by solvent strength whereas selectivity

(ΔCV), the relative retention difference

between any pair of eluting compounds,

is influenced by the solvent type. Solvent

pairings should be based on compound

solubility and solvent miscibility. Refer to

Table 1 for typical normal-phase solvents and

their relative strength and selectivity class.

To ensure accurate method transfer, TLC

plates should be coated with the same silica

as that packed in the flash cartridge. This

is because of differences in silica surface

chemistry between silica manufacturers,

primarily silanol type and ratio.

With silica there are three silanol types,

single (one OH on a single Si), geminal (two

OH groups on a single Si), and bridged

(H-bonded) [1]. The ratio of these three

silanols influences compound retention and

selectivity so developing a method on a

brand X TLC plates and transferring brand Y

flash cartridge may not provide the desired

or expected separation.

When the TLC plate elution studies have

finished, the Rf data need to analysed and

converted in CV. If the compound of interest

elutes between Rf 0.15 and Rf 0.35 (2.9-6.7

CV) and the ΔCV with its nearest eluting

neighbour is two or more, isocratic elution

should work well. However, gradient elution

is now state of the art and can produce

improved separations compared with

isocratic elution, see Figure 1.

Reversed-phase method development:

Method development in reversed-phase

is slightly different than normal-phase.

Although reversed-phase TLC plates

are commercially available, often the

hydrophobic media can have physical issues

when using aqueous mobile phases. These

issues include surface wetting and physical

stability on the solid backing material.

As most synthetic chemistry labs have

Table 1

Solvent Selectivity Class Strength

Methanol II 0.95

Ethanol II 0.88

IPA II 0.82

Acetonitrile VIb 0.65

Ethyl acetate VIa 0.58

THF III 0.57

Acetone VIa 0.56

DCM V 0.42

Chloroform VIII 0.40

Ether I 0.38

Toluene VII 0.29

Cyclohexane ---- 0.04

Hexane ---- 0.01

Heptane ---- 0.01

Iso-octane ---- 0.01

Table 1: Solvent selectivity class and solvent strength table

29chrom doctorseparation science — volume 1 issue 7

access to an HPLC, a better method

development technique is HPLC. Developing

methods using this approach allows for

gradient adjustment and loading studies to

be conducted. The same HPLC methods can

be used on fl ash systems with an increase

in fl ow rate and sample load. The fl ow and

load increases are based on the ratio of fl ash

media mass to method development column.

Making reversed-phase workThe key to getting reversed-phase fl ash

to provide retention and separation is

equilibration. Commercially available

fl ash cartridges are manufactured and

shipped dry. With normal-phase, although

equilibration is preferred, it is not required

because solvent easily passes through the

media’s pores. With reversed-phase, however,

the hydrophobic bonded phase impedes

wetting by aqueous solutions, signifi cantly

reducing the available surface for sample

partitioning.

Three steps to equilibration: Equilibrating

reversed-phase fl ash cartridges requires only

three steps.

1. 100% of the water-miscible organic

solvent (CH3CN, CH3OH, THF, etc.) for

5 CV

2. 50:50 organic/water (including any pH

adjusters) for 5 CV

3. Initial elution conditions for 5 CV

With proper equilibration, the reversed-

phase surface is “wetted” and the maximum

surface area for partitioning will be available.

Adjusting mobile phase pH: This is a very

important step to optimize resolution and

sample load. Because this is purifi cation and

not analysis, inorganic buff ers or modifi ers

should not be used. Instead, volatile buff ers,

acids and bases should be considered.

The key to increasing sample load in

reversed-phase is compound retention.

If trying to purify organic amines, the

mobile phase should have its pH adjusted

into the alkaline range to deprotonate the

compounds. Likewise, acidic samples should

be purifi ed under acidic conditions.

For basic compounds, adjust the mobile

phase pH to two units above the compound’s

pKa (if possible). Typical pH modifi ers include

MEA, DEA, TEA, and NH3 in methanol.

For acidic compounds, adjust the

mobile phase pH to two units below the

compound’s pKa (if possible). Typical pH

modifi ers include formic acid, acetic acid,

and TFA.

Examples of reversed-phase purifi cations

in pH-adjusted solvent systems are shown in

Figures 2 and 3.

ConclusionFlash chromatography can be a time-

consuming process; however, the techniques

discussed will help improve fl ash purifi cation

results by increasing compound load,

resolution and throughput. Through

Figure 1

AUAU

AU

Fractions

Fractions

Fractions

% S

tron

g%

Str

ong

% S

tron

g

1

0

2

3

1

0

2

3

1

0

2

3

1 5

1

1

5

0

6

12

18

24

30

36

42

48

0

7

14

21

28

35

42

49

56

0

4

8

12

16

20

24

28

Figure 1: A comparison of elution modes shows that using gradient elution can radically improve a separation creating more resolution and increased sample load potential. Top = isocratic, middle = linear gradient, bottom = step gradient.


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defi ning clear purifi cation goals, selecting

the right purifi cation and elution modes and

eff ectively developing the method, chemists

can achieve better separations with higher

purity and yield in signifi cantly less time.

Reference[1] U.D. Neue, HPLC Columns Theory, Technology, and

Practice, Wiley-VCH: New York, 1997, p.171

Figure 2

AU

Fractions

% S

tron

g

1 5 10

1

2

3

00

20

40

60

80

1001 2 3

Figure 2: Purifi cation of organic amines by reversed-phase. Cartridge, Biotage SNAP KP-C18-HS 12 g; Sample, (1) Dimethylamino antipyrine, (2) Phenylbenzyl amine, (3) Amitriptyline; Load, 120 mg; Solvents, (A) DI H2O with 0.1% TEA, (B) CH3CN 0.1% TEA; Gradient, 10% B for 12 mL, 10-100% B in 120 mL, 100% B for 24 mL; Flow rate, 12 mL/min.

Figure 3

AU

Fractions

% S

tron

g

1 5

1

2

3

00

20

40

60

80

1001 2 3

Figure 3: Purifi cation of organic acids by reversed-phase. Cartridge, Biotage SNAP KP-C18-HS 12 g; Sample, (1) Homovanillic acid, (2) Nicotinic acid, (3) 3-Nitrobenzoic acid; Load, 120 mg; Mobile phase, Acetonitrile/H2O/TFA (3:2:0.05), Flow rate, 8 mL/min.


June 28 – July 2, 2009

34th International Symposium on High-Performance Liquid Phase Separations and Related TechniquesChairman: Prof. Dr. Christian Huber, Paris-Lodron University Salzburg

Separation Science

ArbeitsKreis

SCIENTIFIC PROGRAMME:

Advances in Liquid Phase Separation Technology Multidimensional and Hyphenated Techniques Fundamental Aspects of Separations Industrial Aspects of Separations Clinical and Pharmaceutical Analysis Life Sciences Food and Environmental Analysis

VENDOR EXHIBITION:

More than 50 companies on display

Contact: Gesellschaft Deutscher Chemiker e.V. Congress Team E-mail: [email protected]

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AnApplication

notes

34 application notes www.sepscience.com

Forensic Toxicology Analysis of Non-derivatized Drugs in Urine by Automated Solid Phase Microextraction (SPME) GCxGC-TOFMS

Company: Leco

Summary: Comprehensive two dimensional gas

chromatography in combination with time-of-� ight mass

spectrometry detection (GCxGC-TOFMS) was used for

drugs of abuse analysis in urine without time consuming sample derivatization.

Methamphetamine, cocaine, diacetylmorphine, codeine, oxycodone, ecstasy,

acetylcodeine, monoacetylmorphine, hydrocodone, and LSD were identi� ed in this

research. This application presents experimental data from the forensics analysis

conducted by automated solid phase microextraction (SPME)-GCxGC-TOFMS.

Click here to view the application

On-Line Two-Dimensional Separation of Intact Proteins

Company: Dionex

Summary: While 2-D-electrophoresis is a powerful tool for protein separation,

it is di� cult to automate, and has limited utility for the analysis of many classes

of proteins. Multidimensional liquid chromatography of peptides, exempli� ed

by shotgun peptide analysis, is highly automated, but requires the proteolytic

digestion of the sample, which greatly increases sample complexity and therefore

the required resolution of subsequent separations. Furthermore, much of the

information on di� erential post-translational modi� cation and isoform expression is lost. The use of Dionex

ProPac and Dionex PepSwift polystyrene divinylbenzne (PS-DVB) columns allow improved resolution, as well

as rapid and automated separation of intact proteins from complex samples.


Forensic Toxicology Analysis of Non-derivatizedDrugs in Urine by Automated Solid PhaseMicroextraction (SPME) GCxGC-TOFMSJohn Heim • LECO Corporation; Saint Joseph, Michigan USA

Key Words: Non-Derivatized Drugs, Automated SPME, GCxGC-TOFMS

1. Introduction

2. Application Objectives

3. Experimental Conditions

4. Results and Discussion

Comprehensive two dimensional gas chromatography incombination with time-of-flight mass spectrometrydetection (GCxGC-TOFMS) was used for drugs of abuseanalysis in urine without time consuming samplederivatization. Methamphetamine, cocaine, diacetyl-morphine, codeine, oxycodone, ecstasy, acetylcodeine,monoacetylmorphine, hydrocodone, and LSD wereidentified in this research.

This application presents experimental data from theforensics analysis conducted by automated solid phasemicroextraction (SPME)-GCxGC-TOFMS. A multiple drugstandard mixture prepared from Sigma-Aldrich standardswas spiked at concentrations from 10 to 1000 ppb into8mL aliquots of urine. Hexachlorobenzene (HCB) wasadded as an internal standard at a concentration of500 ng/mL. Automated solid phase microextraction(SPME) sample preparation was conducted on the non-derivatized spiked urine standards using a GerstelMPS2 autosampler equipped with a SPME prepstationfollowed by thermal desorption of the extracted samplein the GC injection port.

The data from this research will show the identification oftargeted analytes in very complex sample matrices. Theuse of automated SPME applied to non-derivatized urinesamples coupled with comprehensive two dimensionalchromatography and time–of–flight mass spectrometrydetection demonstrates this is a favorable technique forqualitative and quantitative analysis for drug screening inurine.

• Demonstrate the detectability of non-derivatized drugsin complex sample matrices such as urine byautomated SPME combined with GCxGC-TOFMS.

• Show calibration linearity capabilities over the range of10 to 1000 ng/mL for the forensic application of drugscreening in urine.

• Illustrate the feasibility of this analysis for non-derivatized drugs in urine by SPME-GCxGC-TOFMSdescribing the advantages of multidimensionalchromatography (GCxGC) and time-of-flight massspectrometry (TOFMS).

Aliquots of urine spiked with a drug standard mixture atconcentrations from 10, 50, 250, 500, and 1000 ng/mLwere prepared and analyzed without derivatization.Hexachlorobenzene was added to each sample as aninternal standard (ISTD) at 500 ng/mL prior to extraction.Each sample was placed in a 10 mL glass SPMEautosampler vial and sealed. Automated SPME extractionwas conducted using the Gerstel MPS2 Prepstation.

GCxGC-TOFMS results were generated with a LECOPegasus 4D GCxGC-TOFMS equipped with a GerstelMPS2 autosampler and a SPME prepstation. The Pegasus4D instrument was equipped with an Agilent 7890 gaschromatograph featuring a LECO quad jet dual stagethermal modulator and secondary oven. LECOChromaTOF software was used for all acquisition controland data processing. The autosampler was a single railCTC Combi Pal equipped with SPME sample agitator/prepstation and SPME fiber conditioning station.Automated sample extraction was performed using a50/30 µm DVB/Carboxen™/PDMS Stable Flex SPME fiber.The SPME method was developed in the ChromaTOFsoftware autosampler methods section using the CTCCombi PAL option. The sample agitator was set to ON at aspeed of 200 rpm and extraction temperature of 37 C. Thesolid phase microextraction time was set for 30 minutesand sample desorption time in the GC injection port was 2minutes. The fiber was then conditioned in the fiberbakeout station at 270 C for 40 minutes prior to concurrentsample extraction for the next analysis.

A 30 m x 0.25 mm x 0.25 µm film thickness, Rxi-5ms,(Restek Corp) GC capillary column was used as the primarycolumn for the analysis. In the GCxGC configuration, asecond column 1.5 m x 0.18 mm id. x 0.18 µm filmthickness Rtx-200, (Restek Corp) was placed inside theLECO secondary GC oven after the thermal modulator.Helium carrier gas flow rate was set to 1.5 mL/min at acorrected constant flow via pressure ramps. The primarycolumn was programmed with an initial temperature of40 C for 2 minutes and ramped at 6 C/minute to 290 C for10 minutes. The secondary column temperature programwas set to an initial temperature of 50 C for 2 minutes andthen ramped at 6 C/minute to 300 C with a 10 minutehold time. The thermal modulator was set to +25 Crelative to the primary oven and a modulation time of 5seconds was used. The MS mass range was 45-550 m/zwith an acquisition rate of 200 spectra per second. The ionsource chamber was set to 230 C and the detector voltagewas 1650V with an electron energy of -70eV.

Three major points will be discussed including thedetectability of non-derivatized drugs in the complexsample matrix urine. The calibration linearity over therange of 10 to 1000 ng/mL will be shown along with thefeasibility of this analysis for non-derivatized drugs in urineby SPME-GCxGC-TOFMS.

®

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35application notes separation science — volume 1 issue 7

Multiplexed Quantitative Peptide Assays for Protein Biomarkers of Cardiovascular Disease in Human Plasma

Company: Applied Biosystems

Summary: As the study of protein biomarkers increases in importance, extensive

lists of candidate markers are being developed based on protein pathway

information and discovery-based proteomics experiments. Even larger numbers

of candidate genomic markers exist from DNA transcriptional pro� ling based on

microarray-based gene expression studies or other genomic information. This

application note demonstrates the speci� city, sensitivity and reproducibility of this

MRM based strategy in human plasma.


Overview

As the study of protein biomarkersincreases in importance, extensive listsof candidate markers are beingdeveloped based on protein pathwayinformation and discovery-basedproteomics experiments. Even largernumbers of candidate genomicmarkers exist from DNAtranscriptional profiling based onmicroarray-based gene expressionstudies or other genomic information.

While these genomic markers can bevalidated at the RNA level using realtime PCR assays such as TaqMan® kits,and at the protein level using Westernblots, validation at the protein levelrequires specific antibodies to eachprotein which is a very time consumingand expensive task if hundreds ofmarkers are of interest. A much higherthroughput and more universal strategyis needed to narrow the huge numberof candidate biomarkers that are beinggenerated by both genomic andproteomics today at the protein level.The MIDAS™ workflow combinedwith multiplexed high throughputquantitative MRM assays on aMIDAS™ TRAQ system addressesthese challenges.

Many technical challenges exist in thepreliminary validation of putativeprotein biomarkers, such as thedetection of low abundance proteinsin complex tissue or biological fluids,

as well as high-throughput, highprecision quantitation. Validation ofthese markers for clinical use willrequire analysis of extensive candidatemarker panels in thousands of clinicalserum and plasma specimens. In thisstudy, an assay was developed usingmultiple reaction monitoring (MRM)and MIDAS™ workflow on theMIDAS™ TRAQ system to detect andquantitate selected tryptic peptides,where each peptide represents aspecific candidate protein biomarkeridentified for cardiovascular disease.

This application note demonstrates the specificity, sensitivity andreproducibility of this MRM basedstrategy in human plasma.

A number of strategies wereemployed to develop high qualityMRM methods to obtain the bestsensitivity and dynamic range forpeptides from a panel of putativeprotein biomarkers for cardiovasculardisease. For candidate proteinspreviously detected in proteomicsexperiments, MRMs were designedbased on peptide MS/MS spectra.For the remaining candidates,MRMs were developed either by insilico design, based on gene orprotein sequences, or by targeted,direct detection in plasma using theMIDAS™ workflow.

Multiplexed Quantitative Peptide Assays for Protein

Biomarkers of Cardiovascular Disease in Human Plasma

Application Note LC/MS

www.appliedbiosystems.com

Figure 1. Dynamic range of protein abundance in plasma is extremely large1 (inset, blue). This multiple reactionmonitoring (MRM) multiplexed assay profiles 53 plasma proteins (137 MRM transitions) over the first 5 orders ofabundance with minimal sample preparation.

HPLC and UHPLC Methods for Melamine

Company: Grace Davison

Discovery Sciences

Summary: Grace has

developed MS-compatible

LC methods for melamine

using an HILIC media

platform that can be applied

to both traditional HPLC

as well as UHPLC systems. Melamine was analysed

with a standard HPLC system using a 5 μm particle

HILIC phase packed into a 250 x 4.6 mm column.

The 1.5 μm version of this phase was then packed

into a high throughput format conducive to UHPLC

and fast LC systems. Both methods deliver excellent

linearity and use isocratic elution for fast analysis

without the need for re-equilibration.


Grace Davison Discovery Sciences

HPLC and UHPLC Methods for Melamine

Milk, infant formula and other dairy products were recently found contaminated with melamine, following an earlier melamine contamination outbreak in pet food in 2007.

Toxicology studies show ingestion of melamine in large quantities may lead to reproductive damage or bladder cancer due to the formation of bladder/kidney stones. At lower levels, melamine and cyanuric acid are absorbed into the bloodstream. Together, they concentrate and form melamine cyanurate in the urine-filled renal microtubules. Cystallization blocks and damages the renal cells that line the tubes, causing the kidneys to malfunction.

After 2007 and more recent melamine contamination outbreaks, there is an urgent need for analytical methods that can identify and quantify melamine in food. Current melamine detection methods involve LC-MS and GC-MS. GC-MS methods require derivatization, and LC-MS methods generally use gradient conditions that require column clean up and re-equilibration.

Grace has developed MS-compatible LC methods for Melamine using an HILIC media platform that can be applied to both traditional HPLC as well as UHPLC systems. Melamine was analyzed with a standard HPLC system using a 5µm particle HILIC phase packed into a 250 x 4.6mm column. The 1.5µm version of this phase was then packed into a high throughput format conducive to UHPLC and fast LC systems. Both methods deliver excellent linearity and use isocratic elution for fast analysis without the need for re-equilibration.

1

0 2.5 5 7.5 10 12.5 15 17.5 20 Min.0

0.5

1.0

1.52.0mV(x100)

N N

NH2

NH2H

2N N

N N

OH

OHHO N

Melamine

Cyanuric Acid

HPLC Method for Melamine

HPLC Column: Alltima™ HP HILIC, 5µm, 4.6 x 250mm (Part No. 86466)Mobile Phase: Acetonitrile:10mM Ammonium Acetate in Water (95:5)Flow Rate: 1mL/minDetection: UV@240nmColumn Temperature: 30ºCInjection: 40µg/mL x 20µL

0 20 40 60 10080 1200

2

4

6

Peak

Are

a (x

106 m

AU x

sec

.)

Sample Weight (µg)

8

10

16

12

14

1

0 1 2 3 4 5 Min.

-1

123

mAU

0

45

UHPLC Method for Melamine

UHPLC Column: VisionHT™ HILIC, 1.5µm, 2 x 50mm (Part No. 5141919)Mobile Phase: Acetonitrile:10mM Ammonia Acetate in Water (95:5)Flow Rate: 0.2mL/minDetection: UV@240nmColumn Temperature: 30ºCInjection: 50µg/mL x 0.5µL

Compared to the conventional HPLC method, the UHPLC method is 4 times faster. With the use of 1.5μm particles, optimal linear velocities extend over a wider range. Therefore, it is possible to maintain efficiency and resolution while running samples at faster flow rates.

0 0.5 1 1.5 2 2.5 3 Min.

0

10

20

30

40

UHPLC Column: VisionHT™ HILIC, 1.5µm, 2 x 50mm (Part No. 5141919)Mobile Phase: Acetonitrile:Water(20mM Ammonium Formate) (90:10)Flow Rate: 0.2mL/minDetection: UV@240nmColumn Temperature: 30ºCInjection: 50µg/mL x 0.1µL

0 200 400 600

Sample Weight (ng)

Area

(mAU

x s

ec.)

1000800 12000

200

400

600

800

1000

1200

1400

y = 146800x + 87120R2 = 0.998

9 injections in parallel shows good reproducibilityConc.

Inj. (µL)

Weight(ng)

PeakArea

50µg/mL 0.1 5 7.3

50µg/mL 0.5 25 32

50µg/mL 1 50 63

50µg/mL 5 250 318

50µg/mL 10 500 641

50µg/mL 20 1000 1293

y = 1.2924x - 1.8252R2 = 1.000

This HPLC analytical method for melamine fulfills the FDA requirements using a HILIC column and an ionizable mobile phase compatible with mass spec. Low UV detections offers excellent linearity between 40ng and 100µg.

This method exhibits excellent linear response between 5ng and 1000ng for accurate quantitation.

36 technology update www.sepscience.com

Carbohydrates, Organic Acids and Alcohols in Wine

Company: Bischo�

Summary: Bischo� o� ers an application booklet desribing a series of separations

using its ProntoSIL family of columns. Applications include Carbohydrates, Organic

Acids and Alcohols in Wine, Fast Analysis of a pharmaceutical Test standard with

Multiwavelength Detection, Fast Analysis of Parabens II with Multiwavelength

Detection, Aromatic Hydrocarbons according to prEN 12916, Ergosterol in Grass

and Acetaldehyde in Mineral water.


Carbohydrates, Organic Acids and Alcohols in Wine

Carbohydrate H+

Part. Number: 00253776Dimension: 300 x 7.8 mmEluent: 1.25 mM H2SO4Flow: 0.6 ml/minDetection: RITemperature: 45 °CInjection: 50 µlSample: Wine Complett Standard

no. 1

51

etha

nol

~

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 min

22 mV

citric

tarta

ricgl

ucos

em

alic

fruct

ose

succ

inic lac

ticgl

ycer

ol

acet

ic

2,3-b

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Analysis of Pesticide Residues in Apple using Agilent SampliQ QuEChERS European Standard EN Kits by LC/MS/MS Detection

Company: Agilent

Summary: This application note describes the use of a quick, easy, cheap, e� ective,

rugged, and safe (QuEChERS) sample preparation approach described in the

European Committee Standard (EN) for extraction and cleanup of 16 multiple

class pesticide residues of interest in apple. The method employed involves initial

extraction in an aqueous/acetonitrile system, an extraction/partitioning step

after the addition of salt, and then a cleanup step utilizing dispersive solid phase

extraction (dispersive SPE).


Analysis of Pesticide Residues inApple using Agilent SampliQQuEChERS European Standard EN Kitsby LC/MS/MS Detection

Abstract

This application note describes the use of a quick, easy, cheap, effective, rugged, and

safe (QuEChERS) sample preparation approach described in the European Committee

Standard (EN) for extraction and cleanup of 16 multiple class pesticide residues of

interest in apple. The method employed involves initial extraction in an aqueous/ace-

tonitrile system, an extraction/partitioning step after the addition of salt, and then a

cleanup step utilizing dispersive solid phase extraction (dispersive SPE). The two dif-

ferent dispersive SPE clean-up approaches (1 mL and 6 mL sample volume) are evalu-

ated simultaneously after sample extraction. The target pesticides in the apple

extracts are then determined by liquid chromatography coupled to an electrospray ion-

ization tandem mass spectrometer (LC-ESI-MS/MS) operating in positive ion multiple

reaction monitoring (MRM) mode. The method is validated in terms of recovery and

reproducibility. The 5 ng/g of limits of quantitation (LOQ) for pesticides in apple estab-

lished in this application is well below their regulatory maximum residue limits

(MRLs). The spiking levels for the recovery experiments are 10, 50, and 200 ng/g.

Excluding pymetrozine, recoveries of the pesticides ranged between 73 and 111%

(87% on average), and RSDs below 20% (5.8% on average).

Authors

Limian Zhao, David Schultz, and

Joan Stevens

Agilent Technologies, Inc.

2850 Centerville Road

Wilmington, DE 19809

USA

Application NoteFood Safety


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information on product developments?

market trends and opinions?Volume 1 / Issue 3

March 2009

www.sepscience.com

Coupling capillary columns in gas

chromatography

Analytical trends in iso�avone

studies

Minimizing downtime in QA

pharmaceutical laboratories

separation driving analytical chemistry forwardsscience

Volume 1 / Issue 1

Febraury 2009

www.sepscienceasia.comwww.sepscienceasia.com

Volume 1 / Issue 1


Volume 1 / Issue 2

February 2009


Exploiting particle size to reduce

acetonitrile consumption

Multiresidue analysis using SBSE

and GC-MS/MS

Chromatographic methods for

Con�rming biological activity

markers in fruit

Asia Paci�c


Volume 1 / Issue 2

April 2009


固相微萃取-高效液相色谱分析羟烷基喹诺酮

固相萃取和气相色谱与三级四极杆质谱联用测

定熟食品中痕量食品衍生的有害物质

液相色谱填料尺寸对降低

溶剂消耗的影响

中国版

Volume 1 / Issue 5

May 2009

www.sepscience.com

A liquid chromatographer’s

introduction to mass spectrometry

Analysing synthetic polymers with

solvent enhanced light scattering

Minimizing decomposition of

components during GC analysis


Volume 1 / Issue 5

May 2009


RP-HPLC determination

of anti-malarials






37Technology update separation science — volume 1 issue 3

New Kromasil® Eternity™

Designed for long life

How old can a column get? It’s natural to think it depends on how wide the pH variations are or how high the temperature gets. But even under such tough conditions, Kromasil® Eternity™ lives longer than other columns.

It’s all about being designed for long-term survival.

Kromasil® Eternity™ has an organic/inorganic interfacial gradient cover ing the silica, ensuring long life for the column. Check out our website for more information and continuous updates. www.kromasil.com

has

Kromasil®

Eka_Ad_longlife_190x272.indd 1 09-06-02 10.19.03

TuTechnology

update


Key

Email the company

Product information

Applications

Additional information

Oligonucleotide separation technology columns

Manufacturer: Waters

Manufacturer’s description: Oligonucleotide Separation Technology (OST) columns contain

second-generation hybrid-silica BEH Technology particles functionalized with C18. The

separation of detritylated synthetic oligonucleotide samples is based on the well-established

method of ion-pair, reversed-phase chromatography.

This chemistry delivers exceptional sample resolution and superior column life. In addition,

Waters manufacturing and quality control testing procedures help ensure consistent batch-

to-batch and column-to-column performance regardless of application demands.

The availability of 1.7 μm UPLC technology or 2.5 μm HPLC particles in various column

dimensions gives you the flexibility to meet various lab-scale isolation or analysis needs.

Features include:

• Scalable reversed-phase HPLC columns for lab-scale purifications

• Increased sample through-put with maintained component resolution with UPLC

Technology

• LC/UV and LC/MS methods for enhanced quantitative and qualitative analyses

• Exceptional column life using Waters patented Ethylene-Bridged (BEH Technology) Hybrid `

particles

39technology update separation science — volume 1 issue 7


www.sepscience.com

Nitrogen generator

Manufacturer: Parker Hannifi n

Manufacturer’s description: The Parker Balston membrane nitrogen generators can produce up to 75 lpm of pure LC/

MS grade nitrogen at pressures up to 8 bar. Generators are complete systems engineered to transform standard

compressed air into a safe regulated nitrogen supply with minimal operator attention, according to the company.

Typical applications include LC/MS, LC/MS/MS, nebuliser gases for APCI and ESI, ELSD, Turbo Vaps and chemical

solvent evaporation. The membrane nitrogen generators have been tried and tested by all the major LC/MS

manufacturers.

Features

• Recommended and used by all major LC/MS manufacturers

• No electrical requirement, no noise and no moving parts

• Can supply up to 3 LC/MS from one generator

• Utilises Parker’s propriety membrane technology

• Phthalate-free, no organic vapours

• Pliminate inconvenient nitrogen dewars from the laboratory



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Robotic Soliprep – new automation possibilities in ion chromatography

Manufacturer: Metrohm

Manufacturer’s description: Pressure to increase output while reducing

costs requires laboratory automation wherever possible. Metrohm’s

new 815 Robotic Soliprep meets such demands as it allows to

completely automate preparation and analysis of liquid and solid

samples in ion chromatography. The 815 Robotic Soliprep provides

maximum fl exibility as it can be customized on demand to meet

individual customers’ specifi c requirements, according to Metrohm.

The 815 Robotic Soliprep is a state-of-the-art sample processor,

which reliably transfers both liquid and solid samples to an ion

chromatograph. A broad range of working steps such as pulverizing,

homogenizing, extracting, fi ltering or diluting can be safely entrusted

to the 815 Robotic Soliprep and included in the analysis method,

the company states. Due to the incorporation of inline sample

preparation into the analysis system, manual intervention is no

longer required during the determination. The sample rack of the 815

Robotic Soliprep is equipped with various inserts, sample beakers,

syringe fi lters and needles and can be customized on demand to

meet individual customers’ specifi cations. Combined with Metrohm’s

advanced ICs the 815 Robotic Soliprep opens up numerous new

applications and application fi elds. Applications include, to mention

just a few, the analysis of ingredients and contaminants in tablets,

animal feed, sediments or food.

41technology update separation science — volume 1 issue 7

7100 Capillary Electrophoresis system

Manufacturer: Agilent Technologies

Manufacturer’s description: Agilent Technologies has introduced its next generation Agilent 7100 Capillary

Electrophoresis (CE) system, which it states provides at least 10 times more sensitivity than any other commercial CE

instrument.

CE currently attracts attention because the technique uses very small amounts of solvent. The new 7100 system also

requires 25% less bench space, weighs 30% less than its predecessor and uses less power, according to Agilent. The

superior sensitivity is a result of a new detector used in combination with proprietary extended light path capillaries

or a high-sensitivity cell.

The Agilent 7100 offers a wide selection of detectors for flexibility and sensitivity, and the new instrument is

reverse-compatible to the previous Agilent CE platform, so existing methods can continue to be used.

Agilent confirms 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 of use. The new, 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 a new

Chemstation software with an easy-to-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 Agilent 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.

www.sepscience.com

Varian 385-LC ELSD

Manufacturer: Varian

Manufacturer’s description: Evaporative light scattering detectors are ideal for detecting analytes with no UV

chromophore as they do not rely on the optical properties of a compound. The Varian 385-LC Evaporative Light

Scattering Detector is the latest high performance ELS detector from Varian, with almost 20 years’ experience in the

design and manufacture of evaporative light scattering detectors.

The Varian 385-LC Evaporative Light Scattering Detector is the only ELSD that delivers sub-ambient operation,

for unrivalled detection of thermally labile analytes, according to the company. Designed for the analysis of all

compounds, the Varian385-LC delivers evaporation down to 10°C, providing maximum sensitivity for compounds with

significant volatility below 30°C. Programmable control of gas flow and temperature during an injection eliminates

solvent gradient effects and maximizes response for improved accuracy. To detect everything you inject with high

sensitivity, the Varian 385-LC is the ELSD of choice for all applications.

Key Benefits

• High sensitivity provides superb responses for all compounds, down to low nanogram levels.

• Sub-ambient operation using a Peltier cooled evaporation tube delivers temperatures down to 10°C, preventing

degradation of the thermally labile compounds that other ELSDs cannot detect.

• Real-time control during an injection via programmable Dimension Software maintains maximum sensitivity

throughout the run.

• Real-time gas programming eliminates solvent enhancement effects during gradient elution, for excellent

quantification.

• Low dispersion and high-speed data output rates are the perfect match

for Fast LC applications.

• Superb reproducibility below 2% gives reliable and accurate results.

• Multi-vendor software control and data acquisition using Varian’s

Galaxie chromatography data system, and other vendors’ interfaces,

eliminates the need for an analog to digital converter.

• Rapid heating and cooling of the evaporator tube minimizes

equilibration time and increases sample throughput.

• Full DMSO transparency ensures that responses from early eluting

compounds are not hidden.

• Fully integrated with the Varian 920-LC analytical HPLC system for the

complete chromatographic solution.42 technology update

analytica Anacon India is part of the exhibition network analytica worldwide—the most important marketplacein the world for marketable products and solutions in the analysis, laboratory-technology and biotechnologysectors. It is a business and networking platform and a driving force behind future trends and innovations.

www.analyticaindia.com

analytica Anacon India SEPT. 29–OCT. 01, 2009HYDERABAD

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AnaInd_NetAd_210x297_E.qxd 03.04.2009 10:10 Uhr Seite 1


Axima MALDI-TOF MS

Manufacturer: Shimadzu

Manufacturer’s description: Axima Confidence offers powerful MALDI-TOF performance for reliable mass information

and MS/MS derived structural detail, according to Shimadzu. Its linear mode allows the interrogation of high

molecular weight samples, whilst reflectron mode, incorporating the patented curved-field reflectron (CFR), provides

the high resolution and mass accuracy necessary for successful proteomics and life science experiments. It is a high

sensitivity system using a variable repetition rate 50 Hz N2 laser and a variety of target formats to meet all sample

throughput requirements.

The company states it is an affordable robust option for all laboratories requiring routine manual or automated

analysis of a wide variety of sample classes, with excellent sensitivity achieved using near-axis laser irradiation and

advanced ion optics for enhanced ion transmission.

MS/MS may be easily performed using a seamless approach – ions of interest can be isolated using a precursor

ion selection device, incorporated as standard, and data-rich fragment ion spectra quickly and simply acquired.The

newly improved curved field reflectron design augments the low mass fragment region providing useful additional

information.

Key Performance Features

• Near-axis laser irradiation with a 50Hz variable repetition rate N2 laser

• Patented improved curved field reflectron

• Mass range 1-500kDa in linear mode; 1-80kDa in reflectron mode

• Mass resolution 5000 FWHM (ACTH 18-39) in linear mode; 15000 FWHM

(ACTH 7-38) in reflectron mode

• Sensitivity 500 amol (Glu-fibrinopeptide) in reflectron mode

• Mass accuracy <10ppm in reflectron mode with internal calibration

• Precursor ion resolution >200 FWHM

• Seamless MS/MS utilising PSD

• Flexible target formats - ranging from 48 well microscope slide format

targets to 384 well microtitre plate targets

45Technology update separation science — volume 1 issue 7

IntelliTarget

Manufacturer: ACD Labs

Manufacturer’s description: New LC/MS and GC/MS deconvolution software makes confirming the presence of known

compounds in messy samples faster and easier. The new software enables the target analysis of complicated LC/MS

and GC/MS datasets, without the tedious and difficult manual interpretation. ACD/IntelliTarget quickly confirms the

presence or absence of known compounds in samples, even at low concentrations, or when obscured by the presence

of other substances.

ACD/IntelliTarget quickly and accurately deconvolutes LC/MS and GC/MS data and presents a concise report

indicating whether your compounds of interest are ‘Found’ or ‘Not Found’. Specify just one compound or a list of

hundreds, either by formula or by monoisotopic mass. ACD/IntelliXtract then extracts and identifies the ionized

molecule and adducts for each component, even when compounds are present at low concentration, or co-eluting

with other species. ACD/IntelliTarget is an essential tool to ease the workload of experienced spectroscopists, and for

inexperienced users who may find the manual interpretation of data to be overwhelming.

ACD/IntelliTarget is intended for screening, monitoring, and focusing analytical efforts in many fields of study,

including forensic science, environmental monitoring, water analysis, food safety, and drug discovery. It is compatible

with data formats of most major instrument vendors, and can be automated to screen larger batches of samples, or to

monitor samples on-the-fly.

ACE C18-AR - a C18 bonded HPLC column offering alternate selectivity

Manufacturer: Advanced Chromatography Technologies

Manufacturer’s description: ACE C18-AR is a unique C18 bonded HPLC column from Advanced Chromatography

Technologies, which has been developed to provide alternate selectivity to all existing C18 bonded phases. The phase

combines the hydrophobic characteristics of a C18 phase with the enhanced aromatic selectivity of a phenyl phase,

enabling the benefits of both interactions to be fully exploited. According to the company the phase also exhibits

ultra low bleed to ensure LC/MS compatibility and demonstrates hight temperature and pH stability. Additionally, the

ACE C18-AR is suitable for use in highly aqueous (up to 100%) mobile phases to enable the retention and separation

of polar compounds. At launch, materials are available in 3 µm, 5 µm and 10 µm particle sizes and a full range of

column dimensions from high throughput through to preparative scale.

sepa on scienc

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Coupling capillary columns in gas

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pharmaceutical laboratories


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液相色谱-质谱联用新方法的建立

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联用分析太子参中的挥发物

微芯片电泳用于生物医学

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Exploiting particle size to reduce

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Multiresidue analysis using SBSE

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Chromatographic methods for

Con�rming biological activity

markers in fruit

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固相微萃取-高效液相色谱分析羟烷基喹诺酮

固相萃取和气相色谱与三级四极杆质谱联用测

定熟食品中痕量食品衍生的有害物质

液相色谱填料尺寸对降低

溶剂消耗的影响

中国版

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May 2009

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Analysing synthetic polymers with

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RP-HPLC determination

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