the applications book

THE

APPLICATIONSBOOK

September 2013

www.chromatographyonline.com

Introducing the

LCGC EUROPE APP for your iPhone or iPad

Designed for both the iPhone and iPad, LCGC Europe’s new

app may be found on iTunes, under the Business category,

and downloaded for free. Exploring the latest news and

trends for chromatographers has never been easier.

Download it for free today at

http://www.chromatographyonline.com/LCEApp

THE APPLICATIONS BOOK

Food and Beverage5 Thermal Treatment Analysis — Determination of

3,5-stigmastadienes in Olive Oil Using the Agilent Infi nity 1220 LC

System with Diode Array Detector

Sonja Schneider, Agilent Technologies Inc.

6 Determination of Afl atoxins B1, G

1, B

2, and G

2 in Tomato Extract by

HPTLC

CAMAG Laboratory

8 Structural Differences in Modifi ed Starches

Malvern Instruments Ltd.

9 Determination of Pesticide Residues in Whole Milk by QuEChERS

and LC–MS–MS

Xiaoyan Wang, UCT

Industrial10 Glycerol Determination in Biodiesel and Biodiesel Blends

According to ASTM D 7591

J. Gandhi and A. Wille, Metrohm

Medical/Biological14 Extraction of SPICE from Oral Fluid Using ISOLUTE SLE+ Prior to

LC–MS–MS Analysis

Frank Kero and Victor Vandell, Biotage LLC

15 Ultrafast UHPLC–MS–MS Method Development in Therapeutic

Drug Monitoring

Anja Grüning and Gesa Schad, Shimadzu Europa GmbH

17 A Toolbox of Amino Acids for Out-of-the-Box mAb Separations

Tosoh Bioscience

19 Molecular Weight Determination of Low-Molecular-Weight

Heparins: SEC/MALS vs. SEC/UV-RI

Wyatt Technology Corporation

Pharmaceutical/Drug Discovery20 Analysis of Barbiturates in Urine with Agilent 6430 LC–MS–MS

and Poroshell 120 EC-C18

Elijah Steinbauer,1 Pat Friel,1 Rongjie Fu,2 and Andy Zhai,2 1 Toxicology

Laboratory at the Veterans Administration, Portland, Oregon, USA,

2Agilent Technologies

22 Simultaneous Quantitative and Qualitative Measurements in

a Single Workfl ow to Increase Productivity in Primary Drug

Metabolism Investigations

Bruker Daltonics

24 Measuring Antibody Molecular Weight by SEC-MALS

Malvern Instruments Ltd.

25 Sophisticated Antibody Analysis by GPC/SEC with RALS

PSS Polymer Standards Service GmbH

26 Antibody Drug Conjugate (ADC) Analysis

Wyatt Technology Corporation

Cover Photography: Getty Images

CONTENTS

THE APPLICATIONS BOOK – SEPTEMBER 2013 3

4 THE APPLICATIONS BOOK – SEPTEMBER 2013

5IF�1VCMJTIFST�PG�-$t($�&VSPQF�XPVME�MJLF�UP�UIBOL�UIF�NFNCFST�PG�UIF�&EJUPSJBM�"EWJTPSZ�#PBSE�GPS�

UIFJS�DPOUJOVJOH�TVQQPSU�BOE�FYQFSU�BEWJDF��5IF�IJHI�TUBOEBSET�BOE�FEJUPSJBM�RVBMJUZ�BTTPDJBUFE�XJUI�

-$t($�&VSPQF�BSF�NBJOUBJOFE�MBSHFMZ�UISPVHI�UIF�UJSFMFTT�FGGPSUT�PG�UIFTF�JOEJWJEVBMT�

LCGC Europe provides troubleshooting information and application solutions on all aspects of

separation science so that laboratory-based analytical chemists can enhance their practical

LOPXMFEHF�UP�HBJO�DPNQFUJUJWF�BEWBOUBHF��0VS�TDJFOUJGJD�RVBMJUZ�BOE�DPNNFSDJBM�PCKFDUJWJUZ�QSPWJEF�

SFBEFST�XJUI�UIF�UPPMT�OFDFTTBSZ�UP�EFBM�XJUI�SFBM�XPSME�BOBMZTJT�JTTVFT �UIFSFCZ�JODSFBTJOH�UIFJS�

efficiency, productivity and value to their employer.

Editorial Advisory Board

Kevin Altria

(MBYP4NJUI,MJOF �)BSMPX �&TTFY �6,

Daniel W. Armstrong

University of Texas, Arlington, Texas, USA

Michael P. Balogh

Waters Corp., Milford, Massachusetts, USA

Coral Barbas

Faculty of Pharmacy, University of San

Pablo – CEU, Madrid, Spain

Brian A. Bidlingmeyer

Agilent Technologies, Wilmington,

%FMBXBSF �64"

Günther K. Bonn

Institute of Analytical Chemistry and

Radiochemistry, University of Innsbruck,

Austria

Peter Carr

Department of Chemistry, University

of Minnesota, Minneapolis, Minnesota, USA

Jean-Pierre Chervet

"OUFD�-FZEFO �;PFUFSXPVEF �5IF�

Netherlands

Jan H. Christensen

Department of Plant and Environmental

Sciences, University of Copenhagen,

Copenhagen, Denmark

Danilo Corradini

Istituto di Cromatografia del CNR, Rome,

Italy

Hernan J. Cortes

H.J. Cortes Consulting,

Midland, Michigan, USA

Gert Desmet

Transport Modelling and Analytical

Separation Science, Vrije Universiteit,

Brussels, Belgium

John W. Dolan

LC Resources, Walnut Creek, California,

USA

Roy Eksteen

Sigma-Aldrich/Supelco, Bellefonte,

Pennsylvania, USA

Anthony F. Fell

Pharmaceutical Chemistry,

University of Bradford, Bradford, UK

Attila Felinger

Professor of Chemistry, Department of

Analytical and Environmental Chemistry,

University of Pécs, Pécs, Hungary

Francesco Gasparrini

Dipartimento di Studi di Chimica e

Tecnologia delle Sostanze Biologica-

mente Attive, Università “La Sapienza”,

Rome, Italy

Joseph L. Glajch

Momenta Pharmaceuticals, Cambridge,

Massachusetts, USA

Jun Haginaka

School of Pharmacy and Pharmaceutical

4DJFODFT �.VLPHBXB�8PNFO�T�

University, Nishinomiya, Japan

Javier Hernández-Borges

Department of Analytical Chemistry,

Nutrition and Food Science University of

Laguna, Canary Islands, Spain

John V. Hinshaw

Serveron Corp., Hillsboro, Oregon, USA

Tuulia Hyötyläinen

VVT Technical Research of Finland,

Finland

Hans-Gerd Janssen

7BO�U�)PGG�*OTUJUVUF�GPS�UIF�.PMFDVMBS�

Sciences, Amsterdam, The Netherlands

Kiyokatsu Jinno

School of Materials Sciences, Toyohasi

University of Technology, Japan

Huba Kalász

4FNNFMXFJT�6OJWFSTJUZ�PG�.FEJDJOF

Budapest, Hungary

Hian Kee Lee

National University of Singapore,

Singapore

Wolfgang Lindner

Institute of Analytical Chemistry,

University of Vienna, Austria

Henk Lingeman

Faculteit der Scheikunde, Free University,

Amsterdam, The Netherlands

Tom Lynch

BP Technology Centre, Pangbourne, UK

Ronald E. Majors

Agilent Technologies,

8JMNJOHUPO �%FMBXBSF �64"

Phillip Marriot

Monash University, School of Chemistry,

Victoria, Australia

David McCalley

Department of Applied Sciences,

University of West of England, Bristol, UK

Robert D. McDowall

.D%PXBMM�$POTVMUJOH �#SPNMFZ �,FOU �6,

Mary Ellen McNally

%V1POU�$SPQ�1SPUFDUJPO /FXBSL �

%FMBXBSF �64"

Imre Molnár

Molnar Research Institute, Berlin, Germany

Luigi Mondello

Dipartimento Farmaco-chimico, Facoltà

di Farmacia, Università di Messina,

Messina, Italy

Peter Myers

Department of Chemistry,

University of Liverpool, Liverpool, UK

Janusz Pawliszyn

Department of Chemistry, University of

Waterloo, Ontario, Canada

Colin Poole

Wayne State University, Detroit,

Michigan, USA

Fred E. Regnier

Department of Biochemistry, Purdue

University, West Lafayette, Indiana, USA

Harald Ritchie

Thermo Fisher Scientific, Cheshire, UK

Pat Sandra

Research Institute for Chromatography,

Kortrijk, Belgium

Peter Schoenmakers

Department of Chemical Engineering,

Universiteit van Amsterdam, Amsterdam,

The Netherlands

Robert Shellie

Australian Centre for Research on

Separation Science (ACROSS), University

of Tasmania, Hobart, Australia

Yvan Vander Heyden

Vrije Universiteit Brussel,

Brussels, Belgium

Published by

Subscibe on-line at

www.chromatographyonline.com

Group Publisher

Mike Tessalone

[email protected]

Editorial Director

Laura Bush

[email protected]

Editor-in-Chief

Alasdair Matheson

[email protected]

Managing Editor

Kate Mosford

[email protected]

Assistant Editor

Bethany Degg

[email protected]

Sales Manager

Lindsay Jones

[email protected]

Sales Executive

Liz Mclean

[email protected]

Subscriber Customer Service

Visit (chromatographyonline.com)

to request or change a

subscription or call our customer

Service Department on

+001 218 740-6877

Bridgegate Pavilions, 4A

Chester Business Park,

Wrexham Road,

Chester, CH4 9QH

Tel. +44 (0)1244 629 300

Fax +44 (0)1244 678 008

Chief Executive Officer Joe Loggia

Chief Executive Officer Fashion Group, Executive Vice-President Tom Florio

Executive Vice-President, Chief Administrative Officer & Chief Financial Officer Tom Ehardt

Executive Vice-President Georgiann DeCenzo

Executive Vice-President Chris DeMoulin

Executive Vice-President Ron Wall

Executive Vice-President, Business Systems Rebecca Evangelou

Sr Vice-President Tracy Harris

Vice-President, Media Operations Francis Heid

Vice-President, Legal Michael Bernstein

Vice-President, Electronic Information Technology J Vaughn

CORPORATE OFFICE641 Lexington Ave, 8th Fl. New York, New York 10022 USA

‘Like’ our page LCGC Join the LCGC LinkedIn groupFollow us @ LC_GC

46#4$3*15*0/4��-$r($�&VSPQF�JT�GSFF�UP�RVBMJàFE�SFBEFST�JO�&VSPQF� 5P�BQQMZ�GPS�B�GSFF�TVCTDSJQUJPO �PS�UP�DIBOHF�ZPVS�OBNF�PS�BEESFTT �HP�UP�XXX�MDHDFVSPQF�DPN �

DMJDL�PO�4VCTDSJCF �BOE�GPMMPX�UIF�QSPNQUT�

To cancel your subscription or to order back issues, please email your request to

[email protected], putting LCE in the subject line.

Please quote your subscription number if you have it.

MANUSCRIPTS:�'PS�NBOVTDSJQU�QSFQBSBUJPO�HVJEFMJOFT �WJTJU�XXX�MDHDFVSPQF�DPN�PS�DBMM�UIF�&EJUPS �

��"MM�TVCNJTTJPOT�XJMM�CF�IBOEMFE�XJUI�SFBTPOBCMF�DBSF �CVU�UIF�QVCMJTIFS�

BTTVNFT�OP�SFTQPOTJCJMJUZ�GPS�TBGFUZ�PG�BSUXPSL �QIPUPHSBQIT�PS�NBOVTDSJQUT��&WFSZ�QSFDBVUJPO�

is taken to ensure accuracy, but the publisher cannot accept responsibility for the accuracy of

information supplied herein or for any opinion expressed.

DIRECT MAIL LIST: Telephone: +44 (0)1244 629 300, Fax: +44 (0)1244 678 008.

Reprints: Reprints of all articles in this issue and past issues of this publication are available

��NJOJNVN��$POUBDU�#SJBO�,PMC�BU�8SJHIU�T�.FEJB ��5JNCFSMPDI�1MBDF �5IF�8PPEMBOET �59�

��5FMFQIPOF��FYU��&NBJM��CLPMC!XSJHIUTNFEJB�DPN�

©2013 Advanstar Communications Inc. 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

QIPUPDPQZ �SFDPSEJOH �PS�JOGPSNBUJPO�TUPSBHF�BOE�SFUSJFWBM�XJUIPVU�QFSNJTTJPO�JO�XSJUJOH�GSPN�UIF�

publisher.

Authorization to photocopy items for internal/educational or personal use, or the

internal/educational or personal use of specific clients is granted by Advanstar

$PNNVOJDBUJPOT�*OD��GPS�MJCSBSJFT�BOE�PUIFS�VTFST�SFHJTUFSFE�XJUI�UIF�$PQZSJHIU�

$MFBSBODF�$FOUFS ��3PTFXPPE�%S��%BOWFST �."�� GBY��

��PS�WJTJU�IUUQ��XXX�DPQZSJHIU�DPN�POMJOF��'PS�VTFT�CFZPOE�UIPTF�MJTUFE�

BCPWF �QMFBTF�EJSFDU�ZPVS�XSJUUFO�SFRVFTU�UP�1FSNJTTJPO�%FQU��GBY��PS�

email: [email protected].

10% Post

Consumer

Waste


FOOD AND BEVERAGE

3,5-stigmastadienes were analysed in seven olive oil

samples using the Agilent 1220 Infi nity Mobile LC Solution to

differentiate virgin from refi ned or other thermally-treated

olive oil. Because of the robust and rugged 1220 Infi nity

Mobile LC Solution, it is possible to perform olive oil

analysis on-site as a starting point for quality analysis of

virgin olive oils.

Introduction

Virgin olive oil can be created only by mild, cold pressing of the

olives (Olea europea L.). Thermal or chemical treatment is not

allowed in the procedure. There are different analytical methods

to differentiate virgin from refi ned or thermally-treated olive

oils. In addition to the determination of stigmastadienes and

chlorophyll degradation products, the analysis of the concentration

of polymerized triacylglycerides in olive oil is another important

factor. The amount of stigmastadienes in commercially refi ned

vegetable oils is dependent on the conditions applied during the

refi ning process. The determination of stigmastadienes in olive oils

also detects minor amounts of refi ned oils in virgin olive oils and

is, therefore, an important quality characteristic for virgin olive oils.

Because of the ultraviolet (UV) detection of the stigmastadienes

analysis method, the 1220 Infi nity Mobile LC Solution can be used

in a mobile laboratory as a starting point for olive oil quality analysis

before further quality analyses are applied in a stationary laboratory.

Experimental Conditions

Column: Agilent LiChrospher C18, 4 × 250 mm, 5 μm (p/n

79925OD-584), Agilent ZORBAX Extend-C18 RRHT, 4.6 × 50 mm

1.8 μm (p/n 727975-902)

Mobile phase: Acetonitrile/methyl tert-butyl ether (70:30)

Flow: 1 mL/min

Stop time: 30 min or 5 min

Injection volume: 10–50 μL, 20 μL

Column temperature: 25 °C

UV: 235 nm/4 nm Ref.: off

Peak width: >0.05 min (1.0 s response time) (5 Hz)

Sample preparation was carried out according to EN ISO

15788-3:2004 (D) using the internal standard method.

Results

In contrast to virgin olive oils, 3,5-stigmastadienes were detected

in partly refi ned olive oil, see Figure 1. To accelerate analysis time,

the run was shortened to 5 min using a 50-mm, sub-2 μm column

(Agilent ZORBAX Extend-C18 RRHT, 4.6 × 50 mm 1.8 μm), still

obtaining good resolution of the analytes in partly refi ned olive oil.

Conclusion

Seven olive oils were analysed for 3,5-stigmastadiene to determine

refi ning processes or other thermal treatments according to EN

ISO 15788-3:2004 (D). As expected, no 3,5-stigmastadienes were

detected in any of the tested virgin oils. In contrast, in a sample

containing refi ned and virgin oils, the amount of 3,5-stigmastadienes

found was 0.63 mg per kg sample. The analysis time could be

shortened to 5 min using a 50-mm, sub-2 μm column.

References

(1) Quality Analysis of Virgin Olive Oils — Part 3, Agilent Application Note,

Publication Number 5991-1896EN, (2013).

(2) Quality Analysis of Virgin Olive Oils — Part 2, Agilent Application Note,

Publication Number 5991-1895EN, (2013).

(3) Dobarganes et al., Pure & Appl. Chem. 71(2), 349–359 (1999).

(4) L. Brühl and H.J. Fiebig, Fat. Sci. Technol. 97(6), 203–208 (1995).

(5) H.J. Fiebig, Fett/Lipid 101, 442–445 (1999).

Thermal Treatment Analysis — Determination of

3,5-stigmastadienes in Olive Oil Using the Agilent Infi nity 1220

LC System with Diode Array DetectorSonja Schneider, Agilent Technologies Inc.

Agilent Technologies Inc.5301 Stevens Creek Blvd., Santa Clara, California 95051, USA

Tel: (800) 227 9770

Website: www.agilent.com/chem/infi nity-mobile-lc

1a

1

2

3

Time (min)

0 2 4 6 8 10 12 14 16A

bso

rb

an

ce

(m

AU

)

0

10

20

30

40

50

60

70

1a/1

2 3,5-campestadiene

3 3,5-stigmastadiene

3,5-cholestadiene

Figure 1: Detection of 3,5-stigmastadienes in partly refi ned olive oil.


FOOD AND BEVERAGE

Afl atoxins are natural mycotoxins produced by Aspergillus fungi.

High temperatures and humidity favour the occurrence of moulds

and therefore the production of afl atoxins. The contamination of

crops, nuts, dried fruits or vegetables, dried medicinal plants, and

milk is quite common. Because of their strong carcinogenicity,

afl atoxins must be controlled in food and feeds.

Scope

This method is suitable for the quantifi cation of afl atoxins B1, G

1,

B2, and G

2 in tomato extract according to the Test for Afl atoxins

(1) which limits afl atoxin B1 to 5 ppb and the sum of B

1, G

1,

B2, and G

2 to 20 ppb. Chromatography is performed on HPTLC

plates according to Method II.

Required or Recommended CAMAG Devices

Automatic TLC Sampler 4 or Linomat 5, Automatic Developing

Chamber ADC 2 or Twin Trough Chamber 20 cm × 10 cm,

Visualizer, TLC Scanner, and winCATS software.

Sample

Transfer 5 g of a representative powdered sample to a

glass-stoppered fl ask. Add 20 mL of methanol and water (17:3).

Shake vigorously by mechanical means for 30 min and fi lter.

Discard the fi rst 5 mL of the fi ltrate and collect the next 4 mL

portion. Transfer the fi ltrate to a separatory funnel. Add 4 mL of

sodium chloride solution (5 g of sodium chloride in 50 mL of

water) and 2.5 mL of hexane, and shake for 1 min. Allow the

layers to separate and transfer the lower aqueous layer to a

second separatory funnel. Extract the aqueous layer in the

separatory funnel twice, each time with 2.5 mL of methylene

chloride, by shaking for 1 min. Allow the layers to separate each

time. Separate the lower organic layer and collect the combined

organic layers in a 50 mL conical fl ask. Evaporate the organic

solvent on a water bath. Transfer the remaining extract to an

appropriate sample tube and evaporate to dryness on a water

bath. Cool the residue.

If interferences exist in the residue, proceed as directed for

Cleanup with immunoaffinity column (IAC); otherwise, dissolve

the residue obtained in 200 μL of acetonitrile, and shake by

mechanical means if necessary.

Cleanup with Immunoaffi nity Column (IAC)

Dissolve the residue of the above sample solution in 5 mL of

methanol and water (60:40) and then dilute with 5 mL of water.

Apply this extract onto a conditioned IAC. Rinse the IAC twice with

10 mL of phosphate-buffered saline (PBS) solution*, and perform

the elution slowly with 2 mL of methanol. Evaporate the eluate

with nitrogen, and dissolve the residue in 200 μL of acetonitrile.

IAC Preparation

Prior to conditioning, the IAC should be adjusted to room

temperature. For conditioning, apply 10 mL of PBS solution on

each column and pass through at a rate of 2–3 mL/min by gravity.

Leave 0.5 mL of PBS buffer on top of the column until the test

solution is applied.

For this application note, the sample of tomato extract was

extracted using an IAC from R-Biopharm.

Standards

Accurately weighed standard solutions containing 0.05 μg/mL

afl atoxin B1 and afl atoxin G

1 and 0.01 μg/mL afl atoxin B

2 and

afl atoxin G2 in a mixture of chloroform and acetonitrile (9.8:0.2)

were prepared.

Chromatography

Stationary phase: HPTLC Si 60 F254

20 cm × 10 cm (Merck).

Sample application: 10 μL of each test solution and 2, 5, 7.5, and

10 μL of standard are applied as 8 mm bands, a minimum of

2 mm apart, 8 mm from lower edge of plate.

Developing solvent: Chloroform–acetone–water (140:20:0.3)

(v/v/v)

Development: 20 cm × 10 cm Twin Trough Chamber or ADC 2,

saturated for 20 min (fi lter paper), 10 mL developing solvent

per trough, humidity control at 33% relative humidity (using a

saturated solution of MgCl2).

Developing distance: 70 mm from lower edge of plate.

Plate drying: 5 min in a stream of cold air.

Derivatization: Optional: dip (time 0, speed 5) in paraffi n,

n-hexane (2:3), dry in air.

Evaluation: Examination under UV 366 nm.

Densitometry

With CAMAG TLC Scanner and winCATS software in fl uorescence

mode at 366/>400 nm using a mercury lamp; evaluation via peak

area, linear regression.

Determination of Afl atoxins B1, G

1, B

2, and G

2 in Tomato Extract

by HPTLCCAMAG Laboratory


FOOD AND BEVERAGE

Reference

(1) USP 35-2: Test for Aflatoxins of chapter <561> Articles of Botanical Origin.

*Phosphate-buffered saline (PBS) solution: Prepare 10 mM phosphate buffer

solution containing 0.138 M sodium chloride and 0.0027 M potassium

chloride in water, and adjust with 2 M sodium hydroxide to a pH of 7.4. A

suitable powder mixture is available from Sigma as PBS P-3813.

CAMAGSonnenmattstrasse 1, 4132 Muttenz, Switzerland

Tel: +41 61 467 34 34 fax: +41 61 461 07 02

E-mail: [email protected]

Website: http://www.camag.com

1

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

1 2 3 4 5 6 7 8

Figure 1: Image of the derivatized plate under UV 366 nm.

600

500

400

300

200

100

0

0 50 100 150 200

pg

Ab

so

rb

an

ce (

AU

)

250 300 350 400 450

50.0

40.0

35.0

30.0

25.0

20.0

15.0

10.0

5.0

0.00.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45

45.0

50.0

40.0

35.0

30.0

25.0

20.0

15.0

10.0

5.0

0.0

45.0

Ab

so

rb

an

ce (

AU

)

Ab

so

rb

an

ce (

AU

)

(Rf)

30.0 G2

G1

B2

B1

25.0

Ab

so

rb

an

ce (

AU

)

Ab

so

rb

an

ce (

AU

)

20.0

15.0

10.0

5.0

0.0

0.10 0.15 0.20 0.25 0.30 0.35

(Rf)

0.40 0.45 0.50 0.55 0.65

0.0

5.0

10.0

15.0

20.0

25.0

30.0

0.60

Figure 4: Calibration function for afl atoxin B1 measured at 366 nm. Regression via area y = -113.971+1.931x+-0.001x2; r = 0.99998; sdv = 0.84%.

Figure 3: Densitogram of a tomato extract sample (red) and the same sample spiked with 5 ppb of afl atoxins B1 and G1 (blue).

Figure 2: Densitogram of standards afl atoxin G2, G1, B2, and B1.

Table 1: Track assignment.

Track Volume (μL) Sample

1 10 Tomato extract

2 10 Tomato extract with 5 ppb afl atoxins B

1 and G

1

(spiked by overspotting)

3 10 Tomato extract with 25 ppb afl atoxins B

1 and G

1

(spiked by mixing of afl atoxin B1 and G

1 with sample)

4 2 Standards afl atoxin B

1 and G

1 (0.05 μg/mL ´

100 pg absolute)


1 and G

1 (0.05 μg/mL ´

250 pg absolute)

6 7.5 Standards afl atoxin B

1 and G

1 (0.05 μg/mL ´

375 pg absolute)


1 and G

1 (0.05 μg/mL ´

500 pg absolute)

8 10 Tomato paste, spiked with afl atoxins B

1, G

1, B

2,

G2 (B

2, G

2 show only faint zones)

Results


FOOD AND BEVERAGE

Modifi ed starches are important materials used in many applications

including foodstuffs. The starches are modifi ed by a number of

methods — both physical and chemical — to tailor the properties to

the required application. Most commonly the starches are modifi ed

to give a particular texture to a fi nished foodstuff; for example, to give

extra thickening in puddings.

In this application note we show how two modified starch samples

with essentially the same molecular size in solution can be easily

differentiated and characterized by triple detection size-exclusion

chromatography (TD-SEC).

Triple Detection SEC

In the advanced technique of TD-SEC, the sample, after separation on

the chromatography column, is passed though a series of detectors

to provide a complete analysis of the molecules: The low angle light

scattering detector (LALS) provides a direct measure of the molecular

weight; the refractive index (RI) detector measures the concentration;

and the differential viscometer measures the intrinsic viscosity

(IV). From the measured IV and molecular weight (MW) values a

Mark-Houwink (M-H) plot showing structural changes can be made.

Instrumentation and Conditions

SEC system comprising the Viscotek GPCmax (degasser, pump,

autosampler) with the Viscotek TDA detector equipped with the

following detectors: Low angle light scattering; differential viscometer;

RI. The data were all calculated using OmniSEC software.

Discussion

The triple chromatogram of one of the modifi ed starch samples

is shown in Figure 1. The signal-to-noise on all three detectors is

excellent, which ensures the quality of the calculated data. The data

are calculated directly from the chromatograms by the OmniSEC

software and the results for both samples are shown in Table 1. Note

that the hydrodynamic radius (RH) of both samples is within 0.2 nm.

This means that by traditional GPC/SEC techniques the molecular

weights based on retention volumes would be the same. However,

TD-SEC clearly shows the weight average molecular weight of sample

A is only 60% of sample B. We can also see that the viscosity of A,

despite the lower molecular weight, is higher than B.

By looking at the structure plot (M-H plot, Figure 2) of both modified

samples (with a dextran T70 sample as reference), it is clear that the two

modified starches have very different molecular structures. Sample B

has a much more compact structure than sample A; shown by the fact it

appears lower on the M-H plot. This means that despite higher molecular

weight the molecules in sample B are denser — because of the different

modification — resulting in a lower intrinsic viscosity. The dextran T70

material is shown for reference. It indicates, as expected, that modified

starches have a much more compact structure than dextran.

Conclusions

The Viscotek triple detection system provides a convenient and rapid

way to characterize starches and modifi ed starches. The instrument

allows determination of molecular weight and molecular size in a single

run using normal conditions and sample concentrations. The IV and

size data allow differentiation between molecules of differing structures.

The technique is equally applicable to other polysaccharides and all

other synthetic or natural polymers such as proteins and DNA.

Structural Differences in Modifi ed StarchesMalvern Instruments Ltd.

Malvern Instruments Ltd.Enigma Business Park, Grovewood Road, Malvern, UK

Tel: +44 (0) 1684 892456


Website: www.malvern.com

1200

Detecto

r s

ign

al (m

V) 1000

800

600

400

200

-2002 6 10 14 18 22

Retention volume (mL)

RI

Viscometer

LALS

0

-0.2

-0.5

-0.7

-0.9

-1.1

-1.3

-1.5

4.5 5.0 5.5 6.0 6.5

Log molecular weight

Lo

g in

trin

sic

vis

co

sit

y

Dextran

Modified starch A

Modified starch B

Figure 1: Triple chromatogram of a modifi ed starch sample

Figure 2: Mark-Houwink (Structure) plot.

Table 1: Weight average molecular weight, number average

molecular weight, intrinsic viscosity, and hydrodynamic

radius data.

Sample Mw(D) Mn(D) IV(dL/g) RH(nm)

Modifi ed starch A 241.780 123.780 0.117 7.2

Modifi ed starch B 399.020 169.620 0.081 7.4


FOOD AND BEVERAGE

This application note describes a cost-effective and easy to use method

for the fast determination of pesticide residues in whole milk samples.

The method employs the AOAC version of QuEChERS. This procedure

provides better analytical results than either the original or EN versions

of the QuEChERS procedure in extracting a few sensitive pesticides,

such as pymetrozine and hexazinone (Velpar). A sample of 50 mg

primary secondary amine (PSA) and 50 mg C18 are used in dSPE for

the cleanup of whole milk samples. PSA removes organic acids and

carbohydrates, while C18 retains fatty acids and cholesterol. The result

is a clean extract for LC–MS–MS analysis.

QuEChERS Extraction

1) Transfer 15 mL of whole milk into a 50-mL centrifuge tube

(RFV0050CT).

2) Add internal standard to all samples, and appropriate amounts of

pesticide spiking solution to fortifi ed samples.

3) Add 15 mL of acetonitrile (MeCN) with 1% acetic acid.

4) Cap and shake 1 min at 1000 strokes/min using a Spex 2010 Geno/

Grinder.

5) Add salts (6 g MgSO4 and 1.5 g NaOAc) in Mylar pouch

(ECMSSA50CT-MP) to each tube, and vortex for 10 s to break up salt

agglomerates.

6) Shake for 1 min at 1000 strokes/min using Spex Geno/Grinder.

7) Centrifuge the samples at 3830 rcf for 5 min.

Determination of Pesticide

Residues in Whole Milk by

QuEChERS and LC–MS–MSXiaoyan Wang, UCT

UCT, Inc.2731 Bartram Road, Bristol, Pennsylvania19007, USA

Tel: (215) 781 9255


Website: www.unitedchem.com Figure 1: Whole milk samples extracted by the AOAC QuEChERS procedure.

Extraction and Cleanup Products

RFV0050CT 50 mL polypropylene centrifuge tube

ECMSSA50CT-MP 6 g MgSO4 and 1.5 g NaOAc in Mylar pouch

CUMPSC18CT 150 mg MgSO

4, 50 mg PSA, and 50 mg C18

in 2 mL centrifuge tube

Table 1: Accuracy and Precision Data (n = 5).

AnalyteSpiked at 10 ng/g Spiked at 50 ng/g

Recovery (%) RSD (%) Recovery (%) RSD (%)

Methamidophos 85.2 5.8 100.3 5.1

Pymetrozine 93.9 5.2 97.3 5.4

Carbendazim 100.4 3.8 102.8 3.1

Dicrotophos 102.3 2.1 106.5 2.9

Acetachlor 119.9 3.6 128.8 2.9

Thiabendazole 99.8 2.1 103.8 2.3

DIMP 90.3 3.2 93.1 4.7

Tebuthiuron 108.6 3.0 113.3 2.7

Simazine 102.6 1.6 105.1 2.7

Carbaryl 95.6 5.3 97.1 4.0

Atrazine 99.1 2.0 102.8 3.0

DEET 103.6 2.4 106.4 3.4

Pyrimethanil 91.0 4.7 92.3 4.0

Malathion 100.7 3.8 99.1 3.0

Bifenazate 85.6 9.1 81.0 8.7

Tebuconazole 91.0 2.7 91.9 3.5

Cyprodinil 94.2 2.1 95.6 3.1

Diazinon 96.8 2.6 97.7 3.5

Zoxamide 100.4 3.0 101.9 3.0

Pyrazophos 100.3 1.6 104.0 2.0

Profenofos 90.9 2.8 93.0 3.9

Chlorpyrifos 94.2 4.9 87.8 4.5

Abamectin 81.3 7.7 86.6 4.2

Bifenthrin 77.8 3.1 75.8 2.1

Overall mean 96.1 3.7 98.5 3.7

dSPE Cleanup

1) Transfer 1 mL supernatant into a 2-mL dSPE tube (CUMPSC18CT).

2) Shake for 2 min at 1000 strokes/min using Spex Geno/Grinder.

3) Centrifuge at 15300 rcf for 5 min.

4) Transfer 0.3 mL of the cleaned extract into a 2-mL auto-sampler vial.

5) Add 0.3 mL of reagent water, and vortex for 30 s.

6) The samples are ready for LC–MS–MS analysis.

Conclusion

A simple, fast, and cost-effective method has been developed to

determine pesticide residues in whole milk samples. Pesticide residues

in whole milk were extracted using the AOAC version of the QuEChERS

approach, followed by dSPE cleanup using MgSO4, PSA, and C18.

Excellent accuracy and precision were obtained, even for pymetrozine,

a sensitive pesticide with very low recovery when the original or EN

version of the QuEChERS approach is employed. The

overall analytical run time was 20 min with the overall

mean recovery for the 24 pesticides being 96.1% and

98.5% for 10 and 50 ng/mL fortifi ed samples, respectively.

LC–MS–MS conditions and SRM transitions are available

upon request.


INDUSTRIAL

The presented ion chromatographic (IC) method is applicable

to all biodiesel types and blends. Before chromatographic

separation, free glycerol and bound glycerol are isolated by

a straightforward extraction and saponifi cation-extraction

technique. Pulsed amperometric detection (PAD) following

chromatographic separation achieves an outstanding

method detection limit (MDL) of 0.5 ppm by mass for glycerol

and therefore easily fulfi lls ASTM and EN performance

specifi cations. The described method fully complies with

ASTM D 7591.

Biodiesel

The four primary driving forces behind the biofuel boom are

the world’s increasing thirst for petroleum (80 Mbarrels/day),

the diminishing supply of fossil fuels, global warming, and the

intention to reduce the dependence on fuel imports. In addition,

most biofuels are produced by straightforward manufacturing

processes, are readily biodegradable and non-toxic, have

low emission profiles, and can be used as is or blended with

conventional fuels.

Biodiesel is produced by transesterifying the triglycerides in

the parent oil or fat with an alcohol, usually methanol, in the

presence of a catalyst (base, acid, or enzyme) to yield fatty

acid methyl esters (FAME) and free glycerol as coproduct

(Figure 1). As reaction rates under acid or enzyme catalysis are

relatively slow, most producers use the rapid alkali-catalyzed

transesterification.

An incomplete reaction leads to the formation of residual

glycerol intermediates such as mono-, di-, and triacylglycerides

(bonded glycerols). In contrast, complete conversion results in

the formation of highly water-soluble glycerol (free glycerol).

The latter is separated from the final product at the end of the

production process. However, traces of glycerol are frequently

found in the ester phase. Both free and bonded glycerols

(= total glycerol) lead to severe operational problems such as

injector and valve deposits or filter clogging. Accordingly, the US

ASTM D 6751 (1) specifies a maximum total glycerol content of

2400 ppm (0.24%), while the European EN 14214 (2) stipulates

2500 ppm (0.25%). In both standards, the free glycerol content

is limited to 200 ppm (0.02%).

Based on the analysis of biodiesel blends made from

coconut oil, this article demonstrates sensitive analysis of the

free and total glycerol content via simple and innovative ion

chromatography (IC) followed by pulsed amperometric detection

(PAD) according to ASTM D 7591 (3).

Experimental

Instrumentation

The chromatography system consisted of the 850 Professional

IC with Amperometric Detector and the 858 Professional Sample

Processor (all Metrohm AG, Figure 2). For all separations, a

Metrosep Carb 1 - 150/4.0 anion-exchange column was used

with a flow rate of 1 mL/min. The injection valve was fitted with

a 20 μL injection loop and separation was achieved by isocratic

elution employing a 100 mmol/L NaOH eluent.

The amperometric detector consists of a gold working

electrode in combination with a solid-phase reference electrode

and a stainless-steel auxiliary electrode. A triple-step potential

waveform was applied.

Instrument control, data acquisition, and processing were

performed using MagIC Net software (Metrohm).

Glycerol Determination in Biodiesel and Biodiesel Blends

According to ASTM D 7591J. Gandhi and A. Wille, Metrohm

H2C

H2C

HC

OOC OOC

OOC

OOC

R1

R2 3 CH3OH

CH3

CH3

CH3

NaOH

R3

R1

R2

R3

+

H2C

H2C

HC

OH

OH

OH

+OOC

OOC

Triglyceride Methanol Fatty acid methyl esters Glycerol

Figure 1: Base-catalyzed transesterifi cation of a triglyceride with methanol.


INDUSTRIAL

Reagents

Glycerol standard and potassium hydroxide were reagent grade.

They were purchased from Sigma-Aldrich (Milwaukee, Wisconsin,

USA). Synthetic biodiesel blends B2 to B20 were produced by

the mixing of biodiesel made from glycerol-containing coconut-oil

and low-sulphur petroleum diesel, respectively. All standard

solutions and eluents were prepared from deionized water with a

specifi c resistance higher than 18 MΩ·cm.

Extraction and Saponifi cation

While the extraction of free glycerol can be performed by a

simple separating funnel, saponifi cation-extraction for bound

glycerol requires a commercially available refl ux system capable

of heating the reaction mixture to 90 °C.

(a) Free Glycerol

Generally, a high free glycerol content points to incomplete

separation of the ester and glycerol phase. Because of the high

water solubility of the triol — 1000 mg and more will dissolve

in a litre of water — free glycerol can be readily extracted from

biodiesel or biodiesel blends.

The procedure comprises the addition of 45 g of distilled water to

approximately 5 g of sample. After vigorous shaking for 5 min, the

sample is allowed to stand for another 5 min. After phase separation,

an aliquot of the aqueous phase is filled into a chromatography vial

and placed on the Sample Processor for analysis.

(b) Total Glycerol

Total glycerol is the sum of free and bound glycerol. The latter is

the sum of residual mono-, di-, and triglycerides and stems from

incomplete esterifi cation reactions. Glycerides are removed from

the organic phase by saponifi cation reaction with sodium hydroxide

and subsequent extraction of the generated glycerol with water.

In a reflux system, 20 mL of 0.01 mol/L potassium hydroxide

is added to approximately 2 g of sample. The mixture is heated

to reflux for 1 h. After cooling to room temperature, the volume

5

4

3

2

1

0

0 1 2 3 4 5

Time (min)

gly

ce

ro

r;

10

.0 m

g/L

Cu

rre

nt (

μA

)

Figure 2: 850 Professional IC with IC Amperometric Detector and 858 Professional Sample Processor.

Figure 3: Glycerol determination using pulsed amperometric detection.


INDUSTRIAL

of the mixture is made up to 50 mL. The released glycerol is then

extracted into the aqueous phase according to the procedure

described above.

Results

Calibration and Method Detection Limit

Calibration standards range from 0.5 mg/L to 100 mg/L.

Calibration is linear providing a correlation coeffi cient of 0.99996

with a relative standard deviation better than 0.437%.

The detection limit was determined by a 15-fold injection of a

0.5 ppm glycerol standard. The excellent method detection limit

(MDL) of 0.5 ppm glycerol by mass (0.7·10–4%) exceeds by far

the maximum free glycerol content of 200 ppm (0.02%) required

by the ASTM D 6751 or EN 14214.

Free and Total Glycerol Content in Different Biodiesel Blends

and a Coconut-oil Biodiesel Sample

While the total glycerol limit in ASTM D 6157 and EN 14214 is

0.24% and 0.25%, respectively, the allowed maximum content of

free glycerol is only 0.02% in both standards. The pure biodiesel

sample B100 contains 0.027% of free and 0.62% of total

glycerol (Table 1) and therefore exceeds the limits stipulated by

the two standards. Before being used as a fuel or being blended

1700

1600

1500

1400

1300

1200

1100

1000

900

800

Inte

nsit

y (

nA

)

Time (min)

B20 – total glycerol


B20 – free glycerol






0 1 2 3 4 5 6 7 8

Figure 5: Stacked PAD chromatograms of different biodiesel blends (B2…B20).

980

Inten

sit

y (

nA

)

Inten

sit

y (

nA

)

960

940

920

900

880

860

840

820

800

780

760

740

720

1300

1250

1200

1150

1100

1050

1000

950

900

850

800

750

700

650

600

0 1 2 3 4 5

Time (min) Time (min)

0.00056%0.01086%

(a) free glycerol (b) total glycerol

6 7 8 0 1 2 3 4 5 6 7 89 10 11 12

Figure 4: PAD chromatogram with (a) free and (b) total glycerol peaks in a B2 blend.


INDUSTRIAL

with petroleum diesel, the coconut methyl ester fi rst has to be

freed of its excess glyceride and glycerol contents.

According to Table 1 and Figure 4 the free and total glycerol

contents in all investigated biodiesel blends B2 to B20 are below

0.0051% and 0.124%.

Conclusion

Free and bound glycerol is determined by IC and subsequent PAD

in accordance with ASTM D 7591. It is a simple, cost-effective, and

very accurate method that includes a straightforward extraction

and saponifi cation-extraction for determining the free and bound

glycerol content, respectively. With an MDL of 0.5 ppm by mass

for total glycerol, IC–PAD easily exceeds the requirements of ASTM

and DIN standards.

References

(1) ASTM D 6751, Standard specification for biodiesel fuel blend stock (B100)

for middle distillate fuels.

(2) DIN 14214, Automotive fuels — fatty acid methyl esters (FAME) for diesel

engines — requirements and test methods.

(3) ASTM D 7591, Standard test method for determination of free and total

glycerin in biodiesel blends by anion exchange chromatography.

Table 1: Free and total glycerol content in different biodiesel blends.

Blend Statistic ParametersFree Glycerol Total Glycerol

Determined Expected Determined Expected

Mean value [%]weight

, n = 3 0.000559 0.000539a 0.01086 0.012434a

B2 Standard deviation [%]weight

0.000003 0.000344

Relative standard deviation 0.446% 3.168%


, n = 3 0.001322 0.001348a 0.029752 0.031084a


0.000007 0.001263



, n = 3 0.002759 0.002695a 0.061032 0.062168a


0.000321 0.000898



, n = 3 0.005046 0.005390a 0.124133 0.124336a


0.000229 0.001802



, n = 6 0.026950 0.026950 0.621680 0.621680


0.000570 0.019430

(Coconut oil) Relative standard deviation 0.021% 0.031%

acalculated by applying the appropriate dilution factor.

Metrohm International HeadquartersIonenstrasse, 9101 Herisau, Switzerland

Tel: +41 71 353 85 04

Website: www.metrohm.com


MEDICAL/BIOLOGICAL

Synthetic cannabinoids (SPICE) have become an increasing

problem and an ever-changing target during drug screening.

This application note describes a simple but effective method

for extraction of a range of SPICE compounds and metabolites

from oral fl uid (neat or from a commercial collection device),

with high reproducible recoveries and LLOD <1 ng/mL.

Extraction Conditions

Format: ISOLUTE SLE+ 400 μL plate (part number 820-0400-P01).

Sample pre-treatment: Mix oral fl uid sample (200 μL, neat or buffered

from collection kit) with ammonium acetate (100 mM, pH 5, 200 μL).

Sample load: Load pre-treated sample (400 μL) onto the ISOLUTE

SLE+ plate. Apply a short pulse of positive pressure and wait for 5 min.

Analyte elution: Apply ethyl acetate (2 × 700 μL). Apply short

pulses of pressure and collect eluent.

Post extraction: Evaporate to dryness and reconstitute in mobile

phase (500 μL).

HPLC Conditions

Instrument: Agilent 1200 Liquid Handling System (Agilent

Technologies, Berkshire, UK)

Column: Mac-MOD ACE Excel 2 C18-AR, 2.1 mm i.d. × 100 mm

9Mac-MOD Analytical, Chadds Ford, Pennsylvania, USA)

Mobile phase: A: 0.1%formic acid in water; B: 0.1% formic acid in

methanol, Isocratic, 15% A: 85% B at 300 μL min; 9 min run time;

ambient temperature

Injection volume: 10 μL

MS Conditions

System: Applied Biosystems/MDS Sciex 4000 Q-trap mass

spectrometer equipped with a Turbo Ionspray® interface (Applied

Biosystems, Forster City, California, USA)

Ion source temperature: 500 °C

Results

High (>70%), reproducible (RSD<10%) recoveries of a range of

SPICE drugs and metabolites, spiked into oral fl uid samples at

concentrations ranging from 50 ng/mL to 1 ng/mL, were obtained

using ISOLUTE SLE+ supported liquid extraction plates.

Conclusions

ISOLUTE SLE+ supported liquid extraction plates can be

successfully used to extract SPICE and metabolites from oral fl uid

samples. The method is simple, fast, and effective, and can be

applied to both neat samples,

and oral fl uid collected in

commercially available devices.

Extraction of SPICE from Oral Fluid Using ISOLUTE SLE+ Prior

to LC–MS–MS AnalysisFrank Kero and Victor Vandell, Biotage LLC

Biotage ABVimpelgatan 5, Uppsala, Sweden

Tel: +46 18 56 59 00 fax: +46 18 59 19 22


Website: www.biotage.com

Table 1: Retention times and multiple reaction monitoring (MRM) transitions for SPICE drugs and metabolites in positive mode

Turbo Ionspray.

Retention Time

(min)Analyte MRM Transition

Declustering Potential

(DP)

Collision Energy

(CE)

Cell Exit Potential

(CXP)

6.36 JWH-073 328>155 40 30 16

8.14 JWH-018 342>155 40 30 16

3.14 JWH-018 N- (4-hydroxypentyl) 358>155 40 30 16

3.34 JWH-018 5-pentanoic acid 372>155 40 30 16

2.99 JWH-073 N-(3-hydroxybutyl) 344>155 40 30 16

2.55 JWH-250 N-(5-hydroxypentyl) 352>120.9 40 30 16

3.98 JWH-200 385>155 40 30 16

5.32 JWH-250 336>121 40 30 16

3.14 d5-JWH-018 N- (4-hydroxypentyl 363.5>155 40 35 16

4.69 XLR-11 330>125 30 35 16

6.55 UR-144 312.5>125 30 35 16

6.37 UR-144 5-Chloro-pentyl 346.9>125 30 35 16

3.03 UR-144 Pentanoic Acid 342.5>125 30 35 16

3.00 UR-144 5-Hydroxy-pentyl 328.5>125 30 35 16


MEDICAL/BIOLOGICAL

According to the World Health Organization (WHO), heart disease is

the number one cause of death worldwide. As a result, medication

for heart treatment is counted among the most frequently prescribed

therapeutic classes. While most prescription drugs can cause some

adverse reaction in a patient, side effects of cardiovascular agents can

be particularly hard to manage. There may only be a subtle distinction

between a therapeutic dose and a life-threatening one. Therefore,

effi cient drug monitoring is an important tool in enhancement of

drug effi cacy and reduction of the risk of toxic effects resulting in a

balanced treatment.

With the advance of highly sensitive and fast liquid chromatography

tandem mass spectrometry (LC–MS–MS) instruments, triple

quadrupole technology has found its way into clinical drug monitoring.

It is the preferred technique for an increasing number of applications

in the clinical sector, demanding fast and efficient development of new

LC–MS–MS methods. Fast ultrahigh-pressure liquid chromatography

(UHPLC) screening using Shimadzu’s specialized scouting software

in combination with automated MS optimization for multiple reaction

monitoring (MRM) parameters are the perfect platform for rapid

generation of dedicated analytical procedures.

Experimental

For UHPLC method scouting, a Shimadzu Nexera X2 Method Scouting

System was used, consisting of two quaternary solvent pumps

Ultrafast UHPLC–MS–MS Method Development in Therapeutic

Drug MonitoringAnja Grüning and Gesa Schad, Shimadzu Europa GmbH

OH

OH

OH

Metoprolol

Lidocaine

O

H

N

H

H

N

Verapamil

Quinidine

Cl

N

N

HN

N

N

N

N

N

N NN

O

N

C

N

NH2

H3C

H3CO

H3CO

CH3

CH3

CH3

CH3

H3CO OCH

3

OCH3

NH2

O

O

O

Mexiletin

Disopyramide

Amiodarone

Losartan

O

O

O

N

Figure 1: Structures of cardiovascular drugs.

Table 1: Mobile and stationary phases used in method scouting.

Solvent Column

AA: Water Kinetex 2.6μ C18 (Phenomenex)

AB: 5 mM Ammonium acetate;

pH 8

Synergie 2.5μ Fusion-RP

(Phenomenex)

AC: 0.1% Formic acidSynergie 2.5μ Hydro-RP

(Phenomenex)

AD:10 mM Ammonium acetate;

pH 4.5

Shim-pack XR-ODS II 2.2μ

(Shimadzu)

BA: Acetonitrile Shim-pack XR-C8 2.2μ (Shimadzu)

BB: MethanolShim-pack XR-Phenyl 2.2μ

(Shimadzu)

BC: Acetonitrile–Methanol (50:50

v/v)

455.20

455.70

1:455.20>165.051:455.20>165.05

2:455.20>150.05

2:455.20>150.05

2:455.20>150.05

3:455.20>303.253:455.20>303.25

3:455.20>303.25(+)

1:455.20>165.05

165.05

Inten.(x100,000)

5

4

3

2

1

0

150.05303.15

261.25

105.05

135.15

455.90

455.20 (+)

455.80

0.00

0.0 0.1 0.2 0.3 0.4 0.0 0.1 0.2 0.3 0.4 0.0 0.1 0.2 0.3 0.4

1: m/z precursor adjustment

4: m/z product ion adjustment

2: setting Q1 prerod bias

5: setting Q3 prerod bias

3: product ion / CE selection

6: CE fine tuning

0.20

Time (min)

Time (min) Time (min) Time (min)

Mass-to-charge ratio (m/z)Time (min)

0.00 0.25 100 150 200 250 300

Figure 2: Automated multiple reaction monitoring (MRM) optimization on the LCMS 8040.


MEDICAL/BIOLOGICAL

(LC-30AD), an autosampler (Sil-30AC), and a column oven (CTO-20AC)

including a six-column switching valve (FCV-34AH). The system was

also equipped with a Shimadzu LCMS-8040 triple quadrupole mass

spectrometer via an electrospray ionization (ESI) source.

The method scouting system enables screening of a maximum of six

HPLC columns with up to 16 different eluents. The different mobile and

stationary phases used for method scouting for the separation of eight

cardiovascular drugs are displayed in Table 1.

For automated generation of an optimized MRM method the first step

is selection of the precursor ion, followed by mass-to-charge ratio (m/z)

adjustment of the precursor. The collision energy is optimized for the

most abundant fragments and finally the fragment m/z is adjusted. These

optimization steps were performed via flow injection analysis, each taking

30 s (Figure 2).

Method scouting was performed in a 30 h sequence using 5 min

and 2 min gradient runs with varying gradient slope and all possible

combinations of aqueous and organic mobile phases on the six columns

specified in Table 1.

Results

A total of 162 different chromatographic conditions were evaluated for

the best separation and peak intensities (Figure 3).

Final method:

Column: Synergie 2.5μ Hydro-RP, 100 × 2.00 mm

(Phenomenex)

Flow rate: 0.4 mL/min

Temperature: 50 °C

Solvent A: 5 mM Ammonium acetate, pH 8

Solvent B: Methanol

Gradient: 30–85%B in 5 min, 5.01 min to 95%B, 3 min hold,

2 min post time

Conclusion

The Nexera X2 method scouting system in combination with

Shimadzu’s ultrafast LCMS 8040 triple quad mass analyser is a unique

tool for quick and effi cient development of LC–MS–MS applications.

Chromatographic separation of eight cardiovascular drugs as well as

their identifi cation and quantifi cation was established successfully

within two working days.

Shimadzu Europa GmbHAlbert-Hahn-Str. 6–10, D-47269 Duisburg, Germany

Tel: +49 203 76 87 0 fax: +49 203 76 66 25


Website: www.shimadzu.eu

8,000,000

6,000,000

4,000,000

2,000,000

0

20,000,000

15,000,000

10,000,000

30,000,000

25,000,000

20,000,000

15,000,000

10,000,000

5,000,000

5,000,000

0

0

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

Time (min)

(a)

(b)

(c)

Figure 3: (a) Shim-pack C18, 5–95% BA in AC in 5 min; (b) Synergie Hydro-RP, 25–85% BB in AB in 2 min; (c) Synergie Hydro-RP, 30-85% BB in AB in 5 min.


MEDICAL/BIOLOGICAL

Size-exclusion chromatography (SEC) is well-established for mAb

aggregate analysis. As the technique has been used since the early

days of mAb development for pharmaceutical purposes, various

method improvements have evolved. For instance, the benefi ts

of arginine on analytical SEC of mAb aggregate samples are

well-known. Here, we present how SEC of mAb aggregate samples

can take advantage of other amino acid additives in the mobile

phase.

Recently, various approaches to improve analytical SEC have

focused on reducing the analysis time. For instance, this can be

achieved by staggered injection protocols or increased linear flow

rates — possible for columns with outstanding packing quality. On

the other hand, in the light of method optimization, the mobile phase

composition leaves less room for improvement when compared to

other chromatographic modes. As soon as a certain ionic strength

(important to inhibit electrostatic interactions without causing

hydrophobic interactions) and the pH of the mobile phase (to ensure

structural integrity of proteins and the stationary phase) are set, one

might think that the analysis depends solely on the particle size,

packing quality, and column length. However, the mobile phase

composition is not complete until the mentioned parameters have

been set. For example, Arakawa et al. have described the impact

of arginine on aggregate recovery in SEC (1). Confirming that this

effect was not caused by an increased ionic strength, Yumioka et al.

investigated the impact of sodium chloride as a rather chaotropic salt

on mAb aggregate SEC. By increasing the concentration of sodium

chloride, protein recovery was decreased (2). In fact, the arginine

addition ensured proper aggregate elution. This is also true for other

amino acids, as can be seen in Figure 1.

A mAb was aggregated by incubation at 75 °C for 5 min. The

sample was subsequently analysed via TSKgel UltraSW Aggregate

7.8 mm × 30 cm/L with different mobile phases, all of them using

virgin columns. A sample of 0.2 M lysine, arginine, proline, glutamine,

or sodium sulphate was added to 0.1 M sodium phosphate buffer,

pH 6.7, respectively. A flow rate of 1 mL/min was applied, and 20 μL

and 100 μg of the aggregated mAb sample were injected. The

columns were equilibrated for at least 10 column volumes. Figure 1

illustrates the results on aggregate recovery. Glutamine and proline

show a similar behaviour: The aggregates are hardly recovered for the

first two injections, while the aggregate peak suddenly appears for

injection #3 and #4. The rise is not as sudden for sodium sulphate,

but the aggregate peak only achieves its full size for injection #10.

In contrast to these results, lysine shows an even and improved

aggregate recovery compared to arginine. The inter-injection

variability is low, depicting the complete aggregate content for all

of the injections.

Besides aggregate recovery, resolution of the different sample

components, namely the monomer and the different aggregates,

is crucial for accurate analysis. Clearly, there is motivation to

increase resolution. If this was achieved with a simple and

inexpensive mobile phase additive, many applications could

potentially benefit from such an advanced buffer composition.

The impact of arginine in the mobile phase for analytical SEC of

mAb aggregates focusing on the separation performance has

been investigated and reported in the literature (3). Figures 2

and 3 depict the separation profile of an aggregated mAb sample

on TSKgel UltraSW Aggregate using 0.1 M sodium phosphate

buffer, pH 6.7, with an addition of either 0.2 M arginine or 0.2 M

proline.

Ten injections with the respective amino acid buffer were followed

by 10 injections applying sodium phosphate buffer with an addition

of 0.2 M sodium sulphate, to compare the two buffers. Monomer

aggregate resolution as well as monomer fragment resolution

is slightly improved for the two amino acid buffers. Table 1 lists

the resolutions for some amino acid buffers and the results for

the corresponding columns applying sodium phosphate buffer

containing 0.2 M sodium sulphate. New columns were used for

every amino acid.

A Toolbox of Amino Acids for Out-of-the-Box mAb SeparationsTosoh Bioscience

100

Injection number

L-glutamine

L-arginine

L-lysine

L-proline

Sodium

sulphate

Ag

gre

ga

te

re

co

ve

r (

%)

80

60

40

20

0

2 4 6 8 10

Figure 1: Aggregate recovery in analytical SEC on new columns. The mobile phases contain different amino acids: Lysine (yellow), arginine (red), proline (green), and glutamine (blue). Sodium sulphate instead of an amino acid was added as a reference. Lysine and arginine allow almost complete aggregate recovery starting with injection #1, while proline and glutamine lead to reduced aggregate recovery compared to sodium sulphate. Column: TSKgel UltraSW Aggregate; Flow: 1 mL/min; Injected volume: 20 μL; Injected mass: 100 μg; Detection: UV @ 280 nm.


MEDICAL/BIOLOGICAL

Arginine, proline, and glutamine provide slightly increased

monomer aggregate resolution. For arginine, the fragment monomer

resolution is also improved. Although these increases in resolution

are not drastic, they confirm that increased resolution as a result of

the use of an advanced mobile phase is possible and that mobile

phase testing can contribute to a more reliable and robust aggregate

analysis. Depending on the attributes of a particular mAb, one might

consider different amino acids. For mAbs which are especially prone

to unspecific interactions, lysine might be the preferable option, as

it provided the most reliable aggregate recovery beyond the tested

amino acids in this study. On the other hand, if an aggregated

mAb would cause less severe problems as a result of unspecific

interactions, arginine offers highest resolution of all the tested

amino acids and a slightly decreased aggregate recovery for the

first injections, compared to lysine.

References

(1) T. Arakawa et al., J. Pharmaceutical Sciences 99(4), 1674–1692 (2010).

(2) R. Yumioka et al., J. Pharmaceutical Sciences 99(2), 618–620 (2010).

(3) D. Ejima et al., J. Chromatography A 1094(1–2), 49–55 (2005).

Tosoh Bioscience GmbHZettachring 6, 70567 Stuttgart, Germany

Tel: +49 (0)711 13257 0 fax: +49 (0)711 13257 89


Website: www.tosohbioscience.de

80

60

40

20

-20

0

UV

ab

so

rb

an

ce

@ 2

80

nm

(m

AU

)

Time (min)

4 6 8 10 12 14

mo

no

me

r

fra

gm

en

t

ag

gre

ga

te

s

sodiun phosphate

after arginine

proline

100

80

60

40

UV

ab

so

rb

an

ce

@ 2

80

nm

(m

AU

)

20

-20

0

4 6 8

Time (min)

10 12 14

arginine

mo

no

me

r

fra

gm

en

t

ag

gre

ga

te

s

sodiun phosphate

after arginine

Figure 3: A mAb sample on TSKgel UltraSW Aggregate with 0.1 M sodium phosphate buffer containing 0.2 M proline in the mobile phase (blue). After 10 injections, the mobile phase was switched to sodium phosphate buffer with an addition of 0.2 M sodium sulphate (grey). Injection #10 of the corresponding mobile phase is presented in the chromatogram. Column: TSKgel UltraSW Aggregate; Flow: 1 mL/min; Injected volume: 20 μL; Injected mass: 100 μg; Detection: UV @ 280 nm.

Figure 2: A mAb sample on TSKgel UltraSW Aggregate with 0.1 M sodium phosphate buffer containing 0.2 M arginine in the mobile phase (red). After 10 injections, the mobile phase was switched to sodium phosphate buffer with an addition of 0.2 M sodium sulphate (grey). For both mobile phases, injection #10 is shown. Column: TSKgel UltraSW Aggregate; Flow: 1 mL/min; Injected volume: 20 μL; Injected mass: 100 μg; Detection: UV @ 280 nm.

Table 1: The average resolution of 10 injections with the

according mobile phase is listed in the table. Arginine

results in the highest resolution. Column: TSKgel UltraSW

Aggregate; Flow: 1 mL/min; Injected volume: 20 μL; Injected

mass: 100 μg; Detection: UV @ 280 nm.

BufferMean Rs

Monomer–Aggregates

Mean Rs

Monomer–Fragment

Arginine 1.6 3.2

NaP after Arginine 1.4 3.0

Proline 1.5 3.0

NaP after Proline 1.3 3.1

Glutamine 1.4 3.0

NaP after Glutamine 1.3 3.0

Lysine 1.3 3.0

NaP after Lysine 1.4 3.1


MEDICAL/BIOLOGICAL

Low-molecular-weight heparins (LMWHs) are obtained by

fractionation or depolymerization of natural heparins. They are

defi ned as having a mass-average molecular weight of less than

8000 and for which at least 60% of the total weight has a molecular

mass less than 8000.

Size-exclusion chromatography (SEC) has been the most

common way of measuring the molecular weight and molecular

weight distributions of LMWHs by using the two most common

detection technologies: ultraviolet (UV) coupled with refractive

index (RI) detection. However, these detectors embody a relative

method in order to determine molecular weights, requiring

calibration standards. A newer, absolute method involves the use

of multi-angle light scattering (MALS), which does not require

any standards. The European Pharmacopeia (EP) monograph

for LMWH specifi es the use of the UV/RI detection method and

provides a known calibration standard. Many laboratories around

the world have adopted this method.

We previously developed an SEC/MALS method and found it

to be very suitable for the analysis of LMWHs. We have recently

adopted the UV-RI method described in the EP monograph and

compared the molecular weight results generated for LMWH using

each detection type. The adopted method uses an Agilent LC-1200

series HPLC, 0.2 M sodium sulphate pH 5.0 mobile phase, Tosoh

TSK-gel G2000 SWxl column with Tosoh TSK-gel Guard SWxl, Waters

2487 dual wavelength UV detector, and Wyatt Optilab rEX refractive

index detector. For MALS analysis, the UV detector was replaced

with a Wyatt miniDAWN TREOS detector; all other methods aspects

remained the same.

The results indicated that both detection types are suitable

and acceptable for the analysis of LMWHs. The molecular weight

and distribution results generated using each detection type are

comparable. This indicates that a SEC/MALS method could be

adopted in place of the SEC/UV-RI method currently required by

the EP monograph, and that it would result in less time because it

obviates the need for calibration standards.

This note was graciously submitted by Lin Rao and John Beirne

of Scientifi c Protein Laboratories LLC.

Molecular Weight Determination of Low-Molecular-Weight

Heparins: SEC/MALS vs. SEC/UV-RI Wyatt Technology Corporation

LS dRI UV

Define Peaks: LMWH Sample

0.8

0.6

0.4Rela

tive s

cale

0.2

0.0

5.0 10.0

Time (min)

15.0 20.0 25.0 30.0 35.0

Define Peaks: LMWH Sample

1.0

0.5

0.0

Rela

tive s

cale

5.0 10.0

Time (min)

15.0 20.0 25.0 30.0 35.0

LS dRI

Figure 2: Examples of LS and RI traces for an LMWH sample.

Wyatt Technology Corporation6300 Hollister Avenue, Santa Barbara, California 93117, USA

Tel:+1 (805) 681 9009 fax: +1 (805) 681 0123

Website: www.wyatt.com Figure 1: Examples of UV and RI traces for an LMWH sample.


PHARMACEUTICAL/DRUG DISCOVERY

A fast, cost-effective, and highly sensitive method was developed for

the determination of fi ve barbiturates with four internal standards

(ISTDs) in urine using an Agilent 6430 Triple Quadrupole LC–MS

system and an Agilent Poroshell 120 EC-C18 column. The sample

of urine was extracted using an Agilent SPEC-C18AR cartridge.

Results indicate that the method effectively extracts the selected

barbiturates from urine, resolving the target compounds and the

ISTDs in 8.5 min. Though amobarbital and pentobarbital differ

only in the position of a methyl group and are, therefore, diffi cult to

separate, their resolution is suffi cient for routine analysis.

Materials and Methods

All compounds were purchased from Cerilliant Corporation, Round

Rock, Texas, United States.

Sample Preparation

1. Begin by centrifuging the urine sample at 2800 rpm for 5 min.

2. Pipette 1 mL of centrifuged sample into a 13 mm × 100 mm

borosilicate glass tube.

3. Add exactly 35 μL of the working deuterated internal

standard (butalbital-D5, pentobarbital-D5, secobarbital- D5, and

phenobarbital-D5).

4. Pipette 500 μL 0.1 M phosphate buffer into the sample. The

phosphate buffer is prepared by adding 13.61 g KH2PO

4 into

800 mL water, adjusting to pH 6.0 with KOH, then making the

volume up to 1 L.

5. Use a vacuum chamber with the Agilent SPEC-C18AR cartridge

for extraction. Condition the cartridge with 0.2 mL of MeOH and

load the sample solution.

6. Wash the column with 0.5 mL water and dry for 1 min.

Analysis of Barbiturates in Urine with Agilent 6430 LC–MS–MS

and Poroshell 120 EC-C18Elijah Steinbauer,1 Pat Friel,1 Rongjie Fu,2 and Andy Zhai,2 1 Toxicology Laboratory at the Veterans Administration, Portland, Oregon, USA, 2Agilent Technologies

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

Phenobarbital-d5

Phenobarbital

Butalbital-d5

Butalbital

Pentobarbital-d5

Pentobarbital

Amobarbital

Secobarbital-d5

Secobarbital

Counts (%) vs. acquisition time (min)

×1

02

4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6

Figure 1: MRM chromatograms of barbiturates and internal standards using an Agilent Poroshell 120 EC-C18 column.

Table 1: Optimized MRM conditions.

No. CompoundIon pair qualitative and

quantitative analyses

Retention

time (min)

1 Phenobarbital-d5 236.1 & 42.1:236:1 & 193.1 4.825

2 Phenobarbital 231.1 & 42.1:231.1 & 188.2 4.871

3 Butalbital-d5 228.1 & 42.1:228.1 & 185.1 6.105

4 Butalbital 223.2 & 42.1:223.1 & 180.1 6.150

5 Pentobarbital-d5 230.2 & 42.1:230.2 & 187.1 7.348

6 Pentobarbital 225.1 & 42.1:225.1 & 182.1 7.375

7 Amobarbital 225.1 & 42.0:225.1 & 182.1 7.485

8 Secobarbital-d5 242.2 & 42.1:242.2 & 199.1 8.118

9 Secobarbital 237.1 & 263:237.1 & 194.1 8.155



7. Elute the cartridge with 1 mL 90:10 hexane:ethyl acetate mixture.

8. Collect the eluent and dry the sample under nitrogen gas at 35 °C.

9. Reconstitute with 0.5 mL 90:10 water:acetonitrile mixture.

HPLC Conditions

The method was performed on the Agilent 1260 Infi nity LC with a

6430 Triple Quadrupole LC–MS.

Column: Agilent Poroshell 120 EC-C18, 2.1 mm × 100 mm, 2.7 μm

(p/n 695775-902)

Sample preparation: Agilent SPEC-C18CR, 3 mL, 15 mg (p/n

A5321920); Eluent A, 5 mM ammonium acetate; Eluent B, LC–MS

grade acetonitrile



Gradient: Time (min) % B

0 10

10 45

10.5 90

12 90

12.5 10

Temperature: 60 °C

MS Conditions

ESI drying gas: 350 °C, 10 L/min

Nebulizer: 40 psi

Negative ionization mode

Capillary: 4000 V, DEMV 400

Note: If using an instrument that is not confi gured for low delay

volume, use about 1 minute initial hold.

Results and Discussion

The superfi cially porous particles of Poroshell 120 have nearly

identical effi ciency as sub-2 μm totally porous materials and

therefore can be used to provide similarly fast and high resolution

analyses at a lower pressure. A separation of the nine barbiturates

in 8.5 min was achieved on the column with a gradient method

(Figure 1). Reasonable resolution was achieved between the

standard components, except for pentobarbital and amobarbital.

These are the isomers with the same product ions, which could

not be identifi ed by MS. However, they still have some separation

on Poroshell 120 EC-C18 and the resolution for amobarbital and

pentobarbital is suffi cient for routine analysis.

Linearity and Recovery

The stock standards solution, containing phenobarbital, butalbital,

pentobarbital, amobarbital, and secobarbital, was diluted to

a series of linear solutions of 3000 ng/mL, 1500 ng/mL, and

150 ng/mL. In each solution, the ISTDs of phenobarbital-d5,

butalbital-d5, pentobarbital-d5, and secobarbital-d5 were made up

to a concentration of 1000 ng/mL. The method showed excellent

linearity, being very close to 1.0 (from 0.9995 to 0.99998). For

more information, including calibration curves, see the full-length

application note, Agilent publication 5991-2596EN.

The standards, at a concentration of 150 ng/mL, were spiked

into the urine sample blank and processed with the solid-phase

extraction (SPE) procedure. The recoveries were calculated and are

shown in Table 2.

Conclusions

A method was developed for the extraction and separation of

barbiturates using an Agilent SPEC-C18AR for sample extraction

and an Agilent Poroshell 120 EC-C18 column for separation. The

sample preparation method effectively extracted the selected

barbiturates from urine, with suffi cient recoveries and precision.

The column provided good selectivity and good resolution for these

compounds. The method developed on the Agilent 6430 Triple

Quadrupole LC–MS system was suitable for quantitative analysis

of these compounds in urine, especially at low concentration levels.

Agilent Technologies2850 Centerville Road

Wilmington, Delaware 19808, USA

Website: www.agilent.com

Table 2: Recoveries of barbiturates from a urine sample

with SPE.

Compounds Phenobarbital Butalbital Amorbarbital Pentobarbital Secobarbital

Recovery

% (150

ng/mL)

60.6 87.0 125.8 92.7 97.8



The ability to simultaneously collect quantitative and

qualitative information from a DMPK analysis has the potential

to signifi cantly increase productivity in pharmaceutical drug

discovery and development. We present a single workfl ow

allowing P450 drug clearance values to be determined as

well as metabolites identifi ed, profi led, and their structures

elucidated. To be able to do all of this on a high throughput

UHPLC chromatographic timescale is essential for the high

levels of productivity required for today’s DMPK screening

laboratories. Haloperidol provides a good example of what

can be achieved.

HaloperidolC

21H

23NO

2FCl M+H+ = 376.1474

Workfl ow and ProtocolMicrosomal incubations were carried out by Unilabs Bioanalytical

Solutions at 1 μM drug concentration and a protein concentration

of 0.5 mg/mL. Aliquots were taken and quenched with acetonitrile

containing propranolol as an internal standard at eight time points

over a period of 60 min.

ChromatographyColumn: Fortis, 1.7 μm, H

2O, 2.10 mm × 30 mm

Column temperature: 30 °C

MPA: 0.1% formic acid in 95% H2O/CH

3CN

MPB: 100% CH3CN

Gradient: 0.0 0.3 2.0 2.5 2.6 3.0 min

MP %: 95 95 5 5 95 95 %

Flow rate: 300 μL/min


Simultaneous Quantitative and

Qualitative Measurements in

a Single Workfl ow to Increase

Productivity in Primary Drug

Metabolism InvestigationsBruker Daltonics

ID with SF

& MSMS

Acquire MS & auto

MSMS

Metabolite

Detection

Integrate Drug

Peak

Integrate Metabolite

Peak

Peak

Metabolite

Profile Drug

Determine T1/2

Figure 1: In a single workfl ow, data dependent MS–MS spectra identify and elucidate metabolite structures and drug clearance is measured.

IS

0.5 1.0 1.5 2.0 2.5 0

1

2

3

4

.

392

Time (min)

Inten

s ×

10

5

Figure 2: Metabolite detection software compares the data fi le for the drug (in this case t60) with the corresponding control sample. A base peak chromatogram of the difference is created allowing the metabolites to be easily observed and their mass determined to four decimal places.

4.00

3.50

3.00

2.50

2.00

1.50

1.00

0.50

0.00

0 10 20

H/IS 212 354 392

30 40 50 60 70

Figure 3: Time profi les for the disappearance of haloperidol and the appearance of three metabolites.

The high surface area and lipophilic ligand combined with a

hydrophilic end cap give this stationary phase a broad selectivity

and resolving power for the target drug and the metabolites. The

use of small particles allows UHPLC to compress the peak into a

tighter and taller peak, therefore enhancing detection of very low

level analytes.

Metabolite Detection

Metabolite detect software compares the data fi le for the drug (in

this case t60

) with the corresponding control sample. A base peak

chromatogram of the difference is created allowing metabolites m/z

354, 212, and even 392 to be easily observed.

Metaboli te detect ion sof tware is able to detect the

m/z = 392 metabolite even though it co-elutes with the internal

standard.

Drug and Metabolite Profi les

Integration is carried out on the XIC for the measured m/z

of each metabolite +/− 0.005 Da. Plotting the ratio of

metabolite to internal standard (M/IS) versus time produces



the metabolite profi les. Half-life and clearance values are

determined from the natural log (ln) of the drug profi le versus time

plot.

Linearity

MS–MS data was not available for m/z = 392 because of co-elution

with the internal standard. The high quality data available, even for

such a small peak, means SmartFormula is still able to predict the

formula and deduce that it is a mono-oxidative metabolite.

m/z = 392.2422 ∆m = 0.1 mDa (0.3 ppm)

C21

H23

NO3FCl Isotope fi t = 23 ms

Comparison with 3QBoth the AB Sciex API 5000 and Bruker impact QTOF yield

equivalent results for the clearance values. This can be clearly seen

by comparing the ln [Drug]/[IS] versus time plots.

The linearity and gradients of these plots are nearly identical and

result in values for t1/2

of 45 and 47 min, respectively.

The difference in y intercept is a result of a difference in relative

response of the internal standard and has no influence on the

clearance results.

ConclusionsThe Quan–Qual workfl ow is effective and robust using a rapid

analytical method suitable for high throughput screening at 1 μM

drug concentrations.

Metabolite detection software allows metabolites to be rapidly

identified and profiled even when compounds co-elute.

1000000

900000

800000

700000

600000

500000

400000

300000

200000

100000

0

0 10 20 30

Haloperidol

Y=18015x +13547

R2 = 0.9974

40 50 60

Figure 4: Linear calibration of 50 pg/mL to 50 ng/mL (3 decades) was achieved using the XIC for the measured m/z of each metabolite +/- 0.005 Da. R2

= 0.9974.

2.5

Inte

nse×

10

5

2.0

1.5

1.0

0.5

0.0

100 150 200 250 300 350 400

m/z

392.1422

89.0596

107.0704

127.0156

163.1313

185.1147

260.1642

+MS, 2.0-21min #454-464

Figure 6: The structure of metabolite m/z = 392 is easily identifi ed using Smartformula3D to understand the fragmentation pattern

100 150 200 250 300 350

m/z

1.5

123.0239

165.0710

1.0

0.5

0.0

Inte

nse×

10

5

+MS2, (354.1063), 28.9eV, 2.2-2.2min #490-502

O

F

N

Cl

C21

H18

NOFCl

Figure 5: The structure of metabolite m/z = 354 is easily identifi ed using Smartformula3D to understand the fragmentation pattern

1.4

1.2

0.8

0.6

0.4

0.2

0

0 10 20 30 40 50 60 70

1

impact Haloperidol

Y=-0.0147x +1.3022

0.4

0.2

-0.2

-0.4

-0.6

-0.8

0

0 10 20 30 40 50 60 70

Y=-0.0153x +0.2698

3Q Haloperidol

Figure 7: Clearance data from impact.

Figure 8: Clearance data from 3Q.

Bruker Daltonics Inc.40 Manning Road, Billerica, Massachusetts, USA

Tel: (978) 663 3660 fax: (978) 667 5993

Website: www.bruker.com



A purifi ed polyclonal antibody (IgG) is separated and fully

characterized using the Viscotek SEC-MALS 20, allowing

calculation of molecular weight and radius of gyration (Rg).

Therapeutic recombinant antibodies represent a growing proportion

of biopharmaceuticals and are primarily classed as Immunoglobulin

G (IgG). However, proteins have a tendency to aggregate over

time and one challenge for biologic drugs is that the presence

of aggregates will stimulate an immune response. Size-exclusion

chromatography (SEC) is a powerful tool that is commonly used to

look at the aggregation of proteins. While most SEC systems use a

single concentration detector such as ultraviolet (UV), the addition

of light scattering allows the molecular weight of the protein to be

measured independent of its retention volume. The new SEC-MALS

20 detector, which uses multi-angle light scattering (MALS), is

ideal for this application. In addition, the MALS detector makes it

possible to measure the radius of gyration (Rg) of molecules that

scatter light anisotropically.

In this application note, a purified polyclonal antibody (IgG)

is separated using SEC and characterized using the Viscotek

SEC-MALS 20.

Experimental Conditions

Samples were analysed using a Viscotek TDAmax system connected

to Viscotek SEC-MALS 20. The mobile phase was phosphate

buffered saline, which was also used to prepare the IgG for analysis.

Results

The SEC-MALS results are presented in Table 1. The monomer

(15.80 mL) and dimer (14.00 mL) peaks are clearly identifi ed by

the measured molecular weights and low polydispersity (Mw/Mn).

No size (Rg) can be measured for these peaks as they are below

the isotropic scattering threshold of 10–15 nm. Studying Figure 1,

it is just possible to see that the SEC-MALS 20 show the same

response for the monomer peaks at all angles. The aggregate peak

(13.23 mL) is clearly different. The molecular weight is higher and

more polydisperse, which shows that there is a variable composition

of molecules within the aggregate peak. Because it is large, the light

scattering response varies with angle and can be used to measure

the Rg.

Measuring Antibody Molecular Weight by SEC-MALSMalvern Instruments Ltd.

Malvern Instruments Ltd.Enigma Business Park, Grovewood Road, Malvern, UK

Tel: +44 (0) 1684 892456


Website: www.malvern.com

-0.6

026.1

5m

V

8.15 Ret Vol20.04

Figure 1: Overlay of MALS detector responses for IgG.

Table 1: Measured molecular weights of the different peaks

of the IgG sample.

Aggregates Dimer Monomer

Peak RV - (mL) 13.23 14.00 15.80

Mn - (kDa) 674.12 308.6 147.2

Mw - (kDa) 7661.00 309.2 147.4

Mw / Mn 11.364 1.002 1.001

Rg (w) - (nm) 26.6 N/C N/C

Wt Fr (Peak) 0.014 0.065 0.921

Weight Fraction % (Peak) 1.4 6.5 92



Monoclonal antibodies (mABs) are increasingly growing in importance

for the diagnosis and therapy of various diseases, including cancer and

autoimmune and infl ammatory disorders. One essential parameter to

defi ne their quality is the content of aggregates (dimers, trimers, and

higher aggregates). These aggregates can be formed during processing

and purifi cation or are the result of long-term storage. As a result of

aggregation, antibodies lose their pharmaceutical effi cacy and can

facilitate an immunology response.

Antibody fragments which lack the Fc region can be used for the

treatment of diseases. They can also be the result of degradation of full

length antibodies. Therefore, a GPC method, which offers the opportunity

to analyse antibodies and their aggregates, as well as antibody fragments

simultaneously, with superior resolution and high sensitivity is invaluable.

Experimental

GPC/SEC analysis was performed on a PSS SECcurity GPC system,

equipped with a PSS SECcurity SLD1000 light scattering detector, using

the following conditions:

Columns: PSS PROTEEMA, 5 μm, 2 × 300 Å

(8 × 300 mm each) +precolumn

Solvent: 100 mM sodium phosphate pH 6.7 + 0.25 M NaCl


Temperature: 25 °C

Detection: Refractive index (RI), ultraviolet (UV) at

λ = 214 nm, PSS SLD1000 (right-angle light

scattering [RALS]) at λ = 488 nm

Calibration: Light scattering

Injected mass: 60–80 μg

Data acquisition,

calibration, and

evaluation: PSS WinGPC UniChrom 8.1

Results

Figure 1 shows an overlay of elugrams obtained for a full length antibody

and antibody fragments analysed on a single set of columns.

All three detector signals for the analysis of a monoclonal antibody are

shown in Figure 2. The light scattering signal shows improved sensitivity

for high aggregates compared to the other signals.

Conclusion

The GPC/SEC method including UV, RI, and RALS can be used for

the simultaneous determination of aggregate content of monoclonal

antibodies as well as antibody fragments. The column combination

covers the separation range for all three types and provides a high

resolution for the determination of the dimer content. Because of its

molecular weight dependency, the PSS SLD1000 RALS detector

offers high sensitivity for very small quantities of high aggregates and

also allows the determination of the absolute molecular weight of the

antibodies. In addition, it has a unique feature for a light scattering

detector as the wavelength can be altered to increase the sensitivity.

Sophisticated Antibody Analysis by GPC/SEC with RALS

PSS Polymer Standards Service GmbHIn der Dalheimer Wiese 5, D-55120 Mainz, Germany

Tel: +49 6131 962390 fax: +49 6131 962390 11


Website: www.pss-polymer.com

100

80

60

40

20

0

Detecto

r s

ign

al (V

)

Multimers mABs

Fragments

Low-molecular-weight

impurities

FragmentsDimers

10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0

Elution volume (mL)

16 17

125

100

75

50

25

0

18 19 20

Detecto

r r

esp

on

se (

%)

Elution volume (mL)

21 22 23 24 25

2.0

1.5

1.0

0.5

0.0

10.0 12.5 15.0

mABs

Dimers

MultimersRefractive index

UV at 214 nm

Light scattering at 488 nm

Fragments

Detecto

r s

ign

al (V

)

Figure 1: Separation range of the column combination. The red curve shows the UV signal of a full length antibody and its dimers plotted against the elution volume. The blue curve is the elugram of antibody fragments and their high level aggregates.

Figure 2: Sensitive analysis of antibody aggregates. The light scattering signal for the dimer is relatively high compared to that of the mABs because of molar mass dependency and provides improved sensitivity for the detection of high aggregates (inset).



A novel method for Drug Antibody Ratio (DAR) determinations

based on size-exclusion chromatography-multi-angle light

scattering (SEC-MALS) in conjunction with ultraviolet (UV)

absorption and differential refractive index detection.

There has been a signifi cant resurgence in the development of

anti-body-drug conjugates (ADC) as target-directed therapeutic agents

for cancer treatment. Among the factors critical to effective ADC design

is the Drug Antibody Ratio (DAR). The DAR describes the degree of

drug addition which directly impacts both potency and potential toxicity

of the therapeutic, and can have signifi cant effects on properties such

as stability and aggregation. Determination of DAR is, therefore, of

critical importance in the development of novel ADC therapeutics.

DAR is typically assessed by mass spectrometry (MALDI-TOF or

ESI-MS) or UV spectroscopy. Calculations based on UV absorption

are often complicated by similarities in extinction coeffi cients of

the antibody and small molecule. Mass spectrometry, though a

powerful tool for Mw determination, depends on uniform ionization

and recovery between compounds — which is not always the case

for ADCs.

Here we present a method for DAR determination based on

SEC-MALS in conjunction with UV absorption and differential

refractive index detection. Figure 1 shows UV traces for two

model ADCs; molecular weights of the entire ADC complexes are

determined directly from light scattering data.

Component analysis is automated within the ASTRA 6 software

package by using the differential refractive index increments (dn/dc)

and extinction coeffi cients, which are empirically determined for each

specie or mined from the literature, to calculate the molar mass of the

entire complex as well as for each component of the complex.

In this example an antibody has been alkylated with a compound

having a nominal molecular weight of 1250 Da (Figure 2). Molar masses

of the antibody fractions are similar, which indicates that the overall

differences between the two formulations refl ect distinct average DARs

which are consistent with values obtained by orthogonal techniques. Note

that the molar mass traces for the conjugated moiety represent the total

amount of attached pendant groups; the horizontal trends indicate that

modifi cation is uniform throughout the population eluting in that peak.

Antibody Drug Conjugate (ADC) Analysis Wyatt Technology Corporation

Wyatt Technology Corporation6300 Hollister A venue, Santa Barbara, California 93117, USA

Tel: +1 (805) 681 9009 fax: +1 (805) 681 0123

Website: www.wyatt.com

Antibody-Drug Conjugate Analysis

(■) Mw of complex

(+) Mw of antibody

(x) Mw of conjugated drug

1.0x105

1.0x104

9.0 9.5 10.0 10.5 11.0 11.5 12.0

Time (min)

Complex Antibody Drug

DAR

ADC1

ADC2

167.8 (±1.2%)

163.7 (±1.2%)

155.2 (±1.8%)

155.6 (±1.2%)

12.6

8.1 6.5

10.1

Mw (kDa)

Mo

lar M

ass (

g/m

ol)

ADC1

ADC2

2.0x105

Molar mass vs. time

167.8 kDa

ADC1

ADC2

163.7 kDa1.8x105

1.6x105

1.4x105

1.2x105

Mo

lar

Ma

ss (

g/m

ol)

1.0x105

8.0x104

9.0 9.5 10.0 10.5Time (min)

11.0 11.5 12.0

Figure 2: Molar masses for the antibody and total appended drug are calculated in the ASTRA software package based on prior knowledge of each component’s extinction coeffi cent and dn/dc, allowing determination of DAR based on a nominal Mw of 1250 Da for an individual drug.

Figure 1: Molar masses for two distinct ADC formulations are determined using SEC-MALS analysis.

powered by

SPEMSGCHPLC

@ www.chromacademy.com/Premier

New structured courses - Operator and Method Developer - in both HPLC and GC

Full tutor support during your course with assessments and certification

Ask the expert - an answer to your chromatography questions within 24 hrs

Access to our e-learning library with 1000s of pages on HPLC, GC, MS and SPE

All previous CHROMacademy webcasts and accompanying tutorials

Full access to our HPLC and GC interactive troubleshooters

The entire LCGC back catalogue of webcasts and articles

e-Learning for analytical scientists

For our premier members

Find out more about premier membership

the applications book

Documents