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May 2015

Volume 28 Number 5

www.chromatographyonline.com

SAMPLE PREPARATION

PERSPECTIVES

New sample prep instruments

LC TROUBLESHOOTING

Calibration problemsGC CONNECTIONS

Review of new GC products on the market

Up In SmokeAn improved GC–MS method for

cigarette smoke characterization

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LC•GC Europe May 2015

Editorial Policy:

All articles submitted to LC•GC Europe

are subject to a peer-review process in association

with the magazine’s Editorial Advisory Board.

Cover:

Original materials: Cordula Damm/EyeEm/Getty

Images

Features265 Large-Volume Injection LC–MS–MS Methods for Aqueous

Samples and Organic Extracts

B. McKay Allred, Matthew J. Perkins, and Jennifer A. Field

Environmental sample analysis by large-volume injection (LVI) in

combination with liquid chromatography–tandem mass spectrometry

(LC–MS–MS) is described for polar and nonpolar analytes in both

aqueous samples and organic extracts.

272 Simultaneous Determination of Methotrexate and

Sulphasalazine in Plasma by HPLC–DAD

Siji Joseph, Sreelakshmy Menon, and Smriti Khera

A simple and robust high performance liquid chromatography–

diode array detection (HPLC–DAD) method for the simultaneous

determination of methotrexate (MTX) and sulphasalazine (SSZ)

from plasma is presented.

Columns278 LC TROUBLESHOOTING

Calibration Problems — A Case Study

John W. Dolan

Unexpected results from calibration standards create confusion in a

clinical liquid chromatography (LC) method.

282 GC CONNECTIONS

New Gas Chromatography Products, 2014–2015

John V. Hinshaw

John Hinshaw presents his annual review of new developments in the

fi eld of gas chromatography seen at Pittcon and other venues in the

past 12 months.

290 SAMPLE PREPARATION PERSPECTIVES

New Sample Preparation Products and Accessories at Pittcon 2015

Douglas E. Raynie

This yearly report on new products introduced at Pittcon (or in the

preceding year) covers sample preparation instruments.

Departments294 Products

298 Events

COVER STORY258 An Improved GC–MS Method for

Cigarette Smoke Characterization

Using a Novel Cold Trap, Dual

Column, and Cryofocusing System

J.R. Crudo, E. Rouget, and M. Rotach

The development of a novel trapping

system and a modifi ed GC–MS layout

(using dual chromatographic columns

and cryogenic focusing devices) has

enabled a major improvement in the

chromatographic separation of volatile

and semivolatile compounds in cigarette

smoke. This improvement has led to the

potential for identifying compounds which

are usually masked by the solvent peak.

May | 2015

Volume 28 Number 5

252

ES610523_LCE0515_252.pgs 04.30.2015 13:21 ADV blackyellowmagentacyan

©2015 Waters Corporation. Waters and The Science of What’s Possible are registered trademarks of Waters Corporation.

In the hands of leading researchers and clinicians,

scientific technologies from Waters are making a true

difference in the fight against cancer.

PHARMACEUTICAL n HEALTH SCIENCES n FOOD n ENVIRONMENTAL n CHEMICAL MATERIALS



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

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efficiency, productivity and value to their employer.

Kevin AltriaGlaxoSmithKline, Harlow, Essex, UK

Daniel W. ArmstrongUniversity of Texas, Arlington, Texas, USA

Michael P. BaloghWaters Corp., Milford, Massachusetts, USA

Brian A. BidlingmeyerAgilent Technologies, Wilmington,

Delaware, USA

Günther K. BonnInstitute of Analytical Chemistry and

Radiochemistry, University of Innsbruck,

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Peter CarrDepartment of Chemistry, University

of Minnesota, Minneapolis, Minnesota, USA

Jean-Pierre ChervetAntec Leyden, Zoeterwoude, The

Netherlands

Jan H. ChristensenDepartment of Plant and Environmental

Sciences, University of Copenhagen,

Copenhagen, Denmark

Danilo CorradiniIstituto di Cromatografia del CNR, Rome,

Italy

Hernan J. CortesH.J. Cortes Consulting,

Midland, Michigan, USA

Gert DesmetTransport Modelling and Analytical

Separation Science, Vrije Universiteit,

Brussels, Belgium

John W. DolanLC Resources, Walnut Creek, California,

USA

Roy EksteenSigma-Aldrich/Supelco, Bellefonte,

Pennsylvania, USA

Anthony F. FellPharmaceutical Chemistry,

University of Bradford, Bradford, UK

Attila FelingerProfessor of Chemistry, Department of

Analytical and Environmental Chemistry,

University of Pécs, Pécs, Hungary

Francesco GasparriniDipartimento di Studi di Chimica e

Tecnologia delle Sostanze Biologica-

mente Attive, Università “La Sapienza”,

Rome, Italy

Joseph L. GlajchMomenta Pharmaceuticals, Cambridge,

Massachusetts, USA

Jun HaginakaSchool of Pharmacy and Pharmaceutical

Sciences, Mukogawa Women’s

University, Nishinomiya, Japan

Javier Hernández-BorgesDepartment of Analytical Chemistry,

Nutrition and Food Science University of

Laguna, Canary Islands, Spain

John V. HinshawServeron Corp., Hillsboro, Oregon, USA

Tuulia HyötyläinenVVT Technical Research of Finland,

Finland

Hans-Gerd JanssenVan’t Hoff Institute for the Molecular

Sciences, Amsterdam, The Netherlands

Kiyokatsu JinnoSchool of Materials Sciences, Toyohasi

University of Technology, Japan

Huba KalászSemmelweis University of Medicine,

Budapest, Hungary

Hian Kee LeeNational University of Singapore,

Singapore

Wolfgang LindnerInstitute of Analytical Chemistry,

University of Vienna, Austria

Henk LingemanFaculteit der Scheikunde, Free University,

Amsterdam, The Netherlands

Tom LynchBP Technology Centre, Pangbourne, UK

Ronald E. MajorsLCGC columnist and analytical

consultant

West Chester, Pennsylvania, USA

Phillip MarriotMonash University, School of Chemistry,

Victoria, Australia

David McCalleyDepartment of Applied Sciences,

University of West of England, Bristol, UK

Robert D. McDowallMcDowall Consulting, Bromley, Kent, UK

Mary Ellen McNallyDuPont Crop Protection,Newark,

Delaware, USA

Imre MolnárMolnar Research Institute, Berlin, Germany

Luigi MondelloDipartimento Farmaco-chimico, Facoltà

di Farmacia, Università di Messina,

Messina, Italy

Peter MyersDepartment of Chemistry,

University of Liverpool, Liverpool, UK

Janusz PawliszynDepartment of Chemistry, University of

Waterloo, Ontario, Canada

Colin PooleWayne State University, Detroit,

Michigan, USA

Fred E. RegnierDepartment of Biochemistry, Purdue

University, West Lafayette, Indiana, USA

Harald RitchieTrajan Scientific and Medical. Milton

Keynes, UK

Pat SandraResearch Institute for Chromatography,

Kortrijk, Belgium

Peter SchoenmakersDepartment of Chemical Engineering,

Universiteit van Amsterdam, Amsterdam,

The Netherlands

Robert ShellieAustralian Centre for Research on

Separation Science (ACROSS), University

of Tasmania, Hobart, Australia

Yvan Vander HeydenVrije Universiteit Brussel,

Brussels, Belgium

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Cigarette smoke is a complex matrix containing between

4000 and 5300 different identified compounds (1,2,3).

When performing gas chromatography–mass spectrometry

(GC–MS) analysis on cigarette smoke extracts, scientists are

usually confronted with two main difficulties: The first is the

limited chromatographic space, which leads to unresolved

peaks, and the second is the risk of chemical interactions

between the sample and the collection solvent.

Cigarette smoke is known to consist of two distinct parts: the

particulate phase, which can be trapped using a glass fibre

pad filter (as described in ISO 4387 [4] and ISO 22634 [5]),

and the volatile phase, which can be collected in sampling

bags (6,7) or by impingers containing a trapping solvent either

at room temperature (8) or cooled (9,10). A further trapping

method has been detailed using a cryogenic instrument (11),

with a liquid extraction after the smoking run. However, all of

these trapping methods have drawbacks: the glass fibre trap

does not allow the collection of volatile compounds; and the

trapping in sampling bags leads to rapid decomposition of

unstable species, which requires time‑dependent handling

to avoid significant analytical variations. All of the described

trapping methods using solvents result in sample dilution,

which has the potential for chemical interactions between the

trapped compounds and the solvent.

On the chromatographic side, the choice of column

is a critical factor because the samples usually contain

compounds that cover a wide range of volatility and polarity.

Column selection is therefore usually a compromise between

suitability and optimization for a large variety of compounds.

The objective of this project was firstly to develop a

solvent‑free trapping tool for cigarette smoke that is efficient

and simple to use, and secondly to improve separation by

using two different chromatographic columns.

A glassware device was developed to trap cigarette

smoke at a low temperature in a vial compatible with the

headspace sampler. The GC–MS instrument was modified

to improve the chromatographic separation by the addition

of two cryo‑traps, each preceding a chromatographic

column of different polarity (adapted to the type of chemical

compounds), which significantly increased the total peak

capacity of the analysis.

Accordingly, several further technical modifications

were applied to allow the simultaneous use of two

chromatographic columns for the analysis of the samples,

which led to the development of a new alternative analytical

platform described in this article.

ExperimentalThe reference cigarettes 3R4F were supplied by the

University of Kentucky and conditioned according to

the ISO 3402 standard (12) (at least 48 h at 60% relative

humidity and 22 °C). They were inserted into the cigarette

An Improved GC–MS Method for Cigarette Smoke Characterization Using a Novel Cold Trap, Dual Column, and Cryofocusing SystemJ.R. Crudo, E. Rouget, and M. Rotach, Philip Morris International R&D, Philip Morris Products S.A., Neuchâtel, Switzerland.

Cigarette smoke is a highly complex matrix and presents analytical difficulties for the analyst performing compound identifcation by gas chromatography analysis coupled with mass spectrometric detection (GC–MS). The development of a novel trapping system and a modifed GC–MS layout (using dual chromatographic columns and cryogenic focusing devices) has improved the chromatographic separation of volatile and semivolatile compounds found in cigarette smoke. This improvement has led to the potential to identify compounds usually masked by the solvent peak. This approach has also reduced the amount of peak overlapping by increasing the chromatographic peak capacity with the use of two capillary columns chosen for their analytical specifcity.

KEY POINTS• Compounds were trapped at low temperature without

solvent, avoiding dilution, preventing chemical

interaction with solvent and allowing determination of

very volatile species, which are usually masked by the

solvent peak.

• Compounds were sorted by volatility and directed

to chromatographic columns well adapted for their

separation.

• Improvement of the characterization of cigarette smoke

was achieved.

Ph

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holder (Figure 1, part 8 and 9) and were lit with an electric

lighter. Eight puffs were taken following the ISO 3308

standard (13) (one puff per minute, puff volume: 35 mL, puff

duration: 2 s, puff shape: bell curve) using a puff generator

developed in‑house. The cigarette smoke was collected

using two traps mounted sequentially (Figure 1, parts 2

and 8). The first one, a standard glass fibre filter according

to the ISO 3308 standard (part 8 in Figure 1) (Borgwaldt),

collected the particulate phase of the aerosol. The second

trap, a custom‑made glass vial (Figure 2) cooled to a

temperature of ‑150 °C using a flow of liquid nitrogen

regulated by an electronic controller, collected the volatile

compounds representing the vapour phase.

The custom‑designed vial had a Vigreux column‑like

internal geometry to increase flow turbulence and to

maximize contact between the vial surface and the aerosol.

The external dimensions of the vial corresponded to the

model used for headspace analyses with the Turbomatrix

40 Trap (PerkinElmer) headspace sampling device.

To prevent contamination from the laboratory, a glass

tube filled with activated charcoal (part 7 in Figure 1) was

connected to the switch valve (part 6 in Figure 1). Between

each puff, the valve was switched to let pressure equilibrate

and avoid any back flush through the glass fibre filter and

the cigarette.

At the end of each smoking run, the vial was capped with

a Teflon cap. The glass filter contained in the filter holder

was transferred into a separate standard headspace vial

and capped with a Teflon cap.

This trapping mode enabled the analysis of compounds with

a large range of volatility without liquid extraction or dilution.

The use of a headspace sampler gave the opportunity to

sample the volatile and highly volatile compounds, without

extracting the compounds with low volatility.

Headspace Analysis: Headspace sampling was able

to capture a fraction of the headspace volume onto a

headspace trap prior to thermal desorption and injection onto

the chromatographic system via a heated transfer line. The

headspace trap was loaded with three different sorbents:

Tenax GR (PerkinElmer), Carbotrap and Carboxen (Supelco).

The choice of sorbents was made based upon internal

(unpublished) and external studies (14), which demonstrated

that no universal sorbent was adequate to effectively trap

and desorb the compounds of interest belonging to various

chemical classes, and that a mixture of sorbents was required.

After each smoking run, trapped semivolatile (glass fibre

filter in standard headspace vial) and volatile (custom‑made

headspace vial) fractions were submitted for headspace

extraction. The instrument conditions were optimized for

each fraction to ensure the effective transfer of trapped

smoke constituents. During the release phase, vapour

pressure was a key parameter influencing both the diversity

and quantity of compounds extracted (15); fine‑tuning was

required to avoid any segregation of compounds as a result

of differing volatilities. The headspace volumes generated

from each fraction were trapped concomitantly onto the

same headspace trap, and the retained compounds were

then released for injection into the chromatographic system

by thermal desorption at 210 °C.

The GC–MS system (Perkin Elmer Clarus 500) was

modified by the addition of two cryogenic systems (termed

cryo‑traps), developed and manufactured internally at

Philip Morris International for this device (Figure 3), and two

electronically actuated valves (4 ports gas valve, 300 psi,

1/16’’, N9302813, PerkinElmer, 8 ports gas valve, 300 psi,

1/16’’, N9302815, PerkinElmer), which were installed in the

oven to connect the two different chromatographic columns


Crudo et al.

87

6

3

41

2

5

9

Figure 1: HS‑cold trap trapping device: 1: glass cooling mantel; 2: vial; 3: glass head connection; 4: nitrogen evacuation pipe; 5: pipe connection to the puff generator; 6: 3‑way valve; 7: adsorption tube; 8: glass fibre filter holder; 9: cigarette holder.

2

Figure 2: Drawing of the custom‑designed vial.

4

53

2 1

76

Figure 3: Technical scheme of a cryo‑trap: 1: Metal trap formed by two cylinders separated by isolation material; 2: heating device; 3: electrical connection and type K thermocouple; 4: entrance of the capillary column coming from the valve; 5: exit for N2; 6: exit of the capillary column towards the GC; 7: liquid nitrogen entry. The trap is located on the GC, replacing the classical injectors.


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(1st column: 30 m × 0.32 mm, 1‑μm Agilent DB‑WAXetr;

2nd column: 30 m × 0.32 mm, 20‑μm Agilent HP‑PLOT/Q).

The two cryogenic systems, cooled using liquid nitrogen,

enabled the cryo‑focusing of compounds (at a temperature

of ‑80 °C for the one located before the DB‑WAXetr column

and at ‑120 °C for the one located before the HP‑PLOT/Q

column) before introduction to the respective columns. The

two switching valves were controlled by the GC–MS events

software. These modifications enabled a two‑dimensional

analysis of cigarette smoke fractions by GC–MS.

The analytical process was initiated by the operator.

LabVIEW software (National Instruments) then conveyed

this start signal to the GC and to the cryo‑traps, and was

therefore used as a master controller for the analytical

system. The two valves, V1 and V2, were controlled

indirectly via the GC programme sequence.

Improved Gas Chromatography, with Dual Columns and Cryogenic Systems Each analytical run was split in four successive phases to

allow adequate separation of compounds in the two cryogenic

traps and in the two chromatographic columns.

Phase 1: Sample Introduction and Cryo-Focusing

(Figure 4): Following desorption of the headspace trap, which

contained headspace constituents from both semivolatile and

volatile fraction collections, eluted compounds were focused

at ‑80 °C in the cryo‑trap positioned in front of the first column.

Highly volatile compounds, which were not retained at this

temperature, passed directly through the first chromatographic

column. These were trapped by the second cryo‑trap cooled

at ‑120 °C, located in front of the second chromatographic

column, which was maintained at ‑120 °C. These two

different trapping temperatures led to a split of very volatiles

compounds from volatiles, to elute each group of compound

selectively on the two dedicated chromatographic columns.

Phase 2: Release from Cryo-Trap #1 and Elution onto

the First Column: The first cryo‑trap was rapidly heated to

200 °C and a start signal was sent by the software to the

GC–MS system to start the chromatographic run. All

compounds trapped in cryo‑trap #1 were released and

introduced onto the WAX column. Analytes with limited affinity

for the column eluted rapidly and were trapped in the second

cryo‑trap. The remaining analytes continued to be separated

on the WAX column by their different retention behaviours.

Phase 3: Analysis of Semivolatile Compounds: Valve

V2 was switched (by the GC–MS) to isolate cryo‑trap #2

(maintained at ‑120 °C) and the PLOT Q column. This

switch connected the exit of the WAXetr column to the mass

spectrometer and enabled the analysis of the semivolatile

compounds remaining in the chromatographic column.

A first heat cycle of a dedicated double heat cycle GC

oven temperature programme was used with the following

parameters: initial temperature: 41 °C during 5 min, followed

by a temperature increase at a rate of 5 °C/min, up to 250 °C.

Phase 4: Release from Cryo-Trap #2 and Analysis of

Volatile Compounds: After chromatographic analysis of

the semivolatile compounds using the WAX column (30 min

run time), the oven was cooled to 60 °C using CO2 to speed

the process. For the release of the volatiles, valve V1 was

switched to isolate the WAX column and valve V2 was

switched back to direct the carrier gas through cryo‑trap #2,

the PLOT Q column, and into the mass spectrometer. The

volatile compounds were then introduced onto the PLOT

Q column by rapidly heating cryo‑trap #2 to 150 °C and

chromatographically separated using the second heat cycle

of the GC oven temperature programme with the following

parameters: 60 °C during 7.2 min, followed by a temperature

increase at a rate of 7 °C/min, up to 250 °C, final temperature

maintained during 9 min.

At the end of the analysis, the valve V1 was switched

again to allow the carrier gas to pass through both columns

and therefore flush the system. This step was done with

the oven heated to its maximum temperature, and to allow

the valve to be ready for the next run because it was not

possible to switch valve V1 after the end of the GC run.

Data acquisition in scan mode was initiated 15 min after

the start of each run because no meaningful data were

generated during the trapping phase for cryo‑trap #2.

A second pause in acquisition was also made between

46.8 min and 54 min, during the cooling phase of the oven

before the second temperature gradient started.

To compare the described approach with a classical

analysis, the cigarette 3R4F (supplied by University

of Kentucky) was conditioned according to ISO 3402

standard and smoked using the ISO smoking regime

(as described in ISO 3308 standard, 1 puff per min, puff


Crudo et al.

Transfer Line

PLOTColumn

GC oven

WAXColumn

1

1

2

3X

X

X4

2

345

6

78

V2

V1

MS

Highly volatile compounds

Volatile compounds

Semivolatile compounds

Less volatile compounds

Transfer LineHead spaceautosampler

CRY

O-T

RA

P 1

-80o

C

CRY

O-T

RA

P 2

-120

oC

Liquid N2 Liquid N2

Figure 4: Phase 1.

Scan EI+

TIC

1.04e6

1 2 3100

4.17 9.17 14.17 19.17 24.17

Time (min)

29.17 34.17 39.17 44.17

%

0

5 7 8

6

4

Figure 5: Chromatogram of mainstream smoke from the reference cigarette (3R4F supplied by the University of Kentucky). Ten cigarettes were smoked under ISO smoking regime and trapped on a glass fibre filter followed by three impingers containing each 10 mL ethyl acetate cooled at

‑78 °C. One microlitre of sample was injected on‑column in a DB‑FFAP column (30 m × 0.25 mm, 0.25‑μm). Analytes: (1) isoprene, (2) solvent, (3) toluene, (4) limonene, (5) nicotine, (6) neophytadiene, (7) triacetin, and (8) glycerin.


duration: 2 s, puff volume 35 mL, puff profile: bell shape).

The particulate phase of the smoke of 10 cigarettes was

trapped using a glass fibre filter and the gas phase was

bubbled through cooled (‑78 °C) ethyl acetate in three

small impingers mounted in series. The glass fibre filter

was extracted together with the solvent contained in the

impingers and was analyzed by GC–MS in scan mode

following an internal GC–MS method.

Results and DiscussionA typical total ion chromatogram (TIC) for the classical

analysis is presented in Figure 5. One drawback of this

technique is linked to the solvent, which prevents the

detection of coeluting compounds (volatile compounds).

Another issue is compound coelution, which can lead to

uncertainty during the peak identification process.

The innovative aspect of the new platform lies in the

use of a dedicated column selected specifically for

the volatility of each group of compounds. Using this

set‑up, the chromatogram showed first the semivolatile

compounds eluted on the 30 m × 0.32 mm, 1‑μm

column (Figure 6) and later the volatile ones eluted on

the 30 m × 0.32 mm, 20‑μm column (Figure 7). The

split between semivolatile and volatile compounds was

handled using two specific chromatographic columns with

dedicated oven programmes, allowing an improvement in

the chromatography.

A clear improvement in selectivity was apparent when

using the cold trap coupled with the headspace sampling

compared with the traditional analysis. Peaks are properly

resolved and can be identified with improved certainty. In

addition, the second part of the chromatogram (starting

at approximately 53 min) allowed the identification of

numerous volatile compounds that are masked by the

solvent peak when using conventional GC–MS analysis. For

example, Figure 7 demonstrates that compounds such as

chloromethane, HCN, acetaldehyde, ethanol, acetonitrile,

propionaldehyde, and acetone were clearly separated.

263www.chromatographyonline.com

Crudo et al.

21.6

14.4

7.2

016.52 18.03 19.54 21.05 22.56 24.07 25.58 27.09 28.60 30.11 31.63 33.14 34.65 36.16 37.67 39.18 40.69 42.20 43.71 45.22 46.73

Time (min)

1 2

3

5

7

8

9

10

11 14

13

12

15 16 17

6

4

Figure 6: Chromatogram of the semivolatile part of the mainstream smoke from one single reference cigarette (3R4F, supplied by the University of Kentucky) after separation on a DB‑WAXetr column. Analytes: (1) ethylbenzene, (2) limonene, (3) styrene, (4) acetol, (5) 2‑cyclopentene‑1‑one, (6) acetic acid, (7) Propanoic acid, (8) propylene glycol, (9) 2‑furanmethanol, (10) solanone, (11) nicotine, (12) neophytadiene, (13) phenol, (14) triacetin, (15) myosmine, (16) glycerin, and (17) nicotyrine.

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Furthermore, the sensitivity of the technique allows the

analysis of cigarette mainstream smoke from a single

cigarette, therefore reducing the number of cigarettes

required, while increasing the level of information gained.

The automation of the analytical process ensured

reproducible retention times across several different runs

(Figure 8) and displayed very volatile compounds on the

30 m × 0.32 mm, 20‑μm column. The same reproducibility

in retention times was observed on the 30 m × 0.32 mm,

1‑μm column.

ConclusionThe platform for trapping and analysis allowed the

characterization of cigarette smoke in an efficient and

simple way. The trapping device is an improvement

compared to classical liquid trapping because of the

absence of solvent, which reduces sample preparation,

prevents any chemical interaction between solvent and

compounds of interest, and allows determination of

chemical species which are usually masked by the solvent

peak. In addition, no dilution is performed during sample

preparation; therefore, the analysis can be performed

on a single cigarette. Improved sensitivity and resolution

enables a better and more comprehensive characterization

of cigarette mainstream smoke.

The method was focused on qualitative aspects to

facilitate compound identification, rather than to quantify

them. Because compounds may have very different

vapour pressures under the chosen conditions, headspace

sampling would not be suitable for direct quantification of

samples.

The improved chromatographic analysis allowed

an easier identification of compounds because of the

improved peak capacity provided by the hyphenation of

the two columns, which led to a better peak separation.

Retention times were reproducible because the complete

process was fully automated. This reproducibility

also allows the creation of custom libraries to improve

identification certainty.

References(1) R.R. Baker, in Tobacco: production, chemistry and technology, D.L.

Davis and M.T. Nielsen, Eds. (Blackwell Science Ltd, Oxford, 1999),

p. 398–439.

(2) M.F. Borgerding, J.A. Bodnar, H.L. Chung, et al., Food and

Chemical Toxicology 36, 169–182 (1997).

(3) T. Perfetti and A. Rodgman, Beiträge zur Tabakforschung

International 24(5), (May 2011).

(4) International Organization for Standardization (ISO): ISO 4387:2000,

Cigarettes ‑‑ Determination of total and nicotine‑free dry particulate

matter using a routine analytical smoking machine. Geneva,

Switzerland.

(5) International Organization for Standardization (ISO): ISO

22634:2008, Cigarettes ‑‑ Determination of benzo[a]pyrene in

cigarette mainstream smoke ‑‑ Method using gas chromatography–

mass spectrometry. Geneva, Switzerland.

(6) Ji‑Zhou Dong, J. Neil Glass, and Serban C. Moldoveanu, Journal of

Microcolumn Separations 12(3), 142–152 (2000).

(7) J.Z. Dong and S.M. Debusk, Chromatographia 71(3/4), (2010).

(8) N. Mottier, F. Jeanneret, and M. Rotach, Journal of AOAC

International 93(3), (2010).

(9) Health Canada: Determination of Ammonia in Mainstream Tobacco

Smoke, Official Method T‑101 (1999) http://laws‑lois.justice.gc.ca/

eng/regulations/SOR‑2000‑273/page‑14.html

(10) Health Canada: Determination of Selected Carbonyls in Mainstream

Tobacco Smoke, Official Method T‑104 (1999) http://laws‑lois.

justice.gc.ca/eng/regulations/SOR‑2000‑273/page‑14.html

(11) N. Plata, I. Hofer, S. Roudier, and J.P. Schaller, Journal of Aerosol

Science 37(12), 1871–1875, (2006).


Tobacco and tobacco products ‑‑ Atmosphere for conditioning and

testing. Geneva, Switzerland.


Routine Analytical Smoking Machine ‑‑ Definition and Standard

Conditions. Geneva, Switzerland.

(14) M. Schneider and K.‑U. Goss, Analytical Chemistry 81, 3017–3021

(2009).

(15) B. Kolb and L.S. Ettre, Static Headspace-Gas Chromatography:

Theory And Practice (Wiley, UK, 2nd ed., 2006).

Jean-René Crudo is an associate scientist at Philip

Morris International (Research and Development,

Neuchâtel, Switzerland). His analytical focus is on

the development and application of GC–MS methods

focusing on the qualitative and quantitative analysis of

complex matrices.

Emmanuel Rouget is a scientist at Philip Morris

International (Research and Development, Neuchâtel,

Switzerland). His main research interests are related to

the development and application of GC–MS techniques

concerning qualitative and quantitative analysis of

molecules.

Michel Rotach is a senior scientist at Philip Morris

International (Research and Development, Neuchâtel,

Switzerland). His main activities involve providing

scientific advice to sustain the development of Reduced

Risk Product (RRPs), perform data analysis, and provide

scientific guidance.


Crudo et al.

23.5

15.6

7.8

0

54.96 56.66 58.35 60.05 61.75 63.44 65.14 66.84 68.53 70.23 71.92 73.62 75.32 77.01 78.71 80.41 82.11 83.81 85.50 87.20 88.89

Time (min)

1 2

3

57

8

9

10

11

1413

12

15

16

17

18

19

21

22

20

6

4

Figure 7: Chromatogram of the volatile part of the mainstream smoke from one single reference cigarette (3R4F, supplied by the University of Kentucky) after separation on the HP‑PLOT/Q column. Analytes: (1) chloromethan, (2) HCN, (3) acetaldehyde, (4) 2‑methyl‑1‑propene, (5) 1‑3, butadiene, (6) 2‑ butene, (7) ethanol, (8) acetonitrile, (9) 2‑ butyne, (10) furan, (11) propionaldehyde, (12) acetone, (13) acrylonitrile, (14) propanenitrile, (15) metacrolein, (16) methylfuran, (17) mix of butyraldehyde and methyl ethyl ketone, (18) benzene, (19) 2‑pentanone, (20) 2,3‑pentanedione, (21) dimethyl disulphide, and (22) toluene.

75.15 76.15 77.15 78.15

TIC

9.58e9

79.15 80.15 81.15

Time (min)

100

%

2

Figure 8: Overlay of the volatile part of three chromatograms from reference cigarettes (3R4F, supplied by the University of Kentucky).


Environmental sample analysis by large-volume injection

(LVI) in combination with liquid chromatography–tandem

mass spectrometry (LC–MS–MS) is the direct introduction

of large sample volumes (for example, 900–1800 µL) into

an LC system for separation and subsequent detection. The

primary advantages of LVI compared to traditional off-line or

on-line sample preparation techniques, such as solid-phase

extraction (SPE) or liquid–liquid extraction (LLE), include

decreased sample preparation, greater analyte mass

introduced for detection (that is, increased sensitivity),

and less solvent and solid waste. LVI is well-suited for the

concentration and separation of water-soluble analytes

on reversed-phase guard and analytical columns (1),

and has also been extended to organic extracts (2).

Successful combination of LVI with LC–MS–MS requires an

understanding of analyte properties (polarity, stability, and

so on) and sample properties (salinity, particulate matter,

matrix components, and more) (3,4). The unique elements

of several applications are discussed here to highlight

the various capabilities and limitations of LVI and are

divided between aqueous and organic solvent injections.

Finally, a set of loading strategies designed to capture

nonionic analytes from large volumes of organic extracts

on reversed-phase columns is described and experimental

components are listed.

ExperimentalAn Agilent 1100 high performance liquid chromatography

(HPLC) system, upgraded with a 900-µL analytical head

and an extended-seat capillary sample loop, was used for

all methods described below.

Aqueous Samples: Direct InjectionPast examples of aqueous LVI include the analysis

of neurotoxins in drinking water, pharmaceuticals in

wastewater, and corrosion inhibitors in surface water (4).

Here, two LVI methods are presented with a focus on the

important technical features required to inject 1800 µL

onto C18 guard and analytical columns. The LVI methods

highlighted involve the direct injection of raw wastewater

for the determination of illicit drugs (5) and seawater with

25% isopropanol for the determination of the surfactant

components in the oil dispersant Corexit (Nalco) (6).

Before injection, raw wastewater is centrifuged to remove

particulate matter that can potentially clog columns and

other LC components (5). Filtration is a commonly used

alternative, but its suitability is method- and analyte-specific

because filter membranes can serve as a potential sink or

source of analyte contamination (4,7). SPE, however, is not

limited by particulate matter and has been used to analyze

both the aqueous and particulate bound fraction (8). After

centrifugation, the raw wastewater sample is added to

an autosampler vial and spiked with isotopically labelled

internal standards.

The six-port valve connections in the LC autosampler

(Figure 1) are labelled as either mainpass (solid red

channel) or bypass mode (dashed blue channel). The

mobile phase in mainpass mode flows sequentially through

Large-Volume Injection LC–MS–MS Methods for Aqueous Samples and Organic ExtractsB. McKay Allred1, Matthew J. Perkins2, and Jennifer A. Field2, 1Department of Chemistry, Oregon State University, Corvallis,

Oregon, USA, 2Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon, USA.

Environmental sample analysis by large-volume injection (LVI) in combination with liquid chromatography–tandem mass spectrometry (LC–MS–MS) is described for polar and nonpolar analytes in both aqueous samples and organic extracts. LVI is the direct introduction of large sample volumes (for example, 900–1800 µL) into the LC system for separation and subsequent detection. The practical benefts of LVI include faster sample preparation times, lower detection limits, and minimal waste generation.

KEY POINTS

• Environmental sample analysis by LVI in combination

with LC–MS–MS is described for polar and nonpolar

analytes in both aqueous samples and organic

extracts.

• The technique offers several advantages over

traditional small-volume injection and off-line sample

cleanup techniques without diminished method

performance.

• Benefits include faster sample preparation times,

lower detection limits, and minimal waste generation.

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the analytical head, sample loop, needle, extended-seat

capillary sample loop, and then onto the columns. Bypass

mode directs the flow directly onto the column. Initially, the

LC system is operated in bypass mode so that the needle

may be inserted in the sample vial and the first 900-µL

volume is withdrawn. The needle is returned to its seat and

the 900 µL of wastewater is then ejected into a 1400-µL

extended-seat capillary sample loop, which acts as a

storage device during the withdrawal of the second 900 µL.

Afterwards, the six-port valve is switched to mainpass

mode and the cumulative 1800 µL of wastewater sample is

chased by the mobile phase out of the sample loops and

onto C18 guard and analytical columns. After the sample is

out of the sample loops, the six-port valve switches back to

bypass mode. If left in mainpass mode, the gradient would

travel through all of the mainpass tubing before reaching

the column, requiring that the total run time be extended by

several minutes.

Both the wastewater sample and initial mobile-phase

solvents are aqueous with characteristically low eluotropic

strengths on reversed-phase columns, such that

methamphetamine and other illicit drugs and biomarkers are

focused on the head of the C18 guard column. The guard

column protects the analytical column and is preemptively

replaced every 50 sample injections. This is ultimately more

cost and time effective than using individual SPE cartridges

for sample cleanup and analytical column protection.

After loading analytes onto the C18 guard and analytical

columns, a traditional reversed-phase gradient is used to

separate and elute all analytes.

Neither reversed-phase LVI or SPE can selectively remove

matrix components that are coeluted with the analytes

of interest. With LVI, a postcolumn divert valve can be

precisely controlled to divert column effluent containing

sample matrix components away from the detector. The

control of divert valve timing is achieved with greater

precision than the analogous wash steps used in SPE

and can send all unfavourable effluent to waste. As will be

discussed below, the postcolumn divert valve is of even

greater importance with the direct injection of seawater.

An LVI-based method was developed by Place and

colleagues (6) using the direct injection of seawater to

quantify the surfactant components of Corexit: dioctyl

sulphosuccinate (DOSS), Tween 80, Tween 85, and Span

80. Liquid standards of sorbitan monooleate (Span 80;

purity: 70.5%), sorbitan monooleate polyethoxylate (Tween

80; purity: 74%), and sorbitan monooleate polyethoxylate

(Tween 85; purity: 67%) were obtained from Sigma

Aldrich. The direct injection of 1800 µL followed similar

steps as described above for wastewater with two crucial

adjustments. Open seawater does not require centrifugation

and filtration was shown to remove the analytes of interest

from solution. In addition, the Corexit surfactants undergo

hydrophobic exclusion from aqueous solutions and exhibit

significant loss (>50%) to polypropylene or glass container

walls within 8 h of sampling at ambient temperature. To

prevent analyte loss before analysis, seawater is diluted

with isopropanol at the time of collection to give a final

composition of 1:3 isopropanol–seawater. Injections of

1800 µL of the isopropanol-stabilized matrix resulted in

well-resolved peaks for anionic and nonionic surfactant

classes. At higher additions of isopropanol, there is no

improvement of analyte stability in aqueous samples and

peak shape begins to suffer.

Direct injection of seawater into the LC–MS–MS system

is possible without damaging the mass spectrometer

source by using the divert valve to direct the nonvolatile

salts contained in seawater to waste. The 1800-µL injection

volume (3.6 min loading at 0.5 mL/min) is followed by a

5.9-min “wash” under the primary mobile-phase conditions

(5% acetonitrile and 0.5 mM ammonium acetate in water).

This wash is diverted to waste and is sufficient to flush the

nonvolatile salts from the column and away from the mass

spectrometer before elution of the analytes of interest. The

time (5.9 min) used to wash the salts from the seawater

from the columns was evaluated by testing effluent fractions

collected at 30-s intervals with the addition of 100 µL of 1 M

silver nitrate–0.6 M nitric acid. The presence of chloride

in the collected effluent was determined by the formation

of a white silver chloride precipitate, visible in aqueous

solutions containing more than 0.1% seawater. No silver

chloride was observed in the effluent 7.5 min after injection

and a precautionary period of 2 min was added to extend

the loading and wash period to 9.5 min. For this application,

guard columns are replaced approximately every 100

injections and the analytical column was replaced after

approximately 1500 injections. Analysis over two years and

more than 5000 injections did not lead to salt accumulation

or corrosion in the LC–MS–MS system.

Organic Extracts: Orthogonal ChromatographySample treatment to create organic extracts is desirable

if analytes are unstable in aqueous solution, because

of hydrolysis or hydrophobic exclusion, or if the sample

matrices (for example, landfill leachate) are sufficiently

complex that significant sample cleanup is required before

injection. In addition, some analytes do not focus well

on reversed-phase columns, even under high aqueous

conditions, and require a different retention mechanism. For

example, per- and polyfluorinated alkyl substances (PFAS)

present in landfill leachate are commonly concentrated

by SPE before injection (8) because of leachate matrix

complexity. Direct aqueous injection for PFAS analysis is

additionally disadvantageous because polar PFAS are not

retained well on reversed-phase columns and the more

hydrophobic PFAS are lost from aqueous matrices onto

autosampler vials and to the air–water interface. The two

Analytical

head

C18 analytical column

MS–MS

Waste

Waste

Pump

Mainpass

Bypass

C18 guard

Six-port valve

Figure 1: Basic LVI configuration for 1800-µL injections

onto a reversed-phase system. The six-port valve and

injection assembly was adapted from Agilent autosampler

schematics (16).


Allred et al.


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new strategies discussed here for the LVI of PFAS in an organic solvent extract from leachate include orthogonal chromatography (9) and on-line extract dilution.

In-line orthogonal chromatography was developed for PFAS in ethyl acetate–trifluoroethanol extracts of landfill leachates (9). The orthogonal columns system consisted of two zirconium-modified diol (Zr-diol) guard columns (6 µm, 150 Å, 12.5 mm × 4.6 mm, Agilent) placed in-line with a Zorbax Eclipse Plus C18 analytical column (3.5 µm, 95 Å, 75 mm × 4.6 mm, Agilent) (Figure 2). The system proved capable of capturing 70 anionic PFAS from 900-µL injections of the 1200-µL ethyl acetate–trifluoroethanol extract, which is 75% of the extract volume generated.

The PFAS are captured by the Zr-diol columns through interactions between the anionic PFAS and the zirconium d-orbitals during the 900-µL injection of the ethyl acetate–trifluoroethanol extract. The trapped PFAS are then eluted from the Zr-diol guard columns by a mobile phase containing 50% methanol and 5 mM ammonium acetate that then refocuses the majority of analytes on the C18 analytical column. PFAS are then separated by a traditional reversed-phase gradient with an increasing percentage of organic solvent (Figure 3). Without LVI, additional sample preparation steps like solvent transfer and blow-down are necessary to introduce sufficient analyte mass in the small injection volumes typically used (such as 10–20 µL). Moreover, an injection of 10–20 µL of an extract concentrated into about 100 µL is, at most, 20% of the total extract volume generated. Comparatively, LVI capitalizes on the time, effort, and labour costs expended during the creation of organic extracts by analyzing a greater percentage, while also eliminating additional steps that increase the possibility of analyte loss.

Organic Extracts: DilutionOrthogonal chromatography (as described above) is not capable of capturing nonionic PFAS from a large volume of organic extract. Dilution of organic extracts with water is one approach to perform LVI for the analysis of nonionics in organic extracts (for example, pesticides in vegetable [10] or soil extracts [11]). The traditional use of small-volume injections with organic solvent extracts of high eluotropic strength achieve focusing of analytes on reversed-phase columns because of dilution of the small organic extract volume with the larger volume of initial aqueous mobile phase. In the case of PFAS in landfill leachates, an eightfold dilution with water would be needed to retain the most water-soluble, nonionic PFAS on C18 columns (for example,

MeFBSA). For comparable levels of sensitivity using off-line dilution, the 900-µL extract, injected previously, would become at least 7.1 mL and require multiple large autosampler vials, larger extended sample loops, and a longer injection programme. However, as previously mentioned, many PFAS can partition out of the bulk solution under high aqueous conditions. Therefore, on-line dilution strategies are explored here.

The objective of the following dilution-based LVI method is to use the ethyl acetate–trifluoroethanol leachate extracts and the orthogonal Zr-diol–C18 column system for the analysis of nonionic PFAS. Thus, the first alternative to off-line dilution was to inject 900 µL in small volume “packets” segmented by defined volumes (determined by flow rate and time) of the initial aqueous mobile phase. To minimize the overhead time required by the autosampler transport assembly to inject multiple small volumes, one 900-µL volume was drawn up into the sample loop and loaded onto the column in small packets by switching back and forth between mainpass and bypass mode (Figure 2). The volume of packets is determined by the flow rate and time spent in mainpass mode. The flow rate while loading packets onto the Zr-diol guard columns and C18 analytical column was 2 mL/min and the times spent in mainpass and bypass mode were programmed as 0.01 and 0.3 min, respectively (Figure 4). Mainpass and bypass were switched back and forth 18 times over the first 7.5 min where the six-port valve was left in mainpass mode for 1 min the last time. At 8.3 min the gradient was started in bypass mode and the flow rate was decreased to 0.5 mL/min.

The solvents in the mainpass assembly are pressurized when the six-port valve is in mainpass mode. But when in bypass mode, small volumes are lost to the waste line on the six-port valve as the mainpass assembly solvents depressurize and expand. Consequently, the six-port valve leading to waste (Figure 2) was plugged with a blanking nut to avoid losing small volumes of sample while switching between mainpass and bypass mode. Because the waste line was plugged, the 900 µL of mainpass solvent normally ejected to waste in preparation for drawing up sample were instead programmed to eject into an empty sample vial.

120

Time (min)

Resp

on

se (

%)

100

80

60

40

20

00 5

1 2 3 4 5 6

10 15 20 25 30 35

Figure 3: Typical leachate chromatogram for select PFAS using the Zr-diol guard column configuration. Peaks: 1 = PFBA, 2 = PFHxS, 3 = PFOS, 4 = MeFBSAA, 5 = 6:2 DiPAP, 6 = PFHxDA.

Analytical head

Pump

Six-port valve

Waste

Waste

MS–MSC18 analytical

column

MainpassBypass

Zr-diol guard

Zr-diol guard

Figure 2: Schematic of configuration for PFAS analysis in leachate extracts.


Allred et al.


When samples are loaded using this on-line,

packet-dilution configuration, both ionic and nonionic PFAS

are captured and separated from landfill extracts (Figure 4).

The most water-soluble ionic (PFBA) and nonionic

(MeFBSA) PFAS are successfully retained after 18 packet

injections of leachate extract.

The LC pressure isotherm (Figure 4) is an important

tool to aid method development. Pure and mixed solvents

have different viscosities and therefore exert different

corresponding back pressures that can be used to track

gradient and sample delivery. The sawtooth pressure profile

during the first 7.5 min is evidence of extract solvent packets

mixing with the aqueous mobile phase in the Zr-diol guard

columns. While programmed as 0.01 and 0.3 min, the actual

times spent in mainpass and bypass mode were calculated

using the average frequency of packet mixing seen in the

pressure isotherm and were closer to 0.028 and 0.35 min,

respectively. So at 2 mL/min the average packet was

56 µL with 700 µL of aqueous mobile phase on either side.

The volume of mobile phase on both sides of the organic

packets effectively dilutes the extract solvent inside the

Zr-diol guard columns, thus reducing the eluotropic strength

of the sample solvent so that focusing of the nonionic PFAS

occurs on the C18 analytical column. The Zr-diol guard

0 5 10 15 20 25

250 120

100

80

60

40

20

0

200

150

100

50

0

Pre

ssu

re (

bar)

Resp

on

se (

%)

Time (min)

Pressure

Response

1 2 3 4

Figure 4: Pressure isotherm and chromatogram for

mainpass and bypass packet injections. Landfill leachate

peaks for ionic PFAS: 1 = PFBA, 4 = PFOcDA. Nonionic

PFAS peaks: 2 = MeFBSA, 3 = EtFOSA.

C18guard

Pump

C18 analyticalcolumn

Six-port valve

MS–MS

Waste

Waste

MainpassBypass

At-column dilutionhardware changes

Analyticalhead

Figure 5: Divided-flow at-column dilution schematic. The

tubing internal diameters for tubing 1 and 2 were changed

from 0.175 mm to 0.125 and 0.76 mm, respectively.

Allred et al.

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columns were more effective mixing chambers than other

guard columns (such as amino propyl, C18) that required

smaller packet volumes to focus nonionic PFAS on the head

of the C18 analytical column. After loading nonionic PFAS

the analytes were eluted from the Zr-diol guard columns and

C18 analytical column separately; the anionic PFAS were

focused on the Zr-diol columns and nonionic PFAS were

focused on the C18 column.

The second alternative strategy is to trap nonionic PFAS

from an organic extract on reversed-phase columns using

a variation of at-column dilution that divides the flow from a

single pump. At-column dilution typically uses two pumps

that either pushes the sample at a slow flow rate to the tee

junction or pumps a weak solvent at a high flow rate that

dilutes the sample when the two flows combine at the tee

(12). The amount of sample dilution is a function of the two

flow rates. At-column dilution has been applied to load

larger sample volumes onto columns during preparative LC

(13) or analytical LC (for example, pesticides in river water

using on-line SPE with LC–MS–MS) (14). The advantage

of divided-flow at-column dilution is that a second pump

does not need to be purchased and at-column dilution is

achieved using only two tee junctions and tubing of different

internal diameters (Figure 5).

Chromatographic separation was accomplished using a

Zorbax Eclipse Plus C18 guard column (12.5 mm × 4.6 mm,

5-µm Agilent) and a Zorbax Eclipse Plus C18 analytical

column (75 mm × 4.6 mm, 3.5-µm Agilent). The internal

diameter of the tubing between the tee and the autosampler

six-port valve (Figure 5) was decreased from 0.175 to

0.125 mm. The internal diameter of the tubing connecting

the two tees was 0.175 mm and the internal diameter of

the 3-cm length of tubing on the outflow of the second tee

and before the C18 guard column was large (0.76 mm) to

ensure complete mixing of the two recombined flowpaths.

Mobile-phase A consisted of 3% methanol in deionized

water and mobile phase B consisted of 100% methanol

and 10 mM ammonium acetate. The initial aqueous mobile

phase flow rate was 3 mL/min for 3.5 min, during which

time the 900-µL leachate extract was loaded onto the C18

guard column. The C18 guard column effluent was sent to

waste for the first 3.5 min to keep the pressure low during

the high flow rate. After 3.5 min the flow rate dropped to

0.5 mL/min and the guard column effluent was directed

onto the analytical column. The gradient was then increased

to 100% over the next 10 min and held for 5 min. PFAS

were detected using a Quattro Premier XE MS–MS system

(Waters Corporation), operated in multireaction monitoring

mode.

Adequate dilution of the organic extract before the C18

guard column is evidenced by nonionic peak retention and

symmetry (Figure 6). More polar PFAS (such as PFBA) were

not retained using only a C18 guard column. To capture

anionic PFAS additional guard columns with orthogonal

retention mechanisms (for example, Zr-diol) can be added

before or after the C18 guard column with minimal increases

to the back pressure.

While in mainpass mode the flow was divided at the first

tee following the pump (Figure 5), with less mobile phase

being directed through the sample loop. The reduced

flow of mobile phase in the sample loop slowly moved the

sample volume to where it combined with the rest of the

flow at the second tee. The pressure isotherm (Figure 6)

increased 1 min after switching to mainpass mode when

the diluted 900-µL organic solvent extract first reached the

guard column. After 3.5 min the entire sample had passed

through the guard column and the C18 guard column

was once again filled with the aqueous mobile phase.

For 900 µL to pass through the guard column in under

2.5 min the flow rate through the autosampler was at least

0.36 mL/min, which corresponds to a 8.3-fold dilution when

combined at the second tee with the rest of the 3 mL/min.

Reducing the internal diameter of the tubing leading to

the six-port valve from the first tee from 0.175 to 0.125 mm

effectively reduced the percent flow to the six-port valve

from 20% to 12% to achieve the eightfold dilution necessary

for nonionic PFAS retention.

The incidental introduction of foreign matter can adversely

change the internal diameter and by extension the percent

flow to the six-port valve will also change (15). Should the

internal diameter change significantly, samples may not

be sufficiently diluted or may take longer to load onto the

column. The risk of foreign matter introduction can be

reduced by centrifuging or filtering the samples and mobile

phases. Any change of the flow division at the first tee will

be evident in the pressure isotherm profile, and clogged

tubing can be cleared with extended flushing (15) or easily

replaced; the divided-flow at-column dilution tubing used is

relatively short (<5 cm) and therefore inexpensive. Overall,

divided-flow at-column dilution has a simple, more universal

configuration compared to “packet injections,” and it exerts

lower back pressures, is faster, and can be used to inject

more than 900 µL by using the extending capillary (Figure 1)

used in the wastewater and seawater methods described

above.

ConclusionLVI combined with LC–MS–MS offers several advantages

over traditional small-volume injection and off-line

sample cleanup techniques without diminished method

performance. Direct injection LVI often requires little to no

sample preparation and reduces solid and solvent waste

generation associated with traditional off-line sample

preparation. The injection of large sample volumes is

analogous to the concentration step of SPE and increases

1

0 2 4 6 8 10 12 14

2 3 4

70 120

Pressure

Response

100

80

60

40

20

0

60

50

40

30

20

10

0

Pre

ssu

re (

bar)

Resp

on

se (

%)

Time (min)

Figure 6: Divided-flow at-column dilution pressure isotherm

and chromatogram. Peaks 1 = PFHxS, 2 = MeFBSA,

3 = EtFOSA, 4 = PFOcDA.


Allred et al.


the sensitivity of a given analysis by

introducing more mass of the analytes

of interest to the detector. Matrix

components can be separated from

the analytes of interest with greater

specificity using LVI because of the

control afforded by gradient elution

and waste divert valves. Centrifugation

or filtration is necessary before the

direct injection of aqueous samples

with suspended particulate matter,

whereas some SPE-based methods

simultaneously analyze the dissolved

and particulate phases.

When matrix complexity or analyte

stability require the generation of

an organic extract, LVI capitalizes

on the time, effort, and labour costs

expended by injecting a larger

percentage of the total extract

volume. LVI of organic extracts also

shortens sample preparation without

a loss in sensitivity by eliminating

additional steps associated with small

volume injections (such as solvent

evaporation) and the corresponding

opportunities for analyte loss. Direct

injection of organic extracts onto

orthogonal columns was sensitive

and robust, but was limited to

ionic analytes. On-line dilution of

organic extracts enables both ionic

and nonionic analyte retention on

reversed-phase columns and is

preferable to off-line dilution for

analytes unstable in aqueous solution.

Divided-flow at-column dilution

required only one pump, tee junctions,

and tubing and efficiently focused

anionic and nonionic analytes onto a

reversed-phased system.

References(1) T. Reemtsma, L. Alder, and U. Banasiak, J.

Chromatogr. A 1271, 95–104 (2013).

(2) W.J. Backe, T.C. Day, and J.A. Field,

Environ. Sci. Technol. 47, 5226–5234

(2013).

(3) W.J. Backe and J.A. Field, Environ. Sci.

Technol. 46, 6750–6758 (2012).

(4) F. Busetti, W.J. Backe, N. Bendixen, U.

Maier, B. Place, W. Giger, and J.A. Field,

Anal. Bioanal. Chem. 402, 175–186 (2012).

(5) A.C. Chiaia, C. Banta-Green, and J. Field,

Environ. Sci. Technol. 42, 8841–8848

(2008).

(6) B.J. Place, M.J. Perkins, E. Sinclair, A.L.

Barsamian, P.R. Blakemore, and J.A.

Field, Deep Sea Research II (http://dx.doi.

org/10.1016/j.dsr2.2014.01.015i) (2014).

(7) J.W. Martin, K. Kannan, U. Berger, P.D.

Voogt, J. Field, J. Franklin, J.P. Giesy, T.

Harner, D.C.G. Muir, B. Scott, M. Kaiser,

U. Järnberg, K.C. Jones, S.A. Mabury,

H. Schroeder, M. Simcik, C. Sottani, B.V.

Bavel, A. Kärrman, G. Lindström, and

S.V. Leeuwen, Environ. Sci. Technol. 38,

248A–255A (2004).

(8) J.P. Benskin, M.G. Ikonomou, M.B.

Woudneh, and J.R. Cosgrove, J.

Chromatogr. A 1247, 165–170 (2012).

(9) B.M. Allred, J.R. Lang, M.A. Barlaz, and

J.A. Field, J. Chromatogr. A1359, 202–211

(2014).

(10) A.C. Hogenboom, M.P. Hofman, S.J. Kok,

W.M.A. Niessen, and U.A.T. Brinkman, J.

Chromatogr. A 892, 379–390 (2000).

(11) I. Rybar, R. Gora, and M. Hutta, J. Sep. Sci.

30, 3164–3173 (2007).

(12) “At-Column Dilution Application Notes,”

Waters Corp., Milford, Massachusetts, USA,

2003, pp. 1–22.

(13) K.F. Blom, J. Comb. Chem. 4, 295–301

(2002).

(14) H. Sasaki, J. Yonekubo, and K. Hayakawa,

Anal. Sci. 22, 835–840 (2006).

(15) J.W. Dolan, LCGC North Am. 19, 478–482

(2001).

(16) Agilent Technologies, Agilent 1100

Series Standard Micro and Preparative

Autosamplers, Agilent Technologies,

Germany, 2001, pp. 205–206.

B. McKay Allred is with the

Department of Chemistry and Matthew

J. Perkins and Jennifer A. Field are

with the Department of Environmental

and Molecular Toxicology, all at Oregon

State University, in Corvallis, Oregon,

USA. Direct correspondence to:

[email protected]


Allred et al.

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Methotrexate (MTX, Figure 1) is considered an “anchor

drug” in the treatment of rheumatoid arthritis and is among

the most effective disease-modifying antirheumatic drugs

(DMARDs) with less toxicity and better tolerability. However,

MTX alone is often not able to fully control disease activity.

In fact, MTX is used in combination with other nonbiological

DMARDs by 99% of all rheumatologists (1–3). In a 2009

review and meta-analysis by Katchamart and colleagues

(2), results from 19 clinical trials on the efficacy and toxicity

of various nonbiological DMARDs in combination with

MTX, or of MTX alone were compared. It was noted in this

review that several of the combination drug trials (5 out of

19) were recorded evaluating the combination of MTX and

sulphasalazine (SSZ, Figure 1). It has also been reported

elsewhere that the MTX and SSZ combination therapy is

frequently used, and it is inexpensive and efficacious for

the treatment of rheumatoid arthritis (1–6).

The drug concentration of MTX is currently monitored

in clinical practice for the adjustment of treatment (7). The

clinical monitoring of the plasma concentration of SSZ

as well as the patient’s liver function is also performed

for the detection of any immune-allergenic reaction (8).

Additionally, both MTX and SSZ are highly protein bound

(50% for MTX, and 99% for SSZ) and thus are likely to

displace each other through interactions with plasma

proteins (9,10). They also have a common mechanism of

action, that is, inhibition of folate-dependent enzymes (6).

Therefore, the simultaneous monitoring of plasma levels of

MTX and SSZ when used in combination may have both

clinical relevance as well as help further pharmacokinetic

research into any synergistic effects.

The typical oral therapeutic dose of MTX used in

rheumatoid arthritis ranges from 7.5 to 25 mg per week

(2,3,7). Oral doses of SSZ are usually 500 mg twice

daily, but may be increased to 3 g per day depending

on response. However, only 2–13% of this oral dose

reaches the systemic circulation in unchanged form.

Thus, the highest SSZ plasma concentration found in

healthy volunteers ranges from 6 µg/mL to 32 μg/mL

(8). Many methods for the chromatographic analysis of

MTX have been published, and their relative merits have

been reviewed extensively by Rubino and colleagues

(7). Most of these published methods (50 out of 72) used

UV detection for the determination of MTX and the most

commonly used wavelength was between 303 and 313 nm

(in 42 out of 50 assays). Similarly, there are several studies

about the determination of SSZ and its metabolites for

clinical monitoring of its pharmacologic effects in different

biological matrices. Most of these analyses have been

carried out by high performance liquid chromatography

(HPLC) (8). However, to our knowledge, no methods

currently exist for the simultaneous determination of MTX

and SSZ.

Simultaneous Determination of Methotrexate and Sulphasalazine in Plasma by HPLC–DADSiji Joseph1, Sreelakshmy Menon1, and Smriti Khera2, 1Life Science Center, Agilent Technologies India Pvt. Ltd., Bangalore, India, 2Life Science and Diagnostics Group, Agilent Technologies, Inc., Santa Clara, California, USA.

Here we describe a simple and robust high performance liquid chromatography–diode array detection (HPLC–DAD) method for the simultaneous determination of methotrexate (MTX) and sulphasalazine (SSZ) from plasma. MTX and SSZ are used in combination for the treatment of rheumatoid arthritis. Using two detector wavelengths, 304 nm for MTX and 358 nm for SSZ, we were able to selectively quantitate both analytes during the same chromatographic run. The method was validated using quality control samples for critical analytical performance criteria of recovery, reproducibility, selectivity, accuracy, and precision.

KEY POINTS• A simple and robust high performance liquid

chromatography–diode array detection (HPLC–

DAD) method for the simultaneous determination of

methotrexate (MTX) and sulphasalazine (SSZ) from

plasma is described.

• The method is more sensitive, allows a wider

calibration range, and can be performed using a

much smaller (200 µL) volume of plasma sample than

previously reported UV methods for the bioanalysis of

MTX.

• The method was validated consistently with

FDA guidance, using quality control samples for

critical analytical performance criteria of recovery,

reproducibility, selectivity, accuracy, and precision.

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Here, we describe a simple HPLC–diode-array detection

(DAD) method for the simultaneous determination of MTX

and SSZ in plasma. Using two detector wavelengths we

were able to selectively quantitate both analytes during the

same chromatographic run. The chromatographic method

was optimized for selectivity between the two analytes as

well as from background matrix interferences. We have

also compared two sample preparation strategies: off-line

solid-phase extraction (SPE) and protein precipitation.

Although the SPE approach allowed for more-sensitive

detection and a wider calibration range, protein

precipitation is also discussed in brief because of its

simple and inexpensive nature. The bioanalytical method

developed here was validated for the critical analytical

performance criteria recovery, reproducibility, linearity,

sensitivity, selectivity, accuracy, and precision, and those

results are also presented here.

Experimental SectionChemicals and Reagents: MTX, SSZ, methanol

(Chromasolv grade), blank human plasma, ammonium

hydroxide, formic acid, and ammonium acetate used in this

study were all obtained from Sigma-Aldrich India. Milli-Q

grade water (Millipore Corporation) was used throughout

the work. All chemicals used otherwise were of analytical

grade.

Instrumentation: Chromatographic analysis was carried

out using an Agilent 1290 HPLC–DAD system comprising a

binary pump, an autosampler equipped with a thermostat,

a thermostated column compartment, and a diode-array

detector equipped with a 60-mm Max-Light flow cell

(Agilent Technologies). It was operated with ChemStation

Openlab CDS Software Version C.01.05 (Agilent

Technologies).

Calibration Standards: Stock solutions of 2000-ng/µL

MTX and SSZ were prepared by dissolving each compound

in 100:0.1 (v/v) methanol–ammonium hydroxide. A 200-ng/

µL plasma stock solution of a combination of MTX and SSZ

was prepared by spiking the appropriate volume of each

stock solution into plasma. Serial dilutions from this plasma

stock solution were made using blank plasma to achieve

11 concentrations of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 5.0,

10.0, 50.0, and 100 ng/µL. Next, 200-µL aliquots of each of

these plasma calibration standard solutions were extracted

using protein precipitation or SPE as described below to

prepare the calibration standards.

Quality Control Samples: Three sets of quality control

(QC) samples were prepared by spiking stock solutions of

MTX and SSZ into plasma to yield 0.8-, 8.0-, and 80-ng/

µL concentrations to be used as low, middle, and high QC

samples, respectively. QC samples were analyzed in five

replicates each.

Extraction from Human Plasma: Two sample preparation

techniques were evaluated for the extraction of each

Table 1: Accuracy values for each linearity level of MTX and SSZ.

Concentration (ng/µL)Accuracy

MTX SSZ

0.02 85 NA

0.05 95.6 NA

0.1 102 107.1

0.2 101 90

0.5 101.2 86.4

1 99.7 104.5

5 101.4 88.8

10 105.2 89.1

50 106.3 94.4

100 98.4 101.6

NA = not available

H2N NH

2N

NN

N

N

N

O

O

OH

OHO

MTX

Formula

Mol. mass

Formula

Mol. massC

20H

22N

8O

5

C18

H14

N4O

5S

454.44 g/mol 398.39 g/mol

H

H

HO

OH

O

SSZ

N

N

NN

O O

S

Resolution: 3.9 Resolution: 2.5

Resolution: 0.9 Resolution: 2.5

MTX

MTX

SSZ

SSZ

5 6 7

654 7

Time (min)

Time (min)

2

1

0

-1

2

Ab

sorb

an

ce (

mA

U)

3.5

7

7.2

67.2

87.4

27.4

2

3.6

23.6

1

Ab

sorb

an

ce (

mA

U)

3.3

2

1

0

25

Ab

sorb

an

ce (

mA

U)

3.6

1

7.2

7

20

15

10

5

0

2 4 6 8 10 12 14

Time (min)

Figure 1: Structure of methotrexate (MTX) and sulphasalazine

(SSZ).

Figure 2: HPLC chromatogram of standard plasma solution

12.5 ng/µL of MTX and SSZ using SPE (top trace) and protein

precipitation (bottom trace) at 304 nm.

Figure 3: Elution profile of standard plasma solution of 2 ng/µL

of MTX (retention time = 3.6 min) and SSZ (7.2 min) using SPE

at 304 nm overlaid with blank plasma chromatogram.


Khera et al.


calibration standard from plasma, namely, protein

precipitation and SPE. To perform protein precipitation on

a 200-µL plasma sample, double volume of 1:4 (v/v) 0.4 M

zinc sulphate–methanol solution was used. After adding

the precipitating reagent and vortexing for 45 s, the mixture

was centrifuged at 10,000 rpm for 5 min and the resulting

supernatant was used for liquid chromatography (LC)

analyses.

For SPE, 100-mg, 3-mL Bond Elut-C18 cartridges

(Agilent Technologies) were used for the extraction

of analytes from plasma. The SPE cartridges were

preconditioned with 3 × 1 mL of methanol–0.1% ammonium

hydroxide, followed by 3 mL of purified (Milli-Q, Millipore)

water. Then, 200 µL of each plasma standard solution

was loaded onto the SPE cartridge under gentle vacuum.

The cartridges were then washed three times with 1 mL

of mobile-phase A under vacuum for 5 min to near

dryness. The analytes were eluted from SPE cartridges

with 2 × 0.5 mL of methanol–0.1% ammonium hydroxide.

Eluted fractions were concentrated to dryness using a

concentrator (Eppendorf) and further reconstituted with

200 µL of 100:100:0.1 (v/v/v) methanol–water–ammonium

hydroxide.

HPLC–DAD Method: The separation was performed

using a 150 mm × 3.0 mm, 2.7-µm Poroshell Extend-C18

column (Agilent Technologies) that was maintained at 30 °C

using a column oven. Mobile-phase A was water–10 mM

ammonium acetate–0.1% formic acid and mobile-phase

B was methanol–10 mM ammonium acetate –0.1%

formic acid. The mobile phase was freshly prepared and

degassed before use. The chromatographic gradient

consisted of elution with 10–95–95–10% mobile-phase B

in mobile-phase A at 0–10–15–15.1 min at a flow rate of

0.8 mL/min. The column was equilibrated for 5 min using

the initial mobile-phase ratio before every injection. An

injection volume of 5 µL was used for all sample injections

and all injections were preceded with a 10-s needle wash

with methanol–0.1% ammonium hydroxide. The diode-array

detector monitored two UV wavelengths, 304 nm and

358 nm, that corresponded to the absorbance maxima of

MTX and SSZ, respectively.

Results and DiscussionOptimization of Plasma Extraction: The extraction

efficiency for protein precipitation versus SPE was

evaluated by comparing the area under the curve (AUC) of

the analyte peaks from the plasma calibration standards

with the AUC observed for standard solutions at a

concentration of 12.5 ng/µL. The extraction efficiencies

for MTX using protein precipitation and SPE were 17%

and 50%, respectively. The extraction efficiencies for

SSZ using protein precipitation versus SPE were 53%

and 78%, respectively. Thus, the extraction efficiency

proved to be significantly higher using SPE compared with

Table 2: QC sample results summarizing mean recoveries, accuracies, %CV of accuracies, retention time (Rt) RSD, and AUC RSD.

QCTarget

(ng/µL)

MTX (n = 5) SSZ (n = 5)

Mean

Recovery

(ng/µL)

Accuracy

(%)CV (%)

Rt RSD

(%)

AUC

RSD (%)

Mean

Recovery

(ng/µL)

Accuracy

(%)CV (%)

Rt RSD

(%)

AUC RSD

(%)

Low QC 0.8 0.70 87.7 1.02 0.03 0.26 0.82 102.2 0.86 0.09 0.34

Middle QC 8.0 8.1 101.1 0.26 0.03 0.25 7.5 94.2 0.28 0.02 0.41

High QC 80.0 75.5 94.3 0.29 0.03 0.29 78.1 97.6 0.29 0.02 0.29

4

3

2

1

0

4

3

2

1

0

2

Ab

sorb

an

ce (

mA

U)

Ab

sorb

an

ce (

mA

U)

3.6

5 M

TX

3.6

5

3 4 5

Time (min)

Rt of MTX

6 7

2 3 4 5

Time (min)

6 7

12

8

4

0

12

5 7

Rt of SSZ

9 11

5 7 9 11

Ab

sorb

an

ce (

mA

U)

SSZ

Ab

sorb

an

ce (

mA

U)

8

4

0

Time (min)

Time (min)

Figure 4: Elution profile of blank human plasma (top trace)

and plasma spiked with MTX at the LLOQ (bottom trace)

extracted with SPE.

Figure 5: Elution profile of blank human plasma (top trace)

and plasma spiked with SSZ at the LLOQ (bottom trace)

extracted with SPE.


Khera et al.


protein precipitation as expected. Additionally, a detailed

inspection of the chromatographic data over baseline

evidenced a better resolution of the analytes from matrix

interferences using SPE (Figure 2). A matrix peak (retention

time = 3.57 min) eluted just before MTX while using protein

precipitation was found to be completely eliminated with

SPE.

Therefore, SPE allowed better extraction efficiency, better

resolution, and thus more sensitive detection across a

wider calibration range. However, because of its simple and

inexpensive nature, protein precipitation may be used when

the sensitivity requirements of the bioanalytical method are

within the range observed here for protein precipitation.

Chromatographic Method Development: A C18

column was found highly retentive for the analytes, and

the best separation was achieved by using a gradient

of mobile-phase B in mobile-phase A as described

in the experimental section. The use of a low organic

mobile-phase concentration (10%) at the beginning of the

gradient resulted in good resolution of the analytes from

the initial polar matrix background. Also the late-eluted

analyte SSZ peak was well resolved from late-eluted matrix

interferences using a higher organic content (95%) at

the end of the gradient run. Within a run time of 15 min,

baseline separation of the target analytes from each other,

as well as from matrix peaks, was accomplished using

these chromatographic conditions (Figure 3).

Method Validation:

Method Selectivity: The demonstration of selectivity for

the analytes in the presence of matrix interferences

is of high interest for a bioanalytical method. In this

study, the selectivity of the method for the two analytes,

MTX and SSZ, during the chromatographic elution was

examined across the elution window at the lower limit

of quantification (LLOQ). The LLOQ was established as

the lowest concentration on the calibration curve. At the

LLOQ, the analyte response was at least five times higher

compared to the blank response. Figures 4 and 5 provide

chromatograms of MTX and SSZ calibration standards at

the LLOQ, respectively. Minor interferences were observed;

however, these did not impact peak integration and

quantitation of analytes at LLOQ.

Method Linearity: A study of method linearity was

performed by constructing calibration curves consistent

with the Food and Drug Administration’s (FDA) draft

guidance for bioanalytical method validation (11) across

several concentration levels including the LLOQ and

upper limit of quantification (ULOQ) in five replicates.

Calibration curves were constructed for peak area

versus concentration and it was observed that the area

response was linearly and correctly regressed over a wide

concentration range. The linear dynamic range for the

current bioanalytical method is 0.02–100 ng/µL for MTX

and 0.1–100 ng/µL for SSZ. The coefficient of correlation

(R2) was greater than 0.998 in each case. The calibration

curves for both analytes are shown in Figure 6. Observed

accuracy values for each linearity level for MTX and SSZ

are summarized in Table 1. The accuracy of the method at

the LLOQ was 85% for MTX and 107.1% for SSZ. Consistent

with the FDA guidelines, the observed precision standard

deviation at LLOQ was below 20% and for all other

concentration levels including the ULOQ, the precision

standard deviation observed was below 15%.

Limits of Detection and LLOQ: The limits of detection

(LOD) and LLOQ were determined at signal-to-noise ratios

(S/N) at or greater than 3 and 10 for both MTX and SSZ,

respectively. Thus, the LOD and LLOQ for MTX were 0.01

and 0.02 ng/µL with S/N values of 6 and 10, respectively.

For SSZ the LOD and LLOQ were 0.05 and 0.1 ng/µL

with S/N values of 8 and 18, respectively. Figure 7 shows

a typical chromatogram for MTX at the LLOQ overlaid

with blank traces in replicates. The chromatographic

reproducibility at the LLOQ was verified by replicate

injections. The percent of the coefficient of variation (%CV)

of AUC and retention time at the LLOQ were 0.02% and

1.46%, respectively, for MTX, and 0.01% and 0.79%,

respectively, for SSZ.

Analyte Recovery: Analyte recovery was measured

consistent with the FDA guidance for bioanalytical method

validation (12). Thus, three sets of QC samples for each

analyte were analyzed in five replicates. The three sets

of QC samples were selected in such a way that the

concentration range covers the lower, mid, and high region

of the calibration curve. The analyte recoveries from QC

samples were calculated using linearity equations of each

of the analytes. These results were then compared with

theoretical concentrations. The resultant mean recoveries

and %CV are given in Table 2.

Method Reproducibility: The reproducibility of the method

MTX

y = 107.33x +41.177R2 = 0.9986

y = 100.35x -119.77R2 = 0.9987

MTX, Range: 0.02–100 ng/µL

0.02-1ng/µL

0.1-1ng/µL

150

100

1200

1000

8000

60004000

20000

50

Ab

sorb

an

ce (

mA

U)

Ab

sorb

an

ce (

mA

U)

120010008000600040002000

-20000

Ab

sorb

an

ce (

mA

U)

Ab

sorb

an

ce (

mA

U)

0.5

SSZ

0

100

50

0

0

0 0.2 0.4 0.6 0.8 1

0 20 40 60 80 100

1Concentration (ng/µL)

Concentration (ng/µL)

Concentration (ng/µL)

0 20 40 60 80 100Concentration (ng/µL)

SSZ, Range: 0.1–100 ng/µL

1.5

0.5

Ab

sorb

an

ce (

mA

U)

0

3 3.2 3.4 3.6 3.8 4 4.2

Time (min)

3.6

5

1

Figure 6: Linearity curves for MTX (top) and SSZ (bottom). A

zoomed-in view for lower linearity levels is also shown.

Figure 7: Chromatogram for MTX at the LLOQ overlaid with

blank traces in duplicates.


Khera et al.


was determined in accordance with the FDA draft guidance

for bioanalytical method validation (11) by measuring the

accuracy and precision of the method across three QC

samples in the calibration range and using five replicates

at each concentration. The results for intraday precision

and accuracy in plasma quality control samples for MTX

and SSZ are summarized in Table 2. The calculated relative

standard deviation for retention time and area was found

to be within 0.1% and 0.4%, respectively. These results

promise high reproducibility of the method.

ConclusionsA combination regimen of low-dose MTX and high-dose

SSZ is frequently used to combat rheumatoid arthritis.

Therefore, the simultaneous monitoring of plasma levels

of MTX and SSZ has clinical relevance, and also may

aid further pharmacokinetic research into any synergistic

effects. Here, we described a simple, sensitive, and robust

HPLC–DAD method for the simultaneous determination

of MTX and SSZ in plasma. The method is linear within

biologically relevant concentration ranges of 0.02–100 ng/µL

(0.04–220 µM) for MTX and 0.1–100 ng/µL (0.25–251 µM)

for SSZ with a R2 coefficient greater than 0.998 for each.

The method has an LLOQ of 0.02 ng/µL for MTX and an

LLOQ of 0.1 ng/µL for SSZ. In addition, this method to our

knowledge is more sensitive, allows a wider calibration

range, and can be performed using a much smaller

(200 µL) volume of plasma sample than previously reported

UV methods for the bioanalysis of MTX. The method was

validated consistently with FDA guidance, using quality

control samples for critical analytical performance criteria

of recovery, reproducibility, selectivity, accuracy, and

precision.

References(1) J.R. O’Dell, R. Leff, G. Paulsen, C. Haire, J. Mallek, P.J. Eckhoff,

A. Fernandez, K. Blakely, S. Wees, J. Stoner, S. Hadley, J. Felt, W.

Palmer, P. Waytz, M. Churchill, L. Klassen, and G. Moore, Arthritis

Rheum. 46, 1164–1170 (2002).

(2) W. Katchamart, J. Trudeau, and V. Phumethum, Ann. Rheum. Dis.

68, 1105–1112 (2009).

(3) F.M.P. Meier, M. Frerix, W. Hermann, and U. Müller-Ladner,

Immunotherapy 5, 955–974 (2013).

(4) S. Sadray, S. Rezaee, and S. Rezakhah, J. Chromatogr. B 787,

293–302 (2003).

(5) H.A. Capell, R. Madhok, D.R. Porter, R.A. L. Munro, I.B. McInnes,

J.A. Hunter, M. Steven, A. Zoma, E. Morrison, M. Sambrook, F.W.

Poon, R. Hampson, F. McDonald, A. Tierney, N. Henderson, and I.

Ford, Ann. Rheum. Dis. 66, 235–241 (2007).

(6) H.M. James, D. Gillis , P. Hissaria, S. Lester, A.A. Somogyi, L.G.

Cleland, and S.M. Proudman, J. Rheumatol. 35, 562–571 (2008).

(7) F.M. Rubino, J. Chromatogr. B 764, 217–254 (2001).

(8) N. Pastor-Navarro, E. Gallego-Iglesias, A. Maquieira, and R.

Puchades, Anal. Chim. Acta 583, 377–383 (2007).

(9) M.S. Elmasry, I.S. Blagbrough, M.G. Rowan, H.M. Saleh, A.A. Kheir,

and P.J. Rogers, J. Pharmaceut. Biomed. Anal. 54, 646–652 (2011).

(10) U. Klotz, Clin. Pharmacokinet. 10, 285–302 (1985).

(11) US Food and Drug Administration, Guidance for Industry:

Bioanalytical Method Validation, Bio Pharmaceutics Revision 1,

(U.S. Department of Health and Human Services, Food and Drug

Administration; Center for Drug Evaluation and Research [CDER],

Center for Veterinary Medicine [CVM], Rockville, Maryland, USA,

2013).

Siji Joseph and Sreelakshmy Menon are with the Life Science

Center at Agilent Technologies India Pvt. Ltd., in Bangalore, India.

Smriti Khera is with the Life Science and Diagnostics Group at

Agilent Technologies, Inc., in Santa Clara, California, USA. Direct

correspondence to: [email protected]


Khera et al.



LC TROUBLESHOOTING

Recently, I received an inquiry from

a reader regarding a problem he

encountered with a routine liquid

chromatography (LC) method in his

clinical laboratory. He had prepared

a fresh calibration standard (check

sample) for the analyte of interest

(I’ll call it “X” to keep the reader’s

laboratory anonymous), yet when he

assayed a blank sample spiked with

160 ppm of X, he found an indicated

400 ppm. This was puzzling and not

a problem normally encountered,

so he sent the sample to another

laboratory that was analyzing the same

compound by gas chromatography

(GC), and their results showed that

the spiked sample indeed contained

160 ppm of X. At this point he

contacted me to help figure out what

was happening. As we look at possible

causes for and solutions to this

problem, we can use this as a specific

example to which we can apply

general troubleshooting principles.

BackgroundBefore we get further, let’s take a look

at the method, which is designed for

the analysis of X in serum. Samples

are prepared by taking an aliquot of

serum, adding an aliquot of internal

standard (IS), and a small amount

of hydrochloric acid to acidify it. The

solution is vortexed to mix, then an

aliquot of dichloromethane is added,

the solution is vortexed again, and

then centrifuged to separate the two

phases. The dichloromethane phase

is removed, evaporated to dryness,

and reconstituted in the injection

solvent. The separation conditions

comprise a reversed-phase column

(size, stationary phase, and flow

rate were not mentioned) with an

isocratic mobile phase of acetonitrile,

water, and trifluoroacetic acid.

Ultraviolet (UV) detection is used.

The chromatographic conditions give

typical retention times of 9 min (IS)

and 12 min (X), and the chromatogram

is normally free of any other peaks.

Calibration standards are prepared

by spiking a stock solution of X into

serum at 40, 120, and 160 ppm; these

spiked calibrators are then extracted

in the same manner as samples. A

three-point calibration curve is run

and if the regression is acceptable,

this calibration curve is used for three

months. With each batch of samples, a

single injection of blank serum spiked

to 160 ppm is made as a system

suitability test; if this check sample

assays at 160 ppm, the system is

deemed stable and samples are run.

The method had been running

acceptably until he ran out of the

1000 ppm stock of X used for spiking

the check sample. When the new

stock was prepared, the problem of

a 400 ppm assay for the 160 ppm

sample appeared.

Consider the PossibilitiesIn a case like this, I like to divide the

issue up into several possible problem

areas, then see how many of these

possibilities I can eliminate with the

data at hand. This helps to focus my

attention on the source of the problem

so that it can be investigated further,

if necessary, and corrected. We can

broadly, and somewhat arbitrarily,

divide the possible problem areas into

chemistry, hardware, sample-related,

and calibration. Let’s look at each of

these in more detail.

Chemistry: By chemistry, here I

mean the chromatographically related

chemical influences. These are the

nature of the sample, the column,

the mobile phase, and the column

temperature. We can quickly eliminate

these as the likely sources of the

problem. If the column chemistry,

mobile-phase chemistry, or column

temperature had changed, we would

expect a shift in retention for X and

the IS, but this was not observed. The

sample chemistry, or identity, is unlikely

to have changed, because the check

sample had no apparent retention

problems in either the LC or GC assay.

Hardware: LC system hardware

could malfunction in terms of flow

rate, injection problems, or detection.

The flow rate must be correct or the

retention times would shift for both

X and the IS. It is possible that the

autosampler is not working properly,

but this is unlikely to cause the

noted problem, because any volume

error in the autosampler would be

compensated by the use of the IS. The

purpose of the IS is to add it early in

the sample preparation process so that

any loss of sample volume or injection

error would not matter, because it

is the ratio of X/IS that is used in the

calibration process, not the absolute

response of either compound.

Problems related to the detector are

a possible source of error, and should

be checked. Two obvious possibilities

are that the wrong wavelength was

selected or that there is something

wrong with the detector lamp. The

response of X and the IS would be

Calibration Problems — A Case StudyJohn W. Dolan, LC Troubleshooting Editor.

Unexpected results from calibration standards create confusion in a clinical liquid chromatography (LC) method.


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LC TROUBLESHOOTING

expected to change if the detection

wavelength was changed, and a

change in the relative response of

X and the IS would be likely. This

would generate a different X/IS ratio

for a given concentration, which in

turn would change the assay value

for X in the check sample. A change

in lamp energy as the detector lamp

aged could also cause a change in

response, and although I would expect

that such intensity would affect X and

the IS similarly, that is not a certainty.

The proper wavelength should be

verified and the lamp energy should

be compared to normal values to

determine if either of these items could

be the problem source.

Sample-Related Problems: We know

that the identity of the sample is correct

and that the standard was made at

the proper concentration because the

sample assayed at 160 ppm by GC.

The reader did not state if a new batch

of IS stock was made at the same time,

but if we consider the method, either

the new batch of IS was made correctly

or the old one was still good and was

used. The check sample is made by

spiking serum, and serum would never

be injected directly, so it follows that

the check sample was spiked with IS

and extracted in the normal manner.

One of the reasons for adding IS is to

account for the inevitable changes in

sample volume that take place during

sample preparation.

Let’s review the sample cleanup

procedure: 300 µL of serum is

combined with 50 µL of IS and 200 µL

of dilute hydrochloric acid (550 µL

total), centrifuged, and extracted

with 600 µL of dichloromethane.

All of X and the IS should transfer

into the dichloromethane, so the

concentration of X and IS is 550/600

of its concentration in the original

diluted serum. Next, 400 µL of

the dichloromethane is removed,

evaporated to dryness, and

reconstituted in 50 µL of methanol.

This concentrates the dichloromethane

extract by 400/50 or eightfold. With

the extraction, evaporation, and

reconstitution steps, there will be

inevitable volumetric errors introduced,

which is why the IS is added — the

same losses of X and IS should occur,

so the X/IS ratio should stay constant.

All this leads me to conclude that the

GC method would be very unlikely to

give an assay value of 160 ppm of X

by an external standard method, even

if the results were adjusted for the

theoretical changes in concentration.

Instead, I conclude that the IS method

was used for GC, as well, and because

the assay was as expected, it tells

me that the check sample was made

correctly, even though it doesn’t assay

properly by the LC method. The bottom

line here is that it is unlikely that the

current problem lies with the sample or

sample preparation.

Calibration: At this point we’ve

eliminated chemistry problems,

hardware problems (assuming the

detector wavelength is set correctly

and the detector lamp is in acceptable

condition), and sample-related

problems. This leaves calibration

problems as the most likely problem

source (assuming that we haven’t

overlooked something else obvious,

which is always a possibility).

My initial interaction with the reader

simply indicated that the check sample

did not assay correctly by LC, but gave

the expected answer by GC. When

I requested more information about

the method, I learned of the practice

of calibrating every three months and

using the system suitability check

sample to verify that the method

was working properly. Although the

rules are a bit different in the clinical

laboratory industry, this goes strongly

against the analysis of the same drugs

in serum or plasma to support drug

development in the pharmaceutical

industry. The latter techniques fall

under guidelines from the United

States Food and Drug Administration

(FDA). The FDA’s “Guidance for

Industry: Bioanalytical Method

Validation” (1) discusses validation

of methods for the analysis of small

molecular weight drugs in plasma

and other tissues (generally called

“bioanalytical” methods, as opposed to

methods for the analysis of biological

compounds). In this document in the

section titled “Application Of Validated

Method To Routine Drug Analysis”

(pp. 13–14), it is stated:

A calibration curve should be

generated for each analyte to

assay samples in each analytical

run and should be used to

calculate the concentration of the

analyte in the unknown samples

in the run . . . . The calibration

(standard) curve should cover

the expected unknown sample

concentration range in addition

to a calibrator sample at LLOQ

[lower limit of quantification].

It goes on to say:

Once the analytical method has

been validated for routine use, its

accuracy and precision should

be monitored regularly to ensure

that the method continues to

perform satisfactorily. To achieve

this objective, a number of

QC [quality control] samples

prepared separately should be

analyzed with processed test

samples at intervals based on

the total number of samples. . . .

The QC samples in duplicate at

three concentrations . . .

Additionally, it is noted:

A matrix-based standard curve

should consist of a minimum of

six standard points, excluding

blanks (either single or replicate),

covering the entire range.

This says that the calibration curve

should be run with each batch of

samples, not once every three months.

The calibration curve should cover the

expected sample concentration range,

and include the LLOQ. Furthermore,

QC samples should be run at three

concentrations that fall within the

range of sample concentrations. These

guidelines also make good sense from

an analytical chemistry standpoint.

There are just too many potential

problems that can occur which might

cause the calibration curve to be

different on different days. I have

been involved with research and

development (R&D) studies where

the reference standards were so rare

and valuable that it was not possible

to run them every day, but a surrogate

standard was found to verify that the

original calibration was still adequate.

That may seem to align with the current

problem, but in fact the drug X and

its IS are very common compounds

that can be purchased in reference

standard grade for reasonable prices,

so it is hard to justify trimonthly

calibration on economic grounds.

The fact that the check sample was

formulated at 160 ppm and verified

by GC underlines the probability that

ES610495_LCE0515_280.pgs 04.30.2015 13:20 ADV blackmagentacyan


batch by about 2.5 h. It may be very

easy to compensate for this increase

in run time by increasing the flow rate;

with an isocratic run, the separation

should not be affected by the flow rate.

The pressure would rise in proportion

to the increase in flow rate, but it is

fairly rare with conventional LC runs

that pressure is a limiting condition, so

the added pressure is unlikely to be an

issue.

ConclusionsWe have used a specific example of

a method problem to illustrate how to

break down the problem into several

potential problem sources. Most of

these sources could be eliminated by

careful consideration of the method

and how the results deviated from

the expected ones. This left us with

two likely problem sources. First, a

problem with the detector wavelength

setting or detector lamp energy.

These could be quickly checked by

examining the instrument. The second

potential problem source was that the

instrument response to X or the IS had

drifted between the time the original

calibration curve was run and the

problem was noted.

The recommended solution was

to first check for detector problems,

and second rerun the calibration

curve. A more permanent fix to the

problem would be to change the

method to comply better with current

FDA guidelines and general analytical

chemistry practices of running

calibrators contemporaneously with

samples.

References(1) United Stated Food and Drug

Administration, Guidance for Industry:

Bioanalytical Method Validation (FDA,

Rockville, Maryland, USA, 2001).

“LC Troubleshooting” Editor John

Dolan has been writing “LC

Troubleshooting” for LCGC for more

than 30 years. One of the industry’s

most respected professionals, John is

currently the Vice President of and a

principal instructor for LC Resources

in Lafayette, California, USA. He is

also a member of LCGC Europe’s

editorial advisory board. Direct

correspondence about this column via

e-mail to John.Dolan@LCResources.

com. To contact the editor-in-chief,

Alasdair Matheson, please e-mail:

[email protected]

the source of the problem lies with the

calibration curve. My best guess is that

something in the LC system has drifted

over time, most likely the detector

response (or an improper wavelength

setting), and has caused the current

response to the X/IS ratio to be much

larger than it was when the calibration

curve was run originally.

What Now?I recommend that the proper

wavelength setting and detector

lamp performance be verified before

proceeding. After these are found to

be satisfactory, I would generate a

new calibration curve using freshly

prepared standards of X spiked into

blank serum and extracted normally. I

believe that the check sample will now

assay correctly, closing the loop on

identifying the problem source.

Technically, the check sample has

done exactly what it was intended

for — it has alerted the operator to a

problem with the assay before valuable

patient samples were run. However, I

would modify the method to comply

more with the industry standard of

the FDA guidelines (1). This would

require running a calibration curve,

containing samples with at least six

concentrations, each day with each

batch of samples run. In addition, a

set of check samples, or QCs, should

be prepared and included in each

sample batch to show that during

the analysis, the method gives the

expected results for samples of known

concentration. There will be some

documentation required to make these

changes, but the method reliability

will be much improved and should

justify this extra work. The quality of the

results produced should improve as

well. Finally, should the laboratory be

audited by a regulatory agency, there

will be much less likelihood of negative

findings by the auditors.

In terms of day-to-day added work,

there should be only a small impact on

the total batch run time for a potentially

large improvement in data quality. The

calibration and check samples can be

quickly spiked with known amounts

of X and extracted with QC samples

and samples to be analyzed. A total

of six calibrators and six QC samples

(duplicates at three concentrations)

would add 12 samples to the day’s

run. At a 12-min retention time for X,

this would increase the run time for the



GC CONNECTIONS

From 8–12 March 2015 the Pittsburgh

Conference on Analytical Chemistry

and Applied Spectroscopy (Pittcon)

returned to the Morial Convention

Center in New Orleans, Louisiana,

USA, for its 66th annual meeting.

This was the first time since 2008

that the conference had taken

place in New Orleans, and the city

welcomed the 14,272 registered

attendees with open arms, brass

bands, and beignets. There were 919

exhibitors in 1690 booths, and this

year 90 countries were represented.

Although I did not attend many of

the technical sessions, they were

of as high a quality and as well

attended as in previous years. Of

particular interest to the readers

of LCGC was the half-day session

devoted to the presentation of the

2015 LCGC Lifetime Achievement

in Chromatography Award to Jack

Kirkland (Advanced Materials

Technology) and the LCGC Emerging

Leader Award in Chromatography to

Caroline West (Université d’Orléans,

France). Please see the February

2015 issue of LCGC North America

(1) for more information about this

year’s awards.

Pittcon will head to Atlanta in

2016, where conferees will enjoy a

second consecutive year of Southern

hospitality. In 2017, the conference

returns to Chicago.

This annual instalment reviews

gas chromatography (GC)

instrumentation, columns, and

accessories shown at this year’s

Pittcon or introduced during the

previous year. For a review of

new products in other areas of

chromatography, columns, and

related accessories, please see the

additional coverage in the April issue

as well as this issue of LCGC Europe

(2–4), which are also available

on-line at LCGC ’s website.

The information presented here is

based on manufacturers’ replies to

questionnaires, as well as additional

information from manufacturers’

press releases, websites, and

product literature about the past

year’s products, and not on actual

use or experience of the author.

During Pittcon, I took time to stroll

around the convention aisles and see

some of the new products firsthand

as well as discover a number of

items that weren’t covered by the

questionnaires. Every effort has

been made to collect accurate

information, but because of the

preliminary nature of some of the

material, LCGC Europe cannot be

responsible for errors or omissions.

This column instalment cannot be

considered to be a complete record

of all new GC products introduced

this year at Pittcon or elsewhere

because not all manufacturers chose

to respond to the questionnaire or

attend the conference, nor is all of

the submitted information necessarily

included here because of the limited

available space and the editors’

judgment as to its suitability.

Gas Chromatography in 2014–2015

Gas chromatography again

displayed renewed vigour in the past

year, which certainly was evident

at the 2015 Pittcon conference.

Comprehensive GC×GC continues

to yield significant advances, in

particular when combined with mass

spectrometry (MS) detection. MS

detectors for GC alone experienced

no fewer than six new or enhanced

New Gas Chromatography Products, 2014–2015 John V. Hinshaw, GC Connections Editor.

In this instalment, John Hinshaw reviews gas chromatography (GC) instruments, columns, and

accessories that were newly on display at the Pittsburgh Conference in New Orleans, Louisiana, USA,

during March 2015, or were introduced to the marketplace in the preceding year.

Table 1: Companies introducing new

GC products.

Company Name

Agilent Technologies

AFP

Baseline Mocon

DANI

Defiant Technologies

Gow-Mac

Ionicon

JEOL

LECO

Phemonenex

Qmicro

Restek

SGE

Shimadzu

Thermo Scientific

VICI

New Orleans welcomed the 14,272 registered attendees with open arms, brass bands, and beignets. There were 919 exhibitors in 1690 booths, and this year 90 countries were represented.


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GC CONNECTIONS

Table 2: New GC instruments.

Product Company Description

7010 Triple-Quad

GC–MS system

with improved EI

source

Agilent Agilent’s 7010 triple-quadrupole GC–MS system features a redesigned high-efficiency electron

ionization (EI) source that enables attogram detection limits. The system’s new EI source creates

more than 20 times as many ions as the current generation of EI sources, according to Agilent,

to deliver an instrument detection limit (IDL) of 0.5 fg OFN. The new EI source technology is also

available to current and future 7000 C owners in the form of an upgrade package. Specifications:

mode of operation: EI standard, CI optional; ion source temperature: 150–350 °C; dual filaments

for EI; electron energy: 10–300 eV; mass filters: proprietary monolithic hyperbolic gold-coated

quadrupole; mass axis stability: less than ± 0.10 u over 24 h (10–40 °C); quadrupole temperature:

106–200 °C; mass range: m/z 10–1050; resolution: selectable, 0.4–4.0 Da, custom tune; scan rate:

up to 6250 u/s; detector: triple-Axis HED-EM with extended-life EM and dynamically ramped-iris;

MRM speed: 800 transitions/s; minimum MRM dwell: 0.5 ms; collision cell: linear hexapole; collision

cell gas: nitrogen with helium quench gas; collision energy: selectable up to 60 eV.

7200B Q-TOF

GC–MS system

Agilent The Agilent 7200B Series Q-TOF GC–MS system with Agilent MassHunter software replaces the

company’s 7200A offering and provides an improved mass accuracy specification of less than

3 ppm over an extended mass range, with acquisition rates up to 50 Hz. The system must be

combined with the high performance Agilent 7890B gas chromatograph. 7200B specifications:

EI (high sensitivity extraction source), PCI, and NCI ionization mode as standard; ion source

temperatures: 106–350 °C; electron energy: 10–200 eV; removable ion source without breaking

vacuum through an isolation valve; dual filaments for EI source, single filament for CI source; quad

isolation mass range (m/z) 20–1050; resolution (full width at half height) settable from 0.4 to 4.0 Da;

dynamic range (electronic) greater than 105; quadrupole mass axis stability less than ±0.10 Da

over 24 h (10−40 °C); quadrupole temperature: 100–200 °C; collision cell: linear hexapole, nitrogen

collision cell gas; collision energy: selectable up to 60 eV; ion extraction and mirror: two-stage

second-order corrected; TOF flight pathlength: 2 m; microchannel plate/scintillator/PMT detector;

TOF mass range (m/z): 25−1700, extended 15−3000; TOF detector sampling rate ADC: 32 Gbits/s;

autotune or manual tuning; spectra acquisition rate: 1−50 spectra/s; EI instrument detection limit of

240 fg or less of OFN.

AccuTOF-GCx

time-of-flight

GC–MS system

JEOL The fourth-generation AccuTOF-GCx features high sensitivity (S/N > 300 at OFN 1 pg/µL) and

offers improved resolution, accuracy, and sensitivity, while retaining the power and flexibility of

the company’s previous models. The GCx offers both powerful chromatographic separation and

high-resolution mass spectra in combination with comprehensive 2D GC (GC×GC) using the Zoex

thermal modulator. Specifications: mass resolution: 10,000 (FWHM); mass range: 4–6000 (m/z);

data acquisition speed: up to 4 GS/s; spectrum acquisition speed: up to 16,000 spectra/s; spectrum

recording speed: up to 50 spectra/s; sensitivity: 1 pg octafluoronaphthalene (OFN) S/N ≥300.

AQMAlert Ozone

Precursor system

Baseline

Mocon

The company’s AQMAlert multiple-GC field system combines two Series 9100 GC systems with a

Series 9300 Preconcentrator into a photochemical assessment monitoring station (PAMS). The first

GC system uses flame ionization detection (FID) for light hydrocarbon detection; the second GC

system incorporates photoionization detection (PID) for the remaining components. The optional

preconcentrator is a dual-tube desorption system that allows lower detection limits.

Gas

chromatography

cartridge

QMicro The new Qmicro gas chromatography cartridge is based on an innovative micro gas analysis

platform with integrated injector and thermal conductivity detection systems plus columns,

backflush to detector, and temperature programming, all packaged in a small palm-size

oven. Backflush enables protection of sensitive columns by minimizing exposure to harmful

gas components — such as carbon dioxide and water on a molecular sieve 5A column —

thus increasing lifetime. Backflush to detector functionality allows quantification of the total

backflushed sample peak. This enables fast analysis of total C6+ or C9+ content of a natural

gas. The cartridge is based on silicon chips made by MEMS microtechnology and micro

assembly technologies, for virtual zero dead volumes and microscopic small flow channels.

The cartridge is intended for OEM partners and system integrators to integrate micro GC

technology for fast, small, and reliable analyses into instruments and systems.

GCMS-TQ8040 Shimadzu Shimadzu’s GCMS-TQ8040 triple-quadrupole GC–MS system includes the following features:

Smart MRM (multiple reaction monitoring), which can combine over 400 compounds into a single

MRM method without losses in sensitivity or selectivity; MRM analysis at up to 800 transitions/s;

high-speed scanning control at 20,000 u/s; an MRM optimization tool that automatically determines

optimum transitions and collision energies for all compounds in a single sequence; an off-axis

design that eliminates neutral noise; UFsweeper technology that accelerates ions out of the

collision cell to eliminate crosstalk; an automatic adjustment of retention times (AART) function

that updates retention times in both the acquisition and data processing methods after column

maintenance, without changing chromatographic conditions or requiring multiple injections of

standards. The system also has Shimadzu’s Smart Database Series software to create MRM and

scan-MRM methods automatically, and a scan-MRM mode that simultaneously acquires accurate

library-searchable mass spectra and low-level MRM quantitation in a single analysis.

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GC CONNECTIONS

(EI) source. Both of these offerings

work with the company’s gas

chromatographs. Also in the GC–MS

area, the AccuTOF-GCx TOF GC–

MS system from JEOL works with

comprehensive two dimensional (2D)

GC to make a powerful GC×GC–MS

analyzer. LECO displayed its newest

GC×GC–MS system, the Pegasus

GC-HRT 4D. Also coming in with a

new product in this area, Shimadzu

introduced the GCMS-TQ8040

product introductions. Advances

in fast mini- and micro-sized GC

systems were evident, too, as well as

a nice assortment of valves, fittings,

syringes, gas accessories, and

columns.

In the instrument system area,

Agilent Technologies introduced

the 7200B quadrupole time-of-flight

(QTOF) GC–MS system plus the

7010 Triple-Quad GC–MS system

with an improved electron ionization

triple-quadrupole GC–MS system.

Finally, Thermo Scientific had

their TSQ Duo triple-quadrupole

GC–MS–MS system on-hand.

Please see Table 2 for the details

and specifications supplied by the

manufacturers for these high-end

hyphenated GC instruments.

The AQMAlert Ozone Precursor

system from Baseline Mocon

combines two of the company’s

model 9100 GC systems and

Table 2: New GC instruments (continued).


Pegasus

GC-HRT 4D

LECO LECO’s newest GC×GC–MS system combines the company’s Pegasus GC×GC system

with high resolution TOF MS and ChromaTOF-HRT software, which uses high-resolution

deconvolution (HRD) for component detection, National Institute of Standards and

Technology (NIST), and Accurate Mass Library searches, pseudomolecular ions (via

chemical ionization), retention time matching, isotope patterns, and mass accuracy of

deconvoluted fragments within a complete package for data acquisition, processing, and

reporting. According to LECO, the system can produce resolution up to 50,000 FWHM,

mass accuracies less than 1 ppm, and acquisition rates up to 200 spectra/s.

Series 8100

Programmable

GC system

Gow-Mac Gow-Mac’s new Series 8100 GC system is a custom, application-specific system

configurable for research, industrial, laboratory, academic, and quality assurance

(QA) and quality control (QC) environments. The system accommodates up to two

independently controlled detectors that can be operated either individually, in series, or

in parallel depending on the ordered configuration. Detection currently available includes

TCD and FID. Features of the instrument include an ambient plus 5 °C to 450 °C operating

temperature; independently programmed and controlled temperatures at injection

ports, detectors, and column oven; the column oven accommodates up to five packed,

wide-bore capillary, or capillary columns; a column oven temperature programming rate

of 0.1 °C to 40 °C /min in 1 °C increments; an oven cooling rate of 350 °C to 75 °C in

5 min; method storage of three internal methods and an infinite number external methods;

manual differential flow controllers or pressure regulators (detector dependent); and

direct on-column (direct packed–capillary or split–splitless) or gas sample valve injection

methods; and a full array of optional output capabilities that include analog outputs of

0–1 V, 0–1 mV, and 0–10 V VDC, or digital outputs to RS-232, USB, and ethernet utilizing

MODBUS and PROFINET (read only) communication protocols.

TOCAM Defiant

Technologies

The TOCAM miniature GC-based toxic organic chemical monitor for airborne volatile

organic compounds (VOC) includes a microconcentrator, micro-GC column, and two

miniature photoionization detectors for rapid screening as well as detailed analysis of

trapped and desorbed compounds of interest. The first detector responds directly to

desorbed compounds. High levels can trigger a detailed GC analysis onto the second

detector. The portable or mountable instrument features a 10.6-eV detector lamp, a 2.5-m

or 4.6-m GC column, and operates from a 9–2 VDC AC wall adapter.

TSQ Duo

triple-quadrupole

GC–MS–MS

system

Thermo

Scientific

The Thermo Scientific TSQ Duo triple-quadrupole GC–MS–MS bridges from

single-quadrupole full scan and SIM methods to the high selectivity and sensitivity of

SRM (selected ion monitoring) methods. The system operates in both single and triple

quadrupole modes with automatic selected reaction monitoring (AutoSRM) software for

method development and optimization. A selected ion monitoring bridge method migration

tool easily and accurately migrates existing methods to either SIM or SRM methods.

The system uses Thermo Scientific Dionex Chromeleon chromatography data system

software. Other system specifications include a mass analyzer with quadrupole scanning

up to 20,000 u/s; heated, off-axis ion guide for noise reduction and solid, homogeneous,

noncoated, maintenance-free quadrupole rods; automatic tuning down to 0.4 u; selectable

SRM resolution settings in method at autotune value, 0.7, 1.5, and 2.5 u; Thermo Scientific

DynaMax XR detection system, with off-axis 10 kV dynode, discrete dynode electron

multiplier and electrometer, linear range of greater than 107 (0–68 μA); a collision energy

range of 0–60 eV; a mass range of 1.2–1100 u; and scanning capabilities of up to

20,000 u/s with the ability to acquire more than 97 scans/s in FS when scanning over a

range of 125 µ; 1.0 ms minimum SRM dwell times, and up to 300 SRM transitions/s.



a preconcentrator into a photochemical assessment

monitoring station. From Gow-Mac, the Series 8100

programmable GC system represents a new customizable

application-specific laboratory system with multiple inlets,

detectors, and other options. In miniature and micro GC

systems, Defiant Technologies showed the TOCAM toxic

organic chemical monitor, based on a microconcentrator

and micro-GC column for rapid screening and more

detailed analyses. A new entry to micro-GC, QMicro brought

examples of its GC cartridge with integrated sampling valve,

detector, and backflushing options, to be made available to

original equipment manufacturers (OEMs).

Table 3 lists new GC accessories such as autosamplers,

detectors, and more. From DANI, the Peakblade 77

GC×GC modulator is a liquid-nitrogen-free device with

rapid and programmable thermal modulation. Two fast GC

accessories were shown at Pittcon: the fast GC conversion

kit from VICI that integrates the company’s resistively heated

columns and controller with Agilent GC systems, and a fast

GC add-on for Ionicon’s PTR-TOF gas analyzer. Analytical

Flow Products (AFP) introduced a modular multipurpose

valve oven, a miniature version of the company’s multiport

valve, and a new design for zero-dead-volume fittings.

Shimadzu introduced two accessory products: the

AOC-6000 autosampler, which automates calibration

sample preparation, and the ECD-2010 Exceed

electron-capture detector. SGE introduced a new version of

its Diamond headspace syringe line. The EZGC Software

Suite Online from Restek has been expanded with some

additional calculation and translation capabilities.

Only two companies submitted information about new

GC columns, as shown in Table 4. Restek has two new

columns, the Rt-Silica BOND porous-layer open-tubular

(PLOT) column for permanent gas separations and the

Rxi-1301Sil MS column, which targets solvent analyses

with MS detection. Phenomenex introduced a two-column

set that consists of the company’s ZB-CLPesticides-1 and

ZB-CLPesticides-2 columns and is targeted for multiple

polychlorinated biphenyl (PCB) US Environmental Protection

Agency (EPA) methods that use electron-capture detection.

Acknowledgements I would like to thank the manufacturers and distributors that

kindly furnished the requested information, which allowed

a timely report on new product introductions over the past

year. For those manufacturers who did not receive a “New

Products” questionnaire this year and would like to receive

one and be considered for early inclusion into the 2015–2016

new GC and related product introductions review, please

send the name of the primary company contact, the mailing

address, fax number, and e-mail address to Laura Bush,

Editorial Director, LCGC Europe, [email protected], with

the subject line “2016 New GC Products”. The questionnaire

will be sent out in December 2015.

Pittcon will head to Atlanta in 2016, where conferees will enjoy a second consecutive year of Southern hospitality. In 2017, the conference returns to Chicago.

ENGINEERING YOUR SUCCESS.

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Together, we can

convert your GC from helium to hydrogen carrier gas.

Parker hydrogen generators offer the most cost effective and safest solution for supplying carrier gas to GC and GC/MS. In addition, hydrogen carrier gas offers faster analysis, increased resolution and longer column life.

Parker is the leading provider of Analytical Gas Systems for the GC instrument market. Generators are specifically designed to meet the stringent gas requirements for all the leading GC manufactures including Agilent, Perkin Elmer, Bruker, Shimadzu and Thermo Scientific.

Utilising Parker’s range of patented proprietary technologies guarantees ultra high purity gas, silent operation and minimal operator attention.

He



GC CONNECTIONS

Table 3: New GC accessories.


5-mL Diamond

headspace

syringe

SGE SGE enhanced its line of Diamond headspace syringes with a 5-mL model. These syringes

incorporate features for headspace sampling such as a unique “energized” plunger tip

that eliminates dead volume at the end of the plunger stroke and is compatible with a wide

range of solvents and a gas-tight seal that is not affected by temperature cycling up to at

least 150 °C.

AOC-6000 Shimadzu The Shimadzu AOC-6000 system is capable of exchanging syringe modules automatically

between liquid injection, headspace injection, or solid-phase microextraction (SPME)

injection. The autosampler features automated pretreatment with automatic syringe

exchange (10 μL to 1000 μL) and mixing functions that automate the preparation of

calibration curve samples, addition of internal standard substances, and sample dilution.

The device performs sample pretreatment and analysis in parallel so that no time is lost in

the continuous analysis of samples requiring headspace sampling or other time-consuming

pretreatments.

ECD-2010

Exceed

Shimadzu The Shimadzu ECD-2010 Exceed system uses contact-free technology in which the sample gas

flow makes as little contact as possible with the collector electrode or the 63Ni radiation source,

which greatly improves the the ECD cell durability by reducing deposition of sample residue on

the detecor and radiation source. The detector has a limit of detection of 4.0 fg/s and a dynamic

range of 105 for γ-BHC.

EZGC

software suite

Restek Restek has upgraded its EZGC software suite to include a method translator, flow calculator,

and chromatogram modeler. The software application is available in an on-line version. For

new methods, the chromatogram creator uses a database of thousands of compounds to

accurately predict the best column and conditions for a wide range of applications. For

optimization and troubleshooting of established methods, the method translator and flow

calculator helps analysts easily change column formats and carrier gases for faster analyses

or improved capacity, and also calculates splitless hold times for applications using splitless

injections.

Fast GC

conversion kit

for HP6890

and Agilent

7890 systems

VICI This conversion kit from VICI includes the parts and tools to convert HP6890 or Agilent 7890

instruments to fast GC systems. An adapter plate relocates the detector next to the injector to

make room for the high speed components. The kit enables use of VICI’s resistively heated

columns that are wrapped with nickel wire and heated by applying a low-voltage current.

Column temperature is controlled by regulating the amount of current; a small fan provides rapid

cooling of the low-mass column to near-ambient temperatures. The company’s fast temperature

programmer (a separate item) provides precise temperature programming with rapid heating

and cooling in an eight-state profile at up to 1200 °C/min with a 5-m long or 500 °C/min with a

15-m-long nickel-wire or nickel-clad resistively heated column.

fastGC

add-on for

PTR-TOFMS

series

Ionicon Ionicon PTR-TOF systems are capable of measuring trace gas samples in real-time with a

high mass resolving power. The new fastGC module adds an optional chemical preseparation

step before the analysis. The module consists of a short GC column with an advanced heating

concept for ultrafast heating and equally fast cooling rates that makes the preseparation step

nearly real time. The fastGC module is integrated with the PTR-TOF and the normal sample

gas inlet is used, which allows researchers to perform real-time measurements and add fastGC

runs at time points of interest for enhanced separation and identification. Winner of the Gases

& Instrumentation International Magazine’s 2015 Golden Gas Award in the gas chromatography

category.

IMOv

Intelligent

Modular oven

AFP The IMOv Intelligent Modular oven is a modular GC oven system offering flexibility in the

number of column ovens and amount of space for valves and other accessories. The valve

oven is independent from the column, with the choice of one to three column ovens. The valve

oven has a preperforated bottom plate to accommodate AFP’s valves and comes with custom

tube brackets for easy routing. All electronics such as the company’s IVD intelligent valve

driver, electronic relays, electronic pressure control (EPC), flow meters, and communications

ports are accessible from the back of the controlled temperature zone. The system also

features a built-in leak detection system and has software drivers for various third-party

instruments.

IPAPS:

Intelligent

Plasma

Assisted

Purifier system

AFP The Intelligent Plasma Assisted Purifier system from AFP is a gas purifier that uses a plasma to

increase the lifetime for the heated gettering alloy. The purifier features an end-of-life monitoring

system, impurities overload detection, and a capacity of 1 L/min. The device is suitable for gas

purification, zero gas generation for calibration of on-line analyzers, mass spectrometers, and

GC detectors such as plasma emission, helium ionization, discharge ionization, flame ionization,

pulsed-discharge ionization, and electron capture. The purifier can deliver a total outlet impurity

level of less than 1 ppb, with 99.9999% grade inlet gas. It supports AFP’s IMOv and other

accessories through a serial communications link.



GC CONNECTIONS

References

(1) M. L’Heureux, LCGC North Am. 33(2),

108–121 (2015).

(2) M. Swartz, LCGC Europe 28(4),

232–243 (2015).

(3) M.W. Dong, LCGC Europe 28(4),

223–231 (2015).

(4) D.E. Raynie, LCGC Europe 28 (5), In

Press (2015).

John V. Hinshaw is a senior scientist

at Serveron Corporation in Beaverton,

Oregon, USA, and is a member of the

LCGC Europe editorial advisory board.

Direct correspondence about this

column should be addressed

to “GC Connections”, LCGC Europe,

Honeycomb West, Chester Business

Park, Chester, CH4 9QH, UK, or e-mail

the editor-in-chief, Alasdair Matheson,

at [email protected]

Table 3: New GC accessories (continued).


LipSeal fitting AFP AFP introduced a new patented fitting concept that provides two levels of sealing, requires

a much lower torque, and permits an increased number of remakes. Ultrafine-pitch threads

transfer the sealing force to the ferrule and an antifriction and antigalling coating is applied

to the front and the threaded portions of the nut, which reduces the friction by a factor of at

least 10 and eliminates rotation of the ferrule when the nut is tightened. Gold-plated ferrules

optimize sealing performance, and a fine lip is added on the bottom of the detail that creates

a metal-to-metal seal between the tube end and fitting body with no dead volume. The front

portion of the ferrule is longer to occupy more of the empty volume.

MRV: Mini

rotary valve

AFP AFP introduced a smaller form factor rotary valve that includes features from the company’s

larger valve series such as a double stopper to prevent side loading of the rotor, which results

in a longer lifetime; two dowel pins to lock the stator body in place to prevent rotation; and a

treated rotor to enable the use of the high temperature version at ambient temperature without

damaging the valve. The MRV works with the company’s Mini Pneumatic Actuator.

PeakBlade 77

GC×GC

modulation

system

DANI The DANI PeakBlade 77 GC×GC modulator is able to reach down to 77 K without using

liquid nitrogen, which opens up analysis of the most demanding molecules and allows fast

chromatography with sharp peaks. The modulator cuts chromatographic peaks to unfold complex

sample composition. Driven by a cryocooler system, a cool tip connects to the cryofocusing area

of the GC×GC system by heat conduction to allow an effective focusing process into the GC oven.

The system features an innovative heater design that allows ultrafast vaporization of cryofocused

molecules resulting in an ultranarrow peak bandwidth. The modulator can achieve as much as

450 °C difference between temperature of the cold tip and the GC oven when the oven is at

350–400 °C. The system is capable of the analysis of compounds down to C3. The modulator can

use different vaporization temperatures to adapt to different compounds, programmable during

the run. It is possible to change modulation frequency for target peaks or in timed intervals to

better select sensitivity versus peak-cutting. The modulator is available as part of the DANI Master

GC×GC/TOF-MS system or can be sold to support other GC and GC–MS systems on the market.

Table 4: New GC columns.


Rt-Silica BOND Restek The Rt-Silica BOND column is aimed at analysis of permanent gases, chlorofluorocarbons

(CFC), light hydrocarbons, and sulphur analyses. The column has the retention and

capacity of a traditional PLOT column, but the company’s manufacturing process for this

column nearly eliminates particle release, which minimizes detector spikes, maintenance,

and downtime. The silica phase easily stands up to water, eliminating the need for long

bakeouts after injections of water-contaminated samples. Each column is individually

tested with C4 hydrocarbons, including methyl acetylene and 1,3-butadiene, both of which

are extremely sensitive to changes in inertness and selectivity.

Rxi-1301Sil MS Restek The Rxi-1301Sil MS column targets solvent analyses, glycols by MS, and EPA Method

551. It features an arylene-stabilized cyano phase, combining retention and selectivity

for volatiles with the highest maximum temperature and lowest bleed of any cyano

column. This allows for reliable MS analyses for volatiles and polar compounds, fast

elution of less-volatile analytes for faster cycle times, as well as improved robustness.

ZB-CLPesticides-1

and

ZB-CLPesticides-2

column set

Phenomenex Phenomenex’s new columns are intended for application-specific analysis of chlorinated

pesticides, herbicides, and polychlorinated biphenyls (PCB) by GC–ECD. The columns

provide baseline separation of all analytes of interest in under 10 min and are available

as a two-column kit. The column set supports seven EPA methods (8081, 8082, 8151,

504, 505, 508, and 552) without changing columns, which reduces down-time by

eliminating column installations otherwise needed when changing methods. The columns

have temperature limits of 320 °C isothermal and 340 °C programmed temperature. The

ZB-CLPesticides-1 column is available with film thicknesses of 0.25, 0.32, and 0.50 µm,

and the ZB-CLPesticides-2 column is available with 0.20-, 0.25-, 0.42-, and 0.50-µm films.



SAMPLE PREPARATION

PERSPECTIVES

As expected, the new products

introduced in the past year in the

area of chromatographic sample

preparation, while somewhat limited,

mirror the current development in

the field. That is, a few systems were

developed to automate or streamline

the sample preparation process;

new sorptive phases and formats,

including QuEChERS (quick, easy,

cheap, effective, rugged, and safe),

were developed; and accessories and

other stepwise advances in the field

were noted. In late 2014, the LCGC

editorial staff submitted a survey to

vendors of sample preparation products.

Responses to this survey are compiled in

this review. In addition, a keyword search

using the terms “sample preparation”

and “extraction” was conducted for

exhibitors at Pittcon 2015; then each

of these vendors was visited. While

attempts were made to be as inclusive

as possible, we apologize for any

oversight.

Hollow-Fibre Microextraction

Perhaps the highlight among new sample

preparation products is an unheralded

introduction by one of the smallest

vendors. Biomics, Inc., brought forth

devices for hollow‑fibre microextraction

(HFME) at Pittcon 2015. HFME has been

developed for quite some time (more

than a decade) and is performed in a

variety of configurations; for example, it

was reviewed in a 2010 “Sample Prep

Perspectives” column (1). Biomics claims

that their hollow‑fibre product, available in

single‑vial and 96‑well formats, is the only

commercially available HFME product.

Regardless of the validity of this claim,

such products are certainly scarce and

this development by Biomics should drive

the acceptance of the technique. Figure 1

shows an example of the format of a

96‑well plate HFME device. The HFME

approach should work for the isolation of

environmental, pharmaceutical, food, and

nutraceutical samples.

Systems

Several sample preparation systems were

introduced in the past year, typically with

multisample capabilities and generally in

the bioanalytical realm.

Similar to the HFME product

introduction above, Phenomenex

expanded its offerings with the Novum

Simplified Liquid Extraction (SLE)

product line. Available in both cartridge

and 96‑well plate formats, these liquid

extraction products are designed to

replace conventional liquid–liquid

extraction (LLE) in bioanalytical, food

safety, and environmental testing.

In the suggested protocol with the

Novum product, a sample is diluted

with buffer solution and added to the

SLE medium, and after a brief soaking

period, elution with ethyl acetate or

dichloromethane follows — the process

is completed within about 15 min. The

Extrahera system by Biotage supports

both supported liquid extraction and

solid‑phase extraction (SPE), in either

column (1, 3, and 6 mL) or plate formats.

The Extrahera system can also be

used in protein‑crashing applications

and uses positive pressure for more

reproducible flow. Added automation to

the Fotector Plus automated SPE system

(Reeko Instrument USA) provides

capacity to run 48 samples continuously

with positive pressure sampling and

elution modes.

Keeping with developments in the

bioanalytical area, the ECO2Chrom

flash chromatograph from Applied

Separations uses liquid carbon dioxide

to reduce organic solvent use and

lower the analyte concentration time.

The high diffusivity of the mobile phase

allows smaller particle sizes to be used,

allowing for greater efficiency or faster

analysis times for the same efficiency as

with liquid organic solvents. This flash

chromatography system accommodates

multiple sample introduction formats

with time‑ or peak‑triggered fraction

collection. Meanwhile, wet or dry

homogenization of biological samples

can be performed with the Biotage

Bead Ruptor 24. The bead mill uses

24 2‑mL tubes, 12 7‑mL tubes, or

six 30‑mL tubes simultaneously. The

SiliCycle MiniBlock is a general purpose

system that allows flow‑through parallel

processing of chemical reactions,

including derivatizations, peptide

synthesis, and screening, with resin

agitation and washing. The system

operates over a temperature range from

‑20 °C to 120 °C with capacities ranging

from six 40‑mL vials to 48 4‑mL tubes.

Other significant introductions in the

area of sample preparation systems

were updates or product extensions,

especially in systems for environmental

analysis. The Pickering Laboratories

DEXTech system uses columns

with different formats for sample

cleanup in the analysis of dioxins and

polychlorinated biphenyls. Meanwhile,

Horizon Technologies added plungers

for greater flexibility to the SmartPrep

Extractor automated SPE system

and Environmental Express added

chemistries to its SimpleDist system

New Sample Preparation Products and Accessories at Pittcon 2015Douglas E. Raynie, Sample Preparation Perspectives Editor.

This yearly report on new products introduced at Pittcon (or in the preceding year) covers sample preparation instruments.



SAMPLE PREPARATION PERSPECTIVES

Table 1: New sorbent products.

Company Product Format Notes

SiliCycle SiliaQuick QuEChERS

Salt packets with centrifuge tubes for performing QuEChERS method

Available as MgSO4 with primary secondary amine (PSA), carbon black, or C18.

UCT, Inc. Styre Screen HL DVB

SPE cartridges

Cross‑linked divinylbenzene for extraction of acidic, basic, polar, and nonpolar compounds with greater loading capacity than silica‑based phases.

Enviro‑Clean QuEChERS

Salt packets with centrifuge tubes for performing QuEChERS method

Salt ratios optimized for biological samples with limited volumes. Protein precipitation not required for blood samples.

Separation Methods Technologies

SMT MEB Bulk packings

Methyl (1% carbon load), ethyl (2% carbon load), and butyl (4% carbon load) silica‑based phases, 35–50 μm particles, 60‑ or 150‑Å pores. Selective for polar and nonpolar pharmaceuticals, natural products, and very hydrophobic proteins and biomolecules.

Bonna‑Agela Technologies

Cleanert PEP‑2

96‑well, modular microplates

Five phases available:• PVB: functionalized vinyl pyrrolidone

and urea to retain most acidic, basic, and neutral polar compounds without adjusting pH. Design for small sample amounts, resulting in one‑third less evaporation time and reconstitution solvent.

• PWCX: combines weak cation-exchange

and reversed phases using carboxylate radical functional group for improved retention of basic analytes.

• PWAX: combines weak anion-exchange

and reversed phases on polymer support with amino functional group.

• PAX: quaternary ammonium base

functional group with reversed‑phase and strong anion‑exchange modes. Stable from 0–14 pH range.

• PCX: sulpho-functional group

with reversed‑phase and strong cation‑exchange modes. Stable thoughout the pH 0–14 range.

Thermo Scientific

ASE Prep Sorbent cartridges

6‑mL cartridges with 500‑mg resin

Four resins available, designed for cleanup of extracts following accelerated solvent extraction:• Florisil for adsorption of polar

compounds.• Alumina acid for anion exchange and

adsorption of polar compounds.• Alumina base for cation exchange and

adsorption of polar compounds.• Alumina neutral for adsorption of polar

compounds, capable of anion or cation exchange with pH adjustment.



SAMPLE PREPARATION PERSPECTIVES

for the distillation of phenols. The

Omni‑Sampler Plus sample handling

system from Entech Instruments

updated cryogenic preconcentration

for volatile organic compounds onto

glass beads, with mild temperatures

(60–100 °C) for the transfer of C2–

C24 compounds. The Omni‑Sampler

Plus sample handling system has

multiple modes for the determination of

volatile analytes, including headspace

sampling, thermal desorption,

porous cartridge microextraction (a

high‑capacity version of solid‑phase

microextraction [SPME]), and on‑column

trapping. For water analysis, the 4100

Water/Soil Sample Handler from OI

Analytical automates sample handling

and processing in collaboration with the

company’s Eclipse 4660 purge‑and‑trap

concentrator.

Sorbents

Various sorbents, in cartridges or as

bulk phases, have been introduced

in the past year. These phases are

designed for SPE, including dispersive

SPE (dSPE) approaches such as the

QuEChERS method, high sample

capability via polymer supports, and

selectivity in sample cleanup. These

sorbent products are summarized in

Table 1.

Accessories and Other Products

Several other sample preparation

products were recently introduced

to the market. Most notably, Supelco

continues to develop its SPME product

line in the area of biocompatible

SPME. This product extension is more

compatible with biological analyses

such as the direct sampling of small

animals like mice, as well as dried

blood spot analysis, 96‑well plates,

and other microsampling situations.

Since gas chromatography (GC) and

derivatization reactions for GC are

often considered mature technologies,

it is somewhat surprising to see a

new derivatization reagent from Regis

Technologies. N‑Methyl‑N‑(trimethylsilyl)

trifluoroacetamide (MSTFA) with 1%

trimethylsilyl chloride is also marketed

by other vendors for the silylation of

hindered hydroxyl groups that do not

ordinarily react with MSTFA, along with

secondary amines, amides, carboxyls,

and steroids. Thermo Scientific

addresses an expanding number

of application areas for accelerated

solvent extraction (ASE), particularly

polymers, with the offering of ASE

extraction thimbles for samples that

melt at the operating temperatures

used in ASE. The goal is to prevent

the plugging of filters and tubing by

fine particles by using cellulose or

glass fibre filters. The GlycoWorks

RapiFluor‑MS N‑Glycan kit from Waters

is a 96‑well plate product based on

hydrophilic interaction chromatography.

The GlycoWorks kit is used for the

sample preparation of N‑linked glycans

released following rapid deglycosylation

and labelling to provide enhanced

sensitivity for both fluorescence and

mass spectrometric determination.

Sample analysis of glycoproteins can

be completed in less than 1 h. Finally,

J.G. Finneran Associates marketed a

vial loader for 96‑well plates with insert

vial sizes ranging from 350‑μL glass

vials to volumes of 2 mL.

Conclusions

With this review of new product

offerings in the field of chromatographic

sample preparation, the natural

question is: “What’s next?” Based on

this year’s offerings and advancements

in the field, it is anticipated that

commercial developments in the

current year will address several issues.

Sorbent‑based sample preparation

will continue to see significant

commercialization in several areas.

QuEChERS will remain a growing

area and the end of patent protection

for SPME will bring new competitors

to the field and new areas such as

biocompatible SPME and SPME

designed for liquid chromatography

applications. Other advancements

will accommodate serial or parallel

sample processing for increased

throughput. Bioanalytical and food

safety applications will drive these

developments.

References(1) L. Zhao, H.K. Lee, and R.E. Majors, LCGC

North Am. 28(8), 580–591 (2010).

(2) G. Borijijan, Y. Li, J. Gao, and J.J. Bao, J.

Sep. Sci. 37, 1155–1161 (2014).

“Sample Prep Perspectives” editor

Douglas E. Raynie is an Associate

Research Professor at South

Dakota State University, USA. His

research interests include green

chemistry, alternative solvents,

sample preparation, high resolution

chromatography, and bioprocessing in

supercritical fluids. He earned his PhD

in 1990 at Brigham Young University

under the direction of Milton L. Lee.

To contact the editor‑in‑chief, Alasdair

Matheson, please e‑mail: amatheson@

advanstar.com

(a)

(b)(d)

(c)

12

3

4

5

(a)

(b)(d)

(c)

12

3

4

5

(a)

(b)(d)

(c)

12

3

4

5

(a)

(b)(d)

(c)

12

3

4

5

Figure 1: Hollow‑fibre microextraction in a 96‑well format: (a) the plastic base, (b) the attachment of hollow fibre to the plastic base, (c) the 96‑well plate, and (d) an expanded view of the hollow‑fibre device. 1 = hollow‑fibre attachment tip, 2 = donor phase collection tip, 3 = acceptor phase, 4 = donor phase, 5 = hollow fibre. Adapted with permission from reference 2.


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PRODUCTS

2015 GC catalogue

MEGA has presented

its 2015 GC Products

Catalogue. The edition

presents all of the

MEGA GC columns

ranging from the most

commonly used phases

to custom and proprietary

application-specif c

stationary phases. The

catalogue also features the

MEGA-DEX chiral columns

line and the Metal columns for high-temperature GC.

www.mega.mi.it

MEGA s.n.c., Milan, Italy.

2d LC system

The Agilent 1290 Inf nity 2D LC

is a one vendor platform that

allows users to switch between

one-dimensional UHPLC

and the chromatographic

power of heart-cutting,

multiple heart-cutting, and

comprehensive 2D LC.

According to the company, this

instrument achieves ultrahigh peak capacity with a single

2D LC analysis and allows setup of new methods within

seconds.

www.agilent.com/chem/infi nity-2d-lc

Agilent technologies, California, usA.

Automated sample preparation

The PAL RTC system

with robotic tool change

from CTC Analytics

AG automates sample

preparation and sample

injection. According

to the company, it

improves productivity by

automatically changing between different syringes, liquid

injection, headspace, and SPME without the need for

manual intervention. This enables workf ows like liquid–

liquid extraction or SPE to be fully automated.

www.palsystem.com

CtC Analytics AG, Zwingen, switzerland.

Molecular imaging system

The Full Spectrum Molecular Imaging

System from Waters combines advanced

MS imaging technologies to provide

comprehensive large and small molecule

distribution information, according to

the company. Integrating MALDI, DESI,

and ion mobility into a single system

delivers a level of detail and molecular

information beyond any individual

imaging technique. The SYNAPT

HDMS-based system expands imaging

capabilities for the study of molecular distribution in f elds such

as proteomics, metabolomics, cell and tissue biology, research

pathology, and histology.

www.waters.com/msimaging

Waters, Massachusetts, usA.

Meat reference materials

Six new meat mixture

reference materials are

available from LGC to

help protect consumers

from food fraud. The meat

mixtures enable food testing

laboratories to assess the

quality of their measurements

and ensure detection of

substitutions in meat products

at low levels. According to the company, the materials have been

tested by DNA sequencing, a PCR-based and immunoassay

method, to conf rm the expected meat species in samples and

absence of species cross-contamination. Limit of detection is

below 1% of one meat species in the presence of another.

www.lgcstandards.com

LGC, teddington, Middlesex, uK.

HILIC HPLC columns

SeQuant ZIC-HILIC HPLC

columns are offered by

Merck Millipore. HILIC is a

chromatographic technique

for separation of polar and

hydrophilic compounds.

HILIC is a normal-phase

(NPLC) type of separation

but uses reversed-phase

type eluents. It is the method

of choice for separating

charged and neutral hydrophilic compounds, such as acids,

bases, ions, peptides, metabolites, and sugars.

www.merckmillipore.com/chromatography

Merck Millipore, darmstadt, Germany.



ProduCts

thermal desorption tube

cleaner

Markes’ TC-20 tube conditioner

allows up to 20 sorbent-packed

TD tubes to be simultaneously

cleaned at elevated temperatures.

According to the company, the

TC-20 frees up instrument time to

run samples rather than condition

tubes, providing a rapid return on

investment.

www.markes.com

Markes International,

Llantrisant, uK.

uHPLC sEC–MALs detector

Wyatt has launched μDAWN,

a multi-angle light scattering

(MALS) detector that can

reportedly be coupled to any

UHPLC system to determine

absolute molecular weights and

sizes of polymers, peptides,

proteins, or other biopolymers

directly. To accommodate

narrow peaks in UHPLC, the light scattering f ow cell volume has

been reduced from 63 μL to 10 μL. To minimize interdetector

mixing, band broadening is under 7 μL.

www.wyatt.com

Wyatt technology, California, usA.

Evaporative light-scattering

detector

Sedere has introduced the

Sedex 90LT, a low-temperature

evaporative light-scattering

detector (LT-ELSDTM). According

to the company, the detector

provides ppb level sensitivity, four

orders of magnitude dynamic

range, direct linearity on the full

range, excellent efficiency, and

response consistency. The detector

reportedly suits every application in

LC and SFC.

www.sedere.com

sedere sAs, Alfortville, France.

Please submit new product press releases

and catalogue information to

Bethany degg at [email protected]

uHPLC system

As a new UHPLC system

designed for enhanced

levels of performance,

productivity, and usability,

the Thermo Scientif c

Vanquish can be used

standalone or with the

latest mass spectrometer. It

features a clean design that

combines the ruggedness

of an integrated system

with the f exibility and serviceability of a modular system,

according to the company.

www.thermofi sher.com/vanquish

thermo Fisher scientifi c, California, usA.

HPLC

The classif ed directory from LC•GC Europe

To advertise in this section call Elizabeth McLean

today at +44 (0) 1244 629 315 or email

[email protected] to reserve your space

Supplies

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ProduCts

sPE system

The EconoTrace SPE system from

FMS is a parallel SPE system that uses

positive pressure pumping for precise

and accurate delivery of the sample,

conditioning, washing, and elution

solvents. Up to 8 extractions can run

simultaneously. The system uses any

size or type SPE cartridge. According

the company, the system is an economic

solution for consistent, reproducible,

high throughput extractions for

pharmaceutical, food, and water

matrices.

www.fms-inc.com

FMs, Inc., Massachusetts, usA.

Ion chromatography modules

Cecil Instruments has

introduced the IonQuest

ion chromatography

system, which offers

modular and automated

ion chromatography. The

low-drift and ultra-low noise

conductivity detector may

be purchased separately

for use with third party

LC systems. The use of

PowerStream software

helps to achieve ease of use and 21 CFR part 11 compliance.

www.cecilinstruments.com/ionquest-1.html

Cecil Instruments Limited, Cambridge, uK.

dried blood spot autosampler

Flow-through desorption

(FTD) is the concept on

which the new Dried Blood

Spot Autosampler from

Spark Holland is based.

Farewell to punching! Using

a spot recognition camera,

DBS cards are accurately

positioned in a high pressure

clamp for direct, fow-through

desorption of bloodspots into

the analytical system. The company report that the system

provides maximum sensitivity and a fully automated

workfow for DBS analysis.

www.sparkholland.com

spark Holland B.V., Emmen, the Netherlands.

Forensic analysis kits

UCT, LLC has created complete

forensic analytical kits to perform

toxicology extractions and

analyses. The kits contain buffer

reagents, SPE columns, HPLC

columns, hydrolyzing reagents,

and stock drug standards. CoA’s

for all standards as well as

recommended extraction and

analytical procedures are included.

According to the company, these

kits are ideal for start-up labs, labs

developing a new method, or converting from GC–MS to

LC–MS.

www.unitedchem.com

uCt, LLC, Pennsylvania, usA.

sFE–sFC–Ms system

Shimadzu’s Nexera UC

unifed chromatography

system is reportedly

the world’s frst-ever

unifed and fully

automated instrument

combining supercritical

fuid extraction (SFE)

with supercritical fuid

chromatography (SFC). The SFE–SFC–MS platform

merges quick and easy on-line sample preparation with

advanced chromatographic analysis and high sensitivity

detection.

www.shimadzu.eu

shimadzu Europa GmbH, duisburg, Germany.

GC–Ms columns

The Restek Rxi-1301Sil MS

GC columns offer cyano

phase selectivity along with

high thermal stability, which

ensure dependable, accurate

MS results and increased

uptime. The company report

that the columns can improve

the performance of existing

methods for solvents, glycols,

and other polar compounds.

www.restek.com/catalog/view/41734

restek, Bellefonte, Pennsylvania, usA.


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EVENT NEWS

21–25 June 2015 42nd International Symposium on

High Performance Liquid Phase

Separations and Related Techniques

(HPLC 2015)

International Conference Centre,

Geneva, Switzerland

Tel: +41 22 839 84 84

E-mail: [email protected]

Website: www.hplc2015-Geneva.org

28 June–1 July 2015Recent Developments in

Pharmaceutical Analysis

University of Perugia, Perugia, Italy

Tel: +39 0755855131


Website: rdpa2015.chimfarm.unipg.it

30 June–3 July 201521st International Symposium on

Separation Science

Grand Hotel Union, Ljubljana, Slovenia

Tel: +386 1 477 0265


Website: www.isss2015.si

3–6 August 20155th International Network of

Environmental Forensics (INEF)

Victoria University College, Toronto,

Canada


Website: www.inef2015.com

23–28 August 201535th International Symposium on

Halogenated Persistent Organic

Pollutants (Dioxin 2015)

Hotel Maksoud Plaza, Sao Paulo, Brazil

Tel: +55 11 3056 6000


Website: www.dioxin2015.org

18–19 November 2015PEFTEC International Conference

and Exhibition for Petrochemical

Analysis

Antwerp Exhibition Centre,

Antwerp, Belgium

Tel: +44 1727 858840


Website: www.peftec.com

31st Montreux Symposium on LC–MS and MS–MS

The 31st Montreux Symposium on

LC–MS and MS–MS will be hosted

by the International Association

of Environmental Analytical

Chemistry (IAEAC) at the Aldershof

Convention & Exhibition Centre,

Berlin-Aldershof, Germany, on

4–6 November 2015. The Montreux

Symposium series was first established in 1980 by the IAEAC, with the symposium

historically alternating location between Montreux, Switzerland, and the USA.

This year it will be held in Berlin-Aldershof, the location of the science campus of

Humboldt-Universität and of Germany’s leading science and technology park.

The conference symposium will focus on the most recent developments in

liquid chromatography coupled to mass spectrometry (LC–MS) and tandem

mass spectrometry (MS–MS) in the three fields of metabolomics, lipidomics,

and glycomics. These fields were selected because, aside from genomics and

proteomics, the work in these areas is reshaping our understanding of biological

processes and the development of therapies for diseases.

This symposium will bring together scientists working with up-to-date

bioanalytical tools, particularly using separation techniques such as gas and

liquid chromatography, electrophoresis, and ion mobility in combination with

spectroscopy and mass spectrometry. They will join with enabling companies

from those fields, who will have the opportunity to display the latest developments

in all areas of MS and the separation sciences. The organizers promise first-rate

presentations, posters presenting up-to-date research, and an exhibition

highlighting new scientific instrumentation and technical information.

E-mail: [email protected] • Website: www.lcms-montreux2015.de

7th International Symposium on Recent Advances in Food Analysis (RAFA 2015)

The 7th International Symposium on Recent Advances

in Food Analysis (RAFA 2015) will take place at the

Clarion Congress Hotel, Prague, Czech Republic, on

3–6 November 2015. The RAFA 2015 symposium will provide

an overview of the current state-of-the-art on analytical

and bioanalytical food quality, safety control strategies,

and introduce the challenges for novel approaches in this

field. The programme will be tailored to provide networking

opportunities as well as exploring the latest results from the

food analysis community. Presentations will be given by leading scientists

through keynote lectures and contributed oral and poster presentations

covering the topics of general food analysis issues and food quality/safety.

The conference programme will also be accompanied by several satellite

events including workshops on novel analytical strategies and interactive

seminars. An exhibition of recently introduced instrumentation in food analysis

and other valuable equipment will be available during the symposium. Vendor

seminars will also be organized to introduce recent developments and scientific

strategies for advanced food quality and safety control.

Young scientists are encouraged to present their scientific work, with a

number of RAFA 2015 Student Travel Grants available. The prestigious

RAFA Poster Award will also be given for the best poster presentation by a

young scientist, along with other sponsored poster awards. The deadline for

registration at a reduced fee and submission of an abstract for oral presentation

is 31 July 2015; deadline for submission of an abstract for poster presentation is

31 August 2015.

E-mail: [email protected] • Website: www.rafa2015.eu

EVENT NEWS

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