Wine Analysis - transfer.nxtbook.com

SAMPLE PREPARATION

PERSPECTIVES

“Just enough” sample preparation

GC CONNECTIONS

Q&A on gases

HISTORY OF

CHROMATOGRAPHY

MS pioneer Fred McLafferty

Wine AnalysisAnalysing pesticides in red

wine using QuEChERS

March 2013

Volume 16 Number 1

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All articles submitted to LC•GC Asia Pacific

are subject to a peer-review process in association

with the magazine’s Editorial Advisory Board.

Cover:

Original materials: Christian Martinez Kempin

Columns14 HISTORY OF CHROMATOGRAPHY

Man of the Masses

Kate Yu

MS: The Practical Art Editor Kate Yu interviews Fred McLafferty

about his pioneering career in mass spectrometry.

18 SAMPLE PREPARATION PERSPECTIVES

“Just Enough” Sample Preparation: A Proven Trend in Sample

Analysis

Ronald E. Majors

This article discusses the details of how the newer techniques of

sample preparation are simplifi ed by the use of LC–MS–MS and

GC–MS–MS technology.

23 GC CONNECTIONS

Q&A: Gases

John V. Hinshaw

In this month’s instalment, we address common questions about

various gases and their delivery to a gas chromatograph.

27 QUESTIONS OF QUALITY

How Much Value Is There in a Software Operational Qualifi cation?

R.D. McDowall

Answers to common questions about operational qualifi cation software.

32 THE ESSENTIALS

Column Selection for Reversed-Phase HPLC

With so many HPLC columns on the market, we present a simple

guide to what’s important when making your stationary phase and

column dimension choices.

Departments33 Application Notes

COVER STORY6 Determination of Pesticides

in Red Wine by QuEChERS

Extraction, Rapid Mini-Cartridge

Cleanup and

LC–MS–MS Detection

Xiaoyan Wang and Michael

J. Telepchak

A novel, simple, rapid and effective

method to determine pesticide

residues in red wine samples is

described.

March | 2013

Volume 16 Number 1


4 LC•GC Asia Pacific March 2013

The Publishers of LC•GC Asia Pacific would like to thank the members of the Editorial Advisory Board for their continuing support and expert advice. The high standards and editorial quality associated with LC•GC Asia Pacific are maintained largely through the tireless efforts of these individuals.

LCGC Asia Pacific provides troubleshooting information and application solutions on all aspects of separation science so that laboratory-based analytical chemists can enhance their practical knowledge to gain competitive advantage. Our scientific quality and commercial objectivity provide readers with the tools necessary to deal with real-world analysis issues, thereby increasing their efficiency, productivity and value to their employer.

Editorial Advisory Board

Kevin AltriaGlaxoSmithKline, Harlow, Essex, UKDaniel W. ArmstrongUniversity of Texas, Arlington, Texas, USAMichael P. BaloghWaters Corp., Milford, Massachusetts, USA

Coral BarbasFaculty of Pharmacy, University of San Pablo – CEU, Madrid, SpainBrian A. BidlingmeyerAgilent Technologies, Wilmington, Delaware, USAGünther K. BonnInstitute of Analytical Chemistry and Radiochemistry, University of Innsbruck, AustriaPeter CarrDepartment of Chemistry, University of Minnesota, Minneapolis, Minnesota, USAJean-Pierre ChervetAntec Leyden, Zoeterwoude, The NetherlandsJan H. ChristensenDepartment of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, DenmarkDanilo CorradiniIstituto di Cromatografia del CNR, Rome, ItalyHernan J. CortesH.J. Cortes Consulting, Midland, Michigan, USAGert DesmetTransport Modelling and Analytical Separation Science, Vrije Universiteit, Brussels, BelgiumJohn W. DolanLC Resources, Walnut Creek, California, USARoy EksteenTosoh Bioscience LLC, Montgomeryville, Pennsylvania, USAAnthony F. FellPharmaceutical Chemistry, University of Bradford, Bradford, UK

Attila FelingerProfessor of Chemistry, Department of Analytical and Environmental Chemistry,University of Pécs, Pécs, HungaryFrancesco GasparriniDipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente 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, JapanJavier Hernández-BorgesDepartment of Analytical Chemistry, Nutrition and Food Science University of Laguna, Canary Islands, SpainJohn V. HinshawServeron Corp., Hillsboro, Oregon, USATuulia HyötyläinenVVT Technical Research of Finland, Finland

Hans-Gerd JanssenVan’t Hoff Institute for the Molecular Sciences, Amsterdam, The NetherlandsKiyokatsu JinnoSchool of Materials Sciences, Toyohasi University of Technology, JapanHuba KalászSemmelweis University of Medicine, Budapest, HungaryHian Kee LeeNational University of Singapore, SingaporeWolfgang LindnerInstitute of Analytical Chemistry, University of Vienna, AustriaHenk LingemanFaculteit der Scheikunde, Free University, Amsterdam, The NetherlandsTom LynchBP Technology Centre, Pangbourne, UKRonald E. MajorsAgilent Technologies, Wilmington, Delaware, USAPhillip MarriotMonash University, School of Chemistry, Victoria, Australia

David McCalleyDepartment of Applied Sciences, University of West of England, Bristol, UKRobert D. McDowallMcDowall Consulting, Bromley, Kent, UKMary Ellen McNallyDuPont Crop Protection,Newark, Delaware, USAImre MolnárMolnar Research Institute, Berlin, GermanyLuigi MondelloDipartimento Farmaco-chimico, Facoltà di Farmacia, Università di Messina, Messina, ItalyPeter MyersDepartment of Chemistry, University of Liverpool, Liverpool, UK

Janusz PawliszynDepartment of Chemistry, University of Waterloo, Ontario, CanadaColin PooleWayne State University, Detroit, Michigan, USAFred E. RegnierDepartment of Biochemistry, Purdue University, West Lafayette, Indiana, USAHarold RitchieThermo Fisher Scientific, Cheshire, UKPat SandraResearch Institute for Chromatography, Kortrijk, BelgiumPeter SchoenmakersDepartment of Chemical Engineering, Universiteit van Amsterdam, Amsterdam, The NetherlandsRobert ShellieAustralian Centre for Research on Separation Science (ACROSS), University of Tasmania, Hobart, AustraliaYvan Vander HeydenVrije Universiteit Brussel, Brussels, Belgium

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Approximately, 26 billion litres of wine were produced

worldwide and about 24 billion litres were consumed,

according to the International Organization of Vine and Wine,

in 2010 (1). Wine, especially red wine, is a rich source of

polyphenols such as resveratrol, catechin and epicatechin.

These polyphenolic compounds are antioxidants that

protect cells from oxidative damage caused by free radicals.

Research on antioxidants found in red wine has shown that

they may inhibit the development of certain cancers such as

prostate cancer (2). In addition, consumption of red wines

has been believed to have heart-healthy benefits (2). The

application of pesticides such as fungicides and insecticides

to improve grape yields is a common practice in vineyards.

However, the applied pesticides may permeate through plant

tissues and remain in the harvested grapes and subsequent

processed products, such as grape juice and wine. Because

pesticide residues are a potential source of toxic substances

that are harmful to human beings, it is important to test for

the levels of pesticide residues in grapes, juice and wine.

Although the European Union (EU) has set maximum residue

levels (MRLs) for pesticide residues in wine grapes of

0.01–10 mg/kg (3,4), it has not yet established MRLs for

wine. A study of 40 bottles of wine bought within the EU

revealed that 34 of the 40 bottles contained at least one

pesticide. The average number of pesticides per bottle was

KEY POINTS• Over 24 billion litres of wine were consumed in 2010.

• The polyphenols in red wine have been associated

with health benefits.

• Pesticides, fungicides and insecticides are often used

to improve grape yields and a monitoring programme to

analyse these chemicals is essential.

• A simple, fast, novel and effective cleanup method

for pesticides in red wine samples was successfully

developed.

more than four, while the highest number of pesticides found

in a single bottle was 10 (5).

The analysis of pesticide residues in red wine is

challenging because of the complexity of the matrix,

which contains alcohol, organic acids, sugars, phenols

and pigments (such as anthocyanins). Traditional red

wine sample preparation methods include liquid–liquid

extraction (LLE) with different organic solvents (6,7)

and solid-phase extraction (SPE) with reversed-phase

C18 and polymeric sorbents (8–10). However, LLE is

labour-intensive, consumes large amounts of organic

Xiaoyan Wang and Michael J. Telepchak, UCT, Bristol, Pennsylvania, USA.

A simple, rapid and effective method was successfully developed for the determination of pesticide residues in red wine samples. Sample preparation involved extraction of pesticide residues into acetonitrile by QuEChERS (quick, easy, cheap, effective, rugged and safe) and cleanup with a rapid push‑through mini‑cartridge filter instead of dispersive solid‑phase extraction (dSPE). The limit of detection (LOD) and limit of quantification (LOQ) were in the range of 0.01–0.40 and 0.05–1.33 ng/mL, respectively. Six commercially available red wine samples were tested in this study, three of which were found to be positive for the presence of pesticides.

Determination of Pesticides in Red Wineby QuEChERS Extraction, Rapid Mini-Cartridge Cleanup and LC–MS–MS Detection

LC•GC Asia Pacific March 20136

solvents and sometimes forms emulsions, making it

difficult to separate the organic and aqueous phases. In

contrast, SPE uses less solvent without emulsion formation,

but demands more effort for method development.

Other methods such as solid-phase microextraction

(SPME) (11,12), hollow-fibre liquid-phase microextraction

(13) and stir-bar sorptive extraction (SBSE) (14) use

little or no organic solvent but are less reproducible.

Typical instrumental detections systems include gas

chromatography (GC), GC coupled to mass spectrometry

(GC–MS) and liquid chromatography coupled to tandem

mass spectrometry (LC–MS–MS) (6–14).

QuEChERS (quick, easy, cheap, effective, rugged and

safe) is a promising sample preparation method that was first

reported in 2003 by Anastassiades, Lehotay and colleagues

for the determination of pesticide residues in vegetables

and fruits (15). Since then QuEChERS has been widely

used for the analysis of pesticides and other compounds of

concern in various food, oil and beverage matrices (16–18).

The QuEChERS procedure involves extraction of pesticides

from a sample with high water content into acetonitrile with

the addition of salts to separate the phases and partition the

pesticides into the organic layer. This is followed by dispersive

solid-phase extraction (dSPE) to clean up various matrix

coextractives and is achieved by mixing an aliquot of sample

extract with sorbents prepacked in a centrifuge tube.

The aim of this study is to develop a method using

QuEChERS extraction, but an easier and faster cleanup

method compared to dSPE to clean up red wine coextractives.

This novel sample cleanup method is based on a

filter-and-clean concept: The red wine extract is pushed

through a mini-cartridge containing anhydrous magnesium

sulphate and primary secondary amine (PSA) sorbent, residual

water is adsorbed onto the anhydrous magnesium sulphate,

and red wine coextractives are retained by the PSA sorbent.

The purified extract is collected into an autosampler vial and

injected into an LC–MS–MS system for analysis without the

need for further filtration with a syringe filter. This cleanup

procedure is simple and takes less than 1 min per sample.

Red wine extracts were assessed for cleanliness based

on visual appearance and full-scan chromatograms after

cleanup with four traditional dSPE approaches containing

different amounts of PSA sorbent and the rapid mini-cartridge

filtration approach. The rapid mini-cartridge approach

produced a slightly cleaner extract than the dSPE approach

containing the same amount of magnesium sulphate and PSA

sorbent. However, the cleanup procedure with push-through

mini-cartridge filtration was found to be much faster than

dSPE. Eight pesticides belonging to insecticide, fungicide

and parasiticide classes were selected for analysis in this

study. Polarities of the eight selected pesticides were very

different, with the logarithms of the octanol water partition

coefficient (LogP) ranging from -0.779 to 5.004. The classes,

structures, LogP and pKa values are listed in Figure 1. Among

the eight pesticides analysed in this study, cyprodinil was

most often detected on grapes, with chlorpyrifos, diazinone

and methamidophos also frequently found on grapes (19).

The recoveries of planar pesticides included in this study

(carbendazim, thiabendazole, pyrimethanil and cyprodinil) are

often adversely affected by graphitized carbon black (GCB),

a sorbent that is widely used in dSPE to clean up pigmented

samples. In this study, PSA sorbent was used instead of GCB

for cleanup of red wine samples and the recoveries of these

planar pesticides are reported.

Finally, six commercially available red wine samples were

analysed using this simple, rapid and effective sample

preparation method. Carbendazim was detected in three red

wine samples, although the detected concentrations (parts

per billion) are much lower than the European or Japanese

regulated levels (parts per million) in grapes (20,21).

ExperimentalStandards and Reagents: HPLC-grade acetonitrile and

LC–MS-grade methanol were purchased from Spectrum.

Table 1: Retention times, SRM transitions and dwell times for target analytes and internal standard (IS).

Compound Retention (min) Precursor Ion Product Ion 1 CE Product Ion 2 CE S-Lens Dwell Time (s)

Methamidophos 2.78 142.044 94.09 14 125.05 16 59 0.15

Carbendazim 6.48 192.093 132.08 29 160.08 17 81 0.10

Thiabendazole 6.91 202.059 131.06 31 175.07 31 103 0.10

Pyrimethanil 10.43 200.116 107.06 23 183.14 22 66 0.10

Cyprodinil 11.44 226.122 77.03 40 93.05 33 88 0.10

TPP (IS) 11.78 327.093 77.02 37 152.07 33 98 0.10

Diazinone 11.92 305.135 153.09 15 169.08 14 89 0.10

Pyrazophos 12.24 374.103 194.06 20 222.13 20 104 0.10

Chlorpyrifos 13.42 349.989 96.89 32 197.94 17 69 0.10

Table 2: Matrix matched calibration, LODs and LOQs.

Compound Dynamic Linearity

Range (ng/mL)

Regression Equation R2 LOD (ng/mL) LOQ (ng/mL)

Methamidophos 2–400 Y = -6.65815e-005 + 6.0069e-005X 0.9991 0.15 0.49

Carbendazim 2–400 Y = -0.00128523 + .00193304X 0.9981 0.40 1.33

Thiabendazole 2–400 Y = -0.00318887 + 0.00457172X 0.9940 0.09 0.31

Pyrimethanil 2–400 Y = 0.0045227 + 0.00257414X 0.9990 0.01 0.05

Cyprodinil 2–400 Y = -0.000566941 + 0.00578677X 0.9995 0.17 0.57

Diazinone 2–400 Y = -0.00267408 + 0.0199284X 0.9982 0.06 0.21

Pyrazophos 2–400 Y = 0.00283428 + 0.0110651X 0.9976 0.08 0.27

Chlorpyrifos 2–400 Y = 0.000538988 + 0.00136876X 0.9981 0.10 0.32


Wang and Telepchak

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Compound

Methamidophos

Carbendazim

Thiabendazole

Pyrimethanil

Cyprodinil

Diazinone

Pyrazophos

Chlorpyrifos

Fungicide andparasiticide

Fungicide andinsecticide

Fungicide

Fungicide

Fungicide

Insecticide

-0.779

1.52

2.47

2.558

3.012 4.22

1.213.766

2.810

5.004

-1.37

-5.28

4.41

5.66,11.62

3.40,10.53

-0.58H3C CH

3

OCH3

NH2

S

O

HN

HN

N

N

NN H

N

N

NN S

OO

O

O CN

N

N

O

S O

O

PCH3

CH2

CH2

Cl

Cl

ClO

S N

PO

O

CH3

CH3

H2C

H3C

OP

NHH

3C

N

S

NNH

PO

O

Insecticide

Insecticide

Class Structure Log P pKa

Figure 1: Classes, structures, LogP and pKa values of the eight

pesticides selected in this study.

Figure 2: Photograph of red wine extracts without any cleanup,

cleaned up with dSPE A, B, C and D, and cleaned up with rapid

mini-cartridge filtration.

phosphate (TPP, 5000 ppm) was purchased from Cerilliant and

was used as the internal standard (IS) in this study.

A 100 ppm thiabendazole solution was made by mixing

200 µL of the 1000 ppm stock solution with 1.8 mL

acetonitrile. A 2 ppm pesticide working standard solution

was made by adding 80 µL each of the eight 100 ppm

standards with 3.36 mL of acetonitrile. A 5 ppm IS solution

was made by diluting 10 µL of the 5000 ppm TPP stock

solution with 10 mL of acetonitrile.

Sample Preparation:

Extraction: The six red wine samples tested in this study were

provided by coworkers. Portions (10 mL) of the red wine

samples were added into 50-mL polypropylene centrifuge

tubes (UCT). To prepare fortified samples, red wine samples

were spiked with appropriate amounts of the 2 ppm pesticide

working standard solution, vortexed for 30 s and allowed to

equilibrate for 15 min. A 10-mL volume of acetonitrile was

added to each sample and then shaken for 1 min. Salts

(4000 mg of anhydrous magnesium sulphate and 2000 mg of

sodium chloride) packed in a Mylar pouch (UCT) were added,

and the samples were shaken vigorously for 1 min and then

centrifuged at 5000 rpm for 5 min. The upper layer red wine

extract was then ready for cleanup.

Cleanup: Two cleanup methods, traditional dSPE and rapid

push-through mini-cartridge filtration, were compared

for cleanup efficiency of the red wine extract. Four 2-mL

dSPE tubes containing 110 mg MgSO4 and 25 mg PSA

(A); 110 mg MgSO4 and 50 mg PSA (B); 110 mg MgSO4

and 100 mg PSA (C); and 110 mg MgSO4 and 180 mg PSA

(D) were tested to compare the cleanup efficiency against

the rapid push-through mini-cartridge containing 110 mg

MgSO4 and 180 mg PSA (UCT, ECPURMPSMC). For dSPE

cleanup, 1 mL of the red wine extract was transferred into

the 2-mL dSPE tube, shaken for 30 s and then centrifuged

at 10,000 rpm for 5 min. A 0.5-mL volume of the cleaned

extract was transferred into a 2-mL autosampler vial and

10 µL of the 5 ppm TPP (IS) solution was added. For rapid

push-through mini-cartridge cleanup, 1 mL of the red wine

Table 3: Accuracy and precision data.

Compound Fortified at 10 ng/mL Fortified at 50 ng/mL Fortified at 100 ng/mL

Recovery% RSD% (n = 4) Recovery% RSD% (n = 4) Recovery% RSD% (n = 4)

Methamidophos 93.7 3.4 81.6 5.8 84.2 3.5

Carbendazim 105.7 10.8 100.1 10.6 90.5 7.6

Thiabendazole 91.2 4.9 87.9 6.8 85.0 4.0

Pyrimethanil 112.2 2.7 107.0 3.2 102.8 4.9

Cyprodinil 104.3 3.2 99.9 6.1 100.2 4.9

Diazinone 104.9 5.6 102.0 6.6 99.2 6.8

Pyrazophos 99.9 4.0 96.6 5.6 91.3 4.1

Chlorpyrifos 91.7 4.6 99.5 5.2 97.2 3.8

Table 4: Pesticide residues detected in six red wine samples. The minimum reporting limit (MRL) of the method is 2 ng/mL.

Pesticide Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6

Methamidophos < MRL < MRL < MRL < MRL < MRL < MRL

Carbendazim < MRL < MRL < MRL 10.2 ng/mL 8.7 ng/mL 2.3 ng/mL

Thiabendazole < MRL < MRL < MRL < MRL < MRL < MRL

Pyrimethanil < MRL < MRL < MRL < MRL < MRL < MRL

Cyprodinil < MRL < MRL < MRL < MRL < MRL < MRL

Diazinone < MRL < MRL < MRL < MRL < MRL < MRL

Pyrazophos < MRL < MRL < MRL < MRL < MRL < MRL

Chlorpyrifos < MRL < MRL < MRL < MRL < MRL < MRL

Methamidophos, carbendazim, pyrimethanil, diazinone

and chlorpyrifos (all 100 ppm) were purchased from Chem

Service. Thiabendazole (1000 ppm), cyprodinil (100 ppm) and

pyrazophos were purchased from Ultra Scientific. Triphenyl


Wang and Telepchak

cleaned extract was collected in a 2-mL autosampler vial and

10 µL of the 5 ppm TPP (IS) solution was added. For both

cleanup methods, about half the 1-mL portion of the red wine

extract was adsorbed onto the sorbents in the dSPE tube or

mini-cartridge.

Instrumental:

GC–MS: An Agilent 6890 GC system coupled with a model

5975C single-quadrupole mass-selective detector (MSD,

Agilent) was used in this study for the acquisition of full-scan

chromatograms of extracts that were prepared using the

different cleanup methods. The GC system was equipped

with a 30 m × 0.25 mm, 0.25-µm df Rtx-5MS capillary

column integrated with a 10-m guard column (Restek). A

splitless liner with dimensions of 4 mm × 6.5 mm × 78.5 mm

(i.d. × o.d. × L) packed with deactivated glass wool (UCT)

was used to introduce the extract onto the GC column.

Splitless injections (1 µL) at 250 °C were made with a 50-mL/

min split vent at 1 min. Ultrahigh-purity helium at a constant

flow rate of 1.2 mL/min was used as the carrier gas. The oven

temperature was initially held at 40 °C for 1 min; ramped at

10 °C/min to 300 °C and then held for 3 min. The total run

time was 30 min with data acquisition beginning at 4 min. The

detector interface, ion source, and quadrupole temperatures

Figure 3: Full-scan chromatograms of red wine extracts

cleaned up with (a) dSPE A, (b) dSPE B, (c) dSPE C, (d) dSPE D

and (e) rapid mini-cartridge filtration. 100

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Time (min)

9 10 11 12 13 14 15

0.13 0.52 1.14 1.44 1.82

2.25

2.75

2.88

2.84

2.782.81

2.91

2.993.17

3.553.94 4.18 4.78 4.97

6.46

5.05 5.12 6.27 6.71

6.91

6.87

6.80

5.42

5.54 5.85 6.48 6.89 7.61 8.13 8.33 9.00 9.50 9.7710.69

11.74

11.78

11.49

11.92

11.55

11.23

11.09 11.73 12.20 12.83 13.72 14.0714.63

11.93 12.59 13.1813.51

13.42

14.0714.45

12.30

12.24

Pyrazophos

Chlorpyrifos

12.96 13.2213.7314.3714.83

Diazinone

12.76 13.0513.4914.0514.4015.09

12.02 12.6513.24 13.83 14.44 14.82

10.99

11.44

5.83 6.607.13 7.36 8.02 8.21 8.58 9.51 9.71 10.79 10.91

10.43

7.38 7.79 7.92 8.52 9.26 9.57 10.07 10.63 10.90

Thiabendazole

Pyrimethanil

Cyprodinil

TPP (IS)

Carbendazim

6.48 6.52

Methamidophos

2.32

Figure 4: Chromatogram of red wine sample 1 fortified with

pesticides (10 ng/mL).

extract was loaded using a nonsterile latex-free syringe with

a Luer-lock tip (VWR), the loaded syringe was attached to

the mini-cartridge and the extract was pushed through in

a slow, drop-wise fashion. The first 0.5-mL portion of the

11www.chromatographyonline.com

Wang and Telepchak

were set at 280 °C, 250 °C and 150 °C, respectively.

Chromatograms of the red wine extracts were obtained in

full-scan mode with a scanning range of 35–700 amu.

LC–MS–MS: An Accela 1250 LC system coupled to a TSQ

Vantage triple-quadrupole MS system was supplied by

Thermo Fisher Scientific. A PAL autosampler (CTC Analytics)

was equipped for automated sample injections. Xcalibur

(version 2.1) software (Thermo Fisher Scientific) was used for

data acquisition and processing. The separation of the eight

target pesticides was performed on a 100 mm × 2.1 mm,

3-µm dp Sepax HP-C18 column with a 20 mm × 2.1 mm,

3-µm dp Restek C18 guard column. The column temperature

was maintained at room temperature (~20 °C). The injection

volume was 10 µL at 15 °C. Mobile-phase A was 0.1% formic

acid in Milli-Q water (EMD Millipore), and mobile-phase B

was 0.1% formic acid in methanol. A flow rate of 200 µL/min

was used. The gradient programme was as follows: 5% B for

1 min, 5–50% B over 2 min, 50–95% B over 5 min, 95% B for

6 min, 95–5% B in 0.2 min, and 5% B for 2 min.

Tandem MS was operated with heated electrospray

ionization (HESI) in positive mode, and the conditions were

as follows: spray voltage: 3000 V; sheath gas: nitrogen at

40 psi; auxiliary gas: nitrogen at 10 psi; ion transfer capillary

temperature: 350 °C; collision gas: argon at 1.5 mTorr; Q1

peak width: 0.2 Da FWHM (full width half maximum); Q3 peak

width: 0.7 Da FWHM. Optimization of the MS–MS transitions

(collision energies and S-Lens RF values) was performed

individually for each pesticide by infusing 1-μg/mL standard

in acetonitrile at 10 µL/min with 50:50 (v/v) mobile phases

A and B at a flow rate of 200 µL/min. The two most intense

and characteristic precursor–product ion transitions were

chosen for selected reaction monitoring (SRM). Acquisition

was divided into three segments (0–5 min, 5.01–11 min and

11.01–16 min) based on the retention times of the target

analytes. The retention times, precursor and product ions,

collision energies, S-Lens RF values and dwell times are

listed in Table 1.

Results and DiscussionEvaluation of Cleanup Efficiency: Red wine extracts that

underwent cleanup with the rapid mini-cartridge filtration,

and with four traditional dSPE tubes containing 110 mg

anhydrous MgSO4 and different amounts of PSA were

compared as outlined in the “Cleanup” section. Red wine

extracts without any cleanup, with dSPE cleanup A, B, C

and D, and with rapid mini-cartridge filtration are illustrated

in Figure 2. The red colour in the extracts decreases as the

amounts of the PSA sorbent increases in the dSPE tubes. The

samples analysed with dSPE (D) and the rapid mini-cartridge

containing the same amounts of magnesium sulphate and

PSA yielded a similar colourless appearance. Large amounts

of PSA (180 mg) contributed to the efficient removal of

various matrix coextractives such as organic acids, sugars,

phenols and pigments in the red wine. Figure 3 shows the

full-scan chromatograms of four extracts that underwent

cleanup with traditional dSPE [Figures 3(a)–3(d)] and one

with mini-cartridge filtration [Figure 3(e)]. The chromatogram

of rapid mini-cartridge filtration was slightly cleaner than that

with dSPE (D). In addition, the rapid mini-cartridge filtration

approach based on the filter-and-clean concept was simpler

and faster than the dSPE approach, and was therefore

selected for the cleanup of the red wine samples in this study.

Matrix Matched Calibration, LOD and LOQ: Calibration

curves were obtained by analysing matrix matched standards,

which were prepared by spiking appropriate amounts of the

2 ppm pesticide working solution into blank red wine extracts

after cleanup with rapid mini-cartridge filtration. Six matrix

matched calibration standards at concentrations of 2 ng/mL,

10 ng/mL, 40 ng/mL, 100 ng/mL, 200 ng/mL and 400 ng/

mL were analysed. The linear dynamic ranges, regression

equations and correlation coefficients (R2) are listed in

Table 2.

The limit of detection (LOD) and limit of quantification

(LOQ) are the concentrations that give signal-to-noise

ratios (S/N) of 3 and 10, respectively. In this study they were

estimated according to the S/N values of the lowest matrix

matched calibration level of 2 ng/mL. The calculated LOD

ranged from 0.01 ng/mL to 0.40 ng/mL and the LOQ ranged

from 0.05 ng/mL to 1.33 ng/mL (see Table 2). The minimum

reporting limit (MRL) in this study was set at the lowest

calibration level of 2 ng/mL.

Chromatograms: A chromatogram of red wine sample 1

fortified with 10 ng/mL of the target pesticides is shown in

Figure 4. All the peaks, except for methamidophos, were

sharp and offered reliable quantification. The satisfactory

separation of the eight pesticides is also evident in the

chromatogram. This allowed the data acquisition to be

divided into three segments which ensured the optimal

performance of the MS system, including dwell time

(scanning speed) for each analyte.

Accuracy and Precision Data: Red wine samples fortified with

10 ng/mL, 50 ng/mL and 100 ng/mL of the target pesticides

were extracted with QuEChERS and cleaned up using the rapid

mini-cartridge filtration procedure. The recovery and relative

standard deviation (RSD) data are listed in Table 3. Recoveries

of 81.6–112.2%, with an overall recovery of 97.0%, were

achieved with this simple, rapid and easy-to-use procedure.

RSDs of four replicates for each of the three spiking levels were

less than 10.8%, which indicated that this method is suitable for

the determination of pesticide residues in red wine samples.

Application to Red Wine Samples: Six red wine samples

were tested using the newly developed and validated method.

The results of the red wine samples tested are listed in Table 4.

Several of the pesticides were detected at concentrations less

than the method MRL. Carbendazim was the only pesticide

detected above the MRL. The detected carbendazim

concentrations were at 10.2 ng/mL, 8.7 ng/mL and 2.3 ng/

mL in samples 4, 5 and 6, respectively. However, the detected

concentrations are much lower than the European (0.5 mg/kg) or

Japanese (3 mg/kg) regulated levels in grapes.

ConclusionsA simple, fast, novel and effective cleanup method for red

wine samples was successfully developed. Pesticide residues

in red wine samples were extracted using the nonbuffered

QuEChERS procedure. Cleanup was carried out by passing

1 mL of the red wine extract through a mini-cartridge

containing magnesium sulphate and PSA. The magnesium

sulphate adsorbed residual water remaining in the acetonitrile

extract, and the PSA sorbent retained matrix coextractives,

including organic acids, sugars, phenols and pigments. The

cleanup method based on a filter-and-clean concept took less

than 1 min per sample, thereby providing higher throughput

than the traditional dSPE procedure. Cleaned extract was


Wang and Telepchak

injected directly into an LC–MS–MS system for analysis. The

analytical run required only 16 min and the target pesticides

were chromatographically well resolved.

Good sensitivity and selectivity were achieved for the clean

extracts obtained using the rapid mini-cartridge filter and

LC–MS–MS detection. Good linearity, low LODs and LOQs

and satisfactory accuracy and precision data were obtained,

indicating that this method was suitable for the analysis of

pesticide residues in red wine samples. Six commercially

available red wine samples were tested with the newly developed

and validated method. Carbendazim was present in three red

wine samples, although the detected concentrations were far

below the European and Japanese regulated levels in grapes.

AcknowledgmentsThomas August and Lisa Snyder are acknowledged for the

arrangement of the UCT products needed for this study.

Catherine Messinger and Evelyn Scanlon are thanked for

providing red wine samples. Dr Brian Kinsella is thanked

for proofreading the manuscript and providing valuable

discussions and suggestions.

References(1) http://www.oiv.int/oiv/info/enoivbilan2010.

(2) http://www.mayoclinic.com/health/red-wine/HB00089.

(3) Off. J. Eur. Union L70 (2005).

(4) Off. J. Eur. Union L58 (2008).

(5) http://www.pan-europe.info/Resources/Briefings/Message_

in_a_Bottle.pdf.

(6) S. de Melo Abreu, P. Caboni, P. Cabras, V.L. Garau and A. Alves,

Anal. Chim. Acta 291, 573–574 (2006).

(7) J. Oliva, S. Navarro, A. Barba and G. Navarro, J. Chromatogr. A

833, 43–51 (1999).

(8) J.J. Jimenez, J.L. Bernal, M.J. del Nozal, L. Toribio and E. Arias, J.

Chromatogr. A 919(1), 147–156 (2001).

(9) J.F. Wang, L. Luan, Z.Q. Wang, S.R. Jiang and S.P. Pan, Chinese J.

of Anal. Chem. 35(10), 1430–1434 (2007).

(10) A. Economou, H. Botitsi, S. Antoniou and D. Tsipi, J. Chromatogr. A

1216(31), 5856–5867 (2009).

(11) Y. Hu, W.M. Liu, Y.M. Zhou and Y.F. Guan, Se Pu 24(3), 290–293

(2006).

(12) J. Wu, C. Tragas, H. Lord and J. Pawliszyn, J. Chromatogr. A 976,

357–367 (2002).

(13) P. Plaza Bolanos, R. Romero-Gonzalez, A. Garrido Frenich and J.L.

Martinez Vidal, J. Chromatogr. A 1208, 16–24 (2008).

(14) P. Vinas, N. Aguinaga, N. Campillo and M. Hernandez-Cordoba, J.

Chromatogr. A 1194(2), 178–183 (2008).

(15) M. Anastassiades, S.J. Lehotay, D. Stajnbaher and F.J. Schenck, J.

AOAC Int. 86(2), 412–431 (2003).

(16) S.J. Lehotay, Methods in Molecular Biology 747, 65–91 (2011).

(17) S.C. Cunha, S.J. Lehotay, K. Mastovska, J.O. Fernandes, M. Beatriz

and P.P. Oliveira, J. Sep. Sci. 30(4), 320–632 (2007).

(18) M. Whelan, B. Kinsella, A. Furey, M. Moloney, H. Cantwell, S.J.

Lehotay and M. Danaher, J. Chromatogr. A 1217(27), 4612–4622

(2010).

(19) http://www.whatsonmyfood.org/food.jsp?food=GR.

(20) http://www.m5.ws001.squarestart.ne.jp/foundation/fooddtl.php?f_

inq=10800.

(21) http://ec.europa.eu/sanco_pesticides/public/index.

cfm?event=commodity.resultat.

Xiaoyan Wang and Michael J. Telepchak are with

UCT in Bristol, Pennsylvania, USA. Direct correspondence

should be directed to: [email protected]


LCGC Announces Major New Partnership in China


LCGC is pleased to announce a major new partnership in China, with Sepu.net, the China Chromatography Network.

The China Chromatography Network (www.Sepu.net) was established as a chromatography

portal in 1999. Today, it is the largest industry website and the most infuential

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Through this partnership, LCGC Asia Pacifc will now be distributed to more

than 130,000 practicing chromatographers in Greater China.

LC•GC Asia Pacifi c March 201314

HISTORY OF CHROMATOGRAPHY

Kate Yu: What brought you into the

fi eld of mass spectrometry (MS)?

Fred McLafferty: A crazy

coincidence! After a PhD at Cornell

University, (Ithaca, New York, USA)

and a postdoc at the University of

Iowa, (Iowa, USA), I arrived at the Dow

Chemical Co., (Midland, Michigan,

USA), in 1950 for an interview in their

organic chemistry research laboratory.

However, Dow also interviewed me for

their spectroscopy laboratory in the

MS group of Vic Caldecourt and two

instrument operators. Vic had made

the MS analyses so popular within the

company that Dow wanted a chemist

for the increased sample load while

he concentrated on maintaining and

improving the cranky instrumentation,

at which he was terrific. No one,

not even me, understood why I

took the job with absolutely no prior

knowledge (1).

KY: You were in World War II

and were awarded the Combat

Infantryman Badge. What were the

most remarkable experiences you

remember during the war?

FM: I went on active duty in April

1943, just after finishing a BS in

chemistry and mathematics at the

University of Nebraska (Nebraska,

USA). A month before the war ended

in Europe, our 2nd Battalion, 253rd

Infantry, captured a major remaining

German ammunition depot in a fierce

battle against an elite SS unit that

promised Hitler “no retreat”. The

battalion members were awarded

a Presidential Unit Citation. During

this action platoon sergeant John

R. Crews of my rifle company was

awarded the Congressional Medal of

Honor for personally saving part of

another company that was trapped. I

was wounded earlier the same day.

KY: Why did you come back to the

fi eld of chemistry after the war?

FM: I was offered a battlefield

commission from sergeant to officer

rank, so I had a “career choice”, but

I doubt that such a career would

allow the “fun participation” that I am

having at my present age.

KY: Looking back at your career,

who was the most infl uential

person for you?

FM: Professor Franklin A. Long,

who was at the Cornell University

Chemistry Department from 1937 to

1999, was a very special scientific

role model and friend during my

graduate work from 1947 to 1949,

and after I joined the faculty in 1968.

He was a member of the National

Academy of Sciences, Assistant

Director of the US Arms Control and

Disarmament Agency, and on the

Science Advisory Committees for

three US Presidents. But by far the

most important person in my life for

65 years has been my wonderful wife:

Elizabeth “Tibby” Curley McLafferty.

KY: Tell us about your experiences

at Dow Chemical. How did you

develop the GC–MS instrument?

FM: Dow’s spectroscopy laboratory

was one of the best in “modern”

high information methods including

direct reading atomic emission

spectroscopy (AES), X-ray diffraction

(XRD), infrared (IR), nuclear

Man of the Masses“MS: The Practical Art” Editor, Kate Yu, spoke to Fred McLafferty about his pioneering career in mass spectrometry (MS).

Bendix time-of-flight mass spectrometer at the Dow Chemical Company with

Roland Gohlke (foreground) and Fred McLafferty. Published with permission of

Fred McLafferty.



magnetic resonance spectroscopy

(NMR) and MS. Dow was also

unusually supportive of any new MS

applications and related research. At

a 1954 Gordon Research Conference,

friend Steve DalNogare of duPont and

H.N. Wilson from Imperial Chemistry

Industries (ICI) (Manchester, UK),

told me about gas chromatography

(GC) with full details on how to build

one. Soon after, Roland Gohlke,

who worked with me, developed an

improved version to solve problems

in Dow laboratories and plants. For

example, Roland put the column,

a 25-ft copper tubing coil, in a 2L

Dewar for better temperature control

and often used the detergent “Tide”

as a column packing.

In addition, my friends Bill Wiley

and Dan Harrington at nearby

Bendix Research Labs were just

commercializing their time-of-flight

(TOF) MS instrument (10K spectra/s),

and were happy to let us try coupling

our GC to the TOF in February 1956.

It was very exciting to see on the

output oscilloscope the rise and fall

of unit-resolution spectra of acetone,

benzene and toluene, the first

organic compounds run on their TOF

instrument (2).

KY: How did you discover the

famous McLafferty Rearrangement

and what sort of impact did it have

on mass spectrometry?

FM: The infrared capabilities of

Dow’s spectroscopy laboratory

were world-class, helped by the

spectroscopy team’s extensive efforts

to collect a large spectral database of

organic compounds. Consequently,

during any spare MS instrument time

we had, we continued this to build

up an MS spectral database using

the extensive sample collection

available, giving us the mass spectra

of many compound classes. This

made it possible by 1956 to show (3) a

general correlation of novel hydrogen

rearrangements, although individual

cases had previously been reported.

At that time Dow sent me to the

Boston area to set up a corporate

laboratory for basic research.

Although for my personal research I

had no MS instrument, the structural

diversity of our MS database was

already unique, allowing extensive

details of single and double

hydrogen atom rearrangements to be

Time-of-flight mass spectra (oscillograph display) typical of Dow's early GC–MS

capabilities. (a): Mass spectrum of methane (CH4), peaks at m/z = 12–17 (16 tallest).

(b): Mass spectrum of acetone (C3H

6O, MW 58), peaks at 13–59 (contains impurity

peaks, for example, H2O). Published with permission of Fred McLafferty.

m/z = 16

(a)

(b)

Fred McLafferty (far left); Roman Zubarev, Professor of Medical Proteomics

in the Department of Medical Biochemistry and Biophysics at the Karolinska

Institutet (Stockholm, Sweden). Roman also directs the institute’s large

LC–MS proteomics facility; Dr Gary Valaskovic, co-founder and CEO of New Objective

(Woburn, Massachusetts, USA); Neil Kelleher, Professor in Chemistry, Molecular

Biosciences and the Feinberg School of Medicine of Northwestern University

(Chicago, Illinois, USA). Neil also directs the large LC–MS proteomics facility

in Chicago. The photo was taken by Dr Susan Weintraub at the ASMS meeting

(Philadelphia, USA) June 2009. Published with permission of Fred McLafferty.


Fred McLafferty obtained a

BS degree at the University of

Nebraska (Nebraska, USA)

in 1943. He served in France

and Germany with the 253rd

Infantry Regiment of the 63rd

Division and was awarded

the Combat Infantry Badge,

Purple Heart, 5 Bronze Star

Medals and Presidential Unit

Citation. McLafferty then

obtained an MS at Nebraska,

a PhD at Cornell University

(Ithaca, New York, USA),

and postdoctorate at the

University of Iowa (Iowa,

USA), before joining the Dow

Chemical Company in 1950.

At Dow, the mass

spectrometry laboratory he

worked in was one of the

few studying non-petroleum

organic compounds and was

a pioneer in collecting and

correlating reference electron

ionization (EI) mass spectra.

Here he developed the new

“radical-ion” chemistry of

these spectra, including the

McLafferty Rearrangement.

With many contributions by

others, this new chemistry

was key to the acceptance

of MS as a major technique

for molecular structure

characterization. His 1966

book, Interpretation of Mass

Spectra, is still used widely

in its 4th edition, and his

2009 9th edition of Registry

of Mass Spectral Data (1st

edition 1969) is the world’s

largest mass spectral library,

containing 663,000 different

EI mass spectra. In addition to

this, he has co-authored over

500 publications.

McLafferty’s laboratory

introduced and built

gas chromatographs for

Dow in 1954. In 1956 he,

Roland Gohlke and Bendix

researchers performed the

first GC–MS with the Dow GC

and Bendix time-of-flight (TOF)

mass spectrometry (MS). From

1964 to 1968 his laboratory at

Purdue developed collisionally

activated dissociation; ion

structural characterization by

MS–MS; MS–MS of peptide

mixtures; and computer

data acquisition/reduction

and MS instrument control.

Pioneering work at Cornell

included computer

identification of unknown mass

spectra (Probability Based

Matching); LC–MS interfacing;

neutralization—reionization;

high-resolution MS–MS protein

characterization

(top-down proteomics);

electron capture

dissociation; IR gaseous

ion photodissociation

spectroscopy; and

characterization of gaseous

protein conformers.

Among his many honours

are the U.S. National

Academy of Sciences (1982);

American Academy of Arts

and Sciences; Italian National

Academy of Sciences XL.

Am. Chem. Soc. Awards in

Chemical Instrumentation,

Analytical Chemistry & Mass

Spectrometry; J.J. Thomson

Gold Medal (Intern. MS

Soc.); Robert Boyle Gold

Medal (Roy. Soc. Chem); J.

Heyrovsky Medal (Czech

Acad. Sci.); G. Natta Gold

Medal (Italian Chem. Soc.);

Torbern Bergman Medal

(Swedish Chem. Soc.);

Disting. Contrib. Mass Spectr.

(Am. Soc. Mass Spectr.);

and Lavoisier Medal (French

Chem. Soc.).

Dr and Mrs McLafferty

have five children and ten

grandchildren.

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11717



established (4). This helped to show

the key role of odd-electron ions in

MS ion dissociation mechanisms

and convince organic chemists that

mass spectra were based on real

chemistry.

KY: Why did you go back to

university while having a fantastic

career in industry at the time?

FM: The success of Dow’s new basic

research laboratory depended on

selling its research capabilities to

the academic community, as well as

to meet Dow objectives. New hires

found a novel research opportunity

with industrial level support. We did

recruit great people, including later

Nobel Laureate George Olah. But in

trying to sell my industrial research to

academe, they sold me on academia.

In 1964 I went to Purdue University

(West Lafayette, USA), as Professor of

Chemistry.

KY: You’ve made many

great contributions to mass

spectrometry over the years.

What do you think is your greatest

contribution?

FM: In 1950 MS was dominated by

measurements of isotope ratios, atomic

weights and hydrocarbon mixtures,

while my main research efforts since

then have been in “molecular mass

spectrometry”. The current reversal in

dominance came through a long series

of developments that our research

contributed to. These included an

improved understanding of new ion/

radical chemistry; advances in

GC–MS, LC–MS, MS–MS

instrumentation; advances in

techniques, such as collisional

activated dissociation, neutralization–

reionization and electron capture

dissociation; improved computer data

acquisition, reduction and identification

using collected reference data (5);

and developments in top-down

characterization of biomolecules and

gaseous protein conformers (1). It was

the combined efforts of the unusually

cooperative researchers of many MS

laboratories that made these advances

possible.

KY: Among all the awards you have

received, which one was the most

remarkable award to you?

FM: Al Bard and I were elected to the

US National Academy of Sciences in

1982; before that, the only analytical

chemists elected were I.M. “Pete”

Kolthoff (1958) and Charlie Reilley

(1977). Best of all, I have also been

awarded a most remarkable family of

five children and ten grandchildren.

Kate Yu is Senior

Manager, Business

Operations, in the

Pharmaceutical and Life

Sciences department

at Waters Corporation

(Milford, Massachusetts,

USA). Her current

focus is on generics

and biosimilars in

pharmaceutical QC and

manufacture. She has

also been involved in

the field of traditional

medicine for many

years.

Kate joined Waters in

1998 and has spent her

time as an application

scientist. She has a

wealth of experience

in applying LC–MS

technologies into a

variety of applications,

such as metabolite

identification,

metabolomics,

quantitative bioanalysis,

natural products and

environmental.

Before joining Waters,

Kate worked at Sun

Chemical Inc. (New

Jersey, USA), where

she established and

managed an analytical

laboratory that provided

all the analytical

services for the

packaging ink division

of the company.

Kate received her PhD

in Analytical Chemistry

from the University of

Cincinnati (Cincinnati,

Ohio, USA). She studied

for her MS in Chemical

Biology at the Stevens

Institute of Technology

(Hoboken, New Jersey,

USA), and for her BS in

Pharmacy at Shenyang

Pharmaceutical

University (Shenyang,

China).

Kate currently

serves on the Board

of Directors for the

Chinese American

Society for Mass

Spectrometry. She is

the editor of LC•GC’s

regular column MS: The

Practical Art.

KY: What major challenges remain

in mass spectrometry? Which

ones do you think the younger

generation should focus on?

FM: The huge growth of MS removes

its applications further from the basic

research that makes more of this

growth possible. Education awareness

requires updated textbooks, “short

courses”, consultants and, most

importantly, improved communication

in our field. The positions of those

supporting “bottom-up” versus

“top-down” approaches appear to

have shifted little in the last decade.

KY: Where do you think the future

of mass spectrometry lies?

FM: As in 1950, I believe that the future

is still in “molecular mass spectrometry”.

But now we see more clearly the amazing

enhancements possible with far greater

mass range (megadalton+) and resolving

power, coupled separation techniques (for

example, MS with chromatography, ion

mobility) and computerized automation.

These can be applied to an even broader

range of critical areas of basic knowledge

and practical problems.

KY: Is there any advice you

would give to young scientists

embarking on a career in analytical

chemistry?

FM: Young scientists should please

note that analytical chemistry overall has

a great future. In 1984 I proposed the

Analytical Criteria of “6 Ss”: Specificity,

Sensitivity, Speed, Sampling, Simplicity

and $. Improve one or more of these in

an old method, or find a new method

with potential in these criteria — solving

problems has great rewards!

KY: Do you have hobbies outside

your work?

FM: “Hobby” is not a fair descriptor, but

I am lucky to spend most of my time

outside MS with my extended family.

References(1) F.W. McLafferty, Annu. Rev. Anal. Chem. 4,

1–22 (2011).(2) R.S. Gohlke and F.W. McLafferty, J. Am.

Soc. Mass Spectrom. 4, 367–371 (1993).(3) F.W. McLafferty, Anal. Chem. 28,

306–316 (1956).(4) F.W. McLafferty, Anal. Chem. 31, 82–87

(1959).(5) F.W. McLafferty, Registry of Mass Spectral

Data (and Registry combined with NIST), 9th Ed., Wiley-Blackwell: Hoboken, N.J., USA, (2009).

(6) F.W. McLafferty, Science 226, 251–253 (1984).

LC•GC Asia Pacià c March 201318

SAMPLE PREPARATION

PERSPECTIVES

In her February 2012 column instalment

titled “It’s All About Selectivity,” guest

columnist Diane Turner introduced

the topic of how selectivity can be

incorporated throughout the sample

analysis cycle (1). Figure 1, borrowed

from her article, nicely illustrates the

workflow in a typical sample analysis.

In most analytical processes, the

chemist is looking for one or perhaps a

few analytes of interest, often in a very

complex matrix. Having an analytical

method showing sufficient selectivity

to analyse those few compounds of

interest with the precision and accuracy

required at the concentration level

encountered is the desired outcome of

method development. The selectivity

can be achieved anywhere within

the analytical cycle (Figure 1) during

sampling, sample preparation, sample

introduction, analyte separation, at

the detector or even during data

analysis. If the analytes of interest can

be determined with good sensitivity,

the presence of compounds from

the sample matrix can be tolerated

as long as those interferences

do not cause harm (short-term or

long-term) to the analytical instrument

or column or if determined to be

harmful, they can easily be removed.

An example of the latter could be

backflushing after each analysis

to remove high-molecular-weight

contaminants trapped at the head of

a gas chromatography (GC) column.

In Turner’s column (1), she gave very

nice examples of how selectivity can be

achieved at each step of the analytical

cycle for GC.

Having less selectivity in one portion

of the analytical cycle can be made

up for by having greater selectivity

in another portion of the analytical

cycle. For example, if an analyst has

only a fixed-wavelength UV detector

in his or her high performance liquid

chromatography (HPLC) instrument or

a thermal conductivity (TCD) or flame

ionization detector (FID) for the GC

system, there may not be sufficient

detector selectivity to provide the

necessary overall method selectivity to

measure an analyte of interest without

interference from undesired sample

components. Therefore, additional

sample preparation or finding a

separation column that provides

more selectivity during the separation

may be required to make up for the

limitations in the detector. In these

cases, the analyst may spend a great

deal of time and energy performing one

or more sample preparation steps or

optimizing the selectivity of the column

and mobile phase system (HPLC)

to rid it of potential interferences.

On the other hand, if one has a very

sensitive and selective detector, then

perhaps spending a great deal of time

optimizing the sample preparation

or the analytical separation is

unwarranted.

Because achieving selectivity for the

separation column is not an easy task to

predict, sample preparation often gets the

brunt of the job to remove interferences

from the sample of interest. It is

sometimes unfortunate to burden analysts

with this job, but there are time-proven

sample preparation techniques

available. However, with the advent

and widespread use of tandem mass

spectrometry (MS–MS) for both HPLC

and GC with its high degree of selectivity

and sensitivity, sample preparation as

well as the chromatographic separation

can sometimes be simplified as long

as any interferences carried over from

the sample matrix do not interfere with

the separation or detection process. We

term this simplified sample preparation

process just enough sample preparation.

This just enough sample preparation

process doesn’t always provide the

cleanest extract from the sample as

more rigorous approaches such as

multimodal solid-phase extraction

(SPE) or liquid–liquid back extraction

might achieve but as long as the

extractables do not harm the separation

or detection (and, of course, the column

or instrument), that’s okay. In reality, the

sample preparation time can be greatly

reduced as long as the final outcome

meets the needs of the analyst.

Although the mass spectrometer still

“Just Enough” Sample Preparation: A Proven Trend in Sample AnalysisRonald E. Majors, Agilent Technologies, Wilmington, Delaware, USA.

Sample preparation (and to a lesser extent data analysis) has often been considered the rate-determining step and error-prone part of an analytical method. If selectivity can be achieved in other portions of the analytical cycle to meet the needs of the analyst, then the burden placed on sample preparation is decreased. The concept of “just enough” sample preparation is presented here and relies heavily on recent advances in tandem mass spectrometry detection that provides enhanced sensitivity and selectivity, which was unavailable in the past. Even so, more sophisticated sample preparation protocols may still be required, especially if ion suppression or enhancement result from coeluted interferences.


SAMPLE PREPARATION PERSPECTIVES

resulting in poorer analytical precision

and accuracy. If one or two steps meet

the needs of the method that may be

sufficient, but in some cases additional

sample preparation steps may be

needed to get rid of interferences.

The need to eliminate or minimize

interferences is no greater than that

required for liquid chromatography–

mass spectrometry (LC–MS) and

LC–MS–MS (see below).

Figure 3 shows a pictorial

representation of the just enough

sample preparaton concept that actually

applies to the entire analytical cycle,

but is emphasized for the sample

preparation portion. It is here that many

workers are faced with achieving the

bulk of their selectivity enhancement.

Ideally, in an analytical method one

always wants to achieve the best

result with the least amount of effort

and investment. On the other hand,

the actual data requirement may not

require the ideal result but rather an

acceptable result. For example, in

screening hundreds of urine samples

for the presence of drugs of abuse most

samples are negative. Thus, a qualitative

analytical method may be sufficient to

rule out the presence of an illicit drug.

However, if an illegal drug is spotted

during the screening test, then a more

careful and perhaps more sophisticated

look at a positive sample is required for

quantitative analysis.

There are many other factors that

may influence the choice of the sample

preparation techniques used to provide

just enough cleanup. An analyst’s skill

and knowledge are important. The

availability of instrumentation, chemicals,

consumables and other equipment;

the time available to develop a method

and to perform the tasks at hand; the

complexity and nature of the matrix; the

analyte concentration level and stability;

the required sample size; the cost per

sample (budget); and the safety of the

sample preparation technique are just a

few of the many considerations that must

be taken into account. It is the balance

of all of these and other considerations

that come into play.

Ion Suppression in LC–MS and LC–MS–MS An area of potential problem in the just

enough sample preparation approach

is unique to LC–MS and LC–MS–MS.

The impact of unextracted matrix

compounds that may coelute with

represents a much higher priced

detector than a UV or flame ionization

detector, many laboratories are finding

them to be a cost-effective way to

enhance and speed up their analyses,

thereby improving overall productivity

and lowering costs. Of course,

less-expensive selective detectors such

as fluorescence in HPLC and electron

capture in GC still allow the practice

of just enough sample preparation

provided the analytes do not need

derivatization.

The concept of just enough

sample preparation does not imply

one is cutting corners or that more

sophisticated protocols are not

required. It really represents a

continuum of sample preparation

procedures as depicted in Figure 2.

This figure represents just a few of the

many sample preparation methods

that are in widespread use. Starting

at the top of the figure with filtration,

centrifugation and “dilute and shoot”,

moving down the sample preparation

protocols become more selective and

more complex, sometimes requiring a

greater deal of effort and multiple steps

to achieve just enough cleanup to meet

the analytical needs. Minimizing the

number of sample handling steps in

any analytical technique is desirable

since the more times the sample is

transferred, the greater the chance of

analyte loss (or modification), thereby

Sampling and

sample collection

Sample preparation

Sample introduction

Analyteseparation

Analytedetection

Dataanalysis

GC or HPLCCarrier gas (GC)

Mobile phase pump (LC)

Simpler, genericmethodology

Methodology

• Filtration• Direct Injection

• Centrifugation• Dilute and shoot• Sonication• Lyophilization• Protein precipitation

• Distillation

• Soxhlet extraction• Solid-phase microextraction• Supported liquid extraction• Liquid–liquid extraction• Solid-phase extraction• QuEChERS• Turbulent +ow chromatography• Derivatization• Column switching and heart cutting• Immunoaf,nity sorbents• Molecularly imprinted polymers

• Dialysis and ultra,ltration• Liquid–solid extraction and pressurized +uid

extraction

More complicatedmethodology

Greater selectivityOptimal sample clean up

Less selectiveMinimal sample cleanup

Figure 1: Steps in the analytical cycle. Adapted from reference 1.

Figure 2: Just enough sample preparation represents a continuum of methodologies.

LC•GC Asia Paciàc March 201320


syringe pump connected to the column

of fluid. A drop in constant baseline after

a blank sample extract is injected into

the LC system indicates suppression in

ionization of the analyte because of the

presence of the interfering material.

Although beyond the scope of this

article, there are a number of strategies

for reducing ion suppression. Among

them are changing the ionization

mode (such as switching to negative

ionization), sample dilution or volume

reduction, reducing the flow rate,

improving chromatographic selectivity or

performing better sample preparation.

In the latter case, just enough sample

preparation to meet the analytical needs

may be the use of SPE, liquid–liquid

extraction or even additional techniques.

The use of formic acid rather than

trifluoroacetic acid in the HPLC

mobile phase can also help. For more

information, a simple discussion of ion

suppression effects and their elimination

was published earlier (2).

Examples of Just-Enough Sample Preparation Many sample preparation

methodologies have already been

published in earlier instalments of

“Sample Preparation Perspectives”.

Figure 2 provides a number of sample

preparation protocols that could

qualify as just enough procedures. As

mentioned earlier, the fewer sample

preparation steps in an analytical

method the less chance of errors, better

analyte recovery and less time spent

handling samples. However, as one

proceeds down Figure 2, just enough

may require more sophisticated sample

preparation processes.

Let’s look at a few examples of

sample preparation procedures that may

qualify as just enough and see if they

provide acceptable results. In recent

years, for the determination of drugs

and their metabolites in biological fluids

such as plasma, many pharmaceutical

companies have switched their sample

preparation to protein precipitation

(Figure 4) and reversed-phase HPLC

analysis but using a more selective,

sensitive LC-triple-quadrupole MS–MS

detector with multiple reaction monitoring

(MRM) at defined transitions. The first

example shows the direct analysis of

fluticasone proprionate in human plasma

using an LC–triple quadrupole MS

system. This compound is a synthetic

steroid of the glucocorticoid family of

the analytes of interest may end up in

the ionization chamber of the mass

spectrometer. Ion suppression in MS is

one form of a matrix effect that impacts

analyte ionization in the MS source. Most

often a loss in response occurs; hence

the term ion suppression is generally

used. Ion suppression effects impact

reproducibility and signal strength. They

are most noticeable when trace analytes

are in the presence of complex matrices

such as biological fluids. In some cases,

an increasing response of the desired

analyte may occur; ion enhancement or

a stronger-than-expected signal results.

Ion suppression results from the

presence of less volatile compounds

that can change the efficiency of droplet

formation or droplet evaporation, which

in turn affects the amount of charged ion

in the gas phase that ultimately reaches

the detector. Materials shown to cause

ion suppression include salts, ion pairing

reagents, endogenous compounds,

drugs, metabolites and proteins. The

electrospray ionization detector (EID)

is strongly affected by the presence

of certain coeluted compounds.

Atmospheric pressure chemical

ionization detectors are also affected

by ion suppression but to a lesser

extent than the electrospray detector.

The presence of ion suppression can

be determined by the use of infusion.

The infusion experiment involves the

continuous introduction of the standard

solution containing the analyte of interest

and its internal standard by means of the

Effort and investment

Realistic(acceptable)

Unacceptable

Just enough

Ideal

Qu

ality

of

resu

lts

Sample 1) Add organic solvent and

2) Mix 3) Centrifuge or flter

4) Remove supernatant

5) Analyze supernatant, often after dry down and resuspension

Precipitated

Proteins

Protein insolution

Analyte

Otherinterferences

Figure 3: Striking the right balance in sample preparation.

Figure 4: Steps in protein precipitation.



drugs for treating allergic conditions.

When used as a nasal inhaler or

spray, medication goes directly to the

epithelial lining of the nose, and very

little is absorbed into the rest of the

body. Because of its low systemic

levels, a high sensitivity LC–MS assay is

required to determine its concentration

in human plasma. Figure 5 shows the

LC–MS results from a plasma protein

precipitation followed by dilute and shoot

using the MRM transition shown in the

figure caption. In this case, the dilute and

shoot method has more than adequate

sensitivity at the lowest calibration level of

5 pg/mL. Thus, dilute and shoot sample

preparation has an assay performance

well within the accepted regulatory

guidelines and was just enough to meet

the analytical needs.

A second example is also a protein

precipitation but considering an area

where ion suppression comes into

play. The presence of phospholipids

in plasma can cause ion suppression

if analytes of interest coelute in the

portion of a chromatogram where these

phospholipids appear. Phospholipid

MS–MS selectivity can be achieved

by considering the m/e 184 → m/e 184

transition indicative of phospholipid

and lysophosphatidylcholine elution.

Figure 6(a) shows a typical infusion

experiment result that is obtained

from protein-precipitated plasma,

followed by Captiva filtration (Agilent

Technologies, Delaware, USA). If the

drug or its metabolites were to coelute

with these compounds, ion suppression

may occur and the analytical results

would be jeopardized. Thus, in this

case the simple protein precipitation

sample preparation procedure may

not be enough to provide reliable data.

By performing the more complex SPE

[Figure 6(b)] or liquid-liquid extraction

[Figure 6(c)], the extract is now cleaner

and many of the phosphorus-containing

lipids are greatly reduced. To get the

best overall performance, an even more

sophisticated phospholipid reduction

may be achieved with a selective

SPE phase that removes the last

traces of phosphorylated compounds

[Figure 6(d)]. Luckily, a product called

Captiva NDLipids, which is a combined

membrane filtration and phospholipid

removal 96-well plate, performs both

operations at once and thus is a simple

just enough solution to this problem.

QuEChERS (quick, easy, cheap,

effective, rugged and safe) is a sample

preparation technique that was originally

developed for the extraction of pesticides

from fruits and vegetables (4). It is a

relatively simpler sample preparation

procedure with two steps: a salting out

partitioning extraction involving water

and acetonitrile with high concentrations

of salts such as sodium chloride,

magnesium sulphate and buffering

agents, and a dispersive-SPE step in

which an aliquot from the first step is

treated with various sorbents to remove

matrix compounds that could interfere

with subsequent LC–MS, LC–MS–MS,

GC–MS or GC–MS–MS analysis. The

technique has proven to be widely

applicable at trace levels for hundreds

of pesticides in a variety of matrices.

Standard protocols are available that

make it a generic sample preparation

procedure.

Recently, QuEChERS extraction has

expanded well beyond the pesticide

laboratory and has been used for many

matrices ranging from antibiotics in meat

and poultry, veterinary drugs in animal

feed, and environmental contaminants in

soil. In this example, using the protocol

in Figure 7, QuEChERS was used for

the extraction of polycyclic aromatic

hydrocarbons (PAHs) in fish. The PAHs

are a large group of organic compounds

included in the European Union and the

United States Environmental Protection

30

(a)

(b)

(c)

(d)

Co

un

ts (

X 1

06)

20

10

0

30

20

10

0

30

20

10

0

30

20

10

0

5 10 15 20

Time (min)

25 30 35

5.3

5.2

5.1

4.9

4.8

4.7

4.6

4.5

4.4

4.3

4.2

0.8

Acquisition time (min)

Ab

un

dan

ce (

X 1

01)

1.2 1.4 1.6 1.8 2.2 2.421

5

Figure 5: LC–MS analysis of futicasone proprionate in plasma. Shown is an ion

chromatogram (MRM transition 501.2→293.1) for 2.5 fg injected on-column with a

1-fg limit of detection. The standard curve was linear over the range of 5 pg/mL to 50

mg/mL. The plasma sample was precipitated with acetonitrile and then diluted four-

fold with water. Adapted from reference 3.

Figure 6: LC–MS–MS postcolumn infusion studies: (a) protein precipitation (Captiva,

Agilent), (b) solid-phase extraction using a neutral polymeric cartridge, (c) liquid–liquid

extraction with methyl-tert-butyl-ether and (d) lipid-stripped protein precipitation (Captiva

NDLipids, Agilent).



Agency priority pollutant list because

of their mutagenic and carcinogenic

properties. In the marine environment,

PAHs are bioavailable to marine species

via the food chain, as water-borne

compounds and contaminated

sediments. This application shows that

tandem MS detection techniques are

not necessarily required for just enough

sample preparation. Most of the PAHs

are highly fluorescent and thus, as shown

in Figure 8, reversed-phase HPLC was

combined with fluorescence detection

to determine 16 of these compounds at

a spiking level of less than 10 ng/g (5).

QuEChERS extraction provided excellent

recoveries with %RSDs below 2.

Conclusions The selectivity needed for the

determination of targeted analytes in

a complex matrix can be achieved

anywhere in the analytical cycle. With

a focus on the sample preparation

portion, the concept of just enough

sample preparation was presented. This

concept relies heavily on the increased

sensitivity and selectivity that can be

achieved with tandem MS coupled with

chromatographic separation. Provided

that ion suppression and enhancement

contributions are held to a minimum, Just

enough sample preparation can provide

the recoveries, minimum detectable

limits and minimum detectable quantities

consistent with the needs of the assay.

However, as illustrated in the example

of PAH analysis in fish, other selective

detection principles such as fluorescence

can also be used. A note of caution:

in many essays, sample processing

(handling) is still the rate-determining

step and just enough sample preparation

may be insufficient to meet the

needs of the assay. In these cases,

more-sophisticated sample preparation

protocols such as SPE or liquid–liquid

extraction may still be required.

Acknowledgments I would like to acknowledge the

contributions of Trisa Robarge and

Edward Elgart of Agilent Technologies for

their review and input on the contents of

this article.

References(1) D. Turner, LCGC Europe 25(2), 79–87

(2012).

(2) http://www.chromatographyonline.com/lcgc/

article/articleDetail.jsp?id=327354.

(3) Bioanalysis Application Note “Determination

of Fluticasone Proprionate in Human

Plasma,” Agilent Technologies, Santa Clara,

California, USA, Publication #

5990-6380EN, August, 2010.

(4) M. Anastassiades, S.J. Lehotay, D.

Stajnbaher and F.J. Schenck, J. AOAC Int.

86, 412–431 (2003).

(5) B.O. Pule, L.C. Mmualefe and N.

Torto, “Analysis of Polycyclic Aromatic

Hydrocarbons in Fish,” Agilent

Technologies, Santa Clara, California, USA,

Publication #5990-5441EN, January, 2012.

Ronald E. Majors is the editor of

“Sample Preparation Perspectives”

and a senior scientist in the columns

and supplies division at Agilent

Technologies in Wilmington, Delaware,

USA. He is also a member of LC•GC

Asia Pacific’s editorial advisory board.

Direct correspondence about this

column should go to LC•GC Asia Pacific

editor-in-chief, Alasdair Matheson,

at Advanstar Communications, 4A

Bridgegate Pavilion, Chester Business

Park, Wrexham Road, Chester, CH4 9QH,

UK, or e-mail [email protected]

Weigh 5 g of homogenized fsh sample into a 50-mL centrifuge tube

Spike samples with 2000 µL of spiking solution

Add 8 mL of acetonitrile

Shake vigorously 1min

Shake vigorously 1min

Shake 1min, centrifuge at 4000 rpm

Shake 1 min, centrifuge at 4000 rpm

5 min

5 min

Add QuEChERS salt packet

Transfer a 6-mL aliquot to the QuEChERS dispersive SPE 15-mL tube

Filter through a 0.45-µm PVDF syringe flter

Transfer 1 mL of the extract to an autosampler vial

Samples are ready for HPLC–fuorescence analysis

12

10

8

6LU

4

2

0

0

1

2 34

5

6

78

9

1011

12 13

14 15

16

2 4 6Time (min)

8 10 12 14

Figure 7: Flowchart of the QuEChERS AOAC sample preparation procedure. Adapted

from reference 7.

Figure 8: Overlay HPLC–fuorescence chromatograms of a PAH-spiked fsh extract. The

black portion of the chromatogram used 260-nm and 352-nm excitation and emission

wavelengths, respectively, the red portion used 260-nm and 420-nm wavelengths, and the

blue portion used 260-nm and 440-nm wavelengths. For acenaphthylene, UV detection

at 230-nm was used. Column: 50 mm × 4.6 mm, 1.8-µm dp Agilent Zorbax Eclipse PAH

C18; fow rate: 0.8 mL/min; temperature: 18 °C; injection volume: 5 µL; mobile-phase A:

deionized water; mobile-phase B: acetonitrile; gradient: 60% B for 1.5 min, 60–90% B in

6.5 min, 90–100% B in 6 min. Peaks: 1 = naphthalene (20 ng/g), 2 = acenaphthylene

(20 ng/g), 3 = acenaphthene (10 ng/g), 4 = fuorene (10 ng/g), 5 = phenanthrene

(10 ng/g), 6 = anthracene (10 ng/g), 7 = fuoranthene (10 ng/g), 8 = pyrene (10 ng/g),

9 = 1,2-benzanthracene (5 ng/g), 10 = chrysene (10 ng/g), 11 = benzo[e]pyrene (5 ng/g),

12 = benz[e]acenapthylene (5 ng/g), 13 = benzo[k]fuoranthene (5 ng/g), 14 = dibenz[a,h]

anthracene (5 ng/g), 15 = benzo[ghi]perylene (5 ng/g), 16 = indeno[1,2,3-cd]pyrene (5 ng/g).


GC CONNECTIONS

Given the slight chance of a contaminated cylinder, check indicating fi lters every time a tank is replaced or at least once every two weeks if using a gas generator.

Gases for gas chromatography (GC)

have become a hot topic in recent

months, primarily because of concerns

over short supplies of helium.

Already one of the top discussion

items, hydrogen as a carrier gas

has garnered much of the attention

as chromatographers’ awareness of

these issues continue to expand. In

October 2012, on-line web seminars

from Agilent and CHROMacademy

were dedicated to conversion from

helium to hydrogen carrier gas. A web

search on ‘helium hydrogen carrier’

yields guidelines and instructions

from every major GC manufacturer

and supplier, as well as myriad

topical threads on all of the GC blogs,

boards and discussion groups. Go

to Pittcon, Analytica, the Eastern

Analytical Symposium (EAS) or any

other conference where GC is on the

agenda and the helium issue will be

featured prominently.

Among all the discourse I have

noticed that the essential related topic

of good practices for deployment of

carrier gases, and for that matter all

gases used in GC, is largely missing.

Although much of this good advice

is easy enough to find in instrument

installation guides and supplier

catalogues, the connection between

obtaining the information and putting

it into practice is often missed in many

laboratories. Most laboratories will

install gas filters in-line, but many will

fail to obtain the right type of regulator,

make the gas connections correctly,

or maintain the filters and check the

regulators on a regular schedule.

Questions That Should Be Asked Frequently Here are some guidelines and

recommendations about GC gases,

in a question-and-answer format.

This is not an exhaustive list, but

rather it covers some of the more

frequently asked questions as well

as some that are not asked as

often as they should be. The list

starts at the gas source and moves

onward to the instrument. Questions

about the instrument internals are

not addressed because of space

limitations.

What Are the Recommended Gas

Purities for Carrier and Detector

Gases?: The exact requirements

for gas purity should follow the

instrument manufacturer’s guidelines

as found in their site preparation

and installation manuals. If that

information is not available, then

Tables 1 and 2 will serve as a

general guideline. The gas purities

are stated as percent levels rather

than referring to supplier–specific

names, which can be ambiguous or

inconsistent.

How Pure Are My Gases, Really?:

The purity of a gas when it reaches

the back of the instrument depends

on the supply quality, regulators,

filters, fittings and connecting

tubing. Filters will clean up minor

contamination, but they are not

intended to take gas to a higher

purity level. Most of the time the

purity of cylinder gas is as labelled

on the bottle, but occasionally a

contaminated cylinder may make

it to delivery. Although it is bad

practice on the part of cylinder

users, a cylinder might be left open

to the atmosphere for hours when

empty and removed from service.

If not cleaned up by evacuation

and baking before filling with gas

to 2450 psig (166 mPa), such a

cylinder will contain approximately

6000 ppm of air, which degrades

the gas purity to 99.4%. Although

it is extremely unlikely to arrive

in a cylinder at the receiving

dock, this level of contamination

represents a conceivable upper

limit. When placed in service the

resulting onslaught of oxygen, water

and possibly hydrocarbons will

completely exhaust a high-capacity

gas filter before the contaminated

tank is empty.

This potential for contamination is

an excellent reason to use indicating

filters on all gas supply lines. The

indicator will change colour as the

filter reaches capacity. As long as

the colour change is noticed, a new

filter is installed and a pure gas

supply is restored, the GC instrument

will be spared the indignity of gas

contamination and resultant high

detector background, irregular

baselines and accompanying loss of

signal-to-noise and repeatability.

It is possible, but expensive, to

order purity analyses of individual

cylinders. This step is only significant

when it is difficult to observe the

filters or replace the cylinder, such as

at remote unattended locations.

How Often Should the Gas

Delivery System Be Checked?:

Given the slight chance of a

contaminated cylinder, check

Q&A: GasesJohn V. Hinshaw, BPL Global Ltd, Hillsboro, Oregon, USA.

In this month’s instalment, John Hinshaw addresses a number of frequently asked questions about gases and their delivery to a gas chromatography instrument.


GC CONNECTIONS

indicating filters every time a tank

is replaced or at least once every

two weeks if using a gas generator.

That’s also a good time to check the

gas lines for unusual bends or kinks.

Check for leaks any time a fitting is

disconnected, reconnected and of

course on all new connections.

It’s also a good idea to check

regulator function at least once a

year. The easiest way to do this is

to establish a flow of 500 mL/min

or greater on each gas cylinder.

Note the pressure output, then turn

the tank valve off. Wait until the

outlet pressure starts to drop off

and then turn the tank valve back

on. The same pressure should

be re-established. Next, turn the

regulator pressure knob down

and back up while observing how

smoothly the knob moves, how the

output pressure gauge reacts and

that the knob is not screwed in all

the way when the original pressure

is restored. Don’t forget to set the

flow back to normal at the gas

chromatograph.

Check that the correct cylinders

are connected through to the correct

fittings on the instrument. Some

cylinders, such as helium, argon

and nitrogen use the same gas

fitting (at least in the United States);

hydrogen and high-level combustible

gas mixtures may also share a

fitting type. Thus, it is possible

to connect the wrong cylinder.

Chromatographers should not rely on

the uniqueness of cylinder fittings to

ensure the correct gas type.

What Kinds of Regulators Should

Be Used for GC?: Generally,

dual-stage high-purity regulators

with stainless steel diaphragms are

the correct choice in all cases. For

economy, less expensive dual-stage

regulators may be used for detector

air, hydrogen and make-up gas if

separate from the carrier supply.

The added cost of a dual-stage

compared to a single-stage regulator

does not justify the potentially poor

pressure regulation of a single-stage

regulator as the cylinder pressure

decays.

A regulator outlet valve is a handy

accessory that I like to order on all

regulators. However, in laboratories

where the downstream connections

remain in place permanently, the

outlet valve can be omitted.

Always dedicate a regulator to

its intended use. Never change

the cylinder fitting on a regulator

to switch it from inert-gas to

detector-gas service or the other way

around. Making the cylinder fitting

connection correctly so that it will

maintain high pressures is best left to

the regulator manufacturer.

What Types of Fittings and Tubing

Are Required?: The fittings and

ferrules used in any GC installation

should all be new, as should the

tubing. The fittings should be of

a suitable type for high-purity

gas lines, such as those that are

available from instrument company

and supplier catalogues. I always

like to order some spare fittings to

have on hand for the usual problem

connection that refuses to seal, as

well as for later on when the gas

setup needs some modifications.

Only metal tubing should be used

for GC gases, with one exception.

Copper tubing is the easiest to

install, while stainless steel tubing

is more robust and generally can be

more organized visually. Either type

of tubing must be precleaned before

installation, and can be ordered that

way as “GC” or “Chromatography”

cleaned tubing. “Refrigerator”

designated tubing is not suitable.

For installations with tanks or gas

generators dedicated to one or two

instruments, 1/8-in. or 3-mm o.d.

tubing is appropriate. If multiple

instruments share a tank or generator

then consider using ¼-in. or 6-mm

o.d. tubing up to the point where

the flow path splits to the individual

instruments.

Polymer tubing is appropriate only

in one case. If the GC system uses

air-actuated sampling valves then

that air supply can be connected

with polymer tubing. But if the air

tank is shared with a flame ionization

detection (FID) system —which is

not such a good idea, but it is done

— then metal tubing is required

throughout. Polymer tubing may

allow air and airborne contaminants

as well as monomers from the

plastic into the gas stream. This is

unacceptable for carrier and detector

gases, even for FID air.

A nice touch when installing tubing

is to mark either end of each tube

with differently coloured electrical

tape. This makes it clear which

tube goes to which gas inlet and

regulator and avoids some of the

more hazardous mix-ups such as

swapping FID hydrogen and air.

(Yes, I have seen that situation and

the result of igniting the flame was,

well, exciting!)

What Is the Right Way to Make

Gas Connections?: Three gas

connection types are encountered

in a GC system. Occasionally

chromatographers encounter

other fitting types, but these three

are the most common. First is the

high-pressure tank fitting, identified

by a letter and number designation

such as CGA-580, DIN 477-6 or

BS-341-3 for inert gases. With few

exceptions no additional sealing

is required — just assemble the

fitting to the cylinder and tighten to

seal. Most regulator fittings used

in GC have a torque specification

of 40–60 ft-lb (54–81 Nm). Some

types of high pressure fittings,

notably for liquid CO2 in the United

States, require a plastic washer that

is usually supplied with each tank.

If a washer is present then assume

it must be used, and the tightening

torque must be reduced by half. If

not in place, the absence of a washer

will immediately be evident upon

opening the cylinder valve! Never

use polyfluorocarbon tape or liquid

sealant anywhere on a high-pressure

tank fitting, this will just make it

leak. If either side of the fitting is

scratched or deformed then replace

the fitting.

The second type of fitting found

in GC systems is the pipe-thread

type. This fitting involves matching

internally and externally threaded

sides with no washers or ferrules.

After making sure the threads

are clear of old sealing tape,

wrap two layers — no more — of

polyfluorocarbon pipe sealing tape

(available from instrument suppliers)

onto the externally threaded side of

the fitting. Holding the exit end of a

right-hand threaded fitting toward

you, smoothly wrap the tape in a

clockwise direction while stretching it

slightly. Then thread the taped piece

into the internally threaded side and

tighten.

The third type of GC fitting is

the swaged tube fitting. This fitting

consists of a threaded receiving

piece with an internally beveled


GC CONNECTIONS

surface, a matching hexagonal

internally threaded nut, and a one- or

two-piece washer set. The swaged

fitting directly connects the end of a

tube to one side and then includes

one or two additional connections

that may be pipe-thread, another

swaged fitting or a tube. These

fittings can be of different sizes

so that a swaged fitting can be

used with different diameter tubes.

Swaged fittings are available from

several companies as well as in

brass and stainless steel. Always

use matched parts of the same metal

from the same company.

Connecting a new metal swaged

fitting is slightly more complex than

the other two types. First, the tubing

end must be cut squarely and free

of burrs, scratches or cutting debris.

Check that the hexagonal nut threads

onto the fitting smoothly, especially

if reusing the nut or fitting. Using

the tube as a guide, first slide the

nut onto the tubing with the threads

facing outward, then the circular

washer (if needed) with the narrow

side outward and finally the conical

washer with the narrow end facing

outward. Fit the parts together and

hand-tighten.

Now, comes the dexterous part:

while holding the tubing all the way

into the fitting with one hand, take

the right-sized wrench in the other

hand — such as a 7/16-in. wrench for

a 1/8-inch swaged fitting. Hold the

hexagonal part of the fitting with the

wrench. Now, take another wrench in

the other hand and . . . wait hold on,

that’s three hands! In the absence of

a vise, chromatographers soon learn

how to hold a wrench, the fitting

and the tubing in one hand while

tightening the assembly with another

wrench in the other hand. They didn’t

teach that in instrument class.

A metal swaged fitting must

be tightened a certain number

of turns, and not to a particular

torque. For a common type of this

fitting, the 1/8-inch size is tightened

¾ turn when new while the ¼ size

is tightened 1¼ turns. The fitting

manufacturer may make available a

maximum-clearance tool that helps

gauge when the fitting is sufficiently

tightened. In any case, instructions

are available from the manufacturers

and should be followed closely.

Overtightening these fittings will

reduce the number of times they can

be reconnected or even cause them

to fail to seal altogether.

Common capillary column

connections are also of the swaged

type, but instead of hard steel

or brass washers a soft metal or

polymer ferrule is used. Sometimes

the same polymer ferrule type is

used to connect small diameter

tubes inside of an instrument as

well.

Regulator and pipe-thread fittings

may be reused unless damaged.

Swaged fittings, if treated correctly,

also can be reused but must be

carefully examined beforehand.

Do not reuse if the nut does not

thread smoothly onto the fitting or

the existing tube end is distorted or

bulging out of the ferrule. Instead,

substitute all new parts and recut the

tubing to start over.

Always leak-check fittings after

making the connection. In the case

of the regulator or pipe-thread fitting

types, additional tightening up to the

maximum specified level may help

secure the seal, but never exceed

that amount of torque; check the

regulator on another cylinder and

replace as needed.

Many More QuestionsI’ve gotten only halfway down my

list of questions that are or should

be asked about gases for GC. This

discussion will be continued in an

upcoming instalment next year. In the

meantime, readers are encouraged

to submit their questions about

gases or anything else GC-related

to the editor-in-chief amatheson@

advanstar.com

John V. Hinshaw is a senior

research scientist at BPL Global

Ltd., Oregon, USA and is a

member of LC•GC Asia Pacific’s

editorial advisory board. Direct

correspondence about this column

should be addressed to “GC

Connections”, LC•GC Asia Pacific,

4A Bridgegate Pavillion, Chester

Business Park, Wrexham Road,

Chester, CH4 9QH, UK, or email the

editor-in-chief, Alasdair Matheson, at

[email protected]

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QUESTIONS OF QUALITY

Validation of computerized systems

requires that a regulated organization

working under good laboratory

practice (GLP) or good manufacturing

practice (GMP) requirements

implement or develop a system that

follows a predefined life cycle and

generates documented evidence of

the work performed. The extent of work

undertaken on each system depends

on the nature of the software used to

automate the process and the impact

that the records generated by it have

(1–3). In essence, application software

can be classified into one of three

software categories using the approach

described in the ISPE’s Good Automated

Manufacturing Practice (GAMP)

Guidelines, version 5 (“GAMP 5”) (4):

• commercially available nonconfigured

product (category 3)

• commercially available configured

product (category 4)

• custom software and modules

(category 5).

A justified and documented risk

assessment is the key to ensuring that the

validation work can be defended in an

inspection or audit. Therefore, the work

that a laboratory needs to perform will

increase with the increasing complexity of

the software (5). This discussion focuses

on commercially available software,

or GAMP software categories 3 and 4

(5,6). The arguments presented here are

not intended to be applied for custom

software applications or modules (such

as macros or custom code add‑ons to

commercial software), which require a

different approach. Nor will I consider

the role of a supplier audit in leveraging

the testing carried out during product

development and release into a

laboratory’s validation efforts.

Verif cation Stages of a Life CycleTerminology is all important to avoid

misunderstandings. We are looking

at the verification stage of a life cycle

in which the purchased system and

its components are installed and

checked out by the supplier (installation

qualification [IQ] and operational

qualification [OQ]) and then user

acceptance testing is carried out against

the requirements in the user requirement

specification (URS) to demonstrate

that the system is fit for intended use

(performance qualification [PQ]).

In Figure 1, we see three phases of

verification, together with the allocation of

responsibility for each phase. Each phase

is described as follows:

• Installation and integration (IQ) —

In essence, this asks the following

questions: Do you have all of the

items that you ordered? Have they

been installed correctly? Have the

components been connected together

correctly?

• Supplier commissioning (OQ) —

Does the system work as the supplier

expects? Typically, this is performed

on a clean installation of the software,

rather than configuring specifically for

the OQ.

Not covered in Figure 1 is the user task

of configuring the application software.

For a category 3 application this may

be limited to the setup of the user

roles, the associated access privileges

and allocation of these to individual

users. Additional steps for configurable

applications include turning functions on

or off, setting calculations to be used for

specific methods and selecting report

templates to be generated. This turns the

installed software into the as‑configured

application for your laboratory, which the

users are responsible for.

• User acceptance testing (PQ) —

Does the system work as the user

expects against the requirements in

the URS? This will be performed on the

configured application if you are using

a category 4 application.

Following the successful completion of the

user acceptance tests and writing of the

validation summary report, the system is

released for operational use. Please note

that the above descriptions of IQ, OQ and

PQ apply only to software and are not

the same as outlined in the United States

Pharmacopeia (USP) Chapter <1058> on

analytical instrument qualification (AIQ) (7).

In this column, I want to focus on the

supplier commissioning or OQ and ask

the first of the two questions I raised at

the start.

Is OQ Essential to a Validation Project?Before I answer this question, let me

put the question in the heading into

the context of category 3 and category

4 commercially available software.

When looking at the validation of such

How Much Value Is There in a Software Operational Qualif cation?R.D. McDowall, McDowall Consulting, Bromley, Kent, UK.

Software operational qualif cation (OQ) is considered a mandatory item in a computerized system validation project for a regulated laboratory. But we should ask two questions: Is OQ really essential to a validation project for this type of software such as a chromatography data system (CDS)? How much value does a software OQ for commercially available software actually provide to a validation project?



Marketingspecification

Functionalspecification

Moduletesting

Systemtesting and

release

Userrequirementsspecification

Useracceptancetesting (PQ)

Configurationspecification

Configureapplication

Install andcommission(IQ & OQ)

Application

Programming

Supplierdevelopment

life cycle

Laboratoryvalidationlife cycle

Figure 2: Integration of the system life cycles of the supplier and laboratory.

applications we need to consider two

system life cycles that will occur during

the project. The first is the development

of the new version of the product by the

supplier, and the second is that of the

user laboratory carried out to validate

the software for the intended use. This

is discussed in the GAMP 5 section on

life‑cycle models (4).

The main deliverable, from the

perspective of the laboratory, is the CD

or DVD with the application program and

associated goodies on it for installation

to a user’s computer. This optical disk is

the direct link between the two life cycles.

As shown in Figure 2, it is the medium

on which the released application is

transferred to your laboratory computer

system. It is one of the inputs to the

installation and commissioning phase of

the laboratory validation life cycle where

the IQ and OQ will be performed.

Looked at from a software perspective,

what are some of the problems that could

occur in the transfer of the application

program from the supplier’s development

process to the optical and disk and then

installation onto a laboratory’s computer

system? These include

• missing or incomplete transfer of the

whole program to the optical disk

• optical disk is corrupted

• installation on the laboratory computer

system fails

• incomplete installation on the laboratory

computer system.

Typically, most of these problems would

be picked up by the supplier’s service

engineer during the installation of the

software and integration with the rest

of the system components, especially

if there were a utility to check that the

correct software executables had been

installed in the correct directories by

the installation program. Hence the

importance of verifying that the installation

is correct (that is, executing an installation

qualification).

Now we come to the second stage

of the verification process: supplier

commissioning or the operational

qualification. This is necessary as

successful execution of this phase of

the validation is equivalent to saying that

the system works from the supplier’s

perspective. However, there are varying

degrees of OQ offered from suppliers,

ranging from the sublime to the ridiculous

(and expensive).

The aim of the software OQ is to

demonstrate that the system works

from the supplier’s perspective, which

will allow a laboratory to configure the

application and then perform user

acceptance testing, or PQ. In an earlier

Focus on Quality column (8) I discussed

the quality of supplier instrument IQ and

OQ documentation, the same principle

applies to software OQs. Quite simply,

you as the end user of the system are

responsible for the work carried out by the

supplier; this includes the quality of the

documentation, the quality of the tests,

and the quality of the work performed

by the supplier’s engineer. This is not my

opinion, it is the law. In Europe, this is now

very explicit. The new version of Annex 11

to the European Union’s GMP regulations

that was issued last year states that

suppliers have to be assessed and that

there need to be contracts in place for

any work carried out (9,10).

In the US, 21 CFR 211.160(a) (11)

requires that

The establishment of any specifications, standards, sampling plans, test procedures, or other laboratory control mechanisms required by this subpart, including any change in such specification, standards, sampling plans, test procedures, or other laboratory control mechanisms, shall be drafted by the appropriate organizational unit and reviewed and approved by the quality control unit.

Therefore, any supplier of IQ and OQ

documents needs to be approved by

the quality unit before execution, which

does not always occur. Post execution,

the results must also be reviewed

and approved by internal quality unit

personnel. Failure to comply with

this regulation can result in a starring

appearance on the Food and Drug

Administration’s (FDA) wall of shame

or the warning letters section of their

web site, as Spolana found out (12):

“Furthermore, calibration data and results

provided by an outside contractor were

not checked, reviewed and approved by

a responsible Q.C. or Q.A. official.”

Therefore, never accept IQ or OQ

documentation from a supplier without

evaluating and approving it before

execution. Check not only coverage

of testing, but also that test results are

quantified (that is, have supporting

evidence) rather than solely relying on

qualified terms (such as pass or fail).

Quantified results allow for subsequent

review and independent evaluation of the

test results. Furthermore, ensure personnel

involved with IQ and OQ work from the

vendor are trained appropriately by

checking documented evidence of such

training — for example, check that training

certificates to execute the OQ are current

before the work is carried out.

Installation andintegration

(Installationqualification – IQ)

User acceptancetesting

(Performancequalification – PQ)

Suppliercommissioning

(Operationalqualification – OQ)

Responsibility:Supplier

Responsibility:User

Figure 1: Verifcation phases of a system life cycle with an indication of the

responsibility for each phase.


An OQ Case StudyDuring an audit of a supplier of a

configurable software system, it was

discovered that the software OQ

package was the supplier’s complete

internal test suite for the release of

the product. This is offered for sale at

$N (where N is a very large number)

or free if the supplier executes the

protocol which would cost $N × 3, plus

expenses (naturally!). During execution

of the OQ, the application software is

configured with security settings and

systems according to the vendor’s

rather than the user’s requirements.

Following completion of the OQ, the

laboratory would have to change the

configuration of user types and access

privileges, linked equipment and other

software configuration settings to those

actually required before starting the user

acceptance testing.

So let me pose the question: What

is the value of this OQ that is repeating

the execution of the supplier’s complete

internal test suite?

Zero? My thoughts exactly. However,

let me raise another question: How many

laboratories would purchase and execute

this protocol just because it is a general

expectation that they do so?

Let us look in more detail at Figure 2.

Typically, the supplier will take the

released software and burn it onto a

master optical disk that is used to make

the disks sold commercially. We have one

copy stage that will be checked to ensure

the copying is correct. The application is

purchased by a laboratory, as part of the

purchase they have paid for an IQ to be

performed by the supplier. When the IQ is

completed successfully, then the software

has been correctly transferred from

the commercial disk to the laboratory’s

computer system and is the same as

that released and tested by the supplier.

Therefore, why do they need to repeat the

whole internal test suite on the customer’s

site? Where is the value to the validation

project?

Do You Believe in Risk Management?The problem with the pharmaceutical

industry is that it is ultraconservative.

Even when laboratories want to introduce

new ideas and approaches, the

conservative nature of quality assurance

tends to hold them back. However, as

resources are squeezed by the economic

situation, industry must become leaner

and embrace effective risk management

to place resources where they can be

used most effectively. Moreover, clause

1 of the EU’s Annex 11 (9) requires that

risk management principles be applied

throughout the life cycle. In light of this,

let’s look at doing just that for an OQ.

Let’s look at the OQ study above.

We have a situation in which there is a

repeat of the internal test suite on the

customer’s site. Is this justified? Not in my

opinion. What benefit would be gained

from executing the OQ? Not very much,

because the configuration tested is that

of the supplier’s own devising and not the

customer’s. Therefore, why waste time

and money on an OQ that provides no

benefit to the overall validation? What will

you achieve by this? You would generate

a pile of very expensive paper with little

benefit other than ticking a box in the

validation project. It is at this point that

you realize that GMP actually stands for

“Great Mountains of Paper.”

The risk management approach taken

with the project described above was



to document in the validation plan of

the system that a separate OQ was not

going to be performed because of the

reasons outlined above. In fact, the OQ

would be combined with the PQ or user

acceptance testing that was going to

be undertaken. Can this be justified?

Yes, but don’t take my word for it. Have

a look at Annex 15 (13) of the EU GMP

regulations, entitled Qualification and

Validation. Clause 18 states “Although PQ

is described as a separate activity, it may

in some cases be appropriate to perform

it in conjunction with OQ.”

However, you will need to document

and justify the approach taken as noted

in Annex 11 clause 4.1 (9): “The validation

documentation and reports should

cover the relevant steps of the life cycle.

Manufacturers should be able to justify

their standards, protocols, acceptance

criteria, procedures and records based

on their risk assessment.”

Presenting a document to read will be

far easier than squirming in front of an

auditor or inspector, won’t it?

One Size Fits All?

Although the case study discussed above

is an extreme case, can the approach

to dispense with an OQ be taken for all

category 3 and category 4 software?

Well, to quote that classic response of

consultants, “It depends . . . .”

Let us start with category 3 software,

which is nonconfigurable commercially

available software; the business process

automated by the application cannot be

altered. In the life‑cycle model proposed

by GAMP 5 (4,5) for this category of

software there are not separate OQ

and PQ phases but just a single task of

verification against the predefined user

requirements. Does this mean we don’t

need a supplier OQ? Yes and no. You may

think that this is a strange reply, but let’s

think it through.

If a supplier OQ exists, does it test your

user requirements in sufficient detail? If it

does then you don’t need to conduct your

own user acceptance testing (PQ), but you

will need to document the rationale in the

validation plan. This comes back to the

Annex 15 requirement quoted above that

it is permissible to combine the OQ and

PQ phases (13). However, can a supplier

protocol cover all of your requirements

such as user roles and the corresponding

access privileges and backup and

recovery? It may be possible that the

protocol, if written well, can cover these

aspects and you will not need to undertake

any testing yourself as the supplier OQ

protocol will meet your needs. This will

mean a thorough review of the testing

against your requirements to see if the OQ

is worth the cost of purchase. If the OQ

meets the majority of your requirements,

then you can perform a smaller user

acceptance test on those requirements not

tested in the supplier OQ. The goal here

is to leverage as much of the supplier’s

offering as possible, providing that it meets

your requirements. If the OQ does not

meet your requirements, then you will need

to write your own user acceptance tests

and forget the supplier approach.

OQ for Confgurable Software?Let’s return now to consider our approach

to category 4 software, which has tools

provided by the supplier to change the

way the application automates a business

process. There are a wide range of

mechanisms to configure software in

this category ranging from the simplest

approach in which a user selects an

option from a fixed selection (for example,

number of decimal places for reporting

a result, if a software function is turned

on or off or a value is entered into a field

such as password length or expiry) to the

most complex approach in which supplier

language is provided to configure the

application. In the latter situation, this is

akin to writing custom code and it should

be treated as category 5 software (4).

When considering the value of a

supplier OQ, consider how extensively

you will be configuring the application.

This requires knowledge of the

application, but is crucial when evaluating

if there is value in purchasing a supplier

OQ for your application.

My general principle is that the farther

away from the installed software you

intend to configure the application the

lower the value of an extensive supplier

OQ becomes. In these cases, you should

look for a simple supplier OQ that provides

confidence that the system operates in

the default configuration after which you

will spend time configuring the system to

meet your business requirements. In my

opinion, extensive protocols that do not

provide value to you by testing your user

requirements should be avoided.

SummaryA supplier OQ can offer a regulated

laboratory value if the tests carried

out match most, if not all, of the user

requirements, meaning that the user

acceptance testing (PQ) can be

reduced. If the application is extensively

configured then the value of an extensive

supplier OQ falls because typically the

OQ will be run on a default configuration

or one of the supplier’s devising which

will not usually be the same as the

laboratory’s. In this case, use a risk

assessment to document that the

supplier OQ is not useful and integrate

an OQ with the user acceptance testing

(PQ) to complete the verification phase of

the system validation.

References(1) GAMP Good Practice Guide A Risk‑Based

Approach to Compliant Laboratory

Computerized Systems, Second Edition

(International Society of Pharmaceutical

Engineers, Tampa, Florida, USA, 2012).

(2) R.D. McDowall, Quality Assurance Journal

9, 196–227 (2005).

(3) R.D. McDowall, Quality Assurance Journal

12, 64–78 (2009).

(4) Good Automated Manufacturing Practice

(GAMP) Guidelines, version 5 (International

Society of Pharmaceutical Engineers,

Tampa, Florida, USA, 2008).

(5) R.D. McDowall, Spectroscopy 25(4), 22–31

(2010).

(6) R.D. McDowall, Spectroscopy 24(5), 22–31

(2009).

(7) United States Pharmacopeia General

Chapter <1058> “Analytical Instrument

Qualification” (United States Pharmacopeial

Convention, Rockville, Maryland, USA).

(8) R.D. McDowall, Spectroscopy 25(9),

22–31 (2010).

(9) European Commission Health and

Consumers Directorate‑General,

EudraLex: The Rules Governing Medicinal

Products in the European Union. Volume

4, Good Manufacturing Practice Medicinal

Products for Human and Veterinary

Use. Annex 11: Computerized Systems

(Brussels, Belgium, 2011).

(10) R.D. McDowall, Spectroscopy 26(4),

24–33 (2011).

(11) Food and Drug Administration, Current

Good Manufacturing Practice regulations for

finished pharmaceutical products (21 CFR

211.160[a]).

(12) Spolana FDA warning letter, October 2000.

(13) European Commission Health and

Consumers Directorate‑General, EudraLex:

The Rules Governing Medicinal Products

in the European Union. Volume 4, Good

Manufacturing Practice, Medicinal Products

for Human and Veterinary Use. Annex

15: Qualification and Validation (Brussels,

Belgium, 2001).

“Questions of Quality” editor Bob

McDowall is Principal at McDowall

Consulting, Bromley, Kent, UK. He is also

a member of LC•GC Asia Pacific’s Editorial

Advisory Board. Direct correspondence

about this column should be addressed to

“Questions of Quality”, LC•GC Asia Pacific,

4A Bridgegate Pavilion, Chester Business

Park, Wrexham Road, Chester, CH4 9QH,

UK, or e‑mail the editor‑in‑chief Alasdair

Matheson at [email protected]

LC•GC Asia Pacifi c March 201332

THE ESSENTIALS

There are many factors that influence

the performance of a high performance

liquid chromatography (HPLC) stationary

phase, of which the chemical nature of

the bonded phase ligand is important,

but by no means all encompassing.

Minor manufacturing parameters such as

the method of electropolishing the internal

surface of the column can also have an

effect on the selectivity and efficiency

produced by a particular column.

Few of us have time to study each

individual parameter (of which there are

hundreds) and assess their interactive

effects on the selectivity of our stationary

phases. We need readily accessible

measures of column performance to

identify similar or orthogonal chemistries to

those we are currently using, or to gain an

insight into which column types might work

for particular applications.

Several attempts have been made to

produce a “definitive” set of chemical

probes to best characterize the huge

number of stationary phases available

(well over 1000 different types are currently

available). As yet a harmonized set of test

probes and methodologies has not been

identified, however three, independent,

publicly available databases of HPLC

columns exist today:

• ACD Labs Column Selection Database

— based on the early work of Tanaka

and developed by Euerby and Peterson

(1–4), http://www.acdlabs.com/products/

adh/chrom/chromproc/index.php#colsel

• United States Pharmacopeial Convention

(USP) database — based on test probes

established using a National Institute

of Standards and Technology (NIST)

Standard Reference material, http://www.

usp.org/USPNF/columnsDB.html

• The Impurities Working Group of the

Product Quality Research Institute

(PQRI) Drug Substance Technical

Committee — uses probes based on

the hydrophobic subtraction model

of Dolan, Snyder and Carr (a useful

accompanying illustration from reference

1 is shown in Figure 1) (5–8), http://www.

usp.org/USPNF/columnsDB.html

The PQRI database is the best

populated, with 588 columns, and is

a very useful tool to aid HPLC column

selection. A description of the Tanaka test

probes is given below to help understand

the various classifications, with the

analogous PQRI test probes indicated

in parentheses. It should be noted that

the PQRI classification uses different

chemical probes to the Tanaka (now

ACD) database but the results describe a

similar chemical behaviour.

• Retention factor, kPB (not tested in the

PQRI Classification), describes the

hydrophobic retention demonstrated

by the column measured using the

retention of pentylbenzene.

• Hydrophobic selectivity, αCH2 (H),

the retention factor ratio (selectivity)

between pentylbenzene and

butylbenzene reflects the ability of the

phase to separate compounds that

differ by only a single methylene group.

Column hydrophobicity (H) increases

with an increase in total carbon.

Endcapping, because of its low (<10%)

contribution to the overall carbon load

has little effect on retentivity.

• Shape selectivity, αT/0 (S*),

describes the ability of the phase to

discriminate between planar structures

(triphenylene) and those with greater

spatial (hydrodynamic) volume

(o-terphenyl). Column steric interactions

increase as the bonded phase ligands

move closer together on the silica

surface (increased bonded phase

chain length or concentration of the

bonded phase) and for packings with

narrow pore sizes, and has a significant

effect on column selectivity, especially

for molecules of different shapes.

• Hydrogen bonding capacity, αC/P (A

and B), is a measure of the retention

factor ratio (selectivity) between caffeine

and phenol and describes the columns

ability to hydrogen bond with a solute.

The PQRI database further characterizes

hydrogen bonding capacity into

hydrogen-bond acidity (A), the ability

for non-ionized silanols to interact with

bases and hydrogen bond basicity (B),

the ability for surface and bonded-phase

species to further interact with acidic

analyte features.

• Total ion-exchange capacity, αB/PpH

7.6 (C 7.6), is the selectivity between

benzylamine and phenol at a mobile

phase pH of 7.6 and reflects the total

silanol activity of the column, affecting

peak shape and selectivity for polar and

ionizable analytes.

• Acidic ion-exchange capacity, αB/P

pH 2.7 (C 2.7), is measured using

the retention factor ratio between

benzylamine and phenol at pH 2.7 and

reflects the likelihood of peak tailing when

analysing bases at low eluent pH. The

magnitude of the difference between

ion-exchange tests indicates the ability

to discriminate between polar analytes

while maintaining good peak shape.

Most of these groups have used

chemometric approaches to produce

quantitative comparisons between

column characteristics based on

principal component analysis (PCA) or

tools to visualize the relative groupings

of commercially available columns

according to their key descriptors.

ReferencesReferences available in the on-line

edition: www.chromatographyonline.

com/Essentials0313

Column Selection forReversed-Phase HPLCAn excerpt from LC•GC’s e-learning tutorial on column selection for RP-HPLC at CHROMacademy.com

Get the full tutorial at www.CHROMacademy.com/Essentials

(free until 20 April).

More Online:

(hydrophobic)

o o o

o o o

B:|

o o o o

HOH

o o o o oo-BH+

X

(steric) (H-bonding) (ion interaction)

η’H σ’S* β’A α’B κ’C

Figure 1: Schematic representations

of the fi ve interactions described by

the hydrophobic subtraction model

(adapted from reference 5).

LC•GC Asia Pacifi c March 2013 33

ADVERTISEMENT FEATURE

Chlorophyll is one of the most problematic matrix co-extractives in

pesticide residue analysis because of its non-volatile characteristics.

When samples containing chlorophyll are injected into a gas

chromatography (GC) system, chlorophyll accumulates in the

GC inlet and GC column, causing active sites and affecting GC

performance. Graphitized carbon black (GCB) is widely used to

remove chlorophyll from fruit and vegetable samples. However, GCB

will strongly adsorb planar pesticides, such as carbendazim and

thiabendazole, resulting in low recoveries. To resolve this issue, UCT

has invented a novel sorbent, ChloroFiltr®, to remove chlorophyll

from QuEChERS extracts without sacrif cing the recovery of planar

pesticides. ChloroFiltr® should not be used for mycotoxin analysis.

QuEChERS Extraction

1. Weigh 10 g of homogenized spinach sample into a 50-mL centrifuge

tube (ECPAHFR50CT).

2. Spike with 100 µL of 50 ppm triphenyl phosphate internal standard.

3. Vortex for 30 s and equilibrate for 15 min.

4. Add 10 mL of acetonitrile, shake for 1 min.

5. Add salts in Mylar pouch (ECQUUS2-MP), shake vigorously for 1 min.

6. Centrifuge at 5000 rpm for 5 min. The supernatant is ready for

cleanup.

dSPE Cleanup

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

ChloroFiltr® or GCB), shake for 30 s.

2. Centrifuge at 10,000 rpm for 5 min.

3. Transfer 0.4 mL of the cleaned extract into a 2-mL autosampler vial.

4. The sample is ready for liquid chromatography tandem mass

spectrometry (LC–MS–MS) analysis.

LC–MS–MS parameters and multiple reaction monitoring (MRM)

transitions are available upon request.

ChloroFiltr®: A Novel Sorbent for Chlorophyll RemovalXiaoyan Wang and Wayne King, UCT, LLC

UCT, LLC2731 Bartram Road, Bristol, Pennsylvania 19007, USA

tel. 800.385.3153

Email: [email protected]

Website: www.unitedchem.com

Extraction and Clean-up Materials

ECPAHFR50CT 50-mL polypropylene centrifuge tubes

ECQUUS2-MPMylar pouch with 4000 mg MgSO

4 and

2000 mg NaCl

CUMPSC1875CB2CT

dSPE with GCB

2 mL centrifuge tube with 150 mg MgSO4,

50 mg PSA, 50 mg C18, 7.5 mg GCB

CUMPSGGC182CT

dSPE with ChloroFiltr®

2 mL centrifuge tube with 150 mg MgSO4,

50 mg PSA, 50 mg C18, 50 mg ChloroFiltr®

Results

The recoveries of carbendazim, thiabendazole, pyrimethanil and

cyprodinil were adversely affected by GCB, especially thiabendazole

with a much lower recovery of 55.9% compared to 93.2% obtained

by ChloroFiltr®. Diazinon, pyrazophos and chlorpyrifos were less or

not affected by GCB because of the non-planar side chains in their

structures.

Conclusion

ChloroFiltr®, a novel sorbent, is found capable of removing

chlorophyll eff ciently without affecting the recoveries

of planar pesticides. ChloroFiltr® offers a successful

substitute for GCB in chlorophyll removal.

Figure 1: Crude spinach extract (a) cleaned with ChloroFiltr® (b) is less green than that cleaned with graphitized carbon black (GCB) (c), indicating that ChloroFiltr® is more eff cient in chlorophyll clean-up.

Table 1: Comparison of pesticide recoveries and RSDs

obtained by dSPE clean-up of spinach sample using

ChloroFiltr® and GCB (n = 4).

PesticideChloroFiltr® GCB

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

Carbendazim 87.1 1.0 71.2 4.0

Thiabendazole 93.2 1.9 55.9 2.6

Pyrimethanil 97.3 1.2 85.0 1.2

Cyprodinil 91.2 0.5 79.3 3.1

Diazinon 104.5 2.3 100.0 0.6

Pyrazophos 92.0 0.9 92.7 1.6

Chlorpyrifos 95.6 2.5 96.3 2.1

34 LC•GC Asia Pacifi c March 2013

ADVERTISEMENT FEATURE

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

fractionation or depolymerization of natural heparins. They are

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

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

mass less than 8000.

Size-exclusion chromatography (SEC) has been the most

common way of measuring the molecular weight and molecular

weight distributions of LMWHs by using the two most common

detection technologies: ultraviolet (UV) coupled with refractive

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

method in order to determine molecular weights, requiring

calibration standards. A newer, absolute method involves the use

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

any standards. The European Pharmacopeia (EP) monograph

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

provides a known calibration standard. Many laboratories around

the world have adopted this method.

We previously developed an SEC/MALS method and found it

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

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

compared the molecular weight results generated for LMWH using

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

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

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

2487 dual wavelength UV detector, and Wyatt Optilab rEX refractive

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

with a Wyatt miniDAWN TREOS detector; all other methods aspects

remained the same.

The results indicated that both detection types are suitable

and acceptable for the analysis of LMWHs. The molecular weight

and distribution results generated using each detection type are

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

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

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

obviates the need for calibration standards.

This note was graciously submitted by Lin Rao and John Beirne

of Scientif c Protein Laboratories LLC.

Molecular Weight Determination of Low-Molecular-Weight Heparins: SEC/MALS vs. SEC/UV-RIWyatt Technology Corporation

LS dRI UV

Define Peaks: LMWH Sample

0.8

0.6

0.4Rel

ativ

e sc

ale

0.2

0.0

5.0 10.0

Time (min)

15.0 20.0 25.0 30.0 35.0

Define Peaks: LMWH Sample

1.0

0.5

0.0

Rel

ativ

e sc

ale

5.0 10.0

Time (min)

15.0 20.0 25.0 30.0 35.0

LS dRI

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

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

tel. +1 (805) 681 9009 fax +1 (805) 681 0123

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

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