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Comparison of LC/MS/MS and GC/MSD Analyses of Pesticide Residues in Food Products when Using the QuEChERS Sample Preparation Technique

Monika Kansal, A. Carl Sanchez, Michael Rummel, and Art Dixon

Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

QuEChERS, an acronym for Quick, Easy, Cheap, Effective, Rugged, and Safe, is a sample preparation procedure commonly used for the extraction and cleanup of multiple pesticide residues from food and agricultural products. This relatively simple procedure provides extracts suitable for both LC/MS and GC/MS analyses.

In this study the results obtained when analyzing the same extracts from multiple food products using both GC/MSD and LC/MS/MS are presented and compared. The relative strengths of each instrument platform are highlighted and considerations for choosing the most appropriate platform are presented.

Introduction

Results and Discussion

Iceberg Lettuce ExtractionThe total number of possible failures is 125 •100%passingisdifficulttoachieve •Themostproblematicanalytesarediscussedinthisstudy

Analytes LC/MS LC/MS LC/MS GC/MS GC/MS GC/MS

Recovery % RSD Linearity Recovery %RSD LinearityAtrazine 85 2 1.000 105 5% 1.000Azoxystrobin 88 3 0.999 109 11% 0.994Bifenthrin N/A N/A N/A 108 6% 0.997

Carbaryl 94 3 0.999 112 8% 0.999Chlorothalonil N/A N/A N/A 103 7% 0.999Chlorpyrifos 86 6 0.998 99 10% 0.946Chlorpyrifos-methyl 82 3 0.996 115 5% 0.988Cyprodnil 89 2 0.999 107 5% 0.999Dichlorvos 34 37 0.768 106 6% 0.998Endosulfan sulfate N/A N/A N/A 110 5% 0.999Ethion 95 3 0.999 106 7% 0.999Imazalil * 92 3 1.000 87 10% 0.984Imidacloprid 93 3 1.000 N/A N/A N/AKresoxim-methyl 94 2 0.997 107 6% 0.993Linuron 93 3 0.999 N/A N/A N/AL-Cyhalothrin N/A N/A N/A 110 8% 0.997Methamidophos * 74 3 0.999 113 9% 0.995Methomyl 87 2 1.000 N/A N/A N/Ao,p-DDD N/A N/A N/A 100 5% 0.998o-phenolphenyl N/A N/A N/A 103 5% 1.000Permethrins N/A N/A N/A 112 8% 0.998Procymidone N/A N/A N/A 107 5% 0.999Pymetrozine 69 4 1.000 N/A N/A N/ATebuconazole 91 3 0.999 105 7% 0.998Thiabendazole * 95 2 0.997 96 13% 0.986Tolyfluanid 90 2 0.997 95 15% 0.992Trifluralin N/A N/A N/A 101 5% 0.996

Recovery • LC/MS- All analytes (18) passed Association of Analytical Chemists AOAC criteria except Dichlorvos •AllcalculationsbasedonExternalStandardization(ESTD) •PymetrozineislowasexpectedbyAOACmethod • GC/MS- All analytes (23) showed good recovery •AllcalculationsbasedonInternalStandardization(ISTD)% RSD for sample replicates • LC/MS- All analytes passed AOAC criteria except Dichlorvos •Basedon5samplereplicates • GC/MS- All analytes passed AOAC criteria •Basedon5samplereplicatesCorrelation coefficient (linearity) • LC/MS - All analytes passed AOAC criteria except Dichlorvos •AllcalculationsbasedonESTD • GC/MS - Linearity criteria was low for a few analytes •Sensitivityformethamidophos,imazalilandthiabendazolewasinadequateatlow levels

Spinach ExtractionPigmentedmatricesrequiregraphitizedcarbonblack(GCB) •GCBisknowntoadsorbplanarmolecules* •ProcymidonesufferedlossinGC/MSduetomatrixco-elution** •Optimizedanalysisconditionsasdeterminedinthisstudy

Analytes LC/MS LC/MS LC/MS GC/MS GC/MS GC/MS

Recovery %RSD Linearity Recovery %RSD LinearityAtrazine 73 6 0.998 95 3% 0.999Azoxystrobin 88 5 0.999 117 6% 0.996Bifenthrin N/A N/A N/A 92 4% 0.999Carbaryl 73 6 0.999 87 9% 0.999Chlorothalonil * N/A N/A N/A 26* 9% 0.999Chlorpyrifos 63 5 0.997 80 5% 0.999Chlorpyrifos-methyl 75 3 0.997 75 2% 0.999Cyprodnil * 14* 6 0.998 17* 6% 0.999Dichlorvos N/A N/A N/A 81 8% 0.999Endosulfan sulfate N/A N/A N/A 105 6% 0.999Ethion 83 6 0.996 102 4% 0.997Imazalil 82 3 0.998 N/A N/A N/AImidacloprid 89 5 0.999 N/A N/A N/AKresoxim-methyl 97 5 0.996 98 5% 0.999Linuron 74 5 0.997 N/A N/A N/AL-Cyhalothrin N/A N/A N/A 108 9% 0.999Methamidophos 87 6 0.999 N/A N/A N/AMethomyl 85 6 0.999 N/A N/A N/Ao,p-DDD N/A N/A N/A 102 4% 0.998o-phenolphenyl N/A N/A N/A 94 4% 0.998Permethrins N/A N/A N/A 95 4% 0.997Procymidone** N/A N/A N/A 46** 33% 0.999Pymetrozine * 10* 7 0.997 N/A N/A N/ATebuconazole 81 4 0.997 83 3% 0.992Thiabendazole * 12* 6 1.000 N/A N/A N/ATolyfluanid 84 8 0.993 88 7% 0.999Trifluralin N/A N/A N/A 93 3% 0.997

Thiabendazole* Cyprodnil*Pymetrozine*

LC/MS - Challenges and Solutions

Problematic AnalytesInjection solvent mismatch •AOACinjectionsolvent25%Acetonitrile(ACN)peakdistortion Earlyelutersgavesplitpeaks(pymetrozine,methamidophos,thiabendazole) •Reducedsolventstrength-sampleprecipitation FiltermembranesabsorbanalytestovariousdegreesMobile Phase – Limited flexibility •Increaseinitial%organic-earlyelutersnotretained Earlyelutersunretainedinsomepublishedpapers •Decreaseinitial%organic–reconstitutionsolventmismatchMatrix effects •Eachanalyterespondsdifferentlytomatrixinterferences Oneinternalstandardforallanalytesdoesnotgivereliableresults

LC/MS Injection Solvent - Peak Distortion

Earlyelutingpeaksdistortedwith25%acetonitrileinjectionsolvent(assuggestedbyAOAC)

•Initialmobilephase:5mMFormicAcid(FA)in25%methanol(MeOH)

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 min0.00

500.001000.001500.002000.002500.003000.003500.004000.004500.005000.005500.006000.006500.007000.007500.008000.008500.009000.009500.00

1.00e41.04e4

Inte

nsity

, cp

s

3.12

3.5519.4318.0014.00 34.2516.766.26 20.591.29 7.14 33.815.26 13.7911.75 21.68 35.449.08 23.73 30.04 39.4036.7328.69 42.6227.19

Pymetrozine,split peaks

Sample dissolved in 25 % ACN 5mM FA

As per AOAC

Ap

p ID

20

672

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 min111 221 331 442 552 662 772 882 992 1102 1213 1323 1433 1543 1653 1763 1873 1984 2094 2204 2314

050

100150200250300350400450500550600650700750800850900950

1000

Inte

nsity

, cp

s

3. 86

35. 266.1434.338. 08 11.33

7.521. 16

14.782.32 5.76 15. 8711.89 35.399.90 24.79 36. 95

38.4233.2529. 3928.00 40. 1323. 3018. 16 22.23 30.76

28. 1420.47

Methamidophos

Sample dissolved in 25 % ACN 5mM FA

As per AOAC

Ap

p ID

20

66

8

Solution

Challenge

Reducedsolventstrength–25%MeOH

•Peaksplittingissueresolved

•Precipitation

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 min0.0

2000.04000.06000.08000.0

1.0e41.2e41.4e41.6e41.8e42.0e42.2e42.4e42.6e42.8e43.0e43.2e43.4e43.6e43.8e44.0e44.2e4

Inte

nsity

, cp

s

3.45

Pymetrozine

Sample dissolved in 25 % MeOH 5mM FA

Ap

p ID

20

674

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 min111 221 331 442 552 662 772 882 992 1102 1213 1323 1433 1543 1653 1763 1873 1984 2094 2204 2314

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

Inte

nsity

, cp

s

3.82

11.2411.8410.739.03 24.2416.3113.536.86 35.28

Methamidophos

Sample dissolved in 25 % MeOH 5mM FA

Ap

p ID

20

673

LC/MS Injection Solvent – Sample Precipitation(Filtration)

Matrixcomponentsprecipitatewhen25%methanolisused5ng/gCalStandardpreparedin25%Methanol5mMFormicacid •Allfiltermembranesabsorbanalytes–differentforeachfilter

•Belowisthe%losswithfiltrationforthemostproblematicanalytes

Samplesinjectedwithout filtration(centrifuged)

•Noproblemswithcolumn fouling

% Loss withGHP �lter

% Loss withNylon �lter

% Loss withRC �lter

% Loss withPTFE �lter

% Loss withPTVDF �lter

% L

oss

Chlorpyrifos

EthionImazalil 1

Methamidophos 2

Thiabendazole 1

D10-Parathion

% Loss on Filtration-5 ng/g cal standard

Solution

Challenge

LC/MS – Limited Flexibility of Mobile Phase

2 4 6 8 10 12 14 16 18 20 22 24 min114 227 341 454 567 680 793 906 1020 1133 1246 1359

0. 00500. 00

1000. 001500. 002000. 002500. 003000. 003500. 004000. 004500. 005000. 005500. 006000. 006500. 007000. 007500. 008000. 008500. 009000. 009500. 00

1. 00e41. 05e41. 10e41. 15e41. 20e4

Inte

nsity

, cp

s

1. 79

Pymetrozine unretained(to=1.8 mins ), RT=1.79 mins

Column: Prodigy™ ODS-3 5 µm

Dimensions: 150 x 3.0 mm Mobile Phase A: Water with 0.1 % Formic acid

B: Acetonitrile with 0.1 % Formic acid

Gradient: 30 to 100 % B in 8 min, 5.5 min final hold at 100 % B

Flow Rate: 0.3 mL/min

Instrument: MS: API4000™ (ESI+), LC: Agilent® 1200

Sample solvent: 40 % ACN in water with 0.4 % Acetic acid

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 min114 227 341 454 567 680 793 906 1020 1133 1246 1359 1472 1585 1699 1812 1925 2038 2151 2265 2378

0

50010001500200025003000350040004500500055006000650070007500800085008860

Inte

nsity

, cp

s

3. 04

3. 51

Pymetrozine retained RT=3.04 mins, but splitting

Mobile phase/sample solvent mismatch (AOAC 2007)

Column: Luna® C18(2) 3 µm,

Dimensions: 150 x 3.0 mm

Mobile Phase A: Water with 5 mM Formic acidB: Methanol with 5mM Formic acid

Gradient: 25 to 90 % B in 15 mins, 15mins final hold at 100 % B

Flow Rate: 0.3 mL/min

Instrument: MS: API4000™ (ESI+), LC: Agilent 12000

Sample Solvent: 25 % ACN in water with 0.25 % Acetic acid

Different mobile phase conditions (JCA Article (2010))

Ap

p ID

20

675

Ap

p ID

20

670

Effect of Matrix Interference in QC SampleLC/MS Analysis for Lettuce

Carbaryl % RSD=2 %

Ratio IS/TPP % RSD=17 %

Cyprodnil % RSD=2 %

D10-IS % RSD=17 %

TPP% RSD=2 %

Matrix interferences affect the internal standard (d10-parathion as per AOAC) differently than analytes

•%RSDISTD>>%RSDanalyte->matrixeffectswithISTD

•StablelabelISTDforallanalytesnotgenerallyusedinmulti-residuemethods(GC/MS)

• Standard practice for LC/MS due to matrix effects

LC/MS Matrix Effects

Effect of Matrix Interference in QC SampleLC/MS Analysis for Lettuce

Carbaryl % RSD=2 %

Ratio IS/TPP % RSD=17 %

Cyprodnil % RSD=2 %

D10-IS % RSD=17 %

TPP% RSD=2 %

GC/MS - Challenges and Solutions

Problematic AnalytesOverall fewer problems than LC/MS Matrix interferences •Co-elutingmatrixcomponents •SomematricesmoreproblematicthanothersChromatographic degradation (inlet fouling, losses) •Accumulationofmatrixcomponents •Poorpeakshape •Reducedsensitivity

GC/MS Interferences - Methamidophos

3.50 3.52 3.54 3.56 3.58 3.60 3.62 3.64 3.66 3.68 3.70 3.72 3.74 3.76 3.78 3.80 3.82 3.84 min0

100020003000400050006000700080009000

10000110001200013000

Interference

Methamidophos

3.50 3.52 3.54 3.56 3.58 3.60 3.62 3.64 3.66 3.68 3.70 3.72 3.74 3.76 3.78 3.80 3.82 min0

2000

4000

6000

8000

10000

12000

14000

Interference

Early eluter co-elutes with interference

•AnalyzebyLC/MSforoptimumresults

50 ng/g Cal standard (apple extract)

Blank(appleextract)

Ap

p ID

20

676

Ap

p ID

20

671

GC/MS Inlet Losses - Imazalil in Different Matrices

11.60 11.80 12.00 12.20 12.40 12.60 12.80 13.00 13.20 13.40 min0

50100150200250300350400450500550600650700750800850900950

Imazalil

11.60 11.80 12.00 12.20 12.40 12.60 12.80 13.00 13.20 13.40 min

50100150200250300350400450500550600650700750800850900950

Imazalil

Accumulation of matrix components in inlet liner leads to poor peak shape and reduced sensitivity

•AnalyzebyLC/MSforoptimumresults

Lettuce extract 50 ng/g Cal standard

Spinach extract 50 ng/g Cal standard

Advantages of LC/MS Over GC/MS Analysis

Methamidophos, imazalil and thiabendazole show better performance with LC/MS than GC/MS •BetterpeakshapeinLC/MS

•HighersignalintensityinLC/MS

•Goodlinearityand%RSDwithLC/MS

Advantages of LC/MS GC/MS Analysis •Analyze by LC/MS for optimum results

Thiabendazole using LC/MS and GC/MS

Methamidophos using LC/MS and GC/MS

5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 min0.0

2000.04000.06000.08000.01.0e41.2e41.4e41.6e41.8e42.0e42.2e42.4e42.6e42.8e43.0e43.2e43.4e43.6e43.8e4

Inte

nsity

, cp

s

6.40

ThiabendazoleLC/MS 5 ng/g (lettuce extract)

GC/MS 5 ng/g (lettuce extract)

10.60 10.80 11.00 11.20 11.40 11.60 11.80 12.00 12.20 12.40 min

102030405060708090

100110120130140150160170180190

Thiabendazole

3.50 3.52 3.54 3.56 3.58 3.60 3.62 3.64 3.66 3.68 3.70 3.72 3.74 3.76 3.78 min0

100020003000400050006000700080009000

10000110001200013000140001500016000170001800019000

Interference

Methamidophos

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 min0.0

5000.01.0e41.5e42.0e42.5e43.0e43.5e44.0e44.5e45.0e45.5e46.0e46.2e4

Inte

nsity

, cp

s

3.77

MethamidophosLC/MS 100 ng/g (lettuce extract)

GC/MS 100 ng/g (lettuce extract)

Advantages of GC/MS Analysis Over LC/MS AnalysisInternal standardization possible •Ingeneral,GC/MSdoesnotsufferfromcompoundspecificmatrixeffectsSolubility •Nosolubilityproblems,allanalytesaresolubleintolueneandacetonitrileDramatically improved sensitivity for procymidone vs. LC/MS (no fragmentation with CID)Dichlorvos shows reproducible results in GC/MS •LC/MSresultsverypoor

Internal Standardization

Carbaryl % RSD=7 %

Ratio IS/TPP % RSD=14 %

Cyprodnil % RSD=6 %

D10-IS % RSD=8 %

TPP% RSD=7 %

GC/MS Analysis

Carbaryl % RSD=2 %

Ratio IS/TPP % RSD=17 %

Cyprodnil % RSD=2 %

D10-IS % RSD=17 %

TPP% RSD=2 %

LC/MS Analysis

Plot of D10-Parathion(internal standard) GC/MS and LC/MS/MS •InGC/MSmatrixgenerallydoesnotaffectionization • In LC/MS, co-elutingmatrix components affect ionizationof ISTDdifferently than analytes

•%RSDISTD>>%RSDanalyte->matrixeffectswithISTD

Effect of matrix interferences in lettuce QC samples

Analyte results using both GC/MS and LC/MS techniques

Dichlorvos improved performanceDichlorvosshowssignificantmatrixeffectsinLC/MS •ItfailedallcriteriainLC/MSbutpassedallinGC/MS •AnalyzebyGC/MSforoptimumresults

Analytes LC/MS-Lettuce LC/MS-Lettuce LC/MS-Lettuce GC/MS-Lettuce GC/MS-Lettuce GC/MS-Lettuce

Recovery % RSD Linearity Recovery % RSD LinearityAtrazine 85 2 1.000 105 5% 1.000Azoxystrobin 88 3 0.999 109 11% 0.994Carbaryl 94 3 0.999 112 8% 0.999Chlorpyrifos 86 6 0.998 99 10% 0.946Chlorpyrifos-methyl 82 3 0.996 115 5% 0.988Cyprodnil 89 2 0.999 107 5% 0.999Dichlorvos 34 37 0.768 106 6% 0.998Ethion 95 3 0.999 106 7% 0.999Imazalil 92 3 1.000 87 10% 0.984Kresoxim-methyl 94 2 0.997 107 6% 0.993Methamidophos 74 3 0.999 113 9% 0.995Tebuconazole 91 3 0.999 105 7% 0.998

Thiabendazole 95 2 0.997 96 13% 0.986Tolyfluanid 90 2 0.997 95 15% 0.992

Conclusions

Results for all analytes met AOAC performance expectations by carefully optimizing analysis conditionsMost appropriate platform selected for each analyte. •Besttechnique,GCorLC,isanalytespecific •Methamidophos,imazalilandthiabendazole-bestresultswithLC/MS •Dichlorvosandprocymidone-bestresultswithGC/MSInjection solvent and solubility •LC/MS-reconstitutesampleswith25%MeOHwithoutfiltering •GC/MS-nosolubilityproblemsMatrix interferences •ExternalstandardizationforLC/MSanalysis(ifnostablelabelISTDs) •InternalstandardizationforGC/MSanalysis

TrademarksLuna is a registered trademark, and Prodigy is a trademark of Phenomenex. AgilentisaregisteredtrademarkofAgilentTechnologies.API4000isatrademarkofABSCIEXpteLtd.ABSCIEXisbeingusedunderlicense. DisclaimerPhenomenexisnotaffiliatedwithAgilentTechnologies.Comparativeseparationsmaynotberepresentativeofallapplications.

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