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 PO93920312_W_1
Introduction 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.
Results and Discussion Iceberg Lettuce Extraction The total number of possible failures is 125 100 % passing is difficult to achieve The most problematic analytes are discussed in this study Analytes LC/MS LC/MS LC/MS GC/MS GC/MS GC/MS Recovery % RSD Linearity Recovery %RSD Linearity Atrazine 85 2 1.000 105 5% 1.000 Azoxystrobin 88 3 0.999 109 11% 0.994 Bifenthrin N/A N/A N/A 108 6% 0.997 Carbaryl 94 3 0.999 112 8% 0.999 Chlorothalonil N/A N/A N/A 103 7% 0.999 Chlorpyrifos 86 6 0.998 99 10% 0.946 Chlorpyrifos-methyl 82 3 0.996 115 5% 0.988 Cyprodnil 89 2 0.999 107 5% 0.999 Dichlorvos 34 37 0.768 106 6% 0.998 Endosulfan sulfate N/A N/A N/A 110 5% 0.999 Ethion 95 3 0.999 106 7% 0.999 Imazalil * 92 3 1.000 87 10% 0.984 Imidacloprid 93 3 1.000 N/A N/A N/A Kresoxim-methyl 94 2 0.997 107 6% 0.993 Linuron 93 3 0.999 N/A N/A N/A L-Cyhalothrin N/A N/A N/A 110 8% 0.997 Methamidophos * 74 3 0.999 113 9% 0.995 Methomyl 87 2 1.000 N/A N/A N/A o,p-ddd N/A N/A N/A 100 5% 0.998 o-phenolphenyl N/A N/A N/A 103 5% 1.000 Permethrins N/A N/A N/A 112 8% 0.998 Procymidone N/A N/A N/A 107 5% 0.999 Pymetrozine 69 4 1.000 N/A N/A N/A Tebuconazole 91 3 0.999 105 7% 0.998 Thiabendazole * 95 2 0.997 96 13% 0.986 Tolyfluanid 90 2 0.997 95 15% 0.992 Trifluralin 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 All calculations based on External Standardization (ESTD) Pymetrozine is low as expected by AOAC method GC/MS- All analytes (23) showed good recovery All calculations based on Internal Standardization (ISTD) % RSD for sample replicates LC/MS- All analytes passed AOAC criteria except Dichlorvos Based on 5 sample replicates GC/MS- All analytes passed AOAC criteria Based on 5 sample replicates Correlation coefficient (linearity) LC/MS - All analytes passed AOAC criteria except Dichlorvos All calculations based on ESTD GC/MS - Linearity criteria was low for a few analytes Sensitivity for methamidophos, imazalil and thiabendazole was inadequate at low levels
Spinach Extraction Pigmented matrices require graphitized carbon black (GCB) GCB is known to adsorb planar molecules* Procymidone suffered loss in GC/MS due to matrix co-elution** Optimized analysis conditions as determined in this study Analytes LC/MS LC/MS LC/MS GC/MS GC/MS GC/MS Recovery %RSD Linearity Recovery %RSD Linearity Atrazine 73 6 0.998 95 3% 0.999 Azoxystrobin 88 5 0.999 117 6% 0.996 Bifenthrin N/A N/A N/A 92 4% 0.999 Carbaryl 73 6 0.999 87 9% 0.999 Chlorothalonil * N/A N/A N/A 26* 9% 0.999 Chlorpyrifos 63 5 0.997 80 5% 0.999 Chlorpyrifos-methyl 75 3 0.997 75 2% 0.999 Cyprodnil * 14* 6 0.998 17* 6% 0.999 Dichlorvos N/A N/A N/A 81 8% 0.999 Endosulfan sulfate N/A N/A N/A 105 6% 0.999 Ethion 83 6 0.996 102 4% 0.997 Imazalil 82 3 0.998 N/A N/A N/A Imidacloprid 89 5 0.999 N/A N/A N/A Kresoxim-methyl 97 5 0.996 98 5% 0.999 Linuron 74 5 0.997 N/A N/A N/A L-Cyhalothrin N/A N/A N/A 108 9% 0.999 Methamidophos 87 6 0.999 N/A N/A N/A Methomyl 85 6 0.999 N/A N/A N/A o,p-ddd N/A N/A N/A 102 4% 0.998 o-phenolphenyl N/A N/A N/A 94 4% 0.998 Permethrins N/A N/A N/A 95 4% 0.997 Procymidone** N/A N/A N/A 46** 33% 0.999 Pymetrozine * 10* 7 0.997 N/A N/A N/A Tebuconazole 81 4 0.997 83 3% 0.992 Thiabendazole * 12* 6 1.000 N/A N/A N/A Tolyfluanid 84 8 0.993 88 7% 0.999 Trifluralin N/A N/A N/A 93 3% 0.997 Thiabendazole* Pymetrozine* Cyprodnil*
LC/MS - Challenges and Solutions Problematic Analytes Injection solvent mismatch AOAC injection solvent 25 % Acetonitrile (ACN) peak distortion Early eluters gave split peaks (pymetrozine, methamidophos, thiabendazole) Reduced solvent strength - sample precipitation Filter membranes absorb analytes to various degrees Mobile Phase Limited flexibility Increase initial % organic - early eluters not retained Early eluters unretained in some published papers Decrease initial % organic reconstitution solvent mismatch Matrix effects Each analyte responds differently to matrix interferences One internal standard for all analytes does not give reliable results
LC/MS Injection Solvent - Peak Distortion Challenge Early eluting peaks distorted with 25 % acetonitrile injection solvent (as suggested by AOAC) Initial mobile phase: 5 mm Formic Acid (FA) in 25 % methanol (MeOH) Intensity, cps 1000 950 900 850 800 750 700 650 600 550 500 450 400 350 300 250 200 150 100 50 0 2.32 1. 16 3. 86 Methamidophos Sample dissolved in 25 % ACN 5mM FA As per AOAC 6.14 35. 26 34.33 8. 08 11.33 7.52 5.76 18. 16 14.78 15. 87 9.90 11.89 24.79 35.39 36. 95 38.42 33.25 28.00 40. 13 29. 39 23. 30 22.23 30.76 20.47 28. 14 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 min 111 221 331 442 552 662 772 882 992 1102 1213 1323 1433 1543 1653 1763 1873 1984 2094 2204 2314 App ID 20668 Solution Intensity, cps 1.04e4 1.00e4 9500.00 9000.00 8500.00 8000.00 7500.00 7000.00 6500.00 6000.00 5500.00 5000.00 4500.00 4000.00 3500.00 3000.00 2500.00 2000.00 1500.00 1000.00 500.00 0.00 3.12 Pymetrozine, split peaks Reduced solvent strength 25 % MeOH Peak splitting issue resolved Precipitation 3.82 Intensity, cps 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 Sample dissolved in 25 % ACN 5mM FA As per AOAC 3.55 18.0019.43 16.76 14.00 34.25 6.26 20.59 1.29 7.149.08 33.81 5.26 13.79 11.75 21.68 35.44 36.73 23.73 27.1928.69 30.04 39.40 42.62 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 min Methamidophos Sample dissolved in 25 % MeOH 5mM FA 6.86 9.0310.7311.2411.84 13.53 16.31 24.24 35.28 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 min 111 221 331 442 552 662 772 882 992 1102 1213 1323 1433 1543 1653 1763 1873 1984 2094 2204 2314 App ID 20672 App ID 20673 Intensity, cps 4.2e4 4.0e4 3.8e4 3.6e4 3.4e4 3.2e4 3.0e4 2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e4 8000.0 6000.0 4000.0 2000.0 0.0 3.45 Pymetrozine Sample dissolved in 25 % MeOH 5mM FA 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 min App ID 20674
LC/MS Injection Solvent Sample Precipitation (Filtration) Challenge Matrix components precipitate when 25 % methanol is used 5 ng/g Cal Standard prepared in 25 % Methanol 5 mm Formic acid All filter membranes absorb analytes different for each filter Below is the % loss with filtration for the most problematic analytes Solution Samples injected without filtration (centrifuged) No problems with column fouling % Loss % Loss on Filtration-5 ng/g cal standard Chlorpyrifos Ethion Imazalil 1 Methamidophos 2 Thiabendazole 1 D10-Parathion % Loss with GHP filter % Loss with Nylon filter % Loss with RC filter % Loss with PTFE filter % Loss with PTVDF filter
LC/MS Limited Flexibility of Mobile Phase Different mobile phase conditions (JCA Article (2010)) 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 Intensity, cps 1. 20e4 1. 15e4 1. 10e4 1. 05e4 1. 00e4 9500. 00 9000. 00 8500. 00 8000. 00 7500. 00 7000. 00 6500. 00 6000. 00 5500. 00 5000. 00 4500. 00 4000. 00 3500. 00 3000. 00 2500. 00 2000. 00 1500. 00 1000. 00 500. 00 0. 00 1. 79 Pymetrozine unretained (t o =1.8 mins ), RT=1.79 mins 2 4 6 8 10 12 14 16 18 20 22 24 min 114 227 341 454 567 680 793 906 1020 1133 1246 1359 App ID 20670 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 acid B: 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 Intensity, cps 8860 8500 8000 7500 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 3. 04 3. 51 Pymetrozine retained RT=3.04 mins, but splitting 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 min 114 227 341 454 567 680 793 906 1020 1133 1246 1359 1472 1585 1699 1812 1925 2038 2151 2265 2378 App ID 20675
LC/MS Matrix Effects Matrix interferences affect the internal standard (d10-parathion as per AOAC) differently than analytes % RSD ISTD >> % RSD analyte -> matrix effects with ISTD Stable label ISTD for all analytes not generally used in multi-residue methods (GC/MS) Standard practice for LC/MS due to matrix effects Effect of Matrix Interference in QC Sample LC/MS Analysis for Lettuce Carbaryl % RSD=2 % Cyprodnil % RSD=2 % Ratio IS/TPP % RSD=17 % D10-IS % RSD=17 % TPP% RSD=2 %
GC/MS - Challenges and Solutions Problematic Analytes Overall fewer problems than LC/MS Matrix interferences Co-eluting matrix components Some matrices more problematic than others Chromatographic degradation (inlet fouling, losses) Accumulation of matrix components Poor peak shape Reduced sensitivity
GC/MS Interferences - Methamidophos Early eluter co-elutes with interference Analyze by LC/MS for optimum results 50 ng/g Cal standard (apple extract) Blank (apple extract) 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 14000 12000 10000 8000 6000 4000 2000 0 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 3.84 min Interference 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 min App ID 20676 App ID 20671
GC/MS Inlet Losses - Imazalil in Different Matrices Accumulation of matrix components in inlet liner leads to poor peak shape and reduced sensitivity Analyze by LC/MS for optimum results Lettuce extract 50 ng/g Cal standard 950 900 850 800 750 700 650 600 550 500 450 400 350 300 250 200 150 100 50 0 Imazalil 11.60 11.80 12.00 12.20 12.40 12.60 12.80 13.00 13.20 13.40 min Spinach extract 50 ng/g Cal standard 950 900 850 800 750 700 650 600 550 500 450 400 350 300 250 200 150 100 50 Imazalil 11.60 11.80 12.00 12.20 12.40 12.60 12.80 13.00 13.20 13.40 min
Advantages of LC/MS Over GC/MS Analysis Methamidophos, imazalil and thiabendazole show better performance with LC/MS than GC/MS Better peak shape in LC/MS Higher signal intensity in LC/MS Good linearity and % RSD with LC/MS
Advantages of LC/MS GC/MS Analysis Analyze by LC/MS for optimum results Methamidophos using LC/MS and GC/MS LC/MS 100 ng/g (lettuce extract) Intensity, cps 6.2e4 6.0e4 5.5e4 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e4 5000.0 0.0 3.77 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 min GC/MS 100 ng/g (lettuce extract) 19000 18000 17000 16000 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 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 min Thiabendazole using LC/MS and GC/MS LC/MS 5 ng/g (lettuce extract) GC/MS 5 ng/g (lettuce extract) 3.8e4 3.6e4 3.4e4 3.2e4 3.0e4 2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e4 8000.0 6000.0 4000.0 2000.0 0.0 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 6.40 Thiabendazole 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 min Thiabendazole 10.60 10.80 11.00 11.20 11.40 11.60 11.80 12.00 12.20 12.40 min
Advantages of GC/MS Analysis Over LC/MS Analysis Internal standardization possible In general, GC/MS does not suffer from compound specific matrix effects Solubility No solubility problems, all analytes are soluble in toluene and acetonitrile Dramatically improved sensitivity for procymidone vs. LC/MS (no fragmentation with CID) Dichlorvos shows reproducible results in GC/MS LC/MS results very poor
Internal Standardization Plot of D10-Parathion(internal standard) GC/MS and LC/MS/MS In GC/MS matrix generally does not affect ionization In LC/MS, co-eluting matrix components affect ionization of ISTD differently than analytes % RSD ISTD >> % RSD analyte -> matrix effects with ISTD GC/MS Analysis LC/MS Analysis Effect of matrix interferences in lettuce QC samples Cyprodnil % RSD=6 % Carbaryl % RSD=7 % TPP% RSD=7 % Carbaryl % RSD=2 % Cyprodnil % RSD=2 % Ratio IS/TPP % RSD=17 % D10-IS % RSD=17 % Ratio IS/TPP % RSD=14 % TPP% RSD=2 % D10-IS % RSD=8 % Dichlorvos improved performance Dichlorvos shows significant matrix effects in LC/MS It failed all criteria in LC/MS but passed all in GC/MS Analyze by GC/MS for optimum results Analyte results using both GC/MS and LC/MS techniques Analytes LC/MS-Lettuce LC/MS-Lettuce LC/MS-Lettuce GC/MS-Lettuce GC/MS-Lettuce GC/MS-Lettuce Recovery % RSD Linearity Recovery % RSD Linearity Atrazine 85 2 1.000 105 5% 1.000 Azoxystrobin 88 3 0.999 109 11% 0.994 Carbaryl 94 3 0.999 112 8% 0.999 Chlorpyrifos 86 6 0.998 99 10% 0.946 Chlorpyrifos-methyl 82 3 0.996 115 5% 0.988 Cyprodnil 89 2 0.999 107 5% 0.999 Dichlorvos 34 37 0.768 106 6% 0.998 Ethion 95 3 0.999 106 7% 0.999 Imazalil 92 3 1.000 87 10% 0.984 Kresoxim-methyl 94 2 0.997 107 6% 0.993 Methamidophos 74 3 0.999 113 9% 0.995 Tebuconazole 91 3 0.999 105 7% 0.998 Thiabendazole 95 2 0.997 96 13% 0.986 Tolyfluanid 90 2 0.997 95 15% 0.992
Conclusions Results for all analytes met AOAC performance expectations by carefully optimizing analysis conditions Most appropriate platform selected for each analyte. Best technique, GC or LC, is analyte specific Methamidophos, imazalil and thiabendazole - best results with LC/MS Dichlorvos and procymidone - best results with GC/MS Injection solvent and solubility LC/MS -reconstitute samples with 25 % MeOH without filtering GC/MS -no solubility problems Matrix interferences External standardization for LC/MS analysis (if no stable label ISTDs) Internal standardization for GC/MS analysis
Trademarks Luna is a registered trademark, and Prodigy is a trademark of Phenomenex. Agilent is a registered trademark of Agilent Technologies. API 4000 is a trademark of AB SCIEX pte Ltd. AB SCIEX is being used under license. Disclaimer Phenomenex is not affiliated with Agilent Technologies. Comparative separations may not be representative of all applications. 2012 Phenomenex, Inc. All rights reserved.