Analysis of QuEChERS Extracts of a Variety of Foods for Pesticide Residues using Automated SPE Coupled to GC/MS/MS and LC/MS/MS

Mark Hayward,1 Peter Ratsep,2 Jonathan Ho,2 Rick Youngblood,1 and Kim Gamble1

1ITSP Solutions Inc., Hartwell GA USA and 2Shimadzu, Somerset NJ USA

Introduction• QuEChERS extracts of foods and concentrated food ingredients can

contain significant amounts of sugars, sterols, fatty acids, colors, and oils that can interfere with GC/MS/MS or LC/MS/MS analyses of pesticide residues. Effects commonly observed are suppression of pesticide ion formation, instrument fouling, and poor correlation of spiked sample responses with external or non-structurally related internal standards. We took great interest in the announcement (B. Morris et. al. from Hill Labs, NZ, NACRW – 2014) of new custom blends of SPE sorbents designed specifically to remove each of these interfering compound classes resulting in very consistent measurements quantified with external solvent only standards. As a result, we tested these sorbents in a variety of food measurements with focus on minimizing matrix effects. Also, we evaluated this SPE approach for fully automated on-line SPE-GC/MS/MS and SPE-LC/MS/MS.

Technology used and samples• SPE sorbents: Blend of C18, Z-Sep, CarbonX for extraction for

LC/MS/MS (ITSP part# 30-UC18HX-T 30mg - ITSP QuEChERSBlend for LC, 96/Tray) and a blend of MgSO4, PSA, C18, CarbonX for extraction for GC/MS/MS (ITSP part# 45-UMPCX-T - 45mg ITSP QuEChERS Blend for GC, 96/Tray)

• GC/MS/MS: Shimadzu TQ-8040 with 2010 GC• LC/MS/MS: Shimadzu TQ-8050 with Nexera X2 LC• Autosampler: CTC PAL-xt and 6 port injection valve for LC.

This autosampler also is used to automate SPE.• Foods studied: Apples, Lemons, Lettuce, and Wheat• QuEChERS extractions: AOAC 2007.01 (ACN/food ratio is 1/1

for all except wheat 3/1)• Sample source: Same sample (extracts) and standard set (in

ACN) used in the USDA study described in M A Gonzalez-Curbelo et. al., J Chrom A, 1358 (2014) 75-84. Many thanks to Steve Lehotay at USDA.

Study Goals• Apply the SPE approach described by Hill Labs (B.

Morris et. al., NACRW – 2014) to samples obtained from USDA to evaluate its utility

• Use USDA analytical approaches as starting point and optimize both SPE and analysis approaches further

• Measure pesticide variance and SPE recoveries against external and internal standards

• Measure SPE performance at multiple sample dilutions to isolate effects coming from the matrices

• Measure analytical performance using 2 analytical methods (fast/slow) to isolate effects coming from pesticides interfering with each other in the analysis (achieved for GC, fast only for LC – clock ran out before ASMS)

Hill Labs – SPE methods(scavenging, not catch and release)

• For GC/MS/MS:– Condition SPE cartridge with 200 µl ACN (to waste)– Load 100 µl QuEChERS extract (collect)– Elute with 100 µl ACN (collect)– Add 20 µl protectants solution (if not measuring same day)

• For LC/MS/MS:– Condition SPE cartridge with 150 µl ACN (to waste)– Load 150 µl QuEChERS extract (collect)– Elute with 150 µl MeOH/ACN (50/50) (collect)– Add 30 µl protectants solution (if not measuring same day)– Dilute 4x in buffer (ammonium formate)

• Syringe wash (full volume, developed for this study)

ACN 3x, ACN/MeOH/acetone/H2O (1/1/1/1) 10x, ACN 3xThis approach yields measured carryover ≤0.1% for all pesticides

MRMs for LC/MS/MS

LC/MS/MS chromatogram (short)

GC conditions (short)Rate Final Temperature Hold Time

xx 90 3

55 180 0

25 290 4.96

total time: 14 min

Column:Rtx-Hydrogrd (0.18mm x 5m) couple with Rtx-5MS (0.53 mm x 15m; 1.00 mm film)

GC Parameters

Column Oven Temp 90 C

Injection Temp 280 C

Injection mode Splitless

Sampling time 1.00 min

Flow Control Mode Constant Linear Velocity

Pressure 35.4 kPa

Total Flow 4.5 mL/min

Column Flow 1.00 mL/min

Linear Velocity 90.7 cm/sec

Purge flow 0.5 mL/min

Split Flow 3

MS parameters

Ion Source Temp 200 C

Interface Temp 209 C

Sovlent Cut time 2.75 min

Detector Voltage Relative to tuning result: 0.2kV

Threshold 0

All pesticides evaluated with external solvent only standards except fenthion

Tolclofos-methyl

CarbarylPirimiphos-methyl

ChlorpyrifosFenthion

Fenthion-d6

Dichlorofluanid

Triflumizole

FolpetMethidathion

o,p'-DDE

Chlordane

Tricyclazole Fenthion sulfone

GC/MS/MS chromatogram (short)

Dimethoate

Carbofuran

Atrazine

Atrazine-d5

gamma-BHC (Lindane)

Atrazine-d5

Diazinon

Chlorothalonil

Tolclofos-methyl

Carbaryl

Pirimiphos-methyl

Dichlorofluanid

Fenthion

Fenthion-d6

ChlorpyrifosTriflumizole

Folpet o,p'-DDE

Methidathion

Chlordane

Tricyclazole

GC/MS/MS chromatogram (long)

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Recovery vs. Standard Dev. Short Method

ISs & QC

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Recovery vs. Dilution Factor Short method

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Recovery vs. Dilution Factor Long Method

Clearly, both pre-dilution of the extracts and chromatographic (GC) performance play crucial roles in determining recoveries and variability

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Recovery vs. RT (all dilutions)

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1x dilution vs. RT

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2x dilution vs. RT

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3x dilution vs. RT

Examining departures from ideal recovery as a function of RT may allow targeted (pesticide specific) improvement of chromatographic performance

Summary of results• The SPE methodology in production use by Hill Labs

shows great promise and that pesticides in foods can be measured using external solvent only standards

• Matching QuEChERS extract concentration (dilution) to the SPE sorbent capacity is an important part of the sample prep

• Achieving sufficient chromatographic resolution in the analysis is an important part of achieving quantitatively precise and accurate results

• While some recoveries can be lower than ordinarily expected (as low as 20%), they are remarkably reproducible across the concentration range and the 4 foods tested here

• The Hill Labs methodology clearly warrants further study

Next steps: future study• Complete the planned LC/MS/MS long method

testing• Further refine dilution results in the 5-25x range

to firmly establish the amount of dilution required

• Use a pesticide targeted approach to further refine the chromatographic separations to find out if further gains in performance can be achieved

• Adjust MeOH/ACN ratio in SPE elution as means to fine tune SPE selectivity

• Examine additional food types beyond the 4 examined here

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Recovery vs. Dilution Factor Long Method

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Recovery vs. STD Dev. Undiluted Pesticides measured by LC/MS/MS (short method)

When extracts are undiluted, many pesticide recoveries and some pesticide variances are outside the desired range

Pre-dilution prior SPE significantly improves both

It is our belief that focus on improving the LC separation will further improve both recoveries and variance (see GC example)

However, time ran out and we did not have the opportunity to test longer methodology prior to ASMS

Automation of on-line SPE-LC/MS/MS was easily achieved with the PAL system

LC conditions (short)• MPA – 0.1% NH4OAc and

0.1% HOAc buffer• MPB – ACN during peak

elution and 5% HOAc in ACN, acetone, and chloroform for LC & column rinsing steps

• Flow 0.5 ml/min during elution and 1.25 ml/min during rinsing

• Column: PhenomenexProdigy ODS3 150 x 3 mm with 5 mm particles

• Injection volume: 1 ml• Slower method is same as

fast except all times multiplied by 3

Time %MPB0.00 73.00 253.50 304.00 6710.00 7010.50 9511.10 10011.14 Pump B 5% HOAc in ACN11.15 Flow 1.25 ml/min12.71 Pump B acetone14.70 Pump B chloroform16.70 Pump B acetone18.50 Pump B 5% HOAc in ACN20.70 Pump B ACN20.71 721.80 Flow 0.5 ml/min23.00 End

All pesticides evaluated with external solvent only standards except atrazine

Pesticide GC RT Primary MRM Reference MRMDichlorvos 4.513 185.00>93.00

Phthalimde 5.344 147.00>103.00

tetrahydrophthalimide 5.419 151.00>79.00

Omethoate 5.848 156.00>110.00

Ethoprophos 5.988 158.00>97.00 200.00>158.00

Chlorpropham 6.02 213.10>127.10

Dimethoate 6.374 125.00>79.00

Diazinon 6.579 304.10>179.10

Carbofuran 6.375 164.10>149.10

Atrazine 6.403 215.10>200.10

Atrazine-d5 6.389 205.00>105.00 220.10>205.10

gamma-BHC (Lindane) 6.558 219.00>182.20

Chlorothalonil 6.796 266.00>133.00 266.00>231.00

Tolclofos-methyl 7.036 265.00>250.00

Carbaryl 7.037 144.10>115.10 144.10>116.10

Pirimiphos-methyl 7.169 290.00>125.00 305.10>180.10

Chlorpyrifos 7.345 314.00>258.00

Fenthion 7.329 278.00>109.00

Fenthion-d6 7.315 284.00>115.00

Dichlorofluanid 7.278 225.00>124.10 224.00>123.10

Dichlorofluanid 7.277 224.00>123.10

Triflumizole 7.725 278.10>73.00

Folpet 7.762 260.00>130.00 147.00>76.00

o,p'-DDE 7.856 246.00>176.00

Methidathion 7.828 145.00>85.00

trans-Chlordane / cis-Chlordane 7.977 373.00>266.00

Tricyclazole 8.136 189.00>162.00

Fenthion sulfone 8.388 310.00>109.00

Endosulfan sulfate 8.7 387.00>253.00

Triphenyl phosphate 8.87 326.00>169.00

Phosmet 9.06 160.00>77.00

Pyriproxyfen 9.34 226.00>186.00 136.00>78.00

Pyriproxyfen 9.34 136.00>78.00

Cypermethrin-1 10.66 181.10>152.10

Deltamethrin-1 (Tralomethrin deg.-1) 12.22 181.00>152.00 253.00>93.00

Azoxystrobin 12.58 344.10>183.10 344.10>329.10

Azoxystrobin 12.58 344.10>329.10

MRMs for GC/MS/MS