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Dutch Mini-Luke (“NL-”) Extraction Method Followed by LC and GC-MS/MS for Multi- Residue Analysis of Pesticides in Fruits and Vegetables

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Dutch Mini-Luke (“NL-”) Extraction Method Followed by LC and GC-MS/MS for Multi- Residue Analysis of Pesticides in Fruits and Vegetables Dutch mini-Luke (“NL-”) extraction method followed by LC and GC-MS/MS for multi- residue analysis of pesticides in fruits and vegetables Method developed by NVWA, Netherlands Food and Consumer Product Safety Authority, NRL for Pesticide Residues in Food and Feed in collaboration with EURL-FV CONTENTS 1. Aim and scope ................................................................................................................. 2 2. Short description ................................................................................................................ 2 3. Apparatus and consumables ......................................................................................... 2 4. Chemicals ........................................................................................................................... 2 5. Procedure ........................................................................................................................... 3 5.1. Sample preparation ................................................................................................... 3 5.2. Recovery experiments for method validation ...................................................... 3 5.3. Extraction method ...................................................................................................... 3 5.4. Measurement .............................................................................................................. 4 5.5. Instrumentation and analytical conditions for the LC- MS/MS system .............. 4 5.5.1. Acquitty UPLC (Waters) ...................................................................................... 4 5.5.2. XEVO TQ-S triple quadrupole system (Waters) .............................................. 4 5.6. Instrumentation and analytical conditions for the GC- MS/MS system ............ 5 5.6.1. Varian CP-3800 GC (Bruker) .............................................................................. 5 5.6.2. Scion-TQ triple quadrupole system (Bruker) ................................................... 5 6. Validation of the method ................................................................................................ 5 6.1. Recoveries and within-laboratory reproducibility ................................................ 5 6.2. Limits of quantitation .................................................................................................. 6 6.3. Linearity ........................................................................................................................ 6 6.4. Matrix effects ............................................................................................................... 6 7. References ......................................................................................................................... 6 APPENDIX I: MASS TRANSITIONS .......................................................................................... 7 APPENDIX II: VALIDATION RESULTS .................................................................................... 12 Page 1 of 19 1. Aim and scope This report describes a validation data of 175 pesticides using a multiresidue method by LC-MS/MS and GC-MS/MS in lettuce and orange. 2. Short description Homogenous sample is extracted with acetone and partitioned with petroleum ether and dichloromethane. The obtained extract is analysed by GC-MS/MS and LC-MS/MS. 3. Apparatus and consumables Automatic pipettes, suitable for handling volumes of 10 µL to 5000 µL and 1 mL to 3 mL 250 ml PTFE centrifuge tubes 40 ml glass tubes with caps Vortex Turrax homogeniser Centrifuge, suitable for the centrifuge tubes employed in the procedure and capable of achieving at least 3300 rpm Water bath Injection vials, 2 ml, suitable for LC and GC auto-sampler Volumetric flasks 4. Chemicals Acetone p.a. Petroleum ether Dichloromethane Anhydrous sodium sulphate Ammonium formiate Ultra-pure water Methanol HPLC grade Isooctane Toluene Acetic acid Pesticides standards Page 2 of 19 5. Procedure 5.1. Sample preparation Following Document No. SANCO/12571/2013, the sample was perfectly homogenised by grinding finely at its arrival to the laboratory. 5.2. Recovery experiments for method validation The samples employed in validation studies did not contain any of the pesticides analysed. Individual pesticide stock solutions (1000–2000 mg/L) were prepared in toluene or methanol and were stored in screw-capped glass vials in the dark at -20 °C. For spiking, 15 g representative portions of previously homogenised sample were weighed in teflon tubes, where they were fortified homogenously with the appropriate amount of the working standard solution in methanol. The validation method was performed at three fortification levels (0.005, 0.01 and 0.02 mg/Kg). Six replicates were analysed at each level. 5.3. Extraction method 1. Weigh 15 g ± 0.1 g of sample in 250 mL PTFE centrifuge tube. 2. Add 75 µL of 10 mg/L propoxur and HCB-159 (procedure internal standards), 20 mL of acetone and 15 g Na2SO4. 3. Blend the sample using a Turrax homogeniser for 30 s at 1500 rpm (extraction step). 4. Add 20 ml of petroleum ether and 10 ml of dichloromethane. 5. Blend it again by Turrax for 30 s at 1500 rpm (partitioning step). 6. Centrifuge for 3 min at 3300 rpm. 7. Transfer the supernatant into 40 mL glass tube. 8. Evaporate an aliquot of the extract in a water bath programmed at 45ºC and continuing to 63ºC. The last part of the solvent was allowed to evaporate in the air. a. for LC analysis evaporate 0.66 mL extract and reconstitute with 1 mL of 0.025% of acetic acid in methanol containing quinalphos at 0.040 mg/kg (Injection Internal Standard) for LC analysis. Page 3 of 19 b. for GC analysis evaporate 5 mL extract and reconstitute with 1.5 mL of isooctane: toluene (9:1) containing 0.2 μg/mL of HCB-C13 and PCB-209 (Injection Internal Standards). With this treatment, 1 mL of sample extract represents 0.2 g of sample in LC and in GC the final matrix concentration is 1 g/mL. 5.4. Measurement Both LC and GC systems were operated in multiple reaction monitoring mode (MRM). Selected reaction monitoring (SRM) experiments were carried out to obtain the maximum sensitivity for the detection of the target molecules. For confirmation of the studied compounds, two SRM transitions and a correct ratio between the abundances of the two optimised SRM transitions (SRM2/SRM1) were used, along with retention time matching. The mass transitions used are presented in Appendix I. 5.5. Instrumentation and analytical conditions for the LC- MS/MS system 5.5.1. Acquitty UPLC (Waters) Column: Acquity UPLC BEH C18 2.1 mm x 100 mm and 1.7 µm particle size (Waters) Mobile phase A: 300 mg/L ammonium formiate in milliQ water Mobile phase B: methanol Column temperature: 40ºC Flow rate: 0.45 mL/min Injection volume: 1 µL Mobile phase gradient for pesticides analyse Time [min] Mobile phase A Mobile phase B 0 90% 10% 0.25 90% 10% 7.75 0% 100% 8.50 0% 100% 8.51 90% 10% Re-equilibration with initial mobile phase: 1.5 minutes. 5.5.2. XEVO TQ-S triple quadrupole system (Waters) Ionisation mode: Positive mode ESI source gas temperature: 150 °C Desolvation temperature: 600 ºC Desolvation gas flow: 1200 L/h Page 4 of 19 Cone gas flow: 150 L/h Nebuliser gas: nitrogen Capillary voltage: 1.8 kV Collision gas: argon 5.6. Instrumentation and analytical conditions for the GC- MS/MS system 5.6.1. Varian CP-3800 GC (Bruker) Column: Varian VF-5ms 30 m × 0.25 mm ID and 0.25 µm Injection mode: LVI-PTV, solvent vent Open liner with carbofrit Injection volume: 5 µl Injector temperature: held at 80°C (1 min) and then ramped up to 300°C at 200°C/min. This temperature was held for 19.5 min. Carrier gas: helium at constant flow of 1 mL/min Carrier gas purity: 99.999% Oven temperature: 80°C for 1 min, programmed to 180°C at 25°C/min, then to 280°C at 8°C/min and finally to 300°C at 30°C/min and kept at 300ºC for 3.17 min. 5.6.2. Scion-TQ triple quadrupole system (Bruker) Ionisation mode: electron impact ionisation Temperature of the transfer line: 280 °C Temperature of ion source: 250 °C Temperature of manifold: 40 °C Collision gas: argon Collision gas purity: 99.999% Solvent delay: 3.3 minutes 6. Validation of the method 6.1. Recoveries and within-laboratory reproducibility The results corresponding to the mean recovery (n=6) and within- laboratory reproducibility in terms of relative standard deviation (RSDr) at three fortification levels (0.005, 0.01 and 0.02 mg/kg) are summarized in Appendix II, Table 3. Almost all the recoveries results are within the range 70-120% except biphenyl, nitempyram, propamocarb in lettuce and aminopyralid, bifenazate, biphenyl, methamidophos, nitempyram and propamocarb in Page 5 of 19 orange. It could be explained because the high water solubility of methamidophos, nitempyram and propamocarb; high vapour pressure of biphenyl; bifenazate is a strong base and aminopiralid had a poor chromatographic behaviour. 6.2. Limits of quantitation Document N° SANCO/12571/2013 defines limit of quantitation as the lowest validated spike level meeting the method performance acceptability criteria. LOQs are summarized in Appendix II, Table 4. The LOQ for 97% of the pesticides is 0.005 mg/kg in lettuce and 96% in orange. 6.3. Linearity Linearity of the tq-MS systems was evaluated by assessing the signal responses of the target analytes from matrix-matched calibration solutions prepared by spiking blank extracts at seven concentration levels, from 0.0005 to 0.100
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