Validation Data of 127 Pesticides Using a Multiresidue ...

1 1 Validation data of 127 Pesticides Using a Multiresidue Method by LC-MS/MS and GC-MS/MS in Olive Oil UNIVERSITY OF ALMERIA CITE I Dpto. Hidrogeolog a y Qu mica Anal tica Ctra. Sacramento s/n. La Ca ada de San Urbano 04120 ALMER A, SPAIN Tel (+34) 950 01 50 34/55 31 PESTICIDE RESIDUE LABORATORY AGRO-FOOD ANALYSIS SERVICE Direcci n General de Innovaci n Agraria y Ganader a Conseller a de Agricultura, Pesca y Alimentaci n GENERALITAT VALENCIANA Pintor Goya, 8, 46100 Burjassot-VALENCIA, SPAIN Tel (+34) 963 631 551 UNIVERSITY OF ALMERIA CITE I Dpto. Hidrogeolog a y Qu mica Anal tica Ctra. Sacramento s/n. La Ca ada de San Urbano 04120 ALMER A, SPAIN Tel (+34) 950 01 50 34/55 31 PESTICIDE RESIDUE LABORATORY AGRO-FOOD ANALYSIS SERVICE Direcci n General de Innovaci n Agraria y Ganader a Conseller a de Agricultura, Pesca y Alimentaci n GENERALITAT VALENCIANA Pintor Goya, 8, 46100 Burjassot-VALENCIA, SPAIN Tel (+34) 963 631 551 3 CONTENTS 1.

2 Aim and Scope 1 2. Short Description 1 3. Apparatus and Consumables 1 4. Chemicals 1 5. Procedure 2 Sample Preparation 2 Recovery Experiments for Method Validation 2 Extraction 2 Measurement 3 Instrumentation and analytical conditions for the LC-MS/MS system 3 Instrumentation and analytical conditions for the GC-MS/MS system 4 6.

3 Validation of the method 5 Recoveries and within-laboratory reproducibility Limits of Quantification 5 Linearity 5 7. Conclusions 8 8. References 8 Appendix I - Mass Transitions 9 Appendix II - Validation Results 13 Page 1 UNIVERSITY OF ALMERIA CITE I Dpto. Hidrogeolog a y Qu mica Anal tica Ctra. Sacramento s/n. La Ca ada de San Urbano 04120 ALMER A, SPAIN Tel (+34) 950 01 50 34/55 31 PESTICIDE RESIDUE LABORATORY AGRO-FOOD ANALYSIS SERVICE Direcci n General de Innovaci n Agraria y Ganader a Conseller a de Agricultura, Pesca y Alimentaci n GENERALITAT VALENCIANA Pintor Goya, 8, 46100 Burjassot-VALENCIA, SPAIN Tel (+34) 963 631 551 1 1.

4 Aim and Scope This report describes a Validation data of 127 Pesticides Using a Multiresidue method by LC-MS/MS and GC-MS/MS in olive oil. 2. Short Description The homogeneous sample is diluted with n-hexane and extracted with acetonitrile. The purification is carried out Using a SPE cartridge with C18 (500 mg) as sorbent. The extract obtained is analysed by GC-MS/MS and LC-MS/MS. 3. Apparatus and Consumables Automatic pipettes, suitable for handling volumes of 10 L to 500 L and 1 mL to 3 mL. 50 ml centrifuge tubes with screw caps ( Sharlab S. L, Spain, article-no 027 409926). Centrifuge, suitable for the centrifuge tubes employed in the procedure and capable of achieving at least 1000 rpm. C18 SPE cartridges ( Scharlau Extrabond C18 500 mg 6mL). Syringes, 2 mL disposable syringes. Syringe filters, m pore size.

5 Injection vials, 2 ml, suitable for LC and GC auto-sampler. Inserts of 300 L suitable for injection vials ( Waters insert 300 L 6X29 mm with preinstalled plastic spring). Test tubes. Volumetric flasks. Concentration Workstation, TurboVap LV, Zymark. 4. Chemicals n-Hexane 96% HPLC grade Acetonitrile HPLC grade Methanol HPLC grade Cyclohexane GC grade Ultrapure water Pesticides Standards, Dr. Ehrenstorfer, Sigma Aldrich. Page 2 UNIVERSITY OF ALMERIA CITE I Dpto. Hidrogeolog a y Qu mica Anal tica Ctra. Sacramento s/n. La Ca ada de San Urbano 04120 ALMER A, SPAIN Tel (+34) 950 01 50 34/55 31 PESTICIDE RESIDUE LABORATORY AGRO-FOOD ANALYSIS SERVICE Direcci n General de Innovaci n Agraria y Ganader a Conseller a de Agricultura, Pesca y Alimentaci n GENERALITAT VALENCIANA Pintor Goya, 8, 46100 Burjassot-VALENCIA, SPAIN Tel (+34) 963 631 551 25.

6 Procedure Sample preparation Samples were prepared according to the procedure described in the document Method Validation and Quality Control Procedures for Pesticide Residues Analysis in Food and Feed (Document N SANCO/12495/2011). The samples were homogenised by shaking and were stored at room temperature before analysis. As olive oil is a processed sample it should be analysed within the stated shelf life. Recovery Experiments for Method Validation The olive oil employed did not contain any of the Pesticides analysed. Olive oil obtained from organically grown olives is recommended for the analysis. The Validation method was performed at two fortification levels ( mg/Kg and mg/Kg). Five spiked samples were analyzed at each level. Working standard solutions containing all the Pesticides used for the Validation method were prepared in two different solvents, acetonitrile for LC-MS/MS and cyclohexane for GC-MS/MS.

7 Extraction 1. Weigh g g sample in 50 mL centrifuge tube. 2. Add mL of n-hexane. Shake the tube for 20 seconds. 3. Add 6 mL of acetonitrile. 4. Shake vigorously for 10 minutes. 5. Centrifuge for 2 min at 1000 rpm . 6. Condition a C18 SPE cartridge with 5 mL acetonitrile (ACN) 7. Introduce 3 mL of the extract from the upper phase (ACN) into the C18 SPE cartridge. 8. Elute with 3 mL of ACN. 9. Collect the eluate and repeat the elution with 3 mL of ACN. 10. Take 1 mL of the eluate and evaporate to dryness. Page 3 UNIVERSITY OF ALMERIA CITE I Dpto. Hidrogeolog a y Qu mica Anal tica Ctra. Sacramento s/n. La Ca ada de San Urbano 04120 ALMER A, SPAIN Tel (+34) 950 01 50 34/55 31 PESTICIDE RESIDUE LABORATORY AGRO-FOOD ANALYSIS SERVICE Direcci n General de Innovaci n Agraria y Ganader a Conseller a de Agricultura, Pesca y Alimentaci n GENERALITAT VALENCIANA Pintor Goya, 8, 46100 Burjassot-VALENCIA, SPAIN Tel (+34) 963 631 551 3 11.

8 Reconstitute with 1 mL of ACN: H2O 1:9 (v:v). Sake the sample in the vortex and filter with a m syringe filter into an injection vial suitable for HPLC-MS/MS. This extract contains g of sample per mL. 12. Transfer mL eluate into a test tube. Evaporate to dryness 13. Add mL cyclohexane 14. Sake the sample in the vortex. 15. Filter into an injection vial suitable for GC-MS/MS. This extract contains g of sample per mL. Measurement Both LC and GC systems were operated in multiple reaction monitoring mode (SRM). 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.

9 The mass transitions used are presented in Appendix I. Instrumentation and analytical conditions for the LC- MS/MS system LC-MS/MS System 3200 Q TRAP, Applied Biosystems: - Column: Atlantis T3 3 m mm - Column temperature: 40 C - Mobile phase A: 2 mM ammonium formate in water, formic acid. - Mobile phase B: methanol - Injection volume: 5 L - Autosampler temperature: 10 C Page 4 UNIVERSITY OF ALMERIA CITE I Dpto. Hidrogeolog a y Qu mica Anal tica Ctra. Sacramento s/n. La Ca ada de San Urbano 04120 ALMER A, SPAIN Tel (+34) 950 01 50 34/55 31 PESTICIDE RESIDUE LABORATORY AGRO-FOOD ANALYSIS SERVICE Direcci n General de Innovaci n Agraria y Ganader a Conseller a de Agricultura, Pesca y Alimentaci n GENERALITAT VALENCIANA Pintor Goya, 8, 46100 Burjassot-VALENCIA, SPAIN Tel (+34) 963 631 551 4 FLOW RATE AND ELUTION GRADIENT Time (min) A (%) B (%) Flow ( L/min)

10 0,0 95 5 300 1,0 95 5 300 1,1 70 30 300 10,0 0 100 300 13,0 0 100 300 13,1 95 5 300 Instrumentation and analytical conditions for the GC-MS/MS system - GC: Agilent 7890 Series - Autosampler: Agilent 7683 Injector and sample tray - Inlet: Splitless - Carrier gas: Helium - Inlet pressure: psi - Inlet temperature: 250 C - Injection volume: 1 L - Oven temperature program: Rate ( C/min) Value Hold Time (min) Run Time (min) Initial 70 2 2 Ramp 1 25 150 0 Ramp 2 3 200 0 Ramp 3 8 280 10 - Analytical column: Agilent J W HP-5ms 30 m x 250 m x m - Retention time locking: Chlorpyrifos methyl locked to min - Spectrometer: Agilent 7000B Series - Source temperature: 280 C - Quadrupole temperature.