Analysis of 16 Pesticide Residues in Broccoli Using CarbonX dspe QuEChERS AOAC Kits Using GC/MS

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1 Analysis of 16 Pesticide Residues in Broccoli Using CarbonX dspe QuEChERS AOAC Kits Using GC/MS Authors: Conor Smith, Doug Fryer United Science Corp Furuby Rd. Center City, MN USA Abstract This application note describes the use of a quick, easy, cheap, effective, rugged, and safe (QuEChERS) AOAC sample preparation technique used in the extraction and cleanup of 16 pesticide residues in broccoli. The pesticides are all amenable to GC/MS analysis. The method involves the extraction of the pesticides from homogenized broccoli with a buffered aqueous/acetonitrile solvent system with partitioning using added salt. This is followed by cleanup using dispersive solid phase extraction (dspe) with CarbonX for QuEChERS (to remove pigments and sterols), PSA (to remove sugars, fatty acids, and organic acids), and MgSO 4 (to remove leftover water). The pesticides are then analyzed by GC/MS operating in selective ion monitoring (SIM) mode. The method was validated in terms of recovery and reproducibility. All pesticides were evaluated at 50 ng/g. The application produced excellent recoveries for all pesticides, including planar pesticides, without the need for the addition of toxic solvents such as toluene.

2 Reagents and Chemicals All reagents and solvents were high-performance liquid chromatography (HPLC) grade. Methanol (MeOH) was from sigma Aldrich, Acetonitrile (ACN) was from J&W, and glacial acetic acid (HAc) was from Ampresco. All pesticide standards were purchased from Sigma-Aldrich. Solutions and Standards A 1% acetic acid in ACN solution was prepared by adding 10 ml of HAc to 1 L of ACN. Standard stock solution (2 mg/ml) was made in MeOH and stored at -20 C. This solution was diluted to a spiking solution (5 µg/ml in MeOH) for use in the method. Sample Preparation The sample preparation procedure includes sample comminution, extraction and partitioning, and dispersive SPE clean-up. Two 15 g (±0.1 g) amounts of homogenized broccoli sample are placed into two 50 ml centrifuge tubes. For the spiked sample, 150 µl of spiking solution is added, and then the tube is vortexed for 30 seconds. Next, 15 ml of 1% Acetic Acid in ACN is added to each tube. Finally, an extract packet containing 6g of MgSO 4 and 1.5 g NaAcetate is added. (United Science P/N ) is added. Sample tubes are capped tightly, and hand-shaken vigorously for 1 min. Tubes are centrifuged at 4000 rpm for 5 min. Next, the ACN extracts are decanted into separate containers. The volume of ACN extracts (about 10 ml per extract) will be enough for multiple sample tests when using 2 ml dispersive SPE tubes (United Science P/N ). The 2 ml tubes contain 50 mg of PSA, 50 mg of CarbonX for QuEChERS, and 150 mg of anhydrous MgSO 4. We suggest vortexing the tubes for a few seconds before adding the sample, to prevent possible agglomerates. For both the spiked and unspiked extract, 1 ml is added to the 2 ml dspe tubes. At least three tubes are used for the unspiked extract so that there is sufficient cleaned up matrix for the matrix standard. The tubes are tightly capped and vortexed for 1 min. The 2 ml tubes are centrifuged with a micro-centrifuge at 13,000 rpm for 2 min. The spiked samples are then transferred directly into autosampler vials for GC/MS injection. For the matrix standard, 990 µl of cleaned up extract is added to a vial, followed by 10 µl of spiking solution. This mixture is then added into an autosampler vial for GC/MS injection. Recovery and Reproducibility The recovery and reproducibility are evaluated by spiking pesticide standards in comminuted broccoli sample at 50 ng/g. These samples were quantitated against a matrix standard. The ACN extract is spiked with microliter amounts of the pesticide mixture in MeOH. The standard is prepared by first removing the pigment through dspe with the CarbonX for QuEChERS, PSA, and MgSO 4 tube, followed by spiking of the resulting matrix blank. The analysis was performed in replicates of three (n = 3) at each level.

3 Instrument Condition An Agilent 5890 GC/MS was used for this study. GC conditions Inlet: Splitless Inlet liner: Agilent Ultra Inert splitless single taper wool p/n Carrier gas: Helium Inlet pressure: 19.6 psi (constant pressure mode) during run Inlet temperature: 250 ºC Injection volume: 1.0 µl Purge flow to split vent: 30 ml/min at 0.75 min Oven temperature program: 70 ºC (1 min), 50 ºC/min to 150 ºC (0 min), 6 ºC/min to 200 ºC (0 min), 16 ºC/min to 280 ºC (6 min) Column: Agilent J&W HP-5MS Ultra Inert 15 m 0.25 mm, 0.25 µm (p/n 19091S-431UI) MSD MS conditions Tune file: Atune.u Mode: SIM (refer to Table 1 for settings in detail) Source, quad, transfer line temperature: 230 ºC, 150 ºC and 280 ºC respectively Solvent delay: 2.30 min Multiplier voltage: Autotune voltage Table 1 SIM acquisition parameters used for the analysis of 18 pesticides by GC/MS Analyte SIM Collection window (min) RT (min) 1 Dichlorvos σ-Phenylphenol 170.1, Diazinon 137.1, Chlorpyrifos-methyl 285.9, Dichlorobenzophenone 250.0, Chlorpyrifos 196.8, Heptachlor epoxide 352.8, Folpet 259.9, γ-Chlordane 372.8, α-Chlordane 372.8, Dieldrin 262.9, DDE 245.9, Ethion Endosulfan sulfate Permethrin , Coumaphos 362.0,

4 ,6 15, * 11 8 Figure 1. GC/MS Chromatogram of 50 ng/g fortified broccoli sample extract processed with CarbonX dspe method. Peak identification: 1. Dichlorvos 2. Phenylphenol 3. Diazinon 4. Chloropyrifos-methyl 5. Dichlorobenzophenone 6. Chloropyrifos 7. Heptepoxide 8. Folpet 9. γ- Chlordane 10. α-chlordane 11. Dieldrin 12. DDE 13. Ethion 14. Endosulfan Sulfate 15. Permethrin 16. Coumaphos, * Internal Standard Atrazine.

5 Compound %Recovery %RSD Dichlorvos Phenylphenol Atrazine Diazinon Chlorpyrifos-methyl DCBP Chlorpyrifos Heptechlor Epoxide Folpet γ chlordane α chlordane Dieldrin DDE Ethion Endosulfan Sulfate Permethrin Coumaphos Recovery (%) Dichlorvos Phenylphenol Atrazine Broccoli Matrix with CarbonX for QuEChERS dspe Cleanup Figure 2. Pesticide recovery data for CarbonX at 50 ng/g. Note good recovery of planar pesticides Dichlorobenzophenone, Folpet, and Coumaphos without the need for additional solvents. Diazinon Chlorpyrifos-methyl DCBP Chlorpyrifos Heptechlor Epoxide Folpet γ chlordane α chlordane Dieldrin DDE Ethion Endosulfan Sulfate Permethrin Coumaphos

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