Determination of Pesticides in Strawberries: A Comparison of Sample Preparation Techniques SPME and QuEChERS Blair Surridgex, Brenna Stanfordx, Rene Bilodeaup xApplied pMB Chemistry and Biotechnology Camosun College,Victoria BC Laboratories Ltd,, Sidney BC Project Goals 1) To evaluate conditions for SPME-GC/MS for a diverse set of pesticide compounds. 2) To compare extraction methods of SPME to QuEChERS for select pesticides. 3) To evaluate DRS as an option to SIM for the identification of pesticides. Results - Calibration Introduction Calibration solutions were prepared using organic strawberries (Andrew and Williamson) . Example calibration curves for γ-chlordane is provided below for QuEChERS- left and SPME-right. A linear regression model was used with 1/x weighting in each case. For QuEChERS the internal standards cis-permethrin –C6 and chlorpyrifos-D10 were added before GC/MS analysis to correct for instrument variation. These internals standards were evaluated for SPME with limited suggest however more work is required. Ensuring pesticide residues in agricultural products are below acceptable limits is important to both growers and producers as well as the general public. Significant advances have been made to laboratory analysis1,2, however, the cost of analysis remains a barrier for extensive testing of commodities. Also, most tests do not cover an extensive list of pesticides. We have been investigating two methods of extracting of 73 pesticides including organonitrogens, organphosphates, organochlorines, and common contaminants such as PCBs and phthalates. These pesticides were selected as they were most frequently detected in a recently published CFIA report3. The automated solvent less extraction technique headspace SPME (solid-phase micro-extraction) was compared to QuEChERs (Quick, Easy, Cheap, Effective, Rugged and Safe). Analysis was carried out by GC/MS using both SIM and open scan acquisitions with retention time locking. Open scan acquisitions will allow for mass spectral analysis and identification using DRS (Deconvolution Reporting Software). The acetonitrile extracts from QuEChERS were also analyzed by LC-MS/MS, however this data is not reported here. . g-Chlordane g-Chlordane Response Response Ratio r2-0.998 4.00e+004 r2-0.984 Discussion 0.5 The HS SPME automated technique has been shown to be a simple and fast method of measuring a diverse set of organic pesticides and contaminants at low concentrations. 2.00e+004 0 0 1 2 Concentration Ratio 0 3 0 50 100 Concentration The detection limits were estimated as an amount equivalent to a signal-to-noise ratio of 3 to 1. This was calculated from a spiked sample at a low concentration prepared in organic strawberries. In order to calculate the % recovery homogenized strawberries were spiked at 90ng/g before being taken through the procedure. Quantitation was achieved using standards spiked into the final extract before GC/MS analysis. Select Compounds RT (min) Ion (m/z) Headspace SPME Method SPME LOD ng/g) QuEChERS LOD (ng/g) % Recovery QuEChERS SPME for 20minutes with a 65µm PDME fiber at 60oC Desorption in GC inlet at 270oC for 2 minutes Other preliminary extraction tests were carried out on the following vegetables and fruits: Green Beans, Radish, Tomatoes, and Grapes. Only radishes were purchased as an organic product. 12.7 197 0.06 5.4 108 Ethion 15.7 231 3.73 2.8 113 166 177 Fenthion 12.9 278 0.10 1.8 98 155 Malathion 12.7 125 13.24 NA 106 103 Organochlorines QuEChERS Method 15.0 grams of sample + 15mL of 1% AcOH in ACN I and mix Aldrin 12.9 66 0.04 3.3 79 147 a-BHC 9.9 183 0.02 2.9 131 117 b-BHC 10.4 219 0.04 15.4 95 92 14.2 375 0.01 0.5 92 138 141 a-Chlordane 14.4 375 10.9 266 2,4’-DDT 16.4 235 Dieldrin 11.9 263 Buprofezin 15.0 105 0.09 12.8 98 135 Cyprodinil 13.5 224 8.45 2.6 Na 130 Diphenylamine 9.1 169 0.13 2.6 101 93.7 GC : Agilent 7890 with CTC CombiPal including SPME/Headspace Triazine 18.5 239 1.26 3.6 74 132 Column: ZB-5MS, 30m x 0.25mm x 250µm (Phenomenex) Injection volume: 1µL Inlet Conditions: Pulsed Split less at 270oC 400 Retention Time Locking: Methyl chlorpyrifos , 12.7 min at 22.5 psi Permethrin 19.4 183 NA 1.4 105 g-Chlordane Add MgSO4/NaOAc mix then spin at 1500 rcf for 1minute Chlorothalonil Aliquot 1.0mL into tube containing PSA, MgSO4, and C18 Mix and spin at 1500 rcf for 1minute Add internal standards 0.03 1.1 95 1.95 18.8 100 88 0.06 0.7 93 165 3.02 3.1 103 107 Organonitrogens LC-MS/MS Analysis GC/MS Analysis Temperature (oC) The 30µm PDMS fiber gave reasonable results as a first fiber tested. A mixed phase fiber of DVB/PDMS was also evaluated. Most of the compounds dropped in peak area response and some remained constant. % Recovery SPME Chlorpyrifos Analysis by GC/MS (SIM & open scan acquisition) Ionization mode: EI (+) ion Source Temp: 250oC Acquisition: SIM (5 segments) mass scan: 50-400 m/z The reported column oven temperature was found to be optimal for most compounds. The initial column temperature was found to have a significant effect on peak shape and 70 oC was determined to be optimal. Organophosphates 1.0 grams of sample + 3mL of 1% MeOH in NaCl sat’d water Mass Spec : Agilent 5975C The detection limits obtained by SPME were generally 80x lower than QuEChERS. Detection limits ranged from 0.5 to 18.8 ng/g for QuEChERS and 0.01 to 3.7 ng/g for SPME. Internal standards were applied to the QuEChERS method of analysis. The internal standards used for QuEChERS were not suitable for SPME extraction therefore external standard quantitation was used for SPME. The %recovery for QuEChERS was generally better (mean-95%, RSD-9.2%) than SPME (mean -128%, RSD-21%). Reasonable calibration curves were obtained (r2>0.98) for SPME however QuEChERS calibration curves were typically better (r2>0.99). 150 Results – Detection Limits and % Recovery Experimental Methods Approximately 1kg of fresh strawberries were chopped in a Osterizer food processor. A 0.2kg portion was then homogenized thoroughly using a with a Braun hand blender. This material was then frozen before analysis. The initial sample extraction procedure and the aqueous solvent for headspace SPME was reported elsewhere4. A DVB/PDMS fiber was also evaluated under the conditions shown below. The procedure for QuEChERS has been reported elsewhere5. GC/MS Analysis GC/MS Chromatogram of Cyprodinil in a strawberry sample Concentrations: QuEChERS: 582 ppb HS SPME: 700 ppb Column Oven Temperature Program Future Work 1. Different SPME fibers types/sizes and extraction temperatures will be investigated to improve extraction recovery of organonitrogens and organophoshate compounds. One such fiber is the 85µm polyacrylate fiber. 2. Direct immersion SPME technique will also be investigated to improve extraction recovery. To accomplish this the extract containing the extracted material will filtered or spun using a centrifuge. 3. Triphenyl phosphate will be evaluated as an internal standard for SPME and QuEChERs. 4. Application of deconvolution reporting software (DRS) will be investigated further as a method of detection . 5. To apply the developed methods to other fruits and also to vegetables. Acknowledgements Funding for this project was obtained through NSERC’s College and Community Innovation (CCI) Applied Research and Innovation (ARTI) fund. We would like to thank Wendy Riggs and Dr. Harry Hartman from MB Labs Ltd for time taken to set up the project. References 1. 2. 3. 4. 5. Lehoytay, S.,Kok A., Hiemstra M., Bodegraven P. (2005) J. AOAC Int.88, 595 Navalon A., Prieto A., Araujo L., Vilchez J. (2002) J.Chrom. A. 975, 355 CFIA Food Safety Action Plan Report, 2009-2010 Targeted Surveys Chemistry Thermo Scientific Application Note: 51943 AOAC Official 2007.01 method Contaminants Anthracene 11.1 178 0.08 2.8 101 122 Phenanthene 10.9 178 0.04 2.7 92 121 PCB 28 11.9 256 0.03 1.8 85 131 PCB 101 14.3 326 0.04 1.3 86 117 300 200 100 0 0 10 Time (mins) 20 30 LOD – limit of detection RT – retention time m/z – mass to charge This poster was presented at the Western Canada Trace Organics Conference