Summary
The European Standard EN 15662 method for pesticide residues in foods is described for the analysis of 25 pesticides and 2 internal standards from lettuce using a Phenomenex roQ QuEChERS EN 15662 Extraction Kit and roQ dSPE Kit for sample preparation. Analysis was performed by LC/MS/MS using a Luna® C18(2) HPLC column and GC/MS using a ZebronTM ZB-5MSi column. The roQ dSPE Kit effectively removed matrix interferences from the lettuce matrix and featured ease of use aspects as compared to other QuEChERS kits including leak-free tubes and user friendly extraction salt packets. Recoveries of all 25 pesticides were between 70 to 120 % with sample-to-sample variability <15 %. The lower limit of quantification (LLOQ) was well below the maximum residue limit reported in the International Maximum Residue Level (MRL) Database, demonstrating excellent performance of the roQ QuEChERS product for food safety testing.
TN-0052 APPLICATIONSIntroduction
In the past, sample preparation for multiresidue pesticide screening from complex matrices entailed long and laborious extraction procedures using various analyte specific techniques. In recent years sample preparation has been consolidated with the introduction of the QuEChERS method (Quick, Easy, Cheap, Effective, Rugged, and Safe) which has provided useful and somewhat universal protocols for the cleanup of food samples, especially when analyzing multiple classes of compounds. Following the same principles as the AOAC official method 2007.01 for pesticide residues in foods, the EN 15662 QuEChERS method begins with the extraction of pesticides from food samples using acetonitrile. Addition of magnesium sulfate induces phase separation of acetonitrile and water for liquid-liquid partitioning to occur. Buffering salts such as sodium chloride, sodium citrate and disodium citrate hydrogenate sesquihydrate can then be added to control the pH, maintaining a pH level between 4 and 6, for stability of base sensitive pesticides. After extraction, the top organic layer is then treated with a dispersive blend of anhydrous MgSO4 to remove moisture from the acetonitrile and weak anion-exchange SPE sorbent (PSA) to remove fatty acids from the extract while leaving the target analytes in solution. Different blends of SPE sorbents are used depending upon the nature of the sample to achieve sufficient cleanup. Magnesium sulfate aids in removing excess water while reversed phase C18 bonded silica (C18-E) and primary and secondary amine bonded silica (PSA) remove fats and organic acids, respectively. For pigmented samples, graphitized carbon black (GCB) can be added to remove matrix interferences due to pigments. In this study, the roQ brand of QuEChERS kits was selected to cleanup lettuce samples for pesticide analysis. Lettuce is one of the most consumed leafy greens. Besides having a high water content, lettuce contains vitamins, minerals, plus small amounts of sterols, proteins and sugars.1 When analyzing our lettuce samples, 25 pesticides and 2 internal standards were extracted using a roQ dSPE kit containing MgSO4 and PSA (p/n KS0-8928). The split final extracts were further analyzed using LC/MS/MS as well as GC/MS.
Experimental Conditions
Reagents and Chemicals
Pesticide standards and triphenylphosphate (TPP) were obtained from Accustandard (New Haven, CT), Ultra Scientific (N. Kingstown, RI), and Supelco (Bellefonte, PA). HPLC grade water (Milli-Q, Millipore, Billerica, MA) was used to prepare HPLC mobile phase and for sample preparation. Methanol and acetonitrile (ACN) were obtained from Honeywell Burdick & Jackson (Muskegon, MI). Toluene was obtained from Fisher Scientific (Waltham, MA). Acetic acid and formic acid were obtained from Sigma-Aldrich (St. Louis, MO). Solutions and Standards
Standard pesticide mix (80 μg/g) stock solutions were prepared in 0.1 % Formic acid in acetonitrile. A QC spiking and stock solution of 40 μg/mL was prepared by dilution from the standard pesticide mix in acetonitrile containing 0.1 % Acetic acid. Calibration curve standard solutions (5, 10, 50, 100, 250, and 1000 ng/g) were prepared from 2, 10, and 40 μg/mL solutions in acetonitrile containing 0.1% Acetic acid by serial dilution. A 2 % TPP solution in acetonitrile (1 % Acetic acid) was used to determine system suitability. d10-Parathion and d6-α-HCH were used as internal standards. Sample Preparation
Lettuce was chopped into 2-4 cm pieces, placed into a zip-lock bag, and stored in a -80 °C freezer for at least 24 hours prior to further processing. The lettuce was first immersed in liquid nitrogen and homogenized in a blender to generate a powdery consistency.
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