300 mg of bee pollen samples were homogenized in a mortar and pestle, then diluted 1:10 in Milli-Q water. 12 mL of 2% triethylamine in acetonitrile was added to the pollen slurry, vortex mixed, and then added to a QuEChERS AOAC tube 1 (6 mg MgSO4 and 1.5 g sodium acetate). Upon centrifugation, the organic layer was removed and diluted to 15% water content and passed through a Sep-Pak C18 1-cc Cartridge under vacuum. The resulting extract was dried down to near dryness under a gentle stream of nitrogen, and reconstituted in four times the original volume in water.
The samples were then analyzed on an ACQUITY UPLC I-Class System, coupled to Xevo TQ-S. The MRM transition 256.2 → 175.2 was used for quantification, and 256.2 → 209.2 for confirmation. Extraction recovery and matrix suppression were assessed using store-bought organic bee pollen nutritional supplement spiked with 1 ng/g imidacloprid standard, and those results are displayed in Table 1. Both matrix-matched and solvent calibration curves were used for quantification of samples and recovery tests, respectively, and had an r2 > 0.99. Method LOD and LOQ were determined by matrix-matched standards that exceeded a peak-to-peak signal-to-noise (S/N) of 3 and 10, respectively. Well conserved ion ratios were used as an additional criterion for identification.
Of the samples from the exposure area tested, imidacloprid residues were detected in three bee pollen samples, though below the LOQ in matrix, with the exception of one sample that contained 29.3 ng/g. MRMs of the aforementioned sample, one below the LOQ, and a matrix-matched 100 pg/g standard, which surpassed the peak-to-peak S/N ratio of 10, and was considered the method LOQ, as shown in Figure 2 a, b and c.
