Analysis of Pesticide Residues in Pistachios Using QuEChERS Extraction and Cleanup with Supel™ QuE Z-Sep+

Pistachios contain approximately 45% fat, which can result in a significant amount of co- extracted matrix in the acetonitrile extract generated using the QuEChERS procedure. The use of a cleanup sorbent which can reduce this fat is essential to prevent fouling of LC-MS/MS and GC-MS/MS systems, and minimize ion suppression, thus allowing low level detection. In this application, Supel™ QuE Z-Sep+ sorbent was used as part of the QuEChERS method in the analysis of pesticide residues in pistachios. Z-Sep+ is a zirconia and C18 functionalized silica sorbent which acts to retain fatty constituents through both Lewis acid/base and hydrophobic interactions. The selectivity of the zirconia present in Z-Sep+ offers retention of a wider range of fats than C18 alone. In this application, QuEChERS extraction and cleanup using Z-Sep+ sorbent were used before the LC-MS/MS and GC-MS/ MS analysis of pesticide residues in pistachios. The targeted analyte list included pesticides relevant to pistachios.^4,5

Experimental

Pistachios were purchased from a local grocery store. They were frozen with liquid nitrogen (shells on), ground, and spiked at 10 ng/g with the pesticides listed in Tables 2 and 4, and allowed to equilibrate for 1 hour. Samples were then subjected to QuEChERS extraction and cleanup with Z-Sep+ following the procedure in Figure 1. A 100 μL aliquot of the final extract was diluted to 1 mL with 5 mM ammonium formate/0.1% formic acid in water, and analyzed by LC-MS/MS using the conditions shown in Table 1.

The remaining acetonitrile extract was analyzed directly by GC-MS/MS using the conditions shown in Table 3. Spiked samples were quantitated against 5-point matrix-matched calibration curves prepared in unspiked pistachio matrix blanks (after cleanup). No internal standard was used.

Results and discussion

Background

Initially, cleanup using Z-Sep+ sorbent was compared to PSA/C18, a common QuEChERS cleanup sorbent for fat-rich samples. A visual comparison of the QuEChERS extracts (in acetonitrile) is shown in Figure 1. Both cleanups removed some green color, resulting in similar light yellow extracts. GC-MS-scan comparisons (Figure 2) show lower background after Z-Sep+ cleanup compared to PSA/C18. The predominant peaks present in the uncleaned extract are fatty acids and monoglycerides. While PSA/C18 only reduced the levels of these compounds, almost none were detected after Z-Sep+ cleanup.

Pesticide Recovery

Table 5 shows the average %Recovery and %RSD for n=3 replicates of spiked pistachio samples. The majority of the pesticides were analyzed by LC-MS/MS; and those without sufficient response were analyzed by GC-MS/MS. Out of the 30 pesticides analyzed, 22 had recoveries within the generally accepted range of 70-120 %. Reproducibility was good, with RSD values < 20% for all 30 pesticides, and < 10% for many. Two pesticides, etoxazole and trichlorfon, had recoveries < 50%. Trichlorfon was most likely retained by the Z-Sep+ sorbent during the cleanup step. This could be due to the Lewis base character of the phosphate group present in its structure. Etoxazole, on the other hand, does not contain a phosphate group. It is a very lipophilic pesticide, indicated by its log P value of 5.6. Extraction efficiency of this compound from the fatty pistachio matrix was probably very poor using acetonitrile. Spinetoram, with a log P of 6.3, also showed lower recovery (56%) than a majority of the pesticides studied. This trend of decreased recovery for high log P pesticides has been observed by others for high fat matrices.⁶ Recovery of both of these compounds may be increased by addition of a less polar solvent such as ethyl acetate for the extraction; however, an increase in the level of co-extracted background can be expected.

Conclusions

Pistachios, which contain 45% fat, present a challenging matrix when doing pesticide residue analysis. If using QuEChERS extraction, some fat will be co-extracted with the analytes of interest. Thus, the cleanup step must be able to reduce this background. In this application, the use of Supel™ QuE Z-Sep+ was demonstrated for the effective cleanup of these extracts prior to LC-MS/MS and GC-MS/MS analysis. Fatty acid and monoglyceride background were significantly reduced using Z-Sep+, and compared to PSA/C18 cleanup, the resulting extract had lower background; as evidenced by GC-MS-scan data.

Pesticide recovery was within the acceptable range of 70-120% for 22 out of 30 targeted pesticides, with excellent reproducibility demonstrated for spiked replicates.