Sample preparation procedures for pesticide analysis in fruits and vegetables were simplified with the introduction of QuEChERS methodology in 20031. Multi-residue pesticide extraction was accomplished through a 1 minute shaking procedure using acetonitrile as an extraction solvent followed by salt partitioning and dispersive solid phase extraction (SPE) cleanup. This extraction procedure has been successfully applied to a variety of fruits and vegetables. The QuEChERS procedure has grown in popularity and has gained worldwide acceptance during the past 10 years.
It is common to evaluate and validate pesticide residue methods by spiking representative blank matrix samples with a standard mix of pesticides. Pesticide-free produce samples, such as organically grown produce, are often used for this purpose. The pesticides are typically added to a matrix sample 1-2 hours prior to testing in order to allow pesticide incursion. They are then easily extracted using the 1 minute shaking protocol. However, recently, it was noted that existing residues may not be readily accessible for extraction as they can be “enclosed into cells or wax particles” of the produce2. The 1 minute shaking time specified in the QuEChERS protocol was not sufficient to fully extract existing pesticide residues in approximately 50% of tests conducted. It was later observed that a 10-15 minute shaking time with the use of an automated shaker was required to obtain acceptable extraction efficiencies3.
Recently the BenchMixer™ shaker was added to the Supelco/Sigma-Aldrich product listing to compliment the Supel QuE product line. Spinach, a vitamin and mineral-rich green leafy vegetable, was recently cited in the Environmental Working Group’s “Dirty Dozen 2014” for having high levels of pesticide contamination4. For this study, the BenchMixer shaker was used for the extraction and cleanup of existing pesticide residues from non-organically grown spinach. The results were compared to a standard QuEChERS protocol involving the manual shaking of the spinach extracts for 1 minute.
Spinach was extracted with acetonitrile using 1 minute manual shaking (by hand) or 10 minute automated shaking using the BenchMixer vortex shaker. Previous work showed that using the BenchMixer for 1 minute and 5 minutes resulted in incomplete extractions. A minimum of 10 minutes shaking time at the 2250 rpm setting was required for full extraction of existing pesticides. The extraction was performed in Supel QuE 50 mL centrifuge tubes. For each pesticide extraction, 5 replicates were completed. The extraction procedure is presented in Figure 1.
The samples were then analyzed by GC/MS/SIM, using m/z 183 for quantitation, and m/z 163 as a qualifier. Calibration was done using matrix-matched standards. Extracts of organically grown spinach were obtained and were used to construct the calibration curve. The calibration curve standards ranged from 25 ng/mL to 5,000 ng/mL.
Figure 1. QuEChERS Procedure Used for the Extraction of Pesticides from Spinach
Figure 2. GC/MS of Permethrin Pesticide Isomers Extracted from Spinach Using the BenchMixer Shaker
column: SLB®-5ms, 20 m x 0.18 mm I.D., df 0.18 µm (Product No.); oven: 120 °C (1 min), 10 °C/min to 330 °C (3 min); inj. temp.: 250 °C; carrier gas: helium, 0.7 mL/min; detector: Agilent GC 7890 with 5975 MS Detector, selected ion mode (SIM) m/z = 163 (quant), 183 (qual); MSD interface: 325 °C, source = 250 °C, quads = 200 °C; injection: 1 µL; liner: 2 mm I.D., split/splitless type, single taper wool packed FocusLiner™ design (Product No. 2879525-U)
Two permethrin isomers were resolved chromatographically (Figure 2) and quantitated separately. The yield of permethrin isomer 1 was found to be 4.18 ppm using the manual method, but 4.76 ppm using the shaker method (Table 1). Permethrin isomer 2 gave a level of 0.90 ppm from the hand-shaken samples, but a level of 1.03 ppm from BenchMixer shaken samples. For both isomers, the manual method extracted 14% less permethrin than the shaker method. This difference is significant. Similar results were observed in an experiment by Anastassiades et al. when 10-15 minutes of automated shaking was employed for the initial acetonitrile extraction step in the QuEChERS procedure3. The reproducibility of this work was similar for both shaking techniques (2-5%) when hand-shaking was performed by an experienced laboratory technician.
The use of the shaker was convenient as the QuEChERS horizontal rack can hold seven 50 mL tubes during the extraction step, and the rack for 15-mL tubes can hold up to 50 tubes for the cleanup step. Both 50 mL extraction tubes and the new Supel QuE 15 mL cleanup tubes were compatible with the shaker. In addition to producing higher yields of existing pesticides, use of the BenchMixer shaker may also decrease the physical strain experienced by laboratory personnel when performing multiple daily QuEChERS extractions.
Introduction of the BenchMixer shaker into the QuEChERS protocol resulted in a 14% increase in the observed yield of permethrin residues existing in commercially bought spinach. The shaker can be successfully used for analysis of pesticide residues when an extended shaking time of 10-15 minutes is required to achieve better extraction efficiencies.