Recently formulated pesticides are very different in their physical properties from their predecessors, such as 4,4'-DDT (dichlorodiphenyltrichloroethane). Most recently formulated pesticides are smaller in molecular weight and designed to break down rapidly in the environment. Therefore, to successfully identify and quantify these compounds in foods, more careful consideration must be placed on sample preparation for extraction, in addition to the instrument parameters for analysis.
The QuEChERS (quick, easy, cheap, effective, rugged, and safe) SPE (solid-phase extraction) methodology, developed by Anastassiades et al.,1 has become widely used in food safety analyses. The method is:
- Quick—High sample throughput, typically eight samples, can be prepared in just under 30 min
- Easy—The technique requires less handling of extracts than other techniques, and no laborious steps are involved
- Inexpensive—Reduced amounts of sorbent material are required, and less time is needed to process samples compared to other techniques
- Effective—The simple technique gives high and accurate recovery levels for a range of different compound types, e.g., polar pesticides and pH-dependent compounds
- Rugged—The method can detect a large number of pesticides, including pH-dependent and polar pesticides
- Safe—Unlike other techniques, it does not require any chlorinated solvents; extraction is typically carried out using acetonitrile, which is both GC and LC amenable.
The QuEChERS procedure is usually a two-stage process: sample extraction followed by dispersive SPE. In the sample extraction stage, the food sample is homogenized to maximize the available surface area of the sample for better extraction efficiencies. The homogenized sample is placed in the extraction tube containing magnesium sulfate and sodium acetate. Magnesium sulfate ensures that, upon addition of acetonitrile, a phase separation is induced between water and organic solvent, with the pesticides of interest being extracted into the organic phase. When acetonitrile is poured into the extraction tube containing the homogenized sample, an exothermic reaction occurs between the magnesium sulfate and water, which can lead to low recoveries of the pesticides. This effect can be reduced by adding the salt and sample to the extraction tube while this is immersed in an ice bath, or by weighing the sample into an FEP tube and then adding the solvent and salts slowly. The tube is then capped, shaken vigorously, and centrifuged.
The second stage of the QuEChERS method uses dispersive SPE, which involves transferring a portion of the acetonitrile extract to a cleanup tube containing a combination of sorbents for removal of unwanted sample components. This may be followed by solvent exchange to improve compatibility of samples to GC analysis, and additional sample cleanup to reduce matrix effects and therefore improve method robustness.
Internal standards are used to minimize errors that may be introduced in the different steps of the QuEChERS method, as well as to compensate for GC injection variability. Furthermore, the addition of analyte protectants such as sorbitol can be useful for labile pesticides at intermediate pH, which can be prone to decomposition in the GC injector port.
The pesticides analyzed are a mixture of organophosphate, organochlorine, pyretheroid, benzenoid, triazole, and dicarboximide compounds. Lehotay reviewed the LC and GC analyses of pesticides in produce and the type of pesticide that is likely to be found in each matrix.2 The requirements for pesticide residue analysis in fruits and vegetables are established by organizations such as the World Health Organization, Japanese Food Chemical Research Foundation, EEC Directives, and U.S. EPA.3–6 These organizations establish which pesticides need to be determined in different produce and the method regulatory limits (MRLs). The pesticides determined in this study are all listed by the four regulatory organizations, and all have minimum MRLs of 50 ng/g (ppb). The recoveries of the pesticides in grapes are based on this value.
Figure 1 - QuEChERS tubes.
Methodology
The methodology described here is for the preparation of calibration standards and produce sample spike utilizing reagents, equipment, and QuEChERS tubes supplied by Thermo Fisher Scientific (Runcorn, Cheshire, U.K.) (Figure 1).7 A fully validated QuEChERS method in extracting multiresidue pesticides is published in Ref. 8.
Extraction
Figure 2 - Flow diagram of QuEChERS sample preparation methodology used in the analysis of pesticides in grapes.
Figure 2 is a flow diagram showing steps 1–5 of the procedure. The sample of green grapes was homogenized using a blender, and 15 g was weighed into the FEP extraction tube, followed by the addition of 15 mL of 1% acetic acid in acetonitrile. The MgSO4 and sodium acetate mixture in the QuEChERS extraction tube was poured very slowly into the FEP tube. No significant difference in the recoveries was observed when the homogenized grapes were directly weighed into the extraction tube containing the salts that had been placed in an ice bath. This was demonstrated for the grapes matrix only, but it may differ for other fruit and vegetable matrices. The pesticide standard mixture was spiked into the FEP tube for calculating recoveries for produce sample spike. For the calibration standards, the pesticide standard mixture was not spiked at this first stage of the QuEChERS method. The tube was capped, shaken vigorously for 5 min, and centrifuged at 3000 rpm for 5 min. The solids were separated from the supernatant, which contains the pesticides.
Dispersive SPE
The dispersive SPE stage consists of three steps: initial cleanup (steps 6 and 7 in the flow diagram in Figure 2), solvent exchange (steps 8–11), and final cleanup (steps 12–14). Eleven milliliters of the supernatant (acetonitrile extract) was transferred to a stage 2 cleanup tube containing the C18 sorbent, MgSO4, and primary secondary amine (PSA). The sample tube was capped and centrifuged at 3000 rpm for 5 min. Five milliliters of the supernatant was transferred to a 10-mL vial, which was placed into a Dry Block Sample Incubation System (Thermo Fisher Scientific, Bellefonte, PA) for evaporation of the solvent under a stream of nitrogen at 40 ºC for 1 hr. The extract was reconstituted in hexane/acetone (9:1), which is a more acquiescent solvent for GC splitless injection. For the produce sample spike 0.9 mL of hexane/acetone 9:1 was added, and for the calibration sample 0.8 mL was added. This was followed by the addition of internal standard. At this point, calibration standards were also prepared by spiking pesticide standards in various concentrations to each vial. The produce sample spike was prepared in triplicate, whereas seven-point calibration standards were prepared. In the final cleanup step, 1 mL of sample was transferred to a cleanup dispersive SPE tube containing C18, MgSO4, and PSA. This extra cleanup step was performed to reduce the matrix effect. The tube was capped, vortexed, and centrifuged. Next, 500 µL of the supernatant was transferred to a silanized GC vial for GC-MS analysis.
Results and discussion
Figure 3 - Total ion chromatogram (TIC) for the GC-MS analysis of grapes spiked with 1 ng/µL of each pesticide.
Table 1 - Summary of results
In order to assess the method linearity, a calibration curve was constructed for each of the eight pesticides spiked in the sample matrix (Figure 3) using triphenylphosphate as the internal standard. The concentration range studied was 0.005–5 ng/µL. The correlation coefficients (R2) between area ratio of sample and internal standard for all pesticides were higher than 0.99 (Table 1), demonstrating good method linearity. The extraction percent recoveries measured were between 76 and 110%, with an average relative standard deviation (RSD) for three repeated samples of 11.0%.
Conclusion
The QuEChERS sample preparation method provided high recoveries and good reproducibility. The QuEChERS/GC-MS method was found to be linear in the concentration range of 0.005–5 ng/µL spiked matrix, which includes the MRLs of 50 ppb (0.05 ng/µL). This confirms QuEChERS dispersive SPE as a simple, fast, quantitative, and effective sample preparation method for the GC-MS analysis of pesticides in grapes to MRLs in line with the requirements of key regulatory organizations.
References
- Anastassiades , M.; Lehotay, S.J.; Stajnbaher, D.; Schenck, F.J. J. AOAC Int.2003, 86, 412.
- Lehotay, S.J. Analysis in Methods in Biotechnology; Humana Press: New York, NY, 2004.
- CODEX alimentarius; www.codexalimentarius.net/mrls/pestdes/jsp/pest_q-e.jsp.
- Japanese Food Chemical Research Foundation; www.m5.ws001.squarestart.ne.jp/foundation/search.html.
- Informal coordination of MRLs established in Directives 76/895/EEG, 86/362/ EEG, 86/363/EEG and 90/462/EEG (5058/ VI/98); http://ec.europa.eu/food/plant/protection/resources/09-99-2.pdf.
- 40CFR180; www.access.gpo.gov/nara/cfr/waisidx_02/40cfr180_02.html.
- Khan, A.; Pereira, L.; Aspey, S.; Lewis, R. Thermo Fisher Scientific, Runcorn, U.K.; Application note 0709; 2009.
- Butler, J.; Steineger, D.; Philips, E. Thermo Fisher Scientific, Austin, TX; Technical note 10222; 2006.
The authors are with Thermo Fisher Scientific, 112 Chadwick Rd., Astmoor, Runcorn, Cheshire WA7 1PR, U.K.; tel.: +44 (0) 1928 581000; fax: +44 (0) 1928 581078; e-mail: [email protected].