Validação de métodos multirresíduos de extração e desenvolvimento de método de purificação por GPC para análise de resíduos de pesticidas em soja utilizandoGC-MS, GC-MS/MS e LC-MS/MS

Abstract

Due to the complexity of the soybean matrix and the very low pesticide concentrations expected for these samples, it is really necessary to develop efficient and reliable analytical methods to identify and quantify their residues. In this study, an evaluation and validation was performed of the linear range of the analytical curves (7 concentration levels and 6 injections each), detection limit (LOD), quantification limit (LOQ), matrix effect, as well as precision (as RSD%) and accuracy (as recovery percent), for 221 pesticides, analyzed by 2 different extraction methods (modified Luke and Quechers) and also by different chromatographic techniques (GC and LC) coupled with mass spectrometry (MS). Furthermore, a clean up method by gel permeation chromatography (GPC) was developed for soybean extract purification. To that end, milled and homogenized soybean was spiked with 221 pesticides (52 analyzed by GC and 169 by LC), at 3 different spiking levels (10, 50 and 100 μg kg-1, 6 replicates at each level) and extracted by 2 different methods. Applying the modified Luke method, 10 mL of water was added to 5.0 g of soybean matrix and the samples were allowed to stand for 2 hours. A volume of 100 mL of a mixture of acetone, dichloromethane and light petroleum ether (1:1:1) and anhydrous sodium sulfate (15 g) were added, the samples were extracted with a Polytron homogeneiser for 1 min and finally centrifuged for 3 min. Applying the modified Quechers method, 4 mL of water was added to 2.0 g of soybean matrix and the samples were allowed to stand for 2 hours. A volume of 20 mL of acetonitrile was added and the tubes were vigorously shaken by hand for 45 s. The tubes were uncapped, 2 g of anhydrous magnesium sulfate and 2.5 g of sodium acetate were added, the shaking procedure was repeated and the extract was centrifuged for 1 min. The upper layer of the extracts was transferred to another tube, containing 2 g of anhydrous magnesium sulfate, and the shaking and centrifugation procedures were repeated. Prior to GC determination, a volume of 100 μL of each extract (reconstituted in 1 mL dichloromethane) was injected into a GPC system, consisting of a HPLC pump, autosampler, column oven, 2 PLgel columns, DAD detector, ELSD detector, automated fraction collector and computer workstation. Dichloromethane was used as eluent (flow rate, 0.75 mL min-1) and the pesticide fraction was collected between 17.5 and 27.5 min. After evaporation and reconstitution with 1 mL of iso-octane/toluene (9:1), containing 4.0 ng mL-1 bromophos-methyl (internal standard), extracts were analyzed by GC-MS and GC-MS/MS. The clean up procedure was not necessary for LC-MS/MS analysis, so, the extracts were reconstituted in methanol and directly injected into the LC-MS system. In general, LOD, LOQ and r2 results, obtained from GC, were affected by standards prepared in matrix extract compared to the preparation in solvent, which was not observed with results obtained from LC-MS/MS, showing a considerably lower matrix effect. Among the GC-MS techniques, the GC-MS NCI SIM mode was most appropriate for multiresidue pesticide analysis, conciliating sensitivity and acceptable selectivity, and allowing good quantification of 85% of the target compounds. ESI positive mode LC-MS/MS allowed determination of 155 pesticides in one chromatographic analysis, generating LOQm values of 10 or 50 μg kg-1, for ca 80% of the pesticides studied. Hence, it is possible to conclude, that the GPC clean up method developed was efficient to purify soybean extracts and that both extraction methods evaluated showed good performance, depending on which detection technique was to be used. Comparing GC and LC-MS/MS, there is no doubt that the latter was more efficient for pesticide multiresidue analysis, taking into account both robustness and sensitivity. Also, the possibility to perform the determination of a large number of pesticides in just a single chromatographic analysis is another major advantage.