Abad A, Moreno M J, Pelegrí R, Martínez M I, Sáez A, Gamón M, Montoya A
Laboratorio Integrado de Bioingeniería, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain, and Laboratorio Agroalimentario de la Generalitat Valenciana, C/Pintor Goya 8, 46100 Burjassot (Valencia), Spain.
J Agric Food Chem. 2001 Apr;49(4):1707-12. doi: 10.1021/jf0012493.
The N-methylcarbamate pesticide carbaryl is one of the most important insecticides used worldwide. In the present work, the validation of a monoclonal antibody-based enzyme immunoassay (ELISA) for the determination of this compound in fruits and vegetables is described. The immunoassay is a competitive heterologous ELISA in the antibody-coated format, with an I(50) value for standards in buffer of 101.0 +/- 26.9 ng/L and with a dynamic range between 31.6 and 364.0 ng/L. For recovery studies, peppers, cucumbers, strawberries, tomatoes, potatoes, oranges, and apples were spiked with carbaryl at 10, 50, and 200 ppb. After liquid extraction, analyses were performed by ELISA on both extracts purified on solid-phase extraction (SPE) columns and crude, nonpurified extracts. Depending on the crop and the fortification level, recoveries in the 59.0--120.0% range were obtained for purified samples and in the 70.0--137.7% range for crude extracts. The carbaryl immunoassay performance was further validated with respect to high-performance liquid chromatography (HPLC) with postcolumn derivatization and fluorescence detection (EPA Method 531.1). Samples were spiked with carbaryl at several concentrations and analyzed as blind samples by ELISA and HPLC after SPE cleanup. The correlation between methods was excellent (y = 1.04x + 0.71, r(2) = 0.992, n = 33), with HPLC being more precise than ELISA (mean coefficients of variation of 5.2 and 12.0%, respectively). The immunoassay was then applied to the analysis of nonpurified extracts of the same samples. Results also compared very well with those obtained by HPLC on purified samples (y = 1.28x - 0.59, r(2) = 0.987, n = 33) while maintaining similar precision. Therefore, the developed immunoassay is a suitable method for the quantitative and reliable determination of carbaryl in fruits and vegetables even without sample cleanup, which saves time and money and considerably increases sample throughput.
N-甲基氨基甲酸酯类农药西维因是全球使用的最重要的杀虫剂之一。在本研究中，描述了一种基于单克隆抗体的酶免疫分析法（ELISA）用于测定水果和蔬菜中该化合物的验证情况。该免疫分析法是一种以抗体包被形式的竞争性异源ELISA，缓冲液中标准品的半数抑制浓度（I(50)）值为101.0±26.9 ng/L，动态范围在31.6至364.0 ng/L之间。在回收率研究中，向辣椒、黄瓜、草莓、番茄、土豆、橙子和苹果中添加10、50和200 ppb的西维因。液体萃取后，通过ELISA对在固相萃取（SPE）柱上纯化的提取物和未纯化的粗提物进行分析。根据作物和强化水平，纯化样品的回收率在59.0%至120.0%之间，粗提物的回收率在70.0%至137.7%之间。通过柱后衍生化和荧光检测的高效液相色谱法（HPLC）（EPA方法531.1）进一步验证了西维因免疫分析法的性能。向样品中添加几种浓度的西维因，经SPE净化后作为盲样通过ELISA和HPLC进行分析。两种方法之间的相关性极佳（y = 1.04x + 0.71，r(2) = 0.992，n = 33），HPLC比ELISA更精确（平均变异系数分别为5.2%和12.0%）。然后将该免疫分析法应用于相同样品未纯化提取物的分析。结果与HPLC对纯化样品的分析结果也非常吻合（y = 1.28x - 0.59，r(2) = 0.987，n = 33），同时保持了相似的精密度。因此，所开发的免疫分析法是一种适用于水果和蔬菜中西维因定量可靠测定的方法，即使无需样品净化，这既节省了时间和成本，又显著提高了样品通量。
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