新型手性杀虫剂唑虫酯异构体拆分及其在蔬菜中的分析方法建立

以全新手性杀虫剂唑虫酯为研究对象,通过筛选手性色谱柱和优化流动相比例,建立了唑虫酯及其氧化代谢物异构体的拆分方法,在此基础上开发利用高效液相色谱-串联质谱(HPLC-MS/MS)同时测定小白菜和蕹菜中唑虫酯及其氧化产物手性异构体的分析方法。以纤维素-三(3,5-二氯苯基氨基甲酸酯)共价键合手性柱(Chiral INC)(250 mm×4.6 mm, 5 μm)为分析柱,乙腈和2 mmol/L甲酸铵水溶液作为流动相进行梯度洗脱分离,在多反应监测负离子模式下进行检测,唑虫酯4个异构体分离度分别为1.63、2.83和1.74,唑虫酯氧化产物异构体分离度为5.82。通过衍生化的方法进一步确定出峰顺序为RS-唑虫酯、SS-唑虫酯、RR-唑虫酯、SR-唑虫酯、S-唑虫酯氧化产物和R-唑虫酯氧化产物。唑虫酯和其氧化产物的手性异构体分别在1.25~1250 μg/L和2.5~2500 μg/L范围内具有良好的线性关系,相关系数(R²)大于0.99。在蕹菜和小白菜样品中同时添加唑虫酯和唑虫酯氧化产物消旋体进行添加回收试验,添加水平为1、20、400 μg/kg(即唑虫酯异构体为0.25、5、100 μg/kg;唑虫酯氧化代谢产物异构体为0.5、10、200 μg/kg),回收率为72.6%~110.6%,相对标准偏差(RSD)均在9.4%以下,其中日内重复性的RSD在0.5%~9.4%之间;日间重复性的RSD在1.0%~8.6%之间,表明该方法具有良好的回收率和精密度。该研究可为唑虫酯这一新型手性农药的环境行为研究及后续质量控制、药效评价等提供相应的分析技术,为新农药开发应用提供有力的技术支撑。

Chiral separation of new chiral insecticide pyraquinil isomers and establishment of analytical methods in vegetables

Pyraquinil (Pyr), with a new skeleton of pyrazolo1,5-a]quinazoline fused heterocycle, is a new chiral insecticide independently developed by South China Agricultural University in 2017. In previous studies, we found that pyraquinil can effectively control the lepidopteran pest population on cruciferous crops. Remarkably, the insecticidal activity of pyraquinil was 64-fold better than that of fipronil against the fipronil-resistant Plutella xylostella field population. Pesticides with new active mechanisms should be developed in the future to cope with the development of resistance to Plutella xylostella. Therefore, pyraquinil with new active sites has the potential to be the main rotation variety for the control of Plutella xylostella. Thus, pyraquinil has a broad prospect for application in the future. However, a chiral separation and analysis method for pyraquinil and oxidation products using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has not been established yet. Based on the physical and chemical properties of the new chiral pesticide pyraquinil, we screened chiral chromatographic columns and optimized the mobile phase ratio in this study. The separation and analysis methods for pyraquinil were developed based on the QuEChERS (quick, easy, cheap, effective, rugged, safe) system. Simultaneously, we also established oxidation metabolites. These methods were used for the simultaneous determination of the chiral isomers of pyraquinil and oxidation products in pakchoi (Brassica rapa ssp. chinensis L.) and water spinach (Ipomoea aquatica Forsk) via HPLC-MS/MS. The Chiral INC column (250 mm×4.6 mm, 5 μm) was used for separation. Ammonium formate aqueous solution (2 mmol/L) and acetonitrile were used as the mobile phases. The column temperature was 28 ℃. The injection volume was 1 μL, and the flow rate was 0.5 mL/min. MS analysis was performed using an electrospray ionization source in the negative and multiple reaction monitoring modes. We found that under the optimized conditions, the resolution of the four isomers of pyraquinil were 1.63, 2.83, and 1.74, respectively, and the resolution of the isomers of the pyraquinil oxidation product was 5.82, which achieved baseline separation. Then, the absolute configuration and peak order of pyraquinil and oxidation product isomers were determined by derivatization. The order of the peaks was RS-Pyr, SS-Pyr, RR-Pyr, SR-Pyr, S-Pyr+O, and R-Pyr+O. The purification conditions of sample pretreatment were optimized; 1 g (0.835 g MgSO₄+0.150 g PSA+0.015 g GCB) was determined to be the optimal purification agent; and the average recoveries ranged from 80% to 110%. The chiral isomers of pyraquinil and oxidation products showed good linearity in the concentration range of 1.25 to 1250 μg/L and 2.5 to 2500 μg/L respectively. The square of the regression coefficient of the linear equation (R²) was greater than 0.99. The matrix effects of the pyraquinil and oxidation product isomers in pakchoi ranged from 6.1 to 30.6. In the water spinach, the matrix effect of the pyraquinil and oxidation product isomers were in the range of 0.7-26.8. The average recoveries of pyraquinil isomer at three spiked levels of 0.25, 5, 100 μg/kg in samples (pakchoi and water spinach) ranged from 90.2% to 110.6%. The oxidation product isomer average recoveries in samples (pakchoi and water spinach) spiked with 0.5, 10, 200 μg/kg ranged from 72.6% to 100.1%. Further, the relative standard deviations (RSDs) were 0.5%-9.4%. In water spinach, the intra-day and inter-day repeatability RSDs ranged from 0.5% to 8.7% and 1.0%to 8.6%, respectively. In pakchoi, the intra-day and inter-day repeatability RSDs ranged from 0.6% to 9.4% and 1.0% to 7.6%, respectively. These results indicate that the proposed method has satisfactory sensitivity, accuracy, and precision. This study can provide analytic technology for a novel chiral pesticide for environmental behavior studies, quality control, and pharmacodynamics evaluation, as well as significant technical support for the development and application of new pesticides.