联苯肼酯在柑橘上的残留动态及其标准溶液的贮存稳定性

A study on the residual dynamics of bifenazate in citrus and the storage stability of its standard solutions

【目的】为评价联苯肼酯在柑橘中的农药残留安全性,研究联苯肼酯与抗坏血酸还原反应后,标准溶液放置时间及保存温度、光照对检测结果的影响。【方法】建立了高效液相色谱分析方法,并将该分析方法应用于联苯肼酯在柑橘中消解动态和最终残留规律的研究。【结果】还原后的联苯肼酯标准溶液在冷藏条件下贮存,5 d内保持稳定。联苯肼酯在柑橘中均消解较快,在湖南、广州、贵州三地柑橘全果样品中半衰期分别为2.31、2.66、1.92 d。按推荐剂量和推荐使用最大剂量兑水喷雾施药2~3次,联苯肼酯在柑橘全果和橘皮中最高残留量分别为0.093、0.663 mg·kg-1。【结论】采用抗坏血酸还原联苯肼酯检测样品时,处理后的样品应避光冷藏贮存,5 d内分析。联苯肼酯在柑橘中最终残留量均低于国家标准《食品中农药最大残留限量》（GB2763-2014）最大残留限量标准（柑橘0.7 mg·kg-1）,收获的柑橘食用相对安全。

[ Objective ] Bifenazate （C17H20N203）is a novel carbazate acaricide discovered by Uniroyal Chemical （now Chemtura Corporation）. It has been widely used in the prevention and control of phytophagous mites infesting agricultural and ornamental crops. It is one of the best agents to control phytophagous mites in citrus. Therefore, it is important to evaluate bifenazate residues in citrus. In analysis of bife- nazate, ascorbic acid reduction was used for sample processing, but ascorbic acid is unstable and susceptible to breakdown by high temperatures, ultraviolet irradiation, alkaline condition and oxidants. In this concern, we examined the stability of the reduction product of bifenazate by ascorbic acid during storage in order to guarantee the accuracy of the analysis and evaluation. [Methods] In 2014, experiments were carried out in Changsha, Tuyun and Guangzhou to examine the dynamics of bifenazate degradation and the final residues. The tested varieties were Miyagawa No. 1, Citrus unshiu Marc and Luogang orange. For tracing the dynamics of chemical degradation, four plots were set including a control plot, and each plot had protection rows and 3 trees. Trees in plots 1 to 3 were sprayed once with 1 667-time diluent of 30% bifenazate. Samples were collected at 2 h, 1 d, 2 d, 3 d, 5 d, 7 d, 10 d, 14 d, 21 d, 28 d and 35 d after treatment. For final residue tests, samples sprayed with 1667- or 2 500-time diluent of 30% bifenazate for two or three times were collected 20 and 30 days after the final spray. The samples were extracted with acetonitrile and purified using Florisil adsorption column and 0.2% ascorbic acid-acetoni- trile （1：4, V/V）, and detected with HPLC-UVD, which was equipped with an Inertsil ODS-SP chromato- graphic column （250 mm×4,6 mm, 5 μm）. The mobile phase Was methanol： water=65：35 with a flow rate of 1.0 mL·min^-1. The sample injection volume was 20μL and the detection wavelength was 245 nm. The results suggested that the fortified recovery and variation coefficient of the added compounds in whole citrus fruit samples were 8.5%-103.6% and 6.09%-7.49%, respectively, and in citrus peel samples they were 91.1%-109.8% and 2.91%-5.36%, respectively. The recovery rate and relative standard deviation of the method met the requirement for pesticide residue analysis and can be applied for detecting the residue of bifenazate in citrus. The standard matrix liquids （2.0 mg· L^-1） prepared from bifenazate treated citrus fruit or peel were stored under 4 conditions. The first condition was under low light （0.01 Ix） at room temperature; the second was under high light （1 500 lx） at room temperature; the third was cold storage at 4. ℃ under low light （0,01 lx）; and the last at -20 ℃ under low light （0.01 lx）. Samples were collected at 0.1 h, 2 h, 6 h, 12 h, 24 h, 2 d, 3 d, 4 d and 5 d, and the stability of the standard solutions was determined by liquid chromatography. [ Results ] Through the comparative analysis, it was found that the peak area of bifenazate in the standard solutions prepared from orange peel and whole citrus fruit showed a reduction of 6.0% and 9.1%, respectively, after 6 h of storage under high light and room temperature. And the standard solution with ascorbic acid was stable within 5 d of cold storage （4 ℃） under low light （0.01 Ix）. The degradation of bifenazate applied to citrus at the maximal recommended dosage in Hunan, Guangzhou and Guizhou followed the first-order kinetic equation Ct=C0e-kt. The half-life of bifenazate in citrus sampled in Hunan, Guangzhou and Guizhou was 2.31 d, 2.66 d and 1.92 d, respectively. The experiment showed that bifenazate is a kind of pesticide that can be easily degraded. The final residual experiment showed that under the natural conditions in the three provinces, the highest residue of bifenazate in citrus fruit and orange peel harvested more than 20 days after the last application of bifenazate with the recommended dosage and method was 0.093 mg· kg^-1 and 0.663 mg· kg^-1, respectively. [ Conclusion ] In case that the treated samples can not be analyzed in time, the extract Could be stored in cold but should be ana- lyzed within 5 d. Spraying with 1 667-time diluent （recommended using maximum dose） or 2 500-time diluent （the recommended dose） of bffenazate to citrus for 2-3 times, the final residue of bifenazate in citrus sampled 20 d after the last spray was below the maximum residue limit （MRL） of citrus in China （0.7 mg· kg^-1）. Thus, citrus harvested at this time were relatively safe for human consumption. According to the experiment results, it is suggested that the reasonable dosage of bifenazate applied to citrus is a 1 667- time dilnent with a maximum of 3 sprays at intervals of at least 10 d, and the safe interval for harvest is about 20 days. However, the harvest interval should be appropriately extended with the increase in application dosages and times