Residue Analysis of Cyazofamid, and Its Metabolism by Soil Fungus Cunninghamella elegans and Human Liver Microsomes

Abstract

Cyazofamid, a sulfonamide fungicide, has been used for the protection of several vegetables and fruits from various diseases. In the present study, the analytical method of cyazofamid and its metabolite by HPLC and LC-MS/MS was established. And metabolism of cyazofamid by soil fungus Cunninghamella elegans and by in vitro human mimicking system was investigated to understand bioenvironmental fate of cyazofamid. Apple, mandarin, Kimchi cabbage, green pepper, potato, and soybean were chosen as the representative crop samples for analytical study. LOQ and MLOQ for cyazofamid were 2 ng and 0.02 mg/kg, respectively, on HPLC. Recoveries of cyazofamid at three spiking levels were reasonable (75.3 - 98.5%). A rapid and effective LC?MS/MS analytical method of cyazofamid and its metabolite, CCIM (4-chloro-5-p-tolylimidazole-2-carbonitrile), was established for soil and water samples in addition to the crop samples using QuEChERS sample treatment. On LC-MS/MS, MLOQ of cyazofamid and CCIM were 2 ng/g and 5 ng/g for crop/soil samples, respectively, while 0.02 ng/mL and 0.05 ng/mL were achieved, respectively, for water samples. The recoveries of target analytes in crop/environmental samples were 80.2% - 105.1% for cyazofamid and 75.1% - 99.1% for CCIM. CCIM was observed in the metabolism of cyazofamid by artificial gastric juice and intestinal juice. CCHS (4-chloro-2-cyano-5-(4-(hydroxymethyl)phenyl)N,N-dimethyl-imidazole-1-sulfonamide) was identified unambiguously as a noble metabolite in the metabolism of cyazofamid by soil fungus Cunninghamella elegans. It could be degraded to 4-chloro-5-(4-hydroxymethylphenyl)imidazole-2-carbonitrile (CHCN) and further oxidized to 4-(4-chloro-2-cyanoimidazole-5-yl)benzoic acid (CCBA). In vitro metabolism of cyazofamid by HLMs, CCHS was also produced as the only metabolite. Among 10 types of recombinant CYPs (rCYPs), the formation of CCHS was observed only in CYP2B6, 2C9, and 2C19, contributing 27.4%, 66.1% and 6.5% in metabolism. In molecular docking study of crystal structure of cyazofamid with rCYPs 2B6, 2C9, 2C19 and 3A4, the metabolic reactivity order of three rCYPs was well correlated with the distance between the heme iron of rCYPs and hydroxylated carbon of cyazofamid. In phase II metabolism of cyazofamid no conjugate metabolite was observed in reaction with UDP-glucuronosyl transferases (UGTs) however, CCIM was observed in reaction with glutathione S-transferases (GSTs).