| Objective: To explore the possible metabolic pathway in rats through the analysis of trichlorfon in metabolites of SD rats. Methods: 1. Establish animal model: 36 SD rats were randomly divided into control group(n=9) and three exposed group, respectively low dose group(n=9), middle dose group(n=9) and high dose group, and the exposure dose of trichlorfon was 2mg/kg, 10mg/kg, 50mg/kg respectively with mouth feeding lasting 30 days. The rats of control group were fed with distilled water lasting 30 days. Then leave the urine of 24 hours after gave drug, EDTA coagulated whole blood, heart, liver, spleen, lung, kidney and brain tissue at the end day. 2. To analyze the metabolites in urine, blood and tissue of rats by GC-MS detection. 3. To establish the method to analyze 3-trichloroethanol and 2,2-dichloroacetamide in urine and tissue by GC-ECD quantitative detection. The urine was extracted with acetonitrile and the tissue was extracted after homogenizing by ultrasonic. 1,3-Dichloro-2-propanol and 2-chloroacetamide was used as internal standards. The GC was performed on an HP-INNOWAX column(30m×0.32 mm×0.25μm) with ECD detector.The temperature of detector was set at 280 ℃.The temperature of injector was 200 ℃. Splitless mode was used and the injection volume was 1μL.The temperature program of column oven was programmed as follows: 120℃ for 2 min,then increased to 150 ℃ at 10 ℃/min,and kept for 1 min; and then increased to 200℃ at 10℃/min and held for 1min; and finally increased to 240℃at 5℃/min and kept for 2 min. 4. DMP in urine, blood and tissue was detected by GC-FDP detection. 5. The date of trichlorfon metabolites in urine, blood and tissue was analyzed to gain the correlation between them by SPSS 21.0, and to primarily explore the possible metabolic pathway. Results: 1. Trichloroethanol and 2,2-dichloroacetamide were found in urine and kidney by GC-MS, trichloroethanol was found in liver, and the metabolite of 2,2-dichloroacetamide was found at first time. 2. Establish the quantitative analysis method to analyze 3-trichloroethanol and 2,2-dichloroacetamide in urine was established by GC-ECD detection. The calibration curves of trichloroethanol and 2,2-dichloroacetamide were linear in the range of 2.84~210.52 ng/m L and 42.51~411 ng/m L, respectively. The correlation coefficients were above 0.99. The detection limits were 0.82ng/m L and 13.23ng/m L, respectively. The recovery rate was 75.09%~109.55%. 3. Urine, blood and tissue metabolic product content with the increasing doses of trichlorfon exposure increases, the difference was statistically significant(p <0.05), and were significantly associated(p <0.01) and trichlorfon exposure levels, urine, blood levels of the metabolic products of the respective tissue DMP were significantly associated(p <0.05). 4. The metabolic pathways of trichlorfon in vivo: one for trichlorfon generated P-C strand breaks Dimethyl phosphate(DMP) and trichloroethanol, two for trichlorfon metabolized to DDVP, then hydrolyzed to DMP and trichloroacetaldehyde, then trichloroacetaldehyde further metabolized to 2,2- two chloroacetamide. Conclusion: 1. To identify the metabolites of trichloroethanol and 2,2-dichloroacetamide by GC/MC detection. 2. The means involved by the method of analysing trichloroethanol and 2,2-dichloroacetamide in urine and tissues by GC-ECD detection is simple, rapid, practical, and can better extract and isolate the measured substance, and the data is reliable. And it can be used to analyze the content of trichloroethanol and 2,2-dichloroacetamide in urine and tissue. 3. Trichloroethanol and 2,2-dichloroacetamide can reflect the exposure condition of trichlorfon as contact marker. 4. To infer preliminarily the metabolic pathway of trichlorfon in rats in vivo according to the determination results by GC/MS, GC-ECD, GC-FPD. |
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