| Objective:1.To establish the pretreatment and HPLC-MS-MS detection method of cypermethrin and its metabolites in biological samples;2.To study the toxicokinetics of cypermethrin and its metabolites in dogs and establish the dynamic models,equations and parameters.And then discuss the detection time limit of cypermethrin and its metabolites in blood,urine and bile;3.To investigate the relationship between the time of last taking cypermethrin and the concentration of cypermethrin and its metabolites.To establish a model for estimating the time of last taking cypermethrin;Methods:1.Subjects: 6 male dogs offered by the laboratory animal center of Shanxi medical university were recruited to join the experiment,who were healthy and weighed all about 12 kg.No medicine before the test.This research plan had passed the ethical review caarried out by SXMU ethical review committee.2.Specimen Collecting: Gallbladder fistula were operated on the 6 dogs after the general anesthesia.Ensure the bile drainage unblock before their fasting which should be lasted for 12 h.Then 1/4 LD50 doses of cymermethrin were given to the 6 male dogs by oral perfusion and their bile,blood and urine which would be stored at-20? for the determination of cypermethrin and its metabolites were collected at different time just as 0.2h,0.5h,1h,3h,4h,8h,12 h,24h,48 h,72h,96 h,120h,144 h,168h as well as before doesing.3.Extraction and analysis: Cypermethrin and its metabolites in specimens which were added 8ng internal standard were extracted with liquid-liquid extraction(LLE)and determined by HPLC-MS-MS.The qualitative analysis was based on retention time and MRM ions.The quantitative analysis was based on an internal standard method and calibration curve,in which carbamazepine was used as internal standard.4.Statistical methods: WinNonlin pharmacokinetic software was used for dynamics analysis.SPSS 19.0 statistical software was used for variance analysis.Results:1.Extraction and detection: CYM,3-PBA,DCVA could be detected simultaneously by HPLC-MS-MS.The method was validated and proved to be accurate(accuracy within 95.33%-106.6%),precise(RSD<3%)and sensitive.For all analytes,LODs were 0.49ng/m L.Linear curve covered the range 0.2-10?g/mL for cypermethrin and 0.02-4?g/mL for its metabolites,and the correlation index were above 0.99 for all.2.Toxicokinetics: After an oral dose of 1/4 LD50 cypermethrin,in general the concentration of cypermethrin in blood was higher than the concentration in urine,the concentration in urine was higher than the concentration in bile;the concentration of 3-PBA was urine > bile > blood and the concentration of DCVA was bile > urine > blood.In blood and urine,the concentration of 3-PBA was higher than DCVA and CYM in general.The concentration of DCVA was higher than 3-PBA and CYM in bile in general.The Tmax of cypermethrin and its metabolites was 1-2h in bile and urine,while that was 7-8h in blood.The toxicokinetics of cypermethrin and its metabolites in blood,urine and bile met 1st order model,the T1/2k01,T1/2k10 of CYM in the blood,urine and bile respectively were 1.66±1.92 h,0.70±0.47 h,0.43±0.23 h and 5.84±4.26 h,7.99±6.51 h,3.93±0.71h;The T1/2k01 of 3-PBA in the urine and bile respectively were 0.27±0.27 h and 0.64±0.07h;The T1/2k10 of 3-PBA in the blood,urine and bile respectively were 5.33±2.81 h,1.17±0.78 h and 1.36±0.11 h.The T1/2k01,T1/2k10 of DCVA in the blood,urine and bile respectively were 4.29±1.60 h,0.12±0.09 h,0.50±0.28 h and 5.25±2.38 h,1.99±1.12 h and 3.36±1.50 h.3.Estimating time of last taking cypermethrin: By the ratios of one time cypermethrin and its metabolites or two time cypermethrin concertrion in samples,two models for estimating time of last cypermethrin use were established,and an equation was developed to predict elapsed time of cypermethrin use.The equation to predict elapsedtime within a 20% deviation for major data.Conclusion:1.The toxicokinetics model of cypermethrin and its metabolites in dogs was established.The toxicokinetics of cypermethrin in blood and urine met a two compartment model,the toxicokinetics of cypermethrin in bile met a one compartment model;the toxicokinetics of 3-PBA in blood,urine met a one compartment model;the toxicokinetics of 3-PBA in bile met a two compartment model;the toxicokinetics of DCVA in bile,urine met a two compartment model;the toxicokinetics of DCVA in blood met a one compartment model;The data of this study can be used for the forensic identification of cypermethrin poisoning death.2.A preliminary model for estimating time of last taking cypermethrin was est ablished by the concentration of cypermethrin and its metabolites.This study can b e used for predict time of last taking cypermethrin in the forensic cases of identific ation of cypermethrin poisoning. |
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