Study On The Pharmacokinetics Of Diazepam And Its Metabolites In Human

Objective:1. To establish the analytical method of diazepam and its metabolites in body specimens by SPE-HPLC-MS-MS.2. To study the pharmacokinetics of diazepam and its metabolites in human;To explore the detection time limit of diazepam and its metabolites in blood, urine, saliva.3. To investigate the relationship between time of last diazepam use and diazepam/metabolite concentration ratios or metabolite/ metabolite concentration ratios, to establish a model for estimating time of last diazepam use.Methods:1. Subjects: After signing an informed consent, twenty eight participants from Tai yuan were recruited to join the experiment, who was 22 to 26, no medicine within past two weeks, no chronic illness.2. Specimen Collecting: Participants were fasted for twelve hours and then was given an oral dose of 5mg diazepam made in Bei Jing. Before the experiment and at 1h,2h, 4h, 8h, 12 h, 24 h, 2d, 3d, 6d, 12 d, 15 d, 5m L venous blood and saliva, urine were collected and stored at-20℃ for the determination of diazepam and its metabolites.3. Extraction and analysis: Diazepam, nordiazepam, oxazepam, oxazepam glucuronide, temazepam glucuronide in specimens were extracted with solid phase extraction(SPE) 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 estazolam was used as internal standard.4. Statistical methods: SPSS13.0 statistical software was used for t test and variance analysis.Results:1. Extraction and detection The diazepam, nordiazepam, oxazepam, estazolam,oxazepam glucuronide, temazepam glucuronide could be detected simultaneously by a SPE- HPLC-MS-MS. The method was validated and proved to be accurate(accuracy within 80%-120%), precise(RSD<20%) and sensitive. For all analytes, LODs were0.01-0.5ng/ml, recoveries were 84.9%-106%. Linear curve covered the range0.1-100ng/ml for diazepam and nordazepam, 0.5-100ng/ml for oxazepam and oxazepam glucuronide and temazepam glucuronide, and the correlation index were 0.99 for all.2. Pharmacokinetics: After an oral dose of 5mg diazepam, diazepam, nordazepam,oxazepam, oxazepam glucuronide and temazepam glucuronide all could be detected in blood, the metabolites were detected more lately than parent. The detection window ranged from less than an hour to more than 15 d. Diazepam, nordazepam could be detected more lately than oxazepam, oxazepam glucuronide and temazepam glucuronide;Except for oxazepam, diazepam, nordazepam, oxazepam glucuronide and temazepam glucuronide could be detected in urine, of which the detection window was longer than15 d. In the saliva, only diazepam and nordazepam could be detected, and detection window varied from six days to more than fifteen days.The pharmacokinetics of diazepam in blood met a two compartment model, t1/2k01,t1/2k12, t1/2k10 respectively were 0.3h±0.4h, 9.9h±13.6h and 37.2h±23.4h; The pharmacokinetics of nordazepam in the blood, urine and saliva met an one-compartment model, t1/2k01, t1/2k10 respectively were 38.4h±26.9h and 239.1h±289.4h; The pharmacokinetics of oxazepam glucuronide and temazepam glucuronide in the urine met an one-compartment model, t1/2k01, t1/2k10 respectively were 35.3h±32.5h, 234.0h±265.1h and 3.9h±5.2h,7029.4h±32137.7h. Oxazepam were detected in a lower concentration in15 d.The pharmacokinetics of diazepam and its metabolites had showed an individual differences, which might be due to the gene type of CYP2C19.3. Estimating time of last diazepam use: By blood diazepam/nordiazepam concertrion ratios, a model for estimating time of last diazepam use was established, and an equation was developed to predicte elapsed time of diazepam use. Especially the model was developed respectively from male or female blood diazepam/nordazepam concertrion ratios could provided an equation to predict elapsed time with a maximum error value less than 20%.Conclusion:1. A simultaneous analysis of diazepam and its metabolites in biological samples by SPE-HPLC-MS-MS was established with a reasonable LOQ, linear range and recovery,which can be used for determination of diazepam and its metabolites in forensic toxicokinetics study and the forensic identification of diazepam poisoning death.2. The pharmacokinetics model of diazepam and its metabolites in human were established. It was firstly reported that the pharmacokinetics of diazepam and its metabolites(I and II phase) in the blood, saliva, and urine of Chinese people, which met a two and an one compartment model except for oxazepam.3. A preliminary model for estimating time of last diazepam use was establishe d by blood diazepam/nordiazepam concertrion ratios.