| Objective:1. To establish the gas chromatography analysis method of appetite inhibitor sibutramine hydrochloride in biological samples(blood and liver).2. To establish acute poisoning model of sibutramine hydrochloride by irrigating stomach in rats, observe the poisoning performance; Detect the concentration of sibutramine hydrochloride in rat vivo viscera and body fluids, to get important toxicology data and internal distribution law.3. Research the distribution discipline and characteristics of sibutramine hydrochloride in poisoned rat vivo viscera and body fluids, to offer reference and scientific proof for sample collections,determinations,analysis and distinguish of dead reasons in sibutramine hydrochloride poisoning cases.Methods:1. Sample processing methodAfter the PH of the samples were sdjusted into alkaline,extracted by ethyl acetate, blew dry by nitrogen, detected by gas chromatography.2. Instrument analysis methodChromatographic column:Rtx?-1 elastic quartz capillary column(30m×0.32mm×0.25μm).The column temperature was temperature programming.The initial column temperature was 150℃,kept 2 minute,then increased to 170℃at a rate of 10℃/min,kept 3 minute,and then increased to 250℃at a rate of 15℃/min.The temperature of the injector and detector(FID) were both 280℃.Injection was made in the split/splitless mode with split time of 0.5 minute.Nitrogen was used as carrier gas,its pressure was 300 kPa.The air pressure was 35 kPa and the hydrogen was 25 kPa.1μL was injected into the gas chromatograph.3. Qualitative and quantitative analysisStandard series of sibutramine hydrochloride were made up and were determined by the gas chromatograph. Qualitative analysis was based on retention time of sibutramine hydrochloride.Calibration regression were done while concentration of sibutramine hydrochloride as X and peak areas of sibutramine hydrochloride as Y.The regression equation was Y=a+bX,so we can calculate the concentration of sibutramine hydrochloride in biological samples.4. Sampling and testing body distribution12 SD male rats were randomly divided into two groups,experimental group and control group,6 rats in each group. The experimental group was fed with sibutramine hydrochloride from the mouth to the stomach at the dose of 5 times of LD50(LD50 is 78.1 mg/kg), then watched and recorded the toxic reaction and executed by cervical dislocation in an hour.Blood, urine, brain, heart, liver, spleen, lungs, kidney and the muscles of hind leg were got from the dead rats in order to detect the concentration of sibutramine hydrochloride used the detection method above.The control group was fed with physiological saline of the same dose as the experimental group,then got the same specimens as the blank control and used the same detection as the experimental group.Then we could obtain the distribution rule of sibutramine hydrochloride in rats by statistical analysis.Results1.Gas chromatograph analysis results The peak of sibutramine hydrochloride was well under the gas chromatograph method which was built above,and the endogenous impurity did not disturb the qualitative and quantitative detection of sibutramine hydrochloride in samples,the retention time was 3.707 minute.The quantitative analysis range of sibutramine hydrochloride is 1.0～200μg/mL in blood and 5.0～200μg/mL in liver,the linear relationship was well.And the limit of detection was 0.2μg/mL in blood,1.0μg/mLin liver(S/N≥3).The recoveries of different concentration of sibutramine hydrochloride in blood were 99.1±4.9%,101.0±4.7%,101.4±3.4%. The recoveries of different concentration of sibutramine hydrochloride in liver were 94.6±3.7 % ,90.7±2.5%,95.8±2.7%. The standard deviations of blood and liver were both less than 5%.2. Toxic symptomAfter feeding with sibutramine hydrochloride,the rats of the experimental group became restless, ecphysesis and no independent trembling.As time goes by,they felt uneasier and breathed faster.None was dead in one hour after feeding with sibutramine hydrochloride. Apparent anomalies were not seen in the control group.3. Body concentration distributionThe size order of concentration of sibutramine hydrochloride in acute poisoned rats were : liver>lungs>kidney>spleen> brain>heart>muscles>blood,nothing was detected in urine.The concentration of sibutramine hydrochloride in the liver was obviously higher than that in other organs in rats, and certain content of sibutramine hydrochloride could be detected in blood.Conclusion1. This experiment established gas chromatographic analysis method of sibutramine hydrochloride. According to various biological specimens,the sample processing method was simple and fast,the analytical method was precise and reliable.This method was suitable for forensic toxicological analysis of detection of case identification and clinical poisoning detection.2. Distribution results of the poisoned rats that feeding with sibutramine hydrochloride throw the mouth showed that: The highest content of sibutramine hydrochloride was liver, followed by the lung, spleen , kidney and brain, etc,and lower in blood. This was mainly because sibutramine mainly in hepatic metabolism, plasma protein binding rate was higher.3. The experimental results showed that:in the poisoning cases which accidental take or take too much sibutramine hydrochloride, blood can be used as specimens for the qualitative and quantitative analysis.The content of sibutramine hydrochloride in liver, lungs and other organs of content was higher,they were more suitable for the qualitative and quantitative detection of the death cases of sibutramine hydrochloride. Therefore,this experiment provided better detection analysis method for death case of forensic identification involving sibutramine hydrochloride ,had good reference value. |
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