| [Objective]1. To establish the simultaneous determination of6commonly used Benzodiazepines in biological samples.2. To establish an acute poisoned animal model of clonazepam, study the distribution of clonazepam in acute poisoned rats. Obtain the clonazepam distribution in vivo along with the important toxicological data.3. To study the distribution discipline and characteristics of clonazepam in various organs and body fluids of rat models, and provide scientific evidences for forensic identification about sample collections, processing, analysis, distinguish causes of death died of clonazepam intoxication. Provide a simple, reliable analytical method for determination of forbidden addition of clonazepam in tranquilizing mind Traditional Chinese Medicine. Meanwhile, it provides reference for the further analysis and determination of multiple benzodiazepines.[Methods]1. Gas Chromatography (GC) analysis methods:After alkalization procedure, the biological samples were extracted by ethyl acetate in liquid-liquid extraction, then detected on gas chromatography. Chromatographic condition was as follows:a HP-5Elastic quartz capillary column used for compounds separation; a nitrogen phosphorus detector (NPD) was equipped. The oven temperature:By using temperature programming; The initial oven temperature was200℃, hold for1minute, then increased to270℃at a rate of20℃/min, then increased to280℃at a rate of5℃/min with the final temperature hold time of4minutes.The temperature of the injector and detector were both280℃.Injection was performed in spilt mode with a split ratio of5:1; Carrier gas:high purity nitrogen, total flow rate was27mL/min, oven flow rate was4.2mL/min, air flow rate was60mL/min, hydrogen flow rate was2.2mL/min, make up gas flow rate was30mL/min.1μL was injected into the gas chromatographic system.2. Qualitative analysis and quantitative analysi:The retention time (tR) of each benzodiazepine was used to qualitative analysis. Reference mixed standard solutions of diazepam, nitrazepam, clonazepam, estazolam, alprazolam, triazolam were made up and all were simultaneously analysis by gas chromatography to determine the tR value and the order of peaks of each target analytes. Standard working series solutions of clonazepam were prepared, calibration regression were done while concentration of clonazepam as X and peak areas of clonazepam as Y. The regression equation was Y=aX+b, which was used to quantitative analysis the concentration of clonazepam in biological samples.3. Distribution study:12male SD rats were randomly divided into two groups, the experimental group and control group,6rats in each group. The experimental group were given intragastric administration of clonazepam with a dose of LD50(LD50is2000mg/kg), the control group were given intragastric administration of physiological saline, the behavior reactions of each group rats were observed then sacrificed by cervical dislocation in30min. Blood, urine, heart, lungs, liver, spleen, kidney, hind leg muscle, brain were collected immediately at room temperature, then detected the concentration of clonazepam. The specimens of control group were taken out correspondingly and used as blank check. The distribution discipline of clonazepam in rats in vivo was obtained after statistical analysis of the toxicology data.[Results]1. Determination results:The developed method for the simultaneous determination of6benzodiazepines by gas chromatograph was successfully applied, the order of the target analytes was:diazepam, nitrazepam, clonazepam, estazolam, alprazolam, triazolam. The endogenous components has no interfere with the detection. The linear ranges of clonazepam quantitative analysis in blood, urine and liver were1.0-80.0μg/mL,1.0-80.0μg/mL,1.0-80.0μg/g respectively, the linear relationship was well. The limits of detection were0.2μg/mL in blood,1.0μg/mL in urine,1.0μg/g in liver(S/N>3).The spiked samples average recoveries of clonazepam were between97.5%and103.2%. Within-day and between-days relative standard deviation of precision were both less than10%.2. Toxic symptom:After given intragastric administration of clonazepam, the experimental group rats became mental restlessness, convulsions, muscle tremors in10minutes. And then performance quiet gradually, no sense of direction, gait disturbance, hind leg muscle tension, ataxia, no reaction to pain, none was dead in30minutes. No obvious abnormalities were seen in the control group.3. Body concentration distribution:The experimental group results show that clonazepam was widely present in the tissues and body fluids in the acute poisoned rats, meanwhile, the concentration distribution characteristics were as follows:lungs>liver> heart>kidney> spleen> brain>muscles>blood>urine.[Conclusion]1. The developed gas chromatograph method could simultaneous determination of multiple commonly used Benzodiazepines. The method is simple for specimens pretreated, rapid, accurate, reliable and practicable. It is suitable for forensic toxicological analysis and clinical toxicological cases of accidental and suicidal poisoning.2. Distribution in rats those were given intragastric administration of clonazepam showed that:concentrations of clonazepam mainly present in tissues contained rich blood and lipid such as the lungs, liver and heart, while present least in urine.3. The experimental results suggest that:for the cases caused of death suspected clonazepam abuse, postmortem specimens included liver, lungs, kidney and also blood would be selected and suitable for the qualitative and quantitative analysis, as those have a certain amount of clonazepam. When urine was collected for detection, the detection of target analytes should be the metabolites of clonazepam. The established analytical methods and distribution pattern of clonazepam in animal experiment provide an effective practical basis for forensic toxicology contexts. |
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