Simultaneous Determination Of2Anti-obesity Drugs And Distribution Of Fenfluramine In Poisoned Rats

Objective:1. To study the simultaneous determination of fenfluramine hydrochloride andsibutramine hydrochloride in biological samples.2. To establish an acute poisoned model of fenfluramine, study distributionregularity of fenfluramine in rats.3. The distribution characteristics of fenfluramine provide a practical basis forforensic determination cases of fenfluramine poisoned. Provide a simple, reliableanalytical method for health food which is added fenfluramine illegally.Methods:1. Gas Chromatography（GC）methods:After adjusting into alkaline, the samples were extracted by ethyl acetate. Thedetermination was performed on gas chromatography and a TR-5MS capillary column（30m×0.32mm×0.25μm）was used for separation. A flame ionization detector wasequipped. The initial column temperature was80℃, kept2min, then increased to190℃at a rate of13℃/min, and then increased to210℃at a rate of5℃/min,increased to270℃which kept1min at13℃/min at last. The temperature ofinjector and detector were280℃. Injection mode was split/splitless mode，andsplitless time is0.2min. Sample size was1μL.2. Qualitative analysis and quantitative analysisThe retention time of fenfluramine and sibutramine was used to qualitativeanalysis. Standard working solution series of fenfluramine and sibutramine weredetermined by gas chromatography. Concentration of fenfluramine or sibutramine and peak areas was used as horizontal and vertical coordinates. The linear regressionequation was used to quantitative analysis of fenfluramine and sibutramineconcentration in biological samples.3. Distribution studySD rats （male） were randomly divided into experimental and control groups （eachgroup6rats）. The experimental rats were gavage dose of fenfluramine for2times ofLD₅₀(LD₅₀122mg/kg), and the control group rats were given physiological saline atsame time. Experimental rats were executed in30min, and taken blood, urine, brain,heart, lungs, liver, spleen, kidney and muscles to detect the concentration offenfluramine. The samples of control rats were taken as blank check. Obtain thedistribution discipline of fenfluramine in rats by statistical analysis.Results1. Determination resultsThe method for determination in samples presented a perfect separation offenfluramine and sibutramine with retention time of5.8min and12.8min respectively.Endogenous impurity has no interfere to determination. In blood, urine and liver, thelinear range of fenfluramine and sibutramine both are1.0-150.0μg/mL （μg/g）. Thelimit of detection was0.2μg/mL （S/N≥3）. The recoveries of fenfluramine werebetween90%and110%. The inter-day relative standard deviation of precision and theintra-day relative standard deviation of precision were both less than6%.2. Toxic symptomAfter given intragastric administration of fenfluramine, the experimental grouprats became restless, shortness of breath. As time goes by, rats became muscle tremors,sensitive to acoustic stimuli, and breathed faster. none dead in30min. In the controlgroup, no obvious abnormalities were seen.3. DistributionsThe sort of fenfluramine concentration in organs and fluids of acute poisoned rats:lungs>liver>spleen>kidney>brain>urine>heart>muscles>blood.Conclusion 1. The analytical method could determine fenfluramine and sibutraminesimultaneously. This method is rapid, accurate and reliable, which sample processingis simple. It is applicable to forensic toxicology and health food determination.2. Distribution of fenfluramine showed that: concentrations of fenfluramine inlungs, liver, spleen and kidney were high, and lower in blood. This was mainlybecause of high lipid solubility of fenfluramine.3. The results of experimental suggest that: suspected case caused byfenfluramine poisoning, beside blood, lungs, liver, kidney and urine has a certainamount of fenfluramine, and appeared to be suitable for use as samples. The analysismethods of fenfluramine provide a practical basis to forensic identification work.