HPLC法同时测定Beagle犬血浆中茴三硫及其代谢物浓度的方法学研究

Simultaneous Determination of Anethole Trithione and Its Metabolite in Plasma of Beagle Dogs by HPLC

目的:考察高效液相色谱法同时测定Beagle犬血浆中茴三硫(AT)及其代谢物对羟基苯基三硫酮(ATX)浓度的方法学。方法:用乙腈直接沉淀犬血浆样品中的蛋白质,取上清液进样分析。色谱柱为Diamonsil ODS-C18,流动相为甲醇-水(80∶20),流速为1.0mL·min-1,紫外检测波长为346nm。结果:AT和ATX与犬血浆中的内源性物质完全分离且无其它干扰,保留时间分别为5.5、3.4 min。二者检测浓度线性范围分别为0.254～3.392、0.125～1.664μg·mL-1(r分别为0.999 9、0.999 8);平均回收率分别为(93.89±2.39)%、(96.28±2.83)%;日内RSD分别为0.83%～4.11%、0.55%～1.23%,日间RSD分别为0.71%～4.51%、1.74%～3.92%;最低定量浓度分别为0.254、0.125μg·mL-1。结论:方法学考察结果表明,所建立方法简便、准确、重复性好,可用于犬血浆中AT及ATX的浓度测定。

OBJECTIVE： To establish an HPLC method for the determination of anethole trithione （AT） and its metabolite 4-hydroxy-anethole trithione （ATX） in plasma of beagle dogs simultaneously. METHODS： Protein in plasma was deposited directly using acetonitrile and analyzed by supernate. The separation was performed on Diamonsil ODS-C18 column with a mobile phase consisting of methanol and water （80：20） at a flow rate of 1.0 μg·mL^-1 with the detection wavelength of 346 nm. RESULTS： AT and ATX were completely separated without interference. The retention time was 5.5 rain and 3.4 min respectively. The linear range were 0.25-3.392 μg·mL^-1 （r=0.999 9） for AT with average recovery of （93.89±2.39） % and 0.125-1.664 μg·mL^-1 for ATX （r=0,999 8） with average recovery of （96.28±2.83）%. The intra-day RSD and inter-day RSD of AT were 0.83%-44.11% and 0.71% - 4.51% while that of ATX were 0.55 % - 1.23 % and 1.74 % -3.92 %, respectively. The detection limit of AT in human plasma was 0.254μg·mL^-1 and that of ATX was 0.125 μg·mL^-1. CONCLUSION： It is proved that established method is simple, accurate and reproducible for plasma concentration determination of AT and ATX in plasma of beagle dogs.