反相高效液相色谱法测定人血浆中康艾注射液主要有效成分

Determination of Plasma Concentrations of Oxymatrine and Its Active Metabolite Matrine in Kangai Injection by RP-HPLC

目的:建立测定康艾注射液主要有效成分氧化苦参碱(OMT)及其主要活性代谢物苦参碱(MT)人体血药浓度的反相高效液相色谱(RP-HPLC)检测方法。方法:氯仿萃取法处理血浆样品,以Diamonsil C18柱(200 mm×4.6 mm,5μm)为固定相,甲醇-醋酸盐缓冲液系统(V∶V,30∶70)为流动相,流速1.0 mL.min-1,检测波长210 nm,建立同时测定血浆中OMT和MT的HPLC法,并按生物样品的测定要求,对方法的专属性、灵敏度、精密度、稳定性、回收率和线性范围等进行考察。结果:在确定的色谱分离条件下,血浆内源性杂质对样品测定无干扰,OMT和MT的保留时间分别为5.2和7.8 min,二者分别在0.3～19.2 mg.L-1和0.2～12.8 mg.L-1范围内样品峰面积与血药浓度之间呈良好的线性关系,且日内和日间变异系数均小于15%。配对t检验结果表明,室温放置24 h组、反复冻融3次组、-20℃冷冻放置2周组与新鲜制备组间浓度无统计学意义(P>0.05)。结论:所建方法定量准确可靠,适用于康艾注射液主要有效成分及其代谢物血药浓度的测定。

OBJECTIVE： To establish a RP-HPLC method for determining plasma concentrations of oxymatrine（OMT） and its metabolite matrine（MT） in Kangai injection.METHODS： Plasma samples were extracted with chloroform.A reverse phase high performance liquid chromatographic（RP-HPLC） method for simultaneous determination of oxymatrine and matrine in Kangai injection was established with Diamonsil C18 column（particle size 5 μm,200 mm×4.6 mm） used as the stationary phase.The mobile phase was composed of methanol and acetate buffer solution（30∶70,V∶V） at a flow rate of 1.0 mL·min-1.The detection wavelength was set at 210 nm.The specificity,sensitivity,precision,stability,recovery and linear range etc were all determined in accordance with the criteria for determination of biological specimen.RESULTS： Under defined chromatographic separation conditions,endogenous foreign matters showed no interferences on sample determination.The retention time of OMT and MT stood at 5.2 and 7.8 min respectively,and the linear ranges of OMT and MT were 0.3-19.2 mg·L-1 and 0.2-12.8 mg·L-1,respectively,with both inter-day and intra-day coefficients of variation at less than 15%.Pairwise t tests showed no statistically significance in plasma concentrations of OMT and MT among the group stored under room temperature for 24 h,the group undergoing Freeze-thaw for 3 times,the group stored at-20℃ for 2 weeks and the fresh-preparation group（P0.05）.CONCLUSION： The established method is accurate and reliable in quantification and it is thus suitable for determining plasma concentrations of oxymatrine（OMT） and its metabolite matrine（MT） in Kangai injection.