LC-MS/MS法同时测定人尿液中苦杏仁苷和芍药苷及其尿药排泄研究

An LC-MS/MS method for the simultaneous determination of amygdalin and paeoniflorin in human urine and application to urinary excretion study

建立苦杏仁苷和芍药苷尿药浓度的LC-MS/MS同时测定方法,研究健康中国受试者静脉滴注活血通络粉针剂后其体内苦杏仁苷和芍药苷的人体尿药排泄特征。尿液样品经甲醇沉淀蛋白并稀释后,采用Hedera ODS-2色谱柱分离,流动相为乙腈-5 mmol·L-1醋酸铵水溶液（含0.05%甲酸）（20∶80）;质谱采用Turbo-Ionspray电喷雾离子源和正离子多反应监测方式。尿液中杂质不干扰样品的测定,苦杏仁苷和芍药苷的标准曲线线性范围为0.03～40μg·m L-1,线性关系良好;高、中、低三种浓度的日间和日内精密度均小于15.0%;无介质效应、残留效应;稳定性良好。9名健康受试者单次静脉输注活血通络粉针6 g后,苦杏仁苷和芍药苷主要以原形经尿液排出体外,平均累计排泄率分别为79.6%和48.4%。建立的LC-MS/MS分析方法适用于健康中国受试者体内苦杏仁苷和芍药苷的尿药排泄研究。

The study aims to develop an LC-MS/MS method for the simultaneous determination of amygdalin and paeoniflorin in urine samples, and to investigate their urinary excretion characteristics in healthy volunteers after intravenous infusion administration of Huoxue-Tongluo lyophilized powder for injection （HTLPI）. The urine samples were extracted by methanol, and then separated on a Hedera ODS-2 column with a mobile phase of acetonitrile and 5 mmol.L-~ ammonium acetate buffer solution containing 0.05% formic acid （20 ： 80）. Electrospray ionization source was applied and operated in the positive ion mode using MRM. The method exhibited good linearity over the concentration range of 0.03 -40 ~tg.mL-1. The values on both the occasions （intra- and inter-day） were all within 15% at three concentration levels. No matrix effect and carry-over effect were observed. Amygdalin and paeoniflorin were stable in human urine under different storage conditions. Approximately 79.6% of the administered amount of amygdalin was excreted unchanged in urine within 24 h and which was 48.4% for paeoniflorin. The developed LC-MS/MS method can be applied to evaluate the urinary excretion of amygdalin and paeoniflorin.