LC-MS/MS法同时测定大鼠尿液中4种淫羊藿黄酮苷

Simultaneous Quantitative Determination of Four Epimedium Flavonoids in Rat Urine by LC-MS/MS

目的应用高效液相色谱-串联质谱联用(LC-MS/MS)法建立大鼠尿液中朝藿定A、B、C及淫羊藿苷的同时测定方法,初步探讨淫羊藿提取物在大鼠体内的排泄规律。方法以人参皂苷Rg1为内标,选用Agilent Eclipse XDB-C_(18)色谱柱(2.1 mm×150 mm,5μm);流动相:乙腈-0.1%乙酸水(30∶70);流速:0.3 m L·min^(-1);柱温:40℃;采用电喷雾离子源(ESI)负离子多反应检测(MRM)模式测定。结果朝藿定A、B、C以及淫羊藿苷在1~500 ng·m L^(-1)浓度范围内线性关系良好,日内精密度(RSD)均小于7.4%,日间精密度(RSD)均小于12.7%。大鼠口服灌胃给予淫羊藿提取物36 h后朝藿定A、B、C及淫羊藿苷的平均尿累积排泄量/给药量比值不超过0.27‰,0.20‰,0.30‰和0.43‰。结论本研究建立的LC-MS/MS方法简便灵敏快速,适用于尿液中朝藿定A、B、C以及淫羊藿苷的同时测定及淫羊藿提取物尿排泄动力学研究的应用,研究结果显示淫羊藿提取物原形药物基本不经尿液排泄,提示肾排泄不是其主要消除途径。

Objective To establish a liquid chromatography-mass spectrometry/mass spectrometry（LC-MS/MS）method for the simultaneous analysis of epimedin A,epimedin B,epimedin C and icariin in rat urine. Methods Ginsenoside Rg1 was used as the internal standard（IS）. Chromatographic separation was carried out on an Agilent Eclipse XDB-C_（18）column（2.1 mm×150 mm,5 μm） by isocratic elution with 32 % acetonitrile and 68 % water containing 0.1 % acetic acid at a flow rate of 0.3 m L·min^（-1）. The quantification of the analytes was performed by mass spectrometry with electrospray ionization（ESI）inlet and negative ion multiple reaction monitoring（MRM）mode. Results The calibration curves of epimedin A,epimedin B,epimedin C and icariin were linear in the range of 1~500 ng·m L^（-1）. The intra-day precision of this method was less than 7.4 %,and the inter-day precision was less than 12.7 %. The ratio of mean urine cumulative excretion of the four analytes 36 h after intragastric administration to the administration dosage was 0.27 ‰, 0.20 ‰, 0.30 ‰, 0.43 ‰, respectively. Conclusion The established assay method is simple and convenient,which is suitable for the simultaneous measurement of urine epimedin A,epimedin B,epimedin C and icariin concentration in rats,and for the study of urine excretion kinetics of Herba Epimedii extract. The results showed that unchanged analytes of Herba Epimedii extract are hardly eliminated through urine in rats,indicating that renal excretion may not be the major elimination method for Herba Epimedii extract.