LC-MS/MS法测定人血浆中伊伐布雷定及其活性代谢产物的浓度

目的:建立测定人血浆中伊伐布雷定及其活性代谢产物浓度的方法。方法:血样以叔丁基醚提取后,采用液-质联用(LC-MS/MS)法进样测定。色谱柱为Waters C18,流动相为5 mmol/L醋酸铵-甲酸-甲醇(70∶0.1∶30),流速为0.3 ml/min,柱温为30℃,内标为氨溴索;采用电喷雾离子化(ESI)离子源,以多反应监测(MRM)模式检测,伊伐布雷定、去甲伊伐布雷离子对的m/z分别为469.2/177.1、455.2/177.1。结果:伊伐布雷定、去甲伊伐布雷定血药浓度分别在0.48¹20、0.16120、0.16⁴0 ng/ml范围内线性关系良好(r分别为0.999 8和0.999 7);方法回收率分别为96.35%40 ng/ml范围内线性关系良好(r分别为0.999 8和0.999 7);方法回收率分别为96.35%⁹8.33%、96.20%98.33%、96.20%¹02.50%;日内、日间RSD均<7%;血浆样品长期冻存(-20℃冷冻1个月)稳定性良好,反复冻融3次及室温放置4 h条件下,样品浓度均无显著变化。结论:本方法快速、简便、灵敏、准确,可用于伊伐布雷定的药动学研究。

Determination of Ivabradine and Active Metabolites in Human Plasma by LC-MS/MS

OBJECTIVE： To establish a method for the determination of ivabradine and active metabolites in human plasma. METHODS： After extracted with MTBE, LC-MS/MS was adopted for the determination of blood sample. The determination was performed on Waters C18 column with mobile phase consisted of 5 mmol/L ammonium acetate-methanoic acid-methanol （70：0.1：30） at the flow rate of 0.3 ml/min. The column temperature was 30 ℃. The internal standard was ambroxol. Plasma concentrations were determined by MRM mode with ion pairs of 469.2/177.1 （ivabradine） and 455.2/177.1 （N-demethylivabradine）. RESULTS： The linear range of ivabradine and N-demethylivabradine was 0.48-120 ng/ml （r=0.999 8） and 0.16-40 ng/ml （r=0.999 7）, respectively. Method recoveries were 96.35%-98.33% and 96.20%-102.50%, respectively. RSDs of intra-day and inter-day were lower than 7 %. The spiked plasma samples were stable at --20℃ in a long-term stability test. After three freeze-thaw cycles, at indoor temperature stored for 4 h, the concentrations of sample were not obviously changed. CONCLUSIONS： The method is simple, sensitive and accurate, and can be used for pharmacokinetic study of ivabradine.