UPLC-MS/MS测定血浆中伊伐布雷定及其活性代谢产物的含量

Determination of Ivabradine and Its Main Metabolite in Human Plasma by UPLC-MS/MS

关键词:  超高效液相色谱-串联质谱法  伊伐布雷定  代谢产物  血药浓度

Key words:  UPLC-MS/MS  ivabradine  main metabolite  plasma drug concentration

摘要:

目的 建立超高效液相色谱-串联质谱法测定人血浆中伊伐布雷定及其活性代谢产物(N-去甲伊伐布雷定)的含量。方法 选用Waters Acquity BEH C₁₈(50 mm×2.1 mm，1.7 μm)色谱柱，流动相为0.1%甲酸水溶液(A)-乙腈(B)，梯度洗脱；流速为0.4 mL·min^-1；电喷雾离子源，多反应监测。伊伐布雷定：[M+H]⁺，m/z 469.3→177.2，N-去甲伊伐布雷定：[M+H]⁺，m/z 455.2→262.2，卡马西平：[M+H]⁺，m/z 237.1→194.2。结果 伊伐布雷定线性范围为0.2~100 ng·mL^-1(r=0.998 1)，N-去甲伊伐布雷定线性范围为0.05~25 ng·mL^-1(r=0.993 1)；两者日间、日内精密度均<15%，方法回收率>90%，稳定性较好。结论 该方法快速、灵敏、重复性好，适用于血浆中伊伐布雷定及其代谢产物含量测定。

Abstract:

OBJECTIVE To establish a UPLC-MS/MS method for the determination of ivabradine and its main metabolite, N-demethyl ivabradine, in human plasma. METHODS The analytical column was Acquity BEH C₁₈(50 mm×2.1 mm, 1.7 μm). The mobile phase A consisted of water (containing 0.1% formic acid), the mobile phase B consisted of acetonitrile. The analytes were eluted from the column with a linear gradient. The flow rate was 0.4 mL·min^-1. A tandem mass spectrometer equipped with electrospray ionization source was used as detector with multiple reaction monitoring (MRM). The tandem mass ion transitions monitored were 469.3→177.2 for ivabradine, 455.2→262.2 for N-demethyl ivabradine and 237.1→194.2 for carbamazepine. RESULTS The calibration curve was linear over the 0.2–100 ng·mL^-1(r=0.998 1) for ivabradine and 0.05–25 ng·mL^-1 (r=0.993 1) for N-demethyl ivabradine in plasma, respectively. Intra-day and inter-day RSD for assaying the plasma sample were both <15%, absolute recovery were >90%. CONCLUSION The method is proved to be highly sensitive, selective, and suitable for pharmacokinetic investigations of ivabradine and its main metabolite.