高效液相色谱-串联质谱法同时测定人血浆中瑞格列奈和二甲双胍及其在人体药动学中的应用

目的:建立高效液相色谱-串联质谱法(HPLC-MS/MS)同时测定人血浆中瑞格列奈和二甲双胍浓度的方法,并进行人体药动学研究。方法:以乙腈沉淀蛋白处理血浆样品,替米沙坦做内标,采用Kromasil 60-5CN色谱柱(2.1 mm×100 mm, 5 μm),流动相为20 mmol·L^-1醋酸铵水溶液(含0.2%甲酸)-乙腈(45:55);质谱采用电喷雾离子化-多反应离子监测(ESI-MRM)。并测定12名健康志愿者单剂量口服瑞格列奈盐酸二甲双胍复方片(2 mg:500 mg)后瑞格列奈和二甲双胍的血浆浓度。结果:瑞格列奈和二甲双胍分别在0.05~50 μg·L^-1、2~2 000 μg·L^-1范围内线性良好,定量下限分别为0.05 μg·L^-1和2 μg·L^-1,日内日间精密度均小于8%,提取回收率分别为77.32%~80.65%和78.98%~82.46%。瑞格列奈和二甲双胍的主要药动学参数C_max分别为(29.50±9.57),(1 167±197.77)μg·L^-1;t_max分别为(0.83±0.39),(1.88±0.64)h;t_1/2分别为(1.11±0.38),(4.31±0.86)h;AUC_0→∞分别为(56.90±20.31),(7 407.32±1 653.25)μg·L^-1·h^-1。结论:建立的分析方法灵敏、准确可靠,样品处理快速、简便,适用于瑞格列奈/盐酸二甲双胍复方片人体药动学和生物等效性研究。

Simultaneous determination of repaglinide and metformin in human plasma by HPLC-MS/MS and its application in pharmacokinetics

OBJECTIVE To establish an HPLC-MS/MS method for simultaneous determination of repaglinide and metformin in human plasma and to investigate its pharmacokinetics in human.METHODS After protein precipitation by acetonitrile, concentrations of repaglinide and metformin were analyzed on Kromasil 60-5CN column (100 mm×2.1 mm, 5 μm) with aqueous solution (20 mmol·L^-1 ammonium acetate, 0.2% formic acid) and acetonitrile (45:55) as the mobile phase. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI) for repaglinide, metformin and internal standard (telmisartan).RESULTS The calibration curves were linear in concentration ranges of 0.05~50 μg·L^-1 for repaglinide and 2~2 000 μg·L^-1 for metformin. The lower limits of quantification (LOQ) of method were 0.05 μg·L^-1 for repaglinide and 2 μg·L^-1 for metformin, respectively. Intra- and inter-day precision were less than 8%. The extraction recoveries were 77.32%~80.65% for repaglinide and 78.98%~82.46% for metformin, respectively. The main pharmacokinetic parameters of repaglinide and metformin were as follows after a single oral dose of Repaglinide/Metformin hydrochloride tablet in 12 h ealthy subjects: C_max(29.50±9.57) and (1167±197.77)μg·L^-1, t_max (0.83±0.39) and (1.88±0.64)h, t_1/2 (1.11±0.38) and (4.31±0.86)h, AUC_0→∞(56.90±20.31) and (7407.32±1653.25)μg·L^-1·h^-1, respectively.CONCLUSION The method is proved to be rapid,specific, sensitive and accurate, which can be used for clinical pharmacokinetics and bioequivalence studies for the repaglinide /metformin hydrochloride tablets.