LC-MS/MS法同时测定人血浆中阿托伐他汀及其活性代谢物的浓度

Simultaneous determination of atorvastatin and its active metabolites in human plasma by LC-MS/MS

目的建立LC-MS/MS法测定人血浆中阿托伐他汀及其主要活性代谢物2-羟基阿托伐他汀和4-羟基阿托伐他汀的浓度。方法分别以阿托伐他汀-d5,2-羟基阿托伐他汀-d5和4-羟基阿托伐他汀-d5为内标,加入5%甲酸水溶液,用甲基叔丁基醚进行液液萃取后,进样分析。色谱柱为CAPCELL PAK C_(18)(2 mm×100 mm,TYPE:MGⅡ,5μm),流动相为乙腈-5 mmol·L^(-1)乙酸胺水溶液-甲酸=46:54:0.143,流速为0.40 mL·min^(-1)。电喷雾离子源:正离子多反应监测扫描分析。阿托伐他汀的离子对为m/z 559.3→m/z 440.2,2/4-羟基阿托伐他汀的离子对为m/z 559.3→m/z 440.2。考察其专属性、标准曲线与定量下限、准确度和精密度、提取回收率和基质效应、残留效应和稳定性。结果血浆中阿托伐他汀的标准曲线方程为y=4.35×10^(-2)x+3.43×10^(-4)(r=0.9993),在0.05~100 ng·mL^(-1)线性关系良好,定量下限为0.05ng·mL^(-1);2-羟基阿托伐他汀的标准曲线方程为y=1.03×10^(-1)x+6.93×10^(-4)(r=0.998 7),在0.05~50 ng·mL^(-1)线性关系良好,定量下限为0.05ng·mL^(-1);4-阿托伐他汀的标准曲线方程为y=5.35×10^(-1)x-2.26×10^(-3)(r=0.9984),在0.05~5.0 ng·mL^(-1)线性关系良好,定量下限为0.05ng·mL^(-1)。阿托伐他汀、2-羟基阿托伐他汀和4-羟基阿托伐他汀的日内、日间的相对标准偏差(RSD)均小于15%;提取回收率为61.64%~90.29%;基质效应为88.74%~105.62%。结论 LC-MS/MS法快速、灵敏、准确、选择性强、重复性好,适用于人血浆中阿托伐他汀及其活性代谢产物的浓度测定,可以满足临床药物浓度监测以及药代动力学研究的需要。

Objective To develop an LC-MS/MS method for simultaneous determine the concentration of atorvastatin（AT） and its two active metabolites prtho-hydroxyatorvastatin（o-AT） andpara-hydroxyatorvastatin（p-AT） in human plasma.Methods Deuterium-labeled compounds,o-AT-d_5 and p-AT-d_5 were used as the internal standards.The analytes were extracted from human plasma through liquid-liquid extraction method by using tert-butyl ether,after adding 5%formic acid.The chromatographic separation was achieved on a CAPCELL PAK C_（18）（2.0 mm × 100 mm,TYPE：MG Ⅱ,5μm） column with the mobile phase,consisted of a mixture of acetonitrile-5 mmol · L^-1 ammonium acetate buffer-formic acid（46：54：0.143,v/v/v）,in isocratic mode at a flow rate of 0.40 mL · min^-1.Data acquisition was carried out in multiple reaction monitoring（MRM）mode.Analytes were detected on a tandem mass spectrometer,equipped with an electrospray ionization source that was operated in the positive mode,using the transitions of m/z 559.3→m/z 440.2 for atorvastatin,and m/z 575.3→m/z440.2 for both ortho-and para-hydroxyatorvastatin.The selectivity,standard curve,precision and accuracy,extraction recoveries,matrix effect,carry over and stability were investigated.Results The linear range of atorvastatin was0.05-100 ng · mL^-1.The linear regression equation was y =4.35×10^-2x ±3.43 × 10^-4（r =0.999 3） with the lower limit of quantitation（LLOQ） of 0.05 ng · mL^-1.The linear range of ortho-atorvastatin was 0.05-50 ng · mL^-1.The linear regression equation was y = 1.03 × 10^-1x ± 6.93 × 10 -（r = 0.998 7） with the lower limit of quantitation（LLOQ） of 0.05 ng · mL^-1.The linear range of para-atorvastatin was 0.05-5.0 ng · mL^-1.The linear regression equation was y = 5.35 × 10^-1 x-2.26 × 10^-3（r =0.998 4） with the lower limit of quantitation（LLOQ） of0.05 ng · mL^-1.The intra-day and inter-day relative standard deviations（RSD） of LLOQ,low,mediuml,medium2 and high concentrations of atorvastatin were both less than 15%.The extraction recoveries were 61.64%-90.29%.The matrix effects were 88.74%-105.62%.Conclusion The established method is rapid,sensitive,accurate,specific and reliable,and suitablefor simultaneous determination of atorvastatin and its active metabolites（ortho-atorvastatin and para-atorvastatin） in human plasma,which meets the needs of clinical therapeutic drug monitoring and pharmacokinetics research.