超高效液相色谱-质谱联用法同时测定人血浆中美托洛尔及其代谢物的浓度

Simultaneous determination of metoprolol and its metabolites in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry

目的建立超高效液相色谱-质谱联用法(UPLC-MS/MS)定量测定人血浆中美托洛尔及其代谢产物(α-羟基美托洛尔、O-去甲基美托洛尔)的方法。方法采用乙酸乙酯从100μL血浆中提取分析物及内标(d7-美托洛尔),色谱柱:UPLC HSS T3柱(3.0 mm×100 mm,1.8μm),柱温:30℃,流动相:5mmol·L^(-1)醋酸铵+0.2%甲酸与乙腈,梯度洗脱,流速:0.30 m L·min^(-1);质谱采用正离子模式(ESI),多反应监测模式进行检测。结果美托洛尔的标准曲线方程为y=1.56×10^(-1)x-4.80×10^(-2),在0.2~100.0 ng·m L^(-1)范围内线性关系良好,定量下限为0.2 ng·m L^(-1);α-羟基美托洛尔的标准曲线为y=1.06×10^(-1)x^(-1).30×10^(-2),在0.2~100.0 ng·m L^(-1)范围内线性关系良好,定量下限为0.2 ng·m L^(-1);O-去甲基美托洛尔的标准曲线为y=4.40×10^(-2)x^(-1).10×10^(-2),在0.1~50.0 ng·m L^(-1)范围内线性关系良好,定量下限为0.1ng·m L^(-1)。美托洛尔、α-羟基美托洛尔、O-去甲基美托洛尔的平均绝对回收率分别为64.7%,51.7%,55.4%。批间及批内精密度的相对标准差均小于15%,稳定性良好。该方法成功测定了90例服药患者的美托洛尔及其两种代谢产物的稳态血药浓度。结论该方法敏感,快速,血浆消耗量少,可用于临床用药监测及其相关研究。

Objective A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry（ UPLC-MS/MS） assay was developed and validated for the simultaneous quantification of metoprolol and its metabolites（ α-Hydroxy metoprolol and O-desmethyl metoprolol） in human plasma. Methods Sample was prepared by liquid-liquid extraction with ethyl acetate from 100 μL plasma. Chromatographic column： UPLC HSS T3 column（ 3. 0 mm × 100 mm,1. 8 μm）,column temperature： 30℃,mobile phase： 5 mmol · L^（-1） ammonium acetate ＋0. 2% formic acid, gradient acetonitrile at a flow rate of 0. 30 m L·min^（-1）. Mass spectrometry detection was performed through positive ion electrospray ionization（ ESI）,multiple reaction monitoring mode.Results The regression equations of metoprolol,α-Hydroxy metoprolol and O-desmethyl metoprolowere y = 1. 56 × 10^（-1） x-4. 80 × 10^（-2）,y = 1. 06 × 10^（-1） x^（-1）. 30 × 10^（-2）,y = 4. 40 × 10^（-2） x^（-1）. 10 × 10^（-2）,respectively. They all showed good linearities in the ranges of 0. 2^（-1）00. 0 ng·m L^（-1）,0. 2^（-1）00. 0 ng·m L^（-1）,and 0. 1-50. 0 ng·m L^（-1）,respectively. Their lower limits of quantification were0. 2 ng·m L^（-1）,0. 2 ng·m L^（-1）,and 0. 1 ng·m L^（-1）,respectively. Mean absolute recoveries were 64. 7%,51. 7%,55. 4% for metoprolol,α-Hydroxy metoprolol,O-desmethyl metoprolol,respectively. RSD of intra and inter precision and accuracy were less than 15% and stability was good. It was successfully applied to determine steady state concentrations of metoprolol and its two metabolites for 90 patients treated with metoprolol. Conclusion This method is sensitive,less time consuming,less plasma consuming,suitable for clinical monitor or study of metoprolol.