液相色谱-串联质谱法测定人血浆中劳拉西泮的浓度及其在生物等效性研究中的应用

Determination of lorazepam in plasma by LC/MS/MS and its application in the bioequivalence study

目的建立LC/MS/MS法测定人血浆中劳拉西泮含量的方法,研究其片剂生物等效性.方法取血浆样品0.3mL,加入内标地西泮乙腈溶液0.6mL,涡流混合5min,高速离心15min,取上清液20μL进样,流动相:0.04%甲酸-乙腈(35:65,V/V);流速: 0.2mL/min;ZORBAX Eclipse XDB- C18(2.1mm×100mm,3.5μm).采用电喷雾离子源三级四极杆串联质谱,以选择反应监测(SRM)方式进行检测.用于定量分析的离子反应分别为m/z 321→275(劳拉西泮) 和m/z 285 →193(内标:地西泮). 结果劳拉西泮线性范围为0.71～71.33ng·mL-1,定量限为0.71 ng·mL-1,三种浓度的相对回收率为108.8%～112.3%,日内、日间RSD均小于4.1%(n=5).结论本法专属、灵敏、快速,适用于劳拉西泮的生物等效性研究.

OBJECTIVE To establish a LC/MS/MS method for determination of lorazepam in plasma and to study its bioequivalence. METHODS 0.3mL of the plasma sample was pipetted into a conical tube, 0.6mL internal standard solution containing diazepam in acetonitrile was added, the mixture was agitated for 5 min using a vortex agitator and centrifuged for 15 min at high speed. Then 20μL of supernatant was injected into the chromatograph. The mobile phase consisted of 0.04% formic acid -acetonitrile（35： 65）, the flow rate was 0.2mL · min^ -1, the column was ZORBAX Eclipse XDB- C18 （2.1 × 100mm,3.5 μm）. Electrospray ionization source was applied and operated in positive ion mode. Selected reaction monitoring（SRM） mode with the transitions of m/z 321→275 and m/z 285→193 was used to quantify lorazepam and diazepam respectively. RESULTS The linear calibration curve was obtained in the range of 0.71 -71.33 ng · mL^-1 ,The lower limit of quantification was 0.71 ng · mL^-1 ,three level concentration relative recoveries were 108.8% - 112.3% ,the intra-day and inter-day precision （RSD） were below 4.1%. CONCLUSION The method is simple, rapid,accurate and can be used to study bioequivalence of lorazepam.