LC-MS/MS法同时测定人血浆中万古霉素和去甲万古霉素的质量浓度

Simultaneous determination of vancomycin and norvancomycin in human plasma by LC-MS/MS

目的建立一种简单、快速的同时测定人血浆中万古霉素和去甲万古霉素质量浓度的LC-MS/MS方法。方法以利奈唑胺为内标,血浆经高氯酸沉淀蛋白,加入2倍量水,离心,上清液用二氯甲烷萃取后进样分析;色谱柱为Hypersil GOLD aQ,1mL·L-1甲酸水溶液-甲醇梯度洗脱,流速为0.3mL·min-1;离子源为可加热电喷雾离子源,正离子检测,扫描方式为选择反应监测(SRM),万古霉素、去甲万古霉素和利奈唑胺的检测离子对分别为m/z725.7→144.2,m/z718.3→144.2和m/z338.3→296.0。结果各样品分析时间均为8min,血浆中万古霉素和去甲万古霉素的质量浓度在0.01～10μg·mL-1范围内线性关系良好,最低定量限均为10ng·mL-1,两药测定的日内和日间精密度RSD值小于15%,日内和日间相对误差在±15%以内。万古霉素和去甲万古霉素的提取回收率分别为74.16%～76.79%和73.01%～81.11%,基质效应分别为98.86%～108.69%和95.97%～109.64%。万古霉素和去甲万古霉素血浆样品在冻融、室温下保存24h和-80℃保存1个月的条件下稳定。结论建立的LC-MS/MS分析方法简便、快速、灵敏、准确,可同时测定人血浆中万古霉素和去甲万古霉素的质量浓度,并用于临床血药质量浓度的监测。

Objective To establish a simple and rapid LC-MS/MS method for simultaneous determination of the concentrations of vancomycin and norvancomycin in human plasma.Methods Linezolid was used as an internal standard,and plasma sample was deproteinized by adding perchloric acid.After protein precipitation,water was added to increase recovery,and the sample was centrifuged and the resulting supernatant was extracted with dichloromethane before analysis.Separation was achieved on a Hypersil GOLD aQ column with a mobile phase composed of 1 mL·L^-1 formic acid and methanol in gradient elution mode at a flow rate of 0.3 mL·min^-1.The ionization source was heated electrospray source ionization(HESI),and selective reaction monitoring(SRM)in positive ion mode was used to determine the analytes.The precursor-product ion pairs used for SRM were m/z 725.7→144.2,m/z 718.3→144.2 and m/z 338.3→296.0 for vancomycin,norvancomycin and internal standard,respectively.Results Total run time for each sample was 8 min.The proposed method was linear over the range 0.01-10μg·mL^-1 for both vancomycin and norvancomycin,and the lower limit of quantification(LLOQ)was 10 ng·mL^-1 for both of them.Intraday and interday precision were within 15%,and intraday and interday relative errors were within±15%.Recoveries were ranged from 74.16%to 76.79%and from 73.01%to 81.11%for vancomycin and norvancomycin respectively,and matrix effects of vancomycin and norvancomycin were within 98.86%-108.69%and 95.97%-109.64%,respectively.Vancomycin and norvancomycin were stable after freeze-thaw cycles and during storage at room temperature for 24 h and at-80℃for up to 1 month.Conclusion A simple,rapid,sensitive and accurate LC-MS/MS method for simultaneous determination of vancomycin and norvancomycin in human plasma was developed,and was successfully applied to therapeutic drug monitoring.