用HPLC-MS/MS法测定重症感染患儿微量血浆/血清中利奈唑胺和万古霉素浓度

Determination of linezolid and vancomycin in trace plasma/serum of children with severe infection by HPLC-MS/MS

目的建立一种高效液相色谱串联质谱(HPLC-MS/MS)法快速测定重症感染患儿微量血浆/血清中利奈唑胺和万古霉素浓度。方法血浆/血清10μL经甲醇沉淀蛋白后取上清液直接进样分析,内标分别为利奈唑胺-D3和去甲万古霉素;用Kinetex^(R)EVOC18柱(30.0mm×2.1mm,2.6μm)分离,以0.1%甲酸-水和0.1%甲酸-乙腈为流动相,梯度洗脱,流速:0.5mL·min^(-1),柱温:40℃,进样量:2μL,分析时长:2min。电喷雾离子源正离子扫描下,用多反应监测模式进行质谱分析。考察该方法的专属性、定量下限和标准曲线、准确度和精密度、回收率、基质效应、稳定性、血清和血浆样品的交叉验证、溶血效应和高脂效应。结果利奈唑胺、万古霉素及内标利奈唑胺-D3、去甲万古霉素的保留时间分别为1.18、1.03、1.17、1.01min。利奈唑胺在0.2~25.6μg·mL^(-1)内呈良好的线性关系,回归方程是y=8.95×10^(-1)x+3.49×10^(-3)(r=0.9971),定量下限为0.2μg·mL^(-1);万古霉素在1~128μg·mL-内呈良好的线性关系,回归方程是y=3.13×10^(-1)x+6.93×10^(-2)(r=0.9974),定量下限为1μg·mL^(-1);批内、批间精密度的相对标准偏差(RSD)均≤9.55%。2种药物的平均提取回收率均在96.24%~104.57%;内标归一化基质因子的RSD≤7.58%;血浆和血清基质样品可交叉检测,利奈唑胺和万古霉素最高可承受的溶血程度分别为2%和5%,高脂效应均不影响2种抗菌药测定结果。样品的稳定性在试验条件下均良好。该法成功用于我院28例重症感染患儿血浆样本分析。结论本方法所需样本量少,简单、快速、准确、稳定、应用范围广,可应用于利奈唑胺和万古霉素在儿童治疗药物监测及联合用药研究。

Objective To establish a rapid high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method for the determination of linezolid and vancomycin in trace plasma/serum from pediatric patients with severe infection.Methods The plasma/serum specimens(10μL))were precipitated by methanol,then the supernatant was injected for detection directly.The internal standards were linezolid-D3 and norvancomycin.The chromatographic separation was performed with gradient elution on a Kinetex EVO C18 column(30.0 mm×2.1 mm,2.6μm)using water and acetonitrile,each containing 0.1% formic acid,as mobile phase.The flow rate was 0.5 mL·min^(-1)and column temperature was 40℃.The injection volume was 2μL and the total run time was 2 min.For mass spectrometry,electrospray ionization source was chosen,positive ion monitoring was used with multi-reaction monitoring(MRM)mode.The selectivity,lower limit of quantification(LLOQ)&calibration curve,accuracy&precision,recovery,matrix effect,stability,cross detection of plasma and serum samples,evaluation of hemolytic and hyperlipidemic effect were investigated.Results The retention times of linezolid,vancomycin,internal standard linezolid-D3 and norvancomycin were 1.18,1.03,1.17 and 1.01 min,respectively.The calibration curves of linezolid and vancomycin were y=8.95×10^(-1)x+3.49×10^(-3)(r=0.9971)and y=3.13×10^(-1)x+6.93×10^(-2)(r=0.9974),with the linear ranges of 0.2-25.6μg·mL^(-1)and 1-128μg·mL^(-1),and the lower limits of quantification were O.2μg·mL^(-1)and 1μg·mL^(-1),respectively.The intra-run and inter-run precisions relative standard deviation(RSD)were both less than 9.55%.The average extraction recoveries of the two drugs were 96.24%-104.57%.The RSDs of internal standards-normalized matrix effect were no more than 7.58%.Plasma and serum matrix samples could be cross-detected.The maximum tolerable hemolysis degree of linezolid and vancomycin were 2%and 5%,respectively,and the hyperlipidemic effect did not affect the quantitation.The stability of the samples was good under test conditions.This method was successfully applied to the analysis of plasma samples from 28 pediatric patients with severe infection in our hospital.Conclusion This assay is sample-saving,simple,rapid,accurate and robust,widely used,which can be applied to combination medication studies of linezolid and vancomycin and their therapeutic drug monitoring in pediatric patients.