LC-MS/MS检测血清万古霉素浓度方法的建立及评价

摘要

目的 建立检测人血清中万古霉素浓度的液相色谱－串联质谱( LC-MS/MS)法,并与化学发光微粒子免疫法( CMIA)比较方法学性能.方法 血清样品经过甲醇沉淀蛋白质,采用安捷伦Poroshell 120EC C18色谱柱(2.1 mm×50 mm,2.7 μm)梯度洗脱分离,流动相为甲醇(0.1%甲酸)－水(0.1%甲酸),流速为0.5 mL/min,内标采用去甲万古霉素,质谱采用ESI正离子多反应监测扫描模式.用该方法定量检测112例临床服用万古霉素患者血清万古霉素含量,并与临床常用的CMIA所得结果进行比较.结果 万古霉素在1～100 μg/mL内线性关系良好(r2＝0.996 4);方法日内日间精密度、准确度均满足药物定量检测要求,且无基质效应和残留效应. Wilcoxon符号秩和检验结果显示,LC-MS/MS测定结果与CMIA结果比较,差异有统计学意义(P＜0.05);相关性检验结果显示,两者线性相关性良好,Y＝1.06X－0.37(r＝0.986).结论 LC-MS/MS法性能较好,价格优廉,与CMIA法有较好的相关性,且检测在准确度、精密度上优势明显,可用于临床检测万古霉素的血清药物浓度.%Objective To establish a liquid chromatography-tandem mass chromatography ( LC-MS/MS) method for determination of vancomycin concentration in human serum and compare its methodological performance with chemiluminescence microparticle immuno-assay (CMIA). Methods The proteins in serum sample were precipitated by methanol and then vancomycin was eluted and separated on Agilent Poroshell 120EC C18 column (2.1 mm×50 mm, 2.7 μm) with mobile phase of methanol and water in a gradient percentage. Both phases of methanol and water contained 0.1% formic acid. The flow rate was 0.5 mL/min. Norvancomycin was applied as internal standard. Electrospray ionization source was applied and operated in positive ion mode of multiple reaction monitoring (MRM). A total of 112 serum samples collected from the patients taking vancomycin in our hospital, and the results were compared with those of CMIA. Results The method exhibited good linearty within the concentration range of 1 to 100 μg/mL (r2＝ 0.9964).The intra-and inter-day accuracy and precision were all satisfactory for the requirements of quantitative drug testing. No matrix effect and carry-over effect were observed. The results of Wilcoxon symbol rank test showed a difference with statistical significance was found between the serum con-centrations of vancomycin determined by LC-MS/MS and CMIA (P＜0.05), but strong positive correlation and good linear regression were shown between the two methods (Y＝1.06X－0.37, r＝0.986). Conclusion LC-MS/MS should be a cost-saving method superior to CMIA. Its results highly correlated with those of CMIA method. The advantages of LC-MS/MS on accuracy and precision obviously outperformed those of CMIA. Since all the results of LC-MS/MS are comparable to CMIA, this method should be promising to use in determining concentration of vancomycin in serum samples with high clinical value.