LC-MS/MS法测定人血清中丙戊酸钠浓度及临床应用

Determination of Sodium Valproate Concentration in Human Serum by LC-MS/MS

目的建立测定丙戊酸钠血药浓度的高效液相色谱串联质谱法(LC-MS/MS)分析方法,并应用于临床丙戊酸钠浓度的监测。方法以环己烷羧酸为内标,血清样本经乙腈沉淀蛋白处理后,采用ESI离子源,负离子模式下multiple reaction monitoring(MRM)扫描模式。采用Thermo Hypersil BDS C_(18)(10×4.6 mm)色谱柱进行分离,在流速为0.4 mL·min^(-1),柱温为30℃的条件下,以含0.1%甲酸的水溶液和甲醇为流动相进行洗脱。结果丙戊酸钠在5~120μg·mL^(-1)浓度范围具有良好的线性关系,最低定量浓度为5μg·mL^(-1);测得批内、批间准确度和精密度RSD在±15%之内;低、中、高浓度质控样本回收率在标示值的±15%之内;血清基质的内标归一化基质因子的变异系数小于15%,在2~8℃、-40℃存放条件下的稳定性较好。本研究中约有40%患者的丙戊酸钠血药浓度不达标。结论 LC-MS/MS分析方法专属性好,灵敏度高,准确度及精密度均能达到要求,稀释可靠性及稳定性好,可用于临床丙戊酸钠血药浓度监测及临床药代动力学的研究。

Objective To establish and validate an LC-MS/MS method for the determination of sodium valproate in human serum, and apply this method to clinical therapeutic drug monitoring(TDM). Methods Cyclohexane carboxylic acid was used as the internal standard(IS). After protein precipitation with acetonitrile, the determination was performed on a triple quadrupole mass spectrometer that used the ESI technique and operated in the multiple reaction monitoring and negative ion mode. The analyte and IS were separated on a Thermo Hypersil BDS C_(18) column(10×4.6 mm) with a mobile phase composed of 0.1% formic acid-methanol(15∶85) at a flow rate of 0.4 mL·min^(-1). The column temperature was set at 30℃. Results The linear range of sodium valproate was 5~120 μg·m L^(-1). The limit of quantification was 5 μg·m L^(-1).The RSD of accuracy and precision(within-run and between-runs) was less than 15%. The recoveries were within ±15%of the labeled values. The variation coefficient of the internal normalized matrix factor of the serum matrix was less than 15%. The samples were found to be stable when stored at 2~8℃ and-40℃. Approximately 40% of the patients in this study whose serum concentration of sodium valproate were out of the therapeutic ranges. Conclusion The established method has a high specificity and sensitivity, and the accuracy and precision meet the acceptance criteria of bioanalytical methods. The stability and dilution are acceptable. Thus this method could be used for clinical TDM and pharmacokinetic research for determination of VPA in human serum.