用LC-MS/MS法测定人血浆中6-巯基嘌呤的浓度

Determination of the concentration of 6-mercaptopurine in human plasma by LC-MS/MS

目的建立一种测定人血浆中6-巯基嘌呤的液相色谱-串联质谱法。方法用蛋白沉淀法对样品进行前处理,内标为6-巯基嘌呤-^(13) C_(2),^(15) N。色谱柱:AQ-C_(18)(2.1 mm×50.0 mm,3.0μm),流动相:100%水含0.1%甲酸-100%乙腈含0.1%甲酸,流速:0.40 mL·min^(-1),柱温:50℃,自动进样器温度:8℃,进样量:20.0μL。用电喷雾离子化源,负离子模式,多反应监测。考察该方法的专属性、标准曲线和定量下限、精密度与回收率、稀释效应、稳定性。结果6-巯基嘌呤在0.50~200.00 ng·mL^(-1)内,线性关系良好,其标准曲线为y=1.58×10^(-2)x+1.03×10^(-3)(r=0.9981),定量下限为0.50 ng·mL^(-1),日内和日间相对标准偏差为1.99%~9.28%,稀释效应的相对标准偏差为2.97%,回收率可达92%以上。结论本方法专属性强、灵敏度高,适用于6-巯基嘌呤的治疗药物监测研究。

Objective To establish a liquid chromatography-tandem mass spectrometry method for the determination of 6-mercaptopurine in human plasma.Methods Samples were processed by the protein precipitation method.Internal standard was 6-mercaptopurine-^(13) C_(2),^(15) N.The chromatographic column was AQ-C_(18)(2.1 mm×50.0 mm,3.0μm).Mobile phase was 100%water containing 0.1%formic acid and 100%acetonitrile containing 0.1%formic acid.Flow rate was 0.40 mL·min^(-1).The column temperature was 50℃.The automatic sampler temperature was 8℃.The injection volume was 20μL.Electric spray ionization source,negative ion mode,multiple reaction monitoring were applied.The specificity,standard curve and lower limit of quantification,precision and recovery,dilution effect and stability were investigated.Results 6-mercaptopurine had a good linearity within 0.50-200.00 ng·mL^(-1),and the standard curve was y=1.58×10^(-2) x+1.03×10^(-3)(r=0.9981),the lower limit of quantification was 0.50 ng·mL^(-1),the relative standard deviations of intra-day and inter-day were 1.99%-9.28%,the relative standard deviation of dilution effect was 2.97%,and the recovery rates were more than 92%.Conclusion This method has strong specificity and sensitivity and is suitable for therapeutic drug monitoring of 6-mercaptopurine.