液-质联用法同时测定人血浆中霉酚酸及其代谢物浓度及其在肝移植患者药动学研究中的应用

Determination of mycophenolic acid and its metabolites in plasma by liquid chromatographic-tandem mass spectrometry and application in pharmacokinetic study in liver transplant patients

目的：建立LC-MS/MS法同时测定血浆中霉酚酸（MPA）及其代谢物MPAG与AcMPAG浓度,用于治疗药物监测与药动学研究。方法：100μl血浆标本加入300μl含内标吲哚美辛的乙腈沉淀,离心后取上清液进样。色谱柱为Agilent E-clipse XDB-C18柱（3.5μm,2.1 mm×100 mm）,流动相为2 mmol·L-1甲酸铵水溶液-甲醇,采用梯度洗脱的方法,流速0.3 ml·min-1。用多反应监测进行定量,ESI负离子方式进行检测,用于定量分析的检测离子分别为m/z319.1→274.9（MPA）、m/z495.3→174.4（MPAG与AcMPAG）与m/z 356.1→312.0（内标吲哚美辛）。24例肝移植患者,采用包括霉酚酸钠肠溶片（EC-MPS）的三联免疫抑制方案,测定服药后MPA及其代谢物浓度并计算药动学参数。结果：MPA在0.10～50.5μg·ml-1,MPAG在1.13～4501μg·ml-,AcMPAG在0.11～22.4μg·ml-1范围内线性良好（r2〉0.99）。MPA、MPAG、AcMPAG提取回收率为77.3%～92.6%;基质效应为76.0%～86.7%;回收率为94.2%～116.4%;日内及日间变异均小于15%。24例肝移植患者用药1周后MPA、MPAG、AcMPAG的主要药动学参数分别如下：峰浓度Cmax为（18.8±10.9）,（154±118）,（3.07±2.85）μg·ml-1;达峰时间Tmax为（3.82±2.66）,（4.74±2.51）,（4.51±2.72）h;曲线下面积AUC0-12为（45.2±20.3）,（1 456±1 195）,（18.3±16.2）μg·h·ml-1;消除半衰期t1/2为（3.21±2.56）,（9.26±4.33）,（5.57±5.76）h。结论：本研究所建立的方法快速准确、灵敏、专属性强,适用于MPA及其代谢物的血药浓度监测和人体药动学研究。

OBJECTIVE To establish an LC-MS/MS method for the determination of mycophenolic acid（MPA）and its metabolites MPAG and AcMPAG in human plasma and investigate the pharmacokinetic characteristics in Chinese liver-transplanted patients.METHODS 300μl of acetontrile containing indomethacin（internal standard）was added in100μl plasma to precipitate protein.After centrifugation,the supernatant was eluted through Agilent Eclipse XDB-C18（3.5μm,2.1 mm×100mm）column by water（2 mmol·L-1 ammonium acetate）-methanol through a gradient assay,the flow rate was set as 0.3 ml·min-1.Electrospray ionization（ESI）source was applied and operated in the negative ion mode.Multiple reaction monitoring（MRM）mode with the transition of m/z319.1→274.9（MPA）,495.3→174.4（MPAG and AcMPAG）and 356.1→312.0（IS）was used for quantification.Plasma concentration of MPA,MPAG,AcMPAG of 24 liver-transplanted patients received immunosuppressive therapy including enteric-coated mycophenolate sodium were determined and pharmacokinetic parameters were calculated.RESULTS The method was proved to be linear in the range of 0.10-50.5μg·ml-1 for MPA,1.13-450μg·ml-1 for MPAG,0.11-22.4μg·ml-1 for AcMPAG（r2〉0.99）.The extract recovery,matrix effect,recovery of MPA and its metabolites was 77.3%-92.6%,76.0%-86.7% and 94.2%-116.4%,respectively.The within-day and between-day variation were both lower than15%.The pharmacokinetic parameters of MPA and its metabolites of 24 liver transplant patients were as follows：Cmax：（18.8±10.9）,（154±118）,（3.07±2.85）μg·ml-1;tmax：（3.82±2.66）,（4.74±2.51）,（4.51±2.72）h;AUC0-12：（45.2±20.3）,（1 456±1 195）,（18.3±16.2）μg·h·ml-1;t1/2：（3.21±2.56）,（9.26±4.33）,（5.57±5.76）h,respectively.CONCLUSION The method established is rapid,accurate,sensitive and specific,which is suitable for the therapeutic drug monitoring（TDM）and pharmacokinetic study of MPA and its metabolites.