LC-MS/MS法同时测定肾移植患者血浆中霉酚酸及两种代谢物浓度

Simultaneously determination of mycophenolic acid and two metabolites in the plasma of renal transplant patients by LC-MS/MS

目的建立LC-MS/MS法同时测定肾移植患者血浆中霉酚酸(MPA)及其代谢物MPAG和AcMPAG的浓度。方法以稳定同位素(MPA-d3)为内标、甲醇-乙腈溶液(9∶1,V/V)沉淀蛋白,采用色谱柱Kinetex C18(3 mm×100 mm,2.6μm,Phenomenex,USA),流动相为水(0.1%甲酸,10 mmol·L^(-1)乙酸铵)-甲醇(0.1%甲酸)溶液,梯度洗脱,流速0.55 mL·min^(-1)。质谱选用ESI离子源,正离子多反应监测扫描检测,测定肾移植患者服药前及服药后血浆MPA及代谢物MPAG和AcMPAG的浓度。结果MPA在0.2~50μg·mL^(-1),MPAG在1~250μg·mL^(-1),AcMPAG在0.08~20μg·mL^(-1)与测定值线性关系良好,定量限分别为0.2、1和0.08μg·mL^(-1)。MPA及其代谢物日内与日间精密度、准确度、提取回收率、基质效应、稳定性均符合方法学要求。结论本法快速、灵敏、准确,可用于检测肾移植患者血浆中MPA及代谢物MPAG和AcMPAG的浓度。

Objective To determine mycophenolic acid(MPA)and its metabolites MPAG and AcMPAG in the plasma of renal transplant patients by LC-MS/MS.Methods The protein was precipitated in a methanol-acetonitrile solution(9∶1,V/V)with a stable isotope(MPA-d3)as the internal standard.The gradient elution was performed on a Kinetex C18 column(3 mm×100 mm,2.6μm,Phenomenex,USA)at a flow rate of 0.55 mL·min^(-1) with the mobile phase water(0.1%formic acid,10 mmol·L^(-1) ammonium acetate)-methanol(0.1%formic acid)solution.ESI ion source was used for the mass spectrometry and positive ion multiple reaction monitoring(MRM)scanning detection.The concentrations of plasma MPA and metabolites MPAG and ACMPAG were determined before and after the administration.Results The linearity was at 0.2~50μg·mL^(-1) for MPA,1~250μg·mL^(-1) for MPAG and 0.08~20μg·mL^(-1) for AcMPAG.The limits of quantitation were 0.2,1 and 0.08μg·mL^(-1),respectively.The intra-day and inter-day precision,accuracy,extraction recovery rate,matrix effect,and stability of MPA and its metabolites all met the methodological requirements.Conclusion This method is rapid,sensitive and accurate,which is suitable to detect the concentrations of MPA and two metabolites in the plasma of renal transplant patients.