用HPLC-MS/MS法同时测定Caco-2细胞单层转运介质中吗替麦考酚酯和麦考酚酸的浓度

Simultaneous determination of mycophenolate mofetil and mycophenolic acid in transport medium of Caco-2 cell monolayers by HPLC-MS/MS method

目的:建立HPLC-MS/MS法同时测定Caco-2细胞单层转运介质中吗替麦考酚酯(mycophenolate mofetil,MMF)和麦考酚酸(mycophenolic acid,MPA)的浓度,用于药物转运实验研究。方法:采用Zorbax Eclipse XDB-C18色谱柱(50mm×2.1mm,3.5μm);流动相A相:甲醇(含0.05%甲酸),B相:水(含0.05%甲酸);梯度洗脱:0～3.60min,40%A,流速:0.3ml/min,3.61～7.00min,65%A,流速:0.6ml/min,7.01～10.00min,40%A,流速:0.6ml/min;柱温:30℃,以吲哚美辛为内标。质谱检测方式:多种反应监测(MRM),选择监测的离子为:m/z433.8→194.8(MMF),m/z319.1→191.0(MPA)和m/z356.0→312.1(吲哚美辛)。结果:MMF和MPA检测的线性范围均为0.01～20μmol/L,线性回归方程分别为MMF:As/Ai=-0.035 2+0.394 5 c(r=0.999 1);MPA:As/Ai=0.005 5+0.050 8 c(r=0.999 5)。低、中、高浓度(0.1、1.5、15μmol/L)MMF的回收率分别为(103.2±7.5)%、(97.3±3.1)%和(96.0±3.4)%,日内、日间RSD<7.27%;低、中、高浓度(0.1、1.5、15μmol/L)MPA的回收率分别为(106.9±1.0)%、(106.0±5.7)%和(108.4±3.2)%,日内、日间RSD<9.25%(n=5)。结论:本研究建立的测定方法简便、准确、灵敏度高,适用于MMF和MPA体外转运实验研究。

Objective： To establish a HPLC-MS/MS method for simultaneous determination of mycophenolate mofetil（MMF） and mycophenolic acid（MPA） in transport medium of Caco-2 cell monolayer.Methods： The separation was performed on a Zorbax Eclipse XDB-C18 column（50 mm×2.1 mm,3.5 μm） at the temperature of 30 ℃.The mobile phase consisted of phase A（methanol containing 0.05% formic acid） and phase B（water containing 0.05% formic acid）.The gradient elution profile was 40%A at the flow rate of 0.3 ml/min within 0-3.60 min,65%A at the flow rate of 0.6 ml/min within 3.61-7.00 min,40%A at the flow rate of 0.6 ml/min within 7.01-10.00 min.Indomethacin was used as the internal standard.Mass spectrometry was performed in multiple reaction monitoring（MRM） mode,with m/z of MMF 433.8→194.8,m/z of MPA 319.1→191.0 and m/z of indomethacin 356.0→312.1,respectively.Results： Both MMF and MPA were in linearity within the range of 0.01-20 μmol/L.The linear regression equations of MMF and MPA were As/Ai=-0.035 2＋0.394 5 c（r=0.999 1） and As/Ai=0.005 5＋0.050 8 c（r=0.999 5）,respectively.The average recoveries of low,middle and high concentrations（0.1、1.5、15 μmol/L） of MMF were（103.2±7.5）%,（97.3±3.1）% and（96.0±3.4）%,respectively,while the average recoveries of low,middle and high concentrations of MPA were（106.9±1.0）%,（106.0±5.7）% and（108.4±3.2）%,respectively.Conclusion： This method is simple,accurate,sensitive and suitable for analysis of MMF and MPA in in vitro transportation.