A simple, rapid and reliable liquid chromatography-mass spectrometry method for determination of methotrexate in human plasma and its application to therapeutic drug monitoring

摘要

A simple, rapid and reliable liquid chromatography-electrospray ionization tandem mass spectrometry method was established and validated for the determination of methotrexate in human plasma. After a straightforward protein precipitation by acetonitrile-water (70:30, v/v), methotrexate (MTX) and p-aminoacetophenone (used as internal standard, IS) were separated on a Column C-18 column (50x2.1mm, 3 mu m; Column Technology, Fremont, CA, USA) using a gradient elution with mobile phase of acetonitrile and 0.03% acetic acid aqueous solution at a flow rate of 0.5mL/min. The total chromatographic runtime was 5min for each injection. Quantification detection was performed in a triple-quadruple tandem mass spectrometer under positive mode monitoring the following mass transitions: m/z 455.3308.3 for MTX and m/z 136.194.4 for IS. The calibration curve was linear over the range of 0.05-25.0 mu mol/L with a lower limit of quantification of 0.05 mu mol/L. The intra- and interday precisions were <5.2%, the accuracy varied from -4.1 to 4.5%. The recovery was >94%. The LC-MS/MS method showed an excellent agreement with the existing HPLC-UV method using Passing-Bablok regression and Bland-Altman difference plot analysis. The validated LC-MS/MS can be successfully applied to the routine therapeutic drug monitoring of MTX in clinical laboratories. Copyright (c) 2015 John Wiley & Sons, Ltd.