Practical determination of methotrexate in serum of rheumatic patients by LC-MS/MS

A rapid and simple liquid chromatography tandem mass spectrometry method for determination of methotrexate (MTX) in rheumatic patients' serum is described. Serum spiked with pterin as an internal standard was deproteinized with methanol. The separation of MTX from interfering peaks in matrix was achieved on a Luna 3 µm C₁₈ (100 × 4.6 mm i.d.) column with a mixture of 1% acetic acid and acetonitrile (88:12, v/v) within 5 min. Multiple reaction monitoring transitions monitored for MTX were m/z 455.2–308.1. The calibration curve of MTX in serum showed a good linearity (r = 0.999). Limits of detection and quantification of MTX at a signal‐to‐noise ratio of 3 and 10 were 3.0 n m (4.4 fmol/injection) and 10.0 n m (14.5 fmol/injection), respectively. The accuracy and precision for intra‐ and inter‐day assays were 94.6–106.5% and <5.5 and <5.1%, respectively. Furthermore, the proposed method was successfully applied to the sera nine rheumatic patients receiving MTX treatment. Copyright © 2012 John Wiley & Sons, Ltd.