Determination of Methotrexate, 7-Hydroxymethotrexate, and 2,4-Diamino-N10-methylpteroic Acid by LC-MS/MS in Plasma and Cerebrospinal Fluid and Application in a Pharmacokinetic Analysis of High-Dose Methotrexate.

A rapid and robust method for measuring methotrexate (MTX) and its two primary metabolites, 7-hydroxymethotrexate (7-OHMTX) and 2,4-diamino-N10-methylpteroic acid (DAMPA), was developed for use in pharmacokinetic studies of plasma and cerebrospinal fluid samples collected from infants with malignant brain tumors. Sample aliquots (100µL) were prepared for bioanalysis of MTX and metabolites using a Waters Oasis HLB microelution SPE plate. Chromatography was performed using a Phenomenex Synergi Polar-RP 4µ 75 × 2.0mm ID column heated to 40°C. A rapid gradient elution on a Shimadzu HPLC system was used, with mobile phase A consisting of water/formic acid (100/0.1 v/v) and mobile phase B consisting of acetonitrile/formic acid (100/0.1 v/v). Column eluent was analyzed using AB Sciex QTRAP 5500 instrumentation in electrospray ionization mode. The ion transitions (m/z) monitored were 455.2→308.1, 471.1→324.1, and 326.2→175.1 for MTX, 7-OHMTX, and DAMPA respectively. The method was linear over a range of 0.0022 - 5.5 µM for MTX, 0.0085 - 21 µM for 7-OHMTX, and 0.0031 - 7.7 µM for DAMPA. The method was applied to the analysis of serial plasma samples obtained from infants diagnosed with malignant brain tumors receiving high-dose MTX and results were compared to MTX concentrations from a TDx-FLx FPIA.