Optimization and validation of a sensitive liquid chromatography–tandem mass spectrometry method for the simultaneous quantification of methotrexate and 7-hydroxymethotrexate in human serum using hydrophilic interaction chromatography

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC-MS/MS) bioanalytical method was developed and partially validated for the simultaneous quantification of methotrexate (MTX) and its principal metabolite, 7-hydroxymethotrexate (7-OH-MTX), in human K₂EDTA serum using methotrexate-D3 as internal standard. Chromatographic separation was achieved on a Luna 5µ hydrophilic interaction liquid chromatography (HILIC) column (150 × 4.6 mm, 5 µm) using a Shimadzu UFLC system coupled to a Sciex API-4000 triple quadrupole mass spectrometer in positive electrospray ionization–multiple reaction monitoring mode. Analytes were extracted from 250 µL serum by solid-phase extraction using Strata-X C18 cartridges (30 mg/1 mL). The total analytical run time was 2.08 minutes. The method was linear over 2.00–1001 µg/mL for MTX and 2.02–1020 µg/mL for 7-OH-MTX, with coefficients of determination (r 2 ) >0.99 in all batches. Intra- and inter-batch precision (%CV) and accuracy (%nominal) met US FDA and EMA acceptance criteria across all quality control levels. Mean extraction recoveries were 69.25% (MTX) and 66.35% (7-OH-MTX). IS-normalized matrix factors (0.89–1.04 across all lots and QC levels) confirmed minimal ion suppression or enhancement across eight serum lots inclusive of hemolyzed and lipemic matrices. The method is intended for early pharmacokinetic monitoring during high-dose methotrexate therapy. Application to nine patient serum samples collected at 48 h and 72 h post-infusion demonstrated a 74% decline in mean MTX and 7-OH-MTX concentrations between time points, consistent with expected HDMTX elimination kinetics; while 7-OH-MTX was quantifiable within the validated range in five of nine patients at 48 h, MTX concentrations fell below the validated LLOQ at both time points, indicating the present method is suited to early (0–24 h) rather than late-elimination monitoring.