Development and Validation of a High-Performance Liquid Chromatography–Tandem Mass Spectrometry Method for Methotrexate Quantitation in Human Serum

Methotrexate (MTX) is the only chemotherapy drug that is routinely monitored by therapeutic drug monitoring service in Malaysia government hospitals. The patients with MTX plasma concentration exceeding 0.4 μM for 48 h are at higher risk to develop toxicities such as nephrotoxicity, hepatotoxicity, neurotoxicity, mucositis and myelosuppression. The aim of this study was to develop and validate the ultra-high performance liquid chromatography−tandem mass spectrometry (UHPLC−MS/MS) method for MTX quantitation in human serum. Our results show that the retention times of MTX and warfarin (internal standard) were 1.82 and 2.68 min, respectively. The linearity was obtained at the concentrations from 25 to 500 ng/mL with the regression equation of y = 1025x – 11757 and r2 of 0.9901. This analyte was stable in a freezer at –80°C for up to 90 days. As a conclusion, this UHPLC−MS/MS method is an optional technique for clinicians to quantitate the patient’s MTX levels in the hospital.