A rapid LC-MS/MS assay for the measurement of serum methotrexate in patients who have received high doses for chemotherapy

BACKGROUND: Methotrexate is used in high doses to treat a number of cancers, particularly certain haematological malignancies. Monitoring of serum methotrexate concentration is important due to the potential toxicity of methotrexate and the variation in methotrexate pharmacokinetics in different patients on the same treatment regimen. OBJECTIVE: To develop a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for monitoring serum methotrexate in patients on high-dose chemotherapy. METHOD: Isotopically labelled internal standard was added to sample prior to protein precipitation with methanol. Diluted supernatant was injected into a Waters Acquity UPLC system linked to a TQS-Micro mass spectrometer. Separation by chromatography was achieved with a Waters Phenyl Vanguard with a retention time of approximately 0.5 min. The quantifier and qualifier transitions for methotrexate were 455.2>134.1 and 455.2>175.2, respectively. RESULTS: Mean recovery was 111% for three different concentrations of methotrexate spiked into seven different patient samples, with ion suppression <1%. Between-batch and within-batch coefficient of variations were <5% at three different concentrations of methotrexate in fresh frozen plasma. The lower limit of quantification was 0.02 µmol/L and the assay was shown to be linear to approximately 25 µmol/L. The LC-MS/MS assay showed a mean bias of -8.6% compared to an immunoassay, while mean bias compared to weighed in targets in external quality assessment samples was 1.6%. DISCUSSION: A rapid LC-MS/MS assay for methotrexate has been developed and validated. The LC-MS/MS method is likely to offer superior accuracy and specificity to more widely available immunoassays.