Simultaneous Determination of Methotrexate and 7-Hydroxy-Methotrexate by UHPLC-MS Assay Coupled With Multiple Stage Fragmentation to Enhance Sensitivity

A selective ultra-high performance liquid chromatography tandem mass spectrometry cubed (UHPLC/MS) assay for simultaneous determination of methotrexate (MTX) and 7-hydroxy-methotrexate (7-OH-MTX) in human plasma was developed and validated. After protein precipitation with methanol, chromatographic separation of MTX, MTX-d, and 7-OH-MTX was performed on a Waters AcQuity UPLC-BEH column (2.1 × 50 mm I.D., 1.7 µm) with gradient elution. MRM transition was used for the detection of MTX (m/z 455.2→308.0→175.1) and 7-OH-MTX (m/z 471.3→191.0→148.1). The linear range of UHPLC/MS assay for the determination of MTX and 7-OH-MTX was 0.5-300 ng/mL (R ≥ 0.99) and 5-1500 ng/mL (R ≥ 0.99), respectively. The enhanced selectivity and sensitivity are the novelties of the developed UHPLC/MS assay. The analytical method was successfully applied to simultaneous determination of MTX and its major metabolite 7-OH-MTX in real human plasma samples.