Abstract
A selective ultra-high performance liquid chromatography tandem mass spectrometry cubed (UHPLC/MS3) 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-d3, and 7-OH-MTX was performed on a Waters AcQuity UPLC-BEH column (2.1×50mm I.D., 1.7µm) with gradient elution. MRM3 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/MS3 assay for the determination of MTX and 7-OH-MTX was 0.5-300ng/mL (R2≥0.99) and 5-1500ng/mL (R2≥0.99), respectively. The enhanced selectivity and sensitivity are the novelties of the developed UHPLC/MS3 assay. The analytical method was successfully applied to simultaneous determination of MTX and its major metabolite 7-OH-MTX in real human plasma samples.
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