Simultaneous Determination of Methotrexate and 7-Hydroxy-Methotrexate by UHPLC-MS3 Assay Coupled With Multiple Stage Fragmentation to Enhance Sensitivity

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-d(3), and 7-OH-MTX was performed on a Waters AcQuity UPLC-BEH column (2.1 x 50 mm I.D., 1.7 mu m) 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 0.5-300 ng/mL (R-2 >= 0.99) and 5-1500 ng/mL (R-2 >= 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.