Comparison of LC–MS3 and LC–MRM strategy for quantification of methotrexate in human plasma and its application in therapeutic drug monitoring

•The first validated LC-MS3 method for the quantification of methotrexate.•The advantages of this method include high selectivity and high sensitivity.•This work is a proof of concept for using LC-MS3 technique to determine chemicals in biological samples. [Display omitted] A simple, highly selective and high throughput liquid chromatography tandem mass spectrometry cubed (LC/MS3) method was developed and validated for quantification of methotrexate in human plasma. The MS3 detection is a scanning mode of QTrap MS systems or ion trap MS systems. After simple protein precipitation with methanol, methotrexate and methotrexate-d3 were separated on an Agilent Poroshell 120 SB-C18 column (4.6 × 50 mm, 2.7 µm) using isocratic elution with a mobile phase consisting of 60% 0.1% formic acid in water and 40% 0.1% formic acid in acetonitrile. The flow rate is 0.8 mL/min. MS3 detection in positive ion mode used the MRM3 transitions at m/z 455.2→308.2→175.1 for quantification of methotrexate and m/z 458.2→311.2→175.1 for quantification of methotrexate-d3. The total run time was only 3 min for each sample. The LC/MS3 assay was linear in the concentration range 10–3000 ng/mL(R2 ≥ 0.995) and the intra- and inter-day accuracies were