Comparison of LC-MS and LC-MRM strategy for quantification of methotrexate in human plasma and its application in therapeutic drug monitoring.

A simple, highly selective and high throughput liquid chromatography tandem mass spectrometry cubed (LC/MS) method was developed and validated for quantification of methotrexate in human plasma. The MS detection is a scanning mode of QTrap MS systems or ion trap MS systems. After simple protein precipitation with methanol, methotrexate and methotrexate-d 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. MS detection in positive ion mode used the MRM 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-d. The total run time was only 3 min for each sample. The LC/MS assay was linear in the concentration range 10-3000 ng/mL(R ≥ 0.995) and the intra- and inter-day accuracies were< 3.72% and precisions were< 7.78% at all concentrations. The absolute recoveries (%) and matrix effect (%) for methotrexate in human plasma were between 92.6 and 114.3. The novelty of the presented methodology is the MS technique resulting in enhanced selectivity and sensitivity. The application of this LC-MS method was successfully completed on 46 human plasma samples and the quantitative results of identical human plasma samples were compared with another LC-MRM based method. Passing-Bablok regression coefficients demonstrated that there is no significant difference between the LC-MS method and LC-MRM method. Bland-Altman plots showed a concordant results, supporting the developed LC-MS method is a reliable and accurate assay for determination of methotrexate in human plasma. This work is also a proof of concept for using LC-MS technique to determination of chemicals in biological samples.