A highly sensitive, specific and rapid LC-ESI-MS/MS method has been developed and validated for simultaneous quantification of methotrexate (MTX) and tofacitinib (TFB) in rat plasma (50 μL) using phenacetin as an internal standard (IS), as per the US Food and Drug Administration guidelines. After a solid-phase extraction procedure, the separation of the analytes and IS was performed on a Chromolith RP18e column using an isocratic mobile phase of 5 m m ammonium acetate (pH 5.0) and acetonitrile at a ratio of 25:75 (v/v) using flow-gradient with a total run time of 3.5 min. The detection was performed in multiple reaction monitoring mode, using the transitions of m/z 455.2 → 308.3, m/z 313.2 → 149.2 and m/z 180.3 → 110.2 for MTX, TFB and IS, respectively. The calibration curves were linear over the range of 0.49-91.0 and 0.40-74.4 ng/mL for MTX and TFB, respectively. The intra- and interday accuracy and precision values for MTX and TFB were <15% at low quality control (QC), medium QC and high QC and <20% at lower limit of quantification. The validated assay was applied to derive the pharmacokinetic parameters for MTX and TFB post-dosing of MTX and TFB orally and intravenously to rats.
- Method validation
- Rat plasma