Simultaneous measurement of upadacitinib and methotrexate by UPLC-MS/MS and its pharmacokinetic application in rats

Abstract

Upadacitinib, as a selective and reversible Janus kinase (JAK) inhibitor, has been widely used in the treatment of atopic dermatitis, ulcerative colitis and other inflammatory bowel diseases and other immune-mediated diseases. The combination of methotrexate and upadacitinib is a common clinical treatment strategy for rheumatoid arthritis (RA) in recent years. In this study, we established an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for quantitative measurement of upadacitinib and methotrexate, by which we successfully determined pharmacokinetic parameters of them in rat plasma. In order to pretreat the samples, we used acetonitrile as the precipitant, and for the internal standard (IS), we chose tofacitinib. The Acquity BEHC18 (2.1 mm × 50 mm, 1.7 μm) column, with acetonitrile and 0.1% formic acid aqueous solution composed mobile phases, was used to separate upadacitinib, methotrexate and tofacitinib. A Xevo TQ-S triple quadrupole tandem mass spectrometer was used as the detecting instrument in the positive ion mode. For upadacitinib, excellent linearity was shown of this assay in the calibration range with 0.1–200 ng/mL, and as for methotrexate, the range was 0.05–100 ng/mL. As the results indicated, the lower limit of quantification (LLOQ) was respectively 0.1 and 0.05 ng/mL for upadacitinib and methotrexate, the intra- and inter-day precision were ≤ 13.3%, and the accuracy of all the analytes ranged from −4.1% to 12.7%. The recovery of each analyte was > 80.2% in this experiment, and matrix effects we observed were unobvious. The establishment of this method and its successful application in rat plasma can provide a theoretical and technical support for the deeper study of pharmacodynamics and the clinical medication strategies.