Development and validation of a sensitive LC-MS/MS assay for determination of upadacitinib in human plasma and its application in patients with inflammatory bowel disease

Background: Upadacitinib is a selective Janus kinase (JAK) 1 inhibitor approved by the Food and Drug Administration for the treatment of moderate-to-severe inflammatory bowel disease (IBD). We aimed to establish and validate a method for determining Upadacitinib in patients with IBD by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method.

Methods: The mobile phase was 0.1 % formic acid: acetonitrile (35:65, v/v) at a flow rate of 0.40 mL/min. Upadacitinib and its internal standard Upadacitinib ¹⁵N, d₂ were separated by a Waters Xbridge BEH C18 column (4.6 × 100 mm, 2.5 μm) and subjected to mass analysis using positive electrospray ionization (ESI).

Results: The calibration range of Upadacitinib was 0.5-200 ng/mL with the correlation coefficient r² ≥ 0.99. Accuracies ranged from -9.48 % ∼ 8.27 % and the inter- and intra-day precisions were less than 15 % for all analytes in quality control samples. There was no significant matrix effect. The range of extraction recoveries was 87.53-93.47 % for all analytes. Twenty-one plasma samples were obtained from the sixth affiliated hospital of Sun Yat-sen University. The median plasma concentration of Upadacitinib was 7.32 (0.56-26.78) ng/mL.

Conclusion: This newly developed method is sensitive, simple, and successfully applied in determining Upadacitinib in IBD patients to provide reference for safe and effective drug administration in clinical practice.

Inflammatory bowel disease; LC–MS/MS; Therapeutic drug monitoring; Upadacitinib.