Comprehensively validated µSPE-HPLC-MS/MS method for high-throughput clinical monitoring of Janus kinase inhibitor drugs approved for inflammatory bowel disease treatment

Therapeutic drug monitoring (TDM) of Janus kinase inhibitors (JAKis) in inflammatory bowel disease (IBD) remains an evolving field, primarily due to the absence of validated therapeutic targets and heterogeneous pharmacokinetic profiles of individual agents. To study the relevance of systemic exposure from various perspectives, reliable quantification of JAKis is essential. In this work, therefore, a complex validated protocol for quantification of clinically relevant serum concentrations of JAKis, based on a microelution solid-phase extraction (SPE) sample preparation and high-performance liquid chromatography–electrospray ionization triple-quadrupole tandem mass spectrometry (HPLC-ESI-QQQ MS) analysis, was developed. The optimized and systematically validated (following U.S. Food and Drug Administration, FDA, guidelines) workflow provides several important benefits including (i) uniform sample preparation and analysis procedure for all JAKis approved for IBD therapy (tofacitinib TOFA, upadacitinib, UPA, filgotinib, FILGO), (ii) greening of both sample preparation and chromatographic analysis through appropriate solvent selection, (iii) simple and high-throughput microelution SPE sample preparation step for total concentration levels of JAKis, (iv) robust and highly reliable performance for therapeutically relevant JAKis levels. The applicability of the proposed protocol was demonstrated in a pilot real-world exposure monitoring study including serum samples (cohort of 27 patients) from IBD patients treated with tofacitinib (4), upadacitinib (18), and filgotinib (5). Serial opportunistic sampling enabled characterization of exposure variability and detection of treatment interruption. Concentration-matched verification (comparing concentration-matched serum standards with the corresponding clinical samples), further confirmed reliable quantification in authentic biological matrices. The developed workflow provides a robust platform for real-world exposure assessment of JAK inhibitors and may serve as a foundation for future precision dosing strategies in IBD.