Development and validation of a UHPLC-MS/MS method for the simultaneous quantification of five JAK inhibitors (JAKi), venetoclax, and dasatinib in human plasma

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Janus kinase inhibitors (JAKi) are increasingly used in the management of chronic inflammatory and haemato-oncological disorders, including inflammatory bowel disease, rheumatoid arthritis, COVID-19, and Graft versus Host Disease. The complexity of these patients' treatments increases the risk of drug-drug interactions (DDIs), especially since JAKi are primarily metabolized by cytochrome P450 3A4. In this context, therapeutic drug monitoring (TDM) is a valuable approach to optimize treatment, improve safety, and support personalized therapy in these clinically complex patients. A UHPLC-MS/MS method was developed to simultaneously quantify five JAKi – tofacitinib, baricitinib, upadacitinib, filgotinib, and ruxolitinib - in plasma. Venetoclax and dasatinib were included in the method due to their clinical relevance. Sample preparation consisted of protein precipitation with stable isotope-labelled internal standards. Separation was performed using a Luna Omega 1.6 μm Polar C18 100, 100 × 2.1 mm column, with a total run time of 6.5 min. The method showed excellent chromatographic resolution, neither carry-over nor interactions with plasma contaminants or cross-talk between analytes were observed. It was validated over the clinically relevant concentration ranges, demonstrating satisfactory recovery, accuracy, and inter-day precision for all analytes. The robustness and reproducibility of the method achieved good performances, in compliance with FDA and EMA guidelines. The method ensured good accuracy and rapid analysis, resulting suitable for the application in clinical practice.