An ultra-fast UPLC-MS/MS approach for the quantification of baricitinib in the HLM matrix: greenness assessment with application to in vitro and in silico metabolic stability studies

Baricitinib (Olumiant) is a Janus kinase inhibitor utilized for the management of COVID-19, rheumatoid arthritis, and alopecia areata. It received U.S. Food and Drug Administration approval on May 31, 2018. This work developed a sensitive, rapid, environmentally friendly, and reliable UPLC-MS/MS method for quantifying baricitinib in human liver microsomes, utilized to evaluate the in vitro metabolic stability of baricitinib in HLMs. The StarDrop software, with DEREK and P450 metabolic programs, was employed to detect the structural warnings related to BCB and assess the in silico metabolic lability. The validation of the UPLC-MS/MS approach conformed to U.S. Food and Drug Administration standards for bioanalytical approach validation. The present UPLC-MS/MS method exhibited a wide range of linearity (1.0–3000 ng mL⁻¹) and optimum separation of analytes in an ultra-fast separation time (1 min) and was reproducible and accurate in the absence of human liver microsome matrix effects. Baricitinib and encorafenib (the internal standard) were examined employing an isocratic mobile phase technique on a reversed phase (SB C18) column. This study assessed the accuracy and precision of UPLC-MS/MS methodologies for intra- and inter-day evaluations, which ranged from −1.20% to 8.67% and 0.12% to 11.67%, respectively. The intrinsic clearance of baricitinib was quantified at 27.49 mL min⁻¹ kg⁻¹, while the in vitro half-life was established at 29.50 minutes. In silico analysis proposes that slight structural alterations to the pyrrole ring (88%) and the pyrimidine ring (5%) in drug design may increase safety and metabolic stability related to baricitinib. The evaluation of in silico absorption, distribution, metabolism, excretion, and metabolic stability characteristics for baricitinib is crucial for advancing innovative drug discovery focused on enhancing metabolic stability.