Enantioselective Determination of Upadacitinib in Pharmaceutical Formulations Using Reversed-Phase Chiral Liquid Chromatography.

Stereochemical impurities can significantly alter a drug's pharmacological profile and compromise patient safety even in small levels. Therefore, the development of selective chiral analytical methods is critical for quality control assurance. In this study, a novel chiral HPLC method was developed for the enantiomeric separation and quantification of upadacitinib, a selective JAK-1 inhibitor used in the treatment of rheumatoid arthritis, psoriatic arthritis, ulcerative colitis, and Crohn's disease. The Box-Behnken design, a design of experiments approach, was applied to evaluate the effects of mobile phase pH, acetonitrile content, and column temperature on resolution and peak width responses. Based on the design of experiment results, the chromatographic conditions were optimized to achieve optimal separation performance. Chiral separation was achieved under reversed-phase conditions using a Chiralcel OJ-RH column (4.6 × 150 mm, 5 µm) at 40°C. The mobile phase consisted of 20 mM phosphate buffer (pH 6) and acetonitrile (84:16, v/v), delivered isocratically at a flow rate of 0.7 mL/min, with detection performed at 340 nm. Baseline enantiomeric separation was achieved with a resolution value of 2.51. The method was validated according to ICH Q2(R2) guidelines in terms of specificity, linearity, accuracy, precision, and robustness. Good linearity was obtained over the concentration range of 0.1-12.8 µg/mL for upadacitinib and its 3R,4S enantiomer. The validated method was successfully applied to the quantification of upadacitinib in a commercial tablet formulation. Its environmental impact and practicality were evaluated using various tools. This study presents the first validated reversed-phase chiral HPLC method reported for the enantiomeric analysis of upadacitinib.