Box–Behnken Design Optimized LC‐MS/MS Method for Sensitive and Rapid Determination of Baricitinib in Biological Matrix

ABSTRACT A highly sensitive and robust LC–MS/MS method was developed and validated for the quantification of baricitinib in human plasma, employing ruxolitinib as an internal standard (IS). Sample preparation involved simple protein precipitation, and chromatographic separation was achieved on a Luna C18 column (50 × 2.0 mm, 3 µm) using an isocratic mobile phase consisting of 0.1% formic acid in water and acetonitrile (50:50, v/v). Key mass spectrometric parameters—including capillary voltage (3.75 kV), cone voltage (20 V), and collision energy (32 eV)—were systematically optimized using a Box–Behnken design (BBD), ensuring maximum sensitivity and reproducibility. The method exhibited excellent linearity over the concentration range of 1–200 ng/mL (R 2 = 0.99993), with intra‐ and inter‐day precision (%RSD ≤ 4.95%) and accuracy (92.00%–99.06%) within acceptable limits as per FDA and EMA bioanalytical method validation guidelines. The extraction recovery of baricitinib was high and consistent (95.73%–97.38%), while matrix effects were minimal (IS‐normalized matrix factor: 1.067–1.136, %RSD < 5%). Stability studies confirmed that baricitinib remained stable under various storage and processing conditions, including freeze–thaw, short‐term, long‐term, and benchtop stability. The validated method demonstrates its suitability for therapeutic drug monitoring of baricitinib in clinical settings, including treatment of rheumatoid arthritis, pediatric autoinflammatory conditions, and investigational oncology applications.