Design Space–Guided Chiral Chromatographic Method Development for Enantiomeric Resolution and Purity Assessment Thereby Monitoring the Active S-Isomer of Pirtobrutinib by LC-MS/MS

Objective: Quality by Design (QbD) was used to develop, test, and optimize a reliable normal phase high-performance liquid chromatography (NP-HPLC) method for distinguishing R-and S-isomers in bulk pharmaceutical and therapeutic formulations. Separating and quantifying the enantiomers of Jaypirca, the next-generation non-covalent Bruton’s Tyrosine Kinase (BTK) inhibitor, is essential. Methods: A YMC Chiral ART Cellulose-SC column (250×4.6 mm, 5 µm) was utilized for chromatographic separation. At 35 °C, 1.5 ml/min of acetonitrile with 0.1% diethylamine (100:0.1 v/v) was added. R-and S-isomers showed symmetrical peaks at 4.42 and 6.21 min of retention time. The 3² full factorial design showed that acetonitrile composition and flow rate greatly affected retention time and resolution (Model F-values 674.76 and 1177.55; p=0.0001). Results: Models with precision ratios of 61.29 and 75.35 showed high signal dependability and excellent data fit (R²=0.9985 for retention time and 0.9986 for resolution). Under optimal conditions, a resolution of over 5 was regularly reached, observed linearity across 1.0 to 3.5 µg/ml with a R² value of above 0.9999. The quantification limit (LOQ) was 1.0 µg/ml with signal-to-noise ratios of 18.05 and 13.54, while the detection limit (LOD) was 0.5 µg/ml with 6.34 and 4.71. Precision experiments showed %RSD<2% for suitability with system, with accuracy ranging from 98.1 to 101.8%. The Jaypirca tablet assay recovered 99.7% of the active S-isomer with m/z 480.15→400.02. Conclusion: Beyond quality monitoring, chiral separation supports regulatory compliance, stability and racemization studies, impurity profiling, process optimization, and stereoselective synthesis. The validated NP-HPLC method ensures enantiomeric purity and selective production of the pharmacologically active S-isomer.