Enantiomeric Resolution of Odevixibat via Immobilized Polysaccharide Columns: Impact of NP, RP, and PO Modes on LC–MS-Compatible Method Design

Abstract

Odevixibat, a selective and reversible inhibitor of the ileal bile acid transporter (IBAT), received approval from the US Food and Drug Administration (FDA) in 2021 as the first therapeutic option for pruritus associated with progressive familial intrahepatic cholestasis (PFIC). In this study, a comprehensive chiral high-performance liquid chromatography (HPLC) method was developed for the enantiomeric and diastereomeric separation of Odevixibat and its stereoisomeric impurities (RS, RR, SS, and SR) using immobilized polysaccharide-based chiral stationary phases. The various chromatographic modes—including normal-phase (NP), reversed-phase (RP), and polar organic (PO)—were systematically investigated. Optimal resolution was achieved using a CHIRALPAK IA column (4.6 × 250 mm, 3 μm) with a mobile phase of n-hexane/isopropanol/methanol/trifluoroacetic acid (50:35:15:0.1, v/v/v/v) at 40°C and a flow rate of 1.0 mL/min. The method was validated according to ICH Q2(R1) guidelines. Additionally, the CHIRALPAK IM in NP mode effectively resolved closely eluting isomeric impurities, enabling preparative isolation and characterization. The optimized RP-mode method was compatible with LC–MS detection, facilitating trace-level impurity profiling on CHIRALPAK ID-3 and structural characterization of stereoisomers and process-related impurities. Thermodynamic analysis revealed entropy-driven enantioseparations, with the NP-HPLC method on CHIRALPAK IA showing more favorable interactions (ΔG° = 0.06) than the RP-HPLC system (ΔG° = 1.34). The method demonstrated excellent linearity (r² > 0.999) over the concentration range of 0.028–0.211 μg/mL, with recoveries between 87.1%–101.2% and precision within 0.8% RSD. High sensitivity was achieved, with LODs of 0.009–0.025 μg/mL and LOQs of 0.028–0.075 μg/mL. The method proved robust under deliberate variations in column temperature (±1°C), mobile phase composition (±2%), and flow rate (1.0 ± 0.1 mL min⁻¹), showing no significant change in enantioresolution. These complementary approaches are suitable for routine quality control (CHIRALPAK IA), purification (CHIRALPAK IM) process monitoring and LC–MS impurity profiling (CHIRALPAK ID-3), and regulatory submissions, supporting current good manufacturing practices (cGMP) in the development of Odevixibat.