Abstract
In this study, a direct high performance liquid chromatography method was developed to determine the enantiomeric purity of the immunomodulatory drug, ozanimod. A systematic method development process was followed, incorporating risk assessment, identification of critical analytical procedure parameters (APP), initial screening of stationary phases, and software-assisted optimization of method parameters. Eight different polysaccharide-based chiral columns (Lux i-Amylose-1, Lux Amylose-2, Chiralpak AD, Chiralcel OD, Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-3, and Lux Cellulose-4) were selected to assess chiral separation of enantiomers under polar organic elution mode. The most promising results were obtained using a methanol (MeOH)-2-propanol (IPA) mixture on the Chiralpak AD column. Following this, systematic modeling was conducted using DryLab software to optimize method conditions, including isocratic eluent composition (%IPA in MeOH), temperature, and flow rate. Baseline separation was achieved within fifteen minutes using the optimized parameters: Chiralpak AD column thermostated at 10°C, and a mobile phase of MeOH:IPA:diethylamine (DEA), 70:30:0.1 (v/v/v), delivered at a flow rate of 0.8 mL/min. The developed method was validated according to the International Council on Harmonization guideline Q2(R2) for chiral impurity determination in ozanimod samples. Additionally, in silico robustness testing was conducted to determine tolerance limits for critical separation parameters and their impact on enantioresolution. Our findings demonstrate the utility of DryLab, typically employed for reversed-phase achiral separations, in optimizing chiral methods even in polar organic mode. The software's limitations for this purpose are also discussed.