Chirality plays a pivotal role in pharmaceutical efficacy and safety, necessitating precise analytical methods for determining enantiomeric purity. This becomes critically important when a chiral drug is intended for commercialization as a single enantiomer. However, in some cases, there is no significant advantage in doing so, and the drug is commercialized as a racemate instead. Ezetimibe, a lipid-lowering agent, exists in multiple stereoisomeric forms, each potentially exhibiting distinct pharmacological and toxicological profiles. In this study, a rapid and environmentally sustainable supercritical fluid chromatography (SFC) method was developed and validated for the quantification of five key ezetimibe stereoisomers: SSS-EZT, RSR-EZT, SSR-EZT, RRS-EZT, and RRR-EZT. Optimal separation was achieved using a coated amylose-C Neo [amylose tris(3,5-dimethylphenylcarbamate)] chiral stationary phase under isocratic conditions with CO2 and a 35:65 (v/v) isopropanol: methanol mixture as organic modifier. The method demonstrated excellent resolution (Rs ≥ 1.5) within 30 min, significantly reducing analysis time compared to the USP-specified RPLC method. Validation in accordance with ICH guidelines confirmed the method's reliability, with correlation coefficients >0.990 and recovery rates ranging from 93% to 108%. Precision was <5.0% at 50% level and < 2.0% at 100% (SSR-EZT, RSR-EZT, and RRR-EZT at 5 μg/mL, SSS-EZT at 10 μg/mL, and RRS-EZT at 20 μg/mL) and 150% levels. The developed method was successfully applied to the analysis of ezetimibe samples, yielding results (±0.02%) in close agreement with the USP reference method. Furthermore, the analytical method greenness score (AMGS) further confirmed the approach's environmental sustainability. This SFC method offers a robust, efficient, and green alternative for routine stereoisomeric purity analysis of ezetimibe in pharmaceutical development and quality control.
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