Simultaneous separation of ezetimibe and its seven stereoisomers by supercritical fluid chromatography on a polysaccharide-based chiral stationary phase: Optimization and thermodynamic analysis.

The separation behavior of ezetimibe and its seven stereoisomers was investigated using supercritical fluid chromatography (SFC). Polysaccharide-based chiral stationary phases (CSPs) were employed to systematically evaluate the effects of column type, modifier composition and ratio, column temperature and back pressure on the resolution. Optimal separation was achieved on the Chiralpak AD-H column using a mixture of methanol and isopropanol (1:1, v/v) as the organic modifier. The flow rate, column temperature and back pressure were set at 2 mL/min, 40 °C and 2200psi, respectively. The impact of column temperature varied across different enantiomeric pairs, with a notable reversal of elution order observed for the SRR/RSS pair. Method validation demonstrated excellent linearity for ezetimibe and its seven stereoisomers within their individual calibration ranges, which collectively covered 1.46-111.89 μg/mL, with correlation coefficients above 0.999. The limits of detection and quantification for individual analytes ranged from 0.6 to 3.5 μg/mL and from 1.3 to 10.6 μg/mL, respectively. The developed method was successfully applied to the determination of ezetimibe stereoisomers in both bulk drug and tablet samples, confirming its applicability for routine quality control. These results indicate that the developed SFC method is efficient, reliable and suitable for the quantitative determination of ezetimibe and its stereoisomers.