The Applicability of Chromatographic Retention Modeling on Chiral Stationary Phases in Reverse-Phase Mode: A Case Study for Ezetimibe and Its Impurities

Abstract

Mechanistic modeling is useful for predicting and modulating selectivity even in early chromatographic method development. This approach is also in accordance with current analytical quality using design principles and is highly welcomed by the authorities. The aim of this study was to investigate the separation behavior of two different types of chiral stationary phases (CSPs) for the separation of ezetimibe and its related substances using the mechanistic retention modeling approach offered by the Drylab software (version 4.5) package. Based on the obtained results, both CSPs presented with chemoselectivity towards the impurities of ezetimibe. The cyclodextrin-based CSP displayed a higher separation capacity and was able to separate seven related substances from the active pharmaceutical ingredient, while the cellulose-based column enabled the baseline resolution of six impurities from ezetimibe. Generally, the accuracy of predicted retention times was lower for the polysaccharide CSP, which could indicate the presence of additional secondary interactions between the analytes and the CSP. It was also demonstrated that the combination of mechanistic modeling and an experimental design approach can be applied to method development on CSPs in reverse-phase mode. The applicability of the methods was tested on spiked artificial placebo samples, while intraday and long-term (2 years) method repeatability was also challenged through comparing the obtained retention times and resolution values. The results indicated the excellent robustness of the selected setpoints. Overall, our findings indicate that the chiral columns could offer orthogonal selectivity to traditional reverse-phase columns for the separation of structurally similar compounds.

Comprehensive Review on Chiral Stationary Phases in Single-Column Simultaneous Chiral–Achiral HPLC Separation Methods

“…In addition, the same software showed less prediction accuracy in the case of the Chiralcel OD CSP towards the same analytes. The different behaviors of the polysaccharide CSP can be explained by its distinct separation mechanism, involving other types of interactions than those present in a classical achiral RP system [ 90 ]. Virtual and experimental chromatograms obtained for ezetimibe and its related substances on Chiral CD-Ph and Chiralcel OD columns are presented in Figure 3 and Figure 4 , respectively.…”

“… The comparison of ( a ) the virtual chromatogram generated by the DryLab software (version 4.5) and ( b ) the experimental chromatogram obtained in the case of setpoint 1 tested for Chiral CD-Ph column (1—ezetimibe diol; 2—THP (tetrahydropyran) compound; 3—RRS ezetimibe; 4—monofluoro ezetimibe; 5—ezetimibe API; 6—ezetimibe ketone; 7—TBDMS (tert-butyldimethylsilyl) ketone; 8—benzylated ezetimibe). Reprinted from Ferencz et al [ 90 ]; ( c ) Chemical structure of ezetimibe and its impurities. …”

“… The comparison of ( a ) virtual chromatogram generated by the DryLab software (version 4.5) and ( b ) the experimental chromatogram obtained in case of setpoint 1 tested for Chiralcel OD column (1—ezetimibe diol; 2—desfluoro ezetimibe; 3—ezetimibe API; 4—THP (tetrahydropyran) compound; 5—ezetimibe ketone; 6—TBDMS (tert-butyldimethylsilyl) ketone; and 7—benzylated ezetimibe). From Ferencz et al [ 90 ]. …”