Atypical enantioseparation of a non-ionic form of allantoin with Cinchona alkaloid-based zwitterionic chiral stationary phases

Abstract

Allantoin represents a compound widely employed in pharmaceutical and cosmetic fields. Its safety has been acknowledged by regulatory bodies such as the US Food and Drug Administration, the European Commission for Cosmetics and Consumer and Health and European Directorate for the Quality of Medicines & HealthCare. This justifies its wide use in dermatological/cosmetic formulations and allows their safe use.

Allantoin possesses an asymmetric carbon atom, resulting in two enantiomers, with the (S)-enantiomer predominating in plants, although racemization may potentially occur during manufacturing processes. Notably, literature currently lacks enantioselective LC methods for allantoin analysis.

In this study, two zwitterionic Cinchona alkaloid-based chiral stationary phases (CSPs), commercially known as CHIRALPAK® ZWIX(+) (CSP¹) and CHIRALPAK® ZWIX(-) (CSP²), were utilized for the enantioseparation of allantoin under polar-ionic conditions. By employing a mobile phase consisting of acetonitrile/methanol/water/acetic acid (96:2:2:0.1, v/v/v/v), nearly complete baseline separation (with α=1.08) of allantoin enantiomers was achieved in less than 15 min with both CSPs. Due to the “pseudo-enantiomeric” nature of the two chiral selectors (quinine-based in CSP¹ and quinidine-based in CSP²), an inversion of the enantiomer elution order was observed with the two CSPs under identical experimental conditions. Remarkably, this represents a rare instance where these CSPs demonstrate the ability to enantioseparate a non-ionic, non-ionizable species.

The application of a molecular dynamics in silico protocol proved useful in elucidating the retention mechanism in depth, casting light on the central role of the H-bond formation and the involvement of the anionic moiety of the CSP 1.