Separation of N-derivatized di- and tri-peptide stereoisomers by micro-liquid chromatography using a quinidine-based monolithic column – Analysis of l-carnosine in dietary supplements

• N-derivatized peptides were successfully enantioresolved on the poly(MQD-co-HEMA-co-EDMA) monolithic column.
• The monolith exhibited good enantioselectivity and diastereoselectivity for N-derivatized di- and tri-peptides.
• The monolith shows potential as a reliable tool for the quality control of l-carnosine in dietary supplements.

In the present study, a new analytical methodology was developed enabling the enantiomeric determination of N-derivatized di- and tri-peptides in dietary supplements using chiral micro-LC on a monolithic column consisting of poly(O-9-[2-(methacryloyloxy)-ethylcarbamoyl]-10,11-dihydroquinidine-co-2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (poly(MQD-co-HEMA-co-EDMA)). After optimization of the mobile phase conditions, a baseline resolution of the stereoisomers of 24 out of 53 N-derivatized di- and tri-peptides was obtained. 3,5-Dinitrobenzoyl- and 3,5-dichlorobenzoyl-peptide stereoisomers were separated with exceptionally high selectivity and resolution. The monolithic column was then applied to the quantitative analysis of l-carnosine and its enantiomeric impurity in three different commercial dietary supplements. Method validation demonstrated satisfactory results in terms of linearity, precision, selectivity, accuracy and limits of detection and quantification. The determined amounts of l-carnosine in commercial formulations were in agreement with the labeled content for all analyzed samples, and the enantiomeric impurity was found to be below the limit of detection (LOD), showing the potential of the poly(MQD-co-HEMA-co-EDMA) monolithic column as a reliable tool for the quality control of l-carnosine in dietary supplements by micro-LC.