Combined monodimensional chromatographic approaches to monitor the presence of D-amino acids in cheese

The presence of D-amino acids (D-AAs) as a consequence of natural or artificial interventions such as ageing, microorganism action, preservative and conservative processes (alkali or heat treatment), is a scarcely treated aspect from the scientific community. It is also fully documented that even a minor degree of racemisation on the proteins’ AAs is the cause of a reduced digestion of such proteins. Besides interfering with the regular metabolism of L-AAs, D-AAs can also contribute to the development of pathological conditions in humans. So far, nearly all the most important chromatographic techniques were applied to quantify D-AAs in foodstuffs. However, most of them rely upon pre- or post-column derivatization procedures, often combined with sophisticated analytical equipments. Differently, in this paper we propose an easy-to-set up combination of monodimensional chromatographic methods to monitor the variation of the D-Ala, D-Asp and D-Glu content in two commercially available Spanish cheese samples prepared from the same milk mixture and characterized by a different maturity time: no ripening and six months ripening. After the free amino acid mixture was extracted from the two cheese samples, an ion-pairing RP-HPLC achiral protocol was firstly optimized with the objective to avail of a method enabling the complete distinction of Ala, Asp, and Glu from all the other aminoacidic species in the two extracts. An ion-exchange-based chromatographic method was also optimized, thus allowing a profitable fractionation of the two aminoacidic mixtures. With such a procedure, less complex samples to be analyzed with a chiral ligand-exchange chromatography (CLEC) stationary phase based on S-trityl-Lcysteine (L-STC) units were obtained. The optimized CLEC conditions were then applied to the previously identified Ala, Asp and Glu containing fractions as well as to those including all the remaining species. For all the three compounds the enantiomeric excess (ee) was found to decrease passing from the ripened to the fresh cheese. As expected, the largest difference was found for Ala (ee value from 83.0% down to 20.5%), followed progressively by Asp (ee value from 90.5 to 75.0%) and Glu (ee value from 99.0 to 91.8%).