Separation of FLEC diastereomers by CE vs. LC approaches in the context of neurometabolomics

Some of the D-amino acids (D-Ser, D-Asp, D-Glu) have gained an increasing attention during the last decades, due to the discovery of their role as neurotransmitters and their implication in different neurological pathologies (Alzheimer’s disease, schizophrenia etc.). Nevertheless, their use as biomarkers is particularly relevant when correlated with the levels of other neurotransmitters.
In order to develop a fast and efficient separation method widely accessible for the quantitation of these molecules, we used only common separation tools such as RP-18 stationary phases for reversed phase liquid chromatography (RP-LC) or bare fused capillaries for capillary zone electrophoresis (CZE).
For achieving chiral resolution, a derivatization procedure was implemented. (-)-FLEC was the chiral derivatization agent of choice due to its fast and quantitative reaction with primary and secondary amines and the ability of performing in-capillary derivatization. Moreover, the derivatization process implies only a simple mix of the sample and reagent, at room temperature.
The separation of the FLEC derivatives of several biologically relevant D- and L- amino acids (Asp, Glu, Ser, Tyr, Trp, Phe, His) together with certain neurotransmitter molecules have been optimized using CZE or RP-LC, chiral resolution being achievable for all amino acids of interest.
By the CZE approach the running buffer’s pH turned out to be critical in achieving baseline separation of the targeted analytes. The derivatives of most amino acids could be separated using 60mM acetate buffer at pH 5, while for Asp derivatives the separation could be achieved only at pH 4. Being stronger bases, a third run at a more alkaline pH was needed for the separation of the remainder neurotransmitters. Moreover, the implemented in-capillary derivatization allows a fast and fully automated separation procedure.
As for the RP-LC approach 50 mM acetate buffer in combination with an organic modifier (methanol, acetonitrile or tetrahydrofuran (THF)) was tested as mobile phase using gradient elution. Once again, the strong influence of pH on the resolution was observed. The organic modifier nature was of critical importance, where only THF enabled baseline resolution for all amino acid derivatives.