Optimization and validation of a chiral CE-LIF method for quantitation of aspartate, glutamate and serine in murine osteocytic and osteoblastic cells.

Asp, Glu, and D-Ser are chiral amino acids and neurotransmitters binding to the N-methyl-D-aspartate receptor (NMDA) and they participate in glutamate signalization. D-amino acids are increasingly being recognized as important signaling molecules and variations in their levels are considered a marker of different pathologies, however, there is still a lack of knowledge about the role of most of D-amino acids in living organisms such as bone cells. A method for determination of concentrations of L/D-Asp, L/D-Glu and L/D-Ser in two types of bone cell lines: murine osteocytes (MLOY4) and osteoblasts (MC3T3-E1) is presented. It is based on capillary electrophoresis coupled to laser-induced fluorescence detection in normal polarity with 4-fluoro-7-nitro-2,1,3-benzoxadiazole as derivatizing agent suitable for an Argon ion laser source. The electrolyte consists of 137.5 mM borate buffer and 12.5 mM β-cyclodextrins as chiral selectors and the separation lasts 25 min. The method was optimized and validated for specificity, sensitivity, linearity, accuracy, and precision in murine osteocytes and osteoblasts. LLOQ was 0.25 µmol L for the three D-amino acids and linearity was confirmed with r > 0.995 for all D-and L-amino acids. Accuracy ranged between 81.9% and 111.7% and intra-day precision ranged between 1.8% and 10.9%. Concentrations of D- and L- Asp, Glu, and Ser are given and statistical differences between osteocytes and osteoblasts were found. The highest differences corresponded to L- and D-Glu. This method could play a fundamental role in the study of therapeutic targets in the treatment of bone diseases.