Enantioselective two-dimensional high-performance liquid chromatographic determination of N-methyl-D-aspartic acid and its analogues in mammals and bivalves.

A validated and fully automated chiral 2D-HPLC system was developed for the simultaneous determination of N-methyl-d-aspartic acid (NMDA) analogues by combining a long microbore-monolithic ODS column (0.53 mm i.d.× 1,000 mm) and narrowbore-enantioselective columns (1.5mm i.d.×150 or 250 mm). The target analytes, enantiomers of N-methylaspartic acid (NMA) and N-methylglutamic acid (NMG), were precolumn-derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and detected by their fluorescence. The values of the lower limit of quantification for these enantiomers were 2.5 fmol. In the tissues and plasma of rats, neither NMA nor NMG were detected. On the other hand, in the mantle and foot of Scapharca broughtonii, a large amount of NMDA was present (170.1 and 43.5 nmol/g), and the enantiomers of NMG were also observed. Meretrix lusoria contained NMDA (29.3 nmol/g) and NMLG (13.8 nmol/g), and Ruditapes philippinarum contained only NMLG (2.6 nmol/g). The obtained results were confirmed using three different enantioselective columns and also using a 2D-HPLC-MS/MS system. These results indicated that neuroactive d-amino acid, NMDA, and its analogues were present in animals, and their physiological significance is expected to be clarified.

Simultaneous determination of hydrophilic amino acid enantiomers in mammalian tissues and physiological fluids applying a fully automated micro-two-dimensional high-performance liquid chromatographic concept.

A validated two-dimensional HPLC system combining a microbore-monolithic ODS column and a narrowbore-enantioselective column has been established for a sensitive and simultaneous analysis of hydrophilic amino acid enantiomers (His, Asn, Ser, Gln, Arg, Asp, allo-Thr, Glu and Thr) and the non-chiral amino acid, Gly, in biological samples. To accomplish this goal, the amino acids were first tagged with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to the respective fluorescent NBD derivatives which were separated in the first dimension by a micro-reversed-phase column. The automatically collected fractions of the target peaks were then transferred to the second dimension consisting of a Pirkle type enantioselective column generating separation factors higher than 1.13 for all the enantiomeric target analytes. The system was validated using standard amino acids and a rat plasma sample, and analytically satisfactory calibration and precision results were obtained. The present 2D-HPLC system enables the fully automated determination of hydrophilic amino acid enantiomers in mammalian samples. The d-isomers of all the investigated 9 amino acids were found in rat urine but at various enantiomeric ratios.