For a metabolomics study focusing on the analysis of aspartic and glutamic acid enantiomers, a fully automated two-dimensional HPLC system employing a microbore-ODS column and a narrowbore-enantioselective column was developed. By using this system, a detailed distribution of d-Asp and d-Glu besides l-Asp and l-Glu in mammals was elucidated. For the total analysis concept, the amino acids were first pre-column derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to be sensitively and fluorometrically detected. For the non-stereoselective separation of the analytes in the first dimension a monolithic ODS column (750 mm × 0.53. mm i.d.) was adopted, and a self-packed narrowbore-Pirkle type enantioselective column (Sumichiral OA-2500S, 250. mm × 1.5. mm i.d.) was selected for the second dimension. In the rat plasma, RSD values for intra-day and inter-day precision were less than 6.8%, and the accuracy ranged between 96.1% and 105.8%. The values of LOQ of d-Asp and d-Glu were 5. fmol/injection (0.625. nmol/g. tissue). The present method was successfully applied to the simultaneous determination of free aspartic acid and glutamic acid enantiomers in 7 brain areas, 11 peripheral tissues, plasma and urine of Wistar rats. Biologically significant d-Asp values were found in various tissue samples whereas for d-Glu the values were very low possibly indicating less significance.
|ジャーナル||Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences|
|出版ステータス||出版済み - 11 1 2011|
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