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Due to the recent discoveries of several D-amino acids in mammals and also by the elucidation of their physiological functions and/or availabilities as diagnostic markers, the enantioselective analysis of intrinsic amino acids has been the focus of attention. Based on the determination of amino acids with chiral discrimination, the clarification of their distributions, regulation mechanisms, physiological functions and the diagnostic/therapeutic values of a wide variety of D-amino acids is expected. Although various analytical techniques have been reported, the improvement of the selectivity is still in demand especially for the accurate determination of trace levels of D-amino acids in complicated biological matrices. One of the straightforward approaches to address the selectivity issue is the multi-dimensional LC analysis in which several separation modes are combined. In the present review, the two-dimensional (2D) LC methods in combination with the detection using MS/MS are introduced and the applications to various biological samples are summarized. The 2D LC-MS/MS methods were designed by integrating a reversed-phase purification (first dimension), an enantioselective separation (second dimension) and the detection of specific precursor/product ion pairs of analytes using MS/MS. Using these methods, the precise and simultaneous determination of D-alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), leucine (Leu), proline (Pro) and serine (Ser) in human plasma and urine was carried out. The in vivo regulation of D-Ala, Leu, Pro, Ser and tryptophan (Trp) amounts by D-amino acid oxidase (DAO) was clarified by analyzing their concentrations in the plasma and urine of DAO deficient mice. For the accurate analysis of D-amino acid residues in proteins, the hydrolysis using deuterium chloride/deuterium oxide and 2D LC-MS/MS determination were combined, and the stereoinversion of the Ala, Asp, Glu, Pro and Ser residues was evaluated in several proteins.
Ishii et al. (Thu,) studied this question.