Preparation Of Racemic Alpha-Amino Acid Standards For Accurate Mass Spectrometric Analysis Via Racemization Catalyzed By A Hydrophobic Pyridoxal Derivative

Racemic alpha-amino acid standards for chiral metabolomics were prepared from L- alpha-amino acids using a hydrophobic pyridoxal derivative, namely 3-hydroxy-2-methyl-5-((octyloxy)methyl)isonicotinaldehyde (OPy), as the racemization catalyst. Among the 19 tested proteinogenic amino acids, 13 (including the generally unstable asparagine, glutamine, and tryptophan) underwent efficient racemization/epimerization under mildly basic conditions at room temperature, while solid-phase extraction allowed for effective and simple catalyst removal and amino acid recovery, obviating the need for chromatographic separation and recrystallization. Isotopically labeled racemic amino acids are commonly employed as internal standards for highly accurate mass spectrometric analysis. However, as isotopically labeled D-amino acids are often unavailable or highly expensive, the developed method was used to prepare racemic labeled amino acids, which were shown to enhance the repeatability and accuracy of D,L-amino acid quantitation in human urine by liquid chromatography-mass spectrometry (LC-MS). Given that our method should also be applicable to non-proteinogenic alpha-amino acids and the N-termini of peptides, the present study is expected to accelerate the development of LC-MS-based chiral metabolomics.