Development of Precolumn Derivatization–LC/MS for Amino-Acid-Focused Metabolomics

Abstract

Focused metabolomics facilitates the understanding of the current phenotype of living organisms, involves highly practical methods for investigation, and allows for the acquisition of highly quantitative data for important endogenous metabolites. The authors developed novel precolumn derivatization reagents for the analysis of amino acids, which are particularly important endogenous metabolites, through precolumn liquid chromatography/tandem mass spectrometry (LC/MS/MS). One of the reagents facilitated sub-femtomole-to-attomole-scale amino acid detection. We also developed an automated precolumn derivatization amino acid analyzer using another reagent for liquid chromatography/mass spectrometry (LC/MS). With this device, nearly 40 amino acids can be analyzed within a short time (<10 min), compared with the time (~2 h) taken by a conventional automated postcolumn derivatization amino acid analyzer. For highly accurate and precise analysis of free amino acid concentration in human plasma, we validated all steps, from blood collection to measurement, by using the proposed analyzer and developed a test procedure for studying free amino acid metabolomics in human plasma. While calculating the reference intervals of plasma amino acid concentrations in healthy individuals, it was found that the balance of amino acid concentrations in plasma is useful for risk screening of diseases including cancer, diabetes, and myocardial infarction. This functional-group-targeted analysis through LC/MS for amino acid was also effective for dipeptide, tripeptide, and D, L-amino acid analyses and amino acid detection in tissue by imaging mass spectrometry.