Chiral derivatization reagents for enantioselective LC-MS/MS analysis of amino acids with analyte-specific fragmentation

Abstract

Accurate enantioselective analysis of amino acids by liquid chromatography-tandem mass spectrometry (LC–MS/MS) remains challenging, as existing derivatization strategies often compromise either chromatographic resolution or structural discrimination in tandem mass spectrometry. In this study, we developed novel chiral derivatization reagents, (R)- and (S)-Oqic, based on a 1,3-oxazinoquinoline-4-one scaffold, for the simultaneous determination of amino acid enantiomers via LC–MS/MS. The reagents reacted rapidly with amino acids under mild basic conditions to form their chiral derivatives, enabling the simultaneous separation of 37 amino acids (18 pairs of amino acid enantiomers and Gly) on a conventional ODS column within 16 min. The resulting derivatives exhibited unique fragmentation patterns, yielding characteristic fragment ions at m/z 190 and [M + H − 171], with intensity ratios dependent on the tagged amino acid, thereby facilitating reliable analyte discrimination. The method was validated and subsequently applied to the analysis of commercially available fermented dairy products, revealing the presence of d-Ala, d-Ser, d-Asp, d-Glu, and d-Arg in yogurt and a probiotic beverage. The detection of sweet-tasting d-amino acids (d-Ala, d-Ser, and d-Arg) suggests their potential contribution to the sensory properties of fermented foods. Overall, the developed Oqic-based method provides a powerful tool for elucidating bacterial metabolism and food flavor profiles.