dl-Amino Acid Analysis Based on Labeling with Light and Heavy Isotopic Reagents Followed by UPLC-ESI-MS/MS

Abstract

l-Pyroglutamic acid succinimidyl ester (l-PGA-OSu) and its isotopic variant (l-PGA[d5]-OSu) were synthesized and used as the chiral labeling reagents for the enantioseparation of amino acids by reversed-phase UPLC-ESI-MS/MS. The enantiomers of amino acids were labeled with the reagents at 60 °C for 10 min in an alkaline medium. The resulting diastereomers were well separated by the reversed-phase chromatography using an ODS column, packed with small particles (1.7 μm) (Rs = 1.95–8.05). A highly sensitive detection at a low-fmol level (0.5–3.2 fmol) was obtained from the selected reaction monitoring (SRM) chromatograms. An isotope labeling strategy using light and heavy variants for the differential analysis of the dl-amino acids in different sample groups is also presented in this paper. The ratios of d/l-alanine in different yogurt products were successfully determined by the proposed method. The d/l ratios were almost comparable to those obtained from only using light reagent (i.e., l-PGA-OSu). Therefore, the proposed strategy seems to be useful for the differential analysis of dl-amino acids, not only in food products but also in biological samples.