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 10min in an alkaline medium. The resulting diastereomers were well separated by the reversed-phase chromatography using an ODS column, packed with small particles (1.7μm) (Rs=1.95-8.05). A highly sensitive detection at a low-fmol level (0.5-3.2fmol) 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.