Simultaneous Determination of Free DL-Amino Acids in Natto with Novel Fluorescent Derivatization by UPLC-FL

Abstract

A new simple and sensitive method for the simultaneous detection of free DL-amino acids (DL-AA) in natto was established by UPLC-fluorescence (FL) using the newly developed fluorescence derivatization reagent DBD-trans-2-methyl-L-proline (DBD-M-Pro) by precolumn derivatization. DBD-M-Pro reacts with DL-amino acids at 60 °C for 60 min. Using BEH C18 (2.1 mm × 100 mm, 1.7 μm) column gradient analysis for 20 min, the resolution of diastereomers after the derivatization of four DL-amino acids was 1.65–3.50. The detection limit for amino acids was 0.25–2.5 pmol. The average recovery (%) of DL-amino acids was 86.65–118.70%. The method was applied to the detection of chiral amino acids in natto beans and mucus, and four chiral amino acids, DL-Gln, DL-Asp, DL-Glu, and DL-Ala, were simultaneously detected. At the same time, the DL-amino acid content and D/L ratio of three different manufacturers of natto were compared quantitatively.