Evaluation of the fluorescent chiral derivatization reagent DBMA for targeting amino functional groups: An application in the analysis of DL-amino acids in the saliva of patients with diabetes

The study developed a novel chiral fluorescence (FL) derivatization reagent, ( R )-1-(7-( N,N -dimethylsulfamoyl)-benzo[ c ][1,2,5]oxadiazol-4-yl)-2-methylpyrrolidine-2-carboxylic acid (DBMA), targeting the amino functional group. Eighteen DL-amino acids were tested for chiral separation efficiency using ultra-performance liquid chromatography-FL (UPLC-FL). DBMA and DL-amino acids formed diastereomers following reaction conducted at 40°C for 30 min, employing 1-(3-dimethylaminopropyl)-3-ethylcarbohydrazide (EDC) and hydroxybenzotriazole (HOBt) condensation agents. The analysis was performed using a BEH C18 column (2.1 × 100 mm, 1.7 μm) with detection wavelengths set at 460 nm (excitation) and 550 nm (emission). The mobile phase consisted of two components: an aqueous phase containing 10 mM ammonium acetate with 0.05% formic acid (FA), and an organic phase comprising either 0.1% FA in acetonitrile or 0.1% FA in MeOH. The results demonstrated that the Rs was 1.51-4.40, indicating a favorable chiral separation effect. Furthermore, the method was extended to the simultaneous detection of four DL-amino acids (DL-Asp, DL-Thr, DL-Ile, and DL-Lys) from 31 healthy volunteers and 19 diabetic patients. The R 2 was ≥ 0.9991 in the 5-2000 μM concentration range, with a limit of detection ranging from 2.5 to 25 pmol. The intraday and interday precisions ranged from 1.13% to 9.25%. Diabetic patients exhibited notably elevated average concentrations of D-Asp, DL-Thr, L-Ile, and DL-Lys in their saliva, in contrast to healthy volunteers. The disparity was determined to have statistical significance ( p < 0.01). Furthermore, the ratios of D/L-Asp, D/L-Thr, and D/L-Lys were statistically significant ( p < 0.01). DBMA, a novel fluorescent chiral derivatizing agent, enables enantioselective detection of D/L-amino acid biomarkers in biological matrices. This introduces an innovative chiral derivatization agent for studying chiral metabolomics.