Quantitative Analysis of Amino Acid Enantiomers in Human Saliva and the Potential for Early Warning of Diabetes

An ultra-high-performance liquid chromatography with fluorescence detection (UPLC-FL) method was developed and applied for the quantitative analysis of D-alanine (D-Ala) and L-alanine (L-Ala), D-leucine (D-Leu) and L-leucine (L-Leu), and D-valine (D-Val) and L-valine (L-Val) in human saliva. Enantiomers were derivatized using the fluorescent reagent R-(-)-4-(3-isothiocyanatepyrrolidine-l-methyl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R-(-)-DBD-PyNCS] at 55°C in the presence of 3% triethylamine for 20 min, yielding aminothiocarbonyl diastereomeric derivatives. The derivatized products were detected using UPLC-FL at an excitation wavelength (Ex) of 460 nm and a fluorescence wavelength (Em) of 550 nm. The limit of detection was 0.010-0.200 μM, and the resolution (Rs) > 1.5 within 20 min. The R² of D-type amino acids in the range of 0.5-50 μM and L-type amino acids in the range of 1.0-500 μM was greater than or equal to 0.9988. Intraday and interday assay precisions were below 6.94%, with average recoveries ranging from 95.20% to 109.6%. This method was succeeded in quantifying amino acid enantiomers in the saliva of healthy volunteers and patients with diabetes. Significant differences in D-Ala, L-Ala, L-Leu, D-Val, and L-Val levels were observed between the two groups (p < 0.01). The proposed method holds potential for diagnosing diabetes and may assist as an early warning indicator of diabetes.