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

ABSTRACT 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 R 2 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.