Development of a novel method for analysing N-acetyl-DL-leucine enantiomers in human fingernail by UPLC-ESI-MS/MS and the evaluation in diabetes mellitus.

Background: Recent research has been reported that N-acetyl-leucine content is significantly reduced in the saliva of diabetic patients, but no reports of detection in human nails have been found. This study aims to develop a novel method for the chiral separation of N-acetyl-DL-leucine (Ac-DL-Leu) in human fingernails to investigate the differences between healthy volunteers (HVs), prediabetes (PDs) and diabetic patients (DPs), and to verify its effectiveness in early warning of diabetes.

Method: Chiral resolution was performed using DBD-Apy pre-column derivatization on a C18 column (2.1 × 150 mm, 1.9 μm) at 40 °C, and detected by UPLC-ESI-MS/MS.

Results: The resolution and the limit of detection (LOD) of Ac-DL-Leu were 1.75 and 1.50 fmol, respectively. The linear range of Ac-DL-Leu was 10-2000 fmol and the determination coefficient (R) was above 0.9997. The recovery of Ac-DL-Leu in human nails was 96.92-105.69 %. The contents of Ac-D-Leu and Ac-L-Leu were analyzed in 18 HVs, 13 PDs and 16 DPs fingernails. The results showed that their contents were significantly lower in DPs than in PDs and HVs (p < 0.0001).

Conclusions: A method for evaluating the effectiveness of Ac-DL-Leu enantiomers in human fingernails as a biomarker for diabetes was firstly developed.