First observation of N-acetyl leucine and N-acetyl isoleucine in diabetic patient hair and quantitative analysis by UPLC-ESI-MS/MS.

Abstract Background Type 2 diabetes patients (DP) have significantly higher plasma levels of valine, leucine, isoleucine and alanine than the controls. Specific amino acids may acutely and chronically regulate insulin secretion from the pancreatic β-cells. We recently identified a metabolic signature of N -acetyl leucine (Ac-Leu) that strongly predicts diabetes development in mice hair. The Ac-Leu appears to be a potential biomarker candidate related to diabetes. However, the determination of Ac-Leu in human hair has not been reported. We measured the Ac-Leu, and its structure is similar to N -acetyl isoleucine (Ac-Ile) in human hair by ultra-performance liquid chromatography (UPLC) with electrospray ionization tandem mass spectrometry (ESI–MS/MS). The developed method was applied to the determination of Ac-Leu and Ac-Ile in the hair of healthy volunteers (HV) and DP. Methods Ac-Leu, Ac-Ile and N -acetyl norleucine (Ac-Nle, IS) were extracted from human hair samples by a micropulverized extraction procedure, then separated on a C18 column by isocratic elution of acetonitrile–0.1% formic acid in water:0.1% formic acid (14:86, vol./vol.). MRM using the fragmentation transitions of m/z 174.1 → 86.1 in the positive ESI mode was performed to quantify the N -acetyl leucine, N -acetyl isoleucine and IS. Results Ac-Leu, Ac-Ile and Ac-Nle in the human hair samples were completely separated by isocratic elution of a 5.0 min duration wash program using a reversed-phase column, and sensitively detected by LC–MS/MS in the ESI + MRM mode. The amounts of Ac-Leu and Ac-Ile in the hairs of HV and DP were determined. When comparing the concentrations between DP and those from HV, a statistically significant correlation was observed for the Ac-Leu (p  Conclusions The proposed method is useful for the determination of Ac-Leu and Ac-Ile in the hairs of DP and HV. Human hair may serve as a noninvasive biosample for the diagnosis of diabetes.