Simultaneous analysis of monosaccharides using ultra high performance liquid chromatography-high resolution mass spectrometry without derivatization for validation of certified reference materials

Abstract

Monosaccharide composition of biological samples can reflect an individual's health status. Monitoring the concentration of individual monosaccharides in human serum requires a technique for the simultaneous analysis of multiple monosaccharide molecules. Furthermore, certified reference materials (CRMs) for overall monosaccharide composition of human serum are required in order to validate the performance of clinical laboratory instruments. In the present study, we present a novel method for the simultaneous analysis of numerous monosaccharide molecules without the need for derivatization or post-column treatment. We utilized ultra-high-performance liquid chromatography (UHPLC)-quadrupole/orbitrap mass spectrometry incorporating a hydrophilic interaction chromatography (HILIC) column. We optimized the precursor ions, product ions, mobile phase composition and gradient program, flow rate, and column temperature. Seven monosaccharides (D-Ribose, L-Arabinose, D-Xylose, D-Fructose, D-Mannose, D-Galactose and D-Glucose) were able to be separated and quantified. We validated the method and the seven molecules showed favorable limits of detection and quantification, recovery rates, carry-over effects, intraand inter-day accuracy and precision, resolution, and measurement uncertainty. We analyzed human serum samples using the method. To avoid ion suppression and D-d(2)-Glucose peak interference, compounds present at concentrations outside of the calibration range were analyzed from diluted samples. Quantification of serum samples corroborated some previous clinical research, in that increased D-Glucose concentration was associated with increased concentrations of D-Mannose and D-Ribose. We also validated the CRMs, and expect these to have utility as standards for serum monosaccharide profiling, thus contributing to public health.