Simultaneous Quantification of Five Stereoisomeric Hexoses in Nine Biological Matrices Using Ultrahigh Performance Liquid Chromatography with Tandem Mass Spectrometry

Stereoisomeric hexoses are present in almost all biological organisms in the forms of aldoses and ketoses, with diverse physiological and pathophysiological functions. Accurate and simultaneous quantification is vital for understanding their functions individually. However, such analysis remains challenging owing to their highly similar behavior in chromatography and mass spectrometry. By combining the pre-column 3-nitrophenylhydrazine derivatization and ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS), here, we developed a method for simultaneous quantification of five important stereoisomeric hexoses including D-glucose, D-galactose, D-mannose, D-fructose and L-sorbose representing both aldoses and ketoses. The method achieved baseline-separation for all these five derivatized hexoses chromatographically and had high sensitivity (LOD, femtomole on column), excellent linearity (R2 > 0.995) and efficiency with stable-isotope dilution. With this method, we further quantified these hexoses in nine biological matrices including human biofluids (serum, urine and saliva), human cells, human and mouse feces, rat liver tissue, mung-bean seeds and peach pulp. The results provided quantitative data for these hexoses in multiple biological samples and showed significant concentration diversity for these hexoses in different biological samples, which demonstrated the applicability of the method for simultaneous quantification of these hexose phenotypes of biological systems. • An optimized UHPLC-MS/MS method based on 3-nitrophenylhydrazine derivatization. • Simultaneous quantification of five stereoisomeric hexoses. • Quantitative results for these five hexoses in nine different biological matrices.