Selectivity issues in targeted metabolomics: Separation of phosphorylated carbohydrate isomers by mixed-mode hydrophilic interaction/weak anion exchange chromatography

Phosphorylated carbohydrates are important intracellular metabolites and thus of prime interest in metabolomics research. Complications in their analysis arise from the existence of structural isomers that do have similar fragmentation patterns in MS/MS and are hard to resolve chromatographically. Herein, we present selective methods for the liquid chromatographic separation of sugar phosphates, such as hexose and pentose phosphates, 2‐ and 3‐phosphoglycerate, dihydroxyacetone phosphate and glyceraldehyde 3‐phosphate, as well as glucosamine 1‐ and 6‐phosphate utilizing mixed‐mode chromatography with reversed‐phase/weak anion‐exchangers and a charged aerosol detector. The best results were obtained when the reversed‐phase/weak anion‐exchanger column was operated under hydrophilic interaction liquid chromatography elution conditions. The effects of various chromatographic parameters were examined and are discussed on the basis of a simple stoichiometric displacement model for explaining ion‐exchange processes. Employed acidic conditions have led to the complete separation of α‐ and β‐anomers of glucose 6‐phosphate at low temperature. The anomers coeluted in a single peak at elevated temperatures (>40°C) (peak coalescence), while at intermediate temperatures on‐column interconversion with a plateau in‐between resolved anomer peaks was observed with apparent reaction rate constants between 0.1 and 27.8×10^?4 s^?1. Dynamic HPLC under specified conditions enabled to investigate mutarotation of phosphorylated carbohydrates, their interconversion kinetics, and energy barriers for interconversion. A complex mixture of six hexose phosphate structural isomers could be resolved almost completely.