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

Abstract

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.

Technical Challenges in Mass Spectrometry-Based Metabolomics

“…Although several techniques were employed for the metabolic pro ling of the energy metabolism related intermediates using LC/MS, 50,98) there are remaining problems such as broad peak shapes, insu cient separation among structural isomers, and severe ion suppressions from the sample matrix. 99) ese problems allow the opportunity to reconsider the LC/ MS instrumentation for the bioanalysis purposes, since metabolome analysis of biological samples is one of the second largest application of mass spectrometry.…”

“…New hybrid columns for the metabolome analysis of energy metabolism have been developed and commercialized by introducing anion exchange mode into ODS, which is an important step in nding new de facto standards for bioanalysis. 99) It is also known that several metal ions such as Mg 2+ remaining in the sample extracts interact strongly with the di-and trivalent anions resulting in a broader peak shape of citric acid and ATP. 101) is problem could be partly avoided by introducing a trap column or an online solid phase extraction technique for sample injections.…”

LC‐MS‐Based Nontargeted Metabolomics

“…Such polar molecules include examples of practically all primary metabolites such as amines, organic acids, amino acids, and carbohydrates and metabolites involved in a multitude of biochemical pathways and fluxes. Alternative separation modes are urgently sought and as such hydrophilic interaction chromatography (HILIC) [11][12][13][14][15], ion-exchange LC, aqueous normal-phase chromatography [16], or ''mixed mode'' chromatography are increasingly being used coupled to MS [17].…”

Sample‐directed pseudotargeted method for the metabolic profiling analysis of rice seeds based on liquid chromatography with mass spectrometry

“…Metabolomics strategies can be divided into targeted [20][21][22] and non-targeted metabolomics [23,24]. Non-targeted method aims to cover of all the measurable known and unknown metabolites in a biological sample [23].…”