Application of a hybrid zwitterionic hydrophilic interaction liquid chromatography column in metabolic profiling...

Metabolic phenotyping studies using mouse liver extracts as a model, performed on a novel zwitterionic HILIC UHPLC column, which is based on ethylene-bridged hybrid organic/inorganic particles bonded with sulfobetaine groups and packed into column hardware modified with hybrid surface technology are reported. Initially the chromatographic performance was evaluated under different mobile phase conditions using selected metabolite standards. Following optimization of the chromatographic conditions for 88 hydrophilic metabolites both targeted and untargeted profiling analyses were performed on tissue extracts using LC-MS/MS and LC-TOF/MS, respectively. Chromatographic efficiency parameters such as peak resolution, peak shapes, selectivity and precision in retention and peak areas as well as characteristics that are critical for metabolic profiling analysis such as metabolite coverage and retention time distribution were assessed. The hybrid zwitterionic column exhibited efficient chromatographic separations providing analysis of ca 80 hydrophilic metabolites from different chemical classes and polarities. Utilizing a one-dimensional separation both targeted and untargeted profiling provided comprehensive metabolic signatures that enabled the acquisition of the metabolic phenotypes of the tissue extracts.