This application note evaluates the effects that the UPC2 method parameters, such as column chemistry, mobile phase gradient conditions, and eluent solvent composition have on the chromatographic analysis of biologically relevant polar analytes in aqueous based samples.
UPC2-MS shows significant promise as a platform for polar metabolomic profiling, and could allow for the analysis of aqueous biological matrices. Method development for analyzing polar metabolites by UPC2-MS requires the optimization of eluent composition; co-solvent composition, buffer concentration and acidity of the co-solvent.
The addition of water at 5% (v/v) or acidified ammonium formate (Aq), into methanol co-solvent significantly improved the peak shape and analyte retention. Peak shape and analyte retention improved with increasing buffer concentration, however, increasing the methanol concentration above 5% and the acid concentration above 0.2% resulted in deleterious chromatographic effects.
Total analysis times achieved with the ACQUITY UPC2 System were similar to previously reported using HILIC UPLC-MS methods.