Column bleed in the analysis of highly polar substances: an overlooked aspect in HRMS

To close the "analytical gap" in the liquid chromatographic (LC) analysis of highly polar substances, two techniques which have been suggested earlier were tested in terms of retention factors and detection limits: hydrophilic interaction liquid chromatography (HILIC) and mixed-mode chromatography (MMC). A substance mix of 55 analytes ranging from logD - 8.2 to 3.4 and 17 different LC columns, also comprising additional reversed-phase columns were used. Contrary to most reversed-phase columns, column bleed has been identified as an important factor, which may cause serious restrictions during high-resolution mass spectrometric detection (HRMS). We found that highly abundant background masses continuously eluting from the columns heavily influence ion transmission to the detector. As a result, the linear dynamic range as well as the sensitivity decreases and thus limits the HRMS applicability of some columns. We therefore recommend a thorough investigation of ion transmission during HRMS method development. This will help to maintain the high potential of HRMS in terms of qualitative and quantitative screening analysis.