Development of Zwitterionic Hydrophilic Liquid Chromatography (ZICⓇHILIC-MS) metabolomics method for Shotgun analysis of human urine

Urine is a product of the body’s metabolism and the majority of the metabolic products exiting via the renal system are rendered polar in order to be water soluble. Resolution of urinary metabolites for metabolomic studies requires the development of HPLC separation techniques that match this feature of biological chemistry. ZIC –HILIC is an ideal candidate to take forward resolution of such metabolites where reverse phase is unable to give adequate separation. Metabolomic data has to be processed by Shotgun multivariate analysis to sift through thousands of analytes and their variables such as ion intensity.
In the development of ZIC-HILIC separation with mass spectrometric (IT-ToF) detection, methodological variability have to be minimized so that any Shotgun data analysis does not reveal potential biomarker analytes that are artifacts or are adversely affected of the separation and detection technique. Here, we report the development of a ZIC-HILIC mass spectrometry method that is suitable for SIMCA P+ data analysis of urine. Variables such as resolution, run reproducibility and sample storage temperature were evaluated in tandem with SIMCA P+ data analysis and quality control pre-processing.
The developed method couples quality control runs that pre-process and exclude analytes that are insufficiently robust for further candidate biomarker studies. This meant labile analytes that could not be reproduced in 70% of QC runs (which are pools of all samples run that day) were excluded. However, urine samples stored at 4°C for more than 9 months will contain metabolites that will alter and produce small molecule marker artifacts when compared to samples stored at -20°C.
In conclusion, the developed method is a robust method of ZIC-HILIC mass spectrometry shotgun analysis suitable for urinary metabolome discovery of robust biomarkers.