Development of a rapid profiling method for the analysis of polar analytes in urine using HILIC–MS and ion mobility enabled HILIC–MS

Introduction As large scale metabolic phenotyping is increasingly employed in preclinical studies and in the investigation of human health and disease the current LC–MS/MS profiling methodologies adopted for large sample sets can result in lengthy analysis times, putting strain on available resources. As a result of these pressures rapid methods of untargeted analysis may have value where large numbers of samples require screening. Objectives To develop, characterise and evaluate a rapid UHP-HILIC-MS-based method for the analysis of polar metabolites in rat urine and then extend the capabilities of this approach by the addition of IMS to the system. Methods A rapid untargeted HILIC LC–MS/MS profiling method for the analysis of small polar molecules has been developed. The 3.3 min separation used a Waters BEH amide (1 mm ID) analytical column on a Waters Synapt G2-Si Q-Tof enabled with ion mobility spectrometry (IMS). The methodology, was applied to the metabolic profiling of a series of rodent urine samples from vehicle-treated control rats and animals administered tienilic acid. The same separation was subsequently linked to IMS and MS to evaluate the benefits that IMS might provide for metabolome characterisation. Results The rapid HILIC–MS method was successfully applied to rapid analysis of rat urine and found, based on the data generated from the data acquired for the pooled quality control samples analysed at regular intervals throughout the analysis, to be robust. Peak area and retention times for the compounds detected in these samples showed good reproducibility across the batch. When used to profile the urine samples obtained from vehicle-dosed control and those administered tienilic acid the HILIC-MS method detected 3007 mass/retention time features. Analysis of the same samples using HILIC–IMS–MS enabled the detection of 6711 features. Provisional metabolite identification for a number of compounds was performed using the high collision energy MS/MS information compared against the Metlin MS/MS database and, in addition, both calculated and measured CCS values from an experimentally derived CCS database. Conclusion A rapid metabolic profiling method for the analysis of polar metabolites has been developed. The method has the advantages of speed and both reducing sample and solvent consumption compared to conventional profiling methods. The addition of IMS added an additional dimension for feature detection and the identification of metabolites