Optimisation of a dilute-and-shoot UHPLC-MS method for high-throughput urinary metabolomics

Introduction Urinary metabolomics using liquid chromatography-mass spectrometry (LC-MS) holds great potential for biomarker discovery and clinical application. Large-scale studies and clinical trials require rapid, scalable methods. However, existing urine extraction and LC-MS approaches lack standardisation and clear recommendations for high-throughput use. Objectives To optimise a dilute-and-shoot urinary metabolite extraction and untargeted ultra-high-performance LC-MS (UHPLC-MS) method suitable for large-scale studies, and to demonstrate its performance in a clinical cohort. Methods We systematically optimised eight parameters: extraction solvent, dilution factor, chromatographic gradient, injection volume, flow rate, gradient curve factor, full scan automatic gain control (AGC) and MS/MS AGC. Performance was assessed by the number of metabolites detected, peak quality, creatinine performance and peak quality factors (PQFs). The optimised method was applied to 1094 urine samples (862 clinical samples plus QCs) from healthy controls and patients with inflammatory bowel disease (IBD). We compared three normalisation strategies for their effect on data precision: no normalisation, creatinine normalisation and probabilistic quotient normalisation (PQN). Results The optimised method used a C18 column in positive ionisation mode. The mobile phases were water and acetonitrile, each with 0.1% formic acid (v/v). Data acquisition took 6.5 min, and the total run time was 10 min including a cleaning step. In the IBD cohort, over 1500 metabolites were putatively annotated in 182.3 h of instrument time. This saved over 91 h compared with the previously used method. Conclusions This optimised method provides rapid, scalable untargeted urinary metabolomics suitable for large clinical studies and trials.