Multi-reflecting time-of-flight mass spectrometry enables high-accuracy metabolite identification in a demonstration study using serum samples from COVID-19 patients

Abstract Introduction Accurate metabolite identification remains a major challenge in untargeted metabolomics due to overlapping signals, isomeric compounds, and insufficient mass resolution. The multi-reflecting time-of-flight (MRT) platform provides mass resolution exceeding 200,000 Full Width at Half Maximum and sub-ppm mass accuracy, enabling detailed isotopic fine structure analysis. Objectives The performance of the MRT platforms in identifying lipids and metabolites was evaluated using selected serum samples from a group of hospitalized COVID-19 patients. Methods Lipids and polar metabolites were extracted from human serum samples from the Manchester Asthma, Allergy and Thoracic Surgery Biobank and analysed using the MRT platform with data-independent acquisition. Results The MRT platform facilitated the resolution of overlapping lipid species and improved annotation confidence. In a pilot cohort ( n  = 8), metabolomic differences associated with disease severity were observed, alongside annotation of clinically relevant metabolites including sulphated bile acids and retained iohexol associated with renal dysfunction. Conclusions These findings demonstrate that MRT platform enhances confidence in metabolite identification and supports high-throughput metabolomics, offering significant potential for biomarker discovery in clinical studies.