UCL-MetIsoLib: A Public High-Resolution Tandem Mass Spectrometry Library for HILIC-Based Isomer-Resolved Profiling of...

We present UCL-MetIsoLib, a publicly accessible high-resolution tandem mass spectrometry (HRMS/MS) library developed for HILIC-based, ion-pairing-free, isomer-resolved metabolomics using a bioinert UHPLC system and the Acquity Premier BEH Amide column. The platform integrates two complementary methods operating under distinct chromatographic conditions (pH 3.5, ESI⁺; pH 11.0, ESI^-), enabling broad metabolic coverage. A total of 334 metabolites are annotated in the library structure, with thiol derivatization incorporated into the extraction protocol to mitigate redox-driven artifacts. Metabolite identification is supported by 245 authentic reference standards and curated according to MSI Level 1 and Level 2 criteria. Validation followed FDA guidelines for bioanalytical method validation across five biological matrices─urine, plasma, tissues, cells, and patient-derived colorectal organoids. The method demonstrated high precision (<15% RSD intra/inter-day) and recovery (85-115% across all QC levels). To demonstrate biological applicability, UCL-MetIsoLib was applied to a case study comparing healthy and colorectal cancer-derived organoids. The method enabled confident annotation of metabolite isomers, including key glycolytic intermediates such as DHAP and GA3P, as well as sugar phosphates from the glycolysis and pentose phosphate pathways. Metabolic alterations were observed in tumor organoids, including accumulation of nucleotide derivatives and shifts in central carbon metabolism. The library is constantly under expansion and is freely available in its latest version at: https://github.com/kserafimov10/UCLMetIsoLib.