High-resolution LC-MS is an important platform for metabolite detection and quantitation. However, for untargeted Metabolomics rapid, unambiguous and universal compound identification is still challenging. In this work, we report the construction of a library for relevant endogenous metabolite and its’ successful application to human biofluids.
Standards were obtained from the Human Metabolome Database (HMDB). They were injected into an Intensity Solo 2 C18 column via an Elute LC system and detected by a QTOF-MS (impact, compact; all Bruker Daltonics) for acquiring MS/MS library spectra and the retention time determination (RT).
For the analysis of biofluids the same setup was used following a standard operating protocol (SOP) for consistency. Automatic metabolite identification was performed in the MetaboScape software based on matching of multiple parameters: precursor mass accuracy and isotopic pattern, RT, and MS/MS spectrum quality.
In this study a library from over 800 endogenous metabolites was created. It contains MS/MS spectra of 635 compounds acquired in positive mode and 474 negative mode spectra. Up to 5 collision energy levels were applied for each standard giving more than 6000 MS/MS spectra in total. For each metabolite, library fragment spectra were manually curated by confirming each fragment via a molecular formula. For unambiguous identification we determined the RT of each standard. An RT correction method using RT standards was applied to balance effects by variations in experimental conditions like instrument brands, LC columns and gradients. The described procedure was applied to biofluids, e.g. plasma and urine, and metabolite identification results will be presented.