Screening for pharmaco-toxicologically relevant compounds in biosamples using high-resolution mass spectrometry: a 'metabolomic' approach to the discrimination between isomers.

High-resolution mass spectrometry (HRMS) enables the identification of a chemical formula of small molecules through the accurate measurement of mass and isotopic pattern. However, the identification of an unknown compound starting from the chemical formula requires additional tools: (1) a database associating chemical formulas to compound names and (2) a way to discriminate between isomers. The aim of this present study is to evaluate the ability of a novel 'metabolomic' approach to reduce the list of candidates with identical chemical formula. Urine/blood/hair samples collected from real positive cases were submitted to a screening procedure using ESI-MS-TOF (positive-ion mode) combined with either capillary electrophoresis or reversed phase liquid chromatography (LC). Detected peaks were searched against a Pharmaco/Toxicologically Relevant Compounds database (ca 50,500 compounds and phase I and phase II metabolites) consisting of a subset of PubChem compounds and a list of candidates was retrieved. Then, starting from the mass of unknown, mass shifts corresponding to pre-defined biotransformations (e.g. demethylation, glucuronidation, etc.) were calculated and corresponding mass chromatograms were extracted from the total ion current (TIC) in order to search for metabolite peaks. For each candidate, the number of different functional groups in the molecule was automatically calculated using E-Dragon software (Talete srl, Milan, Italy). Then, the presence of metabolites in the TIC was matched with functional groups data in order to exclude candidates with structures not compatible with observed biotransformations (e.g. loss of methyl from a structure not bearing methyls). The procedure was tested on 108 pharmaco-toxicologically relevant compounds (PTRC) and their phase I metabolites were detected in real positive samples. The mean list length (MLL) of candidates retrieved from the database was 7.01 +/- 4.77 (median, 7; range, 1-28) before the application of the 'metabolomic' approach, and after the application it was reduced to 4.08 +/- 3.11 (median 3, range 1-17). HRMS allows a much broader screening for PTRC than other screening approaches (e.g. library search on mass spectra databases). The 'metabolomic' approach enables the reduction of the list of candidate isomers.