Metabolomics workflow as a driven tool for rapid detection of metabolites in doping analysis. Development and validation.

RATIONALE This work demonstrated the high potential of combining high-resolution mass spectrometry with chemometric tools, using metabolomics as a guided tool for anti-doping analysis. The administration of 7-keto-DHEA was studied as a proof-of-concept of the effectiveness of the combination of knowledge-based and machine-learning approaches to differentiate the changes due to the athletic activities from those due to the recourse to doping substances and methods. METHODS Urine samples were collected from 5 healthy volunteers before and after an oral administration by identifying three-time intervals. Raw data were acquired by injecting less than one microliter of derivatized samples into an Agilent Technologies 8890 Gas Chromatograph coupled to an Agilent Technologies 7250 Accurate-Mass Quadrupole Time-of-Flight, by using a low energy electron ionization source, and then they were preprocessed to align peak retention times with the same accurate mass. The resulting data table was subjected to multivariate analysis. RESULTS Multivariate analysis showed a high similarity between the samples belonging to the same collection interval and a clear separation between the different excretion intervals. The discrimination between blank and long excretion groups may suggest the presence of long excretion markers, which are particularly significant in anti-doping analysis. Furthermore, matching the most significant features with some of the metabolites reported in the literature data demonstrated the rationality of the proposed metabolomics-based approach. CONCLUSIONS The application of metabolomics tools as an investigation strategy could reduce the time and resources required to identify and characterize intake markers maximizing the information that can be extracted from the data and extending the research field by avoiding a priori bias. Therefore, metabolic fingerprinting of prohibited substance intakes could be an appropriate analytical approach to reduce the risk of false-positive/negative results, aiding in the interpretation of "abnormal" profiles and discrimination of pseudo-endogenous steroid intake in the anti-doping field.