Servicio de Información Comunitario sobre Investigación y Desarrollo - CORDIS

Chemical synthesis methods for phase II metabolites of anabolic androgenic steroids

The metabolism of xenobiotics, such as anabolic androgenic steroids, as well as endogenous compounds, depends on their physicochemical properties, e.g. polarity and solubility in aqueous phases. Thus, metabolic pathways are described as phase-I and phase-II, the first one of which serves the generation of polar functions in molecules, such as hydroxy groups that are conjugated in a second step of metabolism. For instance, phase-I-metabolites of steroids comprise hydroxy groups obtained by stereospecific reduction of keto functions, reduced carbon-carbon double bonds or keto functions obtained by oxidation of hydroxy groups.

Following the phase-I-metabolism, many compounds (including anabolic and endogenous steroids) are conjugated to glucuronides and/or sulfates. Commonly accepted and employed doping analysis assays are based on the chemical or enzymatic hydrolysis of those conjugates and subsequent measurement of liberated phase-I-metabolites by means of gas chromatography coupled to mass spectrometry. With the EU-funded project, new strategies to identify misuse of anabolic androgenic steroids are developed, based on the analysis of intact phase-II-metabolites by liquid-chromatography-tandem mass spectrometry. Here, comparable to the development, evaluation and validation of other analytical procedures, reference material is of paramount importance. Doping control analysis as well as various fields of analytical chemistry are based on the comparison of analytical results of known compounds and signals/spectra obtained from real-world samples.

In order to be able to generate reference spectra, chemically synthesized, characterized and certified material is necessary. This requires reliable and sophisticated strategies of substance preparation, knowledge about mass spectrometric behaviour as well as different analytical tools providing fundamental data on structure and composition of the synthesized compounds. The possibility to employ exactly defined amounts of phase-I-metabolites of xenobiotics is essential, and with the synthesis of necessary compounds, method development is noticeably facilitated.

Extraction of analytes from biological matrix has often proven to be a crucial step in any kind of analytical chemistry, and the possibility to use synthetically prepared substances of interest enables the frequent control of procedure and instrument parameters. The structures of synthesized materials were elucidated and confirmed by means of nuclear magnetic resonance spectroscopy and mass spectrometry with different ionisation techniques and mass spectral analysers.

Fundamental information on mass spectrometric behaviours after electron impact ionisation, electrospray ionisation and collision-activated dissociation as well as fragmentation pathways was obtained, which are of paramount importance in case of structure determination of related but unknown compounds or metabolites. With every mass spectrometric analysis of new metabolites, important additional information is provided enabling even more comprehensive screenings for prohibited compounds. Doping analysis is primarily based on chromatographic and mass spectrometric assays, and compounds are identified by comparison of retention times and fragment ions of known, prohibited substances with compounds detected in urine samples of athletes.

Besides the use of steroid glucuronides for LC-ESI-MS/MS method development, reference materials are also utilized to control quality and quantity of enzymatic or chemical hydrolysis steps of other, complementary GC-MS procedures.

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German Sport University Cologne
Carl-Diem-Weg 6
50933 Köln
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