Wspólnotowy Serwis Informacyjny Badan i Rozwoju - CORDIS

Chemical synthesis methods for labelled phase I 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. The chemical synthesis of stably deuterium-labelled analogues of endogenous steroids, anabolic steroids, and phase-I-metabolites of anabolic steroids was performed for nandrolone, the nandrolone metabolite 5a-estran-3a-ol-17-one (norandrosterone), testosterone and androstandiol.

Here, molecule structures were temporarily modified in order to acidify protons, enabling the H/D exchange under mild alkaline conditions as used for several other applications in the past. After controlled substitution of hydrogens the deuteria introduced into the steroid scaffold are required to be stably located. Thus, hydrogens located at carbons next to hydroxyl functions were elected for exchange in all compounds labelled in this project. With nandrolone and testosterone, the deuteria were located at C-16 and C-17, with norandrosterone and androstanediol at C-2, C-3 and C-4. With the synthesis of these isotopically labelled analogues of analytes relevant for doping controls, artificial substrates were prepared demonstrating nearly identical physicochemical properties to target compounds of doping analyses.

Hence, they are appropriate in particular as internal standards, ensuring a proper sample preparation and analysis result. Extraction of analytes from biological matrix has often proven to be a crucial step in any kind of analytical chemistry, and the presence of convenient counterparts to compounds 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.

For instance, the dissociation of 17-methylated steroids generates upon trimethylsilylation and EI-mass spectrtometry a characteristic fragment ion at m/z 147 that originates from the steroid D-ring. With testosterone analogues and ESI-CAD mass spectrometry, A- and B-ring fragments are predominant, giving rise to signals at m/z 109 and 97, indicating a 3-keto-4-en-structure of the steroid nucleus. 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. With the knowledge of dissociation behaviour after ionisation by either of the available techniques, identification of analytes is substantiated and supports the conviction, as well as the protection, of athletes.

With commonly accepted gas chromatographic and mass spectrometric procedures, the deuterated analogues of steroids are used frequently owing to the advantages described above, in particular in case of quantitation. With proper internal standards, variations in analysis results owing to difficult sample preparation steps (e.g. solid-phase extraction, liquid-liquid extraction) can be eliminated by correction with a known amount of internal standard.

Reported by

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