Deliverables 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. LCMS methods Liquid chromatography-mass spectrometry (LC-MS) provides an alternative method for doping control. Presently the AAS are determined after hydrolysis of AAS conjugates by GC-MS. However, the GC-MS method is time-consuming and faster methods are required. Furthermore, the sensitivity with GC-MS in analysis of polar AAS may not suffice. LC-MS provides for polar compounds such as AAS glucuronides fast and highly sensitive method. The aim of this work was to develop and evaluate screening and confirmation LC-MS/MS method for twelve AAS glucuronides in urine samples. AAS glucuronides were extracted from urine samples by solid phase extraction (SPE). Extraction method, as well as the HPLC-separation method was optimised and validated. The detection is performed by using tandem mass spectrometer combined with electrospray ionisation (ESI). Novel LC-MS/MS methods were validated with respect to specificity, accuracy, precision and limit of detection. Recovery of the SPE extraction varied between 89-100 %. The quantitative repeatability varied within day from 2 to 10 % (relative standard deviation) and between days from 8 to 32 %. The repeatability of retention time was below 0.1% within day and below 1 % between days. Detection limits in urine were from lng/ml to 40ng/ml, which corresponds to 20-800ng/ml in injected solution. Detection limits in buffer were from 0.1 to 15ng/ml, which corresponds to 2-300ng/ml in injected solution. The results obtained show that the developed LC-MS method provides reliable method for the detection of AAS glucuronides. However, the limits of detection measured with standards prepared in pure solvent were one-two orders of magnitude lower than those obtained with urine samples. This shows that the selectivity of the method is limited due to endogenic compounds in urine samples. This is true in spite of use of tandem mass spectrometry. 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. Enzymatic synthesis methods for phase II metabolites of anabolic androgenic steroids Conjugation with glucuronic acid is the major conjugation reaction in all mammals. Various functional groups have the potential of reacting with glucuronic acid to form O-, S-, N-, and C-glucuronides, which means that a wide variety of compounds are metabolized via the glucuronidation pathway. Glucuronidation is a bimolecular nucleophilic substitution (SN2) reaction, which is catalyzed by uridine diphosphoglucuronosyltransferases (UGTs; Enzyme Classification E.C. 2.4.1.17) and uses uridine-5-diphosphoglucuronic acid (UDPGA) as the co-substrate. The reaction leads to the attachment of the polar sugar moiety to the substrate, which in the case of anabolic androgenic steroids (AAS) is phase-I transformed metabolite of the parent compound, with the immediate inversion of the configuration to yield a beta-glycosidic bond. As a result, these metabolic reactions in most cases terminate the activity of xenobiotics and endobiotics, including AAS. The main site of glucuronidation is the liver, although extra-hepatic glucuronidation has been observed in kidney, intestines, lung, and prostate. UGTs are a family of enzymes bound in the membrane of the endoplasmic reticulum, which catalyze the glucuronidation of various endogenous and exogenous compounds, including steroids. At least 16 different UGTs, ranging from 526 to 533 amino acids in size, are encoded by the human genome. The highly homologous carboxyl terminal is suggested to contain the domain critical for catalysis and for binding of UDPGA, whereas the amino terminal is responsible for the substrate specificity. The expressed UGT proteins have been categorized into two families (UGT1 and UGT2) on the basis of the protein sequence similarity, which is higher than 38% within a single family. According to the sequence homology, the enzyme families are further divided into subfamilies. The most important enzymes involved in steroid glucuronidation are members of subfamilies UGT1A and UGT2B. Scientific result of the enzyme-assisted synthesis offers an elegant and alternative method for the production of anabolic steroid glucuronides parallel to the conventional chemical synthesis pathway. The advantage of the enzyme-mediated synthesis route is the stereo-selectivity encountered in nature for biochemical catalyst, i.e. UGTs. Methods using rat liver preparations as a source of conjugating enzymes have been described for a structurally diverse group of substrates, e.g. p-nitrophenol, a series of nitrogen-containing antidepressants, nitrocatechols, as well as for endogenous steroids such as androsterone, androstanediol, dihydrotestosterone, epitestosterone, and testosterone. Within this project a representative group of anabolic steroids, either the parent steroids or their phase-I transformed metabolites were selected for the enzyme-assisted synthesis, having 3-alpha, 17-alpha, or 17-beta-oriented hydroxyl group(s) in various combinations available as potential glucuronidation sites. Arochlor-induced rat liver microsomal UGTs were used in the synthesis of AAS glucuronides and despite the differences between the conjugation activities, all the tested compounds were capable of glucuronidation in these experiments. Relative glucuronidation rates were higher with rat than human liver microsomal preparations, owing most probably to the enzyme-induction of the rat preparations. In general comparison the activities of liver preparations were significantly higher than those of the tested recombinant UGT isoenzymes, which may be at least partly explained by the better tolerance of tissue preparations to organic solvents in the synthesis mixture. Glucuronide-conjugated metabolites are needed as reference compounds in several fields of analytical research, such as in forensic, metabolic, and pharmaceutical laboratories. In many cases, especially with the new chemical entities, the lack of suitable commercial products represents a major problem in the research progress. Enzyme-assisted synthesis offers a practical pathway for the rapid production of small amounts of AAS glucuronides, such as needed in the build-up of an analytical method. Because the composition of the reaction mixture is relatively simple, and the differences in the optimal conditions for the various substrates are relatively minor, the addition of a new AAS substrate to the test compound set should be straightforward. And furthermore, the substrates are not limited only to AAS, but the methods can be applied to the first steps of conjugation studies also for the substrates with different structures. In future, an appropriate selection of UGT isoenzymes might be used as in vitro model to predict the glucuronidation reactions of a particular xenobiotic in the human body, but for that, the metabolic patterns should be examined in tissues to ensure the equivalence of the isoenzymes. Chemical synthesis methods for non-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. 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. In addition, reference material is essential for the development of analytical assays, regarding validation of procedures, determination of recoveries, reproducibility as well as uncertainties. Here, 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. With the knowledge of dissociation behaviour after ionization by either of the available techniques, identification of analytes is substantiated and supports the conviction, as well as the protection, of athletes. Moreover, the phase-I-metabolites are now conjugated to the corresponding phase-II-metabolites, which are basis for the development of new screening and confirmation procedures of intact glucuronides resulting from anabolic androgenic steroids. Searching for OpenAIRE data... There was an error trying to search data from OpenAIRE No results available
