Detection Of Anabolic Androgenic Steroids Research Articles

Effects of new anti-doping measures on sports performance in elite female athletes.

Studies examining the effects of new anti-doping measures on sports performance in elite athletes are scarce. During the last decade, a number of new anti-doping methods for the detection of anabolic androgenic steroids were developed. We hypothesized that the implementation of these methods may cause a decrease of performance in athletic disciplines where given substances are widely used. For this purpose, the performance results for 10 athletic disciplines from country (Country A), which was widely accused of systematic doping use, were gathered and pooled into the 2012-2015 and 2016-2019 periods. These periods were seen as before and following the implementation periods, where the effects of new anti-doping methods could be manifested. Also, the performance results of their rival athletes from Country B were analyzed for comparison. The incidence of anti-doping rule violations for both countries was followed. The analysis revealed a decrease in performance results of Country A athletes in eight of 10 disciplines. The performance results of Country B's athletes in seven of 10 disciplines did not and in three disciplines did demonstrate statistically significant change. The decrease in performance of Country A athletes followed a high incidence of steroid detection cases by means of new anti-doping methods. It is highly likely that the consequent performance decrease is due to the implementation of new anti-doping methods. The results of the study bring new facts on the effects of new anti-doping methods implementation and demonstrate following decrease in performance results in population of athletes from the country widely accused of doping use.

Detection time comparison of non-hydrolysed sulphated metabolites of metenolone, mesterolone and 17α-methyltestosterone analysed by four different mass spectrometric techniques.

The frequent detection of anabolic androgenic steroids (AAS) indicates their popularity among rule-breaking athletes. The so called long-term metabolites play a crucial role in their detection, and non-hydrolysed sulphated metabolites have gained renewed interest, as research has demonstrated their extended detection time compared to the more conventional markers (e.g., for metenolone and mesterolone). Their potential has been investigated using liquid and gas chromatography-mass spectrometry (LC- and GC-MS). However, due to their complementary nature, chances are that the most promising metabolite on one technique does not necessarily exhibit the same behaviour on the other and vice versa. Therefore, a comparison was carried out where as a trial model, metenolone, mesterolone and 17α-methyltestosterone were selected and the most likely long-term sulphated metabolites identified on four mass spectrometric instruments. Additionally, using a modified sample preparation procedure, comparison between conventional and non-hydrolysed sulphated metabolites between different GC-MS instruments was also included. When focusing on each individual marker, no cases were observed where a single metabolite provided a superior detection time on all instruments. Furthermore, for each AAS, there were incidences where a metabolite provided the best detection time on one instrument but could only be detected for a shorter period or not at all on other instruments. This demonstrates that metabolite detection windows and hence their added-value as target substance are unique and dependent on the analytical technique and not only on their pharmacokinetic behaviour. Consequently, in each case, a metabolite versus instrument evaluation is needed to maximise the probabilities of detecting doping offences.

Detection of anabolic androgenic steroids in serum samples.

When testing for anabolic androgenic steroids (AAS) outside sports communities, for example, in healthcare and forensic medicine, urine is the matrix of choice. However, there are drawbacks with urinary sampling, and serum might be useful as a complementary matrix. The aim was to develop an LC-MS/MS method for serum measuring AAS frequently used outside of sport, including testosterone (T), steroid esters, and eight other synthetic AAS. The sample pretreatment included sample precipitation and evaporation. Limit of quantification for the AAS was 0.05-0.5ng/mL, and linearity was 0.05-20 ng/mL for most of the substances. Generally, the within- and between-day CV results, matrix effect, and process efficiency were <15%. The AAS were stable for at least 6months at -20°C. Serum samples were obtained from previous studies. A novel finding from an administration study was that T enanthate was present in serum even after 5years of storage at -20°C. Serum samples from self-reporting AAS individuals, where T esters were detected, were positive for testosterone using the urinary testosterone/epitestosterone criterion >10. Of those identified as positive in traditional urinary doping tests (n = 15), AAS in serum were found in 80% of the subjects. Our results show that serum may be a valid complementary matrix to urine samples for AAS testing.

Toward Routine Analysis of Anabolic Androgenic Steroids in Urine Using Ion Mobility-Mass Spectrometry.

Anabolic androgenic steroids (AAS) make up one of the most prevalent classes of performance-enhancing drugs banned by the World Anti-Doping Agency (WADA) due to the competitive advantage they can afford athletes. Mass spectrometry-based methods coupled with chromatographic separations have become the gold standard for AAS analysis because of the superior sensitivity and selectivity provided. However, emerging analytical techniques including ion mobility spectrometry (IMS) have been demonstrated in recent applications as a means to further characterize and identify potential unknowns while simultaneously delivering improved sensitivity by filtering noise. Herein we outline the next crucial steps in bringing IMS to the routine drug testing workflow by combining it with established chromatographic and mass spectrometry methods (i.e., LC-IM-MS) for the detection of AAS in human urine. In addition to robust measurement of collision cross sections which can be used for identification purposes, functional group microtrends provide a structural basis on which to elucidate the structure of future novel anabolic agents. Lastly, the developed workflow is tested by analysis of testosterone in a realistic matrix (human urine) and demonstrates a limit of detection of 524 pg/mL, which surpasses the WADA Minimum Required Performance Levels for anabolic steroids. This work is expected to pave the way toward routine incorporation of IMS into analytical drug testing workflows to augment both qualitative and quantitative measure of performance enhancing drugs in the future.

Tailor-Made Immunochromatographic Test for the Detection of Multiple 17α-Methylated Anabolics in Dietary Supplements

In recent years, the undeclared presence of various anabolic androgenic steroids (AAS) in commercial supplements has been confirmed. This fact can be a potential threat to all athletes using these supplements, and therefore, there is of increased interest in the implementation of rapid methods for the detection of AAS. The presented study describes the development of an immunostrip test for the detection of multiple 17α-methylated AAS based on direct and indirect competitive principle using gold nanoparticles as a label. As a capture reagent on test lines conjugated stanazolol to rabbit serum albumin (RSA/ST-3) was used, the intensity of color formed in the test line of the AAS-positive sample was visually distinguishable from that of negative sample within 10 min. The optimized closed direct and indirect format of the test provided a similar visual detection limit (0.7 and 0.9 ng/mL, respectively). The most commonly orally abused AAS (17α-methyltestosterone, methandienone, methyldihydrotestosterone, oxandrolone and oxymetholone) showed a strong cross-reaction. Developed immunostrips were successfully applied to analysis of artificially contaminated dietary supplements with 17α-methylated AASs. The developed immunostrips offer potential as a useful user-friendly method for capturing suspicious dietary supplement samples with different contents of AAS at levels far below the usually used concentrations of AAS.

Bioanalytical Detection of Steroid Abuse in Sports Based on the Androgenic Activity Measurement

The anabolic androgenic steroids (AAS) are the most frequently consumed performance enhancing drugs (PED) in sports. In the anti-doping field, the detection of AAS is carried out by the analysis of the athlete’s urine using methodologies based on liquid/gas chromatography-mass spectrometry. Unfortunately, the detection of unknown compounds is not possible. BDS’s AR CALUX® bio detection technology was studied as an indirect method to detect the administration of a single dose of testosterone (T). Twelve T and placebo single dose administered men volunteers underwent a triple-blind crossover clinical trial. The UGT2B17 deletion was present among the volunteers and evenly distributed in heterozygous (ins/del), wild-type homozygous (ins/ins), and mutated homozygous (del/del) groups. A significant statistical difference in terms of bioluminescence was observed after the testosterone (T) administration for the three types of polymorphic groups. The ratio of means between the pre- and post-T administration periods, depending on the type of polymorphism, was in group ins/ins 3.31 (CI. 95%: 2.07–5.29), group ins/del 4.15 (CI 95%: 3.05–5.67), and group del/del 2.89 (CI 95%: 2.42–3.46). The results of the study are very promising, as they may offer us the possibility of designing a detection approach that, based on intra-individual monitoring of androgenic values, in the UGT2B17 deletion type.

RNA sample preparation applied to gene expression profiling for the horse biological passport.

The improvement of doping control is an ongoing race. Techniques to fight doping are usually based on the direct detection of drugs or their metabolites by analytical methods such as chromatography hyphenated to mass spectrometry after ad hoc sample preparation. Nowadays, omic methods constitute an attractive development and advances have been achieved particularly by application of molecular biology tools for detection of anabolic androgenic steroids (AAS), erythropoiesis-stimulating agent (ESA), or to control human growth hormone misuses. These interesting results across different animal species have suggested that modification of gene expression offers promising new methods of improving the window of detection of banned substances by targeting their effects on blood cell gene expression. In this context, the present study describes the possibility of using a modified version of the dedicated Human IVD (in vitro Diagnostics) PAXgene® Blood RNA Kit for horse gene expression analysis in blood collected on PAXgene® tubes applied to the horse biological passport. The commercial kit was only approved for human blood samples and has required an optimization of specific technical requirements for equine blood samples. Improvements and recommendations were achieved for sample collection, storage and RNA extraction procedure. Following these developments, RNA yield and quality were demonstrated to be suitable for downstream gene expression analysis by qPCR techniques. Copyright © 2017 John Wiley & Sons, Ltd.

Targeted Metabolomics Approach To Detect the Misuse of Steroidal Aromatase Inhibitors in Equine Sports by Biomarker Profiling.

The use of anabolic androgenic steroids (AAS) is prohibited in both human and equine sports. The conventional approach in doping control testing for AAS (as well as other prohibited substances) is accomplished by the direct detection of target AAS or their characteristic metabolites in biological samples using hyphenated techniques such as gas chromatography or liquid chromatography coupled with mass spectrometry. Such an approach, however, falls short when dealing with unknown designer steroids where reference materials and their pharmacokinetics are not available. In addition, AASs with fast elimination times render the direct detection approach ineffective as the detection window is short. A targeted metabolomics approach is a plausible alternative to the conventional direct detection approach for controlling the misuse of AAS in sports. Because the administration of AAS of the same class may trigger similar physiological responses or effects in the body, it may be possible to detect such administrations by monitoring changes in the endogenous steroidal expression profile. This study attempts to evaluate the viability of using the targeted metabolomics approach to detect the administration of steroidal aromatase inhibitors, namely androst-4-ene-3,6,17-trione (6-OXO) and androsta-1,4,6-triene-3,17-dione (ATD), in horses. Total (free and conjugated) urinary concentrations of 31 endogenous steroids were determined by gas chromatography-tandem mass spectrometry for a group of 2 resting and 2 in-training thoroughbred geldings treated with either 6-OXO or ATD. Similar data were also obtained from a control (untreated) group of in-training thoroughbred geldings (n = 28). Statistical processing and chemometric procedures using principle component analysis and orthogonal projection to latent structures-discriminant analysis (OPLS-DA) have highlighted 7 potential biomarkers that could be used to differentiate urine samples obtained from the control and the treated groups. On the basis of this targeted metabolomic approach, the administration of 6-OXO and ATD could be detected for much longer relative to that of the conventional direct detection approach.

Analysis of anabolic androgenic steroids by direct analysis in real time ionization with time-of-flight mass spectrometry

We verified applicability of direct analysis in real time ionization coupled with a time-of-flight mass spectrometer (DART-MS) for detection of anabolic androgenic steroids in supplements and pharmaceuticals. DART-MS was optimized for the analysis of eleven frequently misused anabolic androgenic steroids and their derivatives. Real supplement tablets and injectable oil solutions were used to verify applicability of the method. It was found that anabolic androgenic steroids can be analyzed directly without any sample pre-treatment, which reduces the sample preparation time and hence the overall analysis time. The resulting DART-MS method is suitable for rapid routine qualitative screening of anabolic androgenic steroids in simple sample matrices. It has a great potential in the field of adulterated and counterfeited anabolic steroid pharmaceuticals and supplements.

Mass spectrometric behavior of anabolic androgenic steroids using gas chromatography coupled to atmospheric pressure chemical ionization source. Part I: ionization.

The detection of anabolic androgenic steroids (AAS) is one of the most important topics in doping control analysis. Gas chromatography coupled to (tandem) mass spectrometry (GC-MS(/MS)) with electron ionization and liquid chromatography coupled to tandem mass spectrometry have been traditionally applied for this purpose. However, both approaches still have important limitations, and, therefore, detection of all AAS is currently afforded by the combination of these strategies. Alternative ionization techniques can minimize these drawbacks and help in the implementation of a single method for the detection of AAS. In the present work, a new atmospheric pressure chemical ionization (APCI) source commercialized for gas chromatography coupled to a quadrupole time-of-flight analyzer has been tested to evaluate the ionization of 60 model AAS. Underivatized and trimethylsylil (TMS)-derivatized compounds have been investigated. The use of GC-APCI-MS allowed for the ionization of all AAS assayed irrespective of their structure. The presence of water in the source as modifier promoted the formation of protonated molecules ([M+H](+)), becoming the base peak of the spectrum for the majority of studied compounds. Under these conditions, [M+H](+), [M+H-H2O](+) and [M+H-2·H2O](+) for underivatized AAS and [M+H](+), [M+H-TMSOH](+) and [M+H-2·TMSOH](+) for TMS-derivatized AAS were observed as main ions in the spectra. The formed ions preserve the intact steroid skeleton, and, therefore, they might be used as specific precursors in MS/MS-based methods. Additionally, a relationship between the relative abundance of these ions and the AAS structure has been established. This relationship might be useful in the structural elucidation of unknown metabolites.

Multiplexed immunoassay to detect anabolic androgenic steroids in human serum

A multianalyte enzyme-linked immunosorbent assay (ELISA) has been developed for the simultaneous detection of anabolic androgenic steroids (AAS) in human serum. The multiplexed method was developed according to a planar strategy in which the analytes are identified by their location in the microtiter plate. In the immunochemical procedure established here, human serum samples are mixed with a cocktail of antibodies and added to the distinct sections of a microplate biofunctionalized with different haptenized biomolecules. The cocktail of antibodies consists of a mixture of polyclonal antibodies raised against stanozolol (ST), boldenone (B), and tetrahydrogestrinone (THG). The whole immunochemical analytical procedure takes around 2 h including sample preparation, and many samples can be processed simultaneously to screen for the presence of the three AAS in a single run. Using this ELISA, ST, B, and THG can be detected and quantified individually. When used as a screening method, due to the cross-reactivity profiles of the immunoreagents used, the presence of up to 11 AAS can be detected simultaneously. The detectabilities achieved by this method in human serum are below the MRPLs (minimum required performance limits) proposed by WADA (World Anti-Doping Agency) and reference laboratories of the European Community.

Two-Step Derivatization Procedures For The Ionization Enhancement of Anabolic Steroids in Lc–Esi-Ms For Doping Control Analysis

Two-step derivatization procedures were developed for the enhancement of the positive ESI in LC-MS detection of anabolic androgenic steroids, a class of prohibited substances with limited ionization efficiency in atmospheric pressure interfaces. The developed procedures are based on the esterification of hydroxyl groups of anabolic steroids with picolinic acid, followed by conversion of carbonyl groups to Schiff bases by either Girard's reagent T or 2-hydrazino pyridin. Ionization efficiency for the model derivatized compounds 19-norandrosterone (nandrolone main metabolite) and methasterone was higher by almost two orders of magnitude compared with the respective efficiency of the underivatized compounds. The obtained derivatives provided a significant improvement in the ESI sensitivity, compared with those of underivatized molecules in positive LC-ESI-ion trap-MS full-scan mode.

Detection of anabolic androgenic steroid abuse in doping control using mammalian reporter gene bioassays

Anabolic androgenic steroids (AAS) are a class of steroid hormones related to the male hormone testosterone. They are frequently detected as drugs in sport doping control. Being similar to or derived from natural male hormones, AAS share the activation of the androgen receptor (AR) as common mechanism of action. The mammalian androgen responsive reporter gene assay (AR CALUX ® bioassay), measuring compounds interacting with the AR can be used for the analysis of AAS without the necessity of knowing their chemical structure beforehand, whereas current chemical–analytical approaches may have difficulty in detecting compounds with unknown structures, such as designer steroids. This study demonstrated that AAS prohibited in sports and potential designer AAS can be detected with this AR reporter gene assay, but that also additional steroid activities of AAS could be found using additional mammalian bioassays for other types of steroid hormones. Mixtures of AAS were found to behave additively in the AR reporter gene assay showing that it is possible to use this method for complex mixtures as are found in doping control samples, including mixtures that are a result of multi drug use. To test if mammalian reporter gene assays could be used for the detection of AAS in urine samples, background steroidal activities were measured. AAS-spiked urine samples, mimicking doping positive samples, showed significantly higher androgenic activities than unspiked samples. GC–MS analysis of endogenous androgens and AR reporter gene assay analysis of urine samples showed how a combined chemical–analytical and bioassay approach can be used to identify samples containing AAS. The results indicate that the AR reporter gene assay, in addition to chemical–analytical methods, can be a valuable tool for the analysis of AAS for doping control purposes.