Stanozolol administration combined with exercise leads to decreased telomerase activity possibly associated with liver aging.

Anabolic agents are doping substances which are commonly used in sports. Stanozolol, a 17α­alkylated derivative of testosterone, has a widespread use among athletes and bodybuilders. Several medical and behavioral adverse effects are associated with anabolic androgenic steroids (AAS) abuse, while the liver remains the most well recognized target organ. In the present study, the hepatic effects of stanozolol administration in rats at high doses resembling those used for doping purposes were investigated, in the presence or absence of exercise. Stanozolol and its metabolites, 16­ß­hydroxystanozolol and 3'­hydroxystanozolol, were detected in rat livers using liquid chromatography­mass spectrometry (LC­MS). Telomerase activity, which is involved in cellular aging and tumorigenesis, was detected by examining telomerase reverse transcriptase (TERT) and phosphatase and tensin homolog (PTEN) expression levels in the livers of stanozolol­treated rats. Stanozolol induced telomerase activity at the molecular level in the liver tissue of rats and exercise reversed this induction, reflecting possible premature liver tissue aging. PTEN gene expression in the rat livers was practically unaffected either by exercise or by stanozolol administration.

Inhibitory Effects of Diclofenac on Steroid Glucuronidation In Vivo Do Not Affect Hair-Based Doping Tests for Stanozolol.

In vitro studies show that diclofenac inhibits enzymatic steroid glucuronidation. This study was designed to investigate the influence of diclofenac on the excretion of stanozolol and 3'-hydroxystanozolol via analyses in hair, blood and urine in vivo in a rat study. Brown Norway rats were administered with stanozolol (weeks 1-3) and diclofenac (weeks 1-6). Weekly assessment of steroid levels in hair was complemented with spot urine and serum tests. Levels of both stanozolol and 3'-hydroxystanozolol steadily increased in hair during stanozolol treatment and decreased post-treatment, but remained readily detectable for 6 weeks. In contrast, compared to control rats, diclofenac significantly reduced urinary excretion of 3'-hydroxystanozolol which was undetectable in most samples. This is the first report of diclofenac altering steroid metabolism in vivo, detrimentally affecting detection in urine, but not in hair, which holds considerable advantages over urinalysis for anti-doping tests.

Sensitive detection of 3'-hydroxy-stanozolol glucuronide by liquid chromatography-tandem mass spectrometry.

Stanozolol is one of the most frequently detected anabolic steroids in doping control samples. This compound is metabolized to a large extent and its metabolites can be detected in urine much longer than the parent compound. The main stanozolol metabolites are excreted in urine as glucuronide conjugates and 3'-hydroxy-stanozolol glucuronide (3STANG) is one of the most important in human urine. Therefore enzymatic hydrolysis is usually applied prior to extraction. In this article a method for the sensitive detection of intact 3'-hydroxy-stanozolol glucuronide, by liquid chromatography tandem mass spectrometry, is described. The method takes advantage of an easy and fast sample preparation based on a single solid-phase extraction avoiding enzymatic hydrolysis or derivatization. It allows to detect stanozolol abuse in human urine at 25pgmL(-1). The method was validated according to Eurachem guidelines. The matrix effect, expressed as ion enhancement was +14%. The extraction recovery of the method was 93%. The limit of detection (LOD), whereby all WADA-criteria in chromatography and mass spectrometry are fulfilled, was determined at 50pgmL(-1). Application of the method to an excretion study revealed that the 3'-hydroxy-stanozolol glucuronide could be confirmed for 10 days after oral administration of 2mg of stanozolol, prolonging detection times compared to other metabolites and methodologies by almost 50%.

Use of in vitro technologies to study phase II conjugation in equine sports drug surveillance.

BACKGROUND: Within equine drug surveillance, there is significant interest in analyzing intact phase II conjugates of drugs in urine, but progress has been limited by a lack of reference material. METHOD: In this study, in vitro techniques using equine liver fractions were employed to produce glucuronide and sulfate conjugates of stanozolol, 16ß-hydroxystanozolol and nandrolone, the glucuronide conjugate of morphine and the glutathione metabolite of chlordinitrobenzene for the first time in equine sports drug surveillance. RESULTS: The glucuronide conjugate of the synthetic progestagen altrenogest was also produced in vitro, removing the requirement for sample hydrolysis during routine urinalyses. CONCLUSION: These results highlight the potential of in vitro studies for the production of phase II reference material, allowing the development of assays based on intact conjugates.

Analytical challenges in doping control: Comprehensive two-dimensional gas chromatography with time of flight mass spectrometry, a promising option.

Doping control screening based on the enhanced resolution of comprehensive two-dimensional (2D) gas chromatography hyphenated to time of flight mass spectrometer was investigated. The identification of anabolic agents (clenbuterol, norandrosterone, epimetendiol, two methyltestosterone metabolites and 3'-hydroxystanozolol) contained in a spiked urine sample (2ng/ml) was demonstrated. Special emphasis was given to 3'-hydroxystanozolol, mainly considering the difficulty in its detection. In contrast to conventional GC-MS approaches that must use single-ion monitoring, the GC x GC-TOFMS method enabled the identification of that metabolite through the deconvolution of the full mass spectrum and also resolved the co-eluted peaks of 3'-hydroxystanozolol and an endogenous component.

Simultaneous immunochemical detection of stanozolol and the main human metabolite, 3'-hydroxy-stanozolol, in urine and serum samples.

Two enzyme-linked immunosorbent assays (ELISAs) have been established for the analysis of stanozolol (St) and 3'-hydroxy-stanozolol (3'OH-St), the main metabolite found in humans. The immunizing hapten N2'-(5-valeric acid)-androst-2-eno[3,2-c]-pyrazol-17a-methyl-17b-ol (hapten 8) has been designed with the aid of molecular modeling and theoretical tools to allow immunochemical detection of both compounds. Using an ELISA based on a homologous antisera/coating antigen combination, St can be selectively quantified without significant interference of the St metabolites or other steroids potentially present in the biological samples. On the other hand, St immunoreactivity equivalents due to the additional presence of 3'OH-St can also be quantified using an ELISA based on a heterologous antisera/coating antigen combination, in which the metabolite can be detected with 51% cross-reactivity. Thus, As147/5BSA detects 3'OH-St and St in buffer with IC(50) values of 1.46 and 0.68 microg L(-1), respectively. In contrast, As147/8BSA is highly specific for St with an IC(50) of 0.16 microg L(-1) and a limit of dection of just 0.022 microg L(-1). Performance of both assays in urine and serum samples has been evaluated and demonstrate that inappropriate use of stanozolol by athletes or young people can be detected in these matrices after simple cleanup methods, with IC(50) values below the minimum performance required levels established by the World Antidoping Agency.

[Gas chromatography/tandem mass-spectrometry assay for trace amounts of 3'-hydroxystanozolol].

3'-Hydroxystanosolol detection in biological fluids at pg levels by gas chromatography/tandem mass spectrometry is described. Gas chromatography/high resolution mass spectrometry results can be confirmed with gas chromatography/tandem mass-spectrometry.

Mass spectrometry of stanozolol and its analogues using electrospray ionization and collision-induced dissociation with quadrupole-linear ion trap and linear ion trap-orbitrap hybrid mass analyzers.

Mass spectrometric identification and characterization of growth-promoting anabolic-androgenic steroids in biological matrices has been a major task for doping control as well as food safety laboratories. The fragmentation behavior of stanozolol, its metabolites 17-epistanozolol, 3'-OH-stanozolol, 4alpha-OH-stanozolol, 4beta-OH-stanozolol, 17-epi-16alpha-OH-stanozolol, 16alpha-OH-stanozolol, 16beta-OH-stanozolol, as well as the synthetic analogues 4-dehydrostanozolol, 17-ketostanozolol, and N-methyl-3'-OH-stanozolol, was investigated after positive electrospray ionization and subsequent collision-induced dissociation utilizing a quadrupole-linear ion trap and a novel linear ion trap-orbitrap hybrid mass spectrometer. Stable isotope labeling, H/D-exchange experiments, MS3 analyses and high-resolution/high mass accuracy measurements of fragment ions were employed to allow proposals for charge-driven as well as charge-remote fragmentation pathways generating characteristic product ions of stanozolol at m/z 81, 91, 95, 105, 119, 135 and 297 and 4-hydroxylated stanozolol at m/z 145. Fragment ions were generated by dissociation of the steroidal A- and B-ring retaining the introduced charge within the pyrazole function of stanozolol and by elimination of A- and B-ring fractions including the pyrazole residue. In addition, a charge-remote fragmentation causing the neutral loss of methanol was observed, which was suggested to be composed by the methyl residue at C-18 and the hydroxyl function located at C-17.

Use of ion trap gas chromatography-multiple mass spectrometry for the detection and confirmation of 3'hydroxystanozolol at trace levels in urine for doping control.

Stanozolol, a synthetic anabolic androgenic steroid, is often abused in sports to enhance performance. Consequently, the anti-doping laboratories daily screen for its metabolites (3'hydroxystanozolol and 4beta hydroxystanozolol) in all urines, mainly by GC-MS, after enzymatic hydrolysis and TMS derivatization. A sensitive and specific method by GC-MS(3) has been developed for the identification in urine of 3'hydroxystanozolol at trace levels. Full mass spectra and diagnostic ions are presented and a case report is commented. In this case, it was possible to attest the presence of a low concentration of stanozolol metabolite in a sample obtained from a competition test. This would have not been possible with other analytical techniques used in the laboratory. Through this case report, it was also possible to demonstrate the importance of sampling and the difficulties that has to face the laboratory when the pre-analytical step is not correctly performed.

First synthesis of a pentadeuterated 3'-hydroxystanozolol--an internal standard in doping analysis.

The first synthesis of 16,16,20,20,20-pentadeuterio-3'-hydroxystanozolol (8) in 26% yield over nine steps is described using moderately priced starting materials and economic amounts of reagents. Compound 8 can be used as an internal standard in screening procedures for anabolic steroids as well as for the quantification of stanozolol metabolites via mass spectrometric techniques, such as LC-MS or gas chromatography-mass spectrometry (GC-MS).

Photoaffinity labeling identification of thyroid hormone-regulated glucocorticoid-binding peptides in rat liver endoplasmic reticulum: an oligomeric protein with high affinity for 16beta-hydroxylated stanozolol.

Steroid-binding proteins unrelated to the classical nuclear receptors have been proposed to play a role in non-genomic actions of the17alpha-alkylated testosterone derivative (17alpha-AA) stanozolol (ST). We have previously reported that male rat liver endoplasmic reticulum contains two steroid-binding sites associated with high molecular mass oligomeric proteins: (1) the ST-binding protein (STBP); and (2) the low-affinity glucocorticoid-binding protein (LAGS). To further explore the role of LAGS on the mechanism of action of ST, we have now studied: (1) the interaction of ST and its hydroxylated metabolites with solubilized LAGS and the cytosolic glucocorticoid receptor (GR); and (2) the effects of hormones on the capability of STBP to bind ST. We found that, unlike 17alpha-methyltestosterone, neither ST nor its hydroxylated metabolites bind to GR. However, the 16beta-hydroxylation of ST significantly increases the capability of LAGS to bind ST. Interestingly, 3'-hydroxylation of ST abrogates the capability of LAGS to bind ST. ST (k(i)=30 nM) and 16beta-hydroxystanozolol (k(i)=13 nM) bind with high affinity to LAGS, and are capable of accelerating the rate of dissociation of previously bound dexamethasone from the LAGS. STBP and LAGS are strongly induced by ethinylestradiol. However, unlike STBP, LAGS is regulated by thyroid hormones and growth hormone, which proves that these steroid-binding activities are associated with different binding sites. These findings seem to suggest a novel mechanism for ST whereby membrane-associated glucocorticoid-binding activity is targeted by the 16beta-hydroxylated metabolite of ST. ST and its 16beta-hydroxylated metabolite modulate glucocorticoid activity in the liver through negative allosteric modulation of LAGS, with the result of this interaction an effective increase in classical GR-signaling by increasing glucocorticoid availability to the cytosolic GR.

N-isobutyloxycarbonylation for improved detection of 3'-hydroxystanozolol and its 17-epimer in doping control.

An improved screening method was developed for 3'-hydroxystanozolol and its 17-epimer in human urine involving gas chromatography-mass spectrometry (GC-MS) with N-isobutyloxycarbonyl (isoBOC) and O-trimethylsilyl (TMS) derivatization. A procedure was reported previously for the pentane extraction of many steroids from urine in doping control, but it was not suitable for the detection of stanozolol metabolites. Compared with the n-pentane extraction method, which gave a poor recovery (< 10%), isoBOC extraction resulted in a good recovery (> 80%). The sensitivity and specificity of mixed N-isoBOC-O-TMS derivatization were adequate for the detection of 3'-hydroxystanozolol and its 17-epimer when 3 ml of urine was used with spiking at a level of 2 ng ml-1. When applied to a stanozolol-positive urine sample, the proposed method allowed rapid and sensitive screening for the detection of 3'-hydroxystanozolol and its 17-epimer.

Determination of 16beta-hydroxystanozolol in urine and faeces by liquid chromatography-multiple mass spectrometry.

This paper describes the optimisation of the detection of stanozolol and its major metabolite 16beta-hydroxystanozolol in faeces and urine from cattle. Faeces are extracted directly with diisopropyl ether. Urine is first submitted to an enzymatic hydrolysis and then extracted over a modified diatomaceous earth column (Chem-Elut) with a mixture of diisopropyl ether-isooctane. In a final step an acidic back extraction is performed. For the LC-MS-MS detection two approaches are discussed. In a first approach the final extract is detected without derivatization, while the second approach makes use of a derivatization step for 16beta-hydroxystanozolol. While the MS-MS spectrum without derivatization exhibits extensive fragmentation, the spectrum of the derivative shows two abundant diagnostic ions with much more reproducible ion ratios. The derivatization method and the method without derivatization enable the detection of 16beta-hydroxystanozolol up to 0.03 microg l(-1) in urine and 0.07 microg kg(-1) in faeces. Until now there is no literature available for the detection of 16beta-hydroxystanozolol in faeces and urine at the ppt level.

Multi-laboratory study of the analysis and kinetics of stanozolol and its metabolites in treated calves.

The European Union banned the use of anabolic steroids for cattle fattening in 1988. Analytical techniques able to detect trace amounts of the parent drugs and their metabolites are mandatory for the control of abuse. Stanozolol (Stan) is an anabolic steroid that is often found in injection sites and cocktails. However, it has never been detected in tissues (kidney fat, meat) or excreta (urine, faeces) taken during regulatory inspection. The difference between the structure of Stan and the other steroids (a pyrazole ring fused to the androstane ring system) is probably the cause of this phenomenon. In the multi-laboratory study described here, veal calves were treated with intramuscular doses of Stan. In the excreta of these calves the presence, absence and/or concentration of Stan and of its major metabolites 16 beta-hydroxystanozolol and 3'-hydroxystanozolol were determined. For the determination of these analytes the different laboratories used different extraction and clean-up procedures and also evaluated different analytical techniques such as GC-MS (negative chemical ionization) and LC-MS-MS. The aim of this investigation was to explore which analyte should be validated for veterinary inspection purposes.

Gas chromatographic-mass spectrometric identification of main metabolites of stanozolol in cattle after oral and subcutaneous administration.

An analytical method has been developed in order to control the illegal use of stanozolol as growth promoter in livestock. The procedure was based on enzymatic hydrolysis, purification on a Clean Screen DAU column and derivatization with heptafluorobutyric anhydride prior to GC-MS analysis. This method allowed us to study the metabolism of stanozolol in cattle after oral and subcutaneous administrations. Urinary metabolites were identified by mass spectrometry. Stanozolol and 16-hydroxystanozolol were detected after oral administration, while 16-hydroxystanozolol and 4,16-dihydroxystanozolol were found after subcutaneous administration.