Synthetic Anabolic Steroid Research Articles

Exploring the potential of using ion mobility-mass spectrometry to separate matrix interferences from analytes in food control

During residue analysis in complex matrices for food safety purposes, interfering signals can sometimes overlap with those of the analyte of interest. Access to an additional separation dimension besides chromatographic and mass separation, such as ion mobility, can aid in removing interfering signals, allowing for correct analyte identification in these cases. In our laboratory, during routine LC−MS/MS analysis of liver samples for growth promoter residues, an interfering signal was found that matches the retention time and m/z values for stanozolol, a synthetic anabolic steroid. In the present work, the performance of a liquid chromatography coupled to ion mobility mass spectrometry (LC−IM−MS) method has been evaluated to study whether this LC−MS/MS false positive in liver samples could be eliminated by LC−IM−MS analysis. A cyclic ion mobility system already allowed the separation of stanozolol from the interfering peak after only one pass, showing a significant improvement compared to the conventional LC−MS/MS method. Additionally, collisional cross section (CCS) values were calculated and successfully compared with those from literature for identification purposes, eventually allowing both the identification and quantification of stanozolol in this complex matrix.

Blood plasma proteins and the content of metabolites of carbohydrate and lipid metabolism in the hepatopancreas of fish during the action 19-nortestosterone

The aim: study the effect of the synthetic anabolic steroid 19-nortestosterone on the fractional composition of blood plasma proteins, as well as the total protein and albumin content, indicators of the metabolism of carbohydrates, lipids, macro- and microelements in the fish hepatopancreas. Materials and methods. The experiments were conducted on two-year-old carp (Cyprinus carpio L.), which were kept for 24 hours in aquariums with a volume of 40 dm3 with different concentrations of 19-nortestosterone in water at optimal temperature, oxygen content, and pH. Spectrophotometric methods and an automatic biochemical analyzer were used to determine metabolic indicators in fish, and disk electrophoresis in PAGE was used to determine the fractional composition of proteins. Results. It was established that the steroid hormone 19-nortestosterone, the concentration of which in the water of the fish of the experimental groups was 50 and 200 μg/dm3, respectively, increased the level of total protein and albumin, the concentration of glucose, triglycerides, creatinine, inorganic phosphorus and iron in the hepatopancreas and did not affect the calcium content . In the blood plasma of carp, kept in water with a concentration of 19-nortestosterone of 50 μg/dm3, the content of proteins with a molecular weight of 240-231 increases; 215-179; 169-146 and 105-95 kDa, decreases - 35-33 kDa, fractions of proteins with a molecular weight of 228-220 appear; 116-105 and 48-44 kDa. An increase in the concentration of 19-nortestosterone in water to 200 μg/dm3 increased the content of proteins with a molecular weight of 240-231 and 215-179 kDa in the blood plasma of fish, and decreased it by 35-33 kDa, causing the redistribution of individual protein fractions due to the appearance of proteins with a molecular weight of 240 -228; 116-105 and 48-44 kDa in the absence of proteins 42-41; 38-36 and 24-20 kDa. Conclusions. Based on the obtained results, it was concluded that the anabolic synthetic steroid 19-nortestosterone stimulates the biosynthesis of proteins, the metabolism of carbohydrates and lipids, and its effect on the content of minerals in the hepatopancreas and the fractional composition of blood plasma proteins in carp fish was proven

Severe Drug-Induced Liver Injury in the Military: A Retrospective Review.

Drug-induced liver injury (DILI) is a significant cause of morbidity and mortality. Establishing a diagnosis is challenging due to the broad differential diagnosis of liver injury. We retrospectively reviewed patients with severe idiosyncratic DILI at Walter Reed National Military Medical Center in order to define the scope and patterns of injury in the military population. Using the military health database, we identified a total of 110 patients who had an International Classification of Disease (ICD)-10 code for toxic liver injury in the electronic medical record at Walter Reed National Military Medical Center between 2016 and 2019. Each patient record was reviewed, and all pertinent data for included patients were recorded into a database for analysis. Twenty-seven out of 110 patients with a diagnostic code for toxic liver injury met inclusion criteria for severe idiosyncratic DILI. Nine cases were caused by supplements, including 5 active duty service members using synthetic anabolic steroids or preworkout supplements. The majority of patients were men and one-third were serving on active duty. The ranges of liver enzyme elevation and patterns of liver injury widely varied. Military service members are at particularly high risk for DILI given the frequent use of over-the-counter and other unregulated strength- and performance-enhancing supplements. These injuries not only have significant medical consequences but can profoundly impact military readiness and mission capability. Diagnosis of DILI among active duty service members requires a strong index of suspicion, and inquiry regarding all ingestions is crucial. Educating physicians, providers, and policy makers on the risks of supplement-induced liver injury among service members is crucial. These data will facilitate additional studies exploring susceptibility to severe idiosyncratic DILI among the military population.

The Meat of the Matter: Environmental Dissemination of Beef Cattle Agrochemicals.

Global demand for beef is projected to increase into the foreseeable future (Smith et al., 2018). This article is protected by copyright. All rights reserved.

Protective effect of nanoencapsulated curcumin against boldenone-induced testicular toxicity and oxidative stress in male albino rats

Background Boldenone (BOL) (Equigan) is a synthetic anabolic steroid used mainly by veterinarians to treat and promote horses’ growth. Recently, body builders have started to use it to enhance their physical performance and muscle growth. Equigan is known to induce testicular injury and reduce fertility in males. Current treatments for reduced fertility are very costly. As alternatives, people are investigating naturally occurring bioactive compounds in plants such as curcumin. Objective This study was conducted to evaluate the prophylactic antioxidant effects of nanoencapsulated curcumin (NEC) on BOL-induced testicular toxicity and oxidative stress in male albino rats. Materials and methods NEC was prepared using a novel freeze-drying method. For their characterization, ultraviolet–visible spectrophotometry, transmission electron microscopy, and dynamic light scattering were used. Four groups of male rats were used: the first group served as control, the second group received NEC (100 mg/kg orally, once daily), the third group received BOL (5 mg/kg intramascular, once weekly) for 60 days, and BOL and NEC were concurrently administered in the fourth group. Blood was withdrawn from the rats’ retro-orbital veins 24 h after treatment. Animals were euthanized immediately; the epididymal sperm reserve was separated. Then, one testis from each rat was kept at −80°C for determination of oxidative stress indices, and the other was fixed in 10% formalin solution for histopathological investigation. Results and conclusion Treatment with BOL resulted in significant reproductive damage caused by increased levels of malondialdehyde and nitric oxide and decreased levels of superoxide dismutase and reduced glutathione. Downregulation of the levels of serum testosterone and reduction in semen quantity, sperm count, and motility were also detected in the BOL group. Histopathological examinations showed severe degenerative changes in the testes. Immunohistochemical examination indicated severe reduction in the proliferating cell nuclear antigen-positive spermatogonia in the BOL-treated group as compared with the control. Coadministration of NEC with BOL effectively reduces BOL-induced testicular damage and oxidative stress in male albino rats.

Improving the detection of anabolic steroid esters in human serum by LC–MS

The detection of the abuse of pseudo-endogenous steroids in sport is articulated in two different levels: an initial testing procedure, based on the longitudinal evaluation of the urinary androgenic steroid profile by gas-chromatography mass spectrometry (GC–MSn), and a confirmation analysis, based on the differentiation between the endogenous and exogenous origin of the pseudo-endogenous steroids by gas-chromatography coupled to isotopic ratio mass spectrometry (GC/C/IRMS). The abuse of pharmaceutical preparations displaying a carbon isotopic composition values within a range similar to those reported for endogenous urinary steroids makes more difficult the application of GC/C/IRMS technique. To overcome this limitation, the direct detection of an intact synthetic anabolic steroid ester in blood matrices (plasma and/or serum) could supply the unequivocal proof of exogenous administration of pseudo-endogenous steroids. Here we are presenting a liquid chromatography tandem mass spectrometry (LC–MS/MS) method for the analysis of 14 testosterone (T) esters and 2 nandrolone (Nand) esters in human serum. Sample pre-treatment consisted of protein precipitation, liquid-liquid extraction and derivatization. The formation of three different derivatives (oxime derivatives, Girard P and Girard T hydrazones) is considered, in order to guarantee an improvement in the detection capability of the assay with respect to underivatized compounds. Once the most suitable derivative was selected, the method was validated, according to the World Anti-Doping Agency (WADA) criteria, in terms of specificity, linearity, limit of detection (LOD), extraction recovery, matrix effect (ion suppression/enhancement), carry over and autosampler stability.The formation of Girard P hydrazones of T and Nand esters provides the best results compared to the underivatized compounds, oxime and Girard T derivatives, respectively. The presented analytical method is specific for all considered compounds and linear in the range of concentrations investigated (0.25–10 ng/mL). The LODs are between 0.03 and 0.30 ng/mL, the extraction recovery higher than 70 % for all esters and no remarkable matrix effect, expressed in terms of ion enhancement and ion suppression, was observed.Finally, the developed and validate method was applied in the analysis of serum samples collected after the administration of a single dose (40 mg, 1 capsule) of testosterone undecanoate (Andriol ®) demonstrating its applicability.

SUN-LB12 Prenatal Trenbolone Exposure and External Genital Malformation: A Case Report

Background Trenbolone is a synthetic anabolic steroid used for muscle growth in livestock. Human use is not FDA approved. The active metabolite, 17β trenbolone is 3 times more potent an androgen than testosterone propionate. Prenatal trenbolone exposure in rats results in female offspring with external genital malformations including an increased anogenital distance (AGD). We now report a case of prenatal trenbolone exposure in humans. Clinical Case A 10 hour old baby delivered vaginally at 37 6/7 weeks of gestation with a birth weight of 3615 grams was admitted to the NICU for concern of ambiguous genitalia. Baby was born to 30 year old G6P3 mother who was HIV positive and adequately treated with Triumeq. She had irregular cycles prior to conception and pregnancy recognized at 14 weeks. Unlike her previous pregnancies, during this pregnancy she had acne and mildly increased facial hair. Prenatal cell-free DNA screening was 46XX and ultrasound showed female genitalia. The 9 year old half-sister and 5 year old brother are healthy. No family history of early neonatal deaths. On arrival blood glucose was 55 mg/dL (45-90). On exam baby had no obvious facial dysmorphic features or midline defects. Hand creases were normal and no extremity edema was noted. Genitalia appeared symmetrical and, showed prominent soft tissue of clitoris without corpus enlargement. No gonads were palpable in the groin or labioscrotal folds. Significant posterior labioscrotal fusion was noted. Anogenital ratio was 0.8 (normal <0.5). A single orifice at the base of the clitoral hood was noted, consistent with likely urogenital sinus. Additional history provided by mother included her disclosure of use of 200 mg of IM trenbolone weekly for 5 weeks, for muscle building, prior to her knowledge of being pregnant. Multidisciplinary care included pediatric endocrinology, genetics, pediatric urology, and pediatric infectious disease. Labs at 24 hours of life: Sodium - 139 mmol/L (131-144), Potassium - 5.3 mmol/L (3.2-5.7), Serum glucose - 75 mg/dL (45-90), Random cortisol - 34.1 mcg/dL (1.0-10.0), 17 hydroxyprogesterone - 124 ng/dL (<460), CAH diagnostic panel - normal with exception of elevation in Progesterone - 3400 ng/dL (5-53), Newborn screening - normal, Chromosomal analysis - 46,XX and absent SRY, Ultrasound pelvis - normal Müllerian structures and bilateral intra-abdominal gonads present. Baby was hemodynamically stable, voided well, gained weight by discharge on DOL 4. Baby was stable on follow-up in endocrinology clinic on DOL 10. Surgical repair of anatomic malformation to be performed at 6 months of age by urology team. Conclusion To our knowledge, this is the first report of virilization of female offspring following human prenatal exposure to trenbolone. Women must be cautioned that trenbolone poses risks of external genital malformation to the developing fetus.

Effect of exercise, synthetic anabolic steroids and protein intake on DNA damage in trained and untrained men

BackgroundThe role of exercise in oxidative stress and genomic damage was evaluated as the main target population of ASS and protein supplement in usually physical active individuals. Therefore, the purposes of this study were to determine the effects of exercise and abuse of synthetic anabolic steroids and food supplements on micronuclei frequency in buccal cells and 8-hydroxy-2-deoxyguanosine (8-OHdG) as markers for DNA damage. MethodsThe study included one hundred males aged 17–44 years, distributed into five distinct groups including Group I: 20 bodybuilder individuals with AAS consumption, Group II: 20 bodybuilder individuals with diet supplement (proteins) consumption. Group III: 20 bodybuilders without AAS and diet supplement (proteins) consumption, Group IV: 20 football players without AAS and proteins consumption, and Group V 20 non-athletes without diet supplement or AAS consumption. ResultsThe mean age + standard deviation (SD) of groups I, II, III, IV and V were (27.00 ± 5.53, 27.85 ± 5.60, 29.70 ± 8.04, 22.35 ± 3.89 and 26.05 ± 6.42, respectively. The mean of BMI values was significantly different among groups (p < .0001), being much higher for the group I (26.77 ± 1.64) than for group V (22.78 ± 1.53***). Notably, group I and II exhibited elevation in 8-OHdG concentration (3.19 ± 0.35 and 3.28 ± 1.66, respectively) compared to those from the other groups, not being statistically significant. In addition no statically difference was observed according to frequency of micronuclei in buccal cells. ConclusionOur results outlined that exceeded usage of AAS or food supplement (proteins) exerted no increasing effects of oxidative DNA and/or chromosomal aberration accounting for serum 8-OHdG and micronuclei in buccal cells. These are possibly due to the adaptive responses such as up regulation of DNA repair systems and enhancement of the anti-oxidant capacity in highly-trained individuals.

Separation of Structurally Similar Anabolic Steroids as Cation Adducts in FAIMS-MS

Novel synthetic anabolic androgenic steroids have been developed not only to dodge current antidoping tests at the professional sports level, but also for consumption by noncompetitive bodybuilders. These novel anabolic steroids are commonly referred to as "designer steroids" and pose a significant risk to users because of the lack of testing for toxicity and safety in animals or humans. Manufacturers of designer steroids dodge regulation by distributing them as nutritional or dietary supplements. Improving the throughput and accuracy of screening tests would help regulators to stay on top of illicit anabolic steroids. High-field asymmetric-waveform ion mobility spectrometry (FAIMS) utilizes an alternating asymmetric electric field to separate ions by their different mobilities at high- and low-fields as they travel through the separation space. When coupled to mass spectrometry (MS), FAIMS enhances the separation of analytes from other interfering compounds with little to no increase in analysis time. Here we investigate the effects of adding various cation species to sample solutions for the separation of structurally similar or isomeric anabolic androgenic steroids. FAIMS-MS spectra for these cation-modified samples show an increased number of compensation field (CF) peaks, some of which are confirmed to be unique for one steroid isomer over another. The CF peaks observed upon addition of cation species correspond to both monomer steroid-cation adduct ions and larger multimer ion complexes. Notably, the number of CF peaks and their CF shifts do not appear to have a straightforward relationship with cation size or electronegativity. Future directions aim at investigating the structures for these analyte-cation adduct ions for building a predictive model for their FAIMS separations.

Combined effects of exercise training and high doses of anabolic steroids on cardiac autonomic modulation and ventricular repolarization properties in rats.

Several studies have reported that high doses of synthetic anabolic androgenic steroids (AAS) can have serious negative effects on health, including the cardiovascular system. The aim of this study was to evaluate the combined effects of AAS and exercise training on ventricular repolarization and cardiac autonomic modulation in rats. Male Wistar rats were allocated into 4 groups: sedentary rats treated with vehicle, sedentary rats treated with nandrolone decanoate, swimming-trained rats treated with vehicle, and swimming-trained rats treated with nandrolone decanoate. Ventricular repolarization was evaluated by electrocardiographic analysis of QT interval and QT dispersion. Cardiac autonomic modulation was assessed by heart rate variability. Our results show that AAS increased QT interval and QT dispersion in sedentary rats treated with nandrolone decanoate as compared to sedentary rats treated with vehicle, indicating AAS-induced ventricular repolarization abnormalities. When rats treated with nandrolone decanoate were subjected to concomitant exercise training, ventricular repolarization was normalized. On the other hand, AAS-induced reduction in cardiac parasympathetic modulation was not prevented by exercise training. In conclusion, AAS produced cardiac autonomic dysfunction and ventricular repolarization disturbances in rats. Combining an exercise training protocol during the AAS treatment attenuated the ventricular repolarization abnormalities and did not prevent cardiac autonomic dysfunction.

ANABOLIC ANDROGENIC STEROID ABUSE AND REVERSIBLE CARDIOMYOPATHY: AN EMERGING AND UNDER-RECOGNIZED CARDIOVASCULAR PUBLIC HEALTH PROBLEM AMONG FITNESS ENTHUSIASTS

Emergence of retail websites selling synthetic anabolic androgenic steroid (AAS) and lack of FDA regulations have led to widespread AAS abuse in fitness enthusiasts and is associated with high morbidity and mortality. A 53 year old well built muscular man and fitness trainer using AAS for 20 years

Metabolic study of methylstenbolone in horses using liquid chromatography-high resolution mass spectrometry and gas chromatography-mass spectrometry

Methylstenbolone (2,17α-dimethyl-5α-androst-1-en-17β-ol-3-one) is a synthetic anabolic and androgenic steroid (AAS) sold as an oral ‘nutritional supplement’ under the brand names ‘Ultradrol’, ‘M-Sten’ and ‘Methyl-Sten’. Like other AASs, methylstenbolone is a prohibited substance in both human and equine sports. This paper describes the studies of the in vitro and in vivo metabolism of methylstenbolone in horses using LC/HRMS, GC/MS and GC/MS/MS. Phase I in vitro metabolic study of methylstenbolone was performed using homogenised horse liver. Hydroxylation was the only biotransformation observed. Six in vitro metabolites were detected including four mono-hydroxylated metabolites, namely 16α/β-hydroxymethylstenbolone (M1a, M1b), 20-hydroxymethylstenbolone (M1c), 6-hydroxymethylstenbolone (M1d), and two dihydroxylated methylstenbolone metabolites (M2c–M2d). An in vivo experiment was carried out using two retired thoroughbred geldings. Each horse was administered with 100 mg methylstenbolone supplement by stomach tubing daily for three consecutive days. Methylstenbolone and 14 metabolites were detected in the post-administration urine samples. The proposed in vivo metabolites included 16α/β-hydroxymethylstenbolone (M1a, M1b), 20-hydroxymethylstenbolone (M1c), two dihydroxylated methylstenbolone (M2a, M2b), 17-epi-methylstenbolone (M3), methasterone (M4), 2,17-dimethylandrostane-16,17-diol-3-one (M5), dihydroxylated and reduced methylstenbolone (M6), 2α,17α-dimethylandrostane-3α,17β-diol (M7), 2,17-dimethylandrostane-3,16,17-triol (M8a–M8c) and 2,17-dimethylandrostane-2,3,16,17-tetraol (M9), formed from hydroxylation, reduction and epimerisation. Methylstenbolone and ten of its metabolites could be detected in post-administration plasma samples. The highest concentration of methylstenbolone detected in urine was about 10–36 ng/mL at 3–4 h after the last administration, while the maximum concentration in plasma was about 0.4–0.7 ng/mL at 1 h after the last administration. For controlling the misuse of methylstenbolone, M8c and M9 gave the longest detection time in urine, while M4, M5 and M6 were the longest detecting analytes in plasma. They could be detected for up to 5 and 4.5 days respectively in urine and plasma. Apart from 16α/β-hydroxymethylstenbolone (M1a, M1b), the methylstenbolone metabolites reported herein have never been reported before.

Screening and confirmation of steroids and nitroimidazoles in urine, blood, and food matrices: Sample preparation methods and liquid chromatography tandem mass spectrometric separations

Veterinary drugs containing synthetic anabolic steroid and nitroimidazole active agents are not allowed for their applications in livestock of the European Union (EU). This paper presents analyses of twelve selected steroids and six nitroimidazole antibiotics at low levels (1.56μg/L–4.95μg/L and 0.17μg/kg–2.14μg/kg, respectively) in body fluids and egg incurred samples. Analyses involved clean-up procedures, high performance liquid chromatography (HPLC) separation, and tandem mass spectrometric screening and confirmatory methods. Target steroids and nitroimidazoles in samples were cleaned by two independent supported liquid extraction and solid phase extraction procedures. Separation of the selected compounds was conducted on Kinetex XB C-18 HPLC column using gradient elution. The screening methods utilised supported liquid extraction that enabled fast and cost effective clean-up. The confirmatory methods were improved by extending the number of matrices and compounds, and by introducing an isotope dilution mass spectrometry for nitroimidazoles. The new methods were validated according to the recommendation of the European Union Reference Laboratories and the performance characteristics evaluated met fully the criteria. The methods were applied to incurred samples in the proficiency tests. The obtained results of Z-scores demonstrated the applicability of developed protocols of the methods to real samples. The confirmatory methods were applied to the national monitoring program and natural contamination of prednisolone could be detected in urine at low concentration in few samples.

Danazol As First-Line Therapy for Myelodysplastic Syndromes

▪Introduction: Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematological disorders characterized by ineffective hematopoiesis and variable degrees of peripheral cytopenias. Neither curative nor standard therapy has been available yet for the majority of patients with MDS. Patients with available matched donor may undergo an allogeneic bone marrow transplant with a potential cure rate 30-50%. Danazol is a synthetic anabolic steroid with properties similar to corticosteroids, such as inhibition of interleukin-1 and TNF-α production. Also, has demonstrated activity in immune cytopenias and aplastic anemia but its efficacy in MDS has shown contradictory results. Recently there has been increasing interest in telomere dysfunction in hematological diseases. Short telomeres are the major prognostic risk factor for clonal evolution to myelodysplasia and acute leukemia. Danazol not only prevents telomere loss, but a mean increase occurs with improvement observed early during the course of hormone therapy. In our center with poor access to new strategies and therapies the use of danazol remains as an attractive option for patients with MDS because of its low cost. In this study we aim to document the clinical evolution of patients diagnosed with MDS treated with danazol as first-line therapy in our institution.Methods: We include patients diagnosed with MDS according to WHO criteria treated with danazol between 2005 and 2015. Response was defined as one or more of the following criteria: a rise in the platelet count >25 x 109/I, an increase in hemoglobin >2 g/dl or disappearance of a previous requirement for red blood cell transfusion, or an increase in neutrophil count >0.5 x 109/L. All patients received packed red blood cell and platelet transfusions according to physician criteria. Patients were given intravenous or oral antibiotics as needed as prophylaxis. Criteria for stop danazol included: no response to treatment, toxicity or patient’s refusal to continue treatment. Blood counts were preformed at least 1-month intervals for the first 4 months. We compared those who responded versus no responders using the Student’s t test or Mann-Whitney U test as corresponded.Results: Forty-two patients with MDS were treated with danazol. Median follow-up was 12 months (range 3-75). Median dose used was 400 mg (range 100-600) orally in two divided doses. Mean duration of treatment for all patients was 6 months (range 3-72 months). The distribution by WHO subtypes included 26 refractory cytopenia with multilineage dysplasia (RCMD) (62%), 6 patients with refractory cytopenia with unilineage dysplasia (RCUD), 6 refractory anemia with excess blasts (RAEB), 2 refractory anemia with ring sideroblasts (RARS) and 1 MDS associated with isolated del(5q). Twenty-four (60%) patients presented clinical response. Response for patients with anemia was 23.8% (10/24), increase in absolute neutrophil count occurred in 36.8% (7/19), and 60% (24/40) presented an increase in platelet count. Time to initial response was 2 months (range, 1-8) while time to better response of 3 month (range, 1-8). Response was not associated to any MDS classification or administrated dose. Toxicity was mild and danazol was not discontinued in any patient. Side effects included three patients with gastrointestinal symptoms, and 4 patients reported weight gain. Median overall survival was 24 months (CI95% 5.1-42). Fifteen patients died (35.7%). Five patients progressed to AML.Conclusion: Different agents have been used in MDS. However, for the majority of patients with MDS no curative treatment exists. In conclusion our data suggest that Danazol may be effective in MDS with minimal toxicity, especially in patients with thrombocytopenia. Response was independent of severity, WHO classification and administrated dose. [Display omitted] DisclosuresGomez-Almaguer:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Diagnosis and treatment of autoimmune hemolytic anemia: classic approach and recent advances.

Diagnosis and treatment of autoimmune hemolytic anemia: classic approach and recent advances.

Revisiting the metabolism of 19-nortestosterone using isotope ratio and high resolution/high accuracy mass spectrometry

The synthetic anabolic androgenic steroid 19-nortestosterone is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA) due to its performance-enhancing effects. Today, doping controls focus predominantly on one main urinary metabolite, 19-norandrosterone glucuronide, which offers the required detection windows for an appropriate retrospectivity of sports drug testing programs. As 19-norandrosterone can also be found in urine at low concentrations originating from in situ demethylation of other abundant steroids or from endogenous production, the exogenous source of 19-norandrosterone needs to be verified, which is commonly accomplished by carbon isotope ratio analyses.The aim of this study was to re-investigate the metabolism of 19-nortestosterone in order to probe for additional diagnostic long-term metabolites, which might support the unambiguous attribution of an endo- or exogenous source of detected 19-nortestosterone metabolites. Employing a recently introduced strategy for metabolite identification, threefold deuterated 19-nortestosterone (16,16,17-2H3-NT) was administered to one healthy male volunteer and urine samples were collected for 20 days. Samples were prepared with established methods separating unconjugated, glucuronidated and sulfated steroids, and analytes were further purified by means of high-performance liquid chromatography before trimethylsilylation. Deuterated metabolites were identified using gas chromatograph/thermal conversion/isotope ratio mass spectrometer comprising an additional single quadrupole mass spectrometer. Additional structural information was obtained by gas chromatography/time-of-flight mass spectrometry and liquid chromatography/high resolution mass spectrometry.In general, sulfo-conjugated metabolites were excreted for a longer time period than the corresponding glucuronides. Several unexpected losses of the arguably stable isotope labels were observed and characterized, attributed to metabolic reactions and sample preparation procedures. The detection window of one of the newly detected metabolites was higher than currently used metabolites. The suitability of this metabolite to differentiate between endo- or exogenous sources could however not be verified conclusively.

Establishment of Enhanced Chemiluminescent Immunoassay Formats for Stanozolol Detection in animal-derived foodstuffs and Other Matrices

Stanozolol is one of the most widely used synthetic anabolic steroids but also presents a host of negative side effects. It is necessary to develop a rapid and sensitive method for stanozolol screening to monitor its abuse. In this study, the polyclonal antibody against stanozolol was prepared and a novel indirect competitive chemiluminescence enzyme immunoassay (CLEIA) for stanozolol was developed. The stanozolol hapten was conjugated to proteins to prepare immunogen and coating antigens, firstly. To improve the performances of the method, the chemiluminescent detection system was optimized. The optimal concentration of 4-iodophenol, hydrogen peroxide and luminol was studied and the proper detection time was also investigated. Under the optimal conditions, the IC50 value and the limit of detection (IC20) of the chemiluminescence immunoassay is 0.34 and 0.07 ng/mL in phosphate-buffered saline (PBS), respectively. The accuracy performances of the proposed CLEIA were compared with that of ELISA and HPLC. Different nutraceuticals, foodstuffs, and biological matrices spiked with stanozolol were applied to study the application of the proposed assays, and the average recoveries locate at 92–113 % with the coefficients of variation of 8–13.5 %. The polyclonal antibody-based chemiluminescence immunoassay formats are proved to be a feasible and reliable quantificational method for stanozolol.

Prolifertive effect of 17beta‐trenbolone on human prostate cancer cell line

Trenbolone acetate (TBA) is synthetic anabolic steroid which have used in speed muscle development in cattle. Some of TBA are converted into 17beta-trenbolone (17TB, 17b-hydroxy-estra-4,9,11-trien-3-one), which exerts androgenic activity by binding with androgen receptor like testosterone and dihydrotestosterone (DHT). It has been reported that cross-talk between androgen and cell cycle regulator modulates prostate cancer cell progression. Since 17TB exhibits androgenic activity like DHT, it is important for knowing role of 17TB on prostate cancer cell progression. In this study, we found that 17TB induced prostate cancer cell proliferation with concentration dependent manner (EC50=0.03nM). Furthermore, cell growth by 17TB was completely inhibited in presence of 17TB and 10μM bicalutamide, which acts as an androgen receptor antagonist. To investigate the molecular mechanism of 17TB on prostate cancer cells, western blot analysis was performed. Indeed, 17TB increased cell cycle regulatory proteins cyclin A, cyclin B, cyclin D and it similar with DHT treatment. These data suggest that 17TB promotes 22Rv1 androgen-dependent prostate cancer cell proliferation via regulation of cell cycle.

Micellar electrokinetic chromatography analysis of tetrahydrogestrinone and related anabolic androgenic steroids

Tetrahydrogestrinone and related anabolic androgenic steroids (AASs) have been used extensively for performance enhancement (doping) in sports due to their ability to accelerate protein synthesis and improve physical performance (F. C. W. Wu, Clin. Chem., 1997, 43, 1289–1292). The separation of these AASs was performed by micellar electrokinetic chromatography (MEKC) using borate/borax buffer (200/50 mM, pH 8.6), with sodium cholate (40 mM) as a chiral additive. The analytes included two endogenous steroid hormones (testosterone and epitestosterone) and five synthetic anabolic steroids (methyltestosterone, nandrolone, gestrinone, dihydrogestrinone and tetrahydrogestrinone). Capillary electrophoresis parameters such as the concentration of additives, injection time, temperature, and applied voltage were investigated to improve the separation efficiency. A complete separation was achieved in less than 16 min under the optimized conditions. The RSDs of peak area and migration time were below 3.9% and 0.34%, respectively. The limit of detection (LOD) between 240 and 570 ng mL−1 was obtained for each of the pure standards with a photodiode array (PDA) detector. Lower LODs could be reached when combining with preconcentration. After liquid–liquid extraction, the recoveries of spiked urine samples were in the range from 88% to 99%.

Anti‐Dope Testing in Sport: The History and the Science

This summer, as the XXX modern Olympic Games and the XIV Summer Paralympic Games were held in London, millions watched magnificent efforts from the best athletes in the world. The ability of the athletes to produce their best personal performances in the spotlight and the pressure of the Olympic Games was a testament to the human spirit. But, because the margin between winning and losing is so small, one imprecisely placed foot or hand, one moment of indecision, one gust of wind at the wrong moment, and the gold medal is gone. Behind the scenes at these Olympics—properly so— was a scientific team performing under great pressure to ensure safe and fair competition by testing urine and blood for traces of performance-enhancing prohibited substances and methods. Their efforts were crucial, because, in partnership with the clean athletes, they support the integrity and the spirit of sport. More than 1000 samples were analyzed within a few days after each event for stimulants, steroids, masking agents, recombinant proteins like erythropoietin and growth hormone (GH), and other substances on the World AntiDoping Agency (WADA) Prohibited List (1). The List of Prohibited Substances and Methods is maintained by a committee of internationally recognized scientists and sport administrators. A revised list is released annually by the WADA. In deciding whether to add a compound or method to the list, the committee considers whether there is potential to enhance performance; a potential health risk for the athlete; or if use of the substance or method violates the spirit of sport. If two of the three criteria are met, the substance is added to the Prohibited List. The inclusion of the word “potential” recognizes that a substance may emerge for which there are no toxicological data. For example, the identification of the “designer” steroid tetrahydrogestrinone (THG) in a drop of fluid in a syringe turned in to the U.S. Anti-Doping Agency (USADA) in 2003 illustrates the problem. THG had been developed by Wyeth in the 1960s and taken to clinical trials as a potential anabolic agent, but never brought to market. It was impossible to locate any records from the clinical trials 40 years after the fact. THG had been shown to have anabolic activity (2). There were no publications about toxicological studies, but as a 17 -methyl steroid, comparison to similar structures strongly suggested the potential for some degree of hepatotoxicity. Based on this information, THG was added to the Prohibited List in 2006.