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Boldenone (Bdn) and nandrolone (Ndn) are anabolic androgenic steroids (AASs) that, as our previous studies have shown, may increase the risk of neoplastic transformation of porcine ovarian putative stem cells (poPSCs). The NF-κB pathway may be important in the processes of carcinogenesis and tumour progression. Therefore, in this work, we decided to test the hypothesis of whether Bdn and Ndn can activate the NF-κB pathway by acting through the membrane androgen receptor ZIP-9. For this purpose, the expression profiles of both genes involved in the NF-κB pathway and the gene coding for the ZIP-9 receptor were checked. The expression and localization of proteins of this pathway in poPSCs were also examined. Additionally, the expression of the ZIP-9 receptor and the concentration of the NF-κB1 and 2 protein complex were determined. Activation of the NF-κB pathway was primarily confirmed by an increase in the relative abundances of phosphorylated forms of RelA protein and IκBα inhibitor. Reduced quantitative profiles pinpointed not only for genes representing this pathway but also for unphosphorylated proteins, and, simultaneously, decreased concentration of the NF-κB1 and 2 complex may indicate post-activation silencing by negative feedback. However, the remarkably and sustainably diminished expression levels noticed for the SLC39A9 gene and ZIP-9 protein suggest that this receptor does not play an important role in the regulation of the NF-κB pathway.

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Boldenone is an anabolic-androgenic steroid (AAS) that is prohibited in equine sports. However, in certain situations, it is endogenous, potentially formed by the microbes in urine. An approach to the differentiation based on the detection of the biomarkers Δ1-progesterone, 20(S)-hydroxy-Δ1-progesterone and 20(S)-hydroxyprogesterone was assessed, and their concentrations were monitored in the urine of untreated female horses (n = 291) alongside boldenone, boldienone, testosterone and androstenedione. Using an ultra-sensitive analytical method, boldenone (256 ± 236 pg/mL, n = 290) and the biomarkers (Δ1-progesterone up to 57.6 pg/mL, n = 8; 20(S)-hydroxy-Δ1-progesterone 85.3 ± 181 pg/mL, n = 130; 20(S)-hydroxyprogesterone 43.5 ± 92.1 pg/mL, n = 158) were detected at low concentrations. The ex vivo production of Δ1-steroids was artificially induced following the storage of urine samples at room temperature for 7 days in order to assess the concentrations and ratios of the monitored steroids. The administration of inappropriately stored feed source also resulted in an increase in 20(S)-hydroxy-Δ1-progesterone concentrations and the biomarker ratios. Using the results from different datasets, an approach to differentiation was developed. In situations where the presence of boldenone exceeds a proposed action limit of 5 ng/mL, the presence of the biomarkers would be investigated. If Δ1-progesterone is above 50 pg/mL or if 20(S)-hydroxy-Δ1-progesterone is above 100 pg/mL with the ratio of 20(S)-hydroxy-Δ1-progesterone:20(S)-hydroxyprogesterone greater than 5:1, then this would indicate ex vivo transformation or consumption of altered feed rather than steroid administration. There remains a (small) possibility of a false negative result, but the model increases confidence that adverse analytical findings reported in female horses are caused by AAS administrations.

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Gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) can be used to detect the synthetic forms of endogenous anabolic androgenic steroids (EAAS) by comparing the 13C/12C ratios of the endogenous reference compound to that of the target compound. Isolation and enrichment of the target compound from urinary matrices is an essential prerequisite for the GC/C/IRMS confirmation procedure in doping control analysis. Boldenone (Bo) is a natural anabolic androgenic steroid (AAS) and a derivative of testosterone. The GC/C/IRMS confirmation procedure for Bo and its main metabolite 5ß-androst-1-en-17ß-ol-3-one (BoM) is extremely complicated due to the low concentrations and the enormously complex matrices in urine. The present study demonstrated a sample purification procedure for GC/C/IRMS by using online 2D-HPLC to purify Bo and BoM in urine samples. Bo and BoM with concentrations as low as 2 ng/mL were isolated and enriched with superior purity and selectivity. The validity of the method was verified with the technical document issued by the world anti-doping agency. The online 2D-HPLC purification procedure featured high selectivity for the analytes and no isotopic fractionation in the collection process. The present method can be used as a routine method allowing doping control laboratories to perform Boldenone confirmation.

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INTRODUCTION: The risk of cardiotoxicity is associated with the use of anabolic-androgenic steroids and analgesics, several deaths were attributed to such medications. OBJECTIVES: This study investigates the effects of boldenone (BOLD) and tramadol (TRAM) alone or in combination on the heart. MATERIAL AND METHODS: Forty adult male rats were divided into four groups. Normal control group, BOLD (5 mg/kg, i.m.) per week, tramadol Hcl (TRAM) (20 mg/kg, i.p.) daily and a combination of BOLD (5 mg/kg) and TRAM (20 mg/kg), respectively for two months. Serum and cardiac tissue were extracted for determination of serum, aspartate aminotransferase (AST), creatine phosphokinase (CPK) and lipid profiles, tissue malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), nitric oxide (NO), tumour necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and histopathological examination. Troponin I gene expression was quantified in cardiac tissue using real-time polymerase chain reaction technique. RESULTS: Groups received BOLD and TRAM alone and in combination showed elevated serum biochemical parameters (AST, CPK) and deviations in lipid profiles, elevation in oxidative and inflammatory parameters (MDA, NO, TNF-α and IL-6), and decrease in GSH and SOD, up-regulated cardiac troponin I as well as distorted cardiac histopathological pictures. CONCLUSION: The current study elucidated the risk of administration of these drugs for sustained periods as well as the marked detrimental effects of using these drugs in combination.

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Boldenone and tramadol are abused among large sectors of adolescents. Therefore, the behavioral changes concerned with memory and cognitive functions and neurochemical variations were investigated in the cortex of rats treated with boldenone and/or tramadol. Rats were divided into control and rats treated with boldenone, tramadol, or both drugs. At the end of the treatment period, the memory and cognitive functions were evaluated by the Y-maze test (YMT) and elevated plus maze test (EPMT) and the motor activity was determined by the open field test (OFT). The cortex was dissected to carry out the neurochemical analyses. Rats treated with boldenone and/or tramadol showed impaired memory and cognitive functions and reduced motor activity. A significant increase in lipid peroxidation (MDA), nitric oxide (NO), and a significant decrease in reduced glutathione (GSH) were observed in the cortex of rats treated with boldenone and/or tramadol. The levels of acetylcholinesterase (AChE) and monoamine oxidase (MAO) decreased significantly. Western blot data showed a significant decrease in Bcl2 and a significant increase in caspase-3 and inducible nitric oxide synthase (iNOS) in rats treated with boldenone and/or tramadol. These changes were associated with neuronal death as indicated from the histopathological examination.The present findings indicate that boldenone and/or tramadol induced impairment in memory and cognitive functions. These changes could be mediated by the increase in oxidative stress, neuroinflammation, reduced AChE level, and reduced number of survived neurons in the cortex as indicated from the decreased Bcl2 level and the histological examination.

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Objectives: The testis is the male reproductive gland or gonad having two vital functions: to produce both sperm and androgens, primarily testosterone. The study aimed to investigate the effect of tramadol and boldenone injected alone or in combination for 2 months in rats on testicular function. Materials and Methods: Group 1, normal control; Group 2, tramadol HCl (TRAM) (20 mg/kg bwt.) (IP); Group 3, boldenone undecylenate (BOLD) (5 mg/kg bwt) (i.m); Group 4, combination of TRAM (20 mg/kg bwt.) and BOLD (5 mg/kg); respectively for 2 months. Results: TRAM and BOLD alone and in combination showed deteriorated testicular functions, lowered serum steroid levels (FSH, LH, and testosterone), elevation in oxidative biomarkers (MDA & NO) and reduction in GSH and SOD, down-regulation of StaR and HSD17B3 as well as histopathological testicular assessment using H&E staining revealing massive degenerative changes in the seminiferous epithelium and vacuolar changes of most of the spermatogenic stages in both TRAM and BOLD groups. PAS stain showed an intensive reaction in the interstitial tissue between the tubules in the TRAM group. Masson trichrome stain showed abundant collagen fiber deposits in the tunica albuginea with congested BV in the TRAM group. Conclusion: The study illuminated the hazard of administration of these drugs for a long period as well as the prominent deleterious effects reported on concurrent use of both drugs.

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The illicit abuse of anabolic steroids is associated with brutal aggression, which represents a serious health hazard and social threat. Boldenone is commonly used for doping by athletes and adolescents for esthetic purposes and to enhance performance and endurance during competitions. However, the mechanistic pathways underlying boldenone-induced behavioral deviations and neuronal toxicity have not yet been elucidated. On the other hand, the natural polyphenol curcumin is appreciated for its relative safety, potent antioxidant activity, and anti-inflammatory properties. Therefore, the present study was initiated to explore the signaling pathways underlying boldenone-induced anxiety and aggression in rats, and the protective effects of curcumin. To achieve this aim, male Wistar albino rats were randomly distributed into control, curcumin (100 mg/kg in sesame oil, p.o., once daily), boldenone (5 mg/kg, intramuscular, once weekly), and combination groups. Rats were challenged across the open field, irritability, defensive aggression, and resident-intruder tests. The prefrontal cortex was used to assess serotonin level, oxidative stress markers, and mRNA expression of myeloid differentiation primary response gene (MyD88), TNFR-associated factor 6 (TRAF-6), tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), protein expression of toll-like receptor 4 (TLR4), and phosphorylated nuclear factor-κB transcription factor (NF-κB p65). Unprecedented, the current results showed that boldenone elicited aggression in rats accompanied by depleted serotonin, enhanced oxidative stress, and exaggerated inflammatory response via upregulation of TLR4/MyD88/TRAF-6/NF-κB pathway. Interestingly, curcumin mitigated boldenone-induced neurobehavioral disturbances in rats, normalized the oxidant/antioxidant balance, and suppressed TLR4/MyD88/TRAF-6/NF-κB pathway and its downstream proinflammatory signaling molecules TNF-α and IL-1ß.

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In this work multivariate strategies were employed in order to highlight new potential biomarkers of interest to detect the exogenous treatment of steers intramuscularly treated with boldenone undecylenate. Serum samples collected from treated (n = 4) and control (n = 8) crossbred animals of varying ages and weights were extracted using a simple sample preparation procedure based on salt assisted liquid-liquid extraction. Data acquisition was performed using liquid chromatography and Q-Exactive™ Orbitrap mass spectrometry. Data processing and treatment were performed using two non-targeted workflows: (1) Compound Discoverer software and (2) XCMS package on the open-source R software combined with MetaboAnalyst. Three potential biomarkers were highlighted taking into account the chromatographic shapes, the feature location on the generated s-plots, the fold change, the adjusted p values, the coefficient of variation in the QC samples and the area under the ROC curves. Predicted formulas based on mass accuracy, structural composition and spectra similarity were proposed. A robust statistical model to predict the boldenone treatment was further developed based on the weighted abundances of the selected biomarkers. The requirements for screening methods were successfully fulfilled, together with a wider detection window in comparison with the monitoring of the deconjugated metabolite boldenone, although biomarker identification studies are still ongoing.

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In order to detect the misuse of testosterone (T) or boldenone (Bo) in doping control analysis, the confirmation of atypical findings employing the determination of carbon isotope ratios (CIR) is mandatory for issuing adverse analytical findings. Elevated concentrations of T (or elevated T/epitestosterone ratios) may result from confounding factors such as ethanol intake, and the presence of low urinary concentrations of Bo can originate from endogenous or urinary in situ production of small amounts of the steroid. As pharmaceutical preparations of Bo and T are generally depleted in 13 C, their CIR differ significantly from the 13 C-enriched endogenous steroids. Some rare cases have been reported on pharmaceutical preparations showing 13 C-enriched isotope ratios that complicate the current application of CIR in sports drug testing. Therefore, the CIR of a subset of n = 157 T preparations and n = 39 Bo preparations seized in Switzerland and Germany between 2013 and 2018 was analyzed in order to estimate the possible impact of steroid preparations showing 13 C-enriched isotope ratios on the current approach to detect their misuse. All investigated Bo preparations showed CIR in the expected range between - 26.7 and -30.3‰. Within the T samples, 95% showed the expected values below -26‰ while six samples fall between -25 and -26‰ and one sample was indistinguishable from endogenously produced T with a CIR of -23.3‰.

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Nandrolone (Ndn) and boldenone (Bdn), the synthetic testosterone analogues with strong anabolic effects, despite being recognized as potentially carcinogenic compounds, are commonly abused by athletes and bodybuilders, which includes women, worldwide. This study tested the hypothesis that different doses of Ndn and Bdn can initiate neoplastic transformation of porcine ovarian putative stem cells (poPSCs). Immunomagnetically isolated poPSCs were expanded ex vivo in the presence of Ndn or Bdn, for 7 and 14 days. Results show that pharmacological doses of both Ndn and Bdn, already after 7 days of poPSCs culture, caused a significant increase of selected, stemness-related markers of cancer cells: CD44 and CD133. Notably, Ndn also negatively affected poPSCs growth not only by suppressing their proliferation and mitochondrial respiration but also by inducing apoptosis. This observation shows, for the first time, that chronic exposure to Ndn or Bdn represents a precondition that might enhance risk of poPSCs neoplastic transformation. These studies carried out to accomplish detailed molecular characterization of the ex vivo expanded poPSCs and their potentially cancerous derivatives (PCDs) might be helpful to determine their suitability as nuclear donor cells (NDCs) for further investigations focused on cloning by somatic cell nuclear transfer (SCNT). Such investigations might also be indispensable to estimate the capabilities of nuclear genomes inherited from poPSCs and their PCDs to be epigenetically reprogrammed (dedifferentiated) in cloned pig embryos generated by SCNT. This might open up new possibilities for biomedical research aimed at more comprehensively recognizing genetic and epigenetic mechanisms underlying not only tumorigenesis but also reversal/retardation of pro-tumorigenic intracellular events.

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This study investigated the biochemical and histopathological alterations along with the immunoexpression pattern of heat shock protein 27 (Hsp27) within 6 h postmortem (PM) in skeletal muscle of boldenone (BOL)-treated rats. Forty-eight male rats were divided into two groups; a control group received sesame oil (0.25 mL/kg bwt), and BOL group received 5 mg/kg bwt BOL. Both treatments were intramuscularly injected once a week for eight weeks. Rats were euthanized by cervical dislocation, and the skeletal muscle specimens were collected at zero-time, 2, 4, and 6 h PM for biochemical and histopathological evaluations. The results revealed that BOL treatment significantly increased pH, MDA, ATP, ADP, glycogen, and hydroxyproline values. Still, it decreased the GPX, GST, and lactic acid levels, and Hsp27 immunoexpression compared to the control group. With increasing postmortem interval (PMI), whether control or BOL-treated, a significant reduction in pH value, markers of muscular antioxidant status, ATP, ADP, glycogen, hydroxyproline levels, as well as Hsp27 immunoexpression but a significant increase in lipid peroxidation and lactic acid content were recorded. Of note, the interaction between BOL treatment and PMI had a significant effect on ATP, ADP, lactic acid, hydroxyproline, GST, MDA, and TAC levels. Conclusively, these findings signify BOL exposure's modifying effect on the energy content, oxidative status, and histological architecture of skeletal muscles in the early PMI that reflected in delaying the onset of rigor mortis. For forensic practitioners, these findings should be highly considered at estimating PMI in athletic, AAS-treated patients, and fattening animals.

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The present study was designed to evaluate the effects of boldenone undecylenate (BL) abuse alone and in combination with vitamin C (VC) on the immune responses and thyroid structure and function in rats. Thirty adult male Wistar rats were randomly divided into five equal groups and were subjected to various treatment regimens for eight weeks as follows: control group, vehicle control group, VC group orally received VC (120 mg/Kg BW/day), BL-treated group intramuscularly injected with BL (5 mg/kg BW, once/week), and BL+VC group received BL and VC. At the end of this experiment, blood and tissue samples (thyroid, thymus, and spleen) were subjected to hematological evaluation, biochemical analysis, histopathological, and immunohistochemical examinations. In comparison to controls, BL significantly increased the levels of serum proinflammatory interleukins (IL-1 ß and IL-6), immunoglobulins (IgG and IgM), and complement 3 but reduced anti-inflammatory interleukin-10, lysosome, and nitric oxide. Besides, altered platelet count and leukogram were evident in BL-injected rats. BL notably disturbed thyroid profile as revealed by a significant increase of thyroid-stimulating hormone and thyroid peroxidase antibody. In contrast, both total and free forms of thyroid hormones (tri-iodothyronine and thyroxine), thyroglobulin, and thyroid peroxidase, were significantly decreased. Moreover, BL caused histopathological changes in the thyroid, thymus, and spleen tissues.CD4+ immuno-expression was reduced, but CD8+ immunolabelling was increased in both spleen and thymus. The daily dosing of VC to BL-exposed rats significantly corrected most of the deviations in immune parameters. It restored most of the thyroid architecture and function, revealing a significant protective effect of this vitamin. This experimental study demonstrates that BL abusing disrupts the immune system by different mechanisms and addresses BL, for the first time, as an autoimmune clinical hypothyroidism inducer drug. Additionally, VC is helpful in the management of BL abuse.

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Boldenone Undecylenate (BLD) is a synthetic derivative of testosterone and a widely used anabolic androgenic steroid. The health risk of BLD use as a pharmaceutical or dietary supplement is still underestimated and under-reported. Vitamin C (VC) has been recognized as an antioxidant with prominent hepatorenal protective effects. This study investigated the possible preventive activity of VC against BLD-induced hepatorenal damage. Forty adult male Wistar rats were classified into five groups: control, vehicle control, VC (orally given 120 mg/kg b. wt./day), BLD (intramuscularly injected 5 mg/kg b. wt./week), and BLD + VC-treated groups. The experiment continued for eight weeks. Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. Serum contents of total protein (TP), albumin (ALB), globulin, total cholesterol (TC), triglycerides (TG), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), and very-low-density lipoprotein-cholesterol (VLDL-C) were also assayed. Urea, creatinine, and uric acid levels were determined together with sodium and potassium electrolytes measuring. Moreover, oxidative stress indicators including reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GSR) as well as malondialdehyde (MDA) levels were measured in both hepatic and renal tissues. Corresponding histological examination of renal and hepatic tissues was conducted. Besides, immunohistochemical evaluations for androgen receptors protein (AR) and heat shock protein 90 (Hsp 90) expressions were performed. BLD caused significant rises in serum ALT, AST, TP, ALB, TC, TG, LDL-C, VLDL-C, urea, creatinine, uric acid, potassium, and MDA levels. Further, BLD-injected rats showed significant declines in the serum levels of HDL-C, sodium, GSH, GPx, GST, and GSR. Besides, distinct histopathological perturbations were detected in renal and hepatic tissues of BLD-injected rats. AR and Hsp 90 immunoexpression were increased in hepatic and renal tissues. In contrast, VC significantly reversed the BLD-induced hepatorenal damage in co-treated rats but not ameliorated AR protein overexpression. VC could be an efficient preventive supplement for mitigating BLD-induced hepatorenal damage, possibly via controlling oxidative stress events.

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Androgenic anabolic steroids are the most misused substances in sports because of their performance-enhancing effects. Often synthetic analogues of endogenously present steroids are administered. To determine their endogenous or exogenous origin, Gas Chromatography Combustion Isotope Ratio Mass Spectrometry (GC-C-IRMS) is used in the field of doping control. Compounds subjected to IRMS analysis must be interference-free, with liquid chromatography fraction collection (HPLC-FC) being the crucial clean-up step. However, this clean-up is challenging, particularly for compounds present at low concentrations in samples with pronounced matrix effects. The compounds of interests for IRMS analyses in doping control are testosterone (T) and its main metabolites (androsterone, etiocholanolone, 5α-androstane-3α,17ß-diol, 5ß-androstane-3α,17ß-diol), epitestosterone, 19-norandrosterone (19-NA), boldenone (B) and its main metabolite (BM), formestane (F) and 6αOH-androstenedione (6aOHADION). Currently, the available methods only deal with a selection of the above-mentioned compounds. Some of these compounds (e.g., 19-NA, B, BM, 6aOHADION) are present in very low concentrations, requiring an extensive and dedicated sample clean-up, and this makes it challenging to develop a universal clean-up procedure. Many of these methods require different and multiple offline HPLC-FC setups, which are labour-intensive and time-consuming. That is problematic during, e.g., large sports events, where reporting time is limited (e.g., 72 h). Therefore, in the current work, we developed a uniform online 2D/3D HPLC-FC method, capable of purifying all relevant target compounds in a single run, leading to the fastest clean-up procedure so far (i.e., 31 min for T and its main metabolites; 46 min for 19-NA, F and 6aOHADION; 48 min for B and BM).

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BACKGROUND: This study explored the effect of vitamin C (Vit-C) administration on the reproductive function of adult male Wistar rats injected with boldenone undecylenate (BOL). METHODS: Rats were randomly assigned into control, vehicle control, Vit-C (120 mg/kg b.wt./day, orally), BOL (received 5 mg/kg b.wt./week, IM) and BOL+Vit-C-treated groups. After eight weeks, hormonal assay, semen evaluation, testicular enzymes, and antioxidants biomarkers were assessed. Besides, the histopathological and immunohistochemical investigations of the androgen receptor (AR) expression were performed. RESULTS: The results revealed that serum testosterone, acid phosphatase, sorbitol dehydrogenase, sperm abnormalities, and testicular malondialdehyde were significantly incremented in the BOL-treated group. Testicular weight, sperm count, and sperm motility together with serum levels of luteinizing hormone, follicle-stimulating hormone, and estradiol, and testicular testosterone, catalase, superoxide dismutase, and reduced glutathione showed a significant decrease following BOL treatment. Besides, the AR immunoreactivity was significantly decreased in testicular tissues. Vit-C co-administration with BOL significantly relieved the BOL-induced sperm abnormalities, reduced sperm motility, testicular enzyme leakage, and oxidative damage. However, Vit-C could rescue neither BOL-induced hormonal disturbances nor AR down-regulation. CONCLUSIONS: The results provide further insight into the mechanisms of BOL-induced reproductive dysfunction and its partial recovery by Vit-C.

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Boldenone (1-dehydrotestosterone) is an exogenous anabolic-androgenic steroid (AAS) but is also known to be endogenous in the entire male horse and potentially formed by microbes in voided urine, the gastrointestinal tract, or feed resulting in its detection in urine samples. In this study, equine fecal and urine samples were incubated in the presence of selected stable isotope labeled AAS precursors to investigate whether microbial activity could result in 1-dehydrogenation, in particular the formation of boldenone. Fecal matter was initially selected for investigation because of its high microbial activity, which could help to identify potential 1-dehydrogenated biomarkers that might also be present in low quantities in urine. Fecal incubations displayed Δ1-dehydrogenase activity, as evidenced by the use of isotope labeled precursors to show the formation of boldenone and boldione from testosterone and androstenedione, as well as the formation of Δ1-progesterone and boldione from progesterone. Unlabeled forms were also produced in unspiked fecal samples with Δ1-progesterone being identified for the first time. Subsequent incubation of urine samples with the labeled AAS precursors demonstrated that Δ1-dehydrogenase activity can also occur in this matrix. In all urine samples where labeled boldenone or boldione were detected, labeled Δ1-progesterone was also detected. Δ1-progesterone was not detected any non-incubated urine samples or following an administration of boldenone undecylenate to one mare/filly. Δ1-progesterone appears to be a candidate for further investigation as a suitable biomarker to help evaluate whether boldenone present in a urine sample may have arisen due to microbial activity rather than by its exogenous administration.

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As a result of the findings of scientists working on the biosynthesis and metabolism of steroids in the plant and animal kingdoms over the past five decades, it has become apparent that those compounds that naturally occur in animals can also be found as natural constituents of plants and vice versa, i.e., they have essentially the same fate in the majority of living organisms. This review summarizes the current state of knowledge on the occurrence of animal steroid hormones in the plant kingdom, particularly focusing on progesterone, testosterone, androstadienedione (boldione), androstenedione, and estrogens.

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Boldenone is an anabolic-androgenic steroid that is prohibited in equine sports. Urine from the uncastrated male horse contains boldenone that is thought to be of endogenous origin and thus a threshold ('cut-off') concentration has been adopted internationally for free and conjugated boldenone to help distinguish cases of doping from its natural production. The testis is likely to be a source of boldenone. Qualitative analysis was performed on extracts of equine testicular homogenates (n = 3 horses) incubated non-spiked and in the presence of its potential precursors using liquid chromatography tandem mass spectrometry (LC-MS/MS) and LC high resolution mass spectrometry (LC-HRMS). Samples were analysed both underivatised and derivatised to increase the certainty of identification. In addition to previously reported endogenous steroids, analysis of non-spiked testicular tissue samples demonstrated the presence of boldenone and boldienone at trace levels in the equine testis. Incubation of homogenates with deuterium or carbon isotope labelled testosterone and androstenedione resulted in the matching stable isotope analogues of boldenone and boldienone being formed. Additionally, deuterium and carbon labelled 2-hydroxyandrostenedione was detected, raising the possibility that this steroid is a biosynthetic intermediate. In conclusion, boldenone and boldienone are naturally present in the equine testis, with the biosynthesis of these steroids arising from the conversion of testosterone and androstenedione. However, additional work employing larger numbers of animals, further enzyme kinetic experiments and pure reference standards for 2-OH androstenedione isomers would be required to better characterize the pathways involved in these transformations.

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Boldenone (BOLD), one of androgenic anabolic steroids (AAS), although banned in humans, is still available illegally. AAS abuse has previously been associated with various cardiovascular adverse events including acute myocardial infarction, arrhythmia, and sudden death. In this study, the concentration of BOLD was determined in postmortem specimens from the corpse of a human male who intentionally injected BOLD undecylenate into his shoulder muscle. In addition, the endogenous levels of BOLD in the blood and urine samples of young human males have been reported. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with solid-phase extraction (SPE) was developed and validated for the analysis of BOLD in blood, muscular tissue and urine samples. The validation parameters including linearity, accuracy, precision, matrix effect, and recovery were satisfactory. The concentrations of BOLD in the blood of 20 young human males who didn't take BOLD were under the limit of quantitation (LOQ, 0.5 ng/mL). Additionally, the mean level of BOLD in the urine samples was 3.19 ± 1.65 ng/mL (range: 0.37˜6.02 ng/mL). The concentrations of BOLD in the victim's blood from the femoral vein and heart were 140.44 and 25.74 ng/mL, respectively. On the other hand, those in the muscular tissue from the injection site and the urine sample were 142.3 ng/g and 3474 ng/mL, respectively.

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