Identification of three unexpected new psychoactive substances at an Australian drug checking service.

Drug checking is a harm reduction measure that provides people with the opportunity to confirm the identity and purity of substances before consumption. The CanTEST Health and Drug Checking Service is Australia's first fixed-site drug checking service, where clients can learn about the contents of the samples they provide while receiving tailored harm reduction and health advice. Three samples were recently presented to the service with the expectation of 4-fluoromethylphenidate (4F-MPH) 1, methoxetamine (MXE) 2 and 3-methylmethcathinone (3-MMC) 3. The identity of all three samples did not meet these expectations and remained unknown on-site, as no high confidence identifications were obtained. However, further analysis by nuclear magnetic resonance spectroscopy, high resolution gas chromatography-electron ionisation-mass spectrometry and liquid chromatography-electrospray ionisation-mass spectrometry at the nearby Australian National University allowed for the structure elucidation of the three samples as 4-fluoro-α-pyrrolidinoisohexanophenone (4F-α-PiHP) 4, 1-(4-fluorobenzyl)-4-methylpiperazine (4F-MBZP) 5 and N-propyl-1,2-diphenylethylamine (propylphenidine) 6, respectively. Given all three samples were not of the expected identity and have not yet been described as new psychoactive substances in the literature, this study presents a full characterisation of each compound. As exemplified by this rapid identification of three unexpected new psychoactive substances, drug checking can be used as an effective method to monitor the unregulated drug market.

Comparison of Toxicities among Different Bumped Kinase Inhibitor Analogs for Treatment of Cryptosporidiosis.

Recent advances on the development of bumped kinase inhibitors for treatment of cryptosporidiosis have focused on the 5-aminopyrazole-4-carboxamide scaffold, due to analogs that have less hERG inhibition, superior efficacy, and strong in vitro safety profiles. Three compounds, BKI-1770, -1841, and -1708, showed strong efficacy in C. parvum infected mice. Both BKI-1770 and BKI-1841 had efficacy in the C. parvum newborn calf model, reducing diarrhea and oocyst excretion. However, both compounds caused hyperflexion of the limbs seen as dropped pasterns. Toxicity experiments in rats and calves dosed with BKI-1770 showed enlargement of the epiphyseal growth plate at doses only slightly higher than the efficacious dose. Mice were used as a screen to check for bone toxicity, by changes to the tibia epiphyseal growth plate, or neurological causes, by use of a locomotor activity box. These results showed neurological effects from both BKI-1770 and BKI-1841 and bone toxicity in mice from BKI-1770, indicating one or both effects may be contributing to toxicity. However, BKI-1708 remains a viable treatment candidate for further evaluation as it showed no signs of bone toxicity or neurological effects in mice.

The in vivo metabolism of Jungle Warfare in greyhounds.

Δ6-Methyltestosterone was reported as the main active ingredient of the purported "dietary supplement" Jungle Warfare. This compound is structurally similar to 17α-methyltestosterone, containing an additional Δ6 double bond, and is reported to possess notable androgenic activity, raising concerns over the potential for abuse of Jungle Warfare in sport. The in vivo metabolism of Δ6-methyltestosterone in greyhounds was investigated. Urinary phase I (unconjugated) and phase II (glucuronide) metabolites were detected following oral administration using liquid chromatography-mass spectrometry. No phase II sulfate metabolites were detected. The major phase I metabolite was confirmed as 16α,17ß-dihydroxy-17α-methylandrosta-4,6-dien-3-one by comparison with a synthetically-derived reference material. Minor amounts of the parent drug were also confirmed. Glucuronide conjugated metabolites were also observed, but were found to be resistant to hydrolysis using the Escherichia coli ß-glucuronidase enzyme. Qualitative excretion profiles, limits of detection, and extraction recoveries were determined for the parent drug and the major phase I metabolite. These results provide a method for the detection of Jungle Warfare abuse in greyhounds suitable for incorporation into routine screening methods conducted by anti-doping laboratories.

Engineering Pseudomonas aeruginosa arylsulfatase for hydrolysis of α-configured steroid sulfates.

Steroid sulfate esters are important metabolites for anti-doping efforts in sports, pathology and research. Analysis of these metabolites is facilitated by hydrolysis using either acid or enzymatic catalysis. Although enzymatic hydrolysis is preferred for operating at neutral pH, no known enzyme is capable of hydrolyzing all steroid sulfate metabolites. Pseudomonas aeruginosa arylsulfatase (PaS) is ideal for the hydrolysis of ß-configured steroid sulfates but like other known class I sulfatases it is inefficient at hydrolyzing α-configured steroid sulfates. We have used directed evolution with liquid chromatography mass spectrometry screening to find variants capable of hydrolyzing a α-configured steroid sulfate: etiocholanolone sulfate (ECS). After targeting two regions of PaS, four residues were identified and optimized to yield a final variant with a total of seven mutations (DRN-PaS) capable of hydrolyzing ECS ~80 times faster than the best PaS variant previously available. This DRN-PaS also shows improved activity for other α-configured steroid sulfates. Simultaneous mutagenesis was essential to obtain DRN-PaS due to complementarity between targeted residues.

Energy-Resolved Fragmentation Aiding the Structure Elucidation of Steroid Biomarkers.

The identification and confirmation of steroid sulfate metabolites in biological samples are essential to various fields, including anti-doping analysis and clinical sciences. Ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) is the leading method for the detection of intact steroid conjugates in biofluids, but because of the inherent complexity of biological samples and the low concentration of many targets of interest, metabolite identification based solely on mass spectrometry remains a major challenge. The confirmation of new metabolites typically depends on a comparison with synthetically derived reference materials that encompass a range of possible conjugation sites and stereochemistries. Herein, energy-resolved collision-induced dissociation (CID) is used as part of UHPLC-HRMS/MS analysis to distinguish between regio- and stereo-isomeric steroid sulfate compounds. This wholly MS-based approach was employed to guide the synthesis of reference materials to unambiguously confirm the identity of an equine steroid sulfate biomarker of testosterone propionate administration.

Synthesis of stable isotope labelled steroid bis(sulfate) conjugates and their behaviour in collision induced dissociation experiments.

Steroid bis(sulfate) metabolites derived from the two-fold sulfation of unconjugated precursors represent an important yet understudied portion of the steroid profile. The investigation of these compounds in fields such as medicine or anti-doping science relies on mass spectrometry (MS) as the principal tool to identify and quantify biomarkers of interest and depends in turn on access to steroid reference materials and their stable isotope labelled (SIL) derivatives. A new [18O] stable isotope label for sulfate metabolites is reported, which allows for the selective, late-stage and 'one-pot' synthesis of a variety of SIL-steroid conjugates suitable as MS probes and internal standards. The method is applied to more comprehensively study the MS behaviour of steroid bis(sulfate) compounds through collision-induced dissociation (CID) experiments.

Profiling Urinary Sulfate Metabolites With Mass Spectrometry.

The study of urinary phase II sulfate metabolites is central to understanding the role and fate of endogenous and exogenous compounds in biological systems. This study describes a new workflow for the untargeted metabolic profiling of sulfated metabolites in a urine matrix. Analysis was performed using ultra-high-performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS) with data dependent acquisition (DDA) coupled to an automated script-based data processing pipeline and differential metabolite level analysis. Sulfates were identified through k-means clustering analysis of sulfate ester derived MS/MS fragmentation intensities. The utility of the method was highlighted in two applications. Firstly, the urinary metabolome of a thoroughbred horse was examined before and after administration of the anabolic androgenic steroid (AAS) testosterone propionate. The analysis detected elevated levels of ten sulfated steroid metabolites, three of which were identified and confirmed by comparison with synthesised reference materials. This included 5α-androstane-3ß,17α-diol 3-sulfate, a previously unreported equine metabolite of testosterone propionate. Secondly, the hydrolytic activity of four sulfatase enzymes on pooled human urine was examined. This revealed that Pseudomonas aeruginosa arylsulfatases (PaS) enzymes possessed higher selectivity for the hydrolysis of sulfated metabolites than the commercially available Helix pomatia arylsulfatase (HpS). This novel method provides a rapid tool for the systematic, untargeted metabolic profiling of sulfated metabolites in a urinary matrix.

Circulating Conjugated and Unconjugated Vitamin D Metabolite Measurements by Liquid Chromatography Mass Spectrometry.

CONTEXT: Vitamin D status is conventionally defined by measurement of unconjugated circulating 25-hydroxyvitamin D (25OHD), but it remains uncertain whether this isolated analysis gives sufficient weight to vitamin D's diverse metabolic pathways and bioactivity. Emerging evidence has shown that phase II endocrine metabolites are important excretory or storage forms; however, the clinical significance of circulating phase II vitamin D metabolites remains uncertain. OBJECTIVE: In this study we analyzed the contribution of sulfate and glucuronide vitamin D metabolites relative to unconjugated levels in human serum. METHODS: An optimized enzyme hydrolysis method using recombinant arylsulfatase (Pseudomonas aeruginosa) and beta-glucuronidase (Escherichia coli) was combined with liquid chromatography mass spectrometry (LC-MS/MS) analysis to measure conjugated and unconjugated vitamin D metabolites 25OHD3, 25OHD2, 3-epi-25OHD3, and 24,25(OH)2D3. The method was applied to the analysis of 170 human serum samples from community-dwelling men aged over 70 years, categorized by vitamin D supplementation status, to evaluate the proportions of each conjugated and unconjugated fraction. RESULTS: As a proportion of total circulating vitamin D metabolites, sulfate conjugates (ranging between 18% and 53%) were a higher proportion than glucuronide conjugates (ranging between 2.7% and 11%). The proportion of conjugated 25OHD3 (48 ± 9%) was higher than 25OHD2 conjugates (29.1 ± 10%) across all supplementation groups. Conjugated metabolites correlated with their unconjugated forms for all 4 vitamin D metabolites (r = 0.85 to 0.97). CONCLUSION: Sulfated conjugates form a high proportion of circulating vitamin D metabolites, whereas glucuronide conjugates constitute a smaller fraction. Our findings principally in older men highlight the differences in abundance between metabolites and suggest a combination of both conjugated and unconjugated measurements may provide a more accurate assessment of vitamin D status.

Novel methyllycaconitine analogues selective for the α4ß2 over α7 nicotinic acetylcholine receptors.

Analogues of methyllycaconitine (MLA) based on a (3-ethyl-9-methylidene-3-azabicyclo[3.3.1]nonan-1-yl)methanol template have been designed and synthesised that incorporate the modified ester sidechains distinct from that present in the natural product. Electrophysiology experiments using Xenopus oocytes expressing nicotinic acetylcholine receptors (nAChRs) revealed selected analogues served as non-competitive inhibitors that showed selectivity for the α4ß2 over α7 nAChR subtypes, and selectivity for the (α4)3(ß2)2 over (α4)2(ß2)3 stoichiometry. This study more clearly defines the biological effects of MLA analogues and identifies strategies for the development of MLA analogues as selective ligands for the α4ß2 nAChR subtype.

The in vivo metabolism of Furazadrol in greyhounds.

Samples of the 'dietary supplement' Furazadrol sourced through the internet have been reported to contain the designer anabolic androgenic steroids [1',2']isoxazolo[4',5':2,3]-5α-androstan-17ß-ol (furazadrol F) and [1',2']isoxazolo[4',3':2,3]-5α-androstan-17ß-ol (isofurazadrol IF). These steroids contain an isoxazole fused to the A-ring and were designed to offer anabolic activity while evading detection, raising concerns over the potential for abuse of this preparation in sports. The metabolism of Furazadrol (F:IF, 10:1) was studied by in vivo methods in greyhounds. Urinary phase II Furazadrol metabolites were detected as glucuronides after a controlled administration. These phase II metabolites were subjected to enzymatic hydrolysis by Escherichia coli ß-glucuronidase to afford the corresponding phase I metabolites. Using a library of synthetically derived reference materials, the identities of seven urinary Furazadrol metabolites were confirmed. Major confirmed metabolites were isofurazadrol IF, 4α-hydroxyfurazadrol 4α-HF and 16α-hydroxy oxidised furazadrol 16α-HOF, whereas the minor confirmed metabolites were furazadrol F, 4ß-hydroxyfurazadrol 4ß-HF, 16ß-hydroxyfurazadrol 16ß-HF and 16ß-hydroxy oxidised furazadrol 16ß-HOF. One major hydroxyfurazadrol and two dihydroxyfurazadrol metabolites remained unidentified. Qualitative excretion profiles, limits of detection and extraction recoveries were established for furazadrol F and major confirmed metabolites. These investigations identify the key urinary metabolites of Furazadrol following oral administration, which can be incorporated into routine screening by anti-doping laboratories to aid the regulation of greyhound racing.

AE Succinimide, an Analogue of Methyllycaconitine, When Bound Generates a Nonconducting Conformation of the α4ß2 Nicotinic Acetylcholine Receptor.

Nicotinic acetylcholine (nACh) receptors are pentameric ligand-gated ion channels that mediate fast synaptic transmission. The α4ß2 nACh receptor is highly expressed in the brain and exists in two functional stoichiometries: the (α4)2(ß2)3 and (α4)3(ß2)2 that differ by an ACh-binding site at the α4-α4 interface of (α4)3(ß2)2 receptors. Methyllycaconitine (MLA) is an nACh receptor antagonist, and while potent at both α7 and α4ß2 nACh receptors, it has a higher selectivity for the α7 nACh receptor. The anthranilate-succinimide ester side-chain is important for its activity and selectivity. Here we identify a simplified MLA analogue that contains only the A and E ring skeleton of MLA, AE succinimide, that binds close to the channel lumen to display insurmountable inhibition at α4ß2 nACh receptors. Although inhibition by AE succinimide was found to be voltage-dependent indicating a possible pore channel blocker, substituted-cysteine accessibility experiments indicated it did not bind between 2'-16' region of the channel pore. Instead, we found that upon binding and in the presence of ACh, there is a conformational change to the channel membrane that was identified when the compound was assessed against (α4 V13'C)ß2 nACh receptors. It was found that in the 3:2 stoichiometry the two adjacent α4 subunits containing 13' cysteine mutations formed a disulfide bond and occluded ion conductance. This was reversed by treatment with the reducing agent, dithiothreitol. Thus, AE succinimide has a different mechanism of inhibition to both MLA and other AE analogues, such as AE bicyclic alcohol, in that upon binding to an as yet unidentified site, AE succinimide in the presence of ACh induces a conformational change to the channel that generates a ligand-bound closed state.

In vivo metabolism of the designer anabolic steroid hemapolin in the thoroughbred horse.

Hemapolin (2α,3α-epithio-17α-methyl-5α-androstan-17ß-ol) is a designer steroid that is an ingredient in several "dietary" and "nutritional" supplements available online. As an unusual chemical modification to the steroid A-ring could allow this compound to pass through antidoping screens undetected, the metabolism of hemapolin was investigated by an in vivo equine drug administration study coupled with GC-MS analysis. Following administration of synthetically prepared hemapolin to a thoroughbred horse, madol (17α-methyl-5α-androst-2-en-17ß-ol), reduced and dihydroxylated madol (17α-methyl-5α-androstane-2ß,3α,17ß-triol), and the isomeric enone metabolites 17ß-hydroxy-17α-methyl-5α-androst-3-en-2-one and 17ß-hydroxy-17α-methyl-5α-androst-2-en-4-one, were detected and confirmed in equine urine extracts by comparison with a library of synthetically derived reference materials. A number of additional madol derivatives derived from hydroxylation, dihydroxylation, and trihydroxylation were also detected but not fully identified by this approach. A yeast cell-based androgen receptor bioassay of available reference materials showed that hemapolin and many of the metabolites identified by this study were potent activators of the equine androgen receptor. This study reveals the metabolites resulting from the equine administration of the androgen hemapolin that can be incorporated into routine GC-MS antidoping screening and confirmation protocols to detect the illicit use of this agent in equine sports.

Synthesis of steroid bisglucuronide and sulfate glucuronide reference materials: Unearthing neglected treasures of steroid metabolism.

Doubly or bisconjugated steroid metabolites have long been known as minor components of the steroid profile that have traditionally been studied by laborious and indirect fractionation, hydrolysis and gas chromatography-mass spectrometry (GC-MS) analysis. Recently, the synthesis and characterisation of steroid bis(sulfate) (aka disulfate or bis-sulfate) reference materials enabled the liquid chromatography-tandem mass spectrometry (LC-MS/MS) study of this metabolite class and the development of a constant ion loss (CIL) scan method for the direct and untargeted detection of steroid bis(sulfate) metabolites. Methods for the direct LC-MS/MS detection of other bisconjugated steroids, such as steroid bisglucuronide and mixed steroid sulfate glucuronide metabolites, have great potential to reveal a more complete picture of the steroid profile. However, access to steroid bisglucuronide or sulfate glucuronide reference materials necessary for LC-MS/MS method development, metabolite identification or quantification is severely limited. In this work, ten steroid bisglucuronide and ten steroid sulfate glucuronide reference materials were synthesised through an ordered combination of chemical sulfation and/or enzymatic glucuronylation reactions. All compounds were purified and characterised using NMR and MS methods. Chemistry for the preparation of stable isotope labelled steroid {13C6}-glucuronide internal standards has also been developed and applied to the preparation of two selectively mono-labelled steroid bisglucuronide reference materials used to characterise more completely MS fragmentation pathways. The electrospray ionisation and fragmentation of the bisconjugated steroid reference materials has been studied. Preliminary targeted ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis of the reference materials prepared revealed the presence of three steroid sulfate glucuronides as endogenous human urinary metabolites.

SULFATION PATHWAYS: Alternate steroid sulfation pathways targeted by LC-MS/MS analysis of disulfates: application to prenatal diagnosis of steroid synthesis disorders.

The steroid disulfates (aka bis-sulfates) are a significant but minor fraction of the urinary steroid metabolome that have not been widely studied because major components are not hydrolyzed by the commercial sulfatases commonly used in steroid metabolomics. In early studies, conjugate fractionation followed by hydrolysis using acidified solvent (solvolysis) was used for the indirect detection of this fraction by GC-MS. This paper describes the application of a specific LC-MS/MS method for the direct identification of disulfates in urine, and their use as markers for the prenatal diagnosis of disorders causing reduced estriol production: STSD (steroid sulfatase deficiency), SLOS (Smith-Lemli-Opitz syndrome) and PORD (P450 oxidoreductase deficiency). Disulfates were detected by monitoring a constant ion loss (CIL) from the molecular di-anion. While focused on disulfates, our methodology included an analysis of intact steroid glucuronides and monosulfates because steroidogenic disorder diagnosis usually requires an examination of the complete steroid profile. In the disorders studied, a few individual steroids (as disulfates) were found particularly informative: pregn-5-ene-3ß,20S-diol, pregn-5-ene-3ß,21-diol (STSD, neonatal PORD) and 5α-pregnane-3ß,20S-diol (pregnancy PORD). Authentic steroid disulfates were synthesized for use in this study as aid to characterization. Tentative identification of 5ξ-pregn-7-ene-3ξ,20S-diol and 5ξ-pregn-7-ene-3ξ,17,20S-triol disulfates was also obtained in samples from SLOS affected pregnancies. Seven ratios between the detected metabolites were applied to distinguish the three selected disorders from control samples. Our results show the potential of the direct detection of steroid conjugates in the diagnosis of pathologies related with steroid biosynthesis.

Replacing PAPS: In vitro phase II sulfation of steroids with the liver S9 fraction employing ATP and sodium sulfate.

In vitro technologies provide the capacity to study drug metabolism where in vivo studies are precluded due to ethical or financial constraints. The metabolites generated by in vitro studies can assist anti-doping laboratories to develop protocols for the detection of novel substances that would otherwise evade routine screening efforts. In addition, professional bodies such as the Association of Official Racing Chemists (AORC) currently permit the use of in-vitro-derived reference materials for confirmation purposes providing additional impetus for the development of cost effective in vitro metabolism platforms. In this work, alternative conditions for in vitro phase II sulfation using human, equine or canine liver S9 fraction were developed, with adenosine triphosphate (ATP) and sodium sulfate in place of the expensive and unstable co-factor 3'-phosphoadenosine-5'-phosphosulfate (PAPS), and employed for the generation of six representative steroidal sulfates. Using these conditions, the equine in vitro phase II metabolism of the synthetic or so-called designer steroid furazadrol ([1',2']isoxazolo[4',5':2,3]-5α-androstan-17ß-ol) was investigated, with ATP and Na2 SO4 providing comparable metabolism to reactions using PAPS. The major in vitro metabolites of furazadrol matched those observed in a previously reported equine in vivo study. Finally, the equine in vitro phase II metabolism of the synthetic steroid superdrol (methasterone, 17ß-hydroxy-2α,17α-dimethyl-5α-androstan-3-one) was performed as a prediction of the in vivo metabolic profile.

Epiandrosterone sulfate prolongs the detectability of testosterone, 4-androstenedione, and dihydrotestosterone misuse by means of carbon isotope ratio mass spectrometry.

In the course of investigations into the metabolism of testosterone (T) by means of deuterated T and hydrogen isotope ratio mass spectrometry, a pronounced influence of the oral administration of T on sulfoconjugated steroid metabolites was observed. Especially in case of epiandrosterone sulfate (EPIA_S), the contribution of exogenous T to the urinary metabolite was traceable up to 8 days after a single oral dose of 40 mg of T. These findings initiated follow-up studies on the capability of EPIA_S to extend the detection of T and T analogue misuse by carbon isotope ratio (CIR) mass spectrometry in sports drug testing. Excretion study urine samples obtained after transdermal application of T and after oral administration of 4-androstenedione, dihydrotestosterone, and EPIA were investigated regarding urinary concentrations and CIR. With each administered steroid, EPIA_S was significantly depleted and prolonged the detectability when compared to routinely used steroidal target compounds by a factor of 2 to 5. In order to simplify the sample preparation procedure for sulfoconjugated compounds, enzymatic cleavage by Pseudomonas aeruginosa arylsulfatase was tested and implemented into CIR measurements for the first time. Further simplification was achieved by employing multidimensional gas chromatography to ensure the required peak purity for CIR determinations, instead of sample purification strategies using liquid chromatographic fractionation. Taking into account these results that demonstrate the unique and broad applicability of EPIA_S for the detection of illicit administrations of T or T-related steroids, careful consideration of how this steroid can be implemented into routine doping control analysis appears warranted. Copyright © 2017 John Wiley & Sons, Ltd.

Constant Ion Loss Method for the Untargeted Detection of Bis-sulfate Metabolites.

The untargeted detection of phase II metabolites is a key issue for the study of drug metabolism in biological systems. Sensitive and selective mass spectrometric (MS) techniques coupled to ultrahigh performance liquid chromatographic (UHPLC) systems are the most effective for this purpose. In this study, we evaluate different MS approaches with a triple quadrupole instrument for the untargeted detection of bis-sulfate metabolites. Bis-sulfates of 23 steroid metabolites were synthesized and their MS behavior was comprehensively studied. Bis-sulfates ionized preferentially as the dianion ([M - 2H]2-) with a small contribution of the monoanion ([M - H]-). Product ion spectra generated from the [M - 2H]2- precursor ions were dominated by the loss of HSO4- to generate two product ions, that is, the ion at m/z 97 (HSO4-) and the ion corresponding to the remaining monosulfate fragment. Other product ions were found to be specific for some structures. As an example, the loss of [CH3 + SO3]- was found to be important for several compounds with unsaturation adjacent to the sulfate. On the basis of the common behavior of the bis-sulfate metabolites two alternatives were evaluated for the untargeted detection of bis-sulfate metabolites (i) a precursor ion scan method using the ion at m/z 97 and (ii) a constant ion loss (CIL) method using the loss of HSO4-. Both methods allowed for the untargeted detection of the model compounds. Eight steroid bis-sulfates were synthesized in high purity in order to quantitatively evaluate the developed strategies. Lower limits of detection (2-20 ng/mL) were obtained using the CIL method. Additionally, the CIL method was found to be more specific in the detection of urinary bis-sulfates. The applicability of the CIL approach was demonstrated by determining progestogens altered during pregnancy and by detecting the bis-sulfate metabolites of tibolone.

The use of tandem yeast and mammalian cell in vitro androgen bioassays to detect androgens in internet-sourced sport supplements.

Sport supplements containing steroids never approved for therapeutic use have the potential for abuse by athletes. Most are marketed online and may contain undisclosed steroids yet are readily available despite lacking toxicological or pharmacological evaluation. In this study, 18 supplements purchased online underwent organic solvent extraction to isolate any steroids they contained. From the 18 supplements, 19 steroids were identified and for each, its intrinsic androgenic potency was determined by a yeast cell (Saccharomyces cerevisiae) androgen bioassay and its potential androgenic potency was determined by a liver (HuH7) cell androgen bioassay. The yeast bioassay showed that of the 19 steroids tested, 6 demonstrated strong intrinsic bioactivity, with 4 metabolically activated to even stronger androgens. Moreover, 4 steroids with moderate and 1 with intrinsically weak androgenic bioactivity were activated to more potent androgens. Finally, 8 steroids were metabolically inactivated or deactivated into weaker androgens. Our results show that Internet-sourced sport supplements may contain intrinsically strong androgens, or precursors that can be metabolized to them. These potentially potent pharmacologically active steroids are being used without regulatory control or consumer awareness of their potential adverse effects. Copyright © 2016 John Wiley & Sons, Ltd.

A review of designer anabolic steroids in equine sports.

In recent years, the potential for anabolic steroid abuse in equine sports has increased due to the growing availability of designer steroids. These compounds are readily accessible online in 'dietary' or 'nutritional' supplements and contain steroidal compounds which have never been tested or approved as veterinary agents. They typically have unusual structures or substitution and as a result may pass undetected through current anti-doping screening protocols, making them a significant concern for the integrity of the industry. Despite considerable focus in human sports, until recently there has been limited investigation into these compounds in equine systems. To effectively respond to the threat of designer steroids, a detailed understanding of their metabolism is needed to identify markers and metabolites arising from their misuse. A summary of the literature detailing the metabolism of these compounds in equine systems is presented with an aim to identify metabolites suitable for incorporation into screening protocols by anti-doping laboratories. The future of equine anti-doping research is likely to be guided by the incorporation of alternate testing matrices into routine screening, the improvement of in vitro technologies that can mimic in vivo equine metabolism, and the improvement of instrumentation or analytical methods that allow for the development of untargeted screening, and metabolomics approaches for use in anti-doping screening protocols. Copyright © 2016 John Wiley & Sons, Ltd.

Detection and metabolic investigations of a novel designer steroid: 3-chloro-17α-methyl-5α-androstan-17ß-ol.

In 2012, seized capsules containing white powder were analyzed to show the presence of unknown steroid-related compounds. Subsequent gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) investigations identified a mixture of 3α- and 3ß- isomers of the novel compound; 3-chloro-17α-methyl-5α-androstan-17ß-ol. Synthesis of authentic reference materials followed by comparison of NMR, GC-MS and gas chromatography-tandem mass spectrometry (GC-MS/MS) data confirmed the finding of a new 'designer' steroid. Furthermore, in vitro androgen bioassays showed potent activity highlighting the potential for doping using this steroid. Due to the potential toxicity of the halogenated steroid, in vitro metabolic investigations of 3α-chloro-17α-methyl-5α-androstan-17ß-ol using equine and human S9 liver fractions were performed. For equine, GC-MS/MS analysis identified the diagnostic 3α-chloro-17α-methyl-5α-androstane-16α,17ß-diol metabolite. For human, the 17α-methyl-5α-androstane-3α,17ß-diol metabolite was found. Results from these studies were used to verify the ability of GC-MS/MS precursor-ion scanning techniques to support untargeted detection strategies for designer steroids in anti-doping analyses. Copyright © 2015 John Wiley & Sons, Ltd.