Drug Testing 1esearch article and Analysis Received; ,. June ,01$ Revised; 1 September ,01$ <ccepted: $ September ,01$ =ublished online in 7iley Online Library 3!!!.drugtestinganalysis.com4 >%I 1..1..,?dta.17,@ Anabolic steroids detected in bodybuilding dietary supplements– a significant risk to public health V. Abbate,a A. T. Kicman,b M. Evans-Brown,c J. McVeigh,d D. A. #owan,b #. Wilson,e S. J. #olese and C. J. $alkerb& Twenty-four products suspected of containing anabolic steroids and sold in fitness equipment shops in the (nited Kingdom )(K) ere analyzed for their qualitative and semi-quantitative content using full scan gas chromatography-mass spectrometry ),#-M%*, accurate mass li'uid chromatography-mass spectrometry )-#-M%*, high pressure liquid chromatography ith diode array detection )./-#-"A"*, (V-Vis, and nuclear magnetic resonance )0M1* spectroscopy. 2n addition, 3-ray crystallography enabled the identification of one of the compounds, here reference standard as not available. 4f the 56 products tested, 57 contained steroids including kno n anabolic agents8 9: of these contained steroids that ere different to those indicated on the packaging and one product contained no steroid at all. 4verall, 97 different steroids ere identified8 95 of these are controlled in the (K under the Misuse of "rugs Act 9;<9. %everal of the products contained steroids that may be considered to have considerable pharmaco- logical activity, based on their chemical structures and the amounts present. This could un ittingly e=pose users to a significant risk to their health, hich is of particular concern for na>ve users. #opyright ? 5@96 !ohn $iley A %ons, -td. Additional supporting information may be found in the online version of this article at publisher’s web site. Key ords: anabolic steroids; dietary supplements; image-enhancing drugs; X-ray crystallography; hepatotoxicity; performance-enhancing drugs 2ntroduction Most of the anabolic steroids used in these products are pro- duced in bulk and obtained from wherever they can be cheaply In recent years, a large global uncontrolled market in human and reliably sourced, imported into countries such as the &nited enhancement drugs has developed.[1] This market is a result of States and the United Kingdom where they are processed and entrepreneurs exploiting demand from consumers, and is tightly packaged into finished products and sold in bricks-and-mortar coupled to increased manufacturing capacity of pharmacologically and online fitness equipment shops; their sale as‘dietary supple- active substances in countries with emerging economies, global- ments’ appears to be a ploy to circumvent regulatory systems such ized free trade, and modern communication and transport as those governing medicines. Consumers may find such products networks. Consumers looking to get a better body, empower them- attractive given the marketing practices used by manufacturers and selves and increase their well-being can no! choose from a broad distributors, as well as their ease of availability. %f particular concern range of drugs that claim to allow them to lose weight, brighten here is that potentially vulnerable groups such as teenage boys as their mood, modify their social and sexual behaviour, lighten or well as young men who face gro!ing pressures to conform to a tan their skin, enhance their cognitive function, and build muscle.[1] more stylized muscular body image,[23] may also use such In order to boost sales of these drugs, entrepreneurs have used a products; such a scenario has been seen before in the &nited States range of creative and apparently persuasive marketing strategies,[1] including the sale of these drugs as off-the-shelf‘dietary’,‘food’, or ‘herbal’ ‘supplements’.[1–4] One example of this practice in the &nited Kingdom is the sale of such drugs as bodybuilding‘dietary * Correspondence to: Chris Walker, Drug Control Centre, King’s College London, supplements’ which are often marketed as‘safer’ and‘legal’ !ranklin-Wilkins Building, $%& Stamford Street, London '($ )*H, ,K. ("mail: [email protected] replacements to established anabolic steroids and other [1] performance-enhancing drugs. Similar to the‘legal highs’ a Institute of /harmaceutical Science, King’s College London, Franklin-Wilkins phenomenon,[5,6] and to some degree overlapping,[7–1,] this Building, $%& Stamford Street, London '($ )*H, UK market has been driven by opportunists[13] who have searched b Drug Control Centre, King’s College London, Franklin-Wilkins Building, $%& the scientific and patent literature for active substances, which !ere Stamford Street, London '($ )*H, UK often originally synthesized as part of academic and pharmaceuti- cal research programmes but were not commercialized as c European Monitoring Centre for Drugs and Drug Addiction, Lisbon, /ortugal medicines.[14–,.] Importantly, when such products containing anabolic steroids first emerged in the mid to late 1//.s in the d Centre for Public +ealth, iverpool John Moores University, Liverpool, 2 3(4, UK &nited States, the substances were also selected on the basis that e (/'5C ,K National Crystallography Service, School of Chemistry, University of [21,,,] they were not sub0ect to drug control laws at that time. Southampton, Southampton, '6$7 $#J, ,K Drug 4est. Analysis35@964 Copyright 5 ,.1$ 6ohn 7iley 8 (ons, 9td. Drug Testing and Analysis V. Abbate et al. when supplements containing androstenedione were available monitoring of online shops, bodybuilding !ebsites discussing these from high street and online shops during the late 19/.s and early products, as !ell as from members of the gym community familiar ,...s.[21,,$] The primary aim of the current investigation was to !ith the use of these products. In order to examine any potential identify the specific active substance(s) and estimate the dose pres- variation in composition bet!een different samples of the same ent in a series of bodybuilding‘dietary supplements’ that were product, four products bearing the same name as those purchased suspected to contain anabolic steroids. %ften publications have from the bricks-and-mortar retailers !ere also purchased via the predominantly focused on the accuracy of the container label with Internet, making a total of ,. different products that !ere analy"ed. regard to !hether that steroid is present, without verification of Potassium hydroxide 3'%B4, anhydrous sodium sulfate, sodium amount. Little, if anything, is known about the toxicity or efficacy chloride, ethyl acetate, chloroform and hexane, all analytical of many of these anabolic steroids but, !ith the presumption that reagent grade, and high-performance liquid chromatography they are biologically active on the basis of their structures, knowing 3B=9#4 grade methanol, dichloromethane, acetonitrile, and trifluo- the dose can give an insight into whether these products are likely roacetic acid were purchased from Cisher Scientific 3Loughborough, to be pharmacologically significant. &sing spectroscopic and &'). >odecane, deuterochloroform, D-methyl-N-trimethylsilyl- spectrometric approaches any steroid present in the supplements trifluoroacetamide 31( C<4, ethanethiol, androst-4-ene-3,1+-dione, was characterized and, when estimated to be present in more than adrenosterone (androst-4-ene--,11,1+-trione), dehydroepiandros- 1 mg, the amount also determined. terone 3>BE<4 and ammonium iodide 3DB$I) !ere purchased from Sigma-Aldrich 3Poole, &'4. Cormic acid Analar grade was purchased from F7: 39utterworth, &'4. Curazabol, desoxymethyltestosterone Materials and methods 3>1T, madol) and methasterone were obtained through the World Association of Anti->oping Scientists 37<<>(4 as part of its < total of ,$ products were purchased from two fitness equipment educational programme. =urified !ater was obtained from E9G< shops one in 1erseyside 3n=$ products4 and one in Cheshire (nA-4 Purelab 1axima 3Fivendi Water (ystems, High Wycombe, &'). , and three online shops (nA); n=*; n=*4 3 able 14. The products were Methyl-1-testosterone and [1/,1/,1/- B-]-testosterone (d- 4 was selected after a revie! of information collected from Internet purchased from 9G# =romochem 3 eddington, &'). Table 1. 9ist of the products analyzed with the name of the active ingredient, dose and recommended serving as listed on the packaging. 3H spelling errors in the nomenclature in addition to‘a’ and‘b’ used forα andβ4 I Product Name 1anufacturer?(upplier 9abelled Contents 9abelled >ose Recommended per tablet or serving of tablet capsule 3mg4 or capsule 1 (uper ren-1G Black #hina Labs 1--ethyl-3mthoxy-gona-2,53104 diene-1+-oneH ,) 1/day , 1/-Nor ren Tri-City #hemicals 1/-Norandrosta-4,9-diene--,1+-dione -) ,--/day - ren Bomb Pharma 9abs 1+b-hydoxy-androstan-$-oneH 1. ,/day $ (uper Balo Black #hina Labs polydehydrogenated, polyhydroxylated ,) 1/day halomethetioallocholane ) (traight Epi Black #hina Labs ,,3a-Epithio-1+a-methyletioallo cholane-1+b-ol 1. 1/day * (traight Drol Black #hina Labs 1+a-dimethyl-5a-androst-3-one-1+b-ol 1. 1/day + (traight Phlexed Black #hina Labs 1+a-methyletioallocholan-2-ene-1+b-ol 1. 1/day @ Protodrol I Force Dutrition Protodrol ,) ,/day / Cynostane AI ,-cyano-17a-methyl-1+b-hydroxy-androst---one 1. 1/day 1. Fura"adrol Axis 9abs )a-etioallocholan[,,--c]furazan-1+b- ). ,--/day tetrahydropyranol ether 11 renavol-F Chaparral Labs. Estra-$,9-diene--,1+-dione -. 1--/day 1, &ltra 1ass <=( 1/-Norandrosta-$,9-diene--,1+-dione $. ,--/day 1+a-methyletioallocholan-2-ene-1+b-ol 1. 1- 11-Sterone Competitive Edge 9abs. <ndrost-$-ene-3,11,1+-trione +) --*/day 1$ P-1<G Competitive Edge 9abs. $-Chloro-1+a-methyl-andro-4-ene--,1+b-diol ,) ,--/day 1) Furuza-A Competitive Edge 9abs. )a-etioallocholan[2,--c]furazan-1+b- ). ,--/day tetrahydropyranol ether 1* Epivol Chaparral Labs. Estra-$,9-diene--,1+-dione -. 1-,/day ,a,3a-Epithio-1+a-methyletioallocholanol 1. 1+ 1-L1G Competitive Edge 9abs. 1--ethyl-3methoxy-gona-2,5(1.4 diene-1+-one ,) ,--/day 1@ X- ren Competitive Edge 9abs. 1/-Norandrosta-4,9-diene--,1+-dione -. --*/day 1/ Epivol Jlack Black #hina Labs ,,3a-Epithio-1+a-methyl-)a-androstan-1+b-ol 1.