Analysis ofnon-hormonal Nutritional Supplements for Anabolic-Androgenic Steroids ± Results ofan International Study - PDF

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Analysis ofnon-hormonal al Supplements for Anabolic-Androgenic Steroids ± Results ofan International Study H. Geyer M. K. Parr U. Mareck U. Reinhart Y. Schrader W. Schänzer 14 Abstract Several recent studies

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Analysis ofnon-hormonal al Supplements for Anabolic-Androgenic Steroids ± Results ofan International Study H. Geyer M. K. Parr U. Mareck U. Reinhart Y. Schrader W. Schänzer 14 Abstract Several recent studies have shown evidence of some nutritional supplements containing prohibited anabolic androgenic steroids, so-called prohormones, which were not declared on the label. Therefore, a broad-based investigation of the international nutritional supplement market was initiated to clarify the extent of this problem. From ctober until November 1, 34 non-hormonal nutritional supplements were purchased in 13 countries from 15 different suppliers. Most supplements were bought in shops in the respective countries (57 samples = 91.%) and on the internet (5 samples =.%). 9 supplements were from prohormone-selling companies and 345 supplements came from companies which do not offer prohormones. After isolation from the supplement matrix 11 different anabolic androgenic steroids, mainly prohormones of testosterone and nandrolone, were analysed by gas-chromatography/ mass spectrometry. ut of the 34 samples analysed 94 (14.%) contained anabolic androgenic steroids not declared on the label (ªpositive supplementsº). We could not obtain reliable data for samples (.4%) due to matrix effects. In relation to the total number of products purchased per country, most of the positive supplements were bought in the Netherlands (5.%), in Austria (.7%), in the UK (1.%) and the USA (1.%). According to the label, all positive supplements were from companies located in only five countries: the USA, the Netherlands, the UK, Italy and Germany. 1.1% of the nutritional supplements from prohormone-selling companies contained anabolic androgenic steroids, whereas 9.% of the supplements from companies not selling prohormones were positive. The positive supplements showed anabolic androgenic steroid concentrations of.1 g/g up to 19 g/g. The administration of supplements containing nandrolone prohormones adding up to a total uptake of more than 1 g resulted in positive doping results for norandrosterone for several hours. Key words al supplements doping prohormones anabolic-androgenic steroids Introduction Since 199 anabolic androgenic steroids, so-called prohormones, are available on the US sports nutrition market. These substances are advertised as having enormous properties to increase muscle growth and strength. According to the doping regulations of the IC, these substances belong to the prohibited class of anabolic agents [9]. Several studies have shown that the labelling of prohormone supplements does not reflect their actual content. Many prohormone products contain prohormones as well as concentrations different from those declared on the labels [1,,,,13]. These mislabelling problems indicate an insufficient surveillance and quality control of the production of dietary supplements, especially with respect to the prohormone industry. Based on this knowledge, non-hormonal dietary supplements such as vitamins, minerals, and amino acids have also been analysed for prohormones. In several recent follow-up studies of positive doping cases it could be shown that even non-hormonal nutritional supplements may contain prohormones not declared Affiliation Institute of Biochemistry, German Sport University, Cologne, Germany Correspondence Dr. Hans Geyer Institute of Biochemistry, German Sport University Cologne Carl-Diem-Weg 5933 Köln Germany Phone: + 49() Fax: + 49() Accepted after revision: November, 3 Bibliography Int J Sports Med 4; 5: 14±19 Georg Thieme Verlag Stuttgart New York ISSN 17-4 DI.55/s on the label [5, ]. These prohormones may lead to positive results in doping tests, especially for the nandrolone metabolite norandrosterone. To clarify to which extent nutritional supplements contain not-declared prohormones, a broad-based investigation of the international nutritional supplement market was conducted. Materials and Methods Supplement description From ctober until November 1, 34 non-hormonal nutritional supplements were purchased in 13 different countries (Table 3). They were bought in shops (57 samples = 91.%), on the internet (5 samples =.%) and by telephone order ( samples =.3%). Two samples (.3%) were sent by the IC. The supplements were from 15 companies located in 15 different countries (Table 1). To our knowledge 57 of those companies also sell prohormones. 9 of the supplements (45.%) were purchased from prohormone-selling companies, 345 supplements (54.4%) from companies not selling prohormones. In terms of the formulations of the supplements we analysed 31 capsules, 31 tablets, 7 powders and 15 fluids. Table 1 rigin of the nutritional supplements (according to the label) Country No. of companies No. of samples USA 5 4 Germany UK 5 37 Netherlands 1 7 Italy 15 France Switzerland 14 Norway 7 1 Belgium Spain 5 7 Denmark 4 Sweden 1 Austria 1 5 Finland 1 1 Portugal 1 1 total Analysis of the nutritional supplements The analysis was performed according to the procedure described by Geyer et al. [5] with certain modifications. At least pulverised tablets or the content of at least capsules were homogenised. 1 g of the homogenised supplement is dissolved in 5 ml methanol. l of an internal standard solution (mixture of d3- nortestosterone, d3-testosterone and d3-noretiocholanolone in methanol.5 g/ml each) is added. After shaking for min and centrifugation for 5 min at 3 rpm the methanolic layer is transferred to another test tube and evaporated to dryness with a vacuum rotating evaporator. To the dried residue 5 ml of a sodium hydroxide solution.1 M (the ph is adjusted to values above 11.5) and 5 ml of n-pentane are added. After shaking for 5 min and centrifugation the n-pentane layer is transferred to another test tube. ml of a methanol/water solution (95 : 5, v: v) are added to the n-pentane, shaken for 5 min and centrifuged 5 min at 3 rpm. The n-pentane layer is discarded and l of a solution of 1-(N,N-diisopropylamine)-alkanes in methanol (Dipa 14 ± 3,.5 g/ml each) are added. The methanolic solution is evaporated to dryness in vacuo. The dry residue is derivatised with l of N-methyl-N-trimethylsilyltrifuoracetamide/NH 4 I/Ethanthiol (::3 v:w:v) at C for min. 3 l are injected into the gas chromatograph coupled to a mass selective detector (HP 9/HP 5973) and analysed in the SIM-mode with the characteristic ions for the bis-tms derivatives of the steroids of interest. For the gas-chromatographic separation a crosslinked capillary V1 column (HP 5 MS column, 1.5 m. mm i.d., film thickness.5 m) is used. The oven temperature was programmed from C at 4C/min to 19 C at 5 C/min to 4C and at 4 C/min to 3C. The following anabolic-androgenic steroids were included in the screening: testosterone and prohormones of testosterone: 4-androstene-3,17-dione (4-Andendion), dehydroepiandrosterone (DHEA), 5-androstene-3b,17b-diol (5-Andendiol), 4-androstene-3b,17b-diol (4-Andendiol), and testosterone (Test); nandrolone (19-nortestosterone) and prohormones of nandrolone: 19-nor-4-androstene-3,17-dione (4-Norendion), 19-nor-4-androstene-3b,17b-diol (4-Norendiol), 19-nor-5-androstene- 3b,17b-diol (5-Norendiol) and 19-nortestosterone (Nortest); the prohormone of boldenone: androstadiendion (Adiendione) and the prohormone of 5a-dihydrotestosterone: 5a-androstane-3b,17b-diol (5 a ± Adiol). For confirmation of the presence of the TMS derivatives of 4-androstene-3,17-dione (4-Andendion), 19-nor-4-androstene-3,17- dione (4-Norendion), testosterone (Test) and 19-nortestosterone (Nortest) GC/MS/MS was used additionally to receive more diagnostic ions. The following conditions were applied: GC/MS/MS system: GC Finnigan, GCQ; Injection: l; at 35 C; column: HP Ultra 1, 14 m,.5 mm i.d.,.11 m film thickness; carrier gas: Helium, split 1 :, head pressure psi; temperature programme: C with 4 C/min to 19 C, with 5 C/min to 4C, with 4C/min to 3 C, 3 min hold time; ionisation: 7 ev, electron impact (EI). For identification and quantification the following reference standards were used: DHEA, testosterone, 5a-androstane- 3b,17b-diol and 19-nor-4-androstene-3,17-dione from Sigma, St Louis, USA; 5-androstene-3b,17b-diol and 19-nortestosterone from Serva, Heidelberg, Germany; 4-androstene-3b,17b-diol, 19-nor-5-androstene-3b,17b-diol and androstadiendione from Steraloids, Wilton, USA; 4-androstene-3,17-dione from Schering, Berlin, Germany. The reference standard 19-nor-4-androstene- 3b,17b-diol was synthesized in our laboratory. As internal standard were used d3 ± 19-noretiocholanolone from Steraloids, Wilton, USA, and d3-testosterone and d3-nortestosterone, which were synthesized in our laboratory. 15 Geyer H et al. Anabolic Steroids in al Supplements ¼ Int J Sports Med 4; 5: 14± 19 1 The limit of detection of the method was evaluated in different matrices for all substances mentioned above, and was estimated at about.5 ±.1 g/g. nly concentrations above.1 g/g were considered in this study. The recoveries were determined in different matrices and are listed in the following for a creatine matrix: 5-Norendiol %; 4- Norendiol 7%; DHEA 55%; 4-Andendiol 1%; 4-Norendion 33%; 5-Andendiol 9%; Nordest 3%; 4-Andendion 43%; Test 54%. Excretion studies with nutritional supplements Four male volunteers (age: 4.7. years, height: cm, weight: kg) received several nutritional supplements with known prohormone concentrations. The volunteers were users of the administered supplements. They were informed about the content of low amounts of prohormones in the products and they gave a written consent to participate in an excretion study. Before the administration of the products it could be shown that their urine did not contain norandrosterone and no testosterone/epitestosterone ratio higher than six. Before and several hours after the administration of the supplements, urine samples were collected and analysed with gas chromatography/mass spectrometry for the glucuronides of norandrosterone, testosterone and epitestosterone, according to the screening procedure for anabolic steroids [3]. Results al supplements ut of the 34 samples analysed 94 (14.%) samples contained prohormones not declared on the label. We could not obtain reliable data for samples (.4%) because of matrix effects. ut of all positive supplements 3 samples (4.5%) contained prohormones of nandrolone and testosterone, 4 samples (.1%) only contained prohormones of testosterone, 7 samples (7.5%) only contained prohormones of nandrolone. None of the samples contained the prohormone of boldenone. Most of the positive results were found for capsules. 19.% of the capsules ( of 31), 11.7% of the tablets (7 of 31) and.9% of the powders (5 of 7) showed positive results. In the 15 analysed fluids no anabolic steroids were found. The main ingredients declared on the labels of positive samples are listed in Table. Anabolic androgenic steroids were found in nearly all kinds of nutritional supplements. The distribution of the concentrations of anabolic androgenic steroids in the positive nutritional supplements is shown in Figs. 1±3. 4.5% of the positive supplements showed a total anabolic androgenic steroid concentration higher than 5 g/g. Four samples (4.3%) had a testosterone concentration higher than 4 ng/g. With regard to the prohormones of nandrolone, 1.% of the positive supplements contained more than 1 g/g (Fig. ). number of samples Table Main ingredients declared on the labels of positive samples amino acids/(hydrolysed) proteins minerals herbal extracts vitamins carnitin/acetyl-l-carnitine creatine HMB pyruvate ribose fatty acids/oils caffeine chondroitin (sulfate) lysophosphatidylcholin methylsulfonylmethane enzymes (N-acetyl) glucosamine (sulfate) chrysin melatonin hemoglobin inosine NAC inositol lecithin choline Most positive samples contained DHEA (3.%), 4-Andendion (4.3%), 4-Norendion (7.4%), 4-Andendiol (7.4%) and/or 5-Andendiol (1.1%) (Fig. 5) Fig. 1 Distribution of the total prohormone concentrations (g/g) in positive samples (n = 94). number of samples Fig. Distribution of the nandrolone prohormone concentrations (g/g) in positive samples (n = 94). The number of prohormones per positive sample is presented in Fig. 4. In 47.9% of the positive samples more than 1 prohormone was found. samples (.1%) contained as many as 7 different prohormones. Most of the positive products were bought in the USA and in Germany. In relation to the total number of products purchased, most positive supplements were bought in the Netherlands (5.%), in Austria (.7%), the UK (1.9%) and the USA (1.%, Table 3). Geyer H et al. Anabolic Steroids in al Supplements ¼ Int J Sports Med 4; 5: 14±19 number of samples Fig. 3 Distribution of the testosterone prohormone concentrations (g/g) in positive samples (n = 94). number of supplements Nortest 4-Norendion 4 5-Norendiol 4-Norendiol Test 44 DHEA 4-Andendion 5-Andendiol 4-Andendiol 1 5a-Adiol Adiendione Fig. 5 Number of positive supplements containing specific prohormones. number of samples number of prohormones Fig. 4 Number of detected steroids per positive supplement USA Netherlands Germany UK Italy Fig. rigin of the positive samples according to the label. number of supplements According to the label all positive supplements were from companies located in only five countries: the USA, the Netherlands, the UK, Italy and Germany (Fig. ). 1.1% of the nutritional supplements from prohormone-selling companies contained anabolic androgenic steroids, whereas 9.% of the supplements from companies not selling prohormones were positive (Fig. 7). Excretion studies Excretion studies with supplements which contained 19-nor-4- androstene-3,17-dione (4-Norendion) in amounts of. g, 3. g, 5. g and 4. g resulted in maximum urinary norandrosterone concentrations of.5 ng/ml, 11. ng/ml, 1. ng/ml and 1. ng/ml, respectively, after hours (Fig. ). After the application of various small amounts of prohormones of testosterone the ratio of testosterone/epitestosterone showed the normal variation and did not increase. Discussion Table 3 al supplements containing prohormones, in relation to the total number of supplements purchased in different countries Country No. of products No. of positives Percentage of positives Netherlands 31 5.% Austria 5.7% UK % USA % Italy % Spain % Germany % Belgium 3.7% France 3.7% Norway % Switzerland 13 ± ± Sweden ± ± Hungary ± ± total % 17 The results of this broad-based study, showing that about 15% of the analysed non-hormonal supplements contained undeclared anabolic androgenic steroids, confirm the results of preliminary studies [5, ]. In the present study, about 3% of all positive supplements contained prohormones of nandrolone. This proportion was much higher (3%) in a previous study conducted in our laboratory Geyer H et al. Anabolic Steroids in al Supplements ¼ Int J Sports Med 4; 5: 14± 19 1 number of supplements []. This difference is probably due to the fact that previously investigated nutritional supplements were mainly analysed in follow-up studies of positive norandrosterone cases. For about % of the analysed nutritional supplements we could not obtain reliable data, due to analytical problems with the matrix. Changes 7 negative positive 1 33 non-prohormone sellers prohormone sellers Fig. 7 Number of positive supplements among products from prohormone and non-prohormone sellers. norandrosterone [ng/ml] 15 5, µg 4-Norendion 3,7 µg 4-Norendion 5, µg 4-Norendion 4, µg 4-Norendion 4 time after application [h] Fig. Urinary concentration of norandrosterone after the administration of nutritional supplements containing different amounts of 19-nor-4-androstene-3,17-dione (4-Norendion). Nandrolone H 19-Nor-4-androstene-3,17-dione H H 19-Nor-4-androstene-3, 17 -diol H H 19-Nor-5-androstene-3, 17 -diol 19-Norandrosterone Fig. 9 The urinary norandrosterone as a common metabolite of nandrolone (19-nortestosterone) and nandrolone prohormones. H H of the analytical procedures, especially in the extraction and derivatisation steps, could not improve the results. Momentarily we do not exactly know what kind of matrix is responsible for the analytical problems. The concentrations of the anabolic androgenic steroids in the nutritional supplements are rough estimates. In some matrices and for some anabolic-androgenic steroids, especially DHEA, concentrations lower.1 g/g could be detected. These results were not reported. The amounts of anabolic androgenic steroids in the analysed supplements are much lower than the amounts in the lowest concentrations found in commercially available prohormone supplements. These contain at least g of prohormones (Figs.1±3). According to studies with androstendione [4,11,1], physiological effects or adverse effects after the oral administration of amounts as low as those detected in the positive nutritional supplements, are unlikely but cannot be entirely excluded. We suppose that the low concentrations of anabolic androgenic steroids in non-hormonal nutritional supplements are not intentional admixtures to improve the claimed ªperformance enhancing effectsº of a supplement, but are so-called cross-contaminations. These cross-contaminations are possible, because manufacturers of nutritional supplements do not have to adhere to GMP (Good Manufacturing Practices), as required for the pharmaceutical industry. We suppose that contaminations occur in companies which process and transport prohormones. If the same machines and vessels are used for the processing of other nutritional supplements such as vitamins, minerals, etc., contaminations of these ªnon-hormonalº supplements could result from insufficient cleaning of the processing tools and vessels. Another indicator of cross-contamination is the varying concentration of contaminants found in different products. Such varying concentrations have also been shown in our previous studies [5, ]. In most positive products DHEA, 4-Andendion, 4-Andendiol and 4-Norendion were found (Fig. 5). These anabolic androgenic steroids are probably the most common prohormones on the nutritional supplement market. Testosterone was found in.% of the positive nutritional supplements. Testosterone is not permitted in the nutritional supplement market. Testosterone is probably a by-product of the synthesis of testosterone prohormones, which is not sufficiently separated after synthesis. In previous studies on prohormones testosterone has also been detected in prohormone supplements [1]. In all countries except Switzerland, Sweden and Hungary positive nutritional supplements were among those purchased (Table 3). However, fewer supplements were bought in those countries. In relation to the total number of supplements purchased in different countries, most positive supplements were bought in the Netherlands (5.%), Austria (.7%), the UK (1.9%) and the USA (1.%) (Table 3). The high percentage of positive nutritional supplements from Austria is confirmed in a recently published paper. In this study the analysis of about 5 nutritional supplements found that % of the supplements contained anabolic-androgenic steroids not declared on the label [7]. Geyer H et al. Anabolic Steroids in al Supplements ¼ Int J Sports Med 4; 5: 14±19 As shown in Fig., most positive supplements are from companies which, according to the label, are located in the USA. In most cases the label does not clearly indicate where a nutritional supplement has been produced. According to the legislation it is not necessary for nutritional supplement companies to list this information on the label. Thus, it is possible for a Dutch company to hire a producer in the USA or China, without providing any information to the consumer. such nutritional supplements can lead to positive results in doping tests, especially for the nandrolone metabolite norandrosterone. To minimize the risk of contaminations athletes should only buy nutritional supplements from companies which per
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