- freely available
Nutrients 2017, 9(10), 1093; doi:10.3390/nu9101093
2. Materials and Methods
- Evaluation of marketed dietary supplements for intended use in sports
- Evaluation of any type of prohibited substance, as defined by WADA (World Anti-Doping Agency. 2017 List of prohibited substances and methods. 2017 .)
- Only primary research was allowed, but secondary research was screened (by bibliography)
- No limits were set according language, years considered, or publication status or availability.
- Author/year: authors and year of publication.
- Country: geographical area from which the results obtained in the study come.
- Aim of the study: results that were intended to be achieved with the study.
- Sample: number and type of supplements analyzed.
- Methodology for the analysis of banned substances/metabolites/markers.
- Selected markers: tested substances/metabolites/markers that give positive results in anti-doping controls.
- Main results: final outcomes of the study, in which it is shown whether the proposed objectives have been achieved, and the main results obtained are listed.
- Conclusions: arguments and statements concerning the data obtained in the studies.
4.1. Consumption and Contamination of Ergonutritional Supplements
4.2. Anti-Doping Organizations
Conflicts of Interest
|NSAIDs||Nonsteroidal anti-inflammatory drugs|
|WADA||World Anti-Doping Agency|
|IOC||International Olympic Committee|
|TAS||Court of Arbitration for Sport|
|NADO||National Anti-Doping Organization|
|ASADA||Australian Sports Anti-Doping Authority|
|UKAD||UK Anti-Doping Authority|
|USADA||United States Anti-Doping Agency|
|NZVT||Anti-Doping Authority the Netherlands|
|AEPSAD||Spanish Agency for Health Protection in Sport|
|PSA||Primary secondary amine|
|UHPLC /MS/MS||Ultra-high pressure liquid chromatography tandem mass spectrometry|
|R3-IGF I||Growth factor 1|
|GHRP-2||Growth hormone releasing peptide-2|
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|Author/Year/Country||Aim||Sample||Selected Markers||Main Results||Conclusions|
|Van Thuyne., et al., 2006 ||South Africa||Determine whether the intake of contaminated dietary supplements can make anathlete positive in an anti-doping test.||5 male volunteers (24–55 years old) (11–99.4 kg)||19-nor-4-androstenediona y 4-androsten-3,17-diona||All exceeded the minimum amount established by WADA 2 h post-intake. Two exceeded the minimum amount 36 h post-intake. The maximum value was 54.6 ng/mL (8 h post)||The intake of only micrograms of contaminated substance can provoke a positive in an antidoping test|
|Geyer, H., et al., 2004 ||Germany||Analysis of 634 non-hormonal supplements to identify possible contamination of undeclared prohormones.||634 supplements||Testosterone and its prohormones, nandrolone and its prohormones and baldonone||Of the 634 supplements analyzed, 94 presented unidentified contaminants on their labeling||Despite offering guarantees in terms of pollutants, the population must be cautious about using ergonutricional substances, since not all these products are free of doping substances.|
|Green, H., et al., 2001 ||EEUU||Determine if steroids in supplements meet the Dietary Supplement Health and Education Act (DSHEA) labeling laws.||12 prohormones from 12 different brands, purchased from local stores.||5-androstenediol, 5-androstene-3,17-dione, 5-androstene-3b, 17b-diol, 4-androstene-3,17-dione, 5-androstene-3,17-diol, 19- Androstene-3b, 17b-diol, 4-androstene-3,17-diol, 19-nor-4-androstene-3,17-dione, 19-norandrostenedione, androstenedione, 19-nor-5-androstene-3,17-diol, Tribulus terrestris||Authors found that 11 of 12 brands tested did not meet the labeling requirements set out in the 1994 Dietary Supplement Health and Education Act. One brand contained 10 mg of testosterone, a controlled steroid, another contained 77% more than the label stated, and 11 of 12 contained less than the amount stated on the label.||The current study validates the concerns of physicians and sporting organizations that the labeling of some sports nutritional supplements does not accurately reflect what is contained in the product. This information may be helpful in deterring athletes from using substances that have unsubstantiated efficacy and unknown adverse effects.|
|Kamber, M., et al., 2001 ||Switzerland||Determine whether the products tested contain anabolic steroids or stimulants not indicated or poorly described on the label.||75 products||Anabolic steroids or stimulants not listed or poorly described on labeling||In 7 out of 17 prohormones, different substances than indicated on the labels were found. This corresponds to 41% of the products in this class of supplements and 9% of all analyzed supplements. In two other products (“mental enhancers”), caffeine and ephedrine were found. Both compounds were either not, or not clearly declared, on the labels (e.g., declaration of the plant Ma Huang that contains ephedrine). The concentration of ephedrine in product 56 was so high that an athlete would test positive for doping if only one capsule was consumed just before competition.||It is recommended that athletes use only supplements that are registered in Switzerland (and even these supplements may not be entirely free of contaminates). In light of the easy availability of medicines and nutritional supplements through the Internet, we should strive to inform and educate users (especially adolescents) about nutritional supplements, and support international standards for accurate product labeling.|
|Goel, D.P., et al., 2004 ||Canada||Address and determine the feasibility of conducting clinical tests on a dietary supplement (Cold-FX®) under strict International Olympics Committee (IOC) doping-control procedures and to determine whether ingesting this ginseng extract would result in any doping-control infractions among athletes.||20 men and 20 women||Ginseng Extract (Cold-FX)||No positives were found for prohibited substances in any of the subjects′ urine samples.||Cold-FX® substance is safe. This work could encourage companies to test dietary supplements so that athletes and athletic regulatory bodies have access to competent, comprehensive, credible, and unbiased information on the capacity for nutraceuticals and dietary supplements to induce positive urinalysis tests.|
|Baume, N., et al., 2006 ||Switzerland||To screen the supplements for con-taminations with major anabolic steroid parent compounds, stimulants and traces of testosterone, nandrolone and their precursors||103 supplements, divided into creatine, prohormones, mental enhancers and branched amino acids, all purchased online.||4-Androstenediol, 4-Norandrostenediol, 5-Androstenedione, 5-Norandrostenediol, 19-Noranrostenedione, Androstenediol, Androstenedione, Bolasterone, Boldenone, Clostebol, Dehydroepiandrosterone (DHEA), Dihydrotestosterone (DHT), Drostanolone, Fluoxymesterone, Mesterolone, Metandienone, Metenolone, Methyltestosterone, Norethandrolone, Oxandrolone, Oxymesterone, Stanozol, Oxymetholone, Nandrolone, Testosterone, Testosterone Propionate, Turinabol, 5-Norandrostenedione.||Methandienone was found in 3 of the 103 products. 18% of the products had errors in the labeling, while 18 products were found to contain metabolites of testosterone or nandrolone. The most commonly used contaminant was testosterone and the most contaminated product was prohormonesl.||More studies are needed to analyze contamination in products or poorly labeling, in order to prevent and improve the quality of dietary supplements available in the market.|
|Martello, S., et al., 2007 ||Italy||Validation of a qualitative LC-MS/MS method for the determination of eight doping substances.||64 supplements obtained from stores and court proceedings.||4-androsten-3,17-dione, 4-oestrene-3,17-dione, 5α-androsten-17β-ol-3-one, Boldenone, Nandrolone, Nandrolone Decanolate, Testosterone, Testosterone Decanolate, Ephedrine||This LCMS/MS method was applied to 64 nutritional supplements and 12.5% of tested substances contained prohibited substances (anabolic steroids and ephedrine) not stated on the labeling.||The method reported is sufficiently sensitive, specific and selective for the detection and confirmation of prohibited substances in nutritional supplements. The low levels of the compounds found in the samples may indicate accidental contamination and not intentional admixture. However, athletes should consider only purchasing from companies that perform quality tests on prohormones, and which test for possible contamination during production.|
|Parr, M. K., et al., 2008 ||Germany||Detection of clenbuterol in a sample of a fat burner||Sample of urine, 3 h post ingestion of a supplement tablet||Clenbuterol||After ingesting one tablet the participant reported tremor and delivered a urine sample. This urine was found to contain 2 ng/mL of clenbuterol utilizing LC-MS/MS analysis. Additionally the product itself was analyzed with gas chromatography coupled to mass spectrometry (GC-MS) for clenbuterol, yielding a content of about 30 µg per tablet.||The beta-2 agonist clenbuterol is only legally available on prescription and is classified as a prohibited doping substance in sports. The present case, for the first time, confirms the presence of clenbuterol in a dietary supplement. It again demonstrates the common problem with products on the supplement market, where non-licensed pharmaceuticals and doping substances are easily available. The ingestion of these products, containing additions of therapeutic drugs, can lead to side effects and/or interactions with conventional medicines.|
|Van Poucke, C., et al., 2007 ||Belgium||Determination of anabolic steroids in dietary supplements.||19 dietary supplements obtained via the internet from 12 different companies.||Α and β Boldenone, α and β Nortestosterone, 17α-Hydroxyprogesterone, Algeston acetophenide, Chloromadinon Acetate, Clostebol Acetate, Delmadinone Acetate, Fluoximesterone, Formeblone, Megestrol Acetate, Melengestrol Acetate, Methylboldenone, Methyltestosterone, Norethandrolone, Noretistestosterone, Norgestrel, Oxymetolone, Progesterone, Stanozol, Trenbolone, α and β Zeranol, D-equilenin, Dienestrol, Diethylstolbestrol, Ethinyl estradiol, Estradiol, Hexestrol, Testosterone, 16-dehydroprogesterone, 17α-acetoxyprogesterone, tenbolon 17β-acetate, 20β-hydroxyprogesterone, 3α and Β-hydroxy-5β-estrane-17-one, α-testosterone, ethylstiltranediol, Flugestonacetate, Medroxyprogesterone acetate, Mestranol, Metandriol, Metenolone, Methenolone acetate, Methylandrantranediol, Norethystostosterone acetate, Noretilnodrel, Vinylstestone.||According to the labeling, 15 of the 19 products contained 1–5 prohormones. Eleven products contained at least one anabolic component, all of these products claimed to contain prohormones.||The analysis of the 19 dietary supplements, indicates that the supplements named are not suitable for athletes. In addition to having prohormones that can be activated in the body, anabolic steroids were found in their active form.|
|Parr, M.K., et al., 2011 ||Germany||Identification of Δ6-methyltestosterone in a product named “Jungle Warfare“, which was obtained from a web-based supplement store||1 subject (52 years, 77 kg, 1.70 m)||(Epi-) methyltestosterone||The presence of the study metabolite was confirmed both in the analysis of the supplement and in the urine sample of the study subject.||The Jungle Warfare supplement represents another product labeled as a dietary supplement that contains steroids not approved for medical use.|
|Watson, P., et al., 2009 ||UK||To detect urinary excretion of nandrolone metabolites after ingestion of a precursor of nandrolone.||20 subjects (11 men and 9 women) recreational athletes||19-norandrostenedione (nandrolone)||With the intake of a supplement contaminated with 1 μg, no athlete would give a positive result in a doping control. In the case of 2.5 μg, 5 subjects would give a positive result and 15 subjects, would pass the minimum level (2 ng/mL) allowed, giving a positive result in the test of 5 μg of nandrolone.||Ingestion of trace amounts of 19-norandrostenedione can result in transient elevations of urinary 19-NA and 19-NE concentrations. The addition of as little as 2.5 kg of 19-norandrostenedione to a supplement (0.00005% contamination) appears sufficient to result in a doping violation in some individuals.|
|Parr, M.K., et al., 2007 ||Germany||Check the lack of safety in the production of supplements and obtaining supplements.||2 dietary supplements were analyzed (Stanozol-S and Parabolon-S) obtained by telephone.||Methandienone, norandrostenedione, stanozolol, testosterone, 5α-dihydrotestosterone, boldenone and estrone.||In Parabolon-S, metandienone was found. In addition, Stanozolol-S, stanozol, testosterone, 5α-dihydrotestosterone and boldenone were found.||There is insufficient surveillance of the production and trade of dietary supplements. Consumers should be aware of the enormous health and doping risks connected with the use of such products. New regulations for trade, production and labeling should be adopted. The first step should be a public warning to consumers and the withdrawal of dietary supplements containing prescription drugs.|
|Thevis, M., et al., 2013 ||Germany, EEUU and Switzerland||Study the ability to detect the origin of clenbuterol (therapeutic use or food intake) depending on the presence of racemic mixtures (enantiomers).||6 urine samples, collected from 2 male subjects||Stereoisomers + and – of Clenbuterol||The determination of relative abundances of clenbuterol enantiomers can indicate the ingestion of clenbuterol via contaminated food; however, depletion of (-)-clenbuterol in edible animal tissue is time-dependent and thus results can still be inconclusive as to the inadvertent ingestion of clenbutero, l when clenbuterol administration to animals was conducted until slaughter.|
|Monakhova, Y.B., et al., 2014 ||Germany||Test an magnetic resonance (NMR)-based method with minimal sample preparation for determination of 1,3-dimethylamylamine (DMAA) in sports nutrition and dietary supplements.||16 sports nutrition products and dietary supplements||1,3-dimethylamylamine||9 of the 16 substances were contaminated with DMAA.||Routine application of NMR is an alternative to time-consuming chromatographic methods for DMAA quantification in various kinds of products. 1H NMR spectroscopy has proven to be a robust analytical tool, yielding highly reliable quantitative results regarding DMAA, in a very short time. The approach is advantageous as it minimizes sample preparation and allows for the analysis of a large number of samples without human intervention (120 samples in a batch). The developed NMR method is recommended for use in food testing, customs and doping control laboratories, for the routine control of DMAA.|
|Abbate V., et al., 2014 ||United Kingdom||Determine any steroid present in the supplements, using full scan gas chromatography-mass spectrometry (GC-MS), accurate mass liquid chromatography-mass spectrometry (LC-MS), high pressure liquid chromatography with diode array detection (HPLC-DAD), UV-Vis, and nuclear magnetic resonance (NMR)||A total of 24 products were purchased from two fitness equipment|
shops—one in Merseyside and one in Cheshire—and three online shops.
|Anabolic steroids||Of the 24 products tested, 23 contained steroids, including known anabolic agents; sixteen of these contained steroids that were different to those indicated on the packaging and one product contained no steroids at all. Overall, 13 different steroids were identified; 12 of these are controlled in the UK under the Misuse of Drugs Act 1971. Several of the products contained steroids that may be considered to have considerable pharmacological activity, based on their chemical structures and the amounts present. This could unwittingly expose users to a significant risk to their health, which is of particular concern for naïve users.||The analytical methods used can play an essential role in the public health response to these drugs by providing methodologies to identify and quantify the active substance(s) present. This helps develop our understanding of this market, as well as allowing us to monitor the composition of supplements sold and the hazards that they may pose. When considered with other data, such as prevalence of use, these types of study play a central role n assessing and quantifying the risks to individual and public health.|
|Cooper E.R., et al. 2017 ||Australia||Characterize the androgenic bioactivity of sports supplements available from the Australian market, using yeast and mammalian cell androgen bioassays.||112 sports supplements available from the Australian market, either over the counter or via the Internet.||Androgens (Androgen bioactivity)||All 112 products did not declare an androgen on the label as an included ingredient. Our findings show that 6/112 supplements had strong androgenic bioactivity in the yeast cell bioassay, indicating products spiked or contaminated with androgens. The mammalian cell bioassay confirmed the strong androgenic bioactivity of 5/6 positive supplements. Supplement 6 was metabolized to weaker androgenic bioactivity in the mammalian cells. Further to this, supplement 6 showed a positive result in a yeast cell progestin bioassay.||These findings highlight that nutritional supplements, taken without medical supervision, could expose or predispose users to the adverse consequences of androgen abuse. The findings reinforce the need to increase the awareness of the dangers of nutritional supplements and highlight the challenges that clinicians face in the fast-growing market of nutritional supplements.|
|Cohen E.A., et al., 2013 ||USA||Detect the presence and concentration of N,alpha-Diethylphenylethylamine (N,α-DEPEA) in supplement Craze (Driven Sports, Inc.)||Three samples from different lot numbers of Craze||N,α-DEPEA||The identity of N,α-DEPEA was confirmed using nuclear magnetic resonance and reference standards. Manufacturer recommended servings were estimated to provide 21 to 35 mg of N,α-DEPEA. N,α-DEPEA has never been studied in humans. N,α-DEPEA is a methamphetamine analog; however, its stimulant, addictive and other adverse effects in humans are entirely unknown.||If the findings are confirmed by regulatory authorities, the Food and Drug Administration (FDA) should take immediate action to warn consumers and remove all N,α-DEPEA-containing supplements from the marketplace.|
|Stepan R., et al., 2008 ||Czech Republic||Analytical approach employing ethyl acetate extraction, dispersive solid-phase extraction (SPE) clean-up using PSA followed by an analysis of underivatized compounds, using comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometric detection (GCxGC-TOF MS) is presented.||Two types of commercially available solid nutritional supplements: protein concentrate and creatine monohydrate||Anabolic steroids||Results from this monitoring programme showed a total 6.3% (i.e., three) positive samples. Nandrolone (0.022 mg kg–1), testosterone (0.070 mg kg–1) and DHEA (0.063 mg kg–1) were found in a whey protein gainer, 5-androstan-3,17-dione (0.398 mg kg–1) and 19-norandrostendione (0.304 mg kg–1) in creatine pyruvate, and one sample of synephrine-based ‘fat burner’ contained progesterone (0.102 mg kg–1).||This analytical method, based on the comprehensive two-dimensional gas chromatography with timeof-flight mass spectrometric detection, provides an advantageous strategy for the determination of anabolic androgenic steroids and related compounds in nutritional supplements. The use of dispersive solid-phase extraction (SPE) with primary secondary amine (PSA) for the clean-up of crude extracts prepared from other matrices could likely be an efficient process for removing interferences and should be considered individually, according to the type of co-extracted matrix components.|
|Parr M.K., et al., 2011 ||Germany and Poland||Warn about the presence of designer steroids in some dietary or nutritional supplements.||Reports a few doping cases caused by the use of supplements with doping substances by athletes, noted by two World Anti-Doping Agency (WADA) accredited laboratories (Cologne and Warsaw)||Steroids||Steroids that may be interpreted as metabolites of Δ6-methyltestosterone, were detected. The availability of the athletes‘ supplements allowed for confirmation of this interpretation, as one of the products indeed contained Δ6-methyltestosterone. These findings confirmed the presumption that such products are used by athletes and that their consumption may lead to positive results in doping controls.||Top level athletes use “dietary supplements” that contain so-called designer steroids. The statistics of the World Anti-Doping Agency in recent years has reported some more cases with steroids that are only available in dubious products and not as approved pharmaceuticals. However, people outside of elite sport were also found to have used such designer supplements. Still more education on the health and doping risks of dietary supplement products seems to be necessary for the protection of both athletes and the general public.|
|Stajic A., et al., 2017 ||Serbia||Develop and validate the sensitive and reliable ultra-high pressure liquid chromatography tandem mass spectrometry (UHPLC/MS/MS) method for determination of higenamine in different dietary supplements.||Different dietary supplements of various compositions and pharmaceutical forms were collected. Among all collected supplements, 19 were of interest for higenamine analyses. Dietary supplements were purchased in sport shops, via the Internet or from the local pharmacy. Samples were taken from the original packages, adequately labeled and stored.||Higenamine||According to the results, most of the investigated supplements were free of higenamine, but on the other hand, the presence of higenamine was confirmed in some samples, while it was not declared on the label. Presence of higenamine, a banned substance, was confirmed in two investigated samples.||A sensitive and reliable UHPLC/MS/MS method for higenamine determination in various dietary supplements was developed and validated. This method was successfully applied for the analysis of 19 dietary supplements and, in this way, applicability of the method was confirmed.|
|Kohler M., et al., 2010 ||Germany||Provides an overview the products that were analyzed in the Cologne Doping Control Laboratory in 2009 and gives an overview on the classes of substances and the astonishingly small number of products that contain exactly the labelled substance.||A number of different products were analyzed from various sources, such as customs, police, and national anti-doping authorities, or were bought over-the-counter as nutritional supplements. Most of the commodities contained protein- or peptide-based substances, many of which were not in agreement with their respective labels or contained poorly purified analogues or artefacts.||Long-R3-IGF-I, GHRP-2, Andarine (S-4)||The products analyzed during 2009 showed that black market products nowadays also include different peptide hormone-derived products rather than steroid hormone preparations only. From the confiscated products, only 4 out of 11 contained the substance and amount declared on their label, and long- growth factor 1 (R3-IGF-I) and human growth hormones were the proteins detected (or at least labelled) most frequently (three products each), which may indicate that they are also ordered and used very often. In contrast, the nutritional supplements containing Growth Hormone releasing Peptide-2 (GHRP-2) as well as the glass bottle with Andarine (S-4) were labelled with xenobiotic ingredients, although none of them are approved as regular therapeutic agents yet.||The awareness of new products on the black market and in nutritional supplements is of utmost importance for laboratories to develop detection methods accordingly and screen for new substances as early as possible.|
|Thomas A., et al., 2010 ||Germany||The qualitative identification and quantification of the approximate content of GHRP-2 in tablets offered as nutritional supplements by means of liquid chromatography coupled to high resolution/high accuracy mass spectrometry, is described.||Nutritional supplements in tablets||GHRP-2 and Hexarelin||The presented case report demonstrates the urgency of flexible analytics in doping controls. Although, to date no positive doping cases with GHRP-2 were reported, the fact that the bioactive compound is available as a nutritional supplement, indicates that analytical findings in routinely analyzed plasma or urine samples from elite sportsmen are possible.|
|Kwiatkowska D., et al., 2015 ||Poland and Australia||Analyze urine samples to detecte stimulants and narcotics in anti-doping controls||The urine samples were taken from four athletes during in competition anti-doping control, and nutritional supplement NOXPUMP Pre-Training Formula (Fruit Punch)||Stimulants and narcotics||N,N-dimethyl-2-phenylpropan-1-amine (NN-DMPPA) is a possible new doping agent, detected and identified by the WADA-accredited laboratory in Warsaw (Poland) during routine anti-doping control. The presence of NN-DMPPA in several urine samples and in the supplement, NOXPUMP, was confirmed by GC-MS. In most of the athletes who failed urine drug tests because of the presence of NN-DMPPA, some other banned stimulants were also detected. NN-DMPPA was detected in the supplement NOXPUMP but we cannot exclude its present in other supplements from the black market.||This suggests the use of supplements which are often mixtures of prohibited drugs which may not be listed on the label or even the use of several supplements containing various banned substances. The range of supplements available in stores is constantly growing and while many supplements contain materials (e.g., vitamins, proteins, minerals) with possible useful properties, many pose the risk of unintentional doping with designer agents. This is often caused by the lack of labelling of all contents and/or unusual naming of components on the supplement label. Improved legistation, dealing with the commercialization of the drugs banned for sport, should be enacted.|
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