Creatine and creatine forms intended for sports nutrition.

Creatine is a popular ergogenic supplement in sports nutrition. Yet, supplementation of creatine occasionally caused adverse effects such as gastrointestinal complaints, muscle cramps and an increase in body weight. Creatine monohydrate has already been evaluated by different competent authorities and several have come to the conclusion that a daily intake of 3 g creatine per person is unlikely to pose safety concerns, focusing on healthy adults with exclusion of pregnant and breastfeeding women. Possible vulnerable subgroups were also discussed in relation to the safety of creatine. The present review provides an up-to-date overview of the relevant information with special focus on human studies regarding the safety of creatine monohydrate and other marketed creatine forms, in particular creatine pyruvate, creatine citrate, creatine malate, creatine taurinate, creatine phosphate, creatine orotate, creatine ethyl ester, creatine pyroglutamate, creatine gluconate, and magnesium creatine chelate. Limited data are available with regard to the safety of the latter creatine forms. Considering an acceptable creatine intake of 3 g per day, most of the evaluated creatine forms are unlikely to pose safety concerns, however some safety concerns regarding a supplementary intake of creatine orotate, creatine phosphate, and magnesium creatine chelate are discussed here.

Emerging drugs affecting skeletal muscle function and mitochondrial biogenesis - Potential implications for sports drug testing programs.

RATIONALE: A plethora of compounds potentially leading to drug candidates that affect skeletal muscle function and, more specifically, mitochondrial biogenesis, has been under (pre)clinical investigation for rare as well as more common diseases. Some of these compounds could be the object of misuse by athletes aiming at artificial and/or illicit and drug-facilitated performance enhancement, necessitating preventive and proactive anti-doping measures. METHODS: Early warnings and the continuous retrieval and dissemination of information are crucial for sports drug testing laboratories as well as anti-doping authorities, as they assist in preparation of efficient doping control analytical strategies for potential future threats arising from new therapeutic developments. Scientific literature represents the main source of information, which yielded the herein discussed substances and therapeutic targets, which might become relevant for doping controls in the future. Where available, mass spectrometric data are presented, supporting the development of analytical strategies and characterization of compounds possibly identified in human sports drug testing samples. RESULTS & CONCLUSIONS: Focusing on skeletal muscle and mitochondrial biogenesis, numerous substances exhibiting agonistic or antagonistic actions on different cellular 'control centers' resulting in increased skeletal muscle mass, enhanced performance (as determined with laboratory animal models), and/or elevated amounts of mitochondria have been described. Substances of interest include agonists for REV-ERBα (e.g. SR9009, SR9011, SR10067, GSK4112), sirtuin 1 (e.g. SRT1720, SRT2104), adenosine monophosphate-activated protein kinase (AMPK, e.g. AICAR), peroxisome proliferator-activated receptor (PPAR)δ (e.g. GW1516, GW0742, L165041), and inhibitory/antagonistic agents targeting the methionine-folate cycle (MOTS-c), the general control non-derepressible 5 (GCN5) acetyl transferase (e.g. CPTH2, MB-3), myostatin (e.g. MYO-029), the myostatin receptor (bimagrumab), and myostatin receptor ligands (e.g. sotatercept, ACE-031). In addition, potentially relevant drug targets were identified, e.g. with the sarcoplasmic transmembrane peptide myoregulin and the nuclear receptor corepressor 1 (NCOR-1). The antagonism of these has shown to result in substantially enhanced physical performance in animals, necessitating the monitoring of strategies such as RNA interference regarding these substances. Most drug candidates are of lower molecular mass and comprise non-natural compositions, facts which suggest approaches for their qualitative identification in doping control samples by mass spectrometry. Electrospray ionization/collision-induced dissociation mass spectra of representatives of the aforementioned substances and selected in vitro derived phase-I metabolites support this assumption, and test methods for a subset of these have been recently established. Expanding the knowledge on analytical data will further facilitate the identification of such analytes and related compounds in confiscated material as well as sports drug testing specimens.

Quantifying cobalt in doping control urine samples--a pilot study.

Since first reports on the impact of metals such as manganese and cobalt on erythropoiesis were published in the late 1920s, cobaltous chloride became a viable though not widespread means for the treatment of anaemic conditions. Today, its use is de facto eliminated from clinical practice; however, its (mis)use in human as well as animal sport as an erythropoiesis-stimulating agent has been discussed frequently. In order to assess possible analytical options and to provide relevant information on the prevalence of cobalt use/misuse among athletes, urinary cobalt concentrations were determined by inductively coupled plasma-mass spectrometry (ICP-MS) from four groups of subjects. The cohorts consisted of (1) a reference population with specimens of 100 non-elite athletes (not being part of the doping control system), (2) a total of 96 doping control samples from endurance sport athletes, (3) elimination study urine samples collected from six individuals having ingested cobaltous chloride (500 µg/day) through dietary supplements, and (4) samples from people supplementing vitamin B12 (cobalamin) at 500 µg/day, accounting for approximately 22 µg of cobalt. The obtained results demonstrated that urinary cobalt concentrations of the reference population as well as the group of elite athletes were within normal ranges (0.1-2.2 ng/mL). A modest but significant difference between these two groups was observed (Wilcoxon rank sum test, p < 0.01) with the athletes' samples presenting slightly higher urinary cobalt levels. The elimination study urine specimens yielded cobalt concentrations between 40 and 318 ng/mL during the first 6 h post-administration, and levels remained elevated (>22 ng/mL) up to 33 h. Oral supplementation of 500 µg of cobalamin did not result in urinary cobalt concentrations > 2 ng/mL. Based on these pilot study data it is concluded that measuring the urinary concentration of cobalt can provide information indicating the use of cobaltous chloride by athletes. Additional studies are however required to elucidate further factors potentially influencing urinary cobalt levels.

Traditional Chinese medicine and sports drug testing: identification of natural steroid administration in doping control urine samples resulting from musk (pod) extracts.

The administration of musk extract, that is, ingredients obtained by extraction of the liquid secreted from the preputial gland or resulting grains of the male musk deer (eg, Moschus moschiferus), has been recommended in Traditional Chinese Medicine (TCM) applications and was listed in the Japanese pharmacopoeia for various indications requiring cardiovascular stimulation, anti-inflammatory medication or androgenic hormone therapy. Numerous steroidal components including cholesterol, 5α-androstane-3,17-dione, 5ß-androstane-3,17-dione, androsterone, etiocholanolone, epiandrosterone, 3ß-hydroxy-androst-5-en-17-one, androst-4-ene-3,17-dione and the corresponding urea adduct 3α-ureido-androst-4-en-17-one were characterised as natural ingredients of musk over several decades, implicating an issue concerning doping controls if used for the treatment of elite athletes. In the present study, the impact of musk extract administration on sports drug testing results of five females competing in an international sporting event is reported. In the course of routine doping controls, adverse analytical findings concerning the athletes' steroid profile, corroborated by isotope-ratio mass spectrometry (IRMS) data, were obtained. The athletes' medical advisors admitted the prescription of TCM-based musk pod preparations and provided musk pod samples for comparison purposes to clarify the antidoping rule violation. Steroid profiles, IRMS results, literature data and a musk sample obtained from a living musk deer of a local zoo conclusively demonstrated the use of musk pod extracts in all cases which, however, represented a doping offence as prohibited anabolic-androgenic steroids were administered.

Analysis of octopamine in human doping control samples.

The biogenic amine octopamine [4-(2-amino-1-hydroxyethyl)phenol] is prohibited in sports owing to its stimulating and performance-enhancing properties. Adverse analytical findings in athletes' doping control samples commonly result from surreptitious applications; however, the occurrence of octopamine in nutritional supplements and in selected invertebrates as well as the assumption that its N-methylated analog synephrine [4-(1-hydroxyethyl-2-methylamino)phenol, not banned by anti-doping authorities but currently monitored in prevalence studies) might be converted in-vivo into octopamine have necessitated a study to investigate the elimination of synephrine and octopamine present in over-the-counter products. Urine samples collected after administration of nutritional supplements containing octopamine and/or synephrine as well as urine samples collected after therapeutic application of octopamine- or synephrine-containing drugs were analyzed using a validated solid-phase extraction-based procedure employing a weak cation exchange resin and liquid chromatographic/tandem mass spectrometric detection with electrospray ionization and multiple reaction monitoring. In the case of therapeutic octopamine application, the urinary concentration of the target compound increased from baseline levels below the lower limit of detection to 142 µg/mL, while urine samples collected after synephrine as well as dietary supplement administration did not yield any evidence for elevated renal excretion of octopamine.

Plasticizers excreted in urine: indication of autologous blood transfusion in sports.

BACKGROUND: Misuse of autologous blood transfusions in sports remains undetectable. The metabolites of the plasticizer di-(2-ethylhexyl)phthalate (DEHP) were recently proposed as markers of blood transfusion, based on high urinary concentrations of these compounds observed in patients subjected to blood transfusion. This study evaluates DEHP metabolites in urine for detecting autologous blood transfusion. STUDY DESIGN AND METHODS: One blood bag was drawn from moderately trained subjects and the red blood cells (RBCs) were reinfused after different storage periods. Group 1 (12 subjects) was reinfused after 14 days, and Group 2 (13 subjects), after 28 days of storage. Urine samples were collected before and after reinfusion for determination of the concentrations of three DEHP metabolites, mono-(2-ethylhexyl)phthalate, mono-(2-ethyl-5-hydroxyhexyl)phthalate, and mono-(2-ethyl-5-oxohexyl)phthalate. RESULTS: Concentrations of DEHP metabolites on the days before reinfusion were in agreement with those described after common environmental exposure. A few hours after the reinfusion a significant increase was observed for all metabolites in all volunteers. Concentrations 1 day later were still higher (p < 0.05) than before reinfusion. Variations in urine dilution supported normalization by specific gravity. Concentrations of DEHP metabolites tended to be higher after longer storage times of RBCs. CONCLUSION: Autologous transfusion with RBCs stored in plastic bags provokes an acute increase in the urinary concentrations of DEHP metabolites, allowing the detection of this doping malpractice. The window of detection is approximately 2 days. The method might be applied to urine samples submitted for antidoping testing.

Endocrine characterization of the designer steroid methyl-1-testosterone: investigations on tissue-specific anabolic-androgenic potency, side effects, and metabolism.

Various products containing rarely characterized anabolic steroids are nowadays marketed as dietary supplements. Herein, the designer steroid methyl-1-testosterone (M1T) (17ß-hydroxy-17α-methyl-5α-androst-1-en-3-one) was identified, and its biological activity, potential adverse effects, and metabolism were investigated. The affinity of M1T toward the androgen receptor (AR) was tested in vitro using a yeast AR transactivation assay. Its tissue-specific androgenic and anabolic potency and potential adverse effects were studied in a Hershberger assay (sc or oral), and tissue weights and selected molecular markers were investigated. Determination of M1T and its metabolites was performed by gas chromatography mass spectrometry. In the yeast AR transactivation assay, M1T was characterized as potent androgen. In rats, M1T dose-dependently stimulated prostate and levator ani muscle weight after sc administration. Oral administration had no effect but stimulated proliferation in the prostate and modulated IGF-I and AR expression in the gastrocnemius muscle in a dose-dependent manner. Analysis of tyrosine aminotransferase expression provided evidence for a strong activity of M1T in the liver (much higher after oral administration). In rat urine, 17α-methyl-5α-androstane-3α,17ß-diol, M1T, and a hydroxylated metabolite were identified. In humans, M1T was confirmed in urine in addition to its main metabolites 17α-methyl-5α-androst-1-ene-3α,17ß-diol and 17α-methyl-5α-androstane-3α,17ß-diol. Additionally, the corresponding 17-epimers as well as 17ß-hydroxymethyl-17α-methyl-18-nor-5α-androsta-1,13-dien-3-one and its 17-epimer were detected, and their elimination kinetics was monitored. It was demonstrated that M1T is a potent androgenic and anabolic steroid after oral and sc administration. Obviously, this substance shows no selective AR modulator characteristics and might exhibit liver toxicity, especially after oral administration.

Trafficking of drug candidates relevant for sports drug testing: detection of non-approved therapeutics categorized as anabolic and gene doping agents in products distributed via the Internet.

Identifying the use of non-approved drugs by cheating athletes has been a great challenge for doping control laboratories. This is due to the additional complexities associated with identifying relatively unknown and uncharacterized compounds and their metabolites as opposed to known and well-studied therapeutics. In 2010, the prohibited drug candidates and gene doping substances AICAR and GW1516, together with the selective androgen receptor modulator (SARM) MK-2866 were obtained by the Cologne Doping Control Laboratory from Internet suppliers and their structure, quantity, and formulation elucidated. All three compounds proved authentic as determined by liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry and comparison to reference material. While AICAR was provided as a colourless powder in 100 mg aliquots, GW1516 was obtained as an orange/yellow suspension in water/glycerol (150 mg/ml), and MK-2866 (25 mg/ml) was shipped dissolved in polyethylene glycol (PEG) 300. In all cases, the quantified amounts were considerably lower than indicated on the label. The substances were delivered via courier, with packaging identifying them as containing 'amino acids' and 'green tea extract', arguably to circumvent customs control. Although all of the substances were declared 'for research only', their potential misuse in illicit performance-enhancement cannot be excluded; moreover sports drug testing authorities should be aware of the facile availability of black market copies of these drug candidates.

Seized designer supplement named "1-Androsterone": identification as 3ß-hydroxy-5α-androst-1-en-17-one and its urinary elimination.

New analogues of androgens that had never been available as approved drugs are marketed as "dietary supplement" recently. They are mainly advertised to promote muscle mass and are considered by the governmental authorities in various countries, as well as by the World Anti-doping Agency for sport, as being pharmacologically and/or chemically related to anabolic steroids. In the present study, we report the detection of a steroid in a product seized by the State Bureau of Criminal Investigation Schleswig-Holstein, Germany. The product "1-Androsterone" of the brand name "Advanced Muscle Science" was labeled to contain 100mg of "1-Androstene-3b-ol,17-one" per capsule. The product was analyzed underivatized and as bis-TMS derivative by GC-MS. The steroid was identified by comparison with chemically synthesized 3ß-hydroxy-5α-androst-1-en-17-one, prepared by reduction of 5α-androst-1-ene-3,17-dione with LS-Selectride (Lithium tris-isoamylborohydride), and by nuclear magnetic resonance. Semi-quantitation revealed an amount of 3ß-hydroxy-5α-androst-1-en-17-one in the capsules as labeled. Following oral administration to a male volunteer, the main urinary metabolites were monitored. 1-Testosterone (17ß-hydroxy-5α-androst-1-en-3-one), 1-androstenedione (5α-androst-1-ene-3,17-dione), 3α-hydroxy-5α-androst-1-en-17-one, 5α-androst-1-ene-3α,17ß-diol, and 5α-androst-1-ene-3ß,17ß-diol were detected besides the parent compound and two more metabolites (up to now not finally identified but most likely C-18 and C-19 hydroxylated 5α-androst-1-ene-3,17-diones). Additionally, common steroids of the urinary steroid profile were altered after the administration of "1-Androsterone". Especially the ratios of androsterone/etiocholanolone and 5α-/5ß-androstane-3α,17ß-diol and the concentration of 5α-dihydrotestosterone were influenced. 3α-Hydroxy-5α-androst-1-en-17-one appears to be suitable for the long-term detection of the steroid (ab-)use, as this characteristic metabolite was detectable in screening up to nine days after a single administration of one capsule.

Detection of Δ6-methyltestosterone in a "dietary supplement" and GC-MS/MS investigations on its urinary metabolism.

Since a few years more and more products have appeared on the market for dietary supplements containing steroids that had never been marketed as approved drugs, mostly without proper labeling of the contents. Syntheses and few data on pharmacological effects are available dated back mainly to the 1950s or 1960s. Only little knowledge exists about effects and side effects of these steroids in humans. The present study reports the identification of Δ6-methyltestosterone in a product named "Jungle Warfare", which was obtained from a web-based supplement store. The main urinary metabolites, 17α-hydroxy-17ß-methylandrosta-4,6-dien-3-one (Δ6-epimethyl-testosterone), 17α-methyl-5ß-androstane-3α,17ß-diol (3α,5ß-THMT), and 17ß-methyl-5ß-androstane-3α,17α-diol, as well as the parent compound excreted after a single oral administration were monitored by GC-MS/MS. Δ6-Epimethyltestosterone and 3α,5ß-THMT served for long-term detection (still present in the 181-189 h urine). 17α-Methyltestosterone and its 17-epimer were not detected in the urines (LOD 0.3ng/mL). The highest concentrations were found in the 14-20.5h urine for Δ6-epimethyltestosterone (600 ng/mL), and 3α,5ß-THMT (240 ng/mL) and in the 36-44.5h urine for 17ß-methyl-5ß-androstane-3α,17α-diol (7 ng/mL). For reference methyltestosterone and epimethyltestosterone were dehydrogenated with chloranil. The characterization of the products was performed by GC-MS(/MS) and NMR.

Identification of the growth-hormone-releasing peptide-2 (GHRP-2) in a nutritional supplement.

Black market products of a pharmaceutical nature and nutritional supplements have received substantial and increasing attention because of potential performance enhancement in elite and non-professional sports. In addition, improved general health is claimed for non-competing individuals. The risks and foreseeable dangers of the uncontrolled use of highly potent and non-approved pharmaceutical compounds in healthy individuals are of considerable concern. In the present case report, the emerging drug candidate GHRP-2 with verified growth-hormone-releasing properties was identified and quantified in tablets offered as an over-the-counter nutritional supplement. The impact of this orally active peptide on the hGH/IGF-axis has been established for several years and its illicit use in elite sports has been assumed. As a releasing factor for hGH, GHRP-2 belongs to the list of substances prohibited by the World Anti-Doping Agency (WADA). Unfortunately, to date there is no routinely performed assay for the determination of these peptides potentially occurring in biological fluids of competing athletes, but the present data will facilitate the implementation by providing principle analytical information on liquid chromatographic and mass spectrometric behaviour. Qualitative identification of the target analyte after extraction from the tablet matrix was performed by high resolution/high accuracy mass spectrometry after liquid chromatographic separation under consideration of the accurate masses and the ratios of the protonated molecules and their fragment ions derived from their collisionally induced dissociation. Quantitative results were obtained by means of liquid chromatography coupled to a triple quadrupole mass spectrometer and linear regression using an external calibration curve (with GHRP-2 reference compound) adjusted via internal standard (Hexarelin). Hereby, the content of GHRP-2 was determined with approximately 50 µg per tablet.

Sports-related issues and biochemistry of natural and synthetic anabolic substances.

Testosterone is the principal male sex hormone. As with all natural steroids, it is biosynthesized from cholesterol. Phase I metabolism employs some very specific enzymes and pathways. Phase II metabolism and excretion follow more general patterns. The effects of testosterone are twofold: anabolic and androgenic. Because of its anabolic effects, testosterone is frequently abused in sports. Because of its endogenous nature, testosterone doping is difficult to detect. The standard procedure is based on the evaluation of the urinary steroid profile. Conspicuous samples then are submitted to compound-specific (13)C/(12)C analysis. Synthetic and endogenous steroids differ in this measure. Numerous xenobiotic compounds have been derived from testosterone. The modifications typically aim at a reduction of the androgenic properties while maintaining the anabolic potential. Most of these compounds have been withdrawn from the legal market. However, they are found to be illicitly added to otherwise inefficient nutritional supplements. These products represent a major problem to doping control. Recently, clinical trials with selective androgen receptor modulators have been started.

Confiscated black market products and nutritional supplements with non-approved ingredients analyzed in the Cologne Doping Control Laboratory 2009.

Doping control laboratories are frequently confronted with new substances that may be misused by athletes. Besides new pharmaceuticals, where method development for their detection is dependent on the availability of the substance and corresponding administration studies, some professional and amateur athletes are using illicit 'black market' products, which either differ from known pharmaceuticals but cause similar effects or still are undergoing clinical trials and are therefore rarely available to doping control laboratories. In the Cologne Doping Control Laboratory, different confiscated products and legally obtained nutritional supplements were analyzed in 2009, and various findings were reported including GH-labelled injection vials without any pharmacologically active content; combinations of products indicating the attempt to mask growth hormone abuse; unpurified long-R(3) -IGF-1; nutritional supplements containing the growth hormone releasing peptide-2 (GHRP-2); and ampoules containing the selective androgen receptor modulator Andarine (S-4). This review provides an overview on the substances that were analyzed in 2009. Ingredients relevant for doping control were identified by means of liquid chromatography and mass spectrometry methods. 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.

Dietary supplement use among elite young German athletes.

Little is known about the prevalence and motives of supplement use among elite young athletes who compete on national and international levels. Therefore, the current survey was performed to assess information regarding the past and present use of dietary supplements among 164 elite young athletes (16.6 +/- 3.0 years of age). A 5-page questionnaire was designed to assess their past and present (last 4 weeks) use of vitamins, minerals, carbohydrate, protein, and fat supplements; sport drinks; and other ergogenic aids. Furthermore, information about motives, sources of advice, supplement sources, and supplement contamination was assessed. Eighty percent of all athletes reported using at least 1 supplement, and the prevalence of use was significantly higher in older athletes (p < .05). Among supplement users, minerals, vitamins, sport drinks, energy drinks, and carbohydrates were most frequently consumed. Only a minority of the athletes declared that they used protein/amino acids, creatine, or other ergogenic aids. Major motives for supplement use were health related, whereas performance enhancement and recommendations by others were less frequently reported. Supplements were mainly obtained from parents or by athletes themselves and were mostly purchased in pharmacies, supermarkets, and health-food stores. Among all athletes, only 36% were aware of the problem of supplement contamination. The survey shows that supplement use is common and widespread among German elite young athletes. This stands in strong contrast to recommendations by leading sport organizations against supplement use by underage athletes.

Metabolism of androsta-1,4,6-triene-3,17-dione and detection by gas chromatography/mass spectrometry in doping control.

The urinary metabolism of the irreversible aromatase inhibitor androsta-1,4,6-triene-3,17-dione was investigated. It is mainly excreted unchanged and as its 17beta-hydroxy analogue. For confirmation, 17beta-hydroxyandrosta-1,4,6-trien-3-one was synthesized and characterized by nuclear magnetic resonance (NMR) in addition to the parent compound. In addition, several reduced metabolites were detected in the post-administration urines, namely 17beta-hydroxyandrosta-1,4-dien-3-one (boldenone), 17beta-hydroxy-5beta-androst-1-en-3-one (boldenone metabolite), 17beta-hydroxyandrosta-4,6-dien-3-one, and androsta-4,6-diene-3,17-dione. The identification was performed by comparison of the metabolites with reference material utilizing gas chromatography/mass spectrometry (GC/MS) of the underivatized compounds and GC/MS and GC/tandem mass spectrometry (MS/MS) of their trimethylsilyl (TMS) derivatives. Alterations in the steroid profile were also observed, most obviously in the androsterone/testosterone ratio. Even if not explicitly listed, androsta-1,4,6-triene-3,17-dione is classified as a prohibited substance in sports by the World Anti-Doping Agency (WADA) due to its aromatase-inhibiting properties. In 2006 three samples from human routine sports doping control tested positive for metabolites of androsta-1,4,6-triene-3,17-dione. The samples were initially found suspicious for the boldenone metabolite 17beta-hydroxy-5beta-androst-1-en-3-one. Since metabolites of androst-4-ene-3,6,17-trione were also present in the urine samples, it is presumed that these findings were due to the administration of a product like 'Novedex Xtreme', which could be easily obtained from the sport supplement market.

Development of criteria for the detection of adrenosterone administration by gas chromatography-mass spectrometry and gas chromatography-combustion-isotope ratio mass spectrometry for doping control.

Adrenosterone (androst-4-ene-3,11,17-trione, 11-oxoandrostenedione) is an endogenous steroid hormone that has been promoted as a dietary supplement capable of reducing body fat and increasing muscle mass. It is proposed that adrenosterone may function as an inhibitor of the 11beta-hydroxysteroid dehydrogenase type 1 enzyme (11beta-HSD1), which is primarily responsible for reactivation of cortisol from cortisone. The urinary metabolism of adrenosterone was investigated, after a single oral administration in two male subjects, by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Substantially increased excretion of 11beta-hydroxyandrosterone, 11beta-hydroxyetiocholanolone, 11-oxoandrosterone and 11-oxoetiocholanolone was observed. Minor metabolites such as 3alpha,17beta-dihydroxy-5beta-androstan-11-one, 3alpha-hydroxyandrost-4-ene-11,17-dione and 3alpha,11beta-dihydroxyandrost-4-en-17-one were also identified. The exogenous origin of the most abundant adrenosterone metabolites was confirmed by GC-C-IRMS according to World Anti-Doping Agency criteria. Through analysis of a reference population data set obtained from urine samples provided by elite athlete volunteers (n = 85), GC-MS doping control screening criteria are proposed: 11beta-hydroxyandrosterone concentration greater than 10 000 ng/mL (specific gravity adjusted to 1.020) or 11beta-hydroxyandrosterone/11beta-hydroxyetiocholanolone ratio greater than 20.Urine samples fulfilling these screening criteria may be subjected to GC-C-IRMS analysis for confirmation of adrenosterone administration.

Nutritional supplements cross-contaminated and faked with doping substances.

Since 1999 several groups have analyzed nutritional supplements with mass spectrometric methods (GC/MS, LC/MS/MS) for contaminations and adulterations with doping substances. These investigations showed that nutritional supplements contained prohibited stimulants as ephedrines, caffeine, methylenedioxymetamphetamie and sibutramine, which were not declared on the labels. An international study performed in 2001 and 2002 on 634 nutritional supplements that were purchased in 13 different countries showed that about 15% of the nonhormonal nutritional supplements were contaminated with anabolic-androgenic steroids (mainly prohormones). Since 2002, also products intentionally faked with high amounts of 'classic' anabolic steroids such as metandienone, stanozolol, boldenone, dehydrochloromethyl-testosterone, oxandrolone etc. have been detected on the nutritional supplement market. These anabolic steroids were not declared on the labels either. The sources of these anabolic steroids are probably Chinese pharmaceutical companies, which sell bulk material of anabolic steroids. In 2005 vitamin C, multivitamin and magnesium tablets were confiscated, which contained cross-contaminations of stanozolol and metandienone. Since 2002 new 'designer' steroids such as prostanozol, methasterone, androstatrienedione etc. have been offered on the nutritional supplement market. In the near future also cross-contaminations with these steroids are expected. Recently a nutritional supplement for weight loss was found to contain the beta2-agonist clenbuterol. The application of such nutritional supplements is connected with a high risk of inadvertent doping cases and a health risk. For the detection of new 'designer' steroids in nutritional supplements, mass spectrometric strategies (GC/MS, LC/MS/MS) are presented.

Clenbuterol marketed as dietary supplement.

In several studies it has been demonstrated that products containing pharmaceutically active ingredients are marketed as dietary supplements. Most of these products contain anabolic steroids. Recently products for weight loss containing active drugs have also appeared on the market. In the present case a healthy male ordered the product 'Anabolic burner' via the Internet. The product was received from a German dispatcher and paid by bank transfer to a German bank account. After ingesting one tablet he 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 GC-MS for clenbuterol, yielding a content of about 30 microg per tablet. The beta-2 agonist clenbuterol is only legally available on prescription and is classified as 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.

High amounts of 17-methylated anabolic-androgenic steroids in effervescent tablets on the dietary supplement market.

In numerous studies it has been demonstrated that several nutritional supplements contain prohormones not declared on the label. In the current study two products (effervescent tablets) containing high amounts of the 17-methylated anabolic androgenic steroids metandienone (product 1: 16.8 mg/tablet) and stanozolol (product 2: 14.5 mg/tablet) were identified. Additionally in both products norandrostenedione was detected, in product 2 with minor amounts of several other steroids. The substances identified can cause enormous health risks. In addition, the use of the analyzed tablets can lead to positive doping results for metabolites of the respective steroids in sports. This study again shows the 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. For GC-MS identification of the analytes the trimethylsilyl derivatives of the steroids and the mixed N-t-butyldimethylsilyl,O-trimethylsilyl derivatives were used. The quantitation of metandienone, norandrostenedione, and stanozolol was performed using HPLC-DAD.

Determination of the origin of urinary norandrosterone traces by gas chromatography combustion isotope ratio mass spectrometry.

On the one hand, 19-norandrosterone (NA) is the most abundant metabolite of the synthetic anabolic steroid 19-nortestosterone and related prohormones. On the other hand, small amounts are biosynthesized by pregnant women and further evidence exists for physiological origin of this compound. The World Anti-Doping Agency (WADA) formerly introduced threshold concentrations of 2 or 5 ng of NA per ml of urine to discriminate 19-nortestosterone abuse from biosynthetic origin. Recent findings showed however, that formation of NA resulting in concentrations in the range of the threshold levels might be due to demethylation of androsterone in urine, and the WADA 2006 Prohibited List has defined NA as endogenous steroid. To elucidate the endogenous or exogenous origin of NA, (13)C/(12)C-analysis is the method of choice since synthetic 19-nortestosterone is derived from C(3)-plants by partial synthesis and shows delta(13)C(VPDB)-values of around -28 per thousand. Endogenous steroids are less depleted in (13)C due to a dietary mixture of C(3)- and C(4)-plants. An extensive cleanup based on two high performance liquid chromatography cleanup steps was applied to quality control and doping control samples, which contained NA in concentrations down to 2 ng per ml of urine. (13)C/(12)C-ratios of NA, androsterone and etiocholanolone were measured by gas chromatography/combustion/isotope ratio mass spectrometry. By comparing delta(13)C(VPDB)-values of androsterone as endogenous reference compound with NA, the origin of NA in doping control samples was determined as either endogenous or exogenous.