W. Van Thuyne

Nutritional supplements: prevalence of use and contamination with doping agents

Based upon recent sales numbers, nutritional supplements play a key role in the lifestyle of a substantial proportion of the population. As well as products such as vitamins or minerals, several precursors of anabolic steroids are marketed as nutritional supplements. Another group of commercially available supplements are products for weight loss based upon herbal formulations originating from Ephedra species. Apart from supplements indicating the presence of these active compounds, numerous non-hormonal nutritional supplements were found that were contaminated with non-labelled anabolic steroids. Stimulating agents other than naturally occurring analogues of ephedrine were detected. A major group using dietary supplements are sportsmen, ranging from amateur level to elite athletes. Besides the possible health risks associated with the use of dietary supplements, athletes should take care not to violate the rules of the World Anti-Doping Agency because athletes remain responsible for substances detected in their biofluids, irrespective of their origin. Several analytical methods have been developed to determine the presence of doping agents as contaminants. The present review attempts to address the issues concerning the use of nutritional supplements and the detection of doping agents as contaminants in dietary supplements.

Validation of an extended method for the detection of the misuse of endogenous steroids in sports, including new hydroxylated metabolites

Endogenous steroids are amongst the most misused doping agents in sports. Their presence poses a major challenge for doping control laboratories. Current threshold levels do not allow for the detection of all endogenous steroid misuse due to great interindividual variations in urinary steroid concentrations. A method has been developed and validated to screen for traditionally monitored endogenous steroids in doping control as well as specific hydroxylated/oxygenated metabolites in order to enhance the detection capabilities for the misuse of endogenous steroids.

Implementation of gas chromatography combined with simultaneously selected ion monitoring and full scan mass spectrometry in doping analysis.

A comprehensive screening method for the detection of prohibited substances in doping control is described and validated. This method is capable of detecting over 150 components mentioned on the list of the World Anti-Doping Agency including anabolic androgenic steroids, stimulants and all narcotic agents that are currently analysed using different analytical methods. The analytes are extracted from urine by a combined extraction procedure using freshly distilled diethyl ether and tert-butyl methyl ether as extraction solvents at pH 9.5 and 14 respectively. Prior to GC-MS analysis the residues are combined and derivatised using a mixture of N-methyl-N-trimethylsilyltrifluoroacetamide, NH(4)I and ethanethiol. The mass spectrometer is simultaneously operated in the full scan mode (mass range varies along with GC-oven temperature program) and in the selected ion monitoring mode. The obtained limits of detection are in compliance with the requirements set by the World Anti-Doping Agency. Besides narcotics, stimulants and anabolic androgenic agents, this method is also capable of detecting several agents with anti-estrogenic activity and some beta-agonists. This comprehensive screening method reduces the amount of urine needed and increases the sample throughput without a loss in sensitivity and selectivity.

Urinary concentrations of morphine after the administration of herbal teas containing Papaveris fructus in relation to doping analysis

A quantitative method for the analysis of morphine in human urine in the concentration range between 0.25 and 2 microg/ml is described and validated. Morphine was determined after enzymatic hydrolysis of the urine. After liquid-liquid extraction with dichloromethane-methanol (9:1) at pH 9.5, morphine was derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) and analyzed with GC-MS (full scan). The limit of quantification of the method was 0.25 microg/ml. Two Papaveris fructus containing herbal teas were administered to five male volunteers and urine samples were taken quantitatively during the first 12 h after the administration. The morphine concentration in the tea was 10.4 and 31.5 microg/ml, respectively. Morphine was detected in the urine of all volunteers by 1 h after drinking the tea. Maximum morphine concentrations, 4.3 and 7.4 microg/ml, respectively, were obtained 4-6 h after administration. Doping positive urine samples were delivered for 1-9 h.

Interpretation of urinary concentrations of pseudoephedrine and its metabolite cathine in relation to doping control

Until the end of 2003 a urinary concentration of pseudoephedrine exceeding 25 microg/mL was regarded as a doping violation by the World Anti-Doping Agency. Since its removal from the prohibited list in 2004 the number of urine samples in which pseudoephedrine was detected in our laboratory increased substantially. Analysis of 116 in-competition samples containing pseudoephedrine in 2007 and 2008, revealed that 66% of these samples had a concentration of pseudoephedrine above 25 microg/mL. This corresponded to 1.4% of all tested in competition samples in that period. In the period 2001-2003 only 0.18% of all analysed in competition samples contained more than 25 microg/mL. Statistical comparison of the two periods showed that after the removal of pseudoephedrine from the list its use increased significantly. Of the individual sports compared between the two periods, only cycling is shown to yield a significant increase.Analysis of excretion urine samples after administration of a therapeutic daily dose (240 mg pseudoephedrine) in one administration showed that the threshold of 25 microg/mL can be exceeded. The same samples were also analysed for cathine, which has currently a threshold of 5 microg/mL on the prohibited list. The maximum urinary concentration of cathine also exceeded the threshold for some volunteers. Comparison of the measured cathine and pseudoephedrine concentrations only indicated a poor correlation between them. Hence, cathine is not a good indicator to control pseudopehedrine intake. To control the (ab)use of ephedrines in sports it is recommended that WADA reintroduce a threshold for pseudoephedrine.