P. Van Eenoo

Development and validation of an open screening method for diuretics, stimulants and selected compounds in human urine by UHPLC-HRMS for doping control.

A new doping control screening method for the analysis of diuretics and stimulants using ultra high pressure liquid chromatography-high resolution Orbitrap mass spectrometry has been developed. The screening was performed in full scan MS with scan-to-scan polarity switching which allowed to detect more than 120 target analytes. Sample preparation was limited to 10-fold dilution of the urine into the internal standard solution followed by injection. Total run time per sample was 10 min. Validation of the method yielded detection limits for diuretics between 25 and 250 ng mL(-1) and for stimulants between 5 and 500 ng mL(-1). The screening method has been implemented in routine doping control.

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.

Simultaneous quantitation of ephedrines in urine by gas chromatography-nitrogen-phosphorus detection for doping control purposes

A gas chromatographic method for the simultaneous quantitation of ephedrine, pseudoephedrine, norephedrine (phenylpropanolamine), norpseudoephedrine (cathine) and methylephedrine in urine is described. The method consists of a liquid-liquid extraction with tert.-butyl methyl ether at pH 14. The extracts are analysed on a GC system equipped with an Rtx-5 Amine column and a nitrogen-phosphorus detector. Method validation shows excellent separation, linearity, specificity, accuracy, precision, intra-laboratory repeatability and reproducibility, making the method especially suitable for quantitation of ephedrines in urine samples for doping control purposes. A statistical analysis on the abuse of the different ephedrines in urine from athletes controlled in the Flemish doping control laboratory during the period 1993-2000 is included.

Development and validation of an LC-MS/MS method for the quantification of ephedrines in urine.

The objective of this study was to develop a simple and robust LC-MS/MS method for the quantification of ephedrine type substances in urine. Sample preparation consisted of a 10-fold dilution step of the samples into the internal standard solution (ephedrine-d(3), 4 microg/mL in water). Baseline separation of the diastereoisomers norpseudoephedrine-norephedrine and ephedrine-pseudoephedrine was performed on a C8-column using isocratic conditions followed by positive electrospray ionisation and tandem mass spectrometric detection. The mobile phase consisted of 98/2 (H(2)O/ACN) containing 0.1% HAc and 0.01% TFA. Calibration curves were constructed between 2.5 and 10 microg/mL for norephedrine and norpseudoephedrine and 5 and 20 microg/mL for ephedrine, pseudoephedrine and methylephedrine. The bias ranged from -5.5 to 12% for norephedrine, -4.1 to 8.0 % for norpseudoephedrine, 0.3 to 2.1 % for ephedrine, 1.6 to 2.6 % for pseudoephedrine and 2.9 to 5.0 % for methylephedrine. Precision of the method varied between 2.8 and 10.4% for all compounds and the matrix effect was less than 15%. The applicability of the method has been checked by the analysis of 40 urine samples. The results were compared with those obtained with the common GC-NPD method. Results show a good correlation between both methods with correlation coefficients higher than 0.95 for all analytes.

Qualitative detection of diuretics and acidic metabolites of other doping agents in human urine by high-performance liquid chromatography-tandem mass spectrometry: comparison between liquid-liquid extraction and direct injection.

Direct injection of urine has gained interest in the field of analytical toxicology, including doping control analysis. However, implementation of a direct urinalysis method for the LC-MS/MS detection of 34 diuretics and 9 other doping agents yielded several analytical problems, which were not observed using a traditional liquid-liquid extraction. Therefore a comparative study was made between liquid-liquid extraction and direct injection. Comparison of validation results showed that the liquid-liquid extraction at pH 7 allows to analyze samples without major drawbacks regarding matrix effects. Hence, good sensitivity was observed and detection limits ranged between 1 and 250 ng/mL for all compounds. In the direct injection approach shifted retention times were observed for several acidic and basic compounds due to unwanted matrix effects. This shift was reduced by a 25-fold dilution of the urine samples. Besides the improved retention time stability the diluted samples also exhibited lower ion suppression than the undiluted ones. After 25-fold dilution, detection limits ranged between 10 and 250 ng/mL for all compounds. Since these detection limits are at or below the minimum required performance level, imposed by the World Anti-Doping Agency, the method could be applied to routine anti-doping analysis. Samples, previously declared positive, were reanalysed using both the liquid-liquid extraction and direct injection. With both techniques all 26 samples were found to be positive, showing the applicability of direct injection for the analysis of diuretics.

Prevalence of legal and illegal stimulating agents in sports

This paper reviews the prevalence of legal and illegal stimulants in relation to doping-control analysis. Stimulants are among the oldest classes of doping agents, having been used since ancient times. Despite the ease with which they can be detected and the availability of sensitive detection methods, stimulants are still popular among athletes. Indeed, they remain one of the top three most popular classes of prohibited substances. Because the list of legal and illegal stimulants is extensive only a selection is discussed in detail. The compounds selected are caffeine, ephedrines, amphetamine and related compounds, methylphenidate, cocaine, strychnine, modafinil, adrafinil, 4-methyl-2-hexaneamine, and sibutramine. These compounds are mainly prevalent in sport or are of therapeutic importance. Because stimulants are the oldest doping class the first detection methods were for this group. Several early detection techniques including GC–NPD, GC–ECD, and TLC are highlighted. The more novel detection techniques GC–MS and LC–MS are also discussed in detail. In particular, the last technique has been shown to enable successful detection of stimulants difficult to detect by GC–MS or for stimulants previously undetectable. Because stimulants are also regularly detected in nutritional (food) supplements a section on this topic is also included.

Metabolic studies with promagnon, methylclostebol and methasterone in the uPA+/+-SCID chimeric mice.

The chimeric uPA(+/+)-SCID mouse model, transplanted with human hepatocytes, was previously validated as an alternative tool to study in vivo the human steroid metabolism. This humanized mouse model was now applied, in the framework of anti-doping research, to test different nutritional supplements containing steroids. These steroids, intentionally or accidentally added to a nutritional supplement, usually are derivatives of testosterone. Information about the metabolism of these derivatives, which is important to assure their detection, is quite limited. However, due to ethical constraints, human volunteers cannot be used to perform experimental excretion studies. Therefore the chimeric mice were selected to perform three separated excretion studies with superdrol (methasterone), promagnon and also methylclostebol. The urine of the humanized mice was collected 24h after a single dose administration and analyzed by gas chromatography-mass spectrometry (GC-MS). The results indicated the presence of several metabolites including a 3-keto reduced metabolite and numerous hydroxylated metabolites. Also phase 2 metabolism was investigated to update the complete picture of their metabolism.

Direct quantification of 11‐nor‐Δ9‐tetrahydrocannabinol‐9‐carboxylic acid in urine by liquid chromatography/tandem mass spectrometry in relation to doping control analysis

An accurate and precise method for the quantification of 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid (THCA) in urine by liquid chromatography/tandem mass spectrometry (LC/MS/MS) for doping analysis purposes has been developed. The method involves the use of only 200 microL of urine and the use of D(9)-THCA as internal standard. No extraction procedure is used. The urine samples are hydrolysed using sodium hydroxide and diluted with a mixture of methanol/glacial acetic acid (1:1). Chromatographic separation is achieved using a C8 column with gradient elution. All MS and MS/MS parameters were optimised in both positive and negative electrospray ionisation modes. For the identification and the quantification of THCA three product ions are monitored in both ionisation modes. The method is linear over the studied range (5-40 ng/mL), with satisfactory intra-and inter-assay precision, and the relative standard deviations (RSDs) are lower than 15%. Good accuracy is achieved with bias less than 10% at all levels tested. No significant matrix effects are observed. The selectivity and specificity are satisfactory, and no interferences are detected. The LC/MS/MS method was applied for the analysis of 48 real urine samples previously analysed with a routine gas chromatography/mass spectrometry (GC/MS) method. A good correlation between the two methods was obtained (r(2) > 0.98) with a slope close to 1.

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.