Frans Delbeke

Direct quantification of steroid glucuronides in human urine by liquid chromatography-electrospray tandem mass spectrometry.

A method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the direct quantification of glucuronides of testosterone (TG), epitestosterone (EPG), androsterone (AG) and etiocholanolone (ETG) has been developed. The method allowed for the direct determination of these analytes avoiding hydrolysis and derivatization, which are usual steps in commonly used methods based on gas chromatography-mass spectrometry (GC-MS). The electrospray ionization and the product ion spectra of the glucuronides have been studied in order to obtain the most specific transitions. The use of the selected transitions is necessary for the determination of the analytes at low ng/ml concentration levels. Two different approaches have been tested for sample preparation: direct injection after filtration and acidic liquid-liquid extraction (LLE) with ethyl acetate. Both approaches have been validated obtaining satisfactory values for accuracy and precision with limits of detection lower than 1 ng/ml for TG and EPG. Ion suppression was more pronounced after LLE probably due to the concentration of interferences from acidic urine. 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-MS method. Results have shown a good correlation between both methods with correlation coefficients higher than 0.97. A slope close to 1 was obtained for all analytes except for AG possibly due to losses during the extraction process prior to GC-MS.

Comparison between triple quadrupole, time of flight and hybrid quadrupole time of flight analysers coupled to liquid chromatography for the detection of anabolic steroids in doping control analysis

Triple quadrupole (QqQ), time of flight (TOF) and quadrupole-time of flight (QTOF) analysers have been compared for the detection of anabolic steroids in human urine. Ten anabolic steroids were selected as model compounds based on their ionization and the presence of endogenous interferences. Both qualitative and quantitative analyses were evaluated. QqQ allowed for the detection of all analytes at the minimum required performance limit (MRPL) established by the World Anti-Doping Agency (between 2 and 10 ng mL(-1) in urine). TOF and QTOF approaches were not sensitive enough to detect some of the analytes (3-hydroxy-stanozolol or the metabolites of boldenone and formebolone) at the established MRPL. Although a suitable accuracy was obtained, the precision was unsatisfactory (RSD typically higher than 20%) for quantitative purposes irrespective of the analyser used. The methods were applied to 30 real samples declared positives either for the misuse of boldenone, stanozolol and/or methandienone. Most of the compounds were detected by every technique, however QqQ was necessary for the detection of some metabolites in a few samples. Finally, the possibility to detect non-target steroids has been explored by the use of TOF and QTOF. The use of this approach revealed that the presence of boldenone and its metabolite in one sample was due to the intake of androsta-1,4,6-triene-3,17-dione. Additionally, the intake of methandienone was confirmed by the post-target detection of a long-term metabolite.

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.

No effect of caffeine on exercise performance in high ambient temperature

Caffeine, an adenosine receptor antagonist, has shown to improve performance in normal ambient temperature, presumably via an effect on dopaminergic neurotransmission through the antagonism of adenosine receptors. However, there is very limited evidence from studies that administered caffeine and examined its effects on exercise in the heat. Therefore, we wanted to study the effects of caffeine on performance and thermoregulation in high ambient temperature. Eight healthy trained male cyclists completed two experimental trials (in 30°C) in a double-blind-randomized crossover design. Subjects ingested either placebo (6xa0mg/kg) or caffeine (6xa0mg/kg) 1xa0h prior to exercise. Subjects cycled for 60xa0min at 55% Wmax, immediately followed by a time trial to measure performance. The significance level was set at pxa0<xa00.05. Caffeine did not change performance (pxa0=xa00.462). Rectal temperature was significantly elevated after caffeine administration (pxa0<xa00.036). Caffeine significantly increased B-endorphin plasma concentrations at the end of the time trial (pxa0=xa00.032). The present study showed no ergogenic effect of caffeine when administered 1xa0h before exercise in 30°C. This confirms results from a previous study that examined the effects of caffeine administration on a short (15xa0min) time trial in 40°C. However, caffeine increased core temperature during exercise. Presumably, the rate of increase in core temperature may have counteracted the ergogenic effects of caffeine. However, other factors such as interindividual differences in response to caffeine and changes in neurotransmitter concentrations might also be responsible for the lack of performance improvement of caffeine in high ambient temperature.

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.

Qualitative detection of desmopressin in plasma by liquid chromatography-tandem mass spectrometry

This work describes a liquid chromatography–electrospray tandem mass spectrometry method for detection of desmopressin in human plasma in the low femtomolar range. Desmopressin is a synthetic analogue of the antidiuretic hormone arginine vasopressin and it might be used by athletes as a masking agent in the framework of blood passport controls. Therefore, it was recently added by the World Anti-Doping Agency to the list of prohibited substances in sport as a masking agent. Mass spectrometry characterization of desmopressin was performed with a high-resolution Orbitrap-based mass spectrometer. Detection of the peptide in the biological matrix was achieved using a triple-quadrupole instrument with an electrospray ionization interface after protein precipitation, weak cation solid-phase extraction and high performance liquid chromatography separation with an octadecyl reverse-phase column. Identification of desmopressin was performed using three product ions, m/z 328.0, m/z 120.0, and m/z 214.0, from the parent ion, m/z 535.5. The extraction efficiency of the method at the limit of detection was estimated as 40% (nu2009=u200910), the ion suppression as 5% (nu2009=u200910), and the limit of detection was 50xa0pg/ml (signal-to-noise ratio greater thanxa03). The selectivity of the method was verified against several endogenous and synthetic desmopressin-related peptides. The performance and the applicability of the method were tested by analysis of clinical samples after administration of desmopressin via intravenous, oral, and intranasal routes. Only after intravenous administration could desmopressin be successfully detected.

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.

Quantitative detection of inhaled formoterol in human urine and relevance to doping control analysis

Formoterol is a frequently prescribed β(2)-agonist used for the treatment of asthma. Due to performance-enhancing effects of some β(2) -agonists, formoterol appears on the prohibited list, published by the World Anti-doping Agency (WADA). Its therapeutic use is allowed but restricted to inhalation. Since the data on urinary concentrations originating from therapeutic use is limited, no discrimination can be made between use and misuse when a routine sample is found to contain formoterol. Therefore the urinary excretion of six volunteers after inhalation of 18u2009µg of formoterol was investigated. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of formoterol in urine samples. Sample preparation consists of an enzymatic hydrolysis of the urine samples, followed by a liquid-liquid extraction at pH 9.5 with diethyl ether/isopropanol (5/1, v/v). Analysis was performed using selected reaction monitoring after electrospray ionization. The method was linear in the range of 0.5-50u2009ng/ml. The limit of quantification (LOQ) was 0.5u2009ng/ml. The bias ranged between -1.0 and -6.8 %. Results for the urinary excretion show that formoterol could be detected for 72u2009h. The maximum urinary concentration detected was 8.5u2009ng/ml without and 11.4u2009ng/ml after enzymatic hydrolysis. Cumulative data showed that maximum 11.5% and 23% of the administered dose is excreted as parent drug within the first 12u2009h, respectively, non-conjugated and conjugated. Analysis of 82 routine doping samples, declared positive for formoterol during routine analysis, did not exhibit concentrations which could be attributed to misuse.

Direct quantification of morphine glucuronides and free morphine in urine by liquid chromatography–tandem mass spectrometry

A simple and robust liquid chromatographic-tandem mass spectrometric method was developed and validated for the direct quantification of the structural isomers morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G), and morphine in urine. Method development showed that dilution of the urine samples was mandatory to obtain reproducible chromatography and that the use of deuterated internal standards was needed to compensate for ion suppression. Sample preparation consisted of a 20-fold dilution of urine into the internal standard solution. Chromatography was performed on a C8-column using gradient conditions. The mobile phase consisted of water/methanol, both containing 0.1xa0% acetic acid and 1xa0mM ammonium acetate. Calibration curves were constructed between 0.05 and 2xa0μg/ml. Validation data showed biases ranging from 1.5 to 5.2xa0% for M3G, −2.7 to −5.9xa0% for M6G, and 3.7 to 7.1xa0% for morphine. Imprecision never exceeded 5.2, 5.9, and 5.7xa0% for M3G, M6G, and morphine, respectively. The applicability of the method was checked by the analysis of 20 urine samples that were analyzed concurrently with a routine gas chromatography-mass spectrometry method. Results showed good correlation between the methods with a correlation coefficient of approximately 0.95.

Quantitative detection of inhaled salmeterol in human urine and relevance to doping control analysis.

Salmeterol is a frequently prescribed β2-agonist used for the treatment of asthma. Due to performance-enhancing effects of some β2-agonists, salmeterol appears on the prohibited list published by the World Anti-Doping Agency and its therapeutic use is allowed but restricted to inhalation. Because the data on urinary concentrations originating from therapeutic use are limited, no discrimination can be made between use and abuse when a routine sample is found to contain salmeterol. Therefore, the urinary excretion of 100 μg of inhaled salmeterol was investigated. A liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of urine samples. Sample preparation consists of an enzymatic hydrolysis of the urine samples followed by a liquid-liquid extraction at pH 9.5 with diethyl ether/isopropanol (5/1). Analysis was performed using selected reaction monitoring after electrospray ionization. The method was linear in the range of 0.5-50 ng/mL. The limits of quantification were 500 pg/mL. The inaccuracy ranged between 10.4% and −3.7%. Results show that salmeterol could be detected for 48 hours. The maximum urinary concentration detected was 1.27 ng/mL. Cumulative data showed that only 0.27% of the administered dose is excreted as parent drug within the first 12 hours. Analysis of 47 routine doping samples, declared to contain salmeterol during routine analysis, did not exhibit concentrations that could be considered originating from supratherapeutic doses.