Background: Erythropoietin receptor agonists (ERAs) are substances prohibited in sports and currently monitored in urine and blood. There is a great interest in new matrices like dried blood spots (DBSs). Method: A direct method for the detection of ERAs in DBSs using one single spot of 25 µl has been optimized and validated. Results: Limits of detection close or equal to those required by the World Anti-Doping Agency for serum/plasma samples were achieved, using a volume 20-times lower. All analytes were stable for at least 90 days at room temperature. Conclusion: Method performance was comparable to the requirements established for blood samples and, thus, monitoring of ERAs is reliable in DBSs in the context of doping control.
Body Fluids , Doping in Sports , Doping in Sports/prevention & control , Doping in Sports/methods , Receptors, Erythropoietin , Dried Blood Spot Testing/methods , Plasma
A fast screening method for the detection of more than 60 stimulants in urine was developed. The method consisted of a dilution of the urine (1:5 v/v) and analysis by ultra high performance liquid chromatography coupled to tandem mass spectrometry, using a C18 column (1.7 µm particle size), a mobile phase containing deionized water and acetonitrile with formic acid, and gradient elution. The chromatographic run time was 5 min. The detection was performed in positive mode electrospray ionization, monitoring one or two specific ion transitions for each analyte. Appropriate repeatability was obtained, with relative standard deviation (RSD) values below 25% for most of the analytes. Regarding intermediate precision, estimated during routine work, higher RSDs were obtained, probably due to between-day differences in the status of the mass spectrometer and in the chromatographic system. Matrix effect ranged from 60 to 255% with RSD lower than 35% for the majority of compounds. Despite the matrix effect observed, the signal/noise ratio of the analytes spiked at 50 ng/mL was greater than three in all tested samples, allowing a correct detection of all substances at the minimum required performance levels required by the World Anti-Doping Agency and demonstrating the suitability of the method. The method was tested in administration study samples and satisfactorily in operation for more than one year with routine doping samples. The presence of isomeric stimulants with closely similar chromatographic and/or mass spectrometric properties did not allow the unequivocal identification of these compounds after the first analysis. Different possibilities for separation and identification of isomeric compounds are presented.
Central Nervous System Stimulants/urine , Chromatography, High Pressure Liquid/methods , Tandem Mass Spectrometry/methods , Central Nervous System Stimulants/analysis , Doping in Sports , Humans , Isomerism , Limit of Detection , Reproducibility of Results , Spectrometry, Mass, Electrospray Ionization/methods , Substance Abuse Detection/methods
The use of formoterol in sports is allowed by inhalation at the maximum recommended therapeutic dose. Recently, a threshold concentration of 30 ng.mL(-1) was defined by the World Anti-Doping Agency (WADA) to distinguish between therapeutic and forbidden use of formoterol. The objective of this work was to evaluate that threshold concentration. Concentrations of formoterol were measured in urine samples collected after administration of 18 µg of inhaled formoterol to five healthy volunteers, and in samples collected in routine doping tests belonging to athletes having declared inhaled formoterol use. Formoterol was detected up to 8 h after administration in all volunteers with concentrations up to 19.6 ng.mL(-1) . From 28 routine samples, 27 had less than 10 ng.mL(-1) of formoterol and only in one of the samples the concentration was 25 ng.mL(-1) . Therefore, administration of formoterol by inhalation at the maximum dose allowed by WADA will not produce false positive results using a threshold concentration of 30 ng.mL(-1) , and the experience up to now in routine doping tests indicates that the probability of obtaining urines with concentrations greater than 30 ng.mL(-1) is close to nil. For this reason, sports authorities should re-evaluate the need of a threshold concentration for formoterol and its practical usefulness.
Adrenergic beta-2 Receptor Agonists/urine , Bronchodilator Agents/urine , Ethanolamines/urine , Substance Abuse Detection/methods , Administration, Inhalation , Adrenergic beta-2 Receptor Agonists/administration & dosage , Bronchodilator Agents/administration & dosage , Doping in Sports , Ethanolamines/administration & dosage , Female , Formoterol Fumarate , Humans , Male , Sensitivity and Specificity
The reliability of ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC- MS/MS) for high throughput screening in anti-doping control has been tested. A method to screen for the presence of diuretics and other doping agents in urine has been optimised and validated. The extraction procedure consisted of an alkaline extraction (pH 9.5) with ethyl acetate and salting-out effect (sodium chloride). The extracts were analysed by UPLC-MS/MS. Analysis of 34 forbidden drugs and metabolites was achieved in a total run time of 5 min, using a C18 column (100 mm x 2.1 mm i.d., 1.7 microm particle size) and a mobile phase containing deionised water and acetonitrile with formic acid, with gradient elution at a flow-rate of 0.6 mL min(-1). Identification of the compounds was performed by multiple reaction monitoring, using electrospray ionisation in positive- or negative-ion mode. Precursor and product ions were studied for each compound and cone voltage and collision energy were optimised. Due to the different chemical structure of the compounds under study, extraction recoveries varied from less than 10% to 100% depending on the analyte. The limits of detection ranged from 50 ng mL(-1) to 200 ng mL(-1), and all the compounds comply with the requirements of quality established by the World Anti-doping Agency. Intra-assay precision was evaluated at two concentrations for each compound and, in most cases, a relative standard deviation of the signal ratio lower than 20% was obtained. The method has demonstrated to be reliable when analysing routine samples and the short analysis time resulting from a simple sample preparation and a rapid instrumental analysis allow a fast turn-around time and makes it of great interest for routine anti-doping control purposes.
Central Nervous System Stimulants/urine , Diuretics/urine , Doping in Sports , Spectrometry, Mass, Electrospray Ionization/methods , Substance Abuse Detection/methods , Tandem Mass Spectrometry/methods , Chromatography, High Pressure Liquid , Humans , Reproducibility of Results , Sensitivity and Specificity , Spectrometry, Mass, Electrospray Ionization/instrumentation
