Urine Samples Of Athletes Research Articles

Identification and characterization of higenamine metabolites in human urine by quadrupole-orbitrap LC-MS/MS for doping control

Higenamine is an alkaloid found in aconite, Annona squamosa, nanzhu (sacred bamboo), and other plants. It can be used to treat coughing, asthma, heart failure, and erectile dysfunction as well as aid in weight loss. It is also a banned substance in and out of competition as defined by the World Anti-Doping Agency (WADA). In this work, higenamine metabolic profiles were investigated in detail. Two healthy volunteers (one male and one female) took a higenamine tablet (5 mg), and urine samples were collected for two weeks. Solid-phase extraction (SPE) without enzymatic hydrolysis was used to clean the urine samples, and the urine extracts were then analyzed by liquid chromatography–quadrupole-orbitrap mass spectrometry (quadrupole-orbitrap LC–MS/MS) with accurate mass measurements. Higenamine and 32 metabolites were detected: 6 methylated, 10 sulfated and 16 glucuronidated metabolites. The chemical structures were elucidated by their fragmentation patterns, and accurate molecular formula determination was obtained for these newly reported metabolites. Three metabolic pathways containing methylation, glucuronidation, sulfation, and combinations of these were provided with methylation as the main metabolic pathway. The post-dose detection windows within urine of all 32 metabolites were compared with that of the parent drug, and a new potential biomarker (M7) was suggested for higenamine misuse. All urine samples were processed by two sample preparation methods: the dilute-and-shoot (DS) procedure and acid hydrolysis followed by SPE, and the time periods for a higenamine positive trails of two methods were compared. Although the DS method used to process the urine samples of athletes in the most of WADA-accredited laboratories to detect only free higenamine, acid hydrolysis followed by SPE is preferable and offers routine analysis to avoid false-negative results.

Urinary phenylethylamine metabolites as potential markers for sports drug testing purposes.

The misuse of 2-phenylethylamine (PEA) in sporting competitions is prohibited by the World Anti-Doping Agency. As it is endogenously produced, a method is required to differentiate between naturally elevated levels of PEA and the illicit administration of the drug. In 2015, a sulfo-conjugated metabolite [2-(2-hydroxyphenyl)acetamide sulfate (M1)] was identified, and pilot study data suggested that the ratio M1/PEA could be used as a marker indicating the oral application of PEA. Within this project, the required reference material of M1 was synthesized, single and multiple dose elimination studies were conducted and 369 native urine samples of athletes were analyzed as a reference population. While the oral administration of only 100 mg PEA did not affect urinary PEA concentrations, an increase in urinary concentrations of M1 was observed for all volunteers. However, urinary concentrations of both PEA and M1 showed relatively large inter-individual differences and establishing a cut-off-level for M1/PEA proved difficult. Consequently, a second metabolite, phenylacetylglutamine, was considered. Binary logistic regression demonstrated a significant (P < 0.05) correlation of the urinary M1 and phenylacetylglutamine concentrations with an oral administration of PEA, suggesting that assessing both analytes can assist doping control laboratories in identifying PEA misuse.

Development and validation of a UHPLC-HRMS method for the simultaneous determination of the endogenous anabolic androgenic steroids in human serum

Being performance enhancing hormones, endogenous anabolic androgenic steroids (EAAS) are banned from most competitive sports by the World Anti-doping Agency (WADA). In anti-doping control laboratories, routine assays are mainly performed on urine samples of athletes in and out of competitions. Serum constitutes a promising alternative to urine as it is less subjected to manipulation or contamination that may influence the method sensitivity. The simultaneous determination of EAAS including conjugated metabolites using LC-MS is very challenging due to their contradicting chemical behaviors at the ionization interface of the mass spectrometer. This may prejudice their detection or limit the method sensitivity. Herein, we have addressed these challenges and developed a new method for the simultaneous determination of unconjugated, sulphate- and glucuronide-conjugated EAAS (Androsterone, Etiocholanolone, testosterone, epitestosterone, dihydrotestosterone, dehydroepiandrosterone, androstenedione and 17a-hydroxyprogesterone) in human serum using ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). The use of mass spectrometric detection in full scan mode facilitated the study of the most versatile adducts for detection and quantitation. A solid phase extraction method was developed for the sample preparation prior to analysis. The method limits of quantitation ranged from 0.006 to 7.904 ng/mL and the recoveries ranged from 70.2% to 96.5%. The method calibration was performed in untreated serum representing realistic matrix composition with correlation coeffecients ranged from 0.9859 to 0.9988. Finally, the serum-levels of the investigated steroids were determined in 4 male and 1 female human subjects to provide estimates of baseline levels based on individual values.

A pilot study of non-targeted screening for stimulant misuse using high-resolution mass spectrometry

Forensic and anti-doping laboratories have used strategies based on targeted approaches in data processing, which means that only targeted compounds have been analyzed. The new challenges lie beyond the anticipation of molecular targets, such as the detection of designer drugs. In this research, after automatic extraction of all signals obtained by liquid chromatography–high-resolution mass spectrometry (LC–HRMS), the atypical pattern of stimulant-positive samples is evaluated to indicate potential doping substances by principal component analysis (PCA) when compared to negative samples. The capacity for discrimination of positive samples by PCA was evaluated using 24 samples reported as an adverse analytical finding (AAF) for stimulants from different sports against their corresponding negative controls. The method was then evaluated on routine analysis of 620 samples. AAF samples for stimulants presented an atypical pattern positioning when compared to negative samples in the multidimensional space of PCA. The method was able to identify 22 samples reported as AAF before checking the targeted substance windows. In that scenario, it also allows the detection of designer stimulants when the present procedure is applied complementarily in screening routine analysis. The present approach was applied simultaneously with the targeted procedure through the evaluation of 620 samples without jeopardizing the results deadline. Non-targeted analysis of stimulants by PCA as applied to athlete urine samples was demonstrated for the first time to be capable of flagging positive samples in sport competitions, showing its promise as a complementary screening tool alongside classical methods and as a straightforward approach for selecting samples for long-term storage.

Detection Mycoplasma hominis and genitalium in Urine Samples from Athletes and Non-Athletes by Multiplex- PCR Method

Backgrounds and Objectives: Mycoplasmas are distinguished phenotypically from other bacteria by their minute size and total lack of a cell wall and are involved in urogenital infections in both male and females. The aim of this study was to evaluate the presence of Mycoplasma genus and Mycoplasma homonis and genitalium in urine samples of athletes and non-athletes. Methods: In this study, urine samples were taken from 50 athlete and non-athlete males and were transported to laboratory. After DNA extraction, PCR was performed using specific primers to identify Mycoplasma genus and species hominis and genitalium. Results: Among 100 samples, 5 were positive for Mycoplasma genus which all the 5 isolate belonged to non-athlete persons. In 5 positive samples, 3 belonged to Mycoplasma hominis and 2 belonged to Mycoplasma genitalium. Conclusions: This study showed the presence of Mycoplasma in athletes were less than non-athletes. In addition PCR was a sensitive and precise method to identify Mycoplasma genus and species.

Lead Quantification in Urine Samples of Athletes by Coupling DLLME with UV-Vis Spectrophotometry.

Urine lead level is one of the most employed measures of lead exposure and risk. The urine samples used in this study were obtained from ten healthy male cyclists. Dispersive liquid-liquid microextraction combined with ultraviolet and visible spectrophotometry was utilized for preconcentration, extraction, and determination of lead in urine samples. Optimization of the independent variables was carried out based on chemometric methods in three steps. According to the screening and optimization study, 133μL of CCl4 (extracting solvent), 1.34mL ethanol (dispersing solvent), pH 2.0, 0.00% of salt, and 0.1% O,O-diethyl dithiophosphoric (chelating agent) were used as the optimum independent variables for microextraction and determination of lead. Under the optimized conditions, R 2 was 0.9991, and linearity range was 0.01-100μgL-1. Precision was evaluated in terms of repeatability and intermediate precision, with relative standard deviations being <9.1 and <15.3%, respectively. The accuracy was estimated using urine samples of cyclists as real samples and it was confirmed. The relative error of ≤5% was considered significant in the method specificity study. The lead concentration mean for the cyclists was 3.79μgL-1 in urine samples. As a result, the proposed method is a robust technique to quantify lead concentrations higher than 11.6ngL-1 in urine samples.

Meldonium use by athletes at the Baku 2015 European Games

BackgroundThe aim of this report was to estimate the prevalence of meldonium use in athletes competing in the Baku 2015 European Games to contribute to the surveillance of substances on...

Current status and recent advantages in derivatization procedures in human doping control.

Derivatization is one of the most important steps during sample preparation in doping control analysis. Its main purpose is the enhancement of chromatographic separation and mass spectrometric detection of analytes in the full range of laboratory doping control activities. Its application is shown to broaden the detectable range of compounds, even in LC-MS analysis, where derivatization is not a prerequisite. The impact of derivatization initiates from the stage of the metabolic studies of doping agents up to the discovery of doping markers, by inclusion of the screening and confirmation procedures of prohibited substances in athlete's urine samples. Derivatization renders an unlimited number of opportunities to advanced analyte detection.

Analysis of anabolic androgenic steroids in urine by full-capillary sample injection combined with a sweeping CE stacking method

This study describes an on-line stacking CE approach by sweeping with whole capillary sample filling for analyzing five anabolic androgenic steroids in urine samples. The five anabolic steroids for detection were androstenedione, testosterone, epitestosterone, boldenone, and clostebol. Anabolic androgenic steroids are abused in sport doping because they can promote muscle growth. Therefore, a sensitive detection method is imperatively required for monitoring the urine samples of athletes. In this research, an interesting and reliable stacking capillary electrophoresis method was established for analysis of anabolic steroids in urine. After liquid-liquid extraction by n-hexane, the supernatant was dried and reconstituted with 30 mM phosphate buffer (pH 5.00) and loaded into the capillary by hydrodynamic injection (10 psi, 99.9 s). The stacking and separation were simultaneously accomplished at -20 kV in phosphate buffer (30 mM, pH 5.0) containing 100 mM sodium dodecyl sulfate and 40 % methanol. During the method validation, calibration curves were linear (r≥0.990) over a range of 50-1,000 ng/mL for the five analytes. In the evaluation of precision and accuracy for this method, the absolute values of the RSD and the RE in the intra-day (n=3) and inter-day (n=5) analyses were all less than 6.6 %. The limit of detection for the five analytes was 30 ng/mL (S/N=5, sampling 99.9 s at 10 psi). Compared with simple MECK, this stacking method possessed a 108- to 175-fold increase in sensitivity. This simple and sensitive stacking method could be used as a powerful tool for monitoring the illegal use of doping.

Why Glucocorticosteroids Should Remain in the List of Prohibited Substances: A Sports Medicine Viewpoint

In addition to their therapeutic applications, glucocorticosteroids have been widely used and abused in the belief that these substances may enhance athletic performance. Analysis of athlete urine samples by antidoping laboratories around the world support this conclusion. It is commonly accepted in medical practice to use local glucocorticosteroid injections in the treatment of non-infectious local musculotendinous inflammatory conditions conveying symptom relief and often a speedier return to sporting activity. This practice is not to be considered illicit, but sports physicians must accept that such an intervention is not in itself an immediate cure and that an athlete will still require a period of recuperation before continuing sporting activity. How long such a period of recuperation should last is a matter of conjecture and there is little concrete data to support what is, or what is not, an acceptable period of inactivity. In the interest of athlete safety, we would propose to maintain systemic glucocorticosteroids on the World Anti-Doping Agency&apos;s (WADA) list of prohibited substances, both in and out-of-competition as well as a mandatory period of 48 hours of rest from play after receiving a local glucocorticosteroid injection.

Enantiomeric separation of some common controlled stimulants by capillary electrophoresis with contactless conductivity detection

CE methods with capacitively coupled contactless conductivity detection (C(4)D) were developed for the enantiomeric separation of the following stimulants: amphetamine (AP), methamphetamine (MA), ephedrine (EP), pseudoephedrine (PE), norephedrine (NE) and norpseudoephedrine (NPE). Acetic acid (pH 2.5 and 2.8) was found to be the optimal background electrolyte for the CE-C(4)D system. The chiral selectors, carboxymethyl-β-cyclodextrin (CMBCD), heptakis(2,6-di-O-methyl)-β-cyclodextrin (DMBCD) and chiral crown ether (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18C6H(4)), were investigated for their enantioseparation properties in the BGE. The use of either a single or a combination of two chiral selectors was chosen to obtain optimal condition of enantiomeric selectivity. Enantiomeric separation of AP and MA was achieved using the single chiral selector CMBCD and (hydroxypropyl)methyl cellulose (HPMC) as the modifier. A combination of the two chiral selectors, CMBCD and DMBCD and HPMC as the modifier, was required for enantiomeric separation of EP and PE. In addition, a combination of DMBCD and 18C6H(4) was successfully applied for the enantiomeric separation of NE and NPE. The detection limits of the enantiomers were found to be in the range of 2.3-5.7 μmol/L. Good precisions of migration time and peak area were obtained. The developed CE-C(4)D method was successfully applied to urine samples of athletes for the identification of enantiomers of the detected stimulants.

Stabilization of human urine doping control samples: III. Recombinant human erythropoietin

The tampering of athlete's urine samples by the addition of proteolytic enzymes during the doping control sampling procedure was reported recently. The aim of the current study, funded by the World Anti-Doping Agency (WADA), was the application of a stabilization mixture in urine samples to chemically inactivate proteolytic enzymes and improve the electrophoteric signal of erythropoietin (EPO) in human urine. The stabilization mixture applied was a combination of antibiotics, antimycotic substances and protease inhibitors. A series of incubation experiments were conducted under controlled conditions in the presence and absence of the stabilization mixture in urine aliquots spiked with six proteases. Two different analytical techniques were applied for the qualitative and quantitative EPO measurement: isoelectric focusing (IEF) and chemiluminescent immunoassay respectively. The addition of the chemical stabilization mixture into urine aliquots substantially improved EPO detection in the presence of proteolytic enzymes following incubation at 37 degrees C or storage at -20 degrees C. The results of this study indicated that the stabilization of urine prior to the sample collection procedure with the proposed chemical mixture might prove to be a useful tool for the preservation of anti-doping samples.

The Detection of New Synthetic Drugs in Athlete's Urine Samples

AbstractPharmaceutical preparations marketed for a specific therapeutic application in humans or animals can be abused by some athletes seeking performance enhancement. New drugs are also designed, synthesised and made available in some sport communities. Regularly, the list of compounds tested for in the laboratories involved in athletic drug testing is revised and updated. In this paper, we describe, with selected examples, the detection of new substances in athletes urine samples collected during doping control programmes. Clenbuterol and Zeranol used as a growth-promoters in the cattle industry, found their way in the performance enhancing therapeutic arsenal respectively a stimulant, a bronchodilator and as “anabolic” agents. Clenbuterol had known applications in humans while Zeranol was restricted to the veterinary use. Bromantan, a new substance developed in Russia a few years ago, was distributed in selected groups including athletes. It is now controlled by the sport authorities as a stimulant an...