Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry I. Screening analysis.

The general strategy to perform anti-doping analyses of urine samples starts with the screening for a wide range of compounds. This step should be fast, generic and able to detect any sample that may contain a prohibited substance while avoiding false negatives and reducing false positive results. The experiments presented in this work were based on ultra-high-pressure liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry. Thanks to the high sensitivity of the method, urine samples could be diluted 2-fold prior to injection. One hundred and three forbidden substances from various classes (such as stimulants, diuretics, narcotics, anti-estrogens) were analysed on a C(18) reversed-phase column in two gradients of 9min (including two 3min equilibration periods) for positive and negative electrospray ionisation and detected in the MS full scan mode. The automatic identification of analytes was based on retention time and mass accuracy, with an automated tool for peak picking. The method was validated according to the International Standard for Laboratories described in the World Anti-Doping Code and was selective enough to comply with the World Anti-Doping Agency recommendations. In addition, the matrix effect on MS response was measured on all investigated analytes spiked in urine samples. The limits of detection ranged from 1 to 500ng/mL, allowing the identification of all tested compounds in urine. When a sample was reported positive during the screening, a fast additional pre-confirmatory step was performed to reduce the number of confirmatory analyses.

Cannabis and sport.

BACKGROUND AND OBJECTIVES: Cannabis is on the list of prohibited substances in the practice of sport, although its performance enhancing effect has not yet been proved. Its popularity among the younger generations as a social drug puts cannabis at the top of the list of compounds detected by the anti-doping laboratories accredited by the World Anti-Doping Agency worldwide. The management of the results of urine analysis is quite difficult for the medical and disciplinary committees not only because of the social use of the substance, but also because of the interpretation of the analytical data from urine samples. This paper gives an overview of what is presently known about cannabis in relation with the practice of sport. METHODS: Review of literature on the cannabis and exercise, its effect in the body, and the problems with interpretation of results when it is detected in urine. RESULTS: The paper outlines the major effects of cannabis in the context of its social use and its use for sport activities. The difficulties in the interpretation of urine sample analysis results because of the protracted excretion time of the main metabolite, long after the intake, are described. CONCLUSIONS: There is an urgent need for sport authorities to take measures necessary to avoid players misusing cannabis.

Central nervous system stimulants and sport practice.

BACKGROUND AND OBJECTIVES: Central nervous system (CNS) stimulants may be used to reduce tiredness and increase alertness, competitiveness, and aggression. They are more likely to be used in competition but may be used during training to increase the intensity of the training session. There are several potential dangers involving their misuse in contact sports. This paper reviews the three main CNS stimulants, ephedrine, amfetamine, and cocaine, in relation to misuse in sport. METHODS: Description of the pharmacology, actions, and side effects of amfetamine, cocaine, and ephedrine. RESULTS: CNS stimulants have psychotropic effects that may be perceived to be ergogenic. Some are prescription drugs, such as Ephedra alkaloids, and there are issues regarding their appropriate therapeutic use. Recently attention has been given to their widespread use by athletes, despite the lack of evidence regarding any ergogenic or real performance benefit, and their potentially serious side effects. Recreational drugs, some of which are illegal (cocaine, amfetamines), are commonly used by athletes and cause potential ergolytic effects. Overall, these drugs are important for their frequent use and mention in anti-doping laboratories statistics and the media, and their potentially serious adverse effects. CONCLUSIONS: Doping with CNS stimulants is a real public health problem and all sports authorities should participate in its prevention. Dissemination of information is essential to prevent doping in sport and to provide alternatives. Adequate training and education in this domain should be introduced.

Testosterone and doping control.

BACKGROUND AND OBJECTIVES: Anabolic steroids are synthetic derivatives of testosterone, modified to enhance its anabolic actions (promotion of protein synthesis and muscle growth). They have numerous side effects, and are on the International Olympic Committee's list of banned substances. Gas chromatography-mass spectrometry allows identification and characterisation of steroids and their metabolites in the urine but may not distinguish between pharmaceutical and natural testosterone. Indirect methods to detect doping include determination of the testosterone/epitestosterone glucuronide ratio with suitable cut-off values. Direct evidence may be obtained with a method based on the determination of the carbon isotope ratio of the urinary steroids. This paper aims to give an overview of the use of anabolic-androgenic steroids in sport and methods used in anti-doping laboratories for their detection in urine, with special emphasis on doping with testosterone. METHODS: Review of the recent literature of anabolic steroid testing, athletic use, and adverse effects of anabolic-androgenic steroids. RESULTS: Procedures used for detection of doping with endogenous steroids are outlined. The World Anti-Doping Agency provided a guide in August 2004 to ensure that laboratories can report, in a uniform way, the presence of abnormal profiles of urinary steroids resulting from the administration of testosterone or its precursors, androstenediol, androstenedione, dehydroepiandrosterone or a testosterone metabolite, dihydrotestosterone, or a masking agent, epitestosterone. CONCLUSIONS: Technology developed for detection of testosterone in urine samples appears suitable when the substance has been administered intramuscularly. Oral administration leads to rapid pharmacokinetics, so urine samples need to be collected in the initial hours after intake. Thus there is a need to find specific biomarkers in urine or plasma to enable detection of long term oral administration of testosterone.

Erythropoietin and blood doping.

OBJECTIVE AND METHOD: To outline the direct and indirect approaches in the fight against blood doping in sports, the different strategies that have been used and are currently being used to fight efficiently against blood doping are presented and discussed. RESULTS AND CONCLUSIONS: The paper outlines the different approaches and diagnostic tools that some federations have to identify and target sports people demonstrating abnormal blood profiles. Originally blood tests were introduced for medical reasons and for limiting misuse of recombinant human erythropoietin (rHuEPO). In this way it became possible to prevent athletes with haematocrit levels well above normal, and potentially dangerous for their health, competing in sport. Today, with nearly a decade of blood testing experience, sports authorities should be familiar with some of the limitations and specially the ability of blood tests performed prior to competitions to fight efficiently against the misuse of rHuEPO, blood transfusion, and artificial haemoglobin.

Human growth hormone doping in sport.

BACKGROUND AND OBJECTIVES: Recombinant human growth hormone (rhGH) has been on the list of forbidden substances since availability of its recombinant form improved in the early 1990s. Although its effectiveness in enhancing physical performance is still unproved, the compound is likely used for its potential anabolic effect on the muscle growth, and also in combination with other products (androgens, erythropoietin, etc.). The degree of similarity between the endogenous and the recombinant forms, the pulsatile secretion and marked interindividual variability makes detection of doping difficult. Two approaches proposed to overcome this problem are: the indirect method, which measures a combination of several factors in the biological cascade affected by administration of GH; and the direct method, which measures the difference between the circulating and the recombinant (represented by the unique 22 kD molecule) forms of GH. This article gives an overview of what is presently known about hGH in relation to sport. The available methods of detection are also evaluated. METHODS: Review of the literature on GH in relation to exercise, and its adverse effects and methods of detection when used for doping. RESULTS AND CONCLUSION: The main effects of exercise on hGH production and the use and effects of rhGH in athletes are discussed. Difficulties encountered by laboratories to prove misuse of this substance by both indirect and direct analyses are emphasised. The direct method currently seems to have the best reliability, even though the time window of detection is too short. hGH doping is a major challenge in the fight against doping. The effect of exercise on hGH and its short half-life are still presenting difficulties during doping analysis. To date the most promising method appears to be the direct approach utilising immunoassays.

A validated liquid chromatography-mass spectrometry method for the determination of leukotrienes B4 and B5 produced by stimulated human polymorphonuclear leukocytes.

A validated high-performance liquid chromatography (HPLC)-mass spectrometry method has been developed for the simultaneous assay of leukotrienes (LTs) B4 and B5, derived from omega-6 arachidonic acid and omega-3 polyunsaturated fatty acids (PUFA), respectively, produced by human polymorphonuclear leukocytes (PMNLs) stimulated with calcium ionophore A23187. The HPLC separation of PMNL ether extracts was performed on a reversed-phase column using a gradient elution program of 15 mM ammonium acetate and MeOH. Detection was performed by electrospray ionization-single quadripole mass spectrometry using single ion reaction monitoring in the negative mode at m/z 333.3 [M-H](-) and m/z 335.2 for prostaglandin B2/LTB5 and LTB4, respectively. The calibration curves for LTB4 and LTB5 were linear over the ranges 165-990 and 0.825-13.2 ng/ml, respectively. The lower limit of quantification for LTB5 was 0.66 ng/ml. The mean absolute recoveries for LTB4 and LTB5 were 81+/-4.8% and 82+/-5.9%, respectively. The method is precise with mean interday CVs for LTB4 and LTB5 within 7.1-10.7, and 3.8-9.4%, respectively, and accurate (range of interday deviations for LTB4 and LTB5 were -7.8 to 1, and -5 to 9% , respectively). The method has been validated and is being applied to the simultaneous quantification of the leukotrienes B4 and B5 in stimulated PMNLs in a clinical protocol studying the influence of a diet enriched in omega-3 PUFA on various surrogate markers of inflammation in young cystic fibrosis patients.