Dorota Kwiatkowska

Detection of β-methylphenethylamine, a novel doping substance, by means of UPLC/MS/MS

Novel substances of expected doping activity are constantly introduced to the market. β-Methylphenethylamine (BMPEA) is classified as a doping agent by the World Anti-Doping Agency as it is a positional isomer of amphetamine. In this work, the development and application of a simple and rapid analytical procedure that enables discrimination between both isomers is described. The analytes of interest were extracted from urine by a two-step liquid–liquid extraction and then analyzed by UPLC/MS/MS under isocratic conditions. The entire analytical procedure was validated by evaluating its selectivity, discrimination capabilities, carry-over, sensitivity, and influence of matrix effects on its performance. Application of the method resulted in detection of BMPEA in eight anti-doping samples, including the first report of adverse analytical finding regarding its use. Further analysis showed that BMPEA may be eliminated unchanged along with its phase II conjugates, the hydrolysis of which may considerably improve detection capabilities of the method. Omission of the hydrolysis step may therefore, produce false-negative results. Testing laboratories should also carefully examine their LC/MS/MS-based amphetamine and BMPEA findings as both isomers fragment yielding comparable collision-induced dissociation spectra and their insufficient chromatographic separation may result in misidentification. This is of great importance in case of forensic analyses as BMPEA is not controlled by the public law, and its manufacturing, distribution, and use are legal.

INGESTION OF DESIGNER SUPPLEMENTS PRODUCED POSITIVE DOPING CASES UNEXPECTED BY THE ATHLETES

Since the end of the last century, the sport community has been grappling with contamination and faking of dietary or nutritional supplements. Publication of the results of many studies on this problem has resulted in educational and informative actions on risk connected with consumption of unchecked products. As compared to the past, nowadays athletes have become incomparably more aware of the potential risk of a positive result in anti-doping tests as a consequence of consumption of contaminated dietary supplements. However, this problem is still topical, which is confirmed by regular reports, presented e.g. during the Cologne Workshops on Dope Analysis held annually in Germany. This paper presents a few doping cases caused by the use of supplements with doping substances by athletes, noted by two WADA accredited laboratories (Cologne and Warsaw). Still more education on the health and doping risks of dietary supplement products seems to be necessary for the protection of both athletes and the general public.

Some aspects concerning modifications of the list of prohibited substances and methods in sport.

In 1967 the International Olympic Committee (IOC) founded Medical Commission to organize and supervise fight against doping. At that time, the Commission published the first list of substances prohibited for use in sport to meet the need of anti-doping testing at the 1968 Olympic Games. The Prohibited List included stimulants, sympathomimetic amines, narcotics (narcotic analgesics), antidepressants and tranquilizers. For years the list was expanding and underwent modifications, mainly prior to successive Olympic Games. Starting from 1 January 2004, the World Anti-Doping Agency (WADA) has assumed the role of the main coordinator in fight against doping. WADA significantly modified the list of prohibited substances and methods (the Prohibited List). These modifications initiated changes, whose effects can be observed in three main areas of sport and anti-doping i.e. in: athletes, doping control laboratories, and sport entourage. In Poland, the removal some substances from the List or the addition other compounds to the basic List caused an increase of usage of pseudoephedrine and caffeine by athletes and a decrease of number of positive doping cases with cannabinoids and glucocorticosteroids. The annual modification of the Prohibited List by WADA and subsequent introduction of new examples of prohibited substances strengthened the world anti-doping system. Considering the open character of the list a regular update would be expected, especially indicating prohibited or permitted status of new substances and drugs. It would be advisable to publish, on the WADA website, some additional information regarding those substances which cause the most interpretation problems.

Evaluation of longitudinal steroid profiles from male football players in UEFA competitions between 2008 and 2013

Testosterone and related compounds are the most recurrent doping substances. The steroid profile, consisting of the quantification of testosterone and its metabolites, has been described as the most significant biomarker to detect doping with pseudo-endogenous anabolic steroids. The steroidal module of the Athlete Biological Passport (ABP) was launched by the World Anti-Doping Agency (WADA) in 2014. To assess the value of introducing the module to its anti-doping programme, the Union of European Football Associations (UEFA) decided to analyze retrospectively the steroid profile data of 4195 urine samples, collected from 879 male football players and analyzed in 12 WADA-accredited laboratories between 2008 and mid-2013. This study focused on the evaluation of T/E ratios. The coefficient of variation (CV) and the adaptive model were the two statistical models used to study the longitudinal follow-up. A CV of 46% was determined to be the maximal natural intra-individual variation of the T/E when the sequence consisted of single data points analyzed in different laboratories. The adaptive model showed some profiles with an atypical T/E sequence and also enabled an estimate of the prevalence of external factors impacting the T/E sequences. Despite the limitations of this retrospective study, it clearly showed that the longitudinal and individual follow-up of the T/E biomarker of the players is a good tool for target testing in football. UEFA has therefore decided to implement the steroidal module of the ABP from the start of the next European football season in September 2015. Copyright

The alteration of the urinary steroid profile under the stress

In the second part of twentieth century anabolic-androgenic steroids were introduced into doping practice and received continuously increasing significance. In order to prove the usage of doping substances, the determination of steroid profile in the urine came into practice. Several factors may be responsible for alterations in the normal steroid profile for example age, sex and diet. The aim of this study was to find out, whether the psychological stress may cause modifications in the steroid profile and T/Et ratio. The effect of physical activity was also considered. The steroid profile was determined in the group of 34 students being in non-stress conditions and under stress immediately before an important university exam. The intensity of stress was rated by self-reported questionnaire. The GC/MS method was applied to determine the steroid profile in the urine samples. The results of the experiment have shown that psychological stress may cause significant changes in the steroid profile, especially in females. Physical activity, independently of stress significantly modified the steroid profile. In summary, observed changes in steroid profile suggest, that major fluctuations of T/Et and A/E ratios under the influence of stressogenic factors and physical activity are unlikely. KEY wordS: doping, steroid profile, stress, physical activity dependently on external conditions. This problem is an important one, when anti-doping controls are carried out among competitors exposed to stressful psychological and physical challenges, which might influence on the rate of excretion urinary steroids and affect the doping markers. It is worth to note, that stress effecting on doping markers may give positive false result, as was reported by Armanini et al. The authors described a history of female athlete, Italian Olympic gold medalist, who was accused of growth hormone abuse based on very high the hormone levels in blood samples prior to competition. However, further investigations showed huge variation of endogenous growth hormone in this subject exposed to psychological stress. [1]. As to urinary T/Et status under competition stress condition the only study was carried out [6]. It was found, that pistol shooting resulted in rise of blood testosterone, whereas T/Et in urine was unchanged. Assuming that anticipatory stress in students before exams is similar to that in competitors prior to competition,the purpose of the study was to investigate the effect of highly stressogenic situation of waiting for exam on the steroid profile in the urine of students. Original Paper Biol. Sport 2010;27:3-9

The prevalence of trimetazidine use in athletes in Poland: excretion study after oral drug administration

Stimulants, together with anabolic androgenic steroids, are regarded as one of the most popular doping substances in sport. Owing to a great variety of these substances and new designer drugs being introduced to the market, each year the World Anti-Doping Agency (WADA) updates the list of substances and methods prohibited in sport. On 1 January 2014, a new doping agent - trimetazidine (TMZ) - was added to the WADA Prohibited List. TMZ, a substance prohibited in competition, is classified in the S6b Specified Stimulant Group. TMZ is used as a well-known cardiologic drug with confirmed biochemical and clinical activity. According to knowledge of the pharmacology and mechanism of TMZ action, TMZ can be used by athletes to improve physical efficiency, especially in the case of endurance sports. This study presents the phenomena of TMZ use by Polish athletes involved in anti-doping control in the WADA-accredited laboratory in Warsaw (Poland) between 2008 and 2013. Samples were taken from the athletes of such disciplines as cycling, athletics, and triathlon. Moreover, the elimination study of TMZ has been conducted to establish the change of TMZ concentration in urine sample after oral administration of a single or double (during the long-term therapy) dose. TMZ was monitored in urine samples by gas chromatography-mass spectrometry-nitrogen phosphorus detection (GC-MS-NPD).

Distinction of clenbuterol intake from drug or contaminated food of animal origin in a controlled administration trial – the potential of enantiomeric separation for doping control analysis

ABSTRACT The differentiation of clenbuterol abuse and unintentional ingestion from contaminated meat is crucial with respect to the valuation of an adverse analytical finding in human sports doping control. The proportion of the two enantiomers of clenbuterol may serve as potential discriminating parameter. For the determination of the individual enantiomers, specific methods were developed and validated for the different matrices under investigation based on chiral chromatography coupled to tandem mass spectrometry. Data are presented from the administration to humans of clenbuterol from a pharmaceutical preparation, and from cattle meat and liver containing residues. A shift in the proportion of the enantiomers in cattle meat is detected and this signature is also found in human urine after ingestion. Thus, an altered enantiomeric composition of clenbuterol may be used to substantiate athletes’ claims following adverse analytical findings in doping control. However, in meat, the enantiomeric composition was found to be highly variable. Species as well as tissue dependent variances need to be considered in interpreting enantiomer discrimination. Analysis of post administration urines from a controlled experiment comparing the administration of racemic clenbuterol from a registered pharmaceutical preparation and the administration of residue-containing meat and liver (nonracemic mixture) from treated animals is reported. Furthermore doping control samples from Mexican U17 World Championship 2011 of the Fédération Internationale de Football Association (FIFA), with adverse analytical findings for clenbuterol, were re-analysed. GRAPHICAL ABSTRACT

Isotope-dilution mass spectrometric quantification of the prodrug lisdexamfetamine in human urine in doping control analysis.

RATIONALE Therapeutic approaches concerning attention-deficit hyperactivity disorder (ADHD) commonly include the administration of drugs amplifying cerebral dopamine and norepinephrine signals. Among these, compounds belonging to the Prohibited List as established by the World Anti-Doping Agency (WADA) are present such as amfetamine or methylphenidate, and abuse of these can result in sanctions for athletes. The recently approved therapeutic lisdexamfetamine represents a slow-release prodrug of amfetamine for ADHD treatment. In order to support doping control laboratories in differentiating the abuse of amfetamine from a therapeutic administration of lisdexamfetamine, the determination of the prodrug from urine is desirable. Since approximately 2% of lisdexamfetamine are eliminated intact into urine, a liquid chromatography/high-resolution/high accuracy mass spectrometric method was developed, allowing the target analyte and one of its metabolites (4-hydroxyamfetamine sulfate) to be accurately quantified. METHODS Urine samples were fortified with fourfold deuterated lisdexamfetamine and analyzed directly using ultrahigh-performance liquid chromatography (UHPLC) interfaced via electrospray ionization to a second-generation quadrupole-orbitrap mass spectrometer. The assay was characterized concerning specificity, limits of quantification (0.15-5 ng/mL), intraday and interday imprecision (4-22%), accuracy (90-120%), linearity, and ion suppression/enhancement effects. A patients urine samples were analyzed to provide proof-of-principle data demonstrating that the intact prodrug lisdexamfetamine is detectable in urine up to 11 h post-administration at concentrations up to 80 ng/mL. Moreover, amfetamine and sulfoconjugated 4-hydroxyamfetamine were measured, yielding up to 1146 and 56 ng/mL, respectively. CONCLUSIONS Considering the observed comparably low urinary concentrations of lisdexamfetamine and 4-hydroxyamfetamine sulfate, the preferred minimally labor-intense sample preparation, and the necessity of fast and robust result generation, the employed instrumental setup proved fit-for-purpose in sports drug testing.

In memory of Alfons Bukowski on the centenary of anti-doping research.

Alfons Bukowski (1858-1921) is commonly regarded as the pioneer of anti-doping research. In 1910, he developed a method to detect alkaloids in horse saliva. One hundred years later, this is a good moment to remember Bukowski, an outstanding Polish pharmacist, often mistakenly represented in world literature as a Russian chemist. It is also an occasion to mention that the real driving forces in the history of doping were events related to horse rivalry.

Analytical approach for the determination of steroid profile of humans by gas chromatography isotope ratio mass spectrometry aimed at distinguishing between endogenous and exogenous steroids

The contamination of commonly used supplements by unknown steroids as well as their metabolites (parent compounds) become a challenge for the analytical laboratories. Although the determination of steroids profile is not trivial because of the complex matrix and low concentration of single compound, one of the most difficult current problem is to distinguish, during analytical procedure, endogenous androgens such as testosterone, dehydrotestosterone or dehydroepiandrosterone from their synthetic equivalents. The aim of this work was to develop and validate an analytical procedure for determination of the steroid profile in human urine by gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) toward distinguishing between endogenous and exogenous steroids. Beside the optimization of the experimental parameters for gas chromatography separation and mass spectrometry, attention was focused on urine sample preparation. Using an optimized sample preparation protocol it was possible to achieve better chromatographic resolutions and better sensitivity enabling the determination of 5 steroids, androsterone, etiocholanolone, testosterone, 5-androstandiol, 11-hydroxyandrdostane, pregnandiol, with the expanded uncertainty (k=2) below 1‰. This enable to evaluate the significant shift of the δ(13)C/(12)C [‰] values for each of examined steroids (excluding ERC). The analytical protocol described in this work was successfully used for the confirmation of positive founding urine by evaluation T/E ratio after GC/C/IRMS analysis.