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Doping-related knowledge, beliefs and attitude influence adolescent athletes' susceptibility to prohibited performance-enhancing substances. They might be modified by different cultural backgrounds. This study's aim was to analyse the geographical heterogeneity of doping-related knowledge, beliefs and attitude among adolescent elite athletes.Cross-sectional study.A questionnaire was distributed to athletes participating in the Winter Youth Olympic Games 2020 in Switzerland. Main outcomes ('subjective and actual knowledge', 'beliefs' and 'attitude') were stratified for athletes' region of origin. Geographical heterogeneity was tested with a two-way analysis of variance, and two multiple regression analyses were conducted to assess independent associations of knowledge, age and athletes' geographical region with doping-related beliefs and attitude.533 athletes (54% females, mean age: 16.0 ± 1.0 years), completed the questionnaire (response rate: 33%). Actual knowledge was moderate-to-good (9.2 ± 2.9 correct answers out of 13), and scores of attitude and beliefs showed favourable patterns. Considerable geographical heterogeneity was found for knowledge (p 0.001), beliefs (p = 0.004) and attitude (p 0.001). Higher subjective knowledge and actual knowledge were favourably associated with attitude (β = -0.096, p = 0.049; β = -0.316, p 0.001) and beliefs (β = 0.120, p = 0.016; β = 0.212, p 0.001), independent of age and geographical region.This study demonstrates considerable geographical heterogeneity of doping-related knowledge, beliefs and attitude, which are three essential target factors of doping prevention in adolescent elite athletes. This evidence should encourage medical doctors and other professionals to change their educative anti-doping approach from teaching knowledge about negative consequences into investigating and forming a young athlete's mind-set.

The illicit use of anabolic androgenic steroids (AAS) as performance-enhancing drugs remains a global issue threatening not only the credibility of competitive sports but also public health due to the well-documented adverse effects they elicit. AAS abuse is not restricted only to professional sports, but also extends to recreational athletes and adolescents as well as in livestock production as growth-promoting agents. Testosterone and nandrolone are among the AAS most frequently exploited. Gas chromatography-mass spectrometry is the reference method for AAS detection, but it is strictly laboratory-based and cannot be performed on-site. The great potential of aptamers in bioanalytical applications and specifically for the development of simple analytical tools suitable for on-site analysis has been extensively documented. In this report, we describe the selection and identification of aptamers binding nandrolone, exhibiting affinity dissociation constants in the low nanomolar range. A label-free colorimetric assay based on gold nanoparticles was developed using one of these novel aptamers for the detection of nandrolone and/or its metabolites. The assay could be deployed for the rapid, on-site, facile and cost-effective screening of samples and provide qualitative visual results with a red to purple/blue color change being indicative of a positive result.

The aim of this study was to study and compare attitudes, beliefs and knowledge about doping of footballers, from elite to under-18 categories.The descriptive exploratory design used an instrument combining a validated questionnaire (Performance Enhancement Attitude Scale: PEAS) with qualitative open-ended questions.A total of 1324 Spanish football players (average age 22.56 ± 5.62 years) from 88 football teams that ranged from elite to under-18 categories: Elite (ELI, n = 304), non-elite Professional (PRO, n = 308), top Amateur (AMA, n = 330), elite Under-18 (U18, n = 334) and elite Female (FEM, n = 48) composed the sample.PEAS overall scores (range 17-102, with higher scores representing more permissive attitudes) was 34.02 ± 11.08. The overall scores for all groups analysed were: FEM: 33.75 ± 14.73; ELI: 30.61 ± 9.91; PRO: 34.23 ± 11.13; AMA: 35.05 ± 10.35; and U18: 35.93 ± 11.50. Significant differences were observed between ELI and PRO (p 0.001), ELI and AMA (p 0.001), and ELI and U18 (p 0.001). 95% of participants did not know the meaning of WADA; 97.4% did not know the Prohibited List; 5% admitted having used banned substances and 23.7% knew dopers.This study showed different an important lack of knowledge about doping and an high levels of supplement use in this sample of footballers assessed. It which clearly reinforces the idea of implementing a wide educational doping prevention programme in football environment.

The spectrum of arrhythmias that may be encountered in athletes ranges from isolated ectopic beats to ventricular tachycardia, usually in the context of a structurally normal heart. Anti-arrhythmic therapy in these individuals may be particularly challenging because of the young age, the hypervagotonic state, the desire to maintain a high physical performance, the reluctance to take medications and the need to avoid molecules included in the list of prohibited drugs of the World Anti-Doping Agency. Furthermore, the possible serious adverse effects of anti-arrhythmic drugs should be balanced against the benign nature of arrhythmias in patients with no underlying heart disease. The review summarizes the most common arrhythmias of athletes and the possible therapeutic options, including anti-arrhythmic drugs and non-pharmacological interventions. Eligibility criteria according to current guidelines are also addressed.

Mass spectrometry-based proteomics has been a useful tool for addressing numerous questions in basic biology research for many years. This success, combined with the maturity of mass spectrometric instrumentation, the ever-increasing availability of protein sequence databases derived from genome sequencing, and the growing sophistication of data analysis methods, places proteomics in a position to have an important role in biological forensics. Because proteins contain information about genotype (sequence) and phenotype (expression levels), proteomics methods can both identify biological samples and characterize the conditions that produced them. In addition to serving as a valuable orthogonal method to genomic analyses, proteomics can be used in cases where nucleic acids are absent, degraded, or uninformative. Mass spectrometry provides both broad applicability and exquisite specificity, often without customized detection reagents like primers or antibodies. This review briefly introduces proteomics methods, and surveys a variety of forensic applications (including criminal justice, historical, archaeological, and national security areas). Finally, challenges and crucial areas for further research are addressed.

In anti-doping, a high number of classes of substances are prohibited and laboratories need to detect these at low urinary concentrations. Traditionally, testing is done using complimentary liquid chromatography mass spectrometry and gas chromatography mass spectrometry. High resolution mass spectrometric acquisition has some important advantages over triple quadrupole instruments (e.g., open screening due to full scan high resolution data acquisition with retrospectivity, compatibility with libraries and a straightforward and effortless addition and validation of new compounds in the future). Doping samples can be stored for 10 years and retrospective data analysis can be used to re-evaluate previously acquired data (e.g., searching for prohibited (designer) substances that were unknown at the initial moment of analysis). During the past decade, these advantages have led to the wide-scale transfer of liquid chromatography triple quadrupole mass spectrometry screening to liquid chromatography high resolution mass spectrometry screening for doping control purposes. Up to now, for gas chromatography a similar switch to high resolution screening has not yet occurred, because so far no method has been developed that combines sufficient sensitivity with wide-scale drug detection. In this work, the current gas chromatography triple quadrupole mass spectrometry screening method for human doping control purposes was successfully converted into an equivalent and complete gas chromatography high resolution acquisition screening method. This new screening method on a gas chromatography quadrupole time-of-flight mass spectrometer has been developed and validated. The method is compliant with the World Anti-Doping Agency requirements and allows the detection of 294 target compounds (and 14 internal standards), including diuretics, stimulants, narcotics, beta-2-agonists, beta-blockers, hormone modulators, anabolic agents and the quantification of 14 endogenous steroids in a single fast run (14.1 min).

Doping and the use of performance enhancing drugs (PEDs) are often considered and discussed as a separate issue from other types of substance use, by sporting bodies, politicians, the media, and athletes who use drugs themselves. However, perceptions and understandings of substance use in the sport and fitness world are directly related to those of substance use in the non-sport world. One way the gap between sport and non-sport substance use research can be bridged is to consider sport risk and enabling environments. Similar to non-sport contexts and drug use, it is important to analyse the environments in which doping occurs. This approach allows us to examine the dynamic interplay between risk and enabling factors, as the enabling environment shifts in response to changes produced in the risk environment, and vice versa. There are models of sport environments that have proven effective at both enabling doping by athletes and reducing harms to athletes: systematic doping. This article will use secondary literature in order to review and analyse known cases of systematic doping through the risk and enabling environment frameworks. We argue that these systems responded to anti-doping in ways that protected athletes from the risk factors established by anti-doping policy and that athletes suffered most when these systems were revealed, exposing athletes to the full range of doping harms. Further, we argue that risks within these systems (i.e. extortion, bullying) resulted from the broader prohibitive sport environment that forces doping underground and allows such abuses to occur.

This work presents an ultrafiltration-based, validated method for the screening and confirmation of prohibited growth hormone-releasing hormone (GHRH) analogues (sermorelin/CJC-1293, sermorelin metabolite, CJC-1295 and tesamorelin) in urine by nanoLC-HRMS/MS. Sample preparation avoids the use of laborious antibody-based extraction approaches and consists solely of preconcentration by ultrafiltration. Even in the absence of immuno-affinity purification steps, high sensitivity was still ensured as limits of detection between 5 and 25 pg/mL and limits of identification between 25 and 50 pg/mL were established. The robustness of the miniaturized chromatographic setup was evaluated through the injection of 200 + preconcentrated urinary extracts. In a comparison with immuno-affinity purification, enhanced recoveries (59 - 115%) and similar sensitivity were achieved, yet at lower operational costs. Stability experiments showed the importance of the proper handling of urine samples to avoid degradation of these peptide hormones, especially for sermorelin and its metabolite which were found to rapidly degrade at temperatures 4 °C and pH values 7 in accordance with earlier studies. Without the need for specific antibodies, this method may be expanded to cover emerging peptide drugs (≥ ~3 kDa), as well as their metabolites in the future to facilitate coverage for this class of prohibited substances.

The anti-estrogen clomiphene is prohibited in sports at all times. Yet, adverse analytical findings (AAFs) have increased since 2011. This is possibly due to improved analytical sensitivity, but also contamination of food of animal origin needs to be taken into consideration as a potential source of drug exposure. For instance, studies with laying hens that received orally administered clomiphene have shown a significantly increased egg production rate but, as a consequence, eggs were found to incorporate residues of clomiphene. In order to evaluate if the consumption of clomiphene-contaminated eggs can cause an AAF of a doping control sample, eggs obtained from an animal administration study with clomiphene were consumed by human volunteers. Each volunteer ate two eggs, and urine samples were collected and analyzed using routine doping control procedures. Subsequently, additional volunteers received a microdosed clomiphene capsule to compare the excretion profiles. Maximum urinary concentrations of hydroxy-clomiphene (HC) between 80 and 300 pg mL

Nicotine is classified as a stimulant, and its use is banned in horse racing and equestrian sports by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale, respectively. Because nicotine is a major alkaloid of tobacco leaves, there is a potential risk that doping control samples may be contaminated by tobacco cigarettes or smoke during sample collection. In order to differentiate the genuine doping and sample contamination with tobacco leaves, it is necessary to monitor unique metabolites as biomarkers for nicotine administration and intake. However, little is known about the metabolic fate of nicotine in horses. This is the first report of comprehensive metabolism study of nicotine in horses. Using liquid chromatography/electrospray ionization high-resolution mass spectrometry, we identified a total of 17 metabolites, including one novel horse-specific metabolite (i.e., 4-hydroxy-4-(3-pyridyl)-N-methylbutanamide), in post-administration urine samples after nasoesophageal administration of nicotine to three thoroughbred mares; eight of these compounds were confirmed based on reference standards. Among these metabolites, N-hydroxymethylnorcotinine was the major urinary metabolite in equine, but it could only be tentatively identified by mass spectral interpretation due to the lack of reference material. In addition, we developed simultaneous quantification methods for the eight target analytes in plasma and urine, and applied them to post-administration samples to establish elimination profiles of nicotine and its metabolites. The quantification results revealed that trans-3'-hydroxycotinine could be quantified for the longest period in both plasma (72 h post-administration) and urine (96 h post-administration). Therefore, this metabolite is the most appropriate monitoring target for nicotine exposure for the purpose of doping control due to its long detection times and the availability of its reference material. Further, we identified trans-3'-hydroxycotinine as a unique biomarker allowing differentiation between nicotine administration and sample contamination with tobacco leaves.

Selective androgen receptor modulators (SARMs) are a class of drugs presenting identical anabolic properties to anabolic steroids in addition to marked reduced androgenic effects. These drugs have emerged in the doping area within the early 2000′s. Ligandrol, ostarine, RAD-140 and andarine are the most popular agents belonging to this class. According to the world anti-doping agency (WADA) prohibited list, SARMs are prohibited at all times (i.e. in and out-of-competition) and are listed under the section S1.2 (other anabolic agents). The compilation of the WADA testing figures reports from 2015 to 2019 has indicated a regular increase of adverse analytical findings (AAF) due to SARMs, particularly with ostarine and ligandrol. The implementation of highly sensitive chromatographic anti-doping analyses has induced high-profile challenges of anti-doping rules violations as athletes have claimed in numerous occasions that contamination was the reason for their AAF. Since the early 2000′s, it has been accepted by the Court of Arbitration for Sports (CAS) in Lausanne (Switzerland) that, under some specific circumstances, unusual explanations can be provided to the Panel to explain an AAF. This was the open door for forensic investigations, as it is done in criminal Courts. A forensic approach can include testing for SARMs in food, drinks, but mostly in dietary supplements. As most anti-doping rules violations are only known several weeks after urine collection, this biological matrix is seldom use for further tests, despite the fact that most SARMs can be detected for several weeks in urine. Luckily, hair or nail testing can be a complement to document the claim of the athlete but of course, it cannot be considered as an alternative to urinalysis. This is because a negative hair or nail result cannot exclude the use of the detected drug and cannot overrule the urine result. To date, all methods for SARMs identification in various matrices involve liquid chromatography coupled to tandem mass spectrometry or high-resolution mass spectrometry. The aim of this paper is to review the scientific literature on the analytical possibilities of testing SARMs in dietary supplements, urine and hair or nail clippings after an AAF to document the claims of an athlete or his/her legal team.

The use of performance- and image-enhancing drugs in the past seventy years or so has sparked a number of responses, including heated public debates, the creation of dedicated organisations and drug policies, as well as the emergence of communities of practice and belief in support of or in opposition to the phenomenon. Drug-free, known in the field as ‘natural’, bodybuilding has been developing since the 1970 s as a response to a dominant bodybuilding culture where the use of performance- and image-enhancing drugs has become normalised. Recent years have seen a multiplication of national and international governing bodies, competitions, and participants in drug-free bodybuilding in different parts of the globe. As the field grows, the questions of what constitutes natural bodybuilding and who can authentically represent it become central. Adopting a multi-method, qualitative approach, this article explores the ways organisations and their key figures define and defend their versions of drug-free bodybuilding. The discussion focus is on the policies, meanings and identities embedded in these different versions, and how their production and negotiation makes sense in light of antagonisms between players in the field of natural bodybuilding as well as their relation to drug-enhanced bodybuilding and the wider world of sport. In examining this previously uncharted body culture, the article explores how the use of performance- and image-enhancing drugs provokes responses and processes of contestation and differentiation. In the process, what becomes apparent is the designation and negotiation of drug-free, natural bodies as an ongoing, dynamic, social process.

Objectives Determine whether career paths of elite male professional riders explain the risk of being sanctioned for an Anti-Doping Rules Violation through the International Cycling Union. Design, methods A discrete-time logit model explored the link between career path and ADRV risk in a database of 10,551 riders engaged in the first three world divisions (2005–2016), including 271 sanctioned riders. Results Despite a longer career (7.8 years), sanctioned riders have a precarious path. The odds of finding a sanctioned rider within those who experienced a career interruption is 5.80 times higher than for a non-caught one. 61% of the caught riders have experienced a team change. The odds of finding a caught rider within those who experienced such a change is 1.35 times higher. 44% of caught riders start before 23 years, vs 34% for non-sanctioned ones. The odds of being sanctioned are 1.69 times higher for doped riders beginning before 23. The odds of finding a sanctioned rider are 1.94 times higher among those starting their careers before 2005 (establishment of Pro Tour), than those who started in 2008 or after. In that year, the Cycling Anti-Doping Foundation and the biologic passport were both launched. Conclusions Caught riders could have extended their more precarious careers with doping. The post-2005 generation effect could mean that riders are cleaner or slicker at hiding doping. The higher risk of being caught for riders starting after 23 might indicate that an early professional socialization reduces the risk by teaching them to be cleaner, or better at hiding doping.

Introduction The use of performance enhancing drugs (PEDs) has been reported in several sports. There have been no peer-reviewed articles on the use of PEDs in ultramarathon running. This study was to examine the use of PEDs in ultramarathon running and to identify attitudes and beliefs about the usage of PEDs in the sport. Methods An online survey was developed. The survey was distributed to potential participants through Ultrasignup and the Western States Endurance Run Facebook sites. The survey included 9 demographic questions, 11 PED questions, and a previously validated 17-item performance enhancement attitude scale (PEAS). Results Six hundred nine self-identified ultramarathon runners completed the survey; 8.4% of respondents reported using PEDs during competition or training. Cannabinoids, narcotics, and stimulants were the PEDs that were most frequently reported. There was no difference between sex, age, country of origin, rank, miles/week of training, or longest race between those that reported using PEDs and those that did not report using PEDs. There was, however, a significant difference in athletes who reported they knew another ultramarathon runner who had used PEDs to have significantly higher years of participation and ranked in the top 20th percentile. There additionally was an increased PEAS score of individuals who reported using a PED or individuals that knew an individual who used PEDs. Conclusion PEDs are being used in ultramarathon running. The exact extent of the use of PEDs in ultramarathon running is still unknown and challenging to fully investigate without formal, random testing, which is expensive and technically challenging.

Equine hair is becoming an increasingly popular biological matrix for doping control of horse sports; one of the reasons for this is the significantly longer detection window hair can offer. Hair analysis opens up the opportunity for longitudinal monitoring of drug exposure which would otherwise not be possible with the more traditional and common biological matrices, such as urine and blood. As such, there is a need for more multi-target screening methods covering a broad range of prohibited substances in equine hair at the required sensitivities for equine doping control. This paper describes a sensitive ultra-high performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS) method for the detection of 121 drugs and/or their metabolites in equine hair covering ten classes of prohibited substances with estimated limits of detection between 0.1 and 10 pg/mg. To our knowledge, this is the first report of a screening method in equine hair which can cover such a broad range and well over one hundred prohibited substances in a single analytical run. This method has been validated for its specificity, precision and extraction recovery. Applicability of this method has been demonstrated by: (i) the successful identification of clenbuterol, 2-(1-hydroxyethyl) promazine sulfoxide, acepromazine and tetrahydrozoline in genuine equine mane samples; as well as (ii) the detection of drugs from artificially incurred mane hair samples which have been prepared by soaking blank hair samples in solutions of drug targets.

Anti-salbutamol antibodies remain as important tools for the detection of salbutamol abuse in athletic doping. This study evaluated the feasibility and efficiency of the chicken (Gallus gallus domesticus) as an immunization host to generate anti-salbutamol scFv antibodies by phage display. A phage display antibody library was constructed from a single chicken immunized against salbutamol-KLH conjugate. After a stringent biopanning strategy, a novel scFv clone which was inhibited by free salbutamol recorded the highest affinity. This scFv was expressed as soluble and functional protein in Escherichia coli T7 SHuffle Express B (DE3) strain. Cross-reactivity studies of the scFv towards other relevant β2-agonists revealed that the scFv cross-reacted significantly towards clenbuterol. The determined IC50 of the scFv towards the two β2-agonists were; IC50 salbutamol = ∼0.310 μg/ml, IC50 clenbuterol = ∼0.076 μg/ml. The generated scFv demonstrated poor stability based on accelerated stability studies. The scFv was used to develop an competitive indirect ELISA (LOD = 0.125 μg/ml) for detection of parent salbutamol in spiked human urine (n = 18) with ∼83.4% reliability at the cut-off of 1 μg/ml currently implemented by WADA and may be of potential use in human doping urinalysis.

Xenon is a rare, mostly inert, noble gas that has applications in a wide range of fields, including medicine. Xenon acts on the human body as a useful organ-protective and anesthetic agent and has also been previously studied for potential applications in fields such as optics, aerospace and medical imaging. Recently, it was discovered that xenon can boost erythropoietin production, and it has been used as a performance-enhancing agent in international sports competitions such as the Sochi Olympic Games. Therefore, screening methods to detect the misuse of xenon by analysis of biological samples and to monitor anesthesia kinetics and efficiency are being investigated. The aim of this study was to develop and validate an analytical method to detect xenon in blood samples using gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). Preliminary studies were conducted to determine the best parameters for chromatography and mass spectrometry for xenon. The analysis was performed using the multiple reaction monitoring (MRM) mode using the transitions m/z 129 → 129, 131 → 131 for xenon and 84 → 84, 86 → 86 for krypton, which was chosen as the internal standard. The LOD of GC-MS/MS was found to be 52 pmol on-column. Calibration lines and controls were made to obtain an accuracy profile at a range of 2.08-104 nmol with a β-expectation tolerance interval set at 80% and the acceptability limit set at ±30%. From the accuracy profile, the LOQ of 15 nmol on-column for the range of 2.08-104 nmol was obtained. The method was validated according to the guidelines of the French Society of Pharmaceutical Sciences and Techniques. The detection method was finally validated using blood from test persons subjected to a 15% or 30% xenon mixture with pure oxygen and air for 45 min. Even though the probes were already used for other projects, it was still possible to detect xenon.

Detecting agents allegedly or evidently promoting growth such as human growth hormone (GH) or growth hormone releasing peptides (GHRP) in doping controls has represented a pressing issue for sports drug testing laboratories. While GH is a recombinant protein with a molecular weight of 22 kDa, the GHRPs are short (3–6 amino acids long) peptides with GH releasing properties. The endogenously produced GH (22 kDa isoform) consists of 191 amino acids and has a monoisotopic molecular mass of 22,124 Da. Within this study, a slightly modified form of GH was discovered consisting of 192 amino acids carrying an additional alanine at the N-terminus, leading to a monoisotopic mass of 22,195 Da. This was confirmed by top-down and bottom-up experiments using liquid chromatography coupled to high resolution/high accuracy mass spectrometry. Additionally, three analogues of GHRPs were identified as Gly-GHRP-6, Gly-GHRP-2 and Gly-Ipamorelin, representing the corresponding GHRP extended by a N-terminal glycine residue. The structure of these peptides was characterised by means of high resolution (tandem) mass spectrometry, and for Gly-Ipamorelin and Gly-GHRP-2 their identity was additionally confirmed by custom synthesis. Further, established in-vitro experiments provided preliminary information considering the potential metabolism after administration.

Background This study investigated athlete knowledge of the World Anti-doping Agency (WADA) Prohibited Substances List and the effects of four well-known performance enhancing substances (PES). Methods A sample of 1925 elite and sub-elite athletes (mean age 20.6 years) completed a questionnaire about the banned status of 30 substances/methods and their knowledge of the effects of amphetamines, anabolic steroids, growth hormone and erythropoietin. Results Athletes showed limited understanding of the WADA Prohibited Substances List, scoring 32.2% correct, 36.3% incorrect, and 31.4% indicated they did not know the status of 30 substances. Responses of >50% correct were given for only eight substances/method: anabolic steroids, amphetamines, blood doping, erythropoietin, caffeine, vitamins/minerals, protein powders and iron. Athletes demonstrated moderate knowledge of the desired effects of the four PES (49% correct), but poor knowledge of their adverse effects (29% correct). Age, sex, ethnicity, professional/amateur status, and current competition level were significant predictors of the number of correct responses (r2 = 0.16, p

Anabolic androgenic steroids (AAS) are misused very frequently in sport competitions as performance enhancing agents. One of the doping compounds that has been detected with increased frequency in the last few years is dehydrochloromethyltestosterone (DHCMT, 4-chloro-17β-hydroxy-17α-methylandrosta-1,4-dien-3-one; brand name Oral Turinabol). The long-term DHCMT metabolite 20βOH-NorDHCMT (4-chloro-17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one) was reported earlier to be detectable in urine samples for more than 22 days after DHCMT administration; however, purified reference material was not available so far. In this study we demonstrate a successful combination of Wagner-Meerwein rearrangement of DHCMT to NorDHCMT (4-chloro-17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one) and subsequent whole-cell biotransformation with a recombinant fission yeast strain expressing the human cytochrome P450 enzyme (CYP or P450) CYP21A2 for the synthesis of mg amounts of this metabolite. It was then used as reference for the analysis of a post administration urine of DHCMT. The availability of this reference compound will provide an incontestable proof for DHCMT abuse.

Achievements of supercritical fluid chromatography with mass spectrometric detection made in the field of forensic science during the last decade are reviewed. The main topics include analysis of traditional drugs of abuse (e.g. cannabis, methamphetamine) as well as new psychoactive substances (synthetic cannabinoids, cathinones and phenethylamines), doping agents (anabolic steroids, stimulants, diuretics, analgesics etc.) and chemical warfare agents. Control of food authenticity, detection of adulteration and identification of toxic substances in food are also pointed out. Main aspects of an analytical workflow, such as sample preparation, separation and detection are discussed. A special attention is paid to the performance characteristics and validation parameters of supercritical fluid chromatography-mass spectrometric methods in comparison with other separation techniques.

Exercise-induced bronchoconstriction (EIB) is the transient narrowing of the airways during and after exercise that occurs in response to increased ventilation in susceptible individuals. It occurs across the age spectrum in patients with underlying asthma and can occur in athletes without baseline asthma. The inflammatory mechanisms underlying EIB in patients without asthma may be distinct from those underlying EIB in patients with asthma. This review summarizes mechanistic and clinical data that can guide the choice of chronic and acute pharmacologic therapies targeting control of EIB. Relevant regulations from the World Anti-Doping Agency are also discussed.

Although the use of nutritional supplements by adult athletes has been extensively studied, information on supplements consumption by adolescent athletes is still limited. The present study reports on the use of nutritional supplements contaminated with banned doping substances among 170 recreational adolescent athletes from eleven, randomly selected, gym centres, in Athens, Greece. Nutritional supplements consumption was reported by almost 60% of the study population, with proteins/amino acids and vitamins being the most popular. Nine per cent of the users were found to consume nutritional supplements contaminated with anabolic steroids, prohormones, selective androgen receptor modulators (SARMs) and aromatase inhibitors, all pharmacological substances with endocrine modulating properties not stated on the label. None of these individuals had previously consulted a physician or a nutritionist. A representative sample (ca 15%) of the protein/aminoacids and creatine preparations used by the study population were also tested and found free from doping substances. The majority (63%) of adolescents purchased products from the internet. In conclusion, exercising adolescents can have easy access to contaminated nutritional supplements and "black market" products, which could constitute a risk for public health. Low level of awareness and low involvement of medical care professionals among recreational adolescent athletes is also observed.

A novel assay for the simultaneous determination of ibuprofen (IBU) and its four probable metabolites, 1-hydroxyibuprofen (1-OH IBU), 2-hydroxyibuprofen (2-OH IBU), 3-hydroxyibuprofen (3-OH IBU) and carboxyibuprofen (CBX IBU) in equine urine samples with the application of Gas Chromatography-Electron Ionization-Mass Spectrometry (GC-EI-MS) has been developed and elaborated. The new approach for sample preparation including minimizing matrix effects by the application of weak cation exchange solid-phase extraction together with strong cation exchange solid-phase extraction has been applied. The GC-EI-MS method was validated to demonstrate specificity, matrix effect, linearity, limit of detection (LOD) and quantification (LOQ), precision, trueness, carry-over and stability by using the matrix-matched quality control samples. Additionally, extraction yield was evaluated. The assay achieved the LOQ of 1.75 μg mL-1, 0.62 μg mL-1, 4.15 μg mL-1, 0.58 μg mL-1 and 4.04 μg mL-1 for IBU, 1-OH IBU, 2-OH IBU, 3-OH IBU and CBX IBU, respectively. The developed method has been successfully applied to the excretion study in horses, in which a single oral IBU dose was administered to twelve horses (mares and geldings) and equine urine samples were collected for 5 or 6 days after the drug administration. Data on the detection and determination of three IBU metabolites, 2-OH IBU, 3-OH IBU and CBX IBU in equine urine samples has been presented for the first time. The obtained results indicated the rapid excretion of IBU and its metabolites that were detectable only in the first day after the drug administration. IBU was mainly the most abundant compound detected in equine urine samples (with two exceptions in the case of samples collected from two horses, for which the highest instrumental responses were obtained for CBX IBU). The received results have indicated that two major IBU metabolites, CBX IBU and 2-OH IBU can be important markers for the IBU abuse in view of doping control in equestrian sports.

Measurement of insulin and its therapeutic analogs is important in diabetes, hypoglycemia, sports anti-doping and toxicology. Commercial insulin immunoassays fail to detect commonly prescribed insulin analogs. Because of their unique sequences and masses, these analogs are readily measured and distinguished with mass spectrometric (MS) assays. Reviewed here is an insulin mass spectrometric immunoassay (MSIA) that combines micro-scale immunoaffinity capture with sensitive MS detection of insulin and its therapeutic analogs. An antibody reactive to all insulin analogs was used to affinity capture the insulin analogs. Following elution, insulins were detected with MALDI-TOF MS or LC-MS analysis. Isotopic resolution for insulin was achieved for both MS techniques, and several insulin analogs were detected at unique m/z signals. Porcine insulin, spiked in all samples, served as an internal reference standard for quantification. Linear standard curves spanning three orders of magnitude were obtained, with limits of detection of 15 pM for the MALDI-TOF MS and 1.5 pM for the LC-MS. This insulin assay was capable of detecting and quantifying not only human endogenous insulin, but also most of the therapeutic insulin analogs, which could find use in diagnosis of severe hypoglycemia and in sports anti-doping. Significance Insulin replacement therapy consists of injection of long- or fast-acting insulin analogs with slightly modified primary sequences compared to human insulin. Assays that are capable of detecting all insulin analogs are desired, not only for medical management of diabetes and severe hypoglycemia but also for sports anti-doping and toxicology.

The class of selective androgen receptor modulators (SARMs) has been the subject of intense and dedicated clinical research over the past two decades. Potential therapeutic applications of SARMs are manifold and focus particularly on the treatment of conditions manifesting in muscle loss such as general sarcopenia, cancer-associated cachexia, muscular dystrophy, etc. Consequently, based on the substantial muscle- and bone-anabolic properties of SARMs, these agents constitute substances with significant potential for misuse in sport and have therefore been added to the Word Anti-Doping Agency's (WADA's) Prohibited List in 2008. Since then, numerous adverse analytical findings have been reported for various different SARMs, which has underlined the importance of proactive and preventive anti-doping measures concerning emerging drugs such as these anabolic agents, which have evidently been misused in sport despite the fact that none of these SARMs has yet received full clinical approval. In this review, analytical data on SARMs generated in the context of research conducted for sports drug testing purposes are summarized and state-of-the-art test methods aiming at intact drugs as well as diagnostic urinary metabolites are discussed. Doping control analytical approaches predominantly rely on chromatography hyphenated to mass spectrometry, which have allowed for appropriately covering the considerable variety of pharmacophores present in SARMs such as the non-steroidal representatives ACP-105, BMS-564929, GLPG0492 (DT-200), LG-121071, LGD-2226, LGD-4033/VK 5211, ostarine/enobosarm, RAD-140, S-40503, etc. as well as steroidal compounds such as MK-0773 and YK-11.

Anabolic androgenic steroids (AASs) and structural-like substances are commonly prohibited substances found in doping control studies that can be difficult to accurately detect. In the present study, 11 AASs and 2 structural-like substances that are commonly detected were examined. Currently, such analytes are detected using low resolution GC-MS/MS or LC-MS/MS, with detection not always possible. Herein, the high resolution Quadrupole-Orbitrap liquid chromatography-tandem mass spectrometry LC-MS/MS system Q Exactive was utilized to increase the specificity. This approach was then combined for the first time with parallel reaction monitoring (PRM) during the screening procedure. The results confirmed high specificity, with the LODs of all 13 analytes being at least 25-fold lower than corresponding MRPLs as defined by WADA. Furthermore, the extraction recoveries were above 70% and the intra- and inter-day precisions were lower than 15%. This approach was successfully applied to analyze over 10,000 samples with no false-positive or false-negative results, thus suggesting that Quadrupole-Orbitrap LC-MS/MS when combined with PRM is an effective method for doping control analysis.

Epoxy resin (EP) has been extensively used in the field of insulation for its excellent electrical strength, mechanical property, chemical stability, and low cost. In this paper, computer molecular simulation is used to analyze the influence of nano-POSS (Nano-Polyhedral Oligomericsils Esquioxane) doping on the properties of epoxy composite from the micro point of view, which can provide a scientific basis for the optimization of the epoxy system. Two kinds of nano-POSS fillers with different mass fractions were doped into the base material of diglycidyl ether of bisphenol A (DGEBA) and 3,4-epoxycyclohexylmethyl 3,4-epoxycyclohexanecar (OSC). By molecular dynamics (MD) simulation the influence of nanofillers on the microstructure, thermal and mechanical properties of the composite were analyzed. Simulation results show that the doping of nano-POSS can improve the thermal and mechanical properties of the epoxy composite. Different nano-POSS has little effect on the glass transition temperature (Tg), coefficient of thermal expansion (CTE), and mechanical properties of the epoxy system, while the filling amount has an obvious improvement effect. Compared with EP/methyl-POSS system, the thermal and mechanical properties of the EP/phenyl-POSS system are better. At the same time, the doping of nano-POSS changed the microstructure parameters of epoxy composite. With the increase of nano-POSS filler content, fractional free volume (FFV) and mean square displacement (MSD) of both EP/POSS systems increased after the first drop. Besides, when the content of nano-POSS exceeded a certain range, the aggregation of filler itself hindered the accumulation of epoxy molecular chain segments.

Knowledge of the metabolic profile is essential for doping control analysis in sport since most drugs are excreted after an elaborate biotransformation process. Currently, Zebrafish Water Tank (ZWT) model has been applied to investigate the metabolism of different doping agents. Nevertheless, the class of glucocorticoids has not been subjected to this model for metabolism studies. In the present work, budesonide (BUD) was applied as a pilot to investigate the metabolic pathways of glucocorticoids in the ZWT model. The BUD biotransformation in ZWT model was compared to the described metabolism in humans. Samples from ZWT experiments were collected after BUD administration and analyzed by Liquid Chromatography coupled to High Resolution Mass Spectrometry (LC-HRMS). Following the identification and characterization of all significant metabolites described for humans, it was observed that the ZWT was able to produce in a relevant amount the main target for doping control purposes: the 6β-hydroxy BUD. In addition, prior knowledge about the lack of butyrylcholinesterase activity in the zebrafish organism was considered for the evaluation for the formation of the 16α-hydroxy prednisolone, the most intense BUD metabolite in human urine. Biotransformation of BUD by ZWT focused on metabolites with the acetal fraction preserved, including the intermediate metabolite for the 16α-hydroxy prednisolone pathway. However, an alternative metabolic pathway for the complete biotransformation of the 16α-hydroxy prednisolone intermediate was not observed, leading to the absence of the major human metabolite in the ZWT model. The findings reported in this study elucidate for the first time the application and limitations of the ZWT model to evaluate the metabolism of other glucocorticoids.

Nano-liquid chromatography (nanoLC) has proven itself as a powerful tool and its scope entails various applications in (bio)analytical fields. Operation at low (nL/min) flow rates in combination with reduced inner dimensions (ID 100 µm), leads to significantly enhanced sensitivity when coupled with electrospray ionization-mass spectrometry (ESI-MS). Challenges that remain for the routine implementation of such miniaturized setups are related to clogging of the system and robustness in general, and thus the application of tedious sample preparation steps. To improve ruggedness, a filter placed upstream in the LC prevents particles from entering and clogging the system. This so-called online automatic filtration and filter back-flush (AFFL) system was combined with nanoLC and the direct injection principle for the sensitive confirmatory analysis of fifty different doping-relevant peptides in urine. The presented assay was fully validated for routine purposes according to selectivity and matrix interference, limit of identification (LOI), carryover, matrix effect, sample extract stability, analysis of educational external quality assessment (EQAS) samples, robustness of the online AFFL-setup and retention time stability. It was also fully compliant with the most recent minimum required performance levels (MRPL) and chromatographic/mass spectrometric identification criteria (IDCR), as imposed by the World Anti-Doping Agency (WADA). In the absence of labor-intensive sample preparation, the application of AFFL allowed for the injection of diluted urine samples without any noticeable pressure buildup in the nanoLC system. Contrary to earlier observations by our group and others, the addition of dimethylsulfoxide (DMSO) to the mobile phase did not enhance sensitivity in the presented nanoflow setup, yet was beneficial to reduce carry over. Although the robustness of the presented setup was evaluated only for the analysis of diluted urine samples, it is entirely conceivable that routine applications employing other matrices and currently running on analytical scale LC instruments could be transferred to micro/nanoLC scale systems to reach lower detection limits.

We have developed an analytical procedure to measure the carbon isotopic composition of multiple compounds even when there is a partial overlap in the chromatographic profiles and applied this procedure to measure the carbon isotopic composition of different metabolites in human urine and exhaled breath. Method development and validation was performed with CRM IAEA-600 caffeine after calibration of the reference CO

Androgenic anabolic steroids are the most misused substances in sports because of their performance-enhancing effects. Often synthetic analogues of endogenously present steroids are administered. To determine their endogenous or exogenous origin, Gas Chromatography Combustion Isotope Ratio Mass Spectrometry (GC-C-IRMS) is used in the field of doping control. Compounds subjected to IRMS analysis must be interference-free, with liquid chromatography fraction collection (HPLC-FC) being the crucial clean-up step. However, this clean-up is challenging, particularly for compounds present at low concentrations in samples with pronounced matrix effects. The compounds of interests for IRMS analyses in doping control are testosterone (T) and its main metabolites (androsterone, etiocholanolone, 5α-androstane-3α,17β-diol, 5β-androstane-3α,17β-diol), epitestosterone, 19-norandrosterone (19-NA), boldenone (B) and its main metabolite (BM), formestane (F) and 6αOH-androstenedione (6aOHADION). Currently, the available methods only deal with a selection of the above-mentioned compounds. Some of these compounds (e.g., 19-NA, B, BM, 6aOHADION) are present in very low concentrations, requiring an extensive and dedicated sample clean-up, and this makes it challenging to develop a universal clean-up procedure. Many of these methods require different and multiple offline HPLC-FC setups, which are labour-intensive and time-consuming. That is problematic during, e.g., large sports events, where reporting time is limited (e.g., 72 h). Therefore, in the current work, we developed a uniform online 2D/3D HPLC-FC method, capable of purifying all relevant target compounds in a single run, leading to the fastest clean-up procedure so far (i.e., 31 min for T and its main metabolites; 46 min for 19-NA, F and 6aOHADION; 48 min for B and BM).

Background: Doping is a significant issue in sport. To date, research efforts to inform doping prevention have lacked any guiding framework for research priorities. To ensure future research endeavors are coordinated, sustainable and focused on end-user priorities, a research agenda, informed by practical needs and expert knowledge, is needed. For the first time, this study brought together an international group of practitioners and academic researchers to co-create a research agenda for doping prevention.Methods: The Delphi consensus method was used to identify the most important topics for social science research on doping prevention, as well as specific questions that need addressing. Based on eligibility criteria for key stakeholders in anti-doping, 82 academics, practitioners and representatives of anti-doping organizations were invited to participate in this agenda-setting project. Based upon two substantive reviews of the doping literature and 12 focus groups with elite athletes across five European countries, a questionnaire was developed by social science experts in anti-doping to assess the importance of 15 potential research topics and identify key research questions for each. In Round 1, an expert panel (n = 57) completed this questionnaire. In Round 2, expert panel members (n = 33; 42% attrition from Round 1) ranked the eight topic areas rated as most important in Round 1 on their relative importance, before doing the same for research questions within each topic area. Based on these rankings, a draft research agenda was created. In the final round, expert-panel members (n = 26; 54% attrition from Round 1) rated the degree to which they accepted this draft agenda and the feasibility of its delivery over the next ten years, as well as identifying possible barriers and facilitators to its implementation.Results: The results of Round 1 and Round 2 were used to create a research agenda consisting of 18 research questions stratified across eight topic areas. This draft agenda was either fully (n = 16) or mostly (n = 9) accepted by the expert panel in Round 3 (96.2%). These research topics focus on the effectiveness of anti-doping interventions/education programmes, athlete environment (developmental influences, role and knowledge of the athlete entourage), long-term development of protective and risk factors for doping in athletes and their entourage, athletes' experiences of key procedures and their perceived place in the anti-doping system. Conclusions: For the first time, a rigorous exercise was conducted to create a research agenda for doping prevention research. Adoption and application of this agenda should lead to better coordination, more efficient use of available funding resource, enhanced uptake of research findings by doping-prevention practitioners and in turn more effective anti-doping and clean-sport education.

Following a one-year monitoring program providing unequivocal analytical evidence for a high prevalence in international elite sports, meldonium has been included in the World Anti-Doping Agency's (WADA) list of prohibited substances that came into effect on 1 January 2016. Despite of the polar and hydrophilic nature of the molecule, an unusual long detection window was observed in pilot elimination studies. Consequently, in the present study, urinary excretion profiles after single-dose (5 volunteers, 1×500mg) and multiple-dose oral application (5 volunteers; 2×500mg/day for 6days) were determined in order to facilitate the result management concerning meldonium findings in doping controls. Particularly the option to differentiate between recent use and tapering concentrations was studied. Urinary meldonium concentrations were determined using an analytical approach based on hydrophilic interaction liquid chromatography and high resolution tandem mass spectrometry. The study corroborates the hypothesis of a non-linear, dose-depended and biphasic excretion profile after oral application of meldonium and demonstrates that urinary detection windows are of considerable extent with up to 65 and 117days (concentrations>LOQ of 10ng/mL) following single- and multiple-dose applications, respectively.

Anabolic and androgenic steroids (AASs) are a class of prohibited substances banned in horseracing at all times. The common approach for controlling the misuse of AASs in equine sports is by detecting the presence of AASs and/or their metabolites in urine and blood samples using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). This approach, however, often falls short as the duration of effect for many AASs are longer than their detection time in both urine and blood. As a result, there is a high risk that such AASs could escape detection in their official race-day samples although they may have been used during the long period of training. Hair analysis, on the other hand, can afford significantly longer detection windows. In addition, the identification of synthetic ester derivatives of AASs in hair, particularly for the endogenous ones, can provide unequivocal proof of their exogenous origin. This paper describes the development of a sensitive method (at sub to low parts-per-billion or ppb levels) for detecting 48 AASs and/or their esters in horse hair using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). Decontaminated horse hair was pulverised and subjected to in-situ liquid-liquid extraction in a mixture of hexane - ethyl acetate (7:3, v/v) and phosphate buffer (0.1M, pH 9.5), followed by additional clean-up using mixed-mode solid-phase extraction. The final extract was analysed using UHPLC-HRMS in the positive electrospray ionisation (ESI) mode with both full scan and parallel reaction monitoring (PRM). This method was validated for qualitative identification purposes. Validation data, including method specificity, method sensitivity, extraction recovery, method precision and matrix effect are presented. Method applicability was demonstrated by the successful detection and confirmation of testosterone propionate in a referee hair sample. To our knowledge, this was the first report of a comprehensive screening method for detecting as many as 48 AASs and/or their esters in horse hair. Moreover, retrospective analysis of non-targeted AASs and/or their esters was made feasible by re-examining the full scan UHPLC-HRMS data acquired.

The urinary steroidal fraction has been extensively explored as non-invasive alternative to monitor pathological conditions as well as to unveil the illicit intake of pseudo-endogenous anabolic steroids in sport. However, the majority of previous approaches involved the a priori selection of potentially relevant target analytes. Here we describe the non-targeted analysis of the urinary steroidal profiles. The workflow includes minimal sample pretreatment and normalization according to the specific gravity of urine, a 20 min reverse phase ultra-performance liquid chromatographic separation hyphenated to electrospray time-of-flight mass spectrometry. As initial validation, we analyzed a set of quality control urines spiked with glucurono- and sulfo-conjugated steroids at physiological ranges. We then applied the method for the analysis of samples collected after single transdermal administration of testosterone in hypogonadal men. The method allowed profiling of approximately three thousand metabolic features, including steroids of clinical and forensic relevance. It successfully identified metabolic pathways mostly responsible for groups clustering even in the context of high inter-individual variability and allowed the detection of currently unknown metabolic features correlating with testosterone administration. These outcomes set the stage for future studies aimed at implementing currently monitored urinary steroidal markers both in clinical and forensic analysis.

A fast method for the direct analysis of enzyme-hydrolysed horse urine using an automated on-line solid-phase extraction (SPE) coupled to a liquid-chromatography/high resolution mass spectrometer was developed. Over 100 drugs of diverse drug classes could be simultaneously detected in horse urine at sub to low parts per billion levels. Urine sample was first hydrolysed by β-glucuronidase to release conjugated drugs, followed by centrifugal filtration. The filtrate (1mL) was directly injected into an on-line SPE system consisting of a pre-column filter and a SPE cartridge column for the separation of analytes from matrix components. Through valves-switching, the interfering matrix components were flushed to waste, and the analytes were eluted to a C18 analytical column for refocusing and chromatographic separation. Detections were achieved by full-scan HRMS in alternating positive and negative electrospray ionisation modes within a turn-around time of 16min, inclusive of on-line sample clean-up and post-run mobile phase equilibration. No significant matrix interference was observed at the expected retention times of the targeted masses. Over 90% of the drugs studied gave estimated limits of detection (LoDs) at or below 5ng/mL, with some LoDs reaching down to 0.05ng/mL. Data-dependent acquisition (DDA) was included to provide additional product-ion scan data to substantiate the presence of detected analytes. The resulting product-ion spectra can be searched against an in-house MS/MS library for identity verification. The applicability of the method has been demonstrated by the detection of drugs in doping control samples.

FG-4592 is a hypoxia-inducible factor (HIF) stabilizer, which can increase the number of red blood cells in the body. It has not been approved by regulatory authorities, but is available for purchase on the Internet. Due to its ability to improve the oxygen transportation mechanism in the body, FG-4592 is of interest for doping control laboratories, but prior to this study, little information about its metabolism was available. In this study, the metabolism of FG-4592 was investigated in a human doping control sample and in five in vitro models: human hepatocytes and liver microsomes, equine liver microsomes and S9 fraction and the fungus Cunninghamella elegans. By using liquid chromatography coupled to a Q-TOF mass spectrometer operated in MSE and MSMS modes, twelve different metabolites were observed for FG-4592. One monohydroxylated metabolite was detected in both the human and equine liver microsome incubations. For the fungus Cunninghamella elegans eleven different metabolites were observed of which the identical monohydroxylated metabolite had the highest response. This rich metabolic profile and the higher levels of metabolites produced by Cunninghamella elegans demonstrates its usefulness as a metabolite producing medium. In the doping control urine sample, one metabolite, which was the result of a direct glucuronidation, was observed. No metabolites were detected in neither the human hepatocyte nor in the equine liver S9 fraction incubates.

In June 2012 DMAA (1,3-dimethylamylamine), an ephedrine-like vasoconstricting substance which had been included in many popular sports supplements, became a scheduled substance in Australia, following bans in several other countries. The underlying rationale for this ban was that DMAA use is unsafe. This paper aimed to critically review the available evidence on the acute and/or long-term harms of DMAA. Using five research databases (PubMed, Embase, ProQuest Health and Medical Complete, and Web of Science) and the key terms 'methylhexaneamine', 'DMAA', 'dimethylamylamine', '1,3-dimethylpentylamine' and '2-amino-4-methylhexane', 842 articles were identified once duplicates removed. Sixteen studies met the inclusion criteria and were included in the review. Of the included studies, eight were case studies, which reported on eight patients who presented to emergence departments. All were retrospective in their reporting. The patients displayed various outcomes; while the patients were presenting with serious problems, in most patients conditions subsided on cessation of supplement use. The remaining eight experimental studies were low powered, with a number of studies conducted by a single research group with industry ties, and broadly investigated the effects of DMAA on physiological outcomes. Mixed findings were apparent, although escalations of blood pressure were present on acute dosing, as well as decreases in measures of body weight and body fat. There is a shallow evidence base describing the adverse effects of DMAA and the dose above which such effects may occur. The scheduling of DMAA in many countries may now impede research efforts to determine whether there are safe doses at which DMAA can be consumed.

MicroRNAs are endogenous non-coding RNAs that post-transcriptionally regulate gene expression by specifically binding the target mRNA and by consequently inducing its degradation. miRNAs can be released into the circulation where they remain stable and they can be measured. Their changes reflect individual biologic adaptation to exposures to specific environmental conditions. As such, measurement of circulating microRNAs represents an opportunity to evaluate biologic changes associated with interventions such as exercise and diet. Physical activity is, indeed, a very important modifying factor for circulating miRNAs. Toward their use in clinical settings several issues should be still solved. Their clinical application is hindered by the high heterogeneity of the analytical procedures used for their measurements. Furthermore, several pre-analytical concerns equally reduce the clinical applicability of miRNA. Pre-analytical phase in sports medicine is an important issue both because, often the conditions in which sampling are performed are peculiar (and not always canonical) and because some of the tested parameters, in the case of professional athletes, enters in routine anti-doping testing and, as such, they should be treated according to precise rules in order to avoid any false positive results. Aim of this review is to give an overview of the main available knowledges about the pre-analytical management of the sample for circulating miRNA evaluation along with the importance of miRNA as regulators of the response to physical activity and their possible future use in anti-doping settings.

Growth hormone (GH), or somatotropin, is a protein that may enhance physical performance and facilitate growth and wound healing. For this reason, growth hormones and their recombinant analogues are prohibited in human sports by the World Anti-Doping Agency (WADA) and in horseracing under Article 6 of the International Agreement on Breeding, Racing and Wagering published by the International Federation of Horse Racing Authorities (IFHA). Identifying the illicit use of GHs in both human athletes and racehorses is challenging, especially when differentiating between endogenous and exogenous GHs, and between analogues of GH from different species. This paper describes a multiplexed mass spectrometric method for the simultaneous detection of three recombinant GHs, namely recombinant equine GH (reGH), recombinant human GH (rhGH) and recombinant porcine GH (rpGH), in equine plasma. Recombinant chicken GH (rcGH) was used as an internal standard. The recombinant GHs were extracted from equine plasma by automated C4 solid-phase extraction after ammonium sulfate precipitation, and then cleaned up by chloroform/methanol precipitation before trypsination. Proteotypic peptides were analyzed by liquid chromatography/high-resolution tandem mass spectrometry (LC-MS/HRMS). The limits of detection were estimated to be approximately 0.5ng/mL for reGH, 2.5ng/mL for rhGH and 1.25ng/mL for rpGH. Confirmation at 1ng/mL for reGH and 5ng/mL each for rhGH and rpGH was successfully achieved by comparing the retention times and relative abundances of three major product-ions of the respective standards in accordance with industry criteria published by the Association of Official Racing Chemists. The developed method requires less plasma (2mL) and has a shorter turnaround time as compared with other published mass spectrometric methods, and demonstrates good precision and reproducibility. To our knowledge, this is the first reported method for the simultaneous detection of different recombinant GHs (reGH, rhGH and rpGH) at low ng/mL level in horse plasma samples.

The implementation of global anti-doping regulations was intended to provide a level playing field for all athletes entering sports competitions. However, studies have shown that the worldwide harmonization of rules has not been entirely efficacious. For instance, great variation has been found in how anti-doping organizations implement anti-doping regulations, and it has also been shown that athletes distrust the equivalence of the worldwide rules as regards their effects. The purpose of the present article is to examine how elite athletes from different contexts experience anti-doping procedures and to analyse the legitimacy of anti-doping practice. In order to capture a variety of voices and perspectives, 13 elite athletes from five different continents and three international sports federations were interviewed. The analysis shows that when global anti-doping policy is implemented in different contexts and under different conditions, inequities and structural injustices emerge concerning infrastructure, knowledge and support at the individual athlete level. These consequences may have implications for the legitimacy of anti-doping work, because the existence of procedural justice may be called into questioned. We therefore suggest that anti-doping policy-making should be based on taking into account these different conditions and being aware of the perspectives that underpin regulations intended to be applied global.

The Tokyo Olympic and Paralympic Games in 2021 presented an opportunity for pharmacists to recognize the uniqueness of sports pharmacy as a developing field and to understand the importance of anti-doping education among patient-athletes. Patient-athletes make up a distinct patient population, and pharmacists are well positioned to support athletes’ therapeutic decisions. Pharmacists need to be able to search for and interpret drug information to take care of this special population appropriately. The purpose of this commentary is to facilitate a discussion on what changes or reinforcement might help train pharmacists to become equipped with adequate knowledge and skills to support safe use of drugs among patient-athletes. While effective resources and tools have become more widely available, pharmacists’ awareness of and training in the field of sports pharmacy continue to be needed. No matter where they practice, pharmacists should be ready to promote “the spirit of sport” and defend the importance of “clean” sport for their patient-athletes.

1,3-Dimethylpentylamine (Geranamine) with a similar structure to amphetamine has been used as an athletic performance promoter (doping agent) and also as an indirect sympathomimetic drug to synthesize of 1,3-dimethyl pentyl glycine (13DMPG). Thereafter, two new anticancer platinum complexes as [Pt(DACH)(13DMPG)]NO3 and [Pt(bpy)(13DMPG)]NO3 were synthesized using this ligand and then characterized by spectroscopic methods. ADMET comparative results indicated that they are entirely in the pink area of the Bioavailability Radar, so they can be considered as drug-like and oral medications. Mechanism of tumor inhibition and DNA binding parameters were investigated and the results indicated the higher ability of [Pt(bpy)(13DMPG)]NO3 with the endothermic process for both systems compared with [Pt(DACH)(13DMPG)]NO3. Fluorescence study showed that the quenching mechanism is static for both drugs with large binding constant and high binding (Kb ≈ 8000 M−1 and kq ≈ 5.3 × 1011 M−1 s−1) affinity towards DNA. CD spectra showed the increased intensity of the positive band and the decreased negative band, meaning B-DNA converting to A-DNA form via electrostatic interaction for positively charged complexes. The cytotoxic effect analyzed by MTT assay showed that both compounds have effective antiproliferative on MCF-7 cell line. In addition, the inhibition effect of [Pt(DACH)(13DMPG)]NO3 (IC50 = 17 µM) was shown to be better than [Pt(bpy)(13DMPG)]NO3 (IC50 = 45 µM). According to DFT results, anticancer properties of [Pt(bpy)(13DMPG)]NO3 mainly is more than cisplatin and [Pt(DACH)(13DMPG)]NO3. Docking studies showed that the desolvation energy and hydrogen bond are more effective compared to the other interactions. The torsional free energy, about +1.19 kcal/mol, for both complexes mainly provides groove binding with partially electrostatic and intercalate bindings.

The World Anti-Doping Agency (WADA) publishes yearly their prohibited list, and sets a minimum required performance limit for each substance. To comply with these stringent requirements, the anti-doping laboratories have at least two complementary methods for their initial testing procedure (ITP), one using gas chromatography - mass spectrometry (GC-MS) and the other using liquid chromatography-MS (LC-MS). Anabolic androgenic steroids (AAS) have in previous years consistently been listed as the most frequently detected class of compounds. Over the last decade, evidence has emerged where a longer detection time is attained by focusing on sulfated metabolites of AAS instead of the conventional gluco-conjugated metabolites. Despite a decade of research on sulphated AAS using LC-MS, no LC-MS ITP has been developed that combines this class of compounds with the other mandatory targets. Such combination is essential for economical purposes. Recently, it was demonstrated that the direct injection of non-hydrolysed sulfates is compatible with GC-MS. Using this approach and by taking full use of the open screening capabilities of the quadrupole time of flight MS (QTOF-MS), this work describes for the first time a validated ITP that allows the detection of non-hydrolysed sulfated metabolites of AAS while, simultaneously, remaining capable of detecting a vast range of other classes of compounds, as well as the quantification of endogenous steroids, as required for an ITP compliant with the applicable WADA regulations. The method contains 263 compounds from 9 categories, including stimulants, narcotics, anabolic androgenic steroids and beta-blockers. Additionally, the advantages of the new method were illustrated by analysing excretion samples of drostanolone, mesterolone and metenolone. No negative effects were observed for the conventional markers and the detection time for mesterolone and metenolone increased by up to 150% and 144%, respectively compared to conventional markers.

Stanozolol (STAN) is an androgen anabolic steroid often misused in sports competitions and prohibited at all times by the World Anti-Doping Agency (WADA). It can be long term detected by the analysis of human urine for traces of intact glucuronide metabolites. The Zebrafish Water Tank (ZWT) experimental setup can produce phase I STAN metabolites. In the present study, we investigated the in vivo phase II metabolism of STAN through the ZWT model to determine whether the ZWT produces metabolites relevant for doping control. We added STAN to a 200 mL recipient containing eight fish at 32 ± 1 °C. We analyzed the noninvasive samples (recipient water) both with and without pretreatment using Liquid Chromatography coupled with High-Resolution Mass Spectrometry (LC-HRMS/MS) in positive ionization mode. Our data show that four hydroxylated-sulfate and four hydroxylated-glycoconjugate metabolites were formed, two of the last ones being 3’OH-STAN-Glucuronide and 16β-OH-STAN-Glucuronide. Additionally, two STAN-Glucuronide derivatives were produced: one was confirmed to be 17epi-STAN-N-Glucuronide, and the other was presumed to be STAN-O-Glucuronide. After eight hours of the experiment, STAN-O-Glucuronide was the most intense phase II metabolite produced. The accumulation curves suggest that high concentrations of fish and substrate in water are required to form phase II metabolites.

Analytical procedures to detect the misuse of selective androgen receptor modulators in human urine, targeting either the parent drugs and/or their main metabolites, were developed and validated. In detail, 19 target compounds belonging to 9 different chemical classes were considered: arylpropionamide (i.e., andarine (S4), ostarine (S22), S1, S6, S9 and S23), diarylhydantoin (i.e., GLPG0492), indole (i.e., LY2452473, GSK2881078), isoquinoline-carbonyle (i.e., PF-02620414), phenyl-oxadiazole (i.e., RAD140), pyrrolidinyl-benzonitrile (i.e., LGD4033), quinolinone (i.e., LGD2226, LGD3303), steroidal (i.e., Cl-4AS-1, MK0773 and TFM-4AS-1), and tropanol (i.e., AC-262536 and ACP105) derivatives. The metabolites of the target compounds considered were enzymatically synthesized by using human liver microsomes. Sample pre-treatment included enzymatic hydrolysis followed by liquid-liquid extraction at neutral pH. The instrumental analysis was performed by ultra-high-performance liquid chromatography coupled to either high- or low-resolution mass spectrometry. Validation was performed according to the ISO 17025 and the World Anti-Doping Agency guidelines. The analyses carried out on negative samples confirmed the method's selectivity, not showing any significant interferences at the retention times of the analytes of interest. Detection capability was determined in the range of 0.1-1.0 ng/mL for the screening procedure and 0.2-1.0 ng/mL for the confirmation procedure (except for GLPG0492 and GSK2881078). The recovery was greater than 80 % for all analytes, and the matrix effect was smaller than 35 %. The method also matched the criteria of the World Anti-Doping Agency in terms of repeatability of the relative retention times (CV% < 1.0) and of the relative abundances of the selected ion transitions (performed only in the case of triple quadrupole, CV% < 15), ensuring the correct identification of all the analytes considered. Urine samples containing andarine, ostarine, or LGD4033 were used to confirm the actual applicability of the selected analytical strategies. All target compounds (parent drugs and their main metabolites) were detected and correctly identified.

Replenishing neurons in patients with neurodegenerative diseases is one of the ultimate therapies for these progressive, debilitating and fatal diseases. Electrical stimulation can improve neuron stem cell differentiation but requires a reliable nanopatterned electroconductive substrate. Potential candidate substrates are polycaprolactone (PCL) – polyaniline:camphorsulfonic acid (PANI:CSA) nanofibers, but their nanobiophysical properties need to be finetuned. The present study investigates the use of the pseudo-doping effect on the optimization of the electroconductivity of these polyaniline-based electrospun nanofibers. This was performed by developing a new solvent system that comprises a mixture of hexafluoropropanol (HFP) and trifluoroethanol (TFE). For the first time, an electroconductivity so high as 0.2 S cm−1 was obtained for, obtained from a TFE:HFP 50/50 vol% solution, while maintaining fiber biocompatibility. The physicochemical mechanisms behind these changes were studied. The results suggest HFP promotes changes on PANI chains conformations through pseudo-doping, leading to the observed enhancement in electroconductivity. The consequences of such change in the nanofabrication of PCL-PANI fibers include an increase in fiber diameter (373 ± 172 nm), a decrease in contact angle (42 ± 3°) and a decrease in Young modulus (1.6 ± 0.5 MPa), making these fibers interesting candidates for neural tissue engineering. Electrical stimulation of differentiating neural stem cells was performed using AC electrical current. Positive effects on cell alignment and gene expression (DCX, MAP2) are observed. The novel optimized platform shows promising applications for (1) building in vitro platforms for drug screening, (2) interfaces for deep-brain electrodes; and (3) fully grown and functional neurons transplantation.