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Sports Drug Testing and the Athletes‘ Exposome
Abstract
Similar to the general population, elite athletes are exposed to a complex set of environmental factors including chemicals, radiation, but also biological and physical stressors, which constitute an exposome that is, unlike for the general population, subjected to specific scrutiny for athletes due to applicable anti‐doping regulations and associated (frequent) routine doping controls. Hence, investigations into the athlete’s exposome and how to distinguish between deliberate drug use and different contamination scenarios has become a central topic of anti‐doping research, as a delicate balance is to be managed between the vital and continually evolving developments of sensitive analytical techniques on the one hand, and the risk of the athletes’ exposome potentially causing adverse analytical findings on the other.
... Substances from the World Anti-Doping Agency's (WADA) Prohibited List can be contained in seminal fluid and also enter the female organism during sexual intercourse. This could potentially lead to adverse analytical findings (AAFs) in doping controls, as it is conceivable for a female athlete to excrete prohibited substances into the doping control urine sample via the seminal fluid that entered the body through unprotected intercourse (Thevis et al., 2021). ...
... The mere presence of such substances in a doping control urine sample can lead to an AAF for the athlete, as low concentrations may indicate the tail end of an elimination of a therapeutic (or doping) dose. However, the doping agent (or its marker) could also have entered the athlete's urine through intimate contact or other contamination scenarios (Thevis et al., 2021). In the present study, four substances that represent non-threshold and non-MRL substances were observed: Anastrozole, clomiphene, indapamide, and GW1516. ...
... In addition to seminal fluid, contaminated dietary supplements, medications or environmental factors can also lead to an AAF (Thevis et al., 2021). In 2021, WADA published a technical document for six diuretics, which are known contaminants in oral pharmaceutical products, and set an MRL of 20 ng/mL after minimal amounts of diuretics were found in several case reports (Helmlin et al., 2016;Favretto et al., 2019;Gheddar et al., 2019; World Anti-Doping Agency, 2023a). ...
Exogenous substances, including drugs and chemicals, can transfer into human seminal fluid and influence male fertility and reproduction. In addition, substances relevant in the context of sports drug testing programs, can be transferred into the urine of a female athlete (after unprotected sexual intercourse) and trigger a so-called Adverse Analytical Finding. Here, the question arises as to whether it is possible to distinguish analytically between intentional doping offences and unintentional contamination of urine by seminal fluid. To this end, 480 seminal fluids from non-athletes were analysed to identify concentration ranges and metabolite profiles of therapeutic drugs that are also classified as doping agents. Therefore, a screening procedure was developed using liquid chromatography connected to a triple quadrupole mass spectrometer, and suspect samples (i.e. samples indicating the presence of relevant compounds) were further subjected to liquid chromatography-high-resolution accurate mass (tandem) mass spectrometry. The screening method yielded 90 findings (including aromatase inhibitors, selective estrogen receptor modulators, diuretics, stimulants, glucocorticoids, beta-blockers, antidepressants, and the non-approved PPARδ agonist GW1516) in a total of 81 samples, with 91 % of these suspected cases being verified by the confirmation method. Besides the intact drug, phase-I and -II metabolites were also occasionally observed in the seminal fluid. This study demonstrated that various drugs including those categorized as doping agents partition into seminal fluid. Monitoring substances and metabolites may contribute to a better understanding of the distribution and metabolism of exogenous substances in seminal fluid that may be responsible for the impairment of male fertility. Significance Statement This study demonstrates that doping agents as well as clinically relevant substances are transferred/eliminated into seminal fluid to a substantial extent and that knowledge about drug levels (and potential consequences for the male fertility and female exposure) is limited. The herein generated new dataset provides new insights into an important and yet little explored area of drug deposition and elimination, and hereby a basis for the assessment of contamination cases by seminal fluid in sports drug testing.
... 10 However, the undeniable added value provided by the improved analytical sensitivity of anti-doping laboratories, which has substantially extended the retrospective concerning the use of doping agents, also raised concerns as to the possibility that inadvertent and unknowing contamination scenarios (as opposed to 'doping scenarios') could result in adverse analytical findings (AAFs) of innocent athletes. 11 Awareness of the athlete's exposome, 12 the unique situation of athletes being subject to routine doping controls, and the necessity of complementary strategies in doping control sampling as well as analysis in support of decisionmaking processes have since been frequently thematized as outlined in this brief review. ...
... 17 Acknowledging existing technological limitations in comprehensively profiling the exposome with its inevitable "invisible" (not profiled), "hidden" (not detected), and "dark" (not annotated) features as defined by David et al., 17 the exposome's relevance has received growing attention in various disciplines including acute and long-term toxicological evaluations, 18,19 medical and pathological considerations [20][21][22] and, more recently, in the specific niche context of sports drug testing. 12 In contrast to most medical and toxicological (research) questions with regards to the exposome, elite athletes are frequently blood-and urine-tested with utmost scrutiny concerning trace amounts of a specified subset of drugs, chemicals, and confounding factors; here, AAFs based are on drug or drug metabolite residues at any detectable level (depending on the sample matrix, type of sample collection, i.e., in-vs. out-of-competition, and the identified drug/drug metabolite) might immediately result in sanctions due to an established anti-doping rule violation. ...
... Consequently, in view of the human exposome's extent and the peculiar situation of elite athletes in global anti-doping programs, research, data, and strategies to support result management authorities in identifying and differentiating the inadvertent and unexpected exposure to prohibited substances from findings resulting from the knowing and intentional administration (for performanceenhancing purposes) have become particularly important as recently summarized in a review elsewhere. 12 In brief, at least four categories warrant consideration within the specific focus of the athlete's exposome including (a) food containing prohibited substances because of the natural formation (e.g., cocaine, ephedrine, higenamine, etc., legitimate, e.g., ractopamine, zilpaterol, etc.), or illegal (e.g., clenbuterol, clomiphene, etc.) use during production 23 ; (b) adulterated or contaminated dietary supplements 24 ; (c) drug metabolism (e.g., proguanil/chlorazanil, chlorphenesin/4-chlorophenoxyacetic acid 25 ) or drug contamination (e.g., non-prohibited drugs containing, e.g., diuretics) 26 ; and (d) interindividual drug residue transfer (e.g., via intimate 27 /interpersonal dermal 28 contact). Hence, concerted effort from all partners in sports (i.e., athletes, federations, anti-doping organizations, antidoping laboratories), awareness, and dedicated studies in the context of preventive doping research is required. ...
Test methods in anti‐doping, most of which rely on the most modern mass spectrometric instrumentation, undergo continuous optimization in order to accommodate growing demands as to comprehensiveness, sensitivity, retrospectivity, cost‐effectiveness, turnaround times, etc. While developing and improving analytical approaches is vital for appropriate sports drug testing programs, the combination of today's excellent analytical potential and the inevitable exposure of humans to complex environmental factors, specifically chemicals and drugs at the lowest levels, has necessitated dedicated research, particularly into the elite athlete's exposome. Being subjected to routine doping controls, athletes frequently undergo blood and/or urine tests for a plethora of drugs, chemicals, corresponding metabolic products, and various biomarkers. Due to the applicable anti‐doping regulations, the presence of prohibited substances in an athlete's organism can constitute an anti‐doping rule violation with severe consequences for the individual's career (in contrast to the general population), and frequently the question of whether the analytical data can assist in differentiating scenarios of ‘doping’ from ‘contamination through inadvertent exposure’ is raised. Hence, investigations into the athlete's exposome and how to distinguish between deliberate drug use and potential exposure scenarios have become a central topic of anti‐doping research, aiming at supporting and consolidating the balance between essential analytical performance characteristics of doping control test methods and the mandate of protecting the clean athlete by exploiting new strategies in sampling and analyzing specimens for sports drug‐testing purposes.
... In addition to deliberate doping with SARMs, a problem that has garnered inc attention is athletes' exposome, and especially the risk contaminated supplements athletes [13,14]. As dietary supplements, in contrast to pharmaceuticals, are no sively monitored, they may contain undeclared prohibited ingredients [15]. ...
... In addition to deliberate doping with SARMs, a problem that has garnered increasing attention is athletes' exposome, and especially the risk contaminated supplements pose to athletes [13,14]. As dietary supplements, in contrast to pharmaceuticals, are not extensively monitored, they may contain undeclared prohibited ingredients [15]. ...
RAD140 is a selective androgen receptor modulator which has been abused in sporting competitions. Its use is prohibited by the World Anti-Doping Agency (WADA) for athletes at all times. In addition to its illicit use, adverse analytical findings of RAD140 in doping control samples might result from other scenarios, e.g., the ingestion of contaminated dietary supplements. The differentiation between samples resulting from such contamination scenarios and intentional doping presents a considerable challenge, as little is known about the metabolism and elimination behavior of RAD140 in humans. In this study, six micro-dose excretion studies with five adult male volunteers each were conducted, and urine samples were analyzed by means of LC-HRMS/MS. Multiple metabolites, firstly detected in human urine, are described in this study. The sample preparation included an enzymatic hydrolysis step, which facilitated the estimation of RAD140 concentrations in urine. The elimination profiles and detection times for six metabolites as well as the intact drug are presented. The method was extensively characterized and deemed fit-for-purpose. The metabolite ratios were investigated for their predictive power in estimating the dose of RAD140 intake. The presented data will aid in better case result management in future doping cases involving RAD140.
... By contamination, one can include laced supplements, either voluntarily or by lack of hygiene measures; contaminated meat by growth promoters; poor quality pharmaceuticals with chemical residues, and finally drug transfer during intimate moments or various other scenarios [16]. This has been summarized by Thevis et al. under the concept of "athlete exposome" [17]. ...
Clostebol, the 4-chloro derivative of testosterone, available as Over The Counter product in pharmacies and drugstores in several countries, is mostly commercialized as a cream or spray in the form of acetate ester. As other anabolic steroids, clostebol is listed as a prohibited substance by the World Anti-Doping Agency (WADA). Controlled transdermal application of clostebol acetate has been reported to produce detectable amounts of its metabolites in urine, even after a single exposure. Indeed, a low urine concentration can be interpreted as the tail of a drug voluntarily used to enhance performance or a direct consequence of a contamination. The increased number of adverse analytical findings (AAFs) involving clostebol reported in the last years should lead to highlight the need for athletes to be warned against personal and /or accidental use/exposure of dermal preparation containing this doping agent. Further discussion on possible threshold limits and laboratory testing on different matrices (e.g. hair) to better clarify the origin of minimal amounts of clostebol in urines is advisable.
... A major challenge in that context is the complex environmental influence, which can affect an athlete's career considerably more serious than the general population due to their participation in sports drug testing programs. Prohibited substances can be transferred in trace amounts to athletes, which can then lead to an adverse analytical finding (AAF) [1]. The detection of minute concentrations of doping agents has been of utmost importance to doping controls, and the evolution of analytical techniques has offered increasing sensitivities that allow for the required analytical retrospectivity in sports drug testing. ...
Purpose
Inadvertent and/or unknowing exposure to drugs and drug residues has been frequently debated in situations of so-called adverse analytical finding (AAF) in the context of sports drug testing programs. Transfer of drug residues via unprotected intercourse is a conceivable scenario but scientific data and authentic case reports are scarce. Herein, investigations into two AAFs with the peroxisome proliferator-activated receptor delta (PPARδ) agonist GW1516 are reported and discussed.
Methods
To probe for a contamination scenario involving sexual intercourse, two assays were used to determine semenogelin in human urine, with one employing an immunochromatographic lateral flow approach and another based on liquid chromatography–tandem mass spectrometry. Further, drug-residue testing using patients’ ejaculate was conducted by utilizing liquid chromatography in conjunction with a triple quadrupole mass spectrometer, followed by re-analysis of suspect samples (i.e., samples indicating the presence of relevant compounds) using high resolution/high mass accuracy mass spectrometry.
Results
In one case, but not the other, the possibility of intimate contact as the source of the AAF was confirmed after a thorough investigation of potential contamination scenarios. Subsequent research revealed analytical evidence for the presence of seminal fluid in one of the female athlete’s doping control urine samples, and the analysis of clinical ejaculate specimens provided first data on an authentic concentration level of GW1516 and its metabolites in human seminal fluid.
Conclusions
The combined facts substantiate the possibility of an AAF caused by unprotected sexual intercourse and the plausibility of the case-related arguments.
... Being vigilant when using pharmaceuticals should be a part of active athletes' everyday life in order to avoid unintentional doping, similarly, as has been reported for dietary supplements [e.g., (3)]. The use of pharmaceuticals is thus a part of the athletes' exposome (4), and should also form a part of the athletes' health literacy (5,6). ...
Introduction
The aim of the study was to map the use of pharmaceuticals by Norwegian athletes registered on doping control forms (DCFs) in a five-year period to examine general and some class specific use of pharmaceuticals across sports and athlete levels.
Method
Anonymous data from DCFs collected in 2015-2019 were manually entered into a database using the Anatomical Therapeutic Chemical (ATC) system for classification of the pharmaceuticals. Variables entered were year of control, gender, age group, athlete level, sport, test type, nationality, and pharmaceuticals (and dietary supplements) used.
Results
Pain killers in the ATC groups M01 A (Nonsteroidal anti-inflammatory drugs - NSAIDs) and N02 B (other analgesics), and anti-asthmatics in ATC groups R03 A and R03 B were the most frequently used pharmaceuticals. National level athletes reported more use of pharmaceuticals (1.4 ± 1.7 pharmaceuticals per form) than recreational level athletes (0.9 ± 1.2). The highest proportion of DCFs containing information about at least one pharmaceutical were found in speed skating (79.1%), alpine skiing (74.0%), rowing (72.4%) and cross-country skiing (71.7%). Painkillers were most frequently used in muscular endurance sports (30.4% and 21.2 % for M01A and N02 B, respectively) and ball and team sports (17.9% and 17.0%). Use of hypnotics was reported from ice-hockey players and alpine skiers in around 8% of the cases.
Coclusion
Use of anti-asthmatics was most often reported amongst athletes specially exposed to cold, chemicals and heavy endurance training. Athletes in specialized sports requiring high levels of strength and/or endurance reported a higher use of pharmaceuticals out-of-competition compared to in-competition, while there was no such difference in complex sports, such as team, gymnastic, aiming and combat sports.
... [8] Various anti-doping organizations, such as the International Paralympic Committee, the International Olympic Committee, major event organizations, international sports federations (such as the International Cycling Union), and national anti-doping organizations, are tasked with implementing, adopting, or enforcing anti-doping rules within their respective spheres of authority, and the Code lays out specific rules and principles for them to follow. [26][27][28][29] Every year or so, WADA updates and releases a new version of its list of prohibited drugs. There are certain banned drugs and procedures that are always illegal (in and out of competition) due to the possibility of enhancing performance in future contests or the possibility of masking, and there are other banned substances and methods that are only prohibited during competition. ...
Drug addiction and abuse are major public health issues across the globe that impact millions of people. Substance abuse and dependence seem to have been relatively high among university students and laborers and comparatively low among school students. Drugs used by the person for nonintended reasons, mainly for their psychoactive effects termed as drug abuse. Continued use of alcohol, illegal drugs, or prescription or over-the-counter medications harms health, employment, family, and law. When it comes to illicit substance usage, possession, and trafficking, every nation has its unique set of laws. The frequency of lifetime drug usage in 2015 was 5%, according to the most current World Drug Report. There is reason to be concerned about the high rates of drug and alcohol addiction among youths because of the substantial problems that have been linked to such use, including higher rates of aggression, suicidal attempts, etc., In this narrative review, we have focused on illicit drugs, substance use, its negative impacts among youths and its prevalence among youths nationally and internationally, and also some prevention strategies to control substance use.
... This generally desirable superior detection capability that is vital for appropriate sports drug testing programs can, however, also lead to AAFs, where unintentional and/or unknown exposure of an athlete to drugs or drug residues occurred. Such cases included for instance the use of contaminated dietary supplements or the exchange of contaminated body fluids [3][4][5][6]. ...
Potential scenarios as to the origin of minute amounts of banned substances detected in doping control samples have been a much-discussed problem in anti-doping analysis in recent years. One such debated scenario has been the contamination of female athletes’ urine with ejaculate containing doping agents and/or their metabolites. The aim of this work was to obtain complementary information on whether relevant concentration ranges of doping substances are excreted into the ejaculate and which metabolites can be detected in the seminal fluid (sf) and corresponding blood plasma (bp) samples. A method was established to study the concentration and metabolite profiles of stanozolol and LGD-4033—substances listed under anabolic substances (S1) on the World Anti-Doping Agency’s Prohibited List—in bp and sf using liquid chromatography high-resolution mass spectrometry (LC-HRMS). For sf and bp, methods for detecting minute amounts of these substances were developed and tested for specificity, recovery, linearity, precision, and reliability. Subsequently, sf and bp samples from an animal administration study, where a boar orally received stanozolol at 0.33 mg/kg and LGD-4033 at 0.11 mg/kg, were measured. The developed assays proved appropriate for the detection of the target substances in both matrices with detection limits between 10 and 40 pg/mL for the unmetabolized drugs in sf and bp, allowing to estimate the concentration of stanozolol in bp (0.02–0.40 ng/mL) and in sf (0.01–0.25 ng/mL) as well as of LGD-4033 in bp (0.21–2.00 ng/mL) and in sf (0.03–0.68 ng/mL) post-administration. In addition, metabolites resulting from different metabolic pathways were identified in sf and bp, with sf resembling a composite of the metabolic profile of bp and urine.
Graphical Abstract
The presence of letrozole, an aromatase inhibitor, in an athlete’s sample constitutes one of the more frequent anti-doping rules violation. It is possible to challenge this violation but it is the athletes who have to demonstrate their innocence. The conditions to evidence/establish the absence of fault or negligence hinge on two points: 1. the athletes or their legal representatives have to present verified circumstances of contamination and the source of contamination has to be identified; and 2. there have to be verified claims by the athlete about the fact that the intake of the prohibited substance was not known, i.e. that the violation was not intentional. This corresponds to the suggested shift terminology from “contaminated product” to “unpredictable source of a prohibited substance”. In the recent years, several top athletes challenged their ADRV with a low urine letrozole concentration and requested a hair test. In three cases, letrozole concentration in segmented hair, particularly in the segment corresponding to the urine AAF was significantly lower than 1 pg/mg, which is the limit of quantification of the method. Considering that a ¼ of a 2.5 mg therapeutic dose of letrozole produces a hair concentration of approximately 30 pg/mg, it is easy to establish that the dose that entered in the body of these athletes was incidental. Nevertheless, all three athletes were sentenced a 2-years ban as the source of contamination was not identified. In that sense, the WADA dogma contradicts scientific evidence, and from a forensic perspective, this appears difficult to understand.
The continuous consumption of various foods increases the risk of unintentional exposure to residual contaminants.
Electroanalytical methods have gained significant attention for the detection of performance-enhancing drugs (PEDs) in food matrices due to their high sensitivity, selectivity, and rapid response. This review provides a comprehensive overview of the current state-of-the-art in electroanalytical tools for identifying food-borne PEDs in sports testing. The article discusses various classes of PEDs found in food, including animal-derived, plant-derived, and synthetic nutritional supplements. It also covers the principles and applications of voltammetric techniques, amperometric biosensors, and recent advancements in electrode materials and surface modification strategies. In particular, the use of molecularly imprinted polymers (MIPs) as selective recognition elements in electroanalytical sensors is highlighted, as they offer improved specificity for target PEDs in complex food matrices. The challenges associated with PED detection in food matrices, such as matrix effects and interferences, are addressed, along with future perspectives on method validation, standardization, multiplexed detection, and on-site screening applications. The most significant results highlighted in this review include the achievement of low detection limits, wide linear ranges, and high selectivity for various PEDs in complex food matrices.
The identification of trimetazidine, a medicine used for treating stable angina pectoris and for preventing angina attacks, has been recently observed in doping cases involving high profile athletes from various countries over the world. In all the files where the authors have been involved, the urine concentration of trimetazidine was low (<2 ng/mL), and the athletes argued that contamination was the source of their adverse analytical finding. It is possible to challenge imposed sanctions in relation to an adverse analytical finding, but it is the responsibility of the athlete to demonstrate he/she is innocent and can qualify for no fault or negligence . When the delay between the urine collection and the notification of the violation was not too long (less than 6 months), these athletes requested a head hair test. Trimetazidine was analyzed by an original LC–MS/MS method involving pH 9.5 borate buffer overnight incubation of 20 mg and subsequent solvents extraction in presence of trimetazidine‐D8 used as internal standard. Linearity was verified from 1 to 200 pg/mg ( R ² = 0.9987). Limit of detection of the method was 0.1 pg/mg. The hair specimen of a male subject, collected 4 weeks after single oral ingestion of 20 mg trimetazidine, tested positive at 146 pg/mg in the corresponding segment. Concentrations of trimetazidine measured in several hair specimens ( n = 5) collected from athletes challenging their anti‐doping rule violation were below 1 pg/mg, which is consistent with incidental exposure due to contamination. This is the first evidence that trimetazidine is incorporated in human hair after a single therapeutic dose administration.
Higenamine (HG) is a β 2 receptor agonist and was explicitly added to the Prohibited List of the World Anti‐Doping Agency in 2017. This compound is prohibited in both in‐ and out‐of‐competition athletes and falls under the category of nonthreshold substances. Because of HG presence in numerous plants, as evidenced by a growing body of research data, an exception was made for HG in the TD2017MRPL document, in which adverse analytical findings (AAFs) were not reported if the urinary HG concentration was less than 10 ng/mL. In this study, a comprehensive and systematic analysis of the HG content in five batches of samples from each of the 48 natural spices selected for this investigation was conducted using UPLC–MS/MS technology. Method validation was carried out in accordance with the ICH Analytical Procedures and Methods Validation for Drugs and Biologics Guidance, and the experimental results demonstrated that the method provided appropriate sensitivity, precision, stability, linearity, and accuracy. HG was detected for the first time in Houttuynia cordata , Zingiber officinale , Cinnamomum cassia , Stevia rebaudiana , Piper nigrum , Siraitia grosuenorii , Platycodon grandiflorus , and Myristica fragrans . Furthermore, the content of HG was found to vary significantly among the different plant parts of Nelumbo nucifera , such as rhizomes, leaves, seeds, and plumules. This paper provides systematic and comprehensive data to support the safe use of spices in athletes' diets, thereby reducing the risk of food‐sourced doping violations.
Introduction:
The analysis of doping control samples is preferably performed by mass spectrometry, because obtained results meet the highest analytical standards and ensure an impressive degree of reliability. The advancement in mass spectrometry and all its associated technologies thus allow for continuous improvements in doping control analysis.
Areas covered:
Modern mass spectrometric systems have reached a status of increased sensitivity, robustness, and specificity within the last decade. The improved sensitivity in particular has, on the other hand, also led to the detection of drug residues that were attributable to scenarios where the prohibited substances were not administered consciously but rather by the unconscious ingestion of or exposure to contaminated products. These scenarios and their doubtless clarification represent a great challenge. Here, too, modern MS systems and their applications can provide good insights in the interpretation of dose-related metabolism of prohibited substances. In addition to the development of new instruments itself, software-assisted analysis of the sometimes highly complex data is playing an increasingly important role and facilitating the work of doping control laboratories.
Expert opinion:
The sensitive analysis and evaluation of a higher number of samples in a shorter time is made possible by the ongoing developments in mass spectrometry.
Ice hockey is a high-risk sport known for its dominant macho culture. The purpose of this study was to examine experiences surrounding medication use among male, elite ice hockey players in Norway. A mixed-method design was employed, which first examined medications registered on doping control forms (DCFs) (n = 177) and then involved semi-structured focus group interviews (n = 5) with elite athletes (n = 25). Overall, 68% of the DCFs contained information about ≥1 medication. Among the most registered medications were NSAIDs and hypnotics (20% and 19% of all DCFs, respectively). During the interviews, numerous athletes reported using analgesics to manage injuries and pain caused by the sport, often being motivated by sacrificing themselves for the team during important matches and playoffs. Hypnotics were used due to high cumulative stress due to heavy training and competition load, late-night matches, and playing in a semi-professional league. Athlete support personnel (ASP), including physicians and trainers, were the athletes’ main sources of information. The athletes often displayed a profound and non-critical trust in the advice and products provided to them by their team physician. The findings indicate that male, elite ice hockey players, through their excessive and somewhat ignorant use of medications, expose themselves to health risks and inadvertent doping.
Hair analysis is a reliable and widely used tool to evaluate drug exposure in many fields, including workplace testing, drug abuse history and withdrawal control, post-mortem toxicology, doping control, therapeutic drug monitoring of pharmaceuticals and even environmental exposure to toxic agents. Compounds incorporated into the hair structure resist hair growth and regular washing for several months, leading to a potential chronological trace of exposure, with farther periods corresponding to the hair segments more distant from the hair root. The relentless improvement of analytical procedures and instrumental technologies, together with the continuous introduction of new psychoactive substances, have led to an increasing number of studies and practical applications of hair analysis.
This book is a comprehensive guide to hair analysis from general concepts, ideal for students and those new to the field, to interpretation and advanced methods for experts working in the area. With contributions from world-leading scientists in each field, this book describes state-of-the-art, emerging issues and recent analytical approaches to hair analysis that will serve as an essential tool to clinical and forensic toxicology laboratories across the globe.
Capromorelin is a growth hormone secretagogue. Despite promising results to alleviate muscle-wasting in the elderly, it has not advanced further in human development. Subsequent studies demonstrated capromorelin's ability to increase food intake in animals, leading to approval in the United States and Europe as an appetite stimulant for cats (Elura) and dogs (Entyce). Capromorelin is prohibited in sports due to its ability to stimulate growth hormone production and enhance performance. However, given that its veterinary preparation is formulated as a highly concentrated solution (20 or 30 mg/mL) delivered orally, incidental ingestion or dermal absorption may result in an adverse analytical finding (AAF) by way of direct exposure during oral administration to a pet. An administration study was conducted by either oral or transdermal application of capromorelin solution to mimic the scenario of inadvertent exposure to the drug. Ingestion of 30 μg of capromorelin orally (equivalent to 1 μL of Entyce) resulted in detectable amounts of capromorelin in urine for up to 48 h after administration with a maximum urinary concentration of 7 ng/mL. Importantly, when applied directly to the skin on the hands in larger quantities mimicking a pet administration exposure scenario (30 mg or 1 mL of Entyce), capromorelin was also detected reaching a maximum urinary concentration of 0.7 ng/mL. Athletes and testing authorities should be aware of the risk of an AAF arising due to incidental exposure to veterinary preparations of capromorelin. To our knowledge, before 2022, no positive test for capromorelin had ever been reported.
Clomiphene or clomifene is a selective estrogen receptor modulator (SERM), used to treat female fertility in case of ovulatory dysfunction. In sport, clomiphene is prohibited at all times for use by athletes and is listed in the section S4.2 (hormone and metabolic modulators) by the World Anti-Doping Agency (WADA). Indeed, clomiphene can indirectly increase testosterone levels in the body and can mitigate some side effects of synthetic steroid abuse. Despite its prescription to millions of subjects, its detection in human hair or nail clippings has never been reported. The aim of this study was to develop a specific method to identify clomiphene in hair and nail clippings by LC-MS-MS. The procedure was then applied in a case of challenged doping results. The method involves sonication/incubation for 1 hour of 30 mg of pulverized material in 1 mL of methanol in presence of 2 ng diazepam-d5 used as internal standard. The chromatographic separation was performed using a HSS C18 column with a 15 min gradient elution. After spiking blank hair and nail with the corresponding amounts of clomiphene, linearity was verified from 1 to 500 pg/mg (r² = 0.9994 and 0.9995 for hair and nail, respectively). The limit of detection was estimated at 0.3 pg/mg for both matrices. No interference was noted from endogenous compounds, particularly steroids. Clomiphene was identified at 85 and 20 pg/mg in the pubic hair and the fingernail clippings, respectively, of a male athlete challenging an adverse analytical finding.
Biological testing is a key component of the current anti-doping programme implemented by the authorities to detect doping in sports. Strategies such as longitudinal individualised data analysis and sport-specific analysis have been developed to increase the comprehensiveness of the testing. However, the trends of drug misuse in sports might not be effectively captured through today's testing plan. Wastewater testing, assembling individual-level data of a designated group to produce population-level results in one single aggregated sample, can be employed to as a complementary strategy offering added value for doping control. This paper presents an updated summary of the status of anti-doping testing and analytical methodologies for wastewater. The available literature on wastewater-based analyses of drugs prohibited in sports is reviewed. Publications surrounding sporting activities or competitions and others relevant to sports doping are selected. We debate between potential strategies and major limitations of using wastewater monitoring in anti-doping. Knowledge gaps and research directions, specifically on metabolites, stability, sensitivity, and ethical and legal considerations, are discussed. Choosing different wastewater sampling sites allows target sub-population that involved competing athletes and potentially reveal sport-specific or athlete-level-specific behaviour. Sampling from on-board toilets or athlete villages could target international-level athletes, sampling from the dormitories of national training centres allows monitoring of national-level athletes on a daily basis, and sampling from sports stadiums provides a full picture of drug use in the general population during an event. Confounding occurs as (i) the presence of non-athlete composition and the difficulty of analyses to be completely selective to the athlete population; and (ii) the identification of compounds prescribed legitimately with Therapeutic Use Exemptions, only banned in-competition, and naturally occurring. The practicalities of the approach are contextualised in monitoring the non-threshold substances such as anabolic agents, selective androgen receptor modulators, metabolic modulators, and hypoxia-inducible factor activators.
Roxadustat is an oral inhibitor of hypoxia-inducible factor prolyl hydroxylase which increases erythropoiesis. It can therefore be used as a doping agent. No data are available on how to measure roxadustat in hair and on the concentration found in treated patients. The aim of this study was to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of roxadustat in hair and to apply it to a chronically treated patient. After decontamination with dichloromethane, testosterone-D3 used as an internal standard and phosphate buffer pH 5.0 were added to 20 mg of hair and incubated for 10 min at 95°C. Four ml of dichloromethane were used for extraction and reconstituted into the mobile phase, 10 µl were injected into the chromatographic system. The method was linear in the range 0.5-200 pg/mg, accurate and precise (evaluated at 3 levels) and was successfully applied to measure roxadustat in a brown-haired patient treated pharmacologically with 100-120 mg 3 days a week. Results were stable between 41 and 57 pg/mg in the 6 proximal 1 cm segments. This first method describing the measurement of roxadustat in hair appears to be suitable for the quantification of this compound in clinical or doping control cases.
The presence of a prohibited substance or its metabolites or its markers in an athlete's sample constitutes the more frequent anti-doping rules violation. In the world anti-doping code, it is indicated (point 10.5) that if someone establishes in an individual case that the athlete bears no fault or negligence, then the otherwise applicable period of ineligibility shall be eliminated. The conditions that have to be met to fix the no fault or negligence evidence are described in several other points of the code. The following two points are of paramount importance: 1. the athlete or his/her legal representative must present verified circumstances of contamination and the source of contamination must be identified; and
2. there must be verified claims by the athlete about the fact that he/she did not knowingly take the prohibited substance, i.e., that the violation was not intentional.In recent years, several cases of contamination involving drug transfer during intimate moments have been reported. This later situation was first reported in 2009 with the Richard Gasquet case. Since that time, several athletes have been allowed to return to competition with no charge based on strong evidence that the source of contamination was drug transfer during intimate moments. As some of these cases are public and because the author performed hair tests for the majority of the international athletes involved in such procedures, the strategy of the defence and the scientific bases of discussion are reviewed in this article.
Background
While multiple facets of doping have been subject to intense research, knowledge about the consequences of sanctions following a doping offence for athletes is limited. From a biopsychosocial perspective, an anti-doping rule violation might have a serious adverse effect on an athlete´s health. To encourage more research on this topic, we developed and tested an interview guide.
Methods
Four elite Austrian athletes who had been suspended due to a doping offence first took part in an online brainstorming session on this topic. Based on the existing literature, five fundamental dimensions of life were distinguished: professional and financial situation, mental and psychological well-being, social environment, physical condition, and attitude towards and practice of sport. The participants were asked to report important changes in each dimension. Based on these data, an interview guide was developed. Semi-structured interviews were conducted with the same athletes and evaluated using thematic analysis.
Results and discussion
All athletes reported significant changes in all five dimensions, particularly in psychological well-being. The interview guide captured these changes in a satisfactory manner. Through a process of communicative validation, the interview guide was shown to be appropriate for further research on this topic.
Outlook
Using the interview guide, a larger international sample of athletes will be examined, especially in terms of differences in sport disciplines, achievement levels, doping circumstances, intentionality and socio-demographic factors; such data are essential to develop specific support programs for athletes banned from sport due to a doping offence.
Methodologies in applied sport science have predominantly driven a reductionist grounding to component-specific mechanisms to drive athlete training and care. While linear mechanistic approaches provide useful insights, they have impeded progress in the development of more complex network physiology models that consider the temporal and spatial interactions of multiple factors within and across systems and subsystems. For this, a more sophisticated approach is needed and the development of such a methodological framework can be considered a Sport Grand Challenge. Specifically, a transdisciplinary phenomics-based scientific and modeling framework has merit. Phenomics is a relatively new area in human precision medicine, but it is also a developed area of research in the plant and evolutionary biology sciences. The convergence of innovative precision medicine, portable non-destructive measurement technologies, and advancements in modeling complex human behavior are central for the integration of phenomics into sport science. The approach enables application of concepts such as phenotypic fitness, plasticity, dose-response dynamics, critical windows, and multi-dimensional network models of behavior. In addition, profiles are grounded in indices of change, and models consider the athlete’s performance or recovery trajectory as a function of their dynamic environment. This new framework is introduced across several example sport science domains for potential integration. Specific factors of emphasis are provided as potential candidate fitness variables and example profiles provide a generalizable modeling approach for precision training and care. Finally, considerations for the future are discussed, including scaling from individual athletes to teams and additional factors necessary for the successful implementation of phenomics.
Also in 2021/2022, considerable efforts were invested into advancing human sports drug testing programs, recognizing and taking into account existing as well as emerging challenges in anti‐doping, especially with regards to substances and methods of doping specified in the World Anti‐Doping Agency’s 2022 Prohibited List. In this edition of the annual banned‐substance review, literature on recent developments published between October 2021 and September 2022 is summarized and discussed. Focus is put particularly on enhanced analytical approaches and complementary testing options in human doping controls, appreciating the exigence and mission in anti‐doping and, equally, the contemporary ‘new normal’ considering e.g. the athlete’s exposome vs. analytical sensitivity and applicable anti‐doping regulations for result interpretation and management.
Situations of both, intentional as well as inadvertent or accidental doping, necessitate consideration in today’s doping controls, especially in the light of the substantial consequences that athletes are facing in case of so‐called adverse analytical findings. The aim of this study was to investigate, whether a transdermal uptake of doping substances would be possible. In addition to the period of detectability of the particular substances or respective characteristic metabolites, the possibility of deducing the route of administration by metabolite patterns was also assessed. Twelve male subjects were included in the study. Four common anabolic androgenic steroids (AAS) were dissolved in dimethylsulfoxide (DMSO) to facilitate transdermal administration on different skin regions. One half of the test persons received only oxandrolone (17α‐methyl‐2‐oxa‐4,5α‐dihydrotestosterone), the other half was applied a mixture of oxandrolone, metandienone (17β‐hydroxy‐17‐methylandrosta‐1,4‐dien‐3‐one), clostebol (4‐chlorotestosterone‐17β‐acetate) and dehydrochloromethyltestosterone (DHCMT). Urine samples were collected 1 hour, 6 hours and one sample per day for the next 14 consecutive days. Measurements were conducted on a GC‐MS/MS or LC‐MS/MS system. Substance findings were obtained at least 1 day after application on nearly all skin locations. The results indicated inter‐individual variability in detection windows, also varying between the different analytes and possible impact of skin location and skin thickness, respectively. Nevertheless, a rapid and rather long detectability of all substances (or respective metabolites) was given, in some cases within hours after administration and for up to 10‐14 days. Hence, the transdermal application or exposure to the investigated AAS is a plausible scenario that warrants consideration in anti‐doping.
For decades, blood testing has been an integral part of routine doping controls. The breadth of information contained in blood samples has become considerably more accessible for anti-doping purposes over the last 10 years through technological advancements regarding analytical instrumentation as well as enhanced sample collection systems. Particularly, microsampling of whole blood and serum, for instance as dried blood spots (DBS), has opened new avenues in sports drug testing and substantially increased the availability and cost-effectiveness of doping control specimens. Thus, microvolume blood specimens possess the potential to improve monitoring of blood hormone and drug levels, support evaluation of circulating drug concentrations in competition, and enhance the stability of labile markers and target analytes in blood passport analyses as well as peptide hormone and steroid ester detection. Further, the availability of the fraction of lysed erythrocytes for anti-doping purposes warrants additional investigation, considering the sequestering capability of red blood cells (RBCs) for certain substances, as a complementary approach in support of the clean sport.
RATIONALE
The synthetic β-adrenoreceptor agonist zilpaterol is legitimately used as an animal feed supplement in selected countries due to its known effects on lipolysis and protein biosynthesis. These pharmacological characteristics of zilpaterol have contributed to its classification as doping agent in sport by the World Anti-Doping Agency. However, the use as a feed supplement can lead to residues of the drug in edible tissues and, possibly, also in the urine of consumers.
METHODS
In order to provide urinary elimination profiles of microdosed zilpaterol and to determine whether the ingestion of zilpaterol below or at the acceptable daily intake level of 0.04 μg/kg bodyweight can result in an adverse analytical finding (AAF) in doping controls, healthy volunteers were administered single or multiple oral doses of 0.5 μg or 3 μg zilpaterol to mimic ingestion of contaminated cattle meat. Urine samples were collected and analyzed using a validated HPLC-ESI-MS/MS method and a newly developed chiral HPLC-APCI-MS/MS method.
RESULTS
Urinary peak concentrations of zilpaterol were observed for all volunteers 1.5-12.5 h after ingestion, and maximum levels > 5 ng/mL, which would constitute an AAF in doping controls, were found after the intake of 3 μg of zilpaterol on five consecutive days in one out of five study participants. Noteworthy, the enantiomeric ratio of excreted zilpaterol remained constant over time.
CONCLUSION
This study provides first insights into the urinary excretion of microdosed zilpaterol. Furthermore, a method for the separation of the zilpaterol enantiomers with mass spectrometric detection was successfully developed and applied.
Early November 2020 an Olympic gold medal winner returned during an out‐competition control an adverse analytical finding for dorzolamide, a diuretic mostly used to treat glaucoma. Estimated urine concentrations were 2.2 and 1.6 ng/ml in the A and B specimens, respectively. As the athlete denied any use of dorzolamide, a complex forensic investigation was suggested. It revealed that the athlete was severely injured during a car crash 6 months before where he received 2× 500 ml of red blood cells transfusion. One of the blood donors declared using dorzolamide. A plasma aliquot, stored for legal purposes, was tested by LC‐MS/MS several months later and contained 4.3 ng/ml of dorzolamide. Given the very long half‐life of the drug, up to 150 days and its reported incorporation into erythrocyte, it was accepted by the French antidoping administration (AFLD) that the source of contamination was this blood transfusion and that the antidoping rule violation was unintended.
Most core areas of anti‐doping research exploit and rely on analytical chemistry, applied to studies aiming at further improving the test methods’ analytical sensitivity, the assays’ comprehensiveness, the interpretation of metabolic profiles and patterns, but also at facilitating the differentiation of natural/endogenous substances from structurally identical but synthetically derived compounds and comprehending the athlete’s exposome. Further, a continuously growing number of advantages of complementary matrices such as dried blood spots has been identified and transferred from research to sports drug testing routine applications, with an overall gain of extremely valuable additions to the anti‐doping field. In this edition of the annual banned‐substance review, literature on recent developments in anti‐doping published between October 2020 and September 2021 is summarized and discussed, particularly focusing on human doping controls and potential applications of new testing strategies to substances and methods of doping specified in the World Anti‐Doping Agency’s 2021 Prohibited List.
Lately, the veterinary drug Emidonol® has been discussed as a possible scenario for inadvertent doping in sports. Emidonol® is approved for use in livestock breeding, exhibiting antihypoxic and weak sedative effects. The veterinary drug rapidly dissociates into meldonium, a substance prohibited in sports, and is excreted largely in its unchanged form into urine. To investigate if residues of meldonium in edible produce may result in adverse analytical findings in sports drug testing, a pilot study was conducted with three volunteers consuming a single dose of 100 ml meldonium‐spiked milk at a concentration of 500 ng/ml (Study 1), and multiple doses of 100 ml of meldonium‐spiked milk (500 ng/ml) on five consecutive days (Study 2). In the single dose study, urinary meldonium concentrations peaked between 2 and 6 h post‐administration with maximum values of 7.5 ng/ml, whereas maximum meldonium concentrations of 18.6 ng/ml were determined after multiple doses 4 h post‐administration. All samples were analyzed using an established and validated protocol based on HILIC‐HRMS/MS. The veterinary drug Emidonol® has been discussed as a possible scenario for inadvertent doping in sports. In a recent study, meldonium concentrations determined in raw milk reached up to 701 ng/ml. If consumed, urine collected post‐ingestion can present meldonium residues and adverse analytical findings in doping controls cannot be excluded.
For decades, the class of anabolic androgenic steroids has represented the most frequently detected doping agents in athletes’ urine samples. Roughly 50% of all adverse analytical findings per year can be attributed to anabolic androgenic steroids, of which about 2/3 are synthetic exogenous steroids, where a qualitative analytical approach is sufficient for routine doping controls. For the remaining 1/3 of findings, caused by endogenous steroid-derived analytical test results, a more sophisticated quantitative approach is required, as their sheer presence in urine cannot be directly linked to an illicit administration. Here, the determination of urinary concentrations and concentration ratios proved to be a suitable tool to identify abnormal steroid profiles. Due to the large inter-individual variability of both concentrations and ratios, population-based thresholds demonstrated to be of limited practicability, leading to the introduction of the steroidal module of the Athlete Biological Passport. The passport enabled the generation of athlete-specific individual reference ranges for steroid profile parameters. Besides an increase in sensitivity, several other aspects like sample substitution or numerous confounding factors affecting the steroid profile are addressed by the Athlete Biological Passport-based approach. This narrative review provides a comprehensive overview on current prospects, supporting professionals in sports drug testing and steroid physiology.
Environmental factors contribute to the risk for adverse health outcomes against a background of genetic predisposition. Among these factors, chemical exposures may substantially contribute to disease risk and adverse outcomes. In fact, epidemiological cohort studies have established associations between exposure against individual chemicals and adverse health effects. Yet, in daily life individuals are exposed to complex mixtures in varying compositions. To capture the totality of environmental exposures the concept of the exposome has been developed. Here, we undertake an overview of major exposome projects, which pioneered the field of exposomics and explored the links between chemical exposure and health outcomes using cohort studies. We seek to reflect their achievements with regard to (i) capturing a comprehensive picture of the environmental chemical exposome, (ii) aggregating internal exposures using chemical and bioanalytical means of detection, and (iii) identifying associations that provide novel options for risk assessment and intervention. Various complementary approaches can be distinguished in addressing relevant exposure routes and it emerges that individual exposure histories may not easily be grouped. The number of chemicals for which human exposure can be detected is substantial and highlights the reality of mixture exposures. Yet, to a large extent it depends on targeted chemical analysis with the specific challenges to capture all relevant exposure routes and assess the chemical concentrations occurring in humans. The currently used approaches imply prior knowledge or hypotheses about relevant exposures. Typically, the number of chemicals considered in exposome projects is counted in dozens—in contrast to the several thousands of chemicals for which occurrence have been reported in human serum and urine. Furthermore, health outcomes are often still compared to single chemicals only. Moreover, explicit consideration of mixture effects and the interrelations between different outcomes to support causal relationships and identify risk drivers in complex mixtures remain underdeveloped and call for specifically designed exposome-cohort studies.
Capillary blood sampled as dried blood spot (DBS) has shown substantial potential as test matrix in sports drug testing in various different settings, enabling the analysis of numerous different drugs and/or their respective metabolites. In addition to established beneficial aspects of DBS specimens in general (such as e.g. the minimally invasive and non-intrusive nature, simplified sample transport, etc.), a yet unexplored advantage of DBS in the anti-doping context could be the opportunity of preserving a source of information complementary to routine doping controls performed in urine or venous blood. Whenever follow-up investigations are warranted or required, frequently collected and stored (but yet not analyzed) DBS samples could be target-tested for the compound(s) in question, in order to contribute to results management and decision-making processes.
Higenamine was included in the World Antidoping Agency (WADA) Prohibited Substances and Methods List as a β2‐adrenoceptor agonist in 2017, thereby resulting in its prohibition both in‐ and out‐of‐competition. The present mini‐review describes the physiology and pharmacology of adrenoceptors, summarizes the literature addressing the mechanism of action of higenamine, and extends these findings with previously unpublished in‐silico and in‐vitro work. Studies conducted in isolated in vitro systems, whole animal preparations, and a small number of clinical studies suggest that higenamine acts in part as a β2‐adrenoceptor agonist. In silico predictive tools indicated that higenamine and possibly a metabolite has a high probability of interacting with the β2‐receptor as an agonist. Stable expression of human β2‐receptors in Chinese Hamster Ovary (CHO) cells to measure agonist activity confirmed not only the activity of higenamine at β2, but also closely agreed with the in‐silico prediction of potency for this compound. These data confirm and extend literature findings supporting the inclusion of higenamine in the Prohibited List.
According to the World Anti‐Doping Agency (WADA) regulations, cannabinoids use is prohibited in competition except for cannabidiol (CBD) use. For an adverse analytical finding (AAF) in doping control, cannabinoid misuse is based on identification of the pharmacologically inactive metabolite 11‐nor‐delta‐9‐carboxy‐tetrahydrocannabinol‐9‐carboxylic acid (carboxy‐THC) in urine at a concentration greater than 180 ng/mL. All other (minor) cannabinoids are reported as AAF when identified, except for CBD that has been explicitly excluded from the class of cannabinoids on WADA's Prohibited List since 2018. However, due to the fact that CBD isolated from cannabis plants may contain additional minor cannabinoids, the permissible use of CBD can lead to unintentional violations of anti‐doping regulations.
An assay for the detection of 16 cannabinoids in human urine was established. The sample preparation consisted of enzymatic hydrolysis of glucuronide conjugates, liquid‐liquid extraction, trimethylsilylation, and analysis by gas chromatography/tandem mass spectro‐metry (GC‐MS/MS). Spot urine samples from CBD users, as well as specimens obtained from CBD administration studies conducted with 15 commercially available CBD products were analyzed, and assay characteristics such as selectivity, reproducibility of detection at the Minimum Required Performance Level, limit of detection, and limit of identification were determined.
An ethical committee approved controlled single dose commercially‐available CBD products administration study was conducted to identify 16 cannabinoids in urine samples collected after ingestion or application of the CBD products as well as their presence in spot urine samples of habitual CBD users. Variable patterns of cannabinoids or their metabolites were observed in the urine samples, especially when full spectrum CBD products were consumed. The presence of minor cannabinoids or their metabolites in an athlete´s in‐competition urine sample represents a substantial risk of an anti‐doping rule violation.
The consumption of the offal of noncastrated pigs can lead to the excretion of 19‐norandrosterone (NorA) in urine of humans. In doping control, GC/C/IRMS is the method of choice to differentiate between an endogenous or exogenous origin of urinary NorA. In some cases, after the consumption of wild boar offal, the δ¹³C values of urinary NorA fulfill the criteria of an adverse analytical finding due to differing food sources of boar and consumer. However, consumption of wild boar's offal is not very common in Germany, and thus, the occurrence of such an analytical finding is unlikely. In contrast, the commerce with wild boar meat has increased in Germany within the last years. Up to 20,000 tons of wild boar meat are annually consumed. In order to probe for the probability of the occurrence of urinary NorA after consumption of wild boar meat, human urine samples were tested following the ingestion of commercially available game. In approximately half of the urine samples, traces of NorA were detected postadministration of 200 to 400 g boar meat. The highest urinary concentration was 2.9 ng/ml, and significant amounts were detected up to 9 h after the meal. δ¹³C values ranged from −18.5‰ to −23.5‰, which would have led to at least two adverse analytical findings if the samples were collected in an antidoping context. IRMS analysis on German boar tissue samples showed that δ¹³C values for wild boar's steroids are unpredictable and may vary seasonally.
Analytical chemistry‐based research in sports drug testing has been a dynamic endeavor for several decades, with technology‐driven innovations continuously contributing to significant improvements in various regards including analytical sensitivity, comprehensiveness of target analytes, differentiation of natural/endogenous substances from structurally identical but synthetically derived compounds, assessment of alternative matrices for doping control purposes, etc. The resulting breadth of tools being investigated and developed by anti‐doping researchers has allowed to substantially improve anti‐doping programs and data interpretation in general. Additionally, these outcomes have been an extremely valuable pledge for routine doping controls during the unprecedented global health crisis that severely affected established sports drug testing strategies. In this edition of the annual banned‐substance review, literature on recent developments in anti‐doping published between October 2019 and September 2020 is summarized and discussed, particularly focusing on human doping controls and potential applications of new testing strategies to substances and methods of doping specified the World Anti‐Doping Agency’s 2020 Prohibited List.
Biological systems are disturbed by several factors that are defined by the exposome. Environmental substances, including endocrine disruptors (EDs), represent the chemical exposome. These stressors may alter biological systems, that could lead to toxic health effects. Even if scientific evidence provide links between diverse environmental substances and disorders, innovative approaches, including alternative methods to animal testing, are still needed to address the complexity of the chemical mechanisms of action. Network science appears to be a valuable approach for helping to decipher a comprehensive assessment of the chemical exposome. A computational protein system-system association network (pS-SAN), based on various data sources such as chemical-protein interactions, chemical-system links, and protein-tissue associations was developed. The integrative systems toxicological model was applied to three EDs, to predict potential biological systems they may perturb. The results revealed that several systems may be disturbed by theses EDs, such as the kidney, liver and endocrine systems. The presented network-based approach highlights an opportunity to shift the paradigm of chemical risk assessment towards a better understanding of chemical toxicology mechanisms.
A simple and sensitive procedure for the quantification of meldonium in milk and meat by UHPLC-HRMS is presented. Some products were investigated to contain this substance due to using a veterinary drug called "Emidonol". According to the instruction for this drug, it can be used for injection (for cows) and as an additive in drinking water for chickens. Although meldonium is not a threat for human health, it is strictly prohibited in professional sports according to WADA Prohibited List. Sample preparation conditions were optimized for both matrices that allowed to eliminate matrix effects and achieve reproducible and accurate results. Protein precipitation with dilution were applied for milk samples, while chicken meat and liver were homogenized with quartz sand to achieve satisfactory meldonium recovery. The results of milk and meat samples analysis purchased at the farmers' fair are presented in this article. Meldonium concentration in raw milk was investigated to be up to 880 ng/mL. However, pasteurization can be used for partial cleanup from meldonium (up to 2 times). The same research was conducted for chicken meat and liver. Thermal treatment shows a good result for a meat cleanup. The proposed method was partially validated, limits of detection and quantification were established for each matrix.
A narrative review with an overall aim of indicating the current state of knowledge and the relevance concerning food and supplement contamination and/or adulteration with doping agents and the respective implications for sports drug testing is presented. The identification of a doping agent (or its metabolite) in sports drug testing samples constitutes a violation of the anti-doping rules defined by the World Anti-Doping Agency. Reasons for such Adverse Analytical Findings (AAFs) include the intentional misuse of performance-enhancing/banned drugs; however, also the scenario of inadvertent administrations of doping agents was proven in the past, caused by, amongst others, the ingestion of contaminated dietary supplements, drugs, or food. Even though controversial positions concerning the effectiveness of dietary supplements in healthy subjects exist, they are frequently used by athletes, anticipating positive effects on health, recovery, and performance. However, most supplement users are unaware of the fact that the administration of such products can be associated with unforeseeable health risks and AAFs in sports. In particular anabolic androgenic steroids (AAS) and stimulants have been frequently found as undeclared ingredients of dietary supplements, either as a result of cross-contaminations due to substandard manufacturing practices and missing quality controls or an intentional admixture to increase the effectiveness of the preparations. Cross-contaminations were also found to affect therapeutic drug preparations. While the sensitivity of assays employed to test pharmaceuticals for impurities is in accordance with good manufacturing practice guidelines allowing to exclude any physiological effects, minute trace amounts of contaminating compounds can still result in positive doping tests. In addition, food was found to be a potential source of unintentional doping, the most prominent example being meat tainted with the anabolic agent clenbuterol. The athletes’ compliance with anti-doping rules is frequently tested by routine doping controls. Different measures including offers of topical information and education of the athletes as well as the maintenance of databases summarizing low- or high-risk supplements are important cornerstones in preventing unintentional anti-doping rule violations. Further, the collection of additional analytical data has been shown to allow for supporting result management processes.
Since 2017, higenamine has been added to the World Anti-Doping Agency (WADA) prohibited list as a β2-agonist prohibited at all times for sportspersons. According to WADA’s report, positive cases of higenamine misuse have been increasing yearly. However, higenamine occurs naturally in the Chinese herb lotus plumule—the green embryo of lotus (Nelumbo nucifera Gaertn) seeds—commercially available as concentrated powder on the Asian market. This study evaluated the major phytochemical components of lotus plumule products using an appropriate extraction method, followed by a human study in which the products were orally administered in multiple doses to investigate the risk of doping violations. Comparing various extraction methods revealed that optimized microwave-assisted extraction exhibited the highest extraction efficiency (extraction time, 26 min; power, 1046 W; and temperature, 120 °C). Subsequently, the alkaloids in lotus plumule products were quantitatively confirmed and compared. Human study participants (n = 6) consumed 0.8 g of lotus plumule (equivalent to 679.6 μg of higenamine) three times daily for three consecutive days. All participants’ urinary higenamine concentrations exceeded the WADA reporting cut-off of 10.0 ng/mL. Accordingly, lotus plumule consumption may engender adverse analytical findings regarding higenamine. Athletes should avoid consuming lotus plumule-containing products during in- and out-of-competition periods.
Complementing the human genome with an exposome reflects the increasingly obvious impact of environmental exposure, which far exceeds the role of genetics, on human health. Considering the complexity of exposures and, in addition, the reactions of the body to exposures - i.e., the exposome - reverses classical exposure science where the precise measurement of single or few exposures is associated with specific health or environmental effects. The complete description of an individual's exposome is impossible; even less so is that of a population. We can, however, cast a wider net by foregoing some rigor in assessment and compensating with the statistical power of rich datasets. The advent of omics technologies enables a relatively cheap, high-content description of the biological effects of substances, especially in tissues and biofluids. They can be combined with many other rich data-streams, creating big data of exposure and effect. Computational methods increasingly allow data integration, discerning the signal from the noise and formulating hypotheses of exposure-effect relationships. These can be followed up in a targeted way. With a better exposure element in the risk equation, exposomics - new kid on the block of risk assessment - promises to identify novel exposure (interactions) and health/environment effect associations. This may also create opportunities to prioritize the more relevant chemicals for risk assessment, thereby lowering the burden on hazard assessment in an expo-sure-driven approach. Technological developments and synergies between approaches, quality assurance (ultimately as Good Exposome Practices), and the integration of mechanistic thinking will advance this approach.
Cannabidiol (CBD) is a non-psychoactive cannabinoid, widely marketed to athletes for claimed effects such as decreased anxiety, fear memory extinction, anti-inflammatory properties, relief of pain and for post-exercise recovery. The World Anti-Doping Agency (WADA) has excluded CBD from its list of prohibited substances. Nevertheless, caution is currently advised for athletes intending to use the compound—except CBD, all other cannabinoids are still on the prohibited list. CBD products, specifically non-medicinal, so-called full-spectrum cannabis extracts, may contain significant levels of these substances, but also contaminations of tetrahydrocannabinol (THC) (>2.5 mg/day in >30% of products on the German market) potentially leading to positive doping tests. Labelled claims about CBD content and absence of THC are often false and misleading. Contaminations with the psychoactive THC can result in adverse effects on cognition and, in general, the safety profile of CBD with respect to its toxicity is a controversial topic of discussion. For these reasons, we would currently advise against the use of over-the-counter CBD products, especially those from dubious internet sources without quality control.
Nutrition usually makes a small but potentially valuable contribution to successful performance in elite athletes, and dietary supplements can make a minor contribution to this nutrition programme. Nonetheless, supplement use is widespread at all levels of sport. Products described as supplements target different issues, including (1) the management of micronutrient deficiencies, (2) supply of convenient forms of energy and macronutrients, and (3) provision of direct benefits to performance or (4) indirect benefits such as supporting intense training regimens. The appropriate use of some supplements can benefit the athlete, but others may harm the athlete’s health, performance, and/or livelihood and reputation (if an antidoping rule violation results). A complete nutritional assessment should be undertaken before decisions regarding supplement use are made. Supplements claiming to directly or indirectly enhance performance are typically the largest group of products marketed to athletes, but only a few (including caffeine, creatine, specific buffering agents and nitrate) have good evidence of benefits. However, responses are affected by the scenario of use and may vary widely between individuals because of factors that include genetics, the microbiome and habitual diet. Supplements intended to enhance performance should be thoroughly trialled in training or simulated competition before being used in competition. Inadvertent ingestion of substances prohibited under the antidoping codes that govern elite sport is a known risk of taking some supplements. Protection of the athlete’s health and awareness of the potential for harm must be paramount; expert professional opinion and assistance is strongly advised before an athlete embarks on supplement use.
Crossbred beef steers (n = 240; 12 pens/treatment; initial BW = 305 ± 17.7 kg) were used in a randomized block design feedlot study to evaluate the influence of coated trenbolone acetate (TBA) and estradiol-17β (E2) implants (Merck Animal Health, Madison, NJ) on gain performance, carcass traits, and sera metabolites. The five treatments were no implant (NI), Revalor-XR on d 0 [200 mg TBA + 20 mg E2 (coated); XR], Revalor-XS on d 0 [200 mg TBA + 40 mg E2 (total): 80 mg TBA + 16 mg E2 (non-coated) and 120 mg TBA + 24 mg E2 (coated); XS], Revalor-200 on d 0 [200 mg TBA + 20 mg E2 (non-coated); E200], or Revalor-200 on d 70 (D200). Interim BW and blood were collected on d 0, 14, 35, 70, 105, 140, and 175 prior to feeding, and on d 213 prior to shipping. Following a 24 h clot at 4°C, sera was harvested to quantify circulating E2, IGF-I, NEFA, serum urea-N (SUN) and 17β-trenbolone (17β-TbOH). Implanted steers had greater (P ≤ 0.05) ADG, G:F, and final BW than NI controls. Implants increased (P < 0.05) HCW by 8%, 366 vs. 391, 414, 380, and 396 ± 6.4 kg, for NI vs. XR, XS, E200, and D200, respectively. The greatest (P ≤ 0.05) dressing percentage, yield grade, and calculated empty body fat occurred in XS, which had greater (P < 0.05) rib fat than NI, XR, and D200. Marbling scores in NI were greater (P < 0.05) than E200 and D200; steers in XR and XS were intermediate (P > 0.10), not differing from NI, E200, or D200. An implant × day interaction (P ≤ 0.01) was noted for circulating E2, IGF-I, SUN, and 17β-TbOH. Implanted steers had elevated (P ≤ 0.05) sera E2, IGF-I, and 17β-TbOH, and decreased (P < 0.05) SUN following implantation compared to NI controls. Serum NEFA differed (P < 0.01) over time, but did not differ (P > 0.10) due to implant treatment. These data indicated that the polymer coating applied to the XR implant delayed release of steroidal hormones congruently to D200, with no negative impact on marbling. The greatest dose of E2, contained in XS, provided improvements in gain and carcass weight without detriment to marbling scores compared to NI.
• For more than 50 years, beef cattle producers have safely used growth promotants to enhance muscle leanness, increase average daily gain, stimulate feed intake moderately, and subsequently enhance the rate of body weight gain compared to the amount of food needed to achieve that gain, referred to as feed efficiency.
• Growth-enhancing compounds, including steroidal implants and β-adrenergic agonists, increase production and improve feed efficiency of beef cattle.
• The changes in performance result in an economic benefit to beef cattle producers and impact the relative price competitiveness of beef as compared to other dietary protein sources.
Clenbuterol is a well-established β2-agonist, which is prohibited in sports and strictly regulated for use in the livestock industry. During the last few years clenbuterol-positive results in doping controls and in samples from residents or travellers from a high-risk country were suspected to be related the illegal use of clenbuterol for fattening. A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to detect low clenbuterol residues in hair with a detection limit of 0.02 pg/mg. A sub-therapeutic application study and a field study with volunteers, who have a high risk of contamination, were performed. For the application study, a total dosage of 30 µg clenbuterol was applied to 20 healthy volunteers on 5 subsequent days. One month after the beginning of the application, clenbuterol was detected in the proximal hair segment (0-1 cm) in concentrations between 0.43 and 4.76 pg/mg. For the second part, samples of 66 Mexican soccer players were analyzed. In 89% of these volunteers, clenbuterol was detectable in their hair at concentrations between 0.02 and 1.90 pg/mg. A comparison of both parts showed no statistical difference between sub-therapeutic application and contamination. In contrast, discrimination to a typical abuse of clenbuterol is apparently possible. Due to these findings results of real doping control samples can be evaluated. Copyright © 2014 John Wiley & Sons, Ltd.
Rationale:
Chlorphenesin is an approved biocide frequently used in cosmetics, and its carbamate ester is an approved skeletal muscle relaxant in certain countries for the treatment of discomfort related to skeletal muscle trauma and inflammation. A major urinary metabolite is 4-chlorophenoxy acetic acid (4-CPA), also known as para-chlorophenoxyacetate (pCPA), which is also employed as target analyte in sports drug testing to detect the use of the prohibited nootropic stimulant meclofenoxate. In order to distinguish between 4-CPA resulting from chlorphenesin, chlorphenesin carbamate, and meclofenoxate, urinary metabolite profiles of chlorphenesin after legitimate use were investigated.
Methods:
Human administration studies with commercially available sunscreen containing 0.25% by weight of chlorphenesin were conducted. Six study participants dermally applied 8 g of sunscreen and collected urine samples before and up to 7 days post-application. Another set of six study participants applied 8 g of sunscreen on three consecutive days and urine samples were also taken for up to 5 days after the last dosing. Urine specimens were analyzed by liquid chromatography-high resolution (tandem) mass spectrometry, and urinary metabolites were identified in accordance with literature data by accurate mass analysis of respective precursor and characteristic product ions.
Results:
In accordance with literature data, chlorphenesin yielded the characteristic urinary metabolites chlorphenesin glucuronide, chlorphenesin sulfate, and 3-(4-chlorophenoxy)-2-hydroxypropanoic acid (4-CPP) as well as the common metabolite 4-CPA. 4-CPA and 4-CPP were observed at similar abundances, with urinary concentrations of 4-CPA reaching up to approximately 1500 ng/mL and 2300 ng/mL after single and multiple sunscreen application, respectively.
Conclusion:
4-CPA is a common metabolite of meclofenoxate, chlorphenesin and chlorphenesin carbamate. Monitoring the diagnostic urinary metabolites of chlorphenesin provides conclusive supporting evidence whether chlorphenesin or the prohibited nootropic meclofenoxate was administered.
The selective oestrogen receptor modulator (SERM) clomiphene is therapeutically used to induce ovulation. While prohibited as a doping agent in sports, it is frequently detected in sports drug testing urine samples. Few reports exist on clomiphene’s (illicit) use in the farming industry to increase the egg production rate of laying hens, which creates a risk that eggs as well as edible tissue of these hens contain residues of clomiphene. To investigate the potential transfer of clomiphene into eggs and muscle tissue, laying hens were orally administered with clomiphene citrate at 10 mg/day for 28 days. To determine clomiphene residues in eggs, chicken breast and chicken thigh, the target analyte was extracted from homogenised material with acetonitrile and subjected to ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The test method reached a limit of quantification (LOQ) of 1 µg/kg and was characterised concerning specificity, precision, trueness and linearity. Analyses were performed on whole egg, egg white and yolk separately, and chicken muscle from breast and thigh. Clomiphene was detectable in eggs two days after the beginning of the drug administration period. The drug concentrations increased to 10–20 µg per egg within one week, and after withdrawal of clomiphene, residues decreased after 4 days, but traces of clomiphene were still detectable until the end of the study (14 days after the last administration). In the chicken’s muscle tissue, clomiphene levels up to 150 µg/kg (thigh) and 36 µg/kg (breast) were found. Six days after the last dose, tissue clomiphene concentrations fell below the LOQ. Overall, these results underline the concerns that clomiphene may be transferred into animal-derived food and future research will therefore need to focus on assessing and minimising the risk of unintentional adverse analytical findings in doping controls.
Sports nutrition supplements have previously been reported to contain undeclared doping substances. The use of such supplements can lead to general health risks and may give rise to unintentional doping violations in elite sports. To assess the prevalence of doping substances in a range of high-risk sports nutrition supplements available from Dutch web shops. A total of 66 sports nutrition supplements - identified as potentially high-risk products claiming to modulate hormone regulation, stimulate muscle mass gain, increase fat loss, and/or boost energy - were selected from 21 different brands and purchased from 17 web shops. All products were analyzed for doping substances by the UK life sciences testing company LGC, formerly known as the Laboratory of the Government Chemist, using an extended version of their ISO17025 accredited nutritional supplement screen. A total of 25 out of the 66 products (38%) contained undeclared doping substances, which included high levels of the stimulants oxilofrine, β-methylphenethylamine (BMPEA) and N,β-dimethylphenethylamine (NBDMPEA), the stimulant 4-methylhexan-2-amine (methylhexaneamine, 1,3-dimethylamylamine, DMAA), the anabolic steroids boldione (1,4-androstadiene-3,17-dione) and 5-androstene-3β,17α-diol (17α-AED), the beta-2 agonist higenamine and the beta-blocker bisoprolol. Based upon the recommended dose and the potential variability of analyte concentration, the ingestion of some products identified within this study could pose a significant risk of unintentional doping violations. In addition to inadvertent doping risks, the prescribed use of 3 products (4.5%) could likely impose general health risks.
This review focuses on four new product categories of food supplements: pre‐workout, fat burner/thermogenic, brain/cognitive booster, and hormone/testosterone booster. Many food supplements have been shown to be contaminated with unauthorized substances. In some cases, the ingredients in the new categories of dietary supplements were medicinal products or new synthetic compounds added without performing clinical trials. Some of the new ingredients in dietary supplements are plant materials that are registered in the pharmacopoeia as herbal medicines. In other cases, dietary supplements may contain plant materials that have no history of human use and are often used as materials to “camouflage” stimulants. In the European Union, new ingredients of dietary supplements, according to European Food Safety Authority or unauthorized novel food. Furthermore, selected ingredients in dietary supplements may be prohibited in sports and are recognized as doping agents by World Anti‐Doping Agency.
Current studies on environmental chemistry mainly focus on a single stressor or single group of stressors, which does not reflect the multiple stressors in the dynamic exposome we are facing. Similarly, current studies on environmental toxicology mostly target humans, animals, or the environment separately, which are inadequate to solve the grand challenge of multiple receptors in One Health. Though chemical, biological, and physical stressors all pose health threats, the susceptibilities of different organisms are different. As such, significant relationships and interactions of the chemical, biological, and physical stressors in the environment and their holistic environmental and biological consequences remain unclear. Fortunately, the rapid developments in various techniques, as well as the concepts of multistressors in the exposome and multireceptor in One Health provide the possibilities to understand our environment better. Since the combined stressor is location-specific and mixture toxicity is species-specific, more comprehensive frameworks to guide risk assessment and environmental treatment are urgently needed. Here, three conceptual frameworks to categorize unknown stressors, spatially visualize the riskiest stressors, and investigate the combined effects of multiple stressors across multiple species within the concepts of the exposome and One Health are proposed for the first time.
The detection of clostebol misuse in sports has been growing recently, especially in Italy, due to the ample availability of pharmaceutical formulations containing clostebol acetate (Trofodermin®) and the use of more sensitive instrumentation by the antidoping laboratories. Most of these cases have been claimed to be related to a nonconscious use of the drug or through contact with relatives or teammates using it. We have investigated, through the application of the well‐known and currently used gas chromatographic mass spectrometric procedures, the likelihood of these allegations and have demonstrated that after a single transdermal administration of 5 mg of clostebol acetate and a transient contact with the application area, it is possible to generate adverse analytical findings in antidoping controls. We have reviewed the Phase I and Phase II clostebol metabolism in order to generate evidences that may help the sport authorities reviewing these cases. The main clostebol metabolite (4‐chloro‐androst‐4‐en‐3α‐ol‐17‐one, M1) generally used at the screening level as well as other three metabolites (M2–M4) are mainly excreted as glucuronides, whereas M5 (4ζ‐chloro‐5ζ‐androstan‐3β‐ol‐17‐one) is predominantly excreted as sulfate. Neither the 5α‐reductases activity (impaired by the presence of the chlorine in C4) nor specific sulfotransferases present in the skin allowed a clear distinction of the administration route. Studies with a larger number of volunteers and probably investigating another physiological fluid allowed in antidoping such as blood are needed for a deeper investigation. It is not unreasonable to establish a reporting level for M1, maybe creating some false negatives but excluding nonintentional doping scenarios.
Environmental chemicals comprise a major portion of the human exposome, with some shown to impact the health of susceptible populations, including pregnant women and developing fetuses. The placenta and cord blood serve as important biological windows into the maternal and fetal environments. In this article we review how environmental chemicals (defined here to include man-made chemicals [e.g., flame retardants, pesticides/herbicides, per- and polyfluoroalkyl substances], toxins, metals, and other xenobiotic compounds) contribute to the prenatal exposome and highlight future directions to advance this research field. Our findings from a survey of recent literature indicate the need to better understand the breadth of environmental chemicals that reach the placenta and cord blood, as well as the linkages between prenatal exposures, mechanisms of toxicity, and subsequent health outcomes. Research efforts tailored towards addressing these needs will provide a more comprehensive understanding of how environmental chemicals impact maternal and fetal health.
Higenamine is a β2‐agonist included in the Prohibited List of the World Anti‐Doping Agency (WADA) since 2017. Meanwhile, it exists in plumula nelumbinis, a part of lotus seeds, which is commonly used as an ingredient in cuisines, herbal medicine and nutritional supplements in China and other countries in East Asia. Therefore, the evaluation of the Adverse Analytical Finding (AAF) risk of higenamine caused by plumula nelumbinis products is necessary in doping control. In this study, 14 volunteers orally administered plumula nelumbinis capsules (0.34 g/caplet, 6 caplets/day, 7 days), and another 11 volunteers ingested higenamine tablets (5 mg/piece, 3 pieces/day, 7 days). Urine samples were collected over a period of 14 days. All urine samples were subjected to quantitative dilute‐and‐shoot analysis using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). The analytical results showed that the urinary higenamine concentrations exceeded the WADA reporting limit (10 ng/mL) during the drug period in most sample groups. The maximum higenamine concentration observed in the plumula nelumbinis capsule group was 500 ng/mL. Based on the theory of confidence interval, appropriate data was used to establish mathematical models. The models reflected that the higenamine concentration in urine can be greater than the WADA reporting limit with a high probability after taking plumula nelumbinis capsules. In conclusion, oral administration of the plumula nelumbinis capsule showed a high risk for an AAF due to higenamine.
With advances in analytical technology and new research informing result interpretation, oral fluid (OF) testing has gained acceptance over the past decades as an alternative biological matrix for detecting drugs in forensic and clinical settings. OF testing offers simple, rapid, non-invasive, observed specimen collection. This article offers a review of the scientific literature covering analytical methods and interpretation published over the past two decades for amphetamines, cannabis, cocaine, opioids, and benzodiazepines. Several analytical methods have been published for individual drug classes and, increasingly, for multiple drug classes. The method of OF collection can have a significant impact on the resultant drug concentration. Drug concentrations for amphetamines, cannabis, cocaine, opioids, and benzodiazepines are reviewed in the context of the dosing condition and the collection method. Time of last detection is evaluated against several agencies' cutoffs, including the proposed Substance Abuse and Mental Health Services Administration, European Workplace Drug Testing Society and Driving Under the Influence of Drugs, Alcohol and Medicines cutoffs. A significant correlation was frequently observed between matrices (i.e., between OF and plasma or blood concentrations); however, high intra-subject and inter-subject variability precludes prediction of blood concentrations from OF concentrations. This article will assist individuals in understanding the relative merits and limitations of various methods of OF collection, analysis and interpretation.
Letrozole is an aromatase inhibitor, used to treat post‐menopausal women with hormone receptor‐positive or unknown advanced breast cancer. It is prohibited in sport because it is used together with androgen anabolizing steroids to avoid their adverse effects. In case of adverse analytical finding, it may be important to distinguish between repetitive use due to voluntary administration and occasional use, possibly due to involuntary intake. With the objective to identify the dose capable of producing a positive hair testing, and to apply these results to the scenarios of letrozole inadvertent ingestion by an athlete, this study aims at investigating the urinary excretion and incorporation into hair of single doses of letrozole.
Seven subjects were recruited for an excretion study of letrozole and its metabolite bis(4‐cyanophenyl) methanol (M1) in urine, after the consumption of 0.62 mg, 1.25 mg, and 2.5 mg of letrozole, and to investigate the incorporation in hair after ingestion of 0.62 mg and 2.5 mg of letrozole. Urine and hair samples were also obtained from two women in chronic therapy.
Urinary concentrations of letrozole and its metabolite M1 were lower in subjects administered once with 0.62 mg, 1.25 mg or 2.5 mg letrozole than in women in regular therapy with 2.5 mg/day. In hair collected after single dosage, concentrations of 16‐60 pg/mg were detected while in women in chronic therapy concentrations were higher than 160 pg/mg all along the hair shaft. Hair analysis turned to be a promising possibility for the discrimination of letrozole repetitive use vs occasional/inadvertent administration.
Meldonium is a metabolic drug whose inclusion in the 2016 List of Prohibited Substances and Methods followed the analysis of data collected under the 2015 World Anti‐Doping Agency Monitoring Program.
In the early months of 2016, anti‐doping laboratories reported an unusually high number of cases in which urine samples contained high concentrations of meldonium. Consequently, the meldonium excretion period in healthy athletes and the substance's long‐term urine and blood (plasma) pharmacokinetics became central questions for the anti‐doping community to address, to ensure appropriate assessment of the scientific and medical situation, and also fair treatment of athletes from a result management and legal standpoint.
At the present time, data on meldonium pharmacokinetics is limited to a few studies, with no known data available on long‐term excretion of high oral doses. The primary objective of this open‐label study was to determine long‐term urine and plasma pharmacokinetic parameters of meldonium in healthy volunteers. Study design included single and repeated functional load testing and assessment of L‐carnitine administration on meldonium excretion and pharmacokinetics. Thirty‐two volunteers were equally divided into two groups receiving either 1.0 g or 2.0 g of oral meldonium daily for 3 weeks.
The study found meldonium takes several days to attain a steady state in blood and displays an elimination period over several months after cessation of treatment. Moreover, findings demonstrate that the daily dose, periodicity and duration of treatment with meldonium are the most important factors to consider in calculating the substance's elimination and complete body clearance.
Isotope‐ratio mass spectrometry (IRMS) has been established in doping control analysis to identify the endogenous or exogenous origin of a variety of steroidal analytes including the 19‐norsteroid metabolite norandrosterone (NorA). NorA can be found naturally in human urine in trace amounts due to endogenous demethylation or in‐situ microbial degradation. The administration of nortestosterone (nandrolone) or different prohormones results in the excretion of urinary NorA. Usually, this can be detected by IRMS due to differing δ13C values of synthetic 19‐norsteroids compared to endogenous reference compounds. The consumption of uncastrated pig edible parts like offal or even meat may also lead to a urinary excretion of NorA. In order to determine the δ13C values of such a scenario, urine samples collected after consumption of a wild boar's testicle meal were analyzed. IRMS revealed highly enriched δ13C values for urinary NorA, which could be related to a completely corn‐based nutrition of the animal. Isotopic analysis of the boar's bristles demonstrated a dietary change from C3‐based forage, probably in winter and spring, to a C4‐based diet in the last weeks to months prior to death. These results supported the interpretation of an atypical test result of a Central European athlete's doping control sample with δ13C values for NorA of ‐18 ‰, most probably caused by the consumption of a wild boar's ragout. As stated before, athletes should be fully aware of the risk that consumption of wild boar's edible parts may result in atypical or even adverse analytical findings in sports drug testing.
Diuretic agents are prohibited in sports in‐ and out‐of‐competition according to the regulations of the World Anti‐Doping Agency (WADA) because of their possible masking effects on other doping agents in urine samples, and their ability to produce fast acute weight losses. Despite previous studies reported adverse analytical findings (AAFs) resulting from contaminations at ppm level (μg/g) of medicinal products, and recommended to introduce reporting limits for diuretics in doping controls, these are not adopted in analyses performed by WADA‐accredited laboratories.
We report the case of an athlete with two AAFs for hydrochlorothiazide (HCTZ) at low urinary concentrations (<10 ng/mL), who declared the use of nutritional supplements prepared in a compounding pharmacy. His nutritional supplements were analysed revealing HCTZ presence in different concentrations, at the ppm level (μg/g and ng/mL).
With the aim of testing the plausibility of the observed urinary HCTZ concentrations with the nutritional supplement ingestion, a urinary excretion study with 3 healthy volunteers was performed. HCTZ‐contaminated powder (6.4 μg/g of HCTZ) was administered to each subject in different dosages, reproducing the possible ingestion pattern occurred. Urine specimens were collected before and after ingestion of the powder, up to 24 hours, and underwent liquid‐liquid extraction and liquid chromatography/tandem mass spectrometry determination.
Post‐administration specimens were found to contain HCTZ at concentrations of 5–230 ng/mL, which supported the accidental inadvertent intake of the prohibited substance by the athlete.
This study makes the argument that the introduction of reporting limits for diuretics are warranted in doping control samples, in order to protect against inadvertent AAFs due to contaminated products.
Meldonium is a drug exhibiting cardioprotective and anti-ischemic effects. Due to its potential performance-enhancing benefit in sports, meldonium was added to the World Anti-Doping Agency list of prohibited substances in 2016. Since then, a high number of adverse analytical findings reported on meldonium has questioned meldonium`s detection time in urine. Hence, the objective of the current study was to characterize the pharmacokinetic urinary excretion pattern of meldonium when administered as multiple intravenous injections. Three injections of 250 mg meldonium were given over a time period of five days to six healthy volunteers and urine samples were collected for eight months after the last injection of the drug. For the quantification of meldonium in urine, a liquid chromatography-tandem mass spectrometry method was fully validated according to the World Anti-Doping Agency guidelines in terms of specificity, matrix interferences, intra- and inter-day precision, accuracy, carry-over, robustness, linearity, limit of detection, and limit of quantification. The assay was successfully applied to the pharmacokinetic study. A three-compartment model was found to best describe the pharmacokinetics of meldonium with average alpha, beta, and gamma half-lives of 1.4 h, 9.4 h, and 655 h, respectively. The detection time in urine varied between 94 and 162 days.
Derived from the term exposure, the exposome is an omic-scale characterization of the nongenetic drivers of health and disease. With the genome, it defines the phenome of an individual. The measurement of complex environmental factors that exert pressure on our health has not kept pace with genomics and historically has not provided a similar level of resolution. Emerging technologies make it possible to obtain detailed information on drugs, toxicants, pollutants, nutrients, and physical and psychological stressors on an omic scale. These forces can also be assessed at systems and network levels, providing a framework for advances in pharmacology and toxicology. The exposome paradigm can improve the analysis of drug interactions and detection of adverse effects of drugs and toxicants and provide data on biological responses to exposures. The comprehensive model can provide data at the individual level for precision medicine, group level for clinical trials, and population level for public health. Expected final online publication date for the Annual Review of Pharmacology and Toxicology Volume 59 is January 6, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
A number of high profile revelations concerning anti-doping rule violations over the past 12 months has outlined the importance of tackling prevailing challenges and reducing the limitations of the current anti-doping system. At this time, the necessity to enhance, expand and improve analytical test methods in response to the substances outlined in the World Anti-Doping Agency's (WADA) Prohibited List represents an increasingly crucial task for modern sports drug testing programs. The ability to improve analytical testing methods often relies on the expedient application of novel information regarding superior target analytes for sports drug testing assays, drug elimination profiles, alternative test matrices, together with recent advances in instrumental developments. This annual banned-substance review evaluates literature published between October 2016 and September 2017 offering an in-depth evaluation of developments in these arenas and their potential application to substances reported in WADA's 2017 Prohibited List.
Several banned substances are illegally used by athletes in racemic mixtures for performance enhancement. These include clenbuterol, methyl hexaneamine, methamphetamines, amphetamines. Clenbuterol is present in a large number of doping samples from Olympic and non-Olympic athletes that have adverse analytical findings. In some cases, the presence of these substance could be the result of consumption of meat contaminated with clenbuterol. In other cases, the origin is not clear. In this study, 27 products with racemic clenbuterol were evaluated using a new analytical methodology for the resolution of R-(-) and S-(+)-enantiomers of clenbuterol by Liquid Chromatography-Tandem Mass Spectrometry LC-MS/MS using a chiral column in 15 minutes with good separation. The method here developed can also be used for the analysis of other biological matrix as urine, serum, meat. The resolution between two peaks (Rs) value obtained using chromatographic data was 1.03. Both clenbuterol enantiomers were present in all products analyzed and the ratio was nearly 1. The origin of the product was not important for determining the presence of one or both enantiomers. All products displayed a 50:50 ratio of clenbuterol enantiomers. To the best of our knowledge, clenbuterol ratio determination of a large number of pharmaceutical preparations and black market products have not been reported previously. The information here shown could be use by the National Anti-Doping Organizations and the athletes with AAF attributed to clenbuterol.
Musk, the dried secretion of the musk pod (sac) of adult male musk deer, has been used as traditional Chinese Medicine (TCM) in China and south-east Asian countries for thousands of years. Due to the anabolic steroid component in this TCM, musk preparations have been included in the list of medical products containing prohibited substances employed for doping by the State Food and Drug Administration of China. The application of musk pod formulation was claimed to be responsible for some adverse analytical findings (AAF) in the 2011 FIFA Women's World Cup. Our preliminary study has suggested that musk ingestion did not lead to AAF of doping control with the single dosage of 100 mg. However, the influences of musk administration in large and multi dosage are still unclear. The aim of this study is to further investigate the influences of musk administration for doping control. Wild and domestic deer musk samples were collected. The concentrations and δ13C-values of steroids in musk were analyzed. In an excretion study, 200 and 100 mg of wild and domestic deer musk samples were administrated by 29 subjects respectively. Fluctuations in steroid profile could be observed, and the ratio of 5α-androstane-3α,17β-diol to 5β-androstane-3α,17β-diol was more sensitive than other parameters. In the IRMS test, the ∆δ13C-value between endogenous reference compound and etiocholanolone was a sensitive parameter, and AAFs were obtained. It is the first time to confirm with excretion study that musk administration could lead to positive result of doping control.
Higenamine is a key component of traditional Chinese herbal medicine. The fruit of Nandina domestica (which contains this component) is available as an ingredient in the so-called Nanten-nodo-ame throat lozenge found on the Japanese market, which is an over-the-counter pharmaceutical and is easy to purchase for Japanese athletes. However, higenamine is a non-selective β2-agonist, which is exemplified in the prohibited list of the World Anti-Doping Agency (WADA). Therefore, some have raised a concern regarding the potential cause of increased unintentional higenamine doping cases in the Asian region. This study aimed to investigate components of throat lozenges and develop a mass-spectrometry method for the quantification of higenamine and coclaurine in human urine. Moreover, a population study of Japanese subjects (n = 246) and an excretion study (n = 4) of the corresponding throat-lozenge recipients were performed to test the applicability of the current reporting threshold (i.e., 10 ng/mL) of higenamine set by WADA. The estimates of higenamine and coclaurine were 2.2 ± 0.1 μg/drop (mean of n = 12) and 0.5 ± 0.01 μg/drop (mean of n = 12), respectively. The maximum concentrations of higenamine and coclaurine were 0.2-0.4 and 0.3-1.0 ng/mL, respectively, at 10-12 h after administration of higenamine (nine drops); however, the concentrations in all four volunteers did not reach the positivity criterion of 10 ng/mL. No higenamine and coclaurine could be detected in the Japanese subjects. Therefore, there is no risk of detecting unintentional higenamine doping when the WADA reporting threshold is used.
Today's doping tests involving longitudinal monitoring of steroid profiles are difficult in women. Women have more complex hormonal fluctuations than men and commonly take drugs such as hormonal contraceptives that are shown to affect biomarkers used in these doping tests. In this study we followed six women's urinary steroid profile during one menstrual cycle, including both glucuronides and sulfate conjugated fractions. Additionally, we studied what happens to the steroidal module of the athlete biological passport (ABP) after administration of an emergency contraceptive (levonorgestrel, NorLevo®). The study shows that there are large individual variation in all metabolites included in the ABP and that the administration of emergency contraceptives may lead to suspicious steroid profile findings in the ABP. Urinary epitestosterone concentration increased during the menstrual cycle, leading to a decrease in the testosterone/epitestosterone ratio. The ratios followed in the ABP varied widely throughout the menstrual cycle, the coefficient of variation (CV) ranging from 4 to 99%. There was a 3-fold decrease in epitestosterone 24 hours post administration of the emergency contraceptive pill and also androsterone, etiocholanolone, and 5β- androstan-3α,17β-diol concentrations decreased about 2-fold. When analyzed with the ABP software, one of the six women had an atypical profile after taking the emergency contraceptive. Furthermore, we could not find any alterations in excretion routes (i.e. if the metabolites are excreted as glucuronide or sulfate conjugates) during the menstrual cycle or after administration of emergency contraceptive, indicating no direct effect on phase II enzymes. This article is protected by copyright. All rights reserved.
The differentiation of clenbuterol abuse and unintentional ingestion by 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 from the administration of clenbuterol from a pharmaceutical preparation and from cattle meat and liver containing residues to humans are presented. 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. Post administration urine from a controlled experiment comparing the administration of racemic clenbuterol from a registered pharmaceutical preparation and the administration of residue containing meat and liver from treated animals (non-racemic mixture) 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.
Hydrochlorothiazide (HCTZ, 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide-1,1-dioxide) belongs to the class of diuretic agents that represent one of today’s cornerstones of the treatment of hypertensive patients. In addition to its clinical relevance, HCTZ is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA) at all times and has frequently been detected in sports drug testing urine samples worldwide since its ban was introduced in 1988. Despite these facts, the adverse analytical finding concerning HCTZ in an in-competition routine doping control sample collected in December 2014 was further investigated, particularly motivated by the comparably low urinary concentration of the drug accounting for approximately 5 ng/mL.
The athlete in question did not declare the use of any nutritional supplement or medication other than the ingestion of a non-steroidal anti-inflammatory drug (NSAID) prior to competition. Hence, the drug (formulated as coated tablet) provided by the athlete as well as the corresponding retention sample of the manufacturer were analyzed. Noteworthy, both samples confirmed the presence of about 2 μg of HCTZ per tablet.
In order to further probe for the plausibility of the observed urinary HCTZ concentrations with the scenario of drug ingestion and subsequent doping control sample collection, administration studies with produced HCTZ-spiked placebo-tablets (2.5 μg of HCTZ/tablet) were conducted. Urine specimens were collected prior to and after ingestion of the drug and subjected to routine doping control analytical procedures employing liquid chromatography/tandem mass spectrometry. While blank urine samples returned negative test results, post-administration specimens were found to contain HCTZ at concentrations of approximately 1–16 ng/mL, which supported the athlete’s inadvertent intake of HCTZ via contaminated NSAID tablets.
Due to the substantial sensitivity of test methods employed today by doping control laboratories, even drug contaminations ranging within the good manufacturing practice (GMP) limit of 10 ppm overall carry-over can evidently lead to adverse analytical findings. This calls into question whether selected (classes of) substances such as diuretics should be reported only when exceeding a defined reporting level and/or whether adverse analytical findings of non-threshold substances should be reported with an estimated semi-quantitative concentration of the identified substance to facilitate the result management by anti-doping organizations.
Background:
Autologous blood transfusion (ABT) efficiently increases sport performance and is the most challenging doping method to detect. Current methods for detecting this practice center on the plasticizer di(2-ethlyhexyl) phthalate (DEHP), which enters the stored blood from blood bags. Quantification of this plasticizer and its metabolites in urine can detect the transfusion of autologous blood stored in these bags. However, DEHP-free blood bags are available on the market, including n-butyryl-tri-(n-hexyl)-citrate (BTHC) blood bags. Athletes may shift to using such bags to avoid the detection of urinary DEHP metabolites.
Study design and methods:
A clinical randomized double-blinded two-phase study was conducted of healthy male volunteers who underwent ABT using DEHP-containing or BTHC blood bags. All subjects received a saline injection for the control phase and a blood donation followed by ABT 36 days later. Kinetic excretion of five urinary DEHP metabolites was quantified with liquid chromatography coupled with tandem mass spectrometry.
Results:
Surprisingly, considerable levels of urinary DEHP metabolites were observed up to 1 day after blood transfusion with BTHC blood bags. The long-term metabolites mono-(2-ethyl-5-carboxypentyl) phthalate and mono-(2-carboxymethylhexyl) phthalate were the most sensitive biomarkers to detect ABT with BTHC blood bags. Levels of DEHP were high in BTHC bags (6.6%), the tubing in the transfusion kit (25.2%), and the white blood cell filter (22.3%).
Conclusions:
The BTHC bag contained DEHP, despite being labeled DEHP-free. Urinary DEHP metabolite measurement is a cost-effective way to detect ABT in the antidoping field even when BTHC bags are used for blood storage.
Ether soluble components of musk have been investigated by chromatographic and spectroscopic methods. As indicated in the list of identified constituents (Table II), they were proved to be predominantly steroidal : eleven androstane derivatives, cholesterol and its esters, and cholest-4-en-3-one, in addition to wax and muscone. Characteristic feature of cholesterol esters and wax was that they are the esters derived from very long chain fatty acids of C14 through C40 in which branched ones were predominant except C16 and C18. Wax alcohols were found to have almost exclusively branched chain (C20 to C34).
Chlorazanil (Ordipan, N-(4-chlorophenyl)-1,3,5-triazine-2,4-diamine) is a diuretic agent and as such prohibited in sport according to the regulations of the World Anti-Doping Agency (WADA). Despite its introduction into clinical practice in the late 1950s, the worldwide very first two adverse analytical findings were registered only in 2014, being motive for an in-depth investigation of these cases. Both individuals denied the intake of the drug; however, the athletes did declare the use of the antimalarial prophylactic agent proguanil due to temporary residences in African countries.
Brinzolamide is a highly specific carbonic anhydrase (CA) inhibitor which lowers intraocular pressure (IOP) by reducing the rate of aqueous humour formation. Formulated as a 1 % ophthalmic suspension (Azopt®) and administered twice or three times daily, brinzolamide is indicated for the topical management of primary open-angle glaucoma (POAG) and ocular hypertension (OH) as either monotherapy or adjunctive therapy with topical β-blockers.
As monotherapy in patients with POAG or OH, brinzolamide 1% demonstrated IOP-lowering efficacy that was significantly greater than placebo, equivalent to three-times-daily dorzolamide 2% but significantly lower than twice-daily timolol 0.5%. Brinzolamide 1% was equally effective in twice- and three-times—daily regimens producing diurnal mean IOP reductions from baseline in the range of 13.2–21.8%. When used adjunctively twice daily with timolol 0.5%, brinzolamide 1% was as effective as dorzolamide 2% and superior to placebo in lowering IOP in patients with POAG or OH.
In clinical trials, brinzolamide 1% was well tolerated causing only nonserious adverse effects that were generally local, transient and mild to moderate in severity. The incidence of the most common adverse events associated with the use of brinzolamide 1 % was either similar to (blurred vision and abnormal taste) or significantly lower than (ocular discomfort) with dorzolamide 2%. Topical brinzolamide 1% does not appear to produce the acid-base or electrolyte disturbances and severe systemic adverse effects characteristic of oral CA inhibitors. It can be used in patients unresponsive to β-blockers or in whom β-blockers are contraindicated.
Brinzolamide 1% administered twice daily is among the least costly alternatives and adjuncts to β-blocker therapy for glaucoma and is generally associated with less direct medical cost than dorzolamide.
Conclusion: Brinzolamide 1% ophthalmic suspension administered twice or three times daily, as monotherapy or adjunctive therapy with topical β-blockers, has good IOP-lowering efficacy in patients with POAG or OH that is equivalent to that of dorzolamide 2% (three times daily as monotherapy, twice daily as adjunctive therapy). Brinzolamide is generally well tolerated and does not produce the systemic adverse effects associated with oral CA inhibitors. It can be used in patients who are unresponsive to, intolerant of, or unable to receive, ophthalmic β-blockers. Thus, brinzolamide, either as monotherapy or adjunctive therapy with topical β-blockers, should be regarded as a good second-line option in the pharmacological management of POAG and OH, and may be preferred over dorzolamide because of significantly less ocular discomfort.
Overview of Pharmacodynamic Properties
Brinzolamide is a nonbacteriostatic sulfonamide derivative with higher lipophilicity and lower aqueous solubility than dorzolamide or acetazolamide at physiological pH. As a consequence, brinzolamide forms a suspension at pH 7.4 which is more comfortable to the eye than the acidic pH of dorzolamide solution (pH 5.6).
Brinzolamide is a highly specific, noncompetitive and reversible carbonic anhydrase (CA)-II inhibitor with an ≈4-fold greater in vitro binding affinity for CA-II than dorzolamide. The main local ocular effect of brinzolamide is produced predominantly by inhibition of CA-II in the secretory cells of ciliary processes inside the eye. Inhibition of this isoenzyme reduces the rate of aqueous humour formation, consequently lowering intraocular pressure (IOP).
Following topical instillation, brinzolamide enters the blood circulation but systemic adverse effects with its use do not occur mainly because of incomplete saturation and inhibition of CA-II in erythrocytes and kidneys, and the low affinity of brinzolamide for other CA isoforms in the human body.
Brinzolamide has been shown to produce significant reduction in IOP compared with placebo in the eyes of healthy volunteers and patients with glaucoma (see also Therapeutic Efficacy summary). Significantly greater reductions in both daytime and night-time rates of aqueous humour flow were obtained with brinzolamide compared with both placebo (p < 0.001) and dorzolamide (p < 0.05) in healthy volunteers. However, in the same study, the effects on IOP reduction at both trough and peak times were similar with both drugs.
CA-II is also present in corneal endothelium, where it plays a role in the mechanism responsible for maintaining corneal stroma in a relatively dehydrated state. Inhibition of this mechanism can potentially lead to corneal decompensation and impaired vision. Brinzolamide 1% appears to have no adverse effect on corneal endothelial cell function in patients with normal corneas; no clinically relevant or statistically significant changes from baseline in corneal thickness and corneal endothelial cell density occurred in patients with primary open-angle glaucoma (POAG) or ocular hypertension (OH) during an 18-month clinical trial.
Topically applied brinzolamide significantly (p-values not reported) reduced the optic nerve head blood flow to normal levels in the eyes with hypertensive and preperimetric POAG, but caused no change in the eyes with advanced, perimetric glaucoma compared with normal eyes, in a 1-month clinical trial.
Overview of Pharmacokinetic Properties
Published data relating to the pharmacokinetic properties of brinzolamide in humans are limited. After topical ocular instillation in rabbits, brinzolamide 1% was readily absorbed in the conjunctiva, cornea, iris, ciliary body, aqueous humour, lens, choroid and retina, reaching peak concentrations in the anterior eye segment tissues within 0.5–2 hours. Systemic absorption of brinzolamide does occur, but plasma concentrations in healthy human volunteers are generally below quantitation levels because of the drug’s preferential distribution to erythrocytes. Extensive but saturable binding to CA-II in erythrocytes is the cause of nonlinear whole-blood pharmacokinetics of brinzolamide in rats. Concentration-independent plasma protein binding of brinzolamide in the range of 59–63% has been reported in an in vitro study in human plasma.
Metabolic inactivation of brinzolamide is carried out predominantly in the liver through oxidative O- and N-dealkylation by cytochrome P450 isoenzymes. N-desethyl-brinzolamide binds predominantly to CA-I in erythrocytes and is the major metabolite found in whole human blood (but not in plasma). N-desmethox-ypropyl- and O-desmethyl-brinzolamide have been detected as minor metabolites in urine but not in the whole blood of humans.
In humans, brinzolamide is predominantly excreted in the urine (≈60% unchanged and 20% as the N-desethyl metabolite) and has a long whole-blood half-life (111 days following topical administration of brinzolamide 3% ophthalmic suspension three times daily for 14 days in 15 healthy male volunteers).
Therapeutic Efficacy
The efficacy of brinzolamide 1% ophthalmic suspension administered twice or three times daily as monotherapy or adjunctive therapy with twice-daily timolol 0.5% ophthalmic solution has been evaluated in six randomised, double-blind, multicentre, comparative clinical trials of 2 weeks to 18 months duration in a total of 1735 evaluable patients (previously pharmacologically treated) with POAG or OH. The trials compared the IOP-lowering efficacy of brinzolamide 1% with that of dorzolamide 2% (twice daily in monotherapy, three times daily in adjunctive therapy trials), timolol 0.5% (twice daily) or placebo. The primary efficacy endpoint in most trials was the diurnally corrected (i.e. for trough and peak times) mean IOP reduction from baseline; the longest duration trial reported only the mean reduction from baseline in trough IOP (measured at 8am). In all trials, IOP measurements were performed using Goldmann applanation tonometry. All trials were also preceded by appropriate washout periods, which, in two trials evaluating adjunctive use of brinzolamide 1%, also served as run-in phases for nonblind timolol administration.
As Monotherapy: Overall, in four monotherapy trials in a total of 1414 patients, brinzolamide 1% demonstrated IOP-lowering efficacy that was significantly greater than that of placebo (all p < 0.005), similar to dorzolamide 2%, but significantly lower than timolol 0.5% (p < 0.0002, where reported).
In a placebo-controlled, parallel-group trial in 142 patients with POAG or OH, brinzolamide 1–3% produced clinically relevant and statistically significant reductions in mean baseline IOP (p-values not reported). Brinzolamide 1%, 2% and 3% were equally effective in reducing diurnally corrected mean IOP from baseline and more effective than brinzolamide 0.3% (p < 0.036). The study found brinzolamide 1 % to be the optimal concentration for lowering the elevated IOP in patients with glaucoma when administered twice daily.
The short-term efficacy of brinzolamide 1% administered twice and three times daily was equivalent to that of three-times-daily dorzolamide 2%; mean daily IOP reduction from baseline was in the range of 13.2–21.8% for twice-daily and 13.2–21.5% for three-times-daily brinzolamide 1%, and 15.7–22.9% for dorzolamide 2% in two 3-month clinical trials in a total of 921 patients with POAG or OH (all p < 0.001). Clinically relevant reductions from baseline of the morning trough IOP were maintained long term with brinzolamide 1% throughout an 18-month trial involving 351 patients with POAG or OH (13.2% and 12.6%, respectively, in twice- and three-times-daily regimens; both p < 0.0001 vs baseline), although the effect was significantly lower than with twice-daily timolol 0.5% ophthalmic solution (20.5%; p < 0.0002).
After 3 months of treatment in two monotherapy trials, brinzolamide 1% administered twice or three times daily, respectively, produced IOP response (i.e. an IOP reduction ≥5mm Hg) or control (i.e. IOP of ≤21mm Hg) in up to 75.7% and 80.1% of patients, compared with up to 80.0% of patients who received dorzolamide 2% three times daily and up to 82.0% of patients who received timolol 0.5% twice daily.
As Adjunctive Therapy: Brinzolamide 1% was as effective as dorzolamide 2% (14.1–22.0% vs 14.0–21.2%) and superior to placebo (13.2–16.6% vs 4.4–10.4%; all p ≤ 0.03) in lowering IOP in patients with POAG or OH (n = 321) when used adjunctively with timolol 0.5% ophthalmic solution in twice- and three-times-daily regimens, respectively, at all timepoints during two 3-month trials. Furthermore, in the active-controlled trial (n = 213), a similar proportion of patients achieved IOP reduction or control with brinzolamide plus timolol and dorzolamide plus timolol (50.0–89.3% vs 43.9–85.4%, respectively).
Tolerability
Brinzolamide 1% ophthalmic suspension administered twice or three times daily was well tolerated in randomised, double-blind, multicentre, comparative, mono-therapy clinical trials in a total of 1626 patients with POAG or OH; brinzolamide-related adverse events were nonserious and generally mild to moderate in severity, occurring mostly at the time of eyedrop instillation and usually resolving without treatment.
In the only long-term (18-month) clinical trial, adverse events and inadequate IOP control were the most common reasons for treatment discontinuation and accounted, respectively, for 39% and 17% of discontinuations in the twice-, and 27% and 21% in the three-times-daily brinzolamide groups, and for 30% and 4% in the timolol group.
In all clinical trials, the most common ocular adverse events related to brinzolamide treatment were transient blurring of vision and ocular discomfort (i.e. stinging and burning ocular sensations). The incidence of blurred vision in 3- and 18-month trials was relatively low (3–8% with twice-daily, and 3.6–5.2% with three-times-daily regimens) and not statistically significantly greater than that reported in patients receiving three-times-daily dorzolamide 2% (0.6% and 0.8%) or placebo (1.5%), or twice-daily timolol 0.5% (0% and 5.3%). In clinical trials of up to 18 months duration, ocular discomfort at the time of instillation occurred ≈3–9 times less frequently with brinzolamide 1% than with dorzolamide 2% (p < 0.05). In addition, burning and/or stinging sensation was significantly less intense with brinzolamide 1% compared with dorzolamide 2% (p < 0.0001) in two weekly comfort studies in a total of 198 patients with POAG or OH.
Results of a prospective, nonblind, noncomparative, crossover, multicentre clinical study in 447 evaluable patients with glaucoma indicate that substituting dorzolamide 2% with brinzolamide 1% for mono- or adjunctive therapy may provide improved ocular comfort. One to 3 months after the switch, 69%, 26% and 5% of patients, respectively, reported improvement, no change, and worsening in ocular comfort. Consequently, more than twice as many patients preferred brinzolamide over dorzolamide for their glaucoma therapy (59% vs 26%; p-value not reported).
Brinzolamide did not affect visual acuity, visual fields, cup-to-disc ratio and other ocular signs, and corneal endothelial cell function (see Overview of Pharmacodynamic Properties summary) in patients with POAG or OH receiving treatment for up to 18 months during clinical trials.
Abnormal (i.e. bitter or sour) taste was the only local, nonocular adverse event associated with brinzolamide 1%, which occurred in ≥3% of patients across monotherapy and adjunctive therapy trials. The incidence of abnormal taste with brinzolamide use was dose- and frequency-related (6.8–12.1% with brinzolamide 1% three times daily), and was not statistically significantly different than that with dorzolamide 2%.
Topical brinzolamide 1% does not appear to produce acid-base or electrolyte disturbances and was not associated with clinically significant systemic adverse effects, generally observed with oral CA inhibitors, in clinical trials of up to 18 months duration.
Pharmacoeconomic Analyses
Thus far, pharmacoeconomic analyses relating to the use of brinzolamide in the treatment of POAG and OH have been limited to a cost-minimisation study and two cost analyses.
The European cost-minimisation study showed that initiating medical glaucoma therapy with brinzolamide 1% in patients with POAG or OH was associated with lower (in Italy, Spain and Portugal) or similar (in France) total direct medical costs per patient over 3 months compared with dorzolamide 2%. The analysis was conducted from the perspective of healthcare payers from each country and in the context of second-line use of topical CA inhibitors, and was based on similar week 4 responder rates between brinzolamide and dorzolamide regimens from four randomised, double-blind, comparative brinzolamide clinical trials. The cost benefit of short-term brinzolamide versus dorzolamide therapy was explained by the lower administration rate of brinzolamide (twice daily) with equal efficacy to dorzolamide (three times daily) [see Therapeutic Efficacy summary] and lower rate of therapy discontinuation with brinzolamide than with dorzolamide due to better local tolerability (see Tolerability summary).
Two US cost analyses conducted from a payer’s perspective and covering similar periods (1999 and 1998–2000) both showed that twice-daily therapy with brinzolamide 1% incurred less cost than with dorzolamide 2%. Both studies also found that glaucoma monotherapy was less costly with twice-daily brinzolamide 1% than with brimonidine 2% (twice daily) or latanoprost 0.005% (once daily), but more costly than with generic timolol maleate 0.5% (twice daily).
Dosage and Administration
Brinzolamide 1 % ophthalmic suspension is indicated for the treatment of elevated IOP in patients with POAG or OH. The recommended dosage of brinzolamide in Europe is one drop instilled in the conjunctival sac of the affected eye(s) twice daily (for use as either monotherapy or adjunctive therapy with topical ophthalmic β-blockers). In Europe, brinzolamide monotherapy is also indicated in patients unresponsive to β-blockers or in whom β-blockers are contraindicated. In the US, the recommended administration regimen for brinzolamide as monotherapy is three times daily; formal US recommendations for the use of brinzolamide in combination with other topical antiglaucoma drugs have not been made.
Brinzolamide is not recommended in the US and is contraindicated in Europe for the treatment of patients with severe renal impairment (creatinine clearance <30 mL/min), as brinzolamide and its metabolites are excreted predominantly by the kidney (see Overview of Pharmacokinetic Properties summary). In patients with hepatic impairment, either the use of brinzolamide is not recommended (US) or the drug should be used with caution (Europe). It appears prudent to avoid the use of brinzolamide in patients with previous hypersensitivity reactions to sulfonamides.
Wearers of soft contact lenses should allow a period of at least 15 minutes to elapse between instillation of brinzolamide and the re-insertion of lenses.
Chlorphenesin functions as a biocide in cosmetics and is used at concentrations up to 0.32% in rinse-off products and up to 0.3% in leave-on products. The Cosmetic Ingredient Review Expert Panel (Panel) noted that chlorphenesin was well absorbed when applied to the skin of rats; however, any safety concern was minimized because available data demonstrated an absence of toxicity. The Panel concluded that chlorphenesin is safe in the present practices of use and concentration.
Food supplements are regularly found to contain pharmacologically active substances. Recently, the food supplement Dexaprine was removed from the Dutch market because it was associated with severe adverse events. Reports to the Dutch Poisons Information Center (DPIC) showed that ingestion of as little as half a tablet caused several cases of nausea, agitation, tachycardia, and palpitations and even one case of cardiac arrest. The remaining tablets of four patients were sent in by different healthcare professionals. Analysis by ultra-performance liquid chromatography quadrupole time of flight mass-spectrometry (UPLC-QTOF-MS) confirmed the presence of synephrine, oxilofrine, deterenol, yohimbine, caffeine, and theophylline. Two more compounds were found which were tentatively identified as β-methyl-β-phenylethylamines. This incident is only the next in a series of similar incidents involving dietary supplements with (undeclared) active substances that are either unsafe or have no known safety profile. Copyright © 2014 John Wiley & Sons, Ltd.
The qualitative and quantitative composition of the principal lipid constituents of Siberian musk deer (Moschus moschiferus) preputial gland secretion, main odor carriers and potential precursors of odorous substances, was investigated by means of high-performance liquid chromatography. Free fatty acids and phenols (10%), waxes (38%), and steroids (38%) were found to be the main groups of the secretion lipids. Cholestanol (I), cholesterol (II), androsterone (III), Δ(4)-3α-hydroxy-17-ketoandrostene (IV), 5β, 3α-hydroxy-17-ketoandrostane (V), 5α, 3β, 17α-dihydroxyandrostane (VI), 5β, 3α, 17β-dihydroxyandrostane (VII), and 5β, 3α, 17α-dihydroxyandrostane (VIII) were isolated from the steroid fraction and their structures confirmed by IR, PMR, and mass spectra. 3-Methylpentadecanone (muscone) was not identified among the secretion lipids. Preputial gland secretion stimulated sex behavior of musk deer females.