Investigations into the concentration and metabolite profiles of stanozolol and LGD-4033 in blood plasma and seminal fluid using liquid chromatography high-resolution mass spectrometry.

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.

Mass spectrometry in sports drug testing-Analytical approaches and the athletes' exposome.

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.

Chromatographic-mass spectrometric analysis of the urinary metabolite profile of chlorphenesin observed after dermal application of chlorphenesin-containing sunscreen.

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, which is also employed as a target analyte in sports drug testing to detect the use of the prohibited nootropic stimulant meclofenoxate. 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 after 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 using 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 ~1500 and 2300 ng/mL after single and multiple sunscreen applications, 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 of whether chlorphenesin or the prohibited nootropic meclofenoxate was administered.

Carbon isotope ratios of endogenous steroids found in human serum-method development, validation, and reference population-derived thresholds.

In order to detect the misuse of testosterone (T), urinary steroid concentrations and concentration ratios are quantified and monitored in a longitudinal manner to enable the identification of samples exhibiting atypical test results. These suspicious samples are then forwarded to isotope ratio mass spectrometry (IRMS)-based methods for confirmation. Especially concentration ratios like T over epitestosterone (E) or 5α-androstanediol over E proved to be valuable markers. Unfortunately, depending on the UGT2B17 genotype and/or the gender of the athlete, these markers may fail to provide evidence for T administrations when focusing exclusively on urine samples. In recent years, the potential of plasma steroids has been investigated and were found to be suitable to detect T administrations especially in female volunteers. A current drawback of this approach is the missing possibility to confirm that elevated steroid concentrations are solely derived from an administration of T and cannot be attributed to confounding factors. Therefore, an IRMS method for plasma steroids was developed and validated taking into account the comparably limited sample volume. As endogenous reference compounds, unconjugated cholesterol and dehydroepiandrosterone sulfate were found suitable, while androsterone and epiandrosterone (both sulfo-conjugated) were chosen as target analytes. The developed method is based on multi-dimensional gas chromatography coupled to IRMS in order to optimize the overall assay sensitivity. The approach was validated, and a reference population encompassing n = 65 males and females was investigated to calculate population-based thresholds. As proof-of-concept, samples from volunteers receiving T replacement therapies and excretion study samples were investigated.

UEFA expert group statement on nutrition in elite football. Current evidence to inform practical recommendations and guide future research.

Football is a global game which is constantly evolving, showing substantial increases in physical and technical demands. Nutrition plays a valuable integrated role in optimising performance of elite players during training and match-play, and maintaining their overall health throughout the season. An evidence-based approach to nutrition emphasising, a 'food first' philosophy (ie, food over supplements), is fundamental to ensure effective player support. This requires relevant scientific evidence to be applied according to the constraints of what is practical and feasible in the football setting. The science underpinning sports nutrition is evolving fast, and practitioners must be alert to new developments. In response to these developments, the Union of European Football Associations (UEFA) has gathered experts in applied sports nutrition research as well as practitioners working with elite football clubs and national associations/federations to issue an expert statement on a range of topics relevant to elite football nutrition: (1) match day nutrition, (2) training day nutrition, (3) body composition, (4) stressful environments and travel, (5) cultural diversity and dietary considerations, (6) dietary supplements, (7) rehabilitation, (8) referees and (9) junior high-level players. The expert group provide a narrative synthesis of the scientific background relating to these topics based on their knowledge and experience of the scientific research literature, as well as practical experience of applying knowledge within an elite sports setting. Our intention is to provide readers with content to help drive their own practical recommendations. In addition, to provide guidance to applied researchers where to focus future efforts.

Current Insights into the Steroidal Module of the Athlete Biological Passport.

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.

Detecting the misuse of 7-oxo-DHEA by means of carbon isotope ratio mass spectrometry in doping control analysis.

RATIONALE: The misuse of 7-oxo-DHEA (3ß-hydroxyandrost-5-ene-7,17-dione) is prohibited according to the World Anti-Doping Agency (WADA) code. Nevertheless, it is easily available as a dietary supplement and from black market sources. In two recent doping control samples, significant amounts of its main metabolite 7ß-OH-DHEA were identified, necessitating further investigations. METHODS: As both 7-oxo-DHEA and 7ß-OH-DHEA are endogenously produced steroids and no concentration thresholds applicable to routine doping controls exist, the development and validation of a carbon isotope ratio (CIR) mass spectrometry method ha been desirable. Excretion studies encompassing 7-oxo-DHEA, 7-oxo-DHEA-acetate, and in-house deuterated 7-oxo-DHEA were conducted and evaluated with regard to urinary CIR and potential new metabolites of 7-oxo-DHEA. RESULTS: Numerous urinary metabolites were identified, some of which have not been reported before, while others corroborate earlier findings on the metabolism of 7-oxo-DHEA. The CIRs of both 7-oxo-DHEA and 7ß-OH-DHEA were significantly influenced for more than 50 h after a single oral dose of 100 mg, and a novel metabolite (5α-androstane-3ß,7ß-diol-17-one) was found to prolong this detection time window by approximately 25 h. Applying the validated method to routine doping control specimens presenting atypically high urinary 7ß-OH-DHEA levels clearly demonstrated the exogenous origin of 7-oxo-DHEA and 7ß-OH-DHEA. CONCLUSIONS: As established for other endogenously produced steroids such as testosterone, the CIR allows for a clear differentiation between endo- and exogenous sources of 7-oxo-DHEA and 7ß-OH-DHEA. The novel metabolites detected after administration may help to improve the detection of 7-oxo-DHEA misuse and simplify its detection in doping control specimens.

Analytical challenges in sports drug testing.

Analytical chemistry represents a central aspect of doping controls. Routine sports drug testing approaches are primarily designed to address the question whether a prohibited substance is present in a doping control sample and whether prohibited methods (for example, blood transfusion or sample manipulation) have been conducted by an athlete. As some athletes have availed themselves of the substantial breadth of research and development in the pharmaceutical arena, proactive and preventive measures are required such as the early implementation of new drug candidates and corresponding metabolites into routine doping control assays, even though these drug candidates are to date not approved for human use. Beyond this, analytical data are also cornerstones of investigations into atypical or adverse analytical findings, where the overall picture provides ample reason for follow-up studies. Such studies have been of most diverse nature, and tailored approaches have been required to probe hypotheses and scenarios reported by the involved parties concerning the plausibility and consistency of statements and (analytical) facts. In order to outline the variety of challenges that doping control laboratories are facing besides providing optimal detection capabilities and analytical comprehensiveness, selected case vignettes involving the follow-up of unconventional adverse analytical findings, urine sample manipulation, drug/food contamination issues, and unexpected biotransformation reactions are thematized.

IOC consensus statement: dietary supplements and the high-performance athlete.

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.

IOC Consensus Statement: Dietary Supplements and the High-Performance Athlete.

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 program. Nonetheless, supplement use is widespread at all levels of sport. Products described as supplements target different issues, including the management of micronutrient deficiencies, supply of convenient forms of energy and macronutrients, and provision of direct benefits to performance or indirect benefits such as supporting intense training regimens. The appropriate use of some supplements can offer benefits to the athlete, but others may be harmful to the athlete's health, performance, and/or livelihood and reputation if an anti-doping 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 trialed in training or simulated competition before implementation in competition. Inadvertent ingestion of substances prohibited under the anti-doping 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, and expert professional opinion and assistance is strongly advised before embarking on supplement use.

Expanding analytical options in sports drug testing: Mass spectrometric detection of prohibited substances in exhaled breath.

RATIONALE: Continuously refining and advancing the strategies and methods employed in sports drug testing is critical for efficient doping controls. Besides improving and expanding the spectrum of target analytes, alternative test matrices have warranted in-depth evaluation as they commonly allow for minimal-/non-invasive and non-intrusive sample collection. In this study, the potential of exhaled breath (EB) as doping control specimen was assessed. METHODS: EB collection devices employing a non-woven electret-based air filter unit were used to generate test specimens, simulating a potential future application in doping controls. A multi-analyte sports drug testing approach configured for a subset of 12 model compounds that represent specific classes of substances prohibited in sports (anabolic agents, hormone and metabolic modulators, stimulants, and beta-blockers) was established using unispray liquid chromatography/tandem mass spectrometry (LC/MS/MS) and applied to spiked and elimination study EB samples. The test method was characterized concerning specificity, assay imprecision, and limits of detection. RESULTS: The EB collection device allowed for retaining and extracting all selected model compounds from the EB aerosol. Following elution and concentration, LC/MS/MS analysis enabled detection limits between 5 and 100 pg/filter and imprecisions ranging from 3% to 20% for the 12 selected model compounds. By means of EB samples from patients and participants of administration studies, the elimination of relevant compounds and, thus, their traceability in EB for doping control purposes, was investigated. Besides stimulants such as methylhexaneamine and pseudoephedrine, also the anabolic-androgenic steroid dehydrochloromethyltestosterone, the metabolic modulator meldonium, and the beta-blocker bisoprolol was detected in exhaled breath. CONCLUSIONS: The EB aerosol has provided a promising proof-of-concept suggesting the expansion of this testing strategy as a complement to currently utilized sports drug testing programs.

Mass spectrometric characterization of the selective androgen receptor modulator (SARM) YK-11 for doping control purposes.

RATIONALE: Selective androgen receptor modulators (SARMs) represent an emerging class of therapeutics targeting inter alia conditions referred to as cachexia and sarcopenia. Due to their anabolic properties, the use of SARMs is prohibited in sports as regulated by the World Anti-Doping Agency (WADA), and doping control laboratories test for these anabolic agents in blood and urine. In order to accomplish and maintain comprehensive test methods, the characterization of new drug candidates is critical for efficient sports drug testing. Hence, in the present study the mass spectrometric properties of the SARM YK-11 were investigated. METHODS: YK-11 was synthesized according to literature data and three different stable-isotope-labeled analogs were prepared to support the mass spectrometric studies. Using high-resolution/high-accuracy mass spectrometry following electrospray ionization as well as electron ionization, the dissociation pathways of YK-11 were investigated, and characteristic features of its (product ion) mass spectra were elucidated. These studies were flanked by density functional theory (DFT) computation providing information on proton affinities of selected functional groups of the analyte. RESULTS AND CONCLUSIONS: The steroidal SARM YK-11 was found to readily protonate under ESI conditions followed by substantial in-source dissociation processes eliminating methanol, acetic acid methyl ester, and/or ketene. DFT computation yielded energetically favored structures of the protonated species resulting from the aforementioned elimination processes particularly following protonation of the steroidal D-ring substituent. Underlying dissociation pathways were suggested, supported by stable-isotope labeling of the analyte, and diagnostic product ions for the steroidal nucleus and the D-ring substituent were identified. Further, trimethylsilylated YK-11 and its deuterated analogs were subjected to electron ionization high-resolution/high-accuracy mass spectrometry, complementing the dataset characterizing this new SARM. The obtained fragment ions resulted primarily from A/B- and C/D-ring structures of the steroidal nucleus, thus supporting future studies e.g. concerning metabolic pathways of the substance. Copyright © 2017 John Wiley & Sons, Ltd.

Clinical findings, treatment, and outcome in 11 dairy heifers with breakdown injury due to interosseous medius muscle rupture.

OBJECTIVE: To describe the diagnosis, treatment, and prognosis of fetlock breakdown due to interosseus medius muscle rupture in cattle. STUDY DESIGN: Retrospective clinical study. ANIMALS: Dairy heifers with unilateral or bilateral interosseus medius muscle rupture (n = 11). METHODS: Breakdown injury due to rupture of the interosseus medius muscle was documented clinically, radiographically, and ultrasonographically. Breakdown was bilateral in 4 heifers (3 in forelimbs, 1 in hind limbs) and unilateral in 7 (all hind limbs). One heifer with severe bilateral hind limb breakdown was slaughtered and the remaining 10 were treated by transfixation pin cast (1 heifer), box rest (n = 2), and/or a cast and splint (7). RESULTS: Hyperextension of the fetlock and hyperflexion of the proximal interphalangeal joints during weight bearing were characteristic for interosseus muscle breakdown. Ultrasonographically, the origin and body of the interosseus muscle and the branches to the sesamoid bones were primarily affected by the rupture. Conservative treatment was successful (used for their intended purpose) in 8 of 9 heifers with a median lifespan of 32 months after discharge from the clinic (range 6-83). CONCLUSION: Rupture of the interosseus medius muscle in young cattle may be more common than previously suggested in the literature. Imaging with ultrasound allowed more detailed localization of lesions of the musculo-tendinous structure. Interosseus medius muscle rupture had a favorable prognosis when treated conservatively in these heifers.

Measuring Rabbit (Oryctolagus cuniculus) Tooth Growth and Eruption by Fluorescence Markers and Bur Marks.

Rabbits (Oryctolagus cuniculus) and rodents possess continuously growing teeth, and dental problems are a major health issue in these species. Knowledge of tooth growth characteristics is required to adequately treat dental problems and advise owners concerning diets. Most research was performed using bur marks and measuring eruption and wear manually. However, this method cannot be applied to teeth less rostral than the first premolar; therefore, for evaluation of molars, other methods are needed. We evaluated the use of fluorochromes xylenol orange and calcein green to measure growth rates of rabbit teeth and compared this method to results obtained by manually measuring the distance between a bur mark and the gingival margin of the same tooth (eruption) and by measuring the distance between the bur mark and the apex of the same tooth on computed tomography scans (growth). Apical fluorochrome measurements correlated well with eruption and growth rates obtained with bur marks, whereas measurements coronal to the pulp cavity did not. Growth rates were approximately 1.9 mm/wk for maxillary and 2.2 mm/wk for mandibular incisors. Growth rates of premolars were 2.14 ± 0.28 mm/wk in rabbits on a grass/rice hulls/sand pelleted diet and 0.93 ± 0.18 mm/wk in rabbits on a hay diet. Growth of molars could only be assessed using the measurement in dentin on the wall of the pulp cavity, which does not account for the real growth. However, being similar to this measurement in premolars, one could hypothesize similar growth in molars as in premolars. We conclude that the application of fluorochrome staining can be used to measure tooth growth in teeth that are not accessible for bur marks or in animals that are too small to assess tooth eruption or growth by bur marks.

Urine analysis concerning xenon for doping control purposes.

RATIONALE: On September 1(st) 2014, a modified Prohibited List as established by the World Anti-Doping Agency (WADA) became effective featuring xenon as a banned substance categorized as hypoxia-inducible factor (HIF) activator. Consequently, the analysis of xenon from commonly provided doping control specimens such as blood and urine is desirable, and first data on the determination of xenon from urine in the context of human sports drug testing, are presented. METHODS: In accordance to earlier studies utilizing plasma as doping control matrix, urine was enriched to saturation with xenon, sequentially diluted, and the target analyte was detected as supported by the internal standard d6 -cyclohexanone by means of gas chromatography/triple quadrupole mass spectrometry (GC/MS/MS) using headspace injection. Three major xenon isotopes at m/z 128.9, 130.9 and 131.9 were targeted in (pseudo) selected reaction monitoring mode enabling the unambiguous identification of the prohibited substance. Assay characteristics including limit of detection (LOD), intraday/interday precision, and specificity as well as analyte recovery under different storage conditions were determined. Proof-of-concept data were generated by applying the established method to urine samples collected from five patients before, during and after (up to 48 h) xenon-based general anesthesia. RESULTS: Xenon was traceable in enriched human urine samples down to the detection limit of approximately 0.5 nmol/mL. The intraday and interday imprecision values of the method were found below 25%, and specificity was demonstrated by analyzing 20 different blank urine samples that corroborated the fitness-for-purpose of the analytical approach to unequivocally detect xenon at non-physiological concentrations in human urine. The patients' urine specimens returned 'xenon-positive' test results up to 40 h post-anesthesia, indicating the limits of the expected doping control detection window. CONCLUSIONS: Since xenon has been considered a prohibited substance according to WADA regulations in September 2014, its analysis from common specimens of routine sports drug testing is desirable. In previous studies, its traceability in whole blood and plasma was shown, and herein a complementary approach utilizing doping control urine samples for the GC/MS/MS analysis of xenon was reported.

Determination of Synacthen(®) in dried blood spots for doping control analysis using liquid chromatography tandem mass spectrometry.

Dried blood spot (DBS) sampling, a technique used for taking whole blood samples dried on a filter paper, was initially reported in 1963 by Robert Guthrie. While the diagnostic analysis of metabolic disorders in newborns was the focus of investigations at that time, the number of established applications for preclinical drug development, toxicological studies, and therapeutic drug monitoring increased enormously in the last decades. As a consequence of speed, simplicity, and minimal invasiveness, DBS recommends itself as the preferential technique in sports drug testing. The present approach highlights for the first time the development of a screening assay for the analysis of the synthetic human adrenocorticotropic hormone tetracosactide hexaacetate (Synacthen(®)) in DBS using liquid chromatography tandem mass spectrometry. Highly purified sample extracts were obtained by an advanced sample preparation procedure including the addition of an internal standard (d8-tetracosactide) and immunoaffinity purification. The method's overall recovery was 27.6 %, and the assay's imprecision was calculated between 8.1 and 17.9 % for intraday and 12.9 to 20.5 % for interday measurements. Stability of the synthetic peptide in DBS was shown for at least 10 days at room temperature and presents a major benefit, since a rapid degradation in conventionally applied matrices such as urine or plasma is well known. With a limit of detection of 50 pg/mL, a detection window of several hours is expected considering reported steady-state plasma levels of 300 pg/mL after intramuscular application of Synacthen(®) Depot (1 mg). The analysis of authentic DBS samples within the scope of an administration study with 250 µg Synacthen(®) (short stimulation test) demonstrated the great potential of the developed assay to simplify the analysis of Synacthen(®) for doping control purposes.

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

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

Measuring xenon in human plasma and blood by gas chromatography/mass spectrometry.

RATIONALE: Due to the favorable pharmacokinetic properties and minimal side effects of xenon, its use in modern anesthesia has been well accepted, and recent studies further demonstrated the intra- and postoperative neuro-, cardio-, and reno-protective action of the noble gas. Since the production of the hypoxia-inducible factor 1α (HIF-1α) and its downstream effector erythropoietin as well as noradrenalin reuptake inhibition have been found to play key roles in this context, the question arose as to whether the use of xenon is a matter for doping controls and preventive doping research. The aim of the present study was hence to evaluate whether the (ab)use of xenon can be detected from doping control samples with the instrumentation commonly available in sports drug testing laboratories. METHODS: Plasma was saturated with xenon according to reported protocols, and the target analyte was measured by means of gas chromatography/time-of-flight and triple quadrupole mass spectrometry with headspace injection. Recording the accurate mass of three major xenon isotopes at m/z 128.9048, 130.9045 and 131.9042 allowed for the unequivocal identification of the analyte and the detection assay was characterized concerning limit of detection (LOD), intraday precision, and specificity as well as analyte recovery under different storage conditions. RESULTS: Xenon was detected in fortified plasma samples with detection limits of approximately 0.5 nmol/mL to 50 nmol/mL, depending on the type of mass spectrometer used. The method characteristics of intraday precision (coefficient of variation <20%) and specificity demonstrated the fitness-for-purpose of the analytical approach to unambiguously detect xenon at non-physiological concentrations in human plasma and blood. Eventually, authentic plasma and blood samples collected pre-, intra-, and post-operative (4, 8, and 24 h) were positively analyzed after storage for up to 30 h, and provided proof-of-concept for the developed assay. CONCLUSIONS: If relevant to doping controls, xenon can be determined from plasma and blood samples, i.e. common specimens of routine sports drug testing in the context of Athlete Biological Passport (ABP) analyses. Optimization of sampling and analytical procedures will allow the detection limit to be further improved and potentially enable accurate quantification of the anesthetic agent.