Qualitative and Semiquantitative Analysis of Doping Products Seized at the Swiss Border.

BACKGROUND: Substances developed for therapeutic use are also known to be misused by athletes as doping agents and, outside of regulated sport, for image-enhancement. This has generated a market for counterfeit doping substances. Counterfeit doping agents may be of poor pharmaceutical quality and therefore constitute health risks to consumers. OBJECTIVES: This study aims to investigate the pharmaceutical quality of 1,190 doping products seized at the Swiss border. METHODS: Swiss customs authorities seize incoming shipments potentially containing doping agents. Qualitative and semiquantitative analyses were performed in order to test for prohibited doping substances. The main analytical methods utilized for characterizing confiscated compounds were liquid chromatography-high resolution mass spectrometry, polyacrylamide gel electrophoresis with subsequent in-gel tryptic digestion and identification of peptidic compounds using nanoliquid chromatography-tandem mass spectrometry, and electrochemiluminescence immuno assay. RESULTS: For 889 (75%) of the analyzed products, the label suggested the content of anabolic agents, for 146 samples (12%) peptide hormones or growth factors, and for 113 items (9%) antiestrogens, aromatase inhibitors or other metabolic modulators. For the majority of the investigated products, the pharmaceutical quality was an unsatisfactory standard: nonapproved substances were detected and less than 20% of the products contained the claimed substance in the respective amount. CONCLUSION: A comprehensive sample of confiscated doping products was analyzed, allowing for monitoring of developments regarding the use of doping substances in Switzerland and for anticipating future trends and challenges in sports drug testing. An alarming number of tested products was of substandard pharmaceutical quality.

Detection of the diuretic hydrochlorothiazide in a doping control urine sample as the result of a non-steroidal anti-inflammatory drug (NSAID) tablet contamination.

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 5ng/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 2µg 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 (2.5µg 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 1-16ng/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 10ppm 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.

Seizures of doping substances at the Swiss Border--a descriptive investigation.

This retrospective study evaluates the content, the destination and the source of 960 postal items seized by the Swiss customs authorities at the Swiss border between 2013 and 2014. The packages were seized because they contained at least one prohibited doping product as identified by the Swiss law on encouraging sports and physical activity. A total number of 1825 different doping products were confiscated from these parcels, accounting for an average of 1.9 doping products per seized item. In 74% of the cases, where seizures were made, anabolic androgenic steroids, mostly testosterone esters, were discovered. An obvious trading channel for doping products was identified in this study. The seized compounds were predominately manufactured in Asian countries, but sent to Switzerland mostly via South Eastern Europe countries. Due to the unique collaboration between the Swiss customs authorities and the national anti-doping agency, this study uncovered an alarming trend of illegal doping product trafficked to Switzerland.

Doping mit Anabolika - auch ein Thema für den Hausarzt!

BACKGROUND: Over the last years, various revelations demonstrated that the doping problem is far from being solved. These included the American cyclist Lance Armstrong's disclosure and subsequent conviction for doping abuse over a period of many years. Furthermore, these revelations underlined the importance of strong and independent national antidoping agencies (NADA). During the current revision process of the World Anti-Doping Programme (WADP), Antidoping Switzerland is campaigning for national anti-doping agencies to have the same rights, the same authority and the same degree of responsibility as international sports associations. Further, the newly revised Federal Act on the Promotion of Sport and Exercise (Sport Promotion Act), which entered into force on 1 October 2012, establishes the framework for cooperation with customs officers when suspected doping substances are seized. By the end of 2012 Antidoping Switzerland received about 50 reports from the customs authorities, and in 24 cases an administrative ruling for the seizure and destruction of these doping substances was issued. In addition, there was also a greater cooperation between customs and the Swiss Agency for Therapeutic Products Swissmedic. Two athletes have already been sanctioned under private law for importing doping substances. CONTROLS: Antidoping Switzerland carried out 2'551 controls in 2012. Of these, 1'752 were urine tests, of which 1'089 were conducted out of competition and 663 in competition. The majority of the 799 blood controls were conducted out of competition. In 2012 Antidoping Switzerland lodged about 20 applications on violations of the anti-doping provisions with Swiss Olympic's Disciplinary Chamber for Doping Cases (DC). In numbers, four athletes were banned for two years for using anabolic steroids. A trainer was also suspended for two years for having given an athlete a stimulant before a competition. 2012 was the first year in which two athletes were convicted of import of doping substances (EPO and an anabolic drug respectively) on information provided by customs officers. Both athletes were banned from competition for two years. Legal basis: Of importance to all the physicians looking after athletes is to know the actual legal basis: The international law basis of the Swiss fight against doping are the Council of Europe Convention against Doping of 16 November 1989 and the International Convention against Doping in Sport of the General Conference of the United Nations Educational, Scientific and Cultural Organization (UNESCO) of 19 October 2005. The basis in public law of the Swiss fight against doping is the Federal Act on the Promotion of Gymnastics and Sport of 17 March 1972. Article 11e, paragraph 1 states that "national sport organisations, the relevant umbrella bodies and bodies responsible for sporting events that are supported in the framework of this act […] are required to ensure that the necessary doping controls are carried out in their area of responsibility". Article 11 f, paragraph 1 of the Federal Act also states that "whoever produces, introduces, transfers, sells, prescribes or provides substances for doping purposes or who uses methods for doping purposes for third persons will be liable to a prison sentence or to a fine of up to 100'000 francs". The civil law basis of the Swiss fight against doping consists of the norms established by various actors in the sporting world. These actors are in most cases clubs, associations and foundations in accordance with the stipulations of the Swiss civil code. The Swiss Olympic Association's revised Doping Statute was approved by the Sports Parliament on 15 November 2008. The statute implements the code of the World Anti-Doping Agency (WADA) in Switzerland. In the introduction it defines the anti-doping agencies in our country: Antidoping Switzerland and the Disciplinary Chamber for Doping Offences of the Swiss Olympic Association. The revised Statute entered into force on 1 January 2009. Finally, it is well known that the use of anabolic steroids and other doping substances is a phenomenon not restricted to professional sport only. Also in popular sport, in particular in fitness, the use of anabolic steroids to increase physical performance is very common. To summarize, doping is a widespread phenomenon not only in athletes. In these situations physicians involved have to know not only the medical but also the legal side of prescribing, applying substances to persons actively involved in sport activities. Otherwise it might happen that wittingly or unwittingly the doctor runs into serious problems. All the most valuable information are published at http://www.antidoping.ch.

Development of the role of national anti-doping organisations in the fight against doping: from past to future.

When action against doping began, sport itself was, as a rule, responsible for anti-doping measures and governments often had only a subsidiary function. However, due to doping scandals or doping allegations, the formation of independent anti-doping organisations (NADOs) was already discussed in the 1990s in some countries as guarantors for clean sports. In the course of the doping scandal at the Tour de France in 1998 and the systematic intervention of the French state, the World Anti-Doping Agency (WADA) was founded 1999 at an international level. In the following years, the WADA-model was often copied at a national level and a multitude of independent NADOs came into being. NADOs play a key role worldwide in combating doping. Their influence in developing an international anti-doping policy and implementing it in the form of standards and regulations is today, however, low; they are not directly represented in the decision-making bodies of the WADA. This should be changed with regard to elaborating the new World Anti-Doping Programme for 2014.

Quantification of urinary AICAR concentrations as a matter of doping controls.

Influencing the endurance in elite sports is one of the key points in modern sports science. Recently, a new class of prohibited substances reached in the focus of doping control laboratories and their misuse was classified as gene doping. The adenosine monophosphate activated protein kinase activator 5-amino-4-imidazolecarboxyamide ribonucleoside (AICAR) was found to significantly enhance the endurance even in sedentary mice after treatment. Due to endogenous production of AICAR in healthy humans, considerable amounts were present in the circulation and, thus, were excreted into urine. Considering these facts, the present study was initiated to fix reference values of renally cleared AICAR in elite athletes. Therefore a quantitative analytical method by means of isotope-dilution liquid chromatography (analytical column: C6-phenyl) coupled to tandem mass spectrometry, after a sample preparation consisting of a gentle dilution of native urine, was developed. Doping control samples of 499 athletes were analysed, and AICAR concentrations in urine were determined. The mean AICAR value for all samples was 2,186 ng/mL with a standard deviation of 1,655 ng/mL. Concentrations were found to differ depending on gender, type of sport and type of sample collection (in competition/out of competition). The method was fully validated for quantitative purposes considering the parameters linearity, inter- (12%, 7% and 10%) and intraday precision (14%, 9% and 12%) at low, mid and high concentration, robustness, accuracy (approx. 100%), limit of quantification (100 ng/mL), stability and ion suppression effects, employing an in-house synthesised (13)C(5)-labelled AICAR as internal standard.

The worldwide fight against doping: from the beginning to the World Anti-Doping Agency.

This article describes the worldwide endeavor to combat doping in sports. It describes the historical reasons the movement began and outlines the current status of this effort by international sports groups, governments, and the World Anti-Doping Agency. The purposes, strengths, and limitations of the various entities are illustrated; and recommendations for improvements are made.

Plasma and urine profiles of Delta9-tetrahydrocannabinol and its metabolites 11-hydroxy-Delta9-tetrahydrocannabinol and 11-nor-9-carboxy-Delta9-tetrahydrocannabinol after cannabis smoking by male volunteers to estimate recent consumption by athletes.

Since 2004, cannabis has been prohibited by the World Anti-Doping Agency for all sports competitions. In the years since then, about half of all positive doping cases in Switzerland have been related to cannabis consumption. In doping urine analysis, the target analyte is 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH), the cutoff being 15 ng/mL. However, the wide urinary detection window of the long-term metabolite of Delta(9)-tetrahydrocannabinol (THC) does not allow a conclusion to be drawn regarding the time of consumption or the impact on the physical performance. The purpose of the present study on light cannabis smokers was to evaluate target analytes with shorter urinary excretion times. Twelve male volunteers smoked a cannabis cigarette standardized to 70 mg THC per cigarette. Plasma and urine were collected up to 8 h and 11 days, respectively. Total THC, 11-hydroxy-Delta(9)-tetrahydrocannabinol (THC-OH), and THC-COOH were determined after hydrolysis followed by solid-phase extraction and gas chromatography/mass spectrometry. The limits of quantitation were 0.1-1.0 ng/mL. Eight puffs delivered a mean THC dose of 45 mg. Plasma levels of total THC, THC-OH, and THC-COOH were measured in the ranges 0.2-59.1, 0.1-3.9, and 0.4-16.4 ng/mL, respectively. Peak concentrations were observed at 5, 5-20, and 20-180 min. Urine levels were measured in the ranges 0.1-1.3, 0.1-14.4, and 0.5-38.2 ng/mL, peaking at 2, 2, and 6-24 h, respectively. The times of the last detectable levels were 2-8, 6-96, and 48-120 h. Besides high to very high THC-COOH levels (245 +/- 1,111 ng/mL), THC (3 +/- 8 ng/mL) and THC-OH (51 +/- 246 ng/mL) were found in 65 and 98% of cannabis-positive athletes' urine samples, respectively. In conclusion, in addition to THC-COOH, the pharmacologically active THC and THC-OH should be used as target analytes for doping urine analysis. In the case of light cannabis use, this may allow the estimation of more recent consumption, probably influencing performance during competitions. However, it is not possible to discriminate the intention of cannabis use, i.e., for recreational or doping purposes. Additionally, pharmacokinetic data of female volunteers are needed to interpret cannabis-positive doping cases of female athletes.

Unusual mass spectrometric dissociation pathway of protonated isoquinoline-3-carboxamides due to multiple reversible water adduct formation in the gas phase.

The study of the collision-induced dissociation behavior of various substituted isoquinoline-3-carboxamides, which are amongst a group of drug candidates for the treatment of anemic disorders (e.g., FG-2216), allowed for the formulation of the general mechanisms underlying the unusual fragmentation behavior of this class of compounds. Characterization was achieved with high-resolution/high accuracy LTQ-Orbitrap tandem mass spectrometry of the protonated precursor ions. Presented data were substantiated by the synthesis and analysis of proposed gas-phase intermediate structures and stable isotope labeled analogues, as well as by density functional theory calculations. Exemplary, CID of protonated N-[(1-chloro-4-hydroxy-7-isopropoxy-isoquinolin-3-yl)carbonyl]glycine gives rise to the isoquinoline-3-carboxy-methyleneamide product ion which nominally eliminates a fragment of 11 u. This was attributed to the loss of methyleneamine (-29 u) and a concomitant spontaneous and reversible water addition (+18 u) to the resulting acylium ion to yield the protonated isoquinoline-3-carboxylic acid. The same water addition pattern is observed after loss of propylene (-42 u). A further nominal loss of 10 u is explained by the elimination of carbon monoxide (-28 u) followed by another water adduct formation (+18 u) to yield the protonated 1-chloro-3,4,7-trihydroxy-isoquinoline. The source of the multiple gas-phase water addition remained unclear. This atypical fragmentation pattern proved to be highly characteristic for all studied and differentially substituted isoquinoline-3-carboxamides, and offers powerful analytical tools for the establishment of a LC/MS(/MS) based screening procedure for model HIF-stabilizers and their potential metabolites in clinical, forensic and sports drug testing.

Determination of Synacthen in urine for sports drug testing by means of nano-ultra-performance liquid chromatography/tandem mass spectrometry.

Doping control analysis of performance-enhancing peptides in urine represents a challenging requirement in modern sports drug testing. Low dosing, effective metabolism and short half-life lead to target concentrations in the low fmol/mL range in urine. Synthetic adrenocorticotropic hormone (1-24, Syn-ACTH-en) shares all these characteristics and improved analytical performance is required for its sufficient determination by means of liquid chromatography/tandem mass spectrometry (LC/MS/MS). The desired effects for cheating sportsmen are mainly due to enhanced release of corticosteroids as well as androgenic steroids into the circulation after systemic administration of the drug. Immunoaffinity purification with coated magnetic beads and subsequent liquid chromatography with nano-ultra-performance liquid chromatography (UPLC) coupled to tandem mass spectrometry (high resolution/high mass accuracy) of Synacthen from urinary specimens is described in the present study. The general proof of principle was obtained by analysis of excretion study urine samples and validation was performed with focus on the limit of detection (3 pg/mL), linearity, precision (<20%), recovery ( approximately 30%), robustness, specificity and stability. For all experiments, the ACTH fragment 1-17 was used as the internal standard.

Temporal indication of cannabis use by means of THC glucuronide determination.

According to the regulations of the World Anti-Doping Agency (WADA), the use of cannabinoids is forbidden in competition. In doping controls, the detection of cannabinoid misuse is based on the analysis of the non-psychoactive metabolite 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (carboxy-THC). The determination of values greater than 15 ng/mL in urine represents an adverse analytical finding; however, no accurate prediction of the time of application is possible as the half-life of carboxy-THC ranges between three and four days. Consequently the detection of carboxy-THC in doping control urine samples collected in competition might also result from cannabis use in out-of-competition periods. The analysis of the glucuronide of the pharmacologically active delta 9-tetrahydrocannabinol (THC-gluc) may represent a complementary indicator for the detection of cannabis misuse in competition.An assay for the determination of THC-gluc in human urine was established. The sample preparation consisted of liquid-liquid extraction of urine specimens, and extracts were analysed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Authentic doping-control urine samples as well as specimens obtained from a controlled smoking study were analysed and assay characteristics such as specificity, detection limit (0.1 ng/mL), precision (>90%), recovery ( approximately 80%), and extraction efficiency (90%) were determined.

Detection of the arylpropionamide-derived selective androgen receptor modulator (SARM) S-4 (Andarine) in a black-market product.

Non-steroidal and tissue-selective anabolic agents such as selective androgen receptor modulators (SARMs) represent a promising class of therapeutics for the treatment of various diseases such as sarcopenia or cancer cachexia. Advanced compounds of SARMs are based on an arylpropionamide-derived structure and leading drug candidates have successfully completed phase-II-clinical trials. Although none of these therapeutics have been approved, their performance-enhancing qualities and the black-market availability of these products makes them a viable target for misuse in the athletic community. In 2008, SARMs were added to the Prohibited List established by the World Anti-Doping Agency (WADA). That SARMs are the subject of misuse even without clinical approval was proved for the first time by the detection of the drug candidate Andarine (also referred to as S-4, S-3-(4-acetylamino-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamide), advertised, sold and supplied via the Internet. The oily liquids, declared as green tea extracts and face moisturizer, were assayed using state-of-the-art analytical procedures and S-4 was found at concentrations of approximately 150 mg/mL. The authenticity of the product was demonstrated in comparison to reference material by liquid chromatography, high resolution/high accuracy (tandem) mass spectrometry using positive and negative electrospray ionization, and comparison to reference material. Moreover, an impurity resulting from poor product purification was detected, accounting for approximately 10% of S-4. This consisted of 2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-3-(4-nitro-3-trifluoromethyl-phenylamino)-propionamide. The ease of purchasing non-approved drug candidates that could potentially increase athletic performance demonstrates the need to operate proactively in the continued fight against doping. The early inclusion of emerging drugs into routine sports drug testing procedures is a key element of preventive doping research, limiting the options for cheating athletes who aim to undermine the doping control system.

Mass spectrometric determination of gonadotrophin-releasing hormone (GnRH) in human urine for doping control purposes by means of LC-ESI-MS/MS.

The decapeptide gonadotrophin-releasing hormone (GnRH) is endogenously produced in the hypothalamus and secreted into the microcirculation between hypothalamus and pituitary gland. Here, the bioactive hormone is responsible for the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) into the systemic circulation. Because an intermittent application of exogenous GnRH in young males increases the testosterone plasma level by stimulation of the Leydig cells, the potential misuse of the administered substance offers a reasonable relevancy for doping controls and is prohibited in accordance to the list of banned substances of the World Anti-Doping Agency (WADA). The presented method provides a mass spectrometric approach to determine the nondegraded hormone in regular doping control samples by utilizing a sample preparation procedure with solid phase extraction, immunoaffinity purification and a subsequent separation by liquid chromatography with ESI-MS/MS detection. For liquid chromatography/mass spectrometry two alternative instrumental equipments were tested: the first consisted of an Agilent 1100 liquid chromatograph coupled to an Applied Biosystem Q Trap 4000 mass spectrometer, the second equipment was assembled by a Waters Aquity nano-UPLC coupled to a Thermo LTQ Orbitrap high resolution/high accuracy mass spectrometer. In urine specimens provided from healthy volunteers GnRH was not detected in accordance to the recent literature, but in postadministration samples urinary concentrations between 20 to 100 pg/ml of the intact peptide were determined. The method offered good validation results considering the parameter specificity, linearity (5-300 pg/ml), limit of detection (LOD, approx. 5 pg/ml), precision (inter/intraday, < 20%) and accuracy (105%) using Des-pGlu(1)-GnRH as internal standard to control each sample preparation step.

Determination of benzimidazole- and bicyclic hydantoin-derived selective androgen receptor antagonists and agonists in human urine using LC-MS/MS.

Selective androgen receptor modulators (SARMs) represent a novel class of drugs with tissue-specific agonistic and antagonistic properties, which are prohibited in sports from January 2008 according to the World Anti-Doping Agency. Preventive approaches to restrict the use of SARMs include early implementation of target analytes into doping control screening assays. Five model SARMs were synthesized, four of which are analogs to prostate-specific androgen receptor antagonists with a 5,6-dichloro-benzimidazole nucleus. The fifth SARM is a muscle-tissue specific agonist with a bicyclic hydantoin structure (BMS-564929). Dissociation pathways after negative electrospray ionization were studied using an LTQ-Orbitrap mass analyzer, and diagnostic product ions and common fragmentation patterns were employed to establish a screening procedure that target the intact SARMs as well as putative metabolic products. Sample preparation based on solid-phase extraction and subsequent LC-MS/MS measurement allowed for detection limits of 1-20 ng/mL, intra- and interday precisions of between 2.4 and 13.2% and between 6.5 and 24.2%, respectively. Recoveries varied from 89 to 106%, and tests for ion suppression or enhancement effects were negative for all analytes. [figure: see text]

Mass spectrometry of hydantoin-derived selective androgen receptor modulators.

N-Aryl-hydroxybicyclohydantoins represent a new class of tissue-selective anabolic agents [selective androgen receptor modulators (SARMs)] and are promising therapeutics as well as drugs prohibited in amateur and professional sport. The dissociation behavior after negative and positive electrospray ionization (ESI) and subsequent collision-induced dissociation (CID) was studied with a drug candidate (BMS 564929) as well as structurally related and isotope-labeled analogs using high resolution/high accuracy orbitrap mass spectrometry. Positive ionization and CID yielded characteristic product ions resulting from the cleavage of the hydantoin structure providing information about the proline-derived nucleus as well as the substituted aryl residue at m/z 96 and 193, respectively. Negative ESI and CID (MS/MS) yielded product ions mainly representing losses of water and CO(2), the latter of which is of particular significance as the hydantoin structure does not contain a carboxyl function. Employing MS(n) experiments with accurate mass determination on six model SARMs, dissociation pathways to characteristic product ions were proposed supporting the identification of these drugs, their metabolites or related compounds in future doping control assays.

Aryl-propionamide-derived selective androgen receptor modulators: liquid chromatography-tandem mass spectrometry characterization of the in vitro synthesized metabolites for doping control purposes.

Selective androgen receptor modulators (SARM) are a prominent group of compounds for being misused in sports owing to their advantageous anabolic properties and reduced side effects. To target the preventive doping control analysis in relevant compounds, the challenge is to predict the metabolic fate of a new compound. For aryl-propionamide-derived SARM, an in vitro assay employing microsomal and S9 human liver enzymes was developed to simulate phase-I and phase-II metabolic reactions. In vitro metabolic profiles and the structure-metabolic relationship were compared between four structurally modified substrates. Accurate mass measurements were used to characterize the synthesized metabolites, and also collision-induced dissociation was examined to suggest the methodological approach to monitor the prohibited use of aryl-propionamide-derived drug candidates. Subsequent phase-I and phase-II metabolic reactions were successfully combined in one in vitro assay. The main routes of phase-I modifications involved the hydrolysis of ether linkage, monohydroxylation, and hydrolytic cleavage of the amide bond. Nitro-reduction and deacetylation were reactions observed for substrates possessing the corresponding functionality. SARM metabolites were analyzed in negative ion electrospray ionization and detected as deprotonated species [M-H](-). The main metabolic modifications were observed to occur in the B-ring side, and collision-induced dissociation resulted in the product ions originating from the A-ring side of the compound. These structure-specific ions may be monitored as target ions in the routine doping control.

The public perception of doping in sport in Switzerland, 1995-2004.

The article reports findings on the perception of doping and anti-doping policies from four representative population surveys carried out in 1995, 1998, 2001, and 2004, as well as from a 2005 - 2006 survey of top-level athletes in Switzerland. The results show a growing public awareness for doping issues and increasing support for a comprehensive anti-doping strategy in Switzerland. The vast majority of the Swiss population and top-level athletes are strongly against doping and support a strategy that combines strict prohibition and sanctioning with informational and educational efforts. The perception of the doping issue and the strategic preferences in fighting doping stated by the public are largely in line with the current anti-doping strategy followed by the Swiss authorities. The results thus suggest a successful use of information resources by the authorities to create public awareness and to communicate its strategy.

Screening for 2-quinolinone-derived selective androgen receptor agonists in doping control analysis.

Selective androgen receptor modulators (SARMs) represent a class of emerging drugs with high potential for misuse in sports, and therefore members of this group are banned as anabolic agents by the World Anti-Doping Agency. Preventive approaches to restrict their use include early implementation of target analytes into doping control screening assays and evaluation of the mass spectrometric behavior of these drugs to allow their unequivocal identification as well as the characterization of structurally related compounds and metabolic products. Four model SARMs with the 6-alkylamino-2-quinolinone structure, including the advanced drug candidate LGD-2226, were synthesized. Fragmentation pathways after positive electrospray ionization and collision-induced dissociation were studied using an LTQ Orbitrap mass analyzer, and diagnostic product ions and common dissociation pathways were employed to establish a screening procedure targeting intact quinolinone-based SARMs as well as putative metabolic products such as dealkylated analogues. Therefore, features of a triple quadrupole mass analyzer such as multiple reaction monitoring and precursor ion scanning were utilized. Sample preparation based on commonly employed liquid-liquid extraction and subsequent liquid chromatographic/tandem mass spectrometric measurement allowed for detection limits of 0.01-0.2 ng/mL, and intra- and interday precisions between 3.2 and 8.5% and between 6.3 and 16.6%, respectively. Recoveries varied from 81 to 98%, and tests for ion suppression or enhancement effects were negative for all analytes.

Possible origins of undetectable EPO in urine samples.

BACKGROUND: In order to determine the possible origins of undetectable EPO profiles in athletes' urine, we analyzed the data obtained from a large number of official anti-doping urine tests aimed at detecting recombinant erythropoietin. The following variables were considered as potential causes for lack of EPO detection: athlete's gender, competition effect, urine specific gravity as well as possible usage of proteasic adulterants to evade doping detection. RESULTS: Statistical analyses indicated that undetectable EPO profiles were clearly related to urine properties such as low EPO concentrations or extreme specific gravities. The addition of very small quantities of protease was shown to remove all traces of EPOs in urine. This finding led to the development of a simple, specific and sensitive test that reveals proteasic activity based on albumin digestion. CONCLUSIONS: Urine characteristics clearly affect the detectability of an EPO profile. At the same time, addition of anti-proteases prevents the adulteration of urine. These two findings have clear practical implications with regards to the timing of urine collection as well as the entire anti-doping control procedure.

Bayesian detection of abnormal values in longitudinal biomarkers with an application to T/E ratio.

We developed a test that compares sequential measurements of a biomarker against previous readings performed on the same individual. A probability mass function expresses prior information on interindividual variations of intraindividual parameters. Then, the model progressively integrates new readings to more accurately quantify the characteristics of the individual. This Bayesian framework generalizes the two main approaches currently used in forensic toxicology for the detection of abnormal values of a biomarker. The specificity is independent of the number n of previous test results, with a model that gradually evolves from population-derived limits when n = 0 to individual-based cutoff thresholds when n is large. We applied this model to detect abnormal values in an athlete's steroid profile characterized by the testosterone over epitestosterone (T/E) marker. A cross-validation procedure was used for the estimation of prior densities as well as model validation. The heightened sensitivity/specificity relation obtained on a large data set shows that longitudinal monitoring of an athlete's steroid profile may be used efficiently to detect the abuse of testosterone and its precursors in sports. Mild assumptions make the model interesting for other areas of forensic toxicology.