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.

ABSTRACT: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.For decades, steroidal androgens have been clinically used in the treatment of diseases related to androgen deficiency, including muscle-wasting, osteoporosis, and benign prostate hyperplasia, but recently their suitability for hormone replacement therapy of aging men and regulation of male fertility has been under investigation (NegroVilar, 1999;Gao and Dalton, 2007). Traditional anabolic-androgenic therapies, applying steroid-structured compounds such as testosterone, are often limited due to low oral bioavailability, cross-reactivity with steroid receptors other than the androgen receptor, hepatic toxicity, and other undesirable side effects on the prostate and cardiovascular system (Bhasin and Bremner, 1997). To overcome these drawbacks, a series of nonsteroidal selective androgen receptor modulators (SARM) have been developed. SARM represent enhanced tissue selectivity, binding to the androgen receptor (AR) with affinity similar to testosterone, but exhibiting only partial agonist properties in androgenic tissue. Based on their chemical structure, most prominent pharmacophores of SARM can be categorized at least in four classes: 1) aryl-propionamide, 2) bicyclic hydantoin, 3) quinoline, and 4) tetrahydroquinoline analogs, which all have also entered different stages of clinical studies (Chen et al., 2005a;Thevis and Schänzer, 2007). Among the published data on SARM research, the series of aryl-pr...

Blood doping has been challenging the scientific community since the early 1970's, where it was demonstrated that blood transfusion significantly improves physical performance. Here, we present through 3 applications how statistical classification techniques can assist the implementation of effective tests to deter blood doping in elite sports. In particular, we developed a new indirect and universal test of blood doping, called Abnormal Blood Profile Score (ABPS), based on the statistical classification of indirect biomarkers of altered erythropoiesis. Up to 601 hematological profiles have been compiled in a reference database. Twenty-one of them were obtained from blood samples withdrawn from professional athletes convicted of blood doping by other direct tests. Discriminative training algorithms were used jointly with cross-validation techniques to map these labeled reference profiles to target outputs. The strict cross-validation procedure facilitates the adherence to medico-legal standards mandated by the World Anti Doping Agency (WADA). The test has a sensitivity to recombinant erythropoietin (rhEPO) abuse up to 3 times better than current generative models, independently whether the athlete is currently taking rhEPO or has stopped the treatment. The test is also sensitive to any form of blood transfusion, autologous transfusion included. We finally conclude why a probabilistic approach should be encouraged for the evaluation of evidence in anti-doping area of investigation.

Anabolic agents have been among the most frequently detected drugs in amateur and professional sport. A novel class of therapeutics presumably complementing anabolic steroids in the near future includes so-called selective androgen receptor modulators (SARMs) that have been under clinical investigations for several years. Although not yet commercially available, their potential for misuse in sports is high. Four aryl-propionamide-derived SARMs were synthesized in order to establish a fast and robust screening procedure using liquid chromatography/electrospray ionization tandem mass spectrometry. Synthesized compounds were characterized by high-resolution/high-accuracy mass analysis employing a linear ion trap-Orbitrap hybrid mass spectrometer while routine analyses were conducted on a triple-quadrupole mass spectrometer. Characteristic product ions obtained by collision-induced dissociation were found at m/z 289 and 261 as well as m/z 269 and 241 representing the bisubstituted aniline residues of selected model compounds. Assay validation was performed regarding lower limit of detection (1 ng/mL), recovery (85-105%), intraday precision (7.6-11.6%) and interday precision (9.9-14.4%), and precursor ion scan experiments on diagnostic product ions enabled the detection of a structurally related compound at 50 ng/mL.

The purpose of this study was to analyze the composition of 103 dietary supplements bought on the internet. The supplements were dispatched in four different categories according to their announced contents [creatine, prohormones, “mental enhancers” and branched chain amino acids (BCAA)]. All the supplements were screened for the presence of stimulants and main anabolic steroids parent compounds. At the same time, the research was focused on the precursors and metabolites of testosterone and nandrolone. The study pointed out three products containing an anabolic steroid, metandienone, in a very high amount. The ingestion of such products induced a high quantity of metandienone metabolites in urines that would be considered as a positive antidoping test. The results have also shown that one creatine product and three “mental enhancers” contained traces of hormones or prohormones not claimed on the labels and 14 prohormone products contained substances other than those indicated by the manufacturer. The oral intake of the creatine product revealed the presence of the two main nandrolone metabolites (19‐norandrosterone and 19‐noretiocholanolone) in urine.

We report the findings of the analysis of 75 different nutritional supplements bought through the internet. Seven products (all from the class of prohormones) contained other hormone substances than indicated on the labels, and two further products contained ephedrine and caffeine without a clear indication on the labels.

1H-MR spectra of human skeletal muscle feature peak splittings due to dipolar coupling. Quantitative difference spectroscopy in a double-blind cross-over trial testing oral creatine supplementation revealed that most of the resonances affected by dipolar coupling can be assigned to creatine and/or phosphocreatine. The assignment was performed in two different skeletal muscles and confirmed by measurements at the magic angle where dipolar splittings vanish. Numerical spectral simulations revealed that the observed spectra are consistent with partly-averaged dipolar coupling among methylene and methyl protons of (phospho)creatine. The possible nature of the molecular dynamics leading to incomplete dipolar averaging is discussed.

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.