Identification of erythropoietin mimetic peptide 1 linear form in a sealed vial and its administration study in horses for doping control purpose.

The erythropoietin mimetic peptide 1 linear form (EMP1-linear), GGTYSCHFGPLTWVCKPQGG-NH2 , was identified in an unknown preparation consisting of white crystalline powder contained in sealed glass vials using ultrahigh performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). The white crystalline powder, allegedly used for doping racehorses, was found to contain around 2% (w/w) of EMP1-linear. EMP1-linear can be cyclised in equine plasma at physiological temperature of 37°C by forming an intramolecular disulfide bond to give EMP1, which is a well-known erythropoiesis stimulating agent that can bind to and activate the receptor for cytokine erythropoietin (EPO). Thus, EMP1-linear is a prodrug of EMP1, which is a performance-enhancing doping agent that can be misused in equine sports. In order to identify potential target(s) for detecting the misuse of EMP1-linear in horses, an in vitro metabolic study using horse liver S9 fraction was performed. After incubation, EMP1-linear mainly existed in its cyclic form as EMP1, and four N-terminus truncated in vitro metabolites TYSCHFGPLTWVCKPQGG-NH2 (M1), SCHFGPLTWVCKPQGG-NH2 (M2), WVCKPQGG-NH2 (M3) and VCKPQGG-NH2 (M4) were identified. An intravenous administration study with the preparation of white crystalline powder containing EMP1-linear was also conducted using three retired thoroughbred geldings. EMP1 was detectable only in the postadministration plasma samples, whereas the four identified in vitro metabolites were detected in both postadministration plasma and urine samples. For controlling the misuse of EMP1-linear in horse, its metabolite M3 gave the longest detection time in both plasma and urine and could be detected for up to 4 and 27 h postadministration, respectively.

High-throughput untargeted screening of biotherapeutic macromolecules in equine plasma by UHPLC-HRMS/MS: Application to monoclonal antibodies and Fc-fusion proteins for doping control.

Many innovative biotherapeutics have been marketed in the last decade. Monoclonal antibodies (mAbs) and Fc-fusion proteins (Fc-proteins) have been developed for the treatment of diverse diseases (cancer, autoimmune diseases, and inflammatory disorders) and now represent an important part of targeted therapies. However, the ready availability of such biomolecules, sometimes characterized by their anabolic, anti-inflammatory, or erythropoiesis-stimulating properties, raises concerns about their potential misuse as performance enhancers for human and animal athletes. In equine doping control laboratories, a method has been reported to detect the administration of a specific human biotherapeutic in equine plasma; but no high-throughput method has been described for the screening without any a priori knowledge of human or murine biotherapeutic. In this context, a new broad-spectrum screening method involving UHPLC-HRMS/MS has been developed for the untargeted analysis of murine or human mAbs and related macromolecules in equine plasma. This approach, consisting of a "pellet digestion" strategy performed in a 96-well plate, demonstrates reliable performances at low concentrations (pmol/mL range) with high-throughput capability (≈100 samples/day). Targeting species-specific proteotypic peptides located within the constant parts of mAbs enables the "universal" detection of human biotherapeutics only by monitoring 10 peptides. As proof of principle, this strategy successfully detected different biotherapeutics in spiked plasma samples, and allowed, for the first time, the detection of a human mAb up to 10 days after a 0.12 mg/kg administration to a horse. This development will expand the analytical capabilities of horse doping control laboratories towards protein-based biotherapeutics with adequate sensitivity, throughput, and cost-effectiveness.

Optimization and implementation of four duplex quantitative polymerase chain reaction assays for gene doping control in horseracing.

The concern about gene doping has remained high in horseracing and other equestrian competitions. Our laboratory has previously developed a duplex quantitative polymerase chain reaction (qPCR) assay capable of detecting in equine blood the human erythropoietin (hEPO) transgene and equine tubulin α 4a (TUBA4A) gene as an internal control the latter providing quality control over DNA extraction and qPCR. This study aimed to optimize the method for routine testing of regulatory samples. The use of an automated DNA extraction system has increased the sample throughput, consistency of DNA extraction, and recovery of reference materials. The use of reduced concentration of primers and hydrolysis probe for internal control minimized their competition with transgene amplification and improved the assay sensitivity. Spike-in of an exogenous internal control at low concentration for plasma analysis has also been validated. Using the new workflow, four duplex qPCR assays have been developed for the detection of transgenes, namely, hEPO, human growth hormone (hGH), insulin-like growth factor 1 (hIGF-1), and equine EPO (eEPO). The estimated limits of detection (LODs) of each transgene were 2000 copies/mL of blood and 200 copies/mL of plasma. This method could detect the presence of transgene in blood and plasma collected from a horse administered intramuscularly (IM) with recombinant adeno-associated virus (rAAV) carrying the hEPO transgene. A longer detection time was observed in blood than in plasma. The methods have been applied to the screening of over a thousand official racehorse samples since June 2020 for the presence of these transgenes.

Comprehensive metabolic study of nicotine in equine plasma and urine using liquid chromatography/high-resolution mass spectrometry for the identification of unique biomarkers for doping control.

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

Comprehensive metabolic study of IOX4 in equine urine and plasma using liquid chromatography/electrospray ionization Q Exactive high-resolution mass spectrometer for the purpose of doping control.

IOX4 is a hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) inhibitor, which was developed for the treatment of anemia by exerting hematopoietic effects. The administration of HIF-PHD inhibitors such as IOX4 to horses is strictly prohibited by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale. To the best of our knowledge, this is the first comprehensive metabolic study of IOX4 in horse plasma and urine after a nasoesophageal administration of IOX4 (500 mg/day, 3 days). A total of four metabolites (three mono-hydroxylated IOX4 and one IOX4 glucuronide) were detected from the in vitro study using homogenized horse liver. As for the in vivo study, post-administration plasma and urine samples were comprehensively analyzed with liquid chromatography/electrospray ionization high-resolution mass spectrometry to identify potential metabolites and determine their corresponding detection times. A total of 10 metabolites (including IOX4 glucuronide, IOX4 glucoside, O-desbutyl IOX4, O-desbutyl IOX4 glucuronide, four mono-hydroxylated IOX4, N-oxidized IOX4, and N-oxidized IOX4 glucoside) were found in urine and three metabolites (glucuronide, glucoside, and O-desbutyl) in plasma. Thus, the respective quantification methods for the detection of free and conjugated IOX4 metabolites in urine and plasma with a biphase enzymatic hydrolysis were developed and applied to post-administration samples for the establishment of elimination profiles of IOX4. The detection times of total IOX4 in urine and plasma could be successfully prolonged to at least 312 h.

Sequence determination of phosphorothioated oligonucleotides using MALDI-TOF mass spectrometry for controlling gene doping in equestrian sports.

In human and equestrian sporting events, one method of gene doping is the illegal use of therapeutic oligonucleotides to alter gene expression. In this study, we aimed to identify therapeutic oligonucleotides via sequencing using matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). As a model of therapeutic oligonucleotides, 22 bp-long phosphorothioated oligonucleotides (PSOs) were used. By using a Clarity OTX kit for extracting short-length oligonucleotides, a spectrum of singly charged PSO with a mean intensity of 6.08 × 104 (standard deviation: 4.34 × 103 ) was detected from 500 pmol PSO in 1 ml horse plasma using the linear negative mode of MALDI-TOF MS. In addition, a 17 bp sequence was determined using in-source decay (ISD) mode, indicating that 500 pmol of a PSO in 1 ml plasma is the detection limit for sequencing. Using the determined sequences (17 bp), a targeted gene for PSO was singly identified on the horse reference genome, EquCab2.0, via a GGGenome search. These procedures can be potentially used to identify therapeutic oligonucleotides, whose nucleotides are unknown, for gene doping control.

A fast screening method for the detection of CERA in dried blood spots.

Continuous erythropoietin receptor activator (CERA) is a third-generation erythropoiesis-stimulating agent that was developed for the treatment of anemia. However, misuse of CERA for doping in endurance sports has been reported. Previous studies have shown blood as the matrix of choice for the detection of CERA, due to its high molecular weight. The use of dried blood spots (DBSs) for anti-doping purposes constitutes a complementary approach to the standard urine and venous blood matrices and could facilitate sample collection and increase the number of blood samples available for analysis due to reduced costs of sample collection and transport. Here, we investigated whether CERA could be indirectly detected in extracts of single DBSs using an erythropoietin-specific immunoassay that is capable of providing results within approximately 2 h. Reconstituted DBS samples were prepared from mixtures of red blood cell pellets and serum samples. The samples were collected in a previous clinical study in which six healthy volunteers were injected with a single, 200 µg dose of CERA. Using a commercially available ELISA kit, CERA was detected in the DBSs with a detection window of up to 20 days post-injection. Furthermore, in order to demonstrate the fitness-for-purpose, three authentic doping control serum samples, which were identified as containing CERA, were analyzed by the presented methodological approach on DBS. The testing procedure described here could be used as a fast and cost-effective method for the detection of CERA abuse in sport.

Sports drug testing and the athletes' exposome.

Similar to the general population, elite athletes are exposed to a complex set of environmental factors including chemicals and radiation and 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 antidoping 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 antidoping 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.

Multiplexed detection of agents affecting erythropoiesis (AAEs) and overall strategy for optimizing initial testing procedure.

Erythropoietin receptor agonists (ERAs) are drugs acting on the early erythropoietic stages developed to treat anemia and other erythropoiesis disease and are prohibited by the World Anti-Doping Agency (WADA). As an alternative to ERAs, a new drug, belonging to the transforming growth factor-b inhibitors family, was recently developed to treat diseases linked to ineffective erythropoiesis. This drug, named as Luspatercept (Reblozyl®), is acting on the later stages of erythropoiesis to promote erythrocytes. This drug might be used by cheating athletes either independently or in combination with ERAs. Indeed, it was shown that Luspatercept and recombinant erythropoietin (rEPO) can act synergistically to increase red blood cells production, potentially allowing the use of lower doses for an efficient effect. Our aim was to find a way to combine the detection of ERAs and Luspatercept without impacting the sensitivity and specificity of ERAs detection from the current techniques implemented in antidoping laboratories and to reduce the time of analysis and total sample volume needed. Magnetic beads coated with antibodies were preferred for IP of samples for its potential multiplexing. Then, the following steps of the method were selected considering that SAR/SDS-PAGE are the electrophoretic methods authorized for initial testing procedure by WADA and that biotinylated primary antibodies used for the immunodetection results in the best sensitivity and specificity and is time saving. The method developed in this work for the combined detection of agents affecting erythropoiesis (AAEs) showed specificity, sensitivity, and robustness and is easily and quickly implementable to all antidoping laboratories.

Liquid chromatography-mass spectrometry behavior of Girard's reagent T derivatives of oxosteroid intact phase II metabolites for doping control purposes.

Intact phase II steroid metabolites have poor product ion mass spectra under collision-induced dissociation (CID) conditions. Therefore, we present herein the liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/(MS)) behavior of intact phase II metabolites of oxosteroids after derivatization. Based on the fact that Girard's reagent T (GRT), as derivatization reagent, was both convenient and efficient in terms of the enhancement in the ionization efficiency and the production of diagnostic product ions related to the steroid moiety, the latter was preferably selected between methoxamine and hydroxylamine upon the model compounds of androsterone glucuronide and androsterone sulfate. Sixteen different glucuronides and 29 sulfate conjugated metabolites of anabolic androgenic steroids (AASs), available either as pure reference materials or synthesized/extracted from administration studies, were derivatized with GRT, and their product ion spectra are presented. Product ion spectra include in all cases high number of product ions that in some cases are characteristic for certain structures of the steroid backbone. More specifically, preliminary results have shown major differences in fragmentation pattern for 17α/17ß-isomers of the sulfate conjugates, but limited differentiation for 17α/17ß-isomers of glucuronide conjugates and for 3α/3ß- and 5α/5ß-stereoisomers of both sulfate and glucuronide conjugates. Further to the suggestion of the current work, application on mesterolone administration studies confirmed-according to the World Anti-Doping Agency (WADA) TD2015IDCR-the presence of seven intact phase II metabolites, one glucuronide and six sulfates with use of LC-ESI-MS/(MS).

Analysis of seized peptide and protein-based doping agents using four complimentary methods: Liquid chromatography coupled with time of flight mass spectrometry, liquid chromatography-ultraviolet, Bradford, and immunoassays.

Analysis and identification of seized doping-related products are important tasks for customs or forensic laboratories in order to prevent potentially dangerous and illegal compounds to go into circulation. At the Section of Forensic Chemistry in Copenhagen, we have a workflow consisting of four complimentary validated methods to identify common doping-related substances: liquid chromatography-ultraviolet (LC-UV), LC coupled with time of flight mass spectrometry (LC-TOF-MS), the colorimetric Bradford assay, and an immunoassay. The Bradford assay screens for peptide or proteins in the sample, and the immunoassay confirmed human chorionic gonadotropin (hCG). LC-UV was carried out with a C4 protein column for identification of peptides and proteins from a standard reference library, based on retention times and ratios between peak areas at 220, 254, and 280 nm. LC-TOF-MS was performed using a C18 column, and identification was based on comparison of the retention time and the accurate mass with those of reference standards. In 2019, we received 36 samples for peptide/protein analysis, all of which were tested using the LC-UV, LC-TOF-MS, and colorimetric method, and samples suspected of containing hCG were confirmed with an immunoassay. We found a total of 15 samples containing an illegal doping substance, 12 samples containing substances not prohibited by the Danish Doping List, and nine samples containing no peptides or proteins. In conclusion, the four complimentary methods constitute a suitable approach for identifying common peptide/protein doping substances in the day-to-day routine of a forensic laboratory, with limited sample preparation and interpretation of data.

Cell culture as a toolbox to generate phase I metabolites for antidoping screening.

The knowledge of the biotransformation of compounds prohibited by the World Anti Doping Agency is of high concern as doping analyses are mostly based on the detection of metabolites instead of the parent compounds abused by athletes. While the self-administration of doping-relevant compounds is from an ethical point of view a rather problematic method to investigate metabolism, the usage of cell culture systems allows for studies on biotransformation in vitro. Five cell culture models with different tissue origin (liver, ovary, skin, kidney, and testis) were comparatively incubated with testosterone and epitestosterone as well as with the synthetic testosterone derivatives 17α-methyltestosterone and 4-chlorotestosterone to investigate the impact of synthetic modifications on phase I metabolic pathways. Cell culture supernatants were analyzed by high-performance liquid chromatography-tandem mass spectrometry. All cell lines possessed the default steroid phase I biotransformation reactions. The highest conversion rate was observed in ovarian (BG-1) and liver cells (HepG2). For BG-1 and skin cells (HaCaT), the 5α-reductase products 5α-dihydrotestosterone (for both) and 5α-androstane-3α/ß,17ß-diol (for BG-1 solely) were found to be prevailing after testosterone incubation. In kidney (COS-1) and HepG2 cells, the 17ß-hydroxysteroid dehydrogenase activity was predominant as supported by the observation that the 17α-OH (epitestosterone) and the methyl group (17α-methyltestosterone) impeded the conversion rate in these cell lines. In conclusion, future work should extend the characterization of the BG-1 and HepG2 cells on phase II metabolic pathways to examine whether they are suitable models for the generation of metabolite reference collections comparable to those obtained by human excretion studies.

Detecting the abuse of 19-norsteroids in doping controls: A new gas chromatography coupled to isotope ratio mass spectrometry method for the analysis of 19-norandrosterone and 19-noretiocholanolone.

The detection of 19-norsteroids abuse in doping controls currently relies on the determination of 19-norandrosterone (19-NA) by gas chromatography-tandem mass spectrometry (GC-MS/MS). An additional confirmatory analysis by gas chromatography coupled to isotope ratio mass spectrometry (GC-C-IRMS) is performed on samples showing 19-NA concentrations between 2.5 and 15 ng/ml and not originated from pregnant female athletes or female treated with 19-norethisterone. 19-Noretiocholanolone (19-NE) is typically produced to a lesser extent as a secondary metabolite. The aim of this work was to improve the GC-C-IRMS confirmation procedure for the detection of 19-norsteroids misuse. Both 19-NA and 19-NE were analyzed as target compounds (TCs), whereas androsterone (A), pregnanediol (PD), and pregnanetriol (PT) were selected as endogenous reference compounds (ERCs). The method was validated and applied to urine samples collected by three male volunteers after the administration of nandrolone-based formulations. Before the instrumental analysis, urine samples (<25 ml) were hydrolyzed with ß-glucuronidase from Escherichia coli and extracted with n-pentane. Compounds of interest were purified through a single (for PT) or double (for 19-NE, 19-NA, A, and PD) liquid chromatographic step, to reduce the background noise and eliminate interferences that could have affect the accuracy of δ13 C values. The limit of quantification (LOQ) of 2 ng/ml was ensured for both 19-NA and 19-NE. The 19-NE determination could be helpful in case of "unstable" urine samples, in late excretion phases or when coadministration with 5α-reductase inhibitors occur.

Detecção de esteroides anabólicos androgênicos e seus produtos de biotransformação em dried urine spots por ionização por paper spray acoplada à espectrometria de massas (PS-MS) / Detection of anabolic androgenic steroids and its biotransformation products in dried urine spots by paper spray-mass spectrometry ionization (PS-MS)

Os esteroides anabólicos androgênicos (EAA) são utilizados clinicamente para tratar diferentes doenças, porém propagou-se o uso não terapêutico por atletas de elite e fisiculturistas, com o intuito de aumentar a massa muscular e melhorar o desempenho físico. O uso de substâncias ergogênicas, como fármacos estimulantes e narcóticos analgésicos no esporte, foi proibido em 1967 pelo International Olympic Committee (COI), mas somente em 1976 os EAA entraram para a lista de substâncias proibidas. O uso de EAA está associado a diversos efeitos adversos, principalmente cardiovasculares, neuroendócrinos e distúrbios psiquiátricos, além de dislipidemia, elevação dos marcadores inflamatórios e disfunção endotelial. As análises toxicológicas constam como a maneira mais eficaz de minimizar o doping no esporte. O material é fornecido pelo atleta durante as competições ou treinamentos e previne que os competidores alcancem vantagem competitiva devido ao uso de EAA. A utilização de métodos para amostragem alternativos tem ganhado força, devido à necessidade de técnicas mais práticas que utilizam pouco volume de amostra e possuem facilidade de armazenamento. O dried urine spots é um método no qual pequenas amostras de urina são aplicadas em papéis de filtro para análises qualitativas ou quantitativas. Ele se caracteriza por ser uma técnica rápida, fácil, simples e barata para a coleta, armazenamento e distribuição, além de minimizar os riscos de infecção, podendo ser utilizado na rotina. A técnica de paper spray (PS-MS) foi desenvolvida a partir da relevância de métodos como o dried blood spots por proporcionar análises mais rápidas, apresenta alta especificidade, capacidade de analisar diferentes analitos simultaneamente, baixo limite de detecção e dispensa a necessidade de reagentes específicos. Sendo assim, neste trabalho foi desenvolvido e validado o método de screening de EAAs em dried urine spots por ionização por paper spray acoplada à espectrometria de massas. O método apresentou limites de detecção entre 2-15ng/mL e presença de três interferentes endógenos. Os dez analitos de interesse deste estudo são estáveis por 150 dias em temperatura ambiente. Dessa forma, a análise de EAAs em dried urine spots por PS-MS demonstra grande potencial para se tornar um método alternativo no monitoramento rápido de drogas de abuso

Anabolic androgenic steroids (AAS) are used clinically to treat different diseases, but non-therapeutic use has spread among elite athletes and bodybuilders, with the aim of increase muscle mass and improve physical performance. The International Olympic Committee (IOC) banned the use of ergogenic substances, such as stimulating drugs and analgesic narcotics in sports, in 1967, but only in 1976, AAS were included on the list of prohibited substances. The use of AAS is associated with several adverse effects, mainly cardiovascular, neuroendocrine and psychiatric disorders, in addition to dyslipidemia, elevated inflammatory markers and endothelial dysfunction. Toxicological analyzes are the most effective approach to minimize doping in sport. The material is provided by the athlete during competitions or training and prevents competitors from achieving a competitive advantage due to the use of AAs. The use of alternative sampling methods has gained strength, due to the need for more practical techniques that use low sample volume and can be easily storage. Dried urine spots are a method, which a small amount of urine samples is applied to filter papers for qualitative or quantitative analysis. It is characterized by being a fast, easy, simple and inexpensive technique for collection, storage and distribution, in addition to minimizing the risks of infection, and can be used in the routine. The paper spray technique (PS-MS) was developed based on the relevance of methods such as dried blood spots for providing faster analysis, high specificity, ability to analyze different analytes simultaneously, low detection limit and for eliminating the need for specific reagents. Therefore, this work developed and validated a screening method for AAS in dried urine spots by paper spray-mass spectrometry ionization. The method provided detection limits between 2-15ng/mL and the presence of three endogenous interferents. The ten analytes of interest in this study are stable for 150 days at room temperature. Thus, the analysis of AAS in dried urine spots by PS-MS demonstrates great potential to become an alternative method for the rapid monitoring of drugs of abuse

Searching for new long-term urinary metabolites of metenolone and drostanolone using gas chromatography-mass spectrometry with a focus on non-hydrolysed sulfates.

Sulfated metabolites have been shown to have potential as long-term markers of anabolic-androgenic steroid (AAS) abuse. In 2019, the compatibility of gas chromatography-mass spectrometry (GC-MS) with non-hydrolysed sulfated steroids was demonstrated, and this approach allowed the incorporation of these compounds in a broad GC-MS initial testing procedure at a later stage. However, research is needed to identify which are beneficial. In this study, a search for new long-term metabolites of two popular AAS, metenolone and drostanolone, was undertaken through two excretion studies each. The excretion samples were analysed using GC-chemical ionization-triple quadrupole MS (GC-CI-MS/MS) after the application of three separate sample preparation methodologies (i.e. hydrolysis with Escherichia coli-derived ß-glucuronidase, Helix pomatia-derived ß-glucuronidase/arylsulfatase and non-hydrolysed sulfated steroids). For metenolone, a non-hydrolysed sulfated metabolite, 1ß-methyl-5α-androstan-17-one-3ζ-sulfate, was documented for the first time to provide the longest detection time of up to 17 days. This metabolite increased the detection time by nearly a factor of 2 in comparison with the currently monitored markers for metenolone in a routine doping control initial testing procedure. In the second excretion study, it prolonged the detection window by 25%. In the case of drostanolone, the non-hydrolysed sulfated metabolite with the longest detection time was the sulfated analogue of the main drostanolone metabolite (3α-hydroxy-2α-methyl-5α-androstan-17-one) with a detection time of up to 24 days. However, the currently monitored main drostanolone metabolite in routine doping control, after hydrolysis of the glucuronide with E.coli, remained superior in detection time (i.e. up to 29 days).

Quantification and validation of nine nonsteroidal anti-inflammatory drugs in equine urine using gas chromatography-mass spectrometry for doping control.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used in therapeutic doses in human and veterinary medicine for the treatment of inflammation, pain, and fever. A method for the simultaneous determination of nine NSAIDs, known as therapeutic prohibited substances, in equine urine was developed and fully validated according to the European Commission Decision 2002/657/EC and Association of Official Racing Chemists criteria. The validation was performed for naproxen, flunixin, ketoprofen, diclofenac, eltenac, meclofenamic acid, phenylbutazone, vedaprofen, and carprofen in equine urine in accordance with the International Screening Limits (ISL) regulated by International Federation of Horseracing Authorities. After basic hydrolysis, samples were extracted with a C18 cartridge using automated solid-phase extraction. Several derivatization reagents were investigated, and trimethylphenylammonium hydroxide/methanol (20/80, v/v) was selected. Analyses were carried out using gas chromatography-mass spectrometry with selected ion monitoring mode, but the method can be applied to a large number of analytes. The within-laboratory reproducibility was not more than 12.8% (≤15%), and mean relative recoveries ranged from 91.1% to 104.1% for inter-day and intra-day precision. The decision limits (CCα) and detection capabilities (CCß) were evaluated at concentrations near the ISL for each therapeutic substance. The validation results demonstrated that the method is highly reproducible, easily applicable, and suitable for the analysis of some NSAIDs in equine urine that have not been previously published. Finally, the method was also applied to known positive samples.

Development and validation of a fast gas chromatography combustion isotope ratio mass spectrometry method for the detection of epiandrosterone sulfate in urine.

In doping control, to confirm the exogenous origin of exogenously administered anabolic androgenic steroids (AAS), a gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) analysis is performed. Recently published work suggests that epiandrosterone sulfate (EpiAS) is a promising IRMS target compound for the detection of AAS, capable of prolonging the detection window. However, EpiAS is only excreted in urine in its sulfoconjugated form, while all other IRMS target compounds are excreted glucuronidated, meaning that EpiAS cannot be incorporated in the existing IRMS methods. A separate extensive sample preparation needs to be performed on this compound with a different hydrolysis and extraction procedure and a different liquid chromatography (LC) clean-up. The current work presents a new, fast, and easy to implement EpiAS IRMS method. The approach was based on the direct GC analysis of non-hydrolyzed EpiAS, making the solid phase extraction, hydrolysis, and acetylation step redundant. Sample preparation consisted of a simple liquid-liquid extraction, followed by LC fraction collection. A population study was performed to check compliance with the criteria drafted by the World Anti-Doping Agency (WADA). To verify the applicability of the developed approach, the method was applied to the samples of four administration studies (i.e. dehydroepiandrosterone (DHEA), testosterone gel (T gel), androstenedione (ADION), and intramuscular testosterone undecanoate. In contrast to previously published data, the strength of EpiAS as the target compound and the prolongation of the detection window in comparison with the conventional IRMS target compounds was less pronounced.

Mediterranean diet as a natural supplemental resource for athletes and physical activity.

The WHO Global Action Plan on physical activity underlines the binomial "diet and physical activity" for the maintenance of well-being state. The adequate nutritional intake is required for sport and can be achieved by a well-adjusted diet without adding artificial food supplements, whose abuse can even represent a risk and appear as an antechamber of doping. Within a national doping prevention project, a peer education tool was realized in the form of a book and e-book, based on the principle of the Mediterranean Diet as an effective nutritional support in sport and physical activity. This health-literacy book contains recipes from all Italian regions revised for their capability to satisfy sport nutritional needs.

The prevention of doping and the improper use of drugs and food supplements in sports and physical activities: a survey on the activity of the prevention departments of Italian local health authorities.

INTRODUCTION: Doping is an important public health problem widespread not only among elite athletes, but also among amateur and recreational athletes and the general population. In Italy the introduction of doping prevention within the Essential Levels of Care (LEA) with the DPCM 12/1/2017 represents a crucial step towards the implementation of education and health promotion interventions. In this context, the Departments of Prevention (DP) of the Local Health Authorities (LHA) have to play a fundamental role, becoming the cultural and operational reference on this issue. As part of the "Doping prevention: development of a permanent educational tool coordinated by the National Health Service Prevention Departments" project, funded by the Italian Ministry of Health, a survey was conducted on the activities carried out by the DP regarding doping prevention and improper use and abuse of drugs and food supplements in sports and physical activities, as a basis for the harmonization of organizational structures and prevention programs and the creation of a collaboration network at a regional and national level. METHODS: A semi-structured questionnaire consisting of 11 questions, prepared on an electronic platform, was sent to the DP of all the Italian LHA. RESULTS: A total of 38 DP out of 131 (29%) completed the questionnaire, with representation from all regions. 42.1% of DP carried out or are still running programs for the prevention of doping, a percentage that decreases to 27% considering the programs for the prevention of misuse and abuse of drugs and food supplements in sports and in physical activities; in less than half of the DP, 37.5% and 41.7%, respectively, dedicated funds have been allocated. The professionals most involved in prevention of doping are the Specialists in Sport Medicine (81.3%) followed by Specialists in Hygiene (43.8%) and Psychologists (37.5%), while Health Care Assistants (50%) are the professionals most involved in the prevention of the improper use of drugs and food supplements, followed by Specialists in Hygiene and Specialists in Sport Medicine (40%). Most of the DP (71.9%) believe that the introduction of programs to prevent and counteract doping in the LEA will have repercussions on their approach against doping. CONCLUSIONS: The survey, although conducted on a limited sample, has provided an important framework relating to programs for the prevention of doping and the misuse and abuse of drugs and food supplements in sports and in the physical activities carried out by DP. A remarkable heterogeneity has been highlighted, both at national and regional level. It is urgent to provide DP with homogeneous and effective organizational models and adequate operational tools, paying particular attention to the training of all the professionals involved. It is also essential to implement permanent monitoring tools.

Education to legality and doping.

When planning actions to prevent doping in the general population, public health operators may collide against the interests of criminal organizations involved in illicit trafficking of drugs. In addition to technical and professional expertise, or clinical and pharmacological skills, also a deep knowledge of legal and social issues is strongly required to face the problem and assure the effectiveness of the preventive actions. Sports competitions, athletes training or adapted physical activity may all represent conditions and environments at risk for misusing or abusing drugs and dietary supplements. A correct approach to sport and physical activity implies respect of competition rules, attention to own body limits and knowledge of risk factors. Health education campaigns and preventive actions should also consider education to legality in the different settings. The comprehension of the complex net that is available to access doping, locally or globally through online Internet sites, is essential as well as the awareness of the huge economic burden of crime interests behind the illicit trafficking of drugs. A modern whole rounded approach needs to consider doping not only as a violation of sport rules but also of the own body health, representing almost a form of addiction involving individuals and communities, and being supported by crime. Within this frame, doping is considered not just as a sport violation or a risk factor for individual's health, but as a disease of the society, in the society, against the society. A peculiar equilibrium seems to prevail between crime external pressures and resigned internal acceptance, according to the 'mafia hypothesis' model, where hosts accept parasitism to avoid retaliation. Here, main contributes and topics from the Erice 53rd Course are summarized and reviewed, providing links and references for further studies in the field. Health education and education to legality represent two sides of a same question, concerning both the general population and the health authorities. In conclusion, education to legality is a key component for prevention of doping and a priority for public health operators involved in protecting population health.