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In order to overcome the challenge associated with the screening of Anabolic-Androgenic Steroids abuses in animal competitions, a non-targeted liquid chromatography coupled to high resolution mass spectrometry based metabolomics approach was implemented on equine urine samples to highlight potential biomarkers associated with the administration of such compounds, using testosterone esters as model steroids. A statistical model relying on four potential biomarkers intensity could be defined to predict the status of the samples. With a routine application perspective, the monitoring of the highlighted potential biomarkers was first transferred into high-throughput liquid chromatography-selected reaction monitoring (LC-SRM). The model's performances and robustness of the approach were preserved and providing a first demonstration of metabolomics-based biomarkers integration within a targeted workflow using common benchtop MS instrumentation. In addition, with a view to the widespread implementation of such biomarker-based tools, we have transferred the method to a second laboratory with similar instrumentation. This proof of concept allows the development and application of biomarker-based strategies to meet current doping control needs.

Synthetic androgenic anabolic steroids (AAS) are banned compounds and considered as major threats by both racing and sports international authorities. Hence, doping control laboratories are continually looking into analytical improvements to increase their detection capabilities, notably by means of emerging technologies. To enhance analytical performances for the detection of synthetic AAS such as stanozolol, specific chromatographic procedures have been developed using recent quaternary liquid chromatography technology originally designed for high-throughput standardized proteomics connected to mass spectrometry. Applying the newly designed elution procedures described in this paper to the analyses of stanozolol and its metabolites in complex matrixes revealed improved sensitivity compared to previously described high-throughput methods. Indeed, we report the consistent and reliable detection of 16β-hydroxy-stanozolol down to 10 pg/mL in equine urine and being detectable up-to 3 months after a microdosing administration. Furthermore, a five months long elimination of stanozolol and its metabolites could be monitored on horse mane sections after a single dose administration. Our work highlights novel solutions to detect AAS with improved sensitivity. The application of such developments constitutes new landmarks for doping control laboratories and could be extended to other targeted compounds in residue analysis, toxicology, and metabolomics. Based on this work, the developed chromatographic method is now freely available within the Evosep Plus program.

Ciclesonide (CIC) is the first inhaled highly potent corticosteroid which does not cause any cortisol suppression. It has been developed for the treatment of asthma in human and more recently in equine. CIC is the active compound of Aservo® EquiHaler® (Boehringer Ingelheim Vetmedica GmbH), the pre-filled inhaler generating a medicated mist based on Soft MistTM technology. This prodrug is rapidly converted to desisobutyryl-ciclesonide (des-CIC), the main pharmacologically active compound. Due to its anti-inflammatory properties CIC is prohibited for use in horse competitions. To set up an appropriate control, the determination of detection times and screening limits are required. Therefore, a highly sensitive analytical method based on Supported Liquid Extraction (SLE) combined with Liquid Chromatography - High Resolution tandem Mass Spectrometry (LC-HRMS/MS) was developed to detect CIC and its active metabolite des-CIC in plasma. The lower limit of detection of CIC and des-CIC was approximately 1 pg/mL in plasma. After a pilot study conducted on a single horse at the recommended dose (8 actuations twice daily corresponding to 5.5 mg/day for the first five days, followed by 12 actuations once daily corresponding to 4.1 mg/day in the last five days), the same protocol was applied in the main study using six-horses. In all horses, CIC and des-CIC levels were less than 5 and 10 pg/mL, respectively, at 36 hours after the end of the administration. The outcome of this risk assessment study should be useful to draw any recommendations for horse competitions.

Nowadays, numerous websites attempt to commercialize over the internet various products, regardless of the lack of approval by the EMA or the FDA either for human or veterinary use. These products are often produced after aborted drug development due to insufficient or deleterious biological effects, synthesized based on natural products, or only based on scientific literature. However, the administration of such products is dangerous, considering the lack of official control over the production of these substances and the absence of approval by health authorities. In this short communication, we provide an extensive analysis of three misbranded and adulterated products sold over the internet named TB500, TB1000, and SGF1000. We confirm that the content of TB500/TB1000 products is not systematically consistent with it's former descriptions, but also that SGF1000 is mainly composed of sheep extracellular matrix (ECM) and blood proteins, and the signal corresponding to the purported growth promoters is excessively diluted.

Despite their ban, Anabolic Androgenic Steroids (AAS) are considered as the most important threat for equine doping purposes. In the context of controlling such practices in horse racing, metabolomics has emerged as a promising alternative strategy to study the effect of a substance on metabolism and to discover new relevant biomarkers of effect. Based on the monitoring of 4 metabolomics derived candidate biomarkers in urine, a prediction model to screen for testosterone esters abuse was previously developed. The present work now focuses on assessing the robustness of the associated method and define its scope of application. Several hundred urine samples were selected from 16 different horses of ethically approved animal experiments involving various doping agents’ applications (AAS, SARMS, β-agonists, SAID, NSAID) (n = 349). In addition, 342 urine samples from untreated animals of general equine populations were included in the study. Samples were characterized with the previously described LC-HRMS/MS developed method, with the objective of assessing both its biological and analytical robustness’s. The study concluded that the measurement of the 4 biomarkers involved in the model was fit for purpose. Further, the classification model confirmed its effectiveness in screening for testosterone esters use; and it demonstrated its ability to screen for the administration of other anabolic agents, allowing the development of a global screening tool dedicated to this class of substances. Finally, the results were compared to a direct screening method targeting anabolic agent’s residues, demonstrating complementarity performances of traditional and omics approaches in the screening of anabolic agents in horses.

Short half-life doping substances are, quickly eliminated and therefore difficult to control with traditional analytical chemistry methods. Indirect methods targeting biomarkers constitute an alternative to extend detection time frames in doping control analyses. Gene expression analysis (i.e., transcriptomics) has already shown interesting results in both humans and equines for erythropoietin (EPO), growth hormone (GH), and anabolic androgenic steroid (AAS) misuses. In humans, circulating cell-free microRNAs in plasma were described as new potential biomarkers for control of major doping agent (MDA) abuses. The development of a quantitative polymerase chain reaction (qPCR) method allowing the detection of circulating miRNAs was carried out on equine plasma collected on different type of tubes (EDTA, lithium-heparin [LiHep]). Although analyzing plasma collected in EDTA tubes is a standard method in molecular biology, analyzing plasma collected in LiHep tubes is challenging, as heparin is a reverse transcription (RT) and a PCR inhibitor. Different strategies were considered, and attention was paid on both miRNAs extraction quality and detection sensitivity. The detection of endogenous circulating miRNAs was performed and compared between the different types of tubes. In parallel, homologs of human miRNAs characterized as potential biomarkers of doping were sought in equine databases. The miRNA eca-miR-144, described as potential erythropoiesis stimulating agents (ESAs) administration candidate biomarker was retained and assessed in equine post-administration samples. The results about the qPCR method development and optimization are exposed as well as the equine miRNAs detection. To our knowledge, this work is the first study and the proof of concept of circulating miRNAs detection in plasma dedicated to equine doping control.

According to international sport institutions, the use of peroxisome proliferator activated receptor (PPAR)-δ agonists is forbidden at any time in athlete career due to their capabilities to increase physical and endurance performances. The (PPAR)-δ agonist GW501516 is prohibited for sale but is easily available on internet and can be used by cheaters. In the context of doping control, urine is the preferred matrix because of the non-invasive nature of sampling and providing broader exposure detection times to forbidden molecules but often not detected under its native form due to the organism's metabolism. Even if urinary metabolism of G501516 has been extensively studied in human subjects, knowledge on GW501516 metabolism in horses remains limited. To fight against doping practices in horses' races, GW501516 metabolism has to be studied in horse urine to identify and characterize the most relevant target metabolites to ensure an efficient doping control. In this article, in vitro and in vivo experiments have been conducted using horse S9 liver microsome fractions and horse oral administration route, respectively. These investigations determined that the detection of GW501516 must be performed in urine on its metabolites because the parent molecule was extremely metabolized. To maximize analytical method sensitivity, the extraction conditions have been optimized. In accordance with these results, a qualitative analytical method was validated to detect the abuse of GW501516 based on its most relevant metabolites in urine. This work enabled the Laboratoire des Courses Hippiques (LCH) to highlight two cases of illicit administration of this forbidden molecule in post-race samples.

Bisphosphonates are prohibited drugs according to Article 6 of the International Agreement on Breeding, Racing and Wagering of the International Federation of Horseracing Authorities (IFHA) and the International Equestrian Federation (FEI). These compounds are used for the treatment of lameness, navicular and bone diseases in horses and are divided into two groups: non-nitrogen-containing bisphosphonate drugs (e.g. clodronic acid) and nitrogen-containing bisphosphonate drugs (e.g. zoledronic acid). Their hydrophilic properties and the high affinity for the bone matrix make the control of their use quite difficult. Current analytical strategies to detect such compounds often rely on a solid phase extraction (SPE) followed by detection by means of UHPLC-MS/MS after methylation with chemical reagents. To improve the analysis throughput and to eliminate the need for chemical derivatization, an innovative 96-well SPE followed by ion chromatography-mass spectrometry was developed. Analyses are conducted on an ICS-6000 HPIC system coupled to a TSQ Altis™ (Thermo Scientific™). The use of a 96-well SPE allowed 5-fold sample increase and a 6-fold throughput improvement. While preliminary results conducted on horse plasma exhibited similar performances to the method for the detection of non-nitrogen-containing bisphosphonates, the analytical performances of nitrogen-containing bisphosphonates were greatly improved.

Recombinant human erythropoietin (rHuEPO) belongs to the therapeutic class of erythropoiesis stimulating agents (ESAs) due to its implication in the creation pathway of red blood cells and thus enhancement of oxygenation. Because of this bioactivity, rHuEPO has been considered as a major doping agent in sports competitions for decades. Over the years, doping control laboratories designed several analytical strategies applied to human and animal samples to highlight any misuse. Even though multiple analytical approaches have been reported, none has yet been dedicated to racing camels. Here, we describe an analytical strategy to test camel plasma samples at screening using an ELISA assay and a targeted nano-liquid chromatography-high-resolution tandem mass spectrometry for confirmatory analysis. The method was validated and has been successfully applied to post-race samples, allowing the detection of a positive case of rHuEPO administration.

Clodronate is a non-nitrogen-containing bisphosphonate drug approved in equine veterinary medicine. Clodronate is prohibited for use in competition horses; therefore, to set up an appropriate control, detection times and screening limits are required. The quantitative method in plasma consisted of addition of chloromethylene diphosphonic acid as internal standard. Automated sample preparation comprised a solid phase extraction with weak anion exchange properties on microplate. After methylation of the residue with trimethyl orthoacetate, analysis was conducted by high-performance liquid chromatography-tandem mass spectrometry. Using a weighting factor of 1/(concentration)2 , good linearity was observed in the range of 1 to 500 ng/ml, with low limits of detection and quantification of 0.5 and 1 ng/ml, respectively. Precision and accuracy determined at four concentrations were satisfactory, with an error percentage less than 15%. Absence of carry-over and good stability of clodronic acid in plasma after a long-term storage at -20°C were verified. The method was successfully applied to the quantification of clodronic acid in plasma samples from horses administered with a single intramuscular administration of Osphos® at a mean dose of 1.43 ± 0.07 mg/kg. The observed detection time will be verified in a clinical population study conducted in diseased horses.

With recent advances in analytical chemistry, liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) has become an essential tool for metabolite discovery and detection. Even if most of the common drug transformations have already been extensively described, manual search of drug metabolites in LC-HRMS/MS datasets is still a common practice in toxicology laboratories, complicating metabolite discovery. Furthermore, the availability of free open-source software for metabolite discovery is still limited. In this article, we present MetIDfyR, an open-source and cross-platform R package for in silico drug phase I/II biotransformation prediction and mass-spectrometric data mining. MetIDfyR has proven its efficacy for advanced metabolite identification in semi-complex and complex mixtures in in vitro or in vivo drug studies and is freely available at github.com/agnesblch/MetIDfyR.

After administration to humans or animals, small-molecule drugs most frequently undergo several biochemical transformations by the endogenous enzymatic machinery, called phase I and phase II metabolism. These molecular processes allow organisms to eliminate xenobiotics through modification of their chemical properties and generate metabolites. With recent advances in analytical chemistry, LC-HRMS/MS has become an essential tool for metabolite discovery and detection. Even if most common drug transformations have already been extensively described, manual search of drug metabolites in LC-HRMS/MS datasets is still a common practice in toxicology laboratories, disabling efficient metabolite discovery. Furthermore, the availability of free open-source software for metabolite discovery is still limited. In this article, we present MetIDfyR, an open-source and cross-platform R package for in-silico drug phase I/II biotransformations prediction and mass-spectrometric data mining. MetIDfyR has proven efficacy for advanced metabolite identification in semi-complex and complex mixtures in in-vitro or in-vivo drug studies and is freely available at https://github.com/agnesblch/MetIDfyR.

Cobra (Naja naja kaouthia) venom contains a toxin called α-cobratoxin (α-Cbtx) containing 71 amino acids (MW 7821 Da) with a reported analgesic power greater than morphine. In 2013, the first analytical method for the detection of α-Cbtx in equine plasma was developed by Bailly-Chouriberry et al, allowing the confirmation of the presence of α-Cbtx at low concentrations (1-5 ng/mL or 130-640 fmol/mL) in plasma samples. To increase the method sensitivity and therefore to improve the detection of α-Cbtx in post-administration plasma samples, a nano-liquid chromatography-mass spectrometry/high resolution mass spectrometry (nLC-MS/HRMS) method was developed. This new method allowed us to confirm the presence of α-Cbtx in plasma samples spiked at 100 pg/mL (12.8 fmol/mL) and the detection of α-Cbtx was obtained in plasma samples collected 72 hours post-administration (50 pg/mL or 6.4 fmol/mL) which was defined as the limit of detection (LOD). The presented method is 20-fold more sensitive compared to the method previously described.

Cobalt is an essential mineral micronutrient and is regularly present in equine nutritional and feed supplements. Therefore, cobalt is naturally present at low concentrations in biological samples. The administration of cobalt chloride is considered to be blood doping and is thus prohibited. To control the misuse of cobalt, it was mandatory to establish an international threshold for cobalt in plasma and/or in urine. To achieve this goal, an international collaboration, consisting of an interlaboratory comparison between 5 laboratories for the urine study and 8 laboratories for the plasma study, has been undertaken. Quantification of cobalt in the biological samples was performed by inductively coupled plasma-mass spectrometry (ICP-MS). Ring tests were based on the analysis of 5 urine samples supplemented at concentrations ranging from 5 up to 500 ng/mL and 5 plasma samples spiked at concentrations ranging from 0.5 up to 25 ng/mL. The results obtained from the different laboratories were collected, compiled, and compared to assess the reproducibility and robustness of cobalt quantification measurements. The statistical approach for the ring test for total cobalt in urine was based on the determination of percentage deviations from the calculated means, while robust statistics based on the calculated median were applied to the ring test for total cobalt in plasma. The inter-laboratory comparisons in urine and in plasma were successful so that 97.6% of the urine samples and 97.5% of the plasma samples gave satisfactory results. Threshold values for cobalt in plasma and urine were established from data only obtained by laboratories involved in the ring test. Copyright © 2017 John Wiley & Sons, Ltd.

Since the availability on the European market of the vaccine Improvac® dedicated to male pig immunological castration, the risk of misuse of this product in horses is now considered as a threat for the horseracing industry. Immunological castration is not allowed by the racing codes (immune system, Article 6). Indeed, this vaccination against the hypothalamic hormone luteinizing hormone-releasing hormone or gonadotropin-releasing hormone (GnRH) will prevent the release from the anterior pituitary of luteinizing hormone and follicle stimulating hormone, which are required for the development and activity of gonads in males (testes) and female (ovaries) and therefore all their subsequent physiological functions. This treatment will induce a strong hormonal variation resulting in a behaviour modification of the animals. In this work, four male standardbreds treated with Improvac® vaccine (two intramuscular injections within 4 weeks) were studied. Monitoring of the total scrotal width showed a decrease of the scrotum size (37%) and production of anti-GnRH antibodies was detected up to 200 days after the first injection. Anti-GnRH antibodies were detected in plasma after caprylic acid precipitation followed by an enzyme-linked immunosorbent assay (ELISA) as a rapid and efficient screening method applicable to routine analysis. These results were correlated to a switch of the sexual status from male group to gelding/female group obtained by a steroidomic approach with urine based on ten endogenous compounds. Copyright © 2017 John Wiley & Sons, Ltd.

The improvement of doping control is an ongoing race. Techniques to fight doping are usually based on the direct detection of drugs or their metabolites by analytical methods such as chromatography hyphenated to mass spectrometry after ad hoc sample preparation. Nowadays, omic methods constitute an attractive development and advances have been achieved particularly by application of molecular biology tools for detection of anabolic androgenic steroids (AAS), erythropoiesis-stimulating agent (ESA), or to control human growth hormone misuses. These interesting results across different animal species have suggested that modification of gene expression offers promising new methods of improving the window of detection of banned substances by targeting their effects on blood cell gene expression. In this context, the present study describes the possibility of using a modified version of the dedicated Human IVD (in vitro Diagnostics) PAXgene® Blood RNA Kit for horse gene expression analysis in blood collected on PAXgene® tubes applied to the horse biological passport. The commercial kit was only approved for human blood samples and has required an optimization of specific technical requirements for equine blood samples. Improvements and recommendations were achieved for sample collection, storage and RNA extraction procedure. Following these developments, RNA yield and quality were demonstrated to be suitable for downstream gene expression analysis by qPCR techniques. Copyright © 2017 John Wiley & Sons, Ltd.

Supplemental material, Supplemental Material2 for Tracking main environmental factors masking a minor steroidal doping effect using metabolomic analysis of horse urine by liquid chromatography–high-resolution mass spectrometry by Natali Stojiljkovic, Fanny Leroux, Saša Bubanj, Marie-Agnès Popot, Alain Paris, Jean-Claude Tabet and Christophe Junot in European Journal of Mass Spectrometry

To ensure fair competition and to protect the horse's welfare, horses have to compete on their own merits, without any unfair advantage that might follow the use of drugs. Therefore, regulatory authorities list all substances that are not allowed in competition, including most anabolic-androgenic steroids. As zero-tolerance is retained, the question arose whether the consumption of mouldy feed could lead to the excretion of steroids, due to the biotransformation of plant phytosterols to steroids. A rapid ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analytical method, previously validated according to AORC (Association of Official Racing Chemists) and EC (European Commission) guidelines, was used to measure steroids in different sample types. Multiple mouldy feed samples were tested for the presence of steroids. The effect of digestion was tested by in vitro simulation of the horse's hindgut in batch incubations. In most feed samples no steroids were detected, even when the products were mouldy. Mouldy corn however showed to contain up to 3.0 ± 0.4 µg/kg AED (4-androstenedione), the main testosterone precursor. This concentration increased when mouldy corn (with added phytosterols) was digested in vitro. An herbal phytosupplement also showed to contain α-testosterone. These results demonstrate that it is important to caution against the consumption of any feed or (herbal) supplement of which the detailed ingredients and quantitative analysis are unknown. The consumption of mouldy corn should especially be avoided, not only from a horse health and welfare point of view, but also to avoid possible inadvertent positive doping results. Copyright © 2016 John Wiley & Sons, Ltd.

BACKGROUND Tiludronate is a bisphosphonate drug marketed to treat different bone conditions in horses. OBJECTIVES The goal of this study was to measure the plasma concentrations of tiludronate in a population of race and sport horses under field conditions, and using pharmacokinetic population modelling, to estimate detection times for doping control. STUDY DESIGN Prospective cohort. METHODS This study was conducted under field conditions on 39 race or sport horses diagnosed with bone conditions based on a lameness examination and treated with tiludronate. Each horse received 1 mg/kg of tiludronate (Tildren® ) intravenously (i.v.). Blood samples (from 1 to 4 per horse with a total of 93 samples) were collected around 10, 20, 30, 40 and 50 days after tiludronate administration. Tiludronate was quantified by HPLC/ESI-MSn . Tiludronate concentrations were analysed using nonlinear mixed-effects modelling (population approach). Monte Carlo simulations were then used to compute a prediction interval to estimate the corresponding quantile of horses predicted to have concentrations below some potential screening limits. RESULTS This study highlighted pharmacokinetic differences between healthy experimental horses and the population of horses being treated in the field as well as the effect of level of training on plasma tiludronate. Different detection times were computed corresponding to different possible screening limits. MAIN LIMITATIONS The number of horses in each group was limited, and the specific disease being treated with tiludronate is unknown. CONCLUSIONS This population pharmacokinetic study on tiludronate will enable racing and other sports authorities to provide a detection time reflecting field conditions for the medication control of tiludronate. More generally, our study design and the data modelling serve as an example of how to generate detection times directly from the target horse population rather than from experimental horses.

Doping control is a main priority for regulatory bodies of both the horse racing industry and the equestrian sports. Urine and blood samples are screened for the presence of hundreds of forbidden substances including anabolic-androgenic steroids (AASs). Based on the suspected endogenous origin of some AASs, with β-boldenone as the most illicit candidate, this study aimed to improve the knowledge of the naturally present AAS in horse urine. To this extent, a novel ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated according to the Association of Official Racing Chemists (AORC) and European Commission (EC) guidelines, proving the power of this new method. Low limits of detection (0.2 ng/mL), good reproducibility (percentage of standard deviation (%RSD) 10%), high recovery (94.6 to 117.1%), selectivity and specificity, and a linear response (confirmed with R(2) 0.99 and lack-of-fit analysis) were obtained for all included AASs. With this method, urine samples of 105 guaranteed untreated horses (47 geldings, 53 mares, and 5 stallions serving as a control) were screened for β-boldenone and five related natural steroids: androstadienedione (ADD), androstenedione (AED), alpha-testosterone (αT), beta-testosterone (βT), and progesterone (P). Progesterone, β-testosterone, and α-testosterone were detected in more than half of the horses at low concentrations (2 ng/mL). Occasionally, not only testosterone and progesterone but also low concentrations of AED, ADD, and boldenone (Bol) were found (0.5-5 ng/mL). Graphical Abstract A sensitive, new and fully validated UHPLC-MS/MS method has been developed that is able to quantify low levels of anabolic-androgenic steroids naturally present in urine of untreated horses (mares and geldings).

International audience; Erythropoiesis Stimulating Agents (ESAs) were developed for therapeutic purposes to stimulate red blood cell (RBC) production. Consequently, tissue oxygenation is enhanced as athlete's endurance and ESAs misuse now benefits doping. Our hypothesis is that most of ESAs should have similar mechanisms and thus have the same effects on metabolism. Studying the metabolome variations could allow suspecting the use of any ESAs with a single method by targeting their effects. In this objective, a metabolomic study was carried out on 3 thoroughbred horses with a single administration of 4.2μg/kg of Mircera®, also called Continuous Erythropoiesis Receptor Activator (CERA). Blood and urine samples were collected from D-17to D+74and haematological parameters were followed throughout the study as plasmatic CERA concentration (ELISA). Urine and plasma metabolic fingerprints were recorded by Liquid Chromatography coupled to High Resolution Mass Spectrometry (LC-HRMS) in positive and negative mode. After preprocessing steps, normalized data were analyzed by multivariate statistics to build OPLS models. Hemoglobin concentration and hematocrit showed a significant increase after CERA administration unlike reticulocytes. CERA concentration showed a high intensity peak and then a slow decrease until becoming undetectable after D+31. Models built with multivariate statistics allow a discrimination between pre and post-administration plasma and urine samples until 74days after administration, i.e. 43days longer than ELISA method. By reducing and studying variables (ions), some potential candidate biomarkers were found.

The current detection methods for stanozolol are all based on targeted approach. The present study aimed to assess the global biological effect of stanozolol-treatment by means of chemometric models, after generating and comparing horse urine LC-HRMS fingerprints collected from control and stanozolol-treated horses. The animal study was conducted according to an ethically approved protocol at two different places in France: Chamberet and Coye la Foret. The total duration of the animal phase was seven months and only females were selected to partake. The sixteen mares in this study were not actively racing horses, but were in good physical condition. SIMCA-P+ software and R free software environment were used for multivariate data analysis. Principal Component Analysis (PCA) and Orthogonal Projections to Latent Structures-Discriminant Analysis (OPLS-DA) were applied to build some descriptive and predictive models. The analyzed horse urine fingerprints based on the 220 features selected after suppression of confounding factors show changes in metabolic states after chronic stanozolol treatment. This proof of concept study confirms the power of untargeted approach in doping control since the changes are present over seven months after anabolic administration.

The improvement of doping control is an ongoing race. Techniques to fight doping are usually based on the direct detection of drugs or their metabolites by analytical methods such as chromatography hyphenated to mass spectrometry after ad hoc sample preparation. Nowadays, omic methods constitute an attractive development and advances have been achieved particularly by application of molecular biology tools for detection of anabolic androgenic steroids (AAS), erythropoiesis-stimulating agent (ESA), or to control human growth hormone misuses. These interesting results across different animal species have suggested that modification of gene expression offers promising new methods of improving the window of detection of banned substances by targeting their effects on blood cell gene expression. In this context, the present study describes the possibility of using a modified version of the dedicated Human IVD (in vitro Diagnostics) PAXgene® Blood RNA Kit for horse gene expression analysis in blood collected on PAXgene® tubes applied to the horse biological passport. The commercial kit was only approved for human blood samples and has required an optimization of specific technical requirements for equine blood samples. Improvements and recommendations were achieved for sample collection, storage and RNA extraction procedure. Following these developments, RNA yield and quality were demonstrated to be suitable for downstream gene expression analysis by qPCR techniques. Copyright © 2017 John WileySons, Ltd.

Since the availability on the European market of the vaccine Improvac® dedicated to male pig immunological castration, the risk of misuse of this product in horses is now considered as a threat for the horseracing industry. Immunological castration is not allowed by the racing codes (immune system, Article 6). Indeed, this vaccination against the hypothalamic hormone luteinizing hormone-releasing hormone or gonadotropin-releasing hormone (GnRH) will prevent the release from the anterior pituitary of luteinizing hormone and follicle stimulating hormone, which are required for the development and activity of gonads in males (testes) and female (ovaries) and therefore all their subsequent physiological functions. This treatment will induce a strong hormonal variation resulting in a behaviour modification of the animals. In this work, four male standardbreds treated with Improvac® vaccine (two intramuscular injections within 4 weeks) were studied. Monitoring of the total scrotal width showed a decrease of the scrotum size (37%) and production of anti-GnRH antibodies was detected up to 200 days after the first injection. Anti-GnRH antibodies were detected in plasma after caprylic acid precipitation followed by an enzyme-linked immunosorbent assay (ELISA) as a rapid and efficient screening method applicable to routine analysis. These results were correlated to a switch of the sexual status from male group to gelding/female group obtained by a steroidomic approach with urine based on ten endogenous compounds. Copyright © 2017 John WileySons, Ltd.

A quantitative method, using LC/ESI-MS(n) with a quadrupole linear ion trap mass analyzer, has been developed for the analysis of ipratropium cation in horse plasma and urine. The method applies solid-phase extraction with WCX cartridges for plasma and MM2 cartridges for urine, prior to analysis by LC/ESI-MS(n). The efficiency of extraction combined with the sensitivity and the selectivity of MS(n) allows for the quantification of ipratropium cation at picogram per milliliter levels. The analytical capabilities of the method have been successfully checked by the quantitative analysis of ipratropium cation in post-administration samples collected from horses treated by nebulization.

Growth hormone (GH) is a polypeptide suspected of being used in horse racing to speed up physical performances. Despite scientific advances in the recent years, the control of its administration remains difficult. In order to improve it, a metabolomics study through LC-high resolution mass spectrometry measurements was recently initiated to assess the metabolic perturbations caused by recombinant equine growth hormone administration. Few tens of ions not identified structurally were highlighted as compounds responsible for the modification of metabolic profiling observed in treated animals. This previous work was based on the use of Uptisphere Strategy NEC as the chromatographic column. In parallel, more and more metabolomics studies showed the interest of the use of new chromatographic supports such as hydrophilic interaction chromatography for the analysis of polar compounds. It is in this context that an investigation was conducted on Uptisphere HDO and Luna hydrophilic interaction chromatography stationary phases to generate and process urinary metabolomics fingerprints, which could allow to establish a comparison with Uptisphere Strategy NEC. The chromatographic column the most adapted for the detection of new biomarkers of GH administration has been used to set up a relevant statistical model based on the analysis of more than hundred biological samples.

Summary The aim of this study was to document the 24 h urinary Cortisol concentrations during a rest day and a day that included prolonged exercise. The influence of exercise on disposition (plasma, urine) of a dose of Cortisol (1 mg/kg) was also assessed. Six adult saddle horses were used. In one experiment, urinary Cortisol elimination rates were measured during rest and prolonged exercise (56 km, 12 km/h) corresponding to about 50% of the VO2max. In another experiment, horses received, according to a crossover design, an intravenous (i.v.) bolus administration of Cortisol (1 mg/kg) either at rest or during the same prolonged exercise. The urine Cortisol concentration was lower than 100 ng/ml in the control resting horse. It was increased by exercise but the maximal concentration was lower than 300 ng/ml and returned to control values by 10 h after the end of exercise. After 1 mg/kg Cortisol i.v. Cortisol clearance was decreased by exercise (552 ± 143 vs. 452 ± 59 ml/kg/h, mean ± s.d.), but the difference was not significant The peaks of urinary Cortisol ranged from 760 to 21,967 ng/ml and occurred 0.8 to 6.9 h after Cortisol administration depending on the time of the first urination after administration. The return to values below 100 ng/ml was observed after 21 h in all horses. The 1000 ng/ml threshold for Cortisol control in urine after i.v. Cortisol (1 mg/kg) is specific (conservative) but of low sensitivity.

Summary Reasons for performing study: Equine antidoping rules were established to prevent a horse's performance being altered after the administration of prohibited substances, including approved drugs used for legitimate treatment. Veterinarians have to advise owners or trainers on appropriate withholding times to guarantee that their horses may safely compete after drug administration. In order to propose tailored withdrawal times, several horse organisations released detection time (DT) values, for the main veterinary drugs used in horses. One of the possible limits to the information provided by published DTs in horses is the fact that they are determined from classic pharmacokinetic studies performed at rest under laboratory conditions. In field conditions, training and exercise programmes may have an influence on drug elimination. Methods: Dexamethasone (DMX) and phenylbutazone (PBZ) have been quantified in plasma and urine after solid phase extraction. The kinetic disposition of DXM (8 μg/kg) and PBZ (8 mg/kg) administered by i.v. route in 8 horses, was investigated in rest conditions and during a standardised 3 h test exercise according to a cross-over design. Objectives: The aim of the present study was to compare the kinetic disposition of 2 test drugs, DMX and PBZ in rest vs. exercising conditions. Results: It was shown in 8 horses that a sustained 3 h of mild exercise slightly decreased the plasma clearance of both drugs (about 25% for DXM and 37% for PBZ) and this is mainly explained by the significant decrease of the corresponding hepatic clearance. In addition, as the volume of distribution was correlatively decreased, the plasma terminal half-life, which is a hybrid parameter of plasma clearance and volume of distribution, remains unchanged overall. Conclusion and potential relevance: Establishing DTs or withdrawal times (WTs) are relevant as plasma and urine half-lives, but not clearance, are the main determinants of DT length. Veterinarians may realistically decide upon a WT for a legitimate drug based on the corresponding DT obtained under resting conditions providing this drug has a low hepatic extraction ratio and a safety margin is added to allow for all possible sources of variability.

The insulin-like-growth factor (IGF-I) peptide is considered to be the main indirect marker for growth hormone administration (GH) in a horse. Further to a previous investigation on measurement of IGF-I in plasma samples by mass spectrometry, this study focuses on quantitative and qualitative analysis of intact IGF-I in horse plasma. First, protein-transposing software has been developed for IGF-I to facilitate its quantification by HPLC-electrospray-ion-trap mass spectrometry. Second, product-ion scan experiments on IGF-I have been conducted on standard samples, non-fortified equine plasma samples, fortified plasma samples, and equine GH post-administration samples. This "top-down" approach method enables characterisation of fragment ions corresponding to the carboxy terminal end, which can be useful for the confirmation of the presence of IGF-I in plasma samples.

Faeces, which could be a potential alternative medium for doping control, have been used for the detection of 1,4-androstadiene-3,17-dione administration to the horse. Semi-quantitative analyses of 1,4-androstadiene-3,17-dione, testosterone, 17alpha- and 17beta-boldenone have been conducted in pre- and post-administration faeces, and in controls (untreated stallions, geldings and mares). Sample preparation comprised diethyl ether extraction, lipid removal, HPLC purification and derivatisation. 1,4-Androstadiene-3,17-dione, testosterone, 17alpha- and 17beta-boldenone were analysed by GC-EI/MS/MS. Quantitative limits of detection were 0.1 ng/g for 1,4-androstadiene-3,17-dione, and 0.025 ng/g for testosterone, 17alpha- and 17beta-testosterone. In post-administration samples from geldings and mares, peak levels of 1,4-androstadiene-3,17-dione, 17alpha-, 17beta-boldenone and testosterone were attained 24 h after administration. In untreated geldings and mares (in di- or anoestrus), 17alpha- and 17beta-boldenone and testosterone were not detected. Faeces from females in oestrus had detectable levels of boldenone isomers and testosterone. 1,4-Androstadiene-3,17-dione was undetectable in faeces collected from untreated horses, but the presence of this androgen was recently reported in faeces from untreated swine and it would therefore be advisable to check for its possible presence in a larger number of individual faecal samples.

Since the Australian commercialisation of the recombinant equine growth hormone (reGH) in 1998 (EquiGen-5), Bresagen), this reGH, which differs only from eGH by an additional methionine at the N-terminal end (met-eGH), is worldwide suspected to be administered to racehorses as a doping agent. Indeed, the use of this biological drug is considered as a threat to horseracing since it acts both on growth, development or reproductive functions, and on the improvement of performances. In this work, we describe two reliable techniques based on surface plasmon resonance biosensor immunoassay (SPR-BIA) and solid-phase enzyme-linked immunosorbent assay (ELISA) as new, rapid and efficient long-term screening methods applicable to horseracing antidoping analysis. The ELISA and SPR-BIA tests were applied to octanoic acid purified IgGs from serum/plasma samples collected on two thoroughbreds treated with recombinant equine growth hormone for a period of two weeks. The first kinetic study of serum/plasma antibodies raised as a consequence of recombinant equine growth hormone administrations, which allows the detection from eight days up to 200 days after the beginning of the treatment, was performed. In order to trace the occurrence of anti-reGH antibodies in routine analysis and to monitor the animal level exposure to this forbidden molecule, a random population study was conducted on 233 post-race horses.

Feces are a possible medium to be used for horse doping control. Efficient methods for detecting drugs in feces collected from various animals are routinely applied in institutes of food safety in Belgium. We have already tested whether they are applicable to horse feces. In this report, accelerated solvent extraction (ASE), an efficient method for extracting compounds from solid material, has been tested. ASE has been used to replace the diethyl ether liquid-liquid extraction step present in the method initially set up. This technique has been optimized for detecting several non-steroidal anti-inflammatory drugs (NSAIDs) in horse feces. Extraction recovery and limit of detection have been determined for several NSAIDs, such as meclofenamic acid, flunixin, vedaprofen, celecoxib, carprofen, diclofenac, and ketoprofen. The method has been successfully applied to meclofenamic acid, flunixin, and phenylbutazone post-administration feces samples, and the main metabolites identified in urine were also detected in feces. In the case of meclofenamic acid, the detection profile in feces presented in this report is in accordance with our previous finding in feces obtained with the original method. The use of ASE decreases the time necessary for sample preparation. This method is applicable on a large scale, which is useful for horse doping control.

In order to investigate the detection of boldenone in horse mane samples, a boldenone study was conducted on two horses. The analytical procedure consisted in a hydrolysis using the Sorensen buffer, a liquid-liquid extraction using diethyl ether and a PFPA derivatization. The instrumental method was a gas chromatography sequential mass spectrometry performed on an ion trap instrument in full scan mode. The limit of detection was estimated to 1 pg mg−1. The detection of boldenone in the mane was made possible for up to 12 months after administration.

Background: Horse urine is the medium of choice for the implementation of metabolomic approaches aimed at improving horse doping control. However, drug analysis in this biofluid is a challenging task due to the presence of large amounts of interfering compounds. Methodology & Results: A comparative study of sample preparation has been conducted to evaluate five sample-preparation methods, namely acetonitrile precipitation, proteinase K hydrolysis, membrane filtration and sample dilution with water by factors of five and 20, for metabolome analysis using liquid chromatography coupled to high resolution mass spectrometry. Assessment was performed at both global and targeted levels, by using a few thousand features obtained from peak detection software, and internal standards and 100 annotated or identified metabolites. Conclusion: By considering the number of detected signals, their intensity and their detection repeatability, acetonitrile precipitation was selected as the most efficient sample-preparation method for the analysis of horse urine metabolome in liquid chromatography coupled to high resolution mass spectrometry conditions.

Insulin-like Growth Factor I is a marker of Somatotropin administration. In order to quantify this peptide in horse plasma samples, a high performance liquid chromatography/electrospray mass spectrometry method using R3 Insulin-like Growth Factor I as internal standard was developed. The extraction procedure involves an acid ethanol extraction and an immunoaffinity purification. Quantification is made possible by using deconvolution software and by measuring the insulin like growth factor-I/internal standard ratio. Comparison between high performance liquid chromatography/electrospray mass spectrometry and two immunological methods indicated agreement between methods even though more samples are needed to confirm this finding.

The insulin-like-growth-factor-I (IGF-I) peptide is known to be a marker for growth hormone administration. The development of a quantification method by electrospray ionization mass spectrometry (ESI-MS) coupled with high-performance liquid chromatography (HPLC) is required. This paper describes a method to quantify IGF-I using the internal standard R3 IGF-I in its oxidized forms. A deconvolution software was used to quantify the set of multi-charged molecules recorded on an ESI ion trap mass spectrometer. The results (i.e., linearity, reproducibility and concentration range) were obtained on standard samples and the described LC-ESI-MS method should be applicable to biological samples.

Les nouvelles methodes de detection pouvant etre mises en place dans les laboratoires sont principalement axees autour des spectrometres de masse de haute resolution, notamment les appareils de temps de vol (TOF) dont la technologie s'est considerablement amelioree ces dernieres annees. Ces techniques sont susceptibles d'apporter a la fois une meilleure sensibilite et specificite. En effet l'accroissement de resolution permet d'augmenter significativement le rapport signal/bruit et dans la mesure ou la sensibilite de l'appareil n 'est pas alteree en consequence, le gain en terme de capacite de detection est donc important. Dans le meme esprit, de nouveaux couplages de chromatographie liquide capillaire/ MS/MS, alliant a la fois la resolution chromato graphique des colonnes "micro ou nanobore" aux performances des spectrometres en tandem (MS/MS) recents ou des trappes d'ions permettent d'ameliorer significativement les limites de detection des xenobiotiques dopants. Des exemples de detection de steroides anabolisants dans les milieux biologiques equins illustrent ces nouvelles perspectives analytiques. Le second axe de developpement s'articule autour de la prise en compte de marqueurs biologiques qui peuvent etre alteres par l'administration de biomolecules telles que les hormones de croissance par exemple. Le probleme principal pose par l'administration de ces peptides reside dans le fait que ces molecules elles-meme sont difficiles a detecter du fait de leur poids moleculaire eleve et surtout par le fait que leur demi-vie est tres courte. Ces deux phenomenes ont pour consequence de rendre aleatoire la mise en evidence de la molecule elle-meme, une solution possible a cela consiste a prendre en compte les marqueurs biologiques alteres par l'administration de l'hormone proteique. Ainsi un exemple base sur le suivi de ces marqueurs et en particulier de l'IGFl, apres administration d'hormone de croissance recombinante au cheval est expose.

Cobra venom (Naja kaouthia) contains a toxin called α-cobratoxin (α-Cbtx). This toxin is a natural protein containing 71 amino acids (MW 7821 Da) with a reported analgesic potency greater than morphine. In 2007, in USA, this substance was found in the barns of a thoroughbred trainer and since then till date, the lack of a detection of this molecule has remained a recurring problem for the horseracing industry worldwide. To solve this problem, the first method for the detection of α-cobratoxin in equine plasma has now been developed. Plasma sample (3 mL) was treated with ammonium sulfate at the isoelectric point of α-Cbtx, and the pellet was dissolved in a phosphate buffer and mixed with methanol for precipitation. The supernatant was then concentrated prior to its extraction on WCX SPE cartridges. The eluate was concentrated with two consecutive filtration steps before the trypsin digestion. The samples were analyzed using a LC-MS/MS Q Exactive instrument at 70,000 resolution on the product ions of the doubly charged precursor of the target peptide ((24)TWCDAFCSIR(33)). The method was validated (n = 18) at 5 μg/L (640 pmol/L) according to the Association of Official Racing Chemists (AORC) requirements. The lower limit of detection was 1 μg/L (130 pmol/L). The present method has made it possible for us to confirm the presence of α-Cbtx in a horse plasma sample 24 h post the administration of α-Cbtx. Thus, the present method provides the first sensitive, specific, and reliable analytical method to confirm the presence of α-Cbtx in equine plasma.

Tandem mass spectrometry in negative-ion mode using ammonia as reagent gas has contributed to the differentiation of polyhydroxysteroids and to the study of stereochemical effects. The present study provides additional examples of sterochemical effects. A first observation of intramolecular OH− catalysis yielding stereospecific H2O elimination is proposed.

Recombinant human erythropoietin (rHuEPO) is a 30-34 kDa glycoprotein banned by the racing authorities. For some years this molecule has been detected in race horses in USA and in Europe, and even in racing camels. Although direct methods to differentiate horse endogenous EPO and rHuEPO have been developed either by LC-MS/MS or by isoelectric focusing (IEF) with double-blotting, the short confirmation time of such prohibited hormone in plasma remains a problem for horseracing doping control laboratories. In order to improve the rHuEPOs confirmation process in horse plasma or urine in terms of reliability and delay, a small anti-EPO monolith membrane contained in a disposable column (anti-EPO monolith column) has been successfully used and validated (n = 10). This new sample preparation, combined with LC-FAIMS-MS/MS, has been performed on plasma and urine samples collected from one horse which received an Eprex® treatment during six consecutive days and a second one with a single injection of Aranesp®. This inventive technology allowed the possibility to confirm the presence of rHuEPO within one day with a limit of detection validated for both urine and plasma at 250 pg mL(-1) by means of a disposable, ready to use immunoaffinity column. The lower limit of detection (LLOD) obtained for each matrix was 100 pg mL(-1). These results provide an important improvement for rHuEPO doping control in horseracing especially the possibility to confirm these banned molecules in both matrices, urine and plasma, with a confidence of two specific target peptides.

Doping of horses with recombinant human erythropoietin (rHuEPO) to illegally enhance their endurance capacity in horseracing has been reported during the last years. This leads to increased blood viscosity which can result in sudden death and is of concern for the horse welfare. Additionally, the horse can start production of rHuEPO antibodies, which cross-reacts with endogenous equine EPO and can lead to severe anaemia and even death. In this study, a novel micro-chromatographic method, EPO WGA MAIIA, has been tested for the capability in plasma and urine samples to detect administration of erythropoiesis-stimulating agents, like the rHuEPO glycoprotein varieties Eprex and Aranesp, to horses. After administration of 40 IU Eprex kg(-1) day(-1) to seven horses during 6 days, the presence of Eprex in horse plasma was detected up to 2-5 days after last injection. In urine samples collected from two horses, Eprex was detected up to 3 days. A single injection of Aranesp (0.39 μg/kg) was detected up to 9 days in plasma and up to 8 days, the last day of testing, in the urine sample. The LC-FAIMS-MS/MS system, with 1 day reporting time, confirmed the presence of Eprex up to 1 day after last injection for six out of seven horses and the presence of Aranesp up to 5 days after last injection in plasma samples. The MAIIA system showed to be a promising tool with high sensitivity and extremely short reporting time (1 h).

Growth hormones are proteins produced by the anterior pituitary gland responsible for bone and tissue growth through their effects on carbohydrates, lipids and proteins metabolisms. Despite strict regulations banning the use of recombinant equine growth hormone, this substance is suspected to be misused to improve the horse physical performances. In order to check whether the regulation is fulfilled or not, controls are organized and a new analytical screening tool potentially able to detect such abuse was investigated in this paper. An untargeted metabolomics approach, based on liquid chromatography coupled to high resolution mass spectrometry, was developed and applied to characterize and compare horse urinary and plasmatic metabolic profiles upon reGH administrations. After minimal sample preparation, biological fluids were analyzed by LC-ESI(+/-)-Q-TOF. Data processing was performed by XCMS software and multivariate data analysis applied to the generated data set allowed building OPLS models to discriminate control versus treated populations. Results demonstrated significant metabolic modifications consecutively to the reGH treatment. A comparative study between urinary and plasmatic signatures was performed to evaluate the resulting metabolomic models and to asses their respective interests in the scope of real application for screening reGH administration.

Liquid chromatography-mass spectrometry (LC-MS) has been widely used in doping control laboratories over the last two decades. Currently, simple quadrupole, triple quadrupole and ion trap are the most commonly employed analyzers in toxicological analysis. Nevertheless, the main lack of these technologies is the restricted number of target compounds simultaneously screened without loss of sensitivity. In this article we present an innovative screening approach routinely applied in the French horse doping control laboratory based on high resolution (50000) and high mass accuracy (5 ppm) in full scan MS mode for more than 235 target analytes screened from an initial volume of 5 mL of urine. The sample preparation was classically founded on solid phase extraction by means of reverse phase C18 cartridges. LC-MS analyses were carried out on a Shimadzu binary HPLC pumps linked to a C18 Sunfire column associated with the high resolution exactive benchtop orbitrap mass spectrometer. This screening was performed alternatively in positive-negative ionization mode during the same run. Thus, the identification of compounds of interest was made using their exact mass in positive-negative ionization mode at their expected retention time. All data obtained were processed by ToxID software (ThermoFisherScientific) which is able to identify a molecule by theoretical mass and retention time. In order to illustrate this innovative technology applied in our laboratory, sample preparation, validation data performed on 20 target compounds from 16 different horse urine samples, chromatograms and spectra will be discussed in this paper.

Recombinant human erythropoietins (rHuEPOs) are glycoproteins drugs, produced by the pharmaceutical industry to restore production of red blood cells by stimulating human bone marrow for which this pathology has been diagnosed. It is suspected that these molecules are diverted as doping agents in horseracing to enhance oxygen transport and aerobic power in racehorses. Although indirect double-blotting or direct liquid chromatography-mass spectrometry (LC-MS) methods have been developed to confirm the presence of rHuEPO in a sample, the short detection time (48 h) is still a problem for doping control. In this context, gene profiling investigation through Serial Analysis of Gene Expression (SAGE) has been conducted on seven thoroughbreds treated with Eprex®. This functional genomic method has been performed from total blood cells collected from each animal to assess the mRNA expression consecutive to rHuEPO injections. Sample pooling was chosen as a powerful, cost-effective, and rapid means of identifying the most common and specific changes in terms of gene expression profile and to eliminate individual variation. Consequently, three SAGE libraries were constructed, before, during, and after Eprex® treatment. More than 71 440 mRNA signatures were observed and subjected to statistical analysis; 49 differentially expressed genes were identified and analyzed by qPCR. From the selected gene list, were defined as potential biomarkers in terms of their low inter-individual variation and capacity as strong markers of rHuEPO administration up to 60 days after the beginning of the doping period. In this paper, a new strategy is proposed to the horseracing industry to prevent rHuEPO abuse. Copyright © 2010 John Wiley & Sons, Ltd.

Despite the worldwide existing regulation banning the use of the recombinant equine growth hormone (reGH) as growth promoter, it is suspected to be used in horseracing to improve performances. Various analytical methods previously developed to screen for its misuse have encountered some limitations in terms of detection timeframes, in particular during the first days following reGH administration. A novel strategy involving the characterization of global metabolomic fingerprints in urine samples of non-treated and reGH-treated horses by liquid chromatography–electrospray–high-resolution mass spectrometry (LC-ESI-HRMS) is described and assessed in this paper in order to develop a new screening tool for growth hormone abuse in horseracing. The strategy involves a limited sample preparation of the urine samples and the use of appropriate software for data processing and analysis. As preliminary work, reproducibility of both sample preparation and mass spectrometry (MS) measurements was evaluated in order to demonstrate the reliability of the method. Application of the developed protocol on two horses demonstrated the suitability of the developed strategy and preliminary results showed significant modifications of the metabolome after treatment with reGH.

LC/ESI-MSn methods have been previously set up to detect the administration of (i) Harpagophytum and (ii) preparations containing a plant capable of anti-stress properties: Eleutherococcus senticosus. Harpagoside has been found to be the main indicator of Harpagophytum administration in the horse. These methods have been applied to a large number of horse urine samples of various origins. Regarding the detection of Harpagophytum administration, harpagoside, harpagide and 8-para-coumaroyl harpagide were detected together in only one sample out of 317. Eleutheroside E was found to be the main indicator of Eleutherococcus senticosus administration. It was detected in post-administration samples collected from two horses having received a feed supplement containing Eleutherococcus senticosus for several days. Out of the 382 samples tested, eleutheroside E was found in an unexpected large number of urine samples (39%) of various origins and its presence cannot be only due to the sole use of herbal dietary supplements. Copyright © 2008 John Wiley & Sons, Ltd.