ABSTRACT
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.
Subject(s)
Bone Density Conservation Agents/pharmacokinetics , Diphosphonates/pharmacokinetics , Animals , Bone Density Conservation Agents/blood , Cohort Studies , Diphosphonates/blood , Half-Life , HorsesABSTRACT
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.
Subject(s)
Anabolic Agents/analysis , Androgens/analysis , Animal Feed/analysis , Animal Feed/microbiology , Feces/chemistry , Horses/urine , Steroids/analysis , Aerobiosis , Anabolic Agents/metabolism , Anabolic Agents/urine , Androgens/metabolism , Androgens/urine , Animals , Biotransformation , Chromatography, High Pressure Liquid/methods , Dietary Supplements/analysis , Dietary Supplements/microbiology , Doping in Sports , Horses/metabolism , Mucor/metabolism , Mycobacterium/metabolism , Phytosterols/analysis , Phytosterols/metabolism , Phytosterols/urine , Steroids/metabolism , Steroids/urine , Tandem Mass Spectrometry/methods , Testosterone/analysis , Testosterone/metabolism , Testosterone/urine , Zea mays/chemistry , Zea mays/microbiologyABSTRACT
Traditionally, steroids other than testosterone are considered to be synthetic, anabolic steroids. Nevertheless, in stallions, it has been shown that ß-Bol can originate from naturally present testosterone. Other precursors, including phytosterols from feed, have been put forward to explain the prevalence of low levels of steroids (including ß-Bol and ADD) in urine of mares and geldings. However, the possible biotransformation and identification of the precursors has thus far not been investigated in horses. To study the possible endogenous digestive transformation, in vitro simulations of the horse hindgut were set up, using fecal inocula obtained from eight different horses. The functionality of the in vitro model was confirmed by monitoring the formation of short-chain fatty acids and the consumption of amino acids and carbohydrates throughout the digestion process. In vitro digestion samples were analyzed with a validated UHPLC-MS/MS method. The addition of ß-Bol gave rise to the formation of ADD (androsta-1,4-diene-3,17-dione) or αT. Upon addition of ADD to the in vitro digestions, the transformation of ADD to ß-Bol was observed and this for all eight horses' inocula, in line with previously obtained in vivo results, again confirming the functionality of the in vitro model. The transformation ratio proved to be inoculum and thus horse dependent. The addition of pure phytosterols (50% ß-sitosterol) or phytosterol-rich herbal supplements on the other hand, did not induce the detection of ß-Bol, only low concentrations of AED, a testosterone precursor, could be found (0.1 ng/mL). As such, the digestive transformation of ADD could be linked to the detection of ß-Bol, and the consumption of phytosterols to low concentrations of AED, but there is no direct link between phytosterols and ß-Bol.
Subject(s)
Androstadienes/urine , Androstenedione/urine , Digestion/physiology , Phytosterols/metabolism , Testosterone/analogs & derivatives , Amino Acids/metabolism , Anabolic Agents/metabolism , Androgens/metabolism , Androstadienes/metabolism , Androstenedione/metabolism , Animals , Chromatography, High Pressure Liquid , Dietary Carbohydrates/metabolism , Fatty Acids, Volatile/biosynthesis , Female , Horses , Male , Mycobacterium/metabolism , Steroids/metabolism , Tandem Mass Spectrometry , Testosterone/metabolism , Testosterone/urineABSTRACT
Tiludronate is a non-nitrogen-containing biphosphonate drug approved in equine veterinary medicine for the treatment of navicular disease and bone sparvin in horse. Its hydrophilic properties and its strong affinity for the bone have made the control of its use quite difficult. After an initial step of method development in plasma and urine, due to a strong matrix effect and erratic detection in urine, the final method development was conducted in plasma. After addition of (3-trifluoromethylphenyl) thiomethylene biphosphonic acid as internal standard, automated sample preparation consisted of a filtration on a Nexus cartridge followed by a Solid Phase Extraction on an Oasis WAX cartridge with weak anion exchange properties. After methylation of the residue with trimethyl orthoacetate (TMOA), analysis was conducted by HPLC/ESI-MS(n) on a LTQ mass spectrometer. The method has been validated with a LOD and LOQ of respectively 1 and 2.5ng/mL. Using a weighting factor of 1/concentration(2), a linear model was suitable in the range of 2.5 up to 500ng/mL. Precision and accuracy data determined at two concentrations were satisfactory (i.e. less than 15%). Carryover would have been a problem but this has finally been fixed using the additional steps of washing during robotised SPE extraction and analysis on both the autosampler and the analytical column. The method was successfully employed for the first time to the quantification of tiludronate in plasma samples collected from horses treated with Tildren™ (Intravenous administration at the dose of 0.1mg/kg/day for 10 days).
Subject(s)
Bone Density Conservation Agents/blood , Chromatography, High Pressure Liquid/methods , Diphosphonates/blood , Horses/blood , Spectrometry, Mass, Electrospray Ionization/methods , Animals , Female , Limit of Detection , Solid Phase Extraction , Tandem Mass Spectrometry/methodsABSTRACT
Nandrolone (17ß-hydroxy-4-estren-3-one) is amongst the most misused endogenous steroid hormones in entire male horses. The detection of such a substance is challenging with regard to its endogenous presence. The current international threshold level for nandrolone misuse is based on the urinary concentration ratio of 5α-estrane-3ß,17α-diol (EAD) to 5(10)-estrene-3ß,17α-diol (EED). This ratio, however, can be influenced by a number of factors due to existing intra- and inter-variability standing, respectively, for the variation occurring in endogenous steroids concentration levels in a single subject and the variation in those same concentration levels observed between different subjects. Targeting an efficient detection of nandrolone misuse in entire male horses, an analytical strategy was set up in order to profile a group of endogenous steroids in nandrolone-treated and non-treated equines. Experiment plasma and urine samples were steadily collected over more than three months from a stallion administered with nandrolone laurate (1 mg/kg). Control plasma and urine samples were collected monthly from seven non-treated stallions over a one-year period. A large panel of steroids of interest (n = 23) were extracted from equine urine and plasma samples using a C18 cartridge. Following a methanolysis step, liquid-liquid and solid-phase extractions purifications were performed before derivatization and analysis on gas chromatography-tandem mass spectrometry (GC-MS/MS) for quantification. Statistical processing of the collected data permitted to establish statistical models capable of discriminating control samples from those collected during the three months following administration. Furthermore, these statistical models succeeded in predicting the compliance status of additional samples collected from racing horses.
Subject(s)
Anabolic Agents/blood , Anabolic Agents/urine , Horses/blood , Horses/urine , Nandrolone/blood , Nandrolone/urine , Anabolic Agents/administration & dosage , Animals , Doping in Sports , Gas Chromatography-Mass Spectrometry/methods , Male , Nandrolone/administration & dosage , Tandem Mass SpectrometryABSTRACT
During last decades, the use of natural steroids in racing and food producing animals for doping purposes has been flourishing. The endogenous or exogenous origin of these naturally occurring steroids has since remained a challenge for the different anti-doping laboratories. The administration of these substances to animals is usually made through an intra-muscular pathway with the steroid under its ester form for a higher bioavailability and a longer lasting effect. Detecting these steroid esters would provide an unequivocal proof of an exogenous administration of the considered naturally occurring steroids. A quick analytical method able to detect at trace level (below 50 pg/mL) a large panel of more than 20 steroid esters in serum and plasma potentially used for doping purposes in bovine and equine has been developed. Following a pre-treatment step, the sample is submitted to a solid phase extraction (SPE) before analysis with UPLC-MS/MS. The analytical method's efficiency has been probed through three different in vivo experiments involving testosterone propionate intra-muscular administration to three heifers, 17-estradiol benzoate intra-muscular administration to a bull and a heifer and nandrolone laurate intra-muscular administration to a stallion. The results enabled detecting the injected testosterone propionate and 17-estradiol benzoate 2 and 17 days, respectively, post-administration in bovine and nandrolone laurate up to 14 days post-administration in equine. The corresponding elimination profiles in bovine serum and equine plasma have been established. The first bovine experiment exhibited a maximal testosterone propionate concentration of 400 pg/mL in one of the three heifer serum within 5h post-administration. The second bovine experiment reported a maximal 17-estradiol benzoate concentration of 480 pg/mL in the same matrix recorded 9 days after its administration. The last equine experiment resulted in a maximal nandrolone laurate concentration of 440 pg/mL in horse plasma 24h after administration.
Subject(s)
Cattle/blood , Chromatography, High Pressure Liquid/veterinary , Doping in Sports , Horses/blood , Steroids/blood , Tandem Mass Spectrometry/veterinary , Acetates/chemistry , Acetonitriles/chemistry , Animals , Chromatography, High Pressure Liquid/methods , Estrenes/blood , Hydrogen-Ion Concentration , Linear Models , Male , Reproducibility of Results , Solid Phase Extraction/methods , Solid Phase Extraction/veterinary , Steroids/chemistry , Tandem Mass Spectrometry/methods , Testosterone Propionate/bloodABSTRACT
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.
Subject(s)
Cholinergic Antagonists/blood , Cholinergic Antagonists/urine , Chromatography, High Pressure Liquid/methods , Horses/blood , Horses/urine , Ipratropium/blood , Ipratropium/urine , Animals , Sensitivity and Specificity , Solid Phase Extraction/methods , Spectrometry, Mass, Electrospray Ionization/methodsSubject(s)
Anti-Inflammatory Agents, Non-Steroidal/urine , Clonixin/analogs & derivatives , Doping in Sports/prevention & control , Food Contamination , Horses/urine , Housing, Animal , Poaceae/chemistry , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Anti-Inflammatory Agents, Non-Steroidal/blood , Clonixin/administration & dosage , Clonixin/blood , Clonixin/urine , Drug Administration Schedule/veterinary , Female , Gas Chromatography-Mass Spectrometry/veterinary , Horses/blood , MaleABSTRACT
A sensitive method using LC/ESI-MS(n) has been developed on a quadrupole linear ion trap mass analyser for the detection of nine ß(2) agonists (cimaterol, clenbuterol, fenoterol, formoterol, mabuterol, terbutaline, ractopamine, salbutamol and salmeterol) in horse urine. The method consists of solid-phase extraction on CSDAU cartridges before analysis by LC/ESI-MS(n) . The efficiency of extraction combined with the sensitivity and the selectivity of MS(n) allowed the detection of these compounds at pg/mL levels. Administration studies of fenoterol and formoterol are reported and show their possible detection after inhalation. The method is applicable for screening and confirmatory analysis.
Subject(s)
Adrenergic beta-Agonists/urine , Chromatography, High Pressure Liquid/methods , Doping in Sports/prevention & control , Ethanolamines/urine , Horses/urine , Performance-Enhancing Substances/urine , Spectrometry, Mass, Electrospray Ionization/methods , Adrenergic beta-Agonists/chemistry , Animals , Ethanolamines/chemistry , Performance-Enhancing Substances/chemistry , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
LC/ESI-MS n 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.
Subject(s)
Eleutherococcus/chemistry , Iridoids/urine , Pedaliaceae/chemistry , Plant Extracts/urine , Animals , Chromatography, Liquid/methods , Horses , Mass Spectrometry/methodsABSTRACT
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.
Subject(s)
Androstadienes/administration & dosage , Androstadienes/analysis , Feces/chemistry , Testosterone/analogs & derivatives , Testosterone/analysis , Animals , Female , Gas Chromatography-Mass Spectrometry , Horses , Male , Tandem Mass SpectrometrySubject(s)
Animal Husbandry , Anti-Inflammatory Agents, Non-Steroidal/pharmacokinetics , Horses/metabolism , Meclofenamic Acid/pharmacokinetics , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Anti-Inflammatory Agents, Non-Steroidal/blood , Anti-Inflammatory Agents, Non-Steroidal/urine , Bedding and Linens , Doping in Sports , Female , Infusions, Intravenous/veterinary , Male , Meclofenamic Acid/administration & dosage , Meclofenamic Acid/blood , Meclofenamic Acid/urine , Urinalysis/veterinarySubject(s)
Androgens/pharmacokinetics , Estranes/urine , Gas Chromatography-Mass Spectrometry/veterinary , Horses/metabolism , Nandrolone/pharmacokinetics , Androgens/metabolism , Androgens/urine , Animals , Area Under Curve , Biomarkers/urine , Gas Chromatography-Mass Spectrometry/methods , Horses/urine , Male , Nandrolone/metabolism , Nandrolone/urine , Reference ValuesABSTRACT
Rules of horse racing stipulate that pregnant mares may compete under definite conditions of date, because early pregnant status may be misused for the sake of enhancing physical performance by putative anabolic steroid action. Screening for pregnancy is generally performed by plasma equine gonadotrophin (eCG) immunoassay, which covers the period between Days 40 and 120. In common screening for urinary anabolic steroids performed by gas chromatography-mass spectrometry, inclusion of two complementary criteria, i.e. the evaluation of total conjugates of 5(10)-estrene-3beta,17alpha-diol (EED) and estrone (E1), can easily be performed. Although EED and E1 have no anabolic property per se in the horse, assessing these two markers may be helpful in the period comprised between Days 70 and 250, thereby prolonging the detection period behind that of eCG. Peak values of EED and E1 are then attained, so that visual inspection of chromatographic tracings remains in general sufficient as a diagnostic tool. Comparison of EED and E1 during pregnancy and in an estrus cycle indicates a drastic difference in the attained excretion values, attributable to either the placenta or the ovarian follicle. The identity of EED has been proven by GC-MS(n) in urine and in placental tissue.
Subject(s)
Anabolic Agents/urine , Estrenes/urine , Estrone/urine , Horses/urine , Animals , Doping in Sports , Female , Gas Chromatography-Mass Spectrometry , Placenta , PregnancyABSTRACT
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.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/analysis , Doping in Sports , Feces/chemistry , Horses , Acetone/chemistry , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacokinetics , Desiccation , Female , Male , Methanol/chemistry , Methylene Chloride/chemistry , Solvents/chemistry , Specimen HandlingABSTRACT
REASONS FOR PERFORMING STUDY: Presence of drugs is completely prohibited in post racing urine samples by most of racing and competition authorities, even if environmental contamination might occur. OBJECTIVES: To assess the daily dose of several contaminants absorbed through the diet that would result in detectable concentrations in urine. METHODS: Caffeine, theobromine, theophylline, atropine, scopolamine, bufotenine, DMT or morphine were administered orally to 6 horses, in different dosages, for 3 days before their urine was sampled for regular anti-doping tests. RESULTS: Theobromine, theophylline, bufotenine and morphine daily intake >10 mg, 2 mg, 10 mg and 200 microg, respectively, by a performance horse, were found to result in detectable urinary concentrations. At the 2 tested doses, atropine (5 and 15 mg) and dimethyltryptamine (3 and 10 mg) were not detected in urine. For caffeine and scopolamine, even the lowest dosage tested (5 mg/horse/day and 2 mg/horse/day respectively) induced detectable concentrations of the molecule in urine. CONCLUSIONS: Horses fed dietary contaminants, even at level much below the effective dosage, may be positive to antidoping urine analysis. Further research is needed to gain more confident results on a daily safe intake for caffeine and scopolamine. POTENTIAL RELEVANCE: Selection of feed materials appears to be of great importance to prevent non voluntary positive result to anti-doping tests.
Subject(s)
Food Contamination/analysis , Horses/urine , Physical Conditioning, Animal/physiology , Animals , Atropine/administration & dosage , Atropine/urine , Bufotenin/administration & dosage , Bufotenin/urine , Caffeine/administration & dosage , Caffeine/urine , Cross-Over Studies , Doping in Sports , Dose-Response Relationship, Drug , Horses/metabolism , Morphine/administration & dosage , Morphine/urine , N,N-Dimethyltryptamine/administration & dosage , N,N-Dimethyltryptamine/urine , Scopolamine/administration & dosage , Scopolamine/urine , Theobromine/administration & dosage , Theobromine/urine , Theophylline/administration & dosage , Theophylline/urineABSTRACT
REASONS FOR PERFORMING STUDY: Hyaluronic acid (HA) is an endogenous glycosaminoglycan used in the treatment of joint diseases, but medication control is required by horseracing authorities. Therefore, a medication control policy needs to be established. OBJECTIVES: To establish physiological plasma HA concentrations in post race horses, determine the HA endogenous production rate and document the disposition of HA after i.v. and intra-articular hyaluronic acid administration at recommended therapeutic doses. METHODS: Hyaluronan concentrations in plasma were determined using an ELISA specific test; concentrations in synovial fluid were determined using a radiometric binding assay. RESULTS: The overall mean plasma HA concentration in 120 post competition horses was 89 ng/ml. In a group of 6 experimental horses, synovial fluid control concentration was 328+/-112 microg/ml. After i.v. sodium hyaluronate administration (37.8 mg in toto), the terminal half-life was very short (43+/-29 mins) and after a delay of 3 h, the plasma concentration returned to control values. The endogenous HA production rate was 33-164 mg in toto per day, i.e. 1-4 times the recommended i.v. daily dose. Twenty-four hours after intra-articular administration, HA concentration was not significantly different from control values (328+/-112 microg/ml). CONCLUSIONS AND POTENTIAL RELEVANCE: Due to the rapid disappearance of HA from plasma after i.v. administration and from the joint after intra-articular administration, long-term detection needs a more appropriate approach to be developed.
Subject(s)
Horses/metabolism , Hyaluronic Acid , Synovial Fluid/chemistry , Analysis of Variance , Animals , Cross-Over Studies , Enzyme-Linked Immunosorbent Assay/veterinary , Female , Horses/blood , Hyaluronic Acid/biosynthesis , Hyaluronic Acid/blood , Hyaluronic Acid/pharmacokinetics , Injections, Intra-Articular/veterinary , Injections, Intravenous/veterinary , Male , Physical Conditioning, Animal/physiology , Radiometry , Random Allocation , Reference ValuesABSTRACT
The dietary supplements 19-norandrostenedione and 19-norandrostenediol are potential metabolic precursors of nandrolone. They are considered by law in the United States as prohormones without proven therapeutic, curative or diagnostic properties, and therefore available as over-the-counter drugs. Oral dosages of 0.1-1 mg/kg body weight were readily absorbed in the equine intestinal tract and thereby led to urinary excretion of drastically increased 5alpha-estrane-3beta,17alpha-diol conjugates, which are known to be final metabolites of nandrolone. The actual rules for detection of illicit nandrolone administration to the horse have been found applicable for the detection of surreptitious oral 19-norandrostenedione and 19-norandrostenediol supplementation. Secondary markers of these administrations were high-level excretions of conjugated nandrolone, epinandrolone, 19-noretiocholanolone and 19-norepiandrosterone. No significant increase of circulating, biologically active nandrolone could be firmly evidenced, and it is therefore unclear to what extent continuous long-term administrations may have anabolic action.
Subject(s)
Androstenediol/metabolism , Androstenedione/metabolism , Dietary Supplements , Doping in Sports , Androstenediol/blood , Androstenediol/urine , Androstenedione/analogs & derivatives , Androstenedione/blood , Androstenedione/urine , Animals , Horses , HumansABSTRACT
Insulin-like growth factor-I (IGF -I) is likely to be an indicator of somatotropin (ST) administration in the horse. To investigate the different ways ST administration may be detected, the following aspects of IGF -I concentrations in plasma were studied: (i) the daily variation; (ii) variation following a treadmill test; (iii) concentrations at rest and after exercise; and (iv) concentrations in plasma from two young horses and two adults treated with methionyl equine somatotropin (e ST). In the population of horses at rest, IGF -I mean concentration (SEM) was 261 (104) ng ml(-1). In post race samples, IGF -I mean concentration was 187 (100) ng ml(-1). All of these data indicate that exercise does not modify IGF -I concentration in plasma. The magnitude of the increase in IGF -I following administration of e ST differed according to the age of the horses. The critical value of 700 ng ml(-1)was exceeded for 1 day in adult horses and for at least 11 days in young horses. These results show that IGF -I has potential as an indirect marker of ST administration in horses.
Subject(s)
Doping in Sports , Growth Hormone/administration & dosage , Horses/blood , Insulin-Like Growth Factor I/analysis , Age Factors , Animals , Biomarkers/analysis , Circadian Rhythm/physiology , Exercise Test/veterinary , Female , Growth Hormone/blood , Insulin-Like Growth Factor I/metabolism , Jugular Veins , Male , Physical Conditioning, Animal/physiology , Reference Values , Rest/physiologyABSTRACT
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.