Quantitative analysis of a quaternary ammonium drug: ipratropium bromide by LC/ESI-MS(n) in horse plasma and urine.

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.

Analysis of ß-agonists by HPLC/ESI-MS(n) in horse doping control.

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.

Pharmacokinetics of tiludronate in horses: A field population study.

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.

Urinary excretion of 5(10)-estrene-3beta,17alpha-diol and estrone by the female horse: complementary indicators of early pregnancy screened with regard to a putative anabolic doping practice.

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.

Use of accelerating solvent extraction for detecting non-steroidal anti-inflammatory drugs in horse feces.

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.

Mouldy feed: A possible explanation for the excretion of anabolic-androgenic steroids in horses.

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.

In vitro simulation of the equine hindgut as a tool to study the influence of phytosterol consumption on the excretion of anabolic-androgenic steroids in horses.

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.

Human nutritional supplements in the horse: comparative effects of 19-norandrostenedione and 19-norandrostenediol on the 19-norsteroid profile and consequences for doping control.

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.

Human nutritional supplements in the horse. Dehydroepiandrosterone versus androstenedione: comparative effects on the androgen profile and consequences for doping analysis.

Dehydroepiandrosterone (DHEA) and androstenedione are weak androgens, which need conversion to more potent testosterone in order to enhance anabolic action. Consequences of oral dosing at 1 mg/kg on the urinary and plasma androgen profile of mare and gelding have been evaluated with an analytical method involving conjugate fractionation and selective hydrolysis, group separation, and quantitation by gas chromatography-mass spectrometry with selected ion monitoring of trimethylsilyl ethers. Peak levels of testosterone total conjugates in urine (range 300-6000 microg/L) were attained a few hours after dosing. Renal clearance was fast, so the testosterone detection period lasted only 20 to 33 h, the longest time being generated by androstenedione. The urinary testosterone/epitestosterone ratio for detection of exogenous testosterone in the mare was inoperative after DHEA administration because there was a concomitant increase of epitestosterone, which thereby acted as a masking agent. Androstanediols and androstenediols, as well as some 17-ketosteroids, were additional markers. A transient increase of circulating free testosterone has been evidenced, and this would support possible anabolic/androgenic action by supplementation with DHEA and androstenedione along the oral route.

GC/MS confirmatory method for etorphine in horse urine.

A highly sensitive procedure for GC/MS determination of etorphine in horse urine is described. This assay provides both specificity and reliability and is particularly well suited for the confirmation of radioimmunoassay screening procedures usually used for etorphine. After solvent extraction and purifications, the etorphine is characterized as a pentafluoroacetic derivative (PFAA) by using mass fragmentography. The detection limit is 0.1 ng/mL in urine; the coefficient of variation of the estimations is 10.9%. The procedure has been validated after on-field administration of 5 to 90 micrograms of etorphine to five thoroughbred horses (10 to 180 ng/kg).

Testosterone administration to mares: criteria for detection of testosterone abuse by analysis of metabolites in plasma and urine.

A pharmacological dose of a long-acting testosterone ester, testosterone hexahydrobenzoate, was administered intramuscularly to two mares. The time course for some characteristic metabolites in blood and urine was then studied using an analytical method based on gas chromatography-mass spectrometry associated with stable isotope dilution. Among the plasma analytes, testosterone glucuronide was found to be the most adequate indicator for the monitoring of exogenous testosterone up to 2 weeks postadministration if a threshold value of 200 ng/L was applied. In urine, the simultaneous measurement of the concentrations of testosterone sulfate (TS) and epitestosterone sulfate (ES) allowed the calculation of the concentration ratio, TS/ES, which was independent of urine flow and which offered the possibility of detecting testosterone misuse 20 to 30 days after dosing if a tentative threshold value of 8 was adopted. In addition to this ratio, particularly when the TS/ES ratio was close to the cutoff point, it seemed advisable to take into account the concentrations of 5 alpha-androstane-3 beta, 17 alpha-diol (glucuronide) and its 17 beta-isomer (sulfate), which should not exceed 50 micrograms/L.

IGF -I plasma concentrations in non-treated horses and horses administered with methionyl equine somatotropin.

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.

Hyaluronan in horses: physiological production rate, plasma and synovial fluid concentrations in control conditions and following sodium hyaluronate administration.

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.

Cortisol concentrations in post competition horse urine: a French and British survey.

The purpose of the present report was to estimate the population parameters of cortisol concentrations in urine, an endogenous hormone used as a 'doping' agent and for which an international threshold (1.0 micrograms/ml) has been proposed. Two data bases (French and UK) corresponding to 112 and 142 samples, respectively were considered. Urine was collected under specific post competition conditions. Cortisol concentrations were obtained by validated methods (HPLC for the French samples, and GC-MS for UK samples). No difference was observed between the 2 data sets and statistical analyses were carried out on the two merged files. The overall geometric mean cortisol concentration was 48 ng/ml. Distribution was not Gaussian. A log-normal distribution was not rejected (for P > 0.05). Using the log-normal distribution, it was calculated that the probability of exceeding a cortisol concentration in urine of 1.0 micrograms/ml was 1.1 x 10(-4). It was concluded that the actual international threshold is specific i.e. robust with regard to the risk of erroneously declaring an unmedicated horse as positive.

New approaches to detect cortisol administration in the horse.

The cortisol threshold concentration of 1.0 microg/ml in horse urine adopted by the International Federation of the racing Authorities in 1994 is specific. However, an increase in the sensitivity for the detection of cortisol administration would be helpful. Previous studies have shown that 20beta-dihydrocortisol concentration in urine would be a good indicator of cortisol administration. The purpose of the present work was to estimate the population parameters and the critical values of 20beta-dihydrocortisol and 20beta-dihydrocortisone concentration in urine compared with that of cortisol. Using the same probability (1.1 x 10(-4)) which was used for the establishment of the official cortisol threshold, the critical values of 5000 ng/ml for 20beta-dihydrocortisol and 350 ng/ml for 20beta-dihydrocortisone were obtained. Considering these 2 critical values for 20beta-dihydrocortisol and 20beta-dihydrocortisone, the time during which a horse could be declared positive is significantly increased.

[Determination of the prothrombin time and of the activated cephalin time wit semi-automatic Electra 600 apparatus (author's transl)]. / Détermination du temps de Quick et du temps de céphaline activée avec l' appareillage semi-automatique Electra 600.

This work studies the quality of the response of the Electra 600 for the determination of the prothrombin time (PT) and the activated cephalin time (ACT). The intra-serial precision was good for the PT and the ACT with normal or pathological plasmas (CV 2%). The precision was excellent for the PT (r = 0.98; Electra 600-fibrometer). The correlation coefficient varies between 0.85 and 0.95 for the ACT depending on the nature of the activator chosen. Lactescent plasmas having a protein concentration greater than 85 g/l should be treated manually for measurement of the PT. No interference was noted with other biological substances: eg glucose, bilirubin, hemoglobin. The increasing addition of heparin indicates a correct sensitivity of the response with Electra 600 both for short and long ACT. Autoanalysis renders the determinations of PT and ACT independant of the manipulator, that of the PT being much more rapid than with the manual method

[Enzyme-immuno assay for total estrogens and human placental lactogen. Comparison with radio-immuno-assay in normal pregnancy-monitoring (author's transl)]. / Dosages sériques immuno-enzymatiques des estrogènes totaux et de l'hormone lactogène placentaire. Comparaison avec la méthode radio-immunologique dans la surveillance de la grossesse normale.

The concentrations of estrogens (E) and human placental lactogen (HLP) are estimated in sera by radio immuno-assay (RIA) and enzyme-immuno-assay (EIA). Statistical data indicate mean intra-assay variation coefficients of 7% and 12% for E and HLP tests, respectively. The correlation coefficient (RIA/EIA) are found higher than 0,9% for both hormonal assays. The dilution curves obtained by RIA and EIA are similar. However, Student'test gives a significant difference for E determination. In fact, total E and E 3 only are measured by EIA and RIA, respectively. In most cases biological interferences are negligible except for HLP in presence of higher protein or haemoglobin levels. RIA and EIA are performed to study serum HLP and E levels throughout normal pregnancies. Results allow to use EIA for HLP and E evaluations in pregnancy-monitoring.

Monitoring the endogenous steroid profile disruption in urine and blood upon nandrolone administration: An efficient and innovative strategy to screen for nandrolone abuse in entire male horses.

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.

HPLC/ESI-MS(n) method for non-amino bisphosphonates: application to the detection of tiludronate in equine plasma.

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).