High-performance Liquid Chromatography (HPLC) is an analytical technique used to separate, identify, and quantify components in a mixture. In equine research, HPLC is employed to analyze various biological samples from horses, such as blood, urine, and tissue, to detect and measure compounds like drugs, metabolites, and nutritional components. This technique is valued for its precision, sensitivity, and ability to handle complex matrices, making it suitable for pharmacokinetic studies, monitoring therapeutic drug levels, and assessing metabolic profiles in horses. The application of HPLC in equine studies provides insights into drug metabolism, nutritional status, and the biochemical effects of different treatments. This page compiles peer-reviewed research studies and scholarly articles that explore the applications, methodologies, and findings of HPLC in the context of equine science.
Letendre L, Kvaternick V, Tecle B, Fischer J.A rugged, sensitive and efficient liquid chromatography-tandem mass spectrometry method was developed and validated for the quantitative analysis of firocoxib in urine from 5 to 3000 ng/mL and in plasma from 1 to 3000 ng/mL. The method requires 200 microL of either plasma or urine and includes sample preparation in 96-well solid phase extraction (SPE) plates using a BIOMEK 2000 Laboratory Automated Workstation. Chromatographic separation of firocoxib from matrix interferences was achieved using isocratic reversed phase chromatography on a PHENOMENEX LUNA Phenyl-Hexyl column. The mobile phase w...
Nagata S, Kurosawa M, Kuwajima M.A new enzyme immunoassay (EIA) for the measurement of furosemide in horse plasma is described. The lower limit of detection of this EIA method was 7.8 ng/ml. The intra-and inter-assay coefficients of variation ranged from 2.5% to 4.9% and 7.5% to 9.8%, respectively. Cross-reactivity with other compounds was not observed. There was a high correlation (r2=0.987) between the high-performance liquid chromatography and EIA results obtained for furosemide concentrations in horse plasma. These results indicate that the newly developed EIA method is useful for the quantitative analysis of furosemide i...
Coenen M, Appelt K, Niemeyer A, Vervuert I.Gelatin supplementation is a common measure in an attempt to assist cartilage repair, but little scientific evidence exists to support its efficacy. Objective: To investigate the effects of gelatin administration on post prandial homeostasis. Methods: Twelve Standardbred horses (mean 404 kg bwt) were fed a hay-concentrate diet supplemented by soy bean meal and oil (control [C], n = 6) or with the addition of 60 g gelatin/day (G, n = 6). The horses were trained by an alternate order of interval and prolonged exercise every second day. The velocities of the treadmill corresponding to 2 and 10 mm...
Lindberg JE, Essén-Gustavsson B, Dahlborn K, Gottlieb-Vedi M, Jansson A.High starch intakes increase the risk for metabolic disorders and therefore alternative feedstuffs are of interest. High-fat oat varieties have a lower starch and higher energy content than regular oats and may therefore be useful in this context. Objective: Feeding high fat oats causes no adverse effects on the response to exercise and that the total amount of oats offered could be reduced compared to feeding with regular oats. Methods: Twelve Standardbred trotters were fed regular oats (diet C), high-fat oats (F), and a mixture (50:50) of C and F (M), together with haylage (30:70), in a Lati...
Baragli P, Ducci M, Gatta D, Gazzano A, Sighieri C.Catecholamines (CAT) play an important role in modulating the response to exercise. But the kinetics of CAT changes during exercise are difficult to study due to their short biological half-life. Objective: Learning about variations in plasma CAT levels during training could furnish new information regarding sweating, redistribution of blood flow and energy metabolism. Methods: Four untrained Standardbreds, adapted to treadmill work, were used to determine the influence of training on plasma adrenaline (A) and noradrenaline (NA) kinetics. Horses underwent a standardised exercise test (SET) on ...
Miclo L, Girardet JM, Egito AS, Mollé D, Martin P, Gaillard JL.Highly phosphorylated casein with a low molecular mass was isolated from Haflinger mare's milk by RP-HPLC. It accounts for 4.0% of the casein content. Its mass was determined by LC-ESI-MS before and after treatment by alkaline phosphatase. The molecular mass found for the apo-form (10,591 +/- 2 Da) is in agreement with its primary structure, which was established by ESI-MS/MS from tryptic peptides. It appeared that this short protein (94 amino acid residues) is an internally truncated form of the full-length equine beta-casein (226 residues). This low-Mr variant of equine beta-casein displays ...
Soma LR, Uboh CE, Guan F, McDonnell S, Pack J.Anabolic steroids (ABS) boldenone (BL; 1.1 mg/kg) and stanozolol (ST; 0.55 mg/kg) were administered i.m. to horses and the plasma samples collected up to 64 days. Anabolic steroids and androgenic steroids (ANS) in plasma were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The limit of detection of all analytes was 25 pg/mL. The median absorption (t1/2 partial differential) and elimination (t1/2e) half-lives for BL were 8.5 h and 123.0 h, respectively, and the area under the plasma concentration-time curve (AUCho) was 274.8 ng.h/mL. The median t1/2e for ST was 82.1 ...
Keller KM, Queiroz BD, Keller LA, Ribeiro JM, Cavaglieri LR, Pereyra ML, Dalcero AM, Rosa CA.Feed contamination can lead to nutrient losses and detrimental effects on animal health and production. The purposes of this study were to investigate the mycobiota in equine mixed feeds and to determine natural contamination with aflatoxin B1 (AFB1) and fumonisin B1 (FB1). Fungal enumeration of equine feed samples was done. A commercially available enzyme-linked immunosorbent assay kit was applied to quantify AFB1 and FB1. A comparison between ELISA and HPLC was carried out. Feed mould counts ranged from <1 x 10(2) to 1 x 10(5) cfu/g. The most frequent genus isolated was Aspergillus (40.54...
Thomasy SM, Mama KR, Whitley K, Steffey EP, Stanley SD.In order to evaluate its potential as an adjunct to inhalant anaesthesia in horses, the pharmacokinetics of fentanyl must first be determined. Objective: To describe the pharmacokinetics of fentanyl and its metabolite, N-[1-(2-phenethyl-4-piperidinyl)maloanilinic acid (PMA), after i.v. administration of a single dose to horses that were awake in Treatment 1 and anaesthetised with isoflurane in Treatment 2. Methods: A balanced crossover design was used (n = 4/group). During Treatment 1, horses received a single dose of fentanyl (4 microg/kg bwt, i.v.) and during Treatment 2, they were anaesthet...
Maes A, Weiland L, Sandersen C, Gasthuys F, De Backer P, Croubels S.A sensitive method for the quantification of lidocaine and its metabolites, monoethylglycinexylidide (MEGX) and glycinexylidide (GX), in animal plasma using high-performance liquid chromatography combined with electrospray ionization mass spectrometry is described. The sample preparation includes a liquid-liquid extraction with methyl tert-butylmethyl ether after addition of 2M sodium hydroxide. Ethylmethylglycinexylidide (EMGX) is used as an internal standard. For chromatographic separation, an ODS Hypersil column was used. Isocratic elution was achieved with 0.01 M ammonium acetate and aceto...
Davis JL, Papich MG, Morton AJ, Gayle J, Blikslager AT, Campbell NB.The purpose of this study was to determine the pharmacokinetics of etodolac following oral and intravenous administration to six horses. Additionally, in vitro cyclooxygenase (COX) selectivity assays were performed using equine whole blood. Using a randomized two-way crossover design, horses were administered etodolac (20 mg/kg) orally or intravenously, with a minimum 3-week washout period. Plasma samples were collected after administration for analysis using high pressure liquid chromatography with ultraviolet detection. Following intravenous administration, etodolac had a mean plasma half-li...
Machnik M, Hegger I, Kietzmann M, Thevis M, Guddat S, Schänzer W.The Federation Equestre Internationale has permitted the use of altrenogest in mares for the control of oestrus. However, altrenogest is also suspicious to misuse in competition horses for its potential anabolic effects and suppression of typical male behaviour, and thus is a controlled drug. To investigate the pharmacokinetics of altrenogest in horses we conducted an elimination study. Five oral doses of 44 mug/kg altrenogest were administered to 10 horses at a dose interval of 24 h. Following administration blood and urine samples were collected at appropriate intervals. Altrenogest concentr...
Portier K, Guichardant M, Debouzy JC, Crouzier D, Geraud I, Kirschvink N, Lekeux P, Fellmann N, Coudert J.Whether direct exposure to different concentrations (0%, 13%, 100%) of oxygen may affect horse erythrocyte membrane fluidity (EMF) and fatty acid (FA) composition was studied during 1 (T60) and 2h (T120) exposure. EMF was investigated at the head group level and hydrophobic core thanks to phosphorus nucleus 31 ((31)P) nuclear magnetic resonance ((31)P NMR) and electronic paramagnetic resonance (EPR) using two spin probes: 5-nitroxydestearic acid and 16-doxylstearic acid. Lipid structure of the membranes was studied by gas liquid chromatography. 4-Hydroxy-2E-nonenal was also analyzed as a marke...
Christmann U, Livesey LC, Taintor JS, Waldridge BM, Schumacher J, Grier BL, Hite RD.Lung surfactant function and composition are varied and adapted to the specific respiratory physiology of all mammalian species. Objective: Lung surfactant function and composition are different in neonatal foals as compared to adult horses. Methods: Six adult horses, 7 term foals (<24 hours old), and 4 premature foals were used. Animals were part of the Auburn University teaching herd except for 3 client-owned premature foals. Methods: Bronchoalveolar lavage fluid (BALF) was obtained from all animals. Ultracentrifugation of cell-free BALF separated surfactant into crude surfactant pellets ...
Houghton E, Teale P, Dumasia MC.For almost two decades we have known that enzymatic hydrolysis of "normal" urine samples from the entire male horse using Escherichia coli (E. coli) followed by solvolysis (ethyl acetate:methanol:sulphuric acid) results in the detection of significant amounts of estr-4-ene-3,17-dione (19-norandrost-4-ene-3,17-dione) along with estr-4-en-17beta-ol-3-one (19-nortestosterone, nandrolone) in extracts of the hydrolysed urine and that both steroids are isolated from the solvolysis fraction. This solvolysis process is targeted at the steroid sulphates. Also we have shown that 19-norandrost-4-ene-3,17...
Murchie TA, Macpherson ML, LeBlanc MM, Luznar S, Vickroy TW.Most current treatments for placentitis in mares are empirical with few control studies to evaluate their effectiveness. Objective: To monitor drug concentrations in allantoic fluid of pregnant pony mares using in vivo microdialysis and establish if this method would be useful for determining allantoic concentrations of drugs in normal mares and those with placentitis. Methods: Five late gestational pony mares had microdialysis probes inserted into the allantoic fluid using transabdominal ultrasound-guided allantocentesis. Single injections of penicillin G (22,000 u/kg), gentamicin (6.6 mg/kg ...
Gerber R, Naudé TW, de Kock SS.Two out of a group of 23 mares exposed to tef hay contaminated with Datura ferox (and possibly D. stramonium) developed colic. The 1st animal was unresponsive to conservative treatment, underwent surgery for severe intestinal atony and had to be euthanased. The 2nd was less seriously affected, responded well to analgesics and made an uneventful recovery. This horse exhibited marked mydriasis on the first 2 days of being poisoned and showed protracted, milder mydriasis for a further 7 days. Scopolamine was chemically confirmed in urine from this horse for 3 days following the colic attack, whil...
Stanley SM, Wee WK, Lim BH, Foo HC.Direct-injection LC-LC hybrid tandem MS methods have been developed for undertaking broad-based screening for acidic drugs in protein-precipitated plasma and neutral doping agents in equine urine. In both analyses, analytes present in the matrix were trapped using a HLB extraction column before being refocused and separated on a Chromolith RP-18e monolithic analytical column using a controlled differential gradient generated by proportional dilution of the first column's eluent with water. Each method has been optimised by the adoption of a mobile phase and gradient that was tailored to enhanc...
Milligan M, Kukanich B, Beard W, Waxman S.Lidocaine is administered as an intravenous infusion to horses for a variety of reasons, but no study has assessed plasma lidocaine concentrations during a 12-h infusion to horses. The purpose of this study was to evaluate the plasma concentrations and pharmacokinetics of lidocaine during a 12-h infusion to postoperative horses. A second purpose of the study was to evaluate the in vitro plasma protein binding of lidocaine in equine plasma. Lidocaine hydrochloride was administered as a loading dose, 1.3 mg/kg over 15 min, then by a constant rate IV infusion, 50 microg/kg/min to six postoperativ...
Hackl S, van den Hoven R, Zickl M, Spona J, Zentek J.Few data are available on post-prandial changes of plasma amino acids (AAs) in horses and on the repeatability and the individual variance on different sampling days. The objective of the present study was to measure pre- and post-prandial concentrations of plasma AA in 10 yearling horses. Blood samples were taken on days 1 and 40 of the study before feeding of hay, oats and soya meal and over an 8 h post-prandial period in 2-h intervals. The plasma AAs were measured by high-pressure liquid chromatography after ortho-phthalaldehyde derivatization. Mean fasting concentrations of the AAs were no...
Leung GN, Leung DK, Wan TS, Wong CH.This paper describes a high throughput LC-MS-MS method for the screening of 75 basic drugs in equine plasma at sub-ppb levels. The test scope covers diversified classes of drugs including some alpha- and beta-blockers, alpha- and beta-agonists, antihypotensives, antihypertensives, analgesics, antiarrhythmics, antidepressants, antidiabetics, antipsychotics, antiulcers, anxiolytics, bronchodilators, CNS stimulants, decongestants, sedatives, tranquilizers and vasodilators. A plasma sample was first deproteinated by addition of trichloroacetic acid. Basic drugs were then extracted by solid-phase e...
Colas C, Garcia P, Popot MA, Bonnaire Y, Bouchonnet S.A method has been developed for the analysis and characterization in equine urine and plasma of iridoid glycosides: harpagide, harpagoside and 8-para-coumaroyl harpagide, which are the main active principles of Harpagophytum, a plant with antiinflammatory properties. The method involves liquid chromatography coupled with positive electrospray ionization mass spectrometry. The addition of sodium or lithium chloride instead of formic acid in the eluting solvent has been studied in order to enhance the signal and to modify the ion's internal energy. Fragmentation pathways and associated patterns ...
Womble AY, Giguère S, Lee EA, Vickroy TW.To determine pharmacokinetics of clarithromycin and concentrations in body fluids and bronchoalveolar (BAL) cells of foals. Methods: 6 healthy 2-to 3-week-old foals. Methods: In a crossover design, clarithromycin (7.5 mg/kg) was administered to each foal via IV and intragastric (IG) routes. After the initial IG administration, 5 additional doses were administered IG at 12-hour intervals. Concentrations of clarithromycin and its 14-hydroxy metabolite were measured in serum by use of high-performance liquid chromatography. A microbiologic assay was used to measure clarithromycin activity in seru...
Mills PC, Cross SE.The rate and regional differences for the penetration of fentanyl through equine skin was investigated in vitro using a commercial transdermal therapeutic system (TTS) or 'patch'. Skin collected from the thorax, groin and leg (dorsal metacarpal) regions of five horses was placed in diffusion cells and a fentanyl TTS applied to each skin sample. Drug penetration through each skin sample over 48 h measured using high performance liquid chromatography (HPLC). Cumulative penetration (microg/cm2) was plotted against time (h) and used to regress the steady state flux (microg/cm2/h) of fentanyl throu...
Pfau T, Witte TH, Wilson AM.During locomotion cyclical interchange between different forms of mechanical energy enhances economy; however, 100% efficiency cannot be achieved and ultimately some mechanical work must be performed de novo. There is a metabolic cost associated with fluctuations in mechanical energy, even in the most efficient animals. In this study we investigate the exchanges between different forms of mechanical energy involved in high-speed gallop locomotion in Thoroughbred race horses during over-ground locomotion using innovative, mobile data collection techniques. We use hoof-mounted accelerometers to ...
Fernández-Varón E, Ayala I, Marín P, Carrión A, Martos N, Escudero E, Cárceles CM.Danofloxacin is a fluoroquinolone developed for veterinary medicine showing an excellent activity. However, danofloxacin pharmacokinetics profile have not been studied in horses previously. Objective: To study the pharmacokinetics following i.v., i.m. and intragastric (i.g.) administration of 1.25 mg/kg bwt danofloxacin to 6 healthy horses. Methods: A cross-over design was used in 3 phases (2 x 2 x 2), with 2 washout periods of 15 days (n = 6). Danofloxacin (18%) was administered by i.v. and i.m. routes at single doses of 1.25 mg/kg bwt. For i.g. administration an oral solution was prepared an...
Fidani M, Casagni E, Montana M, Pasello E, Pecoraro C, Gambaro V.Bacteria frequently found in equine urine samples may cause degradation of 17beta-OH steroids. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed to evaluate the microbiological contamination of equine urine as a marker of poor storage conditions. Norethandrolone was used as the internal standard, and the linearity, sensitivity, precision and accuracy of the method were evaluated. 17beta-OH oxidation was demonstrated for testosterone, nandrolone, trenbolone and boldenone, but did not occur in alpha-epimers such as alpha-boldenone and epitestosterone, d...
Songsermsakul P, Sontag G, Cichna-Markl M, Zentek J, Razzazi-Fazeli E.The paper describes a method for the sensitive and selective determination of zearalenone and its metabolites in urine, plasma and faeces of horses by high performance liquid chromatography and atmospheric pressure chemical ionisation (APCI) mass spectrometry (MS). While only one step sample clean-up by an immunoaffinity column (IAC) was sufficient for plasma samples, urine and faeces samples had to be prepared by a combination of a solid-phase extraction (SPE) and an immunoaffinity column. The method allows the simultaneous determination of zearalenone and all of its metabolites; alpha-zearal...
Gokbulut C, Akar F, McKellar QA.Fenbendazole (FBZ), oxfendazole (fenbendazole sulphoxide, FBZSO), and albendazole (ABZ) were administered orally to donkeys at 10mg/kg bodyweight. Blood and faecal samples were collected from 1 to 120 h post-treatment. The plasma and faecal samples were analysed by high performance liquid chromatography (HPLC). The parent molecule and its sulphoxide and sulphone (FBZSO(2)) metabolites did not reach detectable concentrations in any plasma samples following FBZ administration. ABZ was also not detected in any plasma samples, but its sulphoxide and sulphone metabolites were detected, demonstratin...
Fernández-Varón E, Cárceles CM, Marín P, Martos N, Escudero E, Ayala I.To study the pharmacokinetics of difloxacin (5 mg/kg) following IV, IM, and intragastric (IG) administration to healthy horses. Methods: 6 healthy mature horses. Methods: A crossover study design with 3 phases was used (15-day washout periods between treatments). An injectable formulation of difloxacin (5%) was administered IV and IM in single doses (5 mg/kg); for IG administration, an oral solution was prepared and administered via nasogastric tube. Blood samples were collected before and at intervals after each administration. A high-performance liquid chromatography assay with fluorescence ...
Gerber R, Naudé TW, de Kock SS.Two out of a group of 23 mares exposed to tef hay contaminated with Datura ferox (and possibly D. stramonium) developed colic. The 1st animal was unresponsive to conservative treatment, underwent surgery for severe intestinal atony and had to be euthanased. The 2nd was less seriously affected, responded well to analgesics and made an uneventful recovery. This horse exhibited marked mydriasis on the first 2 days of being poisoned and showed protracted, milder mydriasis for a further 7 days. Scopolamine was chemically confirmed in urine from this horse for 3 days following the colic attack, whil...
Burton AJ, Giguère S, Warner L, Alhamhoom Y, Arnold RD.Therapeutic drug monitoring in a small number of foals of various ages indicates that the standard adult dose of 6.6 mg/kg bwt q. 24 h for gentamicin is too low and a dose of 12 mg/kg bwt has been proposed. The pharmacokinetics of this dosage in foals and the ages at which this higher dose should be used have not previously been investigated. Objective: To determine the effect of age on the pharmacokinetics of a single 12 mg/kg bwt i.v. dose of gentamicin in foals. Methods: Six healthy foals were given a single i.v. dose of gentamicin at 1-3 days, 2, 4, 8 and 12 weeks of age. Plasma concentrat...
Yamada T, Aoi S, Adachi M, Kamimura T, Higurashi Y, Wada N, Tsuchiya K, Matsuno F.Horses use the transverse gallop in high-speed running. However, different animals use different gaits, and the gait preference of horses remains largely unclear. Horses have fore-aft asymmetry in their body structure and their center of mass (CoM) is anteriorly located far from the center of the body. Since such a CoM offset affects the running dynamics, we hypothesize that the CoM offset of horses is important in gait selection. In order to verify our hypothesis and clarify the gait selection mechanisms by horses from a dynamic viewpoint, we developed a simple model with CoM offset and inves...
Stojiljkovic N, Leroux F, Bubanj S, Popot MA, Paris A, Tabet JC, Junot C.There is an urgent need to implement holistic and untargeted doping control protocols with improved discriminatory power, compared to conventional methods that only target doping agents. Metabolomics, which aims to characterize all metabolites present in biological matrices, could fulfill this need. In this context, the aim of this study was to evaluate the impact of environmental factors on the ability to obtain a metabolic signature of stanozolol administration in horse doping situation. Urine samples from 16 horses breeded in two different places were collected over a one-year period, befor...
Kondampati KD, Saini SPS, Sidhu PK, Anand A, Kumar D, Beesam S, Bedi JS, Kaur R, Bhardwaj R.The pharmacokinetics of ampicillin-cloxacillin, given as single intravenously dose of 10 mg.kg-1 (5 mg.kg-1 of ampicillin plus 5 mg.kg-1 of cloxacillin) was examined in clinically presented Indian thoroughbred horses (n = 6) in order to design appropriate dosing strategies. Drug concentrations in plasma were determined by high performance liquid chromatography (HPLC) and pharmacokinetic parameters were derived by non-compartmental analysis using WinNonlin software. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ampicillin-cloxacillin against qualit...
Plasma clearance of iohexol is a reliable method to assess the glomerular filtration rate (GFR). The relationship between aging and GFR in horses is unclear. To compare GFR estimated by iohexol clearance in horses of different ages. Twenty-one clinically healthy horses were enrolled. Prospective study. Groups: (A) composed by 8 young horses (≤14 years old) with serum creatinine <1.5 mg/dL, (B) by 7 aged horses (≥20 years old) with serum creatinine <1.5 mg/dL, and (C) by 6 aged horses (≥20 years old) with serum creatinine ≥1.5 mg/dL. Iohexol was injected (75.5 mg/kg) through...
Milligan M, Kukanich B, Beard W, Waxman S.Lidocaine is administered as an intravenous infusion to horses for a variety of reasons, but no study has assessed plasma lidocaine concentrations during a 12-h infusion to horses. The purpose of this study was to evaluate the plasma concentrations and pharmacokinetics of lidocaine during a 12-h infusion to postoperative horses. A second purpose of the study was to evaluate the in vitro plasma protein binding of lidocaine in equine plasma. Lidocaine hydrochloride was administered as a loading dose, 1.3 mg/kg over 15 min, then by a constant rate IV infusion, 50 microg/kg/min to six postoperativ...
Nagata S, Kurosawa M, Kuwajima M.A new enzyme immunoassay (EIA) for the measurement of furosemide in horse plasma is described. The lower limit of detection of this EIA method was 7.8 ng/ml. The intra-and inter-assay coefficients of variation ranged from 2.5% to 4.9% and 7.5% to 9.8%, respectively. Cross-reactivity with other compounds was not observed. There was a high correlation (r2=0.987) between the high-performance liquid chromatography and EIA results obtained for furosemide concentrations in horse plasma. These results indicate that the newly developed EIA method is useful for the quantitative analysis of furosemide i...
Mück WM, Henion JD.The performance of microbore high-performance liquid chromatography and capillary zone electrophoresis, both equipped with on-line tandem mass spectrometric detection capability, was evaluated critically for the determination of endogenous amounts of leucine enkephalin and methionine enkephalin in equine cerebrospinal fluid. Using an identical sample clean-up and enrichment procedure, capillary zone electrophoresis-mass spectrometry is limited in its concentration detection capacity owing to its much smaller injection volume. Leucine enkephalin was identified in post-mortem equine cerebrospina...
Villarino NF, Lopez CM, Sams RA, Bayly WM.The primary objective of this study was to assess the disposition of furosemide in Thoroughbred horses treated intravenously with 1 mg/kg of furosemide 4 and 24 h before supramaximal treadmill exercise without and with controlled access to water, respectively. Another objective was to determine whether furosemide was detectable in the plasma of horses after exposure to supramaximal treadmill exercise. Thoroughbred horses (n = 4-6) were administered single intravenous doses of 1 mg/kg of furosemide at 4 and 24 h before supramaximal exercise on a high-speed treadmill, with controlled ...
Liang FT, Granstrom DE, Timoney JF, Shi YF.We report a simple, economical, and efficient protocol for protein purification from cells. First, proteins of cell lysates were separated by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted to protein-blotting membrane. The blots were stained with Coomassie blue or developed by immunoblotting to visualize specific proteins. The bands corresponding to those visible by immunoblotting were excised from the dye-stained blots and subjected to isoelectric focusing. The focused gel was stained with Coomassie blue. Finally, the stained bands were excise...
Houghton E, Teale P, Dumasia MC.For almost two decades we have known that enzymatic hydrolysis of "normal" urine samples from the entire male horse using Escherichia coli (E. coli) followed by solvolysis (ethyl acetate:methanol:sulphuric acid) results in the detection of significant amounts of estr-4-ene-3,17-dione (19-norandrost-4-ene-3,17-dione) along with estr-4-en-17beta-ol-3-one (19-nortestosterone, nandrolone) in extracts of the hydrolysed urine and that both steroids are isolated from the solvolysis fraction. This solvolysis process is targeted at the steroid sulphates. Also we have shown that 19-norandrost-4-ene-3,17...
Knych HK, Corado CR, McKemie DS, Scholtz E, Sams R.Tramadol is a synthetic opioid used in human medicine, and to a lesser extent in veterinary medicine, for the treatment of both acute and chronic pain. In humans, the analgesic effects are owing to the actions of both the parent compound and an active metabolite (M1). The goal of the current study was to extend current knowledge of the pharmacokinetics of tramadol and M1 following oral administration of three doses of tramadol to horses. A total of nine healthy adult horses received a single oral administration of 3, 6, and 9 mg/kg of tramadol via nasogastric tube. Blood samples were collected...
Macpherson ML, Giguère S, Hatzel JN, Pozor M, Benson S, Diaw M, Sanchez LC, Vickroy TW, Tell L, Wetzlich S, Sims J.The objective of this study was to determine the pharmacokinetics of CCFA in mares with placentitis and evaluate the disposition of the drug in fetal fluids, fetal membranes, colostrum, and serum of foals. A secondary objective was to obtain pilot data regarding the efficacy of CCFA for improving foal survival in mares with placentitis. Twelve pregnant pony mares were enrolled in the study, inoculated with Streptococcus zooepidemicus, intracervically and assigned to one of three groups: CEFT (n = 3; administered CCFA only; 6.6 mg/kg, i.m., q96h); COMBO (n = 6; administered combination therapy ...
Heffron B, Taddei L, Benoit M, Negrusz A.The use of nonsteroidal antiinflammatory drugs in racehorses is allowed under most jurisdictions. Furosemide is administered to treat exercise-induced pulmonary hemorrhage. To help distinguish between therapeutic and illegal uses, racing regulatory bodies have set thresholds in serum for several drugs. The method for the simultaneous detection and quantification of furosemide, flunixin, ketoprofen, phenylbutazone and oxyphenbutazone using 500 µL of serum, and liquid extraction using diethyl ether : hexanes : dichloromethane followed by liquid chromatography tandem mass spectrometry quantitati...
Cloteau C, Dervilly G, Loup B, Delcourt V, Kaabia Z, Bagilet F, Groseille G, Dauriac K, Fisher S, Popot MA, Garcia P, Le Bizec B, Bailly-Chouriberry L.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 focuses on assessing the robustness of the associated method and def...
Guan F, Uboh CE, Soma LR, Robinson M, Maylin GA, Li X.Dermorphin is a unique opioid peptide that is 30-40 times more potent than morphine. It was misused and went undetected in horse racing until 2011 when intelligence obtained from a few North American race tracks suggested its use. To prevent such misuse, a reliable analytical method became necessary for detection and identification of dermorphin in post-race horse samples. This paper describes the first liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for such a purpose. Equine plasma and urine samples were pre-treated with ethylenediamine tetra-acetic acid and urea prior to so...
Corado CR, McKemie DS, Knych HK.Dextromethorphan is an N-methyl-D-aspartate (NMDA) non-competitive antagonist commonly used in human medicine as an antitussive. Dextromethorphan is metabolized in humans by cytochrome P450 2D6 into dextrorphan, which is reported to be more potent than the parent compound. The goal of this study is to describe the metabolism of and determine the pharmacokinetics of dextromethorphan and its major metabolites following oral administration to horses. A total of 23 horses received a single oral dose of 2 mg/kg. Blood samples were collected at time 0 and at various times up to 96 h post drug ad...
Alberghina D, Piccione G, Amorini AM, D'Urso S, Longo S, Picardi M, Tavazzi B, Lazzarino G.This study was designed to examine the influence of sub-maximal exercise on purine and pyrimidine catabolism in horses. Ten horses were initially trained for 12 weeks at the end of which they underwent a standardized exercise test (SET); venous blood samples were taken at rest, 5 and 30 min after the SET. Six untrained healthy horses, from which a blood withdrawal was taken at rest, were used as the control group. Samples were analyzed by HPLC for the simultaneous determination of uric acid, uridine, β-pseudouridine and creatinine in plasma. Glucose and lactate were measured in blood. Trained...
Karatt TK, Sathiq MA, Laya S, Ajeebsanu MPM, Karakka Kal AK, Subhahar MB, Perwad Z.Recently, there has been a report suggesting that ecdysteroids can enhance sports performance, making them relevant substances in doping control. Hence, it is imperative to examine the analytical characteristics of ecdysteroids in biological samples to identify their misuse in competitive sports. Methods: To assess the doping of ecdysteroids such as ecdysone, ecdysterone, ponasterone A, turkesterone, and ajugasterone C, a fast and sensitive extraction and detection method was developed, optimized, and validated using equine urine and plasma. Different extraction techniques, namely, solid-phase...
Takagi M, Uno S, Kokushi E, Sato F, Wijayagunawardane M, Fink-Gremmels J.Mycotoxins may affect animal health, including reproduction. Little is known about the clinical relevance of exposure of horses to contaminated feed. This study aimed at (i) monitoring the levels of the mycotoxins zearalenone (ZEN), with its metabolites α- and β-zearalenol (α- and β-ZOL), and sterigmatocystin (STC) in urine samples from thoroughbred mares in Japan and (ii) relating these findings to the potential effects on reproductive efficacy of breeding mares. Sixty-three urine samples of breeding mares from 59 breeding farms were used. Urine samples and reproductive records were colle...
Ishii H, Shibuya M, Kusano K, Sone Y, Kamiya T, Wakuno A, Ito H, Miyata K, Yamada M, Leung GN.Vadadustat is a newly launched hypoxia-inducible factor stabilizer with anti-anemia and erythropoietic effects; however, its use in horses is expressly forbidden in both racing and equestrian competitions. Following our previous report on the pharmacokinetic study of vadadustat in horse plasma and urine, a long-term longitudinal analysis of vadadustat in horse hair after nasoesophageal administration (3 g/day for 3 days) to three thoroughbred mares is described in this study. Our main objective is to further extend the detection period of vadadustat for the purpose of doping control. Three...
Wong KS, Chan GHM, Ho ENM, Wan TSM.Growth hormone (GH), or somatotropin, is a protein that may enhance physical performance and facilitate growth and wound healing. For this reason, growth hormones and their recombinant analogues are prohibited in human sports by the World Anti-Doping Agency (WADA) and in horseracing under Article 6 of the International Agreement on Breeding, Racing and Wagering published by the International Federation of Horse Racing Authorities (IFHA). Identifying the illicit use of GHs in both human athletes and racehorses is challenging, especially when differentiating between endogenous and exogenous GHs,...
Pesko B, Habershon-Butcher J, Muir T, Gray B, Taylor P, Fenwick S, Hincks P, Scarth J, Paine S.Paracetamol is a widely used, non-opioid analgesic and antipyretic drug. Scientific evidence suggests that it is an effective pain treatment in equine medicine. However, there is very little published information about the pharmacokinetics of the drug in the horse. The aim of the research was to determine the pharmacokinetics of paracetamol in equine plasma and urine to inform treatment of Thoroughbred racehorses. In this multi-dose study, paracetamol was administered orally at 20 mg/kg to six Thoroughbred horses. Pre- and post-administration urine and plasma samples were collected and analys...
Popot MA, Jacobs M, Garcia P, Loup B, Guyonnet J, Toutain PL, Bailly-Chouriberry L, Bonnaire Y.Tiludronate is a bisphosphonate drug marketed to treat different bone conditions in horses. Objective: 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. Methods: 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...
Guan F, Uboh CE, Soma LR, Birks E, Chen J, You Y, Rudy J, Li X.Recombinant human erythropoietin (rhEPO) and darbepoetin alfa (DPO) are protein-based drugs for the treatment of anemia in humans by stimulating erythrocyte production. However, these agents are abused in human and equine sports due to their potential to enhance performance. This paper describes the first method for differentiation and identification of rhEPO and DPO in equine plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The method comprised analyte extraction and enrichment by immunoaffinity separation with anti-rhEPO antibodies, dual digestion by trypsin an...
Caprioli G, Genangeli M, Mustafa AM, Petrelli R, Ricciutelli M, Sagratini G, Sartori S, Laus F, Vittori S, Cortese M.A simple and fast analytical method able to simultaneously identify and quantify 17 endogenous and exogenous steroidal hormones was developed in bovine and equine blood using UHPLC-MS/MS. A total amount of 500 µL of sample was deproteinized with 500 µL of a mixture of methanol and zinc sulfate and evaporated. The mixture was reconstituted with 50 µL of a solution of 25% methanol and injected in the UHPLC-MS/MS triple quadrupole. The correlation coefficients of the calibration curves of the analyzed compounds were in the range of 0.9932-0.9999, and the limits of detection and quantification ...
Roach JM, Pfau T, Bryars J, Unt V, Channon SB, Weller R.Kinematic evaluation of the distal limb of the horse using standard methods is challenging, mainly due to the hoof capsule restricting visualisation, but the recent development of a high-speed fluoroscopy (HSF) system has allowed in vivo cineradiographic assessment of moving skeletal structures at high speeds. The application of this non-invasive method to the equine distal limb is used to describe 'internal' distal limb kinematics including intra-horse and inter-horse variability, and variability between walk and trot. Distal limb kinematic data were collected at walk and trot from six non-la...
Hamamoto-Hardman BD, Steffey EP, McKemie DS, Kass PH, Knych HK.Meperidine is a synthetic opioid that belongs to the phenylpiperidine class and is a weak mu receptor agonist. In horses there are a limited number of published studies describing the analgesic effects of systemically administered meperidine in horses. The objective of this study was to describe the pharmacokinetics, behavioral and physiologic effects and effect on thermal threshold of three doses of intravenously administered meperidine to horses. Eight University owned horses (four mares and four geldings, aged 3-8 years were studied using a randomized balanced 4-way cross-over design. Hors...
Gretler SR, Finno CJ, McKemie DS, Kass PH, Knych HK.To describe the pharmacokinetics and selected pharmacodynamic variables of codeine and its metabolites in Thoroughbred horses following a single oral administration. Methods: Prospective experimental study. Methods: A total of 12 Thoroughbred horses, nine geldings and three mares, aged 4-8 years. Methods: Horses were administered codeine (0.6 mg kg) orally and blood was collected before administration and at various times until 120 hours post administration. Plasma and urine samples were collected and analyzed for codeine and its metabolites by liquid chromatography-mass spectrometry, and plas...
