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.
Byström A, Roepstroff L, Geser-von Peinen K, Weishaupt MA, Rhodin M.Collection is a central term in equine dressage, defined as a shortening of the horse's stride length with retained energy and hind limb activity. How collection is induced by the rider has yet not been investigated objectively. The aim of this study was therefore to compare the movement pattern of high-level dressage riders between free trot (loose reins), passage and a range of three speeds in collected trot. Both at higher speed in collected trot and in passage, the rider's pelvis became more caudally rotated and the rider's lumbar back became more flexed. However, in passage there was also...
Sykes BW, Underwood C, McGowan CM, Mills PC.The objectives of this study were to investigate the impact of formulation (enteric coated and buffered) and feeding on pharmacokinetic variables associated with the oral administration of omeprazole in the horse. Six thoroughbred racehorses were studied in a crossover design. Each received 2 g of an enteric coated or buffered formulation in both the fed and fasted state. Plasma omeprazole concentrations were determined by UHPLC-MS. The effects of feeding or formulation on AUC0-inf_obs, half-life, Tmax or Cmax were not statistically significant. However, a wider-than-expected degree of variati...
Srivastava A, Singh BN, Deepak D, Rawat AK, Singh BR.The discovery of quorum-sensing (QS) systems regulating antibiotic resistance and virulence factors (VFs) has afforded a novel opportunity to prevent bacterial pathogenicity. Dietary molecules have been demonstrated to attenuate QS circuits of bacteria. But, to our knowledge, no study exploring the potential of colostrum hexasaccharide (CHS) in regulating QS systems has been published. In this study, we analyzed CHS for inhibiting QS signaling in Staphylococcus aureus. We isolated and characterized CHS from mare colostrum by high-performance thin-layer chromatography (HPTLC), reverse-phase hig...
Rendle DI, Hughes KJ, Doran GS, Edwards SH.To determine the pharmacokinetics of pergolide after IV administration to horses. Methods: 8 healthy adult horses. Methods: Pergolide mesylate was administered IV at a dose of 20 μg/kg (equivalent to 15.2 μg of pergolide/kg) to each horse, and blood samples were collected over 48 hours. Pergolide concentrations in plasma were determined by means of high-performance liquid chromatography-tandem mass spectrometry, and pharmacokinetic parameters were determined on the basis of noncompartmental methods. Results: After IV administration of pergolide, mean ± SD clearance, elimination half-life, a...
Decloedt A, Bailly-Chouriberry L, Vanden Bussche J, Garcia P, Popot MA, Bonnaire Y, Vanhaecke L.Doping control is a main priority for regulatory bodies of both the horse racing industry and the equestrian sports. Urine and blood samples are screened for the presence of hundreds of forbidden substances including anabolic-androgenic steroids (AASs). Based on the suspected endogenous origin of some AASs, with β-boldenone as the most illicit candidate, this study aimed to improve the knowledge of the naturally present AAS in horse urine. To this extent, a novel ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated according to...
Zielinski J, Mevissen M.Testosterone hydroxylation was investigated in human, canine and equine liver microsomes and in human and canine single CYPs. The contribution of the CYP families 1, 2 and 3 was studied using chemical inhibitors. Testosterone metabolites were analyzed by HPLC. The metabolites androstenedione, 6β- and 11β-hydroxytestosterone were found in microsomes of all species, but the pattern of metabolites varied within species. Androstenedione was more prominent in the animal species, and an increase over time was seen in equines. Testosterone hydroxylation was predominantly catalyzed by the CYP3A subf...
Bishop J, Heffron B, Taddei L, Benoit M, Hurt L, Costello S, Gross M, Negrusz A.Clenbuterol (CLE) is used in horses as a bronchodilator and for its anabolic steroid-like effects. CLE is a Class 3 drug according to current Association of Racing Commissioners International (ARCI) Uniform Classification Guidelines. The Racing Medication and Testing Consortium recommended a urine CLE threshold of 140 pg/mL after careful scientific review of the results of studies describing the disposition of CLE in the horse and this threshold was adopted by the ARCI. Enzyme-linked immunosorbent assay was previously used to screen samples for CLE in Illinois, but could not detect such low co...
Chiavaccini L, Claude AK, Lee JH, Ross MK, Meyer RE, Langston VC.The study objective was to compare butorphanol pharmacokinetics and physiologic effects following intravenous and subcutaneous administration in horses. Ten adult horses received 0.1 mg/kg butorphanol by either intravenous or subcutaneous injections, in a randomized crossover design. Plasma concentrations of butorphanol were measured at predetermined time points using highly sensitive liquid chromatography-tandem mass spectrometry assay (LC-MS/MS). Demeanor and physiologic variables were recorded. Data were analyzed with multivariate mixed-effect model on ranks (P ≤ 0.05). For subcutaneous i...
Hedenström UO, Wattle OS.Ossification of the ungular cartilages (OUC) in the foot of horses has been studied for more than 100 years. There is a high heritability of this condition but its clinical relevance has remained questionable. Nevertheless, modern equine orthopedic literature ranks OUC as one of top 10 causes of lameness in cold-blooded trotters and stallions of these breeds are excluded from breeding if they have more than mild levels of side bones. Cold-blooded trotters have been used for racing for many decades and official sports data have been available since 1923. A decreased performance is often the onl...
Meucci V, Minunni M, Vanni M, Sgorbini M, Corazza M, Intorre L.Detection of nonsteroidal anti-inflammatory drugs (NSAIDs) in equine plasma is a significant analytical problem in veterinary anti-doping controls. Results: A new HPLC method coupled to selective extraction with molecularly imprinted polymers was developed for the simultaneous determination in equine plasma of the NSAIDs phenylbutazone, flunixin, oxyphenbutazone, ketoprofen and naproxen. The analytical performances of the method have been evaluated both in standard solutions and equine plasma samples. Recovery: Molecularly imprinted polymers solid-phase extraction for all NSAIDs was >94% with ...
Hunyadi L, Papich MG, Pusterla N.The purpose of this study was to determine the pharmacokinetics of the FDA-approved labeled dose of diclazuril and compare it to a low dose in plasma and CSF in adult horses. During each research period, six healthy adult horses received 0.5 mg/kg of 1.56% diclazuril pellets (Protazil(TM) , Merck Animal Health) compared to the approved labeled dose of 1 mg/kg orally once in two separate phases. A dose of 0.5 mg/kg was calculated to each horse's weight. Blood was then collected immediately before diclazuril administration and then at regular intervals up to a 168 h. After the last blood collect...
Cagnardi P, Ferraresi C, Zonca A, Pecile A, Ravasio G, Zani DD, Villa R.Tramadol is a synthetic codeine analogue used as an analgesic in human and veterinary medicine. It is not approved for use in horses, but could represent a valid tool for pain treatment in this species. Objective: The serum pharmacokinetic profile and urinary excretion of tramadol and its metabolites (O-desmethyltramadol [M1], N-desmethyltramadol [M2] and N,O-desmethyltramadol [M5]) was investigated in a multidrug anaesthetic and analgesic approach for orchiectomy in horses. The evaluation of the degree of cardiovascular stability, the intraoperative effect and postoperative analgesia obtained...
Swain EA, Magdesian KG, Kass PH, Edman JE, Knych HK.Neonatal foals have unique pharmacokinetics, which may lead to accumulation of certain drugs when adult horse dosage regimens are used. Given its lipophilic nature and requirement for hepatic metabolism, metronidazole may be one of these drugs. The purpose of this study was to determine the pharmacokinetic profiles of metronidazole in twelve healthy foals at 1-2.5 days of age when administered as a single intravenous (IV) and intragastric (IG) dose of 15 mg/kg. Foals in the intravenous group were studied a second time at 10-12 days of age to evaluate the influence of age on pharmacokinetics wi...
Kong N, Yi R, Zhao S, Sandhu J, Lam G, Loganathan D, Morrissey B.A sensitive hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method was developed and validated for the simultaneous detection and quantification of etilefrine and oxilofrine in equine blood plasma and urine. The method is highly sensitive and specific with good precision and accuracy. In plasma the limit of detection and limit of quantification are 0.03 and 0.1 ng/mL, respectively, for both analytes. In urine the limit of detection and limit of quantification are 0.3 and 1 ng/mL, respectively, for both analytes. The suitability of the method for doping con...
Ranheim B, Risberg ÅI, Spadavecchia C, Landsem R, Haga HA.Dexmedetomidine, the most selective α2-adrenoceptor agonist in clinical use, is increasingly being used in both conscious and anaesthetized horses; however, the pharmacokinetics and sedative effects of this drug administered alone as an infusion are not previously described in horses. Seven horses received an infusion of 8 μg dexmedetomidine/kg/h for 150 min, venous blood samples were collected, and dexmedetomidine concentrations were assayed using liquid chromatography-mass spectrometry (LC/MS) and analyzed using noncompartmental pharmacokinetic analysis. Sedation was scored as the distance...
Risberg A, Spadavecchia C, Ranheim B, Krontveit R, Haga HA.Dexmedetomidine and lignocaine IV are used clinically to provide analgesia in horses. The aims of this study were to investigate the antinociceptive effects, plasma concentrations and sedative effects of 2, 4 and 6 µg/kg/h dexmedetomidine IV, with a bolus of 0.96 µg/kg preceding each continuous rate infusion (CRI), and 20, 40 and 60 µg/kg/min lignocaine IV, with a bolus of 550 µg/kg preceding each CRI, in 10 Swiss Warmblood horses. Electrically elicited nociceptive withdrawal reflexes were evaluated by deltoid muscle electromyography. Nociceptive threshold and tolerance were determ...
Burton AJ, Giguère S, Arnold RD.Although gentamicin is highly active against Rhodococcus equi in vitro, its clinical efficacy has been limited presumably due to poor cellular uptake. Encapsulation of drugs in liposomes enhances their cellular uptake. Objective: To compare the disposition of liposomal gentamicin (LG) and free gentamicin (FG) in the plasma, pulmonary epithelial lining fluid and bronchoalveolar cells of healthy foals after i.v. administration or by nebulisation, and to assess the tolerability of the drug after repeated i.v. dosing. Methods: Experimental study. Methods: Eight healthy foals received a single i.v....
Knych HK, Steinmetz SJ, McKemie DS.The anti-anxiety and calming effects following activation of the GABA receptor have been exploited in performance horses by administering products containing GABA. The primary goal of the study reported here was to describe endogenous concentrations of GABA in horses and the pharmacokinetics, selected pharmacodynamic effects, and CSF concentrations following administration of a GABA-containing product. The mean (±SD) endogenous GABA level was 36.4 ± 12.5 ng/mL (n = 147). Sixteen of these horses received a single intravenous and oral dose of GABA (1650 mg). Blood, urine, and cerebrospin...
Santonastaso A, Hardy J, Cohen N, Fajt V.In certain situations, an alternate route for parenteral drug administration in horses may be useful. The intra-osseous (IO) route may provide a safe alternative to the intravenous (i.v.) route for administration of sedatives to horses when the i.v. route is inaccessible or undesirable. Six adult horses were administered xylazine i.v. or IO in a block-randomized crossover design. For the i.v. trial, both jugular veins were catheterized, and one was used for xylazine administration, while the other was used for blood collection. For the IO trial, one jugular vein was catheterized for blood coll...
Stahl J, Kietzmann M.The effect of physical and chemical permeation enhancers on in vitro transdermal permeation of lidocaine was investigated in the horse.Therefore, the effect of six vehicles (phosphate-buffered saline (PBS), 50% ethanol, 50% propylene glycol, 50% isopropylalcohol, 50% isopropylalcohol/isopropylmyristate and 50% dimethylsulfoxide) was examined as well as the effect of microneedle pretreatment with different needle lengths on transdermal drug delivery of lidocaine.The skin was obtained from the thorax of six Warmblood horses and was stored up to two weeks at - 20°C. Franz-type diffusion cells we...
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...
Love EJ, Pelligand L, Taylor PM, Murrell JC, Sear JW.Describe the pharmacokinetics of buprenorphine and norbuprenorphine in horses and to relate the plasma buprenorphine concentration to the pharmacodynamic effects. Methods: Single phase non-blinded study. Methods: Six dedicated research horses, aged 3-10 years and weighing 480-515 kg. Methods: Thermal and mechanical nociceptive thresholds, heart and respiratory rates and locomotor activity were measured before and 15, 30, 45 & 60 minutes and 2, 4, 6, 8, 12 & 24 hours post-administration of 10 μg kg(-1) buprenorphine IV. Intestinal motility was measured 1, 6, 12 & 24 hours af...
Agrawal K, Ebel JG, Bischoff K.Most antidoping method development in the equine industry has been for plasma and urine, though there has been recent interest in the analysis of synovial fluid for evidence of doping by intra-articular corticosteroid injection. Published methods for corticosteroid analysis in synovial fluid are primarily singleplex methods, do not screen for all corticosteroids of interest and are not adequately sensitive. The purpose of this study is to develop a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) screening method for the detection of four of the most common intra-a...
Stojiljkovic N, Paris A, Garcia P, Popot MA, Bonnaire Y, Tabet JC, Junot C.Horse urine is the medium of choice for the implementation of metabolomic approaches aimed at improving horse doping control. However, drug analysis in this biofluid is a challenging task due to the presence of large amounts of interfering compounds. METHODOLOGY & RESULTS: A comparative study of sample preparation has been conducted to evaluate five sample-preparation methods, namely acetonitrile precipitation, proteinase K hydrolysis, membrane filtration and sample dilution with water by factors of five and 20, for metabolome analysis using liquid chromatography coupled to high resolution...
Popot MA, Garcia P, Hubert C, Bolopion A, Bailly-Chouriberry L, Bonnaire Y, Thibaud D, Guyonnet J.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 ...
Doran G, Hughes K, Rendle D, Edwards S.Pergolide is used to treat pituitary pars intermedia dysfunction (equine Cushing's Disease), a neurodegenerative condition associated with loss of dopaminergic inhibition of the pituitary in horses. After oral administration, only low concentrations of the drug are achieved in plasma, making drug detection and quantification difficult. While direct analysis of plasma using sensitive MS/MS techniques is possible, dirty plasma samples and mobile phase buffers can cause instrumentation to become rapidly incapacitated. A method using LC with fluorescence detection was developed for pergolide analy...
Albrecht S, Lane JA, Mariño K, Al Busadah KA, Carrington SD, Hickey RM, Rudd PM.The present study was conducted to obtain a comprehensive overview of oligosaccharides present in the milk of a variety of important domestic animals including cows, goats, sheep, pigs, horses and dromedary camels. Using an analytical workflow that included ultra-performance liquid chromatography-hydrophilic interaction liquid chromatography with fluorescence detection coupled to quadrupole time-of-flight MS, detailed oligosaccharide libraries were established. The partial or full characterisation of the neutral/fucosylated, phosphorylated and sialylated structures was facilitated by sequencin...
Smith KM, Maxwell L, Gull T, Payton ME, Gilmour MA.To establish the effect of storage in a constant-rate infusion (CRI) pump on the sterility and stability of voriconazole 1% solution. Methods: Nine vials of voriconazole (Vfend(®) I.V.) 1% solution were prepared. Approximately half of each solution was used to prime a commercially available CRI pump with attached subpalpebral lavage system (CRI/SPL unit) with the remaining solution stored in the commercial glass vial. Three CRI/SPL units and their three corresponding vials were stored at one of three temperatures: 23 °C, 33 °C, and 40 °C. The CRI pumps ran for 7 days, and the vials were st...
Timms M, Hall N, Levina V, Vine J, Steel R.The growth hormone releasing peptides (GHRPs) hexarelin, ipamorelin, alexamorelin, GHRP-1, GHRP-2, GHRP-4, GHRP-5, and GHRP-6 are all synthetic met-enkephalin analogues that include unnatural D-amino acids. They were designed specifically for their ability to stimulate growth hormone release and may serve as performance enhancing drugs. To regulate the use of these peptides within the horse racing industry and by human athletes, a method is presented for the extraction, derivatization, and detection of GHRPs from equine and human urine. This method takes advantage of a highly specific solid-ph...
Naumann S, Meyer JP, Kiesow A, Mrestani Y, Wohlrab J, Neubert RH.The penetration behavior into human nails and animal hoof membranes of a novel antifungal agent (EV-086K) for the treatment of onychomycosis was investigated in this study. The new drug provides a high lipophilicity which is adverse for penetration into nails. Therefore, four different formulations were developed, with particular focus on a colloidal carrier system (CCS) due to its penetration enhancing properties. On the one hand, ex vivo penetration experiments on human nails were performed. Afterwards the human nail plates were cut by cryomicrotome in order to quantify the drug concentratio...
Sellon DC, Papich MG, Palmer L, Remund B.A two-way cross-over study of the pharmacokinetics of butorphanol after intravenous and intramuscular administration at 0.08 mg/kg in six adult horses was performed. Heparinized venous blood samples were obtained prior to drug administration and at 10, 20, 30, 45, 60, 120, 180, 240, and 360 min after IV injection. Samples were obtained at the same time points and at 6 h and 12 h after IM injection. Physical examination parameters were recorded at each time point. Plasma butorphanol concentrations were determined by high performance liquid chromatography. No significant differences in any physi...
Paine SW, Bright J, Scarth JP, Hincks PR, Pearce CM, Hannan C, Machnik M, Hillyer L.In equine and racing practice, detomidine and butorphanol are commonly used in combination for their sedative properties. The aim of the study was to produce detection times to better inform European veterinary surgeons, so that both drugs can be used appropriately under regulatory rules. Three independent groups of 7, 8 and 6 horses, respectively, were given either a single intravenous administration of butorphanol (100 µg/kg), a single intravenous administration of detomidine (10 µg/kg) or a combination of both at 25 (butorphanol) and 10 (detomidine) µg/kg. Plasma and urine concentratio...
Jacobson GA, Pirie A, Edwards S, Hughes KJ, Rendle DI, Davies NW.Pergolide, an ergot-derived dopamine D2 receptor agonist, is used extensively as an orally administered treatment for pituitary pars intermedia dysfunction (PPID) in horses. One of the barriers associated with pergolide determinations in plasma for pharmacokinetic applications has been the technically demanding requirement for sensitivity. The objective of our work was to develop a simple assay for the determination of pergolide in plasma and demonstrate its potential application in the study of pergolide pharmacokinetics (PK) in horses. A UPLC-MS/MS assay was developed with a simple sample pr...
Manohar M.Previous studies of pulmonary vascular pressures have utilised gradual incremental step exercise protocols, but in competitive racing at the track, horses perform rapid acceleration high-intensity exercise. The rate of rise in pulmonary vascular pressures under conditions of quick onset high-intensity exercise is unknown. Catheter mounted manometers, whose in vivo signals were matched with pressure signals obtained via transducers connected to fluid-filled lumens from same cardiovascular sites, were used to compare right heart and pulmonary vascular pressures in 8 healthy Thoroughbreds perform...
Kelmer G, Tatz AJ, Kdoshim E, Britzi M, Segev G.This prospective experimental study goal was to determine the pharmacokinetics of imipenem after intravenous regional limb perfusion (IV-RLP) in standing horses. Nine horses participated in the study; that was approved by the University Animal Care and Use Committee. One thoracic limb or one pelvic limb of each horse was randomly selected. After the veins were catheterized, an Esmarch bandage tourniquet was applied and the catheter was injected with a solution containing 500mg of imipenem. Synovial fluid samples were collected from the fetlock joint and blood samples were collected from the ju...
Jaglan PS, Roof RD, Yein FS, Arnold TS, Brown SA, Gilbertson TJ.Ceftiofur sodium, a broad spectrum cephalosporin antibiotic approved for veterinary use, is metabolized to desfuroylceftiofur which is conjugated to micro as well as macromolecules. Twelve horses, weighting 442-618 kg, were injected intramuscularly with a single dose of 2.2 mg ceftiofur/kg (1.0 mg/lb) body weight. Blood was collected at various intervals over 24 h after treatment. Three groups of four horses each were euthanized and lungs were collected at 1, 12, and 24 h after treatment. The concentration of desfuroylceftiofur and desfuroylceftiofur conjugates in the plasma and lungs was dete...
Sekkin S, Gokbulut C, Kum C, Karademir U.This study was designed to investigate the plasma disposition and systemic availability of enrofloxacin (ENR) following intramuscular and intravenous administrations. Six donkeys (Equus asinus) were used in this study. The animals were allocated into two groups (intramuscular and intravenous groups). After a 2-week washout period, the experiment was repeated with the groups reversed according to a two-phase crossover design. In phase I, group I received intravenously the commercially available injectable solution of ENR at the dose of 5 mg/kg and group II received intramuscularly the same ENR ...
Cox S, Sommardahl C, Fortner C, Davis R, Bergman J, Doherty T.Non-steroidal anti-inflammatory drugs are inhibitors of cyclooxygenase (COX) in tissues and used as therapeutic agents in different species. Grapiprant, a member of the piprant class of compounds, antagonizes prostaglandin receptors. It is a highly selective EP4 prostaglandin E receptor inhibitor, thereby limiting the potential for adverse effects caused by wider COX inhibition. The objectives of this study were to determine if the approved canine dose would result in measurable concentrations in horses, and to validate a chromatographic method of analysis for grapiprant in urine and plasma. M...
Munsterman AS, Bertone AL, Zachos TA, Weisbrode SE.To determine the effects of pretreatment with alpha-linolenic acid, an omega-3 polyunsaturated fatty acid, on equine synovial explants challenged with lipopolysaccharide (LPS). Methods: 8 mature mixed-breed horses (4 mares and 4 geldings). Methods: Synovial explants were assigned to receive 1 of 7 concentrations of alpha-linolenic acid, ranging from 0 to 300 microg/mL. At each concentration, half of the explants were controls and half were challenged with 0.003 microg of LPS as a model of synovial inflammation. Cell inflammatory response was evaluated by measurement of prostaglandin E2 product...
Lee DH, Birhanu BT, Lee EB, Lee SJ, Boby N, Park YS, Park SC.Cefquinome is administered in horses for the treatment of respiratory infection caused by Streptococcus equi subsp. zooepidemicus, and septicemia caused by Escherichia coli. However, there have been no attempts to use cefquinome against Streptococcus equi subsp. equi (S. equi), the causative agent of strangles. Hence the objective of this study was to calculate an optimal dosage of cefquinome against S. equi based on pharmacokinetics and pharmacodynamics integration. Cefquinome (1.0 mg/kg) was administered by intravenous and intramuscular routes to six healthy thoroughbred foals. Serum cefqui...
Kwok KY, Choi TLS, Kwok WH, Lau MY, Leung EMK, Leung GNW, Wong JKY, Wan TSM, Adrian FF, Prabhu A, Ho ENM.The use of bioactive peptides as a doping agent in both human and animal sports has become increasingly popular in recent years. As such, methods to control the misuse of bioactive peptides in equine sports have received attention. This paper describes a sensitive accurate mass method for the detection of 40 bioactive peptides and two non-peptide growth hormone secretagogues (< 2 kDa) at low pg/mL levels in horse urine using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC/HRMS). A simple mixed-mode cation exchange solid-phase extraction (SPE) cartridge was...
Knych HK, Seminoff K, McKemie DS.Traditional therapeutic options for the treatment of lameness associated with inflammation in performance horses include administration of cyclooxygenase enzyme inhibiting non-steroidal anti-inflammatory drugs (NSAID). As long-term use of these drugs can adversely impact the health of the horse, anti-inflammatories with a more favorable safety profile are warranted. Grapiprant is a newly approved non-cyclooxygenase inhibiting NSAID that has demonstrated efficacy and safety in other species and which may be a valuable alternative to traditional NSAIDs used in the horse. The objectives of the cu...
Gokbulut C, Cirak VY, Senlik B, Yildirim F, McKellar QA.This study aimed to determine the plasma disposition and faecal excretion of netobimin (NTB) and its respective metabolites as well as the efficacy against strongyles in horses following oral administration. Netobimin (10mg/kg) was administered orally to 8 horses. Blood and faecal samples were collected from 1 to 120h post-treatment and analysed by high performance liquid chromatography (HPLC). Using a chiral phase-based HPLC, plasma disposition of ABZSO enantiomers produced was also determined. Faecal strongyle egg counts (EPG) were performed by a modified McMaster's technique before and afte...
Giguère S, Burton AJ, Berghaus LJ, Haspel AD.The objective of this study was to compare the pharmacokinetics of minocycline in foals vs. adult horses. Minocycline was administered to six healthy 6- to 9-week-old foals and six adult horses at a dose of 4 mg/kg intragastrically (IG) and 2 mg/kg intravenously (i.v.) in a cross-over design. Five additional oral doses were administered at 12-h intervals in foals. A microbiologic assay was used to measure minocycline concentration in plasma, urine, synovial fluid, and cerebrospinal fluid (CSF). Liquid chromatography-tandem mass spectrometry was used to measure minocycline concentrations in ...
Steel R, Timms M, Levina V, Vine J.The Dermorphins are a family of peptides that act as potent agonists of the opioid μ receptor. Originally identified as a seven amino acid peptide on the skin of the South American Phyllomedusa frog, peptide chemists have since developed a large number of Dermorphin variants, many with superior opioid activity to the original peptide. Dermorphins are unique among the peptide opioid agonists as they appear to have a limited ability to cross the blood brain barrier, producing effects on both the central and peripheral nervous systems. It is this ability of Dermorphins to provide central anaesth...
Jaussaud P, Guieu D, Bellon C, Barbier B, Lhopital MC, Sechet R, Courtot D, Toutain PL.The pharmacokinetics of tolfenamic acid were studied in five ponies after an intravenous (iv) administration (2 mg/kg bodyweight [bwt]) and in four horses after an oral administration (30 mg/kg bwt) of tolfenamic acid. The plasma levels were determined by high pressure liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). For the iv administration, a three-compartment model was used to represent the plasma concentration-time curve of the drug. The elimination half-life of the compound was 6.1 +/- 1.5 h, the total body clearance was 72.4 +/- 16.7 ml/kg bwt/h and the ste...
von Fellenberg RL, Jordan JC, Ludwig B, Rehm WF.Aditoprim, a broad spectrum antimicrobial agent acting as a reversible dihydrofolate reductase inhibitor, was intravenously injected into four 12 to 24-year old horses at a dosage of 5 mg/kg b. w. Blood samples were collected over a 48-hour period after drug injection, and the separated plasma samples were assayed for aditoprim by high performance liquid chromatography. The body temperature, heart rate, respiration rate, and behaviour were recorded during the experiment. The bilirubin and urea concentrations were also determined in several plasma samples, and liver function tests were carried ...
Ammer H, Henschen A.The major stallion protamine was isolated from sperm cell nuclei by extraction with 6M guanidine/5% mercaptoethanol, alkylation with 4-vinylpyridine and subsequent reversed-phase high-performance liquid chromatography. The primary structure of stallion protamine was determined by N-terminal sequencing of the intact protein and of the fragments obtained from thermolysin cleavage of the S-pyridylethylated and from endoproteinase Lys-C cleavage of the S-aminoethylated protein. Stallion protamine consists of 49 amino-acid residues and shows 49% identity with all other sequenced mammalian type 1 pr...
Matéos A, Girardet JM, Mollé D, Corbier C, Gaillard JL, Miclo L.Equine beta-casein is phosphorylated at variable degrees and isoforms carrying 3 to 7 phosphate groups (3P-7P) have been found in milk, but the phosphorylated amino acid residues of each isoform are not yet identified. In the present work, the different phosphorylation variants were first isolated by ion-exchange chromatography and then hydrolysed by trypsin to generate caseinophosphopeptides (CPPs), each containing all the potential phosphorylation sites. The equine CPPs were prepared by metal oxide affinity chromatography, a method based on the affinity of phosphate groups towards titanium d...
Uniacke-Lowe T, Chevalier F, Hem S, Fox PF, Mulvihill DM.Rennet-induced coagulation of bovine milk is a complex mechanism in which chymosin specifically hydrolyzes κ-casein, the protein responsible for the stability of the casein micelle. In equine milk, this mechanism is still unclear, and the protein targets of chymosin are unknown. To reveal the proteins involved, the rennetability of equine milk by calf chymosin was examined using gel-free and gel-based proteomic analysis and compared to bovine milk. RP-HPLC analysis of bovine and equine milks showed the release of several peptides following chymosin incubation. The hydrolyses of equine and bov...
Flynn H, Cenani A, Brosnan RJ, DiMaio Knych HK, de Araujo Aguiar AJ.To determine the pharmacokinetics and pharmacodynamics of high-concentration formulation of buprenorphine (1.8 mg mL; Simbadol) following subcutaneous (SC) administration in horses. Methods: Prospective, randomized, crossover trial. Methods: A group of six healthy adult horses weighing 521-602 kg. Methods: On three occasions, Simbadol (0.005 mg kg; treatment S5), (0.0025 mg kg; treatment S2.5) or saline (treatment SAL) were administered SC at least 7 days apart in random order. Electrical nociceptive threshold (ENT) measured on the neck region, physiologic variables, locomotor activity, degree...
Zielinski J, Mevissen M.Testosterone hydroxylation was investigated in human, canine and equine liver microsomes and in human and canine single CYPs. The contribution of the CYP families 1, 2 and 3 was studied using chemical inhibitors. Testosterone metabolites were analyzed by HPLC. The metabolites androstenedione, 6β- and 11β-hydroxytestosterone were found in microsomes of all species, but the pattern of metabolites varied within species. Androstenedione was more prominent in the animal species, and an increase over time was seen in equines. Testosterone hydroxylation was predominantly catalyzed by the CYP3A subf...
Joré C, Loup B, Garcia P, Paris AC, Popot MA, Audran M, Bonnaire Y, Varlet-Marie E, Bailly-Chouriberry L.Erythropoiesis Stimulating Agents (ESAs) were developed for therapeutic purposes to stimulate red blood cell (RBC) production. Consequently, tissue oxygenation is enhanced as athlete's endurance and ESAs misuse now benefits doping. Our hypothesis is that most of ESAs should have similar mechanisms and thus have the same effects on metabolism. Studying the metabolome variations could allow suspecting the use of any ESAs with a single method by targeting their effects. In this objective, a metabolomic study was carried out on 3 thoroughbred horses with a single administration of 4.2μg/kg of Mir...
Luo Y, Uboh CE, Soma LR, Guan F, Rudy JA, Tsang DS.This method describes the simultaneous separation, identification, quantification and confirmation of betamethasone (BTM) and dexamethasone (DXM) in equine plasma by liquid chromatography (LC) integrated with multidimensional tandem mass spectrometry. Analytes were directly extracted from equine plasma by methyl tert-butyl ether (MTBE). The residues were reconstituted with sample solvent. LC separation of the analytes was performed on a Hypercarb column using acetonitrile/water/formic acid (95:5:0.5, v/v/v) as the mobile phase. Sample screening, quantification and confirmation were performed i...
Kwok WH, Choi TLS, Kwok KY, Chan GHM, Wong JKY, Wan TSM.The high sensitivity of ultra high performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC-HRMS) allows the identification of many prohibited substances without pre-concentration, leading to the development of simple and fast 'dilute-and-shoot' methods for doping control for human and equine sports. While the detection of polar drugs in plasma and urine is difficult using liquid-liquid or solid-phase extraction as these substances are poorly extracted, the 'dilute-and-shoot' approach is plausible. This paper describes a 'dilute-and-shoot' UHPLC-HRMS screening me...
Caldwell FJ, Mueller PO, Lynn RC, Budsberg SC.To determine whether 1% diclofenac liposomal suspension (DLS) ointment would be absorbed transdermally and attenuate experimentally induced subcutaneous inflammation in horses. Methods: 7 healthy adult horses. Methods: Inflammation was produced by injecting 1% sterile carrageenan into subcutaneously implanted tissue cages 8 hours before (time -8) and at the time of application of test ointment. A crossover design was used. Horses received 1 of 2 treatments (topically administered control or DLS ointments) during 48 hours of carrageenan-induced subcutaneous inflammation. A single application of...
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...
Parsons KJ, Spence AJ, Morgan R, Thompson JA, Wilson AM.Mechanical characterisation of the high speed gallop has significant importance for animal welfare and basic biology. Kinematic parameters such as the velocity of each foot at contact can inform theories of why animals gallop, and supplant epidemiological investigation into the mechanisms of musculoskeletal injury. Objective: To determine the velocity at which the fore and hind hooves of elite galloping horses impact the surface. Methods: High speed videography was used to measure the horizontal and vertical velocity of the hoof immediately prior to impact, and the subsequent sink (vertical) a...
Mangal D, Uboh CE, Soma LR.Oxidative metabolites of arachidonic acid (AA) are implicated in inflammation. Thus, we evaluated cycloxygenases (COXs) and lipoxygenases (LOs) mediated metabolism of AA to eicosanoids in equine plasma. Eicosanoids were extracted from plasma by two liquid-liquid extraction (LLE) steps; first was by chloroform/isopropanol and second by methyl-tert-butyl ether. For identification and quantification of 25 eicosanoids, a highly specific, selective and sensitive stable isotope dilution liquid chromatography (LC) multiple reaction monitoring (MRM) mass spectrometric (MS) method was developed. To avo...
