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Topic:High-performance Liquid Chromatography (HPLC)
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.
Beta-thiopropionyl cytochromes c modified at lysyl residues: preparation and characterization of the monosubstituted horse cytochromes c. beta-Thiopropionyl derivatives of horse cytochrome c singly modified at each of 18 different lysine epsilon-amino groups have been prepared using sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate and purified to homogeneity by high-pressure liquid chromatography. These derivatives were characterized by determination of: (i) the location of the modification; (ii) reduction potentials; (iii) visible and NMR spectra: and by (iv) measurement of electron transfer activity with cytochrome-c oxidase. No significant changes in structure were indicated, except for the ferric forms of the deri...
Acid-base variables during incremental exercise in sprint-trained horses fed a high-fat diet. Seven Arabian horses performed a standard incremental exercise test on a high-speed treadmill at 6% slope then were randomly assigned to two diets, a control diet of ground hay and concentrates and a similar diet with 10% added fat (by weight). Horses were sprint-trained 4 d/wk, and two additional exercise tests were performed at 5-wk intervals. Heart rates and rectal temperatures were monitored and venous blood samples were collected at rest and at each speed increment. Whole blood was analyzed for glucose, lactate, and hemoglobin concentrations, and plasma was analyzed for pH, pCO2, albumin,...
Determination of triamcinolone acetonide in equine serum and urine by liquid chromatography-atmospheric pressure ionization mass spectrometry. Urine and serum samples collected from four standard-bred mares after 30-mg intraarticular administrations of triamcinolone acetonide were analyzed using combined high-performance liquid chromatography-atmospheric pressure ionization mass spectrometry. Maximum triamcinolone acetonide concentrations of 32.3, 14.8, 24.3, and 29.4 ng/mL in the urine and 2.7, 1.9, 2.3, and 2.5 ng/mL in the serum samples were observed. The peak concentrations of the drug were detected approximately 22 h (urine) and 12 h (serum) after administration. The drug elimination profiles for both urine and serum are present...
Detection of clenbuterol (Ventipulmin) in the horse. An enzyme linked immunosorbent assay (ELISA) was developed to detect the beta 2-agonist clenbuterol in equine blood and urine. The antiserum was raised in rabbits, employing clenbuterol-diazo-BSA as antigen. Clenbuterol-diazo-horseradish peroxidase served as enzyme conjugate. The concentration of clenbuterol to decrease tracer binding by 50% (IC50 value) was found to be 27.50 +/- 4.20 pg/well (1.37 ng/ml). The antibody cross-reacted with salbutamol (30%), terbutaline (14%) and cimaterol (1%). Horse serum was used directly to screen for clenbuterol, while urine was employed diluted. Positive sc...
Pharmacokinetics of ketoprofen after multiple intravenous doses to mares. The pharmacokinetics and urinary excretion of ketoprofen in six healthy mares after the first and last of five daily intravenous doses of 2.2 mg of ketoprofen per kg body weight were investigated using a high-performance liquid chromatographic (HPLC) method for determining plasma and urinary ketoprofen concentrations. Plasma ketoprofen concentrations declined triexponentially after each dose with no significant differences in plasma concentrations or pharmacokinetic parameter values between the first and last doses. The harmonic mean of the terminal elimination half-life of ketoprofen after th...
Catecholamine affects acetylcholine release in trachea: alpha 2-mediated inhibition and beta 2-mediated augmentation. We investigated the effects of catecholamines on acetylcholine (ACh) release from equine airway parasympathetic nerves. Trachealis strips were suspended in 2-ml tissue baths with Krebs-Henseleit solution containing atropine (10(-7) M), neostigmine (10(-6) M), and guanethidine (10(-5) M). Electrical field stimulation (20 V, 0.5 ms, 0.5 Hz, for 15 min) was applied, and ACh was measured by high-performance liquid chromatography with electrochemical detection. Epinephrine (Epi) and norepinephrine (NE) inhibited ACh release in a concentration-dependent manner. Inhibition was attenuated by the alpha...
In vitro 19-norandrogen synthesis by equine placenta requires the participation of aromatase. Explants of equine full-term placenta have been shown to synthesize 19-norandrogens from labelled androgens. Steroid metabolites were purified by silica-gel column chromatography then analysed and quantified by c18-reverse-phase HPLC coupled to a radioactive flow detector. 19-Norandrostenedione was subsequently recrystallized to constant specific activity, providing unequivocal evidence of its synthesis by the equine placenta. 19-Norandrostenedione synthesis appeared to be localized in the microsomal fraction. Regardless of the substrate used, formation of 19-norandrogens was far weaker than t...
Acetylcholine release from airway cholinergic nerves in horses with heaves, an airway obstructive disease. The present study was conducted to determine if acetylcholine (ACh) release from airway cholinergic nerves is increased and if modulation of ACh release by prejunctional receptors is altered in horses with heaves, an obstructive airway disease characterized by airway inflammation and bronchospasm. Trachealis strips and bronchial segments of normal horses and horses affected with heaves were suspended in 2-ml tissue baths. ACh release was induced by electrical field stimulation and the bath ACh content was measured by high performance liquid chromatography (HPLC) with electrochemical detection....
[Demonstration of two trimethoprim/sulfonamide combinations in bronchoalveolar lavage fluid of horses and determination of blood levels]. Five healthy horses were given a sulfadoxine/trimethoprim combination (Borgal, Hoechst AG) i.v. on day 1. The next ten days the horses got once a day a sulfadimethoxine/trimethoprim combination orally (Trafigal, Hoechst AG). The doses were given as recommended. One horse received no medicaments for control. On each horse six bronchoalveolar lavages were performed. Blood samples were taken to calculate blood levels and elimination half lives. To determine the amount of substances in lavage fluid and plasma the high performance liquid chromatography (HPLC) was used. Regularly low quantities of s...
Determination of xanthines by high-performance liquid chromatography and thin-layer chromatography in horse urine after ingestion of Guaraná powder. The seeds of Guaraná are rich in xanthines and are used for the preparation of guaraná powder which is very commonly given to horses as a 'tonic' in Brazil. In this paper, the xanthine content of guaraná powder was determined, in addition to its clearance time in horses. Thin-layer chromatography was used as a screening procedure and high-performance liquid chromatography was performed to quantify the drugs in both the powder and urine samples. The guaraná powder was found to contain 2.16, 1.10 and 36.78 mg g-1 of theobromine (TB), theophylline (TP) and caffeine (CF), respectively, and in ...
Horse plasma corticotrophin-releasing hormone (CRH): characterisation and lack of a late gestational rise or a plasma CRH-binding protein. Immunoreactive corticotrophin-releasing hormone (irCRH) was present in methanolic extracts of equine peripheral blood and showed no elevation in maternal peripheral serum in late gestation (0.54 +/- 0.25 pmol/l; mean +/- S.D.) compared with control horses (0.41 +/- 0.15 pmol/l). The irCRH of methanolic extracts of pituitary venous plasma had a similar elution position following reverse-phase HPLC to synthetic human CRH(1-41) and to irCRH released from horse stalk-median eminence tissue incubated in vitro. Gel chromatographic studies showed no evidence for a plasma CRH-binding protein (CRHBP) a...
Pulmonary vascular pressures of thoroughbreds increase rapidly and to a higher level with rapid onset of high-intensity exercise than slow onset. 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...
PGE2 inhibits acetylcholine release from cholinergic nerves in canine but not equine airways. The effects of exogenous prostaglandin E2 (PGE2) and endogenous prostanoids on cholinergic neurotransmission were determined by measurement of acetylcholine (ACh) release from canine and equine airway tissues. Trachealis strips and bronchial segments were suspended in 2 ml tissue baths. ACh release was induced by electrical field stimulation (EFS), and its content in tissue bath liquid was measured by high pressure liquid chromatography (HPLC) with electrochemical detection. In canine airways, exogenous PGE2 (10(-9) to 10(-7) M) inhibited ACh release concentration-dependently, whereas inhibiti...
Disposition of human drug preparations in the horse. III. Orally administered alclofenac. Concentrations of the non-steroidal anti-inflammatory drug (NSAID) alclofenac were determined by a sensitive high performance liquid chromatographic procedure in plasma and urine of horses following oral administration of a dose of 3 g. In plasma, alclofenac was present in detectable concentrations for 72 h. The plasma disposition in individual horses was best described by a bi-compartmental model with two successive rate constants ka1 = 0.05 +/- 0.06 h-1 and ka2 = 0.06 +/- 0.01 h-1. Alclofenac half-lives t1/2 alpha and t1/2 beta were 1.0 +/- 0.8 h and 6.9 +/- 1.5 h, respectively. Maximal conc...
Pharmacokinetics and pharmacodynamics of acepromazine in horses. A specific, sensitive, reverse-phase high-performance liquid chromatographic assay for acepromazine, with analytic sensitivity as low as 5 ng/ml of plasma, and electrochemical detection with an oxidation potential of 0.7 V, was used to study the pharmacokinetics of acepromazine given at a dosage of 0.15 mg/kg of body weight in horses. The relation between effect and pharmacokinetics of the drug was examined. The effects studied included those on blood pressure, pulse, PCV, measures of respiration function, and sedation. Intravenously administered doses led to a biphasic concentration decay pat...
Effects of phenylbutazone on thiamylal disposition and anaesthesia in ponies. Phenylbutazone given during the perisurgical period has been reported to increase the intensity and duration of thiamylal anaesthesia in horses. A possible mechanism of competitive plasma protein binding has been suggested. The purpose of the present study was to experimentally reproduce the phenomenon of increased intensity and/or duration of thiamylal anaesthesia and to determine if there is competitive displacement of plasma protein bound thiamylal by phenylbutazone. Six ponies each received one of three treatments, 11 mg/kg intravenous (i.v.) thiamylal; 8.8 mg/kg i.v. phenylbutazone; and 1...
Comparative aspects of Na(+)-K+ and Ca(2+)-Mg2+ ATPase in erythrocyte membranes of various mammals. This work is a comparative study of Na(+)-K+ and Ca(2+)-Mg2+ ATPase associated with the erythrocyte plasma membranes in different mammals. The method used to test the activity of these enzymes is based on quantitative measurements of ADP released during the reaction with HPLC: the chromatographic type is an Ion-Pair Reversed Phase. We have found that the levels of Ca2+ stimulated ATPase are higher than those of Na(+)-K+ ATPase in red blood cells of all the different mammalian species, with the only exception being lamb erythrocytes where the values of both the ATPase activities are almost equa...
Monitoring furosemide in racehorses participating in an EIPH program. Analytical procedures were developed to monitor furosemide concentrations in post-race serum and urine samples obtained from horses participating in an exercise-induced pulmonary haemorrhage (EIPH) program. High performance liquid chromatography with ultraviolet light detection proved a reliable, sensitive method for measuring urinary furosemide concentrations up to 12 h after administration of either 150 or 250 mg of the drug to race horses. However, this method was unreliable for determination of serum furosemide concentration. High performance liquid chromatography with fluorescence detecti...
The vitamin A and vitamin E status of horses raised in Alberta and Saskatchewan. The purpose of the study was to determine normal baseline levels of vitamin A and vitamin E in clinically normal horses under typical field conditions in Saskatchewan and Alberta. Heparinized blood samples were collected from approximately 400 clinically healthy horses selected from 24 locations in Alberta and Saskatchewan during a two-year period. For each horse, historical information including feed type, vitamin supplementation, time of year, sex, and age were recorded. From each blood sample, the plasma vitamin A (all-transretinol) and vitamin E (alpha-tocopherol) levels were measured usin...
Concentration and molecular weight distribution of hyaluronate in synovial fluid from clinically normal horses and horses with diseased joints. High molecular weight (MW) hyaluronate (HA) is an integral part of synovial fluid (SF), regulating many important physiologic and pathophysiologic mechanisms. Many of its effects depend on, or are reflected in, the concentration and MW of HA. High-performance liquid chromatography was used to assess simultaneously the concentration and MW of HA in SF obtained from horses with various arthritides: acute traumatic arthritis; chronic traumatic arthritis, including degenerative joint disease (DJD); and infectious arthritis. The size-exclusion column was calibrated, using appropriate HA concentrati...
Prolonged presence of isoxsuprine in equine serum after oral administration. 1. Isoxsuprine [1-(4-hydroxyphenyl)-2-(1-methyl-2-phenoxyethylamino)-1- propanol] serum concentrations after single- and multiple-dose administration to horse were investigated using immunoenzymatic ELISA, HPLC-UV and thermospray HPLC-MS methods. 2. Using HPLC-MS, isoxsuprine was detected up to 72 h after a single administration (1.2 mg/kg by gastric probe) and up to 96 h after the end of serial administration (1.2 mg/kg every 12 h for 7 days). 3. ELISA detected the drug up to 96 h after a single dose and up to 6 days after the end of prolonged administration. 4. Isoxsuprine is present in hors...
Identification and verification of the anabolic steroid boldenone in equine blood and urine by HPLC/ELISA. An enzyme linked immunosorbent assay (ELISA) was developed to detect the anabolic steroid boldenone in equine blood and urine. The polyclonal antiserum was raised in rabbits, employing boldenone-17-hemisuccinate-bovine serum albumin as antigen. Boldenone-17-hemisuccinate-horseradish peroxidase served as enzyme conjugate. Sensitivity of the assay was 26.0 +/- 3.0 pg/well. Among the endogenous steroids tested only progesterone and testosterone exhibited moderate cross-reactivities, 3.4 and 2.5%, respectively. These cross-reactivities are of no importance for the boldenone assay. For the reductio...
Concentration of ceftiofur metabolites in the plasma and lungs of horses following intramuscular treatment. 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...
Investigation of the metabolism of azaperone in the horse. Urine samples collected from a horse after intramuscular administration of 40 mg of azaperone were extracted at pH 10 before and after acid hydrolysis. The extracts were concentrated and analysed by LC-MS-MS. Two N-dealkylated metabolites, N-despyridinylazaperol and N-despyridinylazaperone, and a low concentration of azaperone were detected in the unhydrolysed urine. Six metabolites; hydroxyazaperol, two hydroxyazaperones, azaperol, N-despyridinylazaperol and N-despyridinylazaperone were detected in the hydrolysed urine extracts. Using XAD-2 resin extraction, three glucuronide conjugated azape...
A liquid chromatographic method for the determination of fenoprofen in equine plasma and urine. A high performance liquid chromatographic method to measure plasma and urine fenoprofen levels in equine biofluids is described. Liquid-liquid extraction with diethylether was used to isolate the drug from plasma and urine. The accuracy and reproducibility of the method were within acceptable limits over the concentration range 0-10 micrograms/mL and 0-20 micrograms/mL respectively from plasma and urine. Detection limits were 0.05 microgram/mL (2 mL plasma) and 0.2 microgram/mL (0.5 mL urine). This procedure was applied to ascertain the pharmacokinetics of a 3 g dose of fenoprofen calcium in a...
Determination of alclofenac in equine plasma and urine by high-performance liquid chromatography. A high-performance liquid chromatographic method to measure plasma and urinary alclofenac levels in equine biofluids is described. Isolation of the drug from plasma is achieved using liquid-liquid extraction with diethyl ether. Reversed-phase C18 solid phase extraction is used for the extraction of free and conjugated alclofenac from urine. The reproducibility and accuracy of the method were well within acceptable limits over the concentration ranges 0-10 and 0-20 micrograms/ml, respectively, for plasma and urine. Starting with 2 ml of plasma, a concentration of 0.1 microgram/ml could easily b...
Rapid high-performance liquid chromatographic method for the determination of ketamine and its metabolite dehydronorketamine in equine serum. A simple, rapid and sensitive high-performance liquid chromatographic procedure has been developed for the determination of ketamine and dehydronorketamine in equine serum. Sample preparation consisted of mixing equal volumes of serum and acetonitrile-phosphoric acid (85%)-water (20:2:78, v/v/v), followed by ultrafiltration through a 10,000 molecular mass cut-off filter. Separation of these two analytes in the ultrafiltrate was accomplished on a reversed-phase phenyl column eluted with methanol-acetonitrile-phosphate buffer solution. Ketamine and dehydronorketamine were detected by a variable ...
The disposition of suxibuzone in the horse. A high performance liquid chromatographic method is described to determine the anti-inflammatory drug suxibuzone (SXB) and its major metabolites phenylbutazone (PBZ) and oxyphenbutazone (OPBZ) in equine plasma and urine. When suxibuzone (6 mg/kg) was administered intravenously (i.v.) or orally (p.o.) no parent drug was detected in plasma or in urine. The disposition of the metabolite PBZ (i.v.) could be described by a 2 compartment model with a beta half-life varying from 7.40 to 8.35 h. Due to severe side effects the use of i.v. suxibuzone should not be encouraged in the horse. PBZ and OPBZ w...
Purification and characterization of equine complement factor C3. A rapid method for purifying equine C3 which yields milligram quantities of pure C3 is described. Protein from equine plasma was selectively precipitated with polyethylene glycol, and the C3 was purified by anionic and cationic exchange HPLC. The yield from this procedure was 12%. The purified C3 was composed of an alpha chain (118 kD) and a beta chain (68 kD) linked by at least one disulfide bond, and it had an isoelectric point of 4.7. Amino acid analysis indicated a strong conservation of amino acid usage between equine and human C3. The N-terminal sequences of the alpha and beta chains wer...
[Ganglioside GM3 from horse erythrocytes: structure and effect on cell proliferation]. An increase of the mouse fibroblast proliferation by ganglioside GM3 from equine erythrocytes is described. The structure of GM3 has been established on the basis of chemical methods, enzymatic degradation, GC-MS, as well as plasma desorption mass spectrometry and HPLC of 9-anthrylmethyl esters of gangliosides to characterize the long-chain base composition. The oligosaccharide moiety includes an N-glycolylneuraminic acid residue, whereas the main components of the lipid moiety are 20:1 sphingosine and 24:0 fatty acids.
