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Topic:Analytical Methods
Analytical methods in equine research encompass a variety of scientific techniques and tools used to study and evaluate different aspects of horse health, performance, and physiology. These methods help advance our understanding of equine biology, diagnosing conditions, and improving management practices. Common analytical methods include molecular techniques like PCR and ELISA for detecting pathogens and measuring biomarkers, imaging technologies such as ultrasound and MRI for assessing musculoskeletal health, and statistical models for analyzing genetic data and performance metrics. This page compiles peer-reviewed research studies and scholarly articles that explore the development, application, and impact of various analytical methods in equine science.
Stereospecific pharmacokinetics of free and protein-bound ketoprofen in serum and synovial fluid of horses after intravenous and intramuscular administration. To determine intravascular and intrasynovial pharmacokinetics of the R and S enantiomers of ketoprofen after i.v. and i.m. administration to horses. Methods: 6 healthy adult mares. Methods: Horses were weighed and ketoprofen (2.2 mg/kg of body weight) was administered i.v. Blood and synovial fluid samples were obtained and analyzed for concentrations of the R and S enantiomers by means of a modified reverse-phase stereospecific high-pressure liquid chromatographic method. Three weeks later, the procedure was repeated, except that ketoprofen was given IM. Protein binding of ketoprofen enantiome...
Stereochemical structures of synthesized and natural plasmalogalactosylceramides from equine brain. Modified galactosylceramide with a long-chain cyclic acetal at the sugar moiety, plasmalogalactosylceramide, was isolated from equine brain. To identify the isomeric stereostructure of the natural product, the plasmalo derivative was chemically synthesized from galactosylceramide through acetalization. The presence of cyclic acetal linkage, the linked position and length of the acetal chain of the synthesized and natural products were determined by proton nuclear magnetic resonance spectroscopy and fast-atom bombardment-mass spectrometry, as well as gas chromatography-mass spectrometry and gas...
HPLC on Chiralcel OJ-R for enantiomer separation and analysis of ketoprofen, from horse plasma, as the 9-aminophenanthrene derivative. Racemic ketoprofen is a non-steroidal anti-inflammatory drug used to treat musculoskeletal and colic conditions in horses. The enantioselective chiral inversion of ketoprofen administered to horses has been studied by use of cellulose tris(4-methylbenzoate), also known as Chiralcel OJ-R, as chiral stationary phase; acetonitrile - 0.02 M perchlorate buffer (pH 2.0)-methanol, 60:15:25 (v/v/v) was used as mobile phase. Before chromatography, to effect adequate chiral interaction with the chiral stationary phase ketoprofen was derivatized with 9-aminophenanthrene, under acid conditions, after soli...
Evaluation of a haematological analyser (Sysmex F-800) with equine blood. A semiautomatic electronic blood cell counter (Sysmex F-800) was evaluated with equine blood, according to the protocol of the International Committee for Standardization in Haematology (ICSH, 1984). The precision and overall reproducibility were acceptable for all the parameters studied except for the platelet count, in which a coefficient of variation of 18.8% and 21.7% was obtained for within and between batch precision and 26.76% for overall reproducibility. Carry-over for the haematocrit value and platelet count was unsatisfactory, thus the use of a blank diluent sample between different ...
Application of probability techniques to the objective interpretation of veterinary clinical biochemistry data. Methods for the interpretation of veterinary clinical biochemistry have not developed as rapidly as biochemical technology. However, the results of clinical biochemistry tests are only of value when they are interpreted appropriately. A retrospective study was undertaken to investigate the equine biochemistry data which had been stored in a veterinary hospital database. By applying percentile analysis and Bayesian probability methods to the clinical biochemistry and corresponding diagnosis data, a novel method for the interpretation of clinical biochemistry data has been developed. The method ...
Biotransformation of 17-alkyl steroids in the equine: high-performance liquid chromatography-mass spectrometric and gas chromatography-mass spectrometric analysis of fluoxymesterone metabolites in urine samples. In this study the equine metabolism of fluoxymesterone (9alpha-fluoro-11beta-17beta-dihydroxy-17alpha-meth ylandrost-4-ene-3-one) given orally has been investigated. The parent material was not detected, but two major 16-hydroxy metabolites which corresponded to a mono- and a di-hydroxylation product were evident. One of the hydroxylation positions was identified as C-16. Phase II metabolism in the form of glucuronide formation was also common. These steroids will provide target compounds for confirming abuse of this drug in the horse.
Disposition of human drug preparations in the horse. VI. Tiaprofenic acid. Urinary and plasma concentrations of the nonsteroidal anti-inflammatory drug tiaprofenic acid were determined following oral and intramuscular administration of a dose of 1 g to five fasted horses. Quantitation was performed by high-performance liquid chromatography (HPLC). The limit of quantitation (LOQ) was 0.1 microg/ml and 0.5 microg/ml in 2 ml plasma and 1 ml urine, respectively. Assay precision and extraction recovery were between acceptable values. Tiaprofenic acid pharmacokinetics were described by non-compartment analysis of the data. Absorption was faster after oral administration as...
A microtiter plate assay for the determination of uronic acids. The amount of uronic acid residues in samples containing glycosaminoglycans or pectin is an important parameter in the quantitative and structural analysis of these complex carbohydrates. This paper describes a method to determine the content of uronic acids in biological samples, using conventional polystyrene microtiter plates and microtiter plate-reading equipment with standard interference filters (i.e., 540 or 492 nm). This assay is a modification of a commonly used procedure, viz. hydrolysis of uronic acid containing carbohydrate polymers in 80% sulfuric acid containing tetraborate ions ...
Detection of exogenous hydrocortisone in horse urine by gas chromatography-combustion-carbon isotope ratio mass spectrometry. A gas chromatography-combustion-isotope ratio mass spectrometry method for confirmation of hydrocortisone abuse in horseracing and equine sports is proposed. Urinary hydrocortisone was converted to a bismethylenedioxy derivative which presents good gas chromatographic properties and brings an extra carbon contribution of only two carbon atoms. Synthetic hydrocortisone has a different 13C abundance from that of natural urinary horse hydrocortisone and the difference is significant, therefore exogenous and endogenous hydrocortisone can be distinguished.
Evaluation of some parameters influencing the drug delivery from a dry powder inhalation device using an in vitro model of the horse airways. The aim of this study was to determine the effect of breathing pattern, air humidity and position of the device on the delivery of an aerosol generated by a dry powder inhalation (DPI) device (Inhalator M). The in vitro inhalation study was performed using the cascade impaction method (Andersen Sampler) adapted to imitate nasal breathing. The amount of ipratropium found in the device, the artificial upper airways and the six stages of the Andersen Sampler was measured using high precision liquid chromatography. Stage 1 of the Andersen Sampler was considered to be the respirable fraction and st...
Infrared and atomic spectrometry analysis of the mineral composition of a series of equine sabulous material samples and urinary calculi. Atomic spectrometry has been used in 20 samples of equine urinary sabulous deposits in order to detect minor elements accompanying the predominant element, calcium, which is present in the form of calcium carbonate (calcite and/or vaterite). The elements measured have been (besides calcium) magnesium, sodium, potassium, iron, copper and manganese. Phosphates, sulphates and silica are frequently present as minor constituents of equine urinary sabulous deposits and uroliths, but their detection can be difficult by infrared (IR) spectroscopy in the original samples due to overlapping with the ban...
Determination of methocarbamol in equine serum and urine by high-performance liquid chromatography with ultraviolet detection and atmospheric pressure ionization-mass spectrometric confirmation. Urine and serum samples collected from four standard-bred mares after and oral regimen administration of methocarbamol were extracted and analyzed. The method consisted of enzyme hydrolysis followed by a one-step liquid-liquid extraction, separation on a reversed-phase (RP-18) column, and detection using an ultraviolet (UV) detector. The confirmation was carried out using a liquid chromatography-atmospheric pressure ionization-mass spectrometry (LC-API-MS) system. Maximum methocarbamol concentrations of 1498, 1734, 1547, 2322 micrograms/mL in urine and 4.9, 1.7, and 3.6 micrograms/mL in serum ...
Validation of nonradioactive chemiluminescent immunoassay methods for the analysis of thyroxine and cortisol in blood samples obtained from dogs, cats, and horses. The performances of a radioimmunoassay method, a chemiluminescent immunoassay method, and a chemiluminescent-enzyme immunoassay method were evaluated for the analysis of cortisol and total thyroxine in blood samples obtained from dogs, cats, horses, and humans (reference samples). The analysis of cortisol in human and animal samples exhibited good precision, linearity, and recovery. The 3 methods gave comparable values for the ACTH-induced increase and the dexamethasone-induced decrease in cortisol concentrations in animal samples. The recoveries of total thyroxine from human samples, analyzed...
Enzyme immunoassay for measuring 25-hydroxyvitamin D3 in serum. We developed a rapid, competitive enzyme immunoassay (EIA) for measuring 25-hydroxyvitamin D3 [25(OH)D3] in serum. The EIA was based upon 25(OH)D3-3-hemisuccinate covalently coupled to secondary amino groups grafted onto the polystyrene surface of microtiter wells. Optimal coupling conditions were established, and we found that inclusion of 40 mumol/L chloramine T, an agent not previously described for use in coupling to these plates, resulted in both more reproducible coupling as well as more than a twofold increase in the coupling efficiency. Before EIA, 25(OH)D3 was extracted from the serum...
Low-molecular-weight displacers for high-resolution protein separations. The resolving power of displacement chromatography using low-molecular-weight displacers was investigated using a model mixture containing bovine and horse heart cytochrome c. The linear and nonlinear adsorption behavior of these two proteins was examined in cation-exchange chromatography and shown to be quite similar. Furthermore, an analysis of the dynamic affinity of these proteins indicated extremely similar affinities under displacement conditions. Despite the extreme similarities in the adsorption behavior, displacement chromatography using a protected amino acid displacer resulted in ex...
A unique metabolite of nimesulide. Nimesulide is a nonsteroidal anti-inflammatory drug recently detected in equine blood and urine samples taken at the race track. The detection of the drug in a blood sample led to the identification of an unknown thin-layer chromatographic (TLC) spot in track urine samples as a metabolite of nimesulide. Characterization of the unknown TLC spot and comparison with the synthesized compound shows that the unknown TLC spot is a previously unreported equine metabolite of nimesulide. The metabolite was identified as resulting from the reduction of the nitro group on nimesulide to an amino group. Thi...
Determination of flunixin in equine urine and serum by capillary electrophoresis. A capillary electrophoresis (CE) and a solid-phase extraction method was developed for the determination of flunixin in equine urine and serum. The suitable CE run conditions were described. The factors affecting flunixin recovery rates were investigated and optimum solid-phase extraction conditions for flunixin in equine urine and serum were established. Limits of detection and quantitation were 3.4 and 5.6 ng/ml for serum and 16.9 and 33.1 ng/ml for urine, respectively. The recoveries exceeded 96% for urine and 79% for serum. Urine samples from race horses and urine and serum samples from a ...
Multi-element assay of mammary secretions and sera from periparturient mares by inductively coupled argon plasma emission spectroscopy. To document and determine changes in the mineral profiles of sera and mammary secretions from a population of periparturient mares. Methods: 18 clinically normal periparturient Arabian broodmares. Methods: Inductively coupled argon emission spectroscopy was used to measure Ca, Cu, Fe, K, Mg, Mn, Na, P, and Zn concentrations in sera and mammary secretions of periparturient mares. In addition, S was measured in mammary secretions. Results: Serum concentrations of Ca, Cu, Fe, K, Mg, Na, P, and Zn remained constant throughout late pregnancy and the first 7 days of lactation. Compared with values o...
Molten globule state of equine beta-lactoglobulin. The acid-unfolded state of equine beta-lactoglobulin was characterized by means of circular dichroism, nuclear magnetic resonance, analytical gel-filtration chromatography, and analytical centrifugation. The acid-unfolded state of equine beta-lactoglobulin has a substantial secondary structure as shown by the far-ultraviolet circular dichroism spectrum but lacks persistent tertiary packing of the side chains as indicated by the near-ultraviolet circular dichroism and nuclear magnetic resonance spectra. It is nearly as compact as the native conformation as shown by the gel filtration and sedime...
Consideration of the optimum pH for the analysis of serum p-phenylenediamine oxidase activity in thoroughbred horses. The optimum pH for the measurement of serum p-phenylenediamine oxidase (Ox) activity was given (pH 6.6), and the relationship between serum ceruloplasmin (Cp) concentration and its Ox activity was established in healthy adult horses. In adult horses, serum antigenic Cp concentrations were measured by the single radial immunodiffusion (SRID) method with the affinity-purified antibody to equine plasma Cp and compared with its Ox activity. Efficient co-relation between Cp concentration and Ox activity in the sera (r = 0.93) and its Ox/Cp ratio were given. These results might contribute to the cal...
Pharmacokinetics of intravenous and oral prethcamide in horses. The respiratory stimulant prethcamide is a mixture of equal parts of crotethamide and cropropamide. A specific and sensitive gas chromatographic method for the determination of crotethamide and cropropamide in horse plasma and urine is described. Both components of prethcamide were extracted from plasma and urine into dichloromethane. The extracts were analyzed by capillary gas chromatography with thermionic detection in the nitrogen-specific detection mode. The lower limits of quantitation were 4.0 ng ml-1 of plasma and 10.0 ng ml-1 of urine. Calibration curves were linear from 2.0-100 ng ml-...
Quantitative ionspray liquid chromatographic/tandem mass spectrometric determination of reserpine in equine plasma. A method based on ionspray liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed for the determination of reserpine in equine plasma. A comparison was made of the isolation of reserpine from plasma by liquid-liquid extraction and by solid-phase extraction. A structural analog, rescinnamine, was used as the internal standard. The reconstituted extracts were analyzed by ionspray LC/MS/MS in the selected reaction monitoring (SRM) mode. The calibration graph for reserpine extracted from equine plasma obtained using liquid-liquid extraction was linear from 10 to 5000 pg ml-1 and t...
High-performance liquid chromatographic determination of N-alpha-acetyl-L-carnosine in equine plasma. N-alpha-Acetyl-L-carnosine (NAcCAR) in perchloric acid extracts of equine plasma was assayed by high-performance liquid chromatography on a 3 microns Hypersil ODS (150 x 4.6 mm I.D.) column eluted with 5 mM phosphoric acid-1 mM triethylamine, pH 2.58. NAcCAR was isolated by solid-phase extraction on Isolute PRS (propylsulphonyl) columns. The HPLC mean retention time for NAcCAR was 5.9 +/- 0.2 min. The recovery from plasma by solid-phase extraction was 93.9-99.7% and lower limit of detection in plasma was 0.18 microM. The normal NAcCAR concentration in equine plasma was 2.4 +/- 0.3 microM. The ...
High-performance liquid chromatographic determination of imidazole dipeptides, histidine, 1-methylhistidine and 3-methylhistidine in equine and camel muscle and individual muscle fibres. The combined solid-phase extraction (Isolute PRS columns) and reversed-phase gradient HPLC method presented provides a sensitive, reproducible and selective quantification of carnosine, balenine, homocarnosine, histidine, 1-methylhistidine and 3-methylhistidine in equine and camel muscle and individual muscle fibres. Recoveries were 91-115%. Lower limits of detection were 0.005-0.010 mmol kg-1 dry muscle. The compounds were isolated from other physiological amino acids and small peptides and resolved within a single chromatographic run of 55 min. Concentrations of these compounds in equine myo...
Effects of delayed serum separation and long-term storage on the measurement of thyroid hormones in equine blood samples. Studies were conducted to determine the effects of delaying the separation of serum from the clot and of long-term storage of serum samples on the measurement of thyroid hormones in blood from horses using a fluorescence polarization immunoassay. The measured concentrations of T3 and T4 were not affected by leaving serum on the clot for as long as 24 hours at room temperatures. Storage of serum for 19 to 22 months at -20 degrees C resulted in significant increases of measured T4, but not T3. These studies support previous work demonstrating that thyroid hormones are resistant to degradation, i...
Myoglobin oxygen dissociation by multiwavelength spectroscopy. Multiwavelength optical spectroscopy was used to determine the oxygen-binding characteristics for equine myoglobin. Oxygen-binding relationships as a function of oxygen tension were determined for temperatures of 10, 25, 35, 37, and 40 degrees C, at pH 7.0. In addition, dissociation curves were determined at 37 degrees C for pH 6.5, 7.0, and 7.5. Equilibration was achieved with a myoglobin solution, at the desired temperature and pH, and 16 oxygen-nitrogen gas mixtures of known oxygen fraction. Correction for the inevitable presence of metmyoglobin was made by using a three-component least squ...
Local electrostatic potentials in pyridoxal phosphate labelled horse heart cytochrome c. The present work shows the application of an optical label pyridoxal phosphate (PLP) for the experimental determination of local electrostatic potentials in singly substituted cytochromes c modified by pyridoxal phosphate at Lys 79 (PLP-Lys-79-cyt.c) or at Lys 86 (PLP-Lys-86-cyt.c). PLP has also been used to calculate the pKa values of all ionizable groups and the electrostatic potentials in the modified proteins and to analyse their properties. The experimental pKa values for the pyridine nitrogen and phenolic hydroxyl of the bound label were obtained from pH-dependent absorbance and fluoresc...
Solid-phase extraction and derivatisation methods for beta-blockers in human post mortem whole blood, urine and equine urine. This paper details various rapid and sensitive methods for the extraction and derivatisation of propranolol, metoprolol, sotalol, atenolol, pindolol, timolol, oxprenolol, alprenolol and penbutolol in equine urine and in human post mortem whole blood and urine. Three solid-phase extraction methods are described involving the use of either XtrackT XRDAH515, Bond Elut Certify or Sep-Pak C18 cartridges. Two derivatisation methods are also described involving the formation of cyclised silyl or pentafluoropropionate derivatives with either chloromethyldimethylchlorosilane or pentafluoropropionic anh...
