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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.
Circular dichroic properties and conformation of thionicotinamide dinucleotides bound to horse-liver alcohol dehydrogenase. The interaction between horse liver alcohol dehydrogenase and the oxidized and reduced forms of the 3-thionicotinamide--adenine dinucleotide coenzyme analogues (sNAD and sNADH) has been investigated by ultraviolet absorption, fluorescence and circular dichroism. The fluorescence of sNADH is enhanced when bound to the enzyme, and the protein fluorescence is quenched by both sNADH (60--65%) and sNAD (65%). The possible origin of the larger quenching produced by sNAD with respect to that of NAD is discussed. Coenzyme dissociation constants have been determined by monitoring the quenching of the p...
Studies related to the metabolism of anabolic steroids in the horse: a gas chromatographic mass spectrometric method to confirm the administration of 19-nortestosterone or its esters to horses. A method is described to confirm the presence of 19-nortestosterone metabolites in urine after the administration of veterinary preparations of this anabolic steroid to horses. The method is based upon the detection, by gas chromatography mass spectrometry or selected ion monitoring, of an isomer of estrane-3,17-diol in the urine.
Studies related to the metabolism of anabolic steroids in the horse: 19-nortestosterone. 1. The metabolism of 19-nortestosterone in a cross-bred horse has been studied using 14C-labelled material. 2. Two neutral metabolites isolated from urinary extracts by column chromatography were identified as isomers of 3-hydroxyestran-17-one and estrane-3,17-diol by g.l.c.-mass spectrometry. 3. The stereochemistry of the two metabolites has been investigated by comparison of the retention times of their trimethylsilyl derivatives with those of standard steroids of known configuration.
Direct radioimmunoassay of progesterone in mare plasma. A rapid and low cost radioimmunologic procedure for progesterone assay in mare plasma is proposed. Radioimmunoassay is performed directly on 10 microliter of unextracted plasma. Free progesterone is adsorbed on dextran-charcoal, then the aqueous phase is decanted and extracted by 1 ml of scintillation fluid. Counting is performed directly on this two-phase system. Results are comparable to those obtained with radioimmunoassays using extracted plasma.
Chromatographic determination of some corticosteroids, with special reference to horse doping. Some chromatographic procedures, which can be used to detect and determine certain corticosteroids in samples from race horses, are described. These procedures include thin-layer, gas and high pressure liquid chromatography.
Determination of total and ultrafilterable calcium and magnesium in normal equine serum. Total and ultrafilterable calcium (Ca) and magnesium (Mg) values were determined for Shetland pony stallions, stallions, and pregnant and diestrous mares, using a simple, inexpensive, quick procedure to obtain an ultrafiltrate of serum. There was no significant difference between horses and ponies, between stallions and mares, or between pregnant and nonpregnant mares. The percentage of total serum Ca that was ultrafilterable was 63.4+/-1.7 for horses and 64.8+/-2.2 for ponies. The percentage of total serum Mg that was ultrafilterable was 75.6+/-1.5 for horses and 77.0+/-1.7 for ponies. Total ...
Amino acid composition of casein isolated from the milks of different species. Casein was isolated from the milks of the following species: cow, horse, pig, reindeer, caribou, moose, harp seal, musk-ox, polar bear, dall sheep, and fin whale. The caseins were subjected to acid hydrolysis, the resultant amino acids were converted to their n-butyl-N-trifluoroacetyl esters, and the amino acid composition of the caseins was determined by gas chromatographic analysis of these esters. Notable among the results was the close similarity, with respect to amino acid composition, of reindeer and caribou caseins. The results of the amino acid analyses of the other caseins are present...
Isolation, identification and quantitation of serum 5alpha-pregnane-3,20-dione and its relationship to progesterone in the pregnant mare. 5alpha-pregnane-3,20-dione was isolated from pooled pregnant mare serum using Sephadex LH-20 column chromatography and identified by the use of radioimmunoassay, gas-liquid chromatography and gas-liquid chromatography-mass spectrometry analyses. 5beta-pregnane-3,20-dione was not cross-reactive with the radioimmunoassay system and was not detected by gas-liquid chromatography. Peripheral blood levels of progesterone and 5alphs-pregnane-3,20-dione were determined by radioimmunoassay in four Quarter Horse mares for the first 150 days of gestation. Progesterone and 5alpha-pregnane-3,20-dione decli...
Electron capture detection of an apomorphine heptafluorobutyrate derivative at low picogram levels. An electron capturing derivative of apomorphine was prepared by incubating the drug with heptafluorobutyric anhydride (HFBA), triethylamine and heat. Mass spectral analysis suggests that HFBA reacts with both phenolic hydroxyl groups on apomorphine to give a derivative detectable at low picogram levels. This method is sufficiently sensitive for pharmacokinetic studies in the horse and is likely applicable to other dopaminergic analogues of apomorphine.
The excretion of ibuprofen by the horse – a preliminary report. The anti-inflammatory drug Ibuprofen [(+/-)-2-(p-isobutylphenyl) propionic acid] was estimated in the blood and urine of a horse using gas-liquid chromatography of the silylated derivative. Levels of the drug in the two body fluids were measured over a period of about 24 hours after administering a 12 gm dose of Ibuprofen. Plasma peak levels were observed within 30 to 60 min, and the drug was no longer detectable in the plasma by 8 hr. Urinary peak levels were observed 200 to 300 min after dosing, and the drug was no longer detectable in the urine by about 28 hr. It was observed that only 2% t...
The antidoping control in horseraces in Italy. The results and the improvement of the analytical procedures adopted for the control of doping in horses will be reported. This control has been systematically carried out in Italy for about 10 years in the laboratories of Italian Federation of Sport and Medicine in which the biological samples for the control of doping in various sport activities (football, cycling, athletics etc.) are also examined. In this way it is possible to use the same instruments for all these similar problems and compare the results. The analytical procedure is based on the following steps: 1) Extraction of the sampl...
The gas-liquid chromatograph and the electron capture detection in equine drug testing. Three gas-liquid chromatographic (G.L.C.) procedures discussed have been designed around the four "esses" of detection tests--speed, sensitivity, simplicity, and specificity. These techniques are admirably applicable to the very low plasma drug levels encountered in blood testing under pre-race conditions. The methods are equally applicable to post-race testing procedures, where both blood and urine samples are tested. Drugs can only rarely be detected by the electron capture detector (E.C.D.) without a prior derivatization step, which conveys to the drug(s) high electron affinity. Because of ...
Research and identification of tranquillizers – use of retention index. At the request of the Service des Haras, our laboratory works on the toxicological problems of the sport-horse. These studies have resulted in the setting up of an anti-doping control for equestrian competitions of various types, not only flat racing. During events, horses, must be calm and docile to the riders' order. Frequently, the latter use tranquillizers to try and win events. The analytical method for the research and identification of these compounds is described. The technique involves successively: 1. alkalinisation of the sample - saliva, blood or urine after enzymatic hydrolysis. 2...
Less common “doping” agents and substances encountered during routine screening for drugs. The chromatographic and spectroscopic properties of several unusual substances which have been detected in the "alkaloidal" chloroform extract from racehorse urine and saliva samples are reported. Some of these substances have been identified by combined gas chromatography-mass spectrometry and the source of the substance is stated where this is known. Other substances whose identity is not known have been detected and their mass spectra show characteristic amine fragments. The occurrence of these unidentified substances is more frequent in aged urine samples and it would therefore appear that...
Doping control in Japan. An automated extraction procedure for the doping test. Horse racing in Japan consists of two systems, the National (10 racecourses) and the Regional public racing (32 racecourses) having about 2,500 racing meetings in total per year. Urine or saliva samples for dope testing are collected by the officials from thw winner, second and third, and transported to the laboratory in a frozen state. In 1975, 76, 117 samples were analyzed by this laboratory. The laboratory provides the following four methods of analysis, which are variously combined by request. (1) Method for detection of drugs extracted by chloroform from alkalinized sample. (2) Methods fo...
The problem of testing horse kidneys for the presence of antibiotics at meat inspection: how to avoid a false positive reaction. When 33 horse kidneys were tested for the presence of inhibitory substances by the Bacillus subtilis BGA method at pH 8 and the Micrococcus luteus ATCC 9341 method, 24 were positive and 9 negative. The pH of the seeded M. luteus test medium changed from pH 6.6 before incubation to 8.7 after 24 hours incubation at 30 degrees C. When the same 33 kidneys were tested by the B. subtilis BGA method, medium pH 6, and 15 of them also by the M. luteus method using a medium buffered to pH 6, all were negative. The cadmium concentration of the 33 horse kidneys was found to be 70.17 +/- 81.28 mg/kg wet we...
A comparison of techniques for the quantitative analysis of hyaluronic acid in equine synovial fluid. A comparison of methods of preparing the hyaluronic acid of equine synovial fluid for quantitative spectrophotographic analysis is presented. A new method is proposed which appears superior to the previous methods.
Mefanamic acid blood and urine levels in the horse determined by derivative gas-liquid chromatography-electron capture. Mefenamic acid is extracted from biological fluids and is acylated with pentafluoropropionic anhydride to form a derivative possessing high electron affinity. The derivative is analyzed by gas-liquid chromatography with an electron capture detector. The method is particularly valuable for determining drug levels in blood where small sample and/or drug concentrations are available.
Some assay restrictions on inferences made from determining hormones in horses, cows, and their fetuses. Often in developing hormone assays, hormones that may interfere with the assay by cross-reaction are not available for testing the validity of the assay. For example, horse TSH was unavailable to test for cross-reaction in an LH radioimmunoassay (RIA). The authors devised an indirect means of accomplishing the same goal, and the evidence from the indirect test of cross-reaction was at least as persuasive as a direct test might have been. Other examples are given of experiments where extensive effort was devoted to validation of steroid RIA, but there were substantial quantitative differences i...
Purification, characterization, and quantitation of the antigen employed in the immunodiffusion test for diagnosis of equine infectious anemia. Equine infectious anemia (EIA) antigen extracted from the spleen of horses infected with EIA virus was purified by pH treatment, (NH4)2SO4 fractionation and affinity chromatography. The homogeneity of the antigen was indicated by sedimentation rate and sedimentation equilibrium experiments. A S20,w of 0.51 was determined and a molecular weight of 7600 was calculated from sedimentation equilibrium analysis. The amino acid composition of the pure antigen indicated the antigen is an acidic protein. Employing radical immunodiffusion (RID) and pure antigen a method for quantitating antigen content ...
Lipids of human and equine smegma. The lipids of human and equine smegma pools were saponified and the total fatty acids submitted to temperature programmed gas chromatography (GC) analysis. In contrast to the human products, the horse smegma fatty acids contained very low odd saturated as well as olefinic branched chain acid contents. The cyclopropane fatty acid, 9,10-methyleneoctadecanoic acid, occurred in smegma sampled from men over 35 years of age but could not be detected in the pool from persons of 17-20 years of age nor in any of the equine mixtures. The alcoholic fraction from horse smegma contained about 85% sterol, t...
Carbohydrate composition of horse spleen ferritin. The carbohydrate composition of horse spleen ferritin was studied. 1 mol of the apoferritin, the protein moiety of ferritin, contains 25 mol of hexose, 3 mol of hexosamine and 10 mol of fucose. Same carbohydrate composition was detected in the apoferritin from iron rich ferritins. These results indicate that horse spleen ferritin is composed of non-identical subunits as regards its carbohydrate composition.
