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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.
Evaluation of the potential for interference by dimethyl sulfoxide (DMSO) in drug detection in racing animals. Dimethyl sulfoxide (DMSO) had been postulated to be a 'masking agent' when used concurrently with therapeutic or prohibited drugs in racing animals. Eight drugs (flunixin, furosemide, caffeine, apomorphine, phenylbutazone, lidocaine, cocaine, and acepromazine maleate) were administered to six horses singly and with concurrent intravenous DMSO. Urine samples were analyzed for the presence of the drugs and/or their metabolites by thin layer chromatography. Direct comparison of thin layer chromatograms of extracts of positive urine samples with and without DMSO verified that DMSO did not interfer...
Analysis of detomidine in horse blood, plasma and urine samples utilizing a sensitive gas chromatography-mass spectrometry method. Chemical ionization- and electron impact ionization-selective ion monitoring provided a simple and sensitive method for measuring detomidine (Domosedan), a potent sedative-analgesic drug for horses and cattle. Chemical ionization was at least 10 times more sensitive than electron impact ionization. By using propranolol as an internal standard, we found that the recovery of detomidine from the extraction procedure used in this study was greater than 75% for plasma, whole blood, or urine samples. Approximately 68% of detomidine was bound to plasma protein and 53% was bound to red blood cells.
The concentrations of free Mg2+ and free Zn2+ in equine blood plasma. The enzyme phosphoglucomutase can be used as a metal ion indicator to measure the concentrations of free Mg2+ and free Zn2+ in physiological fluids. In horse plasma, the concentration of free Mg2+ is close to 0.5 mM, whereas that of free Zn2+ is about 2 X 10(-10) M, although numerous physiological roles for Zn2+ have been postulated that would require free Zn2+ concentration orders of magnitude higher than this. A titration of plasma with Zn2+ shows that the fractional increase in free Zn2+ is essentially the same as the fractional increase in total exchangeable Zn2+, and the results are consi...
Measurement of flunixin in equine inflammatory exudate and plasma by high performance liquid chromatography. An accurate and reliable method for the separation of flunixin from, and measurement in, equine inflammatory exudate and plasma by high performance liquid chromatography has been developed. Flunixin can be detected in concentrations as low as 0.05 micrograms/ml using an ultraviolet spectrophotometric detector at 285 nm. Samples were acidified with 2M hydrochloric acid and extracted with dichloromethane. The extract was evaporated and reconstituted in acetonitrile. Iminodibenzyl was used as internal standard. The mean recovery of flunixin from plasma was 97.6 +/- 3.9 per cent. Particular advant...
HPLC determination and pharmacokinetics of thiabendazole and its major metabolite 5-OH thiabendazole in equine plasma. Separate high performance liquid chromatographic methods were developed for thiabendazole (TBZ) and 5-hydroxy thiabendazole (5-OH-TBZ) determination in horse plasma using 1-methyl-2-phenyl benzimidazole (MPBZ) as an internal standard. In both methods TBZ and 5-OH-TBZ were extracted from plasma using organic solvents, injected on to a C-18 column, and eluents monitored by a fluorescence detector. However, mobile phase composition, extraction solvent as well as detector wavelength differed in the two methods. The linear range for TBZ was 0.02 to 0.77 microgram ml-1 while that for 5-OH-TBZ was 0....
Screening of amphetamines by gradient microbore liquid chromatography and pre-column technology. Amphetamine-type drugs with a wide polarity range have been screened in both human and horse urine using on-line pre-concentration on pre-columns packed with hydrophobic and cation-exchange sorbents in series and gradient microbore high-performance liquid chromatography. The underivatized amphetamines were identified by UV detection at 210 nm. The method has potential for the automated liquid chromatographic screening of amphetamines in urine, e.g., for doping control.
Principles of drug disposition in the horse. This article is intended to give the reader an understanding of the mathematic and conceptual framework underlying equine pharmacology. The methods by which the veterinary practitioner determines drug concentrations, disposition, and bioavailability are discussed.
Detection and identification of ketamine and its metabolites in horse urine. The possibility exists that ketamine, or ketamine in combination with xylazine, is being used illicitly to affect the performance of racehorses. This study was undertaken to identify the metabolites of ketamine in the urine of adult horses and to evaluate methods for detecting and confirming ketamine administration. Detection of ketamine and two ketamine metabolites is described using thin-layer chromatography (TLC) and their identities are confirmed by comparing their mass spectra and gas chromatographic retention times with those of authentic standards.
Development of a gas chromatographic-mass spectrometric method using multiple analytes for the confirmatory analysis of anabolic steroid residues in horse urine. II. Detection of administration of 19-nortestosterone phenylpropionate to equine male castrates and fillies. Esters of 19-nortestosterone form an important group of anabolic preparations used in veterinary practice. Based upon results from detailed metabolic studies for 19-nortestosterone in the horse, a method to confirm the administration of anabolic preparations of this steroid to castrated male horses and fillies is described; the method is based upon the use of multiple analytes. Following administration of the anabolic preparations, solid-phase extraction of urinary conjugates and the separation of the conjugate groups prior to hydrolysis allow for the determination of specific metabolites conj...
Determination of ampicillin in serum by using simple ultrafiltration technique and liquid chromatographic analysis. A new liquid chromatographic method for determination of ampicillin in canine and equine serum has been developed. The serum sample (500 microL) is vortex-mixed with 20% ethanol (500 microL) and filtered using a 30,000 molecular weight cutoff microseparation tube to separate high molecular weight solutes following low-speed centrifugation. Ampicillin is then separated from other serum components by reverse phase ion-pair liquid chromatography (LC). The ultraviolet (UV) absorbance of the column effluent is monitored at 230 nm. Recoveries of ampicillin from canine serum spiked at concentrations ...
Radioimmunoassay screening for etorphine in racing horses. A commercially available radioimmunoassay kit was used to screen for the presence of etorphine in post-race urines from horses racing in Kentucky. Most horse urines contained small amounts of materials which reacted positively in this immunoassay. These materials are apparently endogenous to the horse and were called apparent etorphine equivalents. The levels of these apparent etorphine equivalents in post-race urines from 70 horses were estimated. Their modal level averaged 0.1 ng/ml, the population distribution was log normal, and individual horses showed levels of up to 0.8 ng/ml.
Development of a gas chromatographic-mass spectrometric method using multiple analytes for the confirmatory analysis of anabolic steroids in horse urine. I. Detection of testosterone phenylpropionate administrations to equine male castrates. A gas chromatographic-mass spectrometric (GC-MS) method using three analytes to detect and confirm the administration to equine male castrates of veterinary pro-drugs based upon esters of testosterone is described. The method involves extraction of steroid conjugates from horse urine by C18-bonded cartridges and fractionation into glucuronic acid and sulpho-conjugates by Sephadex LH-20 column chromatography. After deconjugation, the free neutral steroids were partially purified by thin-layer chromatography and following derivatization (methyloxime-trimethylsilyl ether) were analysed by capilla...
Determination of nefopam in equine plasma by gas chromatography-mass spectrometry with chemical ionization. This study demonstrates the development of a method using gas chromatography-mass spectrometry for determining nefopam, a non-narcotic pain reliever that is sometimes abused in horse doping, in equine plasma. Background […]
Occurrence and distribution of 5-S-cysteinyl derivatives of dopamine, dopa and dopac in the brains of eight mammalian species. The 5-S-cysteinyl derivatives of dopamine, dopa (3,4-dihydroxyphenylalanine) and dopac (3,4-dihydroxyphenylacetic acid) were synthesized and used as reference compounds in high performance liquid chromatography analyses of extracts from various brain regions of eight mammalian species. All three metabolites were detected in the brains of all the species studied. The regional distribution of the metabolites was similar to that of dopamine; the metabolite concentrations ranged from less than 0.1 percent to more than 1 percent of the dopamine level, the highest ratios generally being found in sub...
Isolation of meclofenamic acid and two metabolites from equine urine–a comparison between horse and man. Two metabolites of meclofenamic acid have been isolated from equine urine. Both metabolites are found to be monohydroxylated forms of meclofenamic acid by gas chromatography-mass spectrometry after extractive alkylation. The parent drug and the metabolites are separated by reversed-phase liquid chromatography on a Spherisorb ODS column, using methanol-phosphate buffer eluents and UV detection at 280 nm. The structure of the metabolites is discussed on the basis of LC, TLC and GC-MS data.
Identification of betamethasone and a major metabolite in equine urine. Betamethasone and its major unconjugated metabolite, 6-beta-hydroxybetamethasone, were detected in equine urine by thin-layer chromatography and characterized by micro-liquid chromatography/mass spectrometry (micro-LC/MS). Their structures were confirmed by a combination of infrared spectroscopy and nuclear magnetic resonance spectroscopy.
Analysis of pharmaceutical dosage forms for oxfendazole: II. Simultaneous liquid chromatographic determination of oxfendazole and trichlorfon in equine paste. A reverse phase liquid chromatographic procedure is described for the simultaneous determination of oxfendazole [2-(methoxycarbonylamino)-5-phenylsulfinylbenzimidazole] and trichlorfon [(2,2,2-trichloro-1-hydroxyethyl)phosphonic acid dimethyl ester] in equine paste. The sample is extracted by sonication in methanol. Insoluble excipients are removed by centrifugation and an aliquot plus internal standard are diluted with dilution solvent (water-acetonitrile-phosphoric acid, 80 + 20 + 1). The samples are filtered and injected onto a Partisil-5 ODS-3 column with acetonitrile-0.01 M phosphate buff...
Quantitative methodology for corticosteroids based on chemical oxidation to electrophilic products for electron capture-negative chemical ionization using capillary gas chromatography-mass spectrometry. I. Assessment of feasibility in the analysis of horse urine for dexamethasone. Sensitive and specific methodology based on capillary column gas chromatography-electron capture-negative chemical ionization-mass spectrometry has been developed for the quantitative analysis of corticosteroids from biological fluids. The feasibility of this method is demonstrated in the quantitative analysis of dexamethasone in horse urine following administration of the drug. A structurally similar compound, 6 alpha-methylprednisolone, is added to the urine as an internal standard. The free dexamethasone and the internal standard are extracted and oxidized to high-electron-affinity 1,4-andr...
Methods of assigning accurate values to reference serum. Part 2. The use of definitive methods, reference laboratories, transferred values and consensus values. Eight analytes (Ca, Cl, cholesterol, glucose, Mg, K, Na and urate) have been determined in one horse serum masterlot by up to six different procedures: (i) by so-called definitive methods; (ii) by a group of reference laboratories using a variety of analytical methods; (iii) using the results of two independent external quality assessment schemes; (iv) by transferring values from a human serum standard reference material analysed by definitive methods; (v) by similar transfer of values from several batches of horse reference serum previously analysed by definitive methods; and (vi) as in (v) b...
Concentration of sodium hyaluronate in serum. A radioassay for sodium hyaluronate using high-affinity binding protein from bovine cartilage has been modified for serum analysis. The accuracy of the method was checked by isotope dilution experiments and by recovery studies with exogenous hyaluronate. The between-assay standard deviation in the determination is 15-20%. The concentration of sodium hyaluronate in healthy adults (blood donors) is in the range of 10 to 100 micrograms/l with a mean value in the order of 30 to 40 micrograms/l. This is a lower concentration than previously reported. The same level was found in young people. Higher...
Equine whole saliva: variability of some major constituents. Whole saliva was collected from six horses over a period of five weeks in sufficient volume for the analysis of 10 constituents. There was considerable variation in the concentration of the analytes both between horses and between different days in the same horse. The most variable constituent was sodium, and the least variable was glucose, but this was derived from the sweet used to stimulate salivation. The use of whole saliva as a fluid for investigation would depend on achieving the minimum variability possible.
The isoelectric focusing of keratins in hair followed by silver staining. An isoelectric focusing method followed by silver staining has been developed for the study of keratins which is as effective as two-dimensional electrophoresis and fluorography for hair species identification. Hair from dogs, rabbits, horses, cows, guinea-pigs, donkeys, sheep and cats were successfully identified. Narrow pH ranges were used to observe heterogeneity in human hair. Although this heterogeneity may be affected by environmental conditions, it may be of use in criminalistics.
