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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.
Pharmacokinetic disposition of an immediate-release aminophylline and a sustained-release theophylline formulation in the horse. The pharmacokinetic disposition of theophylline was determined by high-performance liquid chromatographic analysis of plasma samples from six healthy, adult horses following the administration of intravenous aminophylline (dosed at 9.94 mg/kg as theophylline), immediate-release aminophylline tablets (dosed at 9.94 mg/kg as theophylline), and sustained-release theophylline tablets (dosed at 20 mg/kg). The elimination rate constant (lambda z), apparent volume of distribution (Vz), and clearance (Cl) determined by compartmental analysis of the intravenous data were 0.07 +/- 0.01 h-1, 0.80 +/- 0.0...
Thin-layer chromatographic screening procedure for some drugs in horse plasma. A thin-layer chromatographic screening procedure for some basic, neutral and acidic drugs was developed using 3 ml of horse plasma. Chloroform-2-propanol (95:5, v/v) was used as the extraction solvent. The drugs were identified by a high-performance thin-layer chromatographic plate and spraying successively with some detection reagents. In this study, the extraction recovery rates and the detection limits were determined at the same time.
Simultaneous analysis of furosemide and bumetanide in horse plasma using high performance liquid chromatography. A high performance liquid chromatographic method is described for the simultaneous determination of furosemide and bumetanide in horse plasma. The C8 (3 microns) reversed phase column (4.8 x 150 mm) provided clear separation of furosemide and bumetanide with other components present in the horse plasma. The detection limit for both the drugs was 10 ng/mL. Both drugs were stable in plasma (at natural or acidic pH) for up to 24 h. The method is sufficiently sensitive to detect furosemide levels in plasma obtained from horses receiving a therapeutic dose of furosemide.
An improved TLC method for the detection of flunixin in equine serum. A method for flunixin detection in equine serum extracts involving thin layer chromatography, spraying the chromatogram with alkaline sodium hypochlorite solution and heating with a detection limit of 50 ng ml-1 is described.
A new method for hydrolyzing sulfate and glucuronyl conjugates of steroids. A new method for hydrolyzing steroid conjugates (both sulfates and glucuronides conjugates) that is efficient, effective, and inexpensive is described. This method comprises incubation of the conjugates--after salting-out into ethyl acetate or elution from a C18 cartridge--with anhydrous methanolic hydrogen chloride (methanolysis) for 10 min. It has been successfully applied to our routine radioimmunoassay screening and GC/MS confirmation studies of steroids in prerace and postrace equine urine samples. Comparative GC/MS studies on entire (male horse) urine samples showed that methanolysis gav...
Determination of leucine enkephalin and methionine enkephalin in equine cerebrospinal fluid by microbore high-performance liquid chromatography and capillary zone electrophoresis coupled to tandem mass spectrometry. The performance of microbore high-performance liquid chromatography and capillary zone electrophoresis, both equipped with on-line tandem mass spectrometric detection capability, was evaluated critically for the determination of endogenous amounts of leucine enkephalin and methionine enkephalin in equine cerebrospinal fluid. Using an identical sample clean-up and enrichment procedure, capillary zone electrophoresis-mass spectrometry is limited in its concentration detection capacity owing to its much smaller injection volume. Leucine enkephalin was identified in post-mortem equine cerebrospina...
Screening of steroids in horse urine and plasma by using electron impact and chemical ionization gas chromatography-mass spectrometry. Gas chromatography with chemical ionization mass spectrometry and selected-ion monitoring provided a sensitive method for the screening and confirmation of steroids in horse urine and plasma. Chemical ionization mass spectrometry was more sensitive than the electron impact ionization mass spectrometry for most of the steroids except for testosterone, prednisone-metabolite-2 and prednisolone-metabolite-2. The chromatographic conditions used in this study provided clean separation of different natural and synthetic steroids. Approximately 75-85% of the steroids added to plasma and approximately ...
Comparison of the use of mass spectrometry and methylene unit values in the determination of the stereochemistry of estranediol, the major urinary metabolite of 19-nortestosterone in the horse. The stereochemistry of an isomer of 5-estrane-3,17 alpha-diol, the major metabolite of 19-nortestosterone in horse urine has been established by the use of methylene unit (MU) values. The empirical MU values of the bis-trimethylsilyl (TMS) derivatives of the eight available isomers of 5-androstane-3,17-diol and four isomers of 5-estrane-3,17 beta-diol were determined by capillary gas chromatography using three different columns. From this data the theoretical MU values for the bis-TMS derivatives of the four 5-estrane-3,17 alpha-diol isomers were predicted. Comparison of the experimentally det...
Application of high-performance liquid chromatography–inductively coupled plasma mass spectrometry to the investigation of cadmium speciation in pig kidney following cooking and in vitro gastro-intestinal digestion. The speciation of cadmium in retail pig kidney has been examined by size-exclusion chromatography (SEC) coupled directly to inductively coupled plasma mass spectrometry (ICP-MS). Approximately 35% of the cadmium from uncooked kidney was soluble after aqueous extraction at pH 8 and SEC - ICP-MS revealed three discrete peaks whose retention times corresponded to estimated relative molecular masses of 1.2 x 10(6), 7.0 x 10(4) and 6 x 10(3)-9 x 10(3). In the cooked kidney, 35% of the Cd was soluble and was all associated with a peak of a relative molecular mass (Mr) of 6 x 10(3)-9 x 10(3). After s...
GC/MS confirmatory method for etorphine in horse urine. A highly sensitive procedure for GC/MS determination of etorphine in horse urine is described. This assay provides both specificity and reliability and is particularly well suited for the confirmation of radioimmunoassay screening procedures usually used for etorphine. After solvent extraction and purifications, the etorphine is characterized as a pentafluoroacetic derivative (PFAA) by using mass fragmentography. The detection limit is 0.1 ng/mL in urine; the coefficient of variation of the estimations is 10.9%. The procedure has been validated after on-field administration of 5 to 90 microgra...
Analysis of post mortem aqueous humour chemistry in the horse, with particular reference to urea nitrogen and creatinine. The concentrations of several post mortem aqueous humour chemical constituents were compared with ante mortem serum chemical values in the horse. Urea nitrogen and creatinine values in post mortem aqueous humour were good predictors of ante mortem serum values. Aqueous humour urea nitrogen increased only slightly and creatinine did not change significantly for up to 24 h after death. Formulae were derived for calculating estimated ante mortem serum urea nitrogen and creatinine from aqueous humour values obtained after death. These results from normal horses identify analytes that are accurate ...
Screening and confirmation of drugs in horse urine by using a simple column extraction procedure. A simple and reproducible column (Clean Screen-DAU, copolymeric bonded-phase silica column) extraction procedure has been described for the screening and confirmation of drugs in horse urine. The recovery of drugs by the column extraction was better than or comparable to the recovery by the liquid-liquid extraction, which is commonly used in the equine analytical laboratories. The column extraction provided broad coverage of drugs, separated extracts into three fractions (acidic/neutral, steroids, basic), produced a cleaner extract, and eliminated the need for special liquid-liquid extraction ...
Mössbauer spectroscopic study of the initial stages of iron-core formation in horse spleen apoferritin: evidence for both isolated Fe(III) atoms and oxo-bridged Fe(III) dimers as early intermediates. Ferritin stores iron within a hollow protein shell as a polynuclear Fe(III) hydrous oxide core. Although iron uptake into ferritin has been studied previously, the early stages in the creation of the core need to be clarified. These are dealt with in this paper by using Mössbauer spectroscopy, a technique that enables several types of Fe(II) and Fe(III) to be distinguished. Systematic Mössbauer studies were performed on samples prepared by adding 57Fe(II) atoms to apoferritin as a function of pH (5.6-7.0), n [the number of Fe/molecule (4-480)], and tf (the time the samples were held at room ...
The sequence-specific assignment of the 1H-NMR spectrum of an enzyme, horse-muscle acylphosphatase. A complete range of two-dimensional NMR experiments was used for the assignment of the 1H-NMR spectrum of horse muscle acylphosphatase. Firstly the spin systems of some easily identifiable amino acid side chains were assigned. These side chains involved all the aromatic residues and all the leucine, valine, isoleucine, threonine, alanine, proline as well as some of the glycine residues. Analysis of nuclear Overhauser enhancement spectra in our previous work had identified the sequential and long-range patterns characteristics for secondary structure elements. This result had also provided the ...
A spectrophotometric procedure for the determination of objective measurements of equine spermatozoan motility. A spectrophotometric procedure was developed and evaluated for the objective measurement of equine spermatozoan motility. A 100 mul sample of a sperm suspension, prepared by the removal of seminal plasma, was layered under a column of optically clear medium in a specially designed spectrophotometric cuvette maintained at 37 degrees C. Changes in light transmittance above the interface of the sperm suspension and medium were recorded on chart paper. As sperm cells swam into the medium, a decrease in light transmittance was recorded as a deflection on the chart paper. Chart recordings were analy...
Metabolism and pharmacokinetic studies of propionylpromazine in horses. The propionylpromazine concentrations in plasma after intramuscular administration to horses were determined using gas chromatography with nitrogen-phosphorus detection. After hydrolysis by beta-glucuronidase/arylsulphatase, the parent drug and three metabolites were detected in urine. The metabolites were identified as 2-(1-hydroxypropyl)promazine, 2-(1-propenyl)promazine and 7-hydroxypropionylpromazine by gas chromatography-mass spectrometry. No N-demethylated or sulphoxidated metabolites of propionylpromazine were observed in the horse urine.
Pharmacologic effects and detection methods of methylated analogs of fentanyl in horses. Pharmacologic effects of alpha-methylfentanyl and 3-methylfentanyl, analogs of fentanyl, were investigated in mares. The ability of an 125I-labeled fentanyl radioimmunoassay (125I-RIA) to detect these methylated fentanyl analogs in individual and pooled urine samples from horses was evaluated. Also, the ability of 7 fentanyl antibodies to react with fentanyl and fentanyl derivatives (sufentanil, alfentanil, and carfentanil) was investigated. Mares were studied in a locomotor test to determine the amount of stimulation methylated fentanyl analogs might induce. Two mares each were given alpha-me...
Urinary excretion of pentoxifylline and its metabolites by standardbred mares. The urinary excretion of a sustained-release formulation of pentoxifylline was studied in the horse after the oral administration of 4.0 grams of Trental tablets. Urine samples were collected for 24 hours after dosing and analyzed for pentoxifylline and its metabolites using high-performance liquid chromatography coupled with an ultraviolet detector. Six metabolites of pentoxifylline were identified in horse urine in addition to less than 0.2% of unchanged drug. Concomitant use of gas chromatography/mass spectrometry allowed for the elucidation of the chemical structures of the metabolites. Me...
ELISA detection of fentanyl in horse urine and plasma. The prototype of a commercial ELISA test kit designed for fentanyl determination in human urine has been evaluated for screening fentanyl in horse urine and plasma. The measurement of fentanyl after intravenous (2 mg) and intramuscular (0.25 mg) administration in undiluted plasma was not reproducible while accurate quantification of fentanyl in urine greatly depends on the composition of the horse urine. The ELISA assay, however, is simple and could be successfully used for quantitative measurements in diluted urine and for rapid qualitative screening for fentanyl in large numbers of urine sam...
Analysis of N-acetyl-4-O-acetylneuraminic-acid-containing N-linked carbohydrate chains released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Application to the structure determination of the carbohydrate chains of equine fibrinogen. The carbohydrate chains of equine fibrinogen were enzymatically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The oligosaccharides obtained were fractionated by a combination of FPLC and HPLC and analyzed by 500-MHz 1H-NMR spectroscopy. Four monosialo and four disialo diantennary N-acetyllactosamine type of carbohydrate chains occur: (formula; see text)
Purification of chicken liver ferritin by two novel methods and structural comparison with horse spleen ferritin. Ferritin was purified from chicken liver by two different methods: gel filtration on controlled-pore glass beads, and immunoaffinity chromatography employing a chicken ferritin-specific monoclonal antibody that did not cross-react with horse spleen ferritin. This antibody recognizes intact ferritin and an oligomeric 240 kDa form of the molecule after protein transfer to nitrocellulose, but not the 22 kDa chicken ferritin subunit. Chicken liver ferritin purified by these methods exhibited reduced migration on non-denaturing polyacrylamide gels compared with horse spleen ferritin. These results ...
Determination of methandrostenolone and its metabolites in equine plasma and urine by coupled-column liquid chromatography with ultraviolet detection and confirmation by tandem mass spectrometry. Monitoring steroid use requires an understanding of the metabolism in the species in question and development of sensitive methods for screening of the steroid or its metabolites in urine. Qualitative information for confirmation of methandrostenolone and identification of its metabolites was primarily obtained by coupled-column high-performance liquid chromatography-tandem mass spectrometry. The steroids and a sulphuric acid conjugate were isolated and identified by their daughter ion mass spectra in the urine of both man and the horse following administration of methandrostenolone. Spontaneo...
Immunoassay detection of drugs in racing horses. IX. Detection of detomidine in equine blood and urine by radioimmunoassay. Detomidine is a potent non-narcotic sedative agent which is currently in the process of being approved for veterinary clinical use in the United States. Since no effective screening method in horses is available for detomidine, we have developed an 125I radioimmunoassay for detomidine in equine blood and urine as part of a panel of tests for illegal drugs in performance horses. Our 125I radioimmunoassay has an I-50 for detomidine of approximately 2 ng/ml. Our assay shows limited cross-reactivity with the pharmacodynamically similar xylazine, but does not cross-react with acepromazine, epinephr...
Steroids in equine testes: the identification of endogenous 19-hydroxy and 19-nor neutral steroids by gas chromatography–mass spectrometry. After homogenization of testicular tissue from stallions aged 1, 2 and 5 years, the unconjugated and conjugated steroids were isolated by a combined solvent-solid extraction procedure. The conjugates were further separated into glucuronides and sulphates by chromatography using Sephadex LH-20. After enzyme hydrolysis and solvolysis of the respective conjugate classes, the three extracts, unconjugated steroids, aglycones and solvolysed sulphates, were purified by chromatography using Kieselgel 60H columns. Five fractions were resolved from each extract; an aliquot of each fraction was derivatiz...
Application of an affinity electrophoretic and in situ oxidation method to the study of the equine protease inhibitory proteins. An affinity method was developed to investigate the interaction between protease and protease inhibitor by incorporating a protease incubation step into a two-dimensional electrophoretic separation of the plasma protease inhibitory proteins. This involved the application of the isoelectric focusing gel to filter paper saturated in the protease of choice before being placed on the second-dimensional polyacrylamide electrophoresis gel. General protein staining or immunoblotting was used to detect the protein or ligand in the complex. An in situ oxidation method was developed using the reagent ch...
The determination of non-steroidal anti-inflammatory drugs by GC-MS-MS in equine urine. Results are given for a more sensitive screening procedure for non-steroidal anti-inflammatory drugs using GC-MS-MS. By monitoring a selected characteristic reaction for each drug very low detection limits are reached even in a difficult biological matrix such as equine urine. Detection down to 5 ng ml-1 for ibuprofen, ibufenac, alclofenac, fenoprofen, ketoprofen, naproxen and diclofenac is possible in contrast to the 0.5 microgram ml-1 limit for normal GC-MS detection. Examples are given of real positive cases for diclofenac and ibuprofen.
Lipopigments in veterinary pathology: pathogenesis and terminology. The lipopigments are a heterogenous group of pigments whose pathogenesis and terminology is confused. Whereas there is epidemiological and observational evidence that ceroid is derived from degeneration and peroxidation of unsaturated lipid, the assumption that all so-called lipopigments are similarly formed, is questioned. In particular, recent studies have distanced the pathogenesis of the pigment found in the ceroid-lipofuscinoses from that perceived for ceroid. The importance of protein rather than lipid in the pathogenesis of the pigment of ceroid-lipofuscinosis and of age pigment from th...
