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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.
Determination of succinonitrile in horse urine by gas chromatography-nitrogen-phosphorus detector and gas chromatography-mass spectrometry. A chromatographic method was developed to detect and confirm the presence of succinonitrile (SDN) in horse urine samples, for antidoping control. The urine samples (5 ml) were extracted with diethyl ether and screened by gas chromatography-nitrogen-phosphorus detector and the confirmation of the drug's presence was accomplished by using gas chromatography-mass selective detection. The recovery of extraction was 78 and 81% for 1.0 and 2.0 micrograms ml-1 (relative standard deviation, < 10%), respectively. Urine samples collected after the administration of Energisan were positive for SDN (1-30 ...
Preliminary study of the metabolism of 17 alpha-methyltestosterone in horses utilizing gas chromatography-mass spectrometric techniques. Little is known about the metabolism of 17 alpha-alkyl anabolic steroids in horses. In this study, the metabolism of 17 alpha-methyltestosterone is investigated by oral administration of a (1 + 1) mixture of the steroid and its deuteriated analogue. Both compounds were synthesized from dehydroisoandrosterone (DHA), using a Grignard reaction followed by an Oppenauer oxidation. Post-administration urine extracts were analysed by gas chromatography--mass spectrometry (GC-MS) using both electron impact (IE) and chemical ionization (CI). Interpretation of the data was facilitated by observation of ...
Screening of non-steroidal anti-inflammatory drugs, barbiturates and methyl xanthines in equine urine by gas chromatography-mass spectrometry. A gas chromatographic-mass spectrometric (GC-MS) screening procedure for 23 acidic drugs in equine urine is described. With the GC-MS method fifteen anti-inflammatory drugs, five barbiturates and three methyl xanthines can be detected with good sensitivity and selectivity. The method consists of alkaline hydrolysis, extraction with organic solvent using salting-out, clean-up extraction, methylation and screening with GC-MS in selected-ion monitoring mode. The limit of detection is 10 micrograms 1(-1) or lower, for most drugs.
Determination of free malonaldehyde formed in liver microsomes upon CCl4 oxidation. Free malonaldehyde formed in the microsomes prepared from livers of monkey, rat, rabbit, mouse, cow, pig, dog, sheep and horse upon CCl4 oxidation was derivatized by reaction with N-methylhydrazine to form 1-methylpyrazole which was subsequently analyzed by capillary gas chromatography. Among the livers from animals tested, the monkey and rat livers produced the most malonaldehyde upon CCl4 treatment. Horse liver showed the greatest resistance to CCl4 oxidation. The gas chromatography method used in the present study exhibited an accurate and specific measurement of free malonaldehyde that mig...
Equilibrium unfolding studies of horse muscle acylphosphatase. The stability and equilibrium unfolding behaviour of horse muscle acylphosphatase have been studied by denaturing the protein under various conditions of temperature, pH, and urea concentration. Far-ultraviolet circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy indicate that this small monomeric protein unfolds reversibly and cooperatively. Thermodynamic parameters, the Gibbs free energy delta G and enthalpy delta H of unfolding, have been estimated for denaturation of the protein from NMR and CD data as 19 kJ mol-1 and 350 kJ mol-1, respectively. CD and 1H-NMR results s...
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...
Ultrastructure and mineral composition of urinary calculi from horses. Urinary calculi from 17 horses with urolithiasis were examined to study their mineral content and ultrastructure. Among the analytic methods used were X-ray diffractometry, scanning electron microscopy, and electron microprobe analysis. The calculi initially were observed by use of a stereoscopic dissecting microscope and generally were found to have nodular surfaces surrounding a banded or granular-to-chalky interior. Observation by scanning electron microscopy revealed an intricate pattern of irregularly concentric, fine bands and spherules. These had a round, finely banded, globular texture...
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...
FTIR analysis of the interaction of azide with horse heart myoglobin variants. The interaction of azide with variants of horse heart myoglobin (Mb) has been characterized by Fourier transform infrared (FTIR), electron paramagnetic resonance (EPR), and UV-VIS absorption spectroscopy and by molecular modeling calculations. Distal histidine variants (His64Thr, His64Ile, His64Lys) and charged surface variants (Val67Arg, Lys45Glu, Lys45Glu/Lys63Glu) were included in this study. All variants, with the exception of Val67Arg, have a lower azide affinity than the wild-type protein. Analysis of the temperature dependence of the FTIR spectra (277-313 K) revealed that the wild-type ...
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...
Elemental status of grazing animals located adjacent to the London Orbital (M25) motorway. The elemental (Br, Cd, Cr, Cu, Mn, Ni, Pb, Se, V, and Zn) content of blood and wool or hair from animals (sheep, horses and alpacas) exposed to motor vehicle emissions alongside the London Orbital (M25) motorway is reported. Elemental values were determined by inductively coupled plasma mass spectrometry (ICP-MS) quality control assessment using flameless atomic absorption spectroscopy (for Pb, correlation coefficients of whole blood r = +0.87, and hair r = +0.82), and replicate (n = 10) analysis of the international reference material IAEA A13 Animal Blood. For Pb very good agreement was obta...
Molecular entrapment of small molecules within the interior of horse spleen ferritin. A procedure for trapping small molecules inside the interior of horse spleen ferritin (HoSF) and methods for characterizing HoSF and its small entrapped molecules are described. HoSF is first dissociated into subunits by adjustment to pH 2 in the presence of the small molecules to be trapped. The pH of the dissociated HoSF is then increased to 7 at which time the dissociated subunits reassemble reforming the 24-mer HoSF, thereby trapping solvent within its interior. HoSF is then separated from unbound molecules by dialysis, ultrafiltration, and/or ammonium sulfate precipitation. Sephadex G-25 ...
Structure determination of the disialylated poly-(N-acetyllactosamine)-containing O-linked carbohydrate chains of equine chorionic gonadotropin. The disialylated poly-(N-acetyllactosamine)-containing O-linked oligosaccharide alditols, released by alkaline borohydride treatment of the enzymically N-deglycosylated beta-subunit of equine chorionic gonadotropin, were purified by fast protein liquid chromatography (FPLC) on Mono Q and analysed by fast ion bombardment mass spectrometry (FAB-MS) and 1H-NMR spectroscopy. The identified oligosaccharide alditols have the following structure: [Formula: see text]
[Examination of the ovarian activity of mares using progesterone profiles]. By means of clinical and analytical procedures (enzyme immuno assay for progesterone with microtiterplate method) the ovarian activity from 27 mares was tested over a period of several weeks. The measurement of the progesterone level to determine the time of ovulation was proved as suitable in the period of 1-2 days after ovulation. In normocyclic mares (n = 17) a different development of the progesterone profile was detected, so that an insufficient development of the corpus luteum (35%) could be considered. By means of continuous measurement of progesterone (> or = 30 days) six of ten mares ...
Application to cows and horses of Spotchem, a dry-chemistry blood analyzer for use in veterinary clinics. The usefulness of a dry-chemistry blood analyzer, Spotchem SP-4410 (SP-4410) in a veterinary clinic for analysis of bovine and equine blood chemistry was studied. We quantitated total protein (TP), albumin (Alb), total bilirubin (T-Bil), blood urea nitrogen (BUN), total cholesterol (T-Cho), glucose (Glu), calcium (Ca), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), creatinine phosphokinase (CPK), and alkaline phosphatase (ALP) in bovine sera. Each sample was assayed with both the SP-4410 and an automated blood analyzer which served as a wet-chemistry reference system, and t...
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...
Cross-validation of cyanogen bromide-peptide ratios to measure the proportion of type II collagen in pepsin digests of equine articular cartilage, meniscus, and cartilage repair tissue. Collagen type I and type II were purified from equine flexor tendon and articular cartilage, respectively. Equal amounts of these collagens were cleaved with cyanogen bromide, and 11 mixtures containing increasing proportions of type II collagen were separated in seven identical sodium dodecyl sulfate-polyacrylamide gels. The density of bands was measured in wet gels and the peak areas were used to form six ratios of peptide bands that had polynomial relationships with the known proportions of type I and type II collagen in the mixtures. Calibration curves for determining the proportion of typ...
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...
Factors affecting drug withholding time estimates in horses. Although all the factors discussed in this article may have an effect on drug withholding time estimates, the factors that have the potential for the greatest effect or that have been found to cause positive tests in the past are 1. Dosage: Increasing the drug dosage will require a longer withholding time. 2. Dosing interval: Narrowing the dosing interval will require a longer withholding time. 3. Administration route: In general, oral administration results in lower peak plasma concentrations but may result in longer excretion in the urine and therefore longer withholding time. 4. Drug intera...
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...
Comparison of immunoaffinity chromatography combined with gas chromatography-negative ion chemical ionisation mass spectrometry and radioimmunoassay for screening dexamethasone in equine urine. A comparison of the sensitive analytical methods of radioimmunoassay (RIA) and immunoaffinity chromatography (IAC) combined with gas chromatography-negative ion chemical ionisation mass spectrometry for the specific and reliable screening of dexamethasone in equine post-race urine is presented. Results from analyses of samples collected from a mare during 144 hours post administration of 26 mg of dexamethasone sodium phosphate are described.
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 ...
Detection of bicarbonate administration (milkshake) in standardbred horses. Total plasma carbon dioxide (TCO2) concentrations were measured in Standardbred horses to determine criteria to discriminate between normal horses and horses with excessive TCO2 concentrations on raceday. TCO2 concentrations from stabled horses were distributed normally with a mean of 30.2 mmol/L and a standard deviation of 1.2 (n = 192) while pre-race TCO2 concentrations were not normally distributed. The results indicate that about 50 horses per million are likely to have TCO2 concentrations greater than or equal to 35 mmol/L and that it is extremely unlikely that a normal horse would have a...
Stimulated decay of superoxide caused by ferritin-bound copper. The redox interaction between O2.- and ferritin cannot solely be regarded as as a Fe(II) release reaction. We demonstrate that native copper bound to horse spleen ferritin and apoferritin, stimulated the decay of O2.- in a catalytic reaction. Copper was determined by atomic absorption spectrophotometry. Decay of O2.- was monitored spectrophotometrically as the decrease in (A250-A360) at pH 9.5. The catalytic effect was linearly related to the copper content of the protein. Ferritin copper was less efficient than equimolar CuCl2, and iron-poor ferritin was more efficient than iron-rich ferritin...
Nucleation of the iron core occurs at the three-fold channels of horse spleen apoferritin: an EXAFS study on the native and chemically-modified protein. Extended X-ray absorbance fine structure measurements have been carried out on the initial Fe(III)-apoferritin complex at a Fe/subunit ratio of 2 in native and modified horse spleen apoferritin. Analysis of the data indicates that in the native protein the iron forms a protein-bound polynuclear cluster (Fe-Fe distance 3.4 A) with a first coordination sphere constituted by 5-6 low-Z atoms, e.g., nitrogen atoms, carboxylate-like ligands or oxo bridges between the iron atoms. Modification of Cys-126, a residue localized on the outer surface of the hydrophilic three-fold channels, with p-chloromer...
[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.
Effects of sample collection and handling on concentration of osteocalcin in equine serum. A commercially available radioimmunoassay kit for measurement of human osteocalcin was validated for use in horses. For accurate measurement of equine serum osteocalcin, blood samples may be collected at a temperature between 20 and 25 C, then centrifuged within 90 minutes; serum may be stored at -20 C in plastic tubes for up to 26 weeks. Serum may be thawed and refrozen up to 5 times without significant change in measured equine serum osteocalcin concentration. Assay sensitivity was 0.16 ng/ml. Recovery of bovine osteocalcin standard added to equine serum was linear. Intra-assay coefficient o...
