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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.
A specific method for measurement of equine active myeloperoxidase in biological samples and in in vitro tests. An original method called SIEFED (specific immunological extraction followed by enzymatic detection) was developed for the specific detection of the activity of equine myeloperoxidase (MPO). The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by washing (to eliminate proteins and interfering molecules) and measurement of MPO activity using a detection system containing a fluorogenic substrate, hydrogen peroxide, and nitrite as reaction enhancer. The SIEFED technique was applied to study active MPO in horse biological fluids and the ef...
Use of infrared spectroscopy for diagnosis of traumatic arthritis in horses. To evaluate use of infrared spectroscopy for diagnosis of traumatic arthritis in horses. Methods: 48 horses with traumatic arthritis and 5 clinically and radiographically normal horses. Methods: Synovial fluid samples were collected from 77 joints in 48 horses with traumatic arthritis. Paired samples (affected and control joints) from 29 horses and independent samples from an affected (n = 12) or control (7) joint from 19 horses were collected for model calibration. A second set of 20 normal validation samples was collected from 5 clinically and radiographically normal horses. Fourier transfor...
Ferritin-catalyzed consumption of hydrogen peroxide by amine buffers causes the variable Fe2+ to O2 stoichiometry of iron deposition in horse spleen ferritin. Ferritin catalyzes the oxidation of Fe2+ by O2 to form a reconstituted Fe3+ oxy-hydroxide mineral core, but extensive studies have shown that the Fe2+ to O2 stoichiometry changes with experimental conditions. At Fe2+ to horse spleen ferritin (HoSF) ratios greater than 200, an upper limit of Fe2+ to O2 of 4 is typically measured, indicating O2 is reduced to 2H2O. In contrast, a lower limit of Fe2+ to O2 of approximately 2 is measured at low Fe2+ to HoSF ratios, implicating H2O2 as a product of Fe2+ deposition. Stoichiometric amounts of H2O2 have not been measured, and H2O2 is proposed to react ...
Rapid test by liquid chromatography/tandem mass spectrometry to evaluate equine urine reactivity towards 17beta-OH steroids. Bacteria frequently found in equine urine samples may cause degradation of 17beta-OH steroids. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed to evaluate the microbiological contamination of equine urine as a marker of poor storage conditions. Norethandrolone was used as the internal standard, and the linearity, sensitivity, precision and accuracy of the method were evaluated. 17beta-OH oxidation was demonstrated for testosterone, nandrolone, trenbolone and boldenone, but did not occur in alpha-epimers such as alpha-boldenone and epitestosterone, d...
Use of accelerating solvent extraction for detecting non-steroidal anti-inflammatory drugs in horse feces. Feces are a possible medium to be used for horse doping control. Efficient methods for detecting drugs in feces collected from various animals are routinely applied in institutes of food safety in Belgium. We have already tested whether they are applicable to horse feces. In this report, accelerated solvent extraction (ASE), an efficient method for extracting compounds from solid material, has been tested. ASE has been used to replace the diethyl ether liquid-liquid extraction step present in the method initially set up. This technique has been optimized for detecting several non-steroidal ant...
Determination of zearalenone and its metabolites in urine, plasma and faeces of horses by HPLC-APCI-MS. The paper describes a method for the sensitive and selective determination of zearalenone and its metabolites in urine, plasma and faeces of horses by high performance liquid chromatography and atmospheric pressure chemical ionisation (APCI) mass spectrometry (MS). While only one step sample clean-up by an immunoaffinity column (IAC) was sufficient for plasma samples, urine and faeces samples had to be prepared by a combination of a solid-phase extraction (SPE) and an immunoaffinity column. The method allows the simultaneous determination of zearalenone and all of its metabolites; alpha-zearal...
Adverse extrapyramidal effects in four horse given fluphenazine decanoate. 4 racehorses were examined because of markedly abnormal behavior following administration of fluphenazine decanoate. Results: Clinical signs included restlessness, agitation, profuse sweating, hypermetria, aimless circling, intense pawing and striking with the thoracic limbs, and rhythmic swinging of the head and neck alternating with episodes of severe stupor. Fluphenazine was detected in serum or plasma from all 4 horses. The dose of fluphenazine decanoate administered to 3 of the 4 horses was within the range (25 to 50 mg) routinely administered to adult humans. Results: In 2 horses, there ...
Miscibility of binary monolayers at the air-water interface and interaction of protein with immobilized monolayers by surface plasmon resonance technique. The miscibility and stability of the binary monolayers of zwitterionic dipalmitoylphosphatidylcholine (DPPC) and cationic dioctadecyldimethylammonium bromide (DOMA) at the air-water interface and the interaction of ferritin with the immobilized monolayers have been studied in detail using surface pressure-area isotherms and surface plasmon resonance technique, respectively. The surface pressure-area isotherms indicated that the binary monolayers of DPPC and DOMA at the air-water interface were miscible and more stable than the monolayers of the two individual components. The surface plasmon re...
New staining methods for sperm evaluation estimated by microscopy and flow cytometry. New staining methods and automated instruments are now available to evaluate the sperm cell in vitro. Individual compartments of the sperm cell, such as the nucleus and the plasma and acrosomal membranes, may be investigated, as well as the cell function as shown by mitochondria activity and capacitation. Various probes are used and they can be analyzed by direct light or fluorescent microscopy or by flow cytometry. The automated instruments allow objective and accurate analysis and quantification as well as the ability to evaluate large population of cells in a shorter time, thus providing ac...
Application of liquid chromatography-mass spectrometry to the investigation of poisoning by Oenanthe crocata. Liquid chromatography-mass spectrometry (LC-MS) analysis of methanol extracts of Oenanthe crocata roots revealed that oenanthotoxin co-eluted with another major polyalkyne, 2,3-dihydro-oenanthotoxin, using the existing high performance liquid chromatography (HPLC) method (isocratic elution from C18 with aqueous methanol) for investigating Oenanthe poisoning. Positive ES or APCI gave [(M+H)-H(2)O](+) and its methanol adduct as major ion species for oenanthotoxin, whereas 2,3-dihydro-oenanthotoxin formed [M+H](+) and its methanol adduct. The two polyalkynes could be chromatographically resolved ...
Detection of 17alpha-hydroxyprogesterone caproate in equine plasma by gas chromatography/tandem mass spectrometry. A method was developed for the analysis of the synthetic progestin 17alpha-hydroxyprogesterone caproate in equine plasma following its administration by intramuscular injection. The method employed a reversed-phase solid-phase extraction followed by enol-trimethylsilylation and analysis by gas chromatography/tandem mass spectrometry. The intact ester was detectable in the plasma for up to 2 weeks after a single therapeutic dose, and was found to be stable in equine whole blood for at least 2 months.
Comprehensive screening of anabolic steroids, corticosteroids, and acidic drugs in horse urine by solid-phase extraction and liquid chromatography-mass spectrometry. This paper reports two highly efficient liquid chromatography-mass spectrometry (LC-MS) methods for the screening of anabolic steroids, corticosteroids, and acidic drugs for the purpose of doping control in equine sports. Sample extraction was performed using a mixed-mode C8-SCX solid-phase extraction (SPE) cartridge. The first eluted fraction (acidic/neutral fraction) was base-washed and the resulting organic extract was used for the screening of anabolic steroids and corticosteroids by LC-MS using multiple reaction monitoring (MRM) in the positive electrospray ionisation (ESI) mode. The rema...
Determination of amiodarone and desethylamiodarone in horse plasma and urine by high-performance liquid chromatography combined with UV detection and electrospray ionization mass spectrometry. A rapid method for the quantification of amiodarone and desethylamiodarone in animal plasma using high-performance liquid chromatography combined with UV detection (HPLC-UV) is presented. The sample preparation includes a simple deproteinisation step with acetonitrile. In addition, a sensitive method for the quantification of amiodarone and desethylamiodarone in horse plasma and urine using high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is described. The sample preparation includes a solid-phase extraction (SPE) with a SCX c...
Screening for basic drugs in equine urine using direct-injection differential-gradient LC-LC coupled to hybrid tandem MS/MS. A rapid, selective and robust direct-injection LC/hybrid tandem MS method has been developed for simultaneous screening of more than 250 basic drugs in the supernatant of enzyme hydrolysed equine urine. Analytes, trapped using a short HLB extraction column, are refocused and separated on a Sunfire C(18) analytical column using a controlled differential gradient generated by proportional dilution of the first column's eluent with water. Independent data acquisition (IDA) was configured to trigger a sensitive enhanced product ion (EPI) scan when a multiple reaction monitoring (MRM) survey scan s...
Third metacarpal bone mineral density assessment in the standing horse by dual X-ray absorptiometry: suitability, precision and accuracy. Bone mineral density (BMD) is correlated to mechanical properties of bone. In the horse, dual energy X-ray absorptiometry (DXA) has yet only been performed ex-vivo, but a new portable DXA device would be ideal for in-vivo BMD measurement. We explored field suitability, precision and accuracy of this device for in-vivo third metacarpal density assessment. Precision was analysed by calculating measurement variation under repeated measurement tests with (reproducibility) and without (repeatability) limb repositioning. Repeatability and reproducibility were tested ex-vivo, at the same time that in...
Pharmacokinetics of methylprednisolone acetate after intra-articular administration and its effect on endogenous hydrocortisone and cortisone secretion in horses. To determine the pharmacokinetics of methylprednisolone (MP) and develop a pharmacokinetic-pharmacodynamic model of the related changes in plasma concentrations of endogenous hydrocortisone (HYD) and cortisone (COR) following intra-articular administration of methylprednisolone acetate (MPA) in horses. Methods: 6 Thoroughbreds. Methods: In each horse, 200 mg of MPA was injected intrasynovially into a carpal joint, and plasma MP, HYD, and COR concentrations were determined via liquid chromatography-mass spectrometry. Results: A 5-compartment pharmacokinetic-pharmacodynamic model was used to des...
Detection of altrenogest and its metabolites in post administration horse urine using liquid chromatography tandem mass spectrometry–increased sensitivity by chemical derivatisation of the glucuronic acid conjugate. Altrenogest (17alpha-allyl-17beta-hydroxyestra-4,9,11-trien-3-one) is a steroid used for the control of estrus in horses. This drug can potentially be abused in racehorses as the occurrence of estrus can alter their performance. This work describes an analytical method based on liquid chromatography-tandem mass spectrometry for the detection of altrenogest in horse urine down to a concentration of 13 pg/mL (0.042 nM). Furthermore, the qualitative aspect of metabolism of altrenogest in the horse has been studied. The main transformations that were found for this species were conjugation with gl...
Changes in heart rate and heart rate variability in Thoroughbreds during prolonged road transportation. To determine whether evaluation of heart rate (HR) and HR variability (HRV) during prolonged road transportation in horses provides a sensitive index of autonomic stimulation. Methods: Five 2-year-old Thoroughbreds. Methods: ECGs were recorded as horses were transported for 21 hours in a 9-horse van. Heart rate, high-frequency (HF) power, low-frequency (LF) power, and LF-to-HF ratio from Fourier spectral analyses of ECGs were calculated and compared with values recorded during a 24-hour period of stall rest preceding transportation. Results: HR, HF power, and LF power had diurnal rhythms durin...
Detection, quantification and confirmation of anabolic steroids in equine plasma by liquid chromatography and tandem mass spectrometry. Anabolic androgenic steroids are related to the male sex hormones and are abused in equine sports. In an effort to deter the abuse of anabolic steroids, a sensitive LC-MS/MS method was developed for detection, quantification and confirmation of eight major anabolic steroids (testosterone, normethandrolone, nandrolone, boldenone, methandrostenolone, tetrahydrogestrinone (THG), trenbolone, and stanozolol) in equine plasma. Formation of solvent adduct ions of the analytes was observed under electrospray ionization (ESI) conditions, and desolvation of the solvent adduct ions by source collision-in...
Detection of testosterone, nandrolone and precursors in horse hair. Growing interest among several horse-breeder associations has initiated the development of a screening procedure to test for anabolic agents in hair, which has the advantage over blood and urine specimens of allowing long-term detection. An analytical method was established to monitor in tails or manes several anabolic substances available as veterinary medicines or as so-called nutritional supplements (clenbuterol, different esters or prohormones of nandrolone and testosterone). The analytical procedure to detect steroids in hair samples consists of the following steps: decontamination of the...
Twenty-eight element concentrations in mane hair samples of adult riding horses determined by particle-induced X-ray emission. The concentrations of 28 elements (Al, Br, Ca, Cl, Co, Cu, Cr, Fe, Ga, Hg, K, Mg, Mn, Mo, Na, Nb, Ni, P, Pb, Rb, S, Se, Si, Sr, Ti, V, Y, and Zn) were measured in mane hair by the particle-induced X-ray emission method. Except for Br, Cl, K, S, and P, the trace element concentrations in mane hair of horses are similar to literature values for human hair. The values obtained are not dependent on the horse's age, breed, and sex and could be used as reference values in the assessment of diseases and nutritional status in equines.
Deprotonation of the horse liver alcohol dehydrogenase-NAD+ complex controls formation of the ternary complexes. Binding of NAD+ to wild-type horse liver alcohol dehydrogenase is strongly pH-dependent and is limited by a unimolecular step, which may be related to a conformational change of the enzyme-NAD+ complex. Deprotonation during binding of NAD+ and inhibitors that trap the enzyme-NAD+ complex was examined by transient kinetics with pH indicators, and formation of complexes was monitored by absorbance and protein fluorescence. Reactions with pyrazole and trifluoroethanol had biphasic proton release, whereas reaction with caprate showed proton release followed by proton uptake. Proton release (200-55...
Characterization of the stereoselective biotransformation of ketamine to norketamine via determination of their enantiomers in equine plasma by capillary electrophoresis. A robust CE method for the simultaneous determination of the enantiomers of ketamine and norketamine in equine plasma is described. It is based upon liquid-liquid extraction of ketamine and norketamine at alkaline pH from 1 mL plasma followed by analysis of the reconstituted extract by CE in the presence of a pH 2.5 Tris-phosphate buffer containing 10 mg/mL highly sulfated beta-CD as chiral selector. Enantiomer plasma levels between 0.04 and 2.5 microg/mL are shown to provide linear calibration graphs. Intraday and interday precisions evaluated from peak area ratios (n = 5) at the lowest calib...
In vivo biotransformation of metoprolol in the horse and on-column esterification of the aminocarboxylic acid metabolite by alcohols during solid phase extraction using mixed mode columns. The in vivo biotransformation of metoprolol tartrate in the thoroughbred racehorse was studied after administration of a single oral dose. Metoprolol and its basic and bifunctional phase I metabolites were isolated from urine and plasma using mixed mode solid phase extraction (SPE) cartridges. The isolates were derivatised as trimethylsilyl ethers and analysed by capillary column gas chromatography--positive ion electron ionisation and ammonia chemical ionisation mass spectrometry. Metabolism was primarily confined to the oxidative transformations of the p-(2-methoxy)ethyl substituent. Metopro...
Comparative biochemical analyses of venous blood and peritoneal fluid from horses with colic using a portable analyser and an in-house analyser. Fifty-six horses with colic were examined over a period of three months. The concentrations of glucose, lactate, sodium, potassium and chloride, and the pH of samples of blood and peritoneal fluid, were determined with a portable clinical analyser and with an in-house analyser and the results were compared. Compared with the in-house analyser, the portable analyser gave higher pH values for blood and peritoneal fluid with greater variability in the alkaline range, and lower pH values in the acidic range, lower concentrations of glucose in the range below 8.3 mmol/l, and lower concentrations of...
Separation and detection of the isomeric equine conjugated estrogens, equilin sulfate and delta8,9-dehydroestrone sulfate, by liquid chromatography–electrospray-mass spectrometry using carbon-coated zirconia and porous graphitic carbon stationary phases. Equilin-3-sulfate and delta8,9-dehydroestrone-3-sulfate are two isomers found in equine conjugated estrogens that differ in structure only by the position of a double bond in the steroid B-ring. These geometric isomers were not resolved on a C18 column during the analysis of conjugated estrogen drug products by LC-MS using acetonitrile-ammonium acetate buffer as the mobile phase. While no separations of these two isomers were observed on C18 or other alkyl-bonded silica based phases using a variety of mobile phase conditions, partial separations were achieved on phenyl bonded silica phases wit...
Analysis of anabolic steroids in the horse: development of a generic ELISA for the screening of 17alpha-alkyl anabolic steroid metabolites. Due to the potential for misuse of a wide range of anabolic steroids in horse racing, a screening test to detect multiple compounds, via a common class of metabolites, would be a valuable forensic tool. An enzyme-linked immunosorbent assay (ELISA) has been developed to detect 17alpha-alkyl anabolic steroid metabolites in equine urine. 16beta-Hydroxymestanolone (16beta,17beta-dihydroxy-17alpha-methyl-5alpha-androstan-3-one) was synthesised in six steps from commercially available epiandrosterone (3beta-hydroxy-5alpha-androstan-17-one). Polyclonal antibodies were raised in sheep, employing mesta...
The influence of anticoagulants on the measurement of total protein concentration in equine peritoneal fluid. The aim of this study was to evaluate the influence of two commonly used anticoagulants (K3EDTA and lithium heparin) on refractometric and spectrophotometric measurement of total protein (TP) concentration in equine peritoneal fluid samples. The influence of a commercial solution of K3EDTA, a solution of K3EDTA in distilled water and lithium heparin on the refractometric and spectrophotometric (biuret) quantification of TP content in peritoneal fluid samples was assessed. Total protein concentration measured by refractometry was consistently overestimated in samples with commercial K3EDTA. The...
Screening of anabolic steroids in horse urine by liquid chromatography-tandem mass spectrometry. Anabolic steroids have the capability of improving athletic performance and are banned substances in the Olympic games as well as in horseracing and equestrian competitions. The control of their abuse in racehorses is traditionally performed by detecting the presence of anabolic steroids and/or their metabolite(s) in urine samples using gas chromatography-mass spectrometry (GC-MS). However, this approach usually requires tedious sample processing and chemical derivatisation steps and could be very insensitive in detecting certain steroids. This paper describes a high performance liquid chromat...
