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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.
Selective and simultaneous determination of NSAIDs in equine plasma by HPLC with molecularly imprinted solid-phase extraction. Detection of nonsteroidal anti-inflammatory drugs (NSAIDs) in equine plasma is a significant analytical problem in veterinary anti-doping controls. Results: A new HPLC method coupled to selective extraction with molecularly imprinted polymers was developed for the simultaneous determination in equine plasma of the NSAIDs phenylbutazone, flunixin, oxyphenbutazone, ketoprofen and naproxen. The analytical performances of the method have been evaluated both in standard solutions and equine plasma samples. Recovery: Molecularly imprinted polymers solid-phase extraction for all NSAIDs was >94% with ...
Development and validation of a hydrophilic interaction liquid chromatography with tandem mass spectrometry method for the simultaneous detection and quantification of etilefrine and oxilofrine in equine blood plasma and urine. A sensitive hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method was developed and validated for the simultaneous detection and quantification of etilefrine and oxilofrine in equine blood plasma and urine. The method is highly sensitive and specific with good precision and accuracy. In plasma the limit of detection and limit of quantification are 0.03 and 0.1 ng/mL, respectively, for both analytes. In urine the limit of detection and limit of quantification are 0.3 and 1 ng/mL, respectively, for both analytes. The suitability of the method for doping con...
Flow cytometric detection of circulating platelet-derived microparticles in healthy adult horses. To develop a flow cytometric assay to quantify platelet-derived microparticles (PMPs) in equine whole blood and plasma. Methods: Citrate-anticoagulated whole blood from 30 healthy adult horses. Methods: Platelet-poor plasma (PPP) was prepared from fresh whole blood by sequential low-speed centrifugation (twice at 2,500 × g). Samples of fresh whole blood and PPP were removed and stored at 4° and 24°C for 24 hours. Platelet-derived microparticles were characterized in fresh and stored samples on the basis of the forward scatter threshold (log forward scatter < 10(1)) and labeling with anne...
Detection and pharmacokinetics of salbutamol in thoroughbred racehorses following inhaled administration. Salbutamol sulphate (Ventolin Evohaler) was administrated via the inhalation route to six horses at a dose of 0.5 mg every 4 h during the day for 2 days (total dose 4 mg). Urine and blood samples were taken up to 92 h postadministration. Hydrolyzed plasma and urine were extracted using solid phase extraction (SPE). A sensitive tandem mass spectrometric method was developed in this study, achieving a lower limit of quantification (LLOQ) for salbutamol of 10 pg/mL in plasma and urine. The parent drug was identified using UPLC-MS/MS. Most of the determined salbutamol plasma concentrations, post l...
The pharmacokinetics of dexmedetomidine administered as a constant rate infusion in horses. Dexmedetomidine, the most selective α2-adrenoceptor agonist in clinical use, is increasingly being used in both conscious and anaesthetized horses; however, the pharmacokinetics and sedative effects of this drug administered alone as an infusion are not previously described in horses. Seven horses received an infusion of 8 μg dexmedetomidine/kg/h for 150 min, venous blood samples were collected, and dexmedetomidine concentrations were assayed using liquid chromatography-mass spectrometry (LC/MS) and analyzed using noncompartmental pharmacokinetic analysis. Sedation was scored as the distance...
Pharmacokinetic and pharmacodynamic analysis comparing diverse effects of detomidine, medetomidine, and dexmedetomidine in the horse: a population analysis. The present study characterizes the pharmacokinetic (PK) and pharmacodynamic (PD) relationships of the α2-adrenergic receptor agonists detomidine (DET), medetomidine (MED) and dexmedetomidine (DEX) in parallel groups of horses from in vivo data after single bolus doses. Head height (HH), heart rate (HR), and blood glucose concentrations were measured over 6 h. Compartmental PK and minimal physiologically based PK (mPBPK) models were applied and incorporated into basic and extended indirect response models (IRM). Population PK/PD analysis was conducted using the Monolix software implementing t...
The role of the Oregon State University Endophyte Service Laboratory in diagnosing clinical cases of endophyte toxicoses. The Oregon State University Colleges of Veterinary Medicine and Agricultural Sciences instituted the Endophyte Service Laboratory to aid in diagnosing toxicity problems associated with cool-season grasses in livestock. The endophyte (Neotyphodium coenophalum) present in tall fescue (Festuca arundinacea) produces ergopeptine alkaloids, of which ergovaline is the molecule used to determine exposure and toxicity thresholds for the vasoconstrictive conditions "fescue foot" and "summer slump". Another vasoconstrictive syndrome, "ergotism," is caused by a parasitic fungus, Claviceps purpurea, and it...
A fast method for the determination of the PC/LPC ratio in intact serum by MALDI-TOF MS: an easy-to-follow lipid biomarker of inflammation. The PC/LPC ratio of blood serum is increasingly considered to represent an important clinical parameter that reflects various kinds of diseases. Here, a simple and fast method of lipid analyses of "intact" blood serum (i.e. without extraction) by MALDI-TOF mass spectrometry is described. The novel procedure allows the accurate determination of the PC/LPC ratio, utilizing only a tiny amount of blood. The serum is diluted with distilled water and directly applied onto the MALDI target and, after drying, covered by a thin layer of the matrix solution (either 9-aminoacridine or 2,5-dihydroxybenzoi...
Concentrations of strontium, barium, cadmium, copper, zinc, manganese, chromium, antimony, selenium and lead in the equine liver and kidneys. The concentrations of specific elements in the equine liver and kidneys are of practical relevance since horses are not only food-producing animals, but also partially serve as an indicator for the environmental pollution, as the basic feed includes plants like grass, grain and fruits. In this study, the concentrations of strontium (Sr), barium (Ba), cadmium (Cd), copper (Cu), zinc (Zn), manganese (Mn), chromium (Cr), antimony (Sb), selenium (Se) and lead (Pb) were measured in the liver, renal cortex and renal medulla of 21 horses (8 male; 13 female; aged between 5 months-28 years), using indu...
Comparison of two methods for measurement of equine insulin. Diagnosis of equine hyperinsulinemia requires an accurate method for quantification of equine insulin concentrations. The objectives of the current study were to compare 2 commercially available techniques for measurement of equine insulin, the radioimmunoassay (RIA) and chemiluminescent immunoassay (CIA). Recovery was poor for both assays, but worse for the CIA. Serial dilution of a high endogenous insulin sample yielded better linearity for the RIA ( r = 0.99, P < 0.001) than the CIA ( r = 0.92, P = 0.009). Bland-Altman analysis indicated that the CIA was, on average, 91 pmol/l higher tha...
Pharmacokinetics and pharmacodynamics of enalapril and its active metabolite, enalaprilat, at four different doses in healthy horses. Pharmacokinetic and pharmacodynamic of IV enalapril at 0.50 mg/kg, PO placebo and PO enalapril at three different doses (0.50, 1.00 and 2.00 mg/kg) were analyzed in 7 healthy horses. Serum concentrations of enalapril and enalaprilat were determined for pharmacokinetic analysis. Angiotensin-converting enzyme (ACE) activity, serum ureic nitrogen (SUN), creatinine and electrolytes were measured, and blood pressure was monitored for pharmacodynamic analysis. The elimination half-lives of enalapril and enalaprilat were 0.67 and 2.76 h respectively after IV enalapril. Enalapril concentrations ...
Bovine and equine peritubular and intertubular dentin. Dentin contains 1-2μm diameter tubules extending from the pulp cavity to near the junction with enamel. Peritubular dentin (PTD) borders the tubule lumens and is surrounded by intertubular dentin (ITD). Differences in PTD and ITD composition and microstructure remain poorly understood. Here, a (∼200nm)(2), 10.1keV synchrotron X-ray beam maps X-ray fluorescence and X-ray diffraction simultaneously around tubules in 15-30μm thick bovine and equine specimens. Increased Ca fluorescence surrounding tubule lumens confirms that PTD is present, and the relative intensities in PTD and ITD correspon...
Control of the misuse of testosterone in castrated horses based on an international threshold in plasma. Testosterone is an endogenous steroid produced primarily in the testes. Trace levels of testosterone are found in urine samples from geldings, as testosterone is also secreted by the adrenal. An international threshold of free and conjugated testosterone in urine (20 ng/mL) was adopted by the International Federation of Horseracing Authorities (IFHA) in 1996 for controlling testosterone misuse in geldings. In view of the recent popularity of using blood in doping control testing, it is necessary to establish a threshold for testosterone in gelding plasma. A liquid chromatography-mass spectro...
An easy stereoselective synthesis of 5(10)-estrene-3β,17α-diol, a biological marker of pregnancy in the mare. 5(10)-Estrene-3β,17α-diol is an essential reference material for doping analysis in horse-racing laboratories. It is used to detect misuse, for doping purpose, of the pregnancy status in the mare. Its stereoselective synthesis from 17β-estradiol-3-methyl ether (prepared from estrone or 17β-estradiol) was performed in four steps: (1) Mitsunobu inversion of the 17β-alcohol; (2) Birch reduction of the aromatic ring; (3) stereoselective reduction of the 3-ketone via Noyori asymmetric transfer hydrogenation; (4) chemoenzymatic purification.
Blood concentration of copper, cadmium, zinc and lead in horses and its relation to hematological and biochemical parameters. Environmental pollution results in serious health hazards to animals and blood analysis serves as a good alternative for health status assessment. The target of this study was to analyze the concentration of selected metals in equine blood, to analyze the blood parameters and to find possible correlations. Blood samples were collected from the vena jugularis of healthy adult horses. The highest concentration of all elements was found in whole blood (Cu 3.84 ± 0.90 mg L(-1); Cd = 0.81 ± 0.90 mg L(-1); Zn 26.67 ± 14.12 mg L(-1); Pb 9.33 ± 5.76 mg L(-1)). Higher concentrations of copper, ...
A rapid screen for four corticosteroids in equine synovial fluid. Most antidoping method development in the equine industry has been for plasma and urine, though there has been recent interest in the analysis of synovial fluid for evidence of doping by intra-articular corticosteroid injection. Published methods for corticosteroid analysis in synovial fluid are primarily singleplex methods, do not screen for all corticosteroids of interest and are not adequately sensitive. The purpose of this study is to develop a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) screening method for the detection of four of the most common intra-a...
Evaluation of horse urine sample preparation methods for metabolomics using LC coupled to HRMS. Horse urine is the medium of choice for the implementation of metabolomic approaches aimed at improving horse doping control. However, drug analysis in this biofluid is a challenging task due to the presence of large amounts of interfering compounds. METHODOLOGY & RESULTS: A comparative study of sample preparation has been conducted to evaluate five sample-preparation methods, namely acetonitrile precipitation, proteinase K hydrolysis, membrane filtration and sample dilution with water by factors of five and 20, for metabolome analysis using liquid chromatography coupled to high resolution...
HPLC/ESI-MS(n) method for non-amino bisphosphonates: application to the detection of tiludronate in equine plasma. Tiludronate is a non-nitrogen-containing biphosphonate drug approved in equine veterinary medicine for the treatment of navicular disease and bone sparvin in horse. Its hydrophilic properties and its strong affinity for the bone have made the control of its use quite difficult. After an initial step of method development in plasma and urine, due to a strong matrix effect and erratic detection in urine, the final method development was conducted in plasma. After addition of (3-trifluoromethylphenyl) thiomethylene biphosphonic acid as internal standard, automated sample preparation consisted of ...
The analysis of pergolide residues in horse plasma by LC with fluorescence detection. Pergolide is used to treat pituitary pars intermedia dysfunction (equine Cushing's Disease), a neurodegenerative condition associated with loss of dopaminergic inhibition of the pituitary in horses. After oral administration, only low concentrations of the drug are achieved in plasma, making drug detection and quantification difficult. While direct analysis of plasma using sensitive MS/MS techniques is possible, dirty plasma samples and mobile phase buffers can cause instrumentation to become rapidly incapacitated. A method using LC with fluorescence detection was developed for pergolide analy...
A comparative study of free oligosaccharides in the milk of domestic animals. The present study was conducted to obtain a comprehensive overview of oligosaccharides present in the milk of a variety of important domestic animals including cows, goats, sheep, pigs, horses and dromedary camels. Using an analytical workflow that included ultra-performance liquid chromatography-hydrophilic interaction liquid chromatography with fluorescence detection coupled to quadrupole time-of-flight MS, detailed oligosaccharide libraries were established. The partial or full characterisation of the neutral/fucosylated, phosphorylated and sialylated structures was facilitated by sequencin...
Analytical performance of a dry chemistry analyzer designed for in-clinic use. The Heska Dri-Chem 4000 uses dry slide technology to evaluate serum or plasma. No previous independent performance evaluation is published to the authors' knowledge. Objective: The objectives were to (1) characterize analytical performance of a Dri-Chem 4000 by measuring precision and bias, (2) compare analytical performance of that Dri-Chem 4000 unit with a predetermined quality requirement, and (3) determine whether statistical QC of the Dri-Chem 4000 is possible using the 13s control rule. Methods: Sixteen analytes were measured using plasma from dogs, cats, and horses. Coefficient of varia...
Factors influencing total carbon dioxide concentrations in plasma of thoroughbred and standardbred racehorses. Horse racing authorities impose a limit on the concentration of plasma 'total carbon dioxide' (TCO2), typically 36 mM with action taken above 37 mM, as measured by an electrochemical gas analyzer. It is of interest to understand the distribution of TCO2 in a 'normal' population of racehorses and determine probabilities of members of this population exceeding these current regulatory and action limits. TCO2 levels in equine plasma samples have been modelled for 12 months (2011-2012) of thoroughbred (3076 measurements) and standardbred (3788 measurements) data in Australia. The two populations h...
Development and validation of a gas chromatography-flame ionization detection method for quantifying sucrose in equine serum. A simple and accurate method for quantifying sucrose in equine serum that can be applied to sucrose permeability testing in the horse was developed and validated using gas chromatography with flame ionization detection. The assay provided an acceptable degree of linearity, accuracy, and precision at concentrations of sucrose as low as 2.34 μmol/l and as high as 20.45 μmol/l. Percentage recovery of sucrose from serum ranged from 89% to 102%; repeatability and intermediate precision (relative standard deviation) ranged from 3.6% to 6.7% and 4.1% to 9.3%, respectively. The limit of detection wa...
Determination of pergolide in horse plasma by UPLC-MS/MS for pharmacokinetic applications. Pergolide, an ergot-derived dopamine D2 receptor agonist, is used extensively as an orally administered treatment for pituitary pars intermedia dysfunction (PPID) in horses. One of the barriers associated with pergolide determinations in plasma for pharmacokinetic applications has been the technically demanding requirement for sensitivity. The objective of our work was to develop a simple assay for the determination of pergolide in plasma and demonstrate its potential application in the study of pergolide pharmacokinetics (PK) in horses. A UPLC-MS/MS assay was developed with a simple sample pr...
Doping control analyses in horseracing: a clinician’s guide. Doping(1) in sports is highly detrimental, not only to the athletes involved but to the sport itself as well as to the confidence of the spectators and other participants. To protect the integrity of any sport, there must be in place an effective doping control program. In human sports, a 'top-down' and generally unified approach is taken where the rules and regulations against doping for the majority of elite sport events held in any country are governed by the World Anti-Doping Agency (WADA). However, in horseracing, there is no single organisation regulating this form of equestrian sport; i...
Systematic evaluation of evidence on veterinary viscoelastic testing part 1: System comparability. To systematically examine the evidence on system comparability between the thromboelastography and the rotational thromboelastometry viscoelastic point-of-care instruments and to identify knowledge gaps. Methods: Standardized, systematic evaluation of the literature, categorization of relevant articles according to level of evidence and quality, and development of consensus on conclusions for application of the concepts to clinical practice. Methods: Academic and referral veterinary medical centers. Results: Medline via PubMed, CAB abstracts, and Google Scholar were searched. A total of 8 rele...
Troponin assays in the assessment of the equine myocardium. In 2000, troponin assays were adopted as the test of choice for detection of myocardial injury in man. This decision was made after extensive testing and followed a 60 year search for a biomarker of myocardial damage with sufficient analytical sensitivity and specificity. This has led to proliferation of assays for use in human medicine, each requiring extensive testing and validation before it could be made available on the open market for human use. The search for ever-more analytically sensitive assays and for a standard reference material continues. The adoption of troponin testing in vete...
Detection and pharmacokinetics of three formulations of firocoxib following multiple administrations to horses. The use of firocoxib in horses and its ability to affect performance and potential to allow a horse to compete when it otherwise should not, necessitates establishing appropriate withdrawal time guidelines prior to performance. Objective: To describe plasma concentrations and characterise the pharmacokinetics of 3 firocoxib formulations following multiple administrations of the label dose, with respect to recommended plasma thresholds for performance horses. Methods: Balanced 3-way crossover prospective study. Methods: Nine healthy mature horses were administered firocoxib injectable solution ...
Sensitive hydrophilic interaction liquid chromatography/tandem mass spectrometry method for rapid detection, quantification and confirmation of cathinone-derived designer drugs for doping control in equine plasma. Cathinone derivatives are new amphetamine-like stimulants that can evade detection when presently available methods are used for doping control. To prevent misuse of these banned substances in racehorses, development of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method became the impetus for undertaking this study. Methods: Analytes were recovered via liquid-liquid extraction using methyl tert-butyl ether. Analyte separation was achieved on a hydrophilic interaction column using liquid chromatography and mass analysis was performed on a QTRAP mass spectrometer in positive elec...
