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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.
Optimization and validation method to evaluate the residues of β-lactams and tetracyclines in kidney tissue by UPLC-MS/MS. Methods are validated by a process that defines the analytical requirements and confirms that the investigated method is capable of performing consistently. A quantitative and confirmatory method for determining the presence of β-lactam and tetracycline multiresidues in avian, bovine, equine, and swine kidney tissues using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed, optimized, and validated. Analytes were extracted from the kidneys by a mixture of water and acetonitrile, and the extract was then purified with hexane and C18 (dispersive phase). ...
Analytical validation of a high-sensitivity cardiac troponin T assay in horses. Although cardiac troponin T (cTnT) assays have been used to detect myocardial damage in horses, a cTnT assay has not been analytically validated, to our knowledge. The aims of this study were to estimate the precision of a high-sensitivity cTnT assay in horses and determine the effect of hemolysis on the measured cTnT concentration. Serum samples from horses were mixed in 3 different pools. Pool 1 consisted of samples from 3 healthy horses, pool 2 from 6 horses with heart failure or atypical myopathy, and pool 3 from 10 horses with atypical myopathy. The within- and between-run coefficients of...
Identification of the hair of a Holocene “Yukagir horse” (Equus spp.) mummy. No abstract available
Application of the ASVCP guidelines for the establishment of haematologic and biochemical reference intervals in Icelandic horses in Austria. Despite the increasing popularity of Icelandic horses, published reference intervals (RIs) in this breed are rare. Due to their isolation and their small gene pool, alterations in some variables are likely and some possible breed-specific peculiarities have been described. The purpose of the present study was the establishment of comprehensive RIs in Icelandic horses according to recently published guidelines. In a prospective observational study, blood samples were collected from the jugular vein of 142 Icelandic horses into EDTA and serum tubes. Reference intervals were established for haema...
Quantification of hypoglycin A in serum using aTRAQ(®) assay. Hypoglycin A has been recently identified has the causal agent of atypical myopathy (AM) in horses. Its identification and quantification in equine's biological fluids is thus a major concern to confirm maple poisoning and to provide insight into the poorly understood mechanism of hypoglycin A intoxication. Methods: Quantification of hypoglycin A has been achieved with the aTRAQ kit for amino acid analysis of physiological fluids (AB Sciex). Acquisition method on mass spectrometer has been updated to record the hypoglycin A specific MRM transition. Results: Outlined accuracy profiles demonstra...
Metabolic study of androsta-1,4,6-triene-3,17-dione in horses using liquid chromatography/high resolution mass spectrometry. Androsta-1,4,6-triene-3,17-dione (ATD) is an irreversible steroidal aromatase inhibitor and is marketed as a supplement. It has been reported to effectively reduce estrogen biosynthesis and significantly increase the levels of endogenous steroids such as dihydrotestosterone and testosterone in human. ATD abuses have been reported in human sports. Its metabolism in human has been studied, and the in vitro metabolic study of ATD in horses has been reported, however, little is known about its biotransformation and elimination in horses. This paper describes the in vitro and in vivo metabolism stu...
Comparison of milk oligosaccharides pattern in colostrum of different horse breeds. Colostrum oligosaccharides are known to exhibit prebiotic and immunomodulatory properties. Oligosaccharide composition is species-specific, and equine colostrum has been reported to contain unique oligosaccharides. Therefore, equine oligosaccharides (EMOS) from colostrum from different horse breeds were analyzed by CE-LIF, CE-MS(n), HILIC-MS(n), and exoglycosidase degradation. Sixteen EMOS were characterized and quantified, of which half were neutral and half were acidic. EMOS showed about 63% structural overlap with human milk oligosaccharides, known for their bioactivity. Seven EMOS were not...
Opiorphin analysis in equine plasma and urine using hydrophilic interaction LC-MS. Due to opiorphin's analgesic and antidepressant functions, its illicit use is rumored in some racing jurisdictions. Opiorphin is very difficult to detect due to its hydrophilic nature and rapid degradation in plasma and urine samples. METHODOLOGY & RESULTS: We have developed a sensitive, reliable method for opiorphin detection and confirmation in equine samples, using EDTA to inhibit analyte degradation between the time of collection and analysis. Opiorphin was extracted by weak cation exchange followed by analysis using HILIC-MS/MS. The method was validated and the LOD was determined to b...
Chip-based nanoelectrospray ionization with Fourier transform mass spectrometric detection to screen for local anesthetics intended to mask limb sore in walking horses. We report a high-throughput chip-based nanoelectrospray ionization method coupled with Fourier transform mass spectrometry to screen for local anesthetics in samples collected by swabbing. These drugs have been used to mask pain on the limbs of walking horses after forbidden practices of soring or physical abuse. Optimized for lidocaine, the method afforded sub-ppm mass accuracy for nine local anesthetics included in the study. From doped cotton swabs, two third and all of the analytes were detected after adding 10 ng and 100 ng of each drug, respectively. Benzocaine and/or lidocaine were ...
Determination of the unsaturated disaccharides of hyaluronic acid in equine synovial fluid by high-performance liquid chromatography and fluorescence detection. The purpose of this study was to develop and validate an analytical method to determine the presence of hyaluronic acid derived disaccharides in equine synovial fluid. Results: A high-performance liquid chromatography method for the determination of hyaluronic acid derived unsaturated disaccharides in equine synovial fluid was developed and validated. The method is based on the measurement of unsaturated disaccharides released by digestion of linear hyaluronic acid molecules. The method showed linearity (r(2) = 0.996) over the full working concentration range 0.89-30 mg/l. Relative standard ...
Hematologic and biochemical reference intervals for adult Friesian horses from North America. Established breed-specific reference intervals (RI) are an important tool for monitoring the health of horses. There is a lack of published work on breed-specific RI for Friesian horses. Objective: The goal of this project was to determine hematologic and biochemical RI for Friesian horses residing in North America. Methods: Strict inclusion and exclusion criteria were established for selection of reference subjects and for blood specimen collection and handling. Blood samples from 123 healthy, adult (range 3-18 years, median 8 years) Friesian horses of both sexes (70 mares, 45 geldings, and...
Pre-analytical stability of adrenocorticotrophic hormone from healthy horses in whole blood, plasma and frozen plasma samples. The stability of equine adrenocorticotrophic hormone (ACTH) in blood samples is not fully known. The study objectives were to determine ACTH stability (1) in whole blood and plasma over 72 h at either 4 or 21 °C, and (2) in plasma frozen at either -20 or -80 °C over 30 days. Nine horses were sampled and ACTH concentration were measured after storage as whole blood or plasma, at 4, 21, -20 and -80 °C for up to 30 days. The ACTH concentration was significantly reduced at 24 h but remained stable when plasma was frozen at -20 and -80 °C for 30 days. Beyond 24 h, samples stored a...
A Novel Spectral Method for Inferring General Diploid Selection from Time Series Genetic Data. The increased availability of time series genetic variation data from experimental evolution studies and ancient DNA samples has created new opportunities to identify genomic regions under selective pressure and to estimate their associated fitness parameters. However, it is a challenging problem to compute the likelihood of non-neutral models for the population allele frequency dynamics, given the observed temporal DNA data. Here, we develop a novel spectral algorithm to analytically and efficiently integrate over all possible frequency trajectories between consecutive time points. This advan...
A validated UHPLC-MS/MS method to quantify low levels of anabolic-androgenic steroids naturally present in urine of untreated horses. Doping control is a main priority for regulatory bodies of both the horse racing industry and the equestrian sports. Urine and blood samples are screened for the presence of hundreds of forbidden substances including anabolic-androgenic steroids (AASs). Based on the suspected endogenous origin of some AASs, with β-boldenone as the most illicit candidate, this study aimed to improve the knowledge of the naturally present AAS in horse urine. To this extent, a novel ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated according to...
Plasma cholinesterase activity of rats, western grey kangaroos, alpacas, sheep, cattle, and horses. Plasma cholinesterase activity levels of various species may be of interest to toxicologists or pathologists working with chemicals that interfere with the activity of plasma cholinesterase. Methods: We used a pH titration method to measure the plasma cholinesterase activity of six mammalian species. Results: Plasma cholinesterase activity varied up to 50-fold between species: sheep (88 ± 45 nM acetylcholine degraded per ml of test plasma per minute), cattle (94 ± 35), western grey kangaroos (126 ± 92), alpaca (364 ± 70), rats (390 ± 118) and horses (4539 ± 721). Conclusions: We present ...
Confirmation and quantification of clenbuterol in horse urine using liquid chromatography tandem mass spectrometry triple quadrupole. Clenbuterol (CLE) is used in horses as a bronchodilator and for its anabolic steroid-like effects. CLE is a Class 3 drug according to current Association of Racing Commissioners International (ARCI) Uniform Classification Guidelines. The Racing Medication and Testing Consortium recommended a urine CLE threshold of 140 pg/mL after careful scientific review of the results of studies describing the disposition of CLE in the horse and this threshold was adopted by the ARCI. Enzyme-linked immunosorbent assay was previously used to screen samples for CLE in Illinois, but could not detect such low co...
Pharmacokinetics and pharmacodynamics comparison between subcutaneous and intravenous butorphanol administration in horses. The study objective was to compare butorphanol pharmacokinetics and physiologic effects following intravenous and subcutaneous administration in horses. Ten adult horses received 0.1 mg/kg butorphanol by either intravenous or subcutaneous injections, in a randomized crossover design. Plasma concentrations of butorphanol were measured at predetermined time points using highly sensitive liquid chromatography-tandem mass spectrometry assay (LC-MS/MS). Demeanor and physiologic variables were recorded. Data were analyzed with multivariate mixed-effect model on ranks (P ≤ 0.05). For subcutaneous i...
Influence of stage of lactation and year season on composition of mares’ colostrum and milk and method and time of storage on vitamin C content in mares’ milk. Mares' milk is becoming increasingly popular in Western Europe. This study was thus aimed at investigating the impact of stage of lactation and season on chemical composition, somatic cell count and some physicochemical parameters of mares' colostrum and milk, and at developing a method for the determination of vitamin C (ascorbic acid) in mares' milk and to determine its content in fresh and stored milk. Results: The analysis conducted showed an effect of the stage of lactation on contents of selected chemical components and physicochemical parameters of mares' milk. In successive lactation p...
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...
