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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.
Use of Liquid Chromatography–Tandem Mass Spectrometry to Quantify and Confirm the Fentanyl Metabolite N-[1-(2-Phenethy-4-Piperidinyl)] Maloanilinic Acid in Equine Urine for Doping Control. Fentanyl, a powerful synthetic mu opioid receptor agonist, is banned in equine sports by the Association of Racing Commissioners International and the Fédération Équestre Internationale. The presence of fentanyl in equine blood has been confirmed during routine post-race screening for doping substances in the authors' laboratory. While fentanyl can be detected and confirmed in blood, it is rapidly metabolized, and screening for the metabolite N-[1-(2-phenethy-4-piperidinyl)] maloanilinic acid (PMA) in equine urine is expected to allow for a longer detection time. In this study, a quantitati...
Metabolic study of selective androgen receptor modulator LY2452473 in thoroughbred horses for doping control. Since 2010, there has been an increasing number of adverse analytical findings related to selective androgen receptor modulators (SARMs) in competitive sports. It emphasizes the importance of comprehensive doping control analytical procedures that are capable of detecting SARM misuse. Methods: In this study, it is described how LY2452473, a SARM, was metabolized in thoroughbred horses after a single-dose oral administration and in vitro with equine liver microsome preparations. An investigation of the metabolism of LY2452473 in horses' urine, plasma, and hair matrices was carried out during th...
Sample stability and heparin interference in ionized calcium and ionized magnesium measurements in horses using the Stat Profile Prime Plus co-oximetry electrolyte analyzer. The determination of iCa and iMg is important in veterinary medicine, but their immediate determination in whole blood is not always possible. Their stability in other sample types and the existence of interferences must be evaluated before its use. Objective: We aimed to analyze the effects of storage time on the stability of iCa, iMg, and other analytes in whole blood, plasma, and serum samples in horses and assess the interference of heparin in these measurements. Methods: Whole blood, heparin-plasma, and serum samples from 10 horses were stored at 4°C and analyzed 1, 2, 3, 4, 5, 6, 7, 8, ...
Analytical Performance Evaluation of the New GEM® Premier™ 5000 in Comparison to the Epoc® Blood Gas Analyzer in Horses. Different blood gas analyzers are used in equine practice. Every machine needs to be validated, as they have not been designed for use in horses. The aim of this study was to compare the newly marketed GEM5000 machine to the formerly validated epoc machine for blood gas analysis in horses. In this prospective, non-blinded, comparative laboratory analyzer study, 43 equine blood samples were analyzed on both analyzers and values were compared between the two machines via Lin's concordance analysis, Passing-Bablok regression analysis and Bland-Altman plots. Duplicate measurements were conducted o...
Estimating Temporally Variable Selection Intensity from Ancient DNA Data. Novel technologies for recovering DNA information from archaeological and historical specimens have made available an ever-increasing amount of temporally spaced genetic samples from natural populations. These genetic time series permit the direct assessment of patterns of temporal changes in allele frequencies and hold the promise of improving power for the inference of selection. Increased time resolution can further facilitate testing hypotheses regarding the drivers of past selection events such as the incidence of plant and animal domestication. However, studying past selection processes ...
Synopsis of the pharmacokinetics, pharmacodynamics, applications, and safety of firocoxib in horses. According to in vitro and in vivo investigations, firocoxib (FX), a second-generation coxib, is a highly selective COX-2 inhibitor in horses. With a COX-1/COX-2 IC50 ratio of 643 in horses, FX spares the COX-1 inhibitory effects. It is approved for the treatment of musculoskeletal problems and lameness in horses and dogs with osteoarthritis (OA). For the treatment of OA in horses, both an injectable formulation for IV administration at a dose of 0.09 mg/kg for five days and an oral paste formulation at a dose of 0.1 mg/kg for 14 days are licensed. Numerous analytical methods were reported in...
Towards Non-Targeted Screening of Lipid Biomarkers for Improved Equine Anti-Doping. The current approach to equine anti-doping is focused on the targeted detection of prohibited substances. However, as new substances are rapidly being developed, the need for complimentary methods for monitoring is crucial to ensure the integrity of the racing industry is upheld. Lipidomics is a growing field involved in the characterisation of lipids, their function and metabolism in a biological system. Different lipids have various biological effects throughout the equine system including platelet aggregation and inflammation. A certain class of lipids that are being reviewed are the eicosa...
Detection of the growth hormone secretagogue MK-0677 in equine hair following oral administration. MK-0677 (ibutamoren) is an orally active non-peptide growth hormone secretagogue that binds to the ghrelin receptor stimulating the secretion of endogenous growth hormone. It is one of the most prevalent performance-enhancing compounds currently available online and is potentially subject to abuse both in human and equine sports. The aim of the current study was to investigate whether it could be detected in equine hair following oral administration of MK-0677 mesylate to a Thoroughbred racehorse. MK-0677 and its O-dealkylated metabolite were extracted using an existing method for prohibited s...
Cocktail drug usage and etofenamate detection in post-race equine urine sample: A case report. A recent trend in the use of high-resolution accurate mass screening (HRAMS) for doping control testing in both human and animal sports has emerged owing to significant improvement in high-resolution mass spectrometry in terms of sensitivity, mass accuracy, mass resolution and mass stability. Several HRAMS methods have been reported for the detection of multidrug residues in human or equine urine. These improved analytical technologies have led to changes in the use of prohibited substances, and the administration of more than one substance at low concentrations as a "cocktail" has become one ...
Enantioselective CE-MS analysis of ketamine metabolites in urine. The chiral drug ketamine has long-lasting antidepressant effects with a fast onset and is also suitable to treat patients with therapy-resistant depression. The metabolite hydroxynorketamine (HNK) plays an important role in the antidepressant mechanism of action. Hydroxylation at the cyclohexanone ring occurs at positions 4, 5, and 6 and produces a total of 12 stereoisomers. Among those, the four 6HNK stereoisomers have the strongest antidepressant effects. Capillary electrophoresis with highly sulfated γ-cyclodextrin (CD) as a chiral selector in combination with mass spectrometry (MS) was us...
Validation and method comparison for a point-of-care lateral flow assay measuring equine whole blood insulin concentrations. The Wellness Ready Test (WRT) is a lateral flow, stall-side assay that measures equine insulin in whole blood and requires validation before recommending clinical use. We evaluated intra- and inter-assay precision and linearity and compared the WRT with a radioimmunoassay (RIA). Tested concentrations ranged from 695 pmol/L (100 μIU/mL). For 20 replicates at each insulin level, intra-assay CVs of the WRT for insulin were 13.3%, 12.9%, and 15.3% at low (139-278 pmol/L; 20-40 μIU/mL), intermediate (278-417 pmol/L; 40-60 μIU/mL), and high (>417 >60 μIU/mL) concentrations...
Analytical validation of five diagnostic tests for the detection of polymorphonuclear cells in stallion semen. The objectives of this study were to evaluate the ability of five diagnostic tests to detect polymorphonuclear cells (PMNs) in stallion semen, and to determine the concentration of PMNs that affects sperm motility. We hypothesized that all tests have diagnostic value, and even low concentrations of PMNs affect motility. One ejaculate was obtained from six stallions. Aliquots of 50 × 10 purified sperm were incubated, in triplicate, with six concentrations of purified PMNs: 1) no PMNs, 2) 0.25 × 10 PMN/ml, 3) 0.5 × 10 PMN/ml, 4) 2.5 × 10 PMN/ml, 5) 5 × 10 PMN/ml, 6) 10 × 10 PMN...
Evaluation of a Comprehensive Profile of Salivary Analytes for the Diagnosis of the Equine Gastric Ulcer Syndrome. In this report, the measurement of salivary biomarkers as an aid for diagnosis of equine gastric ulcer syndrome (EGUS) was studied. A comprehensive panel of 23 salivary analytes was measured in the saliva of horses affected by EGUS and compared to healthy animals and horses with other diseases clinically similar to EGUS but with a negative diagnosis at gastroscopic examination. A total of 147 horses were included in the study and divided into heathy population (n = 12), the EGUS group (n = 110), and the group of horses with other diseases (n = 25). From the 23 analytes studied, 17 showed incre...
Identification of mare milk adulteration with cow milk by liquid chromatography-high resolution mass spectrometry based on proteomics and metabolomics approaches. Mare milk(MM)production is lower than that of cow milk (CM), making it a premium product. Unfortunately, sellers adulterate MM with cheap CM to unscrupulously gain more profits. This study aimed to analyze MM adulteration with CM using proteomics and metabolomics. Using univariate and multivariate statistical analysis, the relative abundances of certain proteins of caseins and secretoglobin family 1D member were found to be higher with a higher CM admixture percentage. In addition, several metabolites, such as orotic and 4-aminonicotinic acids, increased in the mare-cow milk mixture with an in...
The concentration of lidocaine and mepivacaine measured in synovial fluid of different joints of horses after single intra-articular injection. To determine the synovial fluid (SF) concentrations of lidocaine and mepivacaine after intra-articular injection with clinically relevant doses to the distal interphalangeal (DIP), metacarpophalangeal (MCP), middle carpal (MC), and tarsocrural (TC) joint at two different time points after injection in order to be able to compare concentrations with previously established concentrations associated with cytotoxicity and antimicrobial activity. Unassigned: In the first of two experiments, 20 joints (5 MC, 5 MCP, 10 DIP joints) of five horses under general anesthesia were injected with clinically ...
Administration and detection of gene therapy in horses: A systematic review. Gene therapy uses genetic modification of cells to produce a therapeutic effect. Defective or missing genes can be repaired or replaced, or gene expression can be modified using a variety of technologies. Repair of defective genes can be achieved using specialized gene editing tools. Gene addition promotes gene expression by introducing synthetic copies of genes of interest (transgenes) into cells where they are transcribed and translated into therapeutic proteins. Protein production can also be modified using therapies that regulate gene expression. Gene therapy is currently prohibited in bot...
Hierarchical modeling of elastic moduli of equine hoof wall. This study predicts analytically effective elastic moduli of substructures within an equine hoof wall. The hoof wall is represented as a composite material with a hierarchical structure comprised of a sequence of length scales. A bottom-up approach is employed. Thus, the outputs from a lower spatial scale serve as the inputs for the following scale. The models include the Halpin-Tsai model, composite cylinders model, a sutured interface model, and classical laminate theory. The length scales span macroscale, mesoscale, sub-mesoscale, microscale, sub-microscale, and nanoscale. The macroscale re...
Commercial Hair Analysis in Horses: A Tool to Assess Mineral Intake? The use of hair samples to assess the mineral intake of horses under practical conditions has recently been attracting increased interest. The aims of this study were to compare mineral analysis results in equine mane hair between three commercial laboratories and to determine the relationship between the mineral intake and mineral content in mane hair. Four warmblood horses (14 ± 3 years) were included in the study. Horses were fed hay ad libitum and a commercial mineral supplement to meet nutrient requirements under maintenance conditions. Mane hair from the hairline was collected with a sa...
Generic approach for the discovery of drug metabolites in horses based on data-dependent acquisition by liquid chromatography high-resolution mass spectrometry and its applications to pharmacokinetic study of daprodustat. In drug metabolism studies in horses, non-targeted analysis by means of liquid chromatography coupled with high-resolution mass spectrometry with data-dependent acquisition (DDA) has recently become increasingly popular for rapid identification of potential biomarkers in post-administration biological samples. However, the most commonly encountered problem is the presence of highly abundant interfering components that co-elute with the target substances, especially if the concentrations of these substances are relatively low. In this study, we evaluated the possibility of expanding DDA coverag...
Determination of hair cortisol in horses: comparison of immunoassay vs LC-HRMS/MS. The measure of hair cortisol concentration (HCC) is becoming an emerging approach to monitor mid-/long-term stress in animals, so it is more and more important to develop accurate and reliable methods. In the light of this, the aim of the present study was to compare mane HCCs of 47 horses with different managements, by means of an immunoassay (ELISA) and liquid chromatography coupled to hybrid high-resolution mass spectrometry (LC-HRMS/MS). After the washing step, the ground hair was extracted with methanol. The extract was evaporated and redissolved in two different aqueous solutions, depend...
Additional studies on nicotine exposure in horses: Accurate quantification and elimination profiles of potential biomarkers in plasma and urine. For the purpose of doping control, this is the first report of accurate quantification of four critical structural isomers of nicotine metabolites (trans-3'-hydroxycotinine, cis-3'-hydroxycotinine, 5'-hydroxycotinine, and N'-hydroxymethylnorcotinine) in equine plasma and urine for the establishment of their elimination profiles. Besides, the pharmacokinetic studies of trans-3'-hydroxycotinine and N'-hydroxymethylnorcotinine in equine plasma and urine are also presented for the first time. Methods: The accurate quantification methods of the aforementioned four structural isomers in horse plasma...
Analysis of the effect of 1-Allyl-3-Methylimidazolium chloride on thermodynamic stability, folding kinetics, and motional dynamics of horse cytochrome c. 1-allyl-3-methylimidazolium chloride (AMIMCl) acts as a potential green solvent for proteins. The present work provides a possible pathway by which the structural, kinetic, thermodynamic, and folding properties of horse cytochrome c (cyt c) are affected in green aqueous-AMIMCl systems. Analysis of the effect of AMIMCl on thermodynamic stability, refolding/unfolding kinetics, and motional dynamics of cyt c provided important information, (i) AMIMCl decreases the thermodynamic stability of reduced cyt c and also strengthens the guanidinium chloride (GdmCl)-mediated decrease in thermodynamic stab...
Pharmacokinetic Study of Vadadustat and High-Resolution Mass Spectrometric Characterization of its Novel Metabolites in Equines for the Purpose of Doping Control. Vadadustat, a hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) inhibitor, is a substance which carries a lifetime ban in both horse racing and equestrian competition. A comprehensive metabolic study of vadadustat in horses has not been previously reported. Objective: Metabolism and elimination profiles of vadadustat in equine plasma and urine were studied for the purpose of doping control. Methods: A nasoesophageal administration of vadadustat (3 g/day for 3 days) was conducted on three thoroughbred mares. Potential metabolites were comprehensively detected by differential analysis of ful...
Direct 14C dating of equine products preserved in archaeological pottery vessels from Botai and Bestamak, Kazakhstan. Direct and accurate radiocarbon dating of lipid residues preserved in ceramics is a recently established method that allows direct dating of specific food products and their inception in human subsistence strategies. The method targets individual fatty acids originating from animal fats such as ruminant dairy, ruminant adipose, non-ruminant adipose and aquatic fats. Horse lipid residues found in Central Asian pottery vessels are also directly dateable using this new method. Here we present the identification of equine lipid residues preserved in two pottery assemblages from the Neolithic and E...
A fully automated method to quantitate total carbon dioxide in equine plasma by headspace GCMS. Pre-race dosing of horses with alkalinising agents to manipulate performance has been evident in racing worldwide for over 30 years. To regulate the use of alkalinising agents, racing authorities adopted thresholds for total plasma carbon dioxide (TCO ) in racehorses. Traditionally, racing laboratories have measured plasma TCO using ion selective electrode (ISE) technology, with the Association of Official Racing Chemists (AORC) approving the use of only three ISE instruments for measurement. Because of the manufacture and support of these instruments ceasing, racing laboratories have explor...
Evaluation of hair analysis for determination of trace mineral status and exposure to toxic heavy metals in horses in the Netherlands. Analysis of hair to gain insight into the trace mineral status and exposure to toxic heavy metals of horses is attractive because hair is an easily accessible sample material. To investigate the potential value of hair analysis in horses for determination of trace mineral and heavy metal concentrations, we analyzed mane hair and liver samples from 62 horses presented for slaughter at a facility in the Netherlands that receives horses from all regions of the country. Hair samples were cleaned in warm water. After acid digestion of hair and liver specimens, we quantified, with inductively couple...
Simultaneous quantification of caffeine and its main metabolites by gas chromatography mass spectrometry in horse urine. Caffeine is a naturally occurring alkaloid and it is metabolized to paraxanthine, theophylline and theobromine. Analysis of caffeine and its metabolites is challenging since the metabolites theophylline and paraxanthine generate similar product and precursor ions. In this study, a new method was developed for the simultaneous analysis of caffeine, paraxanthine, theobromine and theophylline in horse urine using gas chromatography-mass spectrometry (GC-MS). Urine samples were treated using solid-phase extraction followed by the elution with dichloromethane-isopropanol (90:10) after the pH was ad...
Application of Fourier transform infrared spectroscopy (FTIR) techniques in the mid-IR (MIR) and near-IR (NIR) spectroscopy to determine n-alkane and long-chain alcohol contents in plant species and faecal samples. n-Alkanes and long-chain alcohols (LCOH) have been used as faecal markers to assess the feeding behaviour of both wild and domestic herbivore species. However, their chemical analysis is time-consuming and expensive, making it necessary to develop more expeditious methodologies to evaluate concentrations of these markers. This work aimed to evaluate the use of Fourier Transform Infrared Spectroscopy (FTIR) technology in the near infrared (NIR) and mid infrared (MIR) intervals, for the determination of n-alkane and LCOH concentrations of different plant species and faecal samples of domestic he...
Characterization of growth hormone secretagogue small molecule ibutamoren (MK-0677) and its possible metabolites in thoroughbred horses for doping control. It is important to remember that performance-enhancing agents such as non-peptide growth hormone secretagogues present a significant risk of abuse. Ibutamoren (MK-0677) is a potent, long-acting, selective non-peptide growth hormone secretagogue that can be taken orally. Methods: This study examines ibutamoren and its metabolites in thoroughbred horses after oral administration. Liquid chromatography/high-resolution mass spectrometry was used to determine the probable structures of the detected metabolites. Results: In this study, 22 metabolites of ibutamoren were identified (17 phase I and 5 p...
Pharmacokinetics of grapiprant and effects on TNF-alpha concentrations following oral administration to horses. Grapiprant is a prostaglandin E2 receptor antagonist that has been found to be an effective anti-inflammatory in dogs and that is devoid of some of the adverse effects associated with traditional NSAIDs that elicit their effects through inhibition of PGE2 production. Previously published reports have described the pharmacokinetics of this drug in horses when administered at 2 mg/kg; however, pharmacodynamic effects in this species have yet to be described. The objective of the current study was to describe the pharmacokinetics and pharmacodynamics of grapiprant at a higher dose. Eight horses...
