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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.
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...
Development of a Microsphere-Based Immunoassay Authenticating A2 Milk and Species Purity in the Milk Production Chain. Processed milk and milk products produced from bovine milk, commonly contain β-casein A1 (βCA1) and β-casein A2 (βCA2). Since the presence of βCA1 is linked to milk intolerance and digestion problems, A2A2 milk, which only contains βCA2, is proposed as a healthier alternative. To support this health claim, the purity of A2A2-milk has to be guaranteed. In the presented study, a multiplex immunoassay, able to distinguish between βCA2 and βCA1, was developed and real-life applicability was shown on raw milk samples from genotyped A1A1, A1A2 and A2A2 cows. Because of its ability to discrim...
A quantitative method for the simultaneous detection of SR9009 and SR9011 in equine plasma using liquid chromatography-electrospray ionization-tandem mass spectrometry. SR9009 and SR9011 are metabolic modulators pharmacologically targeting REV-ERB receptors as synthetic agonists. A liquid chromatography-tandem mass spectrometry method for the detection of SR9009 and SR9011 in equine plasma was developed and validated. Plasma samples were pretreated by protein precipitation with methanol and were loaded onto an ACQUITY ultra performance liquid chromatography high-strength silica C18 column (2.1 × 150 mm, 1.8 μm) for chromatographic separation. The mobile phase consisted of 5-mM ammonium formate (pH 3.0) in distilled water and 0.1% formic acid in ac...
Pharmacokinetics of glaucine after intravenous and oral administrations and detection of systemic aporphine alkaloids after ingestion of tulip poplar shavings in horses. Glaucine, an aporphine alkaloid with anti-tussive, anti-inflammatory, and anti-nociceptive properties, has been identified in post-race samples from racehorses. To investigate pharmacokinetics of glaucine in horses, a three-way crossover study of intravenous and oral glaucine (0.1 mg/kg) and orally administered tulip poplar shavings (50 g shavings = 0.001 mg/kg glaucine) was performed in six horses. A two-compartment model best described IV administration with alpha ( ) and beta ( ) half-life lives of 0.3 (0.1-0.7) and 3.1 (2.4-7.8) h, respectively. The area under the curve ( ) was ...
Equine metabolism of the growth hormone secretagogue MK-0677 in vitro and in urine and plasma following oral administration. Ibutamoren mesylate, or MK-0677, is an orally active, nonpeptide growth hormone secretagogue that has been developed to stimulate excretion of endogenous growth hormone. It has been evaluated for the treatment of a range of clinical conditions but is not available therapeutically. Nonetheless, MK-0677 is widely available to purchase online, sold as 'supplement' products. The mode of action and relative ease of purchase make MK-0677 a potential threat with regard to sports doping. The aim of this study was to investigate the metabolism of MK0677 in the horse following in vitro incubation and or...
Technical Note: A method for determination of titanium dioxide concentration in fecal samples. Titanium dioxide has been used as a marker for determining diet digestibility indirectly, but some authors have expressed difficulty in measuring TiO2 concentrations in fecal material. We developed an accurate and precise method to determine TiO2 concentrations in equine feces. The method includes dry-ashing samples, digestion with (NH4)2SO4 in concentrated sulfuric acid, followed by the addition of H2O2 to produce a yellow to orange color that can be read spectrophotometrically. Accuracy was tested by spike recovery, and precision was tested by examining the coefficient of variation (CV) betw...
Metabolomics in clinical and forensic toxicology, sports anti-doping and veterinary residues. Metabolomics is a multidisciplinary field providing workflows for complementary approaches to conventional analytical determinations. It allows for the study of metabolically related groups of compounds or even the study of novel pathways within the biological system. The procedural stages of metabolomics; experimental design, sample preparation, analytical determinations, data processing and statistical analysis, compound identification and validation strategies are explored in this review. The selected approach will depend on the type of study being conducted. Experimental design influences ...
Identification of potential biomarkers in urine and plasma after consumption of tobacco product in horses. The use of nicotine stimulants in horses is generally banned in horse racing and equestrian sports-accidental consumption of tobacco products is one of the possible causes of nicotine exposure in horses. The authors recently reported a comprehensive metabolic study of nicotine in equines, differentiating between nicotine exposure and sample contamination by means of a nicotine biomarker trans-3'-hydroxycotinine. To identify potential biomarkers for the differentiation of genuine nicotine administration and consumption of tobacco products, tobacco leaves (equivalent to 250 mg of nicotine) wer...
Long-term monitoring of IOX4 in horse hair and its longitudinal distribution with segmental analysis using liquid chromatography/electrospray ionization Q Exactive high-resolution mass spectrometry for the purpose of doping control. IOX4, a hypoxia-inducible factor stabilizer, is classified as a banned substance for horses in both horse racing and equestrian sports. We recently reported the pharmacokinetic profiles of IOX4 in horse plasma and urine and also identified potential monitoring targets for the doping control purpose. In this study, a long-term longitudinal analysis of IOX4 in horse hair after a nasoesophageal administration of IOX4 (500 mg/day for 3 days) to three thoroughbred mares is presented for the first time for controlling the abuse/misuse of IOX4. Six bunches of mane hair were collected at 0 (pre), ...
Measurements of hydrocortisone and cortisone for longitudinal profiling of equine plasma by liquid chromatography-tandem mass spectrometry. The conventional detection of exogenous drugs in equine doping samples has been used for confirmation and subsequent prosecution of participants responsible. In recent years, alternative methods using indirect detection have been investigated due to the expanding number of pharmaceutical agents available with the potential of misuse. The monitoring of endogenous biomarkers such as hydrocortisone (HC) has been studied in equine urine with an international threshold of 1 μg/ml established; however, there is no current threshold for equine plasma. The aim of this research was to investigate pl...
Profiling Urinary Sulfate Metabolites With Mass Spectrometry. The study of urinary phase II sulfate metabolites is central to understanding the role and fate of endogenous and exogenous compounds in biological systems. This study describes a new workflow for the untargeted metabolic profiling of sulfated metabolites in a urine matrix. Analysis was performed using ultra-high-performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS) with data dependent acquisition (DDA) coupled to an automated script-based data processing pipeline and differential metabolite level analysis. Sulfates were identified through -means clustering...
Analytical documentation of an Arabian horse fatality related to Oenanthe crocata poisoning. Analytical detection of Oenanthe crocata toxins in biological samples is challenging because of their instability, the lack of commercially available standards and the exceptionally low detection of these molecules using mass spectrometry. This work aims to report the used analytical methods that allowed identification of the main plant toxins in biological samples from an equid (an Arabian horse) fatality related to hemlock water dropwort (Oenanthe crocata Linnaeus) intake. Using both LC-DAD and LC-HRMS methods allowed identification (i) of oenanthotoxin in roots found on the site, root fragm...
Pharmacokinetics of a single dose of oclacitinib maleate as a top dress in adult horses. The objective of this study was to determine the pharmacokinetic parameters of oclacitinib maleate as a top dress given to adult horses. Six adult horses with a mean weight of 528 kg were administered a single dose of 0.5 mg/kg oclacitinib maleate. Blood was collected prior to drug administration and at 15 min, 30 min, 45 min, 1, 2, 4, 6, 8, 12, 24, 48, and 72 h after treatment. Oclacitinib maleate plasma concentrations were measured by liquid chromatography/mass spectrometry. Pharmacokinetic parameters were found best to fit a one-compartment model. Mean C was 486 ng/ml (range 423-549...
Suspensory ligament size does not change after plantar fasciotomy and neurectomy of the deep branch of the lateral plantar nerve by ultrasonographic assessment. To determine the short-term effect of plantar fasciotomy and neurectomy (PFN) of the deep branch of the lateral plantar nerve on the proximal suspensory ligament (PSL) cross-sectional area (CSA) in horses with hindlimb proximal suspensory desmopathy (PSD). Methods: Analytical, observational, cohort study. Methods: Twenty-one horses. Methods: Records of horses with chronic PSD treated by PFN were included if a preoperative ultrasonographic examination was available and at least one postoperative ultrasonographic examination. One masked observer measured the ultrasonographic cross-sectional area...
Defining the specificity of recombinant human erythropoietin confirmation in equine samples by liquid chromatography-tandem mass spectrometry. The proteotypic human EPO peptides YLLEAK (T4), SLTTLLR (T11), TITADTFR (T14), and VYSNFLR (T17) are often used to confirm the presence of recombinant human EPO (rhEPO) in equine samples. Each of these peptides contains one or more isomeric leucine or isoleucine amino acids, raising the possibility that a simple leucine/isoleucine substitution could lead to a false identification when compared with a rhEPO reference standard. To examine this possibility variants of these four peptides were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These studies indicate that confir...
Detection and residence time of bisphosphonates in bone of horses. Bisphosphonates are potent anti-resorptive agents that have the potential to adversely affect bone healing in equine athletes, and normal bone adaption in young racehorses. A concern exists that bisphosphonate inhibition of normal bone metabolism could lead to increased bone fractures during high-intensity exercise. We found only a single report describing concentrations of tiludronate in the bone of horses, and no studies describing clodronate. Knowledge of the residence time in bone could allow for a better understanding of the long-term effects of these compounds. Our objectives were to dev...
Fast Fluoroalkylation of Proteins Uncovers the Structure and Dynamics of Biological Macromolecules. Covalent labeling of proteins in combination with mass spectrometry has been established as a complementary technique to classical structural methods, such as X-ray, NMR, or cryogenic electron microscopy (Cryo-EM), used for protein structure determination. Although the current covalent labeling techniques enable the protein solvent accessible areas with sufficient spatial resolution to be monitored, there is still high demand for alternative, less complicated, and inexpensive approaches. Here, we introduce a new covalent labeling method based on fast fluoroalkylation of proteins (FFAP). FFAP u...
Reduced rank analysis of morphometric and functional traits in Campolina horses. Multitrait models can increase the accuracy of breeding value prediction and reduce bias due to selection by using traits measured before and after it has occurred. However, as the number of traits grows, a similar trend is expected for the number of parameters to be estimated, which directly affects the computing power and the amount of data required. The aim of the present study was to apply reduced rank (principal components model-PCM) and factor analytical models (FAM), to estimate (co)variance components for nineteen traits, jointly evaluated in a single analysis in Campolina horses. A to...
In vitro studies of hypoxia inducible factor-prolyl hydroxylase inhibitors daprodustat, desidustat, and vadadustat for equine doping control. Performance-enhancing substances and methods have become a serious problem in competitive sports. The hypoxia-inducible factor (HIF) stabilizers can enhance the organism's capacity for molecular oxygen transport and are likely to be abused as performance-enhancing agents in sports. This paper describes the metabolic conversion of the popular hypoxia inducible factor-prolyl hydroxylase (HIF-PH) inhibitors, namely, daprodustat, desidustat, and vadadustat using equine liver microsomes, determined on a QExactive high-resolution mass spectrometer. During this study, a total of 10 metabolites for da...
Development of a Standardized Microflow LC Gradient to Enable Sensitive and Long-Term Detection of Synthetic Anabolic-Androgenic Steroids for High-Throughput Doping Controls. Synthetic androgenic anabolic steroids (AAS) are banned compounds and considered as major threats by both racing and sports international authorities. Hence, doping control laboratories are continually looking into analytical improvements to increase their detection capabilities, notably by means of emerging technologies. To enhance analytical performances for the detection of synthetic AAS such as stanozolol, specific chromatographic procedures have been developed using recent quaternary liquid chromatography technology originally designed for high-throughput standardized proteomics connected...
Quantifying Serum Total Lipids and Tryptophan Concentrations by Raman Spectroscopy During Standardized Obstacle Course in Horses. Raman spectroscopy is an inelastic light-scattering phenomenon that provides vibrational spectrum that contains information relative to chemical bonds and symmetry of a specific molecule, allowing the quali-quantitative simultaneous determination of several components in the biological fluids. Raman spectroscopy measurement returns a spectrum over a wavenumber range constituted by several bands representing biomarkers according to investigated biological matrices. In literature, it has been reported that at the frequencies inside the (1,300-1,360) cm total lipids, and tryptophan have been iden...
Study of cobalt doping control via various routes in thoroughbred horses. Cobalt is a substance that has been abused for athletic performance enhancement and has thus been prohibited by human and animal sports doping control authorities. However, because cobalt is present in humans and animals as a trace element, a certain level of cobalt is naturally present in their excretions. In the racing industry, cobalt is a controlled substance with a threshold concentration specified by the International Agreement on Breeding, Racing and Wagering (IABRW) for international harmonization. Due to environmental and feed consumption differences among countries, regional cobalt c...
Comparison of a point-of-care serum amyloid A analyzer frequently used in equine practice with 2 turbidimetric immunoassays used in human and veterinary medicine. Rapid, accurate detection of serum amyloid A (SAA) is needed in equine practice. We validated a patient-side point-of-care (POC) assay (Stablelab; Zoetis) compared to the turbidimetric immunoassays LZ-SAA (TIA-Hum) and VET-SAA (TIA-Vet; both Eiken Chemical). Analytical performance was assessed at 3 different concentration ranges and with interferences. Inter-method comparison using 49 equine serum samples revealed a significant difference between median SAA results ( < 0.0001), with the strongest bias between the POC and TIA-Vet (median 1,093 vs. 578 mg/L). The median SAA value obtain...
