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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.
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...
Pharmacokinetics of fluoxetine in horses following oral administration. This study aimed to investigate pharmacokinetics of fluoxetine in horses and validate a method for liquid chromatography mass spectrometry analysis of serum levels. Fluoxetine pharmacokinetics were determined using 10 healthy, adult horses. Fluoxetine pharmacokinetics following a single oral dose (0.25 mg/kg) were determined using blood samples collected prior to and at several time points over 7 days following administration. Serum concentrations of fluoxetine and its bioactive metabolite norfluoxetine were measured using liquid chromatography coupled to an accurate mass/high-resolution ma...
LC-HRMS/MS study of the prodrug ciclesonide and its active metabolite desisobutyryl-ciclesonide in plasma after an inhalative administration to horses for doping control purposes. Ciclesonide (CIC) is the first inhaled highly potent corticosteroid that does not cause any cortisol suppression. It has been developed for the treatment of asthma in human and more recently in equine. CIC is the active compound of Aservo® EquiHaler® (Boehringer Ingelheim Vetmedica GmbH), the pre-filled inhaler generating a medicated mist based on Soft Mist™ technology. This prodrug is rapidly converted to desisobutyryl-ciclesonide (des-CIC), the main pharmacologically active compound. Due to its anti-inflammatory properties, CIC is prohibited for use in horse competitions. To set up an ap...
[Determination of donkey skin ingredients in Asini Corii Colla by ultra-high performance liquid chromatography-tandem mass spectrometry]. In recent years, due to the shortage of donkey skin resources, the price of Asini Corii Colla has seen a rapid increase. Consequently, fake gelatin prepared from horse, mules, pig, and cow skin has appeared in the market, resulting in unreliable quality of Asini Corii Colla. Therefore, there is an urgent need to develop an efficient and accurate method for improving the quality of Asini Corii Colla. Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used to determine the donkey skin components in Asini Corii Colla. Accordingly, 0. l g of the evenly mixed sa...
Comprehensive metabolic study of IOX4 in equine urine and plasma using liquid chromatography/electrospray ionization Q Exactive high-resolution mass spectrometer for the purpose of doping control. IOX4 is a hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) inhibitor, which was developed for the treatment of anemia by exerting hematopoietic effects. The administration of HIF-PHD inhibitors such as IOX4 to horses is strictly prohibited by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale. To the best of our knowledge, this is the first comprehensive metabolic study of IOX4 in horse plasma and urine after a nasoesophageal administration of IOX4 (500 mg/day, 3 days). A total of four metabolites (three mono-hydroxylated IOX4 and one ...
Loading equine oocytes with cryoprotective agents captured with a finite element method model. Cryopreservation can be used to store equine oocytes for extended periods so that they can be used in artificial reproduction technologies at a desired time point. It requires use of cryoprotective agents (CPAs) to protect the oocytes against freezing injury. The intracellular introduction of CPAs, however, may cause irreversible osmotic damage. The response of cells exposed to CPA solutions is governed by the permeability of the cellular membrane towards water and the CPAs. In this study, a mathematical mass transport model describing the permeation of water and CPAs across an oocyte membrane...
Detection of Methylphenidate in Equine Hair Using Liquid Chromatography-High-Resolution Mass Spectrometry. Methylphenidate is a powerful central nervous system stimulant with a high potential for abuse in horse racing. The detection of methylphenidate use is of interest to horse racing authorities for both prior to and during competition. The use of hair as an alternative sampling matrix for equine anti-doping has increased as the number of detectable compounds has expanded. Our laboratory developed a liquid chromatography-high-resolution mass spectrometry method to detect the presence of methylphenidate in submitted samples. Briefly, hair was decontaminated, cut, and pulverized prior to liquid-liq...
Accuracy and trending capability of haemoglobin measurement by noninvasive pulse co-oximetry in anaesthetized horses. To assess the accuracy and trending capability of continuous measurement of haemoglobin concentration [Hb], haemoglobin oxygen saturation (SaO) and oxygen content (CaO) measured by the Masimo Radical-7 pulse co-oximeter in horses undergoing inhalational anaesthesia. Methods: Prospective observational clinical study. Methods: A group of 23 anaesthetized adult horses. Methods: In 23 healthy adult horses undergoing elective surgical procedures, paired measurements of pulse co-oximetry-based haemoglobin concentration (SpHb), SaO (SpO), and CaO (SpOC) and simultaneous arterial blood samples were co...
A quantitative PCR screening method for adeno-associated viral vector 2-mediated gene doping. Gene therapy is currently prohibited in human and equine athletes and novel analytical methods are needed for its detection. Most in vivo products use non-integrating, recombinant viral vectors derived from adeno-associated virus (AAV) to deliver transgenes into cells, where they are transcribed and translated into functional proteins. Although the majority of wild-type AAV (WTAAV) DNA is removed from recombinant AAV (rAAV) vectors, some sequences are conserved. The goal of this study was to develop a quantitative polymerase chain reaction (QPCR) screening test targeting conserved AAV sequence...
