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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.
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...
In vitro and in vivo metabolism of the anabolic-androgenic steroid oxandrolone in the horse. Oxandrolone is an anabolic-androgenic steroid with favourable anabolic to androgenic ratio, making it an effective anabolic agent with less androgenic side effects. Although its metabolism has been studied in humans, its phase I and II metabolism has not been previously reported in the horse. The purpose of this study was to investigate the in vitro metabolism of oxandrolone (using both equine liver microsomes and S9) and in vivo metabolism following oral administration (three daily doses of 50 mg of oxandrolone to a single Thoroughbred horse), using both gas and liquid chromatography-mass s...
[Manganese concentrations in whole blood, plasma and serum of adult warmblood horses from 3 locations in Germany]. This study aimed to establish reference intervals for Mn in whole blood, plasma and serum of healthy, adult warmblood horses with known dietary Mn intake and to compare 2 methods of analysis. Methods: Between May 2018 and November 2019 a single blood sample was taken from a total of 270 clinically healthy horses (age: 3-25 years) in 3 stud farms. In lithium-heparin (LH) whole blood, LH plasma and serum Mn concentrations were analyzed by means of atomic absorption spectrometry (AAS) and inductively coupled plasma mass spectrometry (ICP-MS). The reference intervals were calculated according to t...
Detection and confirmation of zilpaterol in equine hair using liquid chromatography-mass spectrometry. Zilpaterol is a β -adrenergic agonist and a repartitioning agent that has a high potential for abuse in equine performance athletes. Analysis of zilpaterol in hair is an alternative sampling matrix that extends detection time periods beyond those found in urine or blood samples. Our laboratory has been screening for zilpaterol in hair for many years and recently detected and confirmed its presence in official samples. Accordingly, a liquid chromatography-mass spectrometry method was developed and validated to detect and confirm zilpaterol in equine hair. Briefly, equine hair was decontaminate...
Robustness of digital PCR and real-time PCR against inhibitors in transgene detection for gene doping control in equestrian sports. Gene doping is a threat to fair competition in sports, both human and equestrian. One method of gene doping is to administer exogenous genetic materials, called transgenes, into the bodies of postnatal humans and horses. Polymerase chain reaction (PCR)-based transgene detection methods such as digital PCR and real-time PCR have been developed for gene doping testing in humans and horses. However, the significance of PCR inhibitors in gene doping testing has not been well evaluated. In this study, we evaluated the effects of PCR inhibitors on transgene detection using digital PCR and real-time ...
In vitro metabolism of the REV-ERB agonist SR-9009 and subsequent detection of metabolites in associated routine equine plasma and urine doping control samples. SR-9009 is a synthetic compound widely available to purchase online as 'supplement' products due to its potential performance-enhancing effects, presenting a significant threat with regard to doping control in sport. In vitro metabolism with equine liver microsomes was performed to identify potential targets for detection of SR-9009. Six metabolites were identified, with the most abundant consisting of N-dealkylated metabolites (M1-M3). The addition of the identified metabolites to high-resolution accurate mass databases resulted in a positive finding for the N-dealkylated metabolite M1 of SR-...
What makes a good fecal egg count technique? The first parasite fecal egg counting techniques were described over 100 years ago, and fecal egg counting remains essential in parasitology research as well as in clinical practice today. Several novel techniques have been introduced and validated in recent years, but this work has also highlighted several current issues in this research field. There is a lack of consensus on which diagnostic parameters to evaluate and how to properly design studies doing so. Furthermore, there is a confusing and sometimes incorrect use of terminology describing performance of fecal egg counting techniques, a...
