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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.
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...
[Methodical investigation of mineral and trace element concentrations in equine feces with special consideration of the sampling location]. Objective measurements of the mineral supply in horses are rarely performed. As a result, incorrect elements or an improper amount of elements are provided. The analysis of feces could represent a novel method to evaluate the nutritive supply. The prerequisite is a knowledge of methodological factors influencing the mineral concentration in the fecal samples. Within the scope of this investigation, the effects of different kinds of mineral supply and the influence of the sampling location on the concentration of minerals in equine feces samples were analyzed. Additionally, the methodical error...
Dose Effect of Milk Thistle (Silybum marianum) Seed Cakes on the Digestibility of Nutrients, Flavonolignans and the Individual Components of the Silymarin Complex in Horses. Milk thistle seeds contain a mixture of flavonoids known as silymarin, which consists of silybin, isosilybin, silychristine, and silydianin. Until now, there has been no evidence of monitoring the digestibility of silymarin complex in horses. The aim of the research was to evaluate the digestibility of silymarin complex and the effect of nutrient digestibility in horses. Different daily feed doses (FD) of milk thistle expeller (0 g, 100 g, 200 g, 400 g, 700 g) were administered to five mares kept under the same conditions and at the same feed rations. Digestibility of silymarin complex was mon...
Stereoselective methadone disposition after administration of racemic methadone to anesthetized Shetland ponies assessed by capillary electrophoresis. The enantioselectivity of the pharmacokinetics of methadone was investigated in anesthetized Shetland ponies after a single intravenous (0.5 mg/kg methadone hydrochloride; n = 6) or constant rate infusion (0.25 mg/kg bolus followed by 0.25 mg/kg/h methadone hydrochloride; n = 3) administration of racemic methadone. Plasma concentrations of l-methadone and d-methadone and their major metabolites, l- and d-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), respectively, were analyzed by CE with highly sulfated γ-cyclodextrin as chiral selector and electrokinetic analyte injection from...
Novel Algorithms for Comprehensive Untargeted Detection of Doping Agents in Biological Samples. To address the limitations of current targeted analytical methods that can only detect known doping agents, a novel methodology that permits untargeted drug detection (UDD) has been developed to help in the fight against doping in sports. Fifty-seven drugs were spiked into blank equine plasma and were treated as unknowns since their exact masses and chromatographic retention times were not utilized for detection. The spiked drugs were extracted from the plasma samples and were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). The acquired LC-HRMS raw ...
Long-term detection of clodronate in equine plasma by liquid chromatography-tandem mass spectrometry. Clodronate is a non-nitrogen-containing bisphosphonate drug approved in equine veterinary medicine. Clodronate is prohibited for use in competition horses; therefore, to set up an appropriate control, detection times and screening limits are required. The quantitative method in plasma consisted of addition of chloromethylene diphosphonic acid as internal standard. Automated sample preparation comprised a solid phase extraction with weak anion exchange properties on microplate. After methylation of the residue with trimethyl orthoacetate, analysis was conducted by high-performance liquid chroma...
miRNAs detection in equine plasma by quantitative polymerase chain reaction for doping control: Assessment of blood sampling and study of eca-miR-144 as potential erythropoiesis stimulating agent biomarker. Short half-life doping substances are, quickly eliminated and therefore difficult to control with traditional analytical chemistry methods. Indirect methods targeting biomarkers constitute an alternative to extend detection time frames in doping control analyses. Gene expression analysis (i.e., transcriptomics) has already shown interesting results in both humans and equines for erythropoietin (EPO), growth hormone (GH), and anabolic androgenic steroid (AAS) misuses. In humans, circulating cell-free microRNAs in plasma were described as new potential biomarkers for control of major doping agen...
Development of a 3′:5′ digital PCR assay to determine horse mRNA integrity. Accurate tools to measure RNA integrity are essential to obtain reliable gene expression data. The reverse transcription quantitative PCR (RT-qPCR) based 3':5' assay permits a direct determination of messenger RNA (mRNA) integrity. However, the use of standard curves and the possible effect of PCR inhibitors make this method cumbersome and prone to variation, especially in small samples. Here we developed a triplex digital PCR (dPCR) 3':5' assay for assessing RNA integrity in equine samples as rapid and simple alternative to RT-qPCR. This dPCR assay not only provides a straight forward analysi...
Quantification of 17 Endogenous and Exogenous Steroidal Hormones in Equine and Bovine Blood for Doping Control with UHPLC-MS/MS. A simple and fast analytical method able to simultaneously identify and quantify 17 endogenous and exogenous steroidal hormones was developed in bovine and equine blood using UHPLC-MS/MS. A total amount of 500 µL of sample was deproteinized with 500 µL of a mixture of methanol and zinc sulfate and evaporated. The mixture was reconstituted with 50 µL of a solution of 25% methanol and injected in the UHPLC-MS/MS triple quadrupole. The correlation coefficients of the calibration curves of the analyzed compounds were in the range of 0.9932-0.9999, and the limits of detection and quantification ...
Internal cavity amplification of shell-like ferritin regulated with the change of the secondary and tertiary structure induced by PEF technology. In this study, the effect of pulsed electric field (PEF) on apparent morphology and molecular structure of shell-like ferritin obtained from horse spleen was determined by circular dichroic (CD), fluorescence spectroscopy, Raman spectroscopy, cold field emission scanning electron microscopy (CF-SEM) and transmission electron microscopy (TEM), and verified by molecule dynamics (MD) simulation. After PEF treatment, the α-helix content of the samples reached a minimum value at 10 kV/cm, which indicated that the ferritin structure has been partially unfolded. However, the α-helix content peaked ...
Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) Spectroscopy for the Quantitative Analysis of Deuterium in Plasma: Application to Total Body Water Determination in Humans and Other Animals. Conventional methods for measuring the concentration of deuterium in body fluids are by either isotope ratio mass spectrometry or Fourier transform infrared transmission (FT-IR) spectroscopy. The latter method is often preferred as it is less expensive and time consuming; however, having a lower sensitivity means a larger sample volume is required. This study investigated an alternative FT-IR spectroscopic method, attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR), which has the potential to provide shorter analysis times while requiring smaller sample volumes. Deu...
Development and validation of a liquid chromatography coupled to mass spectrometer (LC-MS) method for the simultaneous quantification of estrone-3-sulfate, progesterone, estrone and estradiol in serum of mares and American bisons. Steroid concentrations in serum are fluctuating during pregnancy of many mammal species. The current knowledge about endocrinology of gestation is mainly based on immunoassays. However, the lack of specificity of these assays hampers the reliability of the results. In the present work, we developed and validated a methodology associating liquid chromatography (LC) and mass spectrometry (MS) to simultaneously quantify, with high specificity and accuracy, estrone-3-sulfate (E3S), progesterone (PRO), estrone (E1) and estradiol (E2) in serum of two different mammal species. The sample preparation ...
Use of high resolution/accurate mass full scan/data-dependent acquisition for targeted/non-targeted screening in equine doping control. High-resolution mass spectrometry (HRMS) is a very powerful technology for equine doping control analysis. The more recently developed hybrid type of Orbitrap-based HRMS instrument allows for both targeted and non-targeted screening analyses in a single liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) run. In the present study, an LC-HRMS/MS method was developed and validated to detect prohibited substances in equine sports. The substances were recovered from equine plasma by liquid-liquid extraction (LLE) using methyl tert-butyl ether and were separated on a C18 rev...
Towards an untargeted mass spectrometric approach for improved screening in equine antidoping. The emergence of novel doping agents is a continuous issue for analysts who aim to maintain the integrity of horseracing together with the well-being and safety of the animals and riders involved. Untargeted mass spectrometric analysis presents a potential improvement for antidoping as it enables the detection of compounds being indirectly affected by an administered drug. In this study, liquid chromatography-high-resolution mass spectrometry was used to investigate a 12-horse administration study of the synthetic opioid, butorphanol. A mass spectrometric workflow capable of detecting metaboli...
Assay variability and storage stability of the myeloperoxidase index of the ADVIA 2120i hematology analyzer in canine and equine whole blood samples. The myeloperoxidase index (MPXI), on ADVIA hematology analyzers, reflects the mean neutrophil myeloperoxidase staining. It is used as a marker of inflammation in animals and people, but assay variability and storage stability are unknown. Objective: We aimed to determine MPXI precision and stability with refrigerated storage of canine and equine EDTA-anticoagulated blood and compared MPXI results between two analyzers. Methods: Inter-assay coefficients of variations (CVs) were determined from three human-based controls assayed before and after a 20- or 21-day calibration. Blood from 14-16 dogs...
Validation of a flow cytometric assay to detect intraerythrocytic reactive oxygen species in horses. Oxidative stress refers to the accumulation of reactive oxygen species (ROS). Most assays for ROS detection are costly, laborious, and usually use indirect markers. The use of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) is a possible alternative. This substance becomes a fluorochrome when oxidized by ROS, with the resultant fluorescence proportional to ROS concentration. Erythrocytes are highly exposed to ROS, resulting in cell damage and consequently impaired oxygen delivery. The effects of this exposure in physiologic and pathologic conditions necessitate an improvement in ROS detec...
Effects of storage time and temperature on thromboelastographic analysis in dogs and horses. The accessibility of thromboelastography (TEG) to general practitioners is limited by short sample storage times (30 minutes) and storage temperatures (20-23°C). Objective: We aimed to evaluate the stability of canine and equine citrated blood samples when stored for extended periods of time, both at room temperature (RT) (20-23°C) and refrigerator temperature (FT) (2-7.5°C). Methods: Citrated whole blood samples from healthy dogs and horses (n = 10 for each) were stored for 30 minutes (baseline) at RT before TEG analysis. Baseline values for TEG variables R, K, α, MA, LY30, and LY60 w...
Evaluation of Blood Glucose and Lactate Concentrations in Mule and Equine Foals. Information about mule physiology is scarce. Glucose and lactate serve as prognostic tools in neonates; thus, real-time evaluations would be beneficial. Our main objective was to measure glucose and lactate concentrations from healthy mule and equine foals from birth to 720 hours. Glucose and lactate concentrations were evaluated with a benchtop Randox Daytona analyzer (LAB) using plasma and with an Accutrend Plus system (ACP) using whole blood. Data were analyzed using PROC MIXED (P < .05), intraclass coefficient correlation and Bland-Altman analysis. Glucose and lactate concentrations fro...
Ethanol-Soluble Carbohydrates of Cool-Season Grasses: Prediction of Concentration by Near-Infrared Reflectance Spectroscopy (NIRS) and Evaluation of Effects of Cultivar and Management. Ethanol-soluble carbohydrates (ESCs) of cool-season grasses include mono- and disaccharides and sometimes short-chain fructans, which may exacerbate the risk of pasture-associated laminitis. A calibration for prediction of ESC concentrations by near-infrared reflectance spectroscopy (NIRS) was developed from 323 samples of four cool-season grass species (orchardgrass, Kentucky bluegrass, tall fescue, and perennial ryegrass) across 10 cultivars collected in central Kentucky in the morning and afternoon over two growing seasons. The calibration, which had accuracy above 95%, was used to predict ...
Comprehensive characterization of the peroxisome proliferator activated receptor-δ agonist GW501516 for horse doping control analysis. According to international sport institutions, the use of peroxisome proliferator activated receptor (PPAR)-δ agonists is forbidden at any time in athlete career due to their capabilities to increase physical and endurance performances. The (PPAR)-δ agonist GW501516 is prohibited for sale but is easily available on internet and can be used by cheaters. In the context of doping control, urine is the preferred matrix because of the non-invasive nature of sampling and providing broader exposure detection times to forbidden molecules but often not detected under its native form due to the organi...
Determination of vitamin E and its metabolites in equine urine using liquid chromatography-mass spectrometry. Equine neuroaxonal dystrophy/degenerative myeloencephalopathy (eNAD/EDM) is a hereditary, deteriorating central nervous disease in horses. Currently, the only way to confirm eNAD/EDM is through a postmortem histological evaluation of the central nervous system. Vitamin E, specifically the isoform alpha-tocopherol (α-TP), is known to protect eNAD/EDM susceptible horses from developing the clinical phenotype. While vitamin E is an essential nutrient in the diet of horses, there are no diagnostic tests able to quantitate vitamin E and its metabolites in urine. An ultra-performance liquid chromat...
A validated method for the quantification of IOX-2, a potent prolyl hydroxylase inhibitor in equine urine and plasma using liquid chromatography-high-resolution mass spectrometry. IOX-2 is a potent inhibitor of enzyme prolyl hydroxylases-2 (PHD) that plays a critical role in regulating hypoxia inducible factor (HIF) abundance and oxygen homeostasis. Federation for Equestrian Sports has listed HIF activators as prohibited substances to prevent their usage in doping. Consequently, it became essential to develop adequate knowledge and testing methods to detect it in equine sports drug testing samples. The validated method utilizes ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry in order to detect extremely low concentration of the ...
Investigation of Equine In Vivo and In Vitro Derived Metabolites of the Selective Androgen Receptor Modulator (SARM) ACP-105 for Improved Doping Control. Selective Androgen Receptor Modulators (SARMs) have anabolic properties but less adverse effects than anabolic androgenic steroids. They are prohibited in both equine and human sports and there have been several cases of SARMs findings reported over the last few years. The aim of this study was to investigate the metabolite profile of the SARM ACP-105 (2-chloro-4-[(3-endo)-3-hydroxy-3-methyl-8-azabicyclo[3.2.1]oct-8-yl]-3-methylbenzonitrile) in order to find analytical targets for doping control. Oral administration of ACP-105 was performed in horses, where blood and urine samples were collect...
Doping detection in animals: A review of analytical methodologies published from 1990 to 2019. Despite the impressive innate physical abilities of horses, camels, greyhounds, or pigeons, doping agents might be administered to these animals to improve their performance. To control these illegal practices, anti-doping analytical methodologies have been developed. This review compiles the analytical methods that have been published for the detection of prohibited substances administered to animals involved in sports over 30 years. Relevant papers meeting the search criteria that discussed analytical methods aiming to detect and/or quantify doping substances in animal biological matrices pu...
A sensitive HPLC-MS/MS method for the detection, resolution and quantitation of cathinone enantiomers in horse blood plasma and urine. Resolution of cathinone enantiomers in equine anti-doping analysis is becoming more important to distinguish the inadvertent ingestion of plant-based products from those of deliberate administration of designer synthetic analogs. With this in mind, a rapid and sensitive method was developed and validated for the detection, resolution and quantitative determination of cathinone enantiomers in horse blood plasma and urine. The analytes were recovered from the blood plasma and urine matrices by using a liquid-liquid extraction after adjusting the pH to 9. The recovered analytes were derivatized w...
