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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.
High resolution accurate mass screening of prohibited substances in equine plasma using liquid chromatography–Orbitrap mass spectrometry. 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 due to significant improvement in high resolution mass spectrometry in terms of sensitivity, mass accuracy, mass resolution, and mass stability. A number of HRAMS methods have been reported for the detection of multi-drug residues in human or equine urine. As blood has become a common matrix for doping control analysis, especially in equine sports, a sensitive, fast and wide coverage screening method for detecting a large number of drugs in equine ...
A comparison of the microbiome and the metabolome of different regions of the equine hindgut. The microbiome and associated metabolome of faecal samples were compared to those from the caecum and right dorsal colon of horses and ponies euthanised for nonresearch purposes by investigating the microbial population community structure as well as their functional metabolic products. Through the use of 16S rRNA gene dendrograms, the caecum microbiome was shown to cluster separately from the other gut regions. 16S rRNA gene-based quantitative PCR (q-PCR) also demonstrated differences between the caecum and the other gut regions. Metabolites as identified by Fourier transform infrared cluster...
Real time monitoring of propofol blood concentration in ponies anaesthetized with propofol and ketamine. This study examined the pharmacokinetics of propofol by infusion in ponies using an analyser for the rapid measurement of propofol concentrations. The analyser (Pelorus 1000; Sphere Medical Ltd., Cambridge, UK) has a measurement cycle of approximately five minutes. Ten Welsh-cross ponies (weighing 135-300 kg) undergoing minor procedures were studied after premedication with acepromazine 0.03 mg/kg and detomidine 0.015 mg/kg. Anaesthesia was induced with ketamine 2 mg/kg and diazepam 0.03 mg/kg, and maintained with an infusion of propofol at an initial rate of 0.16 mg/kg/min for the first thirt...
The development and validation of a turbulent flow chromatography-tandem mass spectrometry method for the endogenous steroid profiling of equine serum. A method for the detection and quantitation of 35 endogenous steroids in equine serum was developed and validated. Androgens, estrogens, progestins and their metabolites potentially present in serum were simultaneously monitored in one method using on-line sample extraction by turbulent flow chromatography (TFC) on a 2-dimensional liquid chromatography system and detected on a triple-stage quadrupole mass spectrometer by electrospray ionization. Analytes were detected and quantitated by single-reaction monitoring or selected-ion monitoring. Limits of detection (range 0.025-10 ng mL(-1)) and qu...
Evaluation of an automated assay based on monoclonal anti-human serum amyloid A (SAA) antibodies for measurement of canine, feline, and equine SAA. Major acute phase proteins (APPs) have proven diagnostically useful in dogs, cats and horses with routine use facilitated by commercially available automated heterologous assays. An automated assay applicable across all three species would highly facilitate further dissemination of routine use, and the aim of this study was to validate an automated latex agglutination turbidimetric immunoassay based on monoclonal anti-human serum amyloid A (SAA) antibodies for measurement of canine, feline and equine SAA. Serum samples from 60 dogs, 40 cats and 40 horses were included. Intra- and inter-assay i...
Rapid and sensitive analysis of 3,4-methylenedioxypyrovalerone in equine plasma using liquid chromatography-tandem mass spectrometry. 3,4-Methylenedioxypyrovalerone (MDPV) is a psychoactive drug with potent stimulant properties and potential for abuse and drug dependency. MDPV was recently classified as a Class I drug by Racing Commissioners International, indicating that it is a banned substance in equine athletes because it lacks therapeutic value in horses. To enforce this ban, a sensitive and fast liquid chromatography-tandem mass spectrometry method was needed. It is for this reason that this method was developed for quantification and confirmation of MDPV in equine plasma. Sample preparation involved liquid-liquid extr...
Structural elucidation of phase I and II metabolites of bupivacaine in horse urine and fungi of the Cunninghamella species using liquid chromatography/multi-stage mass spectrometry. Bupivacaine is a local anaesthetic prohibited in equine sports. It is highly metabolized in the horse but a thorough description of its metabolite profile is lacking. An administration study should find appropriate analytical targets for doping control. Furthermore, knowledge of an in vitro system for production of metabolites would be beneficial. Methods: Marcain® (bupivacaine hydrochloride) was administered subcutaneously to a horse and urine samples were collected. In vitro metabolic systems consisting of the fungi Cunninghamella elegans and Cunninghamella blakesleeana were incubated with ...
Detection of fluticasone propionate in horse plasma and urine following inhaled administration. Fluticasone propionate (FP) is an anti-inflammatory agent with topical and inhaled applications commonly used in the treatment of asthma in steroid-dependent individuals. The drug is used in racehorses to treat Inflammatory Airway Disease; this work was performed in order to advise on its use and detect potential misuse close to racing. Methods were developed for the extraction and analysis of FP from horse plasma and a carboxylic acid metabolite (FP-17βCOOH) from horse urine. The methods utilize ultra high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) in ...
Detection of recombinant human EPO administered to horses using MAIIA lateral flow isoform test. Doping of horses with recombinant human erythropoietin (rHuEPO) to illegally enhance their endurance capacity in horseracing has been reported during the last years. This leads to increased blood viscosity which can result in sudden death and is of concern for the horse welfare. Additionally, the horse can start production of rHuEPO antibodies, which cross-reacts with endogenous equine EPO and can lead to severe anaemia and even death. In this study, a novel micro-chromatographic method, EPO WGA MAIIA, has been tested for the capability in plasma and urine samples to detect administration of e...
Evaluation of the veterinary application of a point-of-care device measuring white blood cell counts. A point-of-care device (POCD) for measuring total white blood cell count was evaluated for feline, canine, equine and bovine blood samples collected into EDTA. Mean biases were -9.2% (range, -12% to -6.3%) for feline samples, 20.2% (range, 15.3-25.1%) for canine samples, -7.1% (range, -8.3% to -5.9%) for equine samples, and 0.7% (range, -1.1% to 2.5%) for bovine samples. The results were influenced by the presence of nucleated red blood cells. The POCD provided precise, reliable data for feline, equine and bovine samples but the values obtained for the canine counts were overestimations.
Membrane phase behavior during cooling of stallion sperm and its correlation with freezability. Stallion sperm exhibits great male-to-male variability in survival after cryopreservation. In this study, we have investigated if differences in sperm freezability can be attributed to membrane phase and permeability properties. Fourier transform infrared spectroscopy (FTIR) was used to determine supra and subzero membrane phase transitions and characteristic subzero membrane hydraulic permeability parameters. Sperm was obtained from stallions that show differences in sperm viability after cryopreservation. Stallion sperm undergoes a broad and gradual phase transition at suprazero temperatures...
A new analytical method based on anti-EPO monolith column and LC-FAIMS-MS/MS for the detection of rHuEPOs in horse plasma and urine samples. Recombinant human erythropoietin (rHuEPO) is a 30-34 kDa glycoprotein banned by the racing authorities. For some years this molecule has been detected in race horses in USA and in Europe, and even in racing camels. Although direct methods to differentiate horse endogenous EPO and rHuEPO have been developed either by LC-MS/MS or by isoelectric focusing (IEF) with double-blotting, the short confirmation time of such prohibited hormone in plasma remains a problem for horseracing doping control laboratories. In order to improve the rHuEPOs confirmation process in horse plasma or urine in terms of ...
The effects of yohimbine on the pharmacokinetic parameters of detomidine in the horse. To describe the pharmacokinetics of detomidine and yohimbine when administered in combination. Methods: Randomized crossover design. Methods: Nine healthy adult horses aged 9 ± 4 years and weighing of 561 ± 56 kg. Methods: Three dose regimens were employed in the current study. 1) 0.03 mg kg(-1) detomidine IV (D), 2) 0.2 mg kg(-1) yohimbine IV (Y) and 3) 0.03 mg kg(-1) detomidine IV followed 15 minutes later by 0.2 mg kg(-1) yohimbine IV (DY). Each horse received all three dose regimens with a minimum of 1 week in between subsequent regimens. Blood samples were obtained and plasma analyzed f...
Investigation of the presence of endogenous prednisolone in equine urine by high-performance liquid chromatography mass spectrometry and high-resolution mass spectrometry. After the detection of low concentrations of prednisolone in racehorse urine samples collected at Italian racetracks, a study was initiated to investigate the accuracy of the analytical protocol used and the possible endogenous origin of detected prednisolone. Methods: Multiple reaction monitoring (MRM) MS(2) acquisition with a triple quadrupole (n = 780) and full scan MS(2) and MS(3) (n = 180) acquisition with a linear ion trap were checked. As a further confirmation, ten urine samples were analysed by high-resolution mass spectrometry (HRMS). Results: The study showed the difficulty of ident...
Comparison of an inertial sensor system with a stationary force plate for evaluation of horses with bilateral forelimb lameness. To assess the analytic sensitivity of an inertial sensor system for detection of the more severely affected forelimb in horses with bilateral lameness. Methods: 18 adult horses with forelimb lameness. Methods: Horses were fitted with inertial sensors and evaluated for lameness with a stationary force plate as they were trotted in a straight line. Inertial sensor-derived measurements for vertical head movement asymmetry (HMA) and vector sum (VS) of maximum and minimum head height differences between right and left halves of the stride were used to predict differences in mean peak vertical force...
Evaluation of the automated hematology analyzer Sysmex XT-2000iV ™ compared to the ADVIA ® 2120 for its use in dogs, cats, and horses. Part II: Accuracy of leukocyte differential and reticulocyte count, impact of anticoagulant and sample aging. The automated laser-based hematology analyzer Sysmex XT-2000iV™ provides a 5-part differential count and specific cytograms that are of great interest for large veterinary laboratories. The aim of the study was to validate the Sysmex XT-2000iV compared to the laser-based hematology analyzer ADVIA® 2120 and manual differential in dogs, cats, and horses as well as the impact of anticoagulant (heparin, ethylenediamine tetra-acetic acid [EDTA], and citrate) and storage at 22°C and 4°C. Consecutive fresh K(3)-EDTA blood samples from 216 cats, 314 dogs, and 174 horses were included. The impact ...
Evaluation of the automated hematology analyzer Sysmex XT-2000iV™ compared to the ADVIA® 2120 for its use in dogs, cats, and horses: Part I–precision, linearity, and accuracy of complete blood cell count. The automated laser-based hematology analyzer Sysmex XT-2000iV™ providing a complete blood cell count (CBC) and 5-part differential has been introduced in large veterinary laboratories. The aim of the current study was to determine precision, linearity, and accuracy of the Sysmex analyzer. Reference method for the accuracy study was the laser-based hematology analyzer ADVIA® 2120. For evaluation of accuracy, consecutive fresh blood samples from healthy and diseased cats (n = 216), dogs (n = 314), and horses (n = 174) were included. A low intra-assay coefficient of variation (CV) of approxim...
The role of diffusion in ferritin-induced relaxation enhancement of protons. The influence of proton diffusion on nuclear magnetic resonance (NMR) relaxation was investigated in the presence of horse spleen ferritin at 7 T. Binary mixtures of water and glycerol were used to control diffusion within the range of 0.6-2.0 × 10(-9)m(2)/s, which was confirmed by pulsed gradient techniques. The effect of chemical exchange by hydrolysis between water and glycerol on relaxation was characterized with Carr-Purcell-Meiboom-Gill (CPMG) dispersion experiments. The relaxation rate enhancement of the protons due to ferritin was found to be inversely proportional to the diffusion co...
Detection of myo-inositol trispyrophosphate in equine urine and plasma by hydrophillic interaction chromatography-tandem mass spectrometry. Myo-inositol trispyrophosphate (ITPP) is a new drug capable of increasing the amount of oxygen in hypoxic tissues. Studies have shown that administration of ITPP increases the maximal exercise capacity in normal mice as well as mice with severe heart failure. The properties of ITPP make it an ideal candidate as a doping agent to enhance performance in racehorses. While there have been speculations in the horseracing industry that the covert use of ITPP is already widespread, no reported method exists for the detection of ITPP in equine biological samples. ITPP is a difficult-to-detect drug due...
Pharmacokinetics and pharmacodynamics of butorphanol following intravenous administration to the horse. Butorphanol is a narcotic analgesic commonly used in horses. Currently, any detectable concentration of butorphanol in biological samples collected from performance horses is considered a violation. The primary goal of the study reported here was to update the pharmacokinetics of butorphanol following intravenous administration, utilizing a highly sensitive liquid chromatography-mass spectrometry (LC-MS) assay that is currently employed in many drug-testing laboratories. An additional objective was to characterize behavioral and cardiac effects following administration of butorphanol. Ten exer...
Regulatory control of glycopyrrolate in performance horses using validated UHPLC/MS-MS methods. We describe a validated, rapid, sensitive, and specific UHPLC-MS/MS method to detect and quantify glycopyrrolate in 0.5 mL of horse urine. Further, we investigated the elimination of glycopyrrolate in urine after both intravenous and oral administration of clinically relevant doses to Thoroughbred horses. Quantification was performed by weighted, linear regression analysis using a deuterated analogue of glycopyrrolate as internal standard (IS). The method was characterized by a linear range of 5-2500 pg/mL, a lower limit of quantification of 5 pg/mL and a limit of detection of 1 pg/mL. The int...
Quantitative response of volumetric variables measured by a new ultrasound dilution method in a juvenile model of hemorrhagic shock and resuscitation. New volumetric variables of preload, such as total end-diastolic volume index (TEDVI) and active circulation volume index (ACVI) and central blood volume index (CBVI), may represent good indicators of preload and predictors of fluid responsiveness. During acute changes of intravascular volume these variables would allow a more accurate intervention. Objective: The aim of the present study was to investigate the changes in TEDVI, ACVI, CBVI in a juvenile model of hemorrhagic shock and resuscitation. Methods: Twelve anaesthetized ponies (3-8 months of age) were studied at normovolaemia (BASE), a...
Pharmacokinetic profile and pharmacodynamic effects of romifidine hydrochloride in the horse. Romifidine HCl (romifidine) is an α(2)-agonist commonly used in horses. This study was undertaken to investigate the pharmacokinetics (PK) of romifidine following intravenous (i.v.) administration and describe the relationship between PK parameters and simultaneously recorded pharmacodynamic (PD) parameters. Romifidine (80 μg/kg) was administered by i.v. infusion over 2 min to six adult Thoroughbred horses, and plasma samples were collected and analyzed using liquid chromatography-mass spectrometry. Limit of quantification was <0.1 ng/mL. PD parameters and arterial blood gases were measur...
Rapid screening of anabolic steroids in horse urine with ultra-high-performance liquid chromatography/tandem mass spectrometry after chemical derivatisation. Liquid chromatography/mass spectrometry (LC/MS) has been successfully applied to the detection of anabolic steroids in biological samples. However, the sensitive detection of saturated hydroxysteroids, such as androstanediols, by electrospray ionisation (ESI) is difficult because of their poor ability to ionise. In view of this, chemical derivatisation has been used to enhance the detection sensitivity of hydroxysteroids by LC/MS. This paper describes the development of a sensitive ultra-high-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) method for the screening of a...
Pharmacokinetic assessment of ketanserin in the horse. The purpose of this study was to determine the pharmacokinetics (PK) of the 5-HT(2A) receptor antagonist ketanserin in healthy adult horses, and to develop a computational model that could be used to optimize dosing. Plasma concentrations of ketanserin were determined using liquid chromatography with mass spectrometry after single and multiple intravenous administration in the horse. A two-compartment linear pharmacokinetic model described the plasma concentration-time profile of ketanserin after single and multiple doses in healthy horses; the terminal half-life was 11.5 h; steady-state volum...
Validation of a liquid chromatography-tandem mass spectrometry method for quantification of glycopyrrolate in horse plasma. A rapid, sensitive, and specific ultra-high-performance liquid chromatography with heated electrospray ionization-tandem mass spectrometry (UHPLC-HESI-MS-MS) method to detect and quantify glycopyrrolate in horse plasma has been developed and validated. We also determined glycopyrrolate in plasma after oral and intravenous administration of clinically relevant doses to Thoroughbred horses. Calibration was accomplished by weighted, linear regression analysis using a deuterated analogue of glycopyrrolate as internal standard (IS). Glycopyrrolate (GLY) and the IS (GLY-d(3)) were isolated from plas...
Detection of singly- and doubly-charged quaternary ammonium drugs in equine urine by liquid chromatography/tandem mass spectrometry. Quaternary ammonium drugs (QADs) are anticholinergic agents some of which are known to have been abused or misused in equine sports. A recent review of literature shows that the screening methods reported thus far for QADs mainly cover singly-charged QADs. Doubly-charged QADs are extremely polar substances which are difficult to be extracted and poorly retained on reversed-phase columns. It would be ideal if a comprehensive method can be developed which can detect both singly- and doubly-charged QADs. This paper describes an efficient liquid chromatography/tandem mass spectrometry (LC/MS/MS) m...
Ex vivo spontaneous generation of 19-norandrostenedione and nandrolone detected in equine plasma and urine. 19-Norandrostenedione (NAED) and nandrolone are anabolic-androgenic steroids (AASs). Nandrolone was regarded solely as a synthetic AAS until the 1980s when trace concentrations of apparently endogenous nandrolone were detected in urine samples obtained from intact male horses (stallions). Since then, its endogenous origin has been reported in boars and bulls; endogenous NAED and nandrolone have been identified in plasma and urine samples collected from stallions. More recently, however, it was suggested that NAED and nandrolone detected in urine samples from stallions are primarily artifacts d...
Development and validation of a method for Cd, Pb and As analysis in bovine, equine and poultry liver by inductively coupled plasma mass spectrometry. A method for the determination of As, Cd and Pb in bovine, equine and poultry liver by ICP-MS was developed and validated. Samples were digested in a microwave oven using a 10% HNO(3) solution. A set of experiments was made according to a central composite design (CCD) for optimisation of the plasma argon flow, nebuliser argon flow and radiofrequency power applied to the plasma. During the validation, Rh and Ru were evaluated as internal standards and, after validation, the best was Rh for Pb and Cd analysis, but for As better results were obtained without an internal standard. The method allo...
Quantitative analysis of a quaternary ammonium drug: ipratropium bromide by LC/ESI-MS(n) in horse plasma and urine. A quantitative method, using LC/ESI-MS(n) with a quadrupole linear ion trap mass analyzer, has been developed for the analysis of ipratropium cation in horse plasma and urine. The method applies solid-phase extraction with WCX cartridges for plasma and MM2 cartridges for urine, prior to analysis by LC/ESI-MS(n). The efficiency of extraction combined with the sensitivity and the selectivity of MS(n) allows for the quantification of ipratropium cation at picogram per milliliter levels. The analytical capabilities of the method have been successfully checked by the quantitative analysis of ipratr...
