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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.
Correlation of product ion profiles with molecular structures of androgenic and anabolic steroids in ESI MS/MS. Androgenic and anabolic steroids (AASs) are a class of chemical substances closely related to testosterone in molecular structure. They can be abused to enhance performances in human and equine athletes, and are banned by the sports authorities. To assist with method development for doping analyses of AASs, investigations were conducted to correlate their product ion profiles with the molecular structures. Although very similar in chemical structure, AASs generated noticeably different product ion profiles from collision-induced dissociation (CID). On the basis of both outlines of the product ...
The use of a simple backflush technology to improve sample throughput and system robustness in routine gas chromatography tandem mass spectrometry analysis of doping control samples. A simple, low cost system for the backflushing of capillary gas chromatography (GC) columns has been investigated and integrated into a method for the detection of anabolic steroids in equine urine. The modification to the method was simple to make and quick to setup and optimize. The use of backflushing technology was found to offer significant benefits in terms of sample throughput and improved system robustness.
Electrophoretically mediated microanalysis for the evaluation of interspecies variation in cholinesterase metabolism. This study describes an electrophoretically mediated microanalysis method, suitable for the preclinical evaluation of the hydrolysis of ester drugs by the serum of different animals and for further characterization of human-animal correlation. Dog, cat, cow, horse, sheep, rat and human serum were diluted (25%) in the appropriate buffer and replaced the enzyme solution usually used in electrophoretically mediated microanalysis methods for the study of enzyme kinetics. They were then compared in terms of the ability to hydrolyze acetylthiocholine and butyrylthiocholine (0.25 mM) by in-capillary ...
Membrane permeability parameters for freezing of stallion sperm as determined by Fourier transform infrared spectroscopy. Cellular membranes are one of the primary sites of injury during freezing and thawing for cryopreservation of cells. Fourier transform infrared spectroscopy (FTIR) was used to monitor membrane phase behavior and ice formation during freezing of stallion sperm. At high subzero ice nucleation temperatures which result in cellular dehydration, membranes undergo a profound transition to a highly ordered gel phase. By contrast, low subzero nucleation temperatures, that are likely to result in intracellular ice formation, leave membrane lipids in a relatively hydrated fluid state. The extent of free...
Quantitative analysis of lignocaine and metabolites in equine urine and plasma by liquid chromatography-tandem mass spectrometry. In this paper, a method for the sensitive and reproducible analysis of lignocaine and its four principal metabolites, monoethylxylidide (MEGX), glycylxylidide (GX), 3-hydroxylignocaine (3-HO-LIG), 4-hydroxylignocaine (4-HO-LIG) in equine urine and plasma samples is presented. The method uses liquid chromatography coupled to tandem mass spectrometry operating in electrospray ionisation positive ion mode (+ESI) via multiple reaction monitoring (MRM). Sample preparation involved solid-phase extraction using a mixed-mode phase. The internal standard adopted was lignocaine-d(10). Lignocaine and its...
Evaluation of the Limulus amebocyte lysate and recombinant factor C assays for assessment of airborne endotoxin. As a potent inflammatory agent, endotoxin is a key analyte of interest for studies of lung ailments in domestic environments and occupational settings with organic dust. A relatively unexplored advance in endotoxin exposure assessment is the use of recombinant factor C (rFC) from the Limulus pathway in a fluorometric assay. In this study, we compared airborne endotoxin concentrations in laboratory- and field-collected parallel air samples using the kinetic Limulus amebocyte lysate (LAL) assay and the rFC assay. Air sampling was performed using paired Institute of Occupational Medicine (IOM) sa...
Identification of phosphorylation sites of equine beta-casein isoforms. Equine beta-casein is phosphorylated at variable degrees and isoforms carrying 3 to 7 phosphate groups (3P-7P) have been found in milk, but the phosphorylated amino acid residues of each isoform are not yet identified. In the present work, the different phosphorylation variants were first isolated by ion-exchange chromatography and then hydrolysed by trypsin to generate caseinophosphopeptides (CPPs), each containing all the potential phosphorylation sites. The equine CPPs were prepared by metal oxide affinity chromatography, a method based on the affinity of phosphate groups towards titanium d...
Determination of (13)C/(12)C ratios of urinary excreted boldenone and its main metabolite 5beta-androst-1-en-17beta-ol-3-one. Boldenone (androsta-1,4-dien-17beta-ol-3-one, Bo) is an anabolic steroid known to have been used in cattle breeding or equine sport as a doping agent for many years. Although not clinically approved for human application, Bo or its main metabolite 5beta-androst-1-en-17beta-ol-3-one (BM1) were detected in several doping control samples. For more than 15 years the possibility of endogenous Bo production in human beings has been discussed. This is a challenging issue for doping control laboratories as Bo belongs to the list of prohibited substances of the World Anti-Doping Agency and therefore th...
Evaluation of a portable clinical analyzer for the determination of blood gas partial pressures, electrolyte concentrations, and hematocrit in venous blood samples collected from cattle, horses, and sheep. To compare results reported for blood gas partial pressures, electrolyte concentrations, and Hct in venous blood samples collected from cattle, horses, and sheep and analyzed by use of a portable clinical analyzer (PCA) and reference analyzer (RA). Methods: Clinically normal animals (24 cattle, 22 horses, and 22 sheep). Methods: pH; Pco(2); Po(2); total carbon dioxide concentration; oxygen saturation; base excess; concentrations of HCO(3)(-), Na(+), K(+), and ionized calcium; Hct; and hemoglobin concentration were determined with a PCA. Results were compared with those obtained for the same bl...
Determination of non-steroidal anti-inflammatory drugs residues in animal muscles by liquid chromatography-tandem mass spectrometry. A confirmatory method for the determination of residues of nine non-steroidal anti-inflammatory drugs and one metabolite in animal muscles has been developed. After enzymatic hydrolysis samples were extracted with acetonitrile and cleaned up using alumina and C(18) SPE cartridges. Liquid chromatography-tandem mass spectrometry was used for the separation and determination of analytes. The method was validated in bovine muscles, according to the Commission Decision 2002/657/EC criteria. Applicability of the method in the analysis of swine, horse and chicken muscles was checked by precision and ...
Comparative in vitro metabolism of the ‘designer’ steroid estra-4,9-diene-3,17-dione between the equine, canine and human: identification of target metabolites for use in sports doping control. Effective detection of the abuse of androgenic-anabolic steroids in human and animal sports often requires knowledge of the drug's metabolism in order to target appropriate urinary metabolites. 'Designer' steroids are problematic since it is difficult to obtain ethical approval for in vivo metabolism studies due to a lack of a toxicological profile. In this study, the in vitro metabolism of estra-4,9-diene-3,17-dione is reported for the first time. This is also the first study comparing the metabolism of a designer steroid in the three major species subject to sport's doping control; namely th...
Direct injection horse-urine analysis for the quantification and confirmation of threshold substances for doping control. IV. Determination of 3-methoxytyramine by hydrophilic interaction liquid chromatography/quadrupole time-of-flight mass spectrometry. Levodopa and dopamine have been abused as performance-altering substances in horse racing. Urinary 3-methoxytyramine is used as an indicator of dopaminergic manipulation resulting from dopamine or levodopa administration and is prohibited with a urinary threshold of 4 microg mL(-1) (free and conjugated). A simple liquid chromatographic (LC)/mass spectrometric (MS) (LCMS) method was developed and validated for the quantification and identification of 3-methoxytyramine in equine urine. Sample preparation involved enzymatic hydrolysis and protein precipitation. Hydrophilic interaction liquid chro...
Depletion of urinary zilpaterol residues in horses as measured by ELISA and UPLC-MS/MS. Three horses were dosed with dietary zilpaterol and the urine concentrations measured from withdrawal day 0 to withdrawal day 21. The analyses were carried out using both enzyme-linked immunosorbent assay (ELISA) and an ultraperformance liquid chromatography with triple-quadrupole-tandem mass spectrometric detection (UPLC-MS/MS). The UPLC-MS/MS method was developed to provide rapid analysis with positive analyte identification by following three product ions and computing the two independent ion ratios. When urinary zilpaterol concentrations were between 0.2 and 2 ng/mL, the ELISA had interday...
Veterinary medicines and competition animals: the question of medication versus doping control. In racing and other equine sports, it is possible to increase artificially both the physical capability and the presence of a competitive instinct, using drugs, such as anabolic steroids and agents stimulating the central nervous system. The word doping describes this illegitimate use of drugs and the primary motivation of an equine anti-doping policy is to prevent the use of these substances. However, an anti-doping policy must not impede the use of legitimate veterinary medications and most regulatory bodies in the world now distinguish the control of illicit substances (doping control) from...
Alternative instrumentation for the analysis of total carbon dioxide (TCO(2)) in equine plasma. The analysis of total carbon dioxide (TCO(2)) in equine plasma is conventionally done in Australia and elsewhere using Beckman Synchron EL-ISE(R) analysers. This instrument is no longer being manufactured and has not been supported by the supplier since December 2008. For testing for TCO(2) to continue, it is necessary to evaluate and commission alternative instrumentation. In this paper, we compare the Beckman Synchron EL-ISE, the Beckman Synchron CX5, the Beckman UniCel DxC 600 and the Randox Daytona instruments. Results indicate that all four instruments perform in accordance with the manuf...
Doping control analysis of recombinant human erythropoietin, darbepoetin alfa and methoxy polyethylene glycol-epoetin beta in equine plasma by nano-liquid chromatography-tandem mass spectrometry. Recombinant human erythropoietin (rhEPO), darbepoetin alfa (DPO) and methoxy polyethylene glycol-epoetin beta (PEG-EPO) are synthetic analogues of the endogenous hormone erythropoietin (EPO). These erythropoiesis-stimulating agents have the ability to stimulate the production of red blood cells and are commercially available for the treatment of anaemia in humans. These drugs are understood to have performance-enhancing effects on human athletes due to their stimulation of red blood cell production, thereby improving delivery of oxygen to the muscle tissues. Although their effect on horses has...
Evaluation of an in-clinic Serum Amyloid A (SAA) assay and assessment of the effects of storage on SAA samples. An in-clinic assay for equine serum amyloid A (SAA) analysis, Equinostic EVA1, was evaluated for use in a clinical setting. Stability of SAA in serum samples was determined. Methods: Intra- and inter- assay variation of the in-clinic method was determined. The in-clinic method (EVA1) results were compared to a reference method (Eiken LZ SAA) with 62 patient samples. For samples with SAA concentrations within the assay range of EVA1 (10-270 mg/L), differences between the methods were evaluated in a difference plot. Linearity under dilution was evaluated in two samples. Stability of SAA in three...
Screening of drugs in equine plasma using automated on-line solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry. A rapid liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed for the simultaneous screening of 19 drugs of different classes in equine plasma using automated on-line solid-phase extraction (SPE) coupled with a triple quadrupole mass spectrometer. Plasma samples were first protein precipitated using acetonitrile. After centrifugation, the supernatant was directly injected into the on-line SPE system and analysed by a triple quadrupole LC-MS-MS in positive electrospray ionisation (+ESI) mode with selected reaction monitoring (SRM) scan function. On-line extraction and c...
Automated counting of nucleated cells in equine synovial fluid without and with hyaluronidase pretreatment. Microscopy is usually used to obtain manual total and differential cell counts in equine synovial fluid. A faster, more precise method is desirable. Objective: The objectives were to compare an automated impedance method with a manual method for obtaining total and differential cell counts in equine synovial fluid and to evaluate the effect of pretreatment with hyaluronidase on automated results. Methods: Synovial fluid samples (n=48) were collected into EDTA and analyzed within 48 hours. Automated total and differential cell counts were evaluated using a Medonic CA620-VET hematology analyzer ...
Some aspects of doping and medication control in equine sports. This chapter reviews drug and medication control in equestrian sports and addresses the rules of racing, the technological advances that have been made in drug detection and the importance of metabolism studies in the development of effective drug surveillance programmes. Typical approaches to screening and confirmatory analysis are discussed, as are the quality processes that underpin these procedures. The chapter also addresses four specific topics relevant to equestrian sports: substances controlled by threshold values, the approach adopted recently by European racing authorities to control...
Evaluation of shape variability of stallion sperm heads by means of image analysis and Fourier descriptors. This study quantified and evaluated the variability of sperm head shape for 10 different stallions. Sperm head shape characteristics including sperm head length to width ratio, position of the center of gravity, curvature, and degree of roundness were assessed and analysed from images using elliptic Fourier descriptors and inverse Fourier transformation. The first four principal components accounted for 88.46-92.33% of the total variance and provided a good summary of the overall data. In the case of the ejaculate with defective sperm heads the components accounted for 97.35-98.21% of variatio...
Probing the calcium and sodium local environment in bones and teeth using multinuclear solid state NMR and X-ray absorption spectroscopy. Despite the numerous studies of bone mineral, there are still many questions regarding the exact structure and composition of the mineral phase, and how the mineral crystals become organised with respect to each other and the collagen matrix. Bone mineral is commonly formulated as hydroxyapatite, albeit with numerous substitutions, and has previously been studied by (31)P and (1)H NMR, which has given considerable insight into the complexity of the mineral structure. However, to date, there has been no report of an NMR investigation of the other major component of bone mineral, calcium, nor of...
Detection of urine and blood clenbuterol following short-term oral administration in the horse. The pharmacokinetics of clenbuterol in equine urine and blood was investigated. Methods: Urine and blood samples were collected following 3-day multiple oral administrations. The samples were examined using enzyme-linked immunosorbent assay and further confirmed by solid phase extraction and capillary electrophoresis. Results: Urinary clenbuterol was detectable until day 14 after the last dose. The urinary excretion of clenbuterol was characterized by a biphasic pattern. The half-lives of the bi-exponential elimination (t(1/2alpha) and t(1/2beta)) for urinary clenbuterol were about 12.1 and 48...
Direct injection horse urine analysis for the quantification and identification of threshold substances for doping control. III. Determination of salicylic acid by liquid chromatography/quadrupole time-of-flight mass spectrometry. In equine sport, salicylic acid is prohibited with a threshold level of 750 microg mL(-1) in urine; hence, doping control laboratories have to establish quantitative and qualitative methods for its determination. A simple and rapid liquid chromatographic/mass spectrometric method was developed and validated for the quantification and identification of salicylic acid. Urine samples after 900-fold dilution and addition of the internal standard (4-methylsalicylic acid) were directly injected to the liquid chromatography/quadrupole time-of-flight mass spectrometry system. Electrospray ionization i...
CE provides evidence of the stereoselective hydroxylation of norketamine in equines. CE with multiple isomer sulfated-CD as selector was used for the simultaneous analysis of the stereoisomers of ketamine, norketamine, 5,6-dehydronorketamine and hydroxylated metabolites of norketamine in liquid/liquid extracts of (i) in vitro incubations with ketamine or norketamine and equine liver microsomes and (ii) plasma and urine of ponies receiving a target-controlled infusion of ketamine under isoflurane anesthesia. Hydroxynorketamine metabolites with the hydroxy group at the cyclohexanone ring could be shown to be formed stereoselectively both in vitro and in vivo. Due to the lack of ...
Development of a metabonomic approach based on LC-ESI-HRMS measurements for profiling of metabolic changes induced by recombinant equine growth hormone in horse urine. Despite the worldwide existing regulation banning the use of the recombinant equine growth hormone (reGH) as growth promoter, it is suspected to be used in horseracing to improve performances. Various analytical methods previously developed to screen for its misuse have encountered some limitations in terms of detection timeframes, in particular during the first days following reGH administration. A novel strategy involving the characterization of global metabolomic fingerprints in urine samples of non-treated and reGH-treated horses by liquid chromatography-electrospray-high-resolution mass s...
Modern techniques for the determination of anabolic-androgenic steroid doping in the horse. Control of the use of performance-affecting substances in the horse is critical to the integrity of a wide range of equine sports, with major implications for both animal welfare and revenue streams. One class of medications enjoying particular public notoriety is the anabolic-androgenic steroid group, as highlighted by the recent 'Big Brown' affair and Congressional inquiries into the use of steroids in professional sports, including horse racing, in the USA. This review examines the latest developments pertaining to the analytical detection of these substances in equine biological samples an...
Ultra-performance liquid chromatography/tandem mass spectrometry in high-throughput detection, quantification and confirmation of anabolic steroids in equine plasma. An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method for fast-throughput analysis of eight anabolic and androgenic steroids (AAS) in equine plasma is reported. Analytes were recovered by liquid-liquid extraction using methyl tert-butyl ether, separated on a 1.9 microm C(18) reversed-phase column, and analyzed in positive electrospray ionization mode on a triple quadrupole mass spectrometer with selected reaction monitoring (SRM) and full product ion scans. Two SRM ion transitions were monitored for each AAS during screening to obtain highly selective screenin...
