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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.
Metabolism and elimination of the catechol-o-methyltransferase inhibitor tolcapone in the horse. The metabolism of the masking agent tolcapone in the horse has been investigated. This substance was found to have undergone various chemical transformations that produced a large variety of phase I metabolites, as well as glucuronide and sulfate conjugation. Confirmation of the presence of tolcapone and the 3-O-methylated metabolite in the blood samples collected up to 240 minutes and in urine obtained up to 24 hours, was successfully conducted using both gas chromatography- and liquid chromatography-tandem mass spectrometry techniques. The 3-O-methyl tolcapone is the better marker to use i...
High-throughput doping control analysis of 28 amphetamine-type stimulants in equine plasma using hydrophilic interaction liquid chromatography-tandem mass spectrometry. A hydrophilic interaction liquid chromatography-tandem mass spectrometry method (HILIC-MS/MS) was developed for the simultaneous determination of 28 amphetamine-type stimulants (ATSs) in equine plasma for doping control analysis. In this method, stimulants were recovered from equine plasma by liquid-liquid extraction (LLE) at pH 9.5 using methyl tert-butyl ether and detected on a Thermo Finnigan triple quadrupole mass spectrometer operating in positive-ion mode electrospray ionization. All stimulants were eluted within 7 minutes and baseline separation was achieved for isomeric and isobaric ...
Validation of an ELISA for detection of neutrophil gelatinase-associated lipocalin (NGAL) in equine serum. Neutrophil gelatinase-associated lipocalin (NGAL) has been shown to be a useful marker of kidney injury in people and dogs, but has not been described in horses. Objective: The aim of the study was to validate a commercially available porcine-specific ELISA to measure serum concentrations of equine NGAL. Methods: Intra- and interassay imprecisions were evaluated by multiple measurements on equine serum pools. Assay inaccuracy was determined by the linearity under dilution. Overlapping performance was assessed by measuring NGAL concentrations in horses with normal and elevated serum creatinine ...
Detection of phosphorothioated (PS) oligonucleotides in horse plasma using a product ion (m/z 94.9362) derived from the PS moiety for doping control. Clinical research on gene therapy has advanced the field of veterinary medicine, and gene doping, which is the illegal use of gene therapy, has become a major concern in horseracing. Since the International Federation of Horseracing Authorities defined the administration of oligonucleotides and its analogues as a genetic therapy in 2017, the development of therapeutic nucleotide-detection techniques has become an urgent need. Most currently marketed and developed oligonucleotide therapeutics for humans consist of modified nucleotides to increase stability, and phosphorothioate (PS) modificatio...
Concept of sample-specific correction of immunoassay results for precise and accurate IgG quantification in horse plasma. The hyperimmune horse plasma (HHP), prepared through active immunisation of horses with an antigen of interest, is the most common starting material for antitoxin (animal antibody-based therapeutics) production. Precise IgG quantification in plasma is a prerequisite for accurate estimation of the purification process efficiency. Although immunoglobulins from HHP have been purified for over a century, there is still no in vitro method for precise and accurate determination of IgG content in HHP. For this reason, the purification process efficiency has been assessed by antibody activity measurem...
Evaluation of an in-clinic dry chemistry analyzer for canine, equine, and feline plasma samples. Method validation studies characterize the performance of new laboratory methods relative to established methods using quality guidelines in order to define the new method's performance characteristics and to identify differences that could influence data interpretation. We investigated the performance of an in-clinic dry chemistry analyzer (Catalyst One, IDEXX) for measuring 19 routine plasma biochemistry analytes in dogs, cats, and horses. We analyzed 2 levels of quality control material (QCM) in duplicate twice daily for 5 d to determine the coefficient of variation (CV), percent bias, ob...
Biological variation of routine haematology and biochemistry measurands in the horse. Clinical pathology results are typically interpreted by referring to population-based reference intervals. The use of individualised (subject-based) reference intervals is more appropriate for measurands with a high degree of variation between individuals. Objective: To determine the biological variation of routinely analysed equine haematology and biochemistry measurands and calculate indices of individuality and reference change values which enable production of individualised reference intervals, in a group of healthy, privately owned horses. Methods: In a prospective cohort study, thirty-n...
Separation and identification of the epimeric doping agents – Dexamethasone and betamethasone in equine urine and plasma: A reversed phase chiral chromatographic approach. Chirality is one of the most important considerations when controlling doping. The epimeric corticosteroids dexamethasone and betamethasone are significantly potent and long-acting, and they are highly abused in equestrian sports. The scope of this study was to develop a simple and reliable analytical method for simultaneously identifying and separating regularly abused co-eluting corticosteroids in equine urine and plasma. In this paper, we present a simple and rapid method for the chiral separation and identification of epimeric mixtures of dexamethasone and betamethasone using a Thermo Q Ex...
A rapid and sensitive method for the quantitative analysis of ibuprofen and its metabolites in equine urine samples by gas chromatography with tandem mass spectrometry. Ibuprofen is widely used in human and veterinary medicine for the treatment of chronic pain as well as rheumatic and musculoskeletal disorders. However, the analgesic and anti-inflammatory properties of Ibuprofen have contributed to frequent drug abuse in equestrian sports. A sensitive and rapid gas chromatography with tandem mass spectrometry based method with a simple liquid-liquid extraction and derivatization requiring 200 μL volume of sample and 2 mL of extraction solvent for the simultaneous determination of ibuprofen and its metabolites was developed. The proposed procedure was optim...
A mini-STR typing system for degraded equine DNA. Degraded biological samples are a challenge for testing laboratories. Genotyping success can be improved through the use of mini-STRs, by which primers are placed adjacent to the repeat motifs to reduce amplicon size. Here, we present a genetic profiling system comprising 13 autosomal and one X-linked dinucleotide-repeat markers and the SRY gene based on the internationally accepted equine parentage panel. The markers are divided into two panels with all alleles falling at or below 182 bp. The application of this method significantly increases the ability to profile difficult samples and to p...
Comparison of Enterprise Point-of-Care and Nova Biomedical Critical Care Xpress analyzers for determination of arterial pH, blood gas, and electrolyte values in canine and equine blood. Point-of-care analyzers can provide a rapid turnaround time for critical blood test results. Agreement between the Enterprise Point-of-Care (EPOC) and bench-top laboratory analyzers is important to determine the clinical reliability of the EPOC. Objective: The aim of the study was (1) to evaluate the precision (repeatability) of blood gas values measured by the EPOC and (2) to determine the level of agreement between the EPOC and Nova Critical Care Express (Nova CCX) for the assessment of arterial pH, blood gases, and electrolyte variables in canine and equine blood. Methods: Arterial blood sa...
Detection of equine atypical myopathy-associated hypoglycin A in plant material: Optimisation and validation of a novel LC-MS based method without derivatisation. Hypoglycin A (HGA) toxicity, following ingestion of material from certain plants, is linked to an acquired multiple acyl-CoA dehydrogenase deficiency known as atypical myopathy, a commonly fatal form of equine rhabdomyolysis seen worldwide. Whilst some plants are known to contain this toxin, little is known about its function or the mechanisms that lead to varied HGA concentrations between plants. Consequently, reliable tools to detect this amino acid in plant samples are needed. Analytical methods for HGA detection have previously been validated for the food industry, however, these technique...
Post-exercise cardiac troponin I release and clearance in normal Standardbred racehorses. There are currently no studies detailing cardiac troponin I (cTnI) release in normal horses post-exercise using an analytically validated assay. These data are essential for selecting appropriate sampling times in equine athletes with suspected myocardial injury. Objective: To plot the magnitude and time course of cTnI release after maximal effort, using validated cTnI assays. Methods: Descriptive longitudinal study. Methods: Five clinically normal Standardbred racehorses in race training were included in the study. Horses were exercised in harness at near-race intensity. Blood samples were ta...
Monitoring dehydroepiandrosterone (DHEA) in the urine of Thoroughbred geldings for doping control purposes. The use of testosterone and its pro-drugs, such as dehydroepiandrosterone (DHEA), is currently regulated in horseracing by the application of international testosterone thresholds. However, additional steroidomic approaches, such as steroid ratios, to distinguish overall adrenal stimulation from drug administrations and an equine biological passport for longitudinal steroid profiling of individual animals could be advantageous in equine doping testing. Thus, DHEA concentrations and related ratios (testosterone [T] to DHEA and DHEA to epitestosterone [E]) were assessed in the reference populati...
L- and D-threo ethylphenidate concentrations, pharmacokinetics, and pharmacodynamics in horses. Ethylphenidate is a psychostimulant and analog of the commonly prescribed compound, methylphenidate (Ritalin®). There are a limited number of studies describing the disposition and pharmacologic/toxicological effects of ethylphenidate in any species. The abuse potential in equine athletes along with the limited data available regarding administration in horses necessitates further study. The objectives of the current study were to describe drug concentrations, develop an analytical method that could be used to regulate its use, and describe the pharmacodynamic effects of ethylphenidate in hor...
Detection of seventy-two anabolic and androgenic steroids and/or their esters in horse hair using ultra-high performance liquid chromatography-high resolution mass spectrometry in multiplexed targeted MS2 mode and gas chromatography-tandem mass spectrometry. Anabolic and androgenic steroids (AAS) are banned substances in both human and equine sports. They are often administered intramuscularly to horses in esterified forms for the purpose of extending their time of action. The authors' laboratory has previously reported an UHPLC/HRMS method using quadrupole-Orbitrap mass spectrometer in full scan and parallel reaction monitoring (PRM) mode for the detection of 48 AAS and/or their esters in horse hair. However, two injections were required due to the long duty cycle time. In this paper, an UHPLC/HRMS method using multiplexed targeted MS mode was de...
Simultaneous identification of bovine and equine DNA in milks and dairy products inferred from triplex TaqMan real-time PCR technique. Koumiss is a popular dairy product in many lands, traditionally prepared from mare milk with spontaneous fermentation. Mare milk and its fermented derivates are more expensive than cow milk and its fermented derivates, and the possibility exists for producers and dealers to adulterate equine products with bovine items. In this work, we described the development of a triplex real-time PCR based on species-specific TaqMan probes for identification of bovine and equine DNA in milks and dairy products. In addition, a novel designed endogenous control was simultaneously amplified to eliminate possi...
Development of an immunochromatographic assay for the β-adrenergic agonist feed additive zilpaterol. Zilpaterol is a β-adrenergic agonist feed additive approved in the United States to increase weight gain and improve feed efficiency of cattle. A zilpaterol immunochromatographic assay was developed as an economical and user-friendly rapid detection method for zilpaterol and validated using urine and tissue samples derived from animal studies. The assay sensitivity was 1.7-23.2 ng g or mL across a variety of feed and animal matrices and did not cross-react with clenbuterol or ractopamine. No sample pre-treatment of cattle and sheep urine was needed, but horse urine and feed required dilution;...
Doping control analysis of 121 prohibited substances in equine hair by liquid chromatography-tandem mass spectrometry. Equine hair is becoming an increasingly popular biological matrix for doping control of horse sports; one of the reasons for this is the significantly longer detection window hair can offer. Hair analysis opens up the opportunity for longitudinal monitoring of drug exposure which would otherwise not be possible with the more traditional and common biological matrices, such as urine and blood. As such, there is a need for more multi-target screening methods covering a broad range of prohibited substances in equine hair at the required sensitivities for equine doping control. This paper describe...
The content of essential and toxic elements in the hair of the mane of the trotter horses depending on their speed. A study on the Russian trotting breeds was conducted to assess the impact of horses' sporting results and the degree of accumulation of chemical elements in the hair. In the first phase of the research, the elemental composition of the mane hair of trotter horses (n = 215) was studied. Based on these studies, percentile intervals for the distribution of concentrations of chemical elements in the hair have been established, and the values of 25 and 75 percentile adopted as a "physiological standard" have been defined. In the second stage of the research into clinically healthy Russian trott...
A comparison of methods for estimating substitution rates from ancient DNA sequence data. Phylogenetic analysis of DNA from modern and ancient samples allows the reconstruction of important demographic and evolutionary processes. A critical component of these analyses is the estimation of evolutionary rates, which can be calibrated using information about the ages of the samples. However, the reliability of these rate estimates can be negatively affected by among-lineage rate variation and non-random sampling. Using a simulation study, we compared the performance of three phylogenetic methods for inferring evolutionary rates from time-structured data sets: regression of root-to-tip...
Semi-quantitative methods yield greater inter- and intraobserver agreement than subjective methods for interpreting 99m technetium-hydroxymethylene-diphosphonate uptake in equine thoracic processi spinosi. Scintigraphy is a standard diagnostic method for evaluating horses with back pain due to suspected thoracic processus spinosus pathology. Lesion detection is based on subjective or semi-quantitative assessments of increased uptake. This retrospective, analytical study is aimed to compare semi-quantitative and subjective methods in the evaluation of scintigraphic images of the processi spinosi in the equine thoracic spine. Scintigraphic images of 20 Warmblood horses, presented for assessment of orthopedic conditions between 2014 and 2016, were included in the study. Randomized, blinded image ev...
Determination of vitamin B12 in equine urine by liquid chromatography – inductively coupled plasma – mass spectrometry. Regulating authorities in the racing industry have restricted the administration of potentially performance enhancing cobalt salts to horses. There are severe penalties for trainers presenting horses with elevated urine cobalt concentrations, and compliance is ensured via analysis of total urinary cobalt at thresholds of 100 μg/L. When cobalt is present as part of the cobalamin molecule it is not considered performance enhancing. This paper demonstrates that a horse can excrete a significant proportion of a commercially available vitamin B12 injection in urine without metabolic modification...
Development and validation of an ultrahigh performance liquid chromatography-high resolution tandem mass spectrometry assay for nine toxic alkaloids from endophyte-infected pasture grasses in horse serum. Endophyte fungi (e.g. Epichloë ssp. and Neotyphodium ssp.) in symbiosis with pasture grasses (e.g. Festuca arundinacaea and Lolium perenne) can produce toxic alkaloids, which are suspected to be involved in equine diseases such as fescue toxicosis, ryegrass staggers, and equine fescue oedema. The aim of this study was, therefore, to develop and validate a quantification method for these and related alkaloids: ergocristine, ergocryptine, ergotamine, ergovaline, lolitrem B, lysergic acid, N-acetylloline, N-formylloline, peramine, and paxilline in horse serum. Horse serum samples (1.5mL) were wo...
Validation of a paraoxon-based method for measurement of paraoxonase (PON-1) activity and establishment of RIs in horses. Paraoxonase-1 (PON-1) is an antioxidant compound that is considered a negative acute phase protein. No information on the analytic performance of the paraoxon method for measuring PON-1 in horse serum is available. Objective: The aim of this study was to validate a paraoxon-based method to measure PON-1 in horses and to establish RIs in healthy horses and foals. Methods: Horses and foals classified as healthy after physical examination and routine biochemistry were used in the study. Serum PON-1 activity was measured with an automated spectrophotometer and an enzymatic method validated in othe...
Development and validation of rt-qpcr for vesicular stomatitis virus detection (Alagoas vesiculovirus). Vesicular stomatitis is an infectious disease that occurs mainly in countries of the Western Hemisphere and affects cattle, swine and horses. The clinical symptoms in cattle and swine are similar to foot-and-mouth disease and include vesicular ulceration of the tongue and mouth. The disease requires a rapid and accurate differential diagnosis, aiming for immediate implementation of control measures. The objective of the present study was to develop and perform validation tests of multiplex RT-qPCR(s) for the detection of RNA from Alagoas vesiculovirus, considering the parameters of sensitivity...
Detection and pharmacokinetics of grapiprant following oral administration to exercised Thoroughbred horses. Traditional therapeutic options for the treatment of lameness associated with inflammation in performance horses include administration of cyclooxygenase enzyme inhibiting non-steroidal anti-inflammatory drugs (NSAID). As long-term use of these drugs can adversely impact the health of the horse, anti-inflammatories with a more favorable safety profile are warranted. Grapiprant is a newly approved non-cyclooxygenase inhibiting NSAID that has demonstrated efficacy and safety in other species and which may be a valuable alternative to traditional NSAIDs used in the horse. The objectives of the cu...
Analysis of loxoprofen in tablets, patches, and equine urine as tert-butyldimethylsilyl derivative by gas chromatography-mass spectrometry. Loxoprofen is a non-steroidal anti-inflammatory drug of the 2-arylpropionic acid type, which has used to treat musculoskeletal disorders in the horse racing industry. However, it has also used illicitly to mask clinical signs of inflammation and pain in racehorses. Thus, its accurate analysis has become an important issue in horse doping laboratories. In this study, an analytical method of loxoprofen was developed as tert-butyldimethylsilyl (TBDMS) derivative by gas chromatography-mass spectrometry (GC-MS). Characteristic fragment ions of [M-15], [M-57], and [M-139] permitted the accurate and ...
Label-free analysis of physiological hyaluronan size distribution with a solid-state nanopore sensor. Hyaluronan (or hyaluronic acid, HA) is a ubiquitous molecule that plays critical roles in numerous physiological functions in vivo, including tissue hydration, inflammation, and joint lubrication. Both the abundance and size distribution of HA in biological fluids are recognized as robust indicators of various pathologies and disease progressions. However, such analyses remain challenging because conventional methods are not sufficiently sensitive, have limited dynamic range, and/or are only semi-quantitative. Here we demonstrate label-free detection and molecular weight discrimination of HA w...
Simultaneous quantification of free curcuminoids and their metabolites in equine plasma by LC-ESI-MS/MS. The human health benefits attributed to turmeric/curcumin spice has resulted in its wide utilization as a dietary supplement for companion pets and other animals including horses. While the quantification of free curcuminoids (curcumin, demethoxycurcumin, bisdemethoxycurcumin) and their phase-2 metabolites (curcumin-O-sulfate, curcumin-O-glucuronide) have been extensively investigated in human and rodent biological samples (primarily plasma and serum), there is lack of similar data for horses. Herein, we report a validated LC-ESI-MS/MS method for the simultaneous quantification of the aforemen...
