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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.
Development and evaluation of a one-step multiplex real-time TaqMan® RT-qPCR assay for the detection and genotyping of equine G3 and G14 rotaviruses in fecal samples. Equine rotavirus A (ERVA) is the leading cause of diarrhea in neonatal foals and has a negative impact on equine breeding enterprises worldwide. Among ERVA strains infecting foals, the genotypes G3P[12] and G14P[12] are the most prevalent, while infections by strains with other genomic arrangements are infrequent. The identification of circulating strains of ERVA is critical for diagnostic and surveillance purposes, as well as to understand their molecular epidemiology. Current genotyping methods available for ERVA and rotaviruses affecting other animal species rely on Sanger sequencing and ar...
Concentrations of sulphated estrone, estradiol and dehydroepiandrosterone measured by mass spectrometry in pregnant mares. Few studies have provided a longitudinal analysis of systemic concentrations of conjugated oestrogens (and androgens) throughout pregnancy in mares, and those only using immunoassay. The use of liquid chromatography tandem mass spectrometry (LC-MS/MS) will provide more accurate concentrations of circulating conjugated steroids. Objective: To characterise circulating concentrations of individual conjugated steroids throughout equine gestation by using LC-MS/MS. Methods: Longitudinal study and comparison of pregnant mares treated with vehicle or letrozole in late gestation. Methods: Sulphated oe...
Liquid chromatography-mass spectrometry-based determination of ergocristine, ergocryptine, ergotamine, ergovaline, hypoglycin A, lolitrem B, methylene cyclopropyl acetic acid carnitine, N-acetylloline, N-formylloline, paxilline, and peramine in equine hair. Ingestion of hypoglycin A (HGA) in maple seeds or alkaloids produced by symbiotic fungi in pasture grasses is thought to be associated with various syndromes in grazing animals. This article describes analytical methods for monitoring long-term exposure to HGA, its metabolite MCPA-carnitine, as well as ergocristine, ergocryptine, ergotamine, ergovaline, lolitrem B, N-acetylloline, N-formylloline, peramine, and paxilline in equine hair. After extraction of hair samples separation was achieved using two ultra high performance liquid chromatographic systems (HILIC or RP-C18, ammonium formate:acet...
Elucidation of the biosynthetic pathways of boldenone in the equine testis. Boldenone is an anabolic-androgenic steroid that is prohibited in equine sports. Urine from the uncastrated male horse contains boldenone that is thought to be of endogenous origin and thus a threshold ('cut-off') concentration has been adopted internationally for free and conjugated boldenone to help distinguish cases of doping from its natural production. The testis is likely to be a source of boldenone. Qualitative analysis was performed on extracts of equine testicular homogenates (n = 3 horses) incubated non-spiked and in the presence of its potential precursors using liquid chromatog...
Simultaneous quantitation of 9 anabolic and natural steroidal hormones in equine urine by UHPLC-MS/MS triple quadrupole. A new fast and easy analytical procedure for the simultaneous detection and quantification of 9 anabolic steroids (deslorelin, dexamethasone sodium phosphate, prednisolone, methylprednisolone, stanozolol, boldenone, nandrolone, dexamethasone isonicotinate and altrenogest) in horse urine for doping control have been developed by using the ultra-high-performance liquid chromatography coupled with tandem mass spectrometry technique (UHPLC-MS/MS). A total amount of 400 μl of sample was evaporated, restored and injected in the UHPLC-MS/MS. The proposed method was fully validated showing a recove...
Clinical pharmacokinetics and pharmacodynamics of intravenous alfaxalone in young Thoroughbred horses premedicated with medetomidine and midazolam. To investigate the clinical pharmacokinetics and pharmacodynamics of intravenous alfaxalone in young Thoroughbred horses, seven Thoroughbred horses were randomly anaesthetised twice with either 1 or 2 mg/kg of intravenous alfaxalone after premedication with medetomidine (6 µg/kg intravenous) and midazolam (20 µg/kg intravenous). Blood samples were collected at predetermined time points up to two hours after administration. Plasma alfaxalone concentrations were quantified by a liquid chromatography tandem-mass spectrometry method and analysed by non-compartmental pharmacokinetic analy...
Enantioselective capillary electrophoresis for pharmacokinetic analysis of methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in equines anesthetized with ketamine and isoflurane. An enantioselective assay for the determination of methadone and its main metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in equine plasma based on capillary electrophoresis with highly sulfated γ-cyclodextrin as chiral selector and electrokinetic analyte injection is described. The assay is based on liquid/liquid extraction of the analytes at alkaline pH from 0.1 mL plasma followed by electrokinetic sample injection of the analytes from the extract across a buffer plug without chiral selector. Separation occurs cationically at normal polarity in a pH 3 phosphate buffer containin...
Assessment of the CoaguChek-XS portable prothrombin time point-of-care analyzer for horses. Coagulopathies in horses are common and potentially life-threatening. In equine field medicine, a portable point-of-care (POC) prothrombin time (PT) testing device could be useful to identify early changes in extrinsic clotting. The CoaguChek-XS (Roche Diagnostics) is a small, portable POC PT analyzer used in human medicine. Our preliminary study assessed the suitability of CoaguChek-XS for testing PT in horses and established the PT reference interval (PT RI) in healthy horses using this instrument. Blood samples collected from 102 healthy and ill horses were analyzed with the CoaguChek-XS an...
Evaluation of pulse co-oximetry to determine haemoglobin saturation with oxygen and haemoglobin concentration in anaesthetized horses: a retrospective study. This study compared the values of variables measured by pulse co-oximetry (Masimo Radical 7; Masimo Europe Limited, UK) with those measured by a co-oximeter-enabled blood gas analyser (Siemens Rapid-point 500; Siemens Healthcare Limited, UK) in anaesthetized horses. Methods: Retrospective study. Methods: A total of 30 anaesthetized horses. Methods: In total, 47 heparinized arterial blood samples were collected for blood gas analysis to determine haemoglobin concentration (tHb, g L) and percentage of haemoglobin saturation with oxygen (SaO). Arterial haemoglobin saturation with oxygen was deter...
Detection of ESAs in equine urine and blood by SAR-PAGE. Erythropoiesis-stimulating agents (ESAs) have been used in horses for doping purposes to increase the performance of these animals in endurance sports. Currently, enzyme-linked immunosorbent assay (ELISA) and mass spectrometry methods are used to detect ESA abuse in equines. However, the sarcosyl polyacrylamide gel-electrophoresis (SAR-PAGE) technique could also be used, since its application in human doping control is well established and has proven to be more sensitive. In this work, the SAR-PAGE method was used to detect recombinant human erythropoietin (rHuEPO), novel erythropoiesis stimul...
Comparison of two portable clinical analyzers to one stationary analyzer for the determination of blood gas partial pressures and blood electrolyte concentrations in horses. Portable blood gas analyzers are used to facilitate diagnosis and treatment of disorders related to disturbances of acid-base and electrolyte balance in the ambulatory care of equine patients. The aim of this study was to determine whether 2 portable analyzers produce results in agreement with a stationary analyzer. Blood samples from 23 horses hospitalized for various medical reasons were included in this prospective study. Blood gas analysis and electrolyte concentrations measured by the portable analyzers VetStat and epoc were compared to those produced by the cobas b 123 analyzer via conco...
Retraction Note to: Analysis of loxoprofen in tablets, patches, and equine urine as tert-butyldimethylsilyl derivative by gas chromatography-mass spectrometry. The authors have retracted this article [1] because after publication they became aware that the equine urine samples analysed for loxoprofen in this study were in fact equine plasma samples. Therefore the results and conclusions of this article cannot be relied upon. All authors agree to this retraction.
[Comparative analysis of LAMP and Real Time PCR methods to detect pathogens of glanders and meliodosis.]. Results of detection of Burkholderia mallei and Burkholderia pseudomallei DNA strains by LAMP (Loop-mediated Isothermal Amplification) and Real Time PCR are shown. It has been revealed that, in Real Time PCR, primers steadily detected DNA of those microorganism for the sequences of which they were designed. The above mentioned primers did not detect DNA of heterologous strains. During LAMP method no set of primers showed high analytical sensitivity and specificity. Primers did not detected DNA of all the strains under research to target genes of which they were not intended, but they were capa...
Assessment of Gender Effects and Reference Values of Mane Hair Trace Element Content in English Thoroughbred Horses (North Caucasus, Russia) Using ICP-DRC-MS. The objective of the present study was assessment of gender differences in hair trace element content in English Thoroughbred horses (North Caucasus, Russia) using ICP-DRC-MS and calculation of the reference values. Trace element content in mane hair of 190 stallions and 94 mares (3-7 years old) bred in North Caucasus (Russia) was assessed using inductively coupled plasma mass spectrometry. Mane hair Co, Cr, Mn, Li, Si, and Sr levels in mares exceeded those in stallions by 77%, 63%, 64%, 42%, 39%, and 64%, respectively. Hair Fe and Si content was nearly twofold higher in female horses as comp...
Concentrations of indomethacin and its metabolite desmethylindomethacin in plasma and urine after repeated indomethacin topical application to Thoroughbreds. Repeated topical application of indomethacin is common in Japanese racehorses, despite the lack of pharmacokinetic data. Objective: To determine the concentrations of indomethacin and its metabolite, desmethylindomethacin, in plasma and urine of Thoroughbreds topically treated repeatedly with indomethacin. Methods: In vivo experimental. Methods: Seven female Thoroughbreds were topically treated with 50 g of 1% indomethacin cream per horse to the back and hips (500 mg of indomethacin/head/2400 cm , 0.21 g/cm ) for 3 consecutive days. Samples were pretreated by protein precipitation for plas...
Doping control analysis of four JWH-250 metabolites in equine urine by liquid chromatography-tandem mass spectrometry. JWH-250 is a synthetic cannabinoid. Its use is prohibited in equine sport according to the Association of Racing Commissioners International (ARCI) and the Fédération Équestre Internationale (FEI). A doping control method to confirm the presence of four JWH-250 metabolites (JWH-250 4-OH-pentyl, JWH-250 5-OH-pentyl, JWH-250 5-OH-indole, and JWH-250 N-pentanoic acid) in equine urine was developed and validated. Urine samples were treated with acetonitrile and evaporated to concentrate the analytes prior to the analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The chromat...
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...
