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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.
Toltrazuril sulfone sodium salt: synthesis, analytical detection, and pharmacokinetics in the horse. Toltrazuril sulfone (ponazuril) is a triazine-based antiprotozoal agent with clinical application in the treatment of equine protozoal myeloencephalomyelitis (EPM). In this study, we synthesized and determined the bioavailability of a sodium salt formulation of toltrazuril sulfone that can be used for the treatment and prophylaxis of EPM in horses. Toltrazuril sulfone sodium salt was rapidly absorbed, with a mean peak plasma concentration of 2400 ± 169 (SEM) ng/mL occurring at 8 h after oral-mucosal dosing and was about 56% bioavailable compared with the i.v. administration of toltrazuril sul...
[Determination of immunoglobulin G in mare colostrum by high-performance gel permeation chromatography]. A direct high-performance gel permeation chromatographic (HPGPC) method for the determination of immunoglobulin G in mare colostrum was established. HPGPC separation was performed on a TOSOH TSK-G4000PW(XL) column (300 mm x 7.8 mm, 5 microm) with 0.05 mol/L phosphate buffer solution (pH 6.9) as the mobile phase at a flow rate of 0.8 mL/min, and the column temperature was maintained at 25 degrees C. The injection volume was 20 microL. At the detection wavelength of 280 nm, the linear range was from 0.2 to 3.0 g/L (r2 = 0.999 5) with a detection limit of 0.08 mg/L (S/N = 10). The recovery was 97...
Evaluation of commercially available assays for the measurement of equine insulin. Determining circulating equine insulin concentrations is becoming increasingly important in equine clinical practice and research. Most available assays are optimized for human medicine, but there is strong equine cross-reactivity because of the highly conserved nature of insulin. To identify an accurate and reliable assay for equine insulin, 6 commercial immunoassays were evaluated for precision, accuracy, and specificity. Only 1 assay initially reached the requisite standard: Mercodia Equine Insulin Enzyme-linked Immunosorbent assay (ELISA). Plasma matrix interferences were identified when t...
Validation and comparison of two methods of measuring lactate in equine plasma. Some methods of lactate (LA) measurement have not been validated appropriately for use in horses. Objective: To validate 2 LA analysers (YSI 2300 Stat Plus and TDx Lactic Acid Assay) for use with equine plasma and to compare plasma [LA] determined by the 2 methods. Methods: Both instruments were evaluated for linearity, parallelism, recovery and precision using serial dilutions of standard LA solutions and equine plasma and then comparing results with linear regression or paired t tests. Plasma [LA] results were compared in 275 blood samples collected from horses exercising at various intensit...
Simultaneous separation and determination of 16 testosterone and nandrolone esters in equine plasma using ultra high performance liquid chromatography-tandem mass spectrometry for doping control. The potential for using testosterone and nandrolone esters in racehorses to boost the biological concentrations of these steroids and enhance athletic performance is very compelling and should be seriously considered in formulating regulatory policies for doping control. In order to regulate the use of these esters in racehorses, a sensitive and validated method is needed. In this paper, we report such a method for simultaneous separation, screening, quantification and confirmation of 16 testosterone and nandrolone esters in equine plasma by ultra high performance liquid chromatography-tandem ...
A broad-spectrum equine urine screening method for free and enzyme-hydrolysed conjugated drugs with ultra performance liquid chromatography/tandem mass spectrometry. The authors' laboratory at one time employed four liquid chromatography/mass spectrometric (LC/MS) methods for the detection of a large variety of drugs in equine urine. Drug classes covered by these methods included anti-diabetics, anti-ulcers, cyclooxygenase-2 (COX-2) inhibitors, sedatives, corticosteroids, anabolic steroids, sulfur diuretics, xanthines, etc. With the objective to reduce labour and instrumental workload, a new ultra performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method has been developed, which encompasses all target analytes detected by the origina...
Characterization of in vivo plasma metabolites of tepoxalin in horses using LC-MS-MS. Tepoxalin is a veterinary drug registered for use in the dog as a dual inhibitor (cyclooxygenase-5 lipoxygenase). In the horse, it predominantly triggers a strong cyclooxygenase inhibition; this bias seems to be due to the action of its metabolite(s). Among these, only the RWJ-20142 is well known, while to the best of our knowledge no information is available on the other metabolites produced in vivo. Hence, the identification of its main metabolic pathway is pivotal to better understand its clinical activity. A suitable high performance liquid chromatography method has been applied to liquid ...
Easy stereoselective synthesis of 5α-estrane-3β,17α-diol, the major metabolite of nandrolone in the horse. 5α-Estrane-3β,17α-diol is the major metabolite of nandrolone in horse urine. The presence of 5α-estrane-3β,17α-diol in female and gelding urines is prohibited by Racing Rules and its natural presence in male urine led regulation authorities to establish a concentration threshold of 45 ng/mL. This paper describes a rapid, simple and stereoselective synthesis of 5α-estrane-3β,17α-diol, providing horseracing laboratories with an essential reference material for their antidoping performance.
Analysis of gabapentin in equine plasma with measurement uncertainty estimation by liquid chromatography-tandem mass spectrometry. Gabapentin (GPT) is an antiepileptic drug that was approved in 1993 for use in the management of neurotrophic pain and as an adjunctive therapy for refractory partial seizure in humans. It is also being tested in veterinary medicine as an adjunctive medication in the treatment of pain due to laminitis, neuropathic, or chronic pain. Gabapentin is readily available by prescription and even on the internet; therefore, it has the potential of being used in racehorses to mask pain. It is for this reason that a sensitive liquid chromatography-tandem mass spectrometry method has now been developed fo...
Systematic analyses of free ceramide species and ceramide species comprising neutral glycosphingolipids by MALDI-TOF MS with high-energy CID. Free ceramides and glycosphingolipids (GSLs) are important components of the membrane microdomain and play significant roles in cell survival. Recent studies have revealed that both fatty acids and long-chain bases (LCBs) are more diverse than expected, in terms of i) alkyl chain length, ii) hydroxylation and iii) the presence or absence of double bonds. Electrospray ionization mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been well utilized to characterize sphingolipids with high throughput, but reports to date have not ...
A mass spectrometric analysis of 4-hydroxy-2-(E)-nonenal modification of cytochrome c. Cytochrome c is a key mitochondrial respiratory protein that is particularly susceptible to modification during oxidative stress. The nature of this susceptibility is linked to the mitochondrial membrane being rich in esterified linoleic acid, which predisposes this organelle to the formation of lipid peroxidation products such as 4-hydroxy-2-(E)-nonenal (4-HNE). To better understand the nature of cytochrome c modification by 4-HNE, we initiated an in vitro study utilizing a combination of MALDI-TOF mass spectrometry, LC-ESI-MS/MS and isotope labeling to monitor 4-HNE modification of cytochrom...
Determination of angiotensin I-converting enzyme activity in equine blood: lack of agreement between methods of analysis. Angiotensin-I converting enzyme (ACE) is a key regulator of blood pressure, electrolytes and fluid homeostasis through conversion of angiotensin I into angiotensin II. Recently, a genetic polymorphism of the ACE gene, which accounts for 47% of the variation of ACE activity in blood, has been advocated as a biomarker of athletic aptitude. Different methods of analysis and determination of ACE activity in plasma have been used in human and equine research without a consensus of a "gold standard" method. Different methods have often been used interchangeably or cited as being comparable in the ex...
Influence of magnetic field on aqueous two-phase extraction of horse ferritin in the polyethylene glycol/hydroxyethyl starch system. The presented experiments show the model of expectation of equine spleen ferritin extraction in a new aqueous two-phase system which was formed by mixing polyethylene glycol (PEG) and hydroxyethyl starch (HES). The tendency of the protein to migrate in the analyzed systems was dependent on the concentrations of HES and PEG as well as PEG molecular weight. The highest concentration of ferritin in the top phase (rich in PEG) was recorded in the system composed of 6% PEG 3000 and 3% HES. The obtained concentration was 0.88 mg mL(-1). The lowest concentration was 0.42 mg mL(-1) in the system compo...
The use of in vitro technologies and high-resolution/accurate-mass LC-MS to screen for metabolites of ‘designer’ steroids in the equine. Detection of androgenic-anabolic steroid abuse in equine sports requires knowledge of the drug's metabolism in order to target appropriate metabolites, especially where urine is the matrix of choice. Studying 'designer' steroid metabolism is problematic since it is difficult to obtain ethical approval for in vivo metabolism studies due to a lack of toxicological data. In this study, the equine in vitro metabolism of eight steroids available for purchase on the Internet is reported; including androsta-1,4,6-triene-3,17-dione, 4-chloro,17α-methyl-androsta-1,4-diene-3,17β-diol, estra-4,9-diene-...
Analysis of bioactive eicosanoids in equine plasma by stable isotope dilution reversed-phase liquid chromatography/multiple reaction monitoring mass spectrometry. Oxidative metabolites of arachidonic acid (AA) are implicated in inflammation. Thus, we evaluated cycloxygenases (COXs) and lipoxygenases (LOs) mediated metabolism of AA to eicosanoids in equine plasma. Eicosanoids were extracted from plasma by two liquid-liquid extraction (LLE) steps; first was by chloroform/isopropanol and second by methyl-tert-butyl ether. For identification and quantification of 25 eicosanoids, a highly specific, selective and sensitive stable isotope dilution liquid chromatography (LC) multiple reaction monitoring (MRM) mass spectrometric (MS) method was developed. To avo...
Analysis of methyloxime derivatives of intact esters of testosterone and boldenone in equine plasma using ultra high performance liquid chromatography tandem mass spectrometry. Analysis of equine plasma samples to detect the abuse of anabolic steroids can be complicated when the parent steroid is endogenous to the animal. Anabolic steroids are usually administered intramuscularly as synthetic esters and therefore detection of the exogenous esters provides unequivocal proof of illegal administration. An ultra high performance liquid chromatography tandem mass spectrometric (UPLC-MSMS) method for the analysis of esters of testosterone (propionate, phenylpropionate, isocaproate, and decanoate) and boldenone (undecylenate) in equine plasma has been developed. Esters were...
LC-MS/MS method for the simultaneous determination of clarithromycin, rifampicin and their main metabolites in horse plasma, epithelial lining fluid and broncho-alveolar cells. Clarithromycin (CLA) is a well established macrolide antibiotic which is frequently used in therapy of airway diseases in foals. It is extensively metabolized by CYP3A4 resulting in the antimicrobial active metabolite 14-hydroxyclarithromycin (OH-CLA). Rifampicin (RIF) is often comedicated to prevent resistance and augment therapy. RIF is a known inducer for metabolizing enzymes and transporter proteins. Therefore, comedication might bare the risks of pharmacokinetic drug interactions which were investigated in a clinical trial. As no adequate method to determine CLA, RIF and their main metabo...
Assessment of the impact of collection temperature and sampler design on the measurement of exhaled breath condensate pH in healthy horses. The pH measurement of exhaled breath condensate (EBC) may provide a non-invasive method of assessing the lower airways of horses but the methodology used may influence findings. The aim of this study was to investigate the effect of two sampling devices and three methods of condensation surface cooling (ethanol slush, -100°C; dry ice, -75°C; water ice, 0°C) on EBC pH. Each method was tested 30 times using six healthy ponies. Sample pH was determined before and after de-aeration with argon for 10 min. Sampler design was found to significantly affect pH. Samples collected as a liquid had a si...
Use of benchtop exactive high resolution and high mass accuracy orbitrap mass spectrometer for screening in horse doping control. Liquid chromatography-mass spectrometry (LC-MS) has been widely used in doping control laboratories over the last two decades. Currently, simple quadrupole, triple quadrupole and ion trap are the most commonly employed analyzers in toxicological analysis. Nevertheless, the main lack of these technologies is the restricted number of target compounds simultaneously screened without loss of sensitivity. In this article we present an innovative screening approach routinely applied in the French horse doping control laboratory based on high resolution (50000) and high mass accuracy (<5 ppm) in f...
Infrared spectroscopy reveals both qualitative and quantitative differences in equine subchondral bone during maturation. The collagen phase in bone is known to undergo major changes during growth and maturation. The objective of this study is to clarify whether Fourier transform infrared (FTIR) microspectroscopy, coupled with cluster analysis, can detect quantitative and qualitative changes in the collagen matrix of subchondral bone in horses during maturation and growth. Equine subchondral bone samples (n = 29) from the proximal joint surface of the first phalanx are prepared from two sites subjected to different loading conditions. Three age groups are studied: newborn (0 days old), immature (5 to 11 months ol...
Doping control analysis of insulin and its analogues in equine urine by liquid chromatography-tandem mass spectrometry. Insulin and its analogues have been banned in both human and equine sports owing to their potential for misuse. Insulin administration can increase muscle glycogen by utilising hyperinsulinaemic clamps prior to sports events or during the recovery phases, and increase muscle size by its chalonic action to inhibit protein breakdown. In order to control insulin abuse in equine sports, a method to effectively detect the use of insulins in horses is required. Besides the readily available human insulin and its synthetic analogues, structurally similar insulins from other species can also be used a...
Quantitative analysis of collagen fiber organization in injured tendons using Fourier transform-second harmonic generation imaging. Fourier transform-second harmonic generation (FT-SHG) imaging is used as a technique for evaluating collagenase-induced injury in horse tendons. The differences in collagen fiber organization between normal and injured tendon are quantified. Results indicate that the organization of collagen fibers is regularly oriented in normal tendons and randomly organized in injured tendons. This is further supported through the use of additional metrics, in particular, the number of dark (no/minimal signal) and isotropic (no preferred fiber orientation) regions in the images, and the ratio of forward-to-...
Determination of firocoxib in equine plasma using high performance liquid chromatography. A new method of analysis has been developed and validated for the determination of firocoxib, a new nonsteroidal anti-inflammatory drug (NSAID) approved for use in horses and dogs to control pain and inflammation associated with osteoarthritis. Following a liquid extraction using ethyl acetate:hexane (40:60), samples were separated by isocratic reversed-phase HPLC on a Sunfire C(18) column and quantified using UV detection at 290 nm. The mobile phase was a mixture of water with 0.025% trifluoroacetic acid and acetonitrile, with a flow-rate of 1.1 ml/min. The procedure produced a linear curve o...
Detection of prohibited substances in equestrian sport through direct injection of equine serum using micellar LC. Detection of prohibited substances in equestrian sports typically involves time-consuming and tedious sample-preparation methods. Micellar LC (MLC) allows for direct injection of equine serum to detect prohibited NSAIDs. Results: The method was linear over the range of standards examined, with recoveries of 94.2-95.1% for phenylbutazone (12-18 µg/ml), and 83.9-88.7% and 87.9-105.0% for diclofenac and flunixin, respectively (0.1-1.2 µg/ml). The limit of detection was 0.1 µg/ml for all compounds and the limit of quantitation was 0.2 µg/ml for phenylbutazone and 0.3 µg/ml for diclofenac and ...
Metabolism of anabolic steroids and their relevance to drug detection in horseracing. The fight against doping in sport using analytical chemistry is a mature area with a history of approximately 100 years in horseracing. In common with human sport, anabolic/androgenic steroids (AASs) are an important group of potential doping agents. Particular issues with their detection are extensive metabolism including both phase I and phase II. A number of the common AASs are also endogenous to the equine. A further issue is the large number of synthetic steroids produced as pharmaceutical products or as 'designer' drugs intended to avoid detection or for the human supplement market. An u...
Pharmacokinetics of gallium maltolate after intragastric administration in adult horses. To determine the pharmacokinetics of gallium maltolate (GaM) after intragastric administration in adult horses. Methods: 6 adult horses. Methods: Feed was withheld for 12 hours prior to intragastric administration of GaM (20 mg/kg). A single dose of GaM was administered to each horse via a nasogastric tube (time 0). Blood samples were collected at various time points from 0 to 120 hours. Serum was used to determine gallium concentrations by use of inductively coupled plasma-mass spectroscopy. Noncompartmental and compartmental analyses of serum gallium concentrations were performed. Pharmacoki...
Confirmatory analysis of continuous erythropoietin receptor activator and erythropoietin analogues in equine plasma by LC-MS for doping control. Continuous erythropoietin receptor activator (CERA) is the third generation of recombinant human erythropoietin (rhEPO) medication that retains the effect of promoting red blood cell production but has longer duration of action in the body. CERA, rhEPO, and darbepoetin alpha (DPO) can be misused to enhance performance in both human and equine athletes. To deter such misuse, a very selective and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has now been developed for identification of CERA, rhEPO, and DPO in equine plasma. The method employs a new signature tryptic...
Enantiomeric composition analysis of pranoprofen in equine plasma and urine by chiral liquid chromatography-tandem mass spectrometry in selected reaction monitoring mode. The enantioseparation of pranoprofen after its addition in racemic form into equine plasma and urine was conducted by chiral liquid chromatography-tandem mass spectrometry in selected reaction monitoring mode. The methods for the assay of both enantiomers were linear (r≥0.9943) in the low range from 0.001 to 0.1μg/mL and high range from 0.01 to 1.0μg/mL with good precision (% RSD≤5.6) and accuracy (% RE=-5.3 to 1.9). When racemic pranoprofen was orally administered to four horses at a single dose of 3.1mg/kg, the median plasma concentrations of (R)-pranoprofen were lower than the levels ...
The use of in vitro technologies coupled with high resolution accurate mass LC-MS for studying drug metabolism in equine drug surveillance. The detection of drug abuse in horseracing often requires knowledge of drug metabolism, especially if urine is the matrix of choice. In this study, equine liver/lung microsomes/S9 tissue fractions were used to study the phase I metabolism of eight drugs of relevance to equine drug surveillance (acepromazine, azaperone, celecoxib, fentanyl, fluphenazine, mepivacaine, methylphenidate and tripelennamine). In vitro samples were analyzed qualitatively alongside samples originating from in vivo administrations using LC-MS on a high resolution accurate mass Thermo Orbitrap Discovery instrument and by...
