Omneity® Pellets
All-In-One Vitamin & Mineral Pellet
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.
Identification of recombinant equine growth hormone in horse plasma by LC-MS/MS: a confirmatory analysis in doping control. Equine growth hormone (eGH) has been available since 1998 as an approved drug (EquiGen-5, Bresagen) containing recombinant eGH (reGH). It is suspected of being illegally administered to racehorses in order to improve physical performance and to speed-up wound healing. Thus it may be considered a doping agent which would require a sensitive and reliable method of identification and confirmation in order to regulate its use in racehorses. reGH differs from the native eGH by an additional methionine at the N-terminal (met-eGH) and has never been unambiguously detected in any type of biological ma...
Evaluation of a commercially available apparatus for measuring the acute phase protein serum amyloid A in horses. The aim of this study was to investigate the reliability of an immunoturbidometric assay for measuring the acute phase protein serum amyloid A (SAA) in horses in clinical practice. The assay was compared to a previously validated assay, and overlap performance was assessed by measuring the concentration of SAA in clinically healthy horses and horses with inflammatory and non-inflammatory diseases. In pools of serum with low and high SAA concentrations the assay's intra-assay coefficients of variation were 11.7 per cent and 4.6 per cent, and its interassay coefficients of variation were 9.1 per...
Simultaneous doping analysis of main urinary metabolites of anabolic steroids in horse by ion-trap gas chromatography-tandem mass spectrometry. The use of anabolic steroids in racehorses is strictly regulated. We have developed a method for the simultaneous analysis of 11 anabolic steroids: fluoxymesterone, 17alpha-methyltestosterone, mestanolone, methandienone, methandriol, oxymetholone, boldenone, furazabol, methenolone, nandrolone, and stanozolol, for possible application to a doping test in racehorses. We selected 15 kinds of target substances for a doping test from the main metabolites of these anabolic steroids, and established a method for simultaneous analysis. Urine was hydrolyzed and subjected to solid-phase extraction. Then...
Quantification of 19-nortestosterone sulphate and boldenone sulphate in urine from male horses using liquid chromatography/tandem mass spectrometry. Following administration of the anabolic steroid 19-nortestosterone or its esters to the horse, a major urinary metabolite is 19-nortestosterone-17beta-sulphate. The detection of 19-nortestosterone in urine from untreated animals has led to it being considered a naturally occurring steroid in the male horse. Recently, we have demonstrated that the majority of the 19-nortestosterone found in extracts of 'normal' urine from male horses arises as an artefact through decarboxylation of the 19-carboxylic acid of testosterone. The aim of this investigation was to establish if direct analysis of 19-n...
Validation of thromboelastometry in horses. Thromboelastometry is used for identifying or monitoring coagulation abnormalities. It has been validated in several species but not in horses and the characteristics of the equine thromboelastogram have not yet been detailed. Objective: The purpose of this study was to validate a thromboelastometer to be used with equine blood and to define the normal equine thromboelastogram. Methods: A Rotem-gamma thromboelastometer (Pentapharm GmbH, Munich, Germany) was used on 38 citrated blood samples to investigate native coagulation, the intrinsic and extrinsic pathways, the function of fibrinogen (lar...
Development of a liquid chromatography-tandem mass spectrometry method for an euthanasic veterinarian drug: Tanax. A development of a rapid and sensitive LC-MS/MS method for the simultaneous detection of active ingredients of the euthanasic veterinarian drug Tanax mixture is described. The method proposed, with a retention time of few minutes (6 min) was developed for an equine serum sample with solid-phase extraction (S.P.E). This S.P.E. procedure has been revealed useful for the determination of very low concentrations of Tanax analytes (0.05-1 ng/ml). The method was validated in terms of specificity/selectivity, sensitivity, recovery and precision.
Comparison of the Accu-Chek Aviva point-of-care glucometer with blood gas and laboratory methods of analysis of glucose measurement in equine emergency patients. More information is needed regarding accuracy of commonly used methods of glucose measurement in the critically ill horse. Objective: Glucometry will have good agreement with a laboratory standard. Glucometry with plasma will have better agreement than when performed with whole blood. Methods: Fifty sequentially admitted equine emergency patients, aged >1year. Methods: Venous blood was collected at admission and immediately analyzed by point-of-care glucometry on both whole blood (POC/WB) and plasma (POC/PL), a multielectrode blood gas analyzer with whole blood (BLG), and a standard laborat...
Characterization and quantification of fluoxymesterone metabolite in horse urine by gas chromatography/mass spectrometry. Fluoxymesterone, an anabolic steroid with the 17alpha-methyl,17beta-hydroxy group, has been developed as an oral formulation for therapeutic purposes. However, it is also used illegally in racehorses to enhance racing performance. In this study, we detected 9alpha-fluoro-17,17-dimethyl-18-norandrostane-4,13-dien-11beta-ol-3-one by gas chromatography/mass spectrometry (GC/MS), which has not been reported as a fluoxymesterone metabolite so far in horse. It was synthesized for use as a reference standard, and characterized on the basis of (1)H NMR and (13)C NMR spectra, as well as GC/MS EI mass s...
Hair analysis of anabolic steroids in connection with doping control-results from horse samples. Doping control of anabolic substances is normally carried out with urine samples taken from athletes and horses. Investigation of alternative specimens, e.g. hair samples, is restricted to special cases, but can also be worthwhile, in addition to urine analysis. Moreover, hair material is preferred in cases of limited availability or complicated collection of urine samples, e.g. from horses. In this work, possible ways of interpretation of analytical results in hair samples are discussed and illustrated by practical experiences. The results demonstrate the applicability of hair analysis to det...
Detection of urinary metabolites common to structurally related 17alpha-alkyl anabolic steroids in horses and application to doping tests in racehorses: methandienone, methandriol, and oxymetholone. Methandienone, methandriol, and oxymetholone, which are anabolic steroids possessing 17alpha-methyl and 17beta-hydroxy groups, were developed as oral formulations for therapeutic purposes. However, they have been used in racehorses to enhance racing performance. In humans, it has been reported that structurally related anabolic steroids having the 17alpha-methyl and 17beta-hydroxy groups, including 17alpha-methyltestosterone, mestanolone, methandienone, methandriol, and oxymetholone, have metabolites in common. In this study, we found that metabolites common to those of 17alpha-methyltestoster...
Evaluation of three methods for measurement of hemoglobin and calculated hemoglobin parameters with the ADVIA 2120 and ADVIA 120 in dogs, cats, and horses. Besides flow cytometric detection of cellular hemoglobin (HGB) concentration, the ADVIA 2120 uses a novel cyanide-free colorimetric method to determine extracellular total HGB concentration. In human samples, the results are equivalent to those of the cyanmethemoglobin method on the ADVIA 120. Cyanide-free HGB measurement has not been evaluated in animal samples. Objective: The aim of this prospective study was to compare the 3 methods of HGB analysis on the ADVIA 2120 and ADVIA 120 in blood samples from dogs, cats, and horses. Methods: Consecutive fresh K(3)EDTA blood samples from 119 dogs, 1...
Analysis of iridoids from Harpagophytum and eleutherosides from Eleutherococcus senticosus in horse urine. LC/ESI-MS n methods have been previously set up to detect the administration of (i) Harpagophytum and (ii) preparations containing a plant capable of anti-stress properties: Eleutherococcus senticosus. Harpagoside has been found to be the main indicator of Harpagophytum administration in the horse. These methods have been applied to a large number of horse urine samples of various origins. Regarding the detection of Harpagophytum administration, harpagoside, harpagide and 8-para-coumaroyl harpagide were detected together in only one sample out of 317. Eleutheroside E was found to be the main i...
Comparative structural and chemical studies of ferritin cores with gradual removal of their iron contents. Transmission Electron Microscopy (TEM), X-ray Absorption Near Edge Spectroscopy (XANES), Electron Energy-Loss Spectroscopy (EELS), Small-Angle X-ray Scattering (SAXS), and SQUID magnetic studies were performed in a batch of horse spleen ferritins from which iron had been gradually removed, yielding samples containing 2200, 1200, 500, and 200 iron atoms. Taken together, findings obtained demonstrate that the ferritin iron core consists of a polyphasic structure (ferrihydrite, magnetite, hematite) and that the proportion of phases is modified by iron removal. Thus, the relative amount of magneti...
Doping control analysis of insulin and its analogues in equine plasma by liquid chromatography-tandem mass spectrometry. 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 detect effectively the use of insulins in horses would be required. Besides the readily available human insulin and its synthetic analogues, structurally similar insulins from other species can also be used as doping agents. This study describes a method for the simultaneous detection of bovine, porcine and human...
Mass scans from a proton transfer mass spectrometry analysis of air over Mediterranean shrubland browsed by horses. Plants usually emit large amount and varieties of volatiles after being damaged by herbivores. However, analytical methods for measuring herbivore-induced volatiles do not normally monitor the whole range of volatiles and the response to large herbivores such as large mammals is much less studied than the response to other herbivores such as insects. In this paper we present the results of using a highly sensitive proton transfer reaction-mass spectrometry (PTR-MS) technique that allows simultaneous monitoring of leaf volatiles in the pptv range. The resulting mass scans in air over Mediterran...
Direct injection LC/ESI-MS horse urine analysis for the quantification and identification of threshold substances for doping control. I. Determination of hydrocortisone. Two simple and rapid LC/MS methods with direct injection analysis were developed and validated for the quantification and identification of hydrocortisone in equine urine using the same sample preparation but different mass spectrometric systems: ion trap mass spectrometry (IT-MS) and time-of-flight mass spectrometry (TOF-MS). The main advantage of the proposed methodology is the minimal sample preparation procedure, as particle-free diluted urine samples were directly injected into both LC/MS systems. Desonide was used as internal standard (IS). The linear range was 0.25-2.5 microg ml(-1) for...
Differentiation and identification of recombinant human erythropoietin and darbepoetin Alfa in equine plasma by LC-MS/MS for doping control. Recombinant human erythropoietin (rhEPO) and darbepoetin alfa (DPO) are protein-based drugs for the treatment of anemia in humans by stimulating erythrocyte production. However, these agents are abused in human and equine sports due to their potential to enhance performance. This paper describes the first method for differentiation and identification of rhEPO and DPO in equine plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The method comprised analyte extraction and enrichment by immunoaffinity separation with anti-rhEPO antibodies, dual digestion by trypsin an...
Evaluation of equine hemograms using the ADVIA 120 as compared with an impedance counter and manual differential count. The ADVIA 120 is an automated laser cell counter widely used in veterinary medicine. Although specific software for equine samples is available and validated, only a few reports have been published comparing the ADVIA 120 with other methods for equine hemogram evaluation. Objective: The purpose of this study was to compare the hematologic values and reference intervals obtained on the ADVIA 120 with those obtained on an impedance cell counter and manual differential counts in healthy horses. Methods: EDTA-anticoagulated blood samples were obtained from 114 clinically healthy horses of various ...
Optimization of solid-phase extraction for the liquid chromatography-mass spectrometry analysis of harpagoside, 8-para-coumaroyl harpagide, and harpagide in equine plasma and urine. Solid-phase extraction cartridges among those usually used for screening in horse doping analyses are tested to optimize the extraction of harpagoside (HS), harpagide (HG), and 8-para-coumaroyl harpagide (8PCHG) from plasma and urine. Extracts are analyzed by liquid chromatography coupled with multi-step tandem mass spectrometry. The extraction process retained for plasma applies BondElut PPL cartridges and provides extraction recoveries between 91% and 93%, with RSD values between 8 and 13% at 0.5 ng/mL. Two different procedures are needed to extract analytes from urine. HS and 8PCHG are extr...
Intake and excretion of disodium monomethylarsonate in horses: a speciation study. Capillary electrophoresis coupled to inductively coupled plasma mass spectrometry was used in a speciation study on disodium monomethylarsonate (DS-MMA(V)) and its metabolites in horses, to which the drug was administered by intramuscular injection on five consecutive days at a single arsenic dosage of 270 mg day(-1). Samples of urine, whole blood, plasma, and mane hair were analyzed before, during, and after drug administration. The data show that blood clearing and urinary excretion of MMA is a fast process following first-order kinetics with biological half-lives of about 38 h and 44 h for ...
Technical note: a novel method for routine genotyping of horse coat color gene polymorphisms. The aim of this note is to describe a reliable, fast, and cost-effective real-time PCR method for routine genotyping of mutations responsible for most coat color variation in horses. The melanocortin-1 receptor, Agouti-signaling peptide, and membrane-associated transporter protein alleles were simultaneously determined using 2 PCR protocols. The assay described here is an alternative method for routine genotyping of a defined number of polymorphisms. Allelic variants are detected in real time and no post-PCR manipulations are required, therefore limiting costs and possible carryover contaminat...
Specific electrochemical iodination of horse heart myoglobin at tyrosine 103 as determined by Fourier transform ion cyclotron resonance mass spectrometry. The iodination of proteins remains a useful tool in biochemistry for radiolabelling. However, chemical or enzymatic iodination is difficult to control and can give deleterious polyiodination. Previously, we have shown that electrooxidation with nitrite is a rapid method for the selective nitration of tyrosine residues in proteins. In principle, it should be possible to substitute a number of electrooxidisable anions into the tyrosine phenol ring. Electrochemical iodination is more difficult to control than nitration because the rapid anodic oxidation of I(-) leads to persistent formation of th...
Determination of IGF-I in horse plasma by LC electrospray ionisation mass spectrometry. The insulin-like-growth factor (IGF-I) peptide is considered to be the main indirect marker for growth hormone administration (GH) in a horse. Further to a previous investigation on measurement of IGF-I in plasma samples by mass spectrometry, this study focuses on quantitative and qualitative analysis of intact IGF-I in horse plasma. First, protein-transposing software has been developed for IGF-I to facilitate its quantification by HPLC-electrospray-ion-trap mass spectrometry. Second, product-ion scan experiments on IGF-I have been conducted on standard samples, non-fortified equine plasma sa...
Unusual observations during steroid analysis. In September 2005, our laboratory detected the presence of 4-androstene-3,17-dione and androsterone in a standard steroid screen of a post-race gelding urine sample received from an overseas authority. All other urine samples from the same batch tested negative. Subsequent gas chromatography/mass spectrometry (GC/MS) confirmatory analyses, however, repeatedly failed to detect any amount of 4-androstene-3,17-dione and androsterone in the suspicious sample. On the other hand, identical results were obtained when the initial GC/MS screening method was repeated on the suspicious sample as well as ...
Insulins in equine urine: qualitative analysis by immunoaffinity purification and liquid chromatography/tandem mass spectrometry for doping control purposes in horse-racing. Insulin is a peptide hormone consisting of two peptide chains (A- and B-chain) that are cross-linked by two disulfide bonds. To obtain improved pharmacokinetic onset of action profiles of insulin treatment in diabetic patients, recombinant long-, intermediate-, and rapid-acting insulin analogs are produced, in which the C-terminal end of the B-chain plays an especially important role.A review of the veterinary literature reveals the low prevalence of equine type I diabetes mellitus, which indicates that the therapeutic use of insulin in racing horses is unlikely. Although there is no unequivoc...
Joint dependent concentrations of bone alkaline phosphatase in serum and synovial fluids of horses with osteochondral injury: an analytical and clinical validation. Validate use of a commercially available immunoassay for measurement of bone alkaline phosphatase (BAP) in equine serum and synovial fluid (SF), and investigate the effects of osteochondral (OC) injury in horses on BAP concentrations in serum and SF. Methods: SF was collected from 37 joints of 34 Thoroughbred (TB) racehorses undergoing arthroscopic surgery for the removal of OC fragments from either the carpal joints (n=18) or the metacarpo-/metatarsophalangeal (MP) joints (n=19). SF was also obtained from 52 joints of 16 normal TB horses, collected bilaterally from carpal joints of 10 horses ...
Gabapentin in horses: validation of an analytical method for gabapentin quantitation. Gabapentin [1-(aminomethyl)cyclohexaneacetic acid, Neurontin], is a new anticonvulsant used as adjunctive therapy in the treatment of partial seizures in humans not controlled with standard antiseizure drugs, and it has also been used in veterinary medicine. In performance horses, gabapentin is listed as a class 3 performance-enhancing substance by the Association of Racing Commissioners International, and thus is considered to have the potential to influence the outcome of races. Therefore, we developed and validated a sensitive gas chromatographic-mass spectrometric (GC-MS) method for gabape...
Detection of secondary biomarker of met-eGH as a strategy to screen for somatotropin misuse in horseracing. Since the Australian commercialisation of the recombinant equine growth hormone (reGH) in 1998 (EquiGen-5), Bresagen), this reGH, which differs only from eGH by an additional methionine at the N-terminal end (met-eGH), is worldwide suspected to be administered to racehorses as a doping agent. Indeed, the use of this biological drug is considered as a threat to horseracing since it acts both on growth, development or reproductive functions, and on the improvement of performances. In this work, we describe two reliable techniques based on surface plasmon resonance biosensor immunoassay (SPR-BIA)...
Isolation of bicarbonate from equine urine for isotope ratio mass spectrometry. Sodium bicarbonate administration to horses prior to competition in order to enhance the buffer capacity of the organism is considered as a doping offence. The analysis of the isotopic composition of urinary bicarbonate/CO(2) (TCO(2)) may help to identify an exogenous bicarbonate source, as technical sodium bicarbonate exhibits elevated delta(13)C values compared with urinary total carbon. The isolation of TCO(2) from 60 equine urine samples as BaCO(3) followed by an isotopic analysis shows a significant variability of delta(13)C for TCO(2) of more than 10 per thousand. The delta(13)C of total...
