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Topic:High-performance Liquid Chromatography (HPLC)
High-performance Liquid Chromatography (HPLC) is an analytical technique used to separate, identify, and quantify components in a mixture. In equine research, HPLC is employed to analyze various biological samples from horses, such as blood, urine, and tissue, to detect and measure compounds like drugs, metabolites, and nutritional components. This technique is valued for its precision, sensitivity, and ability to handle complex matrices, making it suitable for pharmacokinetic studies, monitoring therapeutic drug levels, and assessing metabolic profiles in horses. The application of HPLC in equine studies provides insights into drug metabolism, nutritional status, and the biochemical effects of different treatments. This page compiles peer-reviewed research studies and scholarly articles that explore the applications, methodologies, and findings of HPLC in the context of equine science.
Concentration of methylprednisolone in the centrodistal joint after administration of methylprednisolone acetate in the tarsometatarsal joint. The centrodistal (CD) and tarsometatarsal (TMT) joints are often injected individually with a corticosteroid to resolve lameness caused by osteoarthritis (OA). There are no data available regarding diffusion of methylprednisolone (MP) from the TMT joint to the CD joint. Objective: A therapeutic concentration of MP diffuses into the CD joint after methylprednisolone acetate (MPA) is administered into the TMT joint. Objective: To measure the concentration of MP in the CD joint after MPA was administered into the TMT joint. Methods: MPA was administered into a TMT joint of 16 horses. At different...
Pharmacokinetics of dexamethasone with pharmacokinetic/pharmacodynamic model of the effect of dexamethasone on endogenous hydrocortisone and cortisone in the horse. A compartmental model was used to describe the pharmacokinetics of dexamethasone (DXM) and changes in the plasma concentration of endogenous cortisone (COR) and hydrocortisone (HYD) following intravenous (i.v.) administration of DXM (0.05 mg/kg) in horses. Quantification of DXM, COR and HYD in equine plasma was achieved using liquid chromatography interfaced with triple spray quadrupole quantum tandem mass spectrometry (LC/TSQ-MS/MS). The median alpha (t(1/2alpha)), beta (t(1/2beta)), and gamma (t(1/2gamma)) half-lives were 0.33, 2.2, and 10.7 h respectively. The area under the DXM plasma conc...
Resolution, quantification and confirmation of betamethasone and dexamethasone in equine plasma by liquid chromatography/tandem mass spectrometry. This method describes the simultaneous separation, identification, quantification and confirmation of betamethasone (BTM) and dexamethasone (DXM) in equine plasma by liquid chromatography (LC) integrated with multidimensional tandem mass spectrometry. Analytes were directly extracted from equine plasma by methyl tert-butyl ether (MTBE). The residues were reconstituted with sample solvent. LC separation of the analytes was performed on a Hypercarb column using acetonitrile/water/formic acid (95:5:0.5, v/v/v) as the mobile phase. Sample screening, quantification and confirmation were performed i...
Pharmacokinetics of R(-) and S(+) carprofen after administration of racemic carprofen in donkeys and horses. To compare plasma disposition of the R(-) and S(+) enantiomers of carprofen after IV administration of a bolus dose to donkeys and horses. Methods: 5 clinically normal donkeys and 3 clinically normal horses. Methods: Blood samples were collected from all animals at time 0 (before) and at 10, 15, 20, 30, and 45 minutes and 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 24, 28, 32, and 48 hours after IV administration of a bolus of carprofen (0.7 mg/kg). Plasma was analyzed in triplicate via high-performance liquid chromatography to determine the concentrations of the carprofen enantiomers. A plasma concent...
Pharmacokinetics of meloxicam in plasma and urine of horses. To determine pharmacokinetic parameters for meloxicam, a nonsteroidal anti-inflammatory drug, in horses. Methods: 8 healthy horses. Methods: In the first phase of the study, horses were administered meloxicam once in accordance with a 2 x 2 crossover design (IV or PO drug administration; horses fed or not fed). The second phase used a multiple-dose regimen (daily oral administration of meloxicam for 14 days), with meloxicam administered at the recommended dosage (0.6 mg/kg). Plasma and urine concentrations of meloxicam were measured by use of validated methods with a limit of quantification of...
Direct detection of boldenone sulfate and glucuronide conjugates in horse urine by ion trap liquid chromatography-mass spectrometry. A study of the equine phase II metabolism of the anabolic agent boldenone is reported. Boldenone sulfate, boldenone glucuronide and their C17-epimers were synthesised as reference standards in our lab and a method was developed for their detection in a horse urine matrix. Solid phase extraction was used to purify the analytes, which were then detected by ion trap LC/MS. Negative and positive ionisation mode MS(2) were used for the detection of sulfate and glucuronide conjugates, respectively. Boldenone sulfate and 17-epiboldenone glucuronide were detected as the major and minor phase II metabo...
Pharmacokinetics and pharmacodynamics of furosemide after oral administration to horses. Furosemide is the most common diuretic drug used in horses. Furosemide is routinely administered as IV or IM bolus doses 3-4 times a day. Administration PO is often suggested as an alternative, even though documentation of absorption and efficacy in horses is lacking. This study was carried out in a randomized, crossover design and compared 8-hour urine volume among control horses that received placebo, horses that received furosemide at 1 mg/kg PO, and horses that received furosemide at 1 mg/kg IV. Blood samples for analysis of plasma furosemide concentrations, PCV, and total solids were obta...
Cefotaxime kinetics in plasma and synovial fluid following intravenous administration in horses. Cefotaxime powder was diluted with sterile water to a concentration of 100 mg/mL. The volume of solution was adjusted for each experimental horse to provide a total dose of 15, 20, and 25 mg/kg and was administered by infusion through a jugular vein catheter over a 10-min period. All three doses were administered to each of the six experimental horses at three different times. Cefotaxime concentrations in plasma and synovial fluid samples were measured by high-performance liquid chromatography (HPLC). Standard compartmental analysis techniques and the WinSAAM modeling program were used to dete...
Utility of high-performance liquid chromatography in the characterization of iatrogenic sodium diatrizoate-induced seizures in a horse. Diagnostic evaluation of a mildly ataxic young gelding Quarterhorse included a contrast myelogram. Unfortunately, sodium diatrizoate was inappropriately administered by intrathecal injection resulting in severe unrelenting seizure activity and necessitated the destruction of the animal. The contrast agent was identified by analyzing cerebrospinal fluid by high-performance liquid chromatography and confirmed with probe mass spectrometric analysis. This contrast agent is highly ionic, has high osmolarity, and also blocks neurotransmission at specific receptors; for these reasons it is contraindi...
Detection of stanozolol and its metabolites in equine urine by liquid chromatography-electrospray ionization ion trap mass spectrometry. The equine phase I and phase II metabolism of the synthetic anabolic steroid stanozolol was investigated following its administration by intramuscular injection to a thoroughbred gelding. The major phase I biotransformations were hydroxylation at C16 and one other site, while phase II metabolism in the form of sulfate and beta-glucuronide conjugation was extensive. An analytical procedure was developed for the detection of stanozolol and its metabolites in equine urine using solid phase extraction, acid solvolysis of phase II conjugates and analysis by positive ion electrospray ionization ion ...
Detection of anti-diabetics in equine plasma and urine by liquid chromatography-tandem mass spectrometry. Anti-diabetics such as sulfonylurea and thiazolidinedione derivatives are hypoglycemic drugs used for the treatment of diabetes. However, they can also be used as a stopper in horseracing. This paper describes a convenient method for the separation and simultaneous detection of 10 anti-diabetic drugs (namely glipizide, glibenclamide, glimepiride, gliclazide, tolazamide, tolbutamide, nateglinide, repaglinide, rosiglitazone and pioglitazone) in equine plasma and urine by LC-MS-MS. Methods: The anti-diabetics were isolated from equine plasma and urine by liquid-liquid extraction with 1,2-dichloro...
Identification of some new clemastine metabolites in dog, horse, and human urine with liquid chromatography/tandem mass spectrometry. The metabolism of clemastine was studied in dogs, horses, and humans after a single dose of Tavegyl. The urine collected was extracted by solid-phase extraction or hydrolyzed with beta-glucuronidase and then extracted by liquid-liquid extraction, prior to analysis for unchanged drug and phase I and II metabolites by liquid chromatography/tandem mass spectrometry. The metabolites were identified by their molecular mass and interpretation of the product ion spectra, since no standard substances were available. Unchanged drug was recovered in urine samples from dogs and humans, but not from horse...
Proteomic tools to characterize the protein fraction of Equidae milk. The principal components of the protein fraction in pony mare's milk have been successfully identified and partially characterized using proteomic tools. Skimmed pony mare's milk was fractionated by either reversed phase-high-performance liquid chromatography (RP-HPLC) on a C4 column or a bi-dimensional separation technique coupling RP-HPLC in the first dimension and sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) in the second dimension (two-dimensional RP-HPLC/SDS-PAGE). The fractions thus obtained were analyzed by Edman N-terminal microsequencing and mass determination, wit...
Detection of endogenous boldenone in the entire male horses. Boldenone (1,2-dehydrotestosterone) is a common veterinary anabolic agent. Its structure is very similar to testosterone. Testosterone is endogenous in the horse, whereas there has been no report concerning the detection of endogenous boldenone. This paper reports the direct observation of sulphate conjugate of boldenone in equine urine from entires. The detection procedures involved solid-phase extraction, immunoaffinity column (IAC) purification, and then LC-MS-MS analysis on a Q-ToF instrument. The identification of boldenone sulphate has provided direct evidence for the endogenous nature o...
Daily rhythm of circulating fat soluble vitamin concentration (A, D, E and K) in the horse. BACKGROUND: Many physiological processes of mammalian species exhibit daily rhythmicity. An intrinsic relationship exists between fat soluble vitamins (A, D, E and K) and several body functions. Few investigations on the rhythmic pattern of vitamins in domestic animals have been carried out. The present study evaluated the circadian rhythmicity of fat soluble vitamins in the horse. METHODS: Blood samples from 5 Thoroughbred mares were collected at four-hour intervals over a 48-hour period (starting at 8:00 hours on day 1 and finishing at 4:00 on day 2) via an intravenous cannula inserted into ...
Detection of enrofloxacin and its metabolite ciprofloxacin in equine hair. Hair analysis to detect drug administration has not been studied extensively in horses. This study aimed to (a) develop an analytical method for enrofloxacin and its metabolite ciprofloxacin in mane and tail hair, (b) relate measured values to doses, routes of administration, hair colour, and (c) demonstrate long-term detectability. Samples were extracted in trifluoroacetic acid at 70 degrees C. Extracts were cleaned-up by solid-phase extraction and analysed by high-performance liquid chromatography with UV-diode array detection. Analyte recoveries were > 87%. Horses were sampled after therape...
The effects of vasoactive amines found in the equine hindgut on digital blood flow in the normal horse. Disturbances of digital blood flow are thought to be fundamental to the pathophysiology of acute laminitis. However, factors linking the initiating events in the equine hindgut with these disturbances in the foot remain to be determined. Objective: Amine compounds, formed by bacteria in the equine hindgut, have digital vasoconstrictor effects in vivo. Methods: Tryptamine (1.6 microg/kg/min) and phenylethylamine (2.13 microg/kg/min) were infused i.v. into standing nonsedated horses. Digital blood flow was measured by Doppler ultrasound and foot surface temperature was monitored. Plasma 5-hydrox...
Detection, quantification, metabolism, and behavioral effects of selegiline in horses. Selegiline ([R]-[-]N,alpha-dimethyl-N-2- propynylphenethylamine or l-deprenyl), an irreversible inhibitor of monoamine oxidase, is a classic antidyskinetic and antiparkinsonian agent widely used in human medicine both as monotherapy and as an adjunct to levodopa therapy. Selegiline is classified by the Association of Racing Commissioners International (ARCI) as a class 2 agent, and is considered to have high abuse potential in racing horses. A highly sensitive LC/MS/MS quantitative analytical method has been developed for selegiline and its potential metabolites amphetamine and methamphetamine...
Detection, quantification, and pharmacokinetics of furosemide and its effects on urinary specific gravity following IV administration to horses. Furosemide is a potent loop diuretic used for the prevention of exercise-induced pulmonary hemorrhage in horses. This drug may interfere with the detection of other substances by reducing urinary concentrations, so its use is strictly regulated. The regulation of furosemide in many racing jurisdictions is based on paired limits of urinary SG (100 ng/ml). To validate this regulatory mechanism, a liquid chromatography/mass spectrometry/mass spectrometry method employing a solid-phase extraction procedure and furosemide-d5 as an internal standard was developed. The method was used to determine th...
Hair analysis as a novel investigative tool for the detection of historical drug use/misuse in the horse: a pilot study. Analysis of human hair for drug residues is being used increasingly as a diagnostic tool in the investigation of drug use and abuse. Hair analysis is complementary to urine/blood testing in that it can provide an extensive historical record of drug use, is noninvasive, impersonal and can facilitate retesting. However, the technique has not been studied in horses. Objective: That the systemic administration of drugs in horses could be identified by the detection of drug residues in hair. Objective: To evaluate hair analysis as a potential retrospective diagnostic test for drug administration in...
Effect of topical application of diclofenac liposomal suspension on experimentally induced subcutaneous inflammation in horses. To determine whether 1% diclofenac liposomal suspension (DLS) ointment would be absorbed transdermally and attenuate experimentally induced subcutaneous inflammation in horses. Methods: 7 healthy adult horses. Methods: Inflammation was produced by injecting 1% sterile carrageenan into subcutaneously implanted tissue cages 8 hours before (time -8) and at the time of application of test ointment. A crossover design was used. Horses received 1 of 2 treatments (topically administered control or DLS ointments) during 48 hours of carrageenan-induced subcutaneous inflammation. A single application of...
Pharmacokinetics and pharmacodynamic effects of amiodarone in plasma of ponies after single intravenous administration. Atrial fibrillation is a well-known heart disease in horses. The common therapy consists of administration of quinidine. More potent antiarrhythmic drugs have become available for human therapy and the use of these as alternatives to quinidine for equine antiarrhythmic therapy is a matter of interest. Amiodarone (AMD) is used in human medicine for treatment of many arrhythmias, including atrial fibrillation. Its disposition in horses has not yet been investigated. The purpose of this study was to measure the effect of single intravenous doses of amiodarone (5 and 7 mg/kg) on the surface electr...
Quantification and confirmation of flunixin in equine plasma by liquid chromatography-quadrupole time-of-flight tandem mass spectrometry. The method describes quantification and confirmation of flunixin in equine plasma by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC/Q-TOF/MS/MS). Samples were screened by enzyme-linked immunosorbent assay (ELISA) and only those samples presumptively declared positive were subjected to quantification and confirmation for the presence of flunixin by this method. The method is also readily adaptable to instrumental screening for the analyte. Flunixin was recovered from plasma by liquid-liquid extraction (LLE). The sample was diluted with 2 ml saturated phosphate buffer (pH ...
Effect of high intensity training on anaerobic capacity of middle gluteal muscle in Thoroughbred horses. We hypothesize that high intensity training for Thoroughbred horses that have been subjected to conventional training could further improve the metabolic properties of the middle gluteal muscle. Nine well-trained horses were subjected to high intensity (80-100% Vdot;O(2)max, 5 minx2) training for 12 weeks. Biopsy samples were obtained from the muscle before and after 4 and 12 weeks of training. Three of the 9 horses did not complete the training programme. In the remaining 6 horses, activities of succinic dehydrogenase (SDH), phosphofructokinase (PFK) and 3-hydroxy acyl CoA dehydrogenase (HAD)...
Liquid chromatography/electrospray ionization tandem mass spectrometric screening and confirmation methods for beta2-agonists in human or equine urine. Electrospray ionization (ESI) mass spectra of 19 common beta(2)-agonists were investigated in terms of fragmentation pattern and dissociation behavior of the analytes, proving the origin of fragment ions and indicating mechanisms of charge-driven and charge-remote fragmentation. Based on these data, liquid chromatographic/ESI tandem mass spectrometric (LC/ESI-MS/MS) screening and confirmation methods were developed for doping control purposes. These procedures employ established sample preparation steps including either acidic or enzymatic hydrolysis, alkaline extraction and, in the case of eq...
Pharmacokinetics and plasma concentrations of acetylsalicylic acid after intravenous, rectal, and intragastric administration to horses. Six healthy adult horses (5 mares and 1 stallion) were given a single dose of acetylsalicylic acid (ASA), 20 mg/kg of body weight, by intravenous (IV), rectal, and intragastric (IG) routes. Serial blood samples were collected via jugular venipuncture over a 36-h period, and plasma ASA and salicylic acid (SA) concentrations were determined by high-performance liquid chromatography. After IV administration, the mean elimination rate constant of ASA (+/- the standard error of the mean) was 1.32 +/- 0.09 h(-1), the mean elimination half-life was 0.53 +/- 0.04 h, the area under the plasma concentra...
Large-volume injection gas chromatography-mass spectrometry for automated broad-spectrum drug screening in horse urine. A rapid, sensitive, and rugged method for detecting drugs and drug metabolites in extracts of horse urine is described. The use of large-volume injection (LVI) gas chromatography-mass spectrometry (GC-MS) for analysis of horse urine extracts allowed automation of the derivatization procedure and reduction of the sample volume from 5 mL to 1 mL of urine. An autosampler and temperature-programmable inlet were used to automatically dissolve the sample extract and form trimethylsilyl derivatives of over 200 analytes. The suitability of this procedure for routine GC-MS detection of approximately 80...
The detection and biotransformation of guanabenz in horses: a preliminary report. Guanabenz (2,6-dichlorobenzylidene-amino-guanidine) is a centrally acting antihypertensive drug whose mechanism of action is via alpha2 adrenoceptors or, more likely, imidazoline receptors. Guanabenz is marketed as an antihypertensive agent in human medicine (Wytensin tablets, Wyeth Pharmaceuticals). Guanabenz has reportedly been administered to racing horses and is classified by the Association of Racing Commissioners International as a class 3 foreign substance. As such, its identification in a postrace sample may result in significant sanctions against the trainer of the horse. The present ...
Seasonal changes in plasma concentrations of cecum-derived amines in clinically normal ponies and ponies predisposed to laminitis. To measure concentrations of amines formed in the cecum of clinically normal ponies, determine amine concentrations in plasma samples collected in spring and winter, and compare concentrations of amines and serotonin in plasma samples obtained from clinically normal ponies and ponies predisposed to laminitis. Methods: Cecal contents obtained from 10 ponies euthanatized at an abattoir and blood samples obtained from 42 adult ponies. Methods: Cecal contents were assayed for amines by high-performance liquid chromatography (HPLC). Blood samples were collected at various times of the year from 20 ...
Quantitative analysis of eumelanin and pheomelanin in humans, mice, and other animals: a comparative review. The color of hair, skin, and eyes in animals mainly depends on the quantity, quality, and distribution of the pigment melanin, which occurs in two types: black to brown eumelanin and yellow to reddish pheomelanin. Microanalytical methods to quantify the amounts of eumelanin and pheomelanin in biological materials were developed in 1985. The methods are based on the chemical degradation of eumelanin to pyrrole-2,3,5-tricarboxylic acid and of pheomelanin to aminohydroxyphenylalanine isomers, which can be analyzed and quantitated by high performance liquid chromatography. This review summarizes a...
