Gas chromatography is an analytical technique used to separate and analyze compounds that can be vaporized without decomposition. In equine research, it is employed to identify and quantify various substances within biological samples, such as blood, urine, and tissue. This method is particularly useful for detecting and measuring volatile organic compounds, metabolites, and residues of medications or performance-enhancing drugs in horses. By providing detailed compositional data, gas chromatography aids in understanding metabolic processes, assessing nutritional status, and ensuring compliance with anti-doping regulations. This page compiles peer-reviewed research studies and scholarly articles that explore the applications, methodologies, and advancements of gas chromatography in equine science.
Bondesson U, Johansson IM.This study demonstrates the development of a method using gas chromatography-mass spectrometry for determining nefopam, a non-narcotic pain reliever that is sometimes abused in horse doping, in equine plasma. Background […]
Johansson IM, Anlér EL, Bondesson U, Schubert B.Two metabolites of meclofenamic acid have been isolated from equine urine. Both metabolites are found to be monohydroxylated forms of meclofenamic acid by gas chromatography-mass spectrometry after extractive alkylation. The parent drug and the metabolites are separated by reversed-phase liquid chromatography on a Spherisorb ODS column, using methanol-phosphate buffer eluents and UV detection at 280 nm. The structure of the metabolites is discussed on the basis of LC, TLC and GC-MS data.
Silberzahn P, Dehennin L, Zwain I, Reiffsteck A.Follicular fluid was obtained from equine follicles at different stages of development as determined by ultrastructural study. Gas chromatography-mass spectrometry associated with stable isotope dilution permitted the demonstration of high levels of 4-estrene-3,7-dione and 17 beta-hydroxy-4-estren-3-one, 17 beta-hydroxy-4-estren-3-one levels often being about 10 times higher than those of testosterone. These findings suggest that in the mare ovary, an aromatizing pathway may proceed using these 19-norsteroids as intermediates. As a consequence of this high level of 19-norsteroids, testosterone...
Potter KA, Leid RW, Kolattukudy PE, Espelie KE.Lipoxygenase products of arachidonic acid are important mediators of inflammation, affecting several aspects of cell function. Monohydroxyeicosatetraenoic acid (mono-HETE) and 5,12-dihydroxyeicosatetraenoic acid (LTB4) enhance migration of both neutrophils and eosinophils in several species. The relative ability of positional isomers of HETE and of LTB4 to affect migration of equine eosinophils was studied. The 5, 8, 9, 11, 12, and 15 isomers of HETE were prepared by autooxidation of arachidonic acid, separated by sequential normal phase and reverse phase high performance liquid chromatography...
Neill SD, O'Brien JJ, McMurray CH, Blanchflower WJ.Cellular fatty acid compositions of contagious equine metritis isolates and three reference Haemophilus equigenitalis cultures were determined by gas chromatography. The chromatographic data were standardised and normalised fatty acid methyl ester (FAME) profiles were produced. The profiles were compared visually and similarity indices were determined using a computer peak matching method. There was little difference between the profiles of the three reference strains, each strain being characterised by three major fatty acids; C 18:1, C 16:0 and 30H-C 14:0. Variations in cultural conditions h...
Silberzahn P, Zwain I, Martin B.Blood testosterone levels were measured by RIA and gas chromatography-mass spectrometry in the pregnant mare. They were found to increase from the very beginning of pregnancy, reaching peak values 10 times higher than the basal values at the seventh month and then to return to basal values by the week after parturition. Testosterone binding by plasma proteins was investigated in nonpregnant and pregnant mares throughout gestation. Equilibrium dialysis and gel equilibration methods did not reveal any blood specific testosterone-binding activity at any gestational stage. Hence, blood testosteron...
Reiffsteck A, Dehennin L, Scholler R.Estrone, 2-methoxyestrone and estradiol-17 beta have been definitely identified in seminal plasma of man, bull, boar and stallion by high resolution gas chromatography associated with selective monitoring of characteristic ions of suitable derivatives. Quantitative estimations were performed by isotope dilution with deuterated analogues and by monitoring molecular ions of trimethylsilyl ethers of labelled and unlabelled compounds. Concentrations of unconjugated and total estrogens are reported together with the statistical evaluation of accuracy and precision.
Muir WW, Sams RA, Huffman RH, Noonan JS.The cardiopulmonary, behavioral, and pharmacokinetic properties of diazepam were determined in horses. Heart rate, cardiac output, mean pulmonary artery, aortic and right atrial blood pressures, respiratory rate, and arterial pH and blood gas values did not change after IV diazepam (0.05, 0.1, 0.2, 0.4 mg/kg) administration. Increasing doses of diazepam resulted in signs of muscle weakness including fixed stance, muscle fasciculations of the head, neck, and thorax muscles, ataxia, and then recumbency. Dosages of diazepam exceeding 0.2 mg/kg produced mild sedation. Behavioral changes persisted ...
Jörg A, Henderson WR, Murphy RC, Klebanoff SJ.Horse eosinophils purified to greater than 98% generated slow reacting substance (SRS) when incubated with the calcium ionophore A23187. On a per cell basis, eosinophils generated four to five times the SRS produced by similarly treated horse neutrophils. Eosinophil SRS production was inhibited by 5,8,11,14-eicosatetraynoic acid and augmented by indomethacin and arachidonic acid, suggesting that it was a product(s) of the lipoxygenase pathway of arachidonic acid metabolism. Compounds with SRS activity were purified by high-pressure liquid chromatography (HPLC) and identified by ultraviolet spe...
Igwe OJ, Blake JW.An analytical gas/liquid chromatographic (GLC) protocol is described for the quantitation of pemoline in biological fluids of the horse. Plasma samples containing known quantities of pemoline and its analog as an internal standard (IS) were deproteinized with 5-sulfosalicylic acid, heated at 80 degree C, and centrifuged. 5-Phenyl-2,4-oxazolidinedione, the hydrolytic product of pemoline in acid medium, was extracted with dichloromethane (DCM). The organic layer was in turn re-extracted with 1% NaHCO3. The aqueous layer was acidified with HCI, and re-extracted with DCM, which was evaporated to d...
Jonckheere JA, Thienpont LM, De Leenheer AP, De Backer P, Debackere M, Belpaire FM.A method has been developed for quantification of bromhexine in plasma using gas chromatography mass spectrometry with selected ion monitoring. A deuterium labelled analogue was synthesized and used as the internal standard. To evaluate the gas chromatographic electron capture detection method described earlier, 23 plasma samples have been analysed by both techniques. Although a good correlation was shown, selected ion monitoring was superior to the electron capture detection method for levels below 3 ng ml-1. The mass spectrometric method has also been used to set up a pharmacokinetic study o...
Houghton E, Oxley GA, Moss MS, Evans S.A method is described to confirm the presence of 19-nortestosterone metabolites in urine after the administration of veterinary preparations of this anabolic steroid to horses. The method is based upon the detection, by gas chromatography mass spectrometry or selected ion monitoring, of an isomer of estrane-3,17-diol in the urine.
Lauer BH, Baker BE.Casein was isolated from the milks of the following species: cow, horse, pig, reindeer, caribou, moose, harp seal, musk-ox, polar bear, dall sheep, and fin whale. The caseins were subjected to acid hydrolysis, the resultant amino acids were converted to their n-butyl-N-trifluoroacetyl esters, and the amino acid composition of the caseins was determined by gas chromatographic analysis of these esters. Notable among the results was the close similarity, with respect to amino acid composition, of reindeer and caribou caseins. The results of the amino acid analyses of the other caseins are present...
Delbeke FT, Debackere M.It is well known that nikethamide (N,N-diethylnicotinamide, CoramineR) is metabolized very rapidly to nicotinamide. Hence, there is difficulty in proving that nikethamide has been used as a doping substance because nicotinamide is a normal physiological metabolite in the organism as well as a vitamin preparation. However, an intermediate metabolite (N-ethylnicotinamide) was found by us in the urine of horses treated with CoramineR. This was characterized by gas chromatography/mass spectrometry, and synthesized and identified as being N-ethylnicotinamide. The excretion and metabolism of niketha...
Nakajima T, Matsumoto T.Horse racing in Japan consists of two systems, the National (10 racecourses) and the Regional public racing (32 racecourses) having about 2,500 racing meetings in total per year. Urine or saliva samples for dope testing are collected by the officials from thw winner, second and third, and transported to the laboratory in a frozen state. In 1975, 76, 117 samples were analyzed by this laboratory. The laboratory provides the following four methods of analysis, which are variously combined by request. (1) Method for detection of drugs extracted by chloroform from alkalinized sample. (2) Methods fo...
Glöckner WM, Newman RA, Dahr W, Uhlenbruck G.Phenol extraction of horse, sheep, cow, pig and human erythrocyte membranes and human milk fat globule membranes gave glycoprotein fractions, all of which were shown by gas chromatography to contain the reduced disaccharide beta-D-galactosyl (1-3)-N-acetyl-D-galactosaminital after treatment with alkaline borohydride. Cow and pig erythrocyte membrane glycoproteins were found however to contain much lower amounts than the erythrocyte membrane glycoproteins of the other species tested. After gel filtration, a tetrasaccharide was isolated from horse and sheep glycoproteins containing the disacchar...
O'Neill HJ, Gershbein LL.The lipids of human and equine smegma pools were saponified and the total fatty acids submitted to temperature programmed gas chromatography (GC) analysis. In contrast to the human products, the horse smegma fatty acids contained very low odd saturated as well as olefinic branched chain acid contents. The cyclopropane fatty acid, 9,10-methyleneoctadecanoic acid, occurred in smegma sampled from men over 35 years of age but could not be detected in the pool from persons of 17-20 years of age nor in any of the equine mixtures. The alcoholic fraction from horse smegma contained about 85% sterol, t...
Dybing O, Peoples SA.The determination of amphetamine in body fluids is of interest in veterinary toxicology because of the possible use of amphetamine in the doping of race horses. Many types of methods for its detection and determination have been developed. In the newest methods gas chromatography and mass spectrometry have been applied, making it possible to detect and identify 1 µg amphetamine in blood samples ( 1970).
Nakajima T, Matsumoto T.Horse racing in Japan consists of two systems, the National (10 racecourses) and the Regional public racing (32 racecourses) having about 2,500 racing meetings in total per year. Urine or saliva samples for dope testing are collected by the officials from thw winner, second and third, and transported to the laboratory in a frozen state. In 1975, 76, 117 samples were analyzed by this laboratory. The laboratory provides the following four methods of analysis, which are variously combined by request. (1) Method for detection of drugs extracted by chloroform from alkalinized sample. (2) Methods fo...
Choi MH, Kim JY, Chung BC.A highly specific method is described for measuring the testosterone:epitestosterone ratio in equine urine by gas chromatography-mass spectrometry (GC-MS) with stable isotope internal standards. The procedure was based on Serdolit Pad-1 resin extraction, enzymatic hydrolysis, and chemical derivatisation prior to instrumental analysis. The mixed derivatives, 3-trimethylsilyl-17-pentafluorophenyldimethylsilyl ether (3-TMS-17-flophemesyl) testosterone and epitestosterone, were found to have excellent analytical properties. The specificity of the derivatisation method exploits a unique feature of ...
Varma KJ, Powers TE, Powers JD.A single-dose pharmacokinetic study of chloramphenicol in propylene glycol was done in 6 horses after 22 mg/kg was administered IV. Serum drug concentrations obtained at various predetermined intervals were determined by an electroncapture gas-chromatographic technique. The time-concentration data were described by a 2-compartment open model, and various pharmacokinetic variables were estimated. The median elimination rate constant was estimated to be -0.0185 minute-1 (-0.0225 to -0.0148 minute-1), and the median half-life was 37.36 minutes (30.74 to 46.90 minutes). The median apparent volume ...
Dehennin L, Petit E, Bonnaire Y, Bruyas JF, Le Bizec B, Plou P.Rules of horse racing stipulate that pregnant mares may compete under definite conditions of date, because early pregnant status may be misused for the sake of enhancing physical performance by putative anabolic steroid action. Screening for pregnancy is generally performed by plasma equine gonadotrophin (eCG) immunoassay, which covers the period between Days 40 and 120. In common screening for urinary anabolic steroids performed by gas chromatography-mass spectrometry, inclusion of two complementary criteria, i.e. the evaluation of total conjugates of 5(10)-estrene-3beta,17alpha-diol (EED) an...
de Brabander HF, van Hende J, Batjoens P, Hendriks L, Raus J, Smets F, Pottie G, van Ginkel L, Stephany RW.When residues of nortestosterone (NT) were found in the urine of cattle, racehorses or bodybuilders, exogenic administration was thought to be proven. In previous literature, no records were found of the endogenic presence of this molecule. In the horse-racing world, Houghton and Courthot found that NT is normally present in the urine of the stallion. Belgian and Dutch researchers found that NT is also present in the urine and edible parts of the intact boar. Vandenbroeck et al. (1991) suggested the endogenous presence of NT (in the beta form) in the pregnant cow. Meyer (1992) reported the pre...
de Rebière de Pouyade G, Salciccia A, Ceusters J, Deby-Dupont G, Serteyn D, Mouithys-Mickalad A.The endothelium plays an active role in ischemia/reperfusion injuries. Herein, we report the effect of a single or successive cycles of anoxia/reoxygenation (A/R) on the mitochondrial respiratory function of equine endothelial cells (cultured from carotids) monitored by high resolution oxymetry, and on their production of reactive oxygen species (ROS). ROS were measured by electron paramagnetic resonance (ESR) using POBN and DMPO spin traps, and by gas chromatography (GC) of ethylene released by ROS-induced α-keto-γ-(methylthio)butyric acid (KMB) oxidation. The oxygen consumption significant...
O'Neill HJ, Gershbein LL.The lipids of human and equine smegma pools were saponified and the total fatty acids submitted to temperature programmed gas chromatography (GC) analysis. In contrast to the human products, the horse smegma fatty acids contained very low odd saturated as well as olefinic branched chain acid contents. The cyclopropane fatty acid, 9,10-methyleneoctadecanoic acid, occurred in smegma sampled from men over 35 years of age but could not be detected in the pool from persons of 17-20 years of age nor in any of the equine mixtures. The alcoholic fraction from horse smegma contained about 85% sterol, t...
Hagedorn HW, Meiser H, Zankl H, Schulz R.Due to its tranquilizing properties, the tricyclic antidepressant doxepin may be misused as a doping agent in competition horses. Therefore, efficient analytical procedures are required to detect this drug in samples submitted for doping control. To screen for parent doxepin in equine blood and urine, a less specific method has been accepted employing gas chromatography (GC) combined with electron impact (EI) mass spectrometry (MS). The aim of this study was identification of doxepin metabolites providing more specific MS data to verify positives resulting from screening. Thus, after a horse w...
Ho EN, Kwok WH, Leung DK, Wan TS, Wong AS.Turinabol (4-chloro-17alpha-methyl-17beta-hydroxy-1,4-androstadien-3-one) is a synthetic oral anabolic androgenic steroid. As in the case of other anabolic steroids, it is a prohibited substance in equine sports. The metabolism of turinabol in human has been reported previously; however, little is known about its metabolic fate in horses. This paper describes the studies of both the in vitro and in vivo metabolism of turinabol in racehorses with an objective to identify the most appropriate target metabolites for detecting turinabol administration. For the in vitro studies, turinabol was incub...
Bonnaire Y, Dehennin L, Plou P, Toutain PL.A pharmacological dose of a long-acting testosterone ester, testosterone hexahydrobenzoate, was administered intramuscularly to two mares. The time course for some characteristic metabolites in blood and urine was then studied using an analytical method based on gas chromatography-mass spectrometry associated with stable isotope dilution. Among the plasma analytes, testosterone glucuronide was found to be the most adequate indicator for the monitoring of exogenous testosterone up to 2 weeks postadministration if a threshold value of 200 ng/L was applied. In urine, the simultaneous measurement ...
Kristinsson J, Thordarson TH, Johannesson T.The pharmacokinetics of lignocaine was studied in four Icelandic horses after infiltration anaesthesia. A total of 240 mg of the drug was injected on either side of the left foreleg, over the medial and lateral branches of the palmar nerve. Blood samples were collected up to seven hours after injection and the concentrations of the drug in plasma were determined by gas chromatography/mass spectrometry. The results showed that lignocaine was rapidly absorbed. A mean maximum concentration of 232 ng/ml was observed after 20 minutes. In three of the horses the decline in the plasma concentration o...
Wong AS, Ho EN, Wan TS, Lam KK, Stewart BD.Oxyguno (4-chloro-17α-methyl-17β-hydroxy-androst-4-ene-3,11-dione) is a synthetic oral anabolic androgenic steroid commercially available without a prescription. Manufacturers of oxyguno claim that its anabolic effect in metabolic enhancement exceeds that of the classic anabolic steroid testosterone by seven times, but its androgenic side-effects are only twelve percent of testosterone. Like other anabolic androgenic steroids, oxyguno is prohibited in equine sports. The metabolism of oxyguno in either human or horse has not been reported and therefore little is known about its metabolic fate...
Igwe OJ, Blake JW.An analytical gas/liquid chromatographic (GLC) protocol is described for the quantitation of pemoline in biological fluids of the horse. Plasma samples containing known quantities of pemoline and its analog as an internal standard (IS) were deproteinized with 5-sulfosalicylic acid, heated at 80 degree C, and centrifuged. 5-Phenyl-2,4-oxazolidinedione, the hydrolytic product of pemoline in acid medium, was extracted with dichloromethane (DCM). The organic layer was in turn re-extracted with 1% NaHCO3. The aqueous layer was acidified with HCI, and re-extracted with DCM, which was evaporated to d...
Roig M, Segura J, Ventura R.A method to quantify metabolites of 17beta-nandrolone (17betaN) in boar and horse urine has been optimized and validated. Metabolites excreted in free form were extracted at pH 9.5 with tert-butylmethylether. The aqueous phases were applied to Sep Pak C18 cartridges and conjugated steroids were eluted with methanol. After evaporation to dryness, either enzymatic hydrolysis with beta-glucuronidase from Escherichia coli or solvolysis with a mixture of ethylacetate:methanol:concentrated sulphuric acid were applied to the extract. Deconjugated steroids were then extracted at alkaline pH with tert-...
Hewetson M, Aaltonen K, Tulamo RM, Sankari S.A simple and accurate method for quantifying sucrose in equine serum that can be applied to sucrose permeability testing in the horse was developed and validated using gas chromatography with flame ionization detection. The assay provided an acceptable degree of linearity, accuracy, and precision at concentrations of sucrose as low as 2.34 μmol/l and as high as 20.45 μmol/l. Percentage recovery of sucrose from serum ranged from 89% to 102%; repeatability and intermediate precision (relative standard deviation) ranged from 3.6% to 6.7% and 4.1% to 9.3%, respectively. The limit of detection wa...
Bosken JM, Lehner AF, Hughes CG, Woods WE, Camargo FC, Harkins JD, Boyles J, Tobin T.Isoxsuprine is used to treat navicular disease and other lower-limb problems in the horse. Isoxsuprine is regulated as a class 4 compound by the Association of Racing Commissioners, International (ARCI) and, thus, requires regulatory monitoring. A gas chromatography-mass spectrometry method utilizing electron impact ionization was developed and validated for the quantitation of isoxsuprine in equine plasma or equine urine. The method utilized robotic solid-phase extraction and tri-methyl silyl ether products of derivatization. Products were bis-trimethylsilyl (TMS) isoxsuprine and tris-TMS rit...
Jonckheere JA, Thienpont LM, De Leenheer AP, De Backer P, Debackere M, Belpaire FM.A method has been developed for quantification of bromhexine in plasma using gas chromatography mass spectrometry with selected ion monitoring. A deuterium labelled analogue was synthesized and used as the internal standard. To evaluate the gas chromatographic electron capture detection method described earlier, 23 plasma samples have been analysed by both techniques. Although a good correlation was shown, selected ion monitoring was superior to the electron capture detection method for levels below 3 ng ml-1. The mass spectrometric method has also been used to set up a pharmacokinetic study o...
Dyke TM, Sams RA.The objective of this study was to determine the urinary excretion of methylxanthines in horses following ingestion of chocolate over eight days. The study was performed in response to gas chromatography-mass spectrometry (GC-MS) confirmation of the presence of caffeine in a positive urine test in a racehorse. The trainer of the horse alleged that he often administered chocolate-coated peanuts as treats to his horses, and he believed that the ingestion of chocolate was responsible for the positive urine test. The urinary excretion of theobromine and caffeine after the ingestion of chocolate-co...
Choi TLS, Wong JKY, Kwok WH, Curl P, Mechie S, Wan TSM.Methylstenbolone (2,17α-dimethyl-5α-androst-1-en-17β-ol-3-one) is a synthetic anabolic and androgenic steroid (AAS) sold as an oral 'nutritional supplement' under the brand names 'Ultradrol', 'M-Sten' and 'Methyl-Sten'. Like other AASs, methylstenbolone is a prohibited substance in both human and equine sports. This paper describes the studies of the in vitro and in vivo metabolism of methylstenbolone in horses using LC/HRMS, GC/MS and GC/MS/MS. Phase I in vitro metabolic study of methylstenbolone was performed using homogenised horse liver. Hydroxylation was the only biotransformation obse...
Benoit M, Lingen K, Taddei LM, Heffron BT, Hurt L, Lokanc JA, Lingner K, Cardenas E, Flores S, Mayer D, Pilipiak D, Folker-Calderon D, Negrusz A.Pyrilamine (mepyramine) is an H1-receptor antagonist used in human and veterinary medicine. It has the potential to produce central nervous system effects in horses and therefore may have some impact on an outcome of a horse race. A single oral dose of pyrilamine (300 mg/horse) was given to three animals. Serum samples were collected before drug administration and at 0.25, 0.5, 1, 2, 4, 6, 24, 48, 72, 96, 120, and 144 h, and 7, 8, 9, 10, 11, 12, and 13 days post-administration. Urine samples were collected at 0-1, 1-2, 2-4, 4-6, 24, 48, 72, 96, 120, and 144 h, and 7, 8, 9, 10, 11, 12, 13 days ...
