Phenylbutazone blood and urine concentrations, pharmacokinetics, and effects on biomarkers of inflammation in horses following intravenous and oral administration of clinical doses.

Phenylbutazone (PBZ) is a potent mon-steroidal anti-inflammatory drug used commonly in performance horses. The objectives of the current study were to describe blood and urine concentrations and the pharmacokinetics of PBZ and its metabolites following intravenous (IV) and oral administration and to describe the duration of pharmacodynamic effect. To that end, 17 horses received an IV administration and 18 horses an oral administration of 2 g of PBZ. Blood and urine samples were collected prior to and for up to 96 hours post drug administration. Whole blood samples were collected at various time points and challenged with lipopolysaccharide or calcium ionophore to induce ex vivo synthesis of eicosanoids. Concentrations of PBZ and eicosanoids were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and non-compartmental pharmacokinetic analysis performed on concentration data from IV and oral administration. Serum concentrations of PBZ and its metabolites were below the limit of quantitation at 96 hours post administration. The volume of distribution at steady state, systemic clearance, and terminal half-life was 0.194 ± 0.019 L/kg, 23.9 ± 4.48 mL/h/kg, and 10.9 ± 5.32 hours, respectively. The terminal half-life following oral administration was 13.4 ± 3.01 (paste) and 15.1 ± 3.96 hours (tablets). Stimulation of PBZ treated whole blood with lipopolysaccharide and calcium ionophore resulted in an inhibition of TXB2 , PGE2 , LTB4 and 15-HETE production for a prolonged period of time post drug administration. The results of this study suggest that PBZ has a prolonged anti-inflammatory following IV or oral administration of 2 g to horses.

Investigation of the role of environmental contamination in the occurrence of residues of the veterinary drug phenylbutazone in cattle.

Phenylbutazone is a non-steroidal anti-inflammatory drug licensed for use in horses to treat musculoskeletal disorders. It is not permitted in the European Union for use in animals destined for the food chain. Official statistics provided by the European Food Safety Authority (EFSA) show that 0.18% of bovines tested in the European Union between 2008 and 2014 for non-steroidal anti-inflammatory drugs were non-compliant, with phenylbutazone representing over 28% of these. Anecdotal evidence suggests animals that have not been treated with the drug may have produced non-compliant samples, possibly through some form of contamination. In this study, ultra-high-performance liquid chromatography coupled with mass-spectrometric detection was applied to bovine plasma samples to determine if detectable residues (CCα = 0.28 ng ml-1) may occur in untreated animals as a result of environmental contamination through normal farming practice. The study demonstrates that waste from animals treated with phenylbutazone, and spread on an area of pasture, can contaminate untreated bovines grazing the pasture many weeks later. It was determined that this contamination, which can persist over a significant period, may be due to the ingestion of as little as 30 µg phenylbutazone by a 500 kg bullock.

Pharmacokinetics of methocarbamol and phenylbutazone in exercised Thoroughbred horses.

Methocarbamol (MCBL) is commonly used in performance horses for the treatment of skeletal muscle disorders. Current regulatory recommendations for show horses and racehorses are based on a single oral dose of 5 g, although doses in excess of this are often administered. The goal of the current study was to characterize the disposition of MCBL following higher dose administration and administration in combination with another commonly used drug in performance horses, phenylbutazone (PBZ). Exercised Thoroughbred horses were administered various doses of MCBL as a sole agent and MCBL in combination with PBZ. Blood samples were collected at various times, concentrations of MCBL and PBZ measured using LC-MS/MS and pharmacokinetic parameters calculated using compartmental analysis. Following administration of 15 g of MCBL, either as part of a single- or multiple-dose regimen, a number of horses exceeded the Association of Racing Commissioners International and the United States Equestrian Federation's recommended regulatory threshold at the recommended withdrawal time. There was not a significant difference between horses that received only MCBL and those that received MCBL and PBZ. Results of the current study support an extended withdrawal guideline when doses in excess of 5 g are administered.

Pharmacokinetics, pharmacodynamics, metabolism, toxicology and residues of phenylbutazone in humans and horses.

The presence of horse meat in food products destined for human consumption and labelled as beef has raised several concerns of public interest. This review deals solely with one aspect of these concerns; samples of equine tissue from horses destined for the human food chain have tested positive for the non-steroidal anti-inflammatory drug, phenylbutazone. The safety of some or all such foods for human consumers is a major concern, because it was shown many years ago that phenylbutazone therapy in humans can be associated with life threatening blood dyscrasias. As an initial basis for assessing the potential toxicity of foods containing phenylbutazone and its metabolites, this article reviews (1) the pharmacokinetic, pharmacodynamic, metabolic and toxicological profiles of phenylbutazone, with particular reference to horses and humans; (2) toxicity data in laboratory animals; (3) phenylbutazone residues in food producing species, and (4) as a preliminary assessment, the potential hazard associated with the consumption of horse meat containing phenylbutazone and its metabolites. Since phenylbutazone cannot be classified as a carcinogenic substance in humans, and noting that blood dyscrasias in humans are likely to be dose and treatment duration-dependent, the illegal and erratic presence of trace amount residues of phenylbutazone in horse meat is not a public health issue.

PAMAM dendrimer-drug interactions: effect of pH on the binding and release pattern.

Understanding the dendrimer-drug interaction is of great importance to design and optimize the dendrimer-based drug delivery system. Using atomistic molecular dynamics (MD) simulations, we have analyzed the release pattern of four ligands (two soluble drugs, namely, salicylic acid (Sal), L-alanine (Ala), and two insoluble drugs, namely, phenylbutazone (Pbz) and primidone (Prim)), which were initially encapsulated inside the ethylenediamine (EDA) cored polyamidoamine (PAMAM) dendrimer using the docking method. We have computed the potential of mean force (PMF) variation with generation 5 (G5)-PAMAM dendrimer complexed with drug molecules using umbrella sampling. From our calculated PMF values, we observe that soluble drugs (Sal and Ala) have lower energy barriers than insoluble drugs (Pbz and Prim). The order of ease of release pattern for these drugs from G5 protonated PAMAM dendrimer was found to be Ala > Sal > Prim > Pbz. In the case of insoluble drugs (Prim and Pbz), because of larger size, we observe much nonpolar contribution, and thus, their larger energy barriers can be reasoned to van der Waals contribution. From the hydrogen bonding analysis of the four PAMAM-drug complexes under study, we found intermolecular hydrogen bonding to show less significant contribution to the free energy barrier. Another interesting feature appears while calculating the PMF profile of G5NP (nonprotonated)-PAMAM-Pbz and G5NP (nonprotonated)-PAMAM-Sal complex. The PMF was found to be less when the drug is bound to nonprotonated dendrimer compared to the protonated dendrimer. Our results suggest that encapsulation of the drug molecule into the host PAMAM dendrimer should be carried out at higher pH values (near pH 10). When such complex enters the human body, the pH is around 7.4 and at that physiological pH, the dendrimer holds the drug tightly. Hence the release of drug can occur at a controlled rate into the bloodstream. Thus, our findings provide a microscopic picture of the encapsulation and controlled release of drugs in the case of dendrimer-based host-guest systems.

Phenylbutazone and flunixin meglumine used singly or in combination in experimental lameness in horses.

REASON FOR PERFORMING STUDY: Using an adjustable heart bar shoe model of foot pain, the objective of this study was to test the hypothesis that the combined use of phenylbutazone (PBZ) and flunixin meglumine (FM) would prove more efficacious in alleviating lameness than either drug alone. MATERIALS AND METHODS: One hour after induction of lameness at weekly intervals, 8 healthy adult Thoroughbred horses randomly underwent one of 4 i.v. treatments: saline (SAL) placebo (1 ml/45 kg bwt), PBZ (4.4 mg/kg bwt), FM (1.1 mg/kg bwt) or PBZ+FM (at the same dosages as given individually). Heart rate (HR) and lameness score (LS) responses were assessed in a blinded manner every 20 min for 5 h after lameness induction and then hourly for 12 h after treatment. Jugular venous blood samples were obtained at -1, 0, 0.05, 1, 2, 4, 6, 8, 10 and 12 h and subsequently analysed for drug concentrations. Repeated measures ANOVA and post hoc Tukey's test were used to identify analgesic effects at a significance level of P<0.05. RESULTS: Heart rate was lower in all nonsteroidal anti-inflammatory drug (NSAID)-treated trials from 2 h to 10 h post treatment (P<0.05). Analgesic effects of FM and PBZ+FM, as evidenced by decreases in HR, lasted for 12 h post treatment (P<0.05). Lameness score decreased earlier in PBZ and PBZ+FM trials than in FM trials (P<0.05) and the analgesic effect on LS lasted for 12 h post treatment for all NSAID trials (P<0.05). Peak PBZ plasma concentration was 73.7 ± 6.0 and 77.9 ± 5.5 µg/ml. Peak FM concentration was 12.0 ± 0.8 and 13.7 ± 1.0 µg/ml. CONCLUSIONS: It was concluded that the combination of PBZ+FM was not more effective than either PBZ or FM alone. These data do not support the hypothesis that the combination is more efficacious at these dosages than either drug alone in this model of acute foot pain.

Muerte súbita asociada a hipersensibilidad tipo I en un equino pura sangre de carrera / Sudden death associated to type I hypersensitivity in a racing horse

El objetivo de este estudio fue reportar un caso de hipersensibilidad tipo I con muerte súbita en un equino Pura Sangre de Carrera en el Hipódromo “La Rinconada” Caracas, Venezuela. Se tomaron muestras de sangre y orina para estudios toxicológicos mediante la técnica de ELISA competitivo. Se le práctico la técnica de necropsia, fueron colectadas muestras de musculo, tejido pulmonar, hepático, renal, gástrico, esplénico, corazón y sistema nervioso central para estudio histopatológico, las muestras fueron procesados por los métodos convencionales histológicos. Los hallazgos de necropsia fueron flebitis severa en vena yugular derecha, con hematoma en el surco yugular. Edema severo de glotis, edema, congestión y hemorragia pulmonar. Hemorragia petequial subendocardica. Bazo esplenocontraido y con focos de necrosis de coagulación. Hidronefrosis aguda con hematuria. Hígado con patrón lobulillar acentuado. El resto de los órganos con evidente congestión y hemorragia. Los cortes histológicos evidenciaron edema, congestión y hemorragia pulmonar severa. Hemorragia subepicardica marcada. Edema subcapsular esplénico y necrosis centro-folicular. Degeneración hidropica tubular, necrosis tubular aguda. Necrosis de corteza renal. Los estudios toxicológicos permitieron la detección de furosemida y fenilbutazona en las muestras de sangre y orina. En conclusión se reporta un síndrome de hipersensibilidad tipo I asociado a la administración de un producto comercial a base de Vitamina E 80mg, Pangamato sódico (B15) 1 mg, Selenio Sódico 0.6 mg, Antioxidantes y Vehículos Solubles c.s.p. con colapso, shock y muerte aguda en un equino Pura Sangre de Carrera mediante un estudio multidisciplinario clínico, anatomopatologico y toxicológico.

The aim of this study was to report a case of type I hypersensitivity to sudden death in a Thoroughbred race horses at the Hippodrome “La Rinconada” Caracas, Venezuela. Samples of blood and urine for toxicology studies using competitive ELISA. He practiced the technique of necropsy, samples were collected from muscle, lung tissue, liver, kidney, stomach, spleen, heart and central nervous system for histopathological examination, samples were processed by conventional histological methods. Autopsy findings were severe phlebitis right jugular vein, with hematoma in the jugular groove. Severe edema of glottis edema, pulmonary congestion and hemorrhage. Subendocardial petechial hemorrhage. Esplenocontraido Spleen foci of necrosis and coagulation. Hydronephrosis with acute hematuria. Liver accentuated lobular pattern. The rest of the organs with obvious congestion and hemorrhage. The histological sections showed edema, severe pulmonary congestion and hemorrhage. Marked subepicardial hemorrhage. Edema and necrosis subcapsular splenic follicular center. Tubular hydropic degeneration, acute tubular necrosis. Necrosis of renal cortex. Toxicological studies allowed the detection of furosemide and phenylbutazone in samples of blood and urine. In conclusion we report type I hypersensitivity syndrome associated with the administration of a commercial product based Vitamin E 80mg, sodium pangamate (B15) 1 mg, 0.6 mg; Sodium Selenium, Soluble Antioxidants and Vehicle qs with collapse, shock and acute death in a race Thoroughbred horses by a multidisciplinary clinical, pathological and toxicological.

Effect of an endurance-like exercise on the disposition and detection time of phenylbutazone and dexamethasone in the horse: application to medication control.

REASONS FOR PERFORMING STUDY: Equine antidoping rules were established to prevent a horse's performance being altered after the administration of prohibited substances, including approved drugs used for legitimate treatment. Veterinarians have to advise owners or trainers on appropriate withholding times to guarantee that their horses may safely compete after drug administration. In order to propose tailored withdrawal times, several horse organisations released detection time (DT) values, for the main veterinary drugs used in horses. One of the possible limits to the information provided by published DTs in horses is the fact that they are determined from classic pharmacokinetic studies performed at rest under laboratory conditions. In field conditions, training and exercise programmes may have an influence on drug elimination. METHODS: Dexamethasone (DMX) and phenylbutazone (PBZ) have been quantified in plasma and urine after solid phase extraction. The kinetic disposition of DXM (8 microg/kg) and PBZ (8 mg/kg) administered by i.v. route in 8 horses, was investigated in rest conditions and during a standardised 3 h test exercise according to a cross-over design. OBJECTIVES: The aim of the present study was to compare the kinetic disposition of 2 test drugs, DMX and PBZ in rest vs. exercising conditions. RESULTS: It was shown in 8 horses that a sustained 3 h of mild exercise slightly decreased the plasma clearance of both drugs (about 25% for DXM and 37% for PBZ) and this is mainly explained by the significant decrease of the corresponding hepatic clearance. In addition, as the volume of distribution was correlatively decreased, the plasma terminal half-life, which is a hybrid parameter of plasma clearance and volume of distribution, remains unchanged overall. CONCLUSION AND POTENTIAL RELEVANCE: Establishing DTs or withdrawal times (WTs) are relevant as plasma and urine half-lives, but not clearance, are the main determinants of DT length. Veterinarians may realistically decide upon a WT for a legitimate drug based on the corresponding DT obtained under resting conditions providing this drug has a low hepatic extraction ratio and a safety margin is added to allow for all possible sources of variability.

Nonsteroidal anti-inflammatory agents and musculoskeletal injuries in Thoroughbred racehorses in Kentucky.

Injuries sustained by horses during racing have been considered as an unavoidable part of horse racing. Many factors may be associated with the musculoskeletal injuries of Thoroughbred race horses. This study surveyed the amounts of nonsteroidal anti-inflammatory agents (NSAIDs) in injured horse's biological system (plasma) at Kentucky racetracks from January 1, 1995 through December 31, 1996. During that period, there were 84 catastrophic cases (euthanized horses) and 126 noncatastrophic cases. Plasma concentrations of NSAIDs were determined by High Performance Liquid Chromatography in injured and control horses. The possible role of anti-inflammatory agents in musculoskeletal injuries of Thoroughbred race horses was investigated by comparing the apparent concentrations of NSAIDs in injured horses to concentrations in control horses. The plasma concentrations of phenylbutazone and flunixin were higher in injured horses than in control horses. Most injured and control horses did not have a detectable level of naproxen in their plasma samples. Further studies must be carried out to determine whether horses with higher plasma concentrations of NSAIDs have an altered risk of musculoskeletal injuries compared with other horses.

Screening, quantification, and confirmation of phenylbutazone and oxyphenbutazone in equine plasma by liquid chromatography-tandem mass spectrometry.

A sensitive liquid chromatographic-tandem mass spectrometric method was developed and validated for screening, quantification, and confirmation of phenylbutazone and oxyphenbutazone in equine plasma. Analytes were recovered from plasma by liquid-liquid extraction followed by separation in a reversed-phase column and identification by mass spectrometry with selected reaction monitoring in negative electrospray ionization mode. Extraction recovery for both analytes was >80%. Limits of detection, quantification, and confirmation for both analytes were 0.01 microg/mL (S/N>or= 3), 0.05 microg/mL, and 0.05 microg/mL, respectively. The assay with d9-labeled phenylbutazone as internal standard (IS) was linear over a range of 0.05-20 microg/mL (r2>0.995). Intra- and interday precision in terms of coefficient of variation was less than 15%. Intra- and interday accuracy (bias%) was within 80-120%. Hemolysis of red blood cells decreased analyte signal intensity but did not affect quantification results because an isotope-labeled IS was used. Analytes were stable in plasma for 24 h at room temperature, 9 days at 4 degrees C, and 45 days at -20 degrees C and -70 degrees C. The method was successfully used in screening, quantification, and confirmation of phenylbutazone in post-competition plasma samples obtained from racehorses. The method is simple, rapid, and reliably reproducible.

Direct-injection screening for acidic drugs in plasma and neutral drugs in equine urine by differential-gradient LC-LC coupled MS/MS.

Direct-injection LC-LC hybrid tandem MS methods have been developed for undertaking broad-based screening for acidic drugs in protein-precipitated plasma and neutral doping agents in equine urine. In both analyses, analytes present in the matrix were trapped using a HLB extraction column before being refocused and separated on a Chromolith RP-18e monolithic analytical column using a controlled differential gradient generated by proportional dilution of the first column's eluent with water. Each method has been optimised by the adoption of a mobile phase and gradient that was tailored to enhance ionisation in the MS source while maintaining good chromatographic behaviour for the majority of the target drugs. The analytical column eluent was fed into the heated nebulizer (HN) part of the Duospray interface attached to a 4000 QTRAP mass spectrometer. Information dependent acquisition (IDA) with dynamic background subtraction (DBS) was configured to trigger a sensitive enhanced product ion (EPI) scan when a multiple reaction monitoring (MRM) survey scan signal exceeded the defined criteria. Ninety-one percent of acidic drugs in protein-precipitated plasma and 80% of the neutral compounds in equine urine were detected when spiked at 10 ng/ml.

Comparison of the effect of Sporobolus virginicus and Rhodes (Chloris gayana) hay diets on the absorption pattern of phenylbutazone in the camel (Camelus dromedarius).

The effect of feeding Sporobolus and Rhodes hay on phenylbutazone (4 g) relative absorption was examined in six camels using a two-period, two-sequence, two-treatment crossover design. Serum concentration of the drug was measured by high performance liquid chromatography. The measured values (means+/-SD) for Rhodes and Sporobolus hay, respectively, were Cmax 35.59+/-22.36 and 36.55+/-18.99 microg/mL, Tmax 26+/-2.53 and 26.3+/-1.97 h and AUC0-72 h 1552+/-872.6 and 1621+/-903.6 microg h/mL. Broad plateau concentrations of phenylbutazone in serum were observed between 12 and 36 h. There was no significant difference in any parameter between the two feeding regimens. Multiple peaks in serum concentration-time curve were observed, regardless of the type of grass available to and the animals prior to drug administration. It was concluded that the phasic absorption of phenylbutazone was a particular feature of hay feeding in camels, and the Sporobolus hay can be fed to camels without any effect on the rate and extent of phenylbutazone absorption compared to Rhodes grass hay.

Evaluation of the protein binding ratio of drugs by a micro-scale ultracentrifugation method.

Ultracentrifugation methods have been widely used for the determination of the free fraction of compounds in plasma, especially for lipophilic compounds. To estimate the effect of contaminated proteins in the "protein-free phase" fraction, 200 microL of human plasma was separated into three layers by ultracentrifugation at 436,000g for 140 min with a table-top ultracentrifuge. Twenty microliters of the middle layer was taken as the protein-free fraction. Major contaminated proteins were analyzed by liquid chromatography/electrospray ionization/tandem mass spectrometry (LC/ESI/MS/MS) and identified as albumin, alpha-1-antitrypsin, alpha-2-HS-glycoprotein, apolipoprotein E, and apolipoprotein A-1. alpha-1-acid glycoprotein was not detected. Contamination of albumin was 0.13% of that in plasma. Simulation analysis demonstrated that at an actual free fraction of 1% (protein binding ratio of 99%), the extent of overestimation of free fraction was just 13% and the apparent free fraction was 1.13%. Human plasma protein binding ratios of 10 drugs estimated by this method correlated well with reported values determined by other methods, such as ultrafiltration and equilibrium dialysis, with a correlation factor of 0.98 and a slope of 0.99. Collectively, our results indicate the reliability of this micro-scale ultracentrifugation technique for the evaluation of the protein binding of drugs despite a little contamination of albumin.

Preliminary studies of offspring exposure to phenylbutazone and ivermectin during the perinatal period in a Holstein cow-calf model.

The pregnant Holstein cow and her newborn calf were evaluated as an animal model to study in utero and for lactational drug transfer and offspring exposure. A nonsteroidal antiinflammatory drug, phenylbutazone, and an antiparasitic drug, ivermectin, were tested in the model. Prior to parturition, pregnant cows were dosed orally to steady state with phenylbutazone at 4 g/day or given a single subcutaneous injection of 200 microg ivermectin/kg body wt. The level of drug transferred to calves exposed in utero, in utero combined with lactational exposure, and via lactational exposure only, was measured from days 1 through 7 postpartum. At birth the plasma level in phenylbutazone-exposed calves was approximately one-half the dam's steady-state level. For ivermectin-exposed calves, plasma levels were at or below the limit of quantitation (0.5 ng/ml) at birth, suggesting that placental transfer of ivermectin is limited in the cow. For both drugs, rapid accumulation of the drug in calf plasma occurred with lactational exposure to a mean daily dose of 2 microg ivermectin/kg body wt or 0.1 mg phenylbutazone/kg body wt/day for the first 7 days of life. The accumulation observed in the newborn calf is attributed to the lipid solubility and long elimination half-lives of these drugs. These results demonstrate that drug transfer and offspring exposure can be studied using the cow-calf model. The data also highlight the importance of considering not only the dose but also physicochemical characteristics and pharmacokinetics of the drug in the offspring when evaluating the safety of a newborn's exposure to a drug in breast milk.

Pharmacokinetic/pharmacodynamic approach to assess irrelevant plasma or urine drug concentrations in postcompetition samples for drug control in the horse.

The current performance of analytical techniques used for drug control in horses lead the Regulatory Authorities to decide whether trace levels of drugs legitimately used for therapeutic medication should or should not be reported. Here, we propose a well-ordered and nonexperimental pharmacokinetic/pharmacodynamic approach for the determination of irrelevant drug plasma (IPC) and urine concentrations (IUC). The published plasma clearance is used to transform an effective (marketed) dose into an effective concentration (EPC). EPC is transformed into an IPC by applying a safety factor (SF). This method is based on several assumptions (eg, drug effects reversibly driven by plasma concentration, linearity of drug disposition). The suitability of the computed IPC and IUC can be checked by calculating the residual amount of drug at IPC and computing a minimal drug withdrawal time. It is concluded that controlling the drug effect (using drug or any analyte concentration as a marker) rather than the drug exposure will be more demanding and also makes urine a less than ideal matrix.

Pharmacokinetics of phenylbutazone in beef steers.

Phenylbutazone was administered intravenously to a group of 11 beef steers at a dosage of 6 mg/kg of body weight. Whole plasma and protein-free plasma were analyzed for phenylbutazone residues. Pharmacokinetic parameters of total and free phenylbutazone in plasma were calculated using a noncompartmental method. In regards to whole plasma data, the mean volume of distribution at steady state (Vss), was 140 mL/kg body weight, with a mean (+/-SEM) terminal elimination half-life (t1/2) of 34 +/- 9 h. The mean clearance was 3.2 mL/h/kg body weight. The Vss, as determined from the protein-free plasma fraction, was 54093 mL/kg body weight. This larger Vss of free phenylbutazone compared with total plasma phenylbutazone was attributed to a high degree of plasma protein binding, as well as the greater penetration of free phenylbutazone into tissues. The mean t1/2 of free phenylbutazone was 35 +/- 12 h. This similarity to the t1/2 estimated from total plasma phenylbutazone data is attributed to an equilibrium between free and plasma phenylbutazone during the terminal elimination phase. The pharmacokinetic parameters of free and total plasma phenylbutazone in beef steers are statistically similar to those previously reported for lactating dairy cows.

Determination by high-performance liquid chromatography of phenylbutazone in samples of plasma from fighting bulls.

The purpose of this study was to investigate the possible presence of phenylbutazone in plasma samples from fighting bulls killed in 2nd and 3rd category bullrings in the province of Salamanca (Spain) in 1998, 1999 and 2000. For quantitative and qualitative determination, a high-performance liquid chromatograph was used, equipped with a photodiode-array detector and setting wavelengths at 240, 254 and 284 nm. The mobile phase optimized for the simultaneous detection of dexamethasone, betamethasone, flunixin and phenylbutazone, was 0.01 M acetic acid pH 3 in methanol (35:65 v/v) at a flow rate of 1 ml/min. Plasma samples were deproteinized with 400 microl of acetonitrile and 20 microl of the supernatant were injected directly into the chromatographic system equipped with a Lichrospher 60 RP select B column and guard column. For the quantitative analysis, standard calibration curves were made in a concentration range between 0.25 and 30 microg/ml, using betamethasone as internal standard. The retention time of phenylbutazone was 8.7 +/- 0.2 min and recovery was 83%. The detection and quantification limits were 0.016 and 0.029, respectively for A=240 nm. The study results show that 17 of the 74 samples analyzed in 1998, 18 of those from 1999 and 10 of those from 2000 were positive for phenylbutazone.

Pharmacokinetics of phenylbutazone and its metabolite oxyphenbutazone in miniature donkeys.

OBJECTIVE: To describe the pharmacokinetics of phenylbutazone and oxyphenbutazone after IV administration in miniature donkeys. ANIMALS: 6 clinically normal miniature donkeys. PROCEDURE: Blood samples were collected before and 5, 10, 20, 30, 45, 60, 90, 120, 180, 240, 300, 360, and 480 minutes after IV administration of phenylbutazone (4.4 mg/kg of body weight). Serum was analyzed in triplicate by use of high-performance liquid chromatography for determination of phenylbutazone and oxyphenbutazone concentrations. The serum concentration-time curve for each donkey was analyzed separately to estimate model-independent pharmacokinetic variables. RESULTS: Serum concentrations decreased rapidly after IV administration of phenylbutazone, and they reached undetectable concentrations within 4 hours. Values for mean residence time ranged from 0.5 to 3.0 hours (median, 1.1 hour), whereas total body clearance ranged from 4.2 to 7.5 ml/kg/min (mean, 5.8 ml/kg/min). Oxyphenbutazone appeared rapidly in the serum; time to peak concentration ranged from 13 to 41 minutes (mean, 26.4 minutes), and peak concentration in serum ranged from 2.8 to 4.0 mg/ml (mean, 3.5 microg/ml). CONCLUSION AND CLINICAL RELEVANCE: Clearance of phenylbutazone in miniature donkeys after injection of a single dose (4.4 mg/kg, IV) is rapid. Compared with horses, miniature donkeys may require more frequent administration of phenylbutazone to achieve therapeutic efficacy.