Pyrilamine in the horse: detection and pharmacokinetics of pyrilamine and its major urinary metabolite O-desmethylpyrilamine.

Pyrilamine is an antihistamine used in human and veterinary medicine. As antihistamines produce central nervous system effects in horses, pyrilamine has the potential to affect the performance of racehorses. In the present study, O-desmethylpyrilamine (O-DMP) was observed to be the predominant equine urinary metabolite of pyrilamine. After intravenous (i.v.) administration of pyrilamine (300 mg/horse), serum pyrilamine concentrations declined from about 280 ng/mL at 5 min postdose to about 2.5 ng/mL at 8 h postdose. After oral administration of pyrilamine (300 mg/horse), serum concentrations peaked at about 33 ng/mL at 30 min, falling to <2 ng/mL at 8 h postdose. Pyrilamine was not detected in serum samples at 24 h postdosing by either route. After i.v. injection of pyrilamine (300 mg/horse) O-DMP was recovered at a level of about 20 microg/mL at 2 h postdose thereafter declining to about 2 ng/mL at 168 h postdose. After oral administration, the O-DMP recovery peaked at about 12 microg/mL at 8 h postdose and declined to <2 ng/mL at 168 h postdose. These results show that pyrilamine is poorly bioavailable orally (18%), and can be detected by sensitive enzyme-linked immunosorbent assay tests in urine for up to 1 week after a single administration. Care should be taken as the data suggest that the withdrawal time for pyrilamine after repeated oral administrations is likely to be at least 1 week or longer.

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.

Intravenous and intratracheal administration of trimetoquinol, a fast-acting short-lived bronchodilator in horses with 'heaves'.

REASON FOR PERFORMING STUDY: Trimetoquinol (TMQ) is a potent beta-adrenoceptor agonist bronchodilator used in human medicine but has not been evaluated for potential use as a therapeutic agent for horses with 'heaves'. OBJECTIVES: To assess the pharmacodynamics of TMQ in horses with 'heaves' to determine potential therapeutic effects. METHODS: Increasing doses of TMQ were administered to horses with 'heaves' by i.v. and intratracheal (i.t.) routes. Doses ranged 0.001-0.2 microg/kg bwt i.v. and 0.01-2 microg/kg bwt i.t. Cardiac and airways effects were assessed by measurement of heart rate (HR) and maximal change in pleural pressure (deltaPplmax), respectively. Side effects of sweating, agitation and muscle trembling were scored subjectively. Duration of action to i.v. (0.2 microg/kg bwt) and i.t. (2 microg/kg bwt) TMQ was evaluated over 6 h. RESULTS: Intravenous TMQ was an exceptionally potent cardiac stimulant. Heart rate increased at 0.01 microg/kg bwt, and was still increasing after administration of highest dose, 0.2 microg/kg bwt. Airway bronchodilation, measured as a decrease in deltaPplmax, also commenced at 0.01 microg/kg bwt. By the i.t. route, TMQ was 50-100-fold less potent than by i.v. Side effects included sweating, agitation and muscle trembling. Overall, the onset of HR and bronchodilator effects was rapid, within about 3 min, but effects were over at 2 h. CONCLUSION: When administered i.v. and i.t., TMQ is a highly potent cardiac stimulant and a modest bronchodilator. It may not be an appropriate pharmacological agent by i.v. and i.t. routes for the alleviation of signs in horses with 'heaves'. Further studies of TMQ by oral and aerosol routes are necessary. POTENTIAL RELEVANCE: In horses, TMQ is a fast-acting bronchodilator with a short duration of action. It could be used as a rescue agent during an episode of 'heaves'. The i.v. and i.t. administration of TMQ is associated with side effects, similar to those reported for all other beta-agonists. However, other routes, such as aerosol and oral, may prove useful and safe for the alleviation of bronchoconstriction typical of 'heaves'.

Effect of competition on performance of thoroughbred racehorses.

The effect of competition and the influence of age and sex on performance were examined in a study of 18 Thoroughbred racehorses. The horses performed two solo and two competitive runs at 1,200 and 1,600 m for a total of eight runs. No group ran faster during competition, which may have been a reflection of the quality of horses used for this study and their susceptibility to stress-induced impairment of performance. Males showed no significant difference between competitive and solo run times, whereas females were consistently slower during competition. Males ran significantly faster than females in all runs. There was no difference in run times due to age, which may have been due to the high mean age (5.9 yr) of the group. The slower competitive run times may have occurred because of an earlier onset of fatigue when compared with solo runs. Plasma lactate was significantly greater for the 1,200-m competitive than for the solo runs.

A GC-MS method for the determination of isoxsuprine in biological fluids of the horse utilizing electron impact ionization.

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 ritodrine, which released intense quantifier ions m/z 178 for isoxsuprine and m/z 236 for ritodrine that were products of C-C cleavage. To our knowledge, this procedure is faster and more sensitive than other methods in the literature. Concentrations in urine and plasma of isoxsuprine were determined from a calibrator curve that was generated along with unknowns. Ritodrine was used as an internal standard and was, therefore, present in all samples, standards, and blanks. Validation data was also collected. The limit of detection of isoxsuprine in plasma was determined to be 2 ng/mL, the limit of quantitation of isoxsuprine in plasma was determined to be < 5 ng/mL. The mean coefficient of determination for the calibrator curves for plasma was 0.9925 +/- 0.0052 and for calibrator curves for urine 0.9904 +/- 0.0075. The recovery efficiencies at concentrations of 50, 200, and 300 ng/mL were 76%, 73%, and 76%, respectively, in plasma and 92%, 89%, and 91% in urine.

Clenbuterol in the horse: confirmation and quantitation of serum clenbuterol by LC-MS-MS after oral and intratracheal administration.

Clenbuterol is a beta2 agonist/antagonist bronchodilator, and its identification in post-race samples may lead to sanctions. The objective of this study was to develop a specific and highly sensitive serum quantitation method for clenbuterol that would allow effective regulatory control of this agent in horses. Therefore, clenbuterol-d9 was synthesized for use as an internal standard, an automated solid-phase extraction method was developed, and both were used in conjunction with a multiple reaction monitoring liquid chromatography-tandem mass spectrometry (LC-MS-MS) method to allow unequivocal identification and quantitation of clenbuterol in 2 mL of serum at concentrations as low as 10 pg/mL. Five horses were dosed with oral clenbuterol (0.8 microg/kg, BID) for 10 days, and serum was collected for 14 days thereafter. Serum clenbuterol showed mean trough concentrations of approximately 150 pg/mL. After the last dose on day 10, serum clenbuterol reached a peak of approximately 500 pg/mL and then declined with a half-life of approximately 7 h. Serum clenbuterol declined to 30 and 10 pg/mL at 48 and 72 h after dosing, respectively. By 96 h after dosing, the concentration was below 4 pg/mL, the limit of detection for this method. Compared with previous results obtained in parallel urinary experiments, the serum-based approach was more reliable and satisfactory for regulation of the use of clenbuterol. Clenbuterol (90 microg) was also administered intratracheally to five horses. Peak serum concentrations of approximately 230 pg/mL were detected 10 min after administration, dropping to approximately 50 pg/mL within 30 min and declining much more slowly thereafter. These observations suggest that intratracheal administration of clenbuterol shortly before race time can be detected with this serum test. Traditionally, equine drug testing has been dependent on urine testing because of the small volume of serum samples and the low concentrations of drugs found therein. Using LC-MS-MS testing, it is now possible to unequivocally identify and quantitate low concentrations (10 pg/mL) of drugs in serum. Based on the utility of this approach, the speed with which new tests can be developed, and the confidence with which the findings can be applied in the forensic situation, this approach offers considerable scientific and regulatory advantages over more traditional urine testing approaches.

Development of a method for the detection and confirmation of the alpha-2 agonist amitraz and its major metabolite in horse urine.

Amitraz (N'-(2,4-dimethylphenyl)-N-[[(2,4-dimethylphenyl)imino]methyl]-N-methyl-methanimidamide) is an alpha-2 adrenergic agonist used in veterinary medicine primarily as a scabicide- or acaricide-type insecticide. As an alpha-2 adrenergic agonist, it also has sedative/tranquilizing properties and is, therefore, listed as an Association of Racing Commissioners International Class 3 Foreign Substance, indicating its potential to influence the outcome of horse races. We identified the principal equine metabolite of amitraz as N-2,4-dimethylphenyl-N'-methylformamidine by electrospray ionization(+)-mass spectrometry and developed a gas chromatographic-mass spectrometric (GC-MS) method for its detection, quantitation, and confirmation in performance horse regulation. The GC-MS method involves derivatization with t-butyldimethylsilyl groups; selected ion monitoring (SIM) of m/z 205 (quantifier ion), 278, 261, and 219 (qualifier ions); and elaboration of a calibration curve based on ion area ratios involving simultaneous SIM acquisition of an internal standard m/z 208 quantifier ion based on an in-house synthesized d(6) deuterated metabolite. The limit of detection of the method is approximately 5 ng/mL in urine and is sufficiently sensitive to detect the peak urinary metabolite at 1 h post dose, following administration of amitraz at a 75-mg/horse intravenous dose.

Detection and confirmation of ractopamine and its metabolites in horse urine after Paylean administration.

We have investigated the detection, confirmation, and metabolism of the beta-adrenergic agonist ractopamine administered as Paylean to the horse. A Testing Components Corporation enzyme-linked imunosorbent assay (ELISA) kit for ractopamine displayed linear response between 1.0 and 100 ng/mL with an I-50 of 10 ng/mL and an effective screening limit of detection of 50 ng/mL. The kit was readily able to detect ractopamine equivalents in unhydrolyzed urine up to 24 h following a 300-mg oral dose. Gas chromatography-mass spectrometry (GC-MS) confirmation comprised glucuronidase treatment, solid-phase extraction, and trimethylsilyl derivatization, with selected-ion monitoring of ractopamine-tris(trimethylsilane) (TMS) m/z 267, 250, 179, and 502 ions. Quantitation was elaborated in comparison to a 445 Mw isoxsuprine-bis(TMS) internal standard monitored simultaneously. The instrumental limit of detection, defined as that number of ng on column for which signal-to-noise ratios for one or more diagnostic ions fell below a value of three, was 0.1 ng, corresponding to roughly 5 ng/mL in matrix. Based on the quantitation ions for ractopamine standards extracted from urine, standard curves showed a linear response for ractopamine concentrations between 10 and 100 ng/mL with a correlation coefficient r > 0.99, whereas standards in the concentration range of 10-1000 ng/mL were fit to a second-order regression curve with r > 0.99. The lower limit of detection for ractopamine in urine, defined as the lowest concentration at which the identity of ractopamine could be confirmed by comparison of diagnostic MS ion ratios, ranged between 25 and 50 ng/mL. Urine concentration of parent ractopamine 24 h post-dose was measured at 360 ng/mL by GC-MS after oral administration of 300 mg. Urinary metabolites were identified by electrospray ionization (+) tandem quadrupole mass spectrometry and were shown to include glucuronide, methyl, and mixed methyl-glucuronide conjugates. We also considered the possibility that an unusual conjugate added 113 amu to give an observed m/z 415 [M+H] species or two times 113 amu to give an m/z 528 [M+H] species with a daughter ion mass spectrum related to the previous one. Sulfate and mixed methyl-sulfate conjugates were revealed following glucuronidase treatment, suggesting that sulfation occurs in combination with glucuronidation. We noted a paired chromatographic peak phenomenon of apparent ractopamine metabolites appearing as doublets of equivalent intensity with nearly identical mass spectra on GC-MS and concluded that this phenomenon is consistent with Paylean being a mixture of RR, RS, SR, and SS diastereomers of ractopamine. The results suggest that ELISA-based screening followed by glucuronide hydrolysis, parent drug recovery, and TMS derivatization provide an effective pathway for detection and GC-MS confirmation of ractopamine in equine urine.

Remifentanil in the horse: identification and detection of its major urinary metabolite.

Remifentanil (4-methoxycarbonyl-4-[(1-oxopropyl)phenylamino]-1-piperidinepropionic acid methyl ester) is a mu-opioid receptor agonist with considerable abuse potential in racing horses. The identification of its major equine urinary metabolite, 4-methoxycarbonyl-4-[(1-oxopropyl)phenylamino]-1-piperidinepropionic+ ++ acid, an ester hydrolysis product of remifentanil is reported. Administration of remifentanil HCl (5 mg, intravenous) produced clear-cut locomotor responses, establishing the clinical efficacy of this dose. ELISA analysis of postadministration urine samples readily detected fentanyl equivalents in these samples. Mass spectrometric analysis, using solid-phase extraction and trimethylsilyl (TMS) derivatization, showed the urine samples contained parent remifentanil in low concentrations, peaking at 1 h. More significantly, a major peak was identified as representing 4-methoxycarbonyl-4-[(1-oxopropyl)phenylamino]-1-piperidinepropionic+ ++ acid, arising from ester hydrolysis of remifentanil. This metabolite reached its maximal urinary concentrations at 1 h and was present at up to 10-fold greater concentrations than parent remifentanil. Base hydrolysis of remifentanil yielded a carboxylic acid with the same mass spectral characteristics as those of the equine metabolite. In summary, these data indicate that remifentanil administration results in the appearance of readily detectable amounts of 4-methoxycarbonyl-4-[(1-oxopropyl)phenylamino]-1-piperidinepropionic+ ++ acid in urine. On this basis, screening and confirmation tests for this equine urinary metabolite should be optimized for forensic control of remifentanil.

Effects of induced alkalosis on performance in thoroughbreds during a 1,600-m race.

There is considerable debate regarding the ergogenic effects of sodium bicarbonate (NaHCO3) on racing performance in horses. Anecdotal evidence suggests that NaHCO3 improves performance by increasing the buffering capacity of the blood and delaying the onset of hydrogen ion-induced fatigue. In a cross-over study, 16 Thoroughbred racehorses were given an aqueous solution of NaHCO3 (0.4 g/kg in 1 litre H2O) or a control treatment (1 litre H2O) before a 1600-m race. Treatments were administered 3 h before the race, which was the time to peak buffering capacity (2.5-3.0 h) determined in a separate study. Before the race, there was a significant increase in venous HCO3- and pH in the NaHCO3-treated horses. After the race, there was a significant increase in venous blood pH and lactate in the NaHCO3-treated horses. Collectively, the data suggest an improved buffering capacity of the blood after NaHCO3 treatment. However, there was no change in race times or venous partial pressure of carbon dioxide. Therefore, the administration of NaHCO3 provided no ergogenic benefit to horses competing in a 1,600-m race.

The correlation of running ability and physiological variables in thoroughbred racehorses.

The running abilities of 25 Thoroughbred racehorses were measured at distances of 1200, 1600 and 20000 m. Various physiological variables were measured subsequently on the treadmill and correlated with running speed. There was a negative correlation for running speed with the velocity (VLa4) and work rate (WLa4) at which blood lactate reaches a steady-state concentration of 4 mmol/litre and a positive correlation with peak plasma lactate, suggesting that plasma lactate concentrations of faster horses rise more rapidly and to higher levels than do those of slower horses. The correlation between running speeds and heart rates (HR) was stronger for unfit than fit horses, suggesting that cardiovascular effects of training are more beneficial to slower horses. The significant correlation between running speeds and V200 suggests that the HR of faster horses increases more rapidly than in slower horses performing similar exercise. The correlation of running speeds and VO2max suggests that the HR of faster horses increases more rapidly than in slower horses performing similar exercise. The correlation of running speeds and VO2max suggests that faster horses utilise more oxygen during maximal intensity exercise. The correlation of running speeds with minimum pH and minimum HCO3- suggests that faster horses maintain speed at higher hydrogen ion (H+) concentrations. Correlations between running speeds and the measured variables were consistently stronger for the longer distance runs. Because VLa4 and WLa4 were obtained during sub-maximal exercise, these variables were determined to be the best correlates of running performance.

Amantadine and equine influenza: pharmacology, pharmacokinetics and neurological effects in the horse.

Amantadine is an antiviral agent effective against influenza A viruses. We investigated 1) the antiviral efficacy, 2) analytical detection, 3) bioavailability and disposition, 4) pharmacokinetic modelling and 5) adverse reactions of amantadine in the horse. In vitro, amantadine and its derivative rimantadine suppressed the replication of recent isolates of equine-2 influenza virus with effective doses (EDs) of less than 30 ng/ml. Rimantadine was more effective than amantadine against most viral isolates; we suggest a minimum plasma concentration of 300 ng/ml of amantadine for therapeutic efficacy. In vivo an i.v. dose of amantadine 15 mg/kg bwt produced mild, transient CNS signs which were no longer apparent after 30 min. Amantadine administered at a dose of 15 mg/kg bwt was established as the maximum safe single i.v. dose. However, if repeated i.v. administration of amantadine is required no more than 10 mg/kg bwt t.i.d. should be used. The maximal safe plasma concentration of amantadine was not evaluated but is probably greater than 2000 ng/ml and possibly greater than 4000 ng/ml. On the other hand, horses with lower seizure thresholds, or those on medications that lower seizure thresholds, may be at increased risk of amantadine-induced seizures, which show few premonitory signs and are rapidly fatal. After i.v. administration of amantadine 10 mg/kg bwt, the disposition kinetics were well fitted by a 2-compartment open model. The estimated peak plasma concentration after this dose was about 4500 ng/ml, the volume of distribution at steady-state (Vdss) was (mean +/- s.d.) 4.9 +/- 1.9 l/kg bwt and the beta phase half-life was 1.83 +/- 0.87 h. Computer projections of plasma amantadine concentrations after i.v. administration of amantadine at a dose of 10 mg/kg bwt t.i.d. at 8 h intervals suggest peak plasma concentrations of 4000-5000 ng/ml and troughs of less than 300 ng/ml will be achieved. Amantadine administered orally at 10 mg/kg bwt and 20 mg/kg bwt showed mean oral bioavailability of about 40-60% and a plasma half life of 3.4 +/- 1.4 h; however, there was substantial inter-animal variation in bioavailability. Projections based on the kinetics observed in individual animals suggest that some animals readily maintain effective plasma concentrations of amantadine after oral administration of 20 mg/kg bwt t.i.d. On the other hand, animals in which amantadine is poorly bioavailable may require up to a 6-fold (120 mg/kg bwt) increase in the oral dose to achieve effective blood concentrations. Withholding food for 15 h did not reduce these inter-animal differences in bioavailability. Our results showed that simple dosing with oral amantadine will not yield effective plasma concentrations in all animals. While i.v. administration yielded more reproducible plasma concentrations, care should be taken to see that the seizure threshold is not exceeded. In acute situations, i.v. administration (5 mg/kg bwt) every 4 h should maintain safe and effective plasma and respiratory tract concentrations of amantadine.

Character and duration of pharmacological effects of intravenous isoxsuprine.

Isoxsuprine is a therapeutic medication used to treat navicular disease and other lower limb problems in horses and is one of the more frequently detected therapeutic agents in racing horses. In a crossover study, horses were administered isoxsuprine i.v. to determine the character and duration of its pharmacological effects. Isoxsuprine significantly increased heart rate 5-150 min following injection. Unrestrained activity following isoxsuprine treatment was significantly greater than control activity for 105 min after treatment. There was an apparent, although statistically nonsignificant, increased cutaneous blood flow resulting in visible water vapour and sweat production 5-60 min after administration. Initially, there was no difference in skin temperature between control and isoxsuprine treatment values; however, skin temperature decreased below control values 45-120 min after injection. Concurrently, there was a significant decrease in rectal temperature reflecting a decrease in body core temperature. Using infrared thermography, a significant decrease in superficial skin temperature of the front legs occurred 30-240 min after treatment. Isoxsuprine also reduced smooth muscle tone, which was apparent by decreased tone of the internal anal sphincter 10-180 min after treatment. It was concluded that the measurable pharmacological effects of i.v. isoxsuprine are short lived, since none of the above responses were apparent 4 h or more after i.v. administration.

Determination of highest no effect dose (HNED) for local anaesthetic responses to procaine, cocaine, bupivacaine and benzocaine.

The highest no effect doses (HNEDs) for the local anaesthetic (LA) effects of procaine, cocaine, bupivacaine and benzocaine were determined using the heat lamp/hoof withdrawal model of Kamerling et al. (1985b) and the abaxial sesamoid block model of local anaesthesia. The heat lamp rapidly (4 or 5 s) increased the temperature of the superficial skin layers of the pastern to about 90 degrees C, at which point the animal sharply withdrew its hoof. Effective LA blockade precluded this response and superficial skin temperatures exceeded 120 degrees C. Thermal stimulus experiments were routinely terminated after 10 s of exposure to prevent undue tissue damage. Following abaxial sesamoid block with bupivacaine, the HNED for that drug was about 0.25 mg/site. Increasing the dose to 2 mg/site apparently produced complete and prolonged LA blockade. Analogous work showed that the HNED for procaine was about 2.5 mg/site. Similarly, the dose response curve for procaine was parallel with that of bupivacaine but was shifted 10-fold to the right. The duration of the LA response following procaine injection was less than for bupivacaine with the statistically significant response following 40 mg/site injection lasting less than 45 min. Cocaine was less potent than procaine, showing a shallower dose response curve. The HNED for cocaine was less than 5 mg/site, although at this dose the duration of action was extremely short (< 7.5 min). Benzocaine had no significant LA action when a dose of 800 mg was applied topically as a 5% preparation. These results show that the HNEDs for bupivacaine and procaine are remarkably low, that cocaine is somewhat less potent as a LA than might be expected, and that 5% topical benzocaine has no significant pharmacology. The small doses of bupivacaine and procaine producing effective local anaesthesia suggests that developing plasma thresholds for these agents is likely to be very challenging.

Regulatory significance of procaine residues in plasma and urine samples: preliminary communication.

Plasma and urinary concentrations of procaine and the duration of response to procaine after its administration as a local anaesthetic to horses were studied. Following injection of a clinical dose of procaine HCl (80 mg), the concentration of procaine in plasma was less than the lower limit of quantitation and unsuitable for threshold determination. Therefore, the urinary concentration of procaine was determined after injection of a dose of 5 mg procaine HCl, the highest no-effect dose (HNED) of this agent. Free unconjugated procaine in equine urine reached a peak concentration of 23.7 ng/mL, while total (unconjugated plus conjugated) procaine peaked at 37.9 ng/mL (mean urine pH of 8.5). Because a basic drug may concentrate substantially in acidic urine, a threshold concentration of 25 ng/mL of unconjugated procaine is a reasonable and conservative threshold for procaine at this time. Horses were administered abaxial sesamoid blocks containing 2% procaine HCl (40, 80, 160 and 320 mg) and 2% procaine HCl (40 and 320 mg) with epinephrine (1:100,000) in local anaesthetic experiments. There was a significant local anaesthetic (LA) effect for all doses of procaine HCl with the duration of effect ranging from 30 min (40 mg) to 60 min (320 mg). The addition of epinephrine significantly increased the duration of local anaesthesia to 180 min for a 40 mg dose and 420 min for a 320 mg dose. Because epinephrine may extend the duration of local anaesthesia beyond a reasonable period of confinement for horses before the starting time of a race, the increased LA effect following the addition of epinephrine to procaine has regulatory significance.

Absence of detectable pharmacological effects after oral administration of isoxsuprine.

Isoxsuprine is reported to be a peripheral vasodilator used in human and veterinary medicine to treat ischaemic vascular disease. In horses, it is generally administered orally to treat navicular disease and other lower limb problems. To define the scope and duration of its pharmacological responses after oral administration, 6 horses were dosed with isoxsuprine HCl (1.2 mg/kg bwt) q. 12 h for 8 days and then tested to assess the duration and extent of pharmacological actions. There was no significant difference between isoxsuprine and control treatment values for heart rate, spontaneous activity, sweat production, anal muscle tone, core and skin temperatures, and cutaneous blood flow. The lack of pharmacological effect following oral administration was in sharp contrast to the marked response following i.v. dosing reported in earlier experiments.

Identification of hydroxyropivacaine glucuronide in equine urine by ESI+/MS/MS.

Ropivacaine is a local anesthetic that has a high potential for abuse in racing horses. It can be recovered from urine collected after administration as a hydroxylated metabolite following beta-glucuronidase treatment of the urine. Based on these findings, it has been inferred that ropivacaine is present in equine urine as a glucuronide metabolite; however, these metabolites have never been directly identified. Using ESI+/MS/MS, the presence of a [M+H]+ molecular ion of m/z 467 was demonstrated in urine corresponding to the calculated mass of a hydroxyropivacaine glucuronide +1. The abundance of this ion diminished after glucuronidase treatment with concomitant appearance of a m/z 291 peak, which is consistent with its hydrolysis to hydroxyropivacaine. In further work, the m/z 467 material was fragmented in the MS/MS system, yielding fragments interpretable as hydroxyropivacaine glucuronide. These data are consistent with the presence of a hydroxyropivacaine glucuronide in equine urine and constitute the first direct demonstration of a specific glucuronide metabolite in equine urine.

Direct MS-MS identification of isoxsuprine-glucuronide in post-administration equine urine.

Isoxsuprine is routinely recovered from enzymatically-hydrolyzed, post-administration urine samples as parent isoxsuprine in equine forensic science. However, the specific identity of the material in horse urine from which isoxsuprine is recovered has never been established, although it has long been assumed to be a glucuronide conjugate (or conjugates) of isoxsuprine. Using ESI/MS/MS positive mode as an analytical tool, urine samples collected 4-8 h after isoxsuprine administration yielded a major peak at m/z 554 that was absent from control samples and resisted fragmentation to daughter ions. Titration of this material with increasing concentrations of sodium acetate yielded m/z peaks consistent with the presence of monosodium and disodium isoxsuprine-glucuronide complexes, suggesting that the starting material was a dipotassium-isoxsuprine-glucuronide complex. Electrospray ionization mass spectrometry negative mode disclosed the presence of a m/z 476 peak that declined following enzymatic hydrolysis and resulted in the concomitant appearance of peaks at m/z 300 and 175. The resulting peaks were consistent with the presence of isoxsuprine (m/z 300) and a glucuronic acid residue (m/z 175). Examination of the daughter ion spectrum of this putative isoxsuprine-glucuronide m/z 476 peak showed overlap of many peaks with those of similar spectra of authentic morphine-3- and morphine-6-glucuronides, suggesting they were derived from glucuronic acid conjugation. These data suggest that isoxsuprine occurs in post-administration urine samples as an isoxsuprine-glucuronide conjugate and also, under some circumstances, as an isoxsuprine-glucuronide-dipotassium complex.

Effect of furosemide on physiologic variables in exercising horses.

Twelve horses (6 Standardbreds and 6 Thoroughbreds) received IM injections of furosemide (250 mg) or physiologic saline solution and performed standard exercise tests, to assess the effects of furosemide and breed on blood gas values, PCV, plasma lactate concentration, and heart rate during exercise. After furosemide administration, arterial and venous blood pH values were significantly (P < 0.05) increased. Partial pressures of O2 and CO2 in arterial blood and of CO2 in venous blood (PaO2, PaCO2, and PVCO2, respectively) were unaffected by furosemide treatment, whereas venous partial pressures of O2 (PVO2) were significantly (P < 0.05) less during exercise after furosemide treatment, suggesting an increase in oxygen uptake by the exercising muscles or a change in cardiac output. A significant (P < 0.05) difference was found between Thoroughbred and Standardbred values for arterial and venous pH, PaO2, PaCO2, plasma lactate concentration, and heart rate, suggesting that Standardbreds exercised at a relatively higher work rate than did Thoroughbreds.

Pharmacokinetics of gentamicin in newborn to 30-day-old foals.

Gentamicin sulfate, equivalent to 4 mg of gentamicin base/kg of body weight, was administered IV to 6 Thoroughbred foals on day 1 (12 to 24 hours of age) and at 5, 10, 15, and 30 days after birth. On day 40 after parturition, gentamicin was given to the mares at a dosage similar to that used in foals. Decay of serum gentamicin concentrations was best described by a 2-compartment model. Among foals, the overall elimination rate constant at 30 days of age was significantly (P less than 0.05) greater than at days 1, 10, and 15. There was, however, no difference in the overall elimination rate constant between foals and mares. The volume of distribution (Vd), determined on the basis of total area under the disposition curve, did not change between day 1 and day 30. Mean values of Vd of foals were between 1.5 and 2.5 times higher than the mean Vd of the mares; however, only values from the foals at days 5 and 10 were significantly greater. Both age and interindividual differences were reflected in the total body clearance (ClB) of gentamicin. Total body clearance of gentamicin of foals on day 1 was less than that of foals on days 5, 10, and 30. Additionally, C1B of gentamicin on day 15 was less than that on day 30. There was no significant difference between ClB of foals and mares except for the day-30 group, which had a higher clearance rate than did the adults. Protein binding of gentamicin was less than 30% in all groups, and there were no apparent age-related differences.