Guanabenz in the horse – A preliminary report on clinical effects and comparison to clonidine and other alpha-2 adrenergic agonists

In veterinary medicine, a number of alpha-2 receptor agonists are marketed as sedatives/hypnotics and analgesics, with their principal use being the chemical restraint of large and small animals. Guanabenz (Wytensin®) is an alpha-2 adrenergic receptor agonist marketed for use in humans as an anti-hypertensive agent. Recent reports indicate that guanabenz has been administered to horses in small doses (0.04 mg/kg) for its anti-hypertensive effects. While this offers both benefits of sedation of the horse as well as amelioration of pulmonary hypertension during running exercise and consequent Exercise-Induced Pulmonary Hemorrhage (EIPH), guanabenz is currently proscribed in most racing jurisdictions and its administration to a racing horse can lead to penalties. The Association of Racing Commissioners International (ARCI) lists guanabenz as an ARCI Class 3 agent; Class 3 agents include bronchodilators, anabolic steroids and other drugs with primary effects on the autonomic nervous system, procaine, antihistamines with sedative properties and diuretics and includes amitraz, clonidine, xylazine, detomidine, medetomidine, and romifidine. Guanabenz is unique among alpha-2 agonists in that it differentiates into E- and Z-forms (Fig. 1), with the Z-form lacking hypotensive properties, yet with both E- and Z-forms able to afford relief to cellular stresses related to inflammation or degenerative diseases. The objective of the study was a preliminary description of the pharmacological properties of guanabenz in comparison with clonidine and a number of other alpha-2 agonists. The goal was clinical evaluation of their sedative, analgesic and related activities with the goal of increasing our understanding of the clinical use of such medications and also as a possible prophylaxis for Exercise-Induced Pulmonary Hemorrhage. The clinical study of guanabenz and clonidine was performed in a complete crossover strategy using quantitative markers of sedation, antinociception, heart rate, blood and urine glucose following administration of each compound in five horses. Amitraz, detomidine, medetomidine, romifidine, and xylazine were studied in one horse each. The sedation was quantified by measuring head droop and locomotor activity, while antinociception was measured by Hoof Withdrawal Reflex Latency. Heart rates, urine glucose, urine production and urine specific gravities were also determined by standard clinical chemistry techniques. Guanabenz serum levels and related urinary guanabenz glucuronide levels were determined by established Liquid Chromatography-tandem Mass Spectrometric (LC-MS) methods. In result the clinically effective doses (0.2 mg/kg) of guanabenz produced a rapid and intense sedative effect, with sagging of the lower lip, sunken eyelids, and marked head droop corresponding to plasma guanabenz concentrations that peaked at 120 ng/mL at 2.5 min post-injection (Fig. 2). The initial head height above the ground is considered 100 %, and head heights fell to values ranging 18–40 % with guanabenz, all of which are greater than a 50 % reduction in head height, considered a full clinically useful sedative effect. Despite the intensity of the sedation, all horses remained standing and were able to walk, and the sedation and head droop responses were rapidly reversed by intravenous administration of the alpha-2 receptor antagonist yohimbine, reversals occurring within 10 min of administration. As a pilot investigation this study was extended to six other members of the alpha-2 agonist group, clonidine administered to five horses, and amitraz, detomidine, medetomidine, romifidine, and xylazine to one horse each. Hoof Withdrawal Reflex Latency evaluation demonstrated the considerable analgesic properties of guanabenz, greater than the corresponding potencies among clonidine, detomidine, romifidine, medetomidine and xylazine. Heart rate monitoring showed guanabenz as possessing capacity for prolonged bradycardia, with effects of a single dose lasting for up to 3.5 hr, in contrast with clonidine (1 hr), amitraz (2 hr), detomidine (<1 hr), medetomidine (1 hr), romifidine (2 hr), and xylazine (<1 hr). Peak urine production following guanabenz administration occurred between 1.5 and 3.0 hr after administration (Fig. 6), as indicated by the steeper decline of the urine volume curve during that period. Urine specific gravity dropped to a low of about 1.006 at 2.0 hr after administration and remained at this level for ~1.0 hr. Urine pH remained at 8, and urine protein was negative throughout testing. The other alpha-2 agonists evaluated also caused an increased urine production with a concomitant decrease in specific gravity. The effect of guanabenz had the longest duration on increased urine volume, lasting about 3.0 hr. Xylazine had the shortest diuretic effect, persisting for only about 1.0 hr. Guanabenz along with romifidine and detomidine induced glucosuria whereas other alpha-2 agonists did not. Hyperglycemia and the corresponding glucosuria resulted in a significant diuresis, as shown by the cumulative urine volume. Guanabenz along with amitraz, detomidine and xylazine also produced measurable sedation presenting as reduced locomotor activity (Table 1). While all alpha-2 agonists showed qualitatively similar pharmacological responses, only guanabenz produced an intense and relatively prolonged antinociceptive response. The study is limited by the number of horses examined (five each for guanabenz and clonidine, five for repeat studies that included yohimbine antagonism, and one each for the other agonists). Study design was focused on clinical evaluation of agonist similarities and differences and thus did not specifically generate data for detailed statistical evaluation. In conclusion these studies show that a 100 mg IV dose of guanabenz rapidly induces clinically useful sedation, analgesia and antinociception effects that are more intense and considerably longer-lasting than those produced by other alpha-2 receptor agonists evaluated. Guanabenz also remains detectable in serum up to 8-hours following administration at doses as low as 0.04 mg/kg. In the work reported here, guanabenz administered at 0.2 mg/kg IV showed peak concentrations in serum of 120 ng/mL at 2.5 min and was detectable for up to 4 hr with its glucuronide metabolite peaking at 120 min post-administration. Although we did not investigate the combination of guanabenz with opioid drugs such as butorphanol for pain management, guanabenz may well be a drug of choice among the other alpha-2 agonists evaluated in this study for administration with opioids for pain management based on maintaining maximum levels of analgesia for longer periods of time. These experiments suggest considerable clinical potential for guanabenz as a sedative and a relatively long-lasting analgesic in equine medicine. Based on these pharmacological properties, guanabenz and related alpha-2 agonists also have considerable potential for clinical use in equine medicin

Guanabenz in the horse – A preliminary report on clinical effects and comparison to clonidine and other alpha-2 adrenergic agonists

In veterinary medicine, a number of alpha-2 receptor agonists are marketed as sedatives/hypnotics and analgesics, with their principal use being the chemical restraint of large and small animals. Guanabenz (Wytensin®) is an alpha-2 adrenergic receptor agonist marketed for use in humans as an anti-hypertensive agent. Recent reports indicate that guanabenz has been administered to horses in small doses (0.04 mg/kg) for its anti-hypertensive effects. While this offers both benefits of sedation of the horse as well as amelioration of pulmonary hypertension during running exercise and consequent Exercise-Induced Pulmonary Hemorrhage (EIPH), guanabenz is currently proscribed in most racing jurisdictions and its administration to a racing horse can lead to penalties. The Association of Racing Commissioners International (ARCI) lists guanabenz as an ARCI Class 3 agent; Class 3 agents include bronchodilators, anabolic steroids and other drugs with primary effects on the autonomic nervous system, procaine, antihistamines with sedative properties and diuretics and includes amitraz, clonidine, xylazine, detomidine, medetomidine, and romifidine. Guanabenz is unique among alpha-2 agonists in that it differentiates into E- and Z-forms (Fig. 1), with the Z-form lacking hypotensive properties, yet with both E- and Z-forms able to afford relief to cellular stresses related to inflammation or degenerative diseases. The objective of the study was a preliminary description of the pharmacological properties of guanabenz in comparison with clonidine and a number of other alpha-2 agonists. The goal was clinical evaluation of their sedative, analgesic and related activities with the goal of increasing our understanding of the clinical use of such medications and also as a possible prophylaxis for Exercise- Induced Pulmonary Hemorrhage. The clinical study of guanabenz and clonidine was performed in a complete crossover strategy using quantitative markers of sedation, antinociception, heart rate, blood and urine glucose following administration of each compound in five horses. Amitraz, detomidine, medetomidine, romifidine, and xylazine were studied in one horse each. The sedation was quantified by measuring head droop and locomotor activity, while antinociception was measured by Hoof Withdrawal Reflex Latency. Heart rates, urine glucose, urine production and urine specific gravities were also determined by standard clinical chemistry techniques. Guanabenz serum levels and related urinary guanabenz glucuronide levels were determined by established Liquid Chromatography-tandem Mass Spectrometric (LC-MS) methods. In result the clinically effective doses (0.2 mg/kg) of guanabenz produced a rapid and intense sedative effect, with sagging of the lower lip, sunken eyelids, and marked head droop corresponding to plasma guanabenz concentrations that peaked at 120 ng/mL at 2.5 min post-injection (Fig. 2). The initial head height above the ground is considered 100 %, and head heights fell to values ranging 18–40 % with guanabenz, all of which are greater than a 50 % reduction in head height, considered a full clinically useful sedative effect. Despite the intensity of the sedation, all horses remained standing and were able to walk, and the sedation and head droop responses were rapidly reversed by intravenous administration of the alpha-2 receptor antagonist yohimbine, reversals occurring within 10 min of administration. As a pilot investigation this study was extended to six other members of the alpha-2 agonist group, clonidine administered to five horses, and amitraz, detomidine, medetomidine, romifidine, and xylazine to one horse each. Hoof Withdrawal Reflex Latency evaluation demonstrated the considerable analgesic properties of guanabenz, greater than the corresponding potencies among clonidine, detomidine, romifidine, medetomidine and xylazine. Heart rate monitoring showed guanabenz as possessing capacity for prolonged bradycardia, with effects of a single dose lasting for up to 3.5 hr, in contrast with clonidine (1 hr), amitraz (2 hr), detomidine (\u3c1 hr), medetomidine (1 hr), romifidine (2 hr), and xylazine (\u3c1 hr). Peak urine production following guanabenz administration occurred between 1.5 and 3.0 hr after administration (Fig. 6), as indicated by the steeper decline of the urine volume curve during that period. Urine specific gravity dropped to a low of about 1.006 at 2.0 hr after administration and remained at this level for ~1.0 hr. Urine pH remained at 8, and urine protein was negative throughout testing. The other alpha-2 agonists evaluated also caused an increased urine production with a concomitant decrease in specific gravity. The effect of guanabenz had the longest duration on increased urine volume, lasting about 3.0 hr. Xylazine had the shortest diuretic effect, persisting for only about 1.0 hr. Guanabenz along with romifidine and detomidine induced glucosuria whereas other alpha-2 agonists did not. Hyperglycemia and the corresponding glucosuria resulted in a significant diuresis, as shown by the cumulative urine volume. Guanabenz along with amitraz, detomidine and xylazine also produced measurable sedation presenting as reduced locomotor activity (Table 1). While all alpha-2 agonists showed qualitatively similar pharmacological responses, only guanabenz produced an intense and relatively prolonged antinociceptive response. The study is limited by the number of horses examined (five each for guanabenz and clonidine, five for repeat studies that included yohimbine antagonism, and one each for the other agonists). Study design was focused on clinical evaluation of agonist similarities and differences and thus did not specifically generate data for detailed statistical evaluation. In conclusion these studies show that a 100 mg IV dose of guanabenz rapidly induces clinically useful sedation, analgesia and antinociception effects that are more intense and considerably longer-lasting than those produced by other alpha-2 receptor agonists evaluated. Guanabenz also remains detectable in serum up to 8-hours following administration at doses as low as 0.04 mg/kg. In the work reported here, guanabenz administered at 0.2 mg/kg IV showed peak concentrations in serum of 120 ng/ mL at 2.5 min and was detectable for up to 4 hr with its glucuronide metabolite peaking at 120 min post-administration. Although we did not investigate the combination of guanabenz with opioid drugs such as butorphanol for pain management, guanabenz may well be a drug of choice among the other alpha-2 agonists evaluated in this study for administration with opioids for pain management based on maintaining maximum levels of analgesia for longer periods of time. These experiments suggest considerable clinical potential for guanabenz as a sedative and a relatively long-lasting analgesic in equine medicine. Based on these pharmacological properties, guanabenz and related alpha-2 agonists also have considerable potential for clinical use in equine medicine

Current therapeutic approaches to equine protozoal myeloencephalitis

Equine protozoal myeloencephalitis is the most important infectious neurologic disease of horses in the Western Hemisphere. Equine protozoal myeloencephalitis can interfere with a horse\u27s ability to race, work, and perform; untreated, EPM can be lethal. Antemortem diagnosis of EPM is challenging, requiring careful evaluation of the animal\u27s history, clinical signs, and laboratory data, with rigorous exclusion of other causes. Therapeutic approaches to EPM are evolving. First-generation therapeutic approaches for EPM were based on the classic anti–Toxoplasma gondii pyrimethamine–sulfonamide combinations; treatment is prolonged and can be associated with a considerable relapse rate, which may be associated with the difficulty in maintaining effective CNS concentrations of pyrimethamine. Second-generation therapeutic approaches are based on diclazuril and related triazine agentsa; in 2001, toltrazuril sulfoneb (ponazuril) became the first FDA-approved treatment for EPM. Triazine agents may have prolonged plasma half-lives, and their therapeutic efficacy would likely be enhanced by application of loading-dose schedules. A pyrimethamine-sulfonamide combination formulationc received FDA approval in 2004 for the treatment of EPM. Additionally, a diclazuril-based topical feed dressing formulationd received FDA approval in 2011. The ideal therapeutic agents for use against EPM would be effective when administered orally, with high efficacy against Sarcocystis neurona and minimal toxicity for horses. This article reviews the current information available for EPM, including the clinical pharmacology and efficacy of FDA-approved and nonapproved investigational medications for the treatment or prophylaxis of EPM. Equine protozoal myeloencephalitis is caused by 2 apicomplexan protozoal parasites: S neurona and, much less commonly, Neospora hughesi. Location of the causative organism in the CNS is random, so clinical signs of EPM are highly variable. Any combination of neurologic signs is possible, although spinal cord involvement is most common. Onset may be gradual or acute, with the usual pattern being mild clinical signs that progress with time. Furthermore, the intracellular localization of the causative organisms in the CNS creates difficulties for chemotherapeutic approaches and may also interfere with host-based immunologic defenses. Antemortem diagnosis of EPM is particularly challenging, requiring careful evaluation of the animal\u27s history, clinical signs, and laboratory data, with rigorous exclusion of other causes. Definitive diagnosis of EPM is dependent on necropsy detection of typical CNS lesions of the disease or presence of the appropriate causative organisms. Although careful clinical examination remains the most important antemortem diagnostic technique for EPM, laboratory methods have been developed to assist clinical diagnosis. As such, for horses with clinical signs consistent with EPM, it is optimal to perform immunoblotting, an indirect florescent antibody test, or ELISA analyses on blood and CSF samples prior to diagnosis and initiation of treatment. Preventative approaches to EPM are not well defined. Prevention of EPM with daily pyrantel tartratee administration at the current labeled dose has not been effective in immunocompetent horses1 or in interferon-γ knockout mice,2 even though the compound is active against S neurona in vitro.3 An EPM vaccine based on homogenates of S neurona merozoites with conditional licensure has been marketed for prevention of EPM, but this vaccine was removed from the market due to lack of efficacy data in prospective studies

Dexamethasone serum concentrations after intravenous administration in horses during race training

Dexamethasone (DXM) sodium phosphate is a widely used corticosteroid for inflammatory conditions in horses, regulated in racing jurisdictions in the USA by a 0.005 ng/ml serum/plasma threshold. This study seeks to describe serum concentrations of DXM at 48 and 72 h after intravenous administration of 20 mg DXM sodium phosphate over 1 to 5 days, and to identify a possible source of DXM overages. 74 horses (39 Thoroughbreds, 13 Standardbreds, 22 Quarter Horses) in active race training received 20 mg DXM sodium phosphate. Serum was collected before injection, at 48 and 72 h post last injection, and analysed by LC/MS-MS (limit of quantification (LOQ) = 2.5 pg/ml). No differences were identified by ANOVA (P≤0.05) for racing breeds, age, gender or the number of days of DXM sodium phosphate administration, so data were pooled for each time point. The DXM serum concentration at 48 h (mean ± standard deviation, range) was 2.18±1.56 pg/ml (&&2.5 to 40 pg/ml). Summary statistics could not be derived for 72 h DXM serum concentration data owing to censored data, but ranged from &2.5 to 95.8 pg/ml. There was one extreme outlier (Tukey) at 48 h, and two extreme outliers at 72 h. A separate study was conducted using sedentary experimental horses to determine the likelihood that positive DXM samples could result from environmental transfer. Urine was collected from a mare 2 to 3 h post administration of 20 mg DXM. Hay with 100 ml of the DXM (17 ng/ml) containing urine was offered to each of six experimental horses and blood was collected at 0, 4, 8, 12, 16, 20 and 24 h. All six horses had plasma DXM concentration above the limit of detection and five of six had plasma DXM concentrations above the LOQ for at least one sample time

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

Clenbuterol is a β2 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 μg/kg, BID) for 10 days, and serum was collected for 14 days thereafter. Serum clenbuterol showed mean trough concentrations of ∼150 pg/mL. After the last dose on day 10, serum clenbuterol reached a peak of ∼500 pg/mL and then declined with a half-life of ∼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 μg) was also administered intratracheally to five horses. Peak serum concentrations of ∼230 pg/mL were detected 10 min after administration, dropping to ∼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

Dexamethasone serum concentrations after intravenous administration in horses during race training

Dexamethasone (DXM) sodium phosphate is a widely used corticosteroid for inflammatory conditions in horses, regulated in racing jurisdictions in the USA by a 0.005 ng/ml serum/plasma threshold. This study seeks to describe serum concentrations of DXM at 48 and 72 h after intravenous administration of 20 mg DXM sodium phosphate over 1 to 5 days, and to identify a possible source of DXM overages. 74 horses (39 Thoroughbreds, 13 Standardbreds, 22 Quarter Horses) in active race training received 20 mg DXM sodium phosphate. Serum was collected before injection, at 48 and 72 h post last injection, and analysed by LC/MS-MS (limit of quantification (LOQ) = 2.5 pg/ml). No differences were identified by ANOVA (P ≤ 0.05) for racing breeds, age, gender or the number of days of DXM sodium phosphate administration, so data were pooled for each time point. Summary statistics could not be derived for 72 h DXM serum concentration data owing to censored data, but ranged from \u3c 2.5 to 95.8 pg/ml. There was one extreme outlier (Tukey) at 48 h, and two extreme outliers at 72 h. A separate study was conducted using sedentary experimental horses to determine the likelihood that positive DXM samples could result from environmental transfer. Urine was collected from a mare 2 to 3 h post administration of 20 mg DXM. Hay with 100 ml of the DXM (17 ng/ml) containing urine was offered to each of six experimental horses and blood was collected at 0, 4, 8, 12, 16, 20 and 24 h. All six horses had plasma DXM concentration above the limit of detection and five of six had plasma DXM concentrations above the LOQ for at least one sample time

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 d6 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 intraveneous 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 1-50 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 \u3e 0.99, whereas standards in the concentration range of 10-1000 ng/mL were fit to a second-order regression curve with r \u3e 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)phyenylamino]-1- piperidinepropionic acid methyl ester) is a μ-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