A 2-week washout period was allowed between each dose. ELISA testing revealed no evidence of albuterol in urine at 24 hours after any single-dose administration. Results indicated that 48 hours or longer should be allowed for albuterol to be cleared from urine after single doses.
When given at the maximum recommended rate of six actuations per dose four times a day for 5 days, urine samples tested by ELISA showed no evidence of albuterol at 48 hours after the final dose.
Testing of nasal swabs by ELISA demonstrated the presence of albuterol for 8 hours after each single dose, and some horses might have detectable levels of albuterol in nasal swabs for several days following administration of multiple doses. As a guideline for withdrawal time, 72 hours or longer should be allowed after administration of aerosol albuterol sulfate to horses before participation in equestrian competitions that are regulated for detection of certain performance-enhancing substances.
However, these recommendations were based on a small sample of horses and the specific ELISA test used and interpreted as described. Factors specific to individual horses may influence these detection times. Onset of action with albuterol is rapid 5 minutes , and its effects can last from 30 minutes to 3 hours.
Because the lung is the target organ for albuterol, administration by this device allows efficient delivery of aerosol albuterol sulfate to the sites of action in the respiratory tract. The efficiency of the inhaler delivery system, therefore, serves to reduce the dose of aerosol albuterol sulfate required to attain a therapeutic response.
This reduction in the required dose is expected to bring the added benefit of reducing both incidence and intensity of adverse responses and, in the case of competition horses, may reduce the possibility of inadvertent medication identifications. ARCI as a Class 3 i. In competition horses, the detection of high-potency medications, such as albuterol, is largely dependent on ELISA testing.
Six mature Thoroughbred mares weighing to kg each were used. Horses were kept in a acre field until they were placed in box stalls and provided with water and hay ad libitum. All horses were acclimated to their stalls 24 hours prior to participation in the study.
The animals were maintained on grass hay, and concentrate feed mixture of oats and an alfalfa-based protein pellet was fed twice a day. Horses were vaccinated annually for tetanus and dewormed every 3 months with a commercial ivermectin product. A routine clinical examination was performed prior to each of the four stages of the study. The horses were managed according to the rules and regulations of the University of Kentucky's Institutional Animal Care and Use Committee, which approved the experimental protocol.
Four dose rates for aerosol albuterol sulfate were administered and evaluated in a series, and all six horses were treated with the same dose of aerosol albuterol sulfate on a given treatment day. A two-week washout period was allowed for each horse before administering the next sequential dose. A venous blood sample was collected into a serum-separator tube from each horse, according to standard postrace testing practice in Kentucky. Complete urine collection was accomplished with a Foley catheter at 0, 1, 2, 4, 6, and 8 hours after administration, and a Harris flush tube 24 Fr x Nasal swabs were collected by inserting a cm inch cotton nasal swab into the ventral nasal meatus of the horse up to approximately 15 cm.
A pretreatment nasal swab sample was taken from each horse; however, this procedure was difficult to accomplish several times daily from the same horse, so a different horse was sampled by nasal swabs at each evaluation one horse per time after treatment with each dose.
The one-step ELISA tests were performed as previously described 7 and according to the manufacturer's instructions, with albuterol standard curves 0. The plates were then placed on a microplate shaker briefly, covered, and incubated at room temperature for 45 minutes.
For nasal swab samples, 5 ml of assay buffer was added to each sample in the vial with extraction by vigorous mixing by vortex. The nasal swab samples were assayed using a buffer standard curve. After incubation of the samples, the wells were inverted, and any remaining liquid was removed by tapping the plate on a lint-free towel.
The plates were inverted and tapped dry between each washing. The optical density OD of each well was read at a wavelength of nm with an automated microplate reader. Samples with OD less than that of the highest standard were diluted appropriately with assay buffer and reassayed.
Because ELISA testing does not specifically identify albuterol, its metabolites, or structurally related substances, all ELISA quantifications were identified as "albuterol equivalents. Estimated concentrations of albuterol equivalents were calculated for the serum samples using the serum matrix standard curve, for the urine samples using the urine matrix standard curve, and for the nasal swab samples using the buffer standard curve.
Where appropriate, statistical analysis was performed by applying the central limit theorem to a sampling distribution of the sample mean in a t -test distribution, with the single assumption that the low sample number is representative of the population in a binomial distribution. The ELISA was highly sensitive for terbutaline, followed by clenbuterol, albuterol, and metaproterenol, respectively.
None of the other compounds listed showed significant cross-reactivity. These data indicate that at least 24 hours should be allowed after intranasal administration of six or fewer actuations for the urine to eliminate albuterol. The mean peak urinary concentrations of albuterol equivalents occurred within 2 to 4 hours after administration and were 1. The mean peak urinary concentrations of albuterol equivalents were 0.
The mean peak urinary concentrations of albuterol equivalents ranged from 15 to Concentrations of albuterol equivalents were During the dosing and for at least 8 hours after the last dose, all urine samples gave strong positive indications of the presence of albuterol.
None of the urine samples had detectable concentrations of albuterol equivalents at 24 hours after the last dose. The data suggest that nasal swabs up to at least 6 hours after treatment can give a positive ELISA response after a single dose of either three or six actuations. The apparent concentrations of albuterol equivalents in the nasal swab extracts following single administrations at one, three, or six actuations are shown in FIGURE Peak concentrations of albuterol equivalents were obtained within 1 to 2 hours post-dosing and were 6.
These data suggest that nasal swabs collected up to at least 24 hours after the last dose may have detectable levels of albuterol equivalents by ELISA. As a bronchodilator, albuterol has the potential to alter the athletic performance of horses, 5 particularly if the animal experiences bronchospasm and bronchoconstriction associated with reversible airway bronchoconstriction. The major therapeutic advantage of the inhaler device used to deliver aerosol albuterol sulfate is that the system delivers active compound directly and deeply into the lung resulting in rapid onset of therapeutic action 5 to 15 minutes.
Administration by this route also bypasses the gastrointestinal tract and liver metabolism, either of which can significantly reduce or alter the bioavailability of an orally administered drug.
Administration of aerosol albuterol sulfate with this inhaler device can significantly reduce the dose required for therapeutic efficacy. These characteristics of the inhaler device provide high therapeutic efficacy and a low incidence of side effects.
Regulatory control of albuterol generally involves ELISA screening of postrace urine samples, followed by mass spectral confirmation of its presence. For all time points, the maximum inhibition of ELISA activity after treatment was not substantially different from baseline values.
In the absence of unusual extraction procedures, the ELISA test used in this study is unlikely to detect albuterol in serum samples after administration of these doses by the equine inhaler device. Conversely, albuterol equivalents were readily detected in urine samples following administration of various recommended doses of aerosol albuterol sulfate.
The mean peak urinary concentrations of albuterol equivalents occurred within 2 to 4 hours after treatment, and the majority of the urine samples did not contain ELISA-detectable concentrations of albuterol equivalents at 24 hours after administration. To be conservative, however, it would be prudent to allow at least an additional 24 hours total of 48 hours or longer for albuterol or its metabolites to clear after single administrations.
The maximum recommended duration of treatment with aerosol albuterol sulfate is four daily doses six actuations per dose for 5 days. Following administration of the maximum recommended dose in the present study, the mean peak urinary concentrations of albuterol equivalents ranged between 15 and After multiple dosing for 5 days, the mean urinary concentrations of albuterol declined rapidly, and none of the urine samples had a detectable level of albuterol equivalents 24 hours after the last dose.
However, because the present study involved a small sample of horses and might not be representative of a larger population, an additional 48 hours total of 72 hours , or longer is recommended for albuterol or its metabolites to be cleared from the urine. ELISA analysis of nasal swab material revealed that peak concentrations of albuterol equivalents were obtained within 1 to 2 hours after administration of a single dose one, three, or six actuations per dose. Although the data used to determine elimination of aerosol albuterol sulfate were based on a single horse per dose at each evaluation time, it is likely that nasal swabs collected up to 6 hours or longer after administration of three or six actuations per day for one day will still yield a positive ELISA response.
The data also suggest that any nasal swab collected 24 hours or longer after the last dose of the multiple treatment regimen may have detectable levels of albuterol equivalents by ELISA. However, 48 hours or longer is recommended as a conservative period for albuterol to be eliminated from the urine following administration of these single daily doses before entering a horse into a competition that is regulated by drug testing.
In interpreting and applying these findings, it must be kept in mind that these results are guidelines only and were developed in a relatively small sample of horses.
The findings are based on the sensitivity of the ELISA test used as described, and this test differs somewhat in sensitivity from the one used at racetracks. Modification of this test or its interpretation to increase its sensitivity has the potential to increase the probability of detection of albuterol.
Beyond this, factors specific to individual horses may also influence detection times. As always, prior to administration of any medication to a horse entering an event that is likely to test for medications or drugs, it is advisable to consult your veterinarian or other experts concerning any unique regulatory or testing characteristics related to the medication, the horse, or the event in question.
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