Research Accomplishment Reports 2007

High Sensitivity Analytical/Toxicological Approaches to Problems in Equine Medicine

T. Tobin
Department of Veterinary Sciences

 

Project Description

The program focuses on developing highly sensitive liquid chromatography tandem mass spectrometry [LC-MS-MS] and other MS analytical methods for characterization of the interaction of high potency xenobiotics with horses and related mammals both in regulatory and non-regulatory contexts. Target xenobiotics include therapeutic pharmaceuticals with potential for improper use in racehorses, and environmental xenobiotics, including ergot alkaloids associated with fescue toxicoses.

In related work, unique chemical syntheses are creating forensically useful stable isotope reference standards for use in racing regulation. In pharmacokinetic/pharmacodynamic work with trimethoquinol (TMQ), an agent with high potential for abuse in racehorses, we developed a highly sensitive and specific LC-MS-MS method for this agent in equine plasma and urine. Using these analytical techniques and working with colleagues at Michigan State University (MSU), we showed that TMQ is, for all practical purposes, pharmacologically inactive in horses following oral or intratracheal administration, but highly active following aerosol administration. Additionally, the TMQ LC-MS-MS method is sufficiently sensitive and specific to enable its effective regulatory control in racehorses.

In other work with colleagues at MSU on the equine therapeutic medication tramadol, a potentially useful equine analgesic, we developed a highly sensitive MS analytical method for this agent in equine plasma and are pursuing pharmacokinetic studies relating blood concentrations of tramadol to its pharmacodynamic responses in horses. This work suggests that the analgesic response to tramadol in the horse is relatively modest.

In toxicodynamic research focused on the ergot alkaloids, we have developed a highly sensitive LC-MS-MS method for ergovaline and ergocryptine in equine plasma and shown that the plasma concentrations of these toxins in horses grazing ergot alkaloid positive pastures are in the very low picogram per milliliter range; this newly developed analytical technology is now being applied more widely in the area of fescue toxicoses research.

In the area of equine medication regulation, we are developing a unique panel of stable isotope reference standards for equine therapeutic medications and/or their target analytes.  Products of the program include stable isotope reference standards for flunixin, ketoprofen, methocarbamol, pyrilamine and, additionally, for urinary metabolite target analytes of several equine therapeutic medications, namely, hydroxyethylpromazine sulfoxide, carboxydetomidine, 3-hydroxylidocaine and 3-hydroxymepivacaine, the target analytes in equine urine of, respectively, the equine tranquilizers, acepromazine and detomidine and the local anesthetics, lidocaine and mepivacaine.

Other work carried out in concert with colleagues at the Livestock Disease Diagnostic Laboratory include development of a MS method for unequivocal identification of ivermectin in the tissues of two canine deaths caused by over dosage with ivermectin, and validation of a method for gabapentin quantitation and a pharmacokinetic and pharmacodynamic evaluation of gabapentin in the horse.

Impact

The immediate impact associated with this project will be the worldwide availability of stable isotope reference standards necessary for quantitative regulation of equine therapeutic medication. In the US, the Racing Medication Testing Consortium and the Association of Racing Commissioners International recognize 50 equine therapeutic medications and are committed to developing quantitative thresholds for these medications in plasma or urine. Forensic quantitation requires use of stable isotope reference standards of either the parent medication [plasma] or the urinary target analyte, often a chemically unique urinary metabolite fragment.

Supported by the National and Local Horsemen's Benevolent and Protective Associations and the Kentucky Science and Engineering Foundation, we have created stable isotope reference standards/target analyte standards for at least eight equine therapeutic medications.  Additionally, we are seeking patent protection and are commencing commercialization of these properties with the goal of creating an intellectual property protected panel of commercially available stable isotope reference standards for use worldwide in equine medication regulation. Commercialization will be based in Kentucky, and in support of this plan we have submitted a Small Business Innovative Research application to the USDA. With respect to our newly developed ability to detect and quantify extremely low concentrations of ergot alkaloids in the plasma of horses grazing ergot alkaloid positive pastures, our new technology will allow confident identification of the specific ergot alkaloids associated with fescue toxicosis, raising the possibility of improved diagnostic techniques, with associated benefits in the areas of toxicosis prevention and/or prophylaxis. In this regard, we have submitted a commercialization proposal to the Kentucky Science and Engineering Foundation to commercialize the improved diagnostic techniques resulting from this work.

With regard to Tramadol, control of pain in the horse by narcotic analgesics is not practical because of the marked locomotor stimulation produced by the opiates in horses. As such, there is considerable interest in the therapeutic potential of Tramadol in equines, and the goal of this project is to identify dosage schedules of Tramadol that produce useful analgesia without locomotor stimulation. Achievement of this goal would result in a substantial improvement in the therapeutically available approaches to equine pain control. The studies on Trimethoquinol (TMQ) were initiated by industry concerns about its misuse as a pre-race bronchodilator in racing horses. Studies on TMQ showed that by the intravenous route it was one of the most potent medications ever evaluated in horses. To allow regulatory control of TMQ we have developed a highly sensitive liquid chromatographic tandem mass spectrometric test, that can identify and confirm TMQ as low as 10 pg per milliliter in plasma or urine.  This technology is sufficiently sensitive and specific to fulfill the primary goal of this research, which is to enable effective regulatory control of TMQ in horse racing.

Publications

Camargo, F.C., Robinson, N.E., Berney, C., Eberhart, S., Baker, S., DeTolve, P., Derksen, F.J., Lehner, A.F., Hughes, C., and Tobin, T. (2007). Trimetoquinol, Inolin ®: Bronchodilator Effects in Horses with Heaves Following Aerosol and Oral Administration, Equine Veterinary Journal, 39(3): 215-220.

Lehner, A.F., Stewart, J., Dafalla, A., Daly, J., Connerly, A.L., Jones, C.N., Thompson, K., Dirikolu, L., and Tobin, T. (2007). Gabapentin in Horses: Validation of an Analytical Method for Gabapentin Quantitation, Journal of  Analtical Toxicology, 31: 555-565. 

Budhraja, A., Camargo, F.C., Hughes, C., Lehner, A.F., Stirling, K., Brennan, N., Dowling, M., and Tobin, T. 2007. Caffeine and theobromine identifications in post-race urines: Threshold levels and regulatory significance of such identifications. Page 87 to 92 in Proc. 53rd Annual Conf AAEP, Orlando FL.

Tobin, T., Karpiesiuk, W., Hughes, C., Lehner, A.F., and Camargo, F. 2007. Synthesis of Deuterated Analytical Standards: Ketoprofen-d3 and Flunixin-d3.   Page 214 to 217 in Proc. 16th International Conference of Racing Analysts and Vets, Tokyo, Japan.