Retrospective on Testing for Drugs in Racing Animals trueadmin
Retrospective on Testing for Drugs in Racing Animals
In the early 1800’s there was not much concern about the use of medications to enhance performance of racing animals. However, during the 1800’s the isolation and purification of both morphine and cocaine was achieved. Near the turn of the century (1890-1910) a group of American trainers came to the European continent and brought with them some of the American “medications”.
Because their success rate was so good at some races in Russia, Russian officials came up with the idea to try to test some of the horses saliva for foreign substances by force feeding the saliva to frogs to see if there was any unusual behavior. There was remarkable change in behavior by the frogs to some samples taken from horses trained by American Jack Keene. Behaviors believed to be symptomatic of frogs being given cocaine. Mr. Keene’s career in Russia ended quickly and he returned to a long term career in the US. (Keeneland Race Track in Kentucky).
The first official drug testing in the US began in the early 1930’s in New York. Shortly after the lab in New York was established, some of their staff were recruited to start the second drug testing lab located in Florida. In the late 1930’s, Truesdail Laboratories was selected by the California Racing Commission as the drug testing laboratory of their samples. Truesdail recruited Mr. Bob Vessiny from the Florida laboratory. Mr. Vessiny previously was brought to Florida from the New York lab. Mr. Vessiny, a founding member of the Association of Official Racing Chemists (AORC) set up Truesdail’s drug testing program and remained active at Truesdail until his death in 2006.
Early equine drug testing in the US followed the Russian precedent of testing saliva. Some old timers still refer to the area set aside at the race track for collecting specimens for drug testing as the “spit box”.
1930’s to 1950’s
From 1930 to the 1950’s the main technology employed for drug testing was called “microcrystalline testing”. Saliva samples were extracted with organic solvents, reacted with specific reagents, and the solvents evaporated. Crystalline residues were examined under a microscope and compared with crystals produced by known drugs.
1950’s to 1960’s
In the 1950’s and 60’s, the use of thin layer chromatography (TLC) grew to prominence. Much research was devoted to develop solvent systems for good chromatography and to produce reagents that reacted with different types of drugs to develop color spots on the TLC plates. TLC gave 2 to 3 orders of magnitude improvement in sensitivity over microcrystalline testing and improved specificity to particular drugs. TLC remained a useful tool for some drugs until recent years.
In the 1980’s the use of drug specific immunoassays joined TLC to provide additional sensitivity required to test for the new highly potent drugs arriving on the scene. The initial kits available were radioimmunoassay (RIA) and these were followed by fluorescent immunoassays (FPIA) and then enzyme linked immunosorbent assays (ELISA). Because of their relative ease of use and lower cost ELISA testing soon dominated the field. ELISA tests are not as widely used now, but testing by ELISA is still used for certain drugs that are difficult to isolate or chromatograph.
In the late 1980’s screening methods employing gas chromatography coupled to mass spectroscopy (GC/MS) began to be used for screening for specific groups of drugs. This was the beginning of what has now been given the generic name of “direct instrumental screening”. Direct instrumental screening with GC/MS is still used by some labs for specific groups of drugs, but most labs are replacing GC/MS with methods using liquid chromatography coupled to mass spectroscopy (LC/MS).
LC/MS as a tool for direct instrumental screening made its entry in the 1990’s. Early bench systems had single stage mass spectrometers. Next ion trap mass spectrometers which tended to be slightly more sensitive than GC/MS systems for most compounds were introduced. Arriving shortly after were LC/MS systems with triple quadrupole mass spectrometers (LC/MS/MS) which were substantially more sensitive and had the further advantage in that compounds did not have to be derivatized before they were analyzed on a GC/MS system. Use of LC/MS/MS for direct instrumental screen quickly dominated the field for direct instrumental screening. It is still used by many labs as their primary method for screening.
However, there have been many technology improvements in LC/MS technology since the turn-of-the-century. On the HPLC side there have been improvements in both the pumping technology and the column technology such that systems described as ultra high performance liquid chromatography have become available that have improved resolution and also decreased analysis times. High resolution mass spectrometers (HRMS) became available in smaller, efficient, benchtop systems, and by 2010 the combined systems of UHPLC/HRMS began to be used for drug screening.
Truesdail Laboratories purchased our first UHPLC/HRMS instrument in 2008 and quickly saw it had significant advantages over LC/MS/MS for screening. To gain maximum sensitivity LC/MS/MS systems must be set up to look for specific characteristics of molecules and the number of compounds that can be sought is finite, and once data is collected it cannot be reexamined for compounds that were not initially sought. High resolution mass spectroscopy scans for all masses over a rather large range and the system can be set up to screen for 100’s of compounds. Additionally, the HRMS collected data can be re-analyzed after the fact for additional compounds.
Truesdail added our second UHPLC/HRMS instrument. Our drug screening protocols routinely screen for over 1800 compounds. Compounds are routinely detected in the picogram per ml range and some compounds are detectable in the high femtogram per ml range.
Recently utilization of the newer technologies for drug testing have led to substantial improvements in regulations in the sport of Horse Racing. Truesdail has, and is committed to continue to be at the forefront of technological and regulatory changes for this industry.
Truesdail was the first U.S. Lab to identify abuse of Cobalt in race horses. Cobalt, sometimes referred to as the “Poor man’s EPO” is a naturally occurring compound that is found in trace levels in all horses blood. It is believed to increase the efficiency of oxygen distribution within a horses system, giving it a significant advantage over its competitors. Unfortunately none of Cobalt’s perceived benefits have any basis in science, there are however, several negative consequences to the use of Cobalt, including; colic symptoms, muscle twitching, excessive sweating, and general discomfort. The long term effects on horses are still largely unknown, in humans excessive Cobalt is known to be a contributing factor to Thyroid Disease and heart and muscle disorders that can lead to death.
Truesdail was one of the first five labs in the U.S. to receive accreditation from the Racing Medication Testing Consortium (RMTC). Truesdail representatives are currently members of the RMTC Scientific Advisory Committee, a committee established to review and discuss the scientific merits of a variety of issues related to drug testing for Race Horses.
Truesdail relocated to a state of the art facility in Irvine, California. Our Drug Testing lab was completely redesigned and optimized for the usage of the newest UHPLC/HRMS LC/MS Systems. You can take a virtual tour of our new facility here.
Dr. Anthony Fontana, Truesdail’s Technical Director and Dr. Norman Hester, Truesdail’s Chief Science Officer joined the RMTC Scientific Advisory Committee. The RMTC SAC provides a dynamic and responsive approach to address the ongoing challenges of a rapidly-evolving medication landscape in horseracing.
2016 & Beyond
Truesdail is constantly conducting Research & Development and implementing new compounds into our screening library. Truesdail’s library currently contains over 1800 compounds for analysis.
Some recent examples include:
Truesdail is working closely with our clients to develop and bring online testing for Glaucine. After several positive sample reports Truesdail assisted impacted jurisdictions with identifying potential sources of barn and feed contamination, this information empowered our customers to educate the impacted trainers and vets to avoid potential sources of contamination to avoid potential positive test results and ensure healthy horses.
Truesdail is investigating the potential for feed source contamination that is resulting in a high number of Methocarbamol in sport with our clients.
We continue to work with our customers, regulatory bodies, and industry stakeholders in the future to ensure a clean and healthy sport that future generations can enjoy.