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 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 was 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 in the US, located in Florida. In the late 1930’s, Truesdail Laboratories was selected by the California Racing Commission to do drug testing of their samples and we recruited Mr. Bob Vessiny from the Florida lab,. Mr. Vessiny had previously been 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 about 6 years ago.
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”).
From the 1930’s to the 1950’s the main technology employed for drug testing was called “microcrystalline testing”. Saliva samples were extracted with organic solvents and reacted with specific reagents, and the solvents evaporated. Crystalline residues were examined under a microscope and compared with crystals produced by known drugs.
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 2-3 orders of magnitude improvement in sensitivity over microcrystalline testing and also improved specificity. 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 GCMS 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 and ion trap mass spectrometers which tended to be slightly more sensitive than GC/MS systems for most compounds. Arriving shortly after were LC/MS systems with triple stage mass spectrometers (LC/MS/MS) which were substantially more sensitive and LC/MS has 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.
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 ultrahigh 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 2010 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 and the collected data can be re-analyzed after the fact for additional compounds. In 2013 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.
| Relative Drug Detection Sensitivities
|Thin Layer Chromatography
|Early LC/MS/MS and UHPLC/HRMS
|Most Common LC/MS/MS and UHPLC/HRMS
|Newest top of the line LC/MS/MS and UHPLC/HRMS