Truesdail Laboratories will supply the containers for taking urine and blood samples. After samples are collected they will be sealed and labeled at the track by Oklahoma Horse Racing Commission personnel, then shipped to Truesdail for analysis. When the samples arrive, Truesdail’s personnel will check all seals and sample numbers against the chain-of-custody document and log the data into our computerized Laboratory Information Management System (LIMS). The chain-of-custody is maintained throughout our entire testing process. Whenever a sample(s) aliquot is handled, a log entry is recorded to document the activity.
Blood and urine samples will be opened, portions removed for analysis, the urine containers resealed with security tape, and then initialed and dated by the person who removed the test portions to maintain chain-of-custody. Subsequent removal of test portions will also be completed with this tape-resealing procedure.
Testing for drug control has been a changing process over its approximately 70-year history. Microcrystalline testing was used in the 1940’s and 50’s. This gave way to thin-layer chromatography that dominated testing until the 1980’s, when drug-specific immunoassays became available to improve sensitivity for many drugs.
In the late 1980’s and 90’s direct instrumental screening by gas chromatography/mass spectroscopy (GC/MS) began to replace thin-layer chromatography. Improvements to liquid chromatography/mass spectroscopy in the 1990’s brought a new tool to use for drug screening.
By the 2000’s, high-performance liquid chromatography coupled with ion trap detectors (LC/MSn) and/or triple-stage mass spectroscopic detectors (LC/MS/MS) provided the most sensitive technology for drug screening. These technologies are still prominently used in many laboratories as a common screening method.
While LC/MS ion trap and LC/MS/MS triple-stage technologies are extremely sensitive, they have limitations. The number of compounds that can be screened is limited by the scan time; and the maximum number of compounds that can be sought in a single run is 200 to 250 without losing sensitivity. LC/MS/MS methods only screen for compounds pre-programmed into the method, and, once generated, the data cannot be re-examined to look for additional compounds.
Two (2) new improvements recently became available for LC/MS testing. On the chromatography side, ultra-high performance liquid chromatography (UHPLC) has become more routine. On the mass spectrometer side, high-resolution mass spectroscopy (HRMS) has become available at a much lower cost than previously allowing a much more flexible screening protocol.
UHPLC was made possible by the development of pumping systems that deliver much higher pressures to chromatography columns. High pressures allow for the use of more tightly packed and narrow columns. The net result is much higher resolution chromatography, sharper peaks, and in most cases improved sensitivity. Also, the time required to analyze a sample has been greatly reduced allowing more samples to be analyzed in a given timeframe. While UHPLC instrumentation is rather expensive, the high throughput of samples has allowed our lab to lower the cost for direct instrumental screening of samples.
Screening by HRMS has overcome the two main limitations of the triple-stage mass spectroscopy listed above. The number of drugs that can be sought in a single run is not limited by scan time or loss of sensitivity issues. The HRMS detector easily detects all unknown peaks and the instrument’s manufacturer has developed comprehensive software to help identify unknowns. Since detection criteria do not need to be programmed into the instrument before the analytical run is made, data collected from HRMS systems can be re-examined at any time after collection to look for new compounds once structural information is available.
The Thermo OrbitrapTM HRMS instruments are manufactured in Germany and European labs were the first to employ this technology. The equine drug-testing laboratory in France quickly adopted the “Orbitrap” technology for its equine drug-screening program. In the U.S., the equine drug-testing lab at the University of California, Davis, was the first university lab to bring this system on-line and Truesdail was the first commercial lab in the U.S. to use this technology for screening.
Since acquiring our first Thermo OrbitrapTM UHPLC/HRMS system, Truesdail began building a target list of compounds of interest to the racing community to be sought in each sample. We began with setting up our target list to include the total list of compounds required by the American Graded Stakes Committee and the list of compounds in ARCI’s (Association of Racing Commissioners International) uniform classification list. We have also included drugs targeted in human Olympic athletes, which could be used on horses. Our target list includes both parent drugs and known drug metabolites. Currently our target list has over 1,800 compounds and is still growing.
In 2011 our new UHPLC/HRMS screening technology was approved during our annual ISO/IEC 17025 audit and added to our scope of accreditation.
Screening of both urine and blood samples provides much redundancy to the testing in that most compounds will be found in both media. However, the TOBA Graded Stakes testing has minimum requirements for detectability for most drugs in urine and thus testing of both urine and blood will be done on all Graded Stakes samples.
There are some differences to be noted in blood versus urine samples. Many drugs are removed from circulation in the blood through a metabolic process, thus we tend to find higher levels of parent drug for some in the blood, and higher levels of metabolites of some in the urine.
Because we expect to find many drugs to have been metabolized, urine samples are subjected to enzyme hydrolysis prior to extraction. Hydrolyzed urine is isolated and separated by solid phase extraction into acidic/neutral and basic fractions covering the same type of drugs as indicated for blood samples.
Although immunoassay testing is decreasing in importance, there are a few drugs that do not extract well and could easily be missed in direct instrumental screens. We propose to use eight (8) immunoassays to supplement our direct instrumental screens.
All ELISA kits depend on drug specific antibodies bound to plastic microtiter wells for detection of drugs. The characteristics of the antibodies attached to the wells of the plate determine which drug(s) the assay will detect. In most respects the kits are the same except for the antibodies in the wells and the drug that is used to coat the enzyme responsible for the color reaction.
Truesdail Laboratories currently runs all kits with at least two standards, even when the manufacturer does not specify quantitation. Quantitative data has allowed us to monitor the behavior of the assay and to validate detection levels on each plate.
ELISAs are currently run on a plate of 96 wells. Calibration samples, QA/QC samples and reruns require about 15% of the plate. Therefore, approximately 80 samples can be screened on a typical 96 well microtiter plate when no samples are pooled.