DRUG DETECTION PROCEDURES
John H. Vine
Racing Analytical Services Ltd
400 Epsom Road, Flemington, Victoria 3031. Australia
Drug testing in the racing industry has undergone significant changes in the last 25 years. These changes have transformed drug detection to the extent that the vast majority of drugs can now be easily detected at physiologically significant levels. In addition, the specificity with which drugs may be detected has all but eliminated the possibility of incorrect identification of a drug.
This is not to say that there are no more challenges facing the racing laboratory. While chemical, low molecular weight drugs are now well covered, the analytical challenge is shifting to the large biopolymer drugs such as erythropoietin (Epo) and growth hormone (GH).
This paper presents a brief overview of modern drug detection procedures and an outline of some new developments in drug detection.
DRUG TESTING STRATEGY
There are thousands upon thousands of potential drugs and the racing laboratory must have a strategy which enables the it to quickly test for the presence of as many of these drugs as possible. This is done by splitting drug testing into two procedures, screening and confirmatory testing.
Drug screening involves the application of a wide range of testing methods intended to quickly sort through the samples to identify those which may contain a drug and eliminate those which clearly do not. Screening methods can be divided into two groups, those based on chemical methods and those based on biological methods.
Chemical Methods Biological Methods
Chemical tests Radioimmunoassay (RIA)
Thin layer chromatography (TLC) Fluorescence polarisation immunoassay (RIA)
Gas chromatography (GC) Enzyme multiplied immunoassay (EMIT)
Liquid chromatography (LC) Enzyme linked immunosorbent assay (ELISA)
Gas chromatography mass spectrometry (GCMS)
Liquid chromatography mass spectrometry (LCMS)
The use of chemical tests and TLC has been all but phased out in many racing laboratories, except for some specialised applications, and screening is now usually based on gas and liquid chromatography. The detector of choice for this screening is the mass spectrometer and many laboratories now use both GCMS and LCMS extensively for screening. The advantages of these methods are their sensitivity, specificity and their largely open-ended nature which provides coverage for a very wide range of drugs.
The biological methods which are based on some form of immunoassay tend to be more specific and are better suited to target screening for particular drugs or groups of drugs. These methods are often very sensitive and are easy to automate so that large numbers of samples can be screened quickly. They are, however, relatively expensive and for that reason, are best suited for use in the detection of drugs which are difficult to detect using chromatographic methods.
Confirmatory methods are now almost universally based on GCMS and LCMS as these methods provide the specificity needed for unequivocal identification of a particular drug. Increasingly multistage mass spectrometry (MSMS) is being used to provide additional sensitivity and specificity.
Other specific methods are used for particular substances, for example quantification of total carbon dioxide (TCO2) in plasma is usually carried out using an ion selective electrode analyser.
Perhaps the most important new development in the last few years has been the advent of practical, reliable and easy to use LCMS. This method provides very facile detection of many drugs and drug groups which are difficult to detect by other methods.
For example, amongst the early successes were the detection of the quaternary ammonium drugs clidinium bromide and propantheline bromide. While these drugs can be detected indirectly by GCMS this is difficult to do in practice and requires specific methods rather than the more general screening procedures. LCMS provides a quick and easy way of detecting these two drugs together with a wide range of related drugs.
More recently LCMS has been used to provide a wide ranging screen for corticosteroids which covers many more of these drugs than did the GCMS method used previously. To cover just the most common corticosteroids in this range using immunoassays would require at least 6 or 7 different immunoassays. Other corticosteroids in this range cannot be detected using immunoassay because no assay is currently available for them. LCMS has also been used to improve the sensitivity of trenbolone detection and, because this method does not require derivatisation steps, to halve the time needed to prepare specimens for analysis.
The other major development in analytical methods has been the increasing use of solid phase extraction in place of the older liquid/liquid methods. These solid phase methods can be automated using robotic systems and in many cases provide better recoveries of drugs from blood and urine than more conventional methods. In particular, the use of mixed phase columns has made it possible to elute widely different types of drugs from a single column.
Perhaps the most challenging problem facing the racing laboratories at the present time is the detection and confirmation of the use of biopolymer drugs. The two drugs currently of greatest concern are erythropoeitin (Epo) and equine growth hormone (eGH). Substances of this type are also of great concern in human sports and the International Olympic Committee has committed many millions of dollars to find ways of detecting Epo and human growth hormone (hGH).
The racing industry's approach has been much more modest, however, research work has been carried out on both Epo and eGH. The former may be detected by a number of commercial immunoassays, however, at the present time confirmatory testing to the standard applied to other drugs is not yet a reality. The Australian thoroughbred industry has funded a project aimed at the detection of eGH and a screening method has been developed. Further work is in progress to provide more definitive identification methods.
ACCREDITATION OF RACING LABORATORIES
The final factor in improving drug detection for the racing industry and providing a quality based service has been the move to national accreditation of racing laboratories. Many laboratories are now accredited by their national laboratory accreditation agency. Through international agreements these accreditations are then accepted in other countries and so gradually a network of internationally recognised racing laboratories is being built up.
This provides the racing industry with a guarantee of performance and analytical capability and means that horses racing in different countries are now subject to consistent drug detection procedures so that results obtained in one country can be relied on and repeated in other countries.
This paper has presented a brief overview of drug detection procedures. While detection of the majority of drugs is now relatively straightforward, the introduction of new drugs, the use of old drugs in new ways and the advent of the biopolymer drugs ensures that there are still challenges to overcome. One thing is certain, as long as money is wagered on races, there will always be a small minority who will try to cheat the system. Drug testing will, therefore, need to keep pace with increasing sophistication in the use of drugs if the integrity of the racing industry is to be protected.