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Chemistry at the races
The work of the Horseracing Forensic laboratory
By Ted Lister, Colin Osborne, Maria Pack
The Royal Society of ChemistryCopyright © 2002 The Royal Society of Chemistry
All rights reserved.
Chemistry at the races
The work of the Horseracing Forensic laboratory
Drug testing of animals
Most people will know that sportsmen and women are regularly tested for drug use both in competition and in training. Drugs are also banned in sporting competitions involving animals, including horses and dogs. Cases where household names fail drug tests hit the headlines, Figure 1. The authorities that administer these sports, for example The Jockey Club of Great Britain for horseracing, have strict rules about drug use. This article concentrates on horseracing, but the principles involved are similar for all such authorities. The Horseracing Forensic Laboratory (HFL), situated near Newmarket, in the heart of racing country, carries out drug testing on horses (and on dogs) competing in the UK and abroad.
There are several reasons for drug testing of animals in competition, these include:
1. To ensure that outcomes of races are not manipulated illegally (for betting purposes, for example).
2. To ensure that the welfare of the animals is not put at risk. For example an injured animal might be given pain-killers to allow it to compete when it is not fit to do so, thereby worsening the injury. Humans may choose whether or not to use a drug in this situation, animals cannot.
3. Stud and animal sales issues. 'Stud' refers to the breeding of racehorses to sell the offspring. Someone buying a valuable horse (or its semen) will need to know that its racing performances have been genuine and not enhanced by the use of drugs.
The rules relating to drug use in a particular sport are made by that sport's governing body. HFL in Newmarket analyses samples of urine (or, occasionally of blood) and reports back to the governing body. Each sample arriving at HFL is coded so that the analysts do not know the identity of the animal concerned, or even the sporting event it has come from. This is important to ensure that there can be no accusations of bias. Any report of a positive test is passed on to the appropriate governing body, which decides whether any further action should be taken. In this way the laboratory can maintain its professional integrity – it is completely independent and impartial.
The rules governing what drugs can be given to horses differ in principle from those governing human athletes. For human athletes, there is a list of banned categories of substances. For horses, the principle is that any drug that is physiologically active is banned. You can find more detail about classes of drugs that are banned in horses and the body systems that they act upon at:
(accessed October 2001).
It is not practical to test every racehorse. At a race meeting it is the stewards in charge of the event who decide which horses should be tested. There is no predictable formula for deciding which horses to test because this could help potential dopers to evade detection. However, winners, and horses that have run much better or much worse than expected, are liable to be selected. In this way the mere risk of a dope test being done has a deterrent effect.
Blood samples can be taken for analysis, but they give only a 'snapshot' of what is in the animal's system at the actual time of sampling – as the blood circulates through the liver, especially, changes will take place to the chemicals in it – see Drug Metabolism. More usually, urine is sampled for analysis. Unlike blood, urine's composition does not constantly vary – once blood has passed through the kidneys and excretion products have been removed into the urine, those products no longer change, they are merely stored in the bladder. Consequently, urine analysis gives information on what has been in the body as well as what is there at the time of sampling.
Drugs can remain in the body for several weeks and can also be identified from their metabolites, the substances that they are converted into in the liver. This means that illegal substances can be detected a considerable time after they have been given.
The sensitivity of modern analysis requires trainers to know exactly what has been fed to a horse. This could even include chocolate fed to it by well-meaning strangers; chocolate contains the stimulant theobromine. Security measures are taken to ensure that strangers cannot gain access to horses, so stable lads might be required to sleep in the stables the night before a race.
Keeping samples secure
After a race, the horses to be sampled are escorted to the doping unit, where a specimen must be collected. This might involve a wait, but rustling straw has been known to encourage horses to urinate. The sample is collected in a plastic bag held in a net on the end of a telescopic pole (rather like a fishing net). The urine is then poured into two identical 250 cm3 polythene bottles. The bottles are then sealed by a vet, packed with a cooling pack like those used in picnic boxes and marked with a barcode before being sent to HFL for analysis.
It is essential that the samples analysed are not tampered with on their way to HFL. There are a number of tamper-evident layers of packaging. These include:
* The address label of the sample box covers the closure, so will be obvious if the box has been opened.
* The sample bottles are sealed in a polythene envelope. If the flap is lifted, the word 'void' shows, and the heat-sealed sides of the envelope have lettering in the polymer so that it is obvious if an attempt has been made to slit the sides to get to the bottles.
* The sample bottles have tamper-evident plastic caps with lugs that must be broken to open them (some drinks sold in supermarkets have this type of cap).
* Once this cap has been removed, a heat-sealed diaphragm must be cut through to allow the contents to be poured out. This seal is applied by the vet, and is so strong that neither the bottle nor the seal breaks even if the bottle is stamped on.
The materials required for taking and packaging samples are sent to the racecourse in the form of a kit, Figure 2, timed to arrive just before the meeting to eliminate the potential for them to be interfered with.
If any of the seals have been tampered with, the lab reports the details to the regulatory authority which then decides whether or not to have the sample analysed.
Analysis of a sample
When the lab receives a sample, one of the two bottles is put in a freezer to allow later analysis, or analysis by another independent lab if the first findings are challenged. The bottle that is to be used is opened, and its contents are poured out – no apparatus such as a pipette is inserted because this could introduce contamination. Two samples are taken, one is analysed by a method called immunoassay, and the other by Gas Chromatography-Mass Spectrometry (GCMS).
Q1. Part of the athlete Diane Modahl's defence against an allegation of drug taking was that her sample had been allowed to stand for some time in a hot laboratory before testing.
a) Why is freezing of the second sample necessary to allow it to be tested later?
b) Explain what might happen if the sample were not frozen and how freezing prevents this.
c) As a rule of thumb, what happens to the rate of a chemical reaction when it is cooled by 10 °C?
d) A typical laboratory might be at a temperature of 20 °C. What would happen to reaction rates if a sample were cooled to 0 °C but remained liquid?
e) Why would freezing the sample so that it became solid at 0 °C be more effective than cooling the sample to a liquid at the same temperature?
In this method, the neat sample is treated with a solution containing antibodies. An antibody is a protein molecule produced by the body that will bind very specifically to another molecule called its antigen. Certain antibodies can be produced that bind specifically to particular drug molecules. Treatment of the sample with antibodies followed by enzymes and other reagents brings about a colour change so that a sample containing the drug targeted by a particular antibody stays colourless, and a sample without it goes yellow. The amount of colour can be read by a colorimeter, a device that shines a beam of light through the sample. More light will pass through a sample containing the drug than one without it. About 30 drugs are tested for by this method. These are ones that, for various reasons, are not easily measured by CCMS or ones present in low levels, for which immunoassay is particularly sensitive.
Q2. a) When a colorimeter is used in the immunoassay test, explain why 'more light will pass through a sample containing the drug than one without it'.
(b) Which colour of light would be most suitable to measure the depth of colour of the yellow solution in the immunoassay test – yellow or blue? Explain your answer.
Gas chromatography-mass spectrometry (GCMS)
Before this test, the urine sample is passed through a cartridge containing about a 10 mm depth of specially treated silica particles. These absorb the groups of compounds likely to contain drugs and their breakdown products, and let through others compounds such as inorganic salts so that they do not interfere with the analysis. The cartridge is then washed with solvents that remove more of the urine components whilst leaving any drugs in the silica. This is in effect a cleaning step. The mixture containing potential drugs is then washed from the silica with solvents of different pHs and separated by gas chromatography (GC).
Here the mixture is injected onto a chromatography column. This is a 25 m-long silica tube with an internal diameter of just 0.25 mm. (This means that it is flexible enough to be coiled into a spiral about 15 cm in diameter and placed in a temperature controlled oven, Figure 3.) The inside of the tube is specially coated and a stream of helium gas flows through the tube at about 1–2 cm3 min-1. This carries the components of the mixture through the tube. Some, however, move more slowly than others, as they tend to 'stick' to the coating of the tube. The 'stickier' they are, the more they tend to bond to the coating and the longer they take to come out of the tube. The time taken for a component to travel the length of the column is called its retention time. Typical fretention times may be several minutes.
Q3. a) Suggest what type of bonding occurs between the molecules in a mixture and the coating of the GC tube to make them 'stick' to it.
(b) Imagine that one type of GC tube coating has many -OH groups. Suggest which of the following molecules would have (i) the longest (ii) the shortest retention time: propan-1-ol, propane, propanone. Explain your answers.
Under a specified set of conditions, any component of the mixture will always have the same retention time. So its retention time can be used to help identify it. This is the gas chromatography (GC) part of GCMS. As each component of the mixture leaves the column, a detector measures its amount. The information is stored in a computer and can be presented as a graph of amount of substance against time. This is called a chromatogram. A typical one is shown in Figure 4. Each peak corresponds to a different component of the mixture. The taller the peak (strictly speaking the greater its area), the more there is of that component. In Figure 4, the numbers printed by some of the peaks are the retention times of those peaks. The computer used to store, manipulate and print the data has been used to mark the retention times of main peaks of the chromatogram to make it easier to interpret.
However, finding a peak in the chromatogram with the same retention time as a known drug is not enough to be certain that that drug is present. Two quite different substances might coincidentally have the same retention time. Here is where the mass spectrometer (MS) part of GCMS comes in. As the components leave the column, they are directed into a mass spectrometer. This fires a beam of electrons at the molecule, which becomes ionised and breaks up into fragments. The instrument separates these fragments by their masses, stores the data, and plots a graph of the relative amount of each fragment against mass, see Figure 5. This is called a mass spectrum.
Q4. In the mass spectrometer, a beam of electrons is used to (i) ionise and (ii) fragment the sample molecules.
a) Explain how the electron beam brings about each of these processes.
b) What will be the charge on the ions formed?
Each substance has its own individual pattern of fragments, so a substance can be identified by comparing its mass spectrum with a mass spectrum known to be of that substance. The computer can easily match a mass spectrum obtained from GCMS with a database of many thousands of previously analysed spectra. A drug will be identified if both the mass spectrum and the retention time match those of a known standard.
In fact, the combination of chromatogram and mass spectrum for each of its peaks generates a three-dimensional data set as shown in Figure 6.
If a drug is detected, another sample of the urine is taken for confirmatory analysis.
Q5. Figure 7 shows the mass spectrum of aspirin and Figure 8 its displayed formula.
a) i) Use the displayed formula to help you suggest what fragments of the aspirin molecule are responsible for the peaks at mass 43 and mass 121.
ii) What causes the peak at mass 1807
b) Suggest why a racehorse might be b) given aspirin.
Many of the screening processes described above are carried out automatically by the instruments themselves under computer control. This means that no single analyst will have supervised the whole screening process for any single sample. If a regulatory authority, such as The Jockey Club, wishes to take disciplinary proceedings as a result of drug testing, it is vital that the analysing laboratory is able to state without fear of contradiction that:
1. There was no way in which samples from different horses had been mixed, confused or contaminated.
2. The materials claimed to be present were actually present.
3. The instrumentation used was not giving false positives (indications that substances were present when they actually were not).
To ensure that this is the case, a further analysis is carried out. This time all the operations are carried out or supervised by a single analyst. This analyst will then be able to give evidence in a court of law, if required.
The analyst prepares a reference sample that contains known drugs to check that the analysis method detects them and so that the mass spectrum of this sample can be compared with that from the horse. He or she then performs the analysis on four samples:
1. a system blank consisting of water or a buffer solution to show that there is no contamination from one liquid to another and that the instrumentation is not giving false readings;
2. a biological blank – urine or blood plasma as appropriate -which is known to be drug free;
3. the sample believed to contain the drug identified during screening; and
4. the reference sample.
In general, the regulatory bodies are interested only in whether a drug substance is or is not present rather than its concentration. Complications may arise if the substance found could reasonably be expected to be present in minute concentrations because it is a naturally occurring compound, eg theobromine or salicylic acid (2-hydroxybenzoic acid). In such cases the regulatory authority prescribes a threshold limit.
Action taken when a test is positive
Once the analyst is satisfied that the positive result from screening is correct, a case file is prepared. This contains the screening tests – immunoassay results, the gas chromatogram and mass spectra – and the confirmatory data. This is checked by another analyst before the report is sent to the regulatory authority, which then decides on the action to be taken.
Few cases of doping are reported in this country and this shows the care with which owners and trainers follow the rules of their regulatory authorities. Table 1 shows some recent figures.
It should be added at this stage that not all cases where drugs are found are deliberate attempts to alter the outcome of a race. Horse owners and trainers occasionally give their horses medications, feedstuffs and supplements that contain ingredients that they did not know were banned, or which are there as contaminants whose presence could not be known until the horses were dope tested.
An example of this was the disqualification of the British showjumper David Broome and his horse, Lannegan, in the 1990 Irish Nations Cup. As was reported in the media at the time, the British team won the trophy and were going to retain it permanently because this would have been the third consecutive win. When Lannegan was tested, a substance called isoxsuprine was detected. lsoxsuprine is routinely used to treat navicular disease, a condition in which the flow of blood to the foot is impaired. It dilates (opens up) blood vessels, improving the circulation of blood and can enable a lame horse to run. This is why it is banned. Investigations followed, which unearthed the fact that a rehydrating drink that had being given to the horse in the days before the race also contained isoxsuprine. The company that manufactured the drink, which should contain only salts and sugar, sent another sample of the drink from the same batch for analysis. This also proved positive. Further inquiries revealed that a shovel used in the department making the drink had also been used in a part of the factory manufacturing isoxsuprine, and that enough had been transferred to the drink to cause the horse to fail a drug test. The manufacturing company that made the drink paid David Broome the prize money that he had lost (see The Human Element, John Emsley, BBC, 1992).
Excerpted from Chemistry at the races by Ted Lister, Colin Osborne, Maria Pack. Copyright © 2002 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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