The selection of horses to be tested is the province of the officials conducting the event. Winners are often tested, as are horses showing abnormal behaviour or performance, but horses can also be chosen at random. Drug testing is usually performed on urine, except where it cannot be obtained. The Equestrian Federation of Australia requires blood for phenylbutazone testing, and five tubes are collected in addition to the urine.l Where urine cannot be obtained, four tubes of blood are usually collected in racing, and 150 millilitres of blood in EFA events.
Collection and packaging of urine samples should be done such that the horse's attendant can witness the whole procedure, including sealing of samples, and testify to the correct procedure being followed by signing the documentation.
The starting point is when the urine pan is rinsed with a control solution. This picks up any contamination.

The bottles for the urine are also rinsed to detect any contamination, after which the urine is collected by a veterinarian or a veterinary assistant watched by the attendant/witness. If urine cannot be collected after a suitable period, say, 60 minutes, blood is taken. The urine is split into two lots and each placed in a bottle. These bottles are then sealed with adhesive paper and signed by the attendant and the person preforming the swab.

The control solution is sealed too. This acts as a check for any inadvertent contamination of the other collection bottles or the urine pan.The sealed bottles are placed into a pack with documents identifying the sample by number only. The drug testing laboratory does not have access to the name of the horse or owner.

An identification slip is placed with the main sample, the reserve sample and the control sample, and all three samples are separately sealed.

When the paperwork is completed and the attendant has put his signature on it, a carbon copy of the document - identifying the particular sample with the horse and signed by both the attendant and person doing the swab - is then given to the attendant.
With the blood samples, the control solution is not required so this is not sealed. The blood samples are split between the two urine bottles.

The urine bottles containing the blood and packs are then sealed in the normal manner. This procedure may seem complicated but it is designed to protect the horse owner and the horse against inadvertent contamination of a sample that could result in a positive swab, or any perception that samples could be tampered with. The samples remain in the custody of the veterinarian or officials conducting the swab and are delivered to the testing laboratory soon after the swab.

At the laboratory, samples are logged into the record book and refrigerated until the analysis begins. One urine sample is separated from the reserve and the control samples, which are then frozen, while work begins on the separated urine sample.
The urine is extracted with a number of chemical solvents that allow the extracted urine to be run in the various tests and that allow detection of various types of drugs or metabolites of drugs.

As different drugs require different methods, a number of screening tests and methods are used. These include gas chromatography (GC), thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA).

The RIA and ELISA tests are quite specific for a particular drug or a small class of drugs whereas the other methods can detect a wide range of drugs. The HPLC screen separates compounds according to size and/or polarity and produces a printed readout that looks much like a slice through the earth's crust. The “hills” and “mountains” correspond to the presence of various compounds in varying amounts, while the “plains” indicate background content.

Drugs or their metabolites are identified by a peak at a defined location that depends on the physical and chemical properties of the compound. While the screening methods may show a certain drug or metabolites of that drug to be present, final confirmation depends on gas chromatogaphy and/or mass spectometry (GC S). The machine costs about half a million dollars. Sometimes, the GC S has a lower sensitivity than the screening methods, so a positive may not be called as the level of drug is below the sensitivity of the GC S. The GC S allows the analyst to fragment a drug molecule and see what it is made of.

A computerised data system collects the data and compares it with a reference library.
When any positive is detected in the urine sample, the governing body of the sport then gives the horse trainer, owner or the rider the opportunity for the reserve and control smaples to be analysed in the presence of an independent analyst. Most owners use this option as a final safeguard and confirmation of the presence, or otherwise, of the drug in both urine samples.

If the reserve sample gives a negative result, a positive swab is not likely to result in an inquiry or penalty for the trainer, owner or rider. Reserve samples are held frozen until required. The laboratory generally does not need to equantify or accurately determine the exact levels of drug in a swab sample.

ESTIMATIONS
However, it does sometimes make estimations to compare a result with evidence relating to administration of the drug at a subsequent inquiry.
Hence in such cases the laboratory must accurately determine the levels in plasma for these horses. Certain substances which occur natually in the horse - for example the male testosterone hormone present in colts and stallions - bicarbonate, cortisol and some substances possible present at low levels through feed intake, may also be determined quantiatively. When these substances are detected, their levels must be compared with levels that could arise naturally in the particular type of horse.
Levels must be abnormally high for a positive to be called

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