Equine Drug Testing

Introduction

The twentieth century saw the development of a multimillion dollar horseracing industry. For competitions, two main elements must be considered in the control of drugs in racehorses:
1. Legitimate treatment, supervised by a veterinarian, may be underway for illness or injury, so that the horse will be fit to perform.
2. Drugs may have been administered to the horse with the intent of altering its performance. This is tested for by analysing official test samples (urine and/or blood), which are taken before and/ or after competition. In some countries, between-competition testing is also organized. Treated horses must not race until they have eliminated any traces of the administered substances and/or their metabolites.
This dual nature of therapeutic medication and drug abuse in racehorses created a difficult regulatory problem. The act of taking substances that influence the physical performance, and are usually banned, is now known as doping. Most racing jurisdictions have strict medication rules, similar to the control of drugs in human athletes, which prohibit the presence of any drug in an official test sample of urine, blood or other body fluids taken before or after a race. The most recent rules of the majority of governing bodies for horse sport conform roughly to those of the Federation Equestre Internationale (FEI). Similar rules have been adopted by the International Federation of Horseracing Authorities (IFHA). These two bodies are the leading international equestrian sports associations. The regulations forbid the presence of any performance-enhancing substances in the body of the animal. The stated objective of such regulations is to protect fair competition or, more specifically, any person betting on horses of unknown or inconsistent performance owing to the influence of drugs. Racing analysts and veterinarians are the forensic and sport medicine experts who are responsible for monitoring the medication of competing horses. The prime responsibility of racing laboratories, together with racing authorities, is to protect the integrity of racing.

Context of Control

Owing to the proliferation of pharmaceuticals in the last 40 years, it has become difficult for the analyst to keep up with new drug developments. Even before new pharmacologically active substances are introduced on the market, whether for human or veterinary uses, they may appear in samples taken during doping controls. Comparing the work of the scientist who has to analyse urine and/or blood samples from competing horses with analysts dealing with clinical, toxicological or forensic samples shows obvious differences. In sport, the laboratory receives coded samples, with the only indication being the type of horse. No additional information is given; one is not provided with a case report, nor advised to look for any particular class of drug because of any evidence such as apparent intoxication. If a doping agent has been taken, the amount in blood is likely to be within the normal, that is the therapeutic, range of concentration. Obviously, we never encounter any drug overdose case where large concentrations of the drug and/ or metabolites are typically present.
A limited volume of urine or blood is provided, generally 40-50 ml, and, for the large majority of cases, there will be no opportunity for a second, follow-up sample. Nevertheless, the exact identity of any banned substance present in the sample must be made unequivocally, and frequently within strict time limits of, usually, between 3 and 10 days, as specified in the contract agreed with the controlling authorities.
Since most of the samples are usually negative, i.e. are found not to contain any substance from a banned class, and since confirmatory tests are relatively complex, time-consuming and expensive, all samples are submitted to a range of screening tests. The screening tests usually rely on one or several techniques: thin-layer chromatography (TLC), gas chromatography (GC), high-performance liquid chromatography (HPLC), mass spectrometry, usually coupled with GC (GC-MS) or HPLC (LC-MS), and immunoassays (RIA and/or ELISA, for example). Only those samples that fail screening tests are then submitted to confirmatory testing. The screening tests are designed to be sensitive, and typically 20% of the samples require some form of confirmatory tests. The confirmatory test relies heavily on GC-MS or LC-MS, together with the use of chemical derivatization and/or chemical ionization to improve both GC and MS characteristics of the substance. The laboratory must frequently identify metabolites of the substance in urine, especially when the parent compound is undetectable. This is evidenced by comparison with with urine and/or blood samples obtained from controlled administration of banned substances in healthy horses.

How Horses are Tested

At each competition, judges or stewards can select for testing not only the winner of a race but also other horses, at random or depending on their individual performance during the race. There are no strict rules for the number of samples to be collected at each competition. The FEI states that, for each competition, a minimum of 5% of the horses shall be controlled. Most of the time, only the winner and an antepost favourite which has lost might be preferentially selected. An official test sample (urine or blood) is collected from the horse by the designated test inspector or under his or her supervision, in order to determine the presence of any prohibited substance. Ideally, this inspector shall be independent from the laboratory, and is responsible for the supervision of the horse before the withdrawal of the samples, the collection itself, the mailing of the samples and the establishment of the related documentation.
The term ‘prohibited substance’ means the substance, the metabolite(s) and the isomers or biological indicators of such substance (including any metabo-lite(s) originating externally), whether or not they are endogenous to the horse, and which are contained in the official list (see below). Horses may compete with the presence of certain substances in their tissues, body fluids or excreta, for which specific instructions to the laboratories have indicated threshold levels or ratios (as reported below), provided the concentration of the substance is not greater than that indicated. When the analysis of a sample gives a positive finding for a substance which may have been endogenously produced, the horse may be submitted to further examination. The same is true for the finding of a substance which could be related to food contamination. In view of the rapid development of new drugs and pharmacological agents, and of the changes in preparing horses for competition, the regulations governing the use of new products and new techniques are kept under continuous review and may be changed at any time.
Immediately after the race ends, the designated horse is conducted to a specially prepared retention area, where the animal is placed in a secure stall and watched until such time as the samples can be obtained with specially designed tools (sampling kits are usually approved by the controlling authorities). Urine is preferred as the laboratory has a better chance of detecting forbidden substances. It is also possible to get a larger volume and it is considered noninvasive. If the horse has not been able to provide any urine within 1 h of the end of the race, venous blood is taken by a veterinarian or any other qualified individual, in the presence of the test inspector. Once the official samples have been collected, the bottles are sealed and labeled with track name, date and a unique number, and kept in a refrigerator until dispatch to the laboratory. The bottles are mailed in a sealed box within 24 h, together with the corresponding documentation labeled in such a way that the accredited laboratory cannot identify the horses. The full documentation is kept apart in a safe place.

Penalties and Arbitration

After a positive test is reported, the racing commission notifies the trainer of the horse that has produced a positive test sample. A hearing is held to assess the penalty to be given, according to the commission’s rules. The owner or trainer has the right to appeal the ruling to the racing commission. Any horse found to have a prohibited substance in any of its tissues, body fluids or excreta at an event is automatically disqualified, together with the competitor, from all competitions at the same event. The classification is adjusted accordingly. If the disqualified horse and competitor are members of a team, the rest of that team is not disqualified. Appeals may be directed to the Court for Arbitration in Sport (CAS) in Lausanne. Any emergency treatment to a competing horse just before an event must be certified, in writing, by the veterinarian, with details of the extraordinary circumstances. In cases of obvious illness or injury during an event, the jury will decide whether the horse may continue in that or subsequent competitions.
Owners or trainers may ask for the sample residue for independent testing to verify a positive test result. A sample residue is the urine or blood remaining after the analysis of an official sample has been completed. The independent analysis can be conducted at a referee laboratory, selected by the owner or trainer. Care should be taken selecting a referee laboratory, as the methods used by the referee laboratory might not be comparable to those used by the primary one. Further, drug breakdown or sample deterioration could cause different analytical results. Consequently, the analytical results of the referee laboratory may not be given equal weight as evidence at a hearing.

Establishing the Presence of Prohibited Substances

Objective

The objective of controlling the use of substances is based on the capability of certain chemicals of giving a horse an advantage, or being disadvantaged, in a race, contrary to the horse’s inherent merits. To establish whether a prohibited substance is present, samples (urine or blood, but can also be any part, or in contact with any part, of the horse) are taken from horses that have run in a race. Authorities may also take samples at any other time, according to their own specific rules. This varies from one country to another.

Sampling and forensic integrity

Any sample shall be collected under strict and secure chain of custody, with splitting into sample A and B. If the A sample is reported to contain prohibited substances, the B sample is usually analysed for those substances, either automatically or optionally at the trainer’s or owner’s request. This is generally done in the same place or in another Association of Official Racing Chemists (AORC) accredited laboratory.

Prohibited Substances

Officially, any xenobiotics are forbidden: horses are allowed only pure water, grass and oats. Exceptions are vitamins and a restricted list of antiparasitic and antimicrobial agents. Thus the lists for these animals are much larger than for humans and are usually given classes responsible for specific therapeutic actions. New additions and details are set by the Advisory Council to the International Federation of Horseracing Authorities on Doping Control.
The following are prohibited substances according to the current FEI veterinary regulations (effective 1 January 1998):
• substances, originating externally, whether they are endogenous or not to the horse and capable at any time of acting on one or more of the following mammalian body systems:
nervous system,
cardiovascular system,
respiratory system,
digestive system,
urinary system,
reproductive system,
musculoskeletal system,
skin (e.g. hypersensitizing agents),
blood system,
immune system, other than those in licensed vaccines,
endocrine system, endocrine secretions and their synthetic counterparts;
• antipyretics, analgesics and antiinflammatory substances;
• cytotoxic substances.
This includes also any masking agents. Maximum threshold levels or ratios have also been established for those substances that are either endogenous or originate from common natural feed sources (see below).
As can be realized, there are quite a large number of substances that have to be tracked during screening and identified during confirmation procedures. It is impossible here to give all compounds that can potentially modify horse performance, but we will give a few examples of those that have been detected and reported in 1998 as the main substances:
16a-hydroxystanozolol
acepromazine and 2-(1-hydroxyethyl)-promazine
phenylbutazone, hydroxyphenylbutazone
oxyphenbutazone
benzydamine metabolite
boldenone
caffeine, theobromine, theophylline, paraxanthine
chlorobutanol
clanobutin
clenbuterol
dexamethazone
diclofenac
dimethylsulfoxide
ephedrine, norephedrine
nortestosterone
fentanyl, desethylfentanyl
flunixin
furosemide
isoxsuprine
ketoprofen, dihydroketoprofen
lidocaine, 3-hydroxylignocaine, lignocaine
methandriol
methocarbamol
methoxytyramine
naproxen
nefopam
nordiazepam
orphenadrine, nororphenadrine polyethylene glycol procaine
propantheline, 9-xanthenoic acid
pyrilamine
romifidine
salicylic acid
strychnine
testosterone
thioridazine, sulforidazine.

Regulatory identification

Different tests are used for different drugs or medicines. A broad range of tests is applied to every sample. Various types of target or special tests are used to screen for drugs and medicines that are not detected in broad-spectrum screening. Target tests are used on a random basis. A finding of a prohibited substance means a finding of the substance itself or a metabolite of the substance, or an isomer of the substance or an isomer of a metabolite.

Regulatory quantification

Thresholds can only be adopted for:
• substances endogenous to the horse;
• substances arising from plants traditionally grazed or harvested as equine feed;
• substances in equine feed arising from contamination during cultivation, processing or treatment, storage or transportation.
For any finding of a prohibited substance of endogenous nature, the authorities may decide, either themselves or at the owner’s or trainer’s request, to examine the horse further.
Laboratory Performance Specification of the International Federation of Horseracing Authorities (IFHA)

AORC laboratories

The aim of future agreements between countries submitting samples to doping control analyses is such that the requested laboratory should present a minimum requirement for quality and performance:
• The laboratory should be accredited according to the current ISO 17025 regulations (previously called ISO guide 25), General requirements for the competence of testing and calibration laboratories, and to the supplementary document ILAC-G7, Accreditation requirements and operating criteria for horseracing laboratories.
• The laboratory should conform to the Guide for establishing the presence of prohibited substances (which is part B of ILAC-G7).
• The laboratory should meet the Performance specification of the International Federation of Horse-racing Authorities (which is part C of ILAC-G7).
• The laboratory shall take part in interlaboratory comparisons (clause 5.9(b) of ISO 17025:1998).
AORC laboratories publish annual summary reports; these show that, in 1997, there were more than 600 000 analyses of saliva, urine and blood samples taken at equine events, with almost half of them performed in the USA. Saliva sampling seems to be mostly restricted to Illinois; and there are twice as many urine samples as blood samples. The number of positive cases is very few, 0.5% on average, with the highest rate in Europe.
Each year, proficiency testing is organized, with each laboratory having to analyse an average of eight samples with analyte concentrations ranging between 0.05 and 2 ug l”1. It is not easy to know which techniques have been used, and little information is available. As a rule, each laboratory has to choose its own combination of techniques to achieve the best results over the number of samples submitted to it. In 1997, 63 different laboratories participated in the AORC proficiency test, which was mailed during the second week of March. Reports were received from 44 laboratories. The detection score was excellent, with the exception of bumetanide (a diuretic) and oxyphenbutazone. General considerations can, however, be given from the authors’ own practical experiences in horse doping detection and other related fields like ‘general unknown analyses’ in forensic toxicology and human doping analyses.
• Each laboratory performs 1-5 (average 2) extraction procedures on the urine samples, prior to the use of one or more chromatographic techniques to detect various group of drugs and drug metabolites.
• A total of 14 different extraction procedures can be identified. The total volume of urine used for the extractions average 11 ml, and range from 3 to 20 ml.
• The base hydrolysis/acid extraction procedure is frequently used, followed by the cation exchange/ reversed phase extraction. The dioctylsulfosuccinate paired ion extraction procedure seems to be no longer in use.
• The most frequently used chromatographic technique is TLC, followed by HPLC/DAD (photo-diode array detector), specially in the USA. Over the years, it appears that there has been a decrease in the use of TLC and an increase in the use of HPLC/DAD and GC/MS.
• When using TLC, mostly on Silica Gel GF 254, several developing solvents combinations are used, together with various visualization reagents, noting that multiple visualizations are not necessarily overlaid on the same TLC plate. In many cases, more than one TLC system is used on the same urine extract, with different visualization techniques used on each plate.
• In HPLC/DAD screening, reverse phase systems with aqueous acid/organic mobile phases and solvent programming is used to analyse acidic extracts for antiinflammatory drugs. Computer programs allow reviewing of the data with respect to their respective retention and ultraviolet spectra data.
• A few laboratories make use of GC/NPD(nitro-gen-phosphorus detector) analysis, mostly on basic extracts with no derivatization. The analyses are performed on capillary columns with temperature programming. Retention times are evaluated manually and stimulants are mostly detected.
• Automated GC-MS screening is made possible with instruments equipped with suitable automatic injection devices and comparison with adequate reference mass spectra databases. Dedicated collections of MS are available from various commercial sources, but none is specifically dedicated to doping, except that of the AORC Drug Library, the 1998 revision of which contains 2104 entries.
• Screening urine samples with immunoassays is quite common and laboratories may rotate the use of up to 55 different drug immunoassays. These are always used in combination with at least one other chromatographic technique.

Special Requirements for Specific Compounds

The concept of threshold values for endogenous substances and substances of dietary origin is now well established in the field of equine drug testing. In order to be legally defensible, and to establish effective but fair means of control, it was imperative that these threshold values be introduced on a sound scientific basis. The corresponding research effort to achieve this objective has been met by a limited number of laboratories. Today, an agreement has been obtained on a ‘no effect threshold’. Implicit in this approach is the requirement that these threshold values reflect drug concentrations that are pharmacologically inactive (Table 1). Substances below the thresholds are not actionable.
A few medications, for example phenylbutazone, are considered as permissible in some countries, depending on their regulations.
Other compounds might originate from natural sources or contamination of feeds and have resulted in controversy. For example, the growing practice of feeding manufactured compound feeds to horses in training has resulted in the detection of substances such as caffeine, theobromine, hordenine, lupanine, etc. in horse body fluids. The presence of these substances in horse feeds could be attributed to the choice of ingredients used in compounding the feed, or from crosscontamination on the production line. The philosophy of some national regulatory agencies in setting the threshold level for some of these substances (i.e. caffeine at 10ngml_1 plasma) is to overcome the problem of contaminated feeds causing ‘unnecessary’ withdrawal of horses from racing, with low, probably insignificant level of xenobiotics.

Table 1 Threshold values below which substances are considered to be inactive in horse doping testing

Elimination Rates for Xenobiotics

Published detection periods for interpretation by the veterinarian allow the estimation for safe withdrawal times of a treated horse with minimal risk of false positives. It is important that such information is based on sound scientific grounds.
The main difficulties in detecting doping agents in horses lies in the lack of reliable sources for pharmacokinetic data and the unknown structure of expected metabolites. Usually, each laboratory has its own source of positive urine and blood samples taken from horses that have received a low dose of one or several chemicals under controlled conditions. General commercial sources are those such as Sigma, Upjohn and Radian, for example. More specific sources can now provide drug standards for metabolites in calibrated solutions: Neogen Corp, Lexington, KY, USA and Gluck Equine Research Centre at the University of Kentucky, also in the USA.
In 1980, the Canadian Pari-Mutuel Agency (CPMA) initiated a research project, which had the following goals:
• to assess analytical methods in current use and determine their efficacy;
• to broaden the analytical coverage of prohibited drugs;
• to study the elimination and metabolism of 125150 drugs in horses.
Much of the invaluable data developed in the CPMA research program are now published and serve as reference for many controlling laboratories. These reports provide methodology for the analysis of individual drugs or groups of related drugs in equine body fluids, and also provide background information for those wishing to perform additional studies on the behavior of drugs administered to horses.

Conclusion

Drug testing in equine sport is a complex and difficult task for the analyst. The number of substances to be characterized is large, and the most modern instrumentation must be used for ultimate sensitivity and specificity. Since many samples prove to be negative, very efficient initial screening procedures are chosen in order to concentrate the laboratory’s remaining facilities and resources on confirmation. Analysts in this field of forensic science, as in all others, need a high level of training and skill.