Although there has been some discussion among industry stakeholders of the need for federal regulation, currently, medication rules for racing animals in the United States are determined by the individual states. States may have a Racing Commission, a Division of Parimutuel Wagering, or some other state agency that is given the authority and responsibility of regulating racing animals, horses and/or Greyhounds. It is the parimutuel wagering component of racing that has resulted in government oversight of these animal sports. Rules can vary significantly from state to state, so it is important to review the regulations in each state.

Most commonly, urine and/or blood samples are collected and analyzed for the presence of nonpermitted substances and overages of permitted medications. Traditionally, urine has been used primarily for regulating nonpermitted substances, whereas blood samples were used to control permitted medications through adoption of regulatory limits or thresholds, but this approach is changing.

The selection of urine as the analytical matrix of choice is often made because it can be collected noninvasively with relative ease, particularly in horses. The large volumes typically voided in horses are also an advantage. In dogs, collection is a bit more difficult and volumes are often limited, but it is still the most common fluid collected in drug testing programs. Because many, if not most, drugs are eliminated in the urine and the kidney concentrates the urine, drugs can generally be detected in urine samples longer than they can be detected in blood samples. One limitation of the use of urine, however, is that it is very difficult to determine the pharmacological significance of the analytical finding, because urine concentrations will vary dramatically with the specific gravity of the urine.

Many medication control programs have been moving toward exclusively using blood samples, and the resulting serum or plasma collected from them, as the testing matrix. Even though, as previously mentioned, the concentration of drugs in the plasma/serum is often less than it is in urine, recent advances in technology have tremendously increased the sensitivity of analytical methods, generally rendering this disadvantage irrelevant. A major advantage of the use of plasma/serum harvested from blood samples is that the concentrations can more easily be correlated to pharmacodynamic effects if the relevant pharmacokinetic/pharmacodynamic studies have been conducted. A disadvantage to their use is the need for venipuncture, which is invasive and requires at the minimum a trained veterinary technician, if not a veterinarian.

Alternative matrixes, such as hair, have been investigated but their use is problematic. It would be extremely difficult to prove the time frame of exposure when a drug is found in a hair sample, and some horses may change hands several times in the course of the year. Saliva has also been used as a testing matrix, but because it is difficult to obtain sufficient volume to allow for a confirmatory analysis, its usefulness is limited.

The goal of an extraction process is to isolate and concentrate the drug, referred to as an analyte, from the sample. One of the simplest and oldest extraction methods is a liquid–liquid extraction (LLE). In LLE a liquid biological matrix, such as urine or plasma, is mixed with an organic solvent that is not miscible with the sample. Most drugs are more soluble in the organic solvent than in the aqueous matrix of the sample, so they concentrate into the solvent during the mixing process. The solvent is separated from the aqueous phase and usually concentrated by evaporation to increase the concentration of the analyte in the final reconstitution buffer, which will increase the sensitivity of the assay. In solid-phase extraction (SPE) the organic matrix is passed through a column containing beads to which functional groups, such as hydrocarbons or ion exchange groups, have been bound. The analytes bind to the beads allowing the organic contaminants to be removed by washing the column. In the final step the column is buffered to a specific pH to facilitate release of the analytes, which are then removed from the column with a small amount of elution buffer. The buffer is generally removed by evaporation and the sample reconstituted in a solvent amendable to analysis. Analytes may also be extracted from the sample matrix by precipitation, which relies on differences in the solubility of the analytes and other compounds in the matrix. This process is commonly used to remove the proteins from plasma or serum, after which the resulting supernatant can be directly analyzed.

It is important to realize that different extraction processes may be more or less efficient at separating drug from the biological matrix. This can have significant regulatory ramifications because the final determination of the amount of drug present in the sample will depend to some extent on the extraction method used. So if two laboratories analyze the same sample using different extraction methods, the final concentrations of the analyte determined by the labs may be slightly different. The extraction efficiency is often determined during validation of an analytical method, but rarely is it factored into the determination of the final concentration in a biological sample.

The goal of the separation process is to isolate analytes from each other after extraction and prior to the detection process. The most common method used in forensic drug testing laboratories for the separation of analytes is chromatography. For a chromatographic separation the extracted sample is reconstituted in a mobile phase and passed through an immiscible stationary phase. Differences in the chemical characteristics of the analytes, such as polarity, size, and charge state, will cause them to have different rates of migration through the stationary phase. These differences, when optimized, result in the analytes moving through the column in discrete waves or bands, which can be collected or diverted to other instruments for further analysis.

One of the oldest types of chromatography is planar chromatography, where the stationary phase is bonded to a piece of glass or paper, and the mobile phase passes over it by either gravity or capillary action. Thin layer chromatography (TLC) is a type of planar chromatography that was used for many years in forensic drug testing laboratories as a primary screening test. Because it is possible for different analytes to have similar migration patterns, the findings are only presumptive and a second confirmatory method must always be employed. TLC is extremely labor intensive and not as sensitive as more modern chromatography methods, and therefore it is not used with much frequency today.