Doping Detection

The term “doping” refers to the attempted use of a prohibited substance or prohibited method to improve athletic performance. Each year, 40 scientists at the UCLA Olympic Analytical Laboratory in Los Angeles analyze about 40,000 urine specimens for traces of banned substances intended to give athletes an unfair edge in competition.

“I personally want to witness a clean sport. I want all the players to be on an even playing field so I can see their natural abilities and a fair competition,” explained Anthony Butch, PhD, professor, pathology and laboratory medicine at UCLA School of Medicine and director of the UCLA Olympic Analytical Laboratory, the largest World Anti-Doping Agency-accredited laboratory.

The lab performs drug testing to detect banned substances in athletes at all levels of competition including the Olympics, college, high school and professional sports. The World Anti-Doping Agency (WADA) updates the list of prohibited substances annually. In 2014, the list included 46 exogenous steroids and 67 stimulants.

Collection Process
The lab works with collection agencies that collect urine samples from sports teams and follows specific privacy guidelines to protect the identity of each individual. “It’s critical for us to know who the client is,” Butch said. “The patient’s identity is coded on the bottle for privacy but we need to know the athlete’s gender and whether the sample is collected in- or out-of-competition.”

Gender identity is important because human chorionic gonadotropin (hCG), a marker for pregnancy, is prohibitive only for males, according to Butch.

Samples are generally delivered to the lab via a commercial carrier. Each box of samples contains a manifest that identifies the samples in the box, date and time the samples were collected, gender of the athletes, and a list of sports and events. Two bottles-Bottle A and Bottle B-are delivered for each client. The lab puts the B bottle in the freezer and does not touch it unless follow-up testing is required.

The lab scientists check the database to see what tests need to be performed for each particular sport. The testing menus vary by client and whether the athlete is in- or out-of-competition. “We start chain of custody documentation as soon as the samples arrive,” Butch said. “Up until the time the samples are disposed of, we follow a strict protocol that is similar to the forensic testing method.”

The chemists are required to follow a sample cleanup procedure. “If we use a clinical analyzer for hCG testing, then no cleanup is required,” Butch said. “Urine is dirty and contains many waste products so most tests need a sample cleanup step.”

The scientists open the samples and measure the specific gravity and pH, and prepare aliquots designated for testing. If looking for anabolic steroids, the lab tests by two different methods.

Most of the testing uses either gas chromatography or liquid chromatography separation techniques and mass spectrometry detection to identify the presence of drugs and other substances in urine. The retention time and relative intensities of ion fragments in the urine samples are compared to reference compounds for the detection of prohibited substances.

“We can program the instrument for the particular group of compounds we are looking for,” Butch said. The lab performs the tests in batches containing 20 to 30 samples. The whole testing process takes a few hours to a couple of days.

The lab will perform other tests by special request including Isotope Ratio Mass Spectrometry to detect testosterone or a testosterone precursor, and tests to detect the use of erythropoietin (EPO), and human growth hormone (hGH).

Confirmation Testing
If a sample screens positive for a banned substance, then confirmation testing is performed on an additional aliquot taken from the A bottle. The confirmation test is optimized for the compound detected in the screen and is usually more sensitive and specific than the screening method.

The mass spectra of the drug in the athlete’s urine sample is compared to the mass spectra of the same drug in a certified reference standard. If the spectra match, then the sample is positive for the drug in the reference standard. The data from the confirmation test is reviewed by at least two certifying scientists before being considered positive.

The positive test or adverse analytical finding is transmitted to the testing authority that submitted the urine sample. It is possible that the athlete has a legitimate medical reason for using the drug detected in the urine sample. For instance, the athlete may have attention deficit disorder and is currently being prescribed amphetamine for the disorder.

“This would be considered a therapeutic use exemption and the athlete would not receive a sanction,” Butch stated. “If the athlete does not have a medical reason for using the drug found in the urine, then the athlete would receive a sanction.”

The testing authority and/or the athlete can request that the B bottle is tested to confirm the results obtained with the A bottle. The athlete also has the right to witness the opening of the B bottle and the entire testing process. The athlete may request that a representative such as a lawyer or forensic toxicologist is present during the entire process. If the test results for the B bottle do not match the results from the A bottle, then the urine sample is considered negative. This situation rarely occurs.

Just like the athletes, the lab professionals don’t want a sample to test positive. “Positive samples are an enormous amount of work,” Butch explained. “Negative samples do not require confirmation testing and can be reported quickly. But the confirmation testing required for a positive test is a lengthy process.”

According to Butch, education is a critical aspect of controlling doping. “We need to do a better job of educating young people to understand that doping is wrong and can be harmful to health,” he shared.

Rebecca Mayer Knutsen is on staff at ADVANCE. Contact: rknutsen@advanceweb.com

Blood Doping
A recent resurgence in the use of blood transfusions for blood doping is likely due to the introduction of efficient erythropoietin (EPO) detection methods, according to the World Anti-Doping Agency. There are two forms of blood doping: autologous and homologous. Autologous blood doping is the transfusion of one’s own blood, which has been stored (refrigerated or frozen) until needed. Homologous blood doping is the transfusion of blood that has been taken from another person with the same blood type.

EPO, a peptide hormone that is produced naturally by the human body, is released from the kidneys and acts on the bone marrow to stimulate red blood cell production. An increase in red blood cells is attractive to athletes because it improves the amount of oxygen that the blood can carry to the body’s muscles. It may also increase the body’s capacity to buffer lactic acid.

An EPO test based on blood and urine matrices was introduced at the 2000 Summer Olympic Games in Sydney and was validated by the International Olympic Committee. Recently, the urine test used for the detection of some new erythropoiesis stimulating agents was adapted for blood testing.

References:

World Anti-Doping Agency

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