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Analytical Chemistry

Drug-testing Lab Stands Ready

Athens lab is set to work nonstop to help ensure a level playing field at the Summer Olympics

by STEPHEN RITTER, C&EN WASHINGTON
August 9, 2004 | A version of this story appeared in Volume 82, Issue 32

TRUTH TEST
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Credit: AGILENT PHOTO
Agilent Technologies' LC-MS system is used in doping-control labs to test for biological agents, such as peptide hormones, that are not amenable to GC-MS analysis.
Credit: AGILENT PHOTO
Agilent Technologies' LC-MS system is used in doping-control labs to test for biological agents, such as peptide hormones, that are not amenable to GC-MS analysis.

The games of the XXVII Olympiad begin in Athens this week, and following the opening ceremonies, drug testing of the athletes will hit a fever pitch.

When it comes to doping, the sports world lost its innocence years ago. Recently, a controversy has swirled around positive drug tests by several U.S. track-and-field athletes. There's also the related federal investigation into a possible conspiracy to distribute anabolic steroids and other performance-enhancing drugs to athletes.

The increased publicity about drugs in athletics may have done some good. "Now that everybody is talking about doping, [track-and-field] is cleaner than it's ever been," commented U.S. 110-meter hurdler Allen Johnson in an interview a couple of weeks ago. Johnson, the 1996 Olympic champion, is a medal hopeful in Athens.

Indeed, the current worldwide antidoping system--the most advanced in the history of sports--seems to be doing well. A primary reason for this is the behind-the-scenes efforts of a dedicated cadre of scientists--chemists, chemical engineers, biochemists, chemical technicians, and others--who play a vital role by designing and carrying out analytical procedures to analyze urine and blood samples.

For the Summer Olympics, this duty has fallen on the shoulders of chemical engineer Costas Georgakopoulos and his staff at the Doping Control Laboratory of Athens. And these aren't just ordinary shoulders.

Georgakopoulos, an Athens native, received an undergraduate degree in 1987 and a Ph.D. degree in 1992, both in chemical engineering, at the National Technical University of Athens. He joined the staff of the Athens lab in 1988, and he became lab director in 1996.

The unique element Georgakopoulos brings to doping control is the perspective of an elite athlete. As a discus thrower, he was a member of the Greek national track-and-field team for more than a decade and was the Greek national record holder from 1983 to 2002. A highlight of his career was an 11th-place finish at the 1984 Olympic Games in Los Angeles.

"The combination of being an athlete and a student was hard, and eventually it led to a not-so-satisfactory performance for both," Georgakopoulos tells C&EN. But he says continuing in athletics and in chemical engineering helped forge a work ethic that is now invaluable to him as he works long hours to ensure that everything runs smoothly in the Athens lab during the coming weeks.

OVERSIGHT for all drug testing at the Olympics and for international sports rests with the World Anti-Doping Agency (WADA), an independent nonprofit organization created in 2000 to govern drug testing and provide drug education for elite athletes. Prior to WADA, the International Olympic Committee (IOC) Medical Commission was in charge of Olympics drug testing, which began in full at the Munich Summer Games in 1972.

Over time, as new drugs have been developed and athletes have become more wily, the list of prohibited substances and methods maintained by IOC and now by WADA has been expanded. Classes of compounds under scrutiny include stimulants, narcotics, anabolic steroids, diuretics, peptide hormones, and more.

In Athens, the top four finishers in individual competitions will be tested, along with at least one other randomly selected athlete in each event. For team sports, athletes will be selected at random for testing throughout the competition, and at least two athletes from each of the top four teams will be tested. All athletes are also subject to random tests during the official Olympic period, which runs from July 30 through Aug. 29. Altogether, this adds up to several thousand samples that will pass through the Athens lab.

The collection process is highly regimented to ensure the authenticity of a sample. The athlete is required to provide a urine sample--and for some sports a blood sample--while in the presence of a doping-control official. For urine, about two-thirds of the sample is poured by the athlete into an "A" bottle and one-third into a "B" bottle. The bottles are sealed in the presence of the athlete, and a secure chain of custody is maintained while the samples are delivered to the lab.

The basic testing procedure starts with an aliquot of the A sample for each class of drugs being tested, Georgakopoulos explains. The samples are filtered, and then they undergo liquid-liquid extraction followed by solid-phase extraction to separate any solids, salts, and water. The extraction process and any additional chemical workup, such as derivatization, are optimized for each class of compounds. The end product is a small amount of residue containing only the compounds or metabolites of interest.

If an initial test of a sample is negative, the technician handling the sample moves on to a different test or to the next sample. If there's a positive result, a more detailed analysis is carried out using a new aliquot of the A sample, Georgakopoulos notes. This follow-up may involve tailoring the chemical workup to make a new or different derivative of the drug or metabolite that was observed.

If the confirmation test is positive, IOC officials inform the athlete or team official, and then the B sample, which has remained sealed, may be opened and tested as well. The result of the B analysis is considered the final identification of the doping agent. If the B sample does not confirm the A sample analysis, no further action will be taken. If the B sample tests positive, a hearing will be held to determine if a rule violation has occurred. For the Olympics, the IOC executive board will decide what sanctions, if any, will be meted out.

Negative test results will be reported within 24 hours, and positive test results will be reported in 36 hours, Georgakopoulos says. One exception will be positive test results for erythropoietin, which take 72 hours. The routine reporting time for test results for WADA labs is 10 working days, he points out.

GAS CHROMATOGRAPHY (GC), liquid chromatography (LC), and mass spectrometry (MS) are the techniques at the heart of drug-testing laboratories. These methods also form the basic setup for forensics and criminal investigation labs as well as labs specializing in environmental testing and food safety.

"Two basic instruments for testing in the Athens lab are GC with nitrogen-phosphorus detectors for stimulants, and GC with quadrupole MS for smaller molecules, such as stimulants, narcotics, steroids, and diuretics," Georgakopoulos notes. "When there is an initial positive test, GC with time-of-flight MS is typically used for a full-scan confirmatory analysis."

Other instruments include GC with combustion isotope ratio magnetic-sector MS. It's used to measure the 13C/12C isotope ratio of CO2 from the combustion of a sample to discriminate between natural and synthetic anabolic steroids, such as testosterone. GC with high-resolution MS is used to detect parts-per-billion or lower levels of synthetic anabolic agents, Georgakopoulos adds. LC with tandem ion-trap mass spectrometry (LC-MS/MS) is used for small polar compounds such as corticosteroids, as well as for direct urine analysis.

For blood, gel-permeation LC with UV detection is used for analysis of some compounds. Enzyme-linked immunosorbent assay (ELISA) and radioimmunoassays are used to measure human growth hormone and other hormones to see if they are within normal levels, he notes. Isoelectric focusing electrophoresis with chemiluminescence detection is used to check on peptide hormones, such as erythropoietin, which boosts red-blood-cell concentration to increase oxygen-carrying capacity, a bonus for endurance athletes.

The major supplier of instrumentation for the Athens lab is Agilent Technologies. The company, spun off from Hewlett-Packard in 1999, has supplied the Olympics and other major sports competitions worldwide for 30 years, according to Stuart P. Cram, a Ph.D. analytical chemist who is worldwide business development manager for the company's Life Science & Chemical Analysis unit.

AGILENT'S FORAY into drug testing began with the Munich Games in 1972, Cram notes. At that time, gas chromatographs did not have very good detectors for drug-testing applications--GC-MS was not what it is today, he says. There was a need for a specific type of GC detector, which gave rise to Hewlett-Packard's developing the nitrogen-phosphorus flame-ionization detector, or NPD, specifically to meet the needs of the Munich games. "That's what got us involved in the Olympics, and we have been at it ever since," Cram says.

There's a healthy competition to get instruments into a doping-control lab, he notes. Of critical importance to choosing an instrument is its reliability. "During the games, you cannot have downtime," Cram says. "You have to count on the instruments running 24/7. Agilent has a proven track record for this, which is one of the reasons we are continually selected to provide instruments for doping-control labs at the Olympic Games, Commonwealth Games, Asia-Pacific Games, and other major competitions."

The other crucial factor for a lab is day-to-day reproducibility of results from sample to sample and from instrument to instrument. This is especially important for confirmatory analysis of a sample because it most likely would be analyzed by a different technician on a different instrument than the original analysis. "The reproducibility has to be lock tight," Cram emphasizes.

The Olympics is not a one-shot event for Agilent, he adds. "Doping control is an ongoing business with many opportunities." For example, the company has been a major supplier to the Department of Defense for military drug testing. The field of forensics and criminal investigation continues to grow globally as well, he says, creating new opportunities. "It's big and it's getting bigger."

Although the Athens lab is outfitted predominantly with Agilent instruments, there are some exceptions. The specialty high-resolution MS, time-of-flight MS, and ion-trap MS instruments are from Waters Corp.'s Micromass line, although the front-end GCs or LCs come from Agilent, Cram explains. The instruments to carry out blood analysis and the automated immunoassays--instruments commonly found in hospital labs--are supplied by a variety of companies, including Agilent, Diagnostic Products Corp., Amersham Biosciences, PerkinElmer, and Beckman Coulter.

The Athens lab, which belongs to the Greek Ministry of Sports, was founded in 1986 and was first accredited by IOC in 1990. The lab also became a national horse-race testing laboratory in 1998. In 2000, it received ISO 17025 certification, the global standard to ensure the technical competence of calibration and testing laboratories. The lab is one of 32 accredited WADA testing labs.

Hatton
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Credit: AGILENT PHOTO
Credit: AGILENT PHOTO

THE COST of doping control for the Olympics in Athens will be nearly $6 million, Georgakopoulos notes. The lab recently moved into a new building, and a number of new instruments have been added. The new instruments have been acquired by various means, he says, including purchase by the lab, government purchase, IOC purchase, and rental. After the games, some of the no-longer-needed instruments will be sold, while others will go to Greek government labs.

In 2001, the number of permanent staff at the lab increased from seven to 16 to begin gearing up for the games, Georgakopoulos says. Since last year, temporary staff members have gradually been added, including 30 scientists and 50 undergraduate and graduate students. In recent weeks, eight staff members from an Athens hospital have joined the lab, as well as 17 scientists from other European WADA labs and the Chinese WADA lab. IOC requires the doping-control staff for the next Summer Olympics--in this case, Beijing in 2008--to participate as a dress rehearsal. Altogether, the total scientific and support staff of the Athens lab has swelled to 145 people for the Olympics.

The lab is prepared to analyze 180 samples per day, working in three shifts around the clock, Georgakopoulos adds. Overall, the Athens lab will analyze about 2,400 urine samples and 600 to 700 blood samples during the Olympics. On top of that, the Paralympic Games for disabled athletes will be held in Athens in September, which will bring another 1,000 samples for the lab to process.

"Certainly, there will be no free time--only time for work and to sleep for a few hours," Georgakopoulos remarks.

"The day before the opening ceremonies, the Greek lab staff should take a deep breath and not plan on breathing again until several days after the closing ceremonies," says Caroline K. Hatton, a Ph.D. organic chemist and director of special projects at the Olympic Analytical Laboratory, the U.S.'s WADA-certified lab located at the University of California, Los Angeles. The lab is one of the world's busiest, processing some 35,000 samples per year for a broad range of international and U.S. clients.

Hatton notes that work in a doping-control lab requires creative thinking at times and is a real team effort. "We must understand the pharmacology, medicine, and epidemiology of drug abuse in sports, and we must be able to work with sports administrators and lawyers," she says. "For doping-control scientists to do a good job, we also must think like crooks. We must imagine ways to cheat our own tests, then find ways to close any loopholes."

A good example she cites is abuse of human chorionic gonadotropin (hCG), a hormone that appears in the urine of a woman when she becomes pregnant. Only men are tested for hCG.

It's basically a pregnancy test on male urine samples, Hatton explains. About the only reason hCG is found in a man's urine is if it is taken to counteract testicular atrophy, a known side effect of anabolic steroid use.

Male urine also is tested for tamoxifen, an antiestrogen drug used to help prevent breast cancer recurrence in women, she adds. It's abused by men to counteract breast enlargement, another known side effect of anabolic steroids.

STEROIDS ARE still the favorite drug of abuse among athletes, Georgakopoulos observes, and designer steroids like tetrahydrogestrinone, uncovered by the UCLA lab last year, will probably continue to be a problem (C&EN, Nov. 17, 2003, page 66). "The classical anabolic agents--stanozolol, methandienone, testosterone, clenbuterol, nandrolone--are still very much in use," he says, "especially in cases where the cheaters feel safe and protected from unannounced antidoping tests." Other older agents, such as recombinant and natural human growth hormone, also continue to be favorites of athletes because it's difficult to distinguish normal versus abnormal levels from person to person.

In the testing lab, the scientists work primarily on detection, but they also are involved with education and research. The enforcement or handing out of sanctions, however, is the purview of WADA, IOC, and national sports-governing bodies. But it's difficult for the lab staff to not get emotional when it comes to the politics of doping.

Drug education is an important avenue to teach about ethics, Georgakopoulos notes, but it also provides information about the evolution of sports technology that allows athletes to improve performance without the use of doping agents. The legal and ethical aspects play an important role, especially to protect athletes against an undocumented charge of using prohibited substances, he says.

"To the people participating in the fight against doping, this protection is imperative," he emphasizes. "Sometimes it allows cheaters to escape, but if the system works properly, these cheaters are expected to be caught in future [tests]."

Over time, a doping-control scientist must grow thick skin and learn to be a bit detached from a lab report of an adverse finding, Hatton adds. "Science merely does fact-finding, then the sports-governing bodies or society must decide what to do with the facts--based on the values they embrace or morals they practice," she says. "This mechanism and this separation should never change. What might change, and in some cases should change, are the values."

Ethical dilemmas abound, she continues, but those individuals involved in doping control have devised rules of conduct--standards for making certain types of decisions. "We definitely have great pride and satisfaction in knowing that a drug test is safe in our hands--done meticulously right--because all athletes, including those who have committed a doping violation, deserve the best. The system must be able to rely on top-quality work. Only then have we done everything we can to protect the honest athletes' freedom to compete without drugs.

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