SCIENCE & TECHNOLOGY TRUTH TEST 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.
DRUG-TESTING LAB STANDS READY Athens lab is set to work nonstop to help ensure a level playing field at the Summer Olympics STEPHEN K. RITTER, C&EN WASHINGTON
T
HE GAMES OF THE XXVII OLYM-
piad begin in Athens this week, and following the opening ceremonies, drug testing of the athletes will hit a fever pitch. W h e n 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 anti-
doping system—the most advanced in the history of sports—seems to be doing well. A primary reason for this is the behindthe-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 ofAthens. 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 trackand-field team for more than a decade and was the Greek national record holder from 1983 to 2002. Ahighlight of his career was an llth-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
'Tor doping-control scientists to do a good job, we must think like crooks. We must imagine ways to cheat our own tests, then find ways to close any loopholes/' 26
C&EN
/ AUGUST
9,
2004
HTTP://WWW.CEN-ONLINE.ORG
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 twothirds 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. T h e 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 ofthe 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 WADAlabs 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 G C with quadrupole MS for smaller molecules, such as stimulants, narcotics, steroids, and diuretics," Georgakopoulos notes. "When there is an initial positive test, G C with time-of-flight MS is typically used for a full-scan confirmatory analysis." Other instruments include G C with combustion isotope ratio magnetic-sector MS. It's used to measure the 13C/12C isotope ratio of C 0 2 from the combustion of a sample to discriminate between natural and synthetic anabolic steroids, such as testosterone. GC with high-resolution MS is used
flWWTWWIVWMWIYlMMB
use
The Loker Hydrocarbon Research Institute and the Department of Chemistry
UNIVERSITY
OF SOUTHERN CALIFORNIA
University Park, Los Angeles, California 90089-1661 For information: Phone:(213)740-5974; Fax:(213)740-5087; e-mail:
[email protected] Invites you to the
2004 Annual George A. Olah Lecture "Supramolecular Chemistry: Self-Assembly via Metal-Ligand Interactions" presented by
Peter J. Stang Distinguished Professor, University of Utah and A Mini-Symposium "Challenges for the 21st Century" (Celebrating the 10* Anniversary of Professor Olah's Nobel Prize in Chemistry) Speakers:
Karl A. Christe, George A, Olah, Roy A. Periana, Nicos Petasis, G. K. Surya Prakash (University of Southern California); Gabor A. Somorjai (University of California, Berkeley)
Friday, October 8, 2004 The Scientific Community is Invited
HTTP://WWW.CEN-0NLINE.ORG
C&EN
/ AUGUST
9,
2004
27
SCIENCE & TECHNOLOGY 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. T h e company, spun off from HewlettPackard 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 drugtesting applications —GC-MS was not what it is today he says. There was a need for a specific type of G C 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 dayto-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 instru28
C&EN
/ AUGUST
9,
2004
weeks, eight staff members from an Athens ment than the original analysis. "The rehospital have joined the lab, as well as 17 sciproducibility has to be lock tight," Cram entists from other European WADA labs emphasizes. and the Chinese WADAlab. I O C requires The Olympics is not a one-shot event the doping-control staff for the next Sumfor Agilent, he adds. "Doping control is an mer Olympics—in this case, Beijing in ongoing business with many opportunities." 2008—to participate as a dress rehearsal. For example, the company has been a maAltogether, the total scientific and support jor supplier to the Department of Defense staff of the Athens lab has swelled to 145 for military drug testing. The field of forenpeople for the Olympics. sics and criminal investigation continues to grow globally as well, he says, creating new The lab is prepared to analyze 180 samopportunities. "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 Georgakopoulos Hatton analysis and the automated immunoassays—instruments commonly pies per day working in three shifts around found in hospital labs—are supplied by a the clock, Georgakopoulos adds. Overall, variety of companies, including Agilent, the Athens lab will analyze about 2,400 Diagnostic Products Corp., Amersham urine samples and 600 to 700 blood samBiosciences, PerkinElmer, and Beckman ples during the Olympics. On top of that, the Paralympic Games for disabled athCoulter. letes will be held in Athens in September, The Athens lab, which belongs to the which will bring another 1,000 samples Greek Ministry of Sports, was founded in for the lab to process. 1986 and was first accredited by I O C in "Certainly, there will be no free time— 1990. The lab also became a national horseonly time for work and to sleep for a few race testing laboratory in 1998. In 2000, hours," Georgakopoulos remarks. it received ISO 17025 certification, the "The day before the opening cereglobal standard to ensure the technical monies, the Greek lab staff should take a competence of calibration and testing labdeep breath and not plan on breathing oratories. The lab is one of 32 accredited again until several days after the closing WADA testing labs. ceremonies," says Caroline K. Hatton, a Ph.D. organic chemist and director of speTHE COST of doping control for the cial projects at the Olympic Analytical LabOlympics in Athens will be nearly $6 miloratory the U.S.'s WADA-certified lab lolion, Georgakopoulos notes. The lab recated at the University of California, Los cently moved into a new building, and a Angeles. T h e lab is one of the world's number of new instruments have been busiest, processing some 35,000 samples added. The new instruments have been acper year for a broad range of international quired by various means, he says, including and U.S. clients. purchase by the lab, government purchase, IOC purchase, and rental. After the games, Hatton notes that work in a doping-consome of the no-longer-needed instruments trol lab requires creative thinking at times will be sold, while others will go to Greek and is a real team effort. "We must undergovernment labs. stand the pharmacology, medicine, and epidemiology of drug abuse in sports, and In 2001, the number ofpermanent staff at the lab increased from seven to 16 to be- we must be able to work with sports administrators and lawyers," she says. "For gin gearing up for the games, Geordoping-control scientists to do a good job, gakopoulos says. Since last year, temporary we also must think like crooks. We must staff members have gradually been added, imagine ways to cheat our own tests, then including 30 scientists and 50 undergradfind ways to close any loopholes." uate and graduate students. In recent HTTP://WWW.CEN-ONLINE.ORG
Agood example she cites is abuse ofhuman 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 cheaters to escape, but if the system works from unannounced antidoping tests." Oth- properly, these cheaters are expected to be er older agents, such as recombinant and caught in future {tests]." natural human growth hormone, also conOver time, a doping-control scientist tinue to be favorites of athletes because it's must grow thick skin and learn to be a bit difficult to distinguish normal versus ab- detached from a lab report of an adverse normal levels from person to person. finding, Hatton adds. "Science merely does In the testing lab, the scientists work fact-finding, then the sports-governing bodprimarily on detection, but they also are in- ies or society must decide what to do with volved with education and research. The en- the facts—based on the values they emforcement or handing out ofsanctions, how- brace or morals they practice," she says. ever, is the purview of WADA, IOC, and "This mechanism and this separation should national sports-governing bodies. But it's never change. What might change, and in difficult for the lab staffto not get emotional some cases should change, are the values." when it comes to the politics of doping. Ethical dilemmas abound, she continDrug education is an important avenue ues, but those individuals involved in dopto teach about ethics, Georgakopoulos ing control have devised rules of conductnotes, but it also provides information standards for making certain types of about the evolution of sports technology decisions. "We definitely have great pride that allows athletes to improve perfor- and satisfaction in knowing that a drug test mance without the use of doping agents. is safe in our hands—done meticulously The legal and ethical aspects play an im- right—because all athletes, including those portant role, especially to protect athletes who have committed a doping violation, against an undocumented charge of using deserve the best. The system must be able prohibited substances, he says. to rely on top-quality work. Only then have "To the people participating in the fight we done everything we can to protect the against doping, this protection is impera- honest athletes' freedom to compete withtive," he emphasizes. "Sometimes it allows out drugs." •
^
Looking for specialty chemicals beyond the ordinary for your next breakthrough discovery? The NEW 2004-2005 Poiysciences, Inc. Catalog is your sourcebook featuring unique monomers and polymers, specialty and fine chemicals, new products, enhanced technical j information, and much more!
The Curie
Emission Spectrofluorometer
Detects picomolar-range concentration of fluorophores in solutions from 200-850 nm Advanced filtering system discriminates between powerful pulsed xenon source wavelengths and weaker spectral emissions from samples
In addition to the products listed in the catalog, we provide custom designed materials and custom variations of current products to suit your specific needs.
^ff^ra Poiysciences, Inc.
Only $ 8 , 4 9 9
Request your FREE catalog today!
Emission Wavelength Range
200-850 nm
Excitation Wavelength Range
200-700 nm
Optical Resolution
10.0 nm (FWHM)
Dimensions
33 cm x 24.9 cm x 12.8 cm
SignahNoise
250:1
Visit us online at r
~" r , /
Chemistry beyond the ordinary
W W W . p o l y SCienCeS.COITI
cean
First in Photonics'*
400 Valley Road, Warrington, PA 18976 HTTP://WWW.CEN-ONLINE.ORG
NEW!
727.733.2447 OceanOptics.com
[email protected] 1-800-523-2575 C & E N / AUGUST 9, 2 0 0 4
29
