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May 2001
Vol. 10, No. 05,
pp 28–30, 33–34.
 
 
 
Today's Chemist at Work
Focus: Gas Chromatography

FEATURE

GC/MS Drug Testers Face Olympian Challenge

opening artAnalytical chemists check athletes for more than 150 illegal substances.

Just before the 2000 Olympic Games in Sydney, the Chinese Olympic team cut 27 of its members for failing drug tests. A few weeks later, both the Romanian and Bulgarian weight-lifting squads were expelled from the games after members of the teams tested positive for diuretics that help athletes lose weight and can conceal the presence of steroids. In November, German freestyle wrestler Alexander Leipold was stripped of his gold medal after authorities confirmed he tested positive for nandrolone, a steroid.

That these athletes were caught using banned substances might suggest that the International Olympic Committee (IOC) has the problem under control. But experts say the number of athletes found using banned substances is only a small fraction of those who use illegal substances and get away with it. “To put it bluntly, only stupid and careless people get caught,” says Charles Yesalis, an epidemiologist at Pennsylvania State University (University Park) and an expert in the study of sports doping.

But that may be changing. New testing methods introduced in Sydney and others under development offer hope that offending athletes may not always escape detection. Combined with rigorous random testing, increasingly sensitive and sophisticated gas chromatography (GC) and mass spectrometry (MS) may soon provide the tools to nab athletes who don’t play by the rules (see box, A Day (and a Half) in the Life).

Figure 1. EPO Spins the Wheels.
Figure 1. EPO Spins the Wheels. Athletes use the hormone erythropoietin (EPO) to increase their red blood cell count, thereby increasing the level and circulation of oxygen throughout their body. This gives them the advantages of extra strength and endurance.
GC/MS Successes …
Analytical chemists certainly have their hands full. The IOC lists more than 150 illegal substances that chemists must try to identify from blood and urine, and perhaps, in the future, from hair. Chief among these are anabolic agents, such as steroids, which increase muscle mass and strength, and peptide hormones, such as erythropoietin (EPO), which raise the level of oxygen-carrying red blood cells (Figure 1).The IOC also bans the use of stimulants such as amphetamines, narcotic analgesics for pain relief, and urine-manipulating agents used to mask the illegal substances.

In addition to having to detect a broad range of compounds, chemists must catch substances that are in the body only briefly—in some cases, a matter of hours. And even if a test were given within that time, the identification of performance-enhancing agents like testosterone or EPO is complicated by the body’s own production of these compounds, albeit at lower levels. Furthermore, because the normal concentrations of these compounds in urine or blood vary from athlete to athlete, chemists must devise clever strategies for identifying the true source of the performance-enhancing agent.

Often, just detecting a banned substance is not enough. For a positive test to withstand an athlete’s legal challenge, IOC chemists must develop secondary, more accurate tests that confirm the drug’s presence. “For confirmation, the test must be 100% accurate, because you’re destroying an athlete’s character,” says Craig Kammerer, a chemist who helped run the UCLA Olympic Analytical Laboratory during the 1984 Olympic Games in Los Angeles.

Compounding these difficulties is the relative lack of scientific literature for a field of analytical chemistry just over 30 years old. Although athletes discovered steroids in the 1950s, it wasn’t until the 1968 Olympic Games in Mexico City that authorities began testing athletes on a preliminary basis to try out recently developed techniques. These first tests attempted to detect only the use of stimulants, because a reliable test for steroids did not yet exist. In any case, these preliminary tests carried no consequences for offending athletes, Kammerer says.

Four years later, in Munich, IOC-accredited labs began testing in earnest, using GC instruments equipped with nitrogen-specific detectors to test for a broad range of stimulants and opioids. Of the 2070 urine samples tested, 7 were found to contain traces of amphetamines or other stimulants. For the 1976 Olympic Games in Montreal, labs began testing for anabolic steroids, using an initial radioimmunoassay to flag potentially tainted specimens and GC/MS for confirmation. Further improvements followed at the 1983 Pan American Games, where benchtop quadrupole GC/MS instruments screened samples of every athlete’s urine for signs of anabolic steroids and their metabolites.

Today, the primary method of choice for IOC testing laboratories is GC/MS. At the Unité d’Analyse du Dopage, an IOC-accredited lab in Lausanne (Switzerland), GC/MS is used in six of the seven standard tests for dopant detection. Identifying and confirming an illegal substance is sufficient in most GC/MS procedures; quantification is required only for naturally occurring substances and other drugs with concentration limits such as caffeine.

… and Limitations
But GC/MS isn’t suitable for every banned substance. Corticosteroids, a class of steroid hormones that produces euphoria and increased motor activity, cannot be reliably detected by GC because the compounds are slightly volatile and can denature when heated. Instead, labs use high-performance liquid chromatography (HPLC) in conjunction with MS and a particle beam interface, a system that minimizes the chance of corticosteroid denaturation.

To test for banned substances in concentrations too low for standard GC/MS detection, IOC labs employ immunoassays, which make use of the body’s ability to recognize and respond to a foreign substance. The hormone human chorionic gonadotropin (hCG) is typical of these low-concentration dopants. It is of particular interest to male athletes because it stimulates the testes to produce natural steroids. An immunoassay quantifies the amount of hCG in the athlete’s urine.

In spite of these efforts, at least two popular performance-enhancing substances, human growth hormone (hGH) and insulin-like growth factor, cannot even be detected with current standard testing procedures. And experts agree that athletes continue to find ways to mask their use of compounds such as testosterone and other steroids that usually can be detected.

New Weapons
But recent events in Sydney illustrate one success story: two tests for EPO—one using blood samples, the other testing urine. After the 1998 scandal surrounding the widespread use of EPO by Tour de France cyclists, researchers worked feverishly to perfect two complementary techniques for detecting the banned substance in time for the Sydney Olympic Games.

By approving one of the EPO tests, the IOC for the first time sanctioned the blood testing of athletes. The test, developed by researchers at the Australian Institute of Sport in Canberra, measures the concentration of EPO in blood as well as four other markers of abnormally high EPO levels by immunoassay and other cellular measurements. Two of these markers leak out of the bone marrow when red blood cells are overproduced; a third is involved in iron metabolism and influences the production of the oxygen-carrying hemoglobin complexes found in red blood cells.

Researchers at the French National Anti-Doping Laboratory in Châtenay-Malabry developed a separate test for EPO in urine. Because EPO produced for pharmaceuticals differs from natural human EPO in the number of attached sugar residues—and thus the two types have different electrical charges—scientists can separate them on a pH gradient through isoelectric focusing. Unfortunately, EPO is present in urine for only about 48–72 hours after injection, whereas the effects remain in blood for the life of the red blood cell, says Larry Bowers of the U.S. Anti-Doping Agency in Boulder (CO).

Researchers hope that other techniques still in development will target hGH, a hormone used therapeutically to treat dwarfism but useful for athletes because of its fat-burning and anabolic properties. One test for hGH, developed at the Ludwig-Maximilian University in Munich, uses an immunoassay to differentiate its two molecular forms: One has a molecular weight 2 kD heavier than the other. The immunoassay can detect illicit doping because 95% of the hGH produced for pharmaceuticals exists in the heavier form, whereas just 50% of natural hGH occurs in the heavier variety. Again, says Bowers, the problem is that hGH is present only very briefly, making detection possible only when the test is administered at random between competitions.

Differentiating between human and pharmaceutical testosterone is another problem. Although the IOC first instituted a test to detect elevated testosterone for the 1984 Olympic Games, athletes have consistently found ways to thwart the procedure, which involves measuring the ratio of testosterone metabolites in urine to another naturally occurring steroid, epitestosterone. To cheat, athletes can inject themselves with epitestosterone—available in an FDA-approved prescription form—in addition to testosterone, leaving the ratio unchanged. Alternatively, athletes dope themselves with human chorionic gonadotropin, which stimulates the body to increase the levels of both testosterone and epitestosterone.

To combat this, researchers at the University of Iowa (Iowa City) advocate measuring the ratio of testosterone to luteinizing hormone (LH), which also controls the natural secretion of testosterone but—unlike epitestosterone—is not yet available in an FDA-approved form. In one study, the scientists showed that measuring the concentration of LH in blood by immunoassay, combined with GC/MS for measuring the level of testosterone in urine, resulted in greater sensitivity for detecting testosterone abuse when compared to the standard measure of testosterone to epitestosterone.

Research at the UCLA Olympic Analytical Laboratory offers another promising avenue for detecting illicit testosterone doping. In one study, scientists relied on differences in 13C abundance to distinguish between human and pharmaceutical testosterone using GC/combustion/isotope ratio MS. Although the method works well in theory, says Kammerer, researchers are still collecting data on what ratio proves that an athlete has abused testosterone.

In another study at the Athletic Drug Testing and Toxicology Laboratory in Indianapolis, researchers developed a method for detecting the sulfate and glucuronide conjugates of steroid metabolites using HPLC/MS. According to Bowers, measuring the concentrations of these compounds offers a way to determine the true causes of “naturally elevated” ratios of testosterone to epitestosterone.

Hair Today
Figure 2. Shave and a Haircut.
Figure 2. Shave and a Haircut. Like the rings of a tree trunk, the hairs on an athlete’s body tell the story of the chemicals that his or her body has carried. If the effect of a chemical is longer lasting than the chemical itself, hair testing may prove invaluable.
Analytical tests of hair samples, however, may offer the best lab-based approach to catching athletes who stop doping themselves in the periods leading up to major competitions. Because drugs and hormones circulating in the bloodstream become permanently incorporated into the keratin matrix of the hair shaft, even a small fragment of hair serves as a time-resolved record of substance abuse (Figure 2).

In a recent study, researchers at the Laboratoire d’Expertises TOXLAB in Paris showed how hair analysis can detect corticosteroid use at a sensitivity reaching 0.1 ng/mg of hair. In the hair of one athlete, the scientists used liquid chromatography-electrospray ionization MS to detect hydrocortisone acetate, a synthetic form of hydrocortisone that breaks down too quickly to be identified in urine. Don Catlin, director of the UCLA Olympic Analytical Laboratory, says the technique can’t yet rule out environmental contaminants as the source of the banned substance, but it “certainly has the potential—it’s making progress, no doubt about it.”

Most experts agree that it will take a significant amount of money to translate progress in hair testing—and other analytical methods—into effective deterrents for doping. Researchers and health policy experts are demanding that the IOC allocate as much as $100 million over five years to develop a comprehensive system for testing athletes. “With out-of-competition, no-announcement testing, and if you really put money into R&D, then athletes would start to think others weren’t also doing it,” says Kammerer. “Now they all think, ‘Everyone does it, so if I don’t, I’m at a disadvantage.’”

Others take a more guarded view, suggesting that no matter what advances are made in testing, athletes and coaches will still try to find ways to circumvent the tests designed to catch them. Yesalis thinks that athletes may soon—perhaps in the next 10 years—use viruses as vectors for delivering genes that offer improved athletic performance. “A good offense will always beat a good defense,” Yesalis says of athletes’ abilities to outwit testing authorities.

But Catlin says the kinds of drugs detected in athletes during the Sydney Olympic Games offer hope that testing authorities may be regaining the upper hand. “In Sydney, we found quite a few [violations], but they were second-line drugs—they weren’t malicious or malignant,” he says. “Compared to the 1984 Olympics, things have vastly improved.”

The ideal system for discouraging drug use, says Catlin, would incorporate voluntary testing on the part of the athletes. “We’re coming to a point where it doesn’t make a lot of sense to chase these athletes all over the world,” he says. “It’s better to get them to come in voluntarily and harness their good energies. By and large, they want a level playing field.”

Further Reading

  • Aguilera, R.; Becchi, M.; Casabianca, H.; Hatton, C. K.; Catlin, D. H.; Starcevic, B.; Pope, H. G., Jr. J. Mass Spectrom. 1996, 31, 169–176.
  • Bévalot, F.; Gaillard, Y.; Lhermitte, M. A.; Pépin, G. J. Chromatogr. B 2000, 740, 227–236.
  • Bowers, L. D. Clin. Chem. 1997, 43, 1299–1304.
  • Lasne, F.; de Ceaurriz, J. Nature 2000, 405, 635.
  • Olympic Movement Anti-Doping Code; International Olympic Committee: Lausanne, Switzerland, April 1, 2000 (available at www.olympic.org/ioc/e/org/medcom/medcom_antidopage_e.html).
  • Parisotto, R.; Gore, C. J.; Emslie, K. R.; Ashenden, M. J.; Brugnara, C.; Howe, C.; Martin, D. T.; Trout, G. J.; Hahn, A. G. Haematologica 2000, 85, 564–572.


John S. MacNeil is a freelance writer living in Alexandria, VA. Send your comments or questions regarding this article to tcaw@acs.org or the Editorial Office 1155 16th st N.W., Washington, DC 20036.

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