Ensuring a level playing field

As memories of the Olympics fade, and thoughts turn to how London 2012 will compare – one thing is certain, the battle against performance enhancing drugs will have to move with the times

TESTING for performance-enhancing substances is now an accepted part of sporting life in major international competitions such as the Olympics, and even during events at the national and collegiate levels. The ability to fight the use of banned drugs in sports - and ensure a level playing field for all participants - depends on tough sanctions supported by a reliable and reproducible scientific system for doping testing.

Liquid chromatography/gas chromatography (LC/GC) coupled to mass spectrometry (MS) has long been the core technology used in doping testing. This is an application that poses many challenges - and opportunities - for scientists and chromatographers. Even as the demands on (and from) regulatory agencies continue to evolve, “underground chemists” try to stay one step ahead by synthesising new and harder-to-detect compounds that can give athletes an unfair advantage.

The death of British cyclist Tommy Simpson during the 1967 Tour de France focused tremendous attention on the issue of doping in sports. It was later discovered that Simpson had taken amphetamines, which caused his heart to give out during his ascent of the formidable 1,909m Mount Ventoux. Stimulants such as amphetamines increase stamina and endurance but also have serious side effects such as dependency, depression and exhaustion. Their use in cycling had become widespread by the end of the 1960s and continued through the 1970s, even though the International Cycling Union introduced a list of banned substances shortly after Simpson’s death.

In 1972, doping testing was introduced at the Munich Olympics. Testing at the international level is now overseen by the World Anti-Doping Agency (WADA), which was established in 20001. Doping testing is also carried out by all the major sporting industry bodies, including Major League Baseball, the National Football League and the National Collegiate Athletic Association in the United States.

As a result, demand for doping testing is expanding. In the 33 WADA-accredited labs around the world, the number of tests increased by more than 14,000 between 2004 and 2005, from 169,187 to 183,337. The corresponding increase in adverse analytical findings - the presence of prohibited substances - grew from 2,909 to 3,909, a 34.4% rise.

Currently, WADA lists 11 categories that include more than 400 banned substances: anabolic agents (Figure 1); hormones and related substances; beta-2 agonists; agents with anti-estrogenic activity; diuretics and other masking agents; stimulants; narcotics; cannabinoids; glucocorticosteroids; alcohol; and beta-blockers. For most of these, LC/GC coupled with some form of MS is the preferred analytical technology. Agilent has a long involvement in doping testing, having developed the GC nitrogen-phosphorus detector (GC/NPD) specifically for use at the Munich Olympics.

Testing done under the auspices of WADA follows a specific procedure. The athlete provides a urine or blood sample in the presence of an anti-doping official; the sample is split, by the athlete, into two - the A and B samples - and these are sealed. The samples are then handled within a strict chain of custody similar to that applied to forensic samples.

Testing starts with the A sample. An aliquot is extracted and derivatised as

Figure 1: Chemical structures of common anabolic substances

necessary to leave a residue that contains the compound or metabolites of interest. The exact procedure depends on the class of drug being tested for in the sample. If the initial test is negative, no further action is taken. With a positive test, a second aliquot of the A sample is taken and subjected to a more-detailed analysis. If this test is positive, the B sample is tested, but only after informing the athlete, who can be present and can also bring along their own expert observer. If the B sample generates a positive result, then the matter is referred to the anti-doping authority. No further action is taken if the B sample test is negative.

The optimal choice of analytical techniques and instruments varies depending on

Figure 2: Chemical structure of the designer steroid THG

the target substance and the task (e.g., screening or confirmation). As a general statement, GC is well suited to the separation of small, volatile compounds while LC is best for larger, non-volatile substances. In recent years, the utility of these separation techniques has been further expanded by the advent of robust tandem MS technologies such as LC or GC combined with a triple-quadrupole mass spectrometer (LC/QQQ and GC/QQQ).

To be more specific, doping control labs typically use GC/NPD for screening of narcotics and some classes of stimulants, while GC/MS is used for broader screens including anabolic steroids, marijuana, marijuana metabolites, and opiates. Larger molecules, such as beta-blockers and glucocorticoids, are often screened for using tandem LC/MS. More recently, laboratories have been applying the rapid, full-scan capability of LC time-of-flight (LC/TOF) and LC quadrupole time-of-flight (LC/QTOF) to screen for presumptive positives. Because TOF instruments always operate in full-scan mode, which collects “all the data, all the time,” these technologies also provide laboratories with the ability to perform retrospective analysis. Immunoassays are used to screen for peptides and hormones such as human chorionic gonadotropin (hCG) and insulin. Isoelectric focusing (IEF) with chemiluminescence detection is used to screen for erythropoietin (EPO), which has become popular for boosting an athlete’s oxygen-carrying capacity.

Confirmation tests are often performed using GC/MS or some form of tandem LC/MS such as LC/QQQ. Recently, some have been using LC/MS ion-trap technology for target confirmation due to its unique ability to generate multiple MS (MSn) spectra and provide a “spectral audit trail.” Similarly, some laboratories leverage the superb mass accuracy of LC/TOF or LC/QTOF to confirm the presence of a target compound through empirical formula determination.

Whatever the method, testing for sports doping places tremendous demands on

Figure 3: Example spectra from accurate mass LC/TOF analysis of 2ng/ml urine extract (1ng/ml 19- norandrosterone). Mass accuracy was better than 2ppm for each analyte3

analytical instruments. Above all, the equipment must be reliable because downtime is unacceptable, especially during a major event. The instrument must be able to process large numbers of samples in the lab with a rapid turnaround time: Those athletes being tested have a right to a prompt result. Day-to-day reproducibility is also crucial, particularly where confirmatory analyses are concerned, to ensure confidence in results that may be challenged.

Steroids are the most commonly abused drugs because they increase muscle growth (mass) and decrease recovery time. More than 70 are on the banned list, the most common of which are stanozolol, testosterone, nandrolone, clenbuterol, and the new “designer steroid” tetrahydrogestrinone (THG) - but new ones are constantly emerging (Figure 2).

Although there are standard techniques, analytical protocols and measurement technology must constantly evolve to keep pace with new trends and demands in doping testing. Examples include the designer steroids clandestine labs synthesise by making a slight chemical modification to a known compound. Consequently, the synthetic steroid has a molecular weight that falls outside the target mass range monitored for anti-doping purposes. One way to detect these compounds is to capitalise on advances in GC/MS technology that allow for simultaneous collection of both selected ion monitoring (SIM) and scan data. This provides an abundance of information to assist in elucidating the molecular structure of the designer steroid. Another useful tool is ion-trap LC/MS with its inherent ability to collect MSn spectra, which provides excellent elucidation of molecular structure2.

Another challenge for doping labs is that some steroids, such as nandrolone and its metabolites, can occur at very low endogenous levels. Consequently, WADA has established a 2ng/ml threshold for these compounds: any concentration above the 2ng/ml level is considered to be suspicious (Figure 3). Traditionally, high-resolution magnetic sector (HRMS) instruments are used to determine these low-level concentrations of steroids. Some laboratories, however, would prefer to avoid HRMS devices due to their complexity and expense. One alternative is GC/QQQ, an older technology that is experiencing a revival because the instrumentation has become more robust – and because it can meet the demanding requirements of steroid testing. Additional alternatives include LC/TOF, LC/QTOF and high-resolution ion traps, which many labs are currently using to detect steroids3.

Many challenges remain in the quest to ensure a level playing field in competitive sports. For example, it is still difficult to distinguish between endogenous and exogenous levels of substances such as human growth hormone (hGH) and EPO. Both natural and recombinant versions of these hormones are popular doping agents.

In the future, athletes may be tempted to try “gene doping”. Research at the University of Pennsylvania has shown that mice administered with the insulin-like growth factor gene (IGF-1) have muscle strength and power up to 30% greater than normal animals, and they also respond better to resistance training. Gene therapy is very difficult to detect - IGF-1, EPO and growth factor are already present in the human body - but test labs are working on ways to detect these forms of doping, too.

Sadly, dopers and cheaters will always try to stay ahead of the game. As a result, some form of testing will remain a fact of life in competitive sports. Fortunately, though, these forces will continue to stimulate the creation of new developments in measurement science and technology.