ac detective
Finding designer steroids Athletes beware! Researchers have a new way to detect previously unidentified doping agents. he abuse of sophisticated designer steroids such as tetrahydrogestrinone (THG) went unnoticed for several years, but thanks to new detection strategies and a little bit of luck, that situation is beginning to change. Even so, specialized underground laboratories continue to stay one step ahead of doping-control authorities. As long as illicit preparations remain unknown, routine sports doping tests do not screen for them. The same problem exists in the veterinary world. In the European Union, hormonal substances used to fatten up cattle are banned from animal feed. But until a new compound is identified, it can be fed to cattle for extended periods of time and not show up in routine testing. To combat the problem, researchers are looking for new ways to detect previously unidentified illicit steroids. In the January 15 issue of Analytical Chemistry (pp 424–431), Michel Nielen and colleagues at the RIKILT Institute of Food Safety (The Netherlands) describe one such effort. By combining an androgen bioassay with LC, the team has developed a simple, inexpensive method that could be used as an early warning system for doping control. In the new approach, urine samples are subjected to enzymatic deconjugation and solid-phase extraction. The extracts are then separated by gradient LC. Every 20 s, a fraction of the LC effluent is transferred to a 96-well plate and tested for androgen activity by a novel yeast reporter gene bioassay. Half of the LC effluent is also collected in a duplicate 96-well plate for identification purposes. Separated sample components that are bioactive in one or more wells from the first plate are subsequently identified by high-resolution LC/quad© 2006 AMERICAN CHEMICAL SOCIETY
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Detecting doping. As illegal laboratories continue to modify the structures of illicit steroids, researchers keep developing new ways to find them.
rupole TOFMS from the same well numbers from the duplicate plate. “The nice thing about the androgen bioassay is that it doesn’t require any reagents,” says Nielen. It is a very inexpensive screening assay, he adds. Only those samples that are relevant are subjected to the more expensive MS identification. Because endogenous androgens in urine can interfere with the screening test, the researchers recommend using the assay after a type of chromatographic separation, such as LC. Nielen and colleagues used a similar approach in 2003 to discover an illegal β-agonist in animal feed. In that case, they used immunoassays and receptor assays against β-agonists. The group then went on to develop an estrogen yeast cell bioassay combined with LC for the detection of estrogen residues in calf urine (Anal. Chem. 2004, 76,
6600–6608). Now, they have modified the estrogen assay for the identification of designer steroid residues in urine by combining an androgen assay with LC. “Especially in the doping field, the abuse of androgens is much more pronounced than the abuse of estrogens. Athletes usually don’t like estrogens because of the side effects,” says Nielen. Thus, an androgen bioassay is much more useful than an estrogen bioassay for doping control, he adds. Although yeast cells are not the same as human cells, they offer several advantages, says Nielen. First of all, they are relatively simple. “A yeast cell will not produce steroid receptors or other kinds of interfering substances by itself,” he says. However, yeast cells can be genetically modified to produce specific steroid receptors. The receptors can be designed to catch estrogens, androgens, corticosteroids, and so forth. Another advantage of using yeast cells is that they can be grown on media that do not contain hormones. In addition, yeast cell bioassays are extremely robust. The researchers demonstrated the potential of the new androgen bioassay combined with LC by testing a urine sample spiked with THG. The next step is to try it on real-world samples. “As we know from our previous work, this type of approach is capable of discovering an entirely new illegal molecule,” says Nielen. “When you apply this method in routine control, then you have a chance of finding the next unknown, which would of course be a headline,” he says. Now it is up to doping-control authorities to adopt methods such as this one to discover unknown illicit substances in urine samples. a —Britt Erickson
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