Next-generation sports drug testing

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  • Published: Mar 16, 2016
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: Next-generation sports drug testing

Masking the proof

It wasn't until the 1920s that restrictions on drug use in sports really came into force, designed to protect the spirit and integrity of sport. Today the use of drugs in sports is strictly regulated under the guidance of the World Anti Doping Agency (WADA).

The problem of doping goes back to the very origins of sport. At the ancient Olympics in Greece, athletes had special diets and stimulating potions, and in ancient Rome gladiators drank strengthening herbal infusions before chariot races. However, it wasn't until the 1920s that restrictions on drug use in sports really came into force, designed to protect the spirit and integrity of sport. Today the use of drugs in sports is strictly regulated under the guidance of the World Anti Doping Agency (WADA).

Asked about doping, most people would likely think of performance-enhancing drugs like steroids, but equally important are substances that hide the intake of such drugs. Vaptans, for example, prescribed to treat low levels of sodium in the blood, are effective masking agents. This is thanks to their ability to increase the passing of urine. In patients with heart failure, these diuretic properties increase the excretion of excess fluids and therefore increase blood sodium levels, but they can also be abused to accelerate the excretion of drugs. Vaptans, such as tolvaptan, are therefore prohibited by WADA.

As well as making it hard to detect performance-enhancing drugs, tolvaptan is itself very difficult to detect; typically less than 1% of the unchanged drug is excreted by the body. One way to overcome this problem is to detect the metabolites of the drug, but tolvaptan’s metabolic pathways in humans are poorly understood.

‘Dilute-and-shoot’

In a study recently published in Drug Testing and Analysis, researchers describe a quick and easy means of detecting tolvaptan, and its metabolites, in human urine. Members of the Norwegian Doping Control Laboratory developed a rapid method based on the dilute-and-shoot approach. As its name suggests, the sample is diluted before being injected into an analytical instrument, in this case high-performance liquid chromatography coupled to tandem mass spectrometry.

To validate the method, the researchers analysed urine samples from 10 healthy volunteers. The samples were spiked with tolvaptan at five concentrations (0.2, 5, 100, 200 and 500 ng/ml). Tolvaptan could be identified in all spiked samples, with a very low limit of detection of 0.2 ng/ml. The validation experiment showed the method to be suitable for detecting tolvaptan in urine, able to meet all of WADA’s technical criteria.

To test the method in a real case, tolvaptan was administered – in a single dose of 15 mg – to a healthy male. Urine samples were collected at various points up to 7 days after intake. Three ions were used for identification, based on the known fragmentation pattern of tolvaptan.

Week-long detection

As expected, the drug was excreted in its unchanged form at very low concentrations, 15 ng/ml at the highest. The urine samples were also analysed for the presence of metabolites, which were easier to detect. The researchers identified several hydroxyl metabolites and one carboxyl metabolite.

Not only were the metabolites excreted at higher concentrations, they were also detectable for longer. The drug itself was only detectable in urine for 24 hours after intake, however some of the metabolites could still be identified 150 hours (almost a week) after intake.

This method extends detection times for tolvaptan by six times. Furthermore, it could easily be incorporated into routine doping analysis as it uses ion transitions, like existing screening methods. Longer term detection of diuretics, as is already the case for steroids, could be of huge benefit for anti-doping agencies.

It’s important to note, however, that this study only included one subject. The authors say further studies are needed to better understand tolvaptan metabolism in humans, as there may be differences between gender (as observed in rats).

Related Links

Drug Test. Analysis, 2016, Early View Article, Analysis of tolvaptan and its metabolites in sports drug testing by high-performance liquid chromatography coupled to tandem mass spectrometry.

Competing against doping

The Story of Anti-Doping

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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