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The smokeless gun: Athletes using smokeless tobacco for nicotine doping

Tobacco is the new sporting stimulant

The World Anti-Doping Agency (WADA) campaigns for global drug-free sport and is supported by funding from national governments and the Olympic Movement. Its key activities include scientific research and the establishment of accredited labs around the world to carry out drug testing on the many thousands of samples provided by athletes each year.

The labs are guided by the WADA Prohibited List, which is the up-to-date bible of banned substances. The list is reviewed annually with new additions published on October 1st and coming into force on the following January 1st.

One of the substances under consideration is nicotine, according to Olivier Rabin, the sporting director of WADA. The growing body of evidence that nicotine can be used to boost performance is behind their deliberations, although it is not cigarettes that are thought to be the problem.

The culprit is smokeless tobacco, which can be chewed, snorted or “dipped”, placing it between the lips and gums. When taken this way, tobacco is not associated with harmful effects on the respiratory tract, which would hinder athletes, but nicotine is still absorbed by the body.

Rabin said recently “it is not our objective to catch athletes who smoke, but those who use nicotine as a means of enhancing their performance,” adding that they would have to find means of distinguishing one from the other.

More support for a smokeless tobacco ban came recently in a study of the urine samples taken from players during the 2009 Ice Hockey World Championships. About 50% of players from 72 tested qualified as consumers of smokeless tobacco products based on threshold levels of nicotine in their system.

Now, a new study published in 2011 supports the widespread use of smokeless tobacco with an extensive study of urine samples from athletes.

Nicotine testing in 2100 urine samples

Francois Marclay, Elia Grata, Laurent Perrenoud and Martial Saugy studied 2185 urine samples that were submitted over a one-year period to their organisation, the Swiss Laboratory for Doping Analyses at the Centre Hospitalier Universitaire Vaudois, in Switzerland.

They adopted a liquid chromatography-mass spectrometry procedure to detect and measure the levels of nicotine and its major and major metabolites cotinine, cotinine N-oxide, trans-3-hydroxycotinine and nicotine N-oxide. The minor tobacco alkaloids anabasine, anatabine and nornicotine were also targeted.

In an effort to speed up sample preparation, they employed a “dilute-and-shoot” approach which is amenable to automation. After adding stable isotope-labelled compounds to act as internal standards, urine samples were diluted 40-fold with acetonitrile and the centrifuged supernatant was retained for analysis.

The degree of dilution was carefully chosen to try and balance out the effects of reduced interference from other compounds in the urine against the reduced sensitivity of the target analytes during mass spectrometric analysis.

One consequence of this strategy was to disregard the phase II glucuronide conjugates of nicotine and its metabolites, which, in some cases, form the major urinary excretion routes. Their inclusion would require a hydrolysis step to be incorporated, slowing down the procedure.

The researchers argued that their primary focus was on the levels of nicotine in the urine of athletes, which would be reflected by the target phase I metabolites that showed good sensitivity in their procedure.

The extracts were injected onto a short hydrophilic interaction chromatography column to separate the analytes. This was accomplished with a high flow rate so that the overall run time was just five minutes without jeopardising the resolution.

The eluting compounds were subjected to electrospray ionisation and analysed by tandem mass spectrometry, yielding detection limits in the region of 1 ng/mL and good analytical characteristics.

Smokeless tobacco doping prevalent in certain sports

The concentrations of the major and minor nicotine metabolites and the other alkaloids across all of the samples ranged from the lower limits of quantitation to 32,223, 6670 and 538 ng/mL, respectively. At least one of the metabolites was detected in 23% of the samples.

Even when accepted threshold levels for active exposure were taken into account (50 ng/mL for nicotine, cotinine and trans-3-hydroxycotinine and 25 ng/mL for the remaining analytes) at least one of the metabolites was present at these levels in more than 15% of cases.

The contribution from secondhand smoke is probably very low, due to increasing legislation for smoke-free environments, and the low likelihood of exposure just before sports practice and subsequent testing. So, these figures are significant.

The World Health Organisation estimates that the proportion of people smoking globally is 25%, so bearing in mind that the Swiss data is for athletes, this “supports the potential use of nicotine in sport with a specific purpose,” announced Marclay.

When the samples were narrowed down from the total of 43 sports to a particular subset, the results were even more startling. For ice hockey, skiing, biathlon, bobsleigh, skating, football, basketball, volleyball, rugby, American football, wrestling and gymnastics, the prevalence ranged from 19-55.6%, far larger than normal recreational use.

This is one of the strongest signs to date that smokeless tobacco is being used routinely in certain sports communities for performance benefits, without the respiratory health problems that accompany smoking.

At this stage, there is no recognised clinical procedure for measuring metabolites to differentiate between different types of nicotine consumption. However, this research team now have a set of quantitative data that is available for retrospective assessment when such a method is developed.

In the meantime, their results add more weight to the argument for adding nicotine to the WADA Prohibited List and they urged the List Expert Group to evaluate its inclusion.

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