Efficient Doping Control Automated Determination of Nicotine and Its Metabolites in Blood

In the race to keep up with new and suspected doping agents, the Centre for Preventive Doping Research at the German Sport University Cologne, Germany is upgrading their laboratory with performance enhancing technologies and automation: The institute is betting on a fully automated Dried Blood Spot (DBS)-LC-MS/MS analysis system.

The pressure to achieve record-breaking results in sports has become extremely intense, advances in technologies that can effectively guide an athlete to peak performance has made achievement of new records an almost routine occurrence. However, even after all aspects of an athlete’s training regime have been optimized and the seemingly endless hours of preparation have been put in, some athletes still cannot achieve the desired results. This is when the temptation to use performance enhancing substances rears its head.

Not every chemical means of enhancing physical or mental performance is currently listed as illegal. But the World Anti-Doping Agency (WADA) (see LAB Info box) has a watchful eye on many substances that they suspect are being used to gain an unfair advantage in sports. In 2015 for instance, the watch list sported names of active pharmaceutical ingredients such as Bupropion, Phenylephrine, Phenylpropanol­amine, Pipradol, and Synephrine, along with stimulants such as caffeine and nicotine that are naturally present in coffee and tobacco.

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Doping with nicotine — more than just a suspicion

In 2011, the German daily newspaper “Süddeutsche Zeitung” reported that “Nicotine doesn’t improve stamina or muscle power, but it affects the brain and places the athlete in a different state of mind”, says the pharmacologist Fritz Sörgel, a recognized doping expert and Head of the Institute of Biomedical and Pharmaceutical research (IBMP) at the University of Nuremberg, Germany. In those sports, in which reaction time and concentration are especially important to performance, an increased nicotine level could help athletes gain an advantage.

While smoking tobacco could have significant detrimental health and performance effects, e-cigarettes or chewing tobacco could present an attractive alternative without the negative side-effects. The same applies to snus, an orally consumed form of tobacco, which is widely used in Norway and Sweden. “The suspicion is that snus is being abused for doping purposes”, states Professor Mario Thevis from the Centre for Preventive Doping Research at the German Sport University Cologne, Germany.

No clinical studies or other well founded data were available concerning the use or effects of nicotine as a performance enhancing drug even though WADA had placed nicotine on the watch list as a suspected doping agent. Prof. Thevis and his colleagues in Cologne along with a scientist from the National Veterinary Institute of the Department of Chemistry in Uppsala, Sweden set out to develop a “fast and inexpensive” method of analysis that would enable the determination of nicotine and its metabolites while also providing insight on how they had been introduced into the body [1]. During their search to find the most suitable analysis technique for their purposes, Dried Blood Spot (DBS) analysis in combination with online solid phase extraction (SPE) and LC-MS/MS soon emerged as the most promising solution.

Cost and speed are the deciding factors

According to the scientists, DBS has proven itself many times, for example in pre-clinical pharmaceutical research; for monitoring of active therapeutic agents; in forensic toxicology; as well as in studies of metabolic disorders. Meanwhile, several examples were also published on DBS being used in doping analysis. According to Mario Thevis and his colleagues, DBS offers a number of benefits when compared with standard strategies for blood sampling: DBS is minimally invasive — a simple finger prick is enough to withdraw a sufficient sample volume (20 μL) for the analysis. Just a few drops of blood absorbed on a suitable, cellulose based medium is all that is needed for a successful determination of the compounds of interest. In addition, blood sampled in this way exhibits good long term stability at room temperature. Samples dry very quickly and the absence of humidity means that enzymes are deactivated, as the scientists point out [1].

To perform DBS analysis, several sample preparation steps are required: “Currently, the pre-analysis workflow includes punching out and dissolving each dried blood spot. Then an extraction is performed with a suitable solvent, occasionally including ultra-sonication. Further clean-up steps involve: Protein precipitation, filtration and transfer of the resulting extract into a sample vial followed by LC-MS/MS analysis”, according to Thevis et al. Automation is an absolute necessity in order to minimize the significant manual workload and to qualify DBS sampling for high throughput analysis in a routine laboratory setting.

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