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Efficient Doping Control

Automated Determination of Nicotine and Its Metabolites in Blood

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The scientists set out to determine nicotine, the main metabolites nornicotine, cotinine and trans-3’-hydrocotinine (trans-3’-HCOT), as well as the alkaloids Anabasine and Anatabine using a fully automated system: A Multi Purpose Sampler (MPS) and a Dried Blood Spot Autosampler (DBSA) coupled to an online SPE system (SPExos) all from Gerstel. This setup was coupled with a high-resolution LC-MS/MS system.

“When using an online SPE system, we can extract the DBS sample, clean up the extract, and proceed directly with the analysis”, explains Prof. Thevis. Automation is only one important aspect of the system, according to the expert: “Not only does the automated DBS sample preparation reduce the workload, it also improves extraction efficiency and limits of detection”. The patented Flow Through Desorption (FTDTM) technology enables good concentration factors to be achieved while minimizing the risk of sample to sample carry over. The Online DBS SPE-LC-MS/MS method was developed using standard solutions at different concentration levels, which were spiked into samples from volunteers who had neither smoked nor consumed snus. The validation was performed following the recommendations of WADA and the European Bioanalysis Forum (EBF). Deuterated analogues were used for quantification of target compounds. In order to investigate potential differences in pharmacokinetics,


Thevis et al. used their method on authentic samples, that is, blood samples taken from cigarette- and e-cigarette smokers, as well as snus users. The project was set up with permission from the local ethics commission, in some countries known as the Institutional Review Board, whose task it is to protect human subjects from harm by overseeing research performed on humans or animals. Written permission was obtained from the volunteers.

Differentiating between normal consumption and doping

When they developed the method, the authors focused on optimizing it for reproducibility and workflow efficiency, according to Thevis and his colleagues [1]. The process steps that held the most promise for improvement was DBS elution, SPE cleanup, as well as Mass Spectrometric Detection. Using the DBS method they developed, the authors succeeded in determining all target analytes with excellent precision and accuracy. The limit of detection for all analytes was 5 ng/mL. The successful analysis of blood samples taken from real smokers as well as e-cigarette and snus users demonstrated that the method could be implemented for routine doping controls as well.

“All target compounds were found in the real samples”, Thevis et al. wrote. Additionally, the statistical evaluation had shown a significant difference in the ratio between nicotine and nornicotine concentrations in the blood depending on whether nicotine was administered via the lungs (inhalational) or via mucous membranes (buccal uptake). This means that based on pharmacokinetic properties, conclusions can be drawn as to the athlete’s method of consumption and maybe the longer term pattern of use.


[1] Laura Tretzel, Andreas Thomas, Thomas Piper, Mikael Hedeland, Hans Geyer, Wilhelm Schänzer, Mario Thevis: Fully automated determination of nicotine and its major metabolites in whole blood by means of a DBS online-SPE LC-HR-MS/MS approach for sports drug testing, Journal of Pharmaceutical and Biomedical Analysis 123 (2016) 132–140

* G. Deußing: Redaktionsbüro Guido Deussing, 41464 Neuss/Germany

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