title: 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.
abstract: Dried blood spots (DBS) represent a sample matrix collected under minimal-invasive, straightforward and robust conditions. DBS specimens have been shown to provide appropriate test material for different analytical disciplines, e.g., preclinical drug development, therapeutic drug monitoring, forensic toxicology and diagnostic analysis of metabolic disorders in newborns. However, the sample preparation has occasionally been reported as laborious and time consuming. In order to minimize the manual workload and to substantiate the suitability of DBS for high sample-throughput, the automation of sample preparation processes is of paramount interest. In the current study, the development and validation of a fully automated DBS extraction method coupled to online solid-phase extraction using the example of nicotine, its major metabolites nornicotine, cotinine and trans-3'-hydroxycotinine and the tobacco alkaloids anabasine and anatabine is presented, based on the rationale that the use of nicotine-containing products for performance-enhancing purposes has been monitored by the World Anti-Doping Agency (WADA) for several years. Automation-derived DBS sample extracts were directed online to liquid chromatography high resolution/high mass accuracy tandem mass spectrometry, and target analytes were determined with support of four deuterated internal standards. Validation of the method yielded precise (CV <7.5% for intraday and <12.3% for interday measurements) and linear (r(2)>0.998) results. The limit of detection was established at 5 ng mL(-1) for all studied compounds, the extraction recovery ranged from 25 to 44%, and no matrix effects were observed. To exemplify the applicability of the DBS online-SPE LC-MS/MS approach for sports drug testing purposes, the method was applied to authentic DBS samples obtained from smokers, snus users, and e-cigarette users. Statistical evaluation of the obtained results indicated differences in metabolic behavior depending on the route of administration (inhalative versus buccal absorption) in terms of the ratio of nicotine and nornicotine.
authors.affiliation: Institute of Biochemistry, Center for Preventive Doping Research, German Sport University Cologne, Germany.
authors.forename: Laura
authors.initials: L
authors.lastname: Tretzel
authors.affiliation: Institute of Biochemistry, Center for Preventive Doping Research, German Sport University Cologne, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Thomas
authors.affiliation: Institute of Biochemistry, Center for Preventive Doping Research, German Sport University Cologne, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Piper
authors.affiliation: National Veterinary Institute, Department of Chemistry, Uppsala, Sweden.
authors.forename: Mikael
authors.initials: M
authors.lastname: Hedeland
authors.affiliation: Institute of Biochemistry, Center for Preventive Doping Research, German Sport University Cologne, Germany.
authors.forename: Hans
authors.initials: H
authors.lastname: Geyer
authors.affiliation: Institute of Biochemistry, Center for Preventive Doping Research, German Sport University Cologne, Germany.
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Schänzer
authors.affiliation: Institute of Biochemistry, Center for Preventive Doping Research, German Sport University Cologne, Germany. Electronic address: thevis@dshs-koeln.de.
authors.forename: Mario
authors.initials: M
authors.lastname: Thevis
mesh_terms: D000470:Alkaloids / Q000737:chemistry; D000691:Anabasine / Q000737:chemistry; D001331:Automation / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D003367:Cotinine / Q000031:analogs & derivatives / Q000737:chemistry; D004300:Doping in Sports / Q000379:methods; D059788:Dried Blood Spot Testing / Q000379:methods*; D066300:Electronic Nicotine Delivery Systems / Q000009:adverse effects; D005260:Female; D006801:Humans; D008297:Male; D009538:Nicotine / Q000031:analogs & derivatives / Q000097:blood* / Q000737:chemistry*; D009862:Online Systems; D011725:Pyridines / Q000737:chemistry; D015203:Reproducibility of Results; D052616:Solid Phase Extraction / Q000379:methods; D013177:Sports; D015813:Substance Abuse Detection / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000470:Alkaloids; D011725:Pyridines; C001381:hydroxycotinine; C012737:anatabine; D009538:Nicotine; C005311:nornicotine; D003367:Cotinine; D000691:Anabasine
keywords: Automated extraction; Dried blood spots; LC-HR-MS/MS; Nicotine; Online solid phase extraction; Sports drug testing