Determination of N-Desmethyl- and N-Bisdesmethyl Metabolites of Sibutramine in Doping Control Analysis Using Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Since January 2006, the list of prohibited substances established by the World Anti-Doping Agency includes the antidepressant/anti-obesity drug Sibutramine. Due to its rapid degradation to its active metabolites N-desmethyl and N-bisdesmethyl sibutramine, reference compounds were synthesized and included into an existing screening assay to allow the unambiguous determination of these metabolic products in human urine using liquid–liquid extraction followed by liquid chromatography/tandem mass spectrometry. Characteristic product ions, obtained after electrospray ionization and collision-induced dissociation, were elucidated using high resolution/high accuracy mass measurements with a hybrid linear ion trap/orbitrap mass analyzer. Based on diagnostic product ions, the extended screening procedure was validated for both sibutramine metabolites using a triple quadrupole mass spectrometer. Items such as lower limits of detection (6–40 ng mL−1), recoveries (39–42%), intraday precision (low: 5.5–10.6%, medium: 4.9–5.9%), high: 12.8–16.4%) and interday precision (low: 15.0–22.8%, medium: 17.7–18.6%), high: 16.5–25.6%) were evaluated and a clinical spot urine sample was analyzed to demonstrate the applicability of the developed assay in sports drug testing.