Development of a liquid chromatography Q Exactive high resolution mass spectrometry method by the Box-Behnken design for the investigation of sibutramine urinary metabolites

Sibutramine is cited by the World Anti-Doping Agency as a stimulant. According to the literature, sibutramine is extensively metabolized into N-desmethyl-sibutramine (M1), N-bisdesmethyl-sibutramine (M2) and monohydroxy derivatives of M1 and M2. Therefore, it is important to verify new sibutramine metabolites through current analytical methodologies, such as liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). Furthermore, the development of a comprehensive approach to investigate sibutramine metabolism can increase the detection window for stimulant misuse and enable advancements in pharmacological studies. This work aimed to develop and evaluate the performance of an LC-HRMS method applying Design of Experiments (DoE) for sibutramine metabolite analysis in human urine. After optimizing the method by DoE, the final chromatographic conditions were based on reversed-phase chromatography using a C18 column with a ramp time of 25 min, a flow rate of 0.17 mL min^-1 and a temperature of 50 °C. Mobile phase A consisted of water with 0.1% formic acid and 5 mM ammonium formate, and mobile phase B consisted of methanol with 0.1% formic acid; the initial gradient percent was 15% B, and the injection volume was 5 μL. In addition to the hydroxylated metabolites previously described in human urine, dihydroxy derivatives of M1 and M2 were observed for the first time. These dihydroxy derivative metabolites can be applied as new targets for doping control.