Detection of main urinary metabolites of β2-agonists clenbuterol, salbutamol and terbutaline by liquid chromatography high resolution mass spectrometry

► Urinary metabolism study of clenbuterol, salbutanol and terbutaline in rat. ► Methodology based on solid-phase extraction followed by LC-Electrospray-TOFMS. ► Use of diagnostic fragments ions and accurate mass shifts from biotransformations. ► Up to eleven metabolites detected, four of them non previously reported in urine. ► Three clenbuterol metabolites were detected over a longer period than parent drug. Clenbuterol, terbutaline and salbutamol are B2-agonists drugs included in the list of banned substances of the World Anti Doping Agency (WADA) prohibited in and out of competition. In this article, the excretion of urinary metabolites of clenbuterol, terbutaline and salbutamol have been studied using liquid chromatography electrospray time-of-flight mass spectrometry (LC-TOFMS), after a single therapeutic dose administration in rats. Urine collected was processed with solid-phase extraction prior to LC-TOFMS analyses using electrospray in the positive ion mode and pseudo MS/MS experiments from in-source collision induced dissociation (CID) fragmentation (without precursor ion isolation). The strategy applied for the identification of metabolites was based on the search of typical biotransformations with their corresponding accurate mass shift and the use of common diagnostic fragment ions from the parent drugs. The approach was satisfactory applied, achieving the identification of 11 metabolites (5 from clenbuterol, 4 from salbutamol and 3 from terbutaline), 4 of them not previously reported in urine. Novel metabolites identified in rat urine included N-oxide-salbutamol, hydroxy-salbutamol, methoxy-salbutamol glucuronide and terbutaline N-oxide, which are all reported here for the first time.