Evaluation of tramadol and its main metabolites in horse plasma by high-performance liquid chromatography/fluorescence and liquid chromatography/electrospray ionization tandem mass spectrometry techniques.

Tramadol is a centrally acting analgesic drug that has been used clinically for the last two decades to treat pain in humans. The clinical response of tramadol is strictly correlated to its metabolism, because of the different analgesic activity of its metabolites. O-Desmethyltramadol (M1), its major active metabolite, is 200 times more potent at the micro-receptor than the parent drug. In recent years tramadol has been widely introduced in veterinary medicine but its use has been questioned in some species. The aim of the present study was to develop a new sensible method to detect the whole metabolic profile of the drug in horses, through plasma analyses by high-performance liquid chromatography (HPLC) coupled with fluorimetric (FL) and photodiode array electrospray ionization mass spectrometric (PDA-ESI-MS) detection, after its sustained release by oral administration (5 mg/kg). In HPLC/FL experiments the comparison of the horse plasma chromatogram profile with that of a standard mixture suggested the identification of the major peaks as tramadol and its metabolites M1 and N,O-desmethyltramadol (M5). LC/PDA-ESI-MS/MS analysis confirmed the results obtained by HPLC/FL and also provided the identification of two more metabolites, N-desmethyltramadol (M2), and N,N-didesmethyltramadol (M3). Another metabolite, M6, was also detected and identified. The present findings demonstrate the usefulness and the advantage of LC/ESI-MS/MS techniques in a search for tramadol metabolites in horse plasma samples.