EVALUATION OF THE METABOLIC PROFILE OF 2-METHIOPROPAMINE BY LC-MS/MS IN MICE AND IN HUMAN LIVER MICROSOMES

Introduction The aim of the present study was to investigate the phase I and phase II metabolism of 2- methiopropamine, a structural analogue of methamphetamine [1-3], after acute administration in mice, in order to select the most appropriate marker(s) of intake, also defining the excretion windows in urine. In vitro metabolism studies were carried out in order to enzymatically synthetize the metabolites of MPA and to set up the most appropriate sample pre-treatment procedures for LC-QqQ analysis. Methods A dose of 10 mg/kg was selected for the in vivo metabolism studies of MPA and urine samples were collected every 3 hours in the range of 0-9 hours after the injection. The In vitro studies were carried out with HLM (human liver microsomes). Samples from both studies were analysed, after extraction with tertbutyl-methyl ether at pH 9, using an Agilent 1200 HPLC equipped with a SUPELCO C18 column coupled to an API4000 triple quadrupole (Sciex) whit ESI source, operating in positive-ion mode. Results To reproduce the in vivo metabolism, the in vitro metabolism protocol was optimized considering different conditions: substrate concentration 20 µM, proteins concentration 0.5 mg/mL, phosphate buffer 0.1 M at pH 7.4, and an incubation time of 4 h at 37°C. Three main metabolites were identified: Nor-MPA, HydroxyMPA, and Nor-hydroxy-MPA. To optimize sample pre-treatment different extraction solvents (tertbutylmethyl ether, diethyl ether and ethylacetate) and pH values (7, 9 and 12) were evaluated. The best recoveries (higher than 70%) for the principal metabolites were obtained at pH 9. In vivo metabolism studies shown the formation of three principal phase I metabolites identified as: Nor-MPA, Hydroxy-MPA, and Oxo-MPA. 2-MPA and its metabolites show a maximum of excretion in the first 3 h from administration to mice showing an increasing conversion into phase II metabolites both glucorono and sulfo-conjugates for MPA, Nor-MPA, Hydroxy-MPA; Nor-Hydroxy-MPA was detected only as a phase II metabolite. Conclusions The excretion profile of 2-MPA after acute administration was evaluated identifying its principal metabolites and their excretion windows in mice. MPA and Nor-MPA are the best markers of intake in the first 0-24 hours after the administration of MPA. The present method was qualitative validated for MPA and Nor-MPA according to WADA technical document[4-5] defining: recovery, LOD, selectivity and repeatability. Novel Aspect There are few studies about MPA metabolism [3]. This is the first study identifying MPA excretion profile including phase I and phase II, also selecting the best markers of intake.