Analysis of primary metabolites of tramadol in rat urine by LC-MSⁿ

Tramadol and its metabolites in rat urine were identified by LC-MSⁿ.  Rat urine samples of 0−36 h were collected after ip 9.0 mg·kg⁻¹ tramadol, then the samples were enriched and purified through solid- phase extraction cartridge.  Purified samples were analyzed by LC-MSⁿ.  Possible metabolites were discovered by comparing the full scan and SIM chromatograms of the test samples with the corresponding blanks and analyzing the retention time, quasi-molecular ion and fragment ion of all chromatograms.  Nine phase I metabolites and four phase II metabolites were identified in rat urine.  One of the metabolites was found first time in living body.  The metabolites were formed via the following metabolic pathways: O-demethylation, N-demethylation, hydroxylation, N-oxidation and conjugation.  The method can be used to identify tramadol and its metabolites in other animals and human.