High-performance liquid chromatographic assay for the simultaneous determination of tramadol and its metabolites in microsomal fractions of human liver

A high-performance liquid chromatographic assay for the quantitative determination of the opioid analgesic tramadol and its metabolites is described. A homologue of tramadol [1-(m-hydroxyphenyl)-2-(N-ethyl-N-methylaminomethyl)cycloheptane-1-ol hydrochloride] is used as internal standard. The assay allows the determination of tramadol O- and N-demethylation activity in vitro in microsomal fractions of human liver. Tramadol and its in vitro generated Phase I metabolites are extracted by a one-step extraction procedure from microsomal incubation mixtures using methylene chloride. Extraction efficiencies of tramadol, O-demethyltramadol and mono-N-demethyltramadol were 70, 91 and 94% respectively. The isocratic high-performance liquid chromatographic system employs a C-18 reversed-phase column. The mobile phase is a mixture of methanol, ammonium hydrogencarbonate solution and ammonium hydroxide solution. Sensitivity of the assay was 0.5, 0.2 and 0.2 mu g/ml for tramadol, O-demethyltramadol and mono-N-demethyltramadol, respectively. Within-run precision of the overall assay was 13, 3.1 and 7.6% for tramadol, O-demethyltramadol and mono-N-demethyltramadol, respectively. Accuracy of the assay was determined as mean differences of concentrations added and found in microsomal fractions. It was -2.4% for tramadol, -0.85% for O-demethyltramadol and 0.32% for mono-N-demethyltramadol.