LC Determination of Tramadol, M1, M2, M4, and M5 in Plasma

A new LC procedure for the determination of tramadol, an analgesic, and its metabolites has been developed and validated. Following a liquid-liquid extraction using ethyl acetate and hexane, samples were separated by RP-LC on a Symmetry C-18 column and quantified using fluorescence detection at an excitation of 202 nm and an emission of 296 nm. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (0.01 M), 0.1% triethylamine (pH 2.9) and acetonitrile, with a flow-rate of 1.1 mL min(-1). The standard curve ranged from 5 to 5,000 ng mL(-1). Intra-and inter-assay variability for all the compounds were less that 10% and the average recovery was greater than 90%. This assay is suitable for use in pharmacokinetic studies.