Development and validation of a rapid HPLC method for simultaneous determination of tramadol, and its two main metabolites in human plasma

Tramadol, an analgesic agent, and its two main metabolites O-desmethyltramadol (M1) and N-desmethyltramadol (M2) were determined simultaneously in human plasma by a rapid and specific HPLC method. The sample preparation was a simple extraction with ethyl acetate. Chromatographic separation was achieved with a ChromolithTM Performance RP-18e 50 mm × 4.6 mm column, using a mixture of methanol:water (13:87, v/v) adjusted to pH 2.5 by phosphoric acid, in an isocratic mode at flow rate of 2 ml/min. Fluorescence detection (λex = 200 nm/λem = 301 nm) was used. The calibration curves were linear (r2 > 0.997) in the concentration range of 2.5–500 ng/ml, 1.25–500 ng/ml and 5–500 ng/ml for tramadol, M1 and M2, respectively. The lower limit of quantification was 2.5 ng/ml for tramadol, 1.25 ng/ml for M1 and 5 ng/ml for M2. The within- and between-day precisions in the measurement of QC samples at four tested concentrations were in the range of 2.5–9.7%, 2.5–9.9% and 5.9–11.3% for tramadol, M1 and M2, respectively. The developed procedure was applied to assess the pharmacokinetics of tramadol and its two main metabolites following administration of 100 mg single oral dose of tramadol to healthy volunteers.