Development and validation of a rapid HPLC- fluorescence method for simultaneous determination of venlafaxine and its major metabolites in human plasma.

Abstract

To develop a simple, rapid and accurate HPLC method for the measurement of the venlafaxine and its main metabolites, O-desmethylvenlafaxine and O,N-didesmethylvenlafaxine in pharmacokinetic studies and therapeutic drug monitoring.Chromatographic separation was achieved with a ChromolithTM Performance RP-18e 100 mm×4.6 mm column equipped with a Fluorescence detectore (λ(ex) 200 nm/λ(em) 300 nm) The mobile phase of methanol:water (35:65, v/v) adjusted to pH 2.5 by phosphoric acid was passed through the column in an isocratic mode at flow rate of 2 ml/min. The sample preparation involved a simple, one-step, extraction with ethyl acetate.The calibration curves were linear in the concentration range of 1-300 ng/ml for all analytes (r2>0.998). The lower limit of quantification was 1 ng/ml for all analytes. Within and between day precisions in the measurement of quality control (QC) of samples were in the range of 1.8-14.1% for all analytes.The developed procedure was used to assess the pharmacokinetics of venlafaxine and its main metabolites following oral administration of 75 mg venlafaxine to a healthy subject.