Rapid determination of venlafaxine and O-desmethylvenlafaxine in human plasma by high-performance liquid chromatography with fluorimetric detection

Abstract

A rapid and sensitive high-performance liquid chromatographic technique for simultaneous measurement of plasma venlafaxine (VEN) and its active metabolite O-desmethylvenlafaxine (ODV) is described. The process begins with the extraction of VEN and ODV, with maprotiline (MAP) as internal standard, from human plasma into an intermediate organic mixture of hexane–isoamyl alcohol and finally into an aqueous solution of 0.05% phosphoric acid. Isocratic separation of VEN, ODV and MAP is carried out by utilizing a reversed-phase butyl-bonded column (C 4/E) with mobile phase consisting of acetonitrile and 40 m M phosphate buffer, pH 6.8 (50:50, v/v). Detection of VEN, ODV and MAP is done by mean of fluorimetry with excitation and emission wavelengths set at 276 and 598 nm, respectively. As low as 1.0 ng/ml VEN is detectable; while the limit of detection for ODV is 5 ng/ml. C.V. (%) of intra-day samples for both VEN and ODV are less than 10% at three concentrations tested (10.0, 50.0, 100.0 ng/ml). Similarly, over the same nominal concentrations, the precision of inter-day (5 days) samples also results in C.V. (%) smaller than 10% for both compounds, except for ODV measured at 10 ng/ml (C.V.<15%). Approximately, 100% VEN can be extracted from plasma; whereas, for ODV the recovery rate is nearly 70%. This present method is rapid, sensitive, accurate and simple for routine clinical monitoring of plasma VEN and its major metabolite ODV.