A highly sensitive HPLC method with automated on-line sample pre-treatment and fluorescence detection for...

A fully automated, rapid and highly sensitive HPLC method with automated sample pre-treatment by column-switching system and fluorescence detection has been developed for the trace quantitative determination of the new antidepressant reboxetine (RBX) in human plasma. A simple pre-column derivatization procedure with 7-flouro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) reagent was employed. Paroxetine (PXT) was used as an internal standard. Plasma samples containing both RBX and PXT, after filtration, were derivatized by heating with NBD-F in borate buffer of pH 8 at 70 degrees C for 30min. The derivatized plasma samples were injected into the HPLC system where an on-line sample clean up was achieved on the pre-treatment column (Co-sense Shim-pack MAYI-ODS) with a washing mobile phase (acetonitrile:2% acetic acid; 40:60, v/v) at a flow rate of 5mLmin(-1) for 1min. After an automated on-line column switching to the analytical Hypersil phenyl 120A column (250mmx4.6mm, 5microm), the separation of the derivatized RBX and PXT was performed using a mobile phase consisting of sodium acetate buffer (pH 3.5):tetrahydrofuran:acetonitrile (55:35:10, v/v/v) at a flow rate of 2.0mLmin(-1). The eluted derivatives were monitored by a fluorescence detector set at an excitation wavelength of 470nm and an emission wavelength of 530nm. Under the optimum chromatographic conditions, a linear relationship with good correlation coefficient (r=0.9995, n=5) was found between the peak area ratio of RBX to PXT and RBX concentration in the range of 2-500ngmL(-1), with limits of detection and quantification of 0.5 and 1.7ngmL(-1), respectively. The intra- and inter-day precisions were satisfactory; the relative standard deviations were 2.25 and 3.01% for the intra- and inter-day precisions, respectively. The accuracy of the method proved as the mean recovery values were 100.11+/-2.24% and 100.99+/-2.98% for the intra- and inter-day assay runs, respectively. The proposed method involved simple and minimum sample preparation procedure and short run-time (<12min) and therefore it can be applied to the routine therapeutic monitoring and pharmacokinetic studies of RBX.