title: A highly sensitive HPLC method with automated on-line sample pre-treatment and fluorescence detection for determination of reboxetine in human plasma.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia. nasrykhalil@hotmail.com
authors.forename: Nasr Y
authors.initials: NY
authors.lastname: Khalil
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000737:chemistry; D053000:Analytic Sample Preparation Methods; D001331:Automation; D001774:Blood Chemical Analysis / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D016014:Linear Models; D009025:Morpholines / Q000097:blood* / Q000737:chemistry; D009862:Online Systems; D000077593:Reboxetine; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence
chemical_list: D009025:Morpholines; C032930:7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole; D000077593:Reboxetine; D009327:4-Chloro-7-nitrobenzofurazan