Efficient high-performance liquid chromatographic assay for the simultaneous determination of metoprolol and two main metabolites in human urine by solid-phase extraction and fluorescence detection

Abstract

An improved, more efficient method for the determination of metoprolol and its two metabolites in human urine is reported. The simultaneous analysis of the zwitterionic metoprolol acidic metabolite ( III, H117/04) with the basic metabolites α-hydroxymetoprolol ( II, H119/66), metoprolol ( I) and guanoxan ( IV, internal standard) was achieved employing solid-phase extraction and isocratic reversed-phase HPLC. The analytes were extracted from urine (100 μl) using C 18 solid-phase extraction cartridges (100 mg), and eluted with aqueous acetic acid (0.1%, v/v)–methanol mixture (40:60, v/v, 1.2 ml). The eluents were concentrated (250 μl) under vacuum, and aliquots (100 μl) were analysed by HPLC with fluorescence detection at 229 nm (excitation) and 309 nm (emission) using simple isocratic reversed-phase HPLC (Novapak C 18 radial compression cartridge, 4 μm, 100×5 mm I.D.). Acetonitrile–methanol–TEA/phosphate buffer pH 3.0 (9:1:90, v/v) was employed as the eluent (1.4 ml/min). All components were fully resolved within 18 min, and the calibration curves for the individual analytes were linear ( r 2≥0.996) within the concentration range of 0.25–40.0 mg/ml. Recoveries for all four analytes were greater than 76% ( n=4). The assay method was validated with intra-day and inter-day variations less than 2.5%.