Determination of metoprolol and its alpha-hydroxide metabolite in serum by reversed-phase high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method for the simultaneous quantitation of metoprolol and alpha-hydroxymetoprolol concentrations in serum is developed. Analysis is performed on a C18 column using a 70:30 mixture of two solutions, A and B respectively. Solution A is 1 L of a 1% acetic acid solution with 25 mL of 0.005 M 1-heptanesulfonic acid. Solution B is 1 L of acetonitrile with 25 mL of 0.005 M 1-heptanesulfonic acid. Column effluent is monitored by fluorescence detection at an excitation wavelength of 225 nm. A 320-nm band pass filter is employed. The limit of sensitivity is approximately 2 ng/mL for both compounds. No potential sources of interference are identified. A coefficient of variation of less than 10% is observed on replicate determinations at 10 ng/mL for both compounds and at 300 and 180 ng/mL for metoprolol and alpha-hydroxymetoprolol, respectively. The method has the advantages of complete resolution of the metabolite of metoprolol, a high degree of reproducibility, adequate sensitivity, and an easily accessible internal standard.