Determination of metoprolol and its two metabolites in human plasma and urine by high performance liquid chromatography with fluorescence detection and its application in pharmacokinetics.

A simple, specific and sensitive HPLC method has been developed for the determination of metoprolol and its two metabolites in human plasma and urine. Separation of metoprolol, α-hydroxymetoprolol, O-desmethylmetoprolol and esmolol (internal standard) was achieved on an Agilent XDB-C18 column (150 mm × 4.6 mm, 5 µm) using fluorescence detection with Ex 216 nm and Em 312 nm. The mobile phase consisted of ACN–H2O-0.1%TFA. The analysis was performed in less than 16 min with a flow rate of 0.8 mL/min. The assay was linear over the concentration range of 5-600 ng/mL and 2.5-300 ng/mL for metoprolol and its metabolites, respectively. The LOQ were 5.0 and 2.5 ng/mL for plasma and urine, respectively. Good precision and accuracy for metoprolol and its two metabolites were obtained. The extraction recoveries were found to be more than 86.91% both in plasma and urine. At the same time, the method was successfully applied to nine healthy volunteers who had been given an oral tablet of 100 mg metoprolol.