HPLC Quantitation of Levobunolol and Its Metabolite, Dihydrolevobunolol in Biological Fluids

In order to quantitate the concentrations of levobunolol and its major metabolite (dihydrolevobunolol) in aqueous humor and in blood after ophthalmic doses, HPLC procedures were developed. The direct injection procedure, after plasma protein precipitation and reduction of levobunolol to dihydrolevobunolol, was found to be suitable for clinical samples. A second procedure, involving extraction with ethyl ether, employed a uv/fluorescence dual detection system to simultaneously monitor levobunolol and dihydrolevobunolol. The sensitivity for levobunolol was 5 ng, and for dihydrolevobunolol, 1 ng. There was no observable interference from the biological fluids, blood and aqueous humor. Both procedures offer high reproducibility, selectivity, and sensitivity, which are essential for pharmacokinetic and metabolism studies.