Determination of penicillin-V in human plasma by high-performance liquid chromatography and solid-phase extraction

A high-performance liquid chromatographic method has been developed for the determination of penicillin-V concentrations between 0.1 and 19 μg/ml in human plasma. Penicillin-V was isolated from plasma by solid-phase extraction on a C₁₈/OH cartridge. The extracts were injected onto a reversed-phase HPLC system. A 125×4 mm C₁₈ column was used to separate penicillin-V from its main metabolites, 5R- and 5S-penicilloic acid and endogenous compounds. The eluent consisted of 66% 0.02 M phosphoric acid buffer, to which tetrabutylammonium dihydrogenphosphate and 34% acetonitrile were added. The column effluent was monitored by ultraviolet spectrophotometry at 269 nm. Using this method, penicillin-V concentrations in plasma could be determined with an accuracy between −5.4 and 5.2% and a precision between 0.8 and 1.6%. The method has proved to be reliable and was used in biovailability studies for the development of a new oral penicillin-V formulation.