Determination of benzylpenicillin and probenecid in human body fluids by high-performance liquid chromatography

A method for the determination of benzylpenicillin and probenecid concentrations in human body fluids using ion-pair reversed-phase chromatography with UV detection has been developed. For plasma samples two extraction techniques were investigated. Precipitation of the plasma proteins with acetonitrile followed by liquid-liquid extraction offered the best results. The limits of detection were 0.5 microgram/ml for benzylpenicillin and 0.25 microgram/ml for probenecid, which offer sufficient sensitivity for application in pharmacokinetic experiments.