A rapid method for the determination of benzylpenicillin in serum by reversed-phase HPLC

A simple procedure for the determination of benzylpenicillin in serum is described. The assay involves the extraction of the drug and the internal standard (phenoxymethylpenicillinic acid) from the sample into dichloromethane, using tetrabutylammonium hydrogen sulfate neutralized with NaOH and buffered with citrate as an ion-pairing reagent. RP-HPLC was performed on a Spherisorb 5 ODS column, eluting the drugs isocratically with 14% acetonitrile in 10 mM ammonium acetate buffer. Monitoring was by UV detection at 208 nm. Our results show that the method is accurate and reproducible, permitting quantification of serum levels of benzylpenicillin without interference from other drugs commonly used in therapy. Analytical recovery was greater than 79.2%.