A reversed phase liquid chromatographic method for the simultaneous determination of several common penicillins in human serum.

A rapid and sensitive high performance liquid chromatographic method is described for the simultaneous determination of benzylpenicillin, ampicillin, phenoxymethylpenicillin, cloxacillin, dicloxacillin and nafcillin in small samples of human serum. The chromatographic system involves the use of a Spherisorb ODS reversed phase column and a gradient elution with 1 mM ammonium acetate buffer/acetonitrile (from 90:10 to 75:25 in 15 min). Detection and quantification are monitored by UV absorption at 208 nm. The compounds are extracted with dichloromethane, using tetrabutylammonium hydrogen sulfate neutralized with sodium hydroxide and buffered with borate as an ion pairing reagent; beta-hydroxyethyltheophylline is added as an internal standard. Our results show that the method is accurate and reproducible, allowing quantification of serum levels of assayed penicillins (0.5-50 micrograms/mL) without interference from other drugs commonly used in therapy. Recoveries were generally greater than 79.4%.