Determination of Penicillinase-Resistant Penicillins In Serum Using High-Pressure Liquid Chromatography

Abstract

A method for the determination of methicillin, oxacillin, cloxacillin, dicloxacillin, and nafcillin in serum using high-pressure liquid chromatography (HPLC) is described. The drugs were extracted from serum using a two-step procedure employing acetonitrile followed by methylene chloride. The extraction procedure concentrated the antibiotics in a smaller volume which allows more accurate determinations of low serum levels. The treated sera were analyzed by HPLC on a reverse-phase column and detected by ultraviolet light absorption at 254 nm. Serum concentrations were measurable as low as 0.5 μg/ml. Recovery procedures showed less than 2.5% variation in peak heights when the antibiotics were extracted from different pools of serum. No interfering absorption was found in extracts of serum samples pooled from healthy volunteers, from a commercial source, or from two serum pools from patients receiving a variety of other drugs. Two spiked serum specimens prepared for each antibiotic were assayed four times by HPLC and by the microbiological agar diffusion method. No significant statistical differences between the methods were observed. Control materials were assayed for between-batch and within-batch reproducibility in the presence or absence of an internal standard. Results for between-batch reproducibility demonstrate CV's of about 5%. This procedure provides a sensitive, specific, accurate, and rapid method for determining antibiotic levels in routine clinical specimens.