High-performance liquid chromatographic analysis of trimethoprim and sulfamethoxazole in microliter volumes of chinchilla middle ear effusion and serum.

A reversed-phase high-performance liquid chromatographic procedure was developed to analyze 25-microliters volumes of chinchilla middle ear effusion and 50-microliters volumes of serum for trimethoprim and sulfamethoxazole. The small sample volumes were dictated by the chinchilla model of otitis media and our need to collect multiple samples over a 12-h drug dosing interval. The drugs were separated on a cyanopropylsilane column using acetonitrile-40 mM sodium phosphate, (16:84, v/v), pH 4.8. Trimethoprim and the internal standard were detected at 230 nm while sulfamethoxazole was detected at 250 nm. Middle ear effusion and serum samples were extracted with ethyl acetate-dichloromethane (25:75, v/v). The limit of quantitation was 0.5 micrograms/ml for sulfamethoxazole and 0.1 micrograms/ml for trimethoprim (coefficient of variation less than 20%), the limit of detection 0.25 and 0.05 micrograms/ml, respectively. Middle ear and serum samples of chinchilla with experimentally induced otitis media receiving 10 mg/kg trimethoprim and 50 mg/kg sulfamethoxazole intramuscularly were collected over a 12-h period and analyzed. All statistics that validate the analytic method are reported.