Liquid-chromatographic determination of tobramycin in serum with spectrophotometric detection

We describe a simple, precise, accurate, and specific liquid-chromatographic procedure for determination of tobramycin in 50 microL of serum. Tobramycin and the internal standard (sisomicin) are quantitatively converted into their trinitrophenyl derivatives by reaction with a water-soluble derivatizing agent (2,4,6-trinitrobenzenesulfonic acid) at 70 degrees C for 30 min. The derivatives are extracted from the crude reaction mixture by using a reversed-phase Bond-Elut C18 column, and separated on a reversed-phase octyl column with a mobile phase consisting of an acetonitrile/phosphate buffer (70/30 by vol) at a flow rate of 3.0 mL/min. The eluted compounds are detected at 340 nm, and quantified from their peak areas. Chromatography is complete in less than 4.5 min at the optimum column temperature of 50 degrees C. The lower limit of detection for tobramycin is less than 0.2 mg/L. Analytical recoveries for tobramycin varied from 94 to 99%, linearity extended to 25 mg/L, and day-to-day precision (CV) was between 4.6 and 5.1%. Numerous drugs and antibiotics tested do not interfere. Results correlate well (r greater than 0.95) with those by radioimmunoassay and EMIT.