Micro-Scale Method for Determination of Tobramycin in Serum Using High-Performance Liquid Chromatography

A rapid, simple, accurate, and micro-scale method for the determination of tobramycin, sisomicin and netilmicin in serum using high-performance liquid chromatography has been developed. The method is sensitive to 0.3 pg/ml using only 20 μl of serum. The serum is deproteinized with methanol containing an internal standard: sisomicin for the tobramycin, netilmicin for the sisomicin, and sisomicin for the netilmicin. After centrifugation, a counter-ion reagent is added to the supernatant, then an aliquot of the solution is injected into the chromatograph. Tobramycin, sisomicin and netilmicin are separated by reversed-phase, ion-pair chromatography and detected by fluorescence using continuous-flow, post-column derivatization with o-phthalaldehyde. For the tobramycin, within-run and day-to-day variation was below 2.5%. Correlation of this method with microbiological assay and homogeneous enzyme immunoassay was good.