High-performance Liquid-chromatographic Assay of Aminoglycoside Antibiotics in Serum

We describe procedures for the high-performance liquid-chromatographic assay of gentamicin, amikacin, and tobramycin in serum. The aminoglycoside antibiotic is extracted from serum by using a CM-Sephadex column and is measured by reversed-phase, ion-pair chromatography. Continuous-flow, post-column derivatization with o-phthalaldehyde is used to form fluorescent products for detection. An internal standard is used in each assay. Synthesis of 1-N-acetylgentamicin C1, the internal standard for the gentamicin assay, is described. For each procedure, concentration and instrument response are linearly related in the range of therapeutically important concentrations. The methods are precise and results correlate well with results obtained by bioassay. The between-day CV (n = 10) for each procedure was less than 4%. No substances have been detected in clinical specimens that interfere with these chromatographic procedures. The possible use of these methods for analysis for other aminoglycoside antibiotics is discussed.