Chemical assay, involving liquid chromatography, for aminoglycoside antibiotics in serum

We describe a chemical assay involving "high-pressure" liquid chromatography for the quantitative determination of three aminoglycosides: netilmicin, tobramycin, and gentamicin. The drugs are separated from serum by means of precipitation of the serum proteins with acetonitrile after dilution with a buffer. The aminoglycosides are quantitatively extracted into the supernate, which is further purified by a two-step partition procedure involving derivatization of the drugs with o-phthalaldehyde. The drug derivatives are separated by reversed-phase chromatography and detected by on-line fluorometry. Sensitivity is 1 mg/L for tobramycin and 0.5 mg/L for netilmicin and gentamicin. Intra- and interassay variation was below 8%. Analytical recovery of each of the three drugs was 92 to 100%. Correlation with microbiological and radioimmunological assay methods was good. The assay is rapid (about 30 min), precise, and specific, and seems suitable for use in a routine clinical laboratory.