Sensitive liquid chromatography assay for the determination of amikacin in human plasma

A selective LC method with on-line post-column derivatization is described for the determination of amikacin in biological fluids. Chromatography was performed on a reversed-phase column, using pentane sulphonic acid as an ion-pairing reagent. For the analysis of biological fluids, amikacin and the internal standard tobramycin were extracted using an ion exchanger (Sephadex). Following complete removal of plasma proteins, the aminoglycosides were eluted with alkaline sodium sulphate solution and injected into the chromatograph. After chromatographic separation the eluent was mixed with the derivatization reagent (o-phthalaldehyde and mercaptoethanol in borate buffer pH 10.4) in a reaction coil at 50 degrees C. Detection was performed by fluorescence (excitation: 340 nm, emission: 418 nm). The overall run time was 8 min, at a flow rate of 1.2 ml min-1. The limit of quantification was 25 ng ml-1 for amikacin in plasma.