Analysis of kanamycin A in human plasma and in oral dosage form by derivatization with 1-naphthyl isothiocyanate and high-performance liquid chromatography

A simple and sensitive HPLC method has been developed for trace determination of kanamycin A by derivatization. Plasma proteins are precipitated by acetonitrile and chemical derivatization is performed on the supernatant containing kanamycin A with 1-naphthyl isothiocyanate in pyridine at 70 degrees C. After the derivatization reaction, a methylamine/acetonitrile solution was added to the reaction mixture to eliminate the excess of derivatizing agent and shorten the analysis time. The resulting derivative was separated using a Lichrocart Purospher STAR RP-18e column and water/methanol (33:67, v/v) as a mobile phase (detection at 230 nm). Optimization conditions for the derivatization of kanamycin A were investigated by HPLC. The linear range for the quantitation of kanamycin A in spiked plasma was over 1.2-40 microg/mL; the detection limit (signal to noise ratio = 3; injection volume, 10 microL) was about 0.3 microg/mL. The relative standard deviation was less than 2.9% for intra-day assay (n = 6) and inter-day assay (n = 6) and relative recoveries were found to be greater than 98%. Preliminary application of the method for monitoring kanamycin A in humans upon intramuscular injection of the injection product demonstrated the usefulness of the assay for clinical studies. The proposed method can also be used to analyze the compound in pharmaceutical formulations.