Development of liquid chromatographic method for the analysis of kanamycin residues in varicella vaccine using phenylisocyanate as a derivatization reagent

A liquid chromatographic method for the determination of the aminoglycoside kanamycin in varicella vaccine is described. Kanamycin sulfate was derived with phenylisocyanate (PIC) and triethylamine for 10 min at 70 degreesC and chromatographed on a alkylamide-bonded column, Suplex pKb-100. A derivative of kanamycin sulfate was attached to four phenylisocynato groups and that molecular mass was confirmed with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). The kanamycin-PIC derivative was found to have a retention time of 11.7 min using an eluent composed of 40% acetonitrile in water at 1.2 ml/min column flow-rate. Detection was at a wavelength of 240 nm. Recoveries ranging from 97.5 to 99.8% were found. The correlation coefficient was greater than 0.9998 over the range between 10 and 100 mug/ml. The method precision of within-day assay showed a 0.5 to 4.0% coefficient of variation (n = 5) ranging from 10 to 70 mug/ml of kanamycin concentration levels. Kanamycin-PIC derivative in reaction solution was stable for 24 h at room temperature. A simple and efficient method for the analysis of the kanamycin in varicella vaccine was developed and validated. (C) 2001 Elsevier Science B.V. All rights reserved.