A Simple and Sensitive HPLC Method for Fluorescence Quantitation of Doxorubicin in Micro-volume Plasma: Applications to Pharmacokinetic Studies in Rats

A validated HPLC method was developed to determine the doxorubicin concentration in a small volume of rat plasma (60 µL) with convenient fluorescence detection. Sample preparation includes a simple one-step liquid-liquid extraction using a minimum amount of organic solvent, with extraction recovery more than 95%. The analysis was accomplished using PerfectSil C18 column maintained at 35 °C and a mobile phase consisted of acetonitrile and water (32:68, v/v; pH=2.6). The flow-rate was kept at 1 mL/min and the column effluent was monitored with a fluorescence detector at an excitation and emission wavelength of 470 and 555 nm, respectively. The detection limit was 5 ng/mL. No analytical interference was observed from endogenous components in the rat plasma. This method was feasibly applied to the pharmacokinetic study of 5 mg/Kg of doxorubicin after the intravenous administration to rats.