Microassay for determination of itraconazole and hydroxyitraconazole in plasma and tissue biopsies

A simple, rapid and sensitive method for the extraction and HPLC analysis of itraconazole and hydroxyitraconazole in tissue and plasma or serum is described. Tissue (5-100 mg) and plasma (0.1 ml) underwent a simple extraction into methanol. Chromatography was performed on a Novapak C18 column using a mobile phase of water-acetonitrile-diethylamine (42:58:0.05, v/v), pH 2.45, with a flow-rate of 1.5 ml/min. Fluorescence was measured at excitation 260 nm and emission 365 nm. The procedure produced a linear curve for the concentration range 10-1000 ng/ml. The development of the assay produced accurate, rapid repeatable results for both tissue and plasma or serum.