HPLC determination of clofazimine in tissues and serum of mice after intravenous administration of nanocrystalline or liposomal formulations.

A simple HPLC method is described for the determination of clofazimine in mouse tissues and in serum. The main application of the method was the determination of the drug in mouse tissues after i.v. administration of nanocrystalline suspensions or liposomal encapsulated clofazimine. Tissues were extracted with a 10-fold (w/v) volume of an extraction solution consisting of methanol/glacial acetic acid 9:1 (v/v). Serum proteins were precipitated with a 2-fold volume of acetonitrile. Isocratic chromatography was performed using an anion exchange column (Nucleosil 100-5 SA, Macherey & Nagel) for separation. The mobile phase was a mixture of acetonitrile and 0.1 mol/l aqueous phosphoric acid (75:25, v/v), adjusted to pH 2.9 with sodium hydroxide solution. Absorption of the eluate was monitored at 495 nm. The assay was precise, simple to perform and fast. Recovery from tissues was > or = 98%, from nanoparticles > or = 98%, and from liposomes > or = 96%. No interference was observed in extracts from mouse liver, spleen, lungs and human serum.