Development of New High Effective Method of Tetraene Macrolide Antibiotic Nystatin Components Separation with Application of HPLC

Valery V. Belakhov, Schulich Faculty of Chemistry, Technion - Israel Institute of Technology, Haifa, Israel
Alexander V. Garabadzhiu, Laboratory Of Molecular Pharmacology, Saint-petersburg State Technological Institute (technical University), Saint-petersburg, Russia

Nystatin was the first polyene macrolide antibiotic discovered and introduced into medical practice for the treatment of many clinical forms of candidiasis caused by the Candida yeast fungi. Commercial tetraene macrolide antibiotic nystatin has been shown to be complex mixture of closely related compounds, it has been separated into three constituents (designated nystatins A₁, A₂, A₃) and heptaene component. Examination of a number of nystatin samples from different sources revealed that there a great variation in the composition of pharmaceutical grade material. It was established that pharmaceutical grade nystatin is expected to consist largely of nystatin A₁. In this view, during the examination of samples of nystatin obtained from various sources it was very important to compare the composition of this tetraene macrolide antibiotic.

In this report we present three different efficient analytical HPLC methods for the separations of components of nystatin, which are all extended to the preparative scale. It was found that although all three presented HPLC methods are rapid and efficient for an analytical separation of nystatin, only the one using a methanol-water-dimethylformamide mixture with neutral pH value is applicable to the preparative purification of nystatin, since it is the only system which does not affect the antifungal activity of the separated components. The using of developed method allows the separation of nystatin complex into five fractions, of which the main component nystatin A₁ can be obtained with excellent purity and high antifungal activity.