Development and validation of three methods: liquid chromatography, flow injection analysis and UV spectrophotometry for the routine control of nystatin capsules

Abstract

Objectives As an alternative to amphotericin B used for selective digestive decontamination, physicians asked the hospital pharmacy for the preparation of nystatin capsules 500 000 IU. Methods Three methods were considered for the routine control: liquid chromatography (LC), flow injection analysis (FIA) and ultraviolet (UV) spectrophotometry. An isocratic LC method with UV detection was developed. A Hypersil BDS C18 column (150 mm×4.6 mm, 5 mm) thermostated at 30°C was used as the stationary phase. The mobile phase was a mixture of 0.05 M ammonium acetate buffer pH 6.0 methanol (35:65, v/v). Flow rate was 1.0 mL/min and run time was 25 min. Detection was operated at 305 nm and spectra were registered between 190 and 400 nm. 10 mL were injected. FIA used LC equipment in which the stationary phase was replaced by a PEEK capillary (164 cm, 0.13 mm internal diameter) with water flow (1.0 mL/min; 25°C). 10 mL were injected. UV spectrophotometry used a double beam spectrophotometer set at 305 nm.Solutions of nystatin (reference and samples) were prepared in methanol for FIA and LC (0.36 mg/mL). For UV spectrophotometry, a subsequent dilution (1:50, v/v) with ammonium acetate buffer/methanol (50:50, v/v) was needed. Results The three methods were validated according to ICH guidelines. In the case of LC, ruggedness of the method was tested. Three batches of hospital preparations were analysed by each method. Conclusions For routine control, UV spectrophotometry or FIA would be the methods of choice (rapid, easy). The LC method could be used to perform stability studies.