Development and Validation of a Stability- Indicating High Pressure Liquid Chromatography Method for Determination of Prostaglandin E1 and its Degradation Products in sn Intracavernous Formulation

Abstract

A new intracavernous formulation to overcome the failure of standard treatments of erectile dysfunction after radical prostatectomy was developed. This formulation contains prostaglandin E1 (pGE1) at 15 µg/ml in association with papaverin and urapidil at 15 mg/ml and 2.5 mg/ml respectively. Since pGE1 was the main compound to be subject to physico-chemical degradation, its degradation and the concomitant apparition of its degradation product prostaglandin A1 (pGA1) are correlated to the stability of the formulation. The low specific absorbance of PGE1 in the ultraviolet region, and the presence of high levels of papaverin and urapidil in the formulation were the principal difficulties to develop a new method for simultaneous determination of pGE1 and pGA1. Many compositions and pH of mobile phase were studied to find the best chromatographic conditions, and the chosen method is a sensitive, precise and accurate ramp reversed-phase high-performance liquid chromatographic (RP-HPLC) assay method. Ramp RP-HPLC separation was achieved on a Kromasil 5 C18 column (250×4.6 mm id, 5 µm particle size) using mobile phase composed of acetonitrile-pH 3 phosphate buffer (37 : 63%, v/v). The detection was performed at 205 and 230 nm for PGE1 and pGA1 respectively, using a multiwave UV detector. The method was validated for specificity, linearity, precision, accuracy, robustness and sensibility. Limits of quantitation were about 3 µg/ml for pGE1 and 0.5 µg/ml for pGA1. The presence of urapidil and papaverin at high concentration did not interfere with determination of pGE1 and pGA1 in the formulation. The method developed which separates all the most degradation products formed under variety of conditions was sensitive, selective, linear, precise and accurate, and thus can be used to study the stability of the formulation.