Development and validation of a stability indicating HPLC-DAD method for the determination of the peptide stigmurin

Abstract

Stigmurin, a synthetic peptide identified in the scorpion Tityus stigmurus, has a high-potential therapeutic use, i. e. antimicrobial, antiparasitic and antiproliferative activities. The physicochemical properties of the peptide guided the HPLC/DAD method development in a traditional and rational approach named one factor at a time. From thequality risk assessment were defined responses and critical parameters of the chromatographic system. A 300-Å C18 column (100 mm × 2.1 mm, 3.5 μm particle size) was used to perform the separation with the temperature kept at 40°C and mobile phase consisting TFA 0,1% in water (58%) and TFA 0,1% in acetonitrile (42%) under isocratic mode. Chromatograms were recorded at the wavelength of 215 nm. The method showed to be linear from 0.8 to 1.2 mg mL−1. The method precision (repeatability and intermediate precision, estimated as the relative standard deviation, RSD (%)), was less than 2.0%. Accuracy, specificity, robustness, and system suitability were also evaluated as specified in the ICH guidelines. A forced degradation study was performed under acid, alkaline, oxidative, thermal and UV light conditions. Stigmurin was stable for 24h at oxidative medium and susceptible to hydrolysis in acid, alkaline, thermal and photodegradation conditions. In all samples, the spectral purity of stigmurin peak was satisfactory and no co-elution was observed.This work proposed a stability indicating method, capable of quantifying the stigmurin peptide, and to detect its degradation products. Finally, the method can be employed to evaluate stigmurin in stability studies performed under regular storage conditions.