Simultaneous determination of praziquantel, fipronil, eprinomectin, (S)-methoprene, their key related substances and butylated hydroxytoluene (BHT) in a topical veterinary drug product by a single stability indicating high performance liquid chromatography method

A simple, robust and QC (quality control) friendly high performance liquid chromatography (HPLC) method was developed and validated for simultaneous determination of four active pharmaceutical ingredient [namely fipronil, (S)-methoprene, eprinomectin, and praziquantel] and their key degradation products in a broadspectrum topical finished product. Typical sample of the finished product contains a total of 30-plus peaks of interest. Analytes were separated on a HALO C18 column (150 mm x 4.6 mm i.d., 2.7 mu m particle size) with a gradient elution at 50 degrees C column temperature and 0.6 mL/min flow rate. Detection wavelength of 245 nm is used for praziquantel, eprinomectin and their degradation products, 265 nm for (S)-methoprene and its degradation products and 280 nm for fipronil and its degradation products and for the antioxidant, BHT. Mobile phase of the method is composed of 10 mM potassium phosphate buffer and 1,4-Dioxane (98/2, v/v, pH 5.0) as mobile phase-A, and EtOH/MeOH/MeCN/IPA (86/4/6/4, v/v/v/v) as mobile phase-B. All analytes of interest were adequately separated by this single HPLC method. The stability-indicating capability of the method has been demonstrated by successfully separating the degradation products in the stressed degraded samples of the finished product. Limit of quantitation (LOQ) and limit of detection (LOD) of the method is 0.3% and 0.1% of target analytical concentration for each individual API in the finished product. This method has been demonstrated to be sensitive, robust, specific, accurate and stability-indicating for analysis of the topical drug product containing praziquantel, fipronil, eprinomectin and (S)-methoprene.

Automated summary

A simple and robust HPLC method was developed for simultaneous determination of four active pharmaceutical ingredients and their degradation products. The method successfully separated the peaks of interest and demonstrated stability-indicating capability by separating the degradation products in stressed samples.