Simultaneous Determination of Fipronil, Permethrin and their Key Related Substances in a Topical Drug Product by a Single Stability Indicating High Performance Liquid Chromatography Method.

Abstract

BACKGROUND The topical veterinary drug product containing fipronil and permethrin provides an effective repellent protection and high insecticidal efficacy for dogs. OBJECTIVE The objective of this study was to develop a stability indicating high performance liquid chromatography (HPLC) method for simultaneous detection and quantification of fipronil, permethrin, their key degradation products and butylated hydroxytoluene (BHT) in a topical drug product. METHOD The two active ingredients, their degradation products, and the antioxidant (BHT) were separated by a gradient elution on a Phenomenex Kinetex C18 column (150 × 3 mm, 2.6 µm particle size) maintained at 37 °C with H2O/acetonitrile/isopropyl alcohol/85% H3PO4 (65.5 + 32.5 + 4/0.0053, v/v/v/v) as mobile phase-A and acetonitrile (100%) as mobile phase-B. The flow rate was 0.9 mL/min and analytes were detected and quantified at 235 nm. RESULTS Specificity of the method was demonstrated by adequate separation of fipronil, permethrin, their degradation products, and BHT in the forced degraded finished product. Linearity of the method was demonstrated in the range of 0.2% to 150% of target analytical concentration of both active ingredients and 50% to 150% for BHT. Excellent recoveries of fipronil, permethrin and BHT in placebo spiked active ingredient solutions in the linearity range showed sufficient accuracy of the method. LOQ and LOD of the method were determined to be 0.2% and 0.07% of the analytical concentration. A robustness study did not identify any critical parameter that adversely effected the separation and quantification. CONCLUSIONS Here, we report the development and validation of a robust, stability indicating HPLC method for identification and assay of fipronil, permethrin, and BHT, including estimation of fipronil's and permethrin's degradation products in a topical drug product for dogs. HIGHLIGHTS The new HPLC method permits the acquisition of data for all analytes of interest for a topical finished drug product containing fipronil, permethrin, and BHT.