Development of a Reversed-Phase UPLC Method for Assay of Fipronil Including Determination of Its Related Substances in Bulk Batches of Fipronil Drug Substance.

Abstract

Fipronil is a commonly used pesticide in the agricultural and animal health industries for the protection of crops and control of fleas, ticks, and chewing lice. It is difficult to obtain reproducible retention time and RRT for a common hydrolytic degradation product of Fipronil with the current European Pharmacopeia (EP) monograph for assay and estimation of related substances of Fipronil. This situation causes misidentification, mislabeling and/or false out-of-specifications results for this hydrolytic degradation product of Fipronil in bulk commercial batches during batch release and/or in the stability samples during the shelf life of a released batch. This study aimed to develop a reversed-phase UPLC method for assay and identification of Fipronil including identification and estimation of its related substances in bulk drug substance batches of Fipronil and provide consistent retention time of the hydrolytic degradation product. Fipronil and its related substances were separated by a gradient elution on a Halo C18 column (50 mm × 2.1 mm i.d., 2.0 µm particle size) maintained at 40 °C with 0.1% H3PO4 in H2O as mobile phase-A and acetonitrile-methanol (50 + 50, v/v) as mobile phase-B. Fipronil and its related substances were detected and quantified at 280 nm with a quantitation limit of 0.05% of the target (analytical) concentration. The UPLC method was able to separate all analytes of interest by gradient elution with a 7 minute total run time (∼40% faster than EP). In this paper, we report the development and validation of a fast, stability indicating reversed phase UPLC method for assay and estimation of related substances of Fipronil in stability samples and bulk batches of Fipronil. The new UPLC method is ∼40% faster than the current EP monograph for Fipronil assay and the new method provides reproducible retention of a common hydrolytic degradation product of Fipronil.