Background 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. Objective 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. Method 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. Results 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). Conclusions 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. Highlights 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.

中文翻译：

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开发用于测定氟虫腈的反相 UPLC 方法，包括测定散装批次的氟虫腈原料药中的相关物质

背景 氟虫腈是农业和动物保健行业中常用的农药，用于保护农作物和控制跳蚤、蜱虫和口虱。现有的欧洲药典（EP）专着很难对氟虫腈的常见水解降解产物获得可重复的保留时间和RRT，用于测定和估计氟虫腈的相关物质。这种情况会导致批量放行期间散装商业批次中和/或放行批次保质期内稳定性样品中氟虫腈水解降解产物的错误识别、贴错标签和/或错误的不合格结果。目的 本研究旨在开发一种用于测定和鉴定氟虫腈的反相 UPLC 方法，包括对氟虫腈原料药批次中的有关物质进行鉴定和评估，并提供一致的水解降解产物保留时间。方法 在保持在 40 °C 的 Halo C18 柱（50 mm × 2.1 mm 内径，2.0 µm 粒径）上，以 0.1% H3PO4 的水溶液为流动相 A，乙腈-甲醇，通过梯度洗脱分离氟虫腈及其有关物质(50 + 50, v/v) 作为流动相-B。在 280 nm 处检测并定量氟虫腈及其相关物质，定量限为目标（分析）浓度的 0.05%。结果 UPLC 方法能够通过梯度洗脱分离所有感兴趣的分析物，总运行时间为 7 分钟（比 EP 快约 40%）。结论 在本文中，我们报告了一种快速、稳定性指示反相 UPLC 方法的开发和验证，该方法用于测定和估计氟虫腈稳定性样品和散装批次中氟虫腈的相关物质。亮点 新的 UPLC 方法比当前用于 Fipronil 测定的 EP 专着快约 40%，并且新方法提供了 Fipronil 常见水解降解产物的可重复保留。