The topical product with three active pharmaceutical ingredients (APIs), namely, esafoxolaner, eprinomectin and praziquantel has demonstrated its efficacy in the treatment of cats with mixed infections with ectoparasites and nematodes and cestodes. A reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed and validated for assay and determination of related substances peaks of three APIs including the assay of antioxidant butylated hydroxytoluene (BHT) in the finished product. Analytes were separated on a short Zorbax SB-C18 column (50 × 4.6 mm I.D., 5 μm particle size, pore size: 80 Å) with gradient elution at 40 °C column temperature. Analytes were detected at 245 nm for praziquantel, esafoxolaner, eprinomectin and their degradation products. BHT and eprinomectin degradation product 8a-oxo-B1a were detected at 280 nm. All analytes of interest were adequately separated within 40 min. The assay for praziquantel, esafoxolaner, eprinomectin and BHT was conducted against their corresponding external reference standards. The related substances peaks of each API were determined by peak area and relative response factor against total peak area of their corresponding API peak in sample solution. This method has been demonstrated to be accurate, robust, specific and stability indicating.

中文翻译：

---

单反相高效液相色谱法同时测定外用成品中的艾沙福拉纳、依普诺菌素、吡喹酮、BHT 及其相关物质。

该外用产品含有三种活性药物成分（API），即艾司福拉纳、依立菌素和吡喹酮，已证明其在治疗猫体外寄生虫、线虫和绦虫混合感染方面的功效。开发并验证了反相高效液相色谱 (RP-HPLC) 方法，用于测定和测定三种 API 的相关物质峰，包括成品中抗氧化剂丁基羟基甲苯 (BHT) 的测定。分析物在短 Zorbax SB-C18 柱（50 × 4.6 mm ID，5 μm 粒径，孔径：80 Å）上分离，在 40 °C 柱温下进行梯度洗脱。在 245 nm 处检测到吡喹酮、艾司福拉纳、依立菌素及其降解产物的分析物。在 280 nm 处检测到 BHT 和依立菌素降解产物 8a-oxo-B1a。所有感兴趣的分析物均在 40 分钟内充分分离。吡喹酮、艾司福拉纳、依立诺菌素和 BHT 的测定是根据其相应的外部参考标准进行的。各API的有关物质峰通过峰面积和相对响应因子相对于样品溶液中相应API峰的总峰面积确定。该方法已被证明准确、稳健、特异、稳定。