Background Eprinomectin is used as an active pharmaceutical ingredient (API) in various drug products by the animal health industry. Several major related impurities of eprinomectin API are not separated and coelute by the current United States Pharmacopeia (USP) method for eprinomectin. Objective The aim was to develop and validate a true and reliable stability-indicating reversed-phase (RP) HPLC method for assay and determination of related substances of eprinomectin in bulk batches of eprinomectin API. Method HPLC analysis is carried out using a Kinetex C8 column (100 mm × 4.6 mm i.d. , 2.6 μm particle size) maintained at 30°C with water–acetonitrile–isopropanol (48 + 42 + 10, v/v/v) as mobile phase A and 100% acetonitrile as mobile phase B. Analytes are separated by gradient elution at a flow rate of 0.7 mL/min and detected by UV at 252 nm. The total run time of the method is 30 min. Eprinomectin assay and estimation of all eprinomectin-related substances are obtained in a single HPLC run. Results The HPLC method was able to separate all analytes of interest by gradient elution. The new method was successfully validated according to current The Internal Council for Hamonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) and International Cooperation on Hamonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) guidelines and was found to be specific, linear, accurate, precise, robust, and sensitive. Conclusions An HPLC method for assay of eprinomectin and estimation of its related substances was successfully developed, validated, and demonstrated to be accurate, robust, specific, and stability-indicating. Highlights The HPLC method presented in this paper is more desirable as compared to USP and suitable for routine analysis of bulk batches of eprinomectin API and stability samples in QC laboratories.

中文翻译：

---

用于测定大批量依立诺菌素原料药中依立诺菌素及其相关化合物的替代和改进的稳定性指示 HPLC 方法

背景 依立菌素被动物保健行业用作各种药品中的活性药物成分 (API)。依立诺菌素 API 的几种主要相关杂质没有被现行的美国药典 (USP) 方法对依立诺菌素进行分离和共洗脱。目的 开发和验证一种真实可靠的反相（RP）高效液相色谱法测定大批量依立诺菌素原料药中依立诺菌素相关物质的含量。方法 HPLC 分析使用保持在 30°C 的 Kinetex C8 色谱柱（100 mm × 4.6 mm id，2.6 μm 粒径），水-乙腈-异丙醇（48 + 42 + 10，v/v/v）作为流动相A 相和 100% 乙腈作为流动相 B。通过梯度洗脱以 0.7 mL/min 的流速分离分析物，并通过 252 nm 的 UV 检测。该方法的总运行时间为 30 分钟。在单次 HPLC 运行中获得所有依立诺菌素相关物质的依立诺菌素测定和估计。结果 HPLC 方法能够通过梯度洗脱分离所有感兴趣的分析物。新方法已根据目前的人用药品技术要求统一内部委员会 (ICH) 和兽药产品注册技术要求统一国际合作 (VICH) 指南成功验证，并被发现具有特异性，线性、准确、精确、稳健和灵敏。结论 成功开发、验证了一种用于测定依立诺菌素及其相关物质的 HPLC 方法，并证明其准确、稳健、特异性和稳定性指示。