Background Ivermectin is a potent semi-synthetic antiparasitic drug used in veterinary medicine. It is widely used for the treatment of parasites. Objective This study aimed to develop a stability-indicating reversed-phase HPLC (RP-HPLC) method for assay and identification of ivermectin including identification and estimation of its related substances in bulk drug substance batches of ivermectin. Method Ivermectin and its related substances were separated on an Ascentis Express C18 column (100 mm × 4.6 mm id, 2.7 µm particle size) maintained at 45°C (column temperature) on an HPLC system with gradient elution. The mobile phase was composed of water − acetonitrile (ACN; 50 + 50, v/v) as mobile phase A, and isopropanol − ACN (15 + 85, v/v) as mobile phase B. Analytes were detected with a detection wavelength of 252 nm and quantitated against an external reference standard of ivermectin with a quantitation limit of 0.1% of the target (analytical) concentration. Results The HPLC method was able to separate all analytes of interest by gradient elution within 25 min. The method was validated according to the guidelines described in the International Conference on Harmonization guideline Q2(R1). Conclusions The HPLC method for assay of ivermectin and estimation of its related substances was successfully developed, validated, and demonstrated to be accurate, robust, specific, and stability indicating. Highlights The performance of the HPLC method is significantly faster and possesses a higher degree of selectivity. Implementation of this method for routine analysis in QC laboratories would save significant time, resources and solvents.

中文翻译：

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用于鉴定、测定和估计伊维菌素原料药相关物质的稳定性指示反相高效液相色谱 (RP-HPLC) 方法的开发和验证

背景 伊维菌素是一种用于兽药的强效半合成抗寄生虫药。它广泛用于治疗寄生虫。目的 本研究旨在开发一种用于伊维菌素测定和鉴定的稳定性指示反相高效液相色谱（RP-HPLC）方法，包括伊维菌素原料药批次中相关物质的鉴定和估计。方法 伊维菌素及其相关物质在采用梯度洗脱的 HPLC 系统上保持在 45°C（柱温）的 Ascentis Express C18 色谱柱（100 mm × 4.6 mm id，2.7 µm 粒径）上进行分离。流动相由作为流动相 A 的水 - 乙腈 (ACN; 50 + 50, v/v) 和作为流动相 B 的异丙醇 - ACN (15 + 85, v/v) 组成。用 252 nm 的检测波长检测分析物，并根据伊维菌素的外部参考标准进行定量，定量限为目标（分析）浓度的 0.1%。结果 HPLC 方法能够在 25 分钟内通过梯度洗脱分离所有感兴趣的分析物。该方法根据国际协调会议指南 Q2(R1) 中描述的指南进行了验证。结论 高效液相色谱法测定伊维菌素及其相关物质的含量得到了成功开发和验证，并证明了其准确、可靠、特异性和稳定性指示。亮点 HPLC 方法的性能明显更快，并且具有更高的选择性。