Ivermectin HPLC Quantification Protocol

Reverse-phase liquid chromatography was carried out. The mobile phase consisted of a solution containing acetonitrile (56%), methanol (37%), and water (7%) in an isocratic process. The stationary phase consisted of a C-18 precolumn and a C-18 column, respectively, a Supelguard reverse-phase chromatographic column C-18 (2 cm long, 4.6 mm internal diameter, and 5 µm average particle diameter) and a C-18 Supelcosil reverse-phase chromatographic column (25 cm long, 4.6 mm internal diameter, and 5 µm average particle diameter).
All HPLC analyses were conducted at room temperature (20°C). Before injecting the samples, the system was equilibrated for 30 minutes under the conditions described above. The flow was set at 1.2 mL/min, and the wavelength at 245 nm. A volume of 100 µL was injected for each sample run.
The ivermectin standard (0.05 µg/mL), the internal standard (0.05 µg/mL), the blank (Figure 1), and the fortified controls (Figure 2) were analyzed first. Subsequently, all samples were run and the standard injections were repeated every three samples.
The analysis was continuously monitored by Accredia, the Italian accreditation body, which certifies the quality of laboratory practices and conformity of the results obtained.