RP-HPLC determination of betulin in oil-in-water emulsion-based feed additives

Betulin, a lupane-type triterpenoid, is of growing interest as an active component of veterinary and feed formulations. For such complex matrices, robust and traceable analytical methods are required to control the betulin content and ensure product quality. In this work, a simple and rapid reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the quantitative determination of betulin in an oil-in-water emulsion-based feed additive. Chromatographic separation was performed on a C18 column using an acetonitrile–water (80:20, v/v) mobile phase in the isocratic mode at 35 °C, with a flow rate of 0.35 mL/min and UV detection at 210 nm; the total run time does not exceed 3 min. Quantification was carried out by external calibration using a certified betulin reference over the concentration range relevant to the declared product content. The method was validated in accordance with national metrological guidelines with respect to selectivity, linearity, accuracy, precision, range, and robustness. The results demonstrate excellent linearity, satisfactory precision, and recoveries close to 100%, as well as the absence of matrix interferences. The developed RP-HPLC procedure is suitable for routine quality control of the novel feed additive and can be adapted to other betulin-containing products.