A confirmation test for the presence of Quillaja saponin and adulteration with Madhuca saponin in poultry feed using high-performance liquid chromatography and quadrupole time-of-flight mass spectrometry

To date, there is a lack of a confirmation test for the presence of Quillaja saponin in poultry feed. This gap persists despite the fact that thousands of tonnes of feed with added saponin are produced and used annually. As a result, even basic tasks such as the analytical confirmation of feed formulations have remained unaddressed. Our work intentionally targets this gap by introducing a presence confirmation. Additionally, poultry feed was examined for the presence of Madhuca longifolia L., as previous studies had demonstrated that commercially available Quillaja saponin extracts are often adulterated with cost-effective Madhuca saponin. Pelleted poultry feed samples were analyzed via high-performance liquid chromatography (HPLC) hyphenated with quadrupole time-of-flight mass spectrometry (QToF-MS) in the negative ionization mode, targeting the main saponin compounds serving as markers for presence confirmation of Quillaja and Madhuca saponin in complex feed matrices. The unequivocal confirmation of Quillaja in feed samples containing additives classified as flavoring substances or sensory additives was enabled for the first time by this newly developed test. Unexpectedly, Madhuca was also found in some samples, clearly revealing adulteration for the first time. This confirmation test may support industry and authorities to confirm the presence of Quillaja saponin in feed and to check poultry feed for adulteration with Madhuca saponin. Furthermore it provides a practical tool for feed manufacturers by offering clarity as often the composition of saponin-containing additives is not fully disclosed by additive suppliers.