Background and Objectives: As a medicinal and food homologous substance, Eupolyphaga steleophaga is renowned for its potential health benefits, including anti-tumor effects, immune system support, and anti-inflammatory properties. Eupolyphaga steleophaga polypeptides have demonstrated significant biological activity, including the regulation of coagulation and lipid metabolism. However, the peptide composition of Eupolyphaga steleophaga requires further clarification to facilitate quality control improvements and a deeper investigation into its pharmacological effects. Therefore, this study aimed to simulate the digestive absorption process of Eupolyphaga steleophaga following oral administration and identify its enzymatic components to enhance quality control. Methods: The digestive absorption process was simulated using artificial gastric fluid and pepsin. A fingerprinting method based on ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS)(Acquire UPLC-Synapt G2-Si HDMS, Waters Corporation, Milford, MA, USA) was developed to identify 63 enzymatic components. The enzymolysis polypeptide fingerprint detection method was used to analyze 10 batches of Eupolyphaga steleophaga sourced from Harbin No. 4 Traditional Chinese Medicine Factory. Chromatographic collection was performed using an ACQUITY UPLC BHE C18 column. Gradient elution was carried out using a mixture of 0.1% formic acid with acetonitrile and 0.1% formic acid with water, with an average flow rate of 0.3 mL/min, a column temperature of 40 °C, and an injection volume of 2 μL. The mass spectrometry (MS) conditions were set as follows: the ion source was operated in positive electrospray ionization (ESI+) mode, with a capillary voltage of 2.8 kV and a sampling cone voltage of 40 V. The ion-source temperature was maintained at 110 °C, while the desolvation temperature was set to 400 °C. The cone gas flow rate was 50 L/h, and the desolvation gas flow rate was 800 L/h. The range for the collection of mass-to-charge ratios (m/z) was between 50 and 1200. Results: The UHPLC-MS method demonstrated high accuracy, repeatability, and stability, successfully identifying 63 enzymatic components of Eupolyphaga steleophaga. Furthermore, polypeptide markers for 63 selected components were identified in all 10 batches of Eupolyphaga steleophaga medicinal materials. This approach was validated by including numerical values such as retention times and peak areas, confirming its reliability for quality control enhancement. Conclusions: This novel UHPLC-MS approach serves as a powerful tool for advancing quality control strategies in veterinary medicine, particularly for animal-derived medicines. It lays a solid foundation for subsequent pharmacological studies of Eupolyphaga steleophaga polypeptides, offering a more reliable means to explore their biological activities and therapeutic potential.

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