Peptide Stability Assay in Simulated Intestinal Fluid

The stability assay of peptides in SIF was conducted according to United States Pharmacopeia & National Formulary (USP 25-NF 20)⁶⁴ and the previous report^{65 (link)} with some modifications as follows. SIF used in this study was prepared by dissolving pancreatin (1.25 mg/mL) (Sigma, P7545, 8 × USP specifications) in 50 mM phosphate buffer at pH 6.8. The solution was vortexed for 1 min, sonicated for 15 min, centrifuged at 20,000 × g for 10 min, and filtered before use. 57 μL of SIF was incubated at 37 °C for 15 min and 3 μL of 1 μM peptide solution in DMSO was added to the warmed SGF. After 1, 2, or 4 h, 60 μL of ice-cold ultra-pure water containing 5% trifluoroacetic acid was added and 120 μL of 30% acetonitrile in ultra-pure water was added. The samples were filtered and injected into UPLC.