Optimized peptide extraction method for analysis of antimicrobial peptide Kn2-7/dKn2-7 stability in human serum by LC–MS

AIM: To develop an extraction protocol and determine stability for antimicrobial peptide (AMP) Kn2-7 and its d-enantiomer dKn2-7 in human serum. MATERIALS & METHODS: We compared use of ethanol, acetonitrile, RapiGest SF Surfactant and 1% formic acid in ethanol for AMP recovery from serum prior to liquid chromatography-mass spectrometry quantification. RESULTS: Precipitation of samples with 1% formic acid in ethanol caused the least amount of AMP loss during extraction from serum. Time-course experiments revealed dKn2-7 was significantly more stable than Kn2-7 in 25% serum, with 78.5% of dKn2-7 and only 1.0% of Kn2-7 remaining after 24 h at 37°C. CONCLUSION: The optimized method significantly increased peptide recovery and allowed more accurate and consistent quantification of Kn2-7 and dKn2-7 serum stability.