SI-traceable purity assignment for peptide sublancin using mass balance approach and isotope dilution mass...

Accurate measurement of the peptide sublancin purity is very important for the application in feed additives. In this study, sublancin was separated and purified from crude samples by using a semi-preparative chromatography. For the purified sublancin sample, a SI-traceable purity assignment strategy was established by using mass balance (MB) and amino acid-based isotope dilution mass spectrometry (AA-IDMS) approaches. The absolute purity measured by MB method was 78.60% ± 0.27% by deducting the amount of all impurities including 5.46% of water, 0.4% of structure-related organic compounds, 15.48% of trifluoroacetate ion residue, and 0.056% of inorganic impurities. Hydrolysis conditions on phenylalanine (Phe), alanine (Ala), leucine (Leu) were optimized and AA-ID-LC-MS/MS measurement of sublancin purity was assigned to be 77.58% ± 0.34%. Additionally, measurement uncertainty was comprehensively evaluated. Therefore, the certified value was assigned to be 78.1% with uncertainty of 1.5%. These established methods can be applicable to SI-traceable determination and development of sublancin certified reference material.