Evaluation of online carbon isotope dilution mass spectrometry for the purity assessment of synthetic peptide standards

We present a novel method for the purity assessment of peptide standards which is applicable to any water soluble peptide. The method is based on the online ¹³C isotope dilution approach in which the peptide is separated from its related impurities by liquid chromatography (LC) and the eluent is mixed post-column with a continuous flow of ¹³C-enriched sodium bicarbonate. An online oxidation step using sodium persulfate in acidic media at 99 °C provides quantitative oxidation to ¹²CO₂ and ¹³CO₂ respectively which is extracted to a gaseous phase with the help of a gas permeable membrane. The measurement of the isotope ratio 44/45 in the mass spectrometer allows the construction of the mass flow chromatogram. As the only species that is finally measured in the mass spectrometer is CO₂, the peptide content in the standard can be quantified, on the base of its carbon content, using a generic primary standard such as potassium hydrogen phthalate. The approach was validated by the analysis of a reference material (NIST 8327), and applied to the quantification of two commercial synthetic peptide standards. In that case, the results obtained were compared with those obtained using alternative methods, such as amino acid analysis and ICP-MS. The results obtained proved the value of the method for the fast, accurate and precise mass purity assignment of synthetic peptide standards.