Use of cation exchange chromatography for human C-peptide isotope dilution – Mass spectrometric assay

An application of ion exchange chromatography for C-peptide analysis is described here. At the stage of C-peptide isolation, a strong cation exchanger (SP HP or MonoS) was used to purify the analyte from ballast proteins and peptides. The conditions of ion-exchange chromatographic separations were optimized using theoretical modeling of the net surface electric charge of the peptide as a function of pH. The purified and concentrated sample was further subjected to LC–MS/MS. In order to improve the reliability of analysis, two fragment ions were monitored simultaneously both for native C-peptide and internal standard, isotopically labeled C-peptides analogues (fragments with m/z of 927.7 and 147.2). Using ion-exchange chromatography, it became possible to process larger sample volumes, important for testing patients with very low C peptide levels, compared to currently used solid phase extraction methods.

► Due to specific nature of C-peptide (low isoelectric point), cation exchange chromatography allows very fast and efficient purification of C-peptide from serum.
► First time for C-peptide analysis, two MRM transitions have been used simultaneously that increases the specificity of C-peptide detection.
► The approach allows proceeding much higher sample volumes compared to previous studies when only hydrophobic interactions are used.