Two‐step ion‐exchange chromatographic purification combined with reversed‐phase chromatography to isolate C‐peptide for mass spectrometric analysis

A liquid chromatography with mass spectrometry on‐line platform that includes the orthogonal techniques of ion exchange and reversed phase chromatography is applied for C‐peptide analysis. Additional improvement is achieved by the subsequent application of cation‐ and anion‐exchange purification steps that allow for isolating components that have their isoelectric points in a narrow pH range before final reversed‐phase mass spectrometry analysis. The utility of this approach for isolating fractions in the desired “pI window” for profiling complex mixtures is discussed.