The Analysis of Insulin-Related Peptides by Reversed-Phase High-Performance Liquid Chromatography

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Abstract
This paper describes the use of high performance liquid chromatography (HPLC) for the rapid analysis and purification of insulin-related peptides prepared by solid-phase synthetic procedures. Examples include the bovine insulin C-peptide (34–45), the porcine insulin C-peptide (41–53) and the insulin B-chain fragment (22–27). Chromatographic elution systems containing reducing reagents like B-mercaptoethanol allow the direct analysis of insulin reduction products. Similar systems should allow the rapid analysis of disulphide bond pairing patterns in appropriate polypeptides and proteins either directly or following proteolytic digestion. Reversed-phase high performance liquid chromatography (RP-HPLC) is a versatile and rapid technique useful for the analysis and purification of biological substances. In a series of recent publication1–4 we have described methods for the analysis of underivatised amino acids, peptides and proteins on reversed-phase packings using ion-pairing or stationary phase modifying reagents as components of the mobile phase. These studies demonstrated that excellent resolution of closely related peptides can be achieved under a variety of elution conditions. The addition of low levels of phosphoric acid, inorganic or organic phosphates to a mobile phase (generally water-organic solvent mixtures), in particular, allows rapid and reproducible analysis of peptidic compounds with high sensitivity detection at wavelengths down to 190nm5,6. It is the purpose of this report to show that these chromatographic conditions allow the facile analysis, and purification, of a variety of insulin-related peptides.