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

Abstract

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.

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

“…For quantitation, the internal standard (IS) was added immediately after thawing. After mixing with methanol (up to 1:4 ratio, v/v), samples were subjected to centrifugation (5 min at 12 000 × g ) to remove precipitated proteins . The supernatant was applied to SepPak C 18 disposable cartridges (Millipore, Billerica, MA).…”

“…Nevertheless, even in its reduced form, the 2D chromatographic approach proved to be effective. Recently, the utility of cation exchange chromatography has been demonstrated . The authors did not use 2D chromatography per se, but a combination of ion‐exchange (IEx) and RP chromatography was utilized.…”

Application of Fused-Core Particle Column in Two Dimensional Reversed Phase - Reversed Phase LC/MS Analysis of Biological Samples. Impact of Extra-Column Volume

“…Direct, highly rugged and accurate analysis of insulin is considerably more challenging due to its higher clearance (and thus lower plasma concentration), and the high variance of measurement by established immunoassays. Sensitive LC/MS analysis has been proposed as a candidate reference method for C-peptide analysis, and has become an invaluable tool for improving our understanding of and diagnosis of diabetes [5,6]. While urinary C-peptide analysis is quite simple [7,8], plasma concentrations (especially fasting) do not exceed 2 ng/ml in non diabetic and Type 2 diabetic subjects.…”

Simultaneous Monitoring of Multiple Transitions in Mass Spectrometric Analysis Improves Limit of Detection for Low Abundance Substances in Complex Biological Samples

“…In our work [5], we successfully used simultaneous monitoring of two mass transitions in order to provide higher specificity and to exclude false-positive signals. We monitored the two transitions 1007.7-142.7 and 1007.7-927.8.…”

“…By utilizing both transitions, there is an opportunity for simultaneous multiple transition signal monitoring. One of the obvious advantages of such an approach is the detection method specificity increases [5].…”