Determination of deamidated isoforms of human insulin using capillary electrophoresis

Abstract

The applicability of capillary zone electrophoresis (CZE) for the separation of the deamidated forms of insulin has been studied. 50 mM NH₄Ac (pH=9) with 20 % v/v isopropylalcohol was found optimal for efficient separation of insulin from its even 10 deamidated forms. The developed method was efficiently applied for monitoring the degradation rate of insulin and the formation of different deamidation isoforms. Two months after the acidification more than thirty peaks can be observed in the electropherogram, because degradation products other than deamidated components were formed as well. The recorded mass spectra enabled us to assign the exact mass of the components, and thus the identification of insulin isoforms could be accomplished. We think that this study provides useful information on how the determination of several deamidation forms can be carried out with CE-MS, but the identification of the exact position of deamidation sites in the insulin molecule remains a challenge.

Keywords: Capillary electrophoresis; Deamidation; Insulin; Isoforms; Mass spectrometry.