Fast Determination of Human Insulin and Its Deamidation Product by Electrospray Ionization Mass Spectrometry

Application of the electrospray ionization (ESI) technique in mass spectrometry (MS) has played an important role in analysis of biological molecules and polymers. The focus of this thesis is to assess and extend the application of this technique in the rapid determination of human insulin and its deamidation product.

Quantitative assessment of deamidation of human insulin generally invokes relatively slow chromatographic analysis. Faster cost-effective analysis using mass spectrometry with a low-resolution analyzer is challenging because the deamidation product is only one Dalton heavier than the 5808-Da protein; low-level contamination has only a small effect on the isotope envelope. Exploiting the chemical differences between insulin and its deamidation product (6 carboxylic acids, vs 7 following deamidation), we have extended an application of an alternative MS method based on the relative intensities of the high charge states in negative ion electrospray, using isopropylamine (IPA) to enhance anion intensities. Source parameters were optimized for high sensitivity and stability of higher charge states.

The intensities of the higher charge states were increased in proportion to the amount of the deamidation product present in the mixtures. From the various ratios of the deamidation product, the study shows that this method was able to determine the ratio of the deamidation product in one minute with LOD = 0.7 % deamidation.