Simultaneous determination of insulin, DesB30 insulin, proinsulin, and C-peptide in human plasma samples by liquid chromatography coupled to high resolution mass spectrometry

The endocrine pancreatic processes comprising the main actions of insulin, proinsulin, and C-peptide, represent a well-investigated system in the human organism because of the widespread epidemic of diabetes mellitus. Besides their medical relevance, insulin and its related compounds have further been of particular interest also to forensic science and doping controls, and in all disciplines, a specific and sensitive determination of the target analytes is critical for accurate result interpretation. In the present study, a method was developed allowing for the simultaneous determination of human insulin (and its synthetic analogues), the degradation product DesB30 human insulin, proinsulin, and C-peptide in plasma samples (100 A mu L) by liquid chromatography coupled to high resolution mass spectrometry. Immunoaffinity extraction employing magnetic nanoparticles and two monoclonal antibodies for insulin and C-peptide were used in combination with two internal standards. The assay was validated for quantitative result evaluation considering the parameters specificity, linearity (0-10 ng/mL), limit of detection (similar to 0.05 ng/mL), precision at high and low levels (6-25%), accuracy at high and low levels (85-127%), recovery (33-44%), ion suppression, and stability. Furthermore, the method was applied to samples from healthy athletes, patients with diabetes mellitus (type II) with and without treatment with insulin, and patients being treated for diabetes mellitus (type I). The results enable a thorough interpretation of the data with respect to endocrine, forensic and doping control samples.