Development and validation of a novel LC-MS/MS assay for C-peptide in human serum

INTRODUCTION: C-peptide is used as a marker of endogenous insulin secretion in the assessment of residual β-cell function in diabetes and in the diagnostic workup of hypoglycemia. Previously developed LC-MS/MS methods to quantify serum concentrations of C-peptide have monitored intact peptide, which ionizes poorly. As a result, methods have leveraged immunoaffinity enrichment or two-dimensional chromatography. In this study, we aimed to use proteolysis during sample preparation to enhance the sensitivity of traditional LC-MS/MS. METHODS: Due to the absence of arginine and lysine residues in C-peptide, we utilized Glu-C as the proteolytic enzyme in the method. After protein precipitation using acetonitrile and solid phase extraction with mixed anion exchange, lower molecular weight polypeptides were reduced, alkylated, and proteolyzed. The two amino-terminal peptide fragments, EAEDLQVGQVE and LGGGPGAGSLQPLALE, were monitored using multiple reaction monitoring in positive ion mode (Acquity ULPC-Xevo TQ-S, Waters). The former peptide was used for quantification and the latter for quality assurance. RESULTS: Glu-C was determined to be a reliable proteolytic enzyme with monotonic digestion kinetics. The assay was linear between 0.1 and 15 ng/mL and had a lower limit of quantification of 0.06 ng/mL. Total imprecision was 7.7 %CV and long-term imprecision at 0.16 ng/mL was 10.0%. Spike-recovery experiments demonstrated a mean recovery of 98.2 % (± 9.1 %) and the method compared favorably with a commercially available immunoassay and a reference measurement procedure. CONCLUSION: Protein precipitation with solid phase extraction and proteolysis with Glu-C is a robust sample preparation method for quantification of C-peptide in human serum by LC-MS/MS.