Development and Validation of a Novel Isotope Dilution-Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry Method for Serum C-Peptide

Background: Mass spectrometry (MS) methods exhibit higher accuracy and comparability in measuring serum C-peptide concentrations than immunoassays.We developed and validated a novel isotope dilution-ultraperformance liquid chromatography-tandem MS (ID-UPLC-MS/MS) assay to measure serum C-peptide concentrations.Methods: Sample pretreatment involved solid-phase extraction, ion-exchange solid-phase extraction, and derivatization with 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (Cayman Chemical, Ann Arbor, Michigan, USA).We used an ExionLC UPLC system (Sciex, Framingham, MA, USA) and a Sciex Triple Quad 6500 + MS/MS system (Sciex) for electrospray ionization in positive-ion mode with multiple charge states of [M+3H]3+ and multiple reaction monitoring transitions.The total run time was 50 mins, and the flow rate was 0.20 mL/min.We evaluated the precision, trueness, linearity, lower limit of quantitation (LLOQ), carryover, and matrix effects.Method comparison with electrochemiluminescence immunoassay (ECLIA) was performed in 138 clinical specimens. Results:The intra-and inter-run precision coefficients of variation were < 5% and the bias values for trueness were < 4%, which were all acceptable.The verified linear interval was 0.050-15 ng/mL, and the LLOQ was 0.050 ng/mL.No significant carryover or matrix effects were observed.The correlation between this ID-UPLC-MS/MS method and ECLIA was good (R = 0.995, slope = 1.564); however, the ECLIA showed a positive bias (51.8%). Conclusions:The developed ID-UPLC-MS/MS assay shows acceptable performance in measuring serum C-peptide concentrations.This will be useful in situations requiring accurate measurement of serum C-peptide in clinical laboratories.