Quantification of serum C-peptide by isotope-dilution liquid chromatography–tandem mass spectrometry: Enhanced detection using chemical modification and immunoaffinity purification

•We developed a highly sensitive method for quantification of serum C-peptide.•This method is based on isotope-dilution mass spectrometry.•We applied chemical modification and immunoaffinity purification to this method.•These resulted in a 23-fold enhancement of the sensitivity.•This method enables high sensitivity and precision covering the reference interval. A method was developed to quantify human serum C-peptide by isotope-dilution mass spectrometry (ID MS). This new approach used immunoaffinity purification and chemical modification to improve the sensitivity which covered the wide range of reference interval of serum C-peptide. The immunoaffinity purification was performed using monoclonal antibody against human C-peptide that was immobilized on magnetic beads, and the purified C-peptide was chemically modified using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) prior to liquid chromatography–tandem mass spectrometry (LC–MS/MS). With this method, the LC–MS/MS peak area increased 23-fold compared with the conventional purification by solid-phase extraction and without chemical modification. The limit of quantification was estimated to be 0.003ng on column, which was lower than previously reported. The validation study showed that (1) the response in the 0.003–2.9ng range on column was linear (regression coefficient, r2=0.9994), (2) the relative standard deviation (RSD) within and between days was inferior to 4.0%, and (3) the spike and recovery test showed the mean recoveries ranging between 99% and 108%. Comparison with an established commercial immunoassay showed high correlation (r2=0.9994) at serum concentration of 0.19–8.49ng/mL. These assessments suggest that this ID MS-based approach can quantify human serum C-peptide with high sensitivity and precision in the reference interval and find a potential use in the reference measurement procedure of serum C-peptide, allowing traceable measurement. This method may also generally be applied to peptide quantification in biological fluids with high sensitivity.