Quantitative analysis of urinary C-peptide by liquid chromatography–tandem mass spectrometry with a stable isotopically labelled internal standard

Sensitive Quantitative Analysis of C-Peptide in Human Plasma by 2-Dimensional Liquid Chromatography–Mass Spectrometry Isotope-Dilution Assay

“…IDAs are used to measure metabolites (17 ), small drug molecules in biological fluids (23,24 ), and large complex molecules such as peptide hormones (6,7,(13)(14)(15)(16)18 ). For reported C-peptide assays, the lowest amount measured was 30 fmoles on column, which was obtained via initial immunoprecipitation/SPE followed by off-line RP chromatography and direct MS detection (7 ); the detection limit was also 30 fmoles for ultrafiltration followed by RP LC/tandem MS (6 ).…”

“…The use of 2D LC/MS reduces matrix effects and increases overall assay sensitivity. Initial prepurification may also lead to analyte loss, with recoveries of 60% for ultrafiltration (6 ) and Ն80% for SPE [Ref. (7 ) and this report].…”

“…IDAs are based on quantitative detection of an analyte signal by MS relative to a heavy-isotope-labeled IS that is added at a known concentration (6,7,17 ). The analyte and IS are of identical amino acid composition and exhibit identical physiochemical properties except that they differ in their m/z ratio (e.g., 1007.7 vs 1017.7 for C-peptide and the [Mϩ30] IS).…”

“…represent the intact molecule (6,7,(13)(14)(15)(16); it thus offers the possibility of absolute specificity. Previously published LC/MS methods for quantifying C-peptide in urine and plasma had low sensitivity and low sample throughput, and required complicated sample preparation and large sample volumes (6,7 ).…”

“…Previously published LC/MS methods for quantifying C-peptide in urine and plasma had low sensitivity and low sample throughput, and required complicated sample preparation and large sample volumes (6,7 ). We describe a novel sample preparation technique and a 2-dimensional (2D) LC/MS IDA method developed to overcome these limitations.…”