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

We describe the first results of a quantitative LC–tandem mass spectrometry method for urinary C-peptide with the use of [2H14]C-peptide as internal standard. LC was based on gradient elution of a Hypersil PEP C18 column. Mass spectrometry was performed in the negative electrospray ionization mode and by monitoring of the transitions at m/z 1514/1334 ([2H14]C-peptide) and 1507/1320 (C-peptide). For sample preparation, we applied ultrafiltration. The analytical performance of the method in terms of measurement precision gave an RSD of <2% (n=10). The overall imprecision was investigated from independent analysis of two urine samples in six-fold and resulted in an RSD<5%. The limit of detection, expressed as signal-to-noise ratio 3, was ∼0.15 ng C-peptide injected. Analysis of 10 random urine samples from laboratory volunteers showed interference-free ion chromatograms at a signal-to-noise ratio of ∼75 on average. The C-peptide concentrations calculated from quantification by the bracketing calibration technique ranged from 32 to 165 ng/ml.