Determination of Cystatin C in human urine by isotope dilution tandem mass spectrometry.

This work presents the development of a methodology for the accurate and precise quantification of the renal biomarker Cystatin C in human urine by Isotope Dilution Mass Spectrometry (IDMS). The procedure is based on the addition of a known quantity of the proteotypic peptide ALDFAVG*EYNK labelled with 13C2-glycine to the urine sample followed by protein hydrolysis using trypsin. Then, preconcentration and purification of the isotope diluted peptide was carried out by a selective monoclonal antibody bound to magnetic beads and final measurement was done after injection of the sample in a HPLC-MS/MS triple quadrupole instrument. The isotopic distribution of the isotope diluted proteotypic peptide was measured by low resolution selected reaction monitoring. Using this aquisition mode, the bandpass of the first quadrupole was widened (FWHM =13 u) so the whole isotopic clusters for both the natural abundance and the labelled peptides entered the collision cell. The proposed acquisition mode provided similar accuracy and precision than the regular SRM mode (FWHM =0.7 u) but a higher sensitivity was observed. The purification of the sample by antibody based enrichment of the target peptide was shown to remove interfering compounds more efficiently in comparison with a sample purification based on semipreparative liquid chromatography. Using 5 ng of the labelled peptide it was possible to quantify Cystatin C in human urine in patients with normal and impaired renal function. Recoveries from 100 to 104% were obtained in samples containing from 90 to 700 µg L-1 of Cystatin C with relative standard deviations from 0.5 to 6%. The stability of Cystatin C in urine samples was evaluated under different storage conditions showing that only when the urine samples were stored at room temperature during more than 10 days, a significant degradation of Cystatin C was observed.

Isotope dilution LC-ESI-MS/MS and low resolution selected reaction monitoring as a tool for the accurate quantification of urinary testosterone.

A new analytical method for the quantification of testosterone in human urine samples by isotope dilution mass spectrometry is proposed. A standard solution of 13C2-testosterone is added to the samples at the beginning of the sample preparation procedure and then the measurements are carried out by UHPLC-ESI-MS/MS. In the proposed method, the resolution of the first quadrupole of the tandem MS instrument is reduced to transmit the whole precursor ion cluster to the collision cell and measure the isotopic distribution of the in-cell product ions with a small number of SRM transitions. The construction of a methodological calibration graph is avoided using a labelled analogue previously characterised in terms of concentration and isotopic enrichment in combination with multiple linear regression. In this way, the molar fractions of natural and labelled testosterone are calculated in each sample injection and the amount of endogenous testosterone computed from the known amount of labelled analogue. Recovery values between 97 and 107% and precisions between 0.4 and 3.7% (as %RSD) were obtained for testosterone concentrations in urine in the range of 1 to 8 ng g-1. The proposed low resolution SRM methodology was compared for the analysis of human urine samples with the traditional IDMS method based on a calibration graph and the IDMS method based on multiple linear regression combined with standard resolution SRM. A similar accuracy and precision was obtained by the three tested approaches. However, using the low resolution SRM method there was no need to resort to calibration graphs or to specific dedicated software to calculate isotopic distributions by tandem MS and a higher sensitivity was obtained. The proposed low resolution SRM method was successfully applied to the analysis of the certified freeze-dried human urine NMIA MX005.