Absolute quantitation of human serum cystatin C: candidate reference method by 15N-labeled recombinant protein isotope dilution UPLC-MS/MS.

Objectives: Serum cystatin C (CysC) is a reliable and ideal endogenous marker for accurately assessing early changes in glomerular filtration rate (GFR), surpassing the limitations of creatinine-based estimated GFR. To improve the precision of GFR calculation, the development of strategies for accurately measuring serum CysC is crucial.

Methods: In this study, the full-length CysC pure product and fully recombinant ¹⁵N-labeled CysC internal standard were subjected to protein cleavage. Subsequently, an LC-MS/MS method was developed for the absolute quantification of serum CysC. The traceability of the method was assigned calibrator using the amino acid reference measurement procedure (RMP). It involved calibrating the instrument using an amino acid reference material with known amino acid concentrations for calibration and comparison purposes.

Results: The total imprecision of the method was determined to be ≤8.2 %, and a lower functional limit of quantification (LLoQ) was achieved. The recoveries ranged from 97.36 to 103.26 %. The relative bias between this candidate RMP for measurement of ERM-DA471-IFCC and the target value was 1.74 %. The linearity response was observed within the concentration range of 0.21-10.13 mg/L, with a high R² value of 0.999. The results obtained using our method was consistent with those obtained using other certified RMPs.

Conclusions: With the establishment of this highly selective and accurate serum CysC measurement method, it is now possible to assess the correlation between immunoassay results of serum CysC and the intended target when discrepancies are suspected in the clinical setting.