Establishment of an absolute quantitative method for measurement of urinary cystatin C by stable isotope dilution ultra high performance liquid chromatography tandem mass spectrometry

Abstract

Urinary Cystatin C (uCysC) is one of the best biomarkers for the diagnosis of early acute kidney injury (AKI). Nowadays, particle-enhanced nephelometric immunoassay (PENIA) and particle-enhanced turbidimetric immunoassay (PETIA) are the preferred methods for uCysC quantification. In this paper, we established a new method for uCysC quantification, which was based on ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). After concentration, denaturation, reduction and alkylation of uCysC, trypsin was added to make uCysC hydrolysate into peptides; a specific peptide (ALDFAVGEYNK) was chosen as a surrogate for uCysC protein and then, uCysC was quantified according to linear regression equations: y = 0.5047x − 0.9037 (R² = 0.989, p < 0.01) for y⁹ (DFAVGEYNK⁺, m/z = 1042.4) and y = 0.5754x − 1.2394 (R² = 0.989, p < 0.01) for y⁶ (VGEYNK⁺, m/z = 709.3). We also conducted method validation for the established method; linear ranges from 0.1 to 40 mg L⁻¹ and 0.5 to 40 mg L⁻¹ were observed for y⁹ and y⁶, respectively. The limit of detection and limit of quantification were 0.012 mg L⁻¹ and 0.068 mg L⁻¹, respectively. With different uCysC concentrations, the recoveries were in the range from 84.4% to 95.0%; the intra-day coefficients of variations (CVs) were in the range from 0.40% to 1.50%, and inter-day CVs were in the range from 0.26% to 0.50%. The carryover rates of uCysC from the highest to the lowest concentrations were in the range from 0.38% to 0.46%. Human albumin, total bilirubin and haemoglobin exhibited no interference for uCysC quantification. UCysC both in spiked and neat urine samples could be stable for at least 24 hours on plate and for 6 days and 15 days at 4 degrees and −20 degrees, respectively.