Absolute quantification of urinary neutrophil gelatinase‐associated lipocalin by ultra‐high‐performance liquid chromatography/tandem mass spectrometry and the diagnostic efficacy of acute kidney injury

Rationale To establish an absolute quantification method for neutrophil gelatinase‐associated lipocalin (NGAL) by ultra‐high‐performance liquid chromatography tandem positive ion electrospray ionization mass spectrometry (UHPLC/MS/MS) and evaluate its diagnostic efficacy for acute kidney injury (AKI). Methods Three target peptides of NGA were prescreened by Skyline software, and two of them could be detected in tryptic peptides of NGAL recombinant protein and human urinary NGAL (uNGAL). Peptide (WYVVGLAGNAILR) was then selected as surrogate peptide. The corresponding isotope‐labeled peptide as the internal standard was next synthesized. Quantification of uNGAL was based on equations of linear regression, and method validation was then conducted. The diagnostic efficacy of uNGAL for AKI was also evaluated using receiver operating characteristic curve (ROC) analysis. Lastly, the UHPLC/MS/MS and the particle‐enhanced turbidimetric immunoassay (PETIA) methods for uNGAL quantification were compared. Results For the y9 and y10 product ions, the linear regression equations were y = 2.5519x–4.6955 (R2 = 0.994, P