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

Abstract

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<.01) and y = 2.4619x-4.3 (R² =0.993, P<.01), respectively, and both of the linear ranges were from 0.5 to 15 mg/L. The limits of detection and quantification were 0.037 mg/L and 0.081 mg/L, respectively. The recoveries were from 97.32% to 107.28% at different uNGAL levels, and the within- and between-day CVs for uNGAL quantification were from 0.22% to 7.65% and from 0.66% to 5.97%, respectively. The carryover rates of uNGAL were in the range of 0.70%-0.99%. The area under the ROC curve (AUC) of uNGAL was 0.96 (P<0.01), and the sensitivity and specificity of uNGAL for AKI diagnosis were 90.0% and 92.5%, respectively. In addition, the UHPLC/MS/MS and PETIA methods showed good agreement for uNGAL quantification (y = 0.7112x-0.0139, P = 0.34).

Conclusions: The UHPLC/MS/MS method for uNGAL quantification has a wide linear range, high sensitivity, precision, and recovery, and low carryover rates, and uNGAL detected by this method had high sensitivity and specificity for AKI diagnosis.