Development and Validation of a Lectin-independent Liquid Chromatography-Tandem Mass Spectrometry Method for Serum Glycosylated Alpha-fetoprotein Analysis and Comparison with a Liquid-phase Binding Assay

Abstract

Background: Alpha-fetoprotein (AFP) and its isoform AFP-L3 are well-established serum biomarkers for hepatocellular carcinoma (HCC), a common malignancy and a leading cause of cancer-related mortality worldwide. Current methods for measuring these biomarkers are primarily lectin-based assays including the liquid-phase binding assay (LiBA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), both of which have limitations in diagnostic sensitivity and clinical utility for samples with low AFP concentrations. We aimed to develop a lectin-independent LC-MS/MS method for quantifying fucosylated AFP proteins (AFP-Fuc%).

Methods: We conducted analytical validation, including method comparisons, over 2 months. The analytical sensitivity and diagnostic performance of this method were evaluated using 525 human serum samples-235 from HCC patients and 290 from non-HCC individuals-and compared with those of LiBA, which measured AFP-L3 levels.

Results: The LC-MS/MS method demonstrated acceptable within-laboratory imprecision (CVs<17.1%) without detectable bias, carryover, or matrix effects. Our method exhibited a broader linear dynamic range (spanning five orders of magnitude) and 10-fold higher analytical sensitivity than LiBA. The diagnostic performance of our method was significantly superior to that of LiBA, particularly in patients with low AFP concentrations (<7 ng/mL, P <0.001), with improved accuracy, sensitivity, and precision at a specificity of 96.2%.

Conclusions: The validated LC-MS/MS method demonstrated robust analytical performance and superior diagnostic accuracy over LiBA for HCC diagnosis while avoiding the inherent limitations of lectin-based assays. Our LC-MS/MS assay shows promise for early HCC detection and may contribute to enhanced patient care.

Keywords: Alpha-fetoprotein; Automation; Fucosylation; Glycopeptide; Hepatocellular carcinoma; Liquid Chromatography; Mass spectrometry.