Development and validation of a multiplexed targeted HILIC-HRMS assay for quantitative analysis of hepatocellular carcinoma circulating biomarkers

= 0.999), and stability (intra-day CV: 0.39%, inter-day CV: 1.46%), moderate matrix effects (14.8%), and recovery (95.82%). Despite analyzing a substantially reduced number of variables with respect to the untargeted approach (24 vs 280), the targeted panel confirmed the diagnostic value of the previously identified signature and remains superior to the AFP classification. Among the measured compounds, LPC 14:0 and LPC 18:2 showed the highest discriminative power (AUC = 0.978, AUC = 0.967). The developed targeted approach demonstrated a promising potential tool for HCC discrimination from other liver diseases, and, by obtaining quantitative values of circulating analytes, could serve as a liquid biopsy approach for early diagnostics and therapy evaluation.