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

Early and accurate diagnosis of hepatocellular carcinoma (HCC) remains a major clinical challenge, particularly in patients with chronic hepatitis C virus (HCV) infection, where standard biomarkers such as alpha-fetoprotein (AFP) often lack sensitivity and specificity. Numerous biomarkers have emerged from untargeted metabolomics and lipidomics approaches; nevertheless, most of these studies provide only relative amounts and do not perform quantitative analysis as further validation. To address this challenge, we developed and validated a targeted method to quantitatively measure panels of biomarkers previously identified as predictive of HCC in untargeted analyses. A high-throughput (6 min) HILIC-HRMS method, based on a multiplexing-HRMS strategy, was developed and validated to measure a panel of lysophosphatidylcholines (LPCs) and carnitines (CARs) in plasma samples from 92 HCC patients. The method showed satisfactory performances in terms of sensitivity (LODavg: 0.0558 ng/mL, LOQavg: 0.667 ng/mL), accuracy (98.75%), linearity (R2 = 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.