Multiplexed HILIC-HRMS Method Quantifies LPC and CAR Biomarkers for HCC

Hepatocellular carcinoma (HCC) remains the predominant form of primary liver cancer, and clinical outcomes continue to be hampered by frequent late-stage diagnosis. Alpha-fetoprotein (AFP), the conventional serum biomarker, suffers from insufficient sensitivity and specificity, particularly in early disease, underscoring the need for improved blood-based diagnostic tools. Liquid biopsy strategies based on circulating tumor DNA have demonstrated potential in this regard, yet they do not capture the metabolic reprogramming characteristic of malignant transformation. Prior work had identified two analyte classes, lysophosphatidylcholines (LPCs) and acylcarnitines (CARs), whose circulating levels distinguished HCC patients from those with chronic hepatitis C infection with greater discriminatory power than AFP. However, those earlier discovery-phase studies relied on relative, semi-quantitative measurements rather than absolute concentrations, limiting their applicability to clinical contexts that require defined reference thresholds and inter-study comparability.

A recent study (and resulting paper¹) conducted by the team responsible for the prior work referenced earlier addressed this limitation through the development of a quantitative assay capable of measuring LPCs and CARs simultaneously within a single analytical run, a nontrivial undertaking given the divergent physicochemical properties of these two compound classes. The method employs hydrophilic interaction liquid chromatography (HILIC), which affords effective separation of polar species and facilitates co-elution with stable isotope-labeled internal standards for accurate quantification, coupled with high-resolution mass spectrometry. Rather than employing conventional selected/multiple reaction monitoring on triple-quadrupole instrumentation, an approach susceptible to isobaric interference when target analytes share common fragment ions, as is the case for LPCs and CARs, the authors implemented a multiplexed targeted-SIM (tSIM-MPX) strategy. This approach leverages high mass-accuracy detection at both the precursor and fragment ion level to achieve enhanced specificity, within a six-minute chromatographic separation that retains resolution of closely related regioisomeric species. Application of this quantitative panel preserved high classification accuracy in distinguishing HCC from chronic HCV infection. Collectively, this work advances a previously discovery-oriented biomarker signature toward an analytically validated, absolute-quantification framework, representing a meaningful step toward its translation into a clinically applicable liquid biopsy assay.

LCGC International spoke to Eduardo Maria Sommella and Danila La Gioia, two of the authors of the previously mentioned paper, about this work.

What were the main analytical challenges in developing a single HILIC–HRMS method for the simultaneous quantification of lysophosphatidylcholines (LPCs) and acylcarnitines (CARs), and how were those challenges addressed?

LPCs and CARs are characterized by markedly different physicochemical behaviors, which creates several analytical challenges for their simultaneous extraction and quantification from biological fluids. This aspect was initially addressed by investigating multiple extraction strategies, ranging from solid-phase extraction (SPE) to protein precipitation with organic solvents, to maximize analyte recovery while minimizing matrix interferences. In parallel, chromatographic behavior also represented a major challenge. Acylcarnitines, particularly short-chain species, are poorly retained in reversed-phase chromatography because of their high polarity, whereas LPCs are strongly retained due to their amphiphilic structure. This creates difficulties for concurrent separation and detection within a single analytical run. HILIC chromatography was therefore selected because it provided efficient retention of polar acylcarnitines while enabling class-based separation of LPCs according to their polar head-group interactions. Careful optimization of the mobile phase composition, gradient profile, extraction solvent, and HRMS acquisition parameters ultimately enabled robust simultaneous quantification of both analyte classes in a rapid 6-minute assay.

Why was hydrophilic interaction chromatography (HILIC) selected instead of reversed-phase LC for this targeted metabolomics/lipidomics workflow?

HILIC offers several advantages over reversed-phase chromatography for the simultaneous analysis of polar metabolites and polar lipids. Polar metabolites such as acylcarnitines are efficiently retained through hydrophilic and ionic interactions, whereas LPCs are separated mainly according to their polar head-group interactions and hydrophobicity. One important advantage of HILIC in quantitative workflows is that analytes often co-elute with their isotopically labelled internal standards, ensuring nearly identical ionization conditions. This improves correction of matrix effects and enhances quantitative reproducibility and accuracy. In contrast, reversed-phase chromatography often results in poor retention of short-chain acylcarnitines and broad retention windows across lipid species, making simultaneous targeted quantification more challenging

Can you explain the advantages of the tSIM-MPX acquisition strategy compared to traditional selected reaction monitoring/multiple reaction monitoring (SRM/MRM) methods on triple quadrupole instruments?

Traditionally, SRM/MRM performed on triple quadrupole instruments remains the gold standard for targeted metabolomics and lipidomics because of its excellent sensitivity and robustness. However, these approaches require extensive optimization of precursor/product ion transitions and collision energies, either experimentally or in silico. In addition, several metabolites and lipids may generate identical fragment ions, potentially reducing specificity in complex biological matrices. The tSIM-MPX strategy addresses these limitations by multiplexing multiple targeted precursor ions within a single acquisition event, thereby improving duty cycle and maintaining sensitivity while reducing cycle time. Moreover, the orbital ion trap analyzer provides high-resolution accurate-mass detection at both precursor and fragment levels, together with full tandem mass spectrometry (MS/MS) fragmentation patterns, greatly improving selectivity, annotation confidence, and quantitative specificity compared with conventional low-resolution SRM approaches, while keeping comparable sensitivity.

During method development, what factors influenced the choice of ammonium acetate-buffered mobile phases over formic acid-based mobile phases?

Different volatile mobile-phase additives were systematically evaluated during method development, including formic acid and ammonium acetate-based conditions. The final selection was driven by the ability to achieve the best compromise between chromatographic peak shape, analyte retention, signal intensity, and signal-to-noise ratio for both analyte classes. Ammonium acetate significantly improved chromatographic performance in HILIC mode and enhanced ionization efficiency for both LPCs and acylcarnitines, resulting in superior sensitivity and reproducibility compared with formic acid-based mobile phases. Additionally, buffered conditions improved retention stability and peak symmetry across repeated injections.

How did acetonitrile (ACN)-based protein precipitation improve chromatographic performance and quantitative reproducibility compared to methanol precipitation or SPE?

Initially, SPE was investigated because of its potential to reduce matrix effects, particularly from abundant plasma phospholipids. However, achieving quantitative and reproducible recovery of multiple compound classes together with their isotopically labelled internal standards proved challenging. ACN-based protein precipitation ultimately provided the best balance between extraction efficiency, simplicity, and reproducibility. Acetonitrile is known to form larger and more compact protein pellets compared with methanol, improving sample clean-up and reducing residual matrix components in the extract. Moreover, both LPCs and acylcarnitines, together with their internal standards, showed high and reproducible recovery values following ACN precipitation, resulting in improved chromatographic performance and quantitative robustness.

What are matrix effects in LC–MS-based bioanalysis, and how did the method design minimize ion suppression and improve quantitative accuracy?

Matrix effects represent one of the major analytical challenges in LC–MS-based bioanalysis and arise when co-eluting endogenous compounds alter analyte ionization efficiency, causing ion suppression or enhancement. In plasma metabolomics and lipidomics, phospholipids are among the primary contributors to matrix effects. Several aspects of the method were specifically optimized to minimize these phenomena. First, ACN-based protein precipitation efficiently reduced protein content and partially removed interfering matrix components. Second, HILIC chromatography improved analyte separation from highly abundant endogenous species, reducing co-elution-related ion suppression. Most importantly, the use of stable-isotope labelled internal standards enabled compensation for residual matrix effects because analytes and internal standards co-eluted under nearly identical ionization conditions. Finally, the high selectivity of Orbitrap high-resolution accurate-mass detection further reduced potential interferences, collectively improving quantitative accuracy and reproducibility,

Why is chromatographic separation still important for distinguishing LPC regioisomers even when using high-resolution accurate-mass spectrometry?

Although high-resolution accurate-mass spectrometry significantly improves selectivity by resolving isobaric species at the mass level, LPC regioisomers remain particularly challenging because they possess identical exact masses and often generate highly similar fragmentation spectra. In these cases, chromatographic separation remains essential to distinguish positional isomers differing in fatty acyl chain localization. HILIC chromatography provided sufficient selectivity to resolve critical regioisomeric LPC species within a short analytical run, improving confidence in analyte identification and preventing potential quantification bias caused by unresolved co-eluting isomers.

How were AGC target values and maximum injection times optimized to mitigate space-charge effects in the orbital ion trap analyzer?

AGC target values and maximum injection times were carefully optimized to balance sensitivity, scan speed, and mass accuracy while minimizing space-charge effects within the orbital ion trap analyzer. Excessive ion accumulation can deteriorate mass accuracy and peak shape because of ion–ion interactions inside the Orbitrap. Therefore, AGC values were empirically optimized to provide sufficient ion populations for sensitive quantification without overfilling the C-trap. Similarly, maximum injection times were adjusted to ensure stable ion accumulation even for low-abundance analytes while maintaining adequate duty cycle for multiplexed targeted acquisition. The combination of optimized AGC settings and tSIM-MPX acquisition enabled robust quantitative performance while preserving high-resolution accurate-mass detection.

What validation parameters would you consider critical when translating a targeted metabolomics assay into a clinically applicable quantitative method under ICH M10 guidelines?

Translation of targeted metabolomics assays into clinically applicable methods requires rigorous analytical validation according to internationally recognized bioanalytical guidelines such as ICH M10. Critical validation parameters include selectivity, sensitivity, linearity, accuracy, precision, recovery, matrix effect evaluation, carryover assessment, and analyte stability under different storage and processing conditions. In addition, calibration strategy and internal standard selection are particularly important in metabolomics because endogenous metabolites are naturally present in biological matrices, making true blank matrices unavailable. Robustness and reproducibility across analytical batches are also essential to ensure inter-study comparability and long-term clinical applicability. Ultimately, clinically translatable metabolomics assays must demonstrate not only strong biological relevance, but also analytical reliability and reproducibility comparable to conventional clinical chemistry methods.

From a metabolomics and lipidomics perspective, why are altered acylcarnitine and LPC profiles considered biologically relevant biomarkers for distinguishing hepatocellular carcinoma from chronic HCV infection?

Acylcarnitines and lysophosphatidylcholines are tightly linked to key metabolic pathways that are profoundly altered during hepatocarcinogenesis. Acylcarnitines reflect mitochondrial fatty acid β-oxidation and energy metabolism, and their dysregulation may indicate mitochondrial dysfunction and metabolic rewiring, both hallmarks of cancer cells. LPCs, on the other hand, are involved in membrane remodeling, inflammation, oxidative stress, and cell signaling pathways that are frequently altered in liver disease progression and tumor development. Importantly, HCC emerges in a chronically damaged liver environment, where metabolic alterations progressively evolve from chronic inflammation and fibrosis toward malignant transformation. Therefore, the combined perturbation of CAR and LPC profiles likely reflects both altered mitochondrial energetics and membrane lipid remodeling associated with tumor progression, making these metabolite classes biologically meaningful circulating biomarkers for distinguishing HCC from chronic HCV infection.

Reference

1. La Gioia, D.; Caponigro, V.; Tornesello, A. L. et al. Development and Validation of a Multiplexed Targeted HILIC-HRMS Assay for Quantitative Analysis of Hepatocellular Carcinoma Circulating Biomarkers. Anal Bioanal Chem. 2026.DOI: 10.1007/s00216-026-06568-1