HIGH THROUGHPUT PLASMA PROFILING OF HUMAN LIVER DISEASE SAMPLES USING RAPID CHROMATOGRAPHY AND A MULTI- REFLECTING TIME-OF-FLIGHT MASS SPECTROMETER

Significance of the topic

Metabolomic profiling of plasma samples from liver disease patients enables the discovery of biomarkers for early diagnosis, disease stratification and therapeutic development. Acute-on-chronic liver failure (ACLF) is marked by systemic inflammation and high mortality with no established early biomarkers. High-throughput, robust analytical platforms are essential to analyze large patient cohorts and to minimize batch-to-batch variability.

Objectives and overview of the study

This study aimed to evaluate and compare two high-throughput chromatographic workflows for plasma metabolome and lipidome profiling in liver disease:

• Rapid microbore metabolic profiling (RAMMP) with HILIC for polar small molecules and RP C8 for lipids.
• Vacuum jacketed column (VJC) technology using identical column dimensions and conditions to RAMMP.

The study assessed analytical reproducibility, mass accuracy and group differentiation across healthy controls, cirrhosis, ACLF and acute liver failure (ALF) cohorts.

Methodology and instrumentation

Sample preparation:

• Lipid extraction: 50 µL plasma precipitated with cold IPA, incubated at 2–8 °C, centrifuged and supernatant collected.
• Small molecule extraction: 50 µL plasma mixed with 20 µL water and 180 µL acetonitrile, incubated, centrifuged and supernatant collected.
• Quality controls prepared by pooling aliquots from all study samples and by phenotypic sub-pooling.

Chromatography and mass spectrometry:

• RAMMP HILIC on ACQUITY BEH Amide (1.0×50 mm), mobile phases water:ACN (95:5) and (5:95) with ammonium formate/formic acid modifiers.
• RAMMP RP lipid on ACQUITY BEH C8 (1.0×50 mm), mobile phases water/IPA/ACN and IPA/ACN with ammonium acetate/acetic acid.
• VJC columns manufactured in-house matching RAMMP dimensions and mobile phases.
• Mass analysis on SELECT SERIES MRT time-of-flight spectrometer in DIA MSE mode (30 Hz scan speed, 170–200 K FWHM resolution). Leucine enkephalin lockmass ensured <1 ppm accuracy.
• Data processing using MassLynx v4.2, MARS and Lipostar2 for peak picking, normalization, database search and statistical analysis.

Main results and discussion

Reproducibility and mass accuracy:

• Overlaid QC injections (n=9) across 168 batches showed tight retention time and intensity clustering for both RAMMP and VJC.
• Mass errors for annotated small molecules and lipids were consistently <1 ppm (often <0.3 ppm), supporting confident compound identification.

Multivariate analysis:

• PCA of lipid data revealed tight QC clustering and clear separation of healthy controls from ALF and ACLF samples, with some overlap among disease groups.
• OPLS-DA and S-plots identified key dysregulated lipid classes (LPC, PC, SM) with significant contribution to group discrimination.
• Overlaid extracted ion chromatograms demonstrated sharper peaks and higher scan density with VJC, enhancing resolution of isobaric lipids (e.g., PC36:1 vs SM42:3).
• Comparison of RAMMP vs VJC lipid PCA showed similar group distributions, indicating minimal impact of column choice on overall statistics.

Benefits and practical applications

• Rapid chromatography reduces analysis time and batch effects, enabling reproducible profiling of large cohorts.
• High resolution and mass accuracy support reliable annotation of metabolites and lipids relevant to liver disease.
• Methodology is applicable to biomarker discovery, clinical research and industrial QA/QC settings.

Future trends and possibilities

Emerging directions include integration of ion mobility to resolve structural isomers, automated sample preparation for higher throughput, real-time quality control feedback and multi-omics data fusion. Advances in machine learning may further enhance biomarker identification and predictive modeling in liver disease research.

Conclusion

This work demonstrates that rapid microbore and vacuum jacketed chromatography coupled with multi-reflecting TOF MS deliver high-throughput, reproducible and accurate metabolomic and lipidomic profiling. Both approaches effectively discriminate liver disease phenotypes and support large-scale biomarker discovery studies.

Reference

1. Liu G, Wang X, Fan X, Luo X. Metabolomics profiles in acute-on-chronic liver failure: Unveiling pathogenesis and predicting progression. Front Pharmacol. 2022;13:953297.
2. King AM, Mullin LG, Wilson ID, et al. Rapid profiling method for the analysis of polar analytes in urine using HILIC–MS and ion mobility. Metabolomics. 2019;15(2).
3. King AM, Trengove RD, Mullin LG, et al. Rapid profiling of lipids in human plasma using ion mobility-enabled RP-UHPLC/MS. J Chromatogr A. 2020;1611:460597.
4. Plumb RS, McDonald T, Rainville PD, et al. High-Throughput UHPLC/MS/MS Metabolic Profiling Using a Vacuum Jacketed Column. Anal Chem. 2021;93(30):10644–10652.
5. Plumb RS, Isaac G, Rainville PD, et al. High-Throughput UHPLC-MS Lipidomics Using Vacuum Jacketed Columns. J Proteome Res. 2022;21(3):691–701.
6. Cooper-Shepherd DA, Wildgoose J, Kozlov B, et al. Novel Hybrid Quadrupole-Multireflecting TOF MS System. J Am Soc Mass Spectrom. 2023;34(2):264–272.