A TRANSLATIONAL LC-MS/MS FRAMEWORK FOR LIPID BIOMARKER IDENTIFICATION AND QUANTIFICATION IN HUMAN PLASMA

Abstract

Lipid metabolism is increasingly recognized as a hallmark of cancer, yet translating lipidomic discoveries into clinically actionable biomarkers remains constrained by analytical variability and limited standardized validation frameworks. This challenge is further compounded by a chicken-or-egg problem, where expensive standards and labelled internal standards are required to identify and quantitate target lipids, but the diagnostic importance of these targets is uncertain until they can be reliably measured. Previous work had indicated the potential of 48 lipid biomarker species for the prediction of breast cancer from plasma samples using high resolution liquid chromatography mass spectrometry. This study aimed to identify each of these 48 species and develop a quantitative method to determine the absolute concentrations of these lipids in plasma to provide the basis for the development of a clinical assay for use in breast cancer detection. In doing so, we present a pragmatic workflow that bridges lipid discovery with lipid identification and robust quantitative analysis. A curated library of 48 lipid species was established using authentic standards to verify plasma lipids through retention-time matching and high-resolution spectral comparison. In plasma, 41 lipids were confidently identified based on co-elution with standards and diagnostic fragment ions. Method qualification, including assessment of accuracy, precision, recovery, and linearity, was performed across all 48 lipids in parallel with identification, and 46 lipids ultimately met all predefined qualification criteria. Notably, practical constraints, including time, cost, and availability of authentic standards, necessitated performing identification and targeted method development in parallel, highlighting challenges inherent to translating lipidomics into commercial or clinical assays. This workflow provides a reproducible framework for harmonizing lipid identification and quantification, enabling the reliable integration of lipidomic data into biomarker discovery and clinical applications.