A Comprehensive GC-MS Toolbox for Targeted and Untargeted Sterol Analysis

Sterols represent a structurally distinct class of lipophilic natural lipids defined by a rigid tetracyclic scaffold. They are essential for cellular architecture and function, serving as key structural components of biological membranes, precursors for bioactive molecules, and regulators of diverse signaling pathways. However, their high structural similarity and closely related physicochemical properties hamper chromatographic resolution, while the limited availability of reference standards restricts accurate identification and quantification. As a consequence, sterols remain underrepresented in many research areas, including human biomarker studies. To address this, we established a comprehensive sterol analytical toolbox that integrates validated commercially available and in-house synthesized reference standards and is released as an open access sterol library for the research community. The presented library comprises 110 sterols of plant, fungal, and mammalian origin, including six sterol acids, seven epoxy sterols, and 22 oxysterols. Each compound was systematically characterized by chromatographic separation on four different GC-columns, determination of relative retention times and Kováts indices, full scan electron ionization (EI) mass spectra, and optimized multiple reaction monitoring transitions for quantitative analysis. The resulting dataset enabled the establishment of sterol-specific EI-MS fragmentation signatures and retention increment principles, providing a robust framework for the reliable identification of known and previously uncharacterized sterols.