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Sterols are an important type of lipids, and play many important roles in physiological and pathological processes. However, comprehensive analysis of sterols especially identification of unknown sterols is challenging. In this work, LC-MS with all ion fragmentation (AIF) technology was developed for untargeted analysis of sterols in biological samples. AIF technology provided holistic and multi-dimensional characterization for both knowns and unknowns sterols, including accurate m/z, isotope pattern, retention time (RT), and co-eluted