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Accurate and efficient analytical detection of these substances in complex biological matrices is critical for disease mechanism research and biomarker discovery. However, challenges including complex biological matrices, large differences in analyte abundance, susceptibility to auto\u2011oxidation, low ionization efficiency, and difficulty in isomer separation pose severe obstacles to reliable analysis, making sample preparation the core step for accurate quantification and valid identification. Continuous innovations have been achieved in sample preparation techniques, such as liquid\u2013liquid extraction, solid\u2011phase extraction, deep eutectic solvent extraction, molecularly imprinted extraction, derivatization, as well as integrated and automated workflows. These advances improve extraction efficiency, selectivity, antioxidant protection, and throughput, while effectively reducing matrix interference and artificial oxidation. This paper systematically reviews the research progress in sample preparation for cholesterol and oxysterol analysis in complex biological matrices from 2015 to 2026, highlights the technical advantages, application scenarios, and current limitations of various pretreatment strategies, and prospects the development trends toward greening, intelligence, and high throughput.<\/jats:p>","DOI":"10.1002\/jssc.70539","type":"journal-article","created":{"date-parts":[[2026,10,10]],"date-time":"2026-10-10T07:07:07Z","timestamp":1791616027000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Recent Advances in Sample Preparation for Cholesterol and Oxysterol Analysis in Complex Biological Matrices"],"prefix":"10.1002","volume":"49","author":[{"given":"Haizhen","family":"Zhang","sequence":"first","affiliation":[{"name":"The MOE Key Laboratory of Standardization of Chinese Medicines Shanghai Key Laboratory of Compound Chinese 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