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Background: Fucosterol is a compound commonly found in algae that has various biological activities. The purpose of this study was to develop a high-performance liquid chromatography (HPLC) validation method for fucosterol and to compare the fucosterol contents of 11 algal species from Ulleungdo, Korea. Method: In this study, we successfully isolated and identified fucosterol from a 70% EtOH extract of Sargassum miyabei, and subsequently conducted specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision analyses for development of an HPLC validation method. Fucosterol contents were compared using the established HPLC validation conditions. Results: We successfully isolated fucosterol from a 70% EtOH extract of S. miyabei and identified it based on spectroscopic analysis. On the basis of HPLC validation using the fucosterol isolated from S. miyabei, we confirmed specificity (8.5 min), linearity (R2 = 0.9998), LOD (3.20 μg mL−1), LOQ (9.77 μg mL−1), accuracy (intra-day and inter-day variation, 90–110%), and precision (RSD, 1.07%). Fucosterol contents in the 11 assessed algal species ranged from 0.22 to 81.67 mg g−1, with the highest content being recorded in a 70% EtOH extract of Desmarestia tabacoides (81.67 mg g−1), followed by that of Agarum clathratum (78.70 mg g−1). Conclusions: The results indicate that 70% EtOH extracts of D. tabacoides and A. clathratum containing fucosterol with various effects can be potential alternative sources of fucosterol.

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