Samples extracted using the organic solvent described above were dissolved in 500 μL methanol–chloroform (1:2, v/v). Then, the samples were applied to a COSMOSIL 5C18-MS-II packed column (4.6 mm I.D. × 150 mm) (Nacalai Tesque, Kyoto, Japan), which was prewashed with methanol–ethanol (3:1, v/v). Samples (100 μL each) were applied to the column and eluted using methanol–ethanol (3:1, v/v). The peaks were detected using UV monitor (280 nm) for quantification of vitamin K1 by HPLC.
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