The three-component mobile phase, methanol-acetonitrile-water, has been frequently used for the separation of natural products in complicated herbal extracts by reversed-phase liquid chromatography, especially when two-component solvent systems do not work. However, the rational for optimization of this three-solvent system is not clear, so far. In this study, the retention behavior of different types of natural products in RP-LC with a methanol-acetonitrile-water elution system was studied. A total of 27 compounds from four classes, including steroids, flavonoids, phenolic acids, and triterpene saponins were analyzed by high-performance liquid chromatography coupled with mass spectrometry or ultraviolet detectors. It was interesting to find that prolonged retention times and improved separation capabilities were obtained when the acetonitrile-to-methanol ratio was 3-16%. This rule applied to all four classes of natural products under different instrumental conditions, and could be valuable for the separation of natural products in complicated samples.
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