An HPLC method was developed for the simultaneous determination of hyoscyamine, scopolamine and anisodamine to investigate medical parts of Herba belladonnae in different harvest time. The solid phase was Shim-pack VP-ODS (4.6 mm × 250 mm, 5 µm), with isocratic elution using 0.1 % phosphoric acid solution (including 0.25 % SDS)-acetonitrile (60:40) as the mobile phase. The flow rate was 1 mL/min and the wavelength was 210 nm. The results of methodology all fit the analytical requests and the recovery were 97.3-103.0 %. Quantitative analysis of the three compounds showed that the contents of hyoscyamine in leaves and flowers were the highest, followed by fruits and roots and the third was stem. The contents of scopolamine in flowers and fruits were the highest. The contents of anisodamine in fruits were the highest. The relative contents of hyoscyamine showed an increasing trend during the growth period, while scopolamine and anisodamine showed an opposite trend, which would reach a balance finally. The method was simple, quick and reliable and could provide a scientific and technical platform for setting up a quality control standard.
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