Abstract:

Gandou decoction (GDD) is a traditional Chinese medicine prescription that has been widely used to treat copper metabolism disorders in China with remarkable clinical effect and lower toxicity. However, evaluation of the complexation ability of copper ions is challenging, which hinders screening and discovery of coordinate active ingredients in GDD. An analytical method is needed to determinate the complexation ability of chemical constituents with copper ions. In this study, a rapid and accurate method based on ultra-high performance liquid chromatography (UHPLC) was developed to determine the complexing ability of rhubarb with copper ions. First, the optimal coordination reaction conditions between active ingredients of rhubarb and copper ions were determined. The samples were separated using an Agilent Eclipse Plus C18 column (50 mm×2.1 mm, 1.8 μm) with 5 μL injection volumes. The mobile phase was gradient eluted with methanol and water containing 0.1% (v/v) phosphoric acid at a flow rate of 0.3 mL/min. The detection wavelength was 254 nm and the column temperature was 30 ℃. Under the optimized chromatographic conditions, the rhubarb constituents were effectively separated. Next, peak areas of rhubarb were calculated before and after the coordination reaction between copper ions. The complexing ability of active ingredients in rhubarb with copper ions was evaluated by calculating the rate of changes of their chromatographic peak areas. Finally, ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was used to identify the coordination active ingredients in rhubarb extract. Focusing on the coordination reaction conditions between active ingredients of rhubarb and copper ions revealed that the active ingredients of rhubarb and copper ions reached equilibrium by coordination reaction at pH 9 for 12 h. Methodological evaluation revealed the good stability and repeatability of the method. Under these conditions, 20 major components of rhubarb were identified by UPLC-Q-TOF-MS. According to the coordination rate of each component and copper ions, eight components with strong coordination were screened out (gallic acid 3-O-β-D-(6'-O-galloyl)-glucopyranoside, aloe emodin-8-O-β-D-glucoside, sennoside B, l-O-galloyl-2-O-cinnamoyl-glucoside, chysophanol-8-O-β-D-(6″-O-acetyl)-glucoside, aloe-emodin, rhein and emodin). The respective complexation rates of the components were 62.50%, 29.94%, 70.58%, 32.77%, 34.61%, 26.07%, 28.73% and 31.78%. Compared with other reported methods, the presently developed method can be used to screen the active ingredients of traditional Chinese medicines that have complexing ability with copper ions, especially in complex mixture systems. This study describes an effective detection technology for evaluating and screening the complexing ability of other traditional Chinese medicines with metal ions.

Key words: ultra-high performance liquid chromatography (UHPLC), coordination reaction, copper ion, rhubarb, hepatolenticular degeneration (HLD)