Abstract: To establish an accurate and reliable determination method for the coupling ratio of the artificial antigen, the HPLC was applied to the determination of free 2-quinoxalinecarboxylic acid of the artificial antigen in this research. The Waters 2695 highly effective liquid phase chromatographs was used by ultraviolet detector 2489 at 320 nm, the mobile phase was methanol∶water(60∶40), column worked at 30 ℃ and the flow rate was 1 mL/min. The results showed that the linearity range of 2-quinoxalinecarboxylic acid was 0.2 to 128 μg/mL,y=17526x+4648,R² was equal to 1.RSD was 0.28%. This method was accurate and reliable which could be used to determinate the coupling ratio of the artificial antigen.

Key words: QCA; HPLC; the coupling ratio