Objective: To develop an HPLC method for quantitative determination of 2-hydroxy-3-methoxy-7-methylanthraquinone (anthraquinone Ⅰ) and 2-hydroxy-1-methoxyanthraquinone (anthraqui-none Ⅱ) in Hedyotis diffusa. Method: A Eclipse XDB-C18 column (4.6 mm×250 mm

5 μm) was adopted with a mobile phase of methanol-water (60：40) at the flow rate of 1.0 mL·min-1

the detection wavelength was set at 272 nm and the column temperature was 25 ℃. Result: The calibration curves were linear in the ranges of 4.0-160 mg·L-1 (r=0.999 6) for anthraquinone Ⅰ and 4.0-160 mg·L-1(r=0.999 5) for anthraquinone Ⅱ. The average recoveries (n=9) of anthraquinone Ⅰ and anthraquinone Ⅱ were 99.27% and 99.08%

respectively. The sample solution was steady within 24 h. Conclusion: The method is precise and accurate for determination of 2-hydroxy-3-methoxy-7-methylanthraquinone and 2-hydroxy-1-methoxyanthraquinone in H. diffusa.