Objective: To establish a determination method for pulsatilla saponin D in Pulsatillae Radix. Method: The content of pulsatilla saponin D was determined by RP-HPLC

chromatographic conditions were as follows:Elite Hypersil ODS2 C18 column(4.6 mm×250 mm

5 μm)

mobile phase of acetonitrile-water-formic acid(38:62:0.1)

flow rate 1.0 mL·min-1

detection wavelength 203 nm

column temperature 25℃.Content difference of pulsatilla saponin D in Pulsatillae Radix from different areas was investigated. Result: Pulsatilla saponin D had good linear relationship in 0.311 2-3.112 μg(r=0.999 9)

RSD of instrument precision was 1.36%

RSD of method repeatability was 1.65%

samples solution was stable within 24 h

the average recovery was 98.07%(RSD 0.64%).The content of pulsatilla saponin D in Pulsatillae Radix from Jilin was the highest(0.256%)

which was 20 folds higher than Pulsatillae Radix from Guangdong. Conclusion: This method was simple

accurate and repeatable.The content of pulsatilla saponin D in Pulsatillae Radix from different areas had much difference

so materials choice should be careful.