|Determination of Ecdysone by HPLC
a. Methanol (HPLC) b. Acetronitrile C. Nanopure water
HPLC (Beckman) Analytical balance (1/10000) Ultrasonic cleaner
0.45μm PTFE filter 50ml volumetric flask Screw cap vials
3) HPLC condition
Column: Hypersil ODS C18 5μm , 4.6×200mm
Mobile Phase: A=water B= Acetronitrile
Time（min） 0.01 18 19 20 25
%A 82 72 72 82 82
%B 18 28 28 18 18
Flow Rate: 1.0ml/min
Accurately weigh 20mg extraction into 50ml volumetric flask. Add MeOH:Water(V/V=1:1) to volume and sonicate to mix, filter through a 0.45μm PTFE filter into a screw cap vial and proceed with the analysis.
Standard preparation /calibration
Prepare a standard solution [approximately 0.1mg/ml in MeOH:Water(V/V=1:1)] using a commercial reference standard of Ecdysone，Sonicate for about 10 minutes and transfer to screw-cap vials and can be stored in the refrigeration.
Determine the peak areas of the Ecdysone on the sample chromatogram and compare to the corresponding peaks in the chromatogram for the reference standard solution. Calculate the weight percent.