|Determination of Sanguinarine and Chelerythrine by HPLC
a. Acetronitrile(HPLC) b. Nanopure water c.NH4H2PO4 (A.R) d.Phosphoric acid(A.R)
HPLC (Shimadzu) Analytical balance (1/10000) Ultrasonic cleaner
0.45μm PTFE filter 50ml volumetric flask Screw cap vials
3) HPLC condition
Column: Kromasil C18 5m 4.6×200mm
Detector: UV 270nm
Mobile Phase: A=0.01mol/L NH4H2PO4 use 2%Phosphoric acidto adjust Ph=3
Flow Rate: 1.0 ml/min
Time(min) %A %B
0.01 67 33
10 62 38
14 62 38
15 67 33
20 67 33
Accurately weigh 30mg extraction into 50ml volumetric flask. Add 1% Phosphoric acid 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 1% Phosphoric acid) using a standard of Sanguinarine and Chelerythrine，Sonicate for about 10 minutes and transfer to screw-cap vials and can be stored in the refrigeration.
Determine the peak areas of the Sanguinarine and Chelerythrine acid on the sample chromatogram and compare to the corresponding peaks in the chromatogram for the standard solution. Calculate the weight percent.