| Determination of THP by HPLC
a. Methanol (HPLC) b. Nanopure water c.Na2HPO4、NaH2PO4(A.R)
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
Detector: UV 280nm
Mobile Phase: A:B:C=1.8:65:35(isocratic)
A=phosphate buffer solution (add 0.98g Na2HPO4 and 2.24g NaH2PO4 into 100ml) B= MeOH
C= Nanopure water
Flow Rate: 1.0ml/min
Accurately weigh 25mg extraction into 50ml volumetric flask. Add methanol 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 0.5mg/ml in methanol) using a commercial reference standard of THP，Sonicate for about 10 minutes and transfer to screw-cap vials and can be stored in the refrigeration.
Determine the peak areas of the THP on the sample chromatogram and compare to the corresponding peaks in the chromatogram for the reference standard solution. Calculate the weight percent.