Determination of Psoralen and Isopsoralen by HPLC 1) Reagents a. Methanol (HPLC) b. Nanopure water
2) Apparatus 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 245nm Mobile Phase: A=Water B= Methanol A/B=48/52(isocratic) Flow Rate: 1.0ml/min Injection: 10μl
4) Procedure Samples Preparation Accurately weigh 30mg 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 0.2mg/ml in water) using a commercial reference standard of Psoralen and Isopsoralen,Sonicate for about 10 minutes and transfer to screw-cap vials and can be stored in the refrigeration.
5) Calculation Determine the peak areas of the Psoralen and Isopsoralen on the sample chromatogram and compare to the corresponding peaks in the chromatogram for the reference standard solution. Calculate the weight percent.
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