Objective: To develop a method for the determination of harpagide and harpagoside in Scrophulariae Radix (Xuanshen) by HPLC-UV under double wavelength, and to study the changes of these two constituents during processing, and to set the limitation of harpagide and harpagoside contents in crude drug and sliced pieces of Xuanshen.
Method: The analyses were performed on an Agilent Technologies ZORBAX SB-C18 (4.6 mm x 250 mm, 5 microm) eluted with acetonitrile-water (containing 0.03% phosphoric acid) in gradient model. The flow rate was 1.0 mL x min(-1) . The column temperature was 25 degrees C. The UV detector wavelength was set at 210 nm before 13 min and then changed to 280 nm.
Result: Harpagide and harpagoside were separated well. The linear calibration curves were obtained over of 0.0549 - 1.46 microg for harpagide (r = 0.9999, n =7) ,0.0225 - 0.900 microg for harpagoside (r = 0.9998, n = 9). The recoveries ( +/- RSD)% were 98.1 (+/- 2.4)% for harpagide and 98.8 (+/- 4.3)% for harpagoside. The contents of harpagide were 0. 277% - 0.620%, harpagoside were 0.078% - 0.362% in Xuanshen, and harpagide were 0.276% - 1.059%, harpagoside were 0. 059% - 0.183% in sliced Xuanshen, respectively. After the processing of Scrophulariae Radix, the content of harpagide increases 13.7% - 96.0%, while harpagoside decreases 11.0%-73.9%.
Conclusion: This method is simple, accurate, and can be used for the quality control of Scrophulariae Radix. We propose that the total content of harpagide and harpagoside in either crude drug or sliced pieces of Scrophulariae Radix should not be less than 0.45%.