Simultaneous Determination of Puerarin,3-Methoxy Puerarin,Mirificin and Daidzin in the Bioactive Fraction of Naodesheng Prescription by RP-HPLC/UV Method

KeyWord:Naodesheng prescription  bioactive fraction  high performance liquid chromatography(HPLC)  isoflavonoid  determination

Abstract:

      A reversed-phase high performance liquid chromatography coupled with UV detection(RP-HPLC/UV) was developed and validated for the simultaneous determination of four isoflavonoids£¬including puerarin(PU),3-methoxy puerarin(MP),mirificin(MI) and daidzin(DA),in the bioactive fraction of Naodesheng prescription,iª±e.a widely used herbal medicine(HM) in China mainly for treatment of cerebral arteriosclerosis,ischemic stroke,sequelae of cerebral hemorrhage and etc.The separation of four compounds was accomplished on a Hyspersil ODS-2 column(250 mm¡Á4.6 mm,5 ¦Ìm i.d.) by using methanol-water(25¡Ã75) as mobile phase with isocratic elution at a flow rate of 0.9 mL/min.The UV detection wavelength was set at 250 nm,and the column temperature was 25 ¡æ.The high resolution and reproducible peaks were achieved for four compounds in 20 min.All the calibration curves showed good linearities(r¡Ý0.999 7) over the test concentration range.The relative standard deviations(RSDs) of the method were less than 2.2% for precision,stability and reproducibility assays,and the spiked recoveries were in the range of 96%-102%.The total contents of the four isoflavonoids were determined to be in the range of 37.3%-37.8%in the bioactive fraction of Naodesheng prescription.