Application of Two Reference Substances for Determination of Multiple Components Method for the Determination of Six Components in ZaoRen AnShen Granules
OBJECTIVE To establish a two reference substances for determination of multiple components method(TRSDMC)for the determination of six components(spinosin,6'''-feruloylspinosin,rosmarinic acid,schisan-drin,γ-schisandrin and salvianolic acid B)of ZaoRen AnShen granules,and to verify the accuracy and feasibility of the method in the quality evaluation of ZaoRen AnShen granules.METHODS HPLC method was used with aceto-nitrile as mobile phase A and 0.2%glacial acetic acid as mobile phase B in gradient elution mode,and the flow rate was 1.0 mL·min-1,the determination wavelengths were 320 nm for spinosin,6'''-feruloylspinosin,rosmarinic acid,salvianolic acid B,250 nm for schisandrin and γ-schisandrin;the column temperature was 28 ℃.The actual retention time of six components in ZaoRen AnShen granules was determined on five different types of C18 chromatographic col-umns.The average retention time of each component in each column was calculated to obtain the standard retention time of each component.Spinosin(peak 1)and γ-schisandrin(peak 5)were selected as the reference substances for the determination.The chromatographic peaks of each component were accurately located by linear calibration using two reference substances,and the position of each component in unknown chromatographic column could be predicted in the same method.The methodology was validated.Taking γ-schisandrin as reference,the content of each compo-nent was determinated.At the same time,the contents of cinnamaldehyde,schizandrin and schisandrin A in ZaoRen AnShen granules were determined by external standard method,and the contents obtained by TRSDMC and external standard method were compared to verify the scientificity and feasibility of TRSDMC.RESULTS The linear calibra-tion using two reference substances can accurately predict the retention time of the tested components on the unknown column.The relative correction factors of spinosin,6'''-feruloylspinosin,rosmarinic acid,schisandrin and salvianolic acid B were 0.7342,0.6063,0.5664,0.9975 and 0.7773,respectively,with RSD values less than 1%.There was no significant difference between the contents obtained by TRSDMC and external standard method for each compo-nent.CONCLUSION TRSDMC for simultaneous determination of 5 components in ZaoRen AnShen granules is fea-sible and accurate.
ZaoRen AnShen granulesTwo reference substances for determination of multiple components method(TRSDM)Ela-tive correction factor quantificationSpinosinγ-schisandrin6'''-feruloylspinosinSalvianolic acid BSchisandrinRosmarinic acid