Establishment of an HPLC Method for Simultaneous Determination of Ten Flavonoids in Germinated Black Wheat
To investigate the changes in the mass fraction of flavonoids in the germination process of black wheat,a high-performance liquid chromatography(HPLC)method was established for simultaneous determination of 10 flavonoids in germinated black wheat.An Agilent 1260 ZORBAX SB-C18 column(4.6 mm×250 mm×5 μm)was used as the stationary phase,with a gradient elution of acetonitrile and 0.8%phosphoric acid aqueous solution as the mobile phase.The flow rate was set at 0.8 mL/min,with a detection wavelength of 225 nm and the column temperature maintained at 25℃.The results showed that the mass concentration of 10 flavonoids,including puerarin,epicatechin,rutin,luteolin,quercetin,apigenin,naringenin,kaempferol,isorhamnetin,and chalcone,exhibited a good linear relationship with peak area(R2≥0.999 1,n=6)within the mass concentration range of 20~100 μg/mL.Precision,repeatability,and stability tests of these compounds showed that the relative standard deviation(RSD)of the peak area was less than 2.00%,and the spiked recovery rate ranged from 98.72%to 102.03%,demonstrating the accuracy and reliability of the established method.Additionally,the extraction method for 10 flavonoids in germinated black wheat was optimized,with the best efficiency achieved using a hydrochloric acid-ethanol solution with a 70%ethanol volume fraction(a solid-to-liquid ratio of 1 g:25 mL),and refluxing at 85℃ for 90 min.The mass fractions of 10 flavonoid were also determined in black wheat germinated for 1,3,5,and 7 d.This research lays a foundation for studying functional bioactive ingredients in germinated black wheat and offers a reference for the quality control and evaluation of its products.
black wheathigh-performance liquid chromatographyflavonoidsgermination