Simultaneous Determination of 11 Flavonoid Aglycones in Pueraria Lobata by Solid Phase Extraction-Liquid Chromatography-Tandem Mass Spectrometry

A method for simultaneous determination of 11 flavonoid aglycones (puerarin, liquiritigenin, daidzein, scutellarein, glycitein, quercetin, tectorigenin, genistein, kaempferol, baicalein and formononetin) in Pueraria lobata by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with solid phase extraction was developed. Pueraria lobata sample of 0.10 g were hydrolyzed with 0.5 g of tert-butylhydroquinone, 36 mL of 50% (φ) ethanol solution and 4 mL of hydrochloric acid for 120 min under the protection of nitrogen to extract 11 flavonoid aglycones. The sample solution was purified by Waters HLB solid phase extraction column with 20% (φ) methanol solution as leacheate and methanol as eluent. Mightysil RP-18 chromatographic column was used as the stationary phase, and the methanol-water system (daidzein, daidzein and formononein) or methanol-0.15% (φ, the below same) formic acid solution system (other 8 flavonoid glycosides and 11 flavonoid aglycones) was used as the mobile phase for gradient elution. 11 flavonoid aglycones were determined by LC-MS/MS. The mixed standard solution series prepared with a mixture of methanol and 0.15% formic acid solution at volume ratio of 4∶6 were used to draw the standard curves, and external standard method was used for quantitative analysis. As shown by the results, linear relationships between values of mass fraction of 11 flavonoid aglycones and their peak area were kept in the range of 0.05%-2.50%, with detection limits (3S/N) of 0.04% for puerarin, daidzein and baicalein, and 0.02% for liquiritigenin, scutellarein, glycitein, quercetin, tectorigenin, genistein, kaempferol and formononetin. Recovery test was made on actual samples at 3 concentration levels by stardand addition method, giving results in the range of 75.0%-94.0%, with RSDs (n=6) of the determined values in the range of 3.5%-12%. This method was applied to Pueraria lobata sample, and puerarin, daidzein, glycitein, genistein and formononetin were detected in varying degrees.