红三叶草提取物中四种异黄酮的HPLC检测方法

Determination of four isoflavones in red clover extract by High-performance liquid chromatography

本研究的目的是建立一种可以同时测定红三叶草提取物中大豆苷元、染料木素、刺芒柄花素和鹰嘴豆素A含量的高效液相色谱检测方法。采用χBridge C18(250 mm×4.6 mm,5μm,Waters)色谱柱,以甲醇(A)和10 mmoL磷酸氢二钠溶液(B)为流动相,流速为1 mL/min,进样量为10μL,在不同的洗脱梯度、检测波长和柱温下检测四种异黄酮的含量。确定了最佳色谱条件:柱温为40℃,检测波长为270 nm,洗脱梯度为:0~5 min,20%~40%A;5~20 min,40%~75%A;20~23 min,75%~100%A;23~26 min,100%A;26~26.5 min,100%~20%A;26.5~35 min,20%A。结果表明:四种异黄酮在浓度为1~50μg/mL时具有良好的线性关系(R2>0.999),保留时间稳定,精密度和稳定性的相对标准偏差(RSD)均小于2%,加标回收率为96%~100.10%。建立的检测方法符合分析方法学评价的要求,可用于同时检测红三叶草提取物中的鹰嘴豆素A等四种异黄酮。

The objective of this study was to establish a High performance liquid chromatography(HPLC)method for simultaneous determination of daidzein,genistein,formononetin and biochanin A in red clover extract.Analytes were separated onχBridge C18(250 mm×4.6 mm,5μm,Waters)column with methanol(A)and 10 mmol disodium hydrogen phosphate solution(B)as mobile phases at a flow rate of 1 mL/min.The injection volume was 10μL.The content of four isoflavones was detected under different elution gradients,detection wavelengths and column temperatures.The optimal chromatographic conditions were determined:the column temperature was 40℃,the detection wavelength was 270 nm,and gradient elution:0~5 min,20%~40%A;5~20 min,40%~75%A;20~23 min,75%~100%A;23~26 min,100%A;26~26.5 min,100%~20%A;26.5~35 min,20%A.The results showed that high correlation coefficients(R2>0.999)for the linear range established for four isoflavones of 1~50μg/mL,the retention times was stable.The relative standard deviation(RSD)of precision and stability were less than 2%,and the recovery rate ranged from 96%to 100.10%.The detection method meet the requirements of analytical methodology evaluation and the detection time was shorter,and could be used to simultaneously detect four isoflavones such as biochanin A in red clover extract.