高效液相色谱法同时测定粮食中6种大豆异黄酮

Determination of 6 kinds of isoflavones in food crops by high performance liquid chromatography

目的建立高效液相色谱法(high performance liquid chromatography,HPLC)测定粮食中6种大豆异黄酮(大豆苷、大豆黄苷、染料木苷、大豆素、大豆黄素和染料木素)含量的分析方法。方法准确称取一定量粉碎混匀后的样品,经80%甲醇提取后取上清液,过0.22μm有机相滤膜上机。采用Thermo Syncronis C18柱(250 mm×4.6 mm,5μm),以0.5%甲酸水溶液(A)、乙腈(B)和甲醇(C)作为流动相进行梯度洗脱,流速0.8m L/min,柱温为35℃,于紫外检测器波长260 nm处检测,大豆异黄酮各组分含量以外标法进行定量。结果本方法在30 min内完成6种大豆异黄酮的分离分析,大豆异黄酮各组分浓度在0.2~50μg/mL范围内呈良好的线性关系(r>0.999),平均加标回收率为96.9%~107.8%,相对标准偏差(relative standard deviation,RSD)为0.6%~5.0%,检出限为0.03~0.1μg/mL,定量限为0.1~0.3μg/mL。结论建立的方法具有较高的灵敏度和重复性,能满足不同粮食种类中大豆异黄酮含量测定。

Objective To establish a method for simultaneous determination of 6 kinds of isoflavones（daidzin, glycitin, genistin, daidzein, glycitein and genistein） in food crops by high performance liquid chromatography（HPLC）. Methods A certain amount of crushed mixed samples were weighed accurately, followed by extracting with 80% methanol, then the supernatant was filtered through 0.22 μm membrane microfilter for analysis. The samples were separated with a Thermo Syncronis C18 column（250 mm×4.6 mm, 5 μm） at the flow rate of 0.8 m L/min by gradient elution using 0.5% formic acid water（solvent A）, acetonitrile（solvent B） and methanol（solvent C）. The temperature of the column oven was maintained at 35 ℃ and the signal was detected with ultraviolet detector in 260 nm, and the contents of 6 kinds of isoflavones were quantitatively analyzed by external standard method. Results The 6 kinds of isoflavones were separated within 30 min, each compound showed a good linear relationship between peak areas and concentrations in the concentration range of 0.2-50 μg/mL（r〉0.999）. The recoveries of 6 kinds of isoflavones in food crops were 96.9%-107.8% with relative standard deviations（RSD） of 0.6%-5.0%. The limits of detection（LODs） were 0.03-0.1 μg/mL and the limit of quantification（LOQs） were 0.1-0.3 μg/mL. Conclusion The established method exhibits high sensitivity and repeatability, which can satisfy the determination of isoflavones in different food crops.