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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 the determination of six isoflavones (daidzin, daidzein, genistin, daidzein, daidzein and genistein) in food by high performance liquid chromatography (HPLC) Methods Accurately weigh a certain amount of crushed and mixed samples, extract the supernatant after 80% methanol, and pass through 0.22μm organic phase filter. A gradient elution was carried out on a Thermo Syncronis C18 column (250 mm × 4.6 mm, 5 μm) with 0.5% formic acid in water (A), acetonitrile (B) and methanol as mobile phase at a flow rate of 0.8 mL / 35 ℃, detected at a UV detector wavelength of 260 nm, the content of each component of soy isoflavones was quantified by external standard method. Results Sixteen isoflavones were separated and analyzed within 30 min. The linear range was 0.2-50 μg / mL for soy isoflavones (r> 0.999). The average recoveries were 96.9 % ~ 107.8%, relative standard deviation (RSD) was 0.6% ~ 5.0%, the detection limit was 0.03 ~ 0.1μg / mL, the limit of quantification was 0.1 ~ 0.3μg / mL. Conclusion The established method has high sensitivity and repeatability, and can meet the determination of soybean isoflavones content in different grain types.