质谱导向高效液相色谱一测多评测定葛根提取物中异黄酮的相对含量  

Determination of Relative Contents of Isoflavones in Radix Puerariae Extract by Mass Spectrometry Guided High Performance Liquid Chromatography Using Quantitative Analysis of Multi-component with Single Marker

作　　者：农金梅 韩默 吴四清 李立兵 邓后勤[1] NONG Jinmei;HAN Mo;WU Siqing;LI Libing;DENG Houqin(College of Food Science and Technology,Hunan Agricultural University,Changsha 410128,China;Hunan Jiuwei Biopharmaceutical Co.Ltd.,Changsha 410125,China)

机构地区：[1]湖南农业大学食品科学技术学院,湖南长沙410128 [2]湖南九维生物医药有限公司,湖南长沙410125

出　　处：《食品科学》2025年第8期259-266,共8页Food Science

摘　　要：建立一种质谱(mass spectrometry,MS)定性鉴别结合高效液相色谱(high performance liquid chromatography,HPLC)法定量分析,并以葛根素为单一外标物的一测多评测定葛根提取物中多种异黄酮类成分相对含量的方法,为保健食品中提取物原料的综合评价提供参考。使用EcoPak C18 Plus色谱柱(4.6 mm×150 mm,5μm),以中药材葛根饮片为对照样优化的色谱条件为:流动相体系0.1%氨水溶液(pH 7.5)-乙腈-甲醇(梯度洗脱),进样体积10μL,检测波长250 nm,柱温(35±0.2)℃,流速0.8 mL/min;MS条件为电喷雾离子源,全离子扫描,负离子模式检测,扫描范围m/z 200~1000,电喷雾电压12000 psi,离子源温度为600℃,载气为氮气。结果表明,葛根饮片中异黄酮化合物的分离度良好,高效液相色谱-紫外(high performance liquid chromatography-ultra violet,HPLC-UV)检测中21个色谱峰重复性较好(相对标准偏差(relative standard deviation,RSD)<0.7%);MS的21个离子流峰的保留时间重复性良好(RSD<1.0%),且两者的保留时间可相对应。结合文献研究鉴定了18个葛根异黄酮化合物发现葛根素质量浓度在0.052~500μg/mL线性范围内线性关系良好(R^(2)=0.9992),检出限为0.015μg/mL(信噪比≥3),同时以葛根素为参照,相对定量了18种葛根异黄酮化合物的含量。植物提取物成分复杂,以质谱定性鉴别具有用量少、操作简单、快速准确的特点,本研究方法可更低成本、方便快捷的综合评价提取物的品质,为葛根类药食同源物质在保健食品开发和应用提供参考。This study developed a method for the qualitative and quantitative analysis of isoflavones in Radix Puerariae extract using mass spectrometry(MS)and high performance liquid chromatography(HPLC).The relative contents of various kinds of isoflavones in the extract were determined using the quantitative analysis of multi-component with single marker(QAMS)method with puerarin as a single external standard.Chromatography was accomplished using a EcoPak C18 Plus column(4.6 mm×150 mm,5μm).Using Radix Puerariae slices as control,the optimized chromatographic conditions were determined as follows:elution with a mobile phase consisting of 0.1%ammonia solution(pH 7.5),acetonitrile and methanol at a flow rate of 0.8 mL/min,an injection volume of 10μL,column temperature of(35±0.2)℃,and detection wavelength of 250 nm.The MS conditions were set as follows:electrospray ionization source(ESI),full scan mode,negative ion mode detection,scan range of m/z 200–1000,electrospray voltage of 12000 psi,ion source temperature of 600℃,and nitrogen as carrier gas.The results showed that the separation of isoflavone compounds from Radix Puerariae slices was good,and 21 peaks were obtained with good reproducibility by HPLC with an ultra violet(HPLC-UV)(relative standard deviations(RSD)<0.7%).The retention time reproducibility of 21 ion current peaks in MS was good(RSD<1.0%),and the retention times of the chromatographic and ion current peaks were matched with each other.Totally 18 isoflavones were identified based on relevant literature.Good linearity was observed within the puerarin range of 0.052–500μg/mL(R^(2)=0.9992),and the detection limit was 0.015μg/mL(RSN≥3).Furthermore,using puerarin as a reference standard,the contents of the 18 isoflavone compounds were relatively quantified.MS is useful for qualitative identification of plant extracts,which has complex composition,with the advantages of low consumption of materials,simple operation,rapidity and accurate results.The method proposed in this study can comprehensively

关 键 词：高效液相色谱-质谱法 葛根 异黄酮 一测多评 

分 类 号：TS207.3[轻工技术与工程—食品科学]