高效液相色谱法测定杜仲中主要成分和食品中大豆异黄酮
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摘要
高效液相色谱法(High Performance Liquid Chromatography,HPLC)是在气相色谱法和经典液相色谱法的基础上发展起来的新型分析分离技术。鉴于其快速、准确、灵敏、简便的特点,至今,己在食品工业、环境监测、生物工程、制药工业、石油化工等领域获得广泛的应用。
本文由绪论和研究报告两部分组成。绪论部分简单概述了高效液相色谱法、色谱技术方面的进展、高效液相色谱在杜仲有效成分分析中的应用和大豆异黄酮的研究进展。研究报告部分建立了杜仲中绿原酸和芦丁HPLC法测定和食品中大豆异黄酮含量的HPLC法测定,包括：HPLC法测定豆奶粉中四种大豆异黄酮含量；不同豆类中四种大豆异黄酮的HPLC法测定；HPLC法测定绿豆芽中四种大豆异黄酮的含量。
1.HPLC法测定杜仲中绿原酸和芦丁
建立HPLC法测定杜仲中绿原酸和芦丁含量,并对不同产地杜仲中绿原酸和芦丁含量进行测定。采用phenomenex C18色谱柱(150 mmx4.6 mm,5μm),以甲醇-冰醋酸(pH=3.10)(45：55)为流动相洗脱,体积流量为0.8 mL/min,紫外检测波长为327 nm,采用外标法测定不同产地杜仲中绿原酸和芦丁的含量。绿原酸和芦丁分别在0.1275-24.0000μg/mL一=0.9996)、0.1250-25.0000μg/mL(rp=0.9996)线性关系良好,平均回收率分别为99.23%和97.13%,RSD分别为1.02%和2.98%。本方法简便、快速、灵敏度高、重现性好,为不同产地杜仲质量标准的进一步研究提供了科学依据。
2.HPLC法测定豆奶粉中四种大豆异黄酮含量
本文建立了同时测定豆奶粉中四种大豆异黄酮(大豆苷、染料木苷、大豆苷元和染料木素)含量的高效液相色谱法,同时对从豆奶粉中提取大豆异黄酮的工艺进行了优化。采用日本岛津LC-20A高效液相色谱仪进行测定的色谱条件为：色谱柱为phenomenex C18(150mm×4.6 mm,5.0μm),采用乙腈-0.2%甲酸为流动相进行梯度洗脱,流速为0.8 mL/min,检测波长为260 nm。大豆苷,染料木苷,大豆苷元,染料木素的线性范围分别是0.74-100.00μg/mL,0.74-200.00/μg/mL,0.]67～500.00 gg/mL,0.198～160.00μg/mL(r>0.9996),线性关系良好；加样回收率(n=9)分别为100.01%,100.01%,99.48%,99.91%,方法简便、灵敏、准确,适用于豆奶粉中这四种大豆异黄酮成分的含量测定和产品质量控制。从豆奶粉中提取大豆异黄酮的最佳工艺为：用70%的乙醇在60℃条件下超声提取40 min。
3.不同豆类中4种大豆异黄酮的HPLC法测定
建立同时测定豆类中大豆苷,染料木苷,大豆苷元,染料木素四种大豆异黄酮成分含量的高效液相色谱法,色谱柱为phenomenex C18(150 mm×4.6 mm,5.0 gm),采用流动相乙腈-0.2%甲酸进行梯度洗脱,流速为0.8 mL/min,检测波长为260 nm。探讨了提取黑豆中四种大豆异黄酮的最佳条件,结果显示,70%的乙醇在60℃超声提取40 min,效果最佳。大豆苷,染料木苷,大豆苷元,染料木素的线性范围分别是0.74～100.00μg/mL,0.74～400.00μg/mL,0.167～500.00μg/mL,0.198～160.00μg/mL(r>0.9996),线性关系良好；加样回收率(n=9)分别为99.81%,99.99%,99.49%,99.89%,本方法简便、灵敏、准确,适用于豆类中这四种大豆异黄酮成分的含量测定和产品质量控制。
4.HPLC法测定绿豆芽中四种大豆异黄酮的含量
本文建立了同时测定绿豆芽中四种大豆异酮(大豆苷、染料木苷、大豆苷元和染料木素)含量的高效液相色谱方法,对从绿豆芽中提取四种大豆异黄酮的工艺进行了优化,最佳工艺为：用60%的乙醇在55℃条件下超声提取60 min,料液比为1：12,同时探究了绿豆发芽不同天数四种大豆异黄酮的含量。采用日本岛津LC-20A高效液相色谱仪进行测定的色谱条件为：色谱柱为phenomenex C18(150 mmx4.6 mm,5.0μm),采用乙腈-0.2%甲酸为流动相进行梯度洗脱,流速为0.8 mL/min,检测波长为260 nm。大豆苷,染料木苷,大豆苷元,染料木素的线性范围分别是0.74～100.00-μg/mL,0.74～200.00-μg/mL,0.167～500.00μg/mL,0.198-160.00μg/mL(r>0.9996),线性关系良好；加样回收率(n=9)分别为99.99%,100.01%,100.07%,99.81%,该方法简便、灵敏、准确,为日常生活中绿豆芽的最佳食用时间提供了科学依据。
High Performance Liquid Chromatography(HPLC)as an important modern measurement of analysis and separation is deveopled on the basis of gas Chromatography and classical Liquid Chromatography. Due to its high-speed, accuracy, sensitivity and convenience, it has been widely used in the areas of foodstuff, environment, biology engineering, pharmacy and petroleum so far.
The thesis includes two sections-review and research report. In the review section, information about high performance liquid chromatography such as characteristic, the development of chromatography technology and application in the effective elments of herb medicine Eucommia ulmoides Oliv by HPLC analysis and isoflavones research was summarized. In the next research report section, analysis methods of determination of chlorogenic acid and rutin in Eucommia ulmoides Oliv and isoflavones in food were reported. That is:Determination of four isoflavones in soybean milk powder by HPLC; Determination of four isoflavones in different beans by HPLC; Determination of four isoflavones in mung bean sprouts by HPLC.
1.Determination of chlorogenic acid and rutin in Eucommia ulmoides Oliv by HPLC
To establish an HPLC method for determination of chlorogenic acid and rutin, and determination chlorogenic acid and rutin in Eucommia ulmoides Oliv from different places. Using a phenomenex C1g column(150 mm×4.6 mm,5μm),the mobile phase of methanol-acetic acid(pH=3.10)(45:55) with elution, the flow rate was 0.8 mL/min, the detection wavelength was at 327 nm, the content of chlorogenic acid and rutin was determined by external standard method. The calibration curves for chlorogenic acid and rutin were linear between 0.1275 and 24.0000μg/mL, between 0.1250 and 25.0000μg/mL(r=0.9996 and r=0.9996 respectively).The average recoveries were 99.23% and 97.13%.RSD were 1.02% and 2.98%. The method is simple, rapid, sensitive and reproducible, it provides scientific proof for quality standard in Eucommia ulmoides Oliv from different places.
2.Determination of four isoflavones in soybean milk powder by HPLC
To develop an HPLC method for determination of four isoflavones, daidzin, genistin, daidzein, genistein in soybean milk powder. An phenomenex C18 column(150 mm×4.6 mm,5.0μm)was applied, a gradient mobile phase composing of acetonitrile and 0.2% formic acid was used, and the flow rate was 0.8 mL/min, detective wavelength was set at 260 nm.The best condition of extracted four isoflavones of soybean milk powder was studied.The results showed the best condition was the ethanol concentration 70%,60℃,extraction for 40 min. The results were obtained that quantitative linear range of four isoflavones were 0.74-100.00μg/mL,0.74-200.00μg/mL,0.167～500.00μg/mL,0.198～160.00μg/mL(r>0.9996)respectively, and the average recoveries(n=9)were 100.01%,100.01%,99.48%.99.91% respectively. A specific, sensitive, simple and reproducible HPLC method has been established for determination of four isoflavones in soybean milk powder.
3.Determination of four isoflavones in different beans by HPLC
To develop an HPLC method for determination of four isoflavones, daidzin, genistin, daidzein, genistein in different beans. An phenomenex C1g column(150 mm×4.6 mm,5.0 vm)was applied, a gradient mobile phase composing of acetonitrile and 0.2% formic acid was used, and the flow rate was 0.8 mL/min, detective wavelength was set at 260 nm.The best condition of extracted four isoflavones of black beans was studied. The results showed the best condition was the ethanol concentration 70%,60℃,extraction for 40 min. The results were obtained that quantitative linear range of four isoflavones were 0.74～100.00μg/mL,0.74～400.00μg/mL,0.167v500.00μg/mL, 0.198～160.00μg/mL(r>0.9996)respectively, and the average recoveries(n=9)were 99.81%. 99.99%,99.49%,99.89% respectively. A specific, sensitive, simple and reproducible HPLC method has been established for determination of four isoflavones in different beans.
4.Determination of four isoflavones in mung bean sprouts by HPLC
To develop an HPLC method for determination of four isoflavones. daidzin, genistin, daidzein, genistein in mung bean sprout. An phenomenex C18 column(150 mm×4.6 mm,5.0μm)was applied, a gradient mobile phase composing of acetonitrile and 0.2% formic acid was used, and the flow rate was 0.8 mL/min, detective wavelength was set at 260 nm.The best condition of extracted four isoflavones of mung bean sprout was studied, the results showed the best condition was the ethanol concentration 60%,55℃,extraction for 60 min with the solid-liquid ratio 1:12(g/mL).The results were obtained that quantitative linear range of four isoflavones were 0.74℃100.00μg/mL,0.74～200.00μg/mL,0.167～500.00μg/mL,0.198～160.00μg/mL(r>0.9996)respectively, and the average recoveries(n=9)were 99.99%,100.01%,100.07%.99.81% respectively. A specific, sensitive, simple and reproducible HPLC method has been established for determination of four isoflavones in mung bean sprouts. It provided scientific bases for the best eating time of mung bean sprout everyday.
本文由绪论和研究报告两部分组成。绪论部分简单概述了高效液相色谱法、色谱技术方面的进展、高效液相色谱在杜仲有效成分分析中的应用和大豆异黄酮的研究进展。研究报告部分建立了杜仲中绿原酸和芦丁HPLC法测定和食品中大豆异黄酮含量的HPLC法测定,包括：HPLC法测定豆奶粉中四种大豆异黄酮含量；不同豆类中四种大豆异黄酮的HPLC法测定；HPLC法测定绿豆芽中四种大豆异黄酮的含量。
1.HPLC法测定杜仲中绿原酸和芦丁
建立HPLC法测定杜仲中绿原酸和芦丁含量,并对不同产地杜仲中绿原酸和芦丁含量进行测定。采用phenomenex C18色谱柱(150 mmx4.6 mm,5μm),以甲醇-冰醋酸(pH=3.10)(45：55)为流动相洗脱,体积流量为0.8 mL/min,紫外检测波长为327 nm,采用外标法测定不同产地杜仲中绿原酸和芦丁的含量。绿原酸和芦丁分别在0.1275-24.0000μg/mL一=0.9996)、0.1250-25.0000μg/mL(rp=0.9996)线性关系良好,平均回收率分别为99.23%和97.13%,RSD分别为1.02%和2.98%。本方法简便、快速、灵敏度高、重现性好,为不同产地杜仲质量标准的进一步研究提供了科学依据。
2.HPLC法测定豆奶粉中四种大豆异黄酮含量
本文建立了同时测定豆奶粉中四种大豆异黄酮(大豆苷、染料木苷、大豆苷元和染料木素)含量的高效液相色谱法,同时对从豆奶粉中提取大豆异黄酮的工艺进行了优化。采用日本岛津LC-20A高效液相色谱仪进行测定的色谱条件为：色谱柱为phenomenex C18(150mm×4.6 mm,5.0μm),采用乙腈-0.2%甲酸为流动相进行梯度洗脱,流速为0.8 mL/min,检测波长为260 nm。大豆苷,染料木苷,大豆苷元,染料木素的线性范围分别是0.74-100.00μg/mL,0.74-200.00/μg/mL,0.]67～500.00 gg/mL,0.198～160.00μg/mL(r>0.9996),线性关系良好；加样回收率(n=9)分别为100.01%,100.01%,99.48%,99.91%,方法简便、灵敏、准确,适用于豆奶粉中这四种大豆异黄酮成分的含量测定和产品质量控制。从豆奶粉中提取大豆异黄酮的最佳工艺为：用70%的乙醇在60℃条件下超声提取40 min。
3.不同豆类中4种大豆异黄酮的HPLC法测定
建立同时测定豆类中大豆苷,染料木苷,大豆苷元,染料木素四种大豆异黄酮成分含量的高效液相色谱法,色谱柱为phenomenex C18(150 mm×4.6 mm,5.0 gm),采用流动相乙腈-0.2%甲酸进行梯度洗脱,流速为0.8 mL/min,检测波长为260 nm。探讨了提取黑豆中四种大豆异黄酮的最佳条件,结果显示,70%的乙醇在60℃超声提取40 min,效果最佳。大豆苷,染料木苷,大豆苷元,染料木素的线性范围分别是0.74～100.00μg/mL,0.74～400.00μg/mL,0.167～500.00μg/mL,0.198～160.00μg/mL(r>0.9996),线性关系良好；加样回收率(n=9)分别为99.81%,99.99%,99.49%,99.89%,本方法简便、灵敏、准确,适用于豆类中这四种大豆异黄酮成分的含量测定和产品质量控制。
4.HPLC法测定绿豆芽中四种大豆异黄酮的含量
本文建立了同时测定绿豆芽中四种大豆异酮(大豆苷、染料木苷、大豆苷元和染料木素)含量的高效液相色谱方法,对从绿豆芽中提取四种大豆异黄酮的工艺进行了优化,最佳工艺为：用60%的乙醇在55℃条件下超声提取60 min,料液比为1：12,同时探究了绿豆发芽不同天数四种大豆异黄酮的含量。采用日本岛津LC-20A高效液相色谱仪进行测定的色谱条件为：色谱柱为phenomenex C18(150 mmx4.6 mm,5.0μm),采用乙腈-0.2%甲酸为流动相进行梯度洗脱,流速为0.8 mL/min,检测波长为260 nm。大豆苷,染料木苷,大豆苷元,染料木素的线性范围分别是0.74～100.00-μg/mL,0.74～200.00-μg/mL,0.167～500.00μg/mL,0.198-160.00μg/mL(r>0.9996),线性关系良好；加样回收率(n=9)分别为99.99%,100.01%,100.07%,99.81%,该方法简便、灵敏、准确,为日常生活中绿豆芽的最佳食用时间提供了科学依据。
High Performance Liquid Chromatography(HPLC)as an important modern measurement of analysis and separation is deveopled on the basis of gas Chromatography and classical Liquid Chromatography. Due to its high-speed, accuracy, sensitivity and convenience, it has been widely used in the areas of foodstuff, environment, biology engineering, pharmacy and petroleum so far.
The thesis includes two sections-review and research report. In the review section, information about high performance liquid chromatography such as characteristic, the development of chromatography technology and application in the effective elments of herb medicine Eucommia ulmoides Oliv by HPLC analysis and isoflavones research was summarized. In the next research report section, analysis methods of determination of chlorogenic acid and rutin in Eucommia ulmoides Oliv and isoflavones in food were reported. That is:Determination of four isoflavones in soybean milk powder by HPLC; Determination of four isoflavones in different beans by HPLC; Determination of four isoflavones in mung bean sprouts by HPLC.
1.Determination of chlorogenic acid and rutin in Eucommia ulmoides Oliv by HPLC
To establish an HPLC method for determination of chlorogenic acid and rutin, and determination chlorogenic acid and rutin in Eucommia ulmoides Oliv from different places. Using a phenomenex C1g column(150 mm×4.6 mm,5μm),the mobile phase of methanol-acetic acid(pH=3.10)(45:55) with elution, the flow rate was 0.8 mL/min, the detection wavelength was at 327 nm, the content of chlorogenic acid and rutin was determined by external standard method. The calibration curves for chlorogenic acid and rutin were linear between 0.1275 and 24.0000μg/mL, between 0.1250 and 25.0000μg/mL(r=0.9996 and r=0.9996 respectively).The average recoveries were 99.23% and 97.13%.RSD were 1.02% and 2.98%. The method is simple, rapid, sensitive and reproducible, it provides scientific proof for quality standard in Eucommia ulmoides Oliv from different places.
2.Determination of four isoflavones in soybean milk powder by HPLC
To develop an HPLC method for determination of four isoflavones, daidzin, genistin, daidzein, genistein in soybean milk powder. An phenomenex C18 column(150 mm×4.6 mm,5.0μm)was applied, a gradient mobile phase composing of acetonitrile and 0.2% formic acid was used, and the flow rate was 0.8 mL/min, detective wavelength was set at 260 nm.The best condition of extracted four isoflavones of soybean milk powder was studied.The results showed the best condition was the ethanol concentration 70%,60℃,extraction for 40 min. The results were obtained that quantitative linear range of four isoflavones were 0.74-100.00μg/mL,0.74-200.00μg/mL,0.167～500.00μg/mL,0.198～160.00μg/mL(r>0.9996)respectively, and the average recoveries(n=9)were 100.01%,100.01%,99.48%.99.91% respectively. A specific, sensitive, simple and reproducible HPLC method has been established for determination of four isoflavones in soybean milk powder.
3.Determination of four isoflavones in different beans by HPLC
To develop an HPLC method for determination of four isoflavones, daidzin, genistin, daidzein, genistein in different beans. An phenomenex C1g column(150 mm×4.6 mm,5.0 vm)was applied, a gradient mobile phase composing of acetonitrile and 0.2% formic acid was used, and the flow rate was 0.8 mL/min, detective wavelength was set at 260 nm.The best condition of extracted four isoflavones of black beans was studied. The results showed the best condition was the ethanol concentration 70%,60℃,extraction for 40 min. The results were obtained that quantitative linear range of four isoflavones were 0.74～100.00μg/mL,0.74～400.00μg/mL,0.167v500.00μg/mL, 0.198～160.00μg/mL(r>0.9996)respectively, and the average recoveries(n=9)were 99.81%. 99.99%,99.49%,99.89% respectively. A specific, sensitive, simple and reproducible HPLC method has been established for determination of four isoflavones in different beans.
4.Determination of four isoflavones in mung bean sprouts by HPLC
To develop an HPLC method for determination of four isoflavones. daidzin, genistin, daidzein, genistein in mung bean sprout. An phenomenex C18 column(150 mm×4.6 mm,5.0μm)was applied, a gradient mobile phase composing of acetonitrile and 0.2% formic acid was used, and the flow rate was 0.8 mL/min, detective wavelength was set at 260 nm.The best condition of extracted four isoflavones of mung bean sprout was studied, the results showed the best condition was the ethanol concentration 60%,55℃,extraction for 60 min with the solid-liquid ratio 1:12(g/mL).The results were obtained that quantitative linear range of four isoflavones were 0.74℃100.00μg/mL,0.74～200.00μg/mL,0.167～500.00μg/mL,0.198～160.00μg/mL(r>0.9996)respectively, and the average recoveries(n=9)were 99.99%,100.01%,100.07%.99.81% respectively. A specific, sensitive, simple and reproducible HPLC method has been established for determination of four isoflavones in mung bean sprouts. It provided scientific bases for the best eating time of mung bean sprout everyday.
引文
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