液相色谱及其质谱联用技术在水稻内源活性物质定量分析中的应用研究
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摘要
植物内源活性物质是植物体内本来存在的或者自身生命活动产生、参与各种生命活动的物质。如何准确快速的定性和定量分析这些内源活性物质,是研究植物内源活性物质的生理功能的重要环节。本论文运用高效液相色谱和电喷雾串联质谱等技术,建立了快速定量分析水稻中尼克酰胺、细胞分裂素、单糖等内源活性物质的方法,这些新发展的定性定量分析方法,对于准确了解这些物质的各种生命活动具有重要意义,具体分为三部分:
一,运用高效液相色谱—电喷雾质谱联用技术,采用选择离子和三级质谱首次建立了水稻组织中尼克酰胺的快速定量方法。样品采用温水提取30分钟,离心,上清液直接进行LC-MS分析。通过对色谱条件和质谱条件的优化,利用反相色谱柱的粗分离和离子阱多级质谱的定性定量优势,测定了水稻中内源NA含量,其线性范围为0.127-65 ng/20μL,检测限为3.175 ng/mL。该方法去除了繁琐的衍生步骤,具有操作简单,分析快速,分辨率和灵敏度高的优点。
二,采用二维高效液相色谱方法,运用中心切割技术,构建了一种新的定量分析玉米素的串联柱模式二维液相色谱定量方法。水稻样品通过简单的80%甲醇水萃取,提取液直接进入一维离子交换色谱柱进行初级分离,利用六通阀将含目标组分切换至二维反相色谱柱进行第二维的分离分析,测定了水稻组织内玉米素的含量,其线性范围为0.0537-27.5 ng,检测限为0.026 ng。二维高效液相色谱定量方法具有纯化能力强,重复性好及操作简单等优势。
三,应用高效液相色谱—蒸发光检测技术新建立了水稻中三种游离糖—葡萄糖、果糖和蔗糖的定量分析方法。水稻样品通过简单的80%乙醇水萃取,氯仿异戊醇纯化步骤后,直接进行液相分离和蒸发光检测,测定了三种糖的含量。葡萄糖、果糖和蔗糖的线性范围分别为0.44-5.275、0.228-5.5和0.422-10.15μg/μL,检测限分别为0.11、0.114和0.212μg/μL。
Plant endogenous coustituents are important physiolygical compounds in plant life cycle.It is essential to quatify and quantify the endogenous constituents in fresh plant tissues.This paper arms to develope some rapid and efficient methods for quatification analyses of some endogenous constistuents,such as nicotianamine, zeatin,glucose,fructose and sucrose.The new methods for separation and determination are significant of plant life activities.It includes three parts as follow:
In the first chapter,a new method for separation and determination of nicotianamine was developed by HPLC-ESI/MS.The fress rice tissues was extracted with ddH_2O at 80℃for 30min and then to centrifigate.The supernatant was separated directed by a reverse phase column,and then the effusion was analyzed by ESI-MS.The linear range is 0.127-65 ng/20μL,and the limite of detection at three times signal to noise(LOD,3*S/N)is 3.175 ng/mL.
In the sencond chapter,the zeatin in rice was analyzed for the first time by 2D-HPLC and using "Heart-cutting" mode.The rice sample extracting with 80% methanol-H_2O was separated primarily in the first dimension,then the aimed part of first dimension run is introduced to separate futher into the second dimension by switching a six port valve.The linear range is 0.0537-27.5 ng,and the LOD(3*S/N) is 0.026 ng.The aavantages of 2D-HPLC are high purification potential, reproducibility,and unattended operation.
The third chapter,a useful HPLC-ELSD method for quatification analyses of sugars-glucose,fructose and sucrose was estabilished.Before analyzed by HPLC-ELSD,the sample first was extracted by 80%ethanol-H_2O at 90℃for 40min and purified by chloroform-Isoamylol(24:1).The linear range of glucose,fructose and sucrose is 0.44-5.275、0.228-5.5 and 0.422-10.15μg/μL,and the LOD(3*S/N)is 0.11、0.114 and 0.212μg/μL.
一,运用高效液相色谱—电喷雾质谱联用技术,采用选择离子和三级质谱首次建立了水稻组织中尼克酰胺的快速定量方法。样品采用温水提取30分钟,离心,上清液直接进行LC-MS分析。通过对色谱条件和质谱条件的优化,利用反相色谱柱的粗分离和离子阱多级质谱的定性定量优势,测定了水稻中内源NA含量,其线性范围为0.127-65 ng/20μL,检测限为3.175 ng/mL。该方法去除了繁琐的衍生步骤,具有操作简单,分析快速,分辨率和灵敏度高的优点。
二,采用二维高效液相色谱方法,运用中心切割技术,构建了一种新的定量分析玉米素的串联柱模式二维液相色谱定量方法。水稻样品通过简单的80%甲醇水萃取,提取液直接进入一维离子交换色谱柱进行初级分离,利用六通阀将含目标组分切换至二维反相色谱柱进行第二维的分离分析,测定了水稻组织内玉米素的含量,其线性范围为0.0537-27.5 ng,检测限为0.026 ng。二维高效液相色谱定量方法具有纯化能力强,重复性好及操作简单等优势。
三,应用高效液相色谱—蒸发光检测技术新建立了水稻中三种游离糖—葡萄糖、果糖和蔗糖的定量分析方法。水稻样品通过简单的80%乙醇水萃取,氯仿异戊醇纯化步骤后,直接进行液相分离和蒸发光检测,测定了三种糖的含量。葡萄糖、果糖和蔗糖的线性范围分别为0.44-5.275、0.228-5.5和0.422-10.15μg/μL,检测限分别为0.11、0.114和0.212μg/μL。
Plant endogenous coustituents are important physiolygical compounds in plant life cycle.It is essential to quatify and quantify the endogenous constituents in fresh plant tissues.This paper arms to develope some rapid and efficient methods for quatification analyses of some endogenous constistuents,such as nicotianamine, zeatin,glucose,fructose and sucrose.The new methods for separation and determination are significant of plant life activities.It includes three parts as follow:
In the first chapter,a new method for separation and determination of nicotianamine was developed by HPLC-ESI/MS.The fress rice tissues was extracted with ddH_2O at 80℃for 30min and then to centrifigate.The supernatant was separated directed by a reverse phase column,and then the effusion was analyzed by ESI-MS.The linear range is 0.127-65 ng/20μL,and the limite of detection at three times signal to noise(LOD,3*S/N)is 3.175 ng/mL.
In the sencond chapter,the zeatin in rice was analyzed for the first time by 2D-HPLC and using "Heart-cutting" mode.The rice sample extracting with 80% methanol-H_2O was separated primarily in the first dimension,then the aimed part of first dimension run is introduced to separate futher into the second dimension by switching a six port valve.The linear range is 0.0537-27.5 ng,and the LOD(3*S/N) is 0.026 ng.The aavantages of 2D-HPLC are high purification potential, reproducibility,and unattended operation.
The third chapter,a useful HPLC-ELSD method for quatification analyses of sugars-glucose,fructose and sucrose was estabilished.Before analyzed by HPLC-ELSD,the sample first was extracted by 80%ethanol-H_2O at 90℃for 40min and purified by chloroform-Isoamylol(24:1).The linear range of glucose,fructose and sucrose is 0.44-5.275、0.228-5.5 and 0.422-10.15μg/μL,and the LOD(3*S/N)is 0.11、0.114 and 0.212μg/μL.
引文
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