人体血液中钙形态分析研究
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摘要
元素的生物活性及毒性不仅与元素的总量有关,更主要的是与元素的赋存形态有关,不同的元素形态具有不同的化学和生物行为。元素的形态不仅决定该元素在环境中的迁移转化规律,而且还决定该元素的生物可利用性和生物效应。因此形态分析对揭示元素在环境和生命科学中的迁移转化规律及其生物化学作用都具有重要意义。
毛细管电泳(Capillary Electrophoresis,CE)是20世纪80年代迅速发展起来的一种新型分离分析技术。被认为是上世纪末在分离分析领域最具影响力的分支之一,也是该领域的公认前沿。毛细管电泳具有分离效率高、分析速度快、分析模式多、试剂用量少、应用范围广、易于自动化等优点,成为复杂体系中成分分析的重要分析方法之一。在生命科学、生物技术、临床医学、药物分析和环境科学等领域显示了极其重要的应用前景。
电感耦合等离子体原子发射光谱(Inductively coupled plasma atomic emission spectrometry,ICP-AES)具有检出限低、精密度高、选择性好、线性范围宽和多元素同时测定等特点,在痕量元素分析领域得到了广泛应用。将CE-ICP-AES结合起来,实现了高效分离与多元素选择性的测定。CE-ICP-AES联用技术在痕量元素形态分析,金属络合物分析,医药,生物,环境等方面开辟了一个新的领域。CE-ICP-AES联用技术结合了CE分离效能高和ICP-AES具有多元素同时测定和检出限低,线性范围宽等优点,为复杂样品的元素形态分析提供了一种强有力的分析手段。
超声波是指频率在20 kHz～106 kHz的机械波,既是一种波动形式,又是一种能量形式。作为一种特殊的能量输入方式,所具有的高效能在材料化学中起到光、电、热方法所无法达到的作用。同时在提取与分离、催化、生物科学等领域发挥着重要作用。毛细管电泳进行复杂体系分析前,必须对样品进行净化处理。本文中自行设计了超声波透析装置,并将它引入血液的形态分析当中,对生物体内的元素形态有着更好的分析。研究超声透析-毛细管电泳和毛细管电泳-等离子体原子发射光谱联用技术在复杂体系形态分析中的应用,可以为人们提供更多有用的信息,在生命科学,环境科学,临床医学,营养学等领域的研究中起着重要的作用,对揭示元素在环境和生命科学中和迁移转化规律及其生物化学作用都具有重要意义。
本论文主要在CE-ICP-AES联用技术及超声透析与CE-ICP-AES联用技术的建立及其在复杂体系血浆,全血等样品元素形态分析中的应用开展工作,并得到如下研究结果:
1.首次把毛细管电泳-电感耦合等离子体原子发射光谱联用技术应用于人体血浆中钙元素的形态分析,对游离态的钙进行了定位表征与定量分析,对其它形态作了一些初步的探讨。在优化条件下,得到人体血浆中钙的主要形态有八种,其中一种为游离钙离子,其10次迁移时间的相对偏差小于2%,血浆样品游离钙的含量为41.9 mg·L-1,该测定方法RSD小于5%,采用以上联用技术测得血浆样品中游离钙回收率为94.5-102%。
2.建立了一种新型的样品前处理方法—超声透析技术,通过在超声透析条件下,对超声透析功率,时间,频率等实验条件进行了优化,得出了在功率为72 W,频率为59kHz,时间为60 min,超声透析的样品效果最佳,在最优化的实验条件下,将此联用技术应用于无机离子Zn2+与ZnEDTA2-及Cu2+与CuEDTA2-的透析采样与形态分析,从分离形态峰可以得出该接口装置用于形态分析是可行的,其峰面积RSD小于5%,检出限小于80μg·L-1。该装置应用于血液透析液钙形态分析,得到五种钙形态,对透析中游离钙进行了定量分析。该方法比常规透析处理样品速度快,操作简单,成本低廉,联用方便,为生物样品的快速分离分析提供新的分析方法。
3.利用毛细管电泳电感耦合等离子体原子发射光谱联用技术,成功地分析了全血中钙的10种形态,其中有5种钙形态的分子量小于15000,其中全血样品中游离钙含量为44.3 mg·L-1,各形态含量的RSD在1.5%-5.1%之间,在样品中加入5 mg·L-1的标准游离钙进行加标回收检测,回收率为96.9%,同时游离钙的检出限为0.027 mg·L-1。
It is a well-known fact that the toxicity, bioavailability, and transport properties of an element are not only dependent upon its total quantification, but also highly lie on the chemical species. The different element speciation have different chemical and biological action. To completely understand the ways in which particular elements will affect living organisms, it is necessary to determine the species within which the elements are found, and to quantify those species. Analysis performed to identify or quantify one or more distinct chemical species in a sample is known as speciation analysis.
The capillary electrophoresis (CE) was developed rapidly in the eighties of the 20th century as a novel separation and analysis technology. It is considered to be one of the strongest influence branches of analysis field at the end of last century, as well as the frontier of this field. It has many advantages: high efficiency, quick analysis speed, many analysis modes, little reagent consumption, wide application, easy automation, etc. Thus it shows extremely important application to life sciences, biotechnology, clinical medicine, medicine analysis and environmental science and so on.
Inductively coupled plasma atomic emission spectrometry (ICP-AES) offers extending good advantages in terms of low detection limit, high precision, wide linear range and simultaneous multi-elements determination, widely used in trace-element analysis fields in recent years. CE coupled with ICP-AES has the individual advantages of capillary electrophoresis and inductively coupled plasma spectrometry, it realized the combination of efficient separation and element alternative measurement. It was considered as a powerful analytical method.
Ultrasonic is a mechanical wave which the frequency is between 20 kHz～106 kHz, is not only a wave, but also an energy. Before analysizing complex system with CE, the samples must be purified. In this paper, an ultrasonic dialysis device has been set up, which had a good efficiency in blood speciation analysis. Studying on the speciation of complex systems by ultrasonic dialysis-CE-ICP-AES, which provided a more important information in the field of biological science, environmental science, clinic medicine and nutrition.
In this paper, the research has been carried on foundation of ultrasonic dialysis capillary electrophoresis and inductively coupling plasma atomic emission spectrometry and their application to the speciation analysis of complex systems, such as biological samples (blood, plasma). The main contents were described as follows:
1. A new method for the determination of free calcium concentration in human plasma was developed by on-line coupling capillary electrophoresis (CE) with inductively coupled plasma optical emission spectrometry (ICP-OES). Baseline separation of calcium-containing species was achieved by CE-ICP-OES in a 120 cm long capillary with 100μm internal diameter, at 20 kV applied voltage, with a 30 mmol/L Tris-HCl buffer at pH 7.4. A total of eight calcium-containing species were found in human plasma; the concentration of free calcium ion was found to be 41.9 mg/L. The precision (RSD, n = 10) ranged from 1.2 to 2.7% for the migration time and 2.8 to 3.9% for the peak area. The developed method was also applied to analyze plasma samples with recovery ranged from 94.5 to 102% for samples spiked with 40.0 mg/L free calcium ion.
2. A new purification method for biological samples- ultrasonic dialysis technique has been developed. The feasibility of this method and its application were tested. The analytical performances have been studied, such as power , time, frequency, etc. The experimental result showed that optimal conditions were power of 72 W,frequency of 59 kHz,time of 60 min. The application of the developed ultrasonic dialysis -CE-ICP-OES for the speciation analysis of Zn2+, ZnEDTA2-, Cu2+and CuEDTA2- was highlighted. The detection limits of these elements were lower than 80μg·L-1, RSDs for those lower than 5%. This device was also used to calcium speciation in the blood dialysate, the result showed five calcium-containing species. The hyphenated technique was simple and convenient, which was fit for the separation and analysis of biological samples.
3. The analytical method for speciation analysis of calcium in human whole blood was first studied, there had 10 species in human whole blood. The concentration of free calcium ion was 44.3 mg·L-1. Five spicies were identified using ultrasonic-assisted dialysis coupled with inductively coupled plasma optical emission spectrometry in the dialysate of the blood, which molecular weight≤15000. The developed method was also applied to analyze samples with recovery was 96.9% for sample spiked with 5.0 mg/L free calcium ion. The precision (RSD, n = 10) ranged from1.5 to5.1% for for the peak area. The detection limit for free calcium was 0.027 mg/L(3σ).
毛细管电泳(Capillary Electrophoresis,CE)是20世纪80年代迅速发展起来的一种新型分离分析技术。被认为是上世纪末在分离分析领域最具影响力的分支之一,也是该领域的公认前沿。毛细管电泳具有分离效率高、分析速度快、分析模式多、试剂用量少、应用范围广、易于自动化等优点,成为复杂体系中成分分析的重要分析方法之一。在生命科学、生物技术、临床医学、药物分析和环境科学等领域显示了极其重要的应用前景。
电感耦合等离子体原子发射光谱(Inductively coupled plasma atomic emission spectrometry,ICP-AES)具有检出限低、精密度高、选择性好、线性范围宽和多元素同时测定等特点,在痕量元素分析领域得到了广泛应用。将CE-ICP-AES结合起来,实现了高效分离与多元素选择性的测定。CE-ICP-AES联用技术在痕量元素形态分析,金属络合物分析,医药,生物,环境等方面开辟了一个新的领域。CE-ICP-AES联用技术结合了CE分离效能高和ICP-AES具有多元素同时测定和检出限低,线性范围宽等优点,为复杂样品的元素形态分析提供了一种强有力的分析手段。
超声波是指频率在20 kHz～106 kHz的机械波,既是一种波动形式,又是一种能量形式。作为一种特殊的能量输入方式,所具有的高效能在材料化学中起到光、电、热方法所无法达到的作用。同时在提取与分离、催化、生物科学等领域发挥着重要作用。毛细管电泳进行复杂体系分析前,必须对样品进行净化处理。本文中自行设计了超声波透析装置,并将它引入血液的形态分析当中,对生物体内的元素形态有着更好的分析。研究超声透析-毛细管电泳和毛细管电泳-等离子体原子发射光谱联用技术在复杂体系形态分析中的应用,可以为人们提供更多有用的信息,在生命科学,环境科学,临床医学,营养学等领域的研究中起着重要的作用,对揭示元素在环境和生命科学中和迁移转化规律及其生物化学作用都具有重要意义。
本论文主要在CE-ICP-AES联用技术及超声透析与CE-ICP-AES联用技术的建立及其在复杂体系血浆,全血等样品元素形态分析中的应用开展工作,并得到如下研究结果:
1.首次把毛细管电泳-电感耦合等离子体原子发射光谱联用技术应用于人体血浆中钙元素的形态分析,对游离态的钙进行了定位表征与定量分析,对其它形态作了一些初步的探讨。在优化条件下,得到人体血浆中钙的主要形态有八种,其中一种为游离钙离子,其10次迁移时间的相对偏差小于2%,血浆样品游离钙的含量为41.9 mg·L-1,该测定方法RSD小于5%,采用以上联用技术测得血浆样品中游离钙回收率为94.5-102%。
2.建立了一种新型的样品前处理方法—超声透析技术,通过在超声透析条件下,对超声透析功率,时间,频率等实验条件进行了优化,得出了在功率为72 W,频率为59kHz,时间为60 min,超声透析的样品效果最佳,在最优化的实验条件下,将此联用技术应用于无机离子Zn2+与ZnEDTA2-及Cu2+与CuEDTA2-的透析采样与形态分析,从分离形态峰可以得出该接口装置用于形态分析是可行的,其峰面积RSD小于5%,检出限小于80μg·L-1。该装置应用于血液透析液钙形态分析,得到五种钙形态,对透析中游离钙进行了定量分析。该方法比常规透析处理样品速度快,操作简单,成本低廉,联用方便,为生物样品的快速分离分析提供新的分析方法。
3.利用毛细管电泳电感耦合等离子体原子发射光谱联用技术,成功地分析了全血中钙的10种形态,其中有5种钙形态的分子量小于15000,其中全血样品中游离钙含量为44.3 mg·L-1,各形态含量的RSD在1.5%-5.1%之间,在样品中加入5 mg·L-1的标准游离钙进行加标回收检测,回收率为96.9%,同时游离钙的检出限为0.027 mg·L-1。
It is a well-known fact that the toxicity, bioavailability, and transport properties of an element are not only dependent upon its total quantification, but also highly lie on the chemical species. The different element speciation have different chemical and biological action. To completely understand the ways in which particular elements will affect living organisms, it is necessary to determine the species within which the elements are found, and to quantify those species. Analysis performed to identify or quantify one or more distinct chemical species in a sample is known as speciation analysis.
The capillary electrophoresis (CE) was developed rapidly in the eighties of the 20th century as a novel separation and analysis technology. It is considered to be one of the strongest influence branches of analysis field at the end of last century, as well as the frontier of this field. It has many advantages: high efficiency, quick analysis speed, many analysis modes, little reagent consumption, wide application, easy automation, etc. Thus it shows extremely important application to life sciences, biotechnology, clinical medicine, medicine analysis and environmental science and so on.
Inductively coupled plasma atomic emission spectrometry (ICP-AES) offers extending good advantages in terms of low detection limit, high precision, wide linear range and simultaneous multi-elements determination, widely used in trace-element analysis fields in recent years. CE coupled with ICP-AES has the individual advantages of capillary electrophoresis and inductively coupled plasma spectrometry, it realized the combination of efficient separation and element alternative measurement. It was considered as a powerful analytical method.
Ultrasonic is a mechanical wave which the frequency is between 20 kHz～106 kHz, is not only a wave, but also an energy. Before analysizing complex system with CE, the samples must be purified. In this paper, an ultrasonic dialysis device has been set up, which had a good efficiency in blood speciation analysis. Studying on the speciation of complex systems by ultrasonic dialysis-CE-ICP-AES, which provided a more important information in the field of biological science, environmental science, clinic medicine and nutrition.
In this paper, the research has been carried on foundation of ultrasonic dialysis capillary electrophoresis and inductively coupling plasma atomic emission spectrometry and their application to the speciation analysis of complex systems, such as biological samples (blood, plasma). The main contents were described as follows:
1. A new method for the determination of free calcium concentration in human plasma was developed by on-line coupling capillary electrophoresis (CE) with inductively coupled plasma optical emission spectrometry (ICP-OES). Baseline separation of calcium-containing species was achieved by CE-ICP-OES in a 120 cm long capillary with 100μm internal diameter, at 20 kV applied voltage, with a 30 mmol/L Tris-HCl buffer at pH 7.4. A total of eight calcium-containing species were found in human plasma; the concentration of free calcium ion was found to be 41.9 mg/L. The precision (RSD, n = 10) ranged from 1.2 to 2.7% for the migration time and 2.8 to 3.9% for the peak area. The developed method was also applied to analyze plasma samples with recovery ranged from 94.5 to 102% for samples spiked with 40.0 mg/L free calcium ion.
2. A new purification method for biological samples- ultrasonic dialysis technique has been developed. The feasibility of this method and its application were tested. The analytical performances have been studied, such as power , time, frequency, etc. The experimental result showed that optimal conditions were power of 72 W,frequency of 59 kHz,time of 60 min. The application of the developed ultrasonic dialysis -CE-ICP-OES for the speciation analysis of Zn2+, ZnEDTA2-, Cu2+and CuEDTA2- was highlighted. The detection limits of these elements were lower than 80μg·L-1, RSDs for those lower than 5%. This device was also used to calcium speciation in the blood dialysate, the result showed five calcium-containing species. The hyphenated technique was simple and convenient, which was fit for the separation and analysis of biological samples.
3. The analytical method for speciation analysis of calcium in human whole blood was first studied, there had 10 species in human whole blood. The concentration of free calcium ion was 44.3 mg·L-1. Five spicies were identified using ultrasonic-assisted dialysis coupled with inductively coupled plasma optical emission spectrometry in the dialysate of the blood, which molecular weight≤15000. The developed method was also applied to analyze samples with recovery was 96.9% for sample spiked with 5.0 mg/L free calcium ion. The precision (RSD, n = 10) ranged from1.5 to5.1% for for the peak area. The detection limit for free calcium was 0.027 mg/L(3σ).
引文
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