植物油和体液等复杂体系中二阶校定量分析应用研究

1、植物油和体液等复杂体系中二阶校正法定量分析应用研究-毕业论文 学校代号 10532 学 号 B1011S0022 分 类 号 密 级 博士学位论文 植物油和体液等复杂体系中二阶校 正法定量分析应用研究 学位申请人姓名 王建瑶 培 养 单 位 化学化工学院 导师姓名及职称 吴海龙 教授 学 科 专 业 分析化学 研 究 方 向 化学计量学 论文提交日期 20 13 年6 月 植物油和体液等复杂体系中二阶校正法定量分析应用研究 摘 要 随着社会的发展,仪器技术的进步，越来越多的分析仪器能够获得高维数据。 在这种形势下，二阶校正方法越来越受到众多分析领域的关注。二阶校正方法被 广泛接受的主要原因，是

2、它能够从许多联用方法，如激发-发射三维荧光、高效液 相色谱-二极管阵列检测器等产生的高维数据中提取有效的信息，即使分析体系中 存在未知干扰，也可以完成对感兴趣物质的定性、定量分析，这种特殊的优势被 Booksh 和 Kowalski 命名为“二阶优势” 。二阶优势的存在使得我们能够从复杂体 系中直接测定分析物，这也成为本论文研究的基石。 本论文侧重于二阶校正方法在植物油和体液等复杂体系中的定量分析应用研 究。这些体系都包含许多种类的化合物，基质极为复杂。分析植物油中的化合物 对建立植物油的质量检测非常重要，而体液中药物和标记物含量水平的检测则与 人体健康程度密切相关。目前，用于植物油和体液等复

3、杂体系分析的最广泛的方 法是液相色谱法，但是这种方法成本高、耗时、需要提取等前处理步骤进行预分 离。因此，本论文旨在发展一种简单、快速、有效且无需提取等预处理步骤的策 略用于定量分析体液和植物油等复杂体系中的化合物。基本的研究内容概括如下： 第二章：提出了高效液相色谱-二极管阵列检测器结合基于交替惩罚三线性分 解（APTLD ）的二阶校正方法，用于快速测定食用植物油中十种合成酚类抗氧化 剂：没食子酸丙酯 （PG ）、3-叔丁基-4-羟基茴香醚 （BHA ） 、3,5-二叔丁基-4-羟 基苯酚甲苯 （BHT ）、2,4,5-三羟基苯丁酮 （THBP ）、叔丁基对苯二酚 （TBHQ ）、 没食子酸

4、辛酯（OG ）、没食子酸月桂酯（DG ）、去甲二氢愈创木酚（NDGA ）、 乙氧喹啉（EQ ）和 2,6-二叔丁基-4-羟甲基苯酚（Ionox- 100 。交替惩罚三线性分 解算法（APTLD ）能够正确的解析包含重叠峰以及基线漂移的二阶数据，从而实 现植物油中抗氧化剂的快速测定。该方法无需提取步骤，避免了分析物的流失， 并能在较短的时间内完成十种物质的洗脱，减少了有机溶剂对环境的污染。我们 计算了预测均方差、线性相关系数、检测下线以及加标回收率等参数用以验证新 方法的有效性。预测结果令人满意，检测下限能够满足大多数国家测定植物油中 酚类抗氧化剂的需求。 第三章：提出了高效液相色谱-荧光检测器

5、结合基于交替三线性分解（ATLD ） 的二阶校正方法，用于同时测定六种植物油中的七种合成酚类抗氧化剂。此方法 无需提取步骤，样品稀释后即可直接进样，在实验过程中也无需清洗色谱柱。所 有物质的洗脱可在短时间内完成。在多种植物油所含未知组分对分析物形成干扰 的情况下，依然可以完成准确的定量分析。此方法的检测下限远远低于抗氧化剂 II 植物油和体液等复杂体系中二阶校正法定量分析应用研究 的常规限量水平，并且比第二章方法的检测下限低一个数量级。 第四章：发展了一种新的算法，自加权整体最小二乘三线性分解算法（SWTT ）， 用于解析三维荧光模拟数据以及实验数据。我们设计了三组模拟数据，分别从噪 声、共线

6、性以及背景干扰强度三个方面对自加权交替三线性分解（SWATLD ），平 行因子分析交替最小二乘（PARAFAC-ALS ）和新算法SWTT 做了比较，并从预测 能力、出错次数以及与真实光谱的拟合程度等几个方面对各个算法做了评价。当 共线性、噪声以及干扰强度水平较低时，三个算法都能得到准确稳定的结果，但 是当其中之一水平较高时，SWATLD 算法就无法得到正确的分辨，而 PARAFAC-ALS 算法则更容易陷入沼泽，运行出错。SWTT 算法通过利用整体最小 二乘和权重因子，提高了稳定性，从一定程度上改善了双因子退化问题，并能得 到更加准确的结果。此外，我们还将三种算法应用于化妆品中雌酮和雌二醇的

7、同 时测定，SWATLD 无法正确分辨，SWTT 和 PARAFAC-ALS 则能够给出合理的结 果，并且两者的结果极为相似。 第五章：一些研究表明，人体体液中异黄蝶呤的增加和癌症之间有一定的联 系，可将其含量作为医学中判断某些癌症病患的重要指标之一。因此，本文通过 结合二阶校正方法和三维荧光技术发展了一种简单、快速并具有高灵敏度和高选 择性的的方法用于测定人体尿液中异黄蝶呤的含量。虽然尿液中其它蝶呤类化合 物的激发、发射光谱与异黄蝶呤的光谱严重重叠，但是由于我们方法具有二阶优 势，在无需任何预处理进行化学或物理分离的情况下就可对异黄蝶呤的含量进行 直接测定。我们还采用常规的高效液相色谱-荧光

8、检测器（HPLC-FLD ）方法对相 同的尿液样本进行了测定，两种方法得到的异黄蝶呤的含量基本一致，证明了我 们方法的可靠性。 第六章：采用交替三线性分解（ATLD ）和交替归一加权残差（ANWE ）法与 激发-发射荧光矩阵相结合，测定细胞培养基中的阿霉素。预测结果令人满意。此 外，还将细胞中的四种内源荧光物加入到细胞培养基，用以模拟细胞内荧光干扰 环境，在此环境下，我们提出的方法依然能成功测定了阿霉素的含量。本方法最 大的优势在于无需对细胞培养基做任何预处理，即可直接取样测定，操作简单， 测定快速，对样本也无破坏性。结果表明二阶校正方法结合激发发射荧光矩阵 （EEM ）适用于培养基中药物的测

9、定，为细胞的体外药物研究提供了一种绿色的 新方法。 第七章：本章通过结合具有高灵敏度的三维荧光技术和具有高选择性的基于 自加权交替归一残差拟合（SWANRF ）算法的二阶校正方法，在背景干扰较强的 化妆品样本中同时测定了丁基羟基茴香醚（BHA ）和没食子酸丙酯（PG ）。我们 采用的这种策略，符合绿色化学的原则，使用极少量的有机溶剂就可完成两种抗 氧化剂的测定。此外，我们的方法无需对样本进行浓缩或洗脱分离的等预处理步 III 植物油和体液等复杂体系中二阶校正法定量分析应用研究 骤，从而减少了分析时间，降低了实验成本。与 HPLC （AOAC 983.15 ，1994）方 法相比，EEM-SWA

10、NRF 方法更加的灵敏、简单、快速、有效且无污染。 关键词：二阶校正方法；高效液相色谱-二极管阵列检测器；高效液相色谱-荧 光检测器；激发-发射荧光矩阵；植物油；合成酚类抗氧化剂；体液； 定量分析 IV 植物油和体液等复杂体系中二阶校正法定量分析应用研究 Abstract As society develops, a wide range of analytical instrumentation is available that enables high dimensionality data to be obtained. In this situation, second-order

11、calibration methods has gained widespread acceptance by the analytical community, because it exploit the intrinsic data structure of hyphenated analytical methods, such as excitation-emission fluorescence matrix spectroscopy and chromatographic separation coupled to diode array detection, to enable

12、one to quantify the analyte, even in the presence of interferences not modelled in the calibration stage. This property of second-order calibration methods has been termed the second -order advantage by Booksh and Kowalski. This advantage makes it possible to directly quantify the interesting analyt

13、es from complex system and provides the main motivation for the present parper . In this paper, second-order calibration methods are employed for the quantitative analysis in complex systems mainly focused on vegetable oil and body fluid . These complex systems are made of complex mixtures of compou

14、nds of a wide range of chemical classes. Characterization of compounds in vegetable oils is of great importance in establishing the oil quality, and it is important to determine the levels of medicines or markers in the body fluid for understanding how healthy people are. The most commonly used anal

15、ytical techniques for analysis of these compounds are liqu id chromatography. These techniques are however expensive, time -consuming and require a pre-separation step before the analysis. Therefore, the aim of this paper is to couple the second-order calibration methods with high-performance liquid

16、 chromatography-diode array detection HPLC-DAD , high-performance liquid chromatography-fluorescence detection HPLC-FLD or excitation -emission fluorescence matrix EEM for the development and optimization of a simple, rapid, efficient, fast and no-extraction methodology for quantitative analysis in

17、complex systems, such as vegetable oil and body fluid . The contents of our studies can be summarized as: Chapter 2:A new chromatographic methodology is presented for fast analysis of ten synthetic phenolic antioxidants in five kinds of oil samples: propyl gallate PG , 2, 4, 5-trihydroxybutyrophenon

18、e THBP , tert-butylhydroquinone TBHQ , nordihydroguaiaretic acid NDGA , ethoxyquin EQ , 3 -tert-butyl -4- hydroxyanisole V 植物油和体液等复杂体系中二阶校正法定量分析应用研究 BHA , octyl gallate OG , 2, 6-di-tert-butyl -4-hydroxymethyphenol Ionox - 100 , dodecyl gallate DG , 3, 5-di-tert-butyl -4-hydroxytoluene BHT . The alt

19、ernating penalty trilinear decomposition APTLD algorithm has shown to be an excellent tool for modelling the second-order data, where overlapping peak and baseline drift were existed, making the fast determination and resolution of the phenolic antioxidants in oils possible. The extraction is unnece

20、ssary and the ten antioxidants can be eluted within 6 mins. For the validation of the method, linearity, root-mean-square error of prediction RMSEP and limit of detection LOD have been performed. The results of overall predictions are desirable and the LOD of our method is enough to detect SPAs in e

21、dible vegetable oils in most cases . Chapter 3: A liquid chromatographic method has been developed, in combination with the second-order calibration method based on the alternating trilinear decomposition ATLD algorithm with fluorescence detection FLD , for the simultaneous determination of seven ph

22、enolic antioxidants in six kinds of oil samples. The extraction is unnecessary and a column washing is also not required. After a simple dilution step, oil samples can be directly injected into the detecting system and the elution is accomplished in a short time 6 min . Overl apped peaks have been s

23、uccessfully resolved and the quantitative results of the analytes of interest have been accurately estimated. The limits of this method are much lower than the imum residue level established for the antioxidants. It is about one -tenth of the value previously proposed by Chapter 2. Chapter 4: A nove

24、l algorithm named as self -weighted total least -squares alternating trilinear decomposition SWTT has been proposed for quantitative analysis of EEM fluorescence data. Based on different input parameters including collinearity, interference intensity and noise level in three simulated data arrays, t

25、he performances of the self -weighted alternating trilinear decomposition SWATLD , the parallel factor analysis -alternating least squares PARAFAC-ALS and the SWTT algorithms were evaluated in terms of predicting ability, error times and consistency of resolved and real profiles. When the collineari

26、ty, interference intensity and noise level of data are low, the results obtained by the three algorithms are nearly the same. However, the SWATLD can not get satisfactory results and the PARAFAC -ALS easily trap into swamps many times when the leve is high. By utilizing the alternating total least-s

27、quares principle and weight factor, the SWTT algorithm can improve the two-factor degeneracy problem, which is difficult to handle for the PARAFAC -ALS algorithm, and obtain more stable and more precise results. In addition, the SWTT and VI 植物油和体液等复杂体系中二阶校正法定量分析应用研究 PARAFAC-ALS method was successful

28、ly applied to the simultaneous analysis f or estriol and estrone in liquid cosmetic samples. Chapter 5: Several studies have shown that an increase in amounts of isoxanthopterin can be associated with the presence of cancer, thus its level is importance parameter in clinical diagnosis. A simple, rap

29、id, sensitive and selective method for determining isoxanthopterin content in human urine samples has been developed using secon-order calibration mehtod coupled with EEM. With the application of a second Corder advantage, the proposed strategy could be utilized for a direct concentration determinat

30、ion of isoxanthopterin without any pretreatment step, even the spectra of isoxanthopterin is highly overlapping with other pterin compounds neopterin, biopterin, pterin and pterin-6-carboxylic acid . The values of the isoxanthopterin from healthy volunteers, which is botained by our method, were in

31、good agreement with those obtained by HPLC -FLD method. Chapter 6: A simple and rapid method is proposed for quantitative analysis of adriamycin in cell culture media without any pretreatment procedure. It combined second-order calibration methods based on both an alternating trilinear decomposition

32、 ATLD and an alternating normalization-weighter error ANWE algorithms combined with excitation-emission fluorescent matrix spectra. The satisfactory results showed that the second-order calibration method can be applied to directly quantify adriamycin in cell culture media. Moreover, in the analysis

33、 of cell culture media including four interferences which are the fluoresc ent materials in cell, the adriamycin also can be accurately determinated . It is found that second-order calibration method is appropriate for quantitative analysis of drug content in cell culture media even in the presence

34、of natural fluorescent interfe rences of cell. Chapter 7: This work presents a novel approach for simultaneous determination of butylated hydroxyanisole BHA and propyl gallate PG in a very interfering environment by combining the sensitivity of molecular fluorescence and the selectivi ty of second-o

35、rder calibration method based on the self -weighted alternating normalized residue fitting SWANRF algorithm . This method uses very small amounts of organic reagents and the quantification of PG and BHA were accomplished using green-chemistry principles. In addition, the proposed method avoids preco

36、ncentration and elution procedures, so it considerably decreases the analytical time and the experimental expenses . Because the instrument involved in the measurement is nonsophisticated, the experiments could be carried out in routine laboratories. Compared to the HPLC method the AOAC official met

37、hod 983.15 , 1994 , the VII 植物油和体液等复杂体系中二阶校正法定量分析应用研究 EEM-SWANRF is a more sensitive, simpler, faster, cleaner and more powerful method. Keywords: Seconder-order method; HPLC-DAD; HPLC-FLD ; Excitation-emission fluorescent matrix; Vegetable oils ; Synthetic phenolic antioxidants; Body fluid ; Quantitative analysis VIII 植物油和体液等复杂体系中二阶校正法定量分析应用研究 目 录 学位论文原创性声明. I 学位论文版权使用授权书. I 摘 要.II Abstract .V 第 1 章 绪 论. 1 1.1 分析化学概论. 1 1.2 化学计量学概论. 2 1.3 张量校正研究进展. 3 1.4 高效液相色谱结合二阶校正方法在复杂体系分析中的应用. 7 1.4.1 高效液相色谱方法. 7 1.4.2 高效液相色谱-二极管阵列检测器（HPLC-DAD ）结合二阶校正方法 . 8 1.4.3 高效液相色谱-快速扫描荧光检测器 （