化学计量学方法在毛细管电泳与荧光光谱定量分析及中药质量控制中的应用研究
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摘要
应用化学计量学方法解析重叠信号,对于建立简单快速的毛细管电泳及荧光光谱分析方法能够起到非常重要的作用;应用化学计量学方法结合中药色谱指纹图谱对于中药质量控制的实际工作有着非常重要的意义。本论文旨在研究毛细管电泳重叠峰的化学计量学分辨方法,简化多组分体系毛细管电泳定量分析的实验步骤,为毛细管电泳定量分析提供新的实验技术和分析方法;探讨化学计量学方法在手性化合物对映异构体荧光光谱分析中的应用,建立高灵敏度的手性化合物定量分析方法;将相关化学计量学方法与中药色谱指纹图谱结合,解决中药色谱指纹图谱进行中药质量控制中有关谱图校正、产品评价和数据压缩的问题。全文共分9章。
第一章概述了小波变换、多元校正和多元分辨方法的研究进展及其在分析化学中的应用现状。此外,还概述了化学计量学方法在中药质量控制中的应用及简要介绍了当前分析化学中常用的手性识别方法。
第二章研究了连续小波变换方法在毛细管电泳重叠峰分辨中的应用。将连续小波变换方法成功应用于重叠的电泳信号峰分辨,拓宽了小波变换方法的应用领域。通过对模拟数据和实验数据的分析,讨论了应用连续小波变换方法分辨毛细管电泳重叠信号峰的可行性。对连续小波变换和常用的数字微分方法进行了比较,结果证明连续小波变换用于处理含噪声电泳重叠峰优于数字微分方法。
第三章则根据第二章得到的结论,将连续小波变换方法应用于手性化合物对映异构体的毛细管电泳定量分析。通过结合毛细管电泳、连续小波变换及实验设计方法可对色氨酸对映异构体进行准确的定量测定。其中连续小波变换方法仍然用于重叠峰的分辨;实验设计方法的使用,则较好地解决了毛细管电泳重现性不高影响定量测定的问题。
第四章中讨论了直观推导式演进特征投影法(HELP)在毛细管电泳重叠峰分辨中的应用。利用HELP方法对复方降压片的毛细管电泳分离中两种组分形成的重叠峰进行了分辨,可定量测定这两种化合物。分辨结果说明HELP方法是一种可行的二阶电泳重叠峰分辨方法。
第五章基于牛血清蛋白(BSA)的手性识别功能,将荧光光谱法,BSA主—客体配合物和人工神经网络(ANN)方法相结合,建立了一种高灵敏测定色氨酸对映异构体组成的新方法。该方法利用BSA与手性色氨酸混合溶液的荧光光谱会随对映异构体组成变化的现象,用人工神经网络建立光谱变化与对映异构体组成之间的数学模型,可对混合物中的色氨酸对映异构体组成进行测定。实验结果表明,该方法能用于测定含L-色氨酸和D-色氨酸总浓度为2.5μmol·L~(-1)的溶液中两种色氨酸异构体的组成。
第六章将遗传算法(GA)与多元线性回归(MLR)方法结合应用于荧光光谱,进行色氨酸对映异构体组成的定量测定。实验同第5章,但采用GA-MLR方法取代ANN方法建立荧光光谱与对映异构体组成之间的数学模型。与第5章相比,对色氨酸对映异构体组成的测定误差更小,充分体现了GA方法在多元校正变量选择中的优势。这是一种很有潜力的手性化合物的定量分析方法。
第七章以中药粉葛为例,讨论了correlation optimized warping方法应用于中药质量控制的可行性。对粉葛样品的讨论结果表明,该方法能够在不影响指纹图谱形状的同时,消除由保留时间漂移引起的对样品质量评价的影响,从而明显提高质量评价的准确性,因此可用于校正中药色谱指纹图谱中的保留时间漂移。此方法的优点在于能够自动运行,可对指纹图谱进行快速处理,有很好的实用价值。
第八章提出了一种根据其色谱指纹图谱应用化学模式识别方法对经醋制与未经醋制加工的延胡索样品进行识别的方法。该方法结合了无监督的主成分分析方法和有监督的人工神经网络方法,能够准确的区别两种延胡索样品。由于醋制与未醋制的延胡索可视作同种药物的质量不同的产品,因此这种方法还能推广应用于中药质量控制中对产品质量的评价。
第九章将一种基于离散小波变换的数据压缩算法成功地应用于对中药粉葛和延胡索的色谱指纹图谱的数据压缩。在不引起指纹图谱改变的前提下,通过对这两种中药的色谱指纹图谱的压缩,可使其占用的存储空间分别减小92.03%和93.91%。这种方法对于中药色谱指纹图谱的储存、传输与应用,以及中药色谱指纹图谱数据库的建立都有着重要意义。
There are nine chapters in this dissertation,which investigates the application of capillary electrophoresis(CE),high performance liquid chromatography and fluorescence spectroscopy combined with chemometric methods including wavelet transform,multivariate calibration and multivariate resolution to the quality control of Traditional Chinese Medicine(TCM)and the simultaneous determination of unseparated compounds.
In chapter 1,the development of wavelet transform,multivariate calibration and multivariate resolution and the application of these chemometric methods in analytical chemistry in recent years were reviewed.In addition,there are a review of the application of chemometrics in the quality control of TCM and a brief introduction of chiral discrimination in the chapter.
In chapter 2,the application of continuous wavelet transform(CWT)to resolving overlapping peaks from CE was described.The proposed approach was studied and examined on both simulated and experimental data.The quantification results are satisfactory.CE combined with CWT has been shown to be a practicable approach for quantitative determining coeluted compounds in overlapping peaks from CE.In addition,the quantification results from CWT were compared with those from numerical differentiation method.CWT has been shown to be superior to numerical differentiation method for processing experimental signals which contain noise.
In chapter 3,CWT combined with experimental design(ED)was applied to determining the enantiomers of tryptophan(Trp).When the baseline separation of L-Trp and D-Trp was not reached by CE,by utilizing CWT to resolve the overlapping peaks of the Trp enantiomers and using ED to overcome the low reproducibility of CE, the composition of the L-Trp and D-Trp were determined precisely.
Chapter 4 is concerned with the application of heuristic evolving latent projections (HELP)method to second-order CE data.The HELP method was applied to resolving the overlapping peaks of chloroquine phosphate and promethazine hydrochloride,the two main components in compound reserpine tablets.Satisfactory resolution results were obtained.
In chapter 5,a novel strategy which combines ordinary fluorescence spectroscopy, guest-host chemistry and artificial neural networks(ANN),was proposed for determining Trp enantiomers.ANN was employed to establish a quantitative model between the fluorescence spectra of the prepared samples and the compositions of Trp enantiomers.The proposed approach was found to be a high-sensitivity method for determining Trp enantiomers.
Chapter 6 is concerned with the application of genetic algorithm-multiple linear regression(GA-MLR)method to the fluorescence spectroscopy for determining the enantiomeric composition of Trp.The difference between the method described in this chapter and the method described in chapter5 is that the calibration model for calculating the concentration of Trp enantiomers was built with GA-MLR instead of ANN.The quantification results of this chapter is better than those of chapter 5,the GA-MLR method being used has been shown superior to the ANN method being used in chapter 5 for determining Trp enantiomers.
It is important to correct the retention time shifts in chromatographic fingerprints of TCM for the quality control of TCM.In Chapter 7,it was investigated whether correlation optimized warping(COW)method is a practicable method for correcting the retention time shifts in chromatographic fingerprints of TCM for quality control purpose.The method was successful applied to 27 Radix Puerariae thomsonii(RPT) samples.
Chapter 8 aims at developing a chemical pattern recognition method to discriminate processed and unprocessed Rhizoma Corydalis(RC)samples.By combining principal component analysis and ANN,the processed and unprocessed samples were successfully discriminated.
In chapter 9,a data compression algorithm based on discrete wavelet transform was proposed to compress the chromatographic fingerprints of TCM.The proposed algorithm was successfully applied to the fingerprints described in chapter 7 and chapter 8.The storage space of the fingerprints of RPT and RC can be reduced by 92.03%and 93.91%respectively.
In conclusion,CWT and HELP methods were applied to the resolution of overlapping peaks from CE and a novel strategy which combines fluorescence spectroscopy and multivariate calibration methods was proposed for determining Trp enantiomers.The proposed methods can be applied to quantitative determining the components in mixtures without separating them.In addition,the correction of the retention time shifts in chromatographic fingerprints of TCM,data compression of chromatographic fingerprints of TCM and the recognition of TCM samples were investigated.The proposed approaches can benefit the quality control of TCM.
第一章概述了小波变换、多元校正和多元分辨方法的研究进展及其在分析化学中的应用现状。此外,还概述了化学计量学方法在中药质量控制中的应用及简要介绍了当前分析化学中常用的手性识别方法。
第二章研究了连续小波变换方法在毛细管电泳重叠峰分辨中的应用。将连续小波变换方法成功应用于重叠的电泳信号峰分辨,拓宽了小波变换方法的应用领域。通过对模拟数据和实验数据的分析,讨论了应用连续小波变换方法分辨毛细管电泳重叠信号峰的可行性。对连续小波变换和常用的数字微分方法进行了比较,结果证明连续小波变换用于处理含噪声电泳重叠峰优于数字微分方法。
第三章则根据第二章得到的结论,将连续小波变换方法应用于手性化合物对映异构体的毛细管电泳定量分析。通过结合毛细管电泳、连续小波变换及实验设计方法可对色氨酸对映异构体进行准确的定量测定。其中连续小波变换方法仍然用于重叠峰的分辨;实验设计方法的使用,则较好地解决了毛细管电泳重现性不高影响定量测定的问题。
第四章中讨论了直观推导式演进特征投影法(HELP)在毛细管电泳重叠峰分辨中的应用。利用HELP方法对复方降压片的毛细管电泳分离中两种组分形成的重叠峰进行了分辨,可定量测定这两种化合物。分辨结果说明HELP方法是一种可行的二阶电泳重叠峰分辨方法。
第五章基于牛血清蛋白(BSA)的手性识别功能,将荧光光谱法,BSA主—客体配合物和人工神经网络(ANN)方法相结合,建立了一种高灵敏测定色氨酸对映异构体组成的新方法。该方法利用BSA与手性色氨酸混合溶液的荧光光谱会随对映异构体组成变化的现象,用人工神经网络建立光谱变化与对映异构体组成之间的数学模型,可对混合物中的色氨酸对映异构体组成进行测定。实验结果表明,该方法能用于测定含L-色氨酸和D-色氨酸总浓度为2.5μmol·L~(-1)的溶液中两种色氨酸异构体的组成。
第六章将遗传算法(GA)与多元线性回归(MLR)方法结合应用于荧光光谱,进行色氨酸对映异构体组成的定量测定。实验同第5章,但采用GA-MLR方法取代ANN方法建立荧光光谱与对映异构体组成之间的数学模型。与第5章相比,对色氨酸对映异构体组成的测定误差更小,充分体现了GA方法在多元校正变量选择中的优势。这是一种很有潜力的手性化合物的定量分析方法。
第七章以中药粉葛为例,讨论了correlation optimized warping方法应用于中药质量控制的可行性。对粉葛样品的讨论结果表明,该方法能够在不影响指纹图谱形状的同时,消除由保留时间漂移引起的对样品质量评价的影响,从而明显提高质量评价的准确性,因此可用于校正中药色谱指纹图谱中的保留时间漂移。此方法的优点在于能够自动运行,可对指纹图谱进行快速处理,有很好的实用价值。
第八章提出了一种根据其色谱指纹图谱应用化学模式识别方法对经醋制与未经醋制加工的延胡索样品进行识别的方法。该方法结合了无监督的主成分分析方法和有监督的人工神经网络方法,能够准确的区别两种延胡索样品。由于醋制与未醋制的延胡索可视作同种药物的质量不同的产品,因此这种方法还能推广应用于中药质量控制中对产品质量的评价。
第九章将一种基于离散小波变换的数据压缩算法成功地应用于对中药粉葛和延胡索的色谱指纹图谱的数据压缩。在不引起指纹图谱改变的前提下,通过对这两种中药的色谱指纹图谱的压缩,可使其占用的存储空间分别减小92.03%和93.91%。这种方法对于中药色谱指纹图谱的储存、传输与应用,以及中药色谱指纹图谱数据库的建立都有着重要意义。
There are nine chapters in this dissertation,which investigates the application of capillary electrophoresis(CE),high performance liquid chromatography and fluorescence spectroscopy combined with chemometric methods including wavelet transform,multivariate calibration and multivariate resolution to the quality control of Traditional Chinese Medicine(TCM)and the simultaneous determination of unseparated compounds.
In chapter 1,the development of wavelet transform,multivariate calibration and multivariate resolution and the application of these chemometric methods in analytical chemistry in recent years were reviewed.In addition,there are a review of the application of chemometrics in the quality control of TCM and a brief introduction of chiral discrimination in the chapter.
In chapter 2,the application of continuous wavelet transform(CWT)to resolving overlapping peaks from CE was described.The proposed approach was studied and examined on both simulated and experimental data.The quantification results are satisfactory.CE combined with CWT has been shown to be a practicable approach for quantitative determining coeluted compounds in overlapping peaks from CE.In addition,the quantification results from CWT were compared with those from numerical differentiation method.CWT has been shown to be superior to numerical differentiation method for processing experimental signals which contain noise.
In chapter 3,CWT combined with experimental design(ED)was applied to determining the enantiomers of tryptophan(Trp).When the baseline separation of L-Trp and D-Trp was not reached by CE,by utilizing CWT to resolve the overlapping peaks of the Trp enantiomers and using ED to overcome the low reproducibility of CE, the composition of the L-Trp and D-Trp were determined precisely.
Chapter 4 is concerned with the application of heuristic evolving latent projections (HELP)method to second-order CE data.The HELP method was applied to resolving the overlapping peaks of chloroquine phosphate and promethazine hydrochloride,the two main components in compound reserpine tablets.Satisfactory resolution results were obtained.
In chapter 5,a novel strategy which combines ordinary fluorescence spectroscopy, guest-host chemistry and artificial neural networks(ANN),was proposed for determining Trp enantiomers.ANN was employed to establish a quantitative model between the fluorescence spectra of the prepared samples and the compositions of Trp enantiomers.The proposed approach was found to be a high-sensitivity method for determining Trp enantiomers.
Chapter 6 is concerned with the application of genetic algorithm-multiple linear regression(GA-MLR)method to the fluorescence spectroscopy for determining the enantiomeric composition of Trp.The difference between the method described in this chapter and the method described in chapter5 is that the calibration model for calculating the concentration of Trp enantiomers was built with GA-MLR instead of ANN.The quantification results of this chapter is better than those of chapter 5,the GA-MLR method being used has been shown superior to the ANN method being used in chapter 5 for determining Trp enantiomers.
It is important to correct the retention time shifts in chromatographic fingerprints of TCM for the quality control of TCM.In Chapter 7,it was investigated whether correlation optimized warping(COW)method is a practicable method for correcting the retention time shifts in chromatographic fingerprints of TCM for quality control purpose.The method was successful applied to 27 Radix Puerariae thomsonii(RPT) samples.
Chapter 8 aims at developing a chemical pattern recognition method to discriminate processed and unprocessed Rhizoma Corydalis(RC)samples.By combining principal component analysis and ANN,the processed and unprocessed samples were successfully discriminated.
In chapter 9,a data compression algorithm based on discrete wavelet transform was proposed to compress the chromatographic fingerprints of TCM.The proposed algorithm was successfully applied to the fingerprints described in chapter 7 and chapter 8.The storage space of the fingerprints of RPT and RC can be reduced by 92.03%and 93.91%respectively.
In conclusion,CWT and HELP methods were applied to the resolution of overlapping peaks from CE and a novel strategy which combines fluorescence spectroscopy and multivariate calibration methods was proposed for determining Trp enantiomers.The proposed methods can be applied to quantitative determining the components in mixtures without separating them.In addition,the correction of the retention time shifts in chromatographic fingerprints of TCM,data compression of chromatographic fingerprints of TCM and the recognition of TCM samples were investigated.The proposed approaches can benefit the quality control of TCM.
引文
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