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Hyphenated Liquid Chromatography Techniques for the Simultaneous Analysis of Aristolochic Acid Analogues and Their Applications

Author: YuanJinBin
Tutor: YaoShouZhuo
School: Hunan University
Course: Analytical Chemistry
Keywords: Aristolochic acid Aristololactam High Performance Liquid Chromatography Traditional Chinese medicine Component analysis Mass Spectrometry Fluorescence detection
CLC: R284
Type: PhD thesis
Year: 2008
Downloads: 309
Quote: 2
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Abstract

Aristolochic acids, including aristolochic acid (aristolochic acids, AAs) and the aristololactam (aristolactams ALs), has been confirmed to have the role of renal toxicity, carcinogenic and mutagenic, containing aristolochic acid medicinal plants and formulations of the substances most countries disabled. China has also been canceled off Akebia, fangchi, and medicinal standards and green wood, and containing aristolochic find bone wind, the teng Zhushalian traditional Chinese medicine preparation in strict accordance with the prescription drug management. However, there are still some low levels of aristolochic acids in Chinese medicine or herbal use. Therefore, in order to ensure the safe use of drugs, we should strengthen drug production, distribution and use of the various aspects of the regulatory. TCM TCM toxicity for a long time, the development of Chinese medicine in the long-term practice, and the theory and technology of the formation of a series of attenuated efficiency. Have confirmed concocted and compatibility to reduce the toxicity of traditional Chinese medicine (TCM) the manshuriensis and fangchi. We should correctly understand the toxic effects of aristolochic acids, under the guidance of Chinese medicine theory, actively carry out experimental research, looking for Chinese herbal medicine containing aristolochic acids attenuated manufacturing dangerous drugs law and ways to play its normal medicinal efficacy to reduce the negative impact of aristolochic acid. This paper first introduces the medicinal plants and their preparations aristolochic acids, a series of liquid chromatography coupled with detection technology. Then, based on the development of chromatographic detection platform, the cytotoxicity of aristolochic acid I cells toxicokinetic discussed and the manshuriensis compatibility process component analysis, to study the compatibility of this from a chemical point of view attenuated the material basis. Specific research work are as follows: 1. In order to meet the demand of medicinal plants and their preparations aristolochic acids screening, we have developed equipment for the most popular and simple analysis by high performance liquid chromatography method. By extraction, separation, and analysis of the optimization of the conditions, we successfully conducted simultaneous analysis of 12 species of medicinal plants and nine aristolochic acids in the two formulations. The law as mobile phase gradient elution of methanol -0.2% HAc Aristolochia substances to obtain a good chromatographic separation. The linear range of the analysis method is about two orders of magnitude, within the concentration range studied, the linear correlation coefficient is more than 0.9950. Day precision and inter-day precision experiments relative standard deviation is less than 4.35%. The average recovery rate falls between 88.4-98.8%. These results indicate that the method is accurate and reliable. Multicomponent analysis method for this experiment in the screening and quantitative analysis of of Aristolochia substances suspected drugs compared to the previous literature has obvious advantages, more effective, more practical. Aristolochic acids weakly polar small molecules, high ionization efficiency in electrospray ionization mass spectrometry (electro-spray ionization / mass spectrometry, ESI / MS), susceptible to ion suppression effects . Matrix effects, ionization mode, and cone voltage, we systematically explore the behavior of aristolochic acids in ESI mass spectrometry ionization, ESI mass fragmentation regularity found, for the first time The system described aristolochic acids ionization mechanism. On this basis, through the optimization of the experimental conditions and instrumental parameters, reduce the effect of ion suppression, detection sensitivity is slightly higher than the literature. Developed by HPLC-DAD-ESI/MS method can also be the retention time of each peak, the UV spectrum, and the relative molecular weight and mass spectral characteristics debris, and thus has a good qualitative ability. Us, this method is applied to the nine kinds of of aristolochic plants and two formulations nine aristolochic acids analysis, the results are satisfactory. To further enhance the sensitivity of the analysis of the substances Aristolochia, we introduce the fluorescence detection. According to the two major categories of aristolochic acids (AAs and ALs) UV fluorescence characteristics and their distribution in the Aristolochia species characteristics (relatively high levels the AAs and trace ALs), DAD and FLD (fluorescence detection, FLD) series use skillfully to achieve a direct analysis of the most samples AAs and ALs. When the analysis of a trace amount of AAs, we propose a simple derivatives, i.e., under acidic conditions, to the nitro group reduction of AAs imino group into the corresponding ALS then analyzed, and thus achieve high AAs were sensitivity analysis. The analysis results show that the different samples, both cheap and wide popularity DAD and FLD detector associated with a nearly perfect combination of aristolochic acids in the natural distribution. This HPLC-DAD-FLD hyphenated techniques applied to the analysis of various Aristolochia plants and their preparations, the results show that the method is good selectivity and high sensitivity. Under normal circumstances, the linear range of up to 3-4 orders of magnitude, and the linear correlation coefficient (r2) greater than 0.9970; detection limit can be as low as 0.2 ng / ml; intra-day precision and inter-day precision RSDs less than 5.74%; aristolochic acids average recovery was 94.5-99.2%. Although renal toxicity of aristolochic acids have been studied more in-depth, but the toxicokinetic data is still limited. Toxicology or pharmacology data To obtain the Class AA substances, the development of a biological samples in the Class AA substances and their metabolites specific sensitive detection analysis, is undoubtedly of great significance. Aristolochic acids (AAs) in vivo metabolites of aristolochic acid amide derivative (ALs). This article first HPLC-DAD-FLD detection technology of AA I and its metabolites in the cellular environment simultaneously sensitive proprietary detection. The linear range of up to three orders of magnitude, and the linear correlation coefficient greater than 0.9994. The various cell sample analysis results show that the method has the suitable precision and sensitivity applicable to the cell toxicokinetic study. Based on the inverted phase contrast microscope and MTT assay, we studied the cytotoxicity of the L-02 AA I normal human liver cells. Meanwhile, the chromatographic analysis of the cell culture solution of AA I and its metabolites. Assay to two metabolites, one for AL I, can be attributed to another the aristololactam derivative. According to the AA I and its metabolites in the cell culture medium concentration - time data were preliminary statistical estimation of AA I toxicokinetic parameters. The 5. Researchers have confirmed through animal experiments compound compatibility able to to reduce toxicity manshuriensis fact. From the chemical point of view in order to study the relevant wood through the compatibility process toxic ingredients or active ingredient changes, we have developed a good qualitative and quantitative ability HPLC-DAD-ESI/MS analysis method. 27 manshuriensis and Coptis component tracking analysis on quantitative analysis of seven major components. Manshuriensis compatibility Coptis Coptis in protoberberine alkaloids fried out significantly reduce aristolochic acids in manshuriensis still can not be ignored. Experimental manshuriensis compatibility rhubarb similar conclusions. Experiments, we also found that boiling water can significantly reduce the aristolochic acids manshuriensis dissolution rate, compared with 70% ethanol extraction method, water reflux extraction liquid in AA I and AA II content decreased by 58.6% This phenomenon indicates that the practice of Chinese medicine medication and 51.5%, there is a reasonable scientific basis. Our study shows that, manshuriensis compatibility attenuated effect does not appear in the role of compatibility attenuated link pharmacy links. To clarify the compatibility attenuated manshuriensis material foundation for further research work on two fronts, continue Prescription herbs active ingredients, the effective part of the separation, analysis, and differential; animal experiments, tissues and organs, the four pharmacological level of subcellular and molecular biology on the efficacy and mechanism of action studies.

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