| Red clover,Radix Puerariae and other herbal medicines that are rich in isoflavones are often used as alternative medicine to relieve symptoms such as hot flashes,palpitation,and irritability caused by hormone-dependent diseases.Isoflavones are plant metabolites that have estrogenic effects,and usually served as active ingredients of herbal medicines.Studying the distribution and determination of these isoflavones in plants and organisms will help us to better control the quality of these herbal medicines.However,the current methods for analyzing isoflavones are mainly reversed high performance liquid chromatography usingwater-organic solvent system.In order to solve the problems of the traditional reversed phase high performance liquid chromatography in isoflavones analysis,such as the difficulty of separation,the high cost of analysis,the complicated and time-consuming sample processing,and the large amount of organic solvents,this study has established a series of micellar liquid chromatography methods.These methods are efficient,low-cost,simple to operate,environment-friendly,and can be used for the detection of isoflavones in herbal medicines and biological samples.The main research works are as follows:(1)A micellar liquid chromatography method using 0.08 M sodium lauryl sulfate and 6.05 m M β-cyclodextrin: methanol(87:13,v/v)(p H 3)as the mobile phase and C18 as the stationary phase for separating 9 kinds of common isoflavones in herbal medicines within 30 minutes was established.The established micellar liquid chromatography method used an isocratic elution mode with a flow rate at 1 m L/min.This study systematically optimized the chromatographic conditions,including p H,the composition and concentration of surfactants,and the types and proportions of organic solvents.The established micellar liquid chromatography method used an isocratic elution mode with a flow rate of 1 m L/min.This study systematically optimized the chromatographic conditions,including p H,the composition and concentration of surfactants,and the types and proportions of organic solvents.The method has been validated according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use guidelines.There had been no reports of using micellar liquid chromatography to detect isoflavones.This method has been successfully applied to the quantitative analysis of isoflavones in red clover and Radix Puerariae.The method is efficient,cheap and simple to operate.Finally,we verified the presence of supramolecular amphiphilic vesicles in the mobile phase by transmission electron microscopy to explain the increase in chromatographic efficiency.(2)The developed mixed micellar liquid chromatography method containing supramolecular amphiphiles was extended to the analysis of more complex matrix samples.The micellar liquid chromatography method was combined with the second-order calibration method based on the multivariate curve resolution-alternating least square algorithm to analyze the isoflavone content in soybeans.The experimental results show that although there are unknown interfering substances in soybean samples that can affect the accuracy of quantification,and the retention time of different samples has also shifted slightly,the multivariate curve resolution-alternating least squares algorithm combined micellar liquid chromatography combined strategy can achieve satisfactory qualitative and quantitative determination of the target isoflavones.The results show that this combined strategy is efficient,cheap,and convenient.With the presence of unknown interferences,it can also achieve satisfactory qualitative and quantitative analysis of isoflavones in soybeans.Further more,the potential for analysis of isoflavones in more herb medicines has been shown.(3)The mixed micellar solution containing sodium lauryl sulfate/β-cyclodextrin supramolecular amphiphile was uesd as the mobile phase,and diluted serum without other treatment was uesd as the samples to analyze the content of genistein formononetin and biochanin A in serum.At the same time,this method was used to monitor the metabolism of these three isoflavones in the serum of mice after oral administration of red clover extract.The results show that this micellar liquid chromatography method has high selectivity and relatively satisfactory sensitivity,and can accurately measure the content of isoflavones in the spiked serum.Although the sensitivity of this method is lower than the commonly used high-performance liquid chromatography-mass spectrometry/mass spectrometry methods,its fast,simple,safe and cheap characteristics make it a possible alternative to daily monitoring of isoflavones in serum. |
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