| Object:Saponins are rich in resources and are of great value in the development of new drugs and functional foods.However,the quality evaluation of traditional Chinese medicines and their preparations containing saponins have always been difficult in the field of traditional Chinese medicine analysis.The main reason is that most of the saponins do not produce ultraviolet absorption or produce weak ultraviolet absorption,and the detection method has low sensitivity.Moreover,the preparation method in the analysis method is complicated and time consuming,resulting in unsatisfactory repeatability and accuracy of the method,and lack of practicality.In this study,taking Astragali radix Pieces-Intermediates(Standard Decoction and Freeze-dried Powder)-Formulated Granules as the research object,a simple and comprehensive saponin quality control method was established by optimizing the sample preparation method and introducing a new analytical instrument QDA.Therefore,a scientific,holistic and practical quality evaluation method for Astragali radix and its preparations is established,which also provides reference for the establishment of other saponin analysis methods.Methods:1)Quality evaluation method of Astragali radix pieces:The sample is prepared by ultrasonic extraction instead of heating and reflux extraction,using ammonia water addition and SPE column enrichment.UPLC-QDA analytical instrument was introduced,and the method of determining the content of astragaloside IV was established by using selective ion monitoring mode and ginsenoside Rgl as internal standard.At the same time,UPLC-PDA was used to establish the method for the determination of flavonoid in Astragali radix and the simultaneous analysis of fingerprints.And methodological investigation and verification of 15 batches of Astragali radix.2)Establish a full-component fingerprint analysis method and target fingerprint of Astragali radix:Using UPLC-QDA,the fingerprint of the Astragali radix was established by using the full scan mode,and the common peak was identified by the mass number and the standard product;A selective ion monitoring mode was used to specifically detect the main saponins of Astragali radix,and a targeted fingerprint of Astragali radix was established.Calculating the similarity and the relative retention time and relative peak area of common peak.3)Establishment of analytical methods for intermediates(standard decoction and freeze-dried powder)and their preparations(formulation granules)of Astragali radix:The sample solution was prepared by ultrasonic extraction,ammonia addition and SPE column enrichment,and the quantitative analysis method of astragaloside ? was established by universal HPLC-ELSD method.HPLC-DAD was used to establish the method for the determination of flavonoids in Astragali radix and the simultaneous analysis of fingerprints.Methodological investigation and analysis of different Astragali radix intermediates and formula particles were carried out.4)Expanding the application of the method:Taking the standard decoction of Platycodon grandiflorum as the research object,the changes of chemical components and contents of saponins before and after ammonia water treatment of Platycodon grandiflorum standard decoction were compared.High performance liquid chromatography-mass spectrometry(UPLC-Orbitrap-MS/MS)was used to identify the components of the changed chromatographic peaks and to explore the chemical mechanism of ammonia water treatment.The sample solution was prepared by ultrasonic extraction,ammonia water addition and SPE column enrichment.The content of platycodin D was determined by UPLC-PDA,and the characteristic map of platycodin was analyzed.Results:1)The established method has high sensitivity and wide linear range.The content of astragaloside ? was 0.04?0.25%,which indicated that the established UPLC-QDA method could be used to determine the content of astragaloside ? in Astragali radix.The established simultaneous determination method of flavonoid content and fingerprint is fast and accurate,which can be used to evaluate the similarities and differences of flavonoids in different batches of Astragali radix.2)UPLC-QDA fingerprint of Astragali radix was established.The similarity of 15 batches of Astragali radix was greater than 0.920.There were 12 common peaks,which were identified by QDA mass spectrometer.Six of them were saponins and six were flavonoids.The results show that the fingerprint method established by UPLC-QDA can simultaneously detect a variety of saponins and flavonoids,and the components corresponding to each common peak can be directly determined by its mass number.A targeted fingerprint for saponin was established.Targeted fingerprints are more specific and more targeted to achieve targeted detection of saponins3)The content of astragaloside IV in the intermediates(standard decoction and freeze-dried powder)of Astragali radix and their preparations(formula granules)was determined by HPLC-ELSD.The results showed that the content of astragaloside IV in different batches of standard decoction,freeze-dried powder and formula granules were 0.13 mg mL-1?0.33 mg mL-1,0.10%?0.19%,0.11%?0.13%,respectively.Quantitative analysis of flavonoids in standard decoction,freeze-dried powder and formula granules by HPLC-DAD showed that the contents of isoflavone glucoside in three formulations were 0.03 mg mL-1?0.19 mg mL-1,0.05%?0.18%,0.11%?0.14%,respectively.Meanwhile,the similarity of flavonoid fingerprints were greater than 0.910,0.700 and 0.990,respectively4)UPLC-PDA analysis of 11 batches of Platycodon grandiflorum Decoction showed that:The method has good reproducibility and high accuracy.The effect of adding ammonia water is to promote the conversion of the platycodin D analog containing acetyl group to platycodin D,thereby increasing the content of platycodin D.The content of Platycodon D in different batches of Platycodon grandiflorum standard decoction ranged from 0.21%to 0.34%,and the similarity of characteristic map was more than 0.800.Conclusion:In this study,the content determination of saponins in Astragali radix and the overall fingerprint analysis method were established.The method is simple and reproducible.For the intermediates of Astragali radix preparations and their preparations(formula granules),a practical method for determination of Astragali radix preparations by HPLC and fingerprint analysis was established to meet the requirements of product quality control in the production process.The method was successfully applied to the determination of Platycodon D in Platycodon grandiflorum standard decoction.Therefore,this study provides theoretical basis and analytical methods for quantitative analysis and fingerprint analysis of saponins. |
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