| Senna granule,as an effective medical option for constipation relief,is now widely used in clinical practice.Senna ethanol extract is the API extracted and concentrated from senna using ethanol for granule production.Sennoside is the main active ingredient of senna as well as the quality control point for the collection of senna herbs and production of senna preparation.At present,the detection of sennoside content in the ethanol extract and senna granule mostly relies on traditional manual extraction method to extract the active ingredient and then the total sennoside content can be measured via UV spectrophotometry.The traditional method does not satisfy the requirements for a high-efficiency and eco-friendly production environment because of its time consuming tests,tedious operation steps,low precision,and the use of solvents that are hazardous to the health of laboratory technicians.Therefore,the development of new testing methods is imperative for senna granule manufacturers.In addition,the components of traditional Chinese medicine are complex.The content of active ingredient of senna herbs fluctuates remarkably depending on the area and period of collection,which significantly affects the production efficiency.In order to help effectively identify the herbs and control the quality of drugs from the very beginning,this study established the first fingerprint of senna herbs through HPLC-Mass Spectrometry and performed herb profile comparison for herb quality identification.In this study,we established a method for qualitative identification of the complex components of the ethanol extract of senna,which have few systematic studies and related reports,through combined application of mobility high resolution liquid chromatography mass spectrometry,which provides new ideas for the development of the medicinal value of senna.The main contents of the paper include:(1)The content of the main components sennoside A and sennoside B were clearly detected,and the corresponding relationship between the total content of sennoside and that of sennoside A and sennoside B in the ethanolic extract of senna leaf was investigated for the first time,and new quality control standard ranges were established,which made it possible for manufacturers to achieve quality control in the production of senna pharmaceuticals.To help improve the efficiency in the traditional production of senna pharmaceuticals using quantitative analysis methods,this study replaced the traditional manual extraction UV method with high performance liquid chromatography(HPLC)analysis method.The parameters of the newly established HPLC method are detailed as follows:an Aglient ZORBAX SB-C18,250 mm × 4.6 mm,3.5 μm column with acetonitrile-0.1%trifluoroacetic acid(14:86)as the mobile phase,flow rate at 1 mL/min,column temperature maintained at 40℃,UV wavelength at 340 nm,and injection volume at 10μL.Compared with the traditional UV method,the HPLC method is simple in sample pretreatment,safe and environmentally friendly in reagents,and the cost of the complete assay is only 50%of that of the traditional UV method,and the detection time is at least 6 hours less than that of the traditional UV method.The spiked recovery of the sample solution by the new method is an astonishing 99%～103%.(2)Using HPLC-MS/MS,the fingerprint profile of senna herb was established for the first time,which helped improve the quality control level for senna pharmaceutical manufacturers.Based on the HPLC method established in this paper,the data of the ethanol extract of the herbs was collected using HPLC-MS/MS,which provides higher separation efficiency.The results showed that the chromatographic peak similarity of narrow senna leaves collected from different areas is greater than 0.98,and the similarity between the pointed senna leaves and narrow senna leaves is 0.95,which confirmed that the HPLC-MS/MS method is effective in the separation of sennoside A and B from other complex components.Comparative experiments with multiple batches showed that the total content of sennoside A and B was about 1.4%in the Chinese domestic herb and 1.6%in the Indian herb.The total content of sennoside A and B was 1.0%in the pointed senna leaf and over 1.3%in the narrow senna leaf.The experiments showed that the quality of the narrow senna herb is better than that of the pointed senna herb in terms of the content of sennoside A and B.And overall,the quality of the Indian senna herb proves to be better than that of its Chinese domestic counterpart.This further confirmed that the newly established herbal fingerprint could be used for effective quality control of the herbs and production.(3)For the first time,a new method was developed for the qualitative identification of the complex components in senna using ultra performance liquid chromatography(UPLC)-ion mobility spectrometry(IMS)-quadruple time-of-flight mass spectrometry(QTOF-MS/MS),and 51 components were identified in the ethanol extract of senna.In addition to the established HPLC and HPLC-MS/MS methods,the UPLC-IMS-QTOF method was used for the first time to further investigate the various components in the ethanol extract of senna and determine the structural formulas,categories and names of these components.Thus,a new method,UPLC-IMS-QTOF,was invented for the qualitative identification of the components.Using the scan mode on HDMSE with Ion Mobility on to collect data generated in the low-energy(parent ion)and high-energy(daughter ion)passageways,four sets of data are to be obtained,including Retention Time,Peak Intensity,Mass Charge Ratio,and Drift Time of Mobility.With any three vectors combined,a three-dimensional spectrum can be formed.Through the 3D spectrums,the low and high energy spectrum corresponding to each retention time point as well as the extraction ion flow and mobility trajectory can be obtained,which can identify isomers or components with the same retention time.As an experiment result,the existence of a variety of complex components such as flavonoids,anthraquinones and naphthoquinones,tannins,and simple aromatic compounds were inferred and confirmed in the ethanol extract of senna leaves based on the mass charge ratio,mass error,extraction ion flow diagrams,fragment analysis and the reference to the related compounds recorded in the Chinese Medicine Library.A total of 10 components were identified in senna leaves,such as tannins and coumarins,which had never been reported before.And 26 flavonoids were detected for the first time.The results of this study provide data support for manufacturers to research and develop new indications and new forms of prepared drugs of senna granules.Results also provide data reference for further research on the pharmacology and toxic side effects of senna herbs,as well as valuable research directions for the development of new medicinal values of senna leaves. |
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