The Study On New Methods Of Quality Analysis For Wenxin Granule

Nowadays,one of the key issues that hindering the upgrading of traditional Chinese medicine(TCM)pharmaceutical technology is that the quality analysis methods of TCM cannot meet the actual needs of industrial production,which makes it difficult to further improve the pharmaceutical quality.The development of appropriate quality analytical methods that are in line with the characteristics of TCM has become a technological breakthrough to improve the safety,effectiveness and batch-to-batch consistency of TCM products.Wenxin granule is a typical TCM solid oral dosage forms and an in-depth studies on its whole production process quality analysis methods is helpful to promote the development of modern TCM industry.Accordingly,this thesis took Wenxin granule as the specific research object,and attempted to focus on developing new quality analysis methods that were urgently needed in the production process of Wenxin granule.Analytical methods related to control and assessment of some critical drug quality attributes,i.e.,accurate and comprehensive identification of chemical constituents,rapid determination of saccharides and rapid screening of possible drug microbial contaminations,were successfully developed based on high-resolution mass spectrometry,Raman spectroscopy,SERS,and informatics techniques of pharmaceutical analysis.The main research contents and results were summarized as follows:(1)Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-QTOF-MS)was used to rapidly identify the chemical constituents of Wenxin granule.Molecular networking was proposed to analyze the high-resolution mass spectrometry data.A total of 163 chemical constituents in Wenxin granule were identified,of which 22 compounds were confirmed by comparison with the reference substances.(2)Codonopsis radix and Polygonati rhizome are the sovereign drug and ministerial drug of Wenxin Granule,respectively.The extraction of Codonopsis radix-Polygonati rhizome is an important procedure in the production of Wenxin granule,which has great impact on the batch-to-batch consistency.Saccharides are the main components of the Codonopsis radix-Polygonati rhizome extraction solution.However,up to now,rapidly detecting saccharides in the in-process extraction solution is still a challenge.Herein,a method based on Raman spectroscopy and competitive adaptive weighted-least squares(CARS-PLS)was established for rapid quantification of saccharides.The experimental results showed that the built quantitative calibration mode had good predictive performance for saccharides.The coefficient of determination of prediction(R_p²)for the validation sets of glucose,maltotriose,Codonopsis radix polysaccharides were?0.9939,and the root-mean-square error of prediction(RMSEP)for the validation sets were?1.1052 mg/m L;for the total saccharides of EPCP,the validation sets of R_p² and RMSEP were 0.9743 and 1.4931mg/m L,respectively.(3)Microbial limit test is an important analytical procedure to ensure the safety of drugs,of which Salmonella should be absent in Wenxin granule according to the pharmacopoeia.However,the current well-established method for the limit test of Salmonella is based on multi-step bacteria culture and identification with biochemical and serotype tests,which usually takes at least 3 days.This long detection period can be used for drug product inspection but is difficult for rapid detection of Salmonella in the production process.In response to this urgent demand by the TCM industry,an aptamer-based surface-enhanced Raman spectroscopy(SERS)method was developed for rapidly detection of Salmonella Enteritidis,in which aptamers were used as the recognition elements for targeting bacteria,4-mercaptobenzoic acid(4-MBA)was used as the Raman probe molecule and Au@Ag nanoparticles was used as the SERS substrate.The method was validated to be high specific with no interference from other five pathogenic bacteria.It could identify S.Enteritidis contaminant at?1 CFU/(10 g)spiked level after 6 h of enrichment.In addition,the method could accurately quatitatively analysis of S.Enteritidis.The experimental results show that when the concentration of S.Enteritidis was in the range of 4.17×10²?1.39×10⁷ CFU/m L,there was a good linear relationship between the 4-MBA Raman signal intensity and the logarithm of the concentration of S.Enteritidis(R² was 0.9873),of which the detection limit was 52 CFU/m L.(4)To further improve the simplicity and applicability of the Salmonella detection method,a label-free SERS method for rapid screening of Salmonella was developed.This method did not require recognition units such as aptamers or antibodies,and only needed to perform SERS detection after culturing in selective medium for 12 h,and directly observed whether there was a characteristic peak at 1222 cm^-1 in the SERS spectrum to determine whether there was Salmonella.Compared with the method in pharmacopeia,the developed SERS method is simple,rapid and reliable,which is a promising alternative for rapidly screening the Salmonella contamination in pharmaceutical production.