| The issue of drug safety has always been a focus of attention in society and academia.Research on related substances is an important part of drug quality control and a key factor in ensuring drug safety.The inspection requirements for related substances in drug quality standards of various countries are gradually improving.Compound aminophenolamine tablets have an important position in the treatment of influenza,and there are many manufacturers.As a compound preparation,it mainly contains acetaminophen,amantadine hydrochloride,artificial bezoar,caffeine,chlorpheniramine maleate,which is suitable for relieving fever,headache,limb aches,runny nose and other influenza-related symptoms.Through literature research on the quality of compound aminophenolamine tablets,it is found that most of the literature focuses on the identification of compound aminophenolamine tablets,the determination of principal component content and other research content,and the control of its chemical composition is mostly focused on the examination of acetaminophen related substances,p-aminophen,while other related substances of acetaminophen,caffeine and chlorpheniramine maleate are less studied.The survey results also show that in the Pharmacopoeia of the People’s Republic of China(2020 edition),the related substance inspection items of the three chemical drugs only control p-aminophen,the related substance of acetaminophen.Therefore,this project systematically studies the related substances of acetaminophen,caffeine and chlorpheniramine maleate in compound aminophenolamine tablets,and makes a preliminary evaluation of the safety of related substances,improves drug impurity information,improves drug quality standards,and then controls drug quality.1.Reserch on the related substances of compound aminophenolamine tablets(1)Screening of detection methods.In this study,thin layer chromatography(TLC method),high performance liquid phase(HPLC method)and high performance liquid-mass spectrometry(HPLC-MS method)were used successively to study three chemical-related substances in compound aminophenolamine tablets.(2)Qualitative and quantitative detection of related substances.Preliminary detection methods for 19 related substances of acetaminophen,caffeine and chlorpheniramine maleate were preliminarily established.7 related substances of acetaminophen,3 related substances of caffeine and 1 related substance of chlorpheniramine maleate were detected in chemical APIs by liquid chromatography-mass chromatography.A total of 2 related substances of acetaminophen,2 related substances of caffeine and 1 related substance of chlorpheniramine maleate were detected in the compound preparation by two preparation methods.The paracetamol-related substances N-(4-hydroxyphenyl)propionamide,4-acetamidophenyl acetate,caffeine-related substances theophylline,1,7-dimethyl-1H-purine-2,6-dione were quantified,and the repeatability,precision,stability and dosing recovery of the above four related substances were completed.At the same time,the stability of destructive samples was investigated under the conditions of strong acid,strong base,oxidation,60°C and 150°C.(3)Separation and preparation of related substances.In this study,chlorpheniramine maleate related substances were separated,purified and enriched by repeated gel column chromatography technology,and the structure of the separated substances was identified by HPLC-MS and nuclear magnetic resonance-hydrogen spectroscopy(~1H-NMR),and the compound was determined to be 2-[1-(4-chlorophenyl)-1-propen-1-yl]pyridine,the related substance of chlorpheniramine maleate.2.Study on the fingerprint of chemical composition of compound aminophenolamine tablets(1)The HPLC method was used to establish a chemical composition fingerprint for the compound preparation before and after the process change.Through the similarity evaluation system of traditional Chinese medicine fingerprint,SPSS data processing system and SIMCA data processing system,the fingerprint was analyzed in detail,and the fingerprint before and after the process change was detected and analyzed through cluster analysis and principal component analysis(PCA),and the samples before and after the change could be divided into three categories,the first category was the test products before the process change.The second and third categories are both test products after process changes.It is explained that the process change has a certain impact on the chemical composition of the compound preparation.3.Toxicity study of compound aminophenolamine tablets related substances(1)In this study,bioinformatics technology was used to conduct preliminary toxicity exploration of related substances,and it was found that acetaminophen-related substance 4-acetaminophenylacetate has strong hepatotoxicity and nephrotoxicity,and caffeine-related substance 1,7-dimethyl-1H-purine-2,6-dione has strong acute toxicity,hepatotoxicity and nephrotoxicity.(2)In vitro cytotoxicity study of related substancesThe MTT method was used to study the effects of four related substances,4-acetylaminophenylacetate,2-(1-(4-chlorophenyl)prop-1-en-1-yl)pyridine,4-(4-chlorobenzoyl)pyridine,and 2,2’-pyridinethylamine,on the survival rate of Hep G2cells.The morphological effects of the four related substances on Hep G2 cells were also investigated.Research has found that 4-acetylaminophenyl acetate and 2-(1-(4-chlorophenyl)prop-1-en-1-yl)pyridine are present between 0.1-100μmol/L.There is a certain toxicity on Hep G2 cells within the concentration range of mol/L,with 2,2’-pyridylethylamine and 4-(4-chlorobenzoyl)pyridine ranging from 0.1-10μmol/L.There is a weak toxicity on Hep G2 cells at a concentration of mol/L,but 2,2’-pyridylethylamine has a strong toxicity at 100μmol/L on Hep G2 cells.Cell morphology studies have also found that the related substances 4-acetylaminophenylacetate and 2-(1-(4-chlorophenyl)prop-1-en-1-yl)pyridine range from 0.1-100μmol/L.The concentration range of mol/L has a certain effect on the morphology of Hep G2 cells,and the degree of influence increases with the increase of concentration,while 2,2’-pyridylethylamine and 4-(4-chlorobenzoyl)pyridine range from 0.1-10μmol/L,there is a slight effect on the morphology of Hep G2 cells,with2,2’-pyridylethylamine at a concentration of 100μmol/L.The concentration has a significant impact on the morphology of Hep G2 cells.It is suggested that the related substances 4-acetylaminophenyl acetate and 2-(1-(4-chlorophenyl)prop-1-en-1-yl)pyridine have certain hepatotoxicity,while the related substances 4-(4-chlorobenzoyl)pyridine have weak hepatotoxicity,while the related substances 2,2’-pyridylethylamine may have stronger hepatotoxicity at high concentrations.(3)Toxicokinetic studies on related substancesFour toxicokinetic detection methods for 4-acetylaminophenylacetate,2-(1-(4-chlorophenyl)prop-1-en-1-yl)pyridine,4-(4-chlorobenzoyl)pyridine,and 2,2’-pyridylethylamine were established,and methodological studies were conducted.The standard curve was reasonable,and the extraction recovery rate and matrix effect were within the required range.After research,it was found that four related substances,4-acetylaminophenylacetate,2-(1-(4-chlorophenyl)prop-1-en-1-yl)pyridine,4-(4-chlorobenzoyl)pyridine,and 2,2’-pyridinethylamine,were not detected in the blood at48 hours,indicating that the four related substances can be completely metabolized within 48 hours.The AUC(0-t)of the four related substances are all relatively high,indicating that the bioavailability of the four related substances is good,indicating that attention should be paid to the toxic effects of the intake of related substances on biological individuals.At the same time,the Tmax of the four related substances is mostly concentrated around half an hour,indicating that the intake of the relevant substances reaches the maximum blood drug concentration within half an hour.This study conducted a systematic study on the detection method and toxicity of chemical substances related to compound paracetamol and amantadine tablets.A detection method for 19 related substances has been established,and quantitative analysis has been conducted on 4 related substances in compound paracetamol and amantadine tablets.Fingerprint analysis was conducted on the chemical components of the compound formulation before and after the process change.And the toxicity of related substances in the chemical drugs was predicted.At the same time,this study conducted systematic in vitro cytotoxicity experiments and in vivo toxicokinetic studies.To lay a scientific foundation for the control of drug production processes and the improvement of quality standards,while also providing research ideas for the detection and safety evaluation of related substances in compound formulations. |
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