The aim of this study was to develop a simple and derivatization free method for the Quantification of S-Epichlorohydrin in R-Epichlorohydrin by using a gas chromatography coupled with flame ionization detector (FID)....The aim of this study was to develop a simple and derivatization free method for the Quantification of S-Epichlorohydrin in R-Epichlorohydrin by using a gas chromatography coupled with flame ionization detector (FID). Enantiopure epichlorohydrin was a valuable epoxide key starting material for preparing optically active Rivaroxaban. The enantiomeric separations of S-Epichlorohydrin and R-Epichlorohydrin were achieved on Gamaa-Dex-225 (30 meters × 0.25 mm I.D, 0.25 μm) column with a total run time of 30 min. Nitrogen was used as a carrier gas with constant pressure 25.0 psi. The critical experimental parameters such as, column selection, flow rate, injection volume and diluent were studied and optimized. Excellent correlation coeffient between peak responses and concentrations was >0.9998. The recoveries of S-Epichlorohydrin spiked in R-Epichlorohydrin were in the range from 98.2% to 102.8%. Limit of quantitation for S-Epichlorohydrin was sufficiently lower than limits specified by ICH. The method has validated as per International Conference on Harmonization (ICH) guidelines. A precise, accurate, linear and robust Gas Chromatography method was developed for the quantification of S-Epichlorohydrin in R-Epichlorohydrin for Rivaroxaban.展开更多
Background:The precise insertion of large DNA fragments(>3–5 kb)remains one of the key obstacles in establishment of genetically modified murine models.Methods:A 21 kb large DNA fragment containing three tandemly ...Background:The precise insertion of large DNA fragments(>3–5 kb)remains one of the key obstacles in establishment of genetically modified murine models.Methods:A 21 kb large DNA fragment containing three tandemly linked copies of the human HRAS gene was inserted into the genome of C57BL/6J mouse,generating a mouse model designated as KI.C57-ras(or named NF-h HRAS).Whole-genome sequencing and Sanger sequencing were utilized to it confirm precise insertion and copy number.The stability of transgene expression among different generations was verified from multiple aspects using by digital PCR,western blot and DNA sequencing.To assess tumor susceptibility in the mouse model,N-Nitroso-N-methylurea(MNU)was administered at a dosage of 75 mg/kg.Histopathological examinations were conducted using hematoxylin and eosin(H&E)staining.Results:The HRAS DNA fragment was inserted into mouse chromosome 15E1 site,locating between 80623202 bp and 80625020 bp.NF-h HRAS mice exhibited stable inheritance and displayed consistent phenotypes across individuals.Moreover,this mouse model exhibited a high susceptibility to carcinogens.Upon administration of MNU the earliest mortality onset was earlier than that of wild-type littermates(day 65 vs.day 78 for male and day 56 vs.day 84 for female).Notably,100%of the NF-h HRAS transgenic mice developed tumors,with approximately 84%of male NF-h HRAS mice exhibiting specific tumor types,such as squamous cell carcinoma or squamous cell papilloma,which was consistent with the previously reported carcinogenic rasH2 mouse model.The types of tumors and the target organs exhibited diversity in NFh HRAS mice,while the spontaneous tumor incidence remained low(1/50).Conclusions:The NF-h HRAS mice demonstrated excellent genetic stability,a reproducible phenotype,and high susceptibility to carcinogens,indicating their potential utility in non-clinical safety evaluations of drugs as per the S1B guidelines issued by the ICH(The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use).展开更多
A stability-indicating liquid chromatographic method has been developed and validated for the determination of Diltiazem Hydrochloride(DTZ) together with its six related substances(Diltiazem sulphoxide,Imp-A,Imp-B,Imp...A stability-indicating liquid chromatographic method has been developed and validated for the determination of Diltiazem Hydrochloride(DTZ) together with its six related substances(Diltiazem sulphoxide,Imp-A,Imp-B,Imp-D,Imp-E,and Imp-F) in a laboratory mixture as well as in a novel tablet formulation developed in-house.Efficient chromatographic separation was achieved on a Hypersil BDS C18(150 mm*4.6 mm,5.0 μm) with mobile phase containing 0.2% Triethylamine(TEA) in gradient combination with acetonitrile(ACN) at a flow rate of 1.0 mL/min and the eluent was monitored at 240 nm.In the developed method,the resolution of DTZ from any pair of impurities was found to be greater than 2.0.The test solution and related substances were found to be stable in the diluent for 24 h.The developed method resolved the drug from its known impurities,stated above,and also from additional impurities generated when the formulation was subjected to forced degradation;the mass balance was found close to 99.9%.Regression analyses indicate correlation coefficient value greater than 0.997 for DTZ and its six known impurities.The LOD for DTZ and the known impurities was at a level below 0.02%.The method has shown good,consistent recoveries for DTZ(99.8-101.2%) and also for its six known impurities(97.2-101.3%).The method was found to be accurate,precise,linear,specific,sensitive,rugged,robust,and stability-indicating.展开更多
文摘The aim of this study was to develop a simple and derivatization free method for the Quantification of S-Epichlorohydrin in R-Epichlorohydrin by using a gas chromatography coupled with flame ionization detector (FID). Enantiopure epichlorohydrin was a valuable epoxide key starting material for preparing optically active Rivaroxaban. The enantiomeric separations of S-Epichlorohydrin and R-Epichlorohydrin were achieved on Gamaa-Dex-225 (30 meters × 0.25 mm I.D, 0.25 μm) column with a total run time of 30 min. Nitrogen was used as a carrier gas with constant pressure 25.0 psi. The critical experimental parameters such as, column selection, flow rate, injection volume and diluent were studied and optimized. Excellent correlation coeffient between peak responses and concentrations was >0.9998. The recoveries of S-Epichlorohydrin spiked in R-Epichlorohydrin were in the range from 98.2% to 102.8%. Limit of quantitation for S-Epichlorohydrin was sufficiently lower than limits specified by ICH. The method has validated as per International Conference on Harmonization (ICH) guidelines. A precise, accurate, linear and robust Gas Chromatography method was developed for the quantification of S-Epichlorohydrin in R-Epichlorohydrin for Rivaroxaban.
基金National Key R&D Program of China,Grant/Award Number:2023YFC3402000National Institutes for Food and Drug Control,State Key Laboratory of Drug Regulatory Science,Grant/Award Number:2023SKLDRS0124。
文摘Background:The precise insertion of large DNA fragments(>3–5 kb)remains one of the key obstacles in establishment of genetically modified murine models.Methods:A 21 kb large DNA fragment containing three tandemly linked copies of the human HRAS gene was inserted into the genome of C57BL/6J mouse,generating a mouse model designated as KI.C57-ras(or named NF-h HRAS).Whole-genome sequencing and Sanger sequencing were utilized to it confirm precise insertion and copy number.The stability of transgene expression among different generations was verified from multiple aspects using by digital PCR,western blot and DNA sequencing.To assess tumor susceptibility in the mouse model,N-Nitroso-N-methylurea(MNU)was administered at a dosage of 75 mg/kg.Histopathological examinations were conducted using hematoxylin and eosin(H&E)staining.Results:The HRAS DNA fragment was inserted into mouse chromosome 15E1 site,locating between 80623202 bp and 80625020 bp.NF-h HRAS mice exhibited stable inheritance and displayed consistent phenotypes across individuals.Moreover,this mouse model exhibited a high susceptibility to carcinogens.Upon administration of MNU the earliest mortality onset was earlier than that of wild-type littermates(day 65 vs.day 78 for male and day 56 vs.day 84 for female).Notably,100%of the NF-h HRAS transgenic mice developed tumors,with approximately 84%of male NF-h HRAS mice exhibiting specific tumor types,such as squamous cell carcinoma or squamous cell papilloma,which was consistent with the previously reported carcinogenic rasH2 mouse model.The types of tumors and the target organs exhibited diversity in NFh HRAS mice,while the spontaneous tumor incidence remained low(1/50).Conclusions:The NF-h HRAS mice demonstrated excellent genetic stability,a reproducible phenotype,and high susceptibility to carcinogens,indicating their potential utility in non-clinical safety evaluations of drugs as per the S1B guidelines issued by the ICH(The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use).
文摘A stability-indicating liquid chromatographic method has been developed and validated for the determination of Diltiazem Hydrochloride(DTZ) together with its six related substances(Diltiazem sulphoxide,Imp-A,Imp-B,Imp-D,Imp-E,and Imp-F) in a laboratory mixture as well as in a novel tablet formulation developed in-house.Efficient chromatographic separation was achieved on a Hypersil BDS C18(150 mm*4.6 mm,5.0 μm) with mobile phase containing 0.2% Triethylamine(TEA) in gradient combination with acetonitrile(ACN) at a flow rate of 1.0 mL/min and the eluent was monitored at 240 nm.In the developed method,the resolution of DTZ from any pair of impurities was found to be greater than 2.0.The test solution and related substances were found to be stable in the diluent for 24 h.The developed method resolved the drug from its known impurities,stated above,and also from additional impurities generated when the formulation was subjected to forced degradation;the mass balance was found close to 99.9%.Regression analyses indicate correlation coefficient value greater than 0.997 for DTZ and its six known impurities.The LOD for DTZ and the known impurities was at a level below 0.02%.The method has shown good,consistent recoveries for DTZ(99.8-101.2%) and also for its six known impurities(97.2-101.3%).The method was found to be accurate,precise,linear,specific,sensitive,rugged,robust,and stability-indicating.
