Study On Key Technology Of Quality Control Of Rubber

Glues Medicines are special crud drugs of Traditional Chinese Medicines (TCM) in China, which are made from hide or bone of animals. Adulteration and counterfeit occured frequently due to lack of materials, which affect the safety and effectiveness of these drugs. These questions flung down a challenge to the quality control of Gules Medicines. There is no specific method for identification of Glues Materials and deerhorn glue currently, because of the specificity of the constituents of drugs from animals and lack of the technologies to protein analysis, The non specific quality standard makes drug administration become difficlut. It is the key to solve these above problem and that establish a specific quality control method based on the species, chemical constituents.In our work, the project aims to search and find the characteristic peptides from the trypsin digested samples of protein in various Glues Medicines by the High Performance Liquid Chromatography-Mass spectrometry (HPLC-MS/MS), to establish a quality and quantity control method of Glues Materials. The work can be the reference to the study of standard of animals drugs.1. In our study, we analyzed the trypsin digested samples by Ultra Performance Liquid Chromatography quaqruple-time-of-flight mass (UPLC-Q/TOF/MS). The UPLC-MS data of the trypsin digested samples were subjected to principal component analysis (PCA) and an orthogonal partial least squares data analysis (OPLS-DA) to find marker peptide.2peptides of deerhorn glue,3peptides of tortoise shell glue,4peptides of Donkey-hide gelatin,4peptides of Bovine-hide gelatin,3peptides of Pig-hide gelatins were found. We had given the amino acid sequence of some marker peptides based on the mass spectrometry of marker peptides and synthetise the marker peptides.2. RRLC-Iontrap Mass spectrometry coupled with MASCOT research was used to establish the mass information based on the marker peptides of tortoise shell glue, deerhorn glue and donkey-hide glue, which could be used to identify the source of Glues.3. Using MRM detection mode of RRLC-QQQ coupled with reference crude drug to establish a quality control method based on the marker peptides of tortoise shell glue (m/z631.3-→546.4,921.4), deerhorn glue (transition m/z765.4→554.0,733.0) and donkey-hide glue (m/z539.8→612.4,923.8). Using MRM detection mode of RRLC-QQQ coupled with marker peptide (m/z641.3) and refernce crude drug of Donkey-hide gelatin (m/z539.8→612.4,923.8), Bovine-hide gelatin (m/z641.3→783.3,726.2) to establish a supplemental testing method for tortoise shell glue, deerhorn glue and donkey-hide glue4. The sample was hydrolyzed for1hour at150℃, and derived with Phenyl isothiocyanate(PITC). The developed method is accurate with high repeatability, which is helpful for the determination of hydroxyproline, glycine, alanine, proline,4amino acid in donkey-hide glue. At last, the limit of hydroxyproline was not less than9.0%, glycine was not less than19.0%, alanine was not less than7.0%, proline was not less than0.5%.The innovation of this thesis is as follows:(1) Identification of Characteristic peptides of different gelatins by tripsin digestion and HPLC-MS/MS, and establishment of reference substance by AA sequence analysis of peptides and chemical synthesis.(2) peptide analysis and statistical analysis are used together for authenticaion of different gelatins, and distinguishing expert system is established through the setting up of a characteristic peptide information database (including the chromatography parameters, mass parameters of characteristic peptides) and mathematical model.(3) The specific authentication methods of Glues Medicines are established for the first time.(4) The specific methods to detect the Counterfeit glues mixed into the real glues are developed for the first time.