| Peptide drugs usually have complex related substances,mainly including synthesis related impurities,degradation impurities,optical impurities and polymers,due to their process characteristics and instability.As the concentration and toxicity of related substances are closely related to the safety of drugs,the analysis of related substances is one of the key items in the quality control of peptide drugs,and its limit requirements and structure confirmation are indispensable indicators in the drug quality standards.RP-HPLC is the most common method for the quality inspection of synthetic substances.With the development of soft ionization technology,RP-HPLC combined with ESI-MS have been applied to the separation and characterization of biological macromolecules.The reports on LC-ESI-MS method for sequence analysis,structural elucidation and Related Substances Research of synthetic peptides are also increasing year by year.However,there are inherent problems in the coupling of traditional non denaturing liquid chromatography and MS.Using two-dimensional liquid chromatography to desalinate samples is an innovative idea to solve the compatibility problem between non-volatile mobile phase additives and MS.In this paper,based on two-dimensional liquid chromatography-mass spectrometry technology,Related Substances Research of synthetic peptides has been studied,aiming to provide a new idea of method development.The research content mainly includes the following three parts.Part 1 The idea of developing 2D-LC-MS analysis method.This method is suitable for the separation,quantitative and qualitative analysis of related substances in peptide drugs,which development idea was summarized from three aspects:the construction of 2D-LCMS instrument system,the selection of analysis conditions(including the selection of interface and mass spectrum parameters,the selection and optimization of primary and secondary liquid phase analysis conditions)and the method development process.In Part 2 and Part 3,two kinds of peptide injection products were taken as examples to introduce the specific process of 2D-LC-MS method development for Related Substances Research based on the above.Part 2 The development and application of 2D-LC-MS method for Related Substances Research in CBTC injection.Firstly,we established a 1D-HPLC method for the separation and quantification of CBTC related substances,based on the reproducing results of HPLC methods described in the literature.For the rapid desalination and qualitative analysis of primary separation components,we used an Agilent C18 column with a length of only 50 mm to separate phosphate and heteropeptide.To development a fast,sensitive and applicable 2D-HPLC-ESI-MS method,the elution procedure,flow rate,mobile phase composition,determination range and scanning resolution of 2D-HPLC were investigated and optimized.Then,a 2D-LC-MS analysis method was obtained by connecting 1D-HPLC and 2D-HPLCESI-MS with six-way values,the specific process of which was as follows:CBTC was separated by 1D-HPLC,its fractions were collected in multiple heart-cutting mode and transferred to desalination by 2D-HPLC in off-line mode,and one or more single impurity was obtained.These single impurities would be ionized by ESI interface and finally determined in MS.The results of methodology validation showed that our method have good specificity and sensitivity for seven known impurities,and the its detection limit can reach 10ng.Finally,several batches of preparation were detected by this 2D-LC-MS method,which proved that this method can be applied to seven known impurities and other unknown impurities of CBTC.In the discussion part,we speculated the chemical structures of the impurities above according to their molecular weight,and summarized the advantages and disadvantages of this method.Part 3 The development and application of 2D-LC-MS method for Related Substances Research in ATSB injection.The process of method development and methodology validation is similar to Part 2.The results of methodology validation showed that our method has good specificity and sensitivity for eight known impurities,and the its detection limit can reach 15ng.In the discussion part,we speculated the chemical structures of the impurities above according to their molecular weight,and compared our method with the traditional LC-MS method using formic acid instead of TFA from three aspects of qualitative,quantitative and preparation detection.In conclusion,based on two-dimensional liquid chromatography-mass spectrometry,the qualitative analysis methods of related substances for CBTC injection and ATSB injection were established,and the compatibility of HPLC analysis conditions and MS were solved.The general method ideas and research strategies of qualitative analysis methods for related substances in peptide drugs were summarized,which had application value in the structure confirmation of peptide drugs with related substances,and provided technical support for accelerating the research and development process of peptide drugs. |
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