Fmoc-protected amino acid purity and related substance analysis method

Abstract

The invention discloses an Fmoc-protected amino acid purity and related substance analysis method. The method comprises the following steps of S1, preparing an Fmoc-Osu impurity control solution, andenabling the ratio of Fmoc-Osu to Fmoc-protected amino acid to be (0.1-1.0): 100 to obtain an Fmoc-Osu impurity control solution; S2, preparing a related impurity control solution, enabling the ratioof single related impurity to Fmoc-protected amino acids to be 0.1-1.0: 1 100, obtaining a plurality of reference substance solutions, etc. According to the invention, a mobile phase, an impurity control solution and the gradient elution conditions are comprehensively designed and optimized, the analysis method is applied to the operation process of the system, the baseline is smooth, no interference peak basically exists in a blank test, the impurity separation effect is prominent, and the specificity is good. The test data shows that the detection limit can achieve 0.002%, the sensitivity isextremely high, the detection result can provide the reliable and effective information for the research, the production and the purification of the product, the solid foundation is established for the protected amino acid quality control standard, and the positive effect is provided for the quality control of the final product.

Description

technical field [0001] The invention relates to the technical field of analysis and detection of Fmoc-protected amino acid purity related substances, in particular to an analysis method for Fmoc-protected amino acid purity and related substances. Background technique [0002] With the development of biotechnology, peptides play an increasingly important role in biomedicine. Protected amino acids are the most basic raw materials for solid-phase synthesis of peptides. Amino acids have α-amino groups and carboxyl groups, and some of the 20 kinds of amino acids also contain side chain active groups, such as: hydroxyl, amino, guanidine and heterocycles, etc. Amino groups and side chain active groups need to be protected in the peptide-binding reaction In general, the protective group should be removed after the peptide is synthesized, otherwise the misconnection of amino acids and many side reactions will occur. [0003] When N-9-fluorenylmethoxycarbonylsuccinimide (Fmoc-Osu) i...

AI Technical Summary

Problems solved by technology

The mobile phase contains triethylamine, which will cause irreversible damage to the chromatographic column and shorten the service life of the chromatographic column; during gradient operation, the phosphoric acid in the mobile phase will cause serious baseline drift; the presence of buffer salt in the mobile phase will make the system baseline Unstable, there are many interference peaks in the blank test, which reduces the sensitivity of the method, and the buffer salt is easy to precipitate, which will cause system pipeline blockage

In addition, the existing technical solutions detect that the purity of the liquid phase is far from the result of the content measured by the titration method. One of the reasons for this situation may be that the method does not completely separate the main peak from the impurities during the detection process. , the main peak contains impurity peaks, so that the purity of the result is high. Such an analysis method for related substances is flawed, and the results obtained are not accurate, and have no substantial guiding effect on production, research and development, and purification.

[0006] In order to develop an analytical method for effectively detecting various potential impurities, technicians have been conducting research experiments for many years, but there has been no significant progress. As an analytical technique with certain technological breakthroughs in recent years, technicians have developed an acetonitrile, Ultrapure water and trifluoroacetic acid are used as mobile phase protection amino acid test methods, but in practical application, in order to ensure the accuracy of test results, the concentration of mobile phase (trifluoroacetic acid) should reach more than 2%, and the mobile phase If the concentration is too high, the mobile phase will be too acidic, and the sample will be partially decomposed in this mobile phase, and other impurities will be produced, which will not only lead to inaccurate test results, but also the mobile phase is highly corrosive and will cause great damage to the instrument. Below this concentration, the separation effect of impurities is poor, and the detection accuracy will decrease again. Therefore, the accuracy and practicability of this test method are not high.

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