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Amino acid analysis refers to the method used to determine the amino acid composition or content of proteins, peptides and other pharmaceutical preparations by physical and chemical means. According to the results of amino acid analysis, proteins and peptides can be identified. Prior to the analysis of amino acids, proteins and peptides must be hydrolyzed into a single amino acid, and the specific hydrolysis method shall be specified under the terms of each variety.
The process of amino acid analysis of proteins and peptides after hydrolysis is the same as that of free amino acids in other pharmaceutical preparations. Reagents are essential in the process of amino acid analysis, such as acetonitrile, phosphoric acid, hydrochloric acid, water and others.
PITC precolumn derivational amino acid analysis: This method uses the reaction of amino acids with phenyl isothiocyanate (PITC). The reaction produces phenylthionyl amino acid (PTC-amino acid), which is an amino acid derivative that responds to ultraviolet light. After separated by reversed-phase high performance liquid chromatography, PTC-amino acid can be detected by UV. In addition, within a certain range, the absorption value of PTC-amino acid is directly proportional to the concentration of amino acids. In the detection process of PITC precolumn derivational amino acid analysis, the generally used regents are sodium acetate solution, acetonitrile, water, triethylamine, n-hexane and others.
AQC precolumn derivational amino acid analysis: According to the reaction of amino acids and 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC), asymmetric urea derivatives (AQC - amino acids) can be obtained. AQC - amino acids can be detected by UV or fluorescence after treatment by reversed-phase high performance liquid chromatography, and the absorption value is directly proportional to the concentration of amino acids in a certain range. In AQC precolumn derivational amino acid analysis method, ammonium acetate, acetonitrile, boric acid and other regents are needed.
OPA and FMOC precolumn derivational amino acid analysis: In this analysis method, primary amino acids, in the presence of sulfhydryl reagents, are first reacted with o-phenyldialdehyde (OPA) to produce OPA - amino acids. After that, adding 9-fluorenyl methyl chloroformate (FMOC), the remaining secondary amino acids continue to react with FMOC to produce FMOC - amino acids. The amino acid derivatives produced by the two reactions are separated by reversed-phase high performance liquid chromatography and detected by UV or fluorescence. In the detection process, many regents are needed, including triethylamine, tetrahydrofuran, acetic acid, sodium hydroxide and others.
Others: In addition to the above analysis methods, FDNB precolumn derivational amino acid analysis method, ninhydrin precolumn derivational amino acid analysis and others are also popular. In those amino acid analysis methods, sodium chloride, hydrochloric acid and many other regents are required.
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