Method for reversed phase separation of derived leucine and isoleucine

Abstract

The invention relates to a method for reversed phase separation of amino acids containing impurities, and mainly solves the technical problems of high cost and high device requirements of present methods for quantitatively determining 18 composite amino acids through pre-column derivatization. The method includes the following steps: (1) preparing a mobile phase A and a mobile phase B needed by analysis, and carrying out ultrasonic mixing, wherein the mobile phase A is an aqueous solution of trifluoroacetic acid, and the mobile phase B is an acetonitrile solution of trifluoroacetic acid; (2) introducing the prepared mobile phases into a chromatographic system, inspecting all systems of a high performance liquid chromatograph to ensure that the chromatograph is normal, setting chromatographic conditions, and balancing a chromatographic column according to the gradient the initial concentrations; (3) dissolving N-(9-fluorenylmethyloxycarbonyl)-L-leucine, N-(9-fluorenylmethyloxycarbonyl)-L-isoleucine and a mixture of the above two substances in acetonitrile, and filtering to obtain single sample liquids and a mixed liquid for later use; and (4) respectively sucking the mixed liquid and the two sample liquids into the chromatographic system after chromatographic system balancing, acquiring chromatographic data, and carrying out area normalization method integration on the obtained chromatograms.

Description

technical field [0001] The invention relates to a method for reverse-phase separation of amino acids containing impurities, in particular to a method for separating amino acids of the main chain with 9-fluorenylmethoxycarbonyl-protected L-leucine (Fmoc-L-Leu-OH) and 9 using a C18 stationary phase. -L-isoleucine (Fmoc-L-Ile-OH) protected by fluorenylmethoxycarbonyl (Fmoc-L-Ile-OH), using high-performance liquid chromatography and reversed-phase chromatography conditions, using water / trifluoroacetic acid and acetonitrile / trifluoroacetic acid as mobile phases , using gradient elution to separate the above two protected amino acids, and can give accurate results. Background technique [0002] Due to the many advantages of peptide drugs, more and more pharmaceutical companies have invested manpower and financial resources in the research and development of peptide drugs, and domestic related industries have also developed rapidly. Drugs, these include leuprolide, thymus α1, trip...

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a method for reverse-phase separation of derivatized leucine and isoleucine, which mainly solves the high cost and equipment requirements of the existing method for quantitative determination of eighteen kinds of compound amino acids through pre-column derivatization high technical issues

The idea of ​​the present invention is to separate N-(9-fluorenylmethoxycarbonyl)-L-leucine and N-(9-fluorenylmethoxycarbonyl)-L-isoleucine by reverse-phase high-performance liquid chromatography, Using water / trifluoroacetic acid and acetonitrile / trifluoroacetic acid as the mobile phase, using a common C18 reversed-phase column, combined with an appropriate gradient, solved the problem of difficult separation of the two derivatized amino acids, and the analytical method has good reproducibility , high accuracy, and no need to add additional equipment, mobile phase preparation is simple, suitable for general HPLC laboratory use

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