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Nesiritide acetate peptide mapping analysis method and application thereof

An analysis method, nesiritide technology, applied in the field of drug analysis, can solve the problems of analysis deviation, time-consuming, and peaks appearing in front, and achieve the effect of short detection time

Active Publication Date: 2020-04-10
SHANGHAI JINGFENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, in the peptide map analysis of nesiritide, trypsin lysis-reverse liquid chromatography is usually used to analyze it. After the analysis, the fragments decomposed by nesiritide need to be collected. However, this process is relatively time consuming
When assigning the peaks of the liquid chromatogram, it is often considered that some small molecular fragments (high polarity) appear behind relatively large molecules (small polarity). However, according to the retention mechanism of reversed-phase chromatography, the polarity Large fragments should be weakly retained on the reversed-phase chromatographic column, and the peaks will elute earlier, so there is a bias in the analysis of the prior art in terms of fragment assignment

Method used

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  • Nesiritide acetate peptide mapping analysis method and application thereof
  • Nesiritide acetate peptide mapping analysis method and application thereof
  • Nesiritide acetate peptide mapping analysis method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0056] This embodiment provides a method for analyzing the peptide map of nesiritide, which specifically includes the following steps:

[0057] (1) Reduction and alkylation of nesiritide samples

[0058] Dilute the sample with water to 1.5mg / mL and add it to a 1.5mL centrifuge tube, add 100μL 0.1mol / L ammonium bicarbonate buffer, then add 11.56μL 0.1mol / L DTT (dithiothreitol), mix well, and Incubate at 56° C. for 1 h, and after incubation, place it in a water bath at 20° C. to cool for 1 min.

[0059] Add 12.6 μL of 0.25 mol / L iodoacetamide solution to the reduced sample, mix well, and incubate at 20° C. in the dark for 1 h.

[0060] After the reaction, use mass spectrometry to confirm that the reduction and alkylation are complete (such as figure 1 shown), four mass spectrum peaks can be observed in the figure, and figure 2 The comparison of the mass spectrum of nesiritide without reduction and alkylation shows that the reduction and alkylation are complete, and the disul...

Embodiment 2

[0074] This embodiment provides a method for analyzing the peptide map of nesiritide, which specifically includes the following steps:

[0075] (1) Reduction and alkylation of nesiritide samples

[0076] Dilute the sample with water to 1.5mg / mL and add it to a 1.5mL centrifuge tube, add 100μL 0.4mol / L ammonium bicarbonate buffer, then add 11.56μL 0.4mol / L DTT (dithiothreitol), mix well, and Incubate at 56° C. for 1 h, and after incubation, place it in a water bath at 25° C. to cool for 1 min.

[0077] Add 12.6 μL of 0.5 mol / L iodoacetamide solution to the reduced sample, mix well, and incubate at 25° C. in the dark for 1 h.

[0078] After the reaction, MS detection was used to confirm that the reduction and alkylation were complete (such as Figure 4 shown), four mass spectrum peaks can be observed in the figure, and figure 2 The comparison of the mass spectrum without reduction and alkylation in the above shows that the reduction and alkylation are complete, and the disulfi...

Embodiment 3

[0089] This embodiment provides a method for analyzing the peptide map of nesiritide, which specifically includes the following steps:

[0090] (1) Reduction and alkylation of nesiritide samples

[0091] Dilute the sample with water to 1.5mg / mL and add it to a 1.5mL centrifuge tube, add 100μL 0.2mol / L ammonium bicarbonate buffer, then add 11.56μL 0.2mol / L DTT, mix well, and incubate at 56°C for 1h, incubate Afterwards, it was placed in a 15°C water bath to cool for 1 min.

[0092] Add 12.6 μL of 0.5 mol / L iodoacetamide solution to the reduced sample, mix well, and incubate at 15° C. in the dark for 1 h.

[0093] After the reaction, MS detection was used to confirm that the reduction and alkylation were complete (such as Figure 4 shown), four mass spectrum peaks can be observed in the figure, and figure 2 The comparison of the mass spectrum without reduction and alkylation in the above shows that the reduction and alkylation are complete, and the disulfide bond of nesiriti...

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Abstract

The invention provides a nesiritide acetate peptide mapping analysis method and application thereof. The method includes the steps of: reducing and alkylating nesiritide acetate, adding endopeptidasefor enzymolysis, then performing detection by LC-UV-MS, and attributing polypeptide fragments and / or amino acid residues formed after enzymolysis of nesiritide acetate according to acquired peptide mapping data. The invention provides the peptide mapping analysis method, after reduction and alkylation of nesiritide acetate, endopeptidase is employed for enzymolysis into a plurality of polypeptidefragments and amino acid residues, and LC-UV-MS detection is used for corresponding the polypeptide fragments and amino acid residues to peaks in peptide mapping correctly, so that whether deletion, misconnection, repetition or other phenomena exist in the polypeptide chain of nesiritide acetate can be accurately judged.

Description

technical field [0001] The invention belongs to the technical field of drug analysis, in particular to a peptide map analysis of recombinant polypeptide drugs, in particular to a peptide map analysis method of nesiritide and its application. Background technique [0002] Heart failure is a clinical syndrome of impaired ventricular filling or ejection due to abnormal cardiac structure and function, and its main clinical manifestations are limited activity tolerance and fluid retention. The traditional treatment for heart failure is "heart strengthening, diuresis, and vasodilator", including diuretics, inotropic drugs, β-blockers, nitrates and other drugs. Although they can relieve the clinical symptoms of heart failure patients, However, the curative effect is limited, and it cannot reverse myocardial fibrosis and ventricular remodeling caused by heart failure. Therefore, it is necessary to find new clinical drugs that can fundamentally treat heart failure. [0003] Nesirit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88
CPCG01N30/88G01N2030/8813
Inventor 李帅苏春所刘美卓芳芳
Owner SHANGHAI JINGFENG PHARMA
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