Identification method and application of insulin mass spectrum peptide graph

A technology for insulin and human insulin, which is applied in the field of establishing the peptide map of insulin enzymatic hydrolysis mass spectrometry, which can solve the problems of difficulty, animal-derived insulin, and other problems.

Active Publication Date: 2019-10-01
天津市药品检验研究院
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the content of recombinant technology is high and the production process is complicated, driven by huge profits, there is a risk that unscrupulous traders use animal-derived insulin to confuse real ones
In the current quality standards, the identification of the authenticity of recombinant insulin is based on the consistency of retention time. However, the molecular structure of different types of recombinant insulin is very similar, and the retention time is very similar. There are certain difficulties in the actual identification process.

Method used

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  • Identification method and application of insulin mass spectrum peptide graph
  • Identification method and application of insulin mass spectrum peptide graph
  • Identification method and application of insulin mass spectrum peptide graph

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Mass spectrometry peptide map of recombinant human insulin, insulin aspart, insulin lispro and porcine insulin injection V8 enzymatic hydrolysis and mass spectrometry peptide map of V8 enzyme combined with lysyl endopeptidase

[0043] Take one injection sample each, transfer it to a 15ml centrifuge tube, adjust the pH value of the sample to 8.5 with a suitable Tris-Hcl buffer, and dilute the volume to 15ml.

[0044] (1) Take 600 μl of the diluted sample, add 200 μl of V8 enzyme (1 mg / ml), overnight at room temperature, and then add 400 μl of Tris-Hcl buffer;

[0045] (2) Take 600 μl of diluted sample, add 200 μl of V8 enzyme (1 mg / ml) and 50 μl of lysyl endopeptidase (20 μg / ml), leave at room temperature overnight, and then add 400 μl of Tris-Hcl buffer. Samples were diluted 20 times before injection. Using Waters ACQUITY UPLC ® CSH TM C18, 1.7μm, 50×2.1mm (I.D) chromatographic column, according to the gradient conditions in the following table, use 0.1% formic acid ...

Embodiment 2

[0055] Determination of Recombinant Human Insulin and Insulin Lispro Injection V8 Enzyme and Lysyl Endopeptidase Enzyme Mass Spectrometry Peptide Mapping

[0056] Take one injection sample each, transfer it to a 15ml centrifuge tube, adjust the pH value of the sample to 9 with a suitable Tris-Hcl buffer, and dilute the volume to 15ml. Take 1000 μl of the diluted sample, add 100 μl of V8 enzyme (1 mg / ml) and 20 μl of lysyl endopeptidase (20 μg / ml), overnight at room temperature, and then add 400 μl of Tris-Hcl buffer. Samples were diluted 10 times before injection. Measured according to the gradient conditions of Example 1, using 0.7% formic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, 0.2ml / min flow rate, 55°C column temperature, 5 μl of sample injection, and collection of ion chromatogram; The four characteristic polypeptide fragments are shown in Table 7:

[0057] Table 7 Peptides hydrolyzed by combination of recombinant human insulin and insuli...

Embodiment 3

[0061] Determination of peptide map of insulin aspart injection and counterfeit insulin aspart injection V8 enzymatic hydrolysis mass spectrometry

[0062] Take one injection sample each, transfer it to a 15ml centrifuge tube, adjust the pH value of the sample to 7.5 with Tris-Hcl buffer, and dilute the volume to 15ml. Take 1000 μl of the diluted sample, add 200 μl of V8 enzyme (1 mg / ml), overnight at room temperature, and then add 400 μl of Tris-Hcl buffer. Samples were diluted 30 times before injection. According to the gradient conditions in Example 1, 0.5% formic acid aqueous solution was used as mobile phase A, acetonitrile was used as mobile phase B, the flow rate was 0.6 ml / min, the column temperature was 50°C, 5 μl was injected for measurement, and the ion chromatogram was collected. The four characteristic polypeptide fragments produced after enzymatic hydrolysis of the sample are shown in Table 8:

[0063] Table 8 Enzymatic hydrolysis peptides of insulin aspart and...

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Abstract

The invention discloses an identification method and an application of an insulin mass spectrum peptide graph. According to the method, a V8 enzyme is adopted, and the V8 enzyme is combined with lysylendopeptidase, specific hydrolysis of insulin is carried out, and a peptide graph containing insulin characteristic fragments is established through a liquid chromatograph-mass spectrometer. The method is characterized in that a buffer solution is used for providing an enzymolysis environment with the pH value of 7.5-9.5, wherein a substrate and the enzyme are reacted overnight according to the mass ratio of insulin to V8 enzyme to lysyl endopeptidase of (100-1000) to (100-500) to 1, the reaction liquid is diluted for 10-30 times, a liquid phase separation mass spectrometry detector is adopted for detection, and a characteristic peptide graphs of a variety of insulin are established. By adopting the mass spectrum peptide graph, various different kinds of insulin can be accurately determined, and the method is accurate and reliable.

Description

technical field [0001] The invention belongs to the technical field of drug analysis, and relates to a method for establishing an insulin enzymatic hydrolysis mass spectrum peptide map and its application. Background technique [0002] Insulin recombinant products are currently the most effective and safest treatment for diabetes. my country has a large population base, a large number of diabetic patients, and a large amount of insulin usage. Although the content of recombinant technology is high and the production process is complicated, driven by huge profits, there is a risk that unscrupulous traders use animal-derived insulin to confuse real ones. In the current quality standards, the identification of the authenticity of recombinant insulin is based on the consistency of retention time. However, the molecular structure of different types of recombinant insulin is very similar, and the retention time is very similar. There are certain difficulties in the actual identifi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/34G01N30/72G01N30/88
CPCG01N30/02G01N30/06G01N30/34G01N30/72G01N30/88G01N2030/067G01N2030/884
Inventor 白海娇韩晓捷伏圣青邵建强
Owner 天津市药品检验研究院
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