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Method for determining nerve growth factor content

A nerve growth factor and content technology, applied in the field of nerve growth factor content, can solve the problems such as the limitation of sensitivity of detection methods that cannot exclude human albumin interference, the inability to quantitatively analyze NGF content, and the influence of quantitative determination of NGF, etc. The effect of improving the detection limit

Active Publication Date: 2007-06-20
STAIDSON BEIJING BIOPHARMACEUTICALS CO LTD
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AI Technical Summary

Problems solved by technology

[0003] The content of NGF in the existing nerve growth factor preparations is 4-30 μg / branch, and human serum albumin is used as a protective agent. Since both human serum albumin and NGF are proteins, the content of human serum albumin in the preparation is much higher than that of NGF, which affects the quantitative determination of NGF
In order to solve this problem, people have tried a variety of detection methods, such as Lowry method, ultraviolet absorption method, Bradford method and ELISA method, etc., but they all failed because the interference of human serum albumin could not be ruled out or the sensitivity of the detection method itself was limited.
We have applied for the analysis of NGF content by gel chromatography, and the mobile phase is 0.01-0.07M phosphate buffer solution, which solves the problem that the NGF content cannot be quantitatively analyzed

Method used

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  • Method for determining nerve growth factor content
  • Method for determining nerve growth factor content
  • Method for determining nerve growth factor content

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: 0.07mol / L sodium dihydrogen phosphate aqueous solution mobile phase was used.

[0036] A chromatographic condition:

[0037] (1) Instrument: use Japan's Shimadzu Shimadzu LC-10Avp high performance liquid chromatograph;

[0038] (2) Gel chromatography column: TSK G3000SWXL 7.8×3.0mm, inner diameter 0.5μm (TOSOH Japan);

[0039] (3) Column temperature: room temperature;

[0040] (4) Detection wavelength: 214nm;

[0041] (5) Flow rate: 0.8ml / min;

[0042] (6) pH value of mobile phase: 7.0.

[0043] Two experimental steps

[0044] (1) Sample processing: Take a nerve growth factor preparation, add 0.5ml of mobile phase precisely, shake gently to dissolve, and use it as the test solution.

[0045] (2) Reference substance treatment: Precisely measure the mouse nerve growth factor reference substance (take the mouse submandibular gland, separate and purify NGF by ion exchange chromatography and molecular sieve, and test the purity according to the method requir...

Embodiment 2

[0048] Example 2: A 2.0 mol / L ammonium acetate aqueous solution was used as the mobile phase.

[0049] 1. Chromatographic conditions: the same as in Example 1.

[0050] Two experimental steps

[0051] (1) The treatment of sample and reference substance is the same as that of Example 1.

[0052] (2) Precisely draw 20 μl of the test solution and the reference solution, respectively, inject them into the liquid chromatograph, and record the chromatogram. Obtain parameters such as retention time, peak height, and peak area.

[0053] Results: The main peak area of ​​nerve growth factor was A=923186, and the area of ​​reference substance was A=921096. The sample content was 100.2% by calculating the peak area by external standard method. The degree of separation between the main peak of nerve growth factor and the main peak of albumin was greater than 1.5, and the retention time of the main peak of the sample was consistent with the retention time of the reference substance. The...

Embodiment 3

[0054] Example 3: A 0.10 mol / L sodium sulfate aqueous solution was used as the mobile phase.

[0055] 1. Chromatographic conditions: the same as in Example 1.

[0056] Two experimental steps

[0057] (1) The treatment of sample and reference substance is the same as that of Example 1.

[0058] (2) Precisely draw 20 μl of the test solution and the reference solution, respectively, inject them into the liquid chromatograph, and record the chromatogram. Obtain parameters such as retention time, peak height, and peak area.

[0059] Results: The main peak area of ​​nerve growth factor was A=3147076, and the area of ​​reference substance was A=3098921. The sample content was 101.6% calculated by the peak area by external standard method. The degree of separation between the main peak of nerve growth factor and the main peak of albumin was greater than 1.5, and the retention time of the main peak of the sample was consistent with the retention time of the reference substance. The...

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Abstract

A method for determining content of nerve growth factor includes chromatograph conditions as follows, gel chromatographic column in molecular weight detection scope of 10000-500000 dalton and with hole diameter of 200-300 angstrom gel column, column temperature in room temperature, detection wavelength of 205-220 nm, flow rate of 0.6-1.0ml / min, flowing phase in pH value of 6.5-7.2 and with salt solution concentration of 0.07-2.0 mol / L.

Description

technical field [0001] The invention relates to a novel method for determining the content of nerve growth factor in preparations by high performance liquid gel chromatography. Background technique [0002] Nerve growth factor (NGF) is one of the most important active components of the nervous system. It can maintain the survival of sympathetic and sensory nerve cells, affect the survival of central neurons, promote the differentiation of nerve cells, and protect the normal function of the nervous system. Important role, my country has approved nerve growth factor preparations as drugs for sale and use. [0003] The content of NGF in the existing nerve growth factor preparation is 4-30 μg / branch, and human albumin is used as a protective agent. Since human albumin and NGF are both proteins, the content of human albumin in the preparation is much higher than that of NGF. NGF, affects the quantitative determination of NGF. In order to solve this problem, people have tried a v...

Claims

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Application Information

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IPC IPC(8): G01N30/02B01J20/281
CPCC07K14/48C07K14/475
Inventor 周志文张洪山王红卫
Owner STAIDSON BEIJING BIOPHARMACEUTICALS CO LTD
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