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Method for quantitatively detecting polypeptide in transfer factor capsule

A quantitative detection method and transfer factor technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of interference detection results, poor reproducibility, inaccurate detection results, etc., to achieve reduced overlap ratio, good peak shape, and improved The effect of detection accuracy

Inactive Publication Date: 2019-01-18
XIAN DAQING PHARMA FACTORY JINHUA ENTERPRISE GROUP CORP
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The prior art uses the Follin-phenol method (Lowry) to measure the polypeptide content in transfer factor capsules, but there are many substances that interfere with this method, such as reducing substances, phenols, citric acid, ammonium sulfate, trishydroxymethylamino Methane buffer, glycine, sugars and glycerin all have interference effects
And it has been reported in the literature that the Follin-phenol method (Lowry) was used to measure the polypeptide content in transfer factor capsules. The excipient mannitol and the free amino acids in it will interfere with the test results, resulting in inaccurate test results and poor reproducibility.

Method used

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  • Method for quantitatively detecting polypeptide in transfer factor capsule
  • Method for quantitatively detecting polypeptide in transfer factor capsule
  • Method for quantitatively detecting polypeptide in transfer factor capsule

Examples

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preparation example Construction

[0050] The preparation method of the test solution for the determination of the total amino acid content of transfer factor capsules of the present invention comprises the following steps:

[0051] a. Accurately weigh about 1.0 g of the contents of the transfer factor capsule, put it in a high-throughput microwave digestion tube, add 6-10 mL of 6mol / L hydrochloric acid solution precisely, weigh it, and put it in a microwave digestion apparatus for hydrolysis according to the procedure in the table below:

[0052] Table 1 Microwave digestion program for determination of total amino acids in transfer factor capsules

[0053] step

climb (min)

keep (min)

temperature(℃)

power (w)

1

5

5

100

1200

2

5

5

150

1200

3

5

15

180

1200

[0054]b. After the digestion process of the contents of the above transfer factor capsules is completed, let cool to room temperature, make up the lost weight with 6mol / L hydr...

Embodiment 1

[0077] Example 1 Screening of Transfer Factor Capsule Microwave Digestion Conditions

[0078] The microwave digestion conditions of transfer factor capsules were screened by 3-factor 3-level orthogonal test. The 3 factors of microwave digestion conditions were digestion sample volume, digestion acid volume and digestion temperature; the optimal level of 3 factors was determined by single factor test. The values ​​are respectively 1.0 g of digested sample volume, 8.0 mL of digested acid volume, and 180°C of digested temperature. See Table 4 for the 3-factor 3-level orthogonal test table designed based on the optimal level values ​​of the above 3 factors, and see Table 5 for the analysis of the orthogonal test results.

[0079] Table 3 Orthogonal test table of transfer factor capsule microwave digestion condition screening

[0080]

[0081]

[0082] Table 4 Analysis table of orthogonal test results for transfer factor capsule microwave digestion conditions screening

[0...

Embodiment 2

[0085] Example 2 Determination of polypeptide content in transfer factor capsules by "amino acid difference method"

[0086] 1. Chromatographic conditions

[0087] Adopt Agilent high-performance liquid chromatography, use octadecylsilane bonded silica gel as filler chromatographic column; use methanol-acetonitrile-water (20:60:20) as mobile phase A, and use 0.1mol / L sodium acetate buffer solution (Adjust the pH value to 6.3 with glacial acetic acid) for gradient elution with mobile phase B; the flow rate is 1.0ml per minute, the detection wavelength is 254nm, the column temperature is 35°C, and the injection volume is 2μl.

[0088] Table 5 Elution Gradient Schedule

[0089]

[0090]

[0091] 2. Determination of standard curve:

[0092] Preparation of reference substance stock solution Accurately weigh 15.0 mg of glutamic acid reference substance, aspartic acid, serine, glycine, histidine, arginine, threonine, alanine, proline, tyrosine, Valine, methionine, cystine, is...

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Abstract

The invention discloses a method for quantitatively detecting polypeptide in a transfer factor capsule. An HPLC-amino acid difference method is used for measurement. The total amino acid content and free amino acid content of the transfer factor capsule are measured by use of an established derivatization-HPLC method respectively, and a difference therebetween is adopted to represent the polypeptide content of a sample. Precolumn derivatization is performed on amino acid by use of a phenyl-isothiocyanate (PITC) derivatization method. A detection chromatographic condition is that octadecyl silane chemically bonded silica is adopted as a filler chromatographic column and gradient elution is performed by taking methanol-acetonitrile-water (20:60:20) as a flowing phase A and taking a 0.1mol / Lsodium acetate buffer solution (the pH value is regulated to be 6.3 by use of glacial acetic acid) as a flowing phase B. The total amino acid content and free amino acid content of the transfer factorcapsule are measured by use of the chromatographic condition respectively, and the difference therebetween is adopted to represent the polypeptide content of the sample. Systematic verification and research results show that the detection method is free of interference of other substances, high in specificity, accurate and reliable, and the polypeptide content of the transfer factor capsule can be effectively detected and monitored.

Description

technical field [0001] The invention belongs to the technical field of multi-component biochemical drug quality analysis research, and in particular relates to a quantitative detection method for polypeptides in transfer factor capsules. Background technique [0002] The main components of transfer factor are polypeptides, amino acids and nucleotides extracted from the spleen of healthy pigs or cattle. It is a natural two-way immunomodulator and an ideal drug for the treatment of related diseases such as low immune function and defects. Small molecule peptides are the functional markers of transfer factor and serve as its main functional components. Accurate determination of the peptide content in transfer factor is of great significance for optimizing the production process of transfer factor and controlling product quality. [0003] The prior art uses the Follin-phenol method (Lowry) to measure the polypeptide content in transfer factor capsules, but there are many substan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/062
Inventor 蔡慧侠
Owner XIAN DAQING PHARMA FACTORY JINHUA ENTERPRISE GROUP CORP
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