Method for determining other amino acids in L-valine raw material by high performance liquid chromatography

A high-performance liquid chromatography and valine technology, which is applied in the field of determination of other amino acids in L-valine raw materials, can solve the problems of inability to clearly display the specific content of various other amino acids, expensive equipment costs and detection costs, and the service life of amino columns. Not long, etc., to solve the reversed-phase service life is not long, improve the effect of chromatographic peak shape, chromatographic peak shape and good resolution

Active Publication Date: 2020-08-07
宜昌三峡普诺丁生物制药有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the production of valine by fermentation method, it is all L-type, no optical resolution is required, but there will be some other amino acids in the finished product, which will have some impact on its clinical application
At the same time, due to the detection of other amino acids of L-valine, the TLC method is used in the 2015 edition of the Chinese Pharmacopoeia and the 42nd edition of the US Pharmacopoeia. The detection sensitivity is poor, and the various amino acids are not clear, and the specific content of various other amino acids cannot be clearly displayed.
Using the phosphate and acetonitrile binary mobile phase system of the 40th edition of the United States Pharmacopoeia, the service life of the amino column is not long, the baseline is unstable, and the baseline separation of L-valine and other amino acids mentioned in the present invention cannot be achieved
The use of amino acid analyzers is expensive in terms of equipment and detection costs, and is not suitable for popularization

Method used

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  • Method for determining other amino acids in L-valine raw material by high performance liquid chromatography
  • Method for determining other amino acids in L-valine raw material by high performance liquid chromatography
  • Method for determining other amino acids in L-valine raw material by high performance liquid chromatography

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Take about 20 mg of L-valine raw material from batch S200308, add 10 ml of mobile phase to dissolve, and use it as the test solution, inject 20 μl of sample, and the chromatographic conditions are as follows:

[0046] Chromatographic column: aminopropyl bonded silica gel column, 250mm×4.6mm, 5μm, Phenomenex Luna NH 2 .

[0047] Mobile phase: a mixed solution of acetonitrile and phosphate buffer with a volume ratio of 72:28, wherein the concentration of phosphate buffer is 15 mmol / L, 0.1% triethylamine is added, and the pH is adjusted to 6.1 with phosphoric acid;

[0048] Phosphate buffer solution: Weigh 1.2g of potassium dihydrogen phosphate and 0.7g of disodium hydrogen phosphate and dissolve them in 1L of water, add 0.1% triethylamine, and adjust the pH to 6.1 with phosphoric acid;

[0049] Equilibrate the column with 50% acetonitrile for at least 30 minutes, then equilibrate the column with the mobile phase for at least 30 minutes.

[0050] The flow rate is 1.0ml / m...

Embodiment 2

[0058] Take about 20 mg of L-valine raw material from batch S200301, add 10 ml of mobile phase to dissolve, and use it as the test solution, inject 20 μl of sample, and the chromatographic conditions are as follows:

[0059] Chromatographic column: aminopropyl bonded silica gel column, 250mm×4.6mm, 5μm, Phenomenex Luna NH 2 .

[0060] Mobile phase: a mixed solution of acetonitrile and phosphate buffer with a volume ratio of 74:26, wherein the concentration of phosphate buffer is 15 mmol / L, 0.3% triethylamine is added, and the pH is adjusted to 6.4 with phosphoric acid;

[0061] Phosphate buffer solution: Weigh 1.2g of potassium dihydrogen phosphate and 0.7g of disodium hydrogen phosphate and dissolve them in 1L of water, add 0.3% triethylamine, and adjust the pH to 6.4 with phosphoric acid;

[0062]Equilibrate the column with 50% acetonitrile for at least 30 minutes, then equilibrate the column with the mobile phase for at least 30 minutes.

[0063] The flow rate is 1.0ml / mi...

Embodiment 3

[0071] Take 200309 batches of L-valine raw materials about 20mg, add mobile phase 10ml to dissolve, as the test solution, inject 20μl, the chromatographic conditions are as follows:

[0072] Chromatographic column: aminopropyl bonded silica gel column, 250mm×4.6mm, 5μm, Phenomenex Luna NH 2 .

[0073] Mobile phase: a mixed solution of acetonitrile and phosphate buffer with a volume ratio of 72:28, wherein the concentration of phosphate buffer is 15 mmol / L, 0.2% triethylamine is added, and the pH is adjusted to 6.2 with phosphoric acid;

[0074] Phosphate buffer solution: Weigh 1.2g of potassium dihydrogen phosphate and 0.7g of disodium hydrogen phosphate and dissolve them in 1L of water, add 0.2% triethylamine, and adjust the pH to 6.2 with phosphoric acid;

[0075] Equilibrate the column with 50% acetonitrile for at least 30 minutes, then equilibrate the column with the mobile phase for at least 30 minutes.

[0076] The flow rate is 1.0ml / min;

[0077] Detection wavelength...

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Abstract

The invention provides a method for determining other amino acids in an L-valine raw material by high performance liquid chromatography. The method comprises the following steps of preparing L-valine,leucine, isoleucine, glycine, alanine and phenylalanine reference substance solutions; detecting through a high performance liquid chromatograph, comparing the appearance time and peak shape of L-valine, leucine, isoleucine, glycine, alanine and phenylalanine in a chromatogram of the test sample with those of L-valine, leucine, isoleucine, glycine, alanine and phenylalanine in a chromatogram of the reference substance solution, and judging whether the test sample solution contains other amino acids or not. According to the method, by screening an aminopropyl bonded silica gel chromatographiccolumn, the separation effects are found, and by screening the wavelengths, the maximum absorption of each amino acid is found, and the retention time of each amino acid peak on the chromatographic column is regulated and controlled by regulating the ratio of triethylamine, the pH value and the ratio of two phases by utilizing an acetonitrile/phosphate buffer solution binary mobile phase, so thatthe hydrolysis and shedding of bonded aminopropyl in the amino column are effectively prevented, and the problems of short reverse-phase service life and unstable baseline of the amino column are solved.

Description

technical field [0001] The invention relates to a method for determining other amino acids in L-valine raw materials, specifically detecting L-norvaline, leucine, isoleucine, Method for residues of glycine, alanine, and phenylalanine. [0002] technical background [0003] L-valine (L-2-amino-3-methylbutyric acid; CAS No.: 72-18-4) is an essential amino acid for human body. It can be used as amino acid medicine, nutritional supplement, amino acid infusion and comprehensive amino acid preparation. main ingredient. which has the formula C 10 h 20 N 2 o 4 , and has the following structural formula: [0004] [0005] L-valine is one of the three branched-chain amino acids and is an essential amino acid. It can treat liver failure and central nervous system dysfunction. As the main component of amino acid infusion and comprehensive amino acid preparations, it has broad market prospects. For the production of valine by fermentation, it is all L-type, and optical resolutio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/32G01N30/34G01N30/54
CPCG01N30/02G01N30/06G01N30/32G01N30/34G01N30/54G01N2030/047G01N2030/324
Inventor 王成张国龙李芹元王孟华曾宪亮
Owner 宜昌三峡普诺丁生物制药有限公司
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