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Separation and liquid chromatography column pre-column derivatization method of biogenic amine in soybean paste

A liquid chromatography column and pre-column derivatization technology, applied in the field of separation and detection of biogenic amines, can solve the problems of low sensitivity, detection by fluorescence detectors, and lack of fluorescence characteristics, etc., to achieve less consumption of reagents and loss of analytes The effect of small size and less interference of spectral peaks

Inactive Publication Date: 2010-08-04
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When selecting a derivatizing agent, the principle of good stability of the derivative product, less impurities, and simple derivatization process should be taken as the principle, but all derivatizing reagents have certain limitations.
Dinitrobenzoyl chloride derivatives and phenyl isothiocyanate derivatives do not have fluorescence properties, so they cannot be detected by fluorescence detectors, and can only be detected by ultraviolet detectors. The sensitivity of the method is lower than that of other derivative reagents

Method used

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  • Separation and liquid chromatography column pre-column derivatization method of biogenic amine in soybean paste
  • Separation and liquid chromatography column pre-column derivatization method of biogenic amine in soybean paste
  • Separation and liquid chromatography column pre-column derivatization method of biogenic amine in soybean paste

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Experimental program
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Effect test

Embodiment 1

[0027] Weigh 4 g of naturally fermented soybean paste from Northeast China into a centrifuge tube, add 6 mL of 5% trichloroacetic acid solution, and homogenize for 3 min at 10,000 r / min. After homogenization, centrifuge at 3500r / min for 5min. Collect the supernatant, repeat the extraction of the residue once with 6mL 5% trichloroacetic acid, combine the extracts, then add 6mL n-hexane to remove lipid impurities, vibrate vigorously and then statically separate the layers, discard the n-hexane layer, and then add 6mL n-hexane The operation was repeated once with alkane, and the lower trichloroacetic acid phase was taken out after the layers were separated. Dissolve 10 mg of dansyl chloride in 1 mL of acetone, prepare fresh and store in the dark. Add 200μL, 2mol / L NaOH solution to 1mL sample solution, then add 300μL saturated NaHCO 3 Solution buffer. Add 1.5 mL of dansyl chloride solution, put the mixed solution in a 40°C incubator for 40 minutes, add 100 μL of ammonia water t...

Embodiment 2

[0029] Weigh and grind 6 g of bean paste produced in Northeast China into a centrifuge tube, add 12 mL of 5% trichloroacetic acid solution, 10000 r / min, homogenize for 3 min. After homogenization, centrifuge at 3500r / min for 5min. Collect the supernatant, repeat the extraction of the residue once with 12mL 5% trichloroacetic acid, combine the extracts, then add 12mL n-hexane to remove lipid impurities, vibrate vigorously and then statically separate the layers, discard the n-hexane layer, then add 12mL n-hexane The operation was repeated once with alkane, and the lower trichloroacetic acid phase was taken out after the layers were separated. Dissolve 10 mg of dansyl chloride in 1 mL of acetone, prepare fresh and store in the dark. Add 400μL, 2mol / L NaOH solution to 2mL sample solution, then add 600μL saturated NaHCO 3 Solution buffer. Add 3 mL of dansyl chloride solution, put the mixed solution in a 40°C incubator for 40 minutes, add 200 μL of ammonia water to remove dansyl...

Embodiment 3

[0031] Weigh 10 g of industrially produced seasoned soybean paste into a centrifuge tube, add 15 mL of 5% trichloroacetic acid solution, and homogenize for 3 min at 10000 r / min. After homogenization, centrifuge at 3500r / min for 5min. Collect the supernatant, repeat the extraction of the residue once with 15mL 5% trichloroacetic acid, combine the extracts, then add 15mL n-hexane to remove lipid impurities, vibrate vigorously and then statically separate the layers, discard the n-hexane layer, and then add 15mL n-hexane The operation was repeated once with alkane, and the lower trichloroacetic acid phase was taken out after the layers were separated. Dissolve 10 mg of dansyl chloride in 1 mL of acetone, prepare fresh and store in the dark. Add 200μL, 2mol / L NaOH solution to 1mL sample solution, then add 300μL saturated NaHCO 3 Solution buffer. Add 1.5 mL of dansyl chloride solution, put the mixed solution in a 40°C incubator for 40 minutes, add 100 μL of ammonia water to remo...

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Abstract

The invention discloses a separation and liquid chromatography column pre-column derivatization method of biogenic amine in a soybean paste, comprising the following steps of: (1) adding a trichloroacetic acid solution to the soybean paste, homogenizing, centrifuging and collecting a supernate; (2) adding normal hexane to the supernate, vibrating and taking out a lower layer trichloroacetic acid phase for later use after static layering; (3) adding an NaOH solution to the trichloroacetic acid phase, then adding an NaHCO3 solution for buffering, and mixing in a dansyl chloride acetone solution to carry out derivatization; and (4) removing dansyl chloride to terminate the reaction to obtain a derivative solution and filtering. The separation method has simple treatment process and low reagent consumption and avoids analyte loss caused by transfer. The soybean paste can reach detection demands after being separated and purified; undesired peak disturbance of a spectrogram is less and the determinand loss is low, and the recovery rate of the biogenic amine can reach more than 80 percent. The method determines the optimum condition for deriving the biogenic amine; dansyl chloride derivative products are stable and the detection sensitivity is high.

Description

technical field [0001] The invention relates to a method for separating and derivatizing biogenic amines, in particular to a method for separating biogenic amines from soybean paste and derivatizing biogenic amines before a liquid chromatography column, belonging to the field of separation and detection of biogenic amines. Background technique [0002] The biogenic amines in soybean paste are mainly produced by the decarboxylation of amino acid decarboxylase produced by microorganisms present therein. The production of biogenic amines requires three conditions: the presence of free amino acids, the precursors for the formation of biogenic amines; the presence of conditions that allow the decarboxylation of amino acids; and an environment suitable for the growth of microorganisms. [0003] The biogenic amines produced by microorganisms during the fermentation of soybean paste include histamine, putrescine, cadaverine, tryptamine, tyramine, β-phenylethylamine, spermine, and sp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/34G01N30/02G01N21/64G01N21/33
Inventor 江连洲韩翠萍王鹏程建军魏冬旭
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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