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Amino acid derivatization method and application thereof

An amino acid and derivatization technology, applied in the field of amino acid detection, can solve the problems of long derivatization time and cumbersome derivatization process, and achieve the effects of short derivatization time, simple and fast derivatization process, and reducing the influence of steric hindrance.

Inactive Publication Date: 2019-07-26
CHINA TOBACCO GUANGDONG IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention provides an amino acid derivatization method, which solves the problems that the existing amino acid derivatization method has a long derivatization time, extracts and separates the derivatized samples, etc., and the derivatization process is cumbersome.

Method used

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  • Amino acid derivatization method and application thereof
  • Amino acid derivatization method and application thereof
  • Amino acid derivatization method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] The amino acids in this example are 20 kinds of natural L-type amino acids, and the preparation method of amino acid derivatization is as follows:

[0056] (1) Preparation of 4-nitrobenzoyl chloride derivatization reagent:

[0057] Take 10 mg of 4-nitrobenzoyl chloride and dissolve it in a certain amount of anhydrous acetonitrile to prepare a 10 mg / L derivatization reagent.

[0058] (2)NH 4 HCO 3 -NH 4 Preparation of OH buffer solution:

[0059] Take 0.39g of NH 4 HCO 3 Dissolve in ultrapure water, add ammonia water, and dissolve in a 50mL volumetric flask to make the buffer solution pH=9.

[0060] (3) Preparation of amino acid standard solution:

[0061] Take 10mg amino acid standard, use 0.1mol / L NH 4 HCO 3 -NH 4 The buffer solution of OH was prepared into a mixed standard solution of 10μg / L for later use.

[0062] (4) Take 900 μL of 10 μg / L amino acid mixed standard solution, add 100 μL of 10 mg / L 4-nitrobenzoyl chloride derivatization reagent, shake the reaction for 1 min, and ...

Embodiment 2

[0064] The derivatized amino acid prepared in Example 1 was sampled and analyzed, and the parent ion and product ion scanning analysis of the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry was performed.

[0065] Chromatographic column: Shim-pack XR-ODS III (2.2μm, 2.0mmi.d.×200mm); injection volume: 20μL; column temperature: 35℃; elution method: gradient elution: mobile phase A: 0.1% An aqueous solution of formic acid, mobile phase B: acetonitrile; flow rate: 0.3 mL / min; time: 20 min. The detection conditions for triple quadrupole mass spectrometry are: ion source: ESI; detection method: multi-reaction monitoring mode; capillary voltage: 3.0kV; ion source temperature: 350°C; desolventizing gas temperature: 400°C; desolventizing gas flow: 650L / hr; Cone air flow: 50L / hr.

[0066] The derivatized amino acid prepared in Example 1 is tested by liquid chromatography and the results are as follows Figure 4 As shown, all 20 amino acids were detected. ...

Embodiment 3

[0076] Determination of amino acid content in tobacco leaf samples:

[0077] 1. Process the tobacco leaf samples according to the requirements of YC / T31-1996, accurately weigh 100mg tobacco leaf samples, add ultrapure water for extraction for 10 minutes, and filter the supernatant obtained after centrifugation with a 0.22μm aqueous phase filter membrane to obtain the extract . Take 100μL extract, use 0.1mol / L NH 4 HCO 3 -NH 3 The buffer solution is diluted to 10mL of the sample to be tested. Take 900μL of sample diluent after centrifugation, add 100μL of 10mg / L 4-nitrobenzoyl chloride derivatization reagent, shake the reaction for 1min, filter by organic filter and combine with liquid chromatography-triple quadrupole mass spectrometry. The MRM multi-channel reaction monitoring method is used for quantitative analysis. The amino acid content and standard addition experiment of tobacco leaf samples are shown in Table 3.

[0078] Table 3 Amino acid content, standard addition recover...

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Abstract

The invention relates to the technical field of amino acid detection methods, in particular to an amino acid derivatization method and an application thereof. The amino acid derivatization method comprises the following step thatan amino acid solution with the pHvalue being 9 is added to a 4-nitrobenzoyl chloride solution to be subjected to an oscillating reaction to obtain derivatized amino acid.Nitro of the derivatization agent 4-nitrobenzoyl chloride of the amino acid derivatization method is in para-position, the impact of steric hindrance can be reduced, and the efficiency of derivatization is improved; in addition, the derivatization process is simple and quick, operation is simple, and derivatization time is short. The derivatized amino acid obtained through theamino acid derivatization method does not need extraction separation, and qualitative and quantitative tests can be carried out directly. According to experimental data, the derivatized amino acid tests by using aconventional chromatographic column combing with a multiple reaction monitoring mode liquid chromatogram-triple quadrupole mass spectrum directly, the response is good, sensitivity is high, and selectivity is good.

Description

Technical field [0001] The invention relates to the technical field of amino acid detection methods, in particular to an amino acid derivatization method and its application. Background technique [0002] Amino acids are both amino (-NH 2 ) And the general term for organic compounds containing carboxyl group (-COOH), which are the basic units of protein. The activity of some proteases is an important factor affecting plant metabolism. As the primary and secondary metabolites in tobacco leaves, amino acids will also undergo enzymatic browning reactions with carbohydrates in the process of aging and degradation to form sugar-amino acid complexes. After further degradation, amino acids will generate ketones, Furans, pyrroles, organic acids and many other aroma components, amino acids will also directly degrade to produce carbonyl aroma components, which will affect the quality of tobacco leaves. [0003] Amino acids are usually detected by liquid chromatography, but because amino aci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/067
Inventor 陈森林陶红林云胡玉玲叶为民李攻科
Owner CHINA TOBACCO GUANGDONG IND
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