Detection method of glucosinolate compounds

A technology of glucosinolates and detection methods, applied in the field of analytical chemistry, which can solve the problems of small sampling volume, many detection steps of fluorescence detection method, and long separation time

Active Publication Date: 2021-09-14
SHANGHAI ACAD OF AGRI SCI
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  • Description
  • Claims
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Problems solved by technology

[0003] At present, there are mainly the following methods for the determination of glucosinolates in cauliflower reported in the literature: (1) near-infrared reflectance spectroscopy (NIRS), which mainly establishes an infrared analysis model through known standard products, compares sample data, and quickly determines the content of glucosinolates ; Near-infrared reflectance spectroscopy can be used for non-destructive testing with a small amount of sampling, but it requires a large number of standard samples to establish a reliable model
(2) Fluorescence detection method, the main principle is that glucosinolate is degraded under the action of myrosinase to obtain glucose, which obtains gluconic acid under the action of glucose oxidase, and then reacts with reagents to generate fluorescent active substances; the fluorescence detection method has many detection steps and will lead to an increase in the error of the test results
(3) Micellar electrokinetic capillary electrophoresis (HPCED), which mainly uses the different charges of glucosinolate molecules, adds surfactants, and separates glucosinolates in a borate buffer system; micellar electrokinetic capillary electrophoresis has few interference factors, Low cost, but long separation time
(4) Gas phase spectrometry (GC), that is, enzymatic hydrolysis of glucosinolates to obtain isothiocyanates, and then determined by gas chromatography; gas chromatography cannot detect glucosinolates that are easily decomposed by heating
(5) Determination of glucosinolates by high-performance liquid chromatography (HPLC) combined with ultraviolet detector (UV) or diode array (DAD), and derivatization modification of isothiocyanates by pre-column derivatization , and then use high performance liquid chromatography (HPLC) to measure; (6) use high performance liquid chromatography (HPLC) combined with mass spectrometry (MS) to measure glucosinolates and isothiocyanates; high performance liquid chromatography can simultaneously determine various The content of glucosinolates, the reproducibility and precision are acceptable, and it is relatively common in practical applications, but the detection limit and sensitivity of this method are poor, and there are limitations in the application of the method on cauliflower varieties with low glucosinolate content

Method used

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  • Detection method of glucosinolate compounds
  • Detection method of glucosinolate compounds
  • Detection method of glucosinolate compounds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Embodiment 1 sample processing

[0109] Weigh 0.20 g sample of crushed cauliflower powder (250-830 μm) after freeze-drying (freeze-drying temperature -50 ° C, time is 48 h), preheat at 75 ° C for 10 min, add 20 mL of 70 vol% methanol aqueous solution preheated to 75 ° C, Extract in a water bath at 75°C for 20 minutes. During the extraction process, oscillate and shake well every 5 minutes. After extraction, extract with ultrasound at room temperature for 10 minutes at a frequency of 40 kHz; centrifuge at 4000 rpm for 5 minutes. Take 100 μL of the supernatant in a 1.5 mL centrifuge tube, add 900 μL of 70% methanol solution, shake and shake well, filter the membrane (0.22 μm), store at 4°C until ready to use.

Embodiment 2

[0110] Embodiment 2 instrument method and standard substance preparation and preservation

[0111] 1. Instruments and equipment

[0112] Waters Acquity UPLC liquid chromatograph (Waters, USA), AB SCIEX 5500 triple quadrupole mass spectrometer (AB SCIEX, USA), vortex mixer, high-speed centrifuge, nitrogen blow-dryer, electronic analytical balance, pipette gun Wait.

[0113] 2. Materials and reagents

[0114] Standard products such as 3-butenyl glucosinolate (Gluconapin), 4-hydroxybenzyl glucosinolate (Sinalbin), and sinigrin (Sinigrin) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; chromatographically pure acetonitrile and methanol were purchased from Shanghai Titan Technology Co., Ltd.

[0115] 3. Preparation and storage of standard solution

[0116] Accurately weigh 5 mg of Gluconapin, Sinalbin, and Sinigrin standards into volumetric flasks, use ultrapure water to make up to 10 mL, and store them as standard stock solutions in the dark at -20°C. Take an app...

Embodiment 3

[0117] Embodiment 3 Qualitative analysis of glucosinolates

[0118] The first ultra-high liquid chromatography and mass spectrometry were used to qualitatively analyze the sample obtained in Example 1.

[0119] The parameters of the first ultra-high liquid chromatography and mass spectrometry include the first ultra-high liquid chromatography parameters and the first mass spectrometry parameters;

[0120] The first ultra-high liquid chromatography parameters include: the chromatographic column is a Merck ZIC-HILIC column, 100mm×2.1mm (i.d.), 3.5μm The temperature of the chromatographic column is preferably 40°C; the mobile phase A is acetonitrile, and the mobile phase B is 5mmol / L ammonium acetate aqueous solution; the flow rate is 0.3mL / min; the injection volume is 3μL; gradient elution conditions: 0~1min, 90% A; 1 ~ 9min, 90% ~ 65% A;

[0121] The first mass spectrometry parameters include: scan mode: precursor ion scan (precursor ion) scan mode, preferably negative ion m...

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Abstract

The invention belongs to the technical field of analytical chemistry, and provides a detection method of glucosinolate compounds. According to the method, common secondary mass spectrum fragments of glucosinolate compounds are adopted, so that the purpose of high-efficiency qualitative analysis is achieved; in the secondary mass spectrum fragments, a set of precursor ion mode scanning qualitative method is constructed based on the fragments with m / z of 96.0, so that the detection sensitivity can be ensured, the qualitative accuracy can be improved, and false positive detection results are reduced; on the basis that the fragments with the m / z of 96.0 are used as quantitative ions, a set of multi-reaction monitoring (MRM) mode scanning quantitative method is constructed by utilizing glucosinolate compound standard substances, quantitative analysis of all glucosinolate compounds in a sample is completed, the problem that quantitative analysis cannot be achieved due to the fact that some standard substances are difficult to obtain is solved, and individual quantification and total amount determination of the compounds in the sample are efficiently completed.

Description

technical field [0001] The invention relates to the technical field of analytical chemistry, in particular to a detection method for glucosinolate compounds. Background technique [0002] Glucosinolates (GLS), referred to as glucosinolates, are important secondary metabolites in cruciferous vegetables. More than 100 glucosinolates have been discovered so far, and the types and contents of glucosinolates in different varieties of vegetables are significantly different. . According to the different R groups in the side chains of glucosinolates, glucosinolates can be divided into three types, namely aliphatic, aromatic and indole glucosinolates. When vegetables containing glucosinolates are eaten or mechanically crushed, endogenous myrosinase is released, which enzymatically hydrolyzes the β-glucosidic bonds in glucosinolates to generate isothiocyanates. Studies have shown that isothiocyanate has anti-cancer, anti-oxidation, anti-diabetic and other effects. Among them, sulfo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/54G01N30/72
CPCG01N30/02G01N30/06G01N30/54G01N30/72
Inventor 鄂恒超赵晓燕周昌艳赵志勇李晓贝张艳梅范婷婷陈磊董慧何香伟李健英彭书婷
Owner SHANGHAI ACAD OF AGRI SCI
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