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A kind of detection method of glucosinolate compound

A glucosinolate and detection method technology, applied in the field of analytical chemistry, can solve the problems of fluorescence detection method with many detection steps, increased error of detection results, failure to detect glucosinolates, etc., achieves reduction of false positive detection results, and simple operation , the effect of improving the qualitative accuracy rate

Active Publication Date: 2022-06-24
SHANGHAI ACAD OF AGRI SCI
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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

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  • A kind of detection method of glucosinolate compound
  • A kind of detection method of glucosinolate compound
  • A kind of detection method of glucosinolate compound

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Example 1 Sample processing

[0109] Weigh 0.20g sample of cauliflower powder (250-830μm) crushed after freeze-drying (freeze-drying temperature -50°C, time is 48h), preheat at 75°C for 10min, add 20mL of 70vol% methanol aqueous solution preheated to 75°C, Extraction was carried out in a water bath at 75°C for 20 minutes. During the extraction process, the samples were shaken and shaken at intervals of 5 minutes. After extraction, ultrasonic-assisted extraction was performed at room temperature for 10 minutes at a frequency of 40 kHz. Centrifugation was performed at 4000 rpm for 5 minutes. Take 100 μL of the supernatant into a 1.5 mL centrifuge tube, add 900 μL of 70% methanol solution by volume, shake well, filter membrane (0.22 μm), store at 4°C and wait for the machine.

Embodiment 2

[0110] Embodiment 2 Instrument method and standard 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, pipetting gun Wait.

[0113] 2. Materials and reagents

[0114] Standard products such as 3-butenyl glucosinolate (Gluconapin), 4-hydroxybenzyl glucosinolate (Sinalbin), and glucosinolate (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 a volumetric flask, dilute to 10 mL with ultrapure water, and store at -20°C as a standard stock solution in the dark. An appropriate amoun...

Embodiment 3

[0117] Example 3 Qualitative analysis of glucosinolates

[0118] The samples obtained in Example 1 were qualitatively analyzed by the first ultra-high liquid chromatography and mass spectrometry.

[0119] The parameters of the first ultra-high liquid chromatography and mass spectrometry analysis include first ultra-high liquid chromatography parameters and 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, 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: scanning mode: precursor ion scanning (precursor ion) mode scanning, preferably negative ion mode...

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Abstract

The invention belongs to the technical field of analytical chemistry and provides a detection method for glucosinolate compounds. In the present invention, the common secondary mass spectrum fragments of glucosinolates are used to achieve high-efficiency qualitative purposes; among these secondary mass spectrum fragments, a set of precursor ion (precursor ion) patterns are constructed based on m / z fragments of 96.0 The scanning qualitative method can not only ensure the detection sensitivity, but also improve the qualitative accuracy and reduce false positive detection results; based on the m / z 96.0 fragments as quantitative ions, a set of multiple reactions can be constructed by using glucosinolate standard products Monitoring (MRM) mode scanning quantitative method, completes the quantitative analysis of all glucosinolates in the sample, solves the problem of inability to quantitatively analyze due to the difficulty in obtaining some standard products, and efficiently completes the individual quantification and total amount of such compounds in the sample Determination.

Description

technical field [0001] The invention relates to the technical field of analytical chemistry, in particular to a method for detecting glucosinolates. Background technique [0002] Glucosinolates (GLS) referred to as glucosinolates are important secondary metabolites in cruciferous vegetables. More than 100 kinds of glucosinolates have been found, and the types and contents of glucosinolates in different varieties of vegetables are significantly different. . According to the different R groups in the side chain of glucosinolates, glucosinolates can be divided into three categories, namely aliphatic, aromatic and indole glucosinolates. When vegetables containing glucosinolates are eaten or mechanically broken, endogenous myrosinase is released, which enzymatically hydrolyzes the β-thiosidic bonds in glucosinolates to generate isothiocyanates. Studies have shown that isothiocyanates have anti-cancer, antioxidant, anti-diabetic and other effects. Among them, sulforaphane, the ...

Claims

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Application Information

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Patent Type & Authority Patents(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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