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A liquid chromatography tandem mass spectrometry method for detecting glucose dehydrogenase in the honey bees and bees

A technology of glucose dehydrogenase and liquid chromatography, which is applied in the field of detection of glucose dehydrogenase in honey by liquid chromatography tandem mass spectrometry, and achieves the effects of strong specificity, high specificity and sensitivity, and high sensitivity

Inactive Publication Date: 2020-11-20
BEE RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the use of this technique for the quantitative detection of glucose dehydrogenase in honey

Method used

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  • A liquid chromatography tandem mass spectrometry method for detecting glucose dehydrogenase in the honey bees and bees
  • A liquid chromatography tandem mass spectrometry method for detecting glucose dehydrogenase in the honey bees and bees
  • A liquid chromatography tandem mass spectrometry method for detecting glucose dehydrogenase in the honey bees and bees

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] 1. Sample source

[0052] A total of 20 samples of real honey were purchased from the market or beekeepers.

[0053] 2. Experimental procedure

[0054] 2.1 Solution preparation

[0055] 5M Urea solution: Weigh 4.5 g of Urea, and dilute to 15 mL with ultrapure water;

[0056] 100 mM DTT: Weigh 308.5 mg DTT, 40 mM NH 4 HCO 3 Dilute the solution to 20 mL, dispense 1 mL into each tube, and store in a -20°C refrigerator until use.

[0057] 40 mM NH 4 HCO 3 Solution: weigh 0.316 g NH 4 HCO 3 , dilute to 100 mL with ultrapure water, and store in a 4°C refrigerator until use.

[0058] 100 mM IAA solution: Weigh 0.39 g of IAA, add 40 mM NH 4 HCO 3 The solution was fixed to 20 mL, and stored in a -20°C refrigerator until use.

[0059] Activation solution: 500 μL ACN, 3 μL TFA, dilute to 1 mL with ultrapure water, store at 4°C.

[0060] Equilibrium solution: 1 μL TFA, dilute to 1 mL with ultrapure water, store at 4°C.

[0061] Eluent: 800 μL ACN, 1 μL TFA, dilute to ...

Embodiment 2

[0078] Example 2 Detection of honey adulteration

[0079] 1. Sample source

[0080] Buy real samples of Chinese honey, Italian honey, and syrups from markets or beekeepers.

[0081] 2. Experimental steps

[0082] (1) Solution preparation

[0083] With embodiment 1.

[0084] (2) Blending of honey: Italian honey is mixed into medium honey in different proportions.

[0085] Mix Italian honey into the honey according to the ratio of 5%, 10%, 20%, and 50%.

[0086] (3) Pretreatment of honey samples

[0087] ① Accurately weigh 10 g of honey to be tested evenly into a 50 mL centrifuge tube, add 10 mL of deionized water, and vortex until the honey is fully dissolved. Centrifuge at 12000 rpm for 10 min at 4°C. Collect the supernatant in a new 2 mL centrifuge tube.

[0088] ② Pipette 200 μL of protein solution and 800 μL of 40 mM NH 4 HCO 3 mix. Add 100 μL of 30 mM DTT solution to the above mixed solution, react at room temperature for 60 min, and then add 500 μL of 100 mM IA...

Embodiment 3

[0094] Example 3 Detection of honey in commodities

[0095] 1. Sample source

[0096] 50 bottles of medium honey from different manufacturers purchased from the mall.

[0097] 2. Experimental steps

[0098] (1) Solution preparation

[0099] With embodiment 1.

[0100] (2) Pretreatment of samples to be tested

[0101] ① Accurately weigh 10 g of honey to be tested evenly into a 50 mL centrifuge tube, add 10 mL of deionized water, and vortex until the honey is fully dissolved. Centrifuge at 12000 rpm for 10 min at 4°C. Collect the supernatant in a new centrifuge tube.

[0102] ② Pipette 200 μL of protein solution and 800 μL of 40 mM NH 4 HCO 3 mix. Add 100 μL of 30 mM DTT solution to the above mixed solution, react at room temperature for 60 min, and then add 500 μL of 100 mM IAA solution and react in dark at room temperature for 60 min.

[0103] ③Add 30 μL of trypsin solution to each sample, and digest at 37°C overnight. When the digestion reaction is complete, add 1 μL...

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Abstract

The invention relates to a method for detecting apis mellifera and apis cerana honey glucose dehydrogenase through liquid chromatography-tandem mass spectrometry. The method comprises the following steps: selecting characteristic peptide fragments respectively belonging to glucose dehydrogenase of apis mellifera honey and apis cerana honey, establishing a liquid chromatography-tandem mass spectrometry detection method, pretreating a sample, performing liquid chromatography separation, performing tandem mass spectrometry detection and performing data analysis, wherein the characteristic peptidefragments comprise an apis mellifera glucose dehydrogenase characteristic peptide fragment, namely ITLDNQSVQVR, and an apis cerana glucose dehydrogenase characteristic peptide fragment, namely FPYQPPFAWEILK. The method for detecting glucose dehydrogenase in honey has high accuracy, precision and sensitivity and strong stability and is suitable for distinguishing apis mellifera honey from apis cerana honey and is especially suitable for identifying apis mellifera honey doped in apis cerana honey. The method has important practical significance for protecting rights and interests of honey consumers and maintaining healthy development of the bee product industry.

Description

technical field [0001] The invention relates to the field of food detection, in particular to a method for detecting glucose dehydrogenase in honey by liquid chromatography tandem mass spectrometry, and the method is suitable for distinguishing middle honey and Italian honey. Background technique [0002] Honey is rich in nutrition, has a sweet taste, and has certain effects such as beautifying, moistening intestines and health care, and is deeply loved by people. In addition, honey is rich in nutrients and active substances, such as phenolic acids, flavonoids, etc., which make honey have certain medicinal value, such as anti-inflammatory, antibacterial, and anti-oxidative effects, and is used in medicine and cosmetics. Compared with Italian honey, Chinese honey has a better taste and is more popular with consumers, but the average yield of each group of Chinese bees is only about 1 / 5 of Italian bees, resulting in a shortage in the market. Currently, the price of Chinese ho...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/88
CPCG01N30/88G01N2030/8831
Inventor 杨术鹏李熠周金慧丛晓蕾张金震杨宇晖黄京平金玥王鹏赵文
Owner BEE RES INST CHINESE ACAD OF AGRI SCI
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