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Method for quantitatively detecting melittin by liquid chromatography-tandem mass spectrometry

A technology of liquid chromatography and tandem mass spectrometry, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of difficult to achieve accurate quantification of melittin and complex matrix, and achieve good accuracy and precision, strong specificity, The effect of high sensitivity

Inactive Publication Date: 2020-10-30
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

Since the content of melittin in skin care products and other products is very low, only at trace levels, and its matrix is ​​very complex, it is difficult to accurately quantify melittin in skin care products and other products by using existing analysis methods

Method used

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  • Method for quantitatively detecting melittin by liquid chromatography-tandem mass spectrometry
  • Method for quantitatively detecting melittin by liquid chromatography-tandem mass spectrometry
  • Method for quantitatively detecting melittin by liquid chromatography-tandem mass spectrometry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Method for Determination of Melittin Content in Bee Venom by Liquid Chromatography Tandem Mass Spectrometry

[0049] 1. Sample source

[0050] A total of more than 140 samples of real bee venom were purchased from the market or beekeepers.

[0051] 2. Experimental procedure

[0052] 2.1 Solution preparation

[0053] 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.

[0054] 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.

[0055] 100 mM IAA solution: Weigh 0.39 g of IAA, add 40 mM NH 4 HCO 3 The solution was adjusted to 20 mL and stored in a -20°C refrigerator until use.

[0056] Activation solution: 500 μL ACN, 3 μL TFA, dilute to 1 mL with ultrapure water, store at 4°C.

[0057] Equilibrium solution: 1 μL TFA, dilute to 1 mL with ultrapure water, store at 4...

Embodiment 2

[0079] Embodiment 2 compares with existing melittin detection method

[0080] 1. Sample source

[0081] Purchase more than 140 samples of real bee venom from the market or beekeepers.

[0082] 2. Experimental steps

[0083] (1) Solution preparation

[0084] With embodiment 1.

[0085] (2) Pretreatment of bee venom samples

[0086] ① Accurately weigh 1 g of the bee venom to be tested evenly into a 10 mL centrifuge tube, add 1 mL of deionized water, and vortex until the bee venom is fully dissolved. Centrifuge at 14000 rpm for 12 min at 4 °C. Collect the supernatant in a new 2 mL centrifuge tube. This supernatant solution can be used as undigested bee venom solution for analysis, and can also be used as protein solution for the next enzyme digestion reaction.

[0087] 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...

Embodiment 3

[0095] Example 3 Quantitative detection of melittin in bee venom products

[0096] 1. Sample source

[0097] Buy products marked with "bee venom" from the market, including toothpaste (10 parts), facial mask (25 parts), plaster (5 parts) and a total of 40 products.

[0098] 2. Experimental steps

[0099] (1) Solution preparation

[0100] With embodiment 1.

[0101] (2) Pretreatment of bee venom samples

[0102] ① Accurately weigh 1 g of the bee venom product to be tested evenly into a 10 mL centrifuge tube, add 1 mL of deionized water, and vortex until the bee venom product is fully dissolved. Centrifuge at 14000 rpm for 12 min at 4 °C. Collect the supernatant in a new 2 mL centrifuge tube.

[0103] 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.

[010...

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Abstract

The invention relates to the field of detection, in particular to a method for quantitatively detecting melittin by liquid chromatography-tandem mass spectrometry, which comprises the step of detecting a characteristic peptide fragment of melittin in a sample to be detected by liquid chromatography-tandem mass spectrometry, wherein the sequence of the characteristic peptide fragment is VLTTGLPALISWIK. The method has high accuracy, precision and sensitivity, is high in stability, is suitable for accurate quantification of melittin in complex matrixes, and has important practical significance for protecting rights and interests of melittin consumers and maintaining healthy development of the bee product industry.

Description

technical field [0001] The invention relates to the detection field, in particular to a method for quantitatively detecting melittin by liquid chromatography tandem mass spectrometry. Background technique [0002] Bee venom is the venom secreted by the venom glands and accessory glands of bee workers. It is a biological weapon released by bees when they sting their enemies. Bee venom is rich in enzymes, polypeptides, biogenic amines, hormones, organic acids, amino acids and other substances, among which melittin (bee venom hemolytic peptide) is the main component of bee venom, which accounts for more than 50% of the weight of dry bee venom . Melittin is a small peptide substance composed of 26 amino acids in an α-helical conformation. It has extensive and strong biological activities, such as antibacterial, anti-inflammatory, analgesic, immune regulation, antiviral and antitumor effects . At present, my country and many countries around the world have approved melittin as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88G01N30/06
CPCG01N30/06G01N30/88
Inventor 杨术鹏李熠蔡冬梅傅怡周金慧杨宇晖
Owner BEE RES INST CHINESE ACAD OF AGRI SCI
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