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Method for detecting CYP2C9 enzyme activity in earthworms by adopting high-performance liquid chromatography-tandem mass spectrometry

A technology of high performance liquid chromatography and tandem mass spectrometry, which is applied in the field of detection of CYP2C9 enzyme activity in earthworms by high performance liquid chromatography and tandem mass spectrometry, which can solve the problems of detection failure and achieve the effect of strong stability, high accuracy and high sensitivity

Active Publication Date: 2017-01-04
CHONGQING ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the content of CYP subenzymes in earthworms is less than that of mammals, and the results of detection failures are mostly reported in the existing literature.

Method used

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  • Method for detecting CYP2C9 enzyme activity in earthworms by adopting high-performance liquid chromatography-tandem mass spectrometry
  • Method for detecting CYP2C9 enzyme activity in earthworms by adopting high-performance liquid chromatography-tandem mass spectrometry
  • Method for detecting CYP2C9 enzyme activity in earthworms by adopting high-performance liquid chromatography-tandem mass spectrometry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Mobile Phase Composition Determination

[0046] Investigate the influence of three groups of mobile phase compositions on the detection sensitivity: the first is that A is pure water in the mobile phase, and B is pure acetonitrile; the second is that A is water (containing 0.1% formic acid by volume fraction), and B is pure Acetonitrile; the third is that A is water (containing 0.1% formic acid by volume fraction), and B is acetonitrile (containing 0.1% formic acid by volume fraction). The results show that the last mobile phase effectively improves the chromatographic peak shape and peak area Therefore, it is determined that A is water (containing 0.1% formic acid by volume fraction), and B is acetonitrile (containing 0.1% formic acid by volume fraction) as the optimal mobile phase.

Embodiment 2

[0048] LC-MS / MS condition setting

[0049] LC-MS / MS is a Thermo Fisher Scientific TSQ Quantum Max triple quadrupole tandem mass spectrometry-HPLC system, wherein the high performance liquid chromatography conditions are:

[0050]a) Chromatographic column: MS Luna C18 column, 2.1mm×50mm, 3.0μm, and equipped with a corresponding C18 guard column; b) The mobile phase is composed of A phase and B phase, and the A phase contains 0.1% formic acid by volume fraction of water, the B phase is acetonitrile containing 0.1% formic acid by volume fraction; c) gradient elution condition: 0-4min, the ratio of mobile phase B rises to 95% from 5%; 4-8min, the proportion of mobile phase B The proportion is reduced from 95% to 5%; 8-10min, the proportion of mobile phase B is 5%; d) column temperature: 50 ℃; e) flow rate: 0.2ml / min; f) automatic sample chamber temperature: 4 ℃, The injection volume was 5 μl.

[0051] The mass spectrometry conditions are:

[0052] a) Ion source: electrospray io...

Embodiment 3

[0059] Matrix effect test

[0060] (1) Configure the stock solution: mix methanol and water at a volume ratio of 1:1 to prepare a dilution solution, dissolve DF in the dilution solution until the final concentration of DF is 1.0mg / ml, and prepare a DF stock solution; 'H-DF was dissolved in methanol until the final concentration of 4'H-DF was 1.0 mg / ml to prepare 4'H-DF stock solution;

[0061] (2) Prepare the working solution: dilute the 4′H-DF stock solution in step (1) to 10ng / ml to obtain the 4′H-DF working solution; dilute the DF stock solution to 5 μg / ml to obtain the DF working solution .

[0062] (3) Mix the 4'H-DF working solution with the DF working solution in a volume ratio of 1:1 in step (2), and carry out LC-MS / MS determination, and its chromatogram is as follows: figure 2 shown.

[0063] (4) Take 1ml of 4′H-DF working solution in a 2ml test tube, dry it under a nitrogen blower, add 250μl of DF working solution, then add 650μl of incubation system and raise th...

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Abstract

The invention relates to a method for detecting CYP2C9 enzyme activity in earthworms by adopting a high-performance liquid chromatography-tandem mass spectrometry and belongs to the technical field of enzyme activity detection. The method specifically comprises the following steps: (1) preparing earthworm microsome protein suspension; (2) adding the earthworm microsome protein suspension prepared in the step (1) into an incubation system containing a probe substrate; raising the temperature to start an enzymatic reaction; after the enzymatic reaction is stopped, detecting a metabolic product, which is generated after the enzymatic reaction is stopped, by adopting the high-performance liquid chromatography-tandem mass spectrometry; calculating the CYP2C9 enzyme activity. The detection method is high in accuracy, precision and sensitivity and high in stability, and can be used for analyzing and detecting the CYP2C9 enzyme activity in the earthworms under different pollution environments, so that a detection method for probing the possibility of taking CYP2C9 as a biological marker to indicate soil pollution is provided.

Description

technical field [0001] The invention belongs to the technical field of enzyme activity detection, and in particular relates to a method for detecting CYP2C9 enzyme activity in earthworms by high performance liquid chromatography tandem mass spectrometry. Background technique [0002] Cytochrome P450 (CYP), as a huge isowork family of enzymes, is a class of important detoxification enzymes containing heme. This family of enzymes transfers electrons to oxidize heterologous substances through iron ions in non-covalently bonded heme, enhances the water solubility of heterologous substances, makes them easier to excrete, and thus plays a detoxification role. At present, CYP subenzyme as an environmental toxicology indicator has been studied more in terrestrial vertebrates and aquatic organisms, compared with soil invertebrates, such as earthworms, but earthworm CYP subenzyme is used to diagnose soil environment Pollution research is becoming more and more extensive. CYP2C9 is a...

Claims

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Application Information

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IPC IPC(8): G01N30/02
CPCG01N30/02
Inventor 杨晓霞龚久平李必全柴勇刘剑飞
Owner CHONGQING ACAD OF AGRI SCI
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