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Method for detecting in-vivo CYP1A2 and CYP3A4 enzyme activity in earthworm through high-performance liquid chromatography-tandem mass spectrometry

A high-performance liquid chromatography and CYP1A2 technology, applied in the field of enzyme activity detection, to achieve rapid detection methods, high throughput, and strong stability

Pending Publication Date: 2019-04-09
CHONGQING ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no literature report on the simultaneous determination of CYP1A2 and CYP3A4 enzyme activities in earthworms. Therefore, there is an urgent need for a method that can perform lower-level quantification with higher specificity in a short period of time from complex biological matrices, and simultaneously detect earthworms. Methods of Enzyme Activity of CYP1A2 and CYP3A4 in Vivo

Method used

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  • Method for detecting in-vivo CYP1A2 and CYP3A4 enzyme activity in earthworm through high-performance liquid chromatography-tandem mass spectrometry
  • Method for detecting in-vivo CYP1A2 and CYP3A4 enzyme activity in earthworm through high-performance liquid chromatography-tandem mass spectrometry
  • Method for detecting in-vivo CYP1A2 and CYP3A4 enzyme activity in earthworm through high-performance liquid chromatography-tandem mass spectrometry

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Mobile Phase Composition Determination

[0046]Investigate the influence of two groups of mobile phase composition on detection sensitivity: the first kind is that A phase is formic acid aqueous solution, the volume fraction of formic acid in this formic acid aqueous solution is 0.05%, and B is methyl alcohol; The second kind is that A phase is formic acid ammonium acetate aqueous solution, The volume fraction of formic acid in this formic acid ammonium acetate aqueous solution is 0.05%, the concentration of ammonium acetate is 1mM, and B is methyl alcohol, the result shows that the second mobile phase has effectively improved the stability of chromatographic peak shape and peak area, therefore, determine with A The phase is ammonium acetate formate aqueous solution (the volume fraction of formic acid in the ammonium acetate formate aqueous solution is 0.05%, and the concentration of ammonium acetate is 1 mM), and B is methanol as the optimal mobile phase.

Embodiment 2

[0048] LC-MS / MS condition setting

[0049] LC-MS / MS is the XEVO TQ-XS triple quadrupole tandem mass spectrometry-HPLC system of Waters Company, wherein the high performance liquid chromatography conditions are:

[0050] a) Chromatographic column: Acquity UPLC C18 column, 2.1mm×50mm, 1.8μm, and equipped with a corresponding C18 guard column; b) The mobile phase is composed of A phase and B phase, wherein, A phase is an aqueous ammonium acetate formic acid solution, and the formic acid The volume fraction of formic acid in the ammonium acetate aqueous solution is 0.05%, the concentration of ammonium acetate is 1mM, and phase B is methanol; c) Gradient elution conditions: 0-0.6min, the proportion of mobile phase B is 0%, 0.6-6min, mobile The ratio of phase B rises from 0% to 50%; 6-8min, the ratio of mobile phase B rises to 95% from 50%; 8-9min, the ratio of mobile phase B drops to 0% from 95%; d) column Temperature: 40°C; e) Flow rate: 0.3mL / min; f) Auto-injection chamber tempe...

Embodiment 3

[0058] Matrix effect test

[0059] (1) Preparation of stock solution

[0060] Weigh 10 mg of acetaminophen powder, dissolve it in 10 mL of methanol until the concentration of acetaminophen is 1.0 mg / mL, take 100 μL of the above solution and dilute it with methanol to a concentration of acetaminophen of 10 μg / mL to prepare acetaminophen stock liquid.

[0061] The purchased 6β-hydroxytestosterone acetone solution (the concentration of 6β-hydroxytestosterone in the solution is 100 μg / mL) was diluted with methanol to a concentration of 10 μg / mL of 6β-hydroxytestosterone to prepare a 6β-hydroxytestosterone stock solution.

[0062] Weigh 0.179g of phenacetin and dissolve it in 10mL of absolute ethanol until the concentration of phenacetin is 0.1M to prepare a phenacetin stock solution.

[0063] Weigh 0.298g of testosterone and dissolve it in 10mL of methanol to a testosterone concentration of 0.1M to prepare a testosterone stock solution.

[0064] (2) Preparation of working solut...

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Abstract

The invention relates to a method for detecting in-vivo CYP1A2 and CYP3A4 enzyme activity in earthworm through high-performance liquid chromatography-tandem mass spectrometry, and belongs to the technical field of the enzyme activity detection. The method comprises the following steps: (1) preparing earthworm microsome protein suspension; (2) adding the earthworm microsome protein suspension prepared in the step (1) in an incubation system containing two specific probe primers, warming to start enzymatic reaction, after the enzymatic reaction is terminated, detecting two specific metabolites produced after the enzymatic reaction is terminated by utilizing high-performance liquid chromatography-tandem mass spectrometry, and respectively computing CYP1A2 and CYP3A4 enzyme activity. The detection method disclosed by the invention is high in accuracy and precision and sensitivity, strong in stability, capable of measuring multiple CYP subenzyme activities in the earthworm body, thereby providing a detection method for exploring response mode on the soil pollutant by different CYP subenzyme of the earthworm.

Description

technical field [0001] The invention belongs to the technical field of enzyme activity detection, and in particular relates to a method for detecting CYP1A2 and CYP3A4 enzyme activities 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. Among them, CYP3A4 and CYP1A2 are two very important CYP subenzymes in the human liver, accounting for 34% and 20% of the total CYP enzymes respectively. At present, CYP subenzymes as environmental toxicology indicators have been studied in terrestrial vertebrates and aquatic organisms, but less in soil inverteb...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
CPCG01N30/02
Inventor 杨晓霞杨俊英龚久平李典晏张雪梅张伟孟霞李必全柴勇刘剑飞
Owner CHONGQING ACAD OF AGRI SCI
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