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Detection method for simultaneously determining metabolic products of seven CYP450 enzyme probe substrates in human liver microsomes

A technology of human liver microsomes and probe substrates, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems that the detection methodology has not been systematically verified, and achieves saving detection time, strong specificity, and fast method Effect

Inactive Publication Date: 2015-08-19
WUXI PEOPLES HOSPITAL
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Problems solved by technology

Journal of Shenyang Pharmaceutical University, 2008); the detection methodology has not undergone strict systematic verification (such as: specificity, standard curve, accuracy, precision, etc.)

Method used

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  • Detection method for simultaneously determining metabolic products of seven CYP450 enzyme probe substrates in human liver microsomes
  • Detection method for simultaneously determining metabolic products of seven CYP450 enzyme probe substrates in human liver microsomes
  • Detection method for simultaneously determining metabolic products of seven CYP450 enzyme probe substrates in human liver microsomes

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Embodiment 1

[0027] Example 1: To investigate the inhibitory effect of baicalin on 7 subtypes of CYP450 enzymes in human liver microsomal.

[0028] (1) Chromatography: The human liver microsomal sample pretreated in step (2) is subjected to liquid chromatography separation: Chromatographic column in chromatographic conditions: ZORBXA Eclipse-plus C18 column; mobile phase: methanol (A): water, water Contains formic acid with a mass concentration of 0.1%; Gradient elution: 0→0.8min, 5%A; 0.8→5.0min, 95%A; 5.0→7.0min, 5%A. Flow rate: 200~300μL / min; Column temperature: 30~35℃; Injection volume: 5~10μL;

[0029] (2) Mass spectrometry: Electrospray ionization positive ion (ESI+); Spray voltage: 4000V; Ion transmission capillary temperature: 350°C; Nebulizing gas pressure: 30psi; Dry gas flow rate: 10L / min; Scanning method: Selective reaction monitoring (SRM); monitoring ion pairs and collision energies: acetaminophen (152 / 110, 16eV), 1-hydroxybupropion (256 / 238, 10eV), 4-hydroxydiclofenac (312 / ...

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Abstract

The invention relates to a detection method for simultaneously determining the metabolic products of seven CYP450 enzyme probe substrate in human liver microsomes, and belongs to the technical field of biological detection. The detection method comprises the following steps: performing incubation on specificity probe substrates of CYP450 enzyme and the human liver microsomes for different periods of reaction time to generate corresponding metabolic products, using Zaltoprofen as an interior label, adopting a high performance liquid chromatography-tandem mass spectrometry after protein precipitation pretreatment, and thus simultaneously detecting the concentrations of the metabolic products of seven probe substrates. The method disclosed by the invention is high in specificity, high in sensitivity and simple and convenient in operation, and has already successfully been applied to the research of baicalin on CYP450 enzyme inhibiting action of the human liver microsomes.

Description

technical field [0001] The invention relates to a method for temporally measuring metabolites of seven CYP450 enzyme probe substrates in human liver microsomes, belonging to the technical field of biological detection. Background technique [0002] In the biotransformation process of drugs, CYP450 enzymes participate in the metabolism of most endogenous and exogenous compounds in vivo, and are key enzymes in drug metabolism, whose activity determines the metabolic rate and clearance rate of drugs; while CYP450 enzymes can be A variety of drugs are inhibited or induced, resulting in metabolic drug interactions, affecting drug efficacy and producing adverse reactions. The "Guidelines for Drug Interaction Research" promulgated by the FDA in 2012 clearly pointed out that when conducting drug interaction research, it is necessary to first examine the effect of drugs on the activity of CYP450 enzymes. Cytochrome P450 (CYP450) is an important oxidase in the human body, including C...

Claims

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Application Information

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IPC IPC(8): G01N30/02
Inventor 黄凯洪远张娜蔡建美贺晴
Owner WUXI PEOPLES HOSPITAL
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