Method for determining coumarin and metabolite of coumarin in mouse blood on basis of UPLC (ultra performance liquid chromatography)-orbitrap HRMS (high-resolution mass spectrum)

A technology of high-resolution mass spectrometry and ultra-high performance liquid chromatography, applied in the field of determination of coumarin and its metabolites in mouse blood based on ultra-high performance liquid chromatography-Orbitrap high-resolution mass spectrometry, can solve the problem that cannot give adduct The problem of structure information, poor stability, and low sensitivity of specificity can be solved, and the effect of high sensitivity, good repeatability, and simple pretreatment can be achieved.

Inactive Publication Date: 2017-06-13
ZHENGZHOU TOBACCO RES INST OF CNTC
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Problems solved by technology

[0003] A variety of detection methods for coumarin compounds have been reported in the literature, such as the traditional detection method fluorescence spectrophotometer method (Miles J S, McLAREN A W, Forrester L M, et al. Identification of the humanliver cytochrome P-450 responsible for coumarin 7-hydroxylase activity[J].Biochemical Journal, 1990, 267(2): 365-371.), this method uses coumarin under the catalysis of coumarin hydroxylase, namely CYP2A5 enzyme, to convert into hydroxycoumarin after the fluorescence peak Qualitative analysis using the change of fluorescence intensity, but the specificity of this method is low, the stability is poor, and the structural information of the adduct cannot be given; the subsequent high-performance liquid chromatography (HPLC) method ( Lake B G, Evans J G, Chapuis F, et al.Studies on the disposition, metabolism and toxicity of coumarin in the rat and Syrian hamster[J]. Food Chem Toxicol, 2002, 40(6): 809-823), high performance liquid chromatography - Ultraviolet detector (HPLC-UV) (Sotaniemi E A, Rautio A, Backstrom M, et al. CYP3A4 and CYP2A6 activities marked by the metabolism of lignocaine and coumarin patients with liver and kidney diseases and epileptic patients[J]. Britishjournal of clinical pharmacology , 1995, 39(1): 71-76.) and high performance liquid chromatography-fluorescence detection (HPLC-RF) (Wang Erhao, Guo Yanlei, Zhang Youjin, Shi Liang, Zhang Yuandong, Wang Yingxiong, Yang Zhu, Yu Chao. HPLC-RF Determination of CYP2A5 enzyme activity in mouse liver microsomes[J]. Chinese Journal of Clinical Pharmacology, 2013, 29(9): 692-624.), although the detection sensitivity has been greatly improved after the introduction of high performance liquid chromatography improved, but also cannot give accurate structural information of the analyte, and the specificity has not been improved; ultra-high performance liquid chromatography-orbitrap high-resolution mass spectrometry (UPL C-Orbitrap HRMS) has the advantages of low detection limit, good selectivity, high sensitivity, and strong stability. Ultra-high performance liquid chromatography-mass spectrometry study of serum metabolomics in patients with drug-induced liver injury [J]. Analytical Chemistry, 2015, 43(9): 1408-1414.), but for the detection of coumarin and its metabolism in mice has not been studied

Method used

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  • Method for determining coumarin and metabolite of coumarin in mouse blood on basis of UPLC (ultra performance liquid chromatography)-orbitrap HRMS (high-resolution mass spectrum)
  • Method for determining coumarin and metabolite of coumarin in mouse blood on basis of UPLC (ultra performance liquid chromatography)-orbitrap HRMS (high-resolution mass spectrum)

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Experimental program
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example 1

[0028] 1. Instruments and materials:

[0029] Ultimate ultra-high performance liquid chromatography, Q-Exactive orbitrap high-resolution mass spectrometer (ThermoScientific, USA); Milli-Q Advantage A10 ultrapure water instrument (Millipore, USA); Agilent ZorbaxEclipse XDB-C 18 (2.1×150 mm, 3.5 μm).

[0030] Female mice (SPF grade C57BL / 6, 7±1 weeks old, body weight 17±1 g, provided by Changzhou Cavens Experimental Animal Co., Ltd.). Methanol, acetonitrile (chromatographically pure, Merck, Germany); formic acid (chromatographically pure, Serva, Germany); coumarin (>98%, TRC Canada); 7-hydroxycoumarin, 4-methylumbelliferone, 5-methanone Oxypsoralen (>99%, Aldrich, USA); Sodium Chloride (analytical grade, Zhengzhou Piney Reagent).

[0031] 2. Sample processing: 0.5 h after intraperitoneal injection of CYP2A5 probe drug coumarin solution (100 mg / kg, 1 mL / 100 g body weight) in mice, 0.5 mL of blood was collected from the canthus of the eye and injected into a 1.5 mL centrifuge ...

example 2

[0033] Example 2: As described in Example 1, the analytical method established in this study was used to detect the content of coumarin and its metabolites in the blood of the control group and CYP2A5 enzyme activity-inhibited mice.

[0034] 1. 100 C57BL / 6 mice were randomly divided into the control group and the 5-methoxypsoralen (5-MOP) group, 50 each, and the two groups were randomly divided into 10 groups with 5 mice in each group. And each group corresponds to a same time point. At 9 am every day, the 5-MOP group was given 5-MOP solution (dissolved in corn oil, 25 mg / kg, 1 mL / 100 g body weight) by intragastric administration, while the control group was given an equal volume of normal saline. Administered three times in a row with an interval of 24 hours each time. Twelve hours before the third dose, the mouse chow was removed. Mice had free access to water during the experiment.

[0035] One hour after the last administration, each mouse was injected intraperitoneall...

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Abstract

The invention provides a method for determining coumarin and metabolite of coumarin in mouse blood on basis of a UPLC (ultra performance liquid chromatography)-orbitrap HRMS (high-resolution mass spectrum). The method is characterized in that with 4-methylumbelliferone as an internal standard, a sample is directly subjected to XDB-C18 chromatographic column separation and mass-spectrographic analysis after protein precipitation performed through acetonitrile, and a Full Ms / ddMS<2> mode including first-level full scanning and second-level data dependent scanning is adopted during mass spectrographic analysis. Coumarin is a specific substrate of a cytochrome P450 2A5 of a mouse, and changes of CYP2A5 enzymatic activity are evaluated by determining generation of 7-hydroxycoumarin in the mouse blood with a liquid chromatography-mass spectrometry technology. The method has good selectivity, high analysis efficiency, is simple to operate, stable, quick, high in sensitivity, good in repetition and suitable for detection and analysis of coumarin and the metabolite of coumarin in the mouse blood.

Description

technical field [0001] The present invention relates to a method for the determination of coumarin in complex matrices of animal blood, specifically a method for the determination of coumarin and its metabolites in mouse blood based on ultra-high performance liquid chromatography-orbitrap high-resolution mass spectrometry (UPLC-Orbitrap HRMS) Determination method, the method adopts the internal standard method to quantify, and detects coumarin and its metabolites in mouse blood by ultra-high performance liquid chromatography-orbitrap high-resolution mass spectrometry (UPLC-Orbitrap HRMS), based on which the coumarin and its metabolites are used Chin as a probe drug to assess changes in cytochrome CYP2A5 enzyme activity. Background technique [0002] Cytochrome P450 (cytochrome P450, CYP) is a type of b-group cytochrome containing iron porphyrin prosthetic group widely present in various tissues of animals, plants and microorganisms, and participates in the metabolism of vari...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06
Inventor 张建勋李琛毛健王丁众赵无垛王荣浩张启东刘珊孙世豪宗永立
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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