CN106841451A - The assay method of cumarin and its metabolin in mouse blood based on Ultra Performance Liquid Chromatography track trap high resolution mass spectrum - Google Patents
The assay method of cumarin and its metabolin in mouse blood based on Ultra Performance Liquid Chromatography track trap high resolution mass spectrum Download PDFInfo
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Abstract
The assay method of cumarin and its metabolin in a kind of mouse blood based on Ultra Performance Liquid Chromatography track trap high resolution mass spectrum, it is characterised in that：It is internal standard with 4 methyl umbelliferones, after sample is with acetonitrile precipitation albumen, is directly entered XDB C18Chromatogram post separation and spectrometer analysis, mass spectrum is using two grades of scanning (Full MS/ddMS of one-level full scan and data dependence2) pattern.Cumarin is mouse cell cytochrome p 450 2A5（Cytochrome P450 2A5, CYP2A5）Specific substrate, by LC-MS technology the generation of 7 Hydroxycoumarins in mouse blood is determined to assess the change of CYP2A5 enzymatic activitys.The method is selectively good, and analysis efficiency is high, and simple to operate, stabilization is quick, and sensitivity is high, reproducible, is suitable for the detection and analysis of cumarin and its metabolin in mouse blood.
Description
Technical field
It is specifically a kind of to be based on ultra high efficiency liquid phase the present invention relates to the assay method of cumarin in animal blood complex matrices
Chromatogram-track trap high resolution mass spectrum（UPLC-Orbitrap HRMS）Mouse blood in cumarin and its metabolin measure side
Method, the method uses inner mark method ration, by Ultra Performance Liquid Chromatography-track trap high resolution mass spectrum（UPLC-Orbitrap
HRMS）Cumarin in mouse blood and its metabolin are detected, is based on this that probe medicament is assessed carefully with cumarin
The change of Cytochrome C YP2A5 enzymatic activitys.
Background technology
Cytochrome P450（CytochromeP450, CYP）It is to be widely present in animals and plants, each tissue of microorganism
One class contains the b races cytochromes of ferriporphyrin prothetic group, the metabolism of various inside and outside source property materials is participated in, in pharmacology and toxicology
Aspect has highly important status.The activity of key metabolic enzymes is set to change by adding inhibitor or derivant, and then
The metabolite growing amount change of its specific substrate is determined, its activity can be more intuitively assessed.Cytochrome P450
2A6 (cytochrome P450 2A6, CYP2A6) is the important drug metabolic enzyme of human body, accounts for mankind's CYP450 enzyme systems
5%, the metabolism such as endoxan, sodium vedproate, caffeine and nicotine is participated in, can be by substantial amounts of nitrosamine procarcinogen in tobacco such as
The activation such as N-Nitrosodimethylamine, NNK, NNN is strong carcinogen.Mouse CYP2A5 is originated from identical generation with mankind CYP2A6
Thank to enzyme, its amino acid sequence and CYP2A6 have 84% homologous, and 26S Proteasome Structure and Function is very much like.Cumarin is the specificity of CYP2A5
Substrate, can be metabolized as umbelliferone discharge by CYP2A5.Cumarin is used as the probe substrate of CYP2A5, by determining it
Specific metabolic product umbelliferone content evaluates its enzymatic activity, is generally acknowledged CYP2A5 method for detecting enzymatic activities.
The detection method of the excessively various coumarin kind compounds of document report, such as traditional detection method sepectrophotofluorometer method
（Miles J S, McLAREN A W, Forrester L M, et al. Identification of the human
liver cytochrome P-450 responsible for coumarin 7-hydroxylase activity[J].
Biochemical Journal, 1990, 267(2): 365-371.）, the method is using cumarin in cumarin hydroxylase
I.e. under the catalysis of CYP2A5 enzymes, the change for being converted into photoluminescence peak after Hydroxycoumarin carrys out qualitative analysis, using fluorescence intensity level
Carry out quantitative analysis, but the method selectivity sensitivity is relatively low, less stable, it is impossible to provide the structural information of adduct；With
High performance liquid chromatography afterwards（HPLC）Method（Lake B G, Evans J G, Chapuis F, et al. Studies on
The disposition, metabolism and hepatotoxicity 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 in
patients with liver and kidney diseases and epileptic patients[J]. British
journal of clinical pharmacology, 1995, 39(1): 71-76.）And high performance liquid chromatography-fluorescence inspection
Survey method（HPLC-RF）（Wang Erhao, Guo Yanlei, Zhang Youjin, Shi Liang, Zhang Yuandong, Wang Yingxiong, Yang Zhu, determine in super HPLC-RF
Enzymatic activity [J] The Chinese Journal of Clinical Pharmacologies of CYP2A5,2013,29 (9) in Mouse Liver Microsomes: 692-624.）,
Although having large increase in terms of detection sensitivity after high performance liquid chromatography is introduced, determinand can not be equally given
Accurate structural information, selectivity also do not improved；Ultra Performance Liquid Chromatography-track trap high resolution mass spectrometry（UPLC-
Orbitrap HRMS）Have the advantages that test limit is low, the good, sensitivity of selectivity is high, stability is strong, it is in recent years to be applied to more
Pharmaceutical Analysis and complex biological matrix detection field（The Android tinkling of pieces of jade, Shi Chen, Zhao Rui wait to be based on Ultra Performance Liquid Chromatography-matter
Drug induced hepatic injury patients serum's metabolism group research [J] analytical chemistry of spectrum, 2015,43 (9): 1408-1414.）,
But do not studied also for cumarin in detection Mice Body and its metabolin aspect.
The content of the invention
The purpose of the present invention is based on above-mentioned prior art situation and uses what UPLC-Orbitrap HRMS methods were set up
A kind of simple, quick, sensitivity method for determining cumarin and its metabolin in mouse blood high.
The purpose of the present invention is achieved through the following technical solutions：It is a kind of based on the small of UPLC-Orbitrap HRMS
The assay method of cumarin and its metabolin in mouse blood, by the collection and pre-treatment of sample, collects blood sample in centrifugation
Guan Zhong is small using Ultra Performance Liquid Chromatography-high resolution mass spectrum analysis detection after adding inner mark solution, protein precipitation to mix centrifugation
Cumarin and its metabolin in mouse blood are m- dense when determining cumarin and its metabolin according to result of the test and drawing corresponding
Line of writing music carries out pharmacokinetic analysis.
The present invention includes step in detail below：
A, sample collection：Mouse takes the mL of blood about 0.5 by the eye corner of the eyes after intraperitoneal injection CYP2A5 probe medicament cumarin solution, puts
In 1.5 mL centrifuge tubes, immediately in 10 min are centrifuged under 4 DEG C, 6 000 rpm；
B, sample pre-treatments：Take in 100 μ L of supernatant serum to 1.5 mL centrifuge tubes, it is 100 ng/mL to add 20 μ L concentration
4-methyl umbelliferone solution and 280 μ L acetonitriles, be vortexed 30 s fully to mix, ultrasonic 10 min, in 4 DEG C, 12 000 rpm
10 min of lower centrifugation, take supernatant, are filtered with 0.22 μm of organic phase pin type filter, are fitted into be measured in chromatogram bottle；
The preparation of c, standard working solution：Using methyl alcohol dissolving, acetonitrile constant volume, cumarin, the 7- hydroxyls for preparing a series of concentration are fragrant
The hybrid standard working solution of legumin；
D, quantitative analysis：Use Ultra Performance Liquid Chromatography-track trap high resolution mass spectrum（UPLC-Orbitrap HRMS）To mixing
Standard working solution is analyzed, and obtains equation of linear regression, then testing sample is measured measures analyte and internal standard peak
The ratio of area, substitutes into unary linear regression equation, tries to achieve the content of object in sample；
Chromatographic condition： Agilent Zorbax Eclipse XDB–C18Chromatographic column（150 mm × 2.1 mm, 3.5 μm）, post
25 DEG C of temperature, sample size 2 μ L, the mL/min of flow velocity 0.2；Mobile phase A：0.1% aqueous formic acid, B:Acetonitrile（A:B=70:30）.
Mass Spectrometry Conditions：Use the electrical heating esi ion source of Q-Exactive track trap high resolution mass spectrum systems（HESI）,
The kV of spray voltage 3.5,320 DEG C of capillary heating temperature, 300 DEG C of heter temperature.Sheath gas（Sheath gas）Flow velocity 1.67
L/min, aids in gas（Aux gas）The L/min of flow velocity 1.Scanning is using Full MS/ddMS2Positive ion mode, one-level full scan model
Enclose 200 ± 100 amu, resolution ratio 7 × 104, the ion maximum ms of injection length 50, automatic growth control（AGC）1×106；Use
Data dependence is scanned（Data dependent）It is two grades of scan patterns, resolution ratio is 1.75 × 104, automatic growth control 1 ×
105, the ms of maximum injection length 50, normalization collision energy（NCE）60%.
Each target compound mass number and predominant secondary daughter ion are shown in Table one.
。
The range of linearity and detection limit of the inventive method：
Cumarin, umbelliferone, 4 methyl umbelliferones are dissolved using methyl alcohol respectively, and acetonitrile constant volume compound concentration is 100 μ g/
The standard reserving solution of mL, adds mouse blank serum, prepares a series of standard liquid of various concentrations, wherein umbelliferone
0.2,0.5,1,2,5,10,20,50,100,200,500,1 000 ng/mL, internal standard 4- methyl are followed successively by with the concentration of cumarin
Umbelliferone concentration is always 5 ng/mL.Using internal standard quanitation, with the ratio between object and interior target chromatographic peak area（Y）To phase
The target concentration answered（X）Carry out linear regression, you can obtain the working curve equation and coefficient correlation of each object.Repeat
The standard solution of sample least concentration 10 times, calculates its standard deviation, the detection with 3 times of standard deviation and 10 times values as method
Limit（LOD）And quantitative limit（LOQ）.As shown in table 2, cumarin and its metabolin are in 0.2 ng/mL ~ 1 000 ng/mL models for result
Enclose interior linear good, between 0.011 ~ 0.016 ng/mL, quantitative limit is between 0.036 ~ 0.052 ng/mL for method detection limit.
。
The rate of recovery and repeatability of the inventive method：
Blank serum is taken, object standard liquid is added, the quality-control sample of high, medium and low 3 kinds of concentration, cumarin and its generation is prepared
Thank to thing concentration for 1,50 and 500 ng/mL, internal standard concentration is 5 ng/mL, carries out pre-treatment using the above method and internal standard is legal
Amount analysis.In interior on the same day, the quality-control sample to high, medium and low 3 concentration carries out 6 repetition sample introductions analyses, quantitative determination respectively
Object content, and then the withinday precision and the rate of recovery of object under 3 concentration levels are calculated, as shown in table 3,3 kinds are dense
The quality-control sample rate of recovery of degree is 90.2% ~ 110.4%, precision（RSD）Between 1.86% ~ 7.08%, illustrate that the method is accurate
Degree is higher, and repeatability preferably, disclosure satisfy that the methodology requirement of trace target analyte detection in complex matrices.
。
The method of the present invention is the detection method of cumarin and its metabolin in a kind of new blood, excellent for blood sample
Changed pre-treating method, and coherent detection condition to Liquid Chromatography-Tandem Mass Spectrometry is optimized, compared with prior art this
Inventive method has following excellent results：
1. compared with traditional detection method fluorescence spectrophotometry, HPLC, HPLC-UV, HPLC-RF etc., UPLC-Orbitrap
HRMS methods analysis efficiency and sensitivity are higher.
2. this method has pre-treatment easy, operates quick and precisely, the good advantage of sensitivity high duplication.
Brief description of the drawings
In Fig. 1 standard liquids（1）In sample blood after 0.5 h of administration（2）Cumarin and its metabolin chromatogram
（A.7- Hydroxycoumarin B. 4-methyl umbelliferones C. cumarins）.
After Fig. 2 mouse peritoneal injection cumarins, control group（■）, 5-MOP groups（●）Cumarin and its metabolin in blood plasma
Concentration time curve.
Specific embodiment
The present invention is described further below in conjunction with example, but is not the limitation present invention.
Example 1：
1. instrument and material：
Ultimate Ultra Performance Liquid Chromatography instruments, Q-Exactive track trap high-resolution mass spectrometers（U.S. Thermo
Scientific）；Milli-Q Advantage A10 type ultra-pure water instrument（U.S. Millipore）；Agilent Zorbax
Eclipse XDB-C18（2.1×150 mm, 3.5 μm）.
Female mice（SPF grades of C57BL/6,7 ± 1 week old, the g of weight 17 ± 1, Changzhou Cavan limited public affairs of this experimental animal
Department provides）.Methyl alcohol, acetonitrile（Chromatographically pure, German Merck）；Formic acid（Chromatographically pure, German Serva）；Cumarin（>98%, Canada
TRC）；Umbelliferone, 4-methyl umbelliferone, 5-MOP（>99%, U.S. Aldrich）；Sodium chloride（Analysis
Pure, Buddhist nun's reagent is sent in Zhengzhou）.
2. sample treatment：Mouse passes through intraperitoneal injection CYP2A5 probe medicament cumarin solution（100 mg/kg, 1 mL/
100 g weights）After 0.5 h, the eye corner of the eyes takes the mL of blood 0.5 and injects 1.5 mL centrifuge tube centrifugal treatings（Condition：4℃、6000
rpm）10 min；The injection of its 100 μ L of supernatant serum is taken afterwards fills 20 μ L, the inner mark solution of 100 ng/mL and 280 μ L second
In 1.5 mL centrifuge tubes of nitrile, 30 s that are vortexed are well mixed, and ultrasonic 10 min；Most at centrifugation under 4 DEG C, 12 000 rpm
After managing 10 min, its supernatant, sample introduction analysis are filtered with 0.22 μm of organic phase pin type filter.
3. assay method：Each 5 μ L of blood sample after the standard liquid of absorption various concentrations and pre-treatment, injection
UPLC-Orbitrap HRMS systems carry out separation analysis, measure the content of cumarin and its metabolin in sample.
Example 2：As described in embodiment 1, the analysis method set up using this research detects control group and CYP2A5 enzymatic activitys
Suppress the content of cumarin and its metabolin in mouse blood.
1. C57BL/6 mouse 100 are taken, control group and 5-MOP is randomly divided into（5-MOP）Group is each 50,
Two groups are only randomly divided into 10 groups, and one same time point of every group of correspondence with every group 5 again respectively.During the daily morning 9,5-
MOP group gavage 5-MOP solution（It is dissolved in corn oil, 25 mg/kg, 1 mL/100 g weights）, then gavage is isometric for control group
Physiological saline.Successive administration three times, every minor tick 24 hours.Before third time administration 12 hours, mouse feed is removed.Experiment periods
Between mouse can free water.
After last time administration 1 hour, every mouse passes through intraperitoneal injection CYP2A5 probe medicament cumarin solution（100
Mg/kg, 1 mL/100 g weights）.First in different time（5、10、15、30、45、60、90、120、180、240 min）
Under, the eye corner of the eyes takes the mL of blood 0.5 and injects 1.5 mL centrifuge tube centrifugal treatings（Condition：4℃、6 000 rpm）10 min；Take it afterwards
The injection of 100 μ L of supernatant serum is filled in the 1.5 mL centrifuge tubes of 20 μ L, the inner mark solution of 100 ng/mL and 280 μ L acetonitriles,
30 s that are vortexed are well mixed, and ultrasonic 10 min；Most after after the min of centrifugal treating 10 under 4 DEG C, 12 000 rpm, 0.22 μ is used
M organic phase pin type filters filter its supernatant, sample introduction analysis.
2. data processing：Experimental data carries out statistical analysis using the softwares of SPSS 11.0, and by non-compartment model
Statistics principle of moment calculates pharmacokinetic parameter.
, as ordinate, with the sampling time as abscissa, control group and 5-MOP groups are small for concentration with cumarin and its metabolin
Cumarin and metabolite concentration versus time curve are as shown in Figure 2 in mouse blood.
As seen from Figure 2, the mean blood plasma concentration of each time point cumarin of 5-MOP groups is obviously higher than control group, and
The mean blood plasma concentration of umbelliferone is significantly lower than control group.To cumarin in mouse blood and metabolite concentration at any time
Between change data carry out non-compartment model analysis, its main pharmacokinetic parameter is shown in Table 4.Result shows that 5-MOP groups are most
Big blood concentrationC maxIt is 1.42 times of control group（P<0.01）, area under the drug-time curve AUC0-∞It is 1.59 times（P<0.05）, it is fragrant
Legumin half-life periodt 1/2It is 1.05 times（P<0.01）, because cumarin metabolism is related to the induction of CYP2A5 enzymatic activitys, it is indicated above 5-
MOP makes CYP2A5 enzymatic activitys in Mice Body substantially reduce, consistent with document（The person of outstanding talent's Herba Apii graveolentis extracts of king two are to CYP2A6 enzymatic activitys
Influence [D] Medical University Of Chongqing, 2014.）.
。
Claims (3)
1. in a kind of mouse blood based on Ultra Performance Liquid Chromatography-track trap high resolution mass spectrum cumarin and its metabolin survey
Determine method, it is characterised in that：By the collection and pre-treatment of sample, blood sample is collected in centrifuge tube, add inner mark solution,
After protein precipitation mixes centrifugation, analyzed using Ultra Performance Liquid Chromatography-track trap high resolution mass spectrum UPLC-Orbitrap HRMS
Cumarin and its metabolin in detection mouse blood, when determining cumarin and its metabolin according to result of the test and drawing corresponding
M- concentration curve carries out pharmacokinetic analysis, specifically includes some steps：
A, sample collection：Mouse takes the mL of blood about 0.5 by the eye corner of the eyes after intraperitoneal injection CYP2A5 probe medicament cumarin solution, puts
In 1.5 mL centrifuge tubes, immediately in 10 min are centrifuged under 4 DEG C, 6000 rpm；
B, sample pre-treatments：Take in 100 μ L of supernatant serum to 1.5 mL centrifuge tubes, it is 100 ng/mL to add 20 μ L concentration
Inner mark solution and 280 μ L acetonitriles, be vortexed 30 s fully to mix, ultrasonic 10 min, under 4 DEG C, 12 000 rpm be centrifuged
10 min, take supernatant, are filtered with 0.22 μm of organic phase pin type filter, are fitted into be measured in chromatogram bottle；
The preparation of c, standard working solution：Using methyl alcohol dissolving, acetonitrile constant volume, cumarin, the 7- hydroxyls for preparing a series of concentration are fragrant
The hybrid standard working solution of legumin；
D, quantitative analysis：Use Ultra Performance Liquid Chromatography-track trap high resolution mass spectrum（UPLC-Orbitrap HRMS）To mixing
Standard working solution is analyzed, and obtains equation of linear regression, then testing sample is measured measures analyte and internal standard peak
The ratio of area, substitutes into unary linear regression equation, tries to achieve the content of object in sample；
Chromatographic condition： Agilent Zorbax Eclipse XDB–C18Chromatographic column, specification 150 mm × 2.1 mm, 3.5 μm；
25 DEG C of column temperature, sample size 2 μ L, the mL/min of flow velocity 0.2；Mobile phase A：0.1% aqueous formic acid, B:Acetonitrile, A:B=70:30；
Mass Spectrometry Conditions：Use the electrical heating esi ion source of Q-Exactive track trap high resolution mass spectrum systems, spray voltage 3.5
KV, 320 DEG C of capillary heating temperature, 300 DEG C of heter temperature, the L/min of sheath gas 1.67,1 L/ of secondary air speed
min；Scanning is using Full MS/ddMS2Positive ion mode, the amu of one-level full scan scope 200 ± 100, resolution ratio 7 × 104, from
The sub- maximum ms of injection length 50, automatic growth control 1 × 106；Scanned using data dependence is two grades of scan patterns, resolution ratio
It is 1.75 × 104, automatic growth control 1 × 105, the ms of maximum injection length 50, normalization collision energy 60%.
2. assay method according to claim 1, it is characterised in that：4-methyl umbelliferone is designated as in described.
3. assay method according to claim 1, it is characterised in that：The collocation method tool of the hybrid standard working solution
Body is as follows：Cumarin, umbelliferone, 4-methyl umbelliferone are dissolved using methyl alcohol respectively, and acetonitrile constant volume compound concentration is 100
The standard reserving solution of μ g/mL, adds mouse blank serum, prepares a series of standard liquid of various concentrations, 7- hydroxyls therein
The concentration of cumarin and cumarin is followed successively by 0.2,0.5,1,2,5,10,20,50,100,200,500,1 000 ng/mL, internal standard
The concentration of 4-methyl umbelliferone is always 5 ng/mL.
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| CN115267012A (en) * | 2022-09-13 | 2022-11-01 | 贵州医科大学 | Qualitative and quantitative analysis method for hematoxylin B-3-O-beta-D-glucoside and metabolite thereof in plasma |
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