CN106290661B - A kind of method that high performance liquid chromatography tandem mass spectrometry detects CYP2C9 enzyme activities in earthworm body - Google Patents
A kind of method that high performance liquid chromatography tandem mass spectrometry detects CYP2C9 enzyme activities in earthworm body Download PDFInfo
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Abstract
The present invention relates to the methods of CYP2C9 enzyme activities in a kind of high performance liquid chromatography tandem mass spectrometry detection earthworm body, belong to technical field of enzyme activity detection, this method specifically comprises the following steps：(1) earthworm microsomal protein suspension is prepared；(2) earthworm microsomal protein suspension obtained in step (1) is added in the incubation system containing probe substrate, heating starts enzymatic reaction, after enzymatic reaction termination, using generated metabolite after the detection of high performance liquid chromatography tandem mass spectrum combined instrument enzymatically reaction terminating, CYP2C9 enzyme activities are calculated.Detection method accuracy, accuracy and the high sensitivity, stability is strong, can be used for the analysis detection of CYP2C9 vigor in earthworm body under different pollution environment, and then indicates that the possibility of soil pollution provides detection method as biomarker to explore CYP2C9.
Description
Technical field
The invention belongs to technical field of enzyme activity detection, and in particular to a kind of high performance liquid chromatography tandem mass spectrometry detection earthworm
The method of CYP2C9 enzyme activities in vermis.
Background technology
Cytochrome P450 (CYP) is used as a huge same work family enzyme, is a kind of important removing toxic substances containing ferroheme
Enzyme.Iron ion in the ferroheme that family's enzyme is combined by non-covalent bond transmits electronics and aoxidizes heterologous object, enhances xenobiotic
Water solubility, make them be easier to excrete, to play detoxication.Currently, the Asias CYP enzyme is as ecotoxicology index
More research is obtained in terrestrial vertebrate organism and aquatile, it is less compared to the research in soil invertebrate biology, such as
Earthworm, but it is more and more extensive using the research of the Asias earthworm CYP enzymatic diagnosis Soil Environmental Pollution.CYP2C9 is an important CYP
Sub- enzyme, can CYP2C9 be used as marker diagnosis Soil Environmental Pollution need further to be studied in earthworm body, stablize, reliable inspection
Survey method becomes the key of this research.The report of the Asias CYP enzyme activity involved in research at present, the assay method of use is mostly probe
Substrate method uses a certain compound for substrate, which can be metabolized by the Asias CYP enzyme, utilize fluophotometer or ultraviolet spectrometry
Photometer detects the production quantity of metabolite, and the vigor of the Asias CYP enzyme is reflected by the production quantity of metabolite.But above-mentioned detection
Means poor sensitivity can not also efficiently separate probe substrate and metabolite.In addition, CYP enzymes itself are a kind of hemachrome enzymes, blood
Red pigment there are the measurement of energy severe jamming fluophotometer or ultraviolet specrophotometer.The Asias CYP enzyme in earthworm body in addition
Content is less relative to mammal, in existing document to have been reported that this result of detection failure more.
Therefore, more low-level can be carried out from complex biological matrix with higher specificity within a short period of time by being badly in need of one kind
Quantitative detection earthworm body in CYP2C9 vigor method.
Invention content
In view of this, the purpose of the present invention is to provide in a kind of high performance liquid chromatography tandem mass spectrometry detection earthworm body
The method of CYP2C9 enzyme activities.
In order to achieve the above objectives, the present invention provides the following technical solutions：
1, a kind of method that high performance liquid chromatography tandem mass spectrometry detects CYP2C9 enzyme activities in earthworm body, including walk as follows
Suddenly：
(1) earthworm microsomal protein suspension is prepared；
(2) earthworm microsomal protein suspension obtained in step (1) is added in the incubation system containing probe substrate,
Heating starts enzymatic reaction, after enzymatic reaction termination, is detected using high performance liquid chromatography tandem mass spectrum combined instrument enzymatically anti-
Generated metabolite after should terminating calculates CYP2C9 enzyme activities.
Further, in step (2), high-efficient liquid phase chromatogram condition is：
A) chromatographic column：MS Luna C18 columns, 2.1mm × 50mm, 3.0 μm, and it is equipped with corresponding C18 guard columns；
B) mobile phase is by A phases and B phase compositions, and the A phases are water or one kind containing the water that volume fraction is 0.1% formic acid,
The B phases are acetonitrile or one kind containing the acetonitrile that volume fraction is 0.1% formic acid；
C) condition of gradient elution：The ratio of 0-4min, Mobile phase B rise to 95% by 5%；4-8min, the ratio of Mobile phase B
Example drops to 5% by 95%；The ratio of 8-10min, Mobile phase B are 5%；
D) column temperature：50℃；
E) flow velocity：0.2ml/min；
F) automatic sampling room temperature：4 DEG C, sampling volume is 5 μ l.
Further, in step (2), Mass Spectrometry Conditions are：
A) ion source：Electron spray ionisation；
B) detection mode：Cation；
C) electron spray voltage：3kV；
D) ion source temperature：350℃；
E) collision gas and air pressure：Argon gas, 1.2mTorr；
F) scan pattern：More reactive ion monitorings；
G) nitrogen sheath, ion scan, auxiliary gas：It is respectively set to 40,0,10 units.
Further, in step (2), the probe substrate is C14H10Cl2NNaO2；The metabolite is that the double chlorine of 4 '-hydroxyls are fragrant
Acid.
Further, in step (2), the earthworm microsomal protein suspension is added in the incubation system containing probe substrate
To a concentration of 10-100 μ g/ml of the microsomal protein.
Further, in step (2), the earthworm microsomal protein suspension is added in the incubation system containing probe substrate
Afterwards, a concentration of 100 μM of the probe substrate.
Further, in step (2), the incubation system includes following component：Buffer solution and NADPH generation systems；It is described
Buffer solution is pH=7.5, the Tris buffer solutions of a concentration of 0.1M；The NADPH generation systems are 5 by volume by A and B:1 group
At the A is G-6-P 20mg/ml, NADP 20mg/ml, magnesium chloride 13.3mg/ml；The B is by glucose-
6- phosphate dehydrogenases are dissolved in the final concentration of 40U/ml to glucose-6-phosphate dehydrogenase (G6PD) in 5mM sodium citrate solutions.
Further, in step (2), the heating is that temperature is risen to 37 ± 0.2 DEG C；The time of enzymatic reacting is 5-
60min；The enzymatic reaction is terminated to be realized by the way that 200 μ l methanol are added.
Further, the time of enzymatic reacting is 15min.
Further, described to detect enzymatically reaction terminating using high performance liquid chromatography tandem mass spectrum combined instrument in step (2)
Further include before generated metabolite afterwards mixtures incubated after terminating enzymatic reaction in the case where centrifugal force is 15000 × g from
Heart 10min takes supernatant for detecting.
The beneficial effects of the present invention are：The present invention has been successfully set up CYP2C9 in LC-MS/MS analysis detection earthworm bodies
The method of enzyme activity, detection method accuracy, accuracy and the high sensitivity, stability is strong, can be used under different pollution environment
The analysis detection of CYP2C9 vigor in earthworm body, and then to explore possibility of the CYPC9 as biomarker instruction soil pollution
Property provides detection method.
Description of the drawings
In order to keep the purpose of the present invention, technical solution and advantageous effect clearer, the present invention provides following attached drawing and carries out
Explanation：
Fig. 1 is that C14H10Cl2NNaO2 generates 4 '-hydroxyl Diclofenac figures after CYP2C9 enzymatics；
Fig. 2 is that C14H10Cl2NNaO2 reacts more with the LC-PDA of the mixed solution of 4 '-hydroxyl Diclofenacs figures with LC-MS/MS
Monitoring figure；
Fig. 3 be add C14H10Cl2NNaO2,4 '-hydroxyl Diclofenacs and incubation system mixed solution LC-PDA figures with
LC-MS/MS multiple-reaction monitoring figures；
Fig. 4 is that addition C14H10Cl2NNaO2,4 '-hydroxyl Diclofenacs, incubation system and heat inactivation earthworm microsomal protein are molten
LC-PDA figures and the LC-MS/MS multiple-reaction monitoring figures of liquid；
Fig. 5 is that CYP2C9 enzyme activity is tried hard in LC-MS/MS detection earthworm bodies；
Fig. 6 is the dependency graph of 4 '-hydroxyl Diclofenac production quantities and microsomal protein concentration；
Fig. 7 is the dependency graph of 4 '-hydroxyl Diclofenac production quantities and incubation time；
Fig. 8 is to be exposed to benzo [a] pyrene to contaminate soil after 14 days, CYP2C9 enzyme activities detection figure in earthworm body.
Specific implementation mode
Below in conjunction with attached drawing, the preferred embodiment of the present invention is described in detail.
Material and reagent：Trifluoroacetic acid aqueous solution, methanol are purchased from Merck companies, chromatographically pure C14H10Cl2NNaO2, formic acid and Tris
It is purchased from Chinese Shanghai Sigma-Aldrich companies, NADPH reaction systems in metabolite 4 '-hydroxyl Diclofenac, incubation reaction
System is purchased from BD genetest, and ultra-pure water is filtered by German Sartorius Arium 611DI ultrapure water systems, C18 mass spectrum colors
It composes column and comes from Phenomenex companies of the U.S., Eisenia Foetida is purchased from Agricultural University Of Shenyang, and under (20 ± 2) DEG C dark condition
It is stored in cleaning soil.
Method And Principle：It can quilt under earthworm CYP2C9 enzyme existence conditions as probe substrate using C14H10Cl2NNaO2 (DF)
4 '-hydroxyl Diclofenacs (4 ' H-DF) (such as Fig. 1) are catalytically conveted to, high performance liquid chromatography tandem mass spectrum combined instrument (LC- is passed through
MS/MS the production quantity of 4 '-hydroxyl Diclofenacs) is measured to reflect CYP2C9 enzyme activities.
Embodiment 1
Phase constituent is flowed to determine
Investigate influence of three groups of flowing phase compositions to detection sensitivity：The first is that A is pure water in mobile phase, and B is pure second
Nitrile；Second is that A is water (formic acid for being 0.1% containing volume fraction), and B is pure acetonitrile；The third is that A is that water (contains volume fraction
Formic acid for 0.1%), B is acetonitrile (formic acid for being 0.1% containing volume fraction), the results showed that finally a kind of mobile phase effectively changes
The stability of chromatographic peak profile and peak area has been apt to it, accordingly, it is determined that using A as water (formic acid for being 0.1% containing volume fraction), B is second
Nitrile (formic acid for being 0.1% containing volume fraction) is used as optimal flow phase.
Embodiment 2
LC-MS/MS condition settings
LC-MS/MS is the triple quadrupole rods tandem mass spectrometries-of Thermo Fisher Scientific TSQ Quantum Max
HPLC system, wherein high-efficient liquid phase chromatogram condition is：
A) chromatographic column：MS Luna C18 columns, 2.1mm × 50mm, 3.0 μm, and it is equipped with corresponding C18 guard columns；B) it flows
Mutually by A phases and B phase compositions, the A phases are containing the water that volume fraction is 0.1% formic acid, and the B phases are to be containing volume fraction
The acetonitrile of 0.1% formic acid；C) condition of gradient elution：The ratio of 0-4min, Mobile phase B rise to 95% by 5%；4-8min, stream
The ratio of dynamic phase B drops to 5% by 95%；The ratio of 8-10min, Mobile phase B are 5%；D) column temperature：50℃；E) flow velocity：
0.2ml/min；F) automatic sampling room temperature：4 DEG C, sampling volume is 5 μ l.
Mass Spectrometry Conditions are：
A) ion source：Electron spray ionisation；B) detection mode：Cation；C) electron spray voltage：3kV；D) ion source temperature：
350℃；E) collision gas and air pressure：Argon gas, 1.2mTorr；F) scan pattern：More reactive ion monitorings；G) nitrogen sheath, ion are swept
It retouches, assist gas：It is respectively set to 40,0,10 units.
Since substrate Diclofenac na concn is higher, quantitative two pole of UV detector still used of C14H10Cl2NNaO2
Pipe array (PDA) is quantitative under 280nm wavelength, and MS/MS is used for detecting metabolite 4 '-hydroxyl Diclofenac.
Each compound chooses 1 pair of parent ion/daughter ion to combining retention time qualitative, and each parameter is listed in table 1, at data
Reason uses the X-Calibur softwares of LC-MS/MS autogamys.
The MS/MS parameters of 1 C14H10Cl2NNaO2 of table (DF) and 4 '-hydroxyl Diclofenacs (4 ' H-DF)
Embodiment 3
Matrix effect is tested
(1) storing solution is configured：By methanol and water by volume 1:1 is mixed to prepare dilution, and DF is dissolved in the dilution
To the final concentration of 1.0mg/ml of DF, DF storing solutions are made；4 ' H-DF are dissolved in methanol to the final concentration of 1.0mg/ of 4 ' H-DF
4 ' H-DF storing solutions are made in ml；
(2) configuration work liquid：4 ' H-DF storing solutions in step (1) are diluted to 10ng/ml, 4 ' H-DF working solutions are made；
DF storing solutions are diluted to 5 μ g/ml, and DF working solutions are made.
(3) by 4 ' H-DF working solutions in step (2) and DF working solutions by volume 1:1 mixing, carries out LC-MS/MS measurement,
Its chromaticness spectrogram is as shown in Figure 2.
(4) it takes 4 ' H-DF working solutions of 1ml in 2ml test tubes, is dried up under nitrogen evaporator, add 250 μ l DF working solutions, it
After add 650 μ l incubation systems after be warming up to 37 DEG C, wherein incubation system includes：590 μ l buffer solutions be (pH=7.5,0.1M's
Tris), (the NADPH generation systems are 5 by volume by A and B to 60 μ l NADPH generation systems:1 composition, the A are grape
Sugar -6- phosphoric acid 20mg/ml, NADP 20mg/ml, magnesium chloride 13.3mg/ml；The B is that glucose-6-phosphate dehydrogenase (G6PD) is molten
To the final concentration of 40U/ml of glucose-6-phosphate dehydrogenase (G6PD) in 5mM sodium citrate solutions), 200 μ l methanol are finally added,
10min is centrifuged in the case where centrifugal force is 15000 × g, 200 μ l supernatants is taken, is detected with LC-MS/MS, chromaticness spectrogram is as schemed
Shown in 3.
(5) it takes 4 ' H-DF working solutions of 1ml in 2ml test tubes, is dried up under nitrogen evaporator, add 250 μ l DF working solutions, it
After add 100 μ l inactivation enzyme solutions (earthworm microsomal protein suspension obtained in embodiment 6 is subjected to inactivation processing), 650 μ l
37 DEG C are warming up to after incubation system, wherein incubation system includes：590 μ l buffer solutions (Tris of pH=7.5,0.1M), 60 μ l
(the NADPH generation systems are 5 by volume by A and B to NADPH generation systems:1 composition, the A are G-6-P
20mg/ml, NADP 20mg/ml, magnesium chloride 13.3mg/ml；The B is that glucose-6-phosphate dehydrogenase (G6PD) is dissolved in 5mM lemons
To the final concentration of 40U/ml of glucose-6-phosphate dehydrogenase (G6PD) in acid sodium solution), 200 μ l methanol are finally added, are in centrifugal force
10min is centrifuged under 15000 × g, 200 μ l supernatants is taken, is detected with LC-MS/MS, chromaticness spectrogram is as shown in Figure 4.
Include three subgraphs in Fig. 2, Fig. 3, Fig. 4, is followed successively by from top to bottom：How anti-LC-PDA chromatograms, LC-MS/MS be
Answer the chromatogram extracted under monitoring pattern：4 '-hydroxyl Diclofenacs (centre), C14H10Cl2NNaO2 (most lower).As shown in Figure 2, PDA
It is only capable of detecting C14H10Cl2NNaO2.Compared to PDA, MS/MS is more sensitive, using retention time (4.98min) and combine parent ion/
Daughter ion being capable of qualitative 4 '-hydroxyl Diclofenac to (312/230).Therefore, the conclusion of document report before this is demonstrated：Ultraviolet inspection
Metabolite can not be detected by surveying device.In addition, according to parent ion/daughter ion pair, infer that (m/z is 4 '-hydroxyl Diclofenac daughter ions
230) be caused by parent ion (m/z 312) loses hydrochloric acid and formic acid, C14H10Cl2NNaO2 daughter ion (m/z 250) be due to
Parent ion (m/z 296) is lost caused by formic acid.The multiple-reaction monitoring pattern extraction figure of the 4 '-hydroxyl Diclofenacs of Fig. 3 and Fig. 4
In, there is by-product peak in retention time about 5.7min, it is shown that matrix effect tentatively judges the by-product for Diclofenac
The conjugate that sodium is formed with G-6-P.
Embodiment 4
The foundation of standard curve：
(1) 4 ' H-DF storing solutions in step (1) in embodiment 3 are diluted to 10ng/ml, 50ng/ml, 250ng/ respectively
64 ' H-DF working solutions are made in ml, 1000ng/ml, 2500ng/ml, 5000ng/ml；DF storing solutions are diluted to 5 μ g/ respectively
6 DF working solutions are made in ml, 10 μ g/ml, 20 μ g/ml, 40 μ g/ml, 80 μ g/ml, 150 μ g/ml.It takes respectively from low to high each
4 ' H-DF working solution 1ml under concentration, are placed in 2ml centrifuge tubes, are dried up under nitrogen evaporator, then corresponding respectively that 250 μ l are added
The DF working solutions of a concentration of 5 μ g/ml, 10 μ g/ml, 20 μ g/ml, 40 μ g/ml, 80 μ g/ml, 150 μ g/ml, obtained 6 to be measured
Liquid.
(2) 650 μ l incubation systems and 100 μ l have been added in 6 centrifuge tubes equipped with prepare liquid into step (1) respectively
37 DEG C are warming up to after the enzyme solutions (earthworm microsomal protein suspension obtained in embodiment 6 is carried out inactivation processing) of inactivation,
Wherein, incubation system includes：590 μ l buffer solutions (Tris of pH=7.5,0.1M), 60 μ l NADPH generation systems are (described
NADPH generation systems are 5 by volume by A and B:1 composition, the A are G-6-P 20mg/ml, NADP 20mg/
Ml, magnesium chloride 13.3mg/ml；The B is that glucose-6-phosphate dehydrogenase (G6PD) is dissolved in 5mM sodium citrate solutions to glucose-
The final concentration of 40U/ml of 6- phosphate dehydrogenases).200 μ l methanol are added into centrifuge tube again, in the case where centrifugal force is 15000 × g
10min is centrifuged, 200 μ l supernatants is taken, is detected with LC-MS/MS, standard curve is made, the data obtained is shown in Table 2.
2 standard curve tables of data of table
As shown in Table 2, the linear relationship of untested compound is good, regression coefficient r2It is all higher than quantifying for 0.99,4 ' H-DF
It is limited to 10ng/ml, quantifying for DF is limited to 5 μ g/ml.
Embodiment 5
LC-MS/MS methodology validations
1, sample preparation
(1) 4 ' H-DF storing solutions in step (1) in embodiment 3 are diluted to 30ng/ml, 500ng/ml, 4000ng/ respectively
34 ' H-DF working solutions are made in ml；DF storing solutions are diluted to 9 μ g/ml, 60 μ g/ml, 120 μ g/ml respectively, and 3 DF work are made
Liquid.It takes 4 ' H-DF working solution 1ml under each concentration respectively from low to high, is placed in 2ml centrifuge tubes, is dried up under nitrogen evaporator, so
The corresponding DF working solutions that a concentration of 9 μ g/ml of 250 μ l, 60 μ g/ml, 120 μ g/ml are added, obtained 3 prepare liquids are pure respectively afterwards
Sample.
(2) according to step (2) the method in embodiment 4,3 matrix samples are made.
2, veracity and precision
3 matrix samples are detected respectively with LC-MS/MS, wherein for each matrix sample, be arranged 5 and put down
Row sample, each sample replication 4 times, repeatedly this process, test result are shown in Table 3 within continuous 4 days.
The veracity and precision of 3 matrix sample of table
As shown in Table 3, all matrix sample accuracy are all higher than 85%, withinday precision ranging from 2.79-10.37%,
Day to day precision ranging from 2.21-10.25%, meet U.S. Food and Drug Administration (FDA) about in method validation to essence
The requirement of density and accuracy.
3, extraction recovery
Pure sample and matrix sample are detected respectively with LC-MS/MS, existed by comparing pure sample and matrix sample
Response on LC-MS/MS, determines the rate of recovery, the results are shown in Table 4.
The rate of recovery ((%, ± SD, n=4)) of 4 target compound of table
As shown in Table 4, in matrix sample, the rate of recovery of 4 ' H-DF is 80-87%, and the rate of recovery of DF is about 80%, is shown
Matrix effect influences quantified results little.
4, stability
By matrix sample respectively at room temperature place 10h, 4 DEG C storage for 24 hours, -30 DEG C storage 30 days after, then use LC-
MS/MS is respectively detected the matrix sample under three kinds of conditions of storage, the results are shown in Table 5.
The stability (rate of recovery, %, ± SD, n=4) of matrix sample, is as a result expressed as adding under the different conditions of storage of table 5
Add the percentage of concentration (respectively under three Quality Control levels of high, medium and low concentration)
As shown in Table 5, matrix sample has good stability.
Embodiment 6
Prepare earthworm microsomal protein suspension
After the earthworm taking-up 20% at 4 DEG C in (V/V) glycerite will be stored in, internal organ are dissected rapidly, use 0.15mol/L
KCl solution cleans internal organ, is subsequently transferred to that 4ml homogenate buffers (sucrose 250mmol/L, Tris 50mmol/L, DTT are housed
1mmol/L, EDTA 1mmol/L, pH=7.5) glass tissue mill in so that it is uniformly crushed, and will finally be homogenized volume
It is set to 6ml.
Microsome is extracted using differential centrifugation：Using low temperature ultracentrifuge, by homogenate at 4 DEG C, centrifugal force is
After centrifuging 15min under the conditions of 15000 × g, retain supernatant, then by supernatant at 4 DEG C, centrifugal force be 15000 × g under the conditions of from
Gained precipitation 3ml is preserved buffer solution (sucrose 50mmol/L, Tris 50mmol/L, DTT 1mmol/L, EDTA by heart 90min
1mmol/L, pH=7.5,20% (V/V) glycerine) it suspends again, earthworm microsome is made and hangs albumen supernatant liquid, is stored at -80 DEG C
It is spare.
Embodiment 7
LC-MS/MS detects CYP2C9 enzyme activities in earthworm body
(1) it takes the centrifuge tube of 2ml, 650 μ l incubation systems and 250 μ l DF solution is added, wherein incubation system includes：
590 μ l buffer solutions (Tris of pH=7.5,0.1M), 60 μ l NADPH generation systems (by A and B pressed by the NADPH generation systems
Volume ratio is 5:1 composition, the A are G-6-P 20mg/ml, NADP 20mg/ml, magnesium chloride 13.3mg/ml；It is described
B be glucose-6-phosphate dehydrogenase (G6PD) is dissolved in 5mM sodium citrate solutions it is final concentration of to glucose-6-phosphate dehydrogenase (G6PD)
40U/ml).At this point, a concentration of 100 μM in mixed solution of DF.Then, by 100 μ l earthworm microsomal protein suspension add from
A concentration of 10 μ g/ml of heart Guan Zhongzhi microsomal proteins, are warming up to 37 DEG C, start enzymatic reaction, reaction time 15min, then to
200 μ l methanol are added in centrifuge tube, centrifuges 10min in the case where centrifugal force is 15000 × g, 200 μ l supernatants is taken, with LC-MS/MS
It is detected, testing result is as shown in Figure 5.It is compared with Fig. 4, Fig. 5 remains to find out matrix effect, the difference is that living since enzyme has
Property, C14H10Cl2NNaO2 can be catalyzed and be converted into 4 '-hydroxyl Diclofenacs, matrix effect weakens.The calculation formula of CYP2C9 enzyme activities
It is as follows：
X=C4′H-DF/(M4′H-DF×Cprotein)
X-CYP2C9 enzyme activities, unit nmol/mgprotein；
C4′H-DF4 ' in reaction system-and the concentration of hydroxyl Diclofenac, unit ng/ml；
M4′H-DFThe molal weight of -4 '-hydroxyl Diclofenac, unit g/mol；
CproteinAlbumen concentration in reaction system, unit mg/ml.
(2) according to the method in (1), change earthworm microsomal protein suspension and microsomal protein after incubation system is added
Concentration is tested namely to 20 μ g/ml, 50 μ g/ml, 100 μ g/ml, 200 μ g/ml, calculates CYP2C9 enzyme activities.Knot
The experimental data in (1) is closed, the correlation of metabolite production quantity and microsomal protein additive amount is obtained, as a result sees Fig. 6, by scheming
6 it is found that the white concentration of microsome is within the scope of 10-100 μ g/ml in incubation system, metabolite production quantity (y) and microsomal protein
Preferable linear relationship, regression equation y=8.273+0.436x, regression coefficient R is presented in concentration (x)2=0.996.
(3) according to the method in (1), change earthworm microsomal protein suspension and microsomal protein after incubation system is added
Then concentration changes time of enzymatic reacting namely to 32 μ g/ml, 64 μ g/ml, make the time be respectively 5min, 15min,
40min, 60min, test, through the sample obtained by the differential responses time, obtain metabolite production quantity and are incubated under various concentration
As a result the correlation of time is shown in Fig. 7, as shown in Figure 7, incubation time is longer, and the production quantity of metabolite is more, but incubation time
More than 15min, the production quantity amplification very little of metabolite (<10%, compare product formation when incubation time 15min).Cause
This, in enzymatic reaction, incubation time 15min is most appropriate.
Embodiment 8
It is exposed to the measurement of earthworm CYP2C9 vigor in low concentration benzo-a-pyrene pyrene contaminated soil
The earthworm sample being exposed in the soil of benzo [a] pyrene contamination after 14 days is taken, is prepared according to the method in embodiment 6
Earthworm microsomal protein suspension.Wherein, the concentration of contamination gradient of benzo [a] pyrene is in soil：0.12、0.24、0.48、
0.96mg/kg, each exposure concentrations setting 6 is parallel, is tested, is obtained under each concentration of contamination, earthworm with LC-MS/MS
The response of CYP2C9 enzyme activities changes, as shown in Figure 8.As shown in Figure 8, low concentration benzo-a-pyrene pyrene (0.12,0.24mg/kg) is aobvious
It writes induction of earthworm CYP2C9 enzyme activities, causes CYP2C9 enzyme activities to be significantly higher than control level, wherein in 0.12mg/kg benzos
[a] pyrene causes CYP2C9 vigor to reach 1.43 times of control level.With the raising of exposure concentrations, earthworm CYP2C9 enzyme activity quilts
Inhibit, is less than control level, causes earthworm CYP2C9 enzyme activities to only reach control level in 0.96mg/kg benzos [a] pyrene
78%.It can be seen that earthworm CYP2C9 enzyme activities can be used as biomarker to diagnose soil benzo [a] pyrene pollution situation.
Finally illustrate, preferred embodiment above is merely illustrative of the technical solution of the present invention and unrestricted, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Claims (5)
1. a kind of method of CYP2C9 enzyme activities in high performance liquid chromatography tandem mass spectrometry detection earthworm body, which is characterized in that packet
Include following steps：
（1）Prepare earthworm microsomal protein suspension；
（2）By step（1）In earthworm microsomal protein suspension obtained be added in the incubation system containing probe substrate to institute
A concentration of 10-100 μ g/ml of microsomal protein are stated, a concentration of 100 μM of the probe substrate, heating starts enzymatic reaction, waits for
After enzymatic reaction terminates, generated metabolism after the detection of high performance liquid chromatography tandem mass spectrum combined instrument enzymatically reaction terminating is utilized
Product calculates CYP2C9 enzyme activities；The probe substrate is C14H10Cl2NNaO2；The metabolite is 4¢Hydroxyl Diclofenac；
High-efficient liquid phase chromatogram condition is：
a）Chromatographic column：MS Luna C18 columns, 2.1 mm × 50 mm, 3.0 mm, and it is equipped with corresponding C18 guard columns；
b）For mobile phase by A phases and B phase compositions, the A phases are water or one kind containing the water that volume fraction is 0.1% formic acid, the B
It is mutually acetonitrile or one kind containing the acetonitrile that volume fraction is 0.1% formic acid；
c）Condition of gradient elution：The ratio of 0-4 min, Mobile phase B rise to 95% by 5%；4-8 min, the ratio of Mobile phase B by
95% drops to 5%；The ratio of 8-10 min, Mobile phase B are 5%；
d）Column temperature：50℃；
e）Flow velocity：0.2 ml/min；
f）Automatic sampling room temperature：4 DEG C, sampling volume is 5 μ l；
Mass Spectrometry Conditions are：
a）Ion source：Electron spray ionisation；
b）Detection mode：Cation；
c）Electron spray voltage：3 kV ；
d）Ion source temperature：350℃；
e）Collision gas and air pressure：Argon gas, 1.2 mTorr；
f）Scan pattern：More reactive ion monitorings；
g）Nitrogen sheath, ion scan, auxiliary gas：It is respectively set to 40,0,10 units.
2. the side of CYP2C9 enzyme activities in a kind of high performance liquid chromatography tandem mass spectrometry detection earthworm body as described in claim 1
Method, which is characterized in that step（2）In, the incubation system includes following component：Buffer solution and NADPH generation systems；It is described slow
Fliud flushing is pH=7.5, the Tris buffer solutions of a concentration of 0.1M；The NADPH generation systems are 5 by volume by A and B:1 composition,
The A is G-6-P 20 mg/ml, NADP 20 mg/ml, 13.3 mg/ml of magnesium chloride；The B is by glucose-
6- phosphate dehydrogenases are dissolved in final concentration of 40 U/ml to glucose-6-phosphate dehydrogenase (G6PD) in 5 mM sodium citrate solutions.
3. the side of CYP2C9 enzyme activities in a kind of high performance liquid chromatography tandem mass spectrometry detection earthworm body as described in claim 1
Method, which is characterized in that step（2）In, the heating is that temperature is risen to 37 ± 0.2 DEG C；The time of enzymatic reacting is 5-60
min；The enzymatic reaction is terminated to be realized by the way that 200 μ l methanol are added.
4. the side of CYP2C9 enzyme activities in a kind of high performance liquid chromatography tandem mass spectrometry detection earthworm body as claimed in claim 3
Method, which is characterized in that the time of enzymatic reacting is 15 min.
5. the side of CYP2C9 enzyme activities in a kind of high performance liquid chromatography tandem mass spectrometry detection earthworm body as described in claim 1
Method, which is characterized in that step（2）In, it is described to detect enzymatically reaction terminating using high performance liquid chromatography tandem mass spectrum combined instrument
Further include before generated metabolite afterwards mixtures incubated after terminating enzymatic reaction in the case where centrifugal force is 15000 × g from
10 min of the heart takes supernatant for detecting.
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