CN106290661A - The method of CYP2C9 enzyme activity in a kind of high performance liquid chromatography tandem mass spectrum method detection Lumbricus body - Google Patents
The method of CYP2C9 enzyme activity in a kind of high performance liquid chromatography tandem mass spectrum method detection Lumbricus body Download PDFInfo
- Publication number
- CN106290661A CN106290661A CN201610994359.4A CN201610994359A CN106290661A CN 106290661 A CN106290661 A CN 106290661A CN 201610994359 A CN201610994359 A CN 201610994359A CN 106290661 A CN106290661 A CN 106290661A
- Authority
- CN
- China
- Prior art keywords
- lumbricus
- detection
- enzyme activity
- mass spectrum
- high performance
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention relates to the method for CYP2C9 enzyme activity in a kind of high performance liquid chromatography tandem mass spectrum method detection Lumbricus body, belong to technical field of enzyme activity detection, the method specifically includes following steps: (1) prepares Lumbricus microsomal protein suspension；(2) the Lumbricus microsomal protein suspension prepared in step (1) is added in the incubation system containing probe substrate, rise startup temperature enzymatic reaction, after enzymatic reaction terminates, utilize high performance liquid chromatography tandem mass spectrum combined instrument to detect enzymatically produced metabolite after reaction terminating, calculate CYP2C9 enzyme activity.This detection method accuracy, degree of accuracy and highly sensitive, stability is strong, can be used for the different analysis detection polluting CYP2C9 vigor in Lumbricus body under environment, and then provides detection method for exploring CYP2C9 as the probability of biomarker instruction soil pollution.
Description
Technical field
The invention belongs to technical field of enzyme activity detection, be specifically related to a kind of high performance liquid chromatography tandem mass spectrum method detection earthworm
The method of CYP2C9 enzyme activity in vermis.
Background technology
Cytochrome P450 (CYP) as a huge same work family enzyme, is a class important removing toxic substances that contains haemachrome
Enzyme.Iron ion transmission electronics oxidation allos thing in the haemachrome that this family's enzyme is combined by non-covalent bond, strengthens xenobiotic
Water solublity, make them be easier to excrete, thus play Detoxication.At present, CYP Asia enzyme is as environmental toxicology index
In terrestrial vertebrate organism and aquatile, obtain more research, compare the research in soil invertebrate biology less, as
Lumbricus, but the research of employing Lumbricus CYP Asia enzymatic diagnosis Soil Environmental Pollution is more and more extensive.CYP2C9 is an important CYP
Sub-enzyme, in Lumbricus body, can CYP2C9 need to be studied further as label diagnosis Soil Environmental Pollution, stablizes, examines reliably
Survey method becomes the key of this research.Relating to the report of CYP Asia enzyme activity at present in research, the assay method of employing mostly is probe
Substrate method, i.e. using a certain compound is substrate, and this substrate can be utilized fluorophotometer or ultraviolet spectrometry by CYP Asia enzymes metabolism
The growing amount of photometer detection metabolite, reflects the vigor of CYP Asia enzyme by the growing amount of metabolite.But above-mentioned detection
Means poor sensitivity, also cannot efficiently separate probe substrate and metabolite.It addition, CYP enzyme itself is an erythrocruorin enzyme, blood
The existence energy severe jamming fluorophotometer of red pigment or the mensuration of ultraviolet spectrophotometer.CYP Asia enzyme in Lumbricus body in addition
Content is less relative to mammal, has been reported that more and detect unsuccessfully this result in existing document.
Therefore, it is badly in need of one and from complex biological substrate, more low-level can be carried out with higher specificity within a short period of time
Detection by quantitative Lumbricus body in the method for CYP2C9 vigor.
Summary of the invention
In view of this, it is an object of the invention to provide in a kind of high performance liquid chromatography tandem mass spectrum method detection Lumbricus body
The method of CYP2C9 enzyme activity.
For reaching above-mentioned purpose, the present invention provides following technical scheme:
1, the method for CYP2C9 enzyme activity in a kind of high performance liquid chromatography tandem mass spectrum method detection Lumbricus body, including walking as follows
Rapid:
(1) Lumbricus microsomal protein suspension is prepared；
(2) the Lumbricus microsomal protein suspension prepared in step (1) is added in the incubation system containing probe substrate,
Rise startup temperature enzymatic reaction, after enzymatic reaction terminates, utilize the detection of high performance liquid chromatography tandem mass spectrum combined instrument the most anti-
After should terminating, produced metabolite, calculates CYP2C9 enzyme activity.
Further, in step (2), high-efficient liquid phase chromatogram condition is:
A) chromatographic column: MS Luna C18 post, 2.1mm × 50mm, 3.0 μm, and it is equipped with corresponding C18 guard column；
B) flowing is by A phase and B phase composition, and described A phase is water or the one containing the water that volume fraction is 0.1% formic acid,
Described B phase is acetonitrile or the one containing the acetonitrile that volume fraction is 0.1% formic acid；
C) condition of gradient elution: 0-4min, the ratio of Mobile phase B is risen to 95% by 5%；4-8min, the ratio of Mobile phase B
Example is dropped to 5% by 95%；8-10min, the ratio of Mobile phase B is 5%；
D) column temperature: 50 DEG C；
E) flow velocity: 0.2ml/min；
F) auto injection room temperature: 4 DEG C, sampling volume is 5 μ l.
Further, in step (2), Mass Spectrometry Conditions is:
A) ion source: electron spray ionisation；
B) detection mode: cation；
C) electron spray voltage: 3kV；
D) ion source temperature: 350 DEG C；
E) collision gas and air pressure: argon, 1.2mTorr；
F) scan pattern: many reactive ions are monitored；
G) nitrogen sheath, ion scan, auxiliary gas: be respectively set to 40,0,10 units.
Further, in step (2), described probe substrate is diclofenac sodium；Described metabolite is that the double chlorine of 4 '-hydroxyl is fragrant
Acid.
Further, in step (2), described Lumbricus microsomal protein suspension adds in the incubation system containing probe substrate
It is 10-100 μ g/ml to described microsomal protein concentration.
Further, in step (2), described Lumbricus microsomal protein suspension adds in the incubation system containing probe substrate
After, the concentration of described probe substrate is 100 μMs.
Further, in step (2), described incubation system includes following component: buffer and NADPH produce system；Described
Buffer is pH=7.5, and concentration is the Tris buffer of 0.1M；It is 5:1 group by volume by A and B that described NADPH produces system
Becoming, described A is G-6-P 20mg/ml, NADP 20mg/ml, magnesium chloride 13.3mg/ml；Described B be by glucose-
6-phosphate dehydrogenase is dissolved in 5mM sodium citrate solution the final concentration of 40U/ml to glucose-6-phosphate dehydrogenase (G6PD).
Further, in step (2), described intensification is for rise to 37 ± 0.2 DEG C by temperature；Described time of enzymatic reacting is 5-
60min；Described enzymatic reaction terminates by adding 200 μ l methanol realizations.
Further, described time of enzymatic reacting is 15min.
Further, in step (2), described high performance liquid chromatography tandem mass spectrum combined instrument is utilized to detect enzymatically reaction terminating
Also include before rear produced metabolite the mixtures incubated after enzymatic reaction is terminated under centrifugal force is 15000 × g from
Heart 10min, takes supernatant for detecting.
The beneficial effects of the present invention is: the present invention has been successfully set up LC-MS/MS and has analyzed CYP2C9 in detection Lumbricus body
The method of enzyme activity, this detection method accuracy, degree of accuracy and highly sensitive, stability is strong, can be used for different pollution under environment
The analysis detection of CYP2C9 vigor in Lumbricus body, and then for exploring the CYPC9 possibility as biomarker instruction soil pollution
Property provides detection method.
Accompanying drawing explanation
In order to make the purpose of the present invention, technical scheme and beneficial effect clearer, the present invention provides drawings described below to carry out
Illustrate:
Fig. 1 is that diclofenac sodium generates 4 '-hydroxyl diclofenac figure after CYP2C9 enzyme catalysis；
Fig. 2 is LC-PDA figure and the many reaction of LC-MS/MS of the mixed solution of diclofenac sodium and 4 '-hydroxyl diclofenac
Monitoring figure；
Fig. 3 be the mixed solution adding diclofenac sodium, 4 '-hydroxyl diclofenac and incubation system LC-PDA figure with
LC-MS/MS multiple-reaction monitoring figure；
Fig. 4 is molten for adding diclofenac sodium, 4 '-hydroxyl diclofenac, incubation system and heat inactivation Lumbricus microsomal protein
LC-PDA figure and the LC-MS/MS multiple-reaction monitoring figure of liquid；
Fig. 5 is CYP2C9 enzyme activity figure in LC-MS/MS detection Lumbricus body；
Fig. 6 is the dependency graph of 4 '-hydroxyl diclofenac growing amount and microsomal protein concentration；
Fig. 7 is the dependency graph of 4 '-hydroxyl diclofenac growing amount and incubation time；
After Fig. 8 is for being exposed to benzo [a] pyrene contamination soil 14 days, CYP2C9 enzyme activity detection figure in Lumbricus body.
Detailed description of the invention
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.
Material and reagent: trifluoroacetic acid aqueous solution, methanol are purchased from Merck company, chromatographically pure diclofenac sodium, formic acid and Tris
It is purchased from Chinese Shanghai Sigma-Aldrich company, NADPH reaction system in metabolite 4 '-hydroxyl diclofenac, incubation reaction
System is purchased from BD genetest, and ultra-pure water is filtered by Germany's Sartorius Arium 611DI ultrapure water system, C18 mass spectrum color
Spectrum post is purchased from Agricultural University Of Shenyang from Phenomenex company of the U.S., Eisenia foetida, and under (20 ± 2) DEG C dark condition
It is stored in cleaning soil.
Method And Principle: employing diclofenac sodium (DF) is as probe substrate, under Lumbricus CYP2C9 enzyme existence condition, can quilt
It is catalytically conveted to 4 '-hydroxyl diclofenac (4 ' H-DF) (such as Fig. 1), by high performance liquid chromatography tandem mass spectrum combined instrument (LC-
MS/MS) growing amount measuring 4 '-hydroxyl diclofenac reflects CYP2C9 enzyme activity.
Embodiment 1
Flowing phase constituent determines
Investigate three groups flowing the phase composition impacts on detection sensitivity: the first be flow mutually in A be pure water, B is pure second
Nitrile；The second be A be water (being the formic acid of 0.1% containing volume fraction), B is pure acetonitrile；The third is that water is (containing volume fraction for A
It is the formic acid of 0.1%), B is acetonitrile (being the formic acid of 0.1% containing volume fraction), and result shows that last a kind of flowing the most effectively changes
It is apt to the stability of chromatographic peak profile and peak area, accordingly, it is determined that with A as water (being the formic acid of 0.1% containing volume fraction), B is second
Nitrile (being the formic acid of 0.1% containing volume fraction) is as optimal flow phase.
Embodiment 2
LC-MS/MS condition setting
LC-MS/MS be the Thermo Fisher Scientific TSQ triple quadrupole rods tandem mass spectrometry of Quantum Max-
HPLC system, wherein, high-efficient liquid phase chromatogram condition is:
A) chromatographic column: MS Luna C18 post, 2.1mm × 50mm, 3.0 μm, and it is equipped with corresponding C18 guard column；B) flowing
By A phase and B phase composition, described A phase is that described B phase is for be containing the water that volume fraction is 0.1% formic acid containing volume fraction
The acetonitrile of 0.1% formic acid；C) condition of gradient elution: 0-4min, the ratio of Mobile phase B is risen to 95% by 5%；4-8min, stream
The ratio of dynamic phase B is dropped to 5% by 95%；8-10min, the ratio of Mobile phase B is 5%；D) column temperature: 50 DEG C；E) flow velocity:
0.2ml/min；F) auto injection room temperature: 4 DEG C, sampling volume is 5 μ l.
Mass Spectrometry Conditions is:
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, 1.2mTorr；F) scan pattern: many reactive ions are monitored；G) nitrogen sheath, ion are swept
Retouch, assist gas: be respectively set to 40,0,10 units.
Owing to substrate diclofenac na concn is higher, therefore UV-detector two pole quantitatively still used of diclofenac sodium
Under 280nm wavelength quantitatively, MS/MS is used for detecting metabolite 4 '-hydroxyl diclofenac pipe array (PDA).
It is qualitative to combining retention time that each compound chooses 1 pair of parent ion/daughter ion, and each parameter is listed in table 1, at data
Reason uses the X-Calibur software of LC-MS/MS autogamy.
Table 1 diclofenac sodium (DF) and the MS/MS parameter of 4 '-hydroxyl diclofenac (4 ' H-DF)
Embodiment 3
Matrix effect is tested
(1) configuration storing solution: methanol is mixed to prepare diluent with water 1:1 by volume, DF is dissolved in described diluent
To the final concentration of 1.0mg/ml of DF, prepare DF storing solution；4 ' H-DF are dissolved in methanol to the final concentration of 1.0mg/ of 4 ' H-DF
Ml, prepares 4 ' H-DF storing solutions；
(2) configuration working solution: 4 ' H-DF storing solutions in step (1) are diluted to 10ng/ml, prepares 4 ' H-DF working solutions；
DF storing solution is diluted to 5 μ g/ml, prepares DF working solution.
(3) 4 ' H-DF working solutions in step (2) are mixed with DF working solution 1:1 by volume, carry out LC-MS/MS mensuration,
Its chromaticness spectrogram is as shown in Figure 2.
(4) take 1ml 4 ' H-DF working solution in 2ml test tube, dry up under Nitrogen evaporator, add 250 μ l DF working solutions, it
Being warming up to 37 DEG C after rear interpolation 650 μ l incubation system, wherein, incubation system includes: 590 μ l buffer be (pH=7.5,0.1M's
Tris), (described NADPH produces system and is made up of for 5:1 by volume A and B 60 μ l NADPH generation systems, and described A is Fructus Vitis viniferae
Sugar-6-phosphoric acid 20mg/ml, NADP 20mg/ml, magnesium chloride 13.3mg/ml；Described 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 solution), finally add 200 μ l methanol,
Under centrifugal force is 15000 × g, centrifugal 10min, takes 200 μ l supernatant, detects with LC-MS/MS, its chromaticness spectrogram such as figure
Shown in 3.
(5) take 1ml 4 ' H-DF working solution in 2ml test tube, dry up under Nitrogen evaporator, add 250 μ l DF working solutions, it
Rear interpolation 100 μ l fermentoid liquid (the Lumbricus microsomal protein suspension prepared in embodiment 6 is carried out inactivation process), 650 μ l
Being warming up to 37 DEG C after incubation system, wherein, incubation system includes: 590 μ l buffer (Tris of pH=7.5,0.1M), 60 μ l
NADPH produces system, and (described NADPH produces system and is made up of for 5:1 by volume A and B, and described A is G-6-P
20mg/ml, NADP 20mg/ml, magnesium chloride 13.3mg/ml；Described B for being dissolved in 5mM Fructus Citri Limoniae by glucose-6-phosphate dehydrogenase (G6PD)
To the final concentration of 40U/ml of glucose-6-phosphate dehydrogenase (G6PD) in acid sodium solution), finally add 200 μ l methanol, at centrifugal force be
Under 15000 × g, centrifugal 10min, takes 200 μ l supernatant, detects with LC-MS/MS, and its chromaticness spectrogram is as shown in Figure 4.
Fig. 2, Fig. 3, Fig. 4 all comprise three subgraphs, is followed successively by from top to bottom: how anti-LC-PDA chromatogram, LC-MS/MS be
Answer under monitoring pattern extract chromatogram: 4 '-hydroxyl diclofenac (middle), diclofenac sodium (under).As shown in Figure 2, PDA
It is only capable of detecting diclofenac sodium.Compare PDA, MS/MS the sensitiveest, use retention time (4.98min) and combine parent ion/
Daughter ion can qualitative 4 '-hydroxyl diclofenac to (312/230).Therefore, the conclusion of document report before this is demonstrated: ultraviolet is examined
Survey device and cannot detect metabolite.It addition, according to parent ion/daughter ion pair, infer that (m/z is 4 '-hydroxyl diclofenac daughter ion
230) be owing to parent ion (m/z is 312) is lost caused by hydrochloric acid and formic acid, diclofenac sodium daughter ion (m/z is 250) be due to
Parent ion (m/z is 296) is lost caused by formic acid.The multiple-reaction monitoring schema extraction figure of the 4 ' of Fig. 3 Yu Fig. 4-hydroxyl diclofenac
In, occur in that by-product peak at retention time about 5.7min, it is shown that matrix effect, tentatively judge that this by-product is 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
Ml, 1000ng/ml, 2500ng/ml, 5000ng/ml, prepare 64 ' H-DF working solutions；DF storing solution is diluted to 5 μ g/ respectively
Ml, 10 μ g/ml, 20 μ g/ml, 40 μ g/ml, 80 μ g/ml, 150 μ g/ml, prepare 6 DF working solutions.Take each the most respectively
4 ' H-DF working solution 1ml under concentration, are placed in 2ml centrifuge tube, dry up under Nitrogen evaporator, and then correspondence adds 250 μ l respectively
Concentration is 5 μ g/ml, 10 μ g/ml, 20 μ g/ml, 40 μ g/ml, 80 μ g/ml, the DF working solution of 150 μ g/ml, prepare 6 to be measured
Liquid.
(2) respectively in step (1) equipped with 6 centrifuge tubes of liquid to be measured having added 650 μ l incubation systems and 100 μ l
It is warming up to 37 DEG C after the enzymatic solution (the Lumbricus microsomal protein suspension prepared in embodiment 6 is carried out inactivation process) of inactivation,
Wherein, incubation system includes: 590 μ l buffer (Tris of pH=7.5,0.1M), and it is (described that 60 μ l NADPH produce system
NADPH produces system and is made up of for 5:1 by volume A and B, and described A is G-6-P 20mg/ml, NADP 20mg/
Ml, magnesium chloride 13.3mg/ml；Described B for glucose-6-phosphate dehydrogenase (G6PD) is dissolved in 5mM sodium citrate solution to glucose-
The final concentration of 40U/ml of 6-phosphate dehydrogenase).200 μ l methanol are added again, under centrifugal force is 15000 × g in centrifuge tube
Centrifugal 10min, takes 200 μ l supernatant, detects with LC-MS/MS, prepares standard curve, and the data obtained is shown in Table 2.
Table 2 standard curve tables of data
As shown in Table 2, the linear relationship of testing compound is good, regression coefficient r2It is all higher than the quantitative of 0.99,4 ' H-DF
Be limited to 10ng/ml, DF is quantitatively limited to 5 μ g/ml.
Embodiment 5
LC-MS/MS Method validation
1, prepared by sample
(1) 4 ' H-DF storing solutions in step (1) in embodiment 3 are diluted to 30ng/ml, 500ng/ml, 4000ng/ respectively
Ml prepares 34 ' H-DF working solutions；DF storing solution is diluted to 9 μ g/ml, 60 μ g/ml, 120 μ g/ml respectively, prepares 3 DF work
Liquid.Take 4 ' H-DF working solution 1ml under each concentration the most respectively, be placed in 2ml centrifuge tube, dry up under Nitrogen evaporator, so
Rear corresponding addition respectively 250 μ l concentration are 9 μ g/ml, 60 μ g/ml, the DF working solution of 120 μ g/ml, prepare 3 liquid to be measured, for pure
Sample.
(2) according to step (2) described method in embodiment 4,3 matrix sample are prepared.
2, veracity and precision
Respectively 3 matrix sample are detected with LC-MS/MS, wherein, for each matrix sample, arrange 5 and put down
Row sample, each sample replication 4 times, continuous 4 days repeat this process, and test result is shown in Table 3.
The veracity and precision of table 3 matrix sample
As shown in Table 3, all matrix sample accuracy are all higher than 85%, and withinday precision scope is 2.79-10.37%,
Day to day precision scope is 2.21-10.25%, meet FDA Food and Drug Administration (FDA) about in method validation to essence
Density and the requirement of accuracy.
3, extraction recovery
Respectively pure sample product and matrix sample are detected with LC-MS/MS, exist with matrix sample by comparing pure sample product
Response on LC-MS/MS, determines the response rate, the results are shown in Table 4.
The response rate ((%, ± SD, n=4)) of table 4 target compound
As shown in Table 4, in matrix sample, the response rate of 4 ' H-DF is that the response rate of 80-87%, DF is about 80%, shows
Matrix effect is little for quantified results impact.
4, stability
By matrix sample the most at room temperature place 10h, 4 DEG C deposit 24h ,-30 DEG C deposit 30 days after, then use LC-
Matrix sample under three kinds of conditions of storage is detected by MS/MS respectively, the results are shown in Table 5.
The stability (response rate, %, ± SD, n=4) of matrix sample under the different condition of storage of table 5, result is expressed as adding
Add the percentage ratio (respectively under three Quality Control levels of high, medium and low concentration) of concentration
As shown in Table 5, the having good stability of matrix sample.
Embodiment 6
Preparation Lumbricus microsomal protein suspension
After the Lumbricus being stored at 4 DEG C in 20% (V/V) glycerite is taken out, dissect rapidly internal organs, use 0.15mol/L
KCl solution cleans internal organs, is subsequently transferred to equipped with 4ml homogenate buffer (sucrose 250mmol/L, Tris 50mmol/L, DTT
1mmol/L, EDTA 1mmol/L, pH=7.5) glass tissue dismembyator in make it uniformly crush, and will finally be homogenized volume
It is set to 6ml.
Differential centrifugation is used to extract microsome: utilizing low temperature supercentrifuge, by homogenate at 4 DEG C, centrifugal force is
Under the conditions of 15000 × g after centrifugal 15min, retain supernatant, then by supernatant at 4 DEG C, centrifugal force be under the conditions of 15000 × g from
Heart 90min, preserves buffer (sucrose 50mmol/L, Tris 50mmol/L, DTT 1mmol/L, EDTA by gained precipitation 3ml
1mmol/L, pH=7.5,20% (V/V) glycerol) Eddy diffusion, prepare Lumbricus microsome and hang albumen supernatant liquid, store at-80 DEG C
Standby.
Embodiment 7
CYP2C9 enzyme activity in LC-MS/MS detection Lumbricus body
(1) taking the centrifuge tube of 2ml, add 650 μ l incubation systems and 250 μ l DF solution, wherein, incubation system includes:
590 μ l buffer (Tris of pH=7.5,0.1M), 60 μ l NADPH produce system, and (described NADPH produces system and is pressed by A and B
Volume ratio is 5:1 composition, and described A is G-6-P 20mg/ml, NADP 20mg/ml, magnesium chloride 13.3mg/ml；Described
B is be dissolved in by glucose-6-phosphate dehydrogenase (G6PD) in 5mM sodium citrate solution to glucose-6-phosphate dehydrogenase (G6PD) final concentration of
40U/ml).Now, DF concentration in mixed solution is 100 μMs.Then, 100 μ l Lumbricus microsomal protein suspensions are added from
Heart Guan Zhongzhi microsomal protein concentration is 10 μ g/ml, is warming up to 37 DEG C, starts enzymatic reaction, and the response time is 15min, then to
Adding 200 μ l methanol in centrifuge tube, under centrifugal force is 15000 × g, centrifugal 10min, takes 200 μ l supernatant, with LC-MS/MS
Detecting, testing result is as shown in Figure 5.Contrasting with Fig. 4, Fig. 5 remains to find out matrix effect, except for the difference that has work due to enzyme
Property, diclofenac sodium can be catalyzed and be converted into 4 '-hydroxyl diclofenac, matrix effect weakens.The computing formula of CYP2C9 enzyme activity
As follows:
X=C4′H-DF/(M4′H-DF×Cprotein)
X CYP2C9 enzyme activity, unit nmol/mg protein；
C4′H-DFThe concentration of 4 '-hydroxyl diclofenac, unit ng/ml in reaction system；
M4′H-DFThe molal weight of 4 '-hydroxyl diclofenac, unit g/mol；
CproteinProtein concentration in reaction system, unit mg/ml.
(2) according to the method in (1), microsomal protein after change Lumbricus microsomal protein suspension addition incubation system
Concentration so that it is be respectively 20 μ g/ml, 50 μ g/ml, 100 μ g/ml, 200 μ g/ml, test, calculates CYP2C9 enzyme activity.Knot
Closing the experimental data in (1), obtain the dependency of metabolite growing amount and microsomal protein addition, result is shown in Fig. 6, by scheming
6 understand, and in incubation system, the white concentration of microsome is in the range of 10-100 μ g/ml, metabolite growing amount (y) and microsomal protein
Concentration (x) presents preferable linear relationship, and regression equation is y=8.273+0.436x, regression coefficient R2=0.996.
(3) according to the method in (1), microsomal protein after change Lumbricus microsomal protein suspension addition incubation system
Concentration so that it is be respectively 32 μ g/ml, 64 μ g/ml, then change time of enzymatic reacting, make the time be respectively 5min, 15min,
40min, 60min, test through the sample of differential responses time gained, obtains metabolite growing amount and hatches under variable concentrations
The dependency of time, result is shown in Fig. 7, and as shown in Figure 7, incubation time is the longest, and the growing amount of metabolite is the most, but incubation time
More than 15min, the growing amount amplification of metabolite the least (< 10%, compare product formation during incubation time 15min).Cause
This, in enzymatic reaction, incubation time 15min is most widely suited.
Embodiment 8
It is exposed to the mensuration of Lumbricus CYP2C9 vigor in low concentration benzo [a] pyrene contaminated soil
Take and be exposed to Lumbricus sample after 14 days in the soil of benzo [a] pyrene contamination, prepare according to the method in embodiment 6
Lumbricus microsomal protein suspension.Wherein, in soil, the concentration of contamination gradient of benzo [a] pyrene is: 0.12,0.24,0.48,
0.96mg/kg, each exposure concentrations arrange 6 parallel, test with LC-MS/MS, obtain under each concentration of contamination, Lumbricus
The response change of CYP2C9 enzyme activity, as shown in Figure 8.As shown in Figure 8, low concentration benzo [a] pyrene (0.12,0.24mg/kg) is aobvious
Write induction of Lumbricus CYP2C9 enzyme activity, cause CYP2C9 enzyme activity to be significantly higher than control level, wherein at 0.12mg/kg benzo
[a] pyrene causes CYP2C9 vigor to reach 1.43 times of control level.Along with the raising of exposure concentrations, Lumbricus CYP2C9 enzyme activity quilt
Suppression, less than control level, causes Lumbricus CYP2C9 enzyme activity to only reach control level at 0.96mg/kg benzo [a] pyrene
78%.As can be seen here, Lumbricus CYP2C9 enzyme activity can diagnose soil benzo [a] pyrene pollution situation as biomarker.
Finally illustrate, preferred embodiment above only in order to technical scheme to be described and unrestricted, although logical
Cross above preferred embodiment the present invention to be described in detail, it is to be understood by those skilled in the art that can be
In form and it is made various change, without departing from claims of the present invention limited range in details.
Claims (10)
1. the method for CYP2C9 enzyme activity in a high performance liquid chromatography tandem mass spectrum method detection Lumbricus body, it is characterised in that bag
Include following steps:
(1) Lumbricus microsomal protein suspension is prepared；
(2) the Lumbricus microsomal protein suspension prepared in step (1) is added in the incubation system containing probe substrate, heat up
Start enzymatic reaction, after enzymatic reaction terminates, utilize the detection of high performance liquid chromatography tandem mass spectrum combined instrument enzymatically to react eventually
After only, produced metabolite, calculates CYP2C9 enzyme activity.
The side of CYP2C9 enzyme activity in a kind of high performance liquid chromatography tandem mass spectrum method the most as claimed in claim 1 detection Lumbricus body
Method, it is characterised in that in step (2), high-efficient liquid phase chromatogram condition is:
A) chromatographic column: MS Luna C18 post, 2.1mm × 50mm, 3.0 μm, and it is equipped with corresponding C18 guard column；
B) flowing is by A phase and B phase composition, and described A phase is water or the one containing the water that volume fraction is 0.1% formic acid, described B
Mutually for acetonitrile or the one containing the acetonitrile that volume fraction is 0.1% formic acid；
C) condition of gradient elution: 0-4min, the ratio of Mobile phase B is risen to 95% by 5%；4-8min, the ratio of Mobile phase B by
95% drops to 5%；8-10min, the ratio of Mobile phase B is 5%；
D) column temperature: 50 DEG C；
E) flow velocity: 0.2ml/min；
F) auto injection room temperature: 4 DEG C, sampling volume is 5 μ l.
The side of CYP2C9 enzyme activity in a kind of high performance liquid chromatography tandem mass spectrum method the most as claimed in claim 1 detection Lumbricus body
Method, it is characterised in that in step (2), Mass Spectrometry Conditions is:
A) ion source: electron spray ionisation；
B) detection mode: cation；
C) electron spray voltage: 3kV；
D) ion source temperature: 350 DEG C；
E) collision gas and air pressure: argon, 1.2mTorr；
F) scan pattern: many reactive ions are monitored；
G) nitrogen sheath, ion scan, auxiliary gas: be respectively set to 40,0,10 units.
The side of CYP2C9 enzyme activity in a kind of high performance liquid chromatography tandem mass spectrum method the most as claimed in claim 1 detection Lumbricus body
Method, it is characterised in that in step (2), described probe substrate is diclofenac sodium；Described metabolite is that the double chlorine of 4 '-hydroxyl is fragrant
Acid.
The side of CYP2C9 enzyme activity in a kind of high performance liquid chromatography tandem mass spectrum method the most as claimed in claim 1 detection Lumbricus body
Method, it is characterised in that in step (2), described Lumbricus microsomal protein suspension adds in the incubation system containing probe substrate
It is 10-100 μ g/ml to described microsomal protein concentration.
The side of CYP2C9 enzyme activity in a kind of high performance liquid chromatography tandem mass spectrum method the most as claimed in claim 1 detection Lumbricus body
Method, it is characterised in that in step (2), described Lumbricus microsomal protein suspension adds in the incubation system containing probe substrate
After, the concentration of described probe substrate is 100 μMs.
The side of CYP2C9 enzyme activity in a kind of high performance liquid chromatography tandem mass spectrum method the most as claimed in claim 1 detection Lumbricus body
Method, it is characterised in that in step (2), described incubation system includes following component: buffer and NADPH produce system；Described slow
Rushing liquid is pH=7.5, and concentration is the Tris buffer of 0.1M；It is 5:1 group by volume by A and B that described NADPH produces system
Becoming, described A is G-6-P 20mg/ml, NADP 20mg/ml, magnesium chloride 13.3mg/ml；Described B be by glucose-
6-phosphate dehydrogenase is dissolved in 5mM sodium citrate solution the final concentration of 40U/ml to glucose-6-phosphate dehydrogenase (G6PD).
The side of CYP2C9 enzyme activity in a kind of high performance liquid chromatography tandem mass spectrum method the most as claimed in claim 1 detection Lumbricus body
Method, it is characterised in that in step (2), described intensification is for rise to 37 ± 0.2 DEG C by temperature；Described time of enzymatic reacting is 5-
60min；Described enzymatic reaction terminates by adding 200 μ l methanol realizations.
The side of CYP2C9 enzyme activity in a kind of high performance liquid chromatography tandem mass spectrum method the most as claimed in claim 8 detection Lumbricus body
Method, it is characterised in that described time of enzymatic reacting is 15min.
CYP2C9 enzyme activity in a kind of high performance liquid chromatography tandem mass spectrum method the most as claimed in claim 1 detection Lumbricus body
Method, it is characterised in that in step (2), described utilizes the detection of high performance liquid chromatography tandem mass spectrum combined instrument enzymatically to react eventually
Also include before produced metabolite after only that the mixtures incubated after enzymatic reaction being terminated is under centrifugal force is 15000 × g
Centrifugal 10min, takes supernatant for detecting.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610994359.4A CN106290661B (en) | 2016-11-11 | 2016-11-11 | A kind of method that high performance liquid chromatography tandem mass spectrometry detects CYP2C9 enzyme activities in earthworm body |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610994359.4A CN106290661B (en) | 2016-11-11 | 2016-11-11 | A kind of method that high performance liquid chromatography tandem mass spectrometry detects CYP2C9 enzyme activities in earthworm body |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106290661A true CN106290661A (en) | 2017-01-04 |
| CN106290661B CN106290661B (en) | 2018-08-03 |
Family
ID=57721383
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610994359.4A Active CN106290661B (en) | 2016-11-11 | 2016-11-11 | A kind of method that high performance liquid chromatography tandem mass spectrometry detects CYP2C9 enzyme activities in earthworm body |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106290661B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108277259A (en) * | 2018-03-30 | 2018-07-13 | 沈阳北创医学检验所有限公司 | The active detection kits of CE2 in a kind of measurement complex biological system |
| CN109596752A (en) * | 2019-02-01 | 2019-04-09 | 重庆市农业科学院 | A kind of method that high performance liquid chromatography tandem mass spectrometry detects CYP1A2 and CYP3A4 enzyme activity in earthworm body |
| CN112098555A (en) * | 2020-09-16 | 2020-12-18 | 重庆市农业科学院 | Method for measuring enzymatic activities of CYP2A6, CYP2B6, CYP2C8 and CYP2D6 in earthworm |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101308119A (en) * | 2008-04-16 | 2008-11-19 | 上海市徐汇区中心医院 | Method for minim hepatic tissue in vitro incubation and detecting CYP450 enzymatic activity |
| CN102080122A (en) * | 2010-11-26 | 2011-06-01 | 吉林大学 | Detecting method for simultaneously measuring metabolic products of probe medicines of five CYP450 enzymes |
| CN102650620A (en) * | 2012-03-15 | 2012-08-29 | 天津医科大学 | Preparation method, detection method and application of probe drug composition for determination of metabolic activity of cytochrome P450 |
| CN104849371A (en) * | 2015-05-22 | 2015-08-19 | 无锡市人民医院 | Detection method for simultaneously determining metabolic products of seven CYP450 enzyme probe substrates in human liver microsomes |
-
2016
- 2016-11-11 CN CN201610994359.4A patent/CN106290661B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101308119A (en) * | 2008-04-16 | 2008-11-19 | 上海市徐汇区中心医院 | Method for minim hepatic tissue in vitro incubation and detecting CYP450 enzymatic activity |
| CN102080122A (en) * | 2010-11-26 | 2011-06-01 | 吉林大学 | Detecting method for simultaneously measuring metabolic products of probe medicines of five CYP450 enzymes |
| CN102650620A (en) * | 2012-03-15 | 2012-08-29 | 天津医科大学 | Preparation method, detection method and application of probe drug composition for determination of metabolic activity of cytochrome P450 |
| CN104849371A (en) * | 2015-05-22 | 2015-08-19 | 无锡市人民医院 | Detection method for simultaneously determining metabolic products of seven CYP450 enzyme probe substrates in human liver microsomes |
Non-Patent Citations (2)
| Title |
|---|
| 《BIOMEDICAL CHROMATOGRAPHY》 * |
| 《DRUG METABOLISM AND DISPOSITION》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108277259A (en) * | 2018-03-30 | 2018-07-13 | 沈阳北创医学检验所有限公司 | The active detection kits of CE2 in a kind of measurement complex biological system |
| CN109596752A (en) * | 2019-02-01 | 2019-04-09 | 重庆市农业科学院 | A kind of method that high performance liquid chromatography tandem mass spectrometry detects CYP1A2 and CYP3A4 enzyme activity in earthworm body |
| CN112098555A (en) * | 2020-09-16 | 2020-12-18 | 重庆市农业科学院 | Method for measuring enzymatic activities of CYP2A6, CYP2B6, CYP2C8 and CYP2D6 in earthworm |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106290661B (en) | 2018-08-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gonzalez et al. | A rapid and reliable method for metabolite extraction in yeast using boiling buffered ethanol | |
| Rumpel et al. | Gas chromatographic analysis of monosaccharides in a forest soil profile: Analysis by gas chromatography after trifluoroacetic acid hydrolysis and reduction–acetylation | |
| CN106290661A (en) | The method of CYP2C9 enzyme activity in a kind of high performance liquid chromatography tandem mass spectrum method detection Lumbricus body | |
| CN104988207B (en) | The α-hydroxybutyrate dehydrogenase reagent and detection method of a kind of stabilization, strong antijamming capability | |
| CN102154442B (en) | Method for detecting 1,5-anhydro sorbitol and related diagnostic kit | |
| CN101003831A (en) | Kit for diagnosing diseases in system of liver and gall | |
| CN104596959A (en) | Method for detecting potassium ion concentration based on DNA enzymes | |
| Yeo et al. | A rapid, automated enzymatic fluorometric assay for determination of D-arabinitol in serum | |
| Qiu et al. | Evaluating the ‘triggering response’in soils, using 13C-glucose, and effects on dynamics of microbial biomass | |
| CN102564979A (en) | Method for determining alcohol concentration by using enzyme cycling method and alcohol determination kit | |
| Pang et al. | Determination and pharmacokinetic study of enasidenib in rat plasma by UPLC–MS/MS | |
| Mostafa et al. | Lucigenin-pyrogallol chemiluminescence for the multiple detection of pyrogallol, cobalt ion, and tyrosinase | |
| CN107703288A (en) | Improve the bile acid detection reagent of reaction stability | |
| Sun et al. | Quantification of 2-NBDG, a probe for glucose uptake, in GLUT1 overexpression in HEK293T cells by LC–MS/MS | |
| Du et al. | Chemiluminescence determination of streptomycin in pharmaceutical preparation and its application to pharmacokinetic study by a flow injection analysis assembly | |
| CN104089956A (en) | Quick water iodine testing kit and testing method thereof | |
| Liu et al. | A sensitive coumarin fluorescence sensor designed for isoprene detection and imaging research in plants | |
| CN109596752A (en) | A kind of method that high performance liquid chromatography tandem mass spectrometry detects CYP1A2 and CYP3A4 enzyme activity in earthworm body | |
| Fan et al. | Rapid and simultaneous quantitation of amoxicillin and clavulanic acid in human plasma and urine by ultra-performance liquid chromatography tandem mass spectrometry and its application to a pharmacokinetic study | |
| Chen et al. | Sequential enzymatic monitoring of glucose, ethanol and glutamate in bioreactor fermentation broth containing a high salt concentration by a multi-channel flow-injection analysis method | |
| Han | Capillary electrophoresis with chemiluminescence detection of rutin and chlorogenic acid based on its enhancing effect for the luminol-ferricyanide system | |
| Akeneev et al. | Determination of tetracycline in honey by voltammetry | |
| CN101806742A (en) | Analysis method for fast detecting degradation rate of organophosphorus degrading bacteria | |
| CN108508129B (en) | Method for measuring biological potency of heparin drugs | |
| Mar et al. | Optical fiber reflectance sensor coupled to a multisyringe flow injection system for preconcentration and determination of 1-naphthylamine in water samples |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |