CN109459518A - The extraction and detection method of lipid in organism liver organization - Google Patents
The extraction and detection method of lipid in organism liver organization Download PDFInfo
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- CN109459518A CN109459518A CN201811603921.1A CN201811603921A CN109459518A CN 109459518 A CN109459518 A CN 109459518A CN 201811603921 A CN201811603921 A CN 201811603921A CN 109459518 A CN109459518 A CN 109459518A
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- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Abstract
This application discloses a kind of extracting methods of lipid in organism liver organization, comprising the following steps: liver organization is taken to carry out tissue homogenate；Methyl tertiary butyl ether(MTBE) is added and methanol carries out lipid extraction；Take upper organic phase that methyl tertiary butyl ether(MTBE) and methanol reextraction are added again；Upper organic phase is taken, lipids extraction is completed.The application gives the detection method of lipid in the organism liver organization based on said extracted method.Present application addresses the lipid quantity that tissue samples dosage is big, extraction efficiency is low, Extraction solvent toxicity is big and existing detection method tests and analyzes out in liver organization existing for current common tissue extraction method is few, the few technical problem of type.
Description
Technical field
This application involves field of biological detection, extraction in particular to lipid in a kind of organism liver organization and
Detection method.
Background technique
The vitals that liver is controlled as body lipid-metabolism and inflammation, research of the people to Liver lipids metabolism group
It is increasingly extensive and deep.With the progress of analytical technology, constantly improve and improve for the analytical technology of lipid compounds, lipid
Class compound can more preferably, more fully be characterized, identify and quantitative analysis.Lipid generation is disclosed based on iipidomics analysis technology
Thank to correlation of the exception with disease, finding potential source biomolecule marker is the hot spot studied at present.According to research mode difference, can incite somebody to action
Lipid-metabolism group credit is non-targeted and targeting lipids metabolism group, and non-targeted lipid-metabolism is from the angle of global analysis by sample
All metabolites identify that the advantage of the research mode is to find discrepant metabolin, disadvantage comprehensively in this
Be to metabolin identification accuracy it is not high, semiquantitative determination can only be carried out.The ideal non-targeted lipid-metabolism group product of imitating are adopted
Collection and preparation method should meet: 1. non-selection to extract in sample containing metabolin as much as possible；2. simplicity is kept away quickly, as far as possible
Exempt from or reduce metabolin loss or degradation；3. method good reproducibility and stability；4. having sample cancellation step, to ensure to be metabolized wheel
Wide authenticity.And the characteristics of targeting metabolism group is then to carry out essence to one group of set metabolite using corresponding standard items
True qualitative and quantitative analysis, the two are combined with each other, so that lipid-metabolism group result of study is more perfect.
Due to biological sample scarcity and can not regaining property and available sample size it is seldom, and matrix is multiple
Miscellaneous, simultaneously because endogenous metabolism species are more, nature difference is big, how to be directed to a trace sample and passes through a kind of pre-treatment side
It is the difficult point studied that method, which is once analyzed and completes the measurement of all kinds of lipid-metabolism objects,.The life more for lipid contents such as liver organizations
Object sample, more common extracting method include classical Folch method and the Bligh-Dyer method that is derived by it, both sides
Method is to have the characteristics that easy to operate, extraction efficiency is high using chloroform and methanol as the two-phase extraction method of organic phase, but chloroform
Have the shortcomings that toxicity is big, be easy to cause the potential hazard to operator.
For tissue samples dosage is big, extraction efficiency is low, Extraction solvent toxicity existing for current common tissue extraction method
Greatly and the lipid quantity that tests and analyzes out in liver organization of existing detection method is few, and the few problem of type not yet mentions at present
Effective solution scheme out.
Summary of the invention
The main purpose of the application is to provide the extraction and detection method of lipid in a kind of organism liver organization, with solution
Certainly tissue samples dosage is big, extraction efficiency is low, Extraction solvent toxicity is big and existing existing for common tissue extraction method at present
The lipid quantity that detection method tests and analyzes out in liver organization is few, the few problem of type.
To achieve the goals above, according to the one aspect of the application, lipid in a kind of organism liver organization is provided
Extracting method, comprising the following steps: take liver organization to carry out tissue homogenate；Methyl tertiary butyl ether(MTBE) is added and methanol carries out lipid
Extraction；Take upper organic phase that methyl tertiary butyl ether(MTBE) and methanol reextraction are added again；Upper organic phase is taken, lipid is completed and mentions
It takes.
Further, the tissue refining step includes: to accurately weigh liver organization in centrifuge tube plus water, in centrifuge tube
Steel ball is added, interval homogenate is cooling when interval, takes out steel ball after homogenate, obtains uniform hepatic homogenate liquid.
Further, be added steel ball quantity be every centrifuge tube 2~3, tissue homogenate when revolving speed be 2000~
3000rpm is homogenized 5~10s every time, is homogenized again after being spaced 5~10s, is homogenized 10~12 times repeatedly, and centrifuge tube is placed in by when interval
It is cooling in ice cube.
Further, the ratio that the methyl tertiary butyl ether(MTBE) and methanol is added is 5:1~6:1.
Further, the extraction step includes: that 30~40s of vortex after methyl tertiary butyl ether(MTBE) and methanol is added, ultrasound 10
~20min is centrifuged 10~15min under 3000~4000rpm revolving speed.
To achieve the goals above, according to further aspect of the application, rouge in a kind of organism liver organization is provided
The detection method of matter carries out pre-treatment to the lipid in organism liver organization using above-mentioned extracting method.
Further, chromatograph-mass spectrometer coupling side is utilized after carrying out pre-treatment to the lipid in the organism liver organization
Method carries out lipid detection.
Further, dry, addition methanol redissolution, in 12300- will be blown to by upper organic phase nitrogen extracted twice
It is centrifuged 1-2min under 13000rpm revolving speed, takes supernatant sample introduction.
Further, it is detected using Q Exactive high-resolution LC-MS instrument.
Further, chromatographic condition are as follows: Hypersil Gold C18 chromatographic column, 50mm × 2.1m × 1.9 μm；Column temperature 40
℃；Positive ion mode: mobile phase A is acetonitrile: 10mM formic acid aqueous ammonium, and 0.1% formic acid is contained in the formic acid aqueous ammonium,
The ratio of acetonitrile and formic acid aqueous ammonium is 1.5:1, and mobile phase D is isopropanol: the ratio of acetonitrile, isopropanol and acetonitrile is 9:1；
Negative ion mode: flow velocity 0.3mL/min；Sampling volume is 2 μ L；Eluent gradient are as follows: mobile phase A is acetonitrile: 10mM acetic acid
The ratio of aqueous ammonium, acetonitrile and formic acid aqueous ammonium is 1.5:1, and mobile phase D is isopropanol: acetonitrile, isopropanol and acetonitrile
Ratio is 9:1；Gradient elution program: 0min, 85%A:15%D；0~2min, 70%A:30%D；2~2.5min, 52%A:
48%D；2.5~11min, 18%A:82%D；11~11.5min, 1%A:99%D；11.5~12min, 1%A:99%D；12
~12.1min, 85%A:15%D, 12.1~15min, 85%A:15%D；Mass Spectrometry Conditions are as follows: ionization mode is electron spray ion
Source, ion source temperature are 375 DEG C, ion spray voltage 3500V, sheath gas 40arb, and auxiliary gas is 10arb, blowback air
It is 50 for 0, S-lens RF；Assist gas pressure: 10arb, spraying gas and collision gas are nitrogen, and scanning mode uses Full MS/
DdMS2+Discovery, positive ion mode and negative ion mode distinguish sample introduction, this mode includes the level-one that resolution ratio is 70000
The second level that the resolution ratio of full scan and data dependence is 17500 scans, and scanning range is 100~1500m/z, and level-one scanning is automatic
Gain control is made as 1.0e6, and the ion implanting time is 100ms；The second level scanning automatic growth control of data dependence is set as 1.0e5,
Maximum ion injection length is set as 100ms, and isolation window is set as 3.0m/z, and collision energy is set as 40eV, and Loop count is set as
3, dynamic excludes to be set as 10.0s.
In the embodiment of the present application, using the methyl tertiary butyl ether(MTBE) of small toxicity and methanol as Extraction solvent to organism liver
Lipid extracts in dirty tissue, by this pretreatment mode, the non-selection quantity for extracting lipid in mouse liver tissue
More, it is more to cover lipoid substance type, has achieved the purpose that easy to operate, rapidly extracting, using the desk-top quadrupole of Q-Exactive
Bar-orbit trap high-resolution LC-MS instrument carries out retrieval analysis to the lipid extracted, and high resolution, mass number are accurate, sweep entirely
It is big to retouch high sensitivity, extraction characteristic peak quantity, detects 30 kinds of lipids, thousands of a lipoid substances, lipid metabolism object identification standard
True property is high.And then solve existing for common tissue extraction method at present that tissue samples dosage is big, extraction efficiency is low, Extraction solvent
The lipid quantity that toxicity is big and existing detection method tests and analyzes out in liver organization is few, the few problem of type.
Detailed description of the invention
The attached drawing constituted part of this application is used to provide further understanding of the present application, so that the application's is other
Feature, objects and advantages become more apparent upon.The illustrative examples attached drawing and its explanation of the application is for explaining the application, not
Constitute the improper restriction to the application.In the accompanying drawings:
Fig. 1 is that sample positive ion mode TIC schemes in the application experimental example 1；
Fig. 2 is that sample negative ion mode TIC schemes in the application experimental example 1；
Fig. 3 is that sample positive ion mode TIC schemes in the application experimental example 2；
Fig. 4 is that sample negative ion mode TIC schemes in the application experimental example 2；
Fig. 5 is that control sample positive ion mode TIC schemes in the application experimental example 3；
Fig. 6 is that control sample negative ion mode TIC schemes in the application experimental example 3；
Fig. 7 is that control sample positive ion mode TIC schemes in the application experimental example 4；And
Fig. 8 is that control sample negative ion mode TIC schemes in the application experimental example 4.
Specific embodiment
In order to make those skilled in the art more fully understand application scheme, below in conjunction in the embodiment of the present application
Attached drawing, the technical scheme in the embodiment of the application is clearly and completely described, it is clear that described embodiment is only
The embodiment of the application a part, instead of all the embodiments.Based on the embodiment in the application, ordinary skill people
Member's every other embodiment obtained without making creative work, all should belong to the model of the application protection
It encloses.
It should be noted that the term " includes " in the description and claims of this application and above-mentioned attached drawing is intended to
In cover it is non-exclusive include, for example, the process for containing series of steps those of is not necessarily limited to be clearly listed step, and
Being may include other steps being not clearly listed or intrinsic for these processes.
It should be noted that in the absence of conflict, the features in the embodiments and the embodiments of the present application can phase
Mutually combination.The application is described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.
Embodiment 1
The extracting method of lipid in a kind of organism liver organization, comprising the following steps:
(1) it accurately weighs liver organization and adds water in centrifuge tube, 2 steel balls are added in each centrifuge tube, when tissue is homogenized
Revolving speed is 2000rpm, is homogenized 10s every time, is homogenized again after being spaced 10s, is homogenized 12 times repeatedly, and centrifuge tube is placed in ice cube when interval
Middle cooling obtains uniform hepatic homogenate liquid；
(2) methyl tertiary butyl ether(MTBE) and methanol, ratio 5:1, vortex 30s, ultrasound are added in hepatic homogenate liquid
10min is centrifuged 15min under 3000rpm revolving speed；
(3) upper organic phase is taken to manage to new EP, repetitive operation step (2) completes reextraction, takes upper organic phase,
Complete lipids extraction.
Organic phase is blown to dry in nitrogen evaporator nitrogen after extracting twice, and methanol is added and redissolves, and 12300rpm is centrifuged 2min, takes
Sample introduction bottle is added in clear liquid.
Embodiment 2
The extracting method of lipid in a kind of organism liver organization, comprising the following steps:
(1) it accurately weighs liver organization and adds water in centrifuge tube, 3 steel balls are added in each centrifuge tube, when tissue is homogenized
Revolving speed is 3000rpm, is homogenized 5s every time, is homogenized again after being spaced 5s, is homogenized 10 times repeatedly, and centrifuge tube is placed in ice cube by when interval
Cooling obtains uniform hepatic homogenate liquid；
(2) methyl tertiary butyl ether(MTBE) and methanol, ratio 6:1, vortex 40s, ultrasound are added in hepatic homogenate liquid
20min is centrifuged 10min under 4000rpm revolving speed；
(3) upper organic phase is taken to manage to new EP, repetitive operation step (2) completes reextraction, takes upper organic phase,
Complete lipids extraction.
Organic phase is blown to dry in nitrogen evaporator nitrogen after extracting twice, and methanol is added and redissolves, and 13000rpm is centrifuged 1min, takes
Sample introduction bottle is added in clear liquid.
Embodiment 3
The detection method of lipid in a kind of organism liver organization, using the extracting method of embodiment 1 to organism liver
Lipid in tissue carries out pre-treatment, is detected using Q Exactive high-resolution LC-MS instrument, wherein
Chromatographic condition are as follows: Hypersil Gold C18 chromatographic column, 50mm × 2.1m × 1.9 μm；40 DEG C of column temperature；Cation
Mode eluent gradient are as follows: mobile phase A is acetonitrile: 10mM formic acid aqueous ammonium contains 0.1% first in the formic acid aqueous ammonium
The ratio of acid, acetonitrile and formic acid aqueous ammonium is 1.5:1, and mobile phase D is isopropanol: the ratio of acetonitrile, isopropanol and acetonitrile is
9:1；Negative ion mode: flow velocity 0.3mL/min；Sampling volume is 2 μ L；Eluent gradient are as follows: mobile phase A is acetonitrile: 10mM
The ratio of ammonium acetate solution, acetonitrile and formic acid aqueous ammonium is 1.5:1, and mobile phase D is isopropanol: acetonitrile, isopropanol and second
The ratio of nitrile is 9:1；Gradient elution program: 0min, 85%A:15%D；0~2min, 70%A:30%D；2~2.5min,
52%A:48%D；2.5~11min, 18%A:82%D；11~11.5min, 1%A:99%D；11.5~12min, 1%A:
99%D；12~12.1min, 85%A:15%D；12.1~15min, 85%A:15%D.
Mass Spectrometry Conditions are as follows: ionization mode is electric spray ion source, and ion source temperature is 375 DEG C, and ion spray voltage is
3500V, sheath gas 40arb, auxiliary gas are 10arb, and blowback air 0, S-lens RF is 50；Assist gas pressure: 10arb, spray
Fog and collision gas are nitrogen, and scanning mode uses Full MS/ddMS2+Discovery, positive ion mode and anion mould
Formula distinguishes sample introduction, this mode includes the second level that the resolution ratio of level-one full scan and data dependence that resolution ratio is 70000 is 17500
Scanning, scanning range are 100~1500m/z, and it is 1.0e6 that level-one, which scans automatic growth control, and the ion implanting time is 100ms；
The second level scanning automatic growth control of data dependence is set as 1.0e5, and maximum ion injection length is set as 100ms, and isolation window is set
For 3.0m/z, collision energy is set as 40eV, and Loop count is set as 3, and dynamic excludes to be set as 10.0s.
The extraction and analysis experiment 1 of lipid in 1 mouse liver tissue of experimental example
1. sample collection
Take out liver organizations from -80 DEG C of refrigerators and be placed in room temperature, accurately weigh liver organization 0.0413,0.0415,0.0421,
0.0415,0.0411,0.0422,0.0416,0.0422,0.0417,0.0415g, 10 samples are respectively placed in 2mL centrifuge tube
In.
2. tissue homogenate
Addition 200uL chromatographic grade water in each centrifuge tube, 3 steel balls of each centrifuge tube addition, tissue homogenate (2000rpm,
10s/10s, 12 times), it is homogenized 10s every time, interval 10s is homogenized again, and when interval is placed in ice cube cooling, steel ball is taken out after homogenate,
Uniform hepatic homogenate liquid is obtained, each centrifuge tube takes out 18ul and mixes, 2 QC parallel groups (QC1, QC2), every 10 samples
Product insert a QC；
3. extracting
QC and 10 sample is separately added into 400uL methyl tertiary butyl ether(MTBE) and 80ul methanol, whirlpool 30s, ultrasonic 10min；
3000rpm is centrifuged 15min, and there is white separating layer in centre, and 200ul upper organic phase methyl tertiary butyl ether(MTBE) is taken to manage to new EP, weight
Multiple 1 extraction；Organic phase is blown to dry in nitrogen evaporator (EYELA MG-2200, Japan) nitrogen after extracting twice, and 100ul methanol is added
It redissolves, 12300rpm is centrifuged 2min, takes supernatant liquor that sample introduction bottle is added (with 250ul internal lining pipe).
4. analysis
The Q Exactive high-resolution LC-MS instrument (LC:UHPLC of chromatograph selection ThermoFisher company
Dionex Ultimate 3000, MS:Q-Orbitrap mass spectrometer, USA)；Chromatographic column selection
ThermoFisher reverse phase C18 chromatographic column (Hypersil Gold C18 50mm × 2.1m × 1.9 μm Column, Thermo
Fisher, USA), column temperature is 40 DEG C；
Positive ion mode: mobile phase A is acetonitrile: water (+0.1% formic acid of 10mM ammonium formate)=60/40 (v/v), mobile phase D
For isopropanol: acetonitrile=90/10 (v/v)；
Negative ion mode flow velocity is 0.3mL/min, and sampling volume is 2 μ L；Eluent gradient are as follows: mobile phase A is acetonitrile: water
(10mM ammonium acetate)=60/40 (v/v), mobile phase D are isopropanol: acetonitrile=90/10 (v/v)；When batch sample introduction, 5-10 is first used
Needle QC stabilizer instrument, every 10 samples, into a needle QC, sampfe order upsets random sample introduction when sample introduction；
Gradient elution program: time (min) A (%) D (%): 0min, 85%A:15%D, 0~2min, 70%A:30%
D, 2~2.5min, 52%A:48%D, 2.5~11min, 18%A:82%D, 11~11.5min, 1%A:99%D, 11.5~
12min, 1%A:99%D, 12~12.1min, 85%A:15%D；12.1~15min, 85%A:15%D.
Wherein, Mass Spectrometry Conditions are as follows: ionization mode is electric spray ion source, and ion source temperature is 375 DEG C, ion spray voltage
For 3500V, sheath gas (Shealth gas) is 40arb, and auxiliary gas (Aux gas) is 10arb, blowback air (Sweep gas)
It is 50 for 0, S-lens RF；Assist gas pressure: 10arb, spraying gas and collision gas are nitrogen, and scanning mode uses Full MS/
The second level of ddMS2+Discovery, level-one full scan (resolution ratio 70000) and data dependence scans (resolution ratio 17500), scanning
Range is m/z100~1500, and it is 1.0e6 that level-one, which scans automatic growth control (AGC), and the ion implanting time (IT) is 100ms；
(AGC) of the second level scanning (dd-MS2) of data dependence is set as 1.0e5, and maximum IT is set as 100ms, and isolation window is set as 3.0m/
Z, (N) CE collision energy are set as 40, Loop count and are set as 3, and dynamic excludes to be set as 10.0s.
5. interpretation of result
Such as (abscissa Time is retention time, ordinate Relaive Abundance to Fig. 1 to sample positive ion mode TIC figure
For relative abundance) shown in: positive ion mode response is up to 4.13E9, can extract 19187 characteristic peaks, 2724 features
Peak is identified, wherein 1026 characteristic peaks have MS2 information.
Such as (abscissa Time is retention time, ordinate Relaive Abundance to Fig. 2 to sample negative ion mode TIC figure
For relative abundance) shown in: the response of negative ion mode highest reaches 1.83E10, can extract 13140 characteristic peaks, 1583 spies
It is identified to levy peak, wherein 921 characteristic peaks have MS2 information.
It can be seen that go out lipid quantity more for the non-targeted iipidomics analysis based on Q Exactive high-resolution LC-MS instrument, contain
30 kinds of lipids of lid (including SM, PS, PE, PC, HexCer, DGTS, Cer, SM, TAG, SQDG, PG, MGDG, G1, DGDG, DAG,
CL, BMP, Acy, MAG, LPE, LPC, LDGTS, CE, ACar, FAHFA, OxFA, OxPC, OxPE, PA, PG), high sensitivity, essence
Exactness is high.
The extraction and analysis experiment 2 of lipid in 2 mouse liver tissue of experimental example
1. sample collection
Take out liver organizations from -80 DEG C of refrigerators and be placed in room temperature, accurately weigh liver organization 0.0456,0.0452,0.0446,
0.0447、0.0452、0.0461、0.0449、0.0443、0.0453、0.0457、0.0456、0.0445、0.0442、0.0457、
0.0442,0.0451,0.0459,0.0461,0.0453,0.0455g, 20 samples are respectively placed in 2mL centrifuge tube.
2. tissue homogenate
Each centrifuge tube addition 200uL chromatographic grade water, 3 steel balls of each centrifuge tube addition, tissue homogenate (2000rpm,
10s/10s, 12 times), it is homogenized 10s every time, interval 10s is homogenized again, and when interval is placed in ice cube cooling, steel ball is taken out after homogenate,
Uniform hepatic homogenate liquid is obtained, each centrifuge tube takes out 18ul and mixes, 3 QC parallel groups (QC1, QC2, QC3), and every 10
A sample inserts a QC；
3. extracting
QC and sample are separately added into 400uL methyl tertiary butyl ether(MTBE) and 80ul methanol, whirlpool 30s, ultrasonic 10min；3000rpm
It is centrifuged 15min, there is white separating layer in centre, takes 200ul upper organic phase methyl tertiary butyl ether(MTBE) to manage to new EP, is repeated 1 times extraction
It takes；Organic phase is blown to dry in nitrogen evaporator (EYELA MG-2200, Japan) nitrogen after extracting twice, and 100ul methanol is added and redissolves,
12300rpm is centrifuged 2min, takes supernatant liquor that sample introduction bottle is added (with 250ul internal lining pipe).
4. analysis
The Q Exactive high-resolution LC-MS instrument (LC:UHPLC of chromatograph selection ThermoFisher company
Dionex Ultimate 3000, MS:Q-Orbitrap mass spectrometer, USA)；Chromatographic column selection
ThermoFisher reverse phase C18 chromatographic column (Hypersil Gold C18 50mm × 2.1m × 1.9 μm Column, Thermo
Fisher, USA), column temperature is 40 DEG C；
Positive ion mode: mobile phase A is acetonitrile: water (+0.1% formic acid of 10mM ammonium formate)=60/40 (v/v), mobile phase D
For isopropanol: acetonitrile=90/10 (v/v)；
Negative ion mode flow velocity is 0.3mL/min, and sampling volume is 2 μ L；Eluent gradient are as follows: mobile phase A is acetonitrile: water
(10mM ammonium acetate)=60/40 (v/v), mobile phase D are isopropanol: acetonitrile=90/10 (v/v)；When batch sample introduction, 5-10 is first used
Needle QC stabilizer instrument, every 10 samples, into a needle QC, sampfe order upsets random sample introduction when sample introduction；
Gradient elution program: time (min) A (%) D (%): 0min, 85%A:15%D, 0~2min, 70%A:30%
D, 2~2.5min, 52%A:48%D, 2.5~11min, 18%A:82%D, 11~11.5min, 1%A:99%D, 11.5~
12min, 1%A:99%D, 12~12.1min, 85%A:15%D；12.1~15min, 85%A:15%D.
Wherein, Mass Spectrometry Conditions are as follows: ionization mode is electric spray ion source, and ion source temperature is 375 DEG C, ion spray voltage
For 3500V, sheath gas (Shealth gas) is 40arb, and auxiliary gas (Aux gas) is 10arb, blowback air (Sweep gas)
It is 50 for 0, S-lens RF；Assist gas pressure: 10arb, spraying gas and collision gas are nitrogen, and scanning mode uses Full MS/
The second level of ddMS2+Discovery, level-one full scan (resolution ratio 70000) and data dependence scans (resolution ratio 17500), scanning
Range is m/z100~1500, and it is 1.0e6 that level-one, which scans automatic growth control (AGC), and the ion implanting time (IT) is 100ms；
(AGC) of the second level scanning (dd-MS2) of data dependence is set as 1.0e5, and maximum IT is set as 100ms, and isolation window is set as 3.0m/
Z, (N) CE collision energy are set as 40, Loop count and are set as 3, and dynamic excludes to be set as 10.0s.
5. interpretation of result
Such as (abscissa Time is retention time, ordinate Relaive Abundance to Fig. 3 to sample positive ion mode TIC figure
For relative abundance) shown in: positive ion mode response is up to 5.04E9, can extract 18765 characteristic peaks, 2810 features
Peak is identified, wherein 1102 characteristic peaks have MS2 information；
Such as (abscissa Time is retention time, ordinate Relaive Abundance to Fig. 4 to sample negative ion mode TIC figure
For relative abundance) shown in: the response of negative ion mode highest reaches 1.47E10, can extract 12950 characteristic peaks, 1497 spies
It is identified to levy peak, wherein 892 characteristic peaks have MS2 information；
It can be seen that go out lipid quantity big for the non-targeted iipidomics analysis based on Q Exactive high-resolution LC-MS instrument, contain
27 kinds of lipids of lid (including SM, PS, PE, PC, HexCer, DGTS, Cer, SM, TAG, SQDG, PG, MGDG, G1, DGDG, DAG,
CL, BMP, Acy, MAG, LPE, LPC, LDGTS, CE, ACar, GlcADG, SHexCer, GM3), high sensitivity, accuracy height.
The extraction effect control experiment of lipid in 3 mouse liver tissue of experimental example
One, experimental method
Laboratory sample is divided into experimental group and control group:
Experimental group is handled according to the method recorded in experimental example 1；
It is constant that other steps are controlled in control group control experimental example 1, organize homogenate and extraction step as follows:
Methanol is added in the centrifuge tube for being loaded with accurate mass liver organization: water (4:3), each centrifuge tube are added 3
Steel ball, tissue homogenate (2000rpm, 10s/10s, 12 time), is homogenized 10s every time, and interval 10s is homogenized again, and when interval is placed in ice cube
Middle cooling takes out steel ball after homogenate, obtains uniform hepatic homogenate liquid, 800uL chloroform recovery is added, homogenate is at 37 degree
20min is incubated, 12300rpm is centrifuged 20min, and lower layer's organic phase is transferred to new EP pipe, and 100ul methanol is added in vacuum drying:
Chloroform (1:1) solution redissolves, and 12300rpm is centrifuged 2min, takes supernatant liquor that sample introduction bottle is added (with 250ul internal lining pipe).
Two, experimental result
Experimental group experimental result is referring to experimental example 1.
Control group: such as (abscissa Time is retention time, ordinate Relaive to Fig. 5 to sample positive ion mode TIC figure
Abundance is relative abundance) shown in: positive ion mode response is up to 1.36E9, can extract 9765 characteristic peaks,
1010 characteristic peaks are identified, wherein 509 characteristic peaks have MS2 information；Sample negative ion mode TIC figure such as Fig. 6 (abscissa
Time is retention time, and ordinate Relaive Abundance is relative abundance) shown in: the response of negative ion mode highest reaches
4.68E9 can extract 6653 characteristic peaks, and 797 characteristic peaks are identified, wherein 263 characteristic peaks have MS2 information.
Three, interpretation of result
Shown according to the comparison of above-mentioned experimental result compared to by the way of chloroform recovery, using liver provided by the present application
Tissue lipid extracting method identifies the characteristic peak quantity come and obviously increases, it was demonstrated that the non-selective extraction of method provided by the present application
The lipid quantity taken out in sample is more, and it is more to cover lipoid substance type, mentions high-efficient, replaces chlorine using the lesser MTBE of toxicity
It is imitative to carry out lipids extraction, reduce the potential hazard to operator.
The extraction and analysis effect comparison experiment of lipid in 4 mouse liver tissue of experimental example
One, experimental method
Laboratory sample is divided into experimental group and control group:
Experimental group is handled according to the method recorded in experimental example 1；
Control that other steps are constant, and instrument condition is as follows in control group control experimental example 1:
The Q Exactive high-resolution LC-MS instrument (LC:UHPLC of chromatograph selection ThermoFisher company
Dionex Ultimate 3000, MS:Q-Orbitrap mass spectrometer, USA)；Chromatographic column selection
ThermoFisher reverse phase C18 chromatographic column (Hypersil Gold C18 50mm × 2.1m × 1.9 μm Column, Thermo
Fisher, USA), column temperature is 40 DEG C；
Positive ion mode: mobile phase A is 0.1% formic acid water, and mobile phase D is acetonitrile；
Negative ion mode flow velocity is 0.3mL/min, and sampling volume is 2 μ L；Eluent gradient are as follows: mobile phase A 0.05%
Ammonium hydroxide, mobile phase D are acetonitrile；When batch sample introduction, 5-10 needle QC stabilizer instrument is first used, every 10 samples, into a needle QC, when sample introduction
Sampfe order upsets random sample introduction；
Gradient elution program: time (min) A (%) D (%): 0min, 85%A:15%D, 0~2min, 70%A:30%
D, 2~2.5min, 52%A:48%D, 2.5~11min, 18%A:82%D, 11~11.5min, 1%A:99%D, 11.5~
12min, 1%A:99%D, 12~12.1min, 85%A:15%D；12.1~15min, 85%A:15%D.
Wherein, Mass Spectrometry Conditions are as follows: ionization mode is electric spray ion source, and ion source temperature is 375 DEG C, ion spray voltage
For 3500V, sheath gas (Shealth gas) is 40arb, and auxiliary gas (Aux gas) is 10arb, blowback air (Sweep gas)
It is 50 for 0, S-lens RF；Assist gas pressure: 10arb, spraying gas and collision gas are nitrogen, and scanning mode uses Full MS/
The second level of ddMS2+Discovery, level-one full scan (resolution ratio 70000) and data dependence scans (resolution ratio 17500), scanning
Range is m/z100~1500, and it is 1.0e6 that level-one, which scans automatic growth control (AGC), and the ion implanting time (IT) is 100ms；
(AGC) of the second level scanning (dd-MS2) of data dependence is set as 1.0e5, and maximum IT is set as 100ms, and isolation window is set as 3.0m/
Z, (N) CE collision energy are set as 30, Loop count and are set as 1, and dynamic excludes to be set as 10.0s.
Two, experimental result
Experimental group experimental result is referring to experimental example 1.
Control group: such as (abscissa Time is retention time, ordinate Relaive to Fig. 7 to sample positive ion mode TIC figure
Abundance is relative abundance) shown in, extractable 12569 characteristic peaks out of positive ion mode, 2012 characteristic peaks are identified,
Wherein 865 characteristic peaks have MS2 information；(abscissa Time is retention time, ordinate to sample positive ion mode TIC such as Fig. 8
Relaive Abundance is relative abundance) shown in shown in, sample negative ion mode it is extractable go out it is extractable go out 9956 features
Peak, 1236 characteristic peaks are identified, wherein 612 characteristic peaks have MS2 information；
Three, interpretation of result
When showing to carry out instrument analysis using the instrument condition of control group according to the comparison of above-mentioned experimental result, identify
Characteristic peak quantity is less slightly compared to experimental group, but still is able to achieve preferable analysis recognition effect, and using in the application experimental example 1
It is higher that the instrument condition of offer carries out lipid identification accuracy and sensitivity when instrument analysis.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field
For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair
Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.
Claims (10)
1. the extracting method of lipid in a kind of organism liver organization, which comprises the following steps:
Liver organization is taken to carry out tissue homogenate；Methyl tertiary butyl ether(MTBE) is added and methanol carries out lipid extraction；Take upper organic phase again
Secondary addition methyl tertiary butyl ether(MTBE) and methanol reextraction；Upper organic phase is taken, lipids extraction is completed.
2. the extracting method of lipid in organism liver organization according to claim 1, which is characterized in that the tissue is even
Starching step includes:
Liver organization is accurately weighed in centrifuge tube plus water, is added steel ball in centrifuge tube, interval homogenate, cooling, homogenate when interval
After take out steel ball, obtain uniform hepatic homogenate liquid.
3. the extracting method of lipid in organism liver organization according to claim 2, which is characterized in that steel ball is added
Quantity is every centrifuge tube 2~3, and revolving speed is 2000~3000rpm when tissue is homogenized, and is homogenized 5~10s every time, is spaced 5~10s
It is homogenized, is homogenized 10~12 times repeatedly again afterwards, centrifuge tube is placed in ice cube when interval cooling.
4. the extracting method of lipid in organism liver organization according to claim 1, which is characterized in that the first is added
The ratio of base tertbutyl ether and methanol is 5:1~6:1.
5. the extracting method of lipid in organism liver organization according to claim 1, which is characterized in that the extraction step
It suddenly include: that 30~40s of vortex, 10~20min of ultrasound, in 3000~4000rpm revolving speed after methyl tertiary butyl ether(MTBE) and methanol is added
10~15min of lower centrifugation.
6. the detection method of lipid in a kind of organism liver organization, which is characterized in that utilize any one of claim 1 to 5 institute
The extracting method stated carries out pre-treatment to the lipid in organism liver organization.
7. the detection method of lipid in organism liver organization according to claim 6, which is characterized in that the biology
Lipid in body liver organization utilizes the progress lipid detection of chromatograph-mass spectrometer coupling method after carrying out pre-treatment.
8. the detection method of lipid in organism liver organization according to claim 7, which is characterized in that will extract twice
Upper organic phase nitrogen afterwards be blown to it is dry, be added methanol redissolve, be centrifuged 1-2min under 12300-13000rpm revolving speed, take supernatant
Sample introduction.
9. the detection method of lipid in organism liver organization according to claim 7, which is characterized in that use Q
Exactive high-resolution LC-MS instrument is detected.
10. the detection method of lipid in organism liver organization according to claim 9, which is characterized in that
Chromatographic condition are as follows: Hypersil Gold C18 chromatographic column, 50mm × 2.1m × 1.9 μm；40 DEG C of column temperature；Positive ion mode:
Mobile phase A is acetonitrile: 10mM formic acid aqueous ammonium, and 0.1% formic acid, acetonitrile and ammonium formate water are contained in the formic acid aqueous ammonium
The ratio of solution is 1.5:1, and mobile phase D is isopropanol: the ratio of acetonitrile, isopropanol and acetonitrile is 9:1；Negative ion mode: stream
Speed is 0.3mL/min；Sampling volume is 2 μ L；Eluent gradient are as follows: mobile phase A is acetonitrile: 10mM ammonium acetate solution, acetonitrile
Ratio with formic acid aqueous ammonium is 1.5:1, and mobile phase D is isopropanol: the ratio of acetonitrile, isopropanol and acetonitrile is 9:1；Gradient
Elution program: 0min, 85%A:15%D；0~2min, 70%A:30%D；2~2.5min, 52%A:48%D；2.5~
11min, 18%A:82%D；11~11.5min, 1%A:99%D；11.5~12min, 1%A:99%D；12~12.1min,
85%A:15%D；12.1~15min, 85%A:15%D.
Mass Spectrometry Conditions are as follows: ionization mode is electric spray ion source, and ion source temperature is 375 DEG C, ion spray voltage 3500V,
Sheath gas is 40arb, and auxiliary gas is 10arb, and blowback air 0, S-lens RF is 50；Assist gas pressure: 10arb, spraying gas and
Collision gas is nitrogen, and scanning mode uses Full MS/ddMS2+Discovery, positive ion mode and negative ion mode difference
Sample introduction, this mode include the second level scanning that the resolution ratio of level-one full scan and data dependence that resolution ratio is 70000 is 17500,
Scanning range is 100~1500m/z, and it is 1.0e6 that level-one, which scans automatic growth control, and the ion implanting time is 100ms；Data according to
Bad second level scanning automatic growth control is set as 1.0e5, and maximum ion injection length is set as 100ms, and isolation window is set as 3.0m/
Z, collision energy are set as 40eV, and Loop count is set as 3, and dynamic excludes to be set as 10.0s.
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|---|---|---|---|---|
| CN112326821A (en) * | 2020-10-27 | 2021-02-05 | 杨振全 | Method for detecting lipid content of biological liver tissue |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103776911A (en) * | 2012-10-22 | 2014-05-07 | 中国科学院大连化学物理研究所 | Method for simultaneous extraction and analysis of metabolite group and lipid group in microtissue |
| CN103884782A (en) * | 2012-12-21 | 2014-06-25 | 中国科学院大连化学物理研究所 | Large-scale blood plasma sphingolipid profile analysis method based on liquid chromatography-mass spectrometry combination |
| US20160102060A1 (en) * | 2004-07-19 | 2016-04-14 | Philera New Zealand Limited | Synthesis of Triethylenetetramines |
-
2018
- 2018-12-26 CN CN201811603921.1A patent/CN109459518A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160102060A1 (en) * | 2004-07-19 | 2016-04-14 | Philera New Zealand Limited | Synthesis of Triethylenetetramines |
| CN103776911A (en) * | 2012-10-22 | 2014-05-07 | 中国科学院大连化学物理研究所 | Method for simultaneous extraction and analysis of metabolite group and lipid group in microtissue |
| CN103884782A (en) * | 2012-12-21 | 2014-06-25 | 中国科学院大连化学物理研究所 | Large-scale blood plasma sphingolipid profile analysis method based on liquid chromatography-mass spectrometry combination |
Non-Patent Citations (2)
| Title |
|---|
| TOMAS CAJKA等: "Comprehensive analysis of lipids in biological systems by liquid chromatography-mass spectrometry", 《TRENDS IN ANALYTICAL CHEMISTRY》 * |
| 朱宗敏等: "高血压病肝阳上亢证大鼠肝脏的脂质组学分析", 《中华中医药杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112326821A (en) * | 2020-10-27 | 2021-02-05 | 杨振全 | Method for detecting lipid content of biological liver tissue |
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