CN109387585A - The method of content of fatty acid in gas-chromatography, mass spectrometric hyphenated technique detection nematode - Google Patents
The method of content of fatty acid in gas-chromatography, mass spectrometric hyphenated technique detection nematode Download PDFInfo
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Abstract
The invention discloses a kind of methods of content of fatty acid in gas-chromatography, mass spectrometric hyphenated technique detection nematode, belong to technical field of analytical chemistry.Method includes the following steps: (1) prepares standard items, it is detected with gas-chromatography, mass spectrometry combination method, and be compared with mass spectrometry database, obtain the corresponding fatty acid component of different retention times, when similarity >=95% is confirmed as this kind of compound；(2) it prepares standard mixed liquor and establishes gas-chromatography, mass spectrometry qualitative-and-quantitative method with internal standard method；(3) nematode sample is pre-processed, analysis sample is obtained；(4) gas-chromatography, mass spectrometry qualitative-and-quantitative method are established using step (2) and analysis detection is carried out to analysis sample to be measured；(5) content for determining each fatty acid in nematode sample is calculated.Detection method detection speed is fast, and detection limits low, high sensitivity, suitable for quickly analyzing the composition of the fatty acid nematode and content.
Description
Technical field
The invention belongs to technical field of analytical chemistry, in particular to a kind of gas-chromatography, mass spectrometric hyphenated technique detect nematode
The method of middle content of fatty acid.
Background technique
Its life cycle of nematode is very of short duration convenient for periodically observation with respect to for other experimental animals；Condition of culture is very
Simply, it is suitable for laboratory cultures；Small, breeding amount is big, guarantees the demand of experiment sample；And it is that one kind has complicated regulation
Multi-celled eukaryotes, mean that it may have similar cellular elements structure and control access with higher mammal.Because it
These unique biological characteristics are widely used in the research of life medical disciplines, are a kind of outstanding model organisms.
The intracorporal polyunsaturated fatty acid synthesis access of nematode combines the process of plant and animal, with higher mammal phase
Than the ability with complete de novo formation polyunsaturated fatty acid is suitble to carry out the relevant all kinds of metabolism groups of free fatty acid
Learn research.Further find the mechanism on higher mammal by studying the intracorporal mechanism of action of the online worm of these substances, for
Further investigation afterwards provides accurate experimental data and theoretical foundation.
Carry out the relevant all kinds of metabolism group researchs of free fatty acid in a deep going way, need it is efficient, stable, can detect simultaneously
The analysis determining method of a variety of fatty acid.It is most common analysis free fatty acid method have high performance liquid chromatography, near-infrared with
And the analysis methods such as gas chromatographic flame ionization detector.However, efficient liquid-phase chromatography method detects free fatty acid, utilize
Time-consuming for column front derivation, and related coefficient is below 80% in the measurement of near-infrared transmittance analysis, as a result relatively poor,
The response time that gas chromatographic flame ionization detector detects free fatty acid is long, and sensitivity is lower.
Summary of the invention
The embodiment of the invention provides a kind of sides of content of fatty acid in gas-chromatography, mass spectrometric hyphenated technique detection nematode
Method, time-consuming, result is relatively poor, the response time is long and sensitivity is lower to solve existing detection and analysis measuring method asks
Topic quickly analyzes fatty acid composition and content in nematode.The technical solution is as follows:
The embodiment of the invention provides a kind of sides of content of fatty acid in gas-chromatography, mass spectrometric hyphenated technique detection nematode
Method, method includes the following steps:
(1) prepare standard items, detected with gas-chromatography, mass spectrometry combination method, obtain each fatty acid retention time and
GC-MS map, and comparing with mass spectrometry database obtains the corresponding fatty acid component of different retention times；
(2) it prepares standard mixed liquor and establishes gas-chromatography, mass spectrometry qualitative-and-quantitative method with internal standard method, it is described qualitative
Quantitative approach is that the mass spectrum of target compound is retrieved and compared in mass spectral database, to be measured while under the conditions of identical GC-MS
Retention time and the mass spectrum comparison that desired fatty acid and analytical standard sample obtain in object are qualitative, and when similarity >=95% is confirmed as
This kind of compound, quantitative approach are internal standard method；
(3) nematode sample is pre-processed, analysis sample is obtained；
(4) using step (2) establish gas-chromatography, mass spectrometry qualitative-and-quantitative method analyzes analysis sample to be measured
Detection, must analyze the retention time and GC-MS map of each fatty acid of sample；
(5) content for determining each fatty acid in nematode sample is calculated, the content of each fatty acid is pressed in the nematode sample
Formula calculates:
Mi: the content of fatty acid methyl ester in sample；As: the peak area of internal standard compound in fatty acid methyl ester standard specimen；Ai: in sample
The peak area of fatty acid methyl ester；Asi: the peak area of internal standard compound in sample；Ar: the peak of fatty acid methyl ester in fatty acid methyl ester standard specimen
Area；Mr: fatty acid methyl ester in fatty acid methyl ester standard specimen.
In a kind of implementation of the invention, standard items described in step (1) are methyl hexadecanoate 16:0, palmitoleic acid
Methyl esters 16:1,18 hard acid methyl esters 18:0, methyl oleate 18:1n9, linolenic acid methyl esters 18:2n6, alpha-linolenic acid methyl esters 18:
3n3, parinaric acid methyl esters 18:4n3, arachidic acid methylester 20:4n6, eicosanoid methyl esters 20:5n3.
In a kind of implementation of the invention, the internal standard compound that the internal standard method is selected is pentadecane alkanoic acid methyl esters C15:0.
In a kind of implementation of the invention, standard Compound mixed solution described in step (2):
Fatty acid methyl ester standard items in step (1) are taken respectively, and the standard inventory of 9 kind of 100 μ g/mL is configured to n-hexane
9 kinds of standard reserving solutions are configured to 9 kinds of fatty acid methyl ester standard mixed liquors of 10 μ g/mL with the n-hexane, are used in combination by liquid
N-hexane dilutes step by step, and it is mixed to obtain 9 kinds of standards that mass concentration gradient is 0.1,0.2,0.5,1,2,5,10,20 μ g/mL
Solution is closed, wherein containing the internal standard standard items C15 of 1 μ g/mL in 9 kinds of standard mixed solutions of each concentration gradient:
0。
In a kind of implementation of the invention, the pretreatment of nematode sample described in step (3):
Adult nematodes are cleaned up into acquisition nematode sample, it is sufficiently mixed that the internal standard solution is added in Xiang Suoshu nematode sample
It is even, add H2SO4/ MeOH solution brings it about esterification reaction of organic acid, and the analysis sample is obtained by extraction to the end of reacting.
In a kind of implementation of the invention, it is described obtain nematode sample method be take adult nematodes in 1.5mL from
In heart pipe, supernatant culture medium is removed with 3500r/min revolving speed centrifugation 2min, adds 1mL PBS to clean, in triplicate.
In a kind of implementation of the invention, the H2SO4/MeOH solution concentration is 1~10%, H2SO4/ MeOH solution
Volume is 750 μ L.
In a kind of implementation of the invention, 55 DEG C~85 DEG C of the esterification bath temperature, the esterification time 1
~3h.
In a kind of implementation of the invention, gas-chromatography described in step (1), gas phase item in mass spectrometry combination method
Part: chromatographic column selects highly polar DB-WAX (30m, internal diameter 0.25mm apply 0.25 μm of thickness)；50 DEG C of chromatographic column initial temperature,
Keep 1min；180 DEG C are warming up to the rate of 20 DEG C/min again, keeps 5min；Finally 230 are warming up to the rate of 8 DEG C/min
DEG C, keep 15min；Carrier gas is high-purity helium, 0.5~1.5mL/min constant current flow velocity, using split sampling, split ratio 20:1~
50:1；1 μ L of sample volume, injector temperature are 200~250 DEG C.
In a kind of implementation of the invention, gas-chromatography described in step (1), Mass Spectrometry Conditions in mass spectrometry combination method
Are as follows: EI electron impact ion source, electron energy 70eV；Ion source temperature: 200~250 DEG C；Interface temperature: 200~250 DEG C, molten
Agent postpones 3.5min, using selection ion scan mode.
Technical solution provided in an embodiment of the present invention has the benefit that
The side of content of fatty acid in a kind of gas-chromatography provided in an embodiment of the present invention, mass spectrometric hyphenated technique detection nematode
In method, with reagent by the free-fat low-kappa number in nematode in preprocessing process, with internal standard method, establish gas-chromatography,
Mass spectrometry qualitative-and-quantitative method, using gas-chromatography, mass spectrometry qualitative-and-quantitative method to reference standards and nematode to be measured
It analyzes sample and carries out analysis detection, calculate the content for determining each fatty acid in nematode sample, main 9 kinds from 15 carbon to 20 carbon rouge
Fat acid can be separated preferably, can be realized satisfactory results in 25min.The detection method has time-consuming short, pollution
The advantages of small, detection limit low, high sensitivity, suitable for quickly analyzing the composition of the fatty acid nematode and content.
Detailed description of the invention
To describe the technical solutions in the embodiments of the present invention more clearly, make required in being described below to embodiment
Attached drawing is briefly described, it should be apparent that, drawings in the following description are only some embodiments of the invention, for
For those of ordinary skill in the art, without creative efforts, it can also be obtained according to these attached drawings other
Attached drawing.
Fig. 1 is the total ion current figure of GC-MS detection provided in an embodiment of the present invention.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with attached drawing to embodiment party of the present invention
Formula is described in further detail.
Detecting instrument: chromatographic system: 1300 series of modular gas chromatograph of TRACE (U.S. Thermo Fisher)；
Mass spectrometer system: ISQTMThe mono- quadrupole rod GC-MS system of LT (U.S. Thermo Fisher)
Instrument testing conditions:
Chromatographic condition
Chromatographic column: DB-WAX (30m × 0.25mm × 0.25 μm, U.S. Agilent)；
Carrier gas: high-purity helium, 1.0mL/min, constant current flow velocity；
Input mode: split sampling, split ratio 50:1；1 μ L of sample volume；
Injector temperature: 220 DEG C；
Temperature programming: 50 DEG C of initial temperature, 1min is kept；180 DEG C are warming up to the rate of 20 DEG C/min again, is kept
5min；230 DEG C finally are warming up to the rate of 8 DEG C/min, keeps 15min.
Mass Spectrometry Conditions
Ionization mode: electron impact ion source (EI), electron energy 70eV；
Ion source temperature: 230 DEG C；
Transmission line interface temperature: 230 DEG C；
Scan pattern: selection ion scan；
Solvent delay: 3.5min.
The embodiment of the invention provides a kind of methods of content of fatty acid in gas-chromatography, mass spectrometric hyphenated technique detection nematode
In, specific steps include:
(1) fatty acid methyl ester standard items: methyl hexadecanoate (16:0) are taken respectively, and Methyl palmitoleinate (16:1), 18 is hard
Sour methyl esters (18:0), methyl oleate (18:1n9), linolenic acid methyl esters (18:2n6), alpha-linolenic acid methyl esters (18:3n3), 18
Carbon tetraenoic acid methyl esters (18:4n3), arachidic acid methylester (20:4n6), eicosanoid methyl esters (20:5n3) (purity >=
98.5%) it, is configured to the standard reserving solution of 9 kind of 100 μ g/mL with n-hexane, is placed in -20 DEG C of refrigerators and saves.
(2) the internal standard product selected using pentadecane alkanoic acid methyl esters C15:0 as internal standard method take internal standard standard items pentadecane
Sour methyl esters C15:0 obtains the inner mark solution of 100 μ g/mL using n-hexane as solvent dissolved dilution.By 9 kinds of standard reserving solution use just oneself
Alkane is configured to 9 kinds of fatty acid methyl ester standard mixed liquors of 10 μ g/mL, and is diluted step by step with n-hexane, obtains mass concentration gradient
For 9 kinds of standard mixed solutions of 0.1,0.2,0.5,1,2,5,10,20 μ g/mL, wherein 9 kinds of standards of each concentration gradient mix
Contain the internal standard standard items C15:0 of 1 μ g/mL in solution.
(3) in 1.5mL centrifuge tube, 3500r/min is centrifuged 2min and removes supernatant culture medium line taking worm, adds 1mL PBS clear
It washes, in triplicate, to guarantee that Escherichia coli are cleaned.
It takes the nematode sample cleaned up in 4mL glass tube, adds 1 μ g internal standard solution (10 μ g/mL C15:0 fatty acid methyls
Rouge) it mixes well.Again plus 750 μ L 5%H2SO4/ MeOH solution, being vortexed to mix is placed on 80 DEG C of water-bath 2h, brings it about esterification
Reaction takes out shaking once every 30min during reaction.Fully reacting to be esterified and after being cooled to room temperature, is added 500 μ L
0.9% NaCl solution is vortexed and mixes.
300 μ L n-hexanes are added in the above solution to be extracted, oscillation mixing 2min, after standing, 3000r/min centrifugation
10min takes supernatant, repeats to extract 3 times, the supernatant extracted three times merging is placed in 1.5mL centrifuge tube, with 12000r/
Min is centrifuged 10min, takes supernatant, is settled to 1ml with n-hexane, obtains analysis sample, -20 DEG C of freezen protectives is placed, to GC-MS
Detection.
(4) fatty acid methyl ester standard items are injected into gas-chromatography, mass spectrometer is measured, scanning acquisition total ion current
Figure.
9 kinds of standard mixed solutions that mass concentration gradient is 0.1,0.2,0.5,1,2,5,10,20 μ g/mL are injected into gas phase
Chromatography, mass spectrometer are measured, scan standard mixed solution GC-MS figure, the GC-MS of reference standard mixed solution
Figure obtains standard curve, establishes GC-MS qualitative-and-quantitative method.
(5) the fatty acid methyl ester total ion current figure detected according to GC-MS, obtains the retention time and GC-MS of each fatty acid
Map, and comparing with mass spectrometry database, obtains the corresponding fatty acid component of different retention times, and when similarity >=95% is true
Think this kind of compound.The retention time and integral area of chromatogram are automatically performed by work station, compose the qualitative color in library using NIST
Spectral peak.The relative amount of each fatty acid component is obtained by area normalization method, and absolute content is by corresponding peak area and internal standard
The ratio of object peak area is obtained multiplied by the concentration of internal standard compound.Table 1 is the GC-MS parameter of 10 kinds of fatty acid methyl esters, referring to table 1, originally
The peak for detecting identifiable fatty acid methyl ester in sample is 10.
1 10 kinds of fatty acid methyl ester GC-MS parameters of table
(6) the broad peak area in the GC-MS map of each fatty acid is integrated and is summed it up respectively, obtain corresponding concentration
GC-MS signal response uses the ratio of target analysis sample object concentration and internal standard compound concentration as abscissa, with GC-MS signal
The ratio between response is used as ordinate, and the standard curve of each target analysis sample object can be obtained.The quantitative internal standard of all fatty acid, line
Property related coefficient and instrument detection limit are shown in Table 2.Wherein concentration of the signal-to-noise ratio (S/N) equal to 3 is defined as instrument detection limit.From table 2
It is found that fatty acid instrument detection limit, between 1.11~27.6ng/mL, the range of quantitative limit is 3.69~92.0ng/mL, linearly
Related coefficient is all larger than 0.99, have preferable linear relationship, the range of linearity is wider (0.1~10 μ g/mL), be able to detect that compared with
The target compound of low concentration.
The linearly dependent coefficient and detection limit, quantitative limit of 29 kinds of fatty acid methyl esters of table
(7) content for determining each fatty acid in nematode sample is calculated, the content of each fatty acid is counted as the following formula in nematode sample
It calculates:
Mi: the content of fatty acid methyl ester in sample, unit mg/L；As: the peak face of internal standard compound in fatty acid methyl ester standard specimen
Product；Ai: the peak area of fatty acid methyl ester in sample；Asi: the peak area of internal standard compound in sample；Ar: rouge in fatty acid methyl ester standard specimen
The peak area of fatty acid methyl esters；Mr: fatty acid methyl ester in fatty acid methyl ester standard specimen, unit mg/L.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of method that gas-chromatography, mass spectrometric hyphenated technique detect content of fatty acid in nematode, which is characterized in that the method
The following steps are included:
(1) standard items are prepared, is detected with gas-chromatography, mass spectrometry combination method, obtains the retention time and GC-MS of each fatty acid
Map, and being compared with mass spectrometry database obtains the corresponding fatty acid component of different retention times；
(2) it prepares standard mixed liquor and establishes gas-chromatography, mass spectrometry qualitative-and-quantitative method, the qualitative, quantitative with internal standard method
Method is that the mass spectrum of target compound is retrieved and compared in mass spectral database, while under the conditions of identical GC-MS, in determinand
Retention time and the mass spectrum comparison that desired fatty acid and analytical standard sample obtain are qualitative, and when similarity >=95% is confirmed as this kind
Compound, quantitative approach are internal standard method；
(3) nematode sample is pre-processed, analysis sample is obtained；
(4) gas-chromatography, mass spectrometry qualitative-and-quantitative method are established using step (2) and analysis inspection is carried out to analysis sample to be measured
It surveys, the retention time and GC-MS map of each fatty acid of sample must be analyzed；
(5) content for determining each fatty acid in nematode sample is calculated, the content of each fatty acid is counted as the following formula in the nematode sample
It calculates:
Mi: the content of fatty acid methyl ester in sample；As: the peak area of internal standard compound in fatty acid methyl ester standard specimen；Ai: fatty in sample
The peak area of sour methyl esters；Asi: the peak area of internal standard compound in sample；Ar: the peak face of fatty acid methyl ester in fatty acid methyl ester standard specimen
Product；Mr: fatty acid methyl ester in fatty acid methyl ester standard specimen.
2. the method that gas-chromatography according to claim 1, mass spectrometric hyphenated technique detect content of fatty acid in nematode, special
Sign is, standard items described in step (1) are methyl hexadecanoate 16:0, Methyl palmitoleinate 16:1,18 hard acid methyl esters 18:0,
Methyl oleate 18:1n9, linolenic acid methyl esters 18:2n6, alpha-linolenic acid methyl esters 18:3n3, parinaric acid methyl esters 18:4n3,
Arachidic acid methylester 20:4n6, eicosanoid methyl esters 20:5n3.
3. the method that gas-chromatography according to claim 1, mass spectrometric hyphenated technique detect content of fatty acid in nematode, special
Sign is that the internal standard compound that the internal standard method is selected is pentadecane alkanoic acid methyl esters C15:0.
4. the method that gas-chromatography according to claim 1, mass spectrometric hyphenated technique detect content of fatty acid in nematode, special
Sign is, standard Compound mixed solution described in step (2):
Fatty acid methyl ester standard items in step (1) are taken respectively, and the standard reserving solution of 9 kind of 100 μ g/mL is configured to n-hexane, it will
9 kinds of standard reserving solutions are configured to 9 kinds of fatty acid methyl ester standard mixed liquors of 10 μ g/mL with the n-hexane, and use just oneself
Alkane dilutes step by step, and it is molten to obtain 9 kinds of standards mixing that mass concentration gradient is 0.1,0.2,0.5,1,2,5,10,20 μ g/mL
Liquid, wherein containing the internal standard standard items C15:0 of 1 μ g/mL in 9 kinds of standard mixed solutions of each concentration gradient.
5. the method that gas-chromatography according to claim 1, mass spectrometric hyphenated technique detect content of fatty acid in nematode, special
Sign is that nematode sample described in step (3) pre-processes:
Adult nematodes are cleaned up into acquisition nematode sample, the internal standard solution is added in Xiang Suoshu nematode sample and is mixed well, then
H is added2SO4/ MeOH solution brings it about esterification reaction of organic acid, and the analysis sample is obtained by extraction to the end of reacting.
6. the method that gas-chromatography according to claim 5, mass spectrometric hyphenated technique detect content of fatty acid in nematode, special
Sign is that the method for obtaining nematode sample is to take adult nematodes in 1.5mL centrifuge tube, with the centrifugation of 3500r/min revolving speed
2min removes supernatant culture medium, adds 1mL PBS to clean, in triplicate.
7. the method that gas-chromatography according to claim 6, mass spectrometric hyphenated technique detect content of fatty acid in nematode, special
Sign is, the H2SO4/ MeOH solution concentration is 1~10%, H2SO4/ MeOH liquor capacity is 750 μ L.
8. the method that gas-chromatography according to claim 7, mass spectrometric hyphenated technique detect content of fatty acid in nematode, special
Sign is, 55 DEG C~85 DEG C of the esterification bath temperature, 1~3h of the esterification time.
9. the method that gas-chromatography according to claim 1, mass spectrometric hyphenated technique detect content of fatty acid in nematode, special
Sign is that gas-chromatography described in step (1), gas phase condition in mass spectrometry combination method: chromatographic column selects highly polar DB-WAX
(30m, internal diameter 0.25mm apply 0.25 μm of thickness)；50 DEG C of chromatographic column initial temperature, keep 1min；Again with the rate of 20 DEG C/min
180 DEG C are warming up to, 5min is kept；230 DEG C finally are warming up to the rate of 8 DEG C/min, keeps 15min；Carrier gas is high-purity helium,
0.5~1.5mL/min constant current flow velocity, using split sampling, split ratio 20:1~50:1；1 μ L of sample volume, injector temperature are
200~250 DEG C.
10. the method that gas-chromatography according to claim 1, mass spectrometric hyphenated technique detect content of fatty acid in nematode,
It is characterized in that, gas-chromatography described in step (1), Mass Spectrometry Conditions in mass spectrometry combination method: EI electron impact ion source, electronics energy
Measure 70eV；Ion source temperature: 200~250 DEG C；Interface temperature: 200~250 DEG C, solvent delay 3.5min, using selection ion
Scanning mode.
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| CN112504989A (en) * | 2020-11-10 | 2021-03-16 | 青岛海关技术中心 | Method for testing, identifying and classifying industrial fatty acid and product thereof |
| CN113030360A (en) * | 2021-02-23 | 2021-06-25 | 上海百趣生物医学科技有限公司 | Free fatty acid high-throughput target detection method and application |
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| CN110427880B (en) * | 2019-08-01 | 2023-05-02 | 齐鲁工业大学 | Nematode fatty acid quantification method and system based on image processing |
| CN110736801A (en) * | 2019-12-02 | 2020-01-31 | 山东农业大学 | method for measuring fatty acid in rice by gas chromatography-mass spectrometry |
| CN111402961A (en) * | 2020-02-28 | 2020-07-10 | 上海鹿明生物科技有限公司 | Multi-species GC-MS endogenous metabolite database and establishment method thereof |
| CN111402961B (en) * | 2020-02-28 | 2020-11-17 | 上海鹿明生物科技有限公司 | Multi-species GC-MS endogenous metabolite database and establishment method thereof |
| CN112504989A (en) * | 2020-11-10 | 2021-03-16 | 青岛海关技术中心 | Method for testing, identifying and classifying industrial fatty acid and product thereof |
| CN113030360A (en) * | 2021-02-23 | 2021-06-25 | 上海百趣生物医学科技有限公司 | Free fatty acid high-throughput target detection method and application |
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| CN115308078A (en) * | 2022-08-09 | 2022-11-08 | 上海海洋大学 | Method for measuring fatty acid mass fractions of different tissues of soft fishes |
| CN115308078B (en) * | 2022-08-09 | 2024-04-02 | 上海海洋大学 | Determination method for mass fractions of fatty acids of different tissues of flexible fish |
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Application publication date: 20190226 |