CN111551663B - Quantitative method for determining soil fatty acid methyl ester and application thereof - Google Patents
Quantitative method for determining soil fatty acid methyl ester and application thereof Download PDFInfo
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- 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
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- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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Abstract
The invention provides a quantitative method for determining soil fatty acid methyl ester and application thereof, wherein 37 fatty acid methyl ester mixed standard substances are used as external standards, operation parameters are determined on a GC-MS (gas chromatography-mass spectrometry) to establish a method for determining the fatty acid methyl ester, a mixed solution of a methyl nonadecanoate internal standard and the 37 fatty acid methyl ester external standards is used for determining a correction factor of the fatty acid methyl ester in the external standards, phospholipid fatty acid in soil is extracted and subjected to methyl esterification, the retention time of the fatty acid methyl ester in the external standards and NIST (NIST) library retrieval are combined, qualitative analysis of types is carried out on the fatty acid methyl ester with the carbon number of 9-19 in a sample to be determined, the correction factor of the fatty acid methyl ester with the carbon number of 9-19 in the sample is determined by using the obtained correction factor of the fatty acid methyl ester in the external standards and combining the chemical structure of the fatty acid methyl ester to be determined, and finally the content of the fatty acid methyl ester with the carbon number of 9-19 in the soil is calculated by the obtained correction factor, so as to realize more accurate characterization of the soil microbial community structure.
Description
Technical Field
The invention relates to the field of chemistry, in particular to a compound detection technology, particularly relates to a technology for detecting a compound by a gas chromatography-mass spectrometer, and particularly relates to a quantitative method for detecting fatty acid methyl ester in soil and application thereof.
Background
Phospholipid fatty acids (PLFAs) are one of the components of cell membranes, and because of the different types of PLFAs contained in different types of microorganisms, the use of PLFAs composition to characterize the soil microbial community structure has been widely used. In order to increase the stability of the substance to be tested and to lower its boiling point to make it more readily vaporizable, it is generally necessary to derivatize the PLFAs into the corresponding methyl ester, i.e. fatty acid methyl ester FAME, and then to test their composition and content. Common instruments for measuring FAME include a gas chromatograph, a gas chromatograph-mass spectrometer, and a liquid chromatograph-mass spectrometer, wherein, the Sherlock Microbiological Identification System (MIS) developed by MIDI in the United states is software for identifying and analyzing the type and content of FAME formed by specific short-chain fatty acids (C9-C20) in microorganisms on a platform of the gas chromatograph (such as GC-6850,7820,6890 and 7890 of Agilent). Although a large number of samples can be tested quickly by using the software, there are cases where FAME to be tested is absent in the database and where chromatographic peak misjudgment occurs, and the cost of purchasing MIDI software is relatively expensive. The liquid chromatogram-mass spectrum combined instrument can analyze the polar phosphoric acid head of the FAME and the nonpolar fatty acid side chain of the FAME, and can display all information carried by original living cell membrane phospholipid molecules. However, since no commercial spectral library is available for user search, the application of the library in FAME detection is not wide enough. The gas chromatography-mass spectrometer (GC-MS) has high sensitivity and low detection limit when used for detecting FAME, has a commercial NIST spectrum library for retrieval, and has good application prospect.
Currently, methods for quantifying FAME mainly include an area normalization method, an internal standard method, an external standard method, and the like. The area normalization method is to calculate the content of all peak-appearing components of the sample to be detected according to 100 percent under the condition that the correction factors of all components are the same, and calculate the percentage content of each component according to the response value. The method is simple and convenient to calculate, and can eliminate errors caused by differences of states and operating conditions of the instrument. However, this method cannot obtain the absolute content of the component to be measured. The internal standard method directly uses the ratio of the response values (peak areas or peak heights) of the component to be detected and the internal standard (generally methyl nonadecanoate) and the content of the internal standard to determine the content of the component to be detected, but does not consider the difference of the responses of different FAMEs to the instrument. The external standard method is to calculate the content of the FAME to be detected in the sample by establishing a standard curve. However, since the FAME mix standard currently marketed cannot contain all of the target FAME extracted from soil, this method cannot accurately quantify the FAME specific to soil.
An article published by Huiyin et al (2014) and the like, namely comparison and quantitative optimization of a phospholipid fatty acid method and a mild alkaline methyl esterification method in soil microbial community structure analysis (environmental chemistry, 33 (5): 760-764), quantifies soil FAME by respectively using an internal standard comparison method (simultaneously using an internal standard and an external standard) and a direct calculation method (according to the principle that the content of a sample is in direct proportion to the peak area), finds that the difference exists between the two methods, calculates a correction coefficient according to the ratio of the two methods, corrects components which can only be used in the direct calculation method, enables FAME without a standard product to be still quantified, and optimizes a quantitative method for determining FAME by using GC-MS. However, this method still has the following disadvantages: firstly, although the internal standard comparison method considers that different FAMEs have different responses to GC-MS, the mass of the internal standard in the default sample and the internal standard and the external standard mixed solution of the formula for quantification is the same, so that the application of the formula is limited; second, the calibration factors for FAMEs that are not included in the external standard are the known average of the calibration factors for all FAMEs, and the averaging process increases the error to some extent. Therefore, it is necessary to further optimize the method for quantifying the methyl ester of soil fatty acid.
Disclosure of Invention
The invention mainly aims to overcome the defects in the prior art and establish a quantitative method for measuring soil fatty acid methyl ester and application thereof, thereby realizing more accurate characterization of soil microbial community structure.
One of the purposes of the invention is to provide a quantitative method for measuring soil fatty acid methyl ester, which comprises the following steps:
1) Determining the operating parameters of the GC-MS: selecting 37 fatty acid methyl ester mixed standard substances as external standards, and determining instrument operation parameters on a GC-MS;
2) Determining a correction factor for each fatty acid methyl ester in the external standard: preparing a mixed solution containing internal standard methyl nonadecanoate and an external standard, determining the mixed solution through the GC-MS operation parameters determined in the step 1), and calculating a correction factor of each fatty acid methyl ester in the external standard by using a correction factor calculation formula, wherein the calculation formula of the correction factor is as follows:
wherein f is x Correction factor for fatty acid methyl esters in external standards, M 0 Mass of internal Standard (. Mu.g), A 0 Response values as internal standards on GC spectra, A x The response value of the fatty acid methyl ester on a GC spectrum is shown; m x The mass (. Mu.g) of the fatty acid methyl ester;
3) Extracting phospholipid fatty acid in soil and carrying out methyl esterification: collecting a soil sample, extracting phospholipid fatty acid in soil by adopting a Bligh-Dyer method, and carrying out methyl esterification on the extracted phospholipid fatty acid;
4) And (3) qualitatively analyzing the sample: adding an internal standard methyl nonadecanoate into the fatty acid methyl ester obtained in the step 3), determining the fatty acid methyl ester to be determined by adopting the GC-MS operation parameters determined in the step 1), performing type qualitative analysis on the fatty acid methyl ester to be determined with the carbon atom number of 9-19, and determining a correction factor of the fatty acid methyl ester to be determined with the carbon atom number of 9-19;
5) Quantitative analysis of the samples: combining the correction factors of the fatty acid methyl esters with the carbon number of 9-19 in the soil determined in the step 4) and the response value of each fatty acid methyl ester on the GC map to calculate the concentration (C, nmol.g) of the fatty acid methyl ester to be detected with the carbon number of 9-19 in the soil -1 ) The calculation formula is as follows:
wherein f is a correction factor of fatty acid methyl ester to be detected, M i Mass of internal Standard (. Mu.g), A i The response value of the internal standard on a GC spectrum, A is the response value of the fatty acid methyl ester on the GC spectrum; m mol The molecular molar mass (μ g. μmol) of the fatty acid methyl ester -1 ) And m is the dried soil mass (g).
Preferably, the determining the operational parameters of the GC-MS in step 1) comprises the following process:
(1) Sucking 0.08ml of the purchased mixed standard substance of 37 fatty acid methyl esters, and diluting the mixed standard substance to 2ml by using normal hexane, namely diluting the mixed standard substance by 25 times, wherein the concentration range after dilution is 6.58-24.9 mu g -1 Then, the measurement is carried out on GC-MS;
(2) Performing GC analysis, and determining fatty acid methyl ester by using a VF-23ms chromatographic column, wherein the carrier gas is He gas, the sample injection is not split, the sample injection volume is 1 mu l, and the sample injection port temperature is 260 ℃; the temperature rise procedure of the chromatographic column box is as follows: the initial temperature is 60 ℃, and the temperature is kept for 1min; at 5 ℃ for min -1 Heating to 170 deg.C, and maintaining for 2min; at 2 ℃ for min -1 Heating to 200 deg.C, and maintaining for 5min;
(3) Performing MS analysis in a full scan mode with a mass-to-charge ratio scan range of 50-500, bombarding an ion source with electrons at an energy of 70eV, a temperature of the ion source of 180 ℃ and a temperature of a mass spectrum interface of 200 ℃.
Preferably, the specific process for preparing the mixed solution containing the internal standard methyl nonadecanoate and the external standard in the step 2) is as follows: methyl nonadecanoate is selected as an internal standard, 0.002g of the internal standard is weighed, and 10ml of n-hexane is used for preparing the internal standard with the concentration of 200 mu g -1 The internal standard solution of (4); 0.2ml of the solution is added to a concentration of 200. Mu.g.ml -1 The internal standard solution of (2) was mixed with 0.2ml of a commercially available mixed external standard solution, and diluted to 5ml with n-hexane to give an internal standard concentration of 8. Mu.g.ml -1 The concentration of the mixed external standard is 6.58-24.9 mu g -1 The mixed solution of (1). 2ml of the mixed solution was aspirated and added to a sample bottle for GC-MS analysis.
Preferably, the extraction of fatty acids from soil by the Bligh-Dyer method in step 3) specifically comprises the following steps:
a. extracting soil lipid: weighing fresh soil, drying the fresh soil to 2.00g, extracting the fresh soil with 15ml of Bligh-Dyer extract, collecting the extract, extracting with 7ml of Bligh-Dyer extract again, mixing the extracts for 2 times, standing overnight, collecting the lower chloroform layer, which is the extracted soil lipid, and performing water bath at 37 ℃ and N 2 Drying;
b. separation of phospholipid fatty acids: dissolving the soil lipid obtained in the step a by using chloroform, adding the soil lipid into a silica gel column, sequentially eluting the silica gel column by using 5ml of chloroform, 5ml of acetone and 5ml of methanol, collecting the eluate of the methanol-eluted silica gel column, and carrying out water bath N at 37 ℃ in water bath 2 Drying by blowing to obtain phospholipid fatty acid;
c. methyl esterification reaction: adding 1ml of toluene-methanol solvent with the volume ratio of 1 -1 Reacting with methanol-KOH solution in water bath at 37 deg.C for 30min, and adding 1ml of 1mol -1 Stopping reaction with acetic acid, adding 2ml chloroform and 2ml water, standing overnight to allow separation, collecting organic phase, and heating in 37 deg.C water bath N 2 And drying by blowing to obtain the fatty acid methyl ester to be detected.
The volume ratio of chloroform, methanol and phosphate buffer in the Bligh-Dyer extracting solution in the step a is 1.
Preferably, the concentration of the internal standard added into the fatty acid methyl ester to be tested in the step 4) is 3.2 mu g -1 The volume was 0.5ml.
Preferably, the qualitative analysis in the step 4) refers to the qualitative analysis of fatty acid methyl ester with 9-19 carbon atoms in the sample to be tested by combining the retention time of the standard, literature data search and NIST library search.
Preferably, the method for determining the correction factor of the fatty acid methyl ester with the carbon number of 9-19 in the sample to be tested in the step 4) is as follows: the fatty acid methyl ester contained in the external standard adopts the correction factor calculated in the step 2), and the principle of determining the correction factor of the fatty acid methyl ester which exists in the sample to be tested and is not contained in the external standard is to adopt the correction factor of the fatty acid methyl ester contained in the external standard with the same carbon atom number, double bond and branched chain number and position.
The invention also provides an application of the quantitative method for determining the soil fatty acid methyl ester.
Compared with the prior art, the invention has the beneficial effects that:
the invention comprehensively utilizes the internal standard and the external standard, considers the different responses of different FAMEs to the instrument, obtains the correction factor of the target FAME which is not contained in the mixed external standard but exists in the soil, and quantifies the FAME more accurately. The invention not only optimizes the characterization of the soil microbial community structure by utilizing the PLFAs technology, but also can be used for measuring FAME with various carbon chain lengths of other samples.
Drawings
FIG. 1 is a chromatogram of an external standard of 37 fatty acid methyl esters according to the present invention;
FIG. 2 is a technical flow diagram of the present invention;
FIG. 3 is a proportion of various groups of soil microorganisms obtained by quantifying FAME using the present invention.
Detailed Description
In order to understand the present invention, the following examples are given to further illustrate the present invention.
The materials and experimental methods involved in the present invention are illustrated below:
the instrument comprises: GC-MS, type Claurs 680-SQ8T, manufactured by PE corporation, USA.
Reagent: 37 FAME mix external standards were purchased from Sigma-Aldrich; methyl nonadecanoate internal standard from Accustandard; both n-hexane and methanol were chromatographically pure and purchased from Tedia; toluene was chromatographically pure, available from Fisher scientific; acetone and chloroform are both HPLC grade and purchased from Nanjing chemical reagents, inc.; a3 ml silica solid phase extraction cartridge (SPE cartridge) was purchased from Waters.
Consumable material: an extraction rack, a polytetrafluoroethylene centrifuge tube, brown screw bottles with different volume specifications (40, 15, 4 and 1.5 ml), a long dropper, a measuring cylinder, a beaker and the like.
Experiment 1: determination of operating parameters for GC-MS
(1) Sucking 0.08ml of the purchased 37 fatty acid methyl ester mixed standard products, and diluting the mixed standard products to 2ml by using normal hexane, namely diluting the mixed standard products by 25 times, wherein the concentration range after dilution is 6.58 to 24.9 mu g -1 Then, the measurement is carried out on GC-MS;
(2) Performing GC analysis, and determining fatty acid methyl ester by using a VF-23ms chromatographic column, wherein the carrier gas is He gas, the sample injection is not split, the sample injection volume is 1 mu l, and the sample injection port temperature is 260 ℃; the temperature rise procedure of the chromatographic column box is as follows: maintaining the initial temperature at 60 deg.C for 1min; at 5 ℃ for min -1 Heating to 170 deg.C, and maintaining for 2min; at 2 ℃ for min -1 Heating to 200 deg.C, and maintaining for 5min;
(3) Performing MS analysis in a full scan mode with a mass-to-charge ratio scan range of 50-500, bombarding an ion source with electrons at an energy of 70eV, a temperature of the ion source of 180 ℃ and a temperature of a mass spectrum interface of 200 ℃. The chromatogram results of the external standard of 37 fatty acid methyl esters are shown in FIG. 1.
Experiment 2: determination of correction factor for fatty acid methyl esters in external standards
Preparing a mixed solution of internal standard methyl nonadecanoate and an external standard, determining the mixed solution through the GC-MS operation parameters determined in the step 1), and calculating a correction factor of each fatty acid methyl ester in the external standard by using a correction factor calculation formula, wherein the calculation formula of the correction factor is as follows:
wherein f is x Correction factor for fatty acid methyl esters in external standards, M 0 Mass (. Mu.g) of internal standard, A 0 Response values as internal standards on GC spectra, A x The response value of the fatty acid methyl ester on a GC spectrum is shown; m x The mass (. Mu.g) of the fatty acid methyl ester was obtained.
The specific process for preparing the mixed solution containing the internal standard methyl nonadecanoate and the external standard is as follows: methyl nonadecanoate is used as an internal standard, 0.002g of internal standard is weighed, and 10ml of n-hexane is used for preparing 200 mu g of internal standard -1 The internal standard solution of (4); 0.2ml of the solution is added to a concentration of 200. Mu.g.ml -1 Was mixed with 0.2ml of a commercially available external standard solution, and diluted to 5ml with n-hexane to give an internal standard concentration of 8. Mu.g.ml -1 The concentration of external standard is 6.58-24.9 μ g -1 The mixed solution of (1). 2ml of the mixed solution was aspirated and added to a sample bottle for GC-MS analysis.
The results of the calibration factor for fatty acid methyl esters in the external standard are shown in table 1.
TABLE 1
Experiment 3: extraction of phospholipid fatty acid in soil and methyl esterification thereof
The method for extracting the phospholipid fatty acid from the soil by adopting the Bligh-Dyer method specifically comprises the following steps:
a. weighing fresh soil, drying to obtain 2.00g, and treating with 15ml Bligh-Dyer extractive solutionExtracting soil, collecting extractive solution, extracting with 7ml Bligh-Dyer extractive solution again, mixing the extractive solutions for 2 times, standing overnight, collecting lower chloroform layer to obtain extracted soil lipid, and heating in water bath N at 37 deg.C 2 Drying;
b. separation of phospholipid fatty acids: dissolving the soil lipid obtained in the step a with chloroform, adding the dissolved soil lipid into a silica gel column, eluting the silica gel column with 5ml of chloroform, 5ml of acetone and 5ml of methanol in sequence, collecting the eluate of the methanol elution silica gel column, and carrying out water bath N at 37 DEG C 2 Drying by blowing to obtain phospholipid fatty acid;
c. methyl esterification reaction: adding 1ml of 1 toluene-methanol solvent with the volume ratio of 1 to the phospholipid obtained in the step b, and then adding 1ml of 0.2mol.l concentration -1 Reacting with methanol-KOH solution in water bath at 37 deg.C for 30min, and adding 1ml of 1mol -1 Stopping the reaction with acetic acid, adding 2ml of chloroform and 2ml of water, standing overnight to allow the mixture to separate, collecting the organic phase, and performing water bath at 37 ℃ in N 2 Drying by blowing to obtain the fatty acid methyl ester to be detected.
The volume ratio of chloroform, methanol and phosphate buffer in the Bligh-Dyer extracting solution in the step a is 1.
Example 1: qualitative analysis of FAME in soil
Soil samples were taken from Shuzeo forest, old poplar forest and young poplar forest in east Taiwan forest farm of salt city of Jiangsu province, and soil depth was 0-20cm. The method for extracting phospholipid fatty acid in soil and carrying out methyl esterification of phospholipid fatty acid in experiment 3 is adopted to obtain FAME to be detected, and 0.5ml of FAME with the concentration of 3.2 mu g.ml is added into the FAME to be detected -1 The method for determining the internal standard solution of methyl nonadecanoate comprises the steps of determining a sample by using GC-MS operation parameters determined in experiment 1, qualitatively analyzing FAME with the carbon atom number of 9-19 in the sample by combining the retention time of an external standard, literature data search and NIST spectrum library search measured in experiment 2, further determining a correction factor of the FAME with the carbon atom number of 9-19 in the sample according to the correction factor of the FAME in the external standard obtained in experiment 2, and determining the correction factor of fatty acid methyl ester which exists in the sample to be detected but does not exist in the external standard by using the correction factor of the fatty acid methyl ester existing in the external standard with the same carbon atom number, double bond and branched chain number and positionAnd (5) performing secondary treatment.
The results of the qualitative analysis of the fatty acid methyl esters in the samples to be tested and the calibration factors are shown in table 2. Among them, 24 FAMEs were detected in total in the soil, 10 types were detected in total which were the same as the external standard FAME types, and 14 types were detected in total which were different from the external standard FAME types.
TABLE 2
Example 2: quantitative analysis of samples
According to the correction factors of various types of fatty acid methyl esters obtained in example 1, the concentrations (C, nmol.g) of the fatty acid methyl esters to be detected in the soil are calculated by using the internal standard and the response value of each fatty acid methyl ester on the GC map -1 ) The calculation formula is as follows:
wherein f is a correction factor of the fatty acid methyl ester to be detected, M i Mass (. Mu.g) of internal standard, A i The response value of the internal standard on a GC spectrum, A is the response value of the fatty acid methyl ester on the GC spectrum; m is a group of mol The molecular molar mass (μ g. μmol) of the fatty acid methyl ester -1 ) And m is the soil mass (g).
Content of phospholipid fatty acids (nmol.g) in different soil samples for characterizing microbial community structure -1 ) The results are shown in Table 3.
TABLE 3
Example 3: soil microbial community structure analysis
Classification of soil microorganisms into general bacteria, G, by phospholipid fatty acid biomarkers - Bacteria, G + The ratio of 5 groups of bacteria, fungi, actinomycetes, and the like to the groups of microorganisms in the soil of aged and young poplars, which are collected from beech forest, eastern forest of salt city, jiangsu province, is shown in FIG. 3.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (2)
1. A quantitative method for measuring soil fatty acid methyl ester is characterized by comprising the following steps:
1) Determining the operating parameters of the GC-MS: selecting 37 fatty acid methyl ester mixed standard substances as external standards, and determining instrument operation parameters on a GC-MS (gas chromatography-mass spectrometry);
2) Determining a correction factor for each fatty acid methyl ester in the external standard: preparing a mixed solution containing a methyl nonadecanoate internal standard and an external standard, determining the mixed solution through the GC-MS operation parameters determined in the step 1), and calculating a correction factor of each fatty acid methyl ester in the external standard by using a correction factor calculation formula, wherein the calculation formula of the correction factor is as follows:f x =(M x ⁄A x )/(M 0 ⁄A 0 )，
wherein,f x a correction factor for fatty acid methyl esters in the external standard;M 0 is the mass of the internal standard, unitIs mu g;A 0 response values of internal standards on GC spectra;A x the response value of the fatty acid methyl ester on a GC spectrum is shown;M x is the mass of the fatty acid methyl ester, and the unit is mug;
3) Extracting phospholipid fatty acid in soil and carrying out methyl esterification: collecting a soil sample, extracting phospholipid fatty acid in soil by adopting a Bligh-Dyer method, and performing methyl esterification on the extracted phospholipid fatty acid;
4) And (3) qualitatively analyzing the sample: adding an internal standard methyl nonadecanoate into the fatty acid methyl ester obtained in the step 3), determining a sample to be detected by adopting the GC-MS operation parameters determined in the step 1), performing type qualitative analysis on the fatty acid methyl ester to be detected with the carbon atom number of 9-19, and determining a correction factor of the fatty acid methyl ester to be detected with the carbon atom number of 9-19;
5) Quantitatively analyzing a sample: combining the correction factor of the fatty acid methyl ester with the carbon number of 9-19 in the soil determined in the step 4) and the response value of each fatty acid methyl ester on the GC map to calculate the concentration of the fatty acid methyl ester to be detected with the carbon number of 9-19 in nmol -1 The calculation formula is as follows:
wherein,fa correction factor for the fatty acid methyl ester to be detected;M i is the mass of the internal standard in μ g;A i response values of the internal standard on a GC spectrum;Athe response value of the fatty acid methyl ester on a GC spectrum is shown;M mol the molecular molar mass of the fatty acid methyl ester is in units of mu g. Mu mol -1 ；mThe unit is g for the dried soil mass;
wherein the determining of the operating parameters of the GC-MS in the step 1) comprises the following processes:
(1) Sucking 0.08ml of the purchased mixed standard substance of 37 fatty acid methyl esters, diluting the mixed standard substance to 2ml by using normal hexane, namely diluting the mixed standard substance by 25 times, wherein the concentration range of the diluted fatty acid methyl esters is 6.58-24.9 mu g -1 Then, the measurement is carried out on GC-MS;
(2) Performing GC analysis, selecting a VF-23ms chromatographic column to determine fatty acid methyl ester, taking He as carrier gas, carrying out no-flow sampling, wherein the sampling volume is 1 mu l, and the temperature of a sampling port is 260 ℃; the temperature rise procedure of the chromatographic column box is as follows: the initial temperature is 60 ℃, and the temperature is kept for 1min;5 degree C.min -1 Raising to 170 ℃ and keeping for 2min;2 degree C.min -1 Raising the temperature to 200 ℃ and keeping the temperature for 5min;
(3) Performing MS analysis by adopting a full scanning mode, wherein the mass-to-charge ratio scanning range is 50-500, electrons bombard an ion source, the electron energy is 70eV, the temperature of the ion source is 180 ℃, and the temperature of a mass spectrum interface is 200 ℃;
wherein, the specific process for preparing the mixed solution containing the internal standard methyl nonadecanoate and the external standard in the step 2) is as follows: methyl nonadecanoate is selected as an internal standard, 0.002g of the internal standard is weighed, and 10ml of n-hexane is used for preparing the internal standard with the concentration of 200 mu g -1 The internal standard solution of (4); 0.2ml of the solution is added to a concentration of 200. Mu.g.ml -1 The internal standard solution of (2) was mixed with 0.2ml of a commercially available mixed external standard solution, and diluted to 5ml with n-hexane to give an internal standard concentration of 8. Mu.g.ml -1 The concentration of the mixed external standard is 6.58-24.9 mu g -1 Sucking 2ml of the mixed solution, and adding the mixed solution into a sample bottle for GC-MS analysis;
wherein, the method for extracting the phospholipid fatty acid in the soil by adopting the Bligh-Dyer method and carrying out methyl esterification in the step 3) specifically comprises the following steps:
a. extracting soil lipid: weighing fresh soil, drying the fresh soil to 2.00g, extracting the fresh soil by using 15ml of Bligh-Dyer extracting solution, collecting the extracting solution, extracting by using 7ml of Bligh-Dyer extracting solution again, combining the extracting solutions for 2 times, standing overnight, and taking a lower chloroform layer, namely extracted soil lipid, which is subjected to 37-degree water bath N, wherein the water bath N is water bath 2 Drying;
b. separation of phospholipid fatty acids: dissolving the soil lipid obtained in the step a with chloroform, adding the soil lipid into a silica gel column, eluting the silica gel column with 5ml of chloroform, 5ml of acetone and 5ml of methanol in sequence, collecting the eluate of the methanol elution silica gel column, and carrying out water bath N at 37 DEG C 2 Drying by blowing to obtain phospholipid fatty acid;
c. methyl esterificationReaction: adding 1ml of toluene-methanol solvent with the volume ratio of 1 to 1 into the phospholipid fatty acid obtained in the step b for dissolving, and then adding 1ml of 0.2mol -1 The methanol-KOH solution is reacted for 30min in water bath at 37 ℃, and finally 1ml of solution with the concentration of 1mol -1 The acetic acid stops reacting, 2ml of chloroform and 2ml of water are added, the mixture is kept stand overnight for layering, an organic phase is taken, and water bath N at 37 ℃ is carried out 2 Drying to obtain fatty acid methyl ester to be detected;
wherein the concentration of the internal standard solution added into the fatty acid methyl ester to be detected in the step 4) is 3.2 mu g -1 The volume is 0.5ml;
wherein, the qualitative analysis in the step 4) refers to the qualitative analysis of the fatty acid methyl ester with the carbon number of 9-19 in the sample to be tested by combining the retention time of the fatty acid methyl ester in the external standard, literature data search and NIST spectral library retrieval;
wherein, the method for determining the correction factor of the fatty acid methyl ester with the carbon number of 9-19 in the sample to be detected in the step 4) comprises the following steps: the fatty acid methyl ester contained in the external standard adopts the correction factor calculated in the step 2), and the principle of determining the correction factor of the fatty acid methyl ester which exists in the sample to be tested and is not contained in the external standard is to adopt the correction factor of the fatty acid methyl ester contained in the external standard with the same carbon number, double bond and branched chain number and position.
2. Use of the quantitative method for determining soil fatty acid methyl esters as claimed in claim 1.
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