CN110736801B - Method for measuring fatty acid in rice by using gas chromatography-mass spectrometry - Google Patents
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Abstract
The invention discloses a method for determining fatty acid in rice by using gas chromatography-mass spectrometry, which comprises the following steps: (1) preparing a sample to be tested: crushing a rice sample, sequentially adding n-hexane, acetyl chloride-methanol solution and dibutyl hydroxy toluene, mixing, heating, and extracting fatty acid and performing methyl esterification treatment; then cooling to room temperature, adding a sodium carbonate solution, uniformly mixing, centrifuging, taking supernatant, and filtering to obtain a sample to be detected; (2) fatty acid component determination: and (3) carrying out quantitative or qualitative determination on the 37 fatty acids of the sample to be detected obtained in the step (1) by adopting a gas chromatography-mass spectrometer. Through the combined cooperation of the extraction method and the determination method, the accurate qualitative and quantitative determination of 37 fatty acids in rice is realized, the problem that the fatty acid components in rice cannot be accurately determined in the prior art is solved, and meanwhile, the determination of more types of fatty acids can be realized on the basis of ensuring the determination accuracy of the fatty acid components.
Description
Technical Field
The invention relates to the technical field of fatty acid determination, in particular to a method for determining fatty acid in rice by using gas chromatography-mass spectrometry.
Background
Oil and fat is the main source of energy for human consumption, and more importantly, it can provide essential fatty acids for human body. The plant fatty acid is an important component of plant nutrient components, the component and content proportion of the plant fatty acid determine the quality and the nutritional value of the plant oil and the biological effect of the plant oil and fat, the plant fatty acid is mainly divided into saturated fatty acid and unsaturated fatty acid, and the determination of the content of the plant fatty acid plays a crucial role in analyzing the quality of the plant nutritional value. Therefore, developing a method capable of accurately detecting plant fatty acid has profound influence on deeply knowing the composition of the plant fatty acid components, further improving the content of oil in plants, further improving the dietary structure of human beings, and promoting the dietary health of human beings.
China is a world large country for rice production and consumption, the planting area is the second world, the total rice production is the first world, and rice is the staple food for people close to 2/3. Most of the lipids in the rice are high-quality fatty acids, the content of unsaturated fatty acids is high, and the rice has high nutritional value. Meanwhile, the lipid content in rice is a main factor influencing the palatability of the rice. Researches show that the lipid content of rice is in very obvious positive correlation with taste quality, and the influence on the taste quality of rice is larger than that of other quality indexes, so that the direct effect is achieved. Therefore, the research on the difference of the fat and fatty acid components in the grains of different rice varieties has important significance for crop breeding workers.
Although the fatty acid is measured by using the gas chromatography-mass spectrometry combined technology with the development of the high-sensitivity mass spectrometry technology, the existing gas chromatography-mass spectrometry combined technology can only realize the measurement of a few types of fatty acids, the accuracy is poor for the measurement of more and complicated fatty acid components, and due to the fact that the types of the fatty acids in the rice are complicated and the contents of the fatty acids are different, related reports for measuring the fatty acid components in the rice rarely occur, and related researches and reports for accurately measuring the fatty acid components in the rice by using the gas chromatography-mass spectrometry combined technology are not provided.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a method for measuring fatty acid in rice by using gas chromatography-mass spectrometry, and the method for extracting fatty acid can avoid the interference of other components in rice and effectively extract fatty acid from rice; the method for detecting the components of the extracted fatty acid by the gas chromatography-mass spectrometry and the MRM detection mode improves the accuracy of fatty acid detection.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a method for determining fatty acid in rice by using gas chromatography-mass spectrometry, which comprises the following steps:
(1) preparing a sample to be tested: crushing a rice sample, sequentially adding n-hexane, acetyl chloride-methanol solution and dibutyl hydroxy toluene, mixing, heating, and extracting fatty acid and performing methyl esterification treatment; then cooling to room temperature of 20-25 ℃, adding a sodium carbonate solution, uniformly mixing, centrifuging, taking supernatant, and filtering to obtain a sample to be detected;
(2) fatty acid component determination: and (3) carrying out quantitative or qualitative determination on the 37 fatty acids of the sample to be detected obtained in the step (1) by adopting a gas chromatography-mass spectrometer.
Preferably, in the step (1), the rice sample, n-hexane, acetyl chloride-methanol solution and dibutyl hydroxy toluene are added in a ratio of (0.05-0.1) g: (2-3) ml: (2-3) ml: (0.2-0.3) mg; the volume fraction of the acetyl chloride-methanol solution was 10%.
Preferably, in the step (1), the rice sample is carried out in liquid nitrogen while being pulverized, and the rice sample is ripe rice dried seeds.
Preferably, in the step (1), the heating condition is water bath heating, the water bath heating temperature is 80 ℃ +/-1 ℃, and the time is 2 hours.
Preferably, shaking is carried out 1 time every 20-25min during the heating in the water bath.
Preferably, in the step (1), the ratio of the amount of the sodium carbonate solution added to the amount of the rice sample added is (2-3) mL: (0.05-0.1) g, the mass concentration of the sodium carbonate solution is 6%.
Preferably, in the step (1), the centrifugation conditions are 2000-4000r/min and the centrifugation time is 10 min.
Preferably, in step (1), the filter is 0.22 μm.
Preferably, in step (2), a standard curve is established for the quantitative determination by 37 fatty acid methyl ester standards, and the fatty acid in the sample is quantitatively detected according to the standard curve.
Preferably, in the step (2), the gas chromatography-mass spectrometry is performed by using an Shimadzu GCMS-TQ8040 triple quadrupole gas chromatography-mass spectrometer.
Preferably, in the step (2), the chromatographic conditions are as follows: the chromatographic column adopts an SH-Rt-2560 capillary column (100.0m multiplied by 0.25mm multiplied by 0.20 mu m); the initial temperature of the column box is 40.0 ℃, and the temperature rising program is as follows: maintaining at 40.0 deg.C for 2min, increasing to 240 deg.C at 4 deg.C/min, and maintaining for 15 min; the temperature of a sample inlet is 250 ℃; the sample introduction mode is non-shunting; the carrier gas is helium, and the initial pressure is 500-900 kPa; the flow control mode is linear speed; the pressure was 177.3 kPa; linear velocity 20.0 cm/s; the purging flow is 3.0 mL/min; the sample size was 1.5. mu.L.
Preferably, in step (2), the mass spectrometry conditions are: an electron ionization source with an ion source temperature of 230 ℃; the interface temperature is 250 ℃; the mass scanning range m/z 40-500; the solvent delay time is 15.5 min; detector voltage: tuning voltage +0.7 kv; the detection mode adopts MRM mode.
Preferably, the mass spectrometry conditions further include a quantitative ion pair and a qualitative ion pair used in fatty acid detection, and the quantitative ion pair and the qualitative ion pair are as follows:
the invention has the beneficial effects that:
1. by the fatty acid extraction method, the interference of other components in the rice can be avoided, and the fatty acid can be effectively extracted from the rice; according to the gas chromatography-mass spectrometry method, particularly the temperature rise process in the chromatographic condition, the MRM detection mode and the adopted quantitative ion pair and qualitative ion pair are adopted in the mass spectrometry condition, and the conditions are matched together to carry out component determination on the extracted fatty acid, so that the accuracy of fatty acid determination is improved.
2. Through the cooperation of the extraction method and the determination method, the accurate qualitative and quantitative determination of 37 fatty acids in rice is realized, the problem that the fatty acid components in rice cannot be accurately determined in the prior art is solved, and the determination of more types of fatty acids can be realized on the basis of ensuring the determination accuracy of the fatty acid components.
3. The method can realize the accurate determination of 37 fatty acids in rice, and the 37 fatty acids comprise: butyric acid, caproic acid, caprylic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, myristoleic acid, pentadecanoic acid, cis-10-pentadecanoic acid, palmitic acid, palmitoleic acid, heptadecanoic acid, cis-10-heptadecenoic acid, stearic acid, elaidic acid, oleic acid, linolenic acid, linoleic acid, arachidic acid, gamma-linoleic acid, cis-11-eicosenoic acid, alpha-linoleic acid, heneicosenoic acid, cis-11, 14-eicosadienoic acid, behenic acid, cis-8, 11, 14-eicosatrienoic acid, erucic acid, cis-11, 14, 17-eicosatrienoic acid, arachidonic acid, triconic acid, cis-13, 16-docosadienoic acid, tetracosanoic acid, cis-5, 8,11,14, 17-eicosapentaenoic acid, cis-15-eicosatetraenoic acid, cis-4, 7,10,13,16, 19-docosahexaenoic acid.
Drawings
FIG. 1 is a TIC chart (1000. mu.g/L) of 37 fatty acid methyl ester standard solutions.
Fig. 2 is a mass chromatogram of methyl decanoate in the fatty acid standard of the present invention.
FIG. 3 is a mass chromatogram of methyl tetradecanoate in the fatty acid standard of the present invention.
FIG. 4 is a mass chromatogram of cis-9-tetradecenoic acid methyl ester in the fatty acid standard substance of the present invention.
FIG. 5 is a mass chromatogram of methyl hexadecanoate in the fatty acid standard of the present invention.
FIG. 6 is a mass chromatogram of cis-9-hexadecenoic acid methyl ester in the fatty acid standard of the present invention.
FIG. 7 is a mass chromatogram of methyl eicosanoate in a fatty acid standard of the present invention.
FIG. 8 is a mass chromatogram of cis, cis-6, 9, 12-octadecatrienoic acid methyl ester in the fatty acid standard substance of the present invention.
FIG. 9 is a mass chromatogram of methyl behenate in a fatty acid standard of the present invention.
FIG. 10 is a mass chromatogram of methyl eicosatricarbonate in a fatty acid standard of the present invention.
FIG. 11 is a standard curve of methyl butyrate in fatty acid standards of the present invention.
FIG. 12 is a standard curve for methyl hexanoate in fatty acid standards of the invention.
Fig. 13 is a standard curve for methyl octanoate in fatty acid standards according to the present invention.
FIG. 14 is a standard curve for methyl heptadecacarbonate in a fatty acid standard of the present invention.
FIG. 15 is a standard curve for methyl eicosarbonate in fatty acid standards according to the present invention.
FIG. 16 is a standard curve for methyl behenate in a fatty acid standard of the present invention.
FIG. 17 is a chromatogram of a sample to be tested for fatty acid according to an embodiment of the present invention.
FIG. 18 is a chromatogram of a fatty acid test sample of a comparative example of the present invention.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As introduced in the background art, due to the fact that the types of fatty acids in rice are complex and the contents of the fatty acids are different, no relevant report on the determination of the fatty acid components in the rice appears, and no relevant research and report on how to accurately determine the fatty acid components in the rice by a gas chromatography-mass spectrometry method are available. Based on the method, when the fatty acid is extracted, a proper amount of normal hexane, acetyl chloride-methanol solution and dibutyl hydroxy toluene are added into a sample to perform fatty acid methyl esterification, then sodium carbonate solution is added to perform centrifugation to obtain the fatty acid, the interference of other components in the rice can be avoided, the fatty acid is effectively extracted from the rice, and when the fatty acid is determined, a gas chromatography-mass spectrometry combined technology is adopted, and the accuracy of fatty acid determination is improved by matching the proper chromatographic conditions and mass spectrometry conditions of the method with the MRM detection mode.
The gas chromatography-mass spectrometry adopts an Shimadzu GCMS-TQ8040 triple quadrupole gas chromatography-mass spectrometry instrument, and has the following characteristics: (1) the performance is excellent: firstly, the sensitivity is high, ions generated in an ion source box can efficiently enter a quadrupole rod by the aid of the patented ion source, neutral noise is reduced by means of a unique quadrupole rod off-axis design, and a patented deflection lens is additionally arranged at the front end of a detector to further reduce noise; the software can set sensitivity modes aiming at samples with different concentrations, and ideal results can be obtained in a wide sensitivity range from low to high; a collision pool adopts the unique UFsweeper technology of Shimadzu, the required length is reduced to the minimum limit, and the high efficiency of CID and the rapid transmission of ions are realized; the GCMS-TQ8040 has the fastest scanning speed (20,000u/sec) in the current similar products, an ASSP technology is provided, data sensitivity and mass spectrogram correctness are guaranteed during high-speed scanning, a special UFsweeper collides with a pool, the fastest MRM speed (888MRM/sec) is realized, the minimum residence time is 0.5ms, and the speed is incomparable; and fourthly, the monitoring mode is a multi-reaction scanning (MRM) mode. (2) The software function is powerful: the retention time is automatically corrected, the relative retention time of the normal alkane, namely the retention index is used for calculation, the retention time automatic correction function (AART) can deduce the current retention time of the target compound by analyzing the normal alkane once without analyzing the standard substance again when the device is changed, the chromatographic column is truncated and the like; Scan/MRM simultaneous measurement (fastt), which can satisfy the requirement of high-sensitivity quantitative analysis and can also perform qualitative analysis.
In one embodiment of the invention, the sample extraction method is given by: crushing a rice sample, sequentially adding n-hexane, acetyl chloride-methanol solution and dibutyl hydroxy toluene, mixing, heating, and extracting fatty acid and performing methyl esterification treatment; and then cooling to room temperature, adding a sodium carbonate solution, uniformly mixing, centrifuging, taking supernatant, and filtering to obtain a fatty acid methyl ester sample. The determination method comprises the following steps: and (3) carrying out quantification or qualitative determination on the fatty acid methyl ester sample extracted in the step (b) by using a gas chromatography-mass spectrometer, establishing a standard curve for the quantification by using 37 fatty acid methyl ester standard substances, and carrying out quantitative detection on the fatty acid in the sample according to the standard curve.
The detection method comprises the following chromatographic conditions: the chromatographic column adopts an SH-Rt-2560 capillary column (100.0m multiplied by 0.25mm multiplied by 0.20 mu m); the initial temperature of the column box is 40.0 ℃, the temperature is kept at 40.0 ℃ for 2min, the temperature is increased to 240 ℃ at the speed of 4 ℃/min, and the temperature is kept for 15 min; the temperature of a sample inlet is 250 ℃; the sample introduction mode is non-shunting; the carrier gas is helium, and the initial pressure is 500-900 kPa; the flow control mode is linear speed; the pressure was 177.3 kPa; linear velocity 20.0 cm/s; the purging flow is 3.0 mL/min; the sample size was 1.5. mu.L.
Detection method mass spectrum conditions: an electron ionization source with an ion source temperature of 230 ℃; the interface temperature is 250 ℃; the mass scanning range m/z 40-500; the solvent delay time is 15.5 min; detector voltage: tuning voltage +0.7 kv; the detection mode was MRM mode, and the quantitative ion pair and the qualitative ion pair used for fatty acid detection were as shown in table 1 below.
Table 1: information on the components of 37 fatty acid methyl esters
The methodology for the detection method established by the present invention was examined as follows:
standard curve and detection limit: preparing 1, 5, 10, 50, 100, 200, 400 μ g/L fatty acid methyl ester mixed standard solution, respectively, taking 1.5 μ L sample, and taking concentration as abscissa and peak area as ordinate to make standard curve, wherein the standard curve of partial compounds is shown in FIGS. 11-16. The detection limits of 37 fatty acid methyl esters were calculated from 50. mu.g/L standard data by a 3-fold signal-to-noise ratio (peak-to-peak), and the detection limits and linear correlation coefficients of each compound are shown in Table 2.
Table 2: correlation coefficient, detection limit and repeatability of each component
And (3) repeatability experiment: and taking 100 mu g/L of standard solution, carrying out continuous sample injection for 6 times, and inspecting the repeatability of the instrument, wherein the measurement result is shown in Table 2.
As can be seen from Table 2, the method of the present invention can achieve accurate determination of fatty acids, and has the advantages of high accuracy, low detection limit, and good repeatability.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention were all commercially available materials that are conventional in the art and are commercially available.
Example (b):
(1) grinding a rice seed sample until the sample is crushed, and accurately weighing 0.1g of the crushed sample in a 15mL dry screw glass tube;
(2) adding 3.0mL of n-hexane, 3mL of 10% acetyl chloride-methanol solution and 0.3mg of dibutyl hydroxy toluene in sequence into the sample, and screwing a glass tube screw cover;
(3) oscillating, mixing, standing in 80 deg.C water bath for 2h, taking out every 20min, shaking for 1 time to realize fatty acid methyl esterification, taking out after water bath, and cooling to room temperature;
(4) transferring the reacted sample solution into a 50mL centrifuge tube, cleaning the glass tube for 2 times by using 3mL sodium carbonate solution, combining the sodium carbonate solution in the 50mL centrifuge tube, fully shaking and uniformly mixing, wherein the concentration of the sodium carbonate solution is 6%;
(5) centrifuging at 4000r/min for 10min, sucking 1mL of supernatant through a glass pipette, and filtering through a 0.22-micron filter membrane to obtain a fatty acid sample to be detected;
(6) putting a fatty acid sample to be detected into a gas chromatography-mass spectrometer, wherein the chromatographic conditions are as follows: the chromatographic column adopts an SH-Rt-2560 capillary column (100.0m multiplied by 0.25mm multiplied by 0.20 mu m); the initial temperature of the column box is 40.0 ℃, and the temperature rising program is as follows: maintaining at 40.0 deg.C for 2min, increasing to 240 deg.C at 4 deg.C/min, and maintaining for 15 min; the temperature of a sample inlet is 250 ℃; the sample introduction mode is non-shunting; the carrier gas is helium, and the initial pressure is 500-900 kPa; the flow control mode is linear speed; the pressure was 177.3 kPa; linear velocity 20.0 cm/s; the purging flow is 3.0 mL/min; the sample size was 1.5. mu.L. Mass spectrum conditions: an electron ionization source with an ion source temperature of 230 ℃; the interface temperature is 250 ℃; the mass scanning range m/z 40-500; the solvent delay time is 15.5 min; detector voltage: tuning voltage +0.7 kv; the detection mode adopts MRM mode, and the quantitative ion pair and the qualitative ion pair are shown in Table 1.
The method comprises the steps of qualitatively determining the components of fatty acid according to a quantitative ion pair and a qualitative ion pair, simultaneously carrying out semi-quantification on the components of the fatty acid, respectively preparing 1, 5, 10, 50, 100, 200 and 400 mu g/L of fatty acid methyl ester mixed standard solution for accurate quantification, taking 1.5 mu L of sample introduction, taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate to make a standard curve, and quantitatively detecting the fatty acid in a sample according to the standard curve.
Comparative example
(1) Grinding a rice seed sample until the sample is crushed, and accurately weighing 0.1g of the crushed sample in a 15mL dry screw glass tube;
(2) sequentially adding 3.0mL of n-hexane, 3mL of acetyl chloride-methanol solution with the volume fraction of 10%, 200 mu L of undecane triglyceride methanol solution with the internal standard concentration of 1.00mg/mL, filling nitrogen, and screwing a glass tube screw cap;
(3) oscillating, mixing, standing in 80 deg.C water bath for 2h, taking out every 20min, shaking for 1 time to realize fatty acid methyl esterification, taking out after water bath, and cooling to room temperature;
(4) transferring the reacted sample solution into a 50mL centrifuge tube, cleaning the glass tube for 2 times by using 3mL sodium carbonate solution, combining the sodium carbonate solution in the 50mL centrifuge tube, fully shaking and uniformly mixing, wherein the concentration of the sodium carbonate solution is 6%;
(5) centrifuging at 4000r/min for 10min, sucking 1mL of supernatant through a glass pipette, and filtering through a 0.22-micron filter membrane to obtain a fatty acid sample to be detected;
(6) putting a fatty acid sample to be detected into a gas chromatography-mass spectrometer, wherein the chromatographic conditions are as follows: the chromatographic column adopts an SH-Rt-2560 capillary column (100.0m multiplied by 0.25mm multiplied by 0.20 mu m); the initial temperature of the column box is 150 ℃; the temperature rising procedure is as follows: maintaining at 150.0 deg.C for 5min, heating to 240 deg.C at 4 deg.C/min, and maintaining for 10 min; the temperature of a sample inlet is 250 ℃; and (3) sample introduction mode: split, split ratio 10: 1; the carrier gas is helium, and the initial pressure is 500-900 kPa; the flow control mode is as follows: linear velocity; the pressure was 238.3 kPa; linear velocity 20.0 cm/s; the purging flow is 3.0 mL/min; the sample volume is 1 mu L; an electron ionization source; the ion source temperature is 230 ℃; the interface temperature is 250 ℃; the monitoring mode is full scanning; the mass scanning range m/z 40-500; the solvent delay time is 10.5 min; detector voltage: tuning voltage +0.1 kv; and the detection mode adopts an SCAN mode, and the peak area is checked according to the result.
The rice seed samples adopted in the comparative example and the example are rice seeds of the same batch, and compared with the example, the extraction method and the detection method of the comparative example are different.
The amount of total fatty acids per gram of sample was calculated for the examples and comparative examples separately, and the amount of total fatty acids extracted by the method of the examples was increased by two-fold compared to the amount of total fatty acids extracted by the method of the comparative examples.
The comparative example detected 18 less components than the example, compared to the example, and the total fatty acid amount of the comparative example was lower than that of the example, and the sensitivity of the example was higher, so that the fatty acid content measured by the fatty acid extraction method of the present invention was more accurate.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (5)
1. A method for measuring fatty acid in rice by using gas chromatography-mass spectrometry is characterized by comprising the following steps:
(1) preparing a sample to be tested: crushing a rice sample, sequentially adding n-hexane, acetyl chloride-methanol solution and dibutyl hydroxy toluene, mixing, heating, and extracting fatty acid and performing methyl esterification treatment; then cooling to room temperature, adding a sodium carbonate solution, uniformly mixing, centrifuging, taking supernatant, and filtering to obtain a sample to be detected; the adding amount ratio of the rice sample, n-hexane, acetyl chloride-methanol solution and dibutyl hydroxy toluene is (0.05-0.1) g: (2-3) ml: (2-3) ml: (0.2-0.3) mg; the volume fraction of the acetyl chloride-methanol solution is 10 percent; the ratio of the addition amount of the sodium carbonate solution to the rice sample was (2-3) mL: (0.05-0.1) g;
(2) fatty acid component determination: carrying out quantitative or qualitative determination on 37 fatty acids of the sample to be detected obtained in the step (1) by using a triple quadrupole gas chromatography-mass spectrometer; the chromatographic conditions are as follows: the chromatographic column adopts an SH-Rt-2560 capillary column; the initial temperature of the column box is 40.0 ℃, and the temperature rising program is as follows: maintaining at 40.0 deg.C for 2min, increasing to 240 deg.C at 4 deg.C/min, and maintaining for 15 min; the temperature of a sample inlet is 250 ℃; the sample introduction mode is non-shunting; the carrier gas is helium, and the initial pressure is 500-900 kPa; the flow control mode is linear speed; the pressure was 177.3 kPa; linear velocity 20.0 cm/s; the purging flow is 3.0 mL/min; the sample injection amount is 1.5 mu L;
mass spectrum conditions: electron bombardment ionization source, ion source temperature 230 ℃; the interface temperature is 250 ℃; the mass scanning range m/z is 40-500; the solvent delay time is 15.5 min; detector voltage: tuning voltage +0.7 kv; the detection mode adopts an MRM mode;
the method comprises the following steps of (1) adopting a quantitative ion pair and a qualitative ion pair in fatty acid detection, wherein the quantitative ion pair and the qualitative ion pair are as follows:
2. the method according to claim 1, wherein in the step (1), the heating condition is water bath heating, and the temperature of the water bath heating is 80 ℃ plus or minus 1 ℃ for 2 hours.
3. The method of claim 2, wherein: shaking for 1 time every 20-25min during heating in water bath.
4. The method of claim 1, wherein: in the step (1), the centrifugation condition is 2000-4000r/min for 10 min.
5. The method of claim 1, wherein: in the step (2), for quantitative determination, a standard curve is established by 37 fatty acid methyl ester standard substances, and the fatty acid in the sample is quantitatively detected according to the standard curve.
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