CN113588854A - Method for detecting fatty acid composition - Google Patents
Method for detecting fatty acid composition Download PDFInfo
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- CN113588854A CN113588854A CN202011518968.5A CN202011518968A CN113588854A CN 113588854 A CN113588854 A CN 113588854A CN 202011518968 A CN202011518968 A CN 202011518968A CN 113588854 A CN113588854 A CN 113588854A
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- 235000014113 dietary fatty acids Nutrition 0.000 title claims abstract description 213
- 229930195729 fatty acid Natural products 0.000 title claims abstract description 213
- 239000000194 fatty acid Substances 0.000 title claims abstract description 213
- 150000004665 fatty acids Chemical class 0.000 title claims abstract description 179
- 238000000034 method Methods 0.000 title claims abstract description 77
- 239000000203 mixture Substances 0.000 title claims abstract description 62
- 230000014759 maintenance of location Effects 0.000 claims abstract description 67
- 238000001514 detection method Methods 0.000 claims abstract description 57
- 239000000126 substance Substances 0.000 claims abstract description 54
- 230000004807 localization Effects 0.000 claims description 22
- -1 C20 fatty acids Chemical class 0.000 claims description 12
- 125000004432 carbon atom Chemical group C* 0.000 claims description 11
- 235000020660 omega-3 fatty acid Nutrition 0.000 claims description 8
- 229940012843 omega-3 fatty acid Drugs 0.000 claims description 8
- 239000002202 Polyethylene glycol Substances 0.000 claims description 6
- 239000007789 gas Substances 0.000 claims description 6
- 229920001223 polyethylene glycol Polymers 0.000 claims description 6
- 239000010453 quartz Substances 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- 235000021323 fish oil Nutrition 0.000 claims description 5
- 238000003556 assay Methods 0.000 claims description 4
- 239000012159 carrier gas Substances 0.000 claims description 4
- 239000001307 helium Substances 0.000 claims description 4
- 229910052734 helium Inorganic materials 0.000 claims description 4
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 239000006014 omega-3 oil Substances 0.000 claims description 4
- 230000000630 rising effect Effects 0.000 claims description 4
- CYQFCXCEBYINGO-IAGOWNOFSA-N delta1-THC Chemical compound C1=C(C)CC[C@H]2C(C)(C)OC3=CC(CCCCC)=CC(O)=C3[C@@H]21 CYQFCXCEBYINGO-IAGOWNOFSA-N 0.000 claims description 3
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 238000004451 qualitative analysis Methods 0.000 abstract 1
- 125000004494 ethyl ester group Chemical group 0.000 description 48
- ITNKVODZACVXDS-YNUSHXQLSA-N ethyl (4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoate Chemical compound CCOC(=O)CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CC ITNKVODZACVXDS-YNUSHXQLSA-N 0.000 description 25
- 230000007774 longterm Effects 0.000 description 23
- 230000000052 comparative effect Effects 0.000 description 21
- 238000002360 preparation method Methods 0.000 description 14
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 description 13
- 238000012360 testing method Methods 0.000 description 13
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- 238000004458 analytical method Methods 0.000 description 10
- 239000000463 material Substances 0.000 description 10
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- 238000010792 warming Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 8
- 229920000064 Ethyl eicosapentaenoic acid Polymers 0.000 description 7
- SSQPWTVBQMWLSZ-AAQCHOMXSA-N ethyl (5Z,8Z,11Z,14Z,17Z)-icosapentaenoate Chemical compound CCOC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CC SSQPWTVBQMWLSZ-AAQCHOMXSA-N 0.000 description 7
- RLCKHJSFHOZMDR-UHFFFAOYSA-N (3R, 7R, 11R)-1-Phytanoid acid Natural products CC(C)CCCC(C)CCCC(C)CCCC(C)CC(O)=O RLCKHJSFHOZMDR-UHFFFAOYSA-N 0.000 description 6
- 238000004817 gas chromatography Methods 0.000 description 6
- 239000003814 drug Substances 0.000 description 5
- 238000012423 maintenance Methods 0.000 description 5
- 125000005907 alkyl ester group Chemical group 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000013558 reference substance Substances 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 3
- 239000011203 carbon fibre reinforced carbon Substances 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- GHVNFZFCNZKVNT-UHFFFAOYSA-N decanoic acid Chemical compound CCCCCCCCCC(O)=O GHVNFZFCNZKVNT-UHFFFAOYSA-N 0.000 description 3
- 235000021588 free fatty acids Nutrition 0.000 description 3
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 3
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- NLZUEZXRPGMBCV-UHFFFAOYSA-N Butylhydroxytoluene Chemical compound CC1=CC(C(C)(C)C)=C(O)C(C(C)(C)C)=C1 NLZUEZXRPGMBCV-UHFFFAOYSA-N 0.000 description 2
- 241000195493 Cryptophyta Species 0.000 description 2
- 241000238557 Decapoda Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 235000010354 butylated hydroxytoluene Nutrition 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000001212 derivatisation Methods 0.000 description 2
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000009659 non-destructive testing Methods 0.000 description 2
- 238000010606 normalization Methods 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 235000019198 oils Nutrition 0.000 description 2
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 150000004666 short chain fatty acids Chemical class 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 2
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 239000005632 Capric acid (CAS 334-48-5) Substances 0.000 description 1
- 239000005635 Caprylic acid (CAS 124-07-2) Substances 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- NHTMVDHEPJAVLT-UHFFFAOYSA-N Isooctane Chemical compound CC(C)CC(C)(C)C NHTMVDHEPJAVLT-UHFFFAOYSA-N 0.000 description 1
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- 235000019483 Peanut oil Nutrition 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 235000020661 alpha-linolenic acid Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 235000005687 corn oil Nutrition 0.000 description 1
- 239000002285 corn oil Substances 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- JVSWJIKNEAIKJW-UHFFFAOYSA-N dimethyl-hexane Natural products CCCCCC(C)C JVSWJIKNEAIKJW-UHFFFAOYSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000019387 fatty acid methyl ester Nutrition 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 235000020778 linoleic acid Nutrition 0.000 description 1
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 1
- 229960004488 linolenic acid Drugs 0.000 description 1
- KQQKGWQCNNTQJW-UHFFFAOYSA-N linolenic acid Natural products CC=CCCC=CCC=CCCCCCCCC(O)=O KQQKGWQCNNTQJW-UHFFFAOYSA-N 0.000 description 1
- 150000004668 long chain fatty acids Chemical class 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 150000004667 medium chain fatty acids Chemical class 0.000 description 1
- 125000004492 methyl ester group Chemical group 0.000 description 1
- 235000021281 monounsaturated fatty acids Nutrition 0.000 description 1
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 229960002446 octanoic acid Drugs 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 235000021313 oleic acid Nutrition 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 230000007180 physiological regulation Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 235000003441 saturated fatty acids Nutrition 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- 235000021391 short chain fatty acids Nutrition 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
Abstract
The invention provides a method for detecting a fatty acid composition, which is used for positioning the fatty acid composition in a segmented manner by using two or more positioning substances by a relative retention time method so as to complete qualitative analysis of the fatty acid composition in a sample to be detected. The method is rapid, simple and convenient, stable in detection capability and good in durability, and is suitable for detecting the fatty acid sample.
Description
Technical Field
The invention belongs to the field of drug analysis, and particularly relates to an analysis and detection method of a fatty acid product.
Background
Fatty Acids (FA) are a class of carboxylic acids with Fatty chains. Fatty acid plays a plurality of key roles in organisms, and the analysis of fatty acid components one by one has great significance for the explanation of pathogenesis of related diseases and clinical diagnosis and treatment. Fatty acid radicals can be classified according to the length of the carbon chain therein: short chain fatty acids, mainly including fatty acids with carbon chain number less than 6; medium chain fatty acids, mainly including fatty acids of the type having 6 to 12 carbon atoms in the carbon chain, such as caprylic acid (C8) and capric acid (C10); the long-chain fatty acid mainly comprises fatty acid with the carbon number more than 12 on the carbon chain. Fatty acids can be further classified into saturated fatty acids and unsaturated fatty acids according to whether the carbon chain of the fatty acid contains a carbon-carbon double bond, wherein the unsaturated fatty acids can be further classified into monounsaturated fatty acids (having only one carbon-carbon double bond on the carbon chain) and polyunsaturated fatty acids (having more than one carbon-carbon double bond on the carbon chain). Wherein the polyunsaturated fatty acid is important for organismThe physiological regulation function is receiving wide attention. Representative of the fish oil drugs on the market
Is a fish oil lipoid blood regulator developed by Kurarin Schker, mainly used for reducing triglyceride level in adult patients, and has been approved as diet auxiliary drug by the United states Food and Drug Administration (FDA).
The annual sales amount of the fatty acid substances is close to 10 billion dollars after the fatty acid substances are listed in Europe and America, and the fatty acid substances have wide application prospects and huge market potential in the field of medicine.
At present, fatty acids are obtained mainly by extraction methods, such as extracting EPA and DHA from fish, shrimp or algae, or by extracting vegetable oils from various plants, such as peanut oil, olive oil, corn oil. Because the fatty acid composition of the raw materials is complex and contains many fatty acid components, the extracted fatty acid product contains a plurality of fatty acid components except for the target fatty acid, but the fatty acid components except for the target fatty acid are substances which cannot ensure medical benefit, and therefore the fatty acid components need to be controlled. Therefore, in order to ensure the quality of fatty acid products, it is necessary to accurately analyze the composition of fatty acid products qualitatively or quantitatively. However, the practical problems are that if an analysis method is established for each component in the fatty acid component one by one, the problems of huge analysis operation amount, complex detection procedure, fussy detection procedure, low efficiency and the like exist.
In order to achieve accurate analysis of fatty acid composition products in the prior art that has been published, some of the following detection methods have been used:
the method comprises the following steps: patent document CN103149187B provides a nondestructive testing method for rapidly determining the content of fatty acid by using front surface fluorescence. Firstly, performing fluorescence scanning on a grease sample, collecting obtained two-dimensional fluorescence spectrum data, and performing regression calculation on the fluorescence data and fatty acid data measured by gas chromatography by using a Partial Least Squares (PLS) method to obtain regression model analysis. The method is simple and rapid and belongs to a nondestructive testing method.
The second method comprises the following steps: patent document CN111521699A provides a fatty acid LC-MS/MS analysis method based on a double-derivatization technology, which comprises preparing a derivatized fatty acid mixed standard solution, detecting each standard curve to be detected by LC-MS/MS, establishing a standard curve of each fatty acid, performing derivatization treatment on the sample to be detected, and detecting by LC-MS/MS to obtain the type and content of the fatty acid in the solution to be detected. The method has high sensitivity, can detect short chain fatty acid, and is suitable for detecting plasma sample.
The third method comprises the following steps: patent document CN105044229B provides a gas chromatography method for detecting fatty acid components in soybean, which first establishes a standard curve and a standard curve equation of five fatty acids, i.e., palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid, performs gas chromatography detection on the total fatty acids in a sample to be detected, qualitatively determines the sample to be detected according to the retention time of various standards, and qualitatively and quantitatively analyzes the content of the five fatty acids in the sample to be detected according to the standard curve.
The above patent documents use different methods for detecting the fatty acid component in the analytical product, but still have many problems: (1) the detection sensitivity of the method I and the method II is high, but the used instruments are expensive and high in maintenance cost, and the produced products need to be continuously detected in industrial mass production, so that the use of the instruments undoubtedly brings higher economic cost; (2) in the first, second and third methods, reference standard curves need to be established separately for all fatty acid components to be detected, so that reference substances of all fatty acid components to be detected need to be obtained, and when the reference substances of some components are difficult to obtain or expensive, the application difficulty of the methods is increased; the first method also needs regression calculation and regression model analysis, and the data processing process is complex; the problems of huge analysis operation amount, complex detection procedure, fussy detection procedure, low efficiency and the like can be caused when the fatty acid component to be detected is increased by the conventional method.
Currently, the united states pharmacopoeia (USP40) provides a method for detecting the content of EPA ethyl ester, DHA ethyl ester and total fatty acid ethyl ester in omega-3 fatty acid ethyl ester by using a gas chromatograph, using DHA ethyl ester as a positioning substance, qualifying other fatty acids by relative retention time, and calculating the content of each fatty acid component by an area normalization method. According to the method, a plurality of fatty acid components are qualitatively determined by using the DHA ethyl ester, so that the problem of high difficulty in obtaining a reference substance of partial fatty acids in the fatty acid composition is solved.
Disclosure of Invention
The invention provides a method for detecting a fatty acid composition, which can be stably used for a long time, has stable detection capability, is quick, simple and convenient and has good durability.
The invention provides a method for detecting a fatty acid composition, which comprises the steps of detecting the fatty acid composition by using a chromatography method to obtain the retention time of each fatty acid component in the composition, selecting two or more fatty acid components in the composition as a positioning substance after obtaining the retention time of each fatty acid component, obtaining the relative retention time of other fatty acid components in the composition relative to a specified positioning substance, and carrying out qualitative determination on the fatty acid components except the positioning substance in the composition according to the relative retention time.
Wherein the fatty acid component in the fatty acid composition has at least two carbon atoms, and the carbon atoms of the fatty acids as the positioning substances are different.
Wherein the specified positioning substance is a positioning substance which has a retention time after the fatty acid component and which is closest to the retention time, and when the fatty acid composition further contains a fatty acid component which has no positioning substance after the retention time, the fatty acid component selects a positioning substance which has a retention time before the specified retention time and which is closest to the retention time.
Wherein, the detection method of the fatty acid composition also comprises the step of determining the structure of the positioning substance.
Wherein the method for detecting the fatty acid composition further comprises determining the structure of the fatty acid component corresponding to the relative retention time before the other fatty acid components in the composition are identified by the relative retention time.
Wherein the fatty acid composition is detected by gas chromatography.
Wherein the fatty acid composition is prepared from fatty acid purified from one of natural fish oil, shrimp oil or algae oil; in some embodiments, the fatty acid is prepared from fatty acids purified from natural fish oil.
And comparing the relative retention time of the fatty acid components in other batches with the relative retention time of the qualified fatty acid components, and characterizing the fatty acid components in other batches, wherein the positioning substance used by the fatty acid components in other batches is the same as the positioning substance used by the qualified fatty acid components.
Wherein the fatty acid composition comprises omega-3 fatty acids; in some embodiments, the omega-3 fatty acid content of the fatty acid composition is 90% to 100%; in some embodiments, the fatty acid composition has an EPA content of 42% to 66% and a DHA content of 34% to 58%; in some embodiments, the fatty acid composition has an EPA content of 42% to 50% and a DHA content of 34% to 41%; in some embodiments, the sum of the content of EPA and DHA in the fatty acid composition is 70% to 100%; in some embodiments, the sum of the content of EPA and DHA in the fatty acid composition is from 75% to 100%; in some embodiments, the sum of the content of EPA and DHA in the fatty acid composition is 76% to 91%; in some embodiments, the sum of the content of EPA and DHA in the fatty acid composition is from 79% to 89%; in some embodiments, the fatty acid composition further comprises one or more components of C18 fatty acids, one or more components of C20 fatty acids other than EPA, and one or more components of C22 fatty acids other than DHA.
In some embodiments, the fatty acid composition comprises omega-3 fatty acids and the positioning substance is a C18 fatty acid, a C20 fatty acid, two or three fatty acid components of C22 fatty acids differing in the number of carbon atoms; in some embodiments, the fatty acid composition comprises omega-3 fatty acids and the localization agent is C18:4n-3, EPA, and DHA.
The amount of the positioning substance can be adjusted according to the amount of the fatty acid component in the fatty acid composition; in some embodiments, the number of localizing substances is five; in some embodiments, the number of localized substances is four; in some embodiments, the number of localizing substances is three; in some embodiments, the number of localizing substances is two.
The invention further provides a detection method of the omega-3 fatty acid composition, wherein the chromatographic conditions are as follows:
a chromatographic column: polyethylene glycol is a quartz capillary column of stationary liquid, or a quartz capillary column with polarity similar to that of polyethylene glycol is selected; the specification is 25m × 0.25mm, 0.2 μm;
temperature rising procedure: the initial temperature is 166-170 ℃, the temperature is maintained for 1-3min, the temperature is raised to 240 ℃ at the rate of 2-4 ℃ per minute, and the temperature is maintained for 6-10 min;
carrier gas: helium gas;
sample inlet temperature: 240 ℃ and 250 ℃;
detector temperature: 260 ℃ and 270 ℃;
flow rate: 1.1-1.3 mL/min;
sample introduction amount: 1 mu L of the solution;
the split ratio is as follows: 10:1-50:1.
In some embodiments, the starting temperature is 166 degrees celsius; in some embodiments, the starting temperature is 167 degrees celsius; in some embodiments, the starting temperature is 168 degrees celsius; in some embodiments, the starting temperature is 169 degrees celsius; in some embodiments, the starting temperature is 170 degrees celsius.
In some embodiments, the maintenance time for the initial temperature is 1 min; in some embodiments, the maintenance time for the initial temperature is 1.5 min; in some embodiments, the maintenance time for the initial temperature is 2 min; in some embodiments, the maintenance time for the initial temperature is 2.5 min; in some embodiments, the initial temperature is maintained for a period of 3 min.
In some embodiments, the ramp rate is to ramp to 240 degrees celsius at a rate of 2 degrees celsius per minute; in some embodiments, the ramp rate is to ramp to 240 degrees celsius at a rate of 2.2 degrees celsius per minute; in some embodiments, the ramp rate is to ramp to 240 degrees celsius at a rate of 2.4 degrees celsius per minute; in some embodiments, the ramp rate is to ramp to 240 degrees celsius at a rate of 2.6 degrees celsius per minute; in some embodiments, the ramp rate is to ramp to 240 degrees celsius at a rate of 2.8 degrees celsius per minute; in some embodiments, the ramp rate is 3.2 degrees celsius per minute to 240 degrees celsius; in some embodiments, the ramp rate is 3.4 degrees celsius per minute to 240 degrees celsius; in some embodiments, the ramp rate is 3.6 degrees celsius per minute to 240 degrees celsius; in some embodiments, the ramp rate is 3.8 degrees celsius per minute to 240 degrees celsius; in some embodiments, the ramp rate is to ramp to 240 degrees celsius at a rate of 2.5 degrees celsius per minute; in some embodiments, the ramp rate is to ramp to 240 degrees celsius at a rate of 3 degrees celsius per minute; in some embodiments, the ramp rate is 3.5 degrees celsius per minute to 240 degrees celsius; in some embodiments, the ramp rate is to ramp to 240 degrees celsius at a rate of 4 degrees celsius per minute.
In some embodiments, the holding time after the temperature is raised to 240 ℃ is 6-9 min; in some embodiments, the holding time after the temperature is raised to 240 ℃ is 6-8 min; in some embodiments, the holding time after the temperature is raised to 240 ℃ is 7-10 min; in some embodiments, the holding time after the temperature is raised to 240 ℃ is 7-9 min; in some embodiments, the holding time after the temperature is raised to 240 ℃ is 8-10 min; in some embodiments, the hold time after warming to 240 degrees celsius is 6 min; in some embodiments, the hold time after warming to 240 degrees celsius is 6.5 min; in some embodiments, the hold time after warming to 240 degrees celsius is 7 min; in some embodiments, the hold time after warming to 240 degrees celsius is 7.5 min; in some embodiments, the hold time after warming to 240 degrees celsius is 8 min; in some embodiments, the hold time after warming to 240 degrees celsius is 8.5 min; in some embodiments, the hold time after warming to 240 degrees celsius is 9 min; in some embodiments, the hold time after warming to 240 degrees celsius is 9.5 min; in some embodiments, the hold time after warming to 240 degrees celsius is 10 min.
In some embodiments, the injection port temperature is 240 degrees celsius; in some embodiments, the sample inlet temperature is 242 degrees celsius; in some embodiments, the sample inlet temperature is 244 degrees celsius; in some embodiments, the sample inlet temperature is 246 degrees celsius; in some embodiments, the sample inlet temperature is 248 degrees celsius; in some embodiments, the sample inlet temperature is 245 degrees celsius; in some embodiments, the injection port temperature is 250 degrees celsius.
In some embodiments, the detector temperature is 260 degrees celsius; in some embodiments, the detector temperature is 262 degrees celsius; in some embodiments, the detector temperature is 264 degrees celsius; in some embodiments, the detector temperature is 266 degrees celsius; in some embodiments, the detector temperature is 268 degrees celsius; in some embodiments, the detector temperature is 265 degrees celsius; in some embodiments, the detector temperature is 270 degrees celsius.
In some embodiments, the flow rate is 1.1 mL/min; in some embodiments, the flow rate is 1.2 mL/min; in some embodiments, the flow rate is 1.3 mL/min.
In some embodiments, the split ratio is from 15:1 to 50: 1; in some embodiments, the split ratio is from 20:1 to 50: 1; in some embodiments, the split ratio is from 25:1 to 50: 1; in some embodiments, the split ratio is 30:1 to 50: 1; in some embodiments, the split ratio is from 35:1 to 50: 1; in some embodiments, the split ratio is from 40:1 to 50: 1; in some embodiments, the split ratio is 45:1 to 50: 1; in some embodiments, the split ratio is 10: 1; in some embodiments, the split ratio is 15: 1; in some embodiments, the split ratio is 20: 1; in some embodiments, the split ratio is 25: 1; in some embodiments, the split ratio is 30: 1; in some embodiments, the split ratio is 35: 1; in some embodiments, the split ratio is 40: 1; in some embodiments, the split ratio is 45: 1; in some embodiments, the split ratio is 50: 1.
In some embodiments, the chromatographic conditions are as follows:
a chromatographic column: a quartz capillary column (25m x 0.25mm, 0.2 μm) with polyethylene glycol as the stationary liquid;
temperature rising procedure: the initial temperature is 168 deg.C, maintained for 3min, and heated to 24 deg.C at a rate of 4 deg.C per minute
Maintaining the temperature at 0 ℃ for 10 min;
carrier gas: helium gas;
sample inlet temperature: 245 ℃;
detector temperature: 265 ℃ C;
flow rate: 1.2 mL/min;
sample introduction amount: 1 mu L of the solution;
the split ratio is as follows: 30:1.
In establishing the fatty acid composition detection method, applicants first consult the relevant method in USP 40: determining a sample to be detected by gas chromatography, taking DHA ethyl ester as a positioning substance, recording the relative retention time of each fatty acid ethyl ester component and the DHA ethyl ester, and comparing the relative retention time with the standard relative retention time of each component respectively to achieve the purpose of qualitative fatty acid component. In the research process, in order to research the influence of the type of a chromatographic column and the change of a chromatograph on the method, the applicant examines the type of the chromatographic column and the type of the chromatograph used in the method and determines the type of the chromatographic column and the type of an instrument used in the method according to the test result. Next, a durability test for the column and the instrument was carried out, and the test results showed that the relative retention time values of the fatty acid ethyl ester components drifted within an acceptable range after the column and the instrument were replaced with different columns and instruments of the same type.
However, the applicant finds that when the method is applied to long-term detection of products, the relative retention time value of the detected fatty acid component has obvious drift, and the farther the detected fatty acid component chromatographic peak is away from the DHA chromatographic peak, the more obvious the drift is, the worse the stability of the relative retention time value is, so that the difficulty in locating the fatty acid component is increased during long-term detection, and the qualitative detection capability of the method is directly influenced.
It can be seen that the relative retention time method described in the united states pharmacopeia fails to meet the ideal detection stability requirements in long-term applications. Based on the above-mentioned problems encountered in the long-term detection, the applicant developed a detection method for localizing a fatty acid component by relative retention time segments using two or more localizing substances. The method can be used for a long time, has more stable detection capability and good durability, and can simultaneously determine a plurality of fatty acid components in the fatty acid composition. In addition, the method is simple and convenient to operate and convenient to transfer; the economic cost generated during application is low, and the method is suitable for application in industrial production.
"fatty acids" as referred to herein include free fatty acids or alkyl esters thereof; in some embodiments, free fatty acids; in some embodiments fatty acid methyl esters; including fatty acid ethyl esters in some embodiments.
A specific fatty acid, e.g., C18:4n-3, C20:4n-3, EPA or DHA, etc., as described herein refers to its free fatty acid or its alkyl ester; in some embodiments, the alkyl ester is a methyl ester; in some embodiments, the alkyl ester is an ethyl ester.
The term "qualitative" as used herein refers to determining the structure or composition of a substance.
The "number of carbon atoms" in the present invention refers to the number of carbon atoms in the carbon chain of the fatty acid component.
The term "proximal localization agent" as used herein refers to a localization agent that has a retention time after a particular fatty acid component that is the least different from the retention time of the particular fatty acid component.
The "C18 fatty acid, C20 fatty acid, and C22 fatty acid" referred to herein are collectively referred to as fatty acids having 18, 20, and 22 carbon atoms in the carbon chain, for example, C18 fatty acid is a fatty acid having 18 carbon atoms in the carbon chain.
The "content" in the present invention refers to an area percentage content (A/A) calculated by an area normalization method.
The invention has the beneficial effects that:
(1) a novel positioning method of the fatty acid composition is provided, and guarantees are provided for detection and quality control of the fatty acid composition;
(2) a segmented positioning method capable of simultaneously positioning a plurality of fatty acid components in a fatty acid composition is established, and the method reduces the use of a reference substance and ensures the reliability of a qualitative and quantitative detection result;
(3) establishing a selection standard of a positioning substance in a segmented positioning method, wherein the selection standard has applicability to fatty acid compositions with different compositions;
(4) the method can ensure the stability and the durability of long-term use, and is more suitable for long-term detection of products.
Detailed Description
In order to further illustrate the present invention and to facilitate an understanding thereof, only some examples are provided and will be described in detail. It will be understood by those skilled in the art that the following examples are not intended to limit the scope of the present invention.
Comparative example 1
(1) Sample preparation
Sample preparation: according to the commercial products
The product composition of (1) and preparing the obtained sample.
(2) Sample detection
The fatty acid composition in the sample was measured according to gas chromatography (chinese pharmacopoeia 2015 edition four parts general rules 0512).
Antioxidant solution: taking a proper amount of 2, 6-di-tert-butyl-p-cresol, precisely weighing, dissolving with isooctane, and preparing into a solution containing about 0.05mg of 2, 6-di-tert-butyl-p-cresol per mL;
test solution: taking a proper amount of samples, precisely weighing a proper amount of samples, dissolving the samples by using an antioxidant solution, and preparing the samples into a solution containing about 2.5mg of samples per mL;
the chromatographic conditions were as follows:
a chromatographic column: a quartz capillary column (25m x 0.25mm, 0.2 μm) with polyethylene glycol as the stationary liquid;
temperature rising procedure: the initial temperature is 168 deg.C, maintained for 3min, and heated to 24 deg.C at a rate of 4 deg.C per minute
Maintaining the temperature at 0 ℃ for 10 min;
carrier gas: helium gas;
sample inlet temperature: 245 ℃;
detector temperature: 265 ℃ C;
flow rate: 1.2 mL/min;
sample introduction amount: 1 mu L of the solution;
the split ratio is as follows: 30: 1;
the determination method comprises the following steps: precisely measuring the test solution, injecting into a gas chromatograph, and recording the chromatogram.
(3) Localized material selection
Identifying the structure of the fatty acid component to be detected through a mass spectrometer, and selecting DHA ethyl ester as a positioning substance from the fatty acid component to be detected.
(4) Durability examination
Durability examination conditions: the starting temperature is +/-2 ℃; the temperature of a sample inlet is +/-5 ℃; flow rate. + -. 0.1 mL/min.
And calculating the difference between the standard value of the relative retention time of each fatty acid component in the sample and the detection value of the relative retention time under different chromatographic conditions, and taking the absolute value of the maximum value as the maximum drift value to investigate the durability of the method. The results of the tests are given in the following table:
TABLE 1 results of the durability test with DHA ethyl ester as the positioning substance
Comparative example 2
(1) Sample preparation
The same batch of samples prepared in the "sample preparation" step of step (1) of comparative example 1 was taken, stored under long-term storage conditions (temperature 25 ℃, humidity 60%), and the samples were taken out to be tested after 3 months of storage.
(2) Sample detection
The procedure of step (2) "sample detection" in comparative example 1 was followed.
(3) Localized material selection
According to the "selection of localization material" step of step (3) in comparative example 1, the same localization material DHA ethyl ester as in comparative example 1 was selected as the localization material.
(4) Long term sample test results
Calculating the difference value between the standard value of the relative retention time and the detection value of the relative retention time of each fatty acid component in the sample, and taking the absolute value of the difference value as a drift value to investigate the drift value of the relative retention time in long-term detection, wherein the test result is shown in the following table:
table 2 long term assay relative retention time drift data with DHA ethyl ester localization
| Fatty acid component | Day 0 | 3 months old |
| Phytanic acid ethyl ester | 0.002 | 0.008 |
| C18:3 n-6 Ethyl ester | 0.001 | 0.007 |
| C18:3 n-4 ethyl ester | 0.001 | 0.007 |
| C18:4n-3 ethyl ester | 0.000 | 0.006 |
| C18:4 n-1 ethyl ester | 0.001 | 0.007 |
| Furoic acid 5 ethyl ester | 0.001 | 0.005 |
| C19:5 n-3 Ethyl ester | 0.001 | 0.005 |
| C20:3 n-6 Ethyl ester | 0.001 | 0.005 |
| Furanoic acid 7 ethyl ester | 0.001 | 0.005 |
| Furanoic acid 8 ethyl ester | 0.000 | 0.004 |
| EPA Ethyl ester | 0.000 | 0.004 |
| Furanoic acid 9 ethyl ester | 0.000 | 0.002 |
| C22:4 n-6 ethyl ester | 0.000 | 0.001 |
| Furancarboxylic acid 10 ethyl ester | 0.000 | 0.001 |
| C22:5 n-6 Ethyl ester | 0.000 | 0.001 |
| Furancarboxylic acid 11 ethyl ester | 0.000 | 0.000 |
| DHA Ethyl ester | — | — |
| C24:6 Ethyl ester | 0.000 | 0.003 |
As can be seen from the above table, the relative retention time values of the fatty acid components measured at 3 months of long-term storage shifted when DHA ethyl ester was used as the targeting substance. Wherein, the drift is larger as the fatty acid chromatographic peak in the sample is farther from the DHA ethyl ester chromatographic peak, the relative retention time drift values of the components are more than 0.005, and the relative retention time drift values of the phytanic acid ethyl ester, the C18:3 n-6 ethyl ester and the C18:3 n-4 ethyl ester are more than or equal to 0.007.
Example 1
(1) Sample preparation
The same lot of the sample prepared in the "sample preparation" step of step (1) of comparative example 1 was taken as a sample to be measured.
(2) Sample detection
The procedure of step (2) "sample detection" in comparative example 1 was followed.
(3) Localized material selection
Identifying the structure of the fatty acid component to be detected by a mass spectrometer, selecting C18:4n-3 ethyl ester from C18 fatty acid ethyl ester components in the fatty acid component to be detected, selecting EPA ethyl ester from C20 fatty acid ethyl ester components, selecting DHA ethyl ester from C22 fatty acid ethyl ester components, and taking the three fatty acid ethyl ester components as positioning substances.
(4) Determining relative retention time of each component
The relative retention time of each fatty acid component to be tested was determined based on the localization substance and the results are given in the following table:
TABLE 3 Standard Table of relative Retention time of Components
Example 2
(1) Sample preparation
The same lot of the sample prepared in the "sample preparation" step of step (1) of comparative example 1 was taken as a sample to be measured.
(2) Sample detection
The procedure of step (2) "sample detection" in comparative example 1 was followed.
(3) Localized material selection
Identifying the structure of the fatty acid component to be detected by a mass spectrometer, selecting C18:4n-3 ethyl ester from C18 fatty acid ethyl ester components in the fatty acid component to be detected, selecting EPA ethyl ester from C20 fatty acid ethyl ester components, selecting DHA ethyl ester from C22 fatty acid ethyl ester components, and taking the three fatty acid ethyl ester components as positioning substances.
(4) Durability examination
The procedure of step (4) "durability examination" in comparative example 1 was followed.
The results of the tests are given in the following table:
TABLE 4 test results of durability by stage positioning
From the data in the table, it can be seen that when C18:4n-3 ethyl ester, EPA ethyl ester and DHA ethyl ester were used as the positioning substances for the stepwise positioning, the relative retention time values of the respective fatty acid components in the sample shifted with the fine adjustment of the detection conditions, and the results of the two positioning methods were not much different when the chromatographic conditions were fine-tuned, as can be seen from the results of the durability of the positioning substance DHA ethyl ester in comparative example 1.
Example 3
(1) Sample preparation
The sample obtained in the "sample preparation" step (1) of comparative example 2 was taken as a sample to be measured.
(2) Sample detection
The procedure of step (2) "sample detection" in comparative example 2 was followed.
(3) Localized material selection
Identifying the structure of the fatty acid component to be detected by a mass spectrometer, selecting C18:4n-3 ethyl ester from C18 fatty acid ethyl ester components in the fatty acid component to be detected, selecting EPA ethyl ester from C20 fatty acid ethyl ester components, selecting DHA ethyl ester from C22 fatty acid ethyl ester components, and taking the three fatty acid ethyl ester components as positioning substances.
(4) Long term sample test results
The procedure of "Long term sample test result" of step (4) in comparative example 2 was followed.
The results of the tests are given in the following table:
TABLE 5 Long term test relative retention time drift data for segmented positioning
| Fatty acid component | Day 0 | 3 months old |
| Phytanic acid ethyl ester | 0.000 | 0.003 |
| C18:3 n-6 Ethyl ester | 0.001 | 0.002 |
| C18:3 n-4 ethyl ester | 0.001 | 0.001 |
| C18:4n-3 ethyl ester | — | — |
| C18:4 n-1 ethyl ester | 0.000 | 0.003 |
| Furoic acid 5 ethyl ester | 0.000 | 0.002 |
| C19:5 n-3 Ethyl ester | 0.000 | 0.001 |
| C20:3n-6 ethyl ester | 0.000 | 0.001 |
| Furanoic acid 7 ethyl ester | 0.000 | 0.001 |
| Furanoic acid 8 ethyl ester | 0.000 | 0.000 |
| EPA Ethyl ester | — | — |
| Furanoic acid 9 ethyl ester | 0.000 | 0.002 |
| C22:4 n-6 ethyl ester | 0.000 | 0.001 |
| Furancarboxylic acid 10 ethyl ester | 0.000 | 0.001 |
| C22:5 n-6 Ethyl ester | 0.000 | 0.001 |
| Furancarboxylic acid 11 ethyl ester | 0.000 | 0.000 |
| DHA Ethyl ester | — | — |
| C24:6 Ethyl ester | 0.000 | 0.003 |
As can be seen from the above table, when the segmented localization is used, the relative retention time drift values of the fatty acid components detected at 3 months are not more than 0.003; even if the peaks of the chromatographic profiles of phytanic acid ethyl ester, C18:3 n-6 ethyl ester, C18:3 n-4 ethyl ester are far from the fatty acid component of the DH A ethyl ester component, the relative retention time shift value at 3 months does not exceed 0.003. The result shows that in the long-term application of the detection method, the drift of the relative retention time of the fatty acid component obtained by the detection of the method is obviously reduced, the fatty acid component to be detected can be better positioned, and the qualitative detection capability of the method is more stable than that of the method which singly uses DHA ethyl ester as a positioning substance and is more suitable for the long-term detection of products.
Example 4
(1) Sample preparation
The same lot of the sample prepared in the "sample preparation" step of step (1) of comparative example 1 was taken as a sample to be measured.
(2) Sample detection
The procedure of step (2) "sample detection" in comparative example 1 was followed.
(3) Localized material selection
Identifying the structure of the fatty acid component to be detected by a mass spectrometer, selecting C18:4 n-1 ethyl ester from C18 fatty acid ethyl ester components in the fatty acid component to be detected, selecting C20:3 n-6 ethyl ester from C20 fatty acid ethyl ester components, selecting C22:5 n-6 ethyl ester from C22 fatty acid ethyl ester components, and taking the three fatty acid ethyl ester components as positioning substances.
(4) Determining relative retention time of each component
The relative retention time of each fatty acid component to be tested was determined based on the localization substance and the results are given in the following table:
TABLE 6 Standard Table of relative Retention time of Components
The long-term 3-month samples in the comparative example 2 are detected by using the method, and the detection results show that the relative retention time drift values of the detected fatty acid components do not exceed 0.005 in 3 months; even if the chromatographic peaks of phytanic acid ethyl ester, C18:3 n-6 ethyl ester and C18:3 n-4 ethyl ester are far away from the fatty acid component of DHA ethyl ester component, the relative retention time drift value at 3 months does not exceed 0.004. The result shows that in the long-term application of the detection method, the qualitative detection capability of the method is more stable than that of the method using DHA ethyl ester alone as a positioning substance, and the method is more suitable for long-term detection of products.
Example 5
(1) Sample preparation
The same lot of the sample prepared in the "sample preparation" step of step (1) of comparative example 1 was taken as a sample to be measured.
(2) Sample detection
The procedure of step (2) "sample detection" in comparative example 1 was followed.
(3) Localized material selection
Identifying the structure of the fatty acid component to be detected by a mass spectrometer, selecting C20:3 n-6 ethyl ester from C20 fatty acid ethyl ester components in the fatty acid component to be detected, selecting DHA ethyl ester from C22 fatty acid ethyl ester components, and taking the two fatty acid ethyl ester components as positioning substances.
(4) Determining relative retention time of each component
The relative retention time of each fatty acid component to be tested was determined based on the localization substance and the results are given in the following table:
TABLE 7 Standard Table of relative Retention time of Components
The long-term 3-month samples in the comparative example 2 are detected by using the method, and the detection results show that the relative retention time drift values of the detected fatty acid components do not exceed 0.005 in 3 months; even if the chromatographic peaks of phytanic acid ethyl ester, C18:3 n-6 ethyl ester and C18:3 n-4 ethyl ester are far away from the fatty acid component of DHA ethyl ester component, the relative retention time drift value at 3 months does not exceed 0.005. The result shows that in the long-term application of the detection method, the qualitative detection capability of the method is more stable than that of the method using DHA ethyl ester alone as a positioning substance, and the method is more suitable for long-term detection of products.
In conclusion, in the long-term detection process of the product, the fatty acid components in the sample are positioned in a segmented manner by adopting the technical scheme of the invention, the measured relative retention time values of the fatty acid components in the sample have small drift, and are all obviously smaller than the drift value when the fatty acid components are positioned by DHA ethyl ester, the detection stability is improved, and the difficulty in qualitative fatty acid component determination is reduced.
Claims (9)
1. A method for detecting a fatty acid composition, comprising detecting the fatty acid composition by chromatography to obtain retention times of respective fatty acid components in the composition, wherein two or more fatty acid components in the composition are selected as a localization substance after obtaining the retention times of the respective fatty acid components, relative retention times of other fatty acid components in the composition with respect to a specified localization substance are obtained, and fatty acid components other than the localization substance in the composition are characterized by using the relative retention times.
2. The detection method according to claim 1, wherein the fatty acid composition has at least two kinds of carbon atoms in the fatty acid composition, and the fatty acids as the localization substances have different carbon atoms.
3. The method according to claim 1, wherein the specific localization substance is a localization substance having a retention time after the fatty acid component and having a closest retention time, and the fatty acid composition further contains a fatty acid component having no localization substance after the retention time, and the fatty acid component is a localization substance having a retention time before the retention time and having a closest retention time.
4. The method of claim 1, further comprising determining the structure of the localization substance and/or the structure of the fatty acid component to which the relative retention time corresponds prior to characterizing the other fatty acid components in the composition with the relative retention time.
5. The assay of claim 1, wherein the fatty acid composition is prepared from fatty acids purified from natural fish oil.
6. The assay of any one of claims 1-5, wherein the fatty acid composition comprises omega-3 fatty acids; preferably, the content of omega-3 fatty acid in the fatty acid composition is 90-100%; more preferably, the content of EPA is 42-66% and the content of DHA is 34-58% in the fatty acid composition; more preferably, the fatty acid composition has an EPA content of 42% to 50% and a DHA content of 34% to 41%.
7. The assay of claim 6, wherein the fatty acid composition further comprises one or more components of C18 fatty acids, one or more components of C20 fatty acids other than EPA, and one or more components of C22 fatty acids other than DHA.
8. The detection method according to claim 2, wherein the localization substance is two or three fatty acid components different in the number of carbon atoms in a C18 fatty acid, a C20 fatty acid, a C22 fatty acid; preferred localization substances are C18:4n-3, EPA and DHA.
9. The detection method according to claim 6, wherein the chromatographic conditions are as follows:
a chromatographic column: polyethylene glycol is a quartz capillary column of stationary liquid, or a quartz capillary column with polarity similar to that of polyethylene glycol is selected; the specification is 25m × 0.25mm, 0.2 μm;
temperature rising procedure: the initial temperature is 166-170 ℃, the temperature is maintained for 1-3min, the temperature is raised to 240 ℃ at the rate of 2-4 ℃ per minute, and the temperature is maintained for 6-10 min;
carrier gas: helium gas;
sample inlet temperature: 240 ℃ and 250 ℃;
detector temperature: 260 ℃ and 270 ℃;
flow rate: 1.1-1.3 mL/min;
sample introduction amount: 1 mu L of the solution;
the split ratio is as follows: 10:1-50:1.
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