CN108181395B - Method for detecting triglyceride components in fish oil on line by adopting high performance liquid chromatography - Google Patents
Method for detecting triglyceride components in fish oil on line by adopting high performance liquid chromatography Download PDFInfo
- Publication number
- CN108181395B CN108181395B CN201711464313.2A CN201711464313A CN108181395B CN 108181395 B CN108181395 B CN 108181395B CN 201711464313 A CN201711464313 A CN 201711464313A CN 108181395 B CN108181395 B CN 108181395B
- Authority
- CN
- China
- Prior art keywords
- fish oil
- liquid chromatography
- triglyceride
- sample
- triglyceride components
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 235000021323 fish oil Nutrition 0.000 title claims abstract description 84
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 37
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 14
- 238000004519 manufacturing process Methods 0.000 claims abstract description 19
- 238000004458 analytical method Methods 0.000 claims abstract description 13
- 238000003908 quality control method Methods 0.000 claims abstract description 8
- 238000004451 qualitative analysis Methods 0.000 claims abstract description 7
- 238000011002 quantification Methods 0.000 claims abstract description 7
- 238000000105 evaporative light scattering detection Methods 0.000 claims abstract description 6
- 239000012085 test solution Substances 0.000 claims abstract description 6
- 239000000047 product Substances 0.000 claims description 25
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 238000004811 liquid chromatography Methods 0.000 claims description 10
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 10
- 238000000926 separation method Methods 0.000 claims description 10
- 239000002994 raw material Substances 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 7
- 238000005516 engineering process Methods 0.000 claims description 7
- 239000013067 intermediate product Substances 0.000 claims description 7
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 4
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 3
- FPWNQPQTICPCOM-UHFFFAOYSA-N acetonitrile;propan-2-ol Chemical group CC#N.CC(C)O FPWNQPQTICPCOM-UHFFFAOYSA-N 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 238000001474 liquid chromatography-evaporative light scattering detection Methods 0.000 claims description 2
- 239000000243 solution Substances 0.000 claims description 2
- 238000012544 monitoring process Methods 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 16
- 230000008569 process Effects 0.000 description 8
- 230000008859 change Effects 0.000 description 7
- 235000014113 dietary fatty acids Nutrition 0.000 description 7
- 229930195729 fatty acid Natural products 0.000 description 7
- 239000000194 fatty acid Substances 0.000 description 7
- 150000004665 fatty acids Chemical class 0.000 description 7
- 239000003921 oil Substances 0.000 description 7
- 235000019198 oils Nutrition 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 238000004587 chromatography analysis Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 230000014759 maintenance of location Effects 0.000 description 5
- 238000004809 thin layer chromatography Methods 0.000 description 5
- 241000251468 Actinopterygii Species 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 235000019688 fish Nutrition 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 150000003626 triacylglycerols Chemical class 0.000 description 4
- 238000000889 atomisation Methods 0.000 description 3
- 238000013375 chromatographic separation Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 238000004817 gas chromatography Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241001454694 Clupeiformes Species 0.000 description 2
- 230000005526 G1 to G0 transition Effects 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- JAZBEHYOTPTENJ-JLNKQSITSA-N all-cis-5,8,11,14,17-icosapentaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O JAZBEHYOTPTENJ-JLNKQSITSA-N 0.000 description 2
- 235000019513 anchovy Nutrition 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 235000020673 eicosapentaenoic acid Nutrition 0.000 description 2
- 229960005135 eicosapentaenoic acid Drugs 0.000 description 2
- JAZBEHYOTPTENJ-UHFFFAOYSA-N eicosapentaenoic acid Natural products CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O JAZBEHYOTPTENJ-UHFFFAOYSA-N 0.000 description 2
- 239000003925 fat Substances 0.000 description 2
- 235000019197 fats Nutrition 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000007670 refining Methods 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- -1 silver ions Chemical class 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000004808 supercritical fluid chromatography Methods 0.000 description 2
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 2
- DVSZKTAMJJTWFG-SKCDLICFSA-N (2e,4e,6e,8e,10e,12e)-docosa-2,4,6,8,10,12-hexaenoic acid Chemical compound CCCCCCCCC\C=C\C=C\C=C\C=C\C=C\C=C\C(O)=O DVSZKTAMJJTWFG-SKCDLICFSA-N 0.000 description 1
- GZJLLYHBALOKEX-UHFFFAOYSA-N 6-Ketone, O18-Me-Ussuriedine Natural products CC=CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O GZJLLYHBALOKEX-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- FOIXSVOLVBLSDH-UHFFFAOYSA-N Silver ion Chemical compound [Ag+] FOIXSVOLVBLSDH-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 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
- 210000004204 blood vessel Anatomy 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000010668 complexation reaction Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000004332 deodorization Methods 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 235000020669 docosahexaenoic acid Nutrition 0.000 description 1
- KAUVQQXNCKESLC-UHFFFAOYSA-N docosahexaenoic acid (DHA) Natural products COC(=O)C(C)NOCC1=CC=CC=C1 KAUVQQXNCKESLC-UHFFFAOYSA-N 0.000 description 1
- 235000020774 essential nutrients Nutrition 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000002960 lipid emulsion Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 150000004668 long chain fatty acids Chemical class 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- LADGBHLMCUINGV-UHFFFAOYSA-N tricaprin Chemical compound CCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCC)COC(=O)CCCCCCCCC LADGBHLMCUINGV-UHFFFAOYSA-N 0.000 description 1
- 125000005457 triglyceride group Chemical group 0.000 description 1
- VLPFTAMPNXLGLX-UHFFFAOYSA-N trioctanoin Chemical compound CCCCCCCC(=O)OCC(OC(=O)CCCCCCC)COC(=O)CCCCCCC VLPFTAMPNXLGLX-UHFFFAOYSA-N 0.000 description 1
- 230000008016 vaporization Effects 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
Images
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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
-
- 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
- G01N2030/8813—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 biological materials
-
- 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
- G01N2030/884—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 organic compounds
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Fats And Perfumes (AREA)
Abstract
The invention discloses a method for detecting triglyceride components in fish oil on line by adopting high performance liquid chromatography. The method comprises the following steps: (1) dissolving the fish oil sample, fixing the volume to a scale, and shaking up to obtain a test solution; (2) and (2) carrying out high performance liquid chromatography analysis on the fish oil sample in the step (1), determining the triglyceride components in the fish oil sample by adopting an evaporative light scattering detector, carrying out qualitative analysis and sectional quantification on the triglyceride components of the fish oil, and carrying out online quality monitoring on the production process of the refined fish oil by comparing the content variation difference of the triglyceride components in the fish oil sample at different production stages. The method is simple to operate, economical, environment-friendly and high in safety, and can be used as an auxiliary means for online quality control of a production process of refined fish oil products.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for detecting triglyceride components in fish oil on line by adopting high performance liquid chromatography.
Background
Fish oil is a generic term for all oil substances in fish bodies, and includes body oil, liver oil and naphtha. Unlike other animal fats and oils or vegetable oils, fish oil is rich in n-3 type unsaturated fatty acids, among which eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are the most studied unsaturated fatty acid components at present and are also essential nutrients for human growth and development. Researches show that the fish oil has the effects of reducing blood fat, softening blood vessels, supplementing brain nutrition, assisting in improving memory and the like. Therefore, products such as healthy dietary supplements, health products, medicines and the like which are prepared by taking fish oil as a raw material and processing and refining through a series of processes such as impurity removal, color removal, deodorization and the like are successfully marketed at home and abroad and are favored by a plurality of consumers. At present, fish oil products on the market are numerous, and the quality control of the fish oil mainly focuses on the composition and content percentage of fatty acid besides basic essential control indexes such as appearance, acid value, peroxide value and the like.
The fatty acid component in fish oil exists mainly in triglyceride form, i.e. the fish oil is synthesized by dehydration and condensation of 1 glycerol molecule and 3 fatty acid molecules, wherein long-chain fatty acid triglyceride is taken as a main component. However, because the types of fatty acids that can be combined on the glycerol skeleton are various, and the physical properties and chemical properties of each triglyceride component in the natural fish oil are close, the types of the triglycerides are very large, and isomers and positional isomers also exist in a large amount. At present, the following chromatographic techniques are mainly used for analyzing triglyceride components in oil substances: thin Layer Chromatography (Thin Layer Chromatography, TLC), Gas Chromatography (Gas Chromatography, GC), High Performance Liquid Chromatography (HPLC), and Supercritical Fluid Chromatography (SFC). Among them, the TLC method has long and wide application, but can only be used as an auxiliary means for qualitative analysis due to low separation and recovery effect and difficult quantification; the GC method is used for analyzing triglyceride components, and the phenomena of high column temperature, tailing of chromatographic peaks, unrepeated retention time and the like are caused by the reasons of high boiling point, difficult volatilization and the like of the triglyceride, so a fatty acid derivatization method is usually adopted for determination; SFC analysis requires special operating conditions and high requirements for experimental equipment, and therefore, is only a supplementary means for chromatographic analysis and is not widely used in analysis of triglycerides. The HPLC technique is not limited by thermal stability and volatility, and is widely used for analysis of triglyceride components. Wherein, the conventional normal phase chromatography has poor separation effect and serious peak overlapping phenomenon, so the use is less; silver ion chromatography is a special normal phase chromatography technology, which separates triglyceride with different double bond numbers and positions based on weak pi complexation adsorption between silver ions and triglyceride unsaturated fatty acid double bonds, and when the number of double bonds is more, the elution is more difficult, and after the number of double bonds exceeds 5, the elution is difficult. While reverse phase chromatography is based on octadecyl bonded silica gel as the stationary phase, the retention time of which increases with the increase of the Equivalent Carbon Number (ECN), i.e. the total Carbon Number of triglyceride acyl groups (CNs), is one of the most predominant methods for separating triglyceride compositions.
The triglyceride component of fish oil is very complex and mostly long-chain. The research results of the literature show that few researches on triglyceride components in fish oil are carried out under the conventional liquid chromatography conditions at present, and qualitative and quantitative analysis on triglyceride is carried out in most researches by adopting a liquid chromatography-mass spectrometry tandem technology, but the mass spectrometry technology has high cost and complex operation, has high technical requirements on experimenters, and has high difficulty in realizing online quality monitoring of products. Therefore, establishing an economic, simple and rapid liquid chromatography detection and analysis method for analyzing the process of manufacturing refined fish oil and the change of triglyceride components of the refined fish oil are particularly important for guiding the optimization of the production process.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for detecting triglyceride components in fish oil on line by adopting high performance liquid chromatography. The method is simple to operate, economical, environment-friendly and high in safety, and can be used as an auxiliary means for online quality control of the production process of fish oil products.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for detecting triglyceride components in fish oil on line by adopting high performance liquid chromatography comprises the following steps:
(1) dissolving the fish oil sample, fixing the volume to a scale, and shaking up to obtain a test solution;
(2) and (2) carrying out high performance liquid chromatography analysis on the fish oil sample in the step (1), determining the triglyceride components in the fish oil sample by adopting an evaporative light scattering detector, carrying out qualitative analysis and sectional quantification on the triglyceride components of the fish oil, and comparing the content variation difference of the triglyceride components in the fish oil sample at different production stages to obtain the fish oil product production online quality control method.
Preferably, in the method, the fish oil sample in the step (1) includes a raw material of deep sea fish oil, an intermediate product of refined fish oil, and a finished product of refined fish oil, and the sample is dissolved in n-hexane to prepare a solution of 0.02 g/mL.
Preferably, in the above method, the liquid chromatography conditions of step (2) are C18(Kromasil 100-5) chromatography column, and the stationary phase is octadecylsilane bonded silica gel adsorption phase with specification of 250mm × 4.6mm, 5 μm;
preferably, in the method, the sample injection volume during the liquid chromatography condition sample measurement in the step (2) is 5-10 μ L, preferably 5 μ L; the temperature of the chromatographic column is 30-35 ℃, and 35 ℃ is preferred;
preferably, in the above method, the mobile phase used in the liquid chromatography conditions of step (2) is a mixed mobile phase system, wherein phase a is acetonitrile-water (2:8), phase B is acetonitrile-isopropanol (1:1), and gradient elution is adopted: 0-5min (40-10% of A, 60-90% of B), 5-15min (10-4% of A, 90-96% of B), 15-30min (4% of A, 96% of B), 30-40min (4-2% of A, 96-98% of B), 40-42min (2-0% of A, 98-100% of B), 42-45min (0% of A, 100% of B), 45-50min (0-40% of A, 100-60% of B) and 50-60min (40% of A, 60% of B);
preferably, in the above method, the flow rate of the mobile phase in the step (2): 1.0-1.2 mL/min, preferably 1.0 mL/min;
preferably, in the above method, the liquid chromatography condition in step (2) is an evaporative light scattering detector, and the conditions include a drift tube temperature of 75 to 85 ℃, an atomization chamber temperature of 40 to 50 ℃, a nitrogen flow rate of 2.0 to 2.6mL/min, and a Gain value of 1, 2, or 4. The optimal conditions are as follows: the drift tube temperature is 85 ℃; the temperature of the atomizing chamber is 40 ℃; the nitrogen flow rate is 2.6 mL/min; the Gain value was 2.
The normal hexane is analytically pure, the acetonitrile and the isopropanol are chromatographically pure, and the water is purified water. The reagents and starting materials used in the present invention are commercially available.
In the invention, the triglyceride component is qualitatively analyzed according to the peak retention time, the number of peaks and the difference of peak heights of chromatographic peaks in the fish oil sample. And dividing chromatographic peaks into 6 sections according to the sequence of the peak appearance time of the chromatographic peaks for subsection quantification, and comparing the difference of the relative peak area percentage of each section in the fish oil samples at different production stages to be used as one of the online quality control means of the production process of the fish oil products.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention applies the conventional separation and detection technology of high performance liquid chromatography-evaporative light scattering method to the component analysis of the triglyceride of the fish oil. The analysis method can better reflect the information of multiple components of the triglyceride in the fish oil, and the chromatographic peaks can be well separated. By comparing the peak retention time, the change of the peak height and the content change trend of the triglyceride component chromatogram in the fish oil samples in different production stages, the method is used as an online quality control means of the production process of the fish oil product, and provides data support for the industrialization of subsequent products and basis for establishing the quality standard of the products.
2. Compared with the traditional thin-layer chromatography which is a simple qualitative analysis technology, the method has the advantages of good separation degree effect and high recovery rate, and can be independently used for qualitative analysis and sectional quantification of triglyceride components; compared with the liquid chromatography-mass spectrometry of the triglyceride quantification technology, the method has the advantages of low economic cost, simplicity, easy operation, low requirement on laboratory equipment configuration and popularization and application.
3. In the chromatographic conditions adopted by the invention, the mobile phase composition is not completely organic reagent, and water is adopted as one of the solvents, so that the method is economical and environment-friendly, and has little harm to operators. The toxicity of the mobile phase system is reduced, and the safety of the experimental operation process is high.
4. The chromatographic column adopted by the invention is a commonly used C18 chromatographic column, and the analysis and detection cost is relatively low.
Drawings
FIG. 1 is a liquid chromatogram of a fish oil sample under different chromatographic conditions in example 1 (A: the chromatographic conditions of this patent and B: the reference chromatographic conditions of the literature);
FIG. 2 is a liquid chromatogram of deep sea fish oil raw materials from different fish sources in example 2 (A: tuna oil, B: anchovy fish oil);
FIG. 3 is a liquid chromatogram of a fish oil sample at different stages of the production process in example 2 (A: intermediate product of refined fish oil, B: finished product of refined fish oil).
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, steps or conditions of the present invention by those of skill in the art without departing from the spirit and scope of the present invention.
Example 1: this example compares the chromatographic conditions of the literature with those of the present patent
Literature (investigation of determination of the content of structural triglycerides in structural fat emulsion injection by the Henschel, Liujin Xiu, Xukansen, ELSD method [ J)]Journal of drug analysis, 2004,24(03): 301-. Literature chromatographic conditions: the chromatographic column is Kromasil 100-5C18(250 mm. times.4.6 mm, 5 μm); the mobile phase is a mixed mobile phase system, wherein the mobile phase A is acetonitrile, the mobile phase B is acetonitrile-acetone-isooctane (29.5: 48.5: 22.0), and gradient elution is adopted, and the conditions are as follows: 0-2min (100-60% of A, 0-40% of B), 2-22min (60-35% of A, 40-65% of B), 22-42min (35-25% of A, 65-75% of B), 42-50min (25-10% of A, 75-90% of B), 50-52min (10-100% of A, 90-0% of B), 52-60min (100% of A, 0% of B), 1.0mL/min of mobile phase flow rate, and 25 ℃ of column temperature; the vaporizing photodetector atomization chamber temperature was 65 ℃ and the nitrogen flow rate was 2.6 mL/min.
The chromatographic conditions of the patent are as follows: the chromatographic column is Kromasil 100-5C18(250 mm. times.4.6 mm, 5 μm); the mobile phase is mixed mobile phase system, wherein the mobile phase A is acetonitrile-water (2:8), the mobile phase B is acetonitrile-isopropanol (1:1), and the mixed mobile phase system adoptsGradient elution, as follows: 0-5min (40-10% of A, 60-90% of B), 5-15min (10-4% of A, 90-96% of B), 15-30min (4% of A, 96% of B), 30-40min (4-2% of A, 96-98% of B), 40-42min (2-0% of A, 98-100% of B), 42-45min (0% of A, 100% of B), 45-50min (0-40% of A, 100-60% of B), 50-60min (40% of A, 60% of B), 1.0mL/min of mobile phase flow rate, and 35 ℃ of column temperature; the drift tube temperature of the evaporation photodetector was 85 ℃, the atomization chamber temperature was 40 ℃, and the nitrogen flow rate was 2.6 mL/min.
(1) Liquid chromatographic separation of fatty acid triglyceride standard under two chromatographic conditions
Respectively weighing appropriate amount of caprylic triglyceride and capric triglyceride, dissolving with n-hexane to obtain mixed standard solution with concentration of about 0.5mg/mL, filtering with 0.45 μm filter membrane, respectively measuring according to the chromatographic conditions (1) and (2) of the example by liquid chromatography, and recording chromatogram.
The results show that: the degrees of separation of the two fatty acid triglyceride standard products under the chromatographic conditions of the patent and the literature are respectively 28.4 and 24.4, which shows that the degrees of separation under the chromatographic conditions of the patent are superior to those in the literature.
(2) Liquid chromatographic separation of triglyceride component of fish oil sample under two chromatographic conditions
The fish oil sample is subjected to chromatographic separation of triglyceride component under the chromatographic conditions of the patent and the literature. By comparing the number of chromatographic peaks, the superiority and inferiority of the separation effect of triglyceride components in fish oil under two conditions were examined. The specific implementation is as follows:
taking about 0.1g of fish oil sample, precisely weighing, placing in a 10mL measuring flask, adding n-hexane for dissolving and fixing the volume to obtain a test solution, and filtering with a 0.45-micron filter membrane for high performance liquid chromatography determination.
Taking the test solution, respectively performing liquid chromatography separation and determination on triglyceride in the fish oil finished product according to two different chromatographic conditions, and recording a chromatogram.
The results show that: under the chromatographic conditions of the patent, the number of chromatographic peaks of the fish oil sample is 38 (except for solvent peaks), while the number of chromatographic peaks under the chromatographic conditions of the literature is 33 (except for solvent peaks), and the separation effect of the peaks under the chromatographic conditions of the patent is obviously better than that of the chromatographic conditions of the literature. The chromatogram is shown in FIG. 1.
Example 2: liquid chromatography analysis of triglyceride content in fish oil samples at different process stages
Respectively taking deep sea fish oil raw materials (tuna oil and anchovy fish oil), refined fish oil intermediate products and refined fish oil finished products 0.1g respectively, precisely weighing, placing in a 10mL measuring flask, adding n-hexane for dissolving and fixing the volume, respectively preparing to obtain test solution, filtering with 0.45 μm filter membrane, and determining with high performance liquid chromatography.
mu.L of each sample solution was taken, injected into a liquid chromatograph, measured under the color change conditions of the patent in example 1, and a chromatogram was recorded.
As can be seen from the results of the chromatographic analyses of 2 fish oil raw materials, the intermediate refined fish oil products and the finished refined fish oil products, as shown in fig. 2 and 3, the difference in triglyceride information among the fish oil raw materials of different fish families is large; the number of chromatographic peaks and the percentage change trend of the relative peak area of the triglyceride in samples in different process stages are obvious, and specific results are shown in tables 1 and 2.
TABLE 1 number of triglyceride chromatogram peaks in fish oil at different production stages
TABLE 2 relative peak area percentages of triglycerides in fish oil samples at different stages of production
As can be seen from Table 1, the number of triglyceride chromatographic peaks of the raw material is 32, while the number of triglyceride chromatographic peaks of the process intermediate product reaches 39, and the number of chromatographic peaks of the final product is 36 after the process treatment for removing impurities.
The peak was divided into 6 fragments according to the elution time of the chromatographic peak, and the relative peak area percentage of each fragment was calculated. As can be seen from the analysis of fig. 2, fig. 3 and table 2, the triglyceride content in the fish oil raw material is mainly concentrated in the 4, 5 and 6 sections, and the content is ranked from high to low as 6>5>4, while the triglyceride content in the 1, 2 and 3 sections is different according to the variety of the fish; the refined fish oil intermediate product and the refined fish oil finished product mainly concentrate in 3, 4, 5 and 6 sections, and the content is 4>5>6>3 from high to low. Compared with the intermediate product, the triglyceride content of the 1 section in the finished product is obviously reduced, and the triglyceride content of the 4 sections in the finished product is increased, which is closely related to the technological process of refining the fish oil.
Therefore, the data analysis can summarize the peak retention time, the change of the peak height and the change trend of the content of the triglyceride component chromatogram in the fish oil sample at different production stages, and the method can be used as an online quality control means of the production process of the fish oil product, and provides data support for the industrialization of subsequent products and basis for establishing the quality standard of the product.
Claims (1)
1. A method for detecting triglyceride components in fish oil on line by adopting high performance liquid chromatography is characterized by comprising the following steps:
(1) dissolving the fish oil sample, fixing the volume to a scale, and shaking up to obtain a test solution;
(2) performing high performance liquid chromatography analysis on the fish oil sample in the step (1), determining the triglyceride components in the fish oil sample by adopting an evaporative light scattering detector, performing qualitative analysis and sectional quantification on the triglyceride components of the fish oil, and comparing the content variation difference of the triglyceride components in the fish oil sample at different production stages to obtain an online quality control method for the production of the fish oil product;
the mobile phase adopted by the liquid chromatogram condition of the step (2) is a mixed mobile phase system, wherein the phase A is acetonitrile-water 2:8, the phase B is acetonitrile-isopropanol 1:1, and gradient elution is adopted: 0-5min A: 40% -10%, B: 60% -90%, 5-15min A: 10% -4%, B: 90% -96%, 15-30min A: 4%, B: 96%, 30-40min A: 4% -2%, B: 96% -98%, 40-42min A: 2% -0%, B: 98% -100%, 42-45min A: 0%, B: 100% 45-50min A: 0% -40%, B: 100% -60%, 50-60min A: 40%, B: 60 percent, and the flow rate is 1.0 mL/min;
the fish oil sample in the step (1) comprises a deep sea fish oil raw material, a refined fish oil intermediate product and a refined fish oil finished product; dissolving a sample by using normal hexane to prepare a solution of 0.02g/mL, and analyzing and determining triglyceride components by adopting a high performance liquid chromatography-evaporative light scattering method separation and detection technology;
the chromatographic column adopted by the liquid chromatographic conditions of the step (2) is C18A chromatographic column with the specification of 250mm multiplied by 4.6mm and 5 mu m, wherein the sample injection volume is 5 mu L when the sample is measured, and the column temperature is 35 ℃;
and (3) under the liquid chromatography condition of the step (2), the detector is an evaporative light scattering detector, the conditions are that the temperature of a drift tube is 75-85 ℃, the temperature of an atomizing chamber is 40-50 ℃, the nitrogen flow rate is 2.0-2.6 mL/min, and the Gain value is 1, 2 or 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711464313.2A CN108181395B (en) | 2017-12-28 | 2017-12-28 | Method for detecting triglyceride components in fish oil on line by adopting high performance liquid chromatography |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711464313.2A CN108181395B (en) | 2017-12-28 | 2017-12-28 | Method for detecting triglyceride components in fish oil on line by adopting high performance liquid chromatography |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108181395A CN108181395A (en) | 2018-06-19 |
| CN108181395B true CN108181395B (en) | 2021-01-08 |
Family
ID=62548681
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711464313.2A Active CN108181395B (en) | 2017-12-28 | 2017-12-28 | Method for detecting triglyceride components in fish oil on line by adopting high performance liquid chromatography |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108181395B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110333300B (en) * | 2019-06-12 | 2022-02-22 | 舟山新诺佳生物工程有限责任公司 | Method for detecting types of unsaponifiable matters and oil esters in fish oil and fat |
| CN111257468A (en) * | 2020-03-02 | 2020-06-09 | 品测(上海)检测科技有限公司 | A kind of determination method of quinolone drug residue in animal fat |
| CN114689756B (en) * | 2022-03-31 | 2023-04-07 | 江南大学 | Analysis method of triglyceride positional isomer |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101348807B (en) * | 2008-08-26 | 2011-07-27 | 江南大学 | Method for enrichment of n-3 polyunsaturated fatty acid glyceride from fish oil |
| CA2855246A1 (en) * | 2011-11-09 | 2013-05-16 | Andrew Boam | Membrane-based processes for reducing at least one impurity and making a concentrate comprising at least one natural component from a non-marine fatty acid oil mixture, and compositions resulting thereof |
| CN104655785B (en) * | 2015-02-13 | 2016-03-02 | 嘉必优生物技术（武汉）股份有限公司 | A kind of method analyzing microbial grease composition |
| CN104698104B (en) * | 2015-03-20 | 2016-05-25 | 中国农业科学院油料作物研究所 | A method for analyzing long-chain polyunsaturated fatty acid triglycerides by liquid chromatography-mass spectrometry |
| CN106770822B (en) * | 2016-12-15 | 2019-05-03 | 南京财经大学 | A kind of extraction method of triglyceride |
-
2017
- 2017-12-28 CN CN201711464313.2A patent/CN108181395B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN108181395A (en) | 2018-06-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Guil-Guerrero et al. | Purification process for cod liver oil polyunsaturated fatty acids | |
| CN108181395B (en) | Method for detecting triglyceride components in fish oil on line by adopting high performance liquid chromatography | |
| WO2019043259A1 (en) | Recovery of acidic cannabinoids from plant material | |
| CN112730696A (en) | Method for detecting 5 cannabinol compounds in cannabis sativa oil by using HPLC (high performance liquid chromatography) method | |
| Wang et al. | A novel and efficient method combining SFE and liquid–liquid extraction for separation of coumarins from Angelica dahurica | |
| KR970002037B1 (en) | PROCESS FOR PREPARING Ñß-LINOLEIC ACID FROM PERILLA | |
| Du et al. | Separation and purification of astaxanthin from Phaffia rhodozyma by preparative high-speed counter-current chromatography | |
| Geeraert et al. | On-column injection in the capillary gas chromatographic analysis of fats and oils | |
| JP6991021B2 (en) | Methods for Producing Positional and / or Enantiomers of Triacylglycerol | |
| Li et al. | Separation of high‐purity eicosapentaenoic acid and docosahexaenoic acid from fish oil by pH‐zone‐refining countercurrent chromatography | |
| EP3783358B1 (en) | Method for analyzing triglyceride, method for separating oil and fat, and method for manufacturing triglyceride | |
| JP2019196948A (en) | Method for analyzing triglyceride, sorting methods of fats and oils, and method for producing triglyceride | |
| CN109265494B (en) | Method for extracting kaempferol glucoside compounds from camellia reticulata | |
| Robinson et al. | The composition of New Zealand milk fat triacylglycerols by reversed-phase high-performance liquid chromatography | |
| Sebedio et al. | Utilization of high-performance liquid chromatography as an enrichment step for the determination of cyclic fatty acid monomers in heated fats and biological samples | |
| Nikolova-Damyanova | Lipid analysis by silver ion chromatography | |
| Montañés et al. | Isolation of non-methylene interrupted or acetylenic fatty acids from seed oils using semi-preparative supercritical chromatography | |
| Xu et al. | One-step simultaneous purification of three water-soluble constituents in crude extracts from Danshen by adsorption chromatography on oligo-β-cyclodextrin substituted agarose gel media | |
| Shuting et al. | Preparative isolation of monomer catechins and oligomer procyanidin fractions from grape seed extracts by high-speed counter-current chromatography | |
| CN102921192A (en) | Method for preparing high purity monounsaturated fatty acid | |
| NL2028630B1 (en) | Method for improving purity of batyl alcohol in shark liver oil | |
| JP7263497B2 (en) | Method for separating and analyzing positional isomers and/or enantiomers of triacylglycerols | |
| Montañés et al. | Advances in analytical and preparative supercritical fluid chromatography. Food and nutraceutical applications | |
| EP3991835A1 (en) | Triglyceride analysis method, oil and fat sorting method, and triglyceride production method | |
| Fritsch et al. | Separation of arachidonic acid metabolites by on-line extraction and reversed-phase high-performance liquid chromatography optimized by computer simulation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |