CN1758060A - Determination method of effective component in aliphatic oil - Google Patents
Determination method of effective component in aliphatic oil Download PDFInfo
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Abstract
This invention discloses a test method for the effective components in fat oil, which applies the HPLC method to reduce the derivation operation steps of a sample and save time and reagents, not necessary to apply the step of extraction and applies suitable chromatograph conditions to separate the target components from impurities completely.
Description
Technical field
The present invention relates to a kind of assay method of effective constituent in the fat oil.
Background technology
Fur seal is the deep-sea mammal, lives in the Arctic Circle with in interior-50 ℃ high and cold waters, is food with famous and precious cod, and subcutaneous have a thick fat deposit, is rich in ω-3 polyenoic fatty acid in the body.With Canada's northern pollution-free marine site fur seal fat is the fur seal fat oil that raw material extracts, wherein contain three kinds of important ω-3 polyenoic fatty acids-EPA (Eicosapentaenoic Acid, eicosapentaenoic acid), DPA (Docosapentaenoic Acid, clupanodonic acid) and DHA (Docosahexaenoic Acid, DHA), and total content more than 20%.Medical circle is verified through years of researches, and these three kinds of fatty acid have multiple unique biological active to human body: EPA has the blood circulation of improvement, softening blood vessel, adjustment blood fat, brings high blood pressure down and blood sugar and antiinflammatory action; DHA and DPA have the nutrition brain, promote fetus and children's brain development, vision protection, adjusting immunity and antitumaous effect that wherein DHA is known as " brain gold " again.People find that again they have therapeutic action to some diseases in the recent period: EPA has been developed the medicine as treatment artery sclerosis and high fat of blood; Find that also seal oil has treatment and prevention effect to type ii diabetes, fatty liver and prostatitis.
Human body self can not synthesize ω-3 polyenoic fatty acid, can only absorb from the external world, though also also have ω-3 polyenoic fatty acid in the cold water fish oil of deep-sea, it is compared with seal oil except that content is generally low, also has following significant difference:
1. the fur seal and the mankind are all mammal, the chemical constitution of glyceride is to human similar in its body, ω-3 polyenoic fatty acid generally is positioned at 1,3 of triglyceride, and fish is rudimentary cold-blooded animal, ω-3 polyenoic fatty acid is positioned at 2 of triglyceride, could utilize through after human liver's metabolism.Therefore ω-3 polyenoic fatty acid of seal oil has better bioavilability by contrast, and can not increase burden of liver.
2. the squalene that also contains 2-3% in the seal oil, this also is not have in the fish oil, this material effectively suppresses the absorption of bad cholesterol in the biology and quickens its metabolism, also has protective effect on cancer risk, in addition skin is also had the effect of moistening.
3. not only be rich in EPA and DHA in the seal oil, and be rich in DPA, and DPA content is very low in most of fish oil.
4. contain cholesterol in the seal oil hardly, and most of fish oil contains more cholesterol.
Therefore, seal oil has higher using value with respect to fish oil, can help people to defeat human " the modern civilization disease " of long-term puzzlement such as diabetes, fatty liver, coronary heart disease.
The assay of fatty acid adopts the column front derivation capillary gas chromatography more in the fur seal wet goods fat oil, in recent years the HPLC determination method since its outer marking quantitative accurately, the method reappearance is good, have enough degree of separation, instrument relatively universal, and can provide the advantage of uniqueness such as foundation more and more to come into one's own for the mesolow liquid phase production.
The base catalysis transesterification method of fat oil all has bibliographical information both at home and abroad, but existing operation steps is all more loaded down with trivial details, need repeatedly extract after deriving, and the processing time is long, and expends a large amount of organic solvents, so the method also needs further to improve.For seal oil, adopt the base catalysis transesterification method of fat oil in the deriving method, complex operation step repeatedly extracts after requiring to derive, and remerges the extract anhydrous sodium sulfate drying, and decompression at last dries up extractant constant volume again.By this step operation, sample of every processing needs 4-6 hour approximately, wastes time and energy, and wastes a large amount of organic solvents, and unfavorable health has also been destroyed environment.And adopt traditional analyte derivative method, and the recovery is low, and only about 83.6%, derivatization reagent also needs matching while using, makes troubles to operation.Therefore at present the assay method of effective constituent also needs further to improve in the fur seal wet goods fat oil, could measure effective constituent EPA, DHA in the fat oil and the content of DPA more exactly.
Summary of the invention
The object of the present invention is to provide a kind of assay method of effective constituent in the fat oil, this method adopts the column front derivation rp-hplc method, simplified the analyte derivative operation steps, need not extraction, time and reagent have been saved, can guarantee the derivatization conditions of complete esterification, adopt suitable chromatographic separation condition, purpose component and the complete baseline separation of impurity.
Another object of the present invention is to provide a kind of assay method of effective constituent in the seal oil.
In order to realize purpose of the present invention, the invention provides a kind of assay method of effective constituent in the fat oil, may further comprise the steps:
1) derivatization treatment of fat oil:
Get fat oil and the alkali metal salt that contains 0.2-1.0mol/L alcohol with 1: the 30-100 volume ratio is mixed and is shaken up, 0-60 ℃ down reaction add sour cessation reaction again to little oil droplet complete obiteration, use pure constant volume, filter, get the fat oil need testing solution that derivation process is crossed;
2) the chromatogram sample introduction is analyzed
Get the fat oil after the derivation process, enter liquid chromatograph, described chromatographic chromatographic condition is:
Moving phase: methanol-water system, acetonitrile tetrahydrofuran water system or acetonitrile methanol water system, the constant gradient wash-out,
Flow velocity: 1.1-1.5ml/min,
Column temperature: 15-45 ℃,
Detect wavelength: 202～230nm,
Sample size: 5～50 μ l;
3) record chromatogram is with the content of external standard method fatty acid.
Wherein, described fat oil is the fat oil that fish oil, vegetable oil or fur seal wet goods contain three kinds of polyenoic fatty acid EPA, DPA and DHA, the effective constituent of being measured in the fat oil is meant three kinds of ω-3 polyenoic fatty acids: EPA-Eicosapentaenoic Acid, eicosapentaenoic acid; DPA-Docosapentaenoic Acid, clupanodonic acid and DHA-Docosahexaenoic Acid, DHA, this method can be measured one or more in these three kinds of fatty acid.This method especially is fit to the mensuration of these three kinds of ω-3 polyenoic fatty acids in the seal oil.
Wherein, the derivatization treatment of step 1) fat oil: get fat oil 80-120 μ l, the alkali metal salt 4-8ml that adds 0.2-1.0mol/L alcohol, shake up, 0-60 ℃ of extremely little oil droplet complete obiteration of reaction down, add 0.2-0.5ml acid cessation reaction again, use pure constant volume, 0.2 μ m-0.3 μ m membrane filtration gets the fat oil need testing solution that derivation process is crossed.
Say further, described derivatization treatment method is: get fat oil 100-120 μ l, the sodium methoxide 4-6ml that adds 0.5-0.8mol/L, shake up, room temperature reaction 15-30 minute, to little oil droplet complete obiteration, add 0.2-0.4ml glacial acetic acid cessation reaction again, use methanol constant volume, 0.2 μ m membrane filtration gets the fat oil need testing solution that derivation process is crossed.
The alkali metal salt of the alcohol described in the derivative reaction can be sodium methoxide, caustic alcohol or potassium methoxide etc., is preferably sodium methoxide, sodium methoxide at 15 days with the basic no change of interior catalysis derivatization ability.Its concentration is 0.2-1.0mol/L, is preferably 0.5-0.8mol/L.
The used acid of cessation reaction is any one in glacial acetic acid, formic acid, trifluoroacetic acid or the sulfuric acid etc., and the used alcohol of constant volume is methyl alcohol, ethanol etc.
In order to make product have better stability, can add the antioxidant of 0.001%-0.005% during constant volume, described antioxidant is a 2,6 ditertiary butyl p cresol or to hydroxyl tert-butyl group anisole.
Preferably, derivatization treatment of the present invention was reacted 5-30 minute down at 0-60 ℃, and for the ease of operation, derivative reaction more preferably at room temperature carries out, and reacts 15 minutes.
The used chromatographic column of chromatograph of the present invention can be: Allsphere Octyl (C8), 3 μ m, 100 , 4.6mm * 150mm; Waters SymmetryShield RP-18,5 μ m, 100 , 3.9mm * 150mm and LichrospherRP-18,5 μ m, 100 , 4.6mm * 250mm.With the RP-18 type, 5 μ m, 100 , 4.6mm * 250mm model is for well.As the Waters high performance liquid chromatograph, Lichrospher high performance liquid chromatograph, Mightsil high performance liquid chromatograph.Preferably, liquid chromatograph is the RP-18 type, 5 μ m, 100 , 4.6mm * 250mm model.
When selecting chromatographic chromatographic condition, find to detect wavelength between 202～230nm all can, wherein the three kinds of fatty acid in 210nm place absorb by force and stable, precision also meets the pharmacopeia requirement, is good so the detection wavelength is selected 210nm for use.
The present invention selects in methanol-water system, acetonitrile tetrahydrofuran water system or three kinds of systems of acetonitrile methanol water system any one to carry out the constant gradient wash-out as moving phase.Through debugging repeatedly, the change ratio finds that finally the acetonitrile methanol water system is best, is that 7: 1: 2 flow velocitys are increased to 1.1-1.5ml/min gradually in its ratio, finds that at last every index reaches best when flow velocity is 1.2ml/min.So moving phase is preferably the acetonitrile methanol water system, three's ratio is 7: 1: 2, and flow velocity is 1.2ml/min, and column temperature is chosen in 15-45 ℃ simultaneously.
For realizing that another object of the present invention provides a kind of assay method of effective constituent in the seal oil, it is characterized in that, may further comprise the steps:
1) derivatization treatment of seal oil:
Get seal oil 80-120 μ l, add 0.2-1.0mol/L sodium methoxide 4-8ml, shake up, 0-60 ℃ of extremely little oil droplet complete obiteration of reaction down, add 0.2-0.5ml glacial acetic acid cessation reaction again, with containing 0.001%-0.005%2, the pure constant volume of 6-BHT, 0.2 μ m-0.3 μ m membrane filtration gets the seal oil need testing solution that derivation process is crossed;
2) the chromatogram sample introduction is analyzed
Get the seal oil need testing solution after the derivation process, enter liquid chromatograph, described chromatographic chromatographic condition is:
Moving phase: methanol-water system, acetonitrile tetrahydrofuran water system or acetonitrile methanol water system, the constant gradient wash-out,
Flow velocity: 1.1-1.5ml/min,
Column temperature: 15-45 ℃,
Detect wavelength: 202-230nm,
Sample size: 5～50 μ l;
3) record chromatogram is with the content of external standard method fatty acid.
Described derivatization treatment method is preferably: get fat oil 100-120 μ l, place the 10-200 measuring bottle, add the sodium methoxide 4-6ml of 0.5-0.8mol/L, shake up room temperature reaction 15-30 minute, to little oil droplet complete obiteration, add 0.2-0.4ml glacial acetic acid cessation reaction again, with containing 0.003%-0.005%2, the methanol constant volume of 6-BHT, 0.2 μ m membrane filtration gets the fat oil need testing solution that derivation process is crossed.
Preferred derivatization treatment method is: get seal oil 120 μ l, add 0.5mol/L sodium methoxide 4ml, shake up, room temperature reaction is to little oil droplet complete obiteration, add 0.2ml glacial acetic acid cessation reaction again, with containing 0.005%2, the methanol constant volume of 6-BHT, 0.2 μ m membrane filtration gets the seal oil need testing solution that derivation process is crossed.
Wherein, the effective constituent of being measured in the seal oil is meant three kinds of ω-3 polyenoic fatty acids: EPA-Eicosapentaenoic Acid, eicosapentaenoic acid; DPA-Docosapentaenoic Acid, clupanodonic acid and DHA-Docosahexaenoic Acid, DHA, this method can be measured one or more in these three kinds of fatty acid.
A kind of assay method of effective ingredient is the result that the technology of the present invention personnel grope assay method for many years in the fat oil of the present invention, has the following advantages:
1. the base catalysis transesterification method of fat oil all has bibliographical information both at home and abroad, but operation steps is all more loaded down with trivial details, repeatedly extracts after all requiring to derive, and remerges the extract anhydrous sodium sulfate drying, and decompression at last dries up extractant constant volume again.By this step operation, sample of every processing needs 4-6 hour approximately, wastes time and energy, and wastes a large amount of organic solvents, and unfavorable health has also been destroyed environment.The present invention is behind optimization order, and sample of every processing only needs about 0.5 hour, and need not extraction, has saved plenty of time and organic solvent.
2. adopt traditional analyte derivative method, the recovery only about 83.6% as a result, and this and its operation steps is more, and sample has loss relevant, and the present invention has simplified operation steps, has improved the recovery, and the recovery is near 100%.
3. existing derivatization reagent needs matching while using, makes troubles to operation.The present invention investigates the stability of derivatization reagent, proves in its 15 days to stablize, and has saved running time and reagent.
4. traditional analyte derivative method requires 10 ℃ of reactions 10 minutes, operation inconvenience.The present invention investigated time of deriving and temperature, at room temperature can carry out the derivatization operation, had found handled easily can guarantee the derivatization conditions of complete esterification again.
5. the present invention screens different chromatographic columns in the process that chromatographic condition is studied, and filters out Lichrospher RP-18 chromatographic column, has sought desirable chromatographic condition, the most at last the complete baseline separation of purpose component and impurity.
6. assay method of the present invention is for further laying the foundation to the seal oil purifying.
Description of drawings
Fig. 1 is that seal oil derivatization degree is investigated, and wherein 3 are the sample of not deriving, and 4 are the sample of deriving, and 5 are the sample of not deriving fully;
Fig. 2 is the spectrogram that adds reference substance in sample and the sample;
Fig. 3 is an Allsphere Octyl post optimal separation chromatogram;
Fig. 4 is a Waters-C18 post optimal separation chromatogram;
Fig. 5 is a Lichrospher RP-18 post optimal separation chromatogram;
Fig. 6 is a methanol-water system optimal separation chromatogram;
Fig. 7 is an acetonitrile tetrahydrofuran water system optimal separation chromatogram;
Fig. 8 is an acetonitrile methanol water system optimal separation chromatogram;
Fig. 9 is the chromatogram under the 1.0ml/min flow velocity;
Figure 10 is a room temperature separate colors spectrogram;
Figure 11 is 30 ℃ of separate colors spectrograms;
Figure 12 is blank test and sample contrast color spectrogram;
Figure 13 is Mightsil RP-18 column chromatography figure.
Embodiment
Following embodiment is for further describing the present invention, but described embodiment only is used to illustrate the present invention rather than restriction the present invention.
The assay method of the described active ingredient of seal oil of present embodiment may further comprise the steps:
1) derivatization treatment of seal oil:
Get seal oil 120 μ l, place the 10ml measuring bottle, add 0.5mol/L sodium methoxide 4ml, shake up, room temperature reaction 15 minutes is to little oil droplet complete obiteration, add 0.2ml glacial acetic acid cessation reaction again, with the methanol constant volume that contains 0.005%BHT, 0.2 μ m membrane filtration gets the seal oil need testing solution that derivation process is crossed;
2) get seal oil need testing solution after the 20 μ l derivation process, enter liquid chromatograph, described chromatographic chromatographic condition is:
Chromatographic column: Lichrospher RP-18,5 μ m, 100A, 4.6mm * 250mm;
Moving phase: acetonitrile: methyl alcohol: water=7: 1: 2, constant gradient wash-out;
Flow velocity: 1.2ml/min;
Column temperature: 45 ℃;
Detect wavelength: 210nm;
Sample size: 20 μ l;
3) record chromatogram, the content of mensuration EPA, DHA and three kinds of fatty acid of DPA.
The percentage composition (W/W) that adopts assay method of the present invention to record three kinds of polyenoic fatty acids in the sample seal oil is: EPA6.92%, the recovery 99.10%; DHA9.76%, the recovery 98.90%; DPA4.18%, the recovery 99.60%.
The assay method of the described active ingredient of seal oil of present embodiment may further comprise the steps:
1) derivatization treatment of seal oil:
Get seal oil 80 μ l, place the 50ml measuring bottle, add 0.8mol/L sodium methoxide 6ml, shake up, 40 ℃ of extremely little oil droplet complete obiterations of reaction down add 0.3ml glacial acetic acid cessation reaction again, with the methanol constant volume that contains 0.003%BHT, 0.2 μ m membrane filtration gets the seal oil need testing solution that derivation process is crossed;
2) get seal oil need testing solution after the 30 μ l derivation process, enter liquid chromatograph, described chromatographic chromatographic condition is:
Chromatographic column: LichrospherRP-18,5 μ m, 100A, 4.6mm * 250mm;
Moving phase: methyl alcohol: water=3: 2, constant gradient wash-out;
Flow velocity: 1.3ml/min;
Column temperature: 30 ℃;
Detect wavelength: 202nm;
Sample size: 30 μ l;
3) record chromatogram, the content of mensuration EPA, DHA and three kinds of fatty acid of DPA
The percentage composition (W/W) that adopts the described assay method of present embodiment to record three kinds of polyenoic fatty acids in the sample seal oil is: EPA7.06%, the recovery 99.70%; DHA9.829%, the recovery 98.70%; DPA 4.29%, the recovery 99.50%.
The assay method of the described active ingredient of seal oil of present embodiment may further comprise the steps:
1) derivatization treatment of seal oil:
Get seal oil 100 μ l, place the 100ml measuring bottle, add 1.0mol/L sodium methoxide 8ml, shake up, 50 ℃ of extremely little oil droplet complete obiterations of reaction down add 0.5ml glacial acetic acid cessation reaction again, with the methanol constant volume that contains 0.001%BHT, 0.2 μ m membrane filtration gets the seal oil need testing solution that derivation process is crossed;
2) get seal oil need testing solution after the 50 μ l derivation process, enter liquid chromatograph, described chromatographic chromatographic condition is:
Chromatographic column: Waters SymmetryShield RP-18,5 μ m, 100 , 3.9mm * 150mm;
Moving phase: acetonitrile: methyl alcohol: water=7: 1: 2, constant gradient wash-out;
Flow velocity: 1.3ml/min;
Column temperature: 15 ℃;
Detect wavelength: 220nm;
Sample size: 50 μ l;
3) record chromatogram, the content of mensuration EPA, DHA and three kinds of fatty acid of DPA.
The percentage composition (W/W) that adopts the described assay method of present embodiment to record three kinds of polyenoic fatty acids in the sample seal oil is: EPA7.15%, the recovery 97.90%; DHA9.75%, the recovery 99.60%; DPA4.21%, the recovery 99.30%.
Embodiment 4
The assay method of the described herring oil effective constituent of present embodiment may further comprise the steps:
1) derivatization treatment of herring oil:
Get herring oil 90 μ l, place the 200ml measuring bottle, add 0.6mol/L caustic alcohol 5ml, shake up, 10 ℃ of extremely little oil droplet complete obiterations of reaction down add 0.35ml hydrochloric acid cessation reaction again, with containing 0.002%2, the methanol constant volume of 6 one BHT, 0.2 μ m membrane filtration gets the herring oil need testing solution that derivation process is crossed;
2) get herring oil need testing solution after the 40 μ l derivation process, inject liquid chromatograph, described chromatographic chromatographic condition is:
Chromatographic column: Waters SymmetryShield RP-18,5 μ m, 100 , 3.9mm * 150mm;
Moving phase: acetonitrile tetrahydrofuran water system=7: 1: 2, constant gradient wash-out;
Flow velocity: 1.4ml/min;
Column temperature: 40 ℃;
Detect wavelength: 230nm;
Sample size: 40 μ l;
3) record chromatogram, the content of mensuration EPA and two kinds of fatty acid of DHA.
The percentage composition (W/W) that adopts the described assay method of present embodiment to record three kinds of polyenoic fatty acids in the sample herring oil is: EPA 6.828%, the recovery 99.80%; DHA 6.35%, the recovery 98.50%.
Experimental example 1
This experimental example is to study the derivatization treatment method of fat oil-seal oil.
1. reagent and sample
Sample: seal oil, originate from Canadian TerraNova fishery company limited.
Reagent: 2,6 ditertiary butyl p cresol (BHT), sodium metal, metallic potassium, glacial acetic acid, absolute ether, normal hexane, anhydrous sodium sulfate, potassium hydroxide, ethanol, phenolphthalein, hydrochloric acid, acetone, that triethylamine is homemade analysis is pure; High pure nitrogen, Fisher company product; Alpha-brominated acetophenone, chromatographically pure, Wako company product.
2. the selection of derivatization method
2.1 alpha-brominated acetophenone derivatization method
Get seal oil 20 μ l, it is fixed accurately to claim, adds the 2ml2.0% potassium hydroxide-ethanol and puts in the 5ml centrifuge tube 100 ℃ of saponification of nitrogen-sealed 30 minutes.Being cooled to after the room temperature with phenolphthalein is indicator, acidic alcohol (4: 1) neutralization, the centrifuging and taking supernatant, nitrogen dries up, and adds the acetone soln (10mg/ml) of the alpha-brominated acetophenone of 100 μ l and the acetone soln (10mg/ml) of 100 μ l triethylamines again, and boiling water bath is 5 minutes after the nitrogen-filled seal.The cooling back adds the acetone soln (2mg/ml) of 160 μ l glacial acetic acid, and boiling water bath is 5 minutes once more.Gained reactant liquor 0.45 μ m membrane filtration, nitrogen dries up, and accurately adds the 0.15ml methanol solution, gets 5 μ l sample introductions.
This derivatization method makes measured object at 254nm place very strong absorption be arranged owing to introduced benzene ring structure at the fatty acid chain c-terminus, so the detectability of several purpose component HPLC can reach 0.25ng, and the method complex steps of deriving, during operating cost, and the recovery is difficult to assurance.
2.2 one step of base catalysis is changeed esterification process
Get the about 0.2g of seal oil, the accurate title, decide, and is dissolved in the 4ml tetrahydrofuran, 8ml mixes with new system 0.5mol/L sodium methoxide solution, and 10 ℃ were reacted about 10 minutes, adds 0.4ml glacial acetic acid cessation reaction, reactant liquor divides three extractions with the 50ml ether after using the 20ml distilled water diluting; Combining extraction liquid is used anhydrous Na
2SO
4And KHCO
3After the drying, decompressing and extracting, methanol constant volume be to 100ml, 0.2 μ m membrane filtration, sample introduction 20 μ l.
This method operation is easy relatively, and utilizes a plurality of pairs of extreme ultraviolet ends (210nm) absorptions that key is stronger in the fatty acid, and the about 7-8ng of detectability of several purpose component HPLC can well satisfy quantitative requirement, so adopt the method to do further research.
3. the research of derivatization step
Former method operation steps is more, and the recovery is lower, and only about 83%.Analyze derivatization reaction mechanism and each reactant and product characteristics and, think that the small amounts of salts that reaction produces does not keep substantially on reversed-phase column, do not have influence to separating, and water just had originally in moving phase, also needn't remove in conjunction with the characteristics of reverse phase separation; Test is found to dissolve in methyl alcohol after the seal oil esterification in addition, adds tetrahydrofuran before deriving and also need not.So change method into the directly constant volume sample introduction of back of having derived, step is as follows:
Get seal oil 80-120 μ l, place bottle, add 0.5-1.0mol/L sodium methoxide 4-8ml, shake up, reacted 5-30 minute down at 0-60 ℃, to little oil droplet complete obiteration, add 0.2-0.5ml glacial acetic acid cessation reaction again, with containing 0.001%-0.005%2, the methanol constant volume of 6-BHT, 0.2 μ m-0.3 μ m membrane filtration gets the seal oil need testing solution that derivation process is crossed.
Through repeatedly repeating, and and the contrast of former method, the result proves that the inventive method is feasible fully, separation to the purpose component has no effect, and simplified operation steps greatly, saved the interpolation of tetrahydrofuran solvent, and extraction step, simultaneously but can guarantee higher sample recovery rate, and save a large amount of organic solvents.
4. temperature is to the shadow noon of derivative reaction
Because the derivatization reaction temperature of each bibliographical information has nothing in common with each other, so the analyte derivative temperature is investigated: get 6 parts of each 120 μ l of seal oil and put respectively in the brown volumetric flask of 10ml, the accurate title, decide, divide equally three groups, operation repetitive is derived by having optimized step, and temperature of reaction is respectively first group of ice bath (0 ℃), second group of room temperature (15 ℃), three groups of 60 ℃ of water-baths.Cessation reaction after deriving 10 minutes, each sample introduction twice is investigated the influence of temperature to derivative reaction.
Find in the test that temperature has certain influence to reaction rate: ice bath group sample oil droplet disappears the earliest, the room temperature group secondly, the steamed bun of 60 ℃ of water-bath groups illustrate that low temperature can quicken this derivatization reaction, but oil droplet disappears all after 10 minutes.Constantly derivatization reaction can be obviously quickened in jolting in addition, and this is because jolting can make the fur seal oil droplet break and be little oil droplet, has increased the contact area of oil with derivatization reagent, has accelerated the carrying out of reaction.
Table 1 temperature is to the result that influences of derivative reaction
| Temperature of reaction/℃ | The EPAM average peak area | The DHAM average peak area | The DPAM |
| 0 | 4398834 | 5708265 | 2288815 |
| 0 | 3982547 | 5758572 | 2175738 |
| 15 | 4247395 | 5719200 | 2186221 |
| 15 | 4196638 | 5684480 | 2172971 |
| 60 | 3938505 | 5622290 | 2246374 |
| 60 | 4207884 | 5660505 | 2281390 |
| Mean value | 4161967 | 5689009 | 2212539 |
| Standard deviation | 4398834 | 52523 | 48729 |
| RSD％ | 4.15 | 0.923 | 2.202 |
As shown in Table 1, temperature changes between 0 ℃ to 60 ℃ to be influenced less to the derivative reaction result, through the rough estimates analysis, wherein EPA methyl esters (EPAM) variation is bigger, so with its peak area serves as to investigate index, result under the temperature of respectively deriving is carried out variance analysis, relatively between each group no difference of science of statistics is arranged, the results are shown in Table 2 and table 3.
Table 2 differential responses temperature influences table as a result to the EPAM peak area
| Temperature of reaction/ | 0 | 15 | 60 | |
| 4401125 | 4240804 | 3920078 | ||
| EPAM | 4396542 | 4253985 | 3956932 | |
| Peak area | 3986839 | 4289578 | 4285512 | |
| 3978254 | 4103698 | 4130256 | ∑ | |
| ∑ x | 16762760 | 16888065 | 16292778 | 49943603 |
| ∑ x 2 | 7.04209×10 13 | 7.13216×10 13 | 6.6449×10 13 | 2.08191×10 14 |
Table 3 differential responses temperature influences analysis of variance table to the EPAM peak area
| Source of variation | Sum of sguares of deviation from mean SS | Degree of freedom v | All square MS | F | P |
| Always | 3.27823×10 11 | 11 | |||
| Between group (between processed group) | 49245919643 | 2 | 24622959822 | 0.7955 | ＞0.05 |
| In the group (error) | 2.78577×10 11 | 9 | 30953040596 |
Because F
0.05 (2.9)=4.26, gained F=0.7955＜F is calculated in variance analysis
0.05 (2.9), P＞0.05, so no difference of science of statistics between three groups of data, although the temperature difference of deriving is described, final reaction all reaches a common balance.Temperature can be chosen between 0-60 ℃, and for the ease of operation, derivative reaction preferably at room temperature carries out.
5. derivatization reagent sodium methoxide study on the stability
Because of the bibliographical information sodium methoxide instability that has, the palpus matching while using is brought big inconvenience to test; But and the document that has we were telling you the sodium methoxide room temperature place the several months and keep stable, so the stability of investigation sodium methoxide.
7 parts of preparation 0.5mol/L sodium methoxide solutions, room temperature (about about 15 ℃) was placed 1,2,3,5,10,15,30 day respectively.Get 7 parts of seal oils, the accurate title, decide, and operation repetitive adds 7 kinds of sodium methoxides of placing different times respectively and derives.Each sample introduction twice is to investigate object with DHA, is index with peak area divided by the merchant of sampling amount, investigates derivatization reagent stability.
By table 5 as seen, sodium methoxide 15 days with the basic no change of interior catalysis derivatization ability, peak area/sampling amount only descends about 1%; Slightly descended by the 30th day, it is about 4.5% that peak area/sampling amount only descends, so sodium methoxide prepared afterwards minimum available 15 days.
Table 5 sodium methoxide room temperature is placed the influence to catalytic capability
| Sodium methoxide standing time/sky | Seal oil sampling amount/g | The DHAM average peak area | Peak area/ |
| 1 | 0.11486 | 5416429 | 47156790 |
| 2 | 0.11473 | 5410270 | 47156781 |
| 3 | 0.11478 | 5396079 | 47012365 |
| 5 | 0.11481 | 5394629 | 46987452 |
| 10 | 0.11483 | 5369028 | 46756323 |
| 15 | 0.11479 | 5359061 | 46685790 |
| 30 | 0.11485 | 5170971 | 45023698 |
6. the derivatization completeness is investigated
Get seal oil and derivatization sample spot on silica gel g thin-layer plate (110 ℃ of activation 1h), sherwood oil (30～60 ℃ of boiling ranges)-ether (9: 1) launches, and spray 0.02%Rhodamin6G ethanol liquid is observed (see figure 1) down in uviol lamp (365nm).The R of fatty acid methyl ester
fValue is greater than triglyceride, and the disappearance of triglyceride point illustrates that derivatization is complete in the back sample of deriving.
Among the figure 4 derived the back sample in the complete obiteration of triglyceride spot, analyte derivative is complete.
7. the establishment of analyte derivative method
In summary, the analyte derivative method is: get seal oil 80-120 μ l, place bottle, add 0.5-1.0mol/L sodium methoxide 4-8ml, shake up, reacted 5-30 minute down at 0-60 ℃, add 0.2-0.5ml glacial acetic acid cessation reaction again, with containing 0.001%-0.005%2, the methanol constant volume of 6-BHT, 0.2 μ m-0.3 μ m membrane filtration gets the seal oil need testing solution that derivation process is crossed.
The best sample derivatization method is: get seal oil 120 μ l, the accurate title, decide, put in the brown volumetric flask of 100ml, add 0.5mol/L sodium methoxide 4ml, constantly jolting, about 15 minutes of room temperature reaction, add 0.2ml glacial acetic acid cessation reaction, with the methanol constant volume that contains 0.005%/BHT, 0.2 μ m membrane filtration gets the seal oil need testing solution that derivation process is crossed.
Experimental example 2
This experimental example is to study the chromatographic condition of liquid chromatograph.
1. instrument and reagent
Instrument: Tianjin, island (Shimadzu) 201OA high performance liquid chromatograph, ultraviolet double-wavelength detecting device, Class-VP chromatographic work station; Waters515 pump, 996 type diode array detector, Millennium32 chromatographic work station; MiIlipore ultrapure water draft machine.
Reagent: high pure nitrogen; Methyl alcohol, acetonitrile chromatographically pure, Fisher company product; Trifluoroacetic acid, chromatographically pure, Merck company product; Alpha-brominated acetophenone, chromatographically pure, Wako company product; EPA methyl esters (EPAM) titer (9.99mg/ml), DHA methyl esters (DHAM) titer (9.81mg/ml), DPA methyl esters (DPAM) titer (9.94mg/ml), Supelco company product.
2. the affirmation at purpose peak
Utilize and add reference substance peak area augmentativity under the same terms in the reference substance and sample retention time counter point and sample the purpose component in the sample is confirmed.
As seen from Figure 2, No. 1 peak is identical with EPAM reference substance retention time in the sample under identical chromatographic conditions, and No. 2 the peak is identical with DHAM reference substance retention time, and No. 3 the peak is identical with the DPAM retention time.Add EPAM, DHAM and DPAM reference substance in sample respectively, then No. 1, No. 2, No. 3 the peak increases respectively, this shows that 1,2, No. 3 peak is respectively EPAM, DHAM and DPAM in the sample.Diode display detecting device is used in the back, has also verified this result by the spectrum comparison.
3. detect the selection of wavelength
The laggard liquid chromatograph of analyte derivative with diode display detector recording chromatogram, extracts the spectrogram of three kinds of fatty acid from 3D figure; With absorptivity and precision is index, selects suitable detection wavelength.
By spectrogram as seen, the very big absorbing wavelength of three kinds of fatty acid is 196.9nm, near the ultraviolet end, and the absorption value instability, so precision is relatively poor, withinday precision RSD reaches more than 3%; And most of material all has absorption more by force at 196.6nm, and the baseline noise is bigger.Strong and stable through attempting finding that the three kinds of fatty acid in 210nm place absorb, precision also meets the pharmacopeia requirement, selects 210nm for use so detect wavelength.
4. the selection of test concentrations and sampling amount
For reducing systematic error, it is quantitatively more accurate to make, and the result of combined standard curve determines that sample determination concentration is about about 0.1-0.3mg/ml; Predetermined purpose component concentration is 5～10%, determines that tentatively sampling amount is 120 μ l, is settled to 100ml.
5. the selection of chromatographic column
According to the design feature and the physicochemical property of purpose component, following three kinds of chromatographic columns are investigated: (1) Allsphere Octyl (C8), 3 μ m, 100 , 4.6mm * 150mm; (2) Waters SymmetryShieldRP-18,5 μ m, 100 , 3.9mm * 150mm; (3) Lichrospher RP-18,5 μ m, 100 , 4.6mm * 250mm.
We begin to select for use Allsphere Octyl post, adjust each phase ratio under acetonitrile-methanol-water system repeatedly, and the purpose component preferably can only reach the separation degree as Fig. 3, so be changed to the Waters-C18 post.
Because the Waters-C18 post is a short column, so it is very fast to go out the peak, post is imitated low (see figure 4), still can not be with purpose component baseline separation.
Use Lichrospher RP-18 post at last instead, found optimal separation condition (see figure 5).Three kinds of fatty acid and impurity is baseline separation fully all, and degree of separation is all greater than 1.5, and peak symmetry is good, so the preferred LichrospherRP-18 post of using.
6. the adjustment of moving phase
Following three kinds of moving phase systems are investigated: (1) methyl alcohol: water system; (2) acetonitrile: tetrahydrofuran: water system; (3) acetonitrile: methyl alcohol: water system.(seeing Fig. 6-8)
Through debugging repeatedly, the change ratio finds that finally system (3) is best, is can make the sample baseline separation at 7: 1: 2 o'clock in its ratio, and the degree of separation of three kinds of purpose components and front and back impurity peaks is all greater than 1: 7, and the peak symmetry factor is between 1 to 1.14, so select it.
7. the selection of flow velocity
Under the 1.0ml/min flow velocity, it is later that the purpose component goes out the peak, and last DPAM wanted 42 minutes just can go out the peak (see figure 9).This not only wastes moving phase, more makes the peak shape broadening, and degree of separation descends, and influences quantitative accuracy.Therefore, flow velocity is increased to 1.1-1.5ml/min gradually, finds that at last every index reaches best when flow velocity is 1.2ml/min, so select this flow velocity for use.
8. the selection of column temperature
Investigate sample respectively at room temperature, separating effect 30 ℃, 45 ℃ the time, the peak shape broadening separates variation when room temperature (about 20 ℃) and 30 ℃, sees Figure 10,11.Reason may be since temperature when low three kinds of fatty acid methyl esters change in the solubleness of stationary phase, the absorption resolving time of itself and stationary phase is elongated, so the peak broadening.Find that through the test contrast 45 ℃ are separated best.
9. the establishment of chromatographic condition and system suitability test
In summary, optimum chromatogram condition is:
Chromatographic column: Lichrospher RP-18,5 μ m, 100A, 4.6mm * 250mm;
Moving phase: acetonitrile: methyl alcohol: water=7: 1: 2, constant gradient wash-out;
Flow velocity: 1.2ml/min; Column temperature: 45 ℃; Detect wavelength: 210nm; Sample size: 20 μ l.
Experimental example 3
This experimental example purpose is for liquid phase chromatogram condition methodology checking of the present invention.
1. the range of linearity
Precision is measured EPA methyl esters titer 0.4ml, DHA methyl esters titer 0.4ml, DPA methyl esters titer 0.2ml puts in the brown volumetric flask of 10ml, and the isopropyl alcohol constant volume is as mixing mark solution; This is mixed mark liquid make 7 doubling dilutions continuously with isopropyl alcohol, the series that gets 8 kinds of variable concentrations is mixed mark liquid; Get 20 μ l sample introductions respectively, sample introduction concentration is returned, investigate linear with peak area.
The result shows that three kinds of fatty acid methyl ester standard items peak area Y and its concentration X are good linear, and linear equation sees the following form 6:
Each component range of linearity of table 6, regression equation and related coefficient
| Component | Linear equation | The range of linearity (μ g/ml) | Correlation coefficient r |
| EPAM | Y＝4.576×10 8x-1.33909×10 -3 | 3.12187-399.6 | 0.99994 |
| DHAM | Y＝3.83614×10 8x-1.02352×10 -3 | 3.06562-392.4 | 0.99996 |
| DPAM | Y＝1.34167×10 7x-2.89529×10 -4 | 1.55312-198.8 | 0.99993 |
2. precision
Get the series for preparing mix mark liquid height, in low three concentration, each continuous sample introduction 5 pin calculates withinday precision, each continuous three days sample introduction calculates day to day precision, the results are shown in following table 7:
Table 7 Precision test result
| Component | Withinday precision %RSD (n=5) | Day to day precision %RSD (n=3) | ||||
| High concentration | Middle concentration | Low concentration | High concentration | Middle concentration | Low concentration | |
| EPAM | 0.080 | 0.094 | 0.737 | 0.975 | 0.880 | 0.965 |
| DHAM | 0.161 | 0.238 | 1.335 | 0.935 | 0.895 | 0.929 |
| DPAM | 0.452 | 1.390 | 1.892 | 1.166 | 1.283 | 1.963 |
3. detectability and quantitative limit
To mix the mark solution with the continuous doubling dilution of isopropyl alcohol after sample introduction, the signal of three kinds of fatty acid methyl esters measuring and the noise signal that blank sample is measured compare, be about at 3: 1 o'clock with signal to noise ratio (S/N ratio), respective concentration is determined detectability, be about at 10: 1 o'clock with signal to noise ratio (S/N ratio), respective concentration is determined quantitative limit, the results are shown in Table 9.
Table 9 each component detectability and quantitative limit
| Component | Detectability (μ g/ml) | Quantitative limit (μ g/ml) |
| EPAM | 0.36 | 1.21 |
| DHAM | 0.35 | 1.18 |
| DPAM | 0.40 | 1.34 |
4. specificity
4.1 blank test
Get the blank sample sample introduction, the record chromatogram, investigating other compositions has noiseless to sample analysis.
Be sample chromatogram figure above among Figure 12, be the blank test chromatogram below, contrast as can be seen glacial acetic acid, sodium acetate, BHT appearance time morning, all noiseless to sample analysis.
4.2 peak purity inspection
The laggard liquid chromatograph of analyte derivative with diode display detector recording chromatogram, calculates peak purity, sees Table 10.
The purity angle of three kinds of fatty acid methyl ester chromatographic peaks that chromatographic work station calculates shows that all less than its purity threshold value the three is the pure material peak.
Three kinds of fatty acid methyl ester purity of table 10 angle result of calculation
| Name | RT | Area | Height | Purity1 | Purity1 Threshold | ||
| 1 | EPAM | 20.526 | 2697638 | 124742 | 0.280 | 0.293 | |
| 2 | DHAM | 25.891 | 6125838 | 211120 | 0.234 | 0.288 | |
| 3 | DPAM | 33.557 | 2327500 | 64490 | 0.220 | 0.386 |
5. durability
5.1 sample test liquid study on the stability
Sample test liquid prepares back 4 ℃ of placements, respectively gets 20 μ l sample introductions 2 times respectively at 0,2,4,8,12,24 hour, investigates its stability.
By following table 11 as seen, sample test liquid is with the prolongation of standing time, and the purpose component concentration has downtrending, but changes not quite, and the RSD of three kinds of major component content is all less than 5%, so can think that it is stable that sample test liquid is placed 24 hours at 4 ℃ in 24 hours.But sample test liquid is placed instability for a long time, and room temperature is placed more can quicken its degraded, so should in time measure behind the analyte derivative.
4 ℃ of shelf-stabilities of table 11 derivatization product are investigated the result
| Standing time h | The EPAM average peak area | The DHAM average peak area | The DPAM |
| 0 | 4715461 | 6388976 | 2345467 |
| 4 | 5233537 | 6193141 | 2358255 |
| 8 | 4839895 | 6207655 | 2351127 |
| 12 | 4722964 | 6211872 | 2365919 |
| 24 | 4727123 | 6193824 | 2353844 |
| Mean value | 4847796 | 6239094 | 2354923 |
| Standard deviation | 224651 | 84195 | 7698 |
| RSD％ | 4.572 | 1.349 | 0.327 |
5.2 different instruments and chromatographic column influence to separating
Tianjin, island (Shimadzu) 2010A high performance liquid chromatograph is changed to the Waters high performance liquid chromatograph, the LichrospherRP-18 chromatographic column is changed to Mightsil RP-18 post of the same type (5 μ m, 100A, 4.6mm * 250mm), investigation method durability.
The result of Figure 13 for obtaining with Mightsil RP-18 post, visible sample all can get good separation two chromatographic columns; The peak purity inspection is carried out on the Waters high performance liquid chromatograph, and the result is the display packing good tolerance also.
6. accuracy
Three kinds of fatty acid titers are done 10 times of dilutions with isopropyl alcohol; Accurately take by weighing 9 parts of each 12 μ l of accurately quantitative seal oil, put in the brown volumetric flask of 10ml, quantitatively add EPAM and DHAM standard dilution 1.2,1.0,0.8ml respectively, add DPAM standard dilution 0.6,0.5,0.4ml; 3 parts of every kind of concentration, the back constant volume sample introduction of deriving is measured the content of three kinds of fatty acid, and measuring mean value with mark-on liquid is M, and raw sample liquid hold-up mean value is P, and adding scalar is A, according to the following formula calculate recovery rate: the recovery=(M-P)/A * 100%
Table 12EPA methyl esters recovery of standard addition result of calculation table
| Sequence number | Background content (μ g/ml) | Add scalar (μ g/ml) | Record content (μ g/ml) | The recovery | Average recovery | RSD％ | |
| 1 | 71.75588 | 79.92 | 150.962 | 99.11％ | 98.04％ | 1.215％ | |
| 2 | 73.4426 | 79.92 | 150.324 | 96.20％ | |||
| 3 | 71.6856 | 79.92 | 150.632 | 98.78％ | |||
| 4 | 72.17756 | 99.9 | 168.251 | 96.17％ | |||
| 5 | 74.00484 | 99.9 | 171.325 | 97.42％ | |||
| 6 | 71.12336 | 99.9 | 170.325 | 99.30％ | |||
| 7 | 72.10728 | 119.8/8 | 190.354 | 98.64％ | |||
| 8 | 72.037 | 119.88 | 190.365 | 98.71％ | |||
| 9 | 71.75588 | 119.88 | 189.325 | 98.074％ |
Table 13DHA methyl esters recovery of standard addition result of calculation table
| Sequence number | Background content (μ g/ml) | Add scalar (μ g/ml) | Record content (μ g/ml) | The recovery | Average recovery | RSD％ | |
| 1 | 99.5475 | 78.48 | 178.562 | 100.68％ | 98.04％ | 1.215％ | |
| 2 | 1014.8875 | 78.48 | 181.123 | 100.96％ | |||
| 3 | 99.45 | 78.48 | 180.025 | 102.67％ | |||
| 4 | 100.1325 | 98.1 | 199.687 | 101.48％ | |||
| 5 | 102.6675 | 98.1 | 202.654 | 101.92％ | |||
| 6 | 98.67 | 98.1 | 200.363 | 103.66％ | |||
| 7 | 100.035 | 117.72 | 218.956 | 101.02％ | |||
| 8 | 99.9375 | 117.72 | 217.023 | 99.46％ | |||
| 9 | 99.5475 | 117.72 | 217.632 | 100.31％ |
Table 14DPA methyl esters recovery of standard addition result of calculation table
| Sequence number | Background content (μ g/ml) | Add scalar (μ g/ml) | Record content (μ g/ml) | The recovery | Average recovery | RSD％ | |
| 1 | 43.45376 | 39.76 | 82.963 | 99.37％ | 98.04％ | 1.215％ | |
| 2 | 44.4752 | 39.24 | 81.964 | 95.54％ | |||
| 3 | 43.4112 | 39.24 | 81.325 | 96.62％ | |||
| 4 | 43.70912 | 49.05 | 91.756 | 97.95％ | |||
| 5 | 44.81568 | 49.05 | 93.432 | 99.12％ | |||
| 6 | 43.07072 | 49.05 | 91.324 | 98.38％ | |||
| 7 | 43.66656 | 58.86 | 101.426 | 98.13％ | |||
| 8 | 43.624 | 58.86 | 100.365 | 96.40％ | |||
| 9 | 43.45376 | 58.86 | 100.658 | 97.19％ |
Experimental example 4
This experimental example is the active ingredient of seal oil analysis on Content.
Accurately take by weighing 5 parts of seal oils, by above-mentioned steps operation, the content of three kinds of fatty acid of external standard method, by external standard method at first calculate the content of the corresponding methyl esters of fatty acid in the seal oil, try to achieve content of fatty acid, still need and carry out following conversion:
EPA content=EPAM content * 302.450/316.478=EPAM content * 0.95567
DHA content=DHAM content * 328.487/342.515=DHAM content * 0.95904
DPA content=DPAM content * 330.530/344.531=DPAM content * 0.95936
Three kinds of polyenoic fatty acid percentage compositions (W/W) table as a result in table 15 sample
| Numbering | EPA content | DHA | DPA content | |
| 1 | 6.92 | 9.76 | 4.18 | |
| 2 | 6.98 | 9.64 | 4.35 | |
| 3 | 7.06 | 9.82 | 4.29 | |
| 4 | 7.15 | 9.75 | 4.21 | |
| 5 | 7.03 | 9.78 | 4.25 | |
| Mean value | 7.028 | 9.75 | 4.256 | |
| RSD | 1.23％ | 0.69％ | 1.57％ |
Claims (10)
1. a kind of assay method of effective constituent in the fat oil is characterized in that, may further comprise the steps:
1) derivatization treatment of fat oil:
Get fat oil and the alkali metal salt that contains 0.2-1.0mol/L alcohol with 1: the 30-100 volume ratio is mixed and is shaken up, 0-60 ℃ down reaction add sour cessation reaction again to little oil droplet complete obiteration, use pure constant volume, filter, get the fat oil need testing solution that derivation process is crossed;
2) the chromatogram sample introduction is analyzed
Get the fat oil after the derivation process, enter liquid chromatograph, described chromatographic chromatographic condition is:
Moving phase: methanol-water system, acetonitrile tetrahydrofuran water system or acetonitrile methanol water system, the constant gradient wash-out,
Flow velocity: 1.1-1.5ml/min,
Column temperature: 15-45 ℃,
Detect wavelength: 202～230nm,
Sample size: 5～50 μ l;
3) record chromatogram is with the content of external standard method fatty acid.
2. a kind of assay method of effective constituent in the fat oil according to claim 1, it is characterized in that, in the base, the derivatization treatment of step 1) fat oil: get fat oil 80-120 μ l, add the alkali metal salt 4-8ml of 0.2-1.0mol/L alcohol, shake up, 0-60 ℃ of extremely little oil droplet complete obiteration of reaction down, add 0.2-0.5ml acid cessation reaction again, use pure constant volume, 0.2 μ m-0.3 μ m membrane filtration gets the fat oil need testing solution that derivation process is crossed.
3. a kind of assay method of effective constituent in the fat oil according to claim 1 and 2, it is characterized in that, described derivatization treatment method is: get fat oil 100-120 μ l, add the sodium methoxide 4-6ml of 0.5-0.8mol/L, shake up, room temperature reaction 15-30 minute, to little oil droplet complete obiteration, add 0.2-0.4ml glacial acetic acid cessation reaction again, use methanol constant volume, 0.2 μ m membrane filtration gets the fat oil need testing solution that derivation process is crossed.
4. according to a kind of assay method of effective constituent in any one described fat oil among the claim 1-3, it is characterized in that the alkali metal salt of described alcohol can be sodium methoxide, caustic alcohol or potassium methoxide, is preferably sodium methoxide; The used alcohol of constant volume is methyl alcohol or ethanol; The used acid of described cessation reaction is any one in glacial acetic acid, formic acid, trifluoroacetic acid or the sulfuric acid.
5. according to a kind of assay method of effective constituent in any one described fat oil among the claim 1-3, it is characterized in that, the antioxidant that can add 0.001%-0.005% during constant volume, described antioxidant are 2,6 ditertiary butyl p cresol or to hydroxyl tert-butyl group anisole.
6. according to a kind of assay method of effective constituent in any one described fat oil among the claim 1-3, it is characterized in that, described derivatization treatment method is: get fat oil 100-120 μ l, the sodium methoxide 4-6ml that adds 0.5-0.8mol/L, shake up, room temperature reaction 15-30 minute, to little oil droplet complete obiteration, add 0.2-0.4ml glacial acetic acid cessation reaction again, with containing 0.003%-0.005% 2, the methanol constant volume of 6-BHT, 0.2 μ m membrane filtration gets the fat oil need testing solution that derivation process is crossed.
7. according to a kind of assay method of effective constituent in any one described fat oil among the claim 1-3, it is characterized in that described liquid chromatograph is the RP-18 type, 5 μ m, 100 , 4.6mm * 250mm model.
8. according to a kind of assay method of effective constituent in any one described fat oil among the claim 1-3, it is characterized in that described moving phase is the acetonitrile methanol water system, three's ratio is 7: 1: 2, and flow velocity is 1.2ml/min.
9. a kind of assay method of effective constituent in the seal oil is characterized in that, may further comprise the steps:
1) derivatization treatment of seal oil:
Get seal oil 80-120 μ 1, add 0.2-1.0mol/L sodium methoxide 4-8ml, shake up, 0-60 ℃ of extremely little oil droplet complete obiteration of reaction down, add 0.2-0.5ml glacial acetic acid cessation reaction again, with the pure constant volume that contains the 0.001%-0.005% 2,6 ditertiary butyl p cresol, 0.2 μ m-0.3 μ m membrane filtration gets the seal oil need testing solution that derivation process is crossed;
2) the chromatogram sample introduction is analyzed
Get the seal oil need testing solution after the derivation process, enter liquid chromatograph, described chromatographic chromatographic condition is:
Moving phase: methanol-water system, acetonitrile tetrahydrofuran water system or acetonitrile methanol water system, the constant gradient wash-out,
Flow velocity: 1.1-1.5ml/min,
Column temperature: 15-45 ℃,
Detect wavelength: 202-230nm,
Sample size: 5～50 μ l;
3) record chromatogram is with the content of external standard method fatty acid.
10. a kind of assay method of effective constituent in the seal oil according to claim 9, it is characterized in that, described derivatization treatment method is: get seal oil 120 μ l, add 0.5mol/L sodium methoxide 4ml, shake up, room temperature reaction is to little oil droplet complete obiteration, add 0.2ml glacial acetic acid cessation reaction again, with the methanol constant volume that contains 0.005% 2,6 ditertiary butyl p cresol, 0.2 μ m membrane filtration gets the seal oil need testing solution that derivation process is crossed.
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