CN106645483A - Method for quantitatively detecting sea cucumber polysaccharide - Google Patents
Method for quantitatively detecting sea cucumber polysaccharide Download PDFInfo
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- CN106645483A CN106645483A CN201611215392.9A CN201611215392A CN106645483A CN 106645483 A CN106645483 A CN 106645483A CN 201611215392 A CN201611215392 A CN 201611215392A CN 106645483 A CN106645483 A CN 106645483A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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Abstract
The invention discloses a method for quantitatively detecting sea cucumber polysaccharide. The method comprises the following steps: adding chondroitin sulfate and fucose into a trifluoroacetic acid aqueous solution, and preparing a standard sample solution; extracting polysaccharide in a sea cucumber or sea cucumber product sample to be detected; performing acid hydrolysis, removing acid, adding ammonia water and internal standard substance lactose, and performing derivatization; detecting various-concentration standard sample solutions and standard solution to be detected containing chondroitin sulfate disaccharide derivatives and fucose derivatives by virtue of liquid-phase chromatography-mass spectrometry, and obtaining a content of chondroitin sulfate and fucose in the sample to be detected. The method is used for rapidly quantitatively determining the sea cucumber polysaccharide, is more accurate and convenient and high in efficiency, and can accurately and quantitatively determine various sea cucumber polysaccharide processed in different ways, and provides an effective means for the quality control of different sea cucumber processed products.
Description
Technical field
The present invention relates to a kind of method of detection large biological molecule, more specifically, is related to the method for quantitative determination sea cucumber polysaccharide.
Background technology
Nutriment species and content are enriched in sea cucumber, and nutrient content of the sea cucumber polysaccharide in sea cucumber accounts for critical role.Sea
The structure of gracilis polysaccharide (sea cucumber polysaccharide) mainly has two classes：One class is glycosaminoglycan extracted from sea cucumber, also known as rock
Algae saccharification chondroitin sulfate, another kind of is sea cucumber fucosan.Although the glycosyl composition of two classes is different, on their sugar chain all
By different degrees of Sulfation.Research finds that sea cucumber polysaccharide regulates and controls to the physiological function of human body, maintains the good state tool of life
Have very important significance, be mainly manifested in opposing thrombus, tumour, blood coagulation and reduce serum cholesterol and improve body immunity
Etc. aspect.
Present society is also more and more to the demand of sea cucumber, and Holothurian machining product is varied, causes product quality also each
There is difference.Therefore, quantitative determination sea cucumber polysaccharide is the requisite measure of the quality control of related processing technology product, is also to containing this
The powerful measure of saccharoidal food quality evaluation.
At present, to the quantitative determination of these sea cucumber polysaccharides, it is concentrated mainly on the inspection of the different monosaccharide components produced to degraded
Analysis is surveyed, the most commonly used is chemical staining method.For example after isolating and purifying, by carbazole method determine glucuronic acid content so as to
Indirect determination acid polysaccharide containing alditol, and amino sugared content is determined by Elson-Morgan methods.But these chemical staining methods lack
Weary specificity, it is difficult to obtain accurate result.Application number 200810016622.8 " sea cucumber is more in a kind of sea cucumber and cucumber product
The assay method of sugared content " has also made relevant report, and the method passes through the steps such as enzymolysis alcohol precipitation, on the basis of fucose content,
It is multiplied by the content that transformation ratio has obtained sea cucumber polysaccharide.But, also lack at present simultaneously to fucosylation chondroitin sulfate and
Fucoidin both polysaccharide carry out quantitative detection method.
The content of the invention
It is an object of the invention to realize the quantitative analysis of sea cucumber polysaccharide, glycosaminoglycan extracted from sea cucumber and sea cucumber rock in sea cucumber are made
Algae glycan can simultaneously, fast and accurately complete quantitative determination.
In order to achieve the above object, the invention provides a kind of method of quantitative determination sea cucumber polysaccharide, comprises the steps：
S1, in the trifluoroacetic acid aqueous solution of 1.3mol/L chondroitin sulfate and fucose are added, prepare a series of concentration
The standard sample solution containing chondroitin sulfate and fucose of gradient；
The concentration of the chondroitin sulfate and fucose is in the range of 0.02～0.4mg/ml；
The chondroitin sulfate and fucose optimal concentration scope are 0.04～0.2mg/ml；
S2, the polysaccharide extracted in testing sample：After testing sample crushing, mixing, then precise is adopted after enzymolysis
Alcohol deposition method is extracted, freezed, and obtains testing sample sea cucumber polysaccharide extract, is added in the testing sample sea cucumber polysaccharide extract
Trifluoroacetic acid aqueous solution, the concentration of the trifluoroacetic acid aqueous solution is 1.3mol/L, obtains testing sample sea cucumber polysaccharide solution；
The testing sample is sea cucumber or cucumber product；
Under preferred embodiment, every milliliter of trifluoroacetic acid aqueous solution addition is described to be measured in the testing sample sea cucumber polysaccharide solution
Sample sea cucumber polysaccharide 0.1～100mg of extract；
S3, each concentration standard liquid obtained in step S1 and testing sample solution obtained in step S2, sealing are accurately measured,
The accurate 1.00ml standard liquids of difference and testing sample solution, sealing hydrolyzes 3h at 105 DEG C, places room temperature, using freezing from
Heart concentration removes solvent, collects solid phase；0.3～1mL methyl alcohol or water are added in the solid matter, then it is dense using refrigerated centrifuge
Contracting removes solvent, in triplicate, reaches deacidification purpose, and the solid phase for obtaining each concentration standard sample solution and testing sample solution is residual
Excess；
S4, the ammonia solvent for being separately added into in the solid phase residue of each sample solution obtained in step S3 equivalent, then add
Enter lactose as internal standard compound, add 1-phenyl-3-methyl-5-pyrazolones ketone (PMP) to perform the derivatization, obtain containing chondroitin sulfate
Plain two sugar derivatives, each concentration standard sample solution of fucose derivative and testing sample solution；
S5, liquid chromatograph mass spectrography detecting step S4 are obtained to spread out containing the sugar derivatives of chondroitin sulfate two, fucose
Each concentration standard sample solution and testing sample solution of biology, obtains the sugar derivatives of chondroitin sulfate two and rock algae in each sample
Sugar derivatives each monitors chromatography of ions peak area；
Testing conditions are：
High performance liquid chromatography-triple quadrupole mass spectrometer；
Chromatographic condition is：
Reverse-phase chromatographic column,
30 DEG C of column temperature,
Mobile phase 20mmol ammonium acetate-acetonitrile (88:12, V/V),
Flow velocity 0.5mL/min；
Mass Spectrometry Conditions：
Ion gun ESI sources,
Positive ion mode detection,
Spray voltage 5.5kV,
Auxiliary plus hot air temperature：600 DEG C,
Nitrogen as collision gas and spraying gas,
Select multiple reaction monitoring (MRM), the ion pair of monitoring include 673.3/175.0,687.3/175.0,495.2/
175.0；
S6, the sulphur that internal standard lactose derivatives, standard specimen polysaccharide are distinguished according to chromatographic peak retention time and monitoring feature ion pair
The sugar derivatives of aching and limp ossein two and fucose derivative, then using the ratio of standard specimen chromatographic peak area and interior standard specimen peak area as
Response, according to response and standard sugar concentration drawing curve, compares working curve, draws chondroitin sulfate in testing sample
The content of element and fucose；
The content of S7, the chondroitin sulfate obtained according to step S6 and fucose, obtains treating test sample with reference to formula (1), (2)
The content of fucosan and fucosylation chondroitin sulfate in product；
X=mCS/ A% (1),
Y=mFuc×M(Fuc-H2O)/M(Fuc)- X × (1-A%) (2),
Wherein, X for fucosylation chondroitin sulfate content (mg/g), Y for fucosan content (mg/g), mCSFor
The content (mg/g) of chondroitin sulfate, m in sampleFucFor the content (mg/g) of fucose in sample, A% is chondroitin sulfate master
Chain is in fucosylation chondroitin sulfate proportion.
The structure composition of fucosan is chondroitin sulfate and fucose；And fucosan is be made up of fucose straight
Chain polysaccharide.
A can be obtained by the fucosylation chondroitin sulfate using hydrogen nuclear magnetic resonance analysis of spectrum after purification, it is also possible to
The catabolite for analyzing fucosylation chondroitin sulfate after purification using chromatographic process is obtained.
Under preferred embodiment, the concrete operations of step S4 are：By the solid phase residue of each sample solution obtained in step S3 point
Following process is not carried out：
The solid phase residue of the sample solution is all added in the ammoniacal liquor of 400 μ L and is dissolved, add 1mg/ml's
The μ L of 1-phenyl-3-methyl-5-pyrazolones ketone methanol solution 400 of lactose solution 100 μ L, 0.3mol/L, sealing, under 70 DEG C of environment
Reaction 30min；After reaction terminates, 1mL methyl alcohol or water, centrifugal concentrating is added to remove solvent, in triplicate；Add the body of 1mL
Product concentration is 1% acetic acid aqueous solution, 1mL chloroform extractions, is stood after concussion, removes chloroform, collects water phase, repeats extraction three times
Afterwards, final water is taken as sample test liquid.
The invention has the beneficial effects as follows：
1st, the bglii fragment of chondroitin sulfate feature two for being produced by detection degraded in the present invention and fucose, can realize same
When detect sea cucumber in fucosylation chondroitin sulfate and fucoidin content.
2nd, the inventive method is used for quantitative determination sea cucumber polysaccharide, accurately, more convenient, and efficiency high, can be to difference
The sea cucumber polysaccharide of the different working processes of species, process is accurately quantitative determined, and is the quality of different Holothurian machining products
Control provides effective means.
3rd, the present invention removes the trifluoroacetic acid and ammoniacal liquor in sample using centrifugal concentrating method, is adapted to batch processing sample,
And blow relative to nitrogen, the method such as vacuum drying, sample is not susceptible to loss, ensure that quantitative determination accurately and reliably.
4th, the present invention establish suitable for sea cucumber polysaccharide detection triple quadrupole bar Mass Spectrometry detection method, it is determined that monitor from
Son is right, it is ensured that the stability of detection and sensitivity.
Description of the drawings
Fig. 1 is the typical hybrid standard sugar juice MRM chromatograms of embodiment 1
Fig. 2 is the typical sea cucumber polysaccharide sample solution MRM chromatograms of embodiment 1
Specific embodiment
Following non-limiting examples can make one of ordinary skill in the art be more fully understood the present invention, but not with
Any mode limits the present invention.
The present invention provides a kind of method that inner mark method ration detects sea cucumber polysaccharide, comprises the following steps that：
S1, the chondroitin sulfate and fucose standard solution of preparing a series of concentration gradients, wherein chondroitin sulfate and
In the range of 0.02～0.4mg/ml, wherein optimal concentration scope is 0.04～0.2mg/ml to fucose concentration；Solution is
The aqueous solution of the trifluoroacetic acid of 1.3mol/L.
S2, by sample blending, after crushing, using alcohol deposition method after enzymolysis the polysaccharide in sample is extracted, and add trifluoroacetic acid
Solution, makes trifluoroacetic acid concentration in solution to be measured be 1.3mol/L.
S3,1.00mL standard liquids and testing sample solution are accurately measured, are sealed, 3h is hydrolyzed at 105 DEG C, place room temperature,
Solvent is removed using refrigerated centrifuge concentration, 0.3～1mL methyl alcohol or water is subsequently adding, then solvent is removed using refrigerated centrifuge concentration,
Operation above in triplicate, reaches deacidification purpose；
The ammonia solvent residue of S4,400 μ L of addition, and the lactose solution of the 1mg/ml of 100 μ L is separately added into, add
1-phenyl-3-methyl-5-pyrazolones ketone (PMP) methanol solution of 400 μ L0.3mol/L, sealing, 70 DEG C of water-bath 30min are added
1mL methyl alcohol or water, centrifugal concentrating removes solvent, in triplicate；1.00mL water is subsequently adding, the chloroform of 1mL is added, after concussion
Stand, remove chloroform, repeat extraction three times, water layer is used as test liquid；
The condition that S5, HPLC-MS are analyzed is as follows：High performance liquid chromatography-triple quadrupole mass spectrometer,
Anti-phase C18 chromatographic columns；Mobile phase is 20mmol ammonium acetate solutions (A)：Acetonitrile (B) isocratic elution.Mass Spectrometry Conditions：Ion gun
ESI sources；Positive ion mode；The ion pair of monitoring includes 673.3/175.0,687.3/175.0 and 495.2/175.0.
S6, the chondroitin sulfate that internal standard lactose, standard specimen polysaccharide are distinguished according to chromatographic peak retention time and monitoring feature ion pair
Plain two bglii fragments and fucose derivative, then the ratio using standard specimen chromatographic peak area and interior standard specimen peak area is used as response,
According to response and standard sugar concentration drawing curve, the content of chondroitin sulfate and fucose in testing sample is calculated.
S7, according to formula (1), (2) to calculate sea cucumber in fucosan and fucosylation chondroitin sulfate content.X
=mCS/A% (1)；Y=mFuc × 0.89-X × (1-A%) (2)
The content (mg/g) of X-fucosylation chondroitin sulfate；The content (mg/g) of Y-fucosan；
The content (mg/g) of chondroitin sulfate in mCS-sample；The content (mg/g) of fucose in mFuc-sample
A-chondroitin sulfate main chain is in fucosylation chondroitin sulfate proportion.
Wherein, M (Fuc-H2O)/M (Fuc)=(164-18)/164=0.89；M represents molal weight, and Fuc represents rock algae
Sugar, H2O represents hydrone.
Step S6 of the present invention it is confirmed that in sea cucumber sample chondroitin sulfate and fucose content, according to fucosylation
The ratio of chondroitin sulfate and fucose in chondroitin sulfate, and the formula in S7 can calculate glycosaminoglycan extracted from sea cucumber and
Fucosan both polysaccharide content respectively.
Embodiment 1
(1) by new fresh sea cucumber freeze-drying, smash to obtain freeze-dried powder.1g samples are taken, the 0.1mol/L of 30 times of volumes is added to
In sodium acetate buffer solution (pH=6.0), and 10% papain is added, 5mmol/L EDTA solution and the Guangs of 5mmol/L half
Propylhomoserin solution, after stirring reaction 24h under 60 DEG C of water-baths, reactant mixture centrifugation (4000r/min, 10min, 20 DEG C).To
3 times of absolute ethyl alcohols, centrifugation (4000r/min, 10min, 20 DEG C) are added to take precipitation in supernatant, i.e. enzymolysis liquid, precipitation is freezed
Thick many candies.
(2) each 10mg of chondroitin sulfate, fucose is accurately weighed in 10ml volumetric flasks, add 5ml water dissolves, use
The trifluoroacetic acid constant volume of 2.6mol/L, mixes, and makes standard mother liquor, is then diluted with the trifluoroacetic acid of 1.3mol/L, is configured to
Series concentration (0.02,0.04,0.06,0.08,0.1,0.2,0.3,0.4mg/ml) hybrid standard sugar juice, each concentration two
It is parallel.
(3) testing sample polysaccharide solution is prepared：Fresh sea cucumber polysaccharide 20mg is accurately weighed in 10ml volumetric flasks, 5ml is added
Water dissolves, with the trifluoroacetic acid constant volume of 2.6mol/L, mix, and obtain testing sample polysaccharide solution；Accurate measuring 0.50ml is to be measured
Sample polysaccharide solution complements to 1ml, pyrohydrolysis to be added in 5ml hydrolysis pipes with the trifluoroacetic acid of 1.3mol/L；Accurate measuring
0.50ml testing samples polysaccharide solution is hydrolyzed in 5ml and adds 1.3mol/Ls of the 0.50ml containing 0.20mg/ml standard sugars in pipe
Trifluoroacetic acid mixed solution, makes mark-on sample, sealing, pyrohydrolysis to be added.
(3) standard solution and sample solution, sealing, at 105 DEG C, hydrolyzes 3h, and using refrigerated centrifuge concentration solvent is removed,
0.5mL methyl alcohol is added, using refrigerated centrifuge concentration solvent is removed, the above is operated in triplicate, reaches deacidification purpose；
(4) the ammonia solvent residue of 400 μ L is added, and is separately added into the lactose solution of the 1mg/ml of 100 μ L, added
1-phenyl-3-methyl-5-pyrazolones ketone (PMP) methanol solution of 400 μ L 0.3mol/L, sealing, 70 DEG C of water-bath 30min are added
1mL methyl alcohol, vacuum refrigeration centrifugation removal of solvent under reduced pressure, in triplicate；1mL water is subsequently adding, the chloroform of 1mL is added, is shaken
After remove chloroform, repeat extraction three times, water layer is used as test liquid；
(5) chromatographic condition：High performance liquid chromatograph (Japanese SHIMADZU companies)；Thermo scientific
Hypersil GOLD (150 × 2.1mm, 5 μm) chromatographic column；30 DEG C of column temperature；Flow velocity 0.5mL/min；Mobile phase is 20mmol acetic acid
Aqueous ammonium (A)：Acetonitrile (B)=88:12 isocratic elutions.Mass Spectrometry Conditions：4000QTRAP triple quadrupoles bar series connection linear ion hydrazine
Mass spectrograph (American AB SCIEX company)；Ion gun ESI sources；Spray voltage 5.5kV；Positive ion mode；Auxiliary plus hot air temperature
600℃；Select multiple reaction monitoring (MRM).Other parameters are as shown in table 1.
The disaccharides of table 1PMP derivatizations and the mass spectral analysis condition of monose
CE:Collision energy, DP:Remove cluster voltage
(6) Fig. 1 is the MRM chromatograms of typical hybrid standard sugar juice, and Fig. 2 is typical sea cucumber polysaccharide sample solution MRM
Chromatogram.Tri- chromatography of ions figure of 673.3/175.0,687.3/175.0 and 495.2/175.0 are selected to come to internal standard breast respectively
The derivative of sugar, the bglii fragment of chondroitin sulfate two and fucose is carried out quantitatively, and working curve and sample-adding reclaim the result such as institute of table 2
Show.The content that chondroitin sulfate and fucose in new fresh sea cucumber sample are calculated according to working curve be respectively 0.32mg/g,
0.30mg/g, RSD are respectively 3.39% and 6.46%.
(7) according to formula (1), (2) to calculate sea cucumber in fucosan and fucosylation chondroitin sulfate content.X
=mCS/A% (1)；Content (the mg/ of Y=mFuc × 0.89-X × (1-A%) (2) X-fucosylation chondroitin sulfate
g)；The content (mg/g) of Y-fucosan；The content (mg/g) of chondroitin sulfate in mCS-sample；Rock algae in mFuc-sample
The content (mg/g) of sugar
A-chondroitin sulfate main chain is in fucosylation chondroitin sulfate proportion.
Wherein, M (Fuc-H2O)/M (Fuc)=(164-18)/164=0.89；M represents molal weight, and Fuc represents rock algae
Sugar, H2O represents hydrone.
According to document report, in the sea cucumber of the species, chondroitin sulfate is shared in fucosylation chondroitin sulfate
Ratio is 68.9%, then be calculated fucosan and fucosylation chondroitin sulfate in new fresh sea cucumber sample by formula (1) (2)
The content of element is respectively 0.464mg/g, 0.123mg/g.
The methodological study result of table 2
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any those familiar with the art in the technical scope of present disclosure, technology according to the present invention scheme and its
Inventive concept equivalent or change in addition, all should be included within the scope of the present invention.
Claims (4)
1. a kind of method of quantitative determination sea cucumber polysaccharide, it is characterised in that comprise the steps：
S1, in the trifluoroacetic acid aqueous solution of 1.3mol/L chondroitin sulfate and fucose are added, prepare a series of concentration gradients
The standard sample solution containing chondroitin sulfate and fucose；
The concentration of the chondroitin sulfate and fucose is in the range of 0.02～0.4mg/ml；
S2, the polysaccharide extracted in testing sample：After testing sample crushing, mixing, precise, then using alcohol precipitation after enzymolysis
Method is extracted, freezed, and obtains testing sample sea cucumber polysaccharide extract, and in the testing sample sea cucumber polysaccharide extract trifluoro is added
Acetic acid aqueous solution, the concentration of the trifluoroacetic acid aqueous solution is 1.3mol/L, obtains testing sample sea cucumber polysaccharide solution；
The testing sample is sea cucumber or cucumber product；
S3, each concentration standard liquid obtained in step S1 and testing sample solution obtained in step S2, sealing are accurately measured, respectively
Accurately 1.00ml standard liquids and testing sample solution, sealing, hydrolyze 3h at 105 DEG C, place room temperature, dense using refrigerated centrifuge
Contracting removes solvent, collects solid phase；0.3～1mL methyl alcohol or water are added in the solid matter, then is removed using refrigerated centrifuge concentration
Solvent is removed, in triplicate, deacidification purpose is reached, the solid phase for obtaining each concentration standard sample solution and testing sample solution is remaining
Thing；
S4, the ammonia solvent for being separately added into in the solid phase residue of each sample solution obtained in step S3 equivalent, add breast
Sugar adds 1-phenyl-3-methyl-5-pyrazolones ketone to perform the derivatization as internal standard compound, obtains spreading out containing chondroitin sulfate disaccharides
Biological, each concentration standard sample solution of fucose derivative and testing sample solution；
S5, liquid chromatograph mass spectrography detecting step S4 are obtained to contain the sugar derivatives of chondroitin sulfate two, fucose derivative
Each concentration standard sample solution and testing sample solution, obtain the sugar derivatives of chondroitin sulfate two and fucose in each sample and spread out
Biological each monitoring chromatography of ions peak area；
Testing conditions are：
High performance liquid chromatography-triple quadrupole mass spectrometer；
Chromatographic condition is：
Reverse-phase chromatographic column,
30 DEG C of column temperature,
Mobile phase 20mmol ammonium acetate-acetonitrile (88:12, V/V),
Flow velocity 0.5mL/min；
Mass Spectrometry Conditions：
Ion gun ESI sources,
Positive ion mode detection,
Spray voltage 5.5kV,
Auxiliary plus hot air temperature：600 DEG C,
Nitrogen as collision gas and spraying gas,
Select multiple reaction monitoring (MRM), the ion pair of monitoring include 673.3/175.0,687.3/175.0,495.2/
175.0；
S6, distinguished according to chromatographic peak retention time and monitoring feature ion pair internal standard lactose derivatives, standard specimen polysaccharide sulfuric acid it is soft
The sugar derivatives of ossein two and fucose derivative, then the ratio using standard specimen chromatographic peak area and interior standard specimen peak area is used as response
Value, according to response and standard sugar concentration drawing curve, compares working curve, draw in testing sample chondroitin sulfate and
The content of fucose；
The content of S7, the chondroitin sulfate obtained according to step S6 and fucose, in obtaining testing sample with reference to formula (1), (2)
The content of fucosan and fucosylation chondroitin sulfate；
X=mCS/ A% (1),
Y=mFuc×M(Fuc-H2O)/M(Fuc)- X × (1-A%) (2),
Wherein, X for fucosylation chondroitin sulfate content (mg/g), Y for fucosan content (mg/g), mCSFor sample
The content (mg/g) of middle chondroitin sulfate, mFucFor the content (mg/g) of fucose in sample, A% exists for chondroitin sulfate main chain
Fucosylation chondroitin sulfate proportion.
2. the method for quantitative determination sea cucumber polysaccharide according to claim 1, it is characterised in that chondroitin sulfate described in step S1
And fucose concentration range is 0.04～0.2mg/ml.
3. the method for quantitative determination sea cucumber polysaccharide according to claim 1, it is characterised in that testing sample sea described in step S2
Every milliliter of trifluoroacetic acid aqueous solution adds the testing sample sea cucumber polysaccharide 0.1～100mg of extract in gracilis polysaccharide solution.
4. the method for quantitative determination sea cucumber polysaccharide according to claim 1, it is characterised in that the concrete operations of step S4 are：
The solid phase residue of each sample solution obtained in step S3 is carried out respectively following process：
The solid phase residue of the sample solution is all added in the ammoniacal liquor of 400 μ L and is dissolved, add the lactose of 1mg/ml
The μ L of 1-phenyl-3-methyl-5-pyrazolones ketone methanol solution 400 of solution 100 μ L, 0.3mol/L, sealing is reacted under 70 DEG C of environment
30min；After reaction terminates, 1mL methyl alcohol or water, centrifugal concentrating is added to remove solvent, in triplicate；The volume for adding 1mL is dense
Spend for 1% acetic acid aqueous solution, 1mL chloroform extractions, stand after concussion, remove chloroform, collect water phase, after repeating extraction three times, take
Final water is used as sample test liquid.
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| GB2572322A (en) * | 2018-03-16 | 2019-10-02 | Institute Of Tech Tralee | A hair treatment composition |
| CN110715997A (en) * | 2018-07-13 | 2020-01-21 | 李绍平 | Polysaccharide determination and analysis method and application thereof |
| CN110724209A (en) * | 2018-07-16 | 2020-01-24 | 北京大学 | Fucosylated chondroitin sulfate oligosaccharide and preparation method, composition and application thereof |
| CN115290766A (en) * | 2022-06-30 | 2022-11-04 | 岛津企业管理（中国）有限公司 | Method for accurately and rapidly determining content of 2-deoxy-2-fluoro-L-fucose in antibody drug |
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| GB2572322A (en) * | 2018-03-16 | 2019-10-02 | Institute Of Tech Tralee | A hair treatment composition |
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| CN110724209B (en) * | 2018-07-16 | 2021-11-19 | 烟台东诚药业集团股份有限公司 | Fucosylated chondroitin sulfate oligosaccharide and preparation method, composition and application thereof |
| CN109613132A (en) * | 2018-12-06 | 2019-04-12 | 上海景峰制药有限公司 | A kind of detection method of heterozygosis chondroitin sulfate |
| CN115290766A (en) * | 2022-06-30 | 2022-11-04 | 岛津企业管理（中国）有限公司 | Method for accurately and rapidly determining content of 2-deoxy-2-fluoro-L-fucose in antibody drug |
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