CN105866262B - A kind of method of quick identification chondroitin sulfate A (CSA) and C - Google Patents
A kind of method of quick identification chondroitin sulfate A (CSA) and C Download PDFInfo
- Publication number
- CN105866262B CN105866262B CN201610171668.1A CN201610171668A CN105866262B CN 105866262 B CN105866262 B CN 105866262B CN 201610171668 A CN201610171668 A CN 201610171668A CN 105866262 B CN105866262 B CN 105866262B
- Authority
- CN
- China
- Prior art keywords
- chondroitin sulfate
- sample
- csa
- water
- extraction
- 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
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 title claims abstract description 52
- 229920001287 Chondroitin sulfate Polymers 0.000 title claims abstract description 52
- 229940059329 chondroitin sulfate Drugs 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 27
- 239000007788 liquid Substances 0.000 claims abstract description 29
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 27
- 239000013558 reference substance Substances 0.000 claims abstract description 22
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000000605 extraction Methods 0.000 claims abstract description 12
- 238000001212 derivatisation Methods 0.000 claims abstract description 11
- 238000005903 acid hydrolysis reaction Methods 0.000 claims abstract description 7
- 238000004458 analytical method Methods 0.000 claims abstract description 6
- 238000005516 engineering process Methods 0.000 claims abstract description 6
- -1 1-phenyl-3-methyl-5-pyrazolones ketone Chemical class 0.000 claims abstract description 5
- 239000000413 hydrolysate Substances 0.000 claims abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 48
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 22
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 21
- 150000002500 ions Chemical class 0.000 claims description 17
- 239000000243 solution Substances 0.000 claims description 17
- 239000002904 solvent Substances 0.000 claims description 14
- 230000007062 hydrolysis Effects 0.000 claims description 12
- 238000006460 hydrolysis reaction Methods 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- 235000009508 confectionery Nutrition 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 10
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 9
- 239000005695 Ammonium acetate Substances 0.000 claims description 9
- 229940043376 ammonium acetate Drugs 0.000 claims description 9
- 235000019257 ammonium acetate Nutrition 0.000 claims description 9
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 8
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 8
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 5
- 230000009514 concussion Effects 0.000 claims description 5
- 229910021529 ammonia Inorganic materials 0.000 claims description 4
- 239000012634 fragment Substances 0.000 claims description 4
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 3
- 239000000908 ammonium hydroxide Substances 0.000 claims description 3
- 150000004676 glycans Chemical class 0.000 claims description 3
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims description 3
- 230000014759 maintenance of location Effects 0.000 claims description 3
- 229920001282 polysaccharide Polymers 0.000 claims description 3
- 239000005017 polysaccharide Substances 0.000 claims description 3
- 239000007921 spray Substances 0.000 claims description 3
- 238000003809 water extraction Methods 0.000 claims description 3
- 229920002567 Chondroitin Polymers 0.000 claims description 2
- 229940051880 analgesics and antipyretics pyrazolones Drugs 0.000 claims description 2
- DLGJWSVWTWEWBJ-HGGSSLSASA-N chondroitin Chemical compound CC(O)=N[C@@H]1[C@H](O)O[C@H](CO)[C@H](O)[C@@H]1OC1[C@H](O)[C@H](O)C=C(C(O)=O)O1 DLGJWSVWTWEWBJ-HGGSSLSASA-N 0.000 claims description 2
- 150000002576 ketones Chemical class 0.000 claims description 2
- 239000000047 product Substances 0.000 abstract description 5
- 235000019441 ethanol Nutrition 0.000 description 8
- KXKPYJOVDUMHGS-OSRGNVMNSA-N chondroitin sulfate Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](OS(O)(=O)=O)[C@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](C(O)=O)O1 KXKPYJOVDUMHGS-OSRGNVMNSA-N 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 238000004587 chromatography analysis Methods 0.000 description 6
- 239000002253 acid Substances 0.000 description 5
- 241000881711 Acipenser sturio Species 0.000 description 4
- 210000000845 cartilage Anatomy 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N monobenzene Natural products C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 4
- 230000001376 precipitating effect Effects 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- 229960001866 silicon dioxide Drugs 0.000 description 3
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 2
- 108090000526 Papain Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 210000000988 bone and bone Anatomy 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000009849 deactivation Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000000151 deposition Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 230000007071 enzymatic hydrolysis Effects 0.000 description 2
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 235000013402 health food Nutrition 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 239000012982 microporous membrane Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229940055729 papain Drugs 0.000 description 2
- 235000019834 papain Nutrition 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102000011413 Chondroitinases and Chondroitin Lyases Human genes 0.000 description 1
- 108010023736 Chondroitinases and Chondroitin Lyases Proteins 0.000 description 1
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- AEMOLEFTQBMNLQ-HNFCZKTMSA-N L-idopyranuronic acid Chemical compound OC1O[C@@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-HNFCZKTMSA-N 0.000 description 1
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000006298 dechlorination reaction Methods 0.000 description 1
- 238000012850 discrimination method Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 229940097043 glucuronic acid Drugs 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a kind of methods for quickly identifying chondroitin sulfate A (CSA) and C, comprising steps of 1, extraction chondroitin sulfate；2, sample to be tested utilizes trifluoroacetic acid hydrolysis, the deacidification of gained hydrolysate；3, step 2 gained residue prepares the derivative of sugar with 1-phenyl-3-methyl-5-pyrazolones ketone (PMP) derivatization；4, sour water solution and derivatization are carried out in the method for step S2 and S3 to various chondroitin sulfate reference substances respectively；5, with the derivatization product of liquid chromatography-mass spectrography technology analysis S3 and S4；6, by analysing and comparing, identify the type of chondroitin sulfate in sample.The strong operability of method of the invention, it is low to sample purity requirement, it is applied widely.
Description
Technical field
The present invention relates to a kind of methods for identifying large biological molecule, more specifically, are related to identifying the side of chondroitin sulfate type
Method.
Background technique
Chondroitin sulfate (chondroitin sulfate) is a kind of glycosaminoglycan, it has very high application value.
It can not only reduce blood lipid, there is preferable effect in the prevention and treatment of the cardiovascular diseases such as coronary heart disease, myocardial infarction, moreover it is possible to adjust and close
Function is saved, promotes the formation and healing of bone, and have certain inhibiting effect to tumour cell and virus.At present in drug, guarantor
Health food and cosmetic industry are widely applied.Especially as Bones and joints health food, at home with sold on international market
Scale is all very huge.
Chondroitin sulfate is by uronic acid (glucuronic acid or iduronic acid) and N- acetylgalactosamine disaccharide unit
The sugar chain of composition replaces through sulfate.Replace situation different with sulfate since two bglii fragments are constituted, chondroitin sulfate can
To be divided into the classifications such as A, B, C, D, E, F, H, K, L and M.The source of different types of chondroitin sulfate and functional activity are not
Together, also there is very big difference in price.Therefore, it is highly desirable to identify the type of chondroitin sulfate.
At present to the discrimination method of chondroitin sulfate type, mainly there are spectroscopic methodology and chromatography.Spectroscopic methodology includes infrared light
Spectrometry and nuclear magnetic resonance spectroscopy, but both spectral methods are very high to the purity requirement of sample, for the sulfuric acid in mixture
Chondroitin has to pass through the purification process of cumbersome time-consuming.Chromatography is usually required using chondrosulphatase.Due to various sulfuric acid
Chondroitinase has specificity to the degradation of variety classes chondroitin sulfate, degrades using these enzymes to sample, then combine
The analytical technologies such as electrophoresis, ion chromatography or liquid chromatogram can accurately determine the type of chondroitin sulfate.But chondrosulphatase
It is expensive, limit the popularization and application of this method.
Summary of the invention
It is an object of the invention to quickly identify chondroitin sulfate A (CSA) and C.
In order to achieve the above object, the present invention provides a kind of method for quickly identifying chondroitin sulfate A (CSA) and C, including as follows
Step:
The chondroitin sulfate of S1, Extraction and discrimination sample prepare Thick many candies dry powder sample；
S2, sample to be tested utilize trifluoroacetic acid hydrolysis, and gained hydrolysate is removed under reduced pressure molten in triplicate using water or methanol
Agent, for deacidification to neutrality；
S3, by residue ammonia solvent obtained by S2,1-phenyl-3-methyl-5-pyrazolones ketone (PMP) derivatization is added,
Test liquid is obtained by extraction；
S4, chondroitin sulfate reference substance is taken to repeat step S2 and S3 with same operation parameter, sulfuric acid is soft in reference substance sample
Content of chondroitin sulfate is of substantially equal in the Thick many candies dry powder sample that the content of ossein is prepared with S1；
S5, the derivative that S3 and S4 is analyzed using liquid chromatography-mass spectrography technology:
LC-MS analysis condition includes: using acetonitrile/20mM ammonium acetate solution as mobile phase, instead using outfit
The liquid chromatography-mass spectrography of phase silicagel column, using positive ion mode, the quasi-molecule of derivative of the selection with two bglii fragment of sulfate
Ion；
S6 selects chromatographic peak retention time and mass spectrometric data to compare, to infer the type of chondroitin sulfate in sample.
In the above method, the S1 is specifically included: being extracted using alcohol deposition method after direct water extraction or enzymatic hydrolysis more in sample
Sugar makes free content of chondroitin sulfate be greater than 5%；
In the above method, the S2 is specifically included: 10~200mg of Thick many candies is taken, with the trifluoro second of 0.1~2mol/L of 1mL
Acid dissolution, 100~120 DEG C of 1~1.5h of heating；0.5~1mL water or methanol is added in acid hydrolysis liquid, and solvent is removed under reduced pressure, and repeats three
It is secondary, it deacidifies to neutrality, reaches deacidification purpose；
In the above method, the S3 is specifically included: the ammonia solvent residue of 200~400 μ L is added, then into tool plug test tube
1-phenyl-3-methyl-5-pyrazolones ketone (PMP) methanol solution of 200~400 μ L 0.3mol/L of addition, 70 DEG C of water-bath 30min,
0.5~1mL methanol or water is added, solvent is removed under reduced pressure；Then 1mL water is added, adds the chloroform of 1mL, dechlorination is removed after concussion
It is imitative, repeat extraction three times, water layer is as test liquid；
In the above method, the S4 is specifically included: 0.5~10mg of various chondroitin sulfate reference substances, respectively according to S2 and
S3 carries out sour water solution and derivatization；
In the above method, the S5 is specifically included: anti-using being equipped with using acetonitrile/20mM ammonium acetate solution as mobile phase
The liquid chromatography-mass spectrography of phase silicagel column；Using positive ion mode, the quasi-molecule of derivative of the selection with two bglii fragment of sulfate
Ion m/z 766.2 ± 0.5.
Wherein, optimal situation are as follows:
In step S2 trifluoroacetic acid concentration be 0.2mol/L, 100 DEG C of hydrolysis temperature, hydrolysis time 1h.
The ion of liquid chromatography-mass spectrography choice of technology m/z 766.2 in step S5.
The method of the present invention is for identifying chondroitin sulfate A (CSA) and C.The invention uses acid degradation polysaccharide, low in cost, operability
By force；It is low to sample purity requirement, it is applied widely.
Detailed description of the invention
Fig. 1 is the selection chromatography of ions figure of 1 chondroitin sulfate C reference substance of embodiment；
Fig. 2 is the selection chromatography of ions figure of 1 chondroitin sulfate A (CSA) of embodiment and the mixture reference substance of C；
Fig. 3 is the selection chromatography of ions figure of 1 sample of embodiment；
Fig. 4 is the selection chromatography of ions figure of 2 sample of embodiment.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, actual conditions are not specified in embodiment
Person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer, being can be with
Conventional products that are commercially available.
The present invention provides a kind of method for quickly identifying chondroitin sulfate A (CSA) and C, the specific steps are as follows:
Step1, the polysaccharide in sample is extracted using alcohol deposition method after direct water extraction or enzymatic hydrolysis, makes free chondroitin sulfate
Content is greater than 5%；
Step2,10~200mg of Thick many candies is taken, is dissolved with the trifluoroacetic acid of 0.1~2mol/L of 1mL, 100~120 DEG C add
1~1.5h of heat；0.5~1mL water or methanol is added in acid hydrolysis liquid, and solvent is removed under reduced pressure, in triplicate, reaches deacidification purpose；
Step3, the ammonia solvent residue that 200~400 μ L are added, then 200~400 μ L are added into tool plug test tube
0.5~1mL water is added in 1-phenyl-3-methyl-5-pyrazolones ketone (PMP) methanol solution of 0.3mol/L, 70 DEG C of water-bath 30min
Or methanol, solvent is removed under reduced pressure；Then 1mL water is added, adds the chloroform of 1mL, chloroform is removed after concussion, repeats extraction three
Secondary, water layer is as test liquid；
Step4,0.5~10mg of various chondroitin sulfate reference substances carry out sour water solution and derivatization according to S2 and S3 respectively；
Step5, LC-MS analysis: using acetonitrile/20mM ammonium acetate solution as mobile phase, reverse phase silica gel is used
Column chromatography；Using positive ion mode, the ion chromatography peak of m/z 766.2 ± 0.5 is extracted；
Step6, selection chromatographic peak retention time and mass spectrometric data and reference substance compare, to infer chondroitin sulfate in sample
Plain type.
Wherein, it is 0.2mol/L that optimal conditions, which is acid concentration, in Step2,100 DEG C of hydrolysis temperature, hydrolysis time 1h；?
The band sulfate disaccharides fragment products ratio highest obtained under optimal conditions, the sensitivity highest of detection.
Embodiment 1
(1) sturgeon cartilage is taken, smashes, be freeze-dried to obtain freeze-dried powder.Sample 5g is weighed in the KH of the pH=8 of 25mL2PO4It is slow
It rushes in solution, after 0.5% trypsin digestion 4h is added, 0.5% papain enzymolysis 3h is added, later with 100
DEG C boiling water enzyme deactivation 5min；Enzymolysis liquid is centrifuged 20min under conditions of 10000r/min and takes supernatant；1.5 are added in supernatant
The ethyl alcohol alcohol precipitation of times volume is stayed overnight；Precipitating is taken with 4000r/min centrifugation 15min after alcohol precipitation, Thick many candies dry powder is lyophilized to obtain in precipitating.
(2) Thick many candies dry powder 20mg is accurately weighed in 5mL hydrolysis pipe, and the 0.2mol/L of 1mL is added in hydrolysis pipe
Trifluoroacetic acid hydrolyze 1h under conditions of 100 DEG C；1mL water is added in acid hydrolysis liquid, and solvent is removed under reduced pressure, in triplicate, deacidification.
(3) be added 400 μ L ammonium hydroxide shake dissolved residue, then to hydrolysis pipe in be added 400 μ L 0.3moL/L 1- benzene
Base -3- methyl -5- pyrazolone (PMP) methanol solution, after mixing under the conditions of 70 DEG C water-bath 30min；1mL methanol is added, subtracts
Pressure removes solvent；Then 1mL water is added, adds the chloroform of 1mL, chloroform is removed after concussion, repeats extraction three times, water layer conduct
Test liquid.
(4) the mixture reference substance of chondroitin sulfate C reference substance and chondroitin sulfate A (CSA) and C, respectively takes 10mg, respectively according to
(2) and (3) carry out sour water solution and derivatization.
(5) take (3) and (4) obtain test liquid and reference substance derivatization product after 0.22 μm of filtering with microporous membrane, into
The analysis of row liquid matter.Liquid matter condition is as follows: Silgreen ODS C18 (250 × 4.6mm, 5 μm) chromatographic column；30 DEG C of column temperature；Flowing
Phase 20mmol ammonium acetate solution-acetonitrile (78:22, V/V)；Flow velocity 1mL/min；The source ion source ESI；Spray voltage 4.5kV；Hair
Capillary temperature is 275 DEG C；Capillary voltage is 37V；Sheath gas: 40Au；Assist gas: 10Au；Positive ion mode detection；Scanning mode
For full scan (Full Scan)；Scanning range is 100-2000 (m/z)；Select the ion of m/z 766.2.
(6) sturgeon cartilage sample chromatogram figure pass through as shown in Figure 3 with chondroitin sulfate C reference substance Fig. 1 and chondroitin sulfate A (CSA) and
Mixture reference substance Fig. 2 of C is compared, it is found that the appearance time of sturgeon cartilage sample is consistent with chondroitin sulfate C reference substance, such as table
Shown in 1, illustrate that chondroitin sulfate present in sturgeon cartilage is chondroitin sulfate C.
Table 1
Embodiment 2
(1) fresh pig trunnion is taken, freeze-dried powder is cleaned, smashes, being freeze-dried and to obtain.Take sample 5g in the pH=8's of 25mL
KH2PO4In buffer solution, after 0.5% trypsin digestion 4h is added, 0.5% papain enzymolysis 3h is added, it
Afterwards with 100 DEG C of boiling water enzyme deactivation 5min；Enzymolysis liquid is centrifuged 20min under conditions of 10000r/min and takes supernatant；In supernatant
The ethyl alcohol alcohol precipitation that 1.5 times of volumes are added is stayed overnight；Precipitating is taken with 4000r/min centrifugation 15min after alcohol precipitation, Thick many candies are lyophilized to obtain in precipitating
Dry powder.
(2) Thick many candies dry powder 20mg is accurately weighed in 5mL hydrolysis pipe, and the 2mol/L of addition 1mL in hydrolysis pipe
Trifluoroacetic acid hydrolyzes 1.5h under conditions of 110 DEG C；0.5mL water is added in acid hydrolysis liquid, and solvent is removed under reduced pressure, in triplicate, removes
Acid.
(3) be added 300 μ L ammonium hydroxide shake dissolved residue, then to hydrolysis pipe in be added 300 μ L 0.3moL/L 1- benzene
Base -3- methyl -5- pyrazolone (PMP) methanol solution, after mixing under the conditions of 70 DEG C water-bath 30min；0.5mL methanol is added,
Solvent is removed under reduced pressure；Then 1mL water is added, adds the chloroform of 1mL, chloroform is removed after concussion, repeats extraction three times, water layer is made
For test liquid.
(4) the mixture reference substance of chondroitin sulfate C reference substance and chondroitin sulfate A (CSA) and C, respectively takes 10mg, respectively according to
(2) and (3) carry out sour water solution and derivatization.
(5) take (3) and (4) obtain test liquid and reference substance derivatization product after 0.22 μm of filtering with microporous membrane, into
The analysis of row liquid matter.Liquid matter condition is as follows: Silgreen ODS C18 (250 × 4.6mm, 5 μm) chromatographic column；30 DEG C of column temperature；Flowing
Phase 20mmol ammonium acetate solution-acetonitrile (78:22, V/V)；Flow velocity 1mL/min；The source ion source ESI；Spray voltage 4.5kV；Hair
Capillary temperature is 275 DEG C；Capillary voltage is 37V；Sheath gas: 40Au；Assist gas: 10Au；Positive ion mode detection；Scanning mode
For full scan (Full Scan)；Scanning range is 100-2000 (m/z)；Select 766.2 ion of m/z.
(6) pig trunnion sample chromatogram as shown in figure 4, through with chondroitin sulfate C reference substance Fig. 1 and chondroitin sulfate A (CSA)
Mixture reference substance Fig. 2 comparison with C is it is found that find the appearance time and chondroitin sulfate A (CSA) and C reference substance one of pig trunnion sample
It causes, as shown in table 1, there are chondroitin sulfate A (CSA)s and C in pig trunnion sample.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art within the technical scope of the present disclosure, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (6)
1. a kind of method for quickly identifying chondroitin sulfate A (CSA) and C, which comprises the steps of:
The chondroitin sulfate of S1, Extraction and discrimination sample prepare Thick many candies dry powder sample；
The polysaccharide in sample is extracted using direct water extraction in the step S1, is greater than free content of chondroitin sulfate
5%；
S2, sample to be tested utilize trifluoroacetic acid hydrolysis, and solvent is removed under reduced pressure using methanol in gained hydrolysate in triplicate, and deacidification is extremely
Neutrality, trifluoroacetic acid concentration 0.2mol/L, 100 DEG C of hydrolysis temperature, hydrolysis time 1h；
The content of chondroitin sulfate of sample to be tested and trifluoroacetic acid, methanol 0.5~10mg:1mL:0.5 of mass volume ratio~
1mL；
S3, by residue ammonia solvent obtained by S2,1-phenyl-3-methyl-5-pyrazolones ketone (PMP) derivatization, extraction is added
Obtain test liquid；
S4, chondroitin sulfate reference substance is taken to repeat step S2 and S3, chondroitin sulfate in reference substance sample with same operation parameter
The Thick many candies dry powder sample that is prepared with S1 of content in content of chondroitin sulfate it is of substantially equal；
S5, the derivative that S3 and S4 is analyzed using liquid chromatography-mass spectrography technology:
LC-MS analysis condition include: using acetonitrile/20mM ammonium acetate solution as mobile phase, ammonium acetate solution and
The volume ratio of acetonitrile is 78:22, and using the liquid chromatography-mass spectrography for being equipped with C18 chromatographic column, using positive ion mode, selection is had
The quasi-molecular ion of the derivative of two bglii fragment of sulfate selects the ion of m/z 766.2 ± 0.5；
S6, selection chromatographic peak retention time and mass spectrometric data compare, to infer the type of chondroitin sulfate in sample.
2. a kind of method for quickly identifying chondroitin sulfate A (CSA) and C according to claim 1, wherein in the step S3
Using 70 DEG C of water-bath 30min of 1-phenyl-3-methyl-5-pyrazolones ketone (PMP) methanol solution of 0.3mol/L, water or first is added
Solvent is removed under reduced pressure in alcohol；The content of chondroitin sulfate and ammonium hydroxide, 1-phenyl-3-methyl-5-pyrazolones ketone (PMP) of sample to be tested
The μ of 0.5~10mg:200 of mass volume ratio~400 μ of L:200~400 L:0.5~1mL of methanol solution, water or methanol.
3. a kind of method for quickly identifying chondroitin sulfate A (CSA) and C according to claim 1, wherein in the step S3
Extraction is, using chloroform, repetition extraction is three times.
4. a kind of method for quickly identifying chondroitin sulfate A (CSA) and C according to claim 3, wherein in the step S3
Extraction adds chloroform, chloroform is removed after concussion, in triplicate, water layer is as test liquid for water is added；The sulfuric acid of sample to be tested
0.5~10mg:1mL:1mL of mass volume ratio of chondroitin content and water, chloroform.
5. the method that one kind according to claim 1 or 2 or 4 quickly identifies chondroitin sulfate A (CSA) and C, wherein it is described to
The content of chondroitin sulfate of test specimens is 0.5~10mg.
6. a kind of method for quickly identifying chondroitin sulfate A (CSA) and C according to claim 1, wherein in the step S5
The liquid matter condition of liquid chromatography-mass spectrography technology is as follows: Silgreen ODS C18 chromatographic column, 250 × 4.6mm, and 5 μm；Column temperature 30
℃；The volume ratio of mobile phase 20mmol ammonium acetate solution-acetonitrile, ammonium acetate solution and acetonitrile is 78:22；Flow velocity 1mL/
min；The source ion source ESI；Spray voltage 4.5kV；Capillary temperature is 275 DEG C；Capillary voltage is 37V；Sheath gas: 40Au；It is auxiliary
Help gas: 10Au；Scanning mode is full scan (Full Scan)；Scanning range is 100-2000m/z.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610171668.1A CN105866262B (en) | 2016-03-23 | 2016-03-23 | A kind of method of quick identification chondroitin sulfate A (CSA) and C |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610171668.1A CN105866262B (en) | 2016-03-23 | 2016-03-23 | A kind of method of quick identification chondroitin sulfate A (CSA) and C |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105866262A CN105866262A (en) | 2016-08-17 |
| CN105866262B true CN105866262B (en) | 2019-04-02 |
Family
ID=56625417
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610171668.1A Active CN105866262B (en) | 2016-03-23 | 2016-03-23 | A kind of method of quick identification chondroitin sulfate A (CSA) and C |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105866262B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109696490A (en) * | 2017-10-24 | 2019-04-30 | 汉寿康运医药科技有限公司 | A kind of identification source of chondroitin sulfate HPLC detection method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1761686A (en) * | 2003-03-20 | 2006-04-19 | 细川密克朗集团 | Proteoglycan isolated from cartilaginous fish and process for producing the same |
| CN103698426A (en) * | 2013-12-12 | 2014-04-02 | 中国海洋大学 | Method for degrading chondroitin sulfate and hyaluronic acid to obtain chondroitin sulfate disaccharide and hyaluronic acid disaccharide and detecting chondroitin sulfate disaccharide and hyaluronic acid disaccharide |
| CN104569274A (en) * | 2015-01-21 | 2015-04-29 | 大连工业大学 | Method for identifying uronic acid-containing polysaccharide in biological tissue |
-
2016
- 2016-03-23 CN CN201610171668.1A patent/CN105866262B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1761686A (en) * | 2003-03-20 | 2006-04-19 | 细川密克朗集团 | Proteoglycan isolated from cartilaginous fish and process for producing the same |
| CN103698426A (en) * | 2013-12-12 | 2014-04-02 | 中国海洋大学 | Method for degrading chondroitin sulfate and hyaluronic acid to obtain chondroitin sulfate disaccharide and hyaluronic acid disaccharide and detecting chondroitin sulfate disaccharide and hyaluronic acid disaccharide |
| CN104569274A (en) * | 2015-01-21 | 2015-04-29 | 大连工业大学 | Method for identifying uronic acid-containing polysaccharide in biological tissue |
Non-Patent Citations (1)
| Title |
|---|
| 银杏叶多糖的分离纯化及鉴定;黄桂宽 等;《中国生化药物杂志》;19961231;第17卷(第4期);第157-159页 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105866262A (en) | 2016-08-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105675779B (en) | A kind of method of quantitative detection acid polysaccharide containing alditol | |
| Chen et al. | Preparation of oligosaccharides from Chinese yam and their antioxidant activity | |
| Chang et al. | Analysis of glycosaminoglycan-derived disaccharides by capillary electrophoresis using laser-induced fluorescence detection | |
| CN103822998A (en) | Method for analyzing amine substances in dansyl chloride derived-plasma based on liquid chromatography mass spectrometry | |
| Gargano et al. | Profiling of a high mannose-type N-glycosylated lipase using hydrophilic interaction chromatography-mass spectrometry | |
| CN104569274A (en) | Method for identifying uronic acid-containing polysaccharide in biological tissue | |
| CN114839282B (en) | Method for quantitatively analyzing impurities in polypeptide drug sample based on enzymolysis method | |
| Yu et al. | Glycosaminoglycans in human cerebrospinal fluid determined by LC-MS/MS MRM | |
| Guo et al. | Marker peptide combination for source identification of gelatins obtained from Equidae hides by LC–MS/MS detection | |
| Ostrowski et al. | Acid phosphomonoesterase of human prostate Carbohydrate content and optical properties | |
| CN105866262B (en) | A kind of method of quick identification chondroitin sulfate A (CSA) and C | |
| CN106770871B (en) | A kind of method of quantitative detection acid polysaccharide containing alditol | |
| Zhu et al. | Determination of porcine derived components in gelatin and gelatin-containing foods by high performance liquid chromatography-tandem mass spectrometry | |
| Zinellu et al. | Reverse injection capillary electrophoresis UV detection for serotonin quantification in human whole blood | |
| Deng et al. | Quantitative estimation of enzymatic released specific oligosaccharides from Hericium erinaceus polysaccharides using CE-LIF | |
| CN106198790A (en) | A kind of horse and mule common characteristic polypeptide and application thereof | |
| Jaravel et al. | Off-line coupling of capillary isotachophoresis separation to IRMPD spectroscopy for glycosaminoglycans analysis: Application to the chondroitin sulfate disaccharides model solutes | |
| Henninot et al. | Characterization of monoclonal antibodies by a fast and easy liquid chromatography–mass spectrometry time-of-flight analysis on culture supernatant | |
| Apffel et al. | Application of capillary electrophoresis, high-performance liquid chromatography, on-line electrospray mass spectrometry and matrix-assisted laser desorption ionization-time of flight mass spectrometry to the characterization of single-chain plasminogen activator | |
| CN115166089A (en) | Method for identifying chlorella pyrenoidosa by using methylated sulfated oligosaccharide group | |
| Raymond et al. | Derivatization of carbohydrates for analysis by liquid chromatography and capillary electrophoresis | |
| Guo et al. | Identification and quantification of uronic acid-containing polysaccharides in tissues of Russian sturgeon (Acipenser gueldenstaedtii) by HPLC–MS/MS and HPLC–MS n | |
| Kang et al. | Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans | |
| CN109541222A (en) | The detection method of Protein Palmitoylation decorating site | |
| CN109358154B (en) | Method for determining monosaccharide composition in acidic polysaccharide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |