CN108802248A - A kind of method for separating and analyzing of the heparitin sulfate disaccharides containing free amine group of derivatization - Google Patents
A kind of method for separating and analyzing of the heparitin sulfate disaccharides containing free amine group of derivatization Download PDFInfo
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- CN108802248A CN108802248A CN201810640001.0A CN201810640001A CN108802248A CN 108802248 A CN108802248 A CN 108802248A CN 201810640001 A CN201810640001 A CN 201810640001A CN 108802248 A CN108802248 A CN 108802248A
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- disaccharides
- heparitin sulfate
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- -1 sulfate disaccharides Chemical class 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 37
- 125000003277 amino group Chemical group 0.000 title claims abstract description 26
- 238000001212 derivatisation Methods 0.000 title claims abstract description 9
- 150000002016 disaccharides Chemical class 0.000 claims abstract description 46
- 239000007788 liquid Substances 0.000 claims abstract description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 26
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 18
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 150000002500 ions Chemical class 0.000 claims description 11
- 238000000926 separation method Methods 0.000 claims description 11
- 150000003863 ammonium salts Chemical class 0.000 claims description 10
- 239000008367 deionised water Substances 0.000 claims description 10
- 229910021641 deionized water Inorganic materials 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000001819 mass spectrum Methods 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical group N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 6
- 239000005695 Ammonium acetate Substances 0.000 claims description 6
- 229940043376 ammonium acetate Drugs 0.000 claims description 6
- 235000019257 ammonium acetate Nutrition 0.000 claims description 6
- 230000002209 hydrophobic effect Effects 0.000 claims description 6
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 5
- 239000012266 salt solution Substances 0.000 claims description 5
- 235000000346 sugar Nutrition 0.000 claims description 5
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims description 4
- 238000000132 electrospray ionisation Methods 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 2
- 125000003158 alcohol group Chemical group 0.000 claims 1
- 150000002576 ketones Chemical class 0.000 claims 1
- 239000003550 marker Substances 0.000 claims 1
- 229920000669 heparin Polymers 0.000 abstract description 33
- 229960002897 heparin Drugs 0.000 abstract description 33
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 abstract description 32
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 abstract description 14
- 239000002628 heparin derivative Substances 0.000 abstract description 6
- 238000001514 detection method Methods 0.000 abstract description 4
- 239000007791 liquid phase Substances 0.000 abstract description 4
- 238000007445 Chromatographic isolation Methods 0.000 abstract description 3
- 239000012472 biological sample Substances 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 238000010183 spectrum analysis Methods 0.000 abstract description 2
- 239000012071 phase Substances 0.000 description 19
- 238000004458 analytical method Methods 0.000 description 11
- 239000000523 sample Substances 0.000 description 8
- 239000003795 chemical substances by application Substances 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 108010083213 heparitinsulfate lyase Proteins 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 3
- 239000013076 target substance Substances 0.000 description 3
- PIGCSKVALLVWKU-UHFFFAOYSA-N 2-Aminoacridone Chemical compound C1=CC=C2C(=O)C3=CC(N)=CC=C3NC2=C1 PIGCSKVALLVWKU-UHFFFAOYSA-N 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 108010022901 Heparin Lyase Proteins 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- KXRNYDKIPJKLTD-UHFFFAOYSA-N cyanoboron Chemical compound [B]C#N KXRNYDKIPJKLTD-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 238000004451 qualitative analysis Methods 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- IAJILQKETJEXLJ-KLVWXMOXSA-N (2s,3r,4r,5r)-2,3,4,5-tetrahydroxy-6-oxohexanoic acid Chemical compound O=C[C@H](O)[C@H](O)[C@@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-KLVWXMOXSA-N 0.000 description 1
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 1
- GDALETGZDYOOGB-UHFFFAOYSA-N Acridone Natural products C1=C(O)C=C2N(C)C3=CC=CC=C3C(=O)C2=C1O GDALETGZDYOOGB-UHFFFAOYSA-N 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 101000637792 Homo sapiens Solute carrier family 35 member G5 Proteins 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 102100032019 Solute carrier family 35 member G5 Human genes 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- FZEYVTFCMJSGMP-UHFFFAOYSA-N acridone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3NC2=C1 FZEYVTFCMJSGMP-UHFFFAOYSA-N 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 239000000538 analytical sample Substances 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 230000008901 benefit Effects 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
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000002270 exclusion chromatography Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- MSWZFWKMSRAUBD-IVMDWMLBSA-N glucosamine group Chemical group OC1[C@H](N)[C@@H](O)[C@H](O)[C@H](O1)CO MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000005040 ion trap Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 229910000069 nitrogen hydride Inorganic materials 0.000 description 1
- 238000006268 reductive amination reaction Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 150000003538 tetroses Chemical class 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of method for separating and analyzing of the heparitin sulfate disaccharides containing free amine group of derivatization, the functional relation of the disaccharides quality and Mass Spectrometer Method peak area can be used for the quantitative detection of heparin and heparitin sulfate disaccharides in practical biological sample.The present invention can detect heparin and heparitin sulfate disaccharides, especially the heparitin sulfate disaccharides containing free amine group in liquid phase-mass spectral analysis, and conventional liquid phase chromatographic isolation can also be used and analyzed in conjunction with ultraviolet and fluorescence detector.This has important role to the disaccharide component of research clinical medicine heparin, heparitin sulfate, heparin derivatives and various biological samples.
Description
Technical field
The invention belongs to drug measurement techniques fields, and in particular to a kind of heparitin sulfate containing free amine group of derivatization
The method for separating and analyzing of disaccharides.
Background technology
Heparin and heparitin sulfate are linear glycosaminoglycans, since heparin has stronger anticoagulation, are widely used
In diseases such as treatment thrombotic disease, myocardial infarctions.Different sulphations, acetylation and free amine group replace mould in heparin
Formula forms different disaccharide units, imparts heparin and the numerous biological function of heparitin sulfate, takes part in vascularization, blood
Liquid solidification, cell adherence and the vital movements such as hyperplasia and metastases.Heparin and heparitin sulfate are by disaccharide unit group repeatedly
At disaccharide unit is formed by hexuronic acid and gucosamine with 1-4 glucosides key connections, wherein 12 kinds of representative disaccharide units
The middle N- Glucose sulfates amine, N- acetyl glucoses amine and the rare gucosamine containing free amine group that there is routine is residual
Base.
Studies have found that, concentration of the heparitin sulfate containing free amine group in breast cancer cell is quite high, can press down recently
The activity of heparitinase processed.The tetrose that the Glucosamine residues containing free amine group are synthesized using artificial chemistry is carried out in vitro in fact
It tests, finds it by the activity of Reverse transcriptase heparitinase to further suppress the transfer [S. of breast cancer cell
Nadanaka, et al. J. Biol. Chem., 2014,289:15231-43].In addition it is reported that, contain free amine group
Heparitin sulfate by inhibit tumor tissues medium vessels generate and vascular endothelial growth factor expression and angiogenesis, to
Inhibit transfer [such as Chen Jinlian world Chinese digestion magazine, 2005,13 (22) of tumour:2685-2688].Containing free amine group
Heparitin sulfate promise to be antitumor activity inhibitor.Therefore either natural heparitin sulfate/heparin or artificial
The derivative of synthesis, building the separation of the heparitin sulfate disaccharides containing free amine group and analysis method has greatly challenge,
It is the basis for the biological function for more fully understanding the heparitin sulfate containing free amine group.
When the constituent content of analysis heparin and heparitin sulfate, 12 kinds of main livers are generated usually using heparinase enzymolysis
Element/heparitin sulfate disaccharides, then analysis measurement is carried out to disaccharides.The technology packet of traditional analysis heparin/heparitin sulfate disaccharides
Include high performance liquid chromatography [Wei Z, et al. J. Biol. Chem., 2005,280 (16):15742], Capillary Electrophoresis
Method [Hitchcock A M, et al. Electrophoresis, 2008,29 (23):4538-4548], exclusion chromatography
[Chuang W L, et al. J Chromatogr A, 2001, 932(1):65-74], exclusion liquid chromatograph mass spectrography
Technology, hydrophilic liquid phase chromatograph-mass spectrometer coupling technology [Gill V L, et al. Anal. Chem., 2013,85:1138-
1145] etc..These analytical technologies are mainly used for analyzing 8 kinds of conventional heparins/heparitin sulfate disaccharides.Further, patent
CN104914205B discloses a kind of non-substituted gucosamine containing N-（GlcNH3 +）The separation of heparitin sulfate disaccharides is analyzed
Method, the mass spectrum peak area correction factor can be used for the quantitative detection of heparitin sulfate and heparin disaccharides in practical biological sample,
But this method is analyzed using amine ion-pairing agent.It generally believes that ion-pairing agent exists at present and pollutes mass spectrographic ion
The risk in source can lead to decline [Yang B, et al. Anal. Biochem., 2011,415 (1) of sensitivity of mass spectrometry:
59-66]。
Therefore, the new method of analysis heparin/heparitin sulfate disaccharides is established, reduces in analytic process and has to mass spectrographic damage
There is important meaning.And 2- amino acridones (2-aminoacridone, AMAC) is fluorescent hydrophobic molecule, can with heparin/
Heparitin sulfate disaccharides occurs reductive amination process and is thus connected on disaccharides.Meanwhile fluorescent hydrophobic molecular marking technique energy
Promote the separation of heparin/heparitin sulfate disaccharides.
Invention content
The object of the present invention is to provide the separation of the heparitin sulfate disaccharides containing free amine group of a kind of derivatization point
Analysis method, the present invention can detect heparitin sulfate disaccharides, especially the sulfuric acid liver containing free amine group in liquid phase-mass spectral analysis
Plain disaccharides can also be used conventional liquid phase chromatographic isolation and be analyzed in conjunction with ultraviolet and fluorescence detector.This is clinical to research
Agents heparin, heparitin sulfate, heparin derivatives and different kind organism sample disaccharide component play an important role.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of method for separating and analyzing of the heparitin sulfate disaccharides containing free amine group of derivatization is as follows：
（1）Ammonium salt reagent is dissolved in deionized water, reagent tune pH value is adjusted to 5.2-6.0 with pH, a concentration of 20mM- is made
The mobile phase A of 80mM；Using alcohol reagent as Mobile phase B.
（2）By heparitin sulfate disaccharides to be measured（It include the disaccharides containing free amine group）Respectively it is dissolved in deionized water,
It is configured to two sugar juices of a concentration of 1mg/mL.12 kinds of disaccharides for measuring equivalent, are configured to mixture of disaccharides, are lyophilized.
（3）The labelled reagent of fluorescent hydrophobic is dissolved in acetic acid：Dimethyl sulfoxide（v/v）=3：In 17 solvent, concentration is made
For the labelled reagent solution of 0.05-0.2mol/L.Sodium cyanoborohydride is dissolved in deionized water, a concentration of 0.5- is made
The sodium cyanoborohydride solution of 2.0mol/L.
（4）With（3）In label solution label（2）In dry mixed disaccharides, react 20min after, be added cyano boron hydrogen
Change sodium solution, is uniformly mixed, 30-50 DEG C of water-bath 2-6 hours.With the dimethyl sulfoxide of 50% (v/v) by the disaccharides of above-mentioned label
Mixture is diluted to the disaccharides concentration of 20-40ng/ μ L, is configured to two sugar juice to be measured of multigroup different disaccharides concentration.
（5）The heparitin sulfate standard disaccharides of multigroup various concentration after label is crossed into ODS-2 HYPERSIL C18 respectively
Chromatographic column：Flow velocity is 0.3mL/min, gradient 0-100min：98%-80% mobile phase As（Ammonium salt solution）, 2%-20% flowing
Phase B（Alcohol reagent）, 100min-120min：80%-50% mobile phase As（Ammonium salt solution）, 20%-50% Mobile phase Bs（Alcohols tries
Agent）；PDA detectors detect separation chromatogram under conditions of 200nm-600nm all bands detect；
（6）By Mass Spectrometer Method Mass Spectrometer Method peak area is obtained, calculates the quality and Mass Spectrometer Method peak area of various disaccharides
Functional relation, then calculate the percentage composition of heparitin sulfate disaccharides.
The ammonium salt reagent is ammonium acetate；It is acetic acid that pH, which adjusts reagent,；Alcohol reagent is methanol.
The labelled reagent is 2- amino acridones.
Step（5）In liquid phase-mass spectrum use reversed-phase liquid chromatography series electrical electrospray ionization trap flight time mass spectrum, setting
Parameter is：Negative ion mode orifice potential is -3.5kV, and dry gas stream speed is 1.5L/min, and Heat block & CDL temperature is
200℃。
The remarkable advantage of the present invention is：Be put forward for the first time it is a kind of can baseline separation analysis simultaneously include 4 kinds containing free ammonia
The method of 12 kinds of heparin/heparitin sulfate disaccharides including the heparitin sulfate disaccharides of base, solving at present can not baseline separation
Analyze the problem of heparitin sulfate disaccharides or analytic process intermediate ion source containing free amine group pollute；The instrument of this method is answered simultaneously
Expanded with range, liquid phase-mass spectrum can be used efficiently, high-resolution to detach heparitin sulfate disaccharides of the analysis containing free amine group
Sample can also utilize conventional liquid phase chromatographic isolation and ultraviolet and fluorescence detector is combined to detect.That this is conducive to is clear, accurately divides
From heparin/heparitin sulfate disaccharides in analysis sample, mitigates the heparitin sulfate disaccharides containing free amine group and divide the analysis of variance
Instrument cost, the heparitin sulfate disaccharides tool containing free amine group in qualitative and quantitative analysis heparin derivatives and different kind organism sample
There is great practical value.
Description of the drawings
Fig. 1 is the extraction ion stream chromatogram of 12 kinds of heparin/heparitin sulfate disaccharides.
Fig. 2 is the ultraviolet detection chromatogram of 12 kinds of two saccharides of heparin/heparitin sulfate.
Fig. 3 is the linear diagram of 12 kinds of heparin/heparitin sulfate disaccharides quality and Mass Spectrometer Method peak area.
Specific implementation mode
The present invention utilizes the liver in reversed-phase liquid chromatography series electrical electrospray ionization trap flight time mass spectrum detection sample to be tested
Element/heparitin sulfate disaccharides composition.Include that 4 kinds of heparitin sulfate disaccharides containing free amine group exist by reversed-phase liquid chromatography separation
Interior 12 kinds of heparin/heparitin sulfate disaccharides, and pass through 12 kinds of heparin of electron spray ion trap flight time mass spectrum pair/sulfuric acid liver
Plain disaccharides carries out qualitative and quantitative analysis, obtains the linear side of each heparin/heparitin sulfate disaccharides quality and Mass Spectrometer Method peak area
Journey, the linear equation can be used for the quantitative detection of heparin in actual sample/heparitin sulfate disaccharides.
A kind of method for separating and analyzing of the heparitin sulfate disaccharides containing free amine group of derivatization is as follows：
（1）Ammonium salt reagent is dissolved in deionized water, reagent tune pH value is adjusted to 5.2-6.0 with pH, a concentration of 20mM- is made
The mobile phase A of 80mM；Using alcohol reagent as Mobile phase B.
（2）By heparitin sulfate disaccharides to be measured（It include the disaccharides containing free amine group）Respectively it is dissolved in deionized water,
It is configured to two sugar juices of a concentration of 1mg/mL.12 kinds of disaccharides for taking equivalent, are configured to mixture of disaccharides, freeze-drying.
（3）The labelled reagent of fluorescent hydrophobic is dissolved in acetic acid：Dimethyl sulfoxide（v/v）=3：In 17 solvent, concentration is made
For the labelled reagent solution of 0.05-0.2mol/L.Sodium cyanoborohydride is dissolved in deionized water, a concentration of 0.5- is made
The sodium cyanoborohydride solution of 2.0mol/L.
（4）With（3）In label solution label（2）In dry mixed disaccharides, react 20min after, be added cyano boron hydrogen
Change sodium solution, is uniformly mixed, 30-50 DEG C of water-bath 2-6 hours.With the dimethyl sulfoxide of 50% (v/v) by the disaccharides of above-mentioned label
Mixture is diluted to the disaccharides concentration of 20-40ng/ μ L, is configured to two sugar juice to be measured of multigroup different disaccharides concentration.
（5）The heparitin sulfate standard disaccharides of multigroup various concentration after label is crossed into ODS-2 HYPERSIL C18 respectively
Chromatographic column：Flow velocity is 0.3mL/min, gradient 0-100min：98%-80% mobile phase As（Ammonium salt solution）, 2%-20% flowing
Phase B（Alcohol reagent）, 100min-120min：80%-50% mobile phase As（Ammonium salt solution）, 20%-50% Mobile phase Bs（Alcohols tries
Agent）；PDA detectors detect separation chromatogram under conditions of 200nm-600nm all bands detect；
（6）It is detected by the high-resolution mass spectrometer under negative ion mode, extracts target substance molecular weight, obtain extraction ion
The Mass Spectrometer Method peak area of the standard disaccharides of flow chromatography figure and each label.
（8）Establish the linear relationship of the quality and Mass Spectrometer Method peak area of label disaccharides, linear equation and phase relation ordered series of numbers
In table 1：
The linear equation of the quality and Mass Spectrometer Method peak area of 10 two kinds of heparitin sulfate disaccharides of table
（9）Using above-mentioned analytical sample to be tested, extract target substance molecular weight, obtain extraction ion stream chromatogram and
Mass Spectrometer Method peak area, passes through（8）In each disaccharides linear equation calculate sample in each disaccharides component.
Technical scheme of the present invention is described further below by specific implementation example, but can not be limited with this
The scope of the present invention.Each reagent is commercially available unless otherwise noted in following embodiment.
Embodiment 1
N- sulfated heparins (2S, 6S, NH are taken off to homemade heparin derivatives using the technical program3 +- HP) and complete de- 2,6, N-
Sulfated heparin (NH3 +- HP) sample disaccharide component carry out instance analysis.
A kind of method for separating and analyzing of the heparitin sulfate disaccharides containing free amine group of derivatization, steps are as follows：
（1）Ammonium acetate is dissolved in deionized water, with acetic acid tune pH value to 5.6, the ammonium acetate solution of a concentration of 40mM is made, makees
For mobile phase A；Using methanol as Mobile phase B.
（3）The heparin derivatives for being taken respectively from system take off N- sulfated heparins and entirely each 20 μ g of de- 2,6, N- sulfated heparins,
After the mixed liquor of heparinase I, heparinaseⅡ, heparinase III thoroughly digests, filtering.The 2- amino of 10 μ L 0.1mol/L is added
(2- amino acridones is dissolved in 3 to acridone solution:17 acetic acid:Dimethyl sulfoxide in the mixed solvent), react 20min.Above-mentioned anti-
The sodium cyanoborohydride solution that 10 μ L 1.0mol/L are added in solution is answered, reacts 4h in 45 DEG C.After reaction with 50% (v/
The heparin derivatives that above-mentioned 2- amino acridones marks are diluted to required concentration by dimethyl sulfoxide v).
（4）Use (5 μm, 250*4.6 mm) the separation analyses of ODS-2 HYPERSIL C18 columns（4）The heparin of middle label spreads out
Biological disaccharides：Flow velocity is 0.3mL/min, gradient 0-100min：98%-80% mobile phase As（The ammonium acetate solution of 40mM,
pH=5.6）, 2%-20% Mobile phase Bs（Methanol）, 100min-120min：80%-50% mobile phase As（The ammonium acetate solution of 40mM, pH=
5.6）, 20%-50% Mobile phase Bs（Methanol）.Mass spectrometry parameters：Negative ion mode orifice potential is -3.5kV, and dry gas stream speed is
1.5L/min, Heat block & CDL temperature are 200 DEG C.
（5）It is detected by the high-resolution mass spectrometer under negative ion mode, extracts target substance molecular weight, extracted
The Mass Spectrometer Method peak area of the standard disaccharides of ion stream chromatogram and each label.
（6）Pass through step（5）The Mass Spectrometer Method peak area of obtained each target disaccharides, according to method meter provided by the invention
Obtained linear equation, calculates separately de- N- sulfated heparins and complete de- 2,6, N- sulfated heparins thoroughly digest the group of disaccharides
At disaccharide component table is as shown in table 2：
2 heparin derivativeization of table thoroughly digests disaccharide component table
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with repair
Decorations should all belong to the covering scope of the present invention.
Claims (5)
1. a kind of method for separating and analyzing of the heparitin sulfate disaccharides containing free amine group of derivatization, it is characterised in that：Specific step
It is rapid as follows：
（1）Ammonium salt reagent is dissolved in deionized water, reagent tune pH value is adjusted to 5.2-6.0 with pH, a concentration of 20mM- is made
The mobile phase A of 80mM；Using alcohol reagent as Mobile phase B；
（2）Heparitin sulfate disaccharides to be measured is respectively dissolved in deionized water, the disaccharides for being configured to a concentration of 1mg/mL is molten
Liquid.12 kinds of disaccharides for taking equivalent, are configured to mixture of disaccharides, freeze-drying；
（3）The labelled reagent of fluorescent hydrophobic is dissolved in acetic acid：Dimethyl sulfoxide（v/v）=3：In 17 solvent, it is made a concentration of
The labelled reagent solution of 0.05-0.2mol/L；Sodium cyanoborohydride is dissolved in deionized water, a concentration of 0.5- is made
The sodium cyanoborohydride solution of 2.0mol/L；
（4）With（3）In label solution label（2）In dry mixed disaccharides, react 20min after, be added sodium cyanoborohydride
Solution is uniformly mixed, 30-50 DEG C of water-bath 2-6 hours；The disaccharides of above-mentioned label is mixed with the dimethyl sulfoxide of 50% (v/v)
Object is diluted to the disaccharides concentration of 20-40ng/ μ L, is configured to two sugar juice to be measured of multigroup different disaccharides concentration；
（5）The heparitin sulfate standard disaccharides of multigroup various concentration after label is crossed into C18 chromatographic columns respectively：Flow velocity is
0.3mL/min, gradient 0-100min：98%-80% mobile phase As, 2%-20% Mobile phase Bs, 100min-120min：80%-
50% mobile phase A, 20%-50% Mobile phase Bs；PDA detectors detect separation color under conditions of 200nm-600nm all bands detect
Spectrogram；Wherein mobile phase A is ammonium salt solution, and Mobile phase B is alcohol reagent；
（6）By Mass Spectrometer Method Mass Spectrometer Method peak area is obtained, calculates the quality and Mass Spectrometer Method peak area of various disaccharides
Functional relation, then calculate the percentage composition of heparitin sulfate disaccharides.
2. according to the method described in claim 1, it is characterized in that：The ammonium salt reagent is ammonium acetate；PH adjusts reagent
Acetic acid；Alcohol reagent is methanol.
3. according to the method described in claim 1, it is characterized in that：The fluorescent hydrophobic marker is 2- aminacrines
Ketone.
4. according to the method described in claim 1, it is characterized in that：The heparitin sulfate disaccharides includes four kinds and contains free amine group
Disaccharides.
5. according to the method described in claim 1, it is characterized in that：Step（5）In liquid phase-mass spectrum use reversed-phase liquid chromatography
Series electrical electrospray ionization trap flight time mass spectrum, setup parameter are：Negative ion mode orifice potential is -3.5kV, dry gas stream speed
For 1.5L/min, Heat block & CDL temperature is 200 DEG C.
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