CN104914205A - Segregation analysis method for heparan disaccharide sulfate containing FlcNH3<+> - Google Patents
Segregation analysis method for heparan disaccharide sulfate containing FlcNH3<+> Download PDFInfo
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Abstract
The invention discloses a segregation analysis method for heparan disaccharide sulfate containing FlcNH3<+>. A mass spectrum peak area correcting factor can be used for the quantitative detection of herparan sulfate and herparan disaccharide in a practical biological sample. According to the method, heparan disaccharide sulfate containing FlcNH3<+> can be detected quickly, the content has an important effect of influence research on the anticoagulation effect of clinical drug (herparan) and the detection on the disaccharide component of a biological sample.
Description
Technical field
The invention belongs to medicine, bulk drug detection field, be specifically related to a kind of containing the non-substituted gucosamine (GlcNH of N-
3 +) method for separating and analyzing of heparitin sulfate disaccharides.
Background technology
Heparin is glycosaminoglycan medicine, has stronger anticoagulation, is the choice drug for the treatment of thrombotic disease at present.Heparitin sulfate and heparin are that the disaccharides structural unit of the repetition of being got up with 1 ~ 4 glycosidic bond link by hexuronic acid and aminoglucose is formed; wherein there are 12 kinds of representative disaccharide unit, to contain N-acetyl glucose amine (GlcNAc) residue, containing N-Glucose sulfate amine (GlcNSO
3) heparitin sulfate/heparin disaccharides of residue is main existence form, and containing the non-substituted gucosamine (GlcNH of N-
3 +) the heparin disaccharides of residue is then rarer.The anticoagulation of heparin is relevant with the highly Sulfated pentasaccharides sequence in unique N-position of in its structure, therefore contains GlcNH in heparin medicine
3 +the disaccharides content of residue can the anticoagulant effect [Robert J. Linhardt, et.al, Analytical Biochemistry 461 (2014) 46 – 48.] of appreciable impact heparin medicine.In addition up-to-date research shows, containing GlcNH in malignant breast cancer cells system
3 +there is the expression of high concentration in rare structure, GlcNH
3 +structure has the activity suppressing heparanase, therefore promises to be antitumor activity inhibitor [S. Nadanaka, et al. J. Biol.Chem., 2014,289:15231-43].To containing GlcNH
3 +the qualitative and quantitative analysis of heparitin sulfate and heparin disaccharides, at the quality control of heparin medicine and stability study and GlcNH
3 +the aspects such as the functional study of rare structure all have great importance.
For heparitin sulfate/heparin disaccharides analyzing detecting method, the strong anion that has conventional at present exchanges high performance liquid chromatography (SAX-HPLC), nuclear magnetic resonance method (NMR), fluorescent marker method [R.J. Linhardt, et.al, Biochem. J. 322 (1997) 499 – 506.], mineral sulfates synthetic method [L. Liu, et.al, Carbohydr.Polym. 106 (2014) 343 – 350.], hydrophilic Interaction Chromatography mass spectrometric hyphenated technique (HILIC-MS) [Robert J. Linhardt, et.al, Analytical Biochemistry 461 (2014) 46 – 48.] etc.But these methods are mainly used in analyzing containing GlcNAc residue and GlcNSO
3the 8 kinds of heparitin sulfates/heparin disaccharides of residue, and owing to containing GlcNH
3 +content pettiness, the separation difficulty of heparitin sulfate/heparin disaccharides in biological sample of residue, to its the research of qualitative and quantitative analysis detection method less.Robert J. Linhardt etc. uses GlcNH
3 +the isotope-labelling method of acetylation again of residue to have determined under High Temperature High Pressure in heparinase hydrolysis products containing GlcNH
3 +the content of residue disaccharides, but the method sample pre-treatments is complicated, needs to use large-scale isotope labeling calculating instrument, and be not in Direct Determination sample containing GlcNH
3 +the component of residue disaccharides, detects in application in actual drug and has very large restriction.
Summary of the invention
The object of the present invention is to provide one containing the non-substituted gucosamine (GlcNH of N-
3 +) method for separating and analyzing of heparitin sulfate disaccharides, can detect N-non-substituted gucosamine heparitin sulfate disaccharides fast, its content has important effect to the influence research of clinical medicine anticoagulant heparin effect and the detection of biological sample disaccharide component etc.
For achieving the above object, the present invention adopts following technical scheme:
A kind of concrete steps of the method for separating and analyzing containing N-non-substituted gucosamine heparitin sulfate disaccharides are as follows:
(1) be dissolved in deionized water by amine ion-pairing agent, regulate reagent adjust pH to 8-8.8 with pH, obtained concentration is the mobile phase A of 15mM-45mM;
(2) amine ion-pairing agent being dissolved in percent by volume is in the acetonitrile solution of 80%, and regulate reagent adjust pH to 8-8.8 with pH, obtained concentration is the Mobile phase B of 15mM-45mM;
(3) to be measured is dissolved in deionized water containing N-non-substituted gucosamine heparitin sulfate disaccharides, be mixed with the solution to be measured that concentration is 250ng/mL, filter, cross ACQUITY UPLC BEH C18 post: flow velocity is 0.1mL/min, gradient is 0-30min:100%-85% mobile phase A, 0%-15% Mobile phase B, 30min-50min:85%-75% mobile phase A, 15%-25% Mobile phase B, 50min-60min:75%-35% mobile phase A, 25%-65% Mobile phase B; Separate colors spectrogram is detected under the condition that PDA detecting device detects in 200nm-600nm all band;
(4) by Mass Spectrometer Method and UV detect, obtain UV detect peak area and Mass Spectrometer Method peak area respectively, calculate Ionization Efficiency and mass spectra peak area correction factor, then calculate the percentage composition containing N-non-substituted gucosamine heparitin sulfate disaccharides.
Described amine ion-pairing agent is hexylamine.
Described pH regulates reagent to be formic acid.
Mass spectrum in step (4) adopts high performance liquid chromatography series electrical electrospray ionization trap flight time mass spectrum, setup parameter is: negative ion mode taper hole voltage is-3.5kV, dry gas flow velocity is 1.5L/min, heatblock & CDL temperature is 100 DEG C.
Remarkable advantage of the present invention is: propose first and a kind ofly simultaneously can comprise the method for 12 kinds of heparitin sulfate disaccharides of four kinds of N-non-substituted gucosamine heparin disaccharides by compartment analysis, solve the sample pretreatment process complex operation step of current heparitin sulfate disaccharides method for separating and analyzing, large by the interference of impurity in actual sample in actual sample analytic process, and for the positively charged separation containing N-non-substituted gucosamine heparin disaccharides, qualitative, quantitative difficulty, the problems such as range of application is limited.For the detection level improving heparitin sulfate disaccharides, ensure that the aspects such as drug safety have great practical value.
Accompanying drawing explanation
Fig. 1 is the UV detect chromatogram of 12 kinds of heparitin sulfate two saccharide.
Fig. 2 is the extraction ion current chromatogram of 12 kinds of heparitin sulfate disaccharides.
Embodiment
The heparitin sulfate disaccharides that the present invention utilizes ion pair reverse high performance liquid chromatography series electrical electrospray ionization trap flight time mass spectrum to detect in testing sample forms.The 12 kinds of heparitin sulfate disaccharides comprising four kinds of N-non-substituted gucosamine heparin disaccharides are separated by the reverse high performance liquid chromatography of ion pair, and by electron spray ion trap flight time mass spectrum, qualitative and quantitative analysis is carried out to 12 kinds of heparitin sulfate disaccharides, obtain the mass spectra peak area correction factor of each heparitin sulfate disaccharides, this correction factor can be used for the quantitative detection of heparitin sulfate disaccharides in actual sample.
A kind of method for separating and analyzing containing N-non-substituted residue heparitin sulfate and heparin disaccharides is as follows:
(1) ion-pairing agent is dissolved in deionized water, regulates reagent adjust pH to 8 ~ 8.8 with pH, the mobile phase A of obtained concentration 15mM-45mM.
(2) ion-pairing agent is dissolved in the acetonitrile solution that percent by volume is 80%, regulates reagent adjust pH to 8 ~ 8.8 with pH, the Mobile phase B of obtained concentration 15mM-45mM.
(3) heparitin sulfate two saccharide to be measured is dissolved in deionized water, is mixed with the solution to be measured that concentration is 250ng/mL, after filtering, use ACQUITY UPLC BEH C18 post (1.7 μm, 2.1*150mm): be 0.1mL/min at flow velocity, gradient is 0 ~ 30min, 100% ~ 85% mobile phase A, 0% ~ 15% Mobile phase B, 30min ~ 50min, 85% ~ 75% mobile phase A, 15% ~ 25% Mobile phase B, 50min ~ 60min, 75% ~ 35% mobile phase A, 25% ~ 65% Mobile phase B.PDA detecting device is detect under the condition of all band detection, obtains UV detect chromatogram when detecting device determined wavelength is 232nm.
(4) by the single sampling system of each standard disaccharides, the appearance time of each standard disaccharides is obtained;
(5) by the appearance time of UV detect chromatogram and each standard disaccharides, the UV detect peak area S of each disaccharides in biased sample is obtained
232.
(6) then, by detecting with high-resolution mass spectrometer in the negative ion mode, extracting target substance molecular weight, obtaining extracting ion current chromatogram.
(7) by the extraction ion current chromatogram of target molecular weight, the Mass Spectrometer Method peak area S of each standard disaccharides is obtained
mS.
(8) the UV detect peak area S of the standard substance obtained by step (5)
232with the Mass Spectrometer Method peak area of each standard disaccharides that step (7) obtains, through Ionization Efficiency K and the mass spectra peak area correction factor f of each standard disaccharides of following formulae discovery;
ki= S232i/SMSi （i=1、2、3……12)
fi=ki/kt （i=1、2、3……12； t=7）
Wherein: i is the numbering of each disaccharides:
The disaccharides structure that i=1 represents is HexA-GlcNH
3 +
The disaccharides structure that i=2 represents is HexA-GlcNAc
The disaccharides structure that i=3 represents is HexA-Glc NH
3 +(6S)
The disaccharides structure that i=4 represents is HexA (2S)-Glc NH
3 +
The disaccharides structure that i=5 represents is HexA-GlcNS
The disaccharides structure that i=6 represents is HexA-GlcNAc (6S)
The disaccharides structure that i=7 represents is HexA (2S)-GlcNAc
The disaccharides structure that i=8 represents is HexA (2S)-Glc NH
3 +(6S)
The disaccharides structure that i=9 represents is HexA-GlcNS (6S)
The disaccharides structure that i=10 represents is HexA (2S)-GlcNS
The disaccharides structure that i=11 represents is HexA (2S)-GlcNAc (6S)
The disaccharides structure that i=12 represents is HexA (2S)-GlcNS (6S)
T is the numbering counting the disaccharides of 1 chosen when calculating mass spectra peak area correction factor, and in t=7 and this method, mass spectrum correction factor is mass spectra peak area correction factor when being standard 1 with HexA (2S)-GlcNAc;
(9) the Mass Spectrometer Method peak area S of each standard disaccharides that the mass spectra peak area correction factor then, obtained by step (8) and step (7) are obtained
mS, the percentage composition P of each disaccharide component in following formulae discovery testing sample;
Pi= S
MSi*fi/（S
MS1*f1+ S
MS2*f2+ S
MS3*f3+……S
MS12*f12）
Below by concrete exemplifying embodiment, technical scheme of the present invention is described further, but can not limits the scope of the invention with this.In following examples, each reagent unless otherwise noted, is commercially available.
embodiment 1
The technical program is utilized to carry out instance analysis to the biological sample of ox kidney heparitin sulfate, marketed drugs liquaemin and the disaccharide component of sample that processes through High Temperature High Pressure (121 DEG C) thereof.
Containing the method for separating and analyzing of the non-substituted residue heparitin sulfate of N-and heparin disaccharides, step is as follows:
(1) n-hexylamine is dissolved in deionized water, with formic acid adjust pH to 8.8, obtained concentration is the mobile phase A of 30mM;
(2) n-hexylamine is dissolved in the acetonitrile solution that percent by volume is 80%, with formic acid adjust pH to 8.8, obtained concentration is the Mobile phase B of 30mM;
(3) each 20 μ g of liquaemin getting commercially available ox kidney heparitin sulfate, commercially available liquaemin respectively and process through High Temperature High Pressure (121 DEG C), after the thorough enzymolysis of mixed liquor of heparinase I, heparinaseⅡ, heparinase III, filter.Use (1.7 μm, ACQUITY UPLC BEH C18 post, 2.1*150mm): be 0.1mLmin at flow velocity, gradient is 0 ~ 30min, 100% ~ 85% mobile phase A, 0% ~ 15% Mobile phase B, 30min ~ 50min, 85% ~ 75% mobile phase A, 15% ~ 25% Mobile phase B, 50min ~ 60min, 75% ~ 35% mobile phase A, 25% ~ 65% Mobile phase B.Mass spectrometry parameters is: negative ion mode taper hole voltage :-3.5kV, and dry gas flow velocity is 1.5L/min, heatblock & CDL temperature is 100 DEG C.By detecting with high-resolution mass spectrometer in the negative ion mode, extracting target substance molecular weight, obtaining extracting ion current chromatogram;
(4) by the extraction ion current chromatogram of target molecular weight, the Mass Spectrometer Method peak area S of each target disaccharides is obtained
mS.
(5) the Mass Spectrometer Method peak area S of each target disaccharides obtained by step (4)
mS, according to the mass spectra peak area correction factor that method provided by the invention calculates, calculate the composition (Pi) of the rear thoroughly enzymolysis disaccharides of ox kidney heparitin sulfate, commercially available liquaemin and High Temperature High Pressure (121 DEG C) process respectively.
The mass spectra peak area correction factor of table 1 12 kinds of heparitin sulfate disaccharides
(6) basis is by mass spectra peak area correction factor provided by the invention, and calculate the composition (Pi) of the rear thoroughly enzymolysis disaccharides of ox kidney heparitin sulfate, commercially available liquaemin and High Temperature High Pressure (121 DEG C) process respectively, disaccharide component table is as shown in table 2:
The each heparan of table 2 thorough enzymolysis disaccharide component table
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (4)
1., containing a method for separating and analyzing for N-non-substituted gucosamine heparitin sulfate disaccharides, it is characterized in that: concrete steps are as follows:
(1) be dissolved in deionized water by amine ion-pairing agent, regulate reagent adjust pH to 8-8.8 with pH, obtained concentration is the mobile phase A of 15mM-45mM;
(2) amine ion-pairing agent being dissolved in percent by volume is in the acetonitrile solution of 80%, and regulate reagent adjust pH to 8-8.8 with pH, obtained concentration is the Mobile phase B of 15mM-45mM;
(3) to be measured is dissolved in deionized water containing N-non-substituted gucosamine heparitin sulfate disaccharides, be mixed with the solution to be measured that concentration is 250ng/mL, filter, cross ACQUITY UPLC BEH C18 post: flow velocity is 0.1mL/min, gradient is 0-30min:100%-85% mobile phase A, 0%-15% Mobile phase B, 30min-50min:85%-75% mobile phase A, 15%-25% Mobile phase B, 50min-60min:75%-35% mobile phase A, 25%-65% Mobile phase B; Separate colors spectrogram is detected under the condition that PDA detecting device detects in 200nm-600nm all band;
(4) by Mass Spectrometer Method and UV detect, obtain UV detect peak area and Mass Spectrometer Method peak area respectively, calculate Ionization Efficiency and mass spectra peak area correction factor, then calculate the percentage composition containing N-non-substituted gucosamine heparitin sulfate disaccharides.
2. method according to claim 1, is characterized in that: described amine ion-pairing agent is hexylamine.
3. method according to claim 1, is characterized in that: described pH regulates reagent to be formic acid.
4. method according to claim 1, it is characterized in that: the mass spectrum in step (4) adopts high performance liquid chromatography series electrical electrospray ionization trap flight time mass spectrum, setup parameter is: negative ion mode taper hole voltage is-3.5kV, dry gas flow velocity is 1.5L/min, heatblock & CDL temperature is 100 DEG C.
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|---|---|---|---|---|
| CN108802248A (en) * | 2018-06-21 | 2018-11-13 | 福州大学 | A kind of method for separating and analyzing of the heparitin sulfate disaccharides containing free amine group of derivatization |
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| CN102759596A (en) * | 2012-07-09 | 2012-10-31 | 山东大学 | Method for detecting low-molecular-weight heparin by combining ion pair reversed phase chronmatogaphy and mass spectrum |
| CN103454372A (en) * | 2013-09-10 | 2013-12-18 | 山东大学 | Ion pair antiphase chromatography and mass spectrometry combined detection method for low-molecule heparin part degradation products |
| CN103630647A (en) * | 2013-12-20 | 2014-03-12 | 山东大学 | Reverse-phase chromatography and mass-spectrometry combined detection method for complete low-molecular-heparin degradation product through precolumn derivatization |
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| CN102759596A (en) * | 2012-07-09 | 2012-10-31 | 山东大学 | Method for detecting low-molecular-weight heparin by combining ion pair reversed phase chronmatogaphy and mass spectrum |
| CN103454372A (en) * | 2013-09-10 | 2013-12-18 | 山东大学 | Ion pair antiphase chromatography and mass spectrometry combined detection method for low-molecule heparin part degradation products |
| CN103630647A (en) * | 2013-12-20 | 2014-03-12 | 山东大学 | Reverse-phase chromatography and mass-spectrometry combined detection method for complete low-molecular-heparin degradation product through precolumn derivatization |
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| AMANDA WEYERS ET AL: "A Structural Analysis of Glycosaminoglycans from Lethal and Nonlethal Breast Cancer Tissues:Toward a Novel Class of Theragnostics for POersonalized Medicine in Oncology?", 《OMICS A JOURNAL OF INTEGRATIVE BIOLOGY》, vol. 16, no. 3, 31 December 2012 (2012-12-31), pages 79 - 89 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108802248A (en) * | 2018-06-21 | 2018-11-13 | 福州大学 | A kind of method for separating and analyzing of the heparitin sulfate disaccharides containing free amine group of derivatization |
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