CN103454372B - Ion pair antiphase chromatography and mass spectrometry combined detection method for low-molecule heparin part degradation products - Google Patents

Ion pair antiphase chromatography and mass spectrometry combined detection method for low-molecule heparin part degradation products Download PDF

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CN103454372B
CN103454372B CN201310409051.5A CN201310409051A CN103454372B CN 103454372 B CN103454372 B CN 103454372B CN 201310409051 A CN201310409051 A CN 201310409051A CN 103454372 B CN103454372 B CN 103454372B
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ion
mobile phase
detection method
ion pair
chromatography
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CN103454372A (en
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迟连利
徐晓晖
李道远
孙晓君
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HEBEI CHANGSHAN BIOCHEMICAL PHARMACEUTICAL CO Ltd
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Shandong University
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Abstract

The invention relates to an ion pair antiphase chromatography and mass spectrometry combined detection method for low-molecule heparin part degradation products. All components of the low-molecule heparin part degradation products are detected in a manner of combining ion pair antiphase chromatography and high-resolution mass spectrometer; all the components in a sample are separated through the ion pair antiphase chromatography, and a precise molecule weight is obtained through the high-resolution mass spectrometer, so that sugar chain sequence information, including structures of two ends, lengths of sugar chains and substitution numbers of acetyl and sulfuric acid, of all the components is calculated, so that the structures of the low-molecule heparin part degradation products can be finely represented. The ion pair antiphase chromatography and mass spectrometry combined detection method can detect the structures of the two ends, the lengths of the sugar chains and the substitution numbers of the acetyl and the sulfuric acid of the low-molecule heparin part degradation products; the problem in the prior art that only an ultraviolet absorption atlas for degradation of low-molecule heparin parts can be obtained is solved; the ion pair antiphase chromatography and mass spectrometry combined detection method has a high practicability value in improvement of the detection level of heparin and guarantee of drug safety.

Description

A kind of ION PAIR Reverse Phase combined gas chromatography mass spectrometry detection method of LMWHs Partial digestion product
Technical field
The present invention relates to a kind of ION PAIR Reverse Phase chromatogram and high resolution mass spectrum coupling detection method of LMWHs Partial digestion product, belong to medicine, bulk drug, raw material detection technique field.
Background technology
Heparin is a kind of glycosaminoglycan anticoagulation antithrombotic reagent, LMWHs is the novel antithrombotic reagent that heparin is prepared from through physical method, chemical method or enzymic degradation, there is the features such as strong polarity, height inhomogeneity, sulfate group instability, be difficult to carry out structural characterization to it.The fingerprint map analyzing of LMWHs Partial digestion fragment is a very important link in its structural characterization, the sequence information of heparin sugar chain can be obtained by it, but LMWHs can strengthen the inhomogeneity of enzymolysis product through heparinase Partial digestion, analysis is made to become more difficult.The method of usual analysis LMWHs Partial digestion fragment has reversed-phased high performace liquid chromatographic and strong anion to exchange high performance liquid chromatography (Chuang W L, et al.Journal ofChromatography A, 2001,932:65-74.), but only can obtain UV detect collection of illustrative plates by these methods, accurate qualitative analysis cannot be carried out to various sugar chain fragment.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of ION PAIR Reverse Phase chromatogram and high resolution mass spectrum coupling detection method of LMWHs Partial digestion product are provided, can be used for analyzing the various components in LMWHs Partial digestion product, qualitative analysis is carried out to various component.
Summary of the invention
The present invention's utilize ION PAIR Reverse Phase chromatogram and high resolution mass spectrum to carry out each component that coupling detects LMWHs Partial digestion product; by each component in ION PAIR Reverse Phase chromatographic resolution sample; and obtain accurate molecular weight by high resolution mass spectrum; calculate the sugar chain sequence information of each component; comprise two-end structure, sugar chain length, acetyl group and sulfate and replace quantity, thus meticulous sign is compared to the structure of each LMWHs Partial digestion product.
Detailed Description Of The Invention
An ION PAIR Reverse Phase combined gas chromatography mass spectrometry detection method for LMWHs Partial digestion product, step is as follows:
(1) amine ion-pairing agent is dissolved in deionized water, obtained amine ion pair concentration is the mobile phase A of 10 ~ 40mM, and pH is adjusted to 5.5 ~ 8.5;
Or, amine ion-pairing agent and hexafluoroisopropanol are dissolved in deionized water, obtained amine ion pair concentration are 10 ~ 40mM, hexafluoroisopropanol concentration is the mobile phase A of 20 ~ 100mM ';
(2) amine ion-pairing agent is dissolved in percent by volume be 75% acetonitrile or percent by volume be in the methanol solution of 75%, obtained amine ion pair concentration is the Mobile phase B of 10 ~ 40mM, and pH is adjusted to 5.5 ~ 8.5;
Or, amine ion-pairing agent and hexafluoroisopropanol are dissolved in percent by volume be 75% acetonitrile or percent by volume be 75% methanol solution, obtained amine ion pair concentration is 10 ~ 40mM, hexafluoroisopropanol concentration is the Mobile phase B of 20 ~ 100mM ';
(3) LMWHs sample to be measured is dissolved in mobile phase A or mobile phase A ', be mixed with the solution to be measured that concentration is 6 ~ 10mg/mL, after filtering, use C 18reverse-phase chromatographic column; Flow velocity 8 ~ 12 μ L/min, gradient is as follows, is percent by volume:
0 ~ 5min, 95% mobile phase A, 5% Mobile phase B; 5 ~ 60min, 65 ~ 95% mobile phase A, 5 ~ 35% Mobile phase B,
Or, 0 ~ 5min, 95% mobile phase A ', 5% Mobile phase B '; 5 ~ 60min, 60 ~ 95% mobile phase A ', 5 ~ 40% Mobile phase B ';
(4) detect with high-resolution mass spectrometer under positive ion mode or negative ion mode, obtain high resolution mass spectrum figure;
(5) by the kind of UV detect chromatogram determination LMWHs, the high resolution mass spectrum figure then obtained according to step (4) obtains the mass-to-charge ratio M of main peaks, the accurate molecular weight m through following formulae discovery component:
Positive ion mode: m=zM-nX-zY
Negative ion mode: m=zM-nX+zY
Wherein: z represents charge number, n represents ion-pairing agent Molecules, and X represents the molecular weight of ion-pairing agent, and Y represents the molecular weight of proton hydrogen;
(6) computer-aid method is used to carry out spectrum unscrambling, detailed process is: by the molecular weight data storehouse of each heparin component of Practical computer teaching, compare with the theoretical molecular in the accurate molecular weight obtained in step (5) and database and obtain error amount I, by the size of error amount I, the data in database are arranged, then, error amount II according to mass spectrometer examination criteria product is compared with error amount I, choose the theoretical sample in error amount II database the most close with error amount I, the structural information of LMWHs sample to be measured can be known by the information of this theoretical sample.
Amine ion-pairing agent in described step (1) is selected from: n-propylamine, Tri-n-Propylamine, n-amylamine, n-butylamine or n-hexylamine.
Amine ion-pairing agent in described step (2) is selected from: n-propylamine, Tri-n-Propylamine, n-amylamine, n-butylamine or n-hexylamine.
High resolution mass spectrum in described step (4) is as adopted ion trap time flight tandem mass spectrometer (IT-TOF), and setup parameter is: positive ion mode spray voltage :+3.6kV; Negative ion mode spray voltage :-3.0kV; Atomising air speed: 0.5L/min or 1.5L/min; Quality of scanning scope: 50 ~ 5000; If adopt level Four bar time flight tandem mass spectrometer (Q-TOF), setup parameter is: positive ion mode spray voltage :+5.5kV; Negative ion mode spray voltage :-4.0kV; Curtain air pressure 25psi; Spray pressure: 30psi; Quality of scanning scope: 50 ~ 4000.
Beneficial effect
The present invention can detect LMWHs Partial digestion product two-end structure, sugar chain length, acetyl group and sulfate replace quantity; solve the problem of the ultraviolet absorpting spectrum that only can obtain LMWHs Partial digestion in prior art, for detection level, the guarantee drug safety improving heparin, there is great practical value.
Accompanying drawing explanation
The total ion current figure of gram match Partial digestion product in Fig. 1, embodiment 1;
The high resolution mass spectrum figure of gram match Partial digestion product in Fig. 2, embodiment 1;
The high resolution mass spectrum figure of gram match Partial digestion product in Fig. 3, embodiment 1;
The total ion current figure of gram match Partial digestion product in Fig. 4, embodiment 2;
The high resolution mass spectrum figure of gram match Partial digestion product in Fig. 5, embodiment 2;
The high resolution mass spectrum figure of gram match Partial digestion product in Fig. 6, embodiment 2.
Embodiment
Below in conjunction with embodiment, the present invention will be further described, but institute of the present invention protection domain is not limited thereto.
Liquid chromatograph is Agilent1100series capillary liquid chromatography instrument, and detecting device is diode array detector, and workstation is Agilent ChemStation; Mass spectrum is Shimadzu IT-TOF type high resolution mass spectrum, and workstation is LC-Solution.
Embodiment 1:
An ION PAIR Reverse Phase combined gas chromatography mass spectrometry detection method for LMWHs Partial digestion product, step is as follows:
N-amylamine is dissolved in deionized water by 1.1, and obtained n-amylamine concentration is the mobile phase A of 15mM, and pH is adjusted to 7.0;
N-amylamine is dissolved in the acetonitrile solution that percent by volume is 75% by 1.2, and obtained n-amylamine concentration is the Mobile phase B of 15mM, and pH is adjusted to 7.0;
Freeze-drying after gram match (purchased from Sanofi-Aventis) parenteral solution dialysis is obtained the bulk drug of Enoxaparin by 1.3, Partial digestion is carried out with Heparinase I, become a cadre after the ultrafiltration membrance filter of catabolite Millipore10KDa, be then mixed with the solution to be measured of 10mg/mL by mobile phase A;
1.4 use the C that packing material size is 5 μm, chromatographic column internal diameter is 0.5mm, chromatogram column length is 250mm 18reverse-phase chromatographic column; At flow velocity 10 μ L/min, sample size is 2.0 μ L, and gradient is: 0 ~ 5min, 95% mobile phase A, 5% Mobile phase B; 5 ~ 60min, 65 ~ 95% mobile phase A, 5 ~ 35% Mobile phase B;
1.5 use Shimadzu IT-TOF high resolution mass spectrum to detect in the positive-ion mode, and obtain high resolution mass spectrum figure, setup parameter is: spray voltage :+3.6kV; Atomising air speed: 0.5L/min; Quality of scanning scope: 50 ~ 5000.
1.6 according to the mass-to-charge ratio M of main peaks obtained in the high resolution mass spectrum figure obtained in step 1.5, the accurate molecular weight m through following formulae discovery component:
m=zM-nX-zY
Wherein: z represents charge number, n represents ion-pairing agent Molecules, and X represents the molecular weight of ion-pairing agent, and Y represents the molecular weight of proton hydrogen.
The molecular weight data storehouse of the 1.7 each components produced by Practical computer teaching Enoxaparin Partial digestion, carries out comparison one by one with the theoretical molecular in the accurate molecular weight obtained in step 1.6 and database, and obtain error amount I, its computing formula is as follows:
Error amount I=(actual measurement molecular weight-theoretical molecular)/theoretical molecular
By the size of error amount I, the data in database are arranged, then, error amount II according to mass spectrometer examination criteria product is compared with error amount I, choose the theoretical sample in error amount II database the most close with error amount I, the structural information of LMWHs sample to be measured can be known by the information of this theoretical sample.In total ion current figure (TIC) to mark the structural information of each component of Enoxaparin sodium sample as shown in table 1.
Table 1
Numbering Oligosaccharides size Sulfate substituent number
1 dp2 *1NS
2 dp2 1NS
3 dp2 *1OS,1NS
4 dp2 1OS,1NS
5 dp2 2OS,1NS
6 dp3 2OS,1NS
7 dp3 3OS,1NS
8 dp4 *2NS
9 dp4 2NS
10 dp4 1OS,1NS
11 dp4 *1OS,2NS
12 dp4 1OS,2NS
13 dp4 *2OS,1NS
14 dp4 2OS,1NS
15 dp4 *2OS,2NS
16 dp4 2OS,2NS
17 dp4 3OS,1NS
18 dp4 4OS
19 dp4 *3OS,2NS
20 dp4 3OS,2NS
21 dp4 4OS,1NS
22 dp4 5OS
23 dp4 *4OS,2NS
24 dp4 4OS,2NS
25 dp5 3OS,1NS
26 dp5 3OS,2NS
27 dp6 *3OS,2NS
28 dp5 4OS,1NS
29 dp5 4OS,2NS
30 dp5 5OS,1NS
31 dp5 5OS,2NS
32 dp6 2OS,1NS
33 dp6 1OS,2NS
34 dp6 2OS,2NS
35 dp6 2OS,3NS
36 dp6 3OS,1NS
37 dp6 *3OS,2NS
38 dp6 3OS,2NS
39 dp6 *3OS,3NS
40 dp6 3OS,3NS
41 dp6 *4OS,2NS
42 dp6 4OS,2NS
43 dp6 *4OS,3NS
44 dp6 4OS,3NS
45 dp6 *5OS,2NS
46 dp6 5OS,2NS
47 dp6 *5OS,3NS
48 dp6 5OS,3NS
49 dp6 6OS,2NS
50 dp7 2OS
51 dp7 5OS,2NS
52 dp7 6OS,2NS
53 dp8 1OS,4NS
54 dp8 3OS,3NS
55 dp8 4OS,2NS
56 dp8 4OS,3NS
57 dp8 *5OS,3NS
58 dp8 5OS,3NS
59 dp8 *5OS,4NS
60 dp8 5OS,4NS
61 dp8 7OS,3NS
62 dp8 7OS,4NS
63 dp8 8OS,4NS
64 dp9 2OS,3NS
65 dp10 5OS,3NS
Note: " * " represents that sugar-chain end contains interior ether structure, " OS " expression "-O-SO 3h ", " NS " expression "-NH-SO 3h ", as " *6OS, 2NS " then represent that this component reduction end is containing 1, ether structure in 6, has 6 "-O-SO 3h " structure, 2 "-NH-SO 3h " structure.
The mass spectrophotometry total ion current figure of bulk drug Partial digestion product of gram match is as Fig. 1, and the high resolution mass spectrum illustrated example of component 24 is as Fig. 2, and the high resolution mass spectrum illustrated example of component 48 is as Fig. 3.65 kinds of key components from disaccharides to ten sugar in gram bulk drug Partial digestion product of match are successfully detected by this method, and to its sugar chain length, end structure and "-O-SO 3h " and "-NH-SO 3h " number determines.
Embodiment 2:
An ION PAIR Reverse Phase combined gas chromatography mass spectrometry detection method for LMWHs Partial digestion product, step is as follows:
N-amylamine and hexafluoroisopropanol are dissolved in deionized water by 2.1, and obtained n-amylamine concentration is 15mM, hexafluoroisopropanol concentration is the mobile phase A of 50mM ';
N-amylamine and hexafluoroisopropanol are dissolved in the acetonitrile solution that percent by volume is 75% by 2.2, and obtained n-amylamine concentration is 15mM, hexafluoroisopropanol concentration is the Mobile phase B of 50mM ';
Freeze-drying after gram match (purchased from Sanofi-Aventis) parenteral solution dialysis is obtained the bulk drug of Enoxaparin by 2.3, Partial digestion is carried out with Heparinase I, become a cadre after the ultrafiltration membrance filter of catabolite Millipore 10KDa, then use mobile phase A ' be mixed with the solution to be measured of 10mg/mL;
2.4 use the C that packing material size is 5 μm, chromatographic column internal diameter is 0.5mm, chromatogram column length is 250mm 18reverse-phase chromatographic column; At flow velocity 10 μ L/min, sample size is 2.0 μ L, and gradient is: 0 ~ 5min, 95% mobile phase A ', 5% Mobile phase B '; 5 ~ 60min, 60 ~ 95% mobile phase A ', 5 ~ 40% Mobile phase B ';
2.5 use Shimadzu IT-TOF high resolution mass spectrum to detect in the positive-ion mode, and obtain high resolution mass spectrum figure, setup parameter is: spray voltage :+3.6kV; Atomising air speed: 1.5L/min; Quality of scanning scope: 50 ~ 5000.
2.6 according to the mass-to-charge ratio M of main peaks obtained in the high resolution mass spectrum figure obtained in step 2.5, the accurate molecular weight m through following formulae discovery component:
m=zM-nX-zY
Wherein: z represents charge number, n represents ion-pairing agent Molecules, and X represents the molecular weight of ion-pairing agent, and Y represents the molecular weight of proton hydrogen.
The molecular weight data storehouse of the 2.7 each components produced by Practical computer teaching Enoxaparin Partial digestion, carries out comparison one by one with the theoretical molecular in the accurate molecular weight obtained in step 2.6 and database, and obtain error amount I, its computing formula is as follows:
Error amount I=(actual measurement molecular weight-theoretical molecular)/theoretical molecular
By the size of error amount I, the data in database are arranged, then, error amount II according to mass spectrometer examination criteria product is compared with error amount I, choose the theoretical sample in error amount II database the most close with error amount I, the structural information of LMWHs sample to be measured can be known by the information of this theoretical sample.In total ion current figure (TIC) to mark the structural information of each component of Enoxaparin sodium sample as shown in table 2.
Table 2
Numbering Oligosaccharides size Sulfate substituent number
1 dp2 *1NS
2 dp2 1NS
3 dp2 *1OS,1NS
4 dp2 1OS,1NS
5 dp2 2OS,1NS
6 dp3 2OS,1NS
7 dp3 3OS,1NS
8 dp4 *2NS
9 dp4 2NS
10 dp4 1OS,1NS
11 dp4 *1OS,2NS
12 dp4 1OS,2NS
13 dp4 *2OS,1NS
14 dp4 2OS,1NS
15 dp4 *2OS,2NS
16 dp4 2OS,2NS
17 dp4 3OS,1NS
18 dp4 4OS
19 dp4 *3OS,2NS
20 dp4 3OS,2NS
21 dp4 4OS,1NS
22 dp4 5OS
23 dp4 *4OS,2NS
24 dp4 4OS,2NS
25 dp5 3OS,1NS
26 dp5 3OS,2NS
27 dp6 *3OS,2NS
28 dp5 4OS,1NS
29 dp5 4OS,2NS
30 dp5 5OS,1NS
31 dp5 5OS,2NS
32 dp6 2OS,1NS
33 dp6 1OS,2NS
34 dp6 2OS,2NS
35 dp6 2OS,3NS
36 dp6 3OS,1NS
37 dp6 *3OS,2NS
38 dp6 3OS,2NS
39 dp6 *3OS,3NS
40 dp6 3OS,3NS
41 dp6 *4OS,2NS
42 dp6 4OS,2NS
43 dp6 *4OS,3NS
44 dp6 4OS,3NS
45 dp6 *5OS,2NS
46 dp6 5OS,2NS
47 dp6 *5OS,3NS
48 dp6 5OS,3NS
49 dp6 6OS,2NS
50 dp7 2OS
51 dp7 5OS,2NS
52 dp7 6OS,2NS
53 dp8 1OS,4NS
54 dp8 3OS,3NS
55 dp8 4OS,2NS
56 dp8 4OS,3NS
57 dp8 *5OS,3NS
58 dp8 5OS,3NS
59 dp8 *5OS,4NS
60 dp8 5OS,4NS
61 dp8 7OS,3NS
62 dp8 7OS,4NS
63 dp8 8OS,4NS
64 dp9 2OS,3NS
65 dp10 5OS,3NS
Note: " * " represents that sugar-chain end contains interior ether structure, " OS " expression "-O-SO 3h ", " NS " expression "-NH-SO 3h ".
The total ion current figure of mass spectrophotometry of the bulk drug Partial digestion product of gram match is as Fig. 4, and the high resolution mass spectrum illustrated example of component 24 is as Fig. 5, and the high resolution mass spectrum illustrated example of component 48 is as Fig. 6.65 kinds of key components from disaccharides to ten sugar in gram bulk drug Partial digestion product of match are successfully detected by this method, and to its sugar chain length, end structure and "-O-SO 3h " and "-NH-SO 3h " number determines.

Claims (2)

1. an ION PAIR Reverse Phase combined gas chromatography mass spectrometry detection method for LMWHs Partial digestion product, step is as follows:
(1) amine ion-pairing agent and hexafluoroisopropanol are dissolved in deionized water, obtained amine ion pair concentration is 10 ~ 40mM, hexafluoroisopropanol concentration is the mobile phase A of 20 ~ 100mM ';
(2) amine ion-pairing agent and hexafluoroisopropanol are dissolved in percent by volume be 75% acetonitrile or percent by volume be 75% methanol solution, obtained amine ion pair concentration is 10 ~ 40mM, hexafluoroisopropanol concentration is the Mobile phase B of 20 ~ 100mM ';
(3) LMWHs sample to be measured is dissolved in mobile phase A ', be mixed with the solution to be measured that concentration is 6 ~ 10 mg/mL, after filtering, use C 18reverse-phase chromatographic column; Flow velocity 8 ~ 12 μ L/min, gradient is as follows, is percent by volume:
0 ~ 5 min, 95% mobile phase A ', 5% Mobile phase B '; 5 ~ 60 min, 60 ~ 95% mobile phase A ', 5 ~ 40% Mobile phase B ';
(4) detect with high-resolution mass spectrometer under positive ion mode or negative ion mode, obtain high resolution mass spectrum figure;
(5) by the kind of UV detect chromatogram determination LMWHs, the high resolution mass spectrum figure then obtained according to step (4) obtains the mass-to-charge ratio M of main peaks, the accurate molecular weight m through following formulae discovery component:
Positive ion mode: m=zM-nX-zY
Negative ion mode: m=zM-nX+zY
Wherein: z represents charge number, n represents ion-pairing agent Molecules, and X represents the molecular weight of ion-pairing agent, and Y represents the molecular weight of proton hydrogen;
(6) computer-aid method is used to carry out spectrum unscrambling, detailed process is: by the molecular weight data storehouse of each heparin component of Practical computer teaching, compare with the theoretical molecular in the accurate molecular weight obtained in step (5) and database and obtain error amount I, by the size of error amount I, the data in database are arranged, then, error amount II according to mass spectrometer examination criteria product is compared with error amount I, choose the theoretical sample in error amount II database the most close with error amount I, the structural information of LMWHs sample to be measured can be known by the information of this theoretical sample.
2. detection method as claimed in claim 1, it is characterized in that, the amine ion-pairing agent in described step (1) is selected from: n-propylamine, Tri-n-Propylamine, n-amylamine, n-butylamine or n-hexylamine.
3 .detection method as claimed in claim 1, is characterized in that, the amine ion-pairing agent in described step (2) is selected from: n-propylamine, Tri-n-Propylamine, n-amylamine, n-butylamine or n-hexylamine.
4 .detection method as claimed in claim 1, is characterized in that, the high resolution mass spectrum in described step (4) adopts ion trap time flight tandem mass spectrometer (IT-TOF), and setup parameter is: positive ion mode spray voltage :+3.6 kV; Negative ion mode spray voltage :-3.0 kV; Atomising air speed: 0.5L/min or 1.5L/min; Quality of scanning scope: 50 ~ 5000; If adopt level Four bar time flight tandem mass spectrometer (Q-TOF), setup parameter is: positive ion mode spray voltage :+5.5 kV; Negative ion mode spray voltage :-4.0 kV; Curtain air pressure 25 psi; Spray pressure: 30 psi; Quality of scanning scope: 50 ~ 4000.
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CN103630647B (en) * 2013-12-20 2015-06-03 山东大学 Reverse-phase chromatography and mass-spectrometry combined detection method for complete low-molecular-heparin degradation product through precolumn derivatization
CN103852542B (en) * 2014-03-31 2017-01-11 苏州英诺凯生物医药科技有限公司 Method for detecting complete degradation product of low molecular weight heparin based on post-column derivatization
CN104914205B (en) * 2015-06-23 2017-01-11 福州大学 Segregation analysis method for heparan disaccharide sulfate containing FlcNH3+
CN105548415A (en) * 2016-01-08 2016-05-04 东营天东制药有限公司 Analysis method for identifying oligosaccharide in low-molecular-weight heparin through high-performance liquid
CN107991414A (en) * 2017-12-28 2018-05-04 山东大学 A kind of electrophoresis hydrophilic interaction combined gas chromatography mass spectrometry detection method of Sulodexide
CN108318602A (en) * 2018-05-03 2018-07-24 东营天东制药有限公司 A kind of method of two sugared content of heparin in quick detection heparin and/or low molecular weight heparin
CN113393904B (en) * 2021-06-22 2022-10-18 山东大学 Method and system for detecting low-molecular heparin sugar chain sequence and sequencing kit

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US8435799B2 (en) * 2008-09-03 2013-05-07 Momenta Pharmaceuticals, Inc. Evaluating heparin preparations for pharmaceutical use
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CN102323355B (en) * 2011-08-22 2013-10-16 深圳市天道医药有限公司 Enzymolysis-HPLC method for detecting enoxaparin
CN102759596B (en) * 2012-07-09 2014-08-20 山东大学 Method for detecting low-molecular-weight heparin by combining ion pair reversed phase chronmatogaphy and mass spectrum
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