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.