CN111721872A - Method for identifying heparin and heparan sulfate and application - Google Patents
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Abstract
The invention provides an identification method and application of heparin and heparan sulfate, belonging to the technical field of medicine detection. According to the invention, the research shows that eight disaccharide components of 0S, NS, 6S, 2S, 2SNS, 6SNS, 2S6S and TriS are rich in heparin drugs, and can be well used for identifying heparin and heparan sulfate as a mixed standard product. Therefore, a set of quantitative detection method based on hydrophilic interaction chromatography-mass spectrometry is established for the eight components, and the method has the advantages of simplicity and convenience in operation, short time consumption, high selectivity, high sensitivity, good specificity, good repeatability and the like. Can be used for simply, conveniently and accurately identifying heparin and heparan sulfate and also provides a method and data reference for constructing a more complete quality control standard system of the glycosaminoglycan drugs, thereby having good practical application value.
Description
Technical Field
The invention belongs to the technical field of medicine detection, and particularly relates to an identification method and application of heparin and heparan sulfate.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Heparin and heparan sulfate are endogenous, complex-structure and polydisperse linear sulfated glycosaminoglycans, which are composed of repeated disaccharide units formed by connecting glucuronic acid or iduronic acid and glucosamine through 1 → 4 glycosidic bonds, variable sulfation and isomerization exist in each unit, the sugar chains are different in length, so that the units become a multicomponent mixture with uneven molecular weight and complex structure, and the multicomponent mixture is usually widely existed on extracellular matrix and cell surface in the form of the sugar chains of proteoglycan. Heparin is a special form of highly sulfated heparan sulfate with a charge density, SO4 2-The quantity of the heparin has the greatest influence on the biological functions of the heparin, and the heparin has higher sulfation degree and is mainly used for preventing and treating artery and vein thrombosis. The sulfated heparan sulfate has low sulfation degree and plays an important role in biological processes such as cell proliferation and differentiation, tumor metastasis, virus infection, inflammation and the like.
At present, because the quality standard of novel glycosaminoglycan drugs requires a determined composition and structure, strict quantitative analysis needs to be carried out on the composition components, and the inventor finds that researches for specifically and quantitatively identifying two similar glycosaminoglycan drugs, namely heparin and heparan sulfate, are rarely reported.
Disclosure of Invention
Aiming at the defects of the prior art at present, the invention provides a method for identifying heparin and heparan sulfate and application thereof, and the invention discovers that 0S, delta UA (1 → 4) GlcNAc; NS, Δ UA (1 → 4) GlcNS; 6S, Δ UA (1 → 4) GlcNAc 6S; 2S, Δ UA2S (1 → 4) GlcNAc; 2SNS, Δ UA2S (1 → 4) GlcNS; 6SNS, Δ UA (1 → 4) GlcNS 6S; 2S6S, Δ UA2S (1 → 4) GlcNAc 6S; and TriS, delta UA2S (1 → 4) GlcNS6S eight disaccharide components are rich in heparin drugs, and can be used as a mixed standard substance for well identifying heparin and heparan sulfate. Therefore, a set of quantitative detection method based on hydrophilic interaction chromatography-mass spectrometry is established for the eight components, and the method has the advantages of simplicity and convenience in operation, short time consumption, high selectivity, high sensitivity, good specificity, good repeatability and the like.
In order to achieve the technical purpose, the technical scheme of the invention is as follows:
in a first aspect of the invention, there is provided the use of a disaccharide component for the identification of heparin and heparan sulphate.
The disaccharide component comprises 0S, Δ UA (1 → 4) GlcNAc; NS, Δ UA (1 → 4) GlcNS; 6S, Δ UA (1 → 4) GlcNAc 6S; 2S, Δ UA2S (1 → 4) GlcNAc; 2SNS, Δ UA2S (1 → 4) GlcNS; 6SNS, Δ UA (1 → 4) GlcNS 6S; 2S6S, Δ UA2S (1 → 4) GlcNAc6S and TriS, Δ UA2S (1 → 4) GlcNS 6S.
The application is carried out in particular in glycosaminoglycan drugs.
In a second aspect of the invention, there is provided a method for the identification of heparin and heparan sulfate, said method comprising the quantitative determination of the disaccharide component.
The disaccharide component comprises 0S, Δ UA (1 → 4) GlcNAc; NS, Δ UA (1 → 4) GlcNS; 6S, Δ UA (1 → 4) GlcNAc 6S; 2S, Δ UA2S (1 → 4) GlcNAc; 2SNS, Δ UA2S (1 → 4) GlcNS; 6SNS, Δ UA (1 → 4) GlcNS 6S; 2S6S, Δ UA2S (1 → 4) GlcNAc6S and TriS, Δ UA2S (1 → 4) GlcNS 6S.
The quantitative determination method comprises analysis by HPLC-ESI-MS/MS.
Specifically, the identification method comprises the following steps:
adding heparinase into a sample to be detected for enzymolysis, and performing HPLC-ESI-MS/MS analysis;
wherein the heparinase comprises heparinase I, heparinase II and heparinase III.
The chromatographic conditions include a column of PC HILIC (4.6 × 250mM, 5 μm, Shiseido, Japan) and a mobile phase of A phase (10mM NH)4Ac-Water) -B phase (10mM NH)4Ac-90% acetonitrile); the flow rate of the mobile phase is 0.6-1.2mL/min (preferably 0.9mL/min), the column temperature is 25-45 deg.C (preferably 40 deg.C); the sample volume is 10 mu L; the gradient elution mode is adopted. By optimizing the test parameters, the invention can optimize the test parameters,finally, the condition parameters are obtained, so that the chromatographic separation effect of each component is better, the baseline is stable, the peak shapes are symmetrical, the retention time of each chromatographic peak is proper, and the separation degree is good.
The gradient elution procedure was as follows: 0-1min, mobile phase A40% → 40%; 1-1.1min, mobile phase A40% → 60%; 1.1-3.5min, mobile phase A60% → 60%; 3.5-3.6min, mobile phase A60% → 40%; 3.6-8min, mobile phase A40% → 40%.
The mass spectrum conditions are as follows: an ion source: electrospray (ESI); the scanning mode is as follows: multiple Reaction Monitoring (MRM); an ionization mode: negative ions; ion source voltage: 4500V; ion source temperature: 600 ℃; air curtain air: 10 psi; atomizing: 45 psi; auxiliary gas: 55 psi. By optimizing the mass spectrum condition, the peak of each pair of ion pairs with high response is ensured, and the quantitative analysis is more convenient.
The identification method further comprises quantifying the disaccharide component by an external standard method.
The identification method also comprises the step of analyzing the content of the disaccharide component in the sample to be detected, thereby quantitatively identifying the heparin and the heparan sulfate.
In a third aspect of the invention, there is provided the use of the above-mentioned authentication method in any one of:
1) controlling the quality of the medicine;
2) evaluating the quality of the medicine;
3) and (5) drug identification.
Wherein the drug is glycosaminoglycan drug, further including but not limited to heparin and heparan sulfate.
The beneficial technical effects of one or more technical schemes are as follows:
the technical scheme provides a heparin and heparan sulfate identification method, and the HPLC-ESI-MS/MS analysis method is adopted to simultaneously determine the content of 8 disaccharide components in heparin drugs.
The liquid chromatography-mass spectrometry quantitative determination method has the advantages of accuracy, reliability, high sensitivity, strong specificity, lower detection limit and quantitative limit and the like, is more effective in analyzing the content of the medicine components, and provides a method and data reference for constructing a more complete quality control standard system of the glycosaminoglycan medicines, so that the method has good value in practical application.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings according to the provided drawings without creative efforts.
FIG. 1 is a liquid chromatogram of a control of eight effective components in example 1 of the present invention; wherein 2SNS/6SNS and 2S/6S are two pairs of isomers, which cannot be separated by chromatography and mass spectrometry, and are quantified together in the invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
As mentioned above, because the quality standards of novel glycosaminoglycans require a certain composition and structure, strict quantitative analysis of the composition is required, and studies for specifically and quantitatively identifying two similar glycosaminoglycans (heparin and heparan sulfate) are rarely reported.
In view of the above, in one embodiment of the present invention, there is provided the use of a disaccharide component for the identification of heparin and heparan sulfate.
The disaccharide component comprises 0S, Δ UA (1 → 4) GlcNAc; NS, Δ UA (1 → 4) GlcNS; 6S, Δ UA (1 → 4) GlcNAc 6S; 2S, Δ UA2S (1 → 4) GlcNAc; 2SNS, Δ UA2S (1 → 4) GlcNS; 6SNS, Δ UA (1 → 4) GlcNS 6S; 2S6S, Δ UA2S (1 → 4) GlcNAc6S and TriS, Δ UA2S (1 → 4) GlcNS 6S.
The application is carried out in particular in glycosaminoglycan drugs.
In a second aspect of the invention, there is provided a method for the identification of heparin and heparan sulfate, said method comprising the quantitative determination of the disaccharide component.
The disaccharide component comprises 0S, Δ UA (1 → 4) GlcNAc; NS, Δ UA (1 → 4) GlcNS; 6S, Δ UA (1 → 4) GlcNAc 6S; 2S, Δ UA2S (1 → 4) GlcNAc; 2SNS, Δ UA2S (1 → 4) GlcNS; 6SNS, Δ UA (1 → 4) GlcNS 6S; 2S6S, Δ UA2S (1 → 4) GlcNAc6S and TriS, Δ UA2S (1 → 4) GlcNS 6S.
The eight components are rich in heparin drugs, and can be used as components to be detected to well identify heparin and heparan sulfate.
The quantitative determination method comprises the following steps:
(1) preparation of a reference solution: precisely measuring appropriate amount of mixed standard solution, adding enzyme reaction buffer solution, mixing, adding mixed heparinase I, heparinase II and heparinase III (10 μ L, 40 μ L and 5 μ L respectively), and reacting in 37 deg.C temperature-controlled oven for 24 hr. Centrifuging at 14000rpm for 10min after reaction is finished, and taking supernatant to obtain a reference substance solution.
(2) Preparation of a test solution: taking 10.0mg of test sample powder, precisely weighing, placing in a 10mL volumetric flask, adding water to dissolve and diluting to a scale, and preparing a sample stock solution with the concentration of 1.00 mg/mL. Precisely measuring 100 μ L of sample stock solution, adding 900 μ L of enzyme reaction buffer solution, mixing, adding 50 μ L of mixed heparinase I, II and III (10 μ L, 40 μ L and 5 μ L respectively), and reacting in 37 deg.C temperature control box for 24 h. Centrifuging at 14000rpm for 10min after the reaction is finished, and taking the supernatant to obtain the test solution.
(3) And (3) determination: respectively and precisely measuring a test solution and a reference solution, injecting the solutions into a high performance liquid chromatography-mass spectrometer for separation, and performing gradient elution to obtain a mobile phase A: 10mM NH4Ac-water, mobile phase B phase: 10mM NH4Ac-90% acetonitrile.
The invention selects and optimizes the chromatographic conditions: the effect of different salt solutions (ammonium acetate and ammonium formate) and the ammonium acetate and ammonium formate buffer ratios (5mM and 10mM), acetic acid and formic acid buffer ratios (0.1%, 0.2% and 0.3%) on the chromatographic separation in the mobile phase was examined. The test result shows that: adding 10mM NH into mobile phase A4The chromatographic separation effect is better when the Ac salt solution is used, the base line is stable, the peak shape is symmetrical, the separation degree is better, and the tailing phenomenon is effectively avoided, so that the mobile phase is determined as follows: phase A (10mM NH)4Ac-Water) -B phase (10mM NH)4Ac-90% acetonitrile).
In still another embodiment of the present invention, the column in the step (3) is PC HILIC (4.6X 250mm, 5 μm, ShiseiDO, Japan); the flow rate of the mobile phase is 0.9 ml/min; the column temperature was 40 ℃; the amount of the sample was 10. mu.L.
The invention tests the influence of three flow rates (0.6, 0.9, 1.2mL/min) on the detection results. The results show that: when the flow rate is 0.9mL/min, the separation effect is optimal, the retention time of each chromatographic peak is proper, the separation degree is good, the base line is stable, and the peak shapes are symmetrical, so that the flow rate is selected to be 0.9 mL/min.
Meanwhile, the invention tests the influence of four different column temperatures (such as 25 ℃, 35 ℃, 40 ℃ and 45 ℃) on the mass spectrum detection result. The result shows that when the column temperature is 40 ℃, the retention time of chromatographic peaks is appropriate, the base line is stable, the resolution of each chromatographic peak is good, and the peak shapes are symmetrical, so the column temperature is selected to be 40 ℃.
The mass spectrum conditions are optimized simultaneously, the mass spectrum conditions of 0S, NS, 6S, 2S, 2SNS, 6SNS, 2S6S and TriS are optimized by adopting a multi-reaction ion detection mode (MRM) of an API4000 type triple quadrupole mass spectrometer, the peak emergence of each pair of ions in high response is ensured, the detection results are shown in Table 1, and specific parent ions and specific daughter ions are found for quantitative analysis from all effective components.
The chromatographic and mass spectrometric parameters of the components are shown in Table 1.
TABLE 18 chromatographic and Mass Spectrometry parameters for disaccharides
In the selection of the analysis time, the invention records the chromatogram map of 30min when selecting the elution time of the chromatogram map. The result shows that no obvious chromatographic peak appears after 8min, and in order to take care of the difference of the batch samples, the characteristic peaks of all the batch samples can be detected, so 8min is selected as the analysis time.
In another embodiment of the present invention, the gradient elution mode in step (3) is: 0-1min, mobile phase A40% → 40%; 1-1.1min, mobile phase A40% → 60%; 1.1-3.5min, mobile phase A60% → 60%; 3.5-3.6min, mobile phase A60% → 40%; 3.6-8min, mobile phase A40% → 40%.
In another embodiment of the present invention, the medium mass spectrum conditions are: an ion source: electrospray (ESI); the scanning mode is as follows: multiple Reaction Monitoring (MRM); an ionization mode: negative ions; ion source voltage: 4500V; ion source temperature: at 450 ℃; air curtain air: 10 psi; atomizing: 45 psi; auxiliary gas: 55 psi.
In the invention, an HPLC-ESI-MS/MS (liquid chromatography-Electron ionization-Mass Spectrometry) analysis method is adopted, a selective ion detection (SIM) mode is tried to determine in the experimental process, the response of each component is lower and the baseline is high, the influence of the matrix is larger, and when a multi-reaction detection (MRM) method scans parent ions and daughter ions of characteristic fragments, the response intensity of an ion peak is found to be obviously higher than that of the selective ion detection (SIM) mode, and the baseline is low, so that the quantitative analysis can be realized. Therefore, the experiment selects a multi-reaction detection (MRM) scanning mode for quantifying 0S, NS, 6S, 2S, 2SNS, 6SNS, 2S6S and TriS, and the conventional liquid phase method for separating 8 components takes long time, is difficult to separate and has high detection limit. Which is not favorable for the experiment.
In another embodiment of the present invention, there is provided a use of the above-mentioned authentication method in any one of:
1) controlling the quality of the medicine;
2) evaluating the quality of the medicine;
3) and (5) drug identification.
Wherein the drug is glycosaminoglycan drug, further including but not limited to heparin and heparan sulfate.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1
A method for quantitatively measuring the liquid quality of a disaccharide mixed standard substance of heparin medicaments comprises the following steps:
the first step is as follows:
precisely measuring appropriate amount of mixed standard solution, adding enzyme reaction buffer solution, mixing, adding mixed heparinase I, heparinase II and heparinase III (10 μ L, 40 μ L and 5 μ L respectively), and reacting in 37 deg.C temperature-controlled oven for 24 hr. Centrifuging at 14000rpm for 10min after reaction is finished, and taking supernatant to obtain a reference substance solution.
The second step is that:
taking 10.0mg of test sample powder, precisely weighing, placing in a 10mL volumetric flask, adding water to dissolve and diluting to a scale, and preparing a sample stock solution with the concentration of 1.00 mg/mL. Precisely measuring 100 μ L of sample stock solution, adding 900 μ L of enzyme reaction buffer solution, mixing, adding 50 μ L of mixed heparinase I, II and III (10 μ L, 40 μ L and 5 μ L respectively), and reacting in 37 deg.C temperature control box for 24 h. Centrifuging at 14000rpm for 10min after the reaction is finished, and taking the supernatant to obtain the test solution.
The third step:
respectively and precisely measuring a test solution and a reference solution, injecting the solutions into a high performance liquid chromatography-mass spectrometer for separation, and performing gradient elution to obtain a mobile phase A: 10mM NH4 Ac-water, mobile phase B phase: 10mM NH4 Ac-90% acetonitrile, column PCHILIC (4.6X 250mM, 5 μm, Shiseido, Japan); the flow rate of the mobile phase is 0.9 ml/min; the column temperature was 40 ℃; the amount of the sample was 10. mu.L. The gradient elution mode is as follows: 0-1min, mobile phase A40% → 40%; 1-1.1min, mobile phase A40% → 60%; 1.1-3.5min, mobile phase A60% → 60%; 3.5-3.6min, mobile phase A60% → 40%; 3.6-8min, mobile phase A40% → 40%.
The mass spectrum conditions are as follows: an ion source: electrospray (ESI); the scanning mode is as follows: multiple Reaction Monitoring (MRM); an ionization mode: negative ions; ion source voltage: 4500V; ion source temperature: at 450 ℃; air curtain air: 10 psi; atomizing: 45 psi; auxiliary gas: 55 psi.
The fourth step:
the method for investigating the feasibility of the established high performance liquid chromatography-tandem mass spectrometry method comprises the steps of linear relation, quantitative limit, precision, repeatability, stability and sample adding recovery rate, wherein
Linear relationship and quantitative limit: precisely sucking a proper amount of mixed reference substance solution, using an enzyme reaction buffer solution as a diluent, preparing 9 series of mixed standard solutions (0.02, 0.04, 0.10, 0.30, 0.60, 1.00, 1.50 and 3.00nM/mL) with gradient concentration, processing according to the method, and injecting samples for analysis. The molar concentration X (nM/mL) of each component to be detected is taken as the abscissa, and the peak area (Y) of the component to be detected is taken as the ordinate, so as to obtain a regression equation and a correlation coefficient, and the result shows that the linear relation of the 8 components in the corresponding range is good. Respectively calculating the lowest limit of quantitation and the detection limit according to the signal-to-noise ratio S/N of the component to be detected being 10 and S/N being 3, and the result is shown in table 2;
TABLE 2 Linear equation, Linear Range, correlation coefficient, LOD and LOQ
Precision: taking 50 mu L of mixed reference solution (the concentration is 1.00nM/mL), processing according to the reference solution preparation method, continuously injecting samples for 6 times, and measuring the peak areas RSD of 0S, NS, 6S, 2S, 2SNS, 6SNS, 2S6S and TriS which are all less than 9.0 percent, wherein the result shows that the precision is good;
repeatability: 6 parts of enoxaparin sodium sample (batch No. E0180000) are prepared according to the preparation method of the sample, and the peak areas of the components are measured by sample injection analysis, so that the peak areas RSD of 0S, NS, 2SNS/6SNS, TriS and 2S6S are 5.6%, 7.1%, 3.3%, 5.3% and 3.5% respectively. The results show good reproducibility.
Stability: the same sample solution is taken and subjected to sample injection detection for 0 hour, 2 hours, 4 hours, 8 hours, 12 hours and 24 hours, the results of 0S, 2S6S, NS, 2SNS/6SNS, TriS and 2S/6S peak areas RSD are 7.0%, 9.5%, 4.6%, 9.9%, 6.3% and 5.9% respectively, and the results show that the sample solution has good stability in 24 hours;
and (3) sample recovery rate: precisely sucking a proper amount of mixed standard solution, using an enzyme reaction buffer solution as a diluent to prepare three quality control solutions of high, medium and low (2.00, 0.50 and 0.06nM/mL), taking 50 mu L of enoxaparin sodium sample stock solution (batch No. E0180000) diluted by 10 times, and precisely adding 20 mu L of high, medium and low quality control solutions (n is 6) respectively. Processing and detecting sample according to the method, and calculating content, recovery rate and RSD. As a result, the average sample recovery rates of 0S, NS, 2S/6S, 2SNS/6SNS, 2S6S and TriS were 88.32%, 87.70%, 90.34%, 87.90%, 89.62%, 82.33%, and RSD were 6.7%, 5.8%, 7.4%, 5.0%, 4.9% and 6.2%, respectively.
Example 2
A method for identifying heparin and heparan sulfate comprises the following steps:
respectively taking 8 batches of heparan sulfate samples and 6 batches of heparin samples, obtaining solutions according to a preparation method of a test solution, carrying out sample injection detection, calculating the content by a regression equation, and selecting an external standard method for quantification by a quantification method, wherein the measurement results of the content of disaccharide in 8 in the heparin and heparan sulfate samples are shown in table 3. The heparan sulfate samples had 0S in the larger proportion, on average about 50%, based on the total content of 8 disaccharides. The 2S6SNS in the heparin sample has a larger ratio, and the average ratio is about 70%, so that the 2S6SNS can be used as a basis for quantitatively identifying heparin and heparan sulfate.
TABLE 3 measurement results of disaccharide content in 8 of heparin and heparan sulfate samples (%)
In conclusion, the invention develops the hydrophilic interaction chromatography-mass spectrometry method with high sensitivity, strong specificity and good mass spectrometry compatibility to simultaneously determine the content of 8 disaccharides rich in heparin and heparan sulfate, can be used for simply, conveniently and accurately identifying the heparin and the heparan sulfate, and also provides a method and data reference for constructing a more complete quality control standard system of glycosaminoglycan drugs.
It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. The use of disaccharide components for the identification of heparin and heparan sulphate;
the disaccharide component comprises 0S, Δ UA (1 → 4) GlcNAc; NS, Δ UA (1 → 4) GlcNS; 6S, Δ UA (1 → 4) GlcNAc 6S; 2S, Δ UA2S (1 → 4) GlcNAc; 2SNS, Δ UA2S (1 → 4) GlcNS; 6SNS, Δ UA (1 → 4) GlcNS 6S; 2S6S, Δ UA2S (1 → 4) GlcNAc6S and TriS, Δ UA2S (1 → 4) GlcNS 6S.
2. Use according to claim 1, in particular in glycosaminoglycans.
3. A method for identifying heparin and heparan sulfate, characterized in that the identification method comprises the steps of carrying out quantitative determination on disaccharide components;
the quantitative determination method comprises the steps of analyzing by adopting HPLC-ESI-MS/MS;
the disaccharide component comprises 0S, Δ UA (1 → 4) GlcNAc; NS, Δ UA (1 → 4) GlcNS; 6S, Δ UA (1 → 4) GlcNAc 6S; 2S, Δ UA2S (1 → 4) GlcNAc; 2SNS, Δ UA2S (1 → 4) GlcNS; 6SNS, Δ UA (1 → 4) GlcNS 6S; 2S6S, Δ UA2S (1 → 4) GlcNAc6S and TriS, Δ UA2S (1 → 4) GlcNS 6S.
4. An authentication method according to claim 3, wherein the authentication method comprises:
adding heparinase into a sample to be detected for enzymolysis, and performing HPLC-ESI-MS/MS analysis;
preferably, the heparinase comprises heparinase I, heparinase II and heparinase III.
5. An identification method according to claim 3, wherein the chromatographic conditions comprise: the chromatographic column is PC HILIC; the mobile phase is as follows: phase A (10mM NH)4Ac-Water) -B phase (10mM NH)4Ac-90% acetonitrile); the flow rate of the mobile phase is 0.6-1.2mL/min (preferably 0.9mL/min), the column temperature is 25-45 deg.C (preferably 40 deg.C); the sample volume is 10 mu L; the gradient elution mode is adopted.
6. The identification method of claim 5, wherein the gradient elution procedure is as follows: 0-1min, mobile phase A40% → 40%; 1-1.1min, mobile phase A40% → 60%; 1.1-3.5min, mobile phase A60% → 60%; 3.5-3.6min, mobile phase A60% → 40%; 3.6-8min, mobile phase A40% → 40%.
7. The identification method of claim 3, wherein the mass spectrometry conditions are: an ion source: electrospray (ESI); the scanning mode is as follows: multiple Reaction Monitoring (MRM); an ionization mode: negative ions; ion source voltage: 4500V; ion source temperature: 600 ℃; air curtain air: 10 psi; atomizing: 45 psi; auxiliary gas: 55 psi.
8. The method of claim 3, further comprising quantifying the disaccharide component using an external standard method.
9. The method of claim 3, further comprising analyzing the content of disaccharide component in the sample to be tested, thereby quantitatively identifying heparin and heparan sulfate.
10. Use of the identification method of any one of claims 3 to 9 in any one of:
1) controlling the quality of the medicine;
2) evaluating the quality of the medicine;
3) drug identification;
preferably, the drug is a glycosaminoglycan drug, including heparin and heparan sulfate.
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