A two-dimensional liquid chromatography-mass spectrometry method for the quality analysis of low molecular weight heparin

Abstract

The present invention discloses a two-dimensional liquid chromatography-mass spectrometry (LC-MS) method for the quality analysis of low molecular weight heparin, which comprises the following steps: 1) Using low molecular weight heparin as a raw material, various low molecular weight heparin oligosaccharides with different degrees of polymerization are prepared by medium pressure column chromatography fractionation; 2) Qualitative analysis of low molecular weight heparin oligosaccharides is carried out by two-dimensional liquid chromatography online connected to mass spectrometry, that is, strong anion exchange chromatography is used in the first-dimensional liquid chromatography to separate low molecular weight heparin oligosaccharides with a single degree of polymerization, each SAX-separated oligosaccharide peak is cut into the second-dimensional molecular sieve chromatography, and finally, online mass spectrometry analysis is carried out; 3) Comparing the SAX standard spectra of different samples with the same degree of polymerization, and semi-quantitatively analyzing each oligosaccharide with a certain degree of polymerization through the peak area. This method can perform rapid and precise structural analysis and quality control of low molecular weight heparin, and has great application potential.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analysis and relates to a two-dimensional liquid chromatography-mass spectrometry method for the quality analysis of low molecular weight heparin. Background Art

[0002] Heparin is not only a commonly used anticoagulant drug in clinical practice, but also has the effect of preventing and treating thrombotic diseases. In the domestic market, the market scale of heparin drugs has been increasing year by year, and by 2018, the market scale has exceeded 8 billion yuan. In recent years, low molecular weight heparin has gradually replaced ordinary heparin as the main force of heparin drugs. Among them, the market sales volume of enoxaparin sodium has been increasing rapidly year by year. By 2018, its domestic market scale has reached 2.05 billion yuan, with an annual growth rate of 35% compared with the sales volume of 1.52 billion yuan in 2017. In 2019, global oral anticoagulant drugs accounted for more than 80% of the antithrombotic market, thus threatening the position of the traditional heparin market. However, clinically, heparin is still an indispensable drug and occupies a special market position. Heparin is a linear glycosaminoglycan composed of uronic acid and glucosamine to form the smallest disaccharide repeating unit, and its anticoagulant activity is specifically related to the binding of the 3-O-sulfated pentasaccharide fragment GlcNAc / NS6S(1-4)GlcA(1-4)GlcNS3S,6S(1-4)IdoA2S(1-4)GlcNS6S to antithrombin. Different molecular weights, disaccharide compositions, degrees of sulfation, and sites result in the heterogeneity of the heparin structure. Enoxaparin sodium is derived from ordinary heparin as the starting material through benzyl ester β-elimination and chemical cleavage, and has the advantages of fewer bleeding side effects and a wider range of applications, but its structure is more complex, and there are difficulties in the structural consistency evaluation of generic drugs, and there is still a lack of effective analysis methods. At the same time, the immunogenicity problem of heparin drugs has been increasingly emphasized, and a more refined structural research method is urgently needed.

[0003] Therefore, it is necessary to develop a two-dimensional liquid chromatography-mass spectrometry method for the quality analysis of low molecular weight heparin. Summary of the Invention

[0004] The object of the present invention is to provide a two-dimensional liquid chromatography-mass spectrometry method for the quality analysis of low molecular weight heparin to solve the above problems.

[0005] The technical solution of the present invention is as follows:

[0006] A two-dimensional liquid chromatography-mass spectrometry method for the quality analysis of low molecular weight heparin, the method comprising:

[0007] (1) Using low molecular weight heparin as a raw material, preparing and fractionating through medium-pressure column chromatography to obtain low molecular weight heparin oligosaccharides with different degrees of polymerization;

[0008] (2) Qualitatively analyze the low-molecular-weight heparin oligosaccharides with various different degrees of polymerization by two-dimensional liquid chromatography-mass spectrometry. Separate them by SAX in the first dimension, cut each SAX-separated oligosaccharide peak into SEC in the second dimension, and finally perform tandem MS analysis to obtain the SAX standard spectra of oligosaccharides with different degrees of polymerization;

[0009] (3) Use another two-dimensional liquid chromatography. Separate the low-molecular-weight heparin to be analyzed by SEC in the first dimension, on-line cut each oligosaccharide with a specific degree of polymerization and introduce it into SAX-UV in the second dimension for analysis, and analyze by comparing with the SAX standard spectra corresponding to each oligosaccharide with a specific degree of polymerization.

[0010] Further, in step (1), the degree of polymerization of the low-molecular-weight heparin oligosaccharides is 2n, where n is any natural number between 2 and 20.

[0011] Further, in step (1), the preparation method of the low-molecular-weight heparin is: use heparin as the raw material to obtain low-molecular-weight heparin through benzyl ester β-elimination and chemical cleavage.

[0012] Further, the low-molecular-weight heparin is enoxaparin sodium or bemiparin sodium.

[0013] Further, in step (1), the preparation by medium-pressure column chromatography means fractionating enoxaparin sodium using a molecular exclusion packed column.

[0014] Further, in step (3), the chromatographic conditions of SAX are the same as those of SAX corresponding to the degree of polymerization in step (2).

[0015] Further, the chromatographic conditions of SAX are as follows: Thermo Scientific ProPac PA1 chromatographic column, injection volume 1 - 20 μL, column temperature 20 - 45 °C, flow rate 0.2 - 1.5 mL / min, mobile phase A is an aqueous solution of 25 mM Na2HPO4, mobile phase B is an aqueous solution of 1.8 M NaCl and 25 mM Na2HPO4, mobile phase gradient program: 0 - 2 min, 10% B phase; 2 - 92 min, 10% → 100% B phase; 92 - 97 min, 100% B phase; 97 - 105 min, 100% → 10% B phase, detection wavelength 232 nm.

[0016] Further, in step (3), the chromatographic conditions of SEC are the same as those of SEC corresponding to the degree of polymerization in step (2).

[0017] Further, the chromatographic conditions of the SEC are as follows: an SEC-UPLC chromatographic column, an injection volume of 1-20 μL, a column temperature of 20-45 °C, a flow rate of 0.1-0.3 mL / min, a mobile phase of a 20% methanol aqueous solution of 50 mM NH4OAc, isocratic elution, and a detection wavelength of 232 nm.

[0018] The present invention provides a two-dimensional liquid chromatography-mass spectrometry coupling method for the quality analysis of low molecular weight heparin, which uses a multi-center cutting two-dimensional liquid chromatography-mass spectrometry coupling system to perform on-line qualitative and quantitative analysis of low molecular weight heparin, and has the following advantages:

[0019] 1) This analysis method has excellent separation ability for low molecular weight heparin and can identify the fine structures of oligosaccharides in low molecular weight heparin with complex structures;

[0020] 2) This analysis method can perform rapid semi-quantitative analysis on each structure of the oligosaccharides in low molecular weight heparin;

[0021] 3) This analysis method uses an on-line two-dimensional method and can perform continuous injection analysis on low molecular weight heparin to meet the rapid analysis requirements of a large number of samples. Description of the Drawings

[0022] Figure 1 is a schematic diagram of the structure of low molecular weight heparin in the present invention, wherein n is any natural number between 2 and 40;

[0023] Figure 2 is the column chromatography chromatogram of low molecular weight heparin in the present invention;

[0024] Figure 3 is a schematic diagram of the principle of the SAX-SEC-MS qualitative experiment in the present invention;

[0025] Figure 4 is a schematic diagram of the SAX central cutting site of enoxaparin sodium oligosaccharide in the SAX-SEC-MS qualitative experiment part of the present invention;

[0026] Figure 5 is the SAX standard chromatogram of enoxaparin sodium tetrasaccharide;

[0027] Figure 6 is the SAX standard chromatogram of enoxaparin sodium hexasaccharide;

[0028] Figure 7 is the SAX standard chromatogram of enoxaparin sodium octasaccharide;

[0029] Figure 8 is the SAX standard chromatogram of enoxaparin sodium decasaccharide;

[0030] Figure 9It is a schematic diagram of the principle of SEC-SAX-UV quantitative experiment in the present invention;

[0031] Figure 10 It is a schematic diagram of the SEC central cleavage site of enoxaparin sodium oligosaccharides in the SEC-SAX-UV quantitative experiment part of the present invention;

[0032] Figure 11 They are the SAX chromatograms of the tetrasaccharide, hexasaccharide, octasaccharide and decasaccharide of enoxaparin sodium sample;

[0033] Figure 12 It is the SAX standard chromatogram of bemiparin sodium tetrasaccharide;

[0034] Figure 13 It is the SAX standard chromatogram of bemiparin sodium hexasaccharide;

[0035] Figure 14 It is the SAX standard chromatogram of bemiparin sodium octasaccharide;

[0036] Figure 15 It is the SAX standard chromatogram of bemiparin sodium decasaccharide;

[0037] Figure 16 They are the SAX chromatograms of the tetrasaccharide, hexasaccharide, octasaccharide and decasaccharide of bemiparin sodium sample; Specific embodiments

[0038] The object of the present invention is to provide a two-dimensional liquid chromatography-mass spectrometry coupling method for the quality analysis of low molecular weight heparin, which specifically includes the following steps:

[0039] (1) Using low molecular weight heparin as raw material, various low molecular weight heparin oligosaccharides with different degrees of polymerization are prepared by medium pressure column chromatography fractionation, specifically oligosaccharides with a degree of polymerization of 2n, where n is any natural number between 2 and 20.

[0040] In this step, for low molecular weight heparin obtained by benzyl ester β-elimination and chemical cleavage from heparin sodium, such as enoxaparin sodium and bemiparin sodium, please refer to Figure 1 , Figure 1 It is a schematic diagram of the structure of low molecular weight heparin in the present invention. Its structure is as Figure 1 shown.

[0041] In this step, it is preferred to use a size exclusion packed column, and more preferably the packing is Gel.

[0042] (2) Qualitative analysis of low molecular weight heparin oligosaccharides using two-dimensional liquid chromatography on-line coupled with mass spectrometry, that is, strong anion exchange chromatography (SAX) is used in the first dimension of liquid chromatography to separate low molecular weight heparin oligosaccharides with a single degree of polymerization. Each SAX-separated oligosaccharide peak is cut into the second dimension of molecular sieve chromatography (SEC). Finally, on-line mass spectrometry (MS) analysis, (SAX-SEC-MS) analysis obtains the SAX standard spectra of oligosaccharides with different degrees of polymerization. In this way, the structural composition of each oligosaccharide chain corresponding to each peak in the SAX chromatogram of each single-degree-of-polymerization oligosaccharide is calibrated, forming the SAX standard spectrum of each degree-of-polymerization oligosaccharide;

[0043] In this step, the SAX column used is Thermo Scientific ProPac PA1 column; the injection volume is 1 - 20 μL; the column temperature is 20 - 45 °C, preferably 40 °C; the flow rate is 0.2 - 1.5 mL / min, preferably 1 mL / min; the mobile phase A is an aqueous solution of 25 mM Na2HPO4, and the mobile phase B is an aqueous solution of 1.8 M NaCl and 25 mM Na2HPO4. The mobile phase gradient program is: 0 - 2 min, 10% B phase; 2 - 92 min, 10% → 100% B phase; 92 - 97 min, 100% B phase; 97 - 105 min, 100% → 10% B phase, and the detection wavelength is 232 nm.

[0044] In this step, the SEC column used is SEC-UPLC column, and the pore size is between and, preferably 125 and are connected in series; the injection volume is 1 - 20 μL; the column temperature is 20 - 45 °C, preferably 25 °C; the flow rate is 0.1 - 0.3 mL / min, preferably 0.15 mL / min; the mobile phase is a 20% methanol aqueous solution of 50 mM NH4OAc; isocratic elution; the detection wavelength is 232 nm.

[0045] (3) Exchange the chromatographic order of the first and second dimensions, that is, use the SEC column in the first dimension to separate low molecular weight heparin. Each degree-of-polymerization oligosaccharide is cut on-line and enters the second dimension. The second dimension uses SAX chromatography and the standard SAX chromatographic conditions corresponding to each degree-of-polymerization oligosaccharide to analyze oligosaccharides with different degrees of polymerization on-line (SEC-SAX-UV), where the SAX chromatographic conditions are the same as those of the corresponding degree-of-polymerization SAX chromatographic conditions in step (2), and analyze by comparing the SAX standard spectra corresponding to each degree-of-polymerization oligosaccharide.

[0046] In this step, both SEC and SAX are the same as those in step (2).

[0047] To make the above objects, features, and advantages of the present invention more apparent and understandable, the technical solution of the present invention will be further described below in conjunction with specific embodiments. However, the present invention is not limited to the listed embodiments and should also include any other well-known changes within the scope of the rights required by the present invention.

[0048] As used herein, the term "one embodiment" or "embodiment" refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The appearances of "in one embodiment" in different places in this specification do not all refer to the same embodiment, nor are they separate or alternative embodiments that are mutually exclusive of other embodiments.

[0049] Example 1

[0050] 1. Preparation of enoxaparin sodium oligosaccharides by column chromatography

[0051] Using a protein purifier, a molecular exclusion packed column ( Gel) was used for column chromatography. Enoxaparin sodium was loaded at a concentration of 500 mg / ml, dissolved in a mobile phase of 0.5 mol / L NaCl solution, and the sample was isocratically eluted and separated at a flow rate of 5 ml / min. A UV detector was used with a detection wavelength of 232 nm.

[0052] Please refer to Figure 2 , Figure 2 which is the column chromatography chromatogram of low molecular weight heparin in the present invention. As Figure 2 shown, the oligosaccharides were collected by online detection above the half-peak width. The collected oligosaccharides had a single degree of polymerization. After rotary evaporation and concentration, gel column chromatography was used for desalting, and then freeze-drying. 35.6 mg of enoxaparin sodium tetrasaccharide, 45.1 mg of hexasaccharide, 36.9 mg of octasaccharide, 31.9 mg of decasaccharide, and 28.3 mg of dodecasaccharide with a single degree of polymerization were prepared.

[0053] 2. Qualitative experiment of enoxaparin sodium oligosaccharides by SAX-SEC-MS

[0054] Samples of enoxaparin sodium oligosaccharides with a single degree of polymerization, specifically enoxaparin sodium tetrasaccharide, hexasaccharide, octasaccharide, and decasaccharide, were accurately weighed at 10 - 15 mg each, and dissolved in the corresponding volume of water to prepare a 20 mg / mL solution. The conditions for the first - dimension SAX chromatography were as follows: injection volume 10 μL; flow rate 1 mL / min; column temperature 40°C; mobile phase A: Weigh 17.907 g of disodium hydrogen phosphate dodecahydrate, dissolve it in 2000 mL of pure water, filter through a 0.22 μm filter membrane, and degas for later use; mobile phase B: Weigh 105.192 g of sodium chloride and dissolve it in 1000 mL of mobile phase A, filter through a 0.22 μm filter membrane, and degas for later use; the mobile - phase gradient program was: 0 - 2 min, 10% B phase; 2 - 92 min, 10% → 100% B phase; 92 - 97 min, 100% B phase; 97 - 105 min, 100% → 10% B phase; detection wavelength 232 nm. Please refer to Figure 3 , Figure 3 which is the schematic diagram of the principle of the SAX - SEC - MS qualitative experiment in the present invention. As shown in Figure 3 it, each chromatographic peak obtained from the first - dimension SAX was cut into the second - dimension SEC - MS for analysis to obtain the SAX standard chromatograms of oligosaccharides with different degrees of polymerization. Please refer to Figure 4 , Figure 4 which is the schematic diagram of the SAX central cutting sites of enoxaparin sodium oligosaccharides in the SAX - SEC - MS qualitative experiment part of the present invention. As shown in Figure 4 it, the central cutting sites of SAX were selected at the peak tips for cutting to ensure that the results were the main components of the target peaks. The conditions for the second - dimension SEC chromatography were as follows: mobile phase: Accurately weigh 3.854 g of ammonium acetate, dissolve it in 800 mL of pure water, mix well, filter through a 0.22 μm filter membrane, then add 200 mL of methanol, and degas by ultrasonic treatment for later use; flow rate 0.15 mL / min; isocratic elution; detection wavelength 232 nm. The conditions for mass spectrometry were: negative - ion mode, fragment voltage 120 V, acquisition range 100 - 2000 m / z.

[0055] Please refer to Figures 5 - 8 , Figure 5 which is the SAX standard chromatogram of enoxaparin sodium tetrasaccharide; Figure 6 which is the SAX standard chromatogram of enoxaparin sodium hexasaccharide; Figure 7 which is the SAX standard chromatogram of enoxaparin sodium octasaccharide; Figure 8 which is the SAX standard chromatogram of enoxaparin sodium decasaccharide. As shown in Figures 5 - 8As shown in the figure, the tetrasaccharide, hexasaccharide, octasaccharide and decasaccharide of enoxaparin sodium are well separated on SAX, among which the tetrasaccharide, hexasaccharide, octasaccharide and decasaccharide of enoxaparin sodium correspond to 23, 39, 46 and 25 chromatographic peaks, respectively, and the standard spectrum obtained by SEC-MS qualitative analysis is obtained. Please refer to Table 1, which is the mass spectrum attribution table corresponding to the separation of the decasaccharide of enoxaparin sodium on SAX. Table 1 describes in detail the fine structure of each sugar chain of enoxaparin sodium oligosaccharide, providing accurate qualitative data for the subsequent analysis of multiple batches of enoxaparin sodium samples.

[0056]

[0057]

[0058]

[0059]

[0060] Table 1

[0061] 3. SEC-SAX-UV quantitative experiment of enoxaparin sodium to be tested

[0062] Weigh 10-15 mg of each of the enoxaparin sodium samples 1 to 6 to be analyzed, and add the corresponding volume of water to prepare a 40 mg / mL solution. Figure 9 , Figure 9 Schematic diagram of the SEC-SAX-UV quantitative experimental principle in the present invention. Figure 9 As shown in the figure, the principle of this part of the experiment is to exchange the chromatographic conditions of the first and second dimensions in the qualitative part, and use SEC chromatography in the first dimension: injection volume 20μL; flow rate 0.15mL / min; column temperature 25℃; mobile phase, accurately weigh 3.854g ammonium acetate, dissolve in 800mL pure water, mix evenly, filter with 0.22μm filter membrane, add 200mL methanol, ultrasonically degas, and set aside; flow rate 0.15mL / min; isocratic elution; detection wavelength 232nm. The tetrasaccharide, hexasaccharide, octasaccharide and decasaccharide peaks obtained on the SEC chromatogram were cut into the second dimension SAX-UV for analysis. Please refer to Figure 10 , Figure 10 Schematic diagram of the SEC central cleavage site of the enoxaparin sodium oligosaccharide in the SEC-SAX-UV quantitative experiment of the present invention. Figure 10As shown, the cleavage site of the first-dimensional SEC can cut the main components of a single degree of polymerization into the second-dimensional SAX for analysis. The second-dimensional SAX-UV chromatographic conditions are as follows: injection volume 10 μL; flow rate 1 mL / min; mobile phase A, weigh 17.907 g of disodium hydrogen phosphate dodecahydrate and dissolve it in 2000 mL of pure water, filter through a 0.22 μm filter membrane, and degas for use; mobile phase B, weigh 105.192 g of sodium chloride and dissolve it in 1000 mL of mobile phase A, filter through a 0.22 μm filter membrane, and degas for use; the mobile phase gradient program is: 0 - 2 min, 10% B phase; 2 - 92 min, 10% → 100% B phase; 92 - 97 min, 100% B phase; 97 - 105 min, 100% → 10% B phase; detection wavelength 232 nm.

[0063] Please refer to Figure 11 , Figure 11 are the SAX chromatograms of the tetrasaccharide, hexasaccharide, octasaccharide, and decasaccharide of the enoxaparin sodium sample. As Figure 11 shown, for the oligosaccharide SAX-UV results of the enoxaparin sodium sample, after comparison with the qualitative standard chromatogram, qualitative and quantitative analysis was performed on 20 chromatographic peaks of the tetrasaccharide. Quantitative analysis was performed on 28 chromatographic peaks of the hexasaccharide, and 27 of them could be qualitatively analyzed. Quantitative analysis was performed on 25 chromatographic peaks of the octasaccharide, and 20 of them could be qualitatively analyzed. Quantitative analysis was performed on 40 chromatographic peaks of the decasaccharide, and 16 of them could be qualitatively analyzed. Please refer to Tables 2 - 5. Table 2 shows the percentage content (%) of the tetrasaccharide in six enoxaparin sodium samples, Table 3 shows the percentage content (%) of the hexasaccharide in six enoxaparin sodium samples, Table 4 shows the percentage content (%) of the octasaccharide in six enoxaparin sodium samples, and Table 5 shows the percentage content (%) of the decasaccharide in six enoxaparin sodium samples. As shown by the percentage content data of each oligosaccharide in Tables 2 - 5, the structural compositions and contents of the six enoxaparin sodium samples are consistent. This method can effectively and intuitively display the structural distribution and corresponding content levels of each enoxaparin sodium oligosaccharide.

[0064]

[0065]

[0066] Table 2

[0067]

[0068]

[0069] Table 3

[0070]

[0071] Table 4

[0072]

[0073]

[0074] Table 5

[0075] Example 2

[0076] 1. Preparation of bemiparin sodium oligosaccharides by column chromatography

[0077] Using a protein purifier, a size exclusion packed column ( Gel) was used for column chromatography. Bemiparin sodium was loaded at a concentration of 500 mg / ml, dissolved in a mobile phase of 0.5 mol / L NaCl solution, and the sample was separated by isocratic elution at a flow rate of 5 ml / min. An ultraviolet detector was used with a detection wavelength of 232 nm. The oligosaccharides were collected by online detection above the half-peak width, and the collected oligosaccharides had a single degree of polymerization. After rotary evaporation and concentration, gel column chromatography was used for desalting, and then freeze-drying was carried out. 19.7 mg of bemiparin sodium tetrasaccharide, 23.1 mg of hexasaccharide, 28.0 mg of octasaccharide, 22.4 mg of decasaccharide, and 18.1 mg of dodecasaccharide with a single degree of polymerization were prepared.

[0078] 2. Qualitative experiment of bemiparin sodium oligosaccharides by SAX-SEC-MS

[0079] Samples of bemiparin sodium oligosaccharides with a single degree of polymerization, specifically bemiparin sodium tetrasaccharide, hexasaccharide, octasaccharide, and decasaccharide, were accurately weighed at 10 - 15 mg each, and dissolved in the corresponding volume of water to prepare a 20 mg / mL solution. The first-dimensional SAX chromatographic conditions were as follows: injection volume 10 μL; flow rate 1 mL / min; column temperature 40 °C; mobile phase A, 17.907 g of disodium hydrogen phosphate dodecahydrate was dissolved in 2000 mL of pure water, filtered through a 0.22 μm filter membrane, and degassed for use; mobile phase B, 105.192 g of sodium chloride was dissolved in 1000 mL of mobile phase A, filtered through a 0.22 μm filter membrane, and degassed for use; the mobile phase gradient program was: 0 - 2 min, 10% B phase; 2 - 92 min, 10% → 100% B phase; 92 - 97 min, 100% B phase; 97 - 105 min, 100% → 10% B phase; detection wavelength 232 nm. Each chromatographic peak obtained from the first-dimensional SAX was cut into the second-dimensional SEC-MS for analysis to obtain the SAX standard spectra of oligosaccharides with different degrees of polymerization. The second-dimensional SEC chromatographic conditions were as follows: mobile phase, accurately weigh 3.854 g of ammonium acetate, dissolve it in 800 mL of pure water, mix well, filter through a 0.22 μm filter membrane, then add 200 mL of methanol, and ultrasonically degas for use; flow rate 0.15 mL / min; isocratic elution; detection wavelength 232 nm. The mass spectrometry conditions were: negative ion mode, fragment voltage 120 V, acquisition range 100 - 2000 m / z.

[0080] Please refer to Figures 12 - 15 , Figure 12It is the SAX standard chromatogram of bemiparin sodium tetrasaccharide; Figure 13 It is the SAX standard chromatogram of bemiparin sodium hexasaccharide; Figure 14 It is the SAX standard chromatogram of bemiparin sodium octasaccharide; Figure 15 It is the SAX standard chromatogram of bemiparin sodium decasaccharide. As Figures 12 - 15 shown, the bemiparin sodium tetrasaccharide, hexasaccharide, octasaccharide and decasaccharide have good separation on SAX. Among them, the bemiparin sodium tetrasaccharide, hexasaccharide, octasaccharide and decasaccharide correspond to 22, 39, 29 and 19 chromatographic peaks respectively, and the standard chromatogram obtained by qualitative analysis through SEC-MS is obtained.

[0081] 3. SEC-SAX-UV quantitative experiment of the bemiparin sodium to be tested

[0082] Precisely weigh 10 - 15 mg of each of the bemiparin sodium samples 1 - 4 to be analyzed, and add water with the corresponding volume to make a 40 mg / mL solution. Exchange the chromatographic conditions of the first and second dimensions in the qualitative part. Use SEC chromatography in the first dimension: injection volume 20 μL; flow rate 0.15 mL / min; column temperature 25 °C; mobile phase, accurately weigh 3.854 g of ammonium acetate, dissolve it in 800 mL of pure water, mix evenly, filter with a 0.22 μm filter membrane, then add 200 mL of methanol, and degas by ultrasonic treatment for later use; flow rate 0.15 mL / min; isocratic elution; detection wavelength 232 nm. Cut the peaks of the tetrasaccharide, hexasaccharide, octasaccharide and decasaccharide obtained from the SEC chromatogram into the second dimension SAX-UV for analysis. The chromatographic conditions of the second dimension SAX-UV: injection volume 10 μL; flow rate 1 mL / min; mobile phase A, weigh 17.907 g of disodium hydrogen phosphate dodecahydrate and dissolve it in 2000 mL of pure water, filter with a 0.22 μm filter membrane, and degas for later use; mobile phase B, weigh 105.192 g of sodium chloride and dissolve it in 1000 mL of mobile phase A, filter with a 0.22 μm filter membrane, and degas for later use; the mobile phase gradient program is: 0 - 2 min, 10% B phase; 2 - 92 min, 10% → 100% B phase; 92 - 97 min, 100% B phase; 97 - 105 min, 100% → 10% B phase; detection wavelength 232 nm.

[0083] Please refer to Figure 16 , Figure 16 It is the SAX chromatogram of the tetrasaccharide, hexasaccharide, octasaccharide and decasaccharide of the bemiparin sodium sample. As Figure 16As shown, the SAX-UV results of oligosaccharides of four bemiparin sodium samples are presented. After comparison with the qualitative standard spectrum, 26 chromatographic peaks of the tetrasaccharide were quantified, and 21 of them could be qualitatively identified. Qualitative and quantitative analyses were performed on 27 chromatographic peaks of the hexasaccharide. 26 chromatographic peaks of the octasaccharide were quantified, and 21 of them could be qualitatively identified. 25 chromatographic peaks of the decasaccharide were quantified, and 19 of them could be qualitatively identified. Please refer to Tables 6-9. Table 6 shows the percentage content (%) of the tetrasaccharide in the four bemiparin sodium samples, Table 7 shows the percentage content (%) of the hexasaccharide in the four bemiparin sodium samples, Table 8 shows the percentage content (%) of the octasaccharide in the four bemiparin sodium samples, and Table 9 shows the percentage content (%) of the decasaccharide in the four bemiparin sodium samples. As shown by the percentage content data of each oligosaccharide in Tables 6-9, the structural compositions and contents of the four bemiparin sodium samples are consistent.

[0084]

[0085] Table 6

[0086]

[0087] Table 7

[0088]

[0089]

[0090] Table 8

[0091]

[0092] Table 9 In summary, the two-dimensional liquid chromatography-mass spectrometry method for low molecular weight heparin quality analysis described in the present invention can perform qualitative and quantitative analysis of the oligosaccharide composition of various low molecular weight heparin samples, enabling consistency evaluation with the structure of the originator drug, and has great application value.

[0093] It should be noted that the above embodiments 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 preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A two-dimensional liquid chromatography-mass spectrometry method for the quality analysis of low molecular weight heparin, characterized in that The specific steps are as follows: (1) Using low molecular weight heparin as a raw material, various low molecular weight heparin oligosaccharides with different degrees of polymerization are prepared by preparative fractionation through medium pressure column chromatography. Among them, the degree of polymerization of the low molecular weight heparin oligosaccharides is 2n, where n is any natural number between 2 and 20; (2) Qualitatively analyzing the various low molecular weight heparin oligosaccharides with different degrees of polymerization by two-dimensional liquid chromatography-mass spectrometry. After separation by the first dimension of SAX, each SAX-separated oligosaccharide peak is cut into the second dimension of SEC, and finally tandem MS analysis is performed to obtain the SAX standard spectra of oligosaccharides with different degrees of polymerization; (3) Using another two-dimensional liquid chromatography, separating the low molecular weight heparin to be analyzed by the first dimension of SEC. Each oligosaccharide of a specific degree of polymerization is on-line cut and enters the second dimension of SAX-UV for analysis. By comparing with the corresponding SAX standard spectra of oligosaccharides of each degree of polymerization for analysis. Among them, the chromatographic conditions of SAX are the same as those of SAX corresponding to the degree of polymerization in step (2). The chromatographic conditions of SAX are: Thermo Scientific ProPac PA1 chromatographic column, injection volume 1 - 20 μL, column temperature 20 - 45 °C, flow rate 0.2 - 1.5 mL / min, mobile phase A is an aqueous solution of 25 mM Na2HPO4, mobile phase B is an aqueous solution of 1.8 M NaCl and 25 mM Na2HPO4, and the mobile phase gradient program is: 0 - 2 min, 10% B phase; 2 - 92 min, 10% → 100% B phase; 92 - 97 min, 100% B phase; 97 - 105 min, 100% → 10% B phase, detection wavelength 232 nm; The chromatographic conditions of SEC are the same as those of SEC corresponding to the degree of polymerization in step (2). The chromatographic conditions of SEC are: SEC-UPLC chromatographic column, injection volume 1 - 20 μL, column temperature 20 - 45 °C, flow rate 0.1 - 0.3 mL / min, mobile phase is a 20% methanol aqueous solution of 50 mM NH4OAc, isocratic elution, detection wavelength 232 nm.

2. The two-dimensional liquid chromatography-mass spectrometry method for low molecular weight heparin quality analysis according to claim 1, wherein In step (1), the preparation method of the low molecular weight heparin is: using heparin as a raw material, obtaining low molecular weight heparin through benzyl ester β-elimination and chemical cleavage.

3. The two-dimensional liquid chromatography-mass spectrometry method for low molecular weight heparin quality analysis according to claim 2, wherein: The low molecular weight heparin is enoxaparin sodium or bemiparin sodium.

4. The two-dimensional liquid chromatography-mass spectrometry method for low molecular weight heparin quality analysis according to claim 3, wherein: In step (1), the preparation by medium pressure column chromatography means fractionating enoxaparin sodium using a size exclusion packed column.

Images