A qualitative and quantitative analysis method for heparin-like components

Abstract

This invention discloses a qualitative and quantitative analysis method for heparin-like components in the biopharmaceutical field. The method includes using a liquid chromatograph, an SAX strong anion exchange column, sodium dihydrogen phosphate solution as mobile phase A, and mobile phase B consisting of mobile phase A containing sodium perchlorate, with gradient elution for monitoring and analysis. This invention improves the separation of heparin-like components by screening chromatographic packing materials, optimizing the chromatographic method, and optimizing the detection wavelength. It allows for accurate qualitative analysis of up to five heparin components using a simple liquid chromatography method. Furthermore, this invention incorporates the simulated separation function of the Glycomapping glycosylation software to reconstruct the elution peaks of components that do not reach baseline separation, and then achieves quantitative analysis of each separated component based on the external standard method. This qualitative and quantitative analysis method provides a feasible solution for component identification and content determination in heparin-like samples, and offers an important means for heparin quality control.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to a qualitative and quantitative analysis method for heparin-like components. Background Technology

[0002] Heparin is an important class of glycosaminoglycan drugs with various biological activities such as preventing thrombosis, anti-inflammation and anti-allergy. It has been used clinically as an anticoagulant in surgical procedures and kidney dialysis for nearly a century.

[0003] Modern heparin (HP) is mainly extracted from the mucosa of the small intestine in pigs. It also contains other heparin-like components such as heparan sulfate (HS), dermatan sulfate (DS), and chondroitin sulfate (CS). Further purification processes produce heparin-like derivatives such as oversulfated chondroitin sulfate (OSCS). These substances are all linear long-chain macromolecular polysaccharides with repeating disaccharide units as their backbone and are rich in sulfated modified polysaccharides.

[0004]

[0005] Heparin and heparan sulfate are polymers composed of disaccharides, specifically uronic acid and glucosamine, linked by 1,4-glycosidic bonds. Both uronic acid and glucosamine have sulfation sites, and the difference between heparin and heparan sulfate lies only in the number of sulfation sites, with heparan sulfate exhibiting a lower degree of sulfation than heparin. Chondroitin sulfate is also polymerized from disaccharides, specifically uronic acid and 4S-acetylgalactosamine linked by 1,3-glycosidic bonds. Both uronic acid and 4S-acetylgalactosamine have sulfation sites. There are several types of chondroitin sulfate, including chondroitin A, chondroitin B, and chondroitin C, which differ in the types of uronic acid and the number of sulfation sites. Chondroitin B, also known as dermatan sulfate, is a polysaccharide composed of iduronic acid and 4S-acetylgalactosamine.

[0006] Heparin-like components share high similarity, but their complex structures and large molecular weights make separation very difficult. However, subtle structural differences lead to significant variations in activity; different components exhibit different activities and therapeutic effects. For example, sulodexide, a drug composed of 80% heparin and 20% dermatan sulfate, demonstrates stronger antithrombotic activity and higher safety.

[0007] Currently, analytical methods for heparin-like components include nuclear magnetic resonance (NMR), capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), and mass spectrometry. NMR is complex and highly specialized, requiring advanced spectral interpretation techniques; capillary electrophoresis is still in the exploratory stage and cannot be widely applied; considering industrial production costs and laboratory operability, HPLC has the greatest potential for widespread application, offering advantages such as low sample volume, rapid detection, accurate and reliable results, and good stability. Existing HPLC methods for analyzing heparin-like substances are mostly used for oligosaccharide analysis. Due to the high negative charge and high molecular weight structure of heparin-like substances, analytical methods for heparin polysaccharides are very limited; only a few SAX columns can separate the three components: chondroitin persulfate (OSCS), dermatan sulfate (DS), and heparin (HP). Chinese patent CN 110907571 A successfully separated three components: heparan sulfate (HS), heparin (HP), and dermatan sulfate (DS), and determined the relative contents of heparin and heparan sulfate. However, the method involves co-eluting of CS and DS components. For complex heparin-like samples, it is impossible to accurately analyze the two components CS and DS, let alone perform qualitative and quantitative analysis of the five heparin components at the same time.

[0008] Therefore, there is an urgent need for a liquid chromatography method that can effectively separate the five heparin components and perform qualitative and quantitative analysis on them, and provide guidance for drug quality control, active pharmaceutical ingredient activity research, new drug development and generic drug development. Summary of the Invention

[0009] To address the above issues and overcome the shortcomings of existing technologies, this invention provides a qualitative and quantitative analysis method for heparin-like components. This invention employs high-performance liquid chromatography (HPLC) for detection and analysis, accurately performing qualitative analysis on five heparin components: heparin (HP), heparan sulfate (HS), dermatan sulfate (DS), chondroitin sulfate (CS), and chondroitin persulfate (OSCS). Furthermore, by reconstructing elution peaks using software, simulated separation of components not yet separated from the baseline is achieved. The five separated components are then quantitatively analyzed using an external standard method, improving the sensitivity of heparin-like drug impurity detection and the accuracy of heparin-like drug activity investigation.

[0010] To achieve the above objectives, the following technical solution was adopted:

[0011] A qualitative analysis method for heparin-like components, wherein the method employs high-performance liquid chromatography (HPLC) for detection and analysis, and the HPLC conditions are as follows:

[0012] The SAX strong anion exchange column was used; the sample injection volume was 3-10 μL; the sample concentration was 1-10 mg / mL; the detection wavelength was 202 nm; mobile phase A was 2-3 mM sodium dihydrogen phosphate solution, and mobile phase B was mobile phase A containing 3-5 M sodium perchlorate. The pH of both mobile phases A and B was adjusted to 3.0 with phosphoric acid; initially, mobile phase A: mobile phase B = 90:10 wasocratic elution was performed for 2-5 min, then mobile phase B was continuously increased from 10% to 90%, and mobile phase A was continuously decreased from 90% to 10% for 40-80 min. After maintaining this ratio, elution was continued for 20-40 min, and then mobile phase A was rapidly increased from 10% to 90% and mobile phase B decreased from 90% to 10% within 0.1-1 min, and elution was continued for 5-30 min, after which the detection was completed.

[0013] Furthermore, during data processing, the component can be identified based on the retention time of the elution peak through standard comparison. Simultaneously, leveraging the advantages of Glycomapping peak shape simulation, simulated separation of multiple components can be achieved, and the content of each component can be analyzed using the external standard method. Using the liquid chromatography detection method provided by this invention, the content of five heparin components can be accurately separated and detected.

[0014] The beneficial effects of this invention are as follows:

[0015] (1) Compared with nuclear magnetic resonance and mass spectrometry, this invention does not require expensive instruments or difficult operation;

[0016] (2) This invention is the first to achieve simultaneous qualitative analysis of five types of heparin components;

[0017] (3) The reconstructed elution peaks can be simulated with the help of software, and the content of each component can be accurately quantitatively analyzed by external standard method. Attached Figure Description

[0018] Figure 1 This is the liquid chromatogram of mobile phase A (blank) in Example 1;

[0019] Figure 2 The liquid chromatogram of heparan sulfate (HS) in Example 1 is shown below.

[0020] Figure 3 The liquid chromatogram of heparin sodium (HP) in Example 1;

[0021] Figure 4 The liquid chromatogram of chondroitin sulfate (CS) in Example 1;

[0022] Figure 5 This is the liquid chromatogram of dermatin sulfate (DS) in Example 1;

[0023] Figure 6The liquid chromatogram of chondroitin persulfate (OSCS) in Example 1;

[0024] Figure 7 This is the liquid chromatogram of the mixed standard std5 in Example 1;

[0025] Figure 8 This is a superimposed liquid chromatogram of the five heparin components in Example 1;

[0026] Figure 9 The liquid chromatogram of the mixture standard std5 in Example 2;

[0027] Figure 10 This is a schematic diagram of the complex mapping simulation reconstructing of the std5 standard effluent peak in Example 2;

[0028] Figure 11 The liquid chromatograms are for different packed columns (New column-1) in Comparative Example 1.

[0029] Figure 12 The liquid chromatograms are for different packed columns (New column-2) in Comparative Example 1;

[0030] Figure 13 The liquid chromatograms are for different packed columns (New column-3) in Comparative Example 1;

[0031] Figure 14 The above is a superimposed liquid chromatogram of methods 4, 8, 9, and 10 in Comparative Example 2;

[0032] Figure 15 The chromatograms of different chromatographic methods in Comparative Example 2 (method-13) are liquid chromatograms.

[0033] Figure 16 The chromatograms of different chromatographic methods in Comparative Example 2 (method-14) are liquid chromatograms.

[0034] Figure 17 The chromatograms are liquid chromatograms of different chromatographic methods (method-15) in Comparative Example 2;

[0035] Figure 18 The chromatograms are liquid chromatograms of different chromatographic methods (method-16) in Comparative Example 2;

[0036] Figure 19 The chromatograms of different chromatographic methods in Comparative Example 2 (method-17) are liquid chromatograms.

[0037] Figure 20The above is a superimposed liquid chromatogram of methods 13-17 in Comparative Example 2;

[0038] Figure 21 The chromatograms are liquid chromatograms of different chromatographic methods (method-20) in Comparative Example 2;

[0039] Figure 22 The chromatograms are liquid chromatograms of different chromatographic methods in Comparative Example 2 (method-22);

[0040] Figure 23 The chromatograms are for different chromatographic methods in Comparative Example 2 (method-21).

[0041] Figure 24 The chromatograms are for different chromatographic methods in Comparative Example 2 (method-23).

[0042] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0043] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used in the following examples are all purchased from commercial channels.

[0046] The instruments used in the examples and comparative examples include: LC-20AD liquid chromatograph

[0047] Heparin sodium standard (HP) was purchased from Beijing Wanjia Standard Material Research Center, product number SH-1304016; heparan sulfate standard (HS) was purchased from Wuhan Kostan Biotechnology Co., Ltd., product number CSGC30767.

[0048] Dermatin sulfate standard (DS), purchased from Beijing Wanjia Standard Material Research Center, product number SH-1171455;

[0049] Chondroitin sulfate standard (CS), purchased from Beijing Wanjia Standard Material Research Center, product number CCAD3011193;

[0050] Chondroitin persulfate standard (OSCS), purchased from Shanghai Zhenzhun Biotechnology Co., Ltd., product number 03044162;

[0051] Sodium dihydrogen phosphate was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0052] Sodium perchlorate was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0053] Phosphoric acid was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0054] Example 1

[0055] Separation effect verification

[0056] (1) Take five 5.0 mL (or 4.0 mL) centrifuge tubes and accurately weigh 1 mg each of heparan sulfate standard (HS), heparin sodium standard (HP), dermatan sulfate standard (DS), chondroitin sulfate standard (CS), and chondroitin persulfate standard (OSCS). Place them in centrifuge tubes, add 1.0 mL of water to each tube, dissolve and mix well, and label them as HS, HP, DS, CS, and OSCS. Filter the solution through a 0.22 μm filter membrane.

[0057] (2) Take one 5.0 mL (or 4.0 mL) centrifuge tube, accurately weigh 1 mg each of heparan sulfate standard (HS), dermatan sulfate standard (DS), chondroitin sulfate standard (CS), and chondroitin persulfate standard (OSCS), and 4 mg of heparin sodium standard (HP), place them in the centrifuge tube, add 1.0 mL of water, dissolve and mix well, and label it as Std5; filter the solution through a 0.22 μm filter membrane;

[0058] (3) Take 7 newly opened and clean liquid chromatography injection bottles, and add water (blank) filtered through a 0.22 μm membrane, heparin sulfate standard (HS) solution, heparin sodium standard (HP) solution, dermatan sulfate standard (DS) solution, chondroitin sulfate standard solution (CS) solution, chondroitin persulfate standard solution (OSCS) solution, and multi-component mixed solution Std5 respectively; put the sealed injection bottles into the liquid chromatograph and inject the samples for measurement;

[0059] (4) The liquid chromatography conditions are as follows:

[0060] Chromatographic column: XB-SAX, 3μm, 4.6×250mm;

[0061] Flow rate: 0.15 mL / min;

[0062] Column temperature: 10℃;

[0063] UV detector wavelengths: 202nm, 210nm;

[0064] Injection volume: 3 μL;

[0065] Mobile phase A: 3mM sodium dihydrogen phosphate solution; Mobile phase B: 4M sodium perchlorate dissolved in mobile phase A.

[0066] Mobile phases A and B are carried out according to the following gradient procedure:

[0067] Time (min) A％ B％ Flow rate (mL / min) 0 90 10 0.15 2 90 10 0.15 60 20 80 0.15 65 20 80 0.15 65.1 90 10 0.7 70 90 10 0.7 70.1 90 10 0.15 75 90 10 0.15

[0068] (5) Experimental results:

[0069] Sample solution Test results blank No peak signal HS There is a clear peak signal HP There is a clear peak signal DS There is a clear peak signal CS There is a clear peak signal OSCS There is a clear peak signal Std5 There is a clear 5-peak signal

[0070] The corresponding chromatogram is attached. Figure 1-8 As shown, after comparing the spectra, it can be concluded that when the analytical method described in this invention is used for detection, the five heparin components can be effectively separated, among which heparan sulfate (HS), dermatan sulfate (DS), and chondroitin persulfate (OSCS) achieve baseline separation, thus realizing the qualitative analysis of the five heparin components.

[0071] Example 2

[0072] Validation of the effect of heparin component content determination

[0073] (1) Prepare 20 mg / mL heparan sulfate (HS) standard solution, heparin sodium (HP) standard solution, dermatan sulfate (DS) standard solution, chondroitin sulfate (CS) standard solution and chondroitin persulfate (OSCS) standard solution respectively, and filter them through a 0.22 μm filter membrane;

[0074] (2) Take a 5.0 mL (or 4.0 mL) centrifuge tube, accurately weigh 1 mg each of heparan sulfate standard (HS), dermatan sulfate standard (DS), chondroitin sulfate standard (CS), and chondroitin persulfate standard (OSCS), and 4 mg of heparin sodium standard (HP), place them in the centrifuge tube, add 1.0 mL of water, dissolve and mix well, label it as Std5, and filter the solution through a 0.22 μm filter membrane;

[0075] (3) Dilute the five standard solutions in step 1 by 2 / 5 / 10 / 20 / 50 times respectively to form standard 1, standard 2, standard 3, standard 4, and standard 5;

[0076] (4) Place the concentration gradient standards from step (3) and the mixed standard std5 from step (2) into a sample vial, and analyze it in a liquid chromatograph. The results are shown in the attached figure. Figure 9 As shown

[0077] The liquid chromatography conditions were the same as in Example 1.

[0078] The std5 data of the mixture standard was imported into Glycomapping software for simulation to reconstruct the elution peaks, as shown in the attached figure. Figure 10 As shown.

[0079] Example 3

[0080] Qualitative and quantitative analysis of heparin-like samples

[0081] Heparin-like samples were purchased from a domestic company, prepared into 5 mg / mL solutions, filtered through a 0.22 μm filter membrane, and subjected to qualitative and quantitative analysis under the liquid chromatography conditions described in Example 1. The concentrations of each component in the three heparin-like samples were calculated based on the integral of the reconstructed elution peak area and the standard curve. The relative contents of each component in the heparin-like samples are shown in Table 1.

[0082] Table 1. Retention time, peak area, concentration, and relative content of each component in heparin samples.

[0083]

[0084] Comparative Example 1

[0085] Comparison of results from different chromatographic columns

[0086] To optimize the density of quaternary ammonium groups in the bonded phase, the chromatographic packing material was modified to varying degrees, resulting in three chromatographic columns, named New column-1, New column-2, and New column-3, respectively.

[0087] (1) Prepare a std5 mixture solution as in Example 1 as the test solution;

[0088] (2) Following the chromatographic conditions of Example 1, using the std5 mixture as the separation target, separation tests were performed using three different chromatographic columns. The analytical results are shown in the appendix. Figure 11-13As can be seen from the figure, using New column-2 and New column-3 in the comparative examples, the chromatographic conditions provided by this invention cannot effectively separate the five components. Only New column-1 can effectively separate the five components and can be used for subsequent qualitative and quantitative analysis.

[0089] Comparative Example 2

[0090] Comparison of results under different chromatographic conditions

[0091] Chromatographic separation conditions were compared, including mobile phase composition, gradient, flow rate, and column temperature.

[0092] (1) Prepare a standard solution std4 containing 8 mg / mL heparin, 1 mg / mL dermatan sulfate, 1 mg / mL chondroitin sulfate and 1 mg / mL persulfate, filter it through a 0.22 μm filter membrane, put it into a sample vial, and place it in a liquid chromatograph for detection under the chromatographic conditions of method 4.

[0093] (2) The liquid chromatography conditions for Method4 are as follows:

[0094] Chromatographic column: XB-SAX, 3μm, 4.6×250mm;

[0095] Flow rate: 0.45 mL / min;

[0096] Column temperature: 40℃;

[0097] UV detector wavelengths: 202nm, 210nm;

[0098] Injection volume: 10 μL;

[0099] Mobile phase A: 3mM sodium dihydrogen phosphate solution; Mobile phase B: 2M sodium perchlorate dissolved in mobile phase A.

[0100] Mobile phases A and B are carried out according to the following gradient procedure:

[0101] Time (min) A％ B％ Flow rate (mL / min) 0 90 10 0.4 2 90 10 0.4 22 0 100 0.4 45 0 100 0.4 45.10 90 10 0.7 51 90 10 0.7 51.10 90 10 0.4 71 90 10 0.4

[0102] (3) Based on the chromatographic conditions of method 4, the elution flow rate was increased to 0.45 mL / min, 0.5 mL / min and 0.6 mL / min respectively (the corresponding chromatographic methods are method 8, menthod 9 and method 10 respectively), and the above std 4 standard solution was detected.

[0103] (4) Prepare the std5 standard solution as in Example 1 and fill it into a sample vial. Increase the concentration of mobile phase B sodium perchlorate to 4M, and perform liquid chromatography detection using methods 13, 14, 15, 16, and 17, respectively. Methods 13-17 are still used. An XB-SAX, 3μm, 4.6×250mm column was used. 10μL of sample was injected at a column temperature of 40℃. The elution gradient is shown in the table below.

[0104]

[0105] (5) Based on Menthod 16, the column temperature was lowered to 10℃ (method 20) and 20℃ (method 22) respectively, and the std 5 standard solution was tested using method 20 and method 22.

[0106] (6) Based on method20, increase the elution flow rate to 0.5 mL / min to obtain menthod21, decrease the elution flow rate to 0.15 mL / min to obtain method23, and test the std5 standard solution.

[0107] (7) Reduce the injection volume of method23 from 10 μL to 3 μL, which is the chromatographic method for qualitative analysis of 5 heparin components in Example 1;

[0108] (8) Detection results: The results corresponding to the high performance liquid chromatography methods (methods 4, 8, 9, and 10) in steps 2 and 3 are attached. Figure 14 As shown, elution was performed using 2M sodium perchlorate mobile phase B. Changing the flow rate had no effect on the separation degree and peak shape of the heparin-like components, and no ideal separation effect was observed (HS and OSCS were separated at the baseline, while HP, CS, and DS components could not be separated). After increasing the concentration of mobile phase B to 4M in step 4, the chromatographic method significantly improved the separation ability of the five heparin-like components, as shown in the attached figure. Figure 15-20 As shown, the separation effect was best under the elution concentration gradient of method 16. Therefore, step 5, based on method 16, further explored the effect of column temperature on the separation effect. The results for different high-performance liquid chromatography methods (methods 20 and 22) are shown in the appendix. Figure 21 , 22 As shown, lowering the column temperature significantly improves the separation effect, with HP, CS, and DS showing a more obvious separation trend. Step 6 adjusts the flow rate based on the above findings; the results for different high-performance liquid chromatography methods (methods 21 and 23) are shown in the appendix. Figure 23 , 24 As shown, reducing the flow rate can optimize the separation effect. Finally, reducing the injection volume to 3 μL slightly improved the separation effect of the five types of heparin. These results indicate that using... When using an XB-SAX column with a 3μm, 4.6×250mm diameter, the optimal separation effect is achieved under the following chromatographic conditions: 3-4M sodium perchlorate mobile phase B, injection volume of 3-10μL, and temperature of 4-30℃. Ideal separation results cannot be achieved under other conditions.

[0109] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0110] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for qualitative and quantitative analysis of heparin-like components, characterized in that: The method employs high-performance liquid chromatography (HPLC) for detection and analysis. The HPLC conditions are as follows: Mobile phase A is a 2-3 mM sodium dihydrogen phosphate solution, and mobile phase B is mobile phase A containing 3-5 M sodium perchlorate. The pH of both mobile phases A and B is adjusted to 3.0 with phosphoric acid. The detection wavelength is 202 nm. The quantitative analysis of the heparin-like components is performed using Glycomapping software, which calculates and simulates the elution peaks of the separated components to reconstruct the chromatographic peaks of the unbaseline separated components. Chromatographic column: Waters® XB-SAX, 3μm, 4.6×250mm; Mobile phases A and B are carried out according to the following gradient procedure: ； The heparin-like components include heparan sulfate, sodium heparin, dermatan sulfate, chondroitin sulfate, and chondroitin persulfate.

2. The qualitative and quantitative analysis method for heparin-like components according to claim 1, characterized in that: The sample injection volume is 3-10 μL; the sample concentration is 1-10 mg / mL.

3. The qualitative and quantitative analysis method for heparin-like components according to claim 1, characterized in that: The column temperature in the liquid chromatography conditions is 4-30℃.

4. The qualitative and quantitative analysis method for heparin-like components according to claim 1, characterized in that: The quantitative analysis of the heparin-like components was achieved using the external standard method.

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