Method for detecting the content of polysulfated mucopolysaccharide based on high performance liquid chromatography

Abstract

This invention belongs to the field of chemical pharmaceutical analysis technology, specifically relating to a method for detecting the content of polysulfated mucopolysaccharides based on high-performance liquid chromatography (HPLC). The invention utilizes HPLC to detect the content of polysulfated mucopolysaccharides in a sample, specifically: using an anion exchange column as the chromatographic column and an anion guard column as the guard column, gradient elution is performed using phosphate solution as mobile phase A and a mixed solution of sodium dihydrogen phosphate, sodium perchlorate, and water as mobile phase B. Detection is then performed using a detector with a detection wavelength of 210 nm to obtain a chromatogram. Finally, the polysulfated mucopolysaccharide content is calculated using the area normalization method. This method features high specificity, high sensitivity, and short analysis time, and is of great significance for the quality control of polysulfated mucopolysaccharide products.

Description

Technical Field

[0001] This invention belongs to the field of chemical drug analysis technology, specifically relating to a method for detecting the content of polysulfated mucopolysaccharides based on high performance liquid chromatography. Background Technology

[0002] Polysulfated mucopolysaccharides were developed by Mobilat Produktions GmbH in Germany. Their structure consists of several disaccharide units linked by sulfonated D-glucuronic acid and N-acetyl-D-galactosamine, with a molecular weight between 5000 and 15000 Daltons. The structural formula of polysulfated mucopolysaccharides is as follows:

[0003]

[0004] Mucopolysaccharide polysulfate cream is primarily used clinically to treat thrombophlebitis (including hemorrhoids), pain and inflammatory diseases based on circulatory disorders (induration and pain after injection), chilblains, hypertrophic scars, keloids (treatment and prevention), progressive palmar-digital keratoderma, sebaceous agenesis, swelling after trauma (contusions, sprains, bruises), hematoma, tenosynovitis, myalgia, arthritis, and muscular torticollis (in infancy). The pharmacological action of mucopolysaccharide polysulfate cream is through its antithrombotic effect on the blood coagulation and fibrinolytic systems. Additionally, it has anti-inflammatory effects by inhibiting various enzymes involved in catabolism and affecting the prostaglandin and complement systems. Mucopolysaccharide polysulfate can also promote connective tissue regeneration by promoting mesenchymal cell synthesis and restoring the ability of intercellular substances to retain water. Therefore, mucopolysaccharide polysulfate cream can prevent the formation of superficial thrombi, promote their absorption, inhibit the development of local inflammation, and accelerate the absorption of hematomas.

[0005] Currently, no patents or literature have been found regarding analytical methods for detecting polysulfated mucopolysaccharide (PSM) content. Therefore, it is necessary to find and research methods with high sensitivity and specificity for detecting PSM content to facilitate effective control of PSM product quality. Summary of the Invention

[0006] One of the objectives of this invention is to provide a method for detecting polysulfated mucopolysaccharide content based on high performance liquid chromatography. This method is highly specific and sensitive, and can achieve qualitative and quantitative detection of polysulfated mucopolysaccharides within 55 minutes.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for determining the content of polysulfonic acid mucopolysaccharides based on high performance liquid chromatography includes the following steps:

[0009] (1) Prepare sample solutions and reference solutions;

[0010] (2) The content of polysulfonic acid mucopolysaccharide in the sample was detected by high performance liquid chromatography. The high performance liquid chromatography method used an anion exchange column as the chromatographic column and an anion guard column as the guard column. Mobile phase A and mobile phase B were used as the mobile phases. The polysulfonic acid mucopolysaccharide was separated by gradient elution and then detected by the detector to obtain a chromatogram.

[0011] The mobile phase A is a phosphate solution; the mobile phase B is a mixed solution of sodium dihydrogen phosphate, sodium perchlorate, and water; in the mobile phase B, the mass-to-volume ratio of sodium dihydrogen phosphate, sodium perchlorate, and water is 1 g: 350 g: 2.5 L; the detection wavelength of the detector is 210 nm.

[0012] Furthermore, the samples to be tested were polysulfated mucopolysaccharide cream and polysulfated mucopolysaccharide.

[0013] Preferably, the chromatographic column is an AG22 anion exchange column with dimensions of 2*250mm; the guard column is an AG23 anion guard column with dimensions of 4*50mm.

[0014] Furthermore, the gradient elution procedure is as follows:

[0015] During the 0-10 minute period, the volume ratio of mobile phase A to mobile phase B is set to 65:35.

[0016] During the 10-35 minute period, the volume ratio of the mobile phase A to the mobile phase B is set to 65-0:35-100.

[0017] During the 35-40 minute interval, the volume ratio of mobile phase A to mobile phase B is set to 0:100.

[0018] For 40-45 minutes, the volume ratio of mobile phase A to mobile phase B is set to 0-65:100-35.

[0019] During the 45-55 minute period, the volume ratio of mobile phase A to mobile phase B is set to 65:35.

[0020] Furthermore, the mobile phase A is composed of sodium dihydrogen phosphate dihydrate and water at a mass-volume ratio of 1 g: 2.5 L.

[0021] Furthermore, the pH value of mobile phase A and mobile phase B is 3.0.

[0022] Preferably, the preparation method of the mobile phase A is as follows: dissolve sodium dihydrogen phosphate dihydrate (e.g., 0.8 g) in purified water (e.g., 2 L), adjust the pH to 3.0 with phosphoric acid, filter and degas using a 0.45 μm filter membrane.

[0023] Furthermore, in the mobile phase B, sodium dihydrogen phosphate is preferably sodium dihydrogen phosphate dihydrate, and sodium perchlorate is preferably sodium perchlorate monohydrate.

[0024] Preferably, the preparation method of the mobile phase B is as follows: dissolve sodium dihydrogen phosphate dihydrate (e.g., 0.8 g) and sodium perchlorate monohydrate (e.g., 280 g) in purified water (e.g., 2 L), adjust the pH to 3.0 with phosphoric acid, filter and degas using a 0.45 μm filter membrane.

[0025] Furthermore, the column temperature is 40°C, and the mobile phase flow rate is 0.5 ml / min.

[0026] Furthermore, in step (1), the injection solution is prepared by using a combination of water, hydrochloric acid solution, sodium nitrite solution and sodium hydroxide solution as solvents in sequence.

[0027] Preferably, the detector is an ultraviolet detector.

[0028] As a preferred option, the injection volume is 20 μl.

[0029] Furthermore, the consistency of the chromatographic retention behavior of the sample and the reference standard is used to determine whether the product contains polysulfated mucopolysaccharides.

[0030] Furthermore, based on the chromatogram obtained in step (2), the content of polysulfonic acid mucopolysaccharide is calculated by using the area normalization method.

[0031] The formula for calculating the content of polysulfated mucopolysaccharides is as follows:

[0032]

[0033] In the formula:

[0034] P – Content of polysulfated mucopolysaccharides, %;

[0035] A1—Peak area of ​​polysulfonic acid mucopolysaccharide in the test solution;

[0036] A2 – Peak area of ​​polysulfonic acid mucopolysaccharide in the reference solution;

[0037] C2—Concentration of polysulfated mucopolysaccharide in the reference solution, mg / mL;

[0038] C 样 — The concentration of polysulfonic acid mucopolysaccharide in the test solution, in mg / mL.

[0039] Furthermore, within the linear range of 4.6 μg / mL to 184.0 μg / mL, Y = 20788X + 8068, where X is the X-axis representing the concentration and Y is the Y-axis representing the peak area.

[0040] As a preferred technical solution, the method includes the following steps:

[0041] Step 1. Accurately weigh 20 mg of dermatin sulfate and polysulfated mucopolysaccharide mixed reference standard, dissolve in 10 ml of purified water, vortex to mix, and obtain the reference solution (a).

[0042] Step 2. Accurately measure 400 μl of the reference solution (a) prepared in Step 1, add it to 100 μl of deionized water, then add 250 μl of 1M hydrochloric acid solution and 50 μl of 250 mg / mL sodium nitrite solution. Mix gently, react at room temperature for 40 min, then add 200 μl of 1M sodium hydroxide solution to terminate the reaction, obtaining reference solution (b).

[0043] Step 3. Accurately weigh 100 mg of the polysulfated mucopolysaccharide sample, dissolve it in 50 mL of purified water, and mix with a vortex mixer until completely dissolved. Mix 500 μL of this solution with 250 μL of 1 M hydrochloric acid solution, then add 50 μL of 250 mg / mL sodium nitrite solution, mix gently, and let stand at room temperature for 40 min. Then add 200 μL of 1 M sodium hydroxide solution to terminate the reaction, obtaining the sample solution; duplicate samples are required.

[0044] Step 4. Using deionized water as a blank, inject the reference solution (b) prepared in Step 2 and the sample solution prepared in Step 3 sequentially, and detect them with a UV detector to obtain chromatograms. Then, calculate the content of polysulfated mucopolysaccharides using the area normalization method.

[0045] The second objective of this invention is to provide an application of a reagent composition in the detection of polysulfated mucopolysaccharide content.

[0046] To achieve the above objectives, the present invention adopts the following technical solution:

[0047] The application of a reagent composition in the detection of polysulfated mucopolysaccharide content, the reagent composition comprising a phosphate solution and a mixed solution of sodium dihydrogen phosphate, sodium perchlorate and water; the method for detecting the polysulfated mucopolysaccharide content is high performance liquid chromatography, wherein the phosphate solution is used as mobile phase A, and the mixed solution of sodium dihydrogen phosphate, sodium perchlorate and water is used as mobile phase B for elution of polysulfated mucopolysaccharide.

[0048] Preferably, the mobile phase A is composed of sodium dihydrogen phosphate dihydrate and water in a mass-to-volume ratio of 1 g: 2.5 L; and the mobile phase B has a mass-to-volume ratio of sodium dihydrogen phosphate dihydrate, sodium perchlorate monohydrate and water of 1 g: 350 g: 2.5 L.

[0049] Preferably, the pH value of both mobile phase A and mobile phase B is 3.0.

[0050] The beneficial effects of this invention are as follows:

[0051] 1. This invention proposes a method for detecting the content of polysulfated mucopolysaccharides using high-performance liquid chromatography (HPLC). This method has the advantages of high sensitivity, strong specificity, and short analysis time, enabling accurate qualitative and quantitative analysis of polysulfated mucopolysaccharides within 55 minutes. This method is of great significance for the quality control of polysulfated mucopolysaccharides in the pharmaceutical field.

[0052] 2. The high-performance liquid chromatography method for detecting polysulfated mucopolysaccharide content proposed in this invention has low detection limits and quantitation limits, with the detection limit as low as 1.5 μg / mL and the quantitation limit as low as 4.6 μg / mL. The results are accurate and reliable, and can effectively control product quality. Attached Figure Description

[0053] Figure 1 The chromatogram is for a blank solution;

[0054] Figure 2 This is the chromatogram of the control solution in Example 1;

[0055] Figure 3 The chromatogram of the test solution;

[0056] Figure 4 The chromatogram of the control solution (d) in Example 2;

[0057] Figure 5 The chromatogram of the control solution (e) in Example 2;

[0058] Figure 6 The chromatogram is the limit of detection.

[0059] Figure 7 The chromatogram for detection at the limit of quantitation;

[0060] Figure 8 The graph shows the linear regression equation for polysulfated mucopolysaccharides. Detailed Implementation

[0061] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0062] In this embodiment of the invention, the method for preparing the mobile phase is as follows:

[0063] Mobile phase A: Dissolve 0.8 g of sodium dihydrogen phosphate dihydrate in 2 L of purified water, adjust the pH to 3.0 with phosphoric acid, filter through a 0.45 μm filter membrane and degas.

[0064] Mobile phase B: Dissolve 0.8 g sodium dihydrogen phosphate dihydrate and 280 g sodium perchlorate monohydrate in 2 L of purified water, adjust the pH to 3.0 with phosphoric acid, filter through a 0.45 μm filter membrane and degas.

[0065] In this embodiment of the invention, the blank solution is deionized water.

[0066] Example 1

[0067] (1) Preparation of reference solution

[0068] Reference solution (a): Accurately weigh 20 mg of the standard and dissolve it in 10 ml of purified water. The standard is a mixture of dermatin sulfate and polysulfated mucopolysaccharide reference standard.

[0069] Reference solution (b): Accurately measure 400 μl of reference solution (a) and add it to 100 μl of deionized water. Add 250 μl of 1M hydrochloric acid solution and 50 μl of 250 mg / mL sodium nitrite solution. Mix gently and react at room temperature for 40 min. Then, add 200 μl of 1M sodium hydroxide solution to terminate the reaction.

[0070] (2) Preparation of test solution

[0071] Test solution: Accurately weigh 100 mg of polysulfated mucopolysaccharide sample, dissolve in 50 mL of purified water, and mix with a vortex mixer until completely dissolved. Mix 500 μL of this solution with 250 μL of 1 M hydrochloric acid solution, then add 50 μL of 250 mg / mL sodium nitrite solution, mix gently, and let stand at room temperature for 40 min. Then add 200 μL of 1 M sodium hydroxide solution to terminate the reaction. Perform duplicate tests.

[0072] (3) Detection: Deionized water was used as a blank for injection. Then, the reference solution (b) prepared in step (1) and the test solution prepared in step (2) were injected into the high performance liquid chromatograph for detection and chromatograms were obtained.

[0073] The instrument parameters are as follows:

[0074] The detector was a UV detector; the chromatographic column was a Thermo Fisher AS11-HC anion exchange column (2 x 250 mm); the guard column was a Thermo Fisher AG23 anion guard column (4 x 50 mm); the detection wavelength was 210 nm; the column temperature was 40 °C; the flow rate was 0.5 ml / min; the run time was 55 min; and the injection volume was 20 μl.

[0075] The gradient procedure is shown in Table 1:

[0076] Table 1. Gradient elution program

[0077] 0-10 65 35 Isocratic elution 10-35 65-0 35-100 Linear gradient elution 35-40 0 100 Isocratic elution 40-45 0-65 100-35 Linear gradient elution 45-55 65 35 Isocratic elution

[0078] (4) Injection requirements:

[0079] Deionized water was used as a blank for injection, and the control solution and the test solution were injected once in sequence.

[0080] (5) System adaptability requirements:

[0081] In the chromatogram obtained from the reference solution (b), the peaks of dermatin sulfate and polysulfated mucopolysaccharide eluted sequentially, and the resolution between the two was not less than 3.0.

[0082] Test solution: Free from impurities other than polysulfated mucopolysaccharides, and the content of polysulfated mucopolysaccharides should be ≥95%.

[0083] (6) Results Processing and Analysis: Record the chromatograms and retention times of the main peak. The area normalization method was used for the chromatograms. The content of polysulfated mucopolysaccharides was calculated using the following formula. Report the average value; the relative average deviation should not exceed 2%.

[0084]

[0085] In the formula:

[0086] P – Content of polysulfated mucopolysaccharides, %;

[0087] A1—Peak area of ​​polysulfonic acid mucopolysaccharide in the test solution;

[0088] A2 – Peak area of ​​polysulfonic acid mucopolysaccharide in the reference solution;

[0089] C2—Concentration of polysulfated mucopolysaccharide in the reference solution, mg / mL;

[0090] C 样 — The concentration of polysulfonic acid mucopolysaccharide in the test solution, in mg / mL.

[0091] Test results: such as Figures 1-3 As shown in Table 2.

[0092] Table 2. Sample test results

[0093]

[0094] Example 2. Specificity

[0095] (1) Preparation of control solution (a): Accurately weigh 250 mg of heparin sodium working standard, hydrolyze it with deionized water and make up to 2.0 mL.

[0096] Control solution (b): Accurately measure 1200 μL of control solution (a) and 300 μL of dermatin sulfate and polysulfated mucopolysaccharide reference standard mixture into a 1.5 mL centrifuge tube and mix thoroughly with a mixer.

[0097] Control solution (d): Accurately measure 400 μL of control solution (a) into 100 μL of deionized water, mix thoroughly with a mixer, then add 250 μL of 1M hydrochloric acid solution and 50 μL of sodium nitrite solution with a concentration of 250 mg / mL, mix gently, and let stand at room temperature for 40 min. Then add 200 μL of 1M sodium hydroxide solution to terminate the reaction.

[0098] Control solution (e): Accurately measure 500 μL of control solution (b), add 250 μL of 1M hydrochloric acid solution and 50 μL of sodium nitrite solution with a concentration of 250 mg / mL. Mix gently, react at room temperature for 40 min, and then add 200 μL of 1M sodium hydroxide solution to terminate the reaction.

[0099] The control solution (e) can only be stored at room temperature for 24 hours.

[0100] (2) Detection

[0101] After the chromatographic system stabilized, blank solution, control solution (d), and control solution (e) were injected sequentially under the same chromatographic conditions as in Example 1.

[0102] (3) Acceptable standards

[0103] In the chromatogram obtained from the control solution (d), there is no peak at the position where heparin elutes. In the chromatogram obtained from the control solution (e), the resolution between the dermatin sulfate peak and the polysulfated mucopolysaccharide peak is not less than 3.0.

[0104] Result: As Figures 4-5 As shown, the control solution (d) had no peak at the position of heparin; the separation degree of the peaks of dermatin sulfate and polysulfated mucopolysaccharide in the control solution (e) was 4.2.

[0105] Example 3. Detection Limit

[0106] Detection limits are generally determined using the signal-to-noise ratio (SNR) method and the slope method. The detection limit refers to the lowest amount of analyte in a sample that can be detected. This experiment uses the SNR method, determining the limit of quantitation at a concentration with a SNR of approximately 3:1. The specific method is as follows:

[0107] (1) Preparation of the test solution

[0108] A 23.0 μg / mL polysulfated mucopolysaccharide solution: Accurately transfer 10 μL of dermatin sulfate and polysulfated mucopolysaccharide reference standard into 990 μL of purified water, and mix thoroughly using a vortex mixer.

[0109] The LOD concentration at a signal-to-noise ratio of approximately 3:1 was obtained from a 23.0 μg / mL polysulfated mucopolysaccharide solution.

[0110] (2) Detection

[0111] After the chromatographic system stabilized, blank solution and polysulfated mucopolysaccharide solution with a concentration of 23.0 μg / mL were injected sequentially, and the chromatographic conditions were the same as in Example 1.

[0112] Result: As Figure 6 As shown, when the signal-to-noise ratio is approximately 3:1, the LOD concentration is 1.5 μg / mL.

[0113] Example 4. Limit of Quantification

[0114] The limit of quantitation (LOQ) is the lowest amount of an analyte in a sample that can be quantitatively determined, and the measurement results should meet the requirements of accuracy and precision. This method uses the signal-to-noise ratio (SNR) method, determining the LQ based on the concentration at a SNR of 10:1.

[0115] (1) Solution preparation

[0116] A 23.0 μg / mL polysulfated mucopolysaccharide solution: Accurately transfer 10 μL of dermatin sulfate and polysulfated mucopolysaccharide reference standard into 990 μL of purified water, and mix thoroughly using a vortex mixer.

[0117] The LOQ concentration was determined from a polysulfated mucopolysaccharide solution with a concentration of 23.0 μg / mL when the signal-to-noise ratio was approximately 10:1.

[0118] (2) Detection

[0119] After the chromatographic system stabilized, blank solution and polysulfated mucopolysaccharide solution with a concentration of 23.0 μg / mL were injected sequentially, and the chromatographic conditions were the same as in Example 1.

[0120] Result: As Figure 7 As shown, the LOQ concentration is 4.6 μg / mL when the signal-to-noise ratio is approximately 10:1.

[0121] Example 5. Linear

[0122] (1) Solution preparation

[0123] Control solutions (d) and (e) were prepared according to the method described in "Example 1. Specificity". Simultaneously, polysulfated mucopolysaccharides with concentrations of 4.6 μg / mL, 11.5 μg / mL, 18.4 μg / mL, 23.0 μg / mL, 27.6 μg / mL, 34.5 μg / mL, 46.0 μg / mL, 92.0 μg / mL, 115.0 μg / mL, and 184.0 μg / mL were prepared according to Table 3.

[0124] Table 3. Methods for preparing linear solutions

[0125]

[0126]

[0127] (3) Detection

[0128] After the chromatographic system stabilized, the following samples were injected and analyzed in the following order: blank, control solution (d), control solution (e), 4.6 μg / mL polysulfated mucopolysaccharide, 11.5 μg / mL polysulfated mucopolysaccharide, 18.4 μg / mL polysulfated mucopolysaccharide, 23.0 μg / mL polysulfated mucopolysaccharide, 27.6 μg / mL polysulfated mucopolysaccharide, 34.5 μg / mL polysulfated mucopolysaccharide, 46.0 μg / mL polysulfated mucopolysaccharide, 92.0 μg / mL polysulfated mucopolysaccharide, 115.0 μg / mL polysulfated mucopolysaccharide, and 184.0 μg / mL polysulfated mucopolysaccharide. Linear regression was performed with peak area as Y and concentration as X-axis, under the same chromatographic conditions as in Example 1.

[0129] Result: As Figure 8 As shown, within the linear range of 4.6 μg / mL to 184.0 μg / mL, Y = 20788X + 8068, R 2 =0.999, the concentration of the test solution and the peak area showed a good linear relationship, which meets the requirements of high performance liquid chromatography for content determination.

[0130] The above results demonstrate that the high-performance liquid chromatography method constructed in this invention can determine the content of polysulfonic acid mucopolysaccharides with high specificity and accuracy, ensuring accurate and reliable results, thereby effectively controlling product quality and ensuring product quality stability.

Claims

1. A method for determining the content of polysulfonic acid mucopolysaccharides based on high performance liquid chromatography, characterized in that, Includes the following steps: (1) Prepare sample solutions and reference solutions; The preparation method of the sample solution includes: accurately weighing 100 mg of polysulfated mucopolysaccharide sample, dissolving it in 50 ml of purified water, and mixing it with a vortex mixer until completely dissolved; taking 500 μl of the solution and mixing it with 250 μl of 1 M hydrochloric acid solution, then adding 50 μl of 250 mg / mL sodium nitrite solution, gently mixing and letting it stand at room temperature for 40 min, and then adding 200 μl of 1 M sodium hydroxide solution to terminate the reaction, thus obtaining the sample solution; (2) The content of polysulfonic acid mucopolysaccharide in the sample solution was detected by high performance liquid chromatography (HPLC). The HPLC method used an AG22 anion exchange column with a size of 2*250 mm and an AG23 anion guard column with a size of 4*50 mm. Mobile phases A and B were used as mobile phases, and polysulfonic acid mucopolysaccharides were separated by gradient elution. The chromatogram was obtained by detection in the detector. The mobile phase A consists of sodium dihydrogen phosphate dihydrate and water at a mass-to-volume ratio of 1 g: 2.5 L; the mobile phase B is a mixed solution of sodium dihydrogen phosphate, sodium perchlorate, and water; in the mobile phase B, the mass-to-volume ratio of sodium dihydrogen phosphate, sodium perchlorate, and water is 1 g: 350 g: 2.5 L; the pH value of the mobile phase A and the mobile phase B is 3.0; the detection wavelength of the detector is 210 nm; the column temperature of the chromatographic column is 40 °C; and the flow rate of the mobile phase is 0.5 ml / min. The gradient elution procedure is as follows: 。 2. The method according to claim 1, characterized in that, Based on the chromatogram obtained in step (2), the content of polysulfonic acid mucopolysaccharide is calculated using the peak area.

3. The method according to claim 1, characterized in that, For polysulfated mucopolysaccharides, the linear range is 4.6 μg / mL to 184.0 μg / mL, Y = 20788X + 8068, where X represents the concentration and Y represents the peak area.

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