Method for determining content of low-molecular-weight fucosylated glycosaminoglycan

Abstract

The present invention belongs to the field of pharmaceutical technology, and relates to a method for determining the content of low-molecular-weight fucosylated glycosaminoglycan, specifically a volumetric method for determining the content of low-molecular-weight fucosylated glycosaminoglycan. The reference substance and the test sample are respectively subjected to cation exchange treatment, and then titrated with an alkaline titrant. The volume of the titrant consumed by the sulfate group and the carboxylate group together is recorded, and the content of fucosylated glycosaminoglycan in the test sample of low-molecular-weight fucosylated glycosaminoglycan is calculated by comparison. The operation of the present invention is simple, economical and efficient, and is suitable for large-scale production of the process.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and relates to a method for determining the content of low molecular weight fucosylated glycosaminoglycan, in particular to a method for determining the content of low molecular weight fucosylated glycosaminoglycan by volumetric method. Background Art

[0002] Fucosylated Glycosaminoglycan (FG) is a glycosaminoglycan with a special chemical structure and pharmacological activity that has only been found in echinoderms. It has a main chain similar to chondroitin sulfate and has glycosaminoglycan analogs substituted with fucosyl (Fuc) side chains (Yoshida et al., Tetrahedron Lett, 1992, 33: 4959-62; et.al, J Biol Chem, 1996, 271: 23973-84). LFG (Low Molecular Weight Fucosylated Glycosaminoglycan, hereinafter referred to as "LFG") is a glycosaminoglycan derivative extracted and depolymerized from the body wall of sea cucumbers, which is polymerized by fucose, N-acetylgalactose and glucuronic acid. LFG is a polysaccharide drug. Due to the complex structure and polydispersity of the polysaccharide itself, quality control is relatively difficult. Sugar content determination is a necessary step in the research of polysaccharide drugs, and is also an important content in quality control and product standard formulation. Due to the diverse sources and different structural types of polysaccharide drugs, there are many methods for determining polysaccharides. Common sugar groups in polysaccharide drugs include neutral hexose, pentose and deoxyhexose, negatively charged uronic acid, sialic acid, positively charged amino sugars, etc., and specific methods can be established for determination according to the different chemical structures of different polysaccharides or hydrolysis into monosaccharides.

[0003] Since LFG is a macromolecular polysaccharide, it is composed of a series of oligosaccharide chains of different molecular weights, which is relatively complex and not suitable for the above-mentioned conventional monosaccharide content determination method. Some studies have used high-performance gel chromatography for quantitative research, but considering that polysaccharides themselves are polymers, there are many factors affecting gel exclusion chromatography in actual analysis and determination, such as poor durability of the chromatographic column, wide peak extension, difficult baseline balance, and poor reproducibility. In view of this, a new content determination method needs to be established. There is no report on the use of volumetric method to determine fucosylated glycosaminoglycans in existing reports. Summary of the invention

[0004] The purpose of the present invention is to provide a method for determining the content of low molecular weight fucosylated glycosaminoglycans, which is used to monitor the quality of a low molecular weight fucosylated glycosaminoglycan, ensure the stability and controllability of the quality of the low molecular weight fucosylated glycosaminoglycan product, and promote the application of low molecular weight fucosylated glycosaminoglycans in the fields of medicine, food, etc.

[0005] The present invention provides a method for determining the content of low molecular weight fucosylated glycosaminoglycan, wherein the fucosylated glycosaminoglycan has the following structure:

[0006]

[0007] In the formula,

[0008] R 1 , R 2 , R 3 , R 4 , R 5 Optionally -H or -SO independently of each other 3 H;

[0009] R 6 Optionally -H, substituted or unsubstituted C1-C6 hydrocarbon group or C7-C12 aryl group;

[0010] R 7 Optional: -H, -SO 3 H, C2-C5 acyl;

[0011] R 8 Optionally, it is a group represented by formula (II), formula (III) or formula (IV):

[0012]

[0013]

[0014] In formula (II), (III) and formula (IV),

[0015] R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 All are as defined above;

[0016] R 9 and R 10 Optionally -H, substituted or unsubstituted C1-C6 hydrocarbon group or C 7 -C 12 Aryl;

[0017] R 11 Optional -NHR12 、-OR 13 , where R 12 and R 13 Optionally -H, substituted or unsubstituted C 1 -C 6 Hydrocarbon or C 7 -C 12 Aryl; and n is a natural number optionally selected from 0 or 1 to 8;

[0018] The measuring method is to decationize the low molecular weight fucosylated glycosaminoglycan reference substance, add titration solution for titration, record the change of conductivity value during titration, and determine the volume of titration solution consumed by the reference substance; draw a titration curve with conductivity as the ordinate and the volume of titration solution as the abscissa; draw the first, second and third best straight lines for the three linear parts of the titration curve, which are a sudden drop, a slight climb and a sharp rise; draw a vertical line to the abscissa at the intersection of the second and third straight lines, and the intersection of the vertical line and the abscissa is the volume of titration solution consumed by sulfate and carboxylate. Calculate the volume of titration solution consumed by sulfate and carboxylate in the reference substance according to the titration curve, and then calculate the ratio R of the mass of the reference substance to the volume of titration solution consumed by sulfate and carboxylate in the reference substance. At the same time, the low molecular weight fucosylated glycosaminoglycan sample to be tested is titrated, and the content of the low molecular weight fucosylated glycosaminoglycan therein is calculated according to R, and the titration solution is an alkaline solution, preferably a sodium hydroxide solution, more preferably a sodium hydroxide solution with a concentration of 0.02-0.1 mol / L.

[0019] In a specific embodiment of the present invention, the following steps are adopted:

[0020] (1) Take an appropriate amount of this product, dissolve it in water and quantitatively dilute it to make a solution containing about 5 mg per 1 ml as the test solution;

[0021] (2) Take 2 ml of the test solution and add it to the cation exchange resin column. Slowly wash it into a small beaker with 15 ml of water. Place it on an electromagnetic stirrer, immerse the electrode, stir, and record the initial reading after the conductivity reading stabilizes.

[0022] (3) Use a pipette to add 50 μL of titrant. When the conductivity reading stabilizes, record and continue titrating until the conductivity value changes slowly. Add 10 μL of titrant. When the conductivity value changes dramatically again, add 50 μL of titrant until the conductivity value increases regularly. At the endpoint (the conductivity value returns to the value at the beginning of the titration), record the volume of titrant consumed.

[0023] Then calculate the content of fucosylated glycosaminoglycan in the test sample as follows:

[0024]

[0025] Where V is the volume of the titrant consumed by the sulfate and carboxylate groups in the test sample;

[0026] R is the ratio of the volume of the titration solution consumed by the sulfate and carboxylate groups in the fucosylated glycosaminoglycan reference substance to the quality of the reference substance;

[0027] m is the weight of the fucosylated glycosaminoglycan sample, calculated on a dry basis.

[0028] The method for rapidly measuring a low molecular weight fucosylated glycosaminoglycan provided by the present invention is simple to operate, accurate, economical and efficient, and very suitable for the promotion and application of product batch inspection and quality control in industrial-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Titration curves of low molecular weight fucosylated glycosaminoglycans. DETAILED DESCRIPTION

[0030] Example 1 Verification of LFG content determination method

[0031] 1. Equipment, reagents, reference substances and samples

[0032] 1.1 Main equipment information

[0033] Table 1 Main equipment information

[0034]

[0035] 1.2 Main reagent information

[0036] Table 2 Main reagent information

[0037]

[0038]

[0039] 1.3 Reference Material Information

[0040] Table 3 Reference Material Information

[0041]

[0042] Note: The content of the reference substance is expressed as HPLC purity (differential detector, area normalization), calculated on a dry basis, and the titration coefficient is obtained after calibration.

[0043] The low molecular weight fucosylated glycosaminoglycan sample used in the examples was extracted from sea cucumber according to the method disclosed in Chinese Patent No. 201410007855.

[0044] 2. Determination method

[0045] 2.1 Resin treatment

[0046] Take a certain amount of 732 strong acid cation exchange resin, rinse it with distilled water (freshly boiled and cooled), soak it for 1 hour to make it fully swell, stir and soak it with 1mol / L HCl solution 4 times the volume of the resin for 1 hour, wash it with distilled water until neutral; stir and soak it with 1mol / L NaOH solution 4 times the volume of the resin for 1 hour, wash it with distilled water until neutral; finally, stir and soak it with 4 times the volume of 1mol / L HCl solution for 1 hour, wash it with distilled water until neutral, and set aside.

[0047] 2.2 Sample solution preparation

[0048] Reference solution: Weigh an appropriate amount of LFG reference substance accurately, dissolve in water and dilute to make a solution containing about 5 mg per 1 ml, shake well, prepare 2 portions in parallel, and set aside.

[0049] Test solution: Weigh an appropriate amount of LFG test sample accurately, dissolve in water and dilute to make a solution containing about 5 mg per 1 ml, shake well, prepare 6 portions in parallel, and set aside.

[0050] 2.3 Test methods

[0051] Prepare 2000ml of freshly boiled pure water and cool it quickly (for use on the same day); slowly pour the treated resin into the chromatography column, open the lower valve switch when filling to avoid bubbles, leave some water on the resin, the resin should be filled evenly, without gaps and bubbles, and the chromatography column should be wrapped with an ice bag filled with crushed ice, and the filling volume should be about 10cm×1cm. Take 2ml of the reference solution and slowly add it to the cooled chromatography column along the wall. After the reference solution is added to the column, turn off the chromatography column switch and let it stand for about 20min to make the ion exchange more complete. Then turn on the chromatography column switch and slowly wash it into a 50ml small beaker (stored in an ice bath) with 15ml of water. The eluent should be titrated immediately with sodium hydroxide titration solution (0.1 mol / L). Put the conductivity electrode, temperature probe, and magnet into a small beaker, place the small beaker in an ice bath, adjust the magnetic stirrer to allow the magnet to stir normally, and record the reading after the conductivity reading stabilizes. Use a pipette to add 50μL of 0.1mol / L sodium hydroxide titrant. When the conductivity reading stabilizes, record the value and continue titrating until the conductivity value changes slowly, then add 10μL of titrant. When the conductivity value changes dramatically again, add 50μL of titrant until the conductivity value increases regularly. At the endpoint (the conductivity value returns to the value at the beginning of the titration), record the volume of sodium hydroxide titrant consumed.

[0052] Titrate the LFG test solution in the same manner as above and record the volume of sodium hydroxide titrant consumed.

[0053] 2.4 Test results

[0054] Table 4 Volumetric method repeatability test results

[0055]

[0056] Note: The coefficients in Table 4 are calculated based on the volume V1 of sodium hydroxide titration solution consumed by the LFG reference substance, and are equivalent to a certain mass of LFG (in terms of dry product) per unit volume of sodium hydroxide titration solution consumed.

[0057] 2.5 Experimental Conclusion

[0058] According to the experimental results, the average value of the content determination results of this method is 104.16%, and the repeatability RSD value is 2.90%. This method is used as a method for determining multi-component samples of fucosylated glycosaminoglycans, and the determination results are accurate. See the titration curve example Figure 1 shown.

[0059] Determination of Example 2 Test Sample

[0060] Weigh appropriate amounts of four batches of LFG test products (20170531, 20170612, 20170616, and 20170623) respectively, accurately weigh, dissolve in water and dilute to make a solution containing about 5 mg per 1 ml, shake well, and set aside.

[0061] Take 2ml of each test solution and add it to the cation exchange resin column, wash it slowly with 15ml water into a small beaker, put it on an electromagnetic stirrer, immerse the electrode, stir, and record the reading after the conductivity reading stabilizes. Use a pipette to add 50μL of 0.1mol / L sodium hydroxide titration solution. When the conductivity reading stabilizes, record and continue titration until the conductivity value changes slowly, add 10μL titration solution, and when the conductivity value changes drastically again, add 50μL titration solution until the conductivity value rises regularly. To the end point (the conductivity value returns to the time when the titration starts), record the volume of sodium hydroxide titration solution consumed, draw a curve with conductivity as the ordinate and the volume of titration solution as the abscissa; draw the first, second, and third best straight lines for the three linear parts of the graph that suddenly drop, climb slightly, and rise sharply; draw a vertical line to the abscissa at the intersection of the second and third straight lines, and the intersection of the vertical line and the abscissa is the volume V of the titration solution consumed by sulfate and carboxylate.

[0062] According to the calculation of LFG reference substance, each consumption of 1ml sodium hydroxide titration solution (0.1mol / L) is equivalent to 31.16mg of LFG, the content of LFG sample is calculated. The calculation results are as follows:

[0063] Table 5 Results of four batches of LFG volumetric determination

[0064]

[0065] Moreover, the reagents, consumables and instruments required for this method are all conventional laboratory equipment. Compared with the prior art HPLC determination method, the determination cost is lower and the operation is simpler.

Claims

1. A method for determining the content of low molecular weight fucosylated glycosaminoglycan, wherein the fucosylated glycosaminoglycan has the following structure: In the formula, R 1 , R 2 , R 3 , R 4 , R 5 Optionally -H or -SO independently of each other 3 H; R 6 Optionally -H, substituted or unsubstituted C1-C6 hydrocarbon group or C7-C12 aryl group; R 7 Optional: -H, -SO 3 H, C2-C5 acyl; R 8 Optionally, it is a group represented by formula (II), formula (III) or formula (IV): In formula (II), (III) and formula (IV), R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 All are as defined above; R 9 and R 10 Optionally -H, substituted or unsubstituted C1-C6 hydrocarbon group or C7-C12 aryl group; R 11 Optional -NHR 12 、-OR 13 , where R 12 and R 13 Optionally -H, substituted or unsubstituted C1-C6 hydrocarbon group or C7-C12 aryl group; and n is a natural number optionally selected from 0 or 1 to 8; Features: The low molecular weight fucosylated glycosaminoglycan reference substance is subjected to cation exchange treatment, and titrated with an alkaline solution as a titrant. During the titration process, the change in conductivity is recorded, and the volume of the titrant consumed by the reference substance is determined; a titration curve is drawn with conductivity as the ordinate and the volume of the titrant as the abscissa; the volume of the titrant consumed by sulfate and carboxylate in the reference substance is calculated according to the titration curve: the first, second, and third best straight lines are drawn for the three linear parts of the titration curve, which are a sudden drop, a slight climb, and a sharp rise, respectively; between the second and third lines, the best straight lines are drawn. The intersection of the three straight lines is a vertical line to the horizontal axis, and the intersection of the vertical line and the horizontal axis is the volume of the titration solution consumed by the sulfate and carboxyl groups. Then the ratio R of the mass of the reference substance to the volume of the titration solution consumed by the sulfate and carboxyl groups in the reference substance is calculated. In the same way, the volume V of the titration solution consumed by the sulfate and carboxyl groups in the low molecular weight fucosylated glycosaminoglycan test sample is titrated and calculated, and the content of the low molecular weight fucosylated glycosaminoglycan in the test sample is calculated according to the R of the reference substance. The calculation formula of the low molecular weight fucosylated glycosaminoglycan content is as follows: Wherein, m is the weight of the low molecular weight fucosylated glycosaminoglycan sample, calculated on a dry basis.

2. The assay method according to claim 1, Features: The alkaline solution is a sodium hydroxide solution.

3. The assay method according to claim 2, Features: The concentration of the sodium hydroxide solution is 0.02 mol / L to 0.1 mol / L.

4. The assay method according to claim 1, Features The steps include: (1) Take an appropriate amount of low molecular weight fucosylated glycosaminoglycan test sample, dissolve it in water and quantitatively dilute it to make a 5 mg / ml solution as the test sample solution; (2) Take 2 ml of the test solution and add it to the cation exchange resin column. Slowly wash it into a small beaker with 15 ml of water. Place it on an electromagnetic stirrer, immerse the electrode, stir, and record the initial reading after the conductivity reading stabilizes. (3) Use a pipette to add 50 μL of titrant. When the conductivity reading stabilizes, record and continue titrating until the conductivity value changes slowly. Add 10 μL of titrant. When the conductivity value changes dramatically again, add 50 μL of titrant until the conductivity value increases regularly. The titration process ends when the conductivity value returns to the value at the beginning of the titration.

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