Method for analyzing content of galactosamine in sulfated polysaccharide

Abstract

The invention discloses an analysis method for determining the content of galactosamine in sulfated polysaccharide based on high-temperature hydrochloric acid hydrolysis and an ion chromatograph method, which comprises the following steps: hydrolyzing a test sample with hydrochloric acid at high temperature to release galactosamine, separating with an ion chromatograph and a sugar analysis column, detecting with a pulsed ampere detector, and determining the content of galactosamine in sulfated polysaccharide according to a galactosamine reference substance. And quantitatively analyzing the content of galactosamine in the test sample. The method disclosed by the invention has the advantages of simplicity, rapidness, accuracy, high efficiency, no need of derivation treatment and the like.

Description

Technical Field

[0001] The invention discloses a method for analyzing the content of galactosamine in sulfated polysaccharides based on high-temperature hydrochloric acid hydrolysis and ion chromatography, and belongs to the field of quantitative analysis and detection in the field of biomedicine technology. Background Art

[0002] Sulfated polysaccharides (SP) are a class of polysaccharides containing sulfate modifications, composed of monosaccharide or oligosaccharide units and sulfate groups. In nature, sulfated polysaccharides are widely present in plants, animals, and humans. Heparin, chondroitin sulfate, dermatan sulfate, heparan sulfate, and sea cucumber glycosaminoglycans, among others, are all very important sulfated polysaccharides. Sulfated polysaccharides are important components of signal transduction on the surfaces of many tissues and cells in organisms, playing important physiological and biochemical roles, such as promoting immune function, anti-oxidation, anti-tumor, hypoglycemic, anti-thrombotic, and anti-coagulation functions. Some sulfated polysaccharides have been developed as pharmaceuticals.

[0003] Galactosamine (GalN), also known as 2-amino-2-deoxy-D-galactose or galactosamine, is a key component of the sugar units of natural polysaccharides and a constituent monosaccharide of numerous sulfated polysaccharides. In sulfated polysaccharides, the amino group of galactosamine is often modified with sulfate groups, and some are also modified with acetyl groups. Detection of monosaccharides such as galactosamine typically requires cleavage (to release galactosamine) and derivatization, otherwise chromatographic separation and detection are difficult. However, the structural characteristics of polysaccharide chains, especially the sulfate modifications on the side chains of sulfated polysaccharides, can affect and interfere with cleavage and derivatization. Consequently, analytical methods are generally subject to poor applicability, low accuracy, and cumbersome testing. Scientifically and accurately determining the galactosamine content in polysaccharides, especially sulfated polysaccharides, has long been a challenge in the field.

[0004] With the advancement of science, a variety of analytical methods have been reported for the detection of monosaccharides in carbohydrate compounds, including colorimetry, gas chromatography, capillary electrophoresis, and high-performance liquid chromatography. Colorimetry is the most classic, such as the Wagner assay for galactosamine. However, this assay suffers from poor specificity and accuracy. Furthermore, gas chromatography, capillary electrophoresis, and high-performance liquid chromatography with ultraviolet detection typically require fragmentation and derivatization, making them less convenient and rapid. High-performance liquid chromatography (HPLC) with differential refractive index and evaporative light scattering detection, while requiring only fragmentation without derivatization, lacks stability and reproducibility compared to UV detection. Furthermore, pulsed amperometric detection and ion chromatography are also a widely used method for monosaccharide determination. High-performance ion chromatography, developed based on high-performance liquid chromatography, is a liquid chromatography method for the analysis of anions and cations. The principle of ion chromatography is the reversible exchange between ions on an ion exchange column and solute ions in the mobile phase, resulting in separation of these ions due to their different affinities for the exchanger. Ion chromatography can use a NaOH solution as the eluent for monosaccharide determination. The OH-ions in the eluent act as eluent ions and provide the high pH environment required for sugar dissociation. High-performance ion chromatography (HPLC) for monosaccharide determination offers the advantages of simple, convenient, and highly sensitive separation of sugars in samples without the need for derivatization. It can also simultaneously determine multiple components.

[0005] However, as mentioned above, sulfated polysaccharides, due to the presence of side-chain sulfate modification, will seriously affect the efficiency of polysaccharide cleavage to release monosaccharides, and will also interfere with subsequent derivatization detection. Therefore, it is necessary to find an accurate, efficient and applicable analytical method to solve the cleavage of sulfated polysaccharides and subsequent inspection and detection. Summary of the Invention

[0006] In view of the above-mentioned defects in the prior art, the present invention aims to provide a method for analyzing the content of galactosamine in sulfated polysaccharides, which uses high-temperature hydrochloric acid hydrolysis to release monosaccharides and then conducts ion chromatography for detection.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The test sample is weighed, placed in a hydrolysis tube, hydrolyzed with hydrochloric acid at high temperature, neutralized, and diluted with water to prepare a test sample solution; galactosamine reference solution of different concentrations is weighed, prepared with water, and diluted; the test sample solution and the galactosamine reference solution are respectively sampled and analyzed by ion chromatography; the galactosamine content in the test sample is calculated by the external standard method based on the chromatographic peak area.

[0009] The method for analyzing the content of galactosamine in sulfated polysaccharides of the present invention is characterized by comprising the following steps:

[0010] S1. Preparation of galactosamine reference solution:

[0011] Accurately weigh the galactosamine reference substance and prepare a galactosamine reference substance stock solution and a series of galactosamine standard curve solutions with water;

[0012] S2. Preparation of test solution:

[0013] Accurately weigh the test sample, place it in a hydrolysis tube, add hydrochloric acid, and hydrolyze it at high temperature. After complete hydrolysis, neutralize it with sodium hydroxide solution, add water to dilute to the volume, and prepare the test sample solution;

[0014] S3. Instrumental analysis:

[0015] The galactosamine reference solution and the test solution were detected and analyzed using an ion chromatograph and a sugar analysis column, with water-sodium hydroxide solution-sodium acetate solution as the mobile phase, Au as the working electrode and Ag / AgCl as the reference electrode.

[0016] S4. Result calculation:

[0017] The galactosamine standard curve solution was injected into the ion chromatograph for analysis in order from low to high concentration, and the standard curve was drawn with the galactosamine concentration as the horizontal axis and the chromatographic peak area as the vertical axis; under the same analytical conditions, the test solution was injected into the ion chromatograph for analysis to obtain the corresponding peak area, and the concentration of galactosamine in the test solution was obtained according to the standard curve. The ratio of this concentration to the prepared test sample concentration was the galactosamine content in the test sample.

[0018] Preferably, the standard curve solution of the galactosamine reference substance in step S1 has a concentration range of 0.5-20 μg / mL.

[0019] Preferably, the preparation of the test solution in step S2 comprises the following steps: weighing 10-20 mg of the test sample, placing it in a hydrolysis tube, adding 1-3 mL of 2-6 mol / L hydrochloric acid solution, and placing it in a 90-120°C oil bath until the test sample is completely dissolved. After reacting for 4-6 hours, neutralizing it with sodium hydroxide solution, and diluting it to the fixed volume with water.

[0020] Preferably, in the instrumental analysis in step S3, the sugar analysis column includes but is not limited to PA10, PA20 and / or PA200, the flow rate is 0.2 - 0.6 mL / min, the column temperature is 20 - 40°C, and the injection volume is 10 - 25 μL.

[0021] Preferably, in the instrumental analysis in step S3, the mobile phase is water, sodium hydroxide solution and sodium acetate solution, which are mixed according to a gradient elution program, wherein: the concentration of the sodium hydroxide solution is 15-35 mmol / L, and the concentration of the sodium acetate solution is 0.5-1.5 mol / L.

[0022] Preferably, in the instrumental analysis of step S3, the gradient elution procedure is calculated as follows, in terms of volume percentage:

[0023] 0-20 min: sodium hydroxide solution 5-15%, sodium acetate solution 0%, water to 100%;

[0024] 20.1 min: Sodium hydroxide solution 5-15%, sodium acetate solution 2-8%, make up to 100% with water;

[0025] 20.1 – 30 min: Sodium hydroxide solution 5 - 15%, sodium acetate solution 2 - 8%, make up to 100% with water;

[0026] 30.1 min: Sodium hydroxide solution 90-99%, sodium acetate solution 0%, water to 100%;

[0027] 30.1 – 50 min: Sodium hydroxide solution 90-99%, sodium acetate solution 0%, make up to 100% with water;

[0028] 50.1 min: Sodium hydroxide solution 5-15%, sodium acetate solution 0%, water to 100%;

[0029] 50.1 – 60 min: Sodium hydroxide solution 5 - 15%, sodium acetate solution 0%, make up to 100% with water.

[0030] More preferably, in the instrumental analysis of step S3, the gradient elution procedure is, in terms of volume percentage, as follows:

[0031] 0-20 min: 25 mmol / L sodium hydroxide solution 10%, water 90%;

[0032] 20.1 min: 25 mmol / L sodium hydroxide solution 10%, 1 mol / L sodium acetate solution 5%, water 90%;

[0033] 20.1 – 30 min: 25 mmol / L sodium hydroxide solution 10%, 1 mol / L sodium acetate solution 5%, water 85%;

[0034] 30.1 min: 25 mmol / L sodium hydroxide solution 95%, water 5%;

[0035] 30.1 – 50 min: 25 mmol / L sodium hydroxide solution 95%, water 5%;

[0036] 50.1 min: 25 mmol / L sodium hydroxide solution 10%, water 90%;

[0037] 50.1 – 60 min: 25 mmol / L sodium hydroxide solution 10%, 90%.

[0038] In the above elution procedure, galactosamine and some neutral monosaccharides are eluted by the mobile phase containing a low percentage of sodium hydroxide solution in the first stage of 0-20 minutes. Other monosaccharides such as glucuronic acid and some acidic monosaccharides are eluted by the mobile phase containing a sodium acetate solution in the second stage of 20-30 minutes. Some other difficult-to-elute substances are eluted by the mobile phase containing a high percentage of sodium hydroxide solution in the third stage of 30-50 minutes. In the fourth stage of 50-60 minutes, the mobile phase is exchanged and equilibrated to prepare for the next injection analysis.

[0039] In addition, the analytical method of the present invention is characterized by its application in the quality analysis of sulfated polysaccharides.

[0040] In the specific examples (Example 1 and Example 2) but not limited to this group of examples, the acid used in the pre-treatment hydrolysis conditions of the sample was changed. Compared with hydrochloric acid, trifluoroacetic acid, another commonly used acid in the literature or in the field of polysaccharide analysis, cannot completely dissociate aminogalactose due to the presence and influence of sulfated modifications on the side chains of sulfated polysaccharides. Therefore, the acid used for the hydrolysis of the sample in this method must be hydrochloric acid.

[0041] In specific examples (Example 1 and Example 3) but not limited to this group of examples, the hydrolysis conditions and instrument analysis conditions of the test sample were changed. The results showed that when the hydrochloric acid hydrolysis conditions and instrument analysis conditions were changed within a certain range, the analysis results remained unchanged, indicating that the durability of this method is good.

[0042] In specific examples (Examples 1, 3, and 4) but not limited to this group of examples, when the test samples were different types of sulfated polysaccharides, the results showed that the results of this method were reproducible and consistent with the literature values, indicating that the method has a wide range of applicability.

[0043] In a specific embodiment (Example 5) but not limited to this group of embodiments, the method for analyzing the content of aminogalactose in the sulfated polysaccharide was studied and investigated for specificity, repeatability, linearity, range, injection precision, accuracy, solution stability, detection limit and quantification limit, and the results all met the methodological validation requirements.

[0044] The outstanding effect of the present invention is: providing a method for analyzing the content of galactosamine in sulfated polysaccharides based on high-temperature hydrochloric acid hydrolysis and ion chromatography, wherein a test sample is hydrolyzed with hydrochloric acid at high temperature to release galactosamine, and an galactosamine reference substance does not need to be hydrolyzed. The galactosamine reference substance and the test sample are separated by an ion chromatograph and a sugar analysis column, and detected by a pulsed amperometric detector, and the galactosamine reference substance and the test sample are analyzed separately to perform quantitative analysis of galactosamine. The method has the advantages of being simple, rapid, accurate, efficient, and not requiring derivatization treatment.

[0045] The specific implementation of the present invention will be further described below in conjunction with the accompanying drawings to make the technical solution of the present invention easier to understand and grasp. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram of the ion chromatogram of the aminogalactose reference substance described in Example 1.

[0047] Figure 2 This is the chromatogram of the low-molecular-weight sea cucumber glycosaminoglycan hydrolyzed solution described in Example 1.

[0048] Figure 3 Schematic diagram of the correlation between the concentration of the aminogalactose reference substance and the chromatographic peak area described in Example 1.

[0049] Figure 4 Schematic diagram of the correlation between the concentration of the aminogalactose reference substance and the chromatographic peak area described in Example 3.

[0050] Figure 5 Schematic diagram of the correlation between the concentration of the aminogalactose reference substance and the chromatographic peak area described in Example 4. DETAILED DESCRIPTION

[0051] Equipment and reagents:

[0052] Equipment: Dionex ICS-5000 ion chromatograph;

[0053] Galactosamine hydrochloride reference substance: national standard substance;

[0054] Fucose reference substance: national standard substance;

[0055] Glucuronic acid reference substance: national standard substance;

[0056] Reagents: sodium hydroxide (Sinopharm, AR), sodium acetate (Sinopharm, AR), hydrochloric acid (Sinopharm, AR), trifluoroacetic acid (Sinopharm, AR), and ultrapure water.

[0057] Example 1 Determination of galactosamine content in low-molecular-weight sea cucumber glycosaminoglycans

[0058] S1. Preparation of galactosamine reference solution:

[0059] Accurately weigh 10.52 mg of galactosamine hydrochloride reference substance and place it in a 100 mL volumetric flask. Dissolve it in water and dilute to the mark. Shake well to obtain a solution with a concentration of 105.2 μg / mL and a galactosamine concentration of 87.4 μg / mL. Accurately measure 0.1, 0.2, 0.4, 0.8, 1.2, 1.6, and 2.0 mL, respectively, and place them in a 10 mL volumetric flask. Dilute to the mark with water and shake well to obtain galactosamine reference substance solutions of varying concentrations (0.8–17.5 μg / mL).

[0060] S2. Preparation of test solution:

[0061] Accurately weigh 10 mg of low-molecular-weight sea cucumber glycosaminoglycan test sample, place it in a hydrolysis tube, add 3 mL of 4 mol / L hydrochloric acid solution, shake until the test sample is completely dissolved, seal the hydrolysis tube, place it in a 120°C oil bath, react for 6 hours, neutralize it with 4 mol / L sodium hydroxide solution, transfer it to a 100 mL volumetric flask, and dilute it to the scale with water; accurately measure 3 mL into a 10 mL volumetric flask, dilute it to the scale with water, and shake well to obtain the product.

[0062] S3. Instrumental analysis:

[0063] The galactosamine reference solution and the test solution were detected and analyzed using an ion chromatograph and a sugar analysis column, with water-sodium hydroxide solution-sodium acetate solution as the mobile phase and a pulsed amperometric detector with Au as the working electrode and Ag / AgCl as the reference electrode, and quantification was performed using the external standard method.

[0064] Chromatographic columns: Dionex Amino Trap Column, Dionex PA20 Guard Column, and Dionex PA20 Analytical Column connected in series;

[0065] Mobile phase: water, sodium hydroxide at a concentration of 25 mmol / L, sodium acetate at a concentration of 1 mol / L;

[0066] Flow rate: 0.4 mL / min;

[0067] Column temperature: 30 °C;

[0068] The ion chromatograph uses gradient elution. In terms of volume percentage, the gradient elution procedure is:

[0069] Table 1 Mobile phase gradient elution program

[0070] Time (min) water(%) 25 mmol / L sodium hydroxide (%) 1 mol / L sodium acetate (%) 0 90 10 0 21 90 10 0 21.1 85 10 5 30 85 10 5 30.1 5 95 0 50 5 95 0 50.1 90 10 0 60 90 10 0

[0071] S4. Result calculation:

[0072] The galactosamine standard curve solution was injected into the ion chromatograph for analysis in order from low to high concentration, and the standard curve was drawn with the galactosamine concentration as the horizontal axis and the chromatographic peak area as the vertical axis; under the same analytical conditions, the test solution was injected into the ion chromatograph for analysis to obtain the corresponding peak area, and the concentration of galactosamine in the test solution was obtained according to the standard curve. The ratio of this concentration to the prepared test sample concentration was the galactosamine content in the test sample.

[0073] With the concentration of the reference substance as the horizontal axis and the chromatographic peak area as the vertical axis, draw a standard curve. Figure 1 .also, Figure 2 The ion chromatogram of the galactosamine reference substance is given. Figure 3 The chromatogram of the hydrolyzed solution of low molecular weight sea cucumber glycosaminoglycans is given.

[0074] Table 2 Reference substance concentration and chromatographic peak area data

[0075] Reference test number Reference substance concentration (µg / mL) Peak area Reference solution 1 0.8738 0.0686 Reference solution 2 1.7476 0.1520 Reference solution 3 3.4953 0.3487 Reference solution 4 6.9906 0.6318 Reference solution 5 10.4858 0.9815 Reference solution 6 13.9811 1.2686 Reference solution 7 17.4764 1.5526

[0076] According to the standard curve, the content of galactosamine was calculated:

[0077] Table 3 Results of galactosamine content in low molecular weight sea cucumber glycosaminoglycan test samples

[0078]

[0079] The results showed that the content of galactosamine in low molecular weight sea cucumber glycosaminoglycans was 18.60%.

[0080] Example 2 Determination of galactosamine content in low-molecular-weight sea cucumber glycosaminoglycans

[0081] S1 and S3 were the same as in Example 1, except that the hydrolysis conditions in S2 were changed to trifluoroacetic acid hydrolysis conditions commonly used in polysaccharide determination, and the test results were compared.

[0082] S2. Preparation of test solution:

[0083] The low-molecular-weight sea cucumber glycosaminoglycan test sample was the same as in Example 1. 10 mg was accurately weighed and placed in a hydrolysis tube. 3 mL of 6 mol / L trifluoroacetic acid solution was added. After the test sample was completely dissolved, it was placed in a 120°C oil bath and reacted for 6 hours. The mixture was neutralized with 4 mol / L sodium hydroxide solution and transferred to a 100 mL volumetric flask. Water was added to dilute to the mark. 3 mL was accurately measured and transferred to a 10 mL volumetric flask. Water was added to dilute to the mark and the mixture was shaken to obtain the product.

[0084] S4. Result calculation:

[0085] The standard curve is the same as that in Example 1.

[0086] Table 4 Results of galactosamine content in low molecular weight sea cucumber glycosaminoglycan test samples

[0087]

[0088] The results show that the galactosamine content of low-molecular-weight sea cucumber glycosaminoglycans is 14.83%. Compared with hydrochloric acid hydrolysis, the content obtained is lower. This is because sulfated polysaccharides are often composed of galactosamine and other sugar units, and the sulfate groups on the side chains seriously affect the release and determination of galactosamine. Trifluoroacetic acid has a weaker hydrolysis degree and cannot completely dissociate galactosamine. This method uses hydrochloric acid hydrolysis conditions to measure the galactosamine content, which is more accurate.

[0089] Example 3 Determination of galactosamine content in low molecular weight sea cucumber glycosaminoglycans

[0090] Compared with Example 1, the hydrolysis conditions in step S2 and the instrumental analysis conditions in step S3 in this example are changed.

[0091] S1. Preparation of galactosamine reference solution:

[0092] Accurately weigh 10.15 mg of galactosamine hydrochloride reference substance and place it in a 100 mL volumetric flask. Dissolve it in water and dilute to the mark. Shake well to obtain a solution with a concentration of 101.5 μg / mL and a galactosamine concentration of 84.3 μg / mL. Accurately measure 0.1, 0.2, 0.4, 0.8, 1.2, 1.6, and 2.0 mL, respectively, and place them in a 10 mL volumetric flask. Dilute to the mark with water and shake well to obtain galactosamine reference substance solutions of varying concentrations (0.8–16.8 μg / mL).

[0093] S2. Preparation of test solution:

[0094] The low-molecular-weight sea cucumber glycosaminoglycan test sample was the same as in Example 1. 20 mg was accurately weighed and placed in a hydrolysis tube. 2 mL of 2 mol / L hydrochloric acid solution was added. After the test sample was completely dissolved, it was placed in a 90°C oil bath and reacted for 5 hours. The mixture was neutralized with 4 mol / L sodium hydroxide solution and transferred to a 100 mL volumetric flask. Water was added to dilute to the mark. 1.5 mL was accurately measured and transferred to a 10 mL volumetric flask. Water was added to dilute to the mark and the mixture was shaken to obtain the product.

[0095] S3. Instrumental analysis:

[0096] The galactosamine reference solution and the test solution were detected and analyzed by ion chromatography and pulsed amperometric detection, using a sugar analysis column, water-sodium hydroxide solution-sodium acetate solution as the mobile phase, Au as the working electrode and Ag / AgCl as the reference electrode, and the external standard method was used for quantification.

[0097] Chromatographic columns: Dionex Amino Trap Column, Dionex PA20 Guard Column, and Dionex PA20 Analytical Column connected in series;

[0098] Mobile phase: sodium hydroxide at a concentration of 20 mmol / L, sodium acetate at a concentration of 1.2 mol / L;

[0099] Flow rate: 0.6 mL / min;

[0100] Column temperature: 40 °C;

[0101] Injection volume: 25 µL.

[0102] The ion chromatograph uses a gradient elution: in volume percentage, the gradient elution procedure is:

[0103] Table 5 Mobile phase gradient elution program

[0104] Time (min) water(%) 20 mmol / L sodium hydroxide (%) 1.2 mol / L sodium acetate (%) 0 88 12 0 21 88 12 0 21.1 84 12 4 30 84 12 4 30.1 4 96 0 50 4 96 0 50.1 88 12 0 60 88 12 0

[0105] S4. Result calculation:

[0106] The galactosamine standard curve solution was injected into the ion chromatograph for analysis in order from low to high concentration, and the standard curve was drawn with the galactosamine concentration as the horizontal axis and the chromatographic peak area as the vertical axis; under the same analytical conditions, the test solution was injected into the ion chromatograph for analysis to obtain the corresponding peak area, and the concentration of galactosamine in the test solution was obtained according to the standard curve. The ratio of this concentration to the prepared test sample concentration was the galactosamine content in the test sample.

[0107] With the concentration of the reference substance as the horizontal axis and the chromatographic peak area as the vertical axis, draw a standard curve. Figure 4 .

[0108] Table 6 Reference substance concentration and chromatographic peak area data

[0109] Reference test number Reference substance concentration (µg / mL) Peak area Reference solution 1 0.8431 0.1319 Reference solution 2 1.6862 0.3133 Reference solution 3 3.3723 0.7897 Reference solution 4 6.7447 1.7027 Reference solution 5 10.1170 2.4110 Reference solution 6 13.4894 3.1722 Reference solution 7 16.8617 3.7903

[0110] According to the standard curve, the content of galactosamine was calculated:

[0111] Table 7 Results of galactosamine content in low molecular weight sea cucumber glycosaminoglycan test samples

[0112]

[0113] The results show that the galactosamine content in the low-molecular-weight sea cucumber glycosaminoglycans is 18.20%, which is basically consistent with the result measured in Example 1. Therefore, the content analysis results remain unchanged when the hydrochloric acid hydrolysis conditions and instrument analysis conditions are changed within a certain range, indicating that the present method has good durability.

[0114] Example 4 Determination of galactosamine content in chondroitin sulfate

[0115] S1. Preparation of galactosamine reference solution:

[0116] Accurately weigh 10.38 mg of galactosamine hydrochloride reference substance and place it in a 100 mL volumetric flask. Dissolve it in water and dilute to the mark. Shake well to obtain a solution with a concentration of 103.8 μg / mL and a galactosamine concentration of 86.2 μg / mL. Accurately measure 0.2, 0.4, 0.8, 1.2, 1.6, and 2.0 mL, respectively, and place them in a 10 mL volumetric flask. Add water to dilute to the mark and shake well to obtain galactosamine reference substance solutions of varying concentrations (1.7–17.2 μg / mL).

[0117] S2. Preparation of test solution:

[0118] Weigh 15 mg of chondroitin sulfate test sample, place it in a hydrolysis tube, add 1 mL of 4 mol / L hydrochloric acid solution, wait until the test sample is completely dissolved, place in a 110°C oil bath, react for 4 hours, transfer to a 100 mL volumetric flask, add water to dilute to the scale, accurately measure 2 mL into a 10 mL volumetric flask, add water to dilute to the scale, shake well, and obtain.

[0119] S3. Instrumental analysis:

[0120] The galactosamine reference solution and the test solution were detected and analyzed by ion chromatography and pulsed amperometric detection, using a sugar analysis column, water-sodium hydroxide solution-sodium acetate solution as the mobile phase, Au as the working electrode and Ag / AgCl as the reference electrode, and the external standard method was used for quantification.

[0121] Chromatographic columns: Dionex Amino Trap Column, Dionex PA20 Guard Column, and Dionex PA20 Analytical Column connected in series;

[0122] Mobile phase: sodium hydroxide at a concentration of 35 mmol / L, sodium acetate at a concentration of 0.8 mol / L;

[0123] Flow rate: 0.4 mL / min;

[0124] Column temperature: 30 °C;

[0125] Injection volume: 10 µL.

[0126] The ion chromatograph uses a gradient elution: in volume percentage, the gradient elution procedure is:

[0127] Table 8 Mobile phase gradient elution program

[0128] Time (min) water(%) 35 mmol / L sodium hydroxide (%) 0.8 mol / L sodium acetate (%) 0 94 6 0 21 94 6 0 21.1 86 6 8 30 86 6 8 30.1 6 94 0 50 6 94 0 50.1 94 6 0 60 94 6 0

[0129] S4. Result calculation:

[0130] The galactosamine standard curve solution is injected into the ion chromatograph for analysis in order from low to high concentration, and the standard curve is drawn with the galactosamine concentration as the horizontal axis and the chromatographic peak area as the vertical axis; under the same analytical conditions, the test solution is injected into the ion chromatograph for analysis to obtain the corresponding peak area, and the concentration of galactosamine in the test solution is obtained according to the standard curve. The ratio of this concentration to the concentration of the prepared test sample is the galactosamine content in the test sample.

[0131] With the concentration of the reference substance as the horizontal axis and the chromatographic peak area as the vertical axis, draw a standard curve. Figure 5 .

[0132] Table 9 Reference substance concentration and chromatographic peak area data

[0133] Reference test number Reference substance concentration (µg / mL) Peak area Reference solution 1 1.7250 0.1580 Reference solution 2 3.4500 0.3572 Reference solution 3 6.8999 0.7411 Reference solution 4 10.3499 1.2730 Reference solution 5 13.7998 1.6541 Reference solution 6 17.2498 2.0973

[0134] According to the standard curve, the content of galactosamine was calculated:

[0135] Table 10. Data on the content of galactosamine in chondroitin sulfate test samples.

[0136]

[0137] The results show that the galactosamine content in chondroitin sulfate is 26%, which is basically consistent with the literature report and the theoretical value of chondroitin sulfate, indicating the accuracy of this method.

[0138] Example 5 Methodological Investigation and Verification

[0139] 1. System suitability test.

[0140] (1) Measurement method

[0141] Specificity: Weigh 10 mg each of fucose and glucuronic acid reference substances; prepare each as follows: place in a 100 mL volumetric flask, dissolve and dilute to the mark with water, shake well, accurately measure 1 mL, place in a 10 mL volumetric flask, dilute to the mark with water, shake well, to a solution concentration of 10 µg / mL; assay according to the ion chromatography conditions of Example 1.

[0142] Injection precision: Prepare the reference solution according to Example 1, weigh and dilute to a concentration of 10.5 μg / mL, repeat the injection 6 times according to the ion chromatography conditions of Example 1, and calculate the relative standard deviation of the peak area.

[0143] (2) Test results and conclusions

[0144] The results of injection precision and specificity tests are shown in Tables 11-12.

[0145] Table 11 System suitability test results

[0146] Injection number 1 2 3 4 5 6 RSD (%) Peak area 0.9815 0.9756 0.9896 0.9745 0.9803 0.9902 0.68

[0147] Table 12 Specificity determination results.

[0148] Sample name Peak time (min) Fucose reference 5.58 Galactosamine reference substance 10.05 Glucuronic acid reference substance 27.49

[0149] According to the test results, the RSD of the reference peak area = 0.68% ≤ 2%, indicating that the method has good system applicability and specificity.

[0150] 2. Linearity and range

[0151] The linear regression equation obtained from the test results of Example 1 is Y = 0.0897X + 0.0097, R 2 =0.9985. The control showed good linearity in the concentration range of 0.5 - 20 µg / mL.

[0152] 3. Precision (repeatability test)

[0153] According to the test results obtained in Example 1, the galactosamine content in the six low-molecular-weight sea cucumber glycosaminoglycans was between 18.48% and 18.80%, with an RSD of 0.64% ≤ 2%, indicating that the analytical method had good repeatability.

[0154] 4. Solution stability

[0155] (1) Measurement method

[0156] The test solution was prepared as in Example 1 to obtain a test solution concentration of 30.27 μg / mL. The solution was placed at room temperature for 0, 2, 4, 8, 12, 18 and 24 hours, respectively. The sample was analyzed according to the chromatographic conditions in Example 1, and the chromatographic peak areas were recorded to calculate the relative standard deviation.

[0157] (2) Test results and conclusions

[0158] The results of the solution stability test are shown in Table 13.

[0159] Table 13 Solution stability test results

[0160] Injection number 0h 2h 4h 8h 12h 18h 24h RSD (%) Peak area 0.5114 0.5233 0.5095 0.5179 0.5241 0.5198 0.5254 1.21

[0161] The test results show that when the test solution is placed at room temperature for 0, 2, 4, 8, 12, 18 and 24 hours, the RSD of the peak area is 1.21% ≤ 2%, indicating that the solution has good stability within 24 hours.

[0162] 5. Accuracy (spike recovery)

[0163] (1) Measurement method

[0164] Preparation of reference solution:

[0165] Accurately weigh 10.52 mg of galactosamine hydrochloride reference substance, place it in a 100 mL volumetric flask, add water to dissolve and dilute to the scale, shake well, and the galactosamine concentration is 87.4 μg / mL.

[0166] Preparation of test solution:

[0167] Take the low-molecular-weight sea cucumber glycosaminoglycan test sample of Example 1, accurately weigh 10.09 mg, place it in a hydrolysis tube, add 3 mL of 6 mol / L hydrochloric acid solution, wait until the test sample is completely dissolved, place it in a 120 ° C oil bath, react for 6 hours, neutralize it with 4 mol / L sodium hydroxide solution, transfer it to a 100 mL volumetric flask, and dilute it to the scale with water.

[0168] Accurately measure 3 mL of the test solution and add 0.3, 0.6, and 0.9 mL of the reference solution to a 10 mL volumetric flask, respectively. Dilute to the mark with water and shake well. Prepare three replicates. Follow the chromatographic conditions in Example 1 for sample analysis, record the peak areas, and calculate the spiked recovery.

[0169] (2) Test results and conclusions

[0170] The results of the spike recovery test are shown in Table 14.

[0171] Table 14 Spike recovery test results

[0172]

[0173] From the test results, we can see that the RSD of the recovery rate is 1.55%, which shows that the accuracy of this method is high.

[0174] 6. Limit of Detection and Limit of Quantitation

[0175] (1) Measurement method

[0176] According to the analytical method of Example 1, the lowest concentration in the linear range of 0.5 μg / mL was taken, and different concentrations were diluted and injected to obtain the signal-to-noise ratio.

[0177] (2) Test results and conclusions

[0178] When the signal-to-noise ratio was 3:1, the detection limit of galactosamine was 0.05 μg / mL, and when the signal-to-noise ratio was 10:1, the quantification limit of galactosamine was 0.1 μg / mL.

[0179] The above embodiment uses hydrochloric acid high-temperature hydrolysis of the sample combined with ion chromatography to determine the galactosamine content in sulfated polysaccharides. No derivatization is required, and the operation is simple and quick. This pretreatment method of hydrochloric acid high-temperature hydrolysis sample overcomes the difficulty of dissociation of sulfated polysaccharides, and completely releases the galactosamine in the sample. The measurement results are true and reliable. In addition, the method has good specificity, a wide linear range, a good linear relationship, and high repeatability and precision of the test results. It is suitable for the analysis and detection of the galactosamine content in sulfated polysaccharides.

[0180] The above embodiments are preferred examples of the present invention and are not limitations on the implementation methods. Without departing from the principles of the present invention, other forms of changes and modifications can be made to the present invention. These obvious changes and modifications still fall within the scope of protection of the present invention.

Claims

1. A method for analyzing the content of galactosamine in sulfated polysaccharides, characterized in that: The steps include: S1. Preparation of galactosamine reference solution: Accurately weigh the galactosamine reference substance and prepare a galactosamine reference substance stock solution and a series of galactosamine standard curve solutions with water; S2. Preparation of test solution: Accurately weigh the test sample, place it in a hydrolysis tube, add hydrochloric acid, and hydrolyze it at high temperature. After complete hydrolysis, neutralize it with sodium hydroxide solution, add water to dilute to the volume, and prepare the test sample solution; S3. Instrumental analysis: The galactosamine reference solution and the test solution were detected and analyzed using an ion chromatograph and a sugar analysis column, with water-sodium hydroxide solution-sodium acetate solution as the mobile phase, Au as the working electrode and Ag / AgCl as the reference electrode. S4. Result calculation: The galactosamine standard curve solution was injected into the ion chromatograph in order of concentration from low to high for analysis, and the standard curve was drawn with the galactosamine concentration as the horizontal axis and the chromatographic peak area as the vertical axis; Under the same analytical conditions, the test solution was injected into the ion chromatograph for analysis to obtain the corresponding peak area. The concentration of galactosamine in the test solution was obtained according to the standard curve. The ratio of this concentration to the concentration of the prepared test sample was the content of galactosamine in the test sample.

2. The method according to claim 1, characterized in that The standard curve solution of the galactosamine reference substance in step S1 has a concentration range of 0.5 - 20 μg / mL.

3. The method according to claim 1, characterized in that The preparation of the test solution in step S2 comprises the following steps: weighing 10-20 mg of the test sample, placing it in a hydrolysis tube, adding 1-3 mL of a 2-6 mol / L hydrochloric acid solution, and placing it in a 90-120°C oil bath until the test sample is completely dissolved. After reacting for 4-6 hours, the solution is neutralized with a sodium hydroxide solution and diluted to volume with water.

4. The method according to claim 1, wherein For the instrumental analysis in step S3, the sugar analysis column includes PA10, PA20 and / or PA200, the flow rate is 0.2 - 0.6 mL / min, the column temperature is 20 - 40°C, and the injection volume is 10 - 25 μL.

5. The method according to claim 1, wherein In the instrumental analysis in step S3, the mobile phase is water-sodium hydroxide solution-sodium acetate solution, and the three are mixed according to a gradient elution program, wherein: the concentration of the sodium hydroxide solution is 15-35 mmol / L, and the concentration of the sodium acetate solution is 0.5-1.5 mol / L.

6. The method according to claim 1 and claim 5, characterized in that The instrumental analysis of step S3, in terms of volume percentage, was performed using the gradient elution procedure: 0-20 min: sodium hydroxide solution 5-15%, sodium acetate solution 0%, water to 100%; 20.1 min: Sodium hydroxide solution 5-15%, sodium acetate solution 2-8%, make up to 100% with water; 20.1 – 30 min: Sodium hydroxide solution 5 - 15%, sodium acetate solution 2 - 8%, make up to 100% with water; 30.1 min: Sodium hydroxide solution 90-99%, sodium acetate solution 0%, water to 100%; 30.1 – 50 min: Sodium hydroxide solution 90-99%, sodium acetate solution 0%, make up to 100% with water; 50.1 min: Sodium hydroxide solution 5-15%, sodium acetate solution 0%, water to 100%; 50.1 – 60 min: Sodium hydroxide solution 5 - 15%, sodium acetate solution 0%, make up to 100% with water.

7. The method according to claim 1 and claims 6-7, characterized in that The instrumental analysis in step S3, in terms of volume percentage, was performed using a mobile phase gradient elution program as follows: 0-20 min: 25 mmol / L sodium hydroxide solution 10%, water 90%; 20.1 min: 25 mmol / L sodium hydroxide solution 10%, 1 mol / L sodium acetate solution 5%, water 90%; 20.1 – 30 min: 25 mmol / L sodium hydroxide solution 10%, 1 mol / L sodium acetate solution 5%, water 85%; 30.1 min: 25 mmol / L sodium hydroxide solution 95%, water 5%; 30.1 – 50 min: 25 mmol / L sodium hydroxide solution 95%, water 5%; 50.1 min: 25 mmol / L sodium hydroxide solution 10%, water 90%; 50.1 – 60 min: 25 mmol / L sodium hydroxide solution 10%, 90%.

8. The method according to claim 1, characterized in that Application in quality analysis of sulfated polysaccharides.

Images