Detection method of glycosaminoglycan

Abstract

The invention provides a method for quantitatively detecting the content of glycosaminoglycans, the method comprises the step of detecting glycosaminoglycans by using a high performance liquid chromatography, and the chromatographic conditions of the high performance liquid chromatography are as follows: a chromatographic column is an HILIC chromatographic column; the mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A is an ammonium formate or ammonium acetate solution, and the mobile phase B is an acetonitrile solution. The detection method provided by the invention is simple to operate, short in detection time, capable of realizing simultaneous detection of various glycosaminoglycans, good in separation effect, high in precision and accuracy, solvent-saving and convenient in sample preparation.

Description

Technical Field

[0001] The present application relates to the field of high performance liquid chromatography detection, and more specifically to a method for detecting glycosaminoglycans. Background Art

[0002] Glycosaminoglycan (GAG) is a type of linear polysaccharide composed of repeated amino sugar and uronic acid disaccharide units. It is widely present in the extracellular matrix and has important biological functions. Hyaluronic acid, chondroitin, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, etc. are all glycosaminoglycan substances. Glycosaminoglycans usually have the functions of joint lubrication, hydration, anti-inflammatory, and immune regulation. They are often added as important active ingredients to food, health products, medicines, and skin care products. For example, adding glycosaminoglycans to collagen powder can not only maintain skin elasticity but also replenish moisture to the skin and promote skin repair.

[0003] When multiple glycosaminoglycans are added to the product at the same time, the content of different types of glycosaminoglycans cannot be accurately determined. For example, the detection of hyaluronic acid usually adopts the sulfuric acid-carbazole method, using borax as a catalyst, and hyaluronic acid is acid-hydrolyzed with sulfuric acid to separate glucuronic acid. Glucuronic acid reacts with carbazole to form an organic complex, which is used to display a unique purple color. The absorbance is measured to determine the content of hyaluronic acid, but this method can also be used for the detection of chondroitin. Chondroitin can also react with carbazole after acid hydrolysis with sulfuric acid to display a unique purple color. This method cannot accurately determine the content of products containing hyaluronic acid and chondroitin at the same time; for the terminal product, sodium hyaluronate is also usually detected by high-performance liquid chromatography, and sodium hyaluronate is enzymatically hydrolyzed into unsaturated disaccharides using a strong cation exchange column and phosphoric acid as the mobile phase for detection, but the unsaturated disaccharides after enzymatic hydrolysis of chondroitin and chondroitin sulfate are isomers with sodium hyaluronate disaccharides, and peak at the same position, so they cannot be separated.

[0004] In the actual production process, different methods are used to detect different types of glycosaminoglycans in the product, which is not only complicated to operate, but also wastes time, manpower and cost. If different types of glycosaminoglycans in the product can be detected simultaneously, the production efficiency will be greatly improved.

[0005] In the prior art, JP7185232B2 provides a method for analyzing glycosaminoglycans, which uses a glycosaminoglycan-specific enzyme to enzymatically hydrolyze samples to obtain various types of polysaccharides, and then uses a liquid chromatography-mass spectrometry method to detect the various polysaccharides. This method requires mass spectrometry to achieve detection, has strict detection conditions, is costly, and is not suitable for large-scale industrial production. Conventional glycosaminoglycan liquid phase detection methods cannot achieve effective separation of different types of glycosaminoglycan substances. The peak positions of various glycosaminoglycan substances are concentrated, and there is interference between them, making it impossible to achieve accurate detection. Summary of the invention

[0006] Aiming at the shortcomings of the glycosaminoglycan detection methods in the prior art. The present application provides a method for detecting glycosaminoglycans by selecting appropriate chromatographic conditions. The present application provides a new method for detecting glycosaminoglycans, which utilizes a hydrophilic interaction chromatographic column in combination with a high performance liquid chromatography with a mixed solvent of ammonium formate or ammonium acetate solution and an organic solvent as the mobile phase for isocratic elution. It can not only quickly and accurately detect the glycosaminoglycan content in the product, but also realize the simultaneous detection of multiple different types of glycosaminoglycans.

[0007] The present application provides a method for detecting multiple types of glycosaminoglycans under the same chromatographic conditions, which can achieve simultaneous detection of glycosaminoglycans including sodium hyaluronate, chondroitin and chondroitin sulfate, and the chromatographic peaks between different glycosaminoglycans can be completely separated. The method has specificity, applicability and repeatability.

[0008] Specifically, this application involves the following technical solutions:

[0009] Item 1. A method for detecting glycosaminoglycans, comprising detecting glycosaminoglycans using high performance liquid chromatography, wherein the chromatographic conditions of the high performance liquid chromatography are as follows:

[0010] The chromatographic column is a hydrophilic interaction chromatographic column;

[0011] The mobile phase includes a mobile phase A and a mobile phase B. The mobile phase A is an ammonium formate solution or an ammonium acetate solution, and the mobile phase B is an acetonitrile solution.

[0012] Item 2. The method for detecting glycosaminoglycans according to Item 1, wherein the hydrophilic interaction chromatography column is a HILIC chromatography column.

[0013] Item 3. The method for detecting glycosaminoglycans according to any one of Items 1 to 2, wherein the concentration of the ammonium formate solution is greater than 1 mmol / L, preferably 5 mmol / L to 20 mmol / L.

[0014] Item 4. The method for detecting glycosaminoglycans according to any one of Items 1 to 3, wherein the pH of the ammonium formate solution is less than 6.0, preferably 3.0 to 4.5.

[0015] Item 5. The method for detecting glycosaminoglycans according to any one of Items 1 to 4, wherein the volume ratio of the mobile phase A to the mobile phase B is greater than 10:90, preferably (15-25):(85-75).

[0016] Item 6. The method for detecting glycosaminoglycans according to any one of Items 1 to 5, wherein the detector in the high performance liquid chromatography is an ultraviolet detector or a diode array detector, and the detection wavelength is 230-235 nm.

[0017] Item 7. The method for detecting glycosaminoglycans according to any one of Items 1 to 6, wherein the flow rate of the mobile phase is 0.5-1.5 ml / min; the column temperature of the chromatographic column in the high performance liquid chromatography is 20-50°C; the injection volume in the high performance liquid chromatography is 5-50 μl; and the elution mode of the high performance liquid chromatography is isocratic elution.

[0018] Item 8. The method for detecting glycosaminoglycan according to any one of Items 1 to 7, wherein the glycosaminoglycan comprises any one, two or three of sodium hyaluronate, chondroitin, and chondroitin sulfate.

[0019] Item 9. A method for detecting glycosaminoglycans, comprising the following steps:

[0020] preparing a solution of a sample to be tested;

[0021] Using the detection method described in any one of items 1 to 8 to measure the solution of the sample to be tested;

[0022] Based on the detection results, any one, two or three of sodium hyaluronate, chondroitin and chondroitin sulfate contained in glycosaminoglycans can be distinguished.

[0023] Item 10. The method according to Item 9, wherein the sodium hyaluronate, chondroitin and chondroitin sulfate contained in the glycosaminoglycan can be distinguished based on the test results, and the separation degree is greater than 1.5; the sodium hyaluronate, chondroitin and chondroitin sulfate contained in the glycosaminoglycan can be distinguished based on the test results, and the detection limit of the sodium hyaluronate is as low as 8.0×10 - 2 ppm; the detection limit of chondroitin is as low as 1.0×10 -1 ppm; the detection limit of chondroitin sulfate is as low as 2.0×10 - 1 ppm. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the chromatogram of the blank solution in Example 1.

[0025] Figure 2 This is the chromatogram of the sodium hyaluronate reference solution.

[0026] Figure 3 This is the chromatogram of the chondroitin reference solution.

[0027] Figure 4 This is the chromatogram of the chondroitin sulfate reference solution.

[0028] Figure 5 The chromatogram of the test solution in Example 1.

[0029] Figure 6 It is the chromatogram of the test solution in Comparative Example 1.

[0030] Figure 7 It is the chromatogram of the test solution in Comparative Example 2.

[0031] Figure 8 It is the chromatogram of the test solution in Comparative Example 5.

[0032] Fig. 9 It is the chromatogram of the test solution in Comparative Example 6. DETAILED DESCRIPTION

[0033] The present application is further described below in conjunction with examples. It should be understood that the examples are only used to further describe and illustrate the present application and are not used to limit the present application.

[0034] Unless otherwise defined, the technical and scientific terms in this specification have the same meaning as those generally understood by those skilled in the art. Although similar or identical methods and materials as described herein may be used in experiments or practical applications, materials and methods are described herein below. In the event of a conflict, the present specification including the definitions therein shall prevail. In addition, materials, methods and examples are provided for illustration only and are not restrictive. The present application is further described below in conjunction with specific embodiments, but is not intended to limit the scope of the present application.

[0035] Chromatography, also known as "chromatographic analysis", "chromatographic analysis" and "chromatography", is a separation and analysis method that is widely used in analytical chemistry, organic chemistry, biochemistry and other fields. Chromatography uses the selective distribution of different substances in different phases to elute the mixture in the stationary phase with a mobile phase. Different substances in the mixture will move along the stationary phase at different speeds, ultimately achieving the effect of separation.

[0036] As used herein, the term "detection limit" refers to the lowest concentration in the analytical sample that can only generate a reliable detection signal but cannot obtain an accurate measurement value. It refers to the minimum detectable amount of the target compound by the instrument detection system.

[0037] As used herein, the term "resolution" refers to the ratio of the difference between the retention values ​​of two adjacent chromatographic peaks to half the sum of the low widths of the two chromatographic peaks. To determine the separation of two adjacent components in a chromatographic column, the resolution R can be used as a separation efficiency indicator of a chromatographic column.

[0038] The present application provides a method for detecting glycosaminoglycans, which comprises detecting glycosaminoglycans using high performance liquid chromatography, and the chromatographic conditions of the high performance liquid chromatography are as follows:

[0039] The chromatographic column is a hydrophilic interaction chromatographic column;

[0040] The mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is ammonium formate solution or ammonium acetate solution, and mobile phase B is acetonitrile solution. Preferably, mobile phase A is ammonium formate solution.

[0041] In a specific embodiment, the hydrophilic interaction chromatographic column is a HILIC chromatographic column. The column length of the HILIC chromatographic column is 50-250 mm. The chromatographic column is a specification provided by the chromatographic manufacturer, and the selection of the column length should be considered comprehensively based on the substance to be measured and the specific chromatographic conditions. At present, HILIC chromatographic columns mainly have four specifications of 50 mm, 100 mm, 150 mm, and 250 mm. When the instrument pressure conditions permit, they are all suitable for high performance liquid chromatography (HPLC) and ultra high performance liquid chromatography (UPLC). Among them, Poroshell 120HILIC-Z2.1*150mm 1.9μm, the chromatographic column has a smaller pore size and is usually a preferred option for ultra high performance liquid chromatography. The advantages are solvent saving and short analysis time.

[0042] In a specific embodiment, the hydrophilic interaction chromatography column of the present application is HILIC-Z, specifically a Poroshell 120HILIC-Z chromatography column.

[0043] In a specific embodiment, the concentration of the ammonium formate solution is greater than 1mmol / L, for example 1mmol / L, 2mmol / L, 3mmol / L, 4mmol / L, 5mmol / L, 6mmol / L, 7mmol / L, 8mmol / L, 9mmol / L, 10mmol / L, 11mmol / L, 12mmol / L, 13mmol / L, 14mmol / L, 15mmol / L, 16mmol / L, 17mmol / L, 18mmol / L, 19mmol / L, 20mmol / L, 21mmol / L, 22mmol / , 23mmol / L, 24mmol / L, 25mmol / L, etc., preferably 5mmol / L to 20mmol / L. , for example 5mmol / L, 6mmol / L, 7mmol / L, 8mmol / L, 9mmol / L, 10mmol / L, 11mmol / L, 12mmol / L, 13mmol / L, 14mmol / L, 15mmol / L, 16mmol, 17mmol / L, 18mmol / L, 19mmol / L, 20mmol / L, or any value between 5mmol / L and 20mmol / L, more preferably between 10mmol / L and 15mmol / L, for example 10mmol / L, 11mmol / L, 12mmol / L, 13mmol / L, 14mmol / L, 15mmol / L, or any value between 10mmol / L and 15mmol / L.

[0044] In a specific embodiment, the pH of the ammonium formate solution is less than 6.0, for example, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, etc. Preferably, the pH is 3.0-4.5, for example, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, or any value between 3.0 and 4.5.

[0045] In a specific embodiment, the volume ratio of mobile phase A to mobile phase B is greater than 10:90, preferably (15-25):(85-75), for example, it can be 15:85, 16:84, 17:83, 18:82, 19:81, 20:80, 21:79, 22:78, 23:77, 24:76, 25:75, or any ratio among (15-25):(80-75).

[0046] In a specific embodiment, the detector in the high performance liquid chromatography is an ultraviolet detector (UV detector) or a diode array detector (DAD detector), and the detection wavelength is 230-235 nm, preferably 232 nm.

[0047] In a specific embodiment, the flow rate of the mobile phase is 0.5-1.5 ml / min, preferably 0.8-1.2 ml / min, for example 0.8 ml / min, 0.9 ml / min, 1.0 ml / min, 1.1 ml / min, 1.2 ml / min, or any value in the range of 0.8-1.2 ml / min. The flow rate of the mobile phase can be adjusted according to the type of the specific chromatographic column selected, the column length and other factors.

[0048] In a specific embodiment, the column temperature of the chromatographic column in the high performance liquid chromatography is 20-50° C., for example, 20° C., 22° C., 25° C., 28° C., 30° C., 32° C., 35° C., 40° C., 45° C., 50° C. Preferably, it is 25-40° C., for example, 25° C., 28° C., 30° C., 32° C., 35° C., 40° C., or any value between 25-40° C.

[0049] In a specific embodiment, the injection volume in the high performance liquid chromatography method is 5 to 50 μl, for example, 5 μl, 10 μl, 15 μl, 20 μl, 25 μl, 30 μl, 35 μl, 40 μl, 45 μl, 50 μl. Preferably, it is 10 to 30 μl, for example, 10 μl, 15 μl, 20 μl, 25 μl, 30 μl, or any value in the range of 10 to 30 μl. When used specifically, it can be adjusted according to the volume of the quantitative loop, the sample concentration and its response value.

[0050] In a specific embodiment, the elution mode of the high performance liquid chromatography is isocratic elution, wherein isocratic elution refers to an elution mode in which the composition ratio and flow rate of the mobile phase remain constant during the analysis cycle of the sample components.

[0051] In a specific embodiment, the glycosaminoglycan includes any one, two or three of sodium hyaluronate, chondroitin, and chondroitin sulfate, preferably sodium hyaluronate, chondroitin and chondroitin sulfate.

[0052] The present application also provides a method for detecting glycosaminoglycans, comprising the following steps:

[0053] preparing a solution of a sample to be tested;

[0054] Using the aforementioned detection method to measure the solution of the sample to be tested;

[0055] Based on the detection results, any one, two or three of sodium hyaluronate, chondroitin and chondroitin sulfate contained in glycosaminoglycans can be distinguished.

[0056] Preferably, sodium hyaluronate, chondroitin and chondroitin sulfate contained in glycosaminoglycan can be distinguished based on the detection result.

[0057] In a specific embodiment, sodium hyaluronate, chondroitin and chondroitin sulfate contained in glycosaminoglycans can be distinguished based on the detection results, and the separation degree is greater than 1.5, for example, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, etc.

[0058] In a specific embodiment, the detection results can distinguish sodium hyaluronate, chondroitin and chondroitin sulfate contained in glycosaminoglycans, and the detection limit of sodium hyaluronate is as low as 8.0×10 -2 ppm, for example, 8.0×10 -2 ppm、8.2×10 -2 ppm、8.4×10 -2 ppm、8.6×10 -2 ppm、8.8×10 -2 ppm、9.0×10 -2 ppm、9.2×10 - 2 ppm、9.4×10 -2 ppm、9.6×10 -2 ppm、9.8×10 -2 ppm, 10.0×10 -2 ppm、10.2×10 -2 ppm、10.4×10 -2 ppm、10.6×10 -2 ppm、10.8×10 -2 ppm, etc.; the detection limit of chondroitin is as low as 1.0×10 -1 ppm, for example, 1.0×10 -1 ppm、1.1×10 -1 ppm、1.2×10 -1 ppm、1.3×10 -1 ppm、1.4×10 -1 ppm、1.5×10 -1 ppm、1.6×10 -1 ppm、1.7×10 -1ppm、1.8×10 -1 ppm、1.9×10 -1 ppm, etc.; the detection limit of chondroitin sulfate is as low as 2.0×10 -1 ppm, for example 2.0×10 -1 ppm、2.1×10 -1 ppm、2.2×10 -1 ppm、2.3×10 -1 ppm、2.4×10 - 1 ppm、2.5×10 -1 ppm、2.6×10 -1 ppm、2.7×10 -1 ppm、2.8×10 -1 ppm、2.9×10 -1 ppm、3.0×10 - 1 ppm、3.1×10 -1 ppm、3.2×10 -1 ppm、3.3×10 -1 ppm、3.4×10 -1 ppm, etc.

[0059] The present application uses a hydrophilic interaction chromatographic column, and adopts a mobile phase composed of a mobile phase A ammonium formate solution and a mobile phase B acetonitrile solution, and accurately controls the mobile phase flow rate (0.5-1.5 mL / min), the volume ratio of A and B ((15-25): (80-75)), and the pH is 3.0-4.5. The conditions make sodium hyaluronate, chondroitin and chondroitin sulfate effectively retained on the chromatographic column and maintain a good peak shape. For example, at a flow rate of 0.5-1.0 ml / min, sodium hyaluronate, chondroitin and chondroitin sulfate in the embodiment have a good peak shape, and the retention times are 9.066 ± 0.2 min and 11.545 ± 0.2 min, 10.701 ± 0.2 min and 13.982 ± 0.2 min, 25.594 ± 0.2 min and 35.037 ± 0.2 min, respectively.

[0060] The instruments and materials used in this application are as follows:

[0061] Instruments and reagents: Ultra-high performance liquid chromatograph Agilent 1260 Prime (Agilent, USA), equipped with DAD detector; chromatographic column: InfinityLab Poroshell 120HILIC-Z (4.6×100 mm, 2.7 μm, Agilent, USA); ammonium formate, HPLC grade, Merck; acetonitrile, HPLC grade, Merck; glycosaminoglycan (Huaxi Biotechnology Co., Ltd.); ultrapure water machine Milli-Q.

[0062] Example

[0063] Example 1-1

[0064] Preparation of control solution: Sodium hyaluronate, chondroitin, and chondroitin sulfate reference substances were weighed into volumetric flasks to obtain reference substance stock solutions containing 1 mg / mL, 2 mg / mL, and 2 mg / mL of sodium hyaluronate, chondroitin, and chondroitin sulfate, respectively. 0.5 mL of the above reference substance solutions were taken into 10 mL volumetric flasks, 5 mL of enzymatic hydrolysis buffer was added, and 1 mL of sodium hyaluronate enzyme (5000 IU / mL) was added. After enzymolysis for 2 hours in a 42°C water bath, the enzyme was boiled for 2 minutes to inactivate the enzyme, and water was added to the scale to obtain a control solution with a sodium hyaluronate, chondroitin, and chondroitin sulfate concentration of 0.05 mg / mL, 0.1 mg / mL, and 0.1 mg / mL, respectively.

[0065] Prepare the test solution: take about 0.2g of type II collagen powder with sodium hyaluronate, chondroitin, and chondroitin sulfate, accurately weigh, place in a 10ml volumetric flask, add 5mL of enzymatic buffer to dissolve it, add 1mL of sodium hyaluronate enzyme (5000IU / mL), enzymatically hydrolyze in a 42℃ water bath for 2 hours, boil for 2 minutes to inactivate the enzyme, and add water to the scale. Prepare two copies in parallel.

[0066] Testing steps:

[0067] The chromatographic conditions are:

[0068] Chromatographic column: InfinityLab Poroshell 120HILIC-Z (4.6×100mm, 2.7μm)

[0069] Mobile phase: Mobile phase A (10 mmol / L ammonium formate, pH adjusted to 3.8 with formic acid): Mobile phase B (acetonitrile) = 20:80

[0070] Flow rate: 1ml / min

[0071] Injection volume: 20 μL

[0072] Column temperature: 30°C

[0073] Detection wavelength: 232nm

[0074] The mobile phase was used as blank solution.

[0075] Take blank solution, sodium hyaluronate reference solution, chondroitin reference solution, chondroitin sulfate reference solution and test solution for injection respectively, and detect the test solution according to the above chromatographic conditions. The chromatograms of the reference and test solutions are as shown in the figure. Figure 1-5 As shown. Among them, Figure 1 is the chromatogram of the blank solution; Figure 2-4 They are the chromatograms of sodium hyaluronate, chondroitin, and chondroitin sulfate reference solution; Figure 5 is the chromatogram of the test solution. Figure 2-4 It can be seen that sodium hyaluronate, chondroitin and chondroitin sulfate in the test sample have good peak shapes, and the retention times are 9.066min and 11.545min, 10.701min and 13.982min, 25.594min and 35.037min, respectively, indicating that sodium hyaluronate, chondroitin and chondroitin sulfate are greatly retained on the chromatographic column, meeting the needs of detection.

[0076] The content of sodium hyaluronate, chondroitin and chondroitin sulfate in the test solution was determined by the external standard method. The content was calculated using the following formula:

[0077] The calculation formula of sodium hyaluronate, chondroitin and chondroitin sulfate content in the test sample is:

[0078]

[0079] Note: Pi---Sodium hyaluronate, chondroitin, chondroitin sulfate in the test sample, %

[0080] cr---sodium hyaluronate, chondroitin, chondroitin sulfate in reference solution, mg / mL

[0081] Ai---Peak area of ​​the test solution

[0082] Ar---Peak area of ​​reference solution

[0083] Ws---sample weight, g

[0084] 10 --- Volume of reference solution, mL

[0085] The contents of sodium hyaluronate, chondroitin and chondroitin sulfate in the test sample in Example 1-1 were determined as shown in Table 1.

[0086] Table 1 Results of the contents of sodium hyaluronate, chondroitin and chondroitin sulfate in Example 1-1

[0087]

[0088]

[0089] Example 1-2

[0090] The ratio of the chromatographic mobile phase A to the mobile phase B in Example 1-1 was changed to 15:85, and the other conditions were the same as those in Example 1-1. The measured contents of sodium hyaluronate, chondroitin, and chondroitin sulfate are shown in Table 2.

[0091] Table 2 Results of the contents of sodium hyaluronate, chondroitin and chondroitin sulfate in Example 1-2

[0092]

[0093] Examples 1-3

[0094] The volume ratio of the chromatographic mobile phase A to the mobile phase B in Example 1-1 was changed to 17:83, and the other conditions were the same as those in Example 1-1. The measured contents of sodium hyaluronate, chondroitin, and chondroitin sulfate are shown in Table 3.

[0095] Table 3 Results of the contents of sodium hyaluronate, chondroitin and chondroitin sulfate in Examples 1-3

[0096]

[0097]

[0098] Examples 1-4

[0099] The ratio of the chromatographic mobile phase A to the mobile phase B in Example 1-1 was changed to 22:78, and the other conditions were the same as those in Example 1-1. The measured contents of sodium hyaluronate, chondroitin, and chondroitin sulfate are shown in Table 4.

[0100] Table 4 Results of the contents of sodium hyaluronate, chondroitin and chondroitin sulfate in Examples 1-4

[0101]

[0102] Examples 1-5

[0103] The volume ratio of the chromatographic mobile phase A to the mobile phase B in Example 1-1 was changed to 25:75, and the other conditions were the same as in Example 1-1. The measured contents of sodium hyaluronate, chondroitin, and chondroitin sulfate are shown in Table 5.

[0104] Table 5 Results of the contents of sodium hyaluronate, chondroitin and chondroitin sulfate in Examples 1-5

[0105]

[0106] Examples 1-6

[0107] The chromatographic flow rate in Example 1-1 was changed to 0.8 ml / min, and other conditions were the same as in Example 1-1. The measured contents of sodium hyaluronate, chondroitin, and chondroitin sulfate are shown in Table 6.

[0108] Table 6 Results of the contents of sodium hyaluronate, chondroitin and chondroitin sulfate in Examples 1-6

[0109]

[0110] Examples 1-7

[0111] The chromatographic flow rate in Example 1-1 was changed to 1.2 ml / min, and other conditions were the same as in Example 1-1. The measured contents of sodium hyaluronate, chondroitin, and chondroitin sulfate are shown in Table 7.

[0112] Table 7 Results of the contents of sodium hyaluronate, chondroitin and chondroitin sulfate in Examples 1-7

[0113]

[0114] Examples 1-8

[0115] The column temperature in Example 1-1 was changed to 25° C., and other conditions were the same as in Example 1-1. The measured contents of sodium hyaluronate, chondroitin, and chondroitin sulfate are shown in Table 8.

[0116] Table 8 Results of the contents of sodium hyaluronate, chondroitin and chondroitin sulfate in Examples 1-8

[0117]

[0118] Examples 1-9

[0119] The column temperature in Example 1-1 was changed to 35° C., and other conditions were the same as in Example 1-1. The measured contents of sodium hyaluronate, chondroitin, and chondroitin sulfate are shown in Table 9.

[0120] Table 9 Results of the contents of sodium hyaluronate, chondroitin and chondroitin sulfate in Examples 1-9

[0121]

[0122] Examples 1-10

[0123] The column temperature in Example 1-1 was changed to 40° C., and other conditions were the same as in Example 1-1. The measured contents of sodium hyaluronate, chondroitin, and chondroitin sulfate are shown in Table 10.

[0124] Table 10 Results of the contents of sodium hyaluronate, chondroitin and chondroitin sulfate in Examples 1-10

[0125]

[0126] Embodiment 1-11

[0127] The concentration of ammonium formate in the chromatographic mobile phase A in Example 1-1 was changed to 5 mmol / L, and the other conditions were the same as those in Example 1-1. The measured contents of sodium hyaluronate, chondroitin, and chondroitin sulfate are shown in Table 11.

[0128] Table 11 Results of the contents of sodium hyaluronate, chondroitin and chondroitin sulfate in Examples 1-11

[0129]

[0130]

[0131] Examples 1-12

[0132] The ammonium formate concentration of the chromatographic mobile phase A in Example 1-1 was changed to 15 mmol / L, and the other conditions were the same as in Example 1-1. The measured contents of sodium hyaluronate, chondroitin, and chondroitin sulfate are shown in Table 12.

[0133] Table 12 Results of the contents of sodium hyaluronate, chondroitin and chondroitin sulfate in Examples 1-12

[0134]

[0135] Examples 1-13

[0136] The ammonium formate concentration of the chromatographic mobile phase A in Example 1-1 was changed to 20 mmol / L, and the other conditions were the same as those in Example 1-1. The measured contents of sodium hyaluronate, chondroitin, and chondroitin sulfate are shown in Table 13.

[0137] Table 13 Results of the contents of sodium hyaluronate, chondroitin and chondroitin sulfate in Examples 1-13

[0138]

[0139] Examples 1-14

[0140] The pH value of the chromatographic mobile phase A of ammonium formate in Example 1-1 was changed to 3.0, and the other conditions were the same as those in Example 1-1. The measured contents of sodium hyaluronate, chondroitin, and chondroitin sulfate are shown in Table 14.

[0141] Table 14 Results of the contents of sodium hyaluronate, chondroitin and chondroitin sulfate in Examples 1-14

[0142]

[0143] Examples 1-15

[0144] The pH value of the chromatographic mobile phase A of ammonium formate in Example 1-1 was changed to 4.5, and the other conditions were the same as those in Example 1-1. The measured contents of sodium hyaluronate, chondroitin, and chondroitin sulfate are shown in Table 15.

[0145] Table 15 Results of the contents of sodium hyaluronate, chondroitin and chondroitin sulfate in Examples 1-15

[0146]

[0147] Examples 1-16

[0148] The injection volume in Example 1-1 was changed to 10uL, and the other conditions were the same as in Example 1-1. The measured contents of sodium hyaluronate, chondroitin, and chondroitin sulfate are shown in Table 16.

[0149] Table 16 Results of the contents of sodium hyaluronate, chondroitin and chondroitin sulfate in Examples 1-16

[0150]

[0151]

[0152] Examples 1-17

[0153] The injection volume in Example 1-17 was changed to 30uL, and the other conditions were the same as in Example 1-1. The measured contents of sodium hyaluronate, chondroitin, and chondroitin sulfate are shown in Table 17.

[0154] Table 17 Results of the contents of sodium hyaluronate, chondroitin and chondroitin sulfate in Examples 1-17

[0155]

[0156] Table 18 Chromatographic conditions used in Examples 1-1 to 1-17

[0157]

[0158]

[0159] Examples 1-18

[0160] The mobile phase A in Example 1-1 was changed to 10 mmol ammonium acetate, and the other conditions were the same as in Example 1-1. The detection results of sodium hyaluronate, chondroitin, and chondroitin sulfate had lower responses. The detection results are shown in Table 19.

[0161] Table 19 Results of the contents of sodium hyaluronate, chondroitin and chondroitin sulfate in Examples 1-18

[0162]

[0163] Example 2-1 Determination of Repeatability

[0164] According to the test sample processing method in Example 1-1, the same batch number sample as in Example 1-1 was taken and 6 copies were prepared in parallel. The reference solution and detection conditions were the same as in Example 1-1. The measurement results are shown in Table 20.

[0165] Table 20 Results of the contents of sodium hyaluronate, chondroitin and chondroitin sulfate in Example 2-1

[0166]

[0167] Example 2-2 Determination of inter-day precision

[0168] According to the test sample processing method in Example 1-1, the same batch number sample as in Example 1-1 was taken and 6 samples were prepared in parallel at different times. The reference solution and detection conditions were the same as in Example 1-1, but different instruments were used. The measurement results are shown in Table 21.

[0169] Table 21 Results of the contents of sodium hyaluronate, chondroitin and chondroitin sulfate in Example 2-2

[0170]

[0171] Determination of Accuracy of Example 2-3

[0172] Take about 0.2 g of type II collagen powder in Example 1-1, weigh it accurately, put it in a 10 ml volumetric flask, add 5 ml of enzymatic hydrolysis buffer to dissolve it, prepare 9 portions in parallel, and each 3 portions form a group. To each group, add 0.4 ml, 0.5 ml and 0.6 ml of the reference substance stock solution described in 1-1, respectively, and then add 1.0 ml of hyaluronidase. Mix well, seal, enzymolyze at 42 ° C for 2 h, boil for 2 min to inactivate the enzyme, and the detection conditions are the same as those in Example 1-1. The measurement results are shown in Table 22.

[0173] Table 22 Results of the contents of sodium hyaluronate, chondroitin and chondroitin sulfate in Examples 2-3

[0174]

[0175] Comparative Example 1

[0176] The chromatographic column in Example 1-1 was changed to a Hi-Plex Ca chromatographic column, the mobile phase was water, and the other conditions were the same as in Example 1-1. The test results showed that the peak shapes of sodium hyaluronate, chondroitin, and chondroitin sulfate were poor, and the retention times were consistent and not separated. The test results of sodium hyaluronate, chondroitin, and chondroitin sulfate were as follows: Figure 6 shown.

[0177] Comparative Example 2

[0178] The chromatographic column in Example 1-1 was changed to an amide column (ACQUITY UPLC BEH Amide 2.1×100 mm 1.7 um), and the other conditions were the same as those in Example 1-1. The test results showed that the peak shapes of sodium hyaluronate, chondroitin, and chondroitin sulfate were not good, the separation was poor, and effective separation could not be achieved. The test results of sodium hyaluronate, chondroitin, and chondroitin sulfate were as follows: Figure 7 shown.

[0179] Comparative Example 3

[0180] The volume ratio of the chromatographic mobile phase A to the mobile phase B in Example 1-1 was changed to 10:90, and the other conditions were the same as in Example 1-1. The test results are shown in Table 23. The results show that no chondroitin sulfate peak was found within 40 minutes.

[0181] Table 23 Results of the content of sodium hyaluronate, chondroitin and chondroitin sulfate in comparative example 3

[0182]

[0183] Comparative Example 4

[0184] The concentration of ammonium formate in mobile phase A in Example 1-1 was adjusted to 1 mmol, and the other conditions were the same as in Example 1-1. Sodium hyaluronate, chondroitin, and chondroitin sulfate may not be completely detected. The test results are shown in Table 24.

[0185] Table 24 Results of the content of sodium hyaluronate, chondroitin and chondroitin sulfate in comparative example 4

[0186]

[0187] Comparative Example 5

[0188] The pH of the mobile phase A ammonium formate in Example 1-1 was adjusted to 6.0, and the other conditions were the same as those in Example 1-1. The test results showed that the peaks of sodium hyaluronate and chondroitin were bifurcated. The test results of sodium hyaluronate, chondroitin, and chondroitin sulfate were as follows: Figure 8 shown.

[0189] Comparative Example 6

[0190] The chromatographic mobile phase in Example 1-1 was changed to A: 0.3% ammonia water; B: 0.3% ammonia water acetonitrile, and different gradient elution conditions of A:B = 18:82, A:B = 15:85, A:B = 12:88, and A:B = 10:90 were respectively performed. The other conditions were the same as in Example 1-1. The test results showed that under the elution condition of A:B = 15:85, the peak shapes of sodium hyaluronate, chondroitin, and chondroitin sulfate were better, but the retention times were consistent, and effective separation could not be achieved. The test results of sodium hyaluronate, chondroitin, and chondroitin sulfate are as follows: Fig. 9 shown.

[0191] The above is only the preferred embodiment of the present application, and does not limit the present application in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present application without departing from the technical solution of the present application still falls within the protection scope of the technical solution of the present application.

Claims

1. A method for detecting glycosaminoglycans, comprising detecting glycosaminoglycans using high performance liquid chromatography, wherein the chromatographic conditions of the high performance liquid chromatography are as follows: The chromatographic column is a hydrophilic interaction chromatographic column; The mobile phase includes a mobile phase A and a mobile phase B. The mobile phase A is an ammonium formate solution or an ammonium acetate solution, and the mobile phase B is an acetonitrile solution.

2. The method for detecting glycosaminoglycans according to claim 1, wherein the hydrophilic interaction chromatography column is a HILIC chromatography column.

3. The method for detecting glycosaminoglycans according to any one of claims 1-2, wherein the concentration of the ammonium formate solution is greater than 1 mmol / L, preferably 5 mmol / L to 20 mmol / L.

4. The method for detecting glycosaminoglycans according to any one of claims 1 to 3, wherein the pH of the ammonium formate solution is less than 6.0, preferably 3.0 to 4.

5.

5. The method for detecting glycosaminoglycans according to any one of claims 1 to 4, wherein the volume ratio of the mobile phase A to the mobile phase B is greater than 10:90, preferably (15-25):(85-75).

6. The method for detecting glycosaminoglycans according to any one of claims 1 to 5, wherein the detector in the high performance liquid chromatography is an ultraviolet detector or a diode array detector, and the detection wavelength is 230-235 nm.

7. The method for detecting glycosaminoglycans according to any one of claims 1-6, wherein the flow rate of the mobile phase is 0.5-1.5 ml / min; the column temperature of the chromatographic column in the high performance liquid chromatography is 20-50°C; the injection volume in the high performance liquid chromatography is 5-50 μl; and the elution mode of the high performance liquid chromatography is isocratic elution.

8. The method for detecting glycosaminoglycan according to any one of claims 1 to 7, wherein the glycosaminoglycan comprises any one, two or three of sodium hyaluronate, chondroitin, and chondroitin sulfate.

9. A method for detecting glycosaminoglycans, comprising the following steps: preparing a solution of a sample to be tested; Using the detection method according to any one of claims 1 to 8 to measure the solution of the sample to be tested; Based on the detection results, any one, two or three of sodium hyaluronate, chondroitin and chondroitin sulfate contained in glycosaminoglycans can be distinguished.

10. The method according to claim 9, wherein: Based on the test results, sodium hyaluronate, chondroitin and chondroitin sulfate contained in glycosaminoglycans can be distinguished, and the separation degree is greater than 1.5; based on the test results, sodium hyaluronate, chondroitin and chondroitin sulfate contained in glycosaminoglycans can be distinguished, and the detection limit of sodium hyaluronate is as low as 8.0×10 - 2 ppm; the detection limit of chondroitin is as low as 1.0×10 -1 ppm; the detection limit of chondroitin sulfate is as low as 2.0×10 - 1 ppm.

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