HPLC (High Performance Liquid Chromatography) detection method for glycyrrhizic acid compound related substances

Abstract

The invention belongs to the field of pharmaceutical analysis, and provides an HPLC (High Performance Liquid Chromatography) detection method for efficiently separating and detecting related substances of glycyrrhizic acid compounds and application of the HPLC detection method. Wherein a chromatographic column is an octadecyl bonded silica gel column, a mobile phase is composed of a phase A and a phase B, the phase A is a 0.1%-0.3% perchloric acid aqueous solution, the phase B is an acetonitrile-tetrahydrofuran solution, and the column temperature is 25 DEG C; the related substance is selected from any one or a combination of a related substance 1, a related substance 2, 18beta-glycyrrhizic acid, a related substance 4, a related substance 5, a related substance 6, a related substance 7, a related substance 8 and a related substance 9. The method disclosed by the invention is simple and easy to implement, can realize efficient separation of the glycyrrhizic acid compounds and related impurities, can be used for conventional analysis and quality control of the glycyrrhizic acid compounds, and meets the requirements of research, development and production.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to an HPLC detection method for glycyrrhizic acid compound-related substances. Background Art

[0002] Glycyrrhizic acid has nonspecific anti-inflammatory, anti-allergic, lysosomal membrane stabilization and immunomodulatory effects, and is commonly used to treat hepatitis clinically. Glycyrrhizic acid can be prepared by deriving glycyrrhizic acid isolated from the leguminous plant licorice. Glycyrrhizic acid has two diastereomers, 18α-glycyrrhizic acid and 18β-glycyrrhizic acid. Due to the different configurations of hydrogen on the 18th C, there are certain differences in the physicochemical properties and pharmacological effects of 18α-glycyrrhizic acid and 18β-glycyrrhizic acid. Compared with 18β-glycyrrhizic acid, 18α-glycyrrhizic acid has many advantages such as good lipophilicity, strong anti-inflammatory activity, high targeting, fewer adverse reactions, and good safety.

[0003] Commonly used glycyrrhizic acid compounds in clinical practice include glycyrrhizic acid, monoammonium glycyrrhizinate, monopotassium glycyrrhizinate, diammonium glycyrrhizinate, dipotassium glycyrrhizinate, disodium glycyrrhizinate, trisodium glycyrrhizinate, magnesium isoglycyrrhizinate, etc. The main differences are the different configurations at the 18th position and the different salt forms. For example, magnesium isoglycyrrhizinate is mainly 18α-glycyrrhizic acid, diammonium glycyrrhizinate is a mixture of 18α-glycyrrhizic acid and 18β-glycyrrhizic acid, and monoammonium glycyrrhizinate is mainly 18β-glycyrrhizic acid. The chemical structures of 18α-glycyrrhizic acid and 18β-glycyrrhizic acid are shown below:

[0004]

[0005] The presence of related substances is directly related to the quality and safety of drugs. Related substances include impurities brought in by drug raw materials, related substances generated during the process, and related substances related to degradation, which will directly affect the purity and quality of the drug, and may even cause serious adverse reactions, affecting the effectiveness and safety of clinical drugs. In order to improve the product quality of glycyrrhizic acid compounds and ensure the safety and effectiveness of clinical drugs, it is necessary to fully study the related substances and develop applicable separation and detection methods to control impurities within the safety limit, achieve controllable drug quality, and ensure the safety and effectiveness of clinical drugs.

[0006] Patents CN117607315A and CN117647603A report an HPLC method for separating and detecting magnesium isoglycyrrhizinate and its related substances, wherein the chromatographic system is isocratic or gradient elution with 0.05M phosphate (pH 7.0) as mobile phase A and acetonitrile as mobile phase B. Isocratic elution has a weak elution ability for small polar impurities, which is not conducive to controlling related substances such as degradation impurities (such as glycyrrhetinic acid). When gradient elution is used, the high salt concentration aqueous phase has poor solubility with the organic phase, and there is a risk of salt precipitation, thereby reducing the service life of the chromatographic column.

[0007] The structural formulas of some glycyrrhizic acid compounds and related substances are shown in the table below:

[0008]

[0009] Summary of the invention

[0010] In view of the problems of poor impurity detection ability and damage to chromatographic columns caused by high-salt mobile phase in the currently reported detection methods for glycyrrhizic acid-related substances, the present invention provides an HPLC detection method for efficiently separating and detecting glycyrrhizic acid-related substances, which comprises the following steps:

[0011] (1) Test solution: Dissolve the test sample in acetonitrile and water and dilute it to a certain concentration as the test solution.

[0012] (2) Chromatographic conditions:

[0013] Chromatographic column: octadecylsilane bonded silica gel column;

[0014] UV detector: detection wavelength 240~250nm;

[0015] Mobile phase composition:

[0016] Mobile phase A: aqueous perchloric acid solution,

[0017] Mobile phase B: acetonitrile-tetrahydrofuran solution;

[0018] Gradient elution was used with the following procedure:

[0019]

[0020] Furthermore, the concentration of the acetonitrile aqueous solution in step (1) is 20% to 40%, and the concentration of the test sample is 1 mg / ml.

[0021] Furthermore, the length of the chromatographic column in step (2) is 150 to 300 mm.

[0022] Furthermore, the chromatographic conditions of step (2) are a column temperature of 30-40° C., a mobile phase flow rate of 0.9-1.1 ml / min, and an injection volume of 40-60 μl.

[0023] Furthermore, the concentration of the perchloric acid solution in step (2) is 0.05% to 0.5%.

[0024] Furthermore, the ratio of acetonitrile to tetrahydrofuran in step (2) is 70:30 to 30:70.

[0025] Furthermore, the concentration of the perchloric acid solution in step (2) is 0.1% to 0.3%.

[0026] Furthermore, the ratio of acetonitrile to tetrahydrofuran in step (2) is 60:40 to 40:60.

[0027] Furthermore, the gradient elution procedure described in step (2) is as follows:

[0028]

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The separation degree between the main component and specific impurities of this method is better, and the separation degree between 18α-glycyrrhizic acid and 18β-glycyrrhizic acid is greater than 2.0.

[0031] (2) The mobile phase system of this method is simple and does not use high-concentration salt solutions. There is no risk of high-concentration salts precipitating in the organic phase during the gradient elution procedure, which can effectively protect the life of the chromatographic column and reduce the cost of analytical testing.

[0032] (3) This method is simple and easy to use, and has lower requirements for the chromatographic column. An ordinary C18 column can achieve good separation effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Attached Figure 1 : The HPLC chromatogram of Example 1.

[0034] Attached Figure 2 : HPLC chromatogram of Example 2

[0035] Attached Figure 3 : HPLC chromatogram of Example 3

[0036] Attached Figure 4 : High performance liquid chromatogram of comparative example 1

[0037] Attached Figure 5 : High performance liquid chromatogram of comparative example 2 DETAILED DESCRIPTION

[0038] To make the purpose, technical solution and advantages of the present invention more clear, the embodiments of the present invention will be described in detail with reference to the accompanying drawings. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other arbitrarily without conflict.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; when the present invention relates to the percentage between liquid and liquid, the percentage is volume / volume percentage; when the present invention relates to the percentage between liquid and solid, the percentage is volume / weight percentage; when the present invention relates to the percentage between solid and liquid, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0040] The reagents and instruments used in the examples without indicating the manufacturer are all conventional products that can be purchased from the market.

[0041] Example 1 Detection method of related substances of monoammonium glycyrrhizinate

[0042] Liquid chromatography conditions:

[0043] Instrument: Shimadzu LC-20AT

[0044] Chromatographic column: Agilent ZOBAX Eclipse XDB C18 (4.6 mm × 250 mm, 5 μm)

[0045] Mobile phase A: 0.2% perchloric acid

[0046] Mobile phase B: acetonitrile-tetrahydrofuran (40:60)

[0047]

[0048] Flow rate: 1ml / min

[0049] UV detector detection wavelength: 244nm

[0050] Injection volume: 50 μl

[0051] Test solution: Take glycyrrhizic acid monoammonium salt, dissolve it in acetonitrile aqueous solution and dilute it to make a solution containing about 1 mg of 18β-glycyrrhizic acid per 1 ml.

[0052] The chromatographic results of the test solution are shown in Figure 1 .

[0053] The results showed that the main peak had a good shape and the main components and known impurities could be well separated under this system.

[0054] Example 2 Detection method of related substances of methyl glycyrrhizinate

[0055] Liquid chromatography conditions:

[0056] Instrument: Shimadzu LC-20AT

[0057] Chromatographic column: Agilent ZOBAX Eclipse XDB C18 (4.6 mm × 250 mm, 5 μm)

[0058] Mobile phase A: 0.1% perchloric acid

[0059] Mobile phase B: acetonitrile-tetrahydrofuran (50:50)

[0060]

[0061] Flow rate: 1ml / min

[0062] UV detector detection wavelength: 244nm

[0063] Injection volume: 50 μl

[0064] Test solution: Take methyl glycyrrhizinate, dissolve it in acetonitrile-water solution and dilute it to make a solution containing about 1 mg of methyl glycyrrhizinate per 1 ml.

[0065] The chromatographic results of the test solution are shown in Figure 2 .

[0066] The results showed that the main peak had a good shape and the main component was well separated from adjacent impurities in this system.

[0067] Example 3 Detection method of magnesium isoglycyrrhizinate related substances

[0068] Liquid chromatography conditions:

[0069] Instrument: Shimadzu LC-20AT

[0070] Chromatographic column: Agilent ZOBAX Eclipse XDB C18 (4.6 mm × 250 mm, 5 μm)

[0071] Mobile phase A: 0.2% perchloric acid

[0072] Mobile phase B: acetonitrile-tetrahydrofuran (50:50)

[0073]

[0074] Flow rate: 1ml / min

[0075] UV detector detection wavelength: 244nm

[0076] Injection volume: 50 μl

[0077] Mixed solution: Take appropriate amounts of magnesium isoglycyrrhizinate, related substance 2, 18β-glycyrrhizic acid, related substance 7 and related substance 8, dissolve and dilute them with acetonitrile aqueous solution to prepare a mixed solution containing approximately 1 mg of magnesium isoglycyrrhizinate and approximately 10 μg of related substance 2, 18β-glycyrrhizic acid, related substance 7 and related substance 8 per 1 ml.

[0078] The chromatographic results of the mixed solution are shown in Figure 3 .

[0079] The results showed that the main peak had a good shape, and the main component and adjacent impurities, as well as the impurities themselves, could be well separated in this system.

[0080] Example 4 Durability of the HPLC method of the present invention

[0081] The chromatographic conditions were the same as in Example 3.

[0082] Solution preparation method: Take appropriate amounts of magnesium isoglycyrrhizinate, related substance 2, 18β-glycyrrhizic acid, related substance 8 and related substance 9, dissolve and dilute them with acetonitrile aqueous solution to prepare a mixed solution containing approximately 1 mg of magnesium isoglycyrrhizinate and approximately 10 μg of related substance 2, 18β-glycyrrhizic acid, related substance 8 and related substance 9 per 1 ml.

[0083] The separation results are shown in the table below.

[0084]

[0085] The results show that after partial adjustment of the chromatographic conditions of Example 3 of the invention, the separation degree of each compound can meet the requirements, and the durability of the invention is good.

[0086] Comparative Example 1 USP2024 reported glycyrrhizic acid monoammonium salt related substances detection method

[0087] Liquid chromatography conditions:

[0088] Instrument: Shimadzu LC-20AT

[0089] Chromatographic column: Waters μBondapak C18 (3.9 mm × 300 mm, 10 μm)

[0090] Mobile phase: acetonitrile-glacial acetic acid-water (38:1:61)

[0091] Flow rate: 2ml / min

[0092] UV detector detection wavelength: 254nm

[0093] Injection volume: 10 μl

[0094] Test solution: Take monoammonium glycyrrhizinate, dissolve it in the mobile phase and dilute it to make a solution containing about 1 mg of 18β-glycyrrhizic acid per 1 ml.

[0095] The chromatographic results of the test solution are shown in Figure 4 .

[0096] The results showed that the impurities peaked densely and could not be effectively separated, and the main component peaked too early, making it impossible to control product quality.

[0097] Comparative Example 2 Related substance detection method reported in patent CN117607315A

[0098] Liquid chromatography conditions:

[0099] Instrument: Shimadzu LC-20AT

[0100] Chromatographic column: Agilent ZOBAX Eclipse XDB C18 (4.6 mm × 250 mm, 5 μm)

[0101] Mobile phase: 0.05M potassium dihydrogen phosphate solution (adjust pH to 7.0 with phosphoric acid)-acetonitrile (80:20)

[0102] Flow rate: 1ml / min

[0103] UV detector detection wavelength: 254nm

[0104] Injection volume: 20 μl

[0105] Test solution: Take 18α-glycyrrhizic acid monoammonium salt, dissolve it in acetonitrile aqueous solution and dilute it to make a solution containing about 1 mg of 18α-glycyrrhizic acid per 1 ml.

[0106] The chromatographic results of the test solution are shown in Figure 5 .

[0107] The results showed that the main peak had severe tailing (tailing factor 2.4), and 18β-glycyrrhizic acid and 18α-glycyrrhizic acid could be separated, but the separation effect was poor (resolution 0.8), and the product quality could not be controlled.

Claims

1. A method for detecting related substances of glycyrrhizic acid compounds, characterized in that: The steps include: (1) Test solution: Dissolve the test sample in acetonitrile and water and dilute it to a certain concentration as the test solution. (2) Chromatographic conditions: Chromatographic column: octadecylsilane bonded silica gel column; UV detector: detection wavelength 240~250nm; Mobile phase composition: Mobile phase A: aqueous perchloric acid solution, Mobile phase B: acetonitrile-tetrahydrofuran solution; Gradient elution was used with the following procedure:

2. The detection method according to claim 1, characterized in that: The concentration of the acetonitrile aqueous solution in step (1) is 20% to 40%, and the concentration of the test sample is 1 mg / ml.

3. The detection method according to claim 1, characterized in that: The length of the chromatographic column described in step (2) is 150 to 300 mm.

4. The detection method according to claim 1, characterized in that: The chromatographic conditions of step (2) are as follows: column temperature of 30-40° C., mobile phase flow rate of 0.9-1.1 ml / min, and injection volume of 40-60 μl.

5. The detection method according to claim 1, characterized in that: The concentration of the perchloric acid solution in step (2) is 0.05% to 0.5%.

6. The detection method according to claim 1, characterized in that: The ratio of acetonitrile to tetrahydrofuran in step (2) is 70:30 to 30:

70.

7. The detection method according to claim 1, characterized in that: The concentration of the perchloric acid solution described in step (2) is 0.1% to 0.3%.

8. The detection method according to claim 1, characterized in that: The ratio of acetonitrile to tetrahydrofuran in step (2) is 60:40 to 40:

60.

9. The detection method according to claim 1, characterized in that: The gradient elution procedure described in step (2) is as follows:

10. The detection method according to claim 1, characterized in that: Glycyrrhizic acid compounds include glycyrrhizic acid, monoammonium glycyrrhizic acid, monopotassium glycyrrhizic acid, diammonium glycyrrhizic acid, dipotassium glycyrrhizic acid, disodium glycyrrhizic acid, trisodium glycyrrhizic acid, magnesium isoglycyrrhizinate, and methyl glycyrrhizinate.

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