A method for separating and detecting diastereomers of glycyrrhizic acid

Abstract

The present invention provides a separation and detection method for glycyrrhizic acid diastereomers, belonging to the technical field of drug separation and detection. The present invention uses octadecyl bonded silica gel as filler, and performs separation and detection of 18α glycyrrhizic acid and 18β glycyrrhizic acid under the following chromatographic conditions: mobile phase A: organic solvent containing tetrahydrofuran, mobile phase B: acid-containing aqueous solution; column temperature: 25-45°C; flow rate: 0.8-1.2mL / min; detection wavelength: 210-460nm; mobile phase elution procedure: isocratic elution. The separation and detection method can effectively separate 18α and 18β diastereomers in glycyrrhizic acid, and perform quantitative detection by area normalization method, the separation degree meets the requirements, and the detection sensitivity is high; it can realize quantitative detection of another impurity when 18α ​​and 18β glycyrrhizic acid are used alone, and can monitor impurities in the quality control of glycyrrhizic acid monoammonium, glycyrrhizic acid diamine or other glycyrrhizic acid products.

Description

Technical Field

[0001] The invention belongs to the technical field of drug separation and detection, and particularly relates to a method for separating and detecting diastereomers of glycyrrhizic acid. Background Art

[0002] Glycyrrhizic acid is the main component of the natural drug licorice. Its structure is shown in the following formulas Ⅰ and Ⅱ. It is a triterpenoid saponin compound of oleanane type, in which the carbon atom at position 18 is chiral carbon. The compound is divided into two configurations: 18α and 18β. The main form of glycyrrhizic acid in nature is 18β-configuration glycyrrhizic acid, and there is also a small amount of 18α-configuration glycyrrhizic acid. The glycyrrhizic acid extracted on the market is mainly used as glycyrrhizic acid monoammonium salt and glycyrrhizic acid diamine salt.

[0003]

[0004] Monoammonium glycyrrhizinate is an important food, medicine and cosmetic raw material refined from cultivated licorice through extraction, acidification, ammoniation and recrystallization. Its applications cover industries such as food, medicine, cosmetics, high-grade cigarettes and toothpaste. In the food industry, monoammonium glycyrrhizinate is mainly used as a sweetener, and its sweetness is 200 times that of sucrose. The main component of monoammonium glycyrrhizinate is 18β glycyrrhizic acid, and 18α glycyrrhizic acid is one of the main impurities that need to be controlled. This requires a suitable detection method to separate and detect the two isomers of 18α and 18β.

[0005] Glycyrrhizic acid diamine salt has strong anti-inflammatory activity and can protect liver cell membranes and improve liver function. The current preparation method of glycyrrhizic acid diamine is to extract glycyrrhizic acid with a solvent and then synthesize it. Its main component is 18α glycyrrhizic acid, and 18β glycyrrhizic acid is one of the main impurities that need to be controlled. This requires a suitable detection method to separate and detect the two isomers of 18α and 18β.

[0006] Monoammonium glycyrrhizinate and diammonium glycyrrhizinate are the most widely used glycyrrhizic acid products on the market, and have very broad prospects in the fields of medicine, food, cosmetics, etc. However, at present, the detection method for glycyrrhizic acid in the Part I of the 2015 edition of the Chinese Pharmacopoeia cannot perform baseline separation detection on the two isomers of 18α and 18β. Various manufacturers cannot perform effective baseline separation on the two isomers by referring to the pharmacopoeia method, which poses a safety hazard to the quality of the product.

[0007] Chinese patent application 201811148975.3 discloses a high pressure liquid phase quantitative detection method and application of 18α- and 18β-diastereomers in glycyrrhetinic acid stearyl ester. The invention uses phenylhexyl bonded silica gel as the stationary phase and performs area normalization quantitative detection of 18α-glycyrrhetinic acid stearyl ester and 18β-glycyrrhetinic acid stearyl ester under the following chromatographic conditions: mobile phase: acetonitrile: ammonium acetate solution = 10-25:75-90; elution mode: isocratic elution; column temperature: 33-38°C; detection wavelength: 226-280nm. The method provided by the invention can efficiently separate the 18α- and 18β-glycyrrhizic acid isomers in stearyl glycyrrhetinic acid ester, and perform quantitative detection by area normalization method. The separation degree meets the requirements and the detection sensitivity is high. However, the application is for quantitative detection of 18α- and 18β-deviators in glycyrrhetinic acid ester, and the separation degree is relatively small, only 1.52, which cannot better meet the separation requirements.

[0008] Therefore, it is necessary to develop a separation and detection method for glycyrrhizic acid diastereomers with high separation and good accuracy, especially for the separation and detection of 18α- and 18β-diastereomers in glycyrrhizic acid and its salts. Summary of the invention

[0009] Based on the deficiencies of the prior art, the present application provides a method for separating and detecting glycyrrhizic acid diastereomers, which can better separate the two glycyrrhizic acid diastereomers 18α and 18β.

[0010] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0011] A method for separating and detecting glycyrrhizic acid epimers comprises the following steps:

[0012] (1) Test solution: Take 18α-glycyrrhizic acid and 18β-glycyrrhizic acid samples and add them to methanol to prepare a mixed solution. After shaking and filtering, the mixed solution is used as the test solution for later use.

[0013] (2) Reference solution: Take 18α-glycyrrhizic acid and 18β-glycyrrhizic acid reference substances and add them to methanol to prepare solutions, which are used as reference solutions for later use;

[0014] (3) Detection: Take 10uL of the test solution and the reference solution respectively and inject them into the high performance liquid chromatography for determination.

[0015] The chromatographic conditions described in step (3) are:

[0016] Chromatographic column: Octadecyl bonded silica gel as filler.

[0017] Mobile phase: Mobile phase A: organic solvent containing tetrahydrofuran, mobile phase B: aqueous solution containing acid;

[0018] Column temperature: 25-45°C;

[0019] Flow rate: 0.8-1.2mL / min;

[0020] Detection wavelength: 210-460nm;

[0021] Mobile phase elution program: isocratic elution.

[0022] Wherein, the organic solvent in the organic solvent containing tetrahydrofuran is selected from one or more of methanol, ethanol and acetonitrile;

[0023] The volume ratio of tetrahydrofuran to the organic solvent is 0-100:100-0;

[0024] Preferably, the mobile phase A is tetrahydrofuran.

[0025] The acid in the acid-containing aqueous solution is selected from any one of formic acid, acetic acid, phosphoric acid or trifluoroacetic acid; preferably phosphoric acid.

[0026] The mass fraction of the acid is 0.01-2%, preferably 0.2%;

[0027] Preferably, the mobile phase B is 0.2% phosphoric acid.

[0028] As a preferred embodiment, the mobile phase A is tetrahydrofuran, and the mobile phase B is 0.2% phosphoric acid.

[0029] The volume ratio of the mobile phase A to the mobile phase B is 40:60.

[0030] As another preferred embodiment, the column temperature is 30° C., the flow rate is 1.0 ml / min, and the detection wavelength is 252 nm.

[0031] The specifications of the chromatographic column are C18*250*4.6mm, 5um.

[0032] Wherein, the 18α glycyrrhizic acid is 18α glycyrrhizic acid monoammonium, 18α glycyrrhizic acid diamine or other 18α glycyrrhizic acid salts; preferably 18α glycyrrhizic acid diamine.

[0033] The 18β-glycyrrhizic acid is 18β-monoammonium glycyrrhizinate, 18β-diamine glycyrrhizinate or other 18β-glycyrrhizinate salts; preferably 18β-diamine glycyrrhizinate.

[0034] As a preferred technical solution, the present invention provides a method for separating and detecting glycyrrhizic acid epimers, comprising the following steps:

[0035] (1) Test solution: Take 18α-diamine glycyrrhizinate and 18β-diamine glycyrrhizinate samples and add them to the same methanol solution to prepare a mixed sample of 1.0 mg / mL solution. After shaking and filtering, use it as the test solution for later use.

[0036] (2) Reference solution: Dissolve 18α-diamine glycyrrhizinate and 18β-diamine glycyrrhizinate in methanol to prepare a 1.0 mg / mL solution as the reference solution for later use.

[0037] (3) Detection: Take 10uL of the test solution and the reference solution respectively and inject them into a high performance liquid chromatograph for area normalization method detection.

[0038] The chromatographic conditions described in step (3) are:

[0039] Chromatographic column: Octadecyl bonded silica gel as filler;

[0040] Instrument: Waters 2695;

[0041] Detector: DAD detector (diode array detector);

[0042] Mobile phase: Mobile phase A: tetrahydrofuran, mobile phase B: 0.2% phosphoric acid, the volume ratio of mobile phase A to mobile phase B is 40:60;

[0043] Column temperature: 30°C;

[0044] Flow rate: 1.0 mL / min;

[0045] Detection wavelength: 252nm;

[0046] Mobile phase elution program: isocratic elution.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] 1. The method provided by the present invention can effectively separate the 18α and 18β diastereomers in glycyrrhizic acid, and perform quantitative detection by area normalization method, the separation degree meets the requirements, and the detection sensitivity is high; the present application uses octadecyl bonded silica gel as the stationary phase, tetrahydrofuran: phosphoric acid aqueous solution 40:60 as the mobile phase, the flow rate is 1.0 ml / min, the column temperature is 30°C, the detection wavelength is 252 nm, and the 18α- and 18β-glycyrrhizic acid diastereomers can be effectively separated, and the separation degree is 1.79, which is better than the existing patented method.

[0049] 2. The present invention can realize the quantitative detection of another impurity when 18α ​​and 18β glycyrrhizic acid are used alone, and can monitor impurities in the quality control of monoammonium glycyrrhizinate, diammonium glycyrrhizinate or other glycyrrhizic acid products. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The HPLC chromatogram of separation of 18β,20α-ammonium glycyrrhizinate in Example 1;

[0051] Figure 2 The HPLC chromatogram of separation of 18α,20β-ammonium glycyrrhizinate in Example 1;

[0052] Figure 3 The HPLC chromatogram of the test sample separated in Example 1;

[0053] Figure 4 The HPLC chromatogram of the test sample separated in Comparative Example 1;

[0054] Figure 5 The HPLC chromatogram of the test sample separated in Comparative Example 2 is shown in FIG. DETAILED DESCRIPTION

[0055] The specific implementation parameter range of the present invention is further described in detail below in conjunction with the examples, so that those skilled in the art can better understand the present invention and implement it. The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0056] Drugs and reagents

[0057]

[0058] Basic Example 18 Preparation Method of α-Glycyrrhizic Acid Glycyrrhizic Acid Diamine

[0059] The preparation method is prepared according to the existing method. The specific method is: add 18β-glycyrrhizic acid to 8 times the amount of 40% sodium hydroxide solution, heat and stir at 80°C for 8 hours, let it cool to room temperature, adjust the solution pH to 2 with hydrochloric acid, extract with n-butanol, evaporate the n-butanol layer to obtain a white powder, namely 18α-glycyrrhizic acid.

[0060] Example 1 A method for separating and detecting glycyrrhizic acid epimers

[0061] The steps include:

[0062] (1) Test solution: Take 18α-glycyrrhizic acid diammonium glycyrrhizinate and 18β-glycyrrhizic acid diammonium glycyrrhizinate and add them to the same methanol solution to prepare a mixed sample of 1.0 mg / mL solution. After shaking and filtering, use it as the test solution for later use.

[0063] (2) Reference solution: Dissolve 18α-glycyrrhizic acid diammonium glycyrrhizinate and 18β-glycyrrhizic acid diammonium glycyrrhizinate in methanol to prepare a 1.0 mg / mL solution as the reference solution for later use;

[0064] (3) Detection: Take 10uL of the test solution and the reference solution respectively and inject them into a high performance liquid chromatograph for area normalization method detection.

[0065] The chromatographic conditions described in step (3) are:

[0066] Chromatographic column: Octadecyl bonded silica gel as filler;

[0067] Instrument: Waters 2695;

[0068] Detector: DAD detector (diode array detector);

[0069] Mobile phase: Mobile phase A: tetrahydrofuran, mobile phase B: 0.2% phosphoric acid, the volume ratio of mobile phase A to mobile phase B is 40:60;

[0070] Column temperature: 30°C;

[0071] Flow rate: 1.0 mL / min;

[0072] Detection wavelength: 252nm;

[0073] Mobile phase elution program: isocratic elution.

[0074] Test results:

[0075] According to the attached Figure 1 It can be seen that two chromatographic peaks were detected in the test solution, with the elution time being 10.1min and 11.1min respectively, and the separation degree of the chromatographic peaks was 2.2, achieving baseline separation (the standard is greater than 1.5); according to the attached Figure 2-3 It can be seen that the liquid phase elution times of the 18α glycyrrhizic acid reference solution and the 18β glycyrrhizic acid reference solution are 10.1 min and 11.1 min, respectively, which are consistent with the elution times of the two chromatographic peaks in the test sample, indicating that this method can effectively separate the 18α and 18β glycyrrhizic acid isomers.

[0076] Comparative Example 1 A method for separating and detecting diastereomers of glycyrrhizic acid (according to the test method for glycyrrhizic acid in the pharmacopoeia)

[0077] The steps include:

[0078] (1) Test solution: Add 18α-glycyrrhizic acid and 18β-glycyrrhizic acid samples to the same methanol solution to prepare a mixed sample of 1.0 mg / mL solution. Shake well, filter, and use as the test solution for later use.

[0079] (2) Reference solution: Take 18α-glycyrrhizic acid and 18β-glycyrrhizic acid respectively, dissolve them in methanol to prepare a 1.0 mg / mL solution as the reference solution for later use;

[0080] (3) Detection: Take 10uL of the test solution and the reference solution respectively and inject them into a high performance liquid chromatograph for area normalization method detection.

[0081] Chromatographic column: Octadecyl bonded silica gel as filler;

[0082] Instrument: Waters 2695;

[0083] Detector: DAD detector (diode array detector);

[0084] Mobile phase: Mobile phase A: acetonitrile, mobile phase B: 0.5% phosphoric acid solution (containing 1 mmol ammonium acetate solution), the volume ratio of mobile phase A to mobile phase B is 33:67;

[0085] Column temperature: 30°C;

[0086] Flow rate: 1.0 mL / min;

[0087] Detection wavelength: 254nm;

[0088] Mobile phase elution program: isocratic elution.

[0089] Test results:

[0090] According to the attached Figure 4 It can be seen that the liquid phase peak times of the test solution, 18α glycyrrhizic acid control solution and 18β glycyrrhizic acid control solution are 8.301min, 8.294min and 8.289min respectively, all of which are single chromatographic peaks, indicating that this method cannot effectively separate 18α and 18β glycyrrhizic acid isomers.

[0091] Comparative Example 2: A method for separating and detecting diastereomers of glycyrrhizic acid

[0092] The difference from Example 1 is that: Pengl-Hexyl (4.6 mmX100 mm.5 um) is used, the column temperature is 35°C, the mobile phase is acetonitrile: 10 mol / L ammonium acetate solution (84:16 V / V) isocratic elution, the flow rate is 1.0 mL / min, the detection wavelength is 252 nm, and the theoretical plate number is not less than 2000 calculated based on 18β-glycyrrhetinic acid stearyl ester, and the 18α and 18β-glycyrrhizic acid isomers cannot be effectively separated.

[0093] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for separating and detecting glycyrrhizic acid epimers, characterized in that: The steps include: (1) Test solution: Add 18α-diamine glycyrrhizinate and 18β-diamine glycyrrhizinate to the same methanol solution to prepare a mixed sample of 1.0 mg / mL solution. Shake well, filter, and use as the test solution for later use. (2) Reference solution: Dissolve 18α-diamine glycyrrhizinate and 18β-diamine glycyrrhizinate in methanol to prepare a 1.0 mg / mL solution as the reference solution for later use. (3) Detection: Take 10uL of the test solution and the reference solution respectively and inject them into the high performance liquid chromatograph for area normalization method detection; The chromatographic conditions in step (3) are: Chromatographic column: Octadecyl bonded silica gel as filler; Instrument: Waters 2695; Detector: DAD detector; Mobile phase: Mobile phase A: tetrahydrofuran, mobile phase B: 0.2% phosphoric acid, the volume ratio of mobile phase A to mobile phase B is 40:60; Column temperature: 30°C; Flow rate: 1.0 mL / min; Detection wavelength: 252nm; Mobile phase elution program: isocratic elution.

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