Separation and content determination method of ginsenoside compound chiral isomer

Abstract

The invention relates to a separation and content determination method for chiral isomers of ginsenoside compounds. The ginsenoside compounds are selected from ginsenoside Rh1, ginsenoside Rh2 and ginsenoside Rg2. The method comprises two steps of sample preparation and chromatographic separation condition setting. Preferably, the normal-phase HPLC chiral separation conditions are as follows: on a # imgabs0 # chromatographic column, the mobile phase is normal hexane-ethanol (v: v = 80: 20), the flow velocity of the mobile phase is 1.0 mL min <-1 >, the column temperature is 25 DEG C, the sample size of a sample solution is 20 [mu] L, and the detection wavelength is 203 nm. The method is simple to operate, low in cost, good in separation effect, high in speed, good in repeatability and high in precision, can be used for separation and optical purity detection of ginsenoside chiral drugs, and provides guarantee for research and development of the chiral drugs.

Description

Technical Field

[0001] The invention relates to the field of analytical chemistry, in particular to a method for separating and determining the content of chiral isomers of ginsenoside compounds. Background Art

[0002] Ginsenosides are the main active ingredients in ginseng, and have multiple pharmacological activities such as anti-tumor, anti-depression, and antioxidant. Ginsenoside compounds such as Rh1, Rh2, Rg2, and Rg3 have chiral carbon at the C-20 position, which makes ginsenoside compounds have two isomers, namely 20R and 20S. It has been reported in the literature that these isomers all show different pharmacological activities and pharmacokinetic properties. For example, (20R)-Rg3 shows better antioxidant, anti-tumor and neuroprotective activities than (20S)-Rg3, but (20S)-Rg3 has better 5-HT3 inhibitory activity than (20R)-Rg3; the pharmacokinetic behavior of (20R)-Rg2 and (20S)-Rg2 in mouse plasma after intravenous injection is significantly different. Although (20R) / (20S) ginsenoside compounds have different pharmacodynamics and pharmacokinetics, due to the difficulty of isomer separation and preparation, there are currently few studies on the effects of C-20 chirality on efficacy and pharmacokinetics.

[0003] Therefore, it is necessary to establish a new chiral isomer separation and content determination method to resolve the 20R and 20S isomers of ginsenoside compounds. Summary of the invention

[0004] The purpose of the present invention is to provide a method for separation and content determination of chiral isomers of ginsenoside compounds, which can effectively separate two C-20 chiral isomers in the ginsenoside Rh1, Rh2, Rg2 racemic isomers and is easy to operate with high precision.

[0005] To achieve the purpose of the present invention, the technical solution is as follows:

[0006] A method for separating chiral isomers of ginsenoside compounds, the method using high performance liquid chromatography for separation, wherein: the mobile phase is a binary mobile phase consisting of n-hexane and an alcohol organic solvent, with a volume ratio of 80:20 to 95:5; a sample to be tested is taken, dissolved and diluted with the mobile phase to prepare a test solution; the ginsenoside compound is selected from one of ginsenosides Rh1, Rh2, and Rg2.

[0007] Further, the test solution was prepared and placed at 4 o CSave.

[0008] Furthermore, the alcohol organic solvent in the mobile phase is selected from isopropanol, ethanol or a combination of the two.

[0009] Furthermore, the concentration of the test solution is 500 μg mL -1 .

[0010] Furthermore, the liquid chromatography conditions are: the flow rate of the mobile phase is 0.5-2.0 mL min -1 ; Column temperature is 20-30 o C; detection wavelength is 190-250 nm; cellulose bio-coated chiral chromatographic column is used.

[0011] Further, the cellulose bio-coated chiral chromatographic column is selected from Chromatographic column; the specifications of the chromatographic column are 250 mm × 4.6 mm, 5 μm.

[0012] Furthermore, the injection volume was 5-20 μL.

[0013] Furthermore, the mobile phase is n-hexane and ethanol in a volume ratio of 80:20.

[0014] Furthermore, the mobile phase flow rate was 1.0 mL min -1 ; Column temperature is 25 o C; the detection wavelength is 203 nm.

[0015] Furthermore, the injection volume was 20 μL.

[0016] The present invention also provides a method for determining the content of chiral isomers of ginsenoside compounds, which adopts the above-mentioned method for separating chiral isomers of ginsenoside compounds, and prepares a reference solution in the same way as the test solution, specifically: taking a single isomer reference of ginsenoside compounds, dissolving and diluting it in a solution composed of n-hexane and an alcohol organic solvent in a volume ratio of 80: 20 to 95: 5, and preparing a reference solution; the concentration of the reference solution is 500 μg mL -1 , placed at 4 o C; inject the test solution and the reference solution into the liquid chromatograph respectively, record the chromatogram, and calculate the isomer content by the peak area according to the external standard method.

[0017] Compared with the prior art, the present invention has the following significant advantages: the present invention is a method for separating and detecting ginsenoside compounds by normal phase high performance liquid chromatography, which can effectively separate two C-20 chiral isomers in the racemic body of ginsenoside compounds (Rh1, Rh2, Rg2). The method has simple solution preparation, convenient operation, fast separation speed and high precision, and can also be used for the determination of the optical purity of a single isomer of ginsenoside, and is suitable for the quality control of such compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a chromatogram of HPLC separation of ginsenoside Rh1 racemate, (20R)-ginsenoside Rh1 reference substance and (20S)-ginsenoside Rh1 reference substance solution in Example 1 of the present invention.

[0019] Figure 2 It is a chromatogram of HPLC separation of ginsenoside Rh2 racemate, (20R)-ginsenoside Rh1 reference substance and (20S)-ginsenoside Rh1 reference substance solution in Example 2 of the present invention.

[0020] Figure 3 It is a chromatogram of HPLC separation of ginsenoside Rg2 racemate, (20R)-ginsenoside Rh1 reference substance and (20S)-ginsenoside Rh1 reference substance solution in Example 3 of the present invention. DETAILED DESCRIPTION

[0021] The embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0022] Example 1

[0023] A method for separating and detecting chiral isomers of ginsenoside compounds comprises the following steps:

[0024] Preparation of test solution: Weigh 5 mg of the purchased ginsenoside Rh1 racemic body, place it in a 10 mL brown volumetric flask, dissolve it in n-hexane-ethanol (v: v = 80: 20), filter, dilute to the mark, shake well, and obtain a concentration of 500 μg mL -1 of the test solution;

[0025] Preparation of reference solution: Weigh 5 mg of the purchased (20R)-ginsenoside Rh1 reference and (20S)-ginsenoside Rh1 reference respectively, place in a 10 mL brown volumetric flask, dissolve in n-hexane-ethanol (v: v = 80: 20), filter and dilute to the mark, shake well, and obtain a concentration of 500 μg mL -1 of reference solution;

[0026] The determination was carried out under the following chromatographic conditions:

[0027] Chromatographic instrument: Agilent 1200 high performance liquid chromatograph;

[0028] Chromatographic column: Chromatographic column (250 mm × 4.6 mm, 5 μm);

[0029] Mobile phase: n-hexane-ethanol (v: v = 80: 20);

[0030] Flow rate: 1.0 mL min-1 ;

[0031] Column temperature: 25 o C;

[0032] Detection wavelength: 203 nm;

[0033] Injection volume: 20 μL;

[0034] The chromatogram obtained is as follows Figure 1 The chromatographic parameters are shown in Table 1.

[0035] Figure 1 In the figure, (a) is the HPLC chromatogram of the separation of ginsenoside Rh1 racemic isomers, the left peak is (20R)-ginsenoside Rh1, and the right peak is (20S)-ginsenoside Rh1; (b) is the HPLC chromatogram of the (20R)-ginsenoside Rh1 reference substance; (c) is the HPLC chromatogram of the (20S)-ginsenoside Rh1 reference substance. The analysis time of the two isomers is within 25 minutes, and the resolution is 3.8.

[0036] Example 2

[0037] A method for separating and detecting chiral isomers of ginsenoside compounds comprises the following steps:

[0038] Preparation of test solution: Weigh 5 mg of purchased ginsenoside Rh2 racemic body, place in a 10 mL brown volumetric flask, dissolve in n-hexane-ethanol (v: v = 80: 20), filter, dilute to scale, shake well, and obtain a concentration of 500 μg mL -1 of the test solution;

[0039] Preparation of reference solution: Weigh 5 mg of the purchased (20R)-ginsenoside Rh2 reference and (20S)-ginsenoside Rh2 reference respectively, place in a 10 mL brown volumetric flask, dissolve in n-hexane-ethanol (v: v = 80: 20), filter and dilute to the mark, shake well, and obtain a concentration of 500 μg mL -1 of reference solution;

[0040] The determination was carried out under the following chromatographic conditions:

[0041] Chromatographic instrument: Agilent 1200 high performance liquid chromatograph;

[0042] Chromatographic column: Chromatographic column (250 mm × 4.6 mm, 5 μm);

[0043] Mobile phase: n-hexane-ethanol (v: v = 80: 20);

[0044] Flow rate: 1.0 mL min -1 ;

[0045] Column temperature: 25 o C;

[0046] Detection wavelength: 203 nm;

[0047] Injection volume: 20 μL;

[0048] The chromatogram obtained is as follows Figure 2 The chromatographic parameters are shown in Table 1.

[0049] Figure 2 In the figure, (a) is the HPLC chromatogram of the separation of the ginsenoside Rh2 racemic isomer, the left peak is (20S)-ginsenoside Rh2, and the right peak is (20R)-ginsenoside Rh2; (b) is the HPLC chromatogram of the (20R)-ginsenoside Rh2 reference substance; (c) is the HPLC chromatogram of the (20S)-ginsenoside Rh2 reference substance. The analysis time of the two isomers is within 25 minutes, and the resolution is 2.6.

[0050] Example 3

[0051] A method for separating and detecting chiral isomers of ginsenoside compounds comprises the following steps:

[0052] Preparation of test solution: Weigh 5 mg of the purchased ginsenoside Rg2 racemic body, place it in a 10 mL brown volumetric flask, dissolve it in n-hexane-ethanol (v: v = 80: 20), filter, dilute to the mark, shake well, and obtain a concentration of 500 μg mL -1 of the test solution;

[0053] Preparation of reference solution: Weigh 5 mg of the purchased (20R)-ginsenoside Rg2 reference and (20S)-ginsenoside Rg2 reference respectively, place in a 10 mL brown volumetric flask, dissolve in n-hexane-ethanol (v: v = 80: 20), filter and dilute to the mark, shake well, and obtain a concentration of 500 μg mL -1 of reference solution;

[0054] The determination was carried out under the following chromatographic conditions:

[0055] Chromatographic instrument: Agilent 1200 high performance liquid chromatograph;

[0056] Chromatographic column: Chromatographic column (250 mm × 4.6 mm, 5 μm);

[0057] Mobile phase: n-hexane-ethanol (v: v = 80: 20);

[0058] Flow rate: 1.0 mL min -1 ;

[0059] Column temperature: 25 o C;

[0060] Detection wavelength: 203 nm;

[0061] Injection volume: 20 μL;

[0062] The chromatogram obtained is as follows Figure 3 The chromatographic parameters are shown in Table 1.

[0063] Figure 3 In the figure, (a) is the HPLC chromatogram of the separation of the ginsenoside Rg2 racemic isomer, the left peak is (20R)-ginsenoside Rg2, and the right peak is (20S)-ginsenoside Rg2; (b) is the HPLC chromatogram of the (20R)-ginsenoside Rg2 reference substance; (c) is the HPLC chromatogram of the (20S)-ginsenoside Rg2 reference substance. The analysis time of the two isomers is within 25 minutes, and the resolution is 4.2.

[0064] Table 1 Chromatographic parameters of Examples 1-3

Claims

1. A method for separating chiral isomers of ginsenoside compounds; characterized in that: The method uses high performance liquid chromatography for separation, wherein: the mobile phase is a binary mobile phase composed of n-hexane and an alcohol organic solvent, and the volume ratio is 80:20 to 95:5; the sample to be tested is taken, dissolved and diluted with the mobile phase to prepare a test solution; the ginsenoside compound is selected from one of ginsenosides Rh1, Rh2, and Rg2.

2. The method for separating chiral isomers of ginsenoside compounds according to claim 1, characterized in that: The alcohol organic solvent in the mobile phase is selected from isopropanol, ethanol or a combination of the two.

3. The method for separating chiral isomers of ginsenoside compounds according to claim 1, characterized in that: The concentration of the test solution is 500 μg mL -1 .

4. The method for separating chiral isomers of ginsenoside compounds according to claim 1, characterized in that: The liquid chromatography conditions are as follows: the flow rate of the mobile phase is 0.5-2.0 mL min -1 ; Column temperature is 20-30 o C; detection wavelength is 190-250 nm; cellulose bio-coated chiral chromatographic column is used.

5. The method for separating chiral isomers of ginsenoside compounds according to claim 4, characterized in that: the cellulose bio-coated chiral chromatographic column is selected from Chromatographic column; the specifications of the chromatographic column are 250 mm × 4.6 mm, 5 μm.

6. The method for separating chiral isomers of ginsenoside compounds according to claim 4, characterized in that: The injection volume was 5-20 μL.

7. The method for separating chiral isomers of ginsenoside compounds according to claim 1, characterized in that: The mobile phase was n-hexane and ethanol in a volume ratio of 80:

20.

8. The method for separating chiral isomers of ginsenoside compounds according to claim 4, characterized in that: The mobile phase flow rate was 1.0 mL min -1 ; Column temperature is 25 o C; the detection wavelength is 203 nm.

9. The method for separating chiral isomers of ginsenoside compounds according to claim 6, characterized in that: The injection volume was 20 μL.

10. A method for determining the content of chiral isomers of ginsenoside compounds, characterized in that: Using the method for separating chiral isomers of ginsenoside compounds as described in any of the above items, take the ginsenoside test sample and the corresponding ginsenoside single isomer reference substance, dissolve them in a solution composed of n-hexane and alcohol organic solvent in a volume ratio of 80: 20 to 95: 5, and dilute them to prepare test sample and reference substance solutions, respectively, with a solution concentration of 500 μg mL -1 ; Inject the test solution and the reference solution into the liquid chromatograph respectively, record the chromatogram, and calculate the isomer content by peak area according to the external standard method.

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