High-purity cyanine standard sample and preparation method thereof

Abstract

The invention relates to the technical field of anthocyanin preparation and purification, in particular to a high-purity anthocyanin standard sample and a preparation method thereof. According to the method, by combining reversed-phase phenyl chromatography and reversed-phase C18 chromatography and optimizing the relationship between the sample loading amount and the purity, the anthocyanin standard sample with the purity higher than 98% is obtained from acidolysis products of grape seed extracts through purification. Due to the fact that the removal effects of the two chromatography methods on different impurities are different, different types of impurities can be removed through combined use of the two chromatography methods, the sample loading capacity is increased, the preparation efficiency is improved, and the recovery rate can be higher than 90%. The method is simple and economical, and the obtained cyanine standard sample is very high in purity, can be used as a reference substance for quality standard research work of cyanine extract containing cyanine, and provides effective assistance for related scientific research work.

Description

Technical Field

[0001] The invention relates to the technical field of cyanidine preparation and purification, in particular to a high-purity cyanidine standard sample and a preparation method thereof. Background Art

[0002] Chromatography is widely used in pharmaceutical, chemical and other fields as a separation method in modern society. By utilizing the differences in adsorption force, distribution coefficient, solubility and other principles, the target compound can be continuously distributed on the chromatographic column to form separable "color bands" (colorless substances will show different absorption peaks under specific detectors). The application field of chromatography technology is very wide, and the requirements for chromatography technology are very different under different needs. Combining chromatographic methods with different separation mechanisms can remove different types of impurities around the target compound, thereby achieving a more efficient purification effect than a single chromatographic method.

[0003] Cyanidin is a parent compound of anthocyanidin compounds, which has strong antioxidant and anti-inflammatory effects, and can promote the regeneration of rhodopsin in retinal cells, prevent myopia, and improve vision. In nature, there are very few free anthocyanidins. In order to obtain a wider range of anthocyanidins, scientists hydrolyze anthocyanidins from plants rich in proanthocyanidins such as grape seeds by acid hydrolysis, and then obtain crude anthocyanidins by alcohol / acid hydrolysis. However, these methods only obtain crude anthocyanidins, and cannot obtain standard samples with a purity higher than 98%, and cannot be used for quality assessment. Due to the complex composition of crude anthocyanidins, a single chromatographic method is not enough to obtain a standard sample of anthocyanidins with a purity higher than 98%. Summary of the invention

[0004] One of the purposes of the present invention is to overcome the disadvantage that the prior art cannot obtain anthocyanidin standard samples with a purity higher than 98% from anthocyanidin extracts, and to provide a method for preparing anthocyanidin standard samples with high chromatographic loading, good mobile phase compatibility and high purity.

[0005] To achieve the above object, the present invention adopts the following technical scheme: a method for preparing a high-purity cyanine standard sample, comprising the following steps:

[0006] S1. Connect a reverse phase phenyl chromatographic column to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 0-45°C; dissolve the anthocyanidin extract in water to prepare a solution with a concentration of 0.5-100 mg / mL, centrifuge to remove the precipitate, use the solution as the first injection solution, and store it at low temperature and away from light for later use;

[0007] A formic acid aqueous solution with a concentration of 0.05% to 5% and acetonitrile are mixed as the first mobile phase through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the first mobile phase is 5% to 20%, and the flow rate of the solvent pump is adjusted to 0.3 to 1.2 mL / min, and the reversed-phase phenyl chromatographic column is balanced to a baseline level; the first injection liquid is injected into the balanced reversed-phase chromatographic column for separation, and the component containing cyanidine is collected with an injection volume of 5 to 200 μL, and stored at -20°C to 10°C for standby use as the second injection liquid;

[0008] S2. Connect a reverse phase C18 chromatographic column to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 0-45°C;

[0009] A formic acid aqueous solution with a concentration of 0.05% to 5% and acetonitrile are mixed as a second mobile phase through a solvent pump of a high performance liquid chromatograph, the volume fraction of acetonitrile in the second mobile phase is 5% to 30%, the flow rate of the solvent pump is adjusted to 0.3 to 1.2 mL / min, the reversed-phase C18 chromatographic column is balanced to a baseline level, all the second injection liquid is injected into the balanced reversed-phase chromatographic column for separation, components containing cyanidine are collected, and freeze-dried in the dark to obtain a cyanidine standard sample with a purity higher than 98 wt%.

[0010] Further improvement of the preparation method of high purity cyanine standard sample:

[0011] Preferably, the preparation method of the anthocyanidin extract in step S1 is as follows:

[0012] S11, adding a methanol / saturated hydrochloric acid mixed solution to the dried grape seed skin for hot reflux extraction, filtering the obtained extraction solution, concentrating to dryness, adding water to dissolve, and filtering to obtain a filtrate;

[0013] S12. Pump the filtrate into a chromatography column for adsorption, control the column temperature of the chromatography column to -5 to 30°C, and after saturated adsorption, wash the chromatography column with water until the pH of the effluent is neutral, then elute the chromatography column with ethanol, collect the eluate, concentrate it to be alcohol-free, and then dry it to obtain anthocyanidin extract.

[0014] Preferably, in step S11, a methanol / saturated hydrochloric acid aqueous solution is added to the dried grape seed skin for hot reflux extraction, and the material-liquid ratio is 1:(1-5).

[0015] Preferably, the volume fraction of methanol in the methanol / saturated hydrochloric acid aqueous solution in step S11 is 90% to 98%.

[0016] Preferably, the chromatography column in step S12 is filled with HPD 300 or HPD 600 macroporous resin.

[0017] Preferably, the reverse phase phenyl chromatographic column in step S1 has a length of 100 to 350 mm, a diameter of 2 to 5 mm, and a filler particle size of 5 to 10 μm.

[0018] Preferably, the first mobile phase in step S1 is formed by mixing a formic acid aqueous solution with a concentration of 1.5% to 3.5% and acetonitrile through a solvent pump of a high performance liquid chromatograph, and the volume fraction of acetonitrile in the first mobile phase is 12% to 17%.

[0019] Preferably, the reverse phase C18 chromatographic column in step S2 has a length of 100 to 350 mm, a diameter of 2 to 5 mm, and a filler particle size of 5 to 10 μm.

[0020] Preferably, the second mobile phase in step S2 is formed by mixing a formic acid aqueous solution with a concentration of 1.5% to 3.5% and acetonitrile through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the second mobile phase is 8% to 14%.

[0021] The second object of the present invention is to provide a high-purity cyanidine standard sample prepared by the preparation method of the high-purity cyanidine standard sample described in any one of the above items.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1) The present invention relates to a method for preparing a cyanidin standard sample with a purity higher than 98%. The method uses an acid-hydrolyzed cyanidin extract of grape seed extract as a raw material, combines reversed-phase phenyl column chromatography and reversed-phase C18 column chromatography into an orthogonal chromatography method, and removes impurities by utilizing the difference in separation effects of the two chromatographic methods, and finally prepares a cyanidin standard with a purity higher than 98% from the cyanidin crude product. In the present invention, the impurities around the cyanidin that are difficult to remove by the first reversed-phase phenyl column chromatography can be completely removed by the second reversed-phase C18 column by utilizing the different separation degrees and orders between impurities and cyanidin under the two reversed-phase chromatography. Compared with a single reversed-phase chromatography, the two reversed-phase chromatography methods complement each other in terms of their ability to remove impurities, so that in the chromatographic preparation process, the loading capacity of the cyanidin extract on the analytical phenyl chromatography column (specifications are 350 mm long, 4.6 mm in diameter, and a filler particle size of 10 microns) is as high as 10 mg / time, which greatly improves the preparation efficiency, and the recovery rate can also be higher than 90%.

[0024] 2) The purity of the anthocyanidin obtained by the present invention is as high as 98%, and it can be used as a standard sample for the quality standard research of anthocyanidin extracts containing anthocyanidin, providing effective support for related scientific research work. The method is simple and economical, has high sample loading capacity, high purity, high recovery rate, and has certain industrial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1The chromatogram is a chromatogram of the grape seed anthocyanidin extract obtained by hydrolysis in Example 1 of the present invention purified on a reverse phase phenyl column.

[0026] Figure 2 This is a chromatographic analysis chart of the anthocyanidin-containing components obtained after the grape seed anthocyanidin extract obtained by hydrolysis in Example 1 of the present invention was purified by a reverse phase phenyl column.

[0027] Figure 3 This is a chromatogram of the purification of the second injection solution on a reverse phase C18 column in Example 1 of the present invention.

[0028] Figure 4 This is a chromatographic analysis chart of the anthocyanidin component obtained after the second sample solution in Example 1 of the present invention is purified by a reverse phase C18 column.

[0029] Figure 5 This is a chromatographic preparation diagram of the grape seed anthocyanidin extract obtained by hydrolysis in Comparative Example 1 purified by a reverse phase phenyl column.

[0030] Figure 6 This is a chromatographic analysis chart of the anthocyanidin component obtained after the grape seed anthocyanidin extract obtained by hydrolysis in Comparative Example 1 was purified by a reverse phase phenyl column.

[0031] Figure 7 This is a chromatographic preparation diagram of the grape seed anthocyanidin extract obtained by hydrolysis in Comparative Example 2 purified by a reverse phase C18 column.

[0032] Figure 8 This is a chromatographic analysis chart of the anthocyanidin-containing components obtained after the grape seed anthocyanidin extract obtained by hydrolysis in Comparative Example 2 was purified by a reverse phase C18 column. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention.

[0034] Preparation Example 1

[0035] This preparation example provides a method for preparing anthocyanidin extract, which comprises the following steps:

[0036] S11, adding a methanol / saturated hydrochloric acid water mixed solution to the dried grape seed skin for hot reflux extraction, the solid-liquid ratio is 1:5, the volume fraction of methanol in the methanol / saturated hydrochloric acid water mixed solution is 98%, the obtained extraction solution is filtered, concentrated to dryness, water is added, ultrasonically dissolved, and then filtered to obtain a filtrate;

[0037] S12. The filtrate is pumped into a chromatography column filled with HPD 300 macroporous resin for adsorption. During the adsorption process, the column temperature is controlled at 30°C. After saturated adsorption, the chromatography column is washed with water until the effluent is neutral, and then the chromatography column is eluted with ethanol. The eluate is collected, concentrated to alcohol-free, and then freeze-dried to obtain anthocyanidin extract.

[0038] Example 1

[0039] This embodiment provides a method for preparing a high-purity cyanine standard sample, which comprises the following steps:

[0040] S1. Connect a reverse phase phenyl chromatographic column (specifications: column length 250 mm, inner diameter 4.6 mm, filler particle size 10 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 7°C; dissolve the cyanidine extract obtained in Preparation Example 1 in water to prepare a solution with a concentration of 100 mg / mL, centrifuge to remove the precipitate, and use the solution as the first injection solution, and store it at -5°C in a dark place for later use;

[0041] A 2.5% formic acid aqueous solution and acetonitrile are mixed through a solvent pump of a high performance liquid chromatograph as a first mobile phase, wherein the volume fraction of acetonitrile in the first mobile phase is 15%, and the flow rate of the solvent pump is adjusted to 1.0 mL / min, and the above-mentioned reverse-phase phenyl chromatographic column is balanced to a baseline level; the first injection liquid is injected into the balanced reverse-phase chromatographic column for separation, with an injection volume of 50 μL, and a component containing cyanidine is collected and stored at -5°C for use as a second injection liquid.

[0042] S2. Connect a reverse phase C18 chromatographic column (specifications: column length 250 mm, inner diameter 4.6 mm, filler particle size 7 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 7°C;

[0043] A 2.5% formic acid aqueous solution and acetonitrile were mixed as the second mobile phase through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the second mobile phase was 12%. The flow rate of the solvent pump was adjusted to 1.0 mL / min, the reversed-phase C18 chromatographic column was balanced to a baseline level, and the second injection liquid was completely injected into the balanced reversed-phase chromatographic column for separation, and the components containing cyanidine were collected and freeze-dried in the dark to obtain a cyanidine standard sample.

[0044] In step S1 of Example 1, the first sample solution is injected into a well-equilibrated reverse phase chromatographic column for separation. The preparation and purification diagram is shown in FIG. Figure 1 As shown. Figure 1It can be seen that the crude anthocyanidin system is very complex, and there are many types of impurities around the chromatographic peak of anthocyanidin (15-18min).

[0045] The component containing cyanidine in step S1 is detected by high performance liquid chromatography method 1, such as Figure 2 As shown; HPLC method 1 is as follows: a reversed-phase phenyl chromatographic column (specifications: column length 250 mm, inner diameter 4.6 mm, filler particle size 10 μm, manufacturer: Jiangsu Hanbang Technology Co., Ltd.) is connected to a HPLC instrument equipped with a dual-channel UV detector, and the wavelength of the dual-channel UV detector is adjusted to 254 nm, the column temperature is controlled to 25°C, and the injection volume is 50 μL; mobile phase: 2% formic acid aqueous solution and acetonitrile are mixed as mobile phase through the solvent pump of the HPLC instrument, and the volume fraction of acetonitrile in the mobile phase is 12%, and the flow rate of the solvent pump is adjusted to 1.0 mL / min. By Figure 2 It can be seen that after the first injection liquid is purified by the reverse phase phenyl column, there are still many kinds of impurities.

[0046] In step S2 of Example 1, the second sample solution is completely injected into a well-balanced reverse phase chromatographic column for separation, and the preparation and purification diagram thereof is shown in FIG3. Figure 3 It can be seen that the separation on the reversed phase C18 column was better than that on the phenyl column.

[0047] The component containing cyanidine in step S2 is detected by high performance liquid chromatography method 2, and the chromatogram obtained by the detection is as follows: Figure 4 As shown. HPLC method 2 is as follows: a reversed-phase C18 chromatographic column (specifications: column length 250 mm, inner diameter 4.6 mm, filler particle size 5 μm, manufacturer: Huapu Science and Technology Co., Ltd. (Beijing)) is connected to a HPLC instrument equipped with a dual-channel UV detector, and the wavelengths of the dual-channel UV detector are adjusted to 254 nm, the column temperature is controlled to 25°C, and the injection volume is 50 μL; mobile phase: 2% formic acid aqueous solution and acetonitrile are mixed through the solvent pump of the HPLC as the mobile phase, the volume fraction of acetonitrile in the mobile phase is 12%, and the flow rate of the solvent pump is adjusted to 1.0 mL / min. By Figure 4 It can be seen that step S2 finally produces 4.8 mg of cyanidin standard sample with a purity of 98.2% and a recovery rate of 94%.

[0048] Preparation Example 2

[0049] This preparation example provides a method for preparing anthocyanidin extract, which comprises the following steps:

[0050] S11, adding a methanol / saturated hydrochloric acid water mixed solution to the dried grape seed skin for hot reflux extraction, the solid-liquid ratio is 1:3, the volume fraction of methanol in the methanol / saturated hydrochloric acid water mixed solution is 90%, the obtained extraction solution is filtered, concentrated to dryness, water is added, ultrasonically dissolved, and then filtered to obtain a filtrate;

[0051] S12. Pump the filtrate into a chromatography column filled with HPD 300 macroporous resin for adsorption. During the adsorption process, control the column temperature at -5°C. After saturated adsorption, wash the chromatography column with water until the effluent is neutral. Then elute the chromatography column with ethanol. Collect the eluate, concentrate it to be alcohol-free, and then freeze-dry it to obtain anthocyanidin extract.

[0052] Example 2

[0053] This embodiment provides a method for preparing a high-purity cyanine standard sample, which comprises the following steps:

[0054] S1. Connect a reverse phase phenyl chromatographic column (specifications: column length 250 mm, inner diameter 4.6 mm, filler particle size 10 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 0°C; dissolve the cyanidine extract obtained in Preparation Example 2 in water to prepare a solution with a concentration of 50 mg / mL, centrifuge to remove the precipitate, use the solution as the first injection solution, and store it at 0°C in the dark for later use;

[0055] A 2.5% formic acid aqueous solution and acetonitrile are mixed as the first mobile phase through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the first mobile phase is 10%, and the flow rate of the solvent pump is adjusted to 0.8 mL / min, and the above-mentioned reversed-phase phenyl chromatographic column is balanced to a baseline level; the first injection liquid is injected into the balanced reversed-phase chromatographic column for separation, with an injection volume of 200 μL, and the component containing cyanidine is collected and stored at 0° C. for use as the second injection liquid;

[0056] S2. Connect a reverse phase C18 chromatographic column (specifications: column length 250 mm, inner diameter 4.6 mm, filler particle size 10 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 20° C.;

[0057] A 2.5% formic acid aqueous solution and acetonitrile are mixed as the second mobile phase through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the second mobile phase is 15%. The flow rate of the solvent pump is adjusted to 0.8 mL / min, the reversed-phase C18 chromatographic column is balanced to a baseline level, and the second injection liquid is fully injected into the balanced reversed-phase chromatographic column for separation, and the components containing cyanidine are collected and freeze-dried in a dark place to obtain a cyanidine standard sample.

[0058] The test showed that 2.9 mg of a cyanidine standard sample with a purity of 98.5% was obtained in this embodiment, and the recovery rate was 95%.

[0059] Preparation Example 3

[0060] This preparation example provides a method for preparing anthocyanidin extract, which comprises the following steps:

[0061] S11, adding a methanol / saturated hydrochloric acid water mixed solution to the dried grape seed skin for hot reflux extraction, the solid-liquid ratio is 1:1, the volume fraction of methanol in the methanol / saturated hydrochloric acid water mixed solution is 94%, the obtained extraction solution is filtered, concentrated to dryness, water is added, ultrasonically dissolved, and then filtered to obtain a filtrate;

[0062] S12. The filtrate is pumped into a chromatography column filled with HPD 600 macroporous resin for adsorption. During the adsorption process, the column temperature is controlled at 15°C. After saturated adsorption, the chromatography column is washed with water until the effluent is neutral, and then the chromatography column is eluted with ethanol. The eluate is collected, concentrated to alcohol-free, and then freeze-dried to obtain anthocyanidin extract.

[0063] Example 3

[0064] This embodiment provides a method for preparing a high-purity cyanine standard sample, which comprises the following steps:

[0065] S1. Connect a reverse phase phenyl chromatographic column (specifications: column length 350 mm, inner diameter 4.6 mm, filler particle size 10 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 20° C.; dissolve the cyanidine extract obtained in Preparation Example 3 in water to prepare a solution with a concentration of 100 mg / mL, centrifuge to remove the precipitate, use the solution as the first injection solution, and store it in the dark at 10° C. for later use;

[0066] A 5% formic acid aqueous solution and acetonitrile are mixed as the first mobile phase through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the first mobile phase is 5%, and the flow rate of the solvent pump is adjusted to 1.2 mL / min, and the above-mentioned reversed-phase phenyl chromatographic column is balanced to a baseline level; the first injection liquid is injected into the balanced reversed-phase chromatographic column for separation, and the injection volume is 100 μL, and the component containing cyanidine is collected and stored at 0° C. for use as the second injection liquid;

[0067] S2. Connect a reverse phase C18 chromatographic column (specifications: column length 350 mm, inner diameter 2 mm, filler particle size 5 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 0°C;

[0068] A 2.5% formic acid aqueous solution and acetonitrile are mixed as the second mobile phase through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the second mobile phase is 5%. The flow rate of the solvent pump is adjusted to 1.2 mL / min, the reversed-phase C18 chromatographic column is balanced to a baseline level, and the second injection liquid is fully injected into the balanced reversed-phase chromatographic column for separation, and the components containing cyanidine are collected and freeze-dried in a dark place to obtain a cyanidine standard sample.

[0069] The test showed that 2.9 mg of a cyanidine standard sample with a purity of 98.2% was obtained in this embodiment, and the recovery rate was 94%.

[0070] Preparation Example 4

[0071] This preparation example provides a method for preparing anthocyanidin extract, which comprises the following steps:

[0072] S1. Adding a methanol / saturated hydrochloric acid water mixed solution to dried grape seed skins for hot reflux extraction, wherein the solid-liquid ratio is 1:5, and the volume fraction of methanol in the methanol / saturated hydrochloric acid water mixed solution is 98%, filtering the obtained extraction solution, concentrating to dryness, adding water, ultrasonically dissolving, and filtering to obtain a filtrate;

[0073] S2. The filtrate is pumped into a chromatography column filled with HPD 300 macroporous resin for adsorption. During the adsorption process, the column temperature is controlled at -5°C. After saturated adsorption, the chromatography column is washed with water until the effluent is neutral, and then the chromatography column is eluted with ethanol. The eluate is collected, concentrated to alcohol-free, and then freeze-dried to obtain anthocyanidin extract.

[0074] Example 4

[0075] This embodiment provides a method for preparing a high-purity cyanine standard sample, which comprises the following steps:

[0076] S1. Connect a reverse phase phenyl chromatographic column (specifications: column length 250 mm, inner diameter 4.6 mm, filler particle size 7 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 45°C; dissolve the cyanidine extract obtained in Preparation Example 4 in water to prepare a solution with a concentration of 0.5 mg / mL, centrifuge to remove the precipitate, use the solution as the first injection solution, and store it at -20°C in the dark for later use;

[0077] A 0.05% formic acid aqueous solution and acetonitrile are mixed as the first mobile phase through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the first mobile phase is 20%, and the flow rate of the solvent pump is adjusted to 0.3 mL / min, and the above-mentioned reversed-phase phenyl chromatographic column is balanced to a baseline level; the first injection liquid is injected into the balanced reversed-phase chromatographic column for separation, with an injection volume of 200 μL, and the component containing cyanidine is collected and stored at -20°C for use as the second injection liquid;

[0078] S2. Connect a reverse phase C18 chromatographic column (specifications: column length 250 mm, inner diameter 4.6 mm, filler particle size 10 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 45° C.

[0079] A 0.05% formic acid aqueous solution and acetonitrile are mixed as the second mobile phase through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the second mobile phase is 30%. The flow rate of the solvent pump is adjusted to 0.3 mL / min, the reversed-phase C18 chromatographic column is balanced to a baseline level, and the second injection liquid is fully injected into the balanced reversed-phase chromatographic column for separation, and the components containing cyanidine are collected and freeze-dried in a dark place to obtain a cyanidine standard sample.

[0080] The test showed that 0.9 mg of a cyanidine standard sample with a purity of 98.2% was obtained in this embodiment, and the recovery rate was 96%.

[0081] Preparation Example 5

[0082] This preparation example provides a method for preparing anthocyanidin extract, which comprises the following steps:

[0083] S11, adding a methanol / saturated hydrochloric acid water mixed solution to the dried grape seed skin for hot reflux extraction, the solid-liquid ratio is 1:5, the volume fraction of methanol in the methanol / saturated hydrochloric acid water mixed solution is 98%, the obtained extraction solution is filtered, concentrated to dryness, water is added, ultrasonically dissolved, and then filtered to obtain a filtrate;

[0084] S12. The filtrate is pumped into a chromatography column filled with HPD 600 macroporous resin for adsorption. During the adsorption process, the column temperature is controlled at 30°C. After saturation of adsorption, the chromatography column is washed with water until the effluent is neutral. The chromatography column is then eluted with ethanol. The eluate is collected, concentrated to alcohol-free, and then freeze-dried to obtain anthocyanidin extract.

[0085] Example 5

[0086] This embodiment provides a method for preparing a high-purity cyanine standard sample, which comprises the following steps:

[0087] S1. Connect a reverse phase phenyl chromatographic column (specifications: column length 100 mm, inner diameter 5 mm, filler particle size 5 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 20°C; dissolve the cyanidine extract obtained in Preparation Example 5 in water to prepare a solution with a concentration of 100 mg / mL, centrifuge to remove the precipitate, use the solution as the first injection solution, and store it at -20°C in the dark for later use;

[0088] A 5% formic acid aqueous solution and acetonitrile are mixed as the first mobile phase through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the first mobile phase is 20%, and the flow rate of the solvent pump is adjusted to 0.3 mL / min, and the above-mentioned reversed-phase phenyl chromatographic column is balanced to a baseline level; the first injection liquid is injected into the balanced reversed-phase chromatographic column for separation, and the injection volume is 5 μL, and the component containing cyanidine is collected and stored at 10° C. for standby use as the second injection liquid;

[0089] S2. Connect a reverse phase C18 chromatographic column (specifications: column length 100 mm, inner diameter 5 mm, filler particle size 5 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 0°C;

[0090] A 5% formic acid aqueous solution and acetonitrile are mixed as the second mobile phase through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the second mobile phase is 30%. The flow rate of the solvent pump is adjusted to 0.3 mL / min, the reversed-phase C18 chromatographic column is balanced to a baseline level, and the second injection liquid is completely injected into the balanced reversed-phase chromatographic column for separation, and the components containing cyanidine are collected and freeze-dried in a dark place to obtain a cyanidine standard sample.

[0091] The test showed that 0.4 mg of a cyanidin standard sample with a purity of 98.2% was obtained in this embodiment, and the recovery rate was 90%.

[0092] Preparation Example 6

[0093] This preparation example provides a method for preparing anthocyanidin extract, which comprises the following steps:

[0094] S11, adding a methanol / saturated hydrochloric acid water mixed solution to the dried grape seed skin for hot reflux extraction, the solid-liquid ratio is 1:5, the volume fraction of methanol in the methanol / saturated hydrochloric acid water mixed solution is 98%, the obtained extraction solution is filtered, concentrated to dryness, water is added, ultrasonically dissolved, and then filtered to obtain a filtrate;

[0095] S12. The filtrate is pumped into a chromatography column filled with HPD 300 macroporous resin for adsorption. During the adsorption process, the column temperature is controlled at 30°C. After saturated adsorption, the chromatography column is washed with water until the effluent is neutral, and then the chromatography column is eluted with ethanol. The eluate is collected, concentrated to alcohol-free, and then freeze-dried to obtain anthocyanidin extract.

[0096] Example 6

[0097] This embodiment provides a method for preparing a high-purity cyanine standard sample, which comprises the following steps:

[0098] S1. Connect a reverse phase phenyl chromatographic column (specifications: column length 250 mm, inner diameter 2 mm, filler particle size 10 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 5°C; dissolve the cyanidine extract obtained in Preparation Example 6 in water to prepare a solution with a concentration of 100 mg / mL, centrifuge to remove the precipitate, use the solution as the first injection solution, and store it at -10°C in the dark for later use;

[0099] A 1.5% formic acid aqueous solution and acetonitrile are mixed as the first mobile phase through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the first mobile phase is 12%, and the flow rate of the solvent pump is adjusted to 1.0 mL / min, and the above-mentioned reversed-phase phenyl chromatographic column is balanced to a baseline level; the first injection liquid is injected into the balanced reversed-phase chromatographic column for separation, and the injection volume is 100 μL, and the component containing cyanidine is collected and stored at -10°C for use as the second injection liquid;

[0100] S2. Connect a reverse phase C18 chromatographic column (specifications: column length 350 mm, inner diameter 2 mm, filler particle size 7 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 5°C;

[0101] A 1.5% formic acid aqueous solution and acetonitrile are mixed as the second mobile phase through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the second mobile phase is 8%. The flow rate of the solvent pump is adjusted to 1.0 mL / min, the above reversed phase C18 chromatographic column is balanced to a baseline level, and the second injection liquid is fully injected into the balanced reversed phase chromatographic column for separation, and the components containing cyanidine are collected and freeze-dried in a dark place to obtain a cyanidine standard sample.

[0102] The test showed that 2.8 mg of a cyanidin standard sample with a purity of 98.6% was obtained in this embodiment, and the recovery rate was 93%.

[0103] Preparation Example 7

[0104] This preparation example provides a method for preparing anthocyanidin extract, which comprises the following steps:

[0105] S11, adding a methanol / saturated hydrochloric acid water mixed solution to the dried grape seed skin for hot reflux extraction, the solid-liquid ratio is 1:5, the volume fraction of methanol in the methanol / saturated hydrochloric acid water mixed solution is 98%, the obtained extraction solution is filtered, concentrated to dryness, water is added, ultrasonically dissolved, and then filtered to obtain a filtrate;

[0106] S12. The filtrate is pumped into a chromatography column filled with HPD 300 macroporous resin for adsorption. During the adsorption process, the column temperature is controlled at 30°C. After saturated adsorption, the chromatography column is washed with water until the effluent is neutral, and then the chromatography column is eluted with ethanol. The eluate is collected, concentrated to alcohol-free, and then freeze-dried to obtain anthocyanidin extract.

[0107] Example 7

[0108] This embodiment provides a method for preparing a high-purity cyanine standard sample, which comprises the following steps:

[0109] S1. Connect a reverse phase phenyl chromatographic column (specifications: column length 250 mm, inner diameter 4.6 mm, filler particle size 10 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 10°C; dissolve the cyanidine extract obtained in Preparation Example 7 in water to prepare a solution with a concentration of 100 mg / mL, centrifuge to remove the precipitate, use the solution as the first injection solution, and store it at 0°C in the dark for later use;

[0110] A 3.5% formic acid aqueous solution and acetonitrile are mixed as the first mobile phase through the solvent pump of the high performance liquid chromatograph, the volume fraction of acetonitrile in the first mobile phase is 17%, the flow rate of the solvent pump is adjusted to 1.0 mL / min, and the above reversed-phase phenyl chromatographic column is balanced to the baseline level; the first injection liquid is injected into the balanced reversed-phase chromatographic column for separation, the injection volume is 20 μL, and the component containing cyanidine is collected and stored at 0° C. for use as the second injection liquid;

[0111] S2. Connect a reverse phase C18 chromatographic column (specifications: column length 350 mm, inner diameter 2 mm, filler particle size 7 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 10°C;

[0112] A 2.5% formic acid aqueous solution and acetonitrile are mixed as the second mobile phase through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the second mobile phase is 14%. The flow rate of the solvent pump is adjusted to 1.0 mL / min, the reversed-phase C18 chromatographic column is balanced to a baseline level, and the second injection liquid is fully injected into the balanced reversed-phase chromatographic column for separation, and the components containing cyanidine are collected and freeze-dried in a dark place to obtain a cyanidine standard sample.

[0113] The test showed that 4.8 mg of a cyanidine standard sample with a purity of 98.2% was obtained in this embodiment, and the recovery rate was 92%.

[0114] Preparation Example 8

[0115] This preparation example provides a method for preparing anthocyanidin extract, which comprises the following steps:

[0116] S11, adding a methanol / saturated hydrochloric acid water mixed solution to the dried grape seed skin for hot reflux extraction, the solid-liquid ratio is 1:5, the volume fraction of methanol in the methanol / saturated hydrochloric acid water mixed solution is 98%, the obtained extraction solution is filtered, concentrated to dryness, water is added, ultrasonically dissolved, and then filtered to obtain a filtrate;

[0117] S12. The filtrate is pumped into a chromatography column filled with HPD 300 macroporous resin for adsorption. During the adsorption process, the column temperature is controlled at 30°C. After saturated adsorption, the chromatography column is washed with water until the effluent is neutral, and then the chromatography column is eluted with ethanol. The eluate is collected, concentrated to alcohol-free, and then freeze-dried to obtain anthocyanidin extract.

[0118] Example 8

[0119] This embodiment provides a method for preparing a high-purity cyanine standard sample, which comprises the following steps:

[0120] S1. Connect a reverse phase phenyl chromatographic column (specifications: column length 250 mm, inner diameter 4.6 mm, filler particle size 10 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 5°C; dissolve the cyanidine extract obtained in Preparation Example 8 in water to prepare a solution with a concentration of 100 mg / mL, centrifuge to remove the precipitate, use the solution as the first injection solution, and store it at 0°C in the dark for later use;

[0121] A 3.5% formic acid aqueous solution and acetonitrile are mixed as the first mobile phase through the solvent pump of the high performance liquid chromatograph, the volume fraction of acetonitrile in the first mobile phase is 12%, the flow rate of the solvent pump is adjusted to 1.0 mL / min, and the above-mentioned reversed-phase phenyl chromatographic column is balanced to the baseline level; the first injection liquid is injected into the balanced reversed-phase chromatographic column for separation, the injection volume is 20 μL, and the component containing cyanidine is collected and stored at 0° C. for use as the second injection liquid;

[0122] S2. Connect a reverse phase C18 chromatographic column (specifications: column length 350 mm, inner diameter 5 mm, filler particle size 5 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 10°C;

[0123] A 2.5% formic acid aqueous solution and acetonitrile are mixed as the second mobile phase through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the second mobile phase is 10%. The flow rate of the solvent pump is adjusted to 1.0 mL / min, the reversed-phase C18 chromatographic column is balanced to a baseline level, and the second injection liquid is fully injected into the balanced reversed-phase chromatographic column for separation, and the components containing cyanidine are collected and freeze-dried in a dark place to obtain a cyanidine standard sample.

[0124] The test showed that 1.8 mg of a cyanidine standard sample with a purity of 98.3% was obtained in this embodiment, and the recovery rate was 95%.

[0125] Preparation Example 9

[0126] This preparation example provides a method for preparing anthocyanidin extract, which comprises the following steps:

[0127] S11, adding a methanol / saturated hydrochloric acid water mixed solution to the dried grape seed skin for hot reflux extraction, the solid-liquid ratio is 1:5, the volume fraction of methanol in the methanol / saturated hydrochloric acid water mixed solution is 98%, the obtained extraction solution is filtered, concentrated to dryness, water is added, ultrasonically dissolved, and then filtered to obtain a filtrate;

[0128] S12. The filtrate is pumped into a chromatography column filled with HPD 300 macroporous resin for adsorption. During the adsorption process, the column temperature is controlled at 30°C. After saturated adsorption, the chromatography column is washed with water until the effluent is neutral, and then the chromatography column is eluted with ethanol. The eluate is collected, concentrated to alcohol-free, and then freeze-dried to obtain anthocyanidin extract.

[0129] Example 9

[0130] This embodiment provides a method for preparing a high-purity cyanine standard sample, which comprises the following steps:

[0131] S1. Connect a reverse phase phenyl chromatographic column (specifications: column length 250 mm, inner diameter 4.6 mm, filler particle size 10 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 5°C; dissolve the cyanidine extract obtained in Preparation Example 9 in water to prepare a solution with a concentration of 100 mg / mL, centrifuge to remove the precipitate, use the solution as the first injection solution, and store it at 0°C in the dark for later use;

[0132] A 3.5% formic acid aqueous solution and acetonitrile are mixed as the first mobile phase through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the first mobile phase is 15%, and the flow rate of the solvent pump is adjusted to 1.0 mL / min, and the above-mentioned reversed-phase phenyl chromatographic column is balanced to a baseline level; the first injection liquid is injected into the balanced reversed-phase chromatographic column for separation, with an injection volume of 100 μL, and the component containing cyanidine is collected and stored at -10°C for use as the second injection liquid;

[0133] S2. Connect a reverse phase C18 chromatographic column (specifications: column length 350 mm, inner diameter 5 mm, filler particle size 10 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 10°C;

[0134] A 2.5% formic acid aqueous solution and acetonitrile are mixed as the second mobile phase through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the second mobile phase is 8%. The flow rate of the solvent pump is adjusted to 1.0 mL / min, the reversed-phase C18 chromatographic column is balanced to a baseline level, and the second injection liquid is fully injected into the balanced reversed-phase chromatographic column for separation, and the components containing cyanidine are collected and freeze-dried in the dark to obtain a cyanidine standard sample.

[0135] The test showed that 4.9 mg of a cyanidine standard sample with a purity of 98.6% was obtained in this embodiment, and the recovery rate was 92%.

[0136] Preparation Example 10

[0137] This preparation example provides a method for preparing anthocyanidin extract, which comprises the following steps:

[0138] S11, adding a methanol / saturated hydrochloric acid water mixed solution to the dried grape seed skin for hot reflux extraction, the solid-liquid ratio is 1:5, the volume fraction of methanol in the methanol / saturated hydrochloric acid water mixed solution is 98%, the obtained extraction solution is filtered, concentrated to dryness, water is added, ultrasonically dissolved, and then filtered to obtain a filtrate;

[0139] S12. The filtrate is pumped into a chromatography column filled with HPD 300 macroporous resin for adsorption. During the adsorption process, the column temperature is controlled at 30°C. After saturated adsorption, the chromatography column is washed with water until the effluent is neutral, and then the chromatography column is eluted with ethanol. The eluate is collected, concentrated to alcohol-free, and then freeze-dried to obtain anthocyanidin extract.

[0140] Example 10

[0141] This embodiment provides a method for preparing a high-purity cyanine standard sample, which comprises the following steps:

[0142] S1. Connect a reverse phase phenyl chromatographic column (specifications: column length 250 mm, inner diameter 4.6 mm, filler particle size 10 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 5°C; dissolve the cyanidine extract obtained in Preparation Example 10 in water to prepare a solution with a concentration of 100 mg / mL, centrifuge to remove the precipitate, use the solution as the first injection solution, and store it at 0°C in the dark for later use;

[0143] A 3.5% formic acid aqueous solution and acetonitrile are mixed as the first mobile phase through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the first mobile phase is 15%, and the flow rate of the solvent pump is adjusted to 1.0 mL / min, and the above-mentioned reversed-phase phenyl chromatographic column is balanced to a baseline level; the first injection liquid is injected into the balanced reversed-phase chromatographic column for separation, with an injection volume of 100 μL, and the component containing cyanidine is collected and stored at -10°C for use as the second injection liquid;

[0144] S2. Connect a reverse phase C18 chromatographic column (specifications: column length 350 mm, inner diameter 5 mm, filler particle size 10 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 5°C;

[0145] A 2.5% formic acid aqueous solution and acetonitrile are mixed as the second mobile phase through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the second mobile phase is 14%. The flow rate of the solvent pump is adjusted to 1.0 mL / min, the reversed-phase C18 chromatographic column is balanced to a baseline level, and the second injection liquid is fully injected into the balanced reversed-phase chromatographic column for separation, and the components containing cyanidine are collected and freeze-dried in a dark place to obtain a cyanidine standard sample.

[0146] The test showed that 4.5 mg of a cyanidine standard sample with a purity of 98.6% was obtained in this embodiment, and the recovery rate was 91%.

[0147] Comparative Example 1

[0148] This comparative example provides a method for preparing anthocyanidin extract, which specifically comprises the following steps:

[0149] S1. Adding a methanol / saturated hydrochloric acid water mixed solution to dried grape seed skins for hot reflux extraction, wherein the solid-liquid ratio is 1:5, and the volume fraction of methanol in the methanol / saturated hydrochloric acid water mixed solution is 98%, filtering the obtained extraction solution, concentrating to dryness, adding water, ultrasonically dissolving, and filtering to obtain a filtrate;

[0150] S2. The filtrate is pumped into a chromatography column filled with HPD 300 macroporous resin for adsorption. During the adsorption process, the column temperature is controlled at 30°C. After saturation adsorption, the chromatography column is washed with water until the effluent is neutral, and then the chromatography column is eluted with ethanol. The eluate is collected, concentrated to alcohol-free, and then freeze-dried to obtain anthocyanidin extract. Water is added to the anthocyanidin extract and ultrasonically dissolved to prepare a solution with a concentration of 100 mg / mL. The precipitate is removed by centrifugation, and the solution is used as the first injection solution. The solution is stored at 0°C in the dark for later use.

[0151] S3, connecting a reverse phase phenyl chromatographic column (specifications: column length 250 mm, inner diameter 4.6 mm, filler particle size 10 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjusting the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and controlling the column temperature to 25° C.;

[0152] A 2% formic acid aqueous solution and acetonitrile are mixed as a mobile phase through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the mobile phase is 12%, and the flow rate of the solvent pump is adjusted to 1.0 mL / min, and the above-mentioned reversed-phase phenyl chromatographic column is balanced to a baseline level; the first injection liquid is injected into the balanced reversed-phase chromatographic column for separation, and the injection volume is 100 μL, and the chromatogram thereof is shown in Figure 5; the component containing cyanidine is collected, stored at 0°C, and freeze-dried in the dark to obtain a cyanidine sample.

[0153] The test showed that 6 mg of cyanidine sample was obtained in this comparative example, and its purity was verified by high performance liquid chromatography 1 in comparative example 1. Figure 6 As shown. Figure 6 It can be clearly seen that the purity of the obtained anthocyanidin sample is surrounded by many impurities and the purity is significantly lower than 98%, so it cannot be used as a standard sample. This shows that even with a lower loading amount of anthocyanidin extract, a single reversed-phase phenyl chromatographic column cannot obtain anthocyanidin standard sample with a purity higher than 98%.

[0154] Comparative Example 2

[0155] This comparative example provides a method for preparing anthocyanidin extract, which specifically comprises the following steps:

[0156] S1. Add a methanol / saturated hydrochloric acid mixed solution with a methanol volume fraction of 98% to the dried grape seed skin for hot reflux extraction, with a solid-liquid ratio of 1:5, filter the obtained extraction solution, concentrate to dryness, add water, ultrasonically dissolve, and then filter to obtain a filtrate;

[0157] S2. The filtrate is pumped into a chromatography column filled with HPD 300 macroporous resin for adsorption. During the adsorption process, the column temperature is controlled at 30°C. After saturation adsorption, the chromatography column is washed with water until the effluent is neutral, and then the chromatography column is eluted with ethanol. The eluate is collected, concentrated to alcohol-free, and then freeze-dried to obtain anthocyanidin extract. Water is added to the anthocyanidin extract and ultrasonically dissolved to prepare a solution with a concentration of 100 mg / mL. The precipitate is removed by centrifugation, and the solution is used as the first injection solution. The solution is stored at 0°C in the dark for later use.

[0158] S3, connect a reverse phase C18 chromatographic column (specifications: column length 250 mm, inner diameter 4.6 mm, filler particle size 10 μm) to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 25°C;

[0159] A 2% formic acid aqueous solution and acetonitrile are mixed as a mobile phase through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the mobile phase is 12%, and the flow rate of the solvent pump is adjusted to 1.0 mL / min, and the above-mentioned reverse-phase phenyl chromatographic column is balanced to a baseline level; the first injection liquid is injected into the balanced reverse-phase chromatographic column for separation, and the injection volume is 100 μL, and the chromatogram thereof is shown in FIG7; the component containing cyanidine is collected, stored at 0° C., and freeze-dried in the dark to obtain a cyanidine sample.

[0160] The test showed that 6 mg of cyanidine sample was obtained in this comparative example, and its purity was verified by high performance liquid chromatography 2 in comparative example 1. Figure 8 As shown. Figure 8 It can be clearly seen that the purity of the obtained anthocyanidin sample is surrounded by many impurities and the purity is significantly lower than 98%, so it cannot be used as a standard sample. This shows that even with a lower loading amount of anthocyanidin extract, a single reversed-phase C18 column cannot obtain anthocyanidin standard sample with a purity higher than 98%.

[0161] Those skilled in the art should understand that the above are only some specific embodiments of the present invention, rather than all embodiments. It should be noted that for those of ordinary skill in the art, many modifications and improvements can be made, and all modifications or improvements that do not exceed the scope of protection of the present invention should be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a high-purity cyanine standard sample, characterized in that: The steps include: S1. Connect a reverse phase phenyl chromatographic column to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 0-45°C; dissolve the anthocyanidin extract in water to prepare a solution with a concentration of 0.5-100 mg / mL, centrifuge to remove the precipitate, use the solution as the first injection solution, and store it at low temperature and away from light for later use; A formic acid aqueous solution with a concentration of 0.05% to 5% and acetonitrile are mixed as the first mobile phase through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the first mobile phase is 5% to 20%, and the flow rate of the solvent pump is adjusted to 0.3 to 1.2 mL / min, and the reversed-phase phenyl chromatographic column is balanced to a baseline level; the first injection liquid is injected into the balanced reversed-phase chromatographic column for separation, and the component containing cyanidine is collected with an injection volume of 5 to 200 μL, and stored at -20°C to 10°C for standby use as the second injection liquid; S2. Connect a reverse phase C18 chromatographic column to a high performance liquid chromatograph equipped with a dual-channel UV detector, adjust the wavelengths of the dual-channel UV detector to 254 nm and 526 nm, respectively, and control the column temperature to 0-45°C; A formic acid aqueous solution with a concentration of 0.05% to 5% and acetonitrile are mixed as a second mobile phase through a solvent pump of a high performance liquid chromatograph, the volume fraction of acetonitrile in the second mobile phase is 5% to 30%, the flow rate of the solvent pump is adjusted to 0.3 to 1.2 mL / min, the reversed-phase C18 chromatographic column is balanced to a baseline level, all the second injection liquid is injected into the balanced reversed-phase chromatographic column for separation, components containing cyanidine are collected, and freeze-dried in the dark to obtain a cyanidine standard sample with a purity higher than 98 wt%.

2. The method for preparing a high-purity cyanine standard sample according to claim 1, characterized in that: The preparation method of the anthocyanidin extract in step S1 is as follows: S11, adding a methanol / saturated hydrochloric acid mixed solution to the dried grape seed skin for hot reflux extraction, filtering the obtained extraction solution, concentrating to dryness, adding water to dissolve, and filtering to obtain a filtrate; S12. Pump the filtrate into a chromatography column for adsorption, control the column temperature of the chromatography column to -5 to 30°C, and after saturated adsorption, wash the chromatography column with water until the pH of the effluent is neutral, then elute the chromatography column with ethanol, collect the eluate, concentrate it to be alcohol-free, and then dry it to obtain anthocyanidin extract.

3. The method for preparing a high-purity cyanine standard sample according to claim 2, characterized in that: In step S11, a methanol / saturated hydrochloric acid water mixed solution is added to the dried grape seed skin for hot reflux extraction, and the material-liquid ratio is 1:(1-5).

4. The method for preparing a high-purity cyanine standard sample according to claim 2, characterized in that: The volume fraction of methanol in the methanol / saturated hydrochloric acid water mixed solution in step S11 is 90% to 98%.

5. The method for preparing a high-purity cyanine standard sample according to claim 2, characterized in that: The chromatography column in step S12 is filled with HPD 300 or HPD 600 macroporous resin.

6. The method for preparing a high-purity cyanine standard sample according to claim 1 or 2, characterized in that: In step S1, the reverse phase phenyl chromatographic column has a length of 100 to 350 mm, a diameter of 2 to 5 mm, and a filler particle size of 5 to 10 μm.

7. The method for preparing a high-purity cyanine standard sample according to claim 1 or 2, characterized in that: The first mobile phase in step S1 is formed by mixing a formic acid aqueous solution with a concentration of 1.5% to 3.5% and acetonitrile through a solvent pump of a high performance liquid chromatograph, and the volume fraction of acetonitrile in the first mobile phase is 12% to 17%.

8. The method for preparing a high-purity cyanine standard sample according to claim 1 or 2, characterized in that: In step S2, the reverse phase C18 chromatographic column has a length of 100 to 350 mm, a diameter of 2 to 5 mm, and a filler particle size of 5 to 10 μm.

9. The method for preparing a high-purity cyanine standard sample according to claim 1 or 2, characterized in that: The second mobile phase in step S2 is formed by mixing a formic acid aqueous solution with a concentration of 1.5% to 3.5% and acetonitrile through a solvent pump of a high performance liquid chromatograph, wherein the volume fraction of acetonitrile in the second mobile phase is 8% to 14%.

10. A high-purity cyanidine standard sample prepared by the method for preparing a high-purity cyanidine standard sample according to any one of claims 1 to 9.

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