A method for determining the content of chemical components in Banxia Baizhu Tianma decoction

Abstract

The invention belongs to the technical field of traditional Chinese medicine detection, and specifically provides a method for determining the content of chemical components in a Banxia Baizhu Tianma decoction. The raw materials of the Banxia Baizhu Tianma decoction include Pinellia, Gastrodia, Atractylodes, Tangerine Peel, Poria, Licorice, Ginger and Jujube, and the method comprises the following steps: S1, preparing a test solution; S2, preparing a reference solution with gastrodin and p-hydroxybenzyl alcohol as reference substances; S3, respectively determining the chromatograms of the test solution and the reference solution by high performance liquid chromatography; refer to the specification for details, the method for determining the content of chemical components in the Banxia Baizhu Tianma decoction provided by the invention adopts acetonitrile and an aqueous solution containing phosphoric acid as mobile phases, and elution is performed under a specific gradient, so as to achieve complete separation of two effective components, namely gastrodin and p-hydroxybenzyl alcohol, and the content testing method has the advantages of short detection time, high separation degree, strong specificity, good peak shape, linear relationship meeting requirements, and high precision, accuracy and stability.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for determining the content of chemical components in Banxia Baizhu Tianma decoction. Background Art

[0002] Banxia Baizhu Tianma Decoction comes from "Medical Enlightenment". It is composed of 8 herbs: Banxia, ​​Tianma, Baizhu, Tangerine Peel, Poria, Licorice, Ginger, and Jujube. It has the effects of dispelling wind and resolving phlegm, strengthening the spleen and stopping dizziness. It is a classic prescription for treating dizziness, mainly suitable for dizziness caused by wind-phlegm disturbance and phlegm turbidity blocking the middle. In the prescription, Banxia dries dampness and resolves phlegm, descends adverse and stops vomiting; Tianma calms the liver and extinguishes wind and stops dizziness. The two are used together as the main medicine, which is the key medicine for treating dizziness and headache caused by wind-phlegm. Baizhu strengthens the spleen and dries dampness; Poria strengthens the spleen and penetrates dampness, and assists in treating the root of phlegm. It is combined with Banxia and Tianma to strengthen the power of resolving phlegm and calming wind, and they are all ministers. Tangerine Peel regulates qi and resolves phlegm, makes qi smooth and phlegm disappear, and is an adjuvant. Licorice is used to harmonize the various herbs, and ginger and jujube are added to harmonize the spleen and stomach. The combination of various herbs has the effects of resolving phlegm and extinguishing wind, strengthening the spleen and removing dampness.

[0003] The main active ingredients in Gastrodia elata are gastrodin and p-hydroxybenzyl alcohol. Gastrodia elata has the effects of dispelling wind, unblocking meridians, calming wind and stopping convulsions. Gastrodin in Gastrodia elata has obvious sedative and hypnotic effects, has obvious inhibitory effects on the autonomous activities of mice, can shorten the time required for mice to fall asleep, and increase the number of sleeping mice. Gastrodin can pass through the blood-brain barrier and degrade into gastrodin aglycone at a high rate in brain tissue. Gastrodin aglycone is a ligand of the Bz receptor of brain cell membrane. Therefore, gastrodin aglycone acts on the γ-aminobutyric acid / Bz receptor, thereby showing sedative and anticonvulsant central inhibitory effects. Gastrodin is p-hydroxymethylbenzene-β-D-pyranoglucoside, and p-hydroxybenzyl alcohol is its aglycone. Literature research shows that there is a dynamic balance between gastrodin and p-hydroxybenzyl alcohol in Gastrodia elata medicinal materials during the processing and decoction process. Modern pharmacological studies have shown that the two have significant therapeutic effects on central nervous system diseases such as insomnia, convulsions, depression, ischemic stroke, anxiety, cognitive impairment, etc., and are an important material basis for the efficacy of Gastrodia elata medicinal materials. Therefore, determining the contents of gastrodin and hydroxybenzyl alcohol and selecting the total amount of gastrodin and p-hydroxybenzyl alcohol as the content index has more pharmacodynamic significance for the quality of Banxia Baizhu Tianma Decoction than controlling them separately with a single index.

[0004] Wang Xun et al. reported the determination of the content of gastrodin and guanosine in Banxia Baizhu Tianma Decoction by HPLC (Chinese Journal of Experimental Traditional Chinese Medicine, 2010, 16(17): 44-47. 2010.17.066.) using a Platisil ODS column (4.6mm×250mm, 5μm); mobile phase A (water) B (acetonitrile); gradient elution 0-30min (2%-5% B); flow rate 0.65mL / min; column temperature 25℃; detection wavelength 260m. A method for the simultaneous determination of gastrodin and guanosine in Banxia Baizhu Tianma Decoction was established. The established method is accurate and rapid, and can be used for the quality control of Banxia Baizhu Tianma Decoction.

[0005] Wang Shuling et al. reported on the HPLC fingerprint and multi-component quantitative analysis of Banxia Baizhu Tianma decoction (Contemporary Chinese Medicine, 2021, 28(08):4-8+17+241.) using Inert Sustain C 18 The chromatographic column (250 mm × 4.6 mm, 5 μm) was used for separation, and acetonitrile-water solution was used as the mobile phase for gradient elution, with a flow rate of 1.0 mL / min, a detection wavelength of 254 m, and a column temperature of 30 ° C. The HPLC fingerprint of Banxia Baizhu Tianma Decoction was established, and the contents of 7 components (gastrodin, liquiritin, naringin, hesperidin, isoliquiritigenin, atractylodes lactone IIIl, and glycyrrhetinic acid) were determined simultaneously.

[0006] The above methods are unable to effectively separate p-hydroxybenzyl alcohol and can only measure the content of gastrodin, which is not conducive to the quality control of Banxia Baizhu Tianma Decoction product. Summary of the invention

[0007] To this end, the present invention proposes a method for determining the content of a Chinese medicine composition corresponding to Baoyin Decoction and a preparation thereof, and further establishes a method for detecting the quality of a Chinese medicine composition corresponding to Baoyin Decoction and a preparation thereof.

[0008] The present invention provides a method for determining the content of chemical components in Banxia Baizhu Tianma Decoction, wherein the raw materials of the Banxia Baizhu Tianma Decoction include Pinellia, Gastrodia, Atractylodes, Citrus aurantium, Poria, Licorice, Ginger and Jujube, and the method comprises the following steps:

[0009] S1. Preparation of test solution;

[0010] S2. Prepare reference solution using gastrodin and p-hydroxybenzyl alcohol as reference substances;

[0011] S3. The chromatograms of the test solution and the reference solution are determined by high performance liquid chromatography, wherein the chromatographic column is filled with octadecylsilane bonded silica gel, acetonitrile and an aqueous solution containing phosphoric acid are used as mobile phases for gradient elution, and the gradient elution program includes: 0→35min→40min→50min→55min; the volume percentage of acetonitrile in the mobile phase is 2%→2%→60%→80%→2%;

[0012] S4. Calculate the contents of gastrodin and p-hydroxybenzyl alcohol in Banxia Baizhu Tianma Decoction by the external standard method according to the concentration of the chemical components in the reference solution in step S2, the peak area of ​​the reference solution in the chromatogram, and the peak area of ​​the components corresponding to the reference solution in Banxia Baizhu Tianma Decoction in the chromatogram.

[0013] Furthermore, the method for preparing the test solution comprises the step 1) of mixing Banxia Baizhu Tianma decoction with an alcohol solvent, subjecting the mixed solution to a first solid-liquid separation, and taking a liquid; preferably, it also comprises the step 2) drying the liquid obtained after the first solid-liquid separation, mixing the dried product with a solvent, subjecting the mixed solution to a second solid-liquid separation, and taking a liquid.

[0014] Furthermore, in step 1), the alcohol solvent is selected from at least one of methanol, ethanol and water; and / or the first solid-liquid separation is centrifugation or filtration; and / or the ratio of the volume of Banxia Baizhu Tianma Decoction to the volume of the mixed solution in step 1) is 1:2-1:5; or the ratio of the mass of Banxia Baizhu Tianma Decoction to the volume of the mixed solution in step 1) is 1g:2-5mL.

[0015] Further, in step 2), the solvent is selected from 25-70% methanol aqueous solution (volume percentage), 25-70% ethanol aqueous solution (volume percentage), acetonitrile-water or water with a volume ratio of 1:99 and 5:95; and / or, the second solid-liquid separation is selected from centrifugation or filtration; and / or, the ratio of the volume of the liquid to the volume of the mixed solution in step 2) is 20-30:5-50.

[0016] Furthermore, in step S3, the detection wavelength of gastrodin and hydroxybenzyl alcohol is 215-225 nm; and / or, the flow rate is 0.9-1.1 ml / min, and the column temperature is 28-32° C.; and / or, the volume percentage of phosphoric acid in the aqueous solution containing phosphoric acid is 0.03-0.08%.

[0017] Furthermore, step S2 is to prepare a mixed reference solution of gastrodin and hydroxybenzyl alcohol using gastrodin and p-hydroxybenzyl alcohol; preferably, the concentration of gastrodin in the mixed reference solution is 0.0004992 mg / ml to 0.2496 mg / ml; the concentration of hydroxybenzyl alcohol is 0.0002114 mg / ml to 0.1057 mg / ml.

[0018] Furthermore, the content determination method also includes:

[0019] S5. Prepare a reference solution using liquiritin, ammonium glycyrrhizinate and hesperidin as reference substances;

[0020] S6. Use high performance liquid chromatography to measure the chromatograms of the test solution and the reference solution respectively; wherein the chromatographic column uses octadecylsilane bonded silica gel as a filler, acetonitrile and an aqueous solution containing phosphoric acid as a mobile phase for gradient elution, and acetonitrile and an aqueous solution containing phosphoric acid as a mobile phase for gradient elution, and the gradient elution program includes: 0→8min→20min→35min; the volume percentage of acetonitrile in the mobile phase is 19%→19%→30%→50%;

[0021] S7. Calculate the contents of liquiritin, glycyrrhizic acid and hesperidin in Banxia Baizhu Tianma Decoction by external standard method according to the concentration of chemical components in the reference solution in step S5, the peak area of ​​the reference solution in the chromatogram and the peak area of ​​the components corresponding to the reference solution in Banxia Baizhu Tianma Decoction in the chromatogram.

[0022] Furthermore, the detection wavelength of liquiritin and glycyrrhizic acid is 235-240nm, and the detection wavelength of hesperidin is 280-290nm; and / or, the flow rate is 0.9-1.1ml / min, and the column temperature is 28-32°C; and / or, the volume percentage of phosphoric acid in the phosphoric acid-containing aqueous solution is 0.03-0.08%; and / or, the gradient program also includes 35min→36min→40min; the volume percentage of acetonitrile in the mobile phase is 50%→100%→19%.

[0023] Further, in step S5, a hesperidin reference solution and a mixed reference solution of liquiritin and glycyrrhizic acid are prepared respectively. Preferably, the concentration of liquiritin in the mixed reference solution of liquiritin and glycyrrhizic acid is 0.002332 mg / ml to 0.2332 mg / ml, and the concentration of glycyrrhizic acid is 0.003748 mg / ml to 0.3748 mg / ml; the concentration of hesperidin in the hesperidin reference solution is 0.001676 mg / ml to 0.1676 mg / ml.

[0024] Furthermore, in terms of weight, the raw materials of the Banxia Baizhu Tianma Decoction include 4.5-6.7 parts of Pinellia, 3.0-4.5 parts of Tianma, 9.0-13.4 parts of Atractylodes, 3.0-4.5 parts of Tangerine Peel, 3.0-4.5 parts of Poria, 1.5-2.2 parts of Licorice, 0.8-1.2 parts of ginger and 4.8-7.2 parts of jujube.

[0025] The Banxia Baizhu Tianma decoction of the present invention can be prepared by conventional methods in the art, for example, mixing Banxia, ​​Tianma, Baizhu, Tangerine peel, Poria, Licorice, Ginger and Jujube, then boiling with water, filtering and obtaining a filtrate. The boiling times are at least 1 time (for example 1-3 times), each time at least 5 times (for example 10-15 times) of water is added, and each time boiling is at least 10 minutes (for example 20-30 minutes).

[0026] The technical solution of the present invention has the following advantages:

[0027] 1. The method for determining the content of chemical components in Banxia Baizhu Tianma Decoction provided by the present invention adopts acetonitrile and an aqueous solution containing phosphoric acid as mobile phases and performs elution under a specific gradient, thereby achieving complete separation of the two effective ingredients, gastrodin and p-hydroxybenzyl alcohol. The content testing method has the advantages of short detection time, high separation degree, strong specificity, good peak shape, linear relationship that meets the requirements, and high precision, accuracy and stability.

[0028] 2. The method for determining the content of chemical components in Banxia Baizhu Tianma Decoction provided by the present invention is based on the effective components of Banxia Baizhu Tianma Decoction, combined with the chemical components and pharmacological effects of each medicinal taste, to establish a method for determining the content of gastrodin, p-hydroxybenzyl alcohol, liquiritin, glycyrrhizic acid, and hesperidin, the main active ingredients of Tianma, Licorice, and Tangerine in Banxia Baizhu Tianma Decoction. Moreover, this method is stable and reliable, has good reproducibility, and high precision, which makes up for the shortcomings of existing quality control methods and has guiding significance for the quality detection and evaluation of Banxia Baizhu Tianma Decoction. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 It is the chromatogram of method 1 of experimental example 1 of the present invention, from bottom to top: Banxia Baizhu Tianma decoction (lacking tangerine peel) 21030402, Banxia Baizhu Tianma decoction 21030102;

[0031] Figure 2 It is the chromatogram of the detection wavelength of 237nm in Experimental Example 1 Method 2 of the present invention; from bottom to top: mixed reference solution, Banxia Baizhu Tianma decoction 21030102;

[0032] Figure 3 It is a chromatogram of the detection wavelength of 284 nm in Experimental Example 1, Method 2 of the present invention; from bottom to top: hesperidin reference substance solution, Banxia Baizhu Tianma decoction 21030102;

[0033] Figure 4 is the chromatogram of the method 3 of Experimental Example 1 of the present invention at a detection wavelength of 237 nm;

[0034] Figure 5 is the chromatogram of the detection wavelength of 284 nm in method 3 of Experimental Example 1 of the present invention;

[0035] Figure 6 is the chromatogram of the detection wavelength of 237 nm in method 4 of Experimental Example 1 of the present invention;

[0036] Figure 7 is the chromatogram of the method 4 of Experimental Example 1 of the present invention at a detection wavelength of 284 nm;

[0037] Figure 8 This is a comparison chart of the specificity of licorice in Banxia Baizhu Tianma Decoction of Experimental Example 3 of the present invention, from bottom to top: blank, Banxia Baizhu Tianma Decoction (lacking licorice) 21113005, Banxia Baizhu Tianma Decoction single licorice 21113002, liquiritin glycyrrhizic acid mixed control, Banxia Baizhu Tianma Decoction benchmark sample 21112901;

[0038] Fig. 9 This is a comparison chart of the specificity of tangerine peel in Banxia Baizhu Tianma Decoction of Experimental Example 3 of the present invention, from bottom to top: blank, Banxia Baizhu Tianma Decoction (lacking tangerine peel) 21113006, Banxia Baizhu Tianma Decoction single tangerine peel 21113003, hesperidin control, Banxia Baizhu Tianma Decoction benchmark sample 21112901;

[0039] Fig.10 The chromatogram of method 1 of experimental example 4 of the present invention, from bottom to top, are: reference solution, Banxia Baizhu Tianma decoction, 1 is gastrodin, 2 is p-hydroxybenzyl alcohol;

[0040] Fig.11 The chromatogram of method 2 of experimental example 4 of the present invention, from bottom to top, are: reference solution, Banxia Baizhu Tianma decoction, 1 is gastrodin, 2 is p-hydroxybenzyl alcohol;

[0041] Fig.12 The chromatogram of method 4 of experimental example 4 of the present invention, from bottom to top, are: reference solution, Banxia Baizhu Tianma decoction, 1 is gastrodin, 2 is p-hydroxybenzyl alcohol;

[0042] Fig.13 This is a comparison chart of the specificity of tangerine peel in Experimental Example 6 of the present invention, from bottom to top: blank, mixed control solution of gastrodin and p-hydroxybenzyl alcohol, Banxia Baizhu Tianma Decoction (without gastrodia elata), gastrodia elata alone, Banxia Baizhu Tianma Decoction (21112901); 1 is gastrodin, 2 is p-hydroxybenzyl alcohol;

[0043] Fig.14 This is the chromatogram of Example 1 of the present invention, from bottom to top, blank, mixed control solution of gastrodin and p-hydroxybenzyl alcohol, and Banxia Baizhu Tianma decoction test sample; 1 is gastrodin, 2 is p-hydroxybenzyl alcohol;

[0044] Fig.15 This is a chromatogram of comparative example 1 of the present invention, which includes, from bottom to top, blank (methanol), a mixed control solution of gastrodin and p-hydroxybenzyl alcohol, and a test sample of a concentrated solution of Banxia Baizhu Tianma decoction; 1 is gastrodin, and 2 is p-hydroxybenzyl alcohol;

[0045] Fig.16 This is the chromatogram of comparative example 2 of the present invention, which includes, from bottom to top, blank (acetonitrile-0.05% phosphoric acid), mixed control solution of gastrodin and p-hydroxybenzyl alcohol, and decoction of Pinellia, Atractylodes and Gastrodia as test samples; 1 is gastrodin, and 2 is p-hydroxybenzyl alcohol. DETAILED DESCRIPTION

[0046] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0047] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0048] Experimental Example 1 Investigation of Chromatographic Conditions

[0049] 1. Drug testing

[0050] Banxia Baizhu Tianma Decoction, prescription: Banxia 5.60g, Tianma 3.73g, Baizhu 11.19g, Fuling 3.73g, Tangerine peel 3.73g, Licorice 1.87g, Ginger 1.0g, Jujube 6.0g. Preparation method: Add water to the above medicinal ingredients and boil twice, add 12 times the amount of water for 30 minutes the first time, add 10 times the amount of water for 25 minutes the second time, filter the decoction, and combine the filtrate to obtain the decoction.

[0051] Negative control (without tangerine peel), prescription: 5.60g of Banxia, ​​3.73g of Gastrodia, 11.19g of Atractylodes, 3.73g of Poria, 1.87g of Licorice, 1.0g of Ginger, 6.0g of Jujube. Preparation method: the same as that of Banxia Baizhu Tianma Decoction.

[0052] 2. Sample preparation

[0053] Preparation of test solution: Accurately measure 5 ml of Banxia Baizhu Tianma decoction into a 10 ml volumetric flask, add methanol, shake well to the scale line, and filter to obtain the filtrate.

[0054] Preparation of mixed reference solution: Take an appropriate amount of hesperidin reference substance, accurately weigh it, and add methanol to make a hesperidin reference substance stock solution. Take an appropriate amount of liquiritin and ammonium glycyrrhizinate reference substances, accurately weigh them, accurately measure an appropriate amount of hesperidin reference substance stock solution, and add 70% ethanol to make a mixed solution containing 20μg of liquiritin, 80μg of glycyrrhizic acid, and 60μg of hesperidin per 1ml.

[0055] Preparation of negative control solution: Accurately measure 5 ml of negative control (lacking orange peel) in a 10 ml volumetric flask, add methanol, shake well to the scale line, and filter to obtain the filtrate.

[0056] 3. Use high performance liquid chromatography to test according to the following methods.

[0057] Method 1: Octadecylsilane bonded silica as filler (Welch XB-C18, 4.6mm×250mm, 5μm); methanol-water (40:60) as mobile phase; column temperature 30℃, flow rate 1.0ml per minute. Detection wavelength 284nm, inject 10μl of the test solution, negative control solution and mixed control solution under item 2 respectively.

[0058] Method 2: Octadecylsilane bonded silica as filler (Welch XB-C18, 4.6mm×250mm, 5μm); acetonitrile was used as mobile phase A, and 0.05% phosphoric acid solution was used as mobile phase B. The flow rate was 1.0ml per minute, the column temperature was 30°C, the detection wavelengths were 237nm and 284nm, and gradient elution was performed according to the provisions in Table 1. 10μl of the test solution and the mixed reference solution under item 2 were injected respectively.

[0059] Table 1 Mobile phase gradient

[0060] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～20 18→22 82→78 20～35 22→50 78→50 35～38 50→80 50→20 38～43 80 20 43～45 80→18 20→82

[0061] Method 3: Octadecylsilane bonded silica as filler (Welch XB-C18, 4.6mm×250mm, 5μm); acetonitrile was used as mobile phase A, and 0.05% by volume phosphoric acid solution was used as mobile phase B. The flow rate was 1.0 ml per minute, the column temperature was 30°C, the detection wavelengths were 237nm and 284nm, and gradient elution was performed according to the provisions in Table 2. 10μl of the test solution and the mixed reference solution under item 2 were injected respectively.

[0062] Table 2 Mobile phase gradient

[0063] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～8 19 81 8～35 19→50 81→50 35～36 50→100 50→0 36～40 100→19 0→81

[0064] Method 4: Octadecylsilane bonded silica as filler (Welch XB-C18, 4.6mm×250mm, 5μm); acetonitrile was used as mobile phase A, and 0.05% by volume phosphoric acid solution was used as mobile phase B. The flow rate was 1.0 ml per minute, the column temperature was 30°C, the detection wavelength was 237nm / 284nm, and gradient elution was performed according to the provisions in Table 3. 10μl of the test solution and the mixed reference solution under item 2 were injected respectively.

[0065] Table 3 Mobile phase gradient

[0066] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～8 19 81 8～20 19→30 81→70 20～35 30→50 70→50 35～36 50→100 50→0 36～40 100→19 0→81

[0067] The results are shown in the table below and Figure 1-7 shown.

[0068] Table 4 Chromatographic conditions investigation results

[0069]

[0070] In method 1, the sample showed a chromatographic peak with the same retention time as hesperidin and the same absorption spectrum. The peak purity and separation could meet the requirements of content determination, but the chromatographic peaks of liquiritin and glycyrrhizic acid were not presented.

[0071] The sample in method 2 presents chromatographic peaks with consistent elution time and absorption spectrum with liquiritin, glycyrrhizic acid and hesperidin, but the purity angles of liquiritin and hesperidin are greater than the purity threshold, and the separation degree of glycyrrhizic acid is <2.0, which does not meet the content analysis requirements.

[0072] In method 3, the separation degree of liquiritin and glycyrrhizic acid peaks meets the requirements, but the purity angle of hesperidin is greater than the purity threshold, and Figure 5 It can be seen that there is a small interference peak after the hesperidin chromatographic peak, which does not meet the content determination requirements.

[0073] In method 4, the peak purity and separation of liquiritin and glycyrrhizic acid can meet the requirements of content determination. The peak purity of hesperidin chromatographic peak is improved compared with method 3 and can meet the requirements of content determination. Compared with method 3, the peak shape of the target peak is better, and the separation degree of hesperidin and adjacent interference peaks can meet the requirements of content determination.

[0074] 4. The determined chromatographic conditions are as follows: octadecylsilane bonded silica gel as filler; acetonitrile as mobile phase A, 0.05% phosphoric acid solution as mobile phase B. Flow rate 1.0 ml per minute, column temperature 30°C, detection wavelength 237 nm / 284 nm, gradient elution according to the provisions in Table 5 below.

[0075] Table 5 Determined mobile phase gradient

[0076] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～8 19 81 8～20 19→30 81→70 20～35 30→50 70→50 35～36 50→100 50→0 36～40 100→19 0→81

[0077] Experimental Example 2 Investigation of the preparation method of the test solution

[0078] During the research of this method, it was found that the sample was easily precipitated during storage after direct dilution with methanol, which would affect subsequent detection. Therefore, the preparation method of the test sample was investigated, mainly different pretreatment methods and different solvents.

[0079] 1. The investigation contents are shown in the following table. The test solutions were prepared by the following six methods respectively, and then measured according to method 4 in experimental example 1.

[0080] Table 6 Investigation of extraction methods

[0081]

[0082] Table 7 Investigation of solvent types after alcohol precipitation

[0083]

[0084]

[0085] The test results are shown in the table below.

[0086] Table 8 Extraction method test results

[0087]

[0088] Table 9 Results of investigation on solvent types after alcohol precipitation

[0089] solvent Hesperidin mg / ml Licorice glycoside mg / ml Glycyrrhizic acid mg / ml 50% methanol 0.1439 0.02916 0.1416 70% ethanol 0.1496 0.02860 0.1530 70% methanol 0.1588 0.03232 0.1568 water 0.1531 0.02957 0.1422 25% methanol 0.1553 0.03168 0.1506 RSD(%) 3.8 5.5 4.6

[0090] From the content results in the table above, it can be seen that the contents of each component are higher after direct dilution with methanol in the 6 preparation methods, but the test solution is prone to precipitation and affects instrument detection, and the RD values ​​of the two extraction methods are 2.9% for liquiritin, 2.1% for glycyrrhizic acid, and 6.0% for hesperidin, with no significant difference. Considering the stability of the test solution, ethanol precipitation is selected for sample preparation. After ethanol precipitation, the sample is extracted with different solvents. The comparison results show that the sample content is higher after dissolution with 70% methanol, and the peak shape of hesperidin in the test sample is better after dissolution with 70% ethanol, with an asymmetry of 0.99, so 70% ethanol is finally determined as the solvent for this method.

[0091] Table 10 Peak results

[0092]

[0093] 2. Determine the method for preparing the test solution: accurately measure 20 ml of the product in a 100 ml volumetric flask, add ethanol to dilute to the mark, shake well, centrifuge, take 25 ml of the supernatant and evaporate to dryness, add 70% ethanol to dissolve the residue and make up to the volume in a 10 ml volumetric flask, shake well, filter and take the filtrate.

[0094] To facilitate practical operation, before conducting methodological verification, the method was adjusted as follows: take about 20 g of this product, weigh it accurately, place it in a 100 ml volumetric flask, add ethanol to dilute to the scale, shake well, centrifuge, take 25 ml of the supernatant and evaporate to dryness, add 70% ethanol to dissolve the residue and make up to the volume in a 10 ml volumetric flask, shake well, filter and obtain the filtrate.

[0095] Experimental Example 3: Methodological Verification

[0096] 1. Linearity and range

[0097] Take an appropriate amount of liquiritin reference substance, accurately weigh it, and add 70% ethanol to prepare liquiritin reference substance solutions with concentrations of 0.002332 mg / ml, 0.004664 mg / ml, 0.01166 mg / ml, 0.02332 mg / ml, 0.1166 mg / ml, and 0.2332 mg / ml, respectively; take an appropriate amount of ammonium glycyrrhizinate reference substance, accurately weigh it, and add 70% ethanol to prepare concentrations of 0.003748 mg / ml, 0.007496 mg / ml, and 0.01874 mg / ml, respectively. , 0.03748 mg / ml, 0.1874 mg / ml, 0.3748 mg / ml glycyrrhizic acid reference solution (weight of glycyrrhizic acid = weight of ammonium glycyrrhizate / 1.0207); take an appropriate amount of hesperidin reference substance, accurately weigh, and add 70% ethanol to make hesperidin reference substance solutions with concentrations of 0.001676 mg / ml, 0.003352 mg / ml, 0.008380 mg / ml, 0.01676 mg / ml, 0.08380 mg / ml, 0.1676 mg / ml, respectively.

[0098] 10 μl of liquiritin reference solution of different concentrations (0.002332 mg / ml, 0.004664 mg / ml, 0.01166 mg / ml, 0.02332 mg / ml, 0.1166 mg / ml, 0.2332 mg / ml) were accurately drawn and injected into the liquid chromatograph, and the sample was injected according to the content determination method. The concentration of liquiritin was used as the abscissa X and the peak area of ​​liquiritin was used as the ordinate Y for linear regression. The regression equation was: Y=358.1170X-0.0598, r=1.000, and the residual square sum was 0.1084. The results showed that the concentration of liquiritin was in a good linear relationship within the range of 0.002332 mg / ml to 0.2332 mg / ml. The results are shown in Table 1.

[0099] Table 11 Liquorice glycoside linear concentration and peak area results

[0100]

[0101] 10 μl of glycyrrhizic acid reference solution of different concentrations (0.003748 mg / ml, 0.007496 mg / ml, 0.01874 mg / ml, 0.03748 mg / ml, 0.1874 mg / ml, 0.3748 mg / ml) were accurately drawn and injected into the liquid chromatograph, and the sample was injected according to the content determination method. The glycyrrhizic acid concentration was used as the abscissa X and the glycyrrhizic acid peak area was used as the ordinate Y for linear regression processing. The regression equation was: Y=99.9263X-0.0170, r=1.000, and the residual square sum was 0.02991. The results showed that the glycyrrhizic acid concentration was in the range of 0.003748 mg / ml to 0.3748 mg / ml and showed a good linear relationship. The results are shown in Table 2.

[0102] Table 12 Glycyrrhizic acid linear concentration and peak area results

[0103]

[0104] 10 μl of hesperidin reference solution of different concentrations (0.001676 mg / ml, 0.003352 mg / ml, 0.008380 mg / ml, 0.01676 mg / ml, 0.08380 mg / ml, 0.1676 mg / ml) were accurately drawn and injected into the liquid chromatograph, and the sample was injected according to the content determination method. The hesperidin concentration was used as the abscissa X and the hesperidin peak area was used as the ordinate Y for linear regression processing. The regression equation was: Y=301.4503X-0.0774, r=1.000, and the residual square sum was 0.009037. The results showed that the hesperidin concentration was in the range of 0.001676 mg / ml to 0.1676 mg / ml and showed a good linear relationship. The results are shown in Table 3.

[0105] Table 13 Hesperidin linear concentration and peak area results

[0106]

[0107] 2. Specificity test

[0108] Licorice glycoside and glycyrrhizic acid mixed reference solution: Take appropriate amount of licorice glycoside and ammonium glycyrrhizinate reference substances, weigh accurately, add 70% ethanol to prepare a mixed solution containing 20 μg of licorice glycoside and 80 μg of glycyrrhizic acid per 1 ml.

[0109] Banxia Baizhu Tianma Decoction (without licorice) prescription: 5.60g of Banxia, ​​3.73g of Tianma, 11.19g of Baizhu, 3.73g of Fuling, 3.73g of Tangerine Peel, 1.0g of ginger, 6.0g of jujube. Preparation method: the same as that of Banxia Baizhu Tianma Decoction.

[0110] Banxia Baizhu Tianma Decoction with licorice alone, prescription: 1.87g licorice. Preparation method: the same as that of Banxia Baizhu Tianma Decoction.

[0111] The reference sample of Banxia Baizhu Tianma Decoction, prescription: 5.60g of Banxia, ​​3.73g of Tianma, 11.19g of Baizhu, 3.73g of Fuling, 3.73g of Tangerine Peel, 1.87g of Licorice, 1.0g of Ginger, 6.0g of Jujube. Preparation method: add water to the above medicinal ingredients and boil them twice, add 12 times the amount of water for 30 minutes for the first time, and add 10 times the amount of water for 25 minutes for the second time, filter the decoction, and combine the filtrate to obtain the decoction.

[0112] Hesperidin reference solution: Take an appropriate amount of hesperidin reference substance, weigh it accurately, and add 70% ethanol to make a solution containing 60 μg of hesperidin per 1 ml.

[0113] Banxia Baizhu Tianma Decoction (without orange peel) prescription: Banxia 5.60g, Tianma 3.73g, Baizhu 11.19g, Fuling 3.73g, Licorice 1.87g, Ginger 1.0g, Jujube 6.0g. Preparation method: the same as Banxia Baizhu Tianma Decoction.

[0114] Banxia Baizhu Tianma Decoction with Tangerine Peel alone, prescription: Tangerine Peel 3.73g. Preparation method: same as that of Banxia Baizhu Tianma Decoction.

[0115] Take Banxia Baizhu Tianma Decoction (lacking licorice), Banxia Baizhu Tianma Decoction with licorice alone, and Banxia Baizhu Tianma Decoction reference samples, and prepare the test solution according to the preparation method of the test solution determined in Item 2 of Experimental Example 2, respectively. Mix the blank solvent (70% ethanol) and the glycyrrhizin glycyrrhizic acid mixed control solution according to the chromatographic conditions determined in Item 4 of Experimental Example 1, and inject and test. The results are as follows Figure 8 As shown. In the chromatogram of the test sample, chromatographic peaks with the same retention time as the reference peaks of liquiritin and glycyrrhizic acid can be found, and the spectral absorption is consistent with the reference peaks of liquiritin and glycyrrhizic acid; no chromatographic peaks are detected in the blank after 5 minutes, and there is no interference with the chromatogram of the test sample. The separation degree of liquiritin and glycyrrhizic acid in the test sample and their adjacent peaks is greater than 1.5. This shows that the specificity of this method is good.

[0116] Take the reference samples of Banxia Baizhu Tianma Decoction (lacking tangerine peel), Banxia Baizhu Tianma Decoction with tangerine peel alone, and Banxia Baizhu Tianma Decoction, and prepare the test solution according to the preparation method of the test solution determined in Item 2 of Experimental Example 2, respectively. Then, inject and test with the blank solvent (70% ethanol) and the hesperidin control solution according to the chromatographic conditions determined in Item 4 of Experimental Example 1, respectively. The results are as follows Fig. 9As shown. A chromatographic peak with the same retention time as the hesperidin reference peak can be found in the chromatogram of the test sample, and the spectral absorption is consistent with the hesperidin reference; no chromatographic peak is detected in the blank after 5 minutes, and there is no interference with the chromatogram of the test sample. The separation degree of hesperidin in the test sample and its adjacent peaks is greater than 1.5. This shows that the specificity of this method is good.

[0117] 3. Precision test

[0118] ①Repeatability

[0119] Take the reference sample of Banxia Baizhu Tianma Decoction, prepare 6 test sample solutions in parallel according to the method determined in Item 2 of Experimental Example 2, and inject and test according to the chromatographic conditions determined in Item 4 of Experimental Example 1. The results are as follows: the average content of liquiritin calculated from the 6 test samples is 0.04038 mg / ml, and the RSD value is 2.2%, which is less than 6.0%; the average content of glycyrrhizic acid calculated from the 6 test samples is 0.06652 mg / ml, and the RSD value is 2.0%, which is less than 6.0%; the average content of hesperidin calculated from the 6 test samples is 0.09509 mg / ml, and the RSD value is 2.0%, which is less than 4.0%, indicating that the method has good repeatability. The results are shown in Table 4.

[0120] Table 14 Repeatability test results

[0121] serial number Licorice glycoside content (mg / g) Glycyrrhizic acid content (mg / g) Hesperidin(mg / g) 1 0.04041 0.06613 0.09614 2 0.03915 0.06555 0.09239 3 0.04170 0.06880 0.09763 4 0.03973 0.06522 0.09370 5 0.04084 0.06683 0.09581 6 0.04048 0.06658 0.09488 Average (mg / ml) 0.04038 0.06652 0.09509 RSD / % 2.2 2.0 2.0

[0122] ②Intermediate precision

[0123] At different times, different analysts used different equipment to prepare 6 samples of the reference sample solution of Banxia Baizhu Tianma Decoction according to the preparation method determined in Item 2 of Experimental Example 2, and the sample was measured according to the chromatographic conditions determined in Item 4 of Experimental Example 1, and the chromatogram was recorded. Results: The RSD value of the liquiritin content in the 6 samples of the intermediate precision was 0.8%, and the RSD value of the liquiritin content in the 12 samples of the repeatability was 5.6%, both less than 6.0%; the RSD value of the glycyrrhizic acid content in the 6 samples of the intermediate precision was 0.6%, and the RSD value of the glycyrrhizic acid content in the 12 samples of the repeatability was 1.5%, both less than 6.0%; the RSD value of the hesperidin content in the 6 samples of the intermediate precision was 0.6%, and the RSD value of the hesperidin content in the 12 samples of the repeatability was 2.0%, both less than 4.0%, indicating that the intermediate precision of this method is good. The results are shown in Table 5.

[0124] Table 15 Precision test results

[0125]

[0126] 4. Accuracy test

[0127] Take 9 portions of the reference sample of Banxia Baizhu Tianma Decoction with a known liquiritin content of 0.04038 mg / g, glycyrrhizic acid content of 0.06652 mg / g, and hesperidin content of 0.09509 mg / g (mean value of the repeatability test results), 10 g each, accurately weighed, and the sample weight is shown in the table. Add the reference substance as specified in Tables 16-18, and prepare the test solution according to the preparation method of the test solution determined in Item 2 of Experimental Example 2. Sample injection and detection are carried out according to the chromatographic conditions determined in Item 4 of Experimental Example 1, and the contents of liquiritin, glycyrrhizic acid, and hesperidin are determined. The recovery rate and RSD value are calculated. The results are shown in the following table.

[0128] Results: The recovery rate of liquiritin ranged from 85.9% to 96.8%, all within the range of 80% to 115%, the average recovery rate was 93%, and the RSD value was 5.0%, indicating that the accuracy of liquiritin was good under this method. The recovery rate of glycyrrhizic acid ranged from 90.3% to 100.7%, all within the range of 80% to 115%, the average recovery rate was 97%, and the RSD value was 3.3%, indicating that the accuracy of glycyrrhizic acid was good under this method. The recovery rate of hesperidin ranged from 90.2% to 99.3%, all within the range of 85% to 110%, the average recovery rate was 96%, and the RSD value was 3.1%, indicating that the accuracy of hesperidin was good under this method.

[0129] Table 16 Liquorice glycoside accuracy determination test results (n = 9)

[0130]

[0131] Table 17 Glycyrrhizic acid accuracy test results (n = 9)

[0132]

[0133] Table 18 Hesperidin accuracy test results (n = 9)

[0134]

[0135]

[0136] Note: Recovery rate (%) = (test sample measured amount - sample weight × test content) / reference sample added amount × 100%

[0137] 5. Durability test

[0138] ①Solution stability

[0139] Take the Banxia Baizhu Tianma Decoction benchmark sample and prepare the test solution according to the preparation method of the test solution determined in Item 2 of Experimental Example 2. The test solution was injected and measured at different time points (0h, 2h, 4h, 8h, 15h, 20h, 24h, 27h) according to the chromatographic conditions determined in Item 4 of Experimental Example 1. The retention time and peak area of ​​liquiritin, glycyrrhizic acid and hesperidin were recorded, and the RSD values ​​were calculated. The results are shown in the table below.

[0140] Results: Within 27 h, the RSD values ​​of the peak area and retention time of the liquiritin peak in the test solution were 0.9% and 1.8%, respectively; the RSD values ​​of the peak area and retention time of the glycyrrhizic acid peak were 0.2% and 0.7%, respectively; the RSD values ​​of the peak area and retention time of the hesperidin peak were 0.5% and 0.6%, respectively, all of which were less than 2.0%, meeting the requirements, indicating that the test solution had good stability within 27 h at room temperature.

[0141] Table 19 Solution stability results

[0142]

[0143] ②Chromatographic columns of different brands

[0144] Take the reference sample of Banxia Baizhu Tianma Decoction, prepare two test sample solutions in parallel according to the test sample preparation method determined in Item 2 of Experimental Example 2, and use different brands of chromatographic columns (Shiseido chromatographic column CAPCELL PAK C18, 4.6mm×250mm, 5μm; Waters chromatographic column) according to the determined chromatographic conditions. XB-C18, 4.6mm×250mm, 5μm; Kromasil chromatographic column Kromasil 100-5-C18, 4.6mm×250mm 5μm) were injected and measured on the same instrument (Agilent 1200) according to the chromatographic conditions determined in Item 4 of Experimental Example 1. The contents of liquiritin, glycyrrhizic acid and hesperidin in the test sample and the RSD values ​​of the contents of different chromatographic columns were calculated.

[0145] Results: The RSD% of liquiritin content was 5.4%, less than 6.0% when using the same chromatograph with different brands of chromatographic columns; the RSD% of glycyrrhizic acid content was 3.8%, less than 6.0%; and the RSD% of hesperidin content was 1.0%, less than 4.0%. This shows that the use of different chromatographic columns has little effect on the determination results of liquiritin, glycyrrhizic acid, and hesperidin.

[0146] Table 20 Durability of chromatographic columns of different brands for instruments of the same brand

[0147]

[0148]

[0149] ③Different brands of instruments

[0150] Take the reference sample of Banxia Baizhu Tianma Decoction, prepare two samples in parallel according to the sample preparation method determined in Item 2 of Experimental Example 2, and use the same brand of chromatographic column to inject and measure on high performance liquid chromatographs of different brands according to the chromatographic conditions determined in Item 4 of Experimental Example 1, and calculate the results of the contents of liquiritin, glycyrrhizic acid, and hesperidin in the sample and the relative average deviation values ​​between different instruments. As shown in Mark 21, the relative average deviation of the content of liquiritin measured by different chromatographic instruments with the same brand of chromatographic column is 5.7%, which is less than 6.0%; the relative average deviation of the content of glycyrrhizic acid is 0.9%, which is less than 6.0%; and the relative average deviation of the content of hesperidin is 0.7%, which is less than 4.0%. This shows that the use of different instruments has little effect on the results of the determination of the contents of liquiritin, glycyrrhizic acid, and hesperidin.

[0151] Table 21 Durability study of instruments of different brands with the same brand of chromatographic column

[0152] Chromatograph name Licorice glycoside content (mg / g) Glycyrrhizic acid content (mg / g) Hesperidin content (mg / g) Waters 0.04523 0.06764 0.09390 Agilent 0.04038 0.06652 0.09509 Relative average deviation % 5.7 0.9 0.7

[0153] ④ Different flow rates

[0154] Take the reference sample of Banxia Baizhu Tianma Decoction, prepare two samples in parallel according to the sample preparation method determined in Item 2 of Experimental Example 2, use different flow rates, and the other chromatographic conditions are sampled and tested according to the chromatographic conditions determined in Item 4 of Experimental Example 1. Use different flow rates for sample injection and determination, and calculate the results of liquiritin, glycyrrhizic acid, and hesperidin content in the sample and the RSD values ​​of the content between different flow rates. As shown in Table 22, the RSD% of liquiritin content determined at different flow rates is 3.4%, less than 6.0%; the RSD% of glycyrrhizic acid content is 4.1%, less than 6.0%; the RSD% of hesperidin content is 2.2%, less than 4.0%. It shows that the use of different flow rates has little effect on the results of the determination of liquiritin, glycyrrhizic acid, and hesperidin.

[0155] Table 22 Durability study at different flow rates

[0156] Flow rate (ml / min) Licorice glycoside content (mg / g) Glycyrrhizic acid content (mg / g) Hesperidin content (mg / g) 0.9 0.04190 0.06510 0.09082 1.0 0.04446 0.07028 0.09335 1.1 0.04204 0.06633 0.08954 RSD% 3.4 4.1 2.2

[0157] ⑤ Different column temperatures

[0158] Take the reference sample of Banxia Baizhu Tianma Decoction, prepare two test sample solutions in parallel according to the test sample preparation method determined in Item 2 of Experimental Example 2, use different column temperatures, and the other chromatographic conditions are sampled and tested according to the chromatographic conditions determined in Item 4 of Experimental Example 1, and calculate the results of the content of liquiritin, glycyrrhizic acid, and hesperidin in the test sample and the RSD values ​​of the content between different column temperatures. As shown in Table 23, the RSD% of the liquiritin content determined at different column temperatures is 0.7%, which is less than 6.0%; the RSD% of the glycyrrhizic acid content is 5.0%, which is less than 6.0%; and the RSD% of the hesperidin content is 0.9%, which is less than 4.0%. This shows that the use of different instruments has little effect on the results of the determination of liquiritin, glycyrrhizic acid, and hesperidin.

[0159] Table 23 Durability study at different flow rates

[0160] Column temperature (℃) Licorice glycoside content (mg / g) Glycyrrhizic acid content (mg / g) Hesperidin content (mg / g) 28 0.04386 0.06526 0.09271 30 0.04446 0.07028 0.09335 32 0.04423 0.06416 0.09186 RSD% 0.7 5.0 0.9

[0161] Experimental Example 4 Investigation of Chromatographic Conditions

[0162] 1. Drug testing

[0163] Banxia Baizhu Tianma Decoction, prescription: Banxia 5.60g, Tianma 3.73g, Baizhu 11.19g, Fuling 3.73g, Tangerine peel 3.73g, Licorice 1.87g, Ginger 1.0g, Jujube 6.0g. Preparation method: Add water to the above medicinal ingredients and boil twice, add 12 times the amount of water for 30 minutes the first time, add 10 times the amount of water for 25 minutes the second time, filter the decoction, and combine the filtrate to obtain the decoction.

[0164] Negative control (without Gastrodia elata), prescription: 5.60g of Banxia, ​​11.19g of Atractylodes macrocephala, 3.73g of Poria, 3.73g of Citrus aurantium, 1.87g of Licorice, 1.0g of Ginger, 6.0g of Jujube. Preparation method: the same as that of Banxia Baizhu Tianma Decoction.

[0165] 2. Sample preparation

[0166] Preparation of test solution: Accurately measure 10 ml of the product in an evaporating dish, evaporate to dryness in a water bath, add 50% methanol to the residue and dissolve it in a 10 ml volumetric flask, shake well, filter, and take the filtrate as the test solution.

[0167] Preparation of reference solution: Take appropriate amounts of gastrodin reference substance and p-hydroxybenzyl alcohol reference substance, accurately weigh them, add a mixed solution of acetonitrile-water (3:97) to make a mixed solution containing 0.05 mg of gastrodin and 0.025 mg of p-hydroxybenzyl alcohol per 1 ml.

[0168] 3. Use high performance liquid chromatography to test according to the following methods.

[0169] Method 1: Use octadecylsilane bonded silica gel as the filler (NanoChrom ChromCore AQ-C18, 4.6mm×250mm, 5μm); use acetonitrile as mobile phase A, water as mobile phase B, column temperature at 30°C, flow rate at 1.0ml / min, perform gradient elution according to the provisions in Table 24, and inject 10μl of the test solution and mixed reference solution under item 2 respectively.

[0170] Table 24 Mobile phase gradient

[0171]

[0172] Method 2: Use octadecylsilane bonded silica gel as the filler (NanoChrom ChromCore AQ-C18, 4.6mm×250mm, 5μm); use acetonitrile as mobile phase A, 0.05% phosphoric acid as mobile phase B, the column temperature is 30°C, the flow rate is 1.0ml / min, and gradient elution is performed according to the provisions in Table 25. Inject 10μl of the test solution under item 2.

[0173] Table 25 Mobile phase gradient

[0174]

[0175] Method 3: Use octadecylsilane bonded silica gel as the filler (NanoChrom ChromCore AQ-C18, 4.6mm×250mm, 5μm); use acetonitrile as mobile phase A, 0.05% phosphoric acid as mobile phase B, the column temperature is 30°C, the flow rate is 1.0ml / min, and gradient elution is performed according to the provisions in Table 26. Inject 10μl of the test solution under item 2.

[0176] Table 26 Mobile phase gradient

[0177]

[0178] Method 4: Acetonitrile-0.05% phosphoric acid (gradient conditions in Table 26) was used as the mobile phase. No chromatographic peak corresponding to p-hydroxybenzyl alcohol was found in the test sample, and the p-hydroxybenzyl alcohol chromatographic peak could not be eluted within 20 min. Therefore, the isocratic elution time of acetonitrile-0.05% phosphoric acid (2:98) was extended to 35 min, and the gradient conditions were changed as follows: octadecylsilane bonded silica gel was used as the filler (Welch AQ-C18, 4.6 mm×250 mm, 5 μm); acetonitrile was used as mobile phase A, 0.05% phosphoric acid was used as mobile phase B, the column temperature was 30°C, the flow rate was 1.0 ml / min, and gradient elution was performed according to the provisions in Table 27 below, and 10 μl of the test sample solution under item 2 was injected.

[0179] Table 27 Mobile phase gradient

[0180]

[0181] The results are shown in the table below and Figure 10-12 shown.

[0182] Table 28 Chromatographic conditions investigation results

[0183]

[0184] From the results of method 1 and method 2, it can be seen that when acetonitrile-water is used as the mobile phase for detection, the peak shape of gastrodin and p-hydroxybenzyl alcohol in the test sample is worse than that when acetonitrile-0.05% phosphoric acid is used as the mobile phase. However, the purity angles of method 1 and method 2 are greater than the purity threshold and do not meet the analysis requirements.

[0185] The elution gradient was adjusted according to the test results of Method 1 and Method 2 to improve the peak purity of gastrodin and p-hydroxybenzyl alcohol in the test sample. Under the gradient conditions of Method 4, the peak shape and separation of gastrodin and p-hydroxybenzyl alcohol were good.

[0186] 4. The determined chromatographic conditions are as follows: octadecylsilane bonded silica gel as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A, 0.05% phosphoric acid as mobile phase B, gradient elution as specified in the table below; column temperature 30°C; flow rate 1.0 ml per minute; detection wavelength 220 nm. The theoretical plate number calculated based on the gastrodin peak should be no less than 5000.

[0187] Table 29 Determined mobile phase gradient

[0188]

[0189] Experimental Example 5 Investigation of the preparation method of the test solution

[0190] 1. During the pretreatment investigation of the test sample, it was found that after the test sample was evaporated, it was dissolved in 50% methanol to prepare the test solution. After a few hours, the test solution in the injection bottle had precipitation and was turbid. The reason was that the sugar content in the test sample was high. After alcohol treatment, sugar components would be precipitated, which was not conducive to the later inspection of the test sample. In order to remove more sugar in the test sample and improve the stability of the test solution, the precipitation in the test solution after alcohol precipitation treatment was investigated, and the difference in the content of gastrodin and p-hydroxybenzyl alcohol in the test sample was investigated. Therefore, different pretreatment methods were designed for investigation, and the influence of different solvents on the test results was investigated at the same time. The design of the pretreatment method investigation test is shown in Table 30 below, and the content detection results are shown in Table 31:

[0191] Table 30 Study on the preparation method of test solution

[0192]

[0193] Table 31 Content test results

[0194]

[0195] From the content results in Table 32, it can be seen that the difference in the content of gastrodin and p-hydroxybenzyl alcohol in the samples with and without alcohol precipitation is small, and considering the length of time for the later sample detection, precipitation is easily generated during the sample method process, and turbidity is not conducive to instrument detection, which may affect the detection accuracy, so alcohol precipitation is preferred. Moreover, after investigation, when 50% methanol, ethanol, 25% methanol, and acetonitrile-water (3: 97) mixed solution are used as solvents, the peak shape and separation degree are less different. Taking the solvent used for the reference substance into consideration, acetonitrile-water (3: 97) mixed solution is selected as the solvent for this method.

[0196] Table 32 Peak results

[0197]

[0198] 3. Determine the method for preparing the test solution: accurately measure 20 ml of the product in a 100 ml volumetric flask, add ethanol to dilute to the scale, shake well, centrifuge, take 25 ml of the supernatant and evaporate to dryness, add acetonitrile-water (3:97) mixed solution to dissolve the residue, transfer to a 5 ml volumetric flask, add acetonitrile-water (3:97) mixed solution to the scale, shake well, filter, and take the filtrate to obtain.

[0199] To facilitate practical operation, before conducting methodological verification, the method was adjusted as follows: take about 20 g of the product, accurately weigh it, place it in a 100 ml volumetric flask, add ethanol to dilute to the scale, shake well, centrifuge, take 25 ml of the supernatant and evaporate to dryness, add acetonitrile-water (3:97) mixed solution to the residue to dissolve it, transfer it to a 5 ml volumetric flask, add acetonitrile-water (3:97) mixed solution to the scale, shake well, filter, and take the filtrate to obtain.

[0200] Experimental Example 6 Methodological Verification of the Determination Method of Gastrodin and p-Hydroxybenzyl Alcohol in Banxia Baizhu Tianma Decoction

[0201] 1. Linearity and range

[0202] Take an appropriate amount of gastrodin reference substance, accurately weigh it, and add acetonitrile-water (3:97) mixed solution to prepare gastrodin reference substance solutions with concentrations of 0.0004992 mg / ml, 0.002496 mg / ml, 0.009984 mg / ml, 0.02496 mg / ml, 0.04992 mg / ml, 0.09984 mg / ml, and 0.2496 mg / ml, respectively; take an appropriate amount of p-hydroxybenzyl alcohol reference substance, accurately weigh it, and add acetonitrile-water (3:97) mixed solution to prepare p-hydroxybenzyl alcohol reference substance solutions with concentrations of 0.0002114 mg / ml, 0.001057 mg / ml, 0.004228 mg / ml, 0.01057 mg / ml, 0.02114 mg / ml, 0.04228 mg / ml, and 0.1057 mg / ml, respectively.

[0203] 10 μl of gastrodin reference solution of different concentrations (0.0004992 mg / ml, 0.002496 mg / ml, 0.009984 mg / ml, 0.02496 mg / ml, 0.04992 mg / ml, 0.09984 mg / ml, 0.2496 mg / ml) were accurately drawn and injected into the liquid chromatograph for sample injection according to the content determination method. The concentration of gastrodin was taken as the abscissa X and the peak area of ​​gastrodin was taken as the ordinate Y for linear regression processing. The regression equation was: Y=18060483.91X+1332.12, r=1.000, and the residual sum of squares was 239782244. The results showed that the concentration of gastrodin was in a good linear relationship within the range of 0.0004992 mg / ml to 0.2496 mg / ml. The results are shown in Table 33.

[0204] Table 33 Gastrodin linear concentration and peak area results

[0205] Concentration mg / ml 0.0004992 0.002496 0.009984 0.02496 0.04992 0.09984 0.2496 Peak area 9394 46029 179294 453330 896217 1817588 4505308

[0206] 10 μl of p-hydroxybenzyl alcohol reference solution of different concentrations (0.0002114 mg / ml, 0.001057 mg / ml, 0.004228 mg / ml, 0.01057 mg / ml, 0.02114 mg / ml, 0.04228 mg / ml, 0.1057 mg / ml) were accurately drawn and injected into the liquid chromatograph for sample injection according to the content determination method. The p-hydroxybenzyl alcohol concentration was taken as the abscissa X and the p-hydroxybenzyl alcohol peak area was taken as the ordinate Y for linear regression processing. The regression equation was: Y=37475010.13X+217.11, r=1.000, and the residual sum of squares was 274937001. The results showed that the p-hydroxybenzyl alcohol concentration was in a good linear relationship within the range of 0.0002114 mg / ml to 0.1057 mg / ml. The results are shown in Table 34.

[0207] Table 34 p-Hydroxybenzyl alcohol concentration and peak area results

[0208] Concentration mg / ml 0.0002114 0.001057 0.004228 0.01057 0.02114 0.04228 0.1057 Peak area 5975 38245 156527 398314 786699 1598962 3956660

[0209] 2. Specificity test

[0210] Gastrodin and p-hydroxybenzyl alcohol mixed reference solution: Take appropriate amounts of gastrodin and p-hydroxybenzyl alcohol reference substances, weigh them accurately, add acetonitrile-water (volume ratio 3:97) mixed solution to prepare a mixed solution containing 0.05 mg of gastrodin and 0.025 mg of p-hydroxybenzyl alcohol per 1 ml.

[0211] Banxia Baizhu Tianma Decoction (without Tianma) prescription: 5.60g of Banxia, ​​11.19g of Baizhu, 3.73g of Fuling, 3.73g of Tangerine Peel, 1.87g of Licorice, 1.0g of Ginger, 6.0g of Jujube. Preparation method: the same as that of Banxia Baizhu Tianma Decoction.

[0212] Banxia Baizhu Tianma Decoction Single Tianma prescription: Tianma 3.73g. Preparation method: Same as the preparation method of Banxia Baizhu Tianma Decoction.

[0213] The standard sample prescription of Banxia Baizhu Tianma Decoction is: 5.60g of Banxia, ​​3.73g of Tianma, 11.19g of Baizhu, 3.73g of Fuling, 3.73g of Tangerine Peel, 1.87g of Licorice, 1.0g of Ginger, and 6.0g of Jujube. Preparation method: Add water to the above medicinal ingredients and boil them twice. The first time, add 12 times the amount of water and boil for 30 minutes. The second time, add 10 times the amount of water and boil for 25 minutes. Filter the decoction and combine the filtrate to obtain the decoction.

[0214] Take blank solvent (acetonitrile-water mixed solution), p-hydroxybenzyl alcohol mixed reference solution of gastrodin, reference sample of Banxia Baizhu Tianma Decoction, single Tianma, Banxia Baizhu Tianma Decoction (without Tianma), prepare test sample and reference solution respectively according to the determined preparation method, and perform sample injection and determination according to the determined chromatographic conditions. Fig.13 It can be seen that there are no chromatographic peaks at the corresponding positions of gastrodin and p-hydroxybenzyl alcohol in the spectrum of the test solution of Banxia Baizhu Tianma Decoction (lacking Gastrodia elata), and there are no chromatographic peaks at the corresponding positions of gastrodin and p-hydroxybenzyl alcohol in the blank spectrum, the blank has no interference, and the separation degree is greater than 1.5, indicating that the method has good specificity.

[0215] 3. Precision test

[0216] ①Repeatability

[0217] The reference sample solution of Banxia Baizhu Tianma Decoction was prepared according to the determined preparation method, and 6 portions were paralleled and sampled and measured according to the determined chromatographic conditions. The concentration results and RSD values ​​of the 6 portions of the test solution were calculated. As shown in Table 35, the average content of gastrodin in the Banxia Baizhu Tianma Decoction test solution was 0.03266 mg / g, and the RSD value was 0.7%, which was less than 4.0%. The average content of p-hydroxybenzyl alcohol was 0.002472 mg / g, and the RSD value was 2.5%, which was less than 6.0%. The average value of the total amount of gastrodin and p-hydroxybenzyl alcohol was 0.03513 mg / g, and the RSD value was 0.6%, which was less than 4.0%, indicating that the method had good repeatability.

[0218] Table 35 Repeatability test results

[0219]

[0220] ②Intermediate precision

[0221] At different times, different analysts used different equipment to prepare 6 portions of the reference sample solution of Banxia Baizhu Tianma Decoction according to the determined preparation method, and the sample was injected and measured according to the determined chromatographic conditions. The concentration results and RSD values ​​of the 12 test solutions were calculated. It can be seen from Table 36 that the average content of gastrodin was 0.03276 mg / g, and the RSD value was 1.1%, which was less than 4.0%. The average content of p-hydroxybenzyl alcohol was 0.002221 mg / g, and the RSD value was 12.4%, which was greater than 6.0%. The average content of gastrodin and p-hydroxybenzyl alcohol was 0.03498 mg / g, and the RSD value was 1.0%, which was less than 4.0%. The results of the total amount of gastrodin and p-hydroxybenzyl alcohol were calculated, and the intermediate precision of this method was good.

[0222] Table 36 Intermediate precision test results

[0223]

[0224] 4. Accuracy test

[0225] Prepare the reference sample solution of Banxia Baizhu Tianma Decoction according to the determined preparation method, and perform 6 replicates. Perform the injection test according to the determined chromatographic conditions. Calculate the concentration results and RSD values ​​of the 6 replicates of the sample solution. The results are shown in Table 37. This repeatability result is used for accuracy data calculation.

[0226] Table 37 Repeatability test results

[0227]

[0228] Take 9 portions of the Banxia Baizhu Tianma Decoction benchmark sample with a known gastrodin content of 0.03266 mg / g and a p-hydroxybenzyl alcohol content of 0.002472 mg / g (data from repeatability test), each 10 g, accurately weighed, and added with reference substances as specified in Table 38 to prepare the test solution, determine the contents of gastrodin and p-hydroxybenzyl alcohol, and calculate the recovery rate.

[0229] Results: The recovery rate of gastrodin ranged from 92.1% to 98.3%. In the range of 85% to 110%, the average recovery rate was 97%, and the recovery rate RSD value was 2.6%, indicating that the accuracy of gastrodin was good under this method. The recovery rate of p-hydroxybenzyl alcohol ranged from 62.2% to 100.3%, and the average recovery rate was 81% and the recovery rate RSD value was 15.6% beyond the limit range of 75% to 120%. Due to the low content of p-hydroxybenzyl alcohol, which was 2.75 μg / g, the peak area was greatly affected by the integration and instrument baseline fluctuation, which may lead to large test errors. The accuracy of p-hydroxybenzyl alcohol was poor under this method. The recovery rate of the total amount of gastrodin and p-hydroxybenzyl alcohol ranged from 89.8% to 98.9%. In the range of 85% to 110%, the average recovery rate was 95%, and the recovery rate RSD value was 3.1%, indicating that the total amount of gastrodin and p-hydroxybenzyl alcohol was good under this method.

[0230] Table 38 Gastrodin accuracy test results

[0231]

[0232] Table 39 p-Hydroxybenzyl alcohol accuracy test results

[0233]

[0234] Table 40 Test results of the total amount of gastrodin and p-hydroxybenzyl alcohol

[0235]

[0236] Note: Recovery rate (%) = (test sample measured amount - sample content) / reference sample added amount × 100%

[0237] 5. Durability test

[0238] ①Solution stability

[0239] The reference sample solution of Banxia Baizhu Tianma Decoction was prepared according to the determined preparation method, and the sample was injected and measured at different time points (0h, 4h, 8h, 13h, 17h, 24h, 31h) according to the determined chromatographic conditions. The retention time and peak area of ​​gastrodin and p-hydroxybenzyl alcohol were recorded. The RSD values ​​were calculated. As shown in Table 41, the RSD values ​​of gastrodin retention time and peak area were 0.7% and 0.6%, respectively, which were both less than 2.0%, the RSD values ​​of p-hydroxybenzyl alcohol retention time and peak area were 0.5% and 3.0%, respectively, and the RSD value of peak area was greater than 2.0%, and the RSD value of the total peak area of ​​gastrodin and p-hydroxybenzyl alcohol was 0.8%, which was less than 2.0%. It shows that the test solution is stable within 31 hours at room temperature.

[0240] Table 41 Solution stability test results

[0241]

[0242] ②Chromatographic columns of different brands

[0243] The reference sample solution of Banxia Baizhu Tianma Decoction was prepared by the determined method and tested using different brands of C18 chromatographic columns (specifications: 4.6 mm×250 mm, 5 μm), and the content results and RSD values ​​detected by different brands of chromatographic columns were calculated.

[0244] Results: The RSDs of gastrodin and p-hydroxybenzyl alcohol contents in the test samples measured by different brands of chromatographic columns were 2.7% and 9.8%, respectively, and the RSD value of the total content of gastrodin and p-hydroxybenzyl alcohol was 1.7, indicating that different brands of chromatographic columns had little effect on the detection results of the total content of gastrodin and p-hydroxybenzyl alcohol in Banxia Baizhu Tianma Decoction, and the chromatographic columns had good durability.

[0245] Table 42 Chromatographic column durability test results

[0246]

[0247] ③Different brands of instruments

[0248] The reference sample solution of Banxia Baizhu Tianma Decoction was prepared by the determined method. According to the determined chromatographic conditions, the same brand of chromatographic column (chromcore AQ 18 4.6mm×250mm, 5μm) was used, and different brands of high performance liquid chromatographs (Agilent 1100 and Waters 2695) were used to detect the test solution, and the content results and RSD values ​​detected by different brands of instruments were calculated.

[0249] Results: The relative average deviations of gastrodin and p-hydroxybenzyl alcohol in the test samples measured by instruments of different brands were 1.5% and 17.6%, respectively, and the relative average deviation of the total content of gastrodin and p-hydroxybenzyl alcohol was 0.4%. This shows that instruments of different brands have little effect on the test results of the total content of gastrodin and p-hydroxybenzyl alcohol in Banxia Baizhu Tianma Decoction, and the durability of the instruments is good.

[0250] Table 43 Test results of different brands of instruments

[0251]

[0252]

[0253] ④ Different flow rates

[0254] The reference sample solution of Banxia Baizhu Tianma Decoction was prepared by the determined method. The test solution was tested at different flow rates according to the determined chromatographic conditions, and the content results and RSD values ​​detected at different flow rates were calculated.

[0255] Results: The relative average deviations of gastrodin and p-hydroxybenzyl alcohol in the test samples measured at different flow rates were 4.5% and 19.5%, respectively, and the relative average deviation of the total content of gastrodin and p-hydroxybenzyl alcohol was 5.6%, indicating that different flow rates had little effect on the detection results of the total content of gastrodin and p-hydroxybenzyl alcohol in Banxia Baizhu Tianmasu Decoction, and the flow rate durability was good.

[0256] Table 44 Test results of different flow rates

[0257]

[0258] ⑤ Different column temperatures

[0259] The reference sample solution of Banxia Baizhu Tianma Decoction was prepared by the determined method. The test solution was tested at different column temperatures according to the determined chromatographic conditions, and the content results and RSD values ​​detected at different column temperatures were calculated.

[0260] Results: The relative average deviations of the total amount of gastrodin and p-hydroxybenzyl alcohol in the test samples measured at different column temperatures were 4.5% and 19.5%, respectively, and the relative average deviation of the total amount of gastrodin and p-hydroxybenzyl alcohol was 5.6%, indicating that different flow rates had little effect on the detection results of the total amount of gastrodin and p-hydroxybenzyl alcohol in Banxia Baizhu Tianmasu Decoction, and the flow rate durability was good.

[0261] Table 45 Test results at different column temperatures

[0262]

[0263] Experimental Example 7 Determination of the contents of gastrodin, p-hydroxybenzyl alcohol, hesperidin, liquiritin and glycyrrhizic acid in different batches of Banxia Baizhu Tianma decoction benchmark samples

[0264] Fifteen batches of Banxia Baizhu Tianma Decoction benchmark samples were taken to prepare test solutions according to the established content determination method and tested. The contents of various components in Banxia Baizhu Tianma Decoction were calculated by the external standard method based on the concentrations of hesperidin, liquiritin, glycyrrhizic acid, gastrodin and p-hydroxybenzyl alcohol in the reference solution, the peak areas of the reference solution in the chromatogram and the peak areas of various components in Banxia Baizhu Tianma Decoction in the chromatogram.

[0265] Table 46 Results of content determination of 15 batches of reference samples

[0266]

[0267]

[0268] The total content of gastrodin and p-hydroxybenzyl alcohol in 15 batches of Banxia Baizhu Tianma Decoction benchmark samples fluctuated in the range of 0.02826 mg / g to 0.03960 mg / g, with an average of 0.03138 mg / g; the content of hesperidin fluctuated in the range of 0.07541 mg / g to 0.1199 mg / g, with an average of 0.1009 mg / g; the content of glycyrrhizin fluctuated in the range of 0.01597 mg / g to 0.04026 mg / g, with an average of 0.02401 mg / g; the content of glycyrrhizic acid ranged from 0.0.03697 mg / g to 0.08282 mg / g, with an average of 0.05908 mg / g.

[0269] Example 1

[0270] Gastrodin and p-hydroxybenzyl alcohol mixed reference solution: Take appropriate amounts of gastrodin and p-hydroxybenzyl alcohol reference substances, weigh them accurately, add acetonitrile-water (3:97) mixed solution to prepare a mixed solution containing 0.05 mg of gastrodin and 0.025 mg of p-hydroxybenzyl alcohol per 1 ml.

[0271] Test solution: Take about 20g of the product, accurately weigh it, put it in a 100ml volumetric flask, add ethanol to dilute to the scale, shake well, centrifuge, take 25ml of the supernatant and evaporate to dryness, add acetonitrile-water (3:97) mixed solution to dissolve the residue, transfer it to a 5ml volumetric flask, add acetonitrile-water (3:97) mixed solution to the scale, shake well, filter, and take the filtrate to obtain.

[0272] The test solution, the mixed reference solution and the blank (acetonitrile and water in a volume ratio of 3:97) were measured according to the gradient elution conditions determined in Experimental Example 4. The contents of gastrodin and p-hydroxybenzyl alcohol in Banxia Baizhu Tianma Decoction were calculated by the external standard method based on the concentrations of gastrodin and p-hydroxybenzyl alcohol in the reference solution, the peak areas of the reference solution in the chromatogram and the peak areas of gastrodin and p-hydroxybenzyl alcohol in Banxia Baizhu Tianma Decoction in the chromatogram.

[0273] Results Fig.14 As shown in Table 47.

[0274] Table 47 Chromatographic conditions investigation results

[0275]

[0276] Note: An angle value less than the threshold value indicates that the peak purity is good and meets the requirements.

[0277] The results showed that the separation degree, peak shape and peak purity of gastrodin and p-hydroxybenzyl alcohol chromatographic peaks from other peaks were good, meeting the requirements of content determination.

[0278] Comparative Example 1

[0279] Referring to the method in the literature "HPLC fingerprint and multi-component quantitative analysis of Banxia Baizhu Tianma Decoction", the contents of gastrodin and p-hydroxybenzyl alcohol in Banxia Baizhu Tianma Decoction were detected. The specific test method is as follows: Test solution: weigh about 2g of Banxia Baizhu Tianma Decoction concentrate, accurately weigh, place in a 20ml volumetric flask, add methanol to the scale, ultrasonically treat at 40kHz and 250W for 30 minutes, cool, make up the reduced mass with methanol, shake well, filter, and take the filtrate.

[0280] Gastrodin and p-hydroxybenzyl alcohol mixed reference solution: Take appropriate amounts of gastrodin and p-hydroxybenzyl alcohol reference substances, weigh them accurately, add methanol mixed solution to make a mixed solution containing 0.05 mg of gastrodin and 0.025 mg of p-hydroxybenzyl alcohol per 1 ml.

[0281] The test was carried out by high performance liquid chromatography according to the following method: Inertsil ODS-3 chromatographic column (250 mm×4.6 mm, 5 μm); gradient elution was adopted; mobile phase: acetonitrile: water = A: B, gradient elution was carried out according to the ratio of acetonitrile, 0-48 min (A% gradually increased: 3%→45%), 48-60 min (A% gradually increased: 45%→100%); flow rate was 1.0 mL / min; injection volume: 10 μL; detection wavelength: 254 nm; column temperature: 30°C.

[0282] Results Fig.15 As shown in Table 48.

[0283] Table 48 Chromatographic conditions investigation results

[0284]

[0285] Note: Peak purity greater than 990 indicates good quality and meets the requirements.

[0286] The results showed that under the chromatographic conditions, the retention time of gastrodin and p-hydroxybenzyl alcohol was short, and the peak shape of gastrodin and p-hydroxybenzyl alcohol in the test solution was poor. In addition, the ultraviolet absorption of gastrodin and p-hydroxybenzyl alcohol was impure, and may contain other chromatographic peaks.

[0287] Comparative Example 2

[0288] References Yan Guo, Cai Ya, Yang Yi, Wu Guanghui, Che Kai, Yin Zhiyou, Jie Guoliang. Analysis of the content and influencing factors of gastrodin and p-hydroxybenzyl alcohol in fresh Gastrodia elata [J]. Food Industry Science and Technology: 1-13 [2020-11-27]. The disclosed method was used to detect the content of gastrodin and p-hydroxybenzyl alcohol in Banxia Baizhu Tianma Decoction. The specific test method is as follows: Test solution: weigh about 20g of the decoction of Banxia Baizhu Tianma Decoction, accurately weigh it, put it in a 100ml volumetric flask, add ethanol to the scale (shake while adding), let it stand for 30 minutes, centrifuge, accurately transfer 50ml of the supernatant, evaporate it in a water bath, dissolve the residue in acetonitrile-0.05% phosphoric acid and make up to 5ml volumetric flask, filter, and take the filtrate.

[0289] Gastrodin and p-hydroxybenzyl alcohol mixed reference solution: Take appropriate amounts of gastrodin and p-hydroxybenzyl alcohol reference substances, weigh them accurately, add acetonitrile-0.05% phosphoric acid mixed solution to prepare a mixed solution containing 0.05 mg of gastrodin and 0.025 mg of p-hydroxybenzyl alcohol per 1 ml.

[0290] The test was performed by high performance liquid chromatography according to the following method: Inertsil ODS-3 column (250 mm×4.6 mm, 5 μm); mobile phase: acetonitrile-0.05% phosphoric acid aqueous solution (3:97, V / V), isocratic elution; flow rate: 1.0 mL·min -1 ; Detection wavelength: 220nm; Injection volume: 20μL; Analysis time: 45min; Column temperature 25℃.

[0291] Results Fig.16 As shown in Table 49.

[0292] Table 49 Chromatographic conditions investigation results

[0293]

[0294] Note: Peak purity greater than 990 indicates good quality and meets the requirements.

[0295] The results showed that the purity of the chromatographic peak of gastrodin in the test sample was poor, and the peak shape of the chromatographic peak of p-hydroxybenzyl alcohol was poor.

[0296] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for determining the content of chemical components in Banxia Baizhu Tianma decoction, characterized in that: The raw materials of the Banxia Baizhu Tianma decoction include Pinellia, Gastrodia, Atractylodes, Tangerine peel, Poria, Licorice, Ginger and Jujube, and the content determination method comprises the following steps: S1. Preparation of test solution; S2. Prepare reference solution using gastrodin and p-hydroxybenzyl alcohol as reference substances; S3. The chromatograms of the test solution and the reference solution were determined by high performance liquid chromatography, wherein the chromatographic column was filled with octadecylsilane bonded silica gel, and acetonitrile and an aqueous solution containing phosphoric acid were used as mobile phases for gradient elution, and the gradient elution program included: 0→35min→40min→50min→55min; the volume percentage of acetonitrile in the mobile phase was 2%→2%→60%→80%→2%; S4, according to the concentration of the chemical components in the reference solution in step S2, the peak area of ​​the reference solution in the chromatogram, and the peak area of ​​the components corresponding to the reference solution in Banxia Baizhu Tianma Decoction in the chromatogram, calculate the contents of gastrodin and p-hydroxybenzyl alcohol in Banxia Baizhu Tianma Decoction by external standard method; The preparation method of the test solution comprises: Step 1), mixing Banxia Baizhu Tianma decoction with an alcohol solvent, and subjecting the mixed solution to a first solid-liquid separation; Step 2), drying the liquid obtained after the first solid-liquid separation, mixing the dried product with the solvent, subjecting the mixed solution to a second solid-liquid separation, and obtaining the liquid; In step 1), the alcohol solvent is selected from methanol or ethanol; The content determination method also includes: S5. Prepare a reference solution using liquiritin, ammonium glycyrrhizinate and hesperidin as reference substances; S6. The chromatograms of the test solution and the reference solution were determined by high performance liquid chromatography, wherein the chromatographic column was filled with octadecylsilane bonded silica gel, acetonitrile and aqueous solution containing phosphoric acid were used as mobile phases for gradient elution, and the gradient elution program included: 0→8min→20min→35min; the volume percentage of acetonitrile in the mobile phase was 19%→19%→30%→50%; S7. Calculate the contents of liquiritin, glycyrrhizic acid and hesperidin in Banxia Baizhu Tianma Decoction by external standard method according to the concentration of chemical components in the reference solution in step S5, the peak area of ​​the reference solution in the chromatogram and the peak area of ​​the components corresponding to the reference solution in Banxia Baizhu Tianma Decoction in the chromatogram.

2. The method for determining the content of chemical components in Banxia Baizhu Tianma decoction according to claim 1, characterized in that: The first solid-liquid separation is centrifugation or filtration; and / or the ratio of the volume of the Banxia Baizhu Tianma decoction to the volume of the mixed solution in step 1) is 1:2-1:5; or the ratio of the mass of the Banxia Baizhu Tianma decoction to the volume of the mixed solution in step 1) is 1g:2-5mL.

3. The method for determining the content of chemical components in Banxia Baizhu Tianma decoction according to claim 1, characterized in that: In step 2), the solvent is selected from 25-70% methanol aqueous solution, 25-70% ethanol aqueous solution, acetonitrile-water or water in a volume ratio of 1:99 and 5:95; and / or, the second solid-liquid separation is selected from centrifugation or filtration; and / or, the ratio of the volume of the liquid to the volume of the mixed solution in step 2) is 20-30:5-50.

4. The method for determining the content of chemical components in Banxia Baizhu Tianma decoction according to any one of claims 1 to 3, characterized in that: In step S3, the detection wavelength of gastrodin and hydroxybenzyl alcohol is 215-225 nm; and / or, the flow rate is 0.9-1.1 ml / min, and the column temperature is 28-32° C.; and / or, the volume percentage of phosphoric acid in the aqueous solution containing phosphoric acid is 0.03-0.08%.

5. The method for determining the content of chemical components in Banxia Baizhu Tianma decoction according to any one of claims 1 to 3, characterized in that: Step S2 is to prepare a mixed reference solution of gastrodin and hydroxybenzyl alcohol using gastrodin and p-hydroxybenzyl alcohol.

6. The method for determining the content of chemical components in Banxia Baizhu Tianma decoction according to claim 5, characterized in that: The concentration of gastrodin in the mixed reference solution is 0.0004992 mg / ml to 0.2496 mg / ml; the concentration of hydroxybenzyl alcohol is 0.0002114 mg / ml to 0.1057 mg / ml.

7. The method for determining the content of chemical components in Banxia Baizhu Tianma decoction according to claim 1, characterized in that: In step S6, the detection wavelength of liquiritin and glycyrrhizic acid is 235-240 nm, and the detection wavelength of hesperidin is 280-290 nm; and / or, the flow rate is 0.9-1.1 ml / min, and the column temperature is 28-32° C.; and / or, the volume percentage of phosphoric acid in the phosphoric acid-containing aqueous solution is 0.03-0.08%; and / or, the gradient elution program also includes 35 min→36 min→40 min; the volume percentage of acetonitrile in the mobile phase is 50%→100%→19%.

8. The method for determining the content of chemical components in Banxia Baizhu Tianma decoction according to claim 1, characterized in that: In step S5, a hesperidin reference solution and a mixed reference solution of liquiritin and glycyrrhizic acid are prepared respectively.

9. The method for determining the content of chemical components in Banxia Baizhu Tianma decoction according to claim 8, characterized in that: The concentration of liquiritin in the mixed reference solution of liquiritin and glycyrrhizic acid is 0.002332 mg / ml to 0.2332 mg / ml, and the concentration of glycyrrhizic acid is 0.003748 mg / ml to 0.3748 mg / ml; the concentration of hesperidin in the hesperidin reference solution is 0.001676 mg / ml to 0.1676 mg / ml.

10. The method for determining the content of chemical components in Banxia Baizhu Tianma decoction according to any one of claims 1 to 3, characterized in that: In terms of weight, the raw materials of the Banxia Baizhu Tianma Decoction include 4.5-6.7 parts of Pinellia, 3.0-4.5 parts of Tianma, 9.0-13.4 parts of Atractylodes, 3.0-4.5 parts of Tangerine Peel, 3.0-4.5 parts of Poria, 1.5-2.2 parts of Licorice, 0.8-1.2 parts of Ginger and 4.8-7.2 parts of Jujube.

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