Quality control method of traditional Chinese medicine compound containing honeysuckle, isatis root and scutellaria and application thereof

Abstract

This invention discloses a method for determining the content of a traditional Chinese medicine compound containing honeysuckle, isatis root, and scutellaria. The method includes the following steps: taking a test solution of the traditional Chinese medicine compound, a first reference solution, and a second reference solution for detection. The chromatographic conditions are as follows: using a chromatographic column packed with octadecyl-bonded silica gel; mobile phase A selected from one or more of acetonitrile, methanol, and tetrahydrofuran; and mobile phase B being an acidic aqueous solution, an alkaline aqueous solution, and / or a buffer salt aqueous solution. The traditional Chinese medicine compound includes honeysuckle, isatis root, scutellaria, indigo leaf, forsythia, platycodon, patchouli, and licorice. The reference standards are baicalin, ammonium glycyrrhizate, honeysuckle saponin B, and dipsacus saponin B. The detection method of this invention is simple, accurate, efficient, stable, precise, reproducible, convenient, and easy to master. It can effectively save energy and reduce analytical costs, while this technology is not disclosed in existing technologies.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a quality control method for a traditional Chinese medicine compound containing honeysuckle, isatis root, and scutellaria, and its application. Background Technology

[0002] Reference samples embody the efficacy and safety of traditional Chinese medicine (TCM) compositions. Quality research is a crucial aspect and key step in the development of TCM compound preparations, and content determination is the primary method for controlling the quality of reference samples. Screening for quality markers related to the efficacy and safety of TCM compositions and establishing content determination methods can quantitatively characterize the quality of reference samples.

[0003] Current content determination methods mainly focus on single components, and there are very few methods that can simultaneously determine the content of multiple active ingredients. The content determination method and its application for a traditional Chinese medicine compound containing honeysuckle, isatis root, and scutellaria baicalensis, as described in this invention, have not been found in the existing technology. Summary of the Invention

[0004] Based on this, the present invention provides a method for determining the content of a traditional Chinese medicine compound containing honeysuckle, isatis root, and scutellaria, the method comprising the following steps:

[0005] Preparation of the test solution of the traditional Chinese medicine compound: Take an appropriate amount of the traditional Chinese medicine compound powder containing honeysuckle, isatis root and scutellaria, place it in a container, add solvent, sonicate, make up to volume, shake well, filter, and take the filtrate to obtain the test solution of the traditional Chinese medicine compound; wherein, the traditional Chinese medicine compound includes honeysuckle, isatis root, scutellaria, indigo leaf, forsythia, platycodon, patchouli and licorice;

[0006] Preparation of the first reference solution: Weigh appropriate amounts of baicalin reference standard and glycyrrhizic acid ammonium reference standard, add methanol to prepare the first reference solution with a reference standard concentration of 0.01-1 mg / ml;

[0007] Preparation of the second reference solution: Weigh appropriate amounts of honeysuckle saponin B reference standard and dipsacus saponin B reference standard, add methanol to prepare the second reference solution with a reference standard concentration of 0.01-1 mg / ml.

[0008] Take the test solution of the traditional Chinese medicine compound, the first reference solution, and the second reference solution for testing.

[0009] The chromatographic conditions for this detection were as follows: a column packed with octadecyl-bonded silica gel was used; mobile phase A was selected from one or more of acetonitrile, methanol, and tetrahydrofuran; mobile phase B was an aqueous acid solution, an aqueous alkaline solution, and / or an aqueous buffer solution; the gradient elution program was: 0–25 min, 81% B → 80% B; 25–45 min, 80% B → 50% B; 45–50 min, 50% B → 20% B; 50–55 min, 20% B → 81% B; 55–60 min, 81% B; the flow rate was 0.5–1.0 ml / min; the column temperature was 25–35℃; and the injection volume was 1–25 μl. Baicalin and glycyrrhizic acid were detected using ultraviolet light, while Lonicera japonica saponin B and Dipsacus asperoides saponin B were detected using an evaporative light scattering detector.

[0010] Furthermore, this information is based on the recorded peak areas in the chromatograms of the test solution and the reference solution of the traditional Chinese medicine compound, and the contents of baicalin and glycyrrhizic acid reference standards in the traditional Chinese medicine compound are calculated using the blank external standard method. The contents of honeysuckle saponin B and dipsacus saponin B reference standards in the traditional Chinese medicine compound are calculated using the logarithmic equation of the two-point external standard method.

[0011] Furthermore, the mass of glycyrrhizic acid is calculated based on the mass of ammonium glycyrrhizate, with a mass ratio of ammonium glycyrrhizate to glycyrrhizic acid of approximately 1.0207.

[0012] Furthermore, the linear equation for this scutellarin is y = 18728134.4265x - 77989.4438, R0 2 =0.9992.

[0013] Furthermore, the linear equation for this glycyrrhizic acid is y = 11474520.1348x + 8136.4234, R0 2 =1.0000.

[0014] Furthermore, the linear equation for this Lonicera japonica saponin B is y = 1.5881x + 7.4300, R0 2 =0.9901.

[0015] Furthermore, the linear equation for this Dipsacus saponin B is y = 1.7258x + 7.7011, R0 2 =0.9958.

[0016] Furthermore, the preparation method of the traditional Chinese medicine compound powder containing honeysuckle, isatis root and scutellaria includes: (a) weighing appropriate amounts of honeysuckle, isatis root, scutellaria, indigo leaf, forsythia, platycodon, patchouli and licorice, adding water for the first heating extraction, filtering to obtain the first filtrate and the residue; (b) adding water for the second heating extraction, filtering to obtain the second filtrate; (c) combining the first filtrate and the second filtrate, drying to obtain the traditional Chinese medicine compound powder.

[0017] Further, in step (a), the volume / mass (ml / g) ratio of the first water to the sum of the masses of honeysuckle, isatis root, scutellaria, indigo leaf, forsythia, platycodon, patchouli and licorice is 5 to 15, for example about 12.

[0018] Furthermore, in step (a), the first heating extraction time is 60 to 120 minutes, for example, about 90 minutes.

[0019] Further, in step (a), the filtration is performed while the filter is hot using a 200-mesh filter cloth.

[0020] Further, in step (b), the volume / mass (ml / g) ratio of the second water to the sum of the masses of honeysuckle, isatis root, scutellaria, indigo leaf, forsythia, platycodon, patchouli and licorice is 5 to 15, for example about 10.

[0021] Furthermore, in step (b), the second heating extraction time is 30 to 90 minutes, for example, about 60 minutes.

[0022] Furthermore, in step (b), the filtration is performed while the filter is hot using a 200-mesh filter cloth.

[0023] Furthermore, in step (c), the drying is either spray drying or freeze drying.

[0024] Furthermore, the mass ratio of the honeysuckle, the isatis root, the scutellaria, the indigo leaf, the forsythia, the platycodon, the patchouli and the licorice is (1-10):(1-10):(1-10):(1-10):(1-10):(1-10):(1-10):(1-10):(1-10):(1-10).

[0025] Furthermore, the mass ratio of the honeysuckle, the isatis root, the scutellaria, the indigo leaf, the forsythia, the platycodon, the patchouli and the licorice is (2-8):(2-8):(1-5):(1-5):(1-5):(1-5):(1-5):(1-5):(1-5).

[0026] Furthermore, the mass ratio of the honeysuckle, the isatis root, the scutellaria, the indigo leaf, the forsythia, the platycodon, the patchouli, and the licorice is approximately 5: approximately 5: approximately 3: approximately 3: approximately 3: approximately 3: approximately 3: approximately 3: approximately 3.

[0027] Furthermore, the weight range of the honeysuckle is 2–8g, for example, about 5g.

[0028] Furthermore, the weight range of the Isatis root is 2–8g, for example, about 5g.

[0029] Furthermore, the weight of the Scutellaria baicalensis ranges from 1 to 5g, for example, about 3g.

[0030] Furthermore, the weight of the Daqingye (Isatis tinctoria) ranges from 1 to 5g, for example, about 3g.

[0031] Furthermore, the weight of the forsythia ranges from 1 to 5g, for example, about 3g.

[0032] Furthermore, the weight of the platycodon root ranges from 1 to 5g, for example, about 3g.

[0033] Furthermore, the mass of the patchouli ranges from 1 to 5g, for example, about 3g.

[0034] Furthermore, the weight of the licorice ranges from 1 to 5g, for example, about 3g.

[0035] Furthermore, in the preparation of the test solution of this traditional Chinese medicine compound, the solvent is an alcohol, such as methanol.

[0036] Furthermore, the volume percentage concentration of methanol is 20% to 50%, for example, about 35%.

[0037] Furthermore, the container is a measuring bottle.

[0038] Furthermore, the mass of the traditional Chinese medicine compound powder is 0.1 to 1 g, for example, about 0.5 g.

[0039] Furthermore, the power of the ultrasonic treatment is 400–600W, for example, about 500W.

[0040] Furthermore, the frequency of the ultrasonic treatment is 30–50 kHz, for example, about 40 kHz.

[0041] Furthermore, the duration of the ultrasonic treatment is 20–40 minutes, for example, about 30 minutes.

[0042] Furthermore, the mass / volume ratio between the traditional Chinese medicine compound powder and the traditional Chinese medicine compound test solution is 0.01 to 0.1, for example, about 0.02, in g / ml.

[0043] Furthermore, the pretreatment method for the baicalin reference standard and the ammonium glycyrrhizate reference standard is as follows: the baicalin reference standard and the ammonium glycyrrhizate reference standard are dried under reduced pressure at about 60°C for about 4 hours.

[0044] Furthermore, the concentration of baicalin in the first reference solution was approximately 0.3 mg / ml.

[0045] Furthermore, the concentration of ammonium glycyrrhizate in the first reference solution was approximately 0.04 mg / ml.

[0046] Furthermore, the concentration of Lonicera japonica saponin B in the second reference solution was approximately 0.3 mg / ml.

[0047] Furthermore, the concentration of bisacodyl B in the second reference solution was approximately 0.03 mg / ml.

[0048] Furthermore, in the preparation of the second reference solution, the volume percentage concentration of methanol is 40% to 60%, for example, about 50%.

[0049] Furthermore, the flow rate is 0.7–0.9 ml / min, for example, about 0.8 ml / min.

[0050] Furthermore, the column temperature is 28–32°C, for example, about 30°C.

[0051] Furthermore, when using ultraviolet detection, the detection wavelength is 200–300 nm, for example, 245 nm.

[0052] Furthermore, when using ultraviolet detection, the injection volume of the test solution is 5–15 μl, for example, about 10 μl.

[0053] Furthermore, when using an evaporative light scattering detector, the injection volume of the test solution is 5–10 μl.

[0054] Furthermore, the injection volume of the first reference solution is 5–15 μl, for example, about 10 μl.

[0055] Furthermore, the injection volume of the second reference solution is approximately 5 μl or approximately 20 μl.

[0056] Furthermore, the chromatographic column is a HALO column. AQ-C18 chromatographic column.

[0057] Furthermore, the specifications of the chromatographic column are as follows: column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm.

[0058] Furthermore, mobile phase A is acetonitrile.

[0059] Furthermore, the aqueous acid solution, aqueous alkaline solution, and / or aqueous buffer salt solution are selected from one or more weak acids and their salts, weak bases and their salts of different concentrations.

[0060] Furthermore, the aqueous acid solution, aqueous alkaline solution, and / or aqueous buffer solution are selected from different concentrations of formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid.

[0061] Furthermore, the acidic aqueous solution is a 0.01% to 1% acidic aqueous solution.

[0062] Furthermore, the aqueous solution of the acid is a 0.01% to 1% aqueous solution of formic acid.

[0063] Furthermore, the aqueous solution of the acid is an aqueous solution of approximately 0.1% formic acid.

[0064] Furthermore, the buffer salt solution is an acetate solution and / or an acetate solution.

[0065] Furthermore, the pH value of this buffer salt solution is no greater than 7.0.

[0066] Furthermore, the detection limit for this baicalin is approximately 0.0004553 mg / ml.

[0067] Furthermore, the detection limit for this glycyrrhizic acid is approximately 0.0001296 mg / ml.

[0068] Furthermore, the detection limit for this Lonicera japonica saponin B was approximately 0.002066 mg / ml.

[0069] Furthermore, the detection limit for Dipsacin B was approximately 0.001751 mg / ml.

[0070] Furthermore, the limit of quantification for this baicalin is approximately 0.001339 mg / ml.

[0071] Furthermore, the limit of quantification for this glycyrrhizic acid is approximately 0.0003239 mg / ml.

[0072] Furthermore, the limit of quantification for this honeysuckle saponin B is approximately 0.006198 mg / ml.

[0073] Furthermore, the limit of quantification for Dipsacus saponin B is approximately 0.006180 mg / ml.

[0074] According to another aspect of the present invention, the above-described assay method is provided for use in the quality detection and / or quality evaluation and / or quality control of traditional Chinese medicine compound containing honeysuckle, isatis root and scutellaria.

[0075] The beneficial effects of this invention are:

[0076] The detection method of the present invention uses two detection methods simultaneously under the same liquid phase detection conditions, which is simple, accurate, efficient, stable, precise, reproducible, convenient and easy to master. It can effectively save energy and reduce analysis costs, while the prior art does not disclose this technology. Attached Figure Description

[0077] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0078] Figure 1 This is a schematic diagram showing the results obtained by ultraviolet detection of a test solution of a traditional Chinese medicine compound containing honeysuckle, isatis root, and scutellaria.

[0079] Figure 2 This is a schematic diagram showing the results obtained by detecting a compound traditional Chinese medicine sample solution containing honeysuckle, isatis root, and scutellaria using an evaporative light scattering detector. Detailed Implementation

[0080] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0081] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0082] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0083] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0084] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0085] As described in the background section, current content determination methods mainly focus on single components, and there are very few methods that can simultaneously determine the content of multiple active ingredients. The present invention does not provide a method for determining the content of a traditional Chinese medicine compound containing honeysuckle, isatis root, and scutellaria, nor its application. To address the above problems, the present invention provides a method for determining the content of a traditional Chinese medicine compound containing honeysuckle, isatis root, and scutellaria, which includes the following steps:

[0086] Preparation of the test solution of the traditional Chinese medicine compound: Take an appropriate amount of the traditional Chinese medicine compound powder containing honeysuckle, isatis root and scutellaria, place it in a container, add solvent, sonicate, make up to volume, shake well, filter, and take the filtrate to obtain the test solution of the traditional Chinese medicine compound; wherein, the traditional Chinese medicine compound includes honeysuckle, isatis root, scutellaria, indigo leaf, forsythia, platycodon, patchouli and licorice;

[0087] Preparation of the first reference solution: Weigh appropriate amounts of baicalin reference standard and glycyrrhizic acid ammonium reference standard, add methanol to prepare the first reference solution with a reference standard concentration of 0.01-1 mg / ml;

[0088] Preparation of the second reference solution: Weigh appropriate amounts of honeysuckle saponin B reference standard and dipsacus saponin B reference standard, add methanol to prepare the second reference solution with a reference standard concentration of 0.01-1 mg / ml.

[0089] Take the test solution of the traditional Chinese medicine compound, the first reference solution, and the second reference solution for testing.

[0090] The chromatographic conditions for this detection were as follows: a column packed with octadecyl-bonded silica gel was used; mobile phase A was selected from one or more of acetonitrile, methanol, and tetrahydrofuran; mobile phase B was an aqueous acid solution, an aqueous alkaline solution, and / or an aqueous buffer solution; the gradient elution program was: 0–25 min, 81% B → 80% B; 25–45 min, 80% B → 50% B; 45–50 min, 50% B → 20% B; 50–55 min, 20% B → 81% B; 55–60 min, 81% B; the flow rate was 0.5–1.0 ml / min; the column temperature was 25–35℃; and the injection volume was 1–25 μl. Baicalin and glycyrrhizic acid were detected using ultraviolet light, while Lonicera japonica saponin B and Dipsacus asperoides saponin B were detected using an evaporative light scattering detector.

[0091] In this invention, when concentration, column temperature, injection volume, flow rate, ratio, time, mass ratio, volume percentage concentration, mass, power, frequency, wavelength, or other values ​​or parameters are expressed as ranges, preferred ranges, or a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “25–35” is disclosed, the described range should be interpreted as including ranges “25–35”, “25–33”, “25–31”, “25–29”, “25–27”, “27–35”, “27–33”, “27–31”, “27–29”, “29–35”, “29–33”, “29–31”, “31–35”, “31–33”, “33–35”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0092] In a preferred embodiment, the information is obtained by calculating the contents of baicalin and glycyrrhizic acid reference standards in the traditional Chinese medicine compound according to the corresponding peak areas in the chromatograms of the test solution and the reference solution, using the blank external standard method, and by calculating the contents of honeysuckle saponin B and dipsacus saponin B reference standards in the traditional Chinese medicine compound according to the logarithmic equation of the two-point external standard method.

[0093] In a preferred embodiment, the mass of glycyrrhizic acid is calculated based on the mass of ammonium glycyrrhizate, with the mass ratio of ammonium glycyrrhizate to glycyrrhizic acid being approximately 1.0207.

[0094] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.0207" includes ±5% of 1.0207, or from 0.969665 to 1.071735.

[0095] In a preferred embodiment, the linear equation for baicalein is y = 18728134.4265x - 77989.4438, R 2 =0.9992.

[0096] In a preferred embodiment, the linear equation for the glycyrrhizic acid is y = 11474520.1348x + 8136.4234, R 2 =1.0000.

[0097] In a preferred embodiment, the linear equation for the honeysuckle saponin B is y = 1.5881x + 7.4300, R 2 =0.9901.

[0098] In a preferred embodiment, the linear equation for the diosgenin B is y = 1.7258x + 7.7011, R 2 =0.9958.

[0099] In a preferred embodiment, the preparation method of the traditional Chinese medicine compound powder containing honeysuckle, isatis root, and scutellaria includes: (a) weighing appropriate amounts of honeysuckle, isatis root, scutellaria, indigo leaf, forsythia, platycodon, patchouli, and licorice, adding water for a first heating extraction, filtering to obtain a first filtrate and a filter residue; (b) adding water for a second heating extraction, filtering to obtain a second filtrate; (c) combining the first filtrate and the second filtrate, drying to obtain the traditional Chinese medicine compound powder.

[0100] In a preferred embodiment, in step (a), the volume / mass (ml / g) ratio of the first water to the sum of the masses of honeysuckle, isatis root, scutellaria, indigo leaf, forsythia, platycodon, patchouli and licorice is 5 to 15, for example about 12.

[0101] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 12" includes ±5% of 12, or from 11.4 to 12.6.

[0102] In a preferred embodiment, in step (a), the first heating extraction time is 60 to 120 minutes, for example, about 90 minutes.

[0103] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 90" includes ±5% of 90, or from 85.5 to 94.5.

[0104] In a preferred embodiment, in step (a), the filtration is performed while the filter is still hot using a 200-mesh filter cloth.

[0105] In a preferred embodiment, in step (b), the volume / mass (ml / g) ratio of the second water to the sum of the masses of honeysuckle, isatis root, scutellaria, indigo leaf, forsythia, platycodon, patchouli and licorice is 5 to 15, for example about 10.

[0106] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0107] In a preferred embodiment, in step (b), the second heating extraction time is 30 to 90 minutes, for example, about 60 minutes.

[0108] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

[0109] In a preferred embodiment, in step (b), the filtration is performed while the filter is still hot using a 200-mesh filter cloth.

[0110] In a preferred embodiment, in step (c), the drying is either spray drying or freeze drying.

[0111] In a preferred embodiment, the mass ratio of the honeysuckle, the isatis root, the scutellaria, the indigo leaf, the forsythia, the platycodon, the patchouli, and the licorice is (1-10):(1-10):(1-10):(1-10):(1-10):(1-10):(1-10):(1-10):(1-10):(1-10).

[0112] In a preferred embodiment, the mass ratio of the honeysuckle, the isatis root, the scutellaria, the indigo leaf, the forsythia, the platycodon, the patchouli and the licorice is (2-8):(2-8):(1-5):(1-5):(1-5):(1-5):(1-5):(1-5):(1-5):(1-5).

[0113] In a preferred embodiment, the mass ratio of the honeysuckle, the isatis root, the scutellaria, the indigo leaf, the forsythia, the platycodon, the patchouli, and the licorice is about 5: about 5: about 3: about 3: about 3: about 3: about 3: about 3: about 3.

[0114] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5" includes 5 ± 5%, or from 4.75 to 5.25; "about 3" includes 3 ± 5%, or from 2.85 to 3.15.

[0115] In a preferred embodiment, the honeysuckle has a mass range of 2 to 8 g, for example, about 5 g.

[0116] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5" includes 5 ± 5%, or from 4.75 to 5.25.

[0117] In a preferred embodiment, the mass of the Isatis root ranges from 2 to 8 g, for example, about 5 g.

[0118] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5" includes 5 ± 5%, or from 4.75 to 5.25.

[0119] In a preferred embodiment, the Scutellaria baicalensis has a mass range of 1 to 5 g, for example, about 3 g.

[0120] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3" includes 3 ± 5%, or from 2.85 to 3.15.

[0121] In a preferred embodiment, the mass of the indigo leaf ranges from 1 to 5 g, for example, about 3 g.

[0122] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3" includes 3 ± 5%, or from 2.85 to 3.15.

[0123] In a preferred embodiment, the mass of the forsythia ranges from 1 to 5 g, for example, about 3 g.

[0124] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3" includes 3 ± 5%, or from 2.85 to 3.15.

[0125] In a preferred embodiment, the weight of the platycodon root ranges from 1 to 5 g, for example, about 3 g.

[0126] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3" includes 3 ± 5%, or from 2.85 to 3.15.

[0127] In a preferred embodiment, the patchouli is in the range of 1 to 5 g, for example, about 3 g.

[0128] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3" includes 3 ± 5%, or from 2.85 to 3.15.

[0129] In a preferred embodiment, the licorice has a mass range of 1 to 5 g, for example, about 3 g.

[0130] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3" includes 3 ± 5%, or from 2.85 to 3.15.

[0131] In a preferred embodiment, the solvent in the preparation of the traditional Chinese medicine compound test solution is an alcohol, such as methanol.

[0132] In a preferred embodiment, the volume percentage concentration of methanol is 20% to 50%, for example, about 35%.

[0133] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 35%" includes 35% ± 5%, or from 33.25% to 36.75%.

[0134] In a preferred embodiment, the container is a measuring bottle.

[0135] In a preferred embodiment, the mass of the traditional Chinese medicine compound powder is 0.1 to 1 g, for example, about 0.5 g.

[0136] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.5" includes ±5% of 0.5, or from 0.475 to 0.525.

[0137] In a preferred embodiment, the power of the ultrasonic treatment is 400 to 600 W, for example, about 500 W.

[0138] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 500" includes ±5% of 500, or from 475 to 525.

[0139] In a preferred embodiment, the frequency of the ultrasonic treatment is 30 to 50 kHz, for example, about 40 kHz.

[0140] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

[0141] In a preferred embodiment, the ultrasonic treatment time is 20 to 40 minutes, for example, about 30 minutes.

[0142] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, or from 28.5 to 31.5.

[0143] In a preferred embodiment, the mass / volume ratio between the traditional Chinese medicine compound powder and the traditional Chinese medicine compound test solution is 0.01 to 0.1, for example, about 0.02, in g / ml.

[0144] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.02" includes ±5% of 0.02, or from 0.019 to 0.021.

[0145] In a preferred embodiment, the pretreatment method for the baicalin reference standard and the ammonium glycyrrhizate reference standard is as follows: the baicalin reference standard and the ammonium glycyrrhizate reference standard are dried under reduced pressure at about 60°C for about 4 hours.

[0146] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 60" includes ±5% of 60, or from 57 to 63; "about 4" includes ±5% of 4, or from 3.8 to 4.2.

[0147] In a preferred embodiment, the concentration of baicalin in the first reference solution is about 0.3 mg / ml.

[0148] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.3" includes ±5% of 0.3, or from 0.285 to 0.315.

[0149] In a preferred embodiment, the concentration of ammonium glycyrrhizate in the first reference solution is about 0.04 mg / ml.

[0150] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.04" includes ±5% of 0.04, or from 0.038 to 0.042.

[0151] In a preferred embodiment, the concentration of Lonicera japonica saponin B in the second reference solution is about 0.3 mg / ml.

[0152] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.3" includes ±5% of 0.3, or from 0.285 to 0.315.

[0153] In a preferred embodiment, the concentration of bisacodyl B in the second reference solution is about 0.03 mg / ml.

[0154] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.03" includes ±5% of 0.03, or from 0.0285 to 0.0315.

[0155] In a preferred embodiment, the methanol volume percentage concentration in the preparation of the second reference solution is 40% to 60%, for example, about 50%.

[0156] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50%" includes 50% ± 5%, or from 47.5% to 52.5%.

[0157] In a preferred embodiment, the flow rate is 0.7 to 0.9 ml / min, for example, about 0.8 ml / min.

[0158] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.8" includes ±5% of 0.8, or from 0.76 to 0.84.

[0159] In a preferred embodiment, the column temperature is 28–32°C, for example, about 30°C.

[0160] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, or from 28.5 to 31.5.

[0161] In a preferred embodiment, when using ultraviolet detection, the detection wavelength is 200-300 nm, for example, 245 nm.

[0162] In a preferred embodiment, when using ultraviolet detection, the injection volume of the test solution is 5 to 15 μl, for example, about 10 μl.

[0163] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0164] In a preferred embodiment, when using an evaporative light scattering detector, the injection volume of the test solution is 5–10 μl.

[0165] In a preferred embodiment, the injection volume of the first reference solution is 5 to 15 μl, for example, about 10 μl.

[0166] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0167] In a preferred embodiment, the injection volume of the second reference solution is about 5 μl or about 20 μl.

[0168] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5" includes 5 ± 5%, or from 4.75 to 5.25; "about 20" includes 20 ± 5%, or from 19 to 21.

[0169] In a preferred embodiment, the chromatographic column is a HALO column. AQ-C18 chromatographic column.

[0170] In a preferred embodiment, the chromatographic column has the following specifications: column length 150 mm, inner diameter 4.6 mm, and particle size 2.7 μm.

[0171] In a preferred embodiment, the mobile phase A is acetonitrile.

[0172] In a preferred embodiment, the acid aqueous solution, alkaline aqueous solution, and / or buffer salt aqueous solution are selected from one or more weak acids and their salts, and weak bases and their salts of different concentrations.

[0173] In a preferred embodiment, the acidic aqueous solution, alkaline aqueous solution, and / or buffer salt aqueous solution is selected from formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid.

[0174] In a preferred embodiment, the acidic aqueous solution is a 0.01% to 1% acidic aqueous solution.

[0175] In a preferred embodiment, the acidic aqueous solution is a 0.01% to 1% formic acid aqueous solution.

[0176] In a preferred embodiment, the acidic aqueous solution is an aqueous solution of about 0.1% formic acid.

[0177] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.1%" includes 0.1% ± 5%, or from 0.095% to 0.105%.

[0178] In a preferred embodiment, the buffer salt solution is an acetate aqueous solution and / or an acetate aqueous solution.

[0179] In a preferred embodiment, the pH value of the buffer saline solution is not greater than 7.0.

[0180] In a preferred embodiment, the detection limit of baicalin is about 0.0004553 mg / ml.

[0181] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.0004553" includes ±5% of 0.0004553, or from 0.000432535 ​​to 0.000478065.

[0182] In a preferred embodiment, the detection limit of glycyrrhizic acid is about 0.0001296 mg / ml.

[0183] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.0001296" includes ±5% of 0.0001296, or from 0.00012312 to 0.00013608.

[0184] In a preferred embodiment, the detection limit of the honeysuckle saponin B is about 0.002066 mg / ml.

[0185] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.002066" includes ±5% of 0.002066, or from 0.0019627 to 0.0021693.

[0186] In a preferred embodiment, the detection limit of the dipsaponin B is about 0.001751 mg / ml.

[0187] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.001751" includes ±5% of 0.001751, or from 0.00166345 to 0.00183855.

[0188] In a preferred embodiment, the limit of quantification for baicalin is about 0.001339 mg / ml.

[0189] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.001339" includes ±5% of 0.001339, or from 0.00127205 to 0.00140595.

[0190] In a preferred embodiment, the limit of quantification for glycyrrhizic acid is about 0.0003239 mg / ml.

[0191] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.0003239" includes ±5% of 0.0003239, or from 0.000307705 to 0.000340095.

[0192] In a preferred embodiment, the limit of quantification for the honeysuckle saponin B is about 0.006198 mg / ml.

[0193] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.006198" includes ±5% of 0.006198, or from 0.0058881 to 0.0065079.

[0194] In a preferred embodiment, the limit of quantification of the dipsaponin B is about 0.006180 mg / ml.

[0195] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.006180" includes ±5% of 0.006180, or from 0.005871 to 0.006489.

[0196] According to another aspect of the present invention, the above-described assay method is provided for use in the quality detection and / or quality evaluation and / or quality control of traditional Chinese medicine compound containing honeysuckle, isatis root and scutellaria.

[0197] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0198] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0199] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0200] Example

[0201] 1. Experimental Materials and Equipment

[0202] 1.1 Experimental Instruments and Equipment

[0203] Information on experimental instruments and equipment is shown in Table 1.

[0204] Table 1 Information on Experimental Instruments and Equipment

[0205]

[0206]

[0207] 1.2 Experimental Reagents

[0208] The experimental reagent information is shown in Table 2.

[0209] Table 2. Experimental Reagent Information

[0210] Equipment Name level factory methanol HPLC Sigma Acetonitrile HPLC Sigma Formic acid HPLC Tianjin Kemio Chemical Reagent Co., Ltd. water Purified water self made

[0211] 1.3 Experimental Control Information

[0212] The experimental control information is shown in Table 3.

[0213] Table 3. Experimental Control Information Table

[0214] name purity% batch number factory ammonium glycyrrhizate 93.2 110731-202423 China National Institutes for Food and Drug Control baicalin 97.2 110715-202223 China National Institutes for Food and Drug Control Honeysuckle saponin B 96.1 111814-202106 China National Institutes for Food and Drug Control Dipsacus saponin B 96.8 111813-201804 China National Institutes for Food and Drug Control

[0215] 1.4. Information on medicinal slices

[0216] Information on the processed medicinal slices is shown in Table 4.

[0217] Table 4. Information on processed medicinal slices

[0218]

[0219]

[0220] 2. Preparation method

[0221] A traditional Chinese medicine compound sample powder containing honeysuckle, isatis root, and scutellaria: Take 5g of honeysuckle, 3g of scutellaria, 3g of indigo leaf, 3g of forsythia, 3g of platycodon, 3g of patchouli, and 3g of licorice, place them in a 0.5ml round-bottom flask, add 12 times the amount of water for the first extraction, and extract for 90 minutes. Add 10 times the amount of water for the second extraction, and extract for 60 minutes. Filter while hot using a 200-mesh filter cloth, combine the extracts, and dry to obtain the final product.

[0222] Isatis root negative sample solution: Take 5g of honeysuckle, 3g of scutellaria, 3g of indigo leaf, 3g of forsythia, 3g of platycodon, 3g of patchouli, and 3g of licorice, and place them in a 0.5ml round-bottom flask. Add 12 times the amount of water for the first extraction and extract for 90 minutes. Add 10 times the amount of water for the second extraction and extract for 60 minutes. Filter the solution while hot using a 200-mesh filter cloth. Combine the extracts to obtain the final solution.

[0223] Negative sample solution of honeysuckle: Take 5g of Isatis root, 3g of Scutellaria baicalensis, 3g of Isatis leaf, 3g of Forsythia suspensa, 3g of Platycodon grandiflorus, 3g of Pogostemon cablin, and 3g of Glycyrrhiza uralensis, and place them in a 0.5ml round-bottom flask. Add 12 times the amount of water for the first extraction and extract for 90 minutes. Add 10 times the amount of water for the second extraction and extract for 60 minutes. Filter the solution while hot using a 200-mesh filter cloth. Combine the extracts to obtain the final solution.

[0224] Scutellaria baicalensis negative sample solution: Take 5g of honeysuckle, 5g of isatis root, 3g of indigo leaf, 3g of forsythia, 3g of platycodon, 3g of patchouli, and 3g of licorice, and place them in a 0.5ml round-bottom flask. Add 12 times the amount of water for the first extraction and extract for 90 minutes. Add 10 times the amount of water for the second extraction and extract for 60 minutes. Filter the solution while hot using a 200-mesh filter cloth. Combine the extracts to obtain the final solution.

[0225] Negative sample solution of Isatis indigotica: Take 5g of Lonicera japonica, 5g of Isatis tinctoria, 3g of Scutellaria baicalensis, 3g of Forsythia suspensa, 3g of Platycodon grandiflorus, 3g of Pogostemon cablin, and 3g of Glycyrrhiza uralensis, and place them in a 0.5ml round-bottom flask. Add 12 times the amount of water for the first extraction and extract for 90 minutes. Add 10 times the amount of water for the second extraction and extract for 60 minutes. Filter the solution while hot using a 200-mesh filter cloth. Combine the extracts to obtain the final solution.

[0226] Forsythia negative sample solution: Take 5g of honeysuckle, 5g of isatis root, 3g of scutellaria, 3g of indigo leaf, 3g of platycodon, 3g of patchouli, and 3g of licorice, and place them in a 0.5ml round-bottom flask. Add 12 times the amount of water for the first extraction and extract for 90 minutes. Add 10 times the amount of water for the second extraction and extract for 60 minutes. Filter the solution while hot using a 200-mesh filter cloth. Combine the extracts to obtain the final solution.

[0227] Negative sample solution for Platycodon grandiflorus: Take 5g of Lonicera japonica, 5g of Isatis indigotica, 3g of Scutellaria baicalensis, 3g of Isatis tinctoria, 3g of Forsythia suspensa, 3g of Pogostemon cablin, and 3g of Glycyrrhiza uralensis, and place them in a 0.5ml round-bottom flask. Add 12 times the amount of water for the first extraction and extract for 90 minutes. Add 10 times the amount of water for the second extraction and extract for 60 minutes. Filter the solution while hot using a 200-mesh filter cloth. Combine the extracts to obtain the final solution.

[0228] Patchouli negative sample solution: Take 5g of honeysuckle, 5g of isatis root, 3g of scutellaria, 3g of indigo leaf, 3g of forsythia, 3g of platycodon, and 3g of licorice, and place them in a 0.5ml round-bottom flask. Add 12 times the amount of water for the first extraction and extract for 90 minutes. Add 10 times the amount of water for the second extraction and extract for 60 minutes. Filter the solution while hot using a 200-mesh filter cloth. Combine the extracts to obtain the final solution.

[0229] Licorice negative sample solution: Take 5g of honeysuckle, 5g of isatis root, 3g of scutellaria, 3g of indigo leaf, 3g of forsythia, 3g of platycodon, and 3g of patchouli, and place them in a 0.5ml round-bottom flask. Add 12 times the amount of water for the first extraction and extract for 90 minutes. Add 10 times the amount of water for the second extraction and extract for 60 minutes. Filter the solution while hot using a 200-mesh filter cloth. Combine the extracts to obtain the final solution.

[0230] Isatis root single-herb sample solution: Take about 0.7g of Isatis root slices, add 50ml of water and reflux for 30 minutes to extract.

[0231] Honeysuckle single-herb sample solution: Take about 0.7g of honeysuckle slices, add 50ml of water and reflux for 30 minutes to obtain the solution.

[0232] Scutellaria baicalensis single-herb sample solution: Take about 0.4g of Scutellaria baicalensis slices, add 50ml of water and reflux for 30 minutes to obtain the solution.

[0233] Single-herb sample solution of Isatis tinctoria leaf: Take about 0.4g of Isatis tinctoria leaf slices, add 50ml of water and reflux for 30 minutes to obtain the solution.

[0234] Forsythia suspensa single-herb sample solution: Take about 0.4g of Forsythia suspensa slices, add 50ml of water and reflux for 30 minutes to extract.

[0235] Single-herb sample solution of Platycodon grandiflorus: Take about 0.4g of Platycodon grandiflorus slices, add 50ml of water and reflux for 30 minutes to obtain the solution.

[0236] Patchouli single-herb sample solution: Take about 0.4g of patchouli slices, add 50ml of water and reflux for 30 minutes to extract.

[0237] Licorice single-herb sample solution: Take about 0.4g of licorice slices, add 50ml of water and reflux for 30 minutes to extract.

[0238] 3. Screening of chromatographic conditions

[0239] Table 5 Elution conditions 1

[0240]

[0241]

[0242] The results showed that under elution condition 1, the chromatogram of the test sample was relatively uniformly distributed in the UV chromatogram; in the evaporative light chromatogram, most peaks in the chromatogram of the test sample were eluted later, and the separation of honeysuckle saponin B and dipsacus saponin B was poor. Therefore, the chromatographic conditions were optimized.

[0243] Table 6 Elution Conditions 2

[0244] Time (min) Acetonitrile (%) 0.1% Formic acid (%) 0～15 5 95 15～40 5→15 15→85 40～55 15→16 85→84 55～75 16→24 84→76 75～95 24→60 76→40 95～100 60→90 40→10 100～105 90→5 10→95

[0245] The results showed that under elution condition 2, the chromatogram of the test sample was relatively uniform in the UV chromatogram, but the peak shape was poor and the overall resolution was poor. In the evaporative light chromatogram, the resolution of Lonicera japonica saponin B and Dipsacus asperoides saponin B was still poor. Therefore, the chromatographic conditions were further optimized.

[0246] Table 7 Elution Conditions 3

[0247]

[0248]

[0249] The results showed that under elution condition 3, the chromatograms of the test sample were relatively uniformly distributed and had good peak shapes in both the UV and evaporative light chromatograms. The separation of Lonicera japonica saponin B and Dipsacus asperoides saponin B was improved, with no obvious peaks. However, the overall separation was still poor and the overall analysis time was too long, resulting in a significant waste of the instrument and organic solvents. Therefore, the chromatographic conditions should be further optimized to shorten the analysis time.

[0250] Table 8 Elution Conditions 4

[0251] Time (min) Acetonitrile (%) 0.1% Formic acid (%) 0～25 19→20 81→80 25～45 20→50 80→50 45～50 50→80 50→20 50～55 80→19 20→81 55～60 19 81

[0252] The results showed that under elution condition 4 (which halved the running time compared to elution conditions 1-3), the peak shapes of each content index were good in both the UV chromatogram and the evaporative light chromatogram. The chromatographic system applicability parameters all met the relevant requirements for content determination by high performance liquid chromatography. In particular, the separation of honeysuckle saponin B and dipsacus saponin B was excellent. Therefore, elution condition 4 in Table 8 can be used as an analytical method for content determination.

[0253] 4. Method to be verified

[0254] Based on the above experimental screening results, the following methods are now identified for verification:

[0255] Scutellaria baicalensis, Glycyrrhiza uralensis, and Lonicera japonica were determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020).

[0256] Chromatographic conditions and system suitability parameters: Using octadecyl-bonded silica gel as the stationary phase (HALO) AQ-C18 (2.7 μm, 4.6 × 150 mm); using acetonitrile as mobile phase A and 0.1% formic acid as mobile phase B, gradient elution was performed according to the specifications in Table 8; the flow rate was 0.8 ml per minute; the column temperature was 30℃; baicalin and glycyrrhizic acid were detected at a wavelength of 245 nm, while honeysuckle saponin B and dipsacus saponin B were detected using an evaporative light scattering detector.

[0257] Preparation of reference solution: Accurately weigh appropriate amounts of baicalin reference standard and ammonium glycyrrhizate reference standard dried under reduced pressure at 60℃ for 4 hours, and add methanol to prepare a mixed solution containing 0.3 mg of baicalin and 0.04 mg of ammonium glycyrrhizate per ml. Accurately weigh appropriate amounts of honeysuckle saponin B reference standard and dipsacus saponin B reference standard, and add 50% methanol to prepare a mixed solution containing 0.3 mg of honeysuckle saponin B and 0.03 mg of dipsacus saponin B per ml. (Weight of glycyrrhizic acid = weight of ammonium glycyrrhizate / 1.0207).

[0258] Preparation of test solution: Take about 0.5g of the powder of the traditional Chinese medicine compound containing honeysuckle, isatis root and scutellaria, put it in a 25ml volumetric flask, add an appropriate amount of 35% methanol, sonicate (power 500w, frequency 40kHz) for 30 minutes, cool, add 35% methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0259] The assay method involves precisely injecting 10 μl of baicalin and glycyrrhizic acid reference solutions, 5 μl and 20 μl of honeysuckle saponin B and dipsacus saponin B reference solutions, 10 μl of the test solution under UV detection conditions, and 5–10 μl of the test solution under evaporative light detection conditions, into a liquid chromatograph for determination. The contents of baicalin and glycyrrhizic acid are calculated using the blank external standard method, and the contents of honeysuckle saponin B and dipsacus saponin B are calculated using the logarithmic equation of the external standard two-point method.

[0260] 5. Verification Content

[0261] 5.1 Content Methodology Validation

[0262] 5.1.1 Specificity

[0263] The blank solvent, reference solution, and the test solution of the traditional Chinese medicine compound containing honeysuckle, isatis root, and scutellaria (where the results of ultraviolet detection are as follows) were prepared. Figure 1 As shown, the results detected using an evaporative light scattering detector are as follows: Figure 2 As shown), each single-herb test solution and each negative test solution were injected and tested. The chromatograms were recorded, and the peak positions of baicalin, glycyrrhizic acid, honeysuckle saponin B, and dipsacus saponin B were recorded. If there is no interference from the blank solvent and negative control, the specificity is strong.

[0264] The results showed that, under UV detection conditions, the chromatograms of Scutellaria baicalensis and Glycyrrhiza uralensis alone and the chromatograms of the test samples showed corresponding peaks at the retention times corresponding to the reference standards of baicalin and glycyrrhizic acid, with no interference from the blank solution and the negative test solution of Scutellaria baicalensis and Glycyrrhiza uralensis. Under evaporative light detection conditions, the chromatograms of Lonicera japonica alone and the chromatograms of the test samples showed corresponding peaks at the retention times corresponding to the reference standards of Lonicera japonica saponin B and Dipsacus asperoides saponin B, with no interference from the blank solution and the negative test solution of Lonicera japonica. In summary, this method has good specificity for the content determination index components.

[0265] 5.1.2 Precision

[0266] 5.1.2.1 Instrument Precision

[0267] Prepare one test solution according to the above-described method, and inject it six times consecutively. Compare the peak areas and retention times of baicalin, glycyrrhizic acid, honeysuckle saponin B, and dipsacus saponin B. If the RSD of the retention time of baicalin and glycyrrhizic acid is ≤2%, and the RSD of the peak area is ≤2%, and the RSD of the retention time of honeysuckle saponin B and dipsacus saponin B is ≤2%, and the RSD of the peak area is ≤5%, then the instrument precision is good.

[0268] The results showed that the RSD of the retention time of baicalin and glycyrrhizic acid peaks was ≤2%, and the RSD of the peak area was ≤2%. The RSD of the retention time of honeysuckle saponin B and dipsacus saponin B peaks was ≤2%, and the RSD of the peak area was ≤5%, indicating that the instrument precision was good.

[0269] 5.1.2.2 Repeatability

[0270] Six parallel samples were prepared from the same batch of granules using the same method for preparing the test solution described above. Each sample was injected and analyzed, and the chromatograms were recorded. The total contents of baicalin, glycyrrhizic acid, honeysuckle saponin B, and dipsacus saponin B were calculated. If the RSD of baicalin and glycyrrhizic acid content in the six samples was ≤2%, and the RSD of the total contents of honeysuckle saponin B and dipsacus saponin B was ≤5%, then the repeatability was good.

[0271] The results showed that the RSD of baicalin and glycyrrhizic acid content in the six samples was ≤2%, and the RSD of total Lonicera japonica saponin B and Dipsacus asperoides saponin B was ≤5%, indicating good reproducibility.

[0272] 5.1.2.3 Intermediate Precision

[0273] Following the above-described method for preparing the test solution, six test solutions were prepared by different personnel. These solutions were then analyzed using an Agilent liquid chromatograph and a Waters liquid chromatograph, respectively. Chromatograms were recorded, and the total contents of baicalin, glycyrrhizic acid, honeysuckle saponin B, and dipsacus saponin B were calculated. If the RSD of baicalin and glycyrrhizic acid in the 12 samples was ≤2%, and the RSD of the total contents of honeysuckle saponin B and dipsacus saponin B was ≤5%, then the intermediate precision was good.

[0274] The results showed that the RSD of baicalin and glycyrrhizic acid content in 12 samples was ≤2% on different instruments, and the RSD of total Lonicera japonica saponin B and Dipsacus asperoides saponin B was ≤5%, indicating good intermediate precision.

[0275] 5.1.3 Sensitivity (Limit of Detection and Limit of Quantification)

[0276] The detection limit refers to the lowest concentration of a substance in a sample that can be qualitatively detected but does not need to be accurately quantified. In chromatographic analysis, the signal-to-noise ratio (S / N) is generally used to define the detection limit. The S / N is the ratio of the response value of the target analyte in the sample to the baseline noise. It is generally considered that an S / N greater than or equal to 3 can be used for qualitative analysis, and an S / N greater than or equal to 10 can be used for quantitative analysis.

[0277] The results showed that the limit of detection (LOD) for baicalin was 0.0004553 mg / ml, with an S / N value of 3.26; the limit of quantification (LOQ) for baicalin was 0.001339 mg / ml, with peak area and RSD% of signal-to-noise ratio (S / N) both ≤10%; the limit of detection (LOD) for glycyrrhizic acid was 0.0001296 mg / ml, with an S / N value of 3.79; the limit of quantification (LOQ) for glycyrrhizic acid was 0.0003239 mg / ml, with peak area and RSD% of signal-to-noise ratio (S / N) both ≤10%; and the *Lonicera japonica* soap was also tested. The detection limit for glycoside B was 0.002066 mg / ml, with an S / N value of 3.05; the quantitation limit for glycoside B from Lonicera japonica was 0.006198 mg / ml, with peak area and RSD% of signal-to-noise ratio (S / N) both ≤10%; the detection limit for glycoside B from Dipsacus asperoides was 0.001751 mg / ml, with an S / N value of 3.05; the quantitation limit for glycoside B from Dipsacus asperoides was 0.006180 mg / ml, with peak area and RSD% of signal-to-noise ratio (S / N) both ≤10%; all met the requirements.

[0278] 5.1.4 Linear

[0279] Prepare at least five reference solutions at different concentrations for determination. Plot the measured response signal as a function of the analyte concentration, observe whether it is linear, and then perform linear regression using the least squares method. The correlation coefficient R0 is calculated. 2 A value not less than 0.99 indicates that the method has good linearity.

[0280] The experimental results are shown in Tables 9, 10, 11 and 12.

[0281] Table 9. Linear experimental results of baicalin content.

[0282]

[0283] Table 10. Results of linear experiments on glycyrrhizic acid content

[0284]

[0285] Table 11. Linear experimental results of saponin B content in Lonicera macrantha.

[0286]

[0287] Table 12. Linear experimental results of Dipsacus asperoides content.

[0288]

[0289] The results showed that the regression equation for baicalin was y = 18728134.4265x - 77989.4438, with R = 0.9996, indicating a good linear relationship for baicalin concentrations within the range of 0.001339–1.033 mg / ml. The regression equation for glycyrrhizic acid was y = 11474520.1348x + 8136.4234, with R = 1.0000, indicating a good linear relationship for glycyrrhizic acid concentrations within the range of 0.0008097–0.4763 mg / ml. The linear relationship was good; the regression equation for Lonicera japonica saponin B was y = 1.5881x + 7.4300, R = 0.9950, and the regression equation for Dipsacus asperoides saponin B was y = 1.7258x + 7.7011, R = 0.9979. Since Lonicera japonica saponin B and Dipsacus asperoides saponin B were detected under evaporative light conditions, according to the "Research Technology for Quality Standards of Traditional Chinese Medicine Preparations", the linear correlation coefficient R of HPLC-ELSD is > 0.990, indicating that the method has good linearity.

[0290] 5.1.5 Durability

[0291] 5.1.5.1 Stability

[0292] Prepare one test solution according to the above-described preparation method, and inject it for detection at 0, 4, 8, 12, 24, 36, 48, and 80 hours. Record the chromatograms and calculate the RSD values ​​of the retention times and peak areas of baicalin, glycyrrhizic acid, honeysuckle saponin B, and dipsacus saponin B. If the RSD of the retention time of baicalin and glycyrrhizic acid is ≤2% and the RSD of the peak area is ≤2%, and the RSD of the retention time of honeysuckle saponin B and dipsacus saponin B is ≤2% and the RSD of the peak area is ≤5%, then the test solution has good stability.

[0293] The results showed that the RSD of the retention time of baicalin and glycyrrhizic acid peaks was ≤2%, and the RSD of the peak area was ≤2%. The RSD of the retention time of honeysuckle saponin B and dipsacus saponin B peaks was ≤2%, and the RSD of the peak area was ≤5%. The test solution showed good stability within 80 h.

[0294] 5.1.5.2 Flow rate durability

[0295] Two test solutions were prepared according to the above-described test solution preparation method, with flow rates set to 0.7 ml / min, 0.8 ml / min, and 0.9 ml / min, respectively. The solutions were injected and analyzed, and the chromatograms were recorded. The total contents of baicalin, glycyrrhizic acid, honeysuckle saponin B, and dipsacus saponin B were calculated. If the RSD of baicalin and glycyrrhizic acid content is ≤2%, and the RSD of the total contents of honeysuckle saponin B and dipsacus saponin B is ≤5%, then the flow rate robustness is good.

[0296] The results showed that under different flow rate conditions, the RSD of baicalin and glycyrrhizic acid content was ≥2%, and the RSD of the total amount of honeysuckle saponin B and dipsacus saponin B was ≤5%, indicating that the change of flow rate had a significant impact on the content of baicalin and glycyrrhizic acid. Therefore, 0.8 ml / min was fixed as the flow rate for determining the content of traditional Chinese medicine compound containing honeysuckle, isatis root and scutellaria.

[0297] 5.1.5.3 Column Temperature Durability

[0298] Two test solutions were prepared according to the above-described test solution preparation method. The column temperatures were set to 28℃, 30℃, and 32℃, respectively. The samples were injected and detected, and the chromatograms were recorded. The total contents of baicalin, glycyrrhizic acid, honeysuckle saponin B, and dipsacus saponin B were calculated. If the RSD of baicalin and glycyrrhizic acid content is ≤2%, and the RSD of the total contents of honeysuckle saponin B and dipsacus saponin B is ≤5%, it indicates that the column temperature durability is good.

[0299] The results showed that under different column temperatures, the RSD of baicalin and glycyrrhizic acid content was ≥2%, and the RSD of the total amount of honeysuckle saponin B and dipsacus saponin B was ≤5%. This indicates that changes in column temperature have a significant impact on the content of baicalin and glycyrrhizic acid. Therefore, 30℃ was fixed as the column temperature for determining the content of traditional Chinese medicine compound containing honeysuckle, isatis root and scutellaria.

[0300] 5.1.5.4. Formic acid concentration durability of the mobile phase

[0301] Two test solutions were prepared according to the above-described method. Formic acid solutions of 0.08%, 0.10%, and 0.12% were used as mobile phases, respectively. The solutions were injected and analyzed, and the chromatograms were recorded. The total contents of baicalin, glycyrrhizic acid, honeysuckle saponin B, and dipsacus saponin B were calculated. If the RSD of baicalin and glycyrrhizic acid contents is ≤2%, and the RSD of the total contents of honeysuckle saponin B and dipsacus saponin B is ≤5%, it indicates that the solution is robust to different formic acid concentrations.

[0302] The results showed that the RSD of baicalin and glycyrrhizic acid content was ≤2%, and the RSD of total Lonicera japonica saponin B and Dipsacus asperoides saponin B was ≤5%, indicating good durability at different formic acid concentrations.

[0303] 5.1.5.5 Durability of chromatographic columns from different manufacturers

[0304] Two test solutions were prepared according to the above-described test solution preparation method. Three different chromatographic columns were used for injection and detection. The total contents of baicalin, glycyrrhizic acid, honeysuckle saponin B, and dipsacus saponin B were calculated. If the RSD of baicalin and glycyrrhizic acid content is ≤2%, and the RSD of the total contents of honeysuckle saponin B and dipsacus saponin B is ≤5%, it indicates that the different chromatographic columns have good durability.

[0305] The results showed that the RSD of baicalin and glycyrrhizic acid content was ≤2%, and the RSD of total Lonicera japonica saponin B and Dipsacus asperoides saponin B was ≤5%, indicating that the chromatographic columns from different manufacturers had good durability. However, based on the results of characteristic chromatogram 2, further investigation was needed on different batches of chromatographic columns.

[0306] 5.1.5.6 Durability of Columns from Different Batch Numbers

[0307] Prepare a test solution according to the conditions defined above, and use three HALO tubes from different batches. Using an AQ-C18 2.7μm 4.6*150mm column, sample was injected and detected. Chromatograms were recorded, and the total contents of baicalin, glycyrrhizic acid, honeysuckle saponin B, and dipsacus saponin B were calculated. If the RSD of baicalin and glycyrrhizic acid content is ≤2%, and the RSD of the total contents of honeysuckle saponin B and dipsacus saponin B is ≤5%, it indicates that the different chromatographic columns have good robustness.

[0308] The results showed that the RSD of baicalin content was ≤2%, the RSD of glycyrrhizic acid content was ≤3% (according to the analytical method validation guidelines, the RSD of the analyte content was ≤3% when the content of the analyte was 1-10 mg / g), and the RSD of the total amount of honeysuckle saponin B and dipsacus saponin B was ≤5%, indicating that different chromatographic columns had good robustness.

[0309] 5.1.6 Content Confirmation Method

[0310] This method is used to determine the total content of baicalin, glycyrrhizic acid, honeysuckle saponin B, and dipsacus saponin B in a traditional Chinese medicine compound containing honeysuckle, isatis root, and scutellaria baicalensis. Method validation demonstrated good precision, stability, specificity, and column robustness. In the robustness test, changes in flow rate and column temperature significantly affected the content of the indicator components. Therefore, a fixed flow rate, column temperature, and column (HALO AQ-C18 2.7μm 4.6x150mm) were established. In summary, the established method meets the relevant requirements for content determination and is suitable for the content determination of traditional Chinese medicine compound containing honeysuckle, isatis root, and scutellaria baicalensis. Specific conditions are as follows:

[0311] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0312] Chromatographic conditions and system suitability parameters: Using octadecyl-bonded silica gel as the stationary phase (HALO) AQ-C18 (2.7 μm, 4.6 × 150 mm); using acetonitrile as mobile phase A and 0.1% formic acid as mobile phase B, gradient elution was performed according to the specifications in Table 8; the flow rate was 0.8 ml per minute; the column temperature was 30℃; baicalin and glycyrrhizic acid were detected at a wavelength of 245 nm, while honeysuckle saponin B and dipsacus saponin B were detected using an evaporative light scattering detector.

[0313] Preparation of reference solution: Accurately weigh appropriate amounts of baicalin reference standard and ammonium glycyrrhizate reference standard (dried under reduced pressure at 60℃ for 4 hours), and add methanol to prepare a mixed solution containing 0.3 mg of baicalin and 0.04 mg of ammonium glycyrrhizate per ml. Accurately weigh appropriate amounts of honeysuckle saponin B reference standard and dipsacus saponin B reference standard, and add 50% methanol to prepare a mixed solution containing 0.3 mg of honeysuckle saponin B and 0.03 mg of dipsacus saponin B per ml. (Weight of glycyrrhizic acid = weight of ammonium glycyrrhizate / 1.0207)

[0314] Preparation of test solution: Take about 0.5g of this product, place it in a 25ml volumetric flask, add an appropriate amount of 35% methanol, sonicate (power 500w, frequency 40kHz) for 30 minutes, cool, add 35% methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0315] The assay method involves precisely injecting 10 μl of baicalin and glycyrrhizic acid reference solutions, 5 μl and 20 μl of honeysuckle saponin B and dipsacus saponin B reference solutions, 10 μl of the test solution under UV detection conditions, and 5–10 μl of the test solution under evaporative light detection conditions, into a liquid chromatograph for determination. The contents of baicalin and glycyrrhizic acid are calculated using the blank external standard method, and the contents of honeysuckle saponin B and dipsacus saponin B are calculated using the logarithmic equation of the external standard two-point method.

[0316] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for determining the content of a traditional Chinese medicine compound containing honeysuckle, isatis root, and scutellaria, characterized in that, The determination method includes the following steps: Preparation of the test solution of the traditional Chinese medicine compound: Take an appropriate amount of the traditional Chinese medicine compound powder containing honeysuckle, isatis root and scutellaria, place it in a container, add solvent, sonicate, make up to volume, shake well, filter, and take the filtrate to obtain the test solution of the traditional Chinese medicine compound; wherein, the traditional Chinese medicine compound includes honeysuckle, isatis root, scutellaria, indigo leaf, forsythia, platycodon, patchouli and licorice; Preparation of the first reference solution: Weigh appropriate amounts of baicalin reference standard and ammonium glycyrrhizate reference standard, add methanol to prepare the first reference solution with a reference standard concentration of 0.01-1 mg / ml; Preparation of the second reference solution: Weigh appropriate amounts of honeysuckle saponin B reference standard and dipsacus saponin B reference standard, add methanol to prepare the second reference solution with a reference standard concentration of 0.01-1 mg / ml. Take the test solution of the traditional Chinese medicine compound, the first reference solution, and the second reference solution for testing. The chromatographic conditions for the detection were as follows: a column packed with octadecyl-bonded silica gel was used; mobile phase A was selected from one or more of acetonitrile, methanol, and tetrahydrofuran; mobile phase B was an aqueous acid solution, an aqueous alkaline solution, and / or an aqueous buffer solution; the gradient elution program was as follows: 0–25 min, 81% B → 80% B; 25–45 min, 80% B → 50% B; 45–50 min, 50% B → 20% B; 50–55 min, 20% B → 81% B; 55–60 min, 81% B; the flow rate was 0.5–1.0 ml / min; the column temperature was 25–35 °C; and the injection volume was 1–25 μl. Baicalin and glycyrrhizic acid were detected using ultraviolet light, while Lonicera japonica saponin B and Dipsacus asperoides saponin B were detected using an evaporative light scattering detector.

2. The determination method according to claim 1, characterized in that, The information is obtained by calculating the contents of baicalin and glycyrrhizic acid reference standards in the traditional Chinese medicine compound according to the corresponding peak areas in the chromatograms of the test solution and the reference solution, using the blank external standard method, and by calculating the contents of honeysuckle saponin B and dipsacus saponin B reference standards in the traditional Chinese medicine compound according to the logarithmic equation of the two-point external standard method. Preferably, the mass of glycyrrhizic acid is calculated based on the mass of ammonium glycyrrhizate, and the mass ratio of ammonium glycyrrhizate to glycyrrhizic acid is approximately 1.0207; Preferably, the linear equation for scutellarin is y = 18728134.4265x - 77989.4438, R 2 =0.9992; Preferably, the linear equation for the glycyrrhizic acid is y = 11474520.1348x + 8136.4234, R 2 =1.0000; Preferably, the linear equation for the honeysuckle saponin B is y = 1.5881x + 7.4300, R 2 =0.9901; Preferably, the linear equation for the saponin B of Dipsacus asperoides is y = 1.7258x + 7.7011, R 2 =0.9958.

3. The determination method according to claim 1, characterized in that, The preparation method of the traditional Chinese medicine compound powder containing honeysuckle, isatis root, and scutellaria includes: (a) weighing appropriate amounts of honeysuckle, isatis root, scutellaria, indigo leaf, forsythia, platycodon, patchouli, and licorice, adding water for the first heating extraction, filtering to obtain a first filtrate and a residue; (b) adding water for the residue for the second heating extraction, filtering to obtain a second filtrate; (c) combining the first filtrate and the second filtrate, drying to obtain the traditional Chinese medicine compound powder. Preferably, in step (a), the volume / mass (ml / g) ratio of the first water to the sum of the masses of honeysuckle, isatis root, scutellaria, indigo leaf, forsythia, platycodon, patchouli and licorice is 5 to 15, for example about 12; Preferably, in step (a), the first heating extraction time is 60–120 min, for example, about 90 min; Preferably, in step (a), the filtration is performed while the filter is hot using a 200-mesh filter cloth; More preferably, in step (b), the volume / mass (ml / g) ratio of the second water to the sum of the masses of honeysuckle, isatis root, scutellaria, indigo leaf, forsythia, platycodon, patchouli and licorice is 5 to 15, for example about 10; More preferably, in step (b), the second heating extraction time is 30 to 90 minutes, for example, about 60 minutes; Preferably, in step (b), the filtration is performed while the filter is still hot using a 200-mesh filter cloth; More preferably, in step (c), the drying is spray drying or freeze drying; Particularly preferably, the mass ratio of the honeysuckle, the isatis root, the scutellaria, the indigo leaf, the forsythia, the platycodon, the patchouli, and the licorice is (1-10):(1-10):(1-10):(1-10):(1-10):(1-10):(1-10):(1-10):(1-10):(1-10); Particularly preferably, the mass ratio of the honeysuckle, the isatis root, the scutellaria, the indigo leaf, the forsythia, the platycodon, the patchouli, and the licorice is (2-8):(2-8):(1-5):(1-5):(1-5):(1-5):(1-5):(1-5):(1-5); Particularly preferably, the mass ratio of the honeysuckle, the isatis root, the scutellaria, the indigo leaf, the forsythia, the platycodon, the patchouli, and the licorice is about 5: about 5: about 3: about 3: about 3: about 3: about 3: about 3; Particularly preferably, the honeysuckle has a mass range of 2–8 g, for example, about 5 g; Particularly preferably, the mass of the Isatis root ranges from 2 to 8 g, for example, about 5 g; Particularly preferably, the weight of the Scutellaria baicalensis ranges from 1 to 5 g, for example, about 3 g; Particularly preferably, the mass of the indigo leaves ranges from 1 to 5 g, for example, about 3 g; Particularly preferably, the mass of the forsythia is in the range of 1 to 5 g, for example, about 3 g; Particularly preferably, the weight of the platycodon root ranges from 1 to 5 g, for example, about 3 g; Particularly preferably, the patchouli has a mass range of 1–5 g, for example, about 3 g; Particularly preferably, the licorice has a mass range of 1 to 5 g, for example, about 3 g.

4. The determination method according to claim 1, characterized in that, In the preparation of the traditional Chinese medicine compound test solution, the solvent is an alcohol, such as methanol; Preferably, the volume percentage concentration of methanol is 20% to 50%, for example, about 35%; Preferably, the container is a measuring bottle; Preferably, the mass of the traditional Chinese medicine compound powder is 0.1-1g, for example, about 0.5g; More preferably, the power of the ultrasonic treatment is 400-600W, for example, about 500W; More preferably, the frequency of the ultrasonic treatment is 30 to 50 kHz, for example, about 40 kHz; More preferably, the ultrasonic treatment time is 20 to 40 minutes, for example, about 30 minutes; More preferably, the mass / volume ratio between the traditional Chinese medicine compound powder and the traditional Chinese medicine compound test solution is 0.01 to 0.1, for example, about 0.02, in g / ml.

5. The determination method according to claim 1, characterized in that, The pretreatment method for the baicalin reference standard and the ammonium glycyrrhizate reference standard is as follows: the baicalin reference standard and the ammonium glycyrrhizate reference standard are dried under reduced pressure at about 60°C for about 4 hours; Preferably, the concentration of baicalin in the first reference solution is about 0.3 mg / ml; Preferably, the concentration of ammonium glycyrrhizate in the first reference solution is about 0.04 mg / ml; More preferably, the concentration of Lonicera japonica saponin B in the second reference solution is about 0.3 mg / ml; More preferably, the concentration of dipsaponin B in the second reference solution is about 0.03 mg / ml; More preferably, in the preparation of the second reference solution, the volume percentage concentration of methanol is 40% to 60%, for example, about 50%.

6. The determination method according to claim 1, characterized in that, The flow rate is 0.7–0.9 ml / min, for example, about 0.8 ml / min; Preferably, the column temperature is 28–32°C, for example, about 30°C; Preferably, when using ultraviolet detection, the detection wavelength of the ultraviolet detection is 200-300 nm, for example, 245 nm; Preferably, when using ultraviolet detection, the injection volume of the test solution is 5 to 15 μl, for example, about 10 μl; Preferably, when using an evaporative light scattering detector, the injection volume of the test solution is 5–10 μl; Preferably, the injection volume of the first reference solution is 5–15 μl, for example, about 10 μl; Preferably, the injection volume of the second reference solution is about 5 μl or about 20 μl; More preferably, the chromatographic column is a HALO column. AQ-C18 chromatographic column; More preferably, the chromatographic column has the following specifications: column length 150 mm, inner diameter 4.6 mm, and particle size 2.7 μm.

7. The determination method according to claim 1, characterized in that, Mobile phase A is acetonitrile; Preferably, the acid aqueous solution, alkaline aqueous solution and / or buffer salt aqueous solution are selected from one or more weak acids and their salts, weak bases and their salts of different concentrations; Preferably, the acidic aqueous solution, alkaline aqueous solution, and / or buffer salt aqueous solution are selected from formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid; More preferably, the acid aqueous solution is a 0.01% to 1% acid aqueous solution; More preferably, the acid aqueous solution is a 0.01% to 1% formic acid aqueous solution; More preferably, the acidic aqueous solution is an aqueous solution of about 0.1% formic acid; Preferably, the buffer salt solution is an acetate aqueous solution and / or an acetate aqueous solution; Preferably, the pH value of the buffer salt solution is not greater than 7.

0.

8. The determination method according to claim 1, characterized in that, The detection limit for baicalin was approximately 0.0004553 mg / ml; Preferably, the detection limit of glycyrrhizic acid is about 0.0001296 mg / ml; Preferably, the detection limit of the honeysuckle saponin B is about 0.002066 mg / ml; Preferably, the detection limit of the saponin B is about 0.001751 mg / ml.

9. The determination method according to claim 1, characterized in that, The limit of quantification for baicalin is approximately 0.001339 mg / ml; Preferably, the limit of quantification for glycyrrhizic acid is about 0.0003239 mg / ml; Preferably, the limit of quantification for Lonicera japonica saponin B is about 0.006198 mg / ml; Preferably, the limit of quantification for the saponin B is about 0.006180 mg / ml.

10. The use of the determination method according to any one of claims 1 to 9 in the quality detection and / or quality evaluation and / or quality control of traditional Chinese medicine compound formulas containing honeysuckle, isatis root and scutellaria.

Images