A method for determining the content of a traditional Chinese medicine composition containing angelica and cinnamon and its application.

Abstract

This invention discloses a method for determining the content of a traditional Chinese medicine composition containing Angelica sinensis and cinnamon. The method includes the following steps: taking a test solution and a reference solution of the traditional Chinese medicine composition for detection; the chromatographic conditions for detection are: 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; obtaining the content information of the reference standard of the traditional Chinese medicine composition based on the detection results; wherein the traditional Chinese medicine composition includes Angelica sinensis, cinnamon, white peony root, ginger, jujube, and licorice; and the reference standard is ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide. 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 the prior art does not disclose this technical content.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a method for determining the content of a traditional Chinese medicine composition containing angelica and cinnamon, 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 composition containing angelica and cinnamon, as described in this invention, have not been found in the prior art. Summary of the Invention

[0004] Based on this, the present invention provides a method for determining the content of a traditional Chinese medicine composition containing angelica and cinnamon, the method comprising the following steps:

[0005] Take the test solution and reference solution of the traditional Chinese medicine composition for testing.

[0006] 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; and the gradient elution program was as follows: 0–10 min, 95% B → 94% B; 10–12 min, 94% B → 88% B; 12–20 min, 88% B; 20–21 min, 88% B → 84% B; 21–38 min, 84% B; 38–48 min, 84% B → 80% B; 48–52 min… n, 80%B→75%B; 52~58min, 75%B→71%B; 58~62min, 71%B→68%B; 62~80min, 68%B→44%B; 80~83min, 44%B→26%B; 83~88min, 26%B→15%B; 88~89min, 15%B→95%B; 89~100min, 95%B; flow rate 0.5~1.5ml / min, column temperature 10~40℃, detection wavelength 180~360nm, injection volume 1~20μl;

[0007] Based on the test results, the content information of the reference standard of the traditional Chinese medicine composition was obtained;

[0008] The traditional Chinese medicine composition includes angelica, cinnamon, white peony root, ginger, jujube, and licorice. The reference standards are ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide.

[0009] Furthermore, this licorice is stir-fried licorice.

[0010] Furthermore, this information is based on the corresponding peak areas in the chromatograms of the test solution and the reference solution of the traditional Chinese medicine composition, calculated using the external standard method to determine the content of the reference standard in the traditional Chinese medicine composition.

[0011] Furthermore, the linear equation for this ferulic acid is y = 47.474x + 18.878, R0 2 =1.0000.

[0012] Furthermore, the linear equation for cinnamaldehyde is y = 90.332x + 44.58, R0 2 =1.0000.

[0013] Furthermore, the linear equation for 6-gingerol is y = 25.992x + 8.2874, R0 2 =0.9999.

[0014] Furthermore, the linear equation for this ligustilide is y = 10.805x + 10.113, R0 2 =0.9999.

[0015] Furthermore, the mass ratio of the angelica, cinnamon, white peony root, ginger, jujube and licorice is (45-60):(30-50):(70-90):(30-50):(25-45):(20-40).

[0016] Furthermore, the mass ratio of the angelica, cinnamon, white peony root, ginger, jujube and licorice is approximately 55.2: 41.4: 82.8: 41.4: 36: 27.6.

[0017] Furthermore, the preparation method of the test solution of the traditional Chinese medicine composition includes: taking an appropriate amount of the decoction of the traditional Chinese medicine composition containing angelica and cinnamon, placing it in a container, adding solvent, sonicating for a period of time, making up the volume, shaking well, centrifuging, taking the supernatant and filtering, taking the filtrate to obtain the test solution of the traditional Chinese medicine composition.

[0018] Furthermore, the container is a volumetric flask.

[0019] Furthermore, the solvent is an alcohol, such as methanol.

[0020] Furthermore, the mass-to-volume ratio of the decoction of the traditional Chinese medicine composition to the solvent is 0.4 to 0.8, for example, about 0.6, in g / ml.

[0021] Furthermore, the decoction of this traditional Chinese medicine composition has a mass of 5 to 30g, for example, about 15g.

[0022] Furthermore, the power of the ultrasonic treatment is 400–600W, for example, about 500W.

[0023] Furthermore, the frequency of the ultrasonic treatment is 30–50 kHz, for example, about 40 kHz.

[0024] Furthermore, the duration of the ultrasonic treatment is 20–40 minutes, for example, about 30 minutes.

[0025] Furthermore, the centrifugation speed is 4000–6000 rpm / min, for example, about 5000 rpm / min.

[0026] Furthermore, the centrifugation time is 5 to 15 minutes, for example, about 10 minutes.

[0027] Furthermore, the preparation method of the decoction of the traditional Chinese medicine composition containing angelica and cinnamon includes: weighing appropriate amounts of angelica, cinnamon, white peony root, ginger, jujube and licorice, crushing them into coarse particles, placing them in a container, adding water to soak them, boiling them to a boil and boiling for a period of time, then keeping them at a gentle boil for a period of time, filtering them with a filter while they are hot to obtain the decoction of the traditional Chinese medicine composition containing angelica and cinnamon.

[0028] Furthermore, the volume / mass ratio of the water to the traditional Chinese medicine composition containing angelica and cinnamon is 5 to 10, for example, about 7.

[0029] Furthermore, the container is either a ceramic decoction pot or an electric ceramic stove.

[0030] Furthermore, the soaking time is 10 to 100 minutes, for example, about 45 minutes.

[0031] Furthermore, the pot should be covered when it boils.

[0032] Furthermore, the power of the container during boiling is 1000-3000W, for example, about 2000W.

[0033] Furthermore, the boiling time is 5 to 15 minutes, for example, about 10 minutes.

[0034] Furthermore, the power of the container at the simmering stage is 500–1500W, for example, about 900W.

[0035] Furthermore, the simmering time is 10 to 100 minutes, for example, about 60 minutes.

[0036] Furthermore, the volume-to-volume ratio of the decoction containing angelica and cinnamon to the water is 0.1 to 0.5, for example, about 0.3.

[0037] Furthermore, the filter is a 200-mesh nylon filter.

[0038] Furthermore, the preparation method of the reference solution includes: weighing appropriate amounts of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide reference standards, and adding methanol to prepare a reference solution with concentrations of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide of 1–100 μg / ml.

[0039] Furthermore, the concentration of ferulic acid in the reference solution was approximately 18 μg / ml.

[0040] Furthermore, the concentration of cinnamaldehyde in the reference solution was approximately 13 μg / ml.

[0041] Furthermore, the concentration of 6-gingerol in the reference solution was approximately 6 μg / ml.

[0042] Furthermore, the concentration of ligustilide in the reference solution was approximately 18 μg / ml.

[0043] Furthermore, the flow rate for this high-performance liquid chromatography detection is 0.8–1.2 ml / min, for example, about 1.0 ml / min.

[0044] Furthermore, the column temperature is 20–30°C, for example, about 25°C.

[0045] Furthermore, between the 0th minute and the 60th minute, the detection wavelength is 280–340 nm, for example, about 316 nm.

[0046] Furthermore, between the 60th and 77th minutes, the detection wavelength is 260–320 nm, for example, about 290 nm.

[0047] Furthermore, between the 77th and 100th minutes, the detection wavelength is 190–230 nm, for example, about 210 nm.

[0048] Furthermore, the injection volume is 5–15 μl, for example, about 10 μl.

[0049] Furthermore, the theoretical plate number of the chromatographic peak corresponding to this ferulic acid is not less than 5000.

[0050] Furthermore, the chromatographic column is an Agilent 5TC-C18 column.

[0051] Furthermore, the specifications of the chromatographic column are as follows: column length 250 mm, inner diameter 4.6 mm, particle size 5 μm.

[0052] Furthermore, mobile phase A is acetonitrile.

[0053] 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.

[0054] 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.

[0055] Furthermore, the acidic aqueous solution is a 0.01% to 1% acidic aqueous solution.

[0056] Furthermore, the acidic aqueous solution is a 0.01% to 1% phosphoric acid aqueous solution.

[0057] Furthermore, the acidic aqueous solution is an aqueous solution of approximately 0.1% phosphoric acid.

[0058] Furthermore, the buffer salt solution is an acetate solution and / or an acetate solution.

[0059] Furthermore, the pH value of this buffer salt solution is no greater than 7.0.

[0060] Furthermore, the detection limit for this ferulic acid is approximately 0.4189 μg / ml.

[0061] Furthermore, the detection limit for cinnamaldehyde is approximately 0.06601 μg / ml.

[0062] Furthermore, the detection limit for 6-gingerol was approximately 0.1146 μg / ml.

[0063] Furthermore, the detection limit for this ligustilide is approximately 0.1788 μg / ml.

[0064] Furthermore, the limit of quantification for this ferulic acid is approximately 1.3960 μg / ml.

[0065] Furthermore, the limit of quantification for cinnamaldehyde is approximately 0.2200 μg / ml.

[0066] Furthermore, the limit of quantification for this 6-gingerol is approximately 0.3818 μg / ml.

[0067] Furthermore, the limit of quantification for this ligustilide is approximately 0.5961 μg / ml.

[0068] 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 compositions containing angelica and cinnamon.

[0069] The beneficial effects of this invention are:

[0070] The detection method of the present invention 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

[0071] 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.

[0072] Figure 1 This is a chromatogram of the content of a reference sample solution of the traditional Chinese medicine composition containing angelica and cinnamon according to the present invention. Detailed Implementation

[0073] 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.

[0074] 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.

[0075] 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.

[0076] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0077] 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.

[0078] 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 composition containing Angelica sinensis and cinnamon, nor its application. To address the above problems, the present invention provides a method for determining the content of a traditional Chinese medicine composition containing Angelica sinensis and cinnamon, which includes the following steps:

[0079] Take the test solution and reference solution of the traditional Chinese medicine composition for testing.

[0080] 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; and the gradient elution program was as follows: 0–10 min, 95% B → 94% B; 10–12 min, 94% B → 88% B; 12–20 min, 88% B; 20–21 min, 88% B → 84% B; 21–38 min, 84% B; 38–48 min, 84% B → 80% B; 48–52 min… n, 80%B→75%B; 52~58min, 75%B→71%B; 58~62min, 71%B→68%B; 62~80min, 68%B→44%B; 80~83min, 44%B→26%B; 83~88min, 26%B→15%B; 88~89min, 15%B→95%B; 89~100min, 95%B; flow rate 0.5~1.5ml / min, column temperature 10~40℃, detection wavelength 180~360nm, injection volume 1~20μl;

[0081] Based on the test results, the content information of the reference standard of the traditional Chinese medicine composition was obtained;

[0082] The traditional Chinese medicine composition includes angelica, cinnamon, white peony root, ginger, jujube, and licorice. The reference standards are ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide.

[0083] In this invention, when concentration, time, flow rate, column temperature, wavelength, injection volume, ratio, power, frequency, mass, rotation speed, 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 "0.5 to 1.5" is disclosed, the described range should be interpreted as including ranges such as "0.5 to 1.5", "0.5 to 1.3", "0.5 to 1.1", "0.5 to 0.9", "0.5 to 0.7", "0.7 to 1.5", "0.7 to 1.3", "0.7 to 1.1", "0.7 to 0.9", "0.9 to 1.5", "0.9 to 1.3", "0.9 to 1.1", "1.1 to 1.5", "1.1 to 1.3", "1.3 to 1.5", etc. When a range of values ​​is described in this document, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0084] In a preferred embodiment, the licorice is stir-fried licorice.

[0085] In a preferred embodiment, the information is obtained by calculating the content of the reference standard in the traditional Chinese medicine composition according to the external standard method based on the corresponding peak areas in the chromatograms of the test solution and the reference solution of the recorded traditional Chinese medicine composition.

[0086] In a preferred embodiment, the linear equation for ferulic acid is y = 47.474x + 18.878, R 2 =1.0000.

[0087] In a preferred embodiment, the linear equation for cinnamaldehyde is y = 90.332x + 44.58, R0 2 =1.0000.

[0088] In a preferred embodiment, the linear equation for 6-gingerol is y = 25.992x + 8.2874, R 2 =0.9999.

[0089] In a preferred embodiment, the linear equation for the ligustilide is y = 10.805x + 10.113, R 2 =0.9999.

[0090] In a preferred embodiment, the mass ratio of the angelica, cinnamon, white peony root, ginger, jujube and licorice is (45-60):(30-50):(70-90):(30-50):(25-45):(20-40).

[0091] In a preferred embodiment, the mass ratio of the angelica, cinnamon, white peony root, ginger, jujube and licorice is about 55.2: about 41.4: about 82.8: about 41.4: about 36: about 27.6.

[0092] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 55.2" includes ±5% of 55.2, or from 52.44 to 57.96; "about 41.4" includes ±5% of 41.4, or from 39.33 to 43.47; "about 82.8" includes ±5% of 82.8, or from 78.66 to 86.94; "about 36" includes ±5% of 36, or from 34.2 to 37.8; "about 27.6" includes ±5% of 27.6, or from 26.22 to 28.98.

[0093] In a preferred embodiment, the preparation method of the test solution of the traditional Chinese medicine composition includes: taking an appropriate amount of the decoction of the traditional Chinese medicine composition containing angelica and cinnamon, placing it in a container, adding solvent, sonicating for a period of time, making up the volume, shaking well, centrifuging, taking the supernatant and filtering, taking the filtrate to obtain the test solution of the traditional Chinese medicine composition.

[0094] In a preferred embodiment, the container is a volumetric flask.

[0095] In a preferred embodiment, the solvent is an alcohol, such as methanol.

[0096] In a preferred embodiment, the mass-to-volume ratio of the decoction of the traditional Chinese medicine composition to the solvent is 0.4 to 0.8, for example, about 0.6, in g / ml.

[0097] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.6" includes ±5% of 0.6, or from 0.57 to 0.63.

[0098] In a preferred embodiment, the decoction of the traditional Chinese medicine composition has a mass of 5 to 30 g, for example, about 15 g.

[0099] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 15" includes ±5% of 15, or from 14.25 to 15.75.

[0100] In a preferred embodiment, the power of the ultrasonic treatment is 400 to 600 W, for example, about 500 W.

[0101] 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.

[0102] In a preferred embodiment, the frequency of the ultrasonic treatment is 30 to 50 kHz, for example, about 40 kHz.

[0103] 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.

[0104] In a preferred embodiment, the ultrasonic treatment time is 20 to 40 minutes, for example, about 30 minutes.

[0105] 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.

[0106] In a preferred embodiment, the centrifugation speed is 4000 to 6000 rpm / min, for example, about 5000 rpm / min.

[0107] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5000" includes ±5% of 5000, or from 4750 to 5250.

[0108] In a preferred embodiment, the centrifugation time is 5 to 15 minutes, for example, about 10 minutes.

[0109] 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.

[0110] In a preferred embodiment, the method for preparing the decoction of the traditional Chinese medicine composition containing angelica and cinnamon includes: weighing appropriate amounts of angelica, cinnamon, white peony root, ginger, jujube and licorice, crushing them into coarse particles, placing them in a container, adding water to soak them, boiling them to a boil and boiling for a period of time, then keeping them at a gentle boil for a period of time, filtering them with a filter while they are hot to obtain the decoction of the traditional Chinese medicine composition containing angelica and cinnamon.

[0111] In a preferred embodiment, the volume / mass ratio of the water to the herbal composition containing angelica and cinnamon is 5 to 10, for example, about 7.

[0112] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 7" includes ±5% of 7, or from 6.65 to 7.35.

[0113] In a preferred embodiment, the container is a ceramic decoction pot or an electric ceramic stove.

[0114] In a preferred embodiment, the soaking time is 10 to 100 minutes, for example, about 45 minutes.

[0115] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 45" includes ±5% of 45, or from 42.75 to 47.25.

[0116] In a preferred embodiment, the pot is covered when it boils.

[0117] In a preferred embodiment, the container has a power of 1000 to 3000W when boiling, for example, about 2000W.

[0118] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2000" includes ±5% of 2000, or from 1900 to 2100.

[0119] In a preferred embodiment, the boiling time is 5 to 15 minutes, for example, about 10 minutes.

[0120] 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.

[0121] In a preferred embodiment, the power of the container at the simmering stage is 500 to 1500 W, for example, about 900 W.

[0122] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 900" includes ±5% of 900, or from 855 to 945.

[0123] In a preferred embodiment, the simmering time is 10 to 100 minutes, for example, about 60 minutes.

[0124] 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.

[0125] In a preferred embodiment, the volume-to-volume ratio of the decoction containing angelica and cinnamon to the water is 0.1 to 0.5, for example, about 0.3.

[0126] 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.

[0127] In a preferred embodiment, the filter is a 200-mesh nylon filter.

[0128] In a preferred embodiment, the method for preparing the reference solution includes: weighing appropriate amounts of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide reference standards, and adding methanol to prepare a reference solution with concentrations of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide of 1–100 μg / ml.

[0129] In a preferred embodiment, the concentration of ferulic acid in the reference solution is about 18 μg / ml.

[0130] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 18" includes ±5% of 18, or from 17.1 to 18.9.

[0131] In a preferred embodiment, the concentration of cinnamaldehyde in the reference solution is about 13 μg / ml.

[0132] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 13" includes ±5% of 13, or from 12.35 to 13.65.

[0133] In a preferred embodiment, the concentration of 6-gingerol in the reference solution is about 6 μg / ml.

[0134] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 6" includes ±5% of 6, or from 5.7 to 6.3.

[0135] In a preferred embodiment, the concentration of ligustilide in the reference solution is about 18 μg / ml.

[0136] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 18" includes ±5% of 18, or from 17.1 to 18.9.

[0137] In a preferred embodiment, the flow rate of the high-performance liquid chromatography (HPLC) detection is 0.8 to 1.2 ml / min, for example, about 1.0 ml / min.

[0138] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.0" includes ±5% of 1.0, or from 0.95 to 1.05.

[0139] In a preferred embodiment, the column temperature is 20–30°C, for example, about 25°C.

[0140] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 25" includes ±5% of 25, or from 23.75 to 26.25.

[0141] In a preferred embodiment, the detection wavelength is 280–340 nm, for example, about 316 nm, between the 0th minute and the 60th minute.

[0142] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 316" includes ±5% of 316, or from 300.2 to 331.8.

[0143] In a preferred embodiment, the detection wavelength is 260–320 nm, for example, about 290 nm, between the 60th and 77th minutes.

[0144] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 290" includes ±5% of 290, or from 275.5 to 304.5.

[0145] In a preferred embodiment, the detection wavelength is 190–230 nm, for example, about 210 nm, between the 77th minute and the 100th minute.

[0146] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 210" includes ±5% of 210, or from 199.5 to 220.5.

[0147] In a preferred embodiment, the injection volume is 5 to 15 μl, for example, about 10 μl.

[0148] 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.

[0149] In a preferred embodiment, the theoretical plate number of the chromatographic peak corresponding to the ferulic acid is not less than 5000.

[0150] In a preferred embodiment, the chromatographic column is an Agilent 5TC-C18 column.

[0151] In a preferred embodiment, the chromatographic column has the following specifications: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0152] In a preferred embodiment, the mobile phase A is acetonitrile.

[0153] 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.

[0154] 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.

[0155] In a preferred embodiment, the acidic aqueous solution is a 0.01% to 1% acidic aqueous solution.

[0156] In a preferred embodiment, the acidic aqueous solution is a 0.01% to 1% aqueous solution of phosphoric acid.

[0157] In a preferred embodiment, the acidic aqueous solution is an aqueous solution of about 0.1% phosphoric acid.

[0158] 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%.

[0159] In a preferred embodiment, the buffer salt solution is an acetate aqueous solution and / or an acetate aqueous solution.

[0160] In a preferred embodiment, the pH value of the buffer saline solution is not greater than 7.0.

[0161] In a preferred embodiment, the detection limit of ferulic acid is about 0.4189 μg / ml.

[0162] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.4189" includes ±5% of 0.4189, or from 0.397955 to 0.439845.

[0163] In a preferred embodiment, the detection limit of cinnamaldehyde is about 0.06601 μg / ml.

[0164] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.06601" includes ±5% of 0.06601, or from 0.0627095 to 0.0693105.

[0165] In a preferred embodiment, the detection limit of 6-gingerol is about 0.1146 μg / ml.

[0166] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.1146" includes ±5% of 0.1146, or from 0.10887 to 0.12033.

[0167] In a preferred embodiment, the detection limit of the ligustilide is about 0.1788 μg / ml.

[0168] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.1788" includes ±5% of 0.1788, or from 0.16986 to 0.18774.

[0169] In a preferred embodiment, the limit of quantification for ferulic acid is about 1.3960 μg / ml.

[0170] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.3960" includes ±5% of 1.3960, or from 1.3262 to 1.4658.

[0171] In a preferred embodiment, the limit of quantification for cinnamaldehyde is about 0.2200 μg / ml.

[0172] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.2200" includes ±5% of 0.2200, or from 0.2090 to 0.2310.

[0173] In a preferred embodiment, the limit of quantification for 6-gingerol is about 0.3818 μg / ml.

[0174] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.3818" includes ±5% of 0.3818, or from 0.36271 to 0.40089.

[0175] In a preferred embodiment, the limit of quantification for the ligustilide is about 0.5961 μg / ml.

[0176] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.5961" includes ±5% of 0.5961, or from 0.566295 to 0.625905.

[0177] 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 compositions containing angelica and cinnamon.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] Example

[0182] 1. Instruments and reagents

[0183] 1.1 Instruments

[0184] High-performance liquid chromatograph, model: Agilent 1290_DAD HPLC (Agilent Technologies); High-performance liquid chromatograph, model: Waters 2695_2996 (Waters Technologies); Chromatographic column: Agilent 5TC-C18(2)250×4.6mm, SN:731424 / 735516 / 735519; Electronic balance, model: BCE224I-1CCN (Sartorius Group, Germany); Electronic balance, model: JY2002 (Shanghai Puchun Measurement Instrument Co., Ltd.); Benchtop low-speed centrifuge, model: TD4N (Changsha Yingtai Instrument Co., Ltd.); CNC ultrasonic cleaner, model: KQ-500DE (Kunshan Ultrasonic Instrument Co., Ltd.); Digital display constant temperature water bath, model: HH-6 (Shanghai Boxun Medical Biological Instrument Co., Ltd.); Diaphragm oil-free vacuum pump, model: MT-10 (Yancheng Xinmingte Glass Instrument Co., Ltd.).

[0185] 1.2 Reagents and Chemicals

[0186] Methanol and acetonitrile were of chromatographic grade; methanol and phosphoric acid were of analytical grade; and water was purified water. Ferulic acid (batch number: 110773-202316, content: 99.3%, China National Institutes for Food and Drug Control); cinnamaldehyde (batch number: 110710-202223, content: 98.9%, China National Institutes for Food and Drug Control); ligustilide (batch number: 111737-202311, content: 100.0%, China National Institutes for Food and Drug Control); 6-gingerol (batch number: 111833-202007, content: 99.3%, China National Institutes for Food and Drug Control).

[0187] 1.3 Sample Information

[0188] The present invention includes the following reference samples: a granule standard sample of a traditional Chinese medicine composition containing Angelica sinensis and Cinnamomum cassia (batch number: 2024062801-L, source: self-made); a granule standard sample of a traditional Chinese medicine composition containing Angelica sinensis and Cinnamomum cassia (batch number: 2024072501-L, source: self-made); a granule standard sample of a traditional Chinese medicine composition containing Angelica sinensis and Cinnamomum cassia (batch number: 2024082904-L, source: self-made); and a negative reference sample of a granule composition containing Angelica sinensis and Cinnamomum cassia lacking Angelica sinensis (batch number: 202409). 0201-L (Source: Self-made); The traditional Chinese medicine composition granules containing Angelica sinensis and cinnamon of the present invention, lacking cinnamon, negative reference sample (batch number: 2024090204-L, source: self-made); The traditional Chinese medicine composition granules containing Angelica sinensis and cinnamon of the present invention, lacking white peony root, negative reference sample (batch number: 2024090202-L, source: self-made); The traditional Chinese medicine composition granules containing Angelica sinensis and cinnamon of the present invention, lacking ginger, negative reference sample (batch number: 2024090301 ... white peony root, negative reference sample (batch number: 2024090202-L, source: self-made); The following are sample references for traditional Chinese medicine compositions containing cinnamon and angelica: granules lacking jujube (batch number: 2024090302-L, source: self-made); granules containing angelica and cinnamon of the present invention lacking roasted licorice (batch number: 2024090203-L, source: self-made); angelica slices (batch number: ZA00323102101-01, origin: Minxian County, Dingxi City, Gansu Province, source: self-made); cinnamon slices (batch number: ZA04223112801, origin: Pingxiang County, Guigang City, Guangxi Zhuang Autonomous Region, source: self-made). Homemade); White peony root slices (batch number: ZA00123121502, place of origin: Qiaocheng District, Bozhou City, Anhui Province, source: homemade); Jujube slices (batch number: ZA00223121402, place of origin: Ningyang County, Tai'an City, Shandong Province, source: homemade); Ginger slices (batch number: ZA00524071501, place of origin: Wanyuan County, Dazhou City, Sichuan Province, source: homemade); Fried licorice root slices (batch number: ZD00423090401-01, place of origin: Hangjin Banner, Ordos City, Inner Mongolia, source: homemade).

[0189] 1.4 Solution Preparation

[0190] (1) Preparation method of reference sample decoction

[0191] Take 55.20g of Angelica sinensis, 41.40g of cinnamon, 82.80g of white peony root, 41.40g of ginger, 36.00g of jujube, and 27.60g of stir-fried licorice root. Grind the above six ingredients into coarse granules, place them in a ceramic decoction pot, add 2000ml of water, cover, and soak for 45 minutes. Boil on an electric ceramic stove at 2000W for about 10 minutes, then simmer at 900W until reduced to 600ml (about 60 minutes). Filter the decoction through a 200-mesh nylon filter while hot to obtain the final product.

[0192] (2) Preparation of the reference solution:

[0193] Take appropriate amounts of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide reference standards, accurately weigh them, and add methanol to prepare a mixed solution containing 18 μg ferulic acid, 13 μg cinnamaldehyde, 6 μg 6-gingerol, and 18 μg ligustilide per 1 ml, which will be used as the reference solution.

[0194] (3) Preparation of the test solution

[0195] Take about 15g of the standard sample decoction, place it in a 25ml volumetric flask, add an appropriate amount of methanol, sonicate (500W, 40kHz) for 30 minutes, cool, make up to volume, shake well, centrifuge at 5000rpm / min for 10min, take the supernatant and filter it, and take the filtrate to obtain the final product.

[0196] (4) Preparation of negative control solution

[0197] Preparation of negative sample solution for Angelica sinensis deficiency: Weigh the other herbs for Angelica sinensis deficiency according to the prescription, prepare negative sample for Angelica sinensis deficiency according to the preparation method of the reference sample, and prepare negative sample solution for Angelica sinensis deficiency according to item "(3)".

[0198] Negative control solutions lacking Angelica sinensis, cinnamon, white peony root, ginger, jujube, and stir-fried licorice were prepared using the same method.

[0199] (5) Blank solvent: methanol.

[0200] 2. Establishment of chromatographic conditions for content determination

[0201] 2.1 Study on chromatographic conditions for content determination

[0202] The proposed initial chromatographic conditions are as follows:

[0203] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512).

[0204] Chromatographic conditions and system suitability tests were performed using octadecyl-bonded silica gel as the stationary phase (Agilent 5TC-C18 4.6×250mm 5μm); acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, with gradient elution performed according to the specifications in Table 1; flow rate was 1 ml per minute; column temperature was 25℃; and full wavelength scanning was performed.

[0205] Table 1 Chromatographic Condition Gradient 1

[0206]

[0207]

[0208] Table 2 summarizes the optimal absorption wavelengths of the reference solutions.

[0209] Table 2 Summary of Optimal Absorption Wavelengths for Reference Solutions

[0210] Reference solution Peak time Optimal absorption wavelength (nm) ferulic acid 34.661 218、236、322 Cinnamaldehyde 61.018 206、222、290 6-Gingerol 77.183 282 ligustilide 84.164 208、282、326

[0211] Given that the detection wavelength for ferulic acid content determination in Angelica sinensis is 316 nm according to the Chinese Pharmacopoeia, and the optimal wavelength for the ferulic acid reference solution is around 316 nm; the optimal wavelength for the cinnamaldehyde reference solution is 290 nm; and the optimal wavelength for the ligustilide reference solution is 210 nm, and considering that the elution time of 6-gingerol is close to that of ligustilide and that it has good absorption at 210 nm, the detection wavelengths for content determination were determined to be 316 nm for ferulic acid, 290 nm for cinnamaldehyde, and 210 nm for 6-gingerol and ligustilide. Due to poor separation of the characteristic peaks at 30–40 minutes, further optimization was performed as shown in gradient 2 in Table 3.

[0212] Table 3 Gradient 2

[0213]

[0214]

[0215] The results showed that the separation degree and response value of ferulic acid, cinnamaldehyde, 6-gingerol and ligustilide under gradient 2 met the requirements of the liquid phase content determination spectrum; given that the peak time of ligustilide was about 85 minutes and had returned to the initial gradient, further optimization was carried out to gradient 3 as shown in Table 4.

[0216] After the above screening, the following chromatographic conditions for content determination were determined:

[0217] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512).

[0218] Chromatographic conditions and system suitability tests were performed using octadecyl-bonded silica gel as the stationary phase (Agilent 5TC-C18 4.6×250mm 5μm); acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in Table 4; flow rate of 1 mL / min; column temperature of 25℃; detection wavelength of 316 nm for 0–60 min, 290 nm for 60–77 min, and 210 nm for 77–100 min. The theoretical plate number, calculated based on the ferulic acid peak, should be no less than 5000.

[0219] Table 4. Final Elution Gradient

[0220] Time (min) Acetonitrile (%) 0.1% Phosphoric Acid (%) 0→10 5→6 95→94 10→12 6→12 94→88 12→20 12 88 20→21 12→16 88→84 21→38 16 84 38→48 16→20 84→80 48→52 20→25 80→75 52→58 25→29 75→71 58→62 29→32 71→68 62→80 32→56 68→44 80→83 56→74 44→26 83→88 74→85 26→15 88→89 85→5 15→95 89→100 5 95

[0221] The results show that, under this gradient, the symmetry and separation of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide in the granules of the traditional Chinese medicine composition containing angelica and cinnamon of the present invention are good, meeting the requirements for liquid phase content determination; therefore, this method is determined as the method for determining ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide in the granules of the traditional Chinese medicine composition containing angelica and cinnamon of the present invention.

[0222] 3. Preparation and investigation of the reference sample test solution

[0223] Based on the results of the study on the preparation process of the reference sample, which is preserved in the form of decoction, the preparation method of the decoction test solution was investigated. Therefore, it was determined that 15g of decoction would be used for the study.

[0224] 3.1 Investigation of Extraction Solvents

[0225] Six portions of the decoction were taken, and the effects of pure water, 50% methanol, and methanol as extraction solvents on the content determination of the granular reference sample of the traditional Chinese medicine composition containing angelica and cinnamon of the present invention were investigated.

[0226] Accurately weigh six portions (approximately 15g each) of the standard sample decoction and place them in 25ml volumetric flasks. Add appropriate amounts of pure water, 50% methanol, and methanol to each flask. Sonicate (500W, 40kHz) for 30 minutes, cool, dilute to volume, shake well, centrifuge at 5000rpm / min for 10 minutes, filter the supernatant, and collect the filtrate. The injection results are as follows:

[0227] Table 5. Summary of particle content detection information of the traditional Chinese medicine composition containing angelica and cinnamon of the present invention treated with different extraction solvents.

[0228]

[0229] Note: Content (mg / g) indicates the content per gram of decoction, the same applies below.

[0230] The results showed that, after different extraction solvent treatments, the content of the Chinese herbal composition containing angelica and cinnamon was higher after methanol treatment. Therefore, methanol is the appropriate solvent for determining the content of the Chinese herbal composition containing angelica and cinnamon in this invention.

[0231] 3.2. Examination of extraction time

[0232] Six portions of the decoction were taken, and the effects of extraction times of 20 minutes, 30 minutes, and 45 minutes on the content determination of the granular reference sample of the traditional Chinese medicine composition containing angelica and cinnamon of the present invention were investigated.

[0233] Accurately weigh six portions (approximately 15g each) of the standard sample decoction and place them in 25ml volumetric flasks. Add an appropriate amount of methanol and sonicate (500W, 40kHz) for 20, 30, and 45 minutes respectively. After cooling, make up to volume, shake well, and centrifuge at 5000rpm / min for 10 minutes. Filter the supernatant and collect the filtrate. Inject the sample; the results are as follows:

[0234] Table 6 Summary of the detection information of the content of the traditional Chinese medicine composition containing angelica and cinnamon granules at different extraction times in this invention.

[0235]

[0236] The results showed that the content of the Chinese herbal composition containing angelica and cinnamon of the present invention was similar after methanol treatment at different extraction times, with extraction times ranging from 20 to 45 minutes. Since the detection value was slightly higher after ultrasonic treatment for 30 minutes, the treatment time for determining the content of the Chinese herbal composition containing angelica and cinnamon of the present invention was 30 minutes.

[0237] 3.3 Sampling Quantity Examination

[0238] Six portions of the decoction were taken, and the effects of sample amounts of 10g, 15g, and 20g on the determination of the content of the reference sample of the traditional Chinese medicine composition containing angelica and cinnamon of the present invention were investigated.

[0239] Take 6 portions of the standard sample decoction, approximately 10g, 15g, and 20g respectively, weigh them accurately, place them in a 25ml volumetric flask, add an appropriate amount of methanol, sonicate (500W, 40kHz) for 30 minutes, cool, make up to volume, shake well, centrifuge at 5000rpm / min for 10 minutes, take the supernatant and filter it, and take the filtrate to obtain the final product.

[0240] The results showed that after methanol treatment, the detection values ​​of the traditional Chinese medicine composition granules containing angelica and cinnamon of the present invention were close when the sample amount was 10-15g. Considering the peak area of ​​6-gingerol and ligustilide, the sample amount was determined to be 15g.

[0241] 3.4 Validation of the preparation method for the reference sample test solution

[0242] In summary, the preparation method of the reference sample is determined as follows: Take about 15g of the reference sample decoction, accurately weigh it, place it in a 25ml volumetric flask, add an appropriate amount of methanol, sonicate (500W, 40kHz) for 30 minutes, cool, make up to volume, shake well, centrifuge at 5000rpm / min for 10min, filter the supernatant, and take the filtrate to obtain the sample.

[0243] 4. Methodological Validation

[0244] 4.1 Specificity

[0245] The blank solvent, reference solution, negative solutions of each herb in the granular reference sample of the traditional Chinese medicine composition containing angelica and cinnamon of the present invention, and the test solution (2024082904-L) of the granular reference sample of the traditional Chinese medicine composition containing angelica and cinnamon of the present invention were injected for analysis. The peak positions of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide should not be interfered with by the negative and blank solvents. Figure 1 This is a chromatogram of the content of a reference sample solution of the traditional Chinese medicine composition containing angelica and cinnamon according to the present invention.

[0246] The blank solvent, reference solution, negative control solutions of each herb, and the reference sample solution of the traditional Chinese medicine composition containing angelica and cinnamon of the present invention were analyzed. The blank solvent and negative control solutions did not interfere with the peak positions of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide, indicating strong specificity.

[0247] 4.2 Precision

[0248] 4.2.1 Repeatability

[0249] Prepare six test solutions according to the above method for preparing test solution (2024082904-L), inject and detect them, record the chromatograms, and calculate the contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide. If the RSD of the contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide is ≤6%, it indicates that the method has good repeatability.

[0250] The results showed that the RSD of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide was ≤6%, indicating good reproducibility according to the analytical method validation guidelines of the 2020 edition of the Chinese Pharmacopoeia 9101.

[0251] 4.2.2 Intermediate Precision

[0252] Two personnel prepared six test solutions (2024082904-L) according to the above test solution preparation method. These solutions were then analyzed using an Agilent high-performance liquid chromatograph and a Waters high-performance liquid chromatograph, respectively. The contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide were calculated. If the RSD of the contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide were ≤6%, it indicated that the intermediate precision of the method was good.

[0253] The results showed that the RSD of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide in the injection results of Agilent and Waters high performance liquid chromatographs was ≤6%, indicating that the intermediate precision of the content detection methods was good.

[0254] 4.2.3 Instrument Precision

[0255] Prepare a test solution (2024082904-L) according to the above test solution preparation method, inject it 6 times consecutively, record the chromatogram, and calculate the RSD values ​​of the retention time and peak area of ​​ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide. If the RSD of the relative retention time is ≤1% and the RSD of the relative peak area is ≤3%, it indicates that the instrument precision is good.

[0256] The retention time RSD of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide peaks was ≤1%, and the peak area RSD was ≤3%, indicating good instrument precision.

[0257] 4.3 Accuracy

[0258] Take nine portions of this product solution (2024082904-L), each approximately 7.5 g, accurately weighed, and place them in separate 25 ml volumetric flasks. Add 1 ml, 2 ml, and 3 ml of each of the four reference solutions: ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide, respectively. Prepare three replicates for each. Calculate the recovery rate of the reference standards. The RSD of the recovery rates of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide should be ≤6%. A recovery rate of 80%–115% indicates good accuracy of the method. The concentrations of the ferulic acid reference solution are: 0.1141 mg / ml; cinnamaldehyde reference solution: 0.1485 mg / ml; 6-gingerol reference solution: 0.0840 mg / ml; and ligustilide reference solution: 0.1195 mg / ml.

[0259] The results showed that the recoveries of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide had an RSD of ≤6%, and the recoveries were all within the range of 80% to 115%. According to the validation guidelines for analytical methods 9101 in the 2020 edition of the Chinese Pharmacopoeia, this method has good accuracy.

[0260] 4.4 Sensitivity (Limit of Quantitation and Limit of Detection)

[0261] The limit of detection (LOD) refers to the lowest concentration of a substance in a sample that can be qualitatively detected but does not require precise quantification. In chromatographic analysis, the signal-to-noise ratio (S / N) is generally used to define the LOD. The S / N is the ratio of the response value of the target analyte in the sample to the baseline noise. Generally, an S / N of ≥3 is considered suitable for qualitative analysis, and an S / N of ≥10 is considered suitable for quantitative analysis (RSD ≤ 10.0%).

[0262] The results showed that the limit of quantification (LOQ) for ferulic acid was 1.3960 μg / ml, for cinnamaldehyde it was 0.2200 μg / ml, for 6-gingerol it was 3818 μg / ml, and for ligustilide it was 0.5961 μg / ml.

[0263] The detection limits for ferulic acid were 0.4189 μg / ml, for cinnamaldehyde 0.06601 μg / ml, for 6-gingerol 0.1146 g / ml, and for ligustilide 0.1788 μg / ml.

[0264] 4.5 Examination of Linear Relationships

[0265] Six control solutions with different concentrations were prepared for determination. The measured response signal was plotted as a function of the analyte concentration to observe whether it was linear, and then linear regression was performed using the least squares method. A correlation coefficient r of not less than 0.9990 indicates that the method has good linearity.

[0266] The results showed that the linear correlation coefficients r of the contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide were all greater than 0.9990, indicating that the method had good linearity.

[0267] 4.6 Durability Test

[0268] 4.6.1 Flow rate durability

[0269] Two test solutions were prepared according to the above method for preparing the test solution (2024082904-L). The mobile phase flow rates were set to 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min, respectively. The samples were injected and the chromatograms were recorded. The contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide were calculated. The relative average deviation of the contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide should be ≤6%, which indicates that the flow rate robustness is good.

[0270] The results showed that at flow rates of 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min, the relative average deviations of the contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide were all ≤6%, indicating that this content detection method has good flow rate robustness.

[0271] 4.6.2 Column Temperature Durability

[0272] Two test solutions were prepared according to the above method for preparing the test solution (2024082904-L). The column temperatures were set to 20℃, 25℃, and 30℃, respectively. The samples were injected and detected, and the chromatograms were recorded. The contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide were calculated. The relative average deviation of the contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide should be ≤6%, which indicates that the column temperature durability is good.

[0273] The results showed that the relative average deviation of the contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide was ≤6% under different column temperature conditions, indicating good column temperature durability.

[0274] 4.6.3 Durability under different acid concentrations

[0275] Two test solutions were prepared according to the above method for preparing the test solution (2024082904-L). Phosphoric acid solutions with concentrations of 0.09%, 0.10%, and 0.11% were prepared, respectively. The solutions were injected and analyzed, and the chromatograms were recorded. The contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide were calculated. The relative average deviation of the contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide should be ≤6%, which indicates good robustness at different acid concentrations.

[0276] The results showed that the relative average deviation of the contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide was ≤6% under different acid concentrations, indicating good durability under different acid concentrations.

[0277] 4.6.4 Durability of Different Chromatographic Columns

[0278] Two test solutions (2024082904-L) were prepared according to the above test solution preparation method. Three different types of Agilent 5TC-C18(2)250×4.6mm chromatographic columns were used for injection and detection. The contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide were calculated. The relative average deviation of the contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide should be ≤6%, which indicates that the different chromatographic columns have good durability.

[0279] The results showed that the relative average deviation of the contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide in different chromatographic columns should be ≤6%, indicating that different chromatographic columns have good durability.

[0280] 4.7 Stability of the test solution

[0281] Prepare one test solution (2024082904-L) according to the above method, and inject it for analysis at 0, 3, 6, 10, 15, 18, 24, 36, 48, and 62 hours. Record the chromatogram and calculate the contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide. If the RSD of the contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide is ≤6%, it indicates that the test solution has good stability.

[0282] Based on the above experimental results, the RSD of the contents of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide in the test solution is ≤6% within 62 hours, indicating that the test solution has good stability.

[0283] 5. Determination of the content of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide in the reference samples

[0284] Based on the above research results and in accordance with the analytical method validation guidelines in General Chapter 9101 of the 2020 edition of the Chinese Pharmacopoeia, the content determination indicators for this method are determined to be ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide. Through method validation, the specificity, accuracy, precision, sensitivity, linearity, column temperature, flow rate, different acid concentrations, different chromatographic columns, and stability of the test solution all meet the requirements; therefore, this detection method is of good quality, as follows.

[0285] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0286] Chromatographic conditions and system suitability tests were performed using octadecyl-bonded silica gel as the stationary phase (Agilent 5TC-C18 4.6×250mm 5μm); acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in Table 4; flow rate of 1 mL / min; column temperature of 25℃; detection wavelength of 316 nm for 0–60 min, 290 nm for 60–77 min, and 210 nm for 77–100 min. The theoretical plate number, calculated based on the ferulic acid peak, should be no less than 5000.

[0287] Preparation of reference solution: Take appropriate amounts of ferulic acid, paeoniflorin, cinnamaldehyde, 6-gingerol, and ligustilide reference standards, accurately weigh them, and add methanol to prepare a mixed solution containing 18 μg ferulic acid, 13 μg cinnamaldehyde, 6 μg 6-gingerol, and 18 μg ligustilide per 1 ml, as the reference solution.

[0288] Preparation of test solution: Weigh approximately 15g of the decoction of the reference sample accurately, place it in a 25ml volumetric flask, add an appropriate amount of methanol, sonicate (500W, 40kHz) for 30 minutes, cool, make up to volume, shake well, centrifuge at 5000rpm / min for 10min, take the supernatant and filter it, and take the filtrate to obtain the test solution.

[0289] The assay involves precisely pipetting 10 μl of both the reference solution and the test solution into a liquid chromatograph and measuring the concentration.

[0290] 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 composition containing angelica and cinnamon, characterized in that, The determination method includes the following steps: Take the test solution and reference solution of the traditional Chinese medicine composition for testing. The preparation method of the test solution of the traditional Chinese medicine composition includes: taking an appropriate amount of the decoction of the traditional Chinese medicine composition containing angelica and cinnamon, placing it in a container, adding methanol, sonicating for a period of time, making up the volume, shaking well, centrifuging, taking the supernatant and filtering, taking the filtrate to obtain the test solution of the traditional Chinese medicine composition. The chromatographic conditions for the detection were as follows: a column packed with octadecyl-bonded silica gel was used; mobile phase A was acetonitrile; mobile phase B was approximately 0.1% aqueous phosphoric acid solution; and the gradient elution program was as follows: 0–10 min, 95% B → 94% B; 10–12 min, 94% B → 88% B; 12–20 min, 88% B; 20–21 min, 88% B → 84% B; 21–38 min, 84% B; 38–48 min, 84% B → 80% B; 48–52 min, 80% B → 75% B; 52–58 min, 75% B → 71% B. 58–62 min, 71% B → 68% B; 62–80 min, 68% B → 44% B; 80–83 min, 44% B → 26% B; 83–88 min, 26% B → 15% B; 88–89 min, 15% B → 95% B; 89–100 min, 95% B; Flow rate: approximately 1 ml / min; Column temperature: approximately 25 °C; Detection wavelength: approximately 316 nm from 0–60 min; Detection wavelength: approximately 290 nm from 60–77 min; Detection wavelength: approximately 210 nm from 77–100 min; Injection volume: 1–20 μl. Based on the test results, the content information of the reference standard of the traditional Chinese medicine composition is obtained; The traditional Chinese medicine composition includes angelica, cinnamon, white peony root, ginger, jujube, and licorice, and the reference standards are ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide. The specifications of the chromatographic column are as follows: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

2. The determination method according to claim 1, characterized in that, The licorice mentioned is stir-fried licorice.

3. The determination method according to claim 1, characterized in that, The information is obtained by calculating the content of the reference standard in the traditional Chinese medicine composition according to the external standard method based on the corresponding peak areas in the chromatograms of the test solution and the reference solution of the recorded traditional Chinese medicine composition.

4. The determination method according to claim 1, characterized in that, The linear equation for ferulic acid is y = 47.474x + 18.878, R0 2 =1.0000.

5. The determination method according to claim 1, characterized in that, The linear equation for cinnamaldehyde is y = 90.332x + 44.58, R0 2 =1.0000.

6. The determination method according to claim 1, characterized in that, The linear equation for 6-gingerol is y = 25.992x + 8.2874, R0 2 =0.9999.

7. The determination method according to claim 1, characterized in that, The linear equation for the ligustilide is y = 10.805x + 10.113, R 2 =0.9999.

8. The determination method according to claim 1, characterized in that, The mass ratio of the Angelica sinensis, the cinnamon, the white peony root, the ginger, the jujube and the licorice is (45-60):(30-50):(70-90):(30-50):(25-45):(20-40).

9. The determination method according to claim 8, characterized in that, The mass ratio of the angelica, cinnamon, white peony root, ginger, jujube and licorice is approximately 55.2: approximately 41.4: approximately 82.8: approximately 41.4: approximately 36: approximately 27.

6.

10. The determination method according to claim 1, characterized in that, The container is a volumetric flask.

11. The determination method according to claim 1, characterized in that, The mass-to-volume ratio of the decoction of the traditional Chinese medicine composition to the methanol is 0.4 to 0.8, with units of g / ml.

12. The determination method according to claim 11, characterized in that, The mass-to-volume ratio of the decoction of the traditional Chinese medicine composition to the methanol is approximately 0.6, expressed in g / ml.

13. The determination method according to claim 1, characterized in that, The decoction of the traditional Chinese medicine composition has a mass of 5-30g.

14. The determination method according to claim 13, characterized in that, The decoction of the traditional Chinese medicine composition weighs approximately 15g.

15. The determination method according to claim 1, characterized in that, The power of the ultrasonic treatment is 400-600W.

16. The determination method according to claim 15, characterized in that, The power of the ultrasonic treatment is approximately 500W.

17. The determination method according to claim 1, characterized in that, The frequency of the ultrasonic treatment is 30–50 kHz.

18. The determination method according to claim 17, characterized in that, The frequency of the ultrasonic treatment is approximately 40 kHz.

19. The determination method according to claim 1, characterized in that, The ultrasonic treatment time is 20 to 40 minutes.

20. The determination method according to claim 19, characterized in that, The ultrasonic treatment lasts for approximately 30 minutes.

21. The determination method according to claim 1, characterized in that, The centrifuge speed is 4000-6000 rpm / min.

22. The determination method according to claim 21, characterized in that, The centrifuge speed is approximately 5000 rpm / min.

23. The determination method according to claim 1, characterized in that, The centrifugation time is 5 to 15 minutes.

24. The determination method according to claim 23, characterized in that, The centrifugation time is approximately 10 minutes.

25. The determination method according to claim 1, characterized in that, The preparation method of the decoction of the traditional Chinese medicine composition containing angelica and cinnamon includes: weighing appropriate amounts of angelica, cinnamon, white peony root, ginger, jujube and licorice, crushing them into coarse particles, placing them in a container, adding water to soak them, boiling them to a boil and boiling for a period of time, then keeping them at a simmer for a period of time, filtering them with a filter while they are hot to obtain the decoction of the traditional Chinese medicine composition containing angelica and cinnamon.

26. The determination method according to claim 25, characterized in that, The volume / mass ratio of the water to the traditional Chinese medicine composition containing angelica and cinnamon is 5 to 10.

27. The determination method according to claim 26, characterized in that, The volume / mass ratio of the water to the traditional Chinese medicine composition containing angelica and cinnamon is approximately 7.

28. The determination method according to claim 25, characterized in that, The container is a ceramic decoction pot or an electric ceramic stove.

29. The determination method according to claim 25, characterized in that, The soaking time is 10 to 100 minutes.

30. The determination method according to claim 29, characterized in that, The soaking time is approximately 45 minutes.

31. The determination method according to claim 25, characterized in that, Cover the pot when it boils.

32. The determination method according to claim 25, characterized in that, The power of the container during boiling is 1000-3000W.

33. The determination method according to claim 32, characterized in that, The container has a power of approximately 2000W when boiling.

34. The determination method according to claim 25, characterized in that, The boiling time is 5 to 15 minutes.

35. The determination method according to claim 34, characterized in that, The boiling time is approximately 10 minutes.

36. The determination method according to claim 25, characterized in that, The power of the container during simmering is 500-1500W.

37. The determination method according to claim 36, characterized in that, The power of the container at the simmering stage is approximately 900W.

38. The determination method according to claim 25, characterized in that, The simmering time is 10 to 100 minutes.

39. The determination method according to claim 38, characterized in that, The simmering time is approximately 60 minutes.

40. The determination method according to claim 25, characterized in that, The volume ratio of the decoction containing angelica and cinnamon to the water is 0.1 to 0.

5.

41. The determination method according to claim 40, characterized in that, The volume ratio of the decoction containing angelica and cinnamon to the water is approximately 0.

3.

42. The determination method according to claim 25, characterized in that, The filter screen is a 200-mesh nylon filter screen.

43. The determination method according to claim 1, characterized in that, The method for preparing the reference solution includes: weighing appropriate amounts of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide reference standards, and adding methanol to prepare the reference solution with concentrations of ferulic acid, cinnamaldehyde, 6-gingerol, and ligustilide of 1–100 μg / ml.

44. The determination method according to claim 1, characterized in that, The concentration of ferulic acid in the reference solution was approximately 18 μg / ml.

45. The determination method according to claim 1, characterized in that, The concentration of cinnamaldehyde in the reference solution was approximately 13 μg / ml.

46. ​​The determination method according to claim 1, characterized in that, The concentration of 6-gingerol in the reference solution was approximately 6 μg / ml.

47. The determination method according to claim 1, characterized in that, The concentration of ligustilide in the reference solution was approximately 18 μg / ml.

48. The determination method according to claim 1, characterized in that, The injection volume is 5–15 μl.

49. The determination method according to claim 48, characterized in that, The injection volume is approximately 10 μl.

50. The determination method according to claim 1, characterized in that, The theoretical plate number of the chromatographic peak corresponding to the ferulic acid is not less than 5000.

51. The determination method according to claim 1, characterized in that, The chromatographic column is an Agilent 5TC-C18 column.

52. The determination method according to claim 1, characterized in that, The detection limit for ferulic acid is approximately 0.4189 μg / ml.

53. The determination method according to claim 1, characterized in that, The detection limit for cinnamaldehyde is approximately 0.06601 μg / ml.

54. The determination method according to claim 1, characterized in that, The detection limit for 6-gingerol was approximately 0.1146 μg / ml.

55. The determination method according to claim 1, characterized in that, The detection limit for the ligustilide was approximately 0.1788 μg / ml.

56. The determination method according to claim 1, characterized in that, The limit of quantification for the ferulic acid is approximately 1.3960 μg / ml.

57. The determination method according to claim 1, characterized in that, The limit of quantification for cinnamaldehyde is approximately 0.2200 μg / ml.

58. The determination method according to claim 1, characterized in that, The limit of quantification for 6-gingerol is approximately 0.3818 μg / ml.

59. The determination method according to claim 1, characterized in that, The limit of quantification for the ligustilide is approximately 0.5961 μg / ml.

60. Use of the determination method according to any one of claims 1 to 59 in the quality detection and / or quality evaluation and / or quality control of traditional Chinese medicine compositions containing angelica and cinnamon.

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