Method for determining content of 15 amino acids in cornu gorais in phlegm-heat-clearing injection

Abstract

The invention discloses a method for determining the content of 15 amino acids in cornu gorais in a phlegm-heat-clearing injection, and the method comprises the following steps: taking a test solution and a reference solution, carrying out pre-column derivatization, and then detecting, the phlegm-heat-clearing injection is composed of scutellaria baicalensis, bear gall powder, cornu gorais, honeysuckle, fructus forsythiae and propylene glycol, the reference substance is 15 amino acids, and the chromatographic conditions of the detection are as follows: a chromatographic column with octadecylsilane chemically bonded silica as a filler is adopted, a mobile phase A is an acid aqueous solution, an alkali aqueous solution and / or a buffer salt aqueous solution, and a mobile phase B is selected from one or more of acetonitrile, methanol, tetrahydrofuran and water; and obtaining content information of 15 amino acids in cornu gorais in the phlegm-heat-clearing injection according to a detection result. The method disclosed by the invention is simple, convenient, efficient and rapid, and provides a theoretical basis for the quality control of the Tweenqing injection and the deep research of a pharmacodynamic material basis by detecting the content of 15 amino acids in cornu gorais in the Tweenqing injection.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a method for determining the content of 15 amino acids from goat horn in Tanreqing injection. Background Technology

[0002] Tanreqing Injection is made from five medicinal herbs: Scutellaria baicalensis, bear bile powder, goat horn, honeysuckle, and forsythia. It has the effects of clearing heat, detoxifying, and resolving phlegm. It has significant efficacy in treating upper respiratory tract infections, acute bronchitis, pneumonia, pulmonary tuberculosis complicated with pulmonary infection, advanced lung cancer complicated with pulmonary infection, and severe asthma complicated with infection.

[0003] There are no existing reports on methods for determining the content of 15 amino acids from goat horn in Tanreqing injection. Summary of the Invention

[0004] Based on this, the present invention provides a method for determining the content of 15 amino acids from goat horn in Tanreqing injection, the method comprising the following steps:

[0005] The test solution and the reference solution were subjected to pre-column derivatization before detection. The phlegm-clearing injection was composed of Scutellaria baicalensis, bear bile powder, goat horn, honeysuckle, forsythia, and propylene glycol. The reference standard consisted of 15 amino acids.

[0006] The chromatographic conditions for this detection are as follows: a column packed with octadecylsilane-bonded silica gel is used; mobile phase A is an aqueous solution of acid, an aqueous solution of alkaline solution, and / or an aqueous solution of buffer salt; mobile phase B is selected from one or more of acetonitrile, methanol, tetrahydrofuran, and water; the gradient elution program is as follows: 0–1 min, 1% B, 1–16 min, 1–40% B, 16–20 min, 40–60% B, 20.5–23 min, 100% B, 23–25 min, 1% B; the flow rate is 0.5–1.5 ml / min; the column temperature is 30–60 °C; the detection wavelength is 300 nm–350 nm during the first 16 min; and the detection wavelength is switched to 250 nm–300 nm after elution of the lysine peak at 16 min.

[0007] Based on the test results, the content information of 15 amino acids from goat horn in the Tanreqing injection was obtained.

[0008] Furthermore, the 15 amino acids are aspartic acid, glutamic acid, serine, glycine, threonine, alanine, arginine, tyrosine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, and proline.

[0009] Furthermore, the preparation method of the test solution includes: weighing an appropriate amount of Tanreqing injection, adding hydrochloric acid solution for dilution, letting it stand, centrifuging and taking the supernatant to obtain the test solution.

[0010] Furthermore, the preparation method of the test solution includes: weighing an appropriate amount of Tanreqing injection, diluting it with hydrochloric acid solution with a concentration of 0.01-0.5 mol / L, for example, about 0.1 mol / L, letting it stand, centrifuging and taking the supernatant to obtain the test solution.

[0011] Furthermore, the method for preparing the reference solution includes: weighing appropriate amounts of aspartic acid, glutamic acid, serine, glycine, threonine, alanine, arginine, tyrosine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, and proline, and adding hydrochloric acid solution with a concentration of 0.01–0.5 mol / L, for example, about 0.1 mol / L, to prepare a reference solution containing 2 μg–2 mg of each amino acid per ml.

[0012] Furthermore, the pre-column derivatization involves first mixing the test solution and / or the reference solution with a phthalaldehyde solution in borate buffer, and then mixing with a fluorenyl chloroformate solution.

[0013] Furthermore, the pH of this borate buffer solution is approximately 10.2.

[0014] Furthermore, the concentration of the borate buffer is approximately 0.4 mol / L.

[0015] Furthermore, the amount of borate buffer added is approximately seven times the amount of the test solution or the reference solution.

[0016] Furthermore, the concentration of the phthalaldehyde solution is approximately 1%.

[0017] Furthermore, the concentration of the 9-fluorenyl methyl chloroformate solution is approximately 0.5%.

[0018] Furthermore, the mixture was mixed with the phthalaldehyde solution 25 times.

[0019] Furthermore, the mixture was mixed with the 9-fluorenyl methyl chloroformate solution 50 times.

[0020] Furthermore, the chromatographic column is an Agilent Poroshell HPH-C. 18 Chromatographic column (100mm × 4.6mm, 2.7μm), Waters Xbridge C 18 Chromatographic column (100 mm × 4.6 mm, 3.5 μm) or Therom Hypersil-ODS column (150 mm × 4.6 mm, 5 μm).

[0021] 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.

[0022] 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.

[0023] Furthermore, the buffer salt solution is an aqueous solution of phosphate and / or an aqueous solution of acetate.

[0024] Furthermore, the pH value of this buffer salt solution is approximately 8.0.

[0025] Furthermore, the buffer salt is disodium hydrogen phosphate and / or sodium dihydrogen phosphate dihydrate.

[0026] Furthermore, the preparation method of the buffer salt solution includes: taking 1.50g of disodium hydrogen phosphate and 0.15g of sodium dihydrogen phosphate dihydrate, and adding water to dissolve them into 1000ml.

[0027] Furthermore, the mobile phase B is a mixed solution of methanol, acetonitrile, and water.

[0028] Furthermore, the mobile phase B is methanol:acetonitrile:water (45:45:10).

[0029] Furthermore, the flow rate is 0.8–1.2 ml / min, for example, about 1.0 ml / min.

[0030] Furthermore, the column temperature is 35–55°C, for example, about 45°C.

[0031] Furthermore, the detection wavelength is 338 nm from 0 to 16 min, and the detection wavelength is changed to 262 nm after 16 min or after the lysine chromatographic peak elution.

[0032] Furthermore, the theoretical plate number of this alanine is not less than 20,000.

[0033] Furthermore, this information is based on the peak areas recorded in the chromatograms of the test solution and the reference solution, and the content of 15 amino acids from goat horn in the Tanreqing injection is calculated using the standard curve method.

[0034] According to another aspect of the present invention, a method for constructing a fingerprint profile of 15 amino acids from goat horn in Tanreqing injection is provided, the method comprising the following steps:

[0035] Preparation of the test solution: Weigh an appropriate amount of Tanreqing injection, dilute with hydrochloric acid solution, let stand, centrifuge and take the supernatant to obtain the test solution;

[0036] Preparation of reference solution: Weigh appropriate amounts of aspartic acid, glutamic acid, serine, glycine, threonine, alanine, arginine, tyrosine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, and proline, and add hydrochloric acid solution with a concentration of 0.01–0.5 mol / L, for example, about 0.1 mol / L, to prepare a reference solution containing 2 μg–2 mg of each amino acid per ml;

[0037] Based on the results of high-performance liquid chromatography (HPLC) analysis of the test solution and the reference solution after pre-column derivatization, the fingerprint spectrum of the traditional Chinese medicine compound was obtained.

[0038] The chromatographic conditions for high performance liquid chromatography (HPLC) detection are as follows: a column packed with octadecylsilane-bonded silica gel is used; mobile phase A is an aqueous solution of acid, an aqueous solution of alkaline solution, and / or an aqueous solution of buffer salt; mobile phase B is selected from one or more of acetonitrile, methanol, tetrahydrofuran, and water; the gradient elution program is as follows: 0–1 min, 1% B, 1–16 min, 1–40% B, 16–20 min, 40–60% B, 20.5–23 min, 100% B, 23–25 min, 1% B; the flow rate is 0.5–1.5 ml / min; the column temperature is 30℃–60℃; the detection wavelength is 300 nm–350 nm during the 0–16 min period; and the detection wavelength is switched to 250 nm–300 nm after elution of the lysine peak at 16 min.

[0039] Furthermore, the Tanreqing injection is prepared from 100g of Scutellaria baicalensis, 30g of bear bile powder, 25g of goat horn, 100g of honeysuckle, 80g of forsythia, and 100ml of propylene glycol.

[0040] Furthermore, the volume of the Tanreqing injection is 1000ml.

[0041] Furthermore, the pre-column derivatization involves first mixing the test solution and / or the reference solution with a phthalaldehyde solution in borate buffer, and then mixing with a fluorenyl chloroformate solution.

[0042] Furthermore, the pH of this borate buffer solution is approximately 10.2.

[0043] Furthermore, the concentration of the borate buffer is approximately 0.4 mol / L.

[0044] Furthermore, the amount of borate buffer added is approximately seven times the amount of the test solution or the reference solution.

[0045] Furthermore, the concentration of the phthalaldehyde solution is approximately 1%.

[0046] Furthermore, the concentration of the 9-fluorenyl methyl chloroformate solution is approximately 0.5%.

[0047] Furthermore, the mixture was mixed with the phthalaldehyde solution 25 times.

[0048] Furthermore, the mixture was mixed with the 9-fluorenyl methyl chloroformate solution 50 times.

[0049] Furthermore, the chromatographic column is an Agilent Poroshell HPH-C. 18 Chromatographic column (100mm × 4.6mm, 2.7μm), Waters Xbridge C 18 Chromatographic column (100 mm × 4.6 mm, 3.5 μm) or Therom Hypersil-ODS column (150 mm × 4.6 mm, 5 μm).

[0050] Furthermore, the flow rate is 0.8–1.2 ml / min, for example, about 1.0 ml / min.

[0051] Furthermore, the column temperature is 35–55°C, for example, about 45°C.

[0052] Furthermore, the detection wavelength is 338 nm from 0 to 16 min, and the detection wavelength is changed to 262 nm after 16 min or after the lysine chromatographic peak elution.

[0053] Furthermore, the fingerprint chromatograms obtained by detecting aspartic acid, glutamic acid, serine, glycine, threonine, alanine, arginine, tyrosine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, and proline included peaks 1-16. Among them, peak 7 was alanine as a reference peak, peak 1 was aspartic acid, peak 2 was glutamic acid, peak 3 was serine, peak 4 was glycine, peak 5 was threonine, peak 6 was arginine, peak 8 was tyrosine, peak 9 was valine, peak 10 was methionine, peak 11 was phenylalanine, peak 12 was isoleucine, peak 13 was leucine, peak 14 was lysine, peak 15 was an unknown chromatographic peak, and peak 16 was proline.

[0054] Furthermore, the retention times of peaks 1-16 were 1.0–1.5 min, 1.5–2.0 min, 4.5–5.0 min, 6.0–6.5 min, 6.5–7.0 min, 7.5–8.0 min, 8.0–8.5 min, 9.5–10.0 min, 12.0–12.5 min, 12.5–13.0 min, 14.0–14.5 min, 14.5–15.0 min, 15.0–15.5 min, 16.0–16.5 min, 16.5–17.0 min, and 19.0–19.5 min, respectively.

[0055] Furthermore, using peak 7 of alanine as the reference peak, the relative retention times of the other 15 peaks were as follows: peak 1 0.15±0.05, peak 2 0.23±0.05, peak 3 0.60±0.05, peak 4 0.77±0.05, peak 5 0.80±0.05, peak 6 0.96±0.05, peak 8 1.22±0.05, peak 9 1.5±0.05, peak 10 1.55±0.05, peak 11 1.74±0.05, peak 12 1.77±0.05, peak 13 1.88±0.05, peak 14 1.95±0.05, peak 15 2.04±0.05, and peak 16 2.37±0.05.

[0056] According to another aspect of the present invention, the above-described method is provided for use in the quality testing and / or quality evaluation and / or quality control of Tanreqing injection.

[0057] The beneficial effects of this invention are:

[0058] Compared to previous methods, the method of this invention has been optimized and supplemented, mainly in the following aspects: ① Optimized mobile phase composition, simplified method, shortened separation time, and reduced total analysis time to 25 min; ② Optimized OPA and FMOC derivatization methods, increased stability of the derivatization process, linear range to 1-250 μg / ml, and compatible with 20 μl autosamplers (UPLC); ③ Determination of 15 amino acids, with the total measurable amino acid content in horn extract reaching over 90%; ④ Optimized pretreatment method to eliminate the influence of interfering substances in the formulation; ⑤ The content determination method has been changed to the standard curve method. Due to the instability of FMOC derivatization, the standard curve method is less likely to detect various problems in the derivatization process, making the determination results more accurate. Attached Figure Description

[0059] 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.

[0060] Figure 1 This diagram illustrates the optimized preparation method of the test solution. In this diagram, A represents direct injection of the injection solution, and B represents injection after dilution five times with 0.1 mol / L hydrochloric acid.

[0061] Figure 2 This is a schematic diagram showing the relationship between derivatization time and peak area (number of mixing cycles). In this diagram, A represents the 14 primary amino acids derived from OPA; B represents proline derived from FMOC.

[0062] Figure 3 This is a schematic diagram of the standard fingerprint of Tanreqing Injection. In the diagram, 1 represents aspartic acid, 2 represents glutamic acid, 3 represents serine, 4 represents glycine, 5 represents threonine, 6 represents arginine, 7 represents alanine, 8 represents tyrosine, 9 represents valine, 10 represents methionine, 11 represents phenylalanine, 12 represents isoleucine, 13 represents leucine, 14 represents lysine, and 16 represents proline. The chromatographic column used is an Agilent Poroshell HPH-C. 18 Chromatographic column (100 mm × 4.6 mm, 2.7 μm). Detailed Implementation

[0063] 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.

[0064] 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.

[0065] 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.

[0066] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0067] 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.

[0068] As described in the background section, there are no reports in the prior art regarding methods for determining the content of 15 amino acids from goat horn in Tanreqing injection. To address this problem, the present invention provides a method for determining the content of 15 amino acids from goat horn in Tanreqing injection, the method comprising the following steps:

[0069] The test solution and the reference solution were subjected to pre-column derivatization before detection. The phlegm-clearing injection was composed of Scutellaria baicalensis, bear bile powder, goat horn, honeysuckle, forsythia, and propylene glycol. The reference standard consisted of 15 amino acids.

[0070] The chromatographic conditions for this detection are as follows: a column packed with octadecylsilane-bonded silica gel is used; mobile phase A is an aqueous solution of acid, an aqueous solution of alkaline solution, and / or an aqueous solution of buffer salt; mobile phase B is selected from one or more of acetonitrile, methanol, tetrahydrofuran, and water; the gradient elution program is as follows: 0–1 min, 1% B, 1–16 min, 1–40% B, 16–20 min, 40–60% B, 20.5–23 min, 100% B, 23–25 min, 1% B; the flow rate is 0.5–1.5 ml / min; the column temperature is 30–60 °C; the detection wavelength is 300 nm–350 nm during the first 16 min; and the detection wavelength is switched to 250 nm–300 nm after elution of the lysine peak at 16 min.

[0071] Based on the test results, the content information of 15 amino acids from goat horn in the Tanreqing injection was obtained.

[0072] In this invention, when flow rate, temperature, wavelength, concentration, time, pressure, proportion, equivalent, concentration, 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 “30–60” is disclosed, the described range should be interpreted as including ranges “30–60”, “30–55”, “30–50”, “30–45”, “30–40”, “30–35”, “35–60”, “40–60”, “45–60”, “50–60”, “55–60”, 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.

[0073] In a preferred embodiment, the 15 amino acids are aspartic acid, glutamic acid, serine, glycine, threonine, alanine, arginine, tyrosine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, and proline.

[0074] In a preferred embodiment, the preparation method of the test solution includes: weighing an appropriate amount of Tanreqing injection, adding hydrochloric acid solution for dilution, letting it stand, centrifuging and taking the supernatant to obtain the test solution.

[0075] In a preferred embodiment, the preparation method of the test solution includes: weighing an appropriate amount of Tanreqing injection, diluting it with hydrochloric acid solution with a concentration of 0.01-0.5 mol / L, for example, about 0.1 mol / L, letting it stand, centrifuging and taking the supernatant to obtain the test solution.

[0076] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.1" includes ±5% of 0.1, or from 0.095 to 0.105.

[0077] In a preferred embodiment, the method for preparing the reference solution includes: weighing appropriate amounts of aspartic acid, glutamic acid, serine, glycine, threonine, alanine, arginine, tyrosine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, and proline, and adding a hydrochloric acid solution with a concentration of 0.01–0.5 mol / L, for example, about 0.1 mol / L, to prepare a reference solution containing 2 μg–2 mg of each amino acid per ml.

[0078] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.1" includes ±5% of 0.1, or from 0.095 to 0.105.

[0079] In a preferred embodiment, the pre-column derivatization includes first mixing the test solution and / or the reference solution with a phthalaldehyde solution in a borate buffer, and then mixing with a fluorenyl chloroformate solution.

[0080] In a preferred embodiment, the pH of the borate buffer is about 10.2.

[0081] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10.2" includes ±5% of 10.2, or from 9.69 to 10.71.

[0082] In a preferred embodiment, the concentration of the borate buffer is about 0.4 mol / L.

[0083] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.4" includes ±5% of 0.4, or from 0.38 to 0.42.

[0084] In a preferred embodiment, the amount of borate buffer added is approximately seven times the amount of the test solution or the reference solution.

[0085] 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.

[0086] In a preferred embodiment, the concentration of the phthalaldehyde solution is about 1%.

[0087] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0088] In a preferred embodiment, the concentration of the 9-fluorenyl methyl chloroformate solution is about 0.5%.

[0089] 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.

[0090] In a preferred embodiment, the mixture is mixed with the phthalaldehyde solution 25 times.

[0091] In a preferred embodiment, the mixture is mixed with the 9-fluorenyl methyl chloroformate solution 50 times.

[0092] In a preferred embodiment, the chromatographic column is an Agilent Poroshell HPH-C column. 18Chromatographic column (100mm × 4.6mm, 2.7μm), Waters Xbridge C 18 Chromatographic column (100 mm × 4.6 mm, 3.5 μm) or Therom Hypersil-ODS column (150 mm × 4.6 mm, 5 μm).

[0093] 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.

[0094] 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.

[0095] In a preferred embodiment, the buffer salt solution is an aqueous solution of phosphate and / or an aqueous solution of acetate.

[0096] In a preferred embodiment, the pH value of the buffer salt solution is about 8.0.

[0097] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 8" includes ±5% of 8, or from 7.6 to 8.4.

[0098] In a preferred embodiment, the buffer salt is disodium hydrogen phosphate and / or sodium dihydrogen phosphate dihydrate.

[0099] In a preferred embodiment, the preparation method of the buffer saline solution includes: taking 1.50g of disodium hydrogen phosphate and 0.15g of sodium dihydrogen phosphate dihydrate, and adding water to dissolve them into 1000ml.

[0100] In a preferred embodiment, the mobile phase B is a mixed solution of methanol, acetonitrile, and water.

[0101] In a preferred embodiment, the mobile phase B is methanol:acetonitrile:water (45:45:10).

[0102] In a preferred embodiment, the flow rate is 0.8 to 1.2 ml / min, for example, about 1.0 ml / min.

[0103] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0104] In a preferred embodiment, the column temperature is 35–55°C, for example, about 45°C.

[0105] 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.

[0106] In a preferred embodiment, the detection wavelength is 338 nm for 0-16 min, and the detection wavelength is changed to 262 nm after 16 min or after the lysine chromatographic peak elution.

[0107] In a preferred embodiment, the theoretical plate number of the alanine is not less than 20,000.

[0108] In a preferred embodiment, the information is calculated by using the standard curve method to determine the content of 15 amino acids from goat horn in the Tanreqing injection, based on the corresponding peak areas in the recorded chromatograms of the test solution and the reference solution.

[0109] According to another aspect of the present invention, a method for constructing a fingerprint profile of 15 amino acids from goat horn in Tanreqing injection is provided, the method comprising the following steps:

[0110] Preparation of the test solution: Weigh an appropriate amount of Tanreqing injection, dilute with hydrochloric acid solution, let stand, centrifuge and take the supernatant to obtain the test solution;

[0111] Preparation of reference solution: Weigh appropriate amounts of aspartic acid, glutamic acid, serine, glycine, threonine, alanine, arginine, tyrosine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, and proline, and add hydrochloric acid solution with a concentration of 0.01–0.5 mol / L, for example, about 0.1 mol / L, to prepare a reference solution containing 2 μg–2 mg of each amino acid per ml;

[0112] Based on the results of high-performance liquid chromatography (HPLC) analysis of the test solution and the reference solution after pre-column derivatization, the fingerprint spectrum of the traditional Chinese medicine compound was obtained.

[0113] The chromatographic conditions for high performance liquid chromatography (HPLC) detection are as follows: a column packed with octadecylsilane-bonded silica gel is used; mobile phase A is an aqueous solution of acid, an aqueous solution of alkaline solution, and / or an aqueous solution of buffer salt; mobile phase B is selected from one or more of acetonitrile, methanol, tetrahydrofuran, and water; the gradient elution program is as follows: 0–1 min, 1% B, 1–16 min, 1–40% B, 16–20 min, 40–60% B, 20.5–23 min, 100% B, 23–25 min, 1% B; the flow rate is 0.5–1.5 ml / min; the column temperature is 30℃–60℃; the detection wavelength is 300 nm–350 nm during the 0–16 min period; and the detection wavelength is switched to 250 nm–300 nm after elution of the lysine peak at 16 min.

[0114] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0115] In a preferred embodiment, the Tanreqing injection is prepared from 100g of Scutellaria baicalensis, 30g of bear bile powder, 25g of goat horn, 100g of honeysuckle, 80g of forsythia, and 100ml of propylene glycol.

[0116] In a preferred embodiment, the volume of the Tanreqing injection is 1000 ml.

[0117] In a preferred embodiment, the pre-column derivatization includes first mixing the test solution and / or the reference solution with a phthalaldehyde solution in a borate buffer, and then mixing with a fluorenyl chloroformate solution.

[0118] In a preferred embodiment, the pH of the borate buffer is about 10.2.

[0119] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10.2" includes ±5% of 10.2, or from 9.69 to 10.71.

[0120] In a preferred embodiment, the concentration of the borate buffer is about 0.4 mol / L.

[0121] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.4" includes ±5% of 0.4, or from 0.38 to 0.42.

[0122] In a preferred embodiment, the amount of borate buffer added is approximately seven times the amount of the test solution or the reference solution.

[0123] 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.

[0124] In a preferred embodiment, the concentration of the phthalaldehyde solution is about 1%.

[0125] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0126] In a preferred embodiment, the concentration of the 9-fluorenyl methyl chloroformate solution is about 0.5%.

[0127] 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.

[0128] In a preferred embodiment, the mixture is mixed with the phthalaldehyde solution 25 times.

[0129] In a preferred embodiment, the mixture is mixed with the 9-fluorenyl methyl chloroformate solution 50 times.

[0130] In a preferred embodiment, the chromatographic column is an Agilent Poroshell HPH-C column. 18 Chromatographic column (100mm × 4.6mm, 2.7μm), Waters Xbridge C 18 Chromatographic column (100 mm × 4.6 mm, 3.5 μm) or Therom Hypersil-ODS column (150 mm × 4.6 mm, 5 μm).

[0131] In a preferred embodiment, the flow rate is 0.8 to 1.2 ml / min, for example, about 1.0 ml / min.

[0132] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0133] In a preferred embodiment, the column temperature is 35–55°C, for example, about 45°C.

[0134] 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.

[0135] In a preferred embodiment, the detection wavelength is 338 nm for 0-16 min, and the detection wavelength is changed to 262 nm after 16 min or after the lysine chromatographic peak elution.

[0136] In a preferred embodiment, the fingerprint spectrum obtained by detecting aspartic acid, glutamic acid, serine, glycine, threonine, alanine, arginine, tyrosine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, and proline includes peaks 1-16, wherein peak 7 is alanine as a reference peak, peak 1 is aspartic acid, peak 2 is glutamic acid, peak 3 is serine, peak 4 is glycine, peak 5 is threonine, peak 6 is arginine, peak 8 is tyrosine, peak 9 is valine, peak 10 is methionine, peak 11 is phenylalanine, peak 12 is isoleucine, peak 13 is leucine, peak 14 is lysine, peak 15 is an unknown chromatographic peak, and peak 16 is proline.

[0137] In a preferred embodiment, the retention times of peaks 1-16 are 1.0-1.5 min, 1.5-2.0 min, 4.5-5.0 min, 6.0-6.5 min, 6.5-7.0 min, 7.5-8.0 min, 8.0-8.5 min, 9.5-10.0 min, 12.0-12.5 min, 12.5-13.0 min, 14.0-14.5 min, 14.5-15.0 min, 15.0-15.5 min, 16.0-16.5 min, 16.5-17.0 min, and 19.0-19.5 min, respectively.

[0138] In a preferred embodiment, the chromatographic peak 7 of alanine is used as the reference peak, and the relative retention times of the other 15 peaks are as follows: peak 1 0.15±0.05, peak 2 0.23±0.05, peak 3 0.60±0.05, peak 4 0.77±0.05, peak 5 0.80±0.05, peak 6 0.96±0.05, peak 8 1.22±0.05, peak 9 1.5±0.05, peak 10 1.55±0.05, peak 11 1.74±0.05, peak 12 1.77±0.05, peak 13 1.88±0.05, peak 14 1.95±0.05, peak 15 2.04±0.05, and peak 16 2.37±0.05.

[0139] According to another aspect of the present invention, the above-described method is provided for use in the quality testing and / or quality evaluation and / or quality control of Tanreqing injection.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] Example

[0144] 1. Experimental equipment and materials

[0145] Agilent 1260 and Agilent 1290 high performance liquid chromatographs (Agilent Technologies, USA), Millipore Milli-Q pure water system (Millipore, USA), Sartorius CP224S and MSU225P-1CE-DU analytical balances (Sartorius GmbH, Germany), ultrasonic instrument (Shanghai Kedao Ultrasonic Instrument Co., Ltd.), and drying oven (BINDER GmbH, Germany).

[0146] Aspartic acid (Asp), glutamic acid (Glu), serine (Ser), glycine (Gly), threonine (Thr), alanine (Ala), arginine (Arg), tyrosine (Tyr), valine (Val), methionine (Met), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), lysine hydrochloride (Lys), proline (Pro) (all of the above are 140624-201506, ≥99.9%), and L-hydroxyproline (111578-201602) were all purchased from the China National Institutes for Food and Drug Control. Tanreqing Injection (provided by Shanghai Kaibao Pharmaceutical Co., Ltd.)

[0147] Phthalate (OPA, Pickering Laboratories); fluorene methyl chloroformate (FMOC, Sigma-Aldrich, 97%); mercaptopropionic acid (AlfaAesar 99%); methanol, acetonitrile, and tetrahydrofuran were chromatographically pure (MERCK); boric acid, sodium hydroxide, and other reagents were all analytically pure and purchased from China National Pharmaceutical Group Shanghai Chemical Reagent Co., Ltd.

[0148] 2. Experimental Methods and Results

[0149] 2.1 Selection of detection wavelength

[0150] Derivatives of primary amino acids (aspartic acid, glutamic acid, serine, glycine, threonine, arginine, alanine, tyrosine, valine, methionine, phenylalanine, isoleucine, leucine, and lysine) showed significant UV absorption at a detection wavelength of 338 nm, while derivatives of secondary amino acids (proline) showed significant UV absorption at a detection wavelength of 262 nm. Furthermore, the derivatizing reagents exhibited low UV absorption at both detection wavelengths. Diode array scanning results were consistent with those described in the literature, and the UV spectra of the 14 primary amino acid derivatives were completely identical. Therefore, 338 nm was used for measurement, and 262 nm was used for measurement of secondary amino acids.

[0151] 2.2 Optimization of chromatographic conditions

[0152] The mobile phase B was selected as methanol:acetonitrile:water (45:45:10). Mobile phase A consisted of four different systems: ① a buffer solution containing 10 mM disodium hydrogen phosphate and 10 mM sodium borate, adjusted to pH 8.0 with hydrochloric acid; ② a solution containing 10 mM disodium hydrogen phosphate, adjusted to pH 8.0 with hydrochloric acid; ③ a solution containing 10 mM disodium hydrogen phosphate and 0.01% triethylamine, adjusted to pH 8.0 with hydrochloric acid; ④ a solution of 1.50 g of disodium hydrogen phosphate and 0.15 g of sodium dihydrogen phosphate (containing two molecules of water of crystallization) (i.e., sodium dihydrogen phosphate dihydrate), dissolved in water to make 1000 ml. The results showed that the mobile phase in method ④ provided the best separation of the 15 amino acids, with the optimal peak shape. Gradient elution according to Table 1 also resulted in good separation of the 15 amino acids.

[0153] Table 1 Gradients for amino acid content determination

[0154]

[0155]

[0156] Using the above mobile phase system, 15 amino acid reference standards and injection samples were determined at column temperatures of 30℃, 35℃, 40℃, 45℃ and 50℃, respectively. The results showed that the separation of each amino acid was better and the peak shape was optimal at a column temperature of 45℃.

[0157] 2.3 Optimization of the preparation method of the test solution

[0158] When the assay was performed using a direct injection method, a very large interference peak was observed between methionine and phenylalanine (e.g., Figure 1 (As shown in A). The pretreatment method was optimized, and the following four pretreatment methods were compared: ① Take this product as the test solution; ② Take 2 ml of this product, place it in a 5 ml volumetric flask, add 0.1 mol / L hydrochloric acid solution to the mark, centrifuge, and collect the supernatant; ③ Take 1 ml of this product, place it in a 5 ml volumetric flask, add 0.1 mol / L hydrochloric acid solution to the mark, centrifuge, and collect the supernatant; ④ Take 1 ml of this product, place it in a stoppered glass bottle, add 5 ml of 6 mol / L hydrochloric acid solution, hydrolyze at 150℃ for 1 hour, cool, transfer to an evaporating dish, wash with 20 ml of water in portions, combine the washings in the evaporating dish, evaporate to dryness, accurately dissolve the residue in 5 ml of 0.1 mol / L hydrochloric acid solution, centrifuge, and collect the supernatant. The results showed that method ③, i.e., diluting the sample 5 times with 0.1 mol / L hydrochloric acid, can completely eliminate the influence of interfering peaks (e.g., ...). Figure 1 (As shown in B). However, some precipitate may form during sample injection. Therefore, method ③ was optimized again. After dilution, the sample was left to stand for 0.5h, 1h, 2h, 4h, and overnight. The supernatant was then centrifuged and placed at room temperature to observe the precipitation. The results showed that overnight standing allowed complete precipitation. Therefore, the final sample solution treatment method is as follows: Take 1ml of this product, place it in a 5ml volumetric flask, add 0.1mol / L hydrochloric acid solution to the mark, let stand overnight, centrifuge, and collect the supernatant.

[0159] 2.4 Optimization of Derivatization Procedure Sample Introduction Method

[0160] The dosage, preparation method, and derivatization time of the derivatization reagents were optimized. The injection volume was calculated based on 250 μg / ml for each of the 15 amino acids, with an injection volume of 1 μl. The reaction ratio of OPA to amino acids was 1:1; the reaction ratio of FMOC to amino acids was 2:1, with the derivatization reagent in excess by 2-5 times compared to the amino acids. The dosage of 1% OPA and 0.5% FMOC was 1 μl each, while borate buffer required more than 5 μl. Considering the peak shape of the amino acids, 7 μl of borate buffer was ultimately selected.

[0161] In this study, the derivatization time of OPA and FMOC was optimized at a rate of 500 μl per minute, with OPA and FMOC derivatization times of 5, 10, 25, 50, 75, and 99 times, respectively. Figure 2 A and Figure 2 (As shown in B), the optimal time for the derivatization reaction was observed by the peak area of ​​the derived amino acids. Finally, OPA was mixed 25 times and FMOC was mixed 50 times.

[0162] Since there are many influencing factors during the derivatization process, the single-point method for calculating the average correction factor may introduce large errors. Therefore, in this study, the standard curve method was used to determine the content of 15 amino acids.

[0163] 2.5 Determination of Reference Frame

[0164] Alanine is one of the main amino acid components in goat horn extract, accounting for 12% of the total amino acid content in the injection. Its content is very stable, with an RSD of only 7.7% in 33 batches of injection. In addition, its chromatographic peak has a moderate retention time among 16 characteristic peaks, so it was selected as a reference.

[0165] 2.6 Methodological Validation

[0166] 2.6.1 Generation of reference maps

[0167] Twenty batches of samples produced in 2017 and 2018 were selected to simulate and generate a control fingerprint spectrum of goat horn in Tanreqing Injection (e.g., Figure 3 (As shown).

[0168] 2.6.2 Specificity

[0169] The reference solution, preparation (batch number 1803221), and blank sample (without goat horn) were pretreated according to the above pretreatment method and analyzed under the above liquid chromatography conditions: The results showed that the chromatogram of the test sample had chromatographic peaks with retention times consistent with those of the reference standards of aspartic acid, glutamic acid, serine, glycine, threonine, alanine, arginine, tyrosine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, and proline, and were well separated from the chromatographic peaks of other components. The blank sample (without goat horn) showed no interference. Peak 15 of the unknown substance was well separated from the chromatographic peaks of other components, and the blank sample (without goat horn) showed no interference.

[0170] 2.6.3 Sample Injection Repeatability

[0171] Accurately pipette 1 μL of the test solution and perform online pre-column derivatization using 1% o-phthalaldehyde (OPA) solution and 0.5% fluorene methyl chloroformate (FMOC) solution. Inject the sample six times consecutively and record the chromatograms. Calculate the similarity using software for evaluating the similarity of chromatographic fingerprints of traditional Chinese medicine. The similarity scores were all above 0.99, indicating that the method has good precision.

[0172] Accurately pipette 1 μl of the reference solution (ST4) and perform online pre-column derivatization using 1% o-phthalaldehyde (OPA) solution and 0.5% fluorene methyl chloroformate (FMOC) solution. Inject the sample six times consecutively and record the peak areas of 15 amino acids. The results showed a precision of 1.6% to 2.1%, indicating that the method has good precision and meets the requirements for quantitative determination.

[0173] 2.6.4 Repeatability Test

[0174] Take six samples (batch number 1803221), record the chromatograms according to the method of this invention, and calculate the similarity using the Chinese medicine chromatographic fingerprint similarity evaluation software. The similarity of the fingerprint chromatograms is above 0.99, indicating that the method has good repeatability.

[0175] Six samples (batch number 1803221) were taken and their contents were determined according to the method of this invention. The results showed that the total RSD of the 15 amino acids was 2.6%. The RSD of each of the 15 amino acids was calculated. Except for methionine (10Met), whose RSD was 6.7%, the RSD of the other amino acids was within 5%, indicating that the method has good repeatability.

[0176] 2.6.5 Stability Test

[0177] Samples (batch number 1803221) were injected and analyzed at 0, 12, 24 and 48 hours. Chromatograms were recorded, and similarity was calculated using software for evaluating the similarity of chromatographic fingerprints of traditional Chinese medicine. The similarity of the fingerprints was above 0.98, indicating that the test solution remained basically stable within 24 hours.

[0178] A sample (batch number 1803221) was taken and a test solution was prepared according to the method of the present invention. The sample was injected and analyzed at 0h, 12h, 24h and 48h after preparation. The results showed that among the 15 amino acids, except for 6-arginine, 8-tyrosine, 10-methionine and 14-lysine, which had larger RSDs due to smaller peak areas (the actual absolute value fluctuation of the peak area was within 2), the RSDs of the other amino acids injected within two days were within 5%, indicating that the method can basically remain stable when the sample is injected within 2 days.

[0179] 2.6.6 Investigation of different chromatographic columns

[0180] Two copies of the sample (batch number 1803221) were taken and analyzed using three different chromatographic columns. The similarity was calculated using software for evaluating the similarity of chromatographic fingerprints of traditional Chinese medicine. The similarity of the fingerprints was above 0.94.

[0181] 2.6.7 Linearity and Linear Range

[0182] Accurately weigh appropriate amounts of aspartic acid, glutamic acid, serine, glycine, threonine, alanine, arginine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, and proline reference standards, and add 0.1 mol / L hydrochloric acid solution to prepare a solution containing 10 mg of each per ml; accurately weigh tyrosine, add 0.1 mol / L hydrochloric acid solution to prepare a solution containing 2 mg per ml, and use these as reference standard stock solutions.

[0183] Accurately measure an appropriate amount of the above reference standards and add 0.1 mol / L hydrochloric acid solution to prepare a solution of 250 μg per ml as ST6. Take 8 ml of ST6 and place it in a 10 ml volumetric flask, then dilute to the mark with 0.1 mol / L hydrochloric acid solution to obtain ST5 (200 μg / ml); take 4 ml of ST6 and place it in a 10 ml volumetric flask, then dilute to the mark with 0.1 mol / L hydrochloric acid solution to obtain ST4 (100 μg / ml); take 2 ml of ST6 and place it in a 10 ml volumetric flask, then dilute to the mark with 0.1 mol / L hydrochloric acid solution to obtain ST3 (50 μg / ml); take 1 ml of ST4 and place it in a 10 ml volumetric flask, then dilute to the mark with 0.1 mol / L hydrochloric acid solution to obtain ST2 (10 μg / ml); take 2 ml of ST2 and place it in a 10 ml volumetric flask, then dilute to the mark with 0.1 mol / L hydrochloric acid solution to obtain ST1 (2 μg / ml).

[0184] Accurately pipette 1 μl each of the above mixed reference solutions ST1-6, and perform pre-column derivatization using a computer-simulated method with 1% o-phthalaldehyde (OPA) solution and 0.5% fluorene methyl chloroformate (FMOC) solution. Inject the solutions into the liquid chromatograph and record the peak areas. Plot a standard curve with the injection concentration (μg / ml) on the x-axis and the peak area on the y-axis. Regression analysis showed that aspartic acid, glutamic acid, serine, glycine, threonine, arginine, alanine, tyrosine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, and proline all exhibited good linear relationships, meeting the requirements for quantitative determination.

[0185] 2.6.8 Accuracy Test

[0186] Accurately pipette appropriate amounts of the reference stock solution and add 0.1 mol / L hydrochloric acid solution to prepare reference solutions containing the concentrations of 15 amino acids per 1 ml as shown in Table 2. (The same stock solution was used as the control, and the results were not multiplied by the control coefficient for ease of calculation).

[0187] Table 215 Concentrations of Amino Acid Sample Control Solutions

[0188]

[0189]

[0190] Take 0.5 ml of this product (batch number 1803221), make nine portions, and place them in 5 ml volumetric flasks. Divide the portions into groups of three: 0.8 ml, 1 ml, and 1.2 ml. Add 0.1 mol / L hydrochloric acid, dilute to the mark, shake well, let stand overnight, centrifuge, and collect the supernatant. Inject each portion into a liquid chromatograph for determination.

[0191] The recoveries of the 15 amino acids ranged from 92.3% to 109.3%. Except for arginine (6Arg, 6.0%), methionine (10Met, 6.0%), and lysine (14Lys, 8.0%), the RSDs of the other 11 amino acids were all within 5%. This indicates that the method has good accuracy and meets the requirements for quantitative determination.

[0192] 2.6.9 Intermediate Precision Examination

[0193] To investigate the effect of random variations on precision, the content of three batches of samples was determined by different analysts on different dates using different instruments and equipment, according to the method of this invention.

[0194] The results showed that the results obtained by different analysts on different dates and with different instruments were consistent, indicating that the intermediate precision of the method was good.

[0195] 2.7 Sample Determination

[0196] A total of 33 batches of Tanreqing injection were analyzed using this method, including 13 batches in 2016, 10 batches in 2017, and 10 batches in 2018. All samples were used to generate control fingerprint chromatograms using the average value method. The similarity between each batch of samples and the control fingerprint chromatograms was calculated. The results are shown in Table 3. The similarity was greater than 0.98 for all samples.

[0197] Table 3. Similarity evaluation results of amino acid fingerprint profiles

[0198]

[0199]

[0200] According to the method of the present invention, 33 batches of samples were measured, and the calculation results of 15 amino acids are shown in Table 4.

[0201] Table 4. Sample Content Determination

[0202]

[0203]

[0204] 2.8 Standards and Limits

[0205] Based on the above methodological investigation and sample test results, the similarity of the horn fingerprint spectrum in Tanreqing Injection is tentatively set at "not less than 0.90".

[0206] 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 15 kinds of amino acids in Shrentqing injection, characterized in that, The method comprises the following steps: The test sample solution and the control sample solution are detected after pre-column derivatization, wherein the Tanreqing injection is composed of Huangqi, Xiongdan powder, Yangga jiang, Jinyinhua, Lianqiao and propylene glycol, and the control sample is 15 kinds of amino acids, The chromatographic conditions of the detection are as follows: a chromatographic column with octadecylsilane bonded silica as the filler is used, the mobile phase A is an acid aqueous solution, an alkali aqueous solution and / or a buffer salt aqueous solution, the mobile phase B is selected from one or more of acetonitrile, methanol, tetrahydrofuran and water, the gradient elution program is as follows: 0-1 min, 1% B, 1-16 min, 1-40% B, 16-20 min, 40-60% B, 20.5-23 min, 100% B, 23-25 min, 1% B, the flow rate is 0.5-1.5 ml / min, the column temperature is 30-60℃, and the detection wavelength is 300 nm-350 nm at 0-16 min, and the detection wavelength is converted to 250 nm-300 nm at 16 min or after the lysine chromatographic peak elutes; According to the detection result, the content information of the 15 kinds of amino acids in the Tanreqing injection is obtained.

2. The method of claim 1, wherein, The 15 kinds of amino acids are aspartic acid, glutamic acid, serine, glycine, threonine, alanine, arginine, tyrosine, valine, methionine, phenylalanine, isoleucine, leucine, lysine and proline; Preferably, the preparation method of the test sample solution comprises the following steps: weighing an appropriate amount of the Tanreqing injection, adding a hydrochloric acid solution to dilute, placing, centrifuging to obtain the supernatant, and obtaining the test sample solution; Preferably, the preparation method of the test sample solution comprises the following steps: weighing an appropriate amount of the Tanreqing injection, adding a hydrochloric acid solution with a concentration of 0.01-0.5 mol / L, for example, about 0.1 mol / L to dilute, placing, centrifuging to obtain the supernatant, and obtaining the test sample solution; Preferably, the preparation method of the control sample solution comprises the following steps: weighing an appropriate amount of aspartic acid, glutamic acid, serine, glycine, threonine, alanine, arginine, tyrosine, valine, methionine, phenylalanine, isoleucine, leucine, lysine and proline, and adding a hydrochloric acid solution with a concentration of 0.01-0.5 mol / L, for example, about 0.1 mol / L to prepare a control sample solution containing each amino acid 2 μg-2 mg per 1 ml; Preferably, the pre-column derivatization comprises the following steps: first mixing the test sample solution and / or the control sample solution with an o-phthaldehyde solution in a boric acid buffer, and then mixing with a 9-fluorenylmethyl chloroformate solution; Preferably, the pH of the boric acid buffer is about 10.2; Preferably, the concentration of the boric acid buffer is about 0.4 mol / L; Preferably, the sample amount of the boric acid buffer is about 7 times the sample amount of the test sample solution or the sample amount of the control sample solution; Preferably, the concentration of the o-phthaldehyde solution is about 1%; Preferably, the concentration of the 9-fluorenylmethyl chloroformate solution is about 0.5%; Preferably, the number of times of mixing with the o-phthaldehyde solution is 25 times. Particularly preferably, the number of times of mixing with the 9-fluorenylmethyl chloroformate solution is 50 times; Especially preferably, the chromatographic column is an Agilent Poroshell HPH-C 18 chromatographic column (100 mm x 4.6 mm, 2.7 μm), Waters Xbridge C 18 chromatographic column (100 mm x 4.6 mm, 3.5 μm), or a Therom Hypersil-ODS chromatographic column (150 mm x 4.6 mm, 5 μm).

3. The method of claim 1, wherein, Preferably, the aqueous acid solution, aqueous alkali solution and / or aqueous buffer salt solution is selected from one or more of weak acids and salts thereof, weak alkalis and salts thereof at different concentrations; More preferably, the aqueous buffer salt solution is a phosphate aqueous solution and / or an acetate aqueous solution; More preferably, the pH value of the aqueous buffer salt solution is about 8.0; More preferably, the buffer salt is sodium phosphate dibasic and / or sodium phosphate dihydrogen dihydrate; More preferably, the preparation method of the aqueous buffer salt solution comprises: taking 1.50 g of sodium phosphate dibasic and 0.15 g of sodium phosphate dihydrogen dihydrate, and dissolving into 1000 ml of water; More preferably, the mobile phase B is a mixed solution of methanol, acetonitrile and water; More preferably, the mobile phase B is methanol: acetonitrile: water (45:45:10). The flow rate is 0.8-1.2 ml / min, for example about 1.0 ml / min; 4. The method of claim 1, wherein, Preferably, the column temperature is 35-55℃, for example about 45℃; Preferably, the detection wavelength is 338 nm at 0-16 min, and is converted to 262 nm after 16 min or the lysine chromatographic peak elutes. The theoretical plate number of alanine is not less than 20000.

5. The method of claim 1, wherein, The information is the content of the 15 kinds of amino acids of the Radix Trachelii in the TCM injection calculated according to the standard curve method according to the corresponding peak areas in the recorded chromatogram of the test product solution and the chromatogram of the control solution.

6. The method of claim 1, wherein, The method comprises the following steps:

7. A method for constructing a fingerprint spectrum of 15 kinds of amino acids in Tan Re Qing injection, characterized in that, Preparation of the test product solution: weigh an appropriate amount of the TCM injection, add hydrochloric acid solution for dilution, place, centrifuge to obtain the supernatant to obtain the test product solution; Preparation of the control solution: weigh an appropriate amount of aspartic acid, glutamic acid, serine, glycine, threonine, alanine, arginine, tyrosine, valine, methionine, phenylalanine, isoleucine, leucine, lysine and proline, and add a hydrochloric acid solution with a concentration of 0.01-0.5 mol / L, for example about 0.1 mol / L to prepare a control solution containing 2 μg-2 mg of each amino acid per 1 ml; According to the results of the high-performance liquid detection of the test product solution and the control solution after pre-column derivatization, the TCM compound fingerprint spectrum is obtained; More preferably, the aqueous buffer salt solution is a phosphate aqueous solution and / or an acetate aqueous solution; More preferably, the pH value of the aqueous buffer salt solution is about 8.0; More preferably, the buffer salt is sodium phosphate dibasic and / or sodium phosphate dihydrogen dihydrate; More preferably, the preparation method of the aqueous buffer salt solution comprises: taking 1.50 g of sodium phosphate dibasic and 0.15 g of sodium phosphate dihydrogen dihydrate, and dissolving into 1000 ml of water; More preferably, the mobile phase B is a mixed solution of methanol, acetonitrile and water; More preferably, the mobile phase B is methanol: acetonitrile: water (45:45:10). The flow rate is 0.8-1.2 ml / min, for example about 1.0 ml / min; Preferably, the column temperature is 35-55℃, for example about 45℃; Preferably, the detection wavelength is 338 nm at 0-16 min, and is converted to 262 nm after 16 min or the lysine chromatographic peak elutes. The theoretical plate number of alanine is not less than 20000. The information is the content of the 15 kinds of amino acids of the Radix Trachelii in the TCM injection calculated according to the standard curve method according to the corresponding peak areas in the recorded chromatogram of the test product solution and the chromatogram of the control solution. The chromatographic conditions of high performance liquid detection are as follows: a chromatographic column with octadecylsilane bonded silica as the filler, mobile phase A is acid aqueous solution, alkali aqueous solution and / or buffer salt aqueous solution, mobile phase B is selected from one or more of acetonitrile, methanol, tetrahydrofuran and water, the gradient elution program is as follows: 0-1 min, 1% B, 1-16 min, 1-40% B, 16-20 min, 40-60% B, 20.5-23 min, 100% B, 23-25 min, 1% B, the flow rate is 0.5-1.5 ml / min, the column temperature is 30-60 ℃, the detection wavelength is 300 nm-350 nm at 0-16 min, and the detection wavelength is converted to 250 nm-300 nm at 16 min or after the lysine chromatographic peak elutes.

8. The method of claim 7, wherein, The Tanreqing injection is prepared from Huangqin 100 g, Xiongdanfen 30 g, Yangtao 25 g, Jinyinhua 100 g, Lianqiao 80 g and propylene glycol 100 ml; Preferably, the volume of the Tanreqing injection is 1000 ml; More preferably, the pre-column derivatization comprises first mixing the test sample solution and / or the control sample solution with an o-phthalaldehyde solution in a boric acid buffer, and then mixing with a 9-fluorenylmethyl chloroformate solution; More preferably, the boric acid buffer has a pH of about 10.2; More preferably, the boric acid buffer has a concentration of about 0.4 mol / L; More preferably, the sample amount of the boric acid buffer is about 7 times the sample amount of the test sample solution or the sample amount of the control sample solution; More preferably, the o-phthalaldehyde solution has a concentration of about 1%; More preferably, the 9-fluorenylmethyl chloroformate solution has a concentration of about 0.5%; More preferably, the o-phthalaldehyde solution is mixed 25 times; More preferably, the 9-fluorenylmethyl chloroformate solution is mixed 50 times; Especially preferably, the chromatographic column is an Agilent Poroshell HPH-C 18 chromatographic column (100 mm x 4.6 mm, 2.7 μm), Waters Xbridge C 18 chromatographic column (100 mm x 4.6 mm, 3.5 μm), or a Therom Hypersil-ODS chromatographic column (150 mm x 4.6 mm, 5 μm); Especially preferably, the flow rate is 0.8-1.2 ml / min, for example about 1.0 ml / min; Especially preferably, the column temperature is 35-55 ℃, for example about 45 ℃; Especially preferably, the detection wavelength is 338 nm at 0-16 min, and the detection wavelength is converted to 262 nm at 16 min or after the lysine chromatographic peak elutes.

9. The construction method according to claim 7 or 8, characterized in that, The obtained fingerprint for detecting aspartic acid, glutamic acid, serine, glycine, threonine, alanine, arginine, tyrosine, valine, methionine, phenylalanine, isoleucine, leucine, lysine and proline comprises peaks 1-16, wherein peak 7 is alanine as a reference peak, peak 1 is aspartic acid, peak 2 is glutamic acid, peak 3 is serine, peak 4 is glycine, peak 5 is threonine, peak 6 is arginine, peak 8 is tyrosine, peak 9 is valine, peak 10 is methionine, peak 11 is phenylalanine, peak 12 is isoleucine, peak 13 is leucine, peak 14 is lysine, peak 15 is an unknown chromatographic peak, and peak 16 is proline; Preferably, the retention time of the peaks No. 1-16 is 1.0-1.5 min, 1.5-2.0 min, 4.5-5.0 min, 6.0-6.5 min, 6.5-7.0 min, 7.5-8.0 min, 8.0-8.5 min, 9.5-10.0 min, 12.0-12.5 min, 12.5-13.0 min, 14.0-14.5 min, 14.5-15.0 min, 15.0-15.5 min, 16.0-16.5 min, 16.5-17.0 min, 19.0-19.5 min, respectively. Preferably, taking the 7th chromatographic peak of alanine as the reference peak, the relative retention time of the other 15 peaks is 1st chromatographic peak 0.15±0.05, 2nd chromatographic peak 0.23±0.05, 3rd chromatographic peak 0.60±0.05, 4th chromatographic peak 0.77±0.05, 5th chromatographic peak 0.80±0.05, 6th chromatographic peak 0.96±0.05, 8th chromatographic peak 1.22±0.05, 9th chromatographic peak 1.5±0.05, 10th chromatographic peak 1.55±0.05, 11th chromatographic peak 1.74±0.05, 12th chromatographic peak 1.77±0.05, 13th chromatographic peak 1.88±0.05, 14th chromatographic peak 1.95±0.05, 15th chromatographic peak 2.04±0.05, 16th chromatographic peak 2.37±0.05, respectively.

10. Use of the method according to any one of claims 1 to 9 in the quality detection and / or quality evaluation and / or quality control of Tanre Qing injection.

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