Quality detection method and application of liver-warming decoction

Abstract

The invention provides a quality detection method and application of a liver-warming decoction, and belongs to the technical field of medicine detection.The quality detection method comprises the steps that the liver-warming decoction is used for preparing a test solution, acetonitrile (A)-0.08 wt%-0.12 wt% formic acid aqueous solution (B) serves as a mobile phase, gradient elution is adopted, and the quality of the liver-warming decoction is detected through ultra-high performance liquid chromatography-tandem mass spectrometry; the obtained detection result can be used for quality evaluation or control of the whole process of research / development / production / clinical application of the liver-warming decoction. According to the present invention, the UPLC-Q-TOF-MS method is adopted to perform qualitative analysis on the substance-based chemical components of the liver-warming decoction, such that the scientific basis is provided for the substance-based material basic research of the liver-warming decoction, and the main chemical components are clarified;

Description

Technical Field

[0001] This invention relates to drug detection technology, and more particularly to a method for quality detection of Nuangan Decoction and its application. Background Technology

[0002] The formula for Warming the Liver Decoction consists of Angelica sinensis, Lycium barbarum, Poria cocos, Foeniculum vulgare, Cinnamomum cassia, Lindera strychnifolia, Aquilaria sinensis, and Zingiber officinale. It is a classic formula created by Zhang Jingyue, a physician of the Ming Dynasty, in his book *Jingyue Quanshu* (Complete Works of Zhang Jingyue), specifically in the section on "Eight New Formulas - Heat Formula." It is used to treat lower abdominal symptoms caused by liver and kidney yin deficiency and stagnation of liver meridians due to cold, such as lower abdominal pain, hernia, and testicular pain. Its core pathogenesis is insufficient liver yang and stagnation of cold pathogens leading to impaired qi circulation. Treatment focuses on warming and tonifying the liver and kidneys, and promoting qi circulation and dispelling cold.

[0003] Nuangan Decoction is made from eight Chinese medicinal herbs. Its complex compound composition contains various active substances, and a comprehensive understanding of its pharmacodynamic material basis has always been a challenge in the modernization of traditional Chinese medicine research. Currently, modern research on Nuangan Decoction mainly focuses on its pharmacological mechanisms and clinical applications, and cannot comprehensively evaluate its quality.

[0004] Traditional analytical methods, such as high-performance liquid chromatography (HPLC), have limited separation efficiency, making it difficult to achieve simultaneous and accurate identification of multiple components, thus hindering quality control and mechanism of action research. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), on the other hand, offers high analytical efficiency and speed, providing accurate molecular structure information and obtaining first-order and multi-level mass spectrometry information for each compound. This provides a superior technical means for multi-index component analysis of complex traditional Chinese medicine systems. Therefore, it is necessary to develop a method for analyzing Nuangan Decoction using UHPLC-MS / MS to more comprehensively evaluate its quality. Summary of the Invention

[0005] To address the above problems, this invention provides a method for quality testing of Nuangan Decoction and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A quality detection method for Nuangan Decoction, wherein the quality detection method involves taking a test solution prepared from Nuangan Decoction, using acetonitrile as mobile phase A and a 0.08-0.12 wt% formic acid aqueous solution as mobile phase B, and using gradient elution followed by ultra-high performance liquid chromatography-tandem mass spectrometry to detect the quality of Nuangan Decoction.

[0008] The elution conditions for the gradient elution are as follows:

[0009] 0–1 min, 1% mobile phase A, 99% mobile phase B;

[0010] 1–10 min, 1% → 11% mobile phase A, 99% → 89% mobile phase B;

[0011] 10–12 min, 11% → 14% mobile phase A, 89% → 86% mobile phase B;

[0012] 12–14 min, 14% → 15% mobile phase A, 86% → 85% mobile phase B;

[0013] 14–22 min, 15% → 34% mobile phase A, 85% → 66% mobile phase B;

[0014] 22-23 min, 34% → 1% mobile phase A, 66% → 99% mobile phase B.

[0015] Furthermore, the flow rate for the high-performance liquid chromatography detection is 0.39–0.41 mL / min.

[0016] Furthermore, the column temperature for the high-performance liquid chromatography detection is 38–41 °C.

[0017] Furthermore, the chromatographic column used in the ultra-high performance liquid chromatography detection is filled with octadecylsilane-bonded silica gel.

[0018] Furthermore, the detection wavelength of the high-performance liquid chromatography is 250–260 nm.

[0019] Furthermore, the mass spectrometry detection is performed in both positive ion mode and negative ion mode.

[0020] Furthermore, the conditions for the mass spectrometry detection are as follows: drying gas temperature 345–355°C, drying gas flow rate 8–12 L / min, nebulizing gas pressure 34–36 psi, sheath gas temperature 345–355°C, sheath gas flow rate 11–13 L / min, capillary voltage in positive ion mode 3400–3600 V, and capillary voltage in negative ion mode 3400–3600 V.

[0021] During the mass spectrometry detection process, the first-stage mass spectrometry uses MS mode with a mass scan range of 100–1200 m / z; the second-stage mass spectrometry uses Auto MSMS mode.

[0022] Furthermore, the test solution is prepared by adding methanol to Nuangan Decoction, sonicating, filtering, evaporating the filtrate to dryness, and then dissolving it in methanol again.

[0023] Furthermore, when Nuangan Decoction is a dry extract powder, the weight-to-volume ratio of the dry extract powder to the test solution is 1.9–2.1 g: 5 mL;

[0024] When Nuangan Decoction is an extract of Nuangan Decoction, the volume ratio of the extract to the test solution is 28-32:5.

[0025] When Nuangan Decoction is a concentrated form of Nuangan Decoction, the volume ratio of the concentrated solution to the test solution is 14-16 mL: 5 mL.

[0026] The application of the test results obtained by the above-mentioned quality testing method in the quality evaluation or control of the entire process of research / development / production / clinical application of Nuangan Decoction.

[0027] The beneficial effects of the quality testing method and application of the liver-warming decoction of the present invention are as follows:

[0028] This invention uses UPLC-Q-TOF-MS to qualitatively analyze the chemical composition of the Nuangan Decoction material reference, providing a scientific basis for the material basis research of the Nuangan Decoction material reference, and elucidating its main chemical composition.

[0029] This invention employs UPLC-Q-TOF-MS to qualitatively analyze the chemical composition of the reference substance of Nuangan Decoction. The analysis is highly efficient and fast, and can obtain accurate molecular structure information of Nuangan Decoction. It can obtain first-level and multi-level mass spectrometry information of each compound, providing a good technical means for the multi-index component analysis of Nuangan Decoction, a complex traditional Chinese medicine system.

[0030] This invention uses UPLC-Q-TOF-MS to qualitatively analyze the chemical composition of the reference substance of Nuangan Decoction, providing a scientific basis for the study of the material basis of Nuangan Decoction and clarifying its main chemical composition.

[0031] The quality testing method of the present invention can simultaneously detect all medicinal ingredients in the whole formula of Nuangan Decoction;

[0032] This invention utilizes the different physicochemical properties of the various active ingredients in Nuangan Decoction and changes the chromatographic conditions to achieve the goal of effectively separating the various active ingredients in Nuangan Decoction.

[0033] By selecting specific chromatographic and mass spectrometric conditions, this invention can obtain a better chromatogram of the warm liver decoction.

[0034] The quality testing method of the present invention also has good feasibility, stability and reproducibility;

[0035] The quality detection method of this invention can detect and clarify the medicinal flavor source and qualitative analysis of 41 peak substances; among them, four alkaloid components were identified, namely betaine, horsfieldine, boridine, and norisoboridine; and 14 chromone components were identified, namely aquilarone, aquilarone isomer, aquilarone A, aquilarone D / E, 5,6,7,8-tetrahydroxy-2-(4-methoxyphenethyl)-5,6,7,8-tetrahydrochromone, and aquilarone. B. 5,6,7,8-tetrahydroxy-2-(4-methoxyphenethyl)-5,6,7,8-tetrahydrochromone isomer, 5,6,7-trihydroxy-5,6,7,8-tetrahydro-2-(2-phenylethyl)chromone, 5,6,7-trihydr oxy-5,6,7,8-tetrahydro-2-(2-phenylethyl)chromone isomer, 5,6,7-trihydroxy-2-(4-methoxyphenethyl)-5,6,7,8-tetrahydrochromone, 8-chloro-5,6,7-trihydroxy-2-(2-ph The following compounds were identified: 2 amino acids (phenylalanine and tryptophan); 7 glycosides (uridine, adenosine, guanosine, syringin, sinaponic acid 1,3'-diglucoside, quercetin-3-O-glucuronide); 3 organic acids (vanillic acid, ferulic acid, and cinnamic acid); 2 phenolic compounds (proanthocyanidins B2 / B1 / B4 and cinnamyl tannin B1); 1 flavonoid (rutin); and 2 lactones (ligustrolide I and ligustrolide H).

[0036] The quality detection method of this invention can be used to obtain the presence and characteristics of common characteristic peaks in the chromatogram of Nuangan Decoction, which can comprehensively monitor the quality of raw materials, semi-finished products and finished products of Nuangan Decoction. By comparing the similarity of chromatographic characteristics, the quality of Nuangan Decoction can be evaluated, and its stability and consistency can be examined, which makes up for the shortcomings of the current quality control methods. At the same time, it can also monitor the stability of the production process of Nuangan Decoction, and ensure its quality is stable, uniform and controllable.

[0037] The quality testing method of this invention has improved the quality control standards of Nuangan Decoction by obtaining its chromatogram, effectively preventing product counterfeiting and ensuring the normal production and distribution order of Nuangan Decoction. Based on this invention, a correlation study can also be conducted between the chromatogram information detected by UPLC-Q-TOF-MS and the pharmacodynamic information, thereby further clarifying the correlation between the intrinsic chemical components of Nuangan Decoction and the efficacy of the preparation.

[0038] The quality testing method described in this invention can be used for quality evaluation or control throughout the entire process of research, development, production, and clinical application of Nuangan Decoction. Attached Figure Description

[0039] Figure 1 The images show the UPLC-UV chromatogram and UPLC-MS total ion chromatogram of Nuangan Decoction in Example 1 of this invention. From top to bottom, they are the UPLC-MS total ion chromatogram (positive ion mode UPLC-MS-TIC chromatogram), the UPLC-MS total ion chromatogram (negative ion mode UPLC-MS-TIC chromatogram), and the UPLC-UV chromatogram of Nuangan Decoction (chromatogram under UV 260nm conditions). Detailed Implementation

[0040] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0041] Example 1: A method for quality testing of Nuangan Decoction and its application

[0042] This embodiment uses a reference material for Nuangan Decoction to test the quality of Nuangan Decoction. The specific preparation method of the reference material for Nuangan Decoction is as follows:

[0043] Weigh out 8 daily prescription doses, each containing 5.63g cinnamon, 7.5g fennel, 9.38g angelica, 11.25g wolfberry, 7.5g lindera, 3.75g agarwood, 7.5g poria cocos, and 4g ginger. Crush the cinnamon and agarwood, and slice the ginger into thick slices. Place all ingredients in a clay pot, add 300mL of water, heat over an open flame, cover and simmer. After boiling over high heat, simmer over low heat for 26 minutes (until reduced to about 150mL). Filter the solution through a 200-mesh filter while hot. Combine the filtrates to obtain the extract. Concentrate the extract under reduced pressure to obtain the concentrate. Freeze-dry the concentrate to obtain the dry powder, which is the material basis for the warm liver decoction (i.e., the dry powder).

[0044] This embodiment describes a quality testing method and application for Warm Liver Decoction, specifically including the following steps:

[0045] S1, Solution Preparation

[0046] S11. Preparation of the test solution

[0047] Material standard: Weigh approximately 2g of the Nuangan Decoction material standard accurately, place it in a stoppered conical flask, accurately add 25mL of methanol, seal tightly, sonicate (power 250W, frequency 40kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in an appropriate amount of methanol, transfer to a 5mL volumetric flask, dilute with methanol to the mark, shake well, filter, and collect the filtrate to obtain the test solution.

[0048] Alternatively, extracts or concentrates can be used in the preparation of the test solution, as follows:

[0049] The method for preparing the extract is as follows: accurately measure 30 mL of extract, evaporate to dryness, add 25 mL of methanol to the residue, sonicate (power 250 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in methanol and transfer it to a 5 mL volumetric flask, dilute with methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0050] The preparation method of the concentrated solution is as follows: accurately measure 15 mL of concentrated solution, evaporate to dryness, add 25 mL of methanol to the residue, sonicate (power 250 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in methanol and transfer it to a 5 mL volumetric flask, dilute with methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0051] S12. Preparation of reference solution

[0052] Take appropriate amounts of agaric tetraol, norisoboridine, ferulic acid and cinnamic acid reference standards, weigh them accurately, and add methanol to prepare a solution containing 0.4 mg of each reference standard per 1 mL.

[0053] S13. Preparation of single-herb solution

[0054] Medicinal materials / processed slices: Take appropriate amounts of each of the following medicinal powders (passed through a No. 3 sieve) from the prescription (about 1g of cinnamon and agarwood, about 1g of crushed ginger, about 2g of angelica and wolfberry, and about 1.5g of fennel, lindera, and poria), accurately weigh them, place them separately in round-bottom flasks, add 50mL of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25mL of methanol to the residue, sonicate (power 250W, frequency 40kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in methanol and transfer it to a 5mL volumetric flask, dilute with methanol to the mark, shake well, filter, and take the filtrate to obtain the solutions of each single herb.

[0055] S2, Quality Inspection

[0056] The test solution was subjected to UPLC-Q-TOF-MS detection to obtain the UPLC-UV chromatogram and UPLC-MS total ion chromatogram (positive and negative ion mode UPLC-MS-TIC chromatograms) of Nuangan Decoction. This is the chromatogram of Nuangan Decoction. See details below. Figure 1 . Figure 1 The substances in chromatographic peaks 1 to 41 marked in the figure were all well separated and detected.

[0057] Meanwhile, UPLC-Q-TOF-MS was performed on each single herb solution and each reference solution, and the results were compared and analyzed with those of the test solution.

[0058] The instruments and equipment used for UPLC-Q-TOF-MS detection are as follows:

[0059] An Agilent 1290 Infinity II ultra-high performance liquid chromatograph (UPLC) connected to a G6530C quadrupole-time-of-flight tandem mass spectrometer (Q-TOF-MS). It is equipped with an independent quaternary pump, autosampler, column oven, diode array detector (DAD), and electrospray ionization source (ESI).

[0060] The chromatographic conditions for UPLC-Q-TOF-MS detection are as follows:

[0061] Chromatographic separation was performed using a column packed with octadecylsilane-bonded silica gel, specifically a Waters ACQUITY UPLC HSS T3 C18 UPLC column (2.1 × 100 mm, 1.8 μm) equipped with an online filter;

[0062] The column temperature is 40℃;

[0063] The flow rate was 0.4 mL / min;

[0064] The detection wavelength is 260nm;

[0065] The injection volume was 3 μL;

[0066] Acetonitrile was used as mobile phase A, and 0.1 wt% formic acid aqueous solution was used as mobile phase B;

[0067] The elution method is gradient elution, and the elution conditions are:

[0068] 0–1 min, 1% mobile phase A, 99% mobile phase B;

[0069] 1–10 min, 1% → 11% mobile phase A, 99% → 89% mobile phase B;

[0070] 10–12 min, 11% → 14% mobile phase A, 89% → 86% mobile phase B;

[0071] 12–14 min, 14% → 15% mobile phase A, 86% → 85% mobile phase B;

[0072] 14–22 min, 15% → 34% mobile phase A, 85% → 66% mobile phase B;

[0073] 22-23 min, 34% → 1% mobile phase A, 66% → 99% mobile phase B.

[0074] The mass spectrometry conditions for UPLC-Q-TOF-MS detection are as follows:

[0075] Mass spectrometry was performed in both positive and negative ion modes, with a drying gas temperature of 350℃, a drying gas flow rate of 10 L / min, a nebulizer gas pressure of 35 psi, a sheath gas temperature of 350℃, a sheath gas flow rate of 12 L / min, and capillary voltages of 3500 V (positive mode) and 3500 V (negative mode). Primary mass spectrometry was performed in MS mode with a mass scan range of 100–1200 m / z. Secondary mass spectrometry was performed in Auto MSMS mode, with the collision voltage adjusted appropriately for different compounds. The obtained LC-MS data were acquired using Agilent MassHunter (B.08.00) software. Data processing was performed using Agilent Qualitative Navigator (B.08.00) and Qualitative Workflows (B.08.00) software.

[0076] Analysis of test results: The Nuangan Decoction's chromatographic reference consists of eight herbs: cinnamon, fennel, angelica, lindera, agarwood, wolfberry, poria, and ginger. By comparing the chromatographic results of the chromatographic reference with those of each individual herb, the source of 41 peak substances and the qualitative analysis of the main chromatographic peaks were clarified. Peaks for all eight herbs could be identified in the chromatographic fingerprint of the chromatographic reference.

[0077] Accurate mass number determination by high-resolution TOF-MS was performed on... Figure 1 Forty-one compounds from peaks 1 to 41 were analyzed and identified, as shown in Table 1. It can be seen that these compounds, in positive mode, can generate the molecular ion peak [M+H]. + , and [M+Na] + [M+K] + In isoadductor mode, a molecular ion peak [MH] can be generated; however, in negative mode, a molecular ion peak can be generated. - and [M+Cl] - [M+HCOO] -Isoadduct ions. Based on this information about adduct ions, the precise molecular weight and molecular formula of the compound can be accurately deduced (mass measurement error ≤ 5 ppm, indicating correct matching results), thus aiding in subsequent structural identification.

[0078] Furthermore, the test solutions were analyzed by Auto MS / MS (positive mode predominant) under the same chromatographic conditions. The structures of some compounds in Table 1 were confirmed, and the results are shown in Table 1. Secondary mass spectrometry analysis, to a certain extent, confirmed the primary mass spectrometry identification results in Table 1.

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089] UPLC-QTOF-MS analysis was performed on the reference solution of Nuangan Decoction, and a total of 41 components were detected. By comparing with literature data, comparing with the reference standard retention time, and analyzing the accurate molecular weight given by UPLC-QTOF-MS, 35 components were identified or inferred, and the source of the compounds was attributed to the medicinal ingredients.

[0090] a) Alkaloids

[0091] Four alkaloid compounds were identified as characteristic components of Lycium barbarum and Lindera strychnifolia, namely betaine, horsile alkaloid, boridine, and norisoboridine. The accurate chemical structure of norisoboridine was confirmed by comparison with standards.

[0092] b. Chromones

[0093] Fourteen chromone compounds were identified, namely aquilarone, aquilarone isomers, aquilarone A, aquilarone D / E, 5,6,7,8-tetrahydroxy-2-(4-methoxyphenethyl)-5,6,7,8-tetrahydrochromone, and aquilarone. B. The isomers of 5,6,7,8-tetrahydroxy-2-(4-methoxyphenethyl)-5,6,7,8-tetrahydrochromone, 5,6,7-trihydroxy-5,6,7,8-tetrahydro-2-(2-phenylethyl)chromone, 5,6,7-trihydroxy-5,6,7,8-tetrahydro-2-(2-phenylethyl)chromone, 5,6,7-trihydroxy-2-(4-methoxyphenethyl)-5,6,7,8-tetrahydrochromone, 8-chloro-5,6,7-trihydroxy-2-(2-phenylethyl)-5,6,7,8-tetrahydrochromone, and 8-chloro-5,6,7-trihydroxy-2-(4-methoxyphenethyl)-5,6,7,8-tetrahydrochromene are mainly classified as agarwood. Among them, agarotetraol's accurate chemical structure was confirmed by comparison with standard samples.

[0094] c. Amino acids

[0095] Two amino acid compounds were identified: phenylalanine and tryptophan, which are mainly attributed to ginger.

[0096] d. Glycosides

[0097] Seven glycoside compounds were identified, namely uridine, adenosine, guanosine, syringin, sinigrin 1,3'-diglucoside, and quercetin-3-O-glucuronide, which are mainly associated with Angelica sinensis and fennel.

[0098] e. Organic acids

[0099] Three organic acid compounds were identified: vanillic acid, ferulic acid, and cinnamic acid. The accurate chemical structures of ferulic acid and cinnamic acid were confirmed by comparison with standards.

[0100] f. Phenols

[0101] Two phenolic compounds were identified: proanthocyanidins B2 / B1 / B4 and cinnamyl tannin B1, which are mainly associated with Lindera strychnifolia.

[0102] g. Flavonoids

[0103] One flavonoid compound was identified, namely rutin, which belongs to Lycium barbarum.

[0104] h, lactones

[0105] Two lactone compounds were identified: ligustilide I and ligustilide H, which are mainly associated with Angelica sinensis.

[0106] The quality testing method for Nuangan Decoction in this embodiment can be used for quality evaluation or control throughout the entire process of Nuangan Decoction research, development, production, and clinical application.

[0107] In this embodiment, the liquid chromatography fingerprint (including but not limited to UPLC-UV chromatogram and / or UPLC-MS total ion chromatogram), primary analysis results (as shown in Table 1 of the embodiment), and secondary mass spectrometry analysis results (as shown in Table 1 of the embodiment) obtained by the quality detection method can all be used for quality evaluation or control throughout the research / development / production / clinical application of Nuangan Decoction. For example, peak number and retention time can be directly used for quality evaluation or control throughout the research / development / production / clinical application of Nuangan Decoction.

[0108] Examples 2-7: Quality testing methods and applications of Nuangan Decoction

[0109] Examples 2-7 are methods for quality testing and application of Warm Liver Decoction. Their steps are basically the same as those in Example 1, with the only difference being the process parameters. See Tables 2-4 for details.

[0110] Table 2. Summary of process parameters in Examples 2-3

[0111]

[0112] Table 3. Summary of process parameters in Examples 4 and 5

[0113]

[0114]

[0115] Table 4. Summary of process parameters in Examples 6 and 7

[0116]

[0117] The process steps and parameters for other parts of Examples 2 to 7 are the same as those in Example 1, and the test results obtained are also similar to those in Example 1, so they will not be repeated here.

[0118] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for quality testing of a liver-warming decoction, characterized in that, The quality detection method involves taking the test solution prepared by Nuangan Decoction, using acetonitrile as mobile phase A and formic acid aqueous solution with a concentration of 0.08-0.12 wt% as mobile phase B, and using gradient elution followed by ultra-high performance liquid chromatography-tandem mass spectrometry to detect the quality of Nuangan Decoction. The elution conditions for the gradient elution are as follows: 0–1 min, 1% mobile phase A, 99% mobile phase B; 1–10 min, 1% → 11% mobile phase A, 99% → 89% mobile phase B; 10–12 min, 11% → 14% mobile phase A, 89% → 86% mobile phase B; 12–14 min, 14% → 15% mobile phase A, 86% → 85% mobile phase B; 14–22 min, 15% → 34% mobile phase A, 85% → 66% mobile phase B; 22-23 min, 34% → 1% mobile phase A, 66% → 99% mobile phase B.

2. The quality testing method for the warm liver decoction according to claim 1, characterized in that, The flow rate for the high-performance liquid chromatography detection is 0.39–0.41 mL / min.

3. The method for quality testing of the warm liver decoction according to claim 1 or 2, characterized in that, The column temperature for the high-performance liquid chromatography detection is 38–41 °C.

4. The quality testing method for the warm liver decoction according to claim 1 or 2, characterized in that, The ultra-high performance liquid chromatography column used for detection is filled with octadecylsilane-bonded silica gel.

5. The method for quality testing of the warm liver decoction according to claim 1 or 2, characterized in that, The detection wavelength for the high-performance liquid chromatography is 250–260 nm.

6. The method for quality testing of the warm liver decoction according to claim 1 or 2, characterized in that, The mass spectrometry detection was performed in both positive ion mode and negative ion mode.

7. The method for quality testing of the warm liver decoction according to claim 1 or 2, characterized in that, The mass spectrometry detection conditions are as follows: drying gas temperature 345–355℃, drying gas flow rate 8–12 L / min, nebulizing gas pressure 34–36 psi, sheath gas temperature 345–355℃, sheath gas flow rate 11–13 L / min, capillary voltage in positive ion mode 3400–3600 V, and capillary voltage in negative ion mode 3400–3600 V. During the mass spectrometry detection process, the first-stage mass spectrometry uses MS mode with a mass scan range of 100–1200 m / z; the second-stage mass spectrometry uses Auto MSMS mode.

8. The method for quality testing of the warm liver decoction according to claim 1 or 2, characterized in that, The test solution was prepared by adding methanol to Nuangan Decoction, sonicating, filtering, evaporating the filtrate to dryness, and then dissolving it in methanol again.

9. The method for quality testing of the warm liver decoction according to claim 8, characterized in that, When Nuangan Decoction is a dry extract powder, the weight-to-volume ratio of the dry extract powder to the test solution is 1.9–2.1 g: 5 mL. When Nuangan Decoction is an extract of Nuangan Decoction, the volume ratio of the extract to the test solution is 28-32:

5. When Nuangan Decoction is a concentrated form of Nuangan Decoction, the volume ratio of the concentrated solution to the test solution is 14-16 mL: 5 mL.

10. The application of the test results obtained by the quality testing method according to any one of claims 1-9 in the quality evaluation or control of the entire process of research / development / production / clinical application of Nuangan Decoction.

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