Establishment of fingerprint of volatile / liposoluble components in Xinuanling capsules

Abstract

The application belongs to the technical field of quality detection of traditional Chinese medicine preparation, and provides a method for establishing volatile / liposoluble component fingerprint spectrum of Xinnaoning capsules. The method comprises establishing volatile component fingerprint spectrum of Xinnaoning capsules and establishing liposoluble component fingerprint spectrum of Xinnaoning capsules, and is to take Xinnaoning capsule drug preparation as a detection object, and to establish a fingerprint spectrum method for the drug preparation by using gas chromatography and mass spectrometry. The method for establishing volatile / liposoluble component fingerprint spectrum of Xinnaoning capsules has the advantages of high stability, high precision and good repeatability. Moreover, the method can evaluate the similarity of liposoluble compounds of Xinnaoning capsules, and provide technical support for realizing more comprehensive and effective control of product quality.

Description

Technical Field

[0001] This invention relates to the field of quality testing technology for traditional Chinese medicine preparations, specifically to a method for establishing fingerprint spectra of volatile / lipid-soluble components in Xinnaoning capsules. Background Technology

[0002] Xinnaoning Capsules are a product of Guizhou Jingcheng Pharmaceutical Co., Ltd., and are listed in the 2020 edition of the Chinese Pharmacopoeia. Composed of Ginkgo biloba leaves, Buxus microphylla, Salvia miltiorrhiza, Litsea cubeba, and Allium macrostemon, it has the effects of promoting blood circulation, regulating qi, clearing the meridians, and relieving pain. Clinically, it is used for chest pain, headache, and dizziness due to qi stagnation and blood stasis, manifested as chest tightness and stabbing pain, palpitations, and vertigo, as well as for coronary heart disease and cerebral arteriosclerosis with the above symptoms. Currently, thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) are commonly used for qualitative and quantitative identification of the active ingredients, such as the TLC qualitative identification of Allium macrostemon and Buxus microphylla in Xinnaoning Capsules, and the HPLC quantitative detection of salvianolic acid B in Salvia miltiorrhiza. However, Xinnaoning Capsules are a compound preparation made from five herbs, containing hundreds of chemical components. Existing detection methods detect only a limited number of active ingredients, making it difficult to achieve more comprehensive and effective quality control of Xinnaoning Capsules.

[0003] Traditional Chinese medicine (TCM) fingerprinting is a spectroscopic or chromatographic representation of the chemical components of TCM obtained using spectroscopic or chromatographic techniques. TCM, especially compound TCM formulas, contains multiple chemical components, and their therapeutic effects are synergistic. Therefore, the reflection of their material basis cannot be explained by just one or a few components; a holistic approach is necessary. TCM fingerprinting possesses characteristics such as holistic, macroscopic, and fuzzy analysis. By describing the overall characteristics and employing appropriate fuzzy processing methods, the goal of overall quality control can be achieved. Compared to existing analytical methods, TCM fingerprinting technology has unique advantages in the evaluation and quality control of multi-component herbs and prepared TCM products. It is beneficial for establishing more accurate, comprehensive, and batch-wide quality control methods for currently marketed prepared TCM products. Therefore, it is necessary to conduct fingerprinting research on the Xinnaoning capsule product. Summary of the Invention

[0004] The purpose of this invention is to provide a method for establishing fingerprint profiles of volatile / lipid-soluble components in Xinnaoning capsules.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] The method for establishing fingerprint profiles of volatile / lipid-soluble components in Xinnaoning capsules according to the present invention includes establishing fingerprint profiles of volatile components and lipid-soluble components in Xinnaoning capsules. The specific steps are as follows:

[0007] (I) Establishment of fingerprint spectrum of volatile components in Xinnaoning capsules

[0008] A1, Preparation of the test solution

[0009] Weigh the powder from the Xinnaoning capsules, add 8-12 times the amount of water, add 1 ml of n-hexane to the volatile oil collector, start timing from boiling, distill to extract the volatile oil for 5-10 hours, collect the n-hexane portion and store it in a small bottle to obtain the small molecule volatile oil of Xinnaoning capsules; take the small molecule volatile oil of Xinnaoning, dissolve and dilute it 100 times with ethyl acetate, add an appropriate amount of anhydrous sodium sulfate to remove water, filter it with a filter membrane to obtain the test solution;

[0010] A2, GC-MS conditions

[0011] Mass spectrometry conditions: EI source, ion source temperature 230℃, quadrupole temperature set to 150℃, full scan mode, scan range: 30-550u, auxiliary heating zone temperature 270℃; solvent delay: 3min; NIST 14.0 standard spectra can be used for search and analysis.

[0012] Chromatographic conditions: Agilent HP-5MS capillary column, 30m×0.25mm×0.25μm, injection port temperature 240-260℃, column flow rate 1.0-2.0ml / min, septum purge flow rate 3ml / min, split ratio 10-30:1, injection volume 1μl;

[0013] Temperature program: Start at 60℃ and hold for 3 min; increase to 210℃ at a rate of 3.5-5℃ / min and hold for 0 min; increase to 240℃ at a rate of 2-3℃ / min and hold for 5 min.

[0014] A3, Determination Method

[0015] According to the GC-MS conditions, accurately pipette 1 μl of the test solution and detect it by GC-MS;

[0016] (II) Establishment of fingerprint spectrum of fat-soluble components in Xinnaoning capsules

[0017] B1, Preparation of the test solution

[0018] Weigh the powder from Xinnaoning capsules, add ethyl acetate at a mass-to-volume ratio of 1:1-3 g / ml, sonicate for 0.5-1.5 h, filter to obtain an ethyl acetate solution of Xinnaoning capsules; take the ethyl acetate solution of Xinnaoning capsules, add an appropriate amount of anhydrous sodium sulfate to remove water, filter using a filter membrane to obtain the test solution;

[0019] B2, GC-MS conditions

[0020] Mass spectrometry conditions: EI source, ion source temperature 230℃, quadrupole temperature set to 150℃, full scan mode, scan range: 30-550u, auxiliary heating zone temperature 270℃; solvent delay: 3min; NIST 14.0 standard spectra were used for search and analysis.

[0021] Chromatographic conditions: Agilent HP-5MS capillary column, 30m×0.25mm×0.25μm, injection port temperature 240-260℃, column flow rate 1.0ml / min, septum purge flow rate 3ml / min, split ratio 10-30:1, injection volume 1μl;

[0022] Temperature rise program: Start at 60℃ and hold for 3 minutes; rise to 210-220℃ at a rate of 3.5-5℃ / min and hold for 0-5 minutes; run at 250℃ for 15 minutes.

[0023] B3, Determination Method

[0024] According to the GC-MS conditions, accurately pipette 1 μl of the test solution and detect it by GC-MS.

[0025] Preferably, in step A1 of the present invention, 10 times the amount of water is added, and the volatile oil is extracted by distillation for 7 hours.

[0026] Preferably, in step A2 of the present invention, the injection port temperature is 250-260℃, the column flow rate is 1.0 ml / min, and the split ratio is 20-30:1.

[0027] Preferably, in step A2 of the present invention, the injection port temperature is 260°C, the column flow rate is 1.0 ml / min, and the split ratio is 20:1.

[0028] Preferably, the heating program in step A2 of the present invention is as follows: the initial temperature is 60°C, held for 3 minutes; the temperature is increased to 210°C at a rate of 3.5, held for 0 minutes; the temperature is increased to 240°C at a rate of 2°C / min, held for 5 minutes.

[0029] Preferably, in step B1 of the present invention, ethyl acetate is added at a mass-to-volume ratio of 1:1-2 g / ml, and the mixture is sonicated for 1-1.5 h.

[0030] Preferably, in step B1 of the present invention, ethyl acetate is added at a mass-to-volume ratio of 1:1 g / ml, and the mixture is sonicated for 1 hour.

[0031] Preferably, in step B2 of the present invention, the injection port temperature is 250-260℃, the column flow rate is 1.0 ml / min, and the split ratio is 20-30:1.

[0032] Preferably, in step B2 of the present invention, the injection port temperature is 250°C, the column flow rate is 1.0 ml / min, and the split ratio is 20:1.

[0033] Preferably, the heating program in step B2 of the present invention is as follows: the initial temperature is 60°C, held for 3 minutes; the temperature is increased to 220°C at a rate of 3.5°C / min and held for 5 minutes; the temperature is increased to 250°C and then run for 15 minutes.

[0034] The beneficial effects of this invention are:

[0035] This invention provides fingerprinting techniques for the volatile and fat-soluble components of Xinnaoning capsules, exhibiting advantages such as high stability, high precision, and good reproducibility. Furthermore, it evaluates the similarity of fat-soluble compounds in Xinnaoning capsules, providing technical support for more comprehensive and effective control of product quality. Attached Figure Description

[0036] Figure 1 GC-MS (temperature program 1) total ion chromatogram of volatile oil in Xinnaoning capsules;

[0037] Figure 2 GC-MS (temperature program 2) total ion chromatogram of volatile oil in Xinnaoning capsules;

[0038] Figure 3 GC-MS (temperature program 3) total ion chromatogram of volatile oil in Xinnaoning capsules;

[0039] Figure 4 Comparison of total ion chromatograms of volatile oils in Xinnaoning capsules (three temperature programs);

[0040] Figure 5 GC-MS (split ratio 10:1) total ion chromatogram of volatile oil in Xinnaoning capsules;

[0041] Figure 6 GC-MS (split ratio 20:1) total ion chromatogram of volatile oil in Xinnaoning capsules;

[0042] Figure 7 GC-MS (split ratio 30:1) total ion chromatogram of volatile oil in Xinnaoning capsules;

[0043] Figure 8 Comparison of GC-MS total ion chromatograms (with different split ratios) of volatile oils in Xinnaoning capsules;

[0044] Figure 9 GC-MS (injection temperature 240℃) total ion chromatogram of volatile oil in Xinnaoning capsules;

[0045] Figure 10GC-MS (injection temperature 250℃) total ion chromatogram of volatile oil in Xinnaoning capsules;

[0046] Figure 11 GC-MS (injection temperature 260℃) total ion chromatogram of volatile oil in Xinnaoning capsules;

[0047] Figure 12 Comparison of GC-MS total ion chromatograms (at different injection temperatures) of volatile oils in Xinnaoning capsules;

[0048] Figure 13 GC-MS (column flow rate 1.0 ml / min) total ion chromatogram of volatile oil in Xinnaoning capsules;

[0049] Figure 14 GC-MS (column flow rate 2.0 ml / min) total ion chromatogram of volatile oil in Xinnaoning capsules;

[0050] Figure 15 Comparison of total ion chromatograms of volatile oils in Xinnaoning capsules (at different column flow rates) by GC-MS.

[0051] Figure 16 GC-MS total ion chromatogram of volatile oil in Xinnaoning capsules;

[0052] Figure 17 GC-MS (temperature program 1) total ion chromatogram of lipid-soluble compounds in Xinnaoning capsules;

[0053] Figure 18 GC-MS (temperature program 2) total ion chromatogram of lipid-soluble compounds in Xinnaoning capsules;

[0054] Figure 19 GC-MS (temperature program 3) total ion chromatogram of lipid-soluble compounds in Xinnaoning capsules;

[0055] Figure 20 Comparison of total ion chromatograms of lipid-soluble compounds in Xinnaoning capsules (three temperature programs);

[0056] Figure 21 GC-MS (split ratio 10:1) total ion chromatogram of lipid-soluble compounds in Xinnaoning capsules;

[0057] Figure 22 GC-MS total ion chromatogram (split ratio 20:1) of lipid-soluble compounds in Xinnaoning capsules;

[0058] Figure 23 GC-MS total ion chromatogram (split ratio 30:1) of lipid-soluble compounds in Xinnaoning capsules;

[0059] Figure 24 Comparison of GC-MS total ion chromatograms (with different split ratios) of lipid-soluble compounds in Xinnaoning capsules;

[0060] Figure 25 GC-MS total ion chromatogram of lipid-soluble compounds in Xinnaoning capsules (injection temperature 240℃);

[0061] Figure 26 GC-MS (injection temperature 250℃) total ion chromatogram of lipid-soluble compounds in Xinnaoning capsules;

[0062] Figure 27 GC-MS total ion chromatogram of lipid-soluble compounds in Xinnaoning capsules (injection temperature 260℃);

[0063] Figure 28 Comparison of total ion chromatograms of lipid-soluble compounds in Xinnaoning capsules (at different injection temperatures) by GC-MS.

[0064] Figure 29 Total ion chromatogram of lipid-soluble compounds in Xinnaoning capsules by GC-MS (column flow rate 1.0 ml / min);

[0065] Figure 30 Total ion chromatogram of lipid-soluble compounds in Xinnaoning capsules by GC-MS (column flow rate 2.0 ml / min);

[0066] Figure 31 Comparison of total ion chromatograms of lipid-soluble compounds in Xinnaoning capsules using GC-MS (different column flow rates);

[0067] Figure 32 GC-MS total ion chromatogram of lipid-soluble compounds in Xinnaoning capsules;

[0068] Figure 33 Precision assessment chromatogram;

[0069] Figure 34 Repeatability test chromatogram;

[0070] Figure 35 Stability study chromatogram;

[0071] Figure 36 GC-MS fingerprint comparison of lipid-soluble compounds in Xinnaoning capsules (26 batches);

[0072] Figure 37 GC-MS fingerprint overlay of lipid-soluble compounds in Xinnaoning capsules (26 batches);

[0073] Figure 38GC-MS fingerprint comparison of lipid-soluble compounds in Xinnaoning capsules (2017 batch);

[0074] Figure 39 GC-MS fingerprint overlay of lipid-soluble compounds in Xinnaoning capsules (2017 batch);

[0075] Figure 40 Comparison of GC-MS fingerprints of lipid-soluble compounds in Xinnaoning capsules (2018 batch);

[0076] Figure 41 GC-MS fingerprint overlay of lipid-soluble compounds in Xinnaoning capsules (2018 batch);

[0077] Figure 42 Comparison of GC-MS fingerprints of lipid-soluble compounds in Xinnaoning capsules (2019 batch);

[0078] Figure 43 GC-MS fingerprint overlay of lipid-soluble compounds in Xinnaoning capsules (2019 batch);

[0079] Figure 44 GC-MS chromatograms of lipid-soluble compounds in Xinnaoning capsules (April 2016 to 2019)

[0080] Figure 45 GC-MS fingerprint overlay of lipid-soluble compounds in Xinnaoning capsules (4 years). Detailed Implementation

[0081] The technical solution of the present invention will be described in detail below with reference to specific embodiments. The following embodiments are for explanation and illustration only, and do not constitute a limitation on the technical solution of the present invention.

[0082] Example 1: Establishment of the fingerprint spectrum of Xinnaoning capsules

[0083] (I) Establishment of fingerprint spectrum of volatile components in Xinnaoning capsules

[0084] 1) Preparation of test solution: Weigh 50g of powder from Xinnaoning capsules, place it in a steam distillation apparatus, add 10 times the amount of water, add 1ml of n-hexane to the volatile oil collector, start timing from boiling, distill and extract the volatile oil for 7 hours, collect the n-hexane portion and store it in a small bottle to obtain the volatile oil of Xinnaoning capsules; take 10μl of Xinnaoning small molecule volatile oil, dissolve it in ethyl acetate and make up to 1ml, add an appropriate amount of anhydrous sodium sulfate to remove water, filter through a 0.22μm filter membrane to obtain the test solution;

[0085] 2) GC-MS conditions: Mass spectrometry conditions: EI source, ion source temperature 230℃, quadrupole temperature set to 150℃, full scan mode, scan range: 30-550u, auxiliary heating zone temperature 270℃; solvent delay: 3min, NIST14.0 standard chromatogram can be used for search and analysis; Chromatographic conditions: Agilent HP-5MS capillary column (30m×0.25mm×0.25μm), injection port temperature 260℃, column flow rate 1.0ml / min, septum purge flow rate: 3ml / min, split ratio 20:1, injection volume 1μl; Temperature program: initial temperature 60℃, hold for 3min; ramp to 210℃ at 3.5℃ / min, hold for 0min; ramp to 240℃ at 2℃ / min, hold for 5min;

[0086] 3) Determination method: According to the GC-MS conditions, accurately pipette 1 μl of the test solution and detect it by GC-MS;

[0087] (II) Establishment of fingerprint spectrum of fat-soluble components in Xinnaoning capsules

[0088] (1) Preparation of test solution: Weigh 50g of powder from Xinnaoning capsules, add 50ml of ethyl acetate, place in an Erlenmeyer flask, sonicate for 1h, filter to obtain ethyl acetate solution of Xinnaoning capsules; take 5ml of ethyl acetate solution of Xinnaoning capsules, add an appropriate amount of anhydrous sodium sulfate to remove water, filter through a 0.22μm filter membrane to obtain test solution;

[0089] (2) GC-MS conditions: Mass spectrometry conditions: EI source, ion source temperature 230℃, quadrupole temperature set to 150℃, full scan mode, scan range: 30-550u, auxiliary heating zone temperature 270℃; solvent delay: 3min, NIST14.0 standard spectrum was used for search and analysis; Chromatographic conditions: Agilent HP-5MS capillary column (30m×0.25mm×0.25μm), injection port temperature 250℃, column flow rate 1.0ml / min, septum purge flow rate: 3ml / min, split ratio 20:1, injection volume 1μl; Temperature program: initial temperature 60℃, hold for 3min; increase to 220℃ at a rate of 3.5℃ / min and hold for 5min; run at 250℃ for 15min;

[0090] (3) Determination method: According to the GC-MS conditions, accurately pipette 1 μl of the test solution and detect it by GC-MS.

[0091] Example 2: Establishment of the fingerprint spectrum of Xinnaoning capsules

[0092] (I) Establishment of fingerprint spectrum of volatile components in Xinnaoning capsules

[0093] 1) Preparation of the test solution: Same as in Example 1;

[0094] 2) GC-MS conditions: Mass spectrometry conditions: same as in Example 1; Chromatographic conditions: Agilent HP-5MS capillary column (30m×0.25mm×0.25μm), injection port temperature 240℃, column flow rate 1.0ml / min, septum purge flow rate: 3ml / min, split ratio 10:1, injection volume 1μl; Temperature program: initial temperature 60℃, hold for 3min; increase to 210℃ at a rate of 3.5℃ / min, hold for 0min; increase to 240℃ at a rate of 2℃ / min, hold for 5min;

[0095] 3) Determination method: According to the GC-MS conditions, accurately pipette 1 μl of the test solution and detect it by GC-MS;

[0096] (II) Establishment of fingerprint spectrum of fat-soluble components in Xinnaoning capsules

[0097] (1) Preparation of the test solution: Same as in Example 1;

[0098] (2) GC-MS conditions: Mass spectrometry conditions: same as in Example 1; Chromatographic conditions: Agilent HP-5MS capillary column (30m×0.25mm×0.25μm), injection port temperature 240℃, column flow rate 1.0ml / min, septum purge flow rate: 3ml / min, split ratio 10:1, injection volume 1μl; Temperature program: initial temperature 60℃, hold for 3min; increase to 220℃ at a rate of 3.5℃ / min and hold for 5min; run at 250℃ for 15min.

[0099] (3) Determination method: According to the GC-MS conditions, accurately pipette 1 μl of the test solution and detect it by GC-MS.

[0100] Example 3: Establishment of the fingerprint spectrum of Xinnaoning capsules

[0101] (I) Establishment of fingerprint spectrum of volatile components in Xinnaoning capsules

[0102] 1) Preparation of the test solution: Same as in Example 1;

[0103] 2) GC-MS conditions: Mass spectrometry conditions: same as in Example 1; Chromatographic conditions: Agilent HP-5MS capillary column (30m×0.25mm×0.25μm), injection port temperature 250℃, column flow rate 1.0ml / min, septum purge flow rate: 3ml / min, split ratio 30:1, injection volume 1μl; Temperature program: initial temperature 60℃, hold for 3min; increase to 210℃ at a rate of 5℃ / min, hold for 0min; increase to 240℃ at a rate of 3℃ / min, hold for 5min;

[0104] 3) Determination method: According to the GC-MS conditions, accurately pipette 1 μl of the test solution and detect it by GC-MS;

[0105] (II) Establishment of fingerprint spectrum of fat-soluble components in Xinnaoning capsules

[0106] (1) Preparation of the test solution: Same as in Example 1;

[0107] (2) GC-MS conditions: Mass spectrometry conditions: same as in Example 1; Chromatographic conditions: Agilent HP-5MS capillary column (30m×0.25mm×0.25μm), injection port temperature 260℃, column flow rate 1.0ml / min, septum purge flow rate: 3ml / min, split ratio 10:1, injection volume 1μl; Temperature program: initial temperature 60℃, hold for 3min; increase to 220℃ at a rate of 3.5℃ / min and hold for 5min; run at 250℃ for 15min.

[0108] (3) Determination method: According to the GC-MS conditions, accurately pipette 1 μl of the test solution and detect it by GC-MS.

[0109] Example 4

[0110] (I) Establishment of fingerprint spectrum of volatile components in Xinnaoning capsules

[0111] 1) Preparation of the test solution: Same as in Example 1;

[0112] 2) GC-MS conditions: Mass spectrometry conditions: same as in Example 1; Chromatographic conditions: same as in Example 1; Temperature program: starting temperature 60℃, hold for 3 min; increase to 210℃ at a rate of 5℃ / min, hold for 0 min; increase to 240℃ at a rate of 2℃ / min, hold for 5 min;

[0113] 3) Determination method: According to the GC-MS conditions, accurately pipette 1 μl of the test solution and detect it by GC-MS;

[0114] (II) Establishment of fingerprint spectrum of fat-soluble components in Xinnaoning capsules

[0115] (1) Preparation of the test solution: Same as in Example 1;

[0116] (2) GC-MS conditions: Mass spectrometry conditions: same as in Example 1; Chromatographic conditions: same as in Example 1; Temperature program: starting temperature is 60℃, hold for 3 min; increase to 210℃ at a rate of 3.5℃ / min and hold for 5 min; run at 250℃ for 15 min;

[0117] (3) Determination method: According to the GC-MS conditions, accurately pipette 1 μl of the test solution and detect it by GC-MS.

[0118] Example 5

[0119] (I) Establishment of fingerprint spectrum of volatile components in Xinnaoning capsules

[0120] 1) Preparation of the test solution: Same as in Example 1;

[0121] 2) GC-MS conditions: Mass spectrometry conditions: same as in Example 1; Chromatographic conditions: same as in Example 1; Temperature program: starting temperature 60℃, hold for 3 min; increase to 210℃ at a rate of 5℃ / min, hold for 0 min; increase to 240℃ at a rate of 3℃ / min, hold for 5 min;

[0122] 3) Determination method: According to the GC-MS conditions, accurately pipette 1 μl of the test solution and detect it by GC-MS;

[0123] (II) Establishment of fingerprint spectrum of fat-soluble components in Xinnaoning capsules

[0124] (1) Preparation of the test solution: Same as in Example 1;

[0125] (2) GC-MS conditions: Mass spectrometry conditions: same as in Example 1; Chromatographic conditions: same as in Example 1; Temperature program: starting temperature is 60℃, hold for 3 min; increase to 220℃ at a rate of 3.5℃ / min and hold for 0 min; run at 250℃ for 15 min;

[0126] (3) Determination method: According to the GC-MS conditions, accurately pipette 1 μl of the test solution and detect it by GC-MS.

[0127] To further verify the reliability of the present invention, the inventors conducted a series of experiments, as follows:

[0128] I. Fingerprint analysis of volatile and fat-soluble components of Xinnaoning capsules

[0129] 1. Experimental Instruments and Materials

[0130] 1.1 Experimental Instruments: Agilent 7890A-5975C gas chromatography-mass spectrometry system (including controller G3430A, sampler G4513A, and disc G4514A); Agilent HP-5MS capillary column (30m × 0.25mm × 0.25μm)

[0131] 1.2 Experimental reagents: Ethyl acetate (GC grade, I866672 649, manufacturer: Merk KGaA); Anhydrous sodium sulfate (batch number: 20130303-1, manufacturer: Guangzhou Chemical Reagent Factory).

[0132] 1.3 Experimental materials: Xinnaoning capsules (batch number: 20181032, provided by Guizhou Jingcheng Pharmaceutical Co., Ltd.).

[0133] 2. Experimental Content and Results

[0134] 2.1 Fingerprint analysis of the volatility of Xinnaoning capsules

[0135] 2.1.1 Examination of the temperature rise process

[0136] 2.1.1.1 Preparation of the test solution

[0137] Weigh 50g of the powder from Xinnaoning capsules and place it in a steam distillation apparatus. Add 10 times the amount of water and 1ml of n-hexane to the volatile oil collector. Start timing from boiling and distill to extract the volatile oil for 7 hours. Collect the n-hexane portion and store it in a small bottle to obtain the small molecule volatile oil from Xinnaoning capsules.

[0138] Take 10 μl of Xinnaoning small molecule volatile oil, dissolve it in ethyl acetate and make up to 1 ml, add an appropriate amount of anhydrous sodium sulfate to remove water, filter through a 0.22 μm filter membrane to obtain the test solution.

[0139] 2.1.1.2 GC-MS Conditions

[0140] (1) Chromatographic conditions: EI source, ion source temperature 230℃, quadrupole temperature set to 150℃, full scan mode, scan range: 30~550u, auxiliary heating zone temperature: 270℃; solvent delay: 3min, NIST14.0 standard spectral library was used for search and analysis.

[0141] (2) Mass spectrometry conditions: Agilent HP-5MS capillary column (30m×0.25mm×0.25μm), injection port temperature 250℃, column flow rate 1.0ml / min, septum purge flow rate: 3ml / min, split ratio 20:1, injection volume 1μl. The following three temperature programs were investigated: Temperature program 1: Initial temperature 60℃, hold for 3 min; increase to 210℃ at a rate of 3.5℃ / min, hold for 0 min; increase to 240℃ at a rate of 2℃ / min, hold for 5 min. Temperature program 2: Initial temperature 60℃, hold for 3 min; increase to 210℃ at a rate of 5℃ / min, hold for 0 min; increase to 240℃ at a rate of 2℃ / min, hold for 5 min. Temperature rise program 3: Start at 60℃ and hold for 3 minutes; rise to 210℃ at a rate of 5℃ / min and hold for 0 minutes; rise to 240℃ at a rate of 3℃ / min and hold for 5 minutes.

[0142] 2.1.1.3 Determination method: Accurately pipette 1 μl of the test solution according to the GC-MS conditions and detect it by GC-MS.

[0143] 2.1.1.4 Measurement Results

[0144] See Figures 1 to 4 By comparing the GC-MS total ion chromatograms under the three temperature programs, the peak distribution in the total ion chromatogram under temperature program 1 was more uniform compared to temperature programs 2 and 3. Therefore, temperature program 1 was selected as the optimal temperature program.

[0145] 2.1.2 Examination of the split ratio

[0146] 2.1.2.1 Preparation of the test solution

[0147] Take 10 μl of the small molecule volatile oil of Xinnaoning capsules prepared under item “2.1.1.1” above, dissolve it in ethyl acetate and make up to 1 ml, add an appropriate amount of anhydrous sodium sulfate to remove water, filter through a 0.22 μm filter membrane to obtain the test solution.

[0148] 2.1.2.2 GC-MS Conditions

[0149] (1) Mass spectrometry conditions

[0150] The EI source was set at 230°C, the quadrupole temperature at 150°C, and the full scan mode was used. The scan range was 30–550 μs, and the auxiliary heating zone temperature was 270°C. The solvent delay was 3 min. The NIST 14.0 standard spectral library was used for search and analysis.

[0151] (2) Chromatographic conditions

[0152] An Agilent HP-5MS capillary column (30m × 0.25mm × 0.25μm) was used. The injection port temperature was 250℃, the column flow rate was 1.0 ml / min, and the septum purge flow rate was 3 ml / min. The temperature program was as follows: initial temperature 60℃, hold for 3 min; ramp to 210℃ at 3.5℃ / min, hold for 0 min; ramp to 240℃ at 2℃ / min, hold for 5 min. Split ratios of 10:1, 20:1, and 30:1 were investigated to optimize the best split ratio.

[0153] 2.1.2.3 Determination method: Accurately pipette 1 μl of the test solution according to the GC-MS conditions and detect it by GC-MS.

[0154] 2.1.2.4 Measurement Results

[0155] See Figures 5 to 8 By comparing the GC-MS total ion chromatograms at three split ratios, it can be seen that when the split ratio is 10:1, the baseline noise of the spectrum is relatively large; when the split ratio is 30:1, the peak response is low; and when the split ratio is 20:1, the baseline noise of the spectrum is relatively small, and the peak response is moderate and more attractive. Therefore, 20:1 is selected as the optimal split ratio.

[0156] 2.1.3 Investigation of injection port temperature

[0157] 2.1.3.1 Preparation of the test solution

[0158] Take 10 μl of the small molecule volatile oil of Xinnaoning capsules prepared under item “2.1.1.1” above, dissolve it in ethyl acetate and make up to 1 ml, add an appropriate amount of anhydrous sodium sulfate to remove water, filter through a 0.22 μm filter membrane to obtain the test solution.

[0159] 2.1.3.2 GC-MS Conditions

[0160] (1) Mass spectrometry conditions

[0161] The EI source was set at 230°C, the quadrupole temperature at 150°C, and the full scan mode was used. The scan range was 30–550 μs, and the auxiliary heating zone temperature was 270°C. The solvent delay was 3 min. The NIST 14.0 standard spectral library was used for search and analysis.

[0162] (2) Chromatographic conditions

[0163] An Agilent HP-5MS capillary column (30m × 0.25mm × 0.25μm) was used. The column flow rate was 1.0 ml / min, the septum purge flow rate was 3 ml / min, and the split ratio was 20:1. The temperature program was as follows: initial temperature 60℃, hold for 3 min; ramp to 210℃ at 3.5℃ / min, hold for 0 min; ramp to 240℃ at 2℃ / min, hold for 5 min. Injector temperatures of 240℃, 250℃, and 260℃ were investigated to optimize the injection temperature.

[0164] 2.1.3.3 Determination method: Accurately pipette 1 μl of the test solution according to the GC-MS conditions and detect it by GC-MS.

[0165] 2.1.3.4 Measurement Results

[0166] See Figures 9 to 12 By comparing the GC-MS total ion chromatograms at three injection port temperatures, the chromatograms at injection port temperatures of 240℃, 250℃ and 260℃ showed little difference. However, the sample residue was less at the injection port temperature of 260℃, so 260℃ was selected as the optimal injection port temperature.

[0167] 2.1.4 Investigation of column flow rate

[0168] 2.1.4.1 Preparation of the test solution

[0169] Take 10 μl of the small molecule volatile oil of Xinnaoning capsules prepared under item “2.1.1.1” above, dissolve it in ethyl acetate and make up to 1 ml, add an appropriate amount of anhydrous sodium sulfate to remove water, filter through a 0.22 μm filter membrane to obtain the test solution.

[0170] 2.1.4.2 GC-MS Conditions

[0171] (1) Mass spectrometry conditions: EI source, ion source temperature 230℃, quadrupole temperature set to 150℃, full scan mode, scan range: 30~550u, auxiliary heating zone temperature: 270℃; solvent delay: 3min, NIST14.0 standard spectral library was used for search and analysis.

[0172] (2) Chromatographic conditions: Agilent HP-5MS capillary column (30m×0.25mm×0.25μm), injection port temperature 250℃, septum purge flow rate: 3ml / min, split ratio 20:1; temperature program: initial temperature 60℃, hold for 3min; increase to 210℃ at 3.5℃ / min, hold for 0min; increase to 240℃ at 2℃ / min, hold for 5min. The optimal column flow rate was investigated at 1.0ml / min and 2.0ml / min respectively.

[0173] 2.1.4.3 Determination method: According to GC-MS conditions, accurately pipette 1 μl of the test solution and detect.

[0174] 2.1.4.4 Measurement Results

[0175] See Figures 13 to 15 By comparing the flow rates of the two columns, a more uniform total ion chromatogram was obtained when the column flow rate was 1.0 ml / min. Therefore, a column flow rate of 1.0 ml / min was selected.

[0176] 2.1.5 Conclusion

[0177] After optimizing the above chromatographic conditions, the optimal GC-MS determination conditions and the GC-MS total ion chromatogram of Xinnaoning volatile oil under the optimal determination conditions were obtained, as follows:

[0178] 2.1.5.1 Optimal GC-MS determination conditions

[0179] (1) Preparation of the test solution

[0180] Weigh 50g of the powder from Xinnaoning capsules and place it in a steam distillation apparatus. Add 10 times the amount of water and 1ml of n-hexane to the volatile oil collector. Start timing from boiling and distill to extract the volatile oil for 7 hours. Collect the n-hexane portion and store it in a small bottle to obtain the small molecule volatile oil from Xinnaoning capsules.

[0181] Take 10 μl of Xinnaoning small molecule volatile oil, dissolve it in ethyl acetate and make up to 1 ml, add an appropriate amount of anhydrous sodium sulfate to remove water, filter through a 0.22 μm filter membrane to obtain the test solution.

[0182] (2) GC-MS conditions

[0183] Chromatographic conditions: EI source, ion source temperature 230℃, quadrupole temperature set to 150℃, full scan mode, scan range: 30~550u, auxiliary heating zone temperature: 270℃; solvent delay: 3min; NIST 14.0 standard spectral library was used for search and analysis. Mass spectrometry conditions: Agilent HP-5MS capillary column (30m×0.25mm×0.25μm), injection port temperature 260℃, column flow rate 1.0ml / min, septum purge flow rate: 3ml / min, split ratio 20:1; temperature program 1: initial temperature 60℃, hold for 3min; ramp to 210℃ at 3.5℃ / min, hold for 0min; ramp to 240℃ at 2℃ / min, hold for 5min.

[0184] (3) Determination method: According to the GC-MS conditions, accurately pipette 1 μl of the test solution and detect it by GC-MS.

[0185] 2.1.5.2 Total ion chromatogram of Xinnaoning volatile oil under optimal determination conditions by GC-MS

[0186] The measurement results are shown below. Figure 16 The GC-MS total ion chromatogram of the volatile oil of Xinnaoning lipid under optimal conditions showed low baseline noise, uniform peak distribution, and good separation.

[0187] 2.2 Fingerprint analysis of the fat-soluble components of Xinnaoning capsules

[0188] 2.2.1 Examination of the programmed heating process

[0189] 2.2.1.1 Preparation of the test solution

[0190] Weigh 50g of the powder from Xinnaoning capsules, add 50ml of ethyl acetate, place in an Erlenmeyer flask, sonicate for 1 hour, filter to obtain an ethyl acetate solution of Xinnaoning capsules; take 5ml of the ethyl acetate solution of Xinnaoning capsules, add an appropriate amount of anhydrous sodium sulfate to remove water, filter through a 0.22μm filter membrane to obtain the test solution.

[0191] 2.2.1.2 GC-MS Conditions

[0192] (1) Chromatographic conditions: EI source, ion source temperature 230℃, quadrupole temperature set to 150℃, full scan mode, scan range: 30~550u, auxiliary heating zone temperature: 270℃; solvent delay: 3min, NIST14.0 standard spectral library was used for search and analysis.

[0193] (2) Mass spectrometry conditions: Agilent HP-5MS capillary column (30m×0.25mm×0.25μm), injection port temperature 250℃, column flow rate 1.0ml / min, septum purge flow rate: 3ml / min, split ratio 20:1, injection volume 1μl. The following three temperature programs were investigated: Program 1: Initial temperature 60℃, hold for 3min; increase to 210℃ at 3.5℃ / min, hold for 5min; run at 250℃ for 15min. Program 2: Initial temperature 60℃, hold for 3min; increase to 220℃ at 3.5℃ / min, hold for 0min; run at 250℃ for 15min. Program 3: Initial temperature 60℃, hold for 3min; increase to 220℃ at 3.5℃ / min, hold for 5min; run at 250℃ for 15min. The optimal temperature program was selected.

[0194] 2.2.1.3 Determination method: Accurately pipette 1 μl of the test solution according to the GC-MS conditions and detect it by GC-MS.

[0195] 2.2.1.4 Measurement Results

[0196] See Figures 17 to 20 By comparing the GC-MS total ion chromatograms under three temperature programs, compared with temperature programs 1 and 2, temperature program 3 showed more chromatographic peaks, more uniform distribution, and a higher response in its total ion chromatogram. Therefore, temperature program 3 was selected as the optimal temperature program.

[0197] 2.2.2 Examination of the split ratio

[0198] 2.2.2.1 Preparation of the test solution

[0199] Prepare the ethyl acetate solution of Xinnaoning capsules according to section “2.2.1.1”; take 5 ml of the ethyl acetate solution of Xinnaoning capsules, add an appropriate amount of anhydrous sodium sulfate to remove water, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0200] 2.2.2.2 GC-MS Conditions

[0201] (1) Mass spectrometry conditions

[0202] The EI source was set at 230°C, the quadrupole temperature at 150°C, and the full scan mode was used. The scan range was 30–550 μs, and the auxiliary heating zone temperature was 270°C. The solvent delay was 3 min. The NIST 14.0 standard spectral library was used for search and analysis.

[0203] (2) Chromatographic conditions

[0204] An Agilent HP-5MS capillary column (30m × 0.25mm × 0.25μm) was used. The injection port temperature was 250℃, the column flow rate was 1.0ml / min, and the septum purge flow rate was 3ml / min. The temperature program was as follows: initial temperature 60℃, hold for 3 min; increase to 220℃ at a rate of 3.5℃ / min and hold for 5 min; then run at 250℃ for 15 min. Split ratios of 10:1, 20:1, and 30:1 were investigated to optimize the best split ratio.

[0205] 2.2.2.3 Determination method: According to the GC-MS conditions, accurately pipette 1 μl of the test solution and detect it by GC-MS.

[0206] 2.2.2.4 Measurement Results

[0207] See Figures 21 to 24 By comparing the GC-MS total ion chromatograms at three split ratios, it can be seen that when the split ratio is 10:1, the baseline noise of the spectrum is relatively large; when the split ratio is 30:1, the peak response is low; and when the split ratio is 20:1, the baseline noise of the spectrum is relatively small, and the peak response is moderate and better. Therefore, 20:1 is selected as the optimal split ratio.

[0208] 2.2.3 Investigation of injection port temperature

[0209] 2.2.3.1 Preparation of the test solution

[0210] Prepare the ethyl acetate solution of Xinnaoning capsules according to section “2.2.1.1”; take 5 ml of the ethyl acetate solution of Xinnaoning capsules, add an appropriate amount of anhydrous sodium sulfate to remove water, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0211] 2.2.3.2 GC-MS Conditions

[0212] (1) Mass spectrometry conditions

[0213] The EI source was set at 230°C, the quadrupole temperature at 150°C, and the full scan mode was used. The scan range was 30–550 μs, and the auxiliary heating zone temperature was 270°C. The solvent delay was 3 min. The NIST 14.0 standard spectral library was used for search and analysis.

[0214] (2) Chromatographic conditions

[0215] An Agilent HP-5MS capillary column (30m × 0.25mm × 0.25μm) was used. The column flow rate was 1.0 ml / min, the septum purge flow rate was 3 ml / min, and the split ratio was 20:1. The temperature program was as follows: initial temperature 60℃, held for 3 min; increased to 220℃ at a rate of 3.5℃ / min and held for 5 min; then increased to 250℃ and ran for 15 min. Injector temperatures of 240℃, 250℃, and 260℃ were investigated to optimize the injection temperature.

[0216] 2.2.3.3 Determination method: According to the GC-MS conditions, accurately pipette 1 μl of the test solution and detect it by GC-MS.

[0217] 2.2.3.4 Measurement Results

[0218] See Figures 25 to 28 By comparing the GC-MS total ion chromatograms at three injection port temperatures, the chromatograms at injection port temperatures of 240℃, 250℃ and 260℃ showed little difference. However, the sample residue was less at the injection port temperature of 250℃, so 250℃ was selected as the optimal injection port temperature.

[0219] 2.2.4 Investigation of column flow rate

[0220] 2.2.4.1 Preparation of the test solution

[0221] Prepare the ethyl acetate solution of Xinnaoning capsules according to section “2.2.1.1”; take 5 ml of the ethyl acetate solution of Xinnaoning capsules, add an appropriate amount of anhydrous sodium sulfate to remove water, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0222] 2.2.4.2 GC-MS Conditions

[0223] (1) Mass spectrometry conditions: EI source, ion source temperature 230℃, quadrupole temperature set to 150℃, full scan mode, scan range: 30~550u, auxiliary heating zone temperature: 270℃; solvent delay: 3min, NIST14.0 standard spectral library was used for search and analysis.

[0224] (2) Chromatographic conditions: Agilent HP-5MS capillary column (30m×0.25mm×0.25μm), injection port temperature 250℃, septum purge flow rate: 3ml / min, split ratio 20:1; temperature program: initial temperature 60℃, hold for 3min; increase to 210℃ at 3.5℃ / min and hold for 5min; run at 250℃ for 15min. The optimal column flow rate was investigated at 1.0ml / min and 2.0ml / min respectively.

[0225] 2.2.4.3 Determination method: According to GC-MS conditions, accurately pipette 1 μl of the test solution and detect.

[0226] 2.2.4.4 Measurement Results

[0227] See Figures 29 to 31 After comparing the two column flow rates, the total ion chromatogram distribution was more uniform when the column flow rate was 1.0 ml / min. Therefore, a column flow rate of 1.0 ml / min was selected.

[0228] 2.2.5 Conclusion

[0229] After optimizing the above chromatographic conditions, the optimal GC-MS determination conditions and the GC-MS total ion chromatogram of Xinnaoning volatile oil under the optimal determination conditions were obtained, as follows:

[0230] 2.2.5.1 Optimal GC-MS determination conditions

[0231] (1) Preparation of the test solution

[0232] Weigh 50g of the powder from Xinnaoning capsules, add 50ml of ethyl acetate, place in an Erlenmeyer flask, sonicate for 1 hour, filter to obtain an ethyl acetate solution of Xinnaoning capsules; take 5ml of the ethyl acetate solution of Xinnaoning capsules, add an appropriate amount of anhydrous sodium sulfate to remove water, filter through a 0.22μm filter membrane to obtain the test solution.

[0233] (2) GC-MS conditions

[0234] Chromatographic conditions: EI source, ion source temperature 230℃, quadrupole temperature set to 150℃, full scan mode, scan range: 30~550u, auxiliary heating zone temperature: 270℃; solvent delay: 3min; NIST14.0 standard spectral library was used for search and analysis. Mass spectrometry conditions: Agilent HP-5MS capillary column (30m×0.25mm×0.25μm), injection port temperature 250℃, septum purge flow rate: 3ml / min, column flow rate 1.0ml / min, split ratio 20:1; temperature program: initial temperature 60℃, hold for 3min; ramp to 220℃ at 3.5℃ / min and hold for 5min; run at 250℃ for 15min.

[0235] (3) Measurement method

[0236] According to the GC-MS conditions, accurately pipette 1 μl of the test solution and detect it by GC-MS.

[0237] 2.2.5.2 Total ion chromatogram of Xinnaoning volatile oil under optimal determination conditions by GC-MS

[0238] The measurement results are shown below. Figure 32 The lipid-soluble compounds of Xinnaoning showed low baseline noise, uniform peak distribution, and good separation in their GC-MS total ion chromatograms under optimal conditions.

[0239] 2.2.7 Methodological Investigation of Fingerprint Spectroscopy of Lipid-Soluble Components in Xinnaoning Capsules

[0240] 2.2.7.1 Precision Test

[0241] Xinnaoning capsules (batch number: 20181032) were used. Samples were prepared according to the method for preparing the test solution. GC-MS analysis was performed under the specified conditions, with an injection volume of 1 μl, injected six times consecutively. The GC-MS fingerprint was measured, and the retention time and peak area of ​​each common chromatographic peak were recorded. Using the retention time and peak area of ​​peak 6 as a reference, the relative retention time and relative peak area of ​​the main chromatographic peaks in the sample were calculated, and the corresponding average value and relative standard deviation (RSD%) were determined. Results are shown below. Figure 33 See Tables 1 to 4.

[0242] Table 1 Precision test - retention time

[0243]

[0244]

[0245] Table 2 Precision Examination - Peak Area

[0246]

[0247] Table 3 Precision Study - Relative Retention Time

[0248]

[0249] Table 4 Precision Study - Relative Retained Peak Area

[0250]

[0251]

[0252] Through analysis Figure 33 The results of the precision tests are shown in Tables 1 to 4. The RSD values ​​for retention time, peak area, and relative retention time are all less than 1.00%, and the RSD values ​​for relative peak area are all less than 15.00%. This indicates that the instrument has good precision. (In GC-MS coupling, an RSD value ≤ 15% for method validation is considered satisfactory.)

[0253] 2.2.6.2 Repeatability Test

[0254] Six aliquots of Xinnaoning capsules (batch number: 20181032) were prepared according to the preparation method for the test solution. GC-MS was performed under 1 μl injection conditions to determine the fingerprint chromatograms. The retention times and peak areas of each common chromatographic peak were recorded. Using the retention time and peak area of ​​peak number 6 as a reference, the relative retention times and relative peak areas of the main chromatographic peaks in the sample were calculated, and the corresponding averages and relative standard deviations (RSD%) were determined. The results are shown below. Figure 34 See Tables 5 to 8.

[0255] Table 5 Repeatability Tests - Retention Time

[0256]

[0257] Table 6 Repeatability Tests - Peak Area

[0258]

[0259]

[0260] Table 7 Repeatability Tests - Relative Retention Time

[0261]

[0262] Table 8 Repeatability Tests - Relative Peak Area

[0263]

[0264]

[0265] Through analysis Figure 34 Tables 5 to 8 show the repeatability test results. The RSD values ​​for retention time, peak area, and relative retention time are all less than 1.00%, and the RSD values ​​for relative peak area are all less than 15.00%. This indicates that the experiment has good repeatability. (For GC-MS coupling, an RSD value ≤ 15% for method validation is considered satisfactory.)

[0266] 2.2.6.3 Stability Test

[0267] Xinnaoning capsules (batch number: 20181032) were used. Samples were prepared according to the preparation method for the test solution. GC-MS analysis was performed under the specified conditions, with an injection volume of 1 μl. Samples were injected at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h to determine the fingerprint chromatogram. The retention time and peak area of ​​each common chromatographic peak were recorded. Using the retention time and peak area of ​​peak 6 as a reference, the relative retention time and relative peak area of ​​the main chromatographic peaks in the sample were calculated, and the corresponding average and relative standard deviation (RSD%) were determined. The results are shown below. Figure 35 See Tables 9 to 12.

[0268] Table 9 Stability Study - Retention Time

[0269]

[0270] Table 10 Stability Study - Peak Area

[0271]

[0272]

[0273] Table 11 Stability Study - Relative Retention Time

[0274]

[0275] Table 12 Stability Study - Relative Peak Area

[0276]

[0277] Analysis of the above graphs and stability test results table shows that the RSD values ​​of the retention times in the stability tests were all less than [value missing].

[0278] The RSD values ​​for peak area and relative retention time were all less than 1.00%, and the RSD values ​​for relative peak area were all less than 15.00%. These results demonstrate that the prepared sample exhibits good stability within 24 hours. (In GC-MS coupling, an RSD value ≤ 15% for method validation is considered satisfactory.)

[0279] 2.2.7 Conclusion

[0280] Using the established GC-MS conditions for Xinnaoning capsules, the precision, repeatability, and stability of the methodology were studied. The results showed that the instrument had good precision, the assay method had good repeatability, and the prepared sample had good stability within 24 hours.

[0281] 2.3 Similarity Study of Fingerprint Spectra of Lipid-Soluble Components

[0282] A similarity study was conducted on 26 batches of Xinnaoning capsules provided by Guizhou Jingcheng Pharmaceutical Co., Ltd. The batch numbers and serial numbers are detailed in Table 13.

[0283] Table 13 Product Number and Batch Number of Xinnaoning Capsules

[0284]

[0285] 2.3.1 Experimental Methods

[0286] 2.3.1.1 GC-MS Conditions

[0287] Chromatographic conditions: EI source, ion source temperature 230℃, quadrupole temperature set to 150℃, full scan mode, scan range: 30~550u, auxiliary heating zone temperature: 270℃; solvent delay: 3min; NIST14.0 standard spectral library was used for search and analysis.

[0288] Mass spectrometry conditions: Agilent HP-5MS capillary column (30m×0.25mm×0.25μm), injection port temperature 250℃, septum purge flow rate: 3ml / min, column flow rate 1.0ml / min, split ratio 20:1; temperature program: initial temperature 60℃, hold for 3min; ramp to 220℃ at 3.5℃ / min and hold for 5min; run at 250℃ for 15min.

[0289] 2.3.1.2 Preparation of the test solution

[0290] Weigh 50g of the powder from Xinnaoning capsules, add 50ml of ethyl acetate, place in an Erlenmeyer flask, sonicate for 1 hour, filter to obtain an ethyl acetate solution of Xinnaoning capsules; take 5ml of the ethyl acetate solution of Xinnaoning capsules, add an appropriate amount of anhydrous sodium sulfate to remove water, filter through a 0.22μm filter membrane to obtain the test solution.

[0291] 2.3.1.3 Determination Method

[0292] According to the GC-MS conditions, accurately pipette 1 μl of the test solution and detect it by GC-MS.

[0293] 2.3.2 Measurement Results

[0294] 2.3.2.1 Similarity evaluation was performed using the similarity software provided by the National Pharmacopoeia Commission.

[0295] (1) Analysis of all samples from 26 batches

[0296] GC-MS analysis was performed on 26 batches of samples to obtain the fingerprint chromatograms of the lipid-soluble components of Xinnaoning capsules. The obtained chromatograms were analyzed using the "Similarity Software Evaluation System for Chromatographic Fingerprint Spectrum of Traditional Chinese Medicine (2012 Edition)" software from the National Pharmacopoeia Commission. The latest batch (20190722) sample S26 was used as the reference chromatogram. The average method was used to generate the reference chromatogram, with a time window width set to 0.2, multi-point correction, and full-spectrum peak matching. See [link to reference chromatogram]. Figures 36 to 37 The similarity was calculated, and the results are shown in Table 14.

[0297] Table 14. Similarity results of GC-MS fingerprint spectra of fat-soluble components in Xinnaoning capsules (26 batches)

[0298]

[0299]

[0300] According to the experimental results, the similarity of all 26 batches of samples was greater than 0.725, with an average similarity of 0.888. Among them, samples S5 (20170723), S12 (20181032), and S16 (20190142) had similarities less than 0.850, while the others were greater than 0.850. This indicates that some sample spectra have certain differences, possibly due to differences between samples from different years, and further analysis of the similarity of samples from the same year is needed.

[0301] (2) Sample analysis in 2017

[0302] Nine batches of samples from 2017 were analyzed by GC-MS to obtain the fingerprint chromatograms of the lipid-soluble components of Xinnaoning capsules. The obtained chromatograms were analyzed using the "Similarity Software Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" software from the National Pharmacopoeia Commission. The latest batch (20171111) sample S9 was used as the reference chromatogram. The average method was used to generate the reference chromatogram, with a time window width set to 0.2, multi-point correction, and full-spectrum peak matching. See [link to reference chromatogram]. Figures 38 to 39 The similarity was calculated, and the results are shown in Table 15.

[0303] Table 15. Similarity results of GC-MS fingerprint spectra of fat-soluble components in Xinnaoning capsules (2017 batches)

[0304]

[0305] According to the experimental results, the similarity of the nine batches of samples in 2017 was ≥0.888, with an average similarity of 0.944. This shows that the difference in the chromatograms was small and the sample similarity was high, indicating that the production process of Xinnaoning Capsules (2017) was stable.

[0306] (3) Sample analysis in 2018

[0307] Five batches of samples from 2018 were analyzed by GC-MS to obtain the fingerprint chromatograms of the lipid-soluble components of Xinnaoning capsules. The obtained chromatograms were analyzed using the "Similarity Software Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" software from the National Pharmacopoeia Commission. The latest batch (20181133) sample S4 was used as the reference chromatogram. The average method was used to generate the reference chromatogram, with a time window width set to 0.2, multi-point correction, and full-spectrum peak matching. See [link to reference chromatogram]. Figures 40 to 41 The similarity was calculated, and the results are shown in Table 16.

[0308] Table 16. Similarity results of GC-MS fingerprint spectra of fat-soluble components in Xinnaoning capsules (2018 batches)

[0309]

[0310] According to the experimental results, the similarity of the five batches of samples in 2018 was ≥0.935, with an average similarity of 0.954. This shows that the difference in the chromatograms was small and the sample similarity was high, indicating that the production process of Xinnaoning Capsules (2018) was stable.

[0311] (4) Sample analysis in 2019

[0312] Eleven batches of samples from 2019 were analyzed by GC-MS to obtain the fingerprint chromatograms of the lipid-soluble components of Xinnaoning capsules. The obtained chromatograms were analyzed using the "Similarity Software Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" software from the National Pharmacopoeia Commission. The latest batch (20190722) sample S11 was used as the reference chromatogram. The average method was used to generate the reference chromatogram, with a time window width set to 0.2, multi-point correction, and full-spectrum peak matching. See [link to reference chromatogram]. Figures 42 to 43 The similarity was calculated, and the results are shown in Table 17.

[0313] Table 17. Similarity results of GC-MS fingerprint spectra of fat-soluble components in Xinnaoning capsules (2018 batches)

[0314]

[0315] According to the experimental results, the similarity of all 11 batches of samples in 2019 was ≥0.775, with an average similarity of 0.937. The results showed that only batch 20190142 had a similarity of less than 0.850, while the similarity of the other 10 batches was greater than 0.850. This indicates that the differences in the chromatograms were small and the sample similarity was high, suggesting that the production process of Xinnaoning Capsules (2019) was stable.

[0316] (5) 4-year comparative chromatogram analysis of Xinnaoning capsules

[0317] Since there was only one batch of samples in 2016, sample number 20160916 was used to represent the samples of 2016. Similarity was analyzed between the samples and the reference chromatograms from 2017, 2018, and 2019. The chromatograms were analyzed using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Software Evaluation System (2012 version)" software from the National Pharmacopoeia Commission. Sample S4 (the most recent year, 2019 reference chromatogram) was used as the reference chromatogram. The reference chromatogram was generated using the averaging method, with a time window width of 0.2, multi-point correction, and full-spectrum peak matching. See [link to reference chromatogram]. Figures 44 to 45 The similarity was calculated, and the results are shown in Table 18.

[0318] Table 18. Similarity results of GC-MS fingerprint spectra of fat-soluble components in Xinnaoning capsules (2018 batches)

[0319]

[0320]

[0321] According to the experimental results, the similarity of the control chromatograms over the four years was greater than 0.906, with an average similarity of 0.942. This indicates that the differences in the chromatograms were small and the sample similarity was high, suggesting that the production process of Xinnaoning capsules was stable and reliable over the past four years.

[0322] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for establishing the fingerprint spectrum of volatile / lipid-soluble components in Xinnaoning capsules, characterized in that, The establishment of fingerprint profiles for volatile components and lipid-soluble components in Xinnaoning capsules is included, with the specific steps as follows: (I) Establishment of fingerprint spectrum of volatile components in Xinnaoning capsules A1, Preparation of the test solution Weigh the powder from the Xinnaoning capsules, add 10 times the amount of water, add 1 ml of n-hexane to the volatile oil collector, start timing from boiling, distill to extract the volatile oil for 7 hours, collect the n-hexane portion and store it in a small bottle to obtain the small molecule volatile oil of Xinnaoning capsules; take the small molecule volatile oil of Xinnaoning, dissolve and dilute it 100 times with ethyl acetate, add an appropriate amount of anhydrous sodium sulfate to remove water, filter it with a filter membrane to obtain the test solution; A2, GC-MS conditions Mass spectrometry conditions: EI source, ion source temperature 230 ℃, quadrupole temperature set to 150 ℃, full scan mode, scan range: 30-550 u, auxiliary heating zone temperature 270 ℃; solvent delay: 3 min; NIST 14.0 standard spectra can be used for search and analysis. Chromatographic conditions: Agilent HP-5MS capillary column, 30m × 0.25 mm × 0.25 μm, injection port temperature 250 °C, column flow rate 1.0 ml / min, septum purge flow rate 3 ml / min, split ratio 20:1, injection volume 1 μl; Temperature program: Start at 60 °C and hold for 3 min; increase to 210 °C at a rate of 3.5 °C / min and hold for 0 min; increase to 240 °C at a rate of 2 °C / min and hold for 5 min. A3, Determination Method According to the GC-MS conditions, accurately pipette 1 μl of the test solution and detect it by GC-MS; (II) Establishment of fingerprint spectrum of fat-soluble components in Xinnaoning capsules B1, Preparation of the test solution Weigh the powder from Xinnaoning capsules, add ethyl acetate at a mass-to-volume ratio of 1:1 g / ml, sonicate for 1 h, filter to obtain an ethyl acetate solution of Xinnaoning capsules; take the ethyl acetate solution of Xinnaoning capsules, add an appropriate amount of anhydrous sodium sulfate to remove water, filter using a filter membrane to obtain the test solution. B2, GC-MS conditions Mass spectrometry conditions: EI source, ion source temperature 230 ℃, quadrupole temperature set to 150 ℃, full scan mode, scan range: 30-550 u, auxiliary heating zone temperature 270 ℃; solvent delay: 3 min; NIST 14.0 standard spectra were used for search and analysis. Chromatographic conditions: Agilent HP-5MS capillary column, 30m × 0.25 mm × 0.25 μm, injection port temperature 250 °C, column flow rate 1.0 ml / min, septum purge flow rate 3 ml / min, split ratio 20:1, injection volume 1 μl; Temperature program: Start at 60 °C and hold for 3 min; increase to 220 °C at a rate of 3.5 °C / min and hold for 5 min; then run at 250 °C for 15 min. B3, Determination Method According to the GC-MS conditions, accurately pipette 1 μl of the test solution and detect it by GC-MS.

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