A method for detecting volatile components in Tongluo Qutong ointment extract by gas chromatography-mass spectrometry

Abstract

This invention relates to a method for detecting the content of volatile components in Tongluo Qutong Gao extract using gas chromatography-mass spectrometry (GC-MS). This method effectively addresses the shortcomings of existing quality evaluation methods and guides the extraction and production process. The technical solution includes the following steps: 1) determining chromatographic conditions; 2) preparing a reference solution; 3) preparing a test solution; 4) establishing and evaluating characteristic chromatograms; and 5) determining the correlation between the volatile components of Tongluo Qutong Gao extract and the medicinal materials. This invention has been validated methodologically for good precision, repeatability, and stability. It is simple, feasible, and can rapidly determine different indicator components with high accuracy, enabling better evaluation of the quality of Tongluo Qutong Gao extract. It represents an innovation in the method for establishing characteristic chromatograms of volatile components in Tongluo Qutong Gao extract.

Description

I. Technical Field

[0001] This invention relates to the field of quality control of traditional Chinese medicine preparations, and in particular to a method for detecting the content of volatile components in Tongluo Qutong Gao extract using gas chromatography-mass spectrometry. II. Background Technology

[0002] Tongluo Qutong Gao is a traditional Chinese medicine rubber plaster for treating osteoarthritis. The prescription contains 11 kinds of Chinese medicinal materials, including Angelica sinensis, Ligusticum chuanxiong, Zanthoxylum bungeanum, black pepper, clove, cinnamon, Piper longum, dried ginger, Kaempferia galanga, safflower, and rhubarb. The extraction method of the medicinal materials is as follows: rhubarb and safflower are crushed into coarse powder and set aside; the remaining nine herbs, including Angelica sinensis, are crushed into coarse powder, and the volatile oil is extracted by distillation and collected for later use; after the residue is drained, it is mixed with the coarse powder of rhubarb and safflower, and soaked in 90% ethanol for 24 hours. After soaking, the residue is percolated and 1000ml of the initial percolate is collected. The residue is then percolated again with 70% ethanol and 2500ml of the subsequent percolate is collected. The percolates are combined and concentrated under reduced pressure to a thick paste with a relative density of not less than 1.25 (60℃) for later use. These medicinal herbs contain a large amount of volatile oils with significant pharmacological effects. Each herb's volatile oil contains a variety of chemical components, forming the essential material basis for the efficacy of the Tongluo Qutong plaster. Representative components include artemisinin found in Angelica sinensis and Ligusticum chuanxiong; p-hepta-phenol in Ligusticum chuanxiong; linalool and α-salicylic acid in Zanthoxylum bungeanum; eugenol in clove; cinnamaldehyde and α-piperene in cinnamon; gingerene, β-sesquiphedrine, and β-bisabolene in dried ginger; geraniol in Piper longum; β-eugenol in pepper; and trans-p-methoxycinnamate in Kaempferia galanga. These volatile oil components possess analgesic, anti-anxiety, sedative-hypnotic, anti-inflammatory, antitumor, and antibacterial pharmacological activities, playing a role in anti-inflammatory analgesia and inhibiting platelet aggregation in clinical practice.

[0003] Tongluo Qutong Gao (a traditional Chinese medicine) extract, as a medicinal material, carries the material basis for the efficacy of drugs and plays a crucial role in the product manufacturing process. Currently, as an intermediate product, the internal control standards for Tongluo Qutong Gao extract are relatively simple, often referencing the evaluation approach of chemical drugs. These standards focus on identifying and controlling the content of medicinal materials with relatively high chemical component content, resulting in isolated indicators and weak quality representativeness. Furthermore, the volatile oil components, which are the main active ingredients in the extract, lack comprehensive control indicators and methods that can represent the overall quality of the prescription medicinal materials. This makes it impossible to directly assess batch-to-batch differences in volatile oil quality, leading to blind spots in the overall quality assessment of the extract. Gas chromatography (GC-MS) is an effective method for identifying and detecting volatile oil substances and is increasingly being used and promoted in drug quality control. Additionally, the characteristic chromatogram of traditional Chinese medicine, as a multi-indicator quality control model, is a method for evaluating complex traditional Chinese medicinal materials and their preparations. It can comprehensively reflect the overall profile characteristics of the evaluated substances and further correlate them with their pharmacological activities. This is essential for improving current quality control methods and ensuring the safety and efficacy of clinical medication. III. Summary of the Invention

[0004] In view of the above situation and to overcome the shortcomings of the existing technology, the purpose of this invention is to provide a method for detecting the content of volatile components in Tongluo Qutong Gao extract using gas chromatography-mass spectrometry, which can effectively improve the deficiencies of existing quality evaluation methods and guide the extraction and production process.

[0005] The technical solution provided by this invention is a method for detecting volatile components in a traditional Chinese medicine extract using gas chromatography-mass spectrometry, comprising the following steps:

[0006] 1) Determination of chromatographic conditions:

[0007] Gas chromatography conditions: Agilent DB-5ms capillary column, 30m × 0.25mm, film thickness 0.25μm; carrier gas: high-purity helium, purity: 99.999%; column temperature programmed: initial temperature 45℃, hold for 2 min, then at 0.8℃ / min. -1 Raise to 52℃ and hold for 15 minutes, at a rate of 1.5℃ / min. -1 Raise to 103℃ and hold for 40 minutes at a rate of 1.7℃ / min. -1 Raise to 160℃, hold for 5 minutes, at a rate of 2.0℃ / min -1 Raise to 210℃ and hold for 30 minutes, at a rate of 4.0℃ / min. -1 Heat to 230℃ and hold for 10 min; injector temperature 220℃; injection volume 1 μL; column flow rate 0.8 ml / min; split ratio 5:1; theoretical plate number based on linalool peak not less than 6000.

[0008] The mass spectrometry conditions were as follows: electron impact ion source; electron energy 70 eV; ion source temperature: 230℃; interface temperature: 230℃; quadrupole temperature 150℃; mass scan range 30-600 m / z full scan; solute delay 5.5 min; subsequent run at 240℃ for 10 min; tuning file was standard tuning.

[0009] 2) Preparation of the reference solution:

[0010] Accurately weigh 10.12 mg of limonene, 10.06 mg of linalool, 10.13 mg of β-caryophyllene, 10.05 mg of eugenol, and 10.15 mg of ethyl p-methoxycinnamate, and place them separately in 10 ml volumetric flasks. Dissolve and dilute to the mark with ethyl acetate to prepare reference stock solutions. Accurately pipette 2 ml of linalool and eugenol reference stock solutions and 1 ml of limonene, β-caryophyllene, and ethyl p-methoxycinnamate reference stock solutions into the same 20 ml volumetric flask. Dilute to the mark with ethyl acetate and shake well to prepare a mixed reference solution.

[0011] 3) Preparation of the test solution:

[0012] Accurately weigh 5g of the Tongluo Qutong Ointment extract sample and place it in a 250ml round-bottom flask. Add 100ml of distilled water and perform the volatile oil determination method. Add water from the top of the measuring apparatus until it fills the graduated part and overflows into the flask. Add 2ml of ethyl acetate, connect the reflux condenser, heat to boiling, and maintain a gentle boil for 5 hours. Cool, separate the ethyl acetate solution, and filter it through a funnel lined with anhydrous sodium sulfate. Place the filtrate in a 10ml volumetric flask and wash the condenser, volatile oil measuring apparatus, and funnel with ethyl acetate in sequence. Combine the washing solution with the above ethyl acetate solution, dilute with ethyl acetate to the mark, shake well, filter, and inject 1μl of the filtrate into the gas chromatograph for determination.

[0013] 4) Establishment and evaluation of feature maps

[0014] Take 16 batches of Tongluo Qutong Ointment extract samples and prepare test solutions according to step 3). Accurately pipette 1 μL and inject it into the gas chromatography-mass spectrometry (GC-MS) instrument. Analyze the samples under the GC-MS conditions of step 1). Based on the relative retention times of each chromatographic peak in the obtained chromatogram, determine the common peaks and select 53 common peaks as characteristic peaks to establish a reference characteristic chromatogram. With peak 23 as the reference peak S, calculate the ratio of the retention time of each chromatographic peak to the retention time of peak S in the same chromatogram to obtain the relative retention time ratio.

[0015] 5) Determine the correlation between the volatile components of the Tongluo Qutong plaster extract and the medicinal materials.

[0016] The test solutions of Tongluo Qutong Gao extract, the test solutions of each single herb in equal amounts to the raw drug of the preparation, and the negative control solution lacking a single herb (the control solution prepared by removing one of the herbs) were taken separately. The characteristic chromatograms of volatile components in Tongluo Qutong Gao extract were compared with the GC-MS characteristic chromatograms of 11 single herbs (Angelica sinensis, Ligusticum chuanxiong, Zanthoxylum bungeanum, black pepper, clove, cinnamon, Piper longum, dried ginger, Kaempferia galanga, Carthamus tinctorius, and rhubarb) and the negative control solution lacking a single herb. The relative retention times of each chromatographic peak were compared to determine the main source of the common peak.

[0017] The GC-MS standard reference characteristic spectrum of the volatile components of the Tongluo Qutong Ointment consists of 53 common peaks. The similarity between the characteristic spectrum of the sample and the characteristic spectrum of the reference is above 0.9. Using the reference standard as a reference, peak 9 is limonene, peak 23 is linalool, peak 24 is β-caryophyllene, peak 44 is eugenol, and peak 53 is ethyl p-methoxycinnamate. Linalool is used as the reference, and peak 23 is used as the reference peak. The standard reference characteristic spectrum has 53 common characteristic peaks. The peak corresponding to the reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within ±1% of the specified value.

[0018] The average relative retention times of the standard control characteristic chromatograms were 0.128 (peak 1), 0.132 (peak 2), 0.159 (peak 3), 0.203 (peak 4), 0.261 (peak 5), 0.286 (peak 6), 0.293 (peak 7), 0.313 (peak 8), 0.356 (peak 9), 0.372 (peak 10), 0.453 (peak 11), 0.466 (peak 12), and 0.488. Peak 13, 0.517, Peak 14, 0.542, Peak 15, 0.636, Peak 16, 0.779, Peak 17, 0.810, Peak 18, 0.822, Peak 19, 0.865, Peak 20, 0.886, Peak 21, 0.912, Peak 22, 1.000, Peak 23, 1.057, Peak 24, 1.078, Peak 25, Peak 26 1.200 (peak 27), 1.264 (peak 28), 1.323 (peak 29), 1.371 (peak 30), 1.392 (peak 31), 1.408 (peak 32), 1.431 (peak 33), 1.502 (peak 34), 1.521 (peak 35), 1.565 (peak 36), 1.587 (peak 37), 1.701 (peak 38), 1.812 (peak 39), 2.060 (Peak 40), 2.152 (Peak 41), 2.169 (Peak 42), 2.180 (Peak 43), 2.233 (Peak 44), 2.316 (Peak 45), 2.326 (Peak 46), 2.386 (Peak 47), 2.397 (Peak 48), 2.420 (Peak 49), 2.457 (Peak 50), 2.526 (Peak 51), 2.569 (Peak 52), 2.618 (Peak 53).

[0019] This invention relates to the application of a gas chromatography-mass spectrometry method for detecting the content of volatile components in Tongluo Qutong Gao extract in the quality standards and quality control of Tongluo Qutong Gao extract.

[0020] This invention has been validated by methodology for its good precision, repeatability, and stability. It is simple, feasible, and can quickly determine different indicator components with high accuracy. It can better evaluate the quality of Tongluo Qutong Gao extract and is an innovation in the method of establishing the characteristic spectrum of volatile components of Tongluo Qutong Gao extract. IV. Description of the attached drawings

[0021] Figure 1 This is a standard characteristic spectrum of the volatile components of the Tongluo Qutong Ointment extract of the present invention.

[0022] Figure 2 This is a superimposed GC-MS characteristic spectrum of volatile components from 16 batches of Tongluo Qutong Ointment extract samples of the present invention, and a control characteristic spectrum (R).

[0023] Figure 3 This is a chromatogram of the reference standard of the present invention.

[0024] Figure 4 This is a comparative chromatogram of the reference medicinal material, the negative control without single medicinal material, and the extract of the Tongluo Qutong Ointment of this invention.

[0025] Figure 5 This is an experimental spectrum for the precision of the feature map of the present invention.

[0026] Figure 6 This is an experimental spectrum showing the stability of the feature spectrum of the present invention.

[0027] Figure 7 This is a repeatability test spectrum of the feature spectrum of the present invention. V. Detailed Implementation Methods

[0028] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings and examples.

[0029] Example 1

[0030] 1) Chromatographic conditions:

[0031] Gas chromatography conditions: Agilent DB-5ms capillary column, 30m × 0.25mm, film thickness 0.25μm; carrier gas: high-purity helium, purity: 99.999%; column temperature programmed: initial temperature 45℃, hold for 2 min, then at 0.8℃ / min. -1 Raise to 52℃ and hold for 15 minutes, at a rate of 1.5℃ / min. -1 Raise to 103℃ and hold for 40 minutes at a rate of 1.7℃ / min. -1 Raise to 160℃, hold for 5 minutes, at a rate of 2.0℃ / min -1 Raise to 210℃ and hold for 30 minutes, at a rate of 4.0℃ / min. -1 Heat to 230℃ and hold for 10 min; injector temperature 220℃; injection volume 1 μL; column flow rate 0.8 ml / min; split ratio 5:1; theoretical plate number based on linalool peak not less than 6000.

[0032] The quantitative mass spectrometry conditions were as follows: electron impact (EI) ion source; electron energy 70 eV; ion source temperature: 230℃; interface temperature: 230℃; quadrupole temperature 150℃; mass scan range m / z (30-600) full scan; solute delay (5.5 min); subsequent run at 240℃ for 10 min; the tuning file was standard tuning, SIM ion monitoring mode, and the specific SIM detection time and quantitative and qualitative ion settings for each analyte are shown in Table 1.

[0033] Table 1 Quantitative and qualitative ions of each component compound

[0034]

[0035] 2) Preparation of the reference solution:

[0036] Accurately weigh 10.12 mg of limonene, 10.06 mg of linalool, 10.13 mg of β-caryophyllene, 10.05 mg of eugenol, and 10.15 mg of ethyl p-methoxycinnamate, and place them separately in 10 ml volumetric flasks. Dissolve and dilute to the mark with ethyl acetate to prepare reference stock solutions. Accurately pipette 2 ml of linalool and eugenol reference stock solutions, and 1 ml of limonene, β-caryophyllene, and ethyl p-methoxycinnamate reference stock solutions, and place them in the same 20 ml volumetric flask. Then accurately add 1 ml of internal standard solution, dilute to the mark with ethyl acetate, and shake well to prepare a mixed reference solution containing internal standard. The internal standard solution is prepared by accurately weighing methyl salicylate, adding ethyl acetate to prepare a solution with a concentration of 0.8 mg / ml, and shaking well.

[0037] 3) Preparation of the test solution:

[0038] Accurately weigh 5g of the Tongluo Qutong Ointment extract sample and place it in a 250ml round-bottom flask. Add 100ml of distilled water and, according to the volatile oil determination method, add water from the top of the measuring apparatus until it fills the graduated portion and overflows into the flask. Then add 3ml of ethyl acetate, connect the reflux condenser, heat to boiling, and maintain a gentle boil for 5 hours. Cool, separate the ethyl acetate solution, and filter it through a funnel lined with anhydrous sodium sulfate. Place the filtrate in a 20ml volumetric flask and wash the condenser, volatile oil measuring apparatus, and funnel sequentially with ethyl acetate. Combine the washings with the ethyl acetate solution above, accurately add 2ml of internal standard solution, dilute to the mark with ethyl acetate, shake well, and filter to obtain the final product.

[0039] 4) Determination of volatile component content

[0040] Sixteen batches of Tongluo Qutong Gao extract samples were taken, and test solutions were prepared according to step 3). The samples were then injected and determined under the GC-MS conditions of step 1). The contents of limonene, linalool, β-caryophyllene, eugenol, and ethyl p-methoxycinnamate in the Tongluo Qutong Gao extract were calculated using the internal standard method. Based on the test results of the 16 batches of samples and the influence of production process conditions, the standard was tentatively set at 50% of the average content of each component in the 16 products. The content standards of the above effective components in each 1g of Tongluo Qutong Gao extract were specified as follows: limonene not less than 0.62mg / g, linalool not less than 1.24mg / g, β-caryophyllene not less than 0.52mg / g, eugenol not less than 1.43mg / g, and ethyl p-methoxycinnamate not less than 0.96mg / g.

[0041] The present invention uses gas chromatography - mass spectrometry to establish the characteristic fingerprint of the extract of Tongluo Qutong Ointment, evaluate the authenticity of the intermediate product derived from medicinal materials from an overall characteristic perspective, the stability of the extraction process, the volatility of the extract quality, and express the consistency of the pharmacodynamic substances in the extracts of different batches of Tongluo Qutong Ointment, so as to supplement and improve the deficiencies existing in the existing quality evaluation methods and guide the extraction production process. The relevant test data are as follows:

[0042] 1. Main instruments

[0043] 7820A - 5977B gas chromatography - mass spectrometry (Agilent, USA), MassHunter workstation, NIST17.0 standard mass spectrometry retrieval library; XPE105 one - hundred - thousandth electronic balance (d = 0.01mg) (Mettler - Toledo, Switzerland); volatile oil detector with relative density < 1.0, 24 / 29 standard mouth plug (Beijing Beibo Bomei Glass Co., Ltd., specification: 5mL);

[0044] 2. Test drugs and reagents

[0045] Eugenol (batch number: 110725 - 201917, National Institutes for Food and Drug Control, purity: 99.1%), Ethyl p - methoxycinnamate (batch number: 110835 - 202005, National Institutes for Food and Drug Control, purity: 99.4%), β - caryophyllene (batch number: 111819 - 202104, National Institutes for Food and Drug Control, purity: 98.0%), Linalool (batch number: 111503 - 202004, National Institutes for Food and Drug Control, purity: 97.8%), Limonene (batch number: 100470 - 201503, National Institutes for Food and Drug Control, purity: 96.0%), Methyl salicylate (batch number: 195005 - 201601, National Institutes for Food and Drug Control, purity: 99.6%), Purified water (batch number: 202212036214HN, source: Hangzhou Wahaha Group Co., Ltd.), Ethyl acetate (batch number: 20220902, source: Tianjin Kemiou Chemical Reagent Co., Ltd., analytical pure)

[0046] Drugs: Tongluo Qutong Ointment Extract (Henan Lingrui Pharmaceutical Co., Ltd., batch numbers: 220802, 220803, 220804, 220805, 220806, 220807, 220808, 220809, 220810, 220903, 220904, 220905, 221006, 221007, 221008, 221009; numbered S1~S16); Angelica sinensis reference material (batch number: 120927-202118), Ligusticum chuanxiong reference material (batch number: 120918-201813), Carthamus tinctorius reference material (batch number: 120907-20171). 3) The following reference materials were purchased from the China National Institutes for Food and Drug Control: Kaempferia galanga (batch number: 121504-201203), Zanthoxylum bungeanum (batch number: 121106-201906), Piper nigrum (batch number: 121441-201902), Piper longum (batch number: 121023-201103), Zingiber officinale (batch number: 120942-201911), Clove (batch number: 121039-201906), Cinnamomum cassia (batch number: 121363-202104), and Rheum palmatum (batch number: 120984-202203).

[0047] 3. Analysis software: 2012 version of the Chromatographic Fingerprint Similarity Evaluation System for Traditional Chinese Medicine (National Pharmacopoeia Commission).

[0048] 4. Selection of test conditions

[0049] 4.1 Selection of Extraction Solvent

[0050] Except for the type and conditions of the extraction solvent, the basic steps for chromatographic detection of the test sample are the same. Take 5g of Tongluo Qutong Gao extract from batch number 220805, accurately weigh it, place it in a 250ml round-bottom flask, add 100ml of distilled water, and perform the test according to the method for determination of volatile oil (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 2204). Add water to the top of the measuring apparatus until it fills the graduated section and overflows into the flask. Add 2 ml each of petroleum ether (60-90%), xylene, and ethyl acetate. Connect the reflux condenser, heat to boiling, and maintain a gentle boil for 5 hours. Cool, separate the extracts, and filter through a funnel lined with an appropriate amount of anhydrous sodium sulfate. Place the filtrate in a 10 ml volumetric flask. Wash the condenser, volatile oil measuring apparatus, and funnel sequentially with appropriate amounts of each extract. Combine the washings with the above extracts, dilute to the mark with each extract, shake well, filter, and inject 1 μl of the filtrate into the gas chromatograph for analysis. The number of identifiable chromatographic peaks, peak shape, peak separation, and peak area per unit mass of the sample were used as evaluation indicators for analysis. The results showed that among the top 5 peaks with larger peak areas (eugenol, linalool, ethyl p-methoxycinnamate, limonene, and β-eugenol) in samples extracted with petroleum ether (60-90) and xylene, the peak areas per unit mass of the sample extracted with petroleum ether (60-90) and xylene were smaller than those extracted with ethyl acetate. Ethyl acetate had the highest number of identifiable peaks. Therefore, ethyl acetate was the preferred extraction solvent.

[0051] 4.2 Selection of Extraction Solvent Amount

[0052] Except for the extraction solvent dosage, the basic steps for chromatographic detection of the test sample are the same as those for the test sample. Take 5g of Tongluo Qutong Gao extract from batch number 220805, accurately weigh it, place it in a 250ml round-bottom flask, add 100ml of distilled water, and perform the test according to the volatile oil determination method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 2204). Add water to the top of the measuring apparatus until it fills the graduated section and overflows into the flask. Then add 1 ml, 2 ml, 3 ml, and 4 ml of ethyl acetate respectively. Connect the reflux condenser, heat to boiling, and maintain a gentle boil for 5 hours. Cool, separate the extract, and filter it through a funnel lined with an appropriate amount of anhydrous sodium sulfate. Place the filtrate in a 10 ml volumetric flask, and wash the condenser, volatile oil measuring apparatus, and funnel sequentially with an appropriate amount of ethyl acetate. Combine the washings with the above extract, dilute to the mark with ethyl acetate, shake well, filter, and inject 1 μl of the filtrate into the gas chromatograph for analysis. The number of identifiable chromatographic peaks, peak shape, peak separation, and peak area per unit mass of the test sample were used as evaluation indicators. The experimental results showed that as the amount of ethyl acetate increased from 1 ml to 2 ml, the number of identifiable chromatographic peaks and the peak area per unit mass of the test sample gradually increased. However, when the amount of ethyl acetate increased to 3 ml and 4 ml, there was no significant increase in the number of identifiable chromatographic peaks and the peak area per unit mass of the test sample. Therefore, ethyl acetate is the preferred extraction solvent with a dosage of 2 ml.

[0053] 4.3 Investigation on the dosage of extract

[0054] Except for the dosage conditions of the extract, the basic steps for chromatographic detection are the same as those for the test sample. Take 3g, 4g, 5g, and 6g of the Tongluo Qutong Gao extract from batch number 220805, respectively, and place them in a 250ml round-bottom flask. Add 100ml of distilled water and perform the test according to the volatile oil determination method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 2204). Add water from the top of the measuring apparatus until it fills the graduated section and overflows into the flask. Then add 2ml of ethyl acetate to each flask, connect the reflux condenser, heat to boiling, and maintain a gentle boil for 5 hours. Cool, separate the extract, and filter it through a funnel lined with an appropriate amount of anhydrous sodium sulfate. Place the filtrate in a 10ml volumetric flask, and wash the condenser, volatile oil measuring apparatus, and funnel sequentially with an appropriate amount of ethyl acetate. Combine the washings with the above extract, dilute to the mark with ethyl acetate, shake well, filter, and inject 1μl of the filtrate into the gas chromatograph for determination. The analysis was conducted using the number of identifiable chromatographic peaks, peak shape, peak separation, and peak area per unit mass of the test sample as evaluation indicators. The results showed that when the amount of Tongluo Qutong Gao extract sample was 3g and 4g, the number of identifiable chromatographic peaks was relatively small. When the amount of Tongluo Qutong Gao extract sample was 5g and 6g, the number of identifiable chromatographic peaks was larger and there was no difference. Therefore, from the perspective of saving sample, the preferred amount of Tongluo Qutong Gao extract sample is 5g.

[0055] 4.4. Examination of extraction time

[0056] Except for the extraction time conditions, the basic steps for chromatographic detection of the test sample are the same as those for the test sample. Take 5g of Tongluo Qutong Gao extract from batch number 220805, accurately weigh it, place it in a 250ml round-bottom flask, add 100ml of distilled water, and perform the test according to the method for determination of volatile oil (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 2204). Add water from the top of the measuring device until it fills the graduated part and overflows into the flask. Then add 2ml of ethyl acetate, connect the reflux condenser, heat to boiling, and maintain a gentle boil for 3, 5, and 7 hours respectively. Cool, separate the extract, filter it through a funnel lined with an appropriate amount of anhydrous sodium sulfate, place the filtrate in a 10ml volumetric flask, and wash the condenser, volatile oil measuring device, and funnel with an appropriate amount of ethyl acetate in sequence. Combine the washing liquid with the above extract, dilute with ethyl acetate to the mark, shake well, filter, and inject 1μl of the filtrate into the gas chromatograph for determination. The number of identifiable chromatographic peaks, peak shape, peak separation, and peak area per unit mass of the test sample were used as evaluation indicators for analysis. The experimental results showed that when the extraction time was 3 hours, the number of identifiable chromatographic peaks was relatively small, and the peak area per unit mass of the test sample was relatively small. When the extraction time was 5 hours and 7 hours, the number of identifiable chromatographic peaks was relatively large and basically consistent, and the peak area per unit mass of the test sample was relatively high and there was no significant difference. Therefore, the optimal extraction time was 5 hours.

[0057] 4.5 Examination of programmed heating

[0058] Except for the programmed temperature conditions, the basic steps for chromatographic detection of the test sample are the same. Take 5g of the Tongluo Qutong Gao extract from batch number 220805, accurately weigh it, place it in a 250ml round-bottom flask, add 100ml of distilled water, and perform the test according to the volatile oil determination method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 2204). Add water to the top of the measuring device until it fills the graduated section and overflows into the flask. Then add 2 ml of ethyl acetate, connect the reflux condenser, heat to boiling, maintain a gentle boil for 5 hours, cool, and separate the extract. Filter the extract through a funnel lined with an appropriate amount of anhydrous sodium sulfate. Place the filtrate in a 10 ml volumetric flask and wash the condenser, volatile oil measuring device, and funnel sequentially with an appropriate amount of ethyl acetate. Combine the washings with the extract, dilute to the mark with ethyl acetate, shake well, filter, and inject 1 μl of the filtrate into the gas chromatography-mass spectrometry (GC-MS) instrument. The GC-MS conditions are as follows: Agilent DB-5ms capillary column (30 m × 0.25 mm, membrane thickness 0.25 μm); carrier gas: high-purity helium; column temperature programmed as shown in Table 7-9; injection volume: 1 μL; column flow rate: 0.8 ml / min; split ratio: 5:1; theoretical plate number (based on linalool peak) not less than 6000.

[0059] The mass spectrometry conditions were as follows: electron impact (EI) ion source; electron energy 70 eV; ion source temperature: 230℃; interface temperature: 230℃; quadrupole temperature 150℃; mass scan range m / z (30-600) full scan; solute delay (5.5 min); subsequent run at 240℃ for 10 min; and the tuning file was standard tuning.

[0060] Table 2. Programmed Heating Method 1

[0061]

[0062] Table 3. Programmed Heating Method 2

[0063]

[0064] Table 4. Programmed Heating Method 3

[0065]

[0066] The analysis was conducted using the number of identifiable chromatographic peaks, peak shape, peak separation, and peak area per unit mass of the test sample as evaluation indicators. The experimental results showed that in temperature-programmed method 1, the peak separation was poor at 17-18 and 55-70 minutes; in temperature-programmed method 2, the peak separation was poor at 78-80 minutes; and in temperature-programmed method 3, the peaks with a total peak area exceeding 1% had good separation, basically meeting the requirement of not less than 1.5. Therefore, temperature-programmed method 3 was the preferred method.

[0067] 4.6 Selection of Reference Frame

[0068] In the characteristic chromatograms established in the experiment, linalool showed good chromatographic peak separation, with a large peak area, moderate retention time, and was common to all samples. Therefore, linalool was selected as the reference peak.

[0069] 5. Establishment of a characteristic spectrum of volatile components of the Tongluo Qutong plaster extract

[0070] 5.1 Chromatographic conditions and system suitability test:

[0071] Gas chromatography conditions: Agilent DB-5ms capillary column (30m × 0.25mm, film thickness 0.25μm); carrier gas was high-purity helium (purity: 99.999%); column temperature was programmed: initial temperature 45℃, hold for 2 min, then increase at 0.8℃ / min. -1 Increase to 52℃, hold for 15 min, then increase to 103℃ at a rate of 1.5℃ / min⁻¹, hold for 40 min, then increase at a rate of 1.7℃ / min⁻¹. -1 Raise to 160℃, hold for 5 minutes, at a rate of 2.0℃ / min -1Raise to 210℃ and hold for 30 minutes, at a rate of 4.0℃ / min. -1 Heat to 230℃ and hold for 10 min; injector temperature 220℃; injection volume 1 μL; column flow rate 0.8 ml / min; split ratio 5:1; theoretical plate number based on linalool peak not less than 6000.

[0072] The mass spectrometry conditions were as follows: electron impact (EI) ion source; electron energy 70 eV; ion source temperature: 230℃; interface temperature: 230℃; quadrupole temperature 150℃; mass scan range m / z (30-600) full scan; solute delay (5.5 min); subsequent run at 240℃ for 10 min; and the tuning file was standard tuning.

[0073] 5.2 Preparation of reference solutions: Accurately weigh 10.12 mg of limonene, 10.06 mg of linalool, 10.13 mg of β-caryophyllene, 10.05 mg of eugenol, and 10.15 mg of ethyl p-methoxycinnamate, and place them separately in 10 ml volumetric flasks. Dissolve and dilute to the mark with ethyl acetate to prepare reference stock solutions. Accurately pipette 2 ml of linalool and eugenol reference stock solutions, and 1 ml of limonene, β-caryophyllene, and ethyl p-methoxycinnamate reference stock solutions, and place them in the same 20 ml volumetric flask. Dilute to the mark with ethyl acetate and mix well to prepare a mixed reference solution.

[0074] 5.3 Preparation of the test solution: Accurately weigh 5g of the Tongluo Qutong Gao extract sample and place it in a 250ml round-bottom flask. Add 100ml of distilled water and perform the test according to the volatile oil determination method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 2204). Add water from the top of the measuring apparatus until it fills the graduated section and overflows into the flask. Add 2ml of ethyl acetate, connect the reflux condenser, heat to boiling, and maintain a gentle boil for 5 hours. Cool, separate the ethyl acetate solution, and filter it through a funnel lined with an appropriate amount of anhydrous sodium sulfate. Place the filtrate in a 10ml volumetric flask and wash the condenser, volatile oil measuring apparatus, and funnel sequentially with an appropriate amount of ethyl acetate. Combine the washings with the above ethyl acetate solution, dilute to the mark with ethyl acetate, shake well, filter, and inject 1μl of the filtrate into the gas chromatograph for determination.

[0075] 5.4 Preparation of individual medicinal materials and negative control solutions: According to the prescription dosage and the sample amount of the test sample, calculate the dosage of each individual medicinal material and raw material in the prescription. Take 3.11g of Angelica sinensis, 1.93g of Ligusticum chuanxiong, 1.93g of Kaempferia galanga, 1.93g of Cinnamomum cassia, 0.93g of Syzygium aromaticum, 2.24g of Zanthoxylum bungeanum, 1.93g of Zingiber officinale, 1.93g of Piper nigrum, 1.93g of Rheum palmatum, and 1.93g of Carthamus tinctorius. Prepare the volatile oil extraction solutions of each individual medicinal material and the negative control sample according to the test sample solution preparation method.

[0076] 5.5 Determination method: Accurately pipette 1 μl each of the reference solution, the herbal material solution, the negative control solution, and the test solution, inject them into the gas chromatography-mass spectrometry (GC-MS) chromatograph, determine the chromatogram, and record the chromatogram after 210 minutes.

[0077] 5.6 Methodological Examination

[0078] 5.6.1 Precision Test

[0079] Prepare the test solution of the same batch of Tongluo Qutong Ointment extract (batch number 220805) according to section "5.3", and inject it continuously 6 times according to the chromatographic and mass spectrometric conditions under section "5.1" to detect the characteristic chromatogram, such as... Figure 5 The retention time and peak area of ​​each common peak were recorded. The relative retention time and relative peak area of ​​each common peak were calculated with reference to the retention time and peak area of ​​the linalool chromatographic peak. The RSD values ​​of the relative retention time and relative peak area of ​​each common peak were less than 3%. The similarity of the characteristic chromatograms of each chromatographic peak was calculated to be greater than 0.99 using similarity evaluation software. The instrument was stable and had good precision. The results are shown in Tables 5, 6 and 7.

[0080] Table 5. Precision test results of Tongluo Qutong Ointment Extract (220805) (relative retention time of index component groups)

[0081]

[0082]

[0083] Table 6. Precision test results of Tongluo Qutong Gao extract (220805) (relative peak area of ​​index component groups)

[0084]

[0085]

[0086] Table 7. Calculation results of similarity of precision test patterns for Tongluo Qutong Gao extract (220805).

[0087]

[0088] 5.6.2 Stability Test

[0089] Prepare the test solution of the same batch of Tongluo Qutong Ointment extract (batch number 220601) according to section "5.3", and inject it at 0h, 2h, 4h, 8h, 12h, and 24h according to the chromatographic and mass spectrometric conditions under section "5.1", and detect the characteristic chromatograms, such as... Figure 6The retention times and peak areas of each common peak were recorded. Using the retention time and peak area of ​​the linalool peak as a reference, the relative retention times and relative peak areas of each common peak were calculated. The RSD values ​​of the relative retention times and relative peak areas of each common peak were all less than 3.0%. The similarity of the characteristic chromatograms of each peak, calculated using similarity evaluation software, was greater than 0.99. The test solution was stable within 24 hours. This indicates that the test sample has good stability. The results are shown in Tables 8, 9, and 10.

[0090] Table 8. Stability test results of Tongluo Qutong Ointment Extract (220805) (relative retention time of index component groups)

[0091]

[0092]

[0093] Table 9. Stability test results of Tongluo Qutong Ointment Extract (220805) (relative peak area of ​​index component groups)

[0094]

[0095]

[0096] Table 10. Calculation results of similarity of stability test chromatograms of Tongluo Qutong Gao extract (220805)

[0097]

[0098] 5.6.3 Repeatability Test

[0099] Take the same batch of Tongluo Qutong Gao extract (sample 220805) and prepare 6 samples according to the method under "5.3" Preparation of Test Solution. Inject them sequentially under the chromatographic and mass spectrometric conditions under "5.1" and detect the characteristic chromatograms respectively. Figure 7 The retention time and peak area of ​​each common peak were recorded. Using the retention time and peak area of ​​the linalool chromatographic peak as a reference, the relative retention time and relative peak area of ​​each common peak were calculated. The RSD values ​​of the relative retention time and relative peak area of ​​each common peak were all less than 3.0%. The similarity of the characteristic chromatograms of each chromatographic peak was calculated using similarity evaluation software and was greater than 0.99, indicating that the experimental method has good repeatability. The results are shown in Tables 11, 12 and 13.

[0100] Table 11 Results of repeatability test of Tongluo Qutong Ointment Extract (220805) (relative retention time of index component groups)

[0101]

[0102]

[0103] Table 12 Results of Repeatability Tests on Tongluo Qutong Ointment Extract (220805) (Relative Peak Area of ​​Indicator Components)

[0104]

[0105]

[0106] Table 13 Results of similarity calculation of repeatability test patterns of Tongluo Qutong Gao extract (220801)

[0107]

[0108] 5.6.4 Establishment of Comparative Fingerprint Patterns

[0109] Establish a characteristic spectrum of volatile components in Tongluo Qutong Gao extract: Sixteen batches of Tongluo Qutong Gao extract samples (batch numbers: 220802, 220803, 220804, 220805, 220806, 220807, 220808, 220809, 220810, 220903, 220904, 220905, 221006, 221007, 221008, 221009) were analyzed according to "5.3". The method described in section "5.1" is used to prepare the test solution. 1 μL of each solution is precisely pipetted into the gas chromatography-mass spectrometry (GC-MS) instrument and analyzed under the GC-MS conditions described in section "5.1". Based on the relative retention times of each peak in the obtained chromatogram, common peaks are identified, and 53 common peaks are selected as characteristic peaks to establish a reference characteristic chromatogram. Peak 23 is used as the reference peak S. The ratio of the retention time of each peak to the retention time of peak S in the same chromatogram is calculated, and the resulting relative retention time ratios are shown in Table 14.

[0110] Table 14. Characteristic peaks and relative retention times of the extract of Tongluo Qutong Gao (n=16)

[0111]

[0112]

[0113] 5.6.5 Identification of common peaks in fingerprint spectra

[0114] Using GC-MS technology, the NIST 17.0 database was searched to identify the common peaks in the characteristic spectra of volatile components in the Tongluo Qutong Gao extract. By comparing with reference standards, peak 9 was identified as limonene, peak 23 as linalool, peak 24 as β-caryophyllene, peak 44 as eugenol, and peak 53 as ethyl p-methoxycinnamate.

[0115] 5.6.6 Determine the correlation between the preparation and the medicinal materials:

[0116] Take samples of the Tongluo Qutong Gao extract test solution, the test solutions of each single herb in equal amounts to the raw drug of the preparation, and the negative control solution lacking a single herb. Use the aforementioned Tongluo Qutong Gao extract characteristic chromatogram detection method to obtain the characteristic chromatograms of the volatile components in the Tongluo Qutong Gao extract (see [link to Tongluo Qutong Gao extract]). Figure 1 ) and 11 single-herb medicinal materials including Angelica sinensis, Ligusticum chuanxiong, Zanthoxylum bungeanum, black pepper, clove, cinnamon, Piper longum, dried ginger, Kaempferia galanga, safflower, and rhubarb (see...) Figure 4 The GC-MS characteristic chromatograms of the sample solution and the negative control solution lacking a single herb were compared, and the relative retention times of each chromatographic peak were compared to determine the main source of the common peak. See Table 15.

[0117] Table 15 Correlation between Preparations and Medicinal Materials

[0118]

[0119]

[0120] The volatile oil chromatographic peaks of single-herb galangal are mainly concentrated in the 135-170 min range among the common peaks in the characteristic chromatogram, including peaks 41, 43, 44, and 53. Comparing the negative and whole-herb chromatograms without galangal, peaks 41 and 44 are present in both the whole-herb chromatogram and the negative sample without galangal, indicating that these two components are provided by galangal, but are not specific components of galangal. Peaks 43 and 53 are only present in single-herb and whole-herb chromatograms, indicating that these two components are specific components of galangal in the Tongluo Qutong Gao extract.

[0121] The volatile oil chromatographic peaks of cinnamon as a single herb were mainly concentrated in the 55-135 min range among the common peaks in the characteristic chromatogram, including peaks 21, 27, 28, 34, and 40. Compared with the results of the whole formula and the whole herb, peak 28 was only present in the whole formula and the single herb, indicating that this component is a specific component of cinnamon in the Tongluo Qutong Gao extract.

[0122] The chromatographic peaks of the volatile oil from Sichuan pepper as a single herb are mainly concentrated in the common peaks of the characteristic chromatograms at 8–35 min, 53–100 min, and 115–170 min, including peaks 1, 2, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 22, 23, 24, 25, 27, 29, 30, 34, 39, 42, 44, and 53. Compared with the results of the whole formula and the missing herb, peaks 2, 11, 13, and 30 only appear in the whole formula and the single herb, indicating that these four components are specific components of Sichuan pepper in the Tongluo Qutong Gao extract.

[0123] The volatile oil chromatographic peaks of pepper as a single herb were mainly concentrated in the common peaks of the characteristic chromatograms at 8–35 min, 53–80 min, and 130–150 min, including peaks 1, 4, 5, 6, 7, 9, 14, 15, 20, 21, 23, 24, 27, 40, 44, and 45. Compared with the results of the whole formula and the missing herb, peak 5 was only present in the whole formula and the single herb, indicating that this component is a specific component of pepper in the Tongluo Qutong Gao extract.

[0124] The chromatographic peaks of the volatile oil from safflower as a single herb are mainly concentrated in the common peaks of the characteristic chromatograms at 53–60 min and 138–160 min, including peaks 20, 21, 23, 43, 44, and 50. Compared with the results of the whole formula and the safflower alone, peak 50 is only present in the whole formula and the single herb, indicating that this component is a specific component of safflower in the Tongluo Qutong Gao extract.

[0125] The volatile oil chromatographic peaks of dried ginger as a single herb were mainly concentrated in the common peaks of the characteristic chromatograms at 8–25 min and 52–135 min, including peaks 1, 3, 7, 9, 10, 19, 23, 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40. Compared with the results of the whole formula and the absence of the herb, peaks 3 and 38 were only present in the whole formula and the single herb, indicating that these two components are specific components of dried ginger in the Tongluo Qutong Gao extract.

[0126] The volatile oil chromatographic peaks of clove as a single herb were mainly concentrated in the common peaks of the characteristic chromatograms at 55–100 min and 140–155 min, including peaks 21, 24, 26, 27, 34, 35, 44, 46, and 48. Compared with the results of the whole formula and the absence of clove, peaks 26, 46, and 48 only appeared in the whole formula and the single herb, indicating that these three components are specific components of clove in the Tongluo Qutong Gao extract.

[0127] The volatile oil chromatographic peaks of Angelica sinensis alone are mainly concentrated in the common peaks of the characteristic chromatograms at 8–23 min, 48–92 min, and 137–170 min, including peaks 1, 4, 7, 9, 17, 32, 42, 44, 52, and 53. Comparing the negative and whole-formula chromatograms without Angelica sinensis, the above peaks are present in both the whole-formula and the negative test sample without Angelica sinensis, indicating that these 10 components are provided by Angelica sinensis, but are not specific components of Angelica sinensis in the Tongluo Qutong Gao extract.

[0128] The volatile oil chromatographic peaks of rhubarb alone were mainly concentrated at 55-60 min and 140-145 min in the common peaks of the characteristic chromatogram, including peaks 22 and 44. Compared with the negative and whole formula chromatograms, the above peaks were present in both the whole formula and the negative sample without rhubarb, indicating that these two components are provided by rhubarb, but are not specific components of rhubarb in the Tongluo Qutong Gao extract.

[0129] The volatile oil chromatographic peaks of Ligusticum chuanxiong (a single herb) were mainly concentrated in the common peaks of the characteristic chromatograms at 8–55 min, 68–87 min, and 138–170 min, including peaks 1, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18, 25, 29, 42, 44, 51, 52, and 53. Compared with the results of the whole herb and the whole formula, peaks 16 and 51 were only present in the whole formula and the single herb, indicating that these two components are specific components of Ligusticum chuanxiong in the Tongluo Qutong Gao extract.

[0130] The chromatographic peaks of the volatile oil from Piper longum alone were mainly concentrated in the common peaks of the characteristic chromatograms at 12–25 min, 56–105 min, and 131–170 min, including peaks 4, 9, 21, 22, 23, 24, 27, 31, 32, 33, 34, 36, 37, 40, 42, 44, 45, 47, 49, and 53. Compared with the results of the whole formula and the absence of the herb, peaks 47 and 49 only appeared in the whole formula and the single herb, indicating that these two components are specific components of Piper longum in the Tongluo Qutong Gao extract.

[0131] 5.6.7 Comparison of characteristic spectra of samples from different batches

[0132] Take 16 different batches of Tongluo Qutong Ointment extract, prepare them according to the method under "5.3" Preparation of Test Solution, inject them, detect the characteristic chromatograms, and calculate the relative retention time and relative peak area of ​​each common peak with the retention time and peak area of ​​linalool as reference, as shown in Table 16.

[0133] Comparing the chromatograms of 16 batches of test samples, the sum of the peak areas of the 53 common fingerprint characteristic peaks of the Tongluo Qutong Gao extract of each batch accounted for more than 80% of the total peak area, as shown in Table 17.

[0134] Table 16-1 Relative retention times of common peaks in 16 batches of Tongluo Qutong Ointment extract samples

[0135]

[0136] Table 16-2 Relative retention times of common peaks in 16 batches of Tongluo Qutong Ointment extract samples

[0137]

[0138] Table 16-3 Relative peak areas of common peaks in 16 batches of Tongluo Qutong ointment extract samples

[0139]

[0140]

[0141] Table 16-4 Relative peak areas of common peaks in 16 batches of Tongluo Qutong Ointment extract samples

[0142]

[0143]

[0144] Table 17 Proportion of the peak areas of the common peaks in 16 batches of Tongluo Qutong Plaster extracts

[0145]

[0146]

[0147] The above results show that the RSD of the relative retention times of the characteristic peaks in each batch of samples is less than 0.40%, indicating that the peak emergence times of the characteristic peaks are relatively stable; while the RSD results of the relative peak areas show that there are large differences in the peak areas of the characteristic peaks per unit mass of the extracts in each batch, reflecting the differences in the contents of the same components in the extracts of different batches. The sources of these components are the medicinal materials in the prescription, and the relative peak area differences of the volatile components transferred from the medicinal materials to the extracts are large, indicating that the uniformity and stability of the quality of the medicinal materials need to be further improved.

[0148] To improve the uniformity and stability of traditional Chinese medicine extracts and preparations, it is necessary to continuously improve and stabilize the quality of medicinal materials, improve and standardize the medicinal material planting techniques from the source, and stabilize the quality of medicinal materials.

[0149] 5.6.8, Similarity analysis

[0150] After converting the volatile oil chromatograms of 16 batches of extracts recorded under the proposed chromatographic conditions into data and exporting them, the data were imported, matched, and corrected using the Similarity Evaluation Software for Traditional Chinese Medicine Chromatographic Fingerprints Version 2012 (National Pharmacopoeia Commission) (see Figure 2 ), and a reference characteristic chromatogram that can comprehensively reflect the characteristic information of all samples, that is, the GC-MS standard reference characteristic chromatogram of Tongluo Qutong Plaster extract (as shown in Figure 1 ) was generated. By comparing with the reference characteristic chromatogram, the similarity of each batch of preparations was calculated, as shown in Table 18.

[0151] Table 18 Calculation results of the similarity of the characteristic chromatograms of 16 batches of Tongluo Qutong Plaster extracts

[0152]

[0153] In this experiment, a study on the characteristic chromatogram of Tongluo Qutong Plaster extract was carried out, and the chromatographic peak data were evaluated using similarity software, and a GC-MS standard reference characteristic chromatogram of Tongluo Qutong Plaster extract was established. The results show that the overall profiles of the volatile oil peak groups in each batch of extracts are basically the same, 53 common peaks were determined, and the similarities are all above 0.9.

[0154] 5.6.9, Specified value of relative retention time

[0155] The characteristic chromatograms of 16 batches of Tongluo Qutong Ointment extract were obtained. Using the retention time of the linalool chromatographic peak as a reference, the relative retention time and RSD of each common peak were calculated. The RSD of the relative retention time of each common peak in the 16 batches was within 0.4%.

[0156] The relative deviation range between the relative retention times of each characteristic peak of the sample to be tested and the relative retention times of each characteristic peak in the standard characteristic spectrum (see Table 19) is shown in Table 20.

[0157] According to Table 20, it is temporarily stipulated that if the relative retention time of each characteristic peak of the Tongluo Qutong Gao extract sample deviates from the relative retention time of each characteristic peak in the standard characteristic spectrum within ±1%, the quality of the Tongluo Qutong Gao extract is qualified; otherwise, it is unqualified.

[0158] Table 19. Standard Characteristic Atlas of Tongluo Qutong Plaster Extract: Relative Retention Time

[0159]

[0160]

[0161] Table 20. Range of relative retention time deviation between the common peaks and corresponding peaks in the standard spectrum of 16 batches of Tongluo Qutong Ointment extract.

[0162]

[0163] Based on the above results, the provisional value for the relative retention time of the standard feature map is:

[0164] The specified values ​​are 0.128 (peak 1), 0.132 (peak 2), 0.159 (peak 3), 0.203 (peak 4), 0.261 (peak 5), 0.286 (peak 6), 0.293 (peak 7), 0.313 (peak 8), 0.356 (peak 9), 0.372 (peak 10), 0.453 (peak 11), 0.466 (peak 12), 0.488 (peak 13), and 0.517 (peak 12). Peak 14), 0.542 (peak 15), 0.636 (peak 16), 0.779 (peak 17), 0.810 (peak 18), 0.822 (peak 19), 0.865 (peak 20), 0.886 (peak 21), 0.912 (peak 22), 1.000 (peak 23), 1.057 (peak 24), 1.078 (peak 25), 1.108 (peak 26), 1.200 (peak 27) 7) 1.264 (peak 28), 1.323 (peak 29), 1.371 (peak 30), 1.392 (peak 31), 1.408 (peak 32), 1.431 (peak 33), 1.502 (peak 34), 1.521 (peak 35), 1.565 (peak 36), 1.587 (peak 37), 1.701 (peak 38), 1.812 (peak 39), 2.060 (peak 40) The peaks are: 2.152 (peak 41), 2.169 (peak 42), 2.180 (peak 43), 2.233 (peak 44), 2.316 (peak 45), 2.326 (peak 46), 2.386 (peak 47), 2.397 (peak 48), 2.420 (peak 49), 2.457 (peak 50), 2.526 (peak 51), 2.569 (peak 52), and 2.618 (peak 53). The test sample's characteristic chromatogram should contain 53 characteristic peaks. The peak corresponding to the reference linalool is designated as the S peak. Calculate the relative retention time of each characteristic peak and the S peak; the relative retention time should be within ±1% of the specified value.

[0165] 6. Determination of the content of five volatile components in the extract of the Tongluo Qutong plaster.

[0166] 6.1 Gas Chromatography Conditions: Agilent DB-5ms capillary column (30m × 0.25mm, film thickness 0.25μm); carrier gas was high-purity helium (purity: 99.999%); column temperature was programmed: initial temperature 45℃, hold for 2 min, then increase at 0.8℃ / min. -1 Raise to 52℃ and hold for 15 minutes, at a rate of 1.5℃ / min. -1 Raise to 103℃ and hold for 40 minutes at a rate of 1.7℃ / min. -1 Raise to 160℃, hold for 5 minutes, at a rate of 2.0℃ / min -1 Raise to 210℃ and hold for 30 minutes, at a rate of 4.0℃ / min. -1Heat to 230℃ and hold for 10 min; injector temperature 220℃; injection volume 1 μL; column flow rate 0.8 ml / min; split ratio 5:1; theoretical plate number based on linalool peak not less than 6000.

[0167] The quantitative mass spectrometry conditions were as follows: electron impact (EI) ion source; electron energy 70 eV; ion source temperature: 230℃; interface temperature: 230℃; quadrupole temperature 150℃; mass scan range m / z (30-600) full scan; solute delay (5.5 min); subsequent run at 240℃ for 10 min; the tuning file was standard tuning, SIM ion monitoring mode, and the specific SIM detection time and quantitative and qualitative ion settings for each analyte are shown in Table 21.

[0168] Table 21 Quantitative and Qualitative Ions of Each Component Compound

[0169]

[0170] 6.2. Internal standard solution

[0171] Accurately weigh an appropriate amount of methyl salicylate, add ethyl acetate to prepare a solution with a concentration of 0.8 mg / ml, shake well, and the solution is obtained.

[0172] 6.3 Preparation of solutions containing internal standard reference material

[0173] Accurately weigh 10.12 mg of limonene, 10.06 mg of linalool, 10.13 mg of β-caryophyllene, 10.05 mg of eugenol, and 10.15 mg of ethyl p-methoxycinnamate, and place them separately in 10 ml volumetric flasks. Dissolve and dilute to the mark with ethyl acetate to prepare the reference stock solutions. Accurately pipette 2 ml of linalool and eugenol reference stock solutions, and 1 ml of limonene, β-caryophyllene, and ethyl p-methoxycinnamate reference stock solutions, and place them in the same 20 ml volumetric flask. Accurately add 1 mL of internal standard solution, dilute to the mark with ethyl acetate, and mix well to prepare the mixed reference solution containing internal standard.

[0174] 6.4 Preparation of test solution containing internal standard:

[0175] Accurately weigh 5g of the Tongluo Qutong Gao extract sample and place it in a 250ml round-bottom flask. Add 100ml of distilled water and perform the test according to the volatile oil determination method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 2204). Add water from the top of the measuring apparatus until it fills the graduated section and overflows into the flask. Then add 3ml of ethyl acetate, connect the reflux condenser, heat to boiling, and maintain a gentle boil for 5 hours. Cool, separate the ethyl acetate solution, and filter it through a funnel lined with an appropriate amount of anhydrous sodium sulfate. Place the filtrate in a 20ml volumetric flask and wash the condenser, volatile oil measuring apparatus, and funnel sequentially with an appropriate amount of ethyl acetate. Combine the washings with the above ethyl acetate solution, accurately add 2ml of internal standard solution, dilute to the mark with ethyl acetate, shake well, and filter to obtain the final product.

[0176] 6.5 Examination of Linear Relationships

[0177] Accurately measure 0.1, 0.5, 1.0, 1.5, 2.0, and 3 mL of the mixed reference standard stock solution into separate 20 mL volumetric flasks. Then, accurately add 0.5 mL of internal standard solution to each flask, dilute to volume with ethyl acetate, and mix well. Inject the sample according to the GC-MS conditions in section "6.1," record the chromatogram, and perform linear regression on the peak area ratio (Y) of the reference standard to the internal standard against the mass concentration (X) of each component to construct a standard curve. Obtain the linear regression equation and the analytical range.

[0178] The limits of detection and quantitation were determined based on the concentrations of each component at peak signal-to-noise ratios of approximately 3:1 and 10:1. The regression equations, linear ranges, limits of detection, and limits of quantitation for limonene, linalool, β-caryophyllene, eugenol, and ethyl p-methoxycinnamate are shown in Table 22. The results indicate that each component exhibits good linearity within its respective range.

[0179] Table 22 Standard Curve and Linear Range

[0180]

[0181]

[0182] 6.6 Sample injection precision, repeatability and stability tests

[0183] Injection precision experiment: Accurately pipette 1 μL of the reference solution under section "6.3", inject it 6 times consecutively according to the GC-MS conditions under section "6.1", measure the peak area, and calculate the RSD value of the ratio of the peak area of ​​each component to the corresponding internal standard peak area.

[0184] Repeatability test: Take six parallel prepared test sample (S1) solutions, prepare six test sample solutions in parallel according to the method under "6.4", inject and determine according to the conditions under "6.1", and calculate the RSD value of each component content (n=6).

[0185] Sample stability test: Take the same sample (S1) solution, prepare the sample solution according to the method in section "6.4", and inject and measure it at 0, 2, 4, 8, 12, and 24 h according to the conditions in section "6.1". Calculate the RSD value of the ratio of the peak area of ​​each component to the corresponding internal standard peak area.

[0186] The results of the injection precision, repeatability, and stability tests are shown in Table 23. The RSD of precision, repeatability, and stability are all less than 5%, which meets the requirements of the Chinese Pharmacopoeia. This indicates that the method has good precision and repeatability, and the test solution is stable within 24 hours.

[0187] Table 23 Precision, repeatability, and stability of the components of the pain-relieving and fitness-enhancing cream samples.

[0188]

[0189] 6.7 Recovery Test

[0190] Accurately weigh appropriate amounts of limonene, linalool, β-caryophyllene, eugenol, and ethyl p-methoxycinnamate reference standards, and prepare a mixed reference solution containing 0.516 mg limonene, 0.903 mg linalool, 0.415 mg β-caryophyllene, 1.322 mg eugenol, and 0.721 mg ethyl p-methoxycinnamate per 1 ml using ethyl acetate. Accurately weigh 2.5 g of the same batch sample (S1) with known content into six parallel portions, and accurately add 1 ml of each of the mixed reference solution of limonene, linalool, β-caryophyllene, eugenol, and ethyl p-methoxycinnamate as described in section "6.7". Prepare a test solution containing the internal standard according to the method described in section "6.4", and determine the sample by GC-MS under the conditions described in section "6.1". Calculate the recovery rate using the following formula. The results are shown in Table 24.

[0191] Table 24 Recovery Rate

[0192]

[0193]

[0194] 6.8 Results of the internal standard method

[0195] Sixteen batches of Tongluo Qutong Gao extract samples were collected, and three test solutions were prepared in parallel for each batch. The test solutions were prepared according to the method in section "6.4", and the samples were injected and determined under the GC-MS conditions in section "6.1". The contents of limonene, linalool, β-eugenol, eugenol and ethyl p-methoxycinnamate in Tongluo Qutong Gao extract were calculated using the internal standard method. The results are shown in Table 25.

[0196] Table 25 Results of component content determination (mg / g, n=3)

[0197]

[0198] 6.9 Content Value Specifications

[0199] The contents of limonene, linalool, β-eugenol, eugenol, and ethyl p-methoxycinnamate in the Tongluo Qutong plaster extract were calculated using the internal standard method with correction factors.

[0200] Based on the test results of 16 batches of samples and the influence of production process conditions, it is tentatively set that 50% of the average content of each component in the 16 products is the standard, and the content standards of the above-mentioned effective components in each 1g of Tongluo Qutong Gao extract are as follows: limonene not less than 0.62mg / g, linalool not less than 1.24mg / g, β-caryophyllene not less than 0.52mg / g, eugenol not less than 1.43mg / g, and ethyl p-methoxycinnamate not less than 0.96mg / g.

[0201] 7 Conclusions

[0202] Using linalool peak as a reference, the experiment established a characteristic spectrum of Tongluo Qutong Gao extract through methodological research and determination of 16 batches of samples. That is, the GC-MS standard control characteristic spectrum of Tongluo Qutong Gao extract consisting of 53 common peaks, where peak S is linalool, peak 9 is limonene, peak 23 is linalool, peak 24 is β-eugenol, peak 44 is eugenol, and peak 53 is ethyl p-methoxycinnamate.

[0203] Experiments have demonstrated that the characteristic chromatogram is highly distinctive and the method is feasible, applicable to product quality control, thereby improving the quality control standards of Tongluo Qutong Gao (a traditional Chinese medicine plaster) extract and providing theoretical basis and technical support for the improvement of drug standards and standardized production. This invention provides a characteristic chromatogram of volatile components in Tongluo Qutong Gao extract and a method for determining the content of five volatile components. It fully reflects the characteristics of volatile components in Tongluo Qutong Gao extract, treating the volatile characteristic chromatogram as a whole. This avoids the one-sidedness of judging the overall quality of a product based on only a few components and reduces the possibility of human interference with a particular indicator. It facilitates comprehensive control of the product quality of Tongluo Qutong Gao extract while saving testing time and costs.

Claims

1. A method for detecting the volatile component content of Tongluo Qutong ointment extract by gas chromatography-mass spectrometry, characterized in that, Comprising the following steps: 1) Chromatographic conditions: Gas chromatography conditions: Agilent DB-5ms capillary chromatographic column, 30 m x 0.25 mm, film thickness 0.25 μm; carrier gas is high-purity helium, purity: 99.999%; column temperature is programmed: initial temperature 45℃, hold for 2 min, increase to 52℃ at 0.8℃ / min, hold for 15 min, increase to 103℃ at 1.5℃ / min, hold for 40 min, increase to 160℃ at 1.7℃ / min, hold for 5 min, increase to 210℃ at 2.0℃ / min, hold for 30 min, increase to 230℃ at 4.0℃ / min, hold for 10 min; injection port temperature 220℃; injection volume 1 μL; column flow 0.8 ml / min; split ratio 5:1, theoretical plate number no less than 6000 according to linalool peak; Quantitative mass spectrometry conditions: electron impact EI ion source; electron energy 70 eV; ion source temperature: 230℃; interface temperature: 230℃; quadrupole temperature 150℃; mass scan range 30-600 m / z full scan; solute delay 5.5 min; tuning file is standard tuning, SIM ion monitoring mode, SIM retention time and quantitative qualitative ions of each test compound component are set as follows: Quantitative qualitative ions of each component compound ； 2) Preparation of reference solution: Accurately weigh 10.12 mg of limonene, 10.06 mg of linalool, 10.13 mg of β-eugenol, 10.05 mg of eugenol, and 10.15 mg of ethyl p-methoxycinnamate, respectively, into 10 ml volumetric flasks, dissolve and dilute to the mark with ethyl acetate as the reference stock solution. Accurately pipette 2 ml of linalool and eugenol reference stock solutions and 1 ml of limonene, β-eugenol, and ethyl p-methoxycinnamate reference stock solutions into the same 20 ml volumetric flask, dilute to the mark with ethyl acetate, and shake well as the mixed reference solution. 3) Preparation of test solution: Accurately weigh 5 g of Tongluo Qutong ointment extract sample into a 250 ml round-bottom flask, add 100 ml of distilled water, and follow the volatile oil determination method test. Add water to the upper end of the tester to fill the marked portion, and overflow into the flask. Add 2 ml of ethyl acetate, connect the reflux condenser, heat to boiling, and maintain gentle boiling for 5 hours. Cool, separate the ethyl acetate liquid, filter through a funnel filled with anhydrous sodium sulfate, and filter the filtrate into a 10 ml volumetric flask. Wash the condenser, volatile oil tester, and funnel with ethyl acetate, combine the washings with the above ethyl acetate liquid, dilute to the mark with ethyl acetate, shake well, filter, and pipette 1 μl of the filtrate into the gas chromatograph mass spectrometer for determination, and obtain the test solution. 4) Establishment and evaluation of characteristic spectrum Take 16 batches of Tongluo Qutong ointment extract sample according to step 3) method to prepare test solution, precision suction 1uL, respectively into gas chromatography mass spectrometry, under the condition of step 1) chromatography mass spectrometry sample analysis, according to the relative retention time of each chromatographic peak in the chromatogram, determine the common peak, and select 53 common peaks as characteristic peaks to establish the control characteristic spectrum; among them, peak 23 is taken as the reference peak S, the relative retention time ratio of each chromatographic peak to the retention time of the reference peak S in the same spectrum is calculated, and the relative retention time ratio obtained is; 5) Determine the correlation between Tongluo Qutong ointment extract volatile components and medicinal materials Respectively take Tongluo Qutong ointment extract test solution, and each single medicinal material test solution with the same amount of crude drug of preparation, and lack of single medicinal material negative control solution, compare the characteristic spectrum of volatile components in Tongluo Qutong ointment extract with the GC-MS characteristic spectrum of 11 single medicinal materials of Angelica sinensis, Ligusticum chuanxiong, Zanthoxylum bungeanum, Piper nigrum, Syzygium aromaticum, Cinnamomum cassia, Amomum villosum, Zingiber officinale, Kaempferia rotunda, Carthamus tinctorius and Rheum officinale and the GC-MS characteristic spectrum of lack of single medicinal material negative control solution, compare the relative retention time of each chromatographic peak to determine the main source of common peaks; 6) Volatile component content determination Take 16 batches of Tongluo Qutong ointment extract samples, prepare test solution according to step 3), and determine the sample under the condition of step 1) GC-MS, and calculate the content of limonene, linalool, beta-eudesmol, eugenol and ethyl p-methoxycinnamate in Tongluo Qutong ointment extract.

2. The method for detecting the volatile component content of the extract of Tongluo Qutong ointment by gas chromatography-mass spectrometry according to claim 1, characterized in that, The GC-MS standard control characteristic spectrum of volatile components in Tongluo Qutong ointment extract is composed of 53 common peaks, the similarity of the characteristic spectrum of the sample to the control characteristic spectrum is more than 0.9, peak 9 is limonene, peak 23 is linalool, peak 24 is beta-eudesmol, peak 44 is eugenol, and peak 53 is ethyl p-methoxycinnamate. Take linalool as the reference, peak 23 as the reference peak S, calculate the relative retention time of each characteristic peak to the reference peak S, and the relative retention time is within ±1% of the specified value.

3. The method for detecting the volatile component content of the extract of Tongluo Qutong ointment by gas chromatography-mass spectrometry according to claim 2, characterized in that, The relative retention time average of the common peaks of the standard control characteristic map is as follows: peak 1 is 0.128, peak 2 is 0.132, peak 3 is 0.159, peak 4 is 0.203, peak 5 is 0.261, peak 6 is 0.286, peak 7 is 0.293, peak 8 is 0.313, peak 9 is 0.356, peak 10 is 0.372, peak 11 is 0.453, peak 12 is 0.466, peak 13 is 0.488, peak 14 is 0.517, peak 15 is 0.542, peak 16 is 0.636, peak 17 is 0.779, peak 18 is 0.810, peak 19 is 0.822, peak 20 is 0.865, peak 21 is 0.886, peak 22 is 0.912, peak 23 is 1.000, peak 24 is 1.057, peak 25 is 1.078, peak 26 is 1.108, peak 27 is 1.200, peak 28 is 1.264, peak 29 is 1.323, peak 30 is 1.371, peak 31 is 1.392, peak 32 is 1.408, peak 33 is 1.431, peak 34 is 1.502, peak 35 is 1.521, peak 36 is 1.565, peak 37 is 1.587, peak 38 is 1.701, peak 39 is 1.812, peak 40 is 2.060, peak 41 is 2.152, peak 42 is 2.169, peak 43 is 2.180, peak 44 is 2.233, peak 45 is 2.316, peak 46 is 2.326, peak 47 is 2.386, peak 48 is 2.397, peak 49 is 2.420, peak 50 is 2.457, peak 51 is 2.526, peak 52 is 2.569, peak 53 is 2.

618.

4. The method for detecting the volatile component content of the extract of Tongluo Qutong ointment by gas chromatography-mass spectrometry according to claim 1, characterized in that, The step 6) further comprises: combining the determination results of the 16 batches of samples and the influence of the production process conditions, and stipulating that the content of the effective components in 1 g Tongluo Qutong ointment extract is: not less than 0.62 mg / g of limonene, not less than 1.24 mg / g of linalool, not less than 0.52 mg / g of β-eugenol, not less than 1.43 mg / g of eugenol, and not less than 0.96 mg / g of ethyl p-methoxycinnamate.

5. The use of the method for detecting the volatile component content of Tongluo Qutong ointment extract by gas chromatography-mass spectrometry in the quality detection of Tongluo Qutong ointment extract medicine according to any one of claims 1-4.

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