A method for establishing a characteristic spectrum of volatile components of a Shufu patch intermediate product and a method for determining the content of the intermediate product using a double internal standard method

Abstract

The present invention provides a method for establishing a characteristic spectrum of volatile components of a Shufu patch intermediate product and a method for determining the content of the intermediate product using a double internal standard method. During the characteristic spectrum establishment process, menthol is used as an internal reference, and through methodological research and 15 batches of sample determination, a characteristic spectrum of volatile components of the Shufu patch intermediate product is established, fully reflecting comprehensive information on the volatile components of the Shufu patch intermediate product. Based on qualitative analysis of the characteristic spectrum and in combination with existing characteristic spectrums, a method for simultaneously determining the contents of nine index components of the Shufu patch intermediate product using a double internal standard method is established, which can make up for the lack of control over multiple volatile chemical components of the Shufu patch intermediate product, thereby more comprehensively detecting and evaluating the quality of the Shufu patch intermediate product, and facilitating improving the quality control level of the product.

Description

Technical Field

[0001] The present invention relates to the technical field of quality detection of Chinese patent medicines, in particular to a method for establishing a characteristic spectrum of volatile components of a Shufu plaster intermediate product and a method for determining the content of the intermediate product using a double internal standard method. Background Art

[0002] The intermediate product of the soothing abdominal patch is a semisolid product composed of the medicinal ingredients ginger powder, camphor, and menthol, along with matrix ingredients such as elastomers, plasticizers, and tackifiers. It is used in the preparation of the soothing abdominal patch. As an intermediate product of the soothing abdominal patch, its quality directly affects the control of subsequent steps and the quality stability and uniformity of the finished product. This intermediate product contains a high number of volatile medicinal ingredients, including camphor and menthol, and ginger powder also contains a high number of volatile ingredients. Volatile drugs are the primary material basis for the efficacy of the product.

[0003] However, the current control of intermediate products (i.e. ginger powder, camphor, and menthol drugs, the same below) mainly focuses on thin-layer chromatography identification. The disadvantages of thin-layer chromatography are unstable reproducibility, low sensitivity, inability to identify trace substances, inability to identify and judge the existence status of effective ingredients as a whole, and failure to reflect the complex and diverse characteristics of Chinese medicine ingredients. In addition, insufficient control over the content of ingredients is not conducive to the quality stability and consistency of intermediate products, and is not conducive to accurately guiding the parameters of subsequent process steps, forming certain operational blind spots, which will cause quality risks to the final product.

[0004] Gas chromatography-mass spectrometry has obvious advantages in volatile component identification and quality control, and is increasingly being promoted and applied. Therefore, in order to further explore the types and substance content of volatile oils contained in the intermediate products of Shufu plaster, promote the continuous stability and refinement of the production process, and ensure product quality, it is necessary to study the types of chemical components of the intermediate products and determine the content of the active ingredients therein. That is, providing a method for establishing a characteristic spectrum of volatile components of the intermediate products of Shufu plaster and a method for determining the content of double internal standards are technical problems that need to be solved urgently. Summary of the Invention

[0005] In view of the above situation, in order to overcome the defects of the existing technology, the purpose of the present invention is to provide a method for establishing a characteristic spectrum of volatile components of the intermediate product of Shufu Patch and a double internal standard method for content determination of the intermediate product, which can effectively solve the problem of lack of control over multiple volatile chemical components of the intermediate product of Shufu Patch and the inability to provide stability and uniformity control of product quality.

[0006] To achieve the above object, the technical solution provided by the present invention is a method for establishing a characteristic spectrum of volatile components of an intermediate product of a Shufu patch, comprising the following steps:

[0007] 1) Preparation of reference substance stock solution:

[0008] Accurately weigh 250.05 mg of camphor reference substance and 60.06 mg of menthol reference substance, respectively, place them in the same 5 ml measuring flask, dissolve them in ethyl acetate and dilute to the mark, shake well, and use them as reference substance stock solution 1; accurately weigh 30.06 mg of trans-nerolidol, 40.12 mg of isoborneol, 60.05 mg of borneol, 20.05 mg of α-terpineol, 20.11 mg of α-gingerene, 20.15 mg of β-bisabolene, and 50.06 mg of α-farnesene, respectively, place them in the same 100 ml measuring flask, dissolve them in ethyl acetate and dilute to the mark, shake well, and use them as reference substance stock solution 2 for later use;

[0009] 2) Preparation of mixed reference solution:

[0010] Accurately pipette 2 ml of reference substance stock solution 1 and reference substance stock solution 2 respectively, place them into the same 25 ml volumetric flask, dilute to the mark with ethyl acetate, and shake well to obtain a mixed reference solution.

[0011] 3) Preparation of test solution:

[0012] Take 2.5g of Shufu patch, cut it into small pieces, remove the cover lining, place it in a 250ml round-bottom flask, add 150ml of water, and test it according to the volatile oil determination method. Add water from the upper end of the measuring instrument to fill the scale part and overflow into the flask, then add 2ml of ethyl acetate, connect a reflux condenser, heat to boiling, and keep it boiling slightly for 4h, let cool, take the ethyl acetate solution, filter it through a funnel covered with anhydrous sodium sulfate, and place the filtrate in a 25ml measuring flask. Use ethyl acetate to wash the condenser, volatile oil measuring instrument, and funnel in sequence. The washing liquid and the above ethyl acetate solution are combined into the same measuring flask, diluted to the scale with ethyl acetate, and shaken to obtain the test solution;

[0013] 4) Preparation of single drug solution:

[0014] According to the composition of the intermediate product of Shufu patch and the amount of the test sample, camphor, menthol and dried ginger were accurately weighed respectively, and placed in a 250ml round-bottom flask respectively. 150ml of water was added and the volatile oil assay was tested according to the volatile oil assay method. Water was added from the upper end of the assay to fill the scale part and overflow into the flask. Then 2ml of ethyl acetate was added, and a reflux condenser was connected. The mixture was heated to boiling and kept at a slight boil for 4 hours. The mixture was cooled and the ethyl acetate solution was separated. The mixture was filtered through a funnel covered with anhydrous sodium sulfate. The filtrate was placed in a 25ml measuring flask. The condenser, volatile oil assay device and funnel were washed with ethyl acetate in sequence. The washing liquid and the above ethyl acetate solution were combined into the same measuring flask, diluted to the scale with ethyl acetate, and shaken to obtain the single drug solutions of the three herbs.

[0015] 5) Characteristic spectrum analysis test: Analyzed by gas chromatography-mass spectrometry:

[0016] The test sample solutions at each level were injected into gas chromatography-mass spectrometry (GC-MS) for analysis. The instrument was a 7820A-5977B gas chromatograph-mass spectrometer (Agilent, USA), a MassHunter workstation, and a NIST 17.0 standard mass spectrum retrieval library.

[0017] GC-MS conditions: HP-INNOWAX column (30 m × 0.25 mm, 0.5 μm); carrier gas: high-purity helium; column temperature: programmed: initial temperature 50 °C, hold for 2 min, increase at 10 °C / min -1 Raise to 76℃, hold for 2min, and heat at 1.3℃ / min -1 Raise to 85℃, maintain for 20min, and heat at 0.4℃ / min -1 Raise to 87℃, maintain for 60min, and heat at 1.0℃ / min -1 Raise to 150℃, hold for 60min, and heat at 1.3℃ / min -1 Heat to 210°C and hold for 60 min; injection port temperature 230°C; column flow rate 0.8 ml / min; split ratio 10:1; theoretical plate number based on camphor peak not less than 5000;

[0018] Qualitative mass spectrometry conditions were as follows: electron impact (EI) ion source; electron energy 70 eV; ion source temperature: 230°C; interface temperature: 230°C; quadrupole temperature 150°C; mass scan range m / z (30-600) full scan; solute delay (4.6 min); post-run at 235°C for 20 min; standard tuning file;

[0019] 6) Establish characteristic maps and determine quantitative detection indicators:

[0020] Fifteen batches of intermediate products of Shufu patch were taken, and sample information was collected according to the above construction method. The chromatogram data of the 15 batches of samples S1-S15 were imported into the Chinese medicine chromatogram similarity evaluation software (2012 version). The S1 sample spectrum was used as the reference spectrum. The average method was used, the time window was set to 0.1 min, and multi-point correction was performed to perform chromatographic peak matching. The superimposed spectrum of the 15 batches of samples and the standard control characteristic spectrum (R) were generated; the mixed reference solution, the test solution and the single drug solution were taken, and the chromatogram and qualitative mass spectrometry conditions of the characteristic spectrum analysis test item were used for injection and determination, and the chromatograms were recorded. The NIST in Msss Hunter mass spectrometry analysis software was used. The MS program identified and compared it with the reference substance. The GC-MS standard reference characteristic spectrum of the intermediate product of the Shufu patch was composed of 9 common peaks. Peak 1 was determined to be camphor, peak 2 to be trans-nerolidol, peak 3 to be menthol, peak 4 to be isoborneol, peak 5 to be borneol, peak 6 to be α-terpineol, peak 7 to be α-gingerene, peak 8 to be β-bisabolene, and peak 9 to be α-farnesene.

[0021] Among the nine common peaks, menthol was used as a reference and peak 3 (S) was used as a reference peak. The relative retention time of each common peak and peak S was calculated. The relative retention time was within ±5% of the specified value. The specified value is shown in Table 1:

[0022] Table 1 Relative retention time specified values ​​of the characteristic spectrum of the intermediate product of Shufu patch

[0023]

[0024] The chromatograms of 15 batches of samples were analyzed using the Chinese medicine chromatographic fingerprint similarity evaluation software (2012 version). The similarity between the characteristic spectra of samples S1 to S15 and the control characteristic spectra was calculated, and the similarity was not less than 0.90.

[0025] The method for establishing the characteristic spectrum of volatile components of the intermediate product of the Shufu patch is used in the quality control of the intermediate product of the Shufu patch.

[0026] A double internal standard method for determining the content of a Shufu patch intermediate product comprises the following steps:

[0027] (1) Preparation of internal standard solution:

[0028] Accurately weigh 10.02 mg of pulegone and 1000.01 mg of 2,4,6-tri-tert-butylphenol, place them separately in a 10 mL volumetric flask, dilute to the mark with ethyl acetate, and shake well to obtain the internal standard solution.

[0029] (2) Preparation of mixed reference solution containing internal standard:

[0030] Accurately pipette 2 ml each of reference stock solution 1 and reference stock solution 2, then accurately pipette 1 ml each of pulegone and 2,4,6-tri-tert-butylphenol internal standard solution, place them in the same 25 ml volumetric flask, dilute to the mark with ethyl acetate, and shake well to prepare the mixed reference solution containing the internal standard.

[0031] (3) Preparation of test solution containing internal standard:

[0032] Take 2.5g of Shufu patch, cut it into small pieces, remove the cover lining, place it in a 250ml round-bottom flask, add 150ml of water, and test according to the volatile oil determination method. Add water from the upper end of the analyzer to fill the scale part and overflow into the flask, then add 2ml of ethyl acetate, connect a reflux condenser, heat to boiling, and keep it boiling slightly for 4h, let it cool, take the ethyl acetate solution, filter it through a funnel covered with anhydrous sodium sulfate, and place the filtrate into a 25ml measuring flask. Wash the condenser, volatile oil analyzer, and funnel with ethyl acetate in sequence. Combine the washing liquid and the above ethyl acetate solution into the same measuring flask, accurately add 1ml each of pulegone and 2,4,6-tri-tert-butylphenol internal standard solution, dilute to the scale with ethyl acetate, and shake well to obtain the internal standard test solution;

[0033] (4) Preparation of negative sample solution:

[0034] According to the prescription composition and production process of the intermediate product of the Shufu plaster, negative samples and blank matrix samples without camphor, menthol and dried ginger are prepared respectively. The negative samples without camphor, menthol and dried ginger and the negative matrix samples are taken respectively and operated according to the method under "(3)" to obtain negative sample solutions with internal standards without camphor, menthol and dried ginger and the negative matrix samples;

[0035] (5) Mass chromatograph-mass spectrometer conditions: The instrument was a 7820A-5977B gas chromatograph-mass spectrometer (Agilent, USA), a MassHunter workstation, and the NIST 17.0 standard mass spectrum retrieval library; gas chromatography-mass spectrometry conditions: HP-INNOWAX column (30 m × 0.25 mm, 0.5 μm); the carrier gas was high-purity helium; the column temperature was programmed: initial temperature 50 °C, maintained for 2 min, and then increased at 10 °C / min. -1 Raise to 76℃, hold for 2min, and heat at 1.3℃ / min -1 Raise to 85℃, maintain for 20min, and heat at 0.4℃ / min -1 Raise to 87℃, maintain for 60min, and heat at 1.0℃ / min -1 Raise to 150℃, hold for 60min, and heat at 1.3℃ / min -1 Heat to 210°C and hold for 60 min; injection port temperature 230°C; column flow rate 0.8 ml / min; split ratio 10:1; theoretical plate number based on camphor peak not less than 5000;

[0036] The quantitative mass spectrometry conditions were as follows: electron impact (EI) ion source; electron energy 70 eV; ion source temperature: 230°C; interface temperature: 230°C; quadrupole temperature 150°C; solvent delay (4.6 min); post-run at 235°C for 20 min; standard tuning file, SIM ion monitoring mode; SIM detection time and quantitative and qualitative ion settings for each compound are shown in Table 2.

[0037] Table 2 Quantitative and qualitative ions of each component compound

[0038]

[0039] (6) Content determination:

[0040] Pulegone was selected as the internal standard for trans-nerolidol, isoborneol, borneol, α-terpineol, α-gingerol, β-bisabolene, and α-farnesene, and 2,4,6-tri-tert-butylphenol was selected as the internal standard for camphor and menthol. 1 μl of the reference solution and the test solution containing the internal standard were accurately aspirated, the chromatograms were recorded, the peak areas of the reference and internal standard substances were measured, and the correction factor f was calculated. The correction factors for trans-nerolidol, isoborneol, borneol, α-terpineol, α-gingerol, β-bisabolene, α-farnesene, camphor, and menthol were 0.48, 0.33, 0.27, 0.20, 0.38, 0.96, 0.27, 0.99, and 0.61, respectively.

[0041] Then take the test sample solution containing the internal standard substance from each batch, inject the sample, record the chromatogram, measure the peak area of ​​the test component and the internal standard substance in the test sample, and use the double internal standard method to calculate the contents of camphor, trans-nerolidol, menthol, isoborneol, borneol, α-terpineol, α-gingerene, β-bisabolene, and α-farnesene in the intermediate product of Shufu patch.

[0042] The calculation formula of the correction factor f is:

[0043]

[0044] Where: A S is the peak area of ​​the internal standard substance; A R is the peak area of ​​the reference substance; c S is the concentration of the internal standard substance; c R is the concentration of the reference substance;

[0045] The content calculation formula is:

[0046]

[0047] Where: A X is the peak area of ​​the test sample; C X is the concentration of the test sample; A' S is the peak area of ​​the internal standard substance; C' S is the concentration of the internal standard substance; f is the internal standard correction factor.

[0048] The double internal standard method for content determination of the intermediate product of the Shufu patch is used in the quality control of the intermediate product of the Shufu patch.

[0049] The characteristic spectrum obtained by gas chromatography-mass spectrometry in the present invention can comprehensively reflect the volatile components of the Shufu patch intermediate product, compensating for the lack of control of volatile components in medicinal materials in current standards. It also enables quantitative detection of key volatile indicator components in the Shufu patch intermediate product, which is conducive to improving the quality control level of the Shufu patch intermediate product. The use of a double internal standard method for multi-index quantification has higher accuracy and significant social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a characteristic spectrum of precision detection of Example 1 of the present invention; wherein, A: overall view; B: partial enlarged view;

[0051] Figure 2 This is a characteristic diagram of repeatability detection in Example 1 of the present invention; wherein, A: overall diagram; B: partial magnified diagram;

[0052] Figure 3 This is a characteristic graph of stability detection in Example 1 of the present invention; wherein A: overall view; B: partial magnified view;

[0053] Figure 4 It is a superposition of the characteristic spectrum of the intermediate product of the Shufu patch of Example 1 of the present invention and the control characteristic spectrum;

[0054] Figure 5 This is the identification of common peaks in the characteristic spectrum of Example 1 of the present invention; wherein: A: mixed reference substance; B: partial enlarged view of the mixed reference substance; C: sample; D: partial enlarged view of the sample; E: camphor; F: menthol; G: dried ginger; H: partial enlarged view of dried ginger; in the figure: 1-camphor, 2-trans-nerolidol, 3-menthol, 4-isoborneol, 5-borneol, 6-α-terpineol, 7-α-gingerene, 8-β-bisabolene, 9-α-farnesene;

[0055] Figure 6 This is a specific profile of the content determination of Example 2 of the present invention; wherein: A: reference substance; B: sample; C: negative sample lacking dried ginger; D: negative sample lacking camphor; E: negative sample lacking menthol; F: blank solvent; G: blank matrix; in the figure: 1-camphor, 2-trans-nerolidol, 3-menthol, 4-isoborneol, 5-borneol, 6-α-terpineol, 7-α-gingerene, 8-β-bisabolene, 9-α-farnesene, 10-pulegone, 11-2,4,6-tri-tert-butylphenol. DETAILED DESCRIPTION

[0056] The specific implementation of the present invention is described in detail below with reference to the accompanying drawings and examples.

[0057] The present invention is specifically implemented by the following examples.

[0058] Example 1

[0059] A method for establishing a characteristic spectrum of volatile components of a Shufu patch intermediate product comprises the following steps:

[0060] 1. Test instruments and materials

[0061] 1.1 Instruments

[0062] 7820A-5977B gas chromatograph-mass spectrometer (Agilent, USA), MassHunter workstation, NIST 17.0 standard mass spectrum retrieval library; XPE105 1 / 100000 electronic balance (d = 0.01 mg) (Mettler-Toledo, Switzerland); volatile oil analyzer with relative density <1.0 and 24 / 29 standard stopper (Beijing North Glass Bomei Glass Co., Ltd., size: 5 mL).

[0063] 1.2. Test drugs and reagents

[0064] Camphor reference substance (batch number: 110747-202011, purity: 98.8%), menthol reference substance (batch number: 110728-202208, purity: 99.6%), pulegone (batch number: 111706-201907, purity: 99.8%), isoborneol reference substance (batch number: 111512-202205; content: 98.7%), borneol reference substance (batch number: 110881-201709; content: 99.6%) were purchased from the China Food and Drug Administration; α-gingerene reference substance (batch number: wkq-0 007937; content: 98.20%), β-bisabolene reference substance (batch number: wkq-0787608; content: 96.50%), and α-farnesene reference substance (batch number: wkq-0027612; content: 99.10%) were all purchased from Sichuan Weikeqi Biotechnology Co., Ltd.; trans-nerolidol reference substance (batch number: A19O7R2317, content: 95%, Shanghai Yuanye Biotechnology Co., Ltd.); α-terpineol reference substance (batch number: AL170602-15; content: 98.05%, Stanford Biotechnology Co., Ltd., USA) were purchased from Chemicals); 2,4,6-tri-tert-butylphenol (batch number le071279711, content: 99.96, Shanghai Haohong Biotechnology Co., Ltd.); purified water (batch number: 202108056214HN, source: Hangzhou Wahaha Group Co., Ltd.), ethyl acetate (batch number: 20220220, source: Tianjin Komeo Chemical Reagent Co., Ltd., analytical grade).

[0065] Medicine: intermediate product of Shufu patch (sample numbers S1 to S15).

[0066] 1.3. Analysis software: Chinese medicine chromatographic fingerprint similarity evaluation system 2012 edition (National Pharmacopoeia Committee).

[0067] 2. Test conditions

[0068] 2.1. Chromatography-mass spectrometry conditions and system suitability test:

[0069] GC-MS conditions: HP-INNOWAX column (30 m × 0.25 mm, 0.5 μm); carrier gas: high-purity helium; column temperature: programmed: initial temperature 50 °C, hold for 2 min, increase at 10 °C / min -1 Raise to 76℃, hold for 2min, and heat at 1.3℃ / min -1 Raise to 85℃, maintain for 20min, and heat at 0.4℃ / min -1 Raise to 87℃, maintain for 60min, and heat at 1.0℃ / min -1 Raise to 150℃, hold for 60min, and heat at 1.3℃ / min -1 Heat to 210°C and hold for 60 min; injection port temperature 230°C; column flow rate 0.8 ml / min; split ratio 10:1; theoretical plate number based on camphor peak not less than 5000;

[0070] The qualitative mass spectrometry conditions were as follows: electron impact (EI) ion source; electron energy 70 eV; ion source temperature: 230°C; interface temperature: 230°C; quadrupole temperature 150°C; mass scan range m / z (30-600) full scan; solute delay (4.6 min); post-run at 235°C for 20 min; and standard tuning file.

[0071] 2.2. Reference substance stock solution

[0072] Accurately weigh 250.05 mg of camphor reference substance and 60.06 mg of menthol reference substance, respectively, place them in the same 5 ml volumetric flask, add ethyl acetate to dissolve and dilute to the scale, shake well, and use them as reference substance stock solution 1; accurately weigh 30.06 mg of trans-nerolidol, 40.12 mg of isoborneol, 60.05 mg of borneol, 20.05 mg of α-terpineol, 20.11 mg of α-gingerene, 20.15 mg of β-bisabolene, and 50.06 mg of α-farnesene, respectively, place them in the same 100 ml volumetric flask, add ethyl acetate to dissolve and dilute to the scale, shake well, and use them as reference substance stock solution 2 for standby use.

[0073] 2.3. Mixed reference solution

[0074] Accurately pipette 2 ml of reference substance stock solution 1 and reference substance stock solution 2 respectively, place them into the same 25 ml volumetric flask, dilute to the scale with ethyl acetate, shake well, and obtain.

[0075] 2.4. Preparation of test solution:

[0076] Take 2.5g of this product, cut it into small pieces, remove the cover and lining, place it in a 250ml round-bottom flask, add 150ml of water, and test according to the volatile oil determination method (Chinese Pharmacopoeia 2020 Edition Part 4 General Chapter 2204). Add water from the upper end of the measuring instrument until the scale is filled and overflows into the flask, then add 2ml of ethyl acetate, connect a reflux condenser, heat to boiling, and keep it boiling for 4 hours, let it cool, take the ethyl acetate solution, filter it through a funnel lined with an appropriate amount of anhydrous sodium sulfate, and place the filtrate in a 25ml measuring flask. Use an appropriate amount of ethyl acetate to wash the condenser, volatile oil measuring instrument, and funnel in turn. Combine the washing liquid and the above ethyl acetate solution into the same measuring flask, add ethyl acetate to dilute to the scale, and shake well.

[0077] 2.5. Single drug solution

[0078] According to the composition of the intermediate product of Shufu patch and the amount of the test sample, camphor, menthol, and dried ginger were accurately weighed and placed in 250ml round-bottom flasks respectively. 150ml of water was added and the volatile oil determination method (Chinese Pharmacopoeia 2020 Edition Part 4 General Chapter 2204) was used for the test. Water was added from the top of the measuring instrument until the scale was filled and overflowed into the flask. Then 2ml of ethyl acetate was added, connected to a reflux condenser, heated to boiling, and kept boiling for 4 hours. The ethyl acetate solution was separated and filtered through a funnel covered with an appropriate amount of anhydrous sodium sulfate. The filtrate was placed in a 25ml measuring flask. The condenser, volatile oil measuring instrument, and funnel were washed in turn with an appropriate amount of ethyl acetate. The washing liquid and the above ethyl acetate solution were combined into the same measuring flask, diluted to the scale with ethyl acetate, and shaken to obtain the 3 separate medicinal material solutions.

[0079] 2.6. Selection of reference objects

[0080] Menthol is one of the main components of the volatile oil in the intermediate product of Shufu patch. It has obvious pharmacological activity, high content, easy to obtain reference materials, and low price. In the spectrum, it has good separation from other components. Therefore, menthol was selected as the internal reference.

[0081] 3. Investigation of characteristic map methodology

[0082] 3.1 Precision

[0083] Prepare the test solution of the same batch of Shufu patch intermediate product (No. S1) according to item "2.4" and inject the sample 6 times continuously according to the chromatography-mass spectrometry conditions under item "2.1" to detect the characteristic spectrum, such as Figure 1The retention time and peak area of ​​each common peak were recorded. The relative retention time and peak area of ​​each common peak were calculated using the retention time and peak area of ​​the menthol chromatographic peak as a reference. The RSD values ​​of the relative retention time and relative peak area of ​​each common peak were all less than 3%. The chromatographic data obtained six times were imported into the "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" for full spectrum peak matching and similarity calculation. The similarity results were all ≥0.999, indicating that the instrument is stable and has good precision. The results are shown in Tables 3, 4, and 5.

[0084] Table 3 Precision test results of intermediate product (S1) of Shufu patch (relative retention time of index component groups)

[0085]

[0086] Table 4 Precision test results of intermediate product (S1) of Shufu patch (relative peak area of ​​index component group)

[0087]

[0088] Table 5 Similarity calculation results of the precision test spectrum of the intermediate product (S1) of Shufu patch

[0089]

[0090]

[0091] 3.2 Repeatability

[0092] Take the same batch of samples of the intermediate product of Shufu patch (No. S1) and prepare 6 portions according to the method of preparation of test solution in "2.4". According to the chromatography-mass spectrometry conditions in "2.1", the samples were injected in sequence and the characteristic spectra were detected respectively. Figure 2 The retention time and peak area of ​​each common peak were recorded. The relative retention time and peak area of ​​each common peak were calculated using the retention time and peak area of ​​the menthol chromatographic peak as a reference. The RSD values ​​of the relative retention time and relative peak area of ​​each common peak were all less than 3.0%. The chromatographic data obtained six times were imported into the "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" for full spectrum peak matching and similarity calculation. The similarity results were all ≥0.999, indicating that the test method has good repeatability. The results are shown in Tables 6, 7, and 8.

[0093] Table 6 Repeatability test results of intermediate product (S1) of Shufu patch (relative retention time of index component groups)

[0094]

[0095] Table 7 Repeatability test results of intermediate product (S1) of Shufu patch (relative peak area of ​​index component group)

[0096]

[0097] Table 8 Similarity calculation results of repeatability test spectrum of intermediate product (S1) of Shufu patch

[0098]

[0099]

[0100] 3.3 Stability

[0101] Prepare the test solution of the same batch of Shufu patch intermediate product (No. S1) according to the "2.4" item, and inject the sample at 0h, 2h, 4h, 8h, 12h, and 24h according to the chromatographic mass spectrometry conditions under "2.1" item, and detect the characteristic spectrum, such as Figure 3 The retention time and peak area of ​​each common peak were recorded. The relative retention time and peak area of ​​each common peak were calculated with reference to the retention time and peak area of ​​the menthol chromatographic peak. The RSD values ​​of the relative retention time and relative peak area of ​​each common peak were all less than 3.0%. The chromatographic data obtained 6 times were imported into the "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" for full spectrum peak matching and similarity calculation. The similarity results were all ≥0.999. The test solution was stable within 24 hours. This indicates that the test sample has good stability. The results are shown in Tables 9, 10, and 11.

[0102] Table 9 Stability test results of intermediate product (S1) of Shufu patch (relative retention time of index component groups)

[0103]

[0104] Table 10 Stability test results of intermediate product (S1) of Shufu patch (relative peak area of ​​index component group)

[0105]

[0106] Table 11 Similarity calculation results of repeatability test spectrum of intermediate product (S1) of Shufu patch

[0107]

[0108]

[0109] 4. Establishment and application of the characteristic map of the intermediate product of Shufu patch

[0110] 4.1. Establishment of the Characteristic Spectrum of the Intermediate Product of Shufu Patch and Similarity Evaluation

[0111] The GC-MS characteristic spectra of 15 batches of Shufu plaster intermediate products were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" in txt format. The chromatogram of sample S1 was selected as the reference spectrum, and the time window width was set to 0.1min. Multi-point calibration and automatic matching were performed on the 15 batches of samples to establish a common fingerprint pattern. The results showed that 9 common peaks were calibrated in the GC-MS characteristic spectra of the 15 batches of Shufu plaster intermediate products, and an overlay spectrum was generated. Figure 4 The similarity was calculated based on the reference fingerprint (R) generated by the average method. The similarity results between the characteristic spectra of 15 batches of Shufu patch intermediate products and the standard characteristic spectra are shown in Table 12.

[0112] Table 12 Calculation results of similarity of characteristic graphs of intermediate products of Shufu patch

[0113]

[0114] The results showed that the overall morphology of the peak groups of each batch of preparations was basically consistent, and 9 common peaks were identified, with similarities all above 0.9.

[0115] 4.2 Identification of common peaks in characteristic spectra

[0116] Take the mixed reference solution under item "2.3", the test solution under item 2.4, and the single drug solution under item 2.5, and inject and measure according to the chromatographic and mass spectrometric conditions of item "2.1", and record the chromatogram. Using the NIST MS program in Msss Hunter mass spectrometry analysis software to identify and compare with the reference, the GC-MS standard reference characteristic spectrum of the intermediate product of the Shufu patch is composed of 9 common peaks, and it is determined that the peak No. 1 is camphor, the peak No. 2 is trans-nerolidol, the peak No. 3 is menthol, the peak No. 4 is isoborneol, the peak No. 5 is borneol, the peak No. 6 is α-terpineol, the peak No. 7 is α-gingerene, the peak No. 8 is β-bisabolene, and the peak No. 9 is α-farnesene. See the mixed reference for details. Figure 5 .

[0117] 4.3. Specification of characteristic spectrum standards

[0118] Based on the relative retention time of each chromatographic peak in the chromatograms obtained from 15 batches of Shufu patch intermediate products, the common peaks were determined, and 9 common peaks were selected as characteristic peaks to establish a control characteristic spectrum; among them, Peak No. 3 was used as the reference peak S, and the retention time of each chromatographic peak was calculated to calculate the retention time ratio of the S peak in the same spectrum. The obtained relative retention time and RSD are shown in Table 13.

[0119] Table 13 Relative retention time of characteristic spectrum of intermediate products of Shufu patch

[0120]

[0121] The relative deviation range of the relative retention time of each characteristic peak of the sample to be tested and the relative retention time of each characteristic peak in the standard characteristic spectrum (see Table 13) is shown in Table 14.

[0122] Table 14 Relative retention time deviation range of common peaks of 15 batches of Shufu patch intermediate products and corresponding peaks in the standard spectrum

[0123]

[0124] According to the durability test results of the characteristic spectrum of volatile components, the relative retention time of each characteristic peak in the characteristic spectrum obtained by different instruments and different brands of chromatographic columns did not exceed ±5% of the mean. Therefore, it is tentatively determined that the characteristic spectrum of the intermediate product of Shufu Patch should have 9 characteristic peaks. The peak corresponding to the reference substance menthol is the S peak. The relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within ±5% of the specified value. The specified values ​​of the relative retention time of the standard characteristic spectrum are: 0.545 (peak 1), 0.680 (peak 2), 1.000 (peak 3), 1.080 (peak 4), 1.273 (peak 5), 1.308 (peak 6), 1.413 (peak 7), 1.432 (peak 8), and 1.631 (peak 9). In addition, combined with the characteristic spectrum similarity analysis, the similarity between the characteristic spectrum of the intermediate product of Shufu Patch and the standard characteristic spectrum should be no less than 0.9.

[0125] Example 2

[0126] The other implementation conditions of this example are consistent with the characteristic spectrum conditions of the volatile components of the intermediate product of the Shufu patch in Example 1. Only the GC-MS conditions have new regulations. Internal standard solution is added to both the test sample and the reference solution.

[0127] A double internal standard method for determining the content of a Shufu patch intermediate product comprises the following steps:

[0128] 5. Content determination method

[0129] 5.1. The conditions for gas quality content determination are as follows:

[0130] The instrument was a 7820A-5977B gas chromatograph-mass spectrometer (Agilent, USA), a MassHunter workstation, and a NIST 17.0 standard mass spectrum retrieval library.

[0131] GC-MS conditions: HP-INNOWAX column (30 m × 0.25 mm, 0.5 μm); carrier gas: high-purity helium; column temperature: programmed: initial temperature 50 °C, hold for 2 min, increase at 10 °C / min -1 Raise to 76℃, hold for 2min, and heat at 1.3℃ / min -1Raise to 85℃, maintain for 20min, and heat at 0.4℃ / min -1 Raise to 87℃, maintain for 60min, and heat at 1.0℃ / min -1 Raise to 150℃, hold for 60min, and heat at 1.3℃ / min -1 Heat to 210°C and hold for 60 min; injection port temperature 230°C; column flow rate 0.8 ml / min; split ratio 10:1; theoretical plate number based on camphor peak not less than 5000;

[0132] The quantitative mass spectrometry conditions were as follows: electron impact (EI) ion source; electron energy 70 eV; ion source temperature: 230°C; interface temperature: 230°C; quadrupole temperature 150°C; solvent delay (4.6 min); post-run at 235°C for 20 min; standard tuning file, SIM ion monitoring mode, SIM detection time for each test compound component and quantitative and qualitative ion settings are shown in Table 15.

[0133] Table 15 Quantitative and qualitative ions of each component compound

[0134]

[0135] The characteristic spectrum contains shared peaks representing information about the three drugs in the Shufu Patch intermediate product. The spectrum identification accuracy is >90%, and the peaks are relatively large, symmetrical, and well resolved. To provide a relatively complete and detailed evaluation of the volatile components in the Shufu Patch intermediate product, assays were planned for the nine shared peaks.

[0136] 5.2. Internal standard solution

[0137] Accurately weigh 10.02 mg of pulegone and 1000.01 mg of 2,4,6-tri-tert-butylphenol, place them separately in a 10 mL volumetric flask, dilute to the mark with ethyl acetate, and shake well to obtain the product.

[0138] 5.3. Reference substance stock solution

[0139] Accurately weigh 250.05 mg of camphor reference substance and 60.06 mg of menthol reference substance, respectively, place them in the same 5 ml measuring flask, dissolve them in ethyl acetate and dilute to the mark, shake well, and use them as reference substance stock solution 1; accurately weigh 30.06 mg of trans-nerolidol, 40.12 mg of isoborneol, 60.05 mg of borneol, 20.05 mg of α-terpineol, 20.11 mg of α-gingerene, 20.15 mg of β-bisabolene, and 50.06 mg of α-farnesene, respectively, place them in the same 100 ml measuring flask, dissolve them in ethyl acetate and dilute to the mark, shake well, and use them as reference substance stock solution 2 for later use;

[0140] 5.4 Mixed reference solution containing internal standard

[0141] Accurately pipette 2 ml each of reference stock solution 1 and reference stock solution 2, and then accurately pipette 1 ml each of pulegone and 2,4,6-tri-tert-butylphenol internal standard solution, place them in the same 25 ml volumetric flask, dilute to the scale with ethyl acetate, and shake well to prepare the mixed reference solution containing the internal standard.

[0142] 5.5 Test solution containing internal standard

[0143] Take 2.5g of Shufu patch, cut it into small pieces, remove the cover lining, place it in a 250ml round-bottom flask, add 150ml of water, and test it according to the volatile oil determination method (Chinese Pharmacopoeia 2020 Edition Part 4 General Chapter 2204). Add water from the upper end of the measuring instrument to fill the scale part and overflow into the flask, then add 2ml of ethyl acetate, connect the reflux condenser, heat to boiling, and keep boiling slightly for 4h, let cool, take the ethyl acetate solution, filter it through a funnel covered with an appropriate amount of anhydrous sodium sulfate, and place the filtrate in a 25ml measuring flask. Use ethyl acetate to wash the condenser, volatile oil measuring instrument, and funnel in turn. The washing liquid and the above ethyl acetate solution are combined into the same measuring flask, and 1ml of pulegone and 2,4,6-tri-tert-butylphenol internal standard solution are accurately added. Dilute to the scale with ethyl acetate and shake well.

[0144] 5.6 Negative sample solution

[0145] Prepare a negative sample that does not contain camphor, menthol, dried ginger, or a blank matrix according to the formula composition and production process of the Shufu patch intermediate product. Separately, take the negative sample that does not contain camphor, menthol, or dried ginger and the negative sample of the blank matrix and follow the procedure under "5.5" to obtain a negative sample solution with internal standard that does not contain camphor, menthol, dried ginger, or a blank matrix.

[0146] 5.7. Exclusivity

[0147] Prepare the test solution according to the test sample preparation method, and examine whether the blank solvent ethyl acetate, negative samples lacking dried ginger medicinal materials, lacking camphor, lacking menthol, and blank matrix will cause interference. Pipette 1ul each of the blank solvent ethyl acetate, negative sample solution containing internal standard, reference solution containing internal standard, test solution containing internal standard, and blank matrix solution containing internal standard, and analyze according to the chromatographic-mass spectrometric conditions described under "5.1". Record the spectrum as follows: Figure 6 As shown in the figure, the results showed that there was no interference from solvent, blank matrix and negative sample, and the method had good specificity.

[0148] 5.8 Linearity and range investigation

[0149] Accurately measure 0.1, 0.5, 1.0, 1.5, 2.0, and 2.5 mL of the mixed reference stock solution and place them in 10 mL volumetric flasks, then accurately add 0.3 mL each of the internal standard solutions pulegone and 2,4,6-tri-tert-butylphenol, make up to volume with ethyl acetate and shake well. Inject and measure according to the GC-MS conditions under "5.1". Record the spectrum. Perform linear regression on the mass concentration (X) of each component using the peak area ratio (Y) of the reference substances camphor and menthol to the internal standard 2,4,6-tri-tert-butylphenol to prepare standard curves for camphor and menthol. Linear regression was performed using the peak area ratios (Y) of the reference substances trans-nerolidol, isoborneol, borneol, α-terpineol, α-gingerene, β-bisabolene, and α-farnesene to the internal standard pulegone against the mass concentration (X) of each component. Standard curves for trans-nerolidol, isoborneol, borneol, α-terpineol, α-gingerene, β-bisabolene, and α-farnesene were generated. The linear regression equation and analytical range were obtained.

[0150] The limits of detection and quantification (LOQs) were determined based on the concentrations of each component at peak signal-to-noise ratios of approximately 3:1 and 10:1, respectively. The regression equations, linear ranges, and limits of detection and quantification for camphor, trans-nerolidol, menthol, isoborneol, borneol, α-terpineol, α-gingerene, β-bisabolene, and α-farnesene are shown in Table 16. The results showed that each component had good linear relationships within their respective ranges.

[0151] Table 16 Standard curves and linear ranges of each component of the intermediate product of Shufu patch

[0152]

[0153] 5.9. Injection precision, repeatability and stability test

[0154] Injection precision test: Accurately aspirate 1 μL of the internal standard reference solution under item "5.4" and perform 6 consecutive injections according to the GC-MS conditions under item "5.1". Determine the peak areas and calculate the RSDs (n=6) of the peak area ratios of the reference substances camphor and menthol to the internal standard 2,4,6-tri-tert-butylphenol. Calculate the RSDs (n=6) of the peak area ratios of the reference substances trans-nerolidol, isoborneol, borneol, α-terpineol, α-gingerene, β-bisabolene, and α-farnesene to the internal standard pulegone.

[0155] Repeatability test: Take sample (S1) and prepare 6 test solutions containing internal standard in parallel according to the method under "5.5". According to the GC-MS conditions under "5.1", the samples were injected and measured. The RSD value of the average content of each component (n=6) was calculated according to the internal standard method.

[0156] Sample stability test: Take the same sample solution (S1) containing the internal standard and place it at room temperature. After 0, 2, 4, 8, 12, and 24 hours, inject the sample according to the GC-MS conditions under "5.1". Measure the peak area and calculate the RSD (n=6) of the peak area ratios of the reference substances camphor and menthol to the internal standard 2,4,6-tri-tert-butylphenol. Also calculate the RSD (n=6) of the peak area ratios of the reference substances trans-nerolidol, isoborneol, borneol, α-terpineol, α-gingerene, β-bisabolene, and α-farnesene to the internal standard pulegone.

[0157] The results of the injection precision, repeatability, and stability tests are shown in Table 17. The precision, repeatability, and stability RSDs meet the requirements of the Chinese Pharmacopoeia, indicating that the method has good precision and repeatability, and the test solution is stable within 24 hours.

[0158] Table 17 Precision, repeatability and stability of each component of the intermediate product of Shufu patch

[0159]

[0160]

[0161] 5.10. Sample recovery test

[0162] Accurately weigh appropriate amounts of camphor, trans-nerolidol, menthol, isoborneol, borneol, α-terpineol, α-gingerol, β-bisabolene, and α-farnesene reference substances and prepare a mixed reference solution containing 45.12 mg of camphor, 0.11 mg of trans-nerolidol, 5.51 mg of menthol, 0.23 mg of isoborneol, 0.33 mg of borneol, 0.18 mg of α-terpineol, 0.10 mg of α-gingerol, 0.05 mg of β-bisabolene, and 0.36 mg of α-farnesene per 1 ml with ethyl acetate. 1.25 g of sample (S1) from the same batch with known content was taken in parallel in 9 portions and divided into three groups. Camphor, trans-nerolidol, menthol, isoborneol, borneol, α-terpineol, α-gingerol, β-bisabolene, and α-farnesene mixed reference solution were precisely added: 1.6 ml, 2 ml, and 2.4 ml, 3 portions each; the test solution containing the internal standard was prepared according to the method under "5.5", and the sample was injected and determined according to the GC-MS conditions under "5.1". The recovery was calculated by the following formula 1. The results are shown in Table 18.

[0163]

[0164] The average recovery rate and RSD of each component met the requirements, demonstrating that the optimized method was accurate and reliable and could be used to determine the contents of nine volatile components in the intermediate product of Shufu patch.

[0165] Table 18 Sample recovery test results

[0166]

[0167]

[0168] 5.11 Calculation of internal standard correction factor

[0169] Select pulegone as the internal standard for trans-nerolidol, isoborneol, borneol, α-terpineol, α-gingerol, β-bisabolene, and α-farnesene, and 2,4,6-tri-tert-butylphenol as the internal standard for camphor and menthol. Accurately pipette 1 μl of each of the reference solution and the test solution containing the internal standard, inject and analyze according to the GC-MS conditions under "5.1", and record the chromatogram. Measure the peak area of ​​the reference and internal standard substances, and calculate the correction factor according to the following formula 2:

[0170]

[0171] Where: A S is the peak area of ​​the internal standard substance; A R is the peak area of ​​the reference substance; c S is the concentration of the internal standard substance; c R is the concentration of the reference substance.

[0172] Then take each batch of test solution containing internal standard substance, inject the sample, record the chromatogram, measure the peak area of ​​the test component and internal standard substance in the test sample, and calculate the content according to the following formula 3:

[0173]

[0174] Where: A X is the peak area of ​​the test sample; C X is the concentration of the test sample; A' S is the peak area of ​​the internal standard substance; C' S is the concentration of the internal standard substance; f is the internal standard correction factor.

[0175] The correction factors of trans-nerolidol, isoborneol, borneol, α-terpineol, α-gingerene, β-bisabolene, α-farnesene, camphor, and menthol were 0.48, 0.33, 0.27, 0.20, 0.38, 0.96, 0.27, 0.99, and 0.61, respectively.

[0176] 5.12 Durability Inspection

[0177] A mixed reference solution containing an internal standard was injected and measured according to the GC-MS conditions in Section "5.1," and correction factors for the nine components were calculated. The effects of different chromatographs, columns, flow rates, inlet temperatures, and gas chromatography-mass spectrometer interface temperatures on the correction factors for the nine components were investigated. The results are shown in Tables 19-1, 19-2, and 20-1, 20-2. The results showed no significant differences in the correction factors for each component (RSDs were all less than 3%).

[0178] Table 19-1 Effects of different chromatographs and columns on the relative correction factors of the various components of the intermediate product of Shufu patch

[0179]

[0180]

[0181] Table 19-2 Effects of different chromatographs and column models on the relative correction factors of the various components in the intermediate product of Shufu Patch

[0182]

[0183] Table 20-1 Effects of different GC-MS conditions on the relative correction factors of each component in the intermediate product of Shufu patch

[0184]

[0185] Table 20-2 Effects of different GC-MS conditions on the relative correction factors of each component in the intermediate product of Shufu patch

[0186]

[0187]

[0188] 5.13. Double internal standard method determination results

[0189] Fifteen batches of Shufu patch intermediate products were taken, and three test sample solutions were prepared in parallel for each batch. The test sample solutions were prepared according to the method under "5.5". The samples were injected and determined according to the GC-MS conditions under "5.1". The double internal standard method was used to calculate the contents of trans-nerolidol, isoborneol, borneol, α-terpineol, α-gingerene, β-bisabolene, α-farnesene, camphor, and menthol in the Shufu patch intermediate products. The results are shown in Table 21.

[0190] Table 21 Determination results of the content of each component (mg / g, n=3)

[0191]

[0192] 5.14 Comparative experiment between double internal standard method and single internal standard method

[0193] The content of each component in 6 identical test samples was determined separately, and the RSD values ​​of the content determined by various methods were calculated, see Table 22.

[0194] Table 22 Comparison of double internal standard and single internal standard content determination results

[0195]

[0196] 6. Conclusion

[0197] The present invention establishes a method for establishing a characteristic spectrum of volatile components of a Shufu patch intermediate product and a method for determining the content using a double internal standard method. During the characteristic spectrum establishment process, menthol is used as an internal reference. Through methodological research and 15 batches of sample determination, a characteristic spectrum of volatile components of the Shufu patch intermediate product is established, which fully reflects comprehensive information on the volatile components of the Shufu patch intermediate product. The volatile characteristic spectrum is viewed as a whole, thereby avoiding the one-sidedness of quality judgment caused by measuring only a small amount of components and reducing the possibility of human interference with a certain indicator.

[0198] Based on qualitative analysis of characteristic spectra and combined with existing characteristic spectra, a dual-internal standard method was established for the simultaneous determination of nine index components in the intermediate product of Shufu Patch. Pulegone was selected as the internal standard for trans-nerolidol, isoborneol, borneol, α-terpineol, α-gingerene, β-bisabolene, and α-farnesene, and 2,4,6-tri-tert-butylphenol was used as the internal standard for camphor and menthol. Relative correction factors were calculated for trans-nerolidol, isoborneol, borneol, α-terpineol, α-gingerene, β-bisabolene, α-farnesene, camphor, and menthol, respectively. The robustness of these nine component correction factors was evaluated under different chromatographic systems, columns, inlet temperatures, and flow rates. The results showed good robustness of the method.

[0199] The correction factor established by the present invention is accurate and reliable, and can be used for simultaneous quantification of multiple components with a maximum difference of 3000 times in the content of the component in the intermediate product. Firstly, it overcomes the problem that the external standard method cannot eliminate the error introduced in the sample processing process due to the complexity of the volatile components of the intermediate product of the Shufu patch, and an internal standard needs to be added to calibrate and eliminate the impact of fluctuations in operating conditions on the analysis results; secondly, it improves the accuracy of the analysis results, overcomes the problem that the content of different components varies greatly, and the deviation of the single internal standard measurement result is large, which will cause the accuracy of the component content to decrease. A new method is provided for the quality control and evaluation of the intermediate product of the Shufu patch, which is simple, accurate, and easy to be further promoted.

[0200] It should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention. Any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for establishing a characteristic spectrum of volatile components of a Shufu patch intermediate product, characterized in that: The following steps are involved: 1) Preparation of reference substance stock solution: Accurately weigh 250.05 mg of camphor reference substance and 60.06 mg of menthol reference substance, respectively, place them in the same 5 ml measuring flask, dissolve them in ethyl acetate and dilute to the mark, shake well, and use them as reference substance stock solution 1; accurately weigh 30.06 mg of trans-nerolidol, 40.12 mg of isoborneol, 60.05 mg of borneol, 20.05 mg of α-terpineol, 20.11 mg of α-gingerene, 20.15 mg of β-bisabolene, and 50.06 mg of α-farnesene, respectively, place them in the same 100 ml measuring flask, dissolve them in ethyl acetate and dilute to the mark, shake well, and use them as reference substance stock solution 2 for later use; 2) Preparation of mixed reference solution: Accurately pipette 2 ml of reference substance stock solution 1 and reference substance stock solution 2 respectively, place them into the same 25 ml volumetric flask, dilute to the mark with ethyl acetate, and shake well to obtain a mixed reference solution. 3) Preparation of test solution: Take 2.5g of Shufu patch, cut it into small pieces, remove the cover lining, place it in a 250ml round-bottom flask, add 150ml of water, and test it according to the volatile oil determination method. Add water from the upper end of the measuring instrument to fill the scale part and overflow into the flask, then add 2ml of ethyl acetate, connect a reflux condenser, heat to boiling, and keep it boiling slightly for 4h, let cool, take the ethyl acetate solution, filter it through a funnel covered with anhydrous sodium sulfate, and place the filtrate in a 25ml measuring flask. Use ethyl acetate to wash the condenser, volatile oil measuring instrument, and funnel in sequence. The washing liquid and the above ethyl acetate solution are combined into the same measuring flask, diluted to the scale with ethyl acetate, and shaken to obtain the test solution; 4) Preparation of single drug solution: According to the composition of the intermediate product of Shufu patch and the amount of the test sample, camphor, menthol and dried ginger were accurately weighed respectively, and placed in a 250ml round-bottom flask respectively. 150ml of water was added and the volatile oil assay was tested according to the volatile oil assay method. Water was added from the upper end of the assay to fill the scale part and overflow into the flask. Then 2ml of ethyl acetate was added, and a reflux condenser was connected. The mixture was heated to boiling and kept at a slight boil for 4 hours. The mixture was cooled and the ethyl acetate solution was separated. The mixture was filtered through a funnel covered with anhydrous sodium sulfate. The filtrate was placed in a 25ml measuring flask. The condenser, volatile oil assay device and funnel were washed with ethyl acetate in sequence. The washing liquid and the above ethyl acetate solution were combined into the same measuring flask, diluted to the scale with ethyl acetate, and shaken to obtain the single drug solutions of the three herbs. 5) Characteristic spectrum analysis test: Analyzed by gas chromatography-mass spectrometry: The test sample solutions at each level were injected into the gas chromatography-mass spectrometry analysis. The gas chromatography-mass spectrometry conditions were as follows: HP-INNOWAX column 30m×0.25mm, 0.5μm; carrier gas was high-purity helium; column temperature was programmed: initial temperature 50℃, maintained for 2min, and then increased at 10℃ / min -1 Raise to 76℃, hold for 2min, and heat at 1.3℃ / min -1 Raise to 85℃, maintain for 20min, and heat at 0.4℃ / min -1 Raise to 87℃, maintain for 60min, and heat at 1.0℃ / min -1 Raise to 150℃, hold for 60min, and heat at 1.3℃ / min -1 Heat to 210°C and hold for 60 min; injection port temperature 230°C; column flow rate 0.8 ml / min; split ratio 10:1; theoretical plate number based on camphor peak not less than 5000; Qualitative mass spectrometry conditions were as follows: electron impact (EI) ion source; electron energy 70 eV; ion source temperature: 230°C; interface temperature: 230°C; quadrupole temperature 150°C; mass scan range m / z 30-600 full scan; The solute delay was 4.6 minutes; the post-run was maintained at 235°C for 20 minutes; the tuning file was standard tuning; 6) Establish characteristic maps and determine quantitative detection indicators: Fifteen batches of intermediate products of Shufu plaster were collected for sample information according to the above-mentioned established method. The chromatogram data of S1-S15 of the 15 batches of samples were imported into the Chinese Medicine Chromatogram Similarity Evaluation Software 2012 version. The S1 sample spectrum was used as the reference spectrum. The average method was used with the time window set to 0.1 min and multi-point correction was performed to perform chromatographic peak matching. The superimposed spectrum of the 15 batches of samples and the standard control characteristic spectrum (R) were generated. The mixed reference solution, the test solution and the single drug solution were taken and sampled according to the chromatographic and qualitative mass spectrometric conditions of the characteristic spectrum analysis test items. The chromatograms were recorded and the NIST in Msss Hunter mass spectrometry analysis software was used. The MS program identified and compared it with the reference substance. The GC-MS standard reference characteristic spectrum of the intermediate product of the Shufu patch was composed of 9 common peaks. Peak 1 was determined to be camphor, peak 2 to be trans-nerolidol, peak 3 to be menthol, peak 4 to be isoborneol, peak 5 to be borneol, peak 6 to be α-terpineol, peak 7 to be α-gingerene, peak 8 to be β-bisabolene, and peak 9 to be α-farnesene.

2. The method for establishing the volatile component characteristic spectrum of the intermediate product of the Shufu patch according to claim 1, wherein Among the nine common peaks, menthol was used as a reference and peak No. 3 was used as a reference peak S. The relative retention time of each common peak and peak S was calculated. The relative retention time was within ±5% of the specified value. The specified value is shown in Table 1: Table 1 Relative retention time specified values ​​of the characteristic spectrum of the intermediate product of Shufu patch The chromatograms of 15 batches of samples were analyzed using the Chinese medicine chromatographic fingerprint similarity evaluation software 2012 version. The similarity between the characteristic spectra of samples S1 to S15 and the control characteristic spectra was calculated, and the similarity was not less than 0.

90.

3. Application of the method for establishing a characteristic spectrum of volatile components of the intermediate product of the Shufu patch according to claim 1 in the quality control of the intermediate product of the Shufu patch.

4. A double internal standard method for determining the content of the intermediate product of Shufu patch, characterized in that: The following steps are involved: (1) Preparation of internal standard solution: Accurately weigh 10.02 mg of pulegone and 1000.01 mg of 2,4,6-tri-tert-butylphenol, place them separately in a 10 mL volumetric flask, dilute to the mark with ethyl acetate, and shake well to obtain the internal standard solution. (2) Preparation of mixed reference solution containing internal standard: Accurately pipette 2 ml each of the reference substance stock solution 1 and the reference substance stock solution 2 described in claim 1, and then accurately pipette 1 ml each of the pulegone and 2,4,6-tri-tert-butylphenol internal standard solutions, place them in the same 25 ml volumetric flask, dilute to the mark with ethyl acetate, and shake well to prepare a mixed reference solution containing the internal standard; (3) Preparation of test solution containing internal standard: Take 2.5g of Shufu patch, cut it into small pieces, remove the cover lining, place it in a 250ml round-bottom flask, add 150ml of water, and test according to the volatile oil determination method. Add water from the upper end of the analyzer to fill the scale part and overflow into the flask, then add 2ml of ethyl acetate, connect a reflux condenser, heat to boiling, and keep it boiling slightly for 4h, let it cool, take the ethyl acetate solution, filter it through a funnel covered with anhydrous sodium sulfate, and place the filtrate into a 25ml measuring flask. Wash the condenser, volatile oil analyzer, and funnel with ethyl acetate in sequence. Combine the washing liquid and the above ethyl acetate solution into the same measuring flask, accurately add 1ml each of pulegone and 2,4,6-tri-tert-butylphenol internal standard solution, dilute to the scale with ethyl acetate, and shake well to obtain the internal standard test solution; (4) Preparation of negative sample solution: According to the prescription composition and production process of the intermediate product of Shufu plaster, negative samples and blank matrix samples without camphor, menthol and dried ginger are prepared respectively. The negative samples without camphor, menthol and dried ginger and the negative matrix samples are taken respectively, and the negative samples without camphor, menthol and dried ginger and the negative matrix samples are operated respectively according to the method under "(3)" to obtain negative sample solutions with internal standards without camphor, menthol and dried ginger and the negative matrix samples; (5) Gas content determination conditions: GC-MS conditions: HP-INNOWAX column 30 m × 0.25 mm, 0.5 μm; carrier gas: high-purity helium; column temperature: programmed: initial temperature 50 °C, hold for 2 min, increase at 10 °C / min -1 Raise to 76℃, hold for 2min, and heat at 1.3℃ / min -1 Raise to 85℃, maintain for 20min, and heat at 0.4℃ / min -1 Raise to 87℃, maintain for 60min, and heat at 1.0℃ / min -1 Raise to 150℃, hold for 60min, and heat at 1.3℃ / min -1 Heat to 210°C and hold for 60 min; injection port temperature 230°C; column flow rate 0.8 ml / min; split ratio 10:1; theoretical plate number based on camphor peak not less than 5000; The quantitative mass spectrometry conditions were as follows: electron impact (EI) ion source; electron energy 70 eV; ion source temperature: 230°C; interface temperature: 230°C; quadrupole temperature 150°C; solvent delay 4.6 min; post-run at 235°C for 20 min; standard tuning file, SIM ion monitoring mode; SIM detection time and quantitative and qualitative ion settings for each compound are shown in Table 2. Table 2 Quantitative and qualitative ions of each component compound (6) Content determination: Pulegone was selected as the internal standard for trans-nerolidol, isoborneol, borneol, α-terpineol, α-gingerol, β-bisabolene, and α-farnesene, and 2,4,6-tri-tert-butylphenol was selected as the internal standard for camphor and menthol. 1 μl of the reference solution and the test solution containing the internal standard were accurately aspirated, the chromatograms were recorded, the peak areas of the reference and internal standard substances were measured, and the correction factor f was calculated. The correction factors for trans-nerolidol, isoborneol, borneol, α-terpineol, α-gingerol, β-bisabolene, α-farnesene, camphor, and menthol were 0.48, 0.33, 0.27, 0.20, 0.38, 0.96, 0.27, 0.99, and 0.61, respectively. Then take the test sample solution containing the internal standard substance from each batch, inject the sample, record the chromatogram, measure the peak area of ​​the test component and the internal standard substance in the test sample, and use the double internal standard method to calculate the contents of camphor, trans-nerolidol, menthol, isoborneol, borneol, α-terpineol, α-gingerene, β-bisabolene, and α-farnesene in the intermediate product of Shufu patch.

5. the double internal standard method assay method of Shufu patch intermediate product according to claim 4, is characterized in that, The calculation formula of the correction factor f is: Where: A S is the peak area of ​​the internal standard substance; A R is the peak area of ​​the reference substance; c S is the concentration of the internal standard substance; c R is the concentration of the reference substance; The content calculation formula is: Where: A X is the peak area of ​​the test sample; C X is the concentration of the test sample; A' S is the peak area of ​​the internal standard substance; C' S is the concentration of the internal standard substance; f is the internal standard correction factor.

6. Application of the double internal standard content determination method of the Shufu patch intermediate product according to claim 4 in the quality control of the Shufu patch intermediate product.

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