Method for establishing characteristic chromatogram of volatile components of intermediate product of abdomen-relaxing plaster and method for measuring content of intermediate product by double internal standard method

Abstract

According to the establishment method of the characteristic spectrum of the volatile components of the intermediate product of the abdomen-relaxing plaster and the double-internal-standard-method content determination method of the intermediate product, in the establishment process of the characteristic spectrum, menthol is used as an internal reference substance, and the characteristic spectrum of the volatile components of the intermediate product of the abdomen-relaxing plaster is established through methodological research and determination of 15 batches of samples; the comprehensive information of the volatile components of the abdomen-relaxing plaster intermediate product is fully embodied, on the basis of qualitative analysis of the characteristic chromatogram, the existing characteristic chromatogram is combined, and a method for simultaneously determining the contents of nine index components of the abdomen-relaxing plaster intermediate product by establishing a double-internal-standard method is established, so that the deficiency of control of various volatile chemical components of the abdomen-relaxing plaster intermediate product can be made up; therefore, the quality of the intermediate product of the abdomen-relaxing plaster can be detected and evaluated more comprehensively, and the quality control level of the product can be improved.

Description

Technical Field

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

[0002] The intermediate product of Shufu Tiegao is a semi-solid composed of drug components such as ginger powder, camphor, and menthol and matrix components such as elastomer, plasticizer, and tackifier, and is used for the preparation of Shufu Tiegao. As an intermediate product of Shufu Tiegao, its quality directly affects the control of the next process and the quality stability and uniformity of the finished product. This intermediate product contains many volatile drug components. Both camphor and menthol are volatile components, and ginger powder also contains many volatile components. Volatile drugs are the main material basis for the product to exert its medicinal effect.

[0003] However, at present, the control of the intermediate product (i.e., ginger powder, camphor, and menthol drugs, the same below) mainly focuses on identification by thin-layer chromatography. The disadvantages of thin-layer chromatography are that the reproducibility is not very stable, the sensitivity is low, it cannot identify trace substances, and it cannot identify and judge the existence state of active ingredients as a whole, and it cannot reflect the complex and diverse characteristics of Chinese medicine components; in addition, the control of the content of components is insufficient, which is not conducive to the quality stability and consistency of the intermediate product, is not conducive to accurately guiding the parameters of the subsequent process, forms a certain operation blind area, and will cause quality risks of the final product.

[0004] Gas chromatography-mass spectrometry has obvious advantages in the identification and quality control of volatile components and is increasingly widely used. Therefore, in order to further explore the types and contents of volatile oils contained in the intermediate product of Shufu Tiegao, promote the continuous stability and refinement of the production process, and ensure the product quality, it is necessary to study the types of chemical components of this intermediate product and determine the content of active ingredients therein. That is, it is an urgent technical problem to provide a method for establishing a characteristic chromatogram of volatile components of an intermediate product of Shufu Tiegao and a double internal standard content determination method. Summary of the Invention

[0005] In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a method for establishing a characteristic chromatogram of volatile components of an intermediate product of Shufu Tiegao and a method for determining the content by double internal standard method of the intermediate product, which can effectively solve the lack of control of various volatile chemical components of the intermediate product of Shufu Tiegao and cannot provide stability and uniformity control for the product quality.

[0006] To achieve the above purpose, the technical solution solved by the present invention is that a method for establishing a characteristic chromatogram of volatile components of an intermediate product of Shufu Tiegao includes 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 separately, place them in the same 5-ml volumetric flask, dissolve with ethyl acetate and dilute to the mark, shake well to obtain 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 - zingiberene, 20.15 mg of β - bisabolene, and 50.06 mg of α - farnesene separately, place them in the same 100-ml volumetric flask, dissolve with ethyl acetate and dilute to the mark, shake well to obtain reference substance stock solution 2 for standby;

[0009] 2) Preparation of mixed reference substance solution:

[0010] Accurately pipette 2 ml each of reference substance stock solution 1 and reference substance stock solution 2 into the same 25-ml volumetric flask, dilute to the mark with ethyl acetate, and shake well to obtain the mixed reference substance solution;

[0011] 3) Preparation of test solution:

[0012] Take 2.5 g of Shufu Tiegao, cut it into small pieces, remove the cover lining, place it in a 250-ml round-bottom flask, add 150 ml of water, conduct the test according to the volatile oil determination method. Add water from the upper end of the measuring device to fill the graduated part and overflow into the flask until it is full, then add another 2 ml of ethyl acetate. Connect the reflux condenser, heat to boiling, and maintain gentle boiling for 4 h. Let it cool, separate the ethyl acetate layer, filter it through a funnel lined with anhydrous sodium sulfate. Transfer the filtrate to a 25-ml volumetric flask, wash the condenser, volatile oil measuring device, and funnel with ethyl acetate in turn. Combine the washing liquid and the above ethyl acetate layer into the same volumetric flask, dilute to the mark with ethyl acetate, and shake well to obtain the test solution;

[0013] 4) Preparation of single-herb solution:

[0014] According to the composition of the intermediate product of Shufu Tiegao and the amount of the test sample taken, accurately weigh 3 herbs, namely camphor, menthol, and dried ginger, separately, place them in 250-ml round-bottom flasks, add 150 ml of water, conduct the test according to the volatile oil determination method. Add water from the upper end of the measuring device to fill the graduated part and overflow into the flask until it is full, then add another 2 ml of ethyl acetate. Connect the reflux condenser, heat to boiling, and maintain gentle boiling for 4 hours. Let it cool, separate the ethyl acetate layer, filter it through a funnel lined with anhydrous sodium sulfate. Transfer the filtrate to a 25-ml volumetric flask, wash the condenser, volatile oil measuring device, and funnel with ethyl acetate in turn. Combine the washing liquid and the above ethyl acetate layer into the same volumetric flask, dilute to the mark with ethyl acetate, and shake well to obtain the single-herb solutions of the 3 herbs;

[0015] 5) Analysis and testing of characteristic chromatogram: Analyze by gas chromatography - mass spectrometry:

[0016] Inject the test sample solutions at all levels into a gas chromatograph - mass spectrometer for analysis. The instrument is a 7820A - 5977B gas chromatograph - mass spectrometer (Agilent, USA), with a MassHunter workstation and a NIST17.0 standard mass spectrometry retrieval library.

[0017] Gas - mass spectrometry conditions: HP - INNOWAX chromatographic column (30m×0.25mm, 0.5μm); the carrier gas is high - purity helium; the column temperature is programmed: the initial temperature is 50°C, held for 2 min, at a rate of 10°C / min -1 rising to 76°C, held for 2 min, at a rate of 1.3°C / min -1 rising to 85°C, held for 20 min, at a rate of 0.4°C / min -1 rising to 87°C, held for 60 min, at a rate of 1.0°C / min -1 rising to 150°C, held for 60 min, at a rate of 1.3°C / min -1 rising to 210°C, held for 60 min; the inlet temperature is 230°C; the column flow rate is 0.8 ml / min; the split ratio is 10:1, and the theoretical plate number is not less than 5000 calculated based on the camphor peak;

[0018] The qualitative mass spectrometry conditions are: electron impact (EI) ion source; electron energy 70 eV; ion source temperature: 230°C; interface temperature: 230°C; quadrupole temperature 150°C; mass scanning range m / z (30 - 600) full scan; solvent delay (4.6 min); post - run held at 235°C for 20 min; the tuning file is the standard tuning;

[0019] 6) Establish a characteristic chromatogram and determine the quantitative detection index:

[0020] Take 15 batches of intermediate products of Shufu Tiegao, collect sample information according to the above construction method, import the chromatogram data of 15 batches of samples S1 - S15 into the Similarity Evaluation Software for Traditional Chinese Medicine Chromatograms (2012 version). Using the S1 sample chromatogram as the reference chromatogram, adopt the average method, set the time window to 0.1 min, perform multi - point calibration, conduct chromatographic peak matching, and generate the superimposed chromatogram of 15 batches of samples and the standard reference characteristic chromatogram (R); take the mixed reference solution, the test solution, and the single - herb solution, inject and measure according to the chromatographic and qualitative mass spectrometry conditions of the characteristic chromatogram analysis test items, record the chromatogram, use the NIST MS program in the Msss Hunter mass spectrometry analysis software to identify and compare with the reference substance. The GC - MS standard reference characteristic chromatogram of this intermediate product of Shufu Tiegao consists of 9 common peaks. Determine that peak 1 is camphor, peak 2 is trans - nerolidol, peak 3 is menthol, peak 4 is isoborneol, peak 5 is borneol, peak 6 is α - terpineol, peak 7 is α - zingiberene, peak 8 is β - bisabolene, and peak 9 is α - farnesene.

[0021] Among the 9 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 the S peak 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 specification values ​​of the characteristic spectrum of the intermediate product of Shufu patch

[0023]

[0024] The similarity evaluation software for Chinese medicine chromatographic fingerprint was used (2012 version) to analyze the spectra of 15 batches of samples, and 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 applied in the quality control of the intermediate product of the Shufu patch.

[0026] A double internal standard method for content determination 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 in 10 mL volumetric flasks, 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 of the reference stock solution 1 and the reference stock solution 2 described in claim 1, and then accurately pipette 1 ml of the internal standard solution of pulegone and 2,4,6-tri-tert-butylphenol, respectively, into 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;

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

[0032] Take 2.5g of Shufu patch, cut into small pieces, remove the cover lining, put into 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 determination device until it fills the scale part and overflows 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 out the ethyl acetate solution, filter it through a funnel covered with anhydrous sodium sulfate, put the filtrate into a 25ml volumetric flask, wash the condenser, volatile oil determination device, and funnel with ethyl acetate in turn, combine the washing liquid and the above ethyl acetate solution into the same volumetric flask, accurately add 1ml each of pulegone and 2,4,6-tri-tert-butylphenol internal standard solution, dilute to the scale with ethyl acetate, shake well, and obtain the internal standard test solution;

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

[0034] According to the formula 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 sample of the blank matrix 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 sample of the blank matrix;

[0035] (5) Gas chromatography-mass spectrometry conditions: The instrument was 7820A-5977B gas chromatography-mass spectrometer (Agilent, USA), MassHunter workstation, NIST17.0 standard mass spectrum retrieval library; gas chromatography-mass spectrometry conditions: HP-INNOWAX chromatographic column (30m×0.25mm, 0.5μm); the carrier gas was high-purity helium; the column temperature was programmed: the initial temperature was 50°C, maintained for 2min, 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 increase the temperature at 0.4℃ / min -1 Raise to 87℃, maintain for 60min, and increase the temperature at 1.0℃ / min -1 Raise to 150℃, maintain for 60min, and increase the temperature at 1.3℃ / min -1 Raise to 210℃, maintain for 60min; injection port temperature 230℃; column flow rate 0.8ml / min; split ratio 10:1, theoretical plate number not less than 5000 based on camphor peak;

[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; the tuning file was standard tuning, SIM ion monitoring mode, and the SIM detection time and quantitative and qualitative ion settings of each compound to be tested were specifically shown in Table 2;

[0037] Table 2 Quantitative and Qualitative Ions of Each Component Compound

[0038]

[0039] (6) Content Determination:

[0040] Piperitone was selected as the internal standard for trans - nerolidol, isoborneol, borneol, α - terpineol, α - zingiberene, 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 substance solution and the test sample solution containing the internal standard were accurately pipetted respectively, the chromatogram was recorded, the peak areas of the reference substance and the internal standard substance were measured, and the correction factor f was calculated. The correction factors for trans - nerolidol, isoborneol, borneol, α - terpineol, α - zingiberene, 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, the test sample solutions containing the internal standard substance of each batch number were injected, the chromatogram was recorded, the peak areas of the components to be measured and the internal standard substance in the test sample were measured, and the double - internal - standard method was used to calculate the contents of camphor, trans - nerolidol, menthol, isoborneol, borneol, α - terpineol, α - zingiberene, β - bisabolene, and α - farnesene in the intermediate product of Shufu Tiegao.

[0042] The calculation formula for the correction factor f is as follows:

[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 as follows:

[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 correction factor of the internal - standard method.

[0048] Application of the double - internal - standard method for content determination of the intermediate product of Shufu Tiegao in the quality control of the intermediate product of Shufu Tiegao.

[0049] The characteristic chromatogram obtained by gas chromatography - mass spectrometry in the present invention can comprehensively reflect the volatile components in the intermediate product of Shufu Tiegao, make up for the lack of control of volatile components in medicinal materials in the current standard, and at the same time realize the quantitative detection of key volatile index components in the intermediate product of Shufu Tiegao, which is beneficial to improving the quality control level of the intermediate product of Shufu Tiegao. The double internal standard method is used for multi - index quantification, with higher accuracy and remarkable social and economic benefits. Description of the Drawings

[0050] Figure 1 It is the characteristic chromatogram of precision detection in Example 1 of the present invention; among them, A: overall view; B: partial enlarged view;

[0051] Figure 2 It is the characteristic chromatogram of repeatability detection in Example 1 of the present invention; among them, A: overall view; B: partial enlarged view;

[0052] Figure 3 It is the characteristic chromatogram of stability detection in Example 1 of the present invention; among them, A: overall view; B: partial enlarged view;

[0053] Figure 4 It is the superposition form of the characteristic chromatogram of the intermediate product of Shufu Tiegao and the control characteristic chromatogram in Example 1 of the present invention;

[0054] Figure 5 It is the identification of common peaks in the characteristic chromatogram in Example 1 of the present invention; among them: A: mixed reference substances; B: partial enlarged view of mixed reference substances; 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 - zingiberene, 8 - β - bisabolene, 9 - α - farnesene;

[0055] Figure 6 It is the specificity chromatogram for content determination in Example 2 of the present invention; among them: A: reference substance; B: sample; C: negative sample without dried ginger; D: negative sample without camphor; E: negative sample without 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 - zingiberene, 8 - β - bisabolene, 9 - α - farnesene, 10 - pulegone, 11 - 2,4,6 - tri - tert - butylphenol. Detailed Embodiments

[0056] The following detailed description of the specific embodiments of the present invention is given in conjunction with the drawings and examples.

[0057] When the present invention is specifically implemented, it is given by the following examples.

[0058] Example 1

[0059] A method for establishing a characteristic spectrum of volatile components of a soothing plaster 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, NIST17.0 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., specification: 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 all purchased from China Food and Drug Inspection Institute; α-gingerene reference substance (batch number: wkq-0 007937; content: 98.20%), β-bisabolene reference substance (batch number: wkq-0787608; content: 96.50%), α-farnesene reference substance (batch number: wkq-0027612; content: 99.10%) were 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%, American Stanford 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 Komio Chemical Reagent Co., Ltd., analytical grade).

[0065] Medicine: intermediate product of soothing abdominal plaster (sample numbers are S1 to S15).

[0066] 1.3 Analytical software: Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines 2012 Edition (National Pharmacopoeia Commission).

[0067] 2. Test conditions

[0068] 2.1 Chromatography - Mass Spectrometry Conditions and System Suitability Test:

[0069] Gas Chromatography - Mass Spectrometry Conditions: HP - INNOWAX chromatographic column (30m × 0.25mm, 0.5μm); carrier gas is high - purity helium; column temperature is programmed temperature rise: initial temperature 50°C, hold for 2min, at a rate of 10°C / min -1 rise to 76°C, hold for 2min, at a rate of 1.3°C / min -1 rise to 85°C, hold for 20min, at a rate of 0.4°C / min -1 rise to 87°C, hold for 60min, at a rate of 1.0°C / min -1 rise to 150°C, hold for 60min, at a rate of 1.3°C / min -1 rise to 210°C, hold for 60min; injection port temperature 230°C; column flow rate 0.8ml / min; split ratio is 10:1, the number of theoretical plates calculated by camphor peak is not less than 5000;

[0070] Qualitative mass spectrometry conditions are: Electron Impact (EI) ion source; electron energy 70eV; ion source temperature: 230°C; interface temperature: 230°C; quadrupole temperature 150°C; mass scanning range m / z (30 - 600) full scan; solvent delay (4.6min); post - run hold at 235°C for 20min; tuning file is standard tuning.

[0071] 2.2 Stock solution of reference substances

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

[0073] 2.3 Mixed reference substance solution

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

[0075] 2.4 Preparation of the test solution:

[0076] Take 2.5 g of this product, cut it into small pieces, remove the cover lining, place it in a 250 ml round-bottom flask, add 150 ml of water, and perform the test according to the volatile oil determination method (General Principles 2204, Part 4 of the Chinese Pharmacopoeia 2020 Edition). Add water from the upper end of the measuring device to fill the graduated part and overflow into the flask. Then add 2 ml of ethyl acetate, connect the reflux condenser, heat to boiling, and maintain gentle boiling for 4 hours. Let it cool, separate the ethyl acetate layer, filter it through a funnel lined with an appropriate amount of anhydrous sodium sulfate. Transfer the filtrate to a 25 ml volumetric flask, wash the condenser, volatile oil measuring device, and funnel successively with an appropriate amount of ethyl acetate. Combine the washing solutions with the above ethyl acetate layer in the same volumetric flask, dilute to the mark with ethyl acetate, and shake well to obtain the solution.

[0077] 2.5 Single-herb solution

[0078] Weigh accurately camphor, menthol, and dried ginger, the three herbs, according to the composition of the intermediate product of Shufu Tiegao and the dosage of the test sample, and place them separately in 250 ml round-bottom flasks. Add 150 ml of water to each flask and perform the test according to the volatile oil determination method (General Principles 2204, Part 4 of the Chinese Pharmacopoeia 2020 Edition). Add water from the upper end of the measuring device to fill the graduated part and overflow into the flask. Then add 2 ml of ethyl acetate, connect the reflux condenser, heat to boiling, and maintain gentle boiling for 4 hours. Let it cool, separate the ethyl acetate layer, filter it through a funnel lined with an appropriate amount of anhydrous sodium sulfate. Transfer the filtrate to a 25 ml volumetric flask, wash the condenser, volatile oil measuring device, and funnel successively with an appropriate amount of ethyl acetate. Combine the washing solutions with the above ethyl acetate layer in the same volumetric flask, dilute to the mark with ethyl acetate, and shake well to obtain the solutions of the three individual herbs.

[0079] 2.6 Selection of reference substance

[0080] Since menthol is one of the main components of the volatile oil of the intermediate product of Shufu Tiegao, it has obvious pharmacological activities, a relatively high content, the reference substance is easily available, and the price is cheap. In the chromatogram, it has good resolution from other components. Therefore, menthol is selected as the internal reference substance.

[0081] 3 Methodology investigation of characteristic chromatogram

[0082] 3.1 Precision

[0083] Prepare the test solution of the same batch of intermediate product of Shufu Tiegao (No. S1) according to item "2.4", and under the chromatographic - mass spectrometry conditions of item "2.1", inject samples continuously for 6 times to detect the characteristic chromatogram. As Figure 1, record the retention time and peak area of each common peak. Using the retention time and peak area of the menthol chromatographic peak as a reference, calculate the relative retention time and relative peak area of each common peak. The RSD values of the relative retention time and relative peak area of each common peak are all less than 3%. Import the spectral data obtained 6 times into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)" for full-spectrum peak matching and calculate the similarity. The results show that the similarity is ≥0.999, indicating that the instrument is stable and the precision is good. The results are shown in Table 3, Table 4, and Table 5.

[0084] Table 3 Results of Precision Test of Intermediate Product (S1) of Shufu Tiegao (Relative Retention Time of Index Component Group)

[0085]

[0086] Table 4 Results of Precision Test of Intermediate Product (S1) of Shufu Tiegao (Relative Peak Area of Index Component Group)

[0087]

[0088] Table 5 Calculation Results of Chromatogram Similarity of Precision Test of Intermediate Product (S1) of Shufu Tiegao

[0089]

[0090]

[0091] 3.2. Repeatability

[0092] Take the same batch of samples (numbered S1) of the intermediate product of Shufu Tiegao, prepare 6 portions respectively according to the method under the preparation of the test solution in "2.4", and inject samples successively according to the chromatographic - mass spectrometry conditions in "2.1" to detect the characteristic chromatograms respectively. Figure 2 , record the retention time and peak area of each common peak. Using the retention time and peak area of the menthol chromatographic peak as a reference, calculate the relative retention time and relative peak area of each common peak. The RSD values of the relative retention time and relative peak area of each common peak are all less than 3.0%. Import the spectral data obtained 6 times into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)" for full-spectrum peak matching and calculate the similarity. The results show that the similarity is ≥0.999, indicating that the test method has good repeatability. The results are shown in Table 6, Table 7, and Table 8.

[0093] Table 6 Results of Repeatability Test of Intermediate Product (S1) of Shufu Tiegao (Relative Retention Time of Index Component Group)

[0094]

[0095] Table 7 Results of Repeatability Test of Intermediate Product (S1) of Shufu Tiegao (Relative Peak Area of Index Component Group)

[0096]

[0097] Table 8 Similarity Calculation Results of the Reproducibility Test Chromatogram of the Intermediate Product (S1) of Shufu Tiegao

[0098]

[0099]

[0100] 3.3 Stability

[0101] Prepare the test solution of the same batch of the intermediate product (No. S1) of Shufu Tiegao according to the method under "2.4", and inject samples at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h respectively according to the chromatographic and mass spectrometric conditions under "2.1" to detect the characteristic chromatogram. As Figure 3 , record the retention time and peak area of each common peak. Taking the retention time and peak area of the menthol chromatographic peak as the reference, calculate the relative retention time and relative peak area of each common peak. The RSD values of the relative retention time and relative peak area of each common peak are all less than 3.0%. Import the chromatogram data obtained 6 times into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" for full-spectrum peak matching and calculate the similarity. The results show that the similarity is all ≥ 0.999, indicating that the test solution is stable within 24 hours. It is suggested that the stability of the test product is good. The results are shown in Table 9, Table 10, and Table 11.

[0102] Table 9 Results of the Stability Test of the Intermediate Product (S1) of Shufu Tiegao (Relative Retention Time of Index Component Group)

[0103]

[0104] Table 10 Results of the Stability Test of the Intermediate Product (S1) of Shufu Tiegao (Relative Peak Area of Index Component Group)

[0105]

[0106] Table 11 Similarity Calculation Results of the Reproducibility Test Chromatogram of the Intermediate Product (S1) of Shufu Tiegao

[0107]

[0108]

[0109] 4. Establishment and Application of the Characteristic Chromatogram of the Intermediate Product of Shufu Tiegao

[0110] 4.1 Establishment and Similarity Evaluation of the Characteristic Chromatogram of the Intermediate Product of Shufu Tiegao

[0111] Import the GC-MS characteristic fingerprints of 15 batches of intermediate products of Shufu Tiegao in txt format into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)". Select the chromatogram of sample S1 as the reference chromatogram, set the time window width to 0.1 min, perform multi-point calibration and automatic matching on the 15 batches of samples, and establish a common pattern for the fingerprints. The results show that a total of 9 common peaks are calibrated in the GC-MS characteristic fingerprints of 15 batches of intermediate products of Shufu Tiegao, and an overlay chromatogram is generated, as shown in Figure 4 . Calculate the similarity based on the reference fingerprint (R) generated by the average method. The similarity results between the characteristic fingerprints of 15 batches of intermediate products of Shufu Tiegao and the standard characteristic fingerprint are shown in Table 12.

[0112] Table 12 Calculation results of the similarity of the characteristic fingerprints of the intermediate products of Shufu Tiegao

[0113]

[0114] The results show that the overall profiles of the peak groups of each batch of preparations are basically the same. Nine common peaks are determined, and the similarities are all above 0.9.

[0115] 4.2 Identification of common peaks in the characteristic fingerprint

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

[0117] 4.3 Provisions for the characteristic fingerprint standard

[0118] According to the relative retention times of each chromatographic peak in the chromatograms obtained from 15 batches of intermediate products of Shufu Tiegao, determine the common peaks, and select 9 common peaks as characteristic peaks to establish a reference characteristic fingerprint; among them, take the No. 3 peak as the reference peak S, calculate the ratio of the retention time of each chromatographic peak to the retention time of the S peak in the same chromatogram, and obtain the relative retention time and RSD, as shown in Table 13.

[0119] Table 13 Relative retention times of the characteristic fingerprints of the intermediate products of Shufu Tiegao

[0120]

[0121] The relative deviation range of the relative retention times of the characteristic peaks of the sample to be tested from those of the characteristic peaks in the standard characteristic chromatogram (see Table 13) is shown in Table 14.

[0122] Table 14 Relative retention time deviation range of the common peaks in the intermediate product of 15 batches of Shufu Tiegao from the corresponding peaks in the standard chromatogram

[0123]

[0124] According to the test results of the durability investigation of the characteristic chromatogram of volatile components, the relative retention times of the characteristic peaks in the characteristic chromatograms detected by different instruments and different brands of chromatographic columns did not exceed ±5% of the mean value. Therefore, it is tentatively determined that: there should be 9 characteristic peaks in the characteristic chromatogram of the intermediate product of Shufu Tiegao. Taking the peak corresponding to the reference substance menthol as the S peak, calculate the relative retention times of each characteristic peak and the S peak, and the relative retention times should be within ±5% of the specified value. The specified values of the relative retention times in the standard characteristic chromatogram 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), 1.631 (peak 9). In addition, combined with the similarity analysis of the characteristic chromatogram, the similarity between the characteristic chromatogram of the intermediate product of Shufu Tiegao and the standard characteristic chromatogram should not be less than 0.9.

[0125] Example 2

[0126] Other implementation conditions in this example are the same as those for the characteristic chromatogram conditions of the volatile components in the intermediate product of Shufu Tiegao in Example 1, and there are only new regulations for the GC-MS conditions. An internal standard solution is added to both the test solution and the reference solution.

[0127] A method for determining the content by double internal standard method of an intermediate product of Shufu Tiegao, comprising the following steps:

[0128] 5. Content determination method

[0129] 5.1. The conditions for gas chromatography-mass spectrometry content determination are as follows:

[0130] The instrument is a 7820A-5977B gas chromatography-mass spectrometry (Agilent, USA), MassHunter workstation, and NIST17.0 standard mass spectrometry retrieval library.

[0131] Gas chromatography-mass spectrometry conditions: HP-INNOWAX chromatographic column (30m×0.25mm, 0.5μm); the carrier gas is high-purity helium gas; the column temperature is programmed temperature rise: the initial temperature is 50°C, held for 2 min, at 10°C / min -1 rising to 76°C, held for 2 min, at 1.3°C / min -1Raise to 85 °C, hold for 20 min, at 0.4 °C / min -1 Raise to 87 °C, hold for 60 min, at 1.0 °C / min -1 Raise to 150 °C, hold for 60 min, at 1.3 °C / min -1 Raise to 210 °C, hold for 60 min; inlet temperature 230 °C; column flow rate 0.8 ml / min; split ratio 10:1, the number of theoretical plates is not less than 5000 calculated by the camphor peak;

[0132] The quantitative mass spectrometry conditions are 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 hold at 235 °C for 20 min; the tuning file is the standard tuning, SIM ion monitoring mode, the SIM detection time and the quantitative and qualitative ion settings of each compound to be measured are shown in Table 15 specifically.

[0133] Table 15 Quantitative and Qualitative Ions of Each Component Compound

[0134]

[0135] The common peaks in the characteristic chromatogram represent the information of 3 drugs in the intermediate product of Shufu Tiegao. The recognition accuracy of the chromatogram is >90%, the peak area is relatively large, the peak shape is symmetrical, and the resolution is good. In order to evaluate the content of volatile components in the intermediate product of Shufu Tiegao relatively completely and in detail, it is proposed to determine the content of 9 common peaks.

[0136] 5.2, Internal Standard Solution

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

[0138] 5.3, Stock Solution of Reference Substances

[0139] Precisely 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, dissolve with ethyl acetate and dilute to the mark, and shake well to obtain Stock Solution of Reference Substances 1; precisely 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-zingiberene, 20.15 mg of β-bisabolene, and 50.06 mg of α-farnesene respectively, place them in the same 100 ml volumetric flask, dissolve with ethyl acetate and dilute to the mark, and shake well to obtain Stock Solution of Reference Substances 2 for standby;

[0140] 5.4, Mixed Stock Solution of Reference Substances Containing Internal Standard

[0141] Precisely pipette 2 ml each of the reference substance stock solution 1 and the reference substance stock solution 2, and then precisely 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 obtain a mixed reference substance solution containing the internal standard.

[0142] 5.5 Test solution containing the internal standard

[0143] Take 2.5 g of Shufu Tiegao, cut it into small pieces, remove the cover lining, place it in a 250-ml round-bottom flask, add 150 ml of water, and conduct the test according to the volatile oil determination method (General Principles 2204, Volume IV, Chinese Pharmacopoeia 2020 Edition). Add water from the upper end of the measuring device to fill the graduated part and overflow into the flask. Then add 2 ml of ethyl acetate, connect the reflux condenser, heat to boiling, and maintain gentle boiling for 4 h. Let it cool, separate the ethyl acetate layer, filter through a funnel lined with an appropriate amount of anhydrous sodium sulfate. Transfer the filtrate to a 25-ml volumetric flask, wash the condenser, the volatile oil measuring device, and the funnel with an appropriate amount of ethyl acetate in sequence. Combine the washing solutions with the above ethyl acetate layer in the same volumetric flask, precisely add 1 ml each of the pulegone and 2,4,6-tri-tert-butylphenol internal standard solutions, dilute to the mark with ethyl acetate, and shake well to obtain the solution.

[0144] 5.6 Negative sample solution

[0145] Prepare a negative sample without camphor, menthol, and dried ginger (blank matrix) according to the prescription composition and production process of the intermediate product of Shufu Tiegao. Take the negative samples without camphor, menthol, and dried ginger and the negative sample of the blank matrix respectively, and operate according to the method under item "5.5" to obtain the negative sample solutions containing the internal standard without camphor, menthol, dried ginger, and blank matrix.

[0146] 5.7 Specificity

[0147] Prepare the test solution according to the test sample preparation method, and investigate whether the blank solvent ethyl acetate, the negative samples lacking dried ginger medicinal materials, camphor, and menthol, and the blank matrix will cause interference. Precisely pipette 1 μl each of the blank solvent ethyl acetate, the negative sample solution containing the internal standard, the reference substance solution containing the internal standard, the test solution containing the internal standard, and the blank matrix solution containing the internal standard, and conduct analysis according to the chromatographic - mass spectrometric conditions described under item "5.1". Record the chromatograms as Figure 6 shown. The results show that the solvent, blank matrix, and negative samples have no interference, and the method has good specificity.

[0148] 5.8 Investigation of linearity and range

[0149] Precisely measure 0.1, 0.5, 1.0, 1.5, 2.0, and 2.5 mL of the mixed reference stock solution respectively, place them in 10-mL volumetric flasks, then precisely add 0.3 mL of the internal standard solutions pulegone and 2,4,6-tri-tert-butylphenol respectively, make up the volume with ethyl acetate and shake well. Inject and measure according to the GC-MS conditions under item "5.1", record the chromatograms, respectively perform linear regression on the peak area ratio (Y) of the reference substances camphor, menthol and the internal standard 2,4,6-tri-tert-butylphenol to the mass concentration (X) of each component, and prepare the standard curves of camphor and menthol. Respectively perform linear regression on the peak area ratio (Y) of the reference substances trans-nerolidol, isoborneol, borneol, α-terpineol, α-zingiberene, β-bisabolene, α-farnesene and the internal standard pulegone to the mass concentration (X) of each component, and prepare the standard curves of trans-nerolidol, isoborneol, borneol, α-terpineol, α-zingiberene, β-bisabolene, α-farnesene. Obtain the linear regression equations and analysis ranges.

[0150] Determine the detection limit and quantification limit according to the concentrations of each component when the peak signal-to-noise ratio is about 3:1 and 10:1 respectively. The regression equations, linear ranges, detection limits and quantification limits of camphor, trans-nerolidol, menthol, isoborneol, borneol, α-terpineol, α-zingiberene, β-bisabolene, α-farnesene are shown in Table 16. The results show that the linear relationships of each component are good within their respective ranges.

[0151] Table 16 Standard Curves and Linear Ranges of Each Component in the Intermediate Product of Shufu Tiegao

[0152]

[0153] 5.9 Injection Precision, Repeatability and Stability Tests

[0154] Injection precision test: Precisely pipette 1 μL of the internal standard-containing reference solution under item "5.4", according to the GC-MS conditions under item "5.1", inject continuously for 6 times, measure the peak areas, and calculate the RSD (n = 6) of the peak area ratios of the reference substances camphor, menthol and the internal standard 2,4,6-tri-tert-butylphenol, and the RSD (n = 6) of the peak area ratios of the reference substances trans-nerolidol, isoborneol, borneol, α-terpineol, α-zingiberene, β-bisabolene, α-farnesene and the internal standard pulegone.

[0155] Repeatability test: Take the sample (S1), prepare 6 portions of the test solution containing the internal standard in parallel according to the method under item "5.5", inject and measure according to the GC-MS conditions under item "5.1", and calculate the RSD value of the average content of each component (n = 6) according to the internal standard method.

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

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

[0158] Table 17 Precision, Repeatability, and Stability of Each Component in Shufu Tiegao Intermediate

[0159]

[0160]

[0161] 5.10, Spiked Recovery Test

[0162] Accurately weigh appropriate amounts of the reference substances camphor, trans-nerylol, menthol, isoborneol, borneol, α-terpineol, α-zingiberene, β-bisabolene, and α-farnesene, and prepare a mixed reference substance solution with ethyl acetate at a concentration of 45.12 mg of camphor, 0.11 mg of trans-nerylol, 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-zingiberene, 0.05 mg of β-bisabolene, and 0.36 mg of α-farnesene per 1 ml. Take 1.25 g of the same batch of samples (S1) with known content in parallel, divide them into three groups, and accurately add 1.6, 2, and 2.4 ml of the mixed reference substance solution of camphor, trans-nerylol, menthol, isoborneol, borneol, α-terpineol, α-zingiberene, β-bisabolene, and α-farnesene to each group (3 portions for each); prepare the sample solution containing the internal standard according to the method under item "5.5", inject samples according to the GC-MS conditions under item "5.1" for determination, and calculate the recovery rate according to the following formula 1. The results are shown in Table 18.

[0163]

[0164] The average recoveries and RSDs of each component meet the requirements, proving that the optimized method is accurate and reliable and can be used for the determination of the contents of 9 volatile components in the intermediate product of Shufu Tiegao.

[0165] Table 18 Results of Spiked Recovery Test

[0166]

[0167]

[0168] 5.11. Calculation of internal standard correction factor

[0169] Piperitone is selected as the internal standard for trans - nerolidol, isoborneol, borneol, α - terpineol, α - zingiberene, β - bisabolene, and α - farnesene, and 2,4,6 - tri - tert - butylphenol is selected as the internal standard for camphor and menthol. 1 μl of the reference solution and the test solution containing the internal standard are accurately pipetted respectively, and injected for analysis under the GC - MS conditions described in "5.1", and the chromatogram is recorded. Measure the peak areas of the reference substance and the internal standard substance, and calculate the correction factor according to the following formula 2:

[0170]

[0171] In the formula: 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 the test solutions of each batch containing the internal standard substance, inject them, record the chromatogram, measure the peak areas of the components to be determined and the internal standard substance in the test sample, and calculate the content according to the following formula 3:

[0173]

[0174] In the formula: 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 correction factor of the internal standard method.

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

[0176] 5.12. Robustness investigation

[0177] Take the mixed reference solution containing the internal standard, inject and measure according to the GC-MS conditions of item "5.1", and calculate the correction factors of the 9 components. Investigate the effects of different chromatographs, different chromatographic columns, different flow rates, injection port temperatures, and gas chromatography-mass interface temperatures on the correction factors of the 9 components. The results are shown in Tables 19-1, 19-2, Tables 20-1, and 20-2. The results show that there is no significant difference in the effects of different chromatographs, different chromatographic columns, different flow rates, injection port temperatures, and gas chromatography-mass interface temperatures on the correction factors of each component (RSD is less than 3%).

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

[0179]

[0180]

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

[0182]

[0183] Table 20-1 Effects of different GC-MS conditions on the relative correction factors of the components to be tested in the intermediate product of Shufu patch

[0184]

[0185] Table 20-2 Effects of different GC-MS conditions on the relative correction factors of the components to be tested in the intermediate product of Shufu patch

[0186]

[0187]

[0188] 5.13. Double internal standard method determination results

[0189] Take 15 batches of Shufu patch intermediate products, prepare 3 test solutions in parallel for each batch, prepare the test solution according to the method under "5.5", and measure the sample under the GC-MS conditions under "5.1". The double internal standard method is used to calculate the contents of trans-nerolidol, isoborneol, borneol, α-terpineol, α-gingerene, β-bisabolene, α-farnesene, camphor and menthol in the intermediate products of Shufu patch. 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 contents of each component in 6 identical test samples were determined separately, and the RSD values of the contents determined by various methods were calculated, as shown in Table 22.

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

[0195]

[0196] 6. Conclusion

[0197] The present invention establishes a method for establishing the characteristic chromatogram of volatile components in the intermediate product of Shufu Tiegao and a method for content determination by double internal standard method. During the establishment of the characteristic chromatogram, menthol is used as the internal reference substance. Through methodological research and the determination of 15 batches of samples, the characteristic chromatogram of volatile components in the intermediate product of Shufu Tiegao is established, which fully reflects the comprehensive information of the volatile components in the intermediate product of Shufu Tiegao. Regarding the volatile characteristic chromatogram as a whole can avoid the one-sidedness of quality judgment caused by only measuring a small number of components and reduce the possibility of artificially interfering with a certain index.

[0198] On the basis of the qualitative analysis of the characteristic chromatogram, combined with the existing characteristic chromatograms, a method for simultaneously determining the contents of 9 index components in the intermediate product of Shufu Tiegao by double internal standard method is established. Pulegone is selected as the internal standard substance for trans-nerolidol, isoborneol, borneol, α-terpineol, α-zingiberene, β-bisabolene, and α-farnesene, and 2,4,6-tri-tert-butylphenol is used as the internal standard substance for camphor and menthol. The relative correction factors of trans-nerolidol, isoborneol, borneol, α-terpineol, α-zingiberene, β-bisabolene, α-farnesene, camphor, and menthol are calculated respectively. The durability of the correction factors of the 9 components is investigated from different aspects such as chromatographic systems, chromatographic columns, inlet temperature, and flow rate, and the results show that the method has good durability.

[0199] The correction factors established by the present invention are accurate and reliable, and can be used for simultaneous quantitative determination of multiple components with a maximum difference of 3000 times in the content of components in this intermediate product. First, it overcomes the problem that due to the complexity of the volatile components in the intermediate product of Shufu Tiegao, the external standard method cannot eliminate the errors introduced in the sample treatment process, and an internal standard substance needs to be added to calibrate and eliminate the influence on the analysis results caused by the fluctuations of operating conditions. Second, it improves the accuracy of the analysis results and overcomes the problem that due to the large difference in the contents of different components, the deviation of the single internal standard determination result is large, which will cause a decrease in the accuracy of the component content. It provides a new method for the quality control and evaluation of the intermediate product of Shufu Tiegao, which is simple, accurate, and easy to be further promoted.

[0200] It should be noted that the above description is only a preferred embodiment of the present invention, and does not impose any formal or substantial limitations on the present invention. It should be pointed out that for those of ordinary skill in the art in this technical field, without departing from the method of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention. Those skilled in the art, without departing from the spirit and scope of the present invention, when making some equivalent changes, modifications and evolutions 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 still fall 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 stock solution: 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; 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 in the same 25 ml volumetric flask, dilute to the mark with ethyl acetate, and shake well to obtain a mixed reference substance solution. 3) Preparation of test solution: Take 2.5g of Shufu patch, cut into small pieces, remove the cover lining, put into 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 determination device until it fills the scale part and overflows 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 out the ethyl acetate liquid, filter it through a funnel covered with anhydrous sodium sulfate, put the filtrate into a 25ml measuring bottle, wash the condenser, volatile oil determination device, and funnel with ethyl acetate in turn, combine the washing liquid and the above ethyl acetate liquid into the same measuring bottle, add ethyl acetate to dilute to the scale, shake well, and the test solution is obtained; 4) Preparation of single drug solution: According to the composition of the intermediate product of the Shufu plaster and the amount of the test sample, accurately weigh camphor, menthol and dried ginger, respectively, and place them in 250ml round-bottom flasks, add 150ml of water, and test according to the volatile oil determination method. Add water from the upper end of the determination device until it fills the scale part and overflows into the flask, then add 2ml of ethyl acetate, connect a reflux condenser, heat to boiling, and keep it boiling slightly for 4 hours, let it cool, take out the ethyl acetate liquid, filter it through a funnel covered with anhydrous sodium sulfate, and place the filtrate in a 25ml volumetric flask. Use ethyl acetate to wash the condenser, volatile oil determination device, and funnel in turn. The washing liquid and the above ethyl acetate liquid are combined into the same volumetric flask, and ethyl acetate is added to dilute to the scale and shake well to obtain the single drug solutions of the three drugs. 5) Characteristic spectrum analysis test: Analyze by gas chromatography: 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); the carrier gas was high-purity helium; the column temperature was programmed: the initial temperature was 50°C, maintained for 2min, 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 increase the temperature at 0.4℃ / min -1 Raise to 87℃, maintain for 60min, and increase the temperature at 1.0℃ / min -1 Raise to 150℃, maintain for 60min, and increase the temperature at 1.3℃ / min -1 Raise to 210℃, maintain for 60min; injection port temperature 230℃; column flow rate 0.8ml / min; split ratio 10:1, theoretical plate number not less than 5000 based on camphor peak; 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; tuning file was standard tuning; 6) Establish characteristic spectrum and determine quantitative detection indicators: Take 15 batches of intermediate products of Shufu plaster, collect sample information according to the above construction method, import the chromatogram data of 15 batches of samples S1-S15 into the Chinese medicine chromatogram similarity evaluation software (2012 version), take the S1 sample spectrum as the reference spectrum, adopt the average method, set the time window to 0.1min, and perform multi-point correction to match the chromatogram peaks, generate the superimposed spectrum of 15 batches of samples and the standard reference characteristic spectrum (R); take the mixed reference solution, the test solution and the single drug solution, and inject and measure according to the chromatogram and qualitative mass spectrometry conditions of the characteristic spectrum analysis test item, record the chromatogram, and use the NIST in Msss Hunter mass spectrometry analysis software 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-pineneol, 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 characteristic spectrum of volatile components of the intermediate product of the Shufu patch according to claim 1, characterized in that: Among the 9 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 the S peak 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 specification values ​​of the characteristic spectrum of the intermediate product of Shufu patch The similarity evaluation software for Chinese medicine chromatographic fingerprint was used (2012 version) to analyze the spectra of 15 batches of samples, and 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 the 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 content determination 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 in 10 mL volumetric flasks, 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 of the reference stock solution 1 and the reference stock solution 2 described in claim 1, and then accurately pipette 1 ml of the internal standard solution of pulegone and 2,4,6-tri-tert-butylphenol, respectively, into 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 into small pieces, remove the cover lining, put into 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 determination device until it fills the scale part and overflows 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 out the ethyl acetate solution, filter it through a funnel covered with anhydrous sodium sulfate, put the filtrate into a 25ml volumetric flask, wash the condenser, volatile oil determination device, and funnel with ethyl acetate in turn, combine the washing liquid and the above ethyl acetate solution into the same volumetric flask, accurately add 1ml each of pulegone and 2,4,6-tri-tert-butylphenol internal standard solution, dilute to the scale with ethyl acetate, shake well, and obtain the internal standard test solution; (4) Preparation of negative sample solution: According to the formula 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 sample of the blank matrix are taken respectively, and the negative samples without camphor, menthol and dried ginger and the negative sample of the blank matrix are operated respectively according to the method under "(3)" to obtain the negative sample solutions with internal standards without camphor, menthol and dried ginger and the negative sample of the blank matrix; (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, maintained for 2 min, 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 increase the temperature at 0.4℃ / min -1 Raise to 87℃, maintain for 60min, and increase the temperature at 1.0℃ / min -1 Raise to 150℃, maintain for 60min, and increase the temperature at 1.3℃ / min -1 Raise to 210℃, maintain for 60min; injection port temperature 230℃; column flow rate 0.8ml / min; split ratio 10:1, theoretical plate number not less than 5000 based on camphor peak; 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; the tuning file was standard tuning, SIM ion monitoring mode, and the SIM detection time and quantitative and qualitative ion settings of each compound to be tested were specifically shown in Table 2; Table 2 Quantitative and qualitative ions of each component compound (6) Content determination: Pulegone was selected as the internal standard of 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 of camphor and menthol. 1 μl of the reference solution and the test solution containing the internal standard were accurately aspirated, and the chromatogram was recorded. The peak areas of the reference and internal standard substances were measured, and the correction factor f was calculated. The correction factors of 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 for content determination of the intermediate product of the Shufu patch according to claim 4, 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 article; 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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