A method for analyzing the characteristic spectral composition of volatile components in a pain-relieving and health-promoting cream and a method for determining the content of its chemical components.

Abstract

This invention provides a method for analyzing the characteristic chromatograms of volatile components in pain-relieving and health-promoting plasters and a method for determining the content of their chemical components. The characteristic chromatograms obtained under gas chromatography-mass spectrometry (GC-MS) conditions can comprehensively reflect the volatile components of the plasters, compensating for the lack of control over volatile components in medicinal materials in current standards. Simultaneously, it enables the quantitative detection of key volatile indicator components in pain-relieving and health-promoting plasters, which is beneficial for improving the quality control level of these plasters. The method employs a multi-indicator quantification approach, saving reference standards and analysis time. This invention creates a rapid, accurate, and low-cost method for quality control and evaluation of pain-relieving and health-promoting plasters. The method simultaneously determines the content of seven indicator components using a multi-indicator quantification approach; that is, only camphor is needed as a reference standard, and the content of the other six components can be calculated using relative correction factors.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine quality testing technology, and in particular to a method for analyzing the characteristic spectral composition of volatile components in a pain-relieving and health-strengthening plaster and a method for determining the content of its chemical components. Background Technology

[0002] The pain-relieving and health-strengthening plaster is a rubber plaster made from seven medicinal herbs: Salvia miltiorrhiza, safflower, chicken blood vine, Clematis chinensis, Angelica pubescens, Sichuan pepper, and gardenia, along with borneol, camphor, and menthol. It is mainly used for neck, shoulder, waist, and leg pain, as well as swelling and pain in the limbs, joints, and muscles caused by falls and sprains.

[0003] As a traditional Chinese medicine rubber plaster, the pain-relieving and health-strengthening plaster contains a variety of Chinese medicinal herbs, among which Sichuan pepper contains volatile oils. The volatile oil monomers are camphor, borneol, and menthol. Modern research has gradually proven that volatile oil components have significant pharmacological activity in anti-inflammatory and analgesic effects, as well as in the treatment of joint and muscle pain. However, their composition is relatively complex, and existing product quality standards do not include quality control for the relevant volatile oils in the medicinal herbs.

[0004] The therapeutic effects of traditional Chinese medicine (TCM) products are the result of the combined action of all chemical substances; that is, the sum of all chemical components forms the material basis for their therapeutic effects. Because the composition of TCM products is quite complex, controlling only a small amount of components in individual or a few medicinal herbs cannot fully reflect the purpose of controlling the overall quality of the TCM and cannot comprehensively reflect the product's quality.

[0005] Characteristic maps of traditional Chinese medicine (TCM) play a crucial role in quality control and pharmacodynamic component research, characterized by their large information content, strong specificity, comprehensiveness, and systematic nature. They are significant for effectively controlling the quality of raw medicinal materials and prepared Chinese medicines, reflecting to a certain extent the chemical basis of TCM, comprehensively illustrating the relative relationships of chemical components, and enabling effective characterization and overall evaluation of the intrinsic quality of TCM. Combined with multi-index quantitative analysis, they can serve as one of the improved methods for TCM quality control.

[0006] Therefore, in order to further explore the mechanism of action of the volatile oil in pain-relieving and health-promoting plaster and promote its application in actual clinical practice, it is necessary to study its chemical composition and determine the content of its effective components. In other words, it is an urgent problem for those skilled in the art to solve the problem of providing a method for establishing a characteristic spectrum of the volatile components of pain-relieving and health-promoting plaster and a method for determining the content. Summary of the Invention

[0007] In view of the above situation and to overcome the defects of the prior art, the present invention provides a method for analyzing the characteristic spectral features of volatile components in pain-relieving and fitness cream and a method for determining the content of its chemical components. This not only makes up for the lack of detection of volatile chemical components in pain-relieving and fitness cream, thereby more comprehensively detecting and evaluating the quality of pain-relieving and fitness cream, but also helps to improve the quality control level of pain-relieving and fitness cream.

[0008] The technical solution includes the following steps:

[0009] The first step was to prepare a reference stock solution. Menthol, camphor, isoborneol, borneol, eucalyptol, limonene, and 4-terpineol were mixed evenly in proportions of 30.0369 mg, 30.0124 mg, 5.1168 mg, 5.0036 mg, 0.3314 mg, 0.3024 mg, and 0.3346 mg per 1 mL, respectively, to prepare the stock solution.

[0010] The second step is to prepare a mixed reference solution. Take each stock solution and dilute it with ethyl acetate to prepare a mixed reference solution with the following concentrations: menthol 6.0074 mg / ml, camphor 6.0025 mg / ml, isoborneol 1.0234 mg / ml, borneol 1.0027 mg / ml, eucalyptol 0.0663 mg / ml, limonene 0.0605 mg / ml, and 4-terpineol 0.0669 mg / ml.

[0011] The third step is to prepare the test solution. Take 140 cm of this product... 2 Cut into small pieces, remove the liner, place in a 250ml round-bottom flask, add 150ml of water, and test according to the method for determination of volatile oil (Chinese Pharmacopoeia 2020 Edition, Part 4, General Chapter 2204).

[0012] Add water to the top of the measuring apparatus until it fills the graduated section and overflows into the flask. Add 2 ml of ethyl acetate, connect the reflux condenser, heat to boiling, and maintain a gentle boil for 4 hours. Cool, separate the ethyl acetate solution, and filter it through a funnel lined with an appropriate amount of anhydrous sodium sulfate. Place the filtrate in a 20 ml volumetric flask, and wash the condenser, volatile oil measuring apparatus, and funnel in sequence with an appropriate amount of ethyl acetate. Combine the washing solution with the above ethyl acetate solution, dilute with ethyl acetate to the mark, shake well, filter, and take 1 μl of the filtrate to obtain the test solution of volatile components of the pain-relieving and health-preserving plaster.

[0013] The fourth step is analysis and testing. Gas chromatography-mass spectrometry (GC-MS) is used for analysis. The mixed reference solution and the test solution from steps two and three are injected into the GC-MS for analysis and determination. The instrument is a 7820A-5977B GC-MS system (Agilent Technologies, USA), a MassHunter workstation, and the NIST 17.0 standard mass spectrometry library.

[0014] Gas chromatography-mass spectrometry (GC-MS) conditions: HP-INNOWAX column (30m × 0.25mm, 0.25μm); carrier gas: high-purity helium; column temperature programmed: initial temperature 50℃, hold for 2 min, increase at 10℃ / min⁻¹ to 76℃, hold for 2.6 min, increase at 1.3℃ / min⁻¹ to 85℃, hold for 20 min, increase at 0.4℃ / min⁻¹ to 87℃, hold for 60 min, increase at 1.2℃ / min⁻¹ to 150℃, hold for 40 min, increase at 1.3℃ / min⁻¹ to 210℃, hold for 60 min; injection port temperature 230℃; column flow rate 1 ml / min; split ratio 6:1; theoretical plate number based on camphor peak not less than 5000.

[0015] The qualitative mass spectrometry conditions were as follows: electron impact (EI) ion source; electron energy 70 eV; ion source temperature: 230℃; interface temperature: 230℃; quadrupole temperature 150℃; mass scan range m / z (30-600) full scan; solute delay (4.6 min); subsequent run at 230℃ for 10 min; tuning file was standard tuning.

[0016] The fifth step is to establish a feature map and determine the quantitative detection index. Take 16 batches of pain relief and fitness cream and obtain the feature map of the pain relief and fitness cream samples according to the above construction method. Then, generate the control fingerprint map by using the median method from the feature map of the 16 batches of pain relief and fitness cream samples.

[0017] The GC-MS standard reference characteristic spectrum of this pain-relieving and health-strengthening ointment consists of 28 common peaks. Compared with the reference standard, peak 9 is limonene, peak 10 is eucalyptol, peak 18 is camphor, peak 23 is 4-terpineol, peak 24 is menthol, peak 25 is isoborneol, and peak 27 is borneol.

[0018] Using camphor as a reference, peak 18 (S) as a reference peak, and the corresponding peak of the reference peak as the S peak, the relative retention time of each characteristic peak and the S peak was calculated. The relative retention time was within ±5% of the specified value, which is shown in Table 1.

[0019] Prepare a test solution from the sample to be tested, and follow the steps of the above construction method to obtain the characteristic spectrum of the sample to be tested. Compare the similarity between the characteristic spectrum of the sample to be tested and the fingerprint spectrum of the pain relief and fitness cream control. The similarity should not be less than 0.90.

[0020] The top seven components with relatively large peak areas and good separation effects in the characteristic spectrum—menthol, camphor, isoborneol, borneol, eucalyptol, limonene, and 4-terpineol—were identified as quantitative detection indicators.

[0021] A method for determining the content of chemical components in a pain-relieving and health-promoting cream, comprising the following steps:

[0022] Step 1: Determine the gas chromatography-mass spectrometry (GC-MS) conditions. The instrument used is a 7820A-5977B GC-MS system (Agilent Technologies, USA), a MassHunter workstation, and the NIST 17.0 standard mass spectrometry database.

[0023] Gas chromatography-mass spectrometry conditions: HP-INNOWAX column (30m × 0.25mm, 0.25μm); carrier gas: high-purity helium; column temperature programmed: initial temperature 50℃, hold for 2 min, then increase at 10℃ / min. -1 Increase to 76℃ and hold for 2.6 minutes, at a rate of 1.3℃ / min. -1 Raise to 85℃ and maintain for 20 minutes, at a rate of 0.4℃ / min. -1 Raise to 87℃ and maintain for 60 minutes, at a rate of 1.2℃ / min. -1 Raise to 150℃ and hold for 40 minutes, at a rate of 1.3℃ / min. -1 Heat to 210℃ and hold for 60 min; injector temperature 230℃; column flow rate 1 ml / min; split ratio 6:1; theoretical plate number based on camphor peak not less than 5000.

[0024] The quantitative mass spectrometry conditions were as follows: electron impact (EI) ion source; electron energy 70 eV; ion source temperature: 230℃; interface temperature: 230℃; quadrupole temperature: 150℃; mass scan range: m / z (30-600) full scan; solute delay (4.6 min); subsequent run at 230℃ for 10 min; the tuning file was standard tuning, SIM ion monitoring mode. Specific SIM detection times and quantitative / qualitative ion settings for each analyte are shown in Table 2.

[0025] Step 2: Content determination. Take 1 μl each of the reference solution and the test solution, and inject them for analysis according to the GC-MS conditions in Step 1. Using camphor as an internal reference, the average relative correction factors for limonene, eucalyptol, 4-terpineol, menthol, isoborneol, and borneol were determined and calculated as follows: 0.6442; 0.0304; 0.0305; 0.6442; 0.4015; and 0.6521.

[0026] Step 3: Using a one-test-multiple-evaluation method, the contents of limonene, eucalyptol, camphor, 4-terpineol, menthol, and borneol (calculated as the sum of borneol and isoborneol contents) in the pain-relieving and health-strengthening cream are calculated through a relative correction factor. Combining the test results of the 16 batches of samples in claim 1 with the influence of production process conditions and storage loss of effective ingredients, a standard is set at 50% of the average content of each component in the 16 products, specifying the content per 100cm³. 2The content standards for the above-mentioned effective ingredients in the pain relief and health-preserving cream are as follows: not less than 0.23 mg of limonene, not less than 0.32 mg of eucalyptol, not less than 31.51 mg of camphor, not less than 0.15 mg of 4-terpineol, not less than 36.37 mg of menthol, and not less than 23.12 mg of borneol (calculated as the sum of borneol and isoborneol content).

[0027] The beneficial effects of this invention are:

[0028] 1. The characteristic chromatograms obtained by the present invention under gas chromatography-mass spectrometry conditions can comprehensively reflect the volatile components of pain relief and health-preserving plaster, making up for the lack of control of volatile components in medicinal materials in the current standards. At the same time, it can realize the quantitative detection of key volatile components in pain relief and health-preserving plaster, which is conducive to improving the quality control level of pain relief and health-preserving plaster.

[0029] 2. This invention employs a one-test-multiple-evaluation method for multi-index quantification, saving reference standards and analysis time. It establishes a rapid, accurate, and low-cost method for quality control and evaluation of pain-relieving and health-enhancing plasters. By simultaneously determining the content of seven indicator components using this method, only camphor is needed as a reference standard; the content of the other six components can be calculated using relative correction factors. Attached Figure Description

[0030] Figure 1 The relative retention time values ​​are specified for the characteristic spectrum of the pain-relieving and health-strengthening ointment in Table 1 of this invention.

[0031] Figure 2 The quantitative and qualitative ions of the compounds in Table 2 of this invention are shown.

[0032] Figure 3 This is a sample spectrum in the experimental specificity of the characteristic spectrum of the present invention.

[0033] Figure 4 The feature map of this invention is missing the pepper map in the experimental specificity of the feature map.

[0034] Figure 5 This is a camphor-deficient spectrum in the experimental specificity of the characteristic spectrum of this invention.

[0035] Figure 6 The menthol spectrum is missing from the experimental specificity of the feature map of this invention.

[0036] Figure 7 This is a borneol-deficient spectrum in the experimental specificity of the feature spectrum of this invention.

[0037] Figure 8 This is to ensure the precision of the present invention.

[0038] Figure 9 Table 3 shows the precision test results (relative retention time of index component groups) of the pain-relieving and health-strengthening ointment (220301) of this invention.

[0039] Figure 10 Table 4 shows the precision test results (relative peak area of ​​index component groups) of the pain relief and fitness cream (220301) of this invention.

[0040] Figure 11 Table 5 shows the similarity calculation results of the precision test spectrum of the pain relief and fitness cream (220301) of this invention.

[0041] Figure 12 This invention is designed for repeatability.

[0042] Figure 13 Table 6 shows the repeatability test results (relative retention time of indicator component groups) of the pain relief and fitness cream (220301) of this invention.

[0043] Figure 14 Table 7 shows the repeatability test results (relative peak area of ​​index component groups) of the pain relief and fitness cream (220301) of this invention.

[0044] Figure 15 Table 8 shows the similarity calculation results of the repeatability test spectrum of the pain relief and fitness cream (220301) of this invention.

[0045] Figure 16 For the stability of this invention.

[0046] Figure 17 Table 9 shows the stability test results (relative retention time of index component groups) of the pain relief and fitness cream (220301) of this invention.

[0047] Figure 18 Table 10 shows the stability test results (relative peak area of ​​index component groups) of the pain relief and fitness cream (220301) of this invention.

[0048] Figure 19 The results of the repeatability test pattern similarity calculation for the pain relief and fitness cream (220301) in Table 11 of this invention are shown.

[0049] Figure 20 This is a superimposed feature map of the pain-relieving and health-strengthening ointment of the present invention and a comparative feature map.

[0050] Figure 21 The similarity calculation results of the feature map of the pain-relieving and fitness cream in Table 12 of this invention are shown.

[0051] Figure 22 This is a mixed control standard for the present invention.

[0052] Figure 23 The relative retention time of the characteristic spectrum of the pain-relieving and fitness cream in Table 13 of this invention.

[0053] Figure 24Table 14 shows the range of relative retention time deviations between the common peaks and the corresponding peaks in the standard spectrum of the pain-relieving and health-strengthening ointment of the present invention in 16 batches.

[0054] Figure 25 The quantitative and qualitative ions of the compounds in Table 15 of this invention are provided.

[0055] Figure 26 Table 16 shows the standard curve and linear range of the ingredients of the pain-relieving and fitness cream of this invention.

[0056] Figure 27 Table 17 shows the precision, repeatability, and stability of the pain-relieving and fitness cream sample components of this invention.

[0057] Figure 28 The results of the sample recovery test are shown in Table 18 of this invention.

[0058] Figure 29 Table 19 shows the effect of different chromatographic instruments and column models on the relative correction factors of each analyte in the pain-relieving and health-preserving ointment.

[0059] Figure 30 Table 20 shows the effect of different GC-MS conditions on the relative correction factors of each component to be tested in the pain relief and fitness cream.

[0060] Figure 31 Table 21 shows the effect of different chromatographic instruments and column models on the relative retention time of each analyte in the pain-relieving and health-strengthening cream.

[0061] Figure 32 Table 22 shows the effect of different GC-MS conditions on the relative retention time of each component in the pain-relieving and health-strengthening ointment.

[0062] Figure 33 The results of the content determination of each component in Table 23 of this invention (mg / 100cm2, n=3) are shown.

[0063] Figure 34 The results of the content determination of each component in Table 24 of this invention (mg / 100cm2, n=3).

[0064] Figure Labels

[0065] 1. Limonene 2. Eucalyptol 3. Camphor 4. 4-Terpinene 5. Menthol 6. Isoboron 7. Borneol. Detailed Implementation

[0066] The following detailed description, in conjunction with the accompanying drawings, provides further illustrative information about specific embodiments of the present invention. All detailed descriptions are exemplary and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0067] When this embodiment is used

[0068] Example 1

[0069] Experimental instruments and materials

[0070] instrument

[0071] 7820A-5977B Gas Chromatography-Mass Spectrometer (Agilent Technologies, USA), MassHunter workstation, NIST 17.0 standard mass spectrometry library; XPE105 0.001 g electronic balance (d = 0.01 mg) (Mettler-Toledo, Switzerland); Volatile oil analyzer with a relative density <1.0 and 24 / 29 standard stopper (Beijing Beibo Bomei Glass Co., Ltd., specification: 5 mL).

[0072] Reagents and reagents

[0073] Camphor reference standard (batch number: 110747-201008, purity: 99.0%, China National Institutes for Food and Drug Control); Menthol reference standard (batch number: 110728-200506, for content determination, China National Institutes for Food and Drug Control); Borneol reference standard (batch number: 110881-201709, China National Institutes for Food and Drug Control, content: 99.6%); Isoborneol reference standard (batch number: 110512-201904, China National Institutes for Food and Drug Control, content: 98.4%); Eucalyptol (batch number: 110788-2021) 08, China National Institutes for Food and Drug Control, purity: 99.4%; 4-terpineol (batch number: TPI-111584, Stanford Chemicals, USA, purity: 97.50%); limonene (batch number: 100470-201503, China National Institutes for Food and Drug Control, purity: 96.0%); purified water (batch number: 202108056214HN, source: Hangzhou Wahaha Group Co., Ltd.); ethyl acetate (batch number: 20220220, source: Tianjin Kemio Chemical Reagent Co., Ltd., analytical grade).

[0074] Pharmaceutical: Pain-relieving and health-strengthening plaster (Henan Lingrui Pharmaceutical Co., Ltd., batch numbers: 220301, 220302, 220303, 220405, 220406, 330406, 220502, 220503, 220504, 220601, 220602, 220603, 220604, 220605, 220606, 220701; product numbers S1~S16)

[0075] Analysis software: 2012 version of the Chromatographic Fingerprint Similarity Evaluation System for Traditional Chinese Medicine (National Pharmacopoeia Commission).

[0076] Experimental conditions

[0077] Chromatographic-mass spectrometry conditions and system suitability testing:

[0078] Gas chromatography conditions: HP-INNOWAX column (30m × 0.25mm, 0.25μm); carrier gas: high-purity helium; column temperature programmed: initial temperature 50℃, hold for 2 min, then increase at 10℃ / min. -1 Increase to 76℃ and hold for 2.6 minutes, at a rate of 1.3℃ / min. -1 Raise to 85℃ and maintain for 20 minutes, at a rate of 0.4℃ / min. -1 Raise to 87℃ and maintain for 60 minutes, at a rate of 1.2℃ / min. -1 Raise to 150℃ and hold for 40 minutes, at a rate of 1.3℃ / min. -1 Heat to 210℃ and hold for 60 min; injector temperature 230℃; column flow rate 1 ml / min; split ratio 6:1; theoretical plate number based on camphor peak not less than 5000.

[0079] The qualitative mass spectrometry conditions were as follows: electron impact (EI) ion source; electron energy 70 eV; ion source temperature: 230℃; interface temperature: 230℃; quadrupole temperature 150℃; mass scan range m / z (30-600) full scan; solute delay (4.6 min); subsequent run at 230℃ for 10 min; and the tuning file was standard tuning.

[0080] Preparation of the reference solution:

[0081] Preparation of reference stock solutions: Accurately weigh appropriate amounts of menthol, camphor, isoborneol, borneol, eucalyptol, limonene, and 4-terpineol reference standards, and add ethyl acetate to prepare a stock solution containing 30.0369 mg, 30.0124 mg, 5.1168 mg, 5.0036 mg, 0.3314 mg, 0.3024 mg, and 0.3346 mg of menthol, camphor, isoborneol, borneol, eucalyptol, limonene, and 4-terpineol per mL, respectively.

[0082] Preparation of mixed reference solution: Take each stock solution and dilute with ethyl acetate to prepare a mixed reference solution with the following concentrations: 6.0074 mg / ml menthol, 6.0025 mg / ml camphor, 1.0234 mg / ml isoborneol, 1.0027 mg / ml borneol, 0.0663 mg / ml eucalyptol, 0.0605 mg / ml limonene, and 0.0669 mg / ml 4-terpineol.

[0083] Preparation of the test solution:

[0084] Take 140cm of this product 2Cut into small pieces, remove the liner, place in a 250ml round-bottom flask, add 150ml of water, and test according to the method for determination of volatile oil (Chinese Pharmacopoeia 2020 Edition, Part 4, General Chapter 2204). Add water from the top of the measuring apparatus until it fills the graduated section and overflows into the flask, then add 2ml of ethyl acetate, connect the reflux condenser, heat to boiling, and maintain a gentle boil for 4 hours. Cool, separate the ethyl acetate solution, filter through a funnel lined with an appropriate amount of anhydrous sodium sulfate, place the filtrate in a 20ml volumetric flask, and wash the condenser, volatile oil measuring apparatus, and funnel sequentially with an appropriate amount of ethyl acetate. Combine the washings with the above ethyl acetate solution, dilute to the mark with ethyl acetate, shake well, filter, and take 1μl of the subsequent filtrate to obtain the test solution of volatile components of the pain-relieving and health-preserving plaster.

[0085] Selection of internal reference materials

[0086] Camphor is chosen as an internal reference because it is the main component of the volatile oil in the pain-relieving and health-preserving plaster, has significant pharmacological activity, is present in high concentrations, is readily available as a reference standard, and is inexpensive. In the chromatogram, it shows good separation from other components.

[0087] Feature mapping methodology examination

[0088] Exclusivity

[0089] Prepare the test solution according to the test solution preparation method, and investigate whether negative samples such as Sichuan pepper, camphor, borneol, and menthol would cause interference. Analyze according to the chromatographic-mass spectrometric conditions described in section "2.1", and record the chromatograms as follows. Figure 3-7 As shown, the results indicate that negative results do not cause interference and the method has good specificity.

[0090] Precision

[0091] Prepare the test solution of the same batch of pain-relieving and health-strengthening cream (batch number 220301) according to section "2.3", and inject it continuously 6 times according to the chromatographic-mass spectrometry conditions under section "2.1" to detect the characteristic chromatogram. Figure 8 The retention time and peak area of ​​each common peak were recorded. Using the retention time and peak area of ​​the camphor chromatographic peak as a reference, the relative retention time and relative peak area of ​​each common peak were calculated. The RSD values ​​of the relative retention time and relative peak area of ​​each common peak were all less than 3%. The chromatographic data obtained from the six tests were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" for full-spectrum peak matching, and the similarity was calculated. 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.

[0092] Repeatability

[0093] Take six samples (batch number 220301) from the same batch of pain-relieving and health-strengthening plaster, and prepare them according to the method described in section "2.3" for the preparation of the test solution. Inject the samples sequentially according to the chromatographic-mass spectrometry conditions described in section "2.1", and detect the characteristic chromatograms of each sample. Figure 12 The retention time and peak area of ​​each common peak were recorded. Using the retention time and peak area of ​​the camphor chromatographic peak as a reference, the relative retention time and relative peak area of ​​each common peak were calculated. The RSD values ​​of the relative retention time and relative peak area of ​​each common peak were all less than 3.0%. The chromatographic data obtained from the six experiments were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" for full-spectrum peak matching, and the similarity was calculated. The similarity results were all ≥0.999, indicating that the experimental method had good repeatability. The results are shown in Tables 6, 7, and 8.

[0094] stability

[0095] Prepare the test solution of the same batch of pain-relieving and health-promoting ointment (batch number 220301) according to section "2.3", and inject it at 0h, 2h, 4h, 8h, 12h, and 24h according to the chromatographic and mass spectrometric conditions under section "2.1", and detect the characteristic chromatograms, such as... Figure 16 The retention times and peak areas of each common peak were recorded. Using the retention time and peak area of ​​the camphor chromatographic peak as a reference, the relative retention times and relative peak areas of each common peak were calculated. The RSD values ​​of the relative retention times and relative peak areas of each common peak were all less than 3.0%. The chromatographic data obtained from the six tests were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" for full-spectrum peak matching, and the similarity was calculated. The similarity results were all ≥0.999, indicating that the test solution was stable within 24 hours. This suggests that the test sample has good stability. The results are shown in Tables 9, 10, and 11.

[0096] Establishment and application of characteristic spectra of pain-relieving and health-strengthening cream

[0097] Establishment and Similarity Evaluation of the Feature Map of Pain-Relieving and Fitness Cream

[0098] The GC-MS characteristic chromatograms of 16 batches of pain-relieving and health-strengthening plasters were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (Version 2012.130723)" in TXT format. The chromatogram of sample S1 was selected as the reference chromatogram, and the time window width was set to 0.1 min. Multi-point calibration and automatic matching were performed on the 16 batches of samples to establish a common fingerprint pattern. The results showed that a total of 28 common peaks were identified in the GC-MS characteristic chromatograms of the 16 batches of pain-relieving and health-strengthening plasters, generating overlay chromatograms. (See figure). Figure 20 Similarity was calculated based on the control fingerprint (R) generated by the median method. The similarity results between the feature maps of 16 batches of pain relief and fitness cream and the median map are shown in Table 12.

[0099] The results showed that the overall peak patterns of the formulations from different batches were basically the same, and 28 common peaks were identified, with a similarity of over 0.9.

[0100] Identification of common peaks in characteristic spectra

[0101] Using GC-MS technology and searching the NIST 17.0 database, the common peaks of the volatile components in the pain-relieving and health-strengthening cream were identified. Combined with comparison with reference standards, peak 9 was determined to be limonene, peak 10 to be eucalyptol, peak 18 to be camphor, peak 23 to be 4-terpineol, peak 24 to be menthol, peak 25 to be isoborneol, and peak 27 to be borneol. The mixed reference standards are shown below. Figure 22 .

[0102] The provisions of the characteristic spectrum standard

[0103] Sixteen batches of pain-relieving and health-strengthening plaster samples were collected, and test solutions were prepared according to the method in section "2.3". 1 μL of each solution was accurately pipetted and injected into the gas chromatography-mass spectrometry (GC-MS) instrument. Analysis was performed under the GC-MS conditions in section "2.1". Based on the relative retention times of each chromatographic peak in the obtained chromatograms, common peaks were identified, and 28 common peaks were selected as characteristic peaks to establish a reference characteristic chromatogram. Peak 18 was used as the reference peak S. The ratio of the retention time of each chromatographic peak to the retention time of peak S in the same chromatogram was calculated. The resulting relative retention times and RSDs are shown in Table 13.

[0104] The relative deviation range between the relative retention times of each characteristic peak of the sample to be tested and the relative retention times of each characteristic peak in the standard characteristic spectrum (see Table 13) is shown in Table 14.

[0105] Based on the robustness test results of the characteristic chromatograms of volatile components, the relative retention times of each characteristic peak in the characteristic chromatograms 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 chromatogram of the pain relief and fitness plaster test sample should have 28 characteristic peaks. The corresponding peak of the reference substance camphor is taken as the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±5% of the specified value. The specified relative retention times for the standard characteristic spectra are: 0.148 (peak 1), 0.154 (peak 2), 0.174 (peak 3), 0.200 (peak 4), 0.209 (peak 5), 0.243 (peak 6), 0.248 (peak 7), 0.261 (peak 8), 0.281 (peak 9), 0.294 (peak 10), 0.318 (peak 11), 0.334 (peak 12), 0.339 (peak 13), and 0.368 (peak 14). 14), 0.384 (peak 15), 0.546 (peak 16), 0.810 (peak 17), 1.000 (peak 18), 1.186 (peak 19), 1.228 (peak 20), 1.255 (peak 21), 1.395 (peak 22), 1.513 (peak 23), 1.883 (peak 24), 2.069 (peak 25), 2.361 (peak 26), 2.443 (peak 27), 2.564 (peak 28). Furthermore, based on the similarity analysis of the feature maps, the similarity between the feature map of the pain-relieving and health-promoting cream sample and the standard feature map should not be less than 0.9.

[0106] Content determination method validation

[0107] The conditions for determining the gas chromatography-mass spectrometry (GC-MS) content are as follows:

[0108] The instrument used was a 7820A-5977B gas chromatograph-mass spectrometer (Agilent Technologies, USA), a MassHunter workstation, and the NIST 17.0 standard mass spectrometry library.

[0109] Gas chromatography-mass spectrometry conditions: HP-INNOWAX column (30m × 0.25mm, 0.25μm); carrier gas: high-purity helium; column temperature programmed: initial temperature 50℃, hold for 2 min, then increase at 10℃ / min. -1 Increase to 76℃ and hold for 2.6 minutes, at a rate of 1.3℃ / min. -1 Raise to 85℃ and maintain for 20 minutes, at a rate of 0.4℃ / min. -1 Raise to 87℃ and maintain for 60 minutes, at a rate of 1.2℃ / min. -1 Raise to 150℃ and hold for 40 minutes, at a rate of 1.3℃ / min. -1 Heat to 210℃ and hold for 60 min; injector temperature 230℃; column flow rate 1 ml / min; split ratio 6:1; theoretical plate number based on camphor peak not less than 5000.

[0110] The quantitative mass spectrometry conditions were as follows: electron impact (EI) ion source; electron energy 70 eV; ion source temperature: 230℃; interface temperature: 230℃; quadrupole temperature 150℃; mass scan range m / z (30-600) full scan; solute delay (4.6 min); subsequent run at 230℃ for 10 min; the tuning file was standard tuning, SIM ion monitoring mode, and the specific SIM detection time and quantitative and qualitative ion settings for each analyte are shown in Table 15.

[0111] The GC-MS conditions for determining the volatile component content in the pain-relieving and health-strengthening ointment were the same as those for the characteristic chromatogram. The top seven components with relatively large peak areas and good separation in the characteristic chromatogram—menthol, camphor, isoborneol, borneol, eucalyptol, limonene, and 4-terpineol—were selected for content determination.

[0112] Linear and range examination

[0113] Accurately pipette 0.3, 1, 2, 2.5, and 3 mL of the reference stock solution from section "2.2.1" into 10 mL volumetric flasks, add ethyl acetate to the mark, shake well, and inject according to the GC-MS conditions from section "5.1". Record the chromatogram, and perform regression analysis with the reference concentration as the abscissa (X) and the peak area as the ordinate (Y) to construct a standard curve, obtaining the linear regression equation and analytical range. The limits of detection (LOD) and quantitation (LOQ) of each component were determined based on the concentrations at peak signal-to-noise ratios of approximately 3:1 and 10:1. The regression equations, linear ranges, LODs, and LOQs of menthol, camphor, isoborneol, borneol, eucalyptol, limonene, and 4-terpineol are shown in Table 16. The results indicate that each component exhibits good linearity within its respective range.

[0114] Sample injection precision, repeatability and stability tests

[0115] Injection precision experiment: Accurately pipette 1 μL of the reference solution under section “2.2.2”, inject it continuously 6 times according to the GC-MS conditions under section “5.1”, measure the peak area, and calculate the RSD value of the peak area of ​​each component.

[0116] Repeatability test: Take six parallel prepared test sample (S1) solutions, prepare six test sample solutions in parallel according to the method in section "2.3", inject and determine according to the conditions in section "5.1", and calculate the RSD value of each component content.

[0117] Sample stability test: Take the same sample (S1) solution, prepare the sample solution according to the method in section "2.3", and inject and measure it at 0, 2, 4, 8, 12, and 24 h according to the conditions in section "5.1". Calculate the RSD value of the peak area of ​​each component.

[0118] The results of the injection precision, repeatability, and stability tests are shown in Table 17. The RSD of precision, repeatability, and stability are all less than 5%, which meets the requirements of the Chinese Pharmacopoeia. This indicates that the method has good precision and repeatability, and the test solution is stable within 24 hours.

[0119] Recovery test

[0120] Take 70 cm of the same batch of samples (S1) with known content. 2 Nine samples were prepared and divided into three groups. The ratio of the amount of reference standard added to the amount of the analyte in the test sample was controlled to be approximately 1:1.2, 1:1.0, and 1:0.8, respectively. Three concentration levels were set: high, medium, and low. Three test sample solutions were prepared in parallel for each concentration according to the test sample solution preparation method. 1 μl of each of the reference standard solution and the test sample solution were accurately pipetted and injected into the gas chromatograph. The chromatograms were recorded, and the content was determined according to the content determination method. The recovery rate was calculated using the following formula. The results are shown in Table 18.

[0121]

[0122] The average recovery rate and RSD of each component met the requirements, proving that the optimized method is accurate and reliable and can be used to determine the content of seven volatile components in pain relief and fitness cream.

[0123] Relative correction factor calculation

[0124] Take 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mL of the reference solution under section "2.2.2" respectively, place them in 2.0 mL volumetric flasks, add ethyl acetate to the mark, shake well, and prepare a series of mixed reference solutions of different concentrations. Analyze according to the GC-MS conditions under section "5.1", inject 1 μL of each solution, and record the peak areas of each component at different concentrations. Using camphor as an internal reference, determine and calculate the average relative correction factor for limonene (0.6442), eucalyptol (0.0304), 4-terpineol (0.0305), menthol (0.6442), isoborneol (0.4015), and borneol (0.6521) in the samples.

[0125] The formula for calculating the relative correction factor is as follows:

[0126] f = C A / A A :C B / A B

[0127] The formula uses a reference standard for calculation, C A : Concentration of analyte A reference standard; A A : Peak area of ​​analyte A (reference standard); C B Concentration of internal reference standard B; A B : Peak area of ​​internal reference standard B; relative correction factor f is the average value of the data obtained from each experiment.

[0128] Durability test

[0129] The Influence of Different Instruments and Columns on Correction Factors

[0130] Take the mixed standard solution and inject it according to the GC-MS conditions in section "5.1" for analysis, and calculate the relative correction factor. Investigate the effects of different chromatographs, column models, flow rates, injection port temperatures, and GC-MS interface temperatures on the relative correction factors of the six components. The results are shown in Tables 19 and 20. The results show that there are no significant differences in the effects of different chromatographs, column models, flow rates, injection port temperatures, and GC-MS interface temperatures on the relative correction factors of each component (RSDs are all less than 3%).

[0131] Chromatographic peak localization

[0132] Take the mixed standard solution and inject it according to the GC-MS conditions in section "5.1" for determination. Calculate the relative retention times of other volatile components with camphor as the internal reference. Investigate the effects of different chromatographs, column models, flow rates, injection port temperatures, and GC-MS interface temperatures on the relative retention times of the analytes. The results are shown in Tables 21 and 22. The results show that there are no significant differences in the relative retention times of each component due to different chromatographs, column models, flow rates, injection port temperatures, and GC-MS interface temperatures (RSDs are all less than 3%).

[0133] Content determination results and application examples

[0134] Comparison of results from the one-test-multiple-evaluation method and the external standard method

[0135] Sixteen batches of pain-relieving and health-promoting plaster samples were collected. Three test solutions were prepared for each batch according to the method described in section "2.3". The solutions were then analyzed under the GC-MS conditions described in section "5.1". The contents of menthol, camphor, isoborneol, borneol, eucalyptol, limonene, and 4-terpineol in the plaster were calculated using both the external standard method and the multiple-samples method. The results are shown in Tables 23 and 24. A paired-samples t-test was performed on the content results obtained by the external standard method and the multiple-samples method. The results showed that P > 0.05, indicating no significant difference between the two methods. This demonstrates that the multiple-samples method can be used to determine the content of the above-mentioned components in the pain-relieving and health-promoting plaster.

[0136] 6.2 Content Value Specifications

[0137] The content of limonene, eucalyptol, camphor, 4-terpineol, menthol, and borneol (calculated as the sum of borneol and isoborneol content) in the pain-relieving and health-strengthening cream was calculated using a single-test, multiple-evaluation method with relative correction factors. Based on the test results of 16 batches of samples and the loss of effective ingredients during production and storage, a provisional standard of 50% of the average content of each component in the 16 products was set, specifying the content per 100cm³. 2 The content standards for the above-mentioned effective ingredients in the pain-relieving and health-strengthening cream are as follows:

[0138] It contains not less than 0.23 mg of limonene, not less than 0.32 mg of eucalyptol, not less than 31.51 mg of camphor, not less than 0.15 mg of 4-terpineol, not less than 36.37 mg of menthol, and not less than 23.12 mg of borneol (calculated as the sum of borneol and isoborneol content).

[0139] This invention establishes a method for constructing a characteristic spectrum of volatile components in a pain-relieving and health-promoting plaster and a method for determining its content. In the process of constructing the characteristic spectrum, camphor is used as an internal reference. Through methodological research and the determination of 16 batches of samples, a characteristic spectrum of volatile components in the pain-relieving and health-promoting plaster is constructed, which fully reflects the characteristics of volatile components in the pain-relieving and health-promoting plaster. The volatile characteristic spectrum is viewed as a whole, which avoids the one-sidedness of judging the overall quality of the product by only measuring a small number of components and reduces the possibility of human interference with a certain indicator.

[0140] Based on the qualitative analysis of characteristic chromatograms and combined with existing characteristic chromatograms, a method for simultaneously determining the content of seven index components in pain-relieving and health-promoting plasters using a single-measurement-multiple-evaluation approach was established. Camphor was used as an internal reference, and the relative correction factors of menthol, isoborneol, borneol, eucalyptol, limonene, and 4-terpineol relative to camphor were calculated. The robustness of the relative correction factors for six components was investigated under different chromatographic systems, columns, injection temperatures, and flow rates. The results showed that the method had good robustness. The content of each component in 16 batches of pain-relieving and health-promoting plaster samples was calculated using the single-measurement-multiple-evaluation approach, and the results were compared with those obtained using the external standard method to verify the accuracy and feasibility of the single-measurement-multiple-evaluation approach. The results showed no significant difference between the calculated values ​​of the single-measurement-multiple-evaluation approach and the measured values ​​of the external standard method. The relative correction factor established in this invention is accurate and reliable. The method of one test and multiple evaluation using camphor as an internal reference effectively solves the difficulties of the shortage of reference standards for some components and the high detection cost in the simultaneous determination of multiple index components of pain relief and fitness cream, greatly reducing the detection cost. It provides a new method for the quality control and evaluation of pain relief and fitness cream. This method is simple, accurate and easy to promote.

Claims

1. A method for analyzing the characteristic spectral composition of a pain-relieving and health-enhancing plaster, characterized in that, Includes the following steps: The first step was to prepare a reference stock solution. Menthol, camphor, isoborneol, borneol, eucalyptol, limonene, and 4-terpineol were mixed evenly in proportions of 30.0369 mg, 30.0124 mg, 5.1168 mg, 5.0036 mg, 0.3314 mg, 0.3024 mg, and 0.3346 mg per 1 mL, respectively, to prepare the stock solution. The second step is to prepare a mixed reference solution. Take each stock solution and dilute it with ethyl acetate to prepare a mixed reference solution with the following concentrations: menthol 6.0074 mg / ml, camphor 6.0025 mg / ml, isoborneol 1.0234 mg / ml, borneol 1.0027 mg / ml, eucalyptol 0.0663 mg / ml, limonene 0.0605 mg / ml, and 4-terpineol 0.0669 mg / ml. The third step is to prepare the test solution. Take 140 cm² of this product, cut it into small pieces, remove the cap, place it in a 250 ml round-bottom flask, add 150 ml of water, and test according to the volatile oil determination method. Add water from the top of the measuring apparatus until it fills the graduated part and overflows into the flask. Then add 2 ml of ethyl acetate, connect the reflux condenser, heat to boiling, and maintain a gentle boil for 4 hours. Cool, separate the ethyl acetate solution, filter it through a funnel lined with an appropriate amount of anhydrous sodium sulfate, place the filtrate in a 20 ml volumetric flask, and wash the condenser, volatile oil measuring apparatus, and funnel with an appropriate amount of ethyl acetate in sequence. Combine the washing solution with the above ethyl acetate solution, dilute with ethyl acetate to the mark, shake well, filter, and take 1 μl of the filtrate to obtain the test solution of volatile components of the pain-relieving and health-preserving plaster. The fourth step is analysis and testing. Gas chromatography-mass spectrometry (GC-MS) is used. The mixed reference solution and test solution from steps two and three are injected into the GC-MS system for analysis. The instrument is a 7820A-5977B GC-MS system with a MassHunter workstation and NIST 17.0 standard mass spectrometry library. The GC-MS conditions are: HP-INNOWAX column, 30 m × 0.25 mm, 0.25 μm; carrier gas is high-purity helium; the column temperature is programmed as follows: initial temperature 50℃, hold for 2 min, increase at 10℃ / min⁻¹ to 76℃, hold for 2.6 min, increase at 1.3℃ / min⁻¹ to 85℃, hold for 20 min, increase at 0.4℃ / min⁻¹ to 87℃, hold for 60 min, increase at 1.2℃ / min⁻¹ to 150℃, hold for 40 min, and increase at 1.3℃ / min⁻¹ to 210℃, hold for 60 min. min; Injector temperature 230℃; Column flow rate 1ml / min; Split ratio 6:1; Theoretical plate number based on camphor peak not less than 5000; Quadrature mass spectrometry conditions: Electron impact ion source; Electron energy 70 eV; Ion source temperature: 230℃; Interface temperature: 230℃; Quadrupole temperature 150℃; Mass scan range 30-600m / z full scan; Solute delay: 4.6min; Follow-up run at 230℃ for 10min; Tuning file is standard tuning; The fifth step is to establish a feature spectrum and determine quantitative detection indicators. Sixteen batches of pain-relieving and health-strengthening ointments were analyzed using the above method to obtain the feature spectrum of the pain-relieving and health-strengthening ointment samples. The feature spectrum of these 16 batches of pain-relieving and health-strengthening ointment samples was then used to generate a control fingerprint spectrum using the median method.

2. The method for analyzing the characteristic spectral composition of analgesic and health-promoting plaster according to claim 1, characterized in that, The GC-MS standard reference characteristic spectrum of the pain-relieving and health-strengthening ointment consists of 28 common peaks. Compared with the reference standard, peak 9 is limonene, peak 10 is eucalyptol, peak 18 is camphor, peak 23 is 4-terpineol, peak 24 is menthol, peak 25 is isoborneol, and peak 27 is borneol. Using camphor as a reference, peak 18 (S) as a reference peak, and the corresponding peak of the reference peak as the S peak, the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within ±5% of the specified value. Prepare a test solution from the sample to be tested, and follow the steps of the above analytical method to obtain the characteristic spectrum of the sample to be tested. Compare the similarity of the characteristic spectrum of the sample to be tested with the fingerprint spectrum of the pain relief and fitness cream control. The similarity should not be less than 0.

90. The top seven components with relatively large peak areas and good separation effects in the characteristic spectrum—menthol, camphor, isoborneol, borneol, eucalyptol, limonene, and 4-terpineol—were identified as quantitative detection indicators.

3. A method for determining the content of chemical components in a pain-relieving and health-promoting cream, characterized in that, Includes the following steps: Step 1: Determine the gas chromatography-mass spectrometry (GC-MS) conditions. The instrument used is a 7820A-5977B GC-MS, a MassHunter workstation, and the NIST 17.0 standard mass spectrometry library. Gas chromatography-mass spectrometry (GC-MS) conditions were as follows: HP-INNOWAX column, 30 m × 0.25 mm, 0.25 μm; carrier gas: high-purity helium; column temperature programmed: initial temperature 50℃, hold for 2 min, then increase at 10℃ / min. -1 Increase to 76℃ and hold for 2.6 minutes, at a rate of 1.3℃ / min. -1 Raise to 85℃ and maintain for 20 minutes, at a rate of 0.4℃ / min. -1 Raise to 87℃ and hold for 60 minutes at a rate of 1.2℃ / min. -1 Raise to 150℃ and hold for 40 minutes, at a rate of 1.3℃ / min. -1 Heat to 210℃ and hold for 60 min; injector temperature 230℃; column flow rate 1 ml / min; split ratio 6:1; theoretical plate number based on camphor peak not less than 5000. The quantitative mass spectrometry conditions were as follows: electron impact ion source; electron energy 70 eV; ion source temperature: 230℃; interface temperature: 230℃; quadrupole temperature 150℃; mass scan range 30-600 m / z full scan; solute delay: 4.6 min; followed by a 10 min hold at 230℃; the tuning file was standard tuning, SIM ion monitoring mode. Step 2: Prepare the test solution. Take 140 cm of this product. 2 Cut into small pieces, remove the cap liner, place in a 250ml round-bottom flask, add 150ml of water, and test according to the volatile oil determination method; add water from the top of the measuring apparatus until it fills the graduated part and overflows into the flask, then add 2ml of ethyl acetate, connect the reflux condenser, heat to boiling, and maintain a gentle boil for 4 hours, cool, separate the ethyl acetate solution, filter through a funnel lined with an appropriate amount of anhydrous sodium sulfate, place the filtrate in a 20ml volumetric flask, and wash the condenser, volatile oil measuring apparatus, and funnel in sequence with an appropriate amount of ethyl acetate. Combine the washing solution with the above ethyl acetate solution, dilute with ethyl acetate to the mark, shake well, filter, and take 1μl of the filtrate to obtain the test solution of volatile components of the pain-relieving and health-preserving plaster; Step 3: Perform content determination. Take 1 μl of the reference solution and the test solution respectively, and inject them for analysis according to the GC-MS conditions in Step 1. Using camphor as an internal reference, use relative correction factors to determine and calculate the average relative correction factor of limonene in the sample as 0.6442, eucalyptol as 0.0304, 4-terpineol as 0.0305, menthol as 0.6442, isoborneol as 0.4015, and borneol as 0.6521. Step four: Using a one-test-multiple-evaluation method, the content of limonene, eucalyptol, camphor, 4-terpineol, menthol, and borneol (calculated as the sum of borneol and isoborneol content) in the pain-relieving and health-strengthening cream is calculated using a relative correction factor. Combining the test results of the 16 batches of samples in claim 1 with the influence of production process conditions and storage loss of effective ingredients, a standard is set at 50% of the average content of each component in the 16 products, specifying the content per 100cm³. 2 The content standards for the above-mentioned effective ingredients in the pain relief and health-preserving cream are as follows: not less than 0.23 mg of limonene, not less than 0.32 mg of eucalyptol, not less than 31.51 mg of camphor, not less than 0.15 mg of 4-terpineol, not less than 36.37 mg of menthol, and not less than 23.12 mg of borneol (calculated as the sum of borneol and isoborneol).

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