A method for identifying components and determining content of Yali Shuang Kouhan drop pills based on gas chromatography technology

Abstract

This invention relates to the field of traditional Chinese medicine detection technology, specifically a method for identifying and determining the content of components in Yanli Shuang oral drops based on gas chromatography. This application uses gas chromatography to determine the content of β-pinene, limonene, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, β-caryophyllene, menthol, borneol, and caryophyllein in Yanli Shuang oral drops. Specific parameters are as follows: chromatographic column: InertCap-Wax (30m × 0.32mm × 0.5μm), injection port temperature: 220℃, pressure: 100kPa; injection volume: 2μL, splitless injection; carrier gas: N2; FID detector: detector temperature: 230℃. This method is simple, convenient to operate, requires no complex pretreatment, can identify a large number of components in Yanli Shuang oral drops, has low detection cost, and better meets the economical and practical needs of routine testing in enterprise laboratories.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine detection technology, specifically a method for identifying and determining the content of components in Yanli Shuang oral drops based on gas chromatography. Background Technology

[0002] Yanlishuang oral drops are a commonly used Miao medicine compound preparation with the effects of dispelling wind and heat, reducing swelling and relieving pain, and clearing the throat. Clinically, it is often used to treat acute / chronic pharyngitis, sore throat, redness and swelling of the pharyngeal mucosa, dry throat, halitosis, etc. The quality control of traditional Chinese medicine relies on the overall characterization of the synergistic effects of multiple components. Yanlishuang oral drops are a Miao medicine compound preparation composed of Artemisia argyi, Artemisia argyi oil, peppermint oil, menthol, and glycyrrhizic acid monoammonium salt. It has many components, but traditional quality control methods are mostly limited to the analysis of single indicator components, making it difficult to comprehensively reflect the group characteristics of volatile active ingredients in the preparation. Patent CN116124922B uses GC-MS to determine the content of four components: camphor, menthone, borneol, and menthol. However, this method still has significant limitations in the industrial application of quality control for traditional Chinese medicine preparations: 1. It only establishes detection standards for four components, making it impossible to evaluate the overall quality of the preparation through the synergistic effect of multiple components, resulting in insufficient quality control coverage. 2. The instrument operating cost is high, and the operation is highly specialized, restricting industrial application. 3. High-throughput detection has poor adaptability. The advantages of GC-MS technology (such as trace detection and identification of unknown substances) are fundamentally mismatched with the core requirements of industrial quality control (high throughput, low cost, and easy replication).

[0003] Gas chromatography (GC) has become the preferred technology for enterprise quality control due to its economy, high efficiency and adaptability.

[0004] Therefore, the development of a gas chromatography method for identifying the components of Yanlishuang oral drops is a key research focus. This involves optimizing chromatographic conditions (column temperature program, carrier gas flow rate), characteristic peak assignment, and common pattern recognition strategies to achieve the simultaneous separation and identification of 12 characteristic volatile components (including menthol, eucalyptol, and α-pinene) in the formulation. Summary of the Invention

[0005] To address the aforementioned technical problems in the existing technology, this invention provides a method for identifying and determining the content of components in Yanli Shuang oral drops based on gas chromatography, as detailed below:

[0006] A method for identifying and determining the components and content of Yanli Shuang oral drops based on gas chromatography is disclosed. The method uses gas chromatography to determine the content of β-pinene, limonene, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, β-caryophyllene, menthol, borneol, and caryophyllein in Yanli Shuang oral drops. Specific parameters are as follows:

[0007] Chromatographic conditions: Column: InertCap-Wax (30m×0.32mm×0.5μm), injection port temperature: 220℃, pressure: 100kPa; injection volume: 2μL, splitless injection; carrier gas: N2; FID detector, detector temperature: 230℃.

[0008] Furthermore, the specific temperature program for the gas chromatography method is as follows:

[0009]

[0010] Furthermore, the gas chromatography method uses an injection volume of 2 μL and employs splitless injection.

[0011] Furthermore, the detector temperature is 230°C.

[0012] Furthermore, the test solution is a 20 mg / mL solution obtained by dissolving the throat lozenges in ethyl acetate. The test solution is specifically prepared by the following method:

[0013] Take 60 pills of Yanli Shuang oral drops, grind them into a fine powder, accurately weigh 0.2g, place them in a 10mL volumetric flask, add ethyl acetate, sonicate for 10min, and make up to volume to obtain a 20mg / mL test solution.

[0014] Furthermore, the reference solution is prepared using a reference stock solution containing β-pinene, limonene, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, β-caryophyllene, menthol, borneol, and caryophyllein. The reference stock solution is prepared by the following method:

[0015] Accurately weigh appropriate amounts of β-pinene, limonene, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, β-caryophyllene, menthol, borneol, and caryophyllein, place them in a 10 mL volumetric flask, add ethyl acetate, sonicate for 10 min to completely dissolve, and dilute to volume to obtain the reference stock solution.

[0016] Compared with the prior art, the technical effects of this invention are reflected in:

[0017] The method established in this application can effectively desorb volatile components embedded in the droplet matrix. Compared with the current method of using gas chromatography-mass spectrometry to detect multiple volatile components in compound preparations, this method is simple, convenient to operate, does not require complex pretreatment, can identify more components in the throat lozenge droplets, has low detection cost, and is more in line with the economical and practical needs of routine testing in enterprise laboratories. In addition, the 12 successfully identified components include: β-pinene, limonene, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, β-caryophyllene, menthol, borneol, and caryophyllein. Among them, menthol and borneol can jointly inhibit the NF-κB and STAT3 pathways, reducing the release of inflammatory factors; isomenthone and menthone relieve smooth muscle spasm through the TRPV1 channel, reducing the foreign body sensation in the throat; terpenes such as limonene and β-pinene destroy the cell membrane of pathogenic microorganisms; glycyrrhizic acid can regulate the body's immune response; and the transdermal binding of menthol and borneol can increase drug utilization. Terpenoids such as β-pinene and limonene, through their anti-inflammatory, antibacterial, and neuromodulatory effects, synergize with the core components of Yanlishuang oral drops, such as menthol and borneol, to jointly achieve the therapeutic goals of Yanlishuang oral drops in dispelling wind and heat, reducing swelling and relieving pain. This further explains the multi-target effect advantage of this compound preparation in treating pharyngitis and provides a scientific basis for the active substances of Yanlishuang oral drops in treating pharyngitis. Attached Figure Description

[0018] Figure 1 This is a GC chromatogram of a mixed reference standard, including: 1. β-pinene, 2. limonene, 3. octanol, 4. menthone, 5. isomenthone, 6. camphor, 7. menthyl acetate, 8. β-caryophyllene, 9. menthone, 10. menthol, 11. borneol, 12. caryophyllein.

[0019] Figure 2 This is the GC chromatogram of the test solution, including: 1. β-pinene, 2. limonene, 3. 3-octanol, 4. menthone, 5. isomenthone, 6. camphor, 7. menthyl acetate, 8. β-caryophyllene, 9. menthone, 10. menthol, 11. borneol, 12. caryophyllein; A: ordinate 3500mV, B: ordinate 440mV. Detailed Implementation

[0020] The technical solution of the present invention will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description made.

[0021] Example:

[0022] 1. Instruments and Materials

[0023] 1.1 Instruments

[0024] Multi-tube vortex oscillator (Typ.VX-III, Beijing Tajin Technology Co., Ltd.), electronic balance (EL204, Mettler Toledo Instruments Shanghai Co., Ltd.), ultrasonic cleaner (CQ250A-TS, Shanghai Yuejin Medical Optical Instrument Factory). Agilent gas chromatograph (GC-7890B).

[0025] 1.2 Materials

[0026] YLS (Yanlishuang) oral drops were supplied by Guizhou Huangguoshushu Shuang Pharmaceutical Co., Ltd. β-pinene (batch number: WP24022005, purity 98%), limonene (batch number: WP24012311, purity 98%), 3-octanol (batch number: WP24112209, purity 98%), isomenthone (batch number: WP24091304, purity 98%), β-caryophyllene (batch number: WP23110105, purity 90%), and menthol (batch number: WP24061004, purity 98%) were all purchased from Sichuan Weikeqi Biotechnology Co., Ltd.; menthone (batch number: 111705) was also supplied. -202106, purity 99%), menthyl acetate (batch number: 190044-202102, purity 98%), and menthol (batch number: 110728-202208, purity 99.6%) were all purchased from the China National Institutes for Food and Drug Control; camphor (batch number: PS020363, purity 99.30%) was purchased from Chengdu Pusi Biotechnology Co., Ltd.; borneol (batch number: MUST-22021501, purity 98%) was purchased from Chengdu Mansite Biotechnology Co., Ltd.; and caryophyllein (batch number: AB2637-0005, purity 98%) was purchased from Chengdu Efa Biotechnology Co., Ltd.

[0027] 2. Methods and Results

[0028] 2.1 Preparation of the test solution

[0029] Take 60 pills of Yanli Shuang oral drops, grind them into a fine powder, accurately weigh 0.2g, place them in a 10mL volumetric flask, add an appropriate amount of ethyl acetate, sonicate for 10min, and make up to volume to obtain a 20mg / mL test solution.

[0030] 2.2 Preparation of reference solution

[0031] Accurately weigh appropriate amounts of β-pinene, limonene, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, β-caryophyllene, menthol, borneol, and caryophyllin, place them in a 10 mL volumetric flask, add an appropriate amount of ethyl acetate, sonicate for 10 min to completely dissolve, and dilute to volume to obtain the reference stock solution.

[0032] 2.3 Gas Chromatography Conditions

[0033] Chromatographic column: InertCap-Wax (30m×0.32mm×0.5μm), injection port temperature: 220℃, pressure: 100kPa; injection volume: 2μL, splitless injection; carrier gas: N2; FID detector, detector temperature: 230℃.

[0034] Table 1. Column Oven Temperature Program

[0035]

[0036] 2.4 Methodological Examination

[0037] 2.4.1 Specificity

[0038] Take the blank solvent, mixed reference solution, and test solution, and analyze them according to the chromatographic conditions under section "2.3". The results are shown in [Figure 1]. Figure 1-2 As shown, the 12 components were well separated, indicating that the method has good specificity.

[0039] 2.4.2 Examination of Linear Relationships

[0040] Take the mixed reference solution from section "2.2" and inject it according to the chromatographic conditions in section "2.3" for determination. Perform linear regression of the peak area (y) of the analyte against its mass concentration (x). The results show that 12 components, including β-pinene, exhibit good linearity within their respective concentration ranges (R0). 2 (≥0.998), the results are shown in Table 4.

[0041] Table 4. Linear equations for the 12 components

[0042]

[0043] 2.4.3 Precision Test

[0044] Take the mixed reference solution under section "2.2" and inject it according to the chromatographic conditions under section "2.3". Inject it 6 times consecutively. The calculated peak area RSD of 12 components, including β-pinene, is between 0.68% and 2.91%, which indicates that the instrument has good precision. The results are shown in Table 5.

[0045] Table 5 Precision test results

[0046]

[0047] 2.4.4 Repeatability Test

[0048] The RSD of the peak areas of 12 components, including β-pinene, in the six parallel samples prepared according to section “2.1” ranged from 0.69% to 2.80%, indicating that the method has good repeatability. The results are shown in Table 6.

[0049] Table 6 Results of Repeatability Testing

[0050]

[0051] 2.4.5 Stability Test

[0052] The test solution was prepared according to section “2.1”, and the sample was injected at 0h, 2h, 4h, 8h, 12h and 24h. The RSD of the peak areas of the four components, including β-pinene, was 0.83% to 2.27%, indicating that the method has good stability within 24h. The results are shown in Table 7.

[0053] Table 7. Results of Stability Study

[0054]

[0055]

[0056] 2.4.6 Recovery Test

[0057] Take about 0.1g of Yanli Shuang oral drops, add the mixed reference solution at a 1:1 ratio to each sample, for a total of 6 samples, and analyze them according to the chromatographic conditions in section "2.3". The results are shown in Table 8. The average recovery rate of 12 components, including β-pinene, was 97.63% to 103.01%, and the RSD was 1.71% to 2.91%, indicating that the recovery rate of this method is good. The results are shown in Table 8.

[0058] Table 8. Recovery results of 12 components (n=6)

[0059]

[0060]

[0061]

[0062] 2.4.7 Content Determination

[0063] Three batches of Litsea cubeba were used to prepare test solutions according to section "2.1", and the chromatographic conditions in section "2.3" were used for determination. The results showed that the contents of β-pinene, limonene, 3-octanol, menthone, isomenthone, camphor, menthyl acetate, β-caryophyllene, menthol, borneol, and caryophyllein in the three batches of Yanli Shuang oral drops ranged from 0.3234 to 0.4150 mg / g, 0.8022 to 1.120 mg / g, 0.4178 to 0.4203 mg / g, and 1.833 to 2.296 mg / g, respectively. g / g, 14.56~17.21mg / g, 5.348~6.888mg / g, 2.103~2.666mg / g, 0.6896~0.8528mg / g , 0.7156~0.9310mg / g, 39.97~41.62mg / g, 92.66~95.23mg / g, 0.7926~0.8991mg / g.

[0064] 3 Discussion

[0065] The method established in this paper can effectively desorb volatile components embedded in the matrix of throat lozenges. Compared with the current method of using gas chromatography-mass spectrometry to detect multiple volatile components in compound preparations, this method is simple, convenient to operate, does not require complex pretreatment, can identify more components in throat lozenges, has low detection cost, and is more in line with the economical and practical needs of routine testing in enterprise laboratories. In addition, the 12 successfully identified components include: β-pinene, limonene, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, β-caryophyllene, menthol, borneol, and caryophyllein. Among them, menthol and borneol can jointly inhibit the NF-κB and STAT3 pathways, reducing the release of inflammatory factors; isomenthone and menthone relieve smooth muscle spasm and reduce the foreign body sensation in the throat through the TRPV1 channel; terpenes such as limonene and β-pinene destroy the cell membrane of pathogenic microorganisms; glycyrrhizic acid can regulate the body's immune response; and the transdermal binding of menthol and borneol can increase drug utilization. Terpenoids such as β-pinene and limonene, through their anti-inflammatory, antibacterial, and neuromodulatory effects, synergize with the core components of Yanlishuang oral drops, such as menthol and borneol, to jointly achieve the therapeutic goals of Yanlishuang oral drops in dispelling wind and heat, reducing swelling and relieving pain. This further explains the multi-target effect advantage of this compound preparation in treating pharyngitis and provides a scientific basis for the active substances of Yanlishuang oral drops in treating pharyngitis.

[0066] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. A method for identifying and determining the content of ingredients in a throat lozenge, characterized in that, The contents of β-pinene, limonene, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, β-caryophyllene, menthol, borneol, and caryophyllin in Yanli Shuang oral drops were determined by gas chromatography. The specific parameters are as follows: Chromatographic conditions: Column: InertCap-Wax, 30 m × 0.32 mm × 0.5 μm; Injector temperature: 220℃; Pressure: 100 kPa; Injection volume: 2 μL, splitless injection; Carrier gas: N2; FID detector, detector temperature: 230℃. The specific temperature program for the gas chromatography method is as follows: The test solution was obtained by dissolving the throat lozenges in ethyl acetate to obtain a solution with a concentration of 20 mg / mL.

2. The method for identifying and determining the content of the ingredients in the throat-soothing oral drops according to claim 1, characterized in that, The test solution is prepared by the following method: Take 60 pills of Yanli Shuang oral drops, grind them into a fine powder, accurately weigh 0.2g, place them in a 10mL volumetric flask, add ethyl acetate, sonicate for 10min, and make up to volume to obtain a 20mg / mL test solution.

3. The method for identifying and determining the content of the ingredients in the throat-soothing oral drops according to claim 1, characterized in that, The reference solution was prepared using a reference stock solution containing β-pinene, limonene, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, β-caryophyllene, menthol, menthol, borneol, and caryophyllein.

4. The method for identifying and determining the content of the ingredients in the throat-soothing oral drops according to claim 3, characterized in that, The reference standard stock solution was prepared by the following method: Accurately weigh appropriate amounts of β-pinene, limonene, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, β-caryophyllene, menthol, borneol, and caryophyllein, place them in a 10 mL volumetric flask, add ethyl acetate, sonicate for 10 min to completely dissolve, and dilute to volume to obtain the reference stock solution.

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