Ingredient identification and content determination method for instant-swallowing and refreshing dropping pill based on gas chromatography technology

Abstract

The invention relates to the technical field of traditional Chinese medicine detection, in particular to a method for identifying components and measuring the content of a tasty and refreshing dropping pill based on a gas chromatography technology. The content of beta-pinene, limonene, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, beta-caryophyllene, pulegone, menthol, borneol and caryophylline in the throat-refreshing dropping pill is measured by adopting a gas chromatography method, and the specific parameters are as follows: a chromatographic column is InertCap-Wax (30m * 0.32 mm * 0.5 mu m), the temperature of a sample inlet is 220 DEG C, and the pressure is 100 kPa; the sample introduction amount is 2 mu L, and split sample introduction is not carried out; carrying gas N2 and an FID (Flame Ionization Detector); the temperature of the detector is 230 DEG C. The method is simple, convenient to operate and low in detection cost, does not need complex pretreatment, can identify more components in the dripping pill with the refreshing and refreshing taste, and better meets the economic and practical requirements of routine detection of enterprises in laboratories.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine detection, and particularly relates to a component identification and content determination method for Yali Shuang Kouhan Drop Pills based on gas chromatography technology. BACKGROUND

[0002] Yali Shuang Kouhan Drop Pills are a compound preparation of Miao medicine commonly used in folk, have the effects of dispelling wind and clearing heat, relieving swelling and pain, and clearing and moistening the throat, and are commonly used in the treatment of acute / chronic pharyngitis, sore throat, red and swollen pharyngeal mucosa, dry throat, and halitosis. The quality control of Chinese patent medicines depends on the overall characterization of the synergistic effect of multiple components. Yali Shuang Kouhan Drop Pills are a compound preparation of Miao medicine composed of moxa tablets, ai-naxiang oil, menthol oil, menthol, and ammonium glycyrrhizinate, and have many components. However, the traditional quality control method is limited to the analysis of a single index component, and it is difficult to comprehensively reflect the group characteristics of volatile active ingredients in the preparation. Patent CN116124922B determines the content of four components, namely camphor, menthone, borneol, and menthol, by using GC-MS. However, this method still has significant limitations in the industrial application scenario of the quality control of traditional Chinese medicine preparations: 1. Only four components are established for detection, and the overall quality of the preparation cannot be evaluated through multiple components, and the quality control coverage is insufficient. 2. The instrument operation cost is high, and the operation is professional and dependent, which restricts the industrial application. 3. The high-throughput detection adaptability is poor, and the technical advantages (such as trace detection and unknown substance identification) of GC-MS are essentially mismatched with the core needs (high-throughput, low cost, and easy replication) of industrial quality control.

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

[0004] Therefore, a gas phase component identification analysis method for Yali Shuang Kouhan Drop Pills is constructed. Through optimization of chromatographic conditions (column temperature program, carrier gas flow rate), characteristic peak attribution, and common mode recognition strategy, synchronous separation and identification of 12 characteristic volatile components (including menthol, eucalyptol, alpha-pinene, etc.) in the preparation are realized, which is the current research direction. SUMMARY

[0005] In order to solve the above technical problems in the prior art, the present application provides a component identification and content determination method for Yali Shuang Kouhan Drop Pills based on gas chromatography technology, which is as follows:

[0006] A component identification and content determination method for Yali Shuang Kouhan Drop Pills based on gas chromatography technology, which determines the content of beta-pinene, limonene, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, beta-caryophyllene, pulegone, menthol, borneol, and caryophyllin in Yali Shuang Kouhan Drop Pills by using gas chromatography, and the specific parameters are as follows:

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

[0008] Further, the temperature programming of the gas chromatography method is specifically as follows:

[0009]

[0010] Further, the injection volume of the gas chromatography method is 2 μL, splitless injection.

[0011] Further, the detector temperature is 230 DEG C.

[0012] Further, the test solution is a solution with a concentration of 20 mg / mL obtained after dissolving Yali Shuang buccal drop pills in ethyl acetate. The test solution is specifically prepared by the following method:

[0013] Take 60 Yali Shuang buccal drop pills, finely grind, accurately take 0.2 g, place in a 10 mL volumetric flask, add ethyl acetate, ultrasonic for 10 min, and then dilute to volume, to obtain a test solution with a concentration of 20 mg / mL.

[0014] Further, the control solution is configured by using a control stock solution containing β-pinene, limonene, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, β-caryophyllene, pulegone, menthol, borneol, caryophyllin. The control stock solution is prepared by the following method:

[0015] Accurately take β-pinene, limonene, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, β-caryophyllene, pulegone, menthol, borneol, caryophyllin, and place them in a 10 mL volumetric flask. Add ethyl acetate, ultrasonic for 10 min to completely dissolve, and then dilute to volume to obtain a control stock solution.

[0016] Compared with the prior art, the technical effects of the present application are embodied in:

[0017] The method developed in this application can effectively desorb volatile components embedded in the pill matrix. Compared to the current method of using gas chromatography-mass spectrometry to detect multiple volatile components in compound preparations, this method is simple, convenient, and does not require complex pretreatment. It can identify a larger number of ingredients in Yanlishuang oral pills, with low detection costs, more in line with the economic and practical needs of routine testing in corporate laboratories. In addition, the 12 ingredients successfully identified include: β-pinene, limonene, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, β-caryophyllene, pulegone, menthol, borneol, and caryophyllin. Among them, menthol, borneol, and linalool can jointly inhibit the NF-κB and STAT3 pathways, reducing the release of inflammatory factors; isomenthone and menthone can relieve smooth muscle spasm through the TRPV1 channel, reducing the foreign body sensation in the throat; terpenoids such as limonene and β-pinene can destroy the cell membranes of pathogenic microorganisms; glycyrrhizic acid can modulate the body's immune response; and the transdermal combination of menthol and borneol can enhance drug utilization. Terpene compounds such as β-pinene and limonene form a synergistic effect with core ingredients such as menthol and borneol in Yanlishuang oral drops through anti-inflammatory, antibacterial and neuromodulatory effects, which can jointly achieve the therapeutic goals of Yanlishuang oral drops of dispersing wind and heat, reducing swelling and relieving pain, further explaining the multi-target action advantages of this compound preparation in treating pharyngitis, and providing a scientific basis for the active substances of Yanlishuang oral drops in treating pharyngitis. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0020] The technical solution of the present invention is further defined below in conjunction with specific implementation methods, but the scope of protection required is not limited to the description.

[0021] Example:

[0022] 1 Instruments and Materials

[0023] 1.1 Instrument

[0024] Multi-tube vortex oscillator (Typ. VX-III, Beijing Tadjin 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 type)

[0025] 1.2 Materials

[0026] Yali Shuang buccal drop pills (YLS) were provided by Guizhou Huangguoshu Lishuang 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%), menthone (batch number: WP24061004, purity 98%) were purchased from Sichuan Weikeqi Biological Technology Co., Ltd.; menthone (batch number: 111705-202106, purity 99%), menthyl acetate (batch number: 190044-202102, purity 98%), menthol (batch number: 110728-202208, purity 99.6%) were purchased from China Institute for Drug Control; camphor (batch number: PS020363, purity 99.30%) was purchased from Chengdu Ps Bio-technology Co., Ltd.; borneol (batch number: MUST-22021501, purity 98%) was purchased from Chengdu Manxite Biological Technology Co., Ltd.; caryophyllin (batch number: AB2637-0005, purity 98%) was purchased from Chengdu Efa Biological Technology Co., Ltd.

[0027] 2 Methods and results

[0028] 2.1 Preparation of test solution

[0029] Take 60 Yali Shuang buccal drop pills, grind them finely, and accurately weigh 0.2 g into a 10 mL volumetric flask. Add ethyl acetate as needed, and ultrasonicate for 10 min. Make up to volume to obtain a test solution of 20 mg / mL.

[0030] 2.2 Preparation of control solution

[0031] Accurately weigh β-pinene, limonene, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, β-caryophyllene, menthone, menthol, borneol, and caryophyllin as needed into a 10 mL volumetric flask. Add ethyl acetate as needed, and ultrasonicate for 10 min to completely dissolve. Make up to volume to obtain a control stock solution.

[0032] 2.3 Gas chromatography conditions

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

[0034] Table 1 Column oven temperature program

[0035]

[0036] 2.4 Methodology investigation

[0037] 2.4.1 Specificity

[0038] The blank solvent, mixed reference solution and sample solution were analyzed under the chromatographic conditions of "2.3", and the results are shown in Table 3. Twelve components were well separated, indicating that the method had good specificity. Figs. 1-2

[0039] 2.4.2 Investigation of linear relationship

[0040] The mixed reference solution under "2.2" was injected and determined under the chromatographic conditions of "2.3". The peak area (y) of the component to be tested was linearly regressed against the mass concentration (x), and the results showed that the linear relationship of β-pinene and other 12 components was good (R 2 ≥ 0.998) in their respective concentration ranges. The results are shown in Table 4.

[0041] Table 4 Linear equation of 12 components

[0042]

[0043] 2.4.3 Precision test

[0044] The mixed reference solution under "2.2" was injected and determined under the chromatographic conditions of "2.3", and the injection was continuously performed for 6 times. The RSD of the peak area of β-pinene and other 12 components was 0.68% to 2.91%, indicating that the precision of the instrument was good. The results are shown in Table 5.

[0045] Table 5 Results of precision investigation

[0046]

[0047] 2.4.4 Reproducibility test

[0048] The RSD of the peak area of β-pinene and other 12 components of the 6 samples prepared in parallel under "2.1" was 0.69% to 2.80%, indicating that the method had good reproducibility. The results are shown in Table 6.

[0049] Table 6 Results of reproducibility investigation ​

[0050]

[0051] 2.4.5 Stability test

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

[0053] Table 7 Stability test results

[0054]

[0055]

[0056] 2.4.6 Sample recovery test

[0057] About 0.1 g of Yali Shuang Kouhan Dropping Pills was taken, and 1:1 mixed with the control sample solution, for a total of 6 samples. The samples were analyzed according to the chromatographic conditions in item 2.3. The results are shown in Table 8. The average sample 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 method had good recovery rate. The results are shown in Table 8.

[0058] Table 8 Sample recovery rate of 12 components (n = 6)

[0059]

[0060]

[0061]

[0062] 2.4.7 Content determination

[0063] The test solution was prepared according to the method of item 2.1, and the determination was performed according to the chromatographic conditions of item 2.3. The results showed that the contents of β-pinene, limonene, 3-octanol, menthone, isomenthone, camphor, menthyl acetate, β-caryophyllene, pulegone, menthol, borneol and caryophyllin in the three batches of Yali Shuang Kouhan Dropping Pills were 0.3234-0.4150 mg / g, 0.8022-1.120 mg / g, 0.4178-0.4203 mg / g, 1.833-2.296 mg / g, 14.56-17.21 mg / g, 5.348-6.888 mg / g, 2.103-2.666 mg / g, 0.6896-0.8528 mg / g, 0.7156-0.9310 mg / g, 39.97-41.62 mg / g, 92.66-95.23 mg / g and 0.7926-0.8991 mg / g, respectively.

[0064] 3Discussion

[0065] The method established in the present application can effectively desorb the volatile components embedded in the matrix of the dropping pills. Compared with the current method of detecting multiple volatile components in compound preparations by gas chromatography-mass spectrometry, the method is simple, easy to operate, does not require complex pretreatment, can identify more components in Yali Shuang Kouhan Dropping Pills, has low detection cost, and is more in line with the economic and practical needs of routine testing in enterprise laboratories. In addition, the 12 components successfully identified include β-pinene, limonene, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, β-caryophyllene, pulegone, menthol, borneol and caryophyllin. Among them, menthol, borneol and can jointly inhibit the NF-κB and STAT3 pathways and reduce the release of inflammatory factors; isomenthone and menthone can relieve smooth muscle spasm through the TRPV1 channel and reduce the foreign body sensation in the pharynx; terpenoids such as limonene and β-pinene can destroy the cell membranes of pathogenic microorganisms; glycyrrhizic acid can regulate the immune response of the body; the transdermal combination of menthol and borneol can increase drug utilization. Terpenoids such as β-pinene and limonene can form a synergistic effect with core components such as menthol and borneol in Yali Shuang Kouhan Dropping Pills through anti-inflammatory, antibacterial and neuroregulatory effects, and can jointly achieve the therapeutic goals of dispersing wind and heat, reducing swelling and relieving pain of Yali Shuang Kouhan Dropping Pills, further explaining the multi-target effect advantage of the compound preparation in treating pharyngitis and providing a scientific basis for the action substances of Yali Shuang Kouhan Dropping Pills in treating pharyngitis.

[0066] Finally, it should be noted that the above examples are only representative of the present application. Obviously, the technical solutions of the present application are not limited to the above examples, but can also have many variations. All variations that can be directly derived or inferred from the content disclosed in the present application by those of ordinary skill in the art should be considered as falling within the scope of protection of the present application.

Claims

1. A method for identifying and determining the ingredients of Yanlishuang oral dripping pills based on gas chromatography technology, characterized in that: The contents of β-pinene, limonene, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, β-caryophyllene, pulegone, menthol, borneol, and dianthin in Yanlishuang oral dripping pills were determined by gas chromatography. The specific parameters are as follows: Chromatographic conditions: chromatographic column: InertCap-Wax (30m×0.32mm×0.5μm), injection port temperature: 220°C, pressure 100kPa; injection volume 2μL, splitless injection; carrier gas N2; FID detector, detector temperature is 230°C.

2. The method for identifying and determining the ingredients of Yanlishuang oral dripping pills based on gas chromatography technology according to claim 1, characterized in that: The temperature programming of the gas chromatography method is as follows:

3. The method for identifying and determining the ingredients of Yanlishuang oral dripping pills based on gas chromatography technology according to claim 1, characterized in that: The injection volume of the gas chromatography method was 2 μL, and the injection was splitless.

4. The method for identifying and determining the ingredients of Yanlishuang oral dripping pills based on gas chromatography technology according to claim 1, characterized in that: The detector temperature was 230°C.

5. The method for identifying and determining the ingredients of Yanlishuang oral dripping pills based on gas chromatography technology according to claim 1, characterized in that: The test solution is a solution with a concentration of 20 mg / mL obtained by dissolving Yanlishuang oral dropping pills in ethyl acetate.

6. The method for identifying and determining the ingredients of Yanlishuang oral dripping pills based on gas chromatography technology according to claim 5, characterized in that: The test solution is prepared by the following method: Take 60 Yanlishuang oral dropping pills, grind them into powder, accurately weigh 0.2 g, place them in a 10 mL volumetric flask, add ethyl acetate, ultrasonicate for 10 min, and dilute to volume to obtain a 20 mg / mL test solution.

7. The method for analyzing the anti-pharyngitis components of Yanlishuang oral dripping pills according to claim 1, characterized in that: The reference substance solution is prepared by using a reference substance stock solution containing β-pinene, limonene, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, β-caryophyllene, pulegone, menthol, borneol, and dianthin.

8. The method for analyzing the anti-pharyngitis components of Yanlishuang oral dripping pills according to claim 7, characterized in that: The reference substance stock solution is prepared by the following method: Accurately weigh appropriate amounts of β-pinene, limonene, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, β-caryophyllene, pulegone, menthol, borneol, and dianthin, place in a 10 mL volumetric flask, add ethyl acetate, sonicate for 10 min to completely dissolve, and dilute to volume to obtain a reference stock solution.

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