Determination of the content of active ingredients in Yanlishuang oral drops and establishment of GC-MS fingerprint

Abstract

The present application discloses a method for determining the content of four active ingredients in Yanli Shuang oral dripping pills in the field of drug detection technology. A method for simultaneously detecting the content of four active ingredients, camphor, menthone, borneol and menthol in Yanli Shuang oral dripping pills, is established by using the GC-MS internal standard method. The method is accurate, simple and has good repeatability, and can be used for the quality control of Yanli Shuang oral dripping pills. The present invention also uses GC-MS to establish a fingerprint of Yanli Shuang oral dripping pills, which has the advantages of saving time and high sensitivity, and provides a scientific basis for clarifying the material basis of drug efficacy and quality control based on volatile components.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a method for determining the content of active ingredients in Yanlishuang oral dripping pills and a method for establishing a GC-MS fingerprint spectrum. Background Art

[0002] Yan Lishuang Oral Drops are a Miao medicine compound preparation produced by Guizhou Huangguoshu Shushuang Pharmaceutical Co., Ltd., which consists of mugwort tablets, mugwort oil, menthol, monoammonium glycyrrhizinate and excipient polyethylene glycol 6000. It has a special aroma, sweet and slightly bitter taste, and has the effects of dispelling wind and heat, reducing swelling and relieving pain, and clearing the throat. It is used for acute pharyngitis, acute attack of chronic pharyngitis, sore throat, red and swollen pharyngeal mucosa, dry throat, bad breath, etc. Mugwort tablets are crystals extracted and processed from the fresh leaves of Blumea balsami era (L.) DC., a plant of the genus Blumea in the Asteraceae family. The main ingredient is borneol, and it contains a small amount of camphor and isoborneol. It has the effects of refreshing the mind, clearing away heat and relieving pain. In the process of refining moxa tablets, the oil obtained by pressing and separating the crude extract is moxa oil, the main components of which are β-pinene, β-caryophyllene, camphor, α-caryophyllene and borneol. It has the effects of clearing away heat and detoxification, anti-inflammatory and analgesic, antibacterial and antipruritic, and local anesthesia. It is used to treat sore throat, mouth sores, skin sunburns, eczema, colitis, and mosquito bites, etc. Peppermint oil is a volatile oil obtained by steam distillation, freezing, and partial brain removal of the fresh stems and leaves of Mentha haplocalyx Briq., a plant of the Lamiaceae family. The main components are menthol, menthone, menthol acetate, and limonene. It can be used to produce a cooling sensation on the skin or mucous membrane to relieve discomfort and pain, anti-inflammatory, antibacterial, and antioxidant, and to promote bile secretion and prevent cholesterol stones.

[0003] The quality research of traditional Chinese medicine has always been the focus and hotspot of the modernization research of traditional Chinese medicine. Some chemical components of traditional Chinese medicine may change during the storage process. The control of these components that are prone to qualitative and quantitative changes has become the main focus of the quality control of traditional Chinese medicine. The Yanlishuang oral dripping pills prepared from mugwort tablets, mugwort oil, menthol oil, menthol, and glycyrrhizic acid monoammonium salt have complex chemical components and strong volatility. Only the joint detection of multiple index components can achieve the quality control of the required effective ingredients. However, the current standards issued by the Bureau only have gas chromatography to determine the content detection indicators and methods of its mugwort tablets and menthol (requires that the product contains no less than 30.0 mg of menthol per gram and no less than 75.0 mg of borneol) and TLC qualitative identification of glycyrrhizic acid monoammonium salt (HPLC is used to determine the content of glycyrrhizic acid monoammonium salt with butyl hydroxybenzoate as the internal standard), which cannot fully control the quality of the compound preparation. Traditional Chinese medicine fingerprint is a multi-index quality control model that comprehensively reflects the types and quantities of chemical components contained by analyzing common peaks, thereby reflecting the quality of medicines. It is widely used in the quality evaluation of traditional Chinese medicine preparations. However, the existing traditional Chinese medicine fingerprint only focuses on multiple components, but does not measure the main components and the coordination of components. At the same time, it only uses fingerprints for qualitative evaluation, while ignoring quantitative evaluation. Summary of the invention

[0004] Aiming at the deficiencies of the prior art, the present invention designs a method for determining the contents of four main medicinal ingredients, namely camphor, menthone, borneol and menthol, in Yanlishuang oral dripping pills by using a GC-MS method.

[0005] One of the purposes of the present invention is to provide a method for determining the content of an active ingredient in a Yanlishuang oral dripping pill, comprising the following steps:

[0006] 1) prepare internal standard solution by taking internal standard substance naphthalene;

[0007] 2) Take the Yanli Shuang oral pills sample to prepare the test solution;

[0008] 3) Take two or more samples of the active ingredient to be tested to prepare reference substance stock solutions, and mix all reference substance stock solutions;

[0009] 4) GC-MS conditions;

[0010] 5) Establishment of SIM mode quantitative method: Accurately pipette the mixed reference solution in step 3) into a brown volumetric flask, add the internal standard solution in step 1) respectively, dilute to the scale with methanol, shake well, and obtain a series of standard solutions; scan in Scan mode, and establish a SIM mode quantitative method for the obtained data to obtain the qualitative and quantitative ion pairs of the active ingredients, and finally obtain the contents of multiple active ingredients at the same time.

[0011] Further, the active ingredients include but are not limited to camphor, menthone, borneol and menthol.

[0012] Further, the gas chromatography conditions of step 4) are as follows: the chromatographic column is a 30m×0.25mm, 0.25μm elastic quartz capillary column, the carrier gas is high-purity helium, the column flow rate is 0.78mL / min, and the programmed temperature conditions are as follows: the initial column temperature is 75°C and maintained for 2min, and the temperature is increased to 110°C at 2.0°C / min, and the analysis is completed in only 19.50min; the injection port temperature is 250°C, the injection volume is 1.0μl, the injection is split, and the split ratio is 50:1;

[0013] Mass spectrometry conditions: the ion source is an electron bombardment ion source, the bombardment energy is 70 Ev, the ion source temperature is 200 °C, the interface temperature is 250 °C, the solvent delay time is 5 min, the scanning mode is Q3 Scan, the mass scanning range is: 45-200 amu, and the detector voltage is 0 kV.

[0014] Furthermore, the steps of the method for determining the content of the four active ingredients in the Yanlishuang oral drops are specifically as follows:

[0015] 1) Preparation of internal standard solution: Take internal standard naphthalene, accurately weigh, place in a 100mL brown volumetric flask, add methanol to dissolve and dilute to scale, shake well, make 800ug / mL internal standard solution;

[0016] 2) Preparation of test solution: Take a sample of Yanli Shuang oral dripping pills, weigh accurately, add methanol, add the internal standard solution in step 1), weigh, weigh accurately, ultrasonically treat for 10 minutes, cool, add methanol to make up the weight loss, add anhydrous sodium sulfate to dehydrate, filter with a 0.22μm microporous membrane, and obtain;

[0017] 3) Preparation of reference substance stock solutions: accurately weigh camphor reference substance, menthone reference substance, borneol reference substance and menthol, place in a brown volumetric flask, add methanol to dissolve and dilute to scale, shake well, and prepare 5 mg / mL camphor reference substance stock solution, 5 mg / mL menthone reference substance stock solution, 20 mg / mL borneol reference substance stock solution, and 8 mg / mL menthol reference substance stock solution respectively;

[0018] 4) Preparation of mixed reference solution: accurately pipette the camphor reference stock solution, menthone reference stock solution, borneol reference stock solution, and menthol reference stock solution prepared in step 3) into a brown volumetric flask, dilute to scale with methanol, and shake well to obtain a mixed reference solution with a camphor concentration of 0.5 mg / mL, a menthone concentration of 0.5 mg / mL, a borneol concentration of 6.0 mg / mL, and a menthol concentration of 2.4 mg / mL;

[0019] 5) Identification and identification of chromatographic peaks: Take the test solution prepared in step 2), add the internal standard solution prepared in step 1) to the mixed reference solution in step 4) and dilute appropriately, inject and measure, identify and identify the chromatographic peaks by combining NIST standard mass spectrometry database retrieval and reference comparison;

[0020] 6) Establishment of SIM mode quantitative method: Accurately pipette multiple groups of mixed reference solution of step 4) into brown volumetric flasks, add internal standard solution of step 1) respectively, dilute to scale with methanol, shake well, and obtain a series of standard solutions; scan in Scan mode, establish and calculate the SIM mode quantitative method for the obtained data, and select the qualitative and quantitative ion pairs as follows: camphor, m / z 95→81 / 108; menthone, m / z 112→69 / 139; borneol, m / z 95→110 / 67; menthol, m / z81→71 / 95, and finally obtain the contents of the four active ingredients at the same time.

[0021] The second object of the present invention is to provide the application of the above method in Yanlishuang oral dripping pills and other drugs containing four ingredients of camphor, menthone, borneol and menthol.

[0022] The working principle and beneficial effects of the present invention are as follows: the present invention establishes a method for determining the contents of camphor, menthol, borneol and menthol, four main active ingredients in Yanli Shuang oral dripping pills, based on the GC-MS method (gas chromatography-mass spectrometry), and can specifically detect the contents of camphor, menthol, borneol and menthol, four main active ingredients in Yanli Shuang oral dripping pills, with good chromatographic peak separation, no interference, good linearity, high accuracy, repeatability, recovery rate and high stability. It can more comprehensively ensure the stable quality of Yanli Shuang oral dripping pills, the consistency of product batches and the safety and effectiveness of clinical medication.

[0023] The third object of the present invention is to provide a method for constructing a GC-MS fingerprint of Yanli Shuang oral dripping pills, comprising the following steps:

[0024] (1) Preparation of test solution: Take Yanli Shuang oral pills, weigh accurately, add methanol, weigh accurately, ultrasonically treat for 10 min, cool, add methanol to make up the weight loss, add anhydrous sodium sulfate to dehydrate, filter with 0.22 μm microporous filter membrane to obtain the test solution; (2) GC-MS analysis: Accurately draw 1.0 μL of the test solution and inject it into the GC-MS chromatograph. The chromatographic conditions are as follows: Chromatographic column: 30 m×0.25 mm, 0.25 μm elastic quartz capillary column SH-Rxi-5Sil MS; helium as carrier gas; column flow rate 1.0 mL / min; programmed temperature conditions: initial column temperature 70 °C, temperature increased to 90 °C at 1.5 °C / min, temperature increased to 120 °C at 3 °C / min, temperature increased to 250 °C at 20 °C / min and maintained for 2 min until analysis is completed; injection port temperature 250 °C; injection volume 1.0 μL; split injection, split ratio 30:1; mass spectrometry conditions: ion source is electron bombardment ion source, bombardment energy is 70 eV; scanning mode Q3 Scan, mass scanning range: 30-700 amu; (3) GC-MS graphs of more than 10 batches of samples and reference fingerprints were measured and fitted to obtain GC-MS superimposed fingerprints.

[0025] The number of samples is preferably more than 10 and less than 20, preferably 12, 13, 14, 15 or 18 batches.

[0026] The fourth object of the present invention is to provide a GC-MS fingerprint of Yanli Shuang oral dropping pills established by the above method.

[0027] The present invention uses GC-MS to establish a fingerprint spectrum of Yanli Shuang oral dripping pills, marks a total of 18 common peaks, identifies 4 characteristic peaks to reflect its material basis and determines the content, combines fuzzy identification with precise quantitative analysis, and provides a method for systematically and rapidly evaluating the quality of the preparation. The method is simple to operate, has good repeatability, and strong specificity. It has the advantages of saving time and high sensitivity compared to the current gas chromatography analysis, and provides a scientific basis for clarifying the material basis of the efficacy and quality control based on volatile components.

[0028] The present invention respectively investigates methanol, anhydrous ethanol, acetonitrile, ethyl acetate, acetone, n-hexane, and dichloromethane as extraction solvents for the test sample, extracts according to the test sample solution preparation method, and analyzes the sample under the same chromatographic conditions. As a result, it is found that the total ion current chromatograms of the several solvent extracts have the same number of chromatographic peaks, and the chromatographic peak separation has no obvious difference. The methanol extract is colorless, transparent and stable, and the extraction efficiency is higher than that of other solvents, and the total ion current chromatogram has a better peak shape, so methanol is selected as the final extraction solvent. When naphthalene is selected as the internal standard, the internal standard peak is completely separated from the peaks of all components in the sample, and the retention time is close to the target component peak.

[0029] The present invention also investigates 0.78mL / min, 1.0mL / min, 1.5mL / min three chromatographic column flow rates respectively, and it is found that as the flow rate increases, the front time is advanced, and the chromatographic peak separation does not change significantly, and the response signal is best when the flow rate is 0.78mL / min, so 0.78mL / min is selected as the chromatographic column flow rate. Then investigate 10:1, 30:1, 50:1 three split ratios, and it is found that when the split ratio is 10:1, the detector is saturated, and 30:1 is better than 50:1 response signal, so the split ratio is selected to be 50:1. Because the sample is a Chinese patent medicine, the matrix is ​​complex, so the program temperature rise mode is selected to investigate, and the sample is peaked in a short time as much as possible. First, 70 ℃, 75 ℃, 80 ℃ are investigated as the initial temperature of the program temperature rise respectively, and it is found that as the initial temperature increases, the separation between borneol and menthol is reduced, and the separation between menthol and internal standard naphthalene increases. Next, the heating rates of 1.0℃ / min, 1.5℃ / min, and 2.0℃ / min were investigated. The results showed that, similar to the initial temperature investigation results, with the increase in heating rate, the separation between borneol and menthol decreased, while the separation between menthol and internal standard naphthalene increased. After comprehensive consideration, when 75℃ was selected as the initial temperature of the programmed temperature and 1.5℃ / min was selected as the heating rate of the programmed temperature, the separation between borneol and menthol, and between menthol and internal standard naphthalene met the analysis requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The total ion current chromatograms of the Yanlishuang oral drops sample (A), mixed reference substance (B), and blank solvent (C) under Scan mode (Note: 1. Camphor; 2. Menthone; 3. Borneol; 4. Menthol; 5. Naphthalene (internal standard)).

[0031] Figure 2 This is the total ion chromatogram of the volatile components of Yanlishuang oral drops;

[0032] Figure 3 This is the GC-MS reference fingerprint of Yanlishuang oral drops.

[0033] Figure 4 Overlay GC-MS fingerprints of 13 batches of Yanlishuang oral drops. DETAILED DESCRIPTION

[0034] The following is further described in detail through specific implementation methods:

[0035] Example 1 Method for determining the content of active ingredients in Yanli Shuang oral dripping pills

[0036] Instruments and equipment: GC-MS combined instrument (GCMS-TQ8040NX, Shimadzu Corporation, Japan); 1 / 10,000 balance (Shanghai Mettler-Toledo Instrument Co., Ltd.); intelligent silent ultrasonic cleaning machine (Xiaomei Ultrasonic Instrument (Kunshan) Co., Ltd.); 0.22μm microporous filter membrane (Nantong Supin Experimental Equipment Co., Ltd.).

[0037] Test drugs and reagents: Yanli Shuang oral drops (Guizhou Huangguoshu Shushuang Pharmaceutical Co., Ltd., batch number S1-S13: 20220414001, 20220415001, 20220418001, 20220421001, 20220422001, 20220425001, 20220427049, 20220428110, 20220505017, 20220506081, 20220509111, 20220510088, 20220511022); methanol (AR, Chengdu Jinshan Chemical Reagent Co., Ltd.); anhydrous sodium sulfate (AR, Chengdu Jinshan Chemical Reagent Co., Ltd. Company); acetone (AR, Chongqing Chuandong Chemical (Group) Co., Ltd.); reference substances: naphthalene (batch number: 84679-1G, mass fraction 99.9%), purchased from Sigma-Aldrich Reagent Company; camphor (batch number: 111749-201702, mass fraction 99.9%); menthone (batch number: 111705-202106, mass fraction 99.1%); borneol (batch number: 111688-201602, mass fraction 99.6%); menthol (batch number: 110728-201707, mass fraction 99.8%), all purchased from China Food and Drug Inspection Institutes for content determination.

[0038] The specific determination method includes the following steps:

[0039] 1) Preparation of internal standard solution: Take about 80 mg of internal standard naphthalene, accurately weigh it, place it in a 100 mL brown volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and make 800 ug / mL internal standard solution.

[0040] 2) Preparation of test solution: Take about 0.15 g of Yanli Shuang oral dropping pills sample, weigh accurately, add 8.75 mL of methanol, add 1.25 mL of the internal standard solution under "1)", weigh, weigh accurately, treat with ultrasound (power 200 W, frequency 40 kHz) for 10 min, cool, make up the weight loss with methanol, add anhydrous sodium sulfate to dehydrate, filter with 0.22 μm microporous membrane to obtain.

[0041] 3) Preparation of reference substance stock solution: Take about 25 mg of camphor reference substance, weigh accurately, place in a 5 mL brown volumetric flask, add methanol to dissolve and dilute to scale, shake well, and prepare 5 mg / mL camphor reference substance stock solution. Prepare 5 mg / mL menthone reference substance stock solution, 20 mg / mL borneol reference substance stock solution, and 8 mg / mL menthol reference substance stock solution in the same way.

[0042] 4) Preparation of mixed reference solution: Accurately pipette 1 mL, 1 mL, 3 mL, and 3 mL of the camphor, menthone, borneol, and menthol reference stock solutions prepared under "3)" into 10 mL brown volumetric flasks, dilute to scale with methanol, and shake well to obtain a mixed reference solution with a camphor concentration of 0.5 mg / mL, a menthone concentration of 0.5 mg / mL, a borneol concentration of 6.0 mg / mL, and a menthol concentration of 2.4 mg / mL.

[0043] 5) Identification and identification of chromatographic peaks: Prepare the test solution according to the method under "2)", add the internal standard solution under "1)" to the mixed reference solution under "4)" and dilute it appropriately, then inject and measure. Combined with the NIST standard mass spectrometry database search and reference comparison, identify and identify the chromatographic peaks.

[0044] 6) Establishment of SIM mode quantitative method: Accurately pipette 0.25mL, 0.50mL, 0.75mL, 1.00mL, 1.25mL, and 1.50mL of the mixed reference solution under item "4)" into a 5mL brown volumetric flask, add 0.625mL of the internal standard solution under item "1)" respectively, dilute to the mark with methanol, and shake well to obtain a series of standard solutions. Scan in Scan mode, and establish and calculate the SIM mode quantitative method for the obtained data. The selected qualitative and quantitative ion pairs are: camphor, m / z 95→81 / 108; menthone, m / z 112→69 / 139; borneol, m / z 95→110 / 67; menthol, m / z 81→71 / 95.

[0045] The detection conditions of GC-MS are as follows: Gas chromatography conditions: the chromatographic column is an elastic quartz capillary column SH-Rxi-5Sil MS (30m×0.25mm, 0.25μm), the carrier gas is high-purity helium (purity ≥99.999%), the column flow rate is 0.78mL / min, and the program temperature condition is: the initial column temperature is 75℃ and maintained for 2min, and the temperature is increased to 110℃ at 2.0℃ / min, and the analysis is completed in only 19.50min. The injection port temperature is 250℃, the injection volume is 1.0μl, and the injection is split, and the split ratio is 50:1.

[0046] Mass spectrometry conditions: the ion source is an electron bombardment ion source, the bombardment energy is 70 Ev, the ion source temperature is 200 °C, the interface temperature is 250 °C, the solvent delay time is 5 min, the scanning mode is Q3 Scan, the mass scanning range is: 45-200 amu, and the detector voltage is 0 kV.

[0047] The following characteristics were examined: linearity, repeatability, precision, stability, and sample recovery.

[0048] 1.1 Linearity

[0049] The samples contained 25.13μg / mL, 50.26μg / mL, 75.39μg / mL, 100.52μg / mL, 125.65μg / mL, 150.78μg / mL of camphor; 28.77μg / mL, 57.54μg / mL, 86.31μg / mL, 115.08μg / mL, 143.85μg / mL, 172.62μg / mL of menthone; 299.70μg / mL, 59 9.40μg / mL, 899.10μg / mL, 1198.80μg / mL, 1498.50μg / mL, 1798.20μg / mL; 121.98μg / mL, 243.96μg / mL, 365.94μg / mL, 487.92μg / mL, 609.90μg / mL, 731.88μg / mL series standard solutions were injected 1μL according to the SIM mode quantitative method to determine the peak area. The ratio of the peak area of ​​each reference substance to the peak area of ​​the internal standard was used as the ordinate (Y), and the ratio of the mass concentration was used as the abscissa (X). The standard curves of camphor, menthone, borneol, and menthol were drawn respectively to obtain the regression equation and its linear range. The results are shown in Table 1. Camphor showed a good linear relationship in the mass concentration range of 25.13 μg / mL-150.78 μg / mL, menthone showed a good linear relationship in the mass concentration range of 28.77 μg / mL-172.62 μg / mL, borneol showed a good linear relationship in the mass concentration range of 299.70 μg / mL-1798.20 μg / mL, and menthol showed a good linear relationship in the mass concentration range of 121.98 μg / mL-731.88 μg / mL. The linear correlation coefficients were all greater than 0.9990.

[0050] Table 1 Correlation coefficients and linear ranges of linear equations for camphor, menthone, borneol and menthol

[0051] 1.2 Precision

[0052] Take an appropriate amount of the same test sample (batch number 20220425001), prepare the test solution according to the test solution preparation method, inject continuously 6 times according to the SIM mode quantitative method, determine the peak area, and calculate the RSD value of the ratio of the peak area of ​​each component in the test solution to the peak area of ​​the internal standard. The results are shown in Table 2. The RSDs of camphor, menthone, borneol and menthol are 2.3%, 2.0%, 2.7% and 2.4%, respectively. The results show that this method has good precision.

[0053] 1.3 Stability

[0054] Take an appropriate amount of the same test sample (batch number 20220425001), prepare the test solution according to the test solution preparation method, store it at room temperature, and measure the peak area by SIM mode quantitative method at 0, 2, 4, 6, 8, and 16 hours, and calculate the RSD value of the ratio of the peak area of ​​each component in the test solution to the peak area of ​​the internal standard. The results are shown in Table 2. The RSDs of camphor, menthone, borneol, and menthol are 2.1%, 2.4%, 2.8%, and 2.6%, respectively. The results show that the components in the test solution are stable within 16 hours at room temperature.

[0055] 1.4 Repeatability

[0056] Take an appropriate amount of the same test sample (batch number is 20220414001), prepare 6 test sample solutions in parallel according to the test sample solution preparation method, sample and measure the peak area according to the SIM mode quantitative method, and calculate the RSD of the content of each component. The results are shown in Table 2. The average content of camphor is 6.551 mg / g, and the RSD value is 1.4% (n=6), the average content of menthone is 5.353 mg / g, and the RSD value is 2.2% (n=6), the average content of borneol is 89.244 mg / g, and the RSD value is 2.9% (n=6), and the average content of menthol is 40.646 mg / g, and the RSD value is 2.4% (n=6), indicating that the method has good repeatability.

[0057] Table 2 Results of precision, stability and repeatability of camphor, menthone, borneol and menthol content determination

[0058] 1.5 Sample recovery rate

[0059] Take 6 portions of Yanli Shuang oral dripping pills with known contents of each component (batch number 20220414001, containing 6.551 mg / g camphor, 5.353 mg / g menthone, 89.244 mg / g borneol, 40.646 mg / g menthol), each portion is about 0.075 g, accurately weighed, prepare the test solution according to the test solution preparation method, add equal amounts of camphor, menthone, borneol and menthol reference substances, and measure the peak area by SIM mode quantitative method. The results are shown in Table 3. The average sample recovery rate of camphor in Yanli Shuang oral dripping pills is 102.02%, and the RSD is 1.3%; the average sample recovery rate of menthone is 96.10%, and the RSD is 1.0%; the average sample recovery rate of borneol is 102.71%, and the RSD is 1.3%; the average sample recovery rate of menthol is 102.58%, and the RSD is 1.1%. The results show that the recovery rate of this method is good.

[0060] Table 3 Results of sample recovery for determination of camphor, menthone, borneol and menthol contents

[0061]

[0062]

[0063] Determination of content in 13 batches of Yanlishuang oral dripping pills

[0064] Thirteen batches of Yanli Shuang oral dripping pills with different batch numbers were taken, and three copies of each batch of samples were prepared in parallel according to the test solution preparation method. The contents of camphor, menthone, borneol and menthol in the 13 batches of samples were calculated by the internal standard method, and the results are shown in Table 4. The results showed that there were certain differences in the contents of camphor, menthone, borneol and menthol in the preparation. The camphor content in multiple batches of samples fluctuated between 5.6025-8.3662 mg / g, the menthone content fluctuated between 4.7871-5.8936 mg / g, the borneol content fluctuated between 88.0034-133.0969, and the menthol content fluctuated between 40.2017-61.9466. The contents of menthol and borneol in the 13 batches of samples all comply with the provisions of the 2020 edition of the "Pharmacopoeia", which stipulate that each gram of Yanlishuang oral drops must contain no less than 30.0 mg of menthol, and no less than 75.0 mg of borneol.

[0065] Table 4 Determination results of the contents of four active ingredients in 13 batches of Yanlishuang oral drops

[0066]

[0067]

[0068] Example 2 Method for constructing GC-MS fingerprint of Yanlishuang oral dripping pills

[0069] GC-MS conditions

[0070] Gas chromatography conditions: the chromatographic column is an elastic quartz capillary column SH-Rxi-5Sil MS (30m×0.25mm, 0.25μm); the carrier gas is high-purity helium (purity ≥99.999%); the column flow rate is 1.0mL / min; the program temperature conditions are: the initial column temperature is 70℃ (maintained for 4min), the temperature is increased to 90℃ (maintained for 5min) at 1.5℃ / min, the temperature is increased to 120℃ at 3℃ / min, the temperature is increased to 250℃ at 20℃ / min, and the temperature is maintained for 2min until the analysis is completed. The injection port temperature is 250℃; the injection volume is 1.0μL; split injection, the split ratio is 30:1. Mass spectrometry conditions: the ion source is an electron bombardment ion source with an impact energy of 70 eV; the ion source temperature is 200°C; the interface temperature is 250°C; the solvent delay time is 2 min, the scanning mode is Q3 Scan, the mass scanning range is 30-700 amu, and the detector voltage is 0.1 kV.

[0071] Preparation of test solution

[0072] Take about 0.15 g of sample, weigh accurately, add 10 mL of methanol, weigh accurately, treat with ultrasound (power 200 W, frequency 40 kHz) for 10 min, cool, make up the weight loss with methanol, add anhydrous sodium sulfate for dehydration, filter with 0.22 μm microporous membrane to obtain.

[0073] According to the GC-MS conditions, the total ion chromatogram of the volatile components of Yanlishuang oral dripping pills was obtained. Figure 2 .

[0074] 2.1 Precision test

[0075] Take an appropriate amount of the same test sample (batch number 20220425001) to prepare the test sample solution. The test conditions were continuously injected and measured 6 times under GC-MS conditions. The peak No. 9 with moderate retention time, good separation, and maximum peak area was used as the reference peak. The consistency of the relative retention time and relative peak area of ​​the chromatographic peaks was investigated, and the relative retention time and relative peak area of ​​the main characteristic peaks were calculated. The RSD of the relative retention time of each characteristic peak was less than 0.1% (n=6), and the RSD of the relative peak area was less than 8% (n=6), as shown in Table 1. The fingerprint spectrum obtained by the first injection was used as a reference to calculate the similarity of the fingerprint spectrum obtained by the next 5 injections, and the similarity was 1.000. The results of the precision test showed that the instrument had good precision, and the peaks of each substance were relatively stable, which could meet the requirements of fingerprint analysis.

[0076] 2.2 Stability test

[0077] Take an appropriate amount of the same test sample (batch number 20220425001) to prepare the test solution, and place it at room temperature for 0, 2, 4, 6, 8, 16, and 24 hours, respectively, and measure the test conditions under GC-MS conditions. Peak 9 is used as the reference peak to calculate the relative retention time of the main characteristic peaks and the RSD of the relative peak area. The results show that the RSD of the relative retention time of each characteristic peak is less than 0.1% (n=7), and the RSD of the relative peak area is less than 8% (n=7), as shown in Table 1. The fingerprint spectrum obtained by the first injection was used as a reference to calculate the similarity of the fingerprint spectrum obtained by the next 6 injections, and the result is a similarity of 1.000. The results of the stability test show that the test solution is basically stable within 24 hours at room temperature.

[0078] 2.3 Repeatability test

[0079] Take an appropriate amount of the same test sample (batch number 20220425001) and prepare 6 test sample solutions in parallel. The test conditions were measured under GC-MS conditions. Peak 9 was used as the reference peak to calculate the relative retention time and relative peak area RSD of the main characteristic peaks. The results showed that the RSD of the relative retention time of each characteristic peak was less than 0.1% (n=6), and the RSD of the relative peak area was less than 7% (n=6), as shown in Table 5. The fingerprint spectrum obtained by the first injection was used as a reference to calculate the similarity of the fingerprint spectrum obtained by the next 5 injections, and the similarity was 1.000. This shows that the method has good repeatability.

[0080] Table 5 Results of GC-MS fingerprint methodological investigation of volatile components of Yanlishuang oral drops

[0081]

[0082] 2.4 Fingerprint establishment and technical parameters

[0083] 13 different batches of Yanli Shuang oral dripping pills were taken to prepare test solutions. The test conditions were tested under GC-MS conditions. The fingerprints of the volatile components of different batches of Yanli Shuang oral dripping pills were detected by GC-MS technology. Peak No. 9 with the largest peak area was used as the reference peak to determine the common characteristic peaks with relatively stable retention time and peak area, and 18 common characteristic peaks were determined. The fingerprints of 13 batches of Yanli Shuang oral dripping pills were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" 2004 A version for analysis, and the control fingerprint (R) was calculated using the average method with a time window width of 0.1. The spectra are detailed in Figure 3 The fingerprints of 13 batches of samples (S1-S13) were compared with R to calculate similarity, and the GC-MS graphs of the fingerprints of 13 batches of samples and the control were fitted to obtain the GC-MS superimposed fingerprints, see Figure 4The results showed that the similarity between the fingerprints of each batch of samples and R was 1.000, indicating that the main components of the volatile components of Yanli Shuang oral dripping pills of different batches were basically the same, and the generated control fingerprints were very representative. The results showed that the quality consistency of Yanli Shuang oral dripping pills produced by Guizhou Huangguoshu Shushuang Pharmaceutical Co., Ltd. was good, and the overall difference was small, indicating that the preparation process of the dripping pills was stable and the quality controllability was good.

[0084] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for determining the content of active ingredients in Yanli Shuang oral dripping pills, characterized in that: The following steps are involved: 1) Take the internal standard naphthalene to prepare the internal standard solution; 2) Take the Yanli Shuang oral pills sample to prepare the test solution; 3) Take two or more samples of the active ingredient to be tested to prepare reference substance stock solutions, and mix all reference substance stock solutions; 4) GC-MS conditions; 5) Establishment of SIM mode quantitative method: Accurately pipette the mixed reference solution in step 3) into a brown volumetric flask, add the internal standard solution in step 1) respectively, dilute to the mark with methanol, and shake well to obtain a series of standard solutions; Scan in Scan mode, and calculate the obtained data using SIM mode quantitative method, and finally obtain the contents of multiple active ingredients at the same time; The gas chromatography conditions were as follows: the chromatographic column was a 30 m×0.25 mm, 0.25 μm elastic quartz capillary column SH-Rxi-5Sil MS, the carrier gas was high-purity helium, the column flow rate was 0.78 mL / min, the programmed temperature conditions were as follows: the initial column temperature was maintained at 75°C for 2 min, the temperature was increased to 110°C at 2.0°C / min, and the analysis was completed in only 19.50 min; the injection port temperature was 250°C, the injection volume was 1.0 μl, the injection was split, and the split ratio was 50:1; Mass spectrometry conditions: ion source: electron bombardment ion source, bombardment energy: 70 Ev, ion source temperature: 200 °C, interface temperature: 250 °C, solvent delay time: 5 min, scanning mode: Q3 Scan, mass scanning range: 45-200 amu, detector voltage: 0 kV; The specific steps of the determination method are: 1) Preparation of internal standard solution: Take the internal standard naphthalene, accurately weigh it, place it in a 100mL brown volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and make an 800ug / mL internal standard solution; 2) Preparation of test solution: Take a sample of Yanli Shuang oral dripping pills, weigh accurately, add methanol, add the internal standard solution in step 1), weigh, weigh accurately, ultrasonically treat for 10 min, cool, add methanol to make up the weight loss, add anhydrous sodium sulfate to dehydrate, filter with a 0.22μm microporous membrane, and obtain; 3) Preparation of reference substance stock solutions: accurately weigh camphor reference substance, menthone reference substance, borneol reference substance and menthol, place in a brown volumetric flask, add methanol to dissolve and dilute to scale, shake well, and prepare 5 mg / mL camphor reference substance stock solution, 5 mg / mL menthone reference substance stock solution, 20 mg / mL borneol reference substance stock solution, and 8 mg / mL menthol reference substance stock solution respectively; 4) Preparation of mixed reference solution: Accurately pipette the camphor reference stock solution, menthone reference stock solution, borneol reference stock solution, and menthol reference stock solution prepared in step 3) into a brown volumetric flask, dilute to scale with methanol, and shake well to obtain a mixed reference solution with a camphor concentration of 0.5 mg / mL, a menthone concentration of 0.5 mg / mL, a borneol concentration of 6.0 mg / mL, and a menthol concentration of 2.4 mg / mL; 5) Identification and identification of chromatographic peaks: Take the test solution prepared in step 2), add the internal standard solution prepared in step 1) to the mixed reference solution in step 4) and dilute appropriately, inject and measure, identify and identify the chromatographic peaks by combining NIST standard mass spectrometry database search and reference comparison; 6) Establishment of SIM mode quantitative method: Accurately pipette multiple groups of mixed reference solution from step 4) into brown volumetric flasks, add internal standard solution from step 1) respectively, dilute to scale with methanol, shake well, and obtain a series of standard solutions; Scanning was performed in Scan mode, and the obtained data were used to establish and calculate the quantitative method in SIM mode. The selected qualitative and quantitative ion pairs were: camphor, m / z 95→81 / 108; menthone, m / z 112→69 / 139; borneol, m / z 95→110 / 67; menthol, m / z 81→71 / 95. Finally, the contents of the four active ingredients were obtained at the same time.

2. The method according to claim 1, characterized in that: Active ingredients include camphor, menthone, borneol and menthol.

3. Application of the method according to claim 2 in Yanlishuang oral dripping pills and other drugs containing four ingredients of camphor, menthone, borneol and menthol.

4. A method for constructing a GC-MS fingerprint of Yanli Shuang oral dripping pills, characterized in that: The following steps are involved: (1) Preparation of test solution: Take Yanli Shuang oral pills, weigh accurately, add methanol, weigh accurately, ultrasonicate for 10 min, cool, add methanol to make up the weight loss, add anhydrous sodium sulfate to dehydrate, filter with 0.22 μm microporous filter membrane to obtain the test solution; (2) GC-MS analysis: Accurately pipette 1.0 μL of the test solution and inject it into the GC-MS chromatograph. The chromatographic conditions are as follows: chromatographic column: 30 m×0.25 mm, 0.25 μm elastic quartz capillary column SH-Rxi-5Sil MS; carrier gas is helium; column flow rate is 1.0 mL / min; programmed temperature conditions: initial column temperature 70℃, increase to 90℃ at 1.5℃ / min, increase to 120℃ at 3℃ / min, increase to 250℃ at 20℃ / min and hold for 2 min until the analysis is completed; injection port temperature 250℃; injection volume 1.0 μL; split injection, split ratio of 30:1; mass spectrometry conditions: ion source is electron bombardment ion source, bombardment energy is 70 eV; scanning mode is Q3 Scan, mass scanning range: 30-700 amu; (3) GC-MS graphs of more than 10 batches of samples and reference fingerprints were fitted to obtain GC-MS superimposed fingerprints.

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