GC-MS (Gas Chromatography-Mass Spectrometer) method for determining effective components in aromatic Wentong prescription

Abstract

The invention discloses a GC-MS (Gas Chromatography-Mass Spectrometer) method for determining effective components in an aromatic Wentong formula. A chromatographic column is a capillary column; a temperature programming method is adopted; the ionization mode in the mass spectrum condition is electron impact ionization, and the acquisition mode is scanning. According to the method disclosed by the invention, the active ingredients of the aromatic Wentong prescription can be effectively separated in a gas chromatograph through a specific heating program in combination with specific sample inlet temperature, chromatographic column temperature and split ratio and can be better focused and enter a mass spectrometer at the same time, and under the participation of specific mass spectrometry conditions, the effective separation of the active ingredients of the aromatic Wentong prescription can be realized. According to the present invention, with the method, the interference of the cinnamyl aldehyde, the alpha-caryophyllene, the methyleugenol and the ligustilide in the chromatogram can be eliminated, the signal strength and the sensitivity are high, the accurate quantification of the four components such as the cinnamyl aldehyde, the alpha-caryophyllene, the methyleugenol and the ligustilide is finally achieved, the reference basis is provided for the quality control of the aromatic warming and dredging formula, and the practical application value is provided.

Description

Technical Field

[0001] The present invention relates to the field of medical detection, and in particular to a GC-MS method for determining effective components in aromatic warming prescriptions. Background Art

[0002] Aromatic warming and promoting circulation is one of the most commonly used therapies in Traditional Chinese Medicine (TCM) for treating diseases of blood stasis caused by invasion of cold pathogens or internal cold due to yang deficiency. Several of the earliest texts of TCM, such as the Wuwei Han Dynasty Medical Bamboo Slips (also known as "Cure for All Diseases"), utilized aromatic warming and promoting mugwort fumigation and aromatic Chinese herbs (such as cinnamon and pepper) to treat diseases related to invasion of cold pathogens. The theory of aromatic warming and promoting circulation for treating diseases related to cold stagnation and blood stasis was comprehensively expounded in the Neijing (Inner Canon of Medicine). Modern medicine has also confirmed that treatment with warming herbs such as cinnamon, fennel, cloves, and Sichuan pepper can significantly relieve muscle spasms and vasoconstriction, improve blood circulation, and alleviate clinical symptoms such as pain and fatigue caused by insufficient blood flow to limbs and organs due to blood stasis.

[0003] With the continuous enrichment and improvement of Traditional Chinese Medicine (TCM) theory and practice, aromatic warming and unblocking therapies and their corresponding TCM preparations have gained increasing attention and expanded in disease prevention and treatment in recent years. For example, the Amber Tongguan Patch from Fujian Zhoujiafu Biopharmaceutical Co., Ltd. is based on the Shenying Pills, Volume 8 of the Song Dynasty's Taiping Huimin Hejiju Fang (prescriptions for the treatment of inflammatory bowel syndromes). It incorporates the TCM "aromatic warming and unblocking" approach, supplemented by modern pharmaceutical technology. Formulated with traditional Chinese medicinal herbs such as cloves, cinnamon, and angelica, it boasts the efficacy of dispelling wind and cold, aromatic warming and unblocking the flow of qi, removing blood stasis and dampness, and dredges the meridians and relieves pain. Based on this formula, the aromatic warming and unblocking formula, formulated according to TCM theory and modified according to symptoms, consists of dried ginger, turmeric, cinnamon, angelica, cloves, Chuanxiong, angelica sinensis, Chuanwu, Caowu, Asarum, Clematis chinensis, Radix Aconiti Lateralis Preparata, Arisaema Confusa, Cercidiphyllum, and Artemisia selengensis. These herbs possess pungent and warm properties, and similarly possess the efficacy of dispelling cold and relieving pain, as well as activating the meridians and activating the collaterals. However, effective quality control methods for these aromatic warming and unblocking formulas are currently lacking. Summary of the Invention

[0004] To solve the above problems, the present invention provides a GC-MS method for determining the effective ingredients in Xiangfang Wentong Fang, which comprises the following steps:

[0005] a. Preparation of test solution: Take the aromatic Wentong formula to be tested, extract it with acetone, add anhydrous sodium sulfate to the supernatant of the extract to absorb water, filter, take the filtrate, and add the internal standard solution to obtain the solution;

[0006] b. Preparation of reference substance working solution: Dissolve cinnamaldehyde, α-caryophyllene, methyl eugenol and / or ligustilide reference substances in acetone, and add internal standard solution to the dissolved solution to obtain the reference substance.

[0007] c. Separately aspirate the test solution and reference working solution and inject them into the GC-MS instrument. The chromatographic conditions are as follows: chromatographic column: capillary column, programmed temperature method; mass spectrometry conditions: ionization mode: electron impact ionization, acquisition mode: scan.

[0008] Furthermore, in step a, the mass volume ratio of the aromatic warming prescription to acetone, anhydrous sodium sulfate and internal standard solution is 50-100 mg: 1-3 ml: 0.1-1 g: 1.1-0.5 ml.

[0009] Furthermore, in step b, the volume ratio of the dissolving solution to the internal standard solution is 0.1-5:0.3; and the concentration of each reference substance in the dissolving solution is 0.01-0.5 mg / mL.

[0010] Furthermore, the internal standard solution is an acetone solution containing naphthalene; the concentration of naphthalene in the acetone solution is 5 to 10 mg / mL.

[0011] Furthermore, in the chromatographic conditions of step c, the chromatographic column is a capillary column filled with (5%-phenyl)-methylpolysiloxane, preferably an HP-5MS chromatographic column, 30m×0.25mm×0.25μm; the heating program is: initial temperature 40°C, hold for 1 minute, increase the temperature to 230°C at a rate of 10°C per minute, and hold for 2 minutes; inlet temperature: 200-300°C; chromatographic column temperature: 50-70°C; split ratio: 10-30:1.

[0012] Furthermore, the injection port temperature is 250° C., the chromatographic column temperature is 60° C., and the split ratio is 20:1.

[0013] Furthermore, the mass spectrometry conditions in step c are: ion source temperature: 200-300° C.; quadrupole temperature: 100-200° C.; solvent delay time: 2-5 minutes.

[0014] Furthermore, the mass spectrometry conditions are as follows: ion source temperature: 230° C.; quadrupole temperature: 150° C.; acquisition mode: scanning; solvent delay time: 3 minutes.

[0015] Furthermore, the chromatogram of the test solution presents a chromatographic peak with the same retention time as that in the chromatogram of the reference working solution, determining that the aromatic warming prescription to be tested contains the component corresponding to the reference; the content of the component corresponding to the reference in the aromatic warming prescription is calculated by peak area according to the internal standard method.

[0016] Furthermore, the aromatic warming prescription is a formula raw material or formula preparation composed of dried ginger, turmeric, cinnamon, angelica dahurica, cloves, ligusticum chuanxiong, angelica sinensis, chuanwu, kusnezoffii, asarum, clematis root, scutellaria baicalensis, arisaema, scutellaria baicalensis and artemisia selengensis; the mass ratio of dried ginger, turmeric, cinnamon, angelica dahurica, cloves, ligusticum chuanxiong, angelica sinensis, chuanwu, kusnezoffii, asarum, clematis root, scutellaria baicalensis, arisaema, scutellaria baicalensis and artemisia selengensis is: 10:10:10:10:10:10:5:5:2:10:10:10:10:5.

[0017] The GC-MS method for determining the effective ingredients in the aromatic warming prescription disclosed by the present invention uses a specific heating program, coordinated with a specific injection port temperature, a chromatographic column temperature, and a split ratio, so that the active ingredients of the aromatic warming prescription can be effectively separated in a gas chromatograph, and simultaneously better focused and introduced into a mass spectrometer. Under the participation of specific mass spectrometry conditions, interference of cinnamaldehyde, α-caryophyllene, methyl eugenol, and ligustilide in a chromatogram can be eliminated, and signal intensity and sensitivity are high, thereby ultimately achieving accurate quantification of the four ingredients, cinnamaldehyde, α-caryophyllene, methyl eugenol, and ligustilide. This method provides a reference basis for quality control of the aromatic warming prescription and has practical application value.

[0018] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0019] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Blank (acetone) solution (A), mixed control solution (B), and test solution (C); 1. Acetone, 2. Internal standard solution naphthalene, 3. Cinnamaldehyde, 4. Methyl eugenol, 5. α-caryophyllene, 6. Ligustilide;

[0021] Figure 2 Standard curve of cinnamaldehyde;

[0022] Figure 3 a-Caryophyllene standard curve;

[0023] Figure 4 Standard curve of methyl eugenol;

[0024] Figure 5 Standard curve of ligustilide. DETAILED DESCRIPTION

[0025] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercial products.

[0026] Example 1 Method for determining the contents of four chemical components in the aromatic warming prescription

[0027] 1. Solution Preparation

[0028] 1) Preparation of internal standard solution

[0029] Take the naphthalene reference substance and dissolve it in acetone to prepare an internal standard solution with a concentration of 7.40 mg / mL;

[0030] 2) Preparation of reference substance working solution

[0031] Take cinnamaldehyde standard reference substance, α-caryophyllene standard reference substance, methyl eugenol standard reference substance, and ligustilide standard reference substance, dissolve and dilute with acetone to prepare a single reference substance stock solution with a cinnamaldehyde concentration of 9.30 mg / mL, an α-caryophyllene concentration of 8.15 mg / mL, a methyl eugenol concentration of 9.00 mg / mL, and a ligustilide concentration of 10.00 mg / mL;

[0032] Take 45.0uL of cinnamaldehyde reference substance mother solution, 30.0uL of methyl eugenol reference substance mother solution, 60.0uL of α-caryophyllene reference substance mother solution, and 300.0uL of ligustilide reference substance mother solution, mix them, and add acetone to make up to 9.0mL to obtain a mixed reference substance solution;

[0033] Take 0.1-2.1 ml of the mixed reference solution and add 0.3 ml of the internal standard solution to obtain the reference working solution;

[0034] 3) Preparation of test solution

[0035] Take 61.00 mg of Xiangfang Wentong Fang powder, add 1.8 mL of acetone to extract for 3 hours, then ultrasonicate for 30 minutes, let it stand, take the supernatant, add 0.5 g of anhydrous sodium sulfate to absorb water, filter, take the filtrate, add 0.2 mL of internal standard solution to obtain the test solution;

[0036] 2. Testing

[0037] Separately aspirate the reference substance working solution and the test substance solution and inject them into the gas chromatography-mass spectrometer;

[0038] Chromatographic conditions: chromatographic column: HP-5MS, 30m×0.25mm×0.25μm; programmed temperature: initial temperature 40°C, hold for 1 minute, increase to 230°C at a rate of 10°C per minute, hold for 2 minutes; injection port temperature: 250°C; column temperature: 60°C; split ratio: 20:1.

[0039] Mass spectrometry conditions: ionization mode: electron impact ionization; ion source temperature: 230°C; quadrupole temperature: 150°C; acquisition mode: scan; solvent delay time: 3 minutes;

[0040] 3. Calculation

[0041] The chromatogram of the test solution shows a chromatographic peak with the same retention time as the chromatogram of the reference working solution, which determines that the sample to be tested contains the components corresponding to the reference. The content of these components is calculated by peak area using the internal standard method.

[0042] The beneficial effects of the present invention are further illustrated by the following test examples:

[0043] Experimental Example 1 Study on the simultaneous determination of the contents of four chemical components in Xiangfang Wentong Fang by GC-MS

[0044] 1. Experimental drugs and instruments

[0045] 1.1 Main test drugs

[0046] Cinnamaldehyde standard reference substance, α-caryophyllene standard reference substance, and ligustilide standard reference substance were provided by Shanghai Yuanye Biological Co., Ltd.; methyl eugenol standard reference substance was provided by MedChenExpress; and the aromatic warming prescription sample was provided by Fujian Zhou Jiafu Biopharmaceutical Co., Ltd. The aromatic warming prescription sample was prepared as follows: 10 g of dried ginger, 10 g of turmeric, 10 g of cinnamon bark, 10 g of angelica dahurica, 10 g of cloves, 10 g of Chuanxiong rhizome, 10 g of angelica sinensis, 5 g of Chuanwu, 5 g of Kusnezoffii, 2 g of asarum, 10 g of clematis root, 10 g of radix polygoni lobatae, 10 g of arisaema, 10 g of scutellaria baicalensis, and 5 g of Artemisia selengensis were mixed and crushed to obtain the product.

[0047] 1.2 Main instruments

[0048] Ultrasonic cleaner (model: KQ-500E, manufacturer: Kunshan Ultrasonic Instrument Co., Ltd.), analytical balance (model: B218895724, manufacturer: Sartorius, Germany), Agilent 7890B gas chromatograph (manufacturer: Agilent Technologies, USA), Agilent 5977A mass spectrometer (manufacturer: Agilent Technologies, USA)

[0049] 2 Experimental content

[0050] 2.1 Solution preparation

[0051] 2.1.1 Preparation of internal standard solution

[0052] Accurately weigh the naphthalene reference substance, dissolve it in acetone, and mix well to prepare an internal standard solution with a mass concentration of 7.40 mg / mL.

[0053] 2.1.2 Preparation of test solution

[0054] Take about 61.00 mg of Xiangfang Wentong Fang powder and place it in a PC tube. Accurately pipette 1.8 mL of acetone into the PC tube, extract for 3 hours, ultrasonicate for 30 minutes, let it stand, take the supernatant, add 0.5 g of anhydrous sodium sulfate to absorb water, filter, take the filtrate, and accurately add 0.2 mL of the internal standard solution in 2.1.1 to obtain the test solution of this experiment.

[0055] 2.1.3 Preparation of single reference substance stock solution

[0056] Accurately weigh 9.30 mg of cinnamaldehyde standard reference substance, 8.15 mg of a-caryophyllene standard reference substance, 9.00 mg of methyl eugenol standard reference substance, and 10.00 mg of ligustilide standard reference substance into a PC tube. Accurately measure 1.0 mL of acetone into the PC tube and shake thoroughly to prepare single reference substance mother solutions with a cinnamaldehyde mass concentration of 9.30 mg / mL, a-caryophyllene mass concentration of 8.15 mg / mL, methyl eugenol mass concentration of 9.00 mg / mL, and ligustilide mass concentration of 10.00 mg / mL, respectively, and use them as stock solutions for later use.

[0057] 2.2 Experimental conditions and optimization

[0058] 2.2.1 Optimization of extraction conditions

[0059] Preliminary experiments determined that the extraction solvents for the volatile components of the aromatic warming prescription include acetone, ethanol, n-heptane, etc. Since volatile substances are easily soluble in organic solvents, this experiment selected acetone based on the extraction efficiency and with reference to the extraction peak areas of the three solvents. There are various extraction methods for volatile Chinese medicines, including saturated grinding, ultrasonication, spray drying, etc. Ultrasonication was selected in this experiment because it is simple and easy. This experiment mainly compared the peak elution efficiency of ultrasonication for 10 minutes, 20 minutes, and 30 minutes. The results showed that the peak elution effect was best at 30 minutes. Therefore, this experiment finally decided to use acetone for extraction and prepare the test solution with an ultrasonication time of 30 minutes.

[0060] 2.2.2 Optimization of instrument conditions

[0061] During the preliminary experiments, it was found that when volatile oils such as cinnamaldehyde, a-caryophyllene, methyl eugenol, and ligustilide were qualitatively and quantitatively analyzed using GC-MS coupling technology, a programmed temperature method was required to effectively separate them in the chromatogram. On this basis, after trial and error, three optimized schemes were finally obtained to quantify the four components of cinnamaldehyde, methyl eugenol, a-caryophyllene, and ligustilide using a programmed temperature method. Scheme 1: Only methyl eugenol, cinnamaldehyde, and ligustilide peaked, and a-caryophyllene did not peak; Scheme 2: The elution time of the two components cinnamaldehyde and a-caryophyllene was too short, and the separation time was poor, which affected the experimental efficiency; Scheme 3: Cinnamaldehyde, a-caryophyllene, methyl eugenol, and ligustilide all peaked, and the phase time was appropriate, and the peak shape was relatively sharp and symmetrical. Therefore, Scheme 3 was selected as the final scheme (see Table 1 for the instrument condition optimization record)

[0062] Table 1 Instrument condition optimization record

[0063]

[0064] 2.2.3 Qualitative experimental instrument conditions

[0065] Chromatographic conditions: HP-5MS column (30 m × 0.25 mm × 0.25 μm); programmed temperature method: initial setting temperature is 40 °C, holding time is 1 minute, heating to 230 °C at a rate of 10 °C / min, holding time is 2 minutes; injection port temperature: 250 °C; column temperature: 60 °C; split ratio: 20:1.

[0066] Mass spectrometry conditions: ionization mode: electron impact ionization; ion source temperature: 230°C; quadrupole temperature: 150°C; acquisition mode: scan; solvent delay time: 3 minutes.

[0067] 2.2.4 Quantitative experimental instrument conditions

[0068] Chromatographic conditions: HP-5MS column (30 m × 0.25 mm × 0.25 μm); programmed temperature: initial temperature 40°C, hold for 1 minute, increase to 230°C at a rate of 10°C per minute, hold for 2 minutes; inlet temperature: 250°C; column temperature: 60°C; split ratio: 20:1.

[0069] Mass spectrometry conditions: ionization mode: electron impact ionization; ion source temperature: 230°C; quadrupole temperature: 150°C; acquisition mode: scan; solvent delay time: 3 minutes.

[0070] 2.3 Methodological Investigation

[0071] 2.3.1 System adaptability experiment

[0072] Take the test solution, single reference solution mixed solution and blank solution (acetone) under 2.1.2 and

[0073] Inject samples and measure the experimental instrument conditions under 2.2.3, and record the chromatogram. Figure 1 .Depend on Figure 1 It can be seen that the blank control and internal standard have no interference with the measured components.

[0074] 2.3.2 Linear Investigation

[0075] Accurately measure 45.0uL, 30.0uL, 60.0uL, and 300.0uL of the mother solutions of cinnamaldehyde, methyl eugenol, α-caryophyllene, and ligustilide reference substances under 2.1.3 into a 10.0mL PC tube. Accurately measure an appropriate amount of acetone to make the volume up to 9.0mL. Take 0.1mL, 0.2mL, 0.4mL, 0.7mL, 1.1mL, 1.6mL, and 2.1mL, respectively, into the PC tube. Add 0.3ml of the internal standard solution under 2.1.1 respectively, shake to mix, and inject according to the chromatographic conditions under 2.2.4. Record the peak area. Linear regression curves were drawn with the mass concentration ratios of cinnamaldehyde to the internal standard, the mass concentration ratios of a-caryophyllene to the internal standard, the mass concentration ratios of methyl eugenol to the internal standard, and the mass concentration ratios of ligustilide to the internal standard (x, mg / mL) as the abscissas, and the peak area ratios of cinnamaldehyde to the internal standard, the peak area ratios of a-caryophyllene to the internal standard, the peak area ratios of methyl eugenol to the internal standard, and the peak area ratios of ligustilide to the internal standard (y) as the ordinates. The results are shown in Table 3. Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 .

[0076] Results: The standard curve regression equations of cinnamaldehyde, methyl eugenol, α-caryophyllene, and ligustilide were y=2.3645x-0.0013, y=3.2673x-0.0014, y=3.3016x+0.0004, and y=1.5718x-0.0031, respectively; the r values were 0.9991, 0.9919, 0.9982, and 0.9960, respectively; and the linear ranges of cinnamaldehyde, methyl eugenol, α-caryophyllene, and ligustilide were good in the ranges of 0.00209-0.0440 mg / mL, 0.00135-0.0284 mg / mL, 0.00245-0.0514 mg / mL, and 0.0150-0.315 mg / mL, respectively.

[0077] Table 2 Linear relationship and investigation results

[0078]

[0079] 2.3.3 Precision investigation

[0080] Accurately measure 15.0 μL, 10.0 μL, 20.0 μL, and 100.0 μL of the stock solutions of the individual reference substances (cinnamaldehyde, methyl eugenol, a-caryophyllene, and ligustilide) under 2.1.3 into PC tubes. Accurately add 0.3 mL of the internal standard solution under 2.1.1. Add an appropriate amount of acetone to 3.0 mL to prepare mixed solutions with concentrations of 0.0465 mg / mL for cinnamaldehyde, 0.0300 mg / mL for methyl eugenol, 0.0543 mg / mL for a-caryophyllene, and 0.3330 mg / mL for ligustilide. Transfer 1.0 mL of this mixed solution to an injection vial and make six consecutive injections of 1.0 μL each time using the chromatographic conditions described in 2.2.4. Record the peak areas of each component and the internal standard. Calculate the RSDs using the peak area ratios of each component to the internal standard. The results are shown in Table 3.

[0081] As shown in Table 3, the RSD values of the ratios of the peak areas of cinnamaldehyde, methyl eugenol, α-caryophyllene, and ligustilide to the peak areas of the internal standard were 1.215%, 1.836%, 2.008%, and 3.607%, respectively. The results showed that the instrument had good precision for cinnamaldehyde, methyl eugenol, α-caryophyllene, and ligustilide.

[0082] Table 3 Precision verification results

[0083]

[0084] 2.3.4 Repeatability study

[0085] Prepare the test solution using six portions of Xiangfang Wentong Recipe from the same batch according to the method in 2.1.2. Prepare six replicates and inject 1.0 μL of each solution. Record the peak area and calculate the RSD. Calculate the RSD of each component relative to the internal standard. The results are shown in Table 4.

[0086] As shown in the table, the RSD values of the ratios of the peak areas of cinnamaldehyde, methyl eugenol, α-caryophyllene, and ligustilide to the internal standard peak area are 3.289%, 5.082%, 2.119%, and 1.807%, respectively. These results indicate that the instrument has good repeatability for cinnamaldehyde, methyl eugenol, α-caryophyllene, and ligustilide.

[0087] Table 4 Repeatability test results

[0088]

[0089]

[0090] 2.3.5 Stability investigation

[0091] A test solution (61.00 mg) of the same batch of Xiangfang Wentong Fang was prepared according to the method in 2.1.2. Samples were injected at room temperature at 0, 2, 4, 6, 8, 12, and 24 hours according to the chromatographic conditions in 2.2.4. The peak area ratios of cinnamaldehyde, methyl eugenol, α-caryophyllene, and ligustilide to the internal standard were recorded. The results are shown in Table 5. The RSDs of the ratios of the peak areas of each component to the internal standard were 6.580%, 4.370%, 6.160%, and 1.807%, respectively, indicating that the test solution had good stability for cinnamaldehyde, methyl eugenol, α-caryophyllene, and ligustilide at room temperature for 24 hours.

[0092] Table 5 Stability test results

[0093]

[0094] 2.3.6 Sample recovery rate investigation

[0095] Accurately measure appropriate amounts of the individual reference substance stock solutions under 2.1.3 to prepare mixed reference substance solutions with concentrations of 0.0837 mg / mL for cinnamaldehyde, 0.0774 mg / mL for α-caryophyllene, 0.0788 mg / mL for methyl eugenol, and 1.125 mg / mL for ligustilide. Accurately weigh approximately 58.90 mg of the same batch of Xiangxiang Wentong Fang sample (six portions) into PC tubes. Add 0.1 mL of each of the above reference substance solutions, 0.2 mL of the internal standard solution under 2.1.1, and acetone to 2.0 mL. Prepare the test solution according to 2.1.2 and inject the sample according to the chromatographic conditions described in 2.2.4. Record the peak area ratios of cinnamaldehyde, methyl eugenol, α-caryophyllene, and ligustilide to the internal standard peak area, and calculate the sample recovery (Tables 6, 7, 8, and 9). The concentrations of the tested components are the ratios of the concentrations of cinnamaldehyde, methyl eugenol, α-caryophyllene and ligustilide to the internal standard concentrations, respectively, in mg / mL. The added concentrations are the ratios of the mass concentrations of the added reference substances cinnamaldehyde, methyl eugenol, α-caryophyllene and ligustilide to the internal standard concentrations, respectively, in mg / mL. The measured concentrations are calculated from the actual recorded peak areas of cinnamaldehyde, methyl eugenol, α-caryophyllene and ligustilide and the standard curve, in mg / mL.

[0096] Table 6 Recovery of cinnamaldehyde

[0097]

[0098] Table 7 Methyl eugenol sample recovery

[0099]

[0100] Table 8a-Caryophyllene sample recovery

[0101]

[0102]

[0103] Table 9 Recovery of ligustilide

[0104]

[0105] 2.3.7 Content determination

[0106] Three batches of aromatic Wentong Fang samples from the same manufacturer were prepared according to the method in 2.1.2. Samples were injected into each batch under the chromatographic conditions in 2.2.4 with a 1.0 μL injection volume. The peak areas were recorded, and the contents of cinnamaldehyde, methyl eugenol, α-caryophyllene, and ligustilide were calculated, as shown in Tables 10, 11, 12, and 13. The contents of cinnamaldehyde, methyl eugenol, α-caryophyllene, and ligustilide were 1.57, 1.64, and 1.57, respectively, with RSDs of 1.57, 1.67, and 1.66, indicating a relatively stable production process.

[0107] Table 10 Determination of cinnamaldehyde content

[0108]

[0109] Table 11 Determination of the content of methyl eugenol

[0110]

[0111] Table 12a-Determination of caryophyllene content

[0112]

[0113] Table 13 Determination of the content of ligustilide

[0114]

[0115] 3 Discussions

[0116] 3.1 Optimization of the determination process of volatile components

[0117] Volatile components primarily refer to volatile oils and volatile small-molecule solids contained in medicinal materials. These components are widely found in various traditional Chinese medicines, including diaphoretic and antipyretic drugs, qi-invigorating and blood-activating drugs, and aromatic and dampness-removing drugs. They primarily include terpenes, aromatic and aliphatic compounds, and their oxygenated derivatives. In recent years, extensive research and practice have been conducted both domestically and internationally on the extraction, analysis, and content determination of the basic substances in volatile Chinese medicinal materials and compound Chinese patent medicines, yielding a series of promising results. For example, supercritical CO2 fluid extraction and microwave extraction have demonstrated excellent extraction and separation effects for volatile components. While each of these methods has its merits, a systematic review of the traditional therapeutic concepts and applications of volatile components in traditional Chinese medicines remains relatively lacking. This study considered both extraction followed by reflux extraction and extraction followed by ultrasonic extraction. Based on the experimental results, extraction followed by ultrasonic extraction was selected for the extraction of volatile components from the aromatic warming formula due to its relative simplicity, solvent conservation, and the ability to extract a relatively large number of components for analysis.

[0118] 3.2 Selection of effective chemical components of aromatic warming prescriptions

[0119] The aromatic warming formula (Aromatic Warming and Unblocking Formula) is composed of dried ginger, turmeric, cinnamon bark, angelica dahurica, cloves, Chuanxiong rhizome, angelica sinensis, Chuanwu (Rhizoma Aconiti Kusnezoffii), asarum, clematis root, radix polygoni multiflori, arisaema, truncatum sinensis, and Artemisia selengensis. This multi-ingredient formula has a complex active chemical composition, with multiple components complementing each other, primarily volatile components. Qualitative screening experiments on the test samples identified volatile components including cinnamaldehyde, 2-methoxy-4-vinylphenol, eugenol, copaene, methyl eugenol, caryophyllene, coumarin, humulene, α-curcumene, turmerone, trimethoxyacetophenone, a new bisabolane-based hemiterpenoid, ligustilide, and trans-ligustilide. Finally, comprehensive content, precision, reproducibility, and stability assessments confirmed that cinnamaldehyde, methyl eugenol, α-caryophyllene, and ligustilide represent the overall quality of the aromatic warming formula.

[0120] 3.3 Optimization of experimental internal standards

[0121] The internal standard method and the external standard method are commonly used to determine drug content. Each method has its own advantages. Studies have found that while the external standard method is simple, it requires very precise injection volume and strict control of chromatographic conditions, as these are used for the standard, otherwise analytical errors can easily occur. The internal standard method offers greater accuracy. This experiment examined the GC / MS chromatograms of dodecane and naphthalene. The results showed that the peak area of naphthalene showed no interference with the other components, indicating good resolution. Using naphthalene as an internal standard for quantitative analysis can reduce the impact of sample handling, instrument response fluctuations, and interfering substances.

[0122] 4 Conclusion

[0123] In this experiment, the contents of cinnamaldehyde, methyl eugenol, a-caryophyllene and ligustilide were used as indicators. The contents of cinnamaldehyde, methyl eugenol, a-caryophyllene and ligustilide in aromatic warming paste were determined by gas chromatography-mass spectrometry, and the contents were calculated by the standard curve method of internal standard method. The results of methodological investigation showed that the linear regression equations of cinnamaldehyde, methyl eugenol, a-caryophyllene and ligustilide were y=2.3645x-0.0013, y=3.2673 ... .0014, y=3.3016x+0.0004, y=1.5718x-0.0031; r values were 0.9991, 0.9919, 0.9982, 0.9960, respectively; linear ranges were 0.00209-0.0440 mg / mL, 0.00135-0.0284 mg / mL, 0.00245-0.0514 mg / mL, 0.0150-0.315 mg / mL, respectively.

[0124] The precision study showed that the RSD values of the ratios of the peak areas of cinnamaldehyde, methyl eugenol, a-caryophyllene and ligustilide to the internal standard peak area were 1.215%, 1.836%, 2.008% and 3.607%, respectively.

[0125] Repeatability test showed that the RSD values of the ratios of the peak areas of cinnamaldehyde, methyl eugenol, a-caryophyllene and ligustilide to the peak area of the internal standard were 3.389%, 5.082%, 2.119% and 1.807%, respectively.

[0126] Stability study showed that the RSD values of cinnamaldehyde, methyl eugenol, a-caryophyllene and ligustilide obtained by sampling and recording the ratio of peak area to internal standard within 24 hours were 6.580%, 4.370%, 6.160% and 1.807%, respectively.

[0127] The recovery rate investigation showed that the recovery rate of cinnamaldehyde was 110.68%-115.60%, with an average recovery rate of 113.05%; the recovery rate of methyl eugenol was 111.07%-119.75%, with an average recovery rate of 115.60%; the recovery rate of a-caryophyllene was 84.00%-91.16%, with an average recovery rate of 87.06%; the recovery rate of ligustilide was 97.38%-106.71%, with an average recovery rate of 101.58%; the RSDs of the recoveries of the four components were 1.84%, 1.33%, 1.41% and 1.77%, respectively.

[0128] From the above results, it can be concluded that the method used in this experiment is convenient, precise, reproducible, stable and feasible. It can be used as a content determination method for the aromatic warming prescription and used for the quality control of the aromatic warming prescription, providing a reference basis for the quality control of the aromatic warming prescription.

Claims

1. A GC-MS method for determining the active ingredients in aromatic warming prescription, characterized in that: It includes the following steps: a. Preparation of test solution: Take the aromatic Wentong formula to be tested, extract it with acetone, add anhydrous sodium sulfate to the supernatant of the extract to absorb water, filter, take the filtrate, and add the internal standard solution to obtain the solution; b. Preparation of reference substance working solution: Dissolve cinnamaldehyde, α-caryophyllene, methyl eugenol and / or ligustilide reference substances in acetone, and add internal standard solution to the dissolved solution to obtain the reference substance. c. Separately aspirate the test solution and reference working solution and inject them into the GC-MS instrument. The chromatographic conditions are as follows: chromatographic column: capillary column; programmed temperature method; mass spectrometry conditions: ionization mode: electron impact ionization, acquisition mode: scan.

2. The GC-MS method according to claim 1, wherein: In step a, the mass volume ratio of the aromatic warming prescription to acetone, anhydrous sodium sulfate and internal standard solution is 50-100 mg: 1-3 ml: 0.1-1 g: 1.1-0.5 ml.

3. The GC-MS method according to claim 1, wherein: In step b, the volume ratio of the dissolving solution to the internal standard solution is 0.1-5:0.3; the concentration of each reference substance in the dissolving solution is 0.01-0.5 mg / mL.

4. The GC-MS method according to any one of claims 1 to 3, characterized in that: The internal standard solution is an acetone solution containing naphthalene; the concentration of naphthalene in the acetone solution is 5-10 mg / mL.

5. The GC-MS method according to claim 1, wherein: In the chromatographic conditions of step c, the chromatographic column is a capillary column filled with (5%-phenyl)-methylpolysiloxane, preferably an HP-5MS chromatographic column, 30m×0.25mm×0.25μm; the heating program is: initial temperature 40°C, hold for 1 minute, heat to 230°C at a rate of 10°C per minute, hold for 2 minutes; injection port temperature: 200-300°C; chromatographic column temperature: 50-70°C; split ratio: 10-30:

1.

6. The GC-MS method according to claim 5, wherein: The injection port temperature is 250° C.; the chromatographic column temperature is 60° C.; and the split ratio is 20:

1.

7. The GC-MS method according to claim 1, wherein: The mass spectrometry conditions in step c are: ion source temperature: 200-300° C.; quadrupole temperature: 100-200° C.; solvent delay time: 2-5 minutes.

8. The GC-MS method according to claim 7, wherein: The mass spectrometry conditions are as follows: ion source temperature: 230° C.; quadrupole temperature: 150° C.; acquisition mode: scan; solvent delay time: 3 minutes.

9. The GC-MS method according to claim 1, wherein: The chromatogram of the test solution shows a chromatographic peak with the same retention time as that in the chromatogram of the reference working solution, confirming that the aromatic warming prescription to be tested contains the component corresponding to the reference; the content of the component corresponding to the reference in the aromatic warming prescription is calculated by peak area using the internal standard method.

10. The GC-MS method according to claim 1, wherein: The aromatic warming prescription is a formula raw material drug or formula preparation composed of dried ginger, turmeric, cinnamon, angelica dahurica, cloves, ligusticum chuanxiong, angelica sinensis, chuanwu, kusnezoffii, asarum, clematis root, pelargonium scabra, arisaema, sinomenine root and artemisia selengensis; the mass ratio of the dried ginger, turmeric, cinnamon, angelica dahurica, cloves, ligusticum chuanxiong, angelica sinensis, chuanwu, kusnezoffii, asarum, clematis root, pelargonium scabra, arisaema, sinomenine root and artemisia selengensis is: 10:10:10:10:10:10:5:5:2:10:10:10:10:5.

Images