Construction method and application of characteristic chromatogram of Liangtoutianjian standard decoction

Abstract

The application discloses a kind of two head tip standard decoction feature spectrum construction method, comprising the following steps: preparation reference solution, the reference solution includes control medicinal material reference solution and control reference solution, the control reference solution includes chicoric acid control product, bamboo joint spice element A control product and coffee acid control product in reference solution;Two head tip standard decoction is prepared for test solution;Take preset amount of reference solution and test solution, inject into liquid chromatograph, the liquid chromatograph is with octadecylsilane bonding silica gel as filler, with acetonitrile as mobile phase A, with phosphoric acid solution as mobile phase B and carries out gradient elution, establishes characteristic spectrum.The application can provide data basis for two head tip standard decoction, medicinal material quality control, effectively guarantee the stability and controllability of two head tip medicinal material, standard decoction product quality.

Description

Technical Field

[0001] The present invention relates to the technical field of quality analysis and detection of traditional Chinese medicines, and in particular to a method for constructing a characteristic spectrum of a Liangtoujian standard decoction and its application. Background Art

[0002] Two-headed thorn (Liangtoujian) is the dried rhizome of Anemone raddeana Regel, a plant in the Ranunculaceae family. It is harvested in summer, the fibrous roots removed, washed, and dried. Also known as Easter flower, wind flower, silver thorn, and bamboo-jointed cyperus, it is pungent, hot, and toxic. It enters the spleen meridian, and its rhizome is used as a medicinal herb, known for its anti-rheumatic and carbuncle-relieving properties. It is used to treat wind-cold-dampness arthralgia, limb cramps, joint pain, and carbuncle ulcers. Two-headed thorn (Liangtoujian) was first recorded as a traditional Chinese medicine in the Ming Dynasty book "Compendium of Materia Medica," written by Liu Wentai. It states: "Two-headed thorn (Liangtoujian) is a type of aconite, with similar seedlings and leaves. Its roots resemble those of Aconitum kusnezoffii, with dark skin and white flesh, thin, and pointed at both ends, hence the name." Two-headed thorn (Liangtoujian) grows wild in mountain forests or shady grasslands at an altitude of approximately 800 meters. It is primarily distributed in the three northeastern provinces, but is also found in Shandong and Hebei. Existing studies on the fingerprint / characteristic spectrum of liangtoujian have shown poor overall separation of chromatographic peaks and limited peak information, failing to fully reflect the intrinsic quality of liangtoujian. For example, Zhang Yanfei et al. used HPLC to establish an HPLC fingerprint spectrum for liangtoujian medicinal materials. This fingerprint identified 11 common peaks, none of which were clearly identified, and the separation of the characteristic peaks was poor. Yan Panfeng et al. used HPLC to determine the spectrum of monosaccharides in liangtoujian, which revealed seven common peaks. However, the overall separation of this fingerprint spectrum was poor and lacked specificity.

[0003] In addition, there is not much research on the quality standards of Liangtoujian. The 2020 edition of the Chinese Pharmacopoeia only includes properties, microscopic identification, thin layer identification, and content determination indicators under the quality standards of Liangtoujian medicinal materials and decoction pieces. It lacks specific characteristic maps, and the quality standards are still incomplete. Moreover, the standard decoction of Liangtoujian is made from Liangtoujian decoction pieces through water extraction, concentration, drying and other processes. Compared with the decoction pieces of medicinal materials, it has lost its inherent morphology and lacks important indicators such as properties and identification in the quality standards. At present, there is no effective quality standard for Liangtoujian standard decoction, which cannot be effectively controlled and comprehensively evaluated. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for constructing a characteristic spectrum of a Liangtoujian standard decoction. The method has good reproducibility, accuracy and reliability, and can provide a data basis for the mass production quality control of the Liangtoujian standard decoction, thereby ensuring the stability and controllability of the quality of the Liangtoujian standard decoction product.

[0005] In order to solve the above technical problems, the present invention provides a method for constructing a characteristic spectrum of a standard decoction of Liangtoujian, comprising the following steps: preparing a reference solution, wherein the reference solution comprises a reference medicinal material reference solution and a reference substance reference solution, wherein the reference substance reference solution comprises a chicoric acid reference substance, a japonicus cyperus A reference substance, and a caffeic acid reference substance;

[0006] Prepare the test solution by taking the standard decoction of Liangtoujian;

[0007] Take a preset amount of reference solution and test solution and inject them into a liquid chromatograph. The liquid chromatograph uses octadecylsilane bonded silica gel as a filler, acetonitrile as mobile phase A, and phosphoric acid solution as mobile phase B for gradient elution to establish a characteristic spectrum.

[0008] In one embodiment, the gradient elution is performed according to the following procedure:

[0009] 0-10 min, mobile phase A from 5% to 17%, mobile phase B from 95% to 83%;

[0010] 10-23 min: mobile phase A from 17% to 29%, mobile phase B from 83% to 71%;

[0011] From 23 to 28 minutes, mobile phase A was maintained at 29% and mobile phase B was maintained at 71%;

[0012] 28-33 min, mobile phase A from 29% to 45%, mobile phase B from 71% to 55%;

[0013] From 33 to 45 minutes, mobile phase A changes from 45% to 80%, and mobile phase B changes from 55% to 20%.

[0014] In one embodiment, the volume concentration of the phosphoric acid solution is 0.15% to 0.25%.

[0015] In one embodiment, the preset amount of the reference solution and the test solution is 0.5 μL to 1.5 μL;

[0016] The liquid chromatograph uses octadecylsilane bonded silica gel as a filler, with a column length of 100 mm, an inner diameter of 2.1 mm, a particle size of 1.7 μm, and a column temperature of 38°C to 42°C;

[0017] When performing gradient elution, the flow rate of the mobile phase is 0.28-0.32 mL / min;

[0018] The ultraviolet detection wavelength of the liquid chromatograph is 200nm to 210nm.

[0019] In one embodiment, the test solution is prepared by the following method:

[0020] Take the standard decoction of Liangtoujian, grind it into powder, weigh 0.49g~0.51g, accurately weigh it, place it in a stoppered conical flask, accurately add 20mL~30mL of 65%~75% methanol, heat and reflux for 15min~45min, filter, evaporate the filtrate to dryness, dissolve the residue in 20mL~21mL of water, shake and extract with water-saturated n-butanol 3 times~5 times, 25mL~26mL each time, combine the n-butanol liquids, evaporate to dryness, dissolve the residue in methanol and transfer it to a 5mL volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate to obtain.

[0021] In one embodiment, the reference substance solution is prepared according to the following method:

[0022] Take appropriate amounts of chicoric acid reference substance, bamboo cyperin A reference substance, and caffeic acid reference substance, accurately weigh them, and add methanol to prepare a mixed solution containing 41.4238 μg of chicoric acid, 17.4902 μg of bamboo cyperin A, and 15.0148 μg of caffeic acid per 1 mL, which is used as the reference substance solution.

[0023] In one embodiment, the control medicinal material reference solution is prepared by the following method:

[0024] Take 0.8g~1.2g of the medicinal material of Liangtoujian, add 20mL~30mL of water, heat and reflux for 15min~45min, centrifuge, take the supernatant, evaporate to dryness, add 20mL~30mL of 65%~75% methanol to the residue, heat and reflux for 15min~45min, filter, evaporate the filtrate to dryness, dissolve the residue in 20mL~21mL of water, shake and extract with water-saturated n-butanol 3 times~5 times, 25mL~26mL each time, combine the n-butanol liquid, evaporate to dryness, dissolve the residue in methanol and transfer it to a 5mL volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate to obtain the reference solution of the control medicinal material.

[0025] In one embodiment, the characteristic spectrum includes 9 common peaks, wherein Peak 1 corresponds to caffeic acid, Peak 2 corresponds to chicoric acid, and Peak 9 corresponds to cyperus rotundus A.

[0026] In one embodiment, in the characteristic spectrum of the Liangtoujian standard decoction, the chicoric acid chromatographic peak is used as the reference peak S1, and the relative retention times of peaks 3 and 4 and the reference peak S1 are consistent with: peak 3 1.24, peak 4 1.59, and the RSD value of the relative retention time of each characteristic peak is less than 3%;

[0027] With the chromatographic peak of rhizoma zedoariae element A as the reference peak S2, the relative retention time of the peaks 5, 6, 7 and 8 conforms to: 0.72 for the peak 5, 0.75 for the peak 6, 0.85 for the peak 7 and 0.91 for the peak 8, and the RSD value of the relative retention time of each characteristic peak is less than 3%.

[0028] Correspondingly, the application further provides a characteristic spectrum of the standard decoction of Aconitum flavum, which is obtained by the above-mentioned method for constructing the characteristic spectrum of the standard decoction of Aconitum flavum.

[0029] And application of the characteristic spectrum of the standard decoction of Aconitum flavum in any one of (1) to (5):

[0030] (1) identification and component detection of the standard decoction of Aconitum flavum;

[0031] (2) identification and component detection of Aconitum flavum medicinal materials;

[0032] (3) differentiation between the standard decoction of Aconitum flavum and the standard decoction of Aconitum coreanum;

[0033] (4) differentiation between the standard decoction of Aconitum flavum and the standard decoction of Aconitum coreanum;

[0034] (5) differentiation between the standard decoction of Aconitum flavum and the standard decoction of rhizoma zedoariae.

[0035] The application has the following beneficial effects:

[0036] The application first establishes the characteristic spectrum of the standard decoction of Aconitum flavum by the method for constructing the characteristic spectrum, which can fully display the chemical component characteristics of the standard decoction of Aconitum flavum, and the characteristic peak information is rich, which can comprehensively reflect the quality information of the standard decoction of Aconitum flavum, so as to achieve the purpose of comprehensively and effectively controlling the quality of the standard decoction of Aconitum flavum. And the application can realize identification and component detection of Aconitum flavum medicinal materials, and differentiation between the standard decoction of Aconitum flavum and the standard decoction of Aconitum coreanum, the standard decoction of Aconitum coreanum or the standard decoction of rhizoma zedoariae, which provides a reference basis for identification of Aconitum flavum medicinal materials and standard decoction. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is the chromatogram of the standard decoction of Aconitum flavum in Example 1 of the application under the detection wavelength of 254 nm;

[0038] Figure 2 is the chromatogram of the standard decoction of Aconitum flavum in Example 1 of the application under the detection wavelength of 280 nm;

[0039] Figure 3 is the chromatogram of the standard decoction of Aconitum flavum in Example 1 of the application under the detection wavelength of 206 nm;

[0040] Figure 4 This is a 3D chromatogram of the Liangtoujian standard decoction in Example 1 of the present invention;

[0041] Figure 5 This is a characteristic chromatogram obtained by method 1 for sample 1 of the Liangtoujian standard decoction in Example 1 of the present invention;

[0042] Figure 6 This is the characteristic chromatogram of sample 2 of the Liangtoujian standard decoction obtained by method 1 in Example 1 of the present invention;

[0043] Figure 7 This is the characteristic chromatogram of sample 3 of the Liangtoujian standard decoction obtained by method 1 in Example 1 of the present invention;

[0044] Figure 8 This is the characteristic chromatogram of sample 3 of the Liangtoujian standard decoction obtained by method 2 in Example 1 of the present invention;

[0045] Figure 9 This is the characteristic chromatogram obtained by method 3 for sample 3 of the Liangtoujian standard decoction in comparative example 1 of the present invention;

[0046] Figure 10 This is the characteristic chromatogram obtained by method 4 for sample 3 of the Liangtoujian standard decoction in Example 1 of the present invention;

[0047] Figure 11 This is the characteristic chromatogram obtained by method 5 for sample 3 of the Liangtoujian standard decoction in Example 1 of the present invention;

[0048] Figure 12 This is the characteristic chromatogram (30° C.) of the Liangtoujian standard decoction sample 3 in Example 1 of the present invention;

[0049] Figure 13 This is the characteristic chromatogram (40° C.) of the Liangtoujian standard decoction sample 3 in Example 1 of the present invention;

[0050] Figure 14 This is the result of investigation of different extraction solvents for the characteristic spectrum of the standard decoction of Liangtoujian in Example 1 of the present invention;

[0051] Figure 15 This is the result of investigating different extraction methods for the characteristic spectrum of the standard decoction of Liangtoujian in Example 1 of the present invention;

[0052] Figure 16 This is the result of investigating the characteristic spectrum of the standard decoction of Liangtoujian at different extraction times in Example 1 of the present invention;

[0053] Figure 17 This is the result of the investigation of the number of extraction times of the characteristic spectrum of the standard decoction of Liangtoujian in Example 1 of the present invention;

[0054] Figure 18 This is the common pattern of the characteristic spectra of 16 batches of Liangtoujian standard decoction in Example 1 of the present invention;

[0055] Figure 19 This is the reference characteristic spectrum of the standard decoction of Liangtoujian in Example 1 of the invention, wherein: Peak 1: Caffeic acid; Peak 2 (S1): Chicoric acid; Peak 9 (S2): Cyperus rotundus A;

[0056] Figure 20 This is the characteristic spectrum of the reference medicinal material of Liangtoujian in Example 1 of the invention, wherein, Peak 1: Caffeic acid; Peak 2 (S1): Chicoric acid; Peak 9 (S2): Bamboo cyperin A;

[0057] Figure 21 The total ion current diagram and ultraviolet absorption chromatogram of the test solution of the Liangtoujian standard decoction in Example 1 of the present invention are shown;

[0058] Figure 22 It is a reference substance confirmation of the characteristic peaks of the characteristic spectrum of the Liangtoujian standard decoction in Example 1 of the present invention;

[0059] Figure 23 This is the characteristic spectrum of the standard decoction of Liangtoujian in Example 1 of the present invention (different chromatographs);

[0060] Figure 24 This is a graph showing the durability test results of the characteristic spectrum of the Liangtoujian standard decoction in Example 1 of the present invention (chromatographic column of the same brand);

[0061] Figure 25 This is a chromatogram showing the durability of the Liangtoujian standard decoction in Example 1 of the present invention at different column temperatures;

[0062] Figure 26 This is a chromatogram showing the durability of the Liangtoujian standard decoction in Example 1 of the present invention at different flow rates;

[0063] Figure 27 This is a comparison chart of the characteristic spectra of the standard decoction of Liangtoujian and the standard decoction of Northeast Nine-section Acorus calamus (Black Mercurial Lotus) in Example 2 of the present invention;

[0064] Figure 28 This is a comparison chart of the characteristic spectra of the standard decoction of Liangtoujian and the standard decoction of Jiujiechangcalamus in Example 3 of the present invention;

[0065] Figure 29 It is a comparative diagram of the characteristic spectra of the standard decoction of Liangtoujian and the standard decoction of Cyperus rotundus in Example 4 of the present invention. DETAILED DESCRIPTION

[0066] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0067] In order to comprehensively reflect the quality information of Liangtoujian standard decoction and achieve comprehensive and effective control of the quality of Liangtoujian standard decoction, the present invention provides a method for constructing a characteristic spectrum of Liangtoujian standard decoction, which is described in detail below:

[0068] Example 1: A method for constructing a characteristic spectrum of a standard decoction of Liangtoujian, the specific implementation steps are as follows:

[0069] 1.1 Instruments, reagents and test drugs

[0070] Instruments: Waters ultra-high performance liquid chromatograph (H-class, Waters Corporation), Thermo ultra-high performance liquid chromatograph (Vanquish, Thermo Fisher Scientific (China) Co., Ltd.), Waters ACQUITY BEH C18 (2.1 mm × 100 mm, 1.7 μm, models: BH-374, BH-307, BH-308), 1 / 10,000 analytical electronic balance (ME204E, Mettler-Toledo), 1 / 10,000 analytical electronic balance (XP26, Mettler-Toledo), electric constant temperature water bath (HWS-28, Shanghai Yiheng Technology Co., Ltd.), CNC ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.), ultrapure water system (Milli-Q Direct, Merck).

[0071] Reagents: Ethanol (Xilong Technology Co., Ltd.) and methanol (Xilong Technology Co., Ltd.) were of analytical grade; phosphoric acid (Tianjin Komiou Chemical Reagent Co., Ltd.) and acetonitrile (Merck Co., Ltd.) for liquid phase were of HPLC grade; and water was ultrapure water (prepared in the laboratory).

[0072] Test drugs: Cyperus rotundus A reference substance (batch number: 111712-201702, content: 96.1%, China Food and Drug Inspection Institute); Caffeic acid reference substance (batch number: 110885-201703, content: 99.7%, China Food and Drug Inspection Institute); Chicoric acid reference substance (batch number: 111752-202104, content: 99.1%, China Food and Drug Inspection Institute); Liangtoujian reference medicinal material (batch number: G1911018, Xihe Town, Shulan City, Jilin Province); 16 batches of Liangtoujian standard decoctions. Table 1 is the origin information table of 16 batches of Liangtoujian decoction pieces and their standard decoctions.

[0073] Table 1 Origin information of 16 batches of Liangtoujian slices and their standard decoctions

[0074]

[0075]

[0076] 1.2 Preparation of reference solution

[0077] Take 1.0 g of the two-headed sharp reference medicinal material, add 25 mL of water, heat and reflux for 30 minutes, centrifuge, take the supernatant, evaporate to dryness, add 25 mL of 70% methanol to the residue, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20 mL of water, shake and extract 4 times with water-saturated n-butanol, 25 mL each time, combine the n-butanol liquid, evaporate to dryness, dissolve the residue in methanol and transfer it to a 5 mL volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate to obtain the reference solution of the reference medicinal material.

[0078] Accurately weigh 2.071 mg of chicoric acid reference substance, 0.874 mg of bamboo cyperin A reference substance, and 0.750 mg of caffeic acid reference substance, place them in a 50 mL volumetric flask, add methanol to make a solution containing 41.4238 μg of chicoric acid, 17.4902 μg of bamboo cyperin A, and 15.0148 μg of caffeic acid per 1 mL, shake well, and obtain the reference substance solution.

[0079] 1.3 Optimization of chromatographic conditions

[0080] (1) Determination of the optimal absorption wavelength

[0081] The detection wavelengths of the characteristic spectrum of the two-headed tip are mainly 206nm, 254nm, and 280nm, which are the maximum absorption wavelengths of the two-headed tip saponins. Therefore, the chromatograms at these different wavelengths are selected for comparison to determine the optimal absorption wavelength.

[0082] Take an appropriate amount of Liangtoujian standard decoction (BT04), grind it into powder, take about 0.5g, weigh it accurately, add 25mL of 70% methanol, weigh it, and treat it ultrasonically (power 250W, frequency 40kHz) for 30 minutes. Take it out, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0083] Chromatographic conditions: A Waters ACQUITY BEH C18 column (2.1 mm × 100 mm, 1.7 μm) was used; acetonitrile was used as mobile phase A, and 0.2% phosphoric acid solution was used as mobile phase B, with gradient elution as specified in Table 2; the flow rate was 0.30 mL / min; the column temperature was 30°C; and the injection volume was 1 μL.

[0084] Table 2 Gradient elution table

[0085]

[0086]

[0087] Depend on Figures 1 to 4The results show that by comparing the chromatograms of the three detection wavelengths, when 206 nm is selected as the detection wavelength, the peak information is richer, the response values ​​of each characteristic peak are larger, the baseline is stable, and the interference is smaller. Therefore, 206 nm is selected as the detection wavelength.

[0088] (2) Optimization of chromatographic conditions

[0089] Method 1: A Waters ACQUITY BEH C18 column (2.1 mm × 100 mm, 1.7 μm) was used; acetonitrile was used as mobile phase A, 0.2% phosphoric acid solution was used as mobile phase B, and the gradient elution was performed according to Table 3 at a flow rate of 0.30 mL / min; the column temperature was 30°C; the injection volume was 1 μL; and the detection wavelength was 206 nm. Results are shown in Figures 5-7 .

[0090] Take an appropriate amount of Liangtoujian standard decoction (BT04), grind it into powder, take about 0.5g, divide it into 3 parallel portions, weigh them accurately, and place them in a stoppered conical flask.

[0091] Sample 1: Accurately add 25 mL of 70% methanol, weigh the sample, and ultrasonically treat the sample (power 250 W, frequency 40 kHz) for 30 minutes. Remove the sample, let it cool, and weigh it again. Make up the lost weight with 70% methanol, shake well, filter, and take the filtrate.

[0092] Sample 2: Accurately add 25 mL of 70% methanol, weigh the weight, and ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes. Take out, let cool, and weigh the weight again. Make up the lost weight with 70% methanol, shake well, filter, and evaporate the filtrate to dryness. Add 20 mL of water to dissolve the residue, and shake and extract with ethyl acetate 3 times, 20 mL each time. Combine the ethyl acetate, evaporate to dryness, dissolve the residue in methanol and transfer it to a 5 mL volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate.

[0093] Sample 3: Accurately add 25 mL of 70% methanol, weigh the weight, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, take out, let cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, evaporate the filtrate to dryness, add 20 mL of water to dissolve the residue, shake and extract 3 times with 20 mL of water-saturated n-butanol, combine the water-saturated n-butanol solution, evaporate to dryness, dissolve the residue with methanol and transfer to a 5 mL volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate.

[0094] Table 3 Characteristic spectrum of Liangtoujian standard decoction gradient elution table

[0095]

[0096] Depend on Figures 5 to 7The results showed that under the current chromatographic conditions, sample three had more chromatographic information and the chromatographic peaks were more evenly distributed. Therefore, sample three was determined as the sample preparation method to further optimize the gradient and increase the chromatographic information.

[0097] Method 2: A Waters ACQUITY BEH C18 column (2.1 mm × 100 mm, 1.7 μm) was used; acetonitrile was used as mobile phase A, 0.2% phosphoric acid solution was used as mobile phase B, and the gradient elution was performed according to Table 4 at a flow rate of 0.30 mL / min; the column temperature was 30°C; the injection volume was 1 μL; and the detection wavelength was 206 nm. Results are shown in Figure 8 .

[0098] Table 4 Gradient elution table

[0099]

[0100] Figure 8 The results showed that under the current chromatographic conditions, the amount of chromatographic information was relatively small, and the next step was to further optimize the gradient to increase the amount of chromatographic information.

[0101] Method 3: A Waters ACQUITY BEH C18 column (2.1 mm × 100 mm, 1.7 μm) was used; acetonitrile was used as mobile phase A, 0.2% phosphoric acid solution was used as mobile phase B, and the gradient elution was performed according to Table 5 at a flow rate of 0.30 mL / min; the column temperature was 30°C; the injection volume was 1 μL; and the detection wavelength was 206 nm. Results are shown in Figure 9 .

[0102] Table 5 Gradient elution table

[0103]

[0104] Figure 9 The results showed that under the current chromatographic conditions, the chromatographic peak separation was poor, and the gradient was further optimized to increase the chromatographic peak separation.

[0105] Method 4: A Waters ACQUITY BEH C18 column (2.1 mm × 100 mm, 1.7 μm) was used; acetonitrile was used as mobile phase A, 0.2% phosphoric acid solution was used as mobile phase B, and the gradient elution was performed according to Table 6 at a flow rate of 0.30 mL / min; the column temperature was 30°C; the injection volume was 1 μL; and the detection wavelength was 206 nm. Results are shown in Figure 10 .

[0106] Table 6 Gradient elution table

[0107]

[0108] Figure 10The results showed that under the current chromatographic conditions, the separation of the chromatographic peaks in the second half was poor, and the gradient should be further optimized to increase the separation of the chromatographic peaks.

[0109] Method 5: A Waters ACQUITY BEH C18 column (2.1 mm × 100 mm, 1.7 μm) was used; acetonitrile was used as mobile phase A, 0.2% phosphoric acid solution was used as mobile phase B, and the gradient elution was performed according to Table 7 at a flow rate of 0.30 mL / min; the column temperature was 30°C; the injection volume was 1 μL; and the detection wavelength was 206 nm. Results are shown in Figure 11 .

[0110] Table 7 Characteristic chromatogram of Liangtoujian standard decoction gradient elution table

[0111]

[0112] Figure 11 The results showed that under the current chromatographic conditions, the chromatographic information content was large, the chromatographic peaks were evenly distributed, and the separation was good. Therefore, method 5 was determined as the chromatographic condition.

[0113] (4) Determination of temperature

[0114] A Waters ACQUITY BEH C18 column (2.1 mm × 100 mm, 1.7 μm) was used; acetonitrile was used as mobile phase A, 0.2% phosphoric acid solution was used as mobile phase B, and the gradient elution was performed according to the gradient specified in Table 8 at a flow rate of 0.30 mL / min; the column temperature was 30°C and 40°C; the injection volume was 1 μL; and the detection wavelength was 206 nm. Figures 12-13 .

[0115] Table 8 Gradient elution table

[0116]

[0117] Figures 12 and 13 The results showed that under the current chromatographic conditions, the chromatographic peak separation at 40°C was better and the peak shape was better, so the column temperature was determined to be 40°C.

[0118] (4) Determination of chromatographic conditions

[0119] A Waters ACQUITY BEH C18 (2.1 mm × 100 mm, 1.7 μm) column was used; acetonitrile was used as mobile phase A, 0.2% phosphoric acid solution was used as mobile phase B, and elution was performed according to the gradient specified in Table 9 at a flow rate of 0.30 mL / min; the column temperature was 40°C; the injection volume was 1 μL; and the detection wavelength was 206 nm.

[0120] Table 9 Gradient elution table

[0121]

[0122] 1.4. Investigation of the pretreatment method of the test solution

[0123] The sample pretreatment methods of the characteristic spectrum of the standard decoction of Liangtoujian were investigated, focusing on the effects of extraction solvent, extraction method, extraction time and extraction solvent dosage on the characteristic spectrum.

[0124] (1) Investigation of extraction solvent

[0125] This experiment investigated the effects of different extraction solvents on the characteristic spectrum of the standard decoction of Liangtoujian. Methanol, 70% methanol, 50% methanol, ethanol, 70% ethanol, and 50% ethanol were selected as extraction solvents. By observing the peak shape and separation effect of the 9 tentative characteristic peaks and calculating the "total peak area / sample weight" value of the 9 characteristic peaks, the effects of different extraction solvents on the characteristic spectrum of the standard decoction of Liangtoujian were compared, and the optimal extraction solvent was selected.

[0126] Take an appropriate amount of the standard decoction of Liangtoujian (No.: BT04), grind it into powder, take about 0.5g, accurately weigh it, and divide it into 6 parallel groups, 2 portions for each group, and place it in a stoppered conical flask. Accurately add 25mL of methanol, 70% methanol, 50% methanol, ethanol, 70% ethanol, and 50% ethanol, weigh the weight, and ultrasonically treat it (power 300W, frequency 40kHz) for 30 minutes. Take it out, let it cool, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, evaporate the filtrate to dryness, dissolve the residue in 20mL of water, and shake and extract it 3 times with water-saturated n-butanol, 25mL each time. Combine the n-butanol liquid, evaporate it to dryness, dissolve the residue in methanol and transfer it to a 5mL volumetric flask, add methanol to the scale, shake it well, filter it, and take the filtrate. According to the chromatographic conditions determined under "1.3", sample analysis was performed, the peak area of ​​each characteristic peak was recorded, and the "total peak area / sample amount" value was calculated. The results are shown in Table 10. Figure 14 .

[0127] Table 10 Results of investigation on characteristic spectra of Liangtoujian standard decoction with different extraction solvents

[0128]

[0129] Figure 14 The results showed that when 70% methanol was used as the extraction solvent, the "total peak area / sample weight" value of each characteristic peak was larger, and the chromatographic peak shape and separation were better. 70% methanol was selected as the extraction solvent for the characteristic spectrum of the Liangtoujian standard decoction.

[0130] (2) Investigation of extraction methods

[0131] This experiment investigated the effects of different extraction methods on the characteristic spectra of the standard decoction of Liangtoujian. Ultrasonic treatment and heating reflux were selected as the extraction methods. The peak shapes and separation effects of the nine temporary characteristic peaks were analyzed, and the "total peak area / sample weight" values ​​of the nine characteristic peaks were calculated to compare the effects of different extraction methods on the characteristic spectra of the standard decoction of Liangtoujian.

[0132] Take an appropriate amount of the standard decoction of Liangtoujian (No.: BT04), grind it into powder, take about 0.5g, accurately weigh it, and place it in two parallel groups, 2 portions in each group, put it in a stoppered conical flask, accurately add 25mL of 70% methanol, weigh it, ultrasonically treat it (power 300W, frequency 40kHz) for 30 minutes, heat it under reflux for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, evaporate the filtrate to dryness, dissolve the residue in 20mL of water, shake it with water-saturated n-butanol for 3 times, 25mL each time, combine the n-butanol liquid, evaporate it to dryness, dissolve the residue in methanol and transfer it to a 5mL volumetric flask, add methanol to the scale, shake it well, filter it, and take the filtrate. According to the chromatographic conditions determined under "1.3", sample analysis was performed, the peak area of ​​each characteristic peak was recorded, and the "total peak area / sample amount" value was calculated. The results are shown in Table 11. Figure 15 .

[0133] Table 11 Results of investigation on characteristic spectrum of Liangtoujian standard decoction by different extraction methods

[0134]

[0135] Figure 15 The results showed that compared with ultrasonic treatment, the "total peak area / sample weight" value of each characteristic peak was higher during heating reflux extraction, so heating reflux was selected as the extraction method.

[0136] (3) Extraction time investigation

[0137] This experiment investigated the effects of different extraction times on the characteristic spectra of the standard decoction of Liangtoujian. Different extraction times of 15 minutes, 30 minutes, and 45 minutes were selected for investigation. The effects of different extraction times on the characteristic spectra of the standard decoction of Liangtoujian were compared by calculating the "total peak area / sample amount" values ​​of the provisional 9 characteristic peaks.

[0138] Take an appropriate amount of the standard decoction of Liangtoujian (No.: BT04), grind it into powder, take about 0.5g, accurately weigh it, and make 3 parallel groups, 2 parts for each group, put it in a stoppered conical flask, accurately add 25mL of 70% methanol, weigh the weight, heat and reflux for 15 minutes, 30 minutes, and 45 minutes respectively, take it out, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, evaporate the filtrate to dryness, dissolve the residue in 20mL of water, shake and extract it 3 times with saturated n-butanol with 25mL each time, combine the n-butanol liquid, evaporate it to dryness, dissolve the residue in methanol and transfer it to a 5mL volumetric flask, add methanol to the scale, shake it well, filter it, and take the filtrate. According to the chromatographic conditions determined under "1.3", sample analysis was performed, the peak area of ​​each characteristic peak was recorded, and the "total peak area / sample amount" value was calculated. The results are shown in Table 12. Figure 16 .

[0139] Table 12 Results of investigation on characteristic spectra of Liangtoujian standard decoction at different extraction times

[0140]

[0141]

[0142] Figure 16 Experimental results: By comparing the effects of different extraction times on the characteristic spectra of the standard decoction of Liangtoujian, it was found that different extraction times had little effect on the characteristic spectra of Liangtoujian, indicating that complete extraction was achieved after 15 minutes of extraction. Considering the influence of the experimental environment, in order to ensure the durability of the method, heating reflux for 30 minutes was chosen.

[0143] (4) Investigation of extraction times

[0144] This experiment investigated the effects of different extraction times on the characteristic spectrum of the Liangtoujian standard decoction. By calculating the "total peak area / sample weight" value of 9 characteristic peaks consistent with the retention time of the characteristic peaks of the standard decoction, the effects of different extraction times on the characteristic spectrum of the Liangtoujian formula granules were compared to determine the optimal extraction times.

[0145] Take an appropriate amount of the standard decoction of Liangtoujian (No.: BT04), grind it into powder, take about 0.5g, accurately weigh it, and divide it into 4 parallel groups, 2 portions for each group, put it into a stoppered conical flask, accurately add 25mL of 70% methanol, weigh it, heat and reflux for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, evaporate the filtrate to dryness, dissolve the residue in 20mL of water, shake and extract it with water-saturated n-butanol 2 times, 3 times, 4 times, and 5 times, 25mL each time, combine the n-butanol liquid, evaporate it to dryness, dissolve the residue in methanol and transfer it to a 5mL volumetric flask, add methanol to the scale, shake it well, filter it, and take the filtrate. According to the chromatographic conditions determined under "1.3", sample analysis was performed, peak area was recorded, and the "total peak area / sample amount" value was calculated. The results are shown in Table 13. Figure 17 .

[0146] Table 13 Investigation results of extraction times of Radix Aconiti Lateralis standard decoction characteristic chromatogram

[0147]

[0148] Figure 17 The results show that, by investigating different extraction times, with the increase of extraction times, the value of "total peak area / sample weight" of the 9 characteristic peaks shows an increasing trend, but after 3 times of extraction, the value of "total peak area / sample weight" basically tends to be stable. Considering comprehensively, the extraction times of 4 times are finally selected.

[0149] (5) Determination of test solution preparation method

[0150] According to the above experimental results, the sample pretreatment method of Radix Aconiti Lateralis standard decoction characteristic chromatogram is determined as follows:

[0151] Take an appropriate amount of Radix Aconiti Lateralis standard decoction, grind it finely, take about 0.5 g, accurately weigh and place it in a conical flask with a stopper, accurately add 25 mL of 70% methanol, heat reflux for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20 mL of water, extract with water-saturated n-butanol 4 times, 25 mL each time, combine the n-butanol liquid, evaporate to dryness, dissolve the residue in methanol and transfer it to a 5 mL volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate, which is obtained.

[0152] 1.5. Determination of characteristic peaks

[0153] Take 16 batches of Radix Aconiti Lateralis standard decoction samples, prepare test solution according to the test solution preparation method determined in "1.4", accurately take the above test solution and reference material solution of control medicinal materials, respectively, according to the chromatographic conditions determined in "1.3", sample and determine, use "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software" to identify common peaks of 16 batches of Radix Aconiti Lateralis standard decoction characteristic chromatogram, select 9 common peaks with larger peak area, better peak shape and separation degree, and higher purity as the characteristic peaks of Radix Aconiti Lateralis standard decoction, as shown in Table 13, take chicoric acid chromatographic peak as reference peak S1, calculate the relative retention time and relative peak area of peaks 1-4 and S1 peak, take rhizocladine A chromatographic peak as reference peak S2, calculate the relative retention time and relative peak area of peaks 5-9 and S2 peak, and use relative retention time to locate the characteristic peaks. Figure 18

[0154] Figure 18 ​The results showed that the characteristic chromatograms of 16 batches of standard decoction of two-headed tip were analyzed, with chicoric acid chromatographic peak as reference peak S1, the relative retention time of peaks 3, 4 and S1 peak was calculated, with rhizocladol A chromatographic peak as reference peak S2, the relative retention time of peaks 5, 6, 7, 8 and S2 peak was calculated, and the average relative retention time of each characteristic peak was: 1.24 (peak 3), 1.59 (peak 4), 0.72 (peak 5), 0.75 (peak 6), 0.85 (peak 7), 0.91 (peak 8).

[0155] 1.6 Preparation of characteristic chromatogram

[0156] The UPLC characteristic chromatograms of 16 batches of standard decoction of two-headed tip were matched using "Chinese medicine chromatographic fingerprint similarity evaluation system", and the control chromatogram was generated by average method, and the control characteristic chromatogram of two-headed tip standard decoction was established, as shown in Figure 19 , the characteristic chromatogram has 9 characteristic peaks, which corresponds to 9 characteristic peaks in the reference material chromatogram of two-headed tip control medicinal material, and the characteristic chromatogram of two-headed tip control medicinal material is shown in Figure 20 . The relative retention time of peaks 3, 4 and S1 peak was calculated with chicoric acid chromatographic peak as reference peak S1, and the relative retention time of peaks 5, 6, 7, 8 and S2 peak was calculated with rhizocladol A chromatographic peak as reference peak S2, and the average relative retention time of each characteristic peak was: 1.24 (peak 3), 1.59 (peak 4), 0.72 (peak 5), 0.75 (peak 6), 0.85 (peak 7), 0.91 (peak 8).

[0157] 1.7 High resolution mass spectrometry identification of characteristic peaks

[0158] (1) Ultra-high performance liquid chromatography conditions

[0159] Except that the mobile phase B is 0.2% formic acid solution, the rest of the chromatographic conditions are the same as those in item

[0160] (2) The mass spectrometry conditions are shown in Table 15:

[0161] Table 15 Mass spectrometry parameter table

[0162]

[0163] (2) Preparation of test solution

[0164] Take two-headed tip standard decoction (number: BT04) and grind it, take about 0.5 g, and prepare the test solution according to the test solution preparation method determined in item

[0165] (3) Sample determination

[0166] Accurately draw 1 μL of the test solution and inject it into the liquid chromatography-mass spectrometer. The test solution is detected using the above-mentioned liquid chromatography conditions and mass spectrometry conditions. The total ion current and ultraviolet absorption chromatogram of the test solution are shown in Figure 21 .

[0167] (4) Result analysis

[0168] Through mass spectrometry precise molecular weight and fragment ion comparison analysis, and matching with the Thermo Fisher Scientific mzVault standard database, three components, caffeic acid (peak 1), chicoric acid (peak 2), and cyperus rotundus A (peak 9), were confirmed in the characteristic spectrum of Liangtoujianbiao decoction.

[0169] 1.8 Confirmation of characteristic peaks by reference substances

[0170] (1) Chromatographic conditions

[0171] Same as under "1.3"

[0172] (2) Preparation of reference solution

[0173] Same as under "1.2"

[0174] (3) Preparation of test solution

[0175] Same as under "1.4"

[0176] (4) Determination method

[0177] Accurately pipette 1 μL of the reference solution and test solution respectively, inject them into the liquid chromatography column, and measure.

[0178] Figure 22 The results showed that the chromatogram of the test sample showed the same chromatographic peak at the corresponding retention time as the chromatogram of the reference sample, and the UV-visible 3D spectral absorption curve of the chromatographic peak in the test sample was basically consistent with that of the reference sample, thereby confirming that characteristic peak 1 was caffeic acid, characteristic peak 2 was chicoric acid, and characteristic peak 9 was cyperus rotundus A.

[0179] 1.9 Methodological Validation

[0180] (1) Precision inspection

[0181] Take an appropriate amount of the standard decoction of Liangtoujian (No.: BT04), grind it into powder, take about 0.5 g, weigh it accurately, prepare the test solution according to the test solution preparation method determined under "1.4", repeat the injection 6 times according to the chromatographic conditions determined under "1.3", use the chicoric acid chromatographic peak as the reference peak S1, calculate the relative retention time and relative peak area of ​​peaks 1 to 4 and S1, use the chromatographic peak of cyperus rotundus A as the reference peak S2, calculate the relative retention time and relative peak area of ​​peaks 5 to 9 and S2, and calculate the RSD value. The experimental results are shown in Tables 16 and 17.

[0182] Table 16 Results of precision investigation of characteristic spectrum of Liangtoujian standard decoction (relative retention time)

[0183]

[0184]

[0185] Table 17 Precision Investigation Results of Characteristic Spectra of Liangtoujian Standard Decoction (Relative Peak Area)

[0186]

[0187] The results showed that the same sample solution was injected six times continuously, with the chromatographic peak of chicoric acid as the reference peak S1 and the chromatographic peak of cyperus rotundus A as the reference peak S2. The RSD values ​​of the relative retention times of the characteristic peaks were in the range of 0.00% to 0.12%, and the RSD values ​​of the relative peak areas were in the range of 0.26% to 1.35%, all less than 3.0%, indicating that the characteristic spectrum method had good precision.

[0188] (2) Stability investigation

[0189] Take an appropriate amount of Liangtoujian standard decoction (No.: BT04), grind it into powder, take about 0.5 g, weigh it accurately, prepare the test solution according to the test solution preparation method determined under "1.4", and according to the chromatographic conditions determined under "1.3", inject the sample at 0, 3, 9, 13, 20, and 24 hours respectively. Take the chicoric acid chromatographic peak as the reference peak S1, calculate the relative retention time and relative peak area of ​​peaks 1 to 4 and S1, take the bamboo cyperin A chromatographic peak as the reference peak S2, calculate the relative retention time and relative peak area of ​​peaks 5 to 9 and S2, calculate the RSD value, and the results are shown in Tables 18 and 19.

[0190] Table 18: Results of stability study of characteristic spectrum of Liangtoujian standard decoction (relative retention time)

[0191]

[0192]

[0193] Table 19: Results of the stability study of the characteristic spectrum of Liangtoujian standard decoction (relative peak area)

[0194]

[0195] The results showed that the same test solution was analyzed at 0, 3, 9, 13, 20 and 24 hours, respectively. The chromatographic peak of chicoric acid was used as the reference peak S1, and the chromatographic peak of cyperus rotundus A was used as the reference peak S2. The RSD values ​​of the relative retention times of the characteristic peaks were in the range of 0.00% to 0.16%, and the RSD values ​​of the relative peak areas were in the range of 0.16% to 2.56%, all of which were less than 3.0%, indicating that the test solution had good relative stability within 24 hours under this method.

[0196] (3) Repeatability study

[0197] Take an appropriate amount of the standard decoction of Liangtoujian (No.: BT04), grind it into powder, take about 0.5 g, weigh it accurately, and prepare 6 test solutions according to the test solution preparation method determined under "1.4". According to the chromatographic conditions determined under "1.3", the samples were analyzed. The chromatographic peak of chicoric acid was used as the reference peak S1, and the relative retention time and relative peak area of ​​peaks 1 to 4 and S1 were calculated. The chromatographic peak of cyperus rotundus A was used as the reference peak S2, and the relative retention time and relative peak area of ​​peaks 5 to 9 and S2 were calculated, and the RSD value was calculated. The experimental results are shown in Tables 20 and 21.

[0198] Table 20 Results of Repeatability Investigation of Characteristic Spectra of Liangtoujian Standard Decoction (Relative Retention Time)

[0199]

[0200]

[0201] Table 21 Repeatability test results of characteristic spectrum of Liangtoujian standard decoction (relative peak area)

[0202]

[0203] The results showed that the same batch of samples were measured six times, with the chromatographic peak of chicoric acid as the reference peak S1 and the chromatographic peak of cyperus rotundus A as the reference peak S2. The RSD values ​​of the relative retention times of the characteristic peaks were in the range of 0.00% to 0.11%, and the RSD values ​​of the relative peak areas were in the range of 0.73% to 2.86%, all less than 3%, indicating that the characteristic spectrum method had good repeatability.

[0204] (4) Intermediate precision inspection

[0205] Other analysts of this project team operated on different dates and under different chromatographs, and took about 0.5 g of the same batch of Liangtoujian standard decoction (No.: BT04), accurately weighed it, and paralleled it into 6 portions. According to the test solution preparation method determined under "1.4", 6 test solutions were prepared. According to the chromatographic conditions determined under "1.3", the samples were injected and analyzed. The chromatographic peak of chicoric acid was used as the reference peak S1, and the relative retention time and relative peak area of ​​peaks 1 to 4 and S1 were calculated. The chromatographic peak of cyperus rotundus A was used as the reference peak S2, and the relative retention time and relative peak area of ​​peaks 5 to 9 and S2 were calculated. The RSD value was also calculated. The experimental results are shown in Tables 22 and 23.

[0206] Table 22 Intermediate precision inspection results of characteristic spectrum of Liangtoujian standard decoction (relative retention time)

[0207]

[0208]

[0209] Table 23 Intermediate precision inspection results of Liangtoujian standard decoction characteristic spectrum (relative peak area)

[0210]

[0211] Figure 23 The results showed that the same batch of samples were measured six times by different analysts on different instruments at different times. The chromatographic peak of chicoric acid was used as the reference peak S1, and the chromatographic peak of cyperus rotundus A was used as the reference peak S2. The RSD values ​​of the relative retention times of the characteristic peaks were in the range of 0.04% to 0.11%, and the RSD values ​​of the relative peak areas were in the range of 1.06% to 1.88%. The RSD values ​​of the relative retention times and the six data of the repeatability test were in the range of 0.10% to 3.77%, and the RSD values ​​of the relative peak areas and the six data of the repeatability test were in the range of 1.04% to 2.31%, indicating that the intermediate precision of the relative retention times and relative peak areas of the characteristic peaks was good.

[0212] (5) Durability inspection

[0213] ①Inspection of chromatographic column

[0214] The effects of three chromatographic columns of the same brand, Waters ACQUITY BEH C18 (2.1 mm × 100 mm, 1.7 μm), on the durability of the characteristic spectrum of a two-pointed standard decoction were compared.

[0215] Take about 0.5g of the standard decoction of Liangtoujian (No.: BT04), accurately weigh it, and prepare the test solution according to the test solution preparation method determined under "1.4". Except for the different chromatographic columns, other chromatographic conditions are the same as those specified under "1.3". Sample analysis is carried out. The chromatographic peak of chicoric acid is used as the reference peak S1, and the relative retention time and relative peak area of ​​peaks 1 to 4 and S1 are calculated. The chromatographic peak of cyperus rotundus A is used as the reference peak S2, and the relative retention time and relative peak area of ​​peaks 5 to 9 and S2 are calculated, and the RSD value is calculated. The experimental results are shown in Table 24, Table 25, Figure 24 .

[0216] Table 24 Characteristic spectrum of Liangtoujian standard decoction Results of the same brand chromatographic column (relative retention time)

[0217]

[0218] Table 25 Characteristic spectrum of Liangtoujian standard decoction Results of the same brand chromatographic column inspection (relative peak area)

[0219]

[0220]

[0221] Figure 24 The results showed that, with the chromatographic peak of chicoric acid as the reference peak S1 and the chromatographic peak of cyperus rotundus A as the reference peak S2, the relative retention time RSD values ​​of each characteristic peak were in the range of 0.00% to 0.85%, and the relative peak area RSD values ​​were in the range of 0.19% to 2.78%, both of which were less than 3%, indicating that the chromatographic columns of the same brand but different batches in this method had good durability.

[0222] ② Investigation of different column temperatures

[0223] The effects of different column temperatures (38℃, 40℃, and 42℃) on the characteristic spectra of Liangtoujian standard decoction were compared.

[0224] Approximately 0.5 g of the standard decoction of Liangtoujian (No. BT04) was accurately weighed and prepared according to the method for preparing the test solution specified in "1.4". The chromatographic conditions were the same as those specified in "1.3", except that the column temperatures were 38°C, 40°C, and 42°C, respectively. The samples were injected and analyzed. The chicoric acid peak was used as the reference peak S1. The relative retention times and peak areas of peaks 1 to 4 relative to S1 were calculated. The cyperin A peak was used as the reference peak S2. The relative retention times and peak areas of peaks 5 to 9 relative to S2 were calculated, and the RSD values ​​were calculated. The experimental results are shown in Tables 26 and 27.

[0225] Table 26 Characteristic spectrum of Liangtoujian standard decoction at different column temperatures (relative retention time)

[0226]

[0227] Table 27 Characteristic spectrum of Liangtoujian standard decoction at different column temperatures (relative peak area)

[0228]

[0229] Figure 25 The results showed that under different column temperatures, the RSD values ​​of the relative retention times of each characteristic peak and the S peak were in the range of 0.06% to 1.46%, and the RSD values ​​of the relative peak areas were in the range of 0.42% to 2.12%, both of which were less than 3%, indicating that when the column temperature was ±2°C, it had little effect on the relative peak areas and relative retention times of each chromatographic peak, indicating that this method can adapt to slight changes in column temperature and has good durability.

[0230] ③ Investigation of different flow rates

[0231] Compare the effects of different flow rates of 0.28mL / min, 0.30mL / min, and 0.32mL / min on the durability of the characteristic spectrum of the Liangtoujian standard decoction.

[0232] Approximately 0.5 g of the standard decoction of Liangtoujian (No. BT04) was accurately weighed and the test solution was prepared according to the test solution preparation method specified in "1.4". 1 μL of each sample was injected according to the chromatographic conditions specified in "1.3". The chicoric acid chromatographic peak was used as the reference peak S1. The relative retention times and relative peak areas of peaks 1 to 4 and S1 were calculated. The chromatographic peak of cyperus rotundus A was used as the reference peak S2. The relative retention times and relative peak areas of peaks 5 to 9 and S2 were calculated, and the RSD values ​​were calculated. The experimental results are shown in Tables 28 and 29.

[0233] Table 28 Results of investigation of characteristic spectrum of Liangtoujian standard decoction at different flow rates (relative retention time)

[0234]

[0235] Table 29: Results of investigation of characteristic spectra of Liangtoujian standard decoction at different flow rates (relative peak area)

[0236]

[0237] Figure 26 The results showed that at different flow rates, the RSD values ​​of the relative retention times of each characteristic peak and the S peak were in the range of 0.06% to 1.77%, and the RSD values ​​of the relative peak areas were in the range of 0.37% to 2.70%, both less than 3%. The characteristic chromatogram profiles were consistent, indicating that when the flow rate was ±0.02 mL / min, the relative peak areas and relative retention times of each chromatographic peak were less affected, indicating that the method can adapt to slight changes in flow rate and has good durability.

[0238] Example 2: Characteristic spectrum identification study of Liangtoujian standard decoction and Northeast Nine-section Acorus calamus (Black Mercury Lotus) standard decoction, the specific implementation steps are as follows:

[0239] The standard decoction of Acorus calamus (Black Mercury Lotus) was prepared according to the preparation process of the standard decoction of Liangtoujian. The test sample was prepared according to the preparation method of the test sample solution specified in "1.4". The sample was injected and determined according to the chromatographic conditions specified in "1.3". The standard decoction of Liangtoujian was compared with the standard decoction of Acorus calamus (Black Mercury Lotus): Figure 27 It can be seen that the characteristic spectrum of Acorus calamus (Black Mercury Lotus) is quite different from that of Acorus tatarinowii, indicating that the chemical components contained in the two are quite different. Figure 27 As can be seen from the chromatograms, there are significant differences between the characteristic spectra of the standard decoctions of Northeast Nine-section Acorus and Liangtoujian: Peaks 1 to 3 and 6 are common peaks of the two. Peaks 4, 5, 7 to 9 are not detected in the characteristic spectra of Northeast Nine-section Acorus, but nine chromatographic peaks (peaks 10 to 18) appear at 6 to 18 minutes and 28 to 29 minutes. The method established in this study can effectively control the quality of the standard decoction of Liangtoujian and effectively distinguish Liangtoujian from the adulterant Northeast Nine-section Acorus (black water lily).

[0240] Example 3: Characteristic spectrum identification study of the standard decoction of Liangtoujian and the standard decoction of Jiujiechangcalamus, the specific implementation steps are as follows:

[0241] Prepare the standard decoction of Nine-section Acorus calamus according to the preparation process of the standard decoction of Liangtoujian, prepare the test sample according to the preparation method of the test sample solution determined in "1.4", and inject and measure according to the chromatographic conditions determined in "1.3". Compare and analyze the standard decoction of Liangtoujian with the standard decoction of Nine-section Acorus calamus: Figure 28 It can be seen that the characteristic spectrum of Acorus calamus is quite different from that of Acorus tatarinowii, indicating that the chemical components of the two are quite different. Figure 28 As can be seen from the figure, peaks 1, 2, 5, and 6 are common characteristic peaks for both. Compared to Liangtoujian, peaks 3, 4, 7, and 8 were not detected in the characteristic spectrum of Jiujie Acorus calamus. However, two highly responsive peaks, 10 to 11, appeared at 10-12 minutes and 16 minutes, respectively. Peak 12 was not transferred to the standard decoction. The method established in this study can effectively control the quality of the Liangtoujian standard decoction and effectively distinguish Liangtoujian from the adulterant Jiujie Acorus calamus.

[0242] Example 4: Characteristic spectrum identification study of Liangtoujian standard decoction and Cyperus rotundus standard decoction, the specific implementation steps are as follows:

[0243] Prepare the standard decoction of Cyperus rotundus according to the preparation process of the standard decoction of Liangtoujian, prepare the test sample according to the preparation method of the test sample solution specified in "1.4", and perform the sample injection and determination according to the chromatographic conditions specified in "1.3". Compare and analyze the standard decoction of Liangtoujian and the standard decoction of Cyperus rotundus: Figure 29 It can be seen that the characteristic spectrum of Cyperus rotundus is quite different from that of Rhizoma Cyperi and Rhizoma Lucidum, indicating that the chemical components contained in the two are quite different. Figure 29 As can be seen from the figure, peaks 2, 4, 6, and 8 are common characteristic peaks for both. The responses of peaks 2, 6, and 8 in the Liangtoujian characteristic spectrum are significantly greater than those of Cyperus rotundus. The responses of peaks 1, 3, 5, 7, and 9 in the Cyperus rotundus characteristic spectrum are very low or absent. However, five peaks 10 to 14 with relatively high responses appear at 12-14 minutes, 17-20 minutes, and 22-24 minutes. The method established in this study can effectively control the quality of the Liangtoujian standard decoction and effectively distinguish Liangtoujian from the adulterated Cyperus rotundus.

[0244] The above is a preferred embodiment of the invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A method for constructing a characteristic spectrum of a standard decoction of Liangtoujian, characterized in that: The following steps are involved: Prepare a reference solution, wherein the reference solution includes a reference medicinal material reference solution and a reference substance reference solution, wherein the reference substance reference solution includes a chicoric acid reference substance, a cyperus rotundus A reference substance, and a caffeic acid reference substance; Prepare the test solution by taking the standard decoction of Liangtoujian; Take a preset amount of reference solution and test solution and inject them into a liquid chromatograph using a Waters ACQUITY BEH C18 column with a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.7 μm. Use acetonitrile as mobile phase A and phosphoric acid solution as mobile phase B for gradient elution to establish a characteristic spectrum; The gradient elution was performed according to the following procedure: 0-10 min, mobile phase A from 5% to 17%, mobile phase B from 95% to 83%; From 10 min to 23 min, mobile phase A changed from 17% to 29%, and mobile phase B changed from 83% to 71%; From 23 to 28 minutes, mobile phase A was maintained at 29% and mobile phase B was maintained at 71%; 28-33 min, mobile phase A from 29% to 45%, mobile phase B from 71% to 55%; 33min~45min, mobile phase A from 45% to 80%, mobile phase B from 55% to 20%; The volume concentration of the phosphoric acid solution is 0.15% to 0.25%; The ultraviolet detection wavelength of the liquid chromatograph is 200nm~210nm; The test solution was prepared by the following method: Take the standard decoction of Liangtoujian, grind it into powder, weigh 0.49g~0.51g, accurately weigh it, place it in a stoppered conical flask, accurately add 20mL~30mL of 65%~75% methanol, heat and reflux for 15min~45min, filter, evaporate the filtrate to dryness, dissolve the residue in 20mL~21mL of water, shake and extract with water-saturated n-butanol 3 times~5 times, 25mL~26mL each time, combine the n-butanol liquids, evaporate to dryness, dissolve the residue in methanol and transfer it to a 5mL volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate to obtain.

2. The method for constructing the characteristic spectrum of the Liangtoujian standard decoction according to claim 1, characterized in that: The preset volume of the reference solution and the test solution is 0.5 μL~1.5 μL; The column temperature of the liquid chromatograph is 38°C to 42°C; When performing the gradient elution, the flow rate of the mobile phase is 0.28-0.32 mL / min.

3. The method for constructing the characteristic spectrum of the Liangtoujian standard decoction according to claim 1, characterized in that: The reference substance solution was prepared according to the following method: Take appropriate amounts of chicoric acid reference substance, bamboo cyperin A reference substance, and caffeic acid reference substance, accurately weigh them, and add methanol to prepare a mixed solution containing 41.4238 μg of chicoric acid, 17.4902 μg of bamboo cyperin A, and 15.0148 μg of caffeic acid per 1 mL, which is used as the reference substance solution.

4. The method for constructing the characteristic spectrum of the Liangtoujian standard decoction according to claim 1, wherein: The control medicinal material reference solution is prepared by the following method: Take 0.8g~1.2g of the medicinal material of Liangtoujian, add 20mL~30mL of water, heat and reflux for 15min~45min, centrifuge, take the supernatant, evaporate to dryness, add 20mL~30mL of 65%~75% methanol to the residue, heat and reflux for 15min~45min, filter, evaporate the filtrate to dryness, dissolve the residue in 20mL~21mL of water, shake and extract with water-saturated n-butanol 3 times~5 times, 25mL~26mL each time, combine the n-butanol liquid, evaporate to dryness, dissolve the residue in methanol and transfer it to a 5mL volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate to obtain the reference solution of the control medicinal material.

5. The method for constructing the characteristic spectrum of the Liangtoujian standard decoction according to claim 1, characterized in that: The characteristic spectrum includes 9 common peaks, wherein Peak 1 corresponds to caffeic acid, Peak 2 corresponds to chicoric acid, and Peak 9 corresponds to cyperin A.

6. The method for constructing the characteristic spectrum of the Liangtoujian standard decoction according to claim 5, characterized in that: In the characteristic spectrum of the Liangtoujian standard decoction, the chicoric acid chromatographic peak is used as the reference peak S1, and the relative retention times of peaks 3 and 4 and reference peak S1 are consistent with: peak 3 1.24, peak 4 1.59, and the RSD value of the relative retention time of each characteristic peak is less than 3%; Taking the chromatographic peak of cyperus rotundus A as the reference peak S2, the relative retention times of peaks 5, 6, 7, and 8 and the reference peak S2 were consistent with: peak 5 0.72, peak 6 0.75, peak 7 0.85, and peak 8 0.

91. The RSD values ​​of the relative retention times of each characteristic peak were <3%.

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