Construction method and application of characteristic chromatogram of anemones raddeanae standard decoction

Abstract

The invention discloses a construction method of a characteristic spectrum of an anemonis raddeanae standard decoction, and the construction method comprises the following steps: preparing a reference substance solution, the reference substance solution comprises a reference substance solution of a reference medicinal material and a reference substance solution of a reference substance, and the reference substance solution of the reference substance comprises a chicoric acid reference substance, a raddeanin A reference substance and a caffeic acid reference substance; taking the rhizoma anemones raddeanae standard decoction to prepare a test solution; a preset amount of the reference substance solution and the test solution are taken and injected into a liquid chromatograph, the liquid chromatograph takes octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and a phosphoric acid solution as a mobile phase B for gradient elution, and the characteristic chromatogram is established. The method can provide a data basis for quality control of the anemonis raddeanae standard decoction and the medicinal material, and effectively ensures the stability and controllability of the product quality of the anemonis raddeanae medicinal material and the standard decoction.

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] The two-headed tip is the dried rhizome of Anemone raddeana Regel, a plant of the Ranunculaceae family. It is dug up in summer, the fibrous roots are removed, washed and dried. The two-headed tip, also known as Easter flower, wind flower, silver anemone and bamboo-jointed cyperus, is pungent and hot in nature; it is toxic and belongs to the spleen meridian. Its rhizome is used as medicine, which has the effect of removing rheumatism and eliminating carbuncle. It is used to treat wind-cold dampness, limb cramps, joint pain, carbuncle ulcer and other symptoms. As a traditional Chinese medicine, the two-headed tip was first recorded in the "Essence of Materia Medica" written by Liu Wentai in the Ming Dynasty. According to the record: "The two-headed tip is a kind of aconite, and the seedlings and leaves are also similar. Its roots are like aconite, with black skin and white flesh, thin and sharp at both ends, so it is named after it." The two-headed tip is mostly wild in mountain forests or shaded grasslands at an altitude of about 800m. It is mainly distributed in the three northeastern provinces, and is also distributed in Shandong and Hebei. The existing research on the fingerprint / characteristic spectrum of Liangtoujian has poor overall separation effect of chromatographic peaks, less peak information, and cannot fully reflect the intrinsic quality of Liangtoujian. For example, Zhang Yanfei et al. used HPLC to establish the HPLC fingerprint spectrum of Liangtoujian medicinal materials. The fingerprint spectrum identified 11 common peaks, none of which were clearly identified, and the separation effect of each characteristic peak was poor. Yan Panfeng et al. used HPLC to determine the spectrum of Liangtoujian monosaccharides. The spectrum had 7 common peaks, but the overall separation effect of the fingerprint spectrum was poor and it was not specific.

[0003] In addition, there are not many studies on the quality standards of Liangtoujian. The 2020 edition of the "Chinese Pharmacopoeia" only has 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 imperfect. 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 effectively control and comprehensively evaluate Liangtoujian standard decoction. 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, which has good reproducibility, is accurate and reliable, 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 product quality of the Liangtoujian standard decoction.

[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 cyperus rotundus A reference substance and a caffeic acid reference substance;

[0006] Take the standard decoction of Liangtoujian to prepare the test solution;

[0007] Preset amounts of reference solution and test solution were taken and injected into a liquid chromatograph, which used octadecylsilane bonded silica gel as a filler, acetonitrile as a mobile phase A, and a phosphoric acid solution as a 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] 0-10min, mobile phase A from 5% to 17%, mobile phase B from 95% to 83%;

[0010] 10min-23min, 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] 28min-33min, mobile phase A from 29% to 45%, mobile phase B from 71% to 55%;

[0013] 33min~45min, mobile phase A changes from 45%→80%, and mobile phase B changes from 55%→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-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, put 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, add 20mL~21mL of water to dissolve the residue, shake and extract with water-saturated n-butanol for 3~5 times, 25mL~26mL each time, combine the n-butanol liquids, evaporate to dryness, add methanol to dissolve the residue 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, cyperus rotundus 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 cyperus rotundus A, and 15.0148 μg of caffeic acid per 1 mL, 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 two-headed pointed medicinal material, 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, add 20mL-21mL of water to dissolve the residue, shake and extract with water-saturated n-butanol for 3 to 5 times, 25mL-26mL each time, combine the n-butanol liquids, evaporate to dryness, add methanol to dissolve the residue and transfer it to a 5mL volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate to obtain a reference medicinal material reference solution.

[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 standard decoction of Liangtoujian, the chromatographic peak of chicoric acid 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 <3%;

[0027] 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, peak 8 0.91, and the RSD values ​​of the relative retention times of each characteristic peak were <3%.

[0028] Correspondingly, the present invention also provides a characteristic spectrum of a Liangtoujian standard decoction, and the characteristic spectrum of the Liangtoujian standard decoction is obtained by using the above-mentioned method for constructing the characteristic spectrum of the Liangtoujian standard decoction.

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

[0030] (1) Authenticity identification and ingredient testing of Liangtoujian standard decoction;

[0031] (2) Authenticity identification and ingredient testing of Liangtoujian medicinal materials;

[0032] (3) Distinguishing between the standard decoction of Liangtoujian and the standard decoction of Northeast Nine-section Acorus Calamus;

[0033] (4) Distinguishing between the standard decoction of Liangtoujian and the standard decoction of Jiujie Acorus calamus;

[0034] (5) Differentiation between the standard decoction of Liangtoujian and the standard decoction of Xiangfu.

[0035] The implementation of the present invention has the following beneficial effects:

[0036] The present invention establishes a characteristic spectrum of the standard decoction of Liangtoujian for the first time through the construction method of the characteristic spectrum, and the characteristic spectrum can fully display the chemical composition characteristics of the standard decoction of Liangtoujian, and the characteristic peak information is rich, which can comprehensively reflect the quality information of the standard decoction of Liangtoujian, thereby achieving the purpose of comprehensively and effectively controlling the quality of the standard decoction of Liangtoujian. It can also realize the authenticity identification and component detection of the medicinal materials of Liangtoujian, as well as the identification and distinction between the standard decoction of Liangtoujian and the standard decoction of Northeast Nine-section Calamus, the standard decoction of Nine-section Calamus or the standard decoction of Cyperus, providing a reference basis for the identification of the medicinal materials of Liangtoujian and the standard decoction. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a chromatogram of the standard decoction of Liangtoujian in Example 1 of the present invention at a detection wavelength of 254 nm;

[0038] Figure 2 is a chromatogram of the standard decoction of Liangtoujian in Example 1 of the present invention at a detection wavelength of 280 nm;

[0039] Figure 3 is a chromatogram of the standard decoction of Liangtoujian in Example 1 of the present invention at a detection wavelength of 206 nm;

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

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

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

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

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

[0045] Fig. 9 This is the characteristic chromatogram of sample 3 of the Liangtoujian standard decoction in Comparative Example 1 of the present invention obtained by method 3;

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

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

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

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

[0050] Fig.14 The characteristic spectrum of the standard decoction of Liangtoujian in Example 1 of the present invention is investigated using different extraction solvents;

[0051] Fig.15 The characteristic spectrum of the standard decoction of Liangtoujian in Example 1 of the present invention is investigated by different extraction methods;

[0052] Fig.16 The characteristic spectrum of the standard decoction of Liangtoujian in Example 1 of the present invention is the result of investigation at different extraction times;

[0053] Fig.17 The characteristic spectrum of the standard decoction of Liangtoujian in Example 1 of the present invention is the result of the investigation of the number of extractions;

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

[0055] Fig.19 It 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] Fig. 20 It 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): cyperus rotundus A;

[0057] Fig.21 It is the total ion flow diagram and ultraviolet absorption chromatogram of the test solution of the standard decoction of Liangtoujian in Example 1 of the present invention;

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

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

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

[0061] Fig.25 It is a chromatogram of the durability of the characteristic spectrum of the Liangtoujian standard decoction in Example 1 of the present invention at different column temperatures;

[0062] Fig.26 It is a chromatogram of the durability of the characteristic spectrum of the standard decoction of Liangtoujian in Example 1 of the present invention at different flow rates;

[0063] Fig. 27 It is a comparison chart of characteristic spectra of the standard decoction of Liangtoujian and the standard decoction of Acorus calamus (black water lily) in Example 2 of the present invention;

[0064] Fig.28 It is a comparison chart of characteristic spectra of the standard decoction of Liangtoujian and the standard decoction of Jiujie Acorus calamus in Example 3 of the present invention;

[0065] Fig.29 It is a comparison chart 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 purpose, 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 realize 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, No.: BH-374, BH-307, BH-308), one-ten-thousandth analytical electronic balance (ME204E, Mettler-Toledo), one-millionth 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 AG).

[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; water was ultrapure water (self-made in the laboratory).

[0072] Test drugs: Bamboo-jointed 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 to a 5 mL volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate to obtain a reference solution for the reference medicinal material.

[0078] Accurately weigh 2.071 mg of chicoric acid reference substance, 0.874 mg of cyperus rotundus 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 cyperus rotundus 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 saponin components. Therefore, the above different wavelength chromatograms 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, treat it with ultrasound (power 250W, frequency 40kHz) for 30 minutes, take it out, cool it, 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 (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 gradient elution was performed according to the provisions in Table 2; the flow rate was 0.30 mL per minute; the column temperature was 30° C.; and the injection volume was 1 μL.

[0084] Table 2 Gradient elution table

[0085]

[0086]

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

[0088] (2) Optimization of chromatographic conditions

[0089] Method 1: Waters ACQUITY BEH C18 (2.1 mm × 100 mm, 1.7 μm) chromatographic 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 3, with 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 to 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 it accurately, and place it in a stoppered conical flask.

[0091] Sample 1: Accurately add 25 mL of 70% methanol, weigh the weight, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, take out, cool, weigh 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, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, take out, 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 with ethyl acetate 3 times, 20 mL each time, combine the ethyl acetate, evaporate to dryness, add methanol to dissolve the residue and transfer 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, 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 water-saturated n-butanol, 20 mL each time, combine the water-saturated n-butanol solutions, evaporate to dryness, add methanol to dissolve the residue and transfer to a 5 mL volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate.

[0094] Table 3 Gradient elution table of Liangtoujian standard decoction characteristic spectrum

[0095]

[0096] Depend on Figure 5 to Figure 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: 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 4, with a flow rate of 0.30 mL / min; column temperature was 30°C; injection volume was 1 μL; 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: 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 5, with a flow rate of 0.30 mL / min; column temperature was 30°C; injection volume was 1 μL; detection wavelength was 206 nm. Results are shown in Fig. 9 .

[0102] Table 5 Gradient elution table

[0103]

[0104] Fig. 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: Waters ACQUITY BEH C18 (2.1 mm × 100 mm, 1.7 μm) chromatographic 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 6, with 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 Fig.10 .

[0106] Table 6 Gradient elution table

[0107]

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

[0109] Method 5: Waters ACQUITY BEH C18 (2.1 mm × 100 mm, 1.7 μm) chromatographic 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 7, with 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 Fig.11 .

[0110] Table 7 Characteristic spectrum of Liangtoujian standard decoction Gradient elution table

[0111]

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

[0113] (4) Determination of temperature

[0114] 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 8, with 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. Results are shown in Figures 12-13 .

[0115] Table 8 Gradient elution table

[0116]

[0117] Figure 12-13 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) chromatographic 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 the extraction solvent, extraction method, extraction time and extraction solvent dosage on the characteristic spectrum.

[0124] (1) Investigation of extraction solvents

[0125] This experiment investigated the effects of different extraction solvents on the characteristic spectra of the standard decoction of Liangtoujian. Methanol, 70% methanol, 50% methanol, ethanol, 70% ethanol, and 50% ethanol were selected as extraction solvents. The peak shapes and separation effects of the 9 tentative characteristic peaks were observed, and the "total peak area / sample weight" values ​​of the 9 characteristic peaks were calculated to compare the effects of different extraction solvents on the characteristic spectra of the standard decoction of Liangtoujian, 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, weigh it accurately, and make 6 parallel groups, 2 portions in each group, put it in a stoppered conical bottle, and accurately add 25mL of methanol, 70% methanol, 50% methanol, ethanol, 70% ethanol, and 50% ethanol, weigh it, and treat it with ultrasound (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, shake and extract it with water-saturated n-butanol 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 bottle, add methanol to the scale, shake it well, filter it, and take the filtrate to obtain it. According to the chromatographic conditions determined under "1.3", sample analysis, record the peak area of ​​each characteristic peak, and calculate the value of "total peak area / sample weight". The results are shown in Table 10. Fig.14 .

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

[0128]

[0129] Fig.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 9 temporary characteristic peaks were analyzed, and the "total peak area / sample weight" values ​​of the 9 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, weigh it accurately, and place it in two parallel groups, 2 portions in each group, put it in a stoppered conical bottle, 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 to extract it 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 bottle, add methanol to the scale, shake it well, filter it, and take the filtrate to obtain it. According to the chromatographic conditions determined under "1.3", inject and analyze, record the peak area of ​​each characteristic peak, and calculate the value of "total peak area / sample weight". The results are shown in Table 11. Fig.15 .

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

[0134]

[0135] Fig.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

[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 Liangtoujian standard decoction (No.: BT04), grind it into powder, take about 0.5g, weigh it accurately, and repeat it in 3 groups, 2 portions in each group, put it in a stoppered conical bottle, accurately add 25mL of 70% methanol, weigh it, 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 bottle, add methanol to the scale, shake it well, filter it, and take the filtrate. According to the chromatographic conditions determined under "1.3", inject and analyze, record the peak area of ​​each characteristic peak, and calculate the value of "total peak area / sample weight". The results are shown in Table 12. Fig.16 .

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

[0140]

[0141]

[0142] Fig.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, heating reflux for 30 minutes was chosen to ensure the durability of the method.

[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 time.

[0145] Take an appropriate amount of Liangtoujian standard decoction (No.: BT04), grind it into powder, take about 0.5g, weigh it accurately, and place it in 4 parallel groups, 2 portions in each group, put it in a stoppered conical bottle, 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 for 2, 3, 4, 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", inject and analyze, record the peak area, and calculate the "total peak area / sample amount" value. The results are shown in Table 13. Fig.17 .

[0146] Table 13 Results of the investigation of the extraction times of the characteristic spectrum of Liangtoujian standard decoction

[0147]

[0148] Fig.17 The results showed that through investigation of different extraction times, it was found that with the increase of extraction times, the "total peak area / sample weight" values ​​of the 9 characteristic peaks showed an increasing trend, but after 3 extractions, the "total peak area / sample weight" values ​​basically tended to be stable. Taking comprehensive considerations, the final number of extractions was selected to be 4 times.

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

[0150] According to the above experimental results, the sample pretreatment method of the characteristic spectrum of Liangtoujian standard decoction is determined as follows:

[0151] Take an appropriate amount of the standard decoction of Liangtoujian, grind it into powder, take about 0.5g, weigh it accurately, put it in a stoppered conical flask, accurately add 25mL of 70% methanol, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20mL of water, shake and extract with water-saturated n-butanol for 4 times, 25mL 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.

[0152] 1.5. Determination of characteristic peaks

[0153] Take 16 batches of Liangtoujian standard decoction samples, prepare the test solution according to the test solution preparation method determined under "1.4", accurately pipette the above test solution and control medicinal material reference solution respectively, and perform sample injection determination according to the chromatographic conditions determined under "1.3". Use the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software" to identify the common peaks of the characteristic spectra of the 16 batches of Liangtoujian standard decoction, and select 9 common peaks with larger peak area, better peak shape and separation, and higher purity as the characteristic peaks of Liangtoujian standard decoction, such as Fig.18 As shown, 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 relative retention time is used to locate the characteristic peak.

[0154] Fig.18The results showed that the characteristic spectra of 16 batches of standard decoction of Liangtoujian were analyzed, and the chromatographic peak of chicoric acid was used as the reference peak S1 to calculate the relative retention times of peaks 3 and 4 with respect to S1. The chromatographic peak of cyperus rotundus A was used as the reference peak S2 to calculate the relative retention times of peaks 5, 6, 7, 8 and S2. The average relative retention times of the characteristic peaks were: 1.24 (peak 3), 1.59 (peak 4), 0.72 (peak 5), 0.75 (peak 6), 0.85 (peak 7), and 0.91 (peak 8).

[0155] 1.6 Preparation of feature maps

[0156] The UPLC characteristic spectra of 16 batches of Liangtoujian standard decoction were matched using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", and a reference spectrum was generated by the average method to establish a reference characteristic spectrum of Liangtoujian standard decoction, such as Fig.19 The characteristic spectrum has 9 characteristic peaks, which correspond to the 9 characteristic peaks in the chromatogram of the reference material of Liangtoujian. The characteristic spectrum of Liangtoujian reference material is shown in Fig. 20 The chromatographic peak of chicoric acid was used as the reference peak S1 to calculate the relative retention time of peak 3, peak 4 and peak S1. The chromatographic peak of cyperus rotundus A was used as the reference peak S2 to calculate the relative retention time of peak 5, peak 6, peak 7, peak 8 and peak S2. 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), and 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 mobile phase B was 0.2% formic acid solution, the other chromatographic conditions were the same as those in item "1.3".

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

[0161] Table 15 Mass spectrometry parameters

[0162]

[0163] (2) Preparation of test solution

[0164] Take an appropriate amount of Liangtoujian standard decoction (No.: BT04), grind it into powder, take about 0.5g, and prepare the test solution according to the test solution preparation method determined under "1.4".

[0165] (3) Sample measurement

[0166] Accurately draw 1 μL of the test solution and inject it into the liquid chromatography-mass spectrometer. Use the above liquid chromatography conditions and mass spectrometry conditions to detect the test solution. The total ion flow diagram and ultraviolet absorption chromatogram of the test solution are shown in Fig.21 .

[0167] (4) Results analysis

[0168] Through mass spectrometry precise molecular weight and fragment ion comparison analysis and matching with the Thermo Fisher 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

[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 "1.4"

[0176] (4) Determination method

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

[0178] Fig. 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 standard decoction of Liangtoujian (No.: BT04), grind it into powder, take about 0.5g, 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", take the chromatographic peak of chicoric acid as the reference peak S1, calculate the relative retention time and relative peak area of ​​peaks 1 to 4 and S1, take 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 spectrum of Liangtoujian standard decoction (relative peak area)

[0186]

[0187] The results showed that the same sample solution was injected 6 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 standard decoction of Liangtoujian (No.: BT04), grind it into powder, take about 0.5g, weigh it accurately, prepare the test solution according to the test solution preparation method determined under "1.4", and inject the sample at 0, 3, 9, 13, 20, and 24 hours according to the chromatographic conditions determined under "1.3". Take the chromatographic peak of chicoric acid as the reference peak S1, calculate the relative retention time and relative peak area of ​​peaks 1 to 4 and S1, take 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, calculate the RSD value, and the results are shown in Tables 18 and 19.

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

[0191]

[0192]

[0193] Table 19 Results of stability study on 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 time of each characteristic peak 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 under this method had good relative stability within 24 hours.

[0196] (3) Repeatability study

[0197] Take an appropriate amount of standard decoction of Liangtoujian (No.: BT04), grind it into powder, take about 0.5g, weigh it accurately, prepare 6 test solutions according to the test solution preparation method determined under "1.4", and perform sample analysis according to the chromatographic conditions determined under "1.3". Take the chromatographic peak of chicoric acid as the reference peak S1, calculate the relative retention time and relative peak area of ​​peaks 1 to 4 and S1, take 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 20 and 21.

[0198] Table 20 Results of the repeatability test of the characteristic spectrum of Liangtoujian standard decoction (relative retention time)

[0199]

[0200]

[0201] Table 21 Results of the repeatability test of the 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 time of each characteristic peak were in the range of 0.00% to 0.11%, and the RSD values ​​of the relative peak area 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 study

[0205] Other analysts of this project team took about 0.5 g of the same batch of Liangtoujian standard decoction (No.: BT04) on different dates and under different chromatographs, accurately weighed it, and prepared 6 parallel 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 with respect to peak 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 with respect to peak S2 were calculated, and the RSD value was calculated. The experimental results are shown in Tables 22 and 23.

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

[0207]

[0208]

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

[0210]

[0211] Fig.23 The results showed that the same batch of samples were measured 6 times by different analysts on different instruments at different 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 time of each characteristic peak were in the range of 0.04% to 0.11%, the RSD values ​​of the relative peak area were in the range of 1.06% to 1.88%, the RSD values ​​of the relative retention time 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 area and the six data of the repeatability test were in the range of 1.04% to 2.31%, indicating that the relative retention time and relative peak area of ​​each characteristic peak had good intermediate precision.

[0212] (5) Durability test

[0213] ① Investigation of chromatographic columns

[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 the double-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, using 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, using 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 Table 24, Table 25, Fig.24 .

[0216] Table 24 Characteristic spectrum of Liangtoujian standard decoction Results of the same brand of chromatographic columns (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] Fig.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%, all of which were less than 3%, indicating that the chromatographic columns of the same brand but different batches of 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] 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 that the column temperature is 38℃, 40℃, and 42℃, the other chromatographic conditions are the same as those specified under "1.3". Sample analysis is performed, and the relative retention time and relative peak area of ​​peaks 1 to 4 and S1 are calculated with the chromatographic peak of chicoric acid as the reference peak S1. The relative retention time and relative peak area of ​​peaks 5 to 9 and S2 are calculated with the chromatographic peak of cyperus rotundus A 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 Tables 26 and 27.

[0225] Table 26 Results of investigation of characteristic spectra of Liangtoujian standard decoction at different column temperatures (relative retention time)

[0226]

[0227] Table 27 Results of the investigation of the characteristic spectrum of Liangtoujian standard decoction at different column temperatures (relative peak area)

[0228]

[0229] Fig.25 The results showed that under different column temperatures, the RSD values ​​of the relative retention time of each characteristic peak and the S peak were in the range of 0.06% to 1.46%, and the RSD of the relative peak area was 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 area and relative retention time of each chromatographic peak, indicating that the 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] Take about 0.5g of the standard decoction of Liangtoujian (No.: BT04), accurately weigh it, prepare the test solution according to the test solution preparation method determined under "1.4", and inject 1μL respectively according to the chromatographic conditions determined under "1.3". Take the chromatographic peak of chicoric acid as the reference peak S1, calculate the relative retention time and relative peak area of ​​peaks 1 to 4 and S1, take 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 28 and 29.

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

[0234]

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

[0236]

[0237] Fig.26 The results showed that under different flow rates, the RSD values ​​of the relative retention time of each characteristic peak and the S peak were in the range of 0.06% to 1.77%, and the RSD of the relative peak area was in the range of 0.37% to 2.70%, all less than 3%, and the characteristic chromatogram profiles were consistent, indicating that when the flow rate was ±0.02mL / min, the relative peak area and relative retention time 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 Sections Acorus Calamus (Black Water Lily) 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 Liangtoujian standard decoction, the test sample was prepared according to the test sample solution preparation method determined in "1.4", and the sample was injected and determined according to the chromatographic conditions determined in "1.3". The Liangtoujian standard decoction was compared with the standard decoction of Acorus calamus (Black Mercury Lotus): Fig. 27 It can be seen that the characteristic spectrum of Acorus calamus (Black Mercury Anemone) is quite different from that of Acorus tatarinowii, indicating that the chemical components contained in the two are quite different. Fig. 27 It can be seen that there are obvious differences in the characteristic spectra of the standard decoction 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 were not detected in the characteristic spectrum of Northeast Nine-section Acorus, but 9 chromatographic peaks (peaks 10 to 18) appeared 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 can effectively distinguish Liangtoujian from the confused Northeast Nine-section Acorus (black water lily).

[0240] Example 3: Study on the identification of characteristic spectra of the standard decoction of Liangtoujian and the standard decoction of Jiujie Acorus calamus, the specific implementation steps are as follows:

[0241] The standard decoction of Acorus calamus 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 determined in "1.4", and the sample was injected and measured according to the chromatographic conditions determined in "1.3". The standard decoction of Liangtoujian was compared with the standard decoction of Acorus calamus: Fig.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 contained in the two are quite different. Fig.28 It can be seen that peaks 1, 2, 5, and 6 are common characteristic peaks of the two; compared with Liangtoujian, peaks 3, 4, 7, and 8 were not detected in the characteristic spectrum of Jiujie Acorus calamus, but two peaks 10 to 11 with higher responses appeared at 10-12min and 16min, among which peak 12 could not be transferred to the standard decoction. The method established in this study can effectively control the quality of Liangtoujian standard decoction, and can effectively distinguish Liangtoujian from the confused product Jiujie Acorus calamus.

[0242] Example 4: Study on the identification of characteristic spectra of standard decoction of Liangtoujian and standard decoction of Cyperus rotundus, 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 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 Cyperus rotundus: Fig.29 It can be seen that the characteristic spectrum of Cyperus rotundus is quite different from that of Rhizoma Cyperi, indicating that the chemical components they contain are quite different. Fig.29 It can be seen that peaks 2, 4, 6, and 8 are common characteristic peaks of the two, among which the responses of peaks 2, 6, and 8 in the characteristic spectrum of Liangtoujian are significantly greater than those of Cyperus rotundus; the characteristic peaks of peaks 1, 3, 5, 7, and 9 in the characteristic spectrum of Cyperus rotundus have very low or no responses, but 5 peaks 10 to 14 with higher responses appear at 12-14min, 17-20min, and 22-24min. The method established in this study can effectively control the quality of Liangtoujian standard decoction and can effectively distinguish Liangtoujian from the confused product Cyperus rotundus.

[0244] The above is a preferred embodiment of the invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the invention. These improvements and modifications are also considered to be within the scope of protection of the 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; Take the standard decoction of Liangtoujian to prepare the test solution; Preset amounts of reference solution and test solution were taken and injected into a liquid chromatograph, which used octadecylsilane bonded silica gel as a filler, acetonitrile as a mobile phase A, and a phosphoric acid solution as a mobile phase B for gradient elution to establish a characteristic spectrum.

2. The method for constructing the characteristic spectrum of the standard decoction of Liangtoujian according to claim 1, characterized in that: The gradient elution was performed according to the following procedure: 0-10min, mobile phase A from 5% to 17%, mobile phase B from 95% to 83%; 10min-23min, mobile phase A from 17% to 29%, mobile phase B from 83% to 71%; From 23 to 28 minutes, mobile phase A was maintained at 29%, and mobile phase B was maintained at 71%; 28min-33min, mobile phase A from 29% to 45%, mobile phase B from 71% to 55%; 33min~45min, mobile phase A changes from 45%→80%, and mobile phase B changes from 55%→20%.

3. The method for constructing the characteristic spectrum of the standard decoction of Liangtoujian according to claim 1, characterized in that: The volume concentration of the phosphoric acid solution is 0.15% to 0.25%.

4. The method for constructing the characteristic spectrum of the standard decoction of Liangtoujian according to claim 1, characterized in that: The preset amount of the reference solution and the test solution is 0.5 μL to 1.5 μL; 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.; When performing gradient elution, the flow rate of the mobile phase is 0.28-0.32 mL / min; The ultraviolet detection wavelength of the liquid chromatograph is 200nm-210nm.

5. The method for constructing the characteristic spectrum of the standard decoction of Liangtoujian according to claim 1, characterized in that: 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, put 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, add 20mL~21mL of water to dissolve the residue, shake and extract with water-saturated n-butanol for 3~5 times, 25mL~26mL each time, combine the n-butanol liquids, evaporate to dryness, add methanol to dissolve the residue and transfer it to a 5mL volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate to obtain.

6. The method for constructing the characteristic spectrum of the standard decoction of Liangtoujian 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, cyperus rotundus 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 cyperus rotundus A, and 15.0148 μg of caffeic acid per 1 mL, as the reference substance solution.

7. The method for constructing the characteristic spectrum of the standard decoction of Liangtoujian according to claim 1, characterized in that: The control medicinal material reference solution is prepared by the following method: Take 0.8g-1.2g of the two-headed pointed medicinal material, 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, add 20mL-21mL of water to dissolve the residue, shake and extract with water-saturated n-butanol for 3 to 5 times, 25mL-26mL each time, combine the n-butanol liquids, evaporate to dryness, add methanol to dissolve the residue and transfer it to a 5mL volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate to obtain a reference medicinal material reference solution.

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

9. The method for constructing the characteristic spectrum of the standard decoction of Liangtoujian according to claim 8, characterized in that: In the characteristic spectrum of the standard decoction of Liangtoujian, the chromatographic peak of chicoric acid 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, peak 8 0.91, and the RSD values ​​of the relative retention times of each characteristic peak were <3%.

10. Use of the characteristic spectrum of the Liangtoujian standard decoction obtained by the method for constructing the characteristic spectrum of the Liangtoujian standard decoction according to any one of claims 1 to 9 in any one of (1) to (5): (1) Authenticity identification and ingredient testing of Liangtoujian standard decoction; (2) Authenticity identification and ingredient testing of Liangtoujian medicinal materials; (3) Distinguishing between the standard decoction of Liangtoujian and the standard decoction of Northeast Nine-section Acorus Calamus; (4) Distinguishing between the standard decoction of Liangtoujian and the standard decoction of Jiujie Acorus calamus; (5) Differentiation between the standard decoction of Liangtoujian and the standard decoction of Xiangfu.

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