Evaluation method of processing degree of vinegar and moxa charcoal

Abstract

The present invention discloses a method for evaluating the processing degree of vinegar and wormwood charcoal, which includes: extracting the vinegar and wormwood charcoal with 20vol% to 90vol% methanol to obtain a test solution; dissolving neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, schaftoside, isochlorogenic acid B, 1,5-O-dicaffeoylquinic acid, isochlorogenic acid A, isochlorogenic acid C, brown cyanidin, and isoeupretinoin with 80vol% to 100vol% methanol to obtain a reference solution; measuring the test solution and the reference solution with a liquid chromatograph to obtain a vinegar and wormwood charcoal fingerprint; and evaluating the processing degree according to the number of chromatographic peaks presented in the fingerprint, and the peak area / sampling amount. The present invention provides a fast, objective and quantitative evaluation method for the quality of vinegar and wormwood charcoal to ensure the effectiveness of clinical medication.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine identification, and in particular to a method for evaluating the processing degree of vinegar, moxa and charcoal. Background Art

[0002] Artemisia argyi Levi, et Vant. is a dried leaf of Artemisia argyi Levi, et Vant. of the Asteraceae family. It has the functions of warming the meridians and stopping bleeding, dispelling cold and relieving pain. It is used for vomiting blood, abalone blood, metrorrhagia, menorrhagia, fetal bleeding, cold pain in the lower abdomen, irregular menstruation, and infertility due to uterine cold. Vinegar wormwood charcoal is a processed product of wormwood, which has the function of warming the meridians and stopping bleeding, and is often used for bleeding due to deficiency and cold. At present, it mainly relies on traditional experience to observe the appearance of charcoal medicines to judge whether their processing degree is "preserved", "not enough" or "too much", lacking a unified and objective quality control standard, and it is difficult to ensure the effectiveness of clinical medication. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide an evaluation method for the processing degree of vinegar and moxa charcoal, which can provide an objective and accurate evaluation standard for the processing degree of vinegar and moxa charcoal.

[0004] In order to solve the above technical problems, the present invention provides a method for evaluating the processing degree of vinegar and moxa charcoal, which comprises:

[0005] Extract the preset sample amount of vinegar moxa charcoal with 20 vol% to 90 vol% methanol to obtain a test solution;

[0006] Dissolve neochlorogenic acid reference substance, chlorogenic acid reference substance, cryptochlorogenic acid reference substance, caffeic acid reference substance, schaftoside reference substance, isochlorogenic acid B reference substance, 1,5-O-dicaffeoylquinic acid reference substance, isochlorogenic acid A reference substance, isochlorogenic acid C reference substance, cyanidin reference substance, and isoeupretinoic acid reference substance in 80 vol% to 100 vol% methanol to obtain a reference substance solution;

[0007] The test solution and the reference solution are measured by liquid chromatography to obtain the fingerprint of vinegar moxa charcoal; wherein the chromatographic column of the liquid chromatography uses octadecylsilane bonded silica gel as the stationary phase, acetonitrile as the mobile phase A, and 0.05 vol% to 0.2 vol% formic acid aqueous solution as the mobile phase B for gradient elution;

[0008] If the fingerprint spectrum shows 15 common peaks in total, and the peak area / sampling amount of peak 13 is 3177.6-13575.5, the peak area / sampling amount of peak 12 is 887.7-3580.2, the peak area / sampling amount of peak 15 is 8950.1-38693.5, the peak area / sampling amount of peak 8 is 6577.8-46481.6, the peak area / sampling amount of peak 10 is 243.5-3922.5, the peak area / sampling amount of peak 9 is 11139.7-86206.2, the peak area / sampling amount of peak 6 is 4505.3-50144.1, and the peak area / sampling amount of peak 5 is 429.6-2110.4, the degree of processing is storage;

[0009] If the number of common peaks in the fingerprint is greater than 15, the retention time of the additional common peaks is ≥42 min, and the peak area / sampling amount of peak 13 is greater than 13575.5, the peak area / sampling amount of peak 12 is greater than 3580.2, the peak area / sampling amount of peak 15 is greater than 38693.5, the peak area / sampling amount of peak 8 is greater than 46481.6, the peak area / sampling amount of peak 10 is greater than 3922.5, the peak area / sampling amount of peak 9 is greater than 86206.2, the peak area / sampling amount of peak 6 is greater than 50144.1, and the peak area / sampling amount of peak 5 is greater than 2110.4, the degree of processing is insufficient;

[0010] If the fingerprint spectrum shows 15 peaks in total, and the peak area / sampling amount of peak 13 is less than 3177.6, the peak area / sampling amount of peak 12 is less than 887.7, the peak area / sampling amount of peak 15 is less than 8950.1, the peak area / sampling amount of peak 8 is less than 6577.8, the peak area / sampling amount of peak 10 is less than 243.5, the peak area / sampling amount of peak 9 is less than 11139.7, the peak area / sampling amount of peak 6 is less than 4505.3, and the peak area / sampling amount of peak 5 is less than 429.6, the degree of processing is too much; or

[0011] If any one or more of peaks 13, 12, 15, 8, 10, 9, 6 and 5 disappear in the fingerprint, the degree of processing is excessive.

[0012] As an improvement of the above technical solution, the gradient elution is performed according to the following procedure:

[0013] 0min~4min, mobile phase A from 8%→10%, mobile phase B from 92%→90%;

[0014] 4min~7min, mobile phase A from 10%→15%, mobile phase B from 90%→85%;

[0015] 7min-12min, mobile phase A from 15% to 18%, mobile phase B from 85% to 82%;

[0016] 12min~18min, mobile phase A changes from 18%→19%, mobile phase B changes from 82%→81%;

[0017] 18min-22min, mobile phase A from 19% to 21%, mobile phase B from 81% to 79%;

[0018] 22min-27min, mobile phase A from 21% to 30%, mobile phase B from 79% to 70%;

[0019] 27min-30min, mobile phase A from 30% to 36%, mobile phase B from 70% to 64%;

[0020] 30min-36min, mobile phase A from 36% to 45%, mobile phase B from 64% to 55%;

[0021] 36min-38min, mobile phase A from 45% to 65%, mobile phase B from 55% to 35%;

[0022] 38min-40min, mobile phase A from 65% to 95%, mobile phase B from 35% to 5%;

[0023] 40min-45min, mobile phase A is 95%, mobile phase B is 5%;

[0024] 45min-46min, mobile phase A from 95% to 8%, mobile phase B from 5% to 92%;

[0025] 46min-50min, mobile phase A is 8%, mobile phase B is 92%;

[0026] As an improvement of the above technical solution, the column length of the chromatographic column is 100 mm to 150 mm, the column diameter is 2 mm to 3 mm, the column temperature is 20° C. to 40° C., and the particle size of the stationary phase is 1.5 μm to 2.1 μm.

[0027] As an improvement of the above technical solution, the column length of the chromatographic column is 150 mm, the column diameter is 2.1 mm, the column temperature is 25° C., and the particle size of the stationary phase is 1.7 μm.

[0028] As an improvement of the above technical solution, the flow rate of the liquid chromatograph is 0.2mL / min to 0.35mL / min, the injection volume of the test solution is 0.5μL to 3μL, and the injection volume of the reference solution is 0.5μL to 3μL.

[0029] As an improvement of the above technical solution, the flow rate of the liquid chromatograph is 0.25 mL / min, the injection volume of the test solution is 1 μL, and the injection volume of the reference solution is 1 μL.

[0030] As an improvement of the above technical solution, the mobile phase B is 0.1 vol% formic acid;

[0031] The detection wavelength of the liquid chromatograph is 320nm-340nm.

[0032] As an improvement of the above technical solution, the detection wavelength of the liquid chromatograph is 325 nm.

[0033] As an improvement of the above technical solution, the preparation method of the test solution is:

[0034] Take 1g of vinegar and wormwood charcoal powder, weigh it accurately, put it in a stoppered conical flask, add 50mL of 80vol% methanol accurately, weigh it, treat it with ultrasound at a power of 300W and a frequency of 45kHz for 30min, take it out, cool it, weigh it again, make up the lost weight with 80vol% methanol, shake it well, filter it through a 0.22μm microporous filter membrane, and take the filtrate to obtain it.

[0035] As an improvement of the above technical solution, the fingerprint spectrum of the vinegar wormwood charcoal includes 15 common peaks, among which peak 1 is neochlorogenic acid, peak 2 is chlorogenic acid, peak 3 is cryptochlorogenic acid, peak 4 is caffeic acid, peak 5 is schaftoside, peak 6 is isochlorogenic acid B, peak 7 is 1,5-O-dicaffeoylquinic acid, peak 8 is isochlorogenic acid A, peak 9 is isochlorogenic acid C, peak 13 is cyanidin, and peak 15 is isocyanidin.

[0036] The implementation of the present invention has the following beneficial effects:

[0037] The present invention is based on the analysis of the material basis of vinegar and wormwood charcoal, constructs a fingerprint spectrum of vinegar and wormwood charcoal, screens out differential characteristic indicators, determines the value range of the peak area / sampling amount of the differential characteristic indicators of vinegar and wormwood charcoal, thereby judging the processing degree of vinegar and wormwood charcoal, providing a rapid, objective and quantitative evaluation method for the quality of vinegar and wormwood charcoal, and ensuring the effectiveness of clinical medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the specific property investigation diagram in Example 1;

[0039] Figure 2 In Example 1 Figure 1 Results of chromatographic peak identification Figure 1 ;

[0040] Figure 3 In Example 1 Figure 1 Results of chromatographic peak identification Figure 2 ;

[0041] Figure 4 In Example 1 Figure 1 Results of chromatographic peak identification Figure 3;

[0042] Figure 5 It is the fingerprint superposition diagram of 16 batches of vinegar and charcoal pieces of mugwort in Example 1;

[0043] Figure 6 It is a comparison diagram of the fingerprints of the wormwood leaf medicinal material and the vinegar wormwood charcoal decoction piece in Example 1;

[0044] Figure 7 It is the fingerprint superposition diagram of 16 batches of Artemisia argyi medicinal materials in Example 2;

[0045] Figure 8 It is the OPLS-DA score graph of each peak in the characteristic spectrum of the wormwood medicinal material and the vinegar wormwood charcoal decoction piece in Example 2;

[0046] Fig. 9 It is a peak area projection importance diagram of each peak in the characteristic spectrum of the wormwood medicinal material and the vinegar wormwood charcoal decoction piece in Example 2;

[0047] Among them: peak 1 is neochlorogenic acid, peak 2 is chlorogenic acid, peak 3 is cryptochlorogenic acid, peak 4 is caffeic acid, peak 5 is schaftoside, peak 6 is isochlorogenic acid B, peak 7 is 1,5-O-dicaffeoylquinic acid, peak 8 is isochlorogenic acid A, peak 9 is isochlorogenic acid C, peak 13 is cyanidin, and peak 15 is isoeupretinoic acid. DETAILED DESCRIPTION

[0048] 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.

[0049] The sources of the instruments, materials and medicinal materials used in the following examples are as follows:

[0050] (1) Instruments: Waters ultra-high performance liquid chromatograph (Waters), PDA detector (Waters), Waters workstation; ME204E 1 / 10,000 balance (Mettler Toledo); XP26 1 / 10,000 balance (Mettler Toledo); 111B 2-liang high-speed Chinese medicine pulverizer (Zhejiang Ruian Yongli Pharmaceutical Machinery Co., Ltd.); CNC KQ-500DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); MiliQ Direct 8 ultrapure water machine (Merck Millipore).

[0051] (2) Materials: Neochlorogenic acid (batch number: 14332, content: 98%, Shanghai Shidande Standard Technology Service Co., Ltd.); Chlorogenic acid (batch number: 110753-201817, content: 96.3%, China Food and Drug Inspection Institute); Cryptochlorogenic acid (batch number: DST220104-035, content: 98%, Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.); Caffeic acid (batch number: 110885-201703, content: 99.7%, China Food and Drug Inspection Institute); Schaftoside (batch number: 111912-202204, content: 94.9%, China Food and Drug Inspection Institute); Isochlorogenic acid B (batch number: wkq210222206, content: 99.85%, Sichuan Weikeqi Biotechnology Co., Ltd.); 1,5-O-dichlorobenzene Caffeoylquinic acid (batch number: MUST-19092911, content: 98.52%, Chengdu Mansite Biotechnology Co., Ltd.); isochlorogenic acid A (batch number: 111782-202208, content: 95.9%, China Food and Drug Inspection Institute); isochlorogenic acid C (batch number: DSTDY003802, content: 99.58%, Desite Biotechnology Co., Ltd.); brown cyanidin (batch number: Z05801909018, content: 98%, Chengdu Ruifensi Biotechnology Co., Ltd.); isozeupetin (batch number: 3670, content: 98%, Shanghai Shidande Standard Technology Service Co., Ltd.); acetonitrile is chromatographically pure (Merck), formic acid is chromatographically pure (Tianjin Komiou Chemical Reagent Co., Ltd.), water is ultrapure water, and other reagents are analytically pure. Artemisia argyi (16 batches), detailed information is shown in Table 1.

[0052] Table 1 Information on the source of medicinal materials

[0053]

[0054] Example 1 Construction of vinegar moxa charcoal fingerprint

[0055] 1. Preparation of test solution

[0056] Take about 1 g of sample powder (passed through No. 3 sieve), weigh accurately, place in a stoppered conical flask, accurately add 50 mL of 80% methanol, weigh the weight, ultrasonically treat (power 300 W, frequency 45 kHz) for 30 min, take out, cool, weigh again, make up the lost weight with 80% methanol, shake well, pass through a 0.22 μm microporous filter membrane, and take the filtrate to obtain.

[0057] 2. Preparation of reference solution

[0058] Take appropriate amount of neochlorogenic acid reference substance, chlorogenic acid reference substance, cryptochlorogenic acid reference substance, caffeic acid reference substance, schaftoside reference substance, isochlorogenic acid B reference substance, 1,5-O-dicaffeoylquinic acid reference substance, isochlorogenic acid A reference substance, isochlorogenic acid C reference substance, brown cyanidin reference substance, and isoeupretinoic acid reference substance, accurately weigh, and add 100 vol% methanol. Each 1 ml contains 43.45 μg of neochlorogenic acid, 87.73 μg of chlorogenic acid, 33.32 μg of cryptochlorogenic acid, 43.55 μg of caffeic acid, 20.60 μg of schaftoside, 20.81 μg of isochlorogenic acid B, 20.54 μg of 1,5-O-dicaffeoylquinic acid, and 20.70 μg of isochlorogenic acid A. The reference solution contained 21.33μg, isochlorogenic acid C 11.59μg, cyanidin 21.25μg, and isoeupretinoic acid 22.54μg.

[0059] 3. Chromatographic conditions

[0060] Chromatographic column: ACQUITY UPLC HSS T3 Column (2.1 mm×150 mm, 1.7 μm); mobile phase: acetonitrile (A)-0.1% formic acid aqueous solution (B); elution mode: gradient elution; flow rate: 0.25 mL / min, column temperature: 25°C, detection wavelength: 325 nm. The mobile phase gradient elution program is shown in Table 2.

[0061] Table 2 Elution program

[0062]

[0063] 4. Determination

[0064] Accurately pipette 1 μL of reference solution and 1 μL of test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0065] 5. Methodological Validation

[0066] (1) Specificity inspection

[0067] Accurately pipette 1 μL of the test solution and blank solvent, inject into the liquid chromatograph, and measure according to the chromatographic conditions under "3". Figure 1 The experimental results show that there are no chromatographic peaks in the blank solvent chromatogram at the retention time corresponding to the characteristic peaks of the test sample chromatogram, indicating that the blank solvent has no interference, indicating that the method has good specificity.

[0068] (2) Precision inspection

[0069] Take the same test solution and inject it continuously for 6 times according to the chromatographic conditions under item "3", record the retention time and peak area, take the chromatographic peak of isochlorogenic acid A as the reference peak (S), and calculate the RSD values ​​of the relative retention time and relative peak area of ​​each characteristic peak, which are all less than 3%, indicating that the precision of the instrument is good.

[0070] (3) Repeatability study

[0071] Take 6 portions of the same batch of wormwood powder, weigh them accurately, prepare the test solution according to the method under "1", and measure them according to the chromatographic conditions under "3". Take the chromatographic peak of isochlorogenic acid A as the reference peak (S), and calculate the RSD values ​​of the relative retention time and relative peak area of ​​each characteristic peak, which are all less than 3%, indicating that the method has good repeatability.

[0072] (4) Stability test

[0073] Accurately aspirate the same test solution, and according to the chromatographic conditions under item "3", inject and measure at 0, 2, 4, 6, 8, 10, 12h, 18h, and 24h, respectively, record the retention time and peak area, take the isochlorogenic acid A chromatographic peak as the reference peak (S), and calculate the RSD of the relative retention time and relative peak area of ​​each characteristic peak, which are all less than 3%, indicating that the test solution has good stability within 24h.

[0074] 6. Identification of chromatographic peaks

[0075] Neochlorogenic acid reference substance, chlorogenic acid reference substance, cryptochlorogenic acid reference substance, caffeic acid reference substance, schaftoside reference substance, isochlorogenic acid B reference substance, 1,5-O-dicaffeoylquinic acid reference substance, isochlorogenic acid A reference substance, isochlorogenic acid C reference substance, brown cyanidin reference substance, and isoeupretinoic acid reference substance were used for positioning. The results showed that peak 1 was neochlorogenic acid, peak 2 was chlorogenic acid, peak 3 was cryptochlorogenic acid, peak 4 was caffeic acid, peak 5 was schaftoside, peak 6 was isochlorogenic acid B, peak 7 was 1,5-O-dicaffeoylquinic acid, peak 8 was isochlorogenic acid A, peak 9 was isochlorogenic acid C, peak 13 was brown cyanidin, and peak 15 was isoeupretinoic acid. Results are shown in Figure 2 to Figure 4 .

[0076] 7. Determination of vinegar and moxa charcoal fingerprint

[0077] Take 16 batches of vinegar moxa charcoal samples respectively, prepare the test solution according to the method under "1", and measure according to the chromatographic conditions under "3" to obtain the fingerprints of 16 batches of vinegar moxa charcoal. The results are shown in Figure 5 The results showed that the fingerprints of the 16 batches of vinegar and wormwood charcoal all showed the same 15 common peaks. Therefore, the above 15 common peaks were selected as the characteristic peaks of the vinegar and wormwood charcoal fingerprint, and peak 8 (isochlorogenic acid A) was used as the reference peak to calculate the relative retention time and relative peak area of ​​each characteristic peak and the S peak, as shown in Tables 3 and 4.

[0078] Table 3 Relative retention time of vinegar and moxa charcoal fingerprints of 16 batches

[0079]

[0080]

[0081] Table 4 Relative peak areas of vinegar and moxa charcoal fingerprints of 16 batches

[0082]

[0083] 8. Comparison of fingerprints of mugwort leaf medicinal materials and vinegar mugwort charcoal slices

[0084] Take about 1g of wormwood leaf powder and vinegar wormwood charcoal powder respectively, accurately weigh them, prepare the test solution according to the method under "1" in Example 1, and closely draw 1μL of the reference solution and different test solutions respectively, and test them according to the method under "3" in Example 1 to obtain fingerprints. Compare the differences between the fingerprints of wormwood leaf and vinegar wormwood charcoal. The results are shown in Figure 6 .

[0085] As can be seen from the figure, the fingerprint of mugwort medicinal material presents 17 characteristic peaks, and the fingerprint of vinegar and mugwort charcoal slices presents 15 characteristic peaks, among which peaks 16 and 17 disappear after the mugwort medicinal material is processed into vinegar and mugwort charcoal slices.

[0086] Embodiment 2 vinegar moxa charcoal processing degree evaluation method

[0087] (1) Vinegar and moxa charcoal processing: According to the Pharmacopoeia vinegar and moxa charcoal processing method, 16 batches of moxa leaves were processed into vinegar and moxa charcoal that met the requirements of the Pharmacopoeia.

[0088] (2) Establishment of fingerprint: 16 batches of mugwort leaves and vinegar mugwort charcoal samples were taken, and the test solution was prepared according to the method under "1" of Example 1, and the fingerprints of the 16 batches of mugwort leaves were obtained according to the chromatographic conditions under "3". Figure 7 ) and vinegar moxa charcoal fingerprint (such as Figure 5 ).

[0089] (3) Obtaining the evaluation index and its value range: The 17 chromatographic peaks in the fingerprints of 16 batches of mugwort leaves and vinegar mugwort charcoal were used to construct a partial orthogonal least squares discriminant analysis (OPLS-DA) model. The peak areas of missing chromatographic peaks were marked with 0. The OPLS-DA score diagram is shown in Figure 8 The peak area projection importance (VIP) of 17 chromatographic peaks in the OPLS-DA model was analyzed (see Fig. 9), the larger the VIP value, that is, the farther the variable is from the X-axis, it indicates that the chromatographic peak contributes more to the classification of mugwort and vinegar mugwort charcoal, that is, it may be the differential component that distinguishes mugwort and vinegar mugwort charcoal. The chromatographic peaks with VIP values ​​greater than 1.0 have an influence on the classification of samples in the following order: peak 13> peak 12> peak 15> peak 17> peak 8> peak 10> peak 9> peak 6> peak 5> peak 16, indicating that the above indicators have a significant effect on the distinction between mugwort and vinegar mugwort charcoal, among which peaks 16 and 17 are peaks that disappear after mugwort is processed into vinegar mugwort charcoal. Therefore, the peak area / sampling amount range of the chromatographic peaks (peak 13, peak 12, peak 15, peak 17, peak 8, peak 10, peak 9, peak 6, peak 5, peak 16) with VIP values ​​greater than 1.0 was established as an evaluation index for judging the processing degree of vinegar and moxa charcoal. According to the minimum and maximum values ​​of the peak areas / sampling amounts of the characteristic peaks of the fingerprint spectra of 16 batches of vinegar and moxa charcoal, the peak area / sampling amount range of peak 13 was determined to be (3177.6-13575.5), the peak area / sampling amount range of peak 12 was (887.7-3580.2), the peak area / sampling amount range of peak 15 was (895 The range of peak area / sampling volume is (0.1-38693.5), the range of peak area / sampling volume of peak 17 is 0, the range of peak area / sampling volume of peak 8 is (6577.8-46481.6), the range of peak area / sampling volume of peak 10 is (243.5-3922.5), the range of peak area / sampling volume of peak 9 is (11139.7-86206.2), the range of peak area / sampling volume of peak 6 is (4505.3-50144.1), the range of peak area / sampling volume of peak 5 is (429.6-2110.4), and the range of peak area / sampling volume of peak 16 is 0.

[0090] (4) Evaluation of processing degree:

[0091] If the fingerprints of the vinegar and moxa charcoal samples to be tested all have peaks 13, 12, 15, 17, 8, 10, 9, 6, 5 and 16, and the peaks mentioned above are all higher than the upper limit of the specified range, then it is judged that the preparation of "vinegar and moxa charcoal" is "not good enough";

[0092] For example, if Peak 13, Peak 12, Peak 15, Peak 5, Peak 10, Peak 8, Peak 9, and Peak 6 of the fingerprint spectrum of the vinegar, wormwood, and charcoal sample are all lower than the lower limit of the specified range of the above peaks, or one or more of the above peaks disappear, it is judged that the "charcoal is fried" too much.

[0093] 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 fingerprint of vinegar moxa charcoal, characterized in that: include: Take 1g of vinegar moxa charcoal powder, weigh it accurately, put it in a stoppered conical flask, add 50mL of 80vol% methanol accurately, weigh it, use 300W power and 45kHz frequency ultrasonic treatment for 30min, take it out, let it cool, weigh it again, make up the lost weight with 80vol% methanol, shake it well, filter it through a 0.22μm microporous membrane, take the filtrate, and get the test solution; The neochlorogenic acid reference substance, chlorogenic acid reference substance, cryptochlorogenic acid reference substance, caffeic acid reference substance, schaftoside reference substance, isochlorogenic acid B reference substance, 1,5-O-dicaffeoylquinic acid reference substance, isochlorogenic acid A reference substance, isochlorogenic acid C reference substance, brown cyanidin reference substance, and isoeupretinoic acid reference substance were dissolved in 100 vol% methanol to obtain 43.45 μg of neochlorogenic acid, 87.73 μg of chlorogenic acid, 33.32 μg of cryptochlorogenic acid, 43.55 μg of caffeic acid, 20.60 μg of schaftoside, 20.81 μg of isochlorogenic acid B, 20.54 μg of 1,5-O-dicaffeoylquinic acid, and 20.70 μg of isochlorogenic acid A. reference solution of 21.33 μg, isochlorogenic acid C 11.59 μg, brown cyanidin 21.25 μg, and isoeupretinoic acid 22.54 μg; The test solution and the reference solution are measured by liquid chromatography to obtain the fingerprint of vinegar moxa charcoal; wherein the chromatographic column of the liquid chromatograph is an ACQUITY UPLC HSS T3 chromatographic column, the column length is 150 mm, the column diameter is 2.1 mm, the particle size of the stationary phase is 1.7 μm, the column temperature of the chromatographic column is 25°C, and the liquid chromatograph uses acetonitrile as mobile phase A and 0.1 vol% formic acid aqueous solution as mobile phase B for gradient elution; the gradient elution is performed according to the following procedure: 0min~4min, mobile phase A from 8%→10%, mobile phase B from 92%→90%; 4min~7min, mobile phase A from 10% to 15%, mobile phase B from 90% to 85%; 7min~12min, mobile phase A from 15% to 18%, mobile phase B from 85% to 82%; 12min~18min, mobile phase A from 18% to 19%, mobile phase B from 82% to 81%; 18min~22min, mobile phase A changed from 19% to 21%, and mobile phase B changed from 81% to 79%; 22min~27min, mobile phase A from 21% to 30%, mobile phase B from 79% to 70%; 27min~30min, mobile phase A from 30% to 36%, mobile phase B from 70% to 64%; 30min~36min, mobile phase A from 36% to 45%, mobile phase B from 64% to 55%; 36min~38min, mobile phase A from 45% to 65%, mobile phase B from 55% to 35%; 38min~40min, mobile phase A from 65% to 95%, mobile phase B from 35% to 5%; 40min~45min, mobile phase A is 95%, mobile phase B is 5%; 45min~46min, mobile phase A from 95% to 8%, mobile phase B from 5% to 92%; 46min~50min, mobile phase A is 8%, mobile phase B is 92%; The detection wavelength of the liquid chromatograph is 325 nm, the flow rate of the liquid chromatograph is 0.25 mL / min, the injection volume of the test solution is 1 μL, and the injection volume of the reference solution is 1 μL.

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