A method for establishing a gynecological menstruation-regulating tablet fingerprint by using high-performance liquid chromatography technology and application thereof

Abstract

The application discloses a method for establishing a gynecological menstruation-regulating tablet fingerprint spectrum by using a high-performance liquid chromatography technology and application, by using the method, a good fingerprint spectrum is obtained, and the chromatographic peak is comprehensive, 12 fingerprint peaks are confirmed, wherein, corresponding attribution peaks of angelica sinensis, fried atractylodes macrocephala koidz, vinegar corydalis and chuanxiong are all in the gynecological menstruation-regulating tablet, and the angelica sinensis, fried atractylodes macrocephala koidz and vinegar corydalis all have exclusive peaks. By comparing with the reference substance, and combining with ultraviolet absorption spectrum characteristics, characteristic peaks of five chemical components, i.e., chlorogenic acid, ferulic acid, chuanxiongolide I, ligusticum lactone and butenyl phthalide are identified. The application further discloses a reference fingerprint spectrum of the gynecological menstruation-regulating tablet obtained by using the method, the gynecological menstruation-regulating tablet fingerprint spectrum detection method and the obtained reference fingerprint spectrum can objectively evaluate the overall quality of the gynecological menstruation-regulating tablet, and provide a research basis for quality control of the gynecological menstruation-regulating tablet.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine analysis and detection, more particularly to a method for establishing a gynecological menstruation regulating tablet fingerprint by using high performance liquid chromatography technology and application thereof. BACKGROUND

[0002] The gynecological menstruation regulating tablet is developed from a traditional Chinese medicine prescription and is made of ten kinds of medicines, including Radix Rehmanniae Preparata, Angelica sinensis, Radix Paeoniae Alba, Ligusticum chuanxiong, Rhizoma Corydalis (vinegar processed), Radix Paeoniae Rubra, Rhizoma Cyperi (vinegar processed), Rhizoma Atractylodis Macrocephalae (fermented with wheat bran), Fructus Jujubae and Radix Glycyrrhizae, and has the effects of nourishing blood and soothing liver, regulating qi and menstruation, and is commonly used for irregular menstruation, irregular menstruation, and abdominal pain during menstruation caused by liver stagnation and blood deficiency.

[0003] At present, the current quality standard of the gynecological menstruation regulating tablet is the first part of Chinese Pharmacopoeia 2020 edition, and the preparation method thereof is mentioned as follows: “fry Rhizoma Atractylodis Macrocephalae, vinegar processed Rhizoma Corydalis, Angelica sinensis and Ligusticum chuanxiong into fine powder, sieve; the rest of vinegar processed Rhizoma Cyperi and the like are added with water and decocted twice, 3 hours for the first time and 2 hours for the second time, filter, combine the filtrates, concentrate to thick paste, add the fine powder of fried Rhizoma Atractylodis Macrocephalae and the like and appropriate amount of excipients, mix uniformly, dry at 60℃ or below, press into 1000 tablets, coat with sugar or film, and obtain the product.” It can be seen that the gynecological menstruation regulating tablet is prepared by mixing the fine powder of fried Rhizoma Atractylodis Macrocephalae, vinegar processed Rhizoma Corydalis, Angelica sinensis and Ligusticum chuanxiong, and the extract of vinegar processed Rhizoma Cyperi and the like and excipients.

[0004] It is also pointed out in the first part of Chinese Pharmacopoeia 2020 edition that, in addition to the content determination of ferulic acid in Angelica sinensis and Ligusticum chuanxiong and paeoniflorin in Radix Paeoniae Alba and Radix Paeoniae Rubra, Rhizoma Corydalis, Angelica sinensis, Radix Paeoniae Alba, Radix Paeoniae Rubra, Rhizoma Atractylodis Macrocephalae and Radix Glycyrrhizae are also subjected to thin layer identification, which can better control the quality of the gynecological menstruation regulating tablet, but cannot comprehensively control the quality status and batch consistency, and cannot control the intermediate of the gynecological menstruation regulating tablet, i.e. the quality status of the fine powder of fried Rhizoma Atractylodis Macrocephalae, vinegar processed Rhizoma Corydalis, Angelica sinensis and Ligusticum chuanxiong. Compared with single component or even multiple component qualitative and quantitative determination, traditional Chinese medicine fingerprint has obvious advantages, especially for traditional Chinese medicine compound preparations with multiple medicines, complex components and unclear material basis, which can realize comprehensive evaluation of the internal quality of traditional Chinese medicine and effective control of the whole material, and is one of the effective means for the quality control of traditional Chinese medicine at present.

[0005] However, there is no report on the method for establishing the fingerprint of Gynecological Tiaojing Tablets and the quality detection method for the mixed fine powder of Angelica sinensis, Bupleurum chinense, and Rhizoma Corydalis and Rhizoma Chuanxiong in Gynecological Tiaojing Tablets. Chinese Patent: CN106018576A discloses a method for establishing the UPLC fingerprint of Gynecological Zaizao Pills. Although some of the medicinal ingredients contained in Gynecological Zaizao Pills are the same as those in Gynecological Tiaojing Tablets, the patent does not provide the attribution of each chromatographic peak to a medicinal ingredient, nor does it identify the chemical components of each chromatographic peak. Therefore, it cannot be applied to the quality analysis of Gynecological Tiaojing Tablets. Similarly, although the method for establishing the UPLC fingerprint of Dingkundan, which contains Angelica sinensis, Bupleurum chinense, Rhizoma Corydalis, and Rhizoma Chuanxiong, is disclosed in the prior art, the attribution of each chromatographic peak to a medicinal ingredient is not provided (Wang Nan. Research on Potential Quality Markers of Dingkundan [D]. Shanxi University [2024-03-15]). Different drug combinations and preparation processes can cause the same medicinal ingredient to exhibit different characteristics on the chromatographic fingerprint. Therefore, independent fingerprint establishment and medicinal ingredient attribution analysis are still needed for the study of Gynecological Tiaojing Tablets to accurately understand the chemical components and their effects in the drug and provide strong support for the quality control of the drug.

[0006] In order to comprehensively control the quality status and batch consistency, and grasp the quality status of the intermediates of Gynecological Tiaojing Tablets, i.e., the mixed fine powder of Angelica sinensis, Bupleurum chinense, Rhizoma Corydalis, and Rhizoma Chuanxiong, it is urgent to establish the fingerprint of Gynecological Tiaojing Tablets and the quality detection method for the mixed fine powder of Angelica sinensis, Bupleurum chinense, Rhizoma Corydalis, and Rhizoma Chuanxiong and the finished product of Gynecological Tiaojing Tablets. SUMMARY

[0007] The purpose of the present application is to overcome the shortcomings of the prior art and provide a method for establishing the fingerprint of Gynecological Tiaojing Tablets using high-performance liquid chromatography technology and applications.

[0008] The first purpose of the present application is to provide a method for establishing the fingerprint of Gynecological Tiaojing Tablets using high-performance liquid chromatography technology.

[0009] The second purpose of the present application is to provide a method for establishing the control fingerprint of Gynecological Tiaojing Tablets.

[0010] The third purpose of the present application is to provide a quality detection method for Gynecological Tiaojing Tablets.

[0011] In order to achieve the above-mentioned purposes, the present application is realized by the following technical solutions:

[0012] A method for establishing the fingerprint of Gynecological Tiaojing Tablets using high-performance liquid chromatography technology, comprising the following steps:

[0013] Reference solution: yangchuanmionl solution;

[0014] Test solution: methanol solution extract of the gynecological menstruation-regulating tablet;

[0015] HPLC chromatographic conditions: a Kromasil C18 chromatographic column was used, acetonitrile was used as the mobile phase A, and a 0.08-0.12% formic acid aqueous solution was used as the mobile phase B, in a gradient elution program, the volume percentage of the mobile phase B in the mobile phase system changed as follows:

[0016] 0-5 min, the mobile phase B decreased from 92% to 82%;

[0017] 5-30 min, the mobile phase B decreased from 82% to 78%;

[0018] 30-35 min, the mobile phase B was 78%;

[0019] 35-40 min, the mobile phase B decreased from 78% to 53%;

[0020] 40-50 min, the mobile phase B decreased from 53% to 45%;

[0021] 50-62 min, the mobile phase B was 45%;

[0022] 62-65 min, the mobile phase B decreased from 45% to 20%;

[0023] 65-75 min, the mobile phase B was 20%;

[0024] 75-85 min, the mobile phase B increased from 20% to 90%;

[0025] The detection wavelength was gradient detection: 0-38 min, the detection wavelength was 277 nm; 38-76 min, the detection wavelength was 254 nm; 76-85 min, the detection wavelength was 277 nm;

[0026] The reference solution and the test solution were respectively subjected to HPLC detection according to the HPLC chromatographic conditions to obtain the fingerprint spectrum of the gynecological menstruation-regulating tablet.

[0027] Preferably, the reference solution is a methanol solution with a concentration of 25-48 μg / ml of ligustilide I.

[0028] More preferably, the reference solution is a methanol solution with a concentration of 39.14-40 μg / ml of ligustilide I.

[0029] Most preferably, the reference solution is a methanol solution with a concentration of 39.14 μg / ml of ligustilide I.

[0030] More preferably, the preparation method of the reference solution is: taking ligustilide I and methanol to prepare a reference solution with a ligustilide I concentration of 25-48 μg / ml.

[0031] Preferably, the preparation method of the test product solution is: taking the gynecological menstruation-regulating tablet to be tested, crushing it, and ultrasonically extracting it with a 65-75% methanol aqueous solution for 30-60 min.

[0032] More preferably, the ultrasonic extraction time is 45 min.

[0033] More preferably, a 70% methanol aqueous solution is used for ultrasonic extraction.

[0034] Preferably, the use amount ratio of the gynecological menstruation-regulating tablet to be tested to the methanol aqueous solution is 1-2 g:10-40 ml.

[0035] More preferably, the use amount ratio of the gynecological menstruation-regulating tablet to be tested to the methanol aqueous solution is 2 g:25 ml.

[0036] Most preferably, the preparation method of the test product solution is: taking the gynecological menstruation-regulating tablet to be tested, crushing it, and ultrasonically extracting it with a 70% methanol aqueous solution for 45 min; after cooling, the weight loss is made up with a 70% methanol aqueous solution, the mixture is shaken, filtered, and the filtrate is taken to prepare the test product solution.

[0037] Further most preferably, the filtering is performed using a filter with a pore size of 0.45 μm.

[0038] Preferably, the gynecological menstruation-regulating tablet is a mixed fine powder that meets the prescription amount ratio of angelica sinensis, wheat-cooked atractylodes, vinegar-cured corydalis and ligusticum.

[0039] More preferably, in the prescription amount ratio, angelica sinensis, wheat-cooked atractylodes, vinegar-cured corydalis and ligusticum are proportioned according to a weight ratio of 144:23:32:16.

[0040] Preferably, a Kromasil 100-5-C18 chromatographic column is used, the column length is 250 mm, the inner diameter is 4.6 mm, and the particle size is 5 μm.

[0041] Preferably, the column temperature of the chromatographic column is 23-27℃.

[0042] More preferably, the column temperature of the chromatographic column is 25℃.

[0043] Preferably, the flow rate of the gradient elution program is 0.9-1.1 ml / min.

[0044] More preferably, the flow rate of the gradient elution program is 1 ml / min.

[0045] Preferably, the injection volume in the HPLC chromatographic condition is 10 μl.

[0046] Preferably, the mobile phase B is 0.10% formic acid aqueous solution.

[0047] Preferably, the theoretical plate number in the established gynecological menstruation regulating tablet fingerprint spectrum by the method is not less than 10000, calculated by the reference solution obtained ligustilide I peak.

[0048] The application of any of the methods in the quality control of gynecological menstruation regulating tablet should also be within the protection scope of the present application.

[0049] Specifically, the application is: performing high performance liquid chromatography on gynecological menstruation regulating tablet standard sample to obtain a control fingerprint spectrum according to any of the methods, performing high performance liquid chromatography on the gynecological menstruation regulating tablet to be tested to obtain a to-be-tested sample fingerprint spectrum, the to-be-tested sample fingerprint spectrum containing 12 fingerprint peaks: 1st peak to 12th peak in turn, with the 4th peak of the reference solution as S peak, and the relative retention time of the rest peaks within ±10% of the specified value, the specified value being: 1st peak 0.27; 2nd peak 0.32; 3rd peak 0.61; S peak 1.00; 5th peak 1.29; 6th peak 1.48; 7th peak 1.68; 8th peak 1.81; 9th peak 1.84; 10th peak 1.89; 11th peak 1.92; 12th peak 1.95, and the 12 fingerprint peaks in the control fingerprint spectrum compared with the corresponding peaks in the to-be-tested sample fingerprint spectrum having a similarity not less than 0.90, and the to-be-tested sample being a qualified product.

[0050] Preferably, the gynecological menstruation regulating tablet is a mixture of fine powder in accordance with the prescription amount ratio of angelica sinensis, wheat-fried atractylodes macrocephala, vinegar-cured corydalis and ligusticum chuanxiong.

[0051] More preferably, the prescription amount ratio is that angelica sinensis, wheat-fried atractylodes macrocephala, vinegar-cured corydalis and ligusticum chuanxiong are proportioned according to the weight ratio of 144:23:32:16.

[0052] A method for establishing a control fingerprint spectrum of gynecological menstruation regulating tablet, the method being: performing high performance liquid chromatography on gynecological menstruation regulating tablet standard sample to obtain a control fingerprint spectrum according to any of the methods, the control fingerprint spectrum containing 12 fingerprint peaks: 1st peak to 12th peak in turn, with the 4th peak of the reference solution as S peak, and the relative retention time of the rest peaks within ±10% of the specified value, the specified value being: 1st peak 0.27; 2nd peak 0.32; 3rd peak 0.61; S peak 1.00; 5th peak 1.29; 6th peak 1.48; 7th peak 1.68; 8th peak 1.81; 9th peak 1.84; 10th peak 1.89; 11th peak 1.92; 12th peak 1.95.

[0053] The fingerprint spectrum obtained by using the method of the present application, by comparing with the reference substance, combined with the ultraviolet absorption spectrum characteristics, identifies 5 characteristic peaks of chemical components in the 12 fingerprint peaks, which are respectively: peak 1 is the characteristic peak of chlorogenic acid; peak 3 is the characteristic peak of ferulic acid; peak 4 is the characteristic peak of senkyunolide I; peak 10 is the characteristic peak of ligustilide; and peak 11 is the characteristic peak of butenphthalide.

[0054] In the fingerprint spectrum obtained by the method of the present application, the four medicines of angelica, fried atractylodes, vinegar cis- and Szechuan lovage have corresponding attribution peaks, and angelica, fried atractylodes and vinegar cis- have exclusive peaks, peak 2, peak 8 and peak 9 are the exclusive peaks of angelica; peak 5 is the exclusive peak of vinegar cis-; and peak 12 is the exclusive peak of fried atractylodes.

[0055] Preferably, the 4th peak corresponding to the reference solution is the S peak, and the relative retention time of each fingerprint peak and the S peak is calculated, and the relative retention time is: peak 1 0.262-0.270; peak 2 0.316-0.324; peak 3 0.584-0.611; S peak 1.000; peak 5 1.247-1.374; peak 6 1.436-1.577; peak 7 1.639-1.782; peak 8 1.765-1.902; peak 9 1.801-1.936; peak 10 1.851-1.983; peak 11 1.875-2.006; and peak 12 1.891-2.033.

[0056] Preferably, 4-8 different batches of gynecological menstruation regulating tablet standard samples are detected by high performance liquid chromatography according to the method, and the fingerprint spectra of different batches of gynecological menstruation regulating tablet standard samples are obtained, and the method of "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" is used to automatically match the spectra by median method and multi-point correction to generate a control fingerprint spectrum.

[0057] More preferably, 5 different batches of gynecological menstruation regulating tablet standard samples are detected by high performance liquid chromatography according to the method.

[0058] Further more preferably, the 12 fingerprint peaks of the control fingerprint spectrum contain 5 characteristic peaks, which are respectively peak 1 chlorogenic acid characteristic peak, peak 3 ferulic acid characteristic peak, peak 4 senkyunolide I characteristic peak, peak 10 ligustilide characteristic peak and peak 11 butenphthalide characteristic peak.

[0059] Specifically, the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)" is used for analysis, a certain batch of gynecological menstruation regulating tablet standard sample fingerprint spectrum is randomly selected as a reference spectrum, the time window width is set to 0.1 min, the median method and multi-point correction are used to automatically match the spectra, and a control fingerprint spectrum is generated.

[0060] Preferably, the gynecological menstruation regulating tablet standard sample is a mixed fine powder in accordance with the prescription amount proportion of angelica, wheat-cooked atractylodes, vinegar corydalis and chuanxiong in the gynecological menstruation regulating tablet.

[0061] More preferably, in the prescription amount proportion, angelica, wheat-cooked atractylodes, vinegar corydalis and chuanxiong are proportioned according to a weight ratio of 144:23:32:16.

[0062] A quality detection method of a gynecological menstruation regulating tablet, comprising the following steps: establishing a fingerprint of a to-be-detected gynecological menstruation regulating tablet according to any of the methods, obtaining a to-be-detected sample fingerprint, the to-be-detected sample fingerprint containing 12 fingerprint peaks in the control fingerprint, and comparing the 12 fingerprint peaks in the control fingerprint with corresponding peaks in the to-be-detected sample fingerprint, and if the similarity is not less than 0.90, the to-be-detected gynecological menstruation regulating tablet is a qualified product.

[0063] Preferably, the similarity of the 12 fingerprint peaks in the control fingerprint and the corresponding peaks in the to-be-detected sample fingerprint is calculated by using the method of traditional Chinese medicine chromatographic fingerprint similarity evaluation system, the similarity of the 12 fingerprint peaks is not less than 0.90, and the to-be-detected sample is a qualified product.

[0064] Preferably, the to-be-detected gynecological menstruation regulating tablet is a mixed fine powder in accordance with the prescription amount proportion of angelica, wheat-cooked atractylodes, vinegar corydalis and chuanxiong in the gynecological menstruation regulating tablet.

[0065] More preferably, in the prescription amount proportion, angelica, wheat-cooked atractylodes, vinegar corydalis and chuanxiong are proportioned according to a weight ratio of 144:23:32:16.

[0066] The application also relates to the use of the gynecological menstruation regulating tablet fingerprint establishing method and the gynecological menstruation regulating tablet quality detection method in the overall evaluation of the gynecological menstruation regulating tablet.

[0067] Compared with the prior art, the application has the following beneficial effects:

[0068] The application establishes an HPLC fingerprint with good separation degree and comprehensive chromatographic peaks for the mixed fine powder of angelica, wheat-cooked atractylodes, vinegar corydalis and chuanxiong in the gynecological menstruation regulating tablet, 12 fingerprint peaks are confirmed, each chromatographic peak is well separated, the baseline is stable, the peak type is good, and the fingerprint has good precision, repeatability, specificity and stability, and through practical application, it is confirmed that the fingerprint establishing method is suitable for the establishment of the gynecological menstruation regulating tablet product fingerprint.

[0069] The gynecological menstruation regulating tablet main component type and content can be detected by using the fingerprint spectrum establishment method, the similarity between the test product fingerprint spectrum and the control fingerprint spectrum can be calculated by using the traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system, and the similarity result can be used for comprehensively evaluating the internal quality of the gynecological menstruation regulating tablet and realizing effective control of the whole chemical substances of the gynecological menstruation regulating tablet.

[0070] The method has good precision and stability, can detect the content of the index component in the gynecological menstruation regulating tablet, and can objectively evaluate the overall quality of the gynecological menstruation regulating tablet by using the obtained control fingerprint spectrum, thereby providing a quality control research basis for the gynecological menstruation regulating tablet. BRIEF DESCRIPTION OF DRAWINGS

[0071] Figure 1 It is a chromatogram obtained by adjusting the elution condition of the liquid chromatographic column.

[0072] Figure 2 It is a chromatogram obtained by adjusting the elution condition of the liquid chromatographic column.

[0073] Figure 3 It is a chromatogram obtained by adjusting the elution condition of the liquid chromatographic column again.

[0074] Figure 4 It is a chromatogram obtained by using different kinds of acid solution as the mobile phase B.

[0075] Figure 5 It is a chromatogram obtained by using different volume percentages of formic acid aqueous solution as the mobile phase B.

[0076] Figure 6 It is a chromatogram obtained by using different column temperatures.

[0077] Figure 7 It is a test product fingerprint spectrum obtained by detecting different wavelengths.

[0078] Figure 8 It is a chromatogram obtained by using different volume fractions of methanol aqueous solution as the extraction solvent.

[0079] Figure 9 It is a medicine taste attribution HPLC contrast spectrum.

[0080] Figure 10 It is a main chemical component confirmation HPLC contrast spectrum of each medicine taste.

[0081] Figure 11 It is a control product and test product ultraviolet spectrum absorption contrast spectrum, wherein the control product ultraviolet spectrum absorption spectrum is located on the left side, the test product ultraviolet spectrum absorption spectrum is located on the right side, and from top to bottom, the ultraviolet spectrum absorption spectra of chlorogenic acid, ferulic acid, chuanxiongol I, ligustilide and butenyl phthalide are arranged.

[0082] Figure 12 The common mode chart of the fingerprint spectrum of the mixed fine powder of Angelica sinensis, Bupleurum chinense, and Rhizoma Chuanxiong in the Gynecological Menstrual Regulating Tablet of different batches according to the prescription amount and proportion.

[0083] Figure 13 The control fingerprint spectrum of the mixed fine powder of Angelica sinensis, Bupleurum chinense, and Rhizoma Chuanxiong in the Gynecological Menstrual Regulating Tablet according to the prescription amount and proportion.

[0084] Figure 14 The HPLC contrast spectrum of the blank solvent, the reference material solution, and the test sample solution, wherein the test sample is the mixed fine powder of Angelica sinensis, Bupleurum chinense, and Rhizoma Chuanxiong in the Gynecological Menstrual Regulating Tablet according to the prescription amount and proportion.

[0085] Figure 15 The fingerprint spectrum of the finished product of the Gynecological Menstrual Regulating Tablet.

[0086] Figure 16 The chromatogram obtained by analyzing the mixed fine powder of Angelica sinensis, Bupleurum chinense, and Rhizoma Chuanxiong in the Gynecological Menstrual Regulating Tablet according to the prescription amount and proportion under the chromatographic conditions disclosed in the Chinese patent CN106018576A. DETAILED DESCRIPTION

[0087] The application will be further described in conjunction with the drawings of the specification and specific embodiments, which are used to explain the application and are not used to limit the scope of the application. The test methods used in the following examples are conventional methods unless otherwise specified; and the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0088] The experimental instruments and reagents used in the following examples are as follows:

[0089] Shimadzu high-performance liquid chromatograph LC-20AD; numerical control ultrasonic cleaner KQ300DE type (Kunshan Ultrasonic Instrument Co., Ltd.); electronic balance AB204-S (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); electronic balance PL203 (Mettler-Toledo Instrument (Shanghai) Co., Ltd.).

[0090] The control substances chlorogenic acid (batch number 110753-202119, purity 96.3%), ferulic acid (batch number 110773-201915, purity 99.4%), and senkyunolide I (batch number 112071-202101, purity 99.2%) were purchased from the China Institute for Drug Control; ligustilide (batch number PS013472, purity 99.4%) and butenolide (batch number PS021016, purity 97.9%) were purchased from Chengdu Pusai Biological Technology Co., Ltd. Water was distilled water; acetonitrile, methanol, and phosphoric acid were chromatographic grade; anhydrous formic acid and glacial acetic acid were analytical pure.

[0091] The samples used in the following examples:

[0092] The Atractylodes Macrocephala Kodo is purchased from Hunan Qianjin Medicine Co., Ltd. The samples with batch number Y090-2208003 and batch number Y090-2305003 are both produced in Bozhou, Anhui; the sample with batch number Y090-2302001 is produced in Jinhua, Zhejiang; the sample with batch number Y090-2306004 is produced in Dongyang, Zhejiang;

[0093] The Rhizoma Corydalis is purchased from Zhongyaokonggu Hunan Minsheng Pharmaceutical Co., Ltd. The samples with batch number Y097-2208003, batch number Y097-2303002 and batch number Y097-2303003 are all produced in Panan, Zhejiang; the sample with batch number Y097-2304004 is produced in Jinhua, Zhejiang;

[0094] The Chuanxiong Rhizoma is purchased from Sichuan Xinhua Chinese Herbal Medicine Co., Ltd. The samples with batch number Y094-2202001, batch number Y094-2212002 and batch number Y094-2303001 are all produced in Pengzhou, Sichuan;

[0095] The Angelica Sinensis is purchased from Longxi Qianjin Medicine Co., Ltd. The samples with batch number Y001-2206017, batch number Y001-2302004, batch number Y001-2305011, batch number Y001-2307017 and batch number Y001-2308020 are all produced in Dingxi, Gansu;

[0096] The negative samples of Atractylodes Macrocephala Kodo (batch number YX20230613-FCBZ), the negative samples of Rhizoma Corydalis (batch number YX20230613-CYHS), the negative samples of Chuanxiong Rhizoma (batch number YX20230613-CX), the negative samples of Angelica Sinensis (batch number YX20230613-DG) and the double negative samples of Chuanxiong Rhizoma and Angelica Sinensis (batch number YX20231008-CXDG) are all self-made.

[0097] The "four-herb mixed fine powder" in the examples refers to the mixed fine powder with the prescription amount and ratio of Angelica Sinensis, Atractylodes Macrocephala Kodo, Rhizoma Corydalis and Chuanxiong Rhizoma in Gynecological Menstrual Regulation Tablets. Specifically, the prescription amount and ratio of Angelica Sinensis, Atractylodes Macrocephala Kodo, Rhizoma Corydalis and Chuanxiong Rhizoma in Gynecological Menstrual Regulation Tablets are as follows: the weight ratio of Angelica Sinensis, Atractylodes Macrocephala Kodo, Rhizoma Corydalis and Chuanxiong Rhizoma is 144:23:32:16.

[0098] Example 1 Influence of different chromatographic conditions on the fingerprint of the four-herb mixed fine powder of Gynecological Menstrual Regulation Tablets

[0099] 1. Preparation of test solution

[0100] Take the mixed fine powder of 2 g according to the prescription amount of Angelica, Fried Atractylodes Rhizome, Vinegar Corydalis and Szechuan Lovage Root in Gynecological Menstrual Regulating Tablets, accurately weigh and place in a conical flask with a stopper, accurately add 25 ml of 70% methanol aqueous solution, tightly seal, weigh after ultrasonic treatment for 45 min, cool and weigh again, make up the weight loss with 70% methanol aqueous solution, shake well, filter with a filter with a pore size of 0.45 μm, take the filtrate, and the test sample solution is obtained.

[0101] 2. Influence of different elution conditions on peak shape

[0102] (1) Experimental method:

[0103] Accurately take 10 μl of the test sample solution prepared in "1. Preparation of test sample solution" and inject it into the high performance liquid chromatograph for high performance liquid chromatography analysis to obtain a chromatogram:

[0104] Use acetonitrile as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B, and elute under the conditions of Table 1, Table 2 or Table 3, respectively;

[0105] Other chromatographic conditions are as follows: octadecylsilane-bonded silica gel as the filler, Kromasil 100-5-C18 chromatographic column (column length 25 cm, inner diameter 4.6 mm, particle size 5 μm); flow rate 1.0 ml / min; detection wavelength 254 nm; column temperature 30°C.

[0106] Table 1 Initial liquid chromatography column elution conditions

[0107]

[0108] Table 2 Liquid chromatography column elution conditions

[0109]

[0110] Table 3 Liquid chromatography column elution conditions

[0111]

[0112] (2) Experimental results

[0113] The results are shown in Table 1, and the chromatogram obtained under the elution conditions is shown in Table 2. Figures 1-3 Figure 1 Under the elution conditions shown in Table 1, the obtained chromatogram is shown in Table 2, and there is basically no chromatographic peak after 75 minutes, and the main chromatographic information is concentrated in the first 60 minutes.

[0114] Under the elution conditions shown in Table 2, the obtained chromatogram is shown in Table 3, and the separation degree of each main chromatographic peak is good, but the distribution is not uniform, and part of the peaks are not eluted after 65 minutes. Figure 2 Under the elution conditions shown in Table 2, the obtained chromatogram is shown in Table 3, and the separation degree of each main chromatographic peak is good, but the distribution is not uniform, and part of the peaks are not eluted after 65 minutes.​

[0115] Under the elution conditions shown in Table 3, the obtained chromatograms are as follows: Figure 3 As shown, the chromatographic peak signals are relatively uniform, most chromatographic peaks are well separated, and the main chromatographic peaks have been basically collected. Therefore, it is preliminarily determined that the elution conditions in Table 3 will be used for subsequent experiments.

[0116] 3. Effects of different types of acids as mobile phase B on chromatographic peak shape and resolution.

[0117] (1) Experimental methods

[0118] Using the elution conditions in Table 3, the test solution was analyzed by high-performance liquid chromatography according to the experimental method in "2. Effect of different elution conditions on peak shape" of this embodiment, except that the mobile phase B was changed:

[0119] Experimental Group 1: Acetonitrile was used as mobile phase A, and 0.1% formic acid aqueous solution was used as mobile phase B;

[0120] Experimental Group 2: Acetonitrile was used as mobile phase A, and 0.1% (v / v) phosphoric acid aqueous solution was used as mobile phase B;

[0121] Experimental Group 3: Acetonitrile was used as mobile phase A, and 0.1% (v / v) aqueous acetic acid solution was used as mobile phase B;

[0122] The effects of different types of acids as mobile phase B on elution results were compared.

[0123] (2) Experimental Results

[0124] Chromatographic results obtained by using different types of acids as mobile phase B are as follows: Figure 4 As shown, the results indicate that experimental group 3 lost chromatographic peaks with retention times of 34–35 minutes; the chromatographic peaks with retention times of around 31 minutes in experimental group 2 had poorer peak shapes. Figure 4 (As indicated by the middle arrow); The peak shapes and resolutions of the main chromatographic peaks in experimental group 1 were good, so the mobile phase was determined to be: acetonitrile as mobile phase A and formic acid aqueous solution with a volume percentage of 0.1% as mobile phase B for subsequent experiments.

[0125] 4. Effects of different volume percentages of formic acid aqueous solution as mobile phase B on chromatographic peak shape and resolution.

[0126] (1) Experimental methods

[0127] Using the elution conditions in Table 3, the test solution was analyzed by high-performance liquid chromatography according to the experimental method in "2. Effect of different elution conditions on peak shape" of this embodiment, except that the mobile phase B was changed:

[0128] Experimental group 1 used a 0.1% (v / v) formic acid aqueous solution as mobile phase B;

[0129] Experimental group 2 used a 0.3% (v / v) formic acid aqueous solution as mobile phase B;

[0130] Experimental group 3 used a 0.5% (v / v) formic acid aqueous solution as mobile phase B;

[0131] The effects of different volume percentages of formic acid aqueous solution as mobile phase B on elution results were compared.

[0132] (2) Experimental Results

[0133] Chromatographic results obtained using aqueous formic acid solutions of different volume percentages as mobile phase B are shown below. Figure 5 As shown, the results indicate that the chromatographic peaks with retention times of approximately 31 minutes in experimental groups 2 and 3 ( Figure 5 The peak shapes (indicated by the middle arrow) were all poor. The main chromatographic peaks in experimental group 1 had better peak shapes and resolution, and the baseline was more stable. Therefore, the mobile phase system was determined to be: acetonitrile as mobile phase A and formic acid aqueous solution with a volume percentage of 0.1% as mobile phase B for subsequent experiments.

[0134] 5. Effect of different column temperatures on elution results

[0135] (1) Experimental methods

[0136] Using the elution conditions in Table 3, the test solution was analyzed by high-performance liquid chromatography (HPLC) according to the experimental method in "2. Effect of different elution conditions on peak shape" of this embodiment, except that the column temperature of the HPLC column was changed:

[0137] The column temperature for experimental group 1 was 40℃; the column temperature for experimental group 2 was 35℃.

[0138] The column temperature in experimental group 3 was 30℃; the column temperature in experimental group 4 was 25℃.

[0139] Compare the effects of different column temperatures on elution results.

[0140] (2) Experimental Results

[0141] Chromatographic values ​​obtained at different liquid chromatography column temperatures are as follows: Figure 6 As shown, the results indicate that the main difference in the liquid chromatograms obtained from different experimental groups lies in the different peak resolutions between 22 and 35 minutes. Figure 6 (The part shown in the Chinese box) Among them, the chromatographic peak resolution of experimental group 4 was the best, so the column temperature was selected as 25℃ for subsequent experiments.

[0142] 6. Effects of different gradient elution conditions

[0143] (1) Experimental methods

[0144] The test solution was analyzed by high performance liquid chromatography according to the experimental method in "2. Effect of different elution conditions on peak shape" of this embodiment. The difference is that the column temperature of the liquid chromatography column was controlled at 25°C and elution was performed according to the conditions in Table 4.

[0145] Table 4 Elution conditions for liquid chromatography column

[0146]

[0147] (2) Experimental Results

[0148] The results showed that under the elution conditions shown in Table 4, the separation between adjacent chromatographic peaks was good, the chromatographic peak signal distribution was relatively uniform, most chromatographic peaks had good separation, no overlap, sharp and symmetrical peak shapes, no leading or trailing peaks, stable baselines, and the main chromatographic peaks were basically collected. Therefore, the elution conditions in Table 4 were determined to be used for subsequent experiments.

[0149] 7. The effect of different detection wavelengths on chromatograms

[0150] (1) Experimental methods

[0151] The fine powder sample of the four herbs of the gynecological menstrual regulating tablet was scanned at wavelengths of 190–400 nm and analyzed by contour viewing. The results showed that the chromatographic peaks in the wavelength range of 210–300 nm contained more information. Therefore, wavelengths of 210 nm, 230 nm, 254 nm, 277 nm and 300 nm were selected for analysis.

[0152] The test solution was analyzed by high-performance liquid chromatography according to the experimental method in "6. Effect of different gradient elution conditions" of this embodiment, only the detection wavelength was changed:

[0153] Experimental group 1 was detected at a wavelength of 210 nm; Experimental group 2 was detected at a wavelength of 230 nm.

[0154] The detection wavelength for experimental group 3 was 254 nm; the detection wavelength for experimental group 4 was 277 nm.

[0155] Experimental group 5 used a detection wavelength of 300 nm; the effects of different detection wavelengths on the chromatogram were compared.

[0156] (2) Experimental Results

[0157] The chromatograms obtained from experimental groups 1-5 are as follows: Figure 7 As shown in the results, compared with the chromatograms obtained from experimental group 1, experimental group 2 and experimental group 3, the chromatogram obtained from experimental group 3 has the most complete peak information. The chromatographic peak (Ligusticum lactone I) in experimental group 4 at about 32 minutes has the highest response value at 277nm. Based on the resolution of each peak, variable wavelength detection was selected. The specific detection wavelengths are shown in Table 5. Table 5 was used as the detection wavelength for subsequent experiments.

[0158] Table 5 Variable wavelength detection conditions

[0159]

[0160] Example 2 A method for detecting four mixed fine powders of gynecological menstruation regulating tablets by high performance liquid chromatography

[0161] The four mixed fine powders of gynecological menstruation regulating tablets were detected by high performance liquid chromatography, and the specific detection method was as follows:

[0162] Octadecylsilane-bonded silica gel was used as the filler, and a Kromasil 100-5-C18 chromatographic column (column length 25 cm, inner diameter 4.6 mm, particle size 5 μm) was used; acetonitrile was used as the mobile phase A, and 0.1% formic acid aqueous solution was used as the mobile phase B, and elution was carried out according to the conditions in Table 4; the flow rate was 1.0 ml per minute; the column temperature was 25°C; and variable wavelength detection was carried out according to the conditions in Table 5.

[0163] Example 3 Influence of different sample processing methods on the fingerprint of four mixed fine powders of gynecological menstruation regulating tablets

[0164] 1. Influence of different concentrations of extraction solvents on chromatographic results

[0165] (1) Experimental method

[0166] The test sample was extracted using different concentrations of methanol aqueous solution: 4 test samples each of 2 g of mixed fine powder corresponding to the prescription amount of angelica, wheat bran-fried atractylodes, vinegar-celery and chuanxiong in gynecological menstruation regulating tablets were accurately weighed and placed in a conical flask with a stopper, and different concentrations of methanol aqueous solution with an extraction volume of 25 mL were accurately added to the conical flask with a stopper:

[0167] In experimental group 1, 30% methanol aqueous solution was added;

[0168] In experimental group 2, 50% methanol aqueous solution was added;

[0169] In experimental group 3, 70% methanol aqueous solution was added;

[0170] In experimental group 4, 100% methanol aqueous solution was added;

[0171] The flask was tightly sealed, the weight was determined, and ultrasonic treatment was carried out for 45 minutes, then the sample was cooled and weighed again. The weight loss of experimental groups 1-4 was made up with the corresponding concentration of methanol aqueous solution, shaken well, filtered with a filter with a pore size of 0.45 μm, and the filtrate was obtained as the test sample;

[0172] The 10 μl of each of the test samples extracted by the methanol aqueous solution of different concentrations was injected into the liquid chromatograph, and the high performance liquid chromatography was analyzed by the method of Example 2.

[0173] (2) Experimental results

[0174] The chromatography results of the methanol aqueous solution of different concentrations as the extraction solvent are shown in Table 1. Figure 8 As shown in Table 1, the results show that the extraction rates of the compounds with the retention time of about 48 minutes and 64 minutes in the chromatograms of Experimental Group 1 and Experimental Group 2 are very low (shown in the middle box), and with the increase of the methanol concentration in the methanol aqueous solution, the extraction rates of the compounds within 30 minutes slightly decrease, while the extraction rates of the compounds after 45 minutes show a trend of increase. Figure 8 As shown in Table 1, the results show that the extraction rates of the compounds with the retention time of about 48 minutes and 64 minutes in the chromatograms of Experimental Group 1 and Experimental Group 2 are very low (shown in the middle box), and with the increase of the methanol concentration in the methanol aqueous solution, the extraction rates of the compounds within 30 minutes slightly decrease, while the extraction rates of the compounds after 45 minutes show a trend of increase. Figure 8 As shown in Table 1, the results show that the extraction rates of the compounds with the retention time of about 48 minutes and 64 minutes in the chromatograms of Experimental Group 1 and Experimental Group 2 are very low (shown in the middle box), and with the increase of the methanol concentration in the methanol aqueous solution, the extraction rates of the compounds within 30 minutes slightly decrease, while the extraction rates of the compounds after 45 minutes show a trend of increase.

[0175] 2. Effect of different ultrasonic treatment time on chromatography results

[0176] (1) Experimental method

[0177] 2 g of the mixed fine powder according to the prescription amount and ratio of Angelica sinensis, Bupleurum chinense, Rhizoma Corydalis and Rhizoma Chuanxiong in the gynecological menstruation regulating tablet was taken as the test sample, and 3 portions of the test sample were accurately weighed and placed in a conical flask with a plug, 25 ml of the methanol aqueous solution with a volume fraction of 70% was accurately added, the flask was tightly plugged, the weight was determined, and different ultrasonic treatment time was carried out:

[0178] Experimental Group 1 was ultrasonically treated for 30 minutes; Experimental Group 2 was ultrasonically treated for 45 minutes;

[0179] Experimental Group 3 was ultrasonically treated for 60 minutes;

[0180] After ultrasonic treatment, the sample was cooled and weighed again, the lost weight was made up with the methanol aqueous solution with a volume fraction of 70%, and the mixture was shaken and filtered through a filter with a pore size of 0.45 μm, and the filtrate was obtained, which was the test sample extracted by different ultrasonic treatment time;

[0181] The 10 μl of each of the test samples extracted by the methanol aqueous solution of different concentrations was injected into the liquid chromatograph, and the high performance liquid chromatography was analyzed by the method of Example 2.

[0182] (2) Experimental results

[0183] The chromatograms of the test samples obtained by different ultrasonic treatment times are not much different. The spectrum obtained by experiment group 3 is taken as the control spectrum, and the method of "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)" is used for full spectrum matching. The similarity calculation method is used to obtain the similarity results of the spectra obtained by experiment groups 1 and 2, and the results are shown in Table 6. The spectrum obtained by experiment group 2 is basically consistent with that of experiment group 3, and the spectrum obtained by experiment group 1 has slightly lower similarity. Therefore, the ultrasonic treatment time of 45 minutes is selected for subsequent experiments.

[0184] Table 6 Similarity results of different ultrasonic treatment times

[0185]

[0186] Preparation method of test sample solution in example 4

[0187] Take 2g of fine powder of the sample to be tested, accurately weigh, and place it in a conical flask with a stopper. Accurately add 25ml of 70% methanol aqueous solution, tightly seal, and weigh after ultrasonic treatment for 45 minutes. After cooling, weigh again, make up the weight loss with 70% methanol aqueous solution, shake well, filter with a filter with a pore size of 0.45μm, and take the filtrate to obtain the test sample solution.

[0188] Example 5: Chromatographic peak assignment and reference selection

[0189] 1. Assignment of chromatographic peaks to medicinal ingredients

[0190] (1) Experimental method

[0191] S1. Preparation of single medicinal ingredient decoction solution: According to the preparation method of example 4, 2g of fine powder of each of angelica, fried atractylodes, vinegar corydalis and chuanxiong was prepared to prepare the single medicinal ingredient decoction solution;

[0192] Preparation of negative sample solution: According to the preparation method of example 4, 1g of angelica negative sample, 2g of fried atractylodes negative sample, 2g of vinegar corydalis negative sample, 2g of chuanxiong negative sample and 1g of chuanxiong angelica double negative sample fine powder were prepared to prepare each negative sample solution. The angelica negative sample is a mixture of fine powder with the prescription amount of fried atractylodes, vinegar corydalis and chuanxiong in gynecological menstruation regulating tablets, the fried atractylodes negative sample is a mixture of fine powder with the prescription amount of angelica, vinegar corydalis and chuanxiong in gynecological menstruation regulating tablets, the vinegar corydalis negative sample is a mixture of fine powder with the prescription amount of angelica, fried atractylodes and chuanxiong in gynecological menstruation regulating tablets, and the chuanxiong angelica double negative sample is a mixture of fine powder with the prescription amount of vinegar corydalis and fried atractylodes in gynecological menstruation regulating tablets;

[0193] Preparation of the test solution: According to the preparation method in Example 4, take 2g of the mixed fine powder that meets the prescription ratio of Angelica sinensis, stir-fried Atractylodes macrocephala, vinegar-processed Corydalis yanhusuo and Ligusticum chuanxiong in the gynecological menstrual regulating tablets to prepare the test solution;

[0194] S2. Accurately pipette 10 μl each of the test solution, single herbal decoction piece solution and negative sample solution obtained in step S1, inject them into the liquid chromatograph, and perform detection according to the method of Example 2 to determine the attribution of each chromatographic peak in the fingerprint spectrum.

[0195] (2) Experimental Results

[0196] The experimental results are shown in Figure 9 The results of the herbal attribution are shown in Table 7. Angelica sinensis, stir-fried Atractylodes macrocephala, vinegar-processed Corydalis yanhusuo, and Ligusticum chuanxiong all have corresponding attributions, and Angelica sinensis, stir-fried Atractylodes macrocephala, and vinegar-processed Corydalis yanhusuo all have unique characteristic peaks. This indicates that the method has high accuracy and specificity, and can effectively identify and distinguish these four herbs in gynecological menstrual regulating tablets.

[0197] Table 7 Results of herbal ingredient attribution

[0198]

[0199] 2. Identification of fingerprint peaks

[0200] (1) Experimental methods

[0201] Based on literature review, peak localization studies were conducted on the main components of each herb: chlorogenic acid, ferulic acid, ligustilide I, ligustilide, and butylphthalide. Chlorogenic acid was assigned to the herbs stir-fried with wheat bran, ligustilide, and angelica sinensis; ferulic acid to ligustilide and angelica sinensis; ligustilide I to ligustilide and angelica sinensis; ligustilide to ligustilide and angelica sinensis; and butylphthalide to ligustilide and angelica sinensis.

[0202] The specific experimental steps are as follows:

[0203] S1. The test solution was prepared according to the preparation method of Example 4, wherein the test sample was 2g of a mixture of fine powders that met the prescription ratio of Angelica sinensis, stir-fried Atractylodes macrocephala, vinegar-processed Corydalis yanhusuo and Ligusticum chuanxiong in the gynecological menstrual regulating tablets.

[0204] Preparation of the reference solution:

[0205] Accurately weigh an appropriate amount of chlorogenic acid reference standard and add methanol to prepare a solution containing 41.60 μg per ml;

[0206] Accurately weigh an appropriate amount of ferulic acid reference standard and add it to a 70% methanol aqueous solution to prepare a solution containing 44.19 μg per ml.

[0207] Accurately weigh a proper amount of ligustilide reference substance, add methanol to make a solution containing 39.14 μg per 1 ml;

[0208] Accurately weigh a proper amount of ligustilide reference substance, add methanol to make a solution containing 39.14 μg per 1 ml;

[0209] Accurately weigh a proper amount of ligustilide reference substance, add methanol to make a solution containing 39.14 μg per 1 ml;

[0210] Accurately weigh a proper amount of ligustilide reference substance, add methanol to make a solution containing 39.14 μg per 1 ml;

[0211] Scan the UV spectrum of the test sample solution and the reference substance solution prepared in step S1 under the condition of 190-400 nm to obtain the UV spectrum absorption contrast chart;

[0212] S3. Identify the main fingerprint peaks by comparing the retention time and the UV spectrum absorption contrast chart of the reference substance solution and the test sample solution.

[0213] (2) Experimental results

[0214] The experimental results are shown in Figure 10 and Figure 11 wherein, Figure 11 the left column is the UV spectrum absorption contrast chart of the reference substance solution, Figure 11 the right column is the UV spectrum absorption contrast chart of the test sample solution, Figure 11 and the middle column from top to bottom is the UV spectrum absorption contrast chart of chlorogenic acid, ferulic acid, ligustilide, ligustrazine and butenplend.

[0215] According to Figure 10 and Figure 11 the chromatographic peak identification results are shown in Table 8.

[0216] Table 8 Chromatographic peak identification results

[0217]

[0218] As Figure 10 can be seen, the ligustilide chromatographic peak has moderate retention time, high response value and good separation degree, so ligustilide is selected as the reference peak and marked as peak S, and the ligustilide reference substance is used as the reference material.

[0219] Example 6 Method for establishing the fingerprint spectrum of gynecological menstruation-regulating tablets

[0220] (1) Preparation of test sample solution

[0221] Accurately weigh out 2 g of the sample powder, place it in a conical flask with a stopper, and precisely add 25 ml of 70% methanol aqueous solution. Seal the flask, weigh it, and then ultrasonically treat it for 45 min. After cooling, re-weigh it, make up the weight loss with 70% methanol aqueous solution, shake well, filter through a filter with a pore size of 0.45 μm, and take the filtrate to obtain the test solution.

[0222] (2) Preparation of the reference solution

[0223] Accurately weigh out an appropriate amount of ligustilide I reference substance, and add methanol to prepare a ligustilide I solution containing 39.14 μg per 1 ml, to obtain the reference solution.

[0224] (3) Determination method

[0225] Accurately pipette 10 μl of the reference solution and the test solution, respectively, inject them into the liquid chromatograph, and determine and record the chromatogram.

[0226] (4) Chromatographic conditions

[0227] Use Kromasil 100-5-C18 (column length 25 cm, inner diameter 4.6 mm, particle size 5 μm) as the chromatographic column; use acetonitrile as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B, elute according to the gradient elution conditions in Table 4; the flow rate is 1.0 ml per minute; perform variable wavelength detection according to the conditions in Table 5; and the column temperature is 25°C. The theoretical plate number should not be less than 10,000, calculated according to the ligustilide I peak.

[0228] Example 7 Establishment of the Control Fingerprint of the Four-Ingredient Mixed Powder of Gynaecological Menstrual Regulation Tablets

[0229] (1) Preparation of multiple batches of samples

[0230] The four-ingredient mixed powder of gynaecological menstrual regulation tablets is composed of Angelica sinensis, Ligusticum chuanxiong, coicis rhizoma prepared with wheat bran, and rhizoma Corydalis, all of which are single base original medicine ingredients. Five batches of four-ingredient mixed powder samples are obtained according to the combinations in Table 9.

[0231] Table 9 Sample combination information table

[0232]

[0233] (2) Marking of common peaks

[0234] The method of Example 6 was used to determine the four-medicinal-ingredient mixed fine powder samples of the five batches of gynecological menstruation-regulating tablets obtained in this example "(1) Multi-batch sample preparation", and the obtained fingerprint spectra were analyzed. The chromatographic peaks with better stability and suitable response values in the fingerprint spectra of the five samples were selected as common peaks. A total of 12 common peaks were labeled, which were the fingerprint peaks in Example 5. The source medicinal ingredients and peak numbers are shown in Table 7.

[0235] The fingerprint spectra of the five samples are shown in Figure 12 , Figure 12 In Table 8, S1 is the fingerprint spectrum of the sample with batch number M; S2 is the fingerprint spectrum of the sample with batch number M'; S3 is the fingerprint spectrum of the sample with batch number XS20231020-1; S4 is the fingerprint spectrum of the sample with batch number XS20231020-2; S5 is the fingerprint spectrum of the sample with batch number XS20231020-3; and R is the control fingerprint spectrum automatically matched by "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" based on S1-S5.

[0236] The results show that the five sample test samples present 12 common characteristic peaks in the fingerprint spectra, among which the peak corresponding to the angelica lactone I reference is the S peak. The relative retention times of each characteristic peak and the S peak were calculated, and the average values of the relative retention times of each characteristic peak were as follows: peak 1, 0.27; peak 2, 0.32; peak 3, 0.61; S peak (peak 4), 1.00; peak 5, 1.29; peak 6, 1.48; peak 7, 1.68; peak 8, 1.81; peak 9, 1.84; peak 10, 1.89; peak 11, 1.92; and peak 12, 1.95.

[0237] (3) Establishment of control fingerprint spectrum

[0238] Using the method of "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)", S1 in this example "(2) Labeling of common peaks" was used as the reference spectrum, the time window width was 0.1 min, the median method and full-spectrum peak automatic matching spectrum were used, and the control fingerprint spectrum was generated. The control fingerprint spectrum is shown in Figure 13 , Figure 13 In Table 8, the 12 common peaks are the fingerprint peaks in Example 5, so according to Figure 10 、 Figure 13 and Table 8, it can be seen that Figure 13 In Table 8, peak 1 is the chlorogenic acid chromatographic peak; peak 3 is the ferulic acid chromatographic peak; peak 4 (S peak) is the angelica lactone I chromatographic peak; peak 10 is the ligusticum lactone chromatographic peak; and peak 11 is the butenyl phthalide chromatographic peak.

[0239] (4) Similarity calculation

[0240] The similarity of the fingerprint chromatograms of the five batches of four-ingredient mixed fine powder samples prepared in "(1) Preparation of multiple batches of samples" to the reference fingerprint chromatogram was calculated using the method of "Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine (2012 Edition)", and the results are shown in Table 10. In Table 10, R is the number of the reference fingerprint chromatogram; S1 is the number of the fingerprint chromatogram of the sample with batch number M; S2 is the number of the fingerprint chromatogram of the sample with batch number M'; S3 is the number of the fingerprint chromatogram of the sample with batch number XS20231020-1; S4 is the number of the fingerprint chromatogram of the sample with batch number XS20231020-2; and S5 is the number of the fingerprint chromatogram of the sample with batch number XS20231020-3.

[0241] Table 10 Similarity results of the fingerprint chromatograms of the four-ingredient mixed fine powder in five batches of Gynecological Menstrual Regulation Tablets

[0242]

[0243] According to the results in Table 10, the similarity of the fingerprint chromatograms of the five batches of four-ingredient mixed fine powder samples prepared in "(1) Preparation of multiple batches of samples" to the reference fingerprint chromatogram is greater than 0.95, i.e., the similarity of the samples between different batches is good.

[0244] Therefore, it is tentatively determined that the test sample fingerprint chromatogram should exhibit chromatographic peaks consistent with the retention times of the reference material chromatographic peaks, and should exhibit 12 common peaks (i.e., fingerprint peaks), and the similarity calculated using the common peaks according to the method of "Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine (2012 Edition)" should be no less than 0.90, then the test sample can be determined as a qualified sample.

[0245] Example 8 Method for establishing the fingerprint chromatogram of the four-ingredient mixed fine powder of Gynecological Menstrual Regulation Tablets and methodological investigation

[0246] 1. Specificity and integrity

[0247] (1) Experimental method

[0248] The fingerprint chromatogram determination method described in Example 6 was used, except that the liquid chromatography elution conditions shown in Table 11 were used for elution.

[0249] A methanol aqueous solution with a volume fraction of 70% was selected as the blank solvent, and the chromatograms of the test sample, the reference material solution, and the blank solvent were established, wherein the test sample was 2 g of the four-ingredient mixed fine powder prepared according to the prescription amount of angelica sinensis, fried atractylodes macrocephala kodo, vinegar corydalis and chuanxiong in Gynecological Menstrual Regulation Tablets.

[0250] Table 11 Liquid chromatography column elution conditions

[0251]

[0252] (2) Experimental results

[0253] The experimental results are shown in Table 6. Figure 14 Figure 14 It can be seen from Table 6 that the blank solvent does not interfere with the fingerprint peaks of the test solution; the test solution has no effective chromatographic peaks after 75 minutes, basically satisfying the principle of maximizing the amount of effective information. It is shown that the specificity and integrity of the fingerprint spectrum determination method described in Example 6 meet the requirements.

[0254] 2. Injection precision

[0255] (1) Experimental method

[0256] The test sample was detected by the method of Example 6. The test sample was 2 g of mixed fine powder in accordance with the prescription amount of Angelica sinensis, Bupleurum chinense, Rhizoma Corydalis and Chuanxiong in Gynecological Menstrual Regulating Tablets, and the sample solution was continuously injected 5 times. The sample numbers were recorded as precision 1 (S2), precision 2 (S3), precision 3 (S4), precision 4 (S5) and precision 5 (S6), respectively. The chromatograms were recorded. The peaks corresponding to the reference peaks were S peaks, and the relative retention times of 12 fingerprint peaks were calculated. The method of Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version) was used to automatically match the repeatability HPLC chromatogram peaks to form a common mode diagram, and a control chromatogram R was established to calculate the similarity.

[0257] (2) Experimental results

[0258] The injection precision results are shown in Table 12, and the fingerprint spectrum similarity results obtained by the injection precision experiment are shown in Table 13. The results show that the similarity of each chromatogram is 1.00 (> 0.95); the experimental data of the 5 groups of precision experiments show that the maximum value of the relative retention time RSD of each fingerprint peak is 0.2% (< 5%), indicating that the injection precision of the fingerprint spectrum determination method described in Example 6 meets the requirements.

[0259] Table 12 Relative retention time of injection precision

[0260]

[0261] Table 13 Similarity results of injection precision

[0262]

[0263]

[0264] 3. Repeatability

[0265] (1) Experimental method

[0266] ​Six test sample solutions were prepared in parallel, each of which was 2 g of a mixture of fine powders corresponding to the prescription amounts of Angelica sinensis, Bupleurum chinense, Corydalis ambigua and Ligusticum chuanxiong in Gynecological Menstrual Regulation Tablets. The fingerprint spectrum determination method described in Example 6 was used, and the sample numbers of the six test sample solutions were recorded as Repetition 1 (S2), Repetition 2 (S3), Repetition 3 (S4), Repetition 4 (S5), Repetition 5 (S6) and Repetition 6 (S7), respectively. The chromatogram was recorded. The peaks corresponding to the reference peaks were S peaks, and the relative retention times of the peaks were calculated. The method of the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version) was used to automatically match the chromatographic peaks of the repetition HPLC chromatograms to form a common mode diagram, and a control chromatogram R was established, and the similarity was calculated.

[0267] (2) Experimental results

[0268] The repetition relative retention time results are shown in Table 14, and the repetition similarity results are shown in Table 15. The results show that the repetition similarity of the six parallel test sample solutions is 1.00 (> 0.95); the experimental data of the six groups of repetition experiments show that the maximum value of the relative retention time RSD of each fingerprint peak is 0.2% (< 5%). This indicates that the repetition of the fingerprint spectrum determination method described in Example 6 meets the requirements.

[0269] Table 14 Repeatability relative retention time results

[0270]

[0271] Table 15 Repeatability similarity results

[0272]

[0273]

[0274] 4. Solution stability

[0275] (1) Experimental method

[0276] The fingerprint spectrum determination method described in Example 6 was used, except that the same test sample was used, which was 2 g of a mixture of fine powders corresponding to the prescription amounts of Angelica sinensis, Bupleurum chinense, Corydalis ambigua and Ligusticum chuanxiong in Gynecological Menstrual Regulation Tablets. The test sample was placed at room temperature for 0 h, 4 h, 8 h, 12 h, 26 h and 36 h, respectively, and then injected. The chromatogram was recorded. The peaks corresponding to the reference peaks were S peaks, and the relative retention times of the peaks were calculated. The Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version) was used, and the fingerprint spectrum at 0 h was used as the reference spectrum to calculate the similarity.

[0277] (2) Experimental results

[0278] The relative retention times of the solutions are shown in Table 16, and the similarity of the solutions is shown in Table 17. In Table 17, R represents the fingerprint spectrum number of the test solution after 0 h at room temperature; S2 represents the fingerprint spectrum number of the test solution after 4 h at room temperature; S3 represents the fingerprint spectrum number of the test solution after 8 h at room temperature; S4 represents the fingerprint spectrum number of the test solution after 12 h at room temperature; S5 represents the fingerprint spectrum number of the test solution after 26 h at room temperature; and S6 represents the fingerprint spectrum number of the test solution after 36 h at room temperature.

[0279] The results showed that, using the fingerprint spectrum at 0 h as the reference spectrum, the similarity of the fingerprint spectra obtained after the test solution was placed at room temperature for 4 h, 8 h, 12 h, 26 h, or 36 h was 1.00 (>0.95); the maximum relative retention time (RSD) of each fingerprint peak was 0.2% (<5%). This indicates that the test solution is stable within 36 h.

[0280] Table 16 Results of relative retention times for the stability of the test solutions

[0281]

[0282] Table 17 Results of Stability Similarity of Test Sample Solutions

[0283]

[0284] Example 9: Robustness of the method for establishing the fingerprint spectrum of the four-ingredient mixed fine powder of gynecological menstrual regulating tablets

[0285] 1. The effect of different column temperatures on the durability of the method for establishing fingerprint spectra of a mixture of four herbs in a gynecological menstrual regulating tablet.

[0286] (1) Experimental methods

[0287] Two g of a finely mixed powder conforming to the prescription ratio of Angelica sinensis, stir-fried Atractylodes macrocephala, vinegar-processed Corydalis yanhusuo, and Ligusticum chuanxiong in the gynecological menstrual regulating tablets was used as the test sample. The effect of different column temperatures on the robustness of the fingerprint chromatogram establishment method for the four-herb mixed powder of the gynecological menstrual regulating tablets was detected. The fingerprint chromatogram determination method described in Example 6 was used, except that experimental groups with different column temperatures were set up:

[0288] The column temperature for experimental group 1 was 23℃; the column temperature for experimental group 2 was 25℃.

[0289] The column temperature in experimental group 3 was 27℃; the effects of different column temperatures on the elution results were compared.

[0290] The peak corresponding to the reference peak is the S peak, and the relative retention time of each fingerprint peak is calculated; the method of "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)" is used to automatically match the chromatographic peaks of the reproducible HPLC chromatogram to form a common mode diagram, and a control chromatogram R is established, and the similarity is calculated.

[0291] (2) Experimental results

[0292] The column temperature durability relative retention time results are shown in Table 18, and the column temperature durability similarity results are shown in Table 19, wherein R is the number of the control fingerprint spectrum; S2 is the number of the fingerprint spectrum obtained in experimental group 1; S3 is the number of the fingerprint spectrum obtained in experimental group 2; and S4 is the number of the fingerprint spectrum obtained in experimental group 3.

[0293] The results show that the peak shape of each fingerprint peak in the spectrum measured under different column temperature conditions is sharp, symmetrical, and has good separation, and the similarity is all 1.00 (>0.90); the experimental data of the three experimental groups show that the maximum value of the relative retention time RSD of each fingerprint peak is 1.9% (<5%). It is shown that the fingerprint spectrum determination method described in Example 6 has good durability under different column temperature (23℃-27℃) conditions.

[0294] Table 18 Column temperature durability relative retention time results

[0295]

[0296]

[0297] 19 Column temperature durability similarity results

[0298]

[0299] 2, Effect of different flow rates on the durability of the fingerprint spectrum establishment method of the four-herb mixed fine powder of the Gynecological Menstrual Regulation Tablet (1) Experimental method

[0300] Take 2g of the mixed fine powder corresponding to the prescription amount and ratio of Angelica sinensis, Bupleurum chinense, Radix Corydalis and Rhizoma Chuanxiong in the Gynecological Menstrual Regulation Tablet as the test sample, and detect the effect of different flow rates on the durability of the fingerprint spectrum establishment method of the four-herb mixed fine powder of the Gynecological Menstrual Regulation Tablet.

[0301] The fingerprint spectrum determination method described in Example 6 is used, except that different flow rates are set for the experimental groups:

[0302] The flow rate of experimental group 1 is 0.9ml / min; the flow rate of experimental group 2 is 1.0ml / min;

[0303] The flow rate of experimental group 3 is 1.1ml / min; and the effects of different flow rates on the elution results are compared.

[0304] The peak corresponding to the reference peak is S peak, and the relative retention time of each fingerprint peak is calculated; the method of "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)" is used to automatically match the repetitive HPLC chromatogram to form a common mode chart, and a control chromatogram R is established, and the similarity is calculated.

[0305] (2) Experimental results

[0306] The flow rate endurance relative retention time results are shown in Table 20, and the flow rate endurance similarity results are shown in Table 21, wherein R is the number of the control fingerprint spectrum; S2 is the number of the fingerprint spectrum obtained in experimental group 1; S3 is the number of the fingerprint spectrum obtained in experimental group 2; and S4 is the number of the fingerprint spectrum obtained in experimental group 3.

[0307] The results show that the peak shape of each fingerprint peak in the spectrum measured under different flow rates is sharp, symmetrical, and has good separation, and the similarity is all 1.00 (>0.90); the experimental data of the three experimental groups show that the maximum value of the relative retention time RSD of each fingerprint peak is 4.9% (<5%). It is shown that the method of Example 6 has good endurance under different flow rates (0.9 ml / min-1.1 ml / min), which meets the requirements.

[0308] Table 20 Flow rate endurance relative retention time results

[0309]

[0310] Table 21 Flow rate endurance similarity results

[0311]

[0312] 3. Influence of different volume percentages of aqueous formic acid as mobile phase B on the endurance of the fingerprint spectrum establishment method for the four-herb mixture fine powder of Gynecological Menstrual Regulation Tablets

[0313] (1) Experimental method

[0314] Take 2 g of the mixture fine powder prepared according to the prescription amount ratio of Angelica sinensis, Bupleurum chinense, Rhizoma Corydalis and Rhizoma Chuanxiong in Gynecological Menstrual Regulation Tablets as the test sample, and detect the influence of different volume percentages of aqueous formic acid as mobile phase B on the endurance of the fingerprint spectrum establishment method for the four-herb mixture fine powder of Gynecological Menstrual Regulation Tablets.

[0315] The fingerprint spectrum determination method described in Example 6 is used, except that different volume percentages of aqueous formic acid are set as the experimental group of mobile phase B:

[0316] Experimental group 1 is 0.08% volume percentage of aqueous formic acid;

[0317] Experimental group 2 is 0.10% volume percentage of aqueous formic acid;

[0318] Experimental group 3 was 0.12% formic acid aqueous solution;

[0319] The effects of different volume percentages of formic acid aqueous solution as mobile phase B on the elution results were compared.

[0320] The peak corresponding to the reference peak was S peak, and the relative retention time of each fingerprint peak was calculated; the method of "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)" was used to automatically match the chromatographic peaks of the reproducible HPLC chromatogram to form a common mode diagram, and a control chromatogram R was established to calculate the similarity.

[0321] (2) Experimental results

[0322] The relative retention time results of different volume percentages of formic acid aqueous solution are shown in Table 22, and the similarity results of different volume percentages of formic acid aqueous solution are shown in Table 23. In Table 23, R is the number of the control fingerprint; S2 is the number of the fingerprint obtained by experimental group 1 with mobile phase B; S3 is the number of the fingerprint obtained by experimental group 2; S4 is the number of the fingerprint obtained by experimental group 3 with mobile phase B.

[0323] The results show that under the condition of different volume percentages of formic acid aqueous solution as mobile phase B, the peak shape of each fingerprint peak in the spectrum is sharp, symmetrical, and has good separation, and the similarity is all 1.00 (> 0.90); the experimental data of the three experimental groups shows that the maximum value of the relative retention time RSD of each fingerprint peak is 0.7% (< 5%). It is shown that the fingerprint determination method described in Example 6 has good durability under the condition of different volume percentages of formic acid aqueous solution (0.08% to 0.12%), which meets the requirements.

[0324] Table 22 Relative retention time results of different volume percentages of formic acid aqueous solution

[0325]

[0326] Table 23 Similarity results of different volume percentages of formic acid aqueous solution

[0327]

[0328] 4, Effect of different chromatographic columns on the durability of the fingerprint chromatography method for four medicinal powder mixtures of Gynecological Menstrual Regulation Tablets

[0329] (1) Experimental method

[0330] Take 2g of mixed fine powder corresponding to the prescription amount of Angelica sinensis, Bupleurum chinense, Radix Corydalis and Rhizoma Chuanxiong in Gynecological Menstrual Regulation Tablets as the test sample, and detect the effect of different chromatographic columns on the durability of the fingerprint chromatography method for four medicinal powder mixtures of Gynecological Menstrual Regulation Tablets.

[0331] The fingerprint spectrum determination method described in Example 6 is used, except that different types of chromatographic columns are set up in the experimental groups:

[0332] The chromatographic column of experimental group 1 is Kromasil 100-5-C18;

[0333] The chromatographic column of experimental group 2 is Agilent Eclipse XDB-C18;

[0334] The chromatographic column of experimental group 3 is SHIMADZU Shim-pack GISS C18;

[0335] The chromatographic column of experimental group 4 is Waters Xselect HSS T3;

[0336] The chromatographic column of experimental group 5 is Inertsil ODS-3;

[0337] The effects of different types of chromatographic columns on the chromatogram are compared.

[0338] The peak corresponding to the reference peak is the S peak, and the relative retention time of each fingerprint peak is calculated; the method of "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)" is used to automatically match the chromatographic peaks of the reproducible HPLC spectrum, form a common mode chart, and establish a control chart R, and calculate the similarity.

[0339] (2) Experimental results

[0340] The results of the relative retention time of the durability of different types of chromatographic columns are shown in Table 24.

[0341] The results show that the peak order and separation of each fingerprint peak in the fingerprint spectrum obtained by the above five experimental groups are different, indicating that the method of Example 6 has poor durability for different types of chromatographic columns, and the chromatographic column needs to be fixed as Kromasil 100-5-C18.

[0342] Table 28 Results of the relative retention time of the durability of different types of chromatographic columns

[0343]

[0344] Example 10 Detection of the Fingerprint Spectrum of Gynecological Menstrual Regulating Tablets

[0345] (1) Experimental method

[0346] The method of Example 6 is used to detect the finished product of gynecological menstrual regulating tablets, and the sample powder to be tested is: an appropriate amount of gynecological menstrual regulating tablet product is removed from the coating, finely ground, and accurately weighed 2.5 g; high performance liquid chromatography fingerprint spectrum is established.

[0347] (2) Experimental results

[0348] The fingerprint spectrum of the finished gynecological menstrual regulating tablets obtained using the method in Example 6 is as follows: Figure 15 As shown, by Figure 15 It can be seen that all 12 fingerprint peaks in the control fingerprint spectrum obtained in Example 7 can be detected. This indicates that the established fingerprint spectrum method can effectively separate and detect the chemical components in the finished gynecological menstrual regulating tablets, and can be applied to the detection and analysis of the finished gynecological menstrual regulating tablets.

[0349] Comparative Example 1: Detection of fingerprint spectrum of the mixed fine powder of four herbs in a gynecological menstrual regulating tablet.

[0350] (1) Experimental methods

[0351] S1. Preparation of reference solution: Take an appropriate amount of ligustrazine lactone I reference standard, accurately weigh it, and add methanol to prepare a solution containing 39.14 μg of ligustrazine lactone I per ml, which is the reference solution.

[0352] Preparation of the test solution: Take 2g of the mixed fine powder that conforms to the prescription ratio of Angelica sinensis, stir-fried Atractylodes macrocephala, vinegar-processed Corydalis yanhusuo and Ligusticum chuanxiong in the gynecological menstrual regulating tablets, place it in a stoppered conical flask, accurately add 25ml of 50% methanol aqueous solution, seal tightly, weigh, sonicate for 1h, cool, weigh again, replenish the lost weight with 50% methanol aqueous solution, shake well, filter through a filter with a pore size of 0.45μm, and take the filtrate to obtain the test solution;

[0353] S2. According to the method for establishing the UPLC fingerprint spectrum of a gynecological regeneration pill disclosed in Chinese Patent CN106018576A, the fingerprint spectrum of the fine powder of the gynecological menstrual regulating tablet was determined. The specific steps are as follows:

[0354] Accurately pipette 2 μl each of the reference solution and the test solution prepared in step S1 and inject them into the liquid chromatograph. Use an ACQUITY UPLC Hss T3 (2.1 × 100 mm, 1.8 μm) column; use 0.1% formic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, and elute according to the specifications in Table 29; the flow rate is 0.2 ml, 0.3 ml or 0.4 ml per minute; the detection wavelength is 280 nm; and the column temperature is 50 °C.

[0355] Table 29 Gradient Elution Conditions

[0356]

[0357] (2) Experimental Results

[0358] Experimental results are as follows Figure 16 As shown, by Figure 16It can be known that according to the chromatographic conditions disclosed in the Chinese patent CN106018576A, only a part of the fingerprint peaks are successfully detected in the chromatogram of the four-medicinal-material mixed fine powder of the gynecological menstruation-regulating tablet under different flow rates, and the rest of the fingerprint peaks are not shown in the chromatogram, and the 12 fingerprint peaks presented in the control fingerprint spectrum obtained in Example 7 cannot be detected. It can be known that the chemical components of different Chinese medicinal materials have significant differences, and therefore the elution conditions of the fingerprint spectra of different medicinal materials and their preparations do not have mutual reference value, and the existing technical disclosure of the fingerprint spectrum establishment method is not sufficient to completely elute other components in the four-medicinal-material mixed fine powder of the gynecological menstruation-regulating tablet.

[0359] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A method for establishing the fingerprint of gynecological regulating menstruation tablets by high performance liquid chromatography, characterized in that, The method comprises the following steps: Reference solution: a ligustilide I solution; Test solution: a methanol solution extract of the gynecological menstruation-regulating tablet, the volume fraction of the methanol solution being 65-75%; HPLC chromatographic conditions: a Kromasil C18 chromatographic column with a column length of 250 mm, an inner diameter of 4.6 mm and a particle size of 5 μm is used, acetonitrile is used as mobile phase A, and a 0.08-0.12% formic acid aqueous solution is used as mobile phase B, in a gradient elution program, the volume fraction of mobile phase B in the mobile phase system changes as follows: 0-5 min, mobile phase B decreases from 92% to 82%; 5-30 min, mobile phase B decreases from 82% to 78%; 30-35 min, mobile phase B is 78%; 35-40 min, mobile phase B decreases from 78% to 53%; 40-50 min, mobile phase B decreases from 53% to 45%; 50-62 min, mobile phase B is 45%; 62-65 min, mobile phase B decreases from 45% to 20%; 65-75 min, mobile phase B is 20%; 75-85 min, mobile phase B increases from 20% to 90%; The detection wavelength is gradient detection: 0-38 min, the detection wavelength is 277 nm; 38-76 min, the detection wavelength is 254 nm; 76-85 min, the detection wavelength is 277 nm; The reference solution and the test solution are subjected to HPLC detection respectively according to the HPLC chromatographic conditions to obtain the fingerprint of the gynecological menstruation-regulating tablet.

2. The method of claim 1, wherein, The reference solution is a methanol solution with a ligustilide I concentration of 25-48 μg / ml.

3. The method of claim 1, wherein, The preparation method of the test solution is as follows: the gynecological menstruation-regulating tablet to be tested is crushed, and then ultrasonic extraction is performed for 30-60 min using a 65-75% methanol aqueous solution.

4. The method of claim 3, wherein, The dosage ratio of the gynecological menstruation-regulating tablet to be tested to the methanol aqueous solution is 1-2 g:10-40 ml.

5. The method of claim 1, wherein, The column temperature of the chromatographic column is 23-27℃.

6. The method of claim 1, wherein, The flow rate of the gradient elution program is 0.9-1.1 ml / min.

7. The method according to any one of claims 1-6 is applied to the quality control of the gynecological menstruation-regulating tablet.

8. A method for establishing a reference fingerprint of a gynecological regulating menstruation tablet, characterized in that, The establishment method is as follows: the standard sample of the gynecological menstruation-regulating tablet is subjected to HPLC detection according to the method of any one of claims 1-6 to obtain a control fingerprint, the control fingerprint contains 12 fingerprint peaks: No. 1 peak to No. 12 peak in sequence, the corresponding No. 4 peak of the reference solution is the S peak, and the relative retention time of the other peaks is within ±10% of the specified value, the specified value is: No. 1 peak 0.27; No. 2 peak 0.32; No. 3 peak 0.61; S peak 1.00; No. 5 peak 1.29; No. 6 peak 1.48; No. 7 peak 1.68; No. 8 peak 1.81; No. 9 peak 1.84; No. 10 peak 1.89; No. 11 peak 1.92; and No. 12 peak 1.

95.

9. The establishment method according to claim 8, characterized by, The standard samples of 4-8 different batches of gynecological menstruation-regulating tablets are detected by the method according to any one of claims 1-6 to obtain the fingerprint of the standard samples of different batches of gynecological menstruation-regulating tablets, and the control fingerprint is generated by the method of "Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine", automatic matching of the fingerprint by median method and multi-point correction.

10. A method for quality detection of a gynecological regulating menstruation tablet, characterized in that, The method comprises the following steps: establishing the fingerprint of the gynecological menstruation-regulating tablets to be detected according to any one of claims 1-6 to obtain the fingerprint of the sample to be detected, the fingerprint of the sample to be detected contains 12 fingerprint peaks in the control fingerprint in claim 8, the similarity between the 12 fingerprint peaks in the control fingerprint in claim 8 and the corresponding peaks in the fingerprint of the sample to be detected is not less than 0.90, and the gynecological menstruation-regulating tablets to be detected are qualified products.

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