A detection method and identification method for characteristic spectrum of aesculus chinensis formula granules

Abstract

The application provides a detection method and identification method of a characteristic spectrum of vinegar stellariae formula granules, wherein the identification method of stellariae includes: obtaining a characteristic spectrum of a to-be-detected object, and comparing the characteristic spectrum with a standard characteristic spectrum of vinegar stellariae formula granules; wherein the characteristic spectrum of the to-be-detected object contains a characteristic peak corresponding to isoquercitrin, and does not contain a characteristic peak corresponding to hydroxyl stellariae; the to-be-detected object is kadsurae; the to-be-detected object contains a characteristic peak corresponding to hydroxyl stellariae, and does not contain a characteristic peak corresponding to isoquercitrin; and when a relative peak area of a peak where chlorogenic acid is located is not greater than 0.2 compared with a peak where apigenin-7-O-beta-D-glucuronide is located, the to-be-detected object is stellariae; when the characteristic spectrum of the to-be-detected object contains characteristic peaks corresponding to isoquercitrin and hydroxyl stellariae, the to-be-detected object is stellariae striata. The application can quickly and effectively distinguish kadsurae, stellariae and stellariae striata.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine detection, and particularly relates to a detection method and identification method for characteristic spectrum of vinegar Daphne formula granules. BACKGROUND

[0002] According to ancient herbal literature, Daphne has Daphne, Chuzhou Daphne, yellow Daphne, Mianzhou Daphne and Jiangzhou Daphne. According to plant description records and modern research, Daphne, Chuzhou Daphne and Mianzhou Daphne recorded in ancient books are Daphne (Daphne genkwa Sieb. et Zucc.) of Thymelaeaceae, named Daphne; yellow Daphne and Jiangzhou Daphne are Wikstroemia chamaedaphne Meisn. of Thymelaeaceae, named yellow Daphne or Wikstroemia. Although they are different in terms of pungent and warm opening and bitter and cold penetration, they have similar functions of breaking accumulation and removing water. It is traditionally believed that they can be used as substitutes, so there is a phenomenon of mixing them in clinical practice. However, in fact, there are still differences in efficacy, and for diseases, the most suitable type of medicinal material should be selected to achieve the best efficacy. In addition, in order to facilitate carrying and storage, reduce the operation of self-cooking by patients, single traditional Chinese medicine is usually prepared into traditional Chinese medicine formula granules; and the traditional Chinese medicine formula granules lose the properties of the original medicinal materials and cannot be distinguished by the naked eye. For example, the medicinal part of Daphne is dried flower buds, and the dried branches are named Daphne, which have the functions of removing water, removing phlegm and relieving cough, but the functions of different parts are slightly different.

[0003] Therefore, it is necessary to distinguish different traditional Chinese medicines and traditional Chinese medicines from different parts of the same traditional Chinese medicine, and there is no corresponding literature published for distinguishing Daphne and Wikstroemia. SUMMARY

[0004] Therefore, the problem to be solved by the present application is that there is no method for distinguishing Daphne and Wikstroemia in the prior art; and the present application provides a detection method and identification method for characteristic spectrum of vinegar Daphne formula granules.

[0005] A method for identifying Daphne, comprising: obtaining a characteristic spectrum of a test object, and comparing the characteristic spectrum with a standard characteristic spectrum of vinegar Daphne formula granules, wherein,

[0006] The characteristic spectrum of the test object contains a characteristic peak corresponding to isoquercitrin and does not contain a characteristic peak corresponding to hydroxydaphnoretin, and the test object is Wikstroemia;

[0007] The test object contains a characteristic peak corresponding to hydroxydaphnoretin and does not contain a characteristic peak corresponding to isoquercitrin, and the relative peak area of the peak of chlorogenic acid is not greater than 0.2 compared with the peak of apigenin-7-O-β-D-glucuronide, and the test object is Daphne;

[0008] When the characteristic map of the test substance contains characteristic peaks corresponding to isoquercitrin and hydroxyl genkwa, the test substance is genkwa strip.

[0009] The characteristic map of the genkwa strip and the gordoniae also contains characteristic peaks corresponding to vitexin, isovitexin and naringin, while the characteristic map of the genkwa does not contain characteristic peaks corresponding to vitexin, isovitexin and naringin.

[0010] The characteristic map of the genkwa also includes characteristic peaks corresponding to ulmussin, apigenin and apigenin-7-O-β-D-glucuronide, and the characteristic map of the genkwa strip and the gordoniae does not contain characteristic peaks corresponding to ulmussin, apigenin and apigenin-7-O-β-D-glucuronide.

[0011] A method for detecting the characteristic map of a vinegar genkwa formula granule, which adopts UPLC for detection, and the chromatographic conditions are as follows:

[0012] The chromatographic column is a chromatographic column with octadecylsilane bonded silica gel as the filler, with a specification of 2.1x100mm, 1.7μm; acetonitrile is used as the mobile phase A, and 0.08%-0.4% formic acid solution is used as the mobile phase B, and elution is carried out according to the following gradient elution program:

[0013]

[0014] Or,

[0015]

[0016] Wherein, t1 is 21-22, t2 is 25-26, t3 is 26-30, and t3>t2.

[0017] A1 is 16-18, A2 is 27-28, and A3 is 75-100.

[0018] The column temperature of the chromatographic column is 20-30℃, the flow rate is 0.3ml / min, the detection wavelength is 300-332nm, and the theoretical plate number calculated according to the apigenin-7-O-β-D-glucuronide peak should not be less than 5000.

[0019] The preparation process of the test solution used in the detection is as follows: take the vinegar genkwa formula granule, add a solvent, weigh, ultrasonic or heating reflux treatment, cool, weigh again, supplement the weight loss with a solvent, mix, filter, and the filtrate is the test solution.

[0020] The solvent is 50% or more of methanol;

[0021] And / or, the power of ultrasonic treatment is 250W, and the ultrasonic treatment time is 30-60min;

[0022] And / or, the heating reflux treatment time is 30-60 min;

[0023] And / or, the solvent addition amount in the preparation process of the sample solution is 75-125 ml / g.

[0024] The application also includes a control medicinal material solution and a control solution;

[0025] The preparation process of the control medicinal material solution is as follows: taking the control medicinal material of flos genitae, adding water, heating reflux, filtering, evaporating the filtrate, adding solvent to the residue, ultrasonic treatment, cooling, shaking, filtering, and taking the subsequent filtrate as the control medicinal material solution.

[0026] The preparation process of the control solution is as follows: taking the control product, adding solvent, and mixing to obtain the control solution; the control product includes at least one of geniposide, apigenin-7-O-β-D-glucuronide, and chlorogenic acid.

[0027] Each 1 ml of the control solution contains 90 μg of geniposide, 100 μg of apigenin-7-O-β-D-glucuronide, and 90 μg of chlorogenic acid.

[0028] The obtained characteristic spectrum includes characteristic peaks corresponding to chlorogenic acid, cryptochlorogenic acid, apigenin-7-O-β-D-glucuronide, geniposide-5-O-alizarin glycoside, luteolin, tilianin, apigenin, hydroxyl geniposide, and geniposide; peak 1 is chlorogenic acid, peak 2 is cryptochlorogenic acid, peak 5 is apigenin-7-O-β-D-glucuronide, peak 6 is geniposide-5-O-alizarin glycoside, peak 8 is luteolin, peak 9 is tilianin, peak 10 is apigenin, peak 11 is hydroxyl geniposide, and peak 12 is geniposide.

[0029] Peak 5 is the S peak, and the relative retention time of other characteristic peaks relative to peak 5 is ±10% of the specified value of each characteristic peak.

[0030] Peak 1: 0.21, peak 2: 0.23, peak 3: 0.69, peak 4: 0.92, peak 5: 1, peak 6: 1.10, peak 7: 1.17, peak 8: 1.36, peak 9: 1.46, peak 10: 1.59, and peak 11: 1.82.

[0031] The technical scheme of the application has the following advantages:

[0032] 1. The application provides a method for identifying Flos Genkwa, which can distinguish Flos Genkwa and Flos Flos Genkwa by obtaining characteristic patterns, and can effectively identify the types of the test substances, realize the differentiation of Flos Genkwa and Flos Flos Genkwa, and the different parts of Flos Genkwa (Flos Genkwa and Flos Genkwa strips), and the effect is remarkable. Specifically, by whether containing isoquercitrin, hydroxyl genkwanin corresponding characteristic peak, and the relative peak area of the peak of chlorogenic acid compared with the peak of apigenin-7-O-β-D-glucuronide, the effective differentiation of Flos Genkwa, Flos Genkwa and Flos Genkwa strips can be realized quickly, and the accuracy of drug use is ensured.

[0033] 2. The characteristic pattern detection method of the vinegar Flos Genkwa formula granules of the application optimizes the chromatographic conditions, can obtain characteristic patterns with short time, stable baseline, many characteristic peaks and good separation degree, better facilitates the differentiation of Flos Genkwa and Flos Flos Genkwa, and is more effectively used for the differentiation of different parts of Flos Genkwa. BRIEF DESCRIPTION OF DRAWINGS

[0034] In order to more clearly illustrate the specific embodiments of the application or the technical solutions in the prior art, the drawings needed in the following specific embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0035] Figure 1 is the standard characteristic pattern of vinegar Flos Genkwa in the embodiment 1 of the application;

[0036] Figure 2 is the fingerprint of vinegar Flos Genkwa and 3 batches of Flos Flos Genkwa in the embodiment 1 of the application;

[0037] Figure 3 is the comparative fingerprint of vinegar Flos Genkwa, Flos Flos Genkwa and the control in the embodiment 1 of the application;

[0038] Figure 4 is the fingerprint of vinegar Flos Genkwa and 3 batches of Flos Genkwa strips in the embodiment 1 of the application;

[0039] Figure 5 is the comparative fingerprint of vinegar Flos Genkwa, Flos Genkwa strips and the control in the embodiment 1 of the application;

[0040] Figure 6 is the fingerprint under the elution gradient program of table 4 in the embodiment 2 of the application;

[0041] Figure 7 is the fingerprint under the elution gradient program of table 5 in the embodiment 2 of the application;

[0042] Figure 8 is the fingerprint under the elution gradient program of table 6 in the embodiment 2 of the application;

[0043] Figure 9 is the fingerprint chromatogram under the elution gradient procedure of Table 7 in Example 2 of the present application;

[0044] Figure 10 is the fingerprint chromatogram under a column temperature of 20℃ in Example 2 of the present application;

[0045] Figure 11 is the fingerprint chromatogram under a column temperature of 25℃ in Example 2 of the present application;

[0046] Figure 12 is the fingerprint chromatogram under a column temperature of 30℃ in Example 2 of the present application;

[0047] Figure 13 is the fingerprint chromatogram under a wavelength of 230nm in Example 2 of the present application;

[0048] Figure 14 is the fingerprint chromatogram under a wavelength of 280nm in Example 2 of the present application;

[0049] Figure 15 is the fingerprint chromatogram under a wavelength of 300nm in Example 2 of the present application;

[0050] Figure 16 is the fingerprint chromatogram under a wavelength of 332nm in Example 2 of the present application;

[0051] Figure 17 is the fingerprint chromatogram under a 0.1% acetic acid-acetonitrile system in Example 2 of the present application;

[0052] Figure 18 is the fingerprint chromatogram under a 0.1% phosphoric acid-acetonitrile system in Example 2 of the present application;

[0053] Figure 19 is the fingerprint chromatogram under a 0.1% formic acid-acetonitrile system in Example 2 of the present application;

[0054] Figure 20 is the fingerprint chromatogram under a water-acetonitrile system in Example 2 of the present application;

[0055] Figure 21 is the fingerprint chromatogram under a 0.1% formic acid-acetonitrile system in Example 2 of the present application;

[0056] Figure 22 is the fingerprint chromatogram under a 0.2% formic acid-acetonitrile system in Example 2 of the present application;

[0057] Figure 23 is the fingerprint chromatogram under a 0.3% formic acid-acetonitrile system in Example 2 of the present application;

[0058] Figure 24 is the fingerprint chromatogram under a 0.4% formic acid-acetonitrile system in Example 2 of the present application;

[0059] Figure 25 is the fingerprint spectrum under ultrasonic treatment in Example 2 of the present application;

[0060] Figure 26 is the fingerprint spectrum under reflux treatment in Example 2 of the present application;

[0061] Figure 27 is the fingerprint spectrum under methanol extraction in Example 2 of the present application;

[0062] Figure 28 is the fingerprint spectrum under 30% methanol extraction in Example 2 of the present application;

[0063] Figure 29 is the fingerprint spectrum under 50% methanol extraction in Example 2 of the present application;

[0064] Figure 30 is the fingerprint spectrum under 70% methanol extraction in Example 2 of the present application. DETAILED DESCRIPTION

[0065] The specific experimental steps or conditions not mentioned in the examples can be carried out according to the conventional experimental steps described in the literature or the operation or conditions. The reagents or instruments used are not marked with the manufacturer, which are conventional reagent products that can be obtained by market purchase. The percentages not mentioned in the present application are all volume percentages.

[0066] Instruments:

[0067] Chromatograph 1: Waters H-CLASS UPLC chromatographic system, including four-element solvent manager (ACQ-QSM), automatic sampler (ACQ-FTN), imported original chromatographic column oven (ACQ-CM), diode array ultraviolet detector (ACQ-PDA), Empower chromatographic management system;

[0068] Chromatograph 2: Agilent 1290Infinity II chromatographic system, including four-element solvent manager (1290Flexible Pump), sample manager (1290Vial sampler), imported original chromatographic column oven (1290MCT), diode array ultraviolet detector (1290PDA FS), OpenLAB CDS chromatographic management system;

[0069] Electronic balance: METTLER TOLEDO (Switzerland Mettler) ME104, XPR2, MS105DU / A; ultrasonic instrument: KQ-300DB Kunshan Ultrasonic Instrument Co., Ltd.; chromatographic column: ACQUITY UPLC BEH C18 2.1*100mm, 1.7μm (batch number: 0432322082, 0436322771, 0327382902).

[0070] Reagents:

[0071] Yuanhua control drug material (batch number: 121062-201904, purchased from China Institute for Food and Drug Control);

[0072] Apigenin-7-O-β-D-glucuronide reference substance (batch number: 10856, for content determination, calculated at 98.0%, purchased from Shanghai Shidanded Standard Technology Service Co., Ltd.);

[0073] Yuanhuacine reference substance (batch number: 111899-201202, purchased from China Institute for Food and Drug Control);

[0074] Chlorogenic acid reference substance (batch number: 110753-202119, purchased from China Institute for Food and Drug Control);

[0075] Yuanhua strip decoction pieces (batch number: 2305001Y, 2305002Y, 2305003Y);

[0076] Jiaohua decoction pieces (batch number: 2305001Y, 2305002Y, 2305003Y);

[0077] Vinegar yuanhua decoction pieces, yuanhua decoction pieces batch number information is shown in Table 1.

[0078] Table 1

[0079]

[0080]

[0081] Yuanhua decoction pieces: remove impurities from the corresponding batch number of yuanhua medicinal materials, dry.

[0082] Vinegar yuanhua decoction pieces: 10 kg of the corresponding batch number of yuanhua decoction pieces is used, 3 kg of vinegar is used, and it is mixed evenly, soaked, and fried with a small fire until the vinegar is absorbed, and the surface is slightly yellow, then it is taken out and cooled.

[0083] Reagents:

[0084] Acetonitrile (Thermo Fisher, chromatographically pure), formic acid (Shanghai Anpu Experimental Technology Co., Ltd., chromatographically pure), water is ultrapure water, and other reagents are analytical pure.

[0085] Example 1

[0086] A detection method of a formula granule of Meconii Herba, which adopts ultra performance liquid chromatography (UPLC) for detection, and the chromatographic conditions are as follows:

[0087] A chromatographic column with octadecylsilane bonded silica gel as the filler (ACQUITY UPLC BEH C18, 2.1x100mm, 1.7μm) is used, acetonitrile is used as the mobile phase A, and 0.1% formic acid solution is used as the mobile phase B, gradient elution is performed according to the following Table 2, the flow rate is 0.3ml per minute, the column temperature is 25°C, and the detection wavelength is 332nm. The theoretical plate number should not be less than 5000 calculated according to the peak of apigenin-7-O-β-D-glucuronide.

[0088] Table 2

[0089]

[0090] Preparation of a control medicinal material solution: 0.5g of Meconii Herba control medicinal material is added into 50ml of water, heated and refluxed for 30 minutes, filtered, and the filtrate is evaporated to dryness. The residue is added into 25ml of 70% methanol, ultrasonically treated (power 250W, frequency 40kHz) for 30 minutes, cooled, shaken uniformly, filtered, and the filtrate is taken as the control medicinal material solution.

[0091] Preparation of a reference solution of a control: appropriate amounts of apigenin control, apigenin-7-O-β-D-glucuronide control, and chlorogenic acid control are added into 70% methanol to prepare a mixed solution containing 90μg of apigenin, 100μg of apigenin-7-O-β-D-glucuronide, and 90μg of chlorogenic acid per 1ml, which is taken as the reference solution of a control.

[0092] Preparation of a test solution: appropriate Meconii Herba formula granules are finely ground, about 0.2g is accurately weighed and placed in a conical flask with a plug, 25ml of 70% methanol is accurately added, the weight is determined, ultrasonically treated (power 250W, frequency 40kHz) for 30 minutes, cooled, the weight is determined again, the lost weight is made up with 70% methanol, shaken uniformly, filtered, and the filtrate is taken as the test solution.

[0093] Determination method: 1μl of the control medicinal material solution, the reference solution of a control, and the test solution is accurately taken respectively, injected into a liquid chromatograph, and determined, and the results are obtained.

[0094] In this example, 18 batches of Meconii Herba decoction pieces are prepared into Meconii Herba decoction piece standard soup, and the specific preparation process is as follows:

[0095] Take the vinegar jinyinhua decoction pieces, add 15 times the amount of water soak for 30 minutes, first boiling water (500W) to turn the fire (200W) decoction 30 minutes, 200 mesh filter cloth while hot filter, drug residue plus 13 times the amount of water to boiling water (500W) to turn the fire (200W) decoction 20 minutes, 200 mesh filter cloth while hot filter, combined with two filtrate, filtrate reduced pressure concentration (50-65 ℃) to the relative density of 1.08-1.12 g / ml of concentrated extract, concentrated extract in the freeze dryer, freeze drying to dry state, take out, weighing, grinding into powder, subpackaged in the westlin bottle, vinegar jinyinhua decoction pieces (lyophilized powder) standard soup is obtained.

[0096] In this embodiment, 3 batches of vinegar jinyinhua medicinal materials (YC16, YC17, YC18) were processed into vinegar jinyinhua decoction pieces, and then prepared into vinegar jinyinhua formula granules (2208001Y, 2208002Y, 2208003Y). The specific preparation process is as follows: take 3000g of vinegar jinyinhua decoction pieces, extract twice, the first time add water quality is 15 times the quality of decoction, soak for 30 minutes, boiling extraction 30 minutes, filter, add water to the drug residue, water quality is 13 times the quality of decoction, boiling extraction 25 minutes, filter. At 70 ℃, reduce pressure concentration to the relative density of 1.03-1.10 g / mL (60-70 ℃) of flow paste, mix evenly, add malt dextrin, dry, crush, the extract rate is 18%-29%, add malt dextrin, mix evenly, dry granulation, into 1000g, subpackaged, vinegar jinyinhua formula granules are obtained.

[0097] The 18 batches of vinegar jinyinhua decoction pieces standard soup prepared above were used to generate standard characteristic spectrum by using the "multi-point correction, MARK peak matching" mode with the "Chinese medicine chromatographic fingerprint similarity evaluation system 2012 edition" prepared by the Pharmacopoeia Commission. After chromatographic matching, 12 common characteristic peaks with suitable response value (peak area), good separation degree and high purity were obtained, and the chromatographic peak order was rearranged as peak 1-peak 12, as shown in the following table. Figure 1

[0098] The characteristic peaks of vinegar jinyinhua decoction pieces standard soup were analyzed by ultra-high performance liquid chromatography-high resolution mass spectrometry. According to the multi-stage mass spectrometry information of the sample, combined with the natural product high resolution mass spectrometry database and related literature, and using UPLC to locate the reference substance, it was determined that peak 1 was chlorogenic acid, peak 2 was cryptochlorogenic acid, peak 5 was apigenin-7-O-β-D-glucuronide, peak 6 was jinyinhua-5-O-alizarin, peak 8 was luteolin, peak 9 was tiliroside, peak 10 was apigenin, peak 11 was hydroxy jinyinhua, and peak 12 was jinyinhua.

[0099] ​Therefore, the standard characteristic spectrum exhibits 12 characteristic peaks, including those corresponding to chlorogenic acid, cryptochlorogenic acid, apigenin-7-O-β-D-glucuronide, genistein-5-O-alizarin, luteolin, tiolinin, apigenin, hydroxygenistein, and genistein. Using the peak corresponding to the apigenin-7-O-β-D-glucuronide reference standard peak as the S peak, the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.21 (peak 1), 0.23 (peak 2), 0.69 (peak 3), 0.92 (peak 4), 1.10 (peak 6), 1.17 (peak 7), 1.36 (peak 8), 1.46 (peak 9), 1.59 (peak 10), and 1.82 (peak 11). Calculate the relative peak area between peak 1 and peak S. The relative peak area should be within the specified range, and the specified value should not be greater than 0.20.

[0100] Based on the characteristic spectral method determined under the above characteristic spectral section, three batches of vinegar-processed Daphne genkwa granules (batch numbers: 2208001Y, 2208002Y, 2208003Y) were tested according to this method, and the test results are shown in Tables 3 and 4 below.

[0101] Table 3

[0102]

[0103] Table 4

[0104]

[0105]

[0106] The results showed that the relative retention times of the three batches of vinegar-processed Daphne genkwa granules were all within the required range.

[0107] Example 2

[0108] The identification of Daphne genkwa was performed using the chromatographic conditions described in Example 1. The specific identification process is as follows:

[0109] Standard decoctions of Daphne genkwa (processed with daphne), vinegar-processed Daphne genkwa (processed with vinegar), and Daphne genkwa strips were prepared using the method described in Example 1. Corresponding test solutions were also prepared using the method described in Example 1. UPLC analysis was then performed under the chromatographic conditions of Example 1. The results are as follows: Figures 2-5 As shown.

[0110] Figure 2 In the diagram, S1 is Daphne genkwa (vinegar-scented daphne), and S2-S4 are Daphne genkwa (yellow daphne). Figure 3 In the formula, S1 is Daphne genkwa, S2 is Daphne genkwa, S3 is vitexin, S4 is isovitexin, S5 is isoquercitrin, and S6 is naringin.Figure 4 In the formula, S1 is Flos Genkwa, S2-S4 are Genkwa strips; Figure 5 In the formula, S1 is Flos Genkwa, S2 is Genkwa strips, S3 is Vitexicarpin, S4 is Isovitexicarpin, S5 is Isoquercitrin, and S6 is Naringin.

[0111] By Figures 2-5 It can be seen that: Flos Genkwa has characteristic peaks of peaks 1-12, and through calculation, the relative peak area of the peak where chlorogenic acid is located is 0.051-0.170 compared with the peak where apigenin-7-O-β-D-glucuronide is located, and both are not greater than 0.2; Flos Genkwa only has characteristic peaks of peaks 1, 2, 6, and 8, does not contain characteristic peaks of peaks 3-5, 7, and 9-12, also has characteristic peaks of vitexicarpin, isovitexicarpin, isoquercitrin, and naringin, and the peak of chlorogenic acid is relatively high, while Flos Genkwa does not have characteristic peaks of vitexicarpin, isovitexicarpin, isoquercitrin, and naringin; Genkwa strips only have characteristic peaks of peaks 1, 2, 6, 8, and 11, do not contain characteristic peaks of peaks 3-5, 7, and 9-10 and 12, also have characteristic peaks of vitexicarpin, isovitexicarpin, isoquercitrin, and naringin. Therefore, by comparing the characteristic spectrum of the test substance with the standard characteristic spectrum of Flos Genkwa, Flos Genkwa, Flos Genkwa, and Genkwa strips can be effectively determined, and this determination method is applicable to medicinal materials, decoction pieces, standard decoctions, and formula granules.

[0112] Example 3

[0113] The difference between this example and Example 1 is that the chromatographic conditions are different, and the specific conditions are as follows:

[0114] 1. Elution gradient

[0115] Take Flos Genkwa formula granules (batch number: 2208001Y) and grind them finely. Prepare a test sample solution according to the preparation method of the test sample solution in Example 1. Use the elution gradient programs in Table 2 and the following Tables 5-8 to perform detection under the same other conditions as in Example 1.

[0116] Table 5

[0117]

[0118] Table 6

[0119]

[0120] Table 7

[0121]

[0122] Table 8

[0123]

[0124] The chromatograms obtained by the gradient elution procedures of Tables 5-8 are shown in Figures 6-9 The gradient elution procedure of Table 5 cannot effectively separate the peaks between Peak 2 and Peak 4, which is not conducive to the differentiation of Flos Daphne, Radix Wikstroemiae and Flos Daphne. The gradient elution procedures of Tables 2 and 6-8 can effectively separate the characteristic peaks 1-12 and the characteristic peaks of isoquercitrin, vitexin, isovitexin and naringin, and therefore can be used to differentiate Flos Daphne, Radix Wikstroemiae and Flos Daphne. The gradient elution procedure of Table 2 has better separation of chromatographic peaks and a more stable baseline, and is therefore preferred.

[0125] 2, Column temperature

[0126] An appropriate amount of Flos Daphne Formula Granules (batch number: 2208001Y) was finely ground, and a test sample solution was prepared according to the preparation method of the test sample solution in Example 1. The gradient elution procedure of Table 5 was used, and the determination was performed under the same other conditions as in Example 1 using different column temperatures (20, 25 and 30°C). The detection results are shown in Figures 10-12 .

[0127] The results of the investigation of different column temperatures show that different column temperatures (25°C, 30°C and 35°C) have certain effects on the retention time of each peak, the total area of the chromatographic peaks, the separation degree and the peak shape, but can be used to differentiate Flos Daphne, Radix Wikstroemiae and Flos Daphne. In the gradient elution procedure of 25°C, each peak is less disturbed by surrounding small chromatographic peaks, and therefore the column temperature of 25°C is preferred.

[0128] 3, Detection wavelength

[0129] An appropriate amount of Flos Daphne Formula Granules (batch number: 2208001Y) was finely ground, and a test sample solution was prepared according to the preparation method of the test sample solution in Example 1. The gradient elution procedure of Table 5 was used, and the determination was performed under the same other conditions as in Example 1 using different detection wavelengths (230 nm, 280 nm, 300 nm and 332 nm). The detection results are shown in Figures 13-16 .

[0130] The results of the investigation of different wavelengths show that the baseline of the chromatographic peaks is not flat and the chromatographic peaks are relatively small at 230 nm-280 nm, which is not conducive to the differentiation of Flos Daphne, Radix Wikstroemiae and Flos Daphne. At 300-332 nm, the chromatographic peaks are more, which is more conducive to the differentiation of Flos Daphne, Radix Wikstroemiae and Flos Daphne. Taking into account the above, 332 nm is preferred as the detection wavelength of the characteristic spectrum of Flos Daphne Formula Granules.

[0131] 4, Mobile phase

[0132] 4.1, Take Yijunhu prescription granules (batch number: 2208001Y) appropriate amount, grind, according to the preparation method of test solution in example 1 into test solution, and use the gradient elution procedure in table 5, respectively using different mobile phase composition (0.1% phosphoric acid-acetonitrile, 0.1% acetic acid-acetonitrile, 0.1% formic acid-acetonitrile, water-acetonitrile) according to the same other conditions in example 1 for determination, the detection results are shown in table 6. Figures 17-20

[0133] The results show that: different kinds of mobile phase of acid can be used for the differentiation of Yijunhu strip, Jiaohua and Yijunhua, but in 0.1% formic acid-acetonitrile, the symmetry of each chromatographic peak is slightly better than that of the same concentration of acetic acid, phosphoric acid and water, especially the separation effect of the position around 8 min is better, and the baseline is more stable under formic acid condition, considering comprehensively, 0.1% formic acid-acetonitrile is preferred as the mobile phase system of Yijunhu prescription granules characteristic chromatogram method.

[0134] 4.2, Take Yijunhu prescription granules (batch number: 2208001Y) appropriate amount, grind, according to the preparation method of test solution in example 1 into test solution, and use the gradient elution procedure in table 5, respectively using different acid concentration of mobile phase (0.1% formic acid-acetonitrile, 0.2% formic acid-acetonitrile, 0.3% formic acid-acetonitrile, 0.4% formic acid-acetonitrile) according to the same other conditions in example 1 for determination, the detection results are shown in table 7. Figures 21-24

[0135] The results show that: changing the pH of mobile phase has no obvious effect on the whole, which can be used for the differentiation of Yijunhu strip, Jiaohua and Yijunhua, and considering that the higher the concentration of formic acid, the lower the pH of mobile phase, the greater the damage to the chromatographic column, and considering comprehensively, 0.1% formic acid-acetonitrile is preferred as the mobile phase system of Yijunhu prescription granules characteristic chromatogram method.

[0136] 5, Test sample processing method

[0137] Take Yijunhu prescription granules (batch number: 2208001Y) appropriate amount, grind, according to the preparation method of test solution below into test solution, according to the same chromatographic conditions in example 1 for determination, the detection results are shown in table 8. Figures 25-26

[0138] Ultrasonic: take the powder of the product about 0.2g, add 70% methanol 25ml, ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes, take out, cool down, reweigh, make up the weight loss with 70% methanol, shake evenly, filter, take the filtrate, that is.

[0139] ​​​Reflux: take about 0.2 g of the powder, add 70% methanol 25 ml, heat and reflux for 30 minutes, remove, cool, re-weigh, make up the weight loss with 70% methanol, shake, filter, take the filtrate, and you get it.

[0140] From the above results, it can be seen that different processing methods can be used for the differentiation of Flos Daphnes Gen-nanensis, Flos Kirilowiae and Flos Daphnes Gen-nanensis.

[0141] 6, Test sample extraction solvent

[0142] Take a suitable amount of Acetum Flos Daphnes Gen-nanensis Granules (batch number: 2208001Y), grind finely, and use different extraction solvents (methanol, 30% methanol, 50% methanol, 70% methanol) to prepare test sample solutions according to the preparation method of test sample solution provided in Example 1. The same chromatographic conditions as in Example 1 were used for determination, and the test results are shown in Table 2. Figures 27-30

[0143] The test results show that: the characteristic peaks in the second half of 30% methanol are not detected, which is not conducive to the differentiation of Flos Daphnes Gen-nanensis, Flos Kirilowiae and Flos Daphnes Gen-nanensis; 50% methanol, 70% methanol and methanol can extract the chromatographic peaks better, which is conducive to the differentiation of Flos Daphnes Gen-nanensis, Flos Kirilowiae and Flos Daphnes Gen-nanensis; among them, the peak type is better when extracted with 70% methanol, and the system suitability parameters are relatively optimal, therefore, 70% methanol is preferred.

[0144] 7, Test sample extraction time

[0145] Take a suitable amount of Acetum Flos Daphnes Gen-nanensis Granules (batch number: 2208001Y), grind finely, and use different ultrasonic treatment times (30, 45, 60 minutes) to prepare test sample solutions according to the preparation method of test sample solution provided in Example 1. The same chromatographic conditions as in Example 1 were used for determination.

[0146] The test results show that when the ultrasonic time is 30-60 minutes, the peak area difference of Acetum Flos Daphnes Gen-nanensis Granules is not large, indicating that the ultrasonic time in the range of 30-60 minutes can be completely extracted, therefore, 30 minutes is selected as the ultrasonic time of Acetum Flos Daphnes Gen-nanensis Granules.

[0147] 8, Test sample sampling amount

[0148] Take a suitable amount of Acetum Flos Daphnes Gen-nanensis Granules (batch number: 2208001Y), grind finely, and use different sampling amounts (0.2 g, 0.25 g, 0.3 g) to prepare test sample solutions according to the preparation method of test sample solution provided in Example 1. The same chromatographic conditions as in Example 1 were used for determination.

[0149] ​The test results show that when the sampling amount is 0.2g-0.3g, the peak area increases in proportion, indicating that when the sampling amount is 0.2g-0.3g, it can be completely extracted. Considering the response of the chromatographic peak, 0.2g is taken as the sampling amount of the Yuchunhua formula granules.

[0150] Example 4

[0151] The method in Example 1 was investigated for precision and stability, and the verification process was as follows:

[0152] 1. Precision

[0153] 1.1 Instrument precision test

[0154] The same sample solution of Yuchunhua formula granules was continuously sampled for 6 times according to the above chromatographic conditions, the relative retention time and relative peak area of each characteristic peak were determined, and the RSD of the relative retention time and relative peak area of each characteristic peak was calculated, as shown in Table 9 and Table 10.

[0155] Table 9

[0156]

[0157] Table 10

[0158]

[0159] The results showed that the relative retention time RSD of each characteristic peak was less than 2%, and the relative peak area RSD was less than 5%, indicating that the precision of the instrument was good.

[0160] 1.2 Method repeatability test

[0161] The same sample of Yuchunhua formula granules was prepared for 6 times according to the sample preparation method of Example 1, and the relative retention time and relative peak area of each common peak were determined according to the chromatographic conditions, and the detection results are shown in Table 11 and Table 12.

[0162] Table 11

[0163]

[0164] Table 12

[0165]

[0166] The results showed that the relative retention time RSD of each characteristic peak was less than 2%, and the relative peak area RSD was less than 5%, indicating that the repeatability of the method was good.

[0167] 1.3 Intermediate precision (different operators)

[0168] The same sample of Yuhua vinegar formula granules was taken by three inspectors at different times, and the sample was prepared according to the sample preparation method of the chromatographic conditions of Example 1, and the relative retention time and relative peak area of each common peak were determined using the same equipment. The test results are shown in Tables 13-14.

[0169] Table 13

[0170]

[0171] Table 14

[0172]

[0173]

[0174] The results show that the relative retention time RSD of each characteristic peak is less than 2%, indicating that the method has good repeatability among different personnel.

[0175] 2. Specificity

[0176] The negative blank sample was prepared by taking about 0.2 g of negative granules (malt dextrin), finely grinding, accurately weighing, placing in a conical flask with a stopper, accurately adding 25 ml of 70% methanol, tightly sealing, weighing, ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 minutes, cooling, weighing again, making up the weight loss with 70% methanol, shaking well, filtering, and taking the filtrate, which was obtained.

[0177] Accurately take 1 μl of the test sample solution and the negative blank sample solution, respectively, and inject them into the high performance liquid chromatograph. The results show that the negative sample has no interference.

[0178] 3. Stability investigation

[0179] The same test sample solution of Yuhua vinegar formula granules was taken, and the sample was injected at 0, 3, 6, 9, 12, 24, 36, and 48 hours according to the chromatographic conditions of Example 1, and the relative retention time and relative peak area of each common peak were determined. The test results are shown in Tables 15-16.

[0180] Table 15

[0181]

[0182] Table 16

[0183]

[0184] The results show that the relative retention time of each characteristic peak is less than 2%, and the RSD of the relative peak area is less than 5%, indicating that the test sample solution is stable within 48 hours and meets the determination requirements.

[0185] Example 5

[0186] The method in Example 1 was subjected to durability investigation, and the verification process was as follows:

[0187] 1. Investigation of different flow rates

[0188] The same Yidianhua granule test solution was taken, and the determination method of Example 1 was used to detect different flow rates (0.28 ml / min, 0.30 ml / min, 0.32 ml / min), and the relative retention time and relative peak area of each characteristic peak were determined, and the results are shown in Tables 17-18.

[0189] Table 17

[0190]

[0191] Table 18

[0192]

[0193] The results showed that the relative retention time RSD values of all characteristic peaks and reference substance S peak were less than 5%, and the relative peak area RSD values of all characteristic peaks and reference substance S peak were not more than 10%, and different flow rates could be used to distinguish Yidianhua strips, Jiaohua and Yidianhua.

[0194] 2. Investigation of different column temperatures

[0195] The same Yidianhua granule test solution was taken, and the determination method of Example 1 was used to detect different column temperatures (23℃, 25℃, 27℃), and the relative retention time and relative peak area of each characteristic peak were determined, and the results are shown in Tables 19-20.

[0196] Table 19

[0197]

[0198] Table 20

[0199]

[0200] The results showed that the relative retention time RSD values of all characteristic peaks and reference substance S peak were less than 5%, and the relative peak area RSD values of all characteristic peaks and reference substance S peak were not more than 10%, and different column temperatures could be used to distinguish Yidianhua strips, Jiaohua and Yidianhua.

[0201] 3. Investigation of different mobile phases

[0202] The same test solution of Meconii Coriandri Granula was detected by mobile phase I (acetonitrile-0.08% formic acid solution), mobile phase II (acetonitrile-0.10% formic acid solution) and mobile phase III (acetonitrile-0.12% formic acid solution) respectively, and the relative retention time and relative peak area of each characteristic peak were determined. The results are shown in Table 21-Table 22.

[0203] Table 21

[0204]

[0205] Table 22

[0206]

[0207] The results show that the relative retention time of each characteristic peak to the reference S peak is less than 5%, and the RSD value of the relative peak area of each characteristic peak to the reference S peak is less than 5%, indicating that the slight change of the formic acid concentration of the mobile phase has no significant effect on the relative retention time and relative retention peak area of the characteristic peak.

[0208] 4. Investigation of different instruments

[0209] At different times, the same Meconii Coriandri Granula was taken 6 times, and the relative retention time and relative peak area of each characteristic peak were determined by the determination method of Example 1 in different equipment. The results are shown in Table 23-Table 24.

[0210] Table 23

[0211]

[0212]

[0213] Table 24

[0214]

[0215] The results show that the RSD of the relative retention time of each characteristic peak is less than 5%, and the RSD of the relative peak area is less than 5%, showing good repeatability in different instruments.

[0216] 5. Investigation of different chromatographic columns

[0217] The same test solution of Meconii Coriandri Granula was taken, and the influence of different batches of chromatographic columns (Waters ACQUITY UPLC BEH C18) on the relative retention time of each characteristic peak was investigated according to the determination method of Example 1. The detection results are shown in Table 25 and Table 26.

[0218] Table 25

[0219]

[0220] Table 26

[0221]

[0222] The results show that the relative retention time RSD values of all characteristic peaks to the reference substance S are less than 5%, the RSD values of relative peak areas of all characteristic peaks to the reference substance S are not more than 10%, and different batches of chromatographic columns have no significant effect on each characteristic peak.

[0223] Obviously, the above examples are only examples for clearly illustrating but not limitation to the embodiments. Other different forms of changes or variations can be made by those skilled in the art on the basis of the above description. All the embodiments do not need and cannot be exhausted here. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method for detecting the characteristic spectrum of a formula granule of Acronychia pedunculata, characterized in that, The UPLC is used for detection, and the chromatographic conditions are as follows: The chromatographic column is a chromatographic column with octadecylsilane bonded silica gel as the filler, the column specification is 2.1*100mm, 1.7um, acetonitrile is used as the mobile phase A, and 0.08%-0.4% formic acid solution is used as the mobile phase B, and elution is carried out according to the following gradient elution program: Or, Or, Or, The column temperature of the chromatographic column is 20-30 DEG C, the detection wavelength is 332nm, and the flow rate is 0.3ml / min; The solvent used in the preparation process of the test solution is 50% or more of methanol; The preparation process of the reference solution is as follows: take the reference standard, add solvent and mix well to obtain the reference solution; the reference standard includes genistein and apigenin-7-O- β -D-glucuronide, chlorogenic acid.

2. The method according to claim 1, wherein the characteristic pattern of the formula granules of Meconps paniculata is shown in Table 1. Theoretical plate count based on apigenin -7- O - β The calculated peak value of -D-glucuronide should be no less than 5000.

3. The method according to claim 2, wherein the preparation process of the test solution is as follows: taking the vinegar jinyinhua formula granules, adding a solvent, weighing, ultrasonic or heating reflux treatment, cooling, weighing again, supplementing the weight loss with a solvent, mixing, filtering, and the filtrate is the test solution. The ultrasonic treatment power is 250W, and the ultrasonic treatment time is 30-60min; 4. The method according to claim 3, wherein the characteristic pattern of the formula granules of Meconps paniculata is shown in Table 1. And / or, the heating reflux treatment time is 30-60min; And / or, in the preparation process of the test solution, the amount of solvent added is 75-125ml / g. Also including a control medicinal material solution; 5. The method according to claim 1, wherein the characteristic pattern of the formula granules of Meconps paniculata is shown in Table 1. The preparation process of the control medicinal material solution is as follows: taking jinyinhua control medicinal material, adding water, heating reflux, filtering, evaporating the filtrate to dryness, adding a solvent to the residue, ultrasonic treatment, cooling, shaking, filtering, and taking the filtrate as the control medicinal material solution. Peak 5 is the S peak, and the relative retention time of other characteristic peaks relative to peak 5 is ±10% of the specified value, and the specified value of each characteristic peak is:

6. The method according to claim 5, wherein the characteristic pattern of the formula granules of Meconps paniculata is shown in Table 1. Each 1 ml of control solution contains 90 μg of genkwanin, 90 μg of apigenin-7-O- β -100 μg of D-glucuronide, 90 μg of chlorogenic acid.

7. The method according to claim 1, wherein the characteristic pattern of the formula granules of Meconps paniculata is shown in Table 1. The obtained characteristic spectrum includes characteristic peaks of chlorogenic acid, cryptochlorogenic acid, apigenin-7-O- glucuronide, luteolin, quercitrin, apigenin, hydroxyyuanhuacine and yuanhuacine, peak 1 is chlorogenic acid, peak 2 is cryptochlorogenic acid, peak 5 is apigenin-7-O- glucuronide, peak 6 is yuanhuacine-5-O-alizarin, peak 8 is luteolin, peak 9 is quercitrin, peak 10 is apigenin, peak 11 is hydroxyyuanhuacine and peak 12 is yuanhuacine. β The obtained characteristic spectrum includes characteristic peaks of chlorogenic acid, cryptochlorogenic acid, apigenin-7-O- glucuronide, luteolin, quercitrin, apigenin, hydroxyyuanhuacine and yuanhuacine, peak 1 is chlorogenic acid, peak 2 is cryptochlorogenic acid, peak 5 is apigenin-7-O- glucuronide, peak 6 is yuanhuacine-5-O-alizarin, peak 8 is luteolin, peak 9 is quercitrin, peak 10 is apigenin, peak 11 is hydroxyyuanhuacine and peak 12 is yuanhuacine. β The obtained characteristic spectrum includes characteristic peaks of chlorogenic acid, cryptochlorogenic acid, apigenin-7-O- glucuronide, luteolin, quercitrin, apigenin, hydroxyyuanhuacine and yuanhuacine, peak 1 is chlorogenic acid, peak 2 is cryptochlorogenic acid, peak 5 is apigenin-7-O- glucuronide, peak Peak 1: 0.21, peak 2: 0.23, peak 3: 0.69, peak 4: 0.92, peak 5: 1, peak 6: 1.10, peak 7: 1.17, peak 8: 1.36, peak 9: 1.46, peak 10: 1.59, peak 11: 1.

82. ​

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