A characteristic map detection method for palm charcoal formula granules and application thereof

Abstract

The present application relates to the technical field of traditional Chinese medicine detection, in particular to a characteristic chromatogram detection method of palm charcoal formula granules and application thereof. The characteristic chromatogram detection method of palm charcoal preparation comprises the following steps: preparation of test sample solution; detection of the test sample solution by ultra-high performance liquid chromatography, and construction of the characteristic chromatogram of palm charcoal preparation according to the measured liquid chromatogram; the conditions of the ultra-high performance liquid chromatography detection include: Waters ACQUITY UPLC HSS T3 chromatographic column with octadecylsilane bonded silica gel as the filler, detection wavelength 285-295 nm, acetonitrile as mobile phase A, and 0.08-0.12% formic acid aqueous solution as mobile phase B; elution according to a specific gradient program. The characteristic peaks obtained by the detection method are more, and the identified characteristic peaks are more, so that the product quality of palm charcoal preparation can be more comprehensively controlled.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine detection, and particularly relates to a characteristic spectrum detection method of palm charcoal formula granules and application thereof. BACKGROUND

[0002] The palm charcoal formula granule is a formula granule prepared by processing and processing the dried petiole of Trachycarpus fortunei (Hook.f.) H.Wendl. according to the main quality indicators of the standard decoction. It tastes bitter and astringent, is flat in nature, and belongs to the lung, liver and large intestine channels, has the effects of astringing and stopping bleeding, and can be used for treating various bleeding such as hemoptysis, epistaxis, hematuria, hematochezia and metrorrhagia. With the continuous widening of the clinical application range, the quality control of the palm charcoal formula granule is also the key to ensure its efficacy, and the characteristic spectrum can not only characterize the stability of the process, but also reflect the overall quality of the finished product.

[0003] At present, the characteristic spectrum method is established under the quality standard of palm charcoal formula granules published by each province, a total of 5 common peaks are confirmed, the protocatechuic acid peak is determined by the reference substance identification method, the protocatechuic acid peak is taken as the S peak, and the relative retention times of the other 4 characteristic peaks are calculated. However, this method has fewer characteristic peaks, and except for protocatechuic acid, other components are not identified, especially peak 3 with the largest peak area is not determined. The method has limitations for the quality control of palm charcoal formula granules, and it is generally believed that more characteristic peaks and more identified characteristic peaks obtained by the characteristic spectrum method can more comprehensively control the quality of the formula granule product. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to overcome the defects of fewer characteristic peaks and identified peaks in the existing characteristic spectrum of palm charcoal preparation, so as to provide a characteristic spectrum detection method of palm charcoal formula granules and application thereof.

[0005] To achieve the above purpose, the present application provides the following technical scheme:

[0006] In the first aspect, the present application provides a characteristic spectrum detection method of palm charcoal preparation, comprising the following steps:

[0007] Preparation of test sample solution;

[0008] Detecting the test sample solution by ultra-high performance liquid chromatography, and constructing the characteristic spectrum of the palm charcoal preparation according to the measured liquid chromatogram;

[0009] The conditions of the ultra-high performance liquid chromatography detection include: Waters ACQUITY UPLC HSS T3 chromatographic column with octadecylsilane-bonded silica gel as the filler, detection wavelength 285-295 nm, acetonitrile as the mobile phase A, and 0.08-0.12% formic acid aqueous solution as the mobile phase B;

[0010] The elution is performed according to the following gradient program:

[0011]

[0012] Preferably, in the conditions of the ultra-high performance liquid chromatography detection, the column length of the chromatographic column is 100 mm, the inner diameter is 2.1 mm, and the particle size is 1.8 μm;

[0013] And / or, the column temperature is 28-32°C, preferably 30°C;

[0014] And / or, the flow rate is 0.25 ml / min-0.40 ml / min, preferably 0.3 ml / min;

[0015] And / or, the injection amount is 1-10 μl, preferably 2 μl;

[0016] And / or, the theoretical plate number calculated according to the protocatechuic acid peak should be not less than 10,000.

[0017] Preferably, the preparation process of the test sample solution is as follows: taking the test sample, finely grinding, accurately weighing, adding solvent, weighing, extracting and processing, taking out, cooling, re-weighing, supplementing the lost weight with the corresponding solvent, shaking, filtering, and taking the filtered solution to obtain the test sample solution.

[0018] Preferably, the test sample is palm charcoal preparation, and optionally, the palm charcoal preparation includes palm charcoal formula granules.

[0019] And / or, the solvent in the test sample solution is aqueous methanol solution with a volume fraction of not more than 50% or water, preferably water;

[0020] And / or, the extraction processing is ultrasonic treatment or / and hot reflux treatment, preferably ultrasonic treatment.

[0021] Preferably, the addition amount of the test sample relative to 10 ml of solvent is 0.1-0.4 g, preferably 0.2 g;

[0022] And / or, the duration of the extraction processing is 10-40 min, preferably 20 min.

[0023] Preferably, the characteristic spectrum detection method further comprises the steps of preparing the reference substance solution by using 5-hydroxymethylfurfural, protocatechuic acid, protocatechuic aldehyde and 4-hydroxybenzoic acid as solvents, and detecting the reference substance solution by the ultra performance liquid chromatography in the characteristic spectrum detection method to obtain the reference substance spectrum.

[0024] Preferably, the reference substance solution is prepared by taking 5-hydroxymethylfurfural, protocatechuic acid, protocatechuic aldehyde and 4-hydroxybenzoic acid respectively, and adding solvents to prepare 5-hydroxymethylfurfural solution containing 10 μg per 1 ml, protocatechuic acid solution containing 20 μg per 1 ml, protocatechuic aldehyde solution containing 50 μg per 1 ml, and 4-hydroxybenzoic acid solution containing 300 μg per 1 ml.

[0025] Preferably, the solvent in the reference substance solution is water.

[0026] Preferably, the characteristic spectrum of the test substance comprises characteristic peaks of peaks 1-11; wherein the retention time of peaks 1, 2, 3 and 4 is consistent with that of the reference substance spectrum of 5-hydroxymethylfurfural, protocatechuic acid, protocatechuic aldehyde and 4-hydroxybenzoic acid respectively.

[0027] Preferably, peak 2 is the S peak, and the relative retention time of peaks 1, 3-11 is calculated, and the specified value of the relative retention time of peaks 1-11 is as follows: the relative retention time of each characteristic peak should be within ±10% of the specified value.

[0028]

[0029] In the second aspect, the application further provides the application of the characteristic spectrum detection method of the palm charcoal preparation in the quality detection of the palm charcoal preparation.

[0030] In the application, the percentage of the solution is volume percentage.

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

[0032] The application provides a characteristic spectrum detection method of palm charcoal preparation, and comprises the following steps: preparation of a test sample solution; performing ultra-high performance liquid chromatography detection on the test sample solution, and constructing a characteristic spectrum of the palm charcoal preparation according to the measured liquid chromatogram; the conditions of the ultra-high performance liquid chromatography detection include: a Waters ACQUITY UPLC HSS T3 chromatographic column with octadecylsilane-bonded silica gel as a filler, a detection wavelength of 285-295 nm, acetonitrile as a mobile phase A, and 0.08-0.12% formic acid aqueous solution as a mobile phase B; and elution is performed according to a specific gradient program. In view of the defects of the existing characteristic spectrum of the palm charcoal preparation, such as few characteristic peaks and few identified peaks, the characteristic spectrum detection method provided by the application has more characteristic peaks and more identified characteristic peaks, realizes more component characterization, can more comprehensively control the product quality of the palm charcoal preparation, and achieves the purposes of controlling the quality of the palm charcoal preparation to be stable and uniform and improving the quality control standard. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the description of the embodiments or the prior art. Obviously, the drawings described below are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0034] Figure 1 is the characteristic spectrum of the palm charcoal preparation granules in Example 1 of the present application;

[0035] Figure 2 is the identification result graph of each characteristic peak in the characteristic spectrum of the palm charcoal preparation granules in Example 1 of the present application;

[0036] Figure 3 is the influence of different organic phases (mobile phase A) on the chromatogram of the palm charcoal preparation granules in Example 2 of the present application;

[0037] Figure 4 is the influence of different aqueous phases (mobile phase B) on the chromatogram of the palm charcoal preparation granules in Example 2 of the present application;

[0038] Figure 5 is the UPLC 3D graph of the palm charcoal preparation granules in Example 2 of the present application and the characteristic spectrum intercepted within the wavelength of 280 nm-300 nm;

[0039] Figure 6 is the characteristic spectrum intercepted at the wavelength of 280 nm in the UPLC 3D graph of the palm charcoal preparation granules in Example 2 of the present application;

[0040] Figure 7is the characteristic spectrum of the UPLC 3D graph of the palm charcoal formula granules in Example 2 of the present application, taken at a wavelength of 283 nm;

[0041] Figure 8 is the characteristic spectrum of the UPLC 3D graph of the palm charcoal formula granules in Example 2 of the present application, taken at a wavelength of 285 nm;

[0042] Figure 9 is the characteristic spectrum of the UPLC 3D graph of the palm charcoal formula granules in Example 2 of the present application, taken at a wavelength of 290 nm;

[0043] Figure 10 is the characteristic spectrum of the UPLC 3D graph of the palm charcoal formula granules in Example 2 of the present application, taken at a wavelength of 295 nm;

[0044] Figure 11 is the characteristic spectrum of the UPLC 3D graph of the palm charcoal formula granules in Example 2 of the present application, taken at a wavelength of 298 nm;

[0045] Figure 12 is the characteristic spectrum of the UPLC 3D graph of the palm charcoal formula granules in Example 2 of the present application, taken at a wavelength of 300 nm;

[0046] Figure 13 is the chromatogram of the palm charcoal formula granules in Example 2 of the present application with different extraction solvents;

[0047] Figure 14 is the chromatogram of the palm charcoal formula granules in Example 2 of the present application with different extraction times;

[0048] Figure 15 is the chromatogram of the palm charcoal formula granules in Example 2 of the present application with different extraction methods;

[0049] Figure 16 is the chromatogram of the palm charcoal formula granules in Example 2 of the present application with different extraction concentrations;

[0050] Figure 17 is the chromatogram of the palm charcoal formula granules in Example 2 of the present application with different injection amounts;

[0051] Figure 18 is the chromatogram of the palm charcoal formula granules in Example 2 of the present application with different injection amounts;

[0052] Figure 19 is the chromatogram of the palm charcoal formula granules in Example 2 of the present application with different injection amounts;

[0053] Figure 20 is the chromatogram of the palm charcoal formula granules in Example 2 of the present application with different injection amounts;

[0054] Figure 21 is the chromatogram of the maltodextrin negative control solution in Example 3 of the present application;

[0055] Figure 22 is the comparison chart of the liquid chromatography in the stability study in Example 3 of the present application;

[0056] Figure 23 is the comparison chart of the liquid chromatography in the different flow rate study in Example 4 of the present application;

[0057] Figure 24 is the comparison chart of the liquid chromatography in the different acid concentration study in Example 4 of the present application;

[0058] Figure 25 is the comparison chart of the liquid chromatography in the different column temperature study in Example 4 of the present application;

[0059] Figure 26 is the comparison chart of the liquid chromatography in the different column study in Example 4 of the present application. DETAILED DESCRIPTION

[0060] The following examples are provided to better further understand the present application, and are not limited to the best mode, and do not constitute a limitation on the content and scope of protection of the present application. Any person under the inspiration of the present application or the combination of the present application with other prior art features will fall within the scope of protection of the present application.

[0061] The specific experimental steps or conditions are not specified in the examples, and can be performed according to the conventional experimental steps described in the literature in the art. The reagents or instruments used are not specified by the manufacturer, and are conventional reagent products that can be obtained by purchase.

[0062] The main test instruments and reagents involved in the following examples and comparative examples are as follows:

[0063] Instruments:

[0064] Waters ACQUITY UPLC H-Class ultra-high performance liquid chromatograph; PDA detector; TUV detector; Empower 3 chromatography workstation; ME104E electronic balance (Mettler Toledo Scientific Instruments (Shanghai) Co., Ltd.), JY20002 electronic balance (Shunyu Hengping Instrument Co., Ltd.), BSA124S electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.), XP26 electronic balance (Mettler Toledo Scientific Instruments (Shanghai) Co., Ltd.), KQ-500DA ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.);

[0065] Chromatographic column:

[0066] Waters ACQUITY UPLC HSS T3 (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm);

[0067] Waters CORTECS UPLC C18 (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm);

[0068] Waters ACQUITY UPLC BEH C18 (column length 100 mm, inner diameter 2.1 mm, particle size 1.7 μm);

[0069] Reagents:

[0070] Protocatechuic acid (batch number: 110809-202207, purity 97.5%, China Institute for Drug Control);

[0071] 5-Hydroxymethylfurfural (batch number: 111626-202316, purity 98.4%, China Institute for Drug Control);

[0072] Protocatechuic aldehyde (batch number: 110810-202210, purity 99.9%, China Institute for Drug Control);

[0073] 4-Hydroxybenzoic acid (batch number: 101149-202204, purity 100%, China Institute for Drug Control);

[0074] Palitonyl Char Formula Granules (batch numbers: K455CP01, K455CP02, K455CP03);

[0075] Reagents: acetonitrile, methanol, formic acid, acetic acid, phosphoric acid are chromatographically pure; water is distilled water; other reagents are analytical pure.

[0076] Example 1

[0077] A characteristic map detection method of Palitonyl Char Formula Granules is determined according to high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition Part 0512), and the specific process is as follows:

[0078] 1. Solution preparation

[0079] Preparation of test sample solution: take Palitonyl Char Formula Granules, grind finely, take about 0.2 g, accurately weigh, put into a conical flask with a plug, tightly plug, weigh, ultrasonic treatment (power 250 W, frequency 40 kHz) for 40 minutes, take out, cool, weigh again, make up the weight loss with the corresponding solvent, shake well, filter, take the filtrate, and the test sample solution is obtained.

[0080] Preparation of control solution: accurately weigh 5-hydroxymethylfurfural, protocatechuic acid, protocatechuic aldehyde, 4-hydroxybenzoic acid (also known as: p-hydroxybenzoic acid) control, add water to prepare 5-hydroxymethylfurfural solution containing 10 μg per 1 ml, protocatechuic acid solution containing 20 μg per 1 ml, protocatechuic aldehyde solution containing 50 μg per 1 ml, 4-hydroxybenzoic acid solution containing 300 μg per 1 ml.

[0081] 2. Detection by ultra-high performance liquid chromatography

[0082] The following chromatographic conditions were used for detection:

[0083] Waters ACQUITY UPLC HSS T3 column (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm) with octadecylsilane-bonded silica gel as filler, acetonitrile as mobile phase A, 0.1% formic acid aqueous solution as mobile phase B, gradient elution according to the following Table 1; flow rate was 0.3 ml / min, column temperature was 30°C, detection wavelength was 290 nm, and theoretical plate number calculated according to protocatechuic acid peak should not be less than 10000.

[0084] Table 1 Elution gradient table

[0085]

[0086]

[0087] 3. Identification of characteristic peaks:

[0088] According to relevant literature, palm charcoal contains cellulose and tannins, and contains organic acids, flavonoids, glycosides, such as protocatechuic acid, protocatechuic aldehyde, etc.

[0089] Therefore, 5-hydroxymethylfurfural solution, protocatechuic acid solution, protocatechuic aldehyde solution, and 4-hydroxybenzoic acid solution were precisely pipetted 3 μl each, and the test solution was injected into the liquid chromatograph (Waters ACQUITY UPLC H-Class (TUV detector)), and the determination was completed, i.e. The determination results are shown in Figure 1 The characteristic chromatogram of palm charcoal formula granules) is shown. By comparing the detection results of the above test solution, the characteristic peaks in the test solution characteristic chromatogram were determined, and the identification results are shown in Figure 2

[0090] ​According to the comparison of the obtained chromatograms by the above method, 11 characteristic peaks can be preliminarily determined. The retention times of peaks 1, 2, 3 and 4 in the test sample chromatogram are consistent with those of 5-hydroxymethylfurfural, protocatechuic acid, protocatechuic aldehyde and 4-hydroxybenzoic acid, respectively. Therefore, it can be confirmed that peak 1 is 5-hydroxymethylfurfural, peak 2 is protocatechuic acid, peak 3 is protocatechuic aldehyde, and peak 4 is 4-hydroxybenzoic acid. Peak 2 (protocatechuic acid) with good peak shape, high response value and relatively central position is determined as the S peak. Figure 1 In the formula, peak 1: 5-hydroxymethylfurfural; peak 2 (S): protocatechuic acid; peak 3: protocatechuic aldehyde; peak 4: 4-hydroxybenzoic acid. The characteristic chromatogram standard of palm charcoal formula granules is that 11 characteristic peaks should be present in the test sample chromatogram, among which peak 2 should have the same retention time as the protocatechuic acid reference peak. The peak corresponding to the retention time of the protocatechuic acid reference peak is the S peak. The relative retention times of each characteristic peak and the S peak are calculated, and the relative retention times should be within ±10% of the specified values. The specified values are 0.77 (peak 1), 1.36 (peak 3), 1.47 (peak 4), 1.64 (peak 5), 2.49 (peak 6), 2.79 (peak 7), 3.15 (peak 8), 3.41 (peak 9), 4.16 (peak 10), and 4.29 (peak 11).

[0091] Example 2

[0092] This example is used to illustrate the establishment of the characteristic chromatogram of palm charcoal formula granules.

[0093] 1. Selection of chromatographic conditions

[0094] 1.1. Selection of mobile phase

[0095] Octadecylsilane-bonded silica gel is used as the filler [chromatographic column: Waters ACQUITY UPLC HSS T3 (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm)]; methanol and acetonitrile are used as mobile phase A, and 0.1 vol% phosphoric acid aqueous solution, 0.1 vol% acetic acid aqueous solution and 0.1 vol% formic acid aqueous solution are used as mobile phase B, gradient elution is performed according to the provisions in Table 1; the flow rate is 0.3 ml per minute; the column temperature is 30°C; the detection wavelength is 290 nm, and the theoretical plate number calculated according to the protocatechuic acid peak should not be less than 10,000. The test results of different organic phases (mobile phase A) are shown in Table 2. Figure 3 The test results of different water phases (mobile phase B) are shown in Table 3. Figure 4

[0096] After comparison, acetonitrile-0.1 vol% formic acid aqueous solution is selected as the mobile phase for gradient elution. In the obtained chromatogram of palm charcoal formula granules, the chromatographic peaks have good separation degree and the chromatogram information is rich. The method has good reproducibility, and therefore this method is determined as the final method for the characteristic chromatogram of palm charcoal formula granules by liquid chromatography.​

[0097] 1.2. Selection of detection wavelength

[0098] The detection was performed according to the liquid phase elution method in Example 1, and the 3D full scan of the palmetto charcoal formula granule test sample solution was performed. The full scan test results are shown in Figure 5 , and the chromatogram in the wavelength range of 280 nm to 300 nm is shown in Figure 6-12 .

[0099] The test results show that in the wavelength range of 283 nm to 298 nm, there is more characteristic spectrum information, and in the range of 285 nm to 295 nm, the sizes of the characteristic peaks are more uniform, and the wavelength of 290 nm is the best. Therefore, the characteristic spectrum is selected at 290 nm for collection.

[0100] 2. Selection of test sample solution

[0101] 2.1. Selection of extraction solvent

[0102] An appropriate amount of palmetto charcoal formula granules (batch number: K455CP02) was finely ground, and about 0.2 g was accurately weighed and placed in a stoppered conical flask. 10 ml of 10 vol% ethanol aqueous solution, 30 vol% ethanol aqueous solution, 50 vol% ethanol aqueous solution, 70 vol% ethanol aqueous solution, 95 vol% ethanol aqueous solution, methanol, 70 vol% methanol aqueous solution, 50 vol% methanol aqueous solution, 30 vol% methanol aqueous solution, 10 vol% methanol aqueous solution, and water were used as extraction solvents, respectively. The flask was tightly sealed, weighed, and ultrasonically treated (power 250 W, frequency 40 kHz) for 40 minutes. After cooling, the weight was re-weighed, the lost weight was made up with the corresponding solvent, shaken well, filtered, and the filtrate was collected to obtain the test sample solution. 2 μl of the filtrate was accurately taken and injected into the ultra-high performance liquid chromatograph, and the detection was performed according to the liquid phase elution method in Example 1 for comparison. The test results are shown in Figure 13 and Tables 2 and 3.

[0103] Table 2. Characteristic peak retention time and relative retention time of each extraction solvent

[0104]

[0105]

[0106] Table 3. Characteristic peak area of each extraction solvent

[0107]

[0108] According to the test results, the peak shape of the chromatographic peaks obtained by different concentrations of ethanol extraction is poor. The peak area of the low concentration (volume fraction ≤ 50%) methanol aqueous solution and pure water sample is higher. Considering the toxicity of the reagent, water is preferred as the extraction solvent.

[0109] 2.2, selection of extraction time

[0110] Take palm charcoal formula granules (batch number: K455CP02) of appropriate amount, grind finely, take 4 parts, about 0.2g each, accurately weigh, put into a conical flask with a plug, accurately add purified water 10ml, tightly plug, weigh, ultrasonic treatment (power 250W, frequency 40kHz) for 10, 20, 30, 40 minutes respectively, take out, cool, weigh again, make up the weight loss with purified water, shake well, filter, take the filtrate, accurately take 2ul of the filtrate, inject into the ultra-high performance liquid chromatograph, and determine according to the liquid phase elution method in example 1, the test results are shown in Figure 14 and table 4, table 5.

[0111] Table 4 characteristic peak retention time and relative retention time of different extraction time

[0112]

[0113]

[0114] Table 5 peak area of characteristic peaks of different extraction time

[0115] Peak area Peak 1 Peak 2 (S) Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 Peak 11 10 min 367159 202840 91309 229350 293230 102825 40933 43757 111308 60558 35470 20 min 377814 219459 95107 235646 330348 110600 41250 44650 113880 67223 41638 30 min 372555 208120 93593 226897 320625 103441 49710 44242 110663 64726 40607 40 min 368970 213017 92509 225779 290654 103958 35305 44088 111135 65955 41972

[0116] According to the test results, the peak shape of the chromatographic peaks obtained by different concentrations of ethanol extraction is poor. The peak area of the low concentration (volume fraction ≤ 50%) methanol aqueous solution and pure water sample is higher. Considering the toxicity of the reagent, water is preferred as the extraction solvent.

[0117] 2.3, selection of extraction method

[0118] Take palm charcoal formula granules (batch number: K455CP02) of appropriate amount, grind finely, take two parts, about 0.2g each, accurately weigh, put into a conical flask with a plug, accurately add purified water 10ml, tightly plug, weigh, ultrasonic treatment (power 250W, frequency 40kHz) for 10 minutes for one part, and reflux extraction for 10 minutes for the other part, take out, cool, weigh again, make up the weight loss with purified water, shake well, filter, take the filtrate, accurately take 2ul of the filtrate, inject into the ultra-high performance liquid chromatograph, and determine according to the liquid phase elution method in example 1, the test results are shown in Figure 15 and table 6, table 7.

[0119] Table 6 characteristic peak retention time and relative retention time of different extraction methods

[0120]

[0121] Table 7 Peak area of characteristic peaks of different extraction methods

[0122] Peak area Peak 1 Peak 2 (S) Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 Peak 11 Ultrasonic 367159 202840 91309 229350 293230 102825 40933 43757 111308 60558 35470 Reflux 413851 233200 100197 244318 312545 118043 43059 46271 121290 71240 45847

[0123] According to the above test results, the peak types of the two extraction methods are not much different. Considering energy saving and simple operation, the ultrasonic extraction method is preferred.

[0124] 2.4, Selection of extraction concentration

[0125] Take palm charcoal formula granules (batch number: K455CP02) of an appropriate amount, grind finely, and take about 0.1 g, 0.2 g, 0.3 g and 0.4 g respectively, and accurately weigh, place in a conical flask with a stopper, accurately add purified water 10 ml respectively, tightly seal, weigh, ultrasonic treatment (power 250 W, frequency 40 kHz) for 40 minutes, take out, cool down, weigh again, make up the weight loss with purified water, shake well, filter, take the filtrate, accurately take 2 μl of the filtrate, inject into the ultra-high performance liquid chromatograph, and determine according to the liquid phase elution method in Example 1, and the test results are shown in Table 7, Table 8 and Table 9. Figure 16

[0126] Table 8 Retention time and relative retention time of characteristic peaks of different extraction concentrations

[0127] Retention time Peak 1 Peak 2 (S) Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 Peak 11 0.1 g / 10 ml 5.083 6.636 8.951 9.709 10.781 16.477 18.352 20.745 22.44 27.441 28.369 0.2 g / 10 ml 5.094 6.646 8.969 9.708 10.806 16.499 18.353 20.754 22.447 27.446 28.378 0.3 g / 10 ml 5.104 6.646 8.967 9.693 10.809 16.473 18.327 20.738 22.439 27.433 28.37 0.4 g / 10 ml 5.092 6.625 8.942 9.656 10.784 16.431 18.277 20.687 22.39 27.394 28.337 Relative retention time Peak 1 Peak 2 (S) Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 Peak 11 0.1 g / 10 ml 0.766 1.000 1.349 1.463 1.625 2.483 2.766 3.126 3.382 4.135 4.275 0.2 g / 10 ml 0.766 1.000 1.35 1.461 1.626 2.483 2.762 3.123 3.378 4.13 4.27 0.3 g / 10 ml 0.768 1.000 1.349 1.458 1.626 2.479 2.758 3.12 3.376 4.128 4.269 0.4 g / 10 ml 0.769 1.000 1.35 1.458 1.628 2.48 2.759 3.123 3.38 4.135 4.277 RSD % 0.196 0 0.043 0.168 0.077 0.083 0.130 0.078 0.076 0.086 0.090

[0128] Table 9 Peak area of characteristic peaks of different extraction concentrations

[0129] Peak area Peak 1 Peak 2 (S) Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 Peak 11 0.1 g / 10 ml 183824 108848 47659 115088 145524 52723 19937 22920 56793 32144 23628 0.2 g / 10 ml 368196 201715 94061 225944 291898 104840 39352 44176 110201 66216 42808 0.3 g / 10 ml 556340 306932 140226 339770 434375 163021 79829 65298 161590 99859 63260 0.4 g / 10 ml 730383 409323 188391 454592 575136 219381 72173 81105 215479 131519 87613

[0130] According to the above test results, the peak types of the four extraction concentrations are not much different. The sampling amount of palm charcoal formula granules is preferably 0.2 g, and 10 ml of extraction solvent is accurately added as the test solution concentration.

[0131] 2.5, Selection of injection amount

[0132] Take palm charcoal formula granules (batch number: K455CP02) of an appropriate amount, grind finely, and take about 0.1 g, 0.2 g, 0.3 g and 0.4 g respectively, and accurately weigh, place in a conical flask with a stopper, accurately add purified water 10 ml respectively, tightly seal, weigh, ultrasonic treatment (power 250 W, frequency 40 kHz) for 40 minutes, take out, cool down, weigh again, make up the weight loss with purified water, shake well, filter, take the filtrate, accurately take 2 μl of the filtrate, inject into the ultra-high performance liquid chromatograph, and determine according to the liquid phase elution method in Example 1, and the test results are shown in Table 7, Table 8 and Table 9. Figure 17

[0133] ​​Table 10 Characteristic peak retention time and relative retention time of different injection amounts

[0134] Retention time Peak 1 Peak 2 (S) Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 Peak 11 10 μl 5.092 6.599 8.899 9.552 10.73 16.311 18.176 20.604 22.321 27.363 28.312 8 μl 5.084 6.591 8.894 9.562 10.724 16.313 18.169 20.587 22.301 27.325 28.28 5 μl 5.073 6.586 8.882 9.571 10.704 16.286 18.148 20.574 22.286 27.326 28.283 2 μl 5.065 6.588 8.895 9.62 10.726 16.308 18.167 20.596 22.31 27.341 28.295 1 μl 5.052 6.564 8.866 9.595 10.68 16.24 18.105 20.538 22.259 27.304 28.267 Relative retention time Peak 1 Peak 2 (S) Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 Peak 11 10 μl 0.772 1.000 1.349 1.447 1.626 2.472 2.754 3.122 3.382 4.147 4.29 8 μl 0.771 1.000 1.349 1.451 1.627 2.475 2.757 3.124 3.384 4.146 4.291 5 μl 0.77 1.000 1.349 1.453 1.625 2.473 2.756 3.124 3.384 4.149 4.294 2 μl 0.769 1.000 1.35 1.46 1.628 2.475 2.758 3.126 3.386 4.15 4.295 1 μl 0.77 1.000 1.351 1.462 1.627 2.474 2.758 3.129 3.391 4.16 4.306 RSD % 0.148 0 0.066 0.431 0.070 0.053 0.061 0.085 0.101 0.135 0.149

[0135] Table 11 Peak area of characteristic peaks of different injection amounts

[0136]

[0137]

[0138] According to the above test results, the peak shape of the five injection amounts is not much different, and the injection amount of 2 microliters is preferred.

[0139] According to the above method, the preferred preparation method of the palm charcoal formula granule test solution is as follows: an appropriate amount of palm charcoal formula granules is finely ground, about 0.2 g is taken and accurately weighed, placed in a conical flask, 10 ml of purified water is accurately added, tightly sealed, the weight is determined, ultrasonic treatment (power 250 W, frequency 40 kHz) is performed for 20 minutes, taken out, cooled, shaken uniformly, filtered, and the filtrate is taken, and the filtrate is obtained.

[0140] Determination: 2 μl of the filtrate is accurately taken and injected into the ultra-high performance liquid chromatograph, and the liquid phase elution method in Example 1 is used for determination.

[0141] 3. Verification of characteristic spectrum

[0142] Three batches of palm charcoal formula granules (batch number: K455CP01, K455CP02, K455CP03, see Table 12 for sample list) are taken respectively, and the liquid chromatogram is obtained according to the liquid phase elution method in Example 1, and the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012.1 version) is used, with S1 liquid chromatogram as the reference spectrum, calculated by the median, the characteristic spectrum of palm charcoal formula granules is obtained (as shown in Figure 1 ), and the common peaks are identified, and 11 common peaks are identified. Among them, the similarity of the three batches of palm charcoal formula granules and palm charcoal characteristic spectrum is as shown in Figure 18 and Table 12. With S peak as the reference, the relative retention time of each characteristic peak is calculated, and the average value of the test results of the three batches of palm charcoal formula granules is taken as the specified value, and the test results are as shown in Table 13.

[0143] Table 12 Similarity of three batches of palm charcoal formula granule samples and palm charcoal characteristic spectrum

[0144] Serial number Batch number Similarity S1 K455CP01 0.969 S2 K455CP02 0.953 S3 K455CP03 0.942 R Standard control chromatogram 1.000

[0145] Table 13 Relative retention time and specified value of palm charcoal formula granules

[0146]

[0147] Example 3

[0148] This example is used to investigate the method of detecting the characteristic map of palm charcoal formula granules.

[0149] (I) Delayed investigation

[0150] According to the method in Example 1, the liquid chromatogram of 2 times the elution time was recorded, and the test results are shown in Figure 19

[0151] According to the above test results, no chromatographic peak appeared after 32 minutes, indicating that the method can completely elute each component.

[0152] (II) Specificity investigation

[0153] According to the preparation method of the test solution in Example 1, blank solvent and malt dextrin negative control solution were prepared, and the chromatogram was recorded according to the liquid phase method in Example 1. Among them, the chromatogram of the blank solvent is shown in Figure 20 , and the chromatogram of the malt dextrin negative control solution is shown in Figure 21 .

[0154] According to the above test results, the blank solvent and the excipient have no interference, indicating that the method has good specificity.

[0155] (III) System suitability

[0156] According to the preparation method of the test solution in Example 1, the test solution was prepared, 2 μl of injection was precisely taken into the liquid chromatograph, a total of 5 times, the chromatogram was recorded, the retention time, peak area, USP separation degree, USP tailing, symmetry factor and USP theoretical plate number of No. 2 peak were recorded, the average value and RSD% value were calculated, and the RSD% value should be ≤2%. The test results are shown in Table 14.

[0157] Table 14 System suitability of palm charcoal formula granules

[0158] System suitability Retention time Area USP resolution USP tailing Symmetry factor USP theoretical plate number 1st injection 6.363 210988 8.736 1.214 1.214 22025 2nd injection 6.408 207507 8.692 1.235 1.235 22830 3rd injection 6.475 212682 8.537 1.251 1.251 22128 4th injection 6.556 210199 8.758 1.246 1.246 22207 5th injection 6.575 210040 8.871 1.269 1.269 21803 Mean value 6.475 210283 8.719 1.243 1.243 22199 RSD % 1.415 0.890 1.390 1.636 1.636 1.731

[0159] According to the above test results, the RSD of the retention time, peak area, USP separation degree, USP tailing, symmetry factor and USP theoretical plate number of each characteristic peak is ≤2%, indicating that the system suitability of this method is passed, and the specified theoretical plate number should not be less than 10000.

[0160] (IV) Stability investigation

[0161] ​Prepare the test sample solution according to the method of preparing the test sample solution in Example 1, and precisely pipette 2 μl of the test sample solution at 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 48 h into the liquid chromatograph, record the chromatogram, take peak 2 as the reference peak, calculate the relative retention time and the relative peak area, and obtain the mean value and the RSD value. The RSD value of the relative retention time should be ≤2%. The test results are shown in Table 15 and Table 16. Figure 22 and Table 15, Table 16.

[0162] Table 15 Relative retention time table for stability of palm charcoal formula granules

[0163]

[0164]

[0165] Table 16 Relative peak area table for stability of palm charcoal formula granules

[0166]

[0167] According to the above test results, the RSD of the relative retention time of each characteristic peak is in the range of 0-1.11%, and the RSD of the relative peak area is in the range of 0-1.99%. The results show that the RSD value of the relative retention time is ≤2%, and the test sample solution of the palm charcoal formula granules adopted in the present application is stable within 48 h.

[0168] (V) Reproducibility investigation

[0169] Prepare the test sample solution according to the method of preparing the test sample solution in Example 1, and precisely pipette 2 μl of the test sample solution at 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 48 h into the liquid chromatograph, record the chromatogram, take peak 2 as the reference peak, calculate the relative retention time and the relative peak area, and obtain the mean value and the RSD value. The RSD value of the relative retention time should be ≤2%. The test results are shown in Table 15 and Table 16.

[0170] Table 17 Retention time and relative retention time table for reproducibility investigation of palm charcoal formula granules

[0171]

[0172] Table 18 Peak area and relative peak area table for reproducibility investigation of palm charcoal formula granules

[0173]

[0174] According to the above test results, the RSD of the relative retention time of each characteristic peak is in the range of 0-1.11%, and the RSD of the relative peak area is in the range of 0-1.99%. The results show that the RSD value of the relative retention time is ≤2%, and the test sample solution of the palm charcoal formula granules adopted in the present application is stable within 48 h.

[0175] (Six) Precision investigation

[0176] Prepare the test solution 1 part according to the preparation method of the test solution in Example 1, precisely pipette 2 μl into the liquid chromatograph, a total of 6 needles, record the chromatogram, take No. 2 peak as the reference peak, calculate the relative retention time, relative peak area, and obtain the mean value and RSD% value, the relative retention time RSD value should be ≤2%. The test results are shown in Tables 19 and 20.

[0177] Table 19 Palms charcoal formula granules precision retention time and relative retention time table

[0178]

[0179] Table 20 Palms charcoal formula granules precision peak area and relative peak area table

[0180]

[0181] According to the above precision investigation results, the relative retention time RSD of each characteristic peak is in the range of 0-0.16%, and the relative peak area RSD is in the range of 0-2.80%, the results show that the relative retention time RSD value is ≤2%, and the precision of the instrument is good.

[0182] (Seven) Intermediate precision investigation

[0183] Take the 6 test solution used in repeatability investigation (instrument: Waters ACQUITY UPLC H-Class, TUV detector) to re-sample and detect on another liquid chromatograph (Waters ACQUITY UPLC H-Class, PDA detector), record the chromatogram, and calculate the relative retention time, relative peak area, and obtain the mean value and RSD% value, the relative retention time RSD% value should be ≤2%. The test results are shown in Tables 21 and 22.

[0184] Table 21 Palms charcoal formula granules intermediate precision retention time and relative retention time table

[0185]

[0186] Table 22 Palms charcoal formula granules intermediate precision peak area and relative peak area table

[0187]

[0188]

[0189] According to the test results, the relative retention time RSD of each characteristic peak in the characteristic spectrum obtained by using another ultra-high performance liquid chromatograph, Waters ACQUITY UPLC H-Class (PDA detector), is in the range of 0% to 0.89%, the relative peak area RSD is in the range of 0% to 1.68%, the relative retention time RSD between different instruments is in the range of 0% to 1.97%, and the relative peak area is in the range of 0% to 2.77%. The experimental results show that the relative retention time RSD value is less than or equal to 2%, and the relative retention time of the characteristic spectrum method between different instruments meets the analysis requirements.

[0190] Example 4

[0191] This example is used to investigate the durability of the characteristic spectrum detection method of palm charcoal formula granules.

[0192] (I) Different flow rate investigation

[0193] The test sample solution of palm charcoal formula granules was prepared according to the preparation method of the test sample solution in Example 1, the influence of 0.20 ml / min, 0.25 ml / min, 0.30 ml / min, 0.35 ml / min and 0.40 ml / min flow rates on the characteristic spectrum was investigated respectively, and the relative retention time was calculated. The test results are shown in Table 23 and Table 24. Figure 23

[0194] Table 23 Relative retention time table of palm charcoal formula granules at different flow rates

[0195]

[0196] Table 24 Relative peak area table of palm charcoal formula granules at different flow rates

[0197]

[0198] According to the experimental results of different flow rates, when the flow rate is 0.25 ml / min to 0.40 ml / min, the relative retention time of each characteristic peak is within the specified value range, and the RSD of the relative peak area obtained by each flow rate is in the range of 0 to 21.839%, which indicates that the present application has good durability to different flow rates, and does not require the relative peak area.

[0199] (II) Different acid concentration investigation

[0200] The test sample solution of palm charcoal formula granules was prepared according to the preparation method of the test sample solution in Example 1, the influence of 0.08 vol% formic acid aqueous solution, 0.10 vol% formic acid aqueous solution and 0.12 vol% formic acid aqueous solution (mobile phase B) concentration on the characteristic spectrum was investigated respectively, and the relative retention time was calculated. The test results are shown in Table 23 and Table 24. Figure 24 ​and Table 25, Table 26.

[0201] Table 25 Relative retention time table of palm charcoal formula granules at different acid concentrations

[0202]

[0203]

[0204] Table 26 Relative peak area table of palm charcoal formula granules at different acid concentrations

[0205]

[0206] According to the above experimental results of different acid concentrations, the relative retention time of each characteristic peak is within the range of ± 10% of the specified value, and the RSD of the relative peak area is within the range of 0-10.05%, and the method has good durability for different acid concentrations.

[0207] (Three) Investigation of different column temperatures

[0208] Prepare the test solution of palm charcoal formula granules according to the preparation method of the test solution in Example 1, respectively investigate the influence of formic acid at 28°C, 30°C and column temperature at 32°C on the characteristic spectrum, and calculate the relative retention time. The test results are as follows Figure 25 and Table 27, Table 28.

[0209] Table 27 Relative retention time table of palm charcoal formula granules at different column temperatures

[0210]

[0211] Table 28 Relative peak area table of palm charcoal formula granules at different column temperatures

[0212]

[0213]

[0214] According to the above experimental results of different column temperatures, the relative retention time of each characteristic peak is within the range of ± 10% of the specified value, and the RSD of the relative peak area is within the range of 0-13.54%, and the method has good durability for different column temperatures.

[0215] (Four) Investigation of different chromatographic columns

[0216] The palm charcoal formula granules test sample solution was prepared according to the preparation method of the test sample solution in Example 1, the effects of different chromatographic columns (Waters CORTECS UPLC C18 chromatographic column, Waters ACQUITY UPLC BEH C18, and Waters ACQUITY UPLC HSS T3) on the characteristic spectrum were investigated respectively, and the relative retention times were calculated. The test results are shown in Table 28 and Table 29. Figure 26 and Table 29.

[0217] Table 29 Relative retention times of palm charcoal formula granules on different chromatographic columns

[0218]

[0219] According to the test results of the investigation of different chromatographic columns, the relative retention times of each characteristic peak obtained by the Waters CORTECS UPLC C18 chromatographic column and the Waters ACQUITY UPLC BEH C18 chromatographic column were not within the range of ± 10% of the specified value. Therefore, in order to ensure the accuracy of the determination results of the characteristic spectrum, the chromatographic column model was specified as Waters ACQUITY UPLC HSS T3.

[0220] Obviously, the above examples are only examples for the purpose of clarity, and are not limitations on the embodiments. Based on the above description, other different forms of changes or variations can also be made by those of ordinary skill in the art. It is not necessary and impossible to exhaust all the embodiments. 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 pattern of a palm charcoal preparation, characterized by, It comprises the following steps: Preparation of the test sample solution; the test sample is palm charcoal preparation, and the palm charcoal preparation is palm charcoal formula granules; the preparation process of the test sample solution is as follows: taking the test sample, grinding, accurately weighing, adding solvent, weighing, extracting and processing, taking out, cooling, re-weighing, supplementing the lost weight with the corresponding solvent, shaking, filtering, and taking the filtrate, to obtain the test sample solution; the solvent in the test sample solution is 50% or less of methanol aqueous solution or water by volume fraction; Preparation of the control sample solution: using 5-hydroxymethyl furfural, protocatechuic acid, protocatechuic aldehyde, and 4-hydroxybenzoic acid to prepare the control sample solution with a solvent; The test sample solution and the control sample solution are detected by ultra-high performance liquid chromatography, and the characteristic spectrum of the palm charcoal preparation is constructed according to the liquid chromatogram of the test sample solution and the control sample spectrum; the characteristic spectrum of the test sample comprises characteristic peaks of peaks 1-11; wherein, the retention time of peaks 1, 2, 3, and 4 is consistent with that of the control sample spectrum of 5-hydroxymethyl furfural, protocatechuic acid, protocatechuic aldehyde, and 4-hydroxybenzoic acid, respectively; The detection conditions of the ultra-high performance liquid chromatography comprise: a Waters ACQUITY UPLC HSS T3 chromatographic column, a detection wavelength of 285-295 nm, and acetonitrile as the mobile phase A and 0.08-0.12% formic acid aqueous solution as the mobile phase B; The elution is performed according to the following gradient program:

2. The feature map detection method of claim 1, wherein, In the detection conditions of the ultra-high performance liquid chromatography, the column length of the chromatographic column is 100 mm, the inner diameter is 2.1 mm, and the particle size is 1.8 μm; And / or, the column temperature is 28-32 ℃; And / or, the flow rate is 0.25 ml / min-0.40 ml / min; And / or, the injection amount is 1-10 μl; And / or, the theoretical plate number calculated according to the protocatechuic acid peak should be not less than 10,000.

3. The feature map detection method of claim 2, wherein, The column temperature is 30 ℃; And / or, the flow rate is 0.3 ml / min; And / or, the injection amount is 2 μl.

4. The feature map detection method of claim 1, wherein, The solvent in the test sample solution is water; And / or, the extraction treatment is ultrasonic treatment or / and hot reflux treatment; And / or, the addition amount of the test sample relative to 10 ml of the solvent is 0.1-0.4 g; And / or, the duration of the extraction treatment is 10-40 min.

5. The feature map detection method of claim 4, wherein, The extraction treatment is ultrasonic treatment; And / or, the addition amount of the test sample relative to 10 ml of the solvent is 0.2 g; And / or, the duration of the extraction treatment is 20 min.

6. The feature map detection method of claim 1, wherein, The preparation process of the control sample solution is as follows: taking 5-hydroxymethyl furfural, protocatechuic acid, protocatechuic aldehyde, and 4-hydroxybenzoic acid, accurately weighing, and adding a solvent to prepare a 5-hydroxymethyl furfural solution containing 10 μg per 1 ml, a protocatechuic acid solution containing 20 μg per 1 ml, a protocatechuic aldehyde solution containing 50 μg per 1 ml, and a 4-hydroxybenzoic acid solution containing 300 μg per 1 ml; And / or, the solvent in the control sample solution is water.

7. The feature map detection method of claim 1, wherein, Taking peak 2 as the S peak, the relative retention times of peaks 1, 3-11 are calculated, and the specified values of the relative retention times of peaks 1-11 are as follows: the relative retention times of the characteristic peaks should be within ±10% of the specified values.

8. The use of the characteristic pattern detection method of the palm charcoal preparation according to any one of claims 1-7 in the quality detection of the palm charcoal preparation.

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