Establishment method of fingerprint spectrum of Chinese traumatic injury pills

Abstract

The invention discloses a method for establishing a fingerprint spectrum of Chinese traumatic injury pills. The method comprises the following steps: preparing a reference solution, preparing a test solution, and obtaining the fingerprint spectrum of the Chinese traumatic injury pill preparation by adopting a high performance liquid chromatography. Wherein in the chromatographic condition and system applicability test, octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A and a 0.1% formic acid solution is used as a mobile phase B for gradient elution, the detection wavelength is 254 nm, and the number of theoretical plates is not less than 10000 according to a geniposide peak. The method can be used for quality control of the Chinese traumatic injury pills, the specificity and effectiveness of quality control of the Chinese traumatic injury pills can be remarkably improved, and safety and reliability of clinical medication are guaranteed.

Description

Technical Field

[0001] The invention belongs to the technical field of medical detection, and specifically relates to a method for establishing a fingerprint spectrum of Chinese Dieda Pills. Background Art

[0002] Zhonghua Dieda Pills is an exclusive product of Guangxi Wuzhou Pharmaceutical (Group) Co., Ltd., and is prepared using 32 kinds of Chinese medicine. Zhonghua Dieda Pills has a large variety of prescription medicinal materials and complex active ingredients. It is relatively difficult to detect the ingredients of the medicinal materials, which increases the difficulty of quality control. Traditional Chinese medicine theory emphasizes the overall effect of Chinese medicine and attaches importance to the synergistic effect in efficacy. If one or two active ingredients in Chinese medicine are used as qualitative and quantitative indicators, it is far from being able to effectively control and evaluate the quality of Chinese medicine. For example, the quality control method of Zhonghua Dieda Pills in the "Chinese Pharmacopoeia" (2020 edition) is like this. Overall, it is difficult to reflect its safety and effectiveness. In recent years, Chinese medicine fingerprints and characteristic fingerprints have been widely used in quality control. Among them, the characteristic fingerprint of Chinese medicine refers to the selection of several highly specific chromatographic peaks or chromatographic peak combination characteristic fingerprints from the fingerprint of Chinese medicine, and the quality of Chinese medicine is monitored by the presence and change of characteristic fingerprint peaks. At present, there is no relevant literature report on the fingerprint of Zhonghua Dieda Pills. It can be seen that exploring a method for establishing a fingerprint spectrum of Zhonghua Dieda Pills and improving the specificity and effectiveness of the quality control of the drug by selecting several chromatographic peaks with strong specificity or chromatographic peak combination characteristic fingerprints is a technical problem that needs to be solved. Summary of the invention

[0003] The present invention aims to provide a method for establishing a fingerprint spectrum of Zhonghua Dieda Pills, aiming to improve the specificity and effectiveness of the quality control of Zhonghua Dieda Pills by selecting a number of chromatographic peaks with strong specificity or chromatographic peak combination characteristic fingerprints.

[0004] The technical solution of the present invention is to provide a method for establishing a fingerprint spectrum of Zhonghua Dieda Pills, characterized in that the method comprises the following steps: 1. Preparation of reference solution: Take an appropriate amount of Gardenia glycoside reference substance, weigh accurately, and add methanol to make a solution containing 0.1 mg of Gardenia glycoside per 1 ml. 2. Preparation of test solution: Cut up the Zhonghua Dieda Pills (Xiao Mi Pills), take about 18 g, weigh accurately, put into a stoppered conical flask, accurately add 150 ml of methanol, weigh the weight, ultrasonicate for 30 min, let cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure 50 ml of the filtrate, concentrate to dryness under reduced pressure, add an appropriate amount of methanol to dissolve the residue, transfer to a 5 ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate; 3. Determination: Accurately pipette 10 µl of the reference solution and the test solution into the HPLC, perform the determination according to the following chromatographic conditions, record the chromatogram, and obtain the fingerprint of Zhonghua Dieda Pills.

[0005] Preferably, the chromatographic conditions are: the chromatographic column is filled with octadecylsilane bonded silica gel (4.6 mm×250 mm, 5 µm), acetonitrile is used as mobile phase A and 0.1% formic acid solution is used as mobile phase B for gradient elution. The gradient elution procedure is as follows, wherein the proportions of the mobile phases are all in volume percentage:

[0006] Preferably, the flow rate is 1.0 ml / min, the column temperature is 25°C, the detection wavelength is 254 nm, and the theoretical plate number, calculated based on the gardenia glycoside peak, should not be less than 10,000.

[0007] Preferably, the method for establishing the fingerprint spectrum of the Chinese Dieda Pills further comprises: comparing multiple Chinese Dieda Pills fingerprint spectrum, selecting common characteristic peaks, and obtaining the characteristic fingerprint spectrum of the Chinese Dieda Pills.

[0008] Preferably, the method for establishing the fingerprint of Zhonghua Dieda Pills is characterized in that the fingerprint or characteristic fingerprint of Zhonghua Dieda Pills contains 10 characteristic peaks, and the retention time of each characteristic peak is: Peak 1: 22.038 min, Peak 2: 28.976 min, Peak 3: 29.614 min, Peak 4: 33.672 min, Peak 5: 41.515 min, Peak 6: 44.71 min, Peak 7: 53.386 min, Peak 8: 65.777 min, Peak 9: 78.672 min, Peak 10: 89.943 min.

[0009] Preferably, the method for establishing the fingerprint of Zhonghua Dieda Pills is characterized in that, in the fingerprint or characteristic fingerprint of Zhonghua Dieda Pills, peak 1 is the gardenia glycoside control peak, and the relative retention time of each characteristic peak is: peak 1: 1, peak 2: 1.315, peak 3: 1.344, peak 4: 1.528, peak 5: 1.884, peak 6: 2.029, peak 7: 2.422, peak 8: 2.985, peak 9: 3.57, peak 10: 4.081.

[0010] The present invention also provides a method for identifying Chinese Dieda Pills, which comprises comparing the fingerprint map or characteristic fingerprint map of the sample to be tested established according to the above method with the standard fingerprint map or characteristic fingerprint map established according to the above method to identify the authenticity of the sample.

[0011] Quality control of Zhonghua Dieda Pills: The chromatogram of the test sample should show 10 chromatographic peaks, one of which should be consistent with the retention time of the reference peak of the reference substance and have a common peak match. According to the similarity evaluation system of the chromatographic fingerprint of traditional Chinese medicine, the fingerprint of the test sample and the reference fingerprint are calculated by similarity. The similarity of the chromatographic peak after 10 minutes is not less than 0.88, and the Zhonghua Dieda Pills are of qualified quality. The present invention treats Zhonghua Dieda Pills as a whole, and by comparing its characteristic peaks, the subtle differences between different batches of products can be found, which is suitable for the identification and control of the quality of Zhonghua Dieda Pills.

[0012] The beneficial technical effects of the present invention are: 1. The present invention adopts the fingerprint spectrum of Zhonghua Dieda Pills as a quality control method for Zhonghua Dieda Pills preparations, which not only avoids the one-sidedness of judging the overall quality of the preparation by only measuring one or two chemical components in the prior art, but also reduces the possibility of artificial control to achieve quality goals.

[0013] 2. In the process of determining the fingerprint spectrum of Zhonghua Dieda Pills by high performance liquid chromatography, the present invention reduces the gradient peaks caused by gradient changes through the selection of mobile phase, separation conditions and parameters, so that the target peaks are well separated from the impurity peaks, and at the same time obtains a good response and a relatively stable retention time, thereby obtaining a good characteristic spectrum.

[0014] 3. The present invention provides an effective method for completely and accurately evaluating the quality of Zhonghua Dieda Pills, thereby ensuring the quality of the product and ensuring the safety, effectiveness and reliability of clinical medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the fingerprint of 10 characteristic peaks of 15 batches of Chinese Dieda Pills samples in Example 1.

[0016] Figure 2 This is the fingerprint of Zhonghua Dieda Pills in Example 2, wherein Peak 1 (S peak) is the Gardenia glycoside peak. Implementation

[0017] The following is further described with reference to examples, but the present invention is not limited to these examples.

[0018] Example 1: Experiment on establishing the HPLC characteristic spectrum of Zhonghua Dieda Pills.

[0019] 1. Chromatographic conditions and system suitability test: The chromatographic column was filled with octadecylsilane bonded silica gel (ZORBAX Eclipse XDB-C18, 4.6 mm×250 mm, 5 µm), acetonitrile was used as mobile phase A and 0.1% formic acid solution was used as mobile phase B for gradient elution. The flow rate was 1.0 ml / min, the column temperature was 25 °C, the detection wavelength was 254 nm, and the theoretical plate number was calculated based on the gardenia glycoside peak and should not be less than 10,000. The gradient elution procedure is as follows, where the mobile phase ratios are all volume percentages:

[0020] 2. Preparation of reference solution: Take an appropriate amount of Gardenia glycoside reference substance, weigh it accurately, and add methanol to make a solution containing 0.1 mg of Gardenia glycoside per 1 ml.

[0021] 3. Preparation of test solution: Cut up Zhonghua Dieda Pills (small honey pills), take about 18 g, accurately weigh, place in a stoppered conical flask, accurately add 150 ml of methanol, weigh, ultrasonically treat for 30 min, cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure 50 ml of the filtrate, concentrate to dryness under reduced pressure, add appropriate amount of methanol to the residue to dissolve, transfer to a 5 ml volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate.

[0022] 4. Establishment of the standard fingerprint of Zhonghua Dieda Pills Fifteen batches of Zhonghua Dieda Pills (small honey pills) were taken (provided by Guangxi Wuzhou Pharmaceutical (Group) Co., Ltd.) and prepared as test samples according to the preparation method of the test sample solution. Under the above-mentioned liquid chromatography conditions, 10 µl of the reference solution and the test sample solution were accurately aspirated and injected into the liquid chromatograph. The fingerprints of the 15 batches of Zhonghua Dieda Pills (small honey pills) were tested and the relevant results were calculated using the fingerprint similarity evaluation software to generate the control fingerprint, as shown in Figure 2. Figure 1 The results show that the similarity of the chromatograms of the 15 batches of samples after 10 minutes is between 0.946 and 0.999. At the same time, the typical chromatographic peaks that appear in the fingerprints of each batch of test samples are taken as the common fingerprint peaks, and there are 10 common peaks after 10 minutes.

[0023] Based on the fingerprints of 15 batches of samples, a characteristic spectrum was formulated: 10 characteristic peaks should be presented in the characteristic spectrum of the test sample, and one of the peaks should be consistent with the retention time of the corresponding reference peak. The reference peak of gardenia glycoside was taken as peak S, and the relative retention time of each characteristic peak and peak S was calculated. The results showed that the RSD of the relative retention time of each common characteristic peak was less than 2%, which met the analysis requirements of the fingerprint spectrum. According to the standard fingerprint spectrum, the relative retention time values ​​of the common characteristic peaks were: peak 1: 1, peak 2: 1.315, peak 3: 1.344, peak 4: 1.528, peak 5: 1.884, peak 6: 2.029, peak 7: 2.422, peak 8: 2.985, peak 9: 3.57, peak 10: 4.081. Among the 10 characteristic peaks, the retention time of peak 1 was consistent with that of the peak corresponding to gardenia glycoside.

[0024] Example 2: Establishment of HPLC characteristic spectrum of Zhonghua Dieda Pills.

[0025] Zhonghua Dieda Pills (small honey pills) were provided by Guangxi Wuzhou Pharmaceutical (Group) Co., Ltd.

[0026] 1. Chromatographic conditions and system suitability test: The chromatographic column was filled with octadecylsilane bonded silica gel (ZORBAX Eclipse XDB-C18, 4.6 mm×250 mm, 5 µm), acetonitrile was used as mobile phase A and 0.1% formic acid solution was used as mobile phase B for gradient elution. The flow rate was 1.0 ml / min, the column temperature was 25 °C, the detection wavelength was 254 nm, and the theoretical plate number was calculated based on the gardenia glycoside peak and should not be less than 10,000. The gradient elution procedure is as follows, where the mobile phase ratios are all volume percentages:

[0027] 2. Preparation of reference solution: Take an appropriate amount of Gardenia glycoside reference substance, weigh it accurately, and add methanol to make a solution containing 0.1 mg of Gardenia glycoside per 1 ml.

[0028] 3. Preparation of test solution: Cut up Zhonghua Dieda Pills (small honey pills), take about 18 g, accurately weigh, place in a stoppered conical flask, accurately add 150 ml of methanol, weigh, ultrasonically treat for 30 min, cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure 50 ml of the filtrate, concentrate to dryness under reduced pressure, add appropriate amount of methanol to the residue to dissolve, transfer to a 5 ml volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate.

[0029] 4. Determination: Accurately pipette 10 µl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and record the chromatogram.

[0030] The characteristic spectrum of the test sample should show 10 characteristic peaks, one of which should have the same retention time as the corresponding reference peak. Figure 2 As shown in the figure, the retention time of each characteristic peak is: peak 1: 22.038 min, peak 2: 28.976 min, peak 3: 29.614 min, peak 4: 33.672 min, peak 5: 41.515 min, peak 6: 44.71 min, peak 7: 53.386 min, peak 8: 65.777 min, peak 9: 78.672 min, peak 10: 89.943 min. The relative retention time of each characteristic peak and peak S was calculated with the reference peak of gardenia glycoside as peak S. The results showed that the RSD of the relative retention time of each common characteristic peak was less than 2%, which met the requirements of fingerprint analysis. According to the standard fingerprint, the relative retention time values ​​are peak 1: 1, peak 2: 1.315, peak 3: 1.344, peak 4: 1.528, peak 5: 1.884, peak 6: 2.029, peak 7: 2.422, peak 8: 2.985, peak 9: 3.57, peak 10: 4.081. Among the 10 characteristic peaks, the retention time of peak 1 is consistent with that of the gardenia glycoside reference peak.

[0031] Although the present invention has been described in detail above by general description, specific embodiments and tests, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention belong to the scope of protection claimed by the present invention.

Claims

1. A method for establishing a fingerprint spectrum of Zhonghua Dieda Pills, characterized in that: The method comprises the following steps: (1) Preparation of reference solution: Take an appropriate amount of Gardenia glycoside reference substance, weigh it accurately, and add methanol to make a solution containing 0.1 mg of Gardenia glycoside per 1 ml. (2) Preparation of test solution: Cut up the Zhonghua Dieda Pills (Xiao Mi Pills), take about 18 g, weigh accurately, place in a stoppered conical flask, add 150 ml of methanol accurately, weigh the weight, ultrasonicate for 30 min, let cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure 50 ml of the filtrate, concentrate to dryness under reduced pressure, add an appropriate amount of methanol to dissolve the residue, transfer to a 5 ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate. (3) Determination: Accurately pipette 10 µl of the reference solution and the test solution into a high performance liquid chromatograph, perform determination according to the following chromatographic conditions, record the chromatogram, and obtain the fingerprint of Zhonghua Dieda Pills. The chromatographic conditions include: the chromatographic column is filled with octadecylsilane bonded silica gel (4.6 mm×250 mm, 5 µm), acetonitrile is used as mobile phase A and 0.1% formic acid solution is used as mobile phase B for gradient elution, the flow rate is 1.0 ml / min, the column temperature is 25 °C, the detection wavelength is 254 nm, and the theoretical plate number is calculated based on the Gardenia glycoside peak, which should not be less than 10,000. The gradient elution procedure is as follows, where the mobile phase ratios are all volume percentages:

2. The method according to claim 1, characterized in that The method further comprises: comparing a plurality of Chinese Dieda Pills fingerprint spectra, selecting common characteristic peaks, and obtaining a characteristic fingerprint spectrum of Chinese Dieda Pills.

3. The method according to claim 2, characterized in that The fingerprint spectrum of the Chinese Dieda Pills or the characteristic fingerprint spectrum of the Chinese Dieda Pills contains 10 characteristic peaks, and the retention time of each characteristic peak is: peak 1: 22.038 min, peak 2: 28.976 min, peak 3: 29.614 min, peak 4: 33.672 min, peak 5: 41.515 min, peak 6: 44.71 min, peak 7: 53.386 min, peak 8: 65.777 min, peak 9: 78.672 min, peak 10: 89.943 min.

4. The method according to claim 3, characterized in that In the fingerprint spectrum of Zhonghua Dieda Pills or the characteristic fingerprint spectrum of Zhonghua Dieda Pills, peak 1 is the gardenia glycoside reference peak.

5. A method for identifying Zhonghua Dieda Pills, the method comprising comparing the fingerprint of the sample to be tested established according to the method of claim 1 with the standard fingerprint established according to the method of claim 1.

6. A method for identifying Zhonghua Dieda Pills, comprising comparing a characteristic fingerprint of a sample to be tested established according to the method described in any one of claims 2 to 4 with a characteristic fingerprint established according to the method described in any one of claims 2 to 4.

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