Pharmaceutical composition as well as construction method and detection method of characteristic chromatogram of pharmaceutical composition

Abstract

The invention provides a pharmaceutical composition and a construction method of a characteristic spectrum of the pharmaceutical composition. The characteristic peak in the characteristic spectrum constructed by the method is used as a measurement index, the pharmaceutical composition can be accurately identified, the component content of the pharmaceutical composition can be determined, the specificity is strong, and the quality control of the pharmaceutical composition is facilitated. Moreover, the construction method is simple and convenient to operate, has the advantages of good reproducibility, high precision, strong stability and the like, and is suitable for popularization and application.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a pharmaceutical composition in the field of traditional Chinese medicine and a construction method and a detection method of its characteristic spectrum. Background Art

[0002] For some products in the field of traditional Chinese medicine, the improvement and improvement of their preparation process may have an impact on product quality, including properties, physical and chemical identification, thin layer identification, routine inspection, content determination, etc. The quality standards of different batches of products need to be unified and need to be further improved. Therefore, establishing a comprehensive and quantifiable comprehensive ingredient quality monitoring method is important for improving product quality stability and consistency. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention proposes a method for constructing a characteristic spectrum of a pharmaceutical composition, a method for identifying the pharmaceutical composition, a method for detecting the pharmaceutical composition, and a method for quality control of the pharmaceutical composition. The characteristic peaks in the characteristic spectrum of the pharmaceutical composition constructed by the method of the present application are used as measurement indicators to accurately identify the pharmaceutical composition and determine the component content of the pharmaceutical composition. It has strong specificity and is helpful for quality control of the pharmaceutical composition. In addition, the construction method is simple to operate, has the advantages of good reproducibility, high precision and strong stability, and is suitable for promotion and application.

[0004] In one aspect of the present invention, the present invention proposes a method for constructing the pharmaceutical composition, characterized in that, according to an embodiment of the present invention, the method comprises: providing a plurality of different batches of pharmaceutical composition samples; pre-treating each sample under the same conditions to obtain a plurality of test solutions; performing high performance liquid chromatography on the plurality of test solutions under the same conditions to obtain a plurality of chromatograms; determining common characteristic peaks by comparing and analyzing the plurality of chromatograms to obtain a characteristic spectrum of the pharmaceutical composition; wherein the mobile phase used in the high performance liquid chromatography detection comprises: mobile phase A, wherein the mobile phase A is 0.05 to 0.15 mass % Phosphoric acid solution; mobile phase B, wherein the mobile phase B is acetonitrile; the elution mode of the mobile phase is gradient elution; wherein the pharmaceutical composition comprises an active ingredient, and the active ingredient is made from the following Chinese medicinal raw materials: Poria, Pinellia, Citrus aurantium, Citrus aurantium, Citrus aurantium, Citrus aurantium, Amomum villosum, Cardamom, Areca nut, Albizzia julibrissin, Ligusticum chuanxiong, Perilla frutescens, Ginger, Periostracum cicadae, Oroxylum indicum, and Magnolia officinalis; preferably, the weight ratio of any one of the raw materials selected from Poria, Pinellia, Citrus aurantium, Citrus aurantium, Citrus aurantium, Citrus aurantium, Citrus aurantium, Amomum villosum, Areca nut, Albizzia julibrissin, Ligusticum chuanxiong, Perilla frutescens, Periostracum cicadae, Oroxylum indicum, and Magnolia officinalis to cardamom is 2:1, and the weight ratio of cardamom to ginger is (3.37-3.38):1. Specifically, in the pharmaceutical composition, the active ingredient is made of the following traditional Chinese medicine raw materials in the following weight percentages: based on the total weight of the traditional Chinese medicine raw materials, 6.826% Poria cocos, 6.826% Pinellia ternata, 6.826% Citrus aurantium, 6.826% Citrus aurantium, 6.826% Citrus aurantium, 6.826% Citrus aurantium, 6.826% Amomum villosum, 3.413% Cardamom, 6.816% Areca catechu, 6.826% Albizzia julibrissin bark, 6.826% Ligusticum chuanxiong, 6.826% Perilla frutescens stem, 1.023% ginger, 6.826% Periostracum cicadae, 6.826% Oroxylum bungeana, and 6.826% Magnolia officinalis.

[0005] The inventors of the present invention used high performance liquid chromatography to detect the pharmaceutical composition and found that the composition and elution conditions of the mobile phase would significantly affect the separation effect. When gradient elution was performed using 0.05-0.15 mass % phosphoric acid solution as mobile phase A and acetonitrile as mobile phase B, more chromatographic peaks with high separation and good peak shape could be obtained.

[0006] Furthermore, the inventors performed the same pretreatment and high-performance liquid chromatography detection on multiple different batches of samples, and determined 11 common characteristic peaks by comparing and analyzing the multiple chromatograms obtained. These 11 characteristic peaks constitute the characteristic spectrum of the pharmaceutical composition, which can be used to identify the pharmaceutical composition, determine the component content of the pharmaceutical composition, and achieve quality control. In addition, the method is simple to operate, has the advantages of good reproducibility, strong specificity, high precision and strong stability, and is suitable for promotion and application.

[0007] According to a specific embodiment of the present invention, the elution mode of the mobile phase is as follows:

[0008] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～15 90～80 10～20 15～25 80～70 20～30 25～35 70～45 30～55 35～45 45～35 55～65 45～60 35 65

[0009] The inventors obtained the above-mentioned preferred elution mode and flow rate through a large number of experiments, thereby all common characteristic peaks can be detected simultaneously, and a characteristic spectrum with high separation and good peak shape can be obtained.

[0010] According to a specific embodiment of the present invention, the flow rate of the mobile phase used in the high performance liquid chromatography detection is 0.8-1.2 mL / min, the column temperature is 25-35°C, the wavelength is 280-285 nm, and the chromatographic column is selected from a C18 chromatographic column, preferably a Welch Ultimate@XB-C18 column. The inventors obtained the above-mentioned preferred chromatographic conditions through a large number of experiments, thereby all common characteristic peaks can be detected simultaneously, and a characteristic spectrum with high separation and good peak shape can be obtained.

[0011] According to a specific embodiment of the present invention, the high performance liquid chromatography detection conditions include: using octadecyl bonded silica gel as a filler, using 0.1 mass % phosphoric acid as mobile phase A, acetonitrile as mobile phase B, gradient elution according to the provisions in the following table, flow rate of 1.0 mL per minute, equilibration for 10 minutes before injection, column temperature of 30°C; detection wavelength of 283 nm. The separation degree of the fifth characteristic peak and the adjacent chromatographic peak should be greater than 1.5.

[0012] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～15 90～80 10～20 15～25 80～70 20～30 25～35 70～45 30～55 35～45 45～35 55～65 45～60 35 65

[0013] According to a specific embodiment of the present invention, the method further comprises: under the same conditions, respectively subjecting a reference solution and a plurality of the test solution to high performance liquid chromatography detection, wherein the reference solution is selected from Solution, Solution, solution or a mixture of the three; specifically, the preparation method of the reference solution comprises: taking An appropriate amount of reference substance was accurately weighed and dissolved in methanol to prepare a solution of about 0.1 mg per 1 mL, which served as the reference solution.

[0014] According to a specific embodiment of the present invention, the number of batches of the plurality of different batches is at least 5 (eg, 5, 6, 7, 8, 9 or 10), so as to ensure the accuracy of the constructed characteristic spectrum.

[0015] According to a specific embodiment of the present invention, the pretreatment comprises: mixing the pharmaceutical composition with methanol and then performing ultrasonic treatment, supplementing the weight of the resulting mixed solution with methanol, shaking, filtering, collecting the filtrate, and obtaining a test solution. The inventors obtained the above-mentioned preferred pretreatment conditions through a large number of experiments, thereby effectively extracting the characteristic components corresponding to the common characteristic peaks with few impurities, thereby helping to obtain a characteristic spectrum with high separation and good peak shape.

[0016] According to a specific embodiment of the present invention, the ultrasonic treatment is carried out at a power of 220 to 280 W and a frequency of 35 to 45 kHz for 5 to 30 minutes, so as to fully extract the components of the pharmaceutical composition of the present invention.

[0017] According to a specific embodiment of the present invention, the pretreatment comprises: grinding the pharmaceutical composition, taking 0.5 g of fine powder, accurately weighing, placing in a stoppered conical flask, accurately adding 50 mL of methanol, weighing, ultrasonically treating (250 W, 40 KHz) for 20 minutes, bringing to room temperature, weighing again, supplementing the lost weight with methanol, shaking, filtering, and taking the filtrate to obtain.

[0018] According to a specific embodiment of the present invention, the concentration of methanol is 90-100% by volume, so as to fully extract the components of the pharmaceutical composition.

[0019] According to a specific embodiment of the present invention, the characteristic spectrum includes 11 common characteristic peaks, with the fifth characteristic peak as the reference peak, and the ratios of the relative retention times of the 11 common characteristic peaks to the retention time of the reference peak are 0.15±8.0%, 0.58±8.0%, 0.93±8.0%, 0.98±8.0%, 1.00, 1.13±8.0%, 1.15±8.0%, 1.27±8.0%, 1.41±8.0%, 1.76±8.0%, and 1.88±8.0%, respectively.

[0020] Specifically, see Figure 1 In the characteristic spectrum, the fifth characteristic peak is used as the reference peak, and the substance corresponding to the fifth characteristic peak has the following structure:

[0021]

[0022] The substance corresponding to the fourth characteristic peak has the following structure:

[0023]

[0024] The substance corresponding to the tenth characteristic peak has the following structure:

[0025]

[0026] The substance corresponding to the eleventh characteristic peak has the following structure:

[0027]

[0028] The relative retention times of the other characteristic peaks are as follows: the relative retention time of peak 1 is 0.15±8.0%, the relative retention time of peak 2 is 0.58±8.0%, the relative retention time of peak 3 is 0.93±8.0%, the relative retention time of peak 4 is 0.98±8.0%, the relative retention time of peak 6 is 1.13±8.0%, the relative retention time of peak 7 is 1.15±8.0%, the relative retention time of peak 8 is 1.27±8.0%, the relative retention time of peak 9 is 1.41±8.0%, the relative retention time of peak 10 is 1.76±8.0%, and the relative retention time of peak 11 is 1.88±8.0%.

[0029] In another aspect of the present invention, the present invention proposes a method for identifying the pharmaceutical composition of the present invention, characterized in that it comprises: pre-treating the sample to be tested to obtain a sample solution; performing high performance liquid chromatography on the sample solution to obtain a chromatogram; analyzing the chromatogram with the characteristic spectrum obtained by the above method to determine whether the sample to be tested is the pharmaceutical composition of the present invention; the detection conditions of the high performance liquid chromatography are as defined in the method for constructing the characteristic spectrum. Thus, the pharmaceutical composition of the present invention can be accurately identified by using the identification method according to the present invention.

[0030] According to a specific embodiment of the present invention, the preprocessing is as defined in the method for constructing the characteristic map described above.

[0031] According to a specific embodiment of the present invention, if the similarity between the chromatogram and the characteristic spectrum is not less than 0.9, the sample to be tested is identified as the pharmaceutical composition of the present invention. Specifically, the similarity is obtained by using the fingerprint similarity evaluation software of the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" V2.0 developed by the Chinese Pharmacopoeia Committee.

[0032] It should be noted that the features and advantages described above for the method for constructing the characteristic spectrum are also applicable to the method for identifying the pharmaceutical composition of the present invention and will not be described in detail here.

[0033] In another aspect of the present invention, the present invention proposes a method for detecting the pharmaceutical composition of the present invention, characterized in that it includes: configuring a known concentration solution of a characteristic component corresponding to at least one characteristic peak in the characteristic spectrum obtained by the method for constructing the characteristic spectrum as a reference solution; pretreating the sample to obtain a test solution; performing high performance liquid chromatography on the reference solution and the test solution to obtain a chromatogram; analyzing the chromatogram of the reference solution and the chromatogram of the test solution to determine the content of at least one characteristic component in the sample; wherein the high performance liquid chromatography detection conditions are as defined in the method for constructing the characteristic spectrum as described above. Thus, the method of the embodiment of the present invention can accurately determine the content of characteristic components in the pharmaceutical composition of the present invention, and has the advantages of good reproducibility, strong specificity, high precision and strong stability, and is suitable for popularization and application.

[0034] According to a specific embodiment of the present invention, the method for preparing the reference solution comprises: taking

[0035]

[0036] An appropriate amount of reference substance was accurately weighed and dissolved in methanol to prepare a solution of about 0.1 mg per 1 mL, which served as the reference solution.

[0037] According to a specific embodiment of the present invention, the preprocessing is as defined in the method for constructing the characteristic map described above.

[0038] It should be noted that the features and advantages described above for the method for constructing the characteristic spectrum are also applicable to the method for detecting the pharmaceutical composition of the present invention and will not be described in detail here.

[0039] In another aspect of the present invention, the present invention proposes a method for quality control, characterized in that it includes: pre-treating a sample of the pharmaceutical composition to obtain a test solution; subjecting the test solution to high performance liquid chromatography to obtain a chromatogram; analyzing the chromatogram with the one obtained by the method for constructing the characteristic spectrum described above to determine whether the quality of the sample to be tested meets the standard; wherein the high performance liquid chromatography detection conditions are as defined in the method for constructing the characteristic spectrum described above. Thus, the method according to an embodiment of the present invention can accurately determine whether the sample of the pharmaceutical composition of the present invention meets the requirements, thereby effectively controlling the quality.

[0040] According to a specific embodiment of the present invention, the preprocessing is as defined in the method for constructing the characteristic map described above.

[0041] According to a specific embodiment of the present invention, if the similarity between the chromatogram and the characteristic spectrum is not less than 0.9, the quality of the pharmaceutical composition is deemed to be up to standard.

[0042] It should be noted that the features and advantages described above for the method for constructing the characteristic spectrum are also applicable to the method for quality control of the pharmaceutical composition and will not be repeated here.

[0043] In another aspect of the present invention, the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition contains an active ingredient, and the active ingredient is made from the following Chinese medicinal raw materials: Poria cocos, Pinellia ternata, Citrus aurantium, Citrus aurantium, Citrus reticulata, Citrus aurantium, Amomum villosum, Cardamom, Areca nut, Albizzia julibrissin, Atractylodes macrocephala, Perilla frutescens, Ginger, Periostracum cicadae, Oroxylum indicum, and Magnolia officinalis; preferably, the weight ratio of any one raw material selected from Poria cocos, Pinellia ternata, Citrus aurantium, Citrus aurantium, Citrus aurantium, Citrus reticulata, Citrus aurantium, Amomum villosum, Areca nut, Albizzia julibrissin, Atractylodes macrocephala, Perilla frutescens, Periostracum cicadae, Oroxylum indicum, and Magnolia officinalis to that of cardamom is 2:1, and the weight ratio of cardamom to ginger is (3.37-3.38):1. Specifically, in the pharmaceutical composition, the active ingredient is made of the following traditional Chinese medicine raw materials in the following weight percentages: based on the total weight of the traditional Chinese medicine raw materials, 6.826% Poria cocos, 6.826% Pinellia ternata, 6.826% Citrus aurantium, 6.826% Citrus aurantium, 6.826% Citrus aurantium, 6.826% Citrus aurantium, 6.826% Amomum villosum, 3.413% Cardamom, 6.826% Areca catechu, 6.826% Albizzia julibrissin bark, 6.826% Ligusticum chuanxiong, 6.826% Perilla frutescens stem, 1.023% ginger, 6.826% Periostracum cicadae, 6.826% Oroxylum indica, and 6.826% Magnolia officinalis.

[0044] According to a specific embodiment of the present invention, the pharmaceutical composition is a granule or a capsule.

[0045] According to a specific embodiment of the present invention, the similarity between the HPLC chromatogram of the pharmaceutical composition and the characteristic spectrum obtained by the method of the first aspect of the present invention is not less than 0.9. Preferably, the HPLC chromatogram of the pharmaceutical composition is obtained using the HPLC detection conditions and pretreatment operations defined in the first aspect of the present invention.

[0046] Specifically, the pharmaceutical composition has a characteristic spectrum constructed by the method of the first aspect of the present invention, which includes 11 characteristic peaks, and the substance corresponding to the fifth characteristic peak has the following structure:

[0047]

[0048] The substance corresponding to the fourth characteristic peak has the following structure:

[0049]

[0050] The substance corresponding to the tenth characteristic peak has the following structure:

[0051]

[0052] The substance corresponding to the eleventh characteristic peak has the following structure:

[0053]

[0054] Among them, taking the fifth characteristic peak as the reference peak, the ratios of the relative retention times of the 11 characteristic peaks to the retention time of the reference peak were 0.15±8.0%, 0.58±8.0%, 0.93±8.0%, 0.98±8.0%, 1.00, 1.13±8.0%, 1.15±8.0%, 1.27±8.0%, 1.41±8.0%, 1.76±8.0%, and 1.88±8.0%, respectively.

[0055] Or, specifically, in the characteristic spectrum of the pharmaceutical composition of the present invention, the relative peak areas of the 11 characteristic peaks are: 0.111, 0.058, 0.225, 0.403, 1.00, 0.248, 0.325, 0.14, 0.078, 0.057, 0.036, respectively.

[0056] In another aspect of the present invention, the present invention also provides a method for preparing the pharmaceutical composition of the present invention, which comprises (or consists of the following):

[0057] (1) Take some amount of Poria cocos, Amomum villosum, and Arisaema cinerea, crush them, sieve them, and mix them evenly;

[0058] (2) taking all the remaining thirteen herbs (areca nut, ginger, pinellia tuber, tangerine peel, julibrissin bark, cicada shell, immature bitter orange, Shenqu, oroxylum, green peel, cardamom, perilla stem, magnolia bark) and the remaining three herbs (poria, amomum villosum, and arisaema) and boiling them in water, combining the decoctions, filtering, and concentrating the filtrate;

[0059] (3) The powder obtained in step (1) and the extract obtained in step (2) are mixed and granulated.

[0060] According to a specific embodiment of the present invention, in the preparation method of the present invention, the granulation in step (3) is wet granulation (extrusion) of the mixed paste powder (i.e., the powder obtained in step (1) and the thick paste obtained in step (2)).

[0061] According to a specific embodiment of the present invention, in the preparation method of the present invention, the partial amount of Poria cocos, Amomum villosum and Arisaema ciliata in step (1) is 15% to 75% of the total amount of each medicinal material, preferably 20% to 70% of the total amount of each medicinal material, and more preferably 25% to 65% of the total amount of each medicinal material.

[0062] According to a specific embodiment of the present invention, in the preparation method of the present invention, the pulverizing in step (1) is pulverizing the medicinal material into fine powder; and / or, according to a specific embodiment of the present invention, in the preparation method of the present invention, the sieving in step (1) is sieving through a 100-120 mesh sieve.

[0063] According to a specific embodiment of the present invention, in the preparation method of the present invention, the number of decoctions in step (2) is 1-5 times, preferably 2-4 times; and / or the decoction time in step (2) is 1-5 hours / time, preferably 2-4 hours / time.

[0064] According to a specific embodiment of the present invention, in the preparation method of the present invention, the filtrate in step (2) is concentrated to a thick paste with a relative density of 1.08 to 1.13 at a temperature of 50°C-80°C, preferably concentrated to a thick paste with a relative density of 1.08 to 1.13 at a temperature of 55°C-75°C.

[0065] In another aspect of the present invention, the present invention provides use of the pharmaceutical composition of the present invention in the preparation of a medicament for treating or preventing throat diseases such as paresthesia, chronic pharyngitis, chronic laryngitis and / or laryngopharyngeal reflux disease.

[0066] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0068] Figure 1 The chromatogram of the test sample using gradient elution condition 1 in the investigation experiment of the HPLC characteristic spectrum of the pharmaceutical composition according to the embodiment of the present invention is shown;

[0069] Figure 2 The chromatogram of the test sample using gradient elution condition 2 in the investigation experiment of the HPLC characteristic spectrum of the pharmaceutical composition according to the embodiment of the present invention is shown;

[0070] Figure 3 The chromatogram of the test sample using gradient elution condition 3 in the investigation experiment of the HPLC characteristic spectrum of the pharmaceutical composition according to the embodiment of the present invention is shown;

[0071] Figure 4 The chromatogram of the test sample using gradient elution condition 5 in the investigation experiment of the HPLC characteristic spectrum of the pharmaceutical composition according to the embodiment of the present invention is shown;

[0072] Figure 5 The chromatogram of the test sample using gradient elution condition 5 in the investigation experiment of the HPLC characteristic spectrum of the pharmaceutical composition according to the embodiment of the present invention is shown;

[0073] Figure 6 The chromatogram of the test sample using gradient elution condition 6 in the investigation experiment of the HPLC characteristic spectrum of the pharmaceutical composition according to the embodiment of the present invention is shown;

[0074] Figure 7 The matching results of common characteristic peaks in characteristic spectra of different batches of pharmaceutical compositions according to the embodiments of the present invention;

[0075] Figure 8 is a solvent chromatogram according to an embodiment of the present invention;

[0076] Fig. 9 This is a characteristic peak attribution diagram of Shimadzu LC-10AVP according to an embodiment of the present invention; the characteristic peaks in the figure are from top to bottom the reference Solution and test sample solution;

[0077] Fig.10 This is a characteristic peak attribution diagram of Thermo U3000 according to an embodiment of the present invention. The characteristic peaks in the diagram are from top to bottom the reference Solution and test sample solution;

[0078] Fig.11 is a solution stability matching diagram according to an embodiment of the present invention;

[0079] Fig.12 is a precision matching diagram according to an embodiment of the present invention;

[0080] Fig.13 is a repetitive experimental matching diagram according to an embodiment of the present invention;

[0081] Fig.14 The intermediate precision Shimadzu-10AT VP HPLC (Welch XB-C18 column) chromatogram according to an embodiment of the present invention;

[0082] Fig.15 It is an intermediate precision Shimadzu-40D HPLC (Welch C18 column) chromatogram according to an embodiment of the present invention;

[0083] Fig.16Thermo U3000 HPLC (Welch Blossmate) according to the intermediate precision of the embodiment of the present invention @ C 18 ) chromatogram;

[0084] Fig.17 is a typical chromatogram of Laboratory 2 according to an embodiment of the present invention;

[0085] Fig.18 It is the characteristic peak of the fingerprint of the pharmaceutical composition according to an embodiment of the present invention. DETAILED DESCRIPTION

[0086] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0087] 1. Preparation Example

[0088] Preparation Example 1

[0089] Prescription: 50 parts of Poria, 50 parts of French Pinellia, 50 parts of Citrus aurantium (fried), 50 parts of Citrus aurantium (fried), 50 parts of Arisaema consanguineum, 50 parts of Tangerine peel, 50 parts of Amomum villosum, 25 parts of Cardamom, 50 parts of Areca catechu, 50 parts of Albizzia julibrissin bark, 50 parts of Ligusticum chuanxiong (fried), 50 parts of Perilla stem, 7.4 parts of ginger, 50 parts of Periostracum cicadae, 50 parts of Oroxylum indica, and 50 parts of Magnolia officinalis (processed).

[0090] The preparation method of the pharmaceutical composition is as follows:

[0091] (1) Take 1 / 2 amount of Poria cocos, Amomum villosum, and Arisaema cinerea, grind them into fine powder, sieve, and mix well;

[0092] (2) taking all the remaining thirteen herbs (areca nut, ginger, pinellia tuber, tangerine peel, julibrissin bark, cicada shell, immature bitter orange, Shenqu, oroxylum, green peel, cardamom, perilla stem, magnolia bark) and the remaining three herbs (poria, amomum villosum, and arisaema) and boiling them twice, each time for 2 hours, combining the decoctions, filtering, and concentrating the filtrate to an extract having a relative density of 1.08 to 1.13 at 70°C;

[0093] (3) The fine powder obtained in step (1) and the extract obtained in step (2) are mixed, wet-extruded and granulated, and dried to obtain granules, which can be loaded into capsules to obtain capsules.

[0094] 2. Experimental Examples

[0095] The test instruments, reagents, samples and reference substances used in the embodiments of the present invention are shown in Tables 1-4.

[0096] Table 1 Test instruments

[0097]

[0098]

[0099] Table 2 Test reagents

[0100] name batch number level Manufacturer Methanol 21100713G108 HPLC OCEANPAK Acetonitrile 21101027G113 HPLC OCEANPAK Phosphoric acid 20210902 HPLC Tianjin Tianli Chemical Reagent Ultrapure water / First-class water self made

[0101] Table 3 Test samples

[0102]

[0103] Table 4 Test reference substances

[0104]

[0105]

[0106] Example 1

[0107] Take 10 pellets of the 6 batches of samples in Table 3 and perform the following operations in Laboratory 1:

[0108] (1) Preparation of reference solution: Take the reference substance (also referred to herein as "Control 1") (also referred to herein as "Control 2") and / or An appropriate amount of (also referred to as "reference substance 3" in this article) was accurately weighed and dissolved in methanol to prepare a solution of 0.1 mg per 1 ml, which served as the reference solution;

[0109] (2) Preparation of test solution: Remove the capsule shell from the sample, grind the contents, take 0.5 g of fine powder, accurately weigh, place in a stoppered conical flask, accurately add 50 mL of 100% methanol, weigh, extract by ultrasonication (250 W, 40 kHz) for 20 min, let cool to room temperature, weigh again, make up the loss with 100% methanol, shake well, filter through a 0.45 μm microporous filter membrane, and take the filtrate to obtain the product.

[0110] (3) Chromatographic conditions: The chromatographic column is Welch Ultimate@XB-C18 (4.6 mm×250 mm, 5 μm), with octadecyl bonded silica as the filler, 0.1 mass% phosphoric acid as the mobile phase A, acetonitrile as the mobile phase B, gradient elution as specified in Table 5-10, flow rate of 1.0 mL per minute, equilibration for 10 minutes before injection, column temperature of 30°C, and detection wavelength of 283 nm. The resolution of the peak of reference 1 and the adjacent chromatographic peak should be greater than 1.5.

[0111] (4) Determination: Accurately pipette 10 μL of each test solution, inject into the liquid chromatograph, measure, and record the chromatogram Figure 1-6 .

[0112] There are more than 20 chromatographic peaks in the chromatogram of the test sample, and baseline separation is achieved between each chromatographic peak. The separation degree between each characteristic peak and the adjacent peak is greater than 1.5. The characteristic peaks better reflect the traceability relationship between the finished product and the medicinal material.

[0113] Table 5 Gradient elution conditions 1

[0114]

[0115] Table 6 Gradient elution conditions 2

[0116]

[0117] Table 7 Gradient elution conditions 3

[0118]

[0119] Table 8 Gradient elution conditions 4

[0120]

[0121] Table 9 Gradient elution conditions 5

[0122]

[0123] Table 10 Gradient elution conditions 6

[0124]

[0125] Example 2 Determining the characteristic spectrum

[0126] The chromatograms of the test solutions of the six batches of samples used in the experiment were analyzed and compared, and the "Chinese Medicine Fingerprint Similarity Software" 2.0 was used for calibration to determine their common characteristic peaks.

[0127] Results: More than 20 chromatographic peaks were separated in the chromatograms of the 6 batches of test solution, of which there were 11 common characteristic peaks, and the common characteristic peak area accounted for more than 85% of the total peak area. See Table 11-13, Figure 7 .

[0128] Taking the peak of reference substance 1 as the reference peak, calculate the relative retention time and peak area ratio. The calculation formula is as follows:

[0129]

[0130] Table 11 Common characteristic peak matching results

[0131]

[0132]

[0133] Table 12 Relative retention time of common characteristic peaks

[0134]

[0135]

[0136] Table 13 Common characteristic peak relative peak area

[0137]

[0138] 1. Attribution of characteristic peaks

[0139] Locate the reference solution: Take reference 1, reference 2, reference 3, (also referred to herein as "reference substance 4"), (also referred to herein as "reference substance 5"), (also referred to herein as "reference substance 6") An appropriate amount of (also referred to as "reference substance 7" in this article) was accurately weighed and dissolved in 100% methanol to prepare a solution containing approximately 0.1 mg per 1 mL, which was used as the positioning reference solution.

[0140] The sample was injected and measured under the above-mentioned chromatographic conditions to be verified (Example 1), and the peaks were compared with those in the chromatogram of the test solution (batch number: JC121030022) to attribute the characteristic peaks. The results are shown in Table 14. Figure 8-10 .

[0141] Table 14 Characteristic peak attribution

[0142]

[0143]

[0144] 2. Methodological investigation

[0145] With reference to the General Rules 9101 "Guidelines for Validation of Analytical Methods" of the 2020 edition of the Chinese Pharmacopoeia and the Technical Requirements for Fingerprint Studies of Traditional Chinese Medicine Injections, characteristic spectrum / fingerprint methodology investigations were conducted, mainly including solution stability, precision, repeatability, intermediate precision, durability and reproducibility tests. The acceptable standards for validation are mainly based on the acceptable range of the content and precision of the components to be tested in the samples of the Technical Requirements for Fingerprint Studies of Traditional Chinese Medicine Injections and the General Rules 9101 "Guidelines for Validation of Analytical Methods" of the 2020 edition of the Chinese Pharmacopoeia.

[0146] (1) Stability test

[0147] The test solution (batch number: JC121030022) was prepared according to the test solution preparation method of Example 1, 10 μL of the test solution was accurately pipetted, and placed at room temperature and protected from light for 0, 6, 12, 24, 36, and 48 h, respectively, for sampling and measurement (chromatographic conditions were the same as in Example 1), and the relative retention time and relative peak area of ​​the common characteristic peaks in the fingerprint were calculated. At the same time, the similarity evaluation was performed using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" V2.0 version to examine the stability of the test solution.

[0148] Verification requirements: The relative retention time RSD of each characteristic peak is ≤1.0%, the relative peak area RSD is ≤3.0%, and the similarity is >0.9. The test solution can be considered stable.

[0149] Verification results: The relative retention time RSD of the 11 common characteristic peaks in the test solution is ≤0.53%, the relative peak area RSD is ≤2.92% (except for peak 1), and the similarity is greater than 0.9, indicating that the test solution is stable within 48 hours. Characteristic peak 1 did not show an obvious degradation trend, which is mainly affected by the separation effect. See Table 15-16, Fig.11 .

[0150] Table 15 Solution stability similarity evaluation

[0151]

[0152]

[0153] Table 16 Solution stability test results

[0154]

[0155] (2) Precision test

[0156] The test solution (batch number: JC121030022) was prepared according to the test solution preparation method of Example 1, 10 μL of the test solution was taken, and the sample was injected 6 times continuously for measurement (chromatographic conditions were the same as in Example 1), and the relative retention time and relative peak area of ​​the common characteristic peaks in the fingerprint were calculated. At the same time, the similarity evaluation was performed using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" V2.0 version to examine the precision of the instrument.

[0157] Verification requirement: The similarity of each characteristic peak is greater than 0.9.

[0158] Verification results: Similarity> 0.9, indicating that the instrument has good precision. See Table 17, Fig.12 .

[0159] Table 17 Precision Similarity Evaluation

[0160]

[0161]

[0162] (3) Repeatability test

[0163] Take the contents of this product (batch number: JC121030022) and prepare 6 test solutions according to the preparation method of the test solution. Take 10μl of each sample for analysis, calculate the relative retention time and relative peak area of ​​the common characteristic peaks in the fingerprint spectrum, and examine the reproducibility of the experimental method. At the same time, the similarity evaluation was performed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" V2.0 version.

[0164] Verification requirements: relative retention time RSD of each characteristic peak ≤ 2.0%, relative peak area RSD ≤ 15%, similarity > 0.9.

[0165] Verification results: The RSD of the relative retention time of the common characteristic peaks in the 6 measurements was ≤0.31%, the RSD of the peak area ratio was ≤12.91%, and the similarity was >0.9, indicating that the repeatability of the determination method was good. See Table 18-19, Fig.13 .

[0166] Table 18 Repeatability similarity evaluation

[0167]

[0168] Table 19 Repeatability test results

[0169]

[0170]

[0171] (4) Intermediate precision test

[0172] The content of this product (batch number: JC121030022) was taken, and different personnel prepared the test solution according to the preparation method of the test solution in Example 1, and 10 μl was respectively injected and analyzed on Shimadzu-10AT VP, Shimadzu-40D and Thermo U3000 high performance liquid chromatographs (chromatographic conditions were the same as in Example 1), and the relative retention time and relative peak area of ​​the common characteristic peaks in the fingerprint were calculated. At the same time, the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" V2.0 version was used to perform a similarity evaluation against the repeatability test (batch number: JC121030022) to examine the intermediate precision of the experimental method.

[0173] Verification requirements: In the fingerprint spectrum, the relative deviation of the relative retention time of the common characteristic peaks is stipulated to be within 5%, the similarity is greater than 0.9, and the separation degree meets the requirements.

[0174] Verification results: The common characteristic peaks were detected by different personnel and instruments, the RSD of relative retention time was ≤3.85%, the similarity was >0.9, and each characteristic peak achieved baseline separation, indicating that the intermediate precision of the determination method was good. See Table 20. Figure 14-16 .

[0175] Table 20 Intermediate precision density similarity evaluation (Mark peak)

[0176]

[0177] (5) Reproducibility test

[0178] Six batches of samples for testing of this product were taken and measured in different laboratories, and characteristic spectra were drawn to examine the reproducibility of the method. Verification requirements: In the characteristic spectra, the relative retention time of the common characteristic peaks is specified to be within the specified range, and the separation degree meets the requirements.

[0179] Verification results: The characteristic spectra of 6 batches of samples measured by Laboratory 2 were compared with the standard characteristic spectra of Laboratory 1 ( Figure 2 ) are completely consistent, the separation meets the requirements, and the relative retention time of the characteristic peaks is completely consistent with that of peak 1, except that peak 1 is not controlled within ±8%, and the other peaks fully meet the requirements, indicating that the method reproducibility is feasible. Peak 1 has different retention times due to different retention times under different chromatographic instruments. It is recommended that the standard relative retention time be revised to 0.15. See Table 21, Fig.17 .

[0180] Table 21 Intermediate precision test results

[0181]

[0182] The common characteristic peaks of the six batches of samples used in the experiment were analyzed. Peaks 3, 5, 6, 7, 13, 16, 17, and 18 in Table 5 were not used as common characteristic peaks because of their small proportion of the total peaks, instability, and poor separation effect. Among them, the known peak 7 (reference substance 5) was weak in the chromatogram, just reaching about 10 times the noise value, and the integration interference was large, so it was not used as a common characteristic peak.

[0183] Peaks 1, 2, 8, 9, 10, 11, 12, 14, 15, 19, and 20 each account for 1.2% to 31.8% of the total peaks, and the chromatographic separation is relatively good. They are intended to be used as characteristic peaks of the sample. Fig.18 .

[0184] 3. Proposed feature map

[0185] There should be 11 characteristic peaks in the characteristic spectrum of the test sample, with the fifth characteristic peak being the S peak, and the relative retention time of each characteristic peak and the S peak should be calculated.

[0186] The relative retention times of the 11 characteristic peaks of this product are: 0.14 (peak 1), 0.58 (peak 2), 0.93 (peak 3), 0.98 (peak 4), 1.00 (peak S), 1.13 (peak 6), 1.15 (peak 7), 1.27 (peak 8), 1.41 (peak 9), 1.76 (peak 10), 1.88 (peak 11), of which peak S is Peak 4 Peak 10 Peak 11 is

[0187]

[0188] 4. Characteristic spectrum limit range

[0189] Comparing the relative retention times of 11 characteristic peaks, the deviations of measurements by different instruments were within ±5% of the mean, and the relative retention times measured by different laboratories were all within ±8% of the above-specified values ​​(except for Peak 1). Peak 1 had differences due to different retention times under different chromatographic instruments, and its relative retention time was revised to 0.15.

[0190] Based on the above results, 11 characteristic peaks should be present in the characteristic spectrum of the test sample. The relative retention time of each characteristic peak and the S peak should be calculated. The relative retention time should be within the specified value ± 8.0%. The specified values ​​are: 0.15 peak (1), 0.58 (peak 2), 0.93 (peak 3), 0.98 (peak 4), 1.00 (peak S), 1.13 (peak 6), 1.15 (peak 7), 1.27 (peak 8), 1.41 (peak 9), 1.76 (peak 10), 1.88 (peak 11), where peak S is Peak 4 Peak 10 Peak 11 is

[0191] 5. Similarity evaluation

[0192] The fingerprint test data of 6 batches of samples were imported into the traditional Chinese medicine fingerprint similarity software. After calculation by the traditional Chinese medicine fingerprint similarity software, the control fingerprint of the sample fingerprint was obtained, the similarity of the MARK peak (characteristic peak) between each batch of samples was calculated, and the similarity of each batch of samples was compared.

[0193] The results showed that characteristic peaks were detected in the fingerprint spectra of samples from different batches, and the similarity of MARK peaks (characteristic peaks) was ≥0.999, with no significant differences, as shown in Table 22.

[0194] Table 22 Similarity evaluation results of fingerprint spectra of different batches of samples

[0195]

[0196] Example 3

[0197] In this embodiment, the characteristic map is constructed according to the following method:

[0198] 1. Preparation of reference solution and test solution: same as in Example 1.

[0199] 2. Chromatographic conditions: The chromatographic column is Welch Ultimate@XB-C18 (4.6mm×250mm, 5μm), with octadecyl bonded silica as the filler, 0.05 mass% phosphoric acid as the mobile phase A, acetonitrile as the mobile phase B, and gradient elution is performed according to the provisions in Table 10, the flow rate is 0.8mL per minute, and the sample is balanced for 10 minutes before injection; the column temperature is 25°C; the detection wavelength is 283nm. The separation degree between the peak of reference substance 1 and the adjacent chromatographic peak should be greater than 1.5.

[0200] 3. Determination: Accurately pipette 10 μL of each test solution, inject into liquid chromatograph, and determine.

[0201] The maximum absorption wavelengths of the main characteristic peaks are all around 280 nm. The characteristic peaks are more obvious at 283 nm, which can better reflect the fingerprint characteristics of the components of the pharmaceutical composition of the present invention as a whole. Therefore, the detection wavelength is preferably 283 nm.

[0202] Example 4

[0203] In this embodiment, the characteristic map is constructed according to the following method:

[0204] 1. The preparation of reference solution is the same as in Example 1.

[0205] 2. Preparation of test solution: remove the capsule shell from the sample, grind the contents, take 0.5 g of fine powder, accurately weigh, place in a stoppered conical flask, accurately add 50 mL of 50% methanol, weigh the weight, ultrasonically extract (250 W, 40 kHz) for 20 minutes, let it cool to room temperature, weigh again, make up the lost weight with methanol, shake well, filter through a 0.45 μm microporous filter membrane, and take the filtrate to obtain.

[0206] 3. Chromatographic conditions: The chromatographic column is Welch Ultimate@XB-C18 (4.6mm×250mm, 5μm), with octadecyl bonded silica as the filler, 0.15 mass% phosphoric acid as the mobile phase A, acetonitrile as the mobile phase B, and gradient elution is performed according to the provisions in Table 10, the flow rate is 1.2mL per minute, and the sample is balanced for 10 minutes before injection; the column temperature is 35°C; the detection wavelength is 285nm. The resolution of the peak of reference substance 1 and the adjacent chromatographic peak should be greater than 1.5.

[0207] 4. Determination: Accurately pipette 10 μL of each test solution, inject into liquid chromatograph, measure, and record the chromatogram.

[0208] The test solutions prepared with methanol (100%) and 50% methanol had the same number of chromatographic peaks, basically similar peak height ratios, and both achieved baseline separation. There was no significant difference in characteristics, but 50% methanol contained 50% water. The test solution prepared with 100% methanol was easier to filter and had a better chromatographic peak shape than that with 50% methanol. Therefore, it was determined that 100% methanol was used as the solvent to prepare the test solution.

[0209] summary

[0210] The characteristic spectrum developed by this method is specific, and the common characteristic peaks basically reflect the main ingredients with ultraviolet absorption and known effective ingredients in the prescription. It has good reproducibility and can be used as a basis for quality control of the pharmaceutical composition, to evaluate the authenticity, excellence and stability of the preparation quality, and to compare the consistency of product quality before and after the change.

[0211] The calculation results of the similarity of the characteristic spectra of each batch of samples show that the characteristic spectra of samples from different batches are not much different, and the product quality is stable and uniform.

[0212] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0213] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for constructing a characteristic spectrum of a pharmaceutical composition, characterized in that: include: Providing a plurality of different batches of pharmaceutical composition samples; Under the same conditions, each sample was pretreated separately to obtain multiple test sample solutions; Under the same conditions, a plurality of the test sample solutions are subjected to high performance liquid chromatography to obtain a plurality of chromatograms; By comparing and analyzing a plurality of the chromatograms, common characteristic peaks are determined to obtain a characteristic spectrum of the pharmaceutical composition; Wherein, the mobile phase used in the high performance liquid chromatography detection includes: Mobile phase A, wherein the mobile phase A is a 0.05-0.15 mass % phosphoric acid solution; Mobile phase B, wherein the mobile phase B is acetonitrile; The elution mode of the mobile phase is gradient elution; Wherein, the pharmaceutical composition contains active ingredients, and the active ingredients are made from the following Chinese medicinal raw materials: Poria cocos, Pinellia ternata, Citrus aurantium, Citrus aurantium, Citrus reticulata, Citrus aurantium, Amomum villosum, Cardamom, Areca nut, Albizzia julibrissin, Ligusticum chuanxiong, Perilla frutescens, ginger, Periostracum cicadae, Oroxylum indicum, and Magnolia officinalis; preferably, the weight ratio of any one raw material selected from Poria cocos, Pinellia ternata, Citrus aurantium, Citrus aurantium, Citrus aurantium, Citrus reticulata, Citrus aurantium, Amomum villosum, Areca nut, Albizzia julibrissin, Ligusticum chuanxiong, Perilla frutescens, Periostracum cicadae, Oroxylum indicum, and Magnolia officinalis to that of cardamom is 2:1, and the weight ratio of cardamom to ginger is (3.37-3.38):

1.

2. The method according to claim 1, characterized in that The elution mode of the mobile phase is as follows: 。 3. The method according to claim 1, characterized in that The high performance liquid chromatography detection uses a mobile phase with a flow rate of 0.8 to 1.2 mL / min, a column temperature of 25 to 35° C., and a wavelength of 280 to 285 nm; and / or, the chromatographic column is selected from a C18 chromatographic column, preferably a Welch Ultimate@XB-C18 column.

4. The method according to claim 1, characterized in that: The pre-processing comprises: The pharmaceutical composition and methanol are mixed and then ultrasonically treated, the resulting mixture is supplemented with methanol to make up the weight, shaken well, filtered, and the filtrate is collected to obtain a test solution; Optionally, the ultrasonic treatment is carried out at a power of 220 to 280 W and a frequency of 35 to 45 kHz for 5 to 30 minutes; Optionally, the concentration of the methanol is 90 to 100 volume %.

5. The method according to claim 1, characterized in that Further including: Under the same conditions, the reference solution and the plurality of test solution are subjected to high performance liquid chromatography detection, preferably, the reference solution is selected from Solution, Solution, solution or a mixture of the three; The number of the multiple different batches is at least 5; Optionally, the characteristic spectrum includes 11 common characteristic peaks, wherein the fifth characteristic peak is taken as the reference peak, and the ratios of the relative retention times of the 11 common characteristic peaks to the retention time of the reference peak are 0.15±8.0%, 0.58±8.0%, 0.93±8.0%, 0.98±8.0%, 1.00, 1.13±8.0%, 1.15±8.0%, 1.27±8.0%, 1.41±8.0%, 1.76±8.0%, and 1.88±8.0%, respectively.

6. A method for identifying the pharmaceutical composition defined in claim 1, characterized in that: include: Pre-treating the sample to be tested to obtain a sample solution; Performing high performance liquid chromatography on the sample solution to obtain a chromatogram; Analyze the chromatogram and the characteristic spectrum obtained by any one of claims 1 to 5 to determine whether the sample to be tested is the pharmaceutical composition defined in claim 1; The detection conditions of the high performance liquid chromatography are as defined in the method according to any one of claims 1 to 3 and 5.

7. The method according to claim 6, characterized in that The pretreatment is as defined in the method of claim 4; If the similarity between the chromatogram and the characteristic spectrum is not less than 0.9, the sample to be tested is determined to be the pharmaceutical composition defined in claim 1.

8. A method for detecting the pharmaceutical composition defined in claim 1, characterized in that: include: A solution of a characteristic component of known concentration corresponding to at least one characteristic peak in the characteristic spectrum obtained by the method according to any one of claims 1 to 5 is prepared as a reference solution; Pre-treating the sample to obtain a test solution; The reference solution and the test solution are respectively subjected to high performance liquid chromatography to obtain a chromatogram; Analyzing the chromatogram of the reference solution and the chromatogram of the test solution to determine the content of at least one characteristic component in the sample; Wherein, the HPLC detection conditions are as defined in the method according to any one of claims 1-3 and 5.

9. The method according to claim 8, characterized in that The pre-treatment is as defined in the method of claim 4.

10. A method of producing a pharmaceutical composition as defined in claim 1, characterized in that include: Pretreating the pharmaceutical composition sample to obtain a test solution; The test solution is subjected to high performance liquid chromatography to obtain a chromatogram; Analyze the chromatogram with the characteristic spectrum obtained by the method according to any one of claims 1 to 5 to determine whether the quality of the sample to be tested meets the standard; Wherein, the HPLC detection conditions are as defined in the method according to any one of claims 1 to 3 and 5; Optionally, the pretreatment is as defined in the method of claim 4; Optionally, if the similarity between the chromatogram and the characteristic spectrum is not less than 0.9, the quality of the pharmaceutical composition is deemed to be up to standard.

11. The pharmaceutical composition according to claim 1, characterized in that The similarity between the high performance liquid chromatogram of the pharmaceutical composition and the characteristic spectrum constructed by the method of any one of claims 1 to 5 is not less than 0.

9.

12. Use of the pharmaceutical composition as defined in claim 11 in the preparation of a medicament for treating or preventing throat diseases such as paresthesia, chronic pharyngitis, chronic laryngitis and / or laryngopharyngeal reflux disease.

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