Construction method, detection method and application of qianglimadetulifaiaoyi preparation fingerprint

Abstract

The application discloses a method for constructing a fingerprint spectrum of a strong Madutu Liayate preparation, a detection method and application. The method comprises the following steps: using high performance liquid chromatography to detect a test solution, and generating a fingerprint spectrum from the detection result; the test solution is a solution containing the strong Madutu Liayate preparation; in the high performance liquid chromatography, a chromatographic column is YMC-Triart C 18 ; the column temperature is 26-40 DEG C; the flow rate is 1.05-1.5 mL / min; the mobile phase A used is acetonitrile, and the mobile phase B used is a phosphoric acid aqueous solution with a volume concentration of 0.06%-0.2%. The application establishes the fingerprint spectrum of the strong Madutu Liayate preparation, can evaluate the quality condition of the strong Madutu Liayate preparation from the overall point of view, avoids the one-sidedness of quality control of a single component, and provides a guarantee for safe use of the strong Madutu Liayate preparation.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine detection technology, specifically to a method for constructing, detecting, and applying a fingerprint spectrum of a strong madodetuliyate preparation. Background Technology

[0002] Traditional Chinese medicine (TCM) herbs have complex compositions, and compound TCM preparations made from multiple herbs are even more complex. TCM fingerprinting can reflect the overall quality of TCM herbs and compound preparations, playing a crucial role in quality control. High-performance liquid chromatography (HPLC) fingerprinting is characterized by high sensitivity and selectivity, enabling the evaluation of the similarity between TCM herbs and compound preparations. The "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" (2012 edition) published by the National Pharmacopoeia Commission is used to comprehensively evaluate the quality of TCM preparations.

[0003] Qianglimadituliyatemi Ointment is a commonly used Uyghur medicine preparation in hospitals. It is listed in the "Standards for Preparations of Uyghur Medical Institutions in Xinjiang Uyghur Autonomous Region" (Standard No.: MZJ-W-0133-2013). It is composed of 14 medicinal materials, including dried ginger, black pepper, long pepper, and cinnamon. It has the effects of strengthening the body, relieving pain, benefiting the brain, and nourishing the brain. It is used to treat hemiplegia, joint and bone pain, dementia, tongue heaviness, backache, and other diseases.

[0004] However, current standard testing items for duramatizine preparations are limited, only including appearance, identification, and inspection items, which cannot accurately and comprehensively control drug quality. Furthermore, there are currently no reports on fingerprint spectroscopy studies for duramatizine preparations. Therefore, it is urgent to establish a fingerprint spectroscopy for duramatizine preparations and improve the quality standards for this preparation, facilitating a systematic and comprehensive evaluation of its overall quality. Summary of the Invention

[0005] The technical problem this invention aims to solve is to overcome the deficiency of existing technologies in the lack of fingerprint spectroscopy for terlimethicone preparations, and to provide a method for constructing, detecting, and applying a fingerprint spectroscopy for terlimethicone preparations. This detection method can simply, quickly, and accurately evaluate the quality of terlimethicone preparations, providing a scientific basis for establishing quality standards for terlimethicone preparations from a holistic perspective. This invention establishes a fingerprint spectroscopy for terlimethicone preparations, which can comprehensively and accurately reflect the quality of terlimethicone preparations, providing a theoretical basis and data reference for its quality control and standard improvement. It can evaluate the quality status of terlimethicone preparations from a holistic perspective, avoiding the one-sidedness of controlling quality with a single component, and ensuring the safe use of terlimethicone preparations.

[0006] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0007] This invention provides a method for constructing a fingerprint spectrum of a strong madrema turi-ayate formulation, which includes the following steps: detecting the test solution using high performance liquid chromatography and generating a fingerprint spectrum from the detection results;

[0008] The test solution is a solution containing the aforementioned strong metrate tartrate preparation;

[0009] In the high-performance liquid chromatography, the chromatographic column was YMC-Triart C. 18 The column temperature was 26-40℃; the flow rate was 1.05-1.5 mL / min; the mobile phase A was acetonitrile, and the mobile phase B was a 0.06%-0.2% (v / v) aqueous solution of phosphoric acid.

[0010] In this invention, the solvent in the test solution can be methanol or an aqueous methanol solution, preferably an aqueous methanol solution. The volume percentage of methanol in the aqueous methanol solution is preferably 60%-80%, for example, 70% or 75%.

[0011] In this invention, the mass-to-volume ratio of the strong madelayate preparation and the solvent in the test solution is preferably 1g:(40-60)mL, for example 1g:45mL or 1g:50mL.

[0012] In this invention, the test solution can be prepared by conventional methods in the art, preferably by the following steps: mixing the strong madrethroid preparation and solvent, ultrasonic treatment, cooling, and filtration.

[0013] The conditions for the ultrasonic treatment can be those conventional in the art. The power of the ultrasonic treatment can be 200-300W, for example, 250W. The frequency of the ultrasonic treatment can be 50-60kHz, for example, 53kHz. The duration of the ultrasonic treatment can be 20-35min, for example, 30min.

[0014] The temperature after cooling can be room temperature, for example, 20-30°C.

[0015] The filtration operation and conditions are conventional in the art, and preferably involve filtration using a 0.45μm microporous membrane. The purpose of the filtration is to remove impurities.

[0016] In this invention, the fingerprint pattern construction method generally also includes the preparation of a mixed reference solution. The solvent in the mixed reference solution is preferably methanol or an aqueous methanol solution, more preferably an aqueous methanol solution. The volume percentage of the aqueous methanol solution is preferably 60%-80%, for example 70% or 75%.

[0017] In this invention, the fingerprint pattern construction method includes one or more of gallic acid, corilagin, cinnamaldehyde, 6-gingerol, ellagic acid, and piperine in the mixed reference solution.

[0018] When the mixed reference solution contains gallic acid, the concentration of gallic acid is preferably 30-40 μg / mL, for example 37 μg / mL.

[0019] When the mixed reference solution contains corilagin, the concentration of corilagin is preferably 30-35 μg / mL, for example 31 or 31.05 μg / mL.

[0020] Wherein, when the mixed reference solution contains cinnamaldehyde, the concentration of cinnamaldehyde is preferably 3-5 μg / mL, for example 4 or 4.12 μg / mL.

[0021] When the mixed reference solution contains 6-gingerol, the concentration of 6-gingerol is preferably 20-30 μg / mL, for example 25.9 or 26 μg / mL.

[0022] When the mixed reference solution contains piperine, the concentration of piperine is preferably 90-100 μg / mL, for example 97 or 97.10 μg / mL.

[0023] When the mixed reference solution contains ellagic acid, the concentration of ellagic acid is preferably 5-10 μg / mL, for example 7 or 7.3 μg / mL.

[0024] In this invention, the injection volume in the high-performance liquid chromatography can be conventional in the art, preferably 5-20 μL, for example 10 μL.

[0025] In this invention, the column temperature in the high-performance liquid chromatography is preferably 28-38°C, for example 30 or 35°C.

[0026] In this invention, the flow rate of the test solution in the high-performance liquid chromatography is preferably 1.1-1.3 mL / min, for example 1.2 mL / min.

[0027] In this invention, the specifications of the chromatographic column in the high-performance liquid chromatography are preferably: column length 150 mm, inner diameter 4.6 mm, and packing particle size 5 μm.

[0028] In this invention, the high-performance liquid chromatograph can be a conventional high-performance liquid chromatograph in the art, such as the Thermo Scientific UlitiMate 3000 high-performance liquid chromatograph, the Agilent 1260 high-performance liquid chromatograph, or the Waters AcQuity*Arc high-performance liquid chromatograph.

[0029] In this invention, the mobile phase B is preferably an aqueous solution of phosphoric acid with a volume concentration of 0.07%-0.16%, more preferably an aqueous solution of phosphoric acid with a volume concentration of 0.1%, where the percentages are the volume percentage of the solute phosphoric acid relative to the volume of the mobile phase B, i.e., the volume percentage concentration.

[0030] In this invention, during the research and development phase, the inventors explored various elution gradients and discovered that the separation of all common peaks could only be achieved when using the following elution gradient: The gradient elution conditions for mobile phase A and mobile phase B are as follows:

[0031] Time (min) Mobile phase A (%) Mobile phase B (%) 0～6 4 96 6～9 4～12 96～88 9～15 12～13 88～87 15～24 13～19 87～81 24～51 19～65 81～35 51～56 65～4 35～96 56～60 4 96

[0032] The percentages in the table represent the volume percentage of each component in the total volume of mobile phase A and mobile phase B, respectively.

[0033] In a preferred embodiment of the present invention, the method for constructing the fingerprint spectrum of the strong morpholine formulation includes the following steps:

[0034] S1. Prepare the test solution;

[0035] S2. Prepare a mixed reference solution; the mixed reference solution includes gallic acid, corilagin, cinnamaldehyde, 6-gingerol, ellagic acid, and piperine, wherein the concentration of gallic acid in the reference solution is 30-40 μg / mL, the concentration of corilagin is 30-35 μg / mL, the concentration of cinnamaldehyde is 3-5 μg / mL, the concentration of 6-gingerol is 20-30 μg / mL, the concentration of piperine is 90-100 μg / mL, and the concentration of ellagic acid is 5-10 μg / mL.

[0036] S3. The test solution and the mixed reference solution are respectively detected by high performance liquid chromatography as described above;

[0037] Based on the retention times of each substance in the mixed reference solution, the peaks in the test solution are assigned, and the detection results of the test solution are used to generate a fingerprint spectrum.

[0038] In this invention, the fingerprint spectrum obtained by the method for constructing the fingerprint spectrum of the strong madoretiate preparation includes 6 common peaks, with peak 11 as the control peak. The relative retention times of the common peaks can be as follows:

[0039] Peak 1 is gallic acid, with a relative retention time of 0.123-0.124 and an RSD of 0.25%.

[0040] Peak 2 was Corilagin, with a relative retention time of 0.392 and an RSD of 0.00%.

[0041] Peak 6 is ellagic acid, with a relative retention time of 0.592 and an RSD of 0.00%.

[0042] Peak 9 is cinnamaldehyde, with a relative retention time of 0.811 and an RSD of 0.00%.

[0043] Peak 10 was 6-gingerol, with a relative retention time of 0.946 and an RSD of 0.00%.

[0044] Peak 11 is piperine, with a relative retention time of 1.00 and an RSD of 0.00%.

[0045] In this invention, the fingerprint spectrum obtained by the method for constructing the fingerprint spectrum of the strong madoretiate preparation further includes the following 5 common peaks, with peak 11 as the control peak. The relative retention times of the common peaks are preferably as follows:

[0046] The relative retention time of peak 3 was 0.439, and the RSD was 0.00%.

[0047] The relative retention time of peak 4 was 0.458–0.459, with an RSD of 0.09%.

[0048] The relative retention time of peak 5 was 0.572–0.573, with an RSD of 0.08%.

[0049] The relative retention time of peak 7 was 0.731, and the RSD was 0.00%.

[0050] The relative retention time of peak 8 was 0.764, and the RSD was 0.00%.

[0051] In this invention, the fingerprint spectrum obtained by the method for constructing the fingerprint spectrum of the strong morpholine preparation includes 11 common peaks, which are more preferably as follows: Figure 1 As shown.

[0052] This invention also provides a method for detecting the quality of a strong metrate preparation. The method involves generating a fingerprint spectrum of the sample to be tested according to the fingerprint spectrum construction method of the strong metrate preparation as described above. The sample to be tested is a qualified product of the strong metrate preparation.

[0053] The present invention also provides a method for constructing a fingerprint spectrum of a thymolide preparation as described above, and the fingerprint spectrum obtained is used as a standard spectrum for the quality control of the thymolide preparation.

[0054] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0055] The reagents and raw materials used in this invention are all commercially available.

[0056] The positive and progressive effects of this invention are as follows:

[0057] 1. The fingerprint spectrum of the strong madelayate preparation established in this invention has a similarity of more than 0.97 with the control spectrum, which can effectively ensure the stability and consistency of its finished product quality and comprehensively monitor the quality of the product.

[0058] 2. The fingerprint spectrum of the strong matte tuliat preparation established by this invention identifies 11 common peaks and calibrates 6 of them. The established fingerprint spectrum has high technical content. Among the 6 common peaks, peak 1 is gallic acid, peak 2 is corilagin, peak 6 is ellagic acid, peak 9 is cinnamaldehyde, peak 10 is 6-gingerol, and peak 11 is piperine. It is suitable for the overall quality control of the strong matte tuliat preparation. Attached Figure Description

[0059] Figure 1 The fingerprint spectrum of the strong madriturizer formulation S11.

[0060] Figure 2 The fingerprint spectrum is for the mixed reference standard.

[0061] Figure 3 The fingerprint spectrum is constructed from 11 batches of the strong madodetuli ayate formulation in Example 5.

[0062] Figure 4 This is a fingerprint comparison chart of the finished product of the Strong Madete Ayate Pill in Example 5.

[0063] Figure 5 This is the fingerprint spectrum obtained from the precision test of the reference standard.

[0064] Figure 6 The fingerprint spectrum is obtained from the precision test of the test sample.

[0065] Figure 7 The fingerprint spectrum is obtained from the stability test of the test sample.

[0066] Figure 8 The fingerprint spectrum obtained from the repeatability test of the test sample.

[0067] Figure 9The fingerprint spectrum is obtained from the intermediate precision test of the test sample. Detailed Implementation

[0068] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0069] The batch numbers and sources of the experimental materials and instruments used in the embodiments and comparative examples of this invention are shown in the table below:

[0070]

[0071]

[0072] Selection of chromatographic conditions for fingerprinting of strong madoreti Ayate pills

[0073] Example 1: Investigation of column temperature

[0074] Example 1-1

[0075] Preparation of the test solution: Weigh approximately 1 g of the finished drug powder (numbered S11, passed through a 24-mesh sieve), accurately weigh it, place it in a brown stoppered conical flask, accurately add 75% methanol aqueous solution, weigh it, sonicate for 30 minutes, cool to room temperature, weigh it again, replenish the lost weight with 75% methanol aqueous solution, shake well, filter it through a filter membrane with a pore size of 0.45 μm, and collect the filtrate to obtain the test solution.

[0076] Preparation of mixed reference solution: Take appropriate amounts of gallic acid, corilagin, cinnamaldehyde, 6-gingerol, and piperine reference standards, dissolve and dilute to volume with 75% methanol aqueous solution to prepare stock solutions for each reference standard; take an appropriate amount of ellagic acid reference standard, dissolve and dilute to volume with methanol to prepare a reference standard stock solution; accurately pipette the above reference standard stock solutions, add 75% methanol aqueous solution to prepare a solution containing 37 μg gallic acid, 31 μg corilagin, 4 μg cinnamaldehyde, 26 μg 6-gingerol, 97 μg piperine, and 7 μg ellagic acid per 1 mL.

[0077] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 1; the detection wavelength was 280 nm; and the flow rate was 1.2 mL / min. -1 Column temperature 30℃; chromatographic column: YMC-Triart C 18The column (4.6 mm × 150 mm, 5 μm) was used; the high-performance liquid chromatograph was a Thermo Scientific UlitiMate 3000; the theoretical plate number calculated based on the gallic acid peak should not be less than 2000.

[0078] Table 1. Mobile Phase Gradient Table

[0079] Time (min) Mobile phase A (%) Mobile phase B (%) 0～6 4 96 6～9 4～12 96～88 9～15 12～13 88～87 15～24 13～19 87～81 24～51 19～65 81～35 51～56 65～4 35～96 56～60 4 96

[0080] Accurately pipette 10 μL each of the mixed reference solution and the test solution into the liquid chromatograph, record the chromatogram for 60 minutes, and calculate the similarity to obtain the result.

[0081] The fingerprint spectrum of S11 obtained from the test is as follows Figure 1 As shown. The fingerprint spectrum of the mixed reference solution is as follows. Figure 2 As shown.

[0082] Examples 1-2

[0083] The only difference from Example 1-1 is that the column temperature is 35°C.

[0084] Comparative Example 1

[0085] The only difference from Example 1-1 is that the column temperature is 25°C.

[0086] Example 2: Investigation of Flow Rate

[0087] The only difference from Example 1-1 is that the flow rate is 1.1 mL / min. -1 .

[0088] Comparative Example 2

[0089] The only difference from Example 1-1 is that the flow rate is 1.0 mL / min. -1 .

[0090] Example 3: Investigation of the Model of High Performance Liquid Chromatograph

[0091] Example 3-1

[0092] The only difference from Example 1-1 is that the high performance liquid chromatograph is an Agilent 1260.

[0093] Example 3-2

[0094] The only difference from Example 1-1 is that the high performance liquid chromatograph is a Waters AcQuity*Arc.

[0095] Investigation of the type of chromatographic column in Comparative Example 3

[0096] Comparative Example 3-1

[0097] The only difference from Example 1-1 is that the chromatographic column is a Waters SunFire C1000. 18 (4.6×150mm, 5μm).

[0098] Comparative Example 3-2

[0099] The only difference from Example 1-1 is that the chromatographic column is a Waters XSelect CSH C. 18 (4.6×250mm, 5μm).

[0100] Investigation of the mobile phase in Comparative Example 4

[0101] Comparative Example 4-1

[0102] The only difference from Example 1-1 is that the mobile phase A is methanol.

[0103] Comparative Example 4-2

[0104] The only difference from Example 1-1 is that the mobile phase B is an aqueous solution of glacial acetic acid with a volume concentration of 0.1%.

[0105] Comparative Example 4-3

[0106] The only difference from Example 1-1 is that the mobile phase B is an aqueous solution of formic acid with a volume concentration of 0.1%.

[0107] Comparative Example 4-4

[0108] The only difference from Example 1-1 is that the mobile phase B is an aqueous solution of phosphoric acid with a volume concentration of 0.05%.

[0109] Example 5 Similarity Evaluation:

[0110] Eleven batches (S1-S11) of the aforementioned strong morphotriate ayate pills were used to prepare eleven batches of test solutions according to the method in Example 1-1, and the solutions were injected and analyzed according to the chromatographic conditions in Example 1-1. The fingerprint chromatograms were recorded. Figure 3 As shown;

[0111] The fingerprint chromatograms of 11 batches of Qianglimadetuliyate pills were imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" and numbered S1-11. Batch QL22091901 (S11) was used as the reference fingerprint. With a time window width of 0.1 min, the average method was used to generate a control fingerprint chromatogram. Figure 4As shown, 11 common peaks were selected for multi-point calibration. Full-spectrum peak matching was used to obtain overlayed fingerprint spectra of 11 batches. The similarity results were all greater than 0.97, indicating good quality consistency among batches. The piperine peak showed suitable separation and relatively stable composition, so it was used as the S peak. The relative peak area and relative retention time of the common peaks were calculated, and the results are shown in [Figure 1]. Figure 4 And Table 7-9.

[0112] Depend on Figure 2 and Figure 4 The comparison revealed that the fingerprint chromatogram of the test sample showed 11 fingerprint peaks, of which 6 peaks corresponded to the retention times of the peaks in the fingerprint chromatogram of the mixed reference standard. These peaks were: peak 1 (gallic acid), peak 2 (corilagin), peak 6 (ellagic acid), peak 9 (cinnamaldehyde), peak 10 (6-gingerol), and peak 11 (piperine). According to the similarity evaluation system for fingerprint chromatograms of traditional Chinese medicine, the similarity between the fingerprint chromatogram of the test sample and the fingerprint chromatogram of the mixed reference standard should not be less than 0.90.

[0113] Depend on Figure 3 It can be seen that the fingerprint comparison spectrum of the finished product of Qianglimadituliyat pill shows a total of 11 fingerprint peaks, and the relative retention times of each peak are as follows:

[0114] Peak 1 is gallic acid, with a relative retention time of 0.123-0.124 and an RSD of 0.25%.

[0115] Peak 2 was Corilagin, with a relative retention time of 0.392 and an RSD of 0.00%.

[0116] The relative retention time of peak 3 was 0.439, and the RSD was 0.00%.

[0117] The relative retention time of peak 4 was 0.458–0.459, with an RSD of 0.09%.

[0118] The relative retention time of peak 5 was 0.572–0.573, with an RSD of 0.08%.

[0119] Peak 6 is ellagic acid, with a relative retention time of 0.592 and an RSD of 0.00%.

[0120] The relative retention time of peak 7 was 0.731, and the RSD was 0.00%.

[0121] The relative retention time of peak 8 was 0.764, and the RSD was 0.00%.

[0122] Peak 9 is cinnamaldehyde, with a relative retention time of 0.811 and an RSD of 0.00%.

[0123] Peak 10 was 6-gingerol, with a relative retention time of 0.946 and an RSD of 0.00%.

[0124] Peak 11 is piperine, with a relative retention time of 1.00 and an RSD of 0.00%.

[0125] Example 1

[0126] The results of the investigation in Examples 1-4 are shown in Tables 2-6.

[0127] Table 2. Investigation at different column temperatures

[0128]

[0129]

[0130] Note: Separation 1 represents the separation between the target peak and its adjacent fingerprint peak; separation 2 represents the separation between the target peak and its adjacent fingerprint peak.

[0131] The results showed that, compared with Examples 1-1 and 1-2, in Comparative Example 1, when the column temperature was 25°C, the resolution 1 and resolution 2 of peak 6 were both less than 1.5. In Examples 1-2, when the column temperature was 35°C, the resolution of peak 7 was slightly worse than that in Example 1-1.

[0132] Table 3. Investigation at different flow velocities

[0133]

[0134]

[0135] Note: Separation 1 represents the separation between the target peak and its adjacent fingerprint peak; separation 2 represents the separation between the target peak and its adjacent fingerprint peak.

[0136] The results showed that, compared with Examples 1-1 and 2-1, in Comparative Example 2, when the flow rate was 1.0 mL·min -1 The resolution of peak 6 is less than 1.5. In Example 2, when the flow rate was 1.1 mL / min... -1 At that time, the separation of peak 4 was slightly worse than that of Example 1-1.

[0137] Table 4. Investigation using different instruments

[0138]

[0139]

[0140] Note: Resolution 1 indicates the separation between the target peak and its adjacent fingerprint peak; Resolution 2 indicates the separation between the target peak and its adjacent fingerprint peak; Resolution indicates the resolution measured when the high performance liquid chromatography instrument is an Agilent.

[0141] The results showed that the resolution of each common peak in the sample fingerprint spectrum met the requirements under the above three types of high performance liquid chromatographs, and the peak area and retention time were appropriate. Therefore, all of the above instruments can be used for the test of this sample.

[0142] Table 5. Investigation of different chromatographic columns

[0143]

[0144]

[0145] Note: Separation 1 represents the separation between the target peak and its adjacent fingerprint peak; separation 2 represents the separation between the target peak and its adjacent fingerprint peak.

[0146] The results showed that, compared with Example 1-1, when Comparative Examples 3-1 and 3-2 used Waters SunFire C 18 With Waters XSelect CSH C 18 The uniform resolution of peak 6 on the chromatographic column was unsatisfactory; YMC-Triart C 18 The resolution of all common peaks on the chromatographic column was satisfactory, and the retention time and peak area were appropriate; therefore, the YMC-Triart C column was selected. 18 The sample was tested.

[0147] Table 6. Investigation of different mobile phases

[0148]

[0149]

[0150]

[0151]

[0152] Conclusion: Compared with Examples 1-1, in Comparative Example 4-1 (methanol-0.1% phosphoric acid aqueous solution), in Comparative Example 4-2 (acetonitrile-0.1% glacial acetic acid aqueous solution), in Comparative Example 4-3 (acetonitrile-0.1% formic acid aqueous solution), and in Comparative Example 4-4 (acetonitrile-0.05% phosphoric acid aqueous solution), the target peak resolution was less than 1.5. Furthermore, the piperine peak could not be eluted when the mobile phase was methanol-0.1% phosphoric acid aqueous solution. In Example 1-1 (acetonitrile-0.1% phosphoric acid aqueous solution), the resolution of each target peak was greater than 1.5, and the retention time and peak area were suitable. Considering all factors, acetonitrile-0.1% phosphoric acid aqueous solution was the optimal mobile phase.

[0153] Example 2

[0154] Table 7. Relative retention times of common peaks in 11 batches of Strong Made Tuli Ayate Pills

[0155]

[0156]

[0157] Table 8. Relative peak area of ​​11 batches of Strong Made Tuli Ayate Pill

[0158]

[0159]

[0160] Table 9. Similarity of fingerprint spectra of 11 batches of Qianglimadituli Ayate pills

[0161] S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 R S1 1.000 0.996 0.996 0.996 0.996 0.998 0.996 0.998 0.989 0.996 0.987 0.998 S2 0.996 1.000 0.996 0.996 0.996 0.998 0.995 0.997 0.982 0.997 0.977 0.996 S3 0.996 0.996 1.000 0.999 1.000 0.999 1.000 0.999 0.992 0.999 0.990 1.000 S4 0.996 0.996 0.999 1.000 1.000 0.999 0.999 0.999 0.988 0.997 0.990 0.999 S5 0.996 0.996 1.000 1.000 1.000 0.999 1.000 0.999 0.989 0.998 0.990 0.999 S6 0.998 0.998 0.999 0.999 0.999 1.000 0.999 0.999 0.989 0.998 0.987 0.999 S7 0.996 0.995 1.000 0.999 1.000 0.999 1.000 0.999 0.992 0.997 0.992 1.000 S8 0.998 0.997 0.999 0.999 0.999 0.999 0.999 1.000 0.992 0.999 0.989 1.000 S9 0.989 0.982 0.992 0.988 0.989 0.989 0.992 0.992 1.000 0.992 0.993 0.993 S10 0.996 0.997 0.999 0.997 0.998 0.998 0.997 0.999 0.992 1.000 0.985 0.998 S11 0.987 0.977 0.990 0.990 0.990 0.987 0.992 0.989 0.993 0.985 1.000 0.991 R 0.998 0.996 1.000 0.999 0.999 0.999 1.000 1.000 0.993 0.998 0.991 1.000

[0162] A direct analysis of the test results from 11 batches of samples revealed similar overall characteristics. Eleven common peaks were identified, and six components were determined: gallic acid (peak 1), corilagin (peak 2), ellagic acid (peak 6), cinnamaldehyde (peak 9), 6-gingerol (peak 10), and piperine (peak 11, S). The 11 batches of finished products showed high similarity, all greater than 0.90. This similarity reflects the stability of the raw materials and processing of the Qiangli Madetuli Ayate pills, indicating good quality consistency among batches.

[0163] The established fingerprinting method for Qianglimatetuliayat pills is stable and reliable, providing a scientific method and reference for the quality analysis and evaluation of Qianglimatetuliayat preparations.

[0164] Example 3: Investigation of Fingerprint Mapping Methodology

[0165] 1. Precision test

[0166] Precision test of the reference standard: A mixed reference standard solution was prepared according to the preparation method of the reference standard in Example 1-1. The solution was injected six times consecutively under the chromatographic conditions of Example 1-1. Using piperine as the S peak, the relative retention time and relative peak area of ​​each common peak were calculated, and the RSD (%, n=6) was calculated. The results showed that the RSD (%) of the relative peak area of ​​each common peak was between 0.00% and 0.79%, and the RSD (%) of the relative retention time was between 0.00% and 0.11%. The similarity was evaluated and calculated using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)". The similarity results were all greater than 0.9. See the results below. Figure 5 (S1-S6 represent 6 injections respectively) and Table 10-12 show that the reference standard has good precision.

[0167] Precision assessment of the test sample: A test sample (Qiangli Madetuli Ayate Pill, batch number S11, QL22091901) was prepared according to the preparation method of the test sample in Example 1-1, and injected continuously 6 times under the chromatographic conditions of Example 1-1 for determination. Using piperine as the S peak, the relative retention time and relative peak area of ​​each common peak were calculated, and the RSD (%, n=6) was calculated. The results showed that the RSD (%) of the relative peak area of ​​each common peak was between 0.00% and 0.47%, and the RSD (%) of the relative retention time was between 0.00% and 0.14%. The similarity was evaluated and calculated using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)". The similarity results were all greater than 0.9. See [the table below for details]. Figure 6 (S1-S6 represent 6 injections respectively) and Table 13-15 show that the sample has good precision.

[0168] Table 10 Relative Peak Area of ​​Precision of Mixed Reference Standards

[0169]

[0170]

[0171] Table 11 Relative Retention Time of Precision of Mixed Reference Standards

[0172] Peak Precision 1 Precision 2 Precision 3 Precision 4 Precision 5 Precision 6 RSD (%) Peak 1 0.124 0.124 0.124 0.124 0.124 0.124 0.00 Peak 2 0.393 0.393 0.393 0.393 0.393 0.392 0.11 Peak 6 0.592 0.592 0.592 0.592 0.592 0.592 0.00 Peak 9 0.811 0.811 0.811 0.811 0.811 0.811 0.00 Peak 10 0.947 0.947 0.947 0.947 0.947 0.947 0.00 Peak 11(S) 1.000 1.000 1.000 1.000 1.000 1.000 0.00

[0173] Table 12 Precision Similarity of Mixed Reference Standards

[0174] S1 S2 S3 S4 S5 S6 Comparison of fingerprint patterns S1 1 1 1 1 1 1 1 S2 1 1 1 1 1 1 1 S3 1 1 1 1 1 1 1 S4 1 1 1 1 1 1 1 S5 1 1 1 1 1 1 1 S6 1 1 1 1 1 1 1 Comparison of fingerprint patterns 1 1 1 1 1 1 1

[0175] Table 13 Relative Peak Area of ​​Precision of Test Samples

[0176]

[0177]

[0178] Table 14 Relative Retention Time of Precision of Test Samples

[0179] Peak Precision 1 Precision 2 Precision 3 Precision 4 Precision 5 Precision 6 RSD (%) Peak 1 0.123 0.123 0.123 0.123 0.123 0.123 0.00 Peak 2 0.392 0.392 0.392 0.391 0.391 0.391 0.14 Peak 3 0.439 0.439 0.439 0.438 0.438 0.438 0.13 Peak 4 0.458 0.458 0.458 0.458 0.457 0.457 0.12 Peak 5 0.572 0.572 0.572 0.572 0.572 0.572 0.00 Peak 6 0.591 0.591 0.591 0.591 0.591 0.590 0.07 Peak 7 0.731 0.731 0.731 0.731 0.731 0.731 0.00 Peak 8 0.764 0.764 0.764 0.764 0.764 0.764 0.00 Peak 9 0.811 0.811 0.811 0.810 0.810 0.810 0.07 Peak 10 0.947 0.947 0.947 0.947 0.947 0.947 0.00 Peak 11(S) 1.000 1.000 1.000 1.000 1.000 1.000 0.00

[0180] Table 15 Precision Similarity of Test Samples

[0181]

[0182]

[0183] 2. Stability test

[0184] Accurately weigh the finished medicinal powder (batch QL22091901, passed through a 24-mesh sieve), prepare the test solution according to the preparation method of the test sample in Example 1-1, and analyze it by injection at 0, 2, 4, 8, 15, 24h, 36h, and 48h according to the chromatographic conditions of Example 1-1. Using piperine as the S peak, calculate the relative retention time and relative peak area of ​​each common peak, and calculate the RSD (%). The results show that the RSD (%) of the relative retention time of each common peak is between 0.00% and 0.44%, and the RSD (%) of the relative peak area is between 0.00% and 3.46%. The similarity was evaluated and calculated using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)". The results show that the similarity is greater than 0.9 for all values. See [the table below for details]. Figure 7 (S1-S8 represent 8 injections respectively) and Table 16-18 show that the sample has good stability within 48 hours.

[0185] Table 16 Relative Peak Areas of Stability of Test Samples

[0186] Peak 0h 2h 4h 8h 15h 24h 36h 48h RSD (%) Peak 1 0.543 0.543 0.542 0.542 0.543 0.542 0.541 0.542 0.14 Peak 2 0.174 0.174 0.175 0.174 0.175 0.175 0.175 0.176 0.41 Peak 3 0.047 0.047 0.047 0.047 0.047 0.047 0.047 0.047 0.00 Peak 4 0.087 0.087 0.087 0.087 0.087 0.087 0.087 0.087 0.00 Peak 5 0.092 0.092 0.092 0.092 0.094 0.093 0.094 0.094 1.07 Peak 6 0.073 0.073 0.073 0.074 0.075 0.075 0.075 0.074 1.26 Peak 7 0.016 0.016 0.016 0.016 0.016 0.016 0.016 0.016 0.00 Peak 8 0.066 0.066 0.066 0.065 0.065 0.065 0.066 0.065 0.81 Peak 9 0.197 0.197 0.198 0.198 0.198 0.198 0.198 0.199 0.33 Peak 10 0.015 0.015 0.015 0.015 0.015 0.016 0.016 0.016 3.46 Peak 11(S) 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.00

[0187] Table 17 Relative retention times of test samples stability

[0188] Peak 0h 2h 4h 8h 15h 24h 36h 48h RSD (%) Peak 1 0.124 0.124 0.123 0.123 0.123 0.124 0.123 0.124 0.44 Peak 2 0.393 0.392 0.392 0.391 0.393 0.393 0.392 0.392 0.19 Peak 3 0.439 0.439 0.439 0.438 0.439 0.440 0.439 0.439 0.13 Peak 4 0.459 0.459 0.458 0.458 0.459 0.459 0.459 0.459 0.11 Peak 5 0.573 0.573 0.573 0.572 0.573 0.573 0.573 0.573 0.07 Peak 6 0.592 0.592 0.591 0.591 0.592 0.592 0.592 0.592 0.08 Peak 7 0.731 0.731 0.731 0.731 0.731 0.731 0.731 0.731 0.00 Peak 8 0.765 0.765 0.765 0.764 0.764 0.765 0.765 0.765 0.07 Peak 9 0.811 0.811 0.811 0.811 0.811 0.811 0.811 0.811 0.00 Peak 10 0.947 0.947 0.947 0.947 0.947 0.947 0.947 0.946 0.04 Peak 11(S) 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.00

[0189] Table 18 Stability Similarity of Test Samples

[0190] S1 S2 S3 S4 S5 S6 S7 S8 Comparison of fingerprint patterns S1 1 1 1 1 1 1 1 1 1 S2 1 1 1 1 1 1 1 1 1 S3 1 1 1 1 1 1 1 1 1 S4 1 1 1 1 1 1 1 1 1 S5 1 1 1 1 1 1 1 1 1 S6 1 1 1 1 1 1 1 1 1 S7 1 1 1 1 1 1 1 1 1 S8 1 1 1 1 1 1 1 1 1 Comparison of fingerprint patterns 1 1 1 1 1 1 1 1 1

[0191] 3. Repeatability test

[0192] Six portions of the finished drug powder (QL22091901, passed through a 24-mesh sieve) were accurately weighed and used to prepare a test solution according to the preparation method of the test sample in Example 1-1. The solution was then injected and analyzed under the chromatographic conditions described in Example 1-1. Using piperine as the S peak, the relative retention time and relative peak area RSD (%) of each common peak were calculated, and the RSD (%, n=6) was also calculated. The results showed that the relative retention time RSD (%) of each common peak was between 0.00% and 0.14%, and the relative peak area RSD (%) was between 0.00% and 3.11%. The similarity was evaluated and calculated using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)". The similarity results were all greater than 0.9. (See attached table for details.) Figure 8 (S1-S6 represent 6 injections respectively) and Table 19-21 show that the repeatability is good.

[0193] Table 19 Relative Peak Area of ​​Test Sample Repeatability

[0194] Peak Repeatability 1 Repeatability 2 Repeatability 3 Repeatability 4 Repeatability 5 Repeatability 6 RSD (%) Peak 1 0.551 0.549 0.547 0.548 0.548 0.547 0.28 Peak 2 0.181 0.182 0.180 0.181 0.179 0.178 0.82 Peak 3 0.047 0.047 0.047 0.048 0.047 0.047 0.87 Peak 4 0.089 0.089 0.088 0.089 0.089 0.088 0.59 Peak 5 0.092 0.091 0.087 0.091 0.090 0.092 2.06 Peak 6 0.080 0.083 0.080 0.083 0.077 0.078 3.11 Peak 7 0.016 0.016 0.016 0.016 0.016 0.016 0.00 Peak 8 0.066 0.066 0.066 0.066 0.066 0.066 0.00 Peak 9 0.191 0.192 0.192 0.192 0.190 0.191 0.43 Peak 10 0.015 0.015 0.015 0.015 0.015 0.015 0.00 Peak 11(S) 1.000 1.000 1.000 1.000 1.000 1.000 0.00

[0195] Table 20 Relative Retention Time of Test Sample Repeatability

[0196]

[0197]

[0198] Table 21 Repeatability Similarity of Test Samples

[0199] S1 S2 S3 S4 S5 S6 Comparison of fingerprint patterns S1 1 1 1 1 1 1 1 S2 1 1 1 1 1 1 1 S3 1 1 1 1 1 1 1 S4 1 1 1 1 1 1 1 S5 1 1 1 1 1 1 1 S6 1 1 1 1 1 1 1 Comparison of fingerprint patterns 1 1 1 1 1 1 1

[0200] 4. Intermediate precision

[0201] Six portions of the finished drug powder (batch QL22091901, passed through a 24-mesh sieve) were accurately weighed and used to prepare a test solution according to the preparation method of the test sample in Example 1-1. The solution was then injected and analyzed under the chromatographic conditions described in Example 1-1. Using piperine as the S peak, the relative retention time and relative peak area of ​​each common peak were calculated, and the RSD (%, n=6) was calculated. The results showed that the RSD (%) of the relative retention time of each common peak was between 0.00% and 0.12%, and the RSD (%) of the relative peak area was between 0.00% and 5.43%. The similarity was evaluated and calculated using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)". The similarity results were all greater than 0.9. (See attached table for details.) Figure 9 (S1-S6 represent 6 injections respectively) and Tables 22-24 show that the intermediate precision is good.

[0202] Table 22 Relative Peak Area of ​​Intermediate Precision of Test Samples

[0203] Peak Intermediate precision Intermediate precision Intermediate precision Intermediate precision Intermediate precision Intermediate precision RSD Peak 1 0.545 0.543 0.545 0.544 0.545 0.541 0.30 Peak 2 0.176 0.177 0.178 0.176 0.178 0.176 0.56 Peak 3 0.047 0.047 0.047 0.047 0.048 0.047 0.87 Peak 4 0.088 0.087 0.088 0.088 0.088 0.087 0.59 Peak 5 0.093 0.093 0.094 0.093 0.094 0.093 0.56 Peak 6 0.071 0.080 0.077 0.072 0.080 0.080 5.43 Peak 7 0.016 0.016 0.016 0.016 0.016 0.016 0.00 Peak 8 0.066 0.066 0.066 0.066 0.066 0.065 0.62 Peak 9 0.196 0.197 0.196 0.196 0.196 0.195 0.33 Peak 10 0.015 0.015 0.015 0.015 0.015 0.015 0.00 Peak 11 1.000 1.000 1.000 1.000 1.000 1.000 0.00

[0204] Table 23 Relative Retention Time of Intermediate Precision of Test Samples

[0205] Peak Intermediate precision Intermediate precision Intermediate precision Intermediate precision Intermediate precision Intermediate precision RSD Peak 1 0.124 0.124 0.124 0.124 0.124 0.124 0.00 Peak 2 0.392 0.393 0.393 0.393 0.393 0.393 0.11 Peak 3 0.439 0.440 0.439 0.439 0.439 0.440 0.12 Peak 4 0.458 0.459 0.459 0.459 0.459 0.459 0.09 Peak 5 0.573 0.573 0.573 0.573 0.573 0.573 0.00 Peak 6 0.592 0.592 0.592 0.592 0.592 0.592 0.00 Peak 7 0.731 0.731 0.731 0.731 0.731 0.731 0.00 Peak 8 0.764 0.765 0.765 0.765 0.765 0.765 0.06 Peak 9 0.811 0.811 0.811 0.811 0.811 0.811 0.00 Peak 10 0.947 0.947 0.947 0.947 0.947 0.947 0.00 Peak 11 1.000 1.000 1.000 1.000 1.000 1.000 0.00

[0206] Table 24 Intermediate Precision Similarity of Test Samples

[0207] S1 S2 S3 S4 S5 S6 Comparison of fingerprint patterns S1 1 1 1 1 1 1 1 S2 1 1 1 1 1 1 1 S3 1 1 1 1 1 1 1 S4 1 1 1 1 1 1 1 S5 1 1 1 1 1 1 1 S6 1 1 1 1 1 1 1 Comparison of fingerprint patterns 1 1 1 1 1 1 1 .

Claims

1. A method for constructing a fingerprint spectrum of a strong morpholine formulation, characterized in that, It includes the following steps: S1. Preparation of test solution: The test solution is a solution containing the strong madelayate preparation; the extraction solvent used in preparing the test solution is a 75% methanol aqueous solution; S2. Preparation of mixed reference solution: The mixed reference solution includes gallic acid, corilagin, cinnamaldehyde, 6-gingerol, ellagic acid and piperine; the dissolving solvent for preparing the mixed reference solution is a 75% methanol aqueous solution; S3. The test solution and the mixed reference solution are respectively detected by high performance liquid chromatography as follows; Chromatographic conditions: octadecylsilane-bonded silica gel as the filler; acetonitrile as the mobile phase A, and 0.1% phosphoric acid aqueous solution as the mobile phase B, gradient elution was carried out according to the following table; the detection wavelength was 280 nm; the flow rate was 1.2 mL·min -1 ; the column temperature was 30°C; the chromatographic column was YMC-Triart C 18 column, 4.6 mm x 150 mm, 5 μm; the high performance liquid chromatograph was Thermo Scientific UlitiMate 3000 type high performance liquid chromatograph; The percentages in the table represent the volume percentage of each component in the total volume of mobile phase A and mobile phase B, respectively. Based on the retention times of each substance in the mixed reference solution, the peaks in the test solution are assigned, and the detection results of the test solution are used to generate a fingerprint spectrum.

2. The method for constructing the fingerprint spectrum of the strong morpholine formulation as described in claim 1, characterized in that, In the high-performance liquid chromatography, the injection volume is 5-20 μL.

3. The method for constructing the fingerprint spectrum of the strong morpholine formulation as described in claim 2, characterized in that, In the high-performance liquid chromatography, the injection volume is 10 μL.

4. The method for constructing the fingerprint spectrum of the strong morpholine formulation as described in claim 1, characterized in that, In the test solution, the mass-to-volume ratio of the strong madaditraayate preparation to the extraction solvent is 1 g: (40-60) mL; And / or, the test solution is prepared by the following steps: mixing the strong madelayate preparation and the extraction solvent, ultrasonicating, cooling, and filtering.

5. The method for constructing the fingerprint spectrum of the strong morpholine formulation as described in claim 4, characterized in that, In the test solution, the mass-to-volume ratio of the strong madelayate preparation to the extraction solvent is 1g:45mL or 1g:50mL.

6. The method for constructing the fingerprint spectrum of the strong morpholine formulation as described in claim 1, characterized in that, The mixed reference solution contained gallic acid at a concentration of 30-40 μg / mL, corilagin at a concentration of 30-35 μg / mL, cinnamaldehyde at a concentration of 3-5 μg / mL, 6-gingerol at a concentration of 20-30 μg / mL, piperine at a concentration of 90-100 μg / mL, and ellagic acid at a concentration of 5-10 μg / mL.

7. The method for constructing the fingerprint spectrum of the strong morpholine formulation as described in claim 6, characterized in that, In the mixed reference solution, the concentration of gallic acid was 37 μg / mL, the concentration of corilagin was 31 or 31.05 μg / mL, the concentration of cinnamaldehyde was 4 or 4.12 μg / mL, the concentration of 6-gingerol was 25.9 or 26 μg / mL, the concentration of piperine was 97 or 97.10 μg / mL, and the concentration of ellagic acid was 7 or 7.3 μg / mL.

8. The method for constructing the fingerprint spectrum of the strong morpholine formulation as described in claim 1, characterized in that, The fingerprint spectrum obtained by the method for constructing the fingerprint spectrum of the aforementioned strong madoretiate preparation includes 6 common peaks, with peak 11 as the control peak. The relative retention times of the common peaks are as follows: Peak 1 is gallic acid, with a relative retention time of 0.123-0.124 and an RSD of 0.25%. Peak 2 was Corilagin, with a relative retention time of 0.392 and an RSD of 0.00%. Peak 6 is ellagic acid, with a relative retention time of 0.592 and an RSD of 0.00%. Peak 9 is cinnamaldehyde, with a relative retention time of 0.811 and an RSD of 0.00%. Peak 10 was 6-gingerol, with a relative retention time of 0.946 and an RSD of 0.00%. Peak 11 is piperine, with a relative retention time of 1.00 and an RSD of 0.00%.

9. The method for constructing the fingerprint spectrum of the strong morpholine formulation as described in claim 8, characterized in that, The fingerprint spectrum obtained by the method for constructing the fingerprint spectrum of the aforementioned strong madoretiate preparation also includes the following 5 common peaks, with peak 11 as the control peak. The relative retention times of the common peaks are as follows: The relative retention time of peak 3 was 0.439, and the RSD was 0.00%. The relative retention time of peak 4 was 0.458–0.459, with an RSD of 0.09%. The relative retention time of peak 5 was 0.572–0.573, with an RSD of 0.08%. The relative retention time of peak 7 was 0.731, and the RSD was 0.00%. The relative retention time of peak 8 was 0.764, and the RSD was 0.00%.

10. A method for detecting the quality of a strong morphine preparation, characterized in that, The detection method includes generating a fingerprint spectrum of the sample to be tested according to the method for constructing a fingerprint spectrum of a thymolide thymolide formulation as described in any one of claims 1-9.

11. A quality control method for a strong morphine preparation, characterized in that, The fingerprint spectrum obtained by the method for constructing the fingerprint spectrum of the strong matte teratate preparation as described in any one of claims 1-9 is used as a standard spectrum for the quality control of the strong matte teratate preparation.

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