Construction method and application of Simiao pill fingerprint spectrum

Abstract

The invention provides a construction method and application of a Simiao pill fingerprint spectrum, and belongs to the technical field of medicine detection.The construction method includes the steps that a test solution is prepared from Simiao pills, acetonitrile (A)-0.08-0.12 wt% phosphoric acid aqueous solution (B) serves as a mobile phase, the fingerprint spectrum of the Simiao pill is obtained through high performance liquid chromatography detection, a standard fingerprint spectrum is established, and the standard fingerprint spectrum is used for determining the content of the Simiao pill. The method is used for quality evaluation or control in the whole research / development / production / clinical application process of the Simiao pill. According to the construction method of the Simiao pill fingerprint spectrum, the main medicinal material components in the Simiao pill can be detected through high performance liquid chromatography, so that the quality condition of the Simiao pill can be reflected more comprehensively.

Description

Technical Field

[0001] The present invention relates to drug detection technology, and in particular to a method for constructing a fingerprint spectrum of Simiao Pills and its application. Background Art

[0002] Simiao Pills is a Chinese patent medicine, a dehumidifying agent, with the effect of clearing away heat and removing dampness. It is mainly used to treat arthralgia caused by damp-heat, with symptoms including redness and swelling of the feet and knees and pain in the tendons and bones. Since the prepared Simiao Pills have lost the morphological characteristics of the original pieces, the qualitative identification and quantitative analysis of the index components alone are difficult to reflect the quality. As a standard reference to measure whether the classic Chinese medicine prescriptions are basically consistent with clinical decoctions, its quality should strengthen specific identification and multi-component and overall quality control.

[0003] The spectrum of Chinese medicine preparations refers to the spectrum that can reflect the information of multiple components and reflect its quality characteristics after the samples of Chinese medicine preparations are properly processed and analyzed by appropriate methods. The characteristic spectrum of Chinese medicine preparations is of great significance for identifying the key quality attributes of Chinese medicine preparations, studying the transfer of quantity values, evaluating the uniformity and stability of the quality of Chinese medicine preparations, and improving the overall quality control level of Chinese medicine preparations.

[0004] "Establishment of HPLC fingerprint of Si Miao Wan" [J]. Chinese patent medicine. Xia Ting et al. (hereinafter referred to as reference) disclosed a method for constructing a fingerprint of Si Miao Wan. The characteristic spectrum recorded in this method has 31 characteristic peaks, including 1 chromatographic peak from Achyranthes bidentata, 27 from Phellodendron chinense, 7 chromatographic peaks from Atractylodes lancea, and 1 common peak between Phellodendron chinense and Atractylodes lancea. However, in the reproducibility experiment, it was proved that the chromatographic peaks from Achyranthes bidentata in the document could not be reproduced, and the quality of Achyranthes bidentata in the Si Miao Wan formula could not be accurately analyzed. Moreover, the number of characteristic peaks separated by this method as a whole is relatively small, which needs further development.

[0005] Therefore, it is necessary to further develop the fingerprint construction method of Simiao Pills in order to achieve more comprehensive control of the overall quality of Simiao Pills and to conduct better and more complete qualitative analysis of Achyranthes bidentata in Simiao Pills. Summary of the invention

[0006] In view of the above problems, the present invention provides a method for constructing a fingerprint spectrum of Simiao Pills and its application.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is:

[0008] A method for constructing a fingerprint spectrum of Simiao Pills, the method comprising the following steps:

[0009] S1. Prepare the test solution with Simiao Pills;

[0010] S2, taking the test solution and performing high performance liquid chromatography to obtain the fingerprint of the Simiao Pills;

[0011] During the high performance liquid chromatography detection process, acetonitrile is used as mobile phase A, 0.08-0.12 wt % phosphoric acid aqueous solution is used as mobile phase B, and elution is performed in a gradient elution manner;

[0012] The elution procedure is:

[0013] The mobile phase is, the elution mode is gradient elution, and the elution program is:

[0014] 0-20min: 7%→10% mobile phase A, 93%→90% mobile phase B;

[0015] 20-30 min: 10% → 18% mobile phase A, 90% → 82% mobile phase B;

[0016] 30-45 min: 18% → 23% mobile phase A, 82% → 77% mobile phase B;

[0017] 45-50 min: 23% → 24% mobile phase A, 77% → 76% mobile phase B;

[0018] 50-55 min: 24% → 48% mobile phase A, 76% → 52% mobile phase B;

[0019] 55-60min: 48%→60% mobile phase A, 52%→40% mobile phase B;

[0020] 60-80 min: 60%→70% mobile phase A, 40%→30% mobile phase B;

[0021] 80-81 min: 70% → 7% mobile phase A, 30% → 93% mobile phase B;

[0022] 81-90min: 7% mobile phase A, 93% mobile phase B.

[0023] Furthermore, in the HPLC detection process, the wavelength detection procedure is:

[0024] 0~56min, 250nm;

[0025] 56~90min, 320nm.

[0026] Furthermore, in the HPLC detection process, the chromatographic column uses octadecylsilane bonded silica gel as a filler.

[0027] Furthermore, the test solution is prepared by adding water-saturated n-butanol to Simiao Pills, extracting with ultrasonic treatment, cooling, filtering, evaporating to dryness, and dissolving the residue with methanol.

[0028] Furthermore, in the process of preparing the test solution, the weight volume ratio of Simiao Pills to water-saturated n-butanol was 0.5-0.7 g:30 mL.

[0029] Furthermore, during the preparation of the test solution, the weight volume ratio of Simiao Pills to the test solution was 0.5-0.7 g:20 mL.

[0030] Furthermore, the ultrasonic treatment time is more than 0.5 h.

[0031] Furthermore, the flow rate of the HPLC detection is 0.9-1.1 mL / min, the column temperature is 28-35°C, and the injection volume is 5-20 μL;

[0032] The construction method also includes using berberine hydrochloride to prepare a reference solution and performing high performance liquid chromatography detection.

[0033] An application of the standard fingerprint of Simiao Pills obtained by the above construction method in the quality evaluation or control of the whole process of research / development / production / clinical application of Simiao Pills.

[0034] Furthermore, a total of 48 common peaks were calibrated in the standard fingerprint of the Simiao Pills, of which peak No. 6 was chlorogenic acid, peak No. 10 was phellodendronine hydrochloride, peak No. 12 was magnolia pine, peak No. 18 was ferulic acid, peak No. 22 was ecdysterone, peak No. 23 was 25R-cyperasterone, peak No. 24 was 25S-cyperasterone, peak No. 31 was jatrorrhizine hydrochloride, peak No. 34 was bamipine hydrochloride, peak No. 35 was berberine hydrochloride, and peak No. 48 was atractylodesin.

[0035] The beneficial effects of the method for constructing the fingerprint spectrum of Simiao Pills of the present invention and its application are as follows:

[0036] The construction method of the present invention is based on references, and improves conditions such as detection wavelength and mobile phase gradient, so that the number of identified characteristic peaks of Achyranthes bidentata is significantly increased, the number of characteristic peaks of Atractylodes lancea is increased by 1, and the number of characteristic peaks of Phellodendron chinense is appropriately reduced, so that the Si Miao Wan can be evaluated more comprehensively;

[0037] The method for constructing the fingerprint spectrum of Simiao Pills of the present invention can detect the main medicinal ingredients in Simiao Pills by high performance liquid chromatography, thereby more comprehensively reflecting the quality status of Simiao Pills;

[0038] The present invention combines the different physical and chemical properties of different effective ingredients in Simiao Pills and adjusts the elution conditions to obtain a standard fingerprint spectrum with good separation, a relatively stable baseline, more peaks and better reflection of the quality of Simiao Pills.

[0039] The present invention obtains a standard fingerprint of the quality of Simiao Pills with a more stable baseline, better peak shape and separation effect by adjusting the mobile phase;

[0040] The present invention can separate different effective components in Simiao Pills well by adjusting the chromatographic column, and the response value and separation degree of the chromatographic peak are good;

[0041] The present invention can more fully extract the medicinal ingredients involved in Simiao Pills by adjusting the preparation method of the test solution, and the concentration of each medicinal ingredient in the obtained test solution can be separated and detected under the same high performance liquid chromatography conditions, and the peaks in the obtained chromatogram are good;

[0042] The present invention effectively optimizes the separation conditions by selecting appropriate process conditions, thereby improving the precision, repeatability, stability and durability of Simiao Pills;

[0043] The standard fingerprint spectrum of Simiao Pills obtained by the present invention can provide effective guarantee for the quality monitoring of Simiao Pills in the whole process of research, development, production and clinical application.

[0044] By using the presence and characteristics of the common characteristic peaks in the standard fingerprint spectrum of Simiao Pills obtained by the present invention, the product quality of Simiao Pills can be fully monitored. By comparing the similarity of the chromatographic fingerprint characteristics, the quality of Simiao Pills can be evaluated, and the stability and uniformity can be examined, which makes up for the shortcomings of the current quality control method. At the same time, the stability of the production process of Simiao Pills can also be monitored to ensure the stability, uniformity and controllability of its quality.

[0045] The standard fingerprint of Simiao Pills obtained by the present invention improves the quality monitoring standards of finished and semi-finished products of Simiao Pills, effectively prevents the occurrence of product counterfeiting incidents, and ensures the normal production and circulation order of Simiao Pills; on the basis of the present invention, the correlation study between fingerprint information and pharmacodynamic activity information can also be carried out, so as to deeply clarify the correlation between the intrinsic chemical components of Simiao Pills and the efficacy of the preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is the characteristic spectrum obtained by detecting the test solution in Example 1 of the present invention, that is, the fingerprint spectrum of Simiao Pills;

[0047] Figure 2 This is the peak attribution investigation result of Phellodendron chinense during the peak attribution investigation in Example 1 of the present invention; Figure 2 From top to bottom in the middle are the fingerprints of Simiao Pills, the fingerprints obtained from the solution of Phellodendron amurense and the fingerprints obtained from the negative test solution of Phellodendron amurense;

[0048] Figure 3 This is the peak attribution investigation result of Achyranthes bidentata during the peak attribution investigation process in Example 1 of the present invention; Figure 3From top to bottom in the middle are the fingerprint of Simiao Pills, the fingerprint of Achyranthes bidentata single herb solution and the fingerprint of Achyranthes bidentata negative test solution;

[0049] Figure 4 is the peak attribution investigation result of Atractylodes lancea during the peak attribution investigation process in Example 1 of the present invention; Figure 4 From top to bottom are the fingerprint of Simiao Pills, the fingerprint obtained from Atractylodes single herb solution and the fingerprint obtained from Atractylodes negative test solution. DETAILED DESCRIPTION

[0050] The technical solutions in the embodiments of the present invention are described clearly and completely below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0051] Example 1 A method for constructing a fingerprint of Simiao Pills and its application

[0052] In this embodiment, Si Miao Wan is used as the sample to be tested, wherein the preparation method of Si Miao Wan is as follows:

[0053] Take 125g of Atractylodes lancea, 125g of Achyranthes bidentata, 250g of Phellodendron chinense (fried with salt) and 250g of Coix seed, grind them into fine powder, sieve, mix well, make pills, and dry to get Simiao Pills.

[0054] This example uses Si Miao Wan as a Si Miao Wan sample to construct a fingerprint of Si Miao Wan. The specific method is as follows:

[0055] S1. Solution preparation

[0056] S11. Preparation method of test solution

[0057] Take an appropriate amount of Simiao Pills, grind them into powder, take about 0.6g, weigh it accurately, put it in a 50mL conical flask, add 30mL of water-saturated n-butanol, ultrasonically treat it (250W, 40KHz) for 1h, cool it, filter it, rinse the filter paper and conical flask with an appropriate amount of methanol, combine the filtrate and washing liquid, evaporate it to dryness, dissolve the residue with an appropriate amount of methanol, transfer it to a 20mL volumetric flask, add methanol to the scale, plug it tightly, shake it well, filter it, and take the filtrate to obtain the test solution.

[0058] S12. Preparation of reference solution

[0059] Take an appropriate amount of berberine hydrochloride, weigh it accurately, and add methanol to make a solution containing 55 μg per 1 mL to obtain the reference solution.

[0060] S2. High performance liquid chromatography qualitative analysis

[0061] The test solution and the reference solution are respectively taken for high performance liquid chromatography detection to obtain corresponding high performance liquid chromatograms and corresponding characteristic spectra.

[0062] Among them, the chromatographic conditions for detecting the reference solution are:

[0063] Chromatographic column: Octadecylsilane bonded silica gel as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm);

[0064] Column temperature: 30°C;

[0065] Flow rate: 1.0 mL / min;

[0066] The detection wavelength program is: 0-56 min, 250 nm; 56-90 min, 320 nm.

[0067] Injection volume: 10 μL;

[0068] The mobile phase was acetonitrile (A)-0.1wt% phosphoric acid aqueous solution (B), the elution method was gradient elution, and the elution program was:

[0069] 0-20min: 7%→10% mobile phase A, 93%→90% mobile phase B;

[0070] 20-30 min: 10% → 18% mobile phase A, 90% → 82% mobile phase B;

[0071] 30-45 min: 18% → 23% mobile phase A, 82% → 77% mobile phase B;

[0072] 45-50 min: 23% → 24% mobile phase A, 77% → 76% mobile phase B;

[0073] 50-55 min: 24% → 48% mobile phase A, 76% → 52% mobile phase B;

[0074] 55-60min: 48%→60% mobile phase A, 52%→40% mobile phase B;

[0075] 60-80 min: 60%→70% mobile phase A, 40%→30% mobile phase B;

[0076] 80-81 min: 70% → 7% mobile phase A, 30% → 93% mobile phase B;

[0077] 81-90min: 7% mobile phase A, 93% mobile phase B.

[0078] S3. Test results

[0079] The characteristic spectrum obtained by testing the test solution is as follows Figure 1 As shown, a total of 48 characteristic peaks are obtained, as shown in the following table:

[0080] Table 1 List of characteristic peaks in the characteristic spectrum obtained from the test solution

[0081]

[0082]

[0083] It can be seen that 48 characteristic peaks can be obtained by qualitatively analyzing Simiao Pills using the construction method of the present invention. Among them, peak 6 is chlorogenic acid, peak 10 is phellodendronine hydrochloride, peak 12 is magnolia pine, peak 18 is ferulic acid, peak 22 is ecdysterone, peak 23 is 25R-cyperidinol, peak 24 is 25S-cyperidinol, peak 31 is jatrorrhizine hydrochloride, peak 34 is palmatine hydrochloride, peak 35 is berberine hydrochloride, and peak 48 is atractylodesin.

[0084] The characteristic spectrum obtained in reference 1 has 31 characteristic peaks, including 1 peak from Achyranthes bidentata, 27 from Phellodendron chinense, 7 from Atractylodes lancea, and 1 peak common to Phellodendron chinense and Atractylodes lancea. However, the reproducibility experiment showed that the chromatographic peak from Achyranthes bidentata in the reference could not be reproduced.

[0085] S5. Investigation of peak attribution

[0086] S51. Preparation of single herbal medicine solutions of Phellodendron chinense, Atractylodes macrocephala and Achyranthes bidentata

[0087] Take 0.2g of Phellodendron chinense, 0.1g of Achyranthes bidentata, and 0.1g of Atractylodes lancea, respectively, and place them in 50mL conical flasks, add 30mL of water-saturated n-butanol, ultrasonically treat (250W, 40KHz) for 1h, cool, filter, rinse the filter paper and conical flask with appropriate amount of methanol, combine the filtrate and washing liquid, evaporate to dryness, dissolve the residue with appropriate amount of methanol, transfer to a 20mL volumetric flask, add methanol to the scale, plug it tightly, shake well, filter, and take the filtrate to obtain the corresponding single drug solution.

[0088] S52. Preparation of negative sample test solution

[0089] Preparation of negative test solution of Phellodendron chinense: Take 0.2g of coix seed control medicinal material, 0.1g of Achyranthes bidentata control medicinal material and 0.1g of Atractylodes lancea control medicinal material, put them in a 50mL conical flask, add 30mL of water-saturated n-butanol, ultrasonically treat (250W, 40KHz) for 1h, cool, filter, rinse the filter paper and conical flask with appropriate amount of methanol, combine the filtrate and washing liquid, evaporate to dryness, dissolve the residue with appropriate amount of methanol, transfer to a 20mL volumetric flask, add methanol to the scale, plug it tightly, shake well, filter, and take the filtrate to obtain the negative test solution of Phellodendron chinense.

[0090] Preparation of negative test solution of Achyranthes bidentata: Take 0.2g of coix seed control medicinal material, 0.2g of Phellodendron chinense control medicinal material and 0.1g of Atractylodes lancea control medicinal material, place them in a 50mL conical flask, add 30mL of water-saturated n-butanol, ultrasonically treat (250W, 40KHz) for 1h, cool, filter, rinse the filter paper and conical flask with appropriate amount of methanol, combine the filtrate and washing liquid, evaporate to dryness, dissolve the residue with appropriate amount of methanol, transfer to a 20mL volumetric flask, add methanol to the scale, plug it tightly, shake well, filter, and take the filtrate to obtain the negative test solution of Phellodendron chinense.

[0091] Preparation of negative test solution of Atractylodes lancea: Take 0.2g of coix seed control medicinal material, 0.2g of Phellodendron chinense control medicinal material and 0.1g of Achyranthes bidentata control medicinal material, put them in a 50mL conical flask, add 30mL of water-saturated n-butanol, ultrasonically treat (250W, 40KHz) for 1h, cool, filter, rinse the filter paper and conical flask with appropriate amount of methanol, combine the filtrate and washing liquid, evaporate to dryness, dissolve the residue with appropriate amount of methanol, transfer to a 20mL volumetric flask, add methanol to the scale, plug it tightly, shake well, filter, and take the filtrate to obtain the negative test solution of Atractylodes lancea.

[0092] Take the test solution, the corresponding single herb solution and the corresponding negative test solution respectively, and perform high performance liquid chromatography detection using the chromatographic conditions in step S2 to obtain the corresponding high performance liquid chromatogram, and determine the source of each characteristic peak. Figure 2 to Figure 4 .

[0093] Depend on Figure 2 to Figure 4 It can be seen that the construction method of the present invention obtains a total of 48 characteristic peaks in the characteristic spectrum, of which 4 characteristic peaks can be proven to be derived from Achyranthes bidentata, 36 from Phellodendron chinense, 9 chromatographic peaks from Atractylodes lancea, and 1 common peak between Achyranthes bidentata and Atractylodes lancea.

[0094] The construction method of the present invention is based on references, and improves conditions such as detection wavelength and mobile phase gradient, so that the number of identified characteristic peaks of Achyranthes bidentata is significantly increased, the number of characteristic peaks of Atractylodes lancea is increased by 1, and the number of characteristic peaks of Phellodendron chinense is appropriately reduced.

[0095] S6. Fingerprint establishment and similarity results

[0096] Fifteen batches of Simiao Pills were used to prepare test solutions for fingerprint detection. The common peaks, similarities and standard fingerprints were obtained through the Chinese medicine chromatographic fingerprint similarity evaluation system (2012,130723 version). Peak 34 corresponded to the retention time of the berberine hydrochloride reference peak. The peak corresponding to the berberine hydrochloride reference peak was taken as the S peak. The relative retention time of each peak and the S peak was calculated. The retention time should be within the range of ±10% of the specified value. The specified value The values ​​are: 0.08 (peak 1), 0.10 (peak 2), 0.12 (peak 3), 0.12 (peak 4), 0.14 (peak 5), 0.16 (peak 6), 0.17 (peak 7), 0.23 (peak 8), 0.26 (peak 9), 0.29 (peak 10), 0.33 (peak 11), 0.34 (peak 12), 0.46 (peak 13), 0.51 (peak 14), 0.52 (peak 15), 0.55 (peak 16), 0.56 (peak 17). Peak 17), 0.59 (Peak 18), 0.60 (Peak 19), 0.61 (Peak 20), 0.62 (Peak 21), 0.63 (Peak 22), 0.65 (Peak 23), 0.67 (Peak 24), 0.67 (Peak 25), 0.70 (Peak 26), 0.72 (Peak 27), 0.73 (Peak 28), 0.74 (Peak 29), 0.82 (Peak 30), 0.84 (Peak 31), 0.86 (Peak 32), 0. 93 (peak 33), 0.99 (peak 34), 1.00 (peak 35), 1.15 (peak 36), 1.16 (peak 37), 1.17 (peak 38), 1.17 (peak 39), 1.19 (peak 40), 1.20 (peak 41), 1.21 (peak 42), 1.24 (peak 43), 1.28 (peak 44), 1.33 (peak 45), 1.34 (peak 46), 1.36 (peak 47), 1.45 (peak 48).

[0097] S7. Application of standard fingerprint

[0098] The standard fingerprint of Simiao Pills obtained in this example can be used for quality evaluation or control in the whole process of research / development / production / clinical application of Simiao Pills.

[0099] Embodiments 2 to 5 Method for constructing fingerprints of Si Miao Wan

[0100] Examples 2 to 5 are respectively methods for constructing a fingerprint of Simiao Pills. According to the optimal construction conditions of the fingerprint of Simiao Pills given in Example 1, the range of construction conditions that can achieve a better fingerprint of Simiao Pills is further explored to obtain the construction methods in Examples 2 to 5. Among them, the construction methods of the fingerprint of Simiao Pills in Examples 2 to 5 are basically the same as those in Example 1, and the difference is only in the difference of some process parameters, as shown in Table 2 for details:

[0101] Table 2 List of process parameters in Examples 2 to 5

[0102]

[0103]

[0104] The contents and results of other parts of Examples 2 to 5 are the same as those of Example 1 and will not be repeated here (since other embodiments of the present invention have been able to fully prove that this method can better construct the fingerprint spectrum of Simiao Pills, no additional drawings will be added here).

[0105] Example 6 Fingerprint Methodology Study

[0106] 1. Precision inspection

[0107] The same test solution was taken and HPLC detection was performed according to the chromatographic conditions of Example 1. The sample was injected six times continuously. Berberine hydrochloride (i.e., peak No. 34) was used as the reference peak. The relative retention time of each common peak was calculated to obtain the corresponding RSD value. The results are shown in Table 3.

[0108] Table 3 Precision relative retention time results summary

[0109]

[0110]

[0111] It can be seen from Table 3 that the construction method of the present invention has good precision.

[0112] 2. Repeatability Study

[0113] The same person prepared 6 test solutions in parallel using the same method, and performed HPLC detection according to the chromatographic conditions of Example 1. Berberine hydrochloride (i.e., peak No. 34) was used as the reference peak, and the relative retention time of each common peak was calculated to obtain the corresponding RSD value. The results are shown in Table 4.

[0114] Table 4 Summary of repeatability relative retention time results

[0115]

[0116]

[0117] It can be seen from Table 4 that the construction method of the present invention has good repeatability.

[0118] 3. Stability inspection

[0119] The same test solution was injected at 0 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h, respectively, and HPLC detection was performed according to the chromatographic conditions of Example 1. Berberine hydrochloride (i.e., peak No. 34) was used as the reference peak, and the relative retention time of each common peak was calculated to obtain the corresponding RSD value. The results are shown in Table 5.

[0120] Table 5 Summary of stability relative retention time results

[0121]

[0122]

[0123] It can be seen from Table 5 that the construction method of the present invention has good stability within 48 hours.

[0124] 4. Durability inspection

[0125] 1) Flow rate investigation

[0126] The same test solution was taken to investigate the durability of different flow rates (0.9, 1.0, 1.1 mL / min). The durability of the chromatographic conditions in Example 1 was adopted (i.e., only the flow rate in the chromatographic conditions of Example 1 was changed). Berberine hydrochloride (i.e., peak No. 34) was used as the reference peak. The relative retention time of each common peak was calculated to obtain the corresponding RSD value. The results are shown in Table 6.

[0127] Table 6 Summary of flow rate durability relative to retention time results

[0128] Peak Flow rate (1.1 mL / min) Flow rate (0.9mL / min) Flow rate 1.0 (mL / min) average value RSD(%) 1 0.09 0.09 0.09 0.09 0.01 2 0.10 0.10 0.10 0.10 0.92 3 0.12 0.12 0.12 0.12 0.13 4 0.15 0.15 0.15 0.15 0.01 5 0.17 0.17 0.17 0.17 0.04 6 0.29 0.29 0.29 0.29 0.81 7 0.33 0.33 0.33 0.33 0.01 8 0.35 0.35 0.35 0.35 0.84 9 0.41 0.40 0.40 0.40 0.81 10 0.46 0.46 0.46 0.46 0.32 11 0.48 0.47 0.47 0.47 0.81 12 0.50 0.50 0.50 0.50 0.33 13 0.51 0.52 0.52 0.52 0.80 14 0.55 0.53 0.54 0.54 1.67 15 0.56 0.55 0.55 0.56 0.79 16 0.59 0.59 0.59 0.59 0.50 17 0.61 0.61 0.60 0.61 0.63 18 0.62 0.62 0.61 0.62 0.59 19 0.63 0.62 0.62 0.62 0.68 20 0.65 0.63 0.63 0.64 0.92 21 0.66 0.65 0.65 0.65 0.58 22 0.68 0.67 0.67 0.67 0.69 23 0.68 0.69 0.69 0.68 0.29 24 0.70 0.69 0.70 0.69 0.47 25 0.71 0.70 0.70 0.71 0.43 26 0.71 0.72 0.71 0.71 0.73 27 0.72 0.73 0.72 0.72 0.52 28 0.74 0.75 0.74 0.74 0.45 29 0.77 0.76 0.76 0.76 0.88 30 0.80 0.82 0.82 0.81 1.25 31 0.87 0.86 0.86 0.86 0.65 32 0.93 0.94 0.93 0.93 0.38 33 0.99 0.99 0.99 0.99 0.11 34 1.00 1.00 1.00 1.00 0.00 35 1.14 1.14 1.16 1.15 0.63 36 1.15 1.15 1.16 1.15 0.68 37 1.17 1.15 1.17 1.16 0.58 38 1.18 1.18 1.17 1.18 0.29 39 1.18 1.18 1.18 1.18 0.07 40 1.21 1.21 1.22 1.21 0.40 41 1.23 1.22 1.23 1.23 0.38 42 1.24 1.24 1.23 1.23 0.01 43 1.26 1.26 1.27 1.26 0.43 44 1.29 1.31 1.32 1.31 0.86 45 1.31 1.34 1.34 1.33 1.10 46 1.37 1.35 1.36 1.36 0.52 47 1.38 1.38 1.38 1.38 0.18 48 1.44 1.44 1.44 1.44 0.10

[0129] It can be seen from the above table that the durability of different flow rates in the method of the present invention is good, and the flow rate durability is less than 5.0%.

[0130] 2) Column temperature investigation

[0131] The same test solution was taken to investigate the durability of different column temperatures (28, 30, 32, 35°C). The durability of the chromatographic conditions in Example 1 was adopted (i.e., only the column temperature in the chromatographic conditions of Example 1 was changed). Berberine hydrochloride (i.e., peak No. 34) was used as the reference peak. The relative retention time of each common peak was calculated to obtain the corresponding RSD value. The results are shown in Table 7.

[0132] Table 7 Column temperature durability relative retention time results summary

[0133]

[0134]

[0135] It can be seen from the above table that the durability of different column temperatures in the method of the present invention is good, and the column temperature durability is less than 5.0%.

[0136] 3) Chromatographic column inspection

[0137] The same test solution was taken to investigate the durability of different chromatographic columns (chromatographic column 038, chromatographic column 052, chromatographic column 066). The durability of the chromatographic conditions in Example 1 was adopted (i.e., only the chromatographic column in the chromatographic conditions of Example 1 was changed). Berberine hydrochloride (i.e., peak No. 34) was used as the reference peak. The relative retention time of each common peak was calculated to obtain the corresponding RSD value. The results are shown in Table 8.

[0138] Table 8 Summary of column durability relative retention time results

[0139]

[0140]

[0141] It can be seen from the above table that the durability of the chromatographic column at different temperatures in the method of the present invention is good, and the durability of the chromatographic column is less than 5.0%.

[0142] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A method for constructing a fingerprint of Simiao Pills, characterized in that: The construction method comprises the following steps: S1. Prepare the test solution with Simiao Pills; S2, taking the test solution and performing high performance liquid chromatography to obtain the fingerprint of the Simiao Pills; During the high performance liquid chromatography detection process, acetonitrile is used as mobile phase A, 0.08-0.12 wt % phosphoric acid aqueous solution is used as mobile phase B, and elution is performed in a gradient elution manner; The elution procedure is: The mobile phase is, the elution mode is gradient elution, and the elution program is: 0-20min: 7%→10% mobile phase A, 93%→90% mobile phase B; 20-30 min: 10% → 18% mobile phase A, 90% → 82% mobile phase B; 30-45 min: 18% → 23% mobile phase A, 82% → 77% mobile phase B; 45-50 min: 23% → 24% mobile phase A, 77% → 76% mobile phase B; 50-55 min: 24% → 48% mobile phase A, 76% → 52% mobile phase B; 55-60min: 48%→60% mobile phase A, 52%→40% mobile phase B; 60-80 min: 60%→70% mobile phase A, 40%→30% mobile phase B; 80-81 min: 70% → 7% mobile phase A, 30% → 93% mobile phase B; 81-90min: 7% mobile phase A, 93% mobile phase B.

2. The method for constructing the fingerprint of Simiao Pills according to claim 1, characterized in that: During the HPLC detection process, the wavelength detection procedure is as follows: 0~56min, 250nm; 56~90min, 320nm.

3. The method for constructing the fingerprint of Simiao Pills according to claim 1 or 2, characterized in that: In the high performance liquid chromatography detection process, the chromatographic column uses octadecylsilane bonded silica gel as a filler.

4. The method for constructing the fingerprint of Simiao Pills according to claim 1 or 2, characterized in that: The test solution is prepared by adding water-saturated n-butanol to Simiao Pills, extracting by ultrasonic treatment, cooling, filtering, evaporating to dryness, and dissolving the residue with methanol.

5. The method for constructing the fingerprint of Simiao Pills according to claim 4, characterized in that: During the preparation of the test solution, the weight volume ratio of Simiao Pills to water-saturated n-butanol was 0.5-0.7 g:30 mL.

6. The method for constructing the fingerprint of Simiao Pills according to claim 4, characterized in that: During the preparation of the test solution, the weight volume ratio of Simiao Pills to the test solution was 0.5-0.7 g:20 mL.

7. The method for constructing the fingerprint of Simiao Pills according to claim 4, characterized in that: The ultrasonic treatment time is more than 0.5 h.

8. The method for constructing the fingerprint of Simiao Pills according to claim 1, 2, 5, 6 or 7, characterized in that: The flow rate of the high performance liquid chromatography detection is 0.9-1.1 mL / min, the column temperature is 28-35° C., and the injection volume is 5-20 μL.

9. An application of the standard fingerprint of Simiao Pills obtained by the construction method described in any one of claims 1 to 8 in the quality evaluation or control of the whole process of research / development / production / clinical application of Simiao Pills.

10. The use according to claim 9, characterized in that: A total of 48 common peaks were calibrated in the standard fingerprint of the Simiao Pills, of which peak No. 6 was chlorogenic acid, peak No. 10 was phellodendronine hydrochloride, peak No. 12 was magnolia pine, peak No. 18 was ferulic acid, peak No. 22 was ecdysterone, peak No. 23 was 25R-cyperasterone, peak No. 24 was 25S-cyperasterone, peak No. 31 was jatrorrhizine hydrochloride, peak No. 34 was bamipine hydrochloride, peak No. 35 was berberine hydrochloride, and peak No. 48 was atractylodesin.

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