Construction method, fingerprint spectrum and application of fingerprint spectrum of Bazhen paste

Abstract

The application discloses a construction method, fingerprint and application of a fingerprint spectrum of Bazhen paste. The construction method comprises the following steps: weighing Bazhen paste to obtain a test sample after the Bazhen paste is added into a methanol solution; weighing 5-hydroxymethyl furfural, paeoniflorin, chlorogenic acid, baicalin, ammonium glycyrrhizinate and glycyrrhizin, and adding the substances into a methanol solution to obtain reference samples; performing high performance liquid chromatography analysis on the test sample and the reference samples respectively to obtain chromatograms of the test sample and the reference samples respectively; determining common peaks from the chromatogram of the test sample, comparing the common peaks with the chromatograms of the reference samples, identifying the chemical components of the common peaks, then selecting reference peaks, calculating the relative retention time of the common peaks, and obtaining the fingerprint spectrum of the Bazhen paste. The construction method of the fingerprint spectrum of the Bazhen paste is beneficial to obtaining a larger number of chromatographic peaks, so that the overall detection of multiple components of the Bazhen paste can be realized, and the number of chromatographic peaks of the liquid chromatography obtained under the condition of gradient elution is large and the resolution is good.

Description

Technical Field

[0001] This application relates to the field of quality control technology for Bazhen Ointment, and in particular to a method for constructing a fingerprint spectrum of Bazhen Ointment, as well as the fingerprint spectrum and its application. Background Technology

[0002] Bazhen Gao (Eight-Treasure Paste) is composed of eight herbs: Codonopsis pilosula, Atractylodes macrocephala (fried), Poria cocos, Glycyrrhiza uralensis, Angelica sinensis, Paeonia lactiflora, Ligusticum chuanxiong, and Rehmannia glutinosa (processed). It has the effect of tonifying qi and nourishing blood, and is suitable for those with deficiency of both qi and blood, sallow complexion, and weakness in the limbs. Currently, the quality control of traditional Chinese medicine compound preparations mainly employs thin-layer chromatography and high-performance liquid chromatography (HPLC) to qualitatively and quantitatively analyze the active ingredients. These techniques primarily target the detection of single active ingredients. However, Chinese medicinal materials contain a wide variety of compounds, and different proportions of herbs, their origins, and pretreatment methods can all affect their composition and efficacy. Therefore, a single or limited number of chemical components cannot fully characterize the pharmacological effects of Chinese medicinal materials. Furthermore, the analysis of Chinese medicinal materials faces challenges such as complex composition and difficulty in separation.

[0003] Bazhen Gao currently only has internal enterprise standards. Its ingredients are relatively complex, and the qualitative identification and quantitative analysis of simple indicator components are insufficient to fully control the quality of Bazhen Gao. Its quality standards should strengthen specific identification and control of multiple components and overall quality. Summary of the Invention

[0004] This invention provides a method for constructing a fingerprint spectrum of Bazhen Ointment, as well as the fingerprint spectrum and its application, to solve the technical problem that existing analytical methods are unable to fully control the internal quality of Bazhen Ointment.

[0005] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0006] In a first aspect, the present invention provides a method for constructing a fingerprint spectrum of Bazhen Ointment, comprising the following steps:

[0007] S1. Weigh out Bazhen Ointment and add it to methanol solution to obtain the test sample; weigh out 5-hydroxymethylfurfural, paeoniflorin, chlorogenic acid, baicalin, ammonium glycyrrhizate and glycyrrhizin respectively, and add them to methanol solution to obtain reference standards containing the above six components respectively.

[0008] S2. Perform high performance liquid chromatography analysis on the test sample and the reference sample obtained in step S1 to obtain chromatograms of the test sample and each of the reference samples respectively.

[0009] The conditions for the high-performance liquid chromatography (HPLC) analysis are as follows: the stationary phase of the column is octadecylsilane-bonded silica gel, and the mobile phase is 0.1%–0.2% (v / v) inorganic acid solution as mobile phase A and acetonitrile as mobile phase B for gradient elution; the gradient elution steps are performed according to the following procedure, expressed as volume fractions:

[0010] From 0 to 10 minutes, mobile phase A decreased from 93% to 85%, while mobile phase B increased from 7% to 15%.

[0011] Within 10–25 minutes, mobile phase A decreased from 85% to 76%, while mobile phase B increased from 15% to 24%.

[0012] Over 25–30 minutes, mobile phase A decreased from 76% to 70%, while mobile phase B increased from 24% to 30%.

[0013] Over 30–35 minutes, mobile phase A decreased from 70% to 55%, while mobile phase B increased from 30% to 45%.

[0014] Over 35–40 minutes, mobile phase A decreased from 55% to 10%, while mobile phase B increased from 45% to 90%.

[0015] 40–50 min, mobile phase A is 10%, mobile phase B is 90%;

[0016] From 50 to 51 minutes, the percentage of mobile phase A increased from 10% to 93%, while the percentage of mobile phase B decreased from 90% to 7%.

[0017] 51–60 min, mobile phase A is 93%, mobile phase B is 7%;

[0018] S3. Determine the common peak from the chromatogram of the test sample, compare the common peak with the chromatogram of the reference standard, identify the chemical components of the common peak, select a reference peak, calculate the relative retention time of the common peak, and construct the fingerprint spectrum of the Bazhen Ointment.

[0019] Furthermore, the fingerprint chromatogram of the Bazhen Ointment was evaluated for similarity. The similarity evaluation method was as follows: the chromatograms of multiple batches of the test samples and the fingerprint chromatogram of the Bazhen Ointment were imported into similarity evaluation software for similarity evaluation analysis, and the similarity was greater than 0.9. The number of common peaks was 19.

[0020] Furthermore, in the step of identifying the chemical components of the common peak, a total of 5 peaks were identified, namely the peaks corresponding to 5-hydroxymethylfurfural, paeoniflorin, chlorogenic acid, ammonium glycyrrhizate, and glycyrrhizin.

[0021] Furthermore, in step S2, during high-performance liquid chromatography analysis, the injection flow rate is 1.0 mL / min to 2.0 mL / min, the column temperature is 25℃ to 35℃, the injection volume is 10 μL to 20 μL, the detection wavelength is 230 nm to 400 nm, and the analysis time is 50 min to 90 min.

[0022] Furthermore, in step S2, the high-performance liquid chromatography analysis is performed with an injection flow rate of 1.0 mL / min, a column temperature of 30℃, an injection volume of 10 μL, a detection wavelength of 230 nm, and an analysis time of 60 min.

[0023] Furthermore, before the high-performance liquid chromatography analysis in step S2, the test sample and the reference sample are filtered through a 0.22μm to 0.45μm microporous membrane.

[0024] Further, the volume percentage of the methanol solution in step S1 is 70% to 100%.

[0025] Further, in step S1, the ratio of the Eight Treasures Paste added to the methanol solution is 1:5 to 10; the ratio of the six ingredients added to the methanol solution is 1:5 to 10.

[0026] In a second aspect, the present invention provides a fingerprint spectrum of Bazhen Ointment obtained by the above-described construction method.

[0027] A third aspect of the present invention provides the application of the fingerprint spectrum of Bazhen Ointment obtained by the above-described construction method in the quality control process of Bazhen Ointment.

[0028] The method for constructing the fingerprint chromatogram of Bazhen Gao (a traditional Chinese medicine) provided by this invention facilitates the acquisition of a greater number of chromatographic peaks, enabling comprehensive detection of multiple components in Bazhen Gao. The gradient elution conditions used in this invention result in a large number of chromatographic peaks with excellent resolution in the liquid chromatography. The fingerprint chromatogram of Bazhen Gao obtained by the above method shows high similarity to the chromatograms of multiple batches of test samples, providing a more comprehensive reflection of the component information contained in Bazhen Gao and better characterizing its quality.

[0029] Bazhen Gao (Eight-Treasure Paste) is mainly made from eight herbs: Codonopsis pilosula, Atractylodes macrocephala (fried), Poria cocos, Glycyrrhiza uralensis, Angelica sinensis, Paeonia lactiflora, Ligusticum chuanxiong, and Rehmannia glutinosa, through a complex process. Its chemical composition is quite complex, and the content determination of a single component is one-sided and cannot reflect the overall composition information of the product. The above-mentioned construction method achieves quality control of the entire Bazhen Gao formula for the first time, rather than identifying a single compound or herb. This allows for more effective guidance on ingredient addition, strict standardization of production operations, and ensures safe and effective use. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 The chromatograms are of 10 batches of Bazhen Ointment test samples in this embodiment of the invention;

[0032] Figure 2 The fingerprints of Bazhen Ointment and the control fingerprints are obtained after peak matching and correction in this embodiment of the invention (S1-S10 are Bazhen Ointment test samples after peak matching and correction, and R is the control fingerprint).

[0033] Figure 3 This is a chromatogram of the common peaks identified in each reference standard and test sample in the embodiments of the present invention;

[0034] Figure 4 This is a chromatogram of the single-decocted sample solutions of eight medicinal materials in the embodiments of the present invention;

[0035] Figure 5 This is a graph showing the results of investigating the influence of different solvents on the construction of fingerprint spectra in an embodiment of the present invention;

[0036] Figure 6 This is a graph showing the results of investigating the effect of different column temperatures on the construction of fingerprint patterns in an embodiment of the present invention;

[0037] Figure 7 This is a graph showing the results of investigating the influence of different chromatographic columns on the construction of fingerprint patterns in an embodiment of the present invention. Detailed Implementation

[0038] To enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0039] It should be noted that the structures, proportions, sizes, etc., shown in the accompanying drawings of this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed in the specification, and are not intended to limit the conditions under which this application can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size should still fall within the scope of the technical content disclosed in this application, provided that they do not affect the effects and purposes that this application can produce.

[0040] A first aspect of this application provides a method for constructing a fingerprint spectrum of Bazhen Ointment, comprising the following steps:

[0041] S1. Weigh out Bazhen Ointment and add it to methanol solution to obtain the test sample; weigh out 5-hydroxymethylfurfural, paeoniflorin, chlorogenic acid, baicalin, ammonium glycyrrhizate and glycyrrhizin respectively, and add them to methanol solution to obtain reference standards containing the above six components respectively.

[0042] S2. Perform high performance liquid chromatography analysis on the test sample and reference standard obtained in step S1 to obtain chromatograms of the test sample and each reference standard.

[0043] The conditions for high-performance liquid chromatography (HPLC) analysis were as follows: the stationary phase of the column was octadecylsilane-bonded silica gel; the mobile phase was 0.1%–0.2% (v / v) inorganic acid solution as mobile phase A; and acetonitrile was used as mobile phase B for gradient elution. The gradient elution procedure was performed according to the following steps, expressed as volume fractions:

[0044] From 0 to 10 minutes, mobile phase A decreased from 93% to 85%, while mobile phase B increased from 7% to 15%.

[0045] Within 10–25 minutes, mobile phase A decreased from 85% to 76%, while mobile phase B increased from 15% to 24%.

[0046] Over 25–30 minutes, mobile phase A decreased from 76% to 70%, while mobile phase B increased from 24% to 30%.

[0047] Over 30–35 minutes, mobile phase A decreased from 70% to 55%, while mobile phase B increased from 30% to 45%.

[0048] Over 35–40 minutes, mobile phase A decreased from 55% to 10%, while mobile phase B increased from 45% to 90%.

[0049] 40–50 min, mobile phase A is 10%, mobile phase B is 90%;

[0050] From 50 to 51 minutes, the percentage of mobile phase A increased from 10% to 93%, while the percentage of mobile phase B decreased from 90% to 7%.

[0051] 51–60 min, mobile phase A is 93%, mobile phase B is 7%;

[0052] S3. Determine the common peaks from the chromatogram of the test sample, compare the common peaks with the chromatogram of the reference standard, identify the chemical components of the common peaks, select the reference peaks, calculate the relative retention times of the common peaks, and construct the fingerprint spectrum of Bazhen Ointment.

[0053] In this embodiment, a 0.2% (v / v) phosphoric acid solution-acetonitrile elution system was used. This resulted in the good separation of most of the active ingredients in the Eight Treasures Ointment, leading to a complete high-performance liquid chromatography (HPLC) chromatogram, which laid the foundation for the construction of fingerprint chromatograms.

[0054] This application uses multiple batches of Bazhen Gao (a traditional Chinese medicine) as samples for high-performance liquid chromatography (HPLC) analysis. During the research, the effects of different mobile phases, different concentrations and types of mixed solvents on the extraction of chemical components in Bazhen Gao were explored, and a suitable chromatographic method for Bazhen Gao samples was developed.

[0055] In this embodiment, similarity evaluation software (2012.130723) was used. A reference chromatogram was selected from the chromatograms of multiple batches of test samples, and a reference fingerprint chromatogram was generated using the averaging method, with a time width of 0.1. The chromatograms of the multiple batches of test samples and the reference fingerprint chromatogram were imported into the similarity evaluation software for similarity evaluation analysis. The similarity was found to be greater than 0.9, indicating that the reference fingerprint chromatogram was representative.

[0056] The chromatograms of the above-mentioned batches of test samples were processed, and a total of 19 common peaks were marked based on factors such as peak shape and retention time. These 19 peaks were numbered from 1 to 19, ascending order, according to their retention times in the reference fingerprint chromatogram. Comparison of these 19 common peaks with the chromatogram of the reference standard allowed for the identification of the chemical components of the common peaks. A reference peak was selected, and the relative retention times of the common peaks were calculated to construct the fingerprint chromatogram of Bazhen Ointment.

[0057] The method for constructing the fingerprint chromatogram of Bazhen Gao (a traditional Chinese medicine) provided in this application is beneficial for obtaining a larger number of chromatographic peaks, thereby enabling the overall detection of multiple components in Bazhen Gao. The gradient elution conditions used in this invention result in a large number of chromatographic peaks with good resolution in the liquid chromatography. The Bazhen Gao fingerprint chromatogram obtained by the above method shows high similarity to the chromatograms of multiple batches of test samples, enabling a more comprehensive reflection of the component information contained in Bazhen Gao and better characterizing its quality.

[0058] Bazhen Gao (Eight-Treasure Paste) is mainly made from eight herbs: Codonopsis pilosula, Atractylodes macrocephala (fried), Poria cocos, Glycyrrhiza uralensis, Angelica sinensis, Paeonia lactiflora, Ligusticum chuanxiong, and Rehmannia glutinosa, through a complex process. Its chemical composition is quite complex, and the content determination of a single component is one-sided and cannot reflect the overall composition information of the product. The above-mentioned construction method achieves quality control of the entire Bazhen Gao formula for the first time, rather than identifying a single compound or herb. This allows for more effective guidance on ingredient addition, strict standardization of production operations, and ensures safe and effective use.

[0059] In this embodiment of the application, during the step of identifying the chemical components of the common peaks, a total of 5 peaks were identified, namely the peaks corresponding to 5-hydroxymethylfurfural, paeoniflorin, chlorogenic acid, ammonium glycyrrhizate, and glycyrrhizin. The identified peaks are 5-hydroxymethylfurfural (peak 1), paeoniflorin (peak 11), chlorogenic acid (peak 8), ammonium glycyrrhizate (peak 16), and glycyrrhizin (peak 12).

[0060] A second aspect of this application provides a fingerprint spectrum of Bazhen Ointment obtained by the above-described method for constructing the fingerprint spectrum of Bazhen Ointment.

[0061] A third aspect of this application provides the application of the fingerprint spectrum of Bazhen Ointment obtained by the above-described method for constructing the fingerprint spectrum of Bazhen Ointment in the quality control process of Bazhen Ointment.

[0062] To better explain and facilitate understanding of this application, a detailed description of the application is provided below with reference to the accompanying drawings and specific embodiments. The chromatographic columns, reagents, and batch numbers of the Bazhen Ointment used in the following embodiments are explained as follows:

[0063] Bazhen Ointment (National Medicine Approval Number Z20093561) is produced by Hunan Kelun Pharmaceutical Co., Ltd.

[0064] Bazhen Ointment (bottled, 150g): A total of 10 batches, with batch numbers X211115, X230201, X230202, X230401, X230402, X230403, X230601, X230601, X240101, X240102, and X240702.

[0065] Medicinal herbs (packaged, sealed): Codonopsis pilosula, Atractylodes macrocephala (fried), Poria cocos, Glycyrrhiza uralensis, Angelica sinensis, Paeonia lactiflora, Ligusticum chuanxiong, Rehmannia glutinosa (processed), available commercially.

[0066] Reagents: Acetonitrile, phosphoric acid, and methanol are all commercially available. 5-Hydroxymethylfurfural, paeoniflorin, chlorogenic acid, baicalin, ammonium glycyrrhizate, and glycyrrhizin are all commercially available.

[0067] Chromatographic columns: CAPCELL PAK C18MGⅡ (4.6mm*250mm, 5μm); ZORBAX SB-C18.

[0068] Example 1

[0069] A method for constructing the fingerprint spectrum of Bazhen Ointment includes the following steps:

[0070] 1. Take 2 mL of each of 10 batches of Bazhen Ointment, dissolve them in 5 times their volume of 70% methanol solution, filter them using a 0.22 μm microporous membrane, and then use them for testing to obtain 10 batches of Bazhen Ointment samples.

[0071] 5-Hydroxymethylfurfural, paeoniflorin, chlorogenic acid, baicalin, ammonium glycyrrhizate, and glycyrrhizin were weighed separately and dissolved in methanol to prepare solutions containing 50 μg, 50 μg, 20 μg, 20 μg, 40 μg, and 50 μg per milliliter, respectively, to obtain reference standards. The peak areas of 5-hydroxymethylfurfural, paeoniflorin, chlorogenic acid, baicalin, ammonium glycyrrhizate, and glycyrrhizin were relatively large and stable; therefore, these six compounds were selected as references for preparing reference standards.

[0072] 2. The above-mentioned test samples and reference standards were analyzed by high-performance liquid chromatography (HPLC). The chromatographic conditions were as follows: column: CAPCELLPAK C18MGⅡ (4.6 mm * 250 mm, 5 μm); mobile phase: 0.2% sulfuric acid aqueous solution (A) - acetonitrile (B); flow rate: 1.0 mL / min; column temperature: 30℃; injection volume: 10 μL; detection wavelength: 230 nm; total analysis time: 60 min. The gradient elution procedure is shown in Table 1.

[0073] Table 1 Gradient elution procedures for test sample and reference sample

[0074]

[0075] 3. Analyze the chromatograms of the 10 batches of test samples obtained, referring to... Figure 1 The chromatographic peaks with good stability and suitable response values ​​in the fingerprint spectra of 10 batches of Bazhen Gao samples were selected as common peaks. The Traditional Chinese Medicine Fingerprint Similarity Evaluation System (2012.130723) software was used to select... Figure 1 The chromatogram of S1 was used as a reference chromatogram, and a reference fingerprint chromatogram was generated using the averaging method. Figure 2 The time width was 0.1, and a total of 19 common peaks were identified. The 19 common peaks were numbered from 1 to 19 in ascending order according to the retention time in the reference fingerprint.

[0076] The similarity between the chromatograms of 10 batches of Bazhen Gao (a traditional Chinese medicine paste) and the reference fingerprint chromatogram (R) is shown in Table 2. The similarity between the two is greater than 0.9, indicating good chromatogram similarity among different batches of Bazhen Gao samples. This suggests that the reference fingerprint chromatogram is representative. Figure 2 R is used as the evaluation standard for Bazhen Ointment samples. Referring to Table 3, peak No. 13, which has the best separation and the largest peak area and peak height, is used as the reference peak. The relative retention time and relative peak area of ​​each common peak are calculated. The calculation results are shown in Tables 4 and 5, respectively.

[0077] Table 210 shows the similarity between the chromatograms of Bazhen Ointment and the control fingerprint chromatograms.

[0078]

[0079]

[0080] Table 3. Peaks, peak areas, and retention times in the comparative fingerprint chromatograms.

[0081]

[0082] Table 4. Relative retention times of the common peaks in batches 410 of Bazhen Gao (a traditional Chinese medicine).

[0083]

[0084]

[0085] Table 5. Relative peak areas of the common peaks in batches 510 of Bazhen Gao (a traditional Chinese medicine).

[0086]

[0087] Compare the chromatogram of the test sample with that of the reference sample, and refer to... Figure 3 Based on the retention times of the chromatographic peaks of each reference standard, five of the 15 common peaks on the chromatogram of the Bazhen Gao sample were identified: 5-hydroxymethylfurfural (peak 1), paeoniflorin (peak 11), chlorogenic acid (peak 8), ammonium glycyrrhizate (peak 16), and glycyrrhizin (peak 12). One additional common peak could not be identified; this may be a component not present individually in the eight herbs of Bazhen Gao, generated during the manufacturing process of traditional Chinese medicine due to reactions between the processing techniques or chemical components. Further investigation is needed.

[0088] The test sample and solutions of eight single herbs (Codonopsis pilosula, Atractylodes macrocephala (fried), Poria cocos, Glycyrrhiza uralensis, Angelica sinensis, Paeonia lactiflora, Ligusticum chuanxiong, and Rehmannia glutinosa) were prepared according to the test sample preparation method of this embodiment. Specifically, the above eight herbs were crushed and then decocted twice with 10 times and 8 times the amount of water, respectively, for 1 hour each time. The decoctions were filtered, and the two filtrates were combined and concentrated to 150 mL to 200 mL for later use. Then, the single-decocted sample solutions were prepared according to the test sample preparation method of this embodiment.

[0089] The above-mentioned single-herb decoction sample solutions were analyzed by high-performance liquid chromatography (HPLC) under the chromatographic conditions of this embodiment, and fingerprint spectra of the eight single herbs were obtained. Figure 4 The fingerprint spectrum, from bottom to top, shows: Codonopsis pilosula, Atractylodes macrocephala (fried), Poria cocos, Glycyrrhiza uralensis, Angelica sinensis, Paeonia lactiflora, Ligusticum chuanxiong, and Rehmannia glutinosa. The 19 common peaks of the fingerprint spectrum of the Eight Treasures Paste identified in this embodiment are compared with the above... Figure 4 The fingerprint spectra of eight single herbs were compared and analyzed. The source herbs of the 19 common peaks in the fingerprint spectra of Bazhen Ointment were assigned. The assignment results are shown in Table 6.

[0090] Table 6. Medicinal Attribution of the 19 Peaks in the Chromatogram of the Test Sample

[0091]

[0092] As shown in Table 6, the 19 common peaks of the Bazhen Gao sample were attributed to eight herbs in Bazhen Gao: Codonopsis pilosula, Atractylodes macrocephala (fried), Poria cocos, Glycyrrhiza uralensis, Angelica sinensis, Paeonia lactiflora, Ligusticum chuanxiong, and Rehmannia glutinosa. This demonstrates that the fingerprinting method can comprehensively control the quality of Bazhen Gao.

[0093] Example 2

[0094] This embodiment examines the methodology of constructing the fingerprint spectrum of Bazhen Ointment, including assessments of precision, repeatability, and stability. The specific methods and conclusions are as follows:

[0095] 1. Precision test

[0096] The Bazhen Ointment test sample was prepared using the method of this embodiment. Six consecutive injections were performed under the same chromatographic conditions. Nineteen common peaks in the chromatogram were manually selected for multi-point correction. After matching the Mark peaks, a reference fingerprint chromatogram was generated using the averaging method with a time width of 0.1. The similarity between the chromatograms in the results was compared. The results showed that the similarity between the six injections was between 99.9% and 100% (see Table 7), indicating that the method of this application has good precision.

[0097] Table 7. Precision Map Similarity Evaluation

[0098]

[0099] 2. Repeatability testing

[0100] Using the method of this embodiment, six groups of test samples were prepared from the same batch of Bazhen Ointment. Under the same chromatographic conditions, each sample was injected and detected. Nineteen common peaks in the chromatograms were manually selected for multi-point correction. After matching the Mark peaks, a reference fingerprint chromatogram was generated using the averaging method, with a time width of 0.1. The similarity between the chromatograms in the results was compared. The results showed that the similarity between the six injections was between 99.9% and 100% (see Table 8), indicating that the method of this application has good repeatability.

[0101] Table 8 Repeatability verification results

[0102]

[0103] 3. Stability test

[0104] The Bazhen Ointment test sample was prepared using the method of this embodiment. Under the same chromatographic conditions, the sample was injected eight times at eight time points (0h, 2h, 4h, 6h, 8h, 10h, 12h, and 24h). Nineteen common peaks in the chromatogram were manually selected for multi-point correction. After matching the Mark peaks, a reference fingerprint chromatogram was generated using the averaging method, with a time width of 0.1. The similarity between the chromatograms in the results was compared. The results showed that the similarity between the eight injections was between 98.7% and 99.9% (see Table 9), indicating that the test sample prepared by the method of this application maintained stable quality within 24 hours after preparation.

[0105] Table 9 Stability Verification Results

[0106]

[0107] Example 3: Parameter Optimization in the Construction of the Eight Treasures Ointment Fingerprint Map

[0108] The study investigated the effects of different brands of chromatographic columns, different column temperatures, different flow rates, and different solvents on fingerprint pattern construction.

[0109] (1) The effect of different solvents

[0110] The effects of different solvents (100% methanol, 70% methanol, 100% ethanol, and 70% ethanol) on the construction of fingerprint spectra were investigated. The results are shown in [link to relevant documentation]. Figure 5 The spectra from bottom to top are: 100% methanol group, 70% methanol group, 100% ethanol group, and 70% ethanol group. Among them, 70% methanol has the best extraction effect, with more peaks and higher separation.

[0111] (2) Effect of different column temperatures

[0112] The effects of different column temperatures (35℃, 30℃, 25℃, 20℃) on the construction of fingerprint spectra were investigated. The results are shown in [link to relevant documentation]. Figure 6 The spectra from bottom to top are: 35℃, 30℃, 25℃, and 20℃, with the 30℃ spectrum showing better separation.

[0113] (3) The influence of different chromatographic columns

[0114] The effects of different chromatographic columns, ZORBAX SB-C18 (S1) and CAPCELLPAK C18MGⅡ (S2), on the construction of fingerprint chromatograms were investigated. The results are shown in [link to relevant documentation]. Figure 7 The chromatograms from bottom to top are: ZORBAX SB-C18 chromatogram and CAPCELLPAK C18MGⅡ chromatogram, with CAPCELLPAK C18MGⅡ showing better separation.

[0115] (4) The effect of different gradient elution conditions

[0116] By setting the proportion of the organic phase from low to high, and based on the chromatographic results, it was confirmed that the peaks of the chemical components in the Bazhen ointment extract are mainly concentrated in the range of 93% 0.2% phosphoric acid aqueous solution - 7% acetonitrile to 70% 0.2% phosphoric acid aqueous solution - 30% acetonitrile. Furthermore, based on the peak density in different retention time regions, the rate of change of the mobile phase proportion per unit time was set to increase the total methodological time, so as to present the peak information in the fingerprint spectrum as completely and clearly as possible. Specific details of some gradient elution settings are shown in Tables 10 to 13.

[0117] Table 10 First Gradient Elution Settings

[0118]

[0119] Table 11 Second gradient elution settings

[0120]

[0121] Table 12 Third Gradient Elution Settings

[0122]

[0123] Table 13 Fourth Gradient Elution Settings

[0124]

[0125] The optimized gradient elution procedure is finally obtained as follows:

[0126] From 0 to 10 minutes, mobile phase A decreased from 93% to 85%, while mobile phase B increased from 7% to 15%.

[0127] Within 10–25 minutes, mobile phase A decreased from 85% to 76%, while mobile phase B increased from 15% to 24%.

[0128] Over 25–30 minutes, mobile phase A decreased from 76% to 70%, while mobile phase B increased from 24% to 30%.

[0129] Over 30–35 minutes, mobile phase A decreased from 70% to 55%, while mobile phase B increased from 30% to 45%.

[0130] Over 35–40 minutes, mobile phase A decreased from 55% to 10%, while mobile phase B increased from 45% to 90%.

[0131] 40–50 min, mobile phase A is 10%, mobile phase B is 90%;

[0132] From 50 to 51 minutes, the percentage of mobile phase A increased from 10% to 93%, while the percentage of mobile phase B decreased from 90% to 7%.

[0133] From 51 to 60 minutes, mobile phase A was 93% and mobile phase B was 7%.

[0134] The fingerprinting method established in this application can simultaneously detect numerous fingerprint components in Bazhen Gao (a traditional Chinese medicine), thus more accurately reflecting the quality of the preparation. Furthermore, the fingerprinting method constructed in this application exhibits good precision, repeatability, and stability, with rich chromatographic peak information and good resolution. The established fingerprint spectrum can effectively characterize the quality of Bazhen Gao, contributing to the improvement of its quality evaluation system and providing a theoretical and practical basis for the comprehensive and effective control of Bazhen Gao's quality.

[0135] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for constructing a fingerprint spectrum of Bazhen Ointment, characterized in that, Includes the following steps: S1. Weigh out Bazhen Ointment and add it to methanol solution to obtain the test sample; weigh out 5-hydroxymethylfurfural, paeoniflorin, chlorogenic acid, baicalin, ammonium glycyrrhizate and glycyrrhizin respectively, and add them to methanol solution to obtain reference standards containing the above six components respectively. S2. Perform high performance liquid chromatography analysis on the test sample and the reference sample obtained in step S1 to obtain chromatograms of the test sample and each of the reference samples respectively. The conditions for the high-performance liquid chromatography (HPLC) analysis were as follows: a CAPCELL PAK C18 MGⅡ column, 4.6 mm × 250 mm, 5 μm; gradient elution using an inorganic acid solution (0.1%–0.2% by volume) as mobile phase A and acetonitrile as mobile phase B; the gradient elution steps were performed according to the following procedure, expressed as volume fractions: From 0 to 10 minutes, mobile phase A decreased from 93% to 85%, while mobile phase B increased from 7% to 15%. Within 10–25 minutes, mobile phase A decreased from 85% to 76%, while mobile phase B increased from 15% to 24%. Over 25–30 minutes, mobile phase A decreased from 76% to 70%, while mobile phase B increased from 24% to 30%. Over 30–35 minutes, mobile phase A decreased from 70% to 55%, while mobile phase B increased from 30% to 45%. Over 35–40 minutes, mobile phase A decreased from 55% to 10%, while mobile phase B increased from 45% to 90%. 40–50 min, mobile phase A is 10%, mobile phase B is 90%; At 50–51 min, mobile phase A increased from 10% to 93%, while mobile phase B decreased from 90% to 7%. 51–60 min, mobile phase A is 93%, mobile phase B is 7%; S3. Determine the common peak from the chromatogram of the test sample, compare the common peak with the chromatogram of the reference standard, identify the chemical components of the common peak, select a reference peak, calculate the relative retention time of the common peak, and construct the fingerprint spectrum of the Bazhen Ointment. The fingerprint chromatogram of the Bazhen Ointment was evaluated for similarity. The similarity evaluation method was as follows: the chromatograms of multiple batches of the test samples and the fingerprint chromatogram of the Bazhen Ointment were imported into similarity evaluation software for similarity evaluation analysis. The similarity was greater than 0.

9. The number of common peaks was 19. In the step of identifying the chemical components of the common peaks, a total of 5 peaks were identified, namely the peaks corresponding to 5-hydroxymethylfurfural, paeoniflorin, chlorogenic acid, ammonium glycyrrhizate, and glycyrrhizin. In step S2, the high-performance liquid chromatography analysis was performed with an injection flow rate of 1.0 mL / min, a column temperature of 30℃, an injection volume of 10 μL, a detection wavelength of 230 nm, and an analysis time of 60 min.

2. The method for constructing the fingerprint spectrum of Bazhen Ointment according to claim 1, characterized in that, Before the high-performance liquid chromatography analysis in step S2, the test sample and the reference sample are filtered through a 0.22μm to 0.45μm microporous membrane.

3. The method for constructing the fingerprint spectrum of Bazhen Ointment according to claim 1 or 2, characterized in that, The volume percentage of the methanol solution in step S1 is 70% to 100%.

4. The method for constructing the fingerprint spectrum of Bazhen Ointment according to claim 1 or 2, characterized in that, In step S1, the Eight Treasures Paste is added to the methanol solution at a mass-to-volume ratio of 1:5 to 10; the six ingredients are added to the methanol solution at a mass-to-volume ratio of 1:5 to 10.

5. The fingerprint spectrum of Bazhen Ointment obtained by the method for constructing the fingerprint spectrum of Bazhen Ointment according to any one of claims 1 to 4.

6. The application of the fingerprint spectrum of Bazhen Ointment obtained by the method for constructing the fingerprint spectrum of Bazhen Ointment according to any one of claims 1 to 4 in the quality control process of Bazhen Ointment.

Images