Construction method and application of the fingerprint spectrum of Naoluotong capsule

Abstract

The application provides a construction method of a fingerprint spectrum of Naoluotong capsules, and comprises the following steps: 1) preparation of a test sample solution and a mixed control sample solution; 2) construction of the fingerprint spectrum of the Naoluotong capsules: the test sample solution and the mixed control sample solution are taken respectively, and high performance liquid chromatography detection is carried out to obtain the fingerprint spectrum of the Naoluotong capsules. The application belongs to the technical field of preparation detection, and provides the construction method of the fingerprint spectrum of the Naoluotong capsules, which is simple and easy to implement, has the advantages of good precision, stability and repeatability, can be used for multi-component fingerprint spectrum research on the Naoluotong capsules, covers three traditional Chinese medicines (salvia miltiorrhiza, astragalus and ligusticum) and three chemical drugs (vitamin B6, tropicamide hydrochloride and methyl hesperidin) in the Naoluotong capsules, and determines 15 common characteristic peaks.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical testing technology, and particularly relates to the method for constructing and applying the fingerprint spectrum of Naoluotong capsules. Background Technology

[0002] Naoluotong Capsules are a compound preparation of traditional Chinese and Western medicine, mainly composed of extracts of three traditional Chinese herbs—Danshen, Huangqi, and Chuanxiong—and three chemical drugs—vitamin B6, tolperidone hydrochloride, and methyl hesperidin. Naoluotong Capsules focus on tonifying Qi and promoting blood circulation, combined with vasodilators and increased cerebral blood flow. It is primarily used to treat Qi deficiency and blood stasis syndrome caused by cerebrovascular diseases such as cerebral infarction, cerebral arteriosclerosis, and post-stroke sequelae, manifesting as symptoms such as headache, dizziness, and limb numbness. Multiple studies have shown that the combined use of Naoluotong Capsules and chemical drugs in the treatment of ischemic cerebral infarction is significantly more effective than chemical drugs alone. This combination therapy can effectively reduce blood viscosity, improve cerebral blood circulation, and promote the recovery of damaged nerve function.

[0003] However, current research on the quality standards of Naoluotong capsules is limited, and the quality control standards for this drug only use tolperone hydrochloride as a single quantitative indicator, which is insufficient to reflect the overall quality of the preparation. In their paper titled "HPLC Determination of Tolperone Hydrochloride Content in Naoluotong Capsules," published in the *West China Journal of Pharmaceutical Sciences*, Ma Zhenghong et al. disclosed a detection method using an ODS column as the stationary phase and methanol-water-triethylamine (160:39:1, pH adjusted to 7.5 with glacial acetic acid) as the mobile phase, with a detection wavelength of 261 nm. This method showed good linearity for tolperone hydrochloride in the range of 0.1–0.6 mg / mL, but it was difficult to detect other active ingredients in Naoluotong capsules. Chinese patent application CN120121747 A discloses a method for simultaneously determining the content of topozone hydrochloride, methyl hesperidin, and vitamin B6 in Naoluotong capsules. The method uses a specific C18 chromatographic column and gradient elution with methanol (A)-0.1% glacial acetic acid solution (B) as the mobile phase, with a detection wavelength of 291 nm. This method achieves the simultaneous determination of the content of the three chemical drug components, topozone hydrochloride, methyl hesperidin, and vitamin B6, in Naoluotong capsules. However, it is difficult to detect the active ingredients of traditional Chinese medicine in Naoluotong capsules.

[0004] Fingerprint spectroscopy is widely used in the overall quality control of traditional Chinese medicine / compound Chinese and Western medicine preparations, reflecting the stability and variability of the overall quality of these preparations. However, there are no reports on fingerprint spectroscopy studies for Naoluotong capsules, lacking a comprehensive detection method to evaluate its intrinsic quality. Therefore, providing a method for constructing and applying the fingerprint spectroscopy of Naoluotong capsules is of great significance. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a method for constructing and applying a fingerprint spectrum of Naoluotong capsules. The method is simple and easy to implement, and has advantages such as good precision, stability, and repeatability, enabling the detection of multiple active ingredients in Naoluotong capsules. Furthermore, the constructed HPLC fingerprint spectrum of Naoluotong capsules shows a rich array of detectable components, identifying 15 common characteristic peaks. These peaks cover three traditional Chinese medicines (Danshen, Huangqi, and Chuanxiong) and three chemical drugs (vitamin B6, tolperidone hydrochloride, and methyl hesperidin) in Naoluotong capsules, better reflecting the integrity, stability, and diversity of the components in Naoluotong capsules.

[0006] The objectives of this invention will be further explained by the following detailed description.

[0007] This invention provides a method for constructing the fingerprint spectrum of Naoluotong capsules, comprising the following steps:

[0008] 1) Preparation of test solution and mixed reference solution: Take the contents of Naoluotong capsules into a container, add methanol, extract by sonication, filter, and filter the filtrate through a filter membrane to obtain the test solution; accurately weigh vitamin B6, tolperone hydrochloride, methyl hesperidin, and salvianolic acid B, and dilute with methanol to prepare mixed reference solution.

[0009] 2) Construction of fingerprint spectrum of Naoluotong capsules: The test solution and the mixed reference solution were taken separately and detected by high performance liquid chromatography to obtain the fingerprint spectrum of Naoluotong capsules;

[0010] The high-performance liquid chromatography detection conditions include:

[0011] Using C 18The chromatographic column was used with acetonitrile as mobile phase A and phosphoric acid solution as mobile phase B, and gradient elution was performed. The gradient elution program included: 0-5 min, mobile phase A 3%, mobile phase B 97%; 5-10 min, mobile phase A 3%→16%, mobile phase B 97%→84%; 10-25 min, mobile phase A 16%, mobile phase B 84%; 25-40 min, mobile phase A 16%→35%, mobile phase B 84%→65%; 40-43 min, mobile phase A 35%→55%, mobile phase B 65%→45%; 43-46 min, mobile phase A 55%→60%, mobile phase B 45%→40%; 46-49 min, mobile phase A 60%→64%, mobile phase B 40%→36%; 49-57 min, mobile phase A 64%→90%, mobile phase B... 36%→10%; 57~61 min, mobile phase A 90%, mobile phase B 10%; 61~61.01 min, mobile phase A 90%→3%, mobile phase B 10%→97%; 61.01~69 min, mobile phase A 3%, mobile phase B 97%; The percentage content of the mobile phase in gradient elution refers to the volume percentage.

[0012] Using the above technical solution, this invention screens and optimizes suitable high-performance liquid chromatography detection conditions, eliminates interference from impurity components, and achieves fast overall peak elution time, good peak shape, resolution, and tailing factor for the target components. This provides a simple and easy method for constructing the fingerprint spectrum of Naoluotong capsules, with advantages such as high precision, stability, and repeatability. It enables multi-component fingerprint studies of Naoluotong capsules, covering three traditional Chinese medicines (Danshen, Huangqi, and Chuanxiong) and three chemical drugs (vitamin B6, tolperidone hydrochloride, and methyl hesperidin), identifying 15 common characteristic peaks.

[0013] Preferably, the fingerprint spectrum of the Naoluotong capsule has 15 common characteristic peaks; among them, peak 1 is vitamin B6, peak 8 is tolperidone hydrochloride, peak 11 is methyl hesperidin; peaks 10 and 12-14 belong to tanshinone, peak 12 is salvianolic acid B; peak 3 belongs to astragalus; peaks 2 and 9 belong to chuanxiong.

[0014] Preferably, the C 18 The chromatographic column used was a Zorbax SB-C 18 The chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 mm. μ m.

[0015] Preferably, the phosphoric acid solution is a 0.3% (v / v) phosphoric acid solution.

[0016] Preferably, the wavelength of the chromatographic detection is 255-265 nm, and the column temperature is 28-32 °C.

[0017] More preferably, the detection wavelength is 258-262 nm and the column temperature is 29-31 °C. Most preferably, the chromatographic detection wavelength is 260 nm and the column temperature is 30 °C.

[0018] Preferably, the flow rate of the mobile phase is 0.8–1.2 mL / min. –1 The injection volume is 4-6 μ L. More preferably, the flow rate of the mobile phase is 1.0 mL·min. –1 The injection volume is 5 μ L.

[0019] Accordingly, the present invention also provides the application of the method for constructing the fingerprint spectrum of the Naoluotong capsule in the quality detection of Naoluotong capsule.

[0020] Furthermore, this invention also provides a quality evaluation method for Naoluotong capsules, comprising the following steps: taking the contents of the Naoluotong capsule to be tested, constructing a fingerprint spectrum of the Naoluotong capsule to be tested according to the construction method described above, and performing similarity analysis using a traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system; the evaluation criterion for the similarity analysis is: if the similarity between the fingerprint spectrum of the Naoluotong capsule to be tested and the control fingerprint spectrum is not less than 0.9, then the quality of the Naoluotong capsule to be tested meets the requirements.

[0021] Using the above technical solution, the present invention establishes a fingerprint spectrum of Naoluotong capsules according to the above construction method. Combined with chemometrics for quality analysis of the sample, it can better reflect the integrity, stability and variability of the chemical components of Naoluotong capsules, which is conducive to the quality control of Naoluotong capsules and further ensures the clinical efficacy of the product.

[0022] Preferably, the quality evaluation method for Naoluotong capsules further includes the following steps: analyzing and evaluating the contents of the Naoluotong capsules to be tested using chemometrics; the chemometrics includes at least one of system cluster analysis, principal component analysis, and orthogonal partial least squares discriminant analysis.

[0023] Compared with the prior art, the beneficial effects of the present invention include:

[0024] (1) This invention screens and optimizes suitable high-performance liquid chromatography (HPLC) detection conditions, eliminates interference from impurity components, and achieves fast overall peak elution time, good peak shape, resolution, and tailing factor for the target components. This provides a method for constructing the fingerprint spectrum of Naoluotong capsules. The constructed fingerprint spectrum of Naoluotong capsules identified 15 common characteristic peaks, indicating a rich variety of target components. These components are attributed to three traditional Chinese medicines (Danshen, Huangqi, and Chuanxiong) and three chemical drugs (vitamin B6, tolperidone hydrochloride, and methyl hesperidin) in Naoluotong capsules. This enables rapid detection of the entire effective component group of Naoluotong capsules, providing a scientific and efficient detection method for comprehensively controlling the intrinsic quality of Naoluotong capsules and ensuring the clinical efficacy of the product.

[0025] (2) The fingerprint spectrum of Naoluotong capsules established by the present invention can quickly and accurately identify the whole composition group of Naoluotong capsules. The method is simple and easy to implement, and has the advantages of good precision, stability and repeatability. It can be used for quality control of the entire production process of Naoluotong capsules. Attached Figure Description

[0026] Figure 1 HPLC fingerprint chromatograms and control chromatograms of 10 batches of Naoluotong capsules.

[0027] Figure 2 HPLC chromatogram of the mixed reference solution; peak 1 is vitamin B6, peak 8 is tolperidone hydrochloride, peak 11 is methyl hesperidin, and peak 12 is salvianolic acid B.

[0028] Figure 3 HPLC chromatogram of the Naoluotong capsule test solution.

[0029] Figure 4 HPLC chromatograms of single medicinal materials and negative sample solutions.

[0030] Figure 5 Dendrogram of phylogenetic cluster analysis of 10 batches of Naoluotong capsules.

[0031] Figure 6 Principal component analysis score charts for 10 batches of Naoluotong capsules.

[0032] Figure 7 VIP values ​​of orthogonal partial least squares discriminant analysis for 10 batches of Naoluotong capsules. Detailed Implementation

[0033] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0034] In this invention, the medicinal materials, reagents, and instruments involved are all commercially available products or can be obtained through conventional technical means in the field. Unless otherwise specified, the percentage content in this invention refers to volume percentage content.

[0035] Specifically, the Agilent 1260 high-performance liquid chromatograph and Zorbax SB-C18 column were purchased from Agilent Technologies China Co., Ltd. Reference standards Vitamin B6 (batch number: 100116-201906, purity: 99.9%), methyl hesperidin (batch number: 111580-201804, purity: 99.2%), and salvianolic acid B (batch number: 111562-202318, purity: 96.6%) were all purchased from the China National Institutes for Food and Drug Control; tolperidone hydrochloride (batch number: Y220321, purity: 99.7%) was purchased from Guangzhou Baiyunshan Hanfang Modern Pharmaceutical Co., Ltd. Naoluotong Capsules (product numbers S1-S10, batch numbers 20200601, 20210402, 20211101, 20220503, 20220802, 20211102, 20211103, 20221101, 20221102, 20221103) are produced by Guangdong Xinbao Pharmaceutical Technology Co., Ltd.

[0036] Preparation of mixed reference solution: Accurately weigh appropriate amounts of reference standards vitamin B6, tolperone hydrochloride, methyl hesperidin, and salvianolic acid B, and dilute with methanol to prepare a solution containing 50 mg each of vitamin B6, tolperone hydrochloride, methyl hesperidin, and salvianolic acid B per mL. μ g of mixed reference solution.

[0037] Example 1: Screening and Optimization of High Performance Liquid Chromatography Detection Conditions

[0038] The chromatographic peaks and peak areas of the main peaks were examined by scanning in the wavelength range of 190 nm to 400 nm using a DAD detector. The results showed that the baseline of the fingerprint chromatogram of Naoluotong capsules was stable at 260 nm, with a large number of peaks and a high response. Therefore, 260 nm was selected as the detection wavelength for the HPLC fingerprint chromatogram of Naoluotong capsules.

[0039] Regarding the selection of mobile phase systems, methanol (acetonitrile)-water, acetonitrile-0.1% formic acid solution, acetonitrile-0.1% acetic acid solution, acetonitrile-0.1% phosphoric acid solution, acetonitrile-0.3% phosphoric acid solution, and acetonitrile-0.5% phosphoric acid solution were investigated. The results showed that when acetonitrile-0.3% phosphoric acid solution was used as the mobile phase system, the number of chromatographic peaks was greater and the peak shape was better. By optimizing the gradient elution program and eliminating interference from other components, the separation degree of the components was improved. The following optimal mobile phase gradient elution program was obtained: 0-5 min, mobile phase A 3%, mobile phase B 97%; 5-10 min, mobile phase A 3%→16%, mobile phase B 97%→84%; 10-25 min, mobile phase A 16%, mobile phase B 84%; 25-40 min, mobile phase A 16%→35%, mobile phase B 84%→65%; 40-43 min, mobile phase A 35%→55%, mobile phase B 65%→45%; 43-46 min, mobile phase A 55%→60%, mobile phase B 45%→40%; 46-49 min, mobile phase A 60%→64%, mobile phase B 40%→36%; 49-57 min, mobile phase A 64%→90%, mobile phase B 36%→10%; 57 min... From 61 to 61.01 min, mobile phase A was 90% and mobile phase B was 10%; from 61 to 61.01 min, mobile phase A decreased from 90% to 3% and mobile phase B decreased from 10% to 97%; from 61.01 to 69 min, mobile phase A was 3% and mobile phase B was 97%. Additionally, Zorbax SB-C... 18 Column temperatures (25, 30, 35 ℃), flow rates (0.9, 1.0, 1.1 mL·min) -1 The results were investigated separately, and the results showed that the column temperature was 30 ℃ and the flow rate was 1.0 mL·min. -1 At this time, the separation of the fingerprint spectrum and the chromatographic peaks for content determination is optimal, the baseline is stable, the retention time is moderate, and the method has good robustness.

[0040] Example 2: Screening and Optimization of Test Solution Preparation Method

[0041] In establishing the fingerprinting and content determination methods, 50% methanol aqueous solution, 75% methanol aqueous solution, and methanol were used as extraction solvents for the test samples. The results showed that 75% methanol aqueous solution had higher extraction efficiency, so it was selected as the extraction solvent. Simultaneously, the differences between ultrasonic and reflux extraction methods were compared. The results showed that ultrasonic extraction yielded more component information, higher extraction rate, better peak shape, and better resolution of each chromatographic peak; therefore, ultrasonic extraction was used. Furthermore, the ultrasonic extraction time (30, 45, and 60 min) was investigated. The results showed that ultrasonic extraction was sufficient for 45 min, and there was no significant difference in extraction rate after 45 min. Therefore, 45 min was selected as the extraction time for the fingerprinting and content determination methods.

[0042] Preparation of the test solution: Accurately weigh 1.0 g of the contents of Naoluotong capsules, place them in a stoppered conical flask, add 50 mL of 75% methanol aqueous solution, weigh, extract ultrasonically for 45 min, cool, weigh again, replenish the lost weight with 75% methanol aqueous solution, shake well, filter, and use 0.45 mL of the filtrate for testing. μ The test solution is obtained by filtration through an organic filter membrane.

[0043] Example 3: Construction and Methodological Investigation of Fingerprint Spectrum of Naoluotong Capsules

[0044] 1. Instrument precision test

[0045] Six test solutions were prepared from the same batch of Naoluotong capsules (S1) according to the method in Example 2. The solutions were injected six times under the chromatographic conditions described in Example 1, and the chromatograms were recorded. Peak 12 (tanshinone B) was used as the reference peak (S), and the relative retention time and relative peak area of ​​each common peak were examined. The results showed that the RSD of the relative retention time of each common peak was between 0.04% and 2.90%, and the RSD of the relative peak area was between 0.20% and 4.80%, indicating good instrument precision.

[0046] 2. Stability Test

[0047] Sample S1 of the same batch of Naoluotong capsules was prepared as a test solution according to the method in Example 2. The solution was injected and analyzed at 0, 24, 48, and 72 h after preparation, under the chromatographic conditions described in Example 1. Peak 12 (tanshinone B) was used as the reference peak (s), and the relative retention time and relative peak area of ​​each common peak were examined. The results showed that the RSD of the relative retention time of each common peak was between 0.00% and 1.94%, and the RSD of the relative peak area was between 0.00% and 4.88%, indicating that the test solution had good stability within 72 h.

[0048] 3 Repeatability Test

[0049] Six test solutions were prepared from the same batch of Naoluotong capsules (S1) according to the method in Example 2, and injected and measured six times under the chromatographic conditions in Example 1. Peak 12 (tanshinone B) was used as the reference peak (s), and the relative retention time and relative peak area of ​​each common peak were examined. The results showed that the RSD of the relative retention time of each common peak was between 0.02% and 2.43%, and the RSD of the relative peak area was between 0.26% and 3.34%, indicating that the method has good repeatability.

[0050] 4. Establishment of fingerprint map

[0051] The method for constructing the fingerprint spectrum of Naoluotong capsules includes the following steps:

[0052] 1) Preparation of the test solution and mixed reference solution: Accurately weigh 1.0 g of the contents of Naoluotong capsules, place them in a stoppered conical flask, add 50 mL of 75% methanol aqueous solution, weigh, extract ultrasonically for 45 min, cool, weigh again, replenish the lost weight with 75% methanol aqueous solution, shake well, filter, and use 0.45 mL of the filtrate for testing. μ The sample solution was obtained by filtration through a polyethersulfone membrane. Appropriate amounts of reference standards vitamin B6, tolperone hydrochloride, methyl hesperidin, and salvianolic acid B were accurately weighed and diluted with methanol to prepare a solution containing 50 mg / mL of each of vitamin B6, tolperone hydrochloride, methyl hesperidin, and salvianolic acid B. μ g of mixed reference solution;

[0053] 2) Construction of the fingerprint chromatogram of Naoluotong capsules: The test solution and the mixed reference solution were taken separately and subjected to high-performance liquid chromatography (HPLC) to obtain the fingerprint chromatogram of Naoluotong capsules; the HPLC detection conditions included:

[0054] Using Zorbax SB-C 18 The chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 mm. μ The chromatographic detection wavelength was 260 nm, the column temperature was 30 ℃, and the flow rate was 1.0 mL·min. -1 The injection volume is 5 μL; Using acetonitrile as mobile phase A and 0.3% (v / v) phosphoric acid solution as mobile phase B, gradient elution was performed according to the following program: 0-5 min, mobile phase A 3%, mobile phase B 97%; 5-10 min, mobile phase A 3%→16%, mobile phase B 97%→84%; 10-25 min, mobile phase A 16%, mobile phase B 84%; 25-40 min, mobile phase A 16%→35%, mobile phase B 84%→65%; 40-43 min, mobile phase A 35%→55%, mobile phase B 65%→45%; 43-46 min, mobile phase A 55%→60%, mobile phase B 45%→40%; 46-49 min, mobile phase A 60%→64%, mobile phase B 40%→36%; 49-57 min, mobile phase A 64%→90%, mobile phase B... 36%→10%; 57~61 min, mobile phase A 90%, mobile phase B 10%; 61~61.01 min, mobile phase A 90%→3%, mobile phase B 10%→97%; 61.01~69 min, mobile phase A 3%, mobile phase B 97%; The percentage content of the mobile phase in gradient elution refers to the volume percentage.

[0055] Ten batches of Naoluotong capsule samples (numbered S1-S10, batch numbers 20200601, 20210402, 20211101, 20220503, 20220802, 20211102, 20211103, 20221101, 20221102, 20221103 respectively) were taken. 1.0 g of the contents of each batch was accurately weighed. Test solutions were prepared according to the method described in this embodiment. The samples were injected and analyzed under the chromatographic conditions described in this embodiment. The chromatograms of each batch were recorded and imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software in CDF format for processing. Using S1 (batch number: 20200601) as the reference chromatogram, with a time window width of 0.1 min, a multi-point correction method was used to match the chromatograms. A reference fingerprint (R) was generated using the median method. HPLC fingerprints and reference fingerprints for 10 batches of Naoluotong capsules were obtained. (See attached image). Figure 1 A total of 15 common peaks were identified.

[0056] The fingerprint data of the above 10 batches of Naoluotong capsules were analyzed, and the similarity between the samples was calculated. The results are shown in Table 1. The similarity between the 10 batches of Naoluotong capsules ranged from 0.974 to 1.000, indicating that the overall quality difference between different batches of Naoluotong capsules was small, and the components contained were basically the same. This shows that the established HPLC fingerprint method is suitable for the qualitative analysis of Naoluotong capsules.

[0057] Table 1. Comparison of similarity among 10 batches of Naoluotong capsules

[0058]

[0059] 5. Identification and Attribution of Common Peaks

[0060] Preparation of single medicinal materials and negative sample solutions: According to the production process and prescription ratio of Naoluotong capsules, samples of each single medicinal material, Danshen, Huangqi, and Chuanxiong, as well as negative samples lacking Danshen, Huangqi, and Chuanxiong, were prepared. Take an appropriate amount and operate according to the preparation method of the test solution to obtain the solution.

[0061] Take the mixed reference solution, the test solution of Naoluotong capsules, the solution of single herb (Danshen), the solution of single herb (Huangqi), the solution of single herb (Chuanxiong), the negative sample solution lacking Danshen, the negative sample solution lacking Huangqi, and the negative sample solution lacking Chuanxiong, respectively, and inject them according to the chromatographic conditions in this embodiment. Record the chromatograms, and the results are shown in [the table below]. Figures 2-4 ,according to Figure 1 The 15 common peaks were numbered, and their identification was performed by comparing the chromatograms of the mixed reference standard and the negative sample, combined with the retention times of the chromatographic peaks. Peak 1 was identified as vitamin B6, peak 8 as tolperidone hydrochloride, peak 11 as methyl hesperidin, and peak 12 as salvianolic acid B. The common peaks were further assigned by comparing the chromatograms of the single herbs and the negative sample. Peaks 10 and 12-14 were assigned to Salvia miltiorrhiza, peak 3 to Astragalus membranaceus, and peaks 2 and 9 to Ligusticum chuanxiong. Peaks 4-7 and 15 did not have corresponding assignments in the fingerprint chromatograms of the extracts of Salvia miltiorrhiza, Astragalus membranaceus, and Ligusticum chuanxiong, but they were stably present in the compound preparation, presumably as products of inter-component reactions during the extraction process.

[0062] Example 4: Quality Evaluation Method for Naoluotong Capsules

[0063] Based on the analysis of the fingerprint data of the above 10 batches of Naoluotong capsules in Example 3, the Naoluotong capsules were analyzed and evaluated by combining chemometrics to further analyze the differences in components between different batches and to more effectively guide the quality assurance of the product.

[0064] Hierarchical cluster analysis (HCA): Using the peak areas of the 15 common peaks in the 10 batches of samples (numbered S1-S10) as variables, the data were imported into SPSS 21.0 statistical software. The between-groups connection method was used, and HCA was performed on the samples using Euclidean distance as the measure. The results showed that when the Euclidean distance was 5, the 10 batches of samples clustered into 3 classes: sample S5 formed one class, samples S1, S2, S6, and S7 formed another class, and samples S3, S4, S8, S9, and S10 formed a third class. See details... Figure 5 .

[0065] Principal component analysis (PCA): Using the peak areas of the 15 common peaks from the 10 batches of samples (numbered S1-S10) as variables, PCA was performed using SIMCA 14.1 software. The results are shown below. Figure 6 The results showed that the 10 batches of samples could be divided into 3 categories: sample S5 was one category, samples S1, S2, S6, and S7 were another category, and samples S3, S4, and S8-S10 were another category, which was consistent with the HCA results.

[0066] Orthogonal partial least squares-discriminant analysis (OPLS-DA): The higher the variable importance in projection (VIP) value, the greater the influence on differences between groups. Variables with VIP > 1 are identified to determine the differential variables affecting the samples. Results are shown in [link to results]. Figure 7 VIP>1 has 4 common characteristic peaks, namely peaks 8, 15, 7 and 2. These 4 components are likely the main chemical components affecting the quality difference. Peak 8 is tolperidone hydrochloride, and the other three are unknown components.

[0067] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A method for constructing the fingerprint spectrum of Naoluotong capsules, characterized in that: Includes the following steps: 1) Preparation of test solution and mixed reference solution: Take the contents of Naoluotong capsules into a container, add methanol, extract by ultrasound, filter, and filter the filtrate through a filter membrane to obtain the test solution; accurately weigh vitamin B6, tolperidone hydrochloride, methyl hesperidin, and salvianolic acid B, and dilute with methanol to prepare mixed reference solution. 2) Construction of fingerprint spectrum of Naoluotong capsules: The test solution and the mixed reference solution were taken separately and detected by high performance liquid chromatography to obtain the fingerprint spectrum of Naoluotong capsules; The high-performance liquid chromatography detection conditions include: Using C 18 The chromatographic column was used with acetonitrile as mobile phase A and phosphoric acid solution as mobile phase B, and gradient elution was performed. The gradient elution program included: 0-5 min, mobile phase A 3%, mobile phase B 97%; 5-10 min, mobile phase A 3%→16%, mobile phase B 97%→84%; 10-25 min, mobile phase A 16%, mobile phase B 84%; 25-40 min, mobile phase A 16%→35%, mobile phase B 84%→65%; 40-43 min, mobile phase A 35%→55%, mobile phase B 65%→45%; 43-46 min, mobile phase A 55%→60%, mobile phase B 45%→40%; 46-49 min, mobile phase A 60%→64%, mobile phase B 40%→36%; 49-57 min, mobile phase A 64%→90%, mobile phase B... 36%→10%; 57~61 min, mobile phase A 90%, mobile phase B 10%; 61~61.01 min, mobile phase A 90%→3%, mobile phase B 10%→97%; 61.01~69 min, mobile phase A 3%, mobile phase B 97%; The phosphoric acid solution is a 0.3% (v / v) phosphoric acid solution; the detection wavelength of the high-performance liquid chromatography is 260 nm.

2. The method for constructing the fingerprint spectrum of the Naoluotong capsule according to claim 1, characterized in that: The fingerprint spectrum of the Naoluotong capsule has 15 common characteristic peaks; among them, peak 1 is vitamin B6, peak 8 is tolperidone hydrochloride, peak 11 is methyl hesperidin; peaks 10 and 12-14 belong to tanshinone, peak 12 is salvianolic acid B; peak 3 belongs to astragalus; peaks 2 and 9 belong to chuanxiong.

3. The method for constructing the fingerprint spectrum of the Naoluotong capsule according to claim 1 or 2, characterized in that: The C 18 The chromatographic column used was a Zorbax SB-C. 18 The chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 mm. μ m.

4. The method for constructing the fingerprint spectrum of the Naoluotong capsule according to claim 1 or 2, characterized in that: The flow rate of the mobile phase is 0.8–1.2 mL / min. –1 The injection volume is 4-6 μ L.

5. The application of the method for constructing the fingerprint spectrum of Naoluotong capsules according to any one of claims 1 to 4 in the quality inspection of Naoluotong capsules.

6. A method for quality evaluation of Naoluotong capsules, characterized in that: The procedure includes the following steps: taking the contents of the Naoluotong capsule to be tested, constructing a fingerprint chromatogram of the Naoluotong capsule to be tested according to the construction method of any one of claims 1 to 4, and performing similarity analysis using a traditional Chinese medicine chromatographic fingerprint chromatogram similarity evaluation system; the evaluation criterion for the similarity analysis is: if the similarity between the fingerprint chromatogram of the Naoluotong capsule to be tested and the control fingerprint chromatogram is not less than 0.9, then the quality of the Naoluotong capsule to be tested meets the requirements.

7. The quality evaluation method for Naoluotong capsules according to claim 6, characterized in that: It also includes the following steps: analyzing and evaluating the contents of the Naoluotong capsules to be tested using chemometrics; the chemometrics includes at least one of system cluster analysis, principal component analysis, and orthogonal partial least squares discriminant analysis.

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