A quality testing method for nasal drops used to treat allergic rhinitis

Abstract

This invention provides a quality testing method for nasal drops used to treat allergic rhinitis, belonging to the field of pharmaceutical testing. Specifically, it includes the following steps: S1. Preparing a test solution; S2. Preparing a mixed reference solution; S3. Separately sampling the reference solution and the test solution for analysis; S4. Generating a reference fingerprint chromatogram. This invention can present all different types of components of the medicinal materials on a single chromatogram through a single test, allowing for simultaneous determination of multiple components and achieving comprehensive and accurate quality control of Artemisia annua and Scutellaria baicalensis nasal drops. The quality assessment method provided by this invention has optimized chromatographic conditions and undergone methodological validation, possessing the advantages of method stability, good repeatability, and stable and reliable test results.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical testing, specifically relating to a quality testing method for nasal drops used to treat allergic rhinitis. Background Technology

[0002] This traditional Chinese medicine compound preparation for treating allergic rhinitis consists of six herbs: Artemisia annua, Ephedra sinica, Xanthium sibiricum, and Scutellaria baicalensis. It possesses the effects of clearing heat and detoxifying, and promoting lung function and nasal passage opening. It is used for acute rhinitis, chronic rhinitis, and allergic rhinitis. Chinese Patent 201710827980.6, entitled "Nasal Preparation for Treating Rhinitis and its Preparation Method and Application," discloses a nasal preparation for treating rhinitis. Compared with existing technologies, the dosage of this invention's nasal preparation for treating rhinitis is only 20% to 30% of that of existing technologies, and it shows significant anti-inflammatory and anti-allergic effects. Furthermore, this invention has a simple preparation process, definite efficacy, no toxic side effects, and the drug acts directly on the nasal mucosa surface, resulting in high bioavailability, rapid onset of action, convenience, and good patient compliance. Different dosage forms can be prepared according to patient needs, making it better suited for treating acute, chronic, and allergic rhinitis. Therefore, exploring the quality control methods for this preparation is particularly important.

[0003] Currently, the standard for this preparation only includes routine inspections, thin-layer chromatography identification, and the determination of artemisinin and baicalin content, which cannot comprehensively reflect the product quality. There is limited literature on the quality of this preparation, and no research reports have been published on its overall quality control. Traditional Chinese medicine compound formulas contain multiple herbs with complex chemical compositions. With increasingly stringent quality control requirements for traditional Chinese medicine, the determination of the content of a single or a few components cannot comprehensively and scientifically evaluate the quality of traditional Chinese medicine. Qualitative and quantitative analysis of multiple indicator components has become crucial for the quality control of traditional Chinese medicine.

[0004] Traditional Chinese medicine (TCM) fingerprinting is a comprehensive and quantifiable quality analysis method that can characterize the overall chemical composition of TCM, comprehensively reflecting the differences in chemical components. It is mainly used to identify the authenticity and quality of raw materials and prepared TCM products, and to evaluate the uniformity and stability of the quality of raw materials, semi-finished products, and finished products in TCM preparations. TCM and its preparations are complex multi-component systems; therefore, quality evaluation should employ appropriate detection methods that provide rich identification information. Establishing TCM fingerprinting can comprehensively reflect the types and quantities of chemical components contained in TCM and its preparations, thus providing an overall description and evaluation of drug quality. By establishing fingerprinting and combining it with multi-component content determination methods for systematic analysis, not only can the consistency of TCM quality be effectively evaluated, but the main factors contributing to quality differences can also be explained, leading to a more objective and comprehensive evaluation of TCM quality.

[0005] Traditional methods for determining the content of multiple components require a large number of reference standards, which are expensive and difficult to obtain. Quantitative analysis of multiple components by a single marker (QAMS) utilizes the intrinsic functional relationships between the active ingredients of traditional Chinese medicine (TCM). By measuring only the content of inexpensive and readily available common components, it achieves simultaneous determination of the content of multiple components. It is characterized by low cost and simple operation, making it suitable for the quality control of TCM compound preparations. To comprehensively and effectively control the quality of the preparation, this invention, for the first time, uses HPLC to establish a fingerprint spectrum of a TCM compound preparation for treating allergic rhinitis, and simultaneously employs QAMS to determine the content of 16 components in the preparation, achieving comprehensive and accurate quality control. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a quality testing method for nasal drops used to treat allergic rhinitis. This method can present different types of components of all medicinal materials in the preparation on a single chromatogram under the same chromatographic conditions, and simultaneously determine the content of 16 components, thereby achieving comprehensive and accurate quality control of Artemisia argyi nasal drops. This testing method is stable, reliable, highly specific, and accurate, and can effectively control the quality of the product.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for quality testing of nasal drops for treating allergic rhinitis, comprising the following steps:

[0009] S1. Preparation of test solution: Accurately measure 2.0 mL of nasal drops, place it in a 10 mL volumetric flask, dilute to the mark with 50% methanol, shake well, filter, and take the filtrate to obtain the test solution;

[0010] S2. Preparation of mixed reference solution: Accurately weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, hyperoside, luteolin, isochlorogenic acid, 1,5-dicaffeoylquinic acid, isochlorogenic acid C, rosmarinic acid, baicalin, buddleja glycoside, isochlorogenic acid, artemisinin, ammonium glycyrrhizate, vitexin and imperatorin reference standards, and add 50% methanol to prepare a mixed reference solution;

[0011] The concentration of the reference standard in the mixed reference solution is 0.04–7.684 mg / mL;

[0012] S3. Take separate pipettes of the mixed reference solution and the test solution, inject them into the liquid chromatograph, detect and analyze the chromatograms, record the chromatograms, and conduct quality evaluation;

[0013] The liquid chromatography conditions are as follows:

[0014] Column: Agilent TCC18 The column has dimensions of 250×4.6mm and a diameter of 5μm.

[0015] Mobile phase: Mobile phase A was 0.1% phosphoric acid solution, and mobile phase B was acetonitrile; mobile phases A and B were eluted using a gradient: 0–10 min, 97%–88% A; 10–30 min, 88%–78% A; 55–95 min, 70%–54% A; 95–115 min, 54%–20% A; 115–120 min, 20% A;

[0016] Volumetric flow rate: 1.0 mL / min;

[0017] Column temperature: 30℃;

[0018] Detection wavelength: 245nm;

[0019] Injection volume: 5 μL.

[0020] S4. Generate a control fingerprint: Export the chromatographic file of the nasal drops obtained in S3 from the instrument and import it into the Chinese medicine chromatographic fingerprint similarity evaluation system; select the chromatographic peaks that are present in the chromatograms of different batches of nasal drops as common peaks; generate a control fingerprint of the nasal drops using the average value calculation method.

[0021] Preferably, the nasal drops are the nasal preparation for treating rhinitis prepared in patent 201710827980.6.

[0022] Preferably, the specific concentrations of each component in the S2 mixed reference solution are as follows: neochlorogenic acid 0.523 mg / mL, chlorogenic acid 0.756 mg / mL, cryptochlorogenic acid 1.046 mg / mL, hyperoside 0.489 mg / mL, luteolin 1.126 mg / mL, isochlorogenic acid B 1.246 mg / mL, 1,5-dicaffeoylquinic acid 0.876 mg / mL, isochlorogenic acid C 0.964 mg / mL, rosmarinic acid 0.222 mg / mL, baicalin 7.684 mg / mL, buddleja glycoside 0.0474 mg / mL, isochlorogenic acid 0.237 mg / mL, artemisinin 1.001 mg / mL, glycyrrhizic acid ammonium 2.056 mg / mL, vitexin 1.174 mg / mL, and imperatorin 0.627 mg / mL.

[0023] Preferably, the chromatogram of S3 identifies 52 common peaks, specifically: the fingerprint spectrum of S3 includes 52 common peaks, namely: peak 8 corresponding to neochlorogenic acid, peak 10 corresponding to chlorogenic acid, peak 11 corresponding to cryptochlorogenic acid, peak 22 corresponding to hyperoside, peak 23 corresponding to luteolin, peak 26 corresponding to isochlorogenic acid B, peak 28 corresponding to 1,5-dicaffeoylquinic acid, peak 29 corresponding to isochlorogenic acid C, peak 30 corresponding to rosmarinic acid, peak 32 corresponding to baicalin, peak 35 corresponding to buddleja glycoside, peak 37 corresponding to isochlorogenic acid, peak 39 corresponding to artemisinin, peak 44 corresponding to ammonium glycyrrhizate, peak 46 corresponding to vitexin, and peak 49 corresponding to imperatorin.

[0024] Preferably, the characteristic peaks in the fingerprint spectrum use the baicalin peak as a reference peak, and the relative retention times of the 16 chromatographic peaks are 0.322, 0.419, 0.431, 0.712, 0.727, 0.741, 0.777, 0.814, 0.879, 1.000, 1.113, 1.279, 1.410, 1.777, 1.845, and 2.178, respectively.

[0025] Preferably, the quality evaluation in S3 involves importing the fingerprint spectrum into a software for evaluating the similarity between the fingerprint spectrum and a control spectrum.

[0026] The present invention also provides the application of the above-mentioned nasal drop quality testing method in the quality control of Artemisia annua nasal drops.

[0027] Preferably, the quality testing method includes a fingerprinting method combined with a multi-component content determination method, which is used in the quality control of Chinese medicinal materials or solid pharmaceutical preparations containing six herbs: Artemisia annua, Ephedra sinica, Xanthium sibiricum, Scutellaria baicalensis, Glycyrrhiza uralensis, and Angelica dahurica.

[0028] It contains at least the following beneficial technical effects:

[0029] This invention provides a quality evaluation method based on high-performance liquid chromatography (HPLC), which presents all different types of components of the medicinal materials in the preparation on a single chromatogram and simultaneously determines the content of 16 components, achieving comprehensive and accurate quality control of Artemisia annua and Scutellaria baicalensis nasal drops. The quality assessment method provided by this invention has undergone optimized screening and methodological validation, exhibiting advantages such as method stability, good repeatability, and stable and reliable measurement results.

[0030] This invention establishes a fingerprint spectrum for Artemisia annua nasal drops, which can comprehensively reflect the types and quantities of chemical components contained in the preparation, thereby providing an overall description and evaluation of the preparation quality.

[0031] This invention establishes a method for determining the content of multiple components in Artemisia annua and Scutellaria baicalensis nasal drops. Combined with fingerprint spectroscopy for systematic analysis, this method can not only effectively evaluate the consistency of traditional Chinese medicine quality, but also interpret the main factors that cause quality differences, and can more objectively and comprehensively evaluate the quality of traditional Chinese medicine.

[0032] This invention establishes a method for determining 16 components in Artemisia annua nasal drops using a single-method, multi-evaluation approach. Using baicalin as an internal reference (S), and utilizing the intrinsic functional relationships between various effective components of traditional Chinese medicine, this method simultaneously determines the content of 16 components in Artemisia annua nasal drops by measuring only the content of the inexpensive and readily available baicalin. It features low cost and simple operation, enabling comprehensive and accurate quality control of Artemisia annua nasal drops. Attached Figure Description

[0033] Figure 1 This is the HPLC chromatogram of the mixed reference standards;

[0034] Figure 2 The HPLC chromatogram of the Artemisia annua nasal drops sample;

[0035] Figure 3 Fingerprint chromatograms (S1-S5) and control chromatograms (R) of Artemisia annua nasal drops;

[0036] Figure 4 Chromatograms of single herbs, corresponding herbs missing, and Artemisia annua and Scutellaria baicalensis nasal drops. Detailed Implementation

[0037] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0038] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0039] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention, and no reference numerals in the claims should be construed as limiting the scope of the claims.

[0040] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can be appropriately combined to form other embodiments that can be understood by those skilled in the art. These other embodiments are also covered within the scope of protection of this invention.

[0041] It should also be understood that the specific embodiments described above are only used to explain the present invention, and the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

[0042] Example 1

[0043] 1. Materials

[0044] 1.1 Instruments

[0045] Agilent 1260 HPLC system (Agilent Technologies, YF-17, YF-18); U3000 HPLC system (Thermo Fisher, YF-8); CPA225D electronic balance (Sartorius, d=0.01mg); AS20500BDT ultrasonic cleaner (60kHz frequency, 500W power, Tianjin Autoshine Instruments Co., Ltd.); UPT-10T UPT laboratory ultrapure water system (Chengdu Ultrapure Technology Co., Ltd.); Agilent XDB C column. 18 (250×4.6mm, 5μm); Chromatographic column YMC-C 18 (250×4.6mm, 5μm); Agilent SB-C column 18 (250×4.6mm, 5μm).

[0046] 1.2 Samples and Reagents

[0047] Chlorogenic acid (batch number 110753-202018, purity 96.1%), hyperoside (batch number 111521-201406, purity 100%), luteolin (batch number 111720-201307, purity 100%), rosmarinic acid (batch number 111871-202007, purity 98.1%), baicalin (batch number 110715-202122, purity 94.2%). %), Buddleja glycoside (111528-202112, purity 98.0%), ammonium glycyrrhizate (batch number 110731-202122, purity 94.4%), vitexin (batch number 111554-201705, purity 98.3%), imperatorin (batch number 110826-201918, purity 99.0%), ferulic acid (batch number 110773-201313, purity %) 99.6%), glycyrrhizin (batch number 111610-201908, purity 95.0%), isoimperatorin (batch number 110827-202113, purity 99.2%), rutin (batch number 100080-202012, purity 92.2%), apigenin (batch number 111901-202004, purity 99.4%), kaempferol (batch number 110861-202013) The following substances were purchased from the China National Institutes for Food and Drug Control: ephedrine hydrochloride (batch number 171241-201809, purity 100%), pseudoephedrine hydrochloride (batch number 171237-201510, purity 99.8%), hyoscyamine (batch number 111879-201102, purity 97.2%), and ursolic acid (batch number 110742-201823, purity 99.9%). Neochlorogenic acid (batch number DSTDX001504), isochlorogenic acid (batch number DSTDX001504), isochlorogenic acid B (batch number DSTTY003703), isochlorogenic acid C (batch number DSTPS0068-0020), 1,5-dicaffeoylquinic acid (batch number DSTDE000602), cryptochlorogenic acid (batch number DST221220-035), and isochlorogenic acid A (batch number DSTDE000602), all with a purity greater than 98%, were purchased from Chengdu Dester Biotechnology Co., Ltd. Artemisinin (batch number DZ20240302, purity 99.71%) was purchased from the Xinjiang Institute of Physics and Chemistry, Chinese Academy of Sciences.

[0048] Artemisia argyi nasal drops, S1-S5, were prepared by the Xinjiang Uygur Autonomous Region Institute of Materia Medica according to the preparation method of the nasal preparation for treating rhinitis described in patent 201710827980.6.

[0049] Acetonitrile, chromatographic grade, Fisher, USA; water was Yibao purified water; all other reagents were analytical grade.

[0050] 2. Methods and Results

[0051] 2.1 Preparation of the reference solution

[0052] Accurately weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, hyperoside, luteolin, isochlorogenic acid B, 1,5-dicaffeoylquinic acid, isochlorogenic acid C, rosmarinic acid, baicalin, buddleja glycoside, isochlorogenic acid, artemisinin, ammonium glycyrrhizate, vitexin, and imperatorin reference standards. Dissolve in 50% methanol and dilute to volume to prepare solutions with mass concentrations of 0.523, 0.756, 1.046, 0.489, and 1.126, respectively.

[0053] Reference stock solutions with concentrations of 1.246, 0.876, 0.964, 0.222, 7.684, 0.0474, 0.237, 1.001, 2.056, 1.174, and 0.627 mg / mL.

[0054] 2.2 Preparation of the test solution

[0055] Accurately measure 2.0 mL of Artemisia annua nasal drops, place it in a 10 mL volumetric flask, dilute to the mark with 50% methanol, shake well, filter, and collect the filtrate to obtain the final product.

[0056] 2.3 Chromatographic conditions

[0057] Chromatographic column Agilent TC C 18 (250×4.6mm, 5μm), mobile phase: 0.1% phosphoric acid solution (A)-acetonitrile (B), gradient elution: 0–10 min, 97%–88% A; 10–30 min, 88%–78% A; 55–95 min, 70%–54% A; 95–115 min, 54%–20% A; 115–120 min, 20% A; flow rate: 1.0 mg / mL; column temperature: 30℃; detection wavelength: 245 nm; injection volume: 5 μL, see [link to table]. Figure 1 and Figure 2 The corresponding relationships of the chromatographic peaks in the figure are as follows: 1. Neochlorogenic acid; 2. Chlorogenic acid; 3. Cryptochlorogenic acid; 4. Hyperoside; 5. Luteinoside; 6. Isochlorogenic acid B; 7. 1,5-Dicaffeoylquinic acid; 8. Isochlorogenic acid C; 9. Rosmarinic acid; 10. Baicalin; 11. Buddleja glycoside; 12. Isochlorogenic acid; 13. Artemisia capillaris acid; 14. Ammonium glycyrrhizate; 15. Vitexin; 16. Imperatorin.

[0058] 2.4 Investigation of fingerprint mapping methodology

[0059] 2.4.1 Precision Test

[0060] Take the test solution (S1) and inject it 6 times consecutively according to the chromatographic method under section "2.3". Using peak 32 (baicalin) as the reference peak, the results showed that the relative retention time RSD of the 52 common peaks was less than 0.17% and the relative peak area RSD was less than 2.9% (n=6), indicating that the instrument precision was good.

[0061] 2.4.2 Stability Test

[0062] Take the test solution (S1) and inject it according to the chromatographic method under section "2.3". Measure at 0, 2, 4, 8, 12 and 24 h respectively. Take peak 32 (baicalin) as the reference peak. The results show that the relative retention time RSD of the 52 common peaks is less than 0.18% and the relative peak area RSD is less than 3.0% (n=6), indicating that the test solution is stable within 24 h.

[0063] 2.4.3 Repeatability Test

[0064] Take the same batch of samples (S1), prepare 6 test solutions according to the method in section "2.2", and inject them according to the chromatographic conditions in section "2.3". With peak 32 (baicalin) as the reference peak, the results showed that the relative retention time RSD of the 52 common peaks was less than 0.23%, and the relative peak area RSD was less than 2.9% (n=6), indicating that the method has good repeatability.

[0065] 2.5 Analysis and Evaluation of Fingerprint Patterns

[0066] 2.5.1 Fingerprint pattern establishment and similarity evaluation

[0067] Five batches of Artemisia annua and Scutellaria baicalensis nasal drops samples were collected. Test solutions were prepared according to the method described in section "2.2", and the samples were injected according to the chromatographic method described in section "2.3". The chromatographic information was imported into the Chinese herbal fingerprinting software (2012 version) for peak matching. The median method was used, with a time window set to 0.2 min. After multi-point correction and automatic peak matching, a reference chromatogram (R) was generated. Peak 32 (baicalin peak) was used as the reference peak (peak S). A total of 52 common peaks were identified. See [link to relevant documentation]. Figure 3 The HPLC fingerprints of five batches of Artemisia annua and Scutellaria baicalensis nasal drops were imported into the software for evaluating the similarity of chromatographic fingerprints of traditional Chinese medicine and compared with the control chromatogram. The results showed that the similarity of each batch of samples (0.942, 0.964, 0.938, 0.942, and 0.938) was greater than 0.9, indicating good similarity and suggesting that the production process of Artemisia annua and Scutellaria baicalensis nasal drops is stable.

[0068] 2.5.2 Chromatographic Peak Assignment Analysis

[0069] Samples S1, Artemisia annua, Ephedra sinica, Angelica dahurica, Xanthium sibiricum, Glycyrrhiza uralensis extract, Scutellaria baicalensis extract, negative samples lacking Artemisia annua, Ephedra sinica, Angelica dahurica, Xanthium sibiricum, Glycyrrhiza uralensis extract, and Scutellaria baicalensis extract were prepared according to the method in section "2.2". Chromatographic conditions in section "2.3" were used for determination. By comparing the chromatograms of the samples, single herbs, and negative samples, 52 common peaks were identified. By comparing the retention times and spectral information with the reference chromatograms, 28 peaks were identified (see Table 1). Figure 4 .

[0070] Table 1 Chromatographic Peak Assignments

[0071]

[0072]

[0073] 2.6 Determination of Multiple Component Content

[0074] 2.6.1 Linear Relationship

[0075] Precisely pipette the reference stock solution from section "2.1" and accurately measure different volumes to prepare a series of mixed reference solutions of different concentrations. Measure the concentrations and plot a standard curve with reference concentration (μg / mL) on the x-axis (X) and peak area on the y-axis (Y). The results showed that all 16 components exhibited good linearity within their respective concentration ranges, as shown in Table 2.

[0076] Table 2. Linear range of 16 components.

[0077]

[0078] 2.6.2 Precision Test

[0079] The mixed reference solution was injected six times consecutively according to the chromatographic method described in section "2.3". The RSDs of the peak areas of the 16 components were 0.65%, 0.93%, 1.83%, 0.68%, 1.16%, 0.98%, 1.70%, 0.38%, 0.55%, 0.56%, 0.72%, 0.55%, 0.48%, 0.46%, 1.43%, and 0.66%, respectively, indicating that the instrument precision was good. The results are shown in Table 3.

[0080] Table 3 Precision test results

[0081]

[0082]

[0083] 2.6.3 Stability Test

[0084] The same test solution (S1) was taken and measured at 0, 2, 4, 8, 12, and 24 h according to the chromatographic conditions under section "2.3". The RSDs of the peak areas of the 16 components were 0.83%, 1.21%, 1.77%, 1.34%, and 1.34%, respectively.

[0085] The values ​​of 1.57%, 2.42%, 1.07%, 1.09%, 1.26%, 1.20%, 1.27%, 1.06%, 0.94%, 0.89%, 1.62%, and 1.25% indicate that the solution is stable after 24 hours of standing. The results are shown in Table 4.

[0086] Table 4. Stability Test Results

[0087]

[0088]

[0089] 2.6.4 Repeatability Test

[0090] Six test solutions were prepared from the same batch of test samples (S1) according to the method in section "2.2" and determined under the chromatographic conditions in section "2.3". The RSDs of the contents of the 16 components were 2.55%, 2.91%, 1.71%, 0.90%, 1.76%, 1.31%, 1.02%, 1.18%, 2.92%, 2.48%, 2.66%, 1.93%, 2.87%, 2.33%, 1.40%, and 0.76%, respectively, indicating that the method has good repeatability. The results are shown in Table 5.

[0091] Table 5 Results of Repeatability Tests

[0092]

[0093]

[0094] 2.6.5 Recovery Test

[0095] Six 1.0 mL portions of the test sample (S1) with known content were accurately measured. Sixteen reference solutions of each component were added at a 1:1 ratio. The test solution was prepared according to the method described in section "2.2" and determined by chromatographic method described in section "2.3". The average recoveries of the 16 components were 101.4%, 99.0%, 101.2%, 98.8%, 100.6%, 101.6%, and 100.2%, respectively. The percentages were 99.6%, 100.8%, 99.8%, 101.5%, 97.5%, 102.0%, 101.4%, 99.3%, and 101.7%, with RSDs of 1.9%, 2.0%, 2.7%, 0.8%, 2.9%, 1.4%, 2.4%, 1.8%, 2.3%, 2.0%, 2.8%, 1.8%, 2.7%, 1.2%, 2.7%, and 1.3%, respectively. The results are shown in Table 6.

[0096] Table 6 Results of the sample recovery test

[0097]

[0098]

[0099] Example 2

[0100] 2.7.1 Establishing QAMS

[0101] 2.7.1 Determination of relative correction factor (f)

[0102] Take the mixed reference solution and inject 1, 2, 4, 5, 8, 10, and 12 μL respectively under the chromatographic conditions described in section "2.1". Use baicalin as an internal reference (S) and calculate the following components according to the formula: neochlorogenic acid (A), chlorogenic acid (B), cryptochlorogenic acid (C), hyperoside (D), luteolin (E), isochlorogenic acid B (F), 1,5-dicaffeoylquinic acid (G), isochlorogenic acid C (H), rosmarinic acid (I), baicalin (J), budesonin (K), isochlorogenic acid (L), artemisinin (M), and glycyrrhizic acid ammonium.

[0103] The relative correction factor f for (N), vitexin (O) and imperatorin (P) S / A f S / B f S / C f S / D f S / E f S / F f S / G f S / H f S / I f S / J f S / K f S / L f S / M f S / N fS / O f S / P The results are shown in Table 7.

[0104] f S / i =f S / f i =(A s ×C i ) / (A i ×C s )

[0105] A s C represents the peak area of ​​the internal reference substance. s For internal reference concentration, A i C represents the peak area of ​​the component to be measured. i The concentration of the component to be measured is given.

[0106] Table 7. f using baicalin as an internal reference si Measurement results

[0107]

[0108]

[0109] 2.7.2 Effect of different volumetric flow rates on f S / i Impact

[0110] The effects of volumetric flow rates of 0.9, 1.0, and 1.1 mL / min on the 16 components were investigated. S / i The effect of different volumetric flow rates on f S / i The RSDs were all less than 3%, indicating that the volumetric flow rate had a significant impact on the efficiency of each component f. S / i No significant effect was observed; results are shown in Table 8.

[0111] Table 8. Effects of different volumetric flow rates on f S / i Impact

[0112]

[0113] 2.7.3 Effect of different column temperatures on f S / i Impact

[0114] The effects of column temperatures of 28, 30, and 32 °C on the 9 components f S / i The effect of different column temperatures on f S / i The RSDs of all components were less than 3%, indicating that column temperature has a significant effect on the f values ​​of each component. S / i No significant effect was observed; results are shown in Table 9.

[0115] Table 9 Effects of different column temperatures on f S / i Impact

[0116]

[0117] 2.7.4 Different instruments for f S / i Impact

[0118] The effects of three chromatographic systems—Thermo Scitific Ultimate 3000 and Agilent 1260—on the f-elements of various components were investigated. S / i The effects of f are shown in Table 10. S / i It adapts well to different instruments.

[0119] Table 10 Different instruments affect f S / i Impact

[0120]

[0121]

[0122] 2.7.5 Different chromatographic columns for f S / i Impact

[0123] The Agilent TC-C was examined. 18 (250×4.6mm, 5μm), YMC-Pack-ODS-A C 18 (250×4.6mm, 5μm), Cosmosil C 18 Three chromatographic columns (250×4.6mm, 5μm) were used to analyze 16 components. S / i The effect of different chromatographic columns on f S / i The RSDs were all less than 3%, indicating that different chromatographic columns have a better effect on the various components f S / i The impact was relatively small; the results are shown in Table 11.

[0124] Table 11 Effects of different chromatographic columns on f S / i Impact

[0125]

[0126]

[0127] 2.7.6 Investigation of chromatographic peak localization of the analyte

[0128] Take the mixed reference solution and determine the concentrations using different instruments and columns under the chromatographic conditions described in section "2.1". Calculate the relative retention times (t) of the other 15 component peaks and the baicalin peak in the preparation. i / s ), and perform chromatographic peak localization on each component, t i / s =t R(i) / t R(s) (i is the analyte, s is the internal reference), results for each analyte t i / s The RSDs are all less than 5%, indicating that t is usable. i / sThe chromatographic peaks of each component were located, and the results are shown in Table 12.

[0129] Table 12 Effects of different chromatographic columns and instruments on t i / s Impact

[0130]

[0131]

[0132] 2.8 Comparison of results from QAMS and external standard method (ESM)

[0133] The contents of 16 components in different batches of Artemisia annua nasal drops were determined by ESM and QAMS methods. The results showed no significant difference between the contents measured by the two methods (P>0.05), indicating that QAMS can be used for the determination of the contents of multiple components in Artemisia annua nasal drops. The results are shown in Table 13.

[0134] Table 13 QAMS and ESM Measurement Results

[0135]

[0136]

[0137]

[0138] In summary, this invention provides a quality evaluation method based on high-performance liquid chromatography (HPLC). Under specific sample solutions and chromatographic conditions, it can present all different types of components of medicinal materials on a single chromatogram through a single detection, allowing for simultaneous determination of multiple components and achieving comprehensive and accurate quality control of Artemisia annua and Scutellaria baicalensis nasal drops. The quality assessment method provided by this invention, with optimized chromatographic conditions and methodological validation, has the advantages of method stability, good repeatability, and stable and reliable measurement results.

[0139] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for detecting the quality of a nasal drop for treating allergic rhinitis, characterized by, It comprises the following steps: S1. Preparation of test solution: accurately measure 2.0 mL of nasal drops, place it in a 10 mL volumetric flask, dilute to the mark with 50% methanol, shake well, filter, and take the filtrate, which is obtained; S2. Preparation of mixed reference solution: accurately weigh new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, hyperoside, luteoloside, isochlorogenic acid, 1,5-dicaffeoylquinic acid, isochlorogenic acid C, rosmarinic acid, bupleurum glycoside, luteoloside, isochlorogenic acid, one branch of artemisia ketone acid, ammonium glycyrrhizinate, vitexicane and imperatorin reference substances, add 50% methanol to prepare a mixed reference solution; The concentration of the reference substances in the mixed reference solution is 0.04-7.68 mg / mL; S3. Take the mixed reference solution and the test solution respectively, inject into the liquid chromatograph for detection, analysis and recording of chromatogram, and quality evaluation; The liquid chromatography conditions are as follows: Column: Agilent TC C 18 Column, dimensions 250 x 4.6 mm, 5 μm; Mobile phase: mobile phase A is 0.1% phosphoric acid solution, and mobile phase B is acetonitrile; mobile phase A and mobile phase B are gradient eluted: 0-10 min, 97%-88% A; 10-30 min, 88%-78% A; 55-95 min, 70%-54% A; 95-115 min, 54%-20% A; 115-120 min, 20% A; Volume flow rate: 1.0 mL / min; Column temperature: 30℃; Detection wavelength: 245 nm; Injection volume: 5 μL; S4. Generation of control fingerprint: the chromatogram file of nasal drops obtained in S3 is exported from the instrument and imported into traditional Chinese medicine chromatography fingerprint similarity evaluation system; select the chromatographic peaks existing in the chromatograms of different batches of nasal drops as common peaks; generate the control fingerprint of nasal drops by using average value calculation method.

2. The quality detection method according to claim 1, characterized in that, The specific concentration of each component in the S2 mixed reference solution is: new chlorogenic acid 0.523 mg / mL, chlorogenic acid 0.756 mg / mL, cryptochlorogenic acid 1.046 mg / mL, hyperoside 0.489 mg / mL, luteoloside 1.126 mg / mL, isochlorogenic acid B 1.246 mg / mL, 1,5-dicaffeoylquinic acid 0.876 mg / mL, isochlorogenic acid C 0.964 mg / mL, rosmarinic acid 0.222 mg / mL, bupleurum glycoside 7.684 mg / mL, luteoloside 0.0474 mg / mL, isochlorogenic acid 0.237 mg / mL, one branch of artemisia ketone acid 1.001 mg / mL, ammonium glycyrrhizinate 2.056 mg / mL, vitexicane 1.174 mg / mL, and imperatorin 0.627 mg / mL.

3. The quality detection method of claim 1, wherein, The fingerprint spectrum of S3 includes 52 common peaks, specifically: peak 8 corresponding to neochlorogenic acid, peak 10 corresponding to chlorogenic acid, peak 11 corresponding to cryptochlorogenic acid, peak 22 corresponding to hyperoside, peak 23 corresponding to luteolin-7-O-glucoside, peak 26 corresponding to isochlorogenic acid B, peak 28 corresponding to 1,5-dicaffeoylquinic acid, peak 29 corresponding to isochlorogenic acid C, peak 30 corresponding to rosmarinic acid, peak 32 corresponding to baicalin, peak 35 corresponding to buddleoside, peak 37 corresponding to isochlorogenic acid, peak 39 corresponding to one artemisia ketone acid, peak 44 corresponding to ammonium glycyrrhizinate, peak 46 corresponding to vitexin, peak 49 corresponding to imperatorin.

4. The quality detection method of claim 1, wherein, The characteristic peaks in the fingerprint spectrum take the baicalin peak as the reference peak, and the relative retention times of 16 chromatographic peaks are 0.322, 0.419, 0.431, 0.712, 0.727, 0.741, 0.777, 0.814, 0.879, 1.000, 1.113, 1.279, 1.410, 1.777, 1.845 and 2.178, respectively.

5. The quality detection method of claim 1, wherein, The quality evaluation of S3 is performed by importing the fingerprint spectrum into traditional Chinese medicine chromatographic fingerprint similarity evaluation software and performing similarity evaluation with the control spectrum.

6. The application of the nasal drop quality detection method according to any one of claims 1-5 in the quality control of Artemisia qinghaiensis nasal drops.

7. Use according to claim 6, characterized in that, The quality detection method includes a fingerprint spectrum combined with a multi-component content determination method, and is applied in the quality control of traditional Chinese medicine material raw materials or solid drug preparations containing Artemisia qinghaiensis, Ephedra, Xanthium sibiricum, Scutellaria baicalensis, Glycyrrhiza and Angelica dahurica.

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