A method for detecting the fingerprint of chuan she gan oral liquid and a quality control method

Abstract

The application discloses a kind of chuan shegan oral liquid fingerprint detection method and quality control method, comprising the following steps: using 40-60% ethanol solution to chuan shegan oral liquid is extracted by ultrasound, after centrifugation, filtration, dilution, obtain the liquid to be measured;Using ultra-high performance liquid chromatograph to detect the liquid to be measured, obtain chromatogram and spectrogram, establish the standard atlas of chuan shegan oral liquid;Chromatographic condition is: mobile phase A is acetonitrile, mobile phase B is 0.4% phosphoric acid aqueous solution, column temperature is 39-41 DEG C;The flow rate of mobile phase: 0.3-0.4mL / min;Detection condition: PDA detector, 3D scanning range 190-400nm and extract maximum value diagram, or using the chromatogram of detection channel for wavelength 264nm, can obtain two kinds of fingerprint. By liquid chromatography, shegan glycoside in chuan shegan and other ten or so compounds are determined at a time, simple and fast, good separation effect, high determination accuracy, good result reproducibility, easy to observe, strong specificity, good chromatographic peak peak shape.

Description

Technical Field

[0001] This invention belongs to the field of detection of effective components in veterinary drugs, specifically relating to a fingerprint spectrum detection method and quality control method for Belamcanda chinensis oral liquid. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Belamcanda chinensis oral liquid is a traditional Chinese veterinary medicine preparation and a new type of veterinary drug. The main content of belamcanda glycosides was determined. In order to better control the quality of the product, the internal quality control standards are more rigorous and scientific, and fingerprint spectroscopy is used for quality control.

[0004] Its preparation process: 1. Extraction of medicinal materials (taking 100g as an example), (1) Remove impurities, dry and crush the medicinal material of Belamcanda chinensis; (2) Weigh 100g of Belamcanda chinensis, add 10 times the amount of 60% ethanol aqueous solution, reflux for 2 hours (extract filtered), then add 8 times the amount of 60% ethanol aqueous solution, reflux for 1.5 hours (extract filtered); (3) Combine the two filtrates and concentrate them to 2.4 times the amount of feed (about 240g). 2. Prescription and preparation process of Belamcanda chinensis oral liquid, (1) Prescription ingredients (310g): 240g of Belamcanda chinensis concentrate; 60g of N,N-dimethylacetamide (about 25% of the total mass of concentrate); 6.2g of Tween 80 (about 2% of the total amount); 0.62g of sodium bisulfite (about 0.2% of the total amount); 3g of water. (2) Formulation process: Solution A: Weigh the prescribed amount of sodium bisulfite, dissolve it in the prescribed amount of purified water, add it to the concentrated Belamcanda chinensis extract, stir well, and set aside. Solution B: Weigh the prescribed amount of N,N-dimethylacetamide, add the prescribed amount of Tween 80, and stir well, and set aside. Slowly pour Solution B into Solution A while stirring, until the concentrate is completely dissolved. After stirring well, let it stand for about 15 minutes, filter (twice), fill, and sterilize to obtain the final product.

[0005] The 2020 edition of the Pharmacopoeia of the People's Republic of China includes Belamcanda chinensis, but only the chromatographic peaks of Belamcanda chinensis and Iris flavin are subject to quality control. The control of the chromatographic peaks of many other components is not carried out, which is a rather one-sided approach and makes it difficult to achieve good quality control of Belamcanda chinensis oral liquid. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a fingerprint spectrum detection method and quality control method for Belamcanda chinensis oral liquid.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0008] In a first aspect, the present invention provides a fingerprint spectrum detection method for Belamcanda chinensis oral liquid, comprising the following steps:

[0009] The oral liquid of Belamcanda chinensis was diluted in a volumetric flask with 40-60% ethanol solution, shaken well, and then subjected to ultrasonic extraction. A portion of the liquid was taken out, centrifuged, filtered, and diluted to obtain the test solution.

[0010] Ultra-high performance liquid chromatography was used to detect the test solution, and chromatograms and spectra were obtained to establish a standard chromatogram for Belamcanda chinensis oral liquid;

[0011] The chromatographic conditions were as follows: mobile phase A was acetonitrile, mobile phase B was 0.4% phosphoric acid aqueous solution, column temperature was 39-41℃, and the flow rate of the mobile phase was 0.3-0.4 mL / min.

[0012] Detection conditions: PDA detector, 3D scanning range 190-400nm, detection wavelength 264nm channel.

[0013] In some embodiments, the gradient elution method for ultra-high performance liquid chromatography detection is as follows:

[0014] The volume fraction of mobile phase A was maintained at 5% for 0-1 min;

[0015] Within 1-3 minutes, the volume fraction of mobile phase A changed from 5% to 15%.

[0016] In 3-4.5 minutes, the volume fraction of mobile phase A changed from 15% to 18%.

[0017] The volume fraction of mobile phase A was maintained at 18% for 4.5-5.5 min.

[0018] In 5.5-8.5 min, the volume fraction of mobile phase A changed from 18% to 20%;

[0019] In 8.5-9.2 minutes, the volume of mobile phase A changed from 20% to 28%.

[0020] 9.2-10 min, the volume fraction of mobile phase A was maintained at 28%;

[0021] In 10-12 minutes, the volume fraction of mobile phase A changed from 28% to 35%.

[0022] The volume fraction of mobile phase A was maintained at 35% for 12-13.5 min.

[0023] In 13.5-15 min, the volume fraction of mobile phase A changed from 35% to 50%;

[0024] For 15-16 minutes, the volume fraction of mobile phase A was maintained at 50%.

[0025] In 16-17 minutes, the volume fraction of mobile phase A changed from 50% to 60%.

[0026] From 17 to 17.5 min, the volume fraction of mobile phase A decreased from 60% to 5%; from 17.5 to 20 min, the volume fraction of mobile phase A remained at 5%.

[0027] Initially, the organic phase of mobile phase A is low; in the middle stage, A gradually increases; and in the later stage, A gradually decreases back to the initial gradient. From 5.5 to 8.5 min, the volume fraction of mobile phase A changes from 18% to 20% to ensure the main component elutes as quickly as possible while maintaining good peak shape and resolution. From 17 to 17.5 min, the volume fraction of mobile phase A changes from 60% to 5% to return to the original gradient and begin the next injection cycle.

[0028] In some embodiments, during ultrasonic extraction, the volume ratio of Belamcanda chinensis oral liquid to extractant is 1:25-100.

[0029] Preferably, the volume ratio of the oral liquid of *Belamcanda chinensis* to the extractant is 1:25-75, more preferably 1:35-65, and even more preferably 1:45-55.

[0030] Preferably, the ultrasonic extraction time is 10-20 minutes. Using ultrasonic extraction can effectively improve the extraction efficiency and the degree of extraction completeness.

[0031] Preferably, the extractant is a 45%-55% ethanol solution. Methanol is highly toxic and harmful to the health of testing personnel; therefore, ethanol, a solvent with low toxicity, should be chosen whenever possible, and the amount of ethanol should be minimized. Using an appropriate concentration of aqueous ethanol solution can save costs and reduce environmental pollution.

[0032] In some embodiments, the centrifugation speed is 8000-12000 rpm / min and the centrifugation time is 12-20 min.

[0033] Preferably, the centrifugation speed is 9000-11000 rpm / min and the centrifugation time is 14-17 min.

[0034] In some embodiments, the injection volume is 1-2 μL.

[0035] In some embodiments, the chromatographic column is a Waters UPLC HSS T3 column. It employs a trifunctional C18 alkyl-bonded phase, which enhances the retention of polar compounds, provides perfect peak shape across the pH range of 2–8, significantly improves separation selectivity and stability, is acid and water resistant, and the column is less prone to collapse when the organic phase ratio is small.

[0036] Secondly, the present invention provides a quality control method for Belamcanda chinensis oral liquid, comprising the following steps:

[0037] A standard fingerprint spectrum for Belamcanda chinensis oral liquid was established based on the above fingerprint spectrum detection method.

[0038] The oral liquid of *Belamcanda chinensis* to be tested was tested according to the fingerprint spectrum detection method described above to obtain the fingerprint spectrum of the oral liquid of *Belamcanda chinensis* to be tested.

[0039] The fingerprint spectrum of the sample to be tested is compared with the standard fingerprint spectrum, and the quality of the sample of Belamcanda chinensis oral liquid is evaluated or controlled by the chromatographic peak parameters in the spectrum.

[0040] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows:

[0041] This invention uses liquid chromatography to simultaneously determine multiple major components, including belamcanda chinensis glycoside and more than ten other compounds, in dandelion. The method is simple, rapid, has good separation effect, high determination accuracy, good reproducibility, is easy to observe, has strong specificity, and produces good chromatographic peak shapes. It meets the requirements of the Chinese Veterinary Pharmacopoeia and saves time and reagents.

[0042] The detection method of this invention establishes a fingerprint spectrum with distinct peak shapes and uniform separation by controlling various process conditions. This systematically reflects the complete chemical composition of *Belamcanda chinensis* oral liquid, and the relative retention times of the obtained common peaks are relatively stable, providing a basis for evaluating the quality of *Belamcanda chinensis* oral liquid. Furthermore, the fingerprint spectrum obtained using this detection method exhibits good separation of each chromatographic peak, with clear separation between peaks, accurately identifying the chromatographic peaks of the main chemical components. It also demonstrates good stability and reproducibility, enabling effective quality control of *Belamcanda chinensis* oral liquid.

[0043] The determination method of the present invention uses 50% ethanol as the extraction solvent to extract the test sample. It has low toxicity, moderate peak area response value of the compound, simple sample processing, and the solvent used is low in toxicity, safe and environmentally friendly, and has little harm to humans and the environment.

[0044] The determination method of this invention uses an ultra-high performance liquid chromatograph in conjunction with a PDA detector to present the results in the form of chromatograms and spectra. It is convenient, fast, and the results are intuitive and easy to interpret. The detection limit is low, which greatly shortens the testing time, greatly increases the number of detection indicators, and improves the testing efficiency, detection sensitivity and testing effect. Attached Figure Description

[0045] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0046] Figure 1 This is the spectrum of the irisin reference standard in Example 1 of the present invention.

[0047] Figure 2 The images show the spectra of 15 compounds in the first batch of oral liquid in Example 1 of this invention, where a is peak 1, b is peak 2, c is peak 3, d is peak 4, e is peak 5, f is peak 6, g is peak 7, h is peak 8, i is peak 9, j is peak 10, k is peak 11, l is peak 12, m is peak 13, n is peak 14, and o is peak 15.

[0048] Figure 3 This is the maximum value image extracted from the first batch of oral liquid photograms in Example 1 of the present invention.

[0049] Figure 4 This is the maximum value image extracted from the second batch of oral liquid photograms in Example 1 of the present invention.

[0050] Figure 5 This is the maximum value image extracted from the third batch of oral liquid photograms in Example 1 of the present invention.

[0051] Figure 6 The chromatogram at 264 nm is the first batch of oral liquid in Example 2 of this invention.

[0052] Figure 7 This is the chromatogram of the second batch of oral liquid at 264 nm in Example 2 of the present invention.

[0053] Figure 8 This is the chromatogram of the third batch of oral liquid in Example 2 of the present invention at 264 nm.

[0054] Figure 9 The image shows the spectra of 12 compounds in the first batch of oral liquid in Example 2 of this invention, where a is peak 1, b is peak 2, c is peak 3, d is peak 4, e is peak 5, f is peak 6, g is peak 7, h is peak 8, i is peak 9, j is peak 10, k is peak 11, and l is peak 12.

[0055] Figure 10 Chromatogram of Belamcanda chinensis reference standard. Detailed Implementation

[0056] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0057] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0058] Example 1

[0059] Simultaneous determination of 15 active ingredients in a Sichuan Belamcanda chinensis oral liquid:

[0060] Test sample: a research and development sample for the company, supplied by Shandong Jinzhuji Pharmaceutical Co., Ltd.

[0061] Phosphoric acid was of superior grade, and ethanol and methanol were of chromatographic grade. Reference standards were mainly purchased from the National Institutes for Food and Drug Control.

[0062] Instruments used: BP211D analytical balance (Sartorius, Germany); Waters Acquity TM Ultra-high performance liquid chromatograph (Waters Corporation, USA), PDA detector, column: Waters UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm).

[0063] A fingerprint chromatogram determination method for Belamcanda chinensis oral liquid, using Belamcanda chinensis glycoside as the reference chromatographic peak, and 14 other chromatographic peaks, for a total of 15 chromatographic peaks.

[0064] The determination method includes the following steps:

[0065] a. Preparation of control stock solution

[0066] Dissolve the irisin reference standard in methanol and dilute to volume to prepare a reference stock solution.

[0067] b. Preparation of the test sample solution: Accurately measure 1 mL of the oral liquid of Belamcanda chinensis and place it in a 50 mL volumetric flask. Dissolve it with an appropriate solvent and dilute to the mark. Weigh it, extract it by sonication for 15 min, weigh it again, and make up the weight loss with an appropriate solvent. Accurately measure 5 mL of the solution, centrifuge it at 10000 rpm for 10 min, filter it, and dilute the filtrate twice with the initial mobile phase (mobile phase A:B volume ratio = 5:95) for liquid chromatography determination.

[0068] Suitable solvents can be methanol, ethanol, or methanol solutions of different concentrations or ethanol solutions of different concentrations. Ultimately, a 50% ethanol solution is preferred.

[0069] c. The instrument used was an ultra-high performance liquid chromatograph (UHPLC), with a photodiode array detector (PDA) and a Waters UPLC HSS T3 column. The column temperature was 39-41℃. Mobile phase A was acetonitrile, and mobile phase B was 0.4% phosphoric acid aqueous solution. The flow rate was 0.35 mL / min, and the injection volume was 0.2-2 μL. A PDA detector was selected, and the maximum value was extracted from the 3D spectrum. The gradient elution program for the mobile phase is shown in Table 1.

[0070] Table 1

[0071] Time (min) Flow rate (ml / min) ％A ％B curve 1 initial 0.350 5.0 95.0 initial 2 1.00 0.350 5.0 95.0 6 3 3.00 0.350 15.0 85.0 6 4 4.50 0.350 18.0 82.0 6 5 5.50 0.350 18.0 82.0 6 6 8.50 0.350 20.0 80.0 6 7 9.20 0.350 28.0 72.0 6 8 10.00 0.350 28.0 72.0 6 9 12.00 0.350 35.0 65.0 6 10 13.50 0.350 35.0 65.0 6 11 15.00 0.350 50.0 50.0 6 12 16.00 0.350 50.0 50.0 6 13 17.00 0.350 60.0 40.0 6 14 17.50 0.350 5.0 95.0 6 15 18.00 0.350 5.0 95.0 6

[0072] The gradient elution program was as follows: 0-1 min, mobile phase A volume fraction maintained at 5%; 1-3 min, mobile phase A volume fraction changed from 5% to 15%; 3-4.5 min, mobile phase A volume fraction changed from 15% to 18%; 4.5-5.5 min, mobile phase A volume fraction maintained at 18%; 5.5-8.5 min, mobile phase A volume fraction changed from 18% to 20%; 8.5-9.2 min, mobile phase A volume fraction changed from 20% to 28%; 9.2-10 min, mobile phase A volume fraction maintained at 28%; 10-12 min... The volume fraction of mobile phase A changes from 28% to 35%; from 12 to 13.5 min, the volume fraction of mobile phase A remains at 35%; from 13.5 to 15 min, the volume fraction of mobile phase A changes from 35% to 50%; from 15 to 16 min, the volume fraction of mobile phase A remains at 50%; from 16 to 17 min, the volume fraction of mobile phase A changes from 50% to 60%; from 17 to 17.5 min, the volume fraction of mobile phase A changes from 60% to 5%; from 17.5 to 20 min, the volume fraction of mobile phase A remains at 5%, and the process returns to the original gradient for the next cycle of sampling.

[0073] Table 2. Chromatographic data of the first batch of Belamcanda chinensis oral liquid. Injection volume (1 μL)

[0074]

[0075] Table 3. Chromatographic data of the second batch of Belamcanda chinensis oral liquid, injection volume (1 μL).

[0076]

[0077]

[0078] Table 4. Chromatographic data of the third batch of Belamcanda chinensis oral liquid, injection volume (2 μL).

[0079]

[0080] d. Comparison of different injection volumes: 0.5 μL, 1 μL, and 2 μL of the mixed control solution and the test sample were injected into the high-performance liquid chromatograph. The chromatographic response was good in all cases. Due to space limitations, only the results of 1-2 μL were listed. Gradient elution and detection were performed under the chromatographic conditions in step c. The retention time and spectrum were used for qualitative identification. The chromatographic parameters such as injection volume, peak area, and retention time of the reference and sample are shown in Tables 2 to 4.

[0081] In the initial pretreatment process, extraction with different concentrations of methanol and ethanol solvents resulted in variations in the number of detected chromatographic peaks and peak areas. Due to the high toxicity of methanol, 50% ethanol was ultimately chosen as the preferred solvent for extraction, which detected 15 compounds.

[0082] e. Results Analysis: The filtrate from step b was injected into a high-performance liquid chromatograph (HPLC). Gradient elution and detection were performed under the chromatographic conditions of step c. The peak areas of each target analyte in the filtrate were measured, and qualitative analysis was conducted using retention time, relative retention time, and spectra. Peak 6, Belamcanda chinensis, was used as the reference peak (S). The relative retention times of other peaks were calculated to establish a fingerprint spectrum, showing a very constant relative retention time. According to the fingerprint spectrum, the chromatogram of the test sample showed the same chromatographic peaks at the corresponding positions as those of the 15 active ingredients in the test sample. The peak shapes were good, and the separation was acceptable, indicating that the target medicinal material could be detected.

[0083] Example 2

[0084] Under identical conditions, multiple wavelengths can be acquired simultaneously. Through multi-channel scanning comparison, the 264nm channel is preferentially selected as the detection wavelength for the active ingredient, Belamcanda chinensis glycoside, showing good chromatographic peak response and resolution. A fingerprint chromatographic determination method for Belamcanda chinensis oral liquid uses Belamcanda chinensis glycoside as the reference chromatographic peak, along with 11 other chromatographic peaks, for a total of 12 chromatographic peaks.

[0085] The optimal wavelength was 264 nm. Chromatograms were extracted at this wavelength, and data processing was performed. Peak 4 (belamcanda chinensis) was used as the reference peak (S). The relative retention times of other peaks were calculated to establish a fingerprint spectrum. The relative retention times were very constant. Specific data are shown in Tables 5-7. According to the fingerprint spectrum, the chromatograms of the test samples showed the same peaks at the corresponding positions as those of the 12 active ingredients in the test samples. The peak shapes were good, and the separation was acceptable, indicating that the target medicinal material could be detected. The peak areas of belamcanda chinensis in the reference solution were compared (chromatograms are shown in Tables 5-7). Figure 10 The amount of belamcanda glycoside in the oral liquid of danshen was calculated by using the method shown in Table 8. The content of each batch was relatively uniform, with an average content of 18.11 mg / ml, which proves that the production process was well optimized.

[0086] Table 5. Chromatographic data of the first batch of oral solutions. Injection volume (1 μL)

[0087]

[0088] Table 6. Chromatographic data of the second batch of oral solutions. Injection volume (1 μL)

[0089]

[0090] Table 7. Chromatographic data of the third batch of oral solutions. Injection volume (2 μL)

[0091]

[0092]

[0093] Table 8. Content of Belamcanda chinensis glycoside, the main component of Belamcanda chinensis oral liquid.

[0094]

[0095] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A fingerprint spectrum detection method for Belamcanda chinensis oral liquid, characterized in that: Includes the following steps: The oral liquid of Belamcanda chinensis was extracted by ultrasonic extraction with 40-50% ethanol solution. After centrifugation, filtration and dilution, the test solution was obtained. During the ultrasonic extraction, the volume ratio of oral liquid of Belamcanda chinensis to extractant was 1:45-55. Ultra-high performance liquid chromatography was used to detect the test solution, and chromatograms and spectra were obtained to establish a standard chromatogram for Belamcanda chinensis oral liquid; The chromatographic conditions were as follows: mobile phase A was acetonitrile, mobile phase B was 0.4% phosphoric acid aqueous solution, column temperature was 39-41℃, mobile phase flow rate was 0.35-0.4 mL / min, and injection volume was 1-2 μL. Detection conditions: PDA detector, 3D scanning range 190-400nm, detection wavelength 264nm channel; The chromatographic column was a Waters UPLC HSS T3 column; The gradient elution method for ultra-high performance liquid chromatography detection is as follows: For 0-1 min, the volume fraction of mobile phase A was maintained at 5%; Within 1-3 minutes, the volume fraction of mobile phase A changed from 5% to 15%. In 3-4.5 minutes, the volume fraction of mobile phase A changed from 15% to 18%. The volume fraction of mobile phase A was maintained at 18% for 4.5–5.5 min. Over 5.5-8.5 minutes, the volume fraction of mobile phase A changed from 18% to 20%. In 8.5-9.2 minutes, the volume of mobile phase A changed from 20% to 28%. 9.2-10 min, the volume fraction of mobile phase A was maintained at 28%; In 10-12 minutes, the volume fraction of mobile phase A changed from 28% to 35%. The volume fraction of mobile phase A was maintained at 35% for 12-13.5 min. In 13.5-15 minutes, the volume fraction of mobile phase A changed from 35% to 50%. For 15-16 minutes, the volume fraction of mobile phase A was maintained at 50%. After 16-17 minutes, the volume fraction of mobile phase A changed from 50% to 60%. Over 17-17.5 minutes, the volume fraction of mobile phase A changed from 60% to 5%. The volume fraction of mobile phase A was maintained at 5% for 17.5-20 min.

2. The fingerprint spectrum detection method for Belamcanda chinensis oral liquid according to claim 1, characterized in that: The ultrasonic extraction time is 10-20 minutes.

3. The fingerprint spectrum detection method for Belamcanda chinensis oral liquid according to claim 1, characterized in that: The extractant is a 45%-50% ethanol solution.

4. The fingerprint spectrum detection method for Belamcanda chinensis oral liquid according to claim 1, characterized in that: The centrifugation speed is 8000-12000 rpm, and the centrifugation time is 12-20 min.

5. A quality control method for a Sichuan Belamcanda chinensis oral liquid, characterized in that: Includes the following steps: According to the fingerprint spectrum detection method of any one of claims 1-4, a 3D scanning range of 190-400nm is used to extract the maximum value map, or a chromatogram with a detection channel of wavelength 264nm is used to obtain two fingerprint spectra, thereby establishing a standard fingerprint spectrum for Belamcanda chinensis oral liquid. The oral liquid of *Belamcanda chinensis* to be tested was tested according to the fingerprint spectrum detection method described above to obtain the fingerprint spectrum of the oral liquid of *Belamcanda chinensis* to be tested. The fingerprint spectrum of the sample to be tested is compared with the standard fingerprint spectrum, and the quality of the sample of Belamcanda chinensis oral liquid is evaluated or controlled by the chromatographic peak parameters in the spectrum.

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