Establishment method of fingerprint spectrum of diaphoresis and antipyretic preparation

Abstract

The invention discloses a method for establishing a fingerprint spectrum of a diaphoresis and antipyretic preparation, which comprises the following steps: preparing a test solution, and carrying out high performance liquid chromatography detection on the test solution, the test solution is prepared by the following steps: adding 65-75% of methanol or ethanol into a diaphoresis and anti-pestilence preparation, and carrying out ultrasonic treatment; the high performance liquid chromatography detection adopts gradient elution, and the gradient elution procedure is as follows: 0-15 min, 6-12% A; 12% A is changed into 20% A, and 20%-22% A is changed into 20% A after 15 min to 45 min and 45 min to 65 min; when the time is 65-85 minutes, 22% A is changed into 70% A; the fingerprint spectrum of the diuresis and antipyretic granules established by the invention can be used for objectively, comprehensively and accurately evaluating the quality of the diuresis and antipyretic granules, and has important significance for controlling the quality of diuresis and antipyretic granules and ensuring the clinical curative effect.

Description

Technical Field

[0001] This invention belongs to the field of component analysis technology, and more specifically, this invention relates to a method for establishing the fingerprint spectrum of a penetrating and detoxifying preparation. Background Technology

[0002] The Toujie Quwen preparation is a pharmaceutically acceptable compound preparation of traditional Chinese medicine, made from 16 herbs including Forsythia suspensa, Cremastra appendiculata, Lonicera japonica, Scutellaria baicalensis, Isatis indigotica, Astragalus membranaceus, Pseudostellaria heterophylla, Poria cocos, Bupleurum chinense, Artemisia annua, Cicadae periostracum, Peucedanum praeruptorum, Fritillaria cirrhosa, Fritillaria thunbergii, Scrophularia ningpoensis, and Prunus mume. It is available in granule, oral liquid, and decoction forms. This preparation has the effects of clearing heat and detoxifying, dispelling wind and releasing the exterior, relieving cough and resolving phlegm, and nourishing qi and yin. It can be used to treat viral pneumonia and other diseases.

[0003] The Toujie Quwen preparation is composed of various medicinal materials with a complex chemical composition, and its pharmacodynamic material basis and mechanism of action are not yet fully understood. Among them, the formula heavily uses Forsythia suspensa, taking advantage of its light and cold nature to enter the lungs and clear lung heat. Modern pharmacological studies have also shown that Forsythia suspensa contains multiple components such as forsythoside and forsythoside A, which have antiviral and anti-inflammatory effects. Therefore, multiple components in Forsythia suspensa may be the potential pharmacodynamic material basis of Toujie Quwen granules.

[0004] Currently, there is limited research on the quality control of Toujie Quwen preparations. Existing quality control methods for hospital preparations mainly involve using two HPLC methods to detect the content of forsythoside, a single component in Forsythia suspensa, and the content of baicalin, a single component in Scutellaria baicalensis. These methods are cumbersome, time-consuming, and costly, and cannot simultaneously control the content of multiple quality control indicators. Therefore, they cannot comprehensively reflect the quality of Toujie Quwen preparations, nor can they meet the needs of research on the pharmacodynamic material basis and mechanism of action of Toujie Quwen preparations.

[0005] Traditional Chinese medicine (TCM) fingerprinting is a holistic quality evaluation method that uses modern analytical techniques to systematically characterize the complex components of TCM. Based on the holistic and comprehensive effects of TCM's multiple components and targets, fingerprinting offers a more comprehensive approach to quality control of non-single-component drugs from a "whole-component" perspective. Combining fingerprinting with multi-component content determination can better control the quality of Toujie Quwen granules. However, on the one hand, Toujie Quwen preparation consists of 16 herbs, resulting in a large formula with significant inter-herb influence and substantial differences in chemical properties among the different herbs. On the other hand, the principal and assistant herbs (Forsythia, Scutellaria, and Lonicera) in Toujie Quwen preparation constitute a relatively low proportion (approximately 20%). Therefore, simultaneously detecting the water-soluble and lipid-soluble components of these herbs is extremely difficult, and achieving complete chromatographic peak separation is challenging, making it difficult to identify consistently occurring common peaks. Summary of the Invention

[0006] Based on this, the purpose of this invention is to provide a method for establishing the fingerprint spectrum of a detoxifying and antipyretic preparation.

[0007] The technical solutions for achieving the above-mentioned objectives include the following.

[0008] A first aspect of the present invention provides a method for establishing a fingerprint spectrum of a penetrating and detoxifying preparation, comprising the following steps: preparing a test solution, and then performing high-performance liquid chromatography (HPLC) on the test solution; the test solution is prepared by the following method: taking the penetrating and detoxifying preparation and adding 65%~75% methanol or ethanol, followed by sonication; the HPLC detection employs gradient elution, the gradient elution program being: 0 min~15 min, 6~12% A; 15 min~45 min, 12% A→20% A, 45 min~65 min, 20~22% A; 65 min~85 min, 22% A→70% A.

[0009] In a second aspect, the present invention provides a fingerprint spectrum of a detoxifying and antipyretic preparation established by the above method.

[0010] In a third aspect, the present invention provides the application of the fingerprint spectrum of the above-mentioned penetrating and detoxifying preparation in the quality control of penetrating and detoxifying preparations.

[0011] In this invention, by selecting appropriate sample preparation methods and HPLC detection conditions, both water-soluble and lipid-soluble components of the Toujie Quwen preparation can be detected simultaneously. The separation of each chromatographic peak is good, with 18 chromatographic peaks exhibiting stable relative retention times. Peaks can be stably eluted from each batch of samples, and the RSD of the relative retention times of the characteristic peaks is less than 0.5%. Thus, a fingerprint chromatogram of Toujie Quwen granules has been successfully established, which can be used to objectively, comprehensively, and accurately evaluate the quality of Toujie Quwen granules. This is of great significance for controlling the quality of Toujie Quwen and ensuring its clinical efficacy. Attached Figure Description

[0012] Figure 1 The chromatograms are of the reference solution and one batch of Toujie Quwen Granules; where A is Toujie Quwen Granules and B is the mixed reference solution; peak 7: forsythoside I, peak 9: forsythoside A, peak 10: (+) pinoresinin-β-D-glucopyranoside, peak 12: 4,5-O-caffeoylquinic acid, peak 13: baicalin, peak 14: forsythoside, peak 17: baicalein, peak 18: forsythoside.

[0013] Figure 2 The fingerprint spectrum of 12 batches of Toujie Quwen Granules.

[0014] Figure 3 The fingerprint spectrum is a control for Toujie Quwen Granules; among them, peak 7: forsythoside I, peak 9: forsythoside A, peak 10: (+) pinoresinin-β-D-glucopyranoside, peak 12: 4,5-O-caffeoylquinic acid, peak 13: baicalin, peak 14: forsythoside, peak 17: baicalein, peak 18: forsythoside.

[0015] Figure 4 The chromatogram is for the specificity test; where A: blank solvent; B: mixed reference standard; C: Toujie Quwen granules.

[0016] Figure 5 HPLC chromatograms of test solution preparations using different extraction solvents.

[0017] Figure 6 HPLC chromatograms of test sample solutions prepared using different extraction methods.

[0018] Figure 7 The images show HPLC chromatograms obtained when different gradient elution programs are used for detection.

[0019] Figure 8 These are HPLC chromatograms obtained when different detection wavelengths are used.

[0020] Figure 9 The images show HPLC chromatograms obtained when different flow rates are used for detection.

[0021] Figure 10 These are HPLC chromatograms obtained when detection is performed at different temperatures. Detailed Implementation

[0022] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0023] Unless otherwise defined, 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. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0024] In some embodiments of the present invention, a method for establishing the fingerprint spectrum of a penetrating and detoxifying preparation is disclosed, comprising the following steps: preparing a test solution, and then performing high-performance liquid chromatography (HPLC) on the test solution; the test solution is prepared by the following method: taking the penetrating and detoxifying preparation and adding 65%~75% methanol or ethanol, followed by sonication; the HPLC detection adopts gradient elution, and the gradient elution program is: 0 min~15 min, 6~12% A; 15 min~45 min, 12% A→20% A, 45 min~65 min, 20~22% A; 65 min~85 min, 22% A→70% A.

[0025] Traditional Chinese medicine (TCM) fingerprint chromatograms have strict quantitative limitations on chromatogram similarity, elution time of all common peaks, and peak area. Even slight changes in the preparation method of the test sample solution and chromatographic conditions can directly affect the elution time and peak area of ​​the overall chromatogram or individual common peaks, as well as the resolution and peak shape of each chromatographic peak. Therefore, when constructing a fingerprint chromatogram for a TCM preparation, the entire method acts as a whole, ultimately affecting whether a qualified fingerprint chromatogram can be obtained. In this invention, by selecting a suitable extraction solvent and extraction method during test sample preparation, and by selecting an appropriate gradient elution program during HPLC detection, along with other chromatographic conditions such as column temperature, flow rate, and detection wavelength, stable and reproducible chromatographic peaks were obtained, thus constructing the fingerprint chromatogram of the TCM preparation.

[0026] In one embodiment, the test solution is prepared by the following method: taking a detoxifying and antipyretic preparation, adding 68%~72% methanol, and sonicating;

[0027] In one embodiment, the mass-to-volume ratio of the detoxifying and detoxifying preparation to 68%~72% methanol is 1 g: 24mL~26mL.

[0028] In one embodiment, the power of the ultrasound is 150 W to 250 W, the frequency of the ultrasound is 70 kHz to 90 kHz, and the duration of the ultrasound is 10 min to 20 min.

[0029] In one embodiment, the ultrasound duration is 14 to 16 minutes.

[0030] In one embodiment, the conditions for the high-performance liquid chromatography detection further include: the chromatographic column is C10. 18 Chromatographic column; detection wavelength: 235 nm to 245 nm; column temperature: 24℃ to 26℃; flow rate: 0.9 mL / min to 1.1 mL / min.

[0031] In one embodiment, the detection wavelength is 238 nm to 242 nm; the column temperature is 25°C to 26°C; and the flow rate is 0.95 mL / min to 1.05 mL / min.

[0032] In one embodiment, the chromatographic column is Hypersil ODS2 C 18 A chromatographic column with dimensions of 4.6 mm × 250 mm and 5 µm.

[0033] In one embodiment, the conditions for high-performance liquid chromatography detection further include: mobile phase A being acetonitrile and mobile phase B being a 0.18%~0.22% aqueous solution of phosphoric acid.

[0034] In one embodiment, the detoxifying and detoxifying preparation is made from raw materials including Forsythia suspensa, Cremastra appendiculata, Lonicera japonica, Scutellaria baicalensis, Isatis indigotica, Astragalus membranaceus, Pseudostellaria heterophylla, Poria cocos, Bupleurum chinense, Artemisia annua, Cicadae periostracum, Peucedanum praeruptorum, Fritillaria cirrhosa, Fritillaria thunbergii, Scrophularia ningpoensis, and Prunus mume.

[0035] In other embodiments of the present invention, fingerprint spectra of the antipyretic preparations established by the above method are disclosed.

[0036] In other embodiments of the present invention, the application of fingerprint spectrum of the penetrating and detoxifying preparation in the quality control of the penetrating and detoxifying preparation is disclosed.

[0037] In the following embodiments of the present invention, the instruments used include: an Agilent 1260 high-performance liquid chromatograph; a Waters 2695 high-performance liquid chromatograph; and an Elite Hypersil ODS2 C. 18 (250 mm×4. 6 mm, 5 μm); Agilent ZORBAX SB-C 18 (250 mm×4. 6 mm, 5 μm); Waters Xbridge C 18 (250 mm × 4.6 mm, 5 μm); CP225D type 1 / 100,000 balance (Sartorius GmbH, Germany); BT 214D type 1 / 10,000 balance (Sartorius GmbH, Germany); Genie U 12 ultrapure water system (Shanghai Lefeng Biotechnology Co., Ltd.).

[0038] In the following embodiments of the present invention, the instruments used include: 9 batches of Toujie Quwen Granules samples from Guangzhou Baiyunshan Hutchison Whampoa Chinese Medicine Co., Ltd., with batch numbers A23A001, A23A002, A23A003, L22A001, L22A002, K22A002, 20220204, 20220205, and 20220206; and 3 batches of Toujie Quwen Granules samples from Guangdong Yifang Pharmaceutical Co., Ltd. (manufactured under contract by Guangzhou Eighth People's Hospital), with batch numbers J2108015, J2108016, and J2108017. Reference standards for forsythoside A (batch number: 111810-202108, purity: 96.2%), forsythoside (batch number: 110821-202117, purity: 94.9%), baicalin (batch number: 110715-202122, purity: 94.2%), and baicalein (batch number: 111595-201808, purity: 97.9%) were purchased from the National Institutes for Food and Drug Control; forsythoside I (batch number: ST20200120-14) 297 (purity: 99.3%), (+) pinoresinin-β-D-glucopyranoside (batch number: ST15550120-14296, purity: 99.3%), forsythoside (batch number: chromatographic grade (Merck, Germany), 4,5-O-caffeoylquinic acid (batch number: RS06601020-9113, purity: 91.3%) were purchased from Shanghai Shidande Biotechnology Co., Ltd.; phosphoric acid was chromatographic grade (Comio Biotechnology); methanol was analytical grade (Guangzhou Chemical Reagent Factory).

[0039] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0040] Example 1: Method for establishing the fingerprint spectrum of a penetrating and detoxifying preparation

[0041] Includes the following steps:

[0042] 1. Preparation of reference solution

[0043] Accurately weigh appropriate amounts of each of the eight reference standards and prepare mixed solutions with methanol at concentrations of 0.2012 mg / mL, 0.3898 mg / mL, 49.85 μg / mL, 46.46 μg / mL, 4.027 μg / mL, 0.1068 mg / mL, 0.1356 mg / mL, and 0.1664 mg / mL, respectively, forsythoside I, forsythoside A, (+)pinoresinin-β-D-glucopyranoside, forsythoside, forsythoside A, 4,5-O-caffeoylquinic acid, baicalin, and baicalein.

[0044] 2. Preparation of the test solution

[0045] Take 12 batches of Toujie Quwen Granules, grind them into a fine powder, accurately weigh 1 g, accurately add 25 mL of 70% methanol, weigh the powder, sonicate (power 200 W, frequency 80 kHz) for 15 min, cool, weigh the powder again, replenish the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the final product.

[0046] 3. HPLC detection

[0047] Chromatographic conditions include:

[0048] Column: Hypersil ODS2 C 18 (250mm×4.6mm, 5μm)

[0049] Mobile phase: Acetonitrile (A) - 0.2% phosphoric acid aqueous solution (B)

[0050] Gradient elution: 0 min ~ 15 min, 6 ~ 12% A; 15 min ~ 45 min, 12% A → 20% A, 45 min ~ 65 min, 20 ~ 22% A; 65 min ~ 85 min, 22% A → 70% A

[0051] Flow rate: 1.0 mL / min

[0052] Detection wavelength: 240 nm

[0053] Column temperature: 25℃

[0054] Injection volume: 10 μL.

[0055] 4. Determination of characteristic peaks and establishment of fingerprint spectrum

[0056] The chromatograms of the reference solution and one batch of Toujie Quwen Granules are shown below. Figure 1 As shown, from Figure 1 It can be seen that the separation of each chromatographic peak is good.

[0057] Based on the stable relative retention times and the common peaks that consistently appeared in all batches of samples, 18 peaks with good repeatability were selected as characteristic peaks. Using peak 7 (forsythoside I) as the S peak (reference peak), the relative retention times of each characteristic peak (peaks 1-6, 8-18) and the S peak were calculated. The results are shown in Table 1.

[0058] Table 1

[0059]

[0060]

[0061] The results in Table 1 show that the RSD of the relative retention times of the 18 characteristic peaks of the 12 batches of Toujie Quwen Granules ranged from 0.09% to 0.45%, all less than 2.0%.

[0062] Therefore, the final stipulation is that the fingerprint spectrum of the test sample should show 18 characteristic peaks. The fingerprint spectra of 12 batches of Toujie Quwen Granules (batch numbers from bottom to top: 20220205, 20220204, 20220206, A232A001, A232A002, A232A003, J2108015, J2108016, J2108017, K22A002, L22A001, L22A002) are as follows: Figure 2 As shown.

[0063] 5. Establish a comparative fingerprint spectrum for Toujie Quwen Granules.

[0064] The similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine (2012 version) was used to synthesize 12 batches of Toujie Quwen granules, and a reference fingerprint chromatogram for Toujie Quwen granules was established. (See attached image.) Figure 3 Eight components were identified: peak 7: forsythoside I, peak 9: forsythoside A, peak 10: (+) pinoresinin-β-D-glucopyranoside, peak 12: 4,5-O-caffeoylquinic acid (from honeysuckle), peak 13: baicalin, peak 14: forsythoside, peak 17: baicalin, and peak 18: forsythoside.

[0065] Example 2: Similarity evaluation of the fingerprint spectrum established in Example 1 of the present invention.

[0066] The fingerprint chromatograms of 12 batches of Toujie Quwen Granules established in Example 1 were evaluated using the 2012 version of the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System". The overall similarity of each chromatogram was calculated, and the results are shown in Table 2.

[0067] Table 2

[0068]

[0069]

[0070] Table 2 shows that the similarity between the fingerprint spectra of the 12 batches of Toujie Quwen Granules and the control fingerprint spectra was greater than 0.90. This indicates that the similarity between the batches of Toujie Quwen Granules is good and the quality is relatively stable.

[0071] Example 3: Methodological Investigation of the Method of the Invention

[0072] 1. Exclusivity

[0073] The blank solvent is a 70% methanol solution. Injecting the sample yields a blank solvent chromatogram.

[0074] Take Toujie Quwen Granules (batch number: 20220205) and inject them according to the method in Example 1, then record the HPLC chromatogram. The results are as follows: Figure 4 As shown, from Figure 4 It can be seen that the identification of the 18 common chromatographic peaks is not affected by factors such as solvents, and has good specificity.

[0075] 2. Precision

[0076] Take the same batch of Toujie Quwen Granules (batch number: 20220205), inject them six times consecutively according to the method in Example 1, and record the HPLC chromatograms. Use the forsythoside I peak (peak 7) as the RSD of the relative retention time of each characteristic peak. The results are shown in Table 3.

[0077] Table 3

[0078]

[0079] The results in Table 3 show that the RSD of the relative retention time of each characteristic peak is between 0.07% and 1.21%, indicating that the instrument has good precision.

[0080] 3. Stability

[0081] Take the same batch of Toujie Quwen Granules (batch number: 20220205), and inject them at 0, 2, 4, 6, 8, 12, 24 and 48 h according to the method in Example 1. Record the HPLC chromatograms. Use the forsythoside I peak 7 as the RSD of the relative retention time of each characteristic peak. The results are shown in Table 4.

[0082] Table 4

[0083]

[0084]

[0085] The results in Table 4 show that the RSD of the relative retention time of each characteristic peak is between 0.08% and 1.09%, indicating that the test solution has good stability and can be detected within 48 hours.

[0086] 4. Repeatability

[0087] Take the same batch of Toujie Quwen Granules (batch number: 20220205) to prepare the test solution, inject it according to the method of Example 1, record the HPLC chromatogram, and use the forsythoside I peak No. 7 as the RSD of the relative retention time of each characteristic peak. The results are shown in Table 5.

[0088] Table 5

[0089]

[0090] The results in Table 5 show that the RSD of the relative retention time of each characteristic peak is between 0.10% and 0.69%, indicating that the method has good repeatability.

[0091] 5. Intermediate precision

[0092] The same batch of Toujie Quwen Granules (batch number: 20220205) was used to prepare test solutions by different personnel (A, B) at different times (I, II). The solutions were injected according to the method in Example 1, and the HPLC chromatograms were recorded. The RSD of the relative retention time of each characteristic peak was calculated using Forsythoside I (peak 7). The results are shown in Table 6.

[0093] Table 6

[0094]

[0095] The results in Table 6 show that the RSD of the relative retention time of each characteristic peak is between 0.04% and 0.59% under different personnel preparing the test solution and different test solution preparation times, indicating that the method has good applicability.

[0096] 6. Durability

[0097] Test solutions were prepared using the same batch of Toujie Quwen Granules (batch number: 20220205), and tested separately using Elite Hypersil ODS2 C. 18 Agilent ZORBAX SB-C 18 Waters Xbridge C 18 Three different types of chromatographic columns were used on an Agilent 1260 high-performance liquid chromatograph and a Waters 2695 high-performance liquid chromatograph, respectively, following the method in Example 1, and HPLC chromatograms were recorded. Forsythoside I (peak 7) was used as the RSD for calculating the relative retention time of each characteristic peak. The results are shown in Table 7.

[0098] Table 7

[0099]

[0100] The results in Table 7 show that when the samples were detected using the above three chromatographic columns on two different high performance liquid chromatographs, namely Agilent 1260 and Waters 2695, the RSD of the relative retention time of each characteristic peak was 1.08%~5.70%, indicating that the method of the present invention has good durability for different instruments and different chromatographic columns. The method of the present invention has the advantages of wide applicability and easy promotion and application.

[0101] Example 4: Comparison of the impact of sample preparation methods on fingerprint pattern establishment

[0102] 1. Extraction solvent

[0103] Take 5 portions of Toujie Quwen Granules (batch number: 20220205), accurately weigh 1 g of each portion, and add 25 mL of 20% methanol, 50% methanol, 70% methanol, methanol, and 70% ethanol respectively. Extract by ultrasonic treatment (power 200W, frequency 80 kHz) for 30 min, shake well, filter, and collect the filtrate. Inject according to the method in Example 1, record the HPLC chromatogram, and the results are shown in [Figure 1]. Figure 5 .

[0104] Figure 5 The results showed that using methanol, 20% methanol, and 50% methanol as extraction solvents resulted in incomplete extraction of chromatographic peaks. While fingerprints obtained using 70% ethanol and 70% methanol as extraction solvents were similar, the peak shape and resolution were better when using 70% methanol as the extraction solvent.

[0105] 2. Extraction method

[0106] Take 5 portions of Toujie Quwen Granules (batch number: 20220205), add 70% methanol, and soak one portion in cold water for 12 hours. Shake well, filter, and take the filtrate to obtain the product.

[0107] Take three portions and sonicate them separately (power 200 W, frequency 80 kHz) for 15 min, 30 min, and 60 min, shake well, filter, and collect the filtrate to obtain the final product.

[0108] The other portion was heated under reflux for 60 minutes, cooled, shaken well, filtered, and the filtrate was collected to obtain the final product.

[0109] The sample was injected according to the method in Example 1, and the HPLC chromatogram was recorded. The results are as follows: Figure 6 .

[0110] from Figure 6 It can be seen that the chromatographic peak around 75 min can only appear stably under the extraction condition of ultrasonic extraction for 15 min, and the key components could not be extracted by the extraction method of cold soaking for 12 h.

[0111] Example 5: Comparison of the effects of HPLC conditions on fingerprint chromatogram establishment

[0112] 1. Gradient elution procedure

[0113] Take Toujie Quwen Granules (batch number: 20220205), prepare the test solution according to the method in Example 1, and inject and detect it according to the method in Example 1 (5 gradient elution programs), and record the HPLC chromatogram. The gradient elution programs are as follows:

[0114] Elution program 1: 0 min ~10 min, 6%A -10%A, 10 min ~16 min, 10%A -10%A, 16 min ~17 min, 10%A -16%A, 17 min ~55 min, 16%A -24%A, 55 min ~65 min, 24%A -70%A.

[0115] Elution program 2: 0 min ~10 min, 6%A -10%A, 10 min ~16 min, 10%A -10%A, 16 min ~20 min, 10%A -20%A, 20 min ~55 min, 20%A -24%A, 55 min ~70 min, 24%A -70%A.

[0116] Elution program 3: 0 min ~15 min, 6%A -12%A, 15 min ~25 min, 12%A -20%A, 25 min ~55 min, 20%A -23%A, 55 min ~65 min, 23%A -70%A.

[0117] Elution program 4: 0 min ~15 min, 6%A -12%A, 15 min ~40 min, 12%A -20%A, 40 min ~65 min, 20%A -23%A, 65 min ~70 min, 23%A -90%A.

[0118] Elution program 5: 0 min ~ 15 min, 6 ~ 12% A; 15 min ~ 45 min, 12% A → 20% A; 45 min ~ 65 min, 20 ~ 22% A; 65 min ~ 85 min, 22% A → 70% A.

[0119] The results are as follows Figure 7 As shown, from Figure 7 It can be seen that, using elution program 1, the chromatographic peaks concentrated between 25 min and 25 min, indicating poor peak resolution in this range; using elution program 2, the chromatographic peaks concentrated between 25 min and 35 min, with unsatisfactory peak elution around 60 min; using elution program 3, the peak elution was too concentrated in the 30 min period, and the chromatogram was too blank around 45 min, with almost no peaks; using elution program 4, the peak separation was poor in the 75 min period. Using elution program 5, the resolution of each chromatographic peak was good, and the baseline was stable.

[0120] 2. Detection wavelength

[0121] Typically, the detection wavelength for forsythoside A is 330 nm, forsythoside is 270 nm, and for baicalin is 280 nm. In this embodiment, a diode array detector was used to perform full-band scanning of the test solution (Toujie Quwen Granules, batch number: 20220205, prepared according to the method of Example 1), and chromatograms of the test solution at wavelengths of 203 nm, 240 nm, 275 nm, and 330 nm were extracted respectively (other conditions were the same as in Example 1).

[0122] The results are as follows Figure 8 The results showed that information from many key chromatographic peaks was missing at detection wavelengths of 203 nm, 275 nm, and 330 nm. However, at a detection wavelength of 240 nm, the amount of chromatographic peak information was the greatest, the separation was better, and the chromatographic baseline was more stable, which could more comprehensively reflect the components of the penetrating and detoxifying preparation.

[0123] 3. Flow rate

[0124] Take Toujie Quwen Granules (batch number: 20220205), prepare the test solution according to the method of Example 1, and inject it for detection according to the method of Example 1 (flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min, respectively), and record the HPLC chromatogram.

[0125] The results are as follows Figure 9 The results showed that when the flow rate was 0.8 mL / min, the separation of peaks 8, 9, and 14 was poor, and the elution times of peaks 17 and 18 were too late. When the flow rate was 1.2 mL / min, the separation of peak 15 was poor, and the column pressure was too high. When the flow rate was 1.0 mL / min, the chromatogram peak shape was good, and the distribution of each characteristic peak was moderate.

[0126] 4. Column temperature

[0127] Take Toujie Quwen Granules (batch number: 20220205), prepare the test solution according to the method of Example 1, and inject the sample for detection according to the method of Example 1 (column temperature of 25℃, 30℃, and 35℃ respectively), and record the HPLC chromatogram.

[0128] The results are as follows Figure 10 The results showed that when the column temperature was 30℃, chromatographic peaks 14 and 15 could no longer be separated from nearby impurity peaks, which would affect the relative retention time of the chromatographic peaks; when the column temperature was 35℃, the separation effect of chromatographic peaks 6, 8, 14 and 15 was not good; when the column temperature was 25℃, the peak shape and resolution of each chromatographic peak were good.

[0129] The results of this embodiment show that the gradient elution program, detection wavelength, flow rate, column temperature and other chromatographic conditions can all affect the separation and peak shape of chromatographic peaks, thereby affecting the establishment of fingerprint chromatograms.

[0130] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0131] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for establishing the fingerprint spectrum of a penetrating and detoxifying preparation, characterized in that, Includes the following steps: A test solution was prepared, and then the test solution was analyzed by high-performance liquid chromatography (HPLC). The test solution was prepared by adding 65% to 75% methanol or ethanol to the detoxifying and detoxifying preparation, and then sonicating. The HPLC analysis used gradient elution, and the gradient elution program was as follows: 0 min to 15 min, 6 to 12% A; 15 min to 45 min, 12% A → 20% A; 45 min to 65 min, 20 to 22% A; 65 min to 85 min, 22% A → 70% A.

2. The method for establishing the fingerprint spectrum of the antipyretic preparation according to claim 1, characterized in that, The test solution was prepared by the following method: taking the antipyretic preparation and adding 68%~72% methanol, followed by ultrasonication; Preferably, the mass-to-volume ratio of the detoxifying and detoxifying preparation to 68%~72% methanol is 1 g: 24 mL~26 mL.

3. The method for establishing the fingerprint spectrum of the antipyretic preparation according to claim 2, characterized in that, The ultrasonic power is 150 W to 250 W, the ultrasonic frequency is 70 kHz to 90 kHz, and the ultrasonic duration is 10 min to 20 min, preferably 14 min to 16 min.

4. The method for establishing the fingerprint spectrum of the antipyretic preparation according to claim 1, characterized in that, The conditions for high-performance liquid chromatography detection also include: the chromatographic column is C10. 18 Chromatographic column; detection wavelength: 235 nm to 245 nm; column temperature: 24℃ to 26℃; flow rate: 0.9 mL / min to 1.1 mL / min.

5. The method for establishing the fingerprint spectrum of the detoxifying and antipyretic preparation according to claim 4, characterized in that, The detection wavelength is 238 nm to 242 nm; the column temperature is 25℃ to 26℃; and the flow rate is 0.95 mL / min to 1.05 mL / min.

6. The method for establishing the fingerprint spectrum of the detoxifying and antipyretic preparation according to claim 4, characterized in that, The chromatographic column was a Hypersil ODS2 C2 column. 18 A chromatographic column with dimensions of 4.6 mm × 250 mm and 5 µm.

7. The method for establishing the fingerprint spectrum of the detoxifying and antipyretic preparation according to claim 1, characterized in that, The conditions for high-performance liquid chromatography detection also include: mobile phase A is acetonitrile, and mobile phase B is a 0.18%~0.22% aqueous solution of phosphoric acid.

8. The method for establishing the fingerprint spectrum of the detoxifying and antipyretic preparation according to any one of claims 1 to 7, characterized in that, The aforementioned detoxifying and detoxifying preparation is made from raw materials including Forsythia suspensa, Cremastra appendiculata, Lonicera japonica, Scutellaria baicalensis, Isatis indigotica, Astragalus membranaceus, Codonopsis pilosula, Poria cocos, Bupleurum chinense, Artemisia annua, Cicadae periostracum, Peucedanum praeruptorum, Fritillaria cirrhosa, Fritillaria thunbergii, Scrophularia ningpoensis, and Prunus mume.

9. The fingerprint spectrum of the antipyretic preparation established by the method according to any one of claims 1 to 8.

10. The application of the fingerprint spectrum of the penetrating and detoxifying preparation according to claim 9 in the quality control of the penetrating and detoxifying preparation.

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