Characteristic chromatogram construction method and application of antipyretic and antitoxic oral liquid

Abstract

The invention relates to the technical field of drug detection, and particularly provides a specific chromatogram construction method and application of a scourge-clearing and detoxification oral liquid. The construction method comprises preparation of a reference substance solution, preparation of a test solution and detection of high performance liquid chromatography. A solution is provided for improving the current situation that the quality of the product on the market is uneven, the vital interests of farmers are protected, and the healthy development of the industry is promoted.

Description

Technical Field

[0001] This application relates to the field of drug detection technology, specifically providing a method for constructing and applying the characteristic spectrum of Qingwen Jiedu oral liquid. Background Technology

[0002] Qingwen Jiedu Oral Liquid is a traditional Chinese veterinary medicine preparation. Its quality standards are currently included in the "Veterinary Drug Quality Standards (2017 Edition, Traditional Chinese Medicine Volume)" and the "Chinese Veterinary Pharmacopoeia" 2020 Edition, Part II. This preparation is made from six traditional Chinese medicinal herbs—Rehmannia glutinosa, Gardenia jasminoides, Scutellaria baicalensis, Forsythia suspensa, Scrophularia ningpoensis, and Isatis indigotica—using modern extraction and processing technology. In the formula, Isatis indigotica detoxifies and soothes the throat, Scutellaria baicalensis clears lung heat, and Forsythia suspensa clears heat from the heart meridian. These three herbs together serve as the principal herbs, clearing excess heat in the upper and middle jiao. Gardenia jasminoides clears heat from the three jiaos and serves as the assistant herb, assisting the principal herbs in clearing heat and detoxifying. Rehmannia glutinosa and Scrophularia ningpoensis cool the blood, nourish yin, and reduce fire, both entering the kidney meridian, and serve as adjuvant herbs. The combined effects of these herbs are to clear heat, purge fire, nourish yin, and detoxify. This formula works by simultaneously supporting the body's resistance and eliminating pathogens, and has the characteristics of "preventing disease before it occurs and preventing its progression once it has occurred". Clinically, it is mainly used to treat fever caused by external pathogens and is widely used in the prevention and control of avian viral diseases. It is one of the representative formulas for clearing heat and detoxifying in the national drug standards.

[0003] The material basis for the disease prevention and treatment and pharmacological effects of traditional Chinese medicine (TCM) lies in its effective components. Isatis root in Qingwen Jiedu oral liquid mainly contains (R,S)-goichun, indigo, indirubin, adenosine, polysaccharides, starch, and amino acids. Among these, (R,S)-goichun exhibits strong antiviral activity and is the controlled component in Isatis root, also considered a characteristic component. Scutellaria baicalensis mainly contains flavonoids (primarily baicalin and baicalein, along with baicalein and wogonin), volatile components, and various amino acids. Forsythia suspensa has a complex chemical composition, mainly containing terpenes, phenylethanol glycosides, lignans, flavonoids, natural alcohols, coumarins, volatile components, and some alcohols, esters, acids, aldehydes, and ketones. Forsythoside is a characteristic component of Forsythia suspensa. The chemical components of gardenia mainly include iridoids, diterpenes, triterpenes, flavonoids, organic acid esters, volatile oils, polysaccharides, and various trace elements. Among them, iridoid glycosides, represented by geniposide, are characteristic components of gardenia. Rehmannia glutinosa contains sterols, mannitol, catalpol, and organic acids such as benzoic acid and phenylacetic acid, as well as polysaccharides and various amino acids. Scrophularia ningpoensis contains alkaloids, iridoids, and volatile oils.

[0004] Due to the complexity of the chemical composition of traditional Chinese medicine (TCM), no single active ingredient in a TCM compound preparation can fully represent its overall therapeutic effect. Characteristic chromatograms of TCM preparations, as a recognized and effective means of identifying the quality of TCM, take a macroscopic perspective, better reflecting the complexity and holistic nature of TCM, and are widely used in the quality control of multiple TCM varieties in Part I of the 2020 edition of the Chinese Pharmacopoeia. A characteristic chromatogram of TCM preparations refers to a chromatogram established by appropriately processing TCM preparation samples and using suitable analytical methods, reflecting information about multiple components and demonstrating their quality characteristics. Characteristic chromatograms of TCM preparations are of great significance for identifying key quality attributes of TCM preparations, studying quantity-quality transfer, evaluating the uniformity and stability of TCM preparation quality, and improving the overall quality control level of TCM preparations.

[0005] The quality control items for Qingwen Jiedu Oral Liquid in the "Veterinary Drug Quality Standards (2017 Edition, Traditional Chinese Medicine Volume)" only include thin-layer chromatography identification of Scutellaria baicalensis, Forsythia suspensa, and Gardenia jasminoides. The 2020 edition of the "Chinese Veterinary Pharmacopoeia" only includes thin-layer chromatography identification of Forsythia suspensa and determination of geniposide and baicalin content under the Qingwen Jiedu Oral Liquid section. Existing literature discloses a method for determining the content of baicalin, geniposide, and forsythoside in Qingwen Jiedu Oral Liquid. Currently, the published standards only involve three medicinal materials: Scutellaria baicalensis, Gardenia jasminoides, and Forsythia suspensa. The quality control level is low, and no relevant research on the quality control and characteristic chromatograms of other medicinal materials has been found. In particular, there are no public reports on the quality control of Isatis indigotica, the principal ingredient in the prescription. This is detrimental to the quality stability control and quality evaluation of the product, and may lead to significant differences in clinical efficacy, thereby harming the interests of farmers.

[0006] In summary, existing quality standards and literature reports do not provide comprehensive quality control for Qingwen Jiedu oral liquid, resulting in inconsistent product quality in the market. Research on the characteristic spectral data of Qingwen Jiedu oral liquid is still lacking, and qualitative methods based on multi-component characteristic spectral data of active ingredients need to be developed.

[0007] In view of the above, this application is hereby submitted. Summary of the Invention

[0008] The purpose of this application is to provide a method for constructing and applying the characteristic spectrum of Qingwen Jiedu oral liquid, to provide a solution for improving the uneven quality of this product in the market, to protect the vital interests of farmers, and to promote the healthy development of the industry.

[0009] For the purposes mentioned above, this application adopts the following technical solution:

[0010] A method for constructing a feature spectrum of Qingwen Jiedu oral liquid, the method comprising the following steps:

[0011] Preparation of reference solution: Weigh appropriate amounts of (R,S)-Gouyichun, geniposide, forsythoside, baicalin, baicalein and wogonin, add solvent to make up to volume, and prepare reference solution;

[0012] Preparation of the test solution: Take an appropriate amount of Qingwen Jiedu oral liquid, add a mixed solution of ethyl acetate and methanol, sonicate, cool, make up to volume, shake well, filter, measure the filtrate, evaporate to dryness, redissolve the residue in methanol aqueous solution, make up to volume, shake well, and obtain the test solution.

[0013] Based on the chromatograms of the reference solution and the test solution detected by high performance liquid chromatography, the characteristic chromatograms of Qingwen Jiedu oral liquid were obtained.

[0014] The detection conditions for the high performance liquid chromatography include: using a column packed with octadecylsilane-bonded silica gel; mobile phase A being methanol and mobile phase B being an aqueous phosphoric acid solution; gradient elution; and detection wavelengths of 241 nm from 0 min to 27 min and 227 nm from 27 min to 70 min.

[0015] Furthermore, the formula of the Qingwen Jiedu oral liquid includes Rehmannia glutinosa, Gardenia jasminoides, Scutellaria baicalensis, Forsythia suspensa, Scrophularia ningpoensis and Isatis indigotica;

[0016] Preferably, the active ingredients of gardenia, scutellaria, forsythia and isatis root include (R,S)-goichun, geniposide, forsythoside, baicalin, baicalein and wogonin.

[0017] Furthermore, in the preparation of the reference solution, the solvent is methanol;

[0018] Preferably, the reference solution contains (R,S)-goichun at a concentration of 0.65 μg / ml, geniposide at a concentration of 97.12 μg / ml, forsythoside at a concentration of 6.37 μg / ml, baicalin at a concentration of 37.54 μg / ml, baicalein at a concentration of 2.42 μg / ml, and wogonin at a concentration of 2.53 μg / ml.

[0019] Further, the concentration of ethyl acetate in the ethyl acetate-methanol mixed solution is 25 v / v% to 75 v / v%, preferably 50 v / v%.

[0020] Preferably, the ultrasound duration is 10–30 minutes, more preferably 20 minutes;

[0021] Preferably, the concentration of the methanol aqueous solution is 30 v / v% to 70 v / v%, more preferably 50 v / v%.

[0022] Furthermore, the chromatographic column of the high performance liquid chromatography is a C18 column packed with octadecylsilane-bonded silica gel, preferably a Kromasil 100-5 C18 column, with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5.0 μm;

[0023] Preferably, the gradient elution program for the high-performance liquid chromatography is as follows: 0–13 min, 11% mobile phase A, 89% mobile phase B; 13–18 min, 11%–33% mobile phase A, 89%–67% mobile phase B; 18–50 min, 33%–70% mobile phase A, 67%–30% mobile phase B; 50–53 min, 70%–80% mobile phase A, 30%–20% mobile phase B; 53–60 min, 80% mobile phase A, 20% mobile phase B; 60–62 min, 80%–11% mobile phase A, 20%–89% mobile phase B; 62–70 min, 11% mobile phase A, 89% mobile phase B; wherein the ratio of mobile phase A to mobile phase B varies within ±1%.

[0024] Preferably, the concentration of the phosphoric acid aqueous solution is 0.02 v / v% to 0.04 v / v%, preferably 0.03 v / v%.

[0025] Preferably, the flow rate of the mobile phase in the high-performance liquid chromatography is 0.9 ml / min to 1.1 ml / min, and more preferably 1.0 ml / min;

[0026] Preferably, the column temperature of the high-performance liquid chromatography is 28℃~32℃, and more preferably 30℃;

[0027] Preferably, the injection volume of the high-performance liquid chromatography is 0.5–20 μl, and more preferably 10 μl.

[0028] Furthermore, the feature map includes 14 common fingerprint peaks, with peak 5 as the reference peak S, and a retention time of 1.0000. The relative retention times of the 14 common feature peaks are as follows: peak 1, retention time 0.3317; peak 2, retention time 0.4588; peak 3, retention time 0.8385; peak 4, retention time 0.9266; peak 5, retention time 1.0000; peak 6, retention time 1.3. Peaks 297 and 7 have a retention time of 1.4937; peak 8 has a retention time of 1.5669; peak 9 has a retention time of 1.7513; peak 10 has a retention time of 1.7870; peak 11 has a retention time of 1.8558; peak 12 has a retention time of 1.9922; peak 13 has a retention time of 2.0907; and peak 14 has a retention time of 2.1943. Their relative retention times should be within ±10% of the specified values.

[0029] Preferably, peak 2 is a characteristic peak of (R,S)-Gouyichun, peak 5 is a characteristic peak of geniposide, peak 7 is a characteristic peak of forsythoside, peak 8 is a characteristic peak of baicalin, peak 12 is a characteristic peak of baicalein, and peak 14 is a characteristic peak of wogonin.

[0030] Furthermore, the retention times of the reference solution chromatogram and the test solution chromatogram are compared for qualitative analysis, and the indicator components in the test solution chromatogram are located to obtain the characteristic chromatogram of Qingwen Jiedu oral liquid.

[0031] A quality control method for a Qingwen Jiedu oral liquid, the quality control method comprising the following steps:

[0032] Based on the above construction method, a characteristic spectrum of the Qingwen Jiedu oral liquid reference sample is established. The Qingwen Jiedu oral liquid test solution is tested according to the detection conditions in the above construction method to obtain the spectrum of the Qingwen Jiedu oral liquid to be tested. The spectrum of the Qingwen Jiedu oral liquid to be tested is compared with the characteristic spectrum of the Qingwen Jiedu oral liquid reference sample. If it meets the requirements, it is a qualified product.

[0033] The above-mentioned method for constructing characteristic spectra or quality control of Qingwen Jiedu oral liquid is applied to the quality monitoring or production quality control of characteristic components of Qingwen Jiedu oral liquid.

[0034] The above-mentioned method for constructing characteristic spectra or quality control methods for Qingwen Jiedu oral liquid is applied to the quality control of preparations containing Isatis root, Scutellaria baicalensis, Forsythia suspensa and Gardenia jasminoides.

[0035] Compared with the prior art, the beneficial effects of this application are as follows:

[0036] 1. This application employs high-performance liquid chromatography (HPLC) characteristic chromatographic method, which is simple to operate, has a short detection time, high sensitivity, is unaffected by temperature and humidity, and has high detection efficiency. For the first time, a characteristic chromatogram of Qingwen Jiedu oral liquid has been constructed for the quality control and qualitative evaluation of this product.

[0037] 2. This application solves the problems of incomplete extraction of characteristic peaks, difficulty in separating multiple characteristic peaks, and interference from impurity peaks by screening the sample processing method and optimizing chromatographic conditions, and by using ethyl acetate-methanol mixed extraction technology and high performance liquid gradient elution method, thus providing a new analytical means for the internal quality control of Qingwen Jiedu oral liquid.

[0038] 3. The characteristic chromatograms provided in this application are the first to conduct quality control on the principal drug Banlangen and its main active ingredient (R,S) - Gaoyichun.

[0039] 4. The characteristic chromatograms provided in this application are the first to demonstrate the quality control of baicalin and wogonin, the main active ingredients in Scutellaria baicalensis.

[0040] 5. This application uses a dual-wavelength switching method to ensure that the main components (R,S)-Gouyichun and Forsythoside have good separation and signal response in the same chromatogram.

[0041] 6. The characteristic spectrum constructed in this application contains approximately 14 common peaks, filling a research gap in the characteristic spectrum of Qingwen Jiedu Oral Liquid. It can simultaneously identify six components in Qingwen Jiedu Oral Liquid: (R,S)-Gouyichun, Gardenoside, Forsythoside, Baicalin, Baicalein, and Wolfberry Extract, effectively characterizing the key chemical components related to the quality of Qingwen Jiedu Oral Liquid. This method can qualitatively evaluate the overall efficacy of Qingwen Jiedu Oral Liquid and significantly improve its quality control level.

[0042] 7. The feature mapping method constructed in this application is simple to operate, and exhibits high stability, precision, and repeatability. It can provide a technical foundation for internal quality control in manufacturing enterprises, monitor drug quality, and ensure batch-to-batch stability; it can also provide an effective analytical means for aquaculture enterprises to distinguish the quality and authenticity of Qingwen Jiedu oral liquid on the market. Attached Figure Description

[0043] Figure 1 The chromatogram of acetonitrile-0.03 phosphate in the mobile phase system of Example 2 is shown.

[0044] Figure 2 The chromatogram of methanol-0.03 phosphoric acid is shown for the mobile phase system in Example 2.

[0045] Figure 3 This is a chromatogram showing the selection of detection wavelength in Example 2;

[0046] Figure 4 Chromatogram 1 for solvent extraction in Example 2;

[0047] Figure 5 Chromatogram 2 for solvent extraction in Example 2;

[0048] Figure 6 The extraction time chromatogram for Example 2;

[0049] Figure 7 The chromatogram for the reconstitution solvent in Example 2 is shown below.

[0050] Figure 8 This is the chromatogram for identifying the characteristic peaks in the characteristic spectrum in Example 3;

[0051] Figure 9 The characteristic chromatograms of 10 batches of our company's Qingwen Jiedu oral liquid in Example 3 are shown.

[0052] Figure 10 Chromatograms of columns with different durability were examined in Example 4;

[0053] Figure 11 This is the chromatogram for the robustness mobile phase ratio assessment in Example 4;

[0054] Figure 12 This is the chromatogram for the durability column temperature investigation in Example 4;

[0055] Figure 13 This is the chromatogram for the determination of the proportion of durable phosphoric acid in Example 4;

[0056] Figure 14 This is the chromatogram for the durability flow rate assessment in Example 4;

[0057] Figure 15 This is the chromatogram for the injection precision assessment in Example 4;

[0058] Figure 16 This is the chromatogram for the repeatability test in Example 4;

[0059] Figure 17 This is the chromatogram for the solution stability study in Example 4;

[0060] Figure 18 This is the chromatogram for the intermediate precision assessment in Example 4;

[0061] Figure 19 The characteristic chromatograms of three batches of commercially available Qingwen Jiedu oral liquid from other manufacturers are shown in Example 5. Detailed Implementation

[0062] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions described in this application will be further described in detail below with reference to specific embodiments.

[0063] The Qingwen Jiedu oral liquid contains six traditional Chinese medicines, with numerous active ingredients and significant differences in polarity among them. It is crucial to ensure that all active ingredients elute while maintaining satisfactory chromatographic peak resolution. If the sample pretreatment or chromatographic conditions are not ideal, the resulting chromatogram will contain numerous extraneous peaks, with severe overlap or tailing between peaks, making it difficult to achieve the required resolution. Furthermore, the large number of active ingredients and their varying maximum UV absorption wavelengths can lead to significant differences in the response values ​​of the chromatographic peaks within the same chromatogram, preventing the effective detection of all active ingredients. In addition, analysis of the characteristic chromatograms of Qingwen Jiedu oral liquid from different manufacturers revealed significant differences in the chemical composition of samples from different manufacturers. The quality of these products directly impacts clinical efficacy and the interests of farmers.

[0064] Based on the above problems, the inventors have developed a method for constructing the characteristic spectrum of Qingwen Jiedu oral liquid, including the following steps:

[0065] Preparation of reference solution: Weigh appropriate amounts of (R,S)-Gouyichun, geniposide, forsythoside, baicalin, baicalein and wogonin, add solvent to make up to volume, and prepare reference solution;

[0066] Preparation of the test solution: Take an appropriate amount of Qingwen Jiedu oral liquid, add a mixed solution of ethyl acetate and methanol, sonicate, cool, make up to volume, shake well, filter, measure the filtrate, evaporate to dryness, redissolve the residue in methanol aqueous solution, make up to volume, shake well, and obtain the test solution.

[0067] Based on the chromatograms of the reference solution and the test solution detected by high performance liquid chromatography, the characteristic chromatograms of Qingwen Jiedu oral liquid were obtained.

[0068] The detection conditions for high performance liquid chromatography include: using a column packed with octadecylsilane-bonded silica gel; mobile phase A being methanol and mobile phase B being an aqueous phosphoric acid solution; gradient elution; detection wavelength of 241 nm from 0 min to 27 min and 227 nm from 27 min to 70 min.

[0069] In this application, the mobile phase does not contain surfactants, which reduces the impact on column life and does not damage the instrument's sealing system.

[0070] In some embodiments, methanol is used as the solvent in the preparation of the reference solution.

[0071] In one embodiment, the reference solution contains (R,S)-goicholine at a concentration of 0.65 μg / ml, geniposide at a concentration of 97.12 μg / ml, forsythoside at a concentration of 6.37 μg / ml, baicalin at a concentration of 37.54 μg / ml, baicalein at a concentration of 2.42 μg / ml, and wogonin at a concentration of 2.53 μg / ml.

[0072] In some embodiments, 1 ml of Qingwen Jiedu oral liquid is accurately measured and placed in a 50 ml volumetric flask. A mixture of ethyl acetate and methanol is added to near the mark, sonicated for 20 min, cooled, diluted to volume, shaken well, and filtered. 25 ml of the filtrate is accurately measured, evaporated to dryness, and the residue is transferred to a 25 ml volumetric flask with a suitable solvent, diluted to volume, and shaken well to obtain the test solution.

[0073] In some embodiments, the concentration of ethyl acetate in the ethyl acetate-methanol mixture is 25 v / v% to 75 v / v, and may be, but is not limited to, 25 v / v%, 30 v / v%, 35 v / v%, 40 v / v%, 45 v / v%, 50 v / v%, 55 v / v%, 60 v / v%, 65 v / v%, 70 v / v%, or 75 v / v.

[0074] In some implementations, the ultrasound duration is 10 to 30 minutes, and may be, but is not limited to, 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes.

[0075] In some embodiments, the concentration of the methanol aqueous solution is 30 v / v% to 70 v / v%, which may be, but is not limited to, 30 v / v%, 35 v / v%, 40 v / v%, 45 v / v%, 50 v / v%, 55 v / v%, 60 v / v%, 65 v / v%, 70 v / v%, or 75 v / v.

[0076] In some implementations, the high-performance liquid chromatography (HPLC) column is a Kromasil 100-5C18 column or an Agilent Eclipse XDB-C18 column.

[0077] In some embodiments, the gradient elution program for high-performance liquid chromatography is as follows: 0–13 min, 11% mobile phase A, 89% mobile phase B; 13–18 min, 11%–33% mobile phase A, 89%–67% mobile phase B; 18–50 min, 33%–70% mobile phase A, 67%–30% mobile phase B; 50–53 min, 70%–80% mobile phase A, 30%–20% mobile phase B; 53–60 min, 80% mobile phase A, 20% mobile phase B; 60–62 min, 80%–11% mobile phase A, 20%–89% mobile phase B; 62–70 min, 11% mobile phase A, 89% mobile phase B; wherein the ratio of mobile phase A to mobile phase B varies within ±1%.

[0078] In some embodiments, the concentration of the aqueous phosphoric acid solution is 0.02 v / v% to 0.04 v / v%, which may be, but is not limited to, 0.02 v / v%, 0.03 v / v%, or 0.04 v / v.

[0079] In some embodiments, the mobile phase flow rate of the high-performance liquid chromatography is 0.9 ml / min to 1.1 ml / min, and may be, but is not limited to, 0.9 ml / min, 1.0 ml / min or 1.1 ml / min.

[0080] In some embodiments, the column temperature of the high-performance liquid chromatography is 28°C to 32°C, and may be, but is not limited to, 28°C, 29°C, 30°C, 31°C or 32°C.

[0081] In some embodiments, the injection volume of high performance liquid chromatography is 0.5 to 20 μl, and may be, but is not limited to, 0.5 μl, 1 μl, 3 μl, 5 μl, 7 μl, 10 μl, 12 μl, 15 μl, 18 μl or 20 μl.

[0082] The mobile phase system, sample preparation method, precision, solution stability, repeatability, intermediate precision, and robustness were thoroughly investigated, and the method is highly reliable.

[0083] In some implementations, the feature spectrum includes 14 common fingerprint peaks, with peak 5 as the reference peak S, and a retention time of 1.0000. The relative retention times of the 14 common feature peaks are as follows: peak 1, retention time 0.3317; peak 2, retention time 0.4588; peak 3, retention time 0.8385; peak 4, retention time 0.9266; peak 5, retention time 1.0000; and peak 6, retention time 1. Peaks 3297 and 7 have a retention time of 1.4937; peak 8 has a retention time of 1.5669; peak 9 has a retention time of 1.7513; peak 10 has a retention time of 1.7870; peak 11 has a retention time of 1.8558; peak 12 has a retention time of 1.9922; peak 13 has a retention time of 2.0907; and peak 14 has a retention time of 2.1943. Their relative retention times should be within ±10% of the specified values.

[0084] Fourteen known peaks and common peaks were identified from samples from different manufacturers and batches, with good separation. The methodology was thoroughly validated and investigated. Characteristic peaks can be located by relative retention time, which can help identify the quality of products on the market and has strong practicality.

[0085] In some embodiments, peak 2 is a characteristic peak of (R,S)-Gouyichun, peak 5 is a characteristic peak of geniposide, peak 7 is a characteristic peak of forsythoside, peak 8 is a characteristic peak of baicalin, peak 12 is a characteristic peak of baicalein, and peak 14 is a characteristic peak of wogonin.

[0086] For the first time, baicalin and wogonin, the active ingredients in Scutellaria baicalensis, were detected, and the separation was good.

[0087] In some embodiments, the retention time of the reference solution spectrum and the test solution spectrum is compared for qualitative analysis, and the indicator components in the test solution spectrum are located to obtain the characteristic spectrum of Qingwen Jiedu oral liquid.

[0088] This application provides a quality control method for Qingwen Jiedu oral liquid, comprising: establishing a characteristic spectrum of a Qingwen Jiedu oral liquid reference sample using the construction method of this application; testing the Qingwen Jiedu oral liquid test solution according to the detection conditions in the construction method of this application to obtain the spectrum of the Qingwen Jiedu oral liquid to be tested; comparing the spectrum of the Qingwen Jiedu oral liquid to be tested with the characteristic spectrum of the Qingwen Jiedu oral liquid reference sample; and selecting the product that meets the requirements.

[0089] Example 1: Test materials used in all examples

[0090] 1. Main Instruments

[0091] An Agilent 1260 high-performance liquid chromatograph (equipped with a G7117C DAD detector, Agilent Technologies, USA), an electronic analytical balance (XSR205DU, Mettler Toledo Technologies), an ultrasonic cleaner (KQ-500DB, Kunshan Ultrasonic Instrument Co., Ltd.), and a C18 column packed with octadecylsilane-bonded silica gel (Kromasil 100-5C18 4mm×250mm, 5μm, Akzonobel, Sweden; Agilent Eclipse XDB-C18 4.6mm×250mm, 5μm, Agilent Technologies).

[0092] 2. Main reagents and standard substances

[0093] Methanol (chromatographic grade, Shanghai Anpu Testing Technology Co., Ltd., batch number: O4561440); Phosphoric acid (chromatographic grade, Tianjin Kemei Chemical Reagent Co., Ltd., batch number: 20231023); Acetonitrile (chromatographic grade, Shanghai Anpu Testing Technology Co., Ltd., batch number: O8371440); (R,S)-Gouyichun (China National Institutes for Food and Drug Control, batch number: 111753-202007, content: 100.0%); Gardenoside (China National Institutes for Food and Drug Control, batch number: 110749- 202320, content: 98.1%); Forsythoside (China National Institutes for Food and Drug Control, batch number: 110821-202318, content: 95.8%); Baicalin (China National Institutes for Food and Drug Control, batch number: 110715-202223, content: 97.2%); Baicalein (China National Institutes for Food and Drug Control, batch number: 111595-202309, content: 98.6%); Wogonin (China National Institutes for Food and Drug Control, batch number: 111514-202207).

[0094] 3 samples

[0095] Qingwen Jiedu Oral Liquid (Luoyang Huizhong Veterinary Medicine Co., Ltd., batch numbers: 20221201, 20221202, 20230301, 20230302, 20230501, 20230601, 20230602, 20230901, 20231101, 20240101); Qingwen Jiedu Oral Liquid (commercially available from Company A, batch number: 2022100501); Qingwen Jiedu Oral Liquid (commercially available from Company B, batch number: 20240301); Qingwen Jiedu Oral Liquid (commercially available from Company C, batch number: 221001).

[0096] Example 2: Establishment of a characteristic chromatogram method for Qingwen Jiedu oral liquid

[0097] 1. Investigation of the mobile phase system

[0098] 1.1 Preparation of reference solution: Accurately weigh appropriate amounts of (R,S)-Gouyichun, Gentianoside, Forsythoside, Baicalin, Baicalein and Wogonin, and add methanol to prepare a reference solution containing (R,S)-Gouyichun 0.65 μg / ml, Gentianoside 97.12 μg / ml, Forsythoside 6.37 μg / ml, Baicalin 37.54 μg / ml, Baicalein 2.42 μg / ml and Wogonin 2.53 μg / ml.

[0099] 1.2 Preparation of the test solution: Accurately measure 1 ml of Qingwen Jiedu oral liquid and place it in a 50 ml volumetric flask. Add methanol solution containing 75% ethyl acetate to near the mark, sonicate for 20 min, cool, dilute to volume, shake well, and filter. Accurately measure 25 ml of the filtrate and place it in a round-bottom flask. Evaporate to dryness. Add 50% methanol to the residue and transfer to a 25 ml volumetric flask. Dilute to volume and shake well to obtain the test solution.

[0100] 1.3 Chromatographic conditions: A Kromasil 100-5C column (184 mm × 250 mm, 5 μm) was used as the packing material, with octadecylsilane-bonded silica gel as the stationary phase; the detection wavelength was 241 nm from 0 min to 27 min and 227 nm from 27 min to 70 min; the column temperature was 30 ℃; and the flow rate was 1.0 ml / min.

[0101] Mobile phase condition 1: Methanol is used as mobile phase A, and 0.03% aqueous phosphoric acid solution is used as mobile phase B.

[0102] The gradient elution procedure is shown in the table below;

[0103] Mobile phase condition 2: Acetonitrile is used as mobile phase A, and 0.03% aqueous phosphoric acid solution is used as mobile phase B.

[0104] The gradient elution procedure is shown in the table below;

[0105] 1.4 Determination: Accurately measure 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, obtain the characteristic chromatograms of the reference solution and the test solution, and compare the peak shape and resolution of the two mobile phases.

[0106] 1.5 Results

[0107] Mobile phase condition two, using acetonitrile as the organic phase, resulted in poor separation of (R,S)-citrinin and excessively long retention time of baicalein. Mobile phase one, however, showed better separation of both known and unknown peaks. Therefore, mobile phase condition one was preferred as the elution condition. Figure 1, Figure 2 .

[0108] 2 Selection of detection wavelength

[0109] 2.1 Preparation of reference solution: Same as in Example 2 "1.1".

[0110] 2.2 Preparation of test solution: Same as in Example 2 "1.2".

[0111] 2.3 Chromatographic conditions:

[0112] A Kromasil 100-5C18 column (4 mm × 250 mm, 5 μm) was used as the packing material, with an octadecylsilane-bonded silica gel column; the column temperature was 30 °C; and the flow rate was 1.0 mL / min. Methanol was used as mobile phase A, and 0.03% aqueous phosphoric acid solution was used as mobile phase B. The gradient elution program is shown in the table below.

[0113] Wavelength condition 1: 227nm;

[0114] Wavelength condition two: 241nm;

[0115] Wavelength condition 3: The detection wavelength is 241nm from 0min to 27min, and 227nm from 27min to 70min;

[0116] 2.4 Results:

[0117] Qingwen Jiedu Oral Liquid contains six traditional Chinese medicines and is a compound preparation with complex components. The maximum absorption of different components varies, and measurements were conducted at 227 nm and 241 nm. Results showed that (R,S)-Gouyichun and geniposide peaked from 0 min to 27 min, with maximum absorption near 241 nm. Forsythoside peaked from 27 min to 70 min, with maximum absorption at 227 nm, but absorption was very weak at 240 nm. (R,S)-Gouyichun and forsythoside are both major active ingredients in this product, but their content is low. To ensure maximum response for both components, a dual-wavelength switching method was used: detection at 241 nm from 0 min to 27 min and at 227 nm from 27 min to 70 min. The results showed good absorption and separation for all components. (See...) Figure 3 .

[0118] 3. Investigation of extraction solvents

[0119] 3.1 Preparation of reference solution: Same as in Example 2, "1.1".

[0120] 3.2 Preparation of the test solution: Accurately measure 1 ml of Qingwen Jiedu oral liquid and place it in a 50 ml volumetric flask. Add methanol solution containing 25% ethyl acetate, methanol solution containing 50% ethyl acetate, methanol solution containing 75% ethyl acetate, water, methanol, and ethyl acetate to near the mark. Sonicate for 20 min, cool, dilute to volume, shake well, and filter. Accurately measure 25 ml of the filtrate and place it in a round-bottom flask. Evaporate to dryness. Add 50% methanol to the residue and transfer to a 25 ml volumetric flask. Dilute to volume and shake well to obtain the test solution.

[0121] 3.3 Chromatographic conditions:

[0122] A Kromasil 100-5C18 column (4 mm × 250 mm, 5 μm) was used as the packing material, with detection wavelengths of 241 nm from 0 min to 27 min and 227 nm from 27 min to 70 min. The column temperature was 30 °C, and the flow rate was 1.0 mL / min. Methanol was used as mobile phase A, and 0.03% aqueous phosphoric acid solution was used as mobile phase B. The gradient elution program is shown in the table below.

[0123] 3.4 Determination: Accurately measure 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and obtain the chromatograms of the reference solution and the characteristic chromatograms of the test solution.

[0124] 3.5 Results:

[0125] Using water as the extraction solvent resulted in poor separation of (R,S)-goicin; using methanol as the extraction solvent resulted in a broad chromatographic peak; using ethyl acetate as the extraction solvent resulted in no peak of baicalin. Considering all factors, a mixed solution of ethyl acetate and methanol was used. The results showed that methanol solutions containing 25%–75% ethyl acetate achieved the desired peak shape and resolution. Considering both peak shape and resolution, a methanol solution containing 75% ethyl acetate was preferred. Figure 4 , Figure 5 .

[0126] 4. Examination of extraction time

[0127] 4.1 Preparation of reference solution: Same as in Example 2, "1.1".

[0128] 4.2 Preparation of the test solution: Accurately measure 1 ml of Qingwen Jiedu oral liquid and place it in a 50 ml volumetric flask. Add methanol solution containing 75% ethyl acetate to near the mark, and shake well. Then, sonicate for 10 min, 20 min, and 30 min respectively. Cool, dilute to volume, shake well, and filter. Accurately measure 25 ml of the filtrate and place it in a round-bottom flask. Evaporate to dryness. Add 50% methanol to the residue and transfer to a 25 ml volumetric flask. Dilute to volume and shake well to obtain the test solution.

[0129] 4.3 Chromatographic conditions: Same as in Example 2, "3.3".

[0130] 4.4 Determination: Accurately measure 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and obtain the characteristic chromatograms of the reference solution and the test solution.

[0131] 4.5 Results: After shaking without sonication, the peak areas of all characteristic peaks were small, indicating incomplete extraction. After sonication for 10 min, 20 min, and 30 min, the peak areas of all characteristic peaks were basically consistent, indicating complete extraction. Considering both extraction completeness and efficiency, 20 min was selected as the optimal extraction time. See Table 1 and... Figure 6 .

[0132] Table 1. Peak area results at different extraction times

[0133] 5. Investigation of Reconstitution Solvents

[0134] 5.1 Preparation of reference solution: Same as in Example 2, "1.1".

[0135] 5.2 Preparation of the test solution: Accurately measure 1 ml of Qingwen Jiedu oral liquid and place it in a 50 ml volumetric flask. Add methanol solution containing 75% ethyl acetate to near the mark, sonicate for 20 min, cool, dilute to volume, shake well, and filter. Accurately measure 25 ml of the filtrate and place it in a round-bottom flask. Evaporate to dryness. Add water, 30% methanol, 50% methanol, 70% methanol, and 100% methanol to the residue, respectively, and transfer to 25 ml volumetric flasks. Dilute to volume and shake well to obtain the test solution.

[0136] 5.3 Chromatographic conditions: Same as in Example 2, "3.3".

[0137] 5.4 Determination: Accurately measure 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and obtain the characteristic chromatograms of the reference solution and the test solution.

[0138] 5.5 Results: When water was used as the resolvation solvent, the baicalin peak was very small; when methanol was used, the peak shape was broader. The peak shapes and resolutions of each characteristic peak were good when the resolvation solvent was 30%, 50%, or 70% methanol. Therefore, a 30%–70% methanol solution is suitable as the resolvation solvent, with a 50% methanol solution being preferred. See [link to product details]. Figure 7 .

[0139] Example 3: Identification of characteristic peaks in the characteristic spectrum

[0140] 1. Preparation of reference solution

[0141] Accurately weigh appropriate amounts of (R,S)-Gouyichun, geniposide, forsythoside, baicalin, baicalein, and wogonin, and add methanol to prepare a mixed reference solution and a positioning solution of each component reference standard containing 0.65 μg / ml of (R,S)-Gouyichun, 97.12 μg / ml of geniposide, 6.37 μg / ml of forsythoside, 37.54 μg / ml of baicalin, 2.42 μg / ml of baicalein, and 2.53 μg / ml of wogonin.

[0142] 2. Preparation of the test solution

[0143] Accurately measure 1 ml of each of 10 batches of our Qingwen Jiedu Oral Solution (batch numbers: 20221201, 20221202, 20230101, 20230301, 20230501, 20230601, 20230602, 20230901, 20231101, 20240101), place them in a 50 ml volumetric flask, add methanol solution containing 75% ethyl acetate to near the mark, sonicate for 20 min, cool, dilute to volume, shake well, and filter. Accurately measure 25 ml of the filtrate into a round-bottom flask, evaporate to dryness, add 50% methanol to the residue, transfer to a 25 ml volumetric flask, dilute to volume, and shake well to obtain the final product.

[0144] 3 Chromatographic conditions

[0145] A Kromasil 100-5C18 column (4 mm × 250 mm, 5 μm) was used as the packing material, with detection wavelengths of 241 nm from 0 min to 27 min and 227 nm from 27 min to 70 min. The column temperature was 30 °C, and the flow rate was 1.0 mL / min. Methanol was used as mobile phase A, and 0.03% aqueous phosphoric acid solution was used as mobile phase B. The gradient elution program is shown in the table below.

[0146] 4. Measurement

[0147] Accurately measure 10 μl each of the mixed reference solution, the positioning solutions of each component reference standard, and the test solution, and inject them into the liquid chromatograph to obtain characteristic chromatograms.

[0148] 5 Results

[0149] Test solutions were prepared using 10 batches of Qingwen Jiedu oral liquid samples, and chromatograms were obtained for 10 batches. Fourteen stable chromatographic peaks were identified as common characteristic peaks. The main characteristic peaks were identified using a reference standard positioning solution: peak 2 was the characteristic peak of (R,S)-Gouyichun; peak 5 was the characteristic peak of geniposide; peak 7 was the characteristic peak of forsythoside; peak 8 was the characteristic peak of baicalin; peak 12 was the characteristic peak of baicalein; and peak 14 was the characteristic peak of wogonin. The geniposide peak with a moderate and stable retention time was selected as the reference peak (S peak). The relative retention times of the 14 common characteristic peaks are as follows: peak 1 (0.3317), peak 2 (0.4588), peak 3 (0.8385), peak 4 (0.9266), peak 5 (1.0000), peak 6 (1.3297), peak 7 (1.4937), peak 8 (1.5669), peak 9 (1.7513), peak 10 (1.7870), peak 11 (1.8558), peak 12 (1.9922), peak 13 (2.0907), and peak 14 (2.1943). Their relative retention times should be within ±10% of the specified value. See [link / reference]. Figure 8 , Figure 9 .

[0150] Example 4: Verification of the Feature Mapping Method

[0151] 1. Durability assessment

[0152] 1.1 Investigation of different chromatographic columns

[0153] (1) Preparation of reference solution: Accurately weigh appropriate amounts of (R,S)-goichun, geniposide, forsythoside, baicalin, baicalein and wogonin, and add methanol to prepare a reference solution containing (R,S)-goichun 0.65 μg / ml, geniposide 97.12 μg / ml, forsythoside 6.37 μg / ml, baicalin 37.54 μg / ml, baicalein 2.42 μg / ml and wogonin 2.53 μg / ml.

[0154] (2) Preparation of the test solution: Accurately measure 1 ml of Qingwen Jiedu oral liquid and place it in a 50 ml volumetric flask. Add methanol solution containing 75% ethyl acetate to near the mark, sonicate for 20 min, cool, make up to volume, shake well, and filter. Accurately measure 25 ml of the filtrate and place it in a round-bottom flask. Evaporate to dryness. Add 50% methanol to the residue and transfer it to a 25 ml volumetric flask. Make up to volume and shake well to obtain the test solution.

[0155] (3) Chromatographic conditions: Column 1: Kromasil 100-5C18 column (4.6mm×250mm, 5μm, SN: P455935); Column 2: Kromasil 100-5C18 column (4.6mm×250mm, 5μm, SN: P495494); Column 3: Agilent Eclipse XDB-C18 column (4.6mm×250mm, 5μm, SN: USWZ001555). All other conditions were the same as in Example 3, "3 Chromatographic Conditions".

[0156] (4) Determination: Accurately measure 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and obtain the characteristic chromatograms of the reference solution and the test solution.

[0157] (5) Results: Using columns from different manufacturers, the peak shapes and resolutions of all characteristic peaks on different batches of Kromasil 100-5C18 columns were good, and the relative retention times were all within the specified range. The relative retention time of peak 2 in the chromatogram of column 3 slightly exceeded the specified range. Therefore, the Kromasil 100-5C18 column (4mm × 250mm, 5μm) was selected. See [link / reference needed]. Figure 10 .

[0158] Table 2 Results of durability studies for different chromatographic columns

[0159] 1.2 Investigation of Mobile Phase Proportion

[0160] (1) Preparation of reference solution: Same as in Example 4 "1.1".

[0161] (2) Preparation of test solution: Same as in Example 4 "1.1".

[0162] (3) Chromatographic conditions:

[0163] Mobile phase ratio 1: organic phase + 1%. Methanol was used as mobile phase A, and 0.03% aqueous phosphoric acid solution was used as mobile phase B. The gradient elution program is shown in the table below. The remaining chromatographic conditions are the same as in Example 3, "3 Chromatographic Conditions".

[0164] Mobile phase ratio 2: Same as Example 3, “3 Chromatographic conditions”.

[0165] Mobile phase ratio 3: organic phase - 1%. Methanol was used as mobile phase A, and 0.03% aqueous phosphoric acid solution was used as mobile phase B. The gradient elution program is shown in the table below. The remaining chromatographic conditions are the same as in Example 3, "3 Chromatographic Conditions".

[0166] (4) Determination: Accurately measure 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and obtain the characteristic chromatograms of the reference solution and the test solution.

[0167] (5) Results: With a change in mobile phase ratio of ±1%, the peak shapes and separation of each characteristic peak in the characteristic chromatogram of Qingwen Jiedu Oral Liquid were good, and the relative retention times were all within the specified range, meeting the requirements. See Figure 11 .

[0168] Table 3 Results of durability studies with different mobile phase ratios

[0169] 1.3 Column Temperature Investigation

[0170] (1) Preparation of reference solution: Same as in Example 4 "1.1".

[0171] (2) Preparation of test solution: Same as in Example 4 "1.1".

[0172] (3) Chromatographic conditions:

[0173] Column temperature 1: 28℃; Column temperature 2: 30℃; Column temperature 3: 32℃. The remaining chromatographic conditions are the same as those in Example 3, "3 Chromatographic Conditions".

[0174] (4) Determination: Accurately measure 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and obtain the characteristic chromatograms of the reference solution and the test solution.

[0175] (5) Results: Using column temperatures of 28℃, 30℃, and 32℃, the peak shapes and separation of each characteristic peak in the characteristic spectrum of Qingwen Jiedu Oral Liquid were good, and the relative retention times were all within the specified range, meeting the requirements. 30℃ was the preferred temperature. See [link to relevant documentation]. Figure 12 .

[0176] Table 4. Durability test results at different column temperatures

[0177] 1.4 Investigation of Phosphoric Acid Ratio

[0178] (1) Preparation of reference solution: Same as in Example 4 "1.1".

[0179] (2) Preparation of test solution: Same as in Example 4 "1.1".

[0180] (3) Chromatographic conditions:

[0181] Phosphoric acid solution ratio 1: 0.02%; phosphoric acid solution ratio 2: 0.03%; phosphoric acid solution ratio 3: 0.04%. The remaining chromatographic conditions are the same as in Example 3, "3 Chromatographic Conditions".

[0182] (4) Determination: Accurately measure 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and obtain the characteristic chromatograms of the reference solution and the test solution.

[0183] (5) Results: Using phosphoric acid solutions of 0.02%, 0.03%, and 0.04%, the peak shapes and resolutions of each characteristic peak in the chromatogram of Qingwen Jiedu Oral Liquid were good, and the relative retention times were all within the specified range, meeting the requirements. 0.03% was the preferred concentration. See [link to relevant documentation]. Figure 13 .

[0184] Table 5 Results of durability studies with different phosphoric acid ratios

[0185] 1.5 Flow velocity investigation

[0186] (1) Preparation of reference solution: Same as in Example 4 "1.1".

[0187] (2) Preparation of test solution: Same as in Example 4 "1.1".

[0188] (3) Chromatographic conditions:

[0189] Flow rate 1: 0.9 ml / min; Flow rate 2: 1.0 ml / min; Flow rate 3: 1.1 ml / min. The remaining chromatographic conditions are the same as in Example 3, "3 Chromatographic Conditions".

[0190] (4) Determination: Accurately measure 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and obtain the characteristic chromatograms of the reference solution and the test solution.

[0191] (5) Results: At flow rates of 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min, the peak shapes and separation of each characteristic peak in the chromatogram of Qingwen Jiedu Oral Liquid were good, and the relative retention times were all within the specified range, meeting the requirements. See [link to relevant documentation]. Figure 14 .

[0192] Table 6 Results of durability tests at different flow velocities

[0193] 2. Sample injection precision test

[0194] (1) Preparation of test solution: Same as in Example 4 "1.1".

[0195] (2) Chromatographic conditions: Same as “3 Chromatographic conditions” in Example 3.

[0196] (3) Determination: Take a sample of Qingwen Jiedu oral liquid and prepare one test solution. Inject the sample six times consecutively to obtain the characteristic spectra of the six test solutions. Calculate the RSD of the relative retention time and relative peak area of ​​each characteristic peak.

[0197] (4) Results: The relative retention time RSD of each characteristic peak was 0.01%–0.18%, and the relative peak area RSD was 0.11%–0.93%, both meeting the requirements. See Figure 15 .

[0198] Table 7 Results of injection precision study (relative retention time)

[0199] Table 8 Results of injection precision investigation (relative peak area)

[0200] 3. Repeatability test

[0201] (1) Preparation of reference solution: Same as in Example 4 "1.1".

[0202] (2) Preparation of test solution: Same as in Example 4 "1.1".

[0203] (3) Chromatographic conditions: Same as in Example 3, “3 Chromatographic conditions”.

[0204] (4) Determination: Take the Qingwen Jiedu oral liquid sample and prepare 6 test solutions. Inject the reference solution and the test solution into the liquid chromatograph to obtain the characteristic chromatograms of the 6 test solutions. Calculate the RSD of the relative retention time and relative peak area of ​​each characteristic peak.

[0205] (5) Results: For the six test solutions, the relative retention time RSD of each characteristic peak was 0.02%–0.15%, and the relative peak area RSD was 0.13%–1.85%, all meeting the requirements. See Figure 16 .

[0206] Table 9. Results of Repeatability Testing (Relative Retention Time)

[0207] Table 10. Repeatability test results (relative peak area)

[0208] 4. Solution stability study

[0209] (1) Preparation of test solution: Same as in Example 4 "1.1".

[0210] (2) Chromatographic conditions: Same as “3 Chromatographic conditions” in Example 3.

[0211] (3) Determination: Take a sample of Qingwen Jiedu oral liquid and prepare one test solution. Inject the sample at 0h, 2h, 4h, 6h and 8h respectively to obtain the characteristic spectra of the six test solutions. Calculate the RSD of the relative retention time and relative peak area of ​​each characteristic peak.

[0212] (4) Results: The relative retention time RSD of each characteristic peak was 0.03%–0.18%, and the relative peak area RSD was 0.08%–1.65%, both meeting the requirements. See Figure 17 .

[0213] Table 11 Results of solution stability study (relative retention time)

[0214] Table 12 Results of solution stability study (relative peak area)

[0215] 5. Intermediate Precision Examination

[0216] (1) Preparation of reference solution: Same as in Example 4 "1.1".

[0217] (2) Preparation of test solution: Same as in Example 4 "1.1".

[0218] (3) Chromatographic conditions: Same as in Example 3, “3 Chromatographic conditions”.

[0219] (4) Determination: Two personnel prepared six aliquots of the reference solution and six aliquots of the test solution on different dates using different instruments. These aliquots were injected into the liquid chromatograph to obtain characteristic chromatograms. The results of the six aliquots prepared by one personnel were the same as those obtained in the repeatability test. The RSD of the relative retention times of each characteristic peak in the twelve aliquots prepared by the two personnel was calculated.

[0220] (5) Results: The relative retention times (RSDs) of the characteristic peaks in the 12 test solutions ranged from 0.32% to 1.83%, all meeting the requirements. See Figure 16 , Figure 18 .

[0221] Table 13 Results of Intermediate Precision Examination - 1

[0222] Table 14 Intermediate Precision Test Results - 2

[0223] Example 5: Application of the Feature Mapping Method

[0224] (1) Preparation of reference solution: Same as in Example 4 "1.1".

[0225] (2) Preparation of the test solution: Take 1 ml of each of the following commercially available Qingwen Jiedu oral liquid samples (Company A, batch number: 2022100501; Company B, batch number: 20240301; Company C, batch number: 221001): Place the sample in a 50 ml volumetric flask, add methanol solution containing 75% ethyl acetate to near the mark, sonicate for 20 min, cool, dilute to volume, shake well, and filter. Accurately measure 25 ml of the filtrate into a round-bottom flask, evaporate to dryness, add 50% methanol to the residue, transfer to a 25 ml volumetric flask, dilute to volume, and shake well to obtain the test solution.

[0226] (3) Chromatographic conditions: Same as in Example 3, “3 Chromatographic conditions”.

[0227] (4) Determination: Accurately measure 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and obtain characteristic chromatograms. The reference solution is used to locate and identify 6 known peaks, and the relative retention time is used to identify the remaining unknown peaks.

[0228] (5) Results: The established characteristic spectrum detection method was used to identify three commercially available Qingwen Jiedu oral liquid samples from different manufacturers. Company A and Company B both contained 14 characteristic peaks, while Company C lacked peak 2 (R,S-gayichun), peak 12 (baicalin peak), and peak 13. The results show significant differences in the chemical composition of samples from different manufacturers. This invention can be used to identify the quality of products from different manufacturers and improve the quality control level of Qingwen Jiedu oral liquid. See [link to relevant documentation]. Figure 19 .

[0229] Table 15 Relative Retention Time of Feature Maps from Different Manufacturers

[0230] Unless otherwise defined, all technical and scientific terms used throughout this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. In case of any inconsistency, the meaning as stated in this application or derived from the content described herein shall prevail. Furthermore, the terminology used in this description is for the purpose of describing embodiments of this application only and is not intended to limit this application.

Claims

1. A method for constructing a characteristic chromatogram of Qingwen Jiedu oral liquid, characterized in that, The construction method comprises the following steps: Preparation of the control solution: appropriate amounts of (R, S)-adonirubin, jatrorrhizine, forsythoside, baicalin, baicalein and wogonin are weighed, a solvent is added to make up the volume, and the control solution is prepared; Preparation of the test solution: an appropriate amount of Qingfeng Jiedu Oral Liquid is taken, a mixed solution of ethyl acetate-methanol is added, and after ultrasonic treatment, it is cooled, made up to volume, shaken, filtered, and the filtrate is taken, evaporated to dryness, redissolved in a methanol aqueous solution, made up to volume, shaken, and the test solution is obtained; According to the control solution spectrum and the test solution spectrum detected by high performance liquid chromatography, the characteristic spectrum of Qingfeng Jiedu Oral Liquid is obtained; The detection conditions of the high performance liquid chromatography comprise: a chromatographic column with octadecylsilane-bonded silica gel as the filler is used; the mobile phase A is methanol, and the mobile phase B is phosphoric acid aqueous solution; gradient elution is used; the detection wavelength is 241 nm at 0 min-27 min, and the detection wavelength is 227 nm at 27 min-70 min.

2. The construction method according to claim 1, characterized in that, The Qingfeng Jiedu Oral Liquid comprises rehmannia, gardenia, scutellaria, forsythia, figwort and isatis root in the prescription; Preferably, the effective components of the gardenia, scutellaria, forsythia and isatis root comprise (R, S)-adonirubin, jatrorrhizine, forsythoside, baicalin, baicalein and wogonin.

3. The construction method of claim 1, wherein, In the preparation of the control solution, the solvent is methanol; Preferably, in the control solution, the concentration of (R, S)-adonirubin is 0.65 μg / ml, the concentration of jatrorrhizine is 97.12 μg / ml, the concentration of forsythoside is 6.37 μg / ml, the concentration of baicalin is 37.54 μg / ml, the concentration of baicalein is 2.42 μg / ml, and the concentration of wogonin is 2.53 μg / ml.

4. The construction method of claim 1, wherein, The concentration of ethyl acetate in the mixed solution of ethyl acetate-methanol is 25v / v%-75v / v%, preferably 50v / v%; Preferably, the ultrasonic treatment time is 10-30 min, preferably 20 min; Preferably, the concentration of the methanol aqueous solution is 30v / v%-70v / v%, preferably 50v / v%.

5. The construction method of claim 1, wherein, The chromatographic column of the high performance liquid chromatography is a C18 chromatographic column with octadecylsilane-bonded silica gel as the filler, preferably a Kromasil 100-5 C18 chromatographic column, with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5.0 μm; Preferably, the gradient elution procedure of the high performance liquid chromatography is: 0-13 min, 11% mobile phase A, 89% mobile phase B; 13-18 min, 11%-33% mobile phase A, 89%-67% mobile phase B; 18-50 min, 33%-70% mobile phase A, 67%-30% mobile phase B; 50-53 min, 70%-80% mobile phase A, 30%-20% mobile phase B; 53-60 min, 80% mobile phase A, 20% mobile phase B; 60-62 min, 80%-11% mobile phase A, 20%-89% mobile phase B; 62-70 min, 11% mobile phase A, 89% mobile phase B; wherein, the proportion of the mobile phase A and the mobile phase B varies in the range of ±1%. Preferably, the concentration of the aqueous phosphoric acid solution is 0.02v / v% to 0.04v / v%, preferably 0.03v / v%; Preferably, the flow rate of the mobile phase of the high performance liquid chromatography is 0.9ml / min to 1.1ml / min, preferably 1.0ml / min; Preferably, the column temperature of the high performance liquid chromatography is 28℃ to 32℃, preferably 30℃; Preferably, the injection volume of the high performance liquid chromatography is 0.5 to 20μl, preferably 10μl.

6. The construction method of claim 1, wherein, The characteristic spectrum includes 14 common fingerprint peaks, with peak No. 5 as the reference peak S peak, and the retention time is 1.0000. The relative retention times of the 14 common characteristic peaks are as follows: peak No. 1, retention time is 0.3317; peak No. 2, retention time is 0.4588; peak No. 3, retention time is 0.8385; peak No. 4, retention time is 0.9266; peak No. 5, retention time is 1.0000; peak No. 6, retention time is 1.3297; peak No. 7, retention time is 1.4937; peak No. 8, retention time is 1.5669; peak No. 9, retention time is 1.7513; peak No. 10, retention time is 1.7870; peak No. 11, retention time is 1.8558; peak No. 12, retention time is 1.9922; peak No. 13, retention time is 2.0907; peak No. 14, retention time is 2.1943. The relative retention times should be within ±10% of the specified values; Preferably, peak No. 2 is the characteristic peak of (R,S)-adiposin, peak No. 5 is the characteristic peak of jatrorrhizine, peak No. 7 is the characteristic peak of forsythoside, peak No. 8 is the characteristic peak of baicalin, peak No. 12 is the characteristic peak of baicalein, and peak No. 14 is the characteristic peak of wogonoside.

7. The construction method of claim 1, wherein, The reference substance solution spectrum is compared with the test substance solution spectrum for qualitative analysis of the retention time, and the index components in the test substance solution spectrum are located, thereby obtaining the characteristic spectrum of Qingwen Jiedu Oral Liquid.

8. A quality control method of Qingwen Jiedu oral liquid, characterized in that, The quality control method comprises the following steps: The characteristic spectrum of the reference sample of Qingwen Jiedu Oral Liquid is established according to the construction method of any one of claims 1 to 7, the test substance solution of the Qingwen Jiedu Oral Liquid to be tested is detected according to the detection conditions in the construction method of any one of claims 1 to 7, the spectrum of the Qingwen Jiedu Oral Liquid to be tested is obtained, and the spectrum of the Qingwen Jiedu Oral Liquid to be tested is compared with the characteristic spectrum of the reference sample of Qingwen Jiedu Oral Liquid. If it meets the requirements, it is a qualified product.

9. The application of the characteristic spectrum construction method of Qingwen Jiedu Oral Liquid according to any one of claims 1 to 7 or the quality control method according to claim 8 in the quality monitoring or production quality control of characteristic components of Qingwen Jiedu Oral Liquid.

10. The application of the characteristic spectrum construction method of Qingwen Jiedu Oral Liquid according to any one of claims 1 to 7 or the quality control method according to claim 8 in the quality control of preparations containing Radix Isatidis, Radix Scutellariae, Forsythia and Gardenia.

Images