Liquid chromatogram characteristic spectrum method for identifying authenticity of veterinary Shuanghuanglian oral liquid
By setting characteristic peaks using liquid chromatography, the problem of distinguishing between honeysuckle and wild honeysuckle, and between forsythia and forsythia leaves in veterinary Shuanghuanglian oral liquid was solved, enabling rapid and accurate identification of authenticity and improving the scientific nature of quality control and the authenticity of the products.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- BAODING JIZHONG PHARMA
- Filing Date
- 2026-02-25
- Publication Date
- 2026-05-12
AI Technical Summary
Existing technologies make it difficult to effectively distinguish and differentiate between honeysuckle and wild honeysuckle, and forsythia and forsythia leaves in veterinary Shuanghuanglian oral liquid, leading to the emergence of counterfeit and substandard products on the market, affecting product quality and safety, and disrupting market order.
Liquid chromatography was used with octadecylsilane-bonded silica gel as the stationary phase and methanol and 0.2% phosphoric acid solution as the mobile phase. Gradient elution was employed, and the detection wavelength was 235 nm. The authenticity of the product was identified by the relative retention time of the characteristic peaks. The characteristic peaks were defined as the specific peaks of Forsythia suspensa and Forsythia suspensa leaves, and Lonicera japonica and Lonicera macrantha.
It enables rapid and accurate identification of the authenticity of Shuanghuanglian oral liquid, can identify illegal production activities, improves the precision and scientific nature of quality control, and ensures the authenticity and safety of the product.
Smart Images
Figure CN122017072A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of high performance liquid chromatography (HPLC), specifically to a liquid chromatographic characteristic chromatographic method for identifying the authenticity of veterinary Shuanghuanglian oral liquid. Background Technology
[0002] Shuanghuanglian oral liquid, a classic Chinese veterinary medicine preparation composed of honeysuckle, scutellaria, and forsythia, has significant effects of relieving exterior syndromes with pungent and cool properties, clearing heat and detoxifying. It is widely used to treat chicken colds and fever and is trusted by farmers. Its efficacy depends on the synergistic effect of active ingredients such as chlorogenic acid, baicalin, and forsythoside in the medicinal materials. However, as market demand expands, the prices of raw materials such as Forsythia fluctuate wildly. In order to reduce costs and make huge profits, some companies have resorted to substituting honeysuckle with wild honeysuckle, forsythia leaves with forsythia fruit, or even directly adding chemical monomers such as chlorogenic acid, baicalin, and forsythoside to produce counterfeit and substandard products. These behaviors not only seriously affect product quality and clinical safety, but also seriously disrupt market order, damage the interests of compliant manufacturers and farmers, and restrict the healthy development of the Chinese veterinary medicine industry. Therefore, in order to effectively curb illegal additives and adulteration, and to maintain a fair and competitive market environment, it is urgent to establish a rapid and accurate method for identifying the authenticity of Shuanghuanglian oral liquid. Summary of the Invention
[0003] The purpose of this invention is to provide a liquid chromatography characteristic chromatographic method for identifying the authenticity of veterinary Shuanghuanglian oral liquid, so as to solve the problems mentioned in the background art.
[0004] To achieve the above objectives, the present invention provides the following technical solution: a liquid chromatography characteristic chromatographic method for identifying the authenticity of veterinary Shuanghuanglian oral liquid, comprising the following steps: S1. Set the chromatographic conditions, using octadecylsilane-bonded silica gel as the stationary phase and methanol and 0.2% phosphoric acid solution as the mobile phase for gradient elution, and perform analysis at the specified detection wavelength; S2. Prepare reference solutions of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, forsythoside E, forsythoside I, forsythoside B, forsythoside H, forsythoside A, forsythoside and baicalin. S3. Accurately measure Shuanghuanglian oral liquid to prepare the test solution; S4. Obtain the chromatogram of the test solution and calculate the relative retention times of peaks 2, 6 and S in the chromatogram. If peak 2 is not detected in the chromatogram, then Forsythia suspensa was not used in the prescription as required. If peak 6 is not detected in the chromatogram, then Lonicera japonica was not used in the prescription as required.
[0005] Preferably, step S1 specifically includes the following steps: S101. Set the chromatographic conditions, using octadecylsilane-bonded silica gel as the stationary phase, methanol as mobile phase A, and 0.2% phosphoric acid as mobile phase B; S102. Perform gradient elution according to the preset specifications, with a flow rate of 0.8 ml per minute, a column temperature of 30℃, a detection wavelength of 235 nm, and a theoretical plate number calculated based on the chlorogenic acid peak of not less than 6000.
[0006] Preferably, step S2 specifically includes the following steps: S201. Take the reference standards of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, forsythoside E, forsythoside I, forsythoside B, forsythoside H, forsythoside A, forsythoside, and baicalin. S202. Prepare reference solutions by adding methanol to each solution containing 40ug of neochlorogenic acid, 40ug of chlorogenic acid, 40ug of cryptochlorogenic acid, 200ug of forsythoside E, 40ug of forsythoside I, 40ug of forsythoside B, 40ug of forsythoside H, 40ug of forsythoside A, 60ug of forsythoside A, and 100ug of baicalin.
[0007] Preferably, step S3 specifically includes the following steps: S301. Measure 1 ml of Shuanghuanglian oral liquid into a 50 ml volumetric flask, add 50% methanol, and treat the resulting solution with ultrasound for 20 min. S302. After cooling, add 50% methanol to the volumetric flask until the liquid level reaches the specified mark. Shake the solution evenly and then filter it. Take the filtrate to obtain the test solution.
[0008] Preferably, step S4 specifically includes the following steps: S401. Obtain the chromatogram of the test solution. The chromatogram should show 12 characteristic peaks, of which 10 characteristic peaks should correspond to the retention times of the reference peaks of the corresponding reference solutions. S402. The peak corresponding to the chlorogenic acid reference peak is called peak S. Calculate the relative retention times of peaks 2 and 6 and peak S in the chromatogram. The relative retention times should be within the specified range.
[0009] Preferably, step S4 further includes the following steps: S403. In this chromatogram, peak 2 is a characteristic peak derived from Forsythia suspensa, which can be transferred to Shuanghuanglian oral liquid and is different from the characteristic peak of Forsythia suspensa leaves; peak 6 is a characteristic peak derived from Lonicera japonica, which can be transferred to Shuanghuanglian oral liquid and is different from the characteristic peak of Lonicera macrantha. S404. If peak 2 is not detected in the chromatogram of the test solution, then Forsythia suspensa was not used in the prescription as required. If peak 6 is not detected in the chromatogram of the test solution, then Lonicera japonica was not used in the prescription as required.
[0010] Compared with the prior art, the beneficial effects of the present invention are: This invention establishes characteristic chromatographic peaks that can simultaneously distinguish between Forsythia suspensa and Forsythia suspensa leaves, and between Lonicera japonica and Lonicera macrantha, fundamentally solving current market technical problems. It can clearly identify illegal production activities and provides a direct and reliable technical means to combat the shady practices in the traditional Chinese veterinary medicine industry. Unlike existing standard methods that only compare overall similarity, this method not only provides exclusive characteristic chromatograms, but also clearly identifies the components and traces the source of 10 main chromatographic peaks in the chromatogram. This upgrades quality control from simple pattern comparison to component analysis, enabling not only the determination of authenticity, but also the analysis of specific illegal production methods, greatly improving the precision and scientific nature of quality evaluation. Attached Figure Description
[0011] Figure 1 This invention provides a schematic diagram comparing the characteristic spectra of honeysuckle and wild honeysuckle using the fingerprinting method in the quality standard of Shuanghuanglian for injection (lyophilized). Figure 2 This invention provides a comparative schematic diagram of the characteristic spectra of Forsythia suspensa leaves using the fingerprinting method in the quality standard of Shuanghuanglian for injection (lyophilized). Figure 3 This is a schematic diagram comparing the characteristic atlases of honeysuckle and wild honeysuckle provided in an embodiment of the present invention; Figure 4 A comparative schematic diagram of the characteristic atlases of Forsythia suspensa and Forsythia suspensa leaves provided in an embodiment of the present invention; Figure 5 For the purposes of this invention, detection maps of characteristic spectra of commercially available Shuanghuanglian oral liquids from 13 companies are provided. Figure 6 An overall method flowchart is provided for embodiments of the present invention. Detailed Implementation
[0012] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0013] Example 1
[0014] Please see Figure 6This invention provides a technical solution: a liquid chromatography characteristic chromatographic method for identifying the authenticity of veterinary Shuanghuanglian oral liquid, comprising the following steps: S1. Set the chromatographic conditions, using octadecylsilane-bonded silica gel as the stationary phase and methanol and 0.2% phosphoric acid solution as the mobile phase for gradient elution, and perform analysis at the specified detection wavelength; S2. Prepare reference solutions of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, forsythoside E, forsythoside I, forsythoside B, forsythoside H, forsythoside A, forsythoside and baicalin. S3. Accurately measure Shuanghuanglian oral liquid to prepare the test solution; S4. Obtain the chromatogram of the test solution and calculate the relative retention times of peaks 2, 6 and S in the chromatogram. If peak 2 is not detected in the chromatogram, then Forsythia suspensa was not used in the prescription as required. If peak 6 is not detected in the chromatogram, then Lonicera japonica was not used in the prescription as required.
[0015] S1 specifically includes the following steps: S101. Set the chromatographic conditions, using octadecylsilane-bonded silica gel as the stationary phase, methanol as mobile phase A, and 0.2% phosphoric acid as mobile phase B; S102. Perform gradient elution according to the preset specifications, with a flow rate of 0.8 ml / min, a column temperature of 30℃, a detection wavelength of 235 nm, and a theoretical plate number calculated based on the chlorogenic acid peak of not less than 6000. The preset specifications are shown in Table 1. Table 1. Preset mobile phase gradient ; S2 specifically includes the following steps: S201. Take the reference standards of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, forsythoside E, forsythoside I, forsythoside B, forsythoside H, forsythoside A, forsythoside, and baicalin. S202. Prepare reference solutions by adding methanol to each solution containing 40ug of neochlorogenic acid, 40ug of chlorogenic acid, 40ug of cryptochlorogenic acid, 200ug of forsythoside E, 40ug of forsythoside I, 40ug of forsythoside B, 40ug of forsythoside H, 40ug of forsythoside A, 60ug of forsythoside A, and 100ug of baicalin. S3 specifically includes the following steps: S301. Accurately measure 1 ml of Shuanghuanglian oral liquid into a 50 ml volumetric flask, add 50% methanol, and treat the resulting solution with ultrasound for 20 min. S302. After cooling, add 50% methanol to the volumetric flask until the liquid level reaches the specified mark. Shake the solution evenly and then filter it. Take the filtrate to obtain the test solution. S4 specifically includes the following steps: S401. Obtain the chromatogram of the test sample solution. The solution in the chromatogram shows 12 characteristic peaks, of which 10 characteristic peaks should correspond to the retention times of the reference peaks of the corresponding reference solution. S402. The peak corresponding to the chlorogenic acid reference peak is the S peak. Calculate the relative retention times of peaks 2 and 6 and peak S in the chromatogram. The relative retention times should be within the specified range. S4 also includes the following steps: S403. In this chromatogram, peak 2 is a characteristic peak derived from Forsythia suspensa, which can be transferred to Shuanghuanglian oral liquid and is different from the characteristic peak of Forsythia suspensa leaves; peak 6 is a characteristic peak derived from Lonicera japonica, which can be transferred to Shuanghuanglian oral liquid and is different from the characteristic peak of Lonicera macrantha. S404. If peak 2 is not detected in the chromatogram of the test solution, then Forsythia suspensa was not used in the prescription as required. If peak 6 is not detected in the chromatogram of the test solution, then Lonicera japonica was not used in the prescription as required.
[0016] Example 2
[0017] Please see Figure 1 - Figure 5 The present invention provides a high-performance liquid chromatography characteristic chromatographic method for identifying the authenticity of Shuanghuanglian oral liquid. The method explores the differences in characteristic peaks by comparing honeysuckle and wild honeysuckle, and forsythia leaves and forsythia. At the same time, a standard sample is prepared by referring to the preparation method of Shuanghuanglian oral liquid in the pharmacopoeia. Negative samples lacking honeysuckle and forsythia are also prepared and compared with the standard sample to realize the transfer of characteristic peaks of medicinal materials to substances and key characteristic peaks of Shuanghuanglian oral liquid, and finally determine the characteristic chromatogram of Shuanghuanglian oral liquid. S01. Preparation of negative samples and standards: Based on the pharmacopoeia preparation method of Shuanghuanglian oral liquid, prepare negative sample solutions and standards for those lacking honeysuckle and forsythia respectively according to the pharmacopoeia. S02. Chromatographic analysis conditions: Octadecylsilane-bonded silica gel was used as the stationary phase, methanol was used as mobile phase A, and 0.2% phosphoric acid was used as mobile phase B. Gradient elution was performed according to the preset specifications. The flow rate was 0.8 ml per minute, the column temperature was 30℃, the detection wavelength was 235 nm, and the theoretical plate number calculated based on the chlorogenic acid peak should not be less than 6000. S03. Preparation of mixed reference solution: Take appropriate amounts of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, forsythoside E, forsythoside I, forsythoside B, forsythoside H, forsythoside A, forsythoside, and baicalin reference standards, and add methanol to prepare solutions containing 40ug of neochlorogenic acid, 40μg of chlorogenic acid, 40ug of cryptochlorogenic acid, 200ug of forsythoside E, 40ug of forsythoside I, 40ug of forsythoside B, 40ug of forsythoside H, 40ug of forsythoside A, 60ug of forsythoside, and 100ug of baicalin per ml, as reference solutions; S04. Preparation of negative test solutions and Shuanghuanglian oral liquid standard test solutions: Accurately measure 1 ml of each negative sample and Shuanghuanglian oral liquid into 50 ml volumetric flasks, add 50% methanol, sonicate for 20 min, cool, and then dilute to the mark with 50% methanol. Shake well, filter, and take the filtrate to obtain the negative test solution.
[0018] S05. Preparation of medicinal material test solutions: Take 0.25g of powdered honeysuckle, wild honeysuckle, forsythia and forsythia leaves respectively. After weighing, put all the powders into a 50ml volumetric flask. Add 50% methanol to the volumetric flask. Treat the resulting solution with ultrasound for 30min. Continue to add 50% methanol to the volumetric flask until the liquid level reaches the specified mark. After shaking thoroughly, filter the solution in the volumetric flask to obtain single medicinal material test solutions of honeysuckle, wild honeysuckle, forsythia and forsythia leaves. S06. Inject the reference solution: Obtain the chromatogram of the reference solution and determine the precise retention times of the 10 target components on the chromatogram; S07. Inject the test solutions of each medicinal material: Obtain the chromatograms of the test solutions of each medicinal material, compare the chromatograms of honeysuckle and wild honeysuckle, and observe the common peaks and the difference peaks; compare the chromatograms of forsythia and forsythia leaves, and observe the common peaks and the difference peaks.
[0019] S08. Inject negative sample solution: After obtaining the chromatogram of the negative sample solution, determine the specific peaks of honeysuckle and forsythia. S09. Inject the Shuanghuanglian oral liquid standard sample test solution: After obtaining the chromatogram, compare it with the results of the reference solution, the test solutions of each medicinal material, and the solutions of each negative sample.
[0020] Depend on Figure 1 As can be seen, by comparing the chromatographic peaks of the negative test for honeysuckle with those of the pharmacopoeia standard prescription, the chromatographic peaks transferred from honeysuckle medicinal material to Shuanghuanglian oral liquid are the chromatographic peaks indicated by the red arrows with retention times of 10 min, 13 min, 15.5 min, 28.5 min, and 30.5 min. All five chromatographic peaks appear in the chromatogram of honeysuckle medicinal material. Therefore, this method cannot accurately identify whether honeysuckle medicinal material or honeysuckle leaves were used. Depend on Figure 2 As can be seen, by comparing the chromatographic peaks of the negative forsythia suspensa sample with those of the pharmacopoeia standard, the chromatographic peaks transferred from forsythia suspensa material to Shuanghuanglian oral liquid are the chromatographic peaks indicated by the red arrows, with retention times of 14 min, 18.5 min, 23.5 min, and 24.5 min. The chromatographic peaks of 18.5 min and 24.5 min appear in the chromatograms of both forsythia suspensa and forsythia suspensa leaves. Therefore, this method cannot accurately identify whether forsythia suspensa material or forsythia suspensa leaves were used. Depend on Figure 3 As can be seen, by comparing the chromatographic peaks of the negative test for honeysuckle with those of the pharmacopoeia standard prescription, the chromatographic peaks transferred from honeysuckle to Shuanghuanglian oral liquid are the six chromatographic peaks indicated by the red arrows, with retention times of 8.8 min, 12.8 min, 13.8 min, 14.4 min, 15.5 min, and 41.9 min. By comparing the chromatograms of honeysuckle and wild honeysuckle, it can be seen that the chromatographic peak at 14.4 min is only present in honeysuckle. Therefore, the chromatographic peak at 14.4 min can be used as the characteristic peak of honeysuckle and the honeysuckle transferred to Shuanghuanglian oral liquid, that is, the characteristic peak for identifying the authenticity of Shuanghuanglian oral liquid. Depend on Figure 4 As can be seen, by comparing the chromatographic peaks of the negative forsythia and the pharmacopoeia standard prescription, the chromatographic peaks transferred from forsythia to Shuanghuanglian oral liquid are the 10 chromatographic peaks marked with red numbers. By comparing the chromatograms of forsythia and forsythia leaves, it can be seen that the chromatographic peak at 8.9 min is only present in forsythia. Therefore, the chromatographic peak at 8.9 min can be used as the characteristic peak of forsythia and forsythia transferred from forsythia to Shuanghuanglian oral liquid, that is, the characteristic peak for identifying the authenticity of Shuanghuanglian oral liquid. For comparative purposes, to further verify the characteristic properties of this method, samples of Shuanghuanglian oral liquid from 13 commercially available brands were collected and tested using both current quality standards and the characteristic chromatographic method described in this invention. The results are shown in [the table below]. Figure 5 ; Depend on Figure 5 It is evident that, among commercially available Shuanghuanglian oral liquids, products from 11 manufacturers (except for manufacturers 10 and 12) met the pharmacopoeia standards. However, when tested using the method of this invention, a comparison revealed that only manufacturer 1's characteristic chromatogram showed two characteristic peaks indicating both Forsythia suspensa and Lonicera japonica (as indicated by red arrows). The other manufacturers' Shuanghuanglian oral liquids did not show the characteristic peak of Forsythia suspensa with a retention time of 8.9 min, and only one manufacturer's Shuanghuanglian oral liquid showed a characteristic peak of Lonicera japonica with a retention time of 14.4 min. Therefore, the quality of commercially available Shuanghuanglian oral liquids is indeed worrying, and this also confirms the objectivity and advantages of the high-performance liquid chromatography characteristic chromatogram method for identifying the authenticity of Shuanghuanglian oral liquids described in this invention.
[0021] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.
[0022] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A liquid chromatography characteristic chromatographic method for identifying the authenticity of veterinary Shuanghuanglian oral liquid, characterized in that, The method includes the following steps: S1. Set the chromatographic conditions, using octadecylsilane-bonded silica gel as the stationary phase and methanol and 0.2% phosphoric acid solution as the mobile phase for gradient elution, and perform analysis at the specified detection wavelength; S2. Prepare reference solutions of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, forsythoside E, forsythoside I, forsythoside B, forsythoside H, forsythoside A, forsythoside and baicalin. S3. Accurately measure Shuanghuanglian oral liquid to prepare the test solution; S4. Obtain the chromatogram of the test solution and calculate the relative retention times of peaks 2, 6 and S in the chromatogram. If peak 2 is not detected in the chromatogram, then Forsythia suspensa was not used in the prescription as required. If peak 6 is not detected in the chromatogram, then Lonicera japonica was not used in the prescription as required.
2. The liquid chromatography characteristic chromatographic method for identifying the authenticity of veterinary Shuanghuanglian oral liquid according to claim 1, characterized in that, S1 specifically includes the following steps: S101. Set the chromatographic conditions, using octadecylsilane-bonded silica gel as the stationary phase, methanol as mobile phase A, and 0.2% phosphoric acid as mobile phase B; S102. Perform gradient elution according to the preset specifications, with a flow rate of 0.8 ml per minute, a column temperature of 30℃, a detection wavelength of 235 nm, and a theoretical plate number calculated based on the chlorogenic acid peak of not less than 6000.
3. The liquid chromatography characteristic chromatographic method for identifying the authenticity of veterinary Shuanghuanglian oral liquid according to claim 1, characterized in that, S2 specifically includes the following steps: S201. Take the reference standards of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, forsythoside E, forsythoside I, forsythoside B, forsythoside H, forsythoside A, forsythoside, and baicalin. S202. Prepare reference solutions by adding methanol to each solution containing 40ug of neochlorogenic acid, 40ug of chlorogenic acid, 40ug of cryptochlorogenic acid, 200ug of forsythoside E, 40ug of forsythoside I, 40ug of forsythoside B, 40ug of forsythoside H, 40ug of forsythoside A, 60ug of forsythoside A, and 100ug of baicalin.
4. The liquid chromatography characteristic chromatographic method for identifying the authenticity of veterinary Shuanghuanglian oral liquid according to claim 1, characterized in that, S3 specifically includes the following steps: S301. Measure 1 ml of Shuanghuanglian oral liquid into a 50 ml volumetric flask, add 50% methanol, and treat the resulting solution with ultrasound for 20 min. S302. After cooling, add 50% methanol to the volumetric flask until the liquid level reaches the specified mark. Shake the solution evenly and then filter it. Take the filtrate to obtain the test solution.
5. The liquid chromatography characteristic chromatographic method for identifying the authenticity of veterinary Shuanghuanglian oral liquid according to claim 1, characterized in that, S4 specifically includes the following steps: S401. Obtain the chromatogram of the test solution. The solution in the chromatogram shows 12 characteristic peaks, of which 10 characteristic peaks should correspond to the retention times of the reference peaks of the corresponding reference solution. S402. The peak corresponding to the chlorogenic acid reference peak is called peak S. Calculate the relative retention times of peaks 2 and 6 and peak S in the chromatogram. The relative retention times should be within the specified range.
6. The liquid chromatography characteristic chromatographic method for identifying the authenticity of veterinary Shuanghuanglian oral liquid according to claim 5, characterized in that, S4 further includes the following steps: S403. In this chromatogram, peak 2 is a characteristic peak derived from Forsythia suspensa, which can be transferred to Shuanghuanglian oral liquid and is different from the characteristic peak of Forsythia suspensa leaves; peak 6 is a characteristic peak derived from Lonicera japonica, which can be transferred to Shuanghuanglian oral liquid and is different from the characteristic peak of Lonicera macrantha. S404. If peak 2 is not detected in the chromatogram of the test solution, then Forsythia suspensa was not used in the prescription as required. If peak 6 is not detected in the chromatogram of the test solution, then Lonicera japonica was not used in the prescription as required.