A method for constructing HPLC characteristic chromatogram of gan'chong granules and application thereof

Abstract

This invention relates to the field of traditional Chinese veterinary medicine analysis technology, and particularly to a method for constructing and applying the HPLC characteristic chromatogram of hepatobiliary granules. The method includes the following steps: 1) preparing a test solution from hepatobiliary granules; 2) preparing reference medicinal material solutions from Artemisia capillaris and Isatis indigotica; 3) preparing reference solutions from neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, guanosine, adenosine, uridine, and (R,S)-gauzecin reference standards; 4) performing liquid chromatography on the above solutions to obtain chromatograms of the test solution, reference solution, and reference medicinal material solution; 5) importing the chromatograms of multiple batches of test solution into a similarity evaluation system for chromatograms of traditional Chinese medicine to generate standard characteristic chromatograms, and identifying the characteristic peaks in the chromatograms by comparing the chromatograms of each reference solution and the reference medicinal material solution, thereby constructing the HPLC characteristic chromatogram of the hepatobiliary granules. The advantages of this invention are that it enables quality evaluation of product authenticity and quality, traces the origin of Isatis root and Artemisia capillaris, and allows for quantitative analysis of the content of (R,S)-glucosinolate, adenosine, uridine and guanosine in Isatis root and the content of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in Artemisia capillaris. Ultimately, this makes the quality evaluation of the product based on evidence, thereby achieving comprehensive quality control of liver and gallbladder granules.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine identification technology, and in particular to a method for constructing HPLC characteristic chromatograms of hepatobiliary granules and its application. Background Technology

[0002] Liver and gallbladder granules belong to the hepatoprotective and choleretic category of veterinary traditional Chinese medicine preparations. They possess the effects of clearing heat and detoxifying, protecting the liver and promoting bile secretion. They are mainly used in livestock and poultry (pigs, chickens, ducks) for liver and kidney damage repair and mycotoxin removal. They were first included in Volume 2 of "Veterinary Drug Local Standards Upgraded to National Standards," and later in the 2017 edition of "Veterinary Drug Quality Standards" (Traditional Chinese Medicine Volume). The draft standard for liver and gallbladder granules was published by the China Institute of Veterinary Drug Control in 2023. Isatis root has anti-inflammatory, antiviral, antipyretic, and immune-enhancing effects, and mainly contains chemical components such as indigo, indirubin, polysaccharides, amino acids, and organic acids. Artemisia capillaris is used to treat wind-dampness, cold-heat evil qi, and jaundice due to heat accumulation, and mainly contains chemical components such as phenolic acids, coumarins, flavonoids, organic acids, and volatile oils.

[0003] According to available information, over 200 companies have already obtained production approval numbers, with a global market size of approximately $275 million, of which the Chinese market accounts for about 40% (i.e., $110 million). Therefore, developing a convenient, efficient, and precise quality control method is of paramount importance.

[0004] The current effective quality standards for liver and gallbladder granules still mainly rely on thin-layer chromatography (TLC) for qualitative identification of Isatis root and Artemisia capillaris. However, this traditional method is increasingly revealing its inherent limitations and technical defects when addressing the needs of modern Chinese medicine quality control. TLC provides limited chemical information, has low resolution, and struggles to comprehensively and accurately reflect the complex chemical components from different medicinal materials in the preparation. This method cannot effectively construct a comprehensive chemical characterization profile of the product, resulting in weak specificity for identifying product authenticity and a lack of objective and precise standards for evaluating the consistency of quality between different production batches.

[0005] Existing standards only specify qualitative identification and lack requirements for the content determination of any key active ingredients. Components such as guanosine, adenosine, uridine, and (R,S)-guanylic acid in Isatis indigotica, and neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid in Artemisia capillaris have all been reported to be related to efficacy, but current technology cannot simultaneously and rapidly quantitatively detect these components. Therefore, the current quality standards evaluate the quality of Isatis indigotica and Artemisia capillaris using thin-layer chromatography, a method that fails to reflect the quality characteristics of the medicinal materials and lacks a comprehensive quality evaluation system. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for constructing HPLC characteristic chromatograms of hepatobiliary granules and its application, so as to realize the quality evaluation of the authenticity and quality of the product, and to trace the source of Isatis root and Artemisia capillaris. At the same time, it can quantitatively analyze the content of (R,S)-guanylic acid, adenosine, uridine and guanosine in Isatis root and the content of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in Artemisia capillaris, so as to make the quality evaluation of the product reliable and thus realize the comprehensive quality control of hepatobiliary granules.

[0007] This invention is achieved through the following technical solution: On one hand, it provides a method for constructing HPLC characteristic chromatograms of hepatobiliary granules, the method comprising the following steps:

[0008] 1) Prepare a test solution from liver and gallbladder granules; 2) Prepare reference herb solutions from Artemisia capillaris and Isatis indigotica respectively; 3) Prepare a reference solution by taking neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, guanosine, adenosine, uridine, and (R,S)-Gauzym reference standard; 4) Perform liquid chromatography on the solutions obtained above to obtain chromatograms of the test solution, reference solution, and reference medicinal material solution, respectively; 5) Import the chromatograms of multiple batches of test sample solutions into the chromatographic feature spectrum similarity evaluation system of traditional Chinese medicine to generate standard feature spectrum. By comparing the chromatograms of each reference solution with the reference medicinal material solution, the characteristic peaks in the spectrum are assigned and identified, thereby constructing the HPLC feature spectrum of the liver and gallbladder granules.

[0009] Further, in step 4), the determination conditions for the liquid chromatography are as follows: Chromatographic column: A reversed-phase chromatographic column packed with octadecylsilane-bonded silica gel; Mobile phase: A gradient elution system comprising organic phase A and aqueous phase B; Detector: Employs a detector capable of wavelength switching or full-wavelength scanning, and detects at at least two preset wavelengths.

[0010] Furthermore, the organic phase A is any one or both of methanol and acetonitrile; the aqueous phase B is an aqueous solution of phosphoric acid with a volume percentage of 0.1%.

[0011] Furthermore, the at least two preset wavelengths include a first wavelength of 245 nm for detecting nucleoside components and a second wavelength of 324 nm for detecting chlorogenic acid components.

[0012] Furthermore, in the gradient elution system, the gradient elution program and the time-wavelength program are as follows:

[0013] Furthermore, the HPLC characteristic chromatogram of the hepatobiliary granules contains 10 characteristic peaks. The peak corresponding to the reference standard chlorogenic acid peak 9 is the S peak. The relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within ±5% of the specified value. The specified values ​​are: peak 1: 0.16, peak 2: 0.19, peak 3: 0.26, peak 4: 0.29, peak 5: 0.42, peak 6: 0.48, peak 7: 0.73, peak 8: 0.77, peak 10: 1.05.

[0014] Furthermore, at least one of the characteristic peaks is identified as originating from Isatis indigotica root, and / or at least one is identified as originating from Artemisia capillaris root.

[0015] Another method for determining the content of multiple components in hepatobiliary granules is provided. Chromatograms of the test solution and the reference solution are obtained according to the above-mentioned method for constructing HPLC characteristic chromatograms of hepatobiliary granules. By comparing the chromatographic peak response values ​​of the target component in the test sample with the chromatographic peak response values ​​of the reference sample, the content of the target component in the hepatobiliary granules is calculated by the external standard method based on the peak area.

[0016] Furthermore, the target component is selected from at least two of guanosine, adenosine, uridine, (R,S)-gawison, cryptochlorogenic acid, chlorogenic acid, and neochlorogenic acid.

[0017] Furthermore, the target components include guanosine, adenosine, uridine, (R,S)-gaurecine, cryptochlorogenic acid, chlorogenic acid, and neochlorogenic acid.

[0018] Beneficial effects The hepatobiliary granule characteristic spectrum established by this invention can simultaneously detect guanosine, uridine, adenosine, (R,S)-gayosin in Isatis root and neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid in Artemisia capillaris. The generated fingerprint spectrum has 10 characteristic peaks, which can effectively characterize the quality of hepatobiliary granules.

[0019] The method for constructing HPLC characteristic chromatograms of hepatobiliary particles provided by this invention mainly involves analysis using a PDA detector in a high-performance liquid chromatograph, while simultaneously running gradient elution and time-wavelength programs. The resulting HPLC chromatograms exhibit stable baselines, high signal-to-noise ratios, sharp and symmetrical peak shapes, and tailing factors all within the range of 0.95-1.05. The columns demonstrate high efficiency, with a theoretical plate number of no less than 10,000. Furthermore, the separation effect is excellent, with the resolution between chromatographic peaks all greater than 1.8.

[0020] The hepatobiliary particle characteristic atlas established by this invention can achieve simple, efficient, accurate and rapid quality detection and analysis of hepatobiliary particles, providing support and guarantee for the quality control of hepatobiliary particles. Attached Figure Description

[0021] Figure 1The HPLC chromatograms of the test solution in the wavelength range of 210-400 nm are presented in superimposed form. Figure 2 The HPLC chromatograms of the test solution were compared when methanol or acetonitrile was used as mobile phase A. Figure 3 HPLC chromatograms of the test solution are shown at column temperatures of 25℃, 30℃, and 35℃. Figure 4 HPLC chromatograms of the test solution were compared at flow rates of 0.8, 1.0, and 1.2 mL / min. Figure 5 It displays the superimposed HPLC chromatograms of six consecutive injections of the same test sample solution; Figure 6 It displays the superimposed HPLC chromatograms of six independently prepared test solutions from the same batch; Figure 7 It displays the superimposed HPLC chromatograms of the same batch of test solutions at time points of 0, 2, 4, 8, 12, and 24 hours; Figure 8 It displays the HPLC chromatograms of 10 different batches of hepatobiliary granule test solutions and the standard characteristic chromatograms (R) generated based on them; Figure 9 The standard characteristic chromatogram (R) was superimposed and compared with the HPLC chromatograms of the reference solutions (guanosine, adenosine, uridine, (R,S)-goichun, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid) and the negative control solutions (Isatis root and Artemisia capillaris). This clarified the component attribution and source of each characteristic peak, verified the specificity and accuracy of the method, and showed no negative interference.

[0022] Figure 10 The standard characteristic spectrum (R) generated based on 10 batches of samples is displayed, and the positions and attributions of the 10 common peaks are clearly marked. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0024] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight.

[0025] Unless otherwise specified, the reagents and raw materials used in the embodiments and comparative examples of this invention are commercially available.

[0026] instrument:

[0027] Reagents: The reference standards for neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, (R,S)-gauzecin, guanosine, adenosine, and uridine, as well as the reference medicinal materials for Isatis root and Artemisia capillaris, all came from the China National Institutes for Food and Drug Control.

[0028] The liver and gallbladder granules are produced by our company, with batch numbers 20230301, 20230701, 20231001, 20231202, 20240101, 20240302, 20240503, 20240602, 20241101, and 20250101.

[0029]

[0030] Example 1 1.1 Sample preparation: Reference solutions: Prepare a 40 μg / mL reference solution by adding 50% methanol to neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid; prepare a 120 μg / mL reference solution by adding methanol to (R,S)-glucose chlorpheniramine.

[0031] Mixed reference solution: Prepare a 60 μg / mL reference solution by adding guanosine, adenosine, and uridine to 50% methanol.

[0032] Preparation of negative control solution: Weigh 10g of Isatis root and Artemisia capillaris powder (passed through a No. 4 sieve), place them in an Erlenmeyer flask, add 50mL of water, heat under reflux for 1h, cool, shake well, filter, and collect the filtrate to obtain the solution.

[0033] Preparation of test solution: Accurately weigh 10g of liver and gallbladder granules, place them in a stoppered conical flask, accurately add 50mL of water, sonicate until completely dissolved, remove, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0034] 1.2. Selection of chromatographic conditions: 1.2.1 Selection of detection wavelength: Accurately weigh 10g of liver and gallbladder granules, prepare the test solution according to the above method, use a PDA detector to perform 210-400nm spectral scanning, collect signals from 0-70min, and perform the remaining chromatographic conditions and elution procedures according to the chromatographic analysis conditions in Example 1. Superimpose and analyze the HPLC chromatograms of the test solution collected at different wavelengths.

[0035] The results show that from 0 to 30 min, high absorption peaks are observed around 5, 8, 10, 13, 20, and 23 min at a wavelength of 245 nm. From 245 nm to 210 nm, although the absorption peaks around 20 and 23 min increase, the absorption peaks around 5, 8, 10, and 13 min decrease. From 245 nm to 400 nm, although the absorption peaks around 5, 8, 10, and 13 min increase, the absorption peaks around 20 and 23 min decrease. From 30 to 70 min, maximum absorption peaks are observed around 40, 50, and 53 min at a wavelength of 324 nm; the absorption peak around 38 min is not significantly different from the maximum absorption peak. Therefore, wavelengths of 245 nm and 324 nm were chosen for further research. Detailed results are attached. Figure 1 .

[0036] 1.2.2 Selection of mobile phase: Accurately weigh 10g of liver and gallbladder granules, and prepare the test solution according to the above method. Select methanol or acetonitrile as mobile phase A, and 0.1% phosphoric acid aqueous solution as mobile phase B. Perform the remaining chromatographic conditions and elution procedures according to the chromatographic analysis conditions in Example 1. Superimpose and analyze the HPLC chromatograms of the test solutions collected under different mobile phases.

[0037] The results showed that methanol, as the mobile phase, eluted significantly more chromatographic peaks than acetonitrile, and the peak heights were also generally higher. Therefore, methanol was subsequently chosen as the mobile phase. See attached results. Figure 2 .

[0038] 1.2.3 Selection of different flow velocities: Accurately weigh 10g of liver and gallbladder granules and prepare the test solution according to the above method. The flow rates are set to 0.8mL / min, 1.0mL / min, and 1.2mL / min, respectively. The remaining chromatographic conditions and elution procedures are the same as those in Example 1. The HPLC chromatograms of the test solution collected at different flow rates are superimposed and analyzed. The resolution, signal-to-noise ratio, tailing factor, and theoretical plate number of the identified peaks are calculated using EMPOWE software.

[0039] The results showed that, compared to flow rates of 0.8 and 1.0 mL / min, peak 7 was not detected at a flow rate of 1.2 mL / min. Specifically, the peak eluted at a flow rate of 1.0 mL / min generally exhibited better resolution (RS > 1.5), sensitivity (S / N > 3), peak shape (0.95 < T < 1.05), and column efficiency (N > 2000) than that eluted at 0.8 mL / min. Therefore, a flow rate of 1.0 mL / min was selected for subsequent studies. The results are shown in Table 1 and Appendix. Figure 3 .

[0040] Table 1. Chromatographic evaluation indicators at different flow rates

[0041] 1.2.4 Selection of different column temperatures: Accurately weigh 10g of liver and gallbladder granules and prepare the test solution according to the above method. The column temperature is set to 25℃, 30℃ and 35℃ respectively. The remaining chromatographic conditions and elution procedures are the same as those in Example 1. The HPLC chromatograms of the test solution collected at different flow rates are compared and analyzed. The resolution, signal-to-noise ratio, tailing factor and theoretical plate number of the identified peaks are calculated by EMPOWE software.

[0042] The results showed that all three temperatures could detect 10 relatively high absorption peaks, and the resolution (RS > 1.5), sensitivity (S / N > 3), and peak shape (0.95 < T < 1.05) of the elution chromatogram at column temperature 30℃ were generally superior to those at column temperatures of 25℃ and 35℃. The column efficiency of peaks 3-7 was better than that at 25℃ and 35℃. Although the column efficiency of peaks 1, 2, and 8-10 was lower than that at 25℃ and 35℃, they still met the detection requirements (N > 2000). Therefore, column temperature 30℃ was selected for further research. The results are shown in Table 2 and Appendix. Figure 4 .

[0043] Table 2. Chromatographic evaluation indicators at different column temperatures

[0044] Chromatographic analysis conditions: The liquid chromatograph was a Waters e2695, the detector was a 2998 PDA, and the column was a C18 column (4.6 × 250 mm, 5 μm) packed with octadecylsilane-bonded silica gel; the flow rate was 1.0 mL / min; and the column temperature was 30 °C. Gradient elution was performed using methanol as mobile phase A and 0.1% phosphoric acid as mobile phase B; the detection wavelengths were 245 nm and 324 nm; the specific gradient elution program is shown in the table below. mobile phase gradient elution table

[0045] Time Wavelength Program

[0046] 1.3 Determination Method The following methodological evaluation focuses on precision, repeatability, and stability to assess whether this method is suitable for constructing subsequent fingerprint profiles.

[0047] 1.3.1 Precision Verification: Accurately weigh 10g of liver and gallbladder granules. Prepare a test solution according to the preparation method and chromatographic conditions described above. Inject the solution six times consecutively and perform instrument precision testing. Using chlorogenic acid (peak 9; S9) as the reference peak, calculate the relative retention time and peak area ratio of the remaining common peaks, and calculate the RSD.

[0048] As shown in Tables 3 and 4, the relative retention time (RSD) of the common peaks of the hepatobiliary granules was ≤1%, and the peak area ratio (RSD) was ≤3%, indicating that the instrument used for detection had good precision and could ensure the consistency and repeatability of the HPLC chromatograms. See the appendix for detailed results. Figure 5 .

[0049] Table 3 Relative Retention Time (Precision)

[0050] Table 4 Peak area ratio (precision)

[0051] 1.3.2 Repeatability Test Based on the above-described preparation method and chromatographic conditions for the test solution, six test solutions of hepatobiliary granules from one batch were prepared and injected into the chromatograph for detection. Based on the detection results, using chlorogenic acid (peak 9; S9) as the reference peak, the relative retention time and peak area ratio of the remaining common peaks were calculated, and the RSD was calculated.

[0052] As shown in Tables 5 and 6, the relative retention time (RSD) of the common peaks of the hepatobiliary granules is ≤1%, and the peak area ratio (RSD) is ≤3%, indicating that the method for detecting the test solution of hepatobiliary granules has good repeatability, and the error of human operation is within a controllable range. The repeatability chromatograms are attached. Figure 6 .

[0053] Table 5 Relative Retention Time (Repeatability)

[0054] Table 6 Peak Area Ratio (Repeatability)

[0055] 1.3.3 Sample stability study Based on the preparation method and chromatographic conditions described above, a sample solution was analyzed by high-performance liquid chromatography (HPLC) at time points of 0, 2, 4, 8, 12, and 24 hours. Using chlorogenic acid (peak 9; S9) as the reference peak, the relative retention time and peak area ratio of the common peaks were calculated, and the RSD was also calculated.

[0056] As shown in Tables 7 and 8, the relative retention time (RSD) of the common peaks of the hepatobiliary granules is ≤1%, and the peak area ratio (RSD) is ≤3%, indicating that the prepared test solution is chemically stable for at least 24 hours and has not undergone degradation. The stability chromatogram is attached. Figure 7 .

[0057] Table 7 Relative retention time (stability)

[0058] Table 8 Peak Area Ratio (Stability)

[0059] First, suitable chromatographic analysis conditions were selected. Then, through precision, stability, and repeatability tests, it was shown that errors caused by the instruments, samples, and personnel operation were within controllable ranges, and the RSDs of the relative retention times and peak area ratios of each common peak met the requirements. Therefore, this method can be used for the subsequent construction of hepatobiliary granule fingerprints.

[0060] Example 2 The specific steps for constructing the HPLC characteristic chromatograms of hepatobiliary granules are as follows: Ten different batches of hepatobiliary granules produced by our company were accurately weighed to 10g each and prepared into a test solution; uridine, adenosine, guanosine, (R,S)-goicin, chlorogenic acid, cryptochlorogenic acid, and neochlorogenic acid were prepared into a reference solution; Isatis root and Artemisia capillaris were prepared into a reference medicinal material solution; the chromatographic conditions and elution procedures were as described in Example 1, and the solution was injected into a high-performance liquid chromatograph for detection. The obtained HPLC chromatogram of the test solution was imported into the Chinese medicine fingerprint chromatogram similarity evaluation system (National Pharmacopoeia Commission) to generate a standard characteristic chromatogram (R).

[0061] 2.1 Generation of fingerprint patterns The HPLC chromatograms of the 10 batches of liver and gallbladder granule test solutions were imported into the fingerprint spectrum similarity evaluation system of the National Pharmacopoeia Commission for Mark matching, generating standard feature spectrum (R), and calculating the similarity between the HPLC chromatogram of each test sample and the standard feature spectrum.

[0062] The results showed that 10 common peaks were identified in the generated fingerprint chromatograms. The similarities between the HPLC chromatograms of each test sample and the standard characteristic chromatogram (R) were 0.997, 0.992, 0.991, 0.993, 0.997, 0.997, 0.996, 0.992, 0.997, 0.993, and 1.000 (generally ≥0.90), indicating that the chemical composition of these 10 batches of hepatobiliary granules was relatively stable and homogeneous. This demonstrates that the established HPLC fingerprint chromatograms for hepatobiliary granules can be used for their identification. Specific data are attached. Figure 8 Table 9.

[0063] Table 9. Similarity calculation of hepatobiliary granule fingerprint patterns

[0064] 2.2 Identification of chromatographic peaks The standard characteristic chromatogram (R), the HPLC chromatograms of the reference solution and the negative control solution generated based on the HPLC chromatograms of different batches of liver and gallbladder granule test solution were superimposed to identify the chromatographic peaks and assign characteristic components.

[0065] The results showed that 10 common peaks were identified in the fingerprint chromatogram. Among them, the retention times of peaks 1, 2, 3, 4, 5, and 6 were consistent with the corresponding chromatographic peaks in the chromatogram of Isatis indigotica reference material, while there were no corresponding chromatographic peaks in the chromatogram of Artemisia capillaris reference material. Therefore, peaks 1, 2, 3, 4, 5, and 6 were identified as characteristic components of Isatis indigotica. Furthermore, the retention times of peaks 3, 4, 5, and 6 were consistent with the retention times of uridine, adenosine, uridine, and (R,S)-gayocin in the reference solution. Thus, peaks 3, 4, 5, and 6 were identified as uridine, adenosine, uridine, and (R,S)-gayocin, respectively. There was no interference from the negative control. Among them, peaks 7, 8, 9, and 10 have the same retention times as the corresponding peaks in the chromatogram of the Artemisia capillaris reference herb solution, and there are no corresponding peaks in the chromatogram of the Isatis indigotica reference herb solution. Therefore, peaks 7, 8, 9, and 10 can be identified as characteristic components of Artemisia capillaris. Furthermore, the retention times of peaks 8, 9, and 10 are consistent with the retention times of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid in the reference solution. Therefore, peaks 8, 9, and 10 can be identified as neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid, respectively. There was no interference from the negative control. See the appendix for specific data. Figure 9 , 10 Table 10. Currently, a preliminary fingerprint spectrum of hepatobiliary granules has been established using this method, which can be used for the quality control of this product.

[0066] Table 10. Components and Peak Assignments in Liver and Gallbladder Granules

[0067] Example 3 The specific steps for identifying the components and determining the content of liver and gallbladder granules are as follows: Six different batches of hepatobiliary granules were randomly selected, and 10g was accurately weighed to prepare a test solution. A mixed control solution of 40μg / mL neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid was prepared; a mixed control solution of 60μg / mL guanosine, adenosine, and uridine was prepared; and a control solution of 120μg / mL (R,S)-gayopicrin was prepared. The chromatographic conditions and elution procedures were all in accordance with the method in Example 1. The above-mentioned hepatobiliary granule test solution and control solution were injected into the liquid chromatograph, and the areas of the characteristic peaks in the obtained HPLC chromatographs were integrated. Finally, the contents of the seven characteristic components were calculated by the external standard method.

[0068] C = × 100% Notes: S1, S2: Peak areas of the reference standard and the test sample; M1, M2: Sampling amounts of the reference standard and the test sample; Standard 1, Standard 2: Labeled amounts of the reference standard and the test sample; N1, N2: Dilution factors of the reference standard and the test sample.

[0069] Table 11 Content of 7 components in liver and gallbladder granules

[0070] The results showed that the HPLC characteristic chromatographic detection method proposed in this invention can simultaneously detect seven characteristic components in six randomly selected batches of hepatobiliary granules, and can be quantified according to standards. The contents of uridine were 0.010-0.151 mg / g, adenosine 0.006-0.073 mg / g, guanosine 0.006-0.085 mg / g, (R,S)-goicol 0.036-0.071 mg / g, neochlorogenic acid 0.469-0.710 mg / g, chlorogenic acid 0.451-0.629 mg / g, and cryptochlorogenic acid 0.605-0.948 mg / g. Specific data are shown in Table 11.

[0071] In summary, the HPLC chromatographic detection method for hepatobiliary granules established in this invention yields HPLC chromatograms with stable baselines and high signal-to-noise ratios; sharp and symmetrical peaks with tailing factors ranging from 0.95 to 1.05; high column efficiency with a theoretical plate number of no less than 10,000; and excellent separation performance with peak resolution greater than 1.8. Furthermore, it simultaneously enables the identification and quantitative analysis of seven characteristic components in hepatobiliary granules: guanosine, uridine, adenosine, (R,S)-guanylic acid, neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid. This provides a more accurate and comprehensive reflection of the quality characteristics of hepatobiliary granules, improves quality control of the product, and ultimately facilitates a comprehensive evaluation of hepatobiliary granules.

[0072] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing HPLC characteristic chromatograms of hepatobiliary granules, characterized in that, The method includes the following steps: 1) Prepare a test solution from liver and gallbladder granules; 2) Prepare reference herb solutions from Artemisia capillaris and Isatis indigotica respectively; 3) Prepare a reference solution by taking neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, guanosine, adenosine, uridine, and (R,S)-Gauzym reference standard; 4) Perform liquid chromatography on the solutions obtained above to obtain chromatograms of the test solution, reference solution, and reference medicinal material solution, respectively; 5) Import the chromatograms of multiple batches of test sample solutions into the chromatographic feature spectrum similarity evaluation system of traditional Chinese medicine to generate standard feature spectrum. By comparing the chromatograms of each reference solution with the reference medicinal material solution, the characteristic peaks in the spectrum are assigned and identified, thereby constructing the HPLC feature spectrum of the liver and gallbladder granules.

2. The method for constructing HPLC characteristic chromatograms of hepatobiliary granules according to claim 1, characterized in that, In step 4), the liquid chromatography determination conditions are as follows: Chromatographic column: A reversed-phase chromatographic column packed with octadecylsilane-bonded silica gel; Mobile phase: A gradient elution system comprising organic phase A and aqueous phase B; Detector: Employs a detector capable of wavelength switching or full-wavelength scanning, and detects at at least two preset wavelengths.

3. The method for constructing HPLC characteristic chromatograms of hepatobiliary granules according to claim 2, characterized in that, The organic phase A is any one or both of methanol and acetonitrile; the aqueous phase B is an aqueous solution of phosphoric acid with a volume percentage of 0.1%.

4. The method for constructing HPLC characteristic chromatograms of hepatobiliary granules according to claim 2, characterized in that, The at least two preset wavelengths include a first wavelength of 245 nm for detecting nucleoside components and a second wavelength of 324 nm for detecting chlorogenic acid components.

5. The method for constructing HPLC characteristic chromatograms of hepatobiliary granules according to claim 4, characterized in that, In the gradient elution system, the gradient elution program and the time-wavelength program are as follows: 。 6. The method for constructing HPLC characteristic chromatograms of hepatobiliary granules according to claim 5, characterized in that, The HPLC chromatogram of the hepatobiliary granules contains 10 characteristic peaks. Peak 9, corresponding to the chlorogenic acid peak of the reference standard, is peak S. The relative retention times of each characteristic peak and peak S are calculated. The relative retention times should be within ±5% of the specified values, which are: peak 1: 0.16, peak 2: 0.19, peak 3: 0.26, peak 4: 0.29, peak 5: 0.42, peak 6: 0.48, peak 7: 0.73, peak 8: 0.77, peak 10: 1.

05.

7. The method for constructing HPLC characteristic chromatograms of hepatobiliary granules according to claim 6, characterized in that, At least one of the characteristic peaks is identified as originating from Isatis indigotica, and / or at least one is identified as originating from Artemisia capillaris.

8. A method for determining the content of multiple components in liver and gallbladder granules, characterized in that, The chromatograms of the test sample solution and the reference solution were obtained according to the method for constructing the HPLC characteristic chromatogram of hepatobiliary granules as described in claims 1-7. The content of the target component in the hepatobiliary granules was calculated by comparing the chromatographic peak response value of the target component in the test sample with the chromatographic peak response value of the reference standard using the external standard method based on the peak area.

9. The method for determining the content of multiple components in hepatobiliary granules according to claim 8, characterized in that, The target ingredient is selected from at least two of guanosine, adenosine, uridine, (R,S)-gautamine, cryptochlorogenic acid, chlorogenic acid, and neochlorogenic acid.

10. The method for determining the content of multiple components in hepatobiliary granules according to claim 9, characterized in that, The target components include guanosine, adenosine, uridine, (R,S)-goicin, cryptochlorogenic acid, chlorogenic acid, and neochlorogenic acid.

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