Method for constructing characteristic chromatogram of Ulmus pumila and Ulmus crassifolia formula granules and identification method

Abstract

This invention relates to the field of traditional Chinese medicine detection technology, specifically disclosing a method for constructing characteristic chromatograms of Sanguisorba officinalis and Sanguisorba longifolia formula granules. The method includes: preparation of a reference solution; preparation of a test solution: taking Sanguisorba officinalis formula granules or Sanguisorba longifolia formula granules, dissolving them in a solvent to obtain a test solution; determining the reference solution and test solution using high-performance liquid chromatography (HPLC) to obtain characteristic chromatograms of Sanguisorba officinalis and Sanguisorba longifolia formula granules; the HPLC chromatographic conditions are: detection wavelength: 225-235 nm for 0-16 min, 240-250 nm for 16.1-50 min; gradient elution using acetonitrile as mobile phase A and 0.15% formic acid solution as mobile phase B. This invention constructs UPLC characteristic chromatograms of Sanguisorba officinalis and Sanguisorba longifolia formula granules, with richer chromatographic peaks and better peak shapes, which can more comprehensively reflect the characteristics of Sanguisorba officinalis and Sanguisorba longifolia traditional Chinese medicine formula granules, enabling more comprehensive overall quality evaluation. The detection time is relatively shorter, and the method is stable, with good precision and reproducibility.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine detection technology, and in particular to a method for constructing and identifying characteristic spectra of Sanguisorba officinalis and Sanguisorba longifolia granules. Background Technology

[0002] Sanguisorba officinalis, or Sanguisorba longifolia, is the dried root of the Rosaceae plant Sanguisorba officinalis or Sanguisorba longifolia. The latter is commonly known as "Mian Sanguisorba officinalis". It is harvested in spring when the plant is sprouting or in autumn after the plant has withered. The fibrous roots are removed, the roots are washed, and then dried, or sliced ​​while fresh and then dried. Sanguisorba longifolia is a variety of Sanguisorba officinalis. The two dominant haplotypes show very little difference, and the loci showing interspecific differences are also intraspecific differences. That is, the DNA barcodes of Sanguisorba officinalis and Sanguisorba longifolia show little interspecific difference, making accurate differentiation between them impossible. Other auxiliary methods (such as chemical composition and morphological identification) are needed for differentiation.

[0003] Traditional Chinese medicine (TCM) granules are characterized by convenient administration, dosage adjustment, standardized specifications, and consistent efficacy with their corresponding traditional medicinal slices. They are produced through water extraction, concentration, drying, and granulation processes. However, TCM granules lose the distinctive features of the original medicinal materials and slices, making identification based on their original shape, color, texture, cross-section, and odor impossible. Furthermore, the efficacy of various original medicinal materials is similar. If the origin and origin of the medicinal materials used in TCM granule production are not consistent, the quality of the final product will be affected, potentially leading to quality differences between different batches of the same variety, impacting the stability of batch-to-batch quality. The species differentiation of multiple original medicinal materials in granule production reflects the consistency and controllability of quality among the three key elements of modern TCM: safety, efficacy, and quality control. The "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules" requires that the standard formulation of multi-origin varieties should distinguish between different origins. Therefore, it is crucial to establish a characteristic spectrum that can distinguish between Sanguisorba officinalis (Diyu) formula granules and Sanguisorba longifolia (Diyu) formula granules for the identification of the two.

[0004] Chinese patent CN113391005A discloses a high-performance liquid chromatography (HPLC) method for detecting and identifying *Sanguisorba officinalis* and its charred decoction pieces, reference extracts, and formulation granules. *Sanguisorba officinalis* and its charred decoction pieces, reference extracts, or formulation granules are used as test samples, with ellagic acid, gallic acid, and 5-hydroxymethylfurfural as reference standards. The HPLC method is used to obtain the characteristic HPLC chromatograms of *Sanguisorba officinalis* and its charred decoction pieces, reference extracts, and formulation granules. The detection time is 60 minutes, and the number of characteristic peaks is only 7. This method can be used for quality control of *Sanguisorba officinalis* and its charred decoction before and after charring. However, it only addresses the identification of different original varieties before and after processing. Summary of the Invention

[0005] To address the above shortcomings, this invention provides a method for constructing and identifying characteristic maps of Sanguisorba officinalis and Sanguisorba longifolia formulation granules, which can distinguish between Sanguisorba officinalis formulation granules and Sanguisorba longifolia formulation granules. The specific technical solution is as follows:

[0006] A method for constructing characteristic maps of Sanguisorba officinalis and Sanguisorba longifolia formulation granules includes the following steps:

[0007] (1) Preparation of reference solution: Take Sanguisorba officinalis reference material or Sanguisorba longifolia reference material and extract it with water to obtain reference solution of reference material; take gallic acid, catechin and ellagic acid, add methanol to make a mixed solution, which is the reference solution of reference material;

[0008] (2) Preparation of test solution: Take Sanguisorba officinalis formula granules or Sanguisorba longifolia formula granules, extract with solvent, the solvent being water or 30% to 50% methanol, to obtain test solution;

[0009] (3) The reference solution and the test solution were determined by high performance liquid chromatography. The corresponding spectra of the reference solution and the test solution were compared to obtain the characteristic spectra of Sanguisorba officinalis and Sanguisorba longifolia formulation granules.

[0010] The chromatographic conditions for the high-performance liquid chromatography method are as follows:

[0011] Detection wavelength: 225-235 nm for 0-16 min, 240-250 nm for 16.1-50 min; gradient elution was performed using acetonitrile as mobile phase A and 0.15% formic acid solution as mobile phase B.

[0012] Preferably, in the above-mentioned method for constructing the characteristic spectrum of Sanguisorba officinalis and Sanguisorba longifolia formulation granules, the preparation of the reference solution in step (1) specifically includes: taking Sanguisorba officinalis or Sanguisorba longifolia reference medicinal material, adding water, with the ratio of reference medicinal material to water being 0.1g:5-12mL, heating under reflux or sonicating for 15-45min, filtering, and taking the filtrate as the reference solution of the reference medicinal material; separately taking gallic acid, catechin, and ellagic acid, adding 50-70% methanol to prepare a mixed solution containing 30-40μg of each per 1mL, which is the reference solution of the reference substance.

[0013] Preferably, in the above-mentioned method for constructing the characteristic spectrum of Sanguisorba officinalis and Sanguisorba longifolia formula granules, the preparation of the test solution in step (2) specifically includes: taking Sanguisorba officinalis formula granules or Sanguisorba longifolia formula granules, adding solvent, the ratio of formula granules to solvent is 0.2g:15-50mL, heating under reflux or ultrasonic treatment for 15-45min, cooling, weighing again, using the test sample extraction solvent to make up for the lost weight, filtering, and taking the filtrate to obtain the solution.

[0014] Preferably, in the above-mentioned method for constructing the characteristic map of Sanguisorba officinalis and Sanguisorba longifolia formulation particles, the gradient elution specifically involves:

[0015] 0–3 min, Phase A: 0–3.5%, Phase B: 100–96.5%;

[0016] 3–7.5 min, Phase A: 3.5%, Phase B: 96.5%;

[0017] 7.5–15 min, Phase A: 3.5–8%, Phase B: 96.5–92%;

[0018] 15–22 min, Phase A: 8–10%, Phase B: 92–90%;

[0019] 22–24 min, Phase A: 10–11%, Phase B: 90–89%;

[0020] 24–26 min, Phase A: 11–13%, Phase B: 89–87%;

[0021] 26–35 min, Phase A: 13–18%, Phase B: 87–82%;

[0022] 35–38 min, Phase A: 18–30%, Phase B: 82–70%;

[0023] 38–50 min, Phase A: 30–33%, Phase B: 70–67%.

[0024] Preferably, in the above-mentioned method for constructing the characteristic chromatograms of the Sanguisorba officinalis and Sanguisorba longifolia formulation granules, the chromatographic conditions are as follows: a chromatographic column with octadecylsilane-bonded silica gel as the packing material, a column length of 150 mm, an inner diameter of 2.1 mm, and a particle size of 1.6 μm; a diode array detector; a column temperature of 40℃~42℃; a flow rate of 0.29~0.31 mL / min; and a theoretical plate number calculated based on the gallic acid peak that should not be less than 5000.

[0025] Preferably, in the above-mentioned method for constructing the characteristic spectrum of Sanguisorba officinalis and Sanguisorba longifolia formulation granules, the detection wavelength is 230 nm for 0-16 min and 245 nm for 16.1-50 min; the column temperature is 40℃ and the flow rate is 0.3 mL / min.

[0026] Preferably, in the above-mentioned method for constructing the characteristic chromatograms of Sanguisorba officinalis and Sanguisorba longifolia formula granules, the similarity of the characteristic chromatograms of Sanguisorba officinalis and Sanguisorba longifolia formula granules is evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system. The test sample (Sanguisorba officinalis formula granules and Sanguisorba longifolia formula granules) shows 13 to 14 characteristic peaks, which should correspond to the retention times of the characteristic peaks in the chromatograms of the reference medicinal materials. Among them, peaks 1, 6, and 12 should correspond to the retention times of the reference peaks of gallic acid, catechin, and ellagic acid. Peak 1 is the gallic acid peak, peak 6 is the catechin peak, and peak 12 is the ellagic acid peak.

[0027] Preferably, in the above-mentioned method for constructing the characteristic spectrum of Sanguisorba officinalis and Sanguisorba longifolia formulation granules, the gallic acid peak is used as the reference peak S1, and the relative retention times of peaks 2, 3, 4, and 5 with peak S1 are calculated. The relative retention times should be within ±10% of the specified values, which are: 1.990 - peak 2, 2.898 - peak 3, 2.974 - peak 4, and 3.527 - peak 5.

[0028] Using the ellagic acid peak as the reference peak S2, calculate the relative retention times of peaks 7, 8, 9, 10, 11, 13, and 14 with peak S2. The relative retention times should be within ±10% of the specified values, which are 0.644 for peak 7, 0.727 for peak 8, 0.851 for peak 9, 0.936 for peak 10, 0.953 for peak 11, 1.034 for peak 13, and 1.506 for peak 14.

[0029] Preferably, in the above-mentioned method for constructing the characteristic spectrum of Sanguisorba officinalis and Sanguisorba longifolia formulation granules, the relative peak area of ​​peak 14 and peak 12 corresponding to the Sanguisorba officinalis formulation granules is 0.022 to 0.148; and the relative peak area of ​​peak 14 and peak 12 of the Sanguisorba longifolia formulation granules is 0 to 0.019.

[0030] This invention also provides a method for identifying the characteristic spectral features of Sanguisorba officinalis and Sanguisorba longifolia granules, and the detection results are analyzed according to the above method.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] This invention constructs UPLC characteristic spectra of Sanguisorba officinalis (Diyu) formula granules and Sanguisorba officinalis (Longleaf Diyu) formula granules, consisting of 14 characteristic peaks. The chromatographic peaks are richer, with good separation of common peaks and good peak shapes, enabling a more comprehensive reflection of the characteristics of Sanguisorba officinalis and Longleaf Diyu traditional Chinese medicine formula granules, and facilitating a more comprehensive overall quality evaluation. The detection time is relatively shorter, the method is stable, and the precision and reproducibility are good. By constructing UPLC characteristic spectra of Sanguisorba officinalis and Sanguisorba officinalis (Longleaf Diyu) formula granules, a rapid and comprehensive detection and identification method for the quality of Sanguisorba officinalis and Longleaf Diyu traditional Chinese medicine formula granules is provided. This identification method can clearly identify the differences between the two and solves the problem of lost microscopic identification points of the original medicinal material decoction pieces. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 Chromatograms of test solutions prepared by different extraction methods;

[0035] Figure 2 Chromatograms of test solutions prepared with different solvent amounts;

[0036] Figure 3 Chromatograms of test solutions prepared for different extraction times;

[0037] Figure 4 Chromatograms of test sample solutions extracted with different solvents (methanol and water);

[0038] Figure 5 Chromatograms of test sample solutions extracted with different solvents (ethanol and water);

[0039] Figure 6 This is a chromatogram for a specific test.

[0040] Figure 7 This is a chromatogram for a precision test;

[0041] Figure 8 This is a chromatogram for stability testing;

[0042] Figure 9 Chromatogram for repeatability test;

[0043] Figure 10 Chromatograms were examined at different column temperatures;

[0044] Figure 11 Chromatograms were examined at different flow rates;

[0045] Figure 12 Chromatograms were examined for different chromatographic columns;

[0046] Figure 13 To examine chromatograms using different instruments;

[0047] Figure 14 A comparative characteristic spectrum of the formula granules of Sanguisorba officinalis (Diyu) is presented;

[0048] Figure 15 A comparative characteristic spectrum of the formulation granules of Sanguisorba officinalis (long-leaved Sanguisorba officinalis);

[0049] Figure 16 The spectrum is an identification diagram of 5 common characteristic peaks.

[0050] Figure 17 The chromatogram of Sanguisorba officinalis particles at a wavelength of 230 nm is shown.

[0051] Figure 18 The chromatogram of Sanguisorba officinalis particles at a wavelength of 245 nm is shown.

[0052] Figure 19 The chromatogram of *Sanguisorba officinalis* particles at a wavelength of 230 nm is shown.

[0053] Figure 20 The chromatogram of long-leaved Sanguisorba officinalis particles was obtained for detection at a wavelength of 245 nm. Detailed Implementation

[0054] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Unless otherwise defined, all technical terms used below have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in the present invention are commercially available or can be prepared by existing methods.

[0055] Instruments and materials:

[0056] Waters H-Plus ultra-high performance liquid chromatograph (Waters Corporation, USA); electronic analytical balance (ML204 / 02, XSR204, Mettler Toledo); electronic balance (E600-2, Changshu Shuangjie Test Instrument Factory); CNC ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.).

[0057] Gallic acid reference standard (batch number 110831-201605, content calculated as 90.8%, stored at 4℃ protected from light) and Sanguisorba officinalis (batch number 121286-201703) reference medicinal material were both sourced from the China National Institutes for Food and Drug Control. Sanguisorba officinalis (long-leaved Sanguisorba officinalis) reference medicinal material (batch number 240100-202210) was sourced from Shanghai Hongyong Biotechnology Co., Ltd. Sanguisorba officinalis (Burnt Sanguisorba officinalis) formula granules and Sanguisorba officinalis (long-leaved Sanguisorba officinalis) formula granules were prepared by Peili (Nanning) Pharmaceutical Co., Ltd.

[0058] Acetonitrile (chromatographic grade), water (ultrapure water), and other reagents were all of analytical grade.

[0059] Example 1

[0060] 1. Proposed ultra-high performance liquid chromatography (UHPLC) detection method

[0061] (1) Preparation of reference solution: Take 0.5g of Sanguisorba officinalis or Sanguisorba longifolia reference material, place it in an Erlenmeyer flask, add 25ml of water, heat under reflux for 30min, filter, and take the filtrate as the reference solution of the reference material. Separately, take appropriate amounts of gallic acid, catechin, and ellagic acid reference standards, add 50% methanol to prepare a mixed solution containing 40μg of each per ml, as the reference solution of the reference standard.

[0062] (2) Preparation of test solution: Take Sanguisorba officinalis (Sanguisorba officinalis) formula granules or Sanguisorba officinalis (Sanguisorba longifolia) formula granules, grind them finely, take about 0.2g, weigh accurately, add 25ml of water, sonicate for 30min, take out, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0063] (3) Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile was used as mobile phase A, and 0.15% formic acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in Table 1. The gradient elution program was continuously optimized and adjusted based on the resolution of each characteristic peak, and the optimal gradient elution program was finally selected. Detection wavelength: 230 nm for 0–16 min, 245 nm for 16.1–50 min; column temperature 40℃, flow rate 0.3 ml / min; the theoretical plate number calculated based on the gallic acid peak should not be less than 5000.

[0064] (4) Determination: Accurately pipette 1 μL of the reference solution and the test solution into the liquid chromatograph, determine and record the chromatogram.

[0065] Table 1 Gradient elution regime

[0066]

[0067] The characteristic chromatograms of the obtained Sanguisorba officinalis and Sanguisorba longifolia formulation granules showed 14 characteristic peaks, of which peak 1 was gallic acid, peak 6 was catechin, and peak 12 was ellagic acid. Using gallic acid as a reference peak S1, the relative retention times of peaks 2, 3, 4, and 5 with peak S1 were calculated. These relative retention times should be within ±10% of a specified value, which is: 1.990 for peak 2, 2.898 for peak 3, 2.974 for peak 4, and 3.527 for peak 5.

[0068] Using the ellagic acid peak as the reference peak S2, calculate the relative retention times of peaks 7, 8, 9, 10, 11, 13, and 14 with peak S2. The relative retention times should be within ±10% of the specified values, which are 0.644 for peak 7, 0.727 for peak 8, 0.851 for peak 9, 0.936 for peak 10, 0.953 for peak 11, 1.034 for peak 13, and 1.506 for peak 14.

[0069] 2. Investigation of the preparation method of the test solution

[0070] 2.1 Selection of Extraction Method

[0071] Using water as the extraction solvent, the effects of ultrasonic extraction for 30 min and reflux extraction for 30 min on the characteristic spectra were compared. The results are as follows: Figure 1As shown in the figure. The results indicate that the chromatogram shapes and the number of chromatographic peaks of ultrasonic and reflux extraction methods are basically the same. Considering that ultrasonic extraction is convenient and simple to operate, ultrasonic extraction method was chosen.

[0072] 2.2 Investigation of Solvent Usage

[0073] Using water as the extraction solvent and employing ultrasonic extraction, the effects of different solvent volumes (15, 25, and 50 ml) on the characteristic chromatograms were investigated. The results are as follows: Figure 2 As shown in the figure. The results indicate that the chromatogram shapes and the number of chromatographic peaks are basically the same after extraction with different solvent volumes. However, the response values ​​of each peak in the chromatogram extracted with 25 ml of solvent are more suitable and the peak shapes are better. Therefore, 25 ml of solvent was selected.

[0074] 2.3 Examination of extraction time

[0075] Ultrasonic extraction was used to compare the effects of different extraction times (15, 30, and 45 min) on the feature maps. The results are as follows: Figure 3 As shown in the figure. The results indicate that the chromatogram shapes and the number of chromatographic peaks are basically the same at different extraction times, and the peak response values ​​at different extraction times are not significantly different. To ensure complete extraction and save time, an extraction time of 30 min was selected.

[0076] 2.4 Investigation of Extraction Solvents

[0077] Different solvents have varying dissolution abilities for the active ingredients in the test samples. Therefore, the extraction effects of solvents with different polarities (water, 30% ethanol, 50% ethanol, 75% ethanol, ethanol, 30% methanol, 50% methanol, 75% methanol, and methanol) on the test samples were investigated. The optimal extraction solvent was selected based on the characteristic features of the chromatographic peaks, the number of peaks, and the absorption intensity. The results are as follows: Figure 4 , Figure 5 As shown. By Figure 5 It can be seen that the ethanol series affects peaks 2, 3, 4, 5, 9, and 10; the higher the purity, the greater the influence. Figure 4 It can be seen that there is no significant difference between the chromatograms of water as the extraction solvent and those of 30%–50% methanol. However, water has no solvent peak, a higher response value, and a better peak shape. Considering cost savings, water is used as the extraction solvent.

[0078] 3. Methodological Validation

[0079] The determination was performed according to the ultra-high performance liquid chromatography detection method proposed in section 1 above.

[0080] In this study, gallic acid was used as reference peak 1, and the relative retention times and relative peak areas of common characteristic peaks 2 to 6 and the reference peak were calculated; ellagic acid was used as reference peak 2, and the relative retention times and relative peak areas of common characteristic peaks 7 to 14 and the reference peak were calculated.

[0081] 3.1 Specificity test

[0082] Take 0.2 g of excipient (maltodextrin) and prepare a negative sample solution according to the test sample method under "1. Proposed Ultra-High Performance Liquid Chromatography Detection Method". Inject 1 μL each of the negative sample solution, the test sample solution of the Sanguisorba officinalis formula granules, and the reference solution into the liquid chromatograph. Analyze under the chromatographic conditions suitable for the system's suitability. The results are shown in the table below. Figure 6 The results showed that the negative sample had no corresponding chromatographic peak at the corresponding position of the analyte in the test sample, indicating that the excipients did not interfere with the detection and that the method has good specificity.

[0083] 3.2 Precision Test

[0084] Take the 0-hour test solution from the stability study and inject it six times consecutively to test the instrument's precision. The results are shown in [Figure number missing]. Figure 7 See Table 2. The results show that the relative retention time (RSD) of each characteristic peak measured after six consecutive injections was less than 3.0%, indicating that the instrument has good precision.

[0085] Table 2. Relative retention time and relative peak area in precision test

[0086]

[0087]

[0088] 3.3 Stability Test

[0089] The *Sanguisorba officinalis* sample solution was prepared according to the method described in section "1. Proposed Ultra-High Performance Liquid Chromatography Detection Method". The stability of the sample solution was investigated at 0, 2, 4, 6, 8, 12, 16, 20, and 24 hours. The results are shown in [Figure number missing]. Figure 8 See Table 3. The relative retention times (RSD) of each characteristic peak were less than 3.0%, indicating that the test solution had good stability within 24 hours.

[0090] Table 3. Relative retention time and relative peak area in stability tests

[0091]

[0092]

[0093] 3.4 Repeatability Test

[0094] Six solutions of Sanguisorba officinalis were prepared according to the method described in section 1. "Proposed Ultra-High Performance Liquid Chromatography Detection Method," and the results were analyzed to examine the repeatability of the experimental method. The results are shown in [Figure Number]. Figure 9 See Table 4. The relative retention times (RSD) of each characteristic peak were less than 3.0%, indicating good repeatability.

[0095] Table 4. Relative retention time and relative peak area in repeatability tests

[0096]

[0097]

[0098] 3.5 Durability Test

[0099] Take a batch of samples, grind them finely, weigh 0.2 g, and prepare the Sanguisorba officinalis test solution according to the method under "1. Proposed Ultra-High Performance Liquid Chromatography Detection Method". Based on the original chromatographic conditions, change the column temperature, flow rate, and different brands of chromatographic columns to determine the robustness of the chromatographic conditions. Statistically record the elution times of each characteristic peak, and calculate the relative retention time and relative peak area (RSD%). The results are shown in [Table missing]. Figures 10-12 See Table 5. The results show that at column temperatures of 40-42℃ and flow rates of 0.29-0.31 ml / min, there were no significant differences in the relative retention times and relative peak areas of the common peaks, indicating good robustness. Different brands of columns, due to variations in the bonding of C18 packing material, had little impact on relative retention times but significantly affected relative peak areas. Therefore, Waters columns are recommended. T3 (2.1*150mm, 1.6μm).

[0100] Table 5. Relative retention times and relative peak areas in durability tests

[0101]

[0102]

[0103] 3.6 Intermediate Precision Test

[0104] Take a batch of samples, grind them finely, weigh 0.2g, and prepare the Sanguisorba officinalis test solution according to the method under "2. Proposed Ultra-High Performance Liquid Chromatography Detection Method". Based on the original chromatographic conditions, use the same brand and specifications of chromatographic columns, and different instruments for injection to investigate the intermediate precision of the method. Count the elution times of each peak, and calculate the RSD values ​​of the relative retention times and relative peak areas. The results are shown in [Figure number missing]. Figure 13See Table 6. The results show that the relative retention time and relative peak area of ​​each common peak are significantly affected by different instrument brands (RSD > 5%), but the relative retention time still fluctuates within ±10% of the standard decoction's specified value, while the relative peak area RSD > 10%. For instruments of the same brand, the relative retention time still fluctuates within ±10% of the average value of 23 batches of *Sanguisorba officinalis* granules, and the relative peak area RSD < 10%. The Agilent 1290DAD instrument's chromatogram shows that peaks 3 and 4 cannot be separated compared to the Waters H-Plus chromatogram. To ensure good separation and peak shape of each characteristic peak, it is recommended to consistently use the Waters H-Plus instrument brand.

[0105] Table 6. Relative retention times and relative peak areas in intermediate precision tests

[0106]

[0107]

[0108] Example 2: Construction of the characteristic spectrum of Sanguisorba officinalis (Diyu) formulation granules

[0109] The characteristic spectra of 23 batches of *Sanguisorba officinalis* (Diyu) formulation granules were determined using the ultra-high performance liquid chromatography (UHPLC) method proposed in Example 1. Based on the principles of stable relative retention times and detectability in all batches with relatively high peak values, 14 peaks with good repeatability were selected as characteristic peaks. The peak corresponding to the gallic acid reference peak was designated as peak S1. The relative retention times and relative peak areas of peaks 2, 3, 4, 5, and 6 with respect to peak S1 were calculated. The peak corresponding to the ellagic acid reference peak was designated as peak S2. The relative retention times and relative peak areas of peaks 7, 8, 9, 10, 11, 13, and 14 with respect to peak S2 were calculated, as shown in Tables 7 and 8.

[0110] The 2012 version of the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System was used to synthesize 23 batches of Sanguisorba officinalis (Diyu) formula granules, and a reference chromatogram of the characteristic chromatogram of Sanguisorba officinalis (Diyu) formula granules was established, such as... Figure 14 As shown.

[0111] Table 7. Relative retention times of 23 batches of Sanguisorba officinalis (Diyu) formulation granules

[0112]

[0113]

[0114] Table 8. Relative peak area of ​​granules from 23 batches of Sanguisorba officinalis (Diyu) formulation.

[0115]

[0116]

[0117] Example 3: Construction of the characteristic spectrum of long-leaved Sanguisorba officinalis (Sanguisorba officinalis) formulation granules

[0118] The characteristic spectra of 18 batches of *Sanguisorba officinalis* (long-leaved *Sanguisorba officinalis*) formulation granules were determined using the ultra-high performance liquid chromatography (UHPLC) method proposed in Example 1. Based on the principles of stable relative retention times and detectability in all batches with relatively high peaks, 14 peaks with good repeatability were selected as characteristic peaks. The peak corresponding to the gallic acid reference peak was designated as peak S1. The relative retention times and relative peak areas of peaks 2, 3, 4, 5, and 6 with respect to peak S1 were calculated. The peak corresponding to the ellagic acid reference peak was designated as peak S2. The relative retention times and relative peak areas of peaks 7, 8, 9, 10, 11, 13, and 14 with respect to peak S2 were calculated. The results are shown in Tables 9 and 10.

[0119] The 2012 version of the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System was used to synthesize 18 batches of *Sanguisorba officinalis* (Diyu) formula granules, and a reference chromatogram of the characteristic chromatogram of *Sanguisorba officinalis* (Diyu) formula granules was established. For example... Figure 15 As shown.

[0120] Table 9. Relative retention times of 18 batches of Sanguisorba officinalis (Sanguisorba longifolia) formulation granules

[0121]

[0122]

[0123] Table 10. Relative Peak Area of ​​18 Batches of Sanguisorba officinalis (Sanguisorba longifolia) Formula Granules

[0124]

[0125]

[0126] Example 4: Identification of Common Characteristic Peaks

[0127] For the unknown characteristic peaks, UHPLC-Q-TOF MS high-resolution mass spectrometry identification and secondary mass spectrometry result analysis were performed, and the results were confirmed with reference standards. Ultimately, peak 1 was identified as gallic acid, peak 2 as gallocatechin, peak 6 as catechin, peak 12 as ellagic acid, and peak 14 as 2,3,8-tri-O-methylellagic acid. Figure 16 As shown.

[0128] Example 5: Establishment of Limits for Relative Retention Time

[0129] Based on the methodological investigation items and validation results, it was finally determined that: the chromatogram of the test sample should show 14 characteristic peaks, and the retention times should correspond to the 14 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peaks 1, 6, and 12 should correspond to the retention times of the reference peaks of gallic acid, catechin, and ellagic acid. The peak corresponding to the gallic acid reference peak is peak S1. The relative retention times of peaks 2, 3, 4, and 5 with peak S1 should be calculated, and their relative retention times should be within ±10% of the specified values, which are 1.990 (peak 2), 2.898 (peak 3), 2.974 (peak 4), and 3.527 (peak 5). The peak corresponding to the ellagic acid reference peak is peak S2. Calculate the relative retention times of peaks 7, 8, 9, 10, 11, 13, and 14 with peak S2. The relative retention times should be within ±10% of the specified values, which are 0.644 (peak 7), 0.727 (peak 8), 0.851 (peak 9), 0.936 (peak 10), 0.953 (peak 11), 1.034 (peak 13), and 1.506 (peak 14).

[0130] Example 6: Identification of Sanguisorba officinalis (Sanguisorba officinalis) Formula Granules and Sanguisorba officinalis (Sanguisorba longifolia) Formula Granules

[0131] From the data in Tables 8 and 10, we can see that:

[0132] (1) The main difference between Sanguisorba officinalis (Sanguisorba officinalis) granules and Sanguisorba officinalis (Sanguisorba longifolia) granules is the relative peak area of ​​peak 14. Based on the actual range of values, the relative peak area limit of Sanguisorba officinalis (Sanguisorba officinalis) granules is defined as 0.022 to 0.148, with the lower limit being the average peak area ratio of peak 14 to peak 12 (S2) of 23 batches of Sanguisorba officinalis (Sanguisorba officinalis) formulation granules minus 2 times SD and the upper limit being the average peak area ratio of peak 14 to peak 12 (S2) of +3 times SD. The relative peak area limit of Sanguisorba officinalis (Sanguisorba officinalis) granules is defined as 0 to 0.019, with the lower limit being the average peak area ratio of peak 14 to peak 12 (S2) of 18 batches of Sanguisorba officinalis (Sanguisorba longifolia) formulation granules minus 1 times SD and the upper limit being the average peak area ratio of peak 14 to peak 12 (S2) of +2.5 times SD.

[0133] (2) The actual range of the relative peak area of ​​peak 14 in 18 batches of Sanguisorba officinalis (Sanguisorba long-leaf) formulation granules is 0 to 0.015 (within the specified range of 0 to 0.019), and the actual range of the relative peak area of ​​peak 14 in 23 batches of Sanguisorba officinalis (Sanguisorba officinalis) granules is 0.045 to 0.128 (within the specified range of 0.022 to 0.148), which can distinguish them from Sanguisorba officinalis (Sanguisorba officinalis) formulation granules and Sanguisorba officinalis (Sanguisorba long-leaf) formulation granules.

[0134] Comparative Example

[0135] The test solutions of Sanguisorba officinalis and Sanguisorba longifolia were prepared using the method described in "1. Proposed Ultra-High Performance Liquid Chromatography Detection Method" in Example 1. Based on the original chromatographic conditions, different detection wavelengths (230 nm for all time and 245 nm for all time) were used to test the chromatograms of the test solutions. The results are shown in [Figure 1]. Figures 17-20 .Depend on Figure 17 and Figure 19 It can be seen that when using a 230nm wavelength for detection throughout the entire time period, the characteristic peak response value is relatively low and the baseline drift is severe after 16 minutes; Figure 18 and Figure 20 It can be seen that when a wavelength of 245nm is used for detection throughout the entire time period, the characteristic peak response value is relatively low before 16 minutes. In summary, it can be seen that the detection wavelength of the present invention (225-235nm for 0-16 minutes, and 240-250nm for 16.1-50 minutes) provides more comprehensive chromatographic peak information and a more stable chromatographic baseline.

[0136] In summary, the method of the present invention can effectively identify Sanguisorba officinalis (Sanguisorba officinalis) formula granules and Sanguisorba officinalis (Sanguisorba longifolia) formula granules, better clarify the differences between traditional Chinese medicines of different origins, facilitate quality control of Sanguisorba officinalis formula granules, and make it easier to use traditional Chinese medicine more accurately.

[0137] It should be noted that the present invention has conducted a large number of research experiments on chromatographic conditions in a long-term and extensive experimental study, but these cannot be described in detail here. The above only uses typical experiments as an illustration.

[0138] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for constructing a characteristic map of Sanguisorba officinalis granule formulation, characterized in that, Includes the following steps: (1) Preparation of reference solution: Take Sanguisorba officinalis reference material and extract it with water to obtain reference solution of reference material; take gallic acid, catechin and ellagic acid, add methanol to make a mixed solution to obtain reference solution of reference material; (2) Preparation of test solution: Take the Sanguisorba officinalis formula granules and extract them with a solvent, wherein the solvent is water or 30%~50% methanol, to obtain the test solution; (3) The reference solution and the test solution were determined by high performance liquid chromatography, and the corresponding spectra of the reference solution and the test solution were compared to obtain the characteristic spectra of the Sanguisorba officinalis formula granules; The chromatographic conditions for the high-performance liquid chromatography method are as follows: A chromatographic column with octadecylsilane-bonded silica gel as the packing material was used. The column length was 150 mm, the inner diameter was 2.1 mm, and the particle size was 1.6 μm. The detection wavelength was 225–235 nm for 0–16 min and 240–250 nm for 16.1–50 min. Gradient elution was performed using acetonitrile as mobile phase A and 0.15% formic acid solution as mobile phase B. The gradient elution specifically refers to: 0~3min, Phase A: 0~3.5%, Phase B: 100~96.5%; 3~7.5 min, Phase A: 3.5%, Phase B: 96.5%; 7.5~15min, Phase A: 3.5~8%, Phase B: 96.5~92%; 15~22min, Phase A: 8~10%, Phase B: 92~90%; 22~24min, Phase A: 10~11%, Phase B: 90~89%; 24~26 min, Phase A: 11~13%, Phase B: 89~87%; 26~35min, Phase A: 13~18%, Phase B: 87~82%; 35~38min, Phase A: 18~30%, Phase B: 82~70%; 38~50min, Phase A: 30~33%, Phase B: 70~67%; The chromatogram of the test sample showed 14 characteristic peaks: peak 1 was gallic acid, peak 2 was gallocatechin, peak 6 was catechin, peak 12 was ellagic acid, and peak 14 was 2,3,8-tri-O-methylellagic acid.

2. The method for constructing the characteristic map of Sanguisorba officinalis formula granules according to claim 1, characterized in that, In step (1), the preparation of the reference solution specifically includes: taking the reference medicinal material of Sanguisorba officinalis, adding water, with the ratio of reference medicinal material to water being 0.1g:5~12mL, heating under reflux or sonication for 15~45min, filtering, and taking the filtrate as the reference solution of the reference medicinal material; separately taking gallic acid, catechin, and ellagic acid, adding 50~70% methanol to prepare a mixed solution containing 30~40μg of each per 1mL, which is the reference solution of the reference substance.

3. The method for constructing the characteristic map of Sanguisorba officinalis formula granules according to claim 1, characterized in that, In step (2), the preparation of the test solution specifically includes: taking the Sanguisorba officinalis formula granules, adding solvent, the ratio of formula granules to solvent being 0.2g:15~50mL, heating under reflux or ultrasonic treatment for 15~45min, filtering, and taking the filtrate to obtain the solution.

4. The method for constructing the characteristic map of Sanguisorba officinalis formula granules according to claim 1, characterized in that, Diode array detector, column temperature 40℃~42℃; flow rate 0.29~0.31mL / min.

5. The method for constructing the characteristic map of Sanguisorba officinalis formula granules according to claim 4, characterized in that, Detection wavelength: 230nm for 0~16min, 245nm for 16.1~50min; Column temperature 40℃, flow rate 0.3ml / min.

6. The method for constructing the characteristic map of Sanguisorba officinalis formula granules according to claim 1, characterized in that, Using gallic acid peak as reference peak S1, calculate the relative retention times of peaks 2, 3, 4, and 5 with peak S1. The relative retention times should be within ±10% of the specified values, which are: 1.990 for peak 2, 2.898 for peak 3, 2.974 for peak 4, and 3.527 for peak 5. Using the ellagic acid peak as the reference peak S2, calculate the relative retention times of peaks 7, 8, 9, 10, 11, 13, and 14 with peak S2. The relative retention times should be within ±10% of the specified values, which are 0.644 for peak 7, 0.727 for peak 8, 0.851 for peak 9, 0.936 for peak 10, 0.953 for peak 11, 1.034 for peak 13, and 1.506 for peak 14.

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