Method for constructing the fingerprint of shuangling zhi xie granules and quality detection

Abstract

This invention belongs to the field of traditional Chinese medicine preparation detection technology, specifically relating to a method for constructing a fingerprint spectrum of Shuangling Zhixie granules and performing quality detection. Utilizing ultra-high performance liquid chromatography (UHPLC), specifically, it includes using octadecylsilane-bonded silica gel as the column packing material, acetonitrile as mobile phase A, and phosphoric acid solution as mobile phase B, performing gradient elution, and using a UV detector to detect and obtain chromatograms. The chromatograms are then processed using fingerprinting software to obtain fingerprint spectra of multiple batches of Shuangling Zhixie granules. The fingerprint spectra of Shuangling Zhixie granules are then imported into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and after multi-point correction, a control fingerprint spectrum of Shuangling Zhixie granules is generated using the average method. The fingerprint spectrum of the Shuangling Zhixie granule sample to be tested is compared with the control fingerprint spectrum of Shuangling Zhixie granules, and the similarity is calculated. This method can more comprehensively and effectively evaluate and control the quality of Shuangling Zhixie granules; the method is simple, stable, and has good repeatability.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine preparation detection technology, specifically relating to a method for constructing a fingerprint spectrum of Shuangling Zhixie Granules and performing quality detection. Background Technology

[0002] Shuangling Antidiarrheal Granules are an improved version of Shuangling Antidiarrheal Oral Liquid. Its ingredients include nine traditional Chinese medicines: Scutellaria baicalensis, Atractylodes macrocephala, Poria cocos, Polyporus umbellatus, Dryopteris crassirhizoma, Pinellia ternata, Citrus reticulata peel, Sanguisorba officinalis, and Cinnamomum cassia. These herbs have the effects of clearing heat and dampness, strengthening the spleen, and stopping diarrhea. This product is mainly used for infantile diarrhea caused by damp-heat accumulation and spleen deficiency, and it has a good therapeutic effect on diarrhea caused by rotavirus enteritis.

[0003] The current quality standard for Shuangling Zhixie Oral Liquid uses thin-layer chromatography (TLC) to identify the four medicinal materials (Scutellaria baicalensis, Atractylodes macrocephala, Citrus reticulata peel, and Cinnamomum cassia) and their main components, and high-performance liquid chromatography (HPLC) to determine the content of baicalin. In existing technology, Wang Jianyong, in his research "Research on Quality Control Methods for Shuangling Zhixie Granules," explored quality control methods for Shuangling Zhixie Granules. He used TLC to qualitatively identify Scutellaria baicalensis, Atractylodes macrocephala, Cinnamomum cassia, and Citrus reticulata peel in Shuangling Zhixie Granules, and used HPLC to determine the content of baicalin in Scutellaria baicalensis. However, this method is difficult to effectively control other components in Shuangling Zhixie Granules such as Sanguisorba officinalis, Poria cocos, Polyporus umbellatus, and Pinellia ternata. The complexity of multiple components in traditional Chinese medicine means that a single component cannot accurately represent the intrinsic quality of the medicine.

[0004] Traditional Chinese medicine (TCM) fingerprinting is a comprehensive and quantifiable identification method, and one of the current quality control models that aligns with the characteristics of TCM in evaluating its authenticity, stability, and consistency. Fingerprinting can comprehensively reflect the components contained in TCM and can characterize the overall quality of TCM products. However, the use of TCM chromatographic fingerprinting as a quality control method for Shuangling Zhixie Granules has not yet been reported. Summary of the Invention

[0005] In view of this, the present invention proposes a fingerprint chromatographic detection method for Shuangling Zhixie Granules based on ultra-high performance liquid chromatography (UPLC), which can more comprehensively and effectively control the quality of Shuangling Zhixie Granules and provide a scientific basis for further exploration and research on the quality of Shuangling Zhixie Granules.

[0006] One of the objectives of this invention is to provide a method for detecting the fingerprint spectrum of Shuangling Zhixie Granules using ultra-high performance liquid chromatography. This method has high specificity and good repeatability, providing technical support for the quality evaluation of Shuangling Zhixie Granules.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for detecting the fingerprint chromatogram of Shuangling Zhixie Granules using ultra-high performance liquid chromatography (UHPLC) is disclosed. The UHPLC method employs octadecylsilane-bonded silica gel as the column packing material, uses mobile phases A and B for gradient elution, and utilizes an ultraviolet (UV) detector to obtain the chromatogram. The chromatogram is then processed using fingerprinting software to obtain the fingerprint chromatogram of Shuangling Zhixie Granules. Mobile phase A is acetonitrile, and mobile phase B is a phosphoric acid solution. The detection wavelength of the UV detector is 200-205 nm.

[0009] Furthermore, the components of the Shuangling Antidiarrheal Granules include Scutellaria baicalensis, Atractylodes macrocephala, Poria cocos, Polyporus umbellatus, Dryopteris crassirhizoma, Pinellia ternata, Citrus reticulata peel, Sanguisorba officinalis, and Cinnamomum cassia.

[0010] Furthermore, the concentration of the phosphoric acid solution is 0.03 vol% - 0.08 vol%, preferably 0.05 vol%.

[0011] Furthermore, the washing time is 78 minutes.

[0012] Furthermore, the gradient elution procedure is as follows:

[0013] For 0-60 min, the volume fraction of mobile phase A is set to 0-26%, and the volume fraction of mobile phase B is set to 100%-74%.

[0014] Set the volume fraction of mobile phase A to 26%-50% and the volume fraction of mobile phase B to 74%-50% for 60-68 minutes.

[0015] Set the volume fraction of mobile phase A to 50%-90% and the volume fraction of mobile phase B to 50%-10% for 68-75 minutes.

[0016] Set the mobile phase A to 90% volume fraction and the mobile phase B to 10% volume fraction for 75-78 minutes.

[0017] Furthermore, the flow rate of the mobile phase is 0.1-0.3 mL / min; the column temperature is 40±2℃.

[0018] Preferably, the flow rate of the mobile phase is 0.2 mL / min, and the column temperature is 40 °C.

[0019] As a preferred option, the injection volume is 1 μL.

[0020] As a preferred option, the chromatographic column uses T3C18 chromatographic column.

[0021] Preferably, the column length is 100 mm, the inner diameter is 2.1 mm, and the packing particle size is 1.6 μm.

[0022] Preferably, the detection wavelength is 203nm.

[0023] Furthermore, the fingerprinting software is a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine.

[0024] Furthermore, the fingerprint spectrum of the product solution to be tested is imported into the Chinese medicine chromatographic fingerprint spectrum similarity evaluation system. After multi-point correction, Mark peak matching is performed to obtain the fingerprint spectrum of the product solution to be tested.

[0025] Furthermore, before performing high performance liquid chromatography analysis, the sample to be tested was prepared using methanol with a concentration of 70 vol% as a solvent.

[0026] Furthermore, the fingerprint spectrum includes 18 characteristic peaks. Taking peak 11 as the reference peak, the relative retention times of peak 1 and the reference peak are 0.063–0.077, peak 2 and the reference peak are 0.081–0.099, peak 3 and the reference peak are 0.162–0.198, peak 4 and the reference peak are 0.261–0.319, peak 5 and the reference peak are 0.522–0.638, peak 6 and the reference peak are 0.693–0.847, peak 7 and the reference peak are 0.729–0.891, peak 8 and the reference peak are 0.747–0.913, peak 9 and the reference peak are 0.765–0.935, and peak 10 and the reference peak are 0.819–1.00. 1. The relative retention times of peak 11 and the reference peak are 1.00; peak 12 and the reference peak are 1.008–1.232; peak 13 and the reference peak are 1.026–1.254; peak 14 and the reference peak are 1.062–1.298; peak 15 and the reference peak are 1.161–1.419; peak 16 and the reference peak have relative retention times of 1.008–1.232. The relative retention times of peaks 17 and 18 were 1.269–1.551, 1.287–1.573, and 1.422–1.738, respectively. Among them, peak 2 was gallic acid, peak 8 was naringin, peak 9 was kaempferol, peak 10 was hesperidin, peak 11 was baicalin, peak 14 was wogonin, peak 15 was baicalein, peak 16 was wogonin, and peak 18 was limonene.

[0027] The components in Shuangling Zhixie Granules can be qualitatively identified based on the relative retention times of 18 characteristic peaks. Simultaneously, the aforementioned method can be used to separate the 18 characteristic peaks in Shuangling Zhixie Granules; the separation time for these 18 characteristic peaks is 78 minutes.

[0028] Furthermore, the consistency of the relative retention behavior of the 18 characteristic peaks in the fingerprint chromatograms of the tested sample and the reference sample was used to determine whether the product contained the 18 characteristic peaks, including the 9 identified chromatographic peaks: peak 2 is gallic acid, peak 8 is rutin, peak 9 is kaempferol, peak 10 is hesperidin, peak 11 is baicalin, peak 14 is wogonin, peak 15 is baicalein, peak 16 is wogonin, and peak 18 is limonene.

[0029] The second objective of this invention is to provide a method for quality testing of Shuangling Zhixie Granules based on the aforementioned method.

[0030] To achieve the above objectives, the present invention adopts the following technical solution:

[0031] A method for quality testing of Shuangling Zhixie Granules based on the aforementioned method, the method comprising the following steps:

[0032] (1) The fingerprint spectrum was obtained by detecting the Shuangling Zhixie Granules sample and the reference standard using the aforementioned method;

[0033] (2) Import the fingerprint spectrum of the test sample and the fingerprint spectrum of the reference sample into the similarity evaluation system of Chinese medicine chromatographic fingerprint spectrum and calculate the similarity; judge the quality stability of Shuangling Zhixie Granules by similarity.

[0034] Furthermore, if the similarity is ≥0.90, it indicates that the Shuangling Zhixie Granules sample is of qualified quality; if the similarity is <0.90, it indicates that the sample is of unqualified quality.

[0035] Furthermore, the comparative fingerprint spectrum is generated by importing multiple batches of the test product solution into the Chinese medicine chromatographic fingerprint spectrum similarity evaluation system, performing Mark peak matching after multi-point correction, and using the mean method; the time window used in the mean method is 0.1 min.

[0036] As a preferred technical solution, the method includes the following steps:

[0037] Step 1. Take a sample of Shuangling Zhixie Granules, grind it into a fine powder, take about 0.5g, weigh it accurately, place it in a stoppered conical flask, accurately add 25mL of 70vol% methanol, weigh it, sonicate it for 30min, cool it, replenish the lost weight with 70vol% methanol, shake it well, centrifuge it at high speed, and take the supernatant as the test sample solution; repeat this method to prepare 10 batches of test sample solutions.

[0038] Step 2. Take appropriate amounts of gallic acid, hesperidin, and baicalin reference standards, and add 70 vol% methanol to prepare a solution containing 40 μg gallic acid, 50 μg hesperidin, and 350 μg baicalin per 1 mL, which will be used as the reference solution.

[0039] Step 3. Take 1 μL each of the 10 batches of sample solutions prepared in Step 1 and the reference solution prepared in Step 2, and inject them into the liquid chromatograph, recording the chromatograms. The chromatographic conditions are: mobile phase A is acetonitrile, mobile phase B is a 0.05 vol% phosphoric acid solution; the chromatographic column is... The chromatographic column is a T3C18, 100 mm in length and 2.1 mm in inner diameter, with a packing particle size of 1.6 μm; the flow rate is 0.2 mL / min; the column temperature is 40 °C; the injection volume is 1 μL; the detection wavelength is 203 nm; and the gradient elution program is as follows:

[0040] For 0-60 min, the volume fraction of mobile phase A is set to 0-26%, and the volume fraction of mobile phase B is set to 100%-74%.

[0041] Set the volume fraction of mobile phase A to 26%-50% and the volume fraction of mobile phase B to 74%-50% for 60-68 minutes.

[0042] Set the volume fraction of mobile phase A to 50%-90% and the volume fraction of mobile phase B to 50%-10% for 68-75 minutes.

[0043] Set the mobile phase A to 90% volume fraction and the mobile phase B to 10% volume fraction for 75-78 minutes.

[0044] Step 4. Import the chromatogram of the sample solution obtained in Step 3 into the Chinese herbal chromatographic fingerprint similarity evaluation system. After multi-point correction, Mark peak matching is performed, and the reference fingerprint is generated using the mean method.

[0045] Step 5. Import the fingerprint spectrum of the sample solution to be tested and the reference fingerprint spectrum into the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint spectrum. After multi-point correction, Mark peak matching is performed to obtain the fingerprint spectrum of the product solution to be tested, and the similarity is calculated. If the similarity is ≥0.90, it indicates that the quality of Shuangling Zhixie Granules sample is qualified.

[0046] The beneficial effects of this invention are as follows:

[0047] 1. The fingerprint spectrum detection method for Shuangling Zhixie Granules provided by this invention can simultaneously separate 18 characteristic peaks (including 9 identified components: gallic acid, rutin, kaempferol, hesperidin, baicalin, wogonin, baicalein, wogonin and limonene) in Shuangling Zhixie Granules within 78 minutes, and perform qualitative analysis on multiple pharmacodynamic components of multiple medicinal materials in Shuangling Zhixie Granules. It has the characteristics of strong specificity, high precision, good repeatability and good stability.

[0048] 2. The fingerprint spectrum detection method for Shuangling Zhixie Granules provided by this invention can more comprehensively and effectively control the quality of Shuangling Zhixie Granules and ensure the uniformity and stability of product quality; at the same time, it provides a scientific basis for further exploration and research on the quality of this preparation. Attached Figure Description

[0049] Figure 1 The UPLC spectrum (gradient 1) of the Shuangling Zhixie Granules sample in Example 1 at a detection wavelength of 203 nm;

[0050] Figure 2 The UPLC spectrum (gradient 1) of the Shuangling Zhixie Granules sample in Example 1 at a detection wavelength of 280 nm is shown.

[0051] Figure 3 The UPLC spectrum of the Shuangling Zhixie Granules sample in Example 1, eluted under gradient condition 2;

[0052] Figure 4 The UPLC spectrum of the Shuangling Zhixie Granules sample in Example 2, using water as the extraction solvent;

[0053] Figure 5 The UPLC spectrum of the Shuangling Zhixie Granules sample in Example 2, using 30% vol methanol as the extraction solvent;

[0054] Figure 6 The UPLC spectrum of the Shuangling Zhixie Granules sample in Example 2, using 50% vol methanol as the extraction solvent;

[0055] Figure 7 The UPLC spectrum of the Shuangling Zhixie Granules sample in Example 2, using 70% vol methanol as the extraction solvent;

[0056] Figure 8 The UPLC spectrum of the Shuangling Zhixie Granules sample in Example 2, using methanol as the extraction solvent;

[0057] Figure 9 These are UPLC spectra extracted by ultrasound at different temperatures in Example 2;

[0058] Figure 10 UPLC diagrams of 10 batches of samples that participated in the establishment of the common mode in Example 4;

[0059] Figure 11 This is the fingerprint chromatogram of Shuangling Zhixie Granules in Example 4;

[0060] Figure 12 This is a UPLC comparison image of the Shuangling Zhixie Granules sample and the blank solvent in Example 4;

[0061] Figure 13This is a UPLC comparison image of the Shuangling Zhixie Granules sample and Scutellaria baicalensis in Example 4;

[0062] Figure 14 This is a UPLC comparison image of the Shuangling Zhixie Granules sample and the tangerine peel in Example 4;

[0063] Figure 15 This is a UPLC comparison image of the Shuangling Zhixie Granules sample and the Diyu sample in Example 4;

[0064] Figure 16 This is a UPLC comparison image of the Shuangling Zhixie Granules sample and Guanzhong in Example 4;

[0065] Figure 17 This is a UPLC comparison image of the Shuangling Zhixie Granules sample and cinnamon in Example 4;

[0066] Figure 18 This is a UPLC comparison image of the Shuangling Zhixie Granules sample and Atractylodes macrocephala in Example 4;

[0067] Figure 19 This is a UPLC comparison image of the Shuangling Zhixie Granules sample and Banxia in Example 4;

[0068] Figure 20 This is a UPLC comparison image of the Shuangling Zhixie Granules sample and Poria cocos in Example 4;

[0069] Figure 21 This is a UPLC comparison image of the Shuangling Zhixie Granules sample and Zhuling in Example 4;

[0070] Figure 22 This is a UPLC comparison image of the Shuangling Zhixie granules sample and the volatile oil inclusion complex in Example 4;

[0071] Figure 23 This is a UPLC comparison image of the Shuangling Zhixie Granules sample and the Shuangling Zhixie Granules without volatile oil inclusion complex in Example 4;

[0072] Figure 24 The figures show the UPLC comparison of blank solvent, gallic acid, baicalin reference standard, wogonin, mixed reference standard, and test sample. S1 is the UPLC figure of blank solvent, S2 is the UPLC figure of gallic acid reference standard, S3 is the UPLC figure of baicalin reference standard solution, S4 is the UPLC figure of wogonin reference standard, S5 is the UPLC figure of mixed reference standard, and S6 is the UPLC figure of test sample.

[0073] Figure 25 For high-precision UPLC spectra;

[0074] Figure 26 For reproducible UPLC spectra;

[0075] Figure 27 UPLC spectra for stability. Detailed Implementation

[0076] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0077] In this embodiment of the invention, the reagent information is as follows: Acetonitrile: chromatographic grade, manufactured by Skysotech; Water: ultrapure water; Methanol: analytical grade, manufactured by Chongqing Chuandong Chemical (Group) Co., Ltd.; Gallic acid, sourced from the China National Institutes for Food and Drug Control, batch number 110831-201906; Rutin, sourced from Shanghai Standard Technology Co., Ltd., batch number 10285; Kaempferol, sourced from Shanghai Hongyong Biotechnology Co., Ltd., batch number 190070-202202; Hesperidin, sourced from the National Institutes for Food and Drug Control, batch number 110721-202019; Baicalin, sourced from the National Institutes for Food and Drug Control, batch number 110715-202223; Wogonin, sourced from the National Institutes for Food and Drug Control, batch number 112002-201702; Baicalein, sourced from the National Institutes for Food and Drug Control, batch number 111595-201808; Wogonin... The following products were supplied by the National Institutes for Food and Drug Control (NIFDC), batch number 111514-201706; limonene was supplied by the NIFDC, batch number 100470-201503; 10 batches of Shuangling Antidiarrheal Granules were supplied by Chongqing Fuling Pharmaceutical Factory Co., Ltd. of Taiji Group, batch numbers: 0523030001, 0523030002, 0523030003, 0523030004, 0523030005, 0523030006, 0523030007, 0523030008, 0523030009, 0523030010.

[0078] In this embodiment of the invention, the instrument information is as follows: the METTLER-TOLEDO XSE205DU electronic balance is manufactured by METTLER GmbH, Switzerland; and the Agilent 1290 ultra-high performance liquid chromatograph is manufactured by Agilent Technologies, USA.

[0079] In this embodiment of the invention, the methodology was studied with reference to the "Guiding Principles for Analytical Method Validation" in Volume IV of the 2020 Chinese Pharmacopoeia, including specificity, precision, repeatability and stability studies.

[0080] Example 1. Selection of chromatographic conditions

[0081] (1) Selection of detection wavelength

[0082] Using acetonitrile-0.05 vol% phosphoric acid as the mobile phase, the chromatographic behavior of Shuangling Zhixie granules was investigated under the same gradient conditions (gradient 1) at two detection wavelengths of 203 nm and 280 nm to select a suitable detection wavelength. The gradient elution program is shown in Table 1.

[0083] Table 1. Gradient Condition 1

[0084] Time (min) 0 45 56 60 63 67 77 79 Acetonitrile (%) 0 17 28 34 50 50 95 95

[0085] Results: The chromatogram is as follows Figures 1-2 As shown, the chromatographic peak information at 203nm is more abundant than that at 280nm. Therefore, 203nm was chosen as the detection wavelength.

[0086] (2) Optimization of the mobile phase elution gradient

[0087] In the selection experiment for the detection wavelength, gradient elution conditions (gradient 1) were used for preliminary screening. Figure 1 It can be seen that when using gradient 1 analysis, the distribution of each chromatographic peak in the chromatogram is not uniform. Therefore, gradient conditions are further screened based on gradient 1. Gradient condition 2 is shown in Table 2.

[0088] Table 2. Gradient Condition 2

[0089] Time (min) 0 60 68 75 78 Acetonitrile (%) 0 26 50 90 90

[0090] Results: The chromatogram is as follows Figure 3 As shown. Under gradient 2 conditions, the chromatographic peaks were well separated, which could fully reflect the overall chemical composition of Shuangling Zhixie Granules; therefore, gradient 2 was determined as the mobile phase gradient condition for fingerprint analysis of Shuangling Zhixie Granules.

[0091] In summary, the following chromatographic conditions were selected for fingerprint analysis of Shuangling Zhixie Granules: chromatographic column T3, 1.6 μm, 2.1*100 mm; using acetonitrile-0.05 vol% phosphoric acid solution as the mobile phase, elution was performed according to the gradient elution program specified in Table 2; the flow rate was 0.2 ml / min; the column temperature was 40 ℃; and the detection wavelength was 203 nm.

[0092] Example 2. Selection of sample pretreatment

[0093] Sample pretreatment selection was performed under the chromatographic conditions selected in Example 1.

[0094] (1) Solvent selection test

[0095] Take this product, grind it into a fine powder, take about 1.0g, accurately weigh 10 portions (1.0178g, 1.0187g, 1.0168g, 1.0016g, 0.9949g, 1.0091g, 1.0114g, 1.0114g, 1.0118g, 1.0171g), place them in stoppered conical flasks, add water, 30 vol% methanol, 50 vol% methanol, 70 vol% methanol, and 25ml of methanol respectively, weigh them, and sonicate (40KHz, 500W) for 60 minutes respectively. After cooling, make up the weight loss with the corresponding solvent, take an appropriate amount, centrifuge at high speed for 5 minutes, take the supernatant and filter it to obtain the product.

[0096] Result: As Figures 4-8 As shown in Table 3, the chromatographic peak information for extraction solvents of methanol, 70 vol% methanol, and 50 vol% methanol is more abundant than that for other extraction solvents. There is no significant difference in the total peak area / weight ratio between extraction solvents of 70 vol% methanol and 50 vol% methanol. Figures 6-7 It can be seen that the peak abundance at 77 minutes in the chromatogram of 70 vol% methanol as the extraction solvent is higher than that of 50 vol% methanol. Taking all factors into consideration, 70 vol% methanol is selected as the extraction solvent.

[0097] Table 3. Effect of different extraction solvents on the extraction efficiency of Shuangling Zhixie Granules

[0098] Extraction solvent water 30 vol% methanol 50 vol% methanol 70 vol% methanol methanol Total peak area / weight 61591.597 61233.832 63810.101 63579.514 41854.838

[0099] (2) Sample size selection test

[0100] Take an appropriate amount of this product, grind it into a fine powder, and accurately weigh 6 portions (0.5109g, 0.5214g, 0.8244g, 0.8043g, 1.0474g, and 1.0564g) of approximately 0.5g, 0.8g, and 1.0g respectively. Place each portion in a stoppered conical flask, add 25ml of 70vol% methanol to each flask, weigh them, and sonicate them separately (500W, 40kHz) for 60 minutes. After cooling, replenish the lost weight with 70vol% methanol, shake well, take an appropriate amount, centrifuge at high speed for 5 minutes, and filter the supernatant to obtain the final product.

[0101] Results: As shown in Table 4, the total peak area / weight ratio was higher when the sample weight was 0.5g than other sample weights. Therefore, 0.5g was selected as the sample weight.

[0102] Table 4. Effect of different weighing methods on the extraction efficiency of Shuangling Zhixie Granules

[0103]

[0104] (3) Ultrasonic temperature selection test

[0105] Take an appropriate amount of this product, grind it into a fine powder, and accurately weigh 12 portions (0.5g each, 0.5103g, 0.5015g, 0.4951g, 0.5257g, 0.5189g, 0.5096g, 0.5113g, 0.5070g, 0.4971g, 0.5347g, 0.5117g, 0.5010g) into stoppered conical flasks. Add 25ml of 70vol% methanol, weigh the flasks, and sonicate them at 20℃, 30℃, 40℃, 50℃, 60℃, and 70℃ (500W power, 40kHz frequency) for 60 minutes each. After cooling, replenish the lost weight with 70vol% methanol, shake well, take an appropriate amount, centrifuge at high speed for 5 minutes, and filter the supernatant to obtain the final product.

[0106] Result: As Figure 9 As shown in Table 5. From Figure 9 Analysis shows that the chromatograms of ultrasonic extraction at 70℃ contain less chromatographic information than those extracted at other temperatures. Table 5 shows that the average total peak area / weight ratio of ultrasonic extraction at 20℃-60℃ is 63330.244 (RSD% = 1.9%), indicating that there is no significant difference in extraction efficiency for Shuangling Zhixie Granules between 20℃ and 60℃. Therefore, considering all factors, no specific control of the ultrasonic temperature is necessary.

[0107] Table 5. Effect of ultrasound at different temperatures on the extraction efficiency of Shuangling Zhixie Granules

[0108] Ultrasonic temperature / °C 20 30 40 50 60 Total peak area / weight 62109.100 62176.420 63186.844 64441.315 64737.541

[0109] (4) Ultrasonic time selection test

[0110] Take an appropriate amount of this product, grind it into a fine powder, take about 0.5g, accurately weigh 6 portions (0.5244g, 0.5309g, 0.5109g, 0.5183g, 0.5250g, 0.5102g), place them in a stoppered conical flask, accurately add 25ml of 70vol% methanol, weigh them, and sonicate them (500W, 40kHz) for 30 minutes, 45 minutes, and 60 minutes respectively. After cooling, make up the weight loss with 70vol% methanol, shake well, take an appropriate amount, centrifuge at high speed for 5 minutes, take the supernatant and filter it to obtain the product.

[0111] Results: As shown in Table 6, the average total peak area / weighing ratio for different ultrasound times was 63475.112 (RSD = 1.5%), indicating that ultrasound time had no significant difference on the extraction efficiency of Shuangling Zhixie Granules. Considering all factors, 30 minutes was selected as the ultrasound time.

[0112] Table 6. Effect of different ultrasound times on the extraction efficiency of Shuangling Zhixie Granules

[0113]

[0114] Example 3. Determination of the characteristic spectrum analysis conditions for Shuangling Zhixie Granules

[0115] (1) Chromatographic conditions

[0116] Based on the results of Example 2, the following chromatographic conditions were selected for fingerprint analysis of Shuangling Zhixie Granules: A T3, 1.6 μm, 2.1*100 mm column was used; acetonitrile was used as mobile phase A and 0.05 vol% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the procedure specified in Table 7; the flow rate was 0.2 ml / min; the column temperature was 40 ℃; and the detection wavelength was 203 nm. The theoretical plate number, calculated based on the baicalin peak, should not be less than 50,000.

[0117] Table 7. Gradient elution program table

[0118] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0-60 0→26 100→74 60-68 26→50 74→50 68-75 50→90 50→10 75-78 90 10

[0119] (2) Prepare the sample to be tested

[0120] Preparation of reference solution: Take appropriate amounts of gallic acid, hesperidin, and baicalin reference standards, add 70 vol% methanol to prepare a solution containing 40 μg gallic acid, 50 μg hesperidin, and 350 μg baicalin per 1 mL.

[0121] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 0.5 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 70 vol% methanol, weigh it, sonicate it (500 W, 40 kHz) for 30 minutes, cool it, make up the weight loss with 70 vol% methanol, shake it well, take an appropriate amount, centrifuge it at high speed for 5 minutes, take the supernatant and filter it to obtain the test solution.

[0122] (3) Determination method

[0123] Accurately pipette 1 μl of the reference solution and the test solution into the liquid chromatograph and measure them to obtain the result.

[0124] Example 4. Establishment of UPLC fingerprint of Shuangling Zhixie Granules

[0125] (1) Establishment of UPLC fingerprint of Shuangling Zhixie Granules

[0126] UPLC fingerprinting of 10 batches of Shuangling Zhixie Granules was performed according to the method and conditions specified in Example 3. 1 μl of each sample was injected for analysis, and the chromatograms were recorded. Information on the 10 batches of Shuangling Zhixie Granules is shown in Table 8.

[0127] Table 8. Information on 10 batches of Shuangling Antidiarrheal Granules

[0128] serial number batch number serial number batch number S1 0523030001 S6 0523030006 S2 0523030002 S7 0523030007 S3 0523030003 S8 0523030008 S4 0523030004 S9 0523030009 S5 0523030005 S10 0523030010

[0129] The chromatograms of 10 batches of samples were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012.130723" software of the National Pharmacopoeia Commission to establish a common pattern. A time window width of 0.1 minutes was selected, and Mark peak matching was performed using the average. The software then generated a "reference fingerprint". The chromatograms of the 10 batches of samples participating in the establishment of the common pattern are shown below. Figure 10 .

[0130] (2) Study of common characteristic peaks

[0131] The chromatograms of the above 10 batches of Shuangling Zhixie Granules samples showed a total of 53 common peaks. Eighteen of these common fingerprint peaks, with larger peak areas and better resolution, were selected. See details below. Figure 11 Peak 11, corresponding to the baicalin reference standard, was identified as the reference peak S. Figure 11 In the middle, peak 2 is gallic acid; peak 8 is rutin; peak 9 is kaempferol; peak 10 is hesperidin; peak 11(S) is baicalin; peak 14 is wogonin; peak 15 is baicalein; peak 16 is wogonin; and peak 18 is limonene.

[0132] The matching results of common characteristic peaks are shown in Tables 9-1 and 9-2. The relative retention times and relative peak area ratios of the characteristic peaks are shown in Tables 10 and 11. As shown in Table 10, the RSD of the relative retention time ratios of the 18 characteristic peaks is less than 2%. The average relative retention time of the common characteristic peaks in the 10 batches of Shuangling Zhixie Granules samples is taken as the specified value of relative retention time. The specified values ​​of the relative retention time between peaks 1 to 18 and peak S are: 0.07 (peak 1), 0.09 (peak 2), 0.18 (peak 3), 0.29 (peak 4), 0.58 (peak 5), 0.77 (peak 6), 0. 81 (peak 7), 0.83 (peak 8), 0.85 (peak 9), 0.91 (peak 10), 1.00 (peak 11), 1.12 (peak 12), 1.14 (peak 13), 1.18 (peak 14), 1.29 (peak 15), 1.41 (peak 16), 1.43 (peak 17), 1.58 (peak 18); As shown in Table 11, the relative peak area ratios of the common characteristic peaks in each batch of samples vary greatly (RSD: 0.4% to 29.5%), all greater than 2%. Although they all have fingerprint spectral characteristics, the relative peak area ratios change irregularly.

[0133] Table 9-1. Matching results of retention time and peak area of ​​common characteristic peaks in 10 batches of Shuangling Zhixie Granules.

[0134]

[0135] Table 9-2. Matching results of retention time and peak area of ​​common characteristic peaks in 10 batches of Shuangling Zhixie Granules.

[0136]

[0137]

[0138] Table 10. Statistical table of relative retention times of common characteristic peaks in 10 batches of Shuangling Zhixie Granules.

[0139] Peak number S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 mean RSD% 1 0.075 0.075 0.075 0.075 0.074 0.074 0.074 0.074 0.074 0.074 0.074 0.53 2 0.093 0.093 0.093 0.092 0.092 0.091 0.091 0.091 0.091 0.091 0.092 1.16 3 0.177 0.177 0.177 0.175 0.175 0.174 0.174 0.174 0.174 0.174 0.175 0.83 4 0.296 0.296 0.296 0.294 0.294 0.293 0.292 0.293 0.293 0.293 0.294 0.50 5 0.577 0.577 0.576 0.575 0.575 0.575 0.575 0.575 0.575 0.575 0.576 0.14 6 0.765 0.764 0.765 0.765 0.765 0.765 0.765 0.765 0.765 0.765 0.765 0.04 7 0.812 0.812 0.812 0.812 0.813 0.813 0.813 0.813 0.813 0.813 0.813 0.06 8 0.834 0.834 0.833 0.833 0.834 0.834 0.834 0.834 0.834 0.834 0.834 0.03 9 0.853 0.853 0.853 0.853 0.854 0.854 0.854 0.854 0.854 0.854 0.854 0.08 10 0.908 0.909 0.908 0.908 0.909 0.909 0.909 0.909 0.909 0.909 0.909 0.05 11(S) 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.00 12 1.121 1.121 1.120 1.121 1.121 1.121 1.121 1.122 1.122 1.122 1.121 0.05 13 1.142 1.142 1.142 1.143 1.143 1.144 1.144 1.144 1.145 1.144 1.143 0.08 14 1.180 1.180 1.180 1.180 1.180 1.181 1.181 1.181 1.182 1.181 1.181 0.06 15 1.291 1.291 1.290 1.290 1.290 1.291 1.291 1.292 1.291 1.292 1.291 0.05 16 1.410 1.409 1.411 1.412 1.413 1.414 1.414 1.415 1.414 1.414 1.412 0.14 17 1.427 1.426 1.428 1.429 1.430 1.431 1.431 1.432 1.432 1.432 1.430 0.15 18 1.572 1.572 1.574 1.575 1.576 1.578 1.578 1.579 1.578 1.578 1.576 0.16

[0140] Table 11. Statistical table of relative peak areas of common characteristic peaks in 10 batches of Shuangling Zhixie Granules.

[0141]

[0142]

[0143] (3) Similarity calculation of 10 batches of Shuangling Zhixie Granules

[0144] The chromatograms of 10 batches of samples were imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (Version 2012.130723)" software of the National Pharmacopoeia Commission to establish control fingerprint chromatograms. A time window width of 0.1 minutes was selected, and Mark peak matching was performed based on the average. The similarity between the fingerprint chromatograms of the 10 batches of Shuangling Zhixie Granules and their control fingerprint chromatograms is shown in Table 12, and the similarity was greater than 0.90 for all of them.

[0145] Table 12. Fingerprint similarity results of 10 batches of Shuangling Zhixie Granules samples

[0146] batch number Sample number Similarity to the control fingerprint 0523030001 S1 0.997 0523030002 S2 0.997 0523030003 S3 1.000 0523030004 S4 1.000 0523030005 S5 1.000 0523030006 S6 1.000 0523030007 S7 1.000 0523030008 S8 1.000 0523030009 S9 1.000 0523030010 S10 1.000

[0147] (4) Study on the attribution of shared peaks

[0148] Following the preparation method of Shuangling Zhixie Granules, samples of single-herb medicinal slices of Sanguisorba officinalis, Citrus reticulata peel, Dryopteris crassirhizoma, Scutellaria baicalensis, Poria cocos, Polyporus umbellatus, Pinellia ternata, Cinnamomum cassia, and Atractylodes macrocephala were prepared. Single-herb medicinal slice sample solutions were prepared according to the dosage ratios specified in the Shuangling Zhixie Granules prescription and the method for treating the test sample solution. Under the planned chromatographic conditions, 1 μl each of the blank solvent, test sample solution, and single-herb medicinal slice sample solution were precisely pipetted and the chromatograms were recorded. The comparison chromatograms are shown below. Figures 12-23 As shown, the peak assignments are shown in Table 13.

[0149] Table 13. Peak Assignment Table of Characteristics of Shuangling Zhixie Granules

[0150] Classification of medicinal materials Peak Burnt Sanguisorba officinalis 1，2，4，5 Dried tangerine peel 8，10，12，17 Guanzhong 3，9 Scutellaria baicalensis 6，7，11，12，13，14，15，16，17 Poria —— Polyporus umbellatus —— Pinellia —— Atractylodes macrocephala —— Cinnamon —— Volatile oil inclusion complex 18

[0151] Example 5. Specificity Test

[0152] The Shuangling Zhixie Granules prescription consists of nine medicinal herbs: Sanguisorba officinalis, Citrus reticulata peel, Dryopteris crassirhizoma, Scutellaria baicalensis, Poria cocos, Polyporus umbellatus, Pinellia ternata, Cinnamomum cassia, and Atractylodes macrocephala. Based on the herbal attribution of characteristic peaks, gallic acid reference solution, baicalin reference solution, wogonin reference solution, a mixed reference solution, and the Shuangling Zhixie Granules test solution were prepared. The preparation method for the Shuangling Zhixie Granules test solution is as described in Example 3; the preparation methods for the other solutions are as follows:

[0153] Gallic acid reference solution: Take an appropriate amount of gallic acid reference standard, accurately weigh it, and add 70 vol% methanol to prepare a solution containing 40 μg per ml.

[0154] Baicalin reference solution: Take an appropriate amount of baicalin reference standard, accurately weigh it, and add 70 vol% methanol to prepare a solution containing 350 μg per ml.

[0155] Baicalin reference solution: Take an appropriate amount of baicalin reference standard, accurately weigh it, and add 70 vol% methanol to prepare a solution containing 80 μg per ml.

[0156] Mixed reference standards: Take appropriate amounts of naringin reference standards, kaempferol reference standards, hesperidin reference standards, baicalin reference standards, wogonin reference standards, and limonene reference standards, respectively, accurately weigh them, and add 70 vol% methanol to prepare solutions containing 30 μg of naringin, 20 μg of kaempferol, 50 μg of hesperidin, 10 μg of baicalin, 10 μg of wogonin, and 20 μg of limonene per 1 ml.

[0157] Under the prescribed chromatographic conditions, 70 vol% methanol blank solvent, reference solution, and Shuangling Zhixie Granules test solution were injected to investigate the specificity of the method. Simultaneously, nine components in the chromatogram of Shuangling Zhixie Granules were identified using the corresponding reference standards: peak 2-gallic acid, peak 8-rutin, peak 9-kaempferol, peak 10-hesperidin, peak 11(S)-baicalin, peak 14-wogonin, peak 15-baicalein, peak 16-wogonin, and peak 18-limonene. The chromatogram is shown below. Figure 24 The chromatogram shows that the blank solution exhibits no interference at the corresponding characteristic peaks, indicating good specificity.

[0158] Example 6. Precision Test

[0159] The test solution was prepared according to the method described in Example 3, and the determination was performed under the proposed chromatographic conditions. The injection was repeated 5 times. The chromatographic overlay chromatogram is shown below. Figure 25In the figure, S1 represents precision 1, S2 represents precision 2, S3 represents precision 3, S4 represents precision 4, and S5 represents precision 5. The similarity and the relative retention time and relative peak area of ​​each characteristic peak and the reference peak (S) were calculated using the Chinese herbal fingerprint similarity evaluation system. The results showed that the similarity of the chromatographic fingerprints from all five injections was greater than 0.90; the relative retention times and relative peak areas of each characteristic peak and the reference peak (S) were basically consistent, and the RSD values ​​were all less than 5%. This indicates good instrument precision. The data are shown in Tables 14 to 16.

[0160] Table 14. Calculation results of similarity in precision tests

[0161] R S1 S2 S3 S4 S5 R 1.000 1.000 1.000 1.000 1.000 1.000 S1 1.000 1.000 1.000 1.000 1.000 1.000 S2 1.000 1.000 1.000 1.000 1.000 1.000 S3 1.000 1.000 1.000 1.000 1.000 1.000 S4 1.000 1.000 1.000 1.000 1.000 1.000 S5 1.000 1.000 1.000 1.000 1.000 1.000

[0162] Table 15. Relative retention time ratios of each characteristic chromatographic peak

[0163]

[0164]

[0165] Table 16. Relative peak area ratios of each characteristic chromatographic peak

[0166]

[0167] Example 7. Repeatability Test

[0168] Prepare six test solutions according to the method described in Example 3, and determine the chromatograms under the proposed chromatographic conditions. See the chromatographic overlay diagram below. Figure 26 In the figure, S1 is the UPLC chromatogram of repeatability 1, S2 is the UPLC chromatogram of repeatability 2, S3 is the UPLC chromatogram of repeatability 3, S4 is the UPLC chromatogram of repeatability 4, S5 is the UPLC chromatogram of repeatability 5, and S6 is the UPLC chromatogram of repeatability 6. The similarity and relative retention times and relative peak areas of each characteristic peak and the reference peak (S) were calculated using the Chinese herbal fingerprint chromatogram similarity evaluation system. The results showed that the similarity of the chromatographic fingerprint chromatograms of the six samples was greater than 0.90; the relative retention times of each characteristic peak and the reference peak (S) were basically consistent, and except for peak 18, the relative peak areas of each characteristic peak and the reference peak (S) were basically consistent, with RSD values ​​all less than 5%, indicating good repeatability. The data are shown in Tables 17 to 19.

[0169] Table 17. Repeatability Similarity Results

[0170] R S1 S2 S3 S4 S5 S6 R 1.000 1.000 1.000 1.000 1.000 1.000 1.000 S1 1.000 1.000 1.000 1.000 1.000 1.000 1.000 S2 1.000 1.000 1.000 1.000 1.000 1.000 1.000 S3 1.000 1.000 1.000 1.000 1.000 1.000 1.000 S4 1.000 1.000 1.000 1.000 1.000 1.000 1.000 S5 1.000 1.000 1.000 1.000 1.000 1.000 1.000 S6 1.000 1.000 1.000 1.000 1.000 1.000 1.000

[0171] Table 18. Relative retention time ratios of each characteristic chromatographic peak

[0172]

[0173] Table 19. Relative peak area ratios of each characteristic chromatographic peak

[0174]

[0175]

[0176] Example 8. Stability Study

[0177] The test solution was prepared according to the method in Example 3, and the sample was injected and analyzed at 0, 2, 4, 6, 8, 10, 12, and 18 hours according to the planned chromatographic conditions. The chromatographic overlay diagram is shown in [Figure number missing]. Figure 27 In the figure, S1 represents stability at 0 h; S2 represents stability at 2 h; S3 represents stability at 4 h; S4 represents stability at 6 h; S5 represents stability at 8 h; S6 represents stability at 10 h; S7 represents stability at 12 h; and S8 represents stability at 18 h. Similarity and the relative retention times and relative peak areas of each characteristic peak and the reference peak (S) were calculated using the traditional Chinese medicine fingerprint similarity evaluation system. The results showed that the similarity of the chromatographic fingerprints of the samples at each time point was greater than 0.90; the relative retention times and relative peak areas of each characteristic peak and the reference peak (S) were basically consistent within 18 hours, and the RSD values ​​were all less than 5%, indicating that the test solution had good stability within 18 hours. The data are shown in Tables 20-22.

[0178] Table 20. Stability Similarity Results

[0179] R S1 S2 S3 S4 S5 S6 S7 S8 R 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 S1 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 S2 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 S3 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 S4 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 S5 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 S6 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 S7 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 S8 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000

[0180] Table 21. Relative retention time ratios of characteristic chromatographic peaks for stable samples

[0181]

[0182]

[0183] Table 22. Relative peak area ratios of characteristic chromatographic peaks for stable samples

[0184]

Claims

1. A method for detecting the fingerprint chromatogram of Shuangling Zhixie Granules using ultra-high performance liquid chromatography, characterized in that, The Shuangling Antidiarrheal Granules are composed of nine medicinal slices: Sanguisorba officinalis, Citrus reticulata peel, Dryopteris crassirhizoma, Scutellaria baicalensis, Poria cocos, Polyporus umbellatus, Pinellia ternata, Cinnamomum cassia, and Atractylodes macrocephala. The ultra-high performance liquid chromatography method uses octadecylsilane-bonded silica gel as the column packing material, performs gradient elution with mobile phases A and B, and uses an ultraviolet detector for detection to obtain a chromatogram. The chromatogram was processed using fingerprinting software to obtain the fingerprint chromatogram of Shuangling Zhixie Granules; the mobile phase A was acetonitrile, and the mobile phase B was a phosphoric acid solution with a concentration of 0.03 vol%-0.08 vol%; the detection wavelength of the ultraviolet detector was 200-205 nm; before performing high performance liquid chromatography analysis, the sample to be tested was prepared using methanol with a concentration of 70 vol% as the extraction solvent. The gradient elution procedure is as follows: For the 0-60 min period, the volume fraction of mobile phase A was set to 0-26%, and the volume fraction of mobile phase B was set to 100%-74%. Set the volume fraction of mobile phase A to 26%-50% and the volume fraction of mobile phase B to 74%-50% for 60-68 minutes. Set the volume fraction of mobile phase A to 50%-90% and the volume fraction of mobile phase B to 50%-10% for 68-75 minutes. Set the mobile phase A to 90% volume fraction and the mobile phase B to 10% volume fraction for 75-78 minutes. The fingerprint spectrum includes 18 characteristic peaks. Peak 11 is used as a reference peak. The relative retention times of peak 1 and the reference peak are 0.063–0.077; peak 2 is 0.081–0.099; peak 3 is 0.162–0.198; peak 4 is 0.261–0.319; peak 5 is 0.522–0.638; peak 6 is 0.693–0.847; peak 7 is 0.729–0.891; peak 8 is 0.747–0.913; peak 9 is 0.765–0.935; and peak 10 is 0.819–1.

001. The relative retention times of peak 11 and the reference peak are 1.00; peak 12 and the reference peak are 1.008–1.232; peak 13 and the reference peak are 1.026–1.254; peak 14 and the reference peak are 1.062–1.298; peak 15 and the reference peak are 1.161–1.419; and peak 16 and the reference peak have relative retention times of [missing values]. The relative retention times of peak 17 and the reference peak were 1.269–1.551, 1.287–1.573, and 1.422–1.738, respectively. Among them, peak 2 was gallic acid, peak 8 was rutin, peak 9 was kaempferol, peak 10 was hesperidin, peak 11 was baicalin, peak 14 was wogonin, peak 15 was baicalein, peak 16 was wogonin, and peak 18 was limonene.

2. The method according to claim 1, characterized in that, The flow rate of the mobile phase is 0.1-0.3 mL / min; the column temperature is 40±2℃.

3. The method according to claim 1, characterized in that, The fingerprinting software is a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine.

4. The method according to claim 1, characterized in that, The consistency of the relative retention behavior of the 18 characteristic peaks in the fingerprint images of the tested sample and the control sample is used to determine whether the product contains the 18 characteristic peaks.

5. A method for quality testing of Shuangling Zhixie Granules, characterized in that, The method includes the following steps: (1) The fingerprint spectrum of Shuangling Zhixie Granules was obtained by using the method described in claim 1; (2) Import the fingerprint spectrum of the test sample and the reference fingerprint spectrum into the similarity evaluation system of Chinese medicine chromatographic fingerprint spectrum and calculate the similarity; judge the quality stability of Shuangling Zhixie Granules by similarity.

6. The method according to claim 5, characterized in that, If the similarity is ≥0.90, it indicates that the quality of the Shuangling Zhixie Granules sample is stable; if the similarity is <0.90, it indicates that the quality of the sample is unstable.

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