A method for determining the fingerprint of Xiao Chaihu granules and the content of multiple components

Abstract

The present invention provides a method for determining the fingerprint and multi-component content of Xiao Chaihu Granules. Using optimized pretreatment and high-performance liquid chromatography, the method enables qualitative analysis of 18 components from three medicinal herbs in Xiao Chaihu Granules using the same chromatographic conditions. Simultaneously, it quantitatively analyzes seven chemical components in Xiao Chaihu Granules: liquiritin, baicalin, wogonin, baicalein, glycyrrhizic acid, saikosaponin B2, and saikosaponin B1. This method addresses the technical issues of Xiao Chaihu Granules' complex chemical composition, the mutual interference of component chromatographic peaks, and the difficulty in comprehensively controlling drug quality. This method allows for more effective quality control of Xiao Chaihu Granules.

Description

Technical Field

[0001] The present invention relates to the field of quality control of traditional Chinese medicines, and in particular to a method for detecting Xiao Chaihu granules. Technical Background

[0002] The Xiao Chaihu Granules of the present invention are a modern dosage form of the classic Xiao Chaihu Decoction in "Treatise on Febrile Diseases". It is a classic prescription in traditional Chinese medicine for resolving Shaoyang meridians. It is composed of seven medicinal materials: bupleurum, scutellaria, pinellia, codonopsis, ginger, liquorice and jujube. It has the effects of dispelling heat from the exterior and soothing the liver and harmonizing the stomach. It is clinically used for exogenous diseases with symptoms such as alternating chills and fever, bitter fullness in the chest and flanks, loss of appetite, bitter taste in the mouth and dry throat.

[0003] The quality standard for Xiao Chaihu Granules is included in the 2020 edition of the Chinese Pharmacopoeia, Volume 1. The current quality standard only includes thin-layer chromatography identification of three medicinal ingredients: scutellaria baicalensis, liquorice, and bupleurum, and content determination of baicalin. The 2025 edition of the Chinese Pharmacopoeia revised and improved this standard, removing the thin-layer chromatography identification of scutellaria baicalensis and adding content determination of glycyrrhizic acid, saikosaponin b2, and saikosaponin b1, as well as fingerprint determination. The 2025 edition of the Chinese Pharmacopoeia uses two different analytical methods for the content determination of the four ingredients: baicalin, glycyrrhizic acid, saikosaponin b2, and saikosaponin b1. The content determination of baicalin and glycyrrhizic acid is performed at different wavelengths. This detection method has shortcomings such as complex and cumbersome operation, low efficiency, long time consumption, and high cost.

[0004] A large number of studies have been reported on the improvement of the quality control of Xiao Chaihu Granules. Among them, the literature "Simultaneous determination of the content of five index components in Xiao Chaihu Granules and study of their characteristic spectra [J]. Acta Pharmaceutica Sinica (Pharmaceutica Sinica), 2012, 47(1): 84-87" used reversed-phase high performance liquid chromatography-diode array detection with acetonitrile-1% phosphoric acid aqueous solution as the mobile phase for linear gradient elution to simultaneously analyze the contents of the five main index components in Xiao Chaihu Granules and study their characteristic spectra. This method can only quantitatively analyze five components, the characteristic spectrum can detect a small number of common peaks, and the separation between some common peaks is poor; the literature "HPLC determination of seven index components in Xiao Chaihu granules [J]. Modern Drugs and Clinic, 2014, 29(2): 162-165" established an HPLC method for the simultaneous determination of glycyrrhizin, baicalin, wogonin, baicalein, ammonium glycyrrhizate, saikosaponin a and wogonin in Xiao Chaihu granules. The running time of this method is 120 minutes, which is time-consuming. It can only perform quantitative analysis on 7 components. It lacks the determination of fingerprint spectrum and cannot more comprehensively reflect the shortcomings of product quality. The literature "One-test-multiple-evaluation method for determination of 7 components in Xiao Chaihu granules [J]. Drug Evaluation Research, 2020, 43(11): 2217-2221" established a one-test-multiple-evaluation method to simultaneously determine the contents of baicalin, wogonin, baicalein, wogonin, glycyrrhizic acid, saikosaponin B2, and saikosaponin B1 in Xiao Chaihu granules. This method can only quantitatively analyze seven components and lacks fingerprint determination, which cannot fully reflect the product quality. The literature "Establishment and Application of Quality Evaluation Method for Xiao Chaihu Granules Based on 'Benchmark Sample' [J]. Chinese Journal of Traditional Chinese Medicine, 2022, 47(1): 85-94" uses UPLC to establish a fingerprint method for the "benchmark sample" of Xiao Chaihu Granules, and simultaneously establishes a method for determining the contents of eight components, including saikosaponin b2, saikosaponin b1, baicalin, wogonin, baicalein, liquiritin, glycyrrhizin G2, and glycyrrhizic acid. The above method requires UPLC for determination, which has high requirements for instruments and operators and high operating costs.

[0005] Through the search of Chinese invention patents, (1) Patent: Detection method of Xiao Chaihu granules (CN112924586B), the qualitative analysis of 15 components and the quantitative analysis of 2 components of Xiao Chaihu granules. The above methods have the following shortcomings: ① The fingerprint spectrum and content determination method use different detection wavelengths, which makes the operation cumbersome and complicated; ② The content determination of the two components uses different detection wavelengths, which makes the detection cumbersome and complicated; ③ Only baicalin and glycyrrhizic acid can be quantitatively analyzed, which cannot more comprehensively reflect the content of specific components. (2) Patent: Fingerprint spectrum detection method of Xiao Chaihu granule compound preparation (CN105486771B), the constructed fingerprint spectrum identified 12 characteristic peaks, which are the exclusive characteristic peaks of Bupleurum, Scutellaria, Licorice, Codonopsis, and Ginger. The above method has the following shortcomings: ① It can only measure the fingerprint spectrum and achieve semi-quantitative evaluation, but cannot quantitatively measure specific components; ② It uses three solutions of methanol, acetonitrile, and 0.2% phosphoric acid as mobile phases, which requires multiple mobile phase channels, consumes a lot of organic reagents, and the detection method is cumbersome and complicated; ③ The detection time is 105 minutes, which is relatively time-consuming and inefficient.

[0006] In view of the shortcomings of the prior art described above, the present invention aims to provide a method for determining the fingerprint and multi-component content of Xiao Chaihu Granules. Using optimized pretreatment and high-performance liquid chromatography, the method enables qualitative analysis of 18 components from the three medicinal herbs in Xiao Chaihu Granules using the same chromatographic conditions. Simultaneously, quantitative analysis is performed on seven chemical components in Xiao Chaihu Granules: liquiritin, baicalin, wogonin, baicalein, glycyrrhizic acid, saikosaponin B2, and saikosaponin B1. This method addresses the technical issues of Xiao Chaihu Granules' complex chemical composition, the mutual interference of component chromatographic peaks, and the difficulty in comprehensively controlling drug quality, thereby enabling more effective quality control of Xiao Chaihu Granules. Summary of the Invention

[0007] The present invention provides a method for determining the fingerprint of Xiao Chaihu granules and the contents of multiple components, comprising the following steps:

[0008] (1) Preparation of reference solution: Take liquiritin, baicalin, wogonin, baicalein, ammonium glycyrrhizate, saikosaponin b2 and saikosaponin b1 reference substances, dissolve them in solvent and adjust to volume to obtain the reference solution;

[0009] (2) Preparation of test solution: Take a sample of Xiao Chaihu granules, extract it with solvent, filter it, and take the filtrate;

[0010] (3) Preparation of single-ingredient decoction piece test solution: Take a single-ingredient decoction piece sample, add solvent to extract, filter, and take the filtrate;

[0011] (4) Preparation of negative test solution: Take each negative sample, extract with solvent, filter, and take the filtrate;

[0012] (5) Fingerprint establishment: Using high performance liquid chromatography, take the reference solution of step (1), the test solution of step (2), the single herbal medicine sample test solution of step (3) and the negative test solution of step (4), inject them into a high performance liquid chromatograph, record the chromatogram, and construct the fingerprint of Xiao Chaihu Granules.

[0013] (6) Content determination: Using the same chromatographic conditions as step (5), take the reference solution of step (1) and the test solution of step (2), inject them into the high performance liquid chromatograph, record the chromatogram, and calculate the contents of the seven components in the test solution by the external standard method.

[0014] In the chromatographic conditions of the high performance liquid chromatography method, mobile phase A is methanol, and mobile phase B is 0.1% phosphoric acid aqueous solution, and gradient elution is performed.

[0015] Preferably, the gradient elution procedure is: 0-10 min, 10%→35% A; 10-60 min, 35%→60% A; 60-80 min, 60%→100% A; 80-85 min, 100%→10% A.

[0016] Preferably, the chromatographic column for the high performance liquid chromatography is a C18 chromatographic column. More preferably, the chromatographic column is a Waters XBridge C18, 150×4.6 mm, 3.5 μm.

[0017] Preferably, the flow rate of the HPLC method is 0.7-0.9 mL / min. More preferably, the flow rate of the HPLC method is 0.8 mL / min.

[0018] Preferably, the column temperature of the HPLC method is 30-45° C. More preferably, the flow rate of the HPLC method is 40° C.

[0019] Preferably, the detection wavelength of the HPLC method is 210-300 nm. More preferably, the detection wavelength of the HPLC method is 276 nm.

[0020] Preferably, the injection volume of the HPLC method is 5 to 20 μL. More preferably, the injection volume of the HPLC method is 10 μL.

[0021] Preferably, the solvent used in step (1) and step (2) is a 50% to 80% ethanol aqueous solution, more preferably, the solvent used is a 70% ethanol aqueous solution.

[0022] Preferably, the extraction method in step (2) is heating reflux method, cold soaking method or ultrasonic extraction method. More preferably, the extraction method is ultrasonic extraction method.

[0023] Further preferably, the ultrasonic extraction time in step (2) is 15 to 40 minutes, preferably 30 minutes. The power of the ultrasonic extraction is 200 to 400 W, preferably 250 W. The frequency of the ultrasonic extraction is 30 to 50 kHz, preferably 40 kHz.

[0024] Preferably, the weight-to-volume ratio of the Xiao Chaihu granule sample to be tested and 50%-80% ethanol in step (2) is equivalent to 1.0-2.4 g of the decoction piece: 25 mL, preferably equivalent to 1.4-1.6 g of the decoction piece: 25 mL.

[0025] The present invention provides a method for constructing a fingerprint spectrum of Xiao Chaihu granules, the construction method comprising the following steps:

[0026] (1) 20 batches of test sample solutions were sampled sequentially to obtain 20 batches of test sample chromatograms. The 20 batches of test sample chromatograms were introduced into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, 18 common peaks were determined, and a Xiao Chaihu granule control fingerprint was generated;

[0027] (2) The common peaks were compared with the chromatograms of the single herbal test sample, negative test sample and reference sample, and the common peaks were assigned. The chemical components of some common peaks were identified. Peak 1 was determined to be liquiritin, peak 2 was baicalin, peak 3 was wogonin, peak 4 was baicalein, peak 5 was glycyrrhizic acid, peak 6 was saikosaponin b2, and peak 7 was saikosaponin b1.

[0028] (3) The quality of Xiao Chaihu Granules was evaluated using the reference fingerprint. According to the Chinese medicine chromatographic fingerprint similarity evaluation system, the similarity between the fingerprint of each batch of test sample and the reference fingerprint was not less than 0.95.

[0029] The beneficial effects of the present invention are:

[0030] 1. The content detection method provided by the present invention has been verified for precision, repeatability, stability, linearity, and sample recovery. The detection results are accurate and reliable, and the contents of seven chemical components in Xiao Chaihu Granules can be accurately quantified simultaneously. This is of great significance for promoting the inspection, control, and evaluation of the quality of Xiao Chaihu Granules.

[0031] 2. The fingerprint obtained by applying the mobile phase, elution conditions and detection wavelength provided by the present invention has a clean background, good peak separation effect, stable baseline, good precision, good repeatability and high stability, which is suitable for the establishment of the fingerprint of Xiao Chaihu Granules.

[0032] 3. The present invention adopts a method combining chemical component content determination with fingerprint spectrum to control the quality of Xiao Chaihu Granules, which can achieve effective and comprehensive control of Xiao Chaihu Granules and provide a basis for the quality control standard of Xiao Chaihu Granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Chromatograms of columns from different manufacturers;

[0034] Figure 2 Chromatograms at different column temperatures;

[0035] Figure 3 Chromatograms at different flow rates;

[0036] Figure 4 The figure is an overlay of HPLC chromatograms of 20 batches of Xiao Chaihu Granules of the present invention (A) and a control fingerprint of Xiao Chaihu Granules (B, where the common peaks 1 to 18 are shown from left to right);

[0037] Figure 5 The peaks of the Xiao Chaihu granules of the present invention are attributed to (Bupleurum), and the numbers in the figure refer to the peak numbers of the chromatographic peaks in the Xiao Chaihu granules that belong to Bupleurum;

[0038] Figure 6 The peaks of Xiao Chaihu granules of the present invention are attributed to Scutellaria baicalensis. The numbers in the figure indicate the peak numbers of the chromatographic peaks in Xiao Chaihu granules that belong to Scutellaria baicalensis.

[0039] Figure 7 The peaks of Xiao Chaihu granules of the present invention are attributed to (Glycyrrhiza uralensis). The numbers in the figure indicate the peak numbers of the chromatographic peaks in Xiao Chaihu granules that belong to Glycyrrhiza uralensis.

[0040] Figure 8 This is the content determination specificity spectrum of the present invention, A is the blank solvent, B is the blank excipient, C is the Xiao Chaihu Granule test solution; D is the mixed reference solution (1. liquiritin; 2. baicalin; 3. wogonin; 4. baicalein; 5. glycyrrhizic acid; 6. saikosaponin b2; 7. saikosaponin b1). DETAILED DESCRIPTION

[0041] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0042] The instruments and reagents used in the present invention are as follows:

[0043] Instruments: Agilent 1260 high performance liquid chromatograph (Agilent Technologies, USA), XP205 analytical balance (d = 0.0001 g, Mettler, Switzerland); PL3002 analytical balance (d = 0.01 g, Mettler, Switzerland); ultrasonic cleaner (Shanghai Kedao Ultrasonic Instrument Co., Ltd.).

[0044] Reagents: Methanol and phosphoric acid were of chromatographic grade, other reagents were of analytical grade, and water was Wahaha purified water.

[0045] Reference substances: baicalin (batch number 110715-202223, content 99.6%), baicalein (batch number 111595-201808, content 97.9%), wogonin (batch number 112002-201702, content 98.5%), liquiritin (batch number 111610-201908, content 95.0%), and ammonium glycyrrhizate (batch number 110731-202122, content 94.4%) were all purchased from the China Food and Drug Inspection Institutes; saikosaponin b1 (batch number PS010830, purity 97.51%) and saikosaponin b2 (batch number PS011487, purity 99.70%) were purchased from Chengdu Pusi Biotechnology Co., Ltd.

[0046] Samples: Multiple batches of Xiao Chaihu Granules were provided by Yunnan Baiyao Co., Ltd. The sample information is shown in Table 1.

[0047]

[0048] Example 1

[0049] 1. Preparation of test solution: Take an appropriate amount of Bupleurum chinense granules, grind into fine powder, take an appropriate amount (equivalent to 1.5g of the decoction piece), accurately weigh, place in a stoppered conical flask, add 25mL of 70% ethanol, stopper, weigh the weight, ultrasonically treat for 30min, cool, weigh again, make up the lost weight with 70% ethanol, shake well, filter through 0.22μm microporous membrane, and take the filtrate to obtain the test solution.

[0050] 2. Chromatographic Conditions

[0051] Mobile phase: methanol (A)-0.1% aqueous phosphoric acid (B); gradient elution: 0–10 min, 10% → 35% A; 10–60 min, 35% → 60% A; 60–80 min, 60% → 100% A; 80–85 min, 100% → 10% A; injection volume: 10 μL; flow rate: 0.8 mL / min; column temperature: 40°C; detection wavelength: 250 nm. Chromatographic columns: ① Waters XBridge C18 (150 mm × 4.6 mm, 3.5 μm); ② Shim-pack CLC-ODS (150 mm × 6.0 mm, 5 μm).

[0052] 3. Determination

[0053] Take the test solution, adopt the above chromatographic conditions, use two chromatographic columns to inject and measure, and record the chromatograms.

[0054] The results are as follows Figure 1 As shown, the common peaks can be well separated and have good peak shapes using chromatographic columns from different manufacturers. The Waters XBridge C18 (150 mm × 4.6 mm, 3.5 μm) is preferred for the determination.

[0055] Example 2

[0056] 1. Preparation of test solution: as shown in Example 1.

[0057] 2. Chromatographic Conditions

[0058] Chromatographic column: Waters XBridge C18 (150 mm × 4.6 mm, 3.5 μm); mobile phase: methanol (A)-0.1% phosphoric acid aqueous solution (B), gradient elution: 0-10 min, 10%→35% A; 10-60 min, 35%→60% A; 60-80 min, 60%→100% A; 80-85 min, 100%→10% A; injection volume: 10 μL; flow rate: 0.8 mL / min; detection wavelength: 250 nm; column temperatures of 30, 40, and 45°C were selected, respectively.

[0059] 3. Determination

[0060] Take the test solution, adopt the above chromatographic conditions, select 30, 40 and 45℃ as the column temperature respectively, inject the sample for measurement, and record the chromatogram.

[0061] The results are as follows Figure 2 As shown in the figure, under different column temperature conditions, each common peak can be well separated and the chromatographic peak shape is good. The column temperature is preferably 40℃ for measurement.

[0062] Example 3

[0063] 1. Preparation of test solution: as shown in Example 1.

[0064] 2. Chromatographic Conditions

[0065] Chromatographic column: Waters XBridge C18 (150 mm × 4.6 mm, 3.5 μm); mobile phase: methanol (A)-0.1% phosphoric acid aqueous solution (B), gradient elution: 0-10 min, 10%→35% A; 10-60 min, 35%→60% A; 60-80 min, 60%→100% A; 80-85 min, 100%→10% A; injection volume: 10 μL; flow rate: 0.8 mL / min; detection wavelength: 250 nm; column temperature: 40°C; flow rates of 0.7, 0.8, and 0.9 mL / min were selected, respectively.

[0066] 3. Determination

[0067] Take the test solution, adopt the above chromatographic conditions, select 0.7, 0.8 and 0.9 mL / min as the flow rate, respectively, inject the sample for measurement, and record the chromatogram.

[0068] The results are as follows Figure 3 As shown in the figure, under different flow rate conditions, each common peak can be well separated and the chromatographic peak shape is good. The preferred flow rate for measurement is 0.8 mL / min.

[0069] Example 4 Construction of fingerprint

[0070] 1. Preparation of Reference and Test Solution

[0071] 1.1 Reference solution

[0072] Take appropriate amounts of baicalin, wogonin, baicalein, liquiritin, ammonium glycyrrhizate, saikosaponin b1 and saikosaponin b2 respectively, weigh them accurately, and add 70% ethanol to prepare a mixed reference solution containing approximately 100 μg of baicalin, 20 μg of wogonin, 20 μg of baicalein, 20 μg of liquiritin, 20 μg of ammonium glycyrrhizate, 20 μg of saikosaponin b1 and 20 μg of saikosaponin b2 per 1 ml.

[0073] 1.2 Test solution

[0074] Take appropriate amount of Bupleurum chinense granules from different batches, grind them into fine powder, take appropriate amount (equivalent to 1.5g of the decoction piece), accurately weigh, place in a stoppered conical flask, add 25mL of 70% ethanol, stopper, weigh, ultrasonically treat for 30min, cool, weigh again, make up the lost weight with 70% ethanol, shake well, filter through a 0.22μm microporous filter membrane, and take the filtrate to obtain the test solution.

[0075] 1.3 Negative test solution

[0076] Take an appropriate amount of each negative particle, grind it into fine powder, take an appropriate amount (approximately equivalent to 1.5g of the decoction piece), accurately weigh it, place it in a stoppered conical flask, add 25mL of 70% ethanol, stopper it, weigh it, ultrasonically treat it for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% ethanol, shake it evenly, filter it through a 0.22μm microporous filter membrane, and take the filtrate to obtain it.

[0077] Take the negative test samples lacking scutellaria baicalensis, pinellia ternata, codonopsis pilosula, ginger, licorice and jujube respectively, grind them into fine powder, take appropriate amount (approximately equivalent to 1.3g of the decoction piece), accurately weigh, place them in stoppered conical flasks, add 25mL of 70% ethanol, stopper, weigh the weight, ultrasonically treat for 30min, cool, weigh again, make up the lost mass with 70% ethanol, shake well, filter through 0.22μm microporous membrane, and take the filtrate to obtain the negative test sample solution lacking scutellaria baicalensis, pinellia ternata, codonopsis pilosula, ginger, licorice and jujube.

[0078] 1.4 Single Herb Piece Test Solution

[0079] Weigh 7 single-ingredient slices according to the prescription ratio, and then decoct and concentrate them according to the preparation process of Xiao Chaihu Granules to obtain the extract of each single-ingredient slice. Take about 0.2 g of the clear paste of each slice, accurately weigh it, and place it in a stoppered conical flask, add 25 mL of 70% ethanol, stopper it, weigh it, ultrasonically treat it for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% ethanol, shake it evenly, filter it through a 0.22 μm microporous filter membrane, and take the filtrate to obtain the test solution of Chaihu slices.

[0080] 2. Chromatographic conditions

[0081] Waters XBridge C18 (4.6 mm × 150 mm, 3.5 μm); mobile phase: methanol (A)-0.1% phosphoric acid aqueous solution (B), gradient elution: 0-10 min, 10%→35% A; 10-60 min, 35%→60% A; 60-80 min, 60%→100% A; 80-85 min, 100%→10% A; injection volume: 10 μL; flow rate: 0.8 mL / min; column temperature: 40°C; detection wavelength: 250 nm.

[0082] 3. Determination

[0083] Take the reference substance and test substance in Example 4 respectively, inject and measure according to the above chromatographic conditions, and record the chromatogram.

[0084] 4. Determination of Common Peaks

[0085] Take 20 batches of Xiao Chaihu Granules respectively, prepare the test solution according to the above method and measure it, and obtain the HPLC chromatogram superposition of 20 batches of Xiao Chaihu Granules, as shown in the following figure: Figure 4 As shown in (A), the HPLC fingerprints of the 20 batches of Xiao Chaihu Granules obtained above were compared using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 edition), and 18 common peaks were identified and a common pattern was obtained (control fingerprint); Figure 4 (B) is the reference fingerprint of Xiao Chaihu Granule of the present invention, Figure 4From left to right in the middle are common peaks 1 to 18.

[0086] 5. Attribution and Identification of Common Peaks

[0087] By comparing with single-ingredient decoction pieces, reference solution and negative test sample, the peak attribution and identification are as follows:

[0088] The common peaks attributed to Bupleurum are peaks 16 to 18, of which peak 16 is the chromatographic peak of saikosaponin b2, and peak 18 is the chromatographic peak of saikosaponin b1; Figure 5 These are the chromatograms of Xiao Chaihu Granules, Chaihu Pieces, Bupleurum Saponin B1 Control, Bupleurum Saponin B2 Control and Bupleurum Negative.

[0089] The common peaks attributed to Scutellaria baicalensis are peaks 2 to 12, of which peak 5 is the chromatographic peak of baicalin, peak 9 is the chromatographic peak of wogonin, and peak 10 is the chromatographic peak of baicalein; Figure 6 These are the chromatograms of Xiao Chaihu Granules, Huangqin slices, baicalin control, wogonin control, baicalein control and Huangqin negative.

[0090] The common peaks attributed to licorice are peaks 1, 13 to 15, of which peak 1 is the chromatographic peak of liquiritin and peak 14 is the chromatographic peak of glycyrrhizic acid; Figure 7 These are the chromatograms of Xiao Chaihu Granules, Licorice Pieces, Liquoricein Control, Glycyrrhizic Acid Control and Licorice Negative.

[0091] 6. Methodological Research

[0092] 6.1 Precision

[0093] Take Xiao Chaihu Granules sample, prepare the test solution according to the above method, and inject it continuously 6 times according to the above chromatographic conditions. Detect the fingerprint spectrum and record the chromatogram. Use baicalin as the reference peak to calculate the RSD values ​​of the relative retention time and relative peak area of ​​each common peak. The results are shown in Tables 2 and 3.

[0094]

[0095]

[0096] As shown in Tables 2 and 3, after six consecutive injections of the Xiao Chaihu Granule sample, the RSD values ​​of the relative retention times of the common peaks were all less than 1%, and the RSD values ​​of the relative peak areas were all less than 3%, indicating good precision.

[0097] 6.2 Repeatability

[0098] Accurately weigh 6 portions of Xiao Chaihu Granules from the same batch and operate according to the method under the item "Preparation of Test Solution in Example 4". Samples were injected separately, fingerprints were detected, and chromatograms were recorded. Baicalin was used as the reference peak to calculate the RSD values ​​of the relative retention time and relative peak area of ​​each common peak. The results are shown in Tables 4-5.

[0099]

[0100]

[0101] As can be seen from Tables 4 and 5, the RSD values ​​of the relative retention times of the common peaks in the six test solutions were all less than 1.0%, and the RSD values ​​of the relative peak areas were all less than 3.0%, indicating good reproducibility.

[0102] 6.3 Intermediate precision

[0103] The same batch of Xiao Chaihu granules samples were tested under the repeatability item by different testers on different days. The chromatograms were recorded, and the RSD values ​​of the relative retention time and relative peak area of ​​the common peaks of the 12 samples were calculated using baicalin as the reference peak. The results are shown in Tables 6 and 7.

[0104]

[0105]

[0106] As can be seen from Tables 6 and 7, the RSD values ​​of the relative retention times of the common peaks of the 12 samples were all less than 1.0%, and the RSD values ​​of the relative peak areas were all less than 3.0%, indicating good intermediate precision.

[0107] 6.4 Solution stability

[0108] Accurately weigh the Xiao Chaihu Granule sample and operate according to the method under the test solution preparation item of Example 4. Samples were injected at 0, 2, 4, 6, 8, 12, 24 and 48 h, the fingerprint was detected, the chromatogram was recorded, and baicalin was used as the reference peak to calculate the relative retention time and RSD value of the relative peak area of ​​each common peak. The results are shown in Tables 8 to 9.

[0109]

[0110]

[0111] Results: As shown in Tables 8 and 9, the RSD values ​​of the relative retention times of the common peaks within 48 hours were all less than 1.0%, and the RSD values ​​of the relative peak areas were all less than 6.0%, indicating that the test solution had good stability within 48 hours.

[0112] In summary, through methodological research, the fingerprint detection method of the present invention has good repeatability, stability and precision, and can more comprehensively control the quality of Xiao Chaihu Granules.

[0113] 7. Similarity results of Xiao Chaihu Granule fingerprint

[0114] Ten batches of Xiao Chaihu granules were prepared according to the test solution preparation method, sampled and measured according to the above chromatographic conditions, and the chromatograms were recorded. The chromatograms were imported into the "Similarity Evaluation System of Chinese Medicine Chromatographic Fingerprints" (2012 edition) for similarity calculation. The similarity results of the fingerprints of each batch of test samples and the control fingerprint are shown in Table 10.

[0115]

[0116] Results: The similarities between the fingerprints of each batch of samples and the control fingerprints were all 1.000 and greater than 0.98, indicating good similarity.

[0117] Example 5 Determination of the content of index components of Xiao Chaihu granules

[0118] 1. Preparation of Reference and Test Solution

[0119] 1.1 Mixed reference solution

[0120] Take appropriate amounts of baicalin, wogonin, baicalein, liquiritin, ammonium glycyrrhizate, saikosaponin b1 and saikosaponin b2 respectively, weigh them accurately, and add 70% ethanol to prepare a mixed reference solution containing approximately 80 μg of baicalin, 20 μg of wogonin, 10 μg of baicalein, 20 μg of liquiritin, 20 μg of ammonium glycyrrhizate, 5 μg of saikosaponin b1 and 10 μg of saikosaponin b2 per 1 ml.

[0121] 1.2 Test solution

[0122] The preparation method of the test solution is the same as that in Example 4.

[0123] 1.3 Blank excipient test solution

[0124] Take about 2 g of sucrose, accurately weigh it, place it in a stoppered conical flask, add 25 mL of 70% ethanol, stopper it tightly, weigh it, ultrasonicate it for 30 minutes, let it cool, weigh it again, make up the lost mass with 70% ethanol, shake it well, filter it through a 0.22 μm microporous filter membrane, and take the filtrate to obtain the blank excipient test solution.

[0125] 2. Chromatographic Conditions

[0126] The chromatographic conditions are the same as those in Example 4.

[0127] 3. Determination

[0128] Take the reference substance and test substance in Example 5 respectively, inject and measure according to the above chromatographic conditions, and record the chromatogram.

[0129] 4. Methodological research

[0130] 4.1 Specificity

[0131] Take 70% ethanol blank solvent, blank excipient, mixed reference solution and test solution respectively, inject them into high performance liquid chromatography, measure and record the chromatogram (such as Figure 8 The results showed that the blank solvent and blank excipients did not interfere with the detection of each component, indicating that the method has good specificity.

[0132] 4.2 Limit of quantification

[0133] An appropriate amount of liquiritin reference solution was diluted stepwise until the signal-to-noise ratio was about 10:1. The sample was injected 6 times continuously. The RSD value of the peak area was 2.11% (see Table 11). The limit of quantification of liquiritin was 0.000387 mg / ml.

[0134]

[0135] An appropriate amount of baicalin reference solution was diluted stepwise until the signal-to-noise ratio was approximately 10:1. The sample was injected six times continuously. The RSD value of the peak area was 1.80% (see Table 12). The limit of quantification of baicalin was 0.000199 mg / ml.

[0136]

[0137] An appropriate amount of wogonin reference solution was taken and diluted stepwise until the signal-to-noise ratio was about 10:1. The sample was injected six times continuously. The RSD value of the peak area was 1.57% (see Table 13). The limit of quantification of wogonin was 0.000201 mg / ml.

[0138]

[0139] An appropriate amount of baicalein reference solution was diluted stepwise until the signal-to-noise ratio was approximately 10:1. The sample was injected six times continuously. The RSD value of the peak area was 1.99% (see Table 14). The limit of quantification of baicalein was 0.000209 mg / ml.

[0140]

[0141] An appropriate amount of glycyrrhizic acid reference solution was diluted stepwise until the signal-to-noise ratio was approximately 10:1. The sample was injected six times continuously. The RSD value of the peak area was 1.93% (see Table 15). The limit of quantification of glycyrrhizic acid was 0.000182 mg / ml.

[0142]

[0143] An appropriate amount of saikosaponin b2 reference solution was diluted stepwise until the signal-to-noise ratio was approximately 10:1. The sample was injected six times continuously. The RSD value of the peak area was 2.05% (see Table 16). The limit of quantification of saikosaponin b2 was 0.000062 mg / ml.

[0144]

[0145] An appropriate amount of saikosaponin b1 reference solution was diluted stepwise until the signal-to-noise ratio was approximately 10:1. The sample was injected six times continuously. The RSD value of the peak area was 2.00% (see Table 17). The limit of quantification of saikosaponin b1 was 0.000060 mg / ml.

[0146]

[0147] 4.3 Linearity and Range

[0148] The concentrations of the glycyrrhizin reference substance were 0.3872, 2.3233, 3.8722, 7.7444, 11.6166, 19.3610, and 27.1054 μg / ml, respectively. Samples were injected under the above chromatographic conditions. A standard curve was drawn with the concentration of the reference substance as the abscissa (X) and the peak area as the ordinate (Y), and regression calculation was performed. The results showed a good linear relationship for glycyrrhizin. The regression equation is shown in Table 18.

[0149]

[0150] Baicalin reference substances were injected at concentrations of 3.97548, 23.85288, 39.7548, 79.5096, 119.2644, 198.774, and 278.2836 μg / ml, respectively, under the aforementioned chromatographic conditions. A standard curve was plotted with the reference substance concentration as the abscissa (X) and the peak area as the ordinate (Y), and regression analysis was performed. The results showed good linearity for baicalin. The regression equation is shown in Table 19.

[0151]

[0152] Wogonin reference standards were injected at concentrations of 0.48216, 2.89296, 4.8216, 9.6432, 14.4648, 24.108, and 33.7512 μg / ml, respectively, under the aforementioned chromatographic conditions. A standard curve was plotted with the reference standard concentration as the abscissa (X) and the peak area as the ordinate (Y), and regression analysis was performed. The results showed good linearity for wogonin. The regression equation is shown in Table 20.

[0153]

[0154] Baicalein reference substances were injected at concentrations of 0.417054, 2.502324, 4.17054, 8.34108, 12.51162, 20.8527, and 29.19378 μg / ml, respectively, under the aforementioned chromatographic conditions. A standard curve was plotted with the reference substance concentration as the abscissa (X) and the peak area as the ordinate (Y), and regression analysis was performed. The results showed good linearity for baicalein. The regression equation is shown in Table 21.

[0155]

[0156] Glycyrrhizic acid reference substance concentrations of 0.911139, 5.466836, 9.111394, 18.22279, 27.22279, 45.55697, and 63.77976 μg / ml were injected according to the above chromatographic conditions. A standard curve was plotted with the concentration of the reference substance as the abscissa (X) and the peak area as the ordinate (Y), and regression calculation was performed. The results showed a good linear relationship for glycyrrhizic acid. The regression equation is shown in Table 22.

[0157]

[0158] The concentrations of saikosaponin b2 reference substance were 0.411163, 2.466977, 4.111628, 8.223256, 12.33488, 20.55814, and 28.7814 μg / ml, respectively. Samples were injected under the above chromatographic conditions. A standard curve was plotted with the concentration of the reference substance as the abscissa (X) and the peak area as the ordinate (Y), and regression calculations were performed. The results showed a good linear relationship for saikosaponin b2. The regression equation is shown in Table 23.

[0159]

[0160] The concentrations of saikosaponin b1 reference substance were 0.402131, 2.412787, 4.021312, 8.042625, 12.06394, 20.10656, and 28.14919 μg / ml, respectively. Samples were injected under the above chromatographic conditions. A standard curve was plotted with the concentration of the reference substance as the abscissa (X) and the peak area as the ordinate (Y), and regression calculations were performed. The results showed a good linear relationship for saikosaponin b1. The regression equation is shown in Table 24.

[0161]

[0162] 4.4 Precision

[0163] Six consecutive injections of reference solutions of liquiritin, baicalin, wogonin, baicalein, glycyrrhizic acid, saikosaponin b2, and saikosaponin b1 were performed according to the above method. Chromatograms were recorded, and the calculated RSD values ​​for the peak areas of liquiritin, baicalin, wogonin, baicalein, glycyrrhizic acid, saikosaponin b2, and saikosaponin b1 were 0.48%, 0.27%, 0.29%, 0.37%, 0.20%, 0.25%, and 0.18%, respectively. This indicates that the precision of this method is good.

[0164] 4.5 Repeatability test

[0165] Six portions of Xiao Chaihu granules from the same batch were prepared according to the test solution preparation method and assayed under the aforementioned chromatographic conditions. The RSDs for liquiritin, baicalin, wogonin, baicalein, glycyrrhizic acid, saikosaponin b2, and saikosaponin b1 were calculated to be 0.11%, 0.10%, 0.08%, 0.08%, 0.17%, 0.16%, and 0.17%, respectively. This indicates good reproducibility of the method.

[0166] 4.6 Sample recovery test

[0167] Nine portions of Xiao Chaihu Granules (approximately 1.5 g each) were accurately weighed. Portions 1–3 were prepared according to the test solution preparation method. Portions 4–9 were added with appropriate amounts of each reference substance and prepared according to the test solution preparation method. Samples were injected and analyzed under the above chromatographic conditions. The calculated average recoveries for liquiritin, baicalin, wogonin, baicalein, glycyrrhizic acid, saikosaponin b2, and saikosaponin b1 were 93.43%, 98.59%, 97.03%, 97.40%, 97.23%, 95.90%, and 108.97%, respectively. The RSDs were 6.73%, 4.05%, 2.70%, 0.74%, 2.52%, 1.31%, and 1.31%, respectively. This indicates that the method has good accuracy.

[0168] 5. Content determination results of Xiao Chaihu granules

[0169] Eleven batches of Xiao Chaihu Granules were prepared according to the test solution preparation method, and the samples were injected and determined according to the above-mentioned chromatographic conditions. The contents of the seven components in the samples, including liquiritin, baicalin, wogonin, baicalein, glycyrrhizic acid, saikosaponin b2 and saikosaponin b1, were calculated. The results are shown in Table 25.

[0170]

[0171] In summary, the present invention provides a method for determining the fingerprint and multi-component content of Xiao Chaihu Granules. Using optimized pretreatment and high-performance liquid chromatography, the same chromatographic conditions allow for qualitative analysis of 18 components from three medicinal herbs in Xiao Chaihu Granules. Simultaneously, quantitative analysis is performed for seven chemical components in Xiao Chaihu Granules: liquiritin, baicalin, wogonin, baicalein, glycyrrhizic acid, saikosaponin B2, and saikosaponin B1. This comprehensive testing of Xiao Chaihu Granules allows for more comprehensive and effective quality control of Xiao Chaihu Granules, providing a scientific basis for drug quality control.

Claims

1. A method for determining the fingerprint of Xiao Chaihu granules and the content of multiple components, characterized in that: The method comprises the following steps: (1) Preparation of reference solution: Take liquiritin, baicalin, wogonin, baicalein, ammonium glycyrrhizate, saikosaponin b2 and saikosaponin b1 reference substances, dissolve them in solvent and adjust to volume to obtain the reference solution; (2) Preparation of test solution: Take a sample of Xiao Chaihu granules, extract it with solvent, filter it, and take the filtrate; (3) Preparation of single-ingredient decoction piece test solution: Take a single-ingredient decoction piece sample, add solvent to extract, filter, and take the filtrate; (4) Preparation of negative test solution: Take each negative sample, extract with solvent, filter, and take the filtrate; (5) Chromatographic conditions: The chromatographic column was a C18 column, the mobile phase was methanol (A)-0.1% phosphoric acid aqueous solution (B), and gradient elution was performed. The elution gradient was 0-10 min, 10%→35% A; 10-60 min, 35%→60% A; 60-80 min, 60%→100% A; 80-85 min, 100%→10% A. The injection volume was 5-20 μL, the flow rate was 0.7-0.9 mL / min, the column temperature was 30-45°C, and the detection wavelength was 210-300 nm. (6) Fingerprint establishment: using the chromatographic conditions of step (5), taking the reference solution of step (1), the test solution of step (2), the single herbal medicine piece test solution of step (3) and the negative test solution of step (4), injecting them into the high performance liquid chromatograph in sequence, recording the chromatogram, and constructing the fingerprint of Xiao Chaihu Granules; (7) Content determination: Using the chromatographic conditions of step (5), take the reference solution of step (1) and the test solution of step (2), inject them into a high performance liquid chromatograph, record the chromatogram, and calculate the contents of the seven components, namely, liquiritin, baicalin, wogonin, baicalein, glycyrrhizic acid, saikosaponin b2, and saikosaponin b1, in the test solution by the external standard method.

2. The method for determining the fingerprint of Xiao Chaihu granules and the content of multiple components according to claim 1, characterized in that: The chromatographic column in step (5) was Waters XBridge C18, 150×4.6 mm, 3.5 μm.

3. The method for determining the fingerprint of Xiao Chaihu granules and the content of multiple components according to claim 1, characterized in that: The solvent used in steps (1)-(4) is a 50% to 80% ethanol aqueous solution, and the extraction method in steps (2)-(4) is a heating reflux method, a cold soaking method or an ultrasonic extraction method.

4. The method for determining the fingerprint of Xiao Chaihu granules and the content of multiple components according to claim 1, wherein: The extraction method in steps (2)-(4) is ultrasonic extraction, wherein the ultrasonic power of the ultrasonic extraction method is 200-400W, the ultrasonic frequency is 30-50kHz, and the ultrasonic time is 15-40min.

5. The method for determining the fingerprint of Xiao Chaihu granules and the content of multiple components according to claim 1, characterized in that: The weight-to-volume ratio of the sample to be tested to 50%-80% ethanol in steps (2)-(4) is equivalent to 1.0-2.4 g:25 mL of the decoction piece, and further equivalent to 1.4-1.6 g:25 mL of the decoction piece.

6. The method for determining the fingerprint of Xiao Chaihu granules and the content of multiple components according to claim 1, characterized in that: Step (6) Fingerprint construction includes the following steps: (1) 20 batches of test sample solutions were sampled sequentially to obtain 20 batches of test sample chromatograms. The 20 batches of test sample chromatograms were introduced into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, 18 common peaks were determined, and a Xiao Chaihu granule control fingerprint was generated; (2) The common peaks were compared with the chromatograms of the single herbal test sample, negative test sample, and reference sample, and the common peaks were assigned. The chemical components of some common peaks were identified, and peak 1 was determined to be liquiritin, peak 2 to be baicalin, peak 3 to be wogonin, peak 4 to be baicalein, peak 5 to be glycyrrhizic acid, peak 6 to be saikosaponin b2, and peak 7 to be saikosaponin b1. (3) The quality of Xiao Chaihu Granules was evaluated using the reference fingerprint. According to the Chinese medicine chromatographic fingerprint similarity evaluation system, the similarity between the fingerprint of each batch of test sample and the reference fingerprint was not less than 0.95.

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