Fingerprint spectrum of common goldenrop particles and determination method for multi-component content of common goldenrop particles

Abstract

The invention provides a common goldenrop particle fingerprint spectrum and multi-component content determination method, which adopts an optimized pretreatment and high performance liquid chromatography method, and can realize qualitative analysis of 18 components of three medicinal materials in common goldenrop particles under the same chromatographic condition. Meanwhile, seven chemical components including liquiritin, baicalin, wogonoside, baicalein, glycyrrhizic acid, saikoside b2 and saikoside b1 in the common goldenrop particles are subjected to quantitative analysis. The technical problems that chemical components of the common goldenrop particles are complex, chromatographic peaks of all the components are prone to mutual interference, and the medicine quality is difficult to comprehensively control are solved, and the quality of the common goldenrop particles can be more effectively controlled.

Description

Technical Field

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

[0002] The Xiao Chaihu Granules of the present invention are a modern dosage form of the classic prescription Xiao Chaihu Decoction in "Treatise on Febrile Diseases", which is a classic prescription of traditional Chinese medicine for reconciling Shaoyang. It is composed of 7 medicinal materials, including bupleurum, scutellaria, ginger pinellia, codonopsis, ginger, liquorice and jujube, and 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 of alternating chills and heat, bitter fullness in the chest and flanks, loss of appetite, bitter taste in the mouth and dry throat, etc.

[0003] The quality standard of Xiao Chaihu Granules is included in the 2020 edition of the Chinese Pharmacopoeia. In the current quality standard, only three medicinal materials, namely, scutellaria, liquorice and bupleurum, were identified by thin-layer chromatography, and baicalin was determined. The 2025 edition of the Chinese Pharmacopoeia revised and improved the standard, removing the thin-layer chromatography identification of scutellaria, adding the determination of glycyrrhizic acid, saikosaponin b2 and saikosaponin b1, and adding the determination of fingerprint spectrum. The 2025 edition of the Chinese Pharmacopoeia uses two different analytical methods for the determination of the four components of baicalin, glycyrrhizic acid, saikosaponin b2 and saikosaponin b1, and the determination of baicalin and glycyrrhizic acid is carried out at different wavelengths. This detection method has the disadvantages of complicated operation, low efficiency, long time consumption and high cost.

[0004] A large number of studies have reported on the research to improve the quality control of Xiaochaihu Granules. Among them, the literature "Simultaneous determination of five marker components in Xiaochaihu Granules and study on its characteristic chromatogram [J]. Acta Pharmaceutica Sinica (Pharmaceutical Journal), 2012, 47(1): 84-87" used reversed-phase high-performance liquid chromatography - diode array detector, with acetonitrile - 1% phosphoric acid aqueous solution as the mobile phase for linear gradient elution, to simultaneously analyze and determine the contents of five main marker components in Xiaochaihu Granules and study its characteristic chromatogram. This method can only perform quantitative analysis on five components, the number of common peaks that can be detected in the characteristic chromatogram is small, and the resolution of some common peaks is not good; the literature "Determination of seven index components in Xiaochaihu Granules by HPLC [J]. Modern Drugs & Clinical, 2014, 29(2): 162-165" established an HPLC method for the simultaneous determination of liquiritin, baicalin, wogonoside, baicalein, ammonium glycyrrhizinate, saikosaponin a, and wogonin in Xiaochaihu Granules. The running time of this method is 120 minutes, which is time-consuming, and it can only perform quantitative analysis on seven components, lacking the determination of fingerprint chromatogram and cannot more comprehensively reflect the shortcomings of product quality; the literature "Determination of seven components in Xiaochaihu Granules by one measure multi-evaluation method [J]. Drug Evaluation Research, 2020, 43(11): 2217-2221" established a one measure multi-evaluation method for the simultaneous determination of the contents of baicalin, wogonoside, baicalein, wogonin, glycyrrhizic acid, saikosaponin B2, and saikosaponin B1 in Xiaochaihu Granules. This method can only perform quantitative analysis on seven components, lacking the determination of fingerprint chromatogram and cannot more comprehensively reflect the product quality; the literature "Establishment and application of a quality evaluation method for Xiaochaihu Granules based on 'benchmark sample' [J]. China Journal of Chinese Materia Medica, 2022, 47(1): 85-94" established a fingerprint chromatogram method for the "benchmark sample" of Xiaochaihu Granules by UPLC, and simultaneously established a method for the determination of the contents of eight components, namely saikosaponin b2, saikosaponin b1, baicalin, wogonoside, baicalein, liquiritin, glycyrrhizin G2, and glycyrrhizic acid. The above methods need to be determined by UPLC, 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), 15 components of Xiao Chaihu granules were qualitatively analyzed and 2 components were quantitatively analyzed. 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 realize 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, uses many 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 time-consuming and inefficient.

[0006] In view of the shortcomings of the prior art described above, the purpose of the present invention is to provide a method for determining the fingerprint spectrum and multi-component content of Xiao Chaihu Granules, using optimized pre-treatment and high performance liquid chromatography methods, and using the same chromatographic conditions to achieve qualitative analysis of 18 components of 3 medicinal materials in Xiao Chaihu Granules; at the same time, quantitative analysis of 7 chemical components in Xiao Chaihu Granules, including liquiritin, baicalin, wogonin, baicalein, glycyrrhizic acid, saikosaponin b2 and saikosaponin b1. The present invention solves the technical problems that the chemical composition of Xiao Chaihu Granules is complex, the chromatographic peaks of each component are easily interfered with each other, and it is difficult to comprehensively control the quality of the drug, and can more effectively control the quality of Xiao Chaihu Granules. Summary of the invention

[0007] The present invention provides a method for determining the fingerprint spectrum 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 glycyrrhizinate, saikosaponin b2 and saikosaponin b1 reference substances, add solvent to dissolve and make up to volume to obtain the reference solution;

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

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

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

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

[0013] (6) Content determination: Using the same chromatographic conditions as in step (5), take the reference substance solution in step (1) and the test sample solution in step (2), inject them into the high performance liquid chromatograph, record the chromatogram, and calculate the contents of 7 components in the test sample solution by the external standard method.

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

[0015] Preferably, the gradient elution program 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 method is a C18 chromatographic column. More preferably, the chromatographic column is Waters XBridge C18, 150×4.6 mm, 3.5 μm.

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

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

[0019] Preferably, the detection wavelength of the high performance liquid chromatography method is 210 - 300 nm. More preferably, the detection wavelength of the high performance liquid chromatography method is 276 nm.

[0020] Preferably, the injection volume of the high performance liquid chromatography method is 5 - 20 μL. More preferably, the injection volume of the high performance liquid chromatography method is 10 μL.

[0021] Preferably, the solvents used in steps (1) and (2) are 50% - 80% ethanol aqueous solution. More preferably, the solvent used is 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, in step (2), the ultrasonic extraction time is 15 to 40 min, preferably 30 min. 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, in step (2), the weight-to-volume ratio of the sample of the Xiaochaihu granules to be tested and 50% - 80% ethanol is equivalent to 1.0 - 2.4 g of the cut crude drug: 25 mL, preferably equivalent to 1.4 - 1.6 g of the cut crude drug: 25 mL.

[0025] The present invention provides a method for constructing a fingerprint of Xiaochaihu granules, and the construction method includes the following steps:

[0026] (1) Inject 20 batches of test solution successively to obtain 20 batches of test chromatograms. Import the 20 batches of test chromatograms into the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints, determine 18 common peaks and generate a control fingerprint of Xiaochaihu granules;

[0027] (2) Compare the common peaks with the chromatograms of the test samples of single herbs, negative test samples and reference substances, attribute each common peak, and identify the chemical components of some common peaks. Determine that peak 1 is liquiritin, peak 2 is baicalin, peak 3 is wogonoside, peak 4 is baicalein, peak 5 is glycyrrhizic acid, peak 6 is saikosaponin b2, and peak 7 is saikosaponin b1.

[0028] (3) Use the control fingerprint to evaluate the quality of Xiaochaihu granules. Calculated according to the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints, the similarity between the fingerprint of each batch of test samples and the control fingerprint is not less than 0.95.

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

[0030] 1. The content detection method provided by the present invention has passed the verifications of precision, repeatability, stability, linearity and recovery of sample addition. The detection results are accurate and reliable. It can accurately quantify the contents of 7 chemical components in Xiaochaihu granules simultaneously, which is of great significance for promoting the inspection, control and evaluation of the quality of Xiaochaihu 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 separation effect of each peak, stable baseline, good precision, good repeatability and high stability, and is suitable for the establishment of the fingerprint of Xiaochaihu granules.

[0032] 3. The present invention adopts a method combining the determination of chemical component content and fingerprint to control the quality of Xiaochaihu granules, which can realize the effective and comprehensive control of Xiaochaihu granules and provide a basis for the quality control standard of Xiaochaihu granules. Description of the Drawings

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

[0034] Figure 2 Chromatograms at different column temperatures;

[0035] Figure 3 Chromatograms at different flow rates;

[0036] Figure 4 Overlay chromatogram (A) of the high-performance liquid chromatography of 20 batches of Xiaochaihu Granules of the present invention and the control fingerprint chromatogram (B, from left to right in the figure are common peaks 1 to 18) of Xiaochaihu Granules;

[0037] Figure 5 Attribution of common peaks of Xiaochaihu Granules of the present invention (Bupleurum chinense), and the numbers in the figure refer to the peak numbers of the chromatographic peaks attributed to Bupleurum chinense in Xiaochaihu Granules;

[0038] Figure 6 Attribution of common peaks of Xiaochaihu Granules of the present invention (Scutellaria baicalensis), and the numbers in the figure refer to the peak numbers of the chromatographic peaks attributed to Scutellaria baicalensis in Xiaochaihu Granules;

[0039] Figure 7 Attribution of common peaks of Xiaochaihu Granules of the present invention (Glycyrrhiza uralensis), and the numbers in the figure refer to the peak numbers of the chromatographic peaks attributed to Glycyrrhiza uralensis in Xiaochaihu Granules;

[0040] Figure 8 Specificity chromatogram for content determination of the present invention, A is the blank solvent, B is the blank excipient, C is the test solution of Xiaochaihu Granules; D is the mixed reference substance solution (1. liquiritin; 2. baicalin; 3. wogonoside; 4. baicalein; 5. glycyrrhizic acid; 6. saikosaponin b2; 7. saikosaponin b1). Detailed Description of the Invention

[0041] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope 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, Inc., USA), XP205 analytical balance (d = 0.0001 g, Mettler-Toledo, Switzerland); PL3002 analytical balance (d = 0.01 g, Mettler-Toledo, Switzerland); ultrasonic cleaner (Shanghai Kedao Ultrasonic Instruments Co., Ltd.).

[0044] Reagents: Methanol and phosphoric acid are of chromatographic purity, other reagents are of analytical purity, and water is Wahaha purified water.

[0045] Reference substances: Baicalin (batch number 110715 - 202223, content 99.6%), Baicalein (batch number 111595 - 201808, content 97.9%), Wogonoside (batch number 112002 - 201702, content 98.5%), Liquiritin (batch number 111610 - 201908, content 95.0%), Ammonium glycyrrhizinate (batch number 110731 - 202122, content 94.4%) reference substances were all purchased from the National Institutes for Food and Drug Control; Saikosaponin b1 (batch number PS010830, purity 97.51%), Saikosaponin b2 (batch number PS011487, purity 99.70%) reference substances were all purchased from Chengdu Push Biotechnology Co., Ltd.

[0046] Samples: Multiple batches of Minor Bupleurum Granules were provided by Yunnan Baiyao Co., Ltd., and the sample information is shown in Table 1.

[0047]

[0048] Example 1

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

[0050] 2. Chromatographic conditions

[0051] 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. The chromatographic column is: 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, under the above - mentioned chromatographic conditions, inject and determine using the two chromatographic columns respectively, and record the chromatogram.

[0054] The results are as Figure 1 shown. Good separation of the common peaks can be achieved with chromatographic columns from different manufacturers, and the chromatographic peaks have good shapes. It is preferred to use Waters XBridge C18 (150 mm × 4.6 mm, 3.5 μm) for determination.

[0055] Example 2

[0056] 1. Preparation of the 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 are respectively selected.

[0059] 3. Determination

[0060] Take the test solution, use the above chromatographic conditions, select column temperatures of 30, 40, and 45 °C respectively, inject the sample for determination, and record the chromatogram.

[0061] The results are as Figure 2 shown. Good separation of the common peaks can be achieved under different column temperature conditions, and the chromatographic peaks have good shapes. It is preferred to use a column temperature of 40 °C for determination.

[0062] Example 3

[0063] 1. Preparation of the 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 are respectively selected.

[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 samples for determination, and record the chromatogram.

[0068] The results are as Figure 3 shown. Under different flow rate conditions, all common peaks can be well separated and the chromatographic peak shapes are good. The preferred flow rate is 0.8 mL / min for determination.

[0069] Construction of the fingerprint of Example 4

[0070] 1. Preparation of reference substance solution and test solution

[0071] 1.1 Reference substance solution

[0072] Take appropriate amounts of baicalin, wogonoside, baicalein, liquiritin, ammonium glycyrrhizinate, saikosaponin b1 and saikosaponin b2 respectively, accurately weigh them, and prepare a mixed reference substance solution containing about 100 μg of baicalin, 20 μg of wogonoside, 20 μg of baicalein, 20 μg of liquiritin, 20 μg of ammonium glycyrrhizinate, 20 μg of saikosaponin b1 and 20 μg of saikosaponin b2 in each 1 ml with 70% ethanol.

[0073] 1.2 Test solution

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

[0075] 1.3 Negative test solution

[0076] Take appropriate amounts of each negative granule, grind them into fine powder, take an appropriate amount (equivalent to about 1.5 g of the crude drug), accurately weigh it, place it in a stoppered conical flask, add 25 mL of 70% ethanol, stopper it tightly, weigh it, ultrasonically treat it for 30 min, let it cool, weigh it again, make up the lost weight with 70% ethanol, shake well, filter through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain it.

[0077] Separate the negative test samples of Scutellaria baicalensis Georgi without roots, Pinellia ternata (Thunb.) Breit. processed with ginger, Codonopsis pilosula (Franch.) Nannf., Zingiber officinale Rosc., Glycyrrhiza uralensis Fisch., and Ziziphus jujuba Mill., grind them into fine powders, take an appropriate amount (equivalent to about 1.3 g of the cut crude drugs), accurately weigh, place them in stoppered conical flasks respectively, add 25 mL of 70% ethanol, stopper tightly, weigh, ultrasonically treat for 30 min, cool, weigh again, make up the lost weight with 70% ethanol, shake well, filter through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain the negative test samples solutions of Scutellaria baicalensis Georgi without roots, Pinellia ternata (Thunb.) Breit. processed with ginger, Codonopsis pilosula (Franch.) Nannf., Zingiber officinale Rosc., Glycyrrhiza uralensis Fisch., and Ziziphus jujuba Mill.

[0078] 1.4 Test sample solutions of single crude drugs

[0079] Weigh 7 single crude drugs according to the prescription ratio, decoct and concentrate them according to the preparation process of Xiaochaihu Granules to obtain the extracts of each single crude drug. Take about 0.2 g of the clear extracts of each crude drug, accurately weigh, place them in stoppered conical flasks respectively, add 25 mL of 70% ethanol, stopper tightly, weigh, ultrasonically treat for 30 min, cool, weigh again, make up the lost weight with 70% ethanol, shake well, filter through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain the test sample solution of Bupleuri Radix crude drug.

[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 sample in Example 4 and inject them for determination according to the above chromatographic conditions, and record the chromatogram.

[0084] 4. Determination of common peaks

[0085] Take 20 batches of Xiaochaihu Granules, prepare test sample solutions according to the above method and conduct determination to obtain the superimposed chromatogram of the high - performance liquid chromatograms of 20 batches of Xiaochaihu Granules, specifically as shown in Figure 4 (A). Then compare the high - performance liquid chromatographic fingerprint spectra of the above - obtained 20 batches of Xiaochaihu Granules with the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" (2012 version) to determine 18 common peaks and obtain the common pattern (reference fingerprint spectrum); Figure 4 (B) is the reference fingerprint spectrum of the Xiaochaihu Granules of the present invention, Figure 4From left to right are common peaks 1 to 18.

[0086] 5. Attribution and Identification of Common Peaks

[0087] By comparing with single-herb slices, reference substance solutions and negative test samples, the attribution and identification of the peaks are as follows:

[0088] The common peaks attributed to Bupleuri Radix are peaks 16 - 18. Among them, peak 16 is the chromatographic peak of saikosaponin b2, and peak 18 is the chromatographic peak of saikosaponin b1. Figure 5 They are the chromatograms of Minor Bupleurum Granules, Bupleuri Radix slices, saikosaponin b1 reference, saikosaponin b2 reference, and Bupleuri Radix negative.

[0089] The common peaks attributed to Scutellariae Radix are peaks 2 - 12. Among them, peak 5 is the chromatographic peak of baicalin, peak 9 is the chromatographic peak of wogonoside, and peak 10 is the chromatographic peak of baicalein. Figure 6 They are the chromatograms of Minor Bupleurum Granules, Scutellariae Radix slices, baicalin reference, wogonoside reference, baicalein reference, and Scutellariae Radix negative.

[0090] The common peaks attributed to Glycyrrhizae Radix are peaks 1, 13 - 15. Among them, peak 1 is the chromatographic peak of liquiritin, and peak 14 is the chromatographic peak of glycyrrhizic acid. Figure 7 They are the chromatograms of Minor Bupleurum Granules, Glycyrrhizae Radix slices, liquiritin reference, glycyrrhizic acid reference, and Glycyrrhizae Radix negative.

[0091] 6. Methodology Research

[0092] 6.1 Precision

[0093] Take the sample of Minor Bupleurum Granules, prepare the test sample solution according to the above method, and inject samples continuously for 6 times under the above chromatographic conditions. Detect the fingerprint, record the chromatogram, take baicalin as the reference peak, and calculate the RSD values of the relative retention time and relative peak area of each common peak. The results are shown in Tables 2 - 3.

[0094]

[0095]

[0096] It can be seen from Tables 2 - 3 that for the sample of Minor Bupleurum Granules injected continuously for 6 times, the RSD values of the relative retention time of each common peak are all less than 1%, and the RSD values of the relative peak area are all less than 3%. This indicates good precision.

[0097] 6.2 Repeatability

[0098] Precisely weigh 6 portions of the same batch of Minor Bupleurum Granules, operate according to the method for preparing the test sample solution in Example 4, inject samples respectively, detect the fingerprint, record the chromatogram, take baicalin as the reference peak, and 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 to 5, the RSD values of the relative retention times of the common peaks in the 6 test solution samples were all less than 1.0%, and the RSD values of the relative peak areas were all less than 3.0%. This indicates good repeatability.

[0102] 6.3 Intermediate precision

[0103] Take samples of Xiao Chaihu Granules from the same batch. Conduct tests according to the repeatability item by different testers on different dates, record the chromatograms, take baicalin as the reference peak, and calculate the RSD values of the relative retention times and relative peak areas of the common peaks in the 12 samples. The results are shown in Tables 6 to 7.

[0104]

[0105]

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

[0107] 6.4 Solution stability

[0108] Precisely weigh samples of Xiao Chaihu Granules, operate according to the method for preparing the test solution in Example 4, inject samples at 0, 2, 4, 6, 8, 12, 24, and 48 h respectively, detect the fingerprint, record the chromatograms, take baicalin as the reference peak, and calculate the RSD values of the relative retention times and relative peak areas of the common peaks. The results are shown in Tables 8 to 9.

[0109]

[0110]

[0111] Results: As can be seen from Tables 8 to 9, the RSD values of the relative retention times of the common peaks were all less than 1.0% within 48 h, and the RSD values of the relative peak areas were all less than 6.0%. This indicates good stability of the test solution within 48 h.

[0112] In summary, through the methodology study, 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 the fingerprint of Xiao Chaihu Granules

[0114] Take 10 batches of Xiaochaihu Granules, prepare them according to the preparation method of the test solution, inject and determine according to the above chromatographic conditions, record the chromatogram, import the chromatogram into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints" (2012 edition) for similarity calculation, and the similarity results of the fingerprint spectra of each batch of test samples and the reference fingerprint spectrum are shown in Table 10.

[0115]

[0116] Results: The similarity between the fingerprint spectra of each batch of samples and the reference fingerprint spectrum is 1.000, all greater than 0.98, indicating good similarity.

[0117] Determination of the Content of Index Components in Xiaochaihu Granules in Example 5

[0118] 1. Preparation of Reference Solution and Test Solution

[0119] 1.1 Mixed Reference Solution

[0120] Accurately weigh appropriate amounts of baicalin, wogonoside, baicalein, liquiritin, ammonium glycyrrhizinate, saikosaponin b1, and saikosaponin b2, dissolve them in 70% ethanol to prepare a mixed reference solution containing about 80 μg of baicalin, 20 μg of wogonoside, 10 μg of baicalein, 20 μg of liquiritin, 20 μg of ammonium glycyrrhizinate, 5 μg of saikosaponin b1, and 10 μg of saikosaponin b2 in 1 ml.

[0121] 1.2 Test Solution

[0122] Prepared according to the same method as the test solution in Example 4.

[0123] 1.3 Test Solution of Blank Excipients

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

[0125] 2. Chromatographic Conditions

[0126] Same as the chromatographic conditions in Example 4.

[0127] 3. Determination

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

[0129] 4. Methodology Study

[0130] 4.1 Specificity

[0131] Take 70% ethanol blank solvent, blank excipients, mixed reference solution and test solution respectively, inject them into a high performance liquid chromatograph, determine and record the chromatogram (as Figure 8 shown). The results show that neither the blank solvent nor the blank excipients interfere with the detection of each component, indicating that this method has good specificity.

[0132] 4.2 Limit of quantitation

[0133] Take an appropriate amount of liquiritin reference solution, serially dilute it until the signal-to-noise ratio is about 10:1, inject it continuously for 6 times, and the RSD value of the peak area is 2.11% (see Table 11). The limit of quantitation of liquiritin is 0.000387 mg / ml.

[0134]

[0135] Take an appropriate amount of baicalin reference solution, serially dilute it until the signal-to-noise ratio is about 10:1, inject it continuously for 6 times, and the RSD value of the peak area is 1.80% (see Table 12). The limit of quantitation of baicalin is 0.000199 mg / ml.

[0136]

[0137] Take an appropriate amount of wogonoside reference solution, serially dilute it until the signal-to-noise ratio is about 10:1, inject it continuously for 6 times, and the RSD value of the peak area is 1.57% (see Table 13). The limit of quantitation of wogonoside is 0.000201 mg / ml.

[0138]

[0139] Take an appropriate amount of baicalein reference solution, serially dilute it until the signal-to-noise ratio is about 10:1, inject it continuously for 6 times, and the RSD value of the peak area is 1.99% (see Table 14). The limit of quantitation of baicalein is 0.000209 mg / ml.

[0140]

[0141] Take an appropriate amount of glycyrrhizic acid reference solution, serially dilute it until the signal-to-noise ratio is about 10:1, inject it continuously for 6 times, and the RSD value of the peak area is 1.93% (see Table 15). The limit of quantitation of glycyrrhizic acid is 0.000182 mg / ml.

[0142]

[0143] Take an appropriate amount of saikosaponin b2 reference solution, serially dilute it until the signal-to-noise ratio is about 10:1, inject it continuously for 6 times, and the RSD value of the peak area is 2.05% (see Table 16). The limit of quantitation of saikosaponin b2 is 0.000062 mg / ml.

[0144]

[0145] Take an appropriate amount of the saikosaponin b1 reference substance solution, and gradually dilute it until the signal-to-noise ratio is about 10:1. Inject continuously for 6 times, and the RSD value of the peak area is 2.00% (see Table 17). The quantitative limit of saikosaponin b1 is 0.000060 mg / ml.

[0146]

[0147] 4.3 Linearity and range

[0148] Take the concentrations of liquiritin reference substance: 0.3872, 2.3233, 3.8722, 7.7444, 11.6166, 19.3610, and 27.1054 μg / ml respectively. Inject according to the above chromatographic conditions. Take the concentration of the reference substance as the abscissa (X) and the peak area as the ordinate (Y) to plot the standard curve and perform regression calculation. The results show that the linear relationship of liquiritin is good. The regression equation is shown in Table 18.

[0149]

[0150] Take the concentrations of baicalin reference substance: 3.97548, 23.85288, 39.7548, 79.5096, 119.2644, 198.774, and 278.2836 μg / ml respectively. Inject according to the above chromatographic conditions. Take the concentration of the reference substance as the abscissa (X) and the peak area as the ordinate (Y) to plot the standard curve and perform regression calculation. The results show that the linear relationship of baicalin is good. The regression equation is shown in Table 19.

[0151]

[0152] Take the concentrations of wogonoside reference substance: 0.48216, 2.89296, 4.8216, 9.6432, 14.4648, 24.108, and 33.7512 μg / ml respectively. Inject according to the above chromatographic conditions. Take the concentration of the reference substance as the abscissa (X) and the peak area as the ordinate (Y) to plot the standard curve and perform regression calculation. The results show that the linear relationship of wogonoside is good. The regression equation is shown in Table 20.

[0153]

[0154] Take the concentrations of baicalein reference substance: 0.417054, 2.502324, 4.17054, 8.34108, 12.51162, 20.8527, and 29.19378 μg / ml respectively. Inject according to the above chromatographic conditions. Take the concentration of the reference substance as the abscissa (X) and the peak area as the ordinate (Y) to plot the standard curve and perform regression calculation. The results show that the linear relationship of baicalein is good. The regression equation is shown in Table 21.

[0155]

[0156] Take the concentrations of glycyrrhizic acid reference substance as 0.911139, 5.466836, 9.111394, 18.22279, 27.22279, 45.55697, and 63.77976 μg / ml respectively. Inject samples according to the above chromatographic conditions. Using the concentration of the reference substance as the abscissa (X) and the peak area as the ordinate (Y), plot the standard curve and perform regression calculation. The results show that the linear relationship of glycyrrhizic acid is good. The regression equation is shown in Table 22.

[0157]

[0158] Take the concentrations of saikosaponin b2 reference substance as 0.411163, 2.466977, 4.111628, 8.223256, 12.33488, 20.55814, and 28.7814 μg / ml respectively. Inject samples according to the above chromatographic conditions. Using the concentration of the reference substance as the abscissa (X) and the peak area as the ordinate (Y), plot the standard curve and perform regression calculation. The results show that the linear relationship of saikosaponin b2 is good. The regression equation is shown in Table 23.

[0159]

[0160] Take the concentrations of saikosaponin b1 reference substance as 0.402131, 2.412787, 4.021312, 8.042625, 12.06394, 20.10656, and 28.14919 μg / ml respectively. Inject samples according to the above chromatographic conditions. Using the concentration of the reference substance as the abscissa (X) and the peak area as the ordinate (Y), plot the standard curve and perform regression calculation. The results show that the linear relationship of saikosaponin b1 is good. The regression equation is shown in Table 24.

[0161]

[0162] 4.4 Precision

[0163] Take the reference substance solutions of liquiritin, baicalin, wogonoside, baicalein, glycyrrhizic acid, saikosaponin b2, and saikosaponin b1. According to the above method, inject samples continuously for 6 times, record the chromatograms, and calculate the RSD values of the peak areas of liquiritin, baicalin, wogonoside, baicalein, glycyrrhizic acid, saikosaponin b2, and saikosaponin b1, which are 0.48%, 0.27%, 0.29%, 0.37%, 0.20%, 0.25%, and 0.18% respectively. It shows that the precision of this method is good.

[0164] 4.5 Repeatability test

[0165] Six portions of the same batch of Xiaochaihu Granules were prepared according to the preparation method of the test solution, and were injected for determination under the above chromatographic conditions. The RSD values of the contents of liquiritin, baicalin, wogonoside, 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, indicating that the method has good repeatability.

[0166] 4.6 Recovery test of added samples

[0167] Nine portions of Xiaochaihu Granules, each about 1.5 g, were accurately weighed. Among them, portions 1 - 3 were prepared according to the preparation method of the test solution, and portions 4 - 9 were respectively added with appropriate amounts of each reference substance and then prepared according to the preparation method of the test solution. They were injected for determination under the above chromatographic conditions. The average recoveries of added samples of liquiritin, baicalin, wogonoside, baicalein, glycyrrhizic acid, saikosaponin b2 and saikosaponin b1 were calculated to be 93.43%, 98.59%, 97.03%, 97.40%, 97.23%, 95.90% and 108.97% respectively, and the RSD values were 6.73%, 4.05%, 2.70%, 0.74%, 2.52%, 1.31% and 1.31% respectively, indicating that the method has good accuracy.

[0168] 5. Determination results of the contents of Xiaochaihu Granules

[0169] Eleven batches of Xiaochaihu Granules were prepared according to the preparation method of the test solution, and were injected for determination under the above chromatographic conditions. The contents of seven components, namely liquiritin, baicalin, wogonoside, baicalein, glycyrrhizic acid, saikosaponin b2 and saikosaponin b1 in the samples were calculated, and the results are shown in Table 25.

[0170]

[0171] In summary, the present invention provides a method for determining the fingerprint and multi - component contents of Xiaochaihu Granules. By using the optimized pretreatment and high - performance liquid chromatography method, qualitative analysis of 18 components from 3 medicinal materials in Xiaochaihu Granules can be achieved under the same chromatographic conditions; meanwhile, quantitative analysis of 7 chemical components, namely liquiritin, baicalin, wogonoside, baicalein, glycyrrhizic acid, saikosaponin b2 and saikosaponin b1 in Xiaochaihu Granules is carried out. A more comprehensive detection of Xiaochaihu Granules is carried out, which can more comprehensively and effectively control the quality of Xiaochaihu Granules and provide a scientific basis for controlling the quality of this medicine.

Claims

1. A method for determining the fingerprint and multi-component content of Xiaochaihu granules, characterized in that, The method comprises the following steps: (1) Preparation of the reference substance solution: Take reference substances of liquiritin, baicalin, wogonoside, baicalein, ammonium glycyrrhizinate, saikosaponin b2 and saikosaponin b1, dissolve them in a solvent and make up the volume to obtain the solution; (2) Preparation of the test sample solution: Take the Xiaochaihu Keli sample, extract it with a solvent, filter it, and take the subsequent filtrate to obtain the solution; (3) Preparation of the test sample solution of single-herb pieces: Take the single-herb piece sample, extract it with a solvent, filter it, and take the subsequent filtrate to obtain the solution; (4) Preparation of the negative test sample solution: Take each negative sample, extract it with a solvent, filter it, and take the subsequent filtrate to obtain the solution; (5) Establishment of the fingerprint: Using high performance liquid chromatography, take the reference substance solution in step (1), the test sample solution in step (2), the test sample solution of single-herb pieces in step (3) and the negative test sample solution in step (4), inject them into the high performance liquid chromatograph in sequence, record the chromatogram, and construct the fingerprint of Xiaochaihu Keli; (6) Content determination: Using the same chromatographic conditions as in step (5), take the reference substance solution in step (1) and the test sample solution in step (2), inject them into the high performance liquid chromatograph, record the chromatogram, and calculate the contents of 7 components in the test sample solution by the external standard method.

2. The method for determining the fingerprint and multi-component content of a Xiaochaihu Granule according to claim 1, wherein In the chromatographic conditions of the high performance liquid chromatography, mobile phase A is methanol and mobile phase B is 0.1% phosphoric acid aqueous solution, and gradient elution is carried out.

3. The method for determining the fingerprint and multi-component content of a Xiaochaihu Granule according to claim 1, wherein The elution gradient is as follows: 0 - 10 min, 10% → 35% A; 10 - 60 min, 35% → 60% A; 60 - 80 min, 60% → 100% A; 80 - 85 min, 100% → 10% A.

4. The method for determining the fingerprint and multi-component content of a Xiaochaihu Granule according to claim 1, wherein The chromatographic column of the high performance liquid chromatography is a C18 chromatographic column, and further the chromatographic column is Waters XBridgeC18, 150×4.6 mm, 3.5 μm.

5. The fingerprint and multi-component content determination method of Xiao Chaihu Granules according to claim 1, characterized in that, The chromatographic conditions of the high performance liquid chromatography further include: the flow rate is 0.7 - 0.9 mL / min; and / or, the column temperature is 30 - 45 °C; and / or, the detection wavelength is 210 - 300 nm, and the injection volume is 5 - 20 μL.

6. The method for determining the fingerprint and multi-component content of a Xiaochaihu Granule according to claim 1, characterized in that, The solvent used in steps (1) - (4) is 50% - 80% ethanol aqueous solution, and the extraction methods in steps (2) - (4) are heating reflux method, cold soaking method or ultrasonic extraction method.

7. The method for determining the fingerprint and multi-component content of a Xiaochaihu Granule according to claim 1, wherein, The extraction method in steps (2) - (4) is ultrasonic extraction method, the ultrasonic power of the ultrasonic extraction method is 200 - 400 W, the ultrasonic frequency is 30 - 50 kHz, and the ultrasonic time is 15 - 40 min.

8. The method for determining the fingerprint and multi-component content of a Xiaochaihu Granule according to claim 1, wherein The weight - volume ratio of the Xiaochaihu Keli sample to be tested in steps (2) - (4) and 50% - 80% ethanol is equivalent to 1.0 - 2.4 g:25 mL of the cut crude drug, and further is equivalent to 1.4 - 1.6 g:25 mL of the cut crude drug.

9. The method for determining the fingerprint and multi-component content of a Xiaochaihu Granule according to claim 1, characterized in that, The construction of the fingerprint in step (5) comprises the following steps: (1) Inject 20 batches of test sample solutions in sequence to obtain 20 batches of test sample chromatograms, import the 20 batches of test sample chromatograms into the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints, determine 18 common peaks and generate the control fingerprint of Xiaochaihu Keli; (2) Compare the common peaks with the chromatograms of the test samples of single herbs, negative test samples and reference substances, assign each common peak, identify the chemical components of some common peaks, and determine that peak 1 is liquiritin, peak 2 is baicalin, peak 3 is wogonoside, peak 4 is baicalein, peak 5 is glycyrrhizic acid, peak 6 is saikosaponin b2, and peak 7 is saikosaponin b1; (3) Evaluate the quality of Xiaochaihu Granules using the reference fingerprint. Calculated according to the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints, the similarity between the fingerprint of each batch of test samples and the reference fingerprint is not less than 0.95.

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