A method for constructing a characteristic spectrum of Uoganning granules

Abstract

The present invention relates to the technical field of traditional Chinese medicine analysis, and in particular to a method for constructing a characteristic spectrum of Uoganning Granules. The method comprises the following steps: 1. preparation of a reference solution; 2. preparation of a test solution; 3. determination conditions; 4. generation of a control characteristic spectrum. The present invention establishes an HPLC characteristic spectrum for Uoganning Granules, identifies 12 characteristic peaks, and more fully demonstrates the chemical characteristics of Uoganning Granules. The RSD values ​​of the relative retention times of the peaks of the present invention are all ≤2.0%, and the RSD values ​​of the relative peak areas are all ≤10.0%. This shows that the method is good, and the major chemical components in Uoganning Granules can be reflected by the method. The overall quality of Uoganning Granules can be controlled more efficiently and quickly.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine analysis, and in particular to a method for constructing a characteristic spectrum of Niaoganning granules. Background Art

[0002] Niaoganning Granules are composed of five herbs: Lygodium japonicum, Herba Lycopodii, Herba Pteris multiflori, Herba Humuli and Viola yedoensis.

[0003] Its quality standards are included in the 2020 edition of the Chinese Pharmacopoeia, Volume 1. The current standards for Nianganning Granules only cover identification of properties and routine granule inspections, lacking a characteristic spectral profile that could comprehensively control the quality of Nianganning Granules. Furthermore, there is little literature on the characteristic spectral profile of Nianganning Granules, and the incomplete quality standards make it difficult to effectively control the quality of this product.

[0004] Urinary Ning Granules are composed of five traditional Chinese medicines, each of which contains multiple active ingredients (such as flavonoids, phenolic acids, alkaloids, etc.). There may be chromatographic peaks with similar or overlapping physical and chemical properties between the ingredients, making separation difficult.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] In order to make up for the deficiencies in the prior art, the present invention provides a method for constructing a characteristic spectrum of Urinary tract Injection Granules.

[0007] The present invention is achieved through the following technical solutions:

[0008] The present invention provides a method for constructing a characteristic spectrum of Niuganning granules, comprising the following steps:

[0009] (1) Preparation of reference solution: Take an appropriate amount of reference substance, weigh accurately, add methanol to make a solution containing 50 μg per 1 mL, shake well, and use it as the reference solution; the reference substances are: aesculetin and luteolin;

[0010] (2) Preparation of test solution: Take an appropriate amount of Nianganning granules, grind them into powder, take 1g, and place it in a 50mL stoppered conical flask; add 40mL of methanol-water-0.1% formic acid solution with a volume ratio of 70:28:2, of which 0.1% formic acid is the volume fraction, and perform ultrasonic assisted extraction for 45 minutes. The ultrasonic parameters are power 250W, frequency 40kHz, and temperature 40℃; centrifuge at 4000rpm for 10 minutes, take the supernatant, and filter it through a 0.45μm microporous membrane to obtain the primary filtrate; add 5mL of methanol, Activate the C18 solid phase extraction column with 5 mL of ultrapure water to keep the column bed moist; slowly load the primary filtrate onto the activated C18 column at a flow rate of 1 mL / min; elute with 5 mL of ultrapure water-methanol solution with a volume ratio of 95:5 to remove impurities; elute the target component with 5 mL of methanol-0.1% formic acid aqueous solution with a volume ratio of 80:20, and collect the eluate; concentrate the eluate to near dryness under nitrogen blow at 40°C, re-dissolve it with 70% methanol and make up to 2 mL, and filter it through a 0.22 μm filter membrane to obtain the test solution;

[0011] (3) Determination: The reference solution and the test solution were injected into the high performance liquid chromatograph according to the following chromatographic conditions, 10 μl of each sample was injected, and the chromatogram was recorded; the chromatographic conditions were: octadecylsilane bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile-tetrahydrofuran-0.1% trifluoroacetic acid (85:10:5) as the mobile phase A, 10 mM phosphoric acid solution-0.05% sodium heptane sulfonate solution (94:5) as the mobile phase B, wherein 0.1% trifluoroacetic acid (85:10:5) was the volume fraction and 0.05% sodium heptane sulfonate solution was the mass fraction; elution was performed according to the specified gradient; the column temperature was 30 °C; the flow rate was 1 mL per minute; the UV-Vis detector had a detection wavelength of 270 nm;

[0012] The gradient conditions are:

[0013] From 0 to 25 min, the volume ratio of mobile phase A:phase B was changed from 5:95 to 10:90;

[0014] From 25 to 50 min, the volume ratio of mobile phase A:phase B was changed from 10:90 to 20:80;

[0015] From 50 to 65 min, the volume ratio of mobile phase A:phase B was changed from 20:80 to 30:70;

[0016] From 65 to 75 min, the volume ratio of mobile phase A:phase B was changed from 30:70 to 60:40;

[0017] From 75 to 78 min, the volume ratio of mobile phase A:phase B was changed from 60:40 to 5:95;

[0018] 78-90 min, mobile phase A:B, volume ratio 5:95;

[0019] (4) Generate a reference characteristic spectrum: Use the “Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine” developed by the Chinese Pharmacopoeia Committee, select the chromatographic peaks that exist in the chromatograms of different batches of Uoganning Granules as common peaks, and use the average value calculation method to generate the reference characteristic spectrum of Uoganning Granules.

[0020] Preferably, the control characteristic spectrum generated in step (4) includes 12 common chromatographic peaks, specifically: Peak 7 corresponding to aesculetin and Peak 11 corresponding to luteolin.

[0021] 1. Preferably, the characteristic peak is based on the chromatographic peak of aesculetin, and the relative retention times of the 12 chromatographic peaks are: peak 1 is 0.20, peak 2 is 0.34, peak 3 is 0.36, peak 4 is 0.40, peak 5 is 0.17, peak 6 is 0.53, peak 8 is 1.44, peak 9 is 1.64, peak 10 is 1.83, peak 11 is 1.92, and peak 12 is 2.24, and the relative retention times are all within ±10% of the specified value.

[0022] The beneficial effects of the present invention are:

[0023] (1) The present invention established an HPLC characteristic spectrum for Niaoganning Granules, identified 12 characteristic peaks, and more fully demonstrated the chemical characteristics of Niaoganning Granules;

[0024] (2) The present invention investigated the sample extraction method and the type and ratio of the mobile phase. Within the investigated range, the chromatographic method showed 13 characteristic peaks, and the relative retention time of each peak had a small change;

[0025] (3) A methodological investigation was conducted on the constructed characteristic spectrum method of Uoganning Granules, including instrument precision experiments, method repeatability experiments, and sample stability experiments. The RSD values ​​of the relative retention time of each peak in each experimental result were all ≤2.0%, and the RSD values ​​of the relative peak area were all ≤10.0%. This shows that the method is good and can reflect the major chemical components in Uoganning Granules. It can control the overall quality of Uoganning Granules more efficiently and quickly. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention will be further described below with reference to the accompanying drawings.

[0027] Figure 1 Specificity chromatogram; where S1: negative solution; S2: luteolin control; S3: aesculetin control; S4: test solution;

[0028] Figure 2Precision test chromatogram; among them, S1~S6: precision 1~6;

[0029] Figure 3 Repeatability test chromatogram; among them, S1~S6: repeatability 1~6;

[0030] Figure 4 Stability test chromatogram; S1~S6: stability 1~6;

[0031] Figure 5 Chromatograms of eight batches of Niaoganning granules samples; among them, S1: 2302013; S2: 2303026; S3: 2304005; S4: 2304011; S5: 2305016; S6: 2307002; S7: 2309015; S8: 2401002;

[0032] Figure 6 Comparative characteristic spectrum of Urinary Ning Granules. DETAILED DESCRIPTION

[0033] To facilitate understanding by those skilled in the art, the present invention is further described below with reference to embodiments and drawings.

[0034] Example 1

[0035] 1. Instruments and test drugs

[0036] 1.1 Instruments and Equipment

[0037] .

[0038] 1.2 Test materials

[0039] .

[0040] 1.3 Reference Material Information

[0041] .

[0042] 1.4 Sample Information

[0043] .

[0044] Example 2 Investigation of the method for constructing the characteristic spectrum of Nianganning granules

[0045] 2.1 Investigation of test sample preparation methods

[0046] 2.1.1 Investigation of extraction solvents for test samples

[0047] Chromatographic conditions

[0048] Chromatographic column: Topsil C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-tetrahydrofuran-0.1% trifluoroacetic acid (85:10:5); mobile phase B: 10 mM phosphoric acid solution-0.05% sodium heptanesulfonate solution (94:5); gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 270 nm. The number of theoretical plates calculated based on the aesculetin peak should be no less than 5000.

[0049] .

[0050] Preparation of reference solution: Take appropriate amount of esculentine and luteolin reference substances, weigh accurately, add methanol to make a solution containing 50ug per 1mL, shake well, and use as reference solution.

[0051] Preparation of test solution

[0052] Take an appropriate amount of Nianganning granules, grind them into powder, take 1g, and place it in a 50mL stoppered conical flask; add 40mL of methanol-water-0.1% formic acid solution with a volume ratio of 70:28:2, methanol-water-0.1% formic acid solution with a volume ratio of 50:50:2, and methanol-water solution with a volume ratio of 70:28, respectively, and perform ultrasound-assisted extraction for 45 minutes. The ultrasound parameters are power 250W, frequency 40kHz, and temperature 40℃; centrifuge at 4000rpm for 10 minutes, take the supernatant, and filter it through a 0.45μm microporous membrane; use Activate the C18 solid phase extraction column with 5 mL of methanol and 5 mL of ultrapure water to keep the column bed moist; slowly load the primary filtrate onto the activated C18 column at a flow rate of 1 mL / min; elute with 5 mL of ultrapure water-methanol solution with a volume ratio of 95:5 to remove impurities; elute the target component with 5 mL of methanol-0.1% formic acid aqueous solution with a volume ratio of 80:20, and collect the eluate; concentrate the eluate at 40°C under nitrogen blowdown to near dryness, redissolve it with 70% methanol and make up to 2 mL, filter it through a 0.22 μm filter membrane to obtain the test solution.

[0053] Assay

[0054] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0055] Solvent investigations revealed that peak separation and peak shape were better when the extraction solvent contained 0.1% formic acid and the solvent volume ratio was 70:28:2. However, when the solvent lacked formic acid, peak shape was poor and peaks were missing, failing to achieve the desired effect. Therefore, a methanol-water-0.1% formic acid solution with a volume ratio of 70:28:2 was selected as the extraction solvent.

[0056] Table 1 Results of relative retention time of solvent investigation

[0057] .

[0058] 2.1.2 Investigation of test sample extraction methods

[0059] Preparation of the test solution: Take an appropriate amount of Nianganning granules, grind them into powder, take 1g, and place it in a 50mL stoppered conical flask; add 40mL of methanol-water-0.1% formic acid solution with a volume ratio of 70:28:2, and perform ultrasonic-assisted extraction (power 250W, frequency 40kHz, temperature 40℃) and heating reflux extraction for 45 minutes respectively; centrifuge at 4000rpm for 10 minutes, take the supernatant, and filter it through a 0.45μm microporous membrane; activate it with 5mL of methanol and 5mL of ultrapure water in sequence. C18 solid phase extraction column, keep the column bed moist; slowly load the primary filtrate onto the activated C18 column at a flow rate of 1 mL / min; elute with 5 mL of ultrapure water-methanol solution with a volume ratio of 95:5 to remove impurities; elute the target component with 5 mL of methanol-0.1% formic acid aqueous solution with a volume ratio of 80:20, and collect the eluate; concentrate the eluate to near dryness under nitrogen blow at 40°C, re-dissolve it with 70% methanol and make up to 2 mL, filter it through a 0.22 μm filter membrane to obtain the test solution.

[0060] The chromatographic conditions are the same as those in item “2.1.1”; the reference solution is the reference solution in item “2.1.1”.

[0061] Assay

[0062] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0063] The results of the extraction method investigation showed that ultrasonic extraction provided better peak separation and a more optimal peak shape. Heating and reflux extraction resulted in poor peak shape and missing peaks, failing to achieve the desired effect. Therefore, ultrasonic extraction was selected as the extraction method for Uoganning Granules.

[0064] Table 2 Results of relative retention time of extraction method

[0065] .

[0066] 2.2 Investigation of chromatographic conditions

[0067] 2.2.1 Investigation of mobile phase types

[0068] Chromatographic conditions 1: Topsil C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-tetrahydrofuran-0.1% trifluoroacetic acid (85:10:5); mobile phase B: 10 mM phosphoric acid solution-0.05% sodium heptanesulfonate solution (94:5); gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 270 nm. The theoretical plate number calculated based on the aesculetin peak should be no less than 5000.

[0069] .

[0070] Chromatographic conditions 2: Topsil C18 (4.6 x 250 mm, 5 μm); acetonitrile-tetrahydrofuran (85:10) as mobile phase A, 10 mM phosphoric acid as mobile phase B, gradient elution as specified in the table below; column temperature 30°C; detection wavelength 270 nm. The theoretical plate number calculated based on the aesculetin peak should be no less than 5000.

[0071] .

[0072] Chromatographic conditions 3: Topsil C18 (4.6 x 250 mm, 5 μm); acetonitrile as mobile phase A, 10 mM phosphoric acid solution-0.05% sodium heptanesulfonate solution (94:5) as mobile phase B, gradient elution as specified in the table below; column temperature 30°C; detection wavelength 270 nm. The theoretical plate number calculated based on the aesculetin peak should be no less than 5000.

[0073] .

[0074] Preparation of reference solution

[0075] Take appropriate amount of esculentine and luteolin reference substances, weigh accurately, add methanol to make a solution containing 50ug per 1mL, shake well, and use it as the reference solution.

[0076] Preparation of test solution

[0077] Take an appropriate amount of Nianganning granules, grind them into powder, take 1g, and place it in a 50mL stoppered conical flask; add 40mL of methanol-water-0.1% formic acid solution with a volume ratio of 70:28:2, and perform ultrasonic-assisted extraction for 45 minutes. The ultrasonic parameters are power 250W, frequency 40kHz, and temperature 40℃. Centrifuge at 4000rpm for 10 minutes, take the supernatant, and filter it through a 0.45μm microporous membrane; activate the C18 solid-phase extraction with 5mL of methanol and 5mL of ultrapure water in sequence. column, keeping the column bed moist; slowly load the primary filtrate onto the activated C18 column at a flow rate of 1 mL / min; elute with 5 mL of ultrapure water-methanol solution with a volume ratio of 95:5 to remove impurities; elute the target component with 5 mL of methanol-0.1% formic acid aqueous solution with a volume ratio of 80:20, and collect the eluate; concentrate the eluate at 40°C with nitrogen blowdown to near dryness, re-dissolve it with 70% methanol and make up to 2 mL, and filter it through a 0.22 μm filter membrane to obtain the test solution.

[0078] Assay

[0079] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0080] The results showed that when elution was performed with acetonitrile-tetrahydrofuran-0.1% trifluoroacetic acid (85:10:5) as mobile phase A and 10mM phosphoric acid solution-0.05% sodium heptane sulfonate solution (94:5) as mobile phase B, the test sample chromatogram presented a total of 12 characteristic peaks, with good peak separation and excellent peak shape; when elution was performed with acetonitrile-tetrahydrofuran (85:10) as mobile phase A and 10mM phosphoric acid solution as mobile phase B or with acetonitrile as mobile phase A and 10mM phosphoric acid solution-0.05% sodium heptane sulfonate solution (94:5) as mobile phase B, all peaks were missing and the corresponding effect was not achieved.

[0081] Table 3 Relative retention time results of mobile phase type investigation

[0082] .

[0083] 2.2.2 Investigation of mobile phase ratio

[0084] Chromatographic conditions

[0085] Chromatographic column: Topsil C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-tetrahydrofuran-0.1% trifluoroacetic acid (85:10:5); mobile phase B: 10 mM phosphoric acid solution-0.05% sodium heptanesulfonate solution (94:5); gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 270 nm. The number of theoretical plates calculated based on the aesculetin peak should be no less than 5000.

[0086] Mobile phase ratio 1:

[0087] .

[0088] Mobile phase ratio 2:

[0089] .

[0090] Mobile phase ratio 3:

[0091] .

[0092] The reference solution is the same as that in item “2.2.1”.

[0093] Preparation of test solution

[0094] Take an appropriate amount of Nianganning granules, grind them into powder, take 1g, and place it in a 50mL stoppered conical flask; add 40mL of methanol-water-0.1% formic acid solution with a volume ratio of 70:28:2, and perform ultrasonic-assisted extraction for 45 minutes. The ultrasonic parameters are power 250W, frequency 40kHz, and temperature 40℃. Centrifuge at 4000rpm for 10 minutes, take the supernatant, and filter it through a 0.45μm microporous membrane; activate the C18 solid-phase extraction with 5mL of methanol and 5mL of ultrapure water in sequence. column, keeping the column bed moist; slowly load the primary filtrate onto the activated C18 column at a flow rate of 1 mL / min; elute with 5 mL of ultrapure water-methanol solution with a volume ratio of 95:5 to remove impurities; elute the target component with 5 mL of methanol-0.1% formic acid aqueous solution with a volume ratio of 80:20, and collect the eluate; concentrate the eluate at 40°C with nitrogen blowdown to near dryness, re-dissolve it with 70% methanol and make up to 2 mL, and filter it through a 0.22 μm filter membrane to obtain the test solution.

[0095] Assay

[0096] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0097] The results showed that when eluted with a mobile phase ratio of 1, the test sample chromatogram showed a total of 12 characteristic peaks, with good peak separation and excellent peak shape; when eluted with a mobile phase ratio of 2 or a mobile phase ratio of 3, each peak was missing and the corresponding effect was not achieved.

[0098] Table 4 Results of relative retention time of mobile phase ratio

[0099] .

[0100] Example 3 Methodological Verification of the Characteristic Spectrum of Nianganning Granules

[0101] 3.1 Exclusivity

[0102] Chromatographic conditions

[0103] Chromatographic column: Topsil C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-tetrahydrofuran-0.1% trifluoroacetic acid (85:10:5); mobile phase B: 10 mM phosphoric acid solution-0.05% sodium heptanesulfonate solution (94:5); gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 270 nm. The number of theoretical plates calculated based on the aesculetin peak should be no less than 5000.

[0104] .

[0105] Preparation of reference solution

[0106] Take appropriate amount of esculentine and luteolin reference substances, weigh accurately, add methanol to make a solution containing 50ug per 1mL, shake well, and use it as the reference solution.

[0107] Preparation of test solution

[0108] Take an appropriate amount of Nianganning granules, grind them into powder, take 1g, and place it in a 50mL stoppered conical flask; add 40mL of methanol-water-0.1% formic acid solution with a volume ratio of 70:28:2, and perform ultrasonic-assisted extraction for 45 minutes. The ultrasonic parameters are power 250W, frequency 40kHz, and temperature 40℃. Centrifuge at 4000rpm for 10 minutes, take the supernatant, and filter it through a 0.45μm microporous membrane; activate the C18 solid-phase extraction with 5mL of methanol and 5mL of ultrapure water in sequence. column, keeping the column bed moist; slowly load the primary filtrate onto the activated C18 column at a flow rate of 1 mL / min; elute with 5 mL of ultrapure water-methanol solution with a volume ratio of 95:5 to remove impurities; elute the target component with 5 mL of methanol-0.1% formic acid aqueous solution with a volume ratio of 80:20, and collect the eluate; concentrate the eluate at 40°C with nitrogen blowdown to near dryness, re-dissolve it with 70% methanol and make up to 2 mL, and filter it through a 0.22 μm filter membrane to obtain the test solution.

[0109] Preparation of negative solution

[0110] Take an appropriate amount of excipients, grind them into powder, take 1g, and place it in a 50mL stoppered conical flask; add 40mL of methanol-water-0.1% formic acid solution with a volume ratio of 70:28:2, and perform ultrasonic-assisted extraction for 45 minutes. The ultrasonic parameters are power 250W, frequency 40kHz, and temperature 40℃; centrifuge at 4000rpm for 10 minutes, take the supernatant, and filter it through a 0.45μm microporous membrane; activate the C18 solid phase extraction column with 5mL of methanol and 5mL of ultrapure water in turn. Keep the column bed moist; slowly load the primary filtrate onto the activated C18 column at a flow rate of 1 mL / min; elute with 5 mL of ultrapure water-methanol solution with a volume ratio of 95:5 to remove impurities; elute the target component with 5 mL of methanol-0.1% formic acid aqueous solution with a volume ratio of 80:20, and collect the eluate; concentrate the eluate to near dryness under nitrogen blow at 40°C, re-dissolve it with 70% methanol and make up to 2 mL, filter it through a 0.22 μm filter membrane to obtain the test solution.

[0111] Assay

[0112] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0113] The results showed that the negative solution had no interference and good specificity. Figure 1 .

[0114] 3.2 Precision

[0115] Grind 1 g of Nianganning Granules (Batch No. 2302013) into powder and prepare the test solution according to the test solution preparation method under "3.1". Inject 6 times according to the chromatographic conditions under "3.1", record the chromatogram, measure the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0116] Table 5 Precision relative retention time results

[0117] .

[0118] Table 6 Precision relative peak area results

[0119] .

[0120] The results showed that the relative retention time RSD of each chromatographic peak was less than 1.0%, and the relative peak area RSD was less than 8.0%, indicating good precision. The chromatogram is attached. Figure 2 .

[0121] 3.3 Repeatability

[0122] Grind Niaoganning Granules (Batch No. 2302013) into powder, take 1 g of the granules, and prepare the test solution according to the test solution preparation method under "3.1". Inject the sample according to the chromatographic conditions under "3.1", record the chromatogram, measure the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0123] Table 7 Repeatability relative retention time results

[0124] .

[0125] Table 8 Repeatability relative peak area results

[0126] .

[0127] The results showed that the relative retention time RSD of each chromatographic peak was less than 1.0%, and the relative peak area RSD was less than 10.0%, with good repeatability. The chromatogram is attached. Figure 3 .

[0128] 3.4 Stability

[0129] Grind 1 g of Nianganning Granules (Batch No. 2302013) into powder and prepare the test solution according to the test solution preparation method under "3.1". Inject the sample at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h according to the chromatographic conditions under "3.1". Record the chromatogram, measure the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0130] Table 9 Stability relative retention time results

[0131] .

[0132] Table 10 Stability relative peak area results

[0133] .

[0134] The results showed that the relative retention time RSD of each chromatographic peak was less than 2.0%, and the relative peak area RSD was less than 10.0%, indicating that the solution was stable. The chromatogram is attached. Figure 4 .

[0135] In summary, the method demonstrated good specificity through specificity testing, with no interference from the negative solution. In precision testing, the relative retention time RSDs for each chromatographic peak were less than 1.0%, and the relative peak area RSDs were less than 8.0%, indicating good precision. In repeatability testing, the relative retention time RSDs for each chromatographic peak were less than 1.0%, and the relative peak area RSDs were less than 10.0%, indicating good repeatability. In stability testing, the relative retention time RSDs for each chromatographic peak were less than 2.0%, and the relative peak area RSDs were less than 10.0%, indicating good solution stability. This method has been methodologically validated, and the results are reliable and accurate.

[0136] Example 4 Construction of the Control Characteristic Spectrum of Nianganning Granules

[0137] Chromatographic conditions

[0138] Chromatographic column: Topsil C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-tetrahydrofuran-0.1% trifluoroacetic acid (85:10:5); mobile phase B: 10 mM phosphoric acid solution-0.05% sodium heptanesulfonate solution (94:5); gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 270 nm. The number of theoretical plates calculated based on the aesculetin peak should be no less than 5000.

[0139] .

[0140] Preparation of reference solution

[0141] Take appropriate amount of esculentine and luteolin reference substances, weigh accurately, add methanol to make a solution containing 50ug per 1mL, shake well, and use it as the reference solution.

[0142] Preparation of test solution

[0143] Take an appropriate amount of Nianganning granules, grind them into powder, take 1g, and place it in a 50mL stoppered conical flask; add 40mL of methanol-water-0.1% formic acid solution with a volume ratio of 70:28:2, and perform ultrasonic-assisted extraction for 45 minutes. The ultrasonic parameters are power 250W, frequency 40kHz, and temperature 40℃. Centrifuge at 4000rpm for 10 minutes, take the supernatant, and filter it through a 0.45μm microporous membrane; activate the C18 solid-phase extraction with 5mL of methanol and 5mL of ultrapure water in sequence. column, keeping the column bed moist; slowly load the primary filtrate onto the activated C18 column at a flow rate of 1 mL / min; elute with 5 mL of ultrapure water-methanol solution with a volume ratio of 95:5 to remove impurities; elute the target component with 5 mL of methanol-0.1% formic acid aqueous solution with a volume ratio of 80:20, and collect the eluate; concentrate the eluate at 40°C with nitrogen blowdown to near dryness, re-dissolve it with 70% methanol and make up to 2 mL, and filter it through a 0.22 μm filter membrane to obtain the test solution.

[0144] Assay

[0145] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0146] The chromatograms of the test samples of 8 batches of Niaoganning Granules were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" and the chromatographic peaks present in the chromatograms of different batches of Niaoganning Granules were selected as common peaks; the control characteristic spectrum was generated using the average calculation method, and the relative retention time and relative peak area of ​​each common peak were calculated. Figure 5 、 Figure 6 .

[0147] Table 11 Relative retention time results of eight batches of Niaoganning granules samples

[0148] .

[0149] Table 12 Relative peak area results of eight batches of Niaoganning granules samples

[0150] .

[0151] The chromatograms of the eight batches of Nianganning Granules samples all showed 12 common peaks. The RSDs of the relative retention times of the 12 common peaks were all less than 2.0%, and the RSDs of the relative peak areas were all less than 10.0%.

[0152] The test sample chromatogram shows a total of 12 characteristic peaks, of which Peak 7 and Peak 11 should correspond to the peaks of the aesculetin and luteolin reference substances, respectively. The peak corresponding to the peak of the aesculetin reference substance is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.20 (peak 1), 0.34 (peak 2), 0.36 (peak 3), 0.40 (peak 4), 0.17 (peak 5), 0.53 (peak 6), 1.44 (peak 8), 1.64 (peak 9), 1.83 (peak 10), 1.92 (peak 11), and 2.24 (peak 12).

Claims

1. A method for constructing a characteristic spectrum of Nianganning granules, characterized in that: The following steps are involved: (1) Preparation of reference solution: Take the reference substance, weigh it accurately, add methanol to make a solution containing 50 μg per 1 mL, shake well, and use it as the reference solution; the reference substances are: aesculetin and luteolin; (2) Preparation of the test solution: Take the Nianganning granules, grind them into powder, and place them in a 50 mL stoppered conical flask; add 40 mL of methanol-water-0.1% formic acid solution with a volume ratio of 70:28:2, and perform ultrasonic-assisted extraction for 45 minutes; centrifuge after ultrasonication, take the supernatant, and filter the supernatant through a 0.45 μm microporous filter membrane to obtain the primary filtrate; activate the C18 solid phase extraction column with 5 mL of methanol and 5 mL of ultrapure water in sequence, keeping the column bed wet; slowly load the primary filtrate onto the activated C18 column at a flow rate of 1 mL / min; elute with ultrapure water-methanol solution to remove impurities; elute the target component with methanol-0.1% formic acid aqueous solution, and collect the eluate; concentrate the eluate to near dryness by nitrogen blowing at 40°C, re-dissolve it with 70% methanol and make it up to 2 mL, filter it through a 0.22 μm filter membrane to obtain the test solution; (3) Determination: The reference solution and the test solution were injected into a high performance liquid chromatograph under the following chromatographic conditions, with 10 μl of each sample injected, and the chromatogram was recorded; the chromatographic conditions were: octadecylsilane bonded silica gel as the filler; acetonitrile-tetrahydrofuran-0.1% trifluoroacetic acid in a volume ratio of 85:10:5 as the mobile phase A, and 10 mM phosphoric acid solution-0.05% sodium heptane sulfonate solution in a volume ratio of 94:5 as the mobile phase B, and elution was performed according to the prescribed gradient; the column temperature was 30°C; the flow rate was 1 mL per minute; the UV-visible detector was used, and the detection wavelength was 270 nm; The elution gradient conditions were: From 0 to 25 min, the volume ratio of mobile phase A:phase B was changed from 5:95 to 10:90; From 25 to 50 min, the volume ratio of mobile phase A:phase B was changed from 10:90 to 20:80; From 50 to 65 min, the volume ratio of mobile phase A:phase B was changed from 20:80 to 30:70; From 65 to 75 min, the volume ratio of mobile phase A:phase B was changed from 30:70 to 60:40; From 75 to 78 min, the volume ratio of mobile phase A:phase B was changed from 60:40 to 5:95; 78-90 min, mobile phase A:B, volume ratio 5:95; (4) Generate a control characteristic spectrum: select the chromatographic peaks that exist in the chromatograms of different batches of Uoganning Granules as common peaks, and use the average value calculation method to generate a control characteristic spectrum of Uoganning Granules.

2. The method for constructing a characteristic spectrum of Nianganning granules according to claim 1, characterized in that: The ultrasonic extraction conditions in step (2) are as follows: ultrasonic parameters: power 250 W, frequency 40 kHz, and temperature 40°C.

3. The method for constructing a characteristic spectrum of Nianganning granules according to claim 1, characterized in that: The centrifugation condition in step (2) is 4000 rpm for 10 minutes.

4. The method for constructing a characteristic spectrum of Nianganning granules according to claim 1, characterized in that: The volume ratio of pure water to methanol solution used for eluting and removing impurities in step (2) is 95:5, and the amount used is 5 mL.

5. The method for constructing a characteristic spectrum of Nianganning granules according to claim 1, characterized in that: In step (2), the volume ratio of methanol-0.1% formic acid aqueous solution for eluting the target component is 80:20, and the amount used is 5 mL.

6. The method for constructing a characteristic spectrum of Nianganning granules according to claim 1, characterized in that: The chromatographic column in step (3) is Topsil C18, with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

7. The method for constructing a characteristic spectrum of Nianganning granules according to claim 1, characterized in that: The control characteristic spectrum generated in step (4) includes 12 common chromatographic peaks, among which: peak 7 corresponds to esculentine, and peak 11 corresponds to luteolin.

8. The method for constructing a characteristic spectrum of Nianganning granules according to claim 1, characterized in that: The characteristic peaks were based on the chromatographic peak of aesculetin. The relative retention times of the 12 chromatographic peaks were: peak 1 was 0.20, peak 2 was 0.34, peak 3 was 0.36, peak 4 was 0.40, peak 5 was 0.17, peak 6 was 0.53, peak 8 was 1.44, peak 9 was 1.64, peak 10 was 1.83, peak 11 was 1.92, and peak 12 was 2.24.

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