Method for constructing specific chromatogram of urine sensitive particles

Abstract

The invention relates to the technical field of traditional Chinese medicine analysis, in particular to a construction method of a specific chromatogram of urine sensitive particles. Comprising the following steps: 1, preparing a reference substance solution; 2, preparing a test solution; 3, determining conditions; and 4, generating a contrast characteristic spectrum. According to the method, the HPLC characteristic chromatogram is established for the urine-sensitive granules, 12 characteristic peaks are defined, and the chemical characteristics of the urine-sensitive granules are relatively fully displayed. The RSD value of the relative retention time of each peak is less than or equal to 2.0%, and the RSD value of the relative peak area is less than or equal to 10.0%. The method is good, and large types of chemical components in the urine sensitive Ning particles can be reflected through the method. And the overall quality of the Uishenning 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 particularly relates to a method for constructing a characteristic chromatogram of Nioganling Granules. Background Art

[0002] Nioganling Granules are composed of five Chinese medicinal herbs, namely Lygodium japonicum (Thunb.) Sw., Glechoma longituba (Nakai) Kupr., Pteris multifida Poir., Humulus scandens (Lour.) Merr., and Viola philippica Cav., and its quality standard is included in the first volume of the Chinese Pharmacopoeia (2020 Edition). In the current standard of Nioganling Granules, there are only character identification and routine inspections of granules, lacking a characteristic chromatogram item that can comprehensively control the quality of Nioganling Granules. Moreover, there is almost no research on the characteristic chromatogram item of Nioganling Granules in the literature, and the quality standard is not perfect enough to effectively control the quality of this variety.

[0003] Nioganling Granules are composed of five traditional Chinese medicines, and each medicinal material contains various active ingredients (such as flavonoids, phenolic acids, alkaloids, etc.). There may be chromatographic peaks with similar or overlapping physicochemical properties among the components, resulting in great separation difficulty.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] In order to make up for the deficiencies in the prior art, the present invention provides a method for constructing a characteristic chromatogram of Nioganling Granules.

[0006] The present invention is realized through the following technical solutions: The present invention provides a method for constructing a characteristic chromatogram of Nioganling Granules, comprising the following steps: (1) Preparation of the reference substance solution: Weigh an appropriate amount of the reference substance accurately, and dissolve it in methanol respectively to make a solution containing 50 μg per 1 mL, shake well, and use it as the reference substance solution; the reference substances are: esculetin and luteoloside; (2) Preparation of the test solution: Weigh an appropriate amount of Nioganling granules and grind them finely. Take 1 g and place it in a 50 mL stoppered conical flask. Add 40 mL of a methanol - water - 0.1% formic acid solution with a volume ratio of 70:28:2 (where 0.1% formic acid is a volume fraction). Perform ultrasonic - assisted extraction for 45 minutes with ultrasonic parameters of power 250 W, frequency 40 kHz, and temperature 40 °C. Centrifuge at 4000 rpm for 10 minutes, take the supernatant, and filter it initially through a 0.45 μm microporous membrane to obtain the initial filtrate. Activate a C18 solid - phase extraction column successively with 5 mL of methanol and 5 mL of ultrapure water to keep the column bed moist. Slowly load the initial filtrate onto the activated C18 column at a flow rate of 1 mL / min. Elute the impurities with 5 mL of a 95:5 ultrapure water - methanol solution. Elute the target components with 5 mL of an 80:20 methanol - 0.1% formic acid aqueous solution, and collect the eluate. Concentrate the eluate to nearly dry by nitrogen blowing at 40 °C, redissolve it with 70% methanol and make the volume up to 2 mL, and filter it through a 0.22 μm membrane to obtain the test solution. (3) Determination: Inject the reference solution and the test solution into a high - performance liquid chromatograph under the following chromatographic conditions, each injecting 10 μL, and record the chromatogram. The chromatographic conditions are as follows: Use octadecylsilane - bonded silica gel as the filler (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm). Use acetonitrile - tetrahydrofuran - 0.1% trifluoroacetic acid (85:10:5) as mobile phase A, and 10 mM phosphoric acid solution - 0.05% sodium heptanesulfonate solution (94:5) as mobile phase B (where 0.1% trifluoroacetic acid (85:10:5) is a volume fraction and 0.05% sodium heptanesulfonate solution is a mass fraction). Perform elution according to the specified gradient. The column temperature is 30 °C, the flow rate is 1 mL per minute, and use an ultraviolet - visible (UV - Vis) detector with a detection wavelength of 270 nm. The gradient conditions are as follows: 0 - 25 min, mobile phase A:B, volume ratio changes from 5:95 to 10:90; 25 - 50 min, mobile phase A:B, volume ratio changes from 10:90 to 20:80; 50 - 65 min, mobile phase A:B, volume ratio changes from 20:80 to 30:70; 65 - 75 min, mobile phase A:B, volume ratio changes from 30:70 to 60:40; 75 - 78 min, mobile phase A:B, volume ratio changes from 60:40 to 5:95; 78 - 90 min, mobile phase A:B, volume ratio is 5:95; (4)Generate a reference characteristic chromatogram: Using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" formulated by the Pharmacopoeia Commission of the People's Republic of China, select the chromatographic peaks that exist in the chromatograms of different batches of Nioganling granules as common peaks, and generate the reference characteristic chromatogram of Nioganling granules by the average value calculation method.

[0007] Preferably, the reference characteristic chromatogram generated in step (4) includes 12 common chromatographic peaks, specifically: peak 7 corresponding to esculetin and peak 11 corresponding to luteoloside.

[0008] 1. Preferably, taking the esculetin chromatographic peak as a reference, the relative retention times of the 12 chromatographic peaks are as follows: 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, peak 12 is 2.24, and their relative retention times are all within the range of ±10% of the specified value.

[0009] Advantages of the present invention: (1)The present invention has established an HPLC characteristic chromatogram for Nioganling granules, identified 12 characteristic peaks, and relatively fully demonstrated the chemical characteristics of Nioganling granules; (2)The present invention has investigated the extraction method of the sample and the types and proportions of the mobile phases. Within the investigated range, 13 characteristic peaks are presented in this chromatographic method, and the relative retention times of each peak vary relatively little; (3)Methodology investigation is carried out on the established characteristic chromatogram method for Nioganling granules, including instrument precision experiment, method repeatability experiment, and sample stability experiment. In the results of each experiment, the RSD values of the relative retention times of each peak are all ≤2.0%, and the RSD values of the relative peak areas are all ≤10.0%. It shows that the method is good, and the major chemical components in Nioganling granules can be reflected by this method. The overall quality of Nioganling granules can be controlled more efficiently and quickly. Description of the Drawings

[0010] The present invention will be further described below with reference to the drawings.

[0011] Figure 1 Specificity chromatogram; wherein, S1: negative solution; S2: luteoloside control; S3: esculetin control; S4: test solution; Figure 2 Precision test chromatogram; wherein, S1~S6: precision 1~6; Figure 3 Repeatability test chromatogram; wherein, S1~S6: repeatability 1~6; Figure 4Stability test chromatogram; where S1 - S6: Stability 1 - 6; Figure 5 Chromatograms of eight batches of Nioganling granules samples; where S1: 2302013; S2: 2303026; S3: 2304005; S4: 2304011; S5: 2305016; S6: 2307002; S7: 2309015; S8: 2401002; Figure 6 Reference characteristic chromatogram of Nioganling granules. Detailed implementation manners

[0012] For the convenience of those skilled in the art to understand, the present invention will be further described below in conjunction with the embodiments and the drawings.

[0013] Embodiment 1 1. Instruments and test drugs 1.1 Instrument equipment 。

[0014] 1.2 Test materials 。

[0015] 1.3 Reference substance information 。

[0016] 1.4 Sample information 。

[0017] Investigation on the construction method of the characteristic chromatogram of Nioganling granules in Embodiment 2 2.1 Investigation on the preparation method of the test sample 2.1.1 Investigation on the extraction solvent of the test sample Chromatographic conditions Chromatographic column: Topsil C18 (4.6x250mm, 5μm); Using acetonitrile - tetrahydrofuran - 0.1% trifluoroacetic acid (85:10:5) as mobile phase A, and 10mM phosphoric acid solution - 0.05% sodium heptanesulfonate solution (94:5) as mobile phase B, and perform gradient elution according to the regulations in the following table; Column temperature is 30°C; Detection wavelength is 270nm. The number of theoretical plates calculated by the peak of aesculin should be not less than 5000.

[0018] 。

[0019] Preparation of the reference solution Weigh appropriate amounts of aesculin and luteoloside reference substances accurately, and dissolve them in methanol respectively to prepare solutions containing 50μg per 1mL, shake well, and use them as the reference solution.

[0020] Preparation of the test sample solution Take an appropriate amount of Nioganling Granules, grind them finely, take 1 g, and place it 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, 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 ultrasonic-assisted extraction for 45 minutes. The ultrasonic parameters are power 250 W, frequency 40 kHz, and temperature 40 °C; centrifuge at 4000 rpm for 10 minutes, take the supernatant, and preliminarily filter it through a 0.45 μm microporous filter membrane; activate the C18 solid-phase extraction column with 5 mL of methanol and 5 mL of ultrapure water in sequence to keep the column bed moist; slowly load the preliminary filtrate onto the activated C18 column at a flow rate of 1 mL / min; elute and remove impurities with 5 mL of ultrapure water-methanol solution with a volume ratio of 95:5; elute the target components 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 nearly dry by nitrogen blowing at 40 °C, redissolve it with 70% methanol and make the volume up to 2 mL, and filter it through a 0.22 μm filter membrane to obtain the test solution.

[0021] Determination method Precisely pipette 10 μl of the reference substance solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0022] The results of solvent investigation showed that when the extraction solvent contained 0.1% formic acid and the volume ratio of the extraction solvent was 70:28:2, the peak separation degree was better and the peak shape was better; when there was no formic acid in the extraction solvent, there were situations of poor peak shape and peak missing, and the corresponding effects could not be achieved. Therefore, the methanol-water-0.1% formic acid solution with a volume ratio of 70:28:2 was selected as the extraction solvent.

[0023] Table 1 Results of relative retention time of solvent investigation 。

[0024] 2.1.2 Investigation of the extraction method of the test sample Preparation of the test solution: Take an appropriate amount of Nioganling Granules, grind them finely, take 1 g, and place it in a 50 mL stoppered conical flask; add 40 mL of a methanol-water-0.1% formic acid solution with a volume ratio of 70:28:2, and perform ultrasonic-assisted extraction (power 250 W, frequency 40 kHz, temperature 40 °C) and heating under reflux for 45 minutes respectively; centrifuge at 4000 rpm for 10 minutes, take the supernatant, and preliminarily filter it through a 0.45 μm microporous membrane; activate the C18 solid-phase extraction column with 5 mL of methanol and 5 mL of ultrapure water in sequence to keep the column bed moist; slowly load the preliminary filtrate onto the activated C18 column at a flow rate of 1 mL / min; elute and remove impurities with 5 mL of an ultrapure water-methanol solution with a volume ratio of 95:5; elute the target components with 5 mL of a methanol-0.1% formic acid aqueous solution with a volume ratio of 80:20, and collect the eluate; concentrate the eluate to nearly dry by nitrogen blowing at 40 °C, redissolve it with 70% methanol and make the volume up to 2 mL, and filter it through a 0.22 μm membrane to obtain the test solution.

[0025] The chromatographic conditions are the same as those under item "2.1.1"; use the reference substance solution under item "2.1.1" as the reference substance solution.

[0026] Assay method Precisely pipette 10 μl of the reference substance solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0027] The results of the investigation on the extraction method showed that the peak resolution obtained by the ultrasonic extraction method was better and the peak shape was more excellent; when heating under reflux extraction was carried out, there were situations of poor peak shape and peak missing, and the corresponding effects could not be achieved. Therefore, ultrasonic extraction was selected as the extraction method for Nioganling Granules.

[0028] Table 2 Results of the relative retention time of the investigation on the extraction method 。

[0029] 2.2 Investigation on chromatographic conditions 2.2.1 Investigation on the type of mobile phase Chromatographic condition 1: Topsil C18 (4.6x250 mm, 5 μm); use acetonitrile-tetrahydrofuran-0.1% trifluoroacetic acid (85:10:5) as mobile phase A, and use 10 mM phosphoric acid solution-0.05% sodium heptanesulfonate solution (94:5) as mobile phase B, and perform gradient elution according to the regulations in the following table; the column temperature is 30 °C; the detection wavelength is 270 nm. The number of theoretical plates calculated by the aesculin peak should be not less than 5000.

[0030] 。

[0031] Chromatographic conditions 2: Topsil C18 (4.6x250mm, 5μm); acetonitrile - tetrahydrofuran (85:10) as mobile phase A, 10 mM phosphoric acid solution as mobile phase B, gradient elution was carried out according to the regulations in the following table; column temperature was 30°C; detection wavelength was 270 nm. The number of theoretical plates calculated based on the scopoletin peak should be not less than 5000.

[0032] 。

[0033] Chromatographic conditions 3: Topsil C18 (4.6x250mm, 5μm); acetonitrile as mobile phase A, 10 mM phosphoric acid solution - 0.05% sodium heptanesulfonate solution (94:5) as mobile phase B, gradient elution was carried out according to the regulations in the following table; column temperature was 30°C; detection wavelength was 270 nm. The number of theoretical plates calculated based on the scopoletin peak should be not less than 5000.

[0034] 。

[0035] Preparation of reference substance solution Appropriately weigh an appropriate amount of scopoletin and cynaroside reference substances, and dissolve them in methanol respectively to make solutions containing 50 μg per 1 mL. Shake well to obtain the reference substance solution.

[0036] Preparation of test solution Take an appropriate amount of Nioganling granules, grind them finely, take 1 g, and place it 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. The ultrasonic parameters are power 250 W, frequency 40 kHz, and temperature 40°C; centrifuge at 4000 rpm for 10 minutes, take the supernatant, and filter it through a 0.45 μm microporous filter membrane for primary filtration; activate the C18 solid-phase extraction column with 5 mL of methanol and 5 mL of ultrapure water in sequence 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 and remove impurities with 5 mL of ultrapure water - methanol solution with a volume ratio of 95:5; elute the target components 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 by nitrogen blowing at 40°C, redissolve it with 70% methanol and make the volume up to 2 mL, and filter it through a 0.22 μm filter membrane to obtain the test solution.

[0037] Determination method Precisely pipette 10 μL each of the reference substance solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0038] The results showed that when eluting with acetonitrile - tetrahydrofuran - 0.1% trifluoroacetic acid (85:10:5) as mobile phase A and 10 mM phosphoric acid solution - 0.05% sodium heptanesulfonate solution (94:5) as mobile phase B, 12 characteristic peaks were presented in the chromatogram of the test sample, and the peak separation was good and the peak shape was excellent; when eluting with acetonitrile - tetrahydrofuran (85:10) as mobile phase A and 10 mM phosphoric acid solution as mobile phase B or with acetonitrile as mobile phase A and 10 mM phosphoric acid solution - 0.05% sodium heptanesulfonate solution (94:5) as mobile phase B, each peak was missing and the corresponding effects could not be achieved.

[0039] Table 3 Results of relative retention time for investigation of mobile phase types 。

[0040] 2.2.2 Investigation of mobile phase ratio Chromatographic conditions Chromatographic column: Topsil C18 (4.6x250 mm, 5 μm); acetonitrile - tetrahydrofuran - 0.1% trifluoroacetic acid (85:10:5) as mobile phase A, 10 mM phosphoric acid solution - 0.05% sodium heptanesulfonate solution (94:5) as mobile phase B, gradient elution was carried out according to the regulations in the following table; column temperature was 30 °C; detection wavelength was 270 nm. The theoretical plate number calculated based on the scopoletin peak should be not less than 5000.

[0041] Mobile phase ratio 1: 。

[0042] Mobile phase ratio 2: 。

[0043] Mobile phase ratio 3: 。

[0044] The reference substance solution was taken from the reference substance solution under item "2.2.1".

[0045] Preparation of test sample solution Take an appropriate amount of Nioganling Granules, grind them finely, take 1 g, and place it in a 50 mL stoppered conical flask; add 40 mL of a 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 a power of 250 W, a frequency of 40 kHz, and a temperature of 40 °C; centrifuge at 4000 rpm for 10 minutes, take the supernatant, and preliminarily filter it through a 0.45 μm microporous filter membrane; activate the C18 solid-phase extraction column with 5 mL of methanol and 5 mL of ultrapure water in sequence to keep the column bed moist; slowly load the preliminary filtrate onto the activated C18 column at a flow rate of 1 mL / min; elute and remove impurities with 5 mL of an ultrapure water-methanol solution with a volume ratio of 95:5; elute the target components with 5 mL of a methanol-0.1% formic acid aqueous solution with a volume ratio of 80:20, and collect the eluate; concentrate the eluate to near dryness by nitrogen blowing at 40 °C, redissolve it with 70% methanol and make the volume up to 2 mL, and filter it through a 0.22 μm filter membrane to obtain the test solution.

[0046] Assay method Precisely pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0047] The results show that when eluting with mobile phase ratio 1, there are 12 characteristic peaks in the chromatogram of the test sample, and the peak resolution is good and the peak shape is excellent; when eluting with mobile phase ratio 2 or mobile phase ratio 3, some peaks are missing and the corresponding effects cannot be achieved.

[0048] Table 4 Results of relative retention time for investigation of mobile phase ratio .

[0049] Methodology verification of the characteristic fingerprint of Nioganling Granules in Example 3 3.1 Specificity Chromatographic conditions Chromatographic column: Topsil C18 (4.6 x 250 mm, 5 μm); use acetonitrile-tetrahydrofuran-0.1% trifluoroacetic acid (85:10:5) as mobile phase A, and 10 mM phosphoric acid solution-0.05% sodium heptanesulfonate solution (94:5) as mobile phase B, and perform gradient elution according to the regulations in the following table; the column temperature is 30 °C; the detection wavelength is 270 nm. The number of theoretical plates calculated based on the esculetin peak should be not less than 5000.

[0050] .

[0051] Preparation of the reference solution Take appropriate amounts of esculetin and luteoloside reference standards, weigh them precisely, and dissolve them in methanol to prepare solutions containing 50 μg per 1 mL respectively. Shake well to obtain the reference solution.

[0052] Preparation of the test solution Take an appropriate amount of Nioganling Granules, grind them finely, take 1 g, and place it in a 50 mL stoppered conical flask; add 40 mL of a 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 a power of 250 W, a frequency of 40 kHz, and a temperature of 40 °C; centrifuge at 4000 rpm for 10 minutes, take the supernatant, and preliminarily filter it through a 0.45 μm microporous filter membrane; activate the C18 solid-phase extraction column with 5 mL of methanol and 5 mL of ultrapure water in sequence to keep the column bed moist; slowly load the preliminary filtrate onto the activated C18 column at a flow rate of 1 mL / min; elute and remove impurities with 5 mL of an ultrapure water-methanol solution with a volume ratio of 95:5; elute the target components with 5 mL of a methanol-0.1% formic acid aqueous solution with a volume ratio of 80:20, and collect the eluate; concentrate the eluate to nearly dry by nitrogen blowing at 40 °C, re-dissolve it with 70% methanol and make the volume up to 2 mL, and filter it through a 0.22 μm filter membrane to obtain the test solution.

[0053] Preparation of negative solution Take an appropriate amount of excipients, grind them finely, take 1 g, and place it in a 50 mL stoppered conical flask; add 40 mL of a 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 a power of 250 W, a frequency of 40 kHz, and a temperature of 40 °C; centrifuge at 4000 rpm for 10 minutes, take the supernatant, and preliminarily filter it through a 0.45 μm microporous filter membrane; activate the C18 solid-phase extraction column with 5 mL of methanol and 5 mL of ultrapure water in sequence to keep the column bed moist; slowly load the preliminary filtrate onto the activated C18 column at a flow rate of 1 mL / min; elute and remove impurities with 5 mL of an ultrapure water-methanol solution with a volume ratio of 95:5; elute the target components with 5 mL of a methanol-0.1% formic acid aqueous solution with a volume ratio of 80:20, and collect the eluate; concentrate the eluate to nearly dry by nitrogen blowing at 40 °C, re-dissolve it with 70% methanol and make the volume up to 2 mL, and filter it through a 0.22 μm filter membrane to obtain the test solution.

[0054] Determination method Precisely pipette 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0055] The results show that the negative solution has no interference and good specificity. The results are shown in the appendix Figure 1 。

[0056] 3.2 Precision Take Nioganling Granules (batch number: 2302013), grind them finely, take 1 g, prepare the test solution according to the preparation method of the test solution under item "3.1", inject 6 needles according to the chromatographic conditions under item "3.1", record the chromatogram, measure the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD.

[0057] Table 5 Results of relative retention time for precision 。

[0058] Table 6 Precision Relative Peak Area Results 。

[0059] The results showed that the RSD of the relative retention time of each chromatographic peak was less than 1.0%, and the RSD of the relative peak area was less than 8.0%, indicating good precision. The chromatogram is shown in the appendix Figure 2 。

[0060] 3.3 Repeatability Take the Yuaoning Granules (batch number: 2302013), grind them finely, take 1 g, a total of 6 portions, prepare the test solution according to the preparation method of the test solution under item "3.1", inject the sample according to the chromatographic conditions under item "3.1", record the chromatogram, determine the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD

[0061] Table 7 Repeatability Relative Retention Time Results 。

[0062] Table 8 Repeatability Relative Peak Area Results 。

[0063] The results showed that the RSD of the relative retention time of each chromatographic peak was less than 1.0%, and the RSD of the relative peak area was less than 10.0%, indicating good repeatability. The chromatogram is shown in the appendix Figure 3 。

[0064] 3.4 Stability Take the Yuaoning Granules (batch number: 2302013), grind them finely, take 1 g, prepare the test solution according to the preparation method of the test solution under item "3.1", inject the sample according to the chromatographic conditions under item "3.1" at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h respectively, record the chromatogram, determine the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD

[0065] Table 9 Stability Relative Retention Time Results 。

[0066] Table 10 Stability Relative Peak Area Results 。

[0067] The results showed that the RSD of the relative retention time of each chromatographic peak was less than 2.0%, and the RSD of the relative peak area was less than 10.0%, indicating good solution stability. The chromatogram is shown in the appendix Figure 4 。

[0068] In summary, through the specificity test of this method, the negative solution showed no interference, indicating good specificity. In the precision test, 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. In the repeatability test, 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%, indicating good repeatability. In the stability test, 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 good solution stability. This method has been verified by methodology, and the results are reliable and accurate.

[0069] Construction of the Control Characteristic Chromatogram of Nioganling Granules in Example 4 Chromatographic Conditions Chromatographic column: Topsil C18 (4.6x250mm, 5μm); mobile phase A was acetonitrile - tetrahydrofuran - 0.1% trifluoroacetic acid (85:10:5), and mobile phase B was 10 mM phosphoric acid solution - 0.05% sodium heptanesulfonate solution (94:5). Gradient elution was carried out according to the regulations in the following table; the column temperature was 30°C; the detection wavelength was 270 nm. The number of theoretical plates calculated based on the peak of aesculin should be not less than 5000.

[0070] 。

[0071] Preparation of the Reference Substance Solution Appropriately weigh aesculin and luteoloside reference substances, and accurately weigh them. Dissolve them in methanol to make solutions containing 50 μg per 1 mL respectively, shake well, and use them as the reference substance solutions.

[0072] Preparation of the Test Solution Take an appropriate amount of Nioganling Granules, grind them finely, take 1 g, and place it 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. The ultrasonic parameters are power 250 W, frequency 40 kHz, and temperature 40°C; centrifuge at 4000 rpm for 10 minutes, take the supernatant, and filter it initially through a 0.45 μm microporous filter membrane; activate the C18 solid-phase extraction column with 5 mL of methanol and 5 mL of ultrapure water in sequence to keep the column bed moist; slowly load the initial filtrate onto the activated C18 column at a flow rate of 1 mL / min; elute and remove impurities with 5 mL of ultrapure water - methanol solution with a volume ratio of 95:5; elute the target components 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 by nitrogen blowing at 40°C, redissolve it with 70% methanol and make the volume up to 2 mL, and filter it through a 0.22 μm filter membrane to obtain the test solution.

[0073] Determination Method Precisely pipette 10 μl of the reference substance solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0074] The chromatograms of the test samples of 8 batches of Nioganling Granules were imported into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints", and the chromatographic peaks that existed in the chromatograms of different batches of Nioganling Granules were selected as the common peaks; the reference fingerprint was generated by the average value calculation method, and the relative retention time and relative peak area of each common peak were calculated. See Appendix Figure 5 、 Figure 6 。

[0075] Table 11 Results of Relative Retention Time of Samples of 8 Batches of Nioganling Granules 。

[0076] Table 12 Results of Relative Peak Area of Samples of 8 Batches of Nioganling Granules 。

[0077] Twelve common peaks were presented in the chromatograms of the samples of 8 batches of Nioganling Granules, and 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%.

[0078] Twelve characteristic peaks were presented in the chromatogram of the test sample. Among them, Peak 7 and Peak 11 should correspond to the reference peaks of aesculin and luteoloside reference substances respectively; the peak corresponding to the reference peak of aesculin reference substance was the S peak, and the relative retention time of each characteristic peak and the S peak was calculated, and its relative retention time should be within the range of ±10% of the specified value. The specified values were: 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), 2.24 (Peak 12).

Claims

1. A method for constructing a characteristic fingerprint of Nioganling Granules, characterized in that, It includes the following steps: (1) Preparation of the reference solution: Take the reference substances, weigh them accurately, and dissolve them in methanol respectively to prepare solutions containing 50 μg per 1 mL. Shake well to obtain the reference solution. The reference substances are aesculin and luteoloside. (2) Preparation of the test solution: Take the Nioganling granules, grind them finely, and place them in a 50 mL stoppered conical flask. Add 40 mL of a methanol-water-0.1% formic acid solution with a volume ratio of 70:28:2, and perform ultrasonic-assisted extraction for 45 minutes. After ultrasonic treatment, centrifuge and take the supernatant. Filter the supernatant through a 0.45 μm microporous 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 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 to remove impurities with an ultrapure water-methanol solution. Elute the target components with a methanol-0.1% formic acid aqueous solution, and collect the eluate. Concentrate the eluate to nearly dry by nitrogen blowing at 40 °C, redissolve it with 70% methanol and make up the volume to 2 mL, and filter through a 0.22 μm membrane to obtain the test solution. (3) Determination: Inject the reference solution and the test solution into a high-performance liquid chromatograph under the following chromatographic conditions, and inject 10 μL each, and record the chromatogram. The chromatographic conditions are as follows: Use octadecylsilane-bonded silica gel as the filler; Use acetonitrile-tetrahydrofuran-0.1% trifluoroacetic acid with a volume ratio of 85:10:5 as mobile phase A, and use a 10 mM phosphoric acid solution-0.05% sodium heptanesulfonate solution with a volume ratio of 94:5 as mobile phase B, and perform elution according to the specified gradient; The column temperature is 30 °C; The flow rate is 1 mL per minute; Ultraviolet-visible detector, the detection wavelength is 270 nm. The elution gradient conditions are as follows: 0 - 25 min, for mobile phase A:B, the volume ratio changes from 5:95 to 10:90; 25 - 50 min, for mobile phase A:B, the volume ratio changes from 10:90 to 20:80; 50 - 65 min, for mobile phase A:B, the volume ratio changes from 20:80 to 30:70; 65 - 75 min, for mobile phase A:B, the volume ratio changes from 30:70 to 60:40; 75 - 78 min, for mobile phase A:B, the volume ratio changes from 60:40 to 5:95; 78 - 90 min, for mobile phase A:B, the volume ratio is 5:95; (4) Generate the reference characteristic chromatogram: Select the chromatographic peaks that exist in the chromatograms of Nioganling granules from different batches as the common peaks, and generate the reference characteristic chromatogram of Nioganling granules by the average value calculation method.

2. The method for constructing the characteristic fingerprint of a urinary sensation-relieving granule according to claim 1, wherein In step (2), the ultrasonic extraction conditions are that the ultrasonic parameters are power 250 W, frequency 40 kHz, and temperature 40 °C.

3. The method for constructing the characteristic spectrum of a urinary sensation-relieving granule according to claim 1, characterized in that, In step (2), the centrifugation conditions are centrifugation at 4000 rpm for 10 minutes.

4. The method for constructing the characteristic fingerprint of a urinary sensation-relieving granule according to claim 1, wherein, In step (2), the volume ratio of the ultrapure water-methanol solution for eluting and removing impurities is 95:5, and the amount used is 5 mL.

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

6. The method for constructing the characteristic spectrum of a urinary infection-relieving granule according to claim 1, wherein In step (3), the chromatographic column is Topsil C18 column with a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

7. The construction method of the characteristic fingerprint of a urinary infection-relieving granule according to claim 1, characterized in that, The control characteristic chromatogram generated in step (4) includes 12 common chromatographic peaks, specifically: peak 7 corresponding to aesculin and peak 11 corresponding to luteoloside.

8. The construction method of the characteristic chromatogram of a urinary infection-relieving granule according to claim 1, characterized in that, Taking the aesculin chromatographic peak as a reference for the characteristic peaks, the relative retention times of the 12 chromatographic peaks are as follows: 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.

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