Construction method of fingerprint spectrum of Yangweikangliu granules and its fingerprint spectrum

Abstract

The present invention relates to the technical field of traditional Chinese medicine analysis, and specifically to a method for constructing a fingerprint spectrum of Yangwei Kangliu Granules and its fingerprint spectrum. The method adopts the HPLC-DAD-ELSD method, wherein the preparation method of the test solution comprises first adding water to part of the Yangwei Kangliu Granules, ultrasonically treating, extracting with water-saturated n-butanol, and combining the n-butanol solution; then taking the remaining Yangwei Kangliu Granules, ultrasonically treating with solvent 1, filtering, and obtaining a filtrate; finally, mixing part of the filtrate with the n-butanol solution, evaporating to dryness, and dissolving the residue with solvent 2. The method of the present invention is simple to operate, accurate and reliable, and has the characteristics of good precision, stability and repeatability. It can overcome the shortcomings of the prior art that the detection index is single and cannot reflect the intrinsic quality of the process control of the Yangwei Kangliu Granules, and realize a comprehensive and accurate evaluation of the preparation quality, thereby more effectively ensuring the preparation quality.

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 fingerprint spectrum of Yangwei Kangliu granules and a fingerprint spectrum thereof. Background Art

[0002] Yangwei Kangliu Granules (reference Chinese invention patent publication number CN101721545A) are an exclusive product developed by Jiangzhong Pharmaceutical Co., Ltd. based on the clinical experience of Professor Sun Guizhi. It is composed of astragalus, ginseng, Panax notoginseng, Houttuynia cordata, Atractylodes macrocephala, Lycium barbarum, Sappan wood, Fagopyrum sappan, Polygonum multiflorum, and fermented Cordyceps sinensis powder. It can nourish qi and blood, promote blood circulation and remove blood stasis, nourish the spleen and kidneys, detoxify and fight cancer. It is clinically used to treat middle and late stage gastric cancer. It can not only prevent tumor recurrence and metastasis, but also prolong the patient's survival period.

[0003] For the quality control of compound Chinese medicine preparations, high performance liquid chromatography and thin layer chromatography are currently used to conduct qualitative and quantitative identification research on the effective ingredients. The fingerprint of Chinese medicine is a spectrum or chromatogram of the chemical components of Chinese medicine obtained by spectral or chromatographic technology. Chinese medicine, especially compound Chinese medicine, contains multiple chemical components, and its therapeutic effect is an overall synergistic effect. Therefore, the reflection of its material basis cannot be explained from only one or several components, but needs to be explained from the overall perspective.

[0004] Traditional Chinese medicine fingerprint has the characteristics of overall, macroscopic and fuzzy analysis. It can achieve the purpose of overall quality control by describing the overall characteristics and taking appropriate fuzzy processing methods. Compared with traditional analysis methods, traditional Chinese medicine fingerprint technology has unique advantages in the evaluation and quality control of multi-component herbs and Chinese patent medicines. It is conducive to establishing a more accurate, comprehensive and cross-batch quality control method for Chinese patent medicines currently circulating on the market. This method has been widely recognized internationally.

[0005] At present, there is no quality testing method for Yangwei Kangliu Granules to ensure the stability of product quality and the safety and effectiveness of clinical use. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a method for constructing a fingerprint spectrum of Yangwei Kangliu Granules. And an HPLC-DAD-ELSD standard fingerprint spectrum of Yangwei Kangliu Granules prepared by the method. In the case of using similarity for evaluation, the quality of Yangwei Kangliu Granules can be comprehensively evaluated and controlled by using its standard fingerprint spectrum, thereby ensuring the stability of product quality and the safety and effectiveness of clinical medication.

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

[0008] A method for constructing a fingerprint spectrum of Yangwei Kangliu granules, the method adopts HPLC-DAD-ELSD method, wherein the preparation method of the test solution comprises the following steps:

[0009] (1) adding water to a portion of the Yangweikangliu granules, subjecting them to ultrasonic treatment, extracting them with water-saturated n-butanol, and combining the n-butanol solutions;

[0010] (2) taking the remaining Yangweikangliu granules, adding solvent 1 for ultrasonic treatment, filtering, and obtaining a filtrate;

[0011] (3) Mix part of the filtrate and the n-butanol solution, evaporate to dryness, and dissolve the residue with solvent 2 to obtain the product.

[0012] Preferably, the portion in step (1) is 45-55% of the mass of the stomach-nourishing and anti-tumor particles.

[0013] Preferably, the ultrasonic conditions in steps (1) and (2) are: frequency 40-60 kHz, power 90-100%, and ultrasonic time 20-50 min.

[0014] Preferably, the extraction times in step (1) is 1-6 times, and after the n-butanol solution is combined, it is washed twice with ammonia test solution to obtain the n-butanol solution.

[0015] Preferably, before filtering in step (2), the weight loss is supplemented with solvent 1, and solvent 1 is methanol.

[0016] Preferably, the portion in step (3) is 45-55% of the volume of the subsequent filtrate, and solvent 2 is methanol.

[0017] Preferably, the mass volume ratio of the total mass of the stomach-nourishing and anti-tumor granules to the solvent 2 is 2 g:5 mL.

[0018] Preferably, the chromatographic conditions in the method include: mobile phase A is acetonitrile, mobile phase B is 0.05-0.15% formic acid aqueous solution, and the gradient elution conditions are as follows:

[0019] 0min, mobile phase A: 3-5%, mobile phase B: 95-97%;

[0020] 15 min, mobile phase A: 3-5%, mobile phase B: 95-97%;

[0021] 30 min, mobile phase A: 9-12%, mobile phase B: 88-91%;

[0022] 80 min, mobile phase A: 18-21%, mobile phase B: 79-82%;

[0023] 90 min, mobile phase A: 31-35%, mobile phase B: 65-69%;

[0024] 110min, mobile phase A: 31-35%, mobile phase B: 65-69%.

[0025] Preferably, the chromatographic conditions include: flow rate 0.8-1.2 ml / min; column temperature 24-30°C; injection volume: 5-50 μL; DAD detection wavelength 250-270 nm; ELSD detector atomization temperature 35-45°C, drift tube temperature 55-65°C, carrier gas flow rate 1.0-1.5 L / min.

[0026] Another object of the present invention is to provide a fingerprint of the stomach-nourishing anti-tumor granules constructed by the above-mentioned construction method, wherein the fingerprint includes an HPLC-DAD fingerprint and an HPLC-ELSD fingerprint; wherein the HPLC-DAD fingerprint has a total of 12 peaks, including a gallic acid peak, a 4-p-coumaric acid peak and a calycoside peak, and the HPLC-ELSD fingerprint has a total of 5 peaks, wherein in order of retention time from small to large, they are notoginseng saponin R1 peak, ginsenoside Rg1 peak, ginsenoside Re peak, ginsenoside Rb1 peak and astragaloside IV peak.

[0027] Beneficial effects of the present invention:

[0028] (1) Based on the conventional HPLC method, the present invention uses a DAD-ELSD detector as a technical means to construct a fingerprint spectrum to study the compound Chinese medicine Yangwei Kangliu Granules, thereby obtaining a number of corresponding fingerprint components, which more accurately reflects the quality of Yangwei Kangliu Granules and is expected to provide a new way to comprehensively and effectively control its quality.

[0029] (2) The present invention provides for the first time a method for establishing the HPLC-DAD-ELSD fingerprint spectrum of Yangwei Kangliu Granules. According to the composition characteristics of the prescription of Yangwei Kangliu Granules, the preparation of the test solution, the optimal mobile phase composition and its gradient elution procedure, flow rate, and detection wavelength are optimized, thereby solving the problems of difficulty in separating characteristic peaks and large interference from impurity peaks. The obtained fingerprint spectrum has high separation degree and good peak shape, and each characteristic peak has achieved baseline separation, which can more comprehensively and accurately evaluate the quality of Yangwei Kangliu Granules.

[0030] (3) When the wavelength of the HPLC-DAD detection method of the present invention is 260nm, the fingerprint spectrum includes 12 peaks, including gallic acid, 4-p-coumaric acid, and calycosin glucoside. The HPLC-ELSD fingerprint spectrum has a total of 5 peaks, which are notoginseng saponin R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and astragaloside IV in order over time. The composition of the fingerprint spectrum is richer and more comprehensive, which further clarifies the chemical substance basis of Yangwei Kangtu Granules.

[0031] (4) The method of the present invention is simple to operate, accurate and reliable, and has the characteristics of good precision, stability and repeatability. It can overcome the shortcomings of the existing technology that the detection index is single and cannot reflect the intrinsic quality of the process control of Yangwei Kangliu Granules, and realize a comprehensive and accurate evaluation of the preparation quality, thereby more effectively ensuring the preparation quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the HPLC-DAD test chromatogram of Example 3 under 0.05% formic acid conditions.

[0033] Figure 2 This is a partial enlarged view of the HPLC-DAD test chromatogram under 0.05% formic acid conditions in Example 3.

[0034] Figure 3 This is the HPLC-ELSD test chromatogram of Example 3 under 0.05% formic acid conditions.

[0035] Figure 4 This is the HPLC-DAD test chromatogram of Example 3 under 0.15% formic acid conditions.

[0036] Figure 5 This is a partial enlarged view of the HPLC-DAD test chromatogram under 0.15% formic acid conditions in Example 3.

[0037] Figure 6 This is the HPLC-ELSD test chromatogram of Example 3 under 0.15% formic acid conditions.

[0038] Figure 7 This is the HPLC-DAD test chromatogram of Example 4 at 23°C.

[0039] Figure 8 This is a partial enlarged view of the HPLC-DAD test chromatogram of Example 4 at 23°C.

[0040] Fig. 9 This is the HPLC-ELSD test chromatogram of Example 4 at 23°C.

[0041] Fig.10 This is the HPLC-DAD test chromatogram of Example 4 at 27°C.

[0042] Fig.11 This is a partial enlarged view of the HPLC-DAD test chromatogram of Example 4 at 27°C.

[0043] Fig.12 This is the HPLC-ELSD test chromatogram of Example 4 at 27°C.

[0044] Fig.13This is the HPLC-DAD test chromatogram of Example 5 under the condition of 0.9 ml / min.

[0045] Fig.14 This is a partial enlarged view of the HPLC-DAD test chromatogram of Example 5 under the condition of 0.9 ml / min.

[0046] Fig.15 This is the HPLC-ELSD test chromatogram of Example 5 under the condition of 0.9 ml / min.

[0047] Fig.16 This is the HPLC-DAD test chromatogram of Example 5 under the condition of 1.1 ml / min.

[0048] Fig.17 This is a partial enlarged view of the HPLC-DAD test chromatogram of Example 5 under the condition of 1.1 ml / min.

[0049] Fig.18 This is the HPLC-ELSD test chromatogram of Example 5 under the condition of 1.1 ml / min.

[0050] Fig.19 This is the HPLC-DAD test chromatogram of Example 6 under 259nm conditions.

[0051] Fig. 20 This is a partial enlarged view of the HPLC-DAD test chromatogram under 259nm conditions in Example 6.

[0052] Fig.21 This is the HPLC-ELSD test chromatogram of Example 6 under 259nm conditions.

[0053] Fig. 22 This is the HPLC-DAD test chromatogram of Example 6 at 261 nm.

[0054] Fig.23 This is a partial enlarged view of the HPLC-DAD test chromatogram under 261nm conditions in Example 6.

[0055] Fig.24 This is the HPLC-ELSD test chromatogram of Example 6 at 261 nm.

[0056] Fig.25 This is the HPLC-DAD test chromatogram of Comparative Example 1.

[0057] Fig.26 This is the HPLC-ELSD test chromatogram of Comparative Example 1.

[0058] Fig. 27 This is the HPLC-DAD test chromatogram of Comparative Example 2.

[0059] Fig.28 This is the HPLC-ELSD test chromatogram of Comparative Example 2.

[0060] Fig.29 This is the HPLC-DAD test chromatogram of Comparative Example 3.

[0061] Fig.30 This is the HPLC-ELSD test chromatogram of Comparative Example 3.

[0062] Fig.31 This is the HPLC-DAD test chromatogram of Comparative Example 4.

[0063] Fig.32 This is the HPLC-ELSD test chromatogram of Comparative Example 4. DETAILED DESCRIPTION

[0064] The present invention is further described below in conjunction with specific implementation methods.

[0065] The stomach-nourishing and anti-tumor granules used in the following embodiments adopt the following prescription: by weight, 15 parts of astragalus, 5 parts of ginseng, 9 parts of atractylodes, 9 parts of wolfberry, 3 parts of fermented cordyceps powder, 3 parts of Panax notoginseng, 3 parts of Sappan wood, 12 parts of Polygonum multiflorum, 12 parts of golden buckwheat, and 15 parts of Hedyotis diffusa.

[0066] Preparation method: Take the above ten herbs in the prescription, add 20 times the total mass of water, decoct 3 times, 1 hour each time, filter, combine the water extracts, concentrate under reduced pressure to a relative density of 1.05-1.30, temperature 60°C, add dextrin, stir rapidly to granulate, stirring speed: 380 rpm, chopping speed: 650 rpm, or spray dry, inlet air 90°C, outlet air 60°C, the obtained powder is granulated by boiling, inlet air 60°C, outlet air 30°C, and granulated.

[0067] Example 1

[0068] Establishment of HPLC-DAD-ELSD fingerprint of Yangweikangliu granules

[0069] 1. Instruments and reagents

[0070] 1.1 Instrument

[0071] 1260 infinityⅡ high performance liquid chromatograph; MSA6.6S-OCE-DM electronic balance; ML204 / 02 electronic balance; SB-500DTY CNC ultrasonic cleaner; HH-6 digital display constant temperature water bath; IQ7000 ultrapure water analyzer.

[0072] 1.2 Drug testing

[0073] Yangwei Kangliu Granules were provided by Jiangzhong Pharmaceutical Co., Ltd., and the sample batch number is shown in Table 1. n-Butanol and ammonia water were of analytical grade, methanol, acetonitrile, and formic acid were of chromatographic grade, and water was ultrapure water.

[0074] Table 1 Sample information of Yangweikangliu granules

[0075]

[0076] 2 Methods and Results

[0077] 2.1 Chromatographic conditions

[0078] Chromatographic column: Shim-pack GISS C18, 5μm, 4.6×250mm; mobile phase: acetonitrile as mobile phase A, 0.10% formic acid aqueous solution as mobile phase B, gradient elution as shown in Table 2; flow rate: 1.0ml / min; column temperature: 25℃; injection volume: 10 μL; DAD acquisition wavelength is 260nm; ELSD detector nebulization temperature is 40℃, drift tube temperature is 60℃, and carrier gas flow rate is 1.2L / min.

[0079] Table 2 Gradient elution

[0080]

[0081] 2.2 Preparation of reference and test solutions

[0082] Accurately weigh 1g of astragaloside IV reference substance, ginsenoside Re reference substance, notoginseng saponin R1 reference substance, ginsenoside Rb1 reference substance, ginsenoside Rg1 reference substance, gallic acid reference substance, p-coumaric acid reference substance, and calycosin glucoside reference substance, place in a 25 ml volumetric flask, add methanol to dissolve, dilute to the scale, shake well, and use as the reference solution.

[0083] Accurately weigh 1 g of Yangweikangliu Granules, accurately weigh it, place it in a stoppered conical flask, accurately add 20 mL of water, seal it, ultrasonically treat it (frequency 59 kHz, power 99%) for 45 min, shake and extract it with water-saturated n-butanol for 5 times, 20 mL each time, combine the n-butanol solution, wash it twice with ammonia test solution, 20 mL each time, and take the n-butanol solution; take 1 g of Yangweikangliu Granule sample powder, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of methanol, seal it, weigh it, ultrasonically treat it (frequency 40 kHz, power 99%) for 30 min, weigh it again, make up the lost weight with methanol, shake it well, filter it, take 25 ml of the filtrate and combine it with the n-butanol solution, evaporate it to dryness, dissolve the residue with an appropriate amount of 50% methanol, transfer it to a 5 mL volumetric flask to make up the volume, shake it well and filter it, take the filtrate, filter it through a 0.45 μm micromembrane, and take the filtrate as the test solution.

[0084] 2.3 Establishment of fingerprint

[0085] The common pattern of the fingerprint spectrum of Yangwei Kangliu Granules was established according to the above method.

[0086] The HPLC fingerprints of 14 batches of Yangweikangliu granules were determined. S1-S14 are batch numbers: GG221121, GG230110, GG230112, GG230111, GG230319, GG230323, GG230525, GG230607, GG230704, GG230709, GG230723, GG230727, GG230908, and GG230303. The fingerprint similarity evaluation software "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.130723 Version)" compiled by the Pharmacopoeia Committee was used for analysis and comparison. The fingerprints included HPLC-DAD fingerprints and HPLC-ELSD fingerprints. The similarity results of the 14 batches of fingerprints are shown in Tables 3 and 4.

[0087] Table 3 Similarity results of HPLC-DAD fingerprints of 14 batches of Yangweikangliu granules

[0088]

[0089] Table 4 Similarity results of HPLC-ELSD fingerprints of 14 batches of Yangweikangliu granules

[0090]

[0091] The 14 batches of fingerprints generated the common pattern spectrum of the HPLC fingerprint spectrum of Yangweikangliu Granules as the reference fingerprint spectrum of Yangweikangliu Granules. It includes the HPLC-DAD reference fingerprint spectrum and the HPLC-ELSD reference fingerprint spectrum, among which the HPLC-DAD reference fingerprint spectrum has 12 peaks in total, and the peak of calycosin isoflavone glucoside is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated, as shown in Table 5; the HPLC-ELSD reference fingerprint spectrum of Yangweikangliu Granules has 5 peaks in total, among which the peak of ginsenoside Rg1 is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated, as shown in Table 6, and the relative retention time should be within the range of ±10% of the specified value.

[0092] Table 5 Relative retention time of HPLC-DAD common patterns

[0093]

[0094] Table 6 Relative retention time of HPLC-ELSD common mode

[0095]

[0096] Through the detection method in this embodiment, HPLC-DAD can be obtained with calycosin glucoside as the reference peak S in the fingerprint spectrum, and the relative retention time of 12 common peaks of Yangweikangliu granules can be determined; HPLC-ELSD can be obtained with ginsenoside Rg1, peak 2, as the reference peak S in the fingerprint spectrum, and the relative retention time of 5 common peaks of Yangweikangliu granules can be determined. The Yangweikangliu granule sample can be fully and quickly detected at one time, which is beneficial to the overall quality control of Yangweikangliu granules, thereby helping to improve the stability and safety of the drug.

[0097] Example 2

[0098] This example provides the process and results of testing the precision, stability and repeatability of the detection method in the above-mentioned Example 1, as follows:

[0099] 1. Precision

[0100] 1.1 Precision test

[0101] Take the same sample solution (batch number: GG23030301), repeat the injection 6 times according to the chromatographic conditions under 2.1 in Example 1, and determine the relative retention time and relative peak area of ​​the common peak. Manually select the common peaks in the spectrum for multi-point correction, match the Mark peak, and use the average method to generate a control fingerprint spectrum with a time width of 0.1 to compare the similarity between the spectra in the data. The relative retention time results of the precision test are shown in Tables 7-8, the relative peak area results of the precision test are shown in Tables 9-10, and the similarity results of the precision test are shown in Tables 11-12.

[0102] Table 7 Relative retention time results of HPLC-DAD precision test

[0103]

[0104] Table 8 Relative retention time results of HPLC-ELSD precision test

[0105]

[0106] Table 9 Relative peak area results of HPLC-DAD precision test

[0107]

[0108] Table 10 Relative peak area results of HPLC-ELSD precision test

[0109]

[0110] Table 11 HPLC-DAD precision test similarity results

[0111]

[0112] Table 12 HPLC-ELSD precision test similarity results

[0113]

[0114] From the above results, it can be seen that the relative retention time and relative peak area RSD of each common peak reference peak S peak are less than 3%, and the similarity with the control fingerprint of Yangwei Kangliu Granule is greater than 0.99, indicating good precision.

[0115] 1.2 Repeatability test

[0116] Take the same sample solution (batch number: GG23030301) to prepare 6 samples, and determine the relative retention time and relative peak area of ​​the common peak according to the chromatographic conditions under 2.1 in Example 1. Manually select the common peaks in the spectrum for multi-point correction, match the Mark peaks, and use the average method to generate a control fingerprint spectrum with a time width of 0.1 to compare the similarity between the spectra in the data. The results of the relative retention time of the repeatability test are shown in Tables 13-14, the results of the relative peak area of ​​the repeatability test are shown in Tables 15-16, and the results of the similarity of the repeatability test are shown in Tables 17-18.

[0117] Table 13 Relative retention time results of HPLC-DAD repeatability test

[0118]

[0119] Table 14 Relative retention time results of HPLC-ELSD repeatability test

[0120]

[0121] Table 15 Relative peak area results of HPLC-DAD repeatability test

[0122]

[0123] Table 16 HPLC-ELSD repeatability test relative peak area results

[0124]

[0125] Table 17 HPLC-DAD repeatability test similarity results

[0126]

[0127] Table 18 Similarity results of HPLC-ELSD repeatability test

[0128]

[0129] From the above results, it can be seen that the relative retention time and relative peak area RSD of each common peak reference peak S are less than 3%, and the similarity with the control fingerprint of Yangwei Kangliu Granule is greater than 0.99, indicating that this method has good repeatability.

[0130] 1.3 Stability test

[0131] Take the same sample solution (batch number: GG23030301), after preparation, according to the chromatographic conditions under 2.1 in Example 1, inject at 0, 2, 4, 6, 8, 12, 24 h, respectively, and determine the relative retention time and relative peak area of ​​the common peak. Manually select the common peaks in the spectrum for multi-point correction, match the Mark peak, and use the average method to generate a control fingerprint spectrum with a time width of 0.1 to compare the similarity between the spectra in the data. The relative retention time results of the stability test are shown in Tables 19-20, the relative peak area results of the stability test are shown in Tables 21-22, and the similarity results of the stability test are shown in Tables 23-24.

[0132] Table 19 Relative retention time results of HPLC-DAD stability test

[0133]

[0134] Table 20 HPLC-ELSD stability test relative retention time results

[0135]

[0136] Table 21 HPLC-DAD stability test relative peak area results

[0137]

[0138] Table 22 HPLC-ELSD stability test relative peak area results

[0139]

[0140] Table 23 Similarity results of HPLC-DAD stability test

[0141]

[0142] Table 24 HPLC-ELSD stability test similarity results

[0143]

[0144] From the above results, it can be seen that the relative retention time and relative peak area RSD of each common peak reference peak S are less than 3%, and the similarity with the control fingerprint of Yangwei Kangliu Granule is greater than 0.99, indicating that the test solution is stable within 24 hours and meets the determination requirements.

[0145] Example 3

[0146] The difference between this embodiment and embodiment 1 is that the concentration of mobile phase B in this embodiment is different, namely 0.05% formic acid and 0.15% formic acid solution. Figure 1 , 2 , 3, 4, 5, and 6. As shown in the figure, 0.05%-0.15% formic acid solution can be effectively used for separating the components of Yangwei Kangtu granules.

[0147] Example 4

[0148] The difference between this embodiment and embodiment 1 is that the column temperature in this embodiment is different, 23°C and 27°C respectively. The Yangwei Kangliu Granule with batch number GG23030301 was used for testing. The test results are as follows: Figure 7 , 8 , 9, 10, 11, and 12. As shown in the figure, the column temperature of 23-27°C is effectively applicable to the separation of the components of Yangwei Kangliu granules.

[0149] Example 5

[0150] The difference between this embodiment and embodiment 1 is that the flow rates in this embodiment are different, 0.9 ml / min and 1.1 ml / min respectively. The Yangwei Kangliu Granule with batch number GG23030301 was used for testing. The test results are as follows: Fig.13 , 14 , 15, 16, 17, and 18. As shown in the figures, the flow rate of 0.9-1.1 ml / min can be effectively applied to the separation of the components of the stomach-nourishing and anti-tumor granules.

[0151] Example 6

[0152] The difference between this embodiment and embodiment 1 is that the wavelengths in this embodiment are different, 259 nm and 261 nm respectively. The Yangwei Kangliu Granule with batch number GG23030301 was used for testing. The test results are as follows: Fig.19 , 20 , 21, 22, 23, and 24. As shown in the figure, the wavelength of 259-261nm can be effectively applied to the separation of the components of the nourishing stomach and anti-tumor particles.

[0153] Comparative Example 1

[0154] The difference between this embodiment and embodiment 1 is that the elution gradient in this embodiment is different, as shown in Table 25. The Yangwei Kangliu Granule with batch number GG23030301 is used for detection. The detection results are as follows: Fig.25 , 26 As shown in the figure, it can be seen that the elution gradient mobile phase is not suitable for the separation of the components of Yangwei Kangliu granules.

[0155] Table 25 Gradient elution table

[0156]

[0157] Comparative Example 2

[0158] The difference between this embodiment and embodiment 1 is that the elution gradient in this embodiment is different, as shown in Table 26. The Yangwei Kangliu Granules with batch number GG23030301 were used for testing. The test results are as follows: Fig. 27 , 28 As shown in the figure, it can be seen that the elution gradient mobile phase is not suitable for the separation of the components of Yangwei Kangliu granules.

[0159] Table 26 Gradient elution table

[0160]

[0161] Comparative Example 3

[0162] The difference between this embodiment and embodiment 1 is that in this embodiment, all samples are subjected to water ultrasonication and n-butanol extraction. The specific test samples are prepared as follows:

[0163] Accurately weigh 1.5 g of Yangwei Kangliu Granules, accurately weigh, place in a stoppered conical flask, accurately add 30 mL of water, seal, ultrasonically treat (frequency 59 kHz, power 99%) for 45 min, shake and extract with water-saturated n-butanol 5 times, 30 mL each time, combine the n-butanol solutions, wash twice with ammonia test solution, 30 mL each time, take the n-butanol solution, evaporate to dryness, dissolve the residue with an appropriate amount of 50% methanol, transfer to a 5 mL volumetric flask to make up the volume, shake and filter, take the filtrate, filter through a 0.45 μm micromembrane, and take the filtrate as the test solution.

[0164] Then, the Yangwei Kangliu Granules with batch number GG23030301 were used for testing. The test results were as follows: Fig.29 , 30 As shown in the figure, this condition can only extract part of the components of Yangwei Kangliu Granules, but cannot extract them completely, and is not suitable for the preparation of the test sample of Yangwei Kangliu Granules.

[0165] Comparative Example 4

[0166] The difference between this embodiment and embodiment 1 is that in this embodiment, all samples are subjected to ultrasonic extraction with methanol, and the specific test samples are prepared as follows:

[0167] Accurately weigh 2 g of Yangwei Kangliu Granule sample powder, accurately weigh, place in a stoppered conical flask, accurately add 100 mL of methanol, seal, weigh, ultrasonically treat (frequency 40 kHz, power 99%) for 30 min, weigh again, make up the lost weight with methanol, shake well, filter, take 75 ml of the filtrate, evaporate to dryness, dissolve the residue with appropriate amount of 50% methanol, transfer to a 5 mL volumetric flask to make up to volume, shake well, filter, take the filtrate, filter through 0.45 μm micromembrane, and take the filtrate as the test solution.

[0168] Then, the Yangwei Kangliu Granules with batch number GG23030301 were used for testing. The test results were as follows: Fig.31 , 32 As shown in the figure, this condition can only extract part of the components of Yangwei Kangliu Granules, but cannot extract them completely, and is not suitable for the preparation of the test sample of Yangwei Kangliu Granules.

[0169] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of Yangwei Kangliu granules, characterized in that: The construction method adopts the HPLC-DAD-ELSD method, wherein the preparation method of the test solution comprises the following steps: (1) adding water to a portion of the Yangweikangliu granules, subjecting them to ultrasonic treatment, extracting them with water-saturated n-butanol, and combining the n-butanol solutions; (2) taking the remaining Yangweikangliu granules, adding solvent 1 for ultrasonic treatment, filtering, and obtaining a filtrate; (3) Mix part of the filtrate and the n-butanol solution, evaporate to dryness, and dissolve the residue with solvent 2 to obtain; The solvent 1 and the solvent 2 are both methanol; The stomach-nourishing and anti-tumor granules are composed of astragalus, ginseng, notoginseng, oldenlandia diffusa, atractylodes, wolfberry, sappan wood, golden buckwheat, bistort and fermented cordyceps powder; The chromatographic conditions in the method include: mobile phase A is acetonitrile, mobile phase B is 0.05-0.15% formic acid aqueous solution, and the gradient elution conditions are as follows: 0min, mobile phase A: 3-5%, mobile phase B: 95-97%; 15 min, mobile phase A: 3-5%, mobile phase B: 95-97%; 30 min, mobile phase A: 9-12%, mobile phase B: 88-91%; 80 min, mobile phase A: 18-21%, mobile phase B: 79-82%; 90 min, mobile phase A: 31-35%, mobile phase B: 65-69%; 110min, mobile phase A: 31-35%, mobile phase B: 65-69%.

2. The construction method according to claim 1, characterized in that: The portion described in step (1) is 45-55% of the mass of the stomach-nourishing anti-tumor granules.

3. The construction method according to claim 1, characterized in that: The ultrasonic conditions in steps (1) and (2) are: frequency 40-60 kHz, power 90-100%, and ultrasonic time 20-50 min.

4. The construction method according to claim 1, characterized in that: The extraction times in step (1) are 1-6 times. After the n-butanol solution is combined, it is washed twice with ammonia test solution to obtain the n-butanol solution.

5. The construction method according to claim 1, characterized in that: Before filtration in step (2), the weight loss is compensated with solvent 1.

6. The construction method according to claim 1, characterized in that: The portion in step (3) is 45-55% of the volume of the subsequent filtrate.

7. The construction method according to claim 1, characterized in that: The mass volume ratio of the total mass of Yangwei Kangliu Granules to the solvent 2 is 2g:5mL.

8. The construction method according to any one of claims 1 to 7, characterized in that: The chromatographic conditions include: flow rate 0.8-1.2 ml / min; column temperature 24-30° C.; injection volume: 5-50 μL; DAD detection wavelength 250-270 nm; ELSD detector atomization temperature 35-45° C., drift tube temperature 55-65° C., and carrier gas flow rate 1.0-1.5 L / min.

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