CN114942291B - Quality detection method of yang-suppressing yin-nourishing particles - Google Patents
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a quality detection method of yang-suppressing and yin-nourishing particles, which comprises a fingerprint detection method and a content determination method; the fingerprint detection method comprises the following steps: step 1, preparing a test solution of particles for suppressing yang and nourishing yin; step 2, preparing a mixed reference substance solution; step 3, respectively precisely sucking the solution of the sample and the reference substance, injecting the solution into a liquid chromatograph, and recording the chromatograms; and 4, deriving a fingerprint instrument of the yang-suppressing and yin-nourishing particles, introducing the fingerprint instrument into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, selecting chromatographic peaks existing in chromatograms of different batches of the yang-suppressing and yin-nourishing particles as common peaks, generating a control fingerprint of the yang-suppressing and yin-nourishing particles by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak. The fingerprint spectrum of the yang-suppressing and yin-nourishing particles established by the invention can comprehensively and objectively evaluate the quality of the yang-suppressing and yin-nourishing particles. The method has the advantages of good stability, high accuracy, good repeatability, high precision and the like.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a quality detection method of yang-suppressing and yin-nourishing particles.
Background
The yang-suppressing yin-nourishing granule is a special hospital preparation in the middle hospital of Jiangsu province, is prepared by teaching Fang Zhouyuan and inheriting academic experience of the first of Tang Shuhua of old traditional Chinese medicine, and combines years of clinical practice according to the methods of clearing liver and tonifying kidney, removing blood stasis and dredging collaterals, and is prepared by selecting six medicines of spanishneedles herb, prepared fleece flower root, cornus officinalis, radix scrophulariae, medicinal cyathula root and rhizoma alismatis. The yang-suppressing yin-nourishing particles have complex components, and cannot comprehensively and objectively detect the quality by means of a single detection method. In order to fully control the clinical effectiveness and safety of the traditional Chinese medicine and maintain the benefit of patients, it is necessary to research and design a detection method capable of comprehensively and accurately detecting the effective components of the particles for suppressing yang and nourishing yin on the basis of the prior art, and then ensuring the quality of the particles. The fingerprint spectrum of the traditional Chinese medicine is taken as a comprehensive and quantifiable identification means, can reflect the characteristics of multiple components of the traditional Chinese medicine, quantitatively detects multiple effective components in the traditional Chinese medicine, can be used for evaluating the authenticity, the effectiveness and the stability of the quality of the traditional Chinese medicine preparation, and comprehensively and accurately controls the quality of the traditional Chinese medicine compound preparation.
The HPLC fingerprint detection method of the yang-suppressing and yin-nourishing particles is established, the main components are identified, and a reference basis can be provided for comprehensively evaluating and controlling the quality of the yang-suppressing and yin-nourishing particles and clinical application.
Disclosure of Invention
The invention aims to: the invention aims to overcome the defects of the prior art, and develop a quality detection method for the yang-suppressing and yin-nourishing particles, which can objectively, correctly and effectively control the quality of the yang-suppressing and yin-nourishing particles and has important guiding significance for ensuring the safety and effectiveness and the clinical quality.
The technical scheme is as follows: in order to achieve the above purpose, the invention adopts the following technical scheme:
a quality detection method of yang-suppressing and yin-nourishing particles comprises the following steps:
step 1: preparation of test solution of yang-suppressing and yin-nourishing particles
Grinding the yang-suppressing and yin-nourishing particles in different batches into powder, sieving with a third sieve, precisely weighing, adding methanol solution, performing ultrasonic extraction, standing for precipitation, and filtering the supernatant with a 0.22 μm filter membrane to obtain the product;
step 2: preparation of mixed control solution
Dissolving reference substances such as morroniside, loganin, ferulic acid, hyperoside, stilbene glucoside, cyasterone, harpagoside, cinnamic acid and emodin in methanol to obtain mixed reference solution, and filtering with 0.22 μm filter membrane;
step 3, respectively sucking the sample solution in the step 1 and the mixed reference substance solution in the step 2, and injecting UPLC to obtain a chromatogram of the sample solution and a chromatogram of the mixed reference substance solution;
step 4, deriving the fingerprint of the test sample solution of the yang-suppressing and yin-nourishing particles obtained in the step 3, and introducing the fingerprint into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2004A; selecting chromatographic peaks existing in chromatograms of different batches of the test sample solutions of the yang-suppressing and yin-nourishing particles as common peaks; generating a control fingerprint of the test sample solution of the yang-suppressing and yin-nourishing particles by using a median calculation method, and calculating the relative retention time and the relative peak area of each common peak; and labeling the chemical components of peaks in the reference fingerprint according to the retention time of the mixed reference solution chromatogram.
As a preferred scheme, the quality detection method of the yang-suppressing and yin-nourishing particles comprises the following steps of:
grinding the yang-suppressing and yin-nourishing particles in different batches into powder, sieving with a third sieve, precisely weighing about 1g of powder, adding 70% methanol solution, ultrasonically extracting for 20min, standing for precipitation, and filtering the supernatant with a 0.22 μm filter membrane to obtain the final product;
as a preferred scheme, the quality detection method of the yang-suppressing and yin-nourishing particles comprises the following steps of:
dissolving morroniside, loganin, ferulic acid, hyperoside, stilbene glycoside, cyamalone, harpagoside, cinnamic acid and emodin reference substances in methanol to obtain a mass concentration of 0.267 mg/mL -1 、0.105mg·mL -1 、0.361mg·mL -1 、0.221mg·mL -1 、0.249mg·mL -1 、0.040mg·mL -1 、0.092mg·mL -1 、0.228mg·mL -1 、0.339mg·mL -1 Filtering with 0.22 μm filter membrane to obtain the final product.
As a preferred scheme, the quality detection method of the yang-suppressing and yin-nourishing particles comprises the following chromatographic conditions in the step 3 and the step 4: watersACQUITY UPLC BEH C18 chromatographic column, 100mm×3.0mm,1.7 μm mobile phase acetonitrile as phase A, aqueous solution containing 0.1% phosphoric acid as phase B, gradient elution, detection wavelength of 250nm; flow rate 0.3mL/min -1 The method comprises the steps of carrying out a first treatment on the surface of the Column temperature was 35 ℃.
As a preferred scheme, the above-mentioned quality detection method of the yang-suppressing and yin-nourishing particles comprises the following gradient elution procedures in steps 3 and 4:
| time/min | A/％ | B/％ |
| 0 | 3 | 97 |
| 5 | 8 | 92 |
| 11 | 30 | 70 |
| 20 | 80 | 20 |
| 21 | 95 | 5 |
| 23 | 95 | 5 |
| 23.5 | 3 | 97 |
| 30 | 3 | 97 |
。
As a preferred scheme, according to the quality detection method of the yang-suppressing and yin-nourishing particles, the reference fingerprint of step 4 has 18 common peaks, wherein the loganin peak No. 7 is used as a reference peak, the peak No. 1 is derived from rhizoma alismatis, the peak No. 3 is derived from prepared fleece-flower root and cornus wine, the peaks No. 2-8 and No. 10 are mainly derived from cornus wine, the peaks No. 9 and 11 are mainly derived from spanishneedles herb, the peak No. 12 is derived from prepared fleece-flower root, the peaks No. 13 and No. 14 are mainly derived from radix cyathulae, the peak No. 15 is derived from rhizoma alismatis, the peaks No. 16 and No. 17 are derived from prepared fleece-flower root and rhizoma alismatis through comparison analysis with the mixed reference solution chromatogram; wherein, the peak 6 is morroniside, the peak 7 is loganin, the peak 11 is hyperin, the peak 12 is stilbene glucoside, the peak 13 is ferulic acid, the peak 14 is amaranth sterone, the peak 16 is harpagoside, the peak 17 is cinnamic acid, and the peak 18 is emodin.
The invention provides a quality detection method of yang-suppressing and yin-nourishing particles, which also comprises a content determination method, and specifically comprises the following steps:
step 1: preparation of test solution of yang-suppressing and yin-nourishing particles
Grinding the yang-suppressing and yin-nourishing particles in different batches into powder, sieving with a third sieve, precisely weighing, adding methanol solution, performing ultrasonic extraction, standing for precipitation, and filtering the supernatant with a 0.22 μm filter membrane to obtain the product;
step 2: preparation of mixed control solution
Precisely weighing appropriate amount of harpagoside, morroniside, loganin, ferulic acid and cyamalone, and adding methanol to obtain mixed reference solution; in addition, a proper amount of stilbene glucoside is precisely weighed, and methanol is added to prepare a stilbene glucoside reference substance solution;
step 3: establishment of a Standard Curve
Taking the mixed reference substance solution in the step 2 and the stilbene glucoside reference substance solution, adding methanol to dilute the mixed reference substance solution in sequence to obtain 5 mixed reference substance solutions with different concentrations, then injecting UPLC, and establishing a standard curve equation by taking the peak area as an ordinate and the concentration as an abscissa;
step 4: and (3) injecting the sample solution in the step (1) into UPLC, substituting the peak area of the chromatogram into the standard curve equation in the step (3), and calculating the content of the corresponding effective components in the sample.
As a preferred scheme, the quality detection method of the yang-suppressing and yin-nourishing particles comprises the following chromatographic conditions in the step 3 and the step 4:
WatersACQUITYUPLC BEH C18, 100mm×3.0mm,1.7 μm as column, mobile phase: acetonitrile as phase A-water containing 0.1% phosphoric acid as phase B, and gradient elution; detection wavelength: ferulic acid and stilbene glycoside are 320nm, morroniside, cuparanthrone, loganin are 250nm, harpagoside and harpagoside are 202nm; the flow rate is 0.3mL/min; the column temperature is 35 ℃; the sample injection amount is 2 mu L; the gradient elution procedure is as follows:
| time/min | A/％ | B/％ |
| 0 | 5 | 95 |
| 15 | 15 | 85 |
| 30 | 30 | 70 |
| 40 | 80 | 20 |
| 42 | 95 | 5 |
。
Preferably, the step 2: the preparation method of the mixed reference substance solution comprises the following steps:
precisely weighing appropriate amount of harpagoside, morroniside, loganin, ferulic acid and cyamalone, and adding methanol to obtain mixed reference substance solutions with concentrations of 0.0194, 0.0114, 0.0174, 0.0134, 0.0005 and 0.0052mg/mL respectively; and precisely weighing proper amount of stilbene glucoside, and adding methanol to prepare a reference substance solution with the concentration of 0.2730 mg/mL.
The preparation method of the yang-suppressing yin-nourishing granule comprises the following steps: decocting herba Bidentis Bipinnatae 810g, radix Polygoni Multiflori Preparata 180g, fructus Corni 108g, radix scrophulariae 180g, alismatis rhizoma 180g, and radix Cyathulae 270g with water for 2 times, filtering, mixing filtrates, concentrating under reduced pressure to appropriate amount, centrifuging, concentrating supernatant to fluid extract with relative density of 1.20 at 60deg.C, and adding pharmaceutically acceptable adjuvants.
Optimization of fingerprint detection conditions
1. Selection of detection wavelength
The mixed reference substance is scanned in full wavelength, and the target component is found to have better absorption at about 250nm and better peak shape. 250nm is selected as the detection wavelength.
2. Selection of mobile phase
The study investigated different mobile phases (acetonitrile-0.2% phosphoric acid aqueous solution, methanol-0.1% phosphoric acid aqueous solution, acetonitrile-0.1% formic acid aqueous solution), and as a result, an acetonitrile-0.1% phosphoric acid aqueous system was adopted, and the separation effect and peak shape were good, and the base line was relatively stable, so that it was selected as the mobile phase.
3. Column temperature and flow rate selection
The study examined the temperature of the various columns (25, 30, 35,40 ℃ and flow rates (0.2, 0.3, 0.4 mL. Min) -1 ) The effect on chromatographic peaks shows that at 250nm wavelength, the column temperature is 35 ℃ and the flow rate is 0.3 mL-min -1 The obtained chromatographic peak has good peak shape, number and separation degree.
The beneficial effects of the invention are as follows:
(1) The invention screens out the best preparation method of the test sample, the preparation method of the reference substance and the best chromatographic analysis conditions through a large number of experiments, and the established fingerprint spectrum and content measurement method of the yang-suppressing and yin-nourishing particles not only can effectively characterize the quality of the yang-suppressing and yin-nourishing particles, but also is beneficial to comprehensively monitoring the quality of the yang-suppressing and yin-nourishing particles.
(2) The method has the advantages of good stability, high precision, high accuracy, good repeatability and the like. The quality of the particles for suppressing yang and nourishing yin can be comprehensively, objectively and accurately evaluated and controlled.
Drawings
FIG. 1 is a chromatogram of UPLC of a mixed control solution.
Fig. 2 is a UPLC superimposed fingerprint and a control fingerprint of 15 batches of yang-suppressing yin-nourishing particles.
Detailed Description
Embodiments of the present invention will be described in detail with reference to examples, which are not to be construed as specific conditions, either as normal conditions or as recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The following examples of drugs and reagents:
the particles (batch numbers 2101001, 2101003, 2102033, 2103009, 2103013, 2104015, 2104017, 2104018, 2105019, 2105020, 2106023, 2107027, 2107028, 2108030, 2109032 and the numbers S1-S15) for the yin-nourishing granules are all from Jiangsu province hospital, and have the specification of 10 g/bag.
The harpagoside reference (lot 111729-201707, purity 96.8%) was purchased from chinese food and drug assay institute; harpagoside reference (lot number MUST-18041504, purity > 98%), loganin reference (lot number MUST-17080704, purity > 98%), stilbene glycoside reference (lot number MUST-18051611, purity > 98%), hyperin reference (lot number MUST-17101605, purity > 98%) were all purchased from Du-Men biosciences, inc.; the emodin control (lot number 200148-200904, purity > 98%), the cuparanthrone control (lot number 200471-200903, purity 91%), the morroniside control (lot number 200514-200704, purity 92.5%), and the ferulic acid control (lot number 200418-170912, purity 97.3%) were all purchased from thailand danding biotechnology limited; cinnamic acid control (lot b21082, purity > 98%) was purchased from Shanghai source leaf biotechnology Co., ltd; methanol, acetonitrile and phosphoric acid are chromatographic purity, the rest reagents are analytical purity, and water is ultrapure water.
Example 1
1. A quality detection method of yang-suppressing and yin-nourishing particles comprises the following steps:
step 1: preparation of test sample solution
Grinding the above particles with total weight of 15 batches of S1-S15 into powder, sieving with a third sieve, precisely weighing about 1g of powder, adding 70% methanol solution, ultrasonically extracting for 20min (power 100W, frequency 40 kHz), cooling, weighing again, supplementing 70% methanol to the weight, shaking uniformly, standing, and collecting supernatant and filtering with 0.22 μm filter membrane.
Step 2: preparation of mixed control solution
Dissolving morroniside, loganin, ferulic acid, hyperoside, stilbene glycoside, cyamalone, harpagoside, cinnamic acid and emodin reference substances in methanol to obtain a mass concentration of 0.267 mg/mL -1 、0.105mg·mL -1 、0.361mg·mL -1 、0.221mg·mL -1 、0.249mg·mL -1 、0.040mg·mL -1 、0.092mg·mL -1 、0.228mg·mL- 1 、0.339mg·mL -1 Filtering with 0.22 μm filter membrane to obtain the final product.
Step 3, respectively sucking the sample solution in the step 1 and the mixed reference substance solution in the step 2, and injecting UPLC to obtain a chromatogram of the sample solution and a chromatogram of the mixed reference substance solution;
step 4, deriving the fingerprint of the test sample solution of the yang-suppressing and yin-nourishing particles obtained in the step 3, and introducing the fingerprint into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2012A; selecting chromatographic peaks existing in chromatograms of different batches of the test sample solutions of the yang-suppressing and yin-nourishing particles as common peaks; generating a control fingerprint of the test sample solution of the yang-suppressing and yin-nourishing particles by an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak; and labeling the chemical components of peaks in the reference fingerprint according to the retention time of the mixed reference solution chromatogram. The yang-suppressing and yin-nourishing particles have 18 common peaks at 250nm, wherein the No. 7 loganin peak is taken as a reference peak, the No. 1 peak is derived from alisma orientale through comparative analysis, the No. 3 peak is derived from prepared fleece-flower root and cornus officinalis, the No. 2-8 and No. 10 peaks are mainly derived from cornus officinalis, the No. 9 and No. 11 peaks are mainly derived from spanishneedles herb, the No. 12 peak is derived from prepared fleece-flower root, the No. 13 and No. 14 peaks are mainly derived from medicinal cyathula root, the No. 15 peak is derived from alisma orientale, the No. 16 and No. 17 peaks are derived from figwort, and the No. 18 peak is derived from prepared fleece-flower root and alisma orientale; wherein, the peak 6 is morroniside, the peak 7 is loganin, the peak 11 is hyperin, the peak 12 is stilbene glucoside, the peak 13 is ferulic acid, the peak 14 is amaranth sterone, the peak 16 is harpagoside, the peak 17 is cinnamic acid, and the peak 18 is emodin.
The chromatographic conditions of the step 3 are as follows:
waters ACQUITYUPLC BEH C A Waters ACQUITYUPLC BEH C chromatographic column (100 mm. Times.3.0 mm,1.7 μm) with acetonitrile (A) -water (B, 0.1% phosphoric acid) as mobile phase, gradient elution procedure as shown in Table 1, detection wavelength 250nm; the flow rate is 0.3mL min < -1 >; column temperature was 35 ℃.
TABLE 1 gradient elution procedure
| Time/min | A/％ | B/％ |
| 0 | 3 | 97 |
| 5 | 8 | 92 |
| 11 | 30 | 70 |
| 20 | 80 | 20 |
| 21 | 95 | 5 |
| 23 | 95 | 5 |
| 23.5 | 3 | 97 |
| 30 | 3 | 97 |
Step 5: similarity evaluation
15 batches of the sample solution of the yang-suppressing and yin-nourishing particles are introduced into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012A edition) for analysis according to sample injection, chromatograms are recorded, and the results show that the similarity of 15 batches of the sample solution of the yang-suppressing and yin-nourishing particles is more than 0.96, and the similarity of the sample solution of the yang-suppressing and yin-nourishing particles is good as shown in table 2.
TABLE 2 similarity of 15 batches of particles for suppressing yang and nourishing yin
2. Fingerprint methodology investigation:
(1) Precision test
Taking a sample of particles (number S1) for suppressing yang and nourishing yin, preparing a sample solution according to the method in the step 1, continuously sampling and measuring for 6 times according to the chromatographic conditions, and calculating that RSD of each common peak relative retention time is smaller than 0.50% (n=6) and RSD of the relative peak area is smaller than 4.60% (n=6) by taking loganin as a reference, so that the method has good precision.
(2) Stability test
Taking a sample of the particles (number S1) for suppressing yang and nourishing yin, preparing a test sample solution according to the method of the step 1, respectively placing the test sample solution for 0, 2, 4, 8, 12 and 24 hours at room temperature, carrying out sample injection measurement according to the chromatographic conditions, taking loganin as a reference, calculating RSD of each common peak relative peak area to be less than 4.70 percent (n=6), and RSD of each common peak relative retention time to be less than 2.90 percent (n=6), wherein the sample solution has good stability in 24 hours at room temperature.
(3) Repeatability test
Taking 6 parts of the particle sample (number S1) for suppressing yang and nourishing yin, preparing a sample solution according to the method in the step 1, carrying out sample injection measurement according to the chromatographic conditions, and calculating to obtain RSD of each common peak relative peak area which is smaller than 1.90% (n=6) and RSD of each common peak relative retention time which is smaller than 0.09% (n=6) by taking loganin as a reference, so that the method has good repeatability.
Example 2
1. The quality detection method of the yang-suppressing and yin-nourishing particles further comprises a content determination method, and specifically comprises the following steps:
step 1: preparation of test solution of yang-suppressing and yin-nourishing particles
Grinding the above particles with total weight of 15 batches of S1-S15 into powder, sieving with a third sieve, precisely weighing about 1g of powder, adding 70% methanol solution, ultrasonically extracting for 20min (power 100W, frequency 40 kHz), cooling, weighing again, supplementing 70% methanol to the weight, shaking uniformly, standing, and collecting supernatant and filtering with 0.22 μm filter membrane.
Step 2: preparation of mixed control solution
Precisely weighing appropriate amounts of harpagoside, morroniside, loganin, ferulic acid and cyamaranth, and adding methanol to prepare mixed reference substance solutions with the concentration of 0.0194mg/mL, 0.0114mg/mL, 0.0174mg/mL, 0.0134mg/mL, 0.0005mg/mL and 0.0052mg/mL respectively; and precisely weighing proper amount of stilbene glucoside, and adding methanol to prepare a reference substance solution with the concentration of 0.2730 mg/mL.
Step 3: establishment of a Standard Curve
Mixing the reference substance solutions in the step 2, adding methanol to dilute the mixed reference substance solutions in 0 time, 2 times, 5 times, 10 times and 20 times in sequence to obtain 5 mixed reference substance solutions with different concentrations, adding methanol to the reference substance solutions in the step 2 to prepare 5 reference substance solutions with the concentrations of 273.000, mug/mL 182.000 mug/mL, 91.000 mug/mL, 36.400 mug/mL and 18.200 mug/mL respectively, then injecting UPLC, and establishing a standard curve equation with the peak area as an ordinate and the mass concentration as an abscissa as shown in the following table 3:
TABLE 3 regression equation, correlation coefficient (r) and linear range
| Component to be measured | Regression equation | r | Linear range/(μg/mL) |
| Habaoside | Y＝6×10 9 X－222.99 | 0.9997 | 0.968～19.360 |
| Harpagoside | Y＝1×10 10 X+208.62 | 0.9999 | 0.568～11.350 |
| Morroniside | Y＝1×10 10 X+1098 | 0.9999 | 0.870～17.390 |
| Loganin | Y＝7×10 9 X+360.27 | 0.9997 | 0.670～13.400 |
| Stilbene glycoside | Y＝8×10 12 X－114904 | 0.9990 | 18.200～273.000 |
| Ferulic acid | Y＝3×10 10 X－1.9523 | 0.9998 | 0.025～0.490 |
| Amaranthrone | Y＝8×10 9 X+62.944 | 0.9999 | 0.259～5.187 |
The chromatographic conditions of this step 3 are:
WatersACQUITYUPLC BEH C18, 100mm×3.0mm,1.7 μm as column, mobile phase: acetonitrile as phase A-water containing 0.1% phosphoric acid as phase B, and gradient elution; detection wavelength: ferulic acid and stilbene glycoside are 320nm, morroniside, cuparanthrone, loganin are 250nm, harpagoside and harpagoside are 202nm; the flow rate is 0.3mL/min; the column temperature is 35 ℃; the sample injection amount is 2 mu L; the gradient elution procedure is as follows:
| time/min | A/％ | B/％ |
| 0 | 5 | 95 |
| 15 | 15 | 85 |
| 30 | 30 | 70 |
| 40 | 80 | 20 |
| 42 | 95 | 5 |
Step 4: and (3) injecting the solutions of the test samples in the step (1) into UPLC, substituting the peak area of the chromatogram into the standard curve equation in the step (3), and calculating the content of the corresponding effective components in the test samples as shown in Table 4.
TABLE 4 content of 7 Components (mg/g) in 15 samples
2. Methodology investigation of content determination:
1. precision experiments
Taking the same mixed reference substance solution, sampling for 6 times, and recording peak area. RSD of harpagoside, morroniside, loganin, stilbene glucoside, harpagoside, ferulic acid and cyasterone are respectively 3.70%, 3.36%, 3.67%, 2.85%, 3.88%, 3.05% and 3.22%, which indicates that the instrument precision is good.
2. Stability test
Sample solutions of the yang-suppressing and yin-nourishing particles are prepared according to the method, sample injection is carried out at 0, 2, 4, 8, 12 and 24 hours respectively, and peak areas are recorded. RSD of harpagoside, morroniside, loganin, stilbene glucoside, ferulic acid and cyasterone are respectively 2.6%, 1.7%, 0.3%, 3.3%, 1.2%, 1.9% and 2.3%, and the result shows that the method has good stability.
3. Repeatability experiments
Taking 6 parts of the yang-suppressing and yin-nourishing particles with the same batch number, preparing a sample solution for sample injection according to the method, and recording peak areas. The results showed that RSD of harpagoside, morroniside, loganin, stilbene glucoside, harpagoside, ferulic acid, cyrtomium fortunei sterone content was 3.59%, 3.66%, 4.07%, 4.89%, 4.71%, 4.70%, 3.96% (n=6), respectively, indicating that the method was good in reproducibility.
4. Sample recovery rate experiment
6 parts of the same batch number of yang-suppressing and yin-nourishing particles with known content are precisely added with harpagoside, morroniside, loganin, stilbene glucoside, ferulic acid, 0.1250, 0.0667, 0.2100, 0.1320, 0.1738, 0.0058 and 0.0570mg, and test sample solutions are prepared according to the content measurement method, and then the content measurement chromatographic condition sample injection measurement is carried out, so that the average sample recovery rates are respectively 105.55%, 95.00%, 101.32%, 101.43%, 97.79%, 105.17%, 102.28% and RSD are respectively 2.90%, 2.53%, 2.47%, 4.11%, 4.83%, 2.07% and 4.13% (n=6), and the method is good in accuracy. The experimental result shows that the fingerprint detection method of the yang-suppressing and yin-nourishing particles, which is established by the invention, has good precision, stability and repeatability, can effectively represent the quality of the yang-suppressing and yin-nourishing particles, and is beneficial to comprehensively monitoring the quality.
Claims (10)
1. The quality detection method of the yang-suppressing and yin-nourishing particles is characterized by comprising the following steps of:
step 1: preparation of test solution of yang-suppressing and yin-nourishing particles
Grinding the yang-suppressing and yin-nourishing particles in different batches into powder, sieving with a third sieve, precisely weighing, adding methanol solution, performing ultrasonic extraction, standing for precipitation, and filtering the supernatant with a 0.22 μm filter membrane to obtain the product;
step 2: preparation of mixed control solution
Dissolving reference substances such as morroniside, loganin, ferulic acid, hyperoside, stilbene glucoside, cyasterone, harpagoside, cinnamic acid and emodin in methanol to obtain mixed reference solution, and filtering with 0.22 μm filter membrane;
step 3, chromatogram determination
Respectively sucking the sample solution in the step 1 and the mixed reference substance solution in the step 2, and injecting UPLC to obtain a chromatogram of the sample solution and a chromatogram of the mixed reference substance solution;
step 4, deriving the fingerprint of the test sample solution of the yang-suppressing and yin-nourishing particles obtained in the step 3, and introducing the fingerprint into a Chinese medicine chromatographic fingerprint similarity evaluation system 2004A version; selecting chromatographic peaks existing in chromatograms of different batches of the test sample solutions of the yang-suppressing and yin-nourishing particles as common peaks; establishing a reference fingerprint of the test sample solution of the yang-suppressing and yin-nourishing particles with a time window width of 0.2min by a median method, and calculating the relative retention time and the relative peak area of each common peak; and labeling the chemical components of peaks in the reference fingerprint according to the retention time of the mixed reference solution chromatogram.
2. The method for detecting the quality of the yang-suppressing and yin-nourishing particles according to claim 1, characterized in that,
the preparation method of the test sample solution of the yang-suppressing and yin-nourishing particles comprises the following steps:
grinding the yang-suppressing and yin-nourishing particles in different batches into powder, sieving with a third sieve, precisely weighing about 1g of powder, adding 70% methanol solution, ultrasonically extracting for 20min, standing for precipitation, and collecting supernatant and filtering with a 0.22 μm filter membrane.
3. The method for detecting the quality of the yang-suppressing and yin-nourishing particles according to claim 1, wherein the preparation method of the mixed reference substance solution in the step 2 is as follows:
dissolving morroniside, loganin, ferulic acid, hyperoside, stilbene glycoside, cyamalone, harpagoside, cinnamic acid and emodin reference substances in methanol to obtain a mass concentration of 0.267 mg/mL -1 、0.105mg·mL -1 、0.361mg·mL -1 、0.221mg·mL -1 、0.249mg·mL -1 、0.040mg·mL -1 、0.092mg·mL -1 、0.228mg·mL -1 、0.339mg·mL -1 Filtering with 0.22 μm filter membrane to obtain the final product.
4. The method for detecting the quality of the particles for suppressing yang and nourishing yin according to claim 1, wherein the chromatographic conditions in the step 3 are as follows: waters ACQUITY UPLC BEH C18 chromatographic column, 100mm×3.0mm,1.7 μm mobile phase acetonitrile as phase A, aqueous solution containing 0.1% phosphoric acid as phase B, gradient elution, detection wavelength of 250nm; flow rate 0.3mL/min -1 The method comprises the steps of carrying out a first treatment on the surface of the Column temperature was 35 ℃.
5. The method for detecting the quality of the particles for suppressing yang and nourishing yin according to claim 4, wherein the gradient elution procedure in the step 3 is as follows:
6. the method for detecting the quality of the yang-suppressing and yin-nourishing particles according to claim 1, wherein the fingerprint spectrum of the step 5 is compared with 18 common peaks, wherein a loganin peak 7 is used as a reference peak, and the loganin peak 7, the loganin peak 11, the hyperin peak 12, the stilbene glycoside 13, the ferulic acid peak 14, the coparasterone peak 16, the harpagoside peak 17, the cinnamic acid peak 18 and the emodin are subjected to comparison analysis by a mixed reference solution chromatogram.
7. The method for detecting the quality of the yang-suppressing and yin-nourishing particles according to claim 1, further comprising a content measuring method, specifically comprising the following steps:
step 1: preparation of test solution of yang-suppressing and yin-nourishing particles
Grinding the yang-suppressing and yin-nourishing particles in different batches into powder, sieving with a third sieve, precisely weighing, adding methanol solution, performing ultrasonic extraction, standing for precipitation, and filtering the supernatant with a 0.22 μm filter membrane to obtain the product;
step 2: preparation of mixed control solution
Precisely weighing appropriate amount of harpagoside, morroniside, loganin, ferulic acid and cyamalone, and adding methanol to obtain mixed reference solution; in addition, a proper amount of stilbene glucoside is precisely weighed, and methanol is added to prepare a stilbene glucoside reference substance solution;
step 3: establishment of a Standard Curve
Taking the mixed reference substance solution in the step 2 and the stilbene glucoside reference substance solution, adding methanol to dilute the mixed reference substance solution in sequence to obtain 5 mixed reference substance solutions with different concentrations, then injecting UPLC, and establishing a standard curve equation by taking the peak area as an ordinate and the concentration as an abscissa;
step 4: and (3) injecting the sample solution in the step (1) into UPLC, substituting the peak area of the chromatogram into the standard curve equation in the step (3), and calculating the content of the corresponding effective components in the sample.
8. The method for detecting the quality of the particles for suppressing yang and nourishing yin according to claim 7, wherein the chromatographic conditions in the step 3 and the step 4 are as follows:
waters ACQUITY UPLC BEH C18, 100mm×3.0mm,1.7 μm as column, mobile phase: acetonitrile as phase A-water containing 0.1% phosphoric acid as phase B, and gradient elution; detection wavelength: ferulic acid and stilbene glycoside are 320nm, morroniside, cuparanthrone, loganin are 250nm, harpagoside and harpagoside are 202nm; the flow rate is 0.3mL/min; the column temperature is 35 ℃; the sample injection amount is 2 mu L; the gradient elution procedure is as follows:
9. the method for detecting the quality of the yang-suppressing and yin-nourishing particles according to claim 7, characterized in that,
the preparation method of the test sample solution of the yang-suppressing and yin-nourishing particles comprises the following steps:
grinding the yang-suppressing and yin-nourishing particles in different batches into powder, sieving with a third sieve, precisely weighing about 1g of powder, adding 70% methanol solution, ultrasonically extracting for 20min, standing for precipitation, and filtering the supernatant with a 0.22 μm filter membrane to obtain the final product;
step 2: preparation of mixed control solution
Precisely weighing appropriate amount of harpagoside, morroniside, loganin, ferulic acid and cyamalone, and adding methanol to obtain mixed reference substance solutions with concentrations of 0.0194, 0.0114, 0.0174, 0.0134, 0.0005 and 0.0052mg/mL respectively; and precisely weighing proper amount of stilbene glucoside, and adding methanol to prepare a reference substance solution with the concentration of 0.2730 mg/mL.
10. The method for detecting the quality of the yang-suppressing and yin-nourishing particles according to any one of claims 1 to 9, characterized in that the method for preparing the yang-suppressing and yin-nourishing particles comprises the steps of: decocting herba Bidentis Bipinnatae, radix Polygoni Multiflori Preparata, fructus Corni, radix scrophulariae, alismatis rhizoma, and radix Cyathulae in water for 1-3 times, filtering, mixing filtrates, concentrating under reduced pressure to appropriate amount, centrifuging, concentrating supernatant to fluid extract with relative density of 1.20 at 60deg.C, and adding pharmaceutically acceptable adjuvants.
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