CN110988238A - Method for constructing HPLC (high performance liquid chromatography) characteristic map of standard decoction of lilac daphne flower bud and method for measuring component content of lilac daphne flower bud - Google Patents
Method for constructing HPLC (high performance liquid chromatography) characteristic map of standard decoction of lilac daphne flower bud and method for measuring component content of lilac daphne flower bud Download PDFInfo
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- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G01N30/02—Column chromatography
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Abstract
本发明涉及一种芫花标准汤剂HPLC特征图谱的构建方法及其成分含量测定方法，所述的芫花标准汤剂的成分含量测定方法，包括如下步骤：（1）分别吸取对照品溶液和待测芫花标准汤剂样品溶液注入高效液相色谱仪，测定相应峰面积；（2）通过外标法计算芫花素的含量；（3）再通过一测多评法计算芹菜素‑7‑O‑β‑葡萄糖醛酸苷、芹菜素、羟基芫花素的含量。本发明通过外标法计算出芫花素的含量，再通过一测多评法计算芹菜素‑7‑O‑β‑葡萄糖醛酸苷、芹菜素、羟基芫花素的含量，能有效简化4中成分的含量测定的操作步骤，减少测定时间，提高工作效率，实现对芫花标准汤剂化学成分的定量分析。
The invention relates to a method for constructing an HPLC characteristic spectrum of a daisy flower standard decoction and a method for determining its component content. The said method for determining the component content of a daisy flower standard decoction comprises the following steps: (1) respectively drawing a reference solution and a The sample solution of the standard decoction of daphnia to be tested was injected into a high performance liquid chromatograph, and the corresponding peak area was determined; (2) the content of davidin was calculated by the external standard method; (3) the apigenin-7 was calculated by the one-measurement-multiple-evaluation method. Contents of ‑O‑β‑glucuronide, apigenin, and hydroxyflorin. The present invention calculates the content of davidin through the external standard method, and then calculates the content of apigenin-7-O-β-glucuronide, apigenin and hydroxy davidin through the one-measurement-multiple-evaluation method, which can effectively simplify 4 The operation steps for the determination of the content of the ingredients in the Chinese herbal medicine can reduce the determination time, improve the work efficiency, and realize the quantitative analysis of the chemical ingredients of the standard decoction of coriander flowers.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicine identification, and particularly relates to a method for constructing a lilac daphne flower bud standard decoction HPLC characteristic spectrum and a method for measuring component content of the lilac daphne flower bud standard decoction.
Background
Flos Genkwa is dry flower bud of flos Genkwa of Thymelaeaceae. Picking in spring when the flower is not open, removing impurities, drying, scraping off the rough skin when the flower is fresh after washing, cleaning and drying; the product has effects of purging pathogenic water, removing toxic substance, and killing parasites, and can be used for treating edema, distention, phlegm and fluid accumulation, asthma, and cough. Flos Genkwa is also named as flos Genkwa, flos Genkwa strip, and herba Houttuyniae. It is recorded in Shen nong Ben Cao Jing, listed as the inferior product. Modern medical research shows that the main drug effect component of the lilac daphne flower bud is flavonoid substance, and has various pharmacological effects of relieving cough, eliminating phlegm, promoting urination, reducing diarrhea, resisting inflammation, resisting tumor, inducing labor, resisting fertility and the like. The chemical components with pharmacological activity are flavonoids such as genkwanin, apigenin, luteolin, etc.
The one-test-multiple-evaluation method (QAMS) can realize simultaneous determination of a plurality of components by only determining one component (the reference substance is easy to obtain), and can effectively solve the difficulty caused by the shortage of the reference substance in the process of quality analysis.
The existing quality control method for the medicine mostly adopts different chromatographic conditions to respectively measure the content of one or two components, and has the defects of complex operation, long detection time, high detection cost and the like.
The standard decoction of lilac daphne flower bud is prepared by extracting a single Chinese medicinal decoction piece with water, concentrating and drying, a method for effectively controlling the quality of the standard decoction of lilac daphne flower bud is not established at present, and a literature report that the characteristic map of the standard decoction of lilac daphne flower bud is simultaneously determined by an HPLC method and the content of 4 chemical components in the standard decoction of lilac daphne flower bud is determined by combining a multi-evaluation method is not found in the prior art.
Disclosure of Invention
The invention aims to provide a method for constructing HPLC (high performance liquid chromatography) characteristic map of a lilac daphne flower bud standard decoction and a method for measuring the content of components of the lilac daphne flower bud standard decoction.
The technical problem to be solved by the invention is realized by the following technical scheme:
a method for constructing HPLC characteristic map of flos genkwa standard decoction comprises the following steps:
(1) accurately weighing flos Genkwa standard decoction powder, and preparing to obtain flos Genkwa standard decoction sample solution;
(2) analyzing the sample solution of the flos Genkwa standard decoction by high performance liquid chromatography to obtain HPLC characteristic map of the flos Genkwa standard decoction.
Preferably, the chromatographic conditions for the high performance liquid chromatograph analysis are as follows: using octadecylsilane chemically bonded silica as a filler, taking methanol as a mobile phase A and 0.05-0.15% phosphoric acid water solution as a mobile phase B at the column temperature of 25-45 ℃, and carrying out gradient elution at the flow rate of 0.6-1.4 mL/min and the detection wavelength of 340-370 nm, wherein the sample amount is 8-12 mu L.
As a preferred embodiment, the gradient elution conditions are: the volume fraction of the mobile phase A is changed to be 20% -43% and the volume fraction of the mobile phase B is changed to be 80% -57% in 0-3 min; the volume fraction of the mobile phase A is changed to 43-45% and the volume fraction of the mobile phase B is changed to 57-55% in 3-25 min; the volume fraction of the mobile phase A is changed to 45-60% and the volume fraction of the mobile phase B is changed to 55-40% in 25-35 min; and 35-50 min, wherein the volume fraction of the mobile phase A is changed to 60-95%, and the volume fraction of the mobile phase B is changed to 40-5%.
As a preferable scheme, the preparation method of the test solution comprises the following steps: grinding standard flos Genkwa decoction, precisely weighing 0.05-0.15 g, precisely adding 20-30 mL of 60-80% ethanol, weighing, ultrasonically treating for 20-40 min, taking out, cooling, supplementing the lost weight with 60-80% ethanol, shaking, and filtering with a filter membrane.
The invention also provides a component content determination method of the lilac daphne flower bud standard decoction, which comprises the following steps:
(1) respectively sucking a reference substance solution and a standard decoction sample solution of flos Genkwa to be detected, injecting into a high performance liquid chromatograph, and measuring corresponding peak areas;
(2) calculating the content of the genkwanin by an external standard method;
(3) and calculating the contents of apigenin-7-O- β -glucuronide, apigenin and hydroxygenkwanin by a one-test-multiple evaluation method.
As a preferable scheme, the preparation method of the reference substance solution comprises the steps of respectively taking apigenin-7-O- β -glucuronide, apigenin, hydroxygenkwanin and genkwanin reference substances, putting the reference substances into a 10mL volumetric flask, adding methanol to a constant volume to reach scales, and preparing reference substance solutions containing 0.04-0.08 mg of apigenin-7-O- β -glucuronide, 0.04-0.08 mg of apigenin, 0.01-0.03 mg of hydroxygenkwanin and 0.01-0.03 mg of genkwanin in each 1mL, so as to obtain the reference substance solution.
As a preferred scheme, the preparation method of the daphne genkwa standard decoction sample solution to be detected comprises the following steps: grinding standard flos Genkwa decoction, precisely weighing 0.05-0.15 g, precisely adding 20-30 mL of 60-80% ethanol, weighing, ultrasonically treating for 20-40 min, taking out, cooling, supplementing the lost weight with 60-80% ethanol, shaking, and filtering with a filter membrane.
Preferably, the chromatographic conditions for the high performance liquid chromatograph analysis are as follows: using octadecylsilane chemically bonded silica as a filler, taking methanol as a mobile phase A and 0.05-0.15% phosphoric acid water solution as a mobile phase B at the column temperature of 25-45 ℃, and carrying out gradient elution at the flow rate of 0.6-1.4 mL/min and the detection wavelength of 340-370 nm, wherein the sample amount is 8-12 mu L.
As a preferred embodiment, the gradient elution conditions are: the gradient elution conditions were: the volume fraction of the mobile phase A is changed to be 20% -43% and the volume fraction of the mobile phase B is changed to be 80% -57% in 0-3 min; the volume fraction of the mobile phase A is changed to 43-45% and the volume fraction of the mobile phase B is changed to 57-55% in 3-25 min; the volume fraction of the mobile phase A is changed to 45-60% and the volume fraction of the mobile phase B is changed to 55-40% in 25-35 min; and 35-50 min, wherein the volume fraction of the mobile phase A is changed to 60-95%, and the volume fraction of the mobile phase B is changed to 40-5%.
A method for constructing HPLC characteristic map of flos Genkwa standard decoction or a method for determining component content of flos Genkwa standard decoction is used for detecting and identifying flos Genkwa standard decoction.
The method has the beneficial effects that (1) an HPLC characteristic map of a daphne genkwa standard decoction is constructed, 6 common peaks are established, chemical components of the daphne genkwa standard decoction are identified, the peak 1 is apigenin-7-O- β -glucuronide, the peak 2 is luteolin, the peak 3 is tilianin, the peak 4 is apigenin, the peak 5 is hydroxydaphnetin, the peak 6 is daphnetin, characteristic peak information of a sample is fully reflected, chemical component characteristics of the daphne genkwa standard decoction are displayed, (2) the HPLC characteristic map constructed by the method can be used for qualitatively identifying the daphne genkwa standard decoction, the method is stable, high in precision and good in reproducibility, (3) the content of the daphne genkwa essence is calculated by an external standard method, the content of the apigenin-7-O- β -glucuronide, the apigenin and hydroxydaphnetin is calculated by a one-many-test method, the operation steps of content determination of the 4 components can be effectively simplified, the working efficiency is improved, quantitative analysis of the daphne genkwa standard decoction components is realized, the qualified content of the daphne genkwa standard decoction, the daphne genkwa essence is obtained, the purity of the daphne genkwa extract, the content of the daphne genkwa standard decoction, the content of the daphne genkwa extract is determined, the content of the daphne genkwa extract, the daphne genkwa extract is determined, the method is determined, the method is accurate, the method is determined, the method is used for identifying the method, the method.
Drawings
Figure 1 is a profile of mobile phase and elution conditions 1.
Fig. 2 is a characteristic diagram of the mobile phase and elution conditions 2.
Fig. 3 is a characteristic diagram of the mobile phase and elution conditions 3.
Fig. 4 is a characteristic diagram of the mobile phase and elution conditions 4.
FIG. 5 is an overlay of HPLC characteristic maps of 15 batches of flos Genkwa standard decoction.
FIG. 6 is HPLC characteristic map of flos Genkwa standard decoction.
FIG. 7 is a graph of the identification of characteristic map control of flos Genkwa standard decoction.
In the figure, the peak 1 is apigenin-7-O- β -glucuronide, the peak 2 is luteolin, the peak 3 is tilianin, the peak 4 is apigenin, the peak 5 is hydroxygenkwanin, and the peak 6 is genkwanin.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The instruments, reagents and reagents used in the invention are as follows:
the instrument comprises the following steps: a Waters e2695 model high performance liquid chromatograph, a Thermo Vanqish model high performance liquid chromatograph, and an Agilent 1290 II model high performance liquid chromatograph; XP26 parts per million electronic balance (mettler-toledo instruments ltd); model ME204E ten thousandth electronic balance (mettler-toledo instruments ltd); one-thousandth balance (mettler-toledo instruments ltd), model KQ500DE digital controlled ultrasonic cleaner; ultrapure water system of the Milli-Qdirect type (Merck Co., Ltd.).
Reagent: methanol (merck, germany, chromatographically pure); methanol (analytical pure, by west longa science ltd); phosphoric acid (Tianjin Kemi Euro Chemicals, Inc., pure chromatography); methanol (analytical pure, by west longa science ltd); the water is laboratory self-made water.
The test reagents comprise genkwanin (China food and drug inspection research institute, content: 94.9%), hydroxygenkwanin (Chengdupifield biotechnology limited, content: 98.92%), apigenin (China food and drug inspection research institute, content: 99.2%), apigenin-7-O- β -glucuronide (Shanghai Shidande biotechnology limited, content: 99.6%), luteolin (China food and drug inspection research institute, content: 99.6%), tilianin (Sichuan Weikeqi biotechnology limited, content: 98.0%), genkwanin control medicinal materials (China food and drug inspection research institute), and standard genkwanin decoction (number: S1-S15, Guangdong pharmaceutical limited, laboratory self-made).
Preparation of standard decoction of flos Genkwa: 100g of daphne genkwa decoction pieces are taken, water is added for decoction twice, 12 times of water is added for the first decoction, the decoction is carried out for 30 minutes after soaking and 30 minutes of decoction, a 350-mesh screen is used for hot filtration, and the filtrate is rapidly cooled by cold water. Adding 10 times of water into the second decoction, decocting for 25 minutes, filtering with a 350-mesh screen while the decoction is hot, rapidly cooling the filtrate with cold water, and combining the two filtrates. And transferring the combined filtrate to a rotary evaporator for concentration (the water bath temperature is 65 ℃, the rotating speed is 90 r/min, the vacuum degree is-0.08 to-0.1 MPa), concentrating to 150mL of fluid extract, subpackaging to 10mL of penicillin bottles with the subpackaging volume of 2mL, transferring to a vacuum cold drying machine for drying after subpackaging is finished, taking out, and rolling an aluminum cover to obtain the penicillin.
Example 1
Method for constructing HPLC (high performance liquid chromatography) characteristic map of flos genkwa standard decoction
1. The chromatographic conditions of the high performance liquid chromatograph analysis are as follows: using octadecylsilane chemically bonded silica as filler, column temperature of 35 deg.C, methanol as mobile phase A, and 0.1% phosphoric acid water solution as mobile phase B, and performing gradient elution at flow rate of 1mL/min, detection wavelength of 350nm, and sample amount of 10 μ L.
2. The gradient elution conditions were: the volume fraction of the mobile phase A is changed to be 20% -43% and the volume fraction of the mobile phase B is changed to be 80% -57% in 0-3 min; the volume fraction of the mobile phase A is changed to 43-45% and the volume fraction of the mobile phase B is changed to 57-55% in 3-25 min; the volume fraction of the mobile phase A is changed to 45-60% and the volume fraction of the mobile phase B is changed to 55-40% in 25-35 min; and 35-50 min, wherein the volume fraction of the mobile phase A is changed to 60-95%, and the volume fraction of the mobile phase B is changed to 40-5%.
3. The preparation method of the test solution comprises the following steps: grinding flos Genkwa standard decoction, collecting 0.1g, precisely weighing, precisely adding 70% ethanol 25mL, weighing, ultrasonic treating for 30min, taking out, cooling, supplementing with 70% ethanol, shaking, and filtering with filter membrane.
4. The control solution is prepared by respectively taking apigenin-7-O- β -glucuronide, luteolin, tiliroside, apigenin, hydroxygenkwanin and genkwanin as appropriate amount, and preparing into control solution containing apigenin-7-O- β -glucuronide 0.06mg, luteolin 0.01mg, tiliroside 0.01mg, apigenin 0.06mg, hydroxygenkwanin 0.02mg and genkwanin 0.02mg per 1 mL.
5. The determination method comprises the following steps: precisely sucking 10 μ L of each of the reference solution and the sample solution, and performing sample injection measurement under the specified chromatographic condition.
6. Methodology investigation:
the preparation method of the test solution is considered as follows: the experiment respectively investigates the influence of different extraction solvents, different extraction modes, different extraction time and different extraction solvent dosage on the characteristic map of the standard lilac daphne flower bud decoction and the content of chemical components in the standard lilac daphne flower bud decoction, determines the optimal conditions by measuring the total peak area/sample weighing amount of each characteristic peak and the content of each component, and has the following selection factors and the following results.
Extracting solvent: methanol, 70% ethanol, ethanol were selected and the results are shown in Table 1.
TABLE 1 examination of extraction solvent
The extraction method comprises the following steps: heating reflux extraction and ultrasonic treatment extraction are selected, and the results are shown in table 2.
TABLE 2 examination of extraction modes
Extraction time: the selection time is 15min, 30min and 45min, and the results are shown in Table 3.
TABLE 3 extraction time survey
The dosage of the extraction solvent is as follows: selecting a material-liquid ratio of 0.1g: 15mL, 0.1g:25mL, 0.1g: 50mL, 0.1g: 100mL, the results are shown in Table 4.
TABLE 4 examination of the amount of extraction solvent used
From the above results, it can be seen that, when 70% ethanol is used as the extraction solvent for extraction, the total peak area/sample weighing amount is the largest and the content of 4 index components is the highest, and 70% ethanol is selected as the extraction solvent; two extraction modes of ultrasonic treatment and heating reflux are adopted, the results of the total peak area/sample weighing and the content of 4 index components are not very different, and the ultrasonic treatment extraction mode is adopted in consideration of the simplicity and convenience of operation; extracting at different extraction times, wherein the differences between the total peak area/sample weighing amount and the content results of the 4 index components are small, and the extraction time is selected to be 30min to ensure complete extraction; along with the increase of the dosage of the extraction solvent, the results of the total peak area/sample weighing and the content of 4 index components have little difference, the dosage of the extraction solvent is 25mL, the extraction is complete, and 25mL is selected for ensuring the durability of the method and selecting proper sample injection concentration.
Therefore, a preparation method using 70% ethanol as an extraction solvent, sonication for 30 minutes, and 0.1g:25mL as a feed-to-liquid ratio as a sample was determined.
7. Optimization of chromatographic conditions
7.1 selection of chromatography columns
As a result of comparison, the Waters HSS T3 column (4.6X 250mm,5.0 μm) showed the best separation and the better peak pattern, so the Waters HSS T3 column (4.6X 250mm,5.0 μm) was selected as the column.
7.2 selection of detection wavelength
The full-wavelength scanning is carried out on the sample by adopting a diode array detector, the base line is stable at 350nm, and the peak area of each peak is higher, so that the detection wavelength is determined to be 350 nm.
7.3 selection of the Mobile phase and elution conditions
The selection of the mobile phase is a key technology for simultaneously determining the characteristic map of the standard decoction of lilac daphne and the content determination of 4 components in the lilac daphne.
The mobile phase and the elution conditions are selected and compared by adopting the following method:
mobile phase condition optimization 1:
using a Waters HSS-T3 column (4.6X 250mm,5.0 μm) as a chromatographic column; gradient elution was performed as specified in table 5 using methanol as mobile phase a and 0.2% formic acid as mobile phase B; the detection wavelength is 332nm, the detection speed is 1.0mL/min, the injection volume is 10 muL, the column temperature is 35 ℃, and the test spectrum is shown in figure 1.
TABLE 5 gradient elution conditions 1
Mobile phase condition optimization 2:
using a Waters HSS-T3 column (4.6X 250mm,5.0 μm) as a chromatographic column; gradient elution was performed as specified in table 6 using methanol as mobile phase a and 0.2% formic acid as mobile phase B; the detection wavelength is 350nm, the detection speed is 1.0mL/min, the sample injection volume is 10 muL, and the test result is shown in figure 2 when the column temperature is 35 ℃.
TABLE 6 gradient elution conditions 2
Mobile phase condition optimization 3:
using a Waters HSS-T3 column (4.6X 250mm,5.0 μm) as a chromatographic column; gradient elution was performed as specified in table 7 with methanol as mobile phase a and 0.2% formic acid as mobile phase B; the detection wavelength is 350nm, the detection speed is 1.0mL/min, the sample injection volume is 10 muL, the column temperature is 35 ℃, and the test result is shown in figure 3.
TABLE 7 gradient elution conditions 3
Mobile phase condition optimization 4:
using a Waters HSS T3 column (4.6X 250mm,5.0 μm) as a chromatographic column; gradient elution was performed as specified in table 8 using methanol as mobile phase a and 0.1% phosphoric acid as mobile phase B; the detection wavelength is 350nm, the detection speed is 1.0mL/min, the sample injection volume is 10 muL, the column temperature is 35 ℃, and the test result is shown in figure 4.
TABLE 8 gradient elution conditions 4
The results show that: the optimum condition 1 separation degree did not meet the requirement. The chromatographic peak separation effect of the optimized condition 2 is better, the peak purity also meets the requirement, but the separation time is longer. The separation effect of the optimized condition 3 is not obvious and is long. And in the optimization condition 4, the individual chromatographic peaks meet the baseline separation requirement and the separation time is short, so the optimization condition 4 is selected for experiments.
8. Characterization atlas methodology validation
8.1 precision test: preparing a test solution from standard decoction of flos Genkwa (batch number: S1), continuously injecting sample for 6 times under the specified chromatographic condition, and examining the relative retention time of the common peak and the consistency of the relative peak area, wherein the relative peak area of the common peak and the relative retention time RSD are less than 2%, which indicates that the precision of the instrument is good.
8.2 stability test: preparing flos Genkwa standard decoction (batch number: S1) into test solution, injecting sample for 0, 2, 4, 8, 16, and 24h under specified chromatographic condition, respectively, measuring the relative retention time of common peak and the consistency of relative peak area, and calculating RSD value. The relative peak area and the relative retention time RSD of the obtained common peak are less than 2 percent, which indicates that the test solution has good stability within 24 hours.
8.3 repeatability tests: taking flos Genkwa standard decoction (batch number: S1), preparing 6 parts of sample solution, performing sample injection measurement under specified chromatographic conditions, examining the relative retention time of common peak and the consistency of relative peak area, and calculating RSD value. The relative peak area RSD of the obtained common peak is less than 3%, and the relative retention time RSD is less than 3%, which indicates that the method has good repeatability.
9. Establishment of characteristic map of standard decoction of lilac daphne
Taking 15 batches of flos Genkwa standard decoction samples, preparing test solution, injecting 10 μ L of sample under specified chromatographic condition, and recording HPLC chromatogram. Introducing the chromatogram into a Chinese medicinal chromatogram fingerprint similarity evaluation system for data matching, establishing characteristic chromatogram of flos Genkwa standard decoction, and generating common pattern as shown in figure 5 and figure 6. Marking 6 common peaks in the standard decoction chromatogram of the lilac daphne flower bud, taking the peak No. 4 as a reference peak S, and calculating the relative retention time of the peaks No. 1 to 5. And calculating the relative retention time RSD value of each characteristic peak. The result shows that the RSD values of the common peaks of the spectrograms of the samples are all less than 3.0 percent; the RSD values of the relative peak areas were greatly different, as shown in tables 9 and 10.
Relative retention time of common peaks of characteristic maps of standard flos Genkwa decoction of Majorana Prain et Burkill 915 batches
Relative peak area of common peak of characteristic spectrum of exterior 1015 batch daphne genkwa standard decoction
The similarity of 15 batches of the daphne genkwa standard decoction samples is respectively calculated, which is shown in table 11, and the similarity of 15 batches of daphne genkwa standard decoction samples is more than 0.90, which indicates that the daphne genkwa standard decoction has better chemical component consistency and more stable quality.
Similarity of standard decoction of flos Genkwa from 1115 batches of them
10. Feature map peak assignment
The test solution and the reference solution are respectively detected under the specified chromatographic condition, 6 compounds are determined in total through the retention time of chromatographic peaks of the compounds and comparison with the reference, and according to the sequence of the retention time, the chromatographic peaks of the test solution and the 6 reference solutions are respectively superimposed, namely apigenin-7-O- β -glucuronide (peak 1), luteolin (peak 2), tilianin (peak 3), apigenin (peak 4), hydroxygenkwanin (peak 5) and genkwanin (peak 6), and the chromatographic peaks of the test solution and the 6 reference solutions are shown in figure 7.
Example 2
Component content determination method for standard decoction of flos Genkwa
1. The chromatographic conditions of the high performance liquid chromatograph analysis are as follows: using octadecylsilane chemically bonded silica as filler, column temperature of 35 deg.C, methanol as mobile phase A, and 0.1% phosphoric acid water solution as mobile phase B, and performing gradient elution at flow rate of 1mL/min, detection wavelength of 350nm, and sample amount of 10 μ L.
2. The gradient elution conditions were: the volume fraction of the mobile phase A is changed to be 20% -43% and the volume fraction of the mobile phase B is changed to be 80% -57% in 0-3 min; the volume fraction of the mobile phase A is changed to 43-45% and the volume fraction of the mobile phase B is changed to 57-55% in 3-25 min; the volume fraction of the mobile phase A is changed to 45-60% and the volume fraction of the mobile phase B is changed to 55-40% in 25-35 min; and 35-50 min, wherein the volume fraction of the mobile phase A is changed to 60-95%, and the volume fraction of the mobile phase B is changed to 40-5%.
3. The preparation method of the standard decoction sample solution of flos Genkwa to be detected comprises the following steps: grinding flos Genkwa standard decoction, collecting 0.1g, precisely weighing, precisely adding 70% ethanol 25mL, weighing, ultrasonic treating for 30min, taking out, cooling, supplementing with 70% ethanol, shaking, and filtering with filter membrane.
4. The control solution is prepared by respectively taking apigenin-7-O- β -glucuronide, apigenin, hydroxygenkwanin and genkwanin as appropriate, placing in a 10mL volumetric flask, adding methanol to constant volume to scale, and preparing into control solution containing apigenin-7-O- β -glucuronide 0.06mg, apigenin 0.06mg, hydroxygenkwanin 0.02mg and genkwanin 0.02mg per 1 mL.
5. Methodology investigation
And 5.1, precisely absorbing 10 mu L of the same standard decoction sample solution of daphne genkwa to be detected respectively, injecting the solution into a high performance liquid chromatograph, repeatedly injecting the sample for 6 times, and recording peak areas, wherein the RSD of the peak area of apigenin-7-O- β -glucuronide is calculated to be 0.47%, the RSD of the peak area of apigenin is 0.24%, the RSD of the peak area of hydroxydaphnetin is 0.30%, and the RSD of the peak area of daphnetin is 0.20%, so that the instrument precision is good.
5.2 Linear relationship, precisely weighing apigenin-7-O- β -glucuronide, apigenin, hydroxygenkwanin, genkwanin reference substance 25.614mg, 7.558mg, 4.036mg and 2.132mg respectively, placing in a 25mL volumetric flask, adding methanol for dissolving, fixing the volume to scale, making a linear mother liquor of the standard decoction of genkwanin, precisely measuring the linear mother liquor of the standard decoction of genkwanin 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0mL in a 10mL volumetric flask, adding methanol for fixing the volume to scale line, thus obtaining a linear mixed reference solution of the standard decoction of genkwanin, precisely sucking and mixing 10 μ L of the mixed reference solution, measuring according to the chromatographic conditions, recording a chromatogram, carrying out regression treatment by using peak area (Y) of each component and concentration (X, 1 μ g/mL) of the reference substance, thus obtaining equation and linear range of each component, and the table 12 shows regression.
Linear relation investigation result of 4 components in standard decoction of Daphne genkwa (Daphne genkwa) in Table 12
And 5.3, repeatability, namely taking the same batch of daphne genkwa standard decoction (batch number: S1), preparing 6 parts of daphne genkwa standard decoction sample solution to be detected in parallel according to the preparation method of the daphne genkwa standard decoction sample solution to be detected, carrying out parallel determination according to the chromatographic conditions, and calculating that the content RSD of apigenin-7-O- β -glucuronide is 1.27%, the content RSD of apigenin is 1.59%, the content RSD of hydroxydaphnetin is 1.66% and the content RSD of daphnetin is 1.15%, thereby indicating that the method has good repeatability.
5.4 stability, precisely absorbing the same standard decoction sample solution of daphne genkwa to be detected, injecting samples after 0, 2, 4, 8, 16 and 24 hours respectively, measuring peak areas, and calculating that the RSD of the peak area of apigenin-7-O- β -glucuronide is 1.48%, the RSD of the peak area of apigenin is 1.55%, the RSD of the peak area of hydroxyl daphne genkwa is 1.51% and the RSD of the peak area of daphne genkwa is 1.36%, thereby indicating that the precision of the instrument is good.
5.5 sample adding and recycling rate, namely precisely weighing 9 parts of standard lilac daphne flower bud decoction (batch number: S1) with known content, respectively and precisely adding a certain amount of reference substance solution, and calculating the recycling rates of apigenin-7-O- β -glucuronide, apigenin, hydroxylilac daphnetin and lilac daphne flower bud element according to the preparation method and chromatographic condition measurement of the standard lilac daphne bud decoction sample solution to be measured, wherein the results are shown in Table 13 and show that the sample adding and recycling rates are good.
TABLE 13 summary of methodological validation results
6. Calculation of Relative Correction Factor (RCF): the multi-evaluation method utilizes the principle that the quantity (quality or concentration) of components in a certain linear range is in direct proportion to a detector, and adopts a multipoint correction method to calculate a correction factor, wherein the formula is as follows:
in the formula ACIs the area of the peak of the internal reference substance CCIs the concentration of internal reference C, AAThe peak area of a certain component A to be measured, CAIs the concentration of a certain component A to be measured.
Taking the reference solution, injecting 10 μ L of the solution under the specified chromatographic condition, measuring, taking genkwanin as an internal reference substance d, and calculating the relative correction factors of apigenin-7-O- β -glucuronide (a), apigenin (b) and hydroxygenkwanin (c) according to a formula, wherein the results are shown in a table 14.
Relative correction factor for 4 components in standard decoction of radix et rhizoma Rhei Palmati and flos Genkwa
Reproducibility study of relative correction factors: the mixed control solution (10. mu.L) was precisely measured and measured under prescribed chromatographic conditions, and the effects of different chromatographic columns, column temperatures and flow rates on the relative correction factors of Waters e2695, Thermo Vanqish, Agilent 1290 II-type high performance liquid chromatograph, Waters HSS T3 (4.6 mm. times.250 mm,5.0 μm) and Agilent TC-C18 (4.6 mm. times.250 mm,5.0 μm) were examined, respectively, and the results are shown in Table 15. Results RSDs were all less than 2%, indicating that different instruments, chromatographic columns, column temperatures and flow rates had no significant effect on the relative correction factors.
TABLE 15 Effect of different conditions on relative correction factor
Positioning of chromatographic peaks to be detected: and calculating the relative retention time of different chromatographs, chromatographic columns, column temperatures and flow rates by using the relative value of the daphnetin retention time as a positioning standard of the component to be detected. The result shows that under the conditions of different chromatographs, chromatographic columns, column temperature and flow rate change, the numerical difference of the relative retention time of the components to be detected is small (RSD is less than 4 percent), and the reproducibility is good. The results are shown in Table 15.
Comparing the results of the multi-evaluation method and the external standard method, namely taking a daphne genkwa standard decoction sample, preparing a daphne genkwa standard decoction sample solution to be detected, analyzing under a specified chromatographic condition, respectively adopting the external standard method and the one-multi evaluation method to calculate the content of apigenin-7-O- β -glucuronide, apigenin, hydroxydaphnetin and daphnetin in the daphne genkwa standard decoction, wherein the results show that the content data measured by the 2 content measurement methods are not different, and the content of 4 target components of apigenin-7-O- β -glucuronide, apigenin, hydroxydaphnetin and daphnetin in the daphne genkwa standard decoction can be simultaneously measured by using one reference substance, and the results are shown in a table 16.
Comparison of results of table 16 external standard method and one-test-multiple evaluation method for determination of 4 components in flos Genkwa standard decoction
As can be seen from the data in Table 15, the RSD of the content of 2 ingredients in 15 batches of the standard Daphne genkwa decoction is less than 4%, which shows that the one-test-multiple evaluation method established in the research can replace an external standard method to control the content of the standard Daphne genkwa decoction.
According to the data, index ranges of genkwa standard decoction contents are established, wherein the total content ranges of apigenin-7-O- β -glucuronide and apigenin are 29.309-49.562 mg/g and the total content ranges of hydroxygenkwa essence and genkwa essence are 3.509-5.197 mg/g in terms of dry products.
Example 3
Identifying flos Genkwa standard decoction by using HPLC characteristic map.
The standard lilac daphne flower bud decoction is prepared by laboratories.
1. The chromatographic conditions of the high performance liquid chromatograph analysis are as follows: using octadecylsilane chemically bonded silica as filler, column temperature of 35 deg.C, methanol as mobile phase A, and 0.1% phosphoric acid water solution as mobile phase B, and performing gradient elution at flow rate of 1mL/min, detection wavelength of 350nm, and sample amount of 10 μ L.
2. The gradient elution conditions were: the volume fraction of the mobile phase A is changed to be 20% -43% and the volume fraction of the mobile phase B is changed to be 80% -57% in 0-3 min; the volume fraction of the mobile phase A is changed to 43-45% and the volume fraction of the mobile phase B is changed to 57-55% in 3-25 min; the volume fraction of the mobile phase A is changed to 45-60% and the volume fraction of the mobile phase B is changed to 55-40% in 25-35 min; and 35-50 min, wherein the volume fraction of the mobile phase A is changed to 60-95%, and the volume fraction of the mobile phase B is changed to 40-5%.
3. The preparation method of the standard decoction sample solution of daphne genkwa to be identified comprises the following steps: grinding flos Genkwa standard decoction, collecting 0.1g, precisely weighing, precisely adding 70% ethanol 25mL, weighing, ultrasonic treating for 30min, taking out, cooling, supplementing with 70% ethanol, shaking, and filtering with filter membrane.
4. The control solution is prepared by respectively taking apigenin-7-O- β -glucuronide, luteolin, tiliroside, apigenin, hydroxygenkwanin and genkwanin as appropriate amount, and preparing into control solution containing apigenin-7-O- β -glucuronide 0.06mg, luteolin 0.01mg, tiliroside 0.01mg, apigenin 0.06mg, hydroxygenkwanin 0.02mg and genkwanin 0.02mg per 1 mL.
5. Precisely sucking 10 μ L of each of the reference solution and the standard decoction sample solution of flos Genkwa to be identified, injecting into liquid chromatograph, and measuring.
6. And (3) measuring results: comparing the characteristic map of the standard decoction sample of the lilac daphne flower bud to be identified with the characteristic map of the standard decoction of the lilac daphne flower bud: 6 characteristic peaks can be detected from the standard decoction of flos Genkwa to be identified, and correspond to the 6 characteristic peaks in the contrast characteristic map of the standard decoction of flos Genkwa, and the sample to be identified is the standard decoction of flos Genkwa.
Example 4
The content of apigenin-7-O- β -glucuronide, apigenin, hydroxygenkwanin and genkwanin in the standard decoction is determined by the component content determination method of the standard decoction.
1. The chromatographic conditions of the high performance liquid chromatograph analysis are as follows: using octadecylsilane chemically bonded silica as filler, column temperature of 35 deg.C, methanol as mobile phase A, and 0.1% phosphoric acid water solution as mobile phase B, and performing gradient elution at flow rate of 1mL/min, detection wavelength of 350nm, and sample amount of 10 μ L.
2. The gradient elution conditions were: the volume fraction of the mobile phase A is changed to be 20% -43% and the volume fraction of the mobile phase B is changed to be 80% -57% in 0-3 min; the volume fraction of the mobile phase A is changed to 43-45% and the volume fraction of the mobile phase B is changed to 57-55% in 3-25 min; the volume fraction of the mobile phase A is changed to 45-60% and the volume fraction of the mobile phase B is changed to 55-40% in 25-35 min; and 35-50 min, wherein the volume fraction of the mobile phase A is changed to 60-95%, and the volume fraction of the mobile phase B is changed to 40-5%.
3. The preparation method of the standard decoction sample solution of flos Genkwa to be detected comprises the following steps: grinding flos Genkwa standard decoction, collecting 0.1g, precisely weighing, precisely adding 70% ethanol 25mL, weighing, ultrasonic treating for 30min, taking out, cooling, supplementing with 70% ethanol, shaking, and filtering with filter membrane.
4. The control solution is prepared by respectively taking apigenin-7-O- β -glucuronide, apigenin, hydroxygenkwanin and genkwanin as appropriate, placing in a 10mL volumetric flask, adding methanol to constant volume to scale, and preparing into control solution containing apigenin-7-O- β -glucuronide 0.06mg, apigenin 0.06mg, hydroxygenkwanin 0.02mg and genkwanin 0.02mg per 1 mL.
5. The determination comprises precisely absorbing 10 μ L of each of the control solution and flos Genkwa standard decoction sample solution to be determined, injecting into liquid chromatograph, determining corresponding peak area, calculating out the content of flos Genkwa essence by external standard method, calculating out the content of apigenin-7-O- β -glucuronide, apigenin and hydroxyflos Genkwa essence by one-time multi-evaluation method, and calculating out the content of apigenin-7-O- β -glucuronide, apigenin and hydroxyflos Genkwa essence by external standard method to verify reliability.
6. The results are shown in Table 17.
TABLE 17 results of measurement of contents
The results show that the determination result of one-test-multiple-evaluation is not obviously different from the determination result of an external standard method, which indicates that the method is more reliable, the total content of apigenin-7-O- β -glucuronide and apigenin is 39.286mg/g, the total content of hydroxygenkwanin and genkwanin is 4.262 mg/g, and the qualified standard decoction of genkwanin is obtained within the specified range.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
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