CN117054565A - Method for constructing UPLC characteristic spectrum of cyrtomium rhizome medicinal material, decoction piece, standard decoction and formula particle - Google Patents
Method for constructing UPLC characteristic spectrum of cyrtomium rhizome medicinal material, decoction piece, standard decoction and formula particle Download PDFInfo
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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
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- 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
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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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
- G01N30/86—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
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Abstract
The invention provides a method for constructing UPLC characteristic patterns of cyrtomium fortunei medicinal materials, decoction pieces, standard decoction and prescription particles, which comprises the following steps: a) Dissolving rhizoma Cyrtomii Falcati raw materials by using a solvent to obtain a liquid to be detected; b) Measuring the liquid to be measured by adopting a high performance liquid chromatography to obtain a UPLC characteristic spectrum of the cyrtomium fortunei raw material; the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; the mobile phase A is methanol, the mobile phase B is water, and the gradient elution is carried out. The invention adopts high performance liquid chromatography, adopts methanol-water as mobile phase for gradient elution, adopts uridine, guanosine, tryptophan and adenosine as reference substances, establishes a UPLC characteristic map of cyrtomium fortunei medicinal materials, decoction pieces, standard decoction pieces and formula particles, has good repeatability and precision, and is stable and reliable in method, and the quality of cyrtomium fortunei medicinal materials, decoction pieces, standard decoction pieces and formula particles can be controlled.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a method for constructing UPLC characteristic patterns of cyrtomium fortunei medicinal materials, decoction pieces, standard decoction pieces and formula particles.
Background
The rhizoma Osmundae is dried rhizome of Nakai, known as rhizoma Osmundae, of the family Umbelliferae, which is obtained by digging rhizoma Cibotii in two seasons of spring and autumn, removing petiole, fibrous root and soil, cleaning, and sun drying; or slicing while fresh, and sun drying. Has effects of clearing heat, detoxicating, stopping bleeding, healing sore and promoting granulation. The cyrtomium fortunei standard decoction is freeze-dried powder prepared by the fixed preparation process after the medicinal materials are processed; the cyrtomium fortunei formula particle is prepared by processing cyrtomium fortunei medicinal materials according to the main quality index of standard decoction. Rhizoma Osmundae is a common traditional Chinese medicine, and is used as an initial drug in Shennong Ben Cao Jing, and has effects of clearing heat, detoxicating, expelling parasites, etc. The morphology, habitat and origin of cyrtomium fortunei recorded in the traditional Chinese medicine of the past generation show that the original plant species of cyrtomium fortunei are numerous. The literature describes that up to 58 species of 18 genus of 11 families were used as raw plants of cyrtomium in our country. Ai Tiemin, etc., showed that 27 plants were used as the source plants for cyrtomium fortunei in the 80 s. The Chinese medicine dictionary records that 7 kinds of cyrtomium fortunei original plants exist, yang Chunyu and the like are investigated and arranged to obtain 38 kinds of cyrtomium fortunei original plants, wherein 18 kinds of cyrtomium fortunei original plants are widely used as cyrtomium fortunei purchased medicines. Even the regulation of Chinese pharmacopoeia on the original plant of cyrtomium fortunei is changed for many times, 2 species of cyrtomium fortunei and osmunda japonica are collected in 1977, only one species of cyrtomium fortunei is recorded in 1995, and the osmunda japonica is added in 2010. Therefore, the cyrtomium fortunei medicinal material is extremely complex.
Lin Yongjiang by adopting UPLC-MS/MS to detect common cold resisting granule of rhizoma Cyrtomii Falcati, octadecylsilane chemically bonded silica is used as filler, acetonitrile with mobile phase of 0.02% formic acid and 0.02% formic acid solution are used for gradient elution, electrospray positive ion mode (ESI+), and Multiple Reaction Monitoring (MRM) is used for quantitative analysis, and can be used for detecting use condition of rhizoma Cyrtomii Falcati in common cold resisting granule. Ma Bingzhi and the like measure 9 single-tooth dogbone samples of different producing places by adopting a UPLC fingerprint method, perform acetonitrile-water gradient elution, establish UPLC fingerprints of the single-tooth dogbone samples of different producing places, perform similarity comparison and further attribute kaempferide-3-0-L rhamnosyl in the UPLC fingerprints.
The fingerprint is an important method for macroscopically controlling the quality of medicinal materials, and related reports on the cyrtomium fortunei fingerprint are not yet available at present. The quality control research of the fingerprint patterns can not only clearly identify various compounds in the fingerprint patterns, but also identify cyrtomium fortunei and mixed and fake products thereof, so that the research work of the fingerprint patterns is necessary to be carried out for comprehensively controlling the quality of cyrtomium fortunei.
Therefore, a characteristic spectrum method of the cyrtomium fortunei is constructed for better quality control of cyrtomium fortunei medicinal materials, decoction pieces, standard decoction and formula particles, and the purpose of scientifically identifying the cyrtomium fortunei and the preparation thereof is achieved.
Disclosure of Invention
In view of the above, the technical problem to be solved by the invention is to provide a method for constructing UPLC characteristic patterns of cyrtomium fortunei medicinal materials, decoction pieces, standard decoction pieces and formula particles, which is stable and reliable and can control the quality of cyrtomium fortunei medicinal materials, decoction pieces, standard decoction pieces and formula particles.
A method for constructing UPLC characteristic spectrum of rhizoma Cyrtomii Falcati medicinal material, decoction piece, standard decoction and prescription granule comprises the following steps:
a) Dissolving rhizoma Cyrtomii Falcati raw materials by using a solvent to obtain a liquid to be detected;
b) Measuring the liquid to be measured by adopting a high performance liquid chromatography to obtain a UPLC characteristic spectrum of the cyrtomium fortunei raw material;
the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase a was acetonitrile, mobile phase B was 0.1% phosphoric acid, and gradient elution was performed.
The invention provides a method for constructing UPLC characteristic patterns of cyrtomium fortunei medicinal materials, decoction pieces, standard decoction and formula particles. The solvent is preferably 70% methanol.
The method has the advantages of large information quantity of the chromatographic peak of the extraction solvent and good effect.
Specifically, the preparation method comprises dissolving rhizoma Cyrtomii Falcati raw material in solvent, extracting, cooling, shaking, and filtering.
The extraction method is ultrasonic extraction or heating reflux extraction; preferably ultrasonic extraction; the ultrasonic power is preferably 600W, and the frequency is preferably 40kHz; the extraction time is preferably 15-45 min; more preferably 15 to 30 minutes.
Wherein, the ratio of the mass g of the cyrtomium rhizome raw material to the volume mL of the solvent is preferably (0.1-0.5): (50-100); more preferably 0.1:50.
the rhizoma Cyrtomii Falcati raw materials comprise rhizoma Cyrtomii Falcati medicinal materials, rhizoma Cyrtomii Falcati decoction pieces, standard decoction and formula granules. The invention is not limited thereto, and the above-mentioned raw materials can be quality-controlled and qualitatively detected by the method of the invention.
The invention also includes preparing a reference solution: dissolving protocatechuic acid with 70% methanol to obtain reference solution;
measuring the reference object solution by adopting a high performance liquid chromatography to obtain a chromatogram of the reference object; and qualitatively determining the components of the UPLC characteristic spectrum of rhizoma Cyrtomii Falcati medicinal materials, decoction pieces, standard decoction and formula particles according to the chromatogram of the reference substance.
Mobile phase a was acetonitrile, mobile phase B was 0.1% phosphoric acid, and gradient elution was performed.
The gradient elution of the invention is preferably specifically:
0-5 min, phase A: 6%, phase B: 94%;
5-10 min, phase A: 6% -15%, phase B: 94% -85%;
10-25 min, phase A: 15% -25%, phase B: 85% -75%;
25-40 min, phase A: 25% -26%, phase B: 75% -74%.
The invention has good baseline separation under the elution gradient, good separation degree of each peak and stable baseline.
1.6 μm, 2.1X106 mm; column temperature was 35 ℃.
The chromatographic column has symmetrical chromatographic peaks and good separation degree under the condition of 35 ℃.
The flow rate of the mobile phase is preferably 0.3ml/min.
The invention discovers that each chromatographic peak is better in separation and symmetrical in peak shape at the flow rate of 0.3ml/min, and is used as the most preferable scheme.
The detection wavelength of the present invention is preferably 250nm or 260nm.
Wherein the detection wavelength of rhizoma Cyrtomii Falcati medicinal material decoction pieces is 250nm, the detection wavelength of standard decoction pieces is 260nm, and the detection wavelength of formula particles is 260nm.
The inventor discovers that the chromatographic information is rich under the wavelength, each component has better absorption, the response value is moderate, the separation degree of each peak is better, and the baseline is stable.
The sample injection amount of the invention is 1 mu L.
The invention has the beneficial effects that under the condition of liquid chromatography, the fingerprint spectrum is used for controlling the substance group of cyrtomium fortunei medicinal materials, decoction pieces, standard decoction and formula particles, and protocatechuic acid is used for positioning the fingerprint spectrum; the cost of detection can be greatly reduced, and qualitative detection is realized.
And (3) evaluating the similarity of the UPLC characteristic patterns of the cyrtomium fortunei medicinal materials, decoction pieces, standard decoction pieces and formula particles by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to obtain the UPLC standard characteristic patterns of the cyrtomium fortunei medicinal materials, decoction pieces, standard decoction pieces and formula particles which are formed by 11 characteristic peaks, wherein the peak 1 is protocatechuic acid.
In the standard decoction feature map of cyrtomium fortunei, protocatechuic acid is used as a reference peak S-peak, the relative retention time of each feature peak and the S-peak is calculated, the relative retention time is within +/-10% of a specified value, and the specified values are respectively: 2.09 (peak 2), 4.40 (peak 3), 4.50 (peak 4), 5.68 (peak 5), 5.84 (peak 6), 5.99 (peak 7), 6.34 (peak 8), 8.36 (peak 9), 8.66 (peak 10), 8.79 (peak 11);
in the characteristic map of the cyrtomium fortunei medicinal material and the decoction piece, protocatechuic acid is used as a reference peak S, the relative retention time of each characteristic peak and the S peak is calculated, the relative retention time is within +/-10% of a specified value, and the specified values are respectively: 2.09 (peak 2), 4.39 (peak 3), 4.49 (peak 4), 5.67 (peak 5), 5.82 (peak 6), 5.97 (peak 7), 6.32 (peak 8), 8.33 (peak 9), 8.63 (peak 10), 8.76 (peak 11);
in the characteristic spectrum of the cyrtomium fortunei formula particle, protocatechuic acid is used as a reference peak S, the relative retention time of each characteristic peak and the S peak is calculated, the relative retention time is within +/-10% of a specified value, and the specified values are respectively:
2.10 (Peak 2), 4.41 (Peak 3), 4.51 (Peak 4), 5.70 (Peak 5), 5.86 (Peak 6), 6.01 (Peak 7), 6.36 (Peak 8), 8.39 (Peak 9), 8.68 (Peak 10), 8.81 (Peak 11).
Quality judgment standard: and taking samples of the cyrtomium fortunei medicinal materials, decoction pieces, standard decoction pieces and formula particles, and operating according to the same method to obtain characteristic patterns of the cyrtomium fortunei medicinal materials, decoction pieces, standard decoction pieces and formula particles, and analyzing the characteristic patterns of the cyrtomium fortunei medicinal materials, decoction pieces, standard decoction pieces, formula particles and the characteristic patterns of the samples by adopting 2012 edition of Chinese pharmacopoeia Committee 'traditional Chinese medicine chromatographic fingerprint similarity evaluation System', wherein the similarity is more than 0.90.
The method provided by the invention can effectively monitor the quality of cyrtomium fortunei medicinal materials, decoction pieces, standard decoction pieces and formula particles in different batches, so that the quality is stable, and the method has the characteristics of high precision, good reproducibility and the like, and is beneficial to comprehensively monitoring the quality of products.
The characteristic patterns of the cyrtomium fortunei medicinal materials, decoction pieces, standard decoction pieces and formula particles established by the invention take uridine, guanosine, tryptophan and adenosine as reference substances, pay attention to the sequence of each characteristic peak and the relativity with medicinal materials and intermediate products, and can comprehensively evaluate the overall quality and appearance characteristics of the products, and the method is scientific and reliable.
The newly established characteristic spectrum method can detect components with larger polarity in the cyrtomium fortunei and the standard decoction thereof. And the preparation method of the sample is simple and easy to operate, and the number of the identified characteristic peaks is relatively large. Can accurately and reliably detect the characteristic patterns of kansui root, cyrtomium rhizome and the preparation thereof. The consistency and the stability of the authenticity and the quality of the cyrtomium fortunei and the preparation thereof can be effectively detected and controlled. Provides a basis for effectively controlling and comprehensively evaluating the quality of the cyrtomium fortunei standard decoction. Ensures uniform and stable quality of rhizoma Osmundae and its standard decoction.
The invention provides a method for constructing UPLC characteristic patterns of cyrtomium fortunei medicinal materials, decoction pieces, standard decoction and prescription particles, which comprises the following steps: a) Dissolving rhizoma Cyrtomii Falcati raw materials by using a solvent to obtain a liquid to be detected; b) Measuring the liquid to be measured by adopting a high performance liquid chromatography to obtain a UPLC characteristic spectrum of the cyrtomium fortunei raw material; the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase a was acetonitrile, mobile phase B was 0.1% phosphoric acid, and gradient elution was performed. The invention adopts high performance liquid chromatography, selects acetonitrile-0.1% phosphoric acid as mobile phase for gradient elution, uses protocatechuic acid as reference substance, establishes the UPLC characteristic spectrum of cyrtomium fortunei medicinal material, decoction piece, standard decoction and prescription granule, has good repeatability and precision, and has stable and reliable method, and can control the quality of cyrtomium fortunei medicinal material, decoction piece, standard decoction and prescription granule.
Drawings
FIG. 1 is a comparison characteristic map of cyrtomium rhizome medicinal material disclosed in example 1 of the present invention;
FIG. 2 is an extraction mode investigation result diagram;
FIG. 3 is a graph of different solvent extraction chromatograms;
FIG. 4 is a graph of different extraction times;
FIG. 5 is a graph of different extraction solvent amounts;
FIG. 6 is a graph of chromatographic peak assignments;
FIG. 7 is a graph of results from various instrument studies;
FIG. 8 is a graph of results from different chromatographic column studies;
FIG. 9-1 is a characteristic map of rhizoma Osmundae;
FIG. 9-2 is a characteristic map of rhizoma Osmundae;
FIG. 10 is a characteristic map of decoction pieces;
FIG. 11 is a characteristic map of decoction pieces;
FIG. 12 is a comparison of feature maps of rhizoma Cyrtomii Falcati decoction pieces feature maps;
FIG. 13 is an extraction method investigation;
extracting solvent from fig. 14;
FIG. 15 different extraction time chromatograms;
FIG. 16 different extraction solvent amount chromatograms;
the chromatographic peak assignment of fig. 17;
fig. 18 different instrument surveys;
fig. 19 different chromatographic column surveys;
FIG. 20 is a characteristic map of rhizoma Cyrtomii Falcati standard decoction;
FIG. 21 is a characteristic map of rhizoma Cyrtomii Falcati standard decoction;
FIG. 22 is a graph of standard rhizoma Osmundae decoction characteristics;
FIG. 23 is a view of ultraviolet absorption spectrum of protocatechuic acid;
FIG. 24 is a graph of different wavelength chromatograms of cyrtomium fortunei formula particles;
FIG. 25 mobile phase species investigation;
FIG. 26 column temperature investigation chromatogram;
Flow rate investigation in FIG. 27;
fig. 28 delay investigation;
FIG. 29 different extraction mode chromatograms;
FIG. 30 different solvent extraction chromatograms;
FIG. 31 different extraction time chromatograms;
FIG. 32 different extraction solvent amount chromatograms;
the chromatographic peak assignment of fig. 33;
fig. 34 different instrument investigation;
fig. 35 different chromatographic column investigation;
FIG. 36 is a graph showing the verification of the characteristic spectrum of the cyrtomium fortunei formula particles;
FIG. 37 is a graph of cyrtomium fortunei formula particle control characteristics;
FIG. 38 is a graph showing the gradient results of different mobile phases of comparative example 1.
Detailed Description
In order to further illustrate the invention, the following embodiment is used for describing the method for constructing the UPLC characteristic spectrum of cyrtomium fortunei medicinal materials, decoction pieces, standard decoction and prescription particles.
High performance liquid chromatograph: an instrument 1; an instrument 2; an instrument 3;
an electronic balance: ME204E/02, MS205DU, XP26 (Metrele Tolyduo instruments Co., ltd.);
ultrapure water machine: cell type 1810A (Shanghai mueller scientific instruments limited);
ultrasonic cleaner: KQ600DB model (600W, 40KHz; kunshan ultrasonic instruments Co., ltd.);
chromatographic column: a chromatographic column 1; a chromatographic column 2; chromatographic column 3
Methanol and acetonitrile are chromatographic purity, water is ultrapure water, and the rest reagents are analytical purity;
Protocatechuic acid reference substance (Chinese food and drug administration institute, lot number: 110809-201906),
rhizoma Osmundae control medicinal material (Sichuan province pharmaceutical inspection institute, lot number: NO. SCZD 003-201905),
lot number of cyrtomium rhizome medicinal materials: YC-01, YC-02, YC-03, YC-04, YC-05, YC-06, YC-07, YC-08, YC-09, YC-10, YC-11, YC-12, YC-13, YC-14, YC-15, YC-16, YC-17, YC-18, YC-19, YC-20
Freeze-dried powder of cyrtomium fortunei standard decoction (prepared by Sichuan New green pharmaceutical industry technology development Co., ltd., lot number: BT-01, BT-02, BT-03, BT-04, BT-05, BT-06, BT-07, BT-08, BT-09, BT-10, BT-11, BT-12, BT-13, BT-14, BT-15, BT-16, BT-17, BT-18, BT-19, BT-20).
Cyrtomium fortunei formula granule (batch number: KL-001, KL-01, KL-02, KL-03).
EXAMPLE 1 chromatographic condition screening of medicinal herbs and decoction pieces
Chromatographic conditions and System applicability test octadecylsilane chemically bonded silica was used as filler (column length 150mm, inner diameter 2.1mm, particle size 1.6 μm); acetonitrile is taken as a mobile phase A; gradient elution was performed with 0.1% formic acid solution as mobile phase B, as specified in the table below; the column temperature is 35 ℃; the flow rate is 0.3ml/min; the detection wavelength was 250nm. The theoretical plate number should be not less than 5000 calculated by protocatechuic acid peak.
Preparation of reference solution Cyrtomium fortunei reference solution 0.5g is taken, placed in an conical flask, added with 70% methanol 50ml, subjected to ultrasonic treatment (power 600W, frequency 40 kHz) for 30 minutes, shaken well, filtered, and the subsequent filtrate is taken as reference solution of reference medicinal material. And (3) taking a proper amount of protocatechuic acid reference substance, precisely weighing, and adding methanol to prepare a solution containing 20 mug per 1ml of the reference substance solution.
Preparation of test solution: taking about 0.5g of medicine powder (passing through a third sieve), placing into a conical flask with a plug, adding 50ml of 70% methanol, performing ultrasonic treatment (power 600W, frequency 40 kHz) for 30 minutes, shaking uniformly, filtering, and taking subsequent filtrate.
Assay: precisely sucking 1 μl of each of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
2 batches of cyrtomium fortunei medicinal materials are synthesized by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), and a comparison characteristic spectrum of the cyrtomium fortunei medicinal materials is established. The results are shown in fig. 1, and fig. 1 is a comparison characteristic map of cyrtomium fortunei medicinal material disclosed in the embodiment 1 of the invention.
And (5) comparing the characteristic patterns.
7 characteristic peaks (generated after vinegar preparation of peak 3) are shown in the characteristic spectrum of the sample, the characteristic peaks correspond to the retention time of 6 characteristic peaks in the chromatogram of the reference substance of the reference medicinal material, the peak corresponding to the peak of the reference substance of the adenosine is an S peak, and the relative retention time of each characteristic peak and the S peak is calculated and is within +/-10% of a specified value. The predetermined values were 0.21 (peak 1), 0.40 (peak 2), 0.50 (peak 3), 0.64 (peak 4), 0.71 (peak 5), and 0.89 (peak 6).
Example 2 extraction method investigation
Compared with the ultrasonic extraction, reflux extraction and ultrasonic extraction after water decoction, the chromatographic peak information is approximately the same, and finally the ultrasonic extraction with simple operation is selected. See fig. 2. Fig. 2 is a view of the results of the extraction mode investigation.
Example 3 extraction solvent investigation:
the results of examining methanol, water, 30% methanol, 50% methanol and 70% methanol as extraction solvents show that the chromatographic peak information obtained by using 70% methanol as extraction solvent is large in amount and good in separation degree, so that 70% methanol is used as extraction solvent. See fig. 3, fig. 3 is a graph of different solvent extraction chromatograms.
Example 4 extraction time investigation
Ultrasonic extraction was compared for 15 minutes, 30 minutes, 45 minutes, and at 30 minutes the sample was completely extracted. The extraction time was determined to be 30 minutes. See fig. 4, fig. 4 is a graph of different extraction times.
Example 5 investigation of the amount of extraction solvent
The amounts of the extraction solvent added were compared with 25ml, 50ml and 100ml, and when the amount of the extraction solvent was 50ml, the chromatographic peak size was appropriate, so that the amount of the extraction solvent was determined to be 50ml. See fig. 5. Fig. 5 is a chromatogram of different extraction solvent amounts.
EXAMPLE 6 methodology investigation
6.1 chromatographic peak assignment
Preparation of test solution: and preparing a cyrtomium fortunei medicinal material sample solution according to a final determined sample preparation method.
Preparation of a reference solution for a control: taking a proper amount of protocatechuic acid reference substance, precisely weighing, adding methanol to prepare reference substance solution containing 20 mug of protocatechuic acid per 1 ml.
Preparation of control medicinal material solution: taking 0.5g of cyrtomium fortunei reference medicine, placing the cyrtomium fortunei reference medicine into a conical flask with a plug, adding 50ml of 70% methanol, performing ultrasonic treatment (with the power of 600W and the frequency of 40 kHz) for 30 minutes, shaking uniformly, filtering, and taking the subsequent filtrate as a reference medicine solution.
Preparation of negative control solution: preparing a negative control solution of the cyrtomium fortunei medicinal material according to the experimental conditions. And (5) positioning the characteristic map peak of the cyrtomium fortunei medicinal material. See fig. 6. Fig. 6 is a graph of the chromatographic peak assignments.
6.2 precision test
Taking sample solution of rhizoma Cyrtomii Falcati (batch number: YC-20), continuously sampling for 6 times according to a formulated experimental method, 1 μl each time, and calculating retention time and peak area of each characteristic peak. See Table 1-1.
TABLE 1-1 precision investigation-retention time
TABLE 1-2 precision investigation-peak area
The results show that the retention time RSD of each characteristic peak of the precision is 0.03-0.15%, and the peak area RSD is 0.48-8.95%. The instrument has good precision.
6.3 repeatability investigation
6 parts of cyrtomium fortunei medicinal material (batch number: YC-20) are precisely weighed, and are prepared and measured according to a planned experimental method. See tables 2-1 and 2-2.
TABLE 2-1 repeatability investigation-relative retention time ratio
TABLE 2 repeatability investigation-relative peak area ratio
The results showed that the relative retention time RSD for each characteristic peak of reproducibility was 0.26% to 0.65% and the relative peak area RSD was 1.90% to 14.92%. The method has good repeatability.
6.4 intermediate precision investigation
6.4.1 different times for different people
Based on the experimental conditions, accurately weighing rhizoma Cyrtomii Falcati (batch number: YC-20), preparing test solution by different personnel and different time, and measuring. See tables 3-1 and 3-2.
TABLE 3 intermediate precision-to-relative retention time ratio
TABLE 3 intermediate precision-to-relative peak area ratio
The result shows that the same sample is measured by different personnel at different times, and the method has better precision.
6.5 different instruments
Based on the experimental conditions set forth above, the instrument 1 is respectively; an instrument 2; the instrument 3 performs the investigation. See FIG. 7. FIG. 7 is a graph of the results of various instrument studies, tables 4-1 and 4-2.
TABLE 4-1 column durability investigation-relative retention time
TABLE 4-2 column durability investigation-relative peak area
The result shows that when the three instruments are used for detection, the RSD of each characteristic peak relative retention time is between 5.76 and 18.80 percent; the RSD of the relative peak area of each characteristic peak is 2.19-10.46%, and the durability of different instruments is poor.
6.6 column durability inspection
Based on the experimental conditions set forth above, the chromatographic column is respectively a chromatographic column 1; a chromatographic column 2; a chromatographic column 3; and (5) performing investigation. See FIG. 8. FIG. 8 is a graph of the results of various chromatographic column studies, tables 5-1 and 5-2.
TABLE 5-1 column durability investigation-relative retention time
TABLE 5-2 column durability investigation-relative peak area
As can be seen from fig. 8, the chromatographic separation of the chromatographic column 1 is good, and therefore, the use of the chromatographic column 1 is recommended.
6.7 stability investigation
Based on the experimental conditions, the same sample solution is taken and measured at the time of 0h,2h,4h,8h,12h and 24h respectively. See tables 6-1 and 6-2.
TABLE 6-1 stability investigation-retention time
TABLE 6-2 stability investigation-peak area
The results show that the RSD of the corresponding characteristic peak retention time is between 0.06 and 0.57 percent, and the sample solution is stable within 24 hours.
In conclusion, the RSD of each characteristic peak relative retention time meets the requirements in the above examinations, and the method is good. The above 11 characteristic peaks were included in the subsequent investigation.
6.8 identification of characteristic spectrum of cyrtomium rhizome medicinal material
The Cyrtomium fortunei medicinal material was examined, and the ratio of the relative retention time to the relative peak area was calculated, and the results are shown in FIGS. 9-10, tables 7-1 and 7-2.
Wherein fig. 9-1 is a characteristic map of cyrtomium fortunei medicinal materials, and the batch numbers are as follows from bottom to top: cyrtomium fortunei reference medicinal materials, YC-01, YC-02, YC-03, YC-04, YC-05, YC-06, YC-07, YC-08, YC-09 and YC-10; fig. 9-2 shows a characteristic map of cyrtomium fortunei medicinal materials, and the lot numbers are as follows from bottom to top: cyrtomium fortunei reference medicinal materials, YC-11, YC-12, YC-13, YC-14, YC-15, YC-16, YC-17, YC-18, YC-19 and YC-20。
TABLE 7-1 Cyrtomium fortunei medicinal material characteristic Spectrum relative retention time
TABLE 7-2 Cyrtomium fortunei medicinal material characteristic map relative peak area
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According to the principle that the relative retention time is stable, the samples in each batch can be detected and the peak is relatively high, 11 peaks with better repeatability are selected as characteristic peaks. The result shows that the relative peak area RSD difference of the characteristic peaks of the medicinal materials is too large, and the relative peak area cannot be specified, so that the medicinal materials are not included in the quality standard text. Finally, the following steps are provided: the characteristic spectrum of the sample should show 11 characteristic peaks and correspond to 11 characteristic peak retention times in the chromatogram of the reference substance of the reference medicinal material, wherein the peak corresponding to the reference substance of protocatechuic acid is S peak, and the relative retention time of each characteristic peak and S peak is calculated and is within + -10% of the specified value. The specified value is: 2.10 (Peak 2), 4.30 (Peak 3), 4.49 (Peak 4), 5.67 (Peak 5), 5.83 (Peak 6), 5.97 (Peak 7), 6.32 (Peak 8), 8.34 (Peak 9), 8.64 (Peak 10), 8.76 (Peak 11).
6.9 Verification of characteristic spectrum of 20-batch cyrtomium fortunei decoction pieces
The 20 lot of cyrtomium fortunei decoction pieces were measured, and the results are shown in FIGS. 10-11, tables 8-1 and 8-2. FIG. 10 is a characteristic map of decoction pieces (lot number from bottom to top: cyrtomium fortunei control medicinal material, YP-01, YP-02, YP-03, YP-04, YP-05, YP-06, YP-07, YP-08, YP-09, and YP-10 in order); FIG. 11 is a characteristic map of decoction pieces (lot number from bottom to top: cyrtomium fortunei control medicinal material, YP-11, YP-12, YP-13, YP-14, YP-15, YP-16, YP-17, YP-18, YP-19, and YP-20).
TABLE 8-1 decoction piece characteristic spectra relative retention time
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TABLE 8-2 decoction piece characteristic spectrum relative peak area
According to the principle that the relative retention time is stable, the samples in each batch can be detected and the peak is relatively high, 11 peaks with better repeatability are selected as characteristic peaks. The result shows that the relative peak area RSD difference of the characteristic peaks of the medicinal materials is too large, and the relative peak area cannot be specified, so that the medicinal materials are not included in the quality standard text. Finally, the following steps are provided: the characteristic spectrum of the sample should show 11 characteristic peaks and correspond to 11 characteristic peak retention times in the chromatogram of the reference substance of the reference medicinal material, wherein the peak corresponding to the reference substance of protocatechuic acid is S peak, and the relative retention time of each characteristic peak and S peak is calculated and is within + -10% of the specified value. The specified value is: 2.09 (Peak 2), 4.39 (Peak 3), 4.49 (Peak 4), 5.67 (Peak 5), 5.82 (Peak 6), 5.97 (Peak 7), 6.32 (Peak 8), 8.33 (Peak 9), 8.63 (Peak 10), 8.76 (Peak 11).
The 20 lot of cyrtomium fortunei medicinal materials are synthesized by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), a comparison characteristic map of the cyrtomium fortunei decoction pieces is established, the result is shown in figure 12, and figure 12 is the comparison characteristic map of the cyrtomium fortunei decoction pieces.
EXAMPLE 7 selection of chromatographic conditions for Cyrtomium fortunei Standard decoction
The chromatographic conditions and the system adaptability test of the standard decoction feature spectrum of cyrtomium fortunei are determined as follows: octadecylsilane chemically bonded silica is used as filler (column length is 150mm, inner diameter is 2.1mm, and granularity is 1.6 μm); acetonitrile is taken as a mobile phase A, 0.1% formic acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the column temperature is 35 ℃; the flow rate is 0.3ml per minute, the detection wavelength is 250nm, and the theoretical plate number is not less than 5000 calculated according to the protocatechuic acid peak.
Preparation of reference solutions
Taking 0.5g of cyrtomium fortunei reference medicine, placing the cyrtomium fortunei reference medicine into a conical flask with a plug, adding 50ml of 70% methanol, carrying out ultrasonic treatment (with the power of 600W and the frequency of 40 kHz) for 30 minutes, shaking uniformly, filtering, and taking the subsequent filtrate as a reference medicine solution. And (3) taking a proper amount of protocatechuic acid reference substance, precisely weighing, and adding methanol to prepare a solution containing 20 mug of protocatechuic acid per 1ml serving as the reference substance solution of the reference substance.
Preparation of test solutions
About 0.2g of cyrtomium fortunei standard decoction is taken, and a sample solution is prepared by the same method as the preparation method of the reference substance solution of the reference medicinal material in the example 1.
Assay
Precisely sucking 1 μl of each of the sample solution and the reference solution, and injecting into a liquid chromatograph for measurement.
7.1 extraction method investigation
Compared with the reflux extraction and the ultrasonic extraction, the chromatographic peak information is approximately the same, and finally the ultrasonic extraction with simple operation is selected. See fig. 13. Fig. 13 is an extraction mode investigation.
7.2 extraction solvent investigation
The results of examining methanol, water, 30% methanol, 50% methanol and 70% methanol as extraction solvents show that the chromatographic peak information obtained by using 70% methanol as extraction solvent is large in amount and good in separation degree, so that 70% methanol is used as extraction solvent. See fig. 14. Fig. 14 extraction solvent investigation.
7.3 extraction time investigation
Ultrasonic extraction was compared for 15 minutes, 30 minutes, 45 minutes and chromatograms were substantially identical, so the extraction time was determined to be 15 minutes, see fig. 15. Fig. 15 different extraction time chromatograms.
7.4 investigation of the extraction solvent quantity
The amounts of the extraction solvent added were compared with 25ml, 50ml and 100ml, and when the amount of the extraction solvent was 50ml, the chromatographic peak size was appropriate, so that the amount of the extraction solvent was determined to be 50ml. See fig. 16. FIG. 16 is a graph of different extraction solvent amounts.
7.5 preparation method of final determination test sample
About 0.2g is put into a conical flask with a plug, 50ml of 70% methanol is added, the mixture is subjected to ultrasonic treatment (power 600W, frequency 40 kHz) for 15 minutes, the mixture is shaken uniformly and filtered, and the subsequent filtrate is obtained.
EXAMPLE 8 standard decoction methodology investigation of Cyrtomium fortunei
8.1 chromatographic peak assignment
Preparation of test solution: and (5) preparing a standard rhizoma cyrtomii decoction sample solution according to a final determined sample preparation method.
Preparation of reference solution: and (3) taking a proper amount of protocatechuic acid reference substance, precisely weighing, and adding methanol to prepare a reference substance solution containing 20 mug of protocatechuic acid per 1 ml.
Preparation of control medicinal material solution: taking 0.5g of cyrtomium fortunei reference medicinal material, placing the cyrtomium fortunei reference medicinal material into a conical flask, adding 50ml of 70% methanol, performing ultrasonic treatment (with the power of 600W and the frequency of 40 kHz) for 30 minutes, shaking uniformly, filtering, and taking the subsequent filtrate as reference medicinal material solution.
Preparation of negative control solution: preparing a negative control solution of the rhizoma dryopteris crassirhizomae standard decoction according to the experimental conditions. And (5) positioning the peak of the characteristic spectrum of the standard decoction of cyrtomium fortunei. See fig. 17. The chromatographic peaks are identified in fig. 17.
8.2 precision test
Sample solution of standard rhizoma Cyrtomii Falcati decoction (batch number: BT-20) is continuously sampled for 6 times according to a formulated experiment method, 1 μl each time, and retention time and peak area of each characteristic peak are calculated. See tables 9-1 and 9-2.
TABLE 9-1 precision investigation-retention time
TABLE 9-2 precision investigation-peak area
The results showed that the retention time RSD of each characteristic peak of the precision was 0.02% -0.17% and the peak area RSD was 0.31% -4.48%. The instrument has good precision.
8.3 repeatability investigation
6 parts of cyrtomium fortunei standard decoction (batch number: BT-20) is precisely weighed, and is prepared and measured according to a planned experimental method. See tables 10-1 and 10-2.
TABLE 10-1 repeatability investigation-relative retention time ratio
TABLE 10-2 repeatability investigation-relative peak area ratio
The results showed that the relative retention time RSD for each characteristic peak of reproducibility was 0.49% -0.62% and the relative peak area RSD was 1.39% -7.73%. The method has good repeatability.
8.4 intermediate precision investigation
8.4.1 different personnel and different time surveys
Based on the experimental conditions, accurately weighing rhizoma Cyrtomii Falcati standard decoction (batch number: BT-20), preparing test solution by different personnel and different time, and measuring. See tables 11-1 and 11-2.
TABLE 11 intermediate precision-to-relative retention time ratio
TABLE 11 intermediate precision-to-relative peak area ratio
The result shows that the same sample is measured by different personnel at different times, and the method has better precision.
8.4.2 different instruments
Based on the experimental conditions set forth above, the instrument 1 is respectively; an instrument 2; the instrument 3 performs the investigation. See FIG. 18, tables 12-1 and 12-2. Fig. 18 is a different instrument view.
Table 12-1 different instrument investigation-relative retention time
Table 12-2 different instrumental investigation-relative peak area
The result shows that when the three instruments are used for detection, the RSD of each characteristic peak relative retention time is between 4.88 and 17.70 percent; the RSD of the relative peak area of each characteristic peak is between 0.86 and 22.77 percent, and the durability of different instruments is poor.
8.5 durability inspection
8.5.1 column durability inspection
Based on the experimental conditions set forth above, the chromatographic column is respectively a chromatographic column 1; a chromatographic column 2; a chromatographic column 3; and (5) performing investigation. See FIG. 19, tables 13-1 and 13-2. FIG. 19 is a different column view.
TABLE 13-1 column durability investigation-relative retention time
TABLE 13-2 column durability investigation-relative peak area
As can be seen from fig. 19, the chromatographic separation of the chromatographic column 1 is good, and therefore, the use of the chromatographic column 1 is recommended.
8.5.2 stability investigation
Based on the experimental conditions, the same sample solution is taken and measured at the time of 0h,2h,4h,8h,12h and 24h respectively. See tables 14-1 and 14-2.
TABLE 14 stability investigation-retention time
TABLE 14-2 stability investigation-peak area
The results show that the RSD of the corresponding characteristic peak retention time is between 0.03 and 0.57 percent, and the sample solution is stable within 24 hours.
In conclusion, the RSD of each characteristic peak relative retention time meets the requirements in the above examinations, and the method is good. The above 11 characteristic peaks were included in the subsequent investigation.
Example 9 determination of characteristic peaks and establishment of control Profile
9.1 The verification result of 20 standard decoction of cyrtomium fortunei adopts the method, the characteristic spectrum analysis is carried out on 20 standard decoction samples, and the relative retention time and the relative peak area ratio are calculated. See FIGS. 20-21, tables 15-1 and 15-2. FIG. 20A is a standard decoction feature map of cyrtomium fortunei (the lot numbers from bottom to top are, in order, cyrtomium fortunei control medicinal material, BT-01, BT-02, BT-03, BT-04, BT-05, BT-06, BT-07, BT-08, BT-09, BT-10); FIG. 21A is a characteristic map of rhizoma Osmundae standard decoction (the lot numbers from bottom to top are, in order, rhizoma Osmundae control medicinal material, BT-11, BT-12, BT-13, BT-14, BT-15, BT-16, BT-17, BT-18, BT-19, and BT-20).
Table 15-1 relative retention time of 20 standard decoction of Cyrtomium fortunei
Table 15-2 relative peak area of 20 standard decoction of Cyrtomium fortunei
According to the principle that the relative retention time is stable, the samples in each batch can be detected and the peak is relatively high, 11 peaks with better repeatability are selected as characteristic peaks. The result shows that when the peak 1 is taken as an S peak, the relative retention time RSD of characteristic peaks of 3 batches of cyrtomium fortunei formula particles is between 0.00 and 0.31 percent, and the relative retention time RSD of 11 characteristic peaks of 3 batches of cyrtomium fortunei formula particles is less than 5 percent.
Finally, the following steps are provided: the sample chromatograph should show 11 characteristic peaks and should correspond to 11 characteristic peaks retention time in the reference chromatograph of the reference medicinal material, the peak corresponding to the reference peak of the protocatechuic acid is S peak, and the relative retention time of each characteristic peak and S peak is calculated, wherein the relative retention time is within + -10% of the specified value. The specified value is: 2.09 (Peak 2), 4.40 (Peak 3), 4.50 (Peak 4), 5.68 (Peak 5), 5.84 (Peak 6), 5.99 (Peak 7), 6.34 (Peak 8), 8.36 (Peak 9), 8.66 (Peak 10), 8.79 (Peak 11). FIG. 22 is a standard decoction of Cyrtomium fortunei with reference to characteristic spectrum, peak 1 (S): protocatechuic acid injection: chromatographic column:T3 2.1×150mm,1.6μ。
EXAMPLE 10 chromatographic conditions for Cyrtomium fortunei formula particles
Chromatographic conditions and System applicability test octadecylsilane chemically bonded silica was used as filler (column length 150mm, inner diameter 2.1mm, particle size 1.6 μm); acetonitrile is taken as a mobile phase A, 0.1% formic acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the column temperature is 35 ℃; the flow rate is 0.3ml/min, the detection wavelength is 250nm, and the theoretical plate number is not less than 5000 calculated according to the protocatechuic acid peak.
10.1 preparation of reference solutions
Taking 0.5g of cyrtomium fortunei reference medicine, placing the cyrtomium fortunei reference medicine into a conical flask with a plug, adding 50ml of 70% methanol, carrying out ultrasonic treatment (with the power of 600W and the frequency of 40 kHz) for 30 minutes, shaking uniformly, filtering, and taking the subsequent filtrate as a reference medicine solution. And (3) taking a proper amount of protocatechuic acid reference substance, precisely weighing, precisely adding methanol to prepare a solution containing 20 mug of protocatechuic acid per 1ml, and taking the solution as the reference substance solution of the reference substance.
10.2 preparation of sample solutions
Taking proper amount of cyrtomium fortunei prescription granule, grinding, taking 0.2g, and preparing into test solution by the same method as the reference solution of the reference medicinal material of the example 1.
10.3 assay
Precisely sucking 1 μl of each of the reference solution and the sample solution, and measuring with a liquid chromatograph.
10.4 wavelength selection
Based on the experimental conditions, the protocatechuic acid reference substance and the sample solution are subjected to full-band scanning by using a diode array detector, and chromatograms of the solutions at wavelengths of 210nm, 230nm, 250nm, 270nm, 290nm and 310nm are respectively extracted. See fig. 23-24. FIG. 23 is a view of ultraviolet absorption spectrum of protocatechuic acid; FIG. 24 shows different wavelength chromatograms of cyrtomium fortunei formula particles.
As a result, the information amount of the chromatographic peak at the detection wavelength of 250nm was large, so that the detection wavelength was determined to be 250nm.
10.5 examination of mobile phase species
On the basis of the experimental conditions set forth above, the mobile phases acetonitrile-water, acetonitrile-0.1% formic acid, acetonitrile-0.1% phosphoric acid were examined, respectively, as shown in FIG. 25. Fig. 25 mobile phase species investigation. The results showed that. When the mobile phase is acetonitrile-0.1% formic acid, the chromatographic peak separation degree is good, so the mobile phase is acetonitrile-0.1% formic acid.
10.6 column temperature investigation
Based on the experimental conditions set forth above, the column temperatures were examined at 25℃and 30℃and 35 ℃. See fig. 26. FIG. 26 column temperature investigation chromatogram.
The results showed that the chromatographic peak separation was good at a column temperature of 35℃and that column Wen Zanding was 35 ℃.
10.7 flow Rate investigation
Based on the experimental conditions set forth above, the flow rates were examined at 0.25ml/min, 0.3ml/min, and 0.35ml/min, respectively. See fig. 27. FIG. 27 flow rate investigation.
The results show that the information content of each characteristic peak chromatogram peak is basically consistent when the flow rates are respectively 0.25ml/min, 0.3ml/min and 0.35 ml/min. At a flow rate of 0.3ml/min, the chromatographic peak separation was good, so the flow rate was determined to be 0.30ml/min.
10.8 delay investigation
Based on the experimental conditions set forth above, the chromatogram acquisition time was set to 2-fold. The results are shown in FIG. 28. Fig. 28 delay profile. The results showed that after 40 minutes of the chromatogram, no color spectrum peaks appear.
In summary, the chromatographic condition and system adaptability test of the characteristic spectrum of the cyrtomium fortunei formula particle are determined as follows: octadecylsilane chemically bonded silica is used as filler (column length is 150mm, inner diameter is 2.1mm, and granularity is 1.6 μm); acetonitrile is taken as a mobile phase A, 0.1% formic acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the column temperature is 35 ℃; the flow rate was 0.3ml/min and the detection wavelength was 260nm. The theoretical plate number should be not less than 5000 calculated by protocatechuic acid peak.
EXAMPLE 11 preparation of sample solution of Cyrtomium fortunei formula particle
11.1 extraction method investigation
Compared with the reflux extraction and the ultrasonic extraction, the chromatographic peak information is approximately the same, and finally the ultrasonic extraction with simple operation is selected. See fig. 29. Fig. 29 different extraction mode chromatograms.
11.2 investigation of extraction solvent
The results of examining methanol, water, 30% methanol, 50% methanol and 70% methanol as extraction solvents show that the chromatographic peak information obtained by using 70% methanol as extraction solvent is large in amount and good in separation degree, so that 70% methanol is used as extraction solvent. See fig. 30. FIG. 30 different solvent extraction chromatograms.
11.3 extraction time investigation
Ultrasonic extraction was compared for 15 minutes, 30 minutes, 45 minutes and chromatograms were substantially identical. The extraction time was determined to be 15 minutes, see fig. 31. Fig. 31 different extraction time chromatograms.
11.4 investigation of the extraction solvent quantity
The amounts of the extraction solvent added were compared with 25ml, 50ml and 100ml, and when the amount of the extraction solvent was 50ml, the chromatographic peak size was appropriate, so that the amount of the extraction solvent was 50ml, see FIG. 32. FIG. 32 different extraction solvent mass chromatograms.
11.5 method for preparing final determination test sample
Grinding the above materials, collecting about 0.2g, placing into conical flask with plug, adding 70% methanol 50ml, ultrasonic treating (power 600W, frequency 40 kHz) for 15 min, shaking, filtering, and collecting filtrate.
EXAMPLE 12 methodology investigation
12.1 chromatographic peak assignment
Preparation of test solution: and preparing a sample solution of the cyrtomium fortunei formula granule according to a final determined sample preparation method.
Preparation of reference solution: taking a proper amount of protocatechuic acid reference substance, precisely weighing, precisely adding methanol to prepare reference substance solution containing 20 mug of protocatechuic acid per 1 ml.
Preparation of control medicinal material solution: taking 0.5g of cyrtomium fortunei reference medicinal material, placing the cyrtomium fortunei reference medicinal material into a conical flask, adding 50ml of 70% methanol, performing ultrasonic treatment (with the power of 600W and the frequency of 40 kHz) for 30 minutes, shaking uniformly, filtering, and taking the subsequent filtrate as reference medicinal material solution.
Preparation of negative control solution: preparing a negative control solution of the cyrtomium fortunei formula particles according to the experimental conditions.
The characteristic spectrum peak of the cyrtomium fortunei formula particle is positioned, as shown in figure 33. The chromatographic peak in FIG. 33 is identified.
12.2 precision test
Taking sample solution of rhizoma Osmundae formula granule (batch number: KL-001), continuously sampling for 6 times according to a formulated experimental method, 1 μl each time, and calculating retention time and peak area of each characteristic peak. See tables 16-1 and 16-2.
TABLE 16-1 precision investigation-retention time
TABLE 16-2 precision investigation-peak area
The results show that the retention time RSD of each characteristic peak of the precision is 0.03-0.15%, and the peak area RSD is 0.40-1.45%. The instrument has good precision.
12.3 repeatability investigation
6 parts of cyrtomium fortunei formula particles (batch number: KL-001) are precisely weighed, and are prepared and measured according to a planned experimental method. See tables 17-1 and 17-2.
TABLE 17-1 repeatability investigation-relative retention time ratio
TABLE 17-2 repeatability investigation-relative peak area ratio
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The results showed that the relative retention time RSD for each characteristic peak of reproducibility was 0.23% to 0.51% and the relative peak area RSD was 0.00% to 5.95%. The method has good repeatability.
12.4 intermediate precision
12.4.1 different people at different times
Based on the experimental conditions, the cyrtomium fortunei particles are respectively and precisely weighed, and sample solutions are prepared by different personnel and different time for measurement. See tables 18-1 and 18-2.
TABLE 18 intermediate precision-to-relative retention time ratio
TABLE 18-2 intermediate precision-to-relative peak area ratio
The result shows that the same sample is measured by different personnel at different times, and the method has better precision.
12.4.2 durability inspection of instrument
Based on the experimental conditions set forth above, the instrument 1 is respectively; an instrument 2; the instrument 3 performs the investigation. See FIG. 34, tables 19-1 and 19-2. Fig. 34 is a different instrument view.
TABLE 19-1 column durability investigation-relative retention time
TABLE 19-2 column durability investigation-relative peak area
The result shows that when the three instruments are used for detection, the RSD of each characteristic peak relative retention time is between 5.39 and 17.01 percent; the RSD of the relative peak area of each characteristic peak is 0.00% -9.49%, and the durability of different instruments is poor.
12.5 durability inspection
12.5.1 chromatographic column durability inspection
Based on the experimental conditions, the chromatographic column is a chromatographic column 1 and a chromatographic column 2; a chromatographic column 3; and (5) performing investigation. See fig. 35, tables 14-15. Fig. 35 is a different chromatographic column.
TABLE 20-1 column durability investigation-relative retention time
TABLE 20-2 column durability investigation-relative peak area
As can be seen from fig. 35, the chromatographic separation of the column 1 is good, and therefore the use of the column 1 is recommended.
12.6 stability investigation
Based on the experimental conditions, the same sample solution is taken and measured at the time of 0h,2h,4h,8h,12h and 24h respectively. See tables 21-1 and 21-2.
TABLE 21 stability investigation-retention time
TABLE 21-2 stability investigation-peak area
The results show that the RSD of the corresponding characteristic peak retention time is between 0.06 and 0.48 percent, and the sample solution is stable within 24 hours.
In conclusion, the RSD of each characteristic peak relative retention time meets the requirements in the above examinations, and the method is good. The above 11 characteristic peaks were included in the subsequent investigation.
Example 13 determination of characteristic peaks and establishment of control Profile
13.1 verification results of Cyrtomium fortunei formula particles
And (3) measuring the characteristic spectrum of 3 batches of samples of the product by adopting a formulated method, and calculating the relative retention time and the relative peak area. See fig. 36, tables 18-19. FIG. 36 is a graph showing the verification of the characteristic spectrum of the cyrtomium fortunei formula granule.
Table 22-1 3 Cyrtomium fortunei formulation particles-relative retention time
Table 22-2 3 Cyrtomium fortunei formulation particles-relative peak area
According to the principle that the relative retention time is stable, the samples in each batch can be detected and the peak is relatively high, 11 peaks with better repeatability are selected as characteristic peaks. The result shows that when the peak 1 is taken as an S peak, the relative retention time RSD of characteristic peaks of 3 batches of cyrtomium fortunei formula particles is between 0.00 and 0.28 percent, and the relative retention time RSD of 11 characteristic peaks of 3 batches of cyrtomium fortunei formula particles is less than 5 percent.
Finally, the following steps are provided: the sample chromatograph should show 11 characteristic peaks and should correspond to 11 characteristic peaks retention time in the reference chromatograph of the reference medicinal material, the peak corresponding to the reference peak of the protocatechuic acid is S peak, and the relative retention time of each characteristic peak and S peak is calculated, wherein the relative retention time is within + -10% of the specified value. The specified value is: 2.10 (Peak 2), 4.41 (Peak 3), 4.51 (Peak 4), 5.70 (Peak 5), 5.86 (Peak 6), 6.01 (Peak 7), 6.36 (Peak 8), 8.39 (Peak 9), 8.68 (Peak 10), 8.81 (Peak 11).
3 batches of cyrtomium fortunei formula particles are synthesized by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), and a comparison pattern of the characteristic pattern of the cyrtomium fortunei formula particles is established. See fig. 37. FIG. 37 is a graph of cyrtomium fortunei formula particle control characteristics; peak 1 (S): protocatechuic acid.
Comparative example 1 different mobile phase gradients:
gradient 1:
gradient 2:
gradient 3:
gradient 4: (gradient of last choice)
As shown in FIG. 38, it can be seen from 38 that the baseline is uneven and the number of peaks is small under the condition of gradient 1; under gradient 2 conditions, peak 1 is absent; under gradient 3 conditions, peak 9.10.11 was absent; under the condition of gradient 4, the number of peaks is more, the separation degree is better, and the peaks are respectively uniform.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A method for constructing UPLC characteristic spectrum of rhizoma Cyrtomii Falcati medicinal material, decoction piece, standard decoction and prescription granule comprises the following steps:
a) Dissolving rhizoma Cyrtomii Falcati raw materials by using a solvent to obtain a liquid to be detected;
b) Measuring the liquid to be measured by adopting a high performance liquid chromatography to obtain a UPLC characteristic spectrum of the cyrtomium fortunei raw material;
the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase a was acetonitrile, mobile phase B was 0.1% phosphoric acid, and gradient elution was performed.
2. The method of claim 1, further comprising preparing a reference solution: dissolving protocatechuic acid with 70% methanol to obtain reference solution; the concentration of the reference solution is 20 mug/mL;
measuring the reference object solution by adopting a high performance liquid chromatography to obtain a chromatogram of the reference object; and qualitatively determining the components of the UPLC characteristic spectrum of rhizoma Cyrtomii Falcati medicinal materials, decoction pieces, standard decoction and formula particles according to the chromatogram of the reference substance.
3. The method according to claim 1, characterized in that the gradient elution is in particular:
0-5 min, phase A: 6%, phase B: 94%;
5-10 min, phase A: 6% -15%, phase B: 94% -85%;
10-25 min, phase A: 15% -25%, phase B: 85% -75%;
25-40 min, phase A: 25% -26%, phase B: 75% -74%.
4. A method according to claim 3, wherein the chromatographic column is a C18 column, having a gauge of 1.6 μm,2.1 x 150mm; column temperature was 35 ℃.
5. The method of claim 4, wherein the mobile phase flow rate is 0.3mL/min; the detection wavelength is 250nm and/or 260nm; the sample loading was 1. Mu.L.
6. The method of claim 1, wherein the similarity of the UPLC characteristic patterns of the cyrtomium fortunei medicinal material, decoction pieces, standard decoction pieces and formula particles is evaluated by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to obtain a standard characteristic pattern of the cyrtomium fortunei medicinal material, decoction pieces, standard decoction pieces and formula particles which consists of 11 characteristic peaks, wherein peak 1 is protocatechuic acid.
7. The method according to claim 6, wherein in the standard decoction feature profile of cyrtomium fortunei, protocatechuic acid is taken as a reference peak S-peak, and the relative retention time of each feature peak and S-peak is calculated, wherein the relative retention time is within +/-10% of a specified value, and the specified values are respectively: 2.09 (peak 2), 4.40 (peak 3), 4.50 (peak 4), 5.68 (peak 5), 5.84 (peak 6), 5.99 (peak 7), 6.34 (peak 8), 8.36 (peak 9), 8.66 (peak 10), 8.79 (peak 11);
In the characteristic map of the cyrtomium fortunei medicinal material and the decoction piece, protocatechuic acid is used as a reference peak S, the relative retention time of each characteristic peak and the S peak is calculated, the relative retention time is within +/-10% of a specified value, and the specified values are respectively: 2.09 (peak 2), 4.39 (peak 3), 4.49 (peak 4), 5.67 (peak 5), 5.82 (peak 6), 5.97 (peak 7), 6.32 (peak 8), 8.33 (peak 9), 8.63 (peak 10), 8.76 (peak 11);
in the characteristic spectrum of the cyrtomium fortunei formula particle, protocatechuic acid is used as a reference peak S, the relative retention time of each characteristic peak and the S peak is calculated, the relative retention time is within +/-10% of a specified value, and the specified values are respectively:
2.10 (Peak 2), 4.41 (Peak 3), 4.51 (Peak 4), 5.70 (Peak 5), 5.86 (Peak 6), 6.01 (Peak 7), 6.36 (Peak 8), 8.39 (Peak 9), 8.68 (Peak 10), 8.81 (Peak 11).
8. The method of claim 1, wherein the solvent of step a) is 70% methanol; the extraction method is ultrasonic extraction or heating reflux extraction; the ultrasonic power is 600W, and the frequency is 40kHz; the extraction time is 15-45 min.
9. The method according to claim 1, wherein the ratio of mass g of cyrtomium fortunei raw material to volume mL of solvent in step a) is (0.1 to 0.5): (50-100).
10. The method of claim 1, wherein the cyrtomium fortunei raw material of step a) is cyrtomium fortunei medicinal material, cyrtomium fortunei decoction pieces, formula granules or decoction.
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