CN117214345A - HPLC (high Performance liquid chromatography) characteristic spectrum detection method for cimicifuga foetida and preparation thereof - Google Patents
HPLC (high Performance liquid chromatography) characteristic spectrum detection method for cimicifuga foetida and preparation thereof Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention provides an HPLC (high Performance liquid chromatography) characteristic spectrum detection method for a honey cimicifuga rhizome and a preparation thereof, which comprises the following steps: a) Dissolving the raw materials of the cimicifuga foetida by adopting 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 an HPLC characteristic spectrum of the raw material of the cimicifuga foetida; the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase a is acetonitrile, mobile phase B is 0.1% formic acid aqueous solution, and gradient elution is performed. According to the invention, a high performance liquid chromatography is adopted, acetonitrile-0.1% formic acid is selected as a mobile phase for gradient elution, 5-hydroxymethylfurfural, caffeic acid, ferulic acid and isoferulic acid are used as reference substances, an HPLC characteristic spectrum of the cimicifuga foetida is established, the repeatability and precision are good, the method is stable and reliable, and the quality of the cimicifuga foetida can be controlled.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to an HPLC (high performance liquid chromatography) characteristic spectrum detection method for cimicifuga foetida and a preparation thereof.
Background
Cimicifugae rhizoma is dried rhizome of Cimicifuga racemosa Cimicifuga heracleifolia Kom., cimicifuga racemosa Cimicifuga dahurica (Turcz.) Maxim or Cimicifuga racemosa Cimicifuga foetida L. Pungent and slightly sweet in flavor and slightly cold in nature. Enter lung, spleen, stomach and large intestine meridians. Has effects of relieving exterior syndrome and promoting eruption, clearing heat and detoxicating, and lifting yang qi, and can be used for treating headache due to wind-heat, odontalgia, aphtha, sore throat, measles, yang toxin and speckle, rectocele, uterine prolapse, etc. Is widely distributed in places such as Sichuan, qinghai, shanxi, henan, hubei, yunnan and the like. Research of domestic and foreign literature shows that the main chemical components of cimicifuga rhizome are triterpenes, glycosides, phenolic acids, chromones and other compounds. The HPLC characteristic spectrum method of cimicifugae rhizoma has few reports at present, and the existing method is difficult to effectively compare and analyze the differences and changes of the cimicifugae rhizoma and the preparation thereof, and is difficult to integrally evaluate and control the quality of the cimicifugae rhizoma and the preparation thereof.
Therefore, the unified method for measuring the characteristic spectrum of the rhizoma cimicifugae and the preparation thereof is favorable for integrally evaluating the scientificity and rationality of the technical process related to the rhizoma cimicifugae, can integrally control the internal quality of the rhizoma cimicifugae and the preparation thereof, and ensures the clinical curative effect of the rhizoma cimicifugae formula particles.
Disclosure of Invention
In view of the above, the technical problem to be solved by the invention is to provide an HPLC (high performance liquid chromatography) characteristic spectrum detection method of the honey-fried cimicifuga rhizome and the preparation thereof, and the HPLC characteristic spectrum method of the honey-fried cimicifuga rhizome constructed by the invention is stable and reliable, and can control the quality of the honey-fried cimicifuga rhizome.
The invention discloses construction and application of a high-efficiency liquid-phase characteristic spectrum detection method of honey-cimicifuga rhizome, which confirms 8 common characteristic peaks, prescribes relative retention time of the common characteristic peaks, establishes a comparison characteristic spectrum, and identifies 4 index components of 5-hydroxymethylfurfural, caffeic acid, ferulic acid and isoferulic acid, fully displays the chemical component characteristics of the honey-cimicifuga rhizome, and comprehensively reflects the quality information of the honey-cimicifuga rhizome; thereby being capable of comprehensively and effectively controlling the quality of the honey cimicifuga rhizome decoction pieces, the standard decoction, the extract prescription granule, the decoction pieces and the related preparations thereof.
The invention provides an HPLC (high Performance liquid chromatography) characteristic spectrum detection method for a honey cimicifuga rhizome and a preparation thereof, which comprises the following steps:
a) Dissolving the raw materials of the cimicifuga foetida by adopting 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 an HPLC characteristic spectrum of the raw material of the cimicifuga foetida;
the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase a is acetonitrile, mobile phase B is 0.1% formic acid aqueous solution, and gradient elution is performed.
The HPLC characteristic spectrum detection method of the honey-cimicifugae rhizoma and the preparation thereof provided by the invention firstly takes the honey-cimicifugae rhizoma raw material, and the solvent is dissolved to obtain the liquid to be detected. The solvent is preferably 30% methanol.
The method has the advantages of large information quantity of the chromatographic peak of the extraction solvent and good effect.
Decocting rhizoma cimicifugae with water, cooling, filtering, evaporating filtrate, dissolving with 30% methanol, and filtering.
The ratio of the mass g of the raw material of the honey cimicifuga rhizome to the volume mL of water is 0.5 (25-50); the decoction time is 20-30 min; more preferably 30min.
The raw materials of the honey cimicifugae include honey cimicifugae granule, decoction pieces, medicinal materials or decoction. 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.
When the raw material of the rhizoma cimicifugae is the standard decoction of the rhizoma cimicifugae, 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 20-30 min; more preferably 30min.
The extraction time is 30min, and the peak shape and the separation degree of the chromatogram are good.
The extraction solvent is 30% methanol.
When the extraction solvent is 30% methanol, each characteristic peak has good peak shape and moderate separation degree.
The method also comprises the preparation of a reference substance solution of the prepared reference substance and the preparation of a reference medicinal material solution.
Preparation of a reference solution for a control: respectively dissolving 5-hydroxymethylfurfural, caffeic acid, ferulic acid and isoferulic acid in 30% methanol to obtain reference solution; the concentration of 5-hydroxymethylfurfural in the reference solution is 20 mug/mL; the concentration in the caffeic acid reference solution is 20 mug/mL; the concentration of the ferulic acid reference substance solution is 20 mug/mL; the method comprises the steps of carrying out a first treatment on the surface of the The concentration of isoferulic acid in the reference solution is 20 mug/mL. The solvent was 30% methanol. When 30% methanol is used as an extraction solvent, the chromatographic peak information amount is large, and the separation effect of each chromatographic peak is good.
Measuring the reference substance solution of the reference substance by adopting a high performance liquid chromatography to obtain a chromatogram of the reference substance; and qualitatively determining the components of the HPLC characteristic spectrum of the cimicifuga foetida according to the chromatogram of the reference substance.
According to the invention, the mobile phase A is acetonitrile, the mobile phase B is 0.1% formic acid aqueous solution, and the gradient elution is carried out.
The gradient elution of the invention is preferably specifically:
0-3 min, phase A: 12%, phase B: 88%;
3-8 min, phase A: 12% -18%, phase B: 88% -82%;
8-15 min, phase A: 18%, phase B: 82%;
15-34 min, phase A: 18% -35%, phase B: 82% -65%;
34-38 min, phase A: 35% -90%, phase B: 65% -10%;
38-48 min, phase A: 90%, phase B: 10%.
The invention has good baseline separation under the elution gradient, good separation degree of each peak and stable baseline.
C18 chromatography column, selectable: agilent C18.6 x 250mm,5 μm, phenomenex C184.6 x 250mm,5 μm, shimeji C18.6 x 250mm,5 μm.
The inventors have found that the chromatographic columns of the above-mentioned specifications for C18 meet the detection requirements of the present invention.
The column temperature was 40 ℃.
The invention has symmetrical peak shape, good separation degree and complete peak generation when the column temperature is 40 ℃.
The flow rate of the mobile phase according to the invention is preferably 1ml/min.
The invention discovers that the separation degree of each chromatographic peak is good and the peak shape is good at the flow rate of 1ml/min, and the invention is used as the most preferable scheme.
The detection wavelength of the present invention is preferably 320nm.
The inventor discovers that the chromatographic information is rich at 320nm, each component has better absorption, the response value is moderate, the chromatographic peak information amount is larger, the separation degree of each peak is better, and the base line is stable.
The sample injection amount of the invention is 5-10 mu L; preferably 10. 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 the cimicifuga foetida, and the fingerprint spectrum is positioned by 5-hydroxymethylfurfural, caffeic acid, ferulic acid and isoferulic acid; the cost of detection can be greatly reduced, and qualitative detection is realized.
The similarity of HPLC characteristic patterns of the cimicifugae rhizoma is evaluated by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to obtain the HPLC standard characteristic patterns of the cimicifugae rhizoma consisting of 8 characteristic peaks, wherein the peak 1: 5-hydroxymethylfurfural; peak 2: caffeic acid; peak 3: ferulic acid; peak 4 (S): isofasafetida acid.
In the standard characteristic spectrum of the honey-cimicifuga rhizome preparation, the relative retention time of each characteristic peak and S peak is calculated by taking isoferulic acid as a reference peak S peak, the relative retention time is within +/-10% of a specified value, and the specified values are respectively: the predetermined values were 1.33 (peak 5), 1.53 (peak 6), 1.67 or 1.68 (peak 7), and 1.71 (peak 8).
Specifically, in a standard characteristic spectrum of the honey-fried cimicifuga rhizome decoction pieces, taking isoferulic acid as a reference peak S-peak, calculating relative retention time of each characteristic peak and the S-peak, wherein the relative retention time is within +/-10% of a specified value, and the specified values are respectively: the predetermined values were 1.33 (peak 5), 1.53 (peak 6), 1.68 (peak 7), and 1.71 (peak 8).
In the honey-cimicifuga standard decoction and the granule characteristic spectrum, the relative retention time of each characteristic peak and the S peak is calculated by taking isoferulic acid as a reference peak S peak, the relative retention time is within +/-10% of a specified value, and the specified values are respectively: the predetermined values were 1.33 (peak 5), 1.53 (peak 6), 1.67 (peak 7), and 1.71 (peak 8).
Quality judgment standard: taking a sample of the cimicifuga foetida, operating according to the same method to obtain a characteristic spectrum of the cimicifuga foetida, and analyzing the standard characteristic spectrum and the sample characteristic spectrum of the cimicifuga foetida by adopting 2012 edition of Chinese pharmacopoeia Committee 'traditional Chinese medicine fingerprint similarity evaluation System', wherein the similarity is more than 0.90.
The method provided by the invention can effectively monitor the quality of different batches of cimicifuga foetida, so that the quality of the cimicifuga foetida 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 map of the honey-like cimicifugae rhizoma established by the invention takes 5-hydroxymethyl furfural, caffeic acid, ferulic acid and isoferulic acid as reference substances, pays attention to the sequence of each characteristic peak and the relativity with medicinal materials and intermediate products, can comprehensively evaluate the overall quality and appearance characteristics of the products, and is scientific and reliable.
The new characteristic spectrum method can detect the components of the cimicifuga foetida and the preparation 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 detect the characteristic spectrum of the cimicifuga foetida and the preparation thereof accurately and reliably. The consistency and the stability of the authenticity and the quality of the cimicifuga foetida and the preparation thereof can be effectively detected and controlled. Provides a basis for effectively controlling and comprehensively evaluating the quality of the honey-fried cimicifuga rhizome preparation. Ensure the uniformity and stability of the quality of the honeyed preparation.
The method is suitable for the detection method of the high-efficiency liquid-phase characteristic spectrum of the rhizoma cimicifugae, can integrally control characteristic components in the rhizoma cimicifugae, ensures the integral stability of the quality of the rhizoma cimicifugae, and has the advantages of simple operation, high precision, good stability, good repeatability and high accuracy.
The invention provides an HPLC (high Performance liquid chromatography) characteristic spectrum detection method for a honey cimicifuga rhizome and a preparation thereof, which comprises the following steps: a) Dissolving the raw materials of the cimicifuga foetida by adopting 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 an HPLC characteristic spectrum of the raw material of the cimicifuga foetida; the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase a is acetonitrile, mobile phase B is 0.1% formic acid aqueous solution, and gradient elution is performed. According to the invention, a high performance liquid chromatography is adopted, acetonitrile-0.1% formic acid is selected as a mobile phase for gradient elution, 5-hydroxymethylfurfural, caffeic acid, ferulic acid and isoferulic acid are used as reference substances, an HPLC characteristic spectrum of the cimicifuga foetida is established, the repeatability and precision are good, the method is stable and reliable, and the quality of the cimicifuga foetida can be controlled.
The invention establishes a high performance liquid characteristic spectrum method for detecting honey cimicifuga rhizome decoction pieces, standard decoction, extract formula granules and related preparations thereof.
In the process of establishing the characteristic spectrum of the honey cimicifuga foetida, 8 common characteristic peaks are confirmed, the relative retention time and the relative peak area of the honey cimicifuga foetida are researched, and the chemical composition stability and the use safety of the honey cimicifuga foetida are ensured. The method for measuring the content of the rhizoma cimicifugae by honey is established, 4 index components are established, the defect that the content measurement of a single component is difficult to reflect the whole content is overcome, the internal quality of the rhizoma cimicifugae and related preparations thereof can be controlled integrally and macroscopically, the curative effect of the medicine is ensured, and the medicine and the related preparations thereof are controlled in a more regular quality.
The method can identify the cimicifugae rhizoma medicinal materials and the related preparations of the cimicifugae rhizoma. The characteristic spectrum of the cimicifugae rhizoma has 7 characteristic peaks, and the characteristic spectrum of the cimicifugae rhizoma decoction pieces, the cimicifugae rhizoma standard decoction and the cimicifugae rhizoma formula granule has 8 characteristic peaks, wherein the main difference is that the peak 1 is 5-hydroxymethylfurfural, so that the difference between the cimicifugae rhizoma before and after processing can be better distinguished.
The method has the advantages of good stability, high precision, good repeatability, convenience and easy grasp.
The invention has the characteristics of strong operability, convenient and stable analysis. The invention uses high performance liquid fingerprint technique to control the quality of cimicifuga rhizome and its preparation, so that the quality of the product is effectively controlled. The invention can analyze the difference and the change of the rhizoma cimicifugae and the preparation thereof, is beneficial to integrally evaluating the scientificity and rationality of the technical process related to the rhizoma cimicifugae, can integrally control the internal quality of the rhizoma cimicifugae and the preparation thereof, and ensures the clinical curative effect of the rhizoma cimicifugae formula particles.
Drawings
FIG. 1 is a diagram of mobile phase selection results;
FIG. 2 is a graph of the selection result of wavelengths;
FIG. 3 is a 3D chromatogram;
FIG. 4 is a graph of flow rate observations;
FIG. 5 is a graph of results of column temperature investigation;
FIG. 6 is a view of a delay study;
FIG. 7 extraction solvent investigation;
FIG. 8 extracts a time study;
FIG. 9 is a graph of solvent addition investigation;
FIG. 10 is a view of the results of the extraction mode investigation;
FIG. 11 identification of cimicifugae rhizoma characteristic spectrum chromatographic peaks;
FIG. 12 is a view of a different instrument;
inspection of different chromatographic columns of the honey-fried cimicifugae rhizoma decoction pieces in FIG. 13;
FIG. 14 is a characteristic map of the honey cimicifugae rhizoma decoction pieces;
FIG. 15 is a control map of the honey cimicifugae rhizoma decoction pieces;
extracting solvent from figure 16;
the extraction mode of fig. 17 is examined;
fig. 18 extraction time investigation;
the extraction solvent addition amount is examined in FIG. 19;
FIG. 20 is a characteristic map of the standard decoction of cimicifugae rhizoma;
FIG. 21 is a honey cimicifugae rhizoma standard soup control map;
FIG. 22 is a characteristic map of the honey cimicifugae rhizoma formulation;
FIG. 23A control map of honey cimicifugae rhizoma particles;
FIG. 24A is a graph showing the comparison of the honey-fried cimicifugae rhizoma decoction pieces,
FIG. 25 is a honey cimicifugae rhizoma standard soup control map;
FIG. 26A control map of honey cimicifugae rhizoma particles;
FIG. 27 is a characteristic spectrum of cimicifugae rhizoma medicinal material, cimicifugae rhizoma decoction pieces, cimicifugae rhizoma standard decoction, cimicifugae rhizoma formula granule;
FIG. 28 is a spectrum of 5-hydroxymethylfurfural;
FIG. 29 is a spectrum of the 5-hydroxymethylfurfural of the cimicifuga rhizome decoction pieces;
FIG. 30 is a spectrum diagram of the honey cimicifugae rhizoma standard soup 5-hydroxymethylfurfural;
FIG. 31 is a spectrum of 5-hydroxymethylfurfural of the honey-cimicifuga rhizome formulation;
FIG. 32 is a graph of 20 batches of honey-fried cimicifugae rhizoma drug profiles;
FIG. 33 contrast profile of cimicifugae rhizoma;
FIG. 34 is a graph showing the results of comparative example 1;
FIG. 35 is a graph showing the results of comparative example 3.
Detailed Description
The invention provides an HPLC characteristic spectrum detection method for honey cimicifugae rhizoma and a preparation thereof, and a person skilled in the art can properly improve the technological parameters by referring to the content of the invention. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and they are intended to be within the scope of the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
In order to further illustrate the invention, the following describes in detail the HPLC characteristic spectrum detection method of the honey cimicifugae rhizoma and the preparation thereof provided by the invention with reference to examples.
Example 1 method for characteristic Spectrum of Honey cimicifugae rhizoma formula granule
1.1 materials, instruments and reagents
High performance liquid chromatograph: agilent 1260 type ultra-high performance liquid chromatograph, waters2695 type high performance liquid chromatograph;
an electronic balance: ME204E, XPE (Metrele Tourette instruments Co., ltd.);
ultrapure water machine: cell type 1810A (Shanghai mueller scientific instruments limited);
ultrasonic cleaner: KQ5200DB model (600W, 40KHz; kunshan ultrasonic instruments Co., ltd.);
electronic constant temperature water bath: DZKW (Beijing Zhongxing Wei industry instruments Co., ltd.)
Chromatographic column: agilent C18.6 x 250mm,5 μm, phenomenex C18.6 x 250mm,5 μm, shimeji C18.6 x 250mm,5 μm
Acetonitrile and formic acid are chromatographic purity, water is ultrapure water, and the rest reagents are analytical purity.
5-hydroxymethylfurfural (China food and drug inspection institute, lot number: 111626-202215, purity: 98.4%)
Caffeic acid (national food and drug institute, lot number: 110885-201703, purity: 99.7%);
ferulic acid (Chinese food and drug assay institute, lot number: 110773-201915, purity: 99.4%);
isofasafetida acid (China food and drug inspection institute, batch No. 111698-201904, purity: 99.3%);
cimicifugae rhizoma control (Shanghai HongYongsheng biotechnology Co., ltd., batch No. 240058-202111);
honey cimicifuga rhizome decoction pieces (batch number: MSM-01-MSM-20).
1.2 chromatographic conditions and System applicability test
Octadecylsilane chemically bonded silica is used as filler (column length is 250mm, inner diameter is 4.6mm, and granularity is 5.0 μm); acetonitrile is taken as a mobile phase A, 0.1% formic acid is taken as a mobile phase B, gradient elution is carried out according to the specification in the table below, and the detection wavelength is 320nm; the flow rate was 1.0ml/min and the column temperature was 40 ℃. The theoretical plate number should be not less than 5000 calculated according to the peak of isoferulic acid. The mobile phase gradient is shown in table 1.
Mobile phase gradients formulated in table 1
1.3 preparation of reference solutions
Taking a proper amount of 5-hydroxymethylfurfural, caffeic acid, ferulic acid and isoferulic acid reference substances, precisely weighing, adding 30% methanol to prepare a solution containing 20 mug per 1ml, and taking the solution as the reference substance solution of the reference substances.
1.4 preparation of sample solutions
Preparation of sample solution the coarse powder of the sample is about 0.5g, put into conical flask with plug, added with 50ml of water, decocted for 30 minutes, cooled, filtered, evaporated to dryness, dissolved with 20ml of 30% methanol, filtered, and the subsequent filtrate is obtained.
1.5 assay
The measurement method comprises respectively precisely sucking 10 μl of reference solution and sample solution, and injecting into liquid chromatograph for measurement.
1.6 chromatographic conditions and System applicability test
1.6.1 mobile phase selection
On the basis of the experimental conditions set forth above, the separation effect of 3 different mobile phases was examined, and the separation effects were: acetonitrile-0.1% acetic acid, acetonitrile-0.1% phosphoric acid, acetonitrile-0.1% formic acid, see FIG. 1. Fig. 1 is a diagram of mobile phase selection results.
The results show that the chromatographic peak baseline is stable under the condition of acetonitrile-0.1% formic acid solution gradient elution, the chromatographic peak is more, and the theoretical plate number, the separation degree and the symmetry are better, so that the acetonitrile-0.1% formic acid solution gradient elution is used as the mobile phase of the honey cimicifuga foetida decoction piece characteristic spectrum measuring method.
1.6.2 wavelength selection
The separation effect of 5 different wavelengths is respectively examined, and the separation effect is respectively: 300nm, 310nm, 320nm, 330nm, 340nm, see FIGS. 2 and 3. Fig. 2 is a graph of the selection result of wavelengths. Fig. 3 is a 3D chromatogram. The result shows that the information amount of the chromatographic peak is larger when the detection wavelength is 320nm, and the base line of the chromatogram is more stable, so that the detection wavelength is determined to be 320nm.
1.6.3 flow Rate investigation
Based on the experimental conditions set forth above, the flow rates were examined at 0.8ml/min, 1.0ml/min, and 1.2ml/min, respectively. See fig. 4. FIG. 4 is a graph of flow rate measurement results. The results show that the flow rates are 0.8ml/min, 1.0ml/min and 1.2ml/min, the peak shapes of the chromatograms are good, and the separation degree is moderate. The flow rate was determined to be 1.0ml/min.
1.6.4 column temperature investigation
Based on the experimental conditions set forth above, the column temperature was examined at 25℃and 30℃and 35℃and 40℃respectively. See fig. 5. FIG. 5 is a graph of results of column temperature investigation.
The result shows that the chromatographic peak shapes are symmetrical and the separation degree is good when the column temperature is 30 ℃, 35 ℃ and 40 ℃. The column temperature was temporarily determined to be 40 ℃.
1.6.5 delay test
Based on the experimental conditions set forth above, a delay test was performed. The result is shown in 6. Fig. 6 is a view for examining the delay. The results showed that the sample was essentially free of chromatographic peaks after 48min, so the sample detection time was set to 48min.
1.6.6 System suitability test results
Octadecylsilane chemically bonded silica is used as a filler; 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 detection wavelength was 320nm. The column temperature is 40 ℃, the flow rate is 1.0ml/min, and the number of theoretical plates is not lower than 5000 according to the calculation of the isoferulic acid. The mobile phase gradient is shown in table 2.
TABLE 2 gradient of mobile phases
1.7 preparation of sample solution
1.7.1 extraction solvent investigation
Taking about 0.5g of cimicifuga foetida decoction pieces, precisely weighing, placing into a conical bottle with a plug, respectively inspecting the sample extraction solvent of 30% methanol, 70% methanol, 10% ethanol, ethanol and water by 25mL, sealing, weighing, performing ultrasonic treatment (power 600W and frequency 40 kHz) for 30min, cooling, weighing again, supplementing the lost weight with the extraction solvent, shaking, filtering, and collecting the filtrate. The results are shown in FIG. 7. Fig. 7 is an extraction solvent investigation diagram.
The result shows that the separation effect of each peak of the chromatogram is better when 30% of methanol is extracted from the solvent, and the tentative extraction solvent is 30% of methanol.
1.7.2 extraction time investigation
Taking about 0.5g of cimicifuga foetida decoction pieces, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of 30% methanol, sealing, weighing, performing ultrasonic treatment (power 600W, frequency 40 kHz) for 15min, 30min and 45min, cooling, weighing again, supplementing the lost weight with extraction solvent, shaking, filtering, and collecting the subsequent filtrate. See fig. 8. Fig. 8 extracts a time study.
The result shows that the chromatogram peak shape and the separation degree are good when the extraction time is 30min. The extraction time was determined to be 30min.
1.7.3 investigation of solvent addition
Taking about 0.5g of cimicifuga foetida decoction pieces, precisely weighing, placing into a conical flask with a plug, precisely adding 10mL, 25mL and 50mL of 30% methanol, sealing, weighing, performing ultrasonic treatment (power 600W, frequency 40 kHz) for 30min, cooling, weighing again, supplementing the lost weight with extraction solvent, shaking, filtering, and collecting the subsequent filtrate. See fig. 9. FIG. 9 is a graph showing the solvent addition amount.
As a result, the amount of the solvent added was 25mL, and the peak shape and the degree of separation of each chromatographic peak were good, so that the amount of the solvent was 25mL.
1.7.4 extraction method investigation
Taking about 0.5g of cimicifugae rhizoma decoction pieces, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of 30% methanol, sealing, weighing, performing ultrasonic treatment and reflux treatment for 30min, cooling, weighing again, supplementing the lost weight with extraction solvent, shaking, filtering, and collecting the filtrate.
Taking about 0.5g of rhizoma cimicifugae decoction pieces, placing into a conical flask with a plug, adding 50ml of water, decocting for 30min, cooling, filtering, evaporating filtrate to dryness, dissolving with 25ml of 30% methanol, filtering, and collecting the subsequent filtrate. See fig. 10. Fig. 10 is an extraction mode investigation result diagram.
The result shows that when the test sample is decocted, ultrasonically and reflux-extracted, the separation effect of each peak is good during the decoction and extraction, so the test sample extraction method is determined as the decoction and extraction.
In summary, the preparation method of the sample solution for the honey-cimicifuga rhizome decoction piece characteristic spectrum is determined as follows: taking about 0.5g of cimicifugae rhizoma decoction pieces, placing into a conical flask with a plug, adding 50ml of water, decocting for 30min, cooling, filtering, evaporating filtrate to dryness, dissolving with 25ml of 30% methanol, filtering, and collecting the subsequent filtrate as sample solution.
1.8 methodology investigation
1.8.1 chromatographic Peak assignment
Preparation of sample solution the sample solution of the honey cimicifugae decoction pieces was prepared according to the experimental conditions set forth above.
Preparation of reference solution A control solution was prepared in an appropriate amount, precisely weighed, and 30% methanol was added to prepare a solution containing 20. Mu.g per 1 ml. And (5) positioning the characteristic spectrum peak of cimicifuga foetida. See fig. 11. Fig. 11 identification of cimicifugae rhizoma characteristic spectrum chromatographic peaks.
The results showed that peak 1 was 5-hydroxymethylfurfural, peak 2 was caffeic acid, peak 3 was ferulic acid, and peak 4 was isoferulic acid, and 8 characteristic peaks in the samples were examined in the following methodological examination.
1.8.2 precision test
1 part of cimicifuga foetida decoction pieces is precisely weighed, a sample is prepared according to a planned experimental method, and continuous sample injection is carried out for 6 times. See table 3.
TABLE 3 precision investigation-relative retention time
The result shows that the RSD value of each peak relative retention time is 0.03% -0.06%, and the instrument has good precision.
1.8.3 repeatability investigation
Precisely weighing 6 parts of cimicifuga foetida decoction pieces, and preparing and measuring according to a planned experimental method. See tables 4-5.
Table 4 repeatability investigation-relative retention time
TABLE 5 repeatability investigation-relative peak area ratio
Characteristic peak
The result shows that the RSD of each characteristic peak relative retention time is 0.02% -0.08%, and the method has good repeatability.
1.8.4 intermediate precision investigation
1.8.4.1 different instruments
Based on the experimental conditions, two parts of the cimicifuga foetida decoction pieces are respectively and precisely weighed, the solution of the sample is prepared, and the measurement is respectively carried out on a Waters and Agilent 1260 high performance liquid chromatograph. See fig. 12, tables 6-7. Fig. 12 shows a different instrument view.
TABLE 6 investigation of different instruments-relative retention time
TABLE 7 investigation of different instruments-characteristic peak to peak area
The results show that when the 2 instruments are used for detecting the test samples, the RSD of each characteristic peak relative retention time is 0.09% -5.78%, which shows that the instrument durability is good.
1.8.4.2 investigation of different personnel and time
Based on the experimental conditions, different people (A, B) respectively and precisely weigh two parts of cimicifuga foetida decoction pieces at different times (T1 and T2), prepare samples to be tested, and measure. See tables 8-9.
TABLE 8 investigation of different personnel versus time-relative Retention time
TABLE 9 investigation of time by different personnel-peak area ratio of characteristic peaks to peak areas
The results show that the RSD of each characteristic peak relative to the retention time is 0.02% -0.13% when different persons measure the same sample at different times, and the method has good stability.
1.8.5 durability inspection
1.8.5.1 chromatographic column durability inspection
Based on the experimental conditions set forth above, the chromatographic columns agilent C184.6 x 250mm,5 μm, phenomenexC C184.6 x 250mm,5 μm, and shimeji C18.6 x 250mm were examined, respectively. See fig. 13, tables 10-11.
Fig. 13 investigation of different chromatographic columns of the honey-fried cimicifuga rhizome decoction pieces.
Table 10 column durability investigation-relative retention time
TABLE 11 durability investigation of chromatographic columns-characteristic peak to peak area
The result shows that the RSD of each characteristic peak of the chromatographic column of different brands relative to the retention time is 2.318-13.503%, which shows that the chromatographic column has better durability.
1.8.5.2 stability investigation
Based on the experimental conditions, the same sample solution is taken and measured at 0h, 2h, 4h, 8h, 12h and 24h respectively. See table 12.
TABLE 12 stability investigation-retention time
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The results showed that the RSD for its corresponding characteristic peak retention time was between 0.07% and 0.34% and the sample solution was stable over 24 hours.
1.8.6 determination of characteristic peaks and establishment of a control Pattern
And (3) measuring the characteristic spectrum of 20 batches of honey cimicifuga rhizome decoction pieces by adopting a formulated method, and calculating the relative retention time and the relative peak area. See fig. 14, table 13. FIG. 14 is a characteristic map of the honey cimicifugae rhizoma decoction pieces; (note: 1 to 20 are MSM-01-MSM19, MSM20, respectively).
TABLE 13 ratio of 20 batches of cimicifugae rhizoma decoction pieces to retention time
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, 8 peaks with better repeatability are selected as characteristic peaks. The RSD of the characteristic peaks from peak 1 to peak 8 with respect to the retention time is 0.09% to 4.49%.
Finally, the following steps are provided: the sample chromatograph should show 8 characteristic peaks, except for peak 1, which should correspond to 7 characteristic peaks in the reference chromatograph of the reference medicinal material, wherein peak 1, peak 2, peak 3 and peak 4 should respectively correspond to the corresponding reference peak retention times of the reference, the peak corresponding to the isoferulic acid reference is S peak, and the relative retention time of each characteristic peak and S peak is calculated, and the relative retention time is within + -10% of the specified value. The predetermined values were 1.33 (peak 5), 1.53 (peak 6), 1.68 (peak 7), and 1.71 (peak 8).
And synthesizing 20 batches of cimicifuga foetida decoction piece characteristic patterns by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), and establishing a reference pattern of the cimicifuga foetida decoction piece characteristic patterns. See fig. 15. FIG. 15 is a control map of the honey cimicifugae rhizoma decoction pieces; wherein peak 1: 5-hydroxymethylfurfural; peak 2: caffeic acid; peak 3: ferulic acid; peak 4 (S): isoferulic acid;
EXAMPLE 2 Standard decoction of Mi cimicifugae
2.1 materials, reagents and instruments
Materials, reagents and instrumentation are the same as 1.1.
Honey cimicifugae rhizoma standard decoction (Sichuan New green pharmaceutical industry technology development Co., ltd., batch number: MSM-BT-01-MSM-BT-20)
2.2 chromatographic conditions and System applicability test
And 1.2 chromatographic conditions and system adaptability tests.
2.3 preparation of reference solutions
And 1.3 preparation of reference solution.
2.4 preparation of sample solutions
2.4.1 extraction solvent investigation
Taking about 0.1g of the product, precisely weighing, placing into a conical flask with a plug, respectively inspecting 25mL of the sample extraction solvent such as methanol, 70% methanol, 50% methanol, 30% methanol, ethanol and water, sealing, weighing, performing ultrasonic treatment (power 600W, frequency 40 kHz) for 30 minutes, cooling, filtering, and collecting the subsequent filtrate. The results are shown in FIG. 16. Extracting solvent from figure 16; the result shows that the separation effect of each peak of the chromatogram is better when 30% of methanol is extracted from the solvent, and the tentative extraction solvent is 30% of methanol.
2.4.2 extraction method investigation
About 0.1g of the product is precisely weighed, placed in a conical flask with a plug, precisely added with 25mL of 30% methanol, sealed, weighed, inspected when the sample is extracted by reflux and ultrasonic for 30 minutes respectively, cooled, weighed again, complemented with 30% methanol by weight, shaken uniformly, filtered, and the subsequent filtrate is taken, thus obtaining the product. See fig. 17. The extraction method of fig. 17 is examined.
The results show that the effect difference is not large when the test sample is subjected to reflux and ultrasonic extraction, and the ultrasonic method is rapid and simple, so the test sample extraction method is determined to be ultrasonic extraction.
2.4.3 extraction time investigation
Taking about 0.1g of the product, precisely weighing, placing into a conical bottle with a plug, precisely adding 25mL of 30% methanol, sealing, weighing, performing ultrasonic treatment, inspecting when the extraction time of the sample is 10min, 30min and 50min, cooling, weighing again, supplementing the lost weight with 30% methanol, shaking uniformly, filtering, and collecting the subsequent filtrate. See fig. 18. Fig. 18 extracts time study.
The result shows that when the extraction time is 30min, the peak shape and the separation degree of the chromatogram are good, and the separation requirement is met, so the extraction time is determined to be 30min.
2.4.4 investigation of the addition amount of extraction solvent
Taking about 0.1g of the product, precisely weighing, placing into a conical bottle with a plug, precisely adding 10mL, 25mL and 50mL of 30% methanol respectively for investigation, sealing, weighing, performing ultrasonic treatment for 30min, cooling, weighing again, supplementing the lost weight with 30% methanol, shaking uniformly, filtering, and taking the subsequent filtrate. See fig. 19. The extraction solvent addition amount was examined in FIG. 19.
As a result, the amount of the solvent added was 25mL, and the peak shape and the degree of separation of each chromatographic peak were good, so that the amount of the solvent was 25mL.
In summary, the preparation method of the honey-cimicifuga rhizome standard decoction characteristic spectrum test sample solution is determined as follows: taking about 0.1g of the powder, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of 30% methanol, weighing, performing ultrasonic treatment for 30min, cooling, weighing again, supplementing the lost weight with 30% methanol, shaking, filtering, and collecting the subsequent filtrate.
2.5 assay
And 1.5 chromatographic conditions and system adaptability tests.
2.6 determination of characteristic peaks and establishment of a control Pattern
And (3) measuring the characteristic spectrum of 20 batches of samples of the product by adopting a formulated method, and calculating the relative retention time and the relative peak area. See fig. 20, table 14. FIG. 20A is a characteristic map of a standard decoction of cimicifuga foetida (note: 1-20 are MSM-BT-01-MSM-BT-19, MSM-BT-20, respectively).
Table 14 ratio of 20 batches of Standard decoction of Miquel cimicifugae to Retention time
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, 8 peaks with better repeatability are selected as characteristic peaks. The RSD of the characteristic peaks from peak 1 to peak 8 is 0.28% to 4.55% relative to the retention time.
Finally, the following steps are provided: the chromatogram of the sample should show 8 characteristic peaks, wherein peak 2, peak 3 and peak 4 should correspond to the retention time of reference peaks of caffeic acid reference, ferulic acid reference and isoferulic acid reference respectively, the peak corresponding to the isoferulic acid reference is S peak, and the relative retention time of each characteristic peak and S peak is calculated and should be within + -10% of the specified value. The predetermined values were 1.33 (peak 5), 1.53 (peak 6), 1.67 (peak 7), and 1.71 (peak 8).
And synthesizing 20 batches of honey cimicifuga rhizome decoction feature maps by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), and establishing a comparison map of the honey cimicifuga rhizome decoction feature maps. See fig. 21. FIG. 21 is a honey cimicifugae rhizoma standard soup control map; wherein peak 2: caffeic acid; peak 3: ferulic acid; peak 4 (S): isofasafetida acid.
EXAMPLE 3 Honey cimicifugae rhizoma formulation granule
3.1 materials, reagents and instruments
Materials, reagents and instrumentation are the same as 1.1.
Honey cimicifugae rhizoma formula granule (Sichuan new green pharmaceutical industry Co., ltd., batch number: KL-1, KL-2, KL-3, KL-4)
3.2 chromatographic conditions and System applicability test
And 1.2 chromatographic conditions and system adaptability tests.
3.3 preparation of reference solutions
And 1.3 preparation of reference solution.
3.4 preparation of sample solutions
About 0.1g of the product powder is temporarily taken, precisely weighed, placed in a conical flask with a plug, added with 50ml of 30% methanol, subjected to ultrasonic treatment for 30 minutes, cooled, filtered, and the subsequent filtrate is taken, thus obtaining the product.
3.5 assay
And 1.5 chromatographic conditions and system adaptability tests.
3.6 determination of characteristic peaks and establishment of a control Pattern
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. 22, table 15. Figure 22 characteristic spectrum of the honey cimicifuga rhizome formula particle (S1-S3 are KL-2, KL-3 and KL-4 in sequence).
TABLE 15 ratio of the relative retention time of the particles of the Miquel cimicifugae formulation
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, 8 peaks with better repeatability are selected as characteristic peaks. The results show that when the peak 4 is taken as an S peak, 8 characteristic peaks of the 3 batches of the honey-cimicifuga rhizome formula particles have a relative retention time RSD ranging from 0.00% to 0.24%.
Finally, the following steps are provided: the chromatogram of the sample should show 8 characteristic peaks except for peak 1 and should correspond to the retention time of 7 characteristic peaks in the chromatogram of the reference substance of the reference medicinal material, wherein peak 2, peak 3 and peak 4 should correspond to the retention time of the reference substances of the caffeic acid reference substance, the isoferulic acid reference substance and the ferulic acid reference substance respectively, the peak corresponding to the isoferulic acid reference substance is S peak, and the relative retention time of each characteristic peak and S peak is calculated and should be within +/-10% of the specified value. The predetermined values were 1.23 (peak 5), 1.39 (peak 6), 1.52 (peak 7), and 1.55 (peak 8).
And synthesizing 3 batches of cimicifuga foetida particle characteristic patterns by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), and establishing a reference pattern of the cimicifuga foetida particle characteristic patterns. See fig. 23. FIG. 23A control map of honey cimicifugae rhizoma particles; wherein, peak 2: caffeic acid; peak 3: ferulic acid; peak 4 (S): isofasafetida acid.
2.3 technical effects
2.3.1 the invention adopts high performance liquid chromatography to establish a characteristic spectrum method for simultaneously measuring the honey cimicifuga rhizome decoction pieces, the honey cimicifuga rhizome standard decoction and the honey cimicifuga rhizome formula granules. The chromatographic conditions of the method are as follows: 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 detection wavelength was 320nm. The number of theoretical plates is not less than 5000 calculated according to the isoferulic acid.
The method can effectively separate the components of the chromatographic peak of the cimicifuga foetida, and successfully identify caffeic acid, ferulic acid and isoferulic acid. And (3) respectively synthesizing the characteristic spectrum of the honey cimicifuga decoction pieces and the honey cimicifuga formula particles into a control spectrum by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition). And establishing a control map of the characteristic maps of the honey-cimicifuga rhizome decoction pieces, the honey-cimicifuga rhizome standard soup and the honey-cimicifuga rhizome granules. See fig. 24-26. Fig. 24, a honey cimicifugae decoction piece control profile, wherein peak 2: caffeic acid; peak 3: ferulic acid; peak 4 (S): isofasafetida acid
Finally, the following steps are provided: the sample chromatograph should show 8 characteristic peaks, except peak 1, and should correspond to 7 characteristic peaks in the reference chromatograph of the control medicinal material, wherein peak 2, peak 3 and peak 4 should correspond to the reference peak retention time of the caffeic acid reference, the ferulic acid reference and the isoferulic acid reference respectively, the peak corresponding to the isoferulic acid reference is S peak, and the relative retention time of each characteristic peak and S peak is calculated, and the relative retention time is within + -10% of the specified value. The predetermined values were 1.33 (peak 5), 1.53 (peak 6), 1.68 (peak 7), and 1.71 (peak 8). Fig. 25 honey cimicifugae rhizoma standard soup control profile, wherein peak 2: caffeic acid; peak 3: ferulic acid; peak 4 (S): isofasafetida acid
Finally, the following steps are provided: the sample chromatograph should show 8 characteristic peaks, except peak 1, and should correspond to 7 characteristic peaks in the reference chromatograph of the control medicinal material, wherein peak 2, peak 3 and peak 4 should correspond to the reference peak retention time of the caffeic acid reference, the ferulic acid reference and the isoferulic acid reference respectively, the peak corresponding to the isoferulic acid reference is S peak, and the relative retention time of each characteristic peak and S peak is calculated, and the relative retention time is within + -10% of the specified value. The predetermined values were 1.33 (peak 5), 1.53 (peak 6), 1.67 (peak 7), and 1.71 (peak 8). FIG. 26A control map of honey cimicifugae rhizoma particles; wherein, peak 2: caffeic acid; peak 3: ferulic acid; peak 4 (S): isofasafetida acid
Finally, the following steps are provided: the chromatogram of the sample should show 8 characteristic peaks except for peak 1 and should correspond to the retention time of 7 characteristic peaks in the chromatogram of the reference substance of the reference medicinal material, wherein peak 2, peak 3 and peak 4 should correspond to the retention time of the reference substances of the caffeic acid reference substance, the isoferulic acid reference substance and the ferulic acid reference substance respectively, the peak corresponding to the isoferulic acid reference substance is S peak, and the relative retention time of each characteristic peak and S peak is calculated and should be within +/-10% of the specified value. The predetermined values were 1.23 (peak 5), 1.39 (peak 6), 1.52 (peak 7), and 1.55 (peak 8).
Example 4 distinguishing cimicifugae rhizoma medicinal materials from cimicifugae rhizoma decoction pieces, cimicifugae rhizoma standard decoction, cimicifugae rhizoma formulation granule
And (3) measuring the characteristic patterns of the cimicifuga foetida medicinal materials, the cimicifuga foetida decoction pieces, the cimicifuga foetida standard decoction and the cimicifuga foetida formula granules by adopting a high performance liquid chromatography method. See fig. 27-31. Wherein, fig. 27 shows characteristic patterns of cimicifugae rhizoma medicinal materials, cimicifugae rhizoma decoction pieces, cimicifugae rhizoma standard decoction and cimicifugae rhizoma formula granules; FIG. 28 is a spectrum of 5-hydroxymethylfurfural; FIG. 29 is a spectrum of 5-hydroxymethylfurfural of cimicifuga foetida decoction pieces. FIG. 30 is a spectrum diagram of the honey cimicifugae rhizoma standard soup 5-hydroxymethylfurfural; FIG. 31 is a spectrum of 5-hydroxymethylfurfural of the honey-like cimicifuga rhizome formulation.
The characteristic spectrum of the cimicifugae rhizoma has 7 characteristic peaks, and the characteristic spectrum of the cimicifugae rhizoma decoction pieces, the cimicifugae rhizoma standard decoction and the cimicifugae rhizoma formula granule has 8 characteristic peaks, wherein the main difference is that the peak 1 is 5-hydroxymethylfurfural, so that the difference between the cimicifugae rhizoma before and after processing can be better distinguished.
And (3) measuring the characteristic spectrum of 20 batches of honey-fried cimicifugae rhizoma medicines by adopting a formulated method, and calculating the relative retention time and the relative peak area. See fig. 32, table 16. FIG. 32 is a graph showing characteristics of 20 batches of the Honey cimicifugae rhizoma medicinal materials (note: 1-20 are S1-S20 respectively).
TABLE 16 20 relative retention time ratio of cimicifugae rhizoma batches
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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, 7 peaks with better repeatability are selected as characteristic peaks, and the relative retention time RSD of 7 characteristic peaks of 20 batches of cimicifugae medicine materials is less than 1.0%.
Finally, the following steps are provided: the chromatogram of the sample should show 7 characteristic peaks and should correspond to the retention time of 7 characteristic peaks in the chromatogram of the reference substance of the reference medicinal material, wherein peak 1, peak 2 and peak 3 should respectively correspond to the retention time of the reference substance of the corresponding reference substance, the peak corresponding to the reference substance of the isoferulic acid is S peak, the relative retention time of each characteristic peak and S peak is calculated, and the relative retention time is within + -10% of the specified value. The predetermined values were 1.33 (peak 4), 1.53 (peak 5), 1.68 (peak 6), and 1.71 (peak 7).
And synthesizing 20 batches of cimicifuga foetida decoction piece characteristic patterns by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), and establishing a reference pattern of the cimicifuga foetida decoction piece characteristic patterns. See fig. 33. FIG. 33 contrast profile of cimicifugae rhizoma; peak 1: caffeic acid; peak 2: ferulic acid; peak 3 (S): isofasafetida acid.
Comparative 1, acetonitrile as mobile phase A and water as mobile phase B, gradient elution is carried out according to the specifications in the following table; the detection wavelength was 320nm. The number of theoretical plates is not less than 5000 calculated according to the isoferulic acid. The chromatogram is shown in FIG. 34, and FIG. 34 is a graph showing the results of comparative example 1.
Comparative 2, acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, gradient elution according to the table; the detection wavelength was 320nm. The number of theoretical plates is not less than 5000 calculated according to the isoferulic acid. The chromatogram is shown in FIG. 35, and FIG. 35 is a graph showing the results of comparative example 2.
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. An HPLC characteristic spectrum detection method of rhizoma cimicifugae and its preparation comprises:
a) Dissolving the raw materials of the cimicifuga foetida by adopting 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 an HPLC characteristic spectrum of the raw material of the cimicifuga foetida;
the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase a is acetonitrile, mobile phase B is 0.1% formic acid aqueous solution, and gradient elution is performed.
2. The method of claim 1, further comprising preparing a reference solution of a control;
preparation of a reference solution for a control: respectively dissolving 5-hydroxymethylfurfural, caffeic acid, ferulic acid and isoferulic acid in 30% methanol to obtain reference solution; the concentration of 5-hydroxymethylfurfural in the reference solution is 20 mug/mL; the concentration in the caffeic acid reference solution is 20 mug/mL; the concentration of the ferulic acid reference substance solution is 20 mug/mL; the method comprises the steps of carrying out a first treatment on the surface of the The concentration of isoferulic acid in the reference solution is 20 mug/mL;
measuring the reference substance solution of the reference substance by adopting a high performance liquid chromatography to obtain a chromatogram of the reference substance; and qualitatively determining the components of the HPLC characteristic spectrum of the cimicifuga foetida according to the chromatogram of the reference substance.
3. The method according to claim 1, characterized in that the gradient elution is in particular:
0-3 min, phase A: 12%, phase B: 88%;
3-8 min, phase A: 12% -18%, phase B: 88% -82%;
8-15 min, phase A: 18%, phase B: 82%;
15-34 min, phase A: 18% -35%, phase B: 82% -65%;
34-38 min, phase A: 35% -90%, phase B: 65% -10%;
38-48 min, phase A: 90%, phase B: 10%.
4. A method according to claim 3, wherein the chromatographic column is a C18 chromatographic column having a specification of 4.6 x 250mm,5 μm and a theoretical plate number of not less than 5000 calculated as isoferulic acid peak and a chromatographic column temperature of 40 ℃.
5. The method of claim 4, wherein the mobile phase flow rate is 1.0mL/min; the detection wavelength is 320nm; the sample loading was 10. Mu.L.
6. The method according to claim 1, wherein the similarity of the HPLC characteristic spectrum of the cimicifuga racemosa is evaluated by using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to obtain an HPLC standard characteristic spectrum of the cimicifuga racemosa consisting of 8 characteristic peaks, wherein peak 1: 5-hydroxymethylfurfural; peak 2: caffeic acid; peak 3: ferulic acid; peak 4 (S): isofasafetida acid.
7. The method according to claim 6, wherein in the standard characteristic spectrum of the honey-cimicifuga rhizome preparation, the relative retention time of each characteristic peak and the S peak is calculated by taking isoferulic acid as a reference peak, the relative retention time is within +/-10% of a specified value, and the specified values are respectively: the predetermined values were 1.33 (peak 5), 1.53 (peak 6), 1.67 or 1.68 (peak 7), and 1.71 (peak 8).
8. The method according to claim 1, wherein step a) is specifically: decocting rhizoma cimicifugae with water, cooling, filtering, evaporating filtrate to dryness, dissolving with 30% methanol, and filtering;
the ratio of the mass g of the honey cimicifuga rhizome raw material to the volume mL of water is 0.5 (25-50); the decoction time is 20-30 min.
9. The method of claim 1, wherein the raw material of cimicifuga foetida comprises particles, pieces, materials or decoction of cimicifuga foetida.
10. A method for identifying cimicifuga foetida medicinal material and cimicifuga foetida preparation, characterized in that the identification is carried out by adopting the method of any one of claims 1 to 9.
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