CN110441444B - Method for constructing UPLC characteristic spectrum of sauropus miq and identification method thereof - Google Patents
Method for constructing UPLC characteristic spectrum of sauropus miq and identification method thereof Download PDFInfo
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Abstract
The invention relates to a method for constructing a UPLC characteristic spectrum of a sauropus herb and an identification method thereof. The method for constructing the UPLC characteristic spectrum of the sauropus spatholobus stem comprises the following steps: (1) precisely weighing a sauropus spatholobus stem medicinal material, and preparing a sauropus spatholobus stem test solution; (2) and analyzing the sauropus test solution by using an ultra performance liquid chromatograph to obtain a sauropus feature map. Reference data of a characteristic spectrum analysis method of sauropus, characteristic peaks are selected from characteristic spectrum angles unique to sauropus, relative retention time and relative peak areas of the characteristic peaks are calculated, a pleopophysique of the characteristic spectrum of the sauropus is formed, quality control of the sauropus is enabled to be measured from a certain original component content, ascending quality detection of the whole medicine is achieved, the defect of single chemical component detection is avoided, and quality of the sauropus can be better evaluated.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicine identification, and particularly relates to a method for constructing a UPLC (unified performance liquid chromatography) characteristic spectrum of a sauropus spatulifolius medicinal material and an identification method thereof.
Background
Sauropus saururi saururus Beille, Sauropus spatulifolius, a plant of the family euphorbiaceae. Collected in summer and autumn and dried in the sun. Folium sauropi (Sauropus spatulifolius Beille), also known as folium agaves americana, folium agaves nigra, folium nelumbinis, folium auriculati, and the like, belongs to the family euphorbiaceae, the genus Sauropus, and is the only medicinal plant in the genus Sauropus. It is recorded in Lingnan Bingyao (records of medicine Collection in Lingnan), sweet, light and flat in nature, enters lung and stomach meridians, and has the functions of clearing lung-heat, treating cough due to internal injury, bronchitis and bronchial asthma, stopping cough due to phlegm-fire, asthma, and treating internal injury, phthisis, aphonia, laryngalgia, etc. The shrub is a evergreen shrub which is as high as 40cm and is mainly distributed in areas such as Guangdong, Guangxi, Hainan and the like in China, and is mostly cultivated or grows in a valley, a mountain slope and a wet fertile forest.
The sauropus makino mainly contains various bioactive components such as monoterpene, sesquiterpene, coumarin and flavonoid glycoside, wherein the flavonoid glycoside component kaempferol-3-O-gentiobioside has the effects of resisting inflammation, relieving cough, relieving spasm, resisting ulcer and the like and is a main effective component of sauropus makino. According to a literature report, ethyl acetate is used as a solvent to research the folium sauropus spatulifolius, 15 common peaks are calibrated by adopting relative retention time, and the similarity of the finger prints of 10 sauropus spatulifolius herbs in different producing areas is more than 0.8; respectively taking the root and the stem of sauropus spatulifolius as research targets, and respectively determining 13 and 14 common peaks through the statistical analysis of HPLC chromatograms of sauropus spatulifolius from different producing areas; the quality of the sauropus culture mass evaluation system is established for comprehensively evaluating the quality, and experimenting in the experiment, the characteristic diagram is sauropus.
Disclosure of Invention
The invention aims to provide a UPLC characteristic spectrum construction method and an identification method of a sauropus herb, aiming at the defects and shortcomings in the prior art.
The technical problem to be solved by the invention is realized by the following technical scheme:
a UPLC characteristic spectrum construction method of sauropus koehne comprises the following steps:
(1) precisely weighing a sauropus spatholobus stem medicinal material, and preparing a sauropus spatholobus stem test solution;
(2) and analyzing the sauropus test solution by using an ultra performance liquid chromatograph to obtain a sauropus feature map.
As a preferred scheme, the chromatographic conditions of the ultra high performance liquid chromatograph analysis are as follows: using octadecylsilane chemically bonded silica as a filler, taking methanol as a mobile phase A, taking a phosphoric acid aqueous solution with the volume fraction of 0.34-0.45% as a mobile phase B, and carrying out gradient elution, wherein the flow rate is 0.25-0.36 ml/min, the detection wavelength is 320-400 nm, and the sample injection amount is 0.5-3 mu l.
As a most preferred scheme, the chromatographic conditions of the ultra high performance liquid chromatograph analysis are as follows: using octadecylsilane chemically bonded silica as a filler, taking methanol as a mobile phase A at a column temperature of 30 ℃, taking a phosphoric acid aqueous solution with a volume fraction of 0.4% as a mobile phase B, and carrying out gradient elution at a flow rate of 0.3ml/min, a detection wavelength of 349nm and a sample injection amount of 1 mul.
As a preferable scheme, the gradient elution condition is 0-10 min, the volume fraction of the mobile phase A is 6%, and the volume fraction of the mobile phase B is 94%; for 10-15 min, the volume fraction of the mobile phase A is changed to 6-7%, and the volume fraction of the mobile phase B is changed to 94-93%; 15-20 min, wherein the volume fraction of the mobile phase A is 7%, and the volume fraction of the mobile phase B is 93%; the volume fraction of the mobile phase A is changed to 7-18% and the volume fraction of the mobile phase B is changed to 93-82% in 20-25 min; in 25-30 min, the volume fraction of the mobile phase A is changed to 18-23%, and the volume fraction of the mobile phase B is changed to 82-77%; 30-40 min, the volume fraction of the mobile phase A is changed to 23-45%, and the volume fraction of the mobile phase B is changed to 77-55%; and (3) changing the volume fraction of the mobile phase A to 45-59% and the volume fraction of the mobile phase B to 55-41% in 40-50 min.
As a preferred scheme, the test solution is prepared by a method comprising the following steps: taking 0.4-0.6 g of sauropus spatulifolius powder, precisely adding 20-30 ml of 45-55% methanol, carrying out ultrasonic treatment for 25-35 minutes, cooling, shaking up, filtering, and taking a subsequent filtrate.
As a most preferred embodiment, the test solution is prepared by a method comprising the steps of: taking 0.5g of sauropus spatulifolius powder, placing the sauropus spatulifolius powder in a conical flask with a plug, precisely adding 25ml of 50% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, shaking up, filtering, and taking a subsequent filtrate.
The invention also provides a method for identifying the sauropus herb, which comprises the following steps:
(1) precisely weighing a sauropus spatulifolius medicinal material to be identified, and preparing a sauropus spatulifolius sample solution to be identified;
(2) precisely sucking a sample solution of the sauropus spatulifolius to be identified, injecting the sample solution into an ultra-high performance liquid chromatograph, and measuring to obtain a UPLC (UPLC) characteristic map of the sauropus spatulifolius to be identified;
(3) comparing the measured UPLC feature spectrum with the constructed UPLC feature spectrum of the sauropus, and if the UPLC feature spectrum is consistent with the UPLC feature spectrum of the sauropus, determining that the sauropus is qualified in quality.
As a preferred scheme, the chromatographic conditions of the ultra high performance liquid chromatograph analysis are as follows: using octadecylsilane chemically bonded silica as a filler, taking methanol as a mobile phase A, taking a phosphoric acid aqueous solution with the volume fraction of 0.34-0.45% as a mobile phase B, and carrying out gradient elution, wherein the flow rate is 0.25-0.36 ml/min, the detection wavelength is 320-400 nm, and the sample injection amount is 0.5-3 mu l.
As a most preferred scheme, the chromatographic conditions of the ultra high performance liquid chromatograph analysis are as follows: using octadecylsilane chemically bonded silica as a filler, taking methanol as a mobile phase A at a column temperature of 30 ℃, taking a phosphoric acid aqueous solution with a volume fraction of 0.4% as a mobile phase B, and carrying out gradient elution at a flow rate of 0.3ml/min, a detection wavelength of 349nm and a sample injection amount of 1 mul.
As a preferred embodiment, the gradient elution conditions are: 0-10 min, wherein the volume fraction of the mobile phase A is 6%, and the volume fraction of the mobile phase B is 94%; for 10-15 min, the volume fraction of the mobile phase A is changed to 6-7%, and the volume fraction of the mobile phase B is changed to 94-93%; 15-20 min, wherein the volume fraction of the mobile phase A is 7%, and the volume fraction of the mobile phase B is 93%; the volume fraction of the mobile phase A is changed to 7-18% and the volume fraction of the mobile phase B is changed to 93-82% in 20-25 min; in 25-30 min, the volume fraction of the mobile phase A is changed to 18-23%, and the volume fraction of the mobile phase B is changed to 82-77%; 30-40 min, the volume fraction of the mobile phase A is changed to 23-45%, and the volume fraction of the mobile phase B is changed to 77-55%; and (3) changing the volume fraction of the mobile phase A to 45-59% and the volume fraction of the mobile phase B to 55-41% in 40-50 min.
As a preferred scheme, the test solution is prepared by a method comprising the following steps: taking 0.4-0.6 g of sauropus spatulifolius powder, precisely adding 20-30 ml of 45-55% methanol, carrying out ultrasonic treatment for 25-35 minutes, cooling, shaking up, filtering, and taking a subsequent filtrate.
As a most preferred embodiment, the test solution is prepared by a method comprising the steps of: taking 0.5g of sauropus spatulifolius powder, placing the sauropus spatulifolius powder in a conical flask with a plug, precisely adding 25ml of 50% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, shaking up, filtering, and taking a subsequent filtrate.
Has the advantages that: (1) determining 8 characteristic peaks in total by measuring 16 sauropus spathulifolius medicinal materials from different sources, identifying chemical components of the 8 common peaks, confirming that the No. 6 peak is a 6-hydroxycoumarin peak, the No. 8 peak is a kaempferol-3-O-gentiobioside peak, and the No. 8 peak is a kaempferol-3-O-gentiobioside peak to prepare reference data of a sauropus spathulifolius medicinal material characteristic spectrum analysis method; (2) selecting characteristic peaks from the specific characteristic spectrum angle of the sauropus, calculating the relative retention time and the relative peak area of the characteristic peaks, and forming a full look of the characteristic spectrum of the sauropus, so that the quality control of the sauropus is improved from the original content measurement of a certain components to the inherent quality detection of the whole medicine, and the defect of single chemical component detection is avoided; (3) the method is rapid, stable and highly specific, can more comprehensively reflect the characteristics of the sauropus, and provides a scientific new method for the quality control of the sauropus.
Drawings
Fig. 1 is a chromatogram peak reference identification chromatogram of sauropus spatulifolius, wherein a is a hydroxycoumarin reference solution, B is a kaempferol-3-O-gentiobioside test solution, and C is a test solution; peak 1 is 6-hydroxycoumarin, Peak 2 is kaempferol-3-O-gentiobioside.
FIG. 2 is a ion flow diagram of kaempferol-3-O-gentiobioside.
FIG. 3 is a mass spectrum of kaempferol-3-O-gentiobioside from sauropus rostratus, wherein B is
The primary mass spectrum and the secondary mass spectrum are C.
Fig. 4 is a mass spectrum of 6-hydroxycoumarin in sauropus spatulifolius, wherein a is an ion flow diagram and B is a primary mass spectrum.
Fig. 5 shows the UPLC profile of 16 batches of sauropus.
Fig. 6 is a UPLC control profile of sauropus spatulifolius in which peak 8 is kaempferol-3-O-gentiobioside.
Fig. 7 is a graph of the cluster analysis of sauropus from different sources.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Construction of characteristic map of sauropus spatulifolius
The main apparatus is as follows: thermo ultra high performance liquid chromatography (Thermo Vanquish, Thermo corporation); DAD Detector, Agilent SB C18Chromatographic column
ACQUITY UPLC I-Class ultra-high performance liquid phase flight time is highA resolution mass spectrometry combination system, wherein the data processing system is a MarkerLynx 4.1 workstation; mettler XP26 parts per million (Mettler Toledo, Switzerland); Milli-Q ultrapure water purification systems (Millipore, USA); model KQ5500DE ultrasonic cleaner (ultrasonic instruments ltd, kunshan).
Sauropus Lindl is obtained from different Chinese medicinal material markets and drugstores in China.
Sauropus spatulifolius source table 1
The main reagents are as follows: methanol and acetonitrile are chromatographically pure (Merk), water is Mili-Q purified water, and the other reagents are analytically pure. Kaempferol-3-O-gentiobioside (batch No. 112025 201601, content: 96.2%) is provided by China food and drug testing research institute; 6-Hydroxycoumarin (batch: Z29D7H27807, content: 98.0%) was supplied from Shanghai-derived Phyllo Biotech Co., Ltd.
Test method
Preparation of a test sample of sauropus
Taking 0.5g of sauropus spatulifolius powder, placing the sauropus spatulifolius powder in a conical flask with a plug, precisely adding 25ml of 50% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, shaking up, filtering, and taking a subsequent filtrate.
Preparation of control solutions
Precisely weighing 3.233mg of kaempferol-3-O-gentiobioside reference substance, putting into a 25ml measuring flask, adding methanol for dissolving, and diluting to scale to obtain a kaempferol-3-O-gentiobioside reference substance solution stock solution with the concentration of 124.4058 mu g/ml. Precisely sucking 0.5ml of the kaempferol-3-O-gentiobioside reference substance solution stock solution into a 10ml measuring flask, adding methanol to a constant volume to scale, and shaking up to prepare a reference substance solution with the concentration of 6.2203 mu g/ml. Accurately weighing 2.126mg of 6-hydroxycoumarin reference substance, placing in a 25ml measuring flask, adding methanol to dissolve and dilute to scale to obtain a 6-hydroxycoumarin reference substance solution stock solution with concentration of 83.3392 μ g/ml. Precisely sucking 1ml of the 6-hydroxycoumarin reference solution stock solution, placing in a 10ml measuring flask, adding methanol to constant volume to scale, shaking up, and making into reference solution with concentration of 8.3339 μ g/ml.
Chromatographic conditions
Adopts the specification of
Agilent SB C18Performing gradient elution on a chromatographic column by taking methanol as a mobile phase A and taking a phosphoric acid aqueous solution with the volume fraction of 0.4% as a mobile phase B at the column temperature of 30 ℃, wherein the flow rate is 0.3ml/min, the detection wavelength is 349nm, and the sample injection amount is 1 mu l.
The gradient elution conditions were: 0-10 min, wherein the volume fraction of the mobile phase A is 6%, and the volume fraction of the mobile phase B is 94%; for 10-15 min, the volume fraction of the mobile phase A is changed to 6-7%, and the volume fraction of the mobile phase B is changed to 94-93%; 15-20 min, wherein the volume fraction of the mobile phase A is 7%, and the volume fraction of the mobile phase B is 93%; the volume fraction of the mobile phase A is changed to 7-18% and the volume fraction of the mobile phase B is changed to 93-82% in 20-25 min; in 25-30 min, the volume fraction of the mobile phase A is changed to 18-23%, and the volume fraction of the mobile phase B is changed to 82-77%; 30-40 min, the volume fraction of the mobile phase A is changed to 23-45%, and the volume fraction of the mobile phase B is changed to 77-55%; and (3) changing the volume fraction of the mobile phase A to 45-59% and the volume fraction of the mobile phase B to 55-41% in 40-50 min.
Conditions of Mass Spectrometry
Adopts the specification of
Agilent SB C18Performing gradient elution by using a chromatographic column, wherein the column temperature is 30 ℃, methanol is used as a mobile phase A, and a formic acid aqueous solution with the volume fraction of 0.05% is used as a mobile phase B; the flow rate is 0.3 ml/min; the detection wavelength was 349nm and the sample size was 1. mu.l.
The gradient elution conditions were: 0-10 min, wherein the volume fraction of the mobile phase A is 6%, and the volume fraction of the mobile phase B is 94%; for 10-15 min, the volume fraction of the mobile phase A is changed to 6-7%, and the volume fraction of the mobile phase B is changed to 94-93%; 15-20 min, wherein the volume fraction of the mobile phase A is 7%, and the volume fraction of the mobile phase B is 93%; the volume fraction of the mobile phase A is changed to 7-18% and the volume fraction of the mobile phase B is changed to 93-82% in 20-25 min; in 25-30 min, the volume fraction of the mobile phase A is changed to 18-23%, and the volume fraction of the mobile phase B is changed to 82-77%; 30-40 min, the volume fraction of the mobile phase A is changed to 23-45%, and the volume fraction of the mobile phase B is changed to 77-55%; and (3) changing the volume fraction of the mobile phase A to 45-59% and the volume fraction of the mobile phase B to 55-41% in 40-50 min.
The nitrogen is used as atomizing and drying gas of the mass spectrum ion source, an electrospray ionization (positive and negative) ion mode is adopted, the spraying voltage is 3.0KV, the Lens voltage is 50V, the desolventizing gas temperature is 400 ℃, the sheath gas pressure is 40Arb, the auxiliary gas pressure is 12Arb, and the mass-to-charge ratio range is 100-1000.
Survey of feature map methodology
And (3) precision test: taking a sample of sauropus spatulifolius S1, preparing a test solution, carrying out continuous sample injection for 6 times under the specified chromatographic condition, and examining the consistency of the relative retention time and the relative peak area of the common peak, wherein the RSD of the relative peak area and the relative retention time of the obtained common peak is less than 2%, which indicates that the instrument precision is good.
And (3) stability test: a sample of sauropus spathula S1 was sampled and measured in a predetermined chromatographic condition at 0, 2, 4, 6, 8, 12, and 20, and the RSD value was calculated by examining the consistency of the relative retention time and the relative peak area of the common peak. The relative peak area and the relative retention time RSD of the obtained common peak are less than 2 percent, which indicates that the test solution has good stability within 20 hours.
And (3) repeatability test: taking a sample of sauropus spathula root medicinal material S1, preparing 6 parts of a test sample solution, carrying out sample injection measurement under specified chromatographic conditions, observing the consistency of relative retention time and relative peak area of a common peak, and calculating an RSD value. The relative peak area RSD of the obtained common peak is less than 3%, and the relative retention time RSD is less than 2%, which indicates that the method has good repeatability.
Characteristic map analysis and evaluation of sauropus spatulifolius
Sample assay
Taking 16 batches of sauropus spathulifolius medicinal material samples, preparing a test solution, and respectively recording a UPLC (ultra performance liquid chromatography) spectrum and a mass spectrum under specified chromatographic and mass spectrum conditions. The chromatogram peak reference chromatogram of sauropus is shown in figure 1.
Determination of common peaks of feature maps of sauropus spatulifolius
Results of the UPLC of 16 sauropus herbs were analyzed by using a traditional Chinese medicine chromatography fingerprint similarity evaluation system (2012.0 edition) recommended by the State pharmacopoeia Committee, and 8 common peaks were determined in total. Among the 8 common peaks, the No. 8 peak (kaempferol-3-O-gentiobioside) has good separation degree and symmetrical peak shape and is one of the main components for resisting inflammation, relieving cough and resisting ulcer in sauropus spatulifolius, so that the No. 8 peak (kaempferol-3-O-gentiobioside) is taken as a reference peak (namely an S peak), the relative retention time and the relative peak area of each common peak are respectively calculated, the relative retention time RSD of each sample common peak is 0.05-1.04%, and the accuracy of common peak selection is shown; the peak areas of the common peaks of different samples are greatly different, which shows that the content of the common peaks of sauropus in different producing areas is different, and is shown in table 2.
Table 216 relative retention time and relative peak area of the characteristic spectrum of sauropus spatulifolius
Mass spectrum identification of characteristic spectrum of sauropus
According to the mass spectrum conditions, the characteristic peak 8 is analyzed, the mass spectrum information of the corresponding peaks of the primary mass spectrum and the secondary mass spectrum of the sample is shown in figure 2, wherein the accurate molecular weight of the peak 8 is 287.055, and the peak is basically consistent with the kaempferol-3-O-gentiobioside according to the judgment of documents and databases, so that the chromatographic peak can be determined to be the kaempferol-3-O-gentiobioside, which is shown in figure 3. Calculating corresponding molecular formula C according to literature report and primary mass spectrum information by using primary mass spectrum and main ion mass-to-charge ratio (163.039) in the sample9H6O3Presumably it should be hydroxycoumarin or the corresponding isomer, see figure 4.
Similarity evaluation of feature spectrum of sauropus
The method comprises the steps of superposing characteristic spectrums of sauropus leaves samples of No. S1-S16 by a traditional Chinese medicine chromatography fingerprint similarity evaluation system (version 2012.0) recommended by the State pharmacopoeia Committee, carrying out common peak identification, generating a comparison spectrum, inspecting consistency of similarity of chromatographic peak, and carrying out similarity evaluation, wherein results are shown in fig. 5, 6 and 3.
Table 3 degree of similarity between different samples of sauropus spathula
Clustering analysis of sauropus spatulifolius from different sources
By performing feature map analysis on 16 batches of sauropus spathulifolius medicinal materials from different sources, 8 common peaks including 6-hydroxycoumarin (peak 7) and kaempferol-3-O-gentiobioside (peak 8) are obtained, the relative peak areas of the common peaks are quantified, the SPSS 22.0 software is used for performing systematic cluster analysis on the common peaks, the Euclidean distance is taken as a measure, the relative peak areas of the 16 batches of sauropus spathulifolius medicinal materials are analyzed, and the result is shown in fig. 7. When the distance was determined to be 5, 16 batches of sauropus. S1-S3, S5-S6, S11-12, S14 and S16 are gathered into a class, and the sources of the sample medicinal materials are Gaoyue city, Qingyuan city and Zhaoqing city; s4, S8, S10 and S13 are clustered, and the main source of the sample medicinal materials is Guangzhou Qingping medicinal material market; the S7, S9 and S15 are grouped into one type, the main sources are Guangzhou Qingping medicinal material market and Zhaoqing city, wherein the S4, S13 and S15 samples are different from the medicinal material sources and are respectively from Gaoqiang city, Qingyuan city and Zhaoqing city, the clustering analysis does not classify the samples into the corresponding type, the relative peak areas of the S4 sample, the peak 2, the peak 3 and the peak 6 are different from the relative peak areas of other samples, the relative peak areas of 8 common peaks of the S13 sample are all larger than those of the same origin, the S15 sample is in a reverse trend, and the relative peak areas are all smaller than those of the same origin. The research of cluster analysis shows that the chemical components of the sauropus, the source of the same source of the. Wherein the processing and drying mode of the sauropus leaves is as follows: taking 3-5 pieces of sauropus, drying in the shade each time, or drying in the shade to seventy-eight percent, bundling the sauropus leaves into a small bundle, covering the small bundle with a cattail mat, and drying in the sun. From the analysis of the sample properties, the samples of the numbers S4, S13 and S15 were softer in texture and higher in moisture content, and it is considered that the difference between the sample contents possibly caused by insufficient drying degree in the manufacturing process of the medicinal material in the manufacturing area has a certain influence on the quality of the sauropus rostratus.
Example 2
The identification method of the sauropus medicinal material comprises the following steps:
(1) precisely weighing a sauropus spatulifolius medicinal material to be identified, and preparing a sauropus spatulifolius sample solution to be identified;
(2) precisely sucking a sample solution of the sauropus spatulifolius to be identified, injecting the sample solution into an ultra-high performance liquid chromatograph, and measuring to obtain a UPLC (UPLC) characteristic map of the sauropus spatulifolius to be identified;
(3) comparing the measured UPLC feature spectrum with the constructed UPLC feature spectrum of the sauropus, and if the UPLC feature spectrum is consistent with the UPLC feature spectrum of the sauropus, determining that the sauropus is qualified in quality.
Chromatographic conditions
Adopts the specification of
Agilent SB C18Performing gradient elution by using a chromatographic column, wherein the column temperature is 30 ℃, methanol is used as a mobile phase A, and a phosphoric acid aqueous solution with the volume fraction of 0.4% is used as a mobile phase B; the flow rate is 0.3 ml/min; the detection wavelength was 349nm and the sample size was 1. mu.l.
Gradient elution conditions
0-10 min, wherein the volume fraction of the mobile phase A is 6%, and the volume fraction of the mobile phase B is 94%; for 10-15 min, the volume fraction of the mobile phase A is changed to 6-7%, and the volume fraction of the mobile phase B is changed to 94-93%; 15-20 min, wherein the volume fraction of the mobile phase A is 7%, and the volume fraction of the mobile phase B is 93%; the volume fraction of the mobile phase A is changed to 7-18% and the volume fraction of the mobile phase B is changed to 93-82% in 20-25 min; in 25-30 min, the volume fraction of the mobile phase A is changed to 18-23%, and the volume fraction of the mobile phase B is changed to 82-77%; 30-40 min, the volume fraction of the mobile phase A is changed to 23-45%, and the volume fraction of the mobile phase B is changed to 77-55%; and (3) changing the volume fraction of the mobile phase A to 45-59% and the volume fraction of the mobile phase B to 55-41% in 40-50 min.
Preparation of a sample solution to be identified as sauropus spatulifolius
Taking 0.5g of sauropus spatulifolius powder, placing the sauropus spatulifolius powder in a conical flask with a plug, precisely adding 25ml of 50% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, shaking up, filtering, and taking a subsequent filtrate.
Sample introduction measurement
The method is used for preparing a solution of a sauropus sample to be identified, and carrying out sample injection measurement under a specified chromatographic condition to obtain a UPLC (UPLC) characteristic spectrum of the sauropus.
The characteristic spectrum of the sauropus, and the characteristic spectrum of the sauropus are consistent with each other and to be identified.
The study establishes a UPLC characteristic spectrum of the sauropus herb and establishes 8 common peaks. The UPLC characteristic spectrum established in the experiment can well reflect the overall appearance of the flavonoid components of the sauropus, and the methodological investigation result shows that the method has good precision, stability and reproducibility, so that the method is accurate and reliable and can be used as the basis for controlling the quality of the sauropus.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (6)
1. A method for constructing a UPLC characteristic spectrum of a sauropus spatulifolius material is characterized by comprising the following steps:
(1) precisely weighing a sauropus spatholobus stem medicinal material, and preparing a sauropus spatholobus stem test solution;
(2) analyzing the sauropus test solution by using an ultra-performance liquid chromatograph to obtain a sauropus feature map;
the chromatographic conditions of the ultra-high performance liquid chromatograph analysis are as follows: performing gradient elution by using octadecylsilane chemically bonded silica as a filling agent, taking methanol as a mobile phase A, taking a phosphoric acid aqueous solution with the volume fraction of 0.34-0.45% as a mobile phase B, wherein the column temperature is 28-32 ℃, the flow rate is 0.25-0.36 ml/min, the detection wavelength is 320-400 nm, and the sample introduction amount is 0.5-3 mu l;
the gradient elution conditions were: 0-10 min, wherein the volume fraction of the mobile phase A is 6%, and the volume fraction of the mobile phase B is 94%; for 10-15 min, the volume fraction of the mobile phase A is changed to 6-7%, and the volume fraction of the mobile phase B is changed to 94-93%; 15-20 min, wherein the volume fraction of the mobile phase A is 7%, and the volume fraction of the mobile phase B is 93%; the volume fraction of the mobile phase A is changed to 7-18% and the volume fraction of the mobile phase B is changed to 93-82% in 20-25 min; in 25-30 min, the volume fraction of the mobile phase A is changed to 18-23%, and the volume fraction of the mobile phase B is changed to 82-77%; 30-40 min, the volume fraction of the mobile phase A is changed to 23-45%, and the volume fraction of the mobile phase B is changed to 77-55%; and (3) changing the volume fraction of the mobile phase A to 45-59% and the volume fraction of the mobile phase B to 55-41% in 40-50 min.
2. The method for constructing a UPLC characteristic spectrum of sauropus spatholobus according to claim 1, wherein the chromatographic conditions of the UPLC analysis are as follows: using octadecylsilane chemically bonded silica as a filler, taking methanol as a mobile phase A at a column temperature of 30 ℃, taking a phosphoric acid aqueous solution with a volume fraction of 0.4% as a mobile phase B, and carrying out gradient elution at a flow rate of 0.3ml/min, a detection wavelength of 349nm and a sample injection amount of 1 mul.
3. The method for constructing a UPLC signature spectrum of sauropus spatholobus according to claim 1, wherein the sample solution is prepared by a method comprising the following steps: taking 0.4-0.6 g of sauropus spatulifolius powder, precisely adding 20-30 ml of 45-55% methanol, carrying out ultrasonic treatment for 25-35 minutes, cooling, shaking up, filtering, and taking a subsequent filtrate.
4. A method for identifying a sauropus spatulifolius material is characterized by comprising the following steps:
(1) precisely weighing a sauropus spatulifolius medicinal material to be identified, and preparing a sauropus spatulifolius sample solution to be identified;
(2) precisely sucking a sample solution of the sauropus spatulifolius to be identified, injecting the sample solution into an ultra-high performance liquid chromatograph, and measuring to obtain a UPLC (UPLC) characteristic map of the sauropus spatulifolius to be identified;
(3) comparing the measured UPLC feature spectrum with a UPLC feature spectrum constructed by the method of any one of claims 1 to 3, wherein if the measured UPLC feature spectrum is consistent with the UPLC feature spectrum, the quality of the sauropus spatulifolius is qualified;
the chromatographic conditions of the ultra-high performance liquid chromatograph analysis are as follows: performing gradient elution by using octadecylsilane chemically bonded silica as a filling agent, taking methanol as a mobile phase A, taking a phosphoric acid aqueous solution with the volume fraction of 0.34-0.45% as a mobile phase B, wherein the column temperature is 28-32 ℃, the flow rate is 0.25-0.36 ml/min, the detection wavelength is 320-400 nm, and the sample introduction amount is 0.5-3 mu l;
wherein, the gradient elution conditions are as follows: 0-10 min, wherein the volume fraction of the mobile phase A is 6%, and the volume fraction of the mobile phase B is 94%; for 10-15 min, the volume fraction of the mobile phase A is changed to 6-7%, and the volume fraction of the mobile phase B is changed to 94-93%; 15-20 min, wherein the volume fraction of the mobile phase A is 7%, and the volume fraction of the mobile phase B is 93%; the volume fraction of the mobile phase A is changed to 7-18% and the volume fraction of the mobile phase B is changed to 93-82% in 20-25 min; in 25-30 min, the volume fraction of the mobile phase A is changed to 18-23%, and the volume fraction of the mobile phase B is changed to 82-77%; 30-40 min, the volume fraction of the mobile phase A is changed to 23-45%, and the volume fraction of the mobile phase B is changed to 77-55%; and (3) changing the volume fraction of the mobile phase A to 45-59% and the volume fraction of the mobile phase B to 55-41% in 40-50 min.
5. The method of identifying a sauropus spatholobus stem as recited in claim 4, wherein the chromatographic conditions of the ultra performance liquid chromatography analysis are as follows: using octadecylsilane chemically bonded silica as a filler, taking methanol as a mobile phase A at a column temperature of 30 ℃, taking a phosphoric acid aqueous solution with a volume fraction of 0.4% as a mobile phase B, and carrying out gradient elution at a flow rate of 0.3ml/min, a detection wavelength of 349nm and a sample injection amount of 1 mul.
6. The method of identifying a sauropus herb as recited in claim 4, wherein the solution of the sauropus herb sample to be identified is prepared by a method comprising: taking 0.4-0.6 g of sauropus spatulifolius powder, precisely adding 20-30 ml of 45-55% methanol, carrying out ultrasonic treatment for 25-35 minutes, cooling, shaking up, filtering, and taking a subsequent filtrate.
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