CN109374787B - Construction method and detection method of radix stemonae tuberose medicinal material UPLC characteristic spectrum - Google Patents
Construction method and detection method of radix stemonae tuberose medicinal material UPLC characteristic spectrum Download PDFInfo
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Abstract
The invention relates to a construction method and a detection method of an UPLC (ultra performance liquid chromatography) characteristic spectrum of a radix stemonae. The characteristic map construction method comprises the following steps: respectively preparing reference substance solutions by using chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid as reference substances; preparing a reference solution of a reference medicinal material by using a radix stemonae tuberose reference medicinal material; respectively taking a stemona tuberosa medicinal material and a standard decoction sample, and preparing a test solution and a standard decoction test solution; respectively sucking reference substance solution, test sample solution, and standard decoction test sample solution, injecting into ultra high performance liquid chromatograph, and measuring; comparing the test sample spectrum of the radix Stemonae, and the standard decoction sample test sample spectrum, and calibrating the characteristic peaks of 8 water-soluble components to obtain the UPLC characteristic spectrum of the radix Stemonae. The characteristic map can be used for qualitatively and quantitatively analyzing the quality of the radix stemonae tuberose medicinal material, can ensure the quality of the traditional decoction of the radix stemonae tuberose prepared by adopting the medicinal material, and is also suitable for detecting other preparations containing the radix stemonae tuberose.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a construction method and a detection method of an UPLC (ultra performance liquid chromatography) characteristic spectrum of a radix stemonae.
Background
Stemona is dried root tuber of Stemona sessilifolia (Stemona sessilifolia Miq.), Stemona tuberosa (Stemona japonica Miq.), or Stemona tuberosa (Stemona tuberosa Lour.) belonging to family Stemonaceae. Collected in spring and autumn, removed fibrous root, cleaned, blanched in boiling water or steamed until there is no heart, taken out, and dried in the sun. The stemona root has the functions of moistening lung, descending qi, relieving cough and killing parasites, is an essential medicine for treating the tuberculosis, can inhibit tubercle bacillus, kill tuberculosis worms, radically eliminate the tubercle bacillus, reduce phlegm, relieve cough, nourish yin, moisten lung, fully nourish lung yin, favorably transform the source, recover the function of dispersing and descending the lung, and is mainly used for treating cough, chronic cough, phthisis and the like. The chemical components of stemona plants are mainly alkaloid, and the rest is diphenyl ethylene compounds and other compounds. The content of alkaloid varies with different varieties (Hujun Pinna, Zhangling, Cynanchum hirsutum, etc. comparison of cough relieving action of 3 kinds of radix Stemonae in Chinese pharmacopoeia [ J ]. Chinese traditional medicine J, 2009, 34(23): 3096-. From the distribution of structure types, the stemona tuberosa is mainly composed of I-type and II-type alkaloids, the stemona tuberosa is mainly composed of II-type alkaloids in the upright stemona tuberosa, the stemona tuberosa is secondarily composed of I-type and IV-type alkaloids, and the stemona tuberosa is mainly composed of II-type and IV-type alkaloids in the vine stemona tuberosa. In the aspect of biological activity research, the alkaloid of types I, II and III all have the function of relieving cough, but the type I is the best. From the research result of the literature, the variety of the stemona tuberosa is superior. Radix stemonae has strong clinical application value and wide market demand, and is developed into traditional Chinese medicine formula granules and related compound preparations to be widely used clinically.
The clinical use of traditional Chinese medicine is mainly traditional decoction. The material basis of the traditional Chinese medicine decoction is the basis for preventing and treating diseases under the guidance of the theory of traditional Chinese medicine. The existing legal standard only carries out quantitative control on a single component, and the dose-effect relationship can not comprehensively reflect the integral action of the traditional Chinese medicine components. At the present stage, under the condition that most of effective components of the traditional Chinese medicine are not clear, the establishment of the traditional Chinese medicine fingerprint spectrum/characteristic spectrum can greatly improve the technical level and the technological content of the quality control of the traditional Chinese medicine.
The quality of the radix stemonae cannot be comprehensively reflected because no content measurement item exists in the radix stemonae of 2015 edition in Chinese pharmacopoeia. The establishment of radix stemonae characteristic maps reported in literature researches at present only aims at medicinal raw materials, most index components take fat-soluble components as research objects, and the radix stemonae characteristic maps are mainly used for qualitative identification of authenticity, production places and quality differences of Chinese medicinal materials, and are difficult to comprehensively reflect the quality characteristics of radix stemonae, and cannot reflect the material basic characteristics of traditional Chinese medicine decoction.
Disclosure of Invention
Based on the method, the invention provides a construction method and a detection method of the radix stemonae UPLC medicinal material characteristic spectrum. The constructed characteristic spectrum of the radix stemonae sessilifoliae medicinal material has characteristic peaks of 8 water-soluble components, can quickly and comprehensively realize quality monitoring of a plurality of characteristic components of the radix stemonae sessilifoliae medicinal material, and can reflect the material basic characteristics of the traditional Chinese medicine decoction of the radix stemonae sessilifoliae.
The specific technical scheme is as follows:
a method for constructing a UPLC characteristic spectrum of a radix stemonae tuberose medicinal material comprises the following steps:
preparation of reference solutions: respectively taking chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid as reference substances, dissolving with solvent, and preparing reference substance solution; adding an extraction solvent into a radix stemonae tuberose reference medicinal material, carrying out ultrasonic treatment to obtain an extracting solution, filtering the extracting solution, and taking a subsequent filtrate as a reference substance solution of the reference medicinal material;
preparation of a test solution: adding an extraction solvent into the stemona tuberosa medicinal material, carrying out ultrasonic extraction to obtain an extracting solution I, filtering the extracting solution I, and taking a subsequent filtrate as a test sample solution I; adding an extraction solvent into a standard decoction sample of the radix stemonae tuberose medicinal material, carrying out ultrasonic extraction to obtain an extracting solution II, filtering the extracting solution II, and taking a subsequent filtrate as a test solution II;
and (3) determination: and injecting the reference substance solution of the reference substance, the reference substance solution of the reference medicinal material, the solution I of the test sample and the solution II of the test sample into an ultra-high performance liquid chromatograph for determination, and calibrating a water-solubility common peak to obtain the UPLC characteristic spectrum of the radix stemonae.
In one embodiment, the chromatographic conditions of the ultra-high performance liquid chromatography comprise:
stationary phase: a chromatographic column using octadecylsilane chemically bonded silica as a filler;
mobile phase: taking acetonitrile as a mobile phase A, taking a phosphoric acid aqueous solution with the volume fraction of 0.05% -0.2% as a mobile phase B, and carrying out gradient elution;
the gradient elution is specifically as follows:
the volume fraction of the mobile phase A is increased to 22 percent from 1 percent, and the volume fraction of the mobile phase B is decreased to 78 percent from 99 percent for 0-10 min;
10min-15min, the volume fraction of the mobile phase A is increased to 50% from 22%, and the volume fraction of the mobile phase B is decreased to 50% from 78%;
15min-18min, the volume fraction of the mobile phase A is increased to 90% from 50%, and the volume fraction of the mobile phase B is decreased to 10%;
the volume fraction of the mobile phase A is kept at 90 percent and the volume fraction of the mobile phase B is kept at 10 percent for 18min-20 min;
20min-20.1min, the volume fraction of the mobile phase A is reduced to 1% from 90%, and the volume fraction of the mobile phase B is increased to 99%;
20.1min-23min, mobile phase A keeps volume fraction at 1%, and mobile phase B keeps volume fraction at 99%.
In one embodiment, the column is a WATERS CORTECS T3.
In one embodiment, the chromatographic conditions of the ultra-high performance liquid chromatography further comprise:
the column temperature is 20-25 ℃, the flow rate is 0.25-0.28 ml per minute, and the detection wavelength is 200-220 nm.
In one embodiment, the chromatographic conditions of the ultra-high performance liquid chromatography are: the mobile phase A is acetonitrile, the mobile phase B is phosphoric acid aqueous solution with the volume fraction of 0.1 percent, the column temperature is 25 ℃, the flow rate is 0.25ml per minute, and the detection wavelength is 210 nm.
In one embodiment, the extraction solvent is 50-80% methanol aqueous solution by volume fraction.
In one embodiment, the time of the ultrasonic extraction is 8min to 20 min.
The invention also provides a detection method of the radix stemonae stemona.
The specific technical scheme is as follows:
a detection method of radix stemonae stemona comprises the following steps:
preparation of reference solutions: respectively taking chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid as reference substances, dissolving with solvent, and preparing reference substance solution; adding an extraction solvent into a radix stemonae tuberose reference medicinal material, carrying out ultrasonic treatment to obtain an extracting solution, filtering the extracting solution, and taking a subsequent filtrate as a reference substance solution of the reference medicinal material;
preparing a sample solution to be tested: adding an extraction solvent into a sample to be detected, carrying out ultrasonic extraction to obtain an extracting solution III, filtering the extracting solution III, and taking a subsequent filtrate as a sample solution to be detected;
and (3) determination: and injecting the reference substance solution of the reference substance, the reference substance solution of the reference medicinal material and the sample solution to be detected into an ultra-high performance liquid chromatograph for determination.
In one embodiment, in the preparation of the test solution, the stemona tuberosa medicinal materials comprise stemona tuberosa medicinal materials in different producing areas, the test raw materials of the test solution are from 3 districts or main producing areas with large output of the stemona tuberosa in China, 15 batches of samples are used, and the test solution has sufficient representativeness.
In one embodiment, the chromatographic conditions of the ultra-high performance liquid chromatography comprise:
taking acetonitrile as a mobile phase A, taking a phosphoric acid aqueous solution with the volume fraction of 0.05% -0.2% as a mobile phase B, and carrying out gradient elution;
the gradient elution is specifically as follows:
the volume fraction of the mobile phase A is increased to 22 percent from 1 percent, and the volume fraction of the mobile phase B is decreased to 78 percent from 99 percent for 0-10 min;
10min-15min, the volume fraction of the mobile phase A is increased to 50% from 22%, and the volume fraction of the mobile phase B is decreased to 50% from 78%;
15min-18min, the volume fraction of the mobile phase A is increased to 90% from 50%, and the volume fraction of the mobile phase B is decreased to 10%;
the volume fraction of the mobile phase A is kept at 90 percent and the volume fraction of the mobile phase B is kept at 10 percent for 18min-20 min;
20min-20.1min, the volume fraction of the mobile phase A is reduced to 1% from 90%, and the volume fraction of the mobile phase B is increased to 99%;
20.1min-23min, mobile phase A keeps volume fraction at 1%, and mobile phase B keeps volume fraction at 99%.
In one embodiment, the column is a WATERS CORTECS T3.
In one embodiment, the chromatographic conditions of the ultra-high performance liquid chromatography further comprise:
the column temperature is 20-25 ℃, the flow rate is 0.25-0.28 ml per minute, and the detection wavelength is 200-220 nm.
In one embodiment, the chromatographic conditions of the ultra-high performance liquid chromatography are: the mobile phase A is acetonitrile, the mobile phase B is phosphoric acid aqueous solution with the volume fraction of 0.1 percent, the column temperature is 25 ℃, the flow rate is 0.25ml per minute, and the detection wavelength is 210 nm.
In one embodiment, the extraction solvent is 50-80% methanol aqueous solution by volume fraction.
In one embodiment, the time of the ultrasonic extraction is 8min to 20 min.
Compared with the prior art, the invention has the following beneficial effects:
the method adopts an ultra-high performance liquid chromatography (UPLC) method to construct the characteristic spectrum of the radix stemonae sessilifoliae medicinal material, introduces the characteristic spectrum of the radix stemonae sessilifoliae standard decoction for research, and researches the water-soluble characteristic components shared by the radix stemonae sessilifoliae standard decoction and the radix stemonae medicinal material (8 components with shared characteristic peaks are confirmed by adopting UPLC-MS and related reference substances and are respectively neochlorogenic acid, 3-coumaroylquinic acid, chlorogenic acid, cryptochlorogenic acid, 4-coumaroylquinic acid, stemonine and stemonine), and the components are used as the basis for determining the characteristic spectrum characteristic peaks of the radix stemonae medicinal material, so that the substance transfer from the medicinal material to the decoction can be well represented, the quality of the medicinal material can be well controlled, and the quality of the decoction can also be ensured; meanwhile, the dual contrast of the reference substance and the reference medicinal materials is adopted, so that the problem of durability deviation inherent in the liquid phase condition fingerprint spectrum can be effectively solved, and the method is more comprehensive.
By adopting the chromatographic conditions, the method can realize better separation of 8 phenolic acids in the characteristic spectrum of the radix stemonae japonicae and water-soluble component chromatographic peaks of alkaloid, and has rich characteristic spectrum information and strong characteristics. And the content determination peak (chlorogenic acid) is used as a reference peak, the relative retention time and the relative peak area limit range of other 7 characteristic peaks are specified, the quantification of a plurality of characteristic components is realized, and the integral action concept of the traditional Chinese medicine is met.
Compared with the conventional HPLC method, the UPLC method is more efficient, rapid and environment-friendly; the characteristic map and the method constructed by the invention have good reproducibility, are accurate and reliable, can quickly and comprehensively realize quality monitoring of a plurality of characteristic components of the radix stemonae tuberosa, improve the quality control level of the radix stemonae tuberosa, improve and stabilize the internal quality of the radix stemonae tuberosa, provide raw materials meeting the standard decoction requirements of the radix stemonae tuberosa clinically, and provide important multi-index parameter basis for the relevant preparation process of the radix stemonae tuberosa.
Drawings
FIG. 1 is a characteristic spectrum of a Stemona tuberosa medicinal material at different wavelengths;
FIG. 2 is a characteristic spectrum of radix Stemonae under full wavelength scanning;
FIG. 3 is a characteristic diagram of a Stemona tuberosa medicinal material under different extraction solvents;
FIG. 4 is a characteristic spectrum of 15 batches of radix Stemonae (Peak 1, neochlorogenic acid; Peak 2, 3-coumaroyl quinic acid; Peak 3, chlorogenic acid; Peak 4, cryptochlorogenic acid; Peak 5, 4-coumaroyl quinic acid; Peak 6, stemonine; Peak 7, stemonine);
FIG. 5 is a characteristic spectrum of 15 batches of radix Stemonae standard decoction samples;
FIG. 6 is a characteristic spectrum of a standard decoction sample of Stemona tuberosa;
FIG. 7 is a characteristic spectrum of a sample of a standard decoction of radix Stemonae and radix Stemonae;
FIG. 8 is a characteristic spectrum of radix Stemonae under different chromatographic columns;
FIG. 9 is a characteristic spectrum of radix Stemonae at different column temperatures;
FIG. 10 is a characteristic spectrum of Stemona tuberosa;
FIG. 11 is a reference characteristic spectrum of radix Stemonae;
FIG. 12 is a comparison of the characteristic spectra of radix Stemonae and radix Stemonae;
FIG. 13 is a comparison of the characteristic spectra of 9 batches of samples to be tested and Stemona tuberosa.
Detailed Description
The method for constructing the UPLC characteristic spectrum of the stemona sessilifolia medicinal material and the detection method thereof are further described in detail with reference to specific examples. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Example 1 construction of UPLC characteristic map of Stemona tuberosa
1. Instruments, reagents and reagents
The instrument is shown in table 1, the reagent is shown in table 2, and the reagent is shown in table 3.
TABLE 1
TABLE 2
TABLE 3
2. The source of the medicinal materials is as follows: in the study, 15 batches of crude medicinal materials of the stemona tuberosa were collected, wherein 6 batches of stemona tuberosa from north Hu Yichang, 3 batches of vitex negundo from north Hu, and 6 batches of stemona tetraphylla were collected. Detailed sources are as follows: but not shown. The details are shown in table 4:
TABLE 4
Remarking: the content of chlorogenic acid is determined by a self-made content determination method.
3. Preparation of reference solution for reference substance, reference solution for reference medicinal material and standard decoction
(1) Taking a chlorogenic acid reference substance: taking a proper amount of chlorogenic acid reference substance, precisely weighing, and adding methanol to obtain reference substance solution containing chlorogenic acid 25 μ g per 1ml as reference substance solution.
Chlorogenic acid control: taking a proper amount of chlorogenic acid reference substance, precisely weighing, and adding methanol to obtain reference substance solution containing chlorogenic acid 25 μ g per 1ml as reference substance solution.
Cryptochlorogenic acid reference: taking a proper amount of cryptochlorogenic acid reference substance, precisely weighing, and adding methanol to obtain reference substance solution containing chlorogenic acid 25 μ g per 1ml as reference substance solution.
(2) Reference solution of control drug: taking 0.25g of radix stemonae tuberose reference medicinal material powder, precisely weighing, placing in a 10ml volumetric flask, adding a proper amount of 80% (volume fraction) methanol aqueous solution, carrying out ultrasonic treatment (power 500W and frequency 40kHz) for 10min, taking an extracting solution, cooling the extracting solution, fixing the volume to a scale by using 80% (volume fraction) methanol aqueous solution, shaking up, centrifuging (8000rpm for 3min) to obtain a supernatant, filtering the supernatant by using a 0.22um microporous membrane, and taking a subsequent filtrate, namely the reference medicinal material solution.
(3) Preparation of standard decoction: taking radix Stemonae, removing impurities, cleaning, moistening, and slicing into thick slices; decocting 100g of radix Stemonae decoction pieces in water twice, adding 9 times of water for the first time, soaking for 30 minutes, boiling with strong fire, keeping slightly boiling with slow fire, decocting for 30 minutes, filtering with 350 mesh sieve, cooling the filtrate rapidly in cold water bath, adding 7 times of water for the second time, boiling with strong fire, keeping slightly boiling with slow fire, decocting for 25 minutes, filtering with 350 mesh sieve, and cooling the filtrate rapidly in cold water bath; mixing the two filtrates, concentrating under reduced pressure (temperature: 50, rotation speed: 70-90 r/min, vacuum degree: 0.08-0.1 MPa) to obtain extract with solid content of 15%, subpackaging into penicillin bottles with each bottle of 2ml, vacuum freeze drying, taking out, and rolling aluminum cap to obtain the final product.
4. Chromatographic conditions
(1) Final defined chromatographic conditions: the chromatographic column uses octadecylsilane chemically bonded silica as filler (column length is 10cm, inner diameter is 2.1mm, particle size is 1.6 μm; water CORTECS T3 column is recommended); acetonitrile was used as mobile phase A, and 0.1% (volume fraction) phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was performed as specified in Table 5; the column temperature was 25 ℃; the flow rate is 0.25 mL/min; the detection wavelength was 210 nm.
TABLE 5
(2) Determination of detection wavelength
And (3) inspecting peak conditions of the radix stemonae sessilifoliae medicinal material under different chromatographic wavelengths through full-wavelength scanning, inspecting chromatographic peak information under different wavelengths through wavelength shifting, and selecting the final detection wavelength.
The specific method comprises the following steps:
taking 0.1g of radix stemonae tuberose (batch number: BB07), grinding, precisely weighing, placing in a 10mL measuring flask, adding a proper amount of 80% (volume fraction) methanol aqueous solution, carrying out ultrasonic treatment (power 500W, frequency 40HKz) for 10min, taking an extracting solution, cooling the extracting solution, fixing the volume to a scale by using 80% (volume fraction) methanol aqueous solution, shaking uniformly, filtering by using a 0.22um filter membrane, and taking a subsequent filtrate as a test solution. The sample solution was injected into a chromatograph, and the absorption spectrum in the range of 190 to 400nm was recorded, as shown in fig. 1 and 2.
The results of fig. 1 and 2 show that: the wavelength is 210mm, the chromatographic peak information is the most extensive, the response of each chromatographic peak is large, and 210mm is selected as the detection wavelength.
(3) Selection of mobile phase
Referring to pharmacopoeia and literature data of various countries, acetonitrile and alkaline ammonia solution are used as mobile phases, but the peak shape of the characteristic spectrum is not good. In this example, an acetonitrile-weak acid system was selected and tested, and it was found that a good peak shape was obtained, and when compared with acetonitrile-0.1% phosphoric acid or acetonitrile-0.1% formic acid, it was found that interference at low wavelengths such as 220nm is large with 0.1% formic acid, and the chromatogram with 0.1% phosphoric acid is more desirable. Therefore, acetonitrile-0.1% phosphoric acid system was chosen.
The experimental results are as follows: when 0.1% (volume fraction) phosphoric acid aqueous solution is selected as the mobile phase, the information of the peak is complete and the separation degree is good.
5. Establishment of method for preparing test solution
(1) Examination of extraction solvent
Taking 0.25g of radix stemonae tuberose medicinal material (batch number: BB07), precisely weighing, respectively adding 80% (volume fraction) of methanol aqueous solution, 50% (volume fraction) of methanol aqueous solution, proper amounts of methanol, water and ethanol, carrying out ultrasonic treatment (power 500W and frequency 40kHz) for 10 minutes, taking an extracting solution, cooling the extracting solution, fixing the volume to a scale by using a corresponding solvent, shaking up, centrifuging (8000rpm for 3 minutes) to obtain a supernatant, filtering the supernatant, and taking a subsequent filtrate to obtain test solution extracted by different solvents.
Performing UPLC test on the sample solution extracted by different solvents by adopting the chromatographic conditions of the 4(1) and the 4(1) to obtain the characteristic maps of 5 radix Stemonae, wherein the characteristic maps are shown in figure 3, and the total peak area/sample weighing amount of the characteristic maps are shown in Table 6.
TABLE 6
And (4) conclusion: by comparison, when 80% (volume fraction) methanol aqueous solution and 50% (volume fraction) methanol aqueous solution are used as extraction solvents, the total peak area/sample weighing amount of the obtained characteristic spectrum is larger, the extraction efficiency is better than that of the extraction solvents in the rest 3, and preferably 80% (volume fraction) methanol aqueous solution is used as the extraction solvent, so that the correlation between the radix stemonae japonicae medicinal material and the standard decoction can be observed more conveniently.
(2) Determination of preparation method of test solution
The preparation method of the finally determined test solution comprises the following steps: taking 0.25g of radix stemonae tuberose medicinal material powder, precisely weighing, placing in a 10ml measuring flask, adding a proper amount of 80% (volume fraction) methanol aqueous solution, carrying out ultrasonic treatment (power 500W and frequency 40kHz) for 10min, taking an extracting solution, cooling the extracting solution, fixing the volume to a scale by using 80% (volume fraction) methanol aqueous solution, shaking up, centrifuging (8000rpm for 3min) to obtain a supernatant, filtering the supernatant by using a 0.22um microporous membrane, and taking a subsequent filtrate, namely a sample solution.
6. Determination of common peak of characteristic spectrum of radix stemonae tuberose medicinal material and standard decoction
Taking 15 batches of radix stemonae tuberose medicinal materials, preparing 15 batches of test solution I according to the preparation method of the test solution in 5(2), injecting the 15 batches of test solution I into a liquid chromatograph, and performing sample injection measurement by adopting the chromatographic conditions in 4(1) to obtain 15 batches of radix stemonae tuberose medicinal material characteristic maps. See fig. 4.
Taking another 15 batches of radix stemonae tuberosa standard decoction samples in proper amount, preparing 15 batches of test solution II according to the preparation method of the test solution in 5(2), injecting the 15 batches of test solution II into a liquid chromatograph, and performing sample injection determination by adopting the chromatographic conditions in 4(1) to obtain the characteristic maps of the 15 batches of radix stemonae tuberosa standard decoction samples. See fig. 5.
Experimental results show that the characteristic spectrum of 15 batches of radix stemonae japonicae medicinal materials has 8 characteristic peaks, and the 8 characteristic peaks can be stably transferred from the radix stemonae japonicae medicinal materials to the standard decoction of the radix stemonae japonicae. That is, the characteristic spectrum of the radix stemonae japonicae corresponds to 8 characteristic peaks in the standard decoction characteristic spectrum, so that the 8 characteristic peaks are selected as the common peaks of the radix stemonae japonicae, and the peak 3 (chlorogenic acid) is used as a reference peak for standard research, and the details are shown in fig. 6 and fig. 7.
7. Methodology validation
7.1 precision
(1) Precision of the instrument
The investigation method comprises the following steps: taking radix Stemonae (BB07), preparing a test solution according to the preparation method of the test solution in 5(2), adopting the chromatographic conditions in 4(1), injecting samples for 6 times, and observing the relative retention time of characteristic peaks and the consistency of relative peak areas, wherein the results are shown in tables 7 and 8, and the sample injection amount is 1 mul each time.
TABLE 7 precision results (relative Retention time)
TABLE 8 precision results (relative peak area)
The experimental results are as follows: the relative retention time and the relative peak area of each peak are all within a specified range, the relative retention time of each peak is within a specified range, and the precision of the instrument is good.
(2) Repeatability survey
The investigation method comprises the following steps: a total of 6 samples (lot: BB07) of the same lot were collected and prepared into a test solution according to the method for preparing a test solution described in 5(2), and 1. mu.l of the sample was introduced under the chromatographic conditions described in 4(1) and analyzed, and the results are shown in tables 9 and 10, in which the relative retention times of characteristic peaks and the areas of relative peaks were examined.
TABLE 9 results of repeated experiments (relative retention time)
TABLE 10 results of repeated experiments (relative peak area)
The experimental results are as follows: the relative retention time and the relative peak area of each peak are all in a specified range, and the repeatability of the instrument is good.
(3) Intermediate precision investigation
The investigation method comprises the following steps: the date was changed, the apparatus was changed, and the person was changed, and 6 parts of the same lot (lot: BB07) were sampled, and a sample solution was prepared according to the method for preparing a sample solution in 5(2), and 1. mu.l of the sample solution was subjected to the chromatographic conditions in 4(1), and the results were shown in tables 11 and 12.
TABLE 11 intermediate precision results (relative retention time)
TABLE 12 intermediate precision experimental results (relative peak area)
The experimental results are as follows: the relative retention time RSD of each characteristic peak is less than 0.13%, the relative retention time and the relative peak area of each peak are in a specified range, and the method has good intermediate precision.
7.2 stability
The investigation method comprises the following steps: a sample (lot No. BB07) of the same lot was sampled and prepared into a sample solution according to the method for preparing a sample solution of 5(2), and the sample solution was sampled once at 0, 2, 4, 6, 8, 12, 16, 20, and 24 hours under the chromatographic conditions of 4(1), measured for 24 hours, and 1. mu.l of the sample solution was sampled, and the relative retention time and the correspondence between the relative peak areas of the characteristic peaks were examined, and the results are shown in Table 13 and Table 14:
TABLE 13 results of stability experiments (relative Retention time)
TABLE 14 results of stability experiments (relative peak area)
The experimental results are as follows: the RSD of the relative retention time of each characteristic peak is 0.69 percent at most, the relative retention time and the relative peak area of each peak are in the specified range, and the stability of the test solution in 24 hours is good.
7.3 durability examination
(1) Chromatographic column investigation
The investigation method comprises the following steps: comparing 3 different columns, each being WATERS cortex T3 (100X 2.1mm,1.6 μm); waters ACQUITY BEH C18 (100X 2.1mm,1.7 μm); waters ACQUITY HSS T3 (100X 2.1mm, 1.8 μm).
Taking radix Stemonae (BB07B1), preparing into test solution according to the preparation method of the test solution in 5(2), and injecting 1 μ l sample under the chromatographic conditions in 4 (1). The chromatogram (FIG. 8) was recorded comparing the separation of peak No. 6 and peak No. 7 with the theoretical plate number of chlorogenic acid peak, and the results are shown in Table 15.
The experimental results are as follows: the water WATERS cortex 3 (100X 2.1mm,1.6 μm) with the best separation and the highest theoretical plate count was chosen.
(2) Selection of column temperature
The investigation method comprises the following steps: the separation effect of the sample was examined with a WATERS CORTECS T3 (2.1X 100mm,1.6 μm) column at a column temperature of 25 ℃ and 20 ℃ respectively.
Taking radix Stemonae (BB07), preparing into test solution according to the preparation method of the test solution in 5(2), and injecting 1 μ l of sample under the chromatographic conditions in 4 (1). The chromatogram was recorded (figure 9),
the experimental results are as follows: the column temperature has a certain influence on the separation effect, the peak is stable between 6 and 7 minutes at 25 ℃, and the column temperature is 25 ℃ or below in the embodiment.
(3) Investigation of flow Rate
The separation of the samples was examined at flow rates of 0.22ml/min, 0.25ml/min and 0.28ml/min using a WATERS CORTECS T3 (2.1X 100mm,1.6 μm) column.
Taking radix Stemonae (BB07), preparing into test solution according to the preparation method of the test solution in 5(2), and injecting 1 μ l of sample under the chromatographic conditions in 4 (1). The chromatogram (FIG. 10) was recorded comparing the separation of peak No. 6 and peak No. 7 with the theoretical plate number of chlorogenic acid peak, and the results are shown in Table 16.
And (4) conclusion: the flow rates of 0.25ml/min and 0.28ml/min are better separated, and the flow rate of 0.25ml/min is selected in the embodiment.
TABLE 16
8. Determination of radix stemonae officinalis medicinal material characteristic map
(1) Analyzing the characteristic spectrum of 15 batches of the stemona tuberosa medicinal material, calculating the relative retention time and the relative peak area of each peak by taking the peak 3 as a reference peak, and obtaining the experimental result shown in tables 17-20.
TABLE 1715 batches of radix Stemonae medicinal materials with retention time
TABLE 1815 relative retention time of radix Stemonae batches
TABLE 2-1915 Peak area of Stemona root batch
TABLE 2015 batch of Stemona sessilifolia drug relative peak area
Analysis and discussion of results
The result shows that the more stable characteristic spectrum of 15 batches of the radix stemonae japonicae medicinal materials has 8 common peaks which are consistent with the common peaks of the characteristic spectrum of the standard decoction of the radix stemonae japonicae.
And taking the contrast peak 3 as a reference peak (S peak), calculating the relative retention time of each characteristic peak and the S peak, wherein the RSD value of the characteristic relative retention time of 15 batches of the stemona tuberosa medicinal materials is 0.35-1.87%. Meets the standard requirements of the characteristic spectrum of the radix stemonae tuberose medicinal material. The average of the relative retention times of the peaks is as follows: 0.83 (peak 1), 0.95 (peak 2), 1.04 (peak 4), 1.18 (peak 5), 1.48 (peak 6), 1.52 (peak 7), 1.66 (peak 8); calculating the relative peak area of each characteristic peak and peak 3, wherein the RSD of the relative peak area of the characteristic peak of 15 batches of radix stemonae tuberosa medicinal materials is 0.233-2.662%, and the results show that the relative peak areas of the characteristic peaks of the radix stemonae tuberosa medicinal materials in different producing areas have certain difference, the relative peak area of peak 1 is 0.921-1.640, the relative peak area of peak 2 is 2.485-5.626, the relative peak area of peak 4 is 0.075-0.641, the relative peak area of peak 5 is 0.377-1.902, the relative peak area of peak 6 is 2.223-12.351, the relative peak area of peak 7 is 0.619-3.431, and the relative peak area of peak 8 is 0.888-2.193.
The method provides excellent and stable raw material medicines for the preparation of standard decoction and formula granules of the stemona japonica, and sets limit standards for the relative peak areas of characteristic peaks of characteristic spectra of the stemona japonica. According to the fluctuation range of the relative peak areas of the peak 3 and other 7 peaks of the radix stemonae sessilifoliae medicinal material in 15 batches of different producing areas, the representativeness of the samples in 15 batches of different producing areas is considered, and the relative peak areas of all characteristic peaks are taken as the lowest value and the highest value, so the characteristic spectrum standard of the provisional radix stemonae sessilifoliae medicinal material is as follows: the characteristic spectrum of the test sample should present 8 characteristic peaks, and should correspond to 8 characteristic peaks in the chromatogram of the reference substance of the reference medicinal material; 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 value is as follows: 0.83 (peak 1), 0.95 (peak 2), 1.04 (peak 4), 1.18 (peak 5), 1.48 (peak 6), 1.52 (peak 7), 1.66 (peak 8); calculating the relative peak area of each characteristic peak and the peak 3, wherein the relative peak area is in a specified range: 0.921 to 1.640 (Peak 1), 2.485 to 5.626 (Peak 2), 0.075 to 0.641 (Peak 4), 0.377 to 1.902 (Peak 5), 2.223 to 12.351 (Peak 6), 0.619 to 3.431 (Peak 7), 0.888 to 2.193 (Peak 8).
(2) Drawing up standard of characteristic map
Matching the characteristic spectrums of 15 batches of the radix stemonae sessilifoliae medicines by using a traditional Chinese medicine chromatography fingerprint similarity evaluation system to generate a reference spectrum, and establishing the reference characteristic spectrum of the radix stemonae sessilifoliae medicines as shown in figure 11.
The standard of the temporary stemona tuberosa medicinal material characteristic map is as follows: 7 characteristic peaks are presented in the chromatogram of the test sample, the peak corresponding to the reference peak of chlorogenic acid is an S peak, the relative retention time of each characteristic peak and the S peak is calculated, the relative retention time is within +/-10% of a specified value, and the specified value is as follows: 0.83 (peak 1), 0.95 (peak 2), 1.04 (peak 4), 1.18 (peak 5), 1.48 (peak 6), 1.52 (peak 7), 1.66 (peak 8); calculating the relative peak area of each characteristic peak and the peak 3, wherein the relative peak area is in a specified range: 0.921 to 1.640 (Peak 1), 2.485 to 5.626 (Peak 2), 0.075 to 0.641 (Peak 4), 0.377 to 1.902 (Peak 5), 2.223 to 12.351 (Peak 6), 0.619 to 3.431 (Peak 7), 0.888 to 2.193 (Peak 8). See table 21 for details.
TABLE 21
9. Identification of characteristic peaks
And performing chemical identification on the characteristic peak on the characteristic spectrum of the radix stemonae sessilifoliae by using an LC-MS technology to determine the chemical components of the radix stemonae sessilifoliae.
9.1 materials and methods
The materials are shown in Table 22.
TABLE 22
9.2 instruments and reagents
The instrument comprises the following steps: SYNAPT G2HDMS ultra-high performance liquid phase time-of-flight high resolution mass spectrometry system (Waters Corporation, Milford, MA, USA), and data processing system is MarkerLynx 4.1 workstation (Waters, Manchester, U.K.), AB135-S electronic analytical balance (Mettler-Toledo), KQ-118B ultrasonic oscillator (Kunshan ultrasonic instruments, Inc.).
Reagent: chromatographic acetonitrile and methanol were purchased from j.t.baker (phillips burg, NJ, USA), chromatographic grade formic acid, phosphoric acid, leucine enkephalin from Sigma Aldrich (MO, USA), experimental ultrapure water (18.2M Ω) was prepared using a Milli-Q water purification system (Millipore, France), and all other reagents were used in analytical purity.
9.3 Experimental methods
Preparing reference substance solution of reference substance and reference substance solution of reference medicinal material: the same as 3.
Preparation of a test solution: the same as 5 (2).
Liquid phase conditions: the same as in 4 (1).
Mass spectrum conditions: the mass spectra were obtained using a Waters SYNAPT G2HDMS system. Nitrogen is used as atomization and taper hole gas of the mass spectrum ion source; electrospray ionization positive and negative ion modes; capillary voltage: 3.0KV (positive)Ion mode)/2.5 KV (negative ion mode); taper hole voltage: 40V; extraction taper hole voltage: 3V; ion source temperature: 100 ℃; desolventizing gas temperature: 400 ℃ (positive ion mode)/350 ℃ (negative ion mode); reverse taper hole air flow: 50L/h; desolventizing air flow rate: 600L/h (positive ion mode)/600L/h (negative ion mode); collision airflow rate: 0.5 mL/min; scanning time: 0.5 s; scanning time interval: 0.02 s; mass to charge ratio range: 50-1200 m/z; the data acquisition form is as follows: a cutium; sensitivity: normal; dynamic range: extended; locking mass number: [ M + H ]]+＝556.2771；[M-H]–＝554.2615。
The experimental results are as follows: the optimized UPLC-Q-TOF/MS analysis method is adopted to detect the radix stemonae tuberose medicinal material sample, and 27 compounds are identified in total by comparing chromatographic peak retention behavior, accurate molecular weight and MSE fragment information of the compounds with standard substances, wherein the compounds mainly comprise 11 phenolic acid compounds and 16 alkaloid compounds. The comparison and verification of the chromatographic retention behavior of the chlorogenic acid, the neochlorogenic acid and the cryptochlorogenic acid with the reference substance and the accurate molecular weight information of the mass spectrum are carried out.
9.5 assignment of characteristic peaks
Finally, the common characteristic peak attribution is determined as follows:
Example 2 detection method of Stemona tuberosa
Stemona is dried root tuber of Stemona sessilifolia (Miq.) Miq, Stemona japonica (Bl.) Miq, or Stemona tuberosa Lour. The embodiment provides an identification method of a radix stemonae stemona medicinal material, which comprises the following steps:
(1) the chromatographic conditions were the same as in 4(1) above.
(2) Preparation of sample solution to be tested
The sample to be detected is a reference medicinal material purchased from China institute for verification, 0.25g of radix stemonae erecta powder is taken, precisely weighed and placed in a 10ml volumetric flask, a proper amount of 80% (volume fraction) methanol aqueous solution is added, ultrasonic treatment is carried out for 10min at the power of 500W and the frequency of 40kHz, an extracting solution is taken, the extracting solution is cooled, the volume is fixed to a scale by 80% (volume fraction) methanol aqueous solution, shaking is carried out uniformly, centrifugation is carried out for 3min at 8000rpm, a supernatant is obtained, the supernatant is filtered by a 0.22um microporous membrane, and a continuous filtrate is taken, namely the sample solution to be detected.
(3) Measurement of
Precisely sucking the sample solution to be detected, injecting into an ultra high performance liquid chromatograph, and measuring to obtain a characteristic spectrum, which is compared with fig. 11, as shown in fig. 12.
The results showed that the Stemona sessilifolia has fewer peaks 1 and 2, and the characteristic peaks can be distinguished from each other by the number of the characteristic peaks. The established characteristic spectrum of the radix stemonae tuberose medicinal material can distinguish the radix stemonae tuberose medicinal material from the radix stemonae stemona sessilifolia medicinal material. Provides a foundation for the research of the stemona tuberosa, and enhances the overall quality control of the raw materials used in the stemona tuberosa formula granules.
Example 4 detection method of Stemona tuberosa
The embodiment provides a method for detecting a radix stemonae stemona, which comprises the following steps:
(1) the chromatographic conditions were the same as in 4(1) above.
(2) Preparation of sample solution to be tested
The method comprises the following steps of taking 9 batches of medicinal materials (BB01B 1-BB 06B1 and BB16B 1-BB 18B1), precisely weighing 0.25g of powder of the 9 batches of medicinal materials, placing the powder into a 10ml volumetric flask, adding an appropriate amount of 80% (volume fraction) methanol aqueous solution, carrying out ultrasonic treatment (power 500W and frequency 40kHz) for 10min, taking an extracting solution, cooling the extracting solution, fixing the volume of the extracting solution to a scale by using the 80% (volume fraction) methanol aqueous solution, shaking up, centrifuging (8000rpm and 3min) to obtain a supernatant, filtering the supernatant by using a 0.22-micrometer microfiltration membrane, and taking a subsequent filtrate to obtain a sample solution to be detected.
(3) Measurement of
Precisely sucking the sample solution to be detected, injecting the sample solution into an ultra high performance liquid chromatograph, and measuring to obtain a characteristic spectrum, as shown in figure 13.
The result shows that 2 characteristic peaks (peak 6 and peak 7) are reduced in the characteristic maps of 9 batches of samples to be detected, which indicates that the radix stemonae japonicae medicinal material characteristic map established in the example 1 can effectively distinguish the radix stemonae japonicae samples, and the medicinal materials with the batch numbers of BB01B 1-BB 06B1 and BB16B 1-BB 18B1 are fake products.
Example 5 detection method of Stemona tuberosa
The embodiment provides a method for detecting a radix stemonae stemona, which comprises the following steps:
(1) the chromatographic conditions were the same as in 4(1) of example 1.
(2) The reference solution was prepared as in 3 of example 1.
(3) Preparation of sample solution to be tested
Taking 0.25g of a sample to be detected, precisely weighing, placing in a 10ml measuring flask, adding a proper amount of 80% methanol, carrying out ultrasonic treatment (power 500W and frequency 40kHz) for 10 minutes, taking an extracting solution, cooling the extracting solution, fixing the volume to the scale with 80% methanol, shaking up, centrifuging (8000rpm for 3 minutes) to obtain a supernatant, filtering the supernatant with a 0.22um microporous membrane, and taking a subsequent filtrate to obtain a sample solution to be detected.
(4) Assay method
Precisely absorbing the reference substance solution and the sample solution to be detected by 1 mu l respectively, injecting the solution into a liquid chromatograph, measuring to obtain a characteristic spectrum, and settling the relative retention time and the relative peak area of the characteristic spectrum of the sample to be detected.
TABLE 23 relative retention time and relative peak area of the characteristic profile of the test article
The data result shows that the sample has 8 identical characteristic peaks, and the relative peak area and the relative retention time of the sample are both in the range specified by the standard, so that the quality of the batch of the radix stemonae tuberose medicinal material is qualified, the quality of the batch of the radix stemonae tuberose medicinal material is stable, and the use requirement of clinical decoction is met.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A construction method of a radix stemonae tuberose medicinal material UPLC characteristic spectrum is characterized by comprising the following steps:
preparation of reference solutions: respectively taking chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid as reference substances, dissolving with solvent, and preparing reference substance solution; adding an extraction solvent into a radix stemonae tuberose reference medicinal material, carrying out ultrasonic treatment to obtain an extracting solution, filtering the extracting solution, and taking a subsequent filtrate as a reference substance solution of the reference medicinal material;
preparation of a test solution: adding an extraction solvent into the stemona tuberosa medicinal material, carrying out ultrasonic extraction to obtain an extracting solution I, filtering the extracting solution I, and taking a subsequent filtrate as a test sample solution I; adding an extraction solvent into a standard decoction sample of the radix stemonae tuberose medicinal material, carrying out ultrasonic extraction to obtain an extracting solution II, filtering the extracting solution II, and taking a subsequent filtrate as a test solution II;
and (3) determination: injecting the reference substance solution of the reference substance, the reference substance solution of the reference medicinal material, the solution I of the test sample and the solution II of the test sample into an ultra-high performance liquid chromatograph for determination, and calibrating a water-solubility common peak to obtain an UPLC characteristic spectrum of the radix stemonae sessilifoliae medicinal material;
the water-soluble common peak comprises characteristic peaks of neochlorogenic acid, 3-coumaryl quinic acid, chlorogenic acid, cryptochlorogenic acid, 4-coumaryl quinic acid, stemonine and stemonine;
the chromatographic conditions of the ultra-high performance liquid chromatography comprise:
stationary phase: a chromatographic column using octadecylsilane chemically bonded silica as a filler;
mobile phase: taking acetonitrile as a mobile phase A, taking a phosphoric acid aqueous solution with the volume fraction of 0.05% -0.2% as a mobile phase B, and carrying out gradient elution;
the gradient elution is specifically as follows:
the volume fraction of the mobile phase A is increased to 22 percent from 1 percent, and the volume fraction of the mobile phase B is decreased to 78 percent from 99 percent for 0-10 min;
10min-15min, the volume fraction of the mobile phase A is increased to 50% from 22%, and the volume fraction of the mobile phase B is decreased to 50% from 78%;
15min-18min, the volume fraction of the mobile phase A is increased to 90% from 50%, and the volume fraction of the mobile phase B is decreased to 10%;
the volume fraction of the mobile phase A is kept at 90 percent and the volume fraction of the mobile phase B is kept at 10 percent for 18min-20 min;
20min-20.1min, the volume fraction of the mobile phase A is reduced to 1% from 90%, and the volume fraction of the mobile phase B is increased to 99%;
20.1min-23min, mobile phase A keeps volume fraction at 1%, and mobile phase B keeps volume fraction at 99%.
2. The method of claim 1, wherein the chromatography column is water cutecs T3.
3. The construction method according to claim 1, wherein the chromatographic conditions of the ultra-high performance liquid chromatography further comprise:
the column temperature is 20-25 ℃, the flow rate is 0.25-0.28 ml per minute, and the detection wavelength is 200-220 nm.
4. The construction method according to claim 3, wherein the chromatographic conditions of the ultra-high performance liquid chromatography further comprise:
the column temperature was 25 ℃, the flow rate was 0.25ml per minute, and the detection wavelength was 210 nm.
5. The construction method according to any one of claims 1 to 4, wherein the extraction solvent is a methanol aqueous solution with a volume fraction of 50-80%, and the ultrasonic extraction time is 8-20 min.
6. A detection method of radix stemonae stemona is characterized by comprising the following steps:
preparation of reference solutions: respectively taking chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid as reference substances, dissolving with solvent, and preparing reference substance solution; adding an extraction solvent into a radix stemonae tuberose reference medicinal material, carrying out ultrasonic treatment to obtain an extracting solution, filtering the extracting solution, and taking a subsequent filtrate as a reference substance solution of the reference medicinal material;
preparing a sample solution to be tested: adding an extraction solvent into a sample to be detected, carrying out ultrasonic extraction to obtain an extracting solution III, filtering the extracting solution III, and taking a subsequent filtrate as a sample solution to be detected;
and (3) determination: injecting the reference substance solution of the reference substance, the reference substance solution of the reference medicinal material and the sample solution to be detected into an ultra-high performance liquid chromatograph for measurement, and comparing the measurement spectrum of the sample solution to be detected with the UPLC characteristic spectrum of the radix stemonae tuberosa medicinal material according to any one of claims 1-5;
the chromatographic conditions of the ultra-high performance liquid chromatography comprise:
stationary phase: a chromatographic column using octadecylsilane chemically bonded silica as a filler;
mobile phase: taking acetonitrile as a mobile phase A, taking a phosphoric acid aqueous solution with the volume fraction of 0.05% -0.2% as a mobile phase B, and carrying out gradient elution;
the gradient elution is specifically as follows:
the volume fraction of the mobile phase A is increased to 22 percent from 1 percent, and the volume fraction of the mobile phase B is decreased to 78 percent from 99 percent for 0-10 min;
10min-15min, the volume fraction of the mobile phase A is increased to 50% from 22%, and the volume fraction of the mobile phase B is decreased to 50% from 78%;
15min-18min, the volume fraction of the mobile phase A is increased to 90% from 50%, and the volume fraction of the mobile phase B is decreased to 10%;
the volume fraction of the mobile phase A is kept at 90 percent and the volume fraction of the mobile phase B is kept at 10 percent for 18min-20 min;
20min-20.1min, the volume fraction of the mobile phase A is reduced to 1% from 90%, and the volume fraction of the mobile phase B is increased to 99%;
20.1min-23min, mobile phase A keeps volume fraction at 1%, and mobile phase B keeps volume fraction at 99%.
7. The method of claim 6, wherein the chromatographic column is a WATERS CORTECS T3.
8. The detection method according to claim 6, wherein the chromatographic conditions of the ultra-high performance liquid chromatography further comprise:
the column temperature is 20-25 ℃, the flow rate is 0.25-0.28 ml per minute, and the detection wavelength is 200-220 nm.
9. The detection method according to claim 8, wherein the chromatographic conditions of the ultra-high performance liquid chromatography further comprise:
the column temperature was 25 ℃, the flow rate was 0.25ml per minute, and the detection wavelength was 210 nm.
10. The detection method according to any one of claims 6 to 9, wherein the extraction solvent is a methanol aqueous solution with a volume fraction of 50% to 80%, and the ultrasonic extraction time is 8min to 20 min.
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