CN114705779A - Method for establishing quantitative fingerprint of fructus evodiae standard decoction - Google Patents
Method for establishing quantitative fingerprint of fructus evodiae standard decoction Download PDFInfo
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Abstract
The invention discloses a method for establishing a quantitative fingerprint of a fructus evodiae standard decoction, which relates to the field of establishment of fingerprints and comprises the following steps: (1) preparing a reference substance solution; (2) preparing a test solution; (3) ultra-high performance liquid chromatography analysis; (4) establishing a quantitative fingerprint, introducing the multi-batch liquid chromatogram into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, matching common peaks, generating a comparison characteristic spectrum, selecting a reference peak, calculating the relative retention time of each characteristic peak, and establishing the quantitative fingerprint of the evodia rutaecarpa standard decoction by combining the content determination of 3 components of limonin, evodiamine and rutaecarpine. The detection of the characteristic spectrum and the content of the standard decoction of the fructus evodiae can be realized simultaneously through one-time analysis, the detection time can be effectively reduced, the working efficiency is improved, and the quality and consistency evaluation of the fructus evodiae formula particles is guaranteed.
Description
Technical Field
The invention relates to the field of establishment of fingerprint spectrums, in particular to a method for establishing a quantitative fingerprint spectrum of a standard decoction of fructus evodiae.
Background
The fructus evodiae is dry and nearly mature fruit of Rutaceae plant Evodia rutaecarpa (Juss.) Benth, Gekko Swinhonis E.rutaceae arpa (Juss.) Benth. var. officinalis (Dode) Huang or Evodia rutaecarpa (Juss.) Benth. var. borheielii (Dode) Huang, and has the effects of dispelling cold, relieving pain, lowering adverse qi, relieving vomit, supporting yang and relieving diarrhea, and can be used for treating syncope headache, cold hernia abdominal pain, cold-dampness beriberi, menstrual abdominal pain, abdominal distention and pain, vomiting, acid regurgitation, and morning diarrhea. Modern researches show that the fructus evodiae mainly contains alkaloids, bitter gourmet powder, volatile oil, flavonoids and the like, wherein the evodiamine and rutaecarpine are main active ingredients. Modern pharmacological research shows that the medicine has the functions of relieving pain, resisting inflammation, ulcer, bacteria and tumor, protecting cardiac vascular system, etc. (Liuli. Chinese herbal medicine 2020,51(10): 2689-2702).
The traditional Chinese medicine formula particle is prepared by carrying out water extraction, separation, concentration, drying and granulation on single traditional Chinese medicine decoction pieces under the guidance of the traditional Chinese medicine theory, and can be taken by patients after being prepared according to the clinical prescription of the traditional Chinese medicine [ national drug administration, bulletin about finishing the test-point work of the traditional Chinese medicine formula particle, 2021-09-20 ]. The decoction pieces are a new decoction piece use form which is suitable for the requirements of modern society, have the advantages of no decoction, easy taking, accurate dosage, safety, sanitation, convenient carrying and the like, not only meet the clinical medication characteristics of diagnosis and treatment based on syndrome differentiation and addition and subtraction according to symptoms of traditional Chinese medicine, but also are beneficial to the standardization of pharmacy preparation and traditional Chinese medicine, and are widely used in modern clinic. The technical requirements for quality control and standard formulation of the traditional Chinese medicine formula granules are defined in the specification that the traditional Chinese medicine formula granules can bear the safety and the effectiveness of traditional Chinese medicine decoction pieces, and standard decoction is required to be used as a bridge. Therefore, the production process and quality standard research of the traditional Chinese medicine formula granule should use standard decoction as a reference, the standard decoction is a material standard for measuring whether the traditional Chinese medicine formula granule is basically consistent with the corresponding clinical decoction of the traditional Chinese medicine decoction pieces, and the quality standard research of the standard decoction is particularly important for the quality control of the traditional Chinese medicine formula granule.
At present, the national pharmacopoeia commission has published the national standard of the evodia rutaecarpa formula particle, the medicinal material source is evodia rutaecarpa base, the quality control method specified in the standard comprises two items of characteristic spectrum and content measurement, when the sample quality is detected, two instruments and two methods are respectively adopted to carry out the quality control on the evodia rutaecarpa formula particle, and the analysis time and the analysis efficiency are restricted. In addition, Zhanghongmei and the like establish UPLC quantitative fingerprint spectrums of the medicinal evodia fruit, and carry out quality evaluation on the medicinal evodia fruit from different producing areas through similarity and content measurement of limonin, evodiamine and rutaecarpine [ Zhanghongmei, Chinese journal of experimental prescriptions, 2014,20(17):69-73 ], but the matching in the text determines 9 common peaks in the fingerprint spectrums, and does not carry out peak identification on the 9 peaks. Chiffon and the like establish a quantitative fingerprint method of the medicinal evodia fruit by adopting an HPLC technology, match 13 common chromatographic peaks and perform mass spectrum qualitative determination, and perform content determination on 4 components of evodiamine, rutaecarpine, dehydroevodiamine and narcissus, but the analysis time and the analysis efficiency are influenced by overlong analysis time of the adopted HPLC technology [ chiffon Chinese herbal medicine, 2021,52(14): 4341-plus 4347 ].
In the existing standards and documents, the standard decoction of fructus evodiae (stone tiger) lacks an effective quality control method, and is difficult to provide reference for the quality control of the fructus evodiae (stone tiger) formula granules. The national standard method of the fructus evodiae formula particle with similar primitive has the problems of long analysis time, low analysis efficiency, incomplete quality control method and the like, and the quality control method of the fructus evodiae medicinal material reported in the literature is mostly alcohol extraction and has long analysis time, so that the quality control requirement of the standard decoction of the fructus evodiae (the caulis spatholobi) cannot be met.
Disclosure of Invention
The invention aims to at least solve one of the technical problems in the prior art, so that the invention establishes a method for quickly and efficiently detecting the quality of the standard decoction of evodia rutaecarpa (stone tiger) by using the UPLC technology, and provides guarantee for the quality and consistency evaluation of the standard decoction and the formula granules of the evodia rutaecarpa (stone tiger).
The technical solution of the invention is as follows:
a method for establishing a quantitative fingerprint of a standard evodia decoction comprises the following steps:
(1) preparing a reference solution;
(2) preparing a plurality of batches of evodia rutaecarpa standard decoction test solution;
(3) analyzing the multiple batches of evodia rutaecarpa standard decoction sample solutions in the step (2) in an ultra-high performance liquid chromatography analyzer respectively to obtain multiple batches of liquid chromatogram maps;
(4) and (3) establishing a quantitative fingerprint, namely introducing the multi-batch liquid chromatogram in the step (3) into software of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, matching common peaks, generating a reference characteristic spectrum, selecting the reference peak, calculating the relative retention time of each characteristic peak, and simultaneously determining the content of 3 components of limonin, evodiamine and rutaecarpine in the same ultra-high performance liquid chromatogram analyzer to establish the quantitative fingerprint of the evodia rutaecarpa standard decoction.
Preferably, the method specifically comprises the following steps:
a: establishing a fingerprint spectrum method:
step a 1: preparation of control solutions: taking appropriate amount of chlorogenic acid and hyperoside reference substance, precisely weighing, and preparing into mixed reference substance solution containing chlorogenic acid and hyperoside with 50-100% methanol (V/V) or 50-100% ethanol (V/V);
step a 2: preparation of a test solution: taking freeze-dried powder of fructus evodiae standard decoction, grinding, precisely weighing, placing into a conical flask with a plug, adding 50-100% methanol solvent or 50-100% ethanol solvent, wherein the ratio of material to liquid (ratio of sample to solvent) is 1: 25-1: 100g/mL, sealing, weighing, refluxing or ultrasonically treating for 10-30min, cooling to room temperature, adding corresponding solvent to compensate weight loss, shaking, centrifuging, collecting supernatant, and filtering to obtain fructus evodiae standard decoction sample solution;
step a 3: ultra Performance Liquid Chromatography (UPLC) analysis: preparing more than 3 different batches of evodia rutaecarpa standard decoction test solution according to the method in the step 2, respectively sucking the reference solution and the test solution, respectively injecting into an ultra-high performance liquid chromatograph, and respectively recording liquid chromatogram;
step a 4: establishing a fingerprint spectrum: introducing the liquid chromatogram of at least more than 3 standard decoction samples of fructus evodiae in different batches into 'Chinese medicine chromatogram characteristic map similarity evaluation system software', matching common peaks, generating a reference characteristic map, and identifying the common peaks; and calculating the relative retention time of each characteristic peak by taking chlorogenic acid and hyperin as reference peaks;
b: establishment of a content determination method:
step b 1: preparation of control solutions: taking appropriate amount of limonin, evodiamine and rutaecarpine as reference substances, precisely weighing, adding 50-100% methanol (V/V) or 50-100% ethanol (V/V) to obtain reference substance mixed solution containing limonin, evodiamine and rutaecarpine;
step b 2: preparing a test solution: taking freeze-dried powder of fructus evodiae standard decoction, grinding, precisely weighing, placing into a conical flask with a plug, adding 50-100% methanol solvent or 50-100% ethanol solvent, wherein the ratio of material to liquid (ratio of sample to solvent) is 1: 25-1: 100g/mL, sealing, weighing, refluxing or ultrasonically treating for 10-30min, cooling to room temperature, adding corresponding solvent to compensate weight loss, shaking, centrifuging, collecting supernatant, and filtering to obtain fructus evodiae standard decoction sample solution;
step b 3: ultra Performance Liquid Chromatography (UPLC) analysis: preparing more than 3 different batches of evodia rutaecarpa standard decoction test solution according to the method in the step 2, respectively sucking the reference solution and the test solution, respectively injecting the reference solution and the test solution into an ultra-high performance liquid chromatograph, and respectively measuring the corresponding peak areas of limonin, evodiamine and rutaecarpine in the reference solution and the test solution;
step b 4: establishment of a content determination method: calculation of Evodia rutaecarpa by external standard methodThe content of limonin, evodiamine and rutaecarpine in standard decoction lyophilized powder for the dogwood fruit is specifically determined by an external standard method (refer to 'Chinese pharmacopoeia' 2020 edition):
wherein A isxThe peak area of a component to be measured in the sample is shown, Rf represents a correction factor, and m represents the sample weighing amount;
as a preferable scheme of the present invention, in the step a1, the concentrations of the prepared mixed reference solution are 20-40 μ g chlorogenic acid and 10-30 μ g hyperoside per 1 mL.
In a preferred embodiment of the present invention, the chromatographic conditions of the ultra performance liquid chromatography analysis in a3 and b3 are: the chromatographic column is Waters Acquity UPLC BEH Shield RP18, the inner diameter is 2.1mm, the column length is 100mm, the particle size is 1.7 mu m, the column temperature is 30-40 ℃, the flow rate is 0.3-0.5mL/min, the sample injection amount is 0.5-2 mu L, the detection wavelength is 210-280nm, the mobile phase A is acetonitrile, the mobile phase B is phosphoric acid solution with the volume concentration of 0.05-0.2%, and the detection data of the whole elution time period are collected by adopting a gradient elution mode.
As a preferable embodiment of the present invention, the conditions of the gradient elution are: 0-4 min: 4% a, 96% B; 4-9 min: 4-11% of A, 89-96% of B; 9-10 min: 11% a, 89% B; 10-14 min: 11-18% of A, 82-89% of B; 14-20 min: 18% a, 82% B; 20-22 min: 18-35% of A, 65-82% of B; 22-28 min: 35-37% of A, 63-65% of B; 28-34 min: 37-39% of A and 61-63% of B; 34-35 min: 39-80% of A, 20-61% of B; 35-38 min: 80% of A, 20% of B, the% are volume percentages.
In the step a4, 11 common peaks of the reference characteristic spectrum are labeled according to the sequence from left to right in the spectrum, wherein the peak 5 is cryptochlorogenic acid, the peak 6 is chlorogenic acid, the peak 8 is dehydroevodiamine, the peak 9 is hyperoside, the peak 10 is narcissin, and the peak 11 is evodiamine.
As a preferred embodiment of the invention, the peak 6 corresponding to the chlorogenic acid control solution is the S1 peak, the peak 9 corresponding to the hyperin control solution is the S2 peak, the relative retention times of the peaks 1,2, 3, 4, 5, 7 are calculated by the peak S1, the relative retention times of the peaks 8, 10, 11 are calculated by the peak S2, and the relative retention times of the characteristic peaks are within +/-10% of the specified value; the relative retention time specified values are: peak 1, 0.28; peak 2, 0.36; peak 3, 0.44; peak 4, 0.83; peak 5, 0.94; peak 6, 1; peak 7, 1.15; peak 8, 0.85; peak 9, 1; peak 10, 1.15; peak 11, 1.76.
As a preferable scheme of the invention, in the step b1, the concentrations of the prepared mixed reference substance solution are 8-200 μ g of limonin, 2-70 μ g of evodiamine and 1-15 μ g of rutaecarpine in each 1 mL.
In the b, the content determination standard of the standard decoction freeze-dried powder of the fructus evodiae is that each 1g of the standard decoction freeze-dried powder contains limonin (C)26H30O8) 2.3-11.4mg of evodiamine (C)19H17N3O) and rutaecarpine (C)18H13N3O) in a total amount of 0.8 to 4.8 mg.
The invention has the beneficial effects that:
1. the UPLC quantitative fingerprint spectrum method established by the evodia rutaecarpa (Sphaeranthus altissima) standard decoction can comprehensively reflect the overall characteristics of the evodia rutaecarpa (Sphaeranthus altissima) standard decoction, can quickly, comprehensively and accurately evaluate the quality of the evodia rutaecarpa (Sphaeranthus altissima) standard decoction, and can be used as one of quality control indexes of the evodia rutaecarpa (Sphaeranthus altissima) standard decoction;
2. the method disclosed by the invention is simple to operate, the chromatographic condition is easy to realize, the precision is high, the stability and the reproducibility are good, the detection of the characteristic spectrum and the content measurement of the standard decoction of the evodia rutaecarpa (stone tiger) can be realized simultaneously through one instrument and one-time analysis, the usage amount of mobile phases such as acetonitrile and the like is reduced, the pollution to the environment is greatly reduced, the method is more environment-friendly, and the detection cost is also reduced; the measuring time can be effectively reduced, and the working efficiency is improved.
Drawings
To more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings used in the detailed description or the prior art will be briefly described below.
FIG. 1 is a superimposed graph of characteristic spectra of 15 batches of standard decoction of evodia rutaecarpa (caulis et folium piperis) Rutaeni;
FIG. 2 is a comparison characteristic spectrum of a standard decoction of evodia rutaecarpa (Bodinieri) Rutaecarpa;
FIG. 3 is a limonin standard curve;
FIG. 4 is a standard curve of evodiamine;
FIG. 5 is a standard curve of rutaecarpine.
Detailed Description
The method for quantifying fingerprint spectrum of the standard decoction of evodia rutaecarpa (stone tiger) of the present invention is further described in detail with reference to the following specific examples.
It should be noted that, the meaning of evodia rutaecarpa (tiger) mentioned in the specification means: the stone tiger is adopted as a basic source.
Example 1: method for establishing UPLC characteristic spectrum of standard decoction of fructus evodiae (caulis et folium piperis)
1.1 instruments, reagents and sources
The instrument comprises the following steps: a Waters Acquity I Class ultra performance liquid chromatograph; agilent Infinity II1290-6545Q-TOF LC-MS Agilent ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometer; ML204T/02 parts per million analytical balance; XSE105 one ten-thousandth analytical balance; KQ-500DV ultrasonic instrument; waters Acquity UPLC BEH Shield RP18 (2.1X 100mm, 1.7 μm) chromatography column and pre-column; a SOrvall ST8 high speed centrifuge;
reagent: acetonitrile (Thermo) and formic acid (Thermo) are of mass spectrum grade; phosphoric acid (Aladdin) and acetonitrile (Fisher) are in chromatographic grade; methanol (General-Reagent) was analytical grade; ultra pure water (Milli-QIQ 7000);
reagent testing: chlorogenic acid (batch No. 110753-202018, specification: 20mg, purity: 96.1%), hyperin (batch No. 111521-201809, specification: 20mg, purity: 94.9%) were purchased from the institute of food and drug assay;
the sample source details are shown in Table 1.
TABLE 115 batch Evodia rutaecarpa (Gekko Swinhonis) Standard decoction sample information
1.2 preparation of the solution
1.2.1 preparation of control solutions
Preparation of a reference solution: precisely weighing appropriate amount of chlorogenic acid reference substance and hyperoside reference substance, and adding methanol to obtain mixed solution containing chlorogenic acid 30 μ g and hyperoside 20 μ g per 1mL as reference substance solution.
1.2.2 preparation of test solutions
Taking a proper amount of fructus evodiae (caulis Entadae) standard decoction lyophilized powder, taking about 0.2g, precisely weighing, placing in a conical flask with a stopper, adding 10mL of methanol, sealing, weighing, ultrasonically treating (power 400W, frequency 40kHz) for 20min, cooling to room temperature, weighing again, supplementing the weight loss with methanol, shaking, and 10000 r.min-1Centrifuging for 10min, and filtering the supernatant to obtain the test solution.
1.3 chromatographic conditions
A chromatographic column: waters Acquity UPLC BEH Shield RP18 column (2.1X 100mm, 1.7 μm)
Mobile phase: a: 0.1% phosphoric acid/water B: acetonitrile
Flow rate: 0.4mL/min
Column temperature: 35 deg.C
Sample introduction amount: 1 μ L
Detection wavelength: 254nm
Gradient elution conditions: 0-4 min: 4% of A; 4-9 min: 4% -11% of A; 9-10 min: 11% of A; 10-14 min: 11% -18% of A; 14-20 min: 18% of A; 20-22 min: 18-35% of A; 22-28 min: 35-37% of A; 28-34 min: 37-39% of A; 34-35 min: 39-80% of A; 35-38 min: 80% of A.
1.4 feature map methodology investigation
1.4.1 precision investigation
Taking lyophilized powder of standard decoction of fructus evodiae (Gekko Swinhonis) of lot number WZY-211031-2, preparing test solution according to the above 1.2.2 process, continuously feeding sample for 6 times, recording characteristic map, and displaying 11 characteristic peaks (such as 11 characteristic peaks identified in the following 1.5, the same applies below). Relative retention times for peaks 1,2, 3, 4, 5, 7 were calculated using peak 6 (chlorogenic acid) as the reference peak (S1), and relative retention times for peaks 8, 10, 11 were calculated using peak 9 (hyperin) as the reference peak (S2). The RSD values of the relative retention times of the 11 characteristic peaks are all less than 1%, the RSD values of the relative peak areas of the characteristic peaks are all less than 3%, the instrument precision is good, and the results are shown in Table 2.
1.4.2 repeatability test
Taking lyophilized powder of standard decoction of fructus evodiae (herba Evodia Rutaecarpae) of lot number WZY-211031-2, preparing 6 test solutions in parallel according to the above 1.2.2 process, injecting sample, recording characteristic map, wherein 11 characteristic peaks are shown in the map. Relative retention times for peaks 1,2, 3, 4, 5, 7 were calculated using peak 6 (chlorogenic acid) as the reference peak (S1), and relative retention times for peaks 8, 10, 11 were calculated using peak 9 (hyperin) as the reference peak (S2). The RSD values of the 11 characteristic peaks are all less than 1%, and the RSD values of the relative peak areas of the characteristic peaks are all less than 2%, which indicates that the method has good repeatability, and the results are shown in Table 2.
1.4.3 stability Studies
Taking lyophilized powder of standard decoction of fructus evodiae (herba Evodia Rutaecarpae) of lot number WZY-211031-2, preparing test solution according to the above 1.2.2 process, injecting into liquid chromatograph for 0h, 2h, 4h, 8h, 12h and 24h, respectively, recording characteristic chromatogram, and displaying 11 characteristic peaks in the chromatogram. Relative retention times for peaks 1,2, 3, 4, 5, 7 were calculated using peak 6 (chlorogenic acid) as the reference peak (S1), and relative retention times for peaks 8, 10, 11 were calculated using peak 9 (hyperin) as the reference peak (S2). The RSD values of the 11 characteristic peaks are all less than 1%, the RSD values of the relative peak areas of the characteristic peaks are all less than 5%, the stability of the test solution in 24h is good, and the results are shown in Table 2.
1.4.4 intermediate precision investigation
Taking the lyophilized powder of the standard evodia rutaecarpa decoction with the batch number of WZY-211031-2, preparing 6 parts of test solution by two experimenters in the same laboratory according to the 1.2.2 process, and obtaining characteristic maps on two Waters Acquity I Class instruments (both are diode array detectors and are 190-400 nm) respectively. The results show that the characteristic maps obtained by different instruments of the same brand have no obvious difference, and the relative retention times of the peaks 1,2, 3, 4, 5 and 7 are calculated by taking the peak 6 (chlorogenic acid) as a reference peak (S1), and the relative retention times of the peaks 8, 10 and 11 are calculated by taking the peak 9 (hyperin) as a reference peak (S2). The relative retention time RSD value of 11 characteristic peaks is less than 5%, and the relative peak area RSD value is less than 10%. Therefore, the method is suitable for the ultra-high performance liquid chromatograph of the same manufacturer, the intermediate precision is good, and the result is shown in table 2.
TABLE 2 Experimental results of precision, repeatability, stability and intermediate precision of characteristic spectrum
1.5 creation of feature map and identification of common peaks
According to the established chromatographic analysis method of 1.3, 1 μ L of each of 15 batches of evodia rutaecarpa standard decoction lyophilized powder test solution (prepared according to the above 1.2.2 process respectively) are precisely absorbed, injected into an ultra-high performance liquid chromatograph, chromatographic peak information is recorded, an overlay chart (figure 1) and a comparison characteristic chart (figure 2) are generated in a traditional Chinese medicine chromatographic characteristic chart similarity evaluation system software (2012 edition), wherein 11 peaks are shared, and the comparison is performed by adopting a liquid chromatography-mass spectrometry technology and combining a literature [ Zhao Xiao Mei et al, evodia rutaecarpa chemical composition analysis based on UPLC-Q-TOF-MS/MS technology, Chinese journal of experimental prescriptions, 2021,27(06):113 and 126 ], a database (database such as PubMed Compound, Chemscope and the like) and a comparison of a comparison product, and 11 shared peaks are identified, and the result is shown in Table 3. The relative retention times of peaks 1,2, 3, 4, 5, 7 were calculated using peak 6 (chlorogenic acid) as the reference peak (S1), the relative retention times of peaks 8, 10, 11 were calculated using peak 9 (hyperin) as the reference peak (S2), and the relative retention times of the control profiles were calculated, the results of which are shown in table 4. The relative retention times of 15 batches of evodia rutaecarpa (Gekko Swinhonis) standard decoction lyophilized powder samples are shown in Table 5.
TABLE 3 common peak identification result of lyophilized powder of standard decoction of fructus evodiae (Gekko Swinhonis)
Indicates the comparison with the control
TABLE 4 relative retention time of control profiles
TABLE 5 statistics of relative retention time for various batches of samples
Example 2: method for determining content of standard decoction of evodia rutaecarpa (caulis et folium piperis) and its application
1.1 instruments, reagents and sources
The instrument comprises the following steps: a Waters Acquity I Class ultra performance liquid chromatograph; ML204T/02 parts per million analytical balance; XSE105 one ten-thousandth analytical balance; KQ-500DV ultrasonic instrument; waters Acquity UPLC BEH Shield RP18 (2.1X 100mm, 1.7 μm) chromatography column and pre-column; a SOrvall ST8 high speed centrifuge;
reagent: formic acid (Macklin) and acetonitrile (Fisher) are in chromatographic grade; methanol (Energy Chemical) was analytical grade; ultrapure water;
reagent testing: limonin (batch No. 110800-201707, specification 20mg, purity 97.9%) was purchased from China institute for food and drug testing; evodiamine (batch No. 110802-201710, specification 20mg, purity 99.6%) was purchased from China institute for food and drug testing; rutaecarpine (batch No. 110801 and 2021109, specification 20mg, purity 99.Macklin 3%) was purchased from China food & drug testing institute.
The samples were sourced as in example 1
1.2 preparation of the solution
1.2.1 preparation of control solutions
Preparing a standard solution of a standard decoction reference substance of evodia rutaecarpa (stone tiger): the limonin, evodiamine and rutaecarpine reference substances are precisely weighed and mixed with methanol to prepare a solution containing 100 micrograms of limonin, 80 micrograms of evodiamine and 50 micrograms of rutaecarpine with the concentration of 1mL respectively as a mixed reference substance solution, and the concentration of the reference substance solution is shown in Table 6.
TABLE 6 concentration of mixed control solution
1.2.2 preparation of test solutions
The test solutions were prepared as in example 1.
1.3 chromatographic conditions
Detection wavelength: 215nm, and the rest of the conditions are the same as those of the chromatography in example 1.
1.4 assay methodology investigation
1.4.1 precision investigation
Taking lyophilized powder of standard decoction of fructus evodiae (herba Gekko Swinhonis) of lot number WZY-211031-2, preparing test solution, and continuously injecting sample for 6 times, wherein the content RSD values of limonin, evodiamine and rutaecarpine are less than 1%, indicating that the precision of the instrument is good, and the results are shown in Table 7.
1.4.2 repeatability test
6 parts of test solution is prepared in parallel by taking the freeze-dried powder of the standard decoction of the evodia rutaecarpa (stone tiger) with the batch number of WZY-211031-2, and the content RSD of the limonin, the evodiamine and the rutaecarpine is less than 2 percent by respective injection analysis, which shows that the method has good repeatability, and the results are shown in a table 7.
1.4.3 stability Studies
Taking lyophilized powder of standard decoction of fructus evodiae (herba Evodia Rutaecarpae) of lot number WZY-211031-2, preparing test solution, standing at room temperature for 0, 2, 4, 8, 12, and 24h, respectively, injecting sample, analyzing, and examining room temperature stability of the test solution. The content RSD of the limonin, the evodiamine and the rutaecarpine is less than 1 percent, which shows that the test solution has good stability in 24 hours, and the result is shown in Table 7.
TABLE 7 content determination precision, repeatability, stability test results
1.4.4 Linear relationship investigation
The concentrations of limonin, evodiamine and rutaecarpine are precisely absorbed respectively as follows: 208.1354. mu.g/mL, 69.5806. mu.g/mL, 15.9198. mu.g/mL; 166.5083 μ g/mL, 55.6644 μ g/mL, 12.7358 μ g/mL; 124.8812 μ g/mL, 41.7483 μ g/mL, 9.5519 μ g/mL; 83.2542 μ g/mL, 27.8322 μ g/mL, 6.3679 μ g/mL; 41.6271 μ g/mL, 13.9161 μ g/mL, 3.1840 μ g/mL; 20.8135 μ g/mL, 6.9581 μ g/mL, 1.5920 μ g/mL; 8.3254 μ g/mL, 2.7832 μ g/mL, 0.6368 μ g/mL mixed control solutions, analysis by injection, and linear regression of solution concentration X with peak area Y to form standard curves of 3 quantitative index components, the results are shown in Table 8 and FIGS. 3-5.
TABLE 8 Standard curves for limonin, evodiamine, and rutaecarpine
1.4.5 sample recovery test
Accurately weighing a proper amount of freeze-dried powder of standard decoction of fructus evodiae (caulis et folium piperis) with known content of lot number WZY-211031-2, accurately adding 3 reference substance mixed solutions with known amount according to concentration levels of about 50 wt%, 100 wt% and 150 wt% of standard content of limonin, evodiamine and rutaecarpine to prepare 9 test solution with low, medium and high concentration respectively, and performing sample injection analysis respectively, wherein the recovery rate of 3 quantitative indexes (recovery rate: (measured amount-content in sample)/added reference substance amount: 100%) is 103.84%, 100.95% and 95.55%, respectively in the range of 95-105%, RSD is less than 2%, and the results are shown in Table 9.
TABLE 9 sample recovery results for limonin, evodiamine, and rutaecarpine
1.4.6 intermediate precision investigation
6 parts of test solution to be tested are respectively prepared in parallel by 2 persons from the same batch of freeze-dried powder of evodia rutaecarpa standard decoction with the number of WZY-211031-2, 12 parts of test solution to be tested are respectively analyzed by sample injection, and the RSD of the content of 3 index components is less than 5 percent, which shows that the method is suitable for an ultra-high performance liquid chromatograph of the same manufacturer, the intermediate precision is good, and the result is shown in Table 10.
TABLE 10 intermediate precision experimental results of limonin, evodiamine and rutaecarpine
1.5 measurement results of the content of each batch of samples
Taking 15 batches of fructus evodiae (caulis Entadae) standard decoction lyophilized powder, measuring by the above method, calculating the content of limonin, evodiamine and rutaecarpine by external standard method, and determining the content of 3 components shown in Table 11. Each 1g of fructus evodiae (caulis Seu folium Ampelopsis Brevipedunculatae) standard decoction contains limonin (C)26H30O8) 2.3-11.4mg of evodiamine (C)19H17N3O) and rutaecarpine (C)18H13N3O) should be 0.8-4.8mg in total.
TABLE 11 content determination results of lyophilized powder of standard decoction of fructus evodiae (Gekko Swinhonis) for each batch
The technical scheme provided by the invention is compared with the prior art as follows:
the table shows that the method has a large number of peak identifications, the information reflected by the characteristic spectrum is relatively comprehensive, and the analysis method can simultaneously detect the characteristic spectrum and the content by one-time analysis, so that the determination time is effectively reduced, and the analysis efficiency is obviously improved.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, so that all equivalent changes or modifications made in accordance with the scope of the present invention shall fall within the scope of the present invention.
Claims (9)
1. A method for establishing a quantitative fingerprint of a standard evodia decoction is characterized by comprising the following steps:
(1) preparing a reference solution;
(2) preparing a plurality of batches of evodia rutaecarpa standard decoction test solution;
(3) analyzing the multiple batches of evodia rutaecarpa standard decoction sample solutions in the step (2) in an ultra-high performance liquid chromatography analyzer respectively to obtain multiple batches of liquid chromatogram maps;
(4) and (3) establishing a quantitative fingerprint, namely introducing the multi-batch liquid chromatogram in the step (3) into software of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, matching common peaks, generating a reference characteristic spectrum, selecting the reference peak, calculating the relative retention time of each characteristic peak, and simultaneously determining the content of 3 components of limonin, evodiamine and rutaecarpine in the same ultra-high performance liquid chromatogram analyzer to establish the quantitative fingerprint of the evodia rutaecarpa standard decoction.
2. The method for establishing the quantitative fingerprint of the fructus evodiae standard decoction according to claim 1, wherein,
a: establishing a fingerprint spectrum method:
step a 1: preparation of control solutions: taking chlorogenic acid and hyperoside as reference substances, and preparing into mixed reference substance solution containing chlorogenic acid and hyperoside with methanol of 50-100% or ethanol of 50-100% as solvent;
step a 2: preparing a test solution: taking freeze-dried powder of fructus evodiae standard decoction, grinding, precisely weighing, placing into a conical flask with a plug, adding 50-100% methanol solvent or 50-100% ethanol solvent, wherein the ratio of material to liquid is 1: 25-1: 100g/mL, sealing, weighing, refluxing or ultrasonically treating for 10-30min, cooling to room temperature, adding corresponding solvent to compensate weight loss, shaking, centrifuging, collecting supernatant, and filtering to obtain fructus evodiae standard decoction sample solution;
step a 3: ultra-high performance liquid chromatography analysis: preparing more than 3 different batches of evodia rutaecarpa standard decoction test sample solutions according to the method in the step a2, respectively sucking the reference substance solution and the test sample solution, respectively injecting the reference substance solution and the test sample solution into an ultra-high performance liquid chromatograph, and respectively recording liquid chromatogram maps;
step a 4: establishing a fingerprint spectrum: introducing the liquid chromatogram of at least more than 3 standard decoction samples of fructus evodiae in different batches into 'Chinese medicine chromatogram characteristic map similarity evaluation system software', matching common peaks, generating a reference characteristic map, and identifying the common peaks; and calculating the relative retention time of each characteristic peak by taking chlorogenic acid and hyperin as reference peaks;
b: establishment of a content determination method:
step b 1: preparation of control solutions: taking appropriate amount of limonin, evodiamine and rutaecarpine as reference substances, precisely weighing, adding 50-100% by volume of methanol or 50-100% by volume of ethanol to obtain reference substance mixed solution containing limonin, evodiamine and rutaecarpine;
step b 2: consistent with step a 2;
step b 3: ultra-high performance liquid chromatography analysis: preparing more than 3 different batches of evodia rutaecarpa standard decoction test solution according to the method in the step 2, respectively sucking the reference solution and the test solution, respectively injecting the reference solution and the test solution into an ultra-high performance liquid chromatograph, and respectively measuring the corresponding peak areas of limonin, evodiamine and rutaecarpine in the reference solution and the test solution;
step b 4: establishment of a content determination method: the content of limonin, evodiamine and rutaecarpine in the freeze-dried powder of the standard decoction of the fructus evodiae is calculated by an external standard method.
3. The method as claimed in claim 2, wherein the concentrations of the mixed control solutions prepared in step a1 are 20-40 μ g chlorogenic acid and 10-30 μ g hyperoside per 1 mL.
4. The method for establishing the quantitative fingerprint of the fructus evodiae standard decoction according to claim 2, wherein the chromatographic conditions of the ultra performance liquid chromatography analysis in the a3 and the b3 are as follows: the chromatographic column is Waters Acquity UPLC BEH Shield RP18, the inner diameter is 2.1mm, the column length is 100mm, the particle size is 1.7 mu m, the column temperature is 30-40 ℃, the flow rate is 0.3-0.5mL/min, the sample injection amount is 0.5-2 mu L, the detection wavelength is 210-280nm, the mobile phase A is acetonitrile, the mobile phase B is phosphoric acid solution with the volume concentration of 0.05-0.2%, and the detection data of the whole elution time period are collected by adopting a gradient elution mode.
5. The method for establishing the quantitative fingerprint of the fructus evodiae standard decoction according to claim 4, wherein the gradient elution condition is as follows: 0-4 min: 4% a, 96% B; 4-9 min: 4-11% of A, 89-96% of B; 9-10 min: 11% a, 89% B; 10-14 min: 11-18% of A, 82-89% of B; 14-20 min: 18% a, 82% B; 20-22 min: 18-35% of A, 65-82% of B; 22-28 min: 35-37% of A, 63-65% of B; 28-34 min: 37-39% of A and 61-63% of B; 34-35 min: 39-80% of A, 20-61% of B; 35-38 min: 80% of A, 20% of B, the% are volume percentages.
6. The method for establishing the quantitative fingerprint of the fructus evodiae standard decoction according to claim 2, wherein in the step a4, 11 common peaks of the reference characteristic spectrum are marked according to the order from left to right in the spectrum, wherein peak 5 is cryptochlorogenic acid, peak 6 is chlorogenic acid, peak 8 is dehydroevodiamine, peak 9 is hyperoside, peak 10 is narcissus, and peak 11 is evodiamine.
7. The method of claim 6, wherein the peak 6 corresponding to chlorogenic acid control solution is the S1 peak, the peak 9 corresponding to hyperin control solution is the S2 peak, the relative retention times of peaks 1,2, 3, 4, 5, and 7 are calculated using the peak S1, the relative retention times of peaks 8, 10, and 11 are calculated using the peak S2, and the relative retention times of characteristic peaks are within ± 10% of the predetermined value; the relative retention time specified values are: peak 1, 0.28; peak 2, 0.36; peak 3, 0.44; peak 4, 0.83; peak 5, 0.94; peak 6, 1; peak 7, 1.15; peak 8, 0.85; peak 9, 1; peak 10, 1.15; peak 11, 1.76.
8. The method of claim 2, wherein in step b1, the concentrations of the prepared mixed control solutions are 8-200 μ g limonin, 2-70 μ g evodiamine, and 1-15 μ g rutaecarpine per 1 mL.
9. The method of claim 2, wherein in b, the content of standard evodia rutaecarpa decoction lyophilized powder is measured with limonin content of 2.3-11.4mg per 1g and total content of evodiamine and rutaecarpine of 0.8-4.8 mg.
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