CN111337580A - HPLC (high Performance liquid chromatography) characteristic spectrum of four-component preparation as well as construction method and application thereof - Google Patents
HPLC (high Performance liquid chromatography) characteristic spectrum of four-component preparation as well as construction method and application thereof Download PDFInfo
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Abstract
The invention relates to a four-component preparation HPLC characteristic spectrum and a construction method thereof, wherein the HPLC standard characteristic spectrum comprises two characteristic spectrums which are respectively collected at the wavelengths of 230nm and 321nm under the same chromatographic condition. Wherein, under the condition of 230nm wavelength, the characteristic spectrum of the test sample presents 5 characteristic peaks with the same retention time as the chromatogram of the radix paeoniae alba reference medicine; under the condition of 321nm wavelength, the characteristic spectrum of the sample presents 7 characteristic peaks which are the same as the retention time of the chromatogram of the rhizoma ligustici wallichii and the angelica sinensis contrast medicinal material. The invention also comprises a construction method of the HPLC characteristic map of the four-component preparation, application of the HPLC characteristic map of the four-component preparation in detection of four-component related preparations and a detection method of the four-component preparation.
Description
Technical Field
The invention relates to the technical field of pharmaceutical analysis, in particular to a four-component preparation HPLC characteristic spectrum and a construction method and application thereof.
Background
The Chinese medicine characteristic spectrum refers to the spectrum or chromatogram of a certain kind of characteristic components shared by one or more Chinese medicinal materials. At present, a characteristic map becomes an internationally recognized effective component for controlling the quality of traditional Chinese medicines or natural medicines, and is a hot problem concerned by the traditional Chinese medicine field at present, and in recent years, people gradually realize that a quality control method formed only aiming at a certain chemical component cannot properly reflect the internal quality of the traditional Chinese medicines, so that the internal quality of a product can be better controlled, and the authenticity and the effectiveness of the product are enhanced.
At present, the quality control of the four-medicine preparation is mainly realized by thin-layer identification of medicinal materials such as angelica, ligusticum wallichii, white paeony root and prepared rehmannia root and control of the content of paeoniflorin in the white paeony root, and the method can detect the product quality to a certain extent but cannot comprehensively reflect the quality condition of the product.
Disclosure of Invention
Aiming at the problem that the quality condition of a product cannot be comprehensively reflected in the prior art, the invention provides a construction method of a four-component preparation HPLC characteristic spectrum.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
the HPLC characteristic spectrum of the four-medicine preparation is characterized in that under the condition of a wavelength of 230nm, a characteristic spectrum of a test sample presents 5 characteristic peaks with the same retention time as that of a chromatogram of a white paeony root control medicinal material; under the condition of 321nm wavelength, the characteristic spectrum of the sample presents 7 characteristic peaks which are the same as the retention time of the chromatogram of the rhizoma ligustici wallichii and the angelica sinensis contrast medicinal material.
And a construction method of HPLC characteristic map of the four-component preparation, which comprises the following steps:
step a, taking 0.2-2.0 g of angelica sinensis reference medicinal material and 0.2-2.0 g of ligusticum wallichii reference medicinal material respectively, putting the angelica sinensis reference medicinal material and the ligusticum wallichii reference medicinal material into a conical flask with a plug, adding water, decocting for 0.5-1 hour, cooling, filtering, evaporating the filtrate to be nearly dry, adding diluted alcohol into residues, transferring the residues into a 5-100 ml measuring flask in batches, adding the diluted alcohol to fix the volume to a scale, shaking up, filtering, and taking the subsequent filtrate as the reference solution of the angelica sinensis and the ligusticum wallichii reference. Preparing reference solution of radix Paeoniae alba reference material by the same method.
B, taking the content of the four-substance preparation, preparing a solution with the concentration of the content of the four-substance preparation being 20-200 mg/mL by using a 10-70% v/v alcohol solution, filtering, and taking a subsequent filtrate as a test solution;
and c, measuring the reference substance solution and the test solution by using a high performance liquid chromatography, wherein the chromatographic conditions are as follows: using a C18 chromatographic column, and adopting an A phase as a mobile phase: and (3) performing gradient elution on a phase B10 → 40:90 → 60, wherein the phase A is methanol, the phase B is 0.05-0.2 wt% of phosphoric acid, the detection wavelength is 230nm and 321nm, the column temperature is 25-45 ℃, the flow rate is 0.9-1.1 mL/min, and the sample injection amount is 5-20 mu L.
And the application of the HPLC characteristic spectrum of the four-component preparation in the detection of the four-component preparation.
The invention adopts the high performance liquid chromatography to establish the construction method of the characteristic spectrum of the four-substance preparation, makes up the defect that the prior art can not comprehensively reflect the quality condition of the product, makes the quality control technology more perfect and scientific, and provides an available quality control mode for the standardized production in the future. The HPLC characteristic spectrum of the four-substance preparation to be detected is compared with the HPLC characteristic spectrum of the four-substance preparation, so that the quality of the product can be better controlled on the whole, and effective guarantee is provided for the production and clinical application of the medicine.
FIG. 1321 nm wavelength condition rhizoma Ligustici Chuanxiong radix Angelicae sinensis reference medicinal material feature map;
FIG. 2321 shows a characteristic spectrum of a sample under a wavelength condition;
FIG. 3230 nm wavelength condition radix Paeoniae alba reference drug characteristic spectrum;
FIG. 4230 nm wavelength condition sample characteristic spectrum;
FIG. 5 white peony specificity study profile;
FIG. 6 is a special survey map of Angelica sinensis and Ligusticum chuanxiong;
figure 7321 nm wavelength four-component preparation feature map precision investigation attached drawing;
FIG. 8230 nm wavelength four-component preparation feature spectrum precision investigation figure;
FIG. 9321 nm wavelength condition four-material preparation characteristic spectrum repeatability investigation spectrogram;
FIG. 10230 nm wavelength condition feature spectrum repeatability investigation spectrogram of four-substance preparation;
FIG. 11321 nm wavelength four-component preparation characteristic map stability investigation spectrogram;
FIG. 12230 nm wavelength four-component preparation characteristic map stability investigation spectrogram.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The embodiment of the invention provides a four-substance preparation HPLC (high performance liquid chromatography) characteristic spectrum, wherein under the condition of a 230nm wavelength, a characteristic spectrum of a test sample presents 5 characteristic peaks with the same chromatographic retention time as a white paeony root reference medicinal material; under the condition of 321nm wavelength, the characteristic spectrum of the sample presents 7 characteristic peaks which are the same as the retention time of the chromatogram of the rhizoma ligustici wallichii and the angelica sinensis contrast medicinal material.
The characteristic map can better control the quality of the product on the whole, and provides effective guarantee for the production and clinical application of the medicine.
The embodiment of the invention also provides a construction method of the HPLC characteristic spectrum of the four-substance preparation, which comprises the following steps:
step a, taking 0.2-2.0 g of angelica sinensis reference medicinal material and 0.2-2.0 g of ligusticum wallichii reference medicinal material respectively, putting the angelica sinensis reference medicinal material and the ligusticum wallichii reference medicinal material into a conical flask with a plug, adding water, decocting for 0.5-1 hour, cooling, filtering, evaporating the filtrate to be nearly dry, adding diluted alcohol into residues, transferring the residues into a 5-100 ml measuring flask in batches, adding the diluted alcohol to fix the volume to a scale, shaking up, filtering, and taking the subsequent filtrate as the reference solution of the angelica sinensis and the ligusticum wallichii reference. Preparing reference solution of radix Paeoniae alba reference material by the same method.
B, taking the content of the four-substance preparation, preparing a solution with the concentration of the content of the four-substance preparation being 20-200 mg/mL by using a 10-70% v/v alcohol solution, filtering, and taking a subsequent filtrate as a test solution;
and c, measuring the reference substance solution and the test solution by using a high performance liquid chromatography, wherein the chromatographic conditions are as follows: using a C18 chromatographic column, and adopting an A phase as a mobile phase: and (3) performing gradient elution on a phase B10 → 40:90 → 60, wherein the phase A is methanol, the phase B is 0.05-0.2 wt% of phosphoric acid, the detection wavelength is 230nm and 321nm, the column temperature is 25-45 ℃, the flow rate is 0.9-1.1 mL/min, and the sample injection amount is 5-20 mu L.
Specifically, in the step C, the inner diameter of the C18 chromatographic column is 4.6mm, the length of the chromatographic column is 250mm, the particle size of the filler is 5 μm, and the preferred inner diameter of the chromatographic column, length of the chromatographic column, particle size of the filler and flow rate are matched, so that the tube wall effect can be avoided.
Further preferably, the time and the mobile phase ratio of the gradient elution are as follows: 0-20 min, phase A: 10-20%, phase B: 90-80%; 20-50 min, phase A: 20-30%, phase B: 80-70%; 50-60 min, phase A: 30-40%, phase B: 70-60%. Because the components in the solution to be detected are complex, the components may not be well separated by using a single mobile phase, and the components with larger differences in the neutral properties of the solution to be detected can be well separated according to the respective properties and the respective proper volume factor k by using the optimal elution time and the optimal mobile phase proportion.
Further preferably, the column temperature is 30 ℃. Too high column temperature will reduce the distribution coefficient K value of each component, decrease the separation degree, too low column temperature will reduce the mass transfer rate, the column efficiency will decrease, and the analysis time will be prolonged.
Further preferably, the flow rate is 1.0mL/min, and the retention time and the separation effect of the solution to be measured are more ideal at the flow rate.
Further preferably, the sample amount is 10 μ L, and the sample amount can enable the peak area to be moderate in size and the measurement result to be more accurate.
The embodiment of the invention also provides application of the HPLC characteristic spectrum of the four-component preparation in detection of the four-component preparation.
The HPLC characteristic spectrum of the four-substance preparation to be detected is compared with the HPLC characteristic spectrum of the four-substance preparation, so that the quality of the product can be better controlled on the whole, and effective guarantee is provided for the production and clinical application of the medicine.
In order to better illustrate the embodiments of the present invention, the following examples are further illustrative.
Example 1
The embodiment provides a method for constructing an HPLC (high performance liquid chromatography) characteristic spectrum of a four-substance preparation, which comprises the following specific steps:
step a, taking 1.0g of each of the angelica sinensis reference medicinal material and the ligusticum wallichii reference medicinal material, putting the angelica sinensis reference medicinal material and the ligusticum wallichii reference medicinal material into a conical flask with a plug, adding water, decocting for 1 hour, cooling, filtering, steaming the filtrate until the filtrate is nearly dry, adding diluted ethanol into residues, transferring the residues into 25ml measuring flasks in batches, adding the diluted ethanol to a constant volume to scale, shaking up, filtering, and taking the subsequent filtrate as a reference solution of the angelica sinensis and the ligusticum wallichii reference medicinal material. Preparing reference solution of radix Paeoniae alba reference material by the same method.
B, taking the contents of the four-substance preparation, preparing a solution with the concentration of the contents of the four-substance preparation being 80mg/mL by using 30% v/v ethanol, filtering, and taking a subsequent filtrate as a test solution;
and C, measuring the reference solution and the test solution by using a high performance liquid chromatography, wherein the chromatographic conditions are that an Agilent ZORBAX Eclipse Plus C18 chromatographic column (4.6 × 250mm 5 mu m) is adopted, and a mobile phase adopts an A phase, namely a B phase 10 → 40:90 → 60 for gradient elution, wherein the A phase is methanol, the B phase is 0.1wt% phosphoric acid, the detection wavelength is 230nm and 321nm, the column temperature is 30 ℃, the flow rate is 1.1mL/min, and the sample introduction amount is 10 mu L.
Example 2
The embodiment provides a method for constructing an HPLC (high performance liquid chromatography) characteristic spectrum of a four-substance preparation, which comprises the following specific steps:
step a, taking 0.2g of angelica sinensis control medicinal material and 0.2g of ligusticum wallichii control medicinal material respectively, putting the angelica sinensis control medicinal material and the ligusticum wallichii control medicinal material into a conical flask with a plug, adding water, decocting for 0.5 hour, cooling, filtering, steaming the filtrate until the filtrate is nearly dry, adding diluted methanol into residues, transferring the residues into a 5ml measuring flask in batches, adding the diluted methanol to a constant volume until the volume is scaled, shaking up, filtering, and taking the subsequent filtrate as the reference solution of the angelica sinensis and ligusticum wallichii control medicinal. Preparing reference solution of radix Paeoniae alba reference material by the same method.
B, taking the contents of the four-substance preparation, preparing a solution with the concentration of the contents of the four-substance preparation being 20mg/mL by using 50% v/v methanol, filtering, and taking a subsequent filtrate as a test solution;
and C, measuring the reference solution and the test solution by using a high performance liquid chromatography, wherein the chromatographic conditions are that a Phenomenex Luna C18 chromatographic column (4.6 × 250mm 5 mu m) is adopted, a mobile phase adopts an A phase, a B phase 10 → 40:90 → 60 is adopted for gradient elution, the A phase is methanol, the B phase is 0.05wt% phosphoric acid, the detection wavelength is 230nm and 321nm, the column temperature is 25 ℃, the flow rate is 0.9mL/min, and the sample injection amount is 20 mu L.
Example 3
The embodiment provides a method for constructing an HPLC (high performance liquid chromatography) characteristic spectrum of a four-substance preparation, which comprises the following specific steps:
step a, taking 2.0g of each of the angelica sinensis reference medicinal material and the ligusticum wallichii reference medicinal material, putting the angelica sinensis reference medicinal material and the ligusticum wallichii reference medicinal material into a conical flask with a plug, adding water, decocting for 1 hour, cooling, filtering, steaming the filtrate until the filtrate is nearly dry, adding diluted ethanol into residues, transferring the residues into 50ml measuring flasks in batches, adding the diluted ethanol to a constant volume to scale, shaking up, filtering, and taking the subsequent filtrate as a reference solution of the angelica sinensis and the ligusticum wallichii reference medicinal material. Preparing reference solution of radix Paeoniae alba reference material by the same method.
B, taking the contents of the four-substance preparation, preparing a solution with the concentration of the contents of the four-substance preparation being 200mg/mL by using 70% v/v ethanol, filtering, and taking a subsequent filtrate as a test solution;
and C, measuring the reference solution and the test solution by using a high performance liquid chromatography, wherein the chromatographic conditions are that an Inertsil ODS-3C 18 chromatographic column (4.6 × 250mm 5 mu m) is adopted, and a mobile phase adopts an A phase, a B phase 10 → 40:90 → 60 for gradient elution, wherein the A phase is methanol, the B phase is 0.2wt% phosphoric acid, the detection wavelength is 230nm and 321nm, the column temperature is 45 ℃, the flow rate is 1.1mL/min, and the sample injection amount is 5 mu L.
Example 4
The embodiment provides an HPLC characteristic spectrum of a four-substance preparation, which comprises the following specific steps:
the four-component preparation HPLC characteristic map is established by adopting the construction method of the four-component preparation characteristic map described in example 2, and is shown in figure 1. The HPLC standard characteristic spectrum is characterized in that under the condition of a wavelength of 230nm, the characteristic spectrum of a test sample presents 5 characteristic peaks with the same retention time as the chromatogram of the radix paeoniae alba reference medicinal material; under the condition of 321nm wavelength, the characteristic spectrum of the sample presents 7 characteristic peaks which are the same as the retention time of the chromatogram of the rhizoma ligustici wallichii and the angelica sinensis contrast medicinal material.
Example 5
This example provides a methodology investigation of the method of construction of the HPLC profile of the four-component formulation described above:
(1) and (3) special investigation: preparation of negative test solution: taking four negative samples of the four preparations including angelica deficient sample, ligusticum wallichii negative sample and white peony root negative sample, preparing negative sample solution according to the preparation method of the test solution in the construction method of the characteristic map of the four preparations described in the embodiment 2, and filtering by using a 0.45-micrometer microporous membrane to obtain the medicine.
According to the chromatographic conditions in the construction method of the characteristic spectrum of the four-substance preparation described in the embodiment 2, the negative sample is taken and is detected by high performance liquid chromatography, the sample and the reference medicinal material have the same peak in the same retention time, and the negative solution has no corresponding peak, so that the specificity of the characteristic spectrum is proved to be good, which is shown in figure 2.
(2) Precision investigation: the same sample solution was taken, sample introduction was repeated 5 times under the chromatographic conditions in the method for constructing the characteristic spectrum of the four-substance preparation described in example 2, similarity analysis was performed by using the national pharmacopoeia committee "traditional Chinese medicine chromatography fingerprint similarity evaluation system (2012 edition)", and the consistency of the relative retention time of the common peaks was examined, and the results are shown in fig. 2, table 1, and table 2, indicating that the method is good in precision.
TABLE 1 precision test Total Peak Retention time-321 nm
| Peak 1 | |
|
|
|
|
| Rhizoma Ligustici Chuanxiong should be used asChinese angelica contrast medicinal material | 6.848 | 14.168 | 23.442 | 24.021 | 45.46 |
| |
6.832 | 14.319 | 23.392 | 24.314 | 45.276 |
| |
6.842 | 14.353 | 23.454 | 24.368 | 45.406 |
| |
6.834 | 14.348 | 23.468 | 24.362 | 45.467 |
| |
6.834 | 14.324 | 23.44 | 24.31 | 45.472 |
| |
6.836 | 14.267 | 23.496 | 24.227 | 45.459 |
| Average | 6.837667 | 14.2965 | 23.44867 | 24.267 | 45.42333 |
| RSD% | 0.09 | 0.49 | 0.15 | 0.54 | 0.17 |
TABLE 2 precision test common peak retention time-230 nm
| Peak 1 | |
|
|
|
|
| Paeonia lactiflora pall pair medicine | 6.675 | 30.088 | 37.232 | 45.460 | 49.065 |
| |
6.681 | 29.976 | 37.115 | 45.276 | 49.081 |
| |
6.702 | 30.053 | 37.214 | 45.406 | 49.188 |
| |
6.695 | 30.077 | 37.246 | 45.467 | 49.233 |
| |
6.695 | 30.068 | 37.241 | 45.472 | 49.214 |
| |
6.690 | 30.136 | 37.300 | 45.459 | 49.136 |
| Average | 6.690 | 30.066 | 37.225 | 45.423 | 49.153 |
| RSD% | 0.15 | 0.17 | 0.16 | 0.17 | 0.14 |
(3) And (3) repeatability inspection: taking the same batch of samples, preparing 6 parts of test solution according to the test preparation method in the construction method of the four-substance preparation characteristic spectrum described in the embodiment 2, performing repeatability investigation under the chromatographic conditions in the construction method of the four-substance preparation characteristic spectrum described in the embodiment 2, performing similarity analysis by using a national pharmacopoeia committee 'traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition)' and investigating the consistency of relative retention time of each chromatographic peak, wherein the results are shown in fig. 3, table 3 and table 4, which indicates that the method has good repeatability.
TABLE 3 repeatability test common peak retention time-321 nm
| Peak 1 | |
|
|
|
|
| Chuandang for the treatment of hepatitis | 6.559 | 13.900 | 23.000 | 23.879 | 44.954 |
| |
6.790 | 14.262 | 23.405 | 24.309 | 45.407 |
| |
6.789 | 14.268 | 23.414 | 24.318 | 45.428 |
| |
6.806 | 14.302 | 23.438 | 24.350 | 45.481 |
| |
6.807 | 14.304 | 23.459 | 24.363 | 45.519 |
| |
6.810 | 14.307 | 23.459 | 24.370 | 45.542 |
| Repeatability 6 | 6.820 | 14.340 | 23.470 | 24.385 | 45.581 |
| Average | 6.769 | 14.240 | 23.378 | 24.282 | 45.416 |
| RSD% | 1.38 | 1.07 | 0.72 | 0.74 | 0.47 |
TABLE 4 repeatability test common peak retention time-230 nm
| Peak 1 | |
|
|
|
|
| Paeonia lactiflora pall pair medicine | 6.688 | 30.183 | 33.255 | 37.360 | 49.334 |
| |
6.653 | 30.086 | 33.135 | 37.213 | 49.149 |
| |
6.650 | 30.120 | 33.163 | 37.241 | 49.175 |
| |
6.669 | 30.137 | 33.177 | 37.281 | 49.219 |
| |
6.672 | 30.143 | 33.194 | 37.300 | 49.248 |
| |
6.678 | 30.158 | 33.225 | 37.326 | 49.268 |
| Repeatability 6 | 6.693 | 30.181 | 33.250 | 37.357 | 49.300 |
| Average | 6.672 | 30.144 | 33.200 | 37.297 | 49.242 |
| RSD% | 0.24 | 0.11 | 0.14 | 0.15 | 0.13 |
(4) And (3) stability investigation: the same test solution was taken, and the detection was performed at 0, 2, 4, 8, 12, and 24 hours under the chromatographic conditions in the method for constructing the four-substance preparation characteristic spectrum described in example 2, and the similarity analysis was performed by using the national pharmacopoeia committee "similarity evaluation system for chromatography fingerprint of chinese medicine (2012 edition)", and the consistency of the relative retention time of the common peaks was examined. The results are shown in FIG. 4, Table 5 and Table 6, which show that the test solution is stable within 24 hours.
TABLE 5 stability test common Peak Retention time-321 nm
| Peak 1 | |
|
|
|
|
| Chuandang for the treatment of hepatitis | 6.848 | 14.168 | 23.442 | 24.021 | 45.460 |
| 0 | 6.832 | 14.319 | 23.392 | 24.314 | 45.276 |
| 2 | 6.842 | 14.353 | 23.454 | 24.368 | 45.406 |
| 5 | 6.834 | 14.324 | 23.440 | 24.310 | 45.472 |
| 8 | 6.836 | 14.267 | 23.496 | 24.227 | 45.459 |
| 12 | 6.849 | 14.152 | 23.442 | 23.994 | 45.466 |
| 24 | 6.851 | 14.327 | 23.416 | 24.550 | 45.386 |
| Average | 6.842 | 14.273 | 23.440 | 24.255 | 45.418 |
| RSD% | 0.11 | 0.57 | 0.14 | 0.81 | 0.16 |
TABLE 6 stability test common Peak Retention time-230 nm
| Peak 1 | |
|
|
|
|
| Paeonia lactiflora pall pair medicine | 6.675 | 30.088 | 37.232 | 44.238 | 49.065 |
| 0 | 6.681 | 29.976 | 37.115 | 44.103 | 49.081 |
| 2 | 6.702 | 30.053 | 37.214 | 44.228 | 49.188 |
| 5 | 6.695 | 30.068 | 37.241 | 44.297 | 49.214 |
| 8 | 6.690 | 30.136 | 37.300 | 44.302 | 49.136 |
| 12 | 6.685 | 30.114 | 37.292 | 44.346 | 49.063 |
| 24 | 6.707 | 30.020 | 37.168 | 44.196 | 49.203 |
| Average | 6.691 | 30.065 | 37.223 | 44.244 | 49.136 |
| RSD% | 0.17 | 0.18 | 0.18 | 0.18 | 0.14 |
The construction method of the characteristic spectrum of the four-substance preparation provided by the embodiment of the invention has the advantages of simple detection method, easy operation, complete retention of corresponding chromatographic peaks of characteristic components, good solution stability, high precision, good reproducibility, certain specificity and good separation effect among the chromatographic peaks in the obtained characteristic spectrum. The HPLC characteristic spectrum of the four-component preparation constructed by the construction method of the characteristic spectrum of the four-component preparation provided by the embodiment of the invention can effectively characterize the quality of the HPLC characteristic spectrum of the four-component preparation, and is beneficial to comprehensively monitoring the quality of a product.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (8)
1. A four-substance preparation HPLC characteristic spectrum comprises the following medicinal materials: the HPLC standard characteristic spectrum is characterized in that the characteristic spectrum of a sample presents 5 characteristic peaks with the same retention time as the chromatogram of a white paeony root reference medicinal material under the condition of a wavelength of 230 nm; under the condition of 321nm wavelength, the characteristic spectrum of the sample presents 7 characteristic peaks which are the same as the retention time of the chromatogram of the rhizoma ligustici wallichii and the angelica sinensis contrast medicinal material.
2. A construction method of HPLC characteristic map of a four-component preparation is characterized by comprising the following steps:
step a, taking 0.2-2.0 g of angelica sinensis reference medicinal material and 0.2-2.0 g of ligusticum wallichii reference medicinal material respectively, putting the angelica sinensis reference medicinal material and the ligusticum wallichii reference medicinal material into a conical flask with a plug, adding water, decocting for 0.5-1 hour, cooling, filtering, evaporating the filtrate to be nearly dry, adding diluted alcohol into residues, transferring the residues into a 5-100 ml measuring flask in batches, adding the diluted alcohol to fix the volume to a scale, shaking up, filtering, and taking the subsequent filtrate as reference substance solution of the angelica sinensis and the ligusticum wallichii reference; preparing a reference substance solution of a radix paeoniae alba reference medicinal material by the same method;
b, taking the content of the four-substance preparation, preparing a solution with the concentration of the content of the four-substance preparation being 20-200 mg/mL by using a 10-70% v/v alcohol solution, filtering, and taking a subsequent filtrate as a test solution;
and c, measuring the reference substance solution and the test solution by using a high performance liquid chromatography, wherein the chromatographic conditions are as follows: using a C18 chromatographic column, and adopting an A phase as a mobile phase: and (3) performing gradient elution on a phase B10 → 40:90 → 60, wherein the phase A is methanol, the phase B is 0.05-0.2 wt% of phosphoric acid, the detection wavelength is 230nm and 321nm, the column temperature is 25-45 ℃, the flow rate is 0.9-1.1 mL/min, and the sample injection amount is 5-20 mu L.
3. The method for constructing an HPLC feature map of a four-substance preparation according to claim 2, wherein the gradient elution in step c is performed for a time and a mobile phase ratio of:
0-20 min, phase A: 10-20%, phase B: 90-80%;
20-50 min, phase A: 20-30%, phase B: 80-70%;
50-60 min, phase A: 30-40%, phase B: 70-60%.
4. The method for constructing an HPLC characteristic map of a four-component preparation according to claim 2, wherein said C18 chromatographic column in step C has an inner diameter of 4.6mm, a column length of 250mm, and a filler particle size of 5 μm.
5. The method for constructing an HPLC profile of a four-substance preparation according to claim 2, wherein said column temperature in step c is 30 ℃.
6. The method of claim 2, wherein said flow rate in step c is 1.0 mL/min.
7. The method for constructing an HPLC profile of a four-substance preparation according to claim 2, wherein said sample volume in step c is 10 μ L.
8. Use of the HPLC profile of a four-substance formulation according to claim 1 for the detection of four-substance formulations.
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