CN115097042B - Method for establishing HPLC fingerprint of spring oral liquid - Google Patents
Method for establishing HPLC fingerprint of spring oral liquid Download PDFInfo
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- CN115097042B CN115097042B CN202210749536.8A CN202210749536A CN115097042B CN 115097042 B CN115097042 B CN 115097042B CN 202210749536 A CN202210749536 A CN 202210749536A CN 115097042 B CN115097042 B CN 115097042B
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention belongs to the field of analysis of traditional Chinese medicine preparations, and particularly discloses a method for establishing an HPLC fingerprint of a spring oral liquid. The invention also discloses an HPLC standard fingerprint of the oral liquid of the spring obtained by the method, and the fingerprint has 28 common peaks. The method has the advantages of simple operation, high stability and good reproducibility, the obtained atlas has a plurality of characteristic peaks, and the quality of the oral liquid in the spring can be comprehensively evaluated and controlled by comparing common peaks in the standard fingerprint atlas, thereby being beneficial to accurately evaluating the internal quality of the product and ensuring the safety and the effectiveness of clinical medication.
Description
Technical Field
The invention relates to a method for establishing an HPLC fingerprint of a spring oral liquid and the fingerprint, belonging to the field of analysis of traditional Chinese medicine preparations.
Background
The oral liquid is prepared from seven Chinese medicinal materials including ginseng, pilose antler, epimedium (roasted), dogwood (roasted with wine), semen cuscutae, black-bone chicken (dehaired, claw and intestine) and actinolite, and is used for treating impotence, spermatorrhea, premature ejaculation, insomnia, amnesia and lumbago due to kidney deficiency caused by kidney yang deficiency and kidney essence deficiency. In the recipe, hairy antler warms kidney and invigorates yang, produces sperm and benefits marrow; ginseng is used as a principal drug for invigorating primordial qi; herba Epimedii and Actinolitum are used as principal drugs for supporting yang and strengthening waist and relieving paralysis; fructus Corni, semen Cuscutae, and gallus Domesticus have effects of nourishing liver and kidney, astringing essence, and relieving seminal emission, and yin-middle-energizer-yang-qi-resolving and inducing infinity, and are used together as adjuvant drugs. The medicines are combined to play the roles of tonifying kidney and strengthening yang, generating essence and nourishing brain, and the clinical effect is remarkable.
The oral liquid for treating the male sexual dysfunction belongs to a Chinese medicinal compound preparation, is prepared from a plurality of Chinese medicinal raw materials, has complex chemical components, and is used for effectively controlling the quality of the oral liquid, thereby being a basis for ensuring the safety and the effectiveness of clinical medication. The prior art lacks comprehensive and comprehensive quality detection means of the oral liquid in the spring, reports on qualitative identification and quantitative analysis of single components, and other medicinal components are not clear, so that the quality of the oral liquid in the spring is difficult to comprehensively evaluate.
The fingerprint spectrum of the traditional Chinese medicine is used as a quality control technology, a multi-component complex system of the traditional Chinese medicine preparation can comprehensively reflect the types and the amounts of chemical components contained in the preparation, and further the quality of the medicine is integrally described and evaluated. At present, related patent publications and literature reports for controlling the quality of the oral liquid in spring by using a traditional Chinese medicine fingerprint spectrum are fewer at home and abroad, luo Xiuqiong and Zeng Fangli only measure the content of icariin in the oral liquid in spring by using an HPLC method, and other components cannot be adhered to, so that the comprehensive evaluation and control of the preparation cannot be realized. Therefore, a new method for establishing the HPLC fingerprint of the oral liquid in the spring needs to be provided, the integrity and the complexity of the chemical components of the oral liquid in the spring can be effectively reflected, and the quality of the preparation can be comprehensively evaluated and controlled.
The traditional Chinese medicine fingerprint is used as a quality control and evaluation technology, has the characteristics of systematicness, integrity and stability, and can more comprehensively control the quality of medicines based on the research of the chemical components of the traditional Chinese medicine, so that the establishment of the traditional Chinese medicine preparation fingerprint has important significance for improving the quality of the traditional Chinese medicine and promoting the modernization of the traditional Chinese medicine. At present, related patents and literature reports for controlling the quality of the oral liquid in the spring by using a traditional Chinese medicine fingerprint spectrum are few, luo Xiuqiong and the like only measure the content of icariin in the oral liquid in the spring by using an HPLC method, and other components cannot be detected, so that the comprehensive evaluation and control of the preparation cannot be realized. Therefore, it is needed to provide a method for establishing a fingerprint of a spring oral liquid, so as to ensure the quality of the product, provide a reliable basis for the authenticity identification and the internal quality control of the spring oral liquid, and provide a reliable analysis method and a reliable technical means for ensuring the stability of the quality of the product and the safety and the effectiveness of clinical medication.
Disclosure of Invention
Aiming at the defects of the existing quality control method of the oral liquid in spring, the invention provides a method for establishing the HPLC fingerprint of the oral liquid in spring.
The invention relates to a method for establishing an HPLC fingerprint of a spring oral liquid, which comprises the following steps:
A. preparation of test solution: taking a spring oral liquid, adding methanol for dilution, filtering, and taking a subsequent filtrate as a sample solution;
determination of hplc chromatographic conditions: acetonitrile as mobile phase a, phosphoric acid solution as mobile phase B, gradient elution:
and C, manufacturing HPLC standard fingerprint patterns: and C, analyzing and comparing the sample solution of the oral liquid according to the chromatographic condition of the step B to obtain the HPLC standard fingerprint of the oral liquid composed of the common characteristic peaks of the samples.
Preferably, the sample solution in step a is prepared as follows: precisely weighing 2mL of the oral liquid of the spring, adding 30% -100% methanol for diluting to a constant volume, filtering, and taking the subsequent filtrate as a test solution.
Further, the sample solution in the step A is prepared according to the following steps: accurately weighing 2mL of the oral liquid of the spring, adding 80% methanol for diluting to a constant volume of 10mL, filtering, and taking the subsequent filtrate as a test solution.
Preferably, the chromatographic column in the HPLC chromatographic conditions described in step B is Agilent extension-C 18 Columns or Agilent ZORBAX SB-C 18 A column; the column had a specification of 4.6X250 mm, 5. Mu.m.
Preferably, the mobile phase B in the step B is a phosphoric acid solution with the volume percentage of 0.05-0.2%; optimally, the mobile phase B in the step B is a phosphoric acid solution with the volume percentage of 0.05-0.2%.
In the chromatographic conditions described in step B, the detection wavelength is 254nm, and the flow rate is: 0.8mL/min, column temperature: 23-30 ℃, sample injection amount: 5. Mu.L.
Preferably, the column temperature in step B is 25 ℃.
The invention further aims to provide the HPLC standard fingerprint spectrum of the spring oral liquid. Preparing 12 batches of the spring oral liquid samples into a test solution according to the method, separating and detecting by HPLC, and analyzing by using software of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2012 edition recommended by the national formulary Committee to obtain the spring oral liquid HPLC standard fingerprint.
The fingerprint obtained by the invention has 28 common characteristic peaks, the 12 # peak is taken as a reference peak, and the calculated relative retention time is respectively as follows in sequence: 0.147, 0.198, 0.286, 0.363, 0.393, 0.412, 0.458, 0.545, 0.700, 0.753, 0.946, 1.000, 1.085, 1.296, 1.321, 1.676, 1.740, 1.791, 2.017, 2.062, 2.110, 2.212, 2.538, 2.578, 2.616, 2.680, 3.253, 3.347.
By reference substance retention time positioning and chromatographic peak analysis, 7 characteristic peaks are identified, wherein the peak number 6 is gallic acid, the peak number 12 is reference peak morroniside, the peak number 14 is loganin, the peak number 17 is rutin, the peak number 18 is hyperin, the peak number 26 is icariine, and the peak number 28 is baohuoside I. Wherein, peaks 6, 7, 9, 11, 12, 14, 15, 17 and 20 are from dogwood, peaks 3, 10, 13, 16 and 18 are from semen cuscutae, and peaks 21, 22, 23, 24, 25, 26, 27 and 28 are from herba epimedii.
Compared with the prior art, the technical scheme of the invention has the following beneficial technical effects:
1) According to the HPLC fingerprint detection method of the spring oral liquid, according to the prescription component composition characteristics of the spring oral liquid, the optimal HPLC condition is optimized, the problems that characteristic peaks are difficult to separate and the interference of impurity peaks is large are solved, the obtained fingerprint has high separation degree and good peak shape, each characteristic chromatographic peak realizes good baseline separation, the stability is good, the characteristic peaks are more, and the quality of the spring oral liquid can be comprehensively and accurately evaluated.
2) The detection method of the invention takes morroniside as a reference peak, determines 28 common peaks as common peaks forming the fingerprint of the oral liquid of the spring, and has more abundant and comprehensive fingerprint composition.
3) The HPLC fingerprint of the oral liquid overcomes the defects that the detection index is single and the internal quality cannot be reflected in the prior art, and the method for establishing the HPLC fingerprint of the oral liquid has high precision, good stability and good repeatability, and can comprehensively evaluate the quality of the oral liquid by comparing the existence of common peaks in the obtained fingerprint, thereby more effectively ensuring the quality of the finished product.
Drawings
FIG. 1 is a superimposed HPLC fingerprint of 12 samples of the spring oral liquid in example 1;
FIG. 2 is a standard control fingerprint of the Yangchun oral liquid of example 1;
FIG. 3 is a graph showing the assignment of common peaks in the oral liquid of example 1;
FIG. 4 is a superimposed HPLC fingerprint of 12 samples of the spring oral liquid in example 2;
FIG. 5 is a standard control fingerprint of the Yangchun oral liquid of example 2;
FIG. 6 is a superimposed HPLC fingerprint of 12 samples of the spring oral liquid in example 3;
FIG. 7 is a standard control fingerprint of the Yangchun oral liquid of example 3;
FIG. 8 is a superimposed HPLC fingerprint of 12 samples of the spring oral liquid in example 4;
FIG. 9 is a standard control fingerprint of the Yangchun oral liquid of example 4;
FIG. 10 is a superimposed HPLC fingerprint of 12 samples of the spring oral liquid in example 5;
fig. 11 is a standard control fingerprint of the yangchun oral liquid of example 5.
Detailed Description
For the purposes of making the objects, technical solutions and advantages of the present application more clear and transparent, the technical solutions of the present invention will be further described with reference to the accompanying drawings and specific embodiments, and it is apparent that the following embodiments are part of, but not all of, the embodiments of the present application. Accordingly, the following detailed description of the embodiments is not intended to limit the scope of the invention, as claimed, but it is intended to cover all such equivalents and modifications as would be included within the scope of the invention.
EXAMPLE 1 establishment of HPLC Standard fingerprint of Potentilla chinensis oral liquid
1 instrument and reagent
1.1 instruments
Waters Acquity Arc high performance liquid chromatograph (united states): 2998PDA detector, quaternary ultra-high pressure gradient pump, empower chromatography workstation.
1.2 reagents
12 batches of the spring oral liquid are provided by Lunan thick general pharmaceutical Co., ltd, and sample batch numbers are shown in Table 1. Acetonitrile is chromatographic pure, water is ultrapure water, and the rest reagents are analytically pure.
Table 1 sample lot number for the test of the oral liquid for spring
2 methods and results
2.1 chromatographic conditions
Agilent Extend-C 18 (4.6X250 mm,5 μm) column; acetonitrile is taken as a mobile phase A, and a phosphoric acid aqueous solution with the volume percentage content of 0.1 percent is taken as a mobile phase B; gradient elution:
flow rate: 0.8mL/min; column temperature: 25 ℃; detection wavelength: 254nm, sample volume: 5. Mu.L.
2.2 preparation of solutions
2.2.1 mixing control solutions
Precisely weighing appropriate amount of reference substances such as gallic acid, morroniside, loganin, rutin, hyperin, icariin and baohuoside I, adding 80% methanol to prepare mixed mother solution containing 54.623 mug, 189.214 mug, 128.578 mug, 54.564 mug, 40.902 mug, 115.834 mug and 9.479 mug of the reference substances per 1mL, precisely sucking 5mL of the mother solution, placing into a 10mL volumetric flask, adding 80% methanol to dilute to constant volume, and shaking uniformly to obtain mixed solution containing 27.3115 mug, 94.607 mug, 64.289 mug, 27.282 mug, 20.451 mug, 57.917 mug and 4.7395 mug of the reference substances per 1 mL.
2.2.2 sample solutions
Precisely measuring 2mL of the oral liquid of the spring, placing the oral liquid in a 10mL volumetric flask, adding 80% methanol for constant volume, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the solution of the test sample.
2.2.3 Single drug test solution
The single medicine extracting solution of dogwood, semen cuscutae and epimedium is prepared according to the prescription process, and the single medicine sample solution is prepared according to the preparation method of the sample solution under the item of 2.2.2.
2.3 construction of finger print and similarity evaluation
2.3.1 construction of finger print
Taking 12 batches of the spring oral liquid samples, preparing a sample solution according to the method under the item "2.2.2", and carrying out detection analysis according to the method under the item "2.1" to obtain 12 batches of sample chromatographic superposition diagrams (figure 1). As can be seen, the major chromatographic peak of the 12 batches of samples peaked within 80 min. And (3) introducing the obtained various chromatograms into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition) for chromatographic peak matching, and fitting to generate a standard control fingerprint (figure 2).
2.3.2 similarity evaluation
And selecting a No. 12 peak with good peak shape and separation degree, no interference chromatographic peak on the left and right and high response value in the chromatogram as a reference peak, determining 28 common peaks as characteristic peaks for forming the fingerprint of the spring oral liquid, and calculating the relative retention time of each characteristic peak (table 2). The result shows that the relative retention time RSD of each common peak is within 0.146%, which indicates that the peak-out time of the oral liquid in spring of different batches is stable.
TABLE 2 relative retention time of fingerprint spectrum common peaks of 12 batches of spring oral liquid
Comparing the chromatograms of 12 batches of samples with standard control fingerprints by using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), and the results show that the similarity of the 12 batches of samples is above 0.995 (Table 3), and the quality stability and uniformity among all samples are good.
TABLE 3 similarity evaluation results of 12 batches of oral liquid for spring
2.4 assignment of common Peak
The single drug test solution and the control solution were taken and analyzed under the chromatographic conditions under the "2.1" item, and the results are shown in FIG. 3. By reference substance retention time positioning and chromatographic peak analysis, 7 characteristic peaks are identified, wherein the peak number 6 is gallic acid, the peak number 12 is reference peak morroniside, the peak number 14 is loganin, the peak number 17 is rutin, the peak number 18 is hyperin, the peak number 26 is icariine, and the peak number 28 is baohuoside I. Wherein, peaks 6, 7, 9, 11, 12, 14, 15, 17 and 20 are from dogwood, peaks 3, 10, 13, 16 and 18 are from semen cuscutae, and peaks 21, 22, 23, 24, 25, 26, 27 and 28 are from herba epimedii.
2.5 methodology investigation
2.5.1 precision test
Taking a sample of the oral liquid (batch number: 24200041) of the spring, preparing a test solution according to the method under the item "2.2.2", continuously injecting 6 needles, measuring according to the chromatographic condition under the item "2.1", and examining the precision. Relative retention times (Table 4) and relative peak areas (Table 5) RSDs for the consensus peaks 1-28 were calculated using peak number 12 as the reference peak. As a result, the relative retention time of each common peak was less than 0.095% and the relative peak area was less than 2.03%, indicating good instrument precision.
TABLE 4 relative retention time of same batch of Potentilla striata oral liquid
TABLE 5 relative peak area of same batch of Potentilla striata oral liquid
2.5.2 stability test
Sample of the oral liquid (batch No. 24200041) of spring was taken, a sample solution was prepared according to the method under item "2.2.2", samples were taken under chromatographic conditions under item "2.1" for 0, 2, 4, 6, 8, 12, 16, 20, 24 hours, peak No. 12 was used as reference peak, and the relative retention time RSD of the common peak No. 1-28 was calculated to be less than 0.3% (Table 6), and the relative peak area RSD was less than 1.727% (Table 7), indicating that the sample solution was stable within 24 hours.
TABLE 6 relative retention time of different time-dependent oral solutions of the same batch
TABLE 7 relative peak areas for different time-dependent spring oral solutions of the same batch
2.4.3 repeatability test
Taking a sample of the oral liquid (batch number: 24200041) of the spring, precisely measuring 6 parts of the sample, preparing 6 parts of the sample solution according to the method under the item "2.2.2", measuring according to the chromatographic condition under the item "2.1", and examining the repeatability. Relative retention time and RSD relative peak area of each common peak were calculated using peak No. 12 as reference peak. As a result, the relative retention time of each chromatographic peak was less than 1.29% (Table 8), and the relative peak area was less than 1.414% (Table 9), indicating good reproducibility of the method.
Table 86 relative retention time of samples of same batch of Yangchun oral liquid
Table 96 relative peak area of test pieces of same batch of Yangchun oral liquid
EXAMPLE 2 establishment of HPLC Standard fingerprint of Potentilla chinensis oral liquid
1 instrument and reagent
1.1 instruments
Waters Acquity Arc high performance liquid chromatograph (united states): 2998PDA detector, quaternary ultra-high pressure gradient pump, empower chromatography workstation.
1.2 reagents
12 batches of the spring oral liquid are provided by Lunan thick general pharmaceutical Co., ltd, and sample batch numbers are shown in Table 1. Acetonitrile is chromatographic pure, water is ultrapure water, and the rest reagents are analytically pure.
2 methods and results
2.1 chromatographic conditions
Agilent Extend-C 18 (4.6X250 mm,5 μm) column; acetonitrile is taken as a mobile phase A, and a phosphoric acid aqueous solution with the volume percentage content of 0.1 percent is taken as a mobile phase B; gradient elution:
flow rate: 0.8mL/min; column temperature: 30 ℃; detection wavelength: 254nm, sample volume: 5. Mu.L.
2.2 preparation of sample solutions
Precisely measuring 2mL of the oral liquid of the spring, placing the oral liquid in a 10mL volumetric flask, adding 30% methanol for constant volume, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the solution of the test sample.
2.3 construction of finger print
Taking 12 batches of the spring oral liquid samples, preparing a test solution according to the method under the item "2.2", and performing detection analysis according to the method under the item "2.1" to obtain 12 batches of sample chromatographic superposition diagrams (figure 4). As can be seen, the major chromatographic peak of the 12 batches of samples peaked within 80 min. And (3) introducing the obtained various chromatograms into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition) for chromatographic peak matching, and fitting to generate a standard control fingerprint (figure 5). And (3) taking the No. 12 peak as a reference peak, and calculating the relative retention time of 28 common peaks of the standard fingerprint spectrum to be respectively: 0.158, 0.208, 0.283, 0.357, 0.384, 0.400, 0.547, 0.677, 0.741, 0.934, 0.958, 1, 1.058, 1.291, 1.312, 1.640, 1.693, 1.752, 1.988, 2.044, 2.100, 2.202, 2.538, 2.579, 2.618, 2.683, 3.150, 3.250. The similarity of the standard control fingerprint of the Yangchun oral liquid established in the embodiment 1 is calculated to be 0.992 by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition).
EXAMPLE 3 establishment of HPLC Standard fingerprint of Potentilla chinensis oral liquid
1 instrument and reagent
1.1 instruments
Waters Acquity Arc high performance liquid chromatograph (united states): 2998PDA detector, quaternary ultra-high pressure gradient pump, empower chromatography workstation.
1.2 reagents
12 batches of the spring oral liquid are provided by Lunan thick general pharmaceutical Co., ltd, and sample batch numbers are shown in Table 1. Acetonitrile is chromatographic pure, water is ultrapure water, and the rest reagents are analytically pure.
2 methods and results
2.1 chromatographic conditions
Agilent ZORBAX SB-C 18 (4.6X250 mm,5 μm) column; acetonitrile is taken as a mobile phase A, and a phosphoric acid aqueous solution with the volume percentage content of 0.2 percent is taken as a mobile phase B; gradient elution:
| time, min | Mobile phase a% | Mobile phase B% |
| 0-60 | 2％→32％ | 98％→68％ |
| 60-65 | 32％→50％ | 68％→50％ |
| 65-70 | 50％ | 50％ |
| 70-71 | 50％→2％ | 50％→98％ |
| 71-80 | 2％ | 98％ |
Flow rate: 0.8mL/min; column temperature: 23 ℃; detection wavelength: 254nm, sample volume: 5. Mu.L.
2.2 preparation of sample solutions
Precisely measuring 2mL of the oral liquid of the spring, placing the oral liquid in a 10mL volumetric flask, adding 50% methanol for constant volume, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the solution of the test sample.
2.3 construction of finger print
Taking 12 batches of the spring oral liquid samples, preparing a test solution according to the method under the item "2.2", and performing detection analysis according to the method under the item "2.1" to obtain 12 batches of sample chromatographic superposition diagrams (figure 6). As can be seen, the major chromatographic peak of the 12 batches of samples peaked within 80 min. And (3) introducing the obtained various chromatograms into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition) for chromatographic peak matching, and fitting to generate a standard control fingerprint (figure 7). And (3) taking the No. 12 peak as a reference peak, and calculating the relative retention time of 28 common peaks of the standard fingerprint spectrum to be respectively: 0.148, 0.196, 0.278, 0.353, 0.383, 0.398, 0.447, 0.537, 0.681, 0.745, 0.971, 1, 1.072, 1.299, 1.320, 1.674, 1.732, 1.788, 2.011, 2.077, 2.132, 2.235, 2.573, 2.614, 2.653, 2.717, 3.299, 3.393. The similarity of the standard control fingerprint of the Yangchun oral liquid established in the embodiment 1 is calculated to be 0.993 by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition).
EXAMPLE 4 establishment of HPLC Standard fingerprint of Potentilla chinensis oral liquid
1 instrument and reagent
1.1 instruments
Waters Acquity Arc high performance liquid chromatograph (united states): 2998PDA detector, quaternary ultra-high pressure gradient pump, empower chromatography workstation.
1.2 reagents
12 batches of the spring oral liquid are provided by Lunan thick general pharmaceutical Co., ltd, and sample batch numbers are shown in Table 1. Acetonitrile is chromatographic pure, water is ultrapure water, and the rest reagents are analytically pure.
2 methods and results
2.1 chromatographic conditions
Agilent Extend-C 18 (4.6X250 mm,5 μm) column; acetonitrile is taken as a mobile phase A, and 0.05 percent of phosphoric acid is taken as a volume percentageThe aqueous solution is mobile phase B; gradient elution:
| time, min | Mobile phase a% | Mobile phase B% |
| 0-60 | 2％→32％ | 98％→68％ |
| 60-65 | 32％→50％ | 68％→50％ |
| 65-70 | 50％ | 50％ |
| 70-71 | 50％→2％ | 50％→98％ |
| 71-80 | 2％ | 98％ |
Flow rate: 0.8mL/min; column temperature: 27 ℃; detection wavelength: 254nm, sample volume: 5. Mu.L.
2.2 preparation of sample solutions
Precisely measuring 2mL of the oral liquid of the spring, placing the oral liquid in a 10mL volumetric flask, adding 70% methanol for constant volume, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the solution of the test sample.
2.3 construction of finger print
Taking 12 batches of the spring oral liquid samples, preparing a test solution according to the method under the item "2.2", and performing detection analysis according to the method under the item "2.1" to obtain 12 batches of sample chromatographic superposition diagrams (figure 8). As can be seen, the major chromatographic peak of the 12 batches of samples peaked within 80 min. And (3) introducing the obtained various chromatograms into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition) for chromatographic peak matching, and fitting to generate a standard control fingerprint (figure 9). And (3) taking the No. 12 peak as a reference peak, and calculating the relative retention time of 28 common peaks of the standard fingerprint spectrum to be respectively: 0.145, 0.200, 0.293, 0.372, 0.403, 0.424, 0.468, 0.551, 0.717, 0.758, 0.948, 1, 1.095, 1.289, 1.320, 1.677, 1.746, 1.791, 2.019, 2.088, 2.121, 2.188, 2.502, 2.541, 2.577, 2.641, 3.201, 3.295. The similarity of the standard control fingerprint of the Yangchun oral liquid established in the embodiment 1 is calculated to be 0.996 by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition).
EXAMPLE 5 establishment of HPLC Standard fingerprint of Potentilla chinensis oral liquid
1 instrument and reagent
1.1 instruments
Waters Acquity Arc high performance liquid chromatograph (united states): 2998PDA detector, quaternary ultra-high pressure gradient pump, empower chromatography workstation.
1.2 reagents
12 batches of the spring oral liquid are provided by Lunan thick general pharmaceutical Co., ltd, and sample batch numbers are shown in Table 1. Acetonitrile is chromatographic pure, water is ultrapure water, and the rest reagents are analytically pure.
2 methods and results
2.1 chromatographic conditions
Agilent Extend-C 18 (4.6X250 mm,5 μm) column; acetonitrile is taken as a mobile phase A, and a phosphoric acid aqueous solution with the volume percentage content of 0.2 percent is taken as a mobile phase B; gradient elution:
| time, min | Mobile phase a% | Mobile phase B% |
| 0-61 | 2％→33％ | 98％→67％ |
| 61-65 | 33％→50％ | 67％→50％ |
| 65-70 | 50％ | 50％ |
| 70-71 | 50％→2％ | 50％→98％ |
| 71-80 | 2％ | 98％ |
Flow rate: 0.8mL/min; column temperature: 28 ℃; detection wavelength: 254nm, sample volume: 5. Mu.L.
2.2 preparation of sample solutions
Precisely measuring 2mL of the oral liquid of the spring, placing the oral liquid in a 10mL volumetric flask, adding 100% methanol for constant volume, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the solution of the test sample.
2.3 construction of finger print
Taking 12 batches of the spring oral liquid samples, preparing a test solution according to the method under the item "2.2", and performing detection analysis according to the method under the item "2.1" to obtain 12 batches of sample chromatographic superposition diagrams (figure 10). As can be seen, the major chromatographic peak of the 12 batches of samples peaked within 80 min. And (3) introducing the obtained various chromatograms into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition) for chromatographic peak matching, and fitting to generate a standard control fingerprint (figure 11). And (3) taking the No. 12 peak as a reference peak, and calculating the relative retention time of 28 common peaks of the standard fingerprint spectrum to be respectively: 0.158, 0.207, 0.282, 0.356, 0.383, 0.399, 0.545, 0.674, 0.740, 0.934, 0.956, 1, 1.058, 1.292, 1.312, 1.641, 1.641, 1.694, 1.753, 1.991, 2.047, 2.103, 2.205, 2.542, 2.583, 2.622, 2.687, 3.220, 3.346. The similarity of the standard control fingerprint of the Yangchun oral liquid established in the embodiment 1 is calculated to be 0.991 by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition).
The foregoing examples are merely preferred embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the invention. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit and scope of the invention.
Claims (6)
1. The method for establishing the HPLC fingerprint of the oral liquid of the spring is characterized by comprising the following steps:
A. preparation of test solution: taking a spring oral liquid, adding methanol for dilution, filtering, and taking a subsequent filtrate as a sample solution;
determination of hplc chromatographic conditions: chromatographic column: agilent extension-C18 column or Agilent ZORBAX SB-C18 column, acetonitrile as mobile phase A,0.05-0.2% phosphoric acid solution as mobile phase B, gradient elution:
and C, manufacturing HPLC standard fingerprint patterns: and C, analyzing and comparing the sample solution of the oral liquid according to the chromatographic condition of the step B to obtain the HPLC standard fingerprint of the oral liquid composed of the common characteristic peaks of the samples.
2. The method of claim 1, wherein the test solution of step a is prepared by: precisely weighing 2mL of the oral liquid of the spring, adding 30% -100% methanol for diluting to a constant volume, filtering, and taking the subsequent filtrate as a test solution.
3. The method of claim 2, wherein the test solution of step a is prepared by: accurately weighing 2mL of the oral liquid of the spring, adding 80% methanol for diluting to a constant volume of 10mL, filtering, and taking the subsequent filtrate as a test solution.
4. The method of claim 1, wherein the chromatographic column has a specification of 4.6x250 mm,5 μm.
5. The method of claim 1, wherein mobile phase B in step B is a 0.1% phosphoric acid solution by volume.
6. The method of claim 1, wherein the chromatographic conditions of step B detect 254nm wavelength, flow rate: 0.8mL/min, column temperature: 23-30 ℃, sample injection amount: 5. Mu.L.
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