CN113945660A - Construction method and application of characteristic spectrum of strong loquat dew - Google Patents
Construction method and application of characteristic spectrum of strong loquat dew Download PDFInfo
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- CN113945660A CN113945660A CN202111202892.XA CN202111202892A CN113945660A CN 113945660 A CN113945660 A CN 113945660A CN 202111202892 A CN202111202892 A CN 202111202892A CN 113945660 A CN113945660 A CN 113945660A
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
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Abstract
The invention relates to the technical field of medicine quality analysis and control, in particular to a construction method and application of a characteristic spectrum of strong loquat dew. The construction method of the characteristic map of the strong loquat dew comprises the following steps: detecting the test solution by using UPLC to obtain the characteristic spectrum of the strong loquat dew; the chromatographic conditions include: octadecylsilane chemically bonded silica is used as a filler for the chromatographic column; taking a phosphoric acid aqueous solution with the volume fraction of 0.35-0.45% as a mobile phase A, and taking acetonitrile as a mobile phase B for gradient elution; the gradient elution includes: and (3) changing the volume fraction of the mobile phase A from 98-99% to 4-5% in 0-10 min. The method for constructing the characteristic spectrum of the strong loquat syrup can identify the main index components in the loquat leaves, the poppy capsules and the platycodon grandiflorum simultaneously, can comprehensively analyze the relative content of the main components in the strong loquat syrup sample, and has very important significance for comprehensively evaluating the quality of the strong loquat syrup.
Description
Technical Field
The invention relates to the technical field of medicine quality analysis and control, in particular to a construction method and application of a characteristic spectrum of strong loquat dew.
Background
The strong loquat syrup is prepared from seven medicinal materials of loquat leaf, poppy shell, stemona root, swallowwort rhizome, white mulberry root-bark, platycodon root and menthol, and has the effects of nourishing yin, astringing lung, relieving cough and eliminating phlegm.
The characteristic spectrum refers to the spectrum or spectrum of a certain kind or several kinds of ingredients which are common in the preparation and have characteristics, and the establishment of the characteristic spectrum has important significance for effectively controlling the quality of the preparation. The quality control method of strong loquat dew in Chinese pharmacopoeia is mainly embodied in the aspect of controlling the morphine content in poppy shell, but lacks the quality control of loquat leaves which are monarch drugs, and can not achieve the aim of comprehensively evaluating and controlling the quality of the preparation.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for constructing a characteristic map of strong loquat dew, so as to solve the technical problems that the full expression cannot be realized and the quality of the strong loquat dew cannot be controlled in the prior art.
The second purpose of the invention is to provide a method for constructing the characteristic comparison map of the strong loquat dew to realize the judgment and evaluation of the quality of the strong loquat dew to be detected.
The third purpose of the invention is to provide the application of the construction method of the characteristic spectrum of the strong loquat dew in the quality detection of the strong loquat dew.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the construction method of the characteristic map of the strong loquat dew comprises the following steps:
detecting the test solution by using UPLC to obtain the characteristic spectrum of the strong loquat dew;
the chromatographic conditions for the UPLC assay include: octadecylsilane chemically bonded silica is used as a filler for the chromatographic column; taking a phosphoric acid aqueous solution with the volume fraction of 0.35-0.45% as a mobile phase A, and taking acetonitrile as a mobile phase B for gradient elution;
the gradient elution comprises: and (3) changing the volume fraction of the mobile phase A from 98-99% to 4-5% for 0-10 min.
In a specific embodiment of the invention, the gradient elution is:
| time: min | Mobile phase A: is based on | Mobile phase B: is based on |
| 0～1 | 99 | 1 |
| 1～2 | 99→90 | 1→10 |
| 2～5 | 90→80 | 10→20 |
| 5～7.5 | 80→45 | 20→55 |
| 7.5～9 | 45→30 | 55→70 |
| 9～10 | 30→5 | 70→95 |
In the embodiment of the invention, the ultraviolet detection wavelength is 205-215 nm, preferably 208-212 nm, and more preferably 210 nm.
In an embodiment of the present invention, the flow rate is 0.3 to 0.5mL/min, preferably 0.35 to 0.45mL/min, and more preferably 0.4 mL/min.
In a specific embodiment of the present invention, the number of theoretical plates should not be less than 5000 on the morphine peak basis.
In the specific embodiment of the invention, the column temperature is 32-38 ℃, and preferably 35 ℃. Further, the specification of the chromatographic column is as follows: the column length was 100mm, the column inner diameter was 2.1mm, and the particle diameter was 1.8. mu.m.
In a specific embodiment of the present invention, the sample volume for detection is 1 to 2 μ L.
In a specific embodiment of the present invention, the method further comprises: detecting a reference substance solution by using the UPLC; the reference substance comprises morphine reference substance, chlorogenic acid reference substance and platycodin D reference substance.
In a specific embodiment of the present invention, the preparation of the test solution comprises:
adding ammonia test solution into strong loquat dew to be tested, extracting for 3 times by adopting water saturated n-butyl alcohol, merging n-butyl alcohol extract, removing and dissolving to be dry, dissolving residues by using a methanol aqueous solution with the volume fraction of 18-22%, fixing the volume, and taking a subsequent filtrate as a test solution.
Wherein, the ammonia test solution can refer to the preparation method in pharmacopeia, such as: taking 400ml of concentrated ammonia solution, and adding water to dissolve the concentrated ammonia solution into 1000ml of concentrated ammonia solution to obtain the ammonia-containing water.
The invention also provides a construction method of the strong loquat syrup control map, which comprises the following steps:
constructing the characteristic spectrum of the strong loquat dew of more than 10 batches by any one of the methods, and synthesizing the contrast spectrum of the strong loquat dew by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2.0 edition).
In a specific embodiment of the present invention, the control map contains 10 common characteristic peaks, and with peak No. 2 as a reference peak, the relative retention times of the 10 common characteristic peaks are as follows:
0.233～0.274、1.000、1.260～1.479、1.359～1.595、1.675～1.966、2.009～2.358、2.066～2.425、2.134～2.505、2.232～2.621、2.418～2.839。
in a specific embodiment of the present invention, in the control map, the peak 2 is a morphine peak, the peak 4 is a chlorogenic acid peak, and the peak 8 is a platycodin D peak.
The invention also provides application of the construction method of any one of the characteristic maps of the strong loquat dew in quality detection of the strong loquat dew.
In a specific embodiment of the present invention, the method of application comprises:
(a) constructing a characteristic map of the strong loquat dew to be detected according to the construction method of the characteristic map of the strong loquat dew;
(b) and (3) analyzing the characteristic spectrum obtained in the step (a) and the control spectrum of the strong loquat distillate by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (version 2.0), and calculating the similarity.
Compared with the prior art, the invention has the beneficial effects that:
(1) the method for constructing the characteristic spectrum of the strong loquat syrup can identify main index components in loquat leaves, poppy shells and platycodon grandiflorum at the same time, can comprehensively analyze the relative content of the main components in a strong loquat syrup sample, and has very important significance for comprehensively evaluating the quality of the strong loquat syrup;
(2) according to the invention, the construction method of the characteristic map of the strong loquat dew is adopted to establish the contrast map of the main components in the strong loquat dew, and the authenticity and quality of the strong loquat dew to be detected can be judged by comparing the characteristic map of the sample of the strong loquat dew to be detected with the contrast map, so that a new method and means are provided for completely and accurately evaluating the quality of the strong loquat dew.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a characteristic diagram of each of 10 batches of strong loquat distillate according to the embodiment of the present invention;
FIG. 2 is a comparison graph of the synthesis of 10 batches of strong loquat syrup according to the present invention;
FIG. 3 is a fingerprint obtained according to the chromatographic conditions of comparative example 1-1 #;
FIG. 4 is a fingerprint obtained according to the chromatographic conditions of comparative example # 1-2;
FIG. 5 shows the fingerprints obtained under the chromatographic conditions of comparative examples 1-3 #;
FIG. 6 is a fingerprint obtained according to the chromatographic conditions of comparative examples 1-4 #;
FIG. 7 is a fingerprint obtained according to the chromatographic conditions of comparative examples 1 to 5 #;
FIG. 8 is a fingerprint obtained according to the method for preparing the sample solution of comparative example 2.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and the detailed description, but those skilled in the art will understand that the following described embodiments are some, not all, of the embodiments of the present invention, and are only used for illustrating the present invention, and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The construction method of the characteristic map of the strong loquat dew comprises the following steps:
detecting the test solution by using UPLC to obtain the characteristic spectrum of the strong loquat dew;
the chromatographic conditions for the UPLC assay include: octadecylsilane chemically bonded silica is used as a filler for the chromatographic column; taking a phosphoric acid aqueous solution with the volume fraction of 0.35-0.45% as a mobile phase A, and taking acetonitrile as a mobile phase B for gradient elution;
the gradient elution comprises: and (3) changing the volume fraction of the mobile phase A from 98-99% to 4-5% for 0-10 min.
In a specific embodiment of the invention, the gradient elution is:
| time: min | Mobile phase A: is based on | Mobile phase B: is based on |
| 0～1 | 99 | 1 |
| 1～2 | 99→90 | 1→10 |
| 2～5 | 90→80 | 10→20 |
| 5～7.5 | 80→45 | 20→55 |
| 7.5～9 | 45→30 | 55→70 |
| 9～10 | 30→5 | 70→95 |
By adopting the mobile phase and the gradient elution program, the situations of uneven base line of the map and the like can be effectively improved while the separation degree is considered.
In the specific embodiment of the invention, the ultraviolet detection wavelength is 205-215 nm.
As in the different embodiments, the wavelength of the ultraviolet detection can be 205nm, 206nm, 207nm, 208nm, 209nm, 210nm, 211nm, 212nm, 213nm, 214nm, 215nm, etc., preferably 208-212 nm, more preferably 210 nm.
When the wavelength of 210nm is adopted for detection, the number of characteristic peaks in the map is the largest, and the separation effect among the characteristic peaks is good.
In a specific embodiment of the present invention, the flow rate is 0.3 to 0.5 mL/min.
As in the different embodiments, the flow rate may be 0.3mL/min, 0.35mL/min, 0.4mL/min, 0.45mL/min, 0.5mL/min, etc., preferably 0.35-0.45 mL/min, more preferably 0.4 mL/min.
In a specific embodiment of the present invention, the number of theoretical plates should not be less than 5000 on the morphine peak basis.
In the specific embodiment of the invention, the column temperature is 32-38 ℃.
As in the different embodiments, the column temperature can be 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃ and so on, preferably 35 ℃.
In a specific embodiment of the invention, the specification of the chromatography column is: the column length was 100mm, the column inner diameter was 2.1mm, and the particle diameter was 1.8. mu.m.
In a specific embodiment of the present invention, the sample volume for detection is 1 to 2 μ L, such as 1 μ L or 2 μ L, preferably 1 μ L.
In a specific embodiment of the present invention, the conditions for UPLC detection include: octadecylsilane chemically bonded silica is used as a filler for the chromatographic column; the column length is 100mm, the column inner diameter is 2.1mm, and the particle size is 1.8 μm; performing gradient elution by taking a phosphoric acid aqueous solution with the volume fraction of 0.4% as a mobile phase A and acetonitrile as a mobile phase B;
the ultraviolet detection wavelength is 210 nm; the flow rate is 0.4 mL/min; the column temperature was 35 ℃; the sample injection amount is 1 mu L; the number of theoretical plates is not less than 5000 calculated according to morphine peak;
the gradient elution is:
| time: min | Mobile phase A: is based on | Mobile phase B: is based on |
| 0～1 | 99 | 1 |
| 1～2 | 99→90 | 1→10 |
| 2～5 | 90→80 | 10→20 |
| 5～7.5 | 80→45 | 20→55 |
| 7.5～9 | 45→30 | 55→70 |
| 9～10 | 30→5 | 70→95 |
The invention adopts Ultra Performance Liquid Chromatography (UPLC) method to determine, can shorten the detection time to 10min, improve the detection efficiency, and can reduce the dosage of mobile phase and reduce the sample volume of the sample solution and the reference substance solution.
In a specific embodiment of the present invention, the method further comprises: detecting a reference substance solution by using the UPLC; the reference substance comprises morphine reference substance, chlorogenic acid reference substance and platycodin D reference substance. Further, in the reference solution, the concentrations of the morphine reference substance, the chlorogenic acid reference substance and the platycodin D are respectively 50 mug/mL, 20 mug/mL and 20 mug/mL.
In a specific embodiment of the present invention, the preparation of the reference solution comprises: morphine reference substance, chlorogenic acid reference substance and platycodin D reference substance are respectively weighed, 18-22% methanol water solution is added, and reference substance solutions containing 50 mu g of morphine reference substance, 20 mu g of chlorogenic acid reference substance and 20 mu g of platycodin D per 1mL are respectively prepared.
In a specific embodiment of the present invention, the preparation of the test solution comprises:
adding ammonia test solution into strong loquat dew to be tested, extracting for 3 times by adopting water saturated n-butyl alcohol, merging n-butyl alcohol extract, removing and dissolving to be dry, dissolving residues by using a methanol aqueous solution with the volume fraction of 18-22%, fixing the volume, and taking a subsequent filtrate as a test solution.
The strong loquat syrup is mainly extracted by water decoction, wherein water-soluble components are more, so that a sample is extracted by using water-saturated n-butyl alcohol, and simultaneously, components with higher polarity in the sample can be retained to the maximum extent by using 18-20% methanol aqueous solution to dissolve the sample, so that the characteristic peak expressed in the characteristic spectrum of a test solution is the maximum.
In the specific embodiment of the invention, the volume ratio of the strong loquat syrup to be detected to the ammonia test solution is 25: 1.
In a specific embodiment of the invention, the extraction with water-saturated n-butanol is performed for 3 times, and the volume ratio of the water-saturated n-butanol used each time to the strong loquat dew to be tested is 1: 1, 0.8: 1 and 0.8: 1 respectively.
In a specific embodiment of the invention, the constant volume is consistent with the volume of the measured strong loquat dew to be measured. That is, when the volume of the strong loquat syrup to be measured which is initially taken is 25mL, the final residue is dissolved in the methanol aqueous solution and the volume is set to 25 mL.
In a specific embodiment of the present invention, the preparing of the test solution specifically comprises:
precisely measuring 25mL of strong loquat syrup to be detected, adding 1mL of ammonia test solution, shaking and extracting with 25mL, 20mL and 20mL of water-saturated n-butyl alcohol respectively for 3 times, combining n-butyl alcohol extracts, recovering solvent to dryness, adding a proper amount of 20 volume percent methanol aqueous solution into residues to dissolve, and fixing the volume to 25mL, shaking up, filtering, and collecting the subsequent filtrate as a test solution.
The invention also provides a construction method of the strong loquat syrup control map, which comprises the following steps:
constructing the characteristic spectrum of the strong loquat dew of more than 10 batches by any one of the methods, and synthesizing the contrast spectrum of the strong loquat dew by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2.0 edition).
In a specific embodiment of the present invention, the control map contains 10 common characteristic peaks, and with peak No. 2 as a reference peak, the relative retention times of the 10 common characteristic peaks are as follows:
0.233～0.274、1.000、1.260～1.479、1.359～1.595、1.675～1.966、2.009～2.358、2.066～2.425、2.134～2.505、2.232～2.621、2.418～2.839。
the common characteristic peaks jointly form the control fingerprint characteristics of the main component of the strong loquat distillate.
In a specific embodiment of the present invention, in the control map, the peak 2 is a morphine peak, the peak 4 is a chlorogenic acid peak, and the peak 8 is a platycodin D peak.
The invention also provides application of the construction method of any one of the characteristic maps of the strong loquat dew in quality detection of the strong loquat dew.
In a specific embodiment of the present invention, the method of application comprises:
(a) constructing a characteristic map of the strong loquat dew to be detected according to the construction method of the characteristic map of the strong loquat dew;
(b) and (3) analyzing the characteristic spectrum obtained in the step (a) and the control spectrum of the strong loquat distillate by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (version 2.0), and calculating the similarity.
In actual operation, the quality of the strong loquat distillate to be tested is evaluated according to the numerical value of the similarity, and if the similarity is required to be more than 0.9, the strong loquat distillate is judged to be a qualified product.
Example 1
The embodiment provides a method for constructing a characteristic spectrum of strong loquat syrup, which comprises the following steps:
(1) preparation of test solution
Precisely measuring 25mL of strong loquat liquid, adding 1mL of ammonia test solution, shaking and extracting with 25mL, 20mL and 20mL of water-saturated n-butyl alcohol respectively for 3 times, combining n-butyl alcohol extracts, recovering solvent to dryness, adding a proper amount of 20% methanol aqueous solution to residues to dissolve the residues, transferring the residues to a 25mL brown measuring flask, adding 20% methanol aqueous solution to the scale, shaking uniformly, filtering, and collecting the subsequent filtrate as a test solution.
(2) Preparation of reference solutions
Accurately weighing appropriate amount of morphine reference substance, chlorogenic acid reference substance, and platycodin D reference substance, and adding 20% methanol water solution to obtain a mixed solution containing 50 μ g of morphine reference substance, 20 μ g of chlorogenic acid reference substance, and 20 μ g of platycodin D per lmL as reference substance solution.
(3) Detection conditions
The chromatographic column uses octadecylsilane chemically bonded silica as filler (ZORBAX Eclipse Plus C18, column length 100mm, column inner diameter 2.1mm, particle size 1.8 μm); the mobile phase A is phosphoric acid aqueous solution with volume fraction of 0.4%, the mobile phase B is acetonitrile, and the gradient elution is carried out, wherein the specific gradient elution procedure is shown in table 1; column temperature: 35 ℃; detection wavelength: 210 nm; flow rate: 0.4 mL/min; sample introduction amount: 1 mu L of the solution; the number of theoretical plates should not be less than 5000 calculated according to morphine peak.
TABLE 1 gradient elution procedure (volume fraction)
(4) Detection step
Precisely absorbing 1 μ L of reference solution and sample solution, respectively, injecting into ultra high performance liquid chromatograph, measuring, and recording chromatogram to obtain UPLC characteristic spectrum of the strong loquat distillate.
Example 2
The embodiment provides a method for constructing a control map of strong loquat syrup, which comprises the following steps:
10 batches (lot numbers are 2012001, 2012003, 2012011, 2012018, 2012019, 2012020, 2012021, 2101001, 2101002 and 2101003 respectively) of strong loquat distillate are detected according to the method in the example 1 to obtain 10 characteristic maps, as shown in figure 1 (wherein each fingerprint map corresponds to synthesized contrast maps with lot numbers of S1: 2012001, S2: 2012003, S3: 2012011, S4: 2012018, S5: 2012019, S6: 2012020, S7: 2012021, S8: 2101001, S9: 2101002, S10: 2101003 and R);
the 10 characteristic maps are introduced into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (version 2.0), and a reference map is synthesized, which is shown in figure 2. The reference map contains 10 common characteristic peaks, and the No. 2 peak is taken as a reference peak, and the relative retention time and the relative peak area of the 10 common characteristic peaks are as follows:
relative retention time: 1# -0.233-0.274, 2# -1.000, 3# -1.260-1.479, 4# -1.359-1.595, 5# -1.675-1.966, 6# -2.009-2.358, 7# -2.066-2.425, 8# -2.134-2.505, 9# -2.232-2.621, 10# -2.418-2.839;
relative peak area: 1# -0.054-0.066, 2# -1.000, 3# -0.158-0.186, 4# -0.115-0.135, 5# -0.080-0.094, 6# -0.589-0.692, 7# -0.263-0.309, 8# -0.138-0.162, 9# -0.123-0.144, 10# -0.061-0.072.
According to the reference solution control, the 2# (S) peak is morphine control peak, the 4# (S) peak is chlorogenic acid control peak, and the 8# (S) peak is platycodin D control peak.
Taking 10 batches of strong loquat distillate (with the batch numbers of 2012002, 2012006, 2012010, 2012013, 2012014, 2101004, 2101005, 2101006, 2101007 and 2101008 in sequence), detecting according to the method in the embodiment 1, introducing 10 measured characteristic maps into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2.0 version), and calculating the similarity with a synthesized comparison map, wherein the similarity sequentially comprises the following steps: 0.92, 0.95, 0.94, 0.95, 0.96 and 0.94, and the similarity is between 0.92 and 0.96, which shows that the fingerprint spectrum measuring method constructed by the invention can be used for the quality control of the strong loquat dew.
The methodology of the method is verified according to the relevant guiding principle in the current pharmacopoeia, and the construction method of the invention is proved to have good specificity, stability, repeatability and the like.
Comparative example 1
Comparative example 1 provides fingerprints for 5 other samples obtained under different chromatographic conditions. The specific process is as in example 1, except that: the chromatographic conditions are different.
The chromatographic conditions for comparative examples 1-1# were: the chromatographic column uses octadecylsilane chemically bonded silica as filler (ZORBAX Eclipse Plus C18, column length 100mm, column inner diameter 2.1mm, particle size 1.8 μm); the mobile phase A is phosphoric acid aqueous solution with the volume fraction of 0.4 percent, the mobile phase B is acetonitrile, and the gradient elution is carried out, wherein the specific gradient elution procedure is shown in a table 2; column temperature: 35 ℃; detection wavelength: 210 nm; flow rate: 0.4 mL/min; sample introduction amount: 1 mu L of the solution;
TABLE 2 gradient elution procedure (volume fraction)
| Time: min | Mobile phase A: is based on | Mobile phase B: is based on |
| 0～1 | 95 | 5 |
| 1～7.5 | 95→78 | 5→22 |
| 7.5～12 | 78→5 | 22→95 |
| 12～15 | 5 | 95 |
The chromatographic conditions for comparative examples 1-2# were: the chromatographic column uses octadecylsilane chemically bonded silica as filler (ZORBAX Eclipse Plus C18, column length 100mm, column inner diameter 2.1mm, particle size 1.8 μm); the mobile phase A is phosphoric acid aqueous solution with the volume fraction of 0.4 percent, the mobile phase B is acetonitrile, and the gradient elution is carried out, wherein the specific gradient elution procedure is shown in a table 3; column temperature: 35 ℃; detection wavelength: 210 nm; flow rate: 0.4 mL/min; sample introduction amount: 1 mu L of the solution;
TABLE 3 gradient elution procedure (volume fraction)
| Time: min | Mobile phase A: is based on | Mobile phase B: is based on |
| 0～1 | 100→99 | 0→1 |
| 1～2 | 99→90 | 1→10 |
| 2～5 | 90→80 | 10→20 |
| 5～8 | 80→45 | 20→55 |
| 8～10 | 45→30 | 55→70 |
| 10～12 | 30→10 | 70→90 |
| 12～15 | 10 | 90 |
The chromatographic conditions for comparative examples 1-3# were: the chromatographic column uses octadecylsilane chemically bonded silica as filler (ZORBAX Eclipse Plus C18, column length 100mm, column inner diameter 2.1mm, particle size 1.8 μm); the mobile phase A is phosphoric acid aqueous solution with the volume fraction of 0.4 percent, the mobile phase B is acetonitrile, and the gradient elution is carried out, wherein the specific gradient elution procedure is shown in a table 4; column temperature: 35 ℃; detection wavelength: 300 nm; flow rate: 0.4 mL/min; sample introduction amount: 1 mu L of the solution;
TABLE 4 gradient elution procedure (volume fraction)
The chromatographic conditions for comparative examples 1-4# were: the detection wavelength of 300nm is changed to 210nm on the basis of the chromatographic condition of the comparative example 3, and other chromatographic conditions are the same as the chromatographic condition of the comparative example 3;
the chromatographic conditions for comparative examples 1-5# were: the chromatographic column uses octadecylsilane chemically bonded silica as filler (ZORBAX Eclipse Plus C18, column length 100mm, column inner diameter 2.1mm, particle size 1.8 μm); the mobile phase A is phosphoric acid aqueous solution with the volume fraction of 0.4 percent, the mobile phase B is acetonitrile, and the gradient elution is carried out, wherein the specific gradient elution procedure is shown in a table 5; column temperature: 30 ℃; detection wavelength: 210 nm; flow rate: 0.4 mL/min; sample introduction amount: 1 mu L of the solution;
TABLE 5 gradient elution procedure (volume fraction)
| Time: min | Mobile phase A: is based on | Mobile phase B: is based on |
| 0～1.5 | 100→99 | 0→1 |
| 1.5～2 | 99→85 | 1→15 |
| 2～5 | 85→80 | 15→20 |
| 5～7.5 | 80→45 | 20→55 |
| 7.5～9 | 45→30 | 55→70 |
| 9～10 | 30→10 | 70→90 |
| 10～12 | 10 | 90 |
The fingerprints of the samples obtained by the above method are shown in fig. 3 to 7, respectively. Different chromatographic conditions are contrasted and examined, so that the chromatographic conditions in the invention can reflect more chromatographic peaks in the sample and have better separation degree.
Comparative example 2
Comparative example 2 provides finger prints of samples from different test sample solutions prepared according to the methods. The specific process is as in example 1, except that: the preparation methods of the test solution are different.
The preparation of the test solution of comparative example 2 includes: precisely measuring 25mL of strong loquat syrup, shaking and extracting with 25mL, 20mL and 20mL of water-saturated n-butyl alcohol respectively for 3 times, combining n-butyl alcohol extracts, recovering solvent to dry, adding a proper amount of 20% methanol aqueous solution into residues to dissolve the residues, transferring the residues into a 25mL brown measuring flask, adding 20% methanol aqueous solution to scale, shaking up, filtering, and collecting the subsequent filtrate as a sample solution.
The fingerprint obtained by the above method is shown in FIG. 8. By comparing and inspecting the preparation methods of different test solution, the preparation method of the test solution can simultaneously extract several components such as morphine, chlorogenic acid, platycodin D and the like, and can more comprehensively characterize and control the quality of the strong loquat syrup.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The construction method of the characteristic map of the strong loquat dew is characterized by comprising the following steps:
detecting the test solution by using UPLC to obtain the characteristic spectrum of the strong loquat dew;
the chromatographic conditions for the UPLC assay include: octadecylsilane chemically bonded silica is used as a filler for the chromatographic column; taking a phosphoric acid aqueous solution with the volume fraction of 0.35-0.45% as a mobile phase A, and taking acetonitrile as a mobile phase B for gradient elution;
the gradient elution comprises: and (3) changing the volume fraction of the mobile phase A from 98-99% to 4-5% for 0-10 min.
2. The method of claim 1, wherein the gradient elution is:
。
3. The method of claim 1, wherein the ultraviolet detection wavelength is 205-215 nm;
preferably, the ultraviolet detection wavelength is 208-212 nm.
4. The method of claim 1, wherein the flow rate is 0.3 to 0.5mL/min, and/or the column temperature is 32 to 38 ℃;
preferably, the flow rate is 0.4 mL/min;
preferably, the specification of the chromatographic column is as follows: the column length was 100mm, the column inner diameter was 2.1mm, and the particle diameter was 1.8. mu.m.
5. The method of claim 1, wherein the conditions for UPLC detection comprise: the chromatographic column takes octadecylsilane chemically bonded silica as a filler, the column length is 100mm, the inner diameter of the column is 2.1mm, and the particle size is 1.8 mu m;
performing gradient elution by taking a phosphoric acid aqueous solution with the volume fraction of 0.4% as a mobile phase A and acetonitrile as a mobile phase B; the ultraviolet detection wavelength is 210 nm; the flow rate is 0.4 mL/min; the column temperature was 35 ℃; the sample injection amount is 1 mu L; the number of theoretical plates is not less than 5000 calculated according to morphine peak;
the gradient elution is:
。
6. The method of any one of claims 1-5, further comprising: detecting a reference substance solution by using the UPLC; the reference substance comprises morphine reference substance, chlorogenic acid reference substance and platycodin D reference substance.
7. The method of claim 1, wherein the preparing of the test solution comprises:
adding ammonia test solution into strong loquat dew to be tested, extracting for 3 times by adopting water saturated n-butyl alcohol, merging n-butyl alcohol extract, removing the solution until the n-butyl alcohol extract is dry, dissolving residues by using a methanol aqueous solution with the volume fraction of 18-22%, fixing the volume, and taking the subsequent filtrate as a test solution;
preferably, the volume ratio of the strong loquat syrup to be detected to the ammonia test solution is 25: 1;
preferably, the volume ratio of the water-saturated n-butanol to the strong loquat dew to be detected in the 3-time extraction with water-saturated n-butanol is 1: 1, 0.8: 1 and 0.8: 1 respectively.
8. The construction method of the strong loquat dew control map is characterized by comprising the following steps:
the method of any one of claims 1-7 is adopted to construct the characteristic spectrum of the strong loquat dew with more than 10 batches, and a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2.0 edition) is adopted to synthesize the contrast spectrum of the strong loquat dew.
9. The method for constructing the control map of loquat dew according to claim 8, wherein the control map comprises 10 common characteristic peaks, and the relative retention time of the 10 common characteristic peaks is as follows in sequence with peak No. 2 as a reference peak:
0.233～0.274、1.000、1.260～1.479、1.359～1.595、1.675～1.966、2.009～2.358、2.066～2.425、2.134～2.505、2.232～2.621、2.418～2.839；
preferably, in the control map, the peak 2 is a morphine peak, the peak 4 is a chlorogenic acid peak, and the peak 8 is a platycodin D peak.
10. The method for constructing the characteristic map of the strong loquat dew as claimed in any one of claims 1 to 7, which is used for quality detection of the strong loquat dew;
preferably, the method of application comprises:
(a) constructing a characteristic map of the strong loquat dew to be detected according to the construction method of the characteristic map of the strong loquat dew;
(b) analyzing the characteristic spectrum obtained in the step (a) and the contrast spectrum of the strong loquat distillate by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, and calculating the similarity.
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