CN112526014A - Jinyinliang oral liquid fingerprint spectrum and establishing method thereof - Google Patents

Jinyinliang oral liquid fingerprint spectrum and establishing method thereof Download PDF

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CN112526014A
CN112526014A CN202011308904.2A CN202011308904A CN112526014A CN 112526014 A CN112526014 A CN 112526014A CN 202011308904 A CN202011308904 A CN 202011308904A CN 112526014 A CN112526014 A CN 112526014A
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peak
retention time
oral liquid
fingerprint
relative retention
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王信
李彩东
赵丽娟
潘新波
张伟
王艳红
郭亮
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Second People's Hospital Of Lanzhou City
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    • 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
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    • 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
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    • G01N30/86—Signal analysis
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Abstract

The invention provides a method for establishing a fingerprint spectrum of a Jinyindan oral liquid, which comprises the following specific steps: (1) preparation of a test solution: carrying out ultrasonic extraction on the Jinyindan oral liquid by adopting methanol and filtering to obtain a test solution; (2) making a fingerprint spectrum: and testing the sample solution by adopting a high performance liquid chromatography, and generating the fingerprint of the Jinyinliandan oral liquid according to the obtained chromatographic data. The invention also provides a fingerprint spectrum of the Jinyinliandan oral liquid. The invention can realize the fingerprint information of effective component groups of the Jinyinliandan oral liquid by using a fingerprint spectrum analysis technology, and provides an effective method for improving the quality control of the Jinyinliandan oral liquid.

Description

Jinyinliang oral liquid fingerprint spectrum and establishing method thereof
Technical Field
The invention belongs to the technical field of medicine detection, and particularly relates to a Jinyindan oral liquid fingerprint spectrum and an establishment method thereof.
Background
The Jinyinli Dan oral liquid is a preparation in the hospital of the second people in Lanzhou, is prepared from more than ten kinds of decoction pieces such as longhairy antenoron herb, virgate wormwood herb, golden thread, giant knotweed rhizome, officinal magnolia bark and the like, has the effects of clearing heat and promoting diuresis, soothing the liver and benefiting gallbladder, promoting qi and removing blood stasis, regulating qi and activating blood, and removing stagnancy and relieving pain, and is mainly used for preventing and treating diseases such as chronic cholecystitis, jaundice, biliary calculus, biliary tract infection, cholecystitis and the like clinically. The Jinyinliandan oral liquid is used as a pure Chinese medicinal preparation, has higher popularity in Gansu province in more than twenty years of clinical application, and is well received by patients and medical workers. However, the fingerprint of Jinyinliandan oral liquid is not established at present.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a fingerprint of a Jinyindan oral liquid and an establishment method thereof. The invention establishes a Jinyinliandan oral liquid fingerprint spectrum research method, and finally applies the Jinyinliandan oral liquid fingerprint spectrum research method to the quality control of the Jinyinliandan oral liquid, thereby better ensuring the consistency of clinical medication, providing an effective way for comprehensively controlling the quality of the Jinyinliandan oral liquid and providing reference for similar research.
The invention provides a method for establishing a fingerprint spectrum of a Jinyindan oral liquid, which comprises the following specific steps:
(1) preparation of a test solution: carrying out ultrasonic extraction on the Jinyindan oral liquid by adopting methanol and filtering to obtain a test solution;
(2) making a fingerprint spectrum: testing the sample solution by high performance liquid chromatography, and generating fingerprint of the Jinyinliang bile oral liquid according to the obtained chromatographic data;
wherein, the high performance liquid chromatography conditions are as follows: feilomen Gemini C18Column, specification: 5 μm, 4.6 × 250mm, moon god C18Column, specification: 5 μm, 4.6 function250mm, Dima second generation diamond C18Column, specification: 5 μm, 4.6X 250mm or Waters symmetry C18Column, specification: 5 μm, 4.6X 250 mm; detection wavelength: 254 nm; sample amount is 5-20 μ l, column temperature: 20-35 ℃, flow rate: 0.9-1.1 ml/min; taking acetonitrile as phase A and phosphoric acid aqueous solution with volume percentage of 0.1 percent as phase B for gradient elution;
preferably, the high performance liquid chromatography conditions are: dima second generation diamond C18Column, specification: 5 μm, 4.6X 250 mm; detection wavelength: 254 nm; sample size 10 μ l, column temperature: 25 ℃, flow rate: 1 ml/min; and taking acetonitrile as a phase A and 0.1% phosphoric acid aqueous solution as a phase B for gradient elution.
Preferably, in the step (1), the preparation method of the jinyinliandan oral liquid comprises the following steps: mixing all the components except the rhubarb, adding 10-12 times of water by weight, soaking for 30-45min, decocting for 1 hour, filtering, adding 8-10 times of water by weight into filter residue, continuing to decoct for 30-45min, filtering, combining decoction, and concentrating under reduced pressure until each milliliter of the decoction is equal to 1.0g of the original medicinal material;
further preferably, the preparation method of the Jinyinliang oral liquid comprises the following steps: mixing all the components except the rhubarb, adding 12 times of water by weight, soaking for 30min, decocting for 1 hour, filtering, adding 10 times of water by weight into filter residue to increase the content of the rhubarb, continuing decocting for 30min, filtering, combining decoction, and concentrating under reduced pressure until each milliliter of the decoction is equivalent to 1.0g of the raw medicinal materials.
Preferably, in the step (1), the filtration is performed by using a microporous membrane, and the pore diameter of the microporous membrane is 0.22-0.45 μm.
Preferably, in step (1), the methanol concentration is 50% by volume.
Preferably, in step (2), the gradient elution procedure is:
0-50min, the fluidity A is 7%, and the fluidity B is 93%;
the fluidity A is changed from 7% to 33% at constant speed and the fluidity B is changed from 93% to 67% at constant speed for 50-60 min;
the fluidity A is changed from 33 percent to 100 percent at constant speed, and the fluidity B is changed from 67 percent to 0 percent at constant speed for 60-67 min;
67min, flowability A is 100%, and flowability B is 0%.
Preferably, the method further comprises the step of preparing a control solution, wherein the preparation method of the control solution comprises the following steps: respectively taking a gallic acid reference substance, a chlorogenic acid reference substance, a schaftoside reference substance, a liquiritin reference substance, a naringin reference substance, a neohesperidin reference substance, a palmatine hydrochloride reference substance, a berberine hydrochloride reference substance and an ammonium glycyrrhizinate reference substance, precisely weighing, and adding pure methanol to respectively prepare berberine hydrochloride 40mg/ml, gallic acid 15mg/ml, schaftoside 80mg/ml, naringin 75mg/ml, neohesperidin 80mg/ml, ammonium glycyrrhizinate 70mg/ml, palmatine hydrochloride 40mg/ml, chlorogenic acid 70mg/ml and liquiritin 70mg/ml solutions to obtain the final product.
The invention also provides a fingerprint of the Jinyinliang oral liquid, which takes the naringin chromatographic peak as a reference peak, and the fingerprint of the Jinyinliang oral liquid comprises 20 common peaks, and specifically comprises the following steps:
the relative retention time of the No. 1 peak is 0.22, the relative retention time of the No. 2 peak is 0.33-0.34, the relative retention time of the No. 3 peak is 0.36-0.37, the relative retention time of the No. 4 peak is 0.44-0.45, the relative retention time of the No. 5 peak is 0.48-0.49, the relative retention time of the No. 6 peak is 0.52-0.53, the relative retention time of the No. 7 peak is 0.54-0.55, the relative retention time of the No. 8 peak is 0.62-0.64, the relative retention time of the No. 9 peak is 0.72-0.74, the relative retention time of the No. 10 peak is 0.84-0.86, the relative retention time of the No. 11 peak is 0.88-0.90, the relative retention time of the No. 12 peak is 0.91-0.92, the relative retention time of the No. 13 peak is 0.93-0.96, the relative retention time of the No. 14 peak is 1.00, the relative retention time of the No. 15 peak is 1.02, the relative retention time of the No. 16 peak is 1.04-1.06, the relative retention time of the 18 th peak is 1.47, the relative retention time of the 19 th peak is 1.49, and the relative retention time of the 20 th peak is 1.50-1.51.
Preferably, the naringin chromatographic peak is taken as a reference peak, and the No. 1 peak in the fingerprint is gallic acid; the 16 peak is palmatine, and the 17 peak is berberine; peak 19 is glycyrrhizic acid.
Preferably, the naringin chromatographic peak is taken as a reference peak, and the No. 1 peak in the fingerprint is from fructus aurantii, rheum officinale and polygonum cuspidatum; peak 6 is mainly from radix aucklandiae and herba Artemisiae Scopariae; peak 9 is from coptis chinensis; peak 11 is from polygonum cuspidatum; the 12-17 component group is derived from Coptidis rhizoma and fructus Aurantii, and the 18-20 component group is derived from Glycyrrhrizae radix.
The invention establishes the high performance liquid phase fingerprint of the Jinyinliang oral liquid and improves the quality control level of the Jinyinliang oral liquid. The invention adopts a Chinese medicine chromatogram fingerprint similarity evaluation system recommended by the State food and drug administration to calculate the similarity, and determines the fingerprint research method of the Jinyinliandan oral liquid. The results obtained were: 20 common peaks are obtained from each batch of Jinyindan oral liquid, the total area is more than 95%, the obtained fingerprint methodology investigation result is good, the similarity calculation is carried out on the fingerprints by adopting the chromatographic fingerprint similarity evaluation software, and the similarity of 10 batches of samples is more than 0.9. The invention can realize the fingerprint information of effective component groups of the Jinyinliandan oral liquid by using a fingerprint spectrum analysis technology, and provides an effective method for improving the quality control of the Jinyinliandan oral liquid.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 shows the common fingerprint peaks of the Jinyindan oral liquid and the attribution of the common fingerprint peaks.
FIG. 2 shows the fingerprint of ten batches of Jinyinliang oral liquid.
FIG. 3 is a chromatogram of directly injected Jinyinliang oral liquid.
FIG. 4 is a chromatogram obtained by removing impurities from a Jinyindan oral liquid with a 50% methanol solution by volume. .
Fig. 5 is a chromatogram when acetonitrile-pure water was used as a mobile phase.
FIG. 6 is a chromatogram when using acetonitrile-0.1% by volume phosphoric acid solution as a mobile phase.
FIG. 7 is a chromatogram using domestic acetonitrile.
Figure 8 is a chromatogram using imported acetonitrile.
FIG. 9 is a chromatogram using an Agilent chromatography column.
FIG. 10 is a chromatogram obtained by using Shimadzu column.
FIG. 11 is a chromatogram using a Dima second generation diamond C18 column.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are commercially available unless otherwise specified.
Example 1
1 Instrument and reagent
Shimadzu 20AT high performance liquid chromatograph; a PDA detector; an electronic analytical balance; a chromatographic column: dima second generation diamond C18(250 mm. times.4.6 mm, 5 μm); ultrasonic cleaner, MiliQ ultra pure water instrument. Comparison products: are purchased from China institute for testing biological products of drugs and drugs for content determination. Reagent: milipore ultrapure water, import acetonitrile (chromatographic purity), methanol, phosphoric acid for domestic chromatographic purity; jinnlihdan oral liquid is produced by the second people hospital preparation center in lanzhou city, lot number (20190328,20190411,20190530,20190620,20190704,20190833,20190930,20191024,20191113,20191219).
The Jinyinliandan oral liquid comprises the following raw material medicines: dandelion, coptis, fructus aurantii, szechwan chinaberry fruit, rhubarb, liquorice, desmodium styracifolium, mangnolia officinalis, polygonum cuspidatum, elecampane, hawthorn, peach kernel, rhizoma cyperi, oriental wormwood, moutan bark, medicated leaven, malt and kadsura longepedunculata; the correctant is simple syrup, and the antiseptic is sodium benzoate.
2 methods and results
2.1 establishment of fingerprint determination conditions
2.1.1 preparation of control solutions: respectively taking appropriate amount of gallic acid reference substance, chlorogenic acid reference substance, schaftoside reference substance, liquiritin reference substance, naringin reference substance, neohesperidin reference substance, palmatine hydrochloride reference substance, berberine hydrochloride reference substance, and ammonium glycyrrhizinate reference substance, precisely weighing, and adding pure methanol to respectively obtain berberine hydrochloride 40mg/ml, gallic acid 15mg/ml, schaftoside 80mg/ml, naringin 75mg/ml, neohesperidin 80mg/ml, ammonium glycyrrhizinate 70mg/ml, palmatine hydrochloride 40mg/ml, chlorogenic acid 70mg/ml, and liquiritin 70mg/ml solutions.
2.1.2 preparation of Jinyinliandan oral liquid: adding 12 times of water into decoction pieces of the whole formula except rhubarb, soaking for 30min, decocting for 1 hour, filtering, adding rhubarb decoction pieces into filter residues, adding 10 times of water, continuing decocting for 30min, filtering, combining decoction, and concentrating under reduced pressure until each milliliter of decoction pieces is equivalent to 1.0g of the original medicinal material.
2.1.3 preparation of test solution:
taking 5ml of each Jinyindan oral liquid of different batches, respectively placing into 10ml volumetric flasks, adding a proper amount of 50% methanol by volume, carrying out ultrasonic treatment for 15min, standing for 1h, fixing the volume to a scale by using 50% methanol by volume, shaking up, and filtering with a 0.45 mu m microporous membrane.
2.1.4 chromatographic conditions Agilent 1260 high performance liquid chromatograph combined with PDA detector, Dima second generation diamond C18Column (5 μm, 250mm × 4.6mm), mobile phase: acetonitrile-phosphoric acid aqueous solution with volume percentage of 0.1%, gradient elution, detection wavelength: 254 nm; sample size 10 μ l, column temperature: 25 ℃, flow rate: 1 ml/min. The gradient elution procedure is shown in table 1.
TABLE 1 HPLC mobile phase gradient elution procedure
Figure BDA0002789165720000051
2.2HPLC fingerprinting
2.2.1 determination of common characteristic peaks and common characteristic peak attribution 10 batches of Jinyindan oral liquid sample solutions are taken, meanwhile, single medicinal materials of the Jinyindan oral liquid are taken to prepare reference solutions, HPLC analysis is respectively carried out according to the chromatographic conditions under the item 2.1, the source of each chromatographic peak is determined, 20 peaks are shared by 10 sample solutions through the reference analysis, and therefore the 20 peaks are determined to be common fingerprint peaks (figure 1).
By comparing with the characteristic peak of the control solution, the result shows that: peak 1 is derived from fructus Aurantii, radix Et rhizoma Rhei, and rhizoma Polygoni Cuspidati; the peak 2 is mainly from herba Desmodii Styracifolii and herba Artemisiae Scopariae; peak 3 is mainly from fructus Aurantii and herba Artemisiae Scopariae; peak 4 is mainly from cortex Magnolia officinalis, rhizoma Polygoni Cuspidati, and radix aucklandiae; peak 5 is mainly from moutan bark and dandelion; peak 6 is mainly from radix aucklandiae and herba Artemisiae Scopariae; peak No. 7 is mainly from Coptidis rhizoma, radix et rhizoma Rhei, and herba Artemisiae Scopariae; peak 8 is mainly from herba Taraxaci and herba Artemisiae Scopariae; peak 9 is from coptis chinensis; peak 10 is from Rheum officinale; peak 11 is from polygonum cuspidatum; 12. peaks 13, 16 and 17 are from Coptidis rhizoma, peak 16 is palmatine, and peak 17 is berberine; peak 14 is derived from fructus Aurantii, and is naringin; no. 15 peak is derived from fructus Aurantii, radix aucklandiae, and herba Artemisiae Scopariae; 18. peaks 19 and 20 are from licorice. In addition, 12-17 component group is from Coptidis rhizoma and fructus Aurantii, 18-20 component group is from Glycyrrhrizae radix, 19 # peak is glycyrrhizic acid, and 1, 6, 9, and 11 common peaks constitute the characteristic identification map of JINYINLIAN oral liquid.
FIG. 1 shows the common fingerprint peaks of the Jinyindan oral liquid and the attribution of the common fingerprint peaks. Wherein, the S1 Jinyinlian oral liquid (S1 is a contrast map generated by 10 batches of Jinyinlian oral liquid fingerprint maps in similarity software), S2 dandelion, S3 coptis root, S4 bitter orange, S5 toosendan fruit, S6 rhubarb, S7 liquorice, S8 desmodium styracifolium, S9 magnolia officinalis, S10 giant knotweed rhizome, S11 costus root, S12 hawthorn, S13 peach kernel, S14 nutgrass galingale rhizome, S15 oriental wormwood, S16 moutan bark and S17 blank solvent.
2.2.2 fingerprint similarity analysis HPLC fingerprints of 10 batches of Jinyindan oral liquid sample solutions are analyzed (figure 2), 20 common peaks are indicated in total, 14 peaks in the fingerprints are used as component naringin, the peak position is relatively middle, the absorption is relatively strong, the separation degree is relatively ideal, therefore 14 peaks of naringin are used as reference peaks, the relative retention time and the relative peak area of 10 batches of samples are calculated (tables 2 and 3), the relative retention time RSD range is less than 3%, and the requirement of the fingerprints is met. The retention times for the 20 common fingerprint peaks were: the retention time of peak 1 is 8.270min, the retention time of peak 2 is 12.698min, the retention time of peak 3 is 13.876min, the retention time of peak 4 is 16.668min, the retention time of peak 5 is 18.191min, the retention time of peak 6 is 19.748min, the retention time of peak 7 is 20.490min, the retention time of peak 8 is 23.674min, the retention time of peak 9 is 27.257min, the retention time of peak 10 is 31.880min, the retention time of peak 11 is 33.030min, the retention time of peak 12 is 34.234min, the retention time of peak 13 is 34.953min, the retention time of peak 14 is 37.459min, the retention time of peak 15 is 38.229min, the retention time of peak 16 is 39.205min, the retention time of peak 17 is 42.073min, the retention time of peak 18 is 54.937min, the retention time of peak 19 is 55.659min, and the retention time of peak 20 is 56.081 min.
The traditional Chinese medicine fingerprint similarity calculation software is used for calculation, and the similarity calculation result shows that the fingerprint similarity of 10 batches of Jinyinliang oral liquid is more than 0.90 (table 4), which indicates that the preparation process of the Jinyinliang oral liquid is stable.
FIG. 2 shows the fingerprint of ten batches of Jinyinliang oral liquid.
TABLE 2 Ten batches of Jinyinliang oral liquid with relative retention time of fingerprint
Figure BDA0002789165720000061
Figure BDA0002789165720000071
TABLE 3 relative peak areas of fingerprint spectra of ten golden capillary bile oral liquids
Figure BDA0002789165720000072
TABLE 4 fingerprint similarity data for ten golden herba Artemisiae Scopariae oral liquid batches
Figure BDA0002789165720000073
Figure BDA0002789165720000081
2.3 methodological investigation
2.3.1 precision experiment: and taking a sample solution of the Jinyindan oral liquid, and continuously carrying out sample injection for 6 times according to the chromatographic condition in 2.1.4, wherein the sample injection amount is 10 mu l, and as a result, the relative retention time of 20 common peaks and the RSD value of the relative peak area are both less than 3%, which indicates that the precision of the instrument is good.
TABLE 5 relative retention times for precision experiments
Figure BDA0002789165720000082
TABLE 6 relative peak area for precision experiments
Figure BDA0002789165720000083
Figure BDA0002789165720000091
2.3.2 stability experiments: sampling herba Artemisiae Scopariae oral liquid and sample solution at the conditions of 2.1.4 chromatography for 0, 8, 12, 24, 48, 96 hr, 10 μ l each time. The results show that the relative retention time of 20 common peaks and the RSD value of the relative peak area are both less than 3%, which indicates that the stability of the test solution of the endotherm-cholangiosa oral liquid in 24 hours is better.
Table 7 stability test relative retention time
Figure BDA0002789165720000092
TABLE 8 relative peak area for stability experiments
Figure BDA0002789165720000093
Figure BDA0002789165720000101
2.3.3 repeatability tests: taking 6 parts of each of the Jinyinliang bile oral liquid medicinal materials in the same batch, preparing the oral liquid according to a preparation method of a test solution, taking 10 mu l of each test solution, and respectively carrying out sample injection analysis by using a high performance liquid chromatograph, wherein the RSD of the 20 common peaks in the result is less than 3% in relative retention time, and the RSD of the relative peak area is less than 5%, which indicates that the method has good repeatability.
TABLE 9 relative retention times for repeated experiments
Figure BDA0002789165720000102
TABLE 10 relative peak area for repeatability experiments
Figure BDA0002789165720000103
Figure BDA0002789165720000111
The optimization test of the method of the invention comprises the following steps:
1. selection of an extraction solvent: the finished Jinyinliandan oral liquid has high concentration degree and is more viscous, the applicant adopts a methanol extraction mode with a certain proportion to carry out appropriate impurity removal, and finds that a 50% methanol solution in volume percentage can better retain chemical information of each part in a finished product compared with a finished product stock solution in a direct sample injection mode, and the peak shape is obviously improved, so that 50% methanol in volume percentage is adopted to carry out impurity removal and extraction.
FIG. 3 is a chromatogram of directly injected Jinyinliang oral liquid.
As can be seen from fig. 3, too much impurities in the golden fimbriae oral liquid cause the peak shape to shift and the peak shape to be not good.
FIG. 4 is a chromatogram obtained by removing impurities from a Jinyindan oral liquid with a 50% methanol solution by volume. .
As can be seen from FIG. 4, the peak shape and the number of peaks (types of components) were good after removing impurities by using a 50% by volume methanol solution.
2. Through exploration of liquid phase conditions, the peak shape can be obviously improved by taking acetonitrile-phosphoric acid solution with volume percentage of 0.1% as a water phase, and a mode of using buffer salt solution when detecting berberine and other components by a pharmacopoeia method is avoided (the biggest defect of a buffer salt elution mode is that repeated blockage of a flow path is easily caused).
Fig. 5 is a chromatogram when acetonitrile-pure water was used as a mobile phase.
As can be seen from fig. 5, when acetonitrile-pure water was used as the mobile phase, the peak shape was slightly inferior and the slit width was slightly wide.
FIG. 6 is a chromatogram when using acetonitrile-0.1% by volume phosphoric acid solution as a mobile phase.
As can be seen from fig. 6, the use of acetonitrile-0.1% by volume phosphoric acid solution as the mobile phase can significantly improve the peak shape.
3. The methanol as an organic phase can not realize the separation of complex components of the product, and domestic acetonitrile generates stubborn ghost peaks in a chromatogram map of 47.5 minutes to seriously influence the separation, so that imported acetonitrile is adopted for analysis.
FIG. 7 is a chromatogram using domestic acetonitrile.
As can be seen from FIG. 7, a persistent ghost peak appeared at a chromatogram of 47.5 minutes using domestic acetonitrile.
Figure 8 is a chromatogram using imported acetonitrile.
As can be seen from fig. 8, the use of imported acetonitrile better solves the ghost peak problem.
4. Firoma gemini C is investigated by chromatographic column18Column (5 μm, 4.6 × 250mm), moon god C18Column (5 μm, 4.6X 250mm), Dima second generation diamond C18Column (5 μm, 4.6X 250mm), Waters symmetry C18Column (5 μm, 4.6X 250mm), Shimadzu C18Column (5 μm, 4.6X 250mm), Agilent Eclipse plus C18The column (5 μm, 4.6 × 250mm) found that Agilent and Shimadzu columns can not achieve the expected separation effect, and other chromatographic columns can achieve the satisfactory separation effect by adjusting the liquid phase conditions. From the aspect of cost performance, Dima second generation diamond C is adopted18And (3) a column.
FIG. 9 is a chromatogram using an Agilent chromatography column.
As can be seen from FIG. 9, the Agilent chromatographic column has a clear peak shape for the separation effect of the product, and the separation degree is poor in about 40 minutes.
FIG. 10 is a chromatogram obtained by using Shimadzu column.
As can be seen from FIG. 10, Shimadzu column was difficult to separate the product and the obtained fraction was small.
FIG. 11 is a chromatogram using a Dima second generation diamond C18 column.
As can be seen from FIG. 11, the Dima second generation diamond C18 pillar can achieve better separation effect.
Discussion of 3
The transformation of individual components: the peak A (shown in figure 1) in the Jinyindan oral liquid is not directly transferred from the dissolved components in each decoction piece, and the source and transformation relationship of the peak A and the dissolved components in each decoction piece need further research. The peak B in the peach kernel decoction pieces (see figure 1) almost disappears in the process of producing finished Jinyinliandan oral liquid products, and the transformation relation is proved to be irrelevant to the peak A in the finished products preliminarily, and the transformation of the component represented by the peak B needs to be further researched.
The contribution of the medicinal slices of medicated leaven, malt, toosendan fruit and the like in the medicinal materials of the Jinyinli gall oral liquid prescription to the chemical components of the finished oral liquid cannot be reflected in the chromatogram of the HPLC combined PDA detector, but the contribution does not mean that the medicinal components do not play a role, which indicates that the medicinal components are not suitable for being detected under the test conditions used in the text.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (9)

1. A method for establishing a fingerprint spectrum of a Jinyinliang oral liquid is characterized by comprising the following steps: the method comprises the following specific steps:
(1) preparation of a test solution: carrying out ultrasonic extraction on the Jinyindan oral liquid by adopting methanol and filtering to obtain a test solution;
(2) making a fingerprint spectrum: testing the sample solution by high performance liquid chromatography, and generating fingerprint of the Jinyinliang bile oral liquid according to the obtained chromatographic data;
wherein, the high performance liquid chromatography conditions are as follows: feilomen Gemini C18Column, specification: 5 μm, 4.6 × 250mm, moon god C18Column, specification: diamond of Dima second generation 5 μm, 4.6X 250mm18Column, specification: 5 μm, 4.6X 250mm or Waters symmetry C18Column, specification: 5 μm, 4.6X 250 mm; detection wavelength: 254 nm; sample amount is 5-20 μ l, column temperature: 20-35 ℃, flow rate: 0.9-1.1 ml/min; taking acetonitrile as phase A and phosphoric acid aqueous solution with volume percentage of 0.1 percent as phase B for gradient elution;
preferably, the high performance liquid chromatography conditions are: dima second generation diamond C18Column, specification: 5 μm, 4.6X 250 mm; detection wavelength: 254 nm; sample size 10 μ l, column temperature: 25 ℃, flow rate: 1 ml/min; and taking acetonitrile as a phase A and taking a phosphoric acid aqueous solution with the volume percentage of 0.1% as a phase B for gradient elution.
2. The method for establishing the fingerprint of the Jinyinliang bile oral liquid according to claim 1, which is characterized in that: in the step (1), the preparation method of the Jinyinliang oral liquid comprises the following steps: mixing all the components except the rhubarb, adding 10-12 times of water by weight, soaking for 30-45min, decocting for 1 hour, filtering, adding 8-10 times of water by weight into filter residue, continuing to decoct for 30-45min, filtering, combining decoction, and concentrating under reduced pressure until each milliliter of the decoction is equal to 1.0g of the original medicinal material.
3. The method for establishing the fingerprint of the Jinyinliang oral liquid according to claim 1 or 2, which is characterized in that: in the step (1), the filtration is carried out by adopting a microporous filter membrane, and the pore diameter of the microporous filter membrane is 0.22-0.45 μm.
4. The method for establishing the fingerprint of the Jinyinliang oral liquid according to claim 1 or 2, which is characterized in that: in the step (1), the volume percentage concentration of the methanol is 50%.
5. The method for establishing the fingerprint of the Jinyinliang oral liquid according to claim 1 or 2, which is characterized in that: in the step (2), the gradient elution procedure is as follows:
0-50min, the fluidity A is 7%, and the fluidity B is 93%;
the fluidity A is changed from 7% to 33% at constant speed and the fluidity B is changed from 93% to 67% at constant speed for 50-60 min;
the fluidity A is changed from 33 percent to 100 percent at constant speed, and the fluidity B is changed from 67 percent to 0 percent at constant speed for 60-67 min;
67min, flowability A is 100%, and flowability B is 0%.
6. The method for establishing the fingerprint of the Jinyinliang oral liquid according to claim 1 or 2, wherein the fingerprint comprises the following steps: the method also comprises the step of preparing a reference substance solution, wherein the preparation method of the reference substance solution comprises the following steps: respectively taking a gallic acid reference substance, a chlorogenic acid reference substance, a schaftoside reference substance, a liquiritin reference substance, a naringin reference substance, a neohesperidin reference substance, a palmatine hydrochloride reference substance, a berberine hydrochloride reference substance and an ammonium glycyrrhizinate reference substance, precisely weighing, and adding pure methanol to respectively prepare berberine hydrochloride 40mg/ml, gallic acid 15mg/ml, schaftoside 80mg/ml, naringin 75mg/ml, neohesperidin 80mg/ml, ammonium glycyrrhizinate 70mg/ml, palmatine hydrochloride 40mg/ml, chlorogenic acid 70mg/ml and liquiritin 70mg/ml solutions to obtain the final product.
7. The fingerprint of the Jinyindan oral liquid is characterized in that: the naringin chromatographic peak is taken as a reference peak, and the fingerprint of the Jinyinliang gall oral liquid comprises 20 common peaks which are as follows:
the relative retention time of the No. 1 peak is 0.22, the relative retention time of the No. 2 peak is 0.33-0.34, the relative retention time of the No. 3 peak is 0.36-0.37, the relative retention time of the No. 4 peak is 0.44-0.45, the relative retention time of the No. 5 peak is 0.48-0.49, the relative retention time of the No. 6 peak is 0.52-0.53, the relative retention time of the No. 7 peak is 0.54-0.55, the relative retention time of the No. 8 peak is 0.62-0.64, the relative retention time of the No. 9 peak is 0.72-0.74, the relative retention time of the No. 10 peak is 0.84-0.86, the relative retention time of the No. 11 peak is 0.88-0.90, the relative retention time of the No. 12 peak is 0.91-0.92, the relative retention time of the No. 13 peak is 0.93-0.96, the relative retention time of the No. 14 peak is 1.00, the relative retention time of the No. 15 peak is 1.02, the relative retention time of the No. 16 peak is 1.04-1.06, the relative retention time of the 18 th peak is 1.47, the relative retention time of the 19 th peak is 1.49, and the relative retention time of the 20 th peak is 1.50-1.51.
8. The fingerprint of Jinyindan oral liquid according to claim 7, wherein: taking a naringin chromatographic peak as a reference peak, and taking a peak No. 1 in a fingerprint as gallic acid; the 16 peak is palmatine, and the 17 peak is berberine; peak 19 is glycyrrhizic acid.
9. The fingerprint of Jinyindan oral liquid according to claim 7, wherein: taking naringin chromatographic peak as reference peak, wherein the No. 1 peak in fingerprint is derived from fructus Aurantii, radix et rhizoma Rhei, and rhizoma Polygoni Cuspidati; peak 6 is mainly from radix aucklandiae and herba Artemisiae Scopariae; peak 9 is from coptis chinensis; peak 11 is from polygonum cuspidatum; the 12-17 component group is derived from Coptidis rhizoma and fructus Aurantii, and the 18-20 component group is derived from Glycyrrhrizae radix.
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