CN114280209A

CN114280209A - Method for establishing fingerprint of heat-clearing cough-relieving oral liquid for children and fingerprint thereof

Info

Publication number: CN114280209A
Application number: CN202111598283.0A
Authority: CN
Inventors: 潘燕; 胡林水; 胡嘉蓉; 吴明; 雷蔚雯
Original assignee: Zhejiang Conba Pharmaceutical Co ltd
Current assignee: Zhejiang Conba Pharmaceutical Co ltd
Priority date: 2021-12-24
Filing date: 2021-12-24
Publication date: 2022-04-05
Anticipated expiration: 2041-12-24
Also published as: CN114280209B

Abstract

The invention discloses a method for establishing a fingerprint of an oral liquid for clearing heat and relieving cough for children and the fingerprint thereof, which comprises the following steps: (1) preparing different test solution; (2) respectively carrying out sample injection analysis on different sample solutions by adopting HPLC (high performance liquid chromatography), and confirming that 28 common chromatographic peaks exist; (3) introducing HPLC (high performance liquid chromatography) fingerprint data of different test solution into 2012 edition of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and generating an HPLC fingerprint comparison map of the oral liquid for clearing heat and relieving cough of children by using an averaging method; (4) and (4) introducing HPLC fingerprints of different test solution samples into a 2012 version of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system to perform similarity analysis with a control map. The invention has high stability, good accuracy and repeatability, can be used for monitoring the production process of the oral liquid for clearing heat and relieving cough for children, and can provide certain theoretical basis and support for establishing a quality control system in the whole process.

Description

Method for establishing fingerprint of heat-clearing cough-relieving oral liquid for children and fingerprint thereof

Technical Field

The invention relates to an oral liquid for clearing heat and relieving cough for children, in particular to an establishment method of a fingerprint of an oral liquid for clearing heat and relieving cough for children and a fingerprint thereof.

Background

The oral liquid for clearing heat and relieving cough for children is a pure traditional Chinese medicine preparation prepared by adding flavor to famous prescription Maxingshigan decoction in Han Dynasty' Shang Lun (treatise on Cold-induced diseases), is recorded in the first part of 2020 edition of Chinese pharmacopoeia, consists of seven traditional Chinese medicines of ephedra, fried bitter apricot seed, gypsum, liquorice, scutellaria baicalensis, isatis root and asiatic moonseed rhizome, and has the effects of clearing heat and freeing lung, relieving asthma and relieving sore throat. Can be used for treating common cold caused by infantile wind heat, with symptoms of fever, aversion to cold, cough, yellow phlegm, short breath, asthma, dry mouth, hoarseness, and sore throat.

The quality of the medicine is the premise of ensuring the safety and effectiveness of clinical medication, and the method for evaluating the quality of the oral liquid preparation for clearing heat and relieving cough for children in the Chinese pharmacopoeia at present takes the ephedrine hydrochloride content as an index for control, but the oral liquid for clearing heat and relieving cough for children has complex components, the quality of the oral liquid for clearing heat and relieving cough for children cannot be integrally described and evaluated only by content measurement of a single component, and the overall quality of the oral liquid for clearing heat and relieving cough for children is difficult to effectively reflect.

As a comprehensive quantifiable identification means, the traditional Chinese medicine fingerprint spectrum has the characteristics of large information amount, strong characteristics, integrity, fuzziness and the like, can comprehensively reflect the types and the quantities of chemical components contained in the traditional Chinese medicine, is one of quality control modes conforming to the characteristics of the traditional Chinese medicine at present, and has increasingly wide application in the quality control of traditional Chinese medicine preparations.

At present, no patent and literature report related to the establishment of the fingerprint of the heat-clearing cough-relieving oral liquid for children is found at home and abroad.

Disclosure of Invention

The invention aims to provide the method for establishing the fingerprint of the pediatric heat-clearing cough-relieving oral liquid, which has high stability, good accuracy and good repeatability, can be used for monitoring the production process of the pediatric heat-clearing cough-relieving oral liquid, provides a certain theoretical basis and support for establishing a whole-process quality control system, and ensures the safety and effectiveness of clinical medication.

The invention also provides a fingerprint of the oral liquid for clearing heat and relieving cough for children.

In order to achieve the purpose, the invention adopts the following technical scheme: a method for establishing a fingerprint of an oral liquid for clearing heat and relieving cough of children comprises the following steps:

(1) different test solutions were prepared.

(2) And respectively carrying out sample injection analysis on different sample solutions by adopting HPLC (high performance liquid chromatography), obtaining HPLC fingerprint spectrums of the different sample solutions, and confirming that 28 common chromatographic peaks exist.

(3) Introducing the HPLC fingerprint data of different test solution into 2012 edition of the traditional Chinese medicine chromatogram fingerprint similarity evaluation system, taking 1 of the test solution fingerprints as reference spectra, setting the time window width to be 0.1min, and generating the HPLC fingerprint comparison spectrum (R) of the oral liquid for clearing heat and relieving cough of children by using a mean value method through multi-point correction and data matching.

(4) And (3) introducing the HPLC fingerprint data of different test solution to be tested into a 2012 version of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system to perform similarity analysis with a reference spectrum (R).

Preferably, in step (1), the sample solution is prepared by the following method: precisely sucking 3-10 mL of a test article, placing the test article in a 100mL volumetric flask, adding 50mL of 30-90% methanol aqueous solution, carrying out ultrasonic treatment at room temperature for 30-60 min, cooling to room temperature, fixing the volume, shaking up, and filtering.

Preferably, the test solution is prepared by the following method: precisely sucking 5mL of a test article, placing the test article in a 100mL volumetric flask, adding 50mL of 50% methanol aqueous solution by volume fraction, carrying out ultrasonic treatment at room temperature for 40min, cooling to room temperature, fixing the volume, shaking up, and filtering.

Preferably, in the step (1), the test sample is an oral liquid for clearing heat and relieving cough for children, an extracted concentrated filtrate or a refined solution. The preparation process of the oral liquid for clearing heat and relieving cough for children is derived from the item of the quality standard [ preparation method ] of the oral liquid for clearing heat and relieving cough for children in the 'Chinese pharmacopoeia' 2020 edition, and comprises the following steps: decocting ephedra and gypsum in water for 30 minutes, adding the rest five medicines such as fried bitter apricot kernel and the like, decocting twice, 2 hours for the first time and 1 hour for the second time, combining the decoctions, filtering, concentrating the filtrate under reduced pressure to a proper amount, standing, filtering, adding 200g of honey, 100g of cane sugar and 3g of sodium benzoate into the filtrate, boiling to dissolve, adding water to 1000mL, stirring uniformly, refrigerating for 24-48 hours, filtering, filling and sealing, and sterilizing to obtain the traditional Chinese medicine. … …, filtering decoction, concentrating the filtrate under reduced pressure to a proper amount, standing, filtering to obtain concentrated filtrate (intermediate I), … …, adding 200g of honey, 100g of cane sugar and 3g of sodium benzoate into the filtrate, boiling to dissolve, adding water to 1000mL, stirring uniformly, refrigerating for 24-48 hours, and filtering to obtain refined solution (intermediate II); the most preferable test sample in the invention is the oral liquid for clearing heat and relieving cough for children.

Preferably, in step (2), the HPLC chromatographic conditions are as follows: the chromatographic column adopts octadecylsilane chemically bonded silica as a filler, and Agilent InfinityLab Poroshell 120EC-C18 as a chromatographic column, and the specification is 150mm multiplied by 4.6mm and 2.7 mu m; the column temperature is 30 ℃, the flow rate is 1.0mL/min, and the sample injection amount is 20 mu L; detecting wavelength of 0 min-31 min 210nm, 31 min-65 min270nm, starting DAD detection, taking acetonitrile-methanol (89: 11) as a mobile phase A, taking 0.01mol/L potassium dihydrogen phosphate solution (pH3.2) as a mobile phase B, and carrying out gradient elution (50 mm multiplied by 4.6mm I.D, impurity trapping column indirect between a pump and a sample injector) for 0-5 min, wherein A: B is 2%: 98% → 3%: 97%; 5-15 min, wherein A and B are 3 percent to 97 percent to → 5 percent to 95 percent; 15-25 min, wherein A and B are 5 percent to 95 percent to 10 percent to 90 percent; 25-35 min, wherein A and B are 10 percent to 90 percent to → 15 percent to 85 percent; 35-60 min, wherein A and B are 15 percent to 85 percent to 25 percent to 75 percent; 60-68 min, wherein A and B are 25 percent to 75 percent to → 41 percent to 59 percent; 68-75 min, wherein A and B are 41 percent to 59 percent to → 41 percent to 59 percent. In the prior art, acetonitrile-0.1% phosphoric acid, acetonitrile-0.05 mol/L ammonium dihydrogen phosphate, (acetonitrile-methanol) -0.01mol/L potassium dihydrogen phosphate and the like are mostly used for gradient elution in the separation and content determination of single medicinal materials in the oral liquid for clearing heat and relieving cough for children, and the applicant discovers that in the chromatogram of elution of the acetonitrile-0.1% phosphoric acid and the acetonitrile-0.05 mol/L ammonium dihydrogen phosphate systems, the number of chromatographic peaks is small, and the chromatogram is insufficient to comprehensively represent the material basis of the oral liquid for clearing heat and relieving cough for children by examining the elution effects of the three systems; although the number of chromatographic peaks in the chromatogram eluted by the acetonitrile-0.05 mol/L ammonium dihydrogen phosphate system is similar to that of the [ acetonitrile-methanol (89: 11) ] -0.01mol/L potassium dihydrogen phosphate, but the separation degree of the peaks is poor, so that the [ acetonitrile-methanol (89: 11) ] -0.01mol/L potassium dihydrogen phosphate is used as an elution solvent in the invention, and the chromatographic peaks obtained under the conditions are remarkably superior to other elution systems.

Preferably, in step (2), the 28 common chromatographic peaks are respectively: peak 1 with a relative retention time of 0.205, peak 2 with a relative retention time of 0.297, peak 3 with a relative retention time of 0.317, peak 4 with a relative retention time of 0.391, peak 5 with a relative retention time of 0.446, peak 6 with a relative retention time of 0.487, peak 7 with a relative retention time of 0.495, peak 8 with a relative retention time of 0.514, peak 9 with a relative retention time of 0.521, peak 10 with a relative retention time of 0.627, peak 11 with a relative retention time of 0.761, peak 12 with a relative retention time of 0.770, peak 13 with a relative retention time of 0.785, peak 14 with a relative retention time of 0.801, peak 15 with a relative retention time of 0.816, peak 16 with a relative retention time of 0.853, peak 17 with a relative retention time of 0.883, peak 18 with a relative retention time of 1.000, peak 18 with a relative retention time of 1.051, peak 18 with a relative retention time of 1.20, The peak 21 with a relative retention time of 1.110, the peak 22 with a relative retention time of 1.126, the peak 23 with a relative retention time of 1.138, the peak 24 with a relative retention time of 1.149, the peak 25 with a relative retention time of 1.172, the peak 26 with a relative retention time of 1.249, the peak 27 with a relative retention time of 1.257, and the peak 28 with a relative retention time of 1.318, wherein 8 chromatographic peaks are identified as 1 peak (R, S) -gomphrenaline, 2 peak ephedrine hydrochloride, 3 peak pseudoephedrine hydrochloride, 7 peak amygdalin, 12 peak glycyrrhizin, 18 peak baicalin, 27 peak baicalein, and 28 peak ammonium glycyrrhizinate, respectively.

A fingerprint of infantile heat clearing and cough relieving oral liquid is obtained by establishing method.

Therefore, the invention has the following beneficial effects: provides a method for establishing the fingerprint of the oral liquid for clearing heat and relieving cough of children, establishes the fingerprint of key process intermediates of the oral liquid for clearing heat and relieving cough of children, including extracting concentrated filtrate (intermediate I) and refined process (intermediate II) on the basis of the fingerprint of the oral liquid for clearing heat and relieving cough of children, and qualitatively and quantitatively analyzing a plurality of medicinal effect components of a plurality of medicinal materials in each process step, comprehensively reflecting the quality information of the oral liquid for clearing heat and relieving cough for children, ensuring the uniform and stable product quality, determining a key process control link according to the variation trend and degree of the relative peak area of a reference substance, establishing a corresponding key point quality control standard, and being used for monitoring the production process of the oral liquid for clearing heat and relieving cough for children, provides a certain theoretical basis and support for the establishment of a whole-process quality control system, and ensures the safety and effectiveness of clinical medication.

Drawings

FIG. 1 is HPLC fingerprint of 6 batches of the final product of the oral liquid for clearing heat and relieving cough in example 1.

FIG. 2 is a HPLC fingerprint of the mixed control solution.

Fig. 3 is an overlay of HPLC fingerprints of finished products of the 6 batches of the pediatric heat clearing and cough relieving oral liquid in example 1.

FIG. 4 is a superimposed chromatogram of HPLC fingerprint of 6 infantile heat-clearing cough-relieving oral liquid finished products in a repeatability test.

FIG. 5 is a superimposed chromatogram of HPLC fingerprint of 6 parts of oral liquid product for clearing heat and relieving cough in stability test.

FIG. 6 is a comparison of HPLC fingerprint of the final product of the oral liquid for clearing heat and relieving cough for children and HPLC chromatogram of the herba Ephedrae extractive solution.

FIG. 7 is a comparison of HPLC fingerprint spectrum of the finished product of the oral liquid for clearing heat and relieving cough of children and HPLC chromatogram of the extract of scutellaria baicalensis.

FIG. 8 is a comparison of HPLC fingerprint of the finished product of the oral liquid for clearing heat and relieving cough for children and HPLC chromatogram of the extract of rhizoma Menispermi.

FIG. 9 is a comparison of HPLC finger print of the final product of the oral liquid for clearing heat and relieving cough of children and HPLC chromatogram of the extractive solution of parched semen Armeniacae amarum.

FIG. 10 is a comparison of HPLC fingerprint of the finished product of the oral liquid for clearing heat and relieving cough of children and HPLC chromatogram of the radix isatidis extract.

FIG. 11 is a comparison of HPLC fingerprint of the final product of the oral liquid for clearing heat and relieving cough of children and HPLC chromatogram of the extract of licorice.

FIG. 12 is a comparison of HPLC fingerprint of the final product of the oral liquid for clearing heat and relieving cough of children and HPLC chromatogram of the extract of Gypsum Fibrosum.

FIG. 13 is HPLC fingerprint of 3 batches of the concentrated filtrate of the pediatric heat-clearing cough-relieving oral liquid extract in example 2.

FIG. 14 is the superimposed chromatogram of HPLC fingerprint of 3 batches of the concentrated filtrate extracted from the pediatric heat-clearing cough-relieving oral liquid in example 2.

Fig. 15 shows HPLC fingerprints of refined solutions of 6 batches of the pediatric heat clearing and cough relieving oral liquid in example 3.

Fig. 16 is an overlay of HPLC fingerprints of refined solutions of 6 batches of the pediatric heat clearing and cough relieving oral liquid in example 3.

Detailed Description

The invention is further described with reference to the following figures and detailed description.

The instruments and reagents used in the following examples are as follows:

the instrument comprises the following steps: electronic balance (XSE205DU, mettler-toledo); a pH meter (Seven Compact S210, Mettler-Torlo); high performance liquid chromatography (Agilent 1260, Agilent, DAD detector); ultrasonic cleaning apparatus (YM-1001LUS, Ally).

Reagent testing: (R, S) -Notopterygium (batch No.: 111753-202007, China institute for testing food and drug);

ephedrine hydrochloride (batch No. 171241) -201809, China institute for testing food and drug);

pseudoephedrine hydrochloride (batch No. 171237-201510, China institute for food and drug testing);

amygdalin (batch No. 110820) 201808, China institute for testing food and drug);

liquiritin (batch number: 111610) -201908, China institute for testing food and drug);

baicalin (batch No. 110715-201821, China institute for food and drug testing);

baicalein (batch number: 111595-201808, China institute for food and drug testing);

ammonium glycyrrhizinate (batch No. 110731-202021, China institute for testing food and drug).

Reagent: the chromatographic methanol and acetonitrile are purchased from Merck company of America, the phosphoric acid is purchased from Scharlur company, the monopotassium phosphate is purchased from chemical reagent of national drug group, and the used water is purified water (self-made).

According to the preparation process of the oral liquid for clearing heat and relieving cough for children, extracting and concentrating filtrate (intermediate I, 1 lot) and sampling are obtained, the extracted and concentrating filtrate is averagely divided into two parts to be respectively subjected to subsequent production, refined solution (intermediate II, 2 lots) is obtained and sampled, the production is continued until the oral liquid for clearing heat and relieving cough for children (finished product, 2 lots) is sampled, namely, the extracted and concentrating filtrate (intermediate I) of 1 batch of oral liquid for clearing heat and relieving cough for children respectively corresponds to 2 batches of refined solution (intermediate II) and 2 batches of finished product, and 5 batches of samples are counted. The above steps are circulated for 3 times, and 15 batches of samples are collected and produced by Zhejiang Connbei pharmaceutical Co. The basic information and the corresponding relation of the 15 batches of the infant heat-clearing cough-relieving oral liquid test samples are shown in table 1.

TABLE 1 basic information and corresponding relation of samples for oral liquid for clearing heat and relieving cough in children

Example 1

Method for establishing fingerprint spectrum of finished product of children heat-clearing cough-relieving oral liquid

(1) Preparing a test solution: taking 6 batches of the oral liquid finished products for clearing heat and relieving cough of children in the table 1, respectively preparing different test solution, wherein the specific preparation method of the test solution comprises the following steps: precisely sucking 5mL of the solution, placing the solution in a 100mL volumetric flask, adding 50mL of 50% methanol, performing ultrasonic treatment (frequency of 45kHz) at room temperature for 40min, cooling to room temperature, fixing the volume, shaking up, and filtering with a 0.45-micrometer organic microporous filter membrane; meanwhile, preparing a mixed reference substance solution, wherein the specific preparation method of the mixed reference substance solution comprises the following steps: accurately weighing appropriate amount of (R, S) -goitrin, ephedrine hydrochloride, pseudoephedrine hydrochloride, amygdalin, liquiritin, baicalin, baicalein, and ammonium glycyrrhizinate as reference substances, and making into reference substance solutions containing 0.007mg, 0.02mg, 0.01mg, 0.09mg, 0.02mg, 0.38mg, 0.01mg, and 0.04mg per 1 mL.

(2) HPLC chromatographic conditions: the chromatographic column adopts octadecylsilane chemically bonded silica as a filler, and Agilent InfinityLab Poroshell 120EC-C18 as a chromatographic column, and the specification is 150mm multiplied by 4.6mm and 2.7 mu m; the column temperature is 30 ℃, the flow rate is 1.0mL/min, and the sample injection amount is 20 mu L; detecting the wavelength of 0 min-31 min 210nm, 31 min-65 min270nm, starting DAD detection, taking acetonitrile-methanol (89: 11) as a mobile phase A, taking 0.01mol/L potassium dihydrogen phosphate solution (pH3.2) as a mobile phase B, and carrying out gradient elution (50 mm multiplied by 4.6mm I.D, impurity trapping column indirect between a pump and a sample injector) for 0-5 min, wherein A: B is 2%: 98% → 3%: 97%; 5-15 min, wherein A and B are 3 percent to 97 percent to → 5 percent to 95 percent; 15-25 min, wherein A and B are 5 percent to 95 percent to 10 percent to 90 percent; 25-35 min, wherein A and B are 10 percent to 90 percent to → 15 percent to 85 percent; 35-60 min, wherein A and B are 15 percent to 85 percent to 25 percent to 75 percent; 60-68 min, wherein A and B are 25 percent to 75 percent to → 41 percent to 59 percent; 68-75 min, wherein A and B are 41 percent to 59 percent to → 41 percent to 59 percent.

And respectively carrying out sample injection analysis on different test solution and reference solution by HPLC according to the above chromatographic conditions to obtain HPLC fingerprints (shown in figure 1) of different test solution and fingerprints (shown in figure 2) of mixed reference solution.

(3) Introducing HPLC (high performance liquid chromatography) fingerprint data of 6 batches of the finished product of the infantile heat-clearing and cough-relieving oral liquid into a 2012 edition of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, taking fingerprint of 210610-1 batches of the test solution as a reference spectrum, setting the time window width to be 0.1min, performing multi-point correction and data matching, and generating a superposition spectrum and a control spectrum (R) of the HPLC fingerprint of the infantile heat-clearing and cough-relieving oral liquid by using an averaging method (shown in figure 3).

(4) As can be seen from fig. 1, the separation degree of the 18 th peak (baicalin peak) is good, the peak area is large, and the retention time is moderate, so that the baicalin peak is selected as a reference peak, the relative peak area and the relative retention time of each common peak and the reference peak are calculated, and the calculation results are shown in tables 2 and 3; similarity analysis is carried out on HPLC fingerprint spectrums and a reference spectrum (R) of 6 batches of finished products of the children heat-clearing cough-relieving oral liquid, and analysis results are shown in table 4.

TABLE 26 batches of infantile heat-clearing cough-relieving oral liquid with fingerprint spectrum having common peak relative peak area

TABLE 36 Final oral liquid for clearing away heat and relieving cough for children with common peak of fingerprint relative retention time

TABLE 46 fingerprint similarity of oral liquid for clearing away heat and relieving cough for children

From table 2, it can be seen that the relative peak area range of each common chromatographic peak is less than 0.01, from table 3, the relative retention time RSD of each common chromatographic peak is less than 3%, from table 4, the fingerprint similarity of the test solution of the 6 batches of the oral liquid for clearing heat and relieving cough for children is 1.000, which indicates that the similarity of the oral liquid for clearing heat and relieving cough for children between different batches is high, and the process is relatively stable.

(ii) fingerprinting methodology investigation

1. Specificity test

Taking a blank auxiliary material sample of the oral liquid for clearing heat and relieving cough for children and the oral liquid (210610-1) for clearing heat and relieving cough for children, and preparing a test solution according to the preparation method of the test solution in the embodiment 1; preparing each reference solution and a mixed reference solution according to the preparation method of the reference solution in example 1, and injecting the blank auxiliary material sample solution of the infantile heat clearing and cough relieving oral liquid, the infantile heat clearing and cough relieving oral liquid (210610-1), each reference solution and the mixed reference solution respectively according to the chromatographic conditions in example 1 to obtain an HPLC chromatogram. The result shows that the blank auxiliary material sample solution of the oral liquid for clearing heat and relieving cough for children has no interference on the fingerprint detection of the oral liquid for clearing heat and relieving cough for children, and the method is feasible.

The control solutions were aligned and compared with each other (fig. 2), confirming that 11.498min peak was (R, S) -feiyichun (peak 1), 16.674min peak was ephedrine hydrochloride (peak 2), 17.786min peak was pseudoephedrine hydrochloride (peak 3), 27.723min peak was amygdalin (peak 7), 43.142min peak was glycyrrhizin (peak 12), 56.062min peak was baicalin (peak 18), 70.461min peak was baicalein (peak 27), 73.865min peak was ammonium glycyrrhizinate (peak 28), and relative retention times were 0.205, 0.297, 0.317, 0.495, 0.770, 1.000, 1.257, and 1.318, respectively, with baicalin as reference peak (S). The HPLC finger print with baicalin as reference peak determines 28 common peaks.

2. Repeatability test

Taking the same finished oral liquid (batch No. 210610-1) for clearing heat and relieving cough of children, preparing 6 parts of test solution with serial numbers of S2-S7 in parallel according to the preparation method of the test solution in the example 1, injecting samples according to the chromatographic conditions in the example 1 to obtain HPLC fingerprint spectrums of the 6 parts of test solution, taking the baicalin peak as a reference peak, calculating the relative peak area and the relative retention time of each common peak and the reference peak, and obtaining the calculation results shown in tables 5 and 6; the HPLC fingerprint data of 6 parallel finished products of the pediatric heat-clearing cough-relieving oral liquid are led into a 2012-version traditional Chinese medicine chromatography fingerprint similarity evaluation system to perform similarity analysis (as shown in figure 4) with a control map, and the analysis results are shown in table 7.

TABLE 5 relative peak area of each common peak in fingerprint repeatability test of infantile heat-clearing and cough-relieving oral liquid

TABLE 6 repeated tests of finger print of oral liquid for clearing away heat and relieving cough for children

TABLE 7 repeated fingerprint similarity of oral liquid for clearing away heat and relieving cough for children

As can be seen from tables 5 to 7, the fingerprint similarity of the test solution of the 6 infantile heat-clearing and cough-relieving oral liquid is 1.000 and is more than 0.999, the relative peak area range of each common chromatographic peak is less than 0.01, and the relative retention time RSD is less than 3%, which indicates that the method has good repeatability.

3. Stability test

Taking the same batch of finished products of the pediatric heat-clearing cough-relieving oral liquid (batch No. 210610-1), preparing the test solution according to the test solution in the example 1 (the preparation method is used for preparing the test solution, the sample injection time is 0 hour, 6 hours, 12 hours, 18 hours, 24 hours and 36 hours according to the chromatographic condition in the example 1, obtaining HPLC fingerprints of 6 test solutions, calculating the relative peak areas and the relative retention times of all common peaks and reference peaks by taking a baicalin peak as a reference peak, and leading the HPLC fingerprint data of the 6 parallel finished products of the pediatric heat-clearing cough-relieving oral liquid into a 2012 version of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to perform similarity analysis (shown in figure 5) with the reference spectrum, wherein the analysis results are shown in a table 10.

TABLE 8 fingerprint chromatogram solution stability test of oral liquid for clearing away Heat and relieving cough for children

TABLE 9 Final oral liquid for clearing away Heat and relieving cough for children fingerprint chromatogram solution stability test

TABLE 10 stability fingerprint similarity of heat-clearing cough-relieving oral liquid for children

From tables 8-10, the fingerprint similarity of the test solution of the 6 infantile heat-clearing and cough-relieving oral liquid finished product is 1.000 and is greater than 0.999, the relative peak area range of each common chromatographic peak is less than 0.01, the relative retention time RSD is less than 3%, and the stability of the solution in 36h is good.

4. Correlation and common peak attribution analysis of finished product of oral liquid for clearing heat and relieving cough of children and each single medicinal material

Preparing each single medicinal material extract of ephedra herb, fried bitter apricot seed, gypsum, liquorice, baical skullcap root, indigowoad root and asiatic moonseed root according to the preparation process of the oral liquid for clearing heat and relieving cough of children, and respectively carrying out sample injection analysis on each single medicinal material extract by HPLC (the chromatographic conditions are the same as in example 1) to obtain the HPLC color spectrum of each single medicinal material extract; comparing and analyzing the HPLC fingerprint of the finished product of the children heat-clearing cough-relieving oral liquid and the HPLC chromatogram of each single medicinal material extract by a DAD detector, comparing the retention time of chromatographic peaks, and finally confirming the attribution peak of the common chromatographic peak on the medicinal material chromatogram in the fingerprint of the children heat-clearing cough-relieving oral liquid.

4.1 common Peak assignment analysis in Ephedra

The comparison of HPLC finger print of the final product of the oral liquid for clearing heat and relieving cough and HPLC chromatogram of herba Ephedrae extractive solution is shown in FIG. 6. As can be seen from FIG. 6, the 2, 3, 5 peaks among the common peaks are derived from Ephedra sinica Stapf

4.2 common Peak assignment analysis in Scutellaria baicalensis

The comparison of HPLC fingerprint of the infantile heat-clearing and cough-relieving oral liquid product and HPLC chromatogram of Scutellariae radix extract is shown in FIG. 7. As can be seen from fig. 7, the No. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 peaks among the common peaks are derived from scutellaria baicalensis.

4.3 common Peak assignment analysis in Asiatic moonseed rhizome

The comparison of HPLC fingerprint common mode of the infantile heat-clearing and cough-relieving oral liquid product and HPLC chromatogram of rhizoma Menispermi extract is shown in FIG. 8. As can be seen from fig. 8, the 9-, 10-and 11-peaks among the common peaks are derived from asiatic moonseed rhizome.

4.4 common Peak assignment analysis in Fried bitter apricot kernels

The comparison of HPLC fingerprint of the infantile heat-clearing cough-relieving oral liquid product and HPLC chromatogram of parched semen Armeniacae amarum extractive solution is shown in FIG. 9. As can be seen from FIG. 9, the peaks No. 6 and 7 among the common peaks are derived from roasted bitter apricot seeds.

4.5 common Peak assignment analysis in Isatis root

The comparison of HPLC fingerprint of the infantile heat-clearing and cough-relieving oral liquid product and HPLC chromatogram of radix Isatidis extract is shown in FIG. 10. As can be seen from fig. 10, the peak No. 1 among the common peaks is derived from isatis root.

4.6 common Peak assignment analysis in Glycyrrhiza uralensis

The comparison of HPLC fingerprint of the infantile heat-clearing and cough-relieving oral liquid product and HPLC chromatogram of Glycyrrhrizae radix extract is shown in FIG. 11. As can be seen from fig. 11, among the common peaks, peaks 12, 13, 19, and 28 are derived from glycyrrhiza.

4.7 common Peak assignment analysis in Gypsum

The comparison of HPLC fingerprint of the infantile heat-clearing and cough-relieving oral liquid product and HPLC chromatogram of Gypsum Fibrosum extract is shown in FIG. 12. As can be seen from fig. 12, no common peak is from gypsum.

The analysis of the results shows that 28 common peaks in the fingerprint of the oral liquid for clearing heat and relieving cough of children are obtained, wherein

peaks

2, 3 and 5 are from ephedra medicinal materials; 13. 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27 are from scutellaria baicalensis; 9. no. 10 and No. 11 are from rhizoma Menispermi; 6. no. 7 peak is derived from parched semen Armeniacae amarum; the No. 1 peak is derived from radix Isatidis; 12. 13, 19, 28 Feng from radix Glycyrrhizae; 4. no. 8 has no herb attribution, and possibly, the new ingredients are generated in the decoction process. Therefore, the fingerprint spectrum of the oral liquid for clearing heat and relieving cough for children can basically represent the material basis of the oral liquid for clearing heat and relieving cough for children.

Example 2

Establishment method for extracting concentrated filtrate fingerprint (intermediate I)

3mL of the 3 batches of the extract concentrated filtrate (intermediate I) of the pediatric heat clearing and cough relieving oral liquid in Table 1 are taken, the test solution is prepared according to the preparation method of the test solution in example 1, and the HPLC fingerprint (shown in figure 13) of the extract concentrated filtrate (intermediate I) of the pediatric heat clearing and cough relieving oral liquid in 3 batches is obtained by sample injection analysis according to the chromatographic conditions in example 1.

Introducing HPLC fingerprint data of 3 batches of concentrated filtrate (intermediate I) extracted from the oral liquid for clearing heat and relieving cough of children into a 2012 edition of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system for similarity analysis with a control map (as shown in FIG. 14); calculating the relative peak area and the relative retention time of each common peak and the reference peak, wherein the calculation results are shown in tables 11 and 12; the results of the similarity analysis are shown in Table 13.

TABLE 113 batches of the concentrated filtrates from the pediatric heat-clearing cough-relieving oral liquid (intermediate I) extraction with the peak relative area

TABLE 123 batches of the relative retention times of the common peaks of the concentrated filtrates (intermediate I) extracted from the pediatric heat-clearing and cough-relieving oral liquid

TABLE 133 concentrated filtrate (intermediate I) for heat-clearing and cough-relieving oral liquid for children with fingerprint similarity

From tables 11-13, the similarity of the fingerprint spectrum of the concentrated filtrate (intermediate I) extracted from 3 batches of the oral liquid for clearing heat and relieving cough for children and the comparison spectrum is 1.000 and is more than 0.999, the relative peak area range of each common chromatographic peak is less than 0.01, and the relative retention time RSD is less than 3%, which indicates that the similarity between different batches is high and the process is relatively stable.

Example 3

Establishment of fingerprint of refined liquid (intermediate II) of children heat-clearing cough-relieving oral liquid

5mL of 6 batches of the refined preparation solution (intermediate II) of the pediatric heat-clearing cough-relieving oral liquid in Table 1 are taken, the test solution is prepared according to the preparation method of the test solution in example 1, and the HPLC fingerprint (shown in figure 15) of the 6 batches of the refined preparation solution (intermediate II) of the pediatric heat-clearing cough-relieving oral liquid is obtained by sample injection analysis according to the chromatographic conditions in example 1.

Introducing HPLC fingerprint data of 6 batches of refined oral liquid (intermediate II) for clearing heat and relieving cough for children into a 2012 edition of Chinese medicinal chromatogram fingerprint similarity evaluation system for similarity analysis with a control chromatogram (as shown in FIG. 16); calculating the relative peak area and relative retention time of each common peak and the reference peak, wherein the calculation results are shown in tables 14 and 15; the results of the similarity analysis are shown in Table 16.

TABLE 146 batches of refined preparation liquid (intermediate II) for children for clearing away Heat and relieving cough

TABLE 156 batches of refined liquid (intermediate II) for children's heat-clearing cough-relieving oral liquid with common peak relative retention time

TABLE 166 refined liquid (intermediate II) for infantile heat-clearing cough-relieving oral liquid with fingerprint similarity

From tables 14 to 16, the similarity between the fingerprint of the refined compound liquid (intermediate II) of 6 batches of the children heat-clearing cough-relieving oral liquid and the comparison map is 1.000 and more than 0.999, the relative peak area range of each common chromatographic peak is less than 0.01, and the relative retention time RSD is less than 3%, which indicates that the similarity between different batches is high and the process is relatively stable.

The same batch of medicinal materials are prepared into a concentrated extract filtrate (intermediate I), a refined compound solution (intermediate II) of the oral liquid for clearing heat and relieving cough for children and a finished product of the oral liquid for clearing heat and relieving cough for children according to the preparation process of the oral liquid for clearing heat and relieving cough for children, and the change of the relative peak area of each common peak is shown in tables 17-19.

TABLE 1728 changes in relative peak area of each common peak (one)

TABLE 1828 changes in relative peak area of each common peak (two)

TABLE 1928 variation of relative peak area of each common peak (III)

From tables 17 to 19, the change rule of the relative peak area of each common peak of the samples in different process procedures is as follows: from the extraction and concentration process to the fine preparation process (from the intermediate I to the intermediate II), the relative peak areas of the other common peaks are not obviously changed except that the relative peak areas of the No. 5 peak and the No. 6 peak are slightly increased; from the refined preparation process to the finished product of the oral liquid for clearing heat and relieving cough for children (from the intermediate II to the finished product), the relative peak areas of all common peaks have no obvious change.

The test results show that the establishing method of the fingerprint of the oral liquid for clearing heat and relieving cough for children, the fingerprint and the application thereof comprehensively analyze the whole components of the oral liquid for clearing heat and relieving cough for children, can basically represent and reflect the effective components and material basis of the oral liquid for clearing heat and relieving cough for children, can have certain identification significance on the quality control of different process links of the preparation by establishing the fingerprint of the oral liquid for clearing heat and relieving cough for children and a process intermediate thereof, provides certain theoretical basis and support for the establishment of a whole-process quality control system, can comprehensively reflect the quality information of the oral liquid for clearing heat and relieving cough for children, and ensures the uniform and stable product quality.

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.

Claims

1. A method for establishing a fingerprint of an oral liquid for clearing heat and relieving cough for children is characterized by comprising the following steps:

(1) preparing different test solution;

(2) respectively carrying out sample injection analysis on different sample solutions by adopting HPLC (high performance liquid chromatography), obtaining HPLC fingerprint spectrums of the different sample solutions, and confirming that 28 common chromatographic peaks exist;

(3) introducing HPLC (high performance liquid chromatography) fingerprint data of different test solution into 2012 edition of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, taking 1 of the test solution fingerprint as a reference map, setting the time window width to be 0.1min, and generating an HPLC fingerprint comparison map (R) of the oral liquid for clearing heat and relieving cough of the children by using a mean value method through multipoint correction and data matching;

2. The method for establishing the fingerprint of the oral liquid for clearing heat and relieving cough of children according to claim 1, wherein in the step (1), the test solution is prepared by the following method: precisely sucking 3-10 mL of a test article, placing the test article in a 100mL volumetric flask, adding 50mL of 30-90% methanol aqueous solution, carrying out ultrasonic treatment at room temperature for 30-60 min, cooling to room temperature, fixing the volume, shaking up, and filtering.

3. The method for establishing the fingerprint of the oral liquid for clearing heat and relieving cough of children according to claim 2, wherein the test solution is prepared by the following method: precisely sucking 5mL of a test article, placing the test article in a 100mL volumetric flask, adding 50mL of 50% methanol aqueous solution by volume fraction, carrying out ultrasonic treatment at room temperature for 40min, cooling to room temperature, fixing the volume, shaking up, and filtering.

4. The method for establishing the fingerprint of the oral liquid for clearing heat and relieving cough for children according to claim 1, wherein in the step (1), the sample is the oral liquid for clearing heat and relieving cough for children, extracted concentrated filtrate or refined liquid.

5. The method for establishing the fingerprint of the oral liquid for clearing heat and relieving cough in children according to claim 1, wherein in the step (2), HPLC chromatographic conditions are as follows: the chromatographic column adopts octadecylsilane chemically bonded silica as a filler, and Agilent InfinityLab Poroshell 120EC-C18 as a chromatographic column, and the specification is 150mm multiplied by 4.6mm and 2.7 mu m; the column temperature is 30 ℃, the flow rate is 1.0mL/min, and the sample injection amount is 20 mu L; detecting the wavelength of 0 min-31 min 210nm, 31 min-65 min270nm, starting DAD detection, taking acetonitrile-methanol (89: 11) as a mobile phase A, taking 0.01mol/L potassium dihydrogen phosphate solution (pH3.2) as a mobile phase B, and carrying out gradient elution (50 mm multiplied by 4.6mm I.D, impurity trapping column indirect between a pump and a sample injector) for 0-5 min, wherein A: B is 2%: 98% → 3%: 97%; 5-15 min, wherein A and B are 3 percent to 97 percent to → 5 percent to 95 percent; 15-25 min, wherein A and B are 5 percent to 95 percent to 10 percent to 90 percent; 25-35 min, wherein A and B are 10 percent to 90 percent to → 15 percent to 85 percent; 35-60 min, wherein A and B are 15 percent to 85 percent to 25 percent to 75 percent; 60-68 min, wherein A and B are 25 percent to 75 percent to → 41 percent to 59 percent; 68-75 min, wherein A and B are 41 percent to 59 percent to → 41 percent to 59 percent.

6. The method for establishing the fingerprint of the oral liquid for clearing heat and relieving cough in children according to claim 1, wherein in the step (2), 28 common chromatographic peaks respectively comprise: peak 1 with a relative retention time of 0.205, peak 2 with a relative retention time of 0.297, peak 3 with a relative retention time of 0.317, peak 4 with a relative retention time of 0.391, peak 5 with a relative retention time of 0.446, peak 6 with a relative retention time of 0.487, peak 7 with a relative retention time of 0.495, peak 8 with a relative retention time of 0.514, peak 9 with a relative retention time of 0.521, peak 10 with a relative retention time of 0.627, peak 11 with a relative retention time of 0.761, peak 12 with a relative retention time of 0.770, peak 13 with a relative retention time of 0.785, peak 14 with a relative retention time of 0.801, peak 15 with a relative retention time of 0.816, peak 16 with a relative retention time of 0.853, peak 17 with a relative retention time of 0.883, peak 18 with a relative retention time of 1.000, peak 18 with a relative retention time of 1.051, peak 18 with a relative retention time of 1.20, The peak 21 with a relative retention time of 1.110, the peak 22 with a relative retention time of 1.126, the peak 23 with a relative retention time of 1.138, the peak 24 with a relative retention time of 1.149, the peak 25 with a relative retention time of 1.172, the peak 26 with a relative retention time of 1.249, the peak 27 with a relative retention time of 1.257, and the peak 28 with a relative retention time of 1.318, wherein 8 chromatographic peaks are identified as 1 peak (R, S) -gomphrenaline, 2 peak ephedrine hydrochloride, 3 peak pseudoephedrine hydrochloride, 7 peak amygdalin, 12 peak glycyrrhizin, 18 peak baicalin, 27 peak baicalein, and 28 peak ammonium glycyrrhizinate, respectively.

7. A fingerprint of an oral liquid for clearing heat and relieving cough for children obtained by the method for establishing the fingerprint of the oral liquid for clearing heat and relieving cough as claimed in any one of claims 1 to 6.