CN113125569B

CN113125569B - Fingerprint spectrum determination method and quality control method based on Guyinshi material

Info

Publication number: CN113125569B
Application number: CN201911391325.6A
Authority: CN
Inventors: 张�杰; 陈阳
Original assignee: HUNAN YINENG BIOLOGICAL PHARMACEUTICAL CO Ltd
Current assignee: HUNAN YINENG BIOLOGICAL PHARMACEUTICAL CO Ltd
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2022-07-22
Anticipated expiration: 2039-12-30
Also published as: CN113125569A

Abstract

The invention provides a fingerprint spectrum measuring method and a quality control method based on a Guyinjian substance. In the process of establishing the fingerprint spectrum of the Guyinhuan decoction substance standard, 12 common characteristic peaks are confirmed, the problems that the fingerprint characteristic peaks are difficult to separate and the impurity peaks are interfered are solved, the chemical composition stability and the use safety of the substance standard are ensured, an important reference basis is provided for the quality control of subsequent preparations, the quality of the Guyinhuan decoction substance standard is effectively controlled, and the more normal quality control of a classical famous prescription is realized.

Description

Fingerprint spectrum determination method and quality control method based on Guyinshi material

Technical Field

The invention relates to the field of medicine quality control, in particular to a fingerprint spectrum measuring method and a quality control method of a Guyinshan material standard.

Background

Guyin Jian is from Jingyue quan Shu (Ming Zhang Jingyue): it is indicated for yin deficiency with slippery diarrhea, stranguria with turbid urine, and channel water deficiency with insecurity. The formula mainly controls liver and kidney, and is convenient for ginseng, prepared rhizome of rehmannia, radix rehmanniae, Chinese yam, rhizoma dioscoreae, fructus corni, fructus aurantii, polygala tenuifolia, radix glycyrrhizae, fructus schizandrae, fructus schisandrae, semen cuscutae, fructus lycii and semen lycii are fried to be fragrant. Decoct the above-mentioned herbs with water for seven minutes, and take the decoction at a high temperature. Is one of 100 catalogs published by the State administration of traditional Chinese medicine (the first catalog in ancient classical famous parties).

The classic famous formula of the traditional Chinese medicine is a prominent representative of a traditional Chinese medicine prescription, bears the deep and thick deposition of brilliant civilization of the traditional Chinese medicine for thousands of years, is a summary of clinical experience of the past generations after the traditional Chinese medicine theory is trained for thousands of years, and is the most essential part in the great treasury of the traditional Chinese medicine Wei. The traditional Chinese medicine classic name prescription is deeply researched and developed and is a gold key for excavating traditional Chinese medicine treasury. The traditional Chinese medicine decoction with the famous classic formula is used as the most common preparation formulation of the traditional Chinese medicine clinical medication, has the advantages of reasonable formula, quick response, obvious curative effect, easy absorption and the like, and is deeply trusted by patients. But the disadvantages of easy mildewing, spoilage and deterioration, bitter taste and large amount of the soup after being prepared, carried, decocted temporarily and placed for a long time, difficult unification of standards, serious influence on the clinical curative effect and the like do not meet the living requirements of modern people. In order to maintain the advantages of the decoction and overcome various defects of the decoction, the national food and drug administration and supervision and management headquarter proposes and adopts the concept of the material standard of the classic famous prescription in registration and management regulation of 6 months in 2018, and clearly indicates that the key quality attribute of the preparation of the classic famous prescription is consistent with the key quality attribute determined by the material standard of the classic famous prescription. The material standard represents the whole internal quality of the preparation, and is basically consistent with other quality control indexes of the preparation except for a forming process. Therefore, the material standard is a real object contrast of the inherent quality of the preparation and is a reference object for the optimization of the large-scale production process and the establishment of the quality standard thereof. The material standard is the standard of classical famous prescriptions and even the research and development of all traditional Chinese medicines, and is a reference substance for ensuring the safety and effectiveness of the medicine. The production process route, parameter optimization and quality standard of the compound preparation of the classical famous prescription are made by taking the substance reference of the classical famous prescription as reference. The material standard is a link for clinical, enterprise and scientific research, and is the standard for inheriting, applying and developing traditional Chinese medicine.

The fingerprint spectrum is based on the recognition of the whole action of the Chinese medicine substance group, and the spectrum or chromatogram of the chemical components of the Chinese medicine is obtained by means of the spectrum and the chromatographic technology, so that the fingerprint spectrum is a feasible mode for realizing the identification of the authenticity of the Chinese medicine, the evaluation of quality consistency and the stability of products, and has the characteristics of large information amount, strong characteristics, integrity, fuzziness and the like. The traditional Chinese medicine fingerprint spectrum can comprehensively reflect the relative relation of chemical components contained in medicinal materials, reflects the complexity and the correlation of the traditional Chinese medicine components, adapts to the traditional theory of traditional Chinese medicines, can really and effectively characterize, comprehensively evaluate and comprehensively control the internal quality of the traditional Chinese medicines, and is particularly suitable for controlling the quality of the traditional Chinese medicines and traditional Chinese medicine products under the condition that the effective components are not completely clear or are not completely clear. The traditional Chinese medicine fingerprint spectrum can be used for inspecting factors such as the production area, the harvesting season, the harvesting part, the processing and the storage time of the traditional Chinese medicine, so as to provide a basis for identifying the authenticity of the raw material medicinal materials before production, and can be further used for the quality control in the production process of the traditional Chinese medicine: tracking the change of some chemical components in the preparation, and monitoring the quality consistency and stability between the raw medicinal materials and the finished product and between batches of the finished product. Compared with a quality analysis method for measuring the content of index components, the fingerprint can comprehensively reflect the types and the quantity of chemical components of the traditional Chinese medicine, can realize comprehensive evaluation on the internal quality of the traditional Chinese medicine and effective control on the whole substances of the traditional Chinese medicine under the condition that the effective components of a compound preparation of the traditional Chinese medicine are not completely clarified, and is one of the effective means for controlling the quality of the traditional Chinese medicine and the preparation thereof at present. The yin-strengthening decoction medicine has various raw materials and complex material reference components, in the prior art, only single component is subjected to quality analysis, a comprehensive and systematic quality control method for reflecting the quality conditions of the main material reference components in the yin-strengthening decoction medicine and a finished product is not available, the production process and the product quality cannot be effectively controlled, and the clinical curative effect cannot be better ensured.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a fingerprint spectrum measuring method and a quality control method of a Guyinshan material standard, which are used for comprehensively controlling the quality of raw medicinal materials and finished products and ensuring the stable quality of the products. The specific scheme is as follows:

a fingerprint spectrum measuring method of a Guyinshi material standard is characterized in that: the method comprises the following steps:

(1) preparing a test solution: taking the reference powder of the solid yin decoction material, taking methanol as an extraction solvent, carrying out ultrasonic treatment, and filtering to obtain a subsequent filtrate for later use;

preparation of mixed control solution: mixing the reference substances, dissolving in methanol to obtain mixed reference substance solution;

preparing a negative control solution of the single decoction piece: taking negative powder of each single decoction piece, performing ultrasonic treatment by using methanol as an extraction solvent, and filtering to obtain a subsequent filtrate for later use;

(2) respectively sucking the test sample solution, the mixed reference substance solution and the single-herb negative reference solution, injecting the two solutions into a high performance liquid chromatograph for determination, and respectively obtaining liquid chromatographs of the test sample solution, the mixed reference substance solution and the single-herb negative reference solution;

(3) adopting traditional Chinese medicine chromatogram fingerprint similarity evaluation system software prepared by the State pharmacopoeia Commission to carry out data matching on the liquid chromatogram of the test solution, the mixed reference solution and the single-flavor decoction piece negative reference solution, thus obtaining a standard fingerprint;

the chromatographic conditions of the step (2) are as follows:

a chromatographic column: agilent 5TC-C18 chromatographic column, 4.6mm × 250mm, 5 μm;

mobile phase: acetonitrile-0.3% phosphoric acid solution;

flow rate: 1.0 ml/min;

wavelength: 250 nm;

sample introduction amount: 20 mu l of the mixture;

column temperature: at 40 ℃;

the number of theoretical plates is not less than 7000 calculated according to the hyperin peak;

the gradient elution procedure is shown in table 1.

TABLE 1 fingerprint gradient elution procedure

The reference substance is 5-hydroxymethyl furfural, morroniside, chlorogenic acid, loganin, hyperoside, 3, 6' -mustard acyl sucrose, ammonium glycyrrhizinate and schizandrol A reference substance.

Further, the standard fingerprint comprises 12 common peaks: peak 1 is from radix rehmanniae Preparata and fructus Schisandrae, Peak 3, 5, 6, 8 is from Corni fructus, Peak 4, 7 is from semen Cuscutae, Peak 9 is from cortex et radix Polygalae, Peak 10, 11 is from Glycyrrhrizae radix, Peak 12 is from fructus Schisandrae, and Peak 7 is used as reference peak.

Further, in the standard fingerprint, the peak 1 is 5-hydroxymethylfurfural, the peak 3 is morroniside, the peak 4 is chlorogenic acid, the peak 6 is loganin, the peak 7 is hyperin, the peak 9 is 3, 6' -erucyl sucrose, the peak 11 is glycyrrhizic acid, and the peak 12 is schizandrol A.

Further, the volume fraction of the methanol in the step (1) is 50-75%.

Further, the filtration in the step (1) is a filtration with a microporous filter membrane, and specifically, the microporous filter membrane is a 0.45 μm filter membrane.

Further, the concentration is a concentration under reduced pressure.

Further, the drying is vacuum drying or freeze drying, specifically, freeze drying.

Furthermore, the reference powder of the yin-fixing decoction material in the step (1) is obtained by decocting, concentrating and drying the yin-fixing decoction prescription recorded in the ancient catalogues of famous and famous users (the first batch) published by the national traditional Chinese medicine administration.

Furthermore, the decoction meets the requirements of the Chinese medicine decoction room management standard issued by the State administration of Chinese medicine and drug administration.

Further, the method for extracting with methanol as a solvent in the step (1) comprises the following steps: taking 1 part by weight of the basic powder of the solid yin decoction material, adding 25 parts by volume of methanol, carrying out ultrasonic treatment, and filtering to obtain a subsequent filtrate for later use;

further, the corresponding relationship between the parts by weight and the parts by volume is as follows: g/ml.

Further, the volume fraction of the methanol in the step (1) is 50-75%, preferably 50%.

Further, the ultrasonic treatment parameter in the step (1) is power 500W and frequency 40 KHz.

Furthermore, the preparation method of the single-flavor decoction piece negative powder is the same as the preparation method of the solid yin decoction substance reference powder.

Furthermore, the preparation method of the negative control solution of the single decoction piece is the same as that of the standard test sample solution of the solid yin decoction substance.

The invention also provides a quality control method of the yin-strengthening decoction substance standard, which is characterized by comprising the following steps:

(1) establishing a standard fingerprint of the Guyinshi material according to the fingerprint measuring method;

(2) taking the Guyinhao standard granule sample solution, and detecting according to the chromatographic conditions in the step (2) in the fingerprint spectrum determination method to obtain a fingerprint spectrum;

(3) and (3) comparing the fingerprint obtained in the step (2) with the standard fingerprint obtained in the step (1), wherein a qualified product is obtained if the fingerprint is qualified, and an unqualified product is obtained if the fingerprint is not qualified.

The met requirements include: 12 characteristic peaks should be presented in the sample fingerprint, wherein 8 peaks should be respectively retained for the same time as the corresponding reference peaks in the standard fingerprint; the peak corresponding to the hyperin reference substance is an S peak, and the relative retention time of each characteristic peak and the S peak is within +/-10% of the retention time value of the standard fingerprint; according to the similarity evaluation system of the traditional Chinese medicine chromatogram fingerprint, the similarity of the fingerprint of the test sample and the standard fingerprint is calculated and is not lower than 0.90.

The preparation method of the test solution of the Guyinshi standard granules comprises the following steps: grinding the decoction of GUYIN standard granules with different dosages, precisely weighing about 3g, placing in a 50ml measuring flask, adding appropriate amount of 50% methanol, ultrasonic treating (power 500W, frequency 40kHz) for 30 min for dissolving, cooling, adding 50% methanol to 50ml scale, shaking, filtering, and collecting the filtrate.

Compared with the prior art, the invention has the following beneficial effects:

(1) the fingerprint spectrum of the basis of the solid yin decoction substance is established, the defect that the whole content is difficult to reflect by measuring the content of a single component is overcome, the internal quality of the basis of the solid yin decoction substance can be controlled integrally and macroscopically, the curative effect of the medicament is ensured, and the more formal quality control of the classical famous prescription is realized.

(2) The method adopts a gradient elution method in the process of establishing the fingerprint spectrum, and solves the problems of difficult separation of the characteristic peaks of the fingerprint and interference of impurity peaks.

(3) In the process of establishing the fingerprint spectrum of the Guyinjian material standard, 12 common characteristic peaks are confirmed, the relative retention time, the relative peak area and the similarity of the characteristic peaks are researched, the chemical composition stability and the use safety of the material standard are ensured, and an important reference basis is improved for the quality control of subsequent preparations.

(4) The fingerprint of each effective component in the fixed-yin decoction substance standard is taken as a whole to be treated, the front-back sequence and the mutual relation of each characteristic peak are emphasized, the one-sidedness of the whole quality of the fixed-yin decoction substance standard determined by measuring only one or two chemical components is avoided, the possibility of manual treatment for reaching the standard of the quality is reduced, and a new method and means are provided for completely and accurately evaluating the quality of the fixed-yin decoction substance standard.

(5) The method has the advantages of good stability, high precision, good reproducibility, convenience and easiness in mastering.

Drawings

FIG. 1 is a graph of 200-400nm full wavelength scan in example 1.

FIG. 2 is a comparative HPLC spectrum for different wavelengths in example 1.

FIG. 3 is a comparative chromatogram of different gradient HPLC in example 1.

FIG. 4 is a comparative HPLC chromatogram of columns from different manufacturers in example 1.

FIG. 5 is a comparative chart of HPLC at different column temperatures in example 1.

FIG. 6 is a comparative HPLC profile for different flow rates in example 1.

Figure 7 is a reference HPLC profile of 20 lots of material from example 1.

Figure 8 shows the reference fingerprints (after matching) for 20 batches of material in example 1.

Figure 9 shows the reference fingerprints (consensus pattern) for 20 batches of material from example 1.

FIG. 10 is a comparative HPLC profile of the combined control and whole formula of example 1.

FIG. 11 is a comparison of the negative control fingerprints of the whole formula and the single herb decoction pieces in example 1.

Detailed Description

The following description describes alternative embodiments of the invention to teach one of ordinary skill in the art how to make and use the invention. Some conventional aspects have been simplified or omitted for the purpose of teaching the technical solutions of the present invention. Those skilled in the art will appreciate that variations or substitutions from these embodiments will fall within the scope of the invention.

Example 1 method of determining fingerprint of Guyinshan material basis

1.1 instruments and reagents

The instrument comprises: waters 2695-; a SHIMADZU AUW120D one in ten thousand electronic balance (SHIMADZU corporation, japan), and a BSA124S one in ten thousand electronic balance (Sartorius corporation, germany).

Reagent preparation: 5-hydroxymethylfurfural (batch No. 111626-201711 with purity of 90.8%), morroniside (batch No. 111998-201602 with purity of 96.3%), chlorogenic acid (batch No. 110753-201817 with purity of 96.8%), loganin (batch No. 111640-201707 with purity of 99.2%), hyperin (batch No. 111521-201004 with purity of 93.9%), 3, 6' -dipasyl sucrose (batch No. 111848-201604 with purity of 96.7%, China food and drug testing institute), ammonium glycyrrhizinate (batch No. 110731-201720 with purity of 97.7%), schisandrinol A (batch No. 110857-201714 with purity of 99.9%) controls were purchased from China food and drug testing institute; methanol (chromatographically pure, TEDIA corporation, usa); acetonitrile (chromatographically pure, TEDIA corporation, usa); phosphoric acid (super pure, chemical reagents of national drug group, ltd.); purified water (Hangzhou child Haha group Co., Ltd.).

1.2 sample preparation:

(1) preparation of a sample: weighing eight medicinal material decoction pieces in a prescription amount for 2 days according to the prescription proportion, decocting twice, soaking in water 7 times of the first time for 30 minutes, heating with strong fire to boil, decocting with slow fire for 30 minutes, filtering with a 120-mesh nylon filter cloth, and reserving decoction; adding 6 times of water into the medicine residues, heating the medicine residues to boil with strong fire, decocting the medicine residues with slow fire for 20 minutes, filtering the medicine residues by using a 120-mesh nylon filter cloth, combining decoction liquids obtained in the two times, concentrating the decoction liquids under reduced pressure to obtain fluid extract with the relative density of 1.10-1.15 (20 ℃), taking out the fluid extract, freeze-drying the fluid extract, and grinding the fluid extract into powder to obtain yin-consolidating decoction substance reference freeze-dried powder, wherein 20 batches of substance reference freeze-dried powders of different batches are prepared according to the method for later use.

(2) Preparation of a test solution: respectively taking about 0.4g of 20 batches of substance standard freeze-dried powder, precisely weighing, placing in a 10ml measuring flask, adding a proper amount of 50% methanol, carrying out ultrasonic treatment (power 500W and frequency 40kHz) for 30 minutes, cooling, adding 50% methanol for diluting to a scale, shaking uniformly, filtering, and taking a subsequent filtrate to obtain a sample solution for later use.

(3) Preparation of mixed control solution: taking a proper amount of 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, hyperoside, 3, 6 '-brassica juncea acyl sucrose, ammonium glycyrrhizinate and schizandrol A as reference substances, precisely weighing, adding 50% methanol to prepare a mixed solution containing 41 mu g of 5-hydroxymethylfurfural, 104 mu g of morroniside, 37 mu g of chlorogenic acid, 77 mu g of loganin, 24 mu g of hyperoside, 20 mu g of 3, 6' -brassica juncea acyl sucrose, 47 mu g of ammonium glycyrrhizinate and 17 mu g of schizandrol A in each 1ml, shaking uniformly, and filtering to obtain the final product.

(4) Preparing a negative sample of the single decoction piece: weighing a yin-strengthening decoction substance reference prescription which is lack of each single decoction piece in the prescription for 2 days according to the prescription proportion, decocting twice, adding 7 times of water for the first time, soaking for 30 minutes, heating to boiling with strong fire, decocting for 30 minutes with slow fire, filtering with a 120-mesh nylon filter cloth, and reserving a decoction; adding 6 times of water into the decoction dregs, heating the decoction dregs to boiling with strong fire, decocting the decoction dregs for 20 minutes with slow fire, filtering the decoction dregs by using a 120-mesh nylon filter cloth, combining decoction liquids obtained in the two times of decoction, concentrating the decoction liquids under reduced pressure to obtain a fluid extract with the relative density of 1.10-1.15 (20 ℃), taking out the fluid extract, freeze-drying the fluid extract, and grinding the fluid extract into powder to obtain negative freeze-dried powder of each single decoction piece for later use.

(5) Preparing a negative control solution of the single decoction piece: taking about 0.4g of material-based negative freeze-dried powder lacking each single-ingredient decoction piece, accurately weighing, putting into a 10ml measuring flask, adding a proper amount of 50% methanol, carrying out ultrasonic treatment (power 500W and frequency 40kHz) for 30 minutes, cooling, adding 50% methanol to dilute to a scale, shaking up, filtering, and taking subsequent filtrate to obtain a negative control solution of each single-ingredient decoction piece for later use.

1.3 chromatographic conditions

And (3) chromatographic column: agilent 5TC-C18 chromatographic column, 4.6mm × 250mm, 5 μm;

mobile phase: acetonitrile-0.3% phosphoric acid solution;

flow rate: 1.0 ml/min;

wavelength: 250 nm;

sample introduction amount: 20 mu l of the solution;

column temperature: 40 ℃;

the gradient elution procedure is shown in table 1.

TABLE 1 fingerprint gradient elution procedure

1.3.1 selection of detection wavelength

The DAD detector is used to perform full-wavelength scanning on the test sample in the range of 200-400nm, and chromatograms at the wavelengths of 250nm, 278nm and 330nm are compared, and the results are shown in figures 1 and 2. (A: wavelength 250nm B: wavelength 278nm C: wavelength 330nm)

As can be seen from fig. 1 and 2: the number, the absorption intensity and the separation degree of chromatographic peaks under the three wavelengths are different, and the number of the chromatographic peaks is the largest when the wavelength is 250nm, the absorption intensity of each chromatographic peak is relatively average and the separation degree is good, so that the wavelength of 250nm is finally selected as the optimal detection wavelength.

1.3.2 selection of the Mobile phase

And (3) screening water-acetonitrile of a mobile phase system, 0.3% phosphoric acid solution-acetonitrile, 0.3% glacial acetic acid solution-acetonitrile, 0.3% formic acid solution-acetonitrile and 0.3% trifluoroacetic acid solution-acetonitrile. As a result, the separation degree of the 0.3% phosphoric acid solution and the acetonitrile is good, and the characteristic peak is obvious, and finally, the 0.3% phosphoric acid solution and the acetonitrile are determined to be a mobile phase system. Secondly, elution gradients with different proportions are optimized, wherein the

elution gradients

1, 2 and 3 are shown in the following tables 2-1, 2-2 and 2-3, and the results are shown in figure 3. (A: gradient 1B: gradient 2C: gradient 3)

TABLE 2-1 FIXED YIN decoction Standard HPLC fingerprinting gradient 1 elution TABLE

TABLE 2-2 FIXED YIN POTTERY SUBSTANCE REFERENCE HPLC FINGERPRINT GRADIENT 2 elution TABLE

TABLE 2-3 FIXED YIN POWDER MATERIALS REFERENCE HPLC FINGERPRINT GRADIENT 3 elution TABLE

As can be seen from fig. 3: the chromatographic peak separation degree is good under the condition of the elution gradient 3, and the retention time is proper, so the gradient 3 is finally selected as the optimal elution gradient.

The results of comparison of the columns of Agilent 5TC-C18, Kromasil C18, Phenomenex C18, respectively, are shown in FIG. 4. (A: Agilent 5TC-C18 column B: Kromasil C18 column C: Phenomenex C18 column)

As can be seen from fig. 4: the sample has good resolution of each chromatographic peak under Agilent 5TC-C18 chromatographic column, obvious characteristic peak and good peak shape. Therefore, the Agilent 5TC-C18 chromatographic column is finally selected as the chromatographic column of HPLC fingerprint.

1.3.3 selection of column temperature

The column temperature conditions of 30 ℃, 35 ℃ and 40 ℃ were screened and optimized, respectively, and the results are shown in fig. 5. (A: 30 ℃ C.: 35 ℃ C.: 40 ℃ C.)

As can be seen from fig. 5: the chromatographic peak separation degree is good under the condition of 40 ℃ of the column temperature, and the influence of interference factors is small. Therefore, the column temperature of 40 ℃ is finally selected as the optimal column temperature condition of the HPLC fingerprint.

1.3.4 selection of flow Rate

The flow rate conditions of 0.8ml/min, 1.0ml/min and 1.2ml/min were screened and optimized, respectively, and the results are shown in FIG. 6. (A: 0.8ml/min B: 1.0ml/min C: 1.2ml/min)

As can be seen from fig. 6: the chromatographic peak separation degree is good under the condition of the flow rate of 1.0ml/min, and the influence of interference factors is small. Therefore, the flow rate of 1.0ml/min is finally selected as the optimal flow rate condition for HPLC fingerprint.

1.4 methodological inspection

1.4.1 Instrument precision test

Taking the sample solution prepared by the method under item 1.2.1(2), continuously injecting samples for 5 times according to the chromatographic condition under item 1.3, injecting 20 mu l of sample each time, taking the retention time and the peak area of a hyperin chromatographic peak (No. 7 peak) as references, calculating the relative retention time and the RSD% value of the relative peak area of each common peak, wherein the relative retention time and the RSD% of the relative peak area of each common peak are both less than 3.0%, and the instrument has good precision.

1.4.2 stability test

Sampling the sample solution prepared by the method under item 1.2.1(2) for 1 time at 0, 2, 6, 12, 24, 40, 60 and 72h respectively according to the chromatographic condition under item 1.3, wherein each sampling is performed by 20 mu l, the retention time and the peak area of a hyperin chromatographic peak (peak No. 7) are taken as reference, and the relative retention time and the RSD% of the relative peak area of each common peak are calculated, wherein the relative retention time and the RSD% of the relative peak area of each common peak are both less than 3.0%, which indicates that the sample solution has good stability in 72 h.

1.4.3 repeatability test

5 parts of test solution is prepared according to the method under item 1.2.1(2), 20 mu l of sample is injected each time according to the chromatographic condition under item 1.3 and the chromatographic condition under item 1.3, the relative retention time of each common peak and the RSD% value of the relative peak area are calculated by taking the retention time and the peak area of the hyperin chromatographic peak (peak No. 7) as reference, and the relative retention time and the RSD% of the relative peak area of each common peak are both less than 3.0%, thus indicating that the method has good repeatability.

1.5 establishment of HPLC fingerprint and analysis of technical parameters

1.5.1 atlas Collection

Taking 20 batches of substance standards prepared by the method under item 1.2.1(1), preparing a sample solution according to the method under item 1.2.1(2), measuring according to the chromatographic conditions under item 1.3, carrying out sample injection of 20 mu l, and recording HPLC fingerprint, wherein the result is shown in figure 7.

1.5.2 selection of reference peaks

Firstly, hyperin is the main active ingredient of dodder as a monarch drug, and secondly, the hyperin is a known chemical ingredient and has stable chemical property, the chromatographic peak of the hyperin is in each chromatographic peak, the separation degree is better, and the peak area is larger, so that a No. 7 peak (hyperin) is selected as a reference peak of a fingerprint.

1.5.3 calibration of common peaks and calculation of relative retention time and relative peak area of common peaks

Data matching is carried out on 20 batches of substance reference spectra by adopting traditional Chinese medicine chromatogram fingerprint similarity evaluation system software (2012 edition) prepared by the State pharmacopoeia Commission, and the result is shown in figure 8. As can be seen from fig. 8, 12 peaks are shared by 20 lots of material basis, and thus 12 peaks were determined as shared chromatographic peaks, and the shared pattern was determined by the median method, and the results are shown in fig. 9, and the relative retention time and the relative peak area of the shared peaks were calculated by using the retention time and the chromatographic peak area of the hyperin peak (peak No. 7) as reference peaks, and the results are shown in tables 3 and 4.

TABLE 3 relative retention time of common peaks for Guyinyan decoction materials

TABLE 4 relative peak area of reference common peak of Guyinjian material

As can be seen from tables 3 and 4: the relative retention time RSD% of common peaks of 20 batches of solid yin decoction materials is less than or equal to 0.27%, the relative peak area RSD% is less than 3%, the fluctuation of the relative peak area RSD% is large and is caused by medicinal material decoction pieces of different sources, so that the relative peak area value is not specified in the standard, and only the number, the relative retention time and the similarity of characteristic peaks are specified, therefore, the method can be used for HPLC fingerprint spectrum determination of the solid yin decoction material.

1.5.4 identification of characteristic peaks

Firstly, a mixed reference substance comparison method is adopted to carry out characteristic peak identification, 20 mul of each of the test solution and the mixed reference substance solution is precisely absorbed and injected into a high performance liquid chromatograph, and a chromatogram is recorded, wherein the result is shown in figure 10. A (1: 5-hydroxymethylfurfural 3: morroniside 4: chlorogenic acid 6: logenin 7: hyperin 9: 3, 6' -digonoylsucrose 11: glycyrrhizic acid 12: schisandrin A)

As can be seen from FIG. 10, the peak 1 is determined to be 5-hydroxymethylfurfural, the peak 3 is determined to be morroniside, the peak 4 is determined to be chlorogenic acid, the peak 6 is loganin, the peak 7 is hyperin, the peak 9 is 3, 6' -erucyl sucrose, the peak 11 is glycyrrhizic acid, and the peak 12 is schisandrin A.

1.5.5 characteristic Peak assignment analysis

Taking 20 μ l of each of the yin-securing decoction substance standard test solution under item 1.2.1(2) and the single-herb decoction piece negative control solution prepared by the method under item 1.2.1(3), and determining according to the chromatographic conditions under item 1.3 to obtain HPLC (high performance liquid chromatography) spectra of each single-herb decoction piece negative control solution in the whole formula and the formula, wherein the results are shown in figure 11. (A: whole formula B: radix rehmanniae Preparata negative control C: Corni fructus negative control D: semen Cuscutae negative control E: cortex et radix Polygalae negative control F: radix Glycyrrhizae Preparata negative control G: fructus Schisandrae chinensis negative control H: rhizoma Dioscoreae negative control I: Ginseng radix negative control)

As can be seen from fig. 11: by comparing the relative retention time, the source of the common peak in the decoction for consolidating yin standard is traced back, and the peak 1 is from prepared rehmannia root and schisandra fruit, the peak 2 is from prepared rehmannia root, the

peaks

3, 5, 6 and 8 are from dogwood, the

peaks

4 and 7 are from dodder, the peak 9 is from polygala root, the

peaks

10 and 11 are from prepared licorice root, and the peak 12 is from schisandra fruit.

Standard similarity evaluation of 1.5.620 batches of Guyinshan material

The similarity evaluation system software (2012 version) of the traditional Chinese medicine chromatogram fingerprint spectrum, which is prepared by the national pharmacopoeia commission, is adopted, the chromatographic peak marked on the reference chromatogram is taken as a matching point, multi-point correction is carried out, and the result similarity is calculated, and the result is shown in table 5.

TABLE 520 batch Guyinjin substance benchmark similarity calculation results

As can be seen from table 5: the similarity of the standard fingerprint of 20 batches of decoction-strengthening materials is 0.963-0.996, the average value is 0.985, and the result shows that: the quality consistency of 20 batches of the decoction-strengthening material is higher.

Example 2 quality control method of Guyinyan decoction Standard granule

(1) Preparing a Guyinhuan decoction substance standard according to the method in 1.2.1(1) in example 1, and establishing a standard fingerprint of the Guyinhuan decoction substance standard by using the measuring method in example 1;

(2) taking the test solution of the Guyinshi standard granules, and obtaining a fingerprint spectrum according to the determination method in the embodiment 1;

(3) and (3) comparing the fingerprint obtained in the step (2) with the standard fingerprint obtained in the step (1), wherein the qualified product is obtained if the standard fingerprint is in line with the standard fingerprint, and the unqualified product is obtained if the standard fingerprint is not in line with the standard fingerprint.

The compliant requirements include: 12 characteristic peaks should be presented in the fingerprint of the test sample, wherein 8 peaks should be respectively kept for the same time as the corresponding reference peaks in the standard fingerprint; the peak corresponding to the hyperin reference substance is an S peak, and the relative retention time of each characteristic peak and the S peak is within +/-10% of the retention time value of the standard fingerprint; according to the similarity evaluation system of the traditional Chinese medicine chromatogram fingerprint, the similarity of the fingerprint of the test sample and the standard fingerprint is calculated and is not lower than 0.90.

Preparing a test solution of the Guyinshu standard granules: grinding the Guyinjian standard granules under the condition of different filling amounts, precisely weighing about 3g, placing into a 50ml measuring flask, adding a proper amount of 50% methanol, performing ultrasonic treatment (power 500W and frequency 40kHz) for 30 minutes to dissolve, cooling, adding 50% methanol to scale, shaking up, filtering, and taking the subsequent filtrate to obtain the Chinese medicinal composition.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.

Claims

1. A fingerprint spectrum measuring method of a Guyinshu material reference is characterized in that: the method comprises the following steps:

(1) preparation of a test solution: taking the reference powder of the decoction of solid yin, taking methanol as an extraction solvent, carrying out ultrasonic treatment, and filtering to obtain a subsequent filtrate;

preparation of mixed control solution: mixing the reference substances, dissolving in methanol to obtain mixed reference substance solution; preparing a negative control solution of the single decoction piece: taking each single decoction piece negative powder, taking methanol as an extraction solvent, performing ultrasonic treatment, and filtering to obtain a subsequent filtrate;

(2) respectively sucking the test sample solution, the mixed reference substance solution and the single-herb negative reference solution, injecting into a high performance liquid chromatograph for determination, and respectively obtaining liquid chromatographs of the test sample solution, the mixed reference substance solution and the single-herb negative reference solution;

the chromatographic conditions of the step (2) are as follows:

a chromatographic column: agilent 5TC-C18 column, 4.6mm × 250mm, 5 μm;

mobile phase: acetonitrile-0.3% phosphoric acid solution;

flow rate: 1.0 ml/min;

wavelength: 250 nm;

sample injection amount: 20 mu l of the solution;

column temperature: at 40 ℃;

the gradient elution procedure is shown in table 1:

TABLE 1 fingerprint gradient elution procedure

The reference substance is 5-hydroxymethyl furfural, morroniside, chlorogenic acid, loganin, hyperoside, 3, 6' -mustard acyl sucrose, ammonium glycyrrhizinate and schizandrol A reference substance;

the reference powder of the yin-consolidating decoction material in the step (1) is prepared by decocting, concentrating and drying yin-consolidating decoction prescriptions recorded in catalogues (the first batch) of classical famous formulae published by the State administration of traditional Chinese medicine.

2. The fingerprint spectrum measuring method according to claim 1, wherein: the standard fingerprint comprises 12 common peaks: peak 1 is from radix rehmanniae Preparata and fructus Schisandrae, Peak 3, 5, 6, 8 is from Corni fructus, Peak 4, 7 is from semen Cuscutae, Peak 9 is from cortex et radix Polygalae, Peak 10, 11 is from Glycyrrhrizae radix, Peak 12 is from fructus Schisandrae, and Peak 7 is used as reference peak.

3. The fingerprint spectrum measuring method according to claim 1, wherein: in the standard fingerprint, the No. 1 peak is 5-hydroxymethylfurfural, the No. 3 peak is morroniside, the No. 4 peak is chlorogenic acid, the No. 6 peak is loganin, the No. 7 peak is hyperin, the No. 9 peak is 3, 6' -brassicanyl sucrose, the No. 11 peak is glycyrrhizic acid, and the No. 12 peak is schisandrin A.

4. The fingerprint spectrum measuring method according to claim 1, wherein: the volume fraction of the methanol in the step (1) is 50-75%.

5. The fingerprint spectrum measuring method according to claim 1, wherein: the filtration in the step (1) is microporous membrane filtration.

6. The fingerprint spectrum measuring method according to claim 1, wherein: the concentration is a reduced pressure concentration.

7. The fingerprint spectrum measuring method according to claim 1, wherein: the drying is vacuum drying or freeze drying.

8. A quality control method based on a decoction substance for consolidating yin is characterized by comprising the following steps:

(1) establishing a reference standard fingerprint of the decoction of solid-decocted material according to the determination method of any one of claims 1 to 7;

(2) taking the decoction of Guyinhuan standard particles as a test sample solution, and detecting the test sample solution according to the chromatographic conditions in the step (2) of the determination method of any one of claims 1 to 7 to obtain a fingerprint;