CN112444590A

CN112444590A - Method for constructing UPLC fingerprint of gallnut medicinal material, fingerprint constructed by method and application of fingerprint

Info

Publication number: CN112444590A
Application number: CN201910821950.3A
Authority: CN
Inventors: 顾芹英; 张云天; 何雪霞; 翟燕娟
Original assignee: Jiangyin Tianjiang Pharmaceutical Co Ltd
Current assignee: Jiangyin Tianjiang Pharmaceutical Co Ltd
Priority date: 2019-09-02
Filing date: 2019-09-02
Publication date: 2021-03-05
Anticipated expiration: 2039-09-02
Also published as: CN112444590B

Abstract

The invention relates to a method for constructing a UPLC fingerprint of a nutgall medicinal material, a fingerprint constructed by the method and application of the fingerprint. Which comprises the following steps: 1) preparing a plurality of test solutions of the nutgall medicinal material and a reference solution of the index components; 2) respectively carrying out ultra-high performance liquid chromatography analysis on the test solution and the reference solution prepared in the step 1) to obtain corresponding ultra-high performance liquid chromatograms; and (3) determining a plurality of batches of nutgall medicinal materials, and analyzing and comparing to obtain the ultra-high performance liquid chromatography fingerprint spectrum of the common peak. The method can realize the control of the internal quality of the whole Chinese gall medicinal material and provide a new analysis means for the internal quality control of the Chinese gall medicinal material.

Description

Method for constructing UPLC fingerprint of gallnut medicinal material, fingerprint constructed by method and application of fingerprint

Technical Field

The invention belongs to the technical field of pharmaceutical analysis, and relates to a method for constructing an ultra-high performance liquid chromatography (UPLC) fingerprint of a gallnut medicinal material, a fingerprint constructed by the method, and application of the fingerprint.

Background

Galla chinensis is gallnut on leaf of Rhus chinensis Mill, Rhus potaninii Maxim, Rhus cornuta Maxim, or Rhus cornuta punjabensis Stew. var. sinica (Diels) Rehd. et Wils. of Anacardiaceae, mainly formed by parasitizing Galla chinensis Melaphoris chinensis Baker (Bell) Baker, also called Baichong cang, Wood aconite, etc. The distribution of Chinese gall is very extensive, most of the distribution is concentrated in the south of the Yangtze river, and Guizhou, Sichuan and the like are the main production areas of Chinese gall.

Collecting Galla chinensis in autumn, boiling in boiling water or steaming until the surface becomes grey, killing aphids therein, taking out, and drying. According to different shapes, gallnut is mainly divided into 'belly folds' and 'corner folds'. The belly is in an oblong or spindle-shaped bag shape, the length of the belly is 2.5-9 cm, and the diameter of the belly is 1.5-4 cm. The angle is rhombus, and has irregular obtuse angle branch, obvious soft hair and thinner wall. Galla chinensis is sour, astringent and cold. It enters lung, large intestine and kidney meridians. Galla chinensis has effects of astringing lung, lowering fire, relieving diarrhea with astringents, arresting sweating, stopping bleeding, astringing dampness, and healing sore. It is mainly used for treating chronic cough due to lung deficiency, chronic diarrhea and dysentery, spontaneous perspiration and night sweat, diabetes, hemafecia and hemorrhoidal bleeding, traumatic hemorrhage, carbuncle, swelling, sore, and skin wet rot. Modern pharmacological research shows that the gallnut has the pharmacological actions of relieving diarrhea, resisting bacteria, resisting inflammation, resisting oxidation, resisting virus, resisting senility, reducing blood sugar and the like (Korean Jianjun, Ningna. the research on the pharmacological actions of the gallnut is advanced [ J ]. Huaihai medicine, 2015,33(04):415 and 416), and the gallnut is mainly applied to the aspects of pediatrics, gynecology and the like in clinic.

The current research shows that the chemical components of the gallnut mainly comprise tannin (also called tannin, tannic acid), gallic acid, quintuce oil (comprising oleic acid, linoleic acid, linolenic acid, lauric acid) and the like, and in addition, the gallnut also contains microelements such as copper, zinc, iron, calcium and the like (oryzalin, separation and identification of a gallnut compound [ D ]. Shenyang, Liaoning university, 2012; Lichun, Dingwei Jia. gallnut chemical component research [ J ]. Chinese herbal medicine, 2008,39(08): 1129-. The highest content of tannin can reach 70 percent, and because gallnut is produced in China, gallnut tannin is called Chinese tannin internationally (zhuibigchen, Liushiwuwuwu, and the like.) the content of tannin in gallnut formula particles is compared [ J ] clinical medicine practice, 2002,21(9): 691-.

Gallnut is a forest special product in China, more than ten kinds of gallnuts are reported at present, commercial gallnuts have tripe type, horn type, double flower type and the like, and the quality difference is large, so the quality of commercial medicinal materials is not good and bad, common horn type and tripe type are listed in pharmacopoeia of the people's republic of China published in 1985, but the study on the gallnut is mostly concentrated on the tripe type medicinal materials at present, and the study on the horn type medicinal materials is less. Therefore, it is important to establish a method for comprehensively, rapidly and objectively evaluating the intrinsic quality of the plant.

The traditional Chinese medicine fingerprint spectrum or fingerprint spectrum is a comprehensive and quantifiable identification means, can systematically, integrally and exclusively represent the internal characteristics of the traditional Chinese medicine, can be used for evaluating the quality of the traditional Chinese medicine, traditional Chinese medicine preparations and semi-finished products, is an effective means for controlling the integral quality of the traditional Chinese medicine, and is accepted by the world health organization.

So far, the gallnut medicinal material has less research on the fingerprint spectrum which can reveal the inherent quality of the gallnut medicinal material. The method comprises the steps of establishing gallnut medicinal material fingerprints (guo Xiaorui, li, Sunzi. gallnut HPLC fingerprints research [ J ]. Chinese pharmacy, 2013,24(27): 2546-; 2 batches of the product are less than 0.900, which are non-recommended products; chenhong and the like utilize UPLC Waters ultra-high performance liquid chromatograph (Chenhong, Longyuan, Zhang Dan. Galla chinensis fingerprint chromatogram research [ J ]. Huaxi pharmaceutical journal, 2010,25(03): 373) 374) and Waters Symmetry Shield C18(150mm multiplied by 3.9mm, 5 μm) chromatographic columns, and acetonitrile (containing 0.1% trifluoroacetic acid) -water (containing 0.1% trifluoroacetic acid) solution is taken as a mobile phase, characteristic peaks of 3 batches of Galla chinensis raw material medicinal materials, 3 batches of semi-finished products and 3 batches of finished products are measured under the wavelength of 280nm, and the similarity of the Galla chinensis medicinal materials, the semi-finished products and the finished products is measured by adopting the cosine of an included angle, and experiments prove that the calculation method of the included angle has higher calculation accuracy and precision, is very simple, convenient and rapid, and the cosine of the included angle between each batch of samples and a reference mode is more than 0.9, and has good correlation. CN104198574A discloses a gallnut dual fingerprint quality control method, which comprises the steps of measuring inorganic elements of gallnut medicinal materials from each source by adopting an ICP-MS method to obtain ICP-MS analysis data, establishing a fingerprint of the inorganic elements of the gallnut medicinal materials, measuring organic components of the gallnut medicinal materials from each source by adopting an HPLC method, establishing a chromatographic fingerprint of the organic components of the gallnut medicinal materials, and then comprehensively analyzing the fingerprint and cluster analysis of the inorganic elements of the gallnut medicinal materials and the chromatographic fingerprint and cluster analysis of the organic components of the gallnut medicinal materials to evaluate the quality of the gallnut medicinal materials.

The researches mainly adopt HPLC (high performance liquid chromatography) for detection, the detection time is as long as 120min or more, the detection time is long, excessive organic solvents are used, the environmental pollution is serious, only 3 batches of raw material medicines are researched in only one detection research on the fingerprint UPLC of the gallnut, the representative results are not obtained, the chromatographic peak separation degree is not ideal, and the internal quality of the gallnut medicinal material cannot be comprehensively disclosed.

Therefore, it is necessary to establish a new analysis means for controlling the internal quality of the gallnut medicinal material, which has strong specificity, high stability and good repeatability, so that the analysis means is not only suitable for Ganba medicinal material, but also suitable for Jiakuan medicinal material, and the aim of identifying the gallnut medicinal materials from different producing areas and different sources is achieved.

Disclosure of Invention

Problems to be solved by the invention

In order to solve the technical problems in the prior art, the invention provides a method for constructing an ultra-high performance liquid chromatography fingerprint of a Chinese gall medicinal material, and the fingerprint constructed by the method is applied to quality detection of the Chinese gall medicinal material and a preparation containing the Chinese gall medicinal material. The construction method of the invention enables the quality control of the Chinese gall medicinal material to be improved from the original content measurement of certain components or the measurement of a few common peaks to the control of the internal quality of the whole Chinese gall medicinal material, can comprehensively reflect the characteristics of the Chinese gall medicinal material, provides a new analysis means for the internal quality control of the Chinese gall medicinal material, and achieves the purpose of identifying the Chinese gall medicinal materials from different production places and different sources.

Means for solving the problems

In one technical scheme, the invention provides a method for constructing an ultra-high performance liquid chromatography fingerprint of a gallnut medicinal material, which comprises the following steps:

1) preparing a plurality of test solutions of the nutgall medicinal material and a reference solution of the index components;

2) respectively carrying out ultra-high performance liquid chromatography analysis on the test solution and the reference solution prepared in the step 1) to obtain corresponding ultra-high performance liquid chromatograms; and (3) determining a plurality of batches of Chinese gall, such as more than 7 batches of Chinese gall, and analyzing and comparing to obtain the ultra-high performance liquid chromatography fingerprint spectrum of the common peak.

In one embodiment, the marker ingredient is at least one selected from the group consisting of gallic acid, gallic acid ester, and penta-galloylglucose, and more preferably, the marker ingredient is gallic acid.

In another embodiment, the construction method further comprises a step 3) of collecting mass spectrum data by adopting a liquid chromatography-mass spectrometry technology according to the ultra-high performance liquid chromatography conditions in the step 2), and assigning common peaks of the collected mass spectrum data.

In another embodiment, the step 1) of preparing the test solution comprises extracting Galla chinensis with solvent, shaking, filtering, and collecting the filtrate.

In another embodiment, the solvent is methanol, water, 30-70% methanol in water or 30-70% ethanol in water by volume.

In another embodiment, the extraction time is 15-60min, preferably 60 min.

In another embodiment, the extraction mode is sonication or shaking extraction or heating reflux, preferably the extraction mode is sonication.

In another embodiment, the conditions of the ultra high performance liquid chromatography in step 2) are as follows: using a reverse phase chromatography column, preferably a Water acquisition BEH Shield RP18 chromatography column; gradient elution is carried out with methanol as mobile phase A and 0.05-0.2 vol% phosphoric acid water solution or water as mobile phase B, preferably, the mobile phase B is selected from 0.1 vol% phosphoric acid water solution.

In another embodiment, the procedure for gradient elution is as follows:

0min → 15min → 20min → 25min → 35min → 45min → 55min → 56min, mobile phase a: 10% → 30% → 31% → 33% → 38% → 45% → 63% → 10%. The percentage of mobile phase is volume percent and the sum of the volume percentages of mobile phase a and mobile phase B is 100%.

In another embodiment, the column temperature of the chromatography column is 20-30 ℃; the flow rate of the mobile phase is 0.25-0.35ml/min, and the preferable flow rate is 0.28-0.32 ml/min; the detection wavelength is 273nm or 280 nm.

In another embodiment, the step 1) prepares a plurality of test solution of Ganbtimes medicinal materials, the ultra performance liquid chromatography fingerprint spectrum containing 14 common peaks obtained in the step 2) takes peak 2 gallic acid as a reference peak, and the relative retention time is respectively as follows: peak 1:0.67 (+ -5%), Peak 2: 1.00(± 5%), peak 3: 2.42(± 5%), peak 4: 3.54(± 5%), peak 5: 4.37(± 5%), peak 6: 5.17(± 5%), peak 7: 5.52(± 5%), peak 8: 6.84(± 5%), Peak 9: 7.69(± 5%), peak 10: 8.35(± 5%), peak 11: 10.28(± 5%), peak 12: 10.78(± 5%), peak 13: 11.61(± 5%), peak 14: 14.29(± 5%). Further, among the 14 common peaks, peak 2 is gallic acid, peak 3 is gallic acid methyl ester, and peak 14 is pentagallic acid acyl glucose.

In another embodiment, the step 1) prepares a plurality of test solution of the horn-fold medicinal material, and the hplc fingerprint obtained in the step 2) and containing 10 common peaks takes the peak 2 gallic acid as a reference peak, and the relative retention time is respectively as follows: peak 1:0.66 (+ -5%), Peak 2: 1.00(± 5%), peak 3: 2.34(± 5%), peak 4: 3.43(± 5%), peak 5: 5.00(± 5%), Peak 6: 5.29(± 5%), peak 7: 6.55(± 5%), Peak 8: 7.95(± 5%), peak 9: 9.69(± 5%), peak 10: 13.52 (+ -5%). Further, of the 10 common peaks, peak 2 is gallic acid, peak 3 is gallic acid methyl ester, and peak 10 is pentagallic acid acyl glucose.

In another technical scheme, the invention provides an ultra-high performance liquid chromatography fingerprint spectrum of a gallnut medicinal material, which is obtained by the construction method.

In one embodiment, the invention provides an ultra high performance liquid chromatography fingerprint of Gastrodia elata medicinal material, which comprises 14 common peaks.

In another embodiment, the invention provides an ultra high performance liquid chromatography fingerprint of a gonioapparent medicinal material, which comprises 10 common peaks.

In another technical scheme, the invention also provides application of the ultra-high performance liquid chromatography fingerprint spectrum disclosed by the invention in detection of the gallnut medicinal material and the preparation containing the gallnut medicinal material.

In one embodiment, the detection in the above application includes identifying the authenticity of the medicinal material, evaluating the quality of the medicinal material, and analyzing the source and the origin of the medicinal material. Comparing the ultra-high performance liquid chromatogram of the medicinal material to be detected with the ultra-high performance liquid chromatogram characteristic chromatogram of the gallnut medicinal material.

In another embodiment, in the above application, the similarity of 17 batches of Ganbao herbs is above 0.940, specifically the similarities are 0.959, 0.953, 0.960, 0.993, 0.993, 0.993, 0.987, 0.972, 0.972, 0.979, 0.975, 0.942, 0.948, 0.970, 0.974, 0.971, and 0.973, respectively.

In another embodiment, in the above application, the similarity of 7 batches of horny tripe herbs is above 0.920, specifically the similarities are 0.951, 0.945, 0.948, 0.936, 0.920, 0.947 and 0.955 respectively.

ADVANTAGEOUS EFFECTS OF INVENTION

The invention adopts the ultra-high performance liquid chromatography and reasonably controls the chromatographic conditions to determine 14 common peaks of the Gancao medicinal material and 10 common peaks of the Jiaozao medicinal material, thereby forming the overall appearance of the fingerprint of the Chinese gall medicinal material, and increasing the quality control of the Chinese gall medicinal material from the content measurement of certain components or the measurement of a few common peaks to the control of the internal quality of the whole Chinese gall medicinal material, thus more comprehensively reflecting the characteristics of the Chinese gall medicinal material, avoiding the defect of measurement of a single peak or a few peaks, providing a new analytical means for the internal quality control of the Chinese gall medicinal material and achieving the purpose of identifying the Chinese gall medicinal materials with different producing areas and different sources.

The method is simple, good in reproducibility, accurate and reliable, convenient to operate, and less in time, solvent consumption and environmental pollution compared with HPLC.

Drawings

FIG. 1 is a full-wavelength scanning 3D UPLC chromatogram of Gadol medicinal materials.

FIG. 2 is a UPLC chromatogram of Ganbao medicinal materials under 273nm and 280nm wavelength conditions.

FIG. 3 is a UPLC chromatogram of Ganbao medicinal materials under different extraction solvent conditions.

FIG. 4 is a UPLC chromatogram of Ganbao medicinal materials under different extraction modes.

FIG. 5 is a UPLC chromatogram of Ganbao medicinal materials at different extraction times.

FIG. 6 is UPLC superposition spectrum of 17 batches of Ganbtimes medicinal materials.

FIG. 7 is UPLC superposition spectrum of 7 batches of horn-fold medicinal materials.

Fig. 8 is a UPLC fingerprint of a control wherein: peak 2 gallic acid, Peak 3 gallic acid methyl ester, Peak 14 is pentagallic acid acyl glucose (Ganbao medicinal material).

Fig. 9 is a UPLC fingerprint of a control wherein: peak 2 gallic acid, Peak 3 gallic acid methyl ester, Peak 10 pentagallic acid acyl glucose (horn times medicinal material).

FIG. 10 is UPLC integral chromatogram of Ganbtimes medicinal material.

FIG. 11 is a UPLC chromatogram of Ganbao medicinal materials under different chromatographic column conditions.

FIG. 12 is a UPLC chromatogram of Ganbao medicinal materials under different column temperatures.

FIG. 13 is a UPLC chromatogram of Ganbao medicinal materials under different flow rates.

Detailed Description

In the following detailed description, numerous specific details are set forth in order to provide a better understanding of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, methods, means, devices and steps which are well known to those skilled in the art have not been described in detail so as not to obscure the invention.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In the present specification, the numerical range represented by "numerical value a to numerical value B" means a range including the end point numerical value A, B.

In the present specification, the meaning of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

It should be understood that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

In the present specification, reference to "one or some particular/preferred embodiments", "another or some other particular/preferred embodiments", "one or another embodiment", or the like, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.

The terms "comprises" and "comprising," and any variations thereof, in the description and claims of this invention and the above-described drawings are intended to cover non-exclusive inclusions. For example, a process, method, or system, article, or apparatus that comprises a list of steps or elements is not limited to only those steps or elements listed, but may alternatively include other steps or elements not listed, or inherent to such process, method, article, or apparatus.

The invention provides a method for constructing a UPLC fingerprint spectrum of a Chinese gall medicinal material, which comprises the following steps:

2) respectively carrying out ultra-high performance liquid chromatography analysis on the test solution and the reference solution prepared in the step 1) to obtain corresponding ultra-high performance liquid chromatograms; and (3) determining a plurality of batches of Chinese gall medicinal materials such as more than 7 batches of Chinese gall medicinal materials, and analyzing and comparing to obtain the ultra-high performance liquid chromatography fingerprint spectrum of the common peak of the Chinese gall medicinal materials.

In a specific embodiment of the present invention, the gallnut herbs in step 1) may be at least one selected from maxa orellana herbs, jiaolox herbs and beihua herbs, and are preferably selected from maxa orellana herbs. Although the peak positions and peak areas of chromatographic peaks of characteristic components in the gallnut medicinal materials are slightly different, main common peaks can be clearly identified, so that the three varieties are all suitable for the construction method of the UPLC fingerprint spectrum.

In one embodiment of the invention, the step 1) of preparing the test solution comprises extracting the gallnut medicinal material with a solvent. In addition, the preparation process also comprises the steps of crushing, sieving, weighing and fixing the volume of the test medicinal materials, shaking up after extraction, filtering and taking the subsequent filtrate. According to the solubility of chemical components of the Chinese gall, the preferable solvent comprises methanol, water, 30-70% methanol aqueous solution or 30-70% ethanol aqueous solution by volume concentration, and the percentage is volume percentage. Further preferably, the solvent is methanol to obtain higher extraction efficiency. In one or more preferred embodiments, the mass-to-volume ratio of the nutgall medicinal material to the solvent is 1 g: 80-160 ml, preferably the mass-to-volume ratio is 1 g: 100-130 ml, more preferably 1 g: 125ml to achieve sufficient extraction and meet the concentration requirements for detection.

Besides the extraction solvent, different extraction methods also affect the extraction effect of the medicinal materials. In a specific embodiment of the present invention, the above extraction is selected from any one of sonication or shaking extraction or heating reflux. In a preferred embodiment, the extraction is ultrasonic extraction, and has the advantages of high efficiency, short time, low temperature, wide adaptability and the like. The power of the ultrasonic wave is 200-300W, and the preferable power is 250W; the frequency of the ultrasonic wave is 30-50 kHz, and more preferably 40 kHz.

In addition, the extraction time will also affect the extraction effect of the herbs. In a specific embodiment of the present invention, when the extraction is ultrasonic extraction, the extraction time is 15 to 60min, more preferably 30 to 60min, and preferably 60min in consideration of the inspection period, extraction sufficiency, and the like.

In one embodiment of the present invention, the index component in step 1) is at least one selected from gallic acid, gallic acid ester, and penta-gallic acid acyl glucose. One or more of the three index components can be optionally selected by the technology in the field as a reference substance for constructing the UPLC fingerprint of the Chinese gall medicinal material. More preferably, the index component is gallic acid. Gallic acid is also called "gallic acid" or "gallic acid". The name is 3,4, 5-trihydroxybenzoic acid. Molecular formula C₇H₆O₅。

In a particular embodiment of the invention, the preparation of the control solution in step 1) comprises a solvent dissolution step, preferably a methanol dissolution step. In addition, the preparation also comprises the related steps of weighing, volume fixing, filtering and the like of the reference substance.

In a specific embodiment of the present invention, the conditions of the hplc analysis in step 2) of the above construction method are as follows: and (2) carrying out gradient elution by adopting a reversed-phase chromatographic column and taking methanol-phosphoric acid aqueous solution or water as a mobile phase, wherein the flow rate is 0.25-0.35mL/min, and the column temperature is 20-30 ℃, so as to realize a better separation effect.

In a preferred embodiment, the reverse phase column is packed with octadecylsilane bonded silica, preferably a Water acquisition beam Shield RP18 column. The present invention compares four different chromatographic columns: the method comprises the following steps: waters ACQUITY BEH Shield RP18 (100X 2.1mm, 1.7 μm) (Ser.No.: 01733805218321), Waters ACQUITY BEH Shield RP18 (100X 2.1mm, 1.7 μm) (Ser.No.: 01733805218379), Waters ACQUITY

BEH C18(Waters,100×2.1mm,1.7μm)、ACQUITY

The effect of four chromatographic columns T3(Waters,100 multiplied by 2.1mm,1.6 mu m) on the durability of the Chinese gall medicinal material characteristic map shows that compared with a Water ACQUITY BEH Shield RP18 chromatographic column (100 multiplied by 2.1mm, 1.7 mu m), the peak packing phenomenon is easy to occur by adopting other types of chromatographic columns.

In a preferred embodiment, the ratio of phosphoric acid in the phosphoric acid aqueous solution is 0.05 to 0.2% by volume. In a more preferred embodiment, the phosphoric acid in the above-mentioned aqueous phosphoric acid solution is present in a proportion of 0.1% by volume.

In a preferred embodiment, the procedure of gradient elution described above is as shown in table 1, the sum of the volume percentages of mobile phase a and mobile phase B being 100%:

TABLE 1 gradient elution Table

Taking the test sample solution, performing gradient elution according to the table, and recording the absorption spectrum in the range of 190-400 nm. The invention finds that the response of each chromatographic peak of a gallnut medicinal material sample solution is better under the wavelength of 273nm or 280nm, and the detection wavelength is preferably 273 nm.

In the embodiment of the invention, in the step 2), 1 μ l of each of the test solution and the reference solution is precisely absorbed and injected into the ultra high performance liquid chromatograph.

In an embodiment of the present invention, a test solution is a solution of a maxa medicinal material, step 2) a plurality of batches, for example, 17 batches of the maxa medicinal material are tested to obtain a corresponding ultra-high performance liquid chromatogram, the ultra-high performance liquid chromatogram is introduced into a "traditional Chinese medicine chromatography fingerprint similarity evaluation system" issued by the national pharmacopoeia committee, a reference fingerprint is generated by a median, a UPLC fingerprint common mode of the maxa medicinal material is generated after multipoint correction, wherein 14 common peaks are included, No. 2 peak (peak 2) gallic acid is used as a reference peak, and the relative retention times are respectively: peak 1:0.67 (+ -5%), Peak 2: 1.00(± 5%), peak 3: 2.42(± 5%), peak 4: 3.54(± 5%), peak 5: 4.37(± 5%), peak 6: 5.17(± 5%), peak 7: 5.52(± 5%), peak 8: 6.84(± 5%), Peak 9: 7.69(± 5%), peak 10: 8.35(± 5%), peak 11: 10.28(± 5%), peak 12: 10.78(± 5%), peak 13: 11.61(± 5%), peak 14: 14.29(± 5%). And superposing ultra-high performance liquid chromatograms of a plurality of batches of Gancao medicinal materials, inspecting the consistency of chromatographic peak similarity, and evaluating the similarity. The results show that the total content is more than 0.900, 14 common peaks can stably appear in each tripe times of medicinal material samples, and the goodness of fit is good. In one embodiment of the present invention, the similarity of 17 batches of Ganbao herbs is above 0.940, and specifically the similarities are 0.959, 0.953, 0.960, 0.993, 0.993, 0.993, 0.987, 0.972, 0.972, 0.979, 0.975, 0.942, 0.948, 0.970, 0.974, 0.971 and 0.973, respectively.

In another embodiment of the present invention, the sample solution is a solution of gonioapparent medicinal materials, step 2) a plurality of batches, for example, 7 batches of gonioapparent medicinal materials are tested to obtain corresponding ultra-high performance liquid chromatograms, which are introduced into a "traditional Chinese medicine chromatography fingerprint similarity evaluation system" issued by the national pharmacopoeia committee, a reference fingerprint is generated by using a median, and a UPLC fingerprint common mode of the gonioapparent medicinal materials is generated after multipoint correction, wherein the reference fingerprint common mode comprises 10 common peaks, and the relative retention times of the reference peaks are respectively: peak 1:0.66 (+ -5%), Peak 2: 1.00(± 5%), peak 3: 2.34(± 5%), peak 4: 3.43(± 5%), peak 5: 5.00(± 5%), Peak 6: 5.29(± 5%), peak 7: 6.55(± 5%), Peak 8: 7.95(± 5%), peak 9: 9.69(± 5%), peak 10: 13.52 (+ -5%). And superposing ultra-high performance liquid chromatograms of a plurality of batches of angle-fold medicinal materials, inspecting the consistency of chromatographic peak similarity, and evaluating the similarity. The results show that the total content is more than 0.900, 10 common peaks can stably appear in each angular time medicinal material sample, and the goodness of fit is good. In one embodiment of the present invention, the similarity of 7 batches of horny tripe herbs is above 0.920, and specifically the similarities are 0.951, 0.945, 0.948, 0.936, 0.920, 0.947 and 0.955 respectively.

In another embodiment of the invention, the construction method further comprises a step 3) of adopting a liquid chromatography-mass spectrometry technology, collecting high-resolution mass spectrometry data according to the ultra-high performance liquid chromatography conditions of the step 2), conjointing the common peaks of the nutgall by conjointing the molecular formula of the compound in the nutgall medicinal material with isotope analysis and comparing the common peaks with the common peaks in the literature and the related reference substances.

The conditions of the mass spectrum detection of the invention are as follows: the ion source is an electrospray ion source ESI, the temperature of a dry gas is 200-400 ℃, the preferred temperature is 300 ℃, the flow rate is 1-20L/min, the preferred flow rate is 8L/min, and the pressure of an atomizer is 10-100psi, the preferred flow rate is 35 psi; the capillary voltage under the positive ion mode is 4000V, the capillary voltage under the negative ion mode is 3500V, the outlet voltage of the capillary is 175V, and the taper hole voltage is 65V; data acquisition is carried out by adopting a high-resolution mode, wherein the mass-to-charge ratio acquisition range is m/z 100-2000, the sampling speed is 1spectra/s, and the sampling time is 1000 ms/spectra; the negative ions were mass number calibrated in real time using TFANH4(112.985587) and HP-0921(1033.988109), the positive ions were mass number calibrated in real time using purine (121.050873) and HP-0921(922.009798), and the atomization pressure was 5 psi.

In an embodiment of the present invention, it is found that in the ultra-high performance liquid chromatogram of the tripeptide medicinal material, the retention times and the ultraviolet spectra of the

peaks

2, 3 and 14 are respectively identical to those of gallic acid, methyl gallate and penta-gallic acid acyl glucose in the reference substance, so that the

peaks

2, 3 and 14 in the fingerprint of the test sample are respectively identified as gallic acid, methyl gallate and penta-gallic acid acyl glucose. In addition, 11 common peaks were identified using LC-MS. The invention finds that in the ultra-high performance liquid chromatogram of the diagonal fold medicinal material, the retention time and the ultraviolet spectrum of the peak 2, the peak 3 and the peak 10 are respectively matched with those of gallic acid, gallic acid methyl ester and pentagallic acid acyl glucose in the reference substance, so that the peak 2, the peak 3 and the peak 10 in the fingerprint of the test product are respectively identified as gallic acid, gallic acid methyl ester and pentagallic acid acyl glucose. In addition, 7 common peaks were identified using LC-MS.

The construction method has good precision and repeatability, and the test solution is stable within at least 12 hours.

In another technical scheme, the invention provides an ultra-high performance liquid chromatography fingerprint spectrum of a gallnut medicinal material, which is obtained by the construction method. In one embodiment, the invention provides an ultra high performance liquid chromatography fingerprint of Gastrodia elata medicinal material, which comprises 14 common peaks. In another embodiment, the invention provides an ultra high performance liquid chromatography fingerprint of a gonioapparent medicinal material, which comprises 10 common peaks.

The technical solution of the present invention will be further described with reference to specific examples. It will be readily understood by those skilled in the art that the specific experimental conditions and results thereof described in the following examples are illustrative of the present invention only and should not be, and should not be construed as, limiting the invention. Changes and substitutions in detail and form may be made without departing from the spirit and scope of the invention, but it is intended that such changes and substitutions fall within the scope of the invention. In addition, unless otherwise specified, instruments, materials, reagents and the like used in the following examples are available by conventional commercial means.

The first embodiment is as follows: galla chinensis medicinal material UPLC fingerprint construction

1. Instrument, reagents and sample:

waters Acquity UPLC ultra high performance liquid chromatograph (Waters corporation); empower 3 workstation (Waters corporation); agilent G6530Accurate-Mass Q-TOF Mass spectrometer (Agilent Corp.); agilent Mass Hunter Workstation data collection and qualitative analysis software (Agilent corporation); electronic analytical balance (mettler-toledo instruments (shanghai) ltd); KQ-250B ultrasonic cleaning machine (Kunshan ultrasonic instruments Co., Ltd.); pure water system (Sartorius corporation); centrifuge model AS165W (shikawa (shanghai) commercial limited). Methanol (chromatographic purity, Thermo Fisher corporation); the water is ultrapure water; phosphoric acid (chromatographically pure, aladdin company); other reagents were analytically pure.

The gallic acid control was purchased from China food and drug assay research institute and was numbered 110831-201605. The gallic acid methyl ester reference substance and the pentagallic acid acyl glucose reference substance are purchased from Shanghai Shidan Biotechnology Limited company and numbered as 6313 and 3367 respectively. Gallnut medicinal materials are respectively collected in Shaanxi, Sichuan, Chongqing, Hubei and Guizhou areas of main production, and insect gall on leaves of Rhus chinensis Chenensis Mill, Rhus potaninii Maxim or Rhus chinensis punjabensis Stew.var.sinica (Diels) Red et Wils is identified by Tianjiang pharmaceutical industry GmbH of Jiangyin, and accords with various regulations under the term of five-fold item of the edition of Chinese pharmacopoeia 2015 for 24 batches in total.

2. Preparation of control solutions:

taking appropriate amount of gallic acid control, precisely weighing, and adding 50% methanol to obtain solution containing 40 μ g per 1 ml. Accurately weighing appropriate amount of methyl gallate and penta-gallate acyl glucose reference substances, and adding methanol to obtain solutions with concentration of 0.020mg/ml and 0.150mg/ml respectively.

3. Preparation of a test solution:

precisely weighing Galla chinensis powder (sieving with a sieve of four numbers) about 0.2g, placing into a conical flask with a plug, precisely adding methanol 25ml, sealing, weighing, ultrasonic processing (power 250W, frequency 40kHz) for 60min, cooling, weighing again, reducing weight with methanol, shaking, filtering, and collecting the filtrate.

4. Determination of chromatographic conditions:

4.1 determination of the detection wavelength

Taking the sample solution, performing gradient elution according to the table 1, and recording an absorption spectrum within the range of 190-400 nm.

As shown in the graph 1 and the graph 2, the response of each spectrum peak of the gallnut medicinal material sample solution is better under the wavelength of 273nm or 280nm, the detection wavelength is determined by referring to Chinese pharmacopoeia content, and 273nm is selected as the detection wavelength.

4.2 optimization of the Mobile phase

Methanol-0.1% phosphoric acid was selected as the mobile phase.

The chromatographic conditions were finally determined as: chromatography on an Acquity UPLC beam Shield RP18(100 × 2.1mm, 1.7 μm) column; gradient elution was performed as specified in table 1 using methanol as mobile phase a and 0.1% phosphoric acid solution as mobile phase B; the column temperature was 25 ℃; the flow rate is 0.30 ml/min; the detection wavelength was 273 nm.

4.3 preparation of test solutions

(1) Examination of extraction solvent

The influence of different extraction solvents on the fingerprint of the Chinese gall medicinal material is respectively considered, 30% methanol, 50% methanol, 70% methanol, 30% ethanol, 50% ethanol, 70% ethanol, ethanol and water are selected as the extraction solvents, and the fingerprint of the different extraction solvents is compared through the temporarily identified total peak area/sample weighing amount and chromatogram of 14 chromatographic peaks.

The investigation results of different extraction solvents of the fingerprint spectrum of the gallnut medicinal material obtained by injecting sample under the chromatographic condition of the item 4.2 are shown in a table 2 and a figure 3.

TABLE 2 inspection of fingerprint extraction solvent (peak area/sample weighing) for Galla chinensis medicinal materials

As a result, as shown in FIG. 3, methanol was selected as the extraction solvent because the extraction efficiency was high in all of chromatographic peaks 1 to 14.

(2) Examination of extraction methods

The influence of different extraction modes on the fingerprint of the Chinese gall medicinal material is respectively considered, ultrasonic treatment, heating reflux and shaking extraction are selected as the extraction modes, and the fingerprint results of the different extraction modes are compared through the temporarily identified total peak area/sample weighing amount of 14 chromatographic peaks and the chromatogram.

Weighing Galla chinensis powder (about 0.2g, sieving with a sieve of four numbers), weighing accurately, placing into a conical flask with a plug, adding methanol 25ml accurately, sealing, weighing, performing ultrasonic treatment (power 250W, frequency 40kHz), heating under reflux, shaking for 60min, cooling, weighing again, reducing weight with methanol, shaking, filtering, and collecting the filtrate.

The results of investigation of the fingerprint spectrum of the gallnut medicinal material obtained by injecting sample under the chromatographic condition of item 4.2 are shown in table 3 and fig. 4.

TABLE 3 inspection of fingerprint extraction of Galla chinensis (peak area/sample weighing)

As a result, as shown in FIG. 4, the number of chromatographic peaks obtained by the three treatment methods was consistent, and the extraction efficiency of the ultrasonic treatment method was slightly higher, so that the ultrasonic treatment was selected as the sample solution extraction method.

(3) Investigation of extraction time

The influence of different extraction time on the fingerprint of the Chinese gall medicinal material is respectively considered, four different extraction time of 15 minutes, 30 minutes, 45 minutes and 60 minutes are selected and considered, and the fingerprint results at different extraction time are compared through the temporarily identified total peak area/sample weighing amount and chromatogram of 10 chromatographic peaks.

Taking about 0.2g of nutgall medicinal material powder (passing through a sieve of No. four), weighing the powder in parallel, precisely weighing the powder, putting the powder into a conical flask with a plug, adding 25ml of methanol, weighing the powder, performing ultrasonic treatment (power of 250W and frequency of 40kHz) for 15 minutes, 30 minutes, 45 minutes and 60 minutes respectively, cooling the mixture, weighing the powder again, supplementing the lost weight with the methanol, shaking the mixture evenly, filtering the mixture, and taking the subsequent filtrate as a test solution. The results of investigation of fingerprint spectrum of Galla chinensis by injecting sample under the chromatographic condition of '4.2' are shown in Table 4 and FIG. 5.

TABLE 4 Galla chinensis medicinal material fingerprint extraction time investigation (peak area/sample weighing)

As a result, as shown in FIG. 5, the number of chromatographic peaks of the sample was consistent with each other at different extraction times, but the extraction efficiency was the highest at 60min, and thus the extraction time was set to 60 min.

5 establishment of fingerprint

5.1 precisely sucking 1 μ l of each of the reference solution and the sample solution, and injecting into a liquid chromatograph.

5.2 evaluation of similarity of Gancao medicinal materials 17 batches of Gancao medicinal material samples are superposed by using a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012.0 edition) issued by the State pharmacopoeia Committee, as shown in figure 6, the consistency of the similarity of chromatographic peak is inspected, the similarity evaluation is carried out, the result is shown in table 5, the result display is more than 0.900, the similarity of the sample fingerprint and the comparison map is higher, 14 common peaks can stably appear in each sample, and the goodness of fit is better, so the map can be used as the fingerprint of the Chinese gall medicinal materials.

TABLE 517 batch of medicinal materials (Ganbao) similarity evaluation results

5.3 evaluation of similarity of Angle-fold medicinal materials adopts a Chinese medicine chromatogram fingerprint similarity evaluation system (2012.0 edition) issued by the State pharmacopoeia Committee to superpose characteristic spectrums of 7 batches of angle-fold medicinal material samples, see figure 7, examine the consistency of chromatographic peak similarity and carry out similarity evaluation, the result is shown in table 6, the result display is more than 0.900, the similarity of the sample fingerprint and a comparison spectrum is higher, 10 common peaks can stably appear in each sample, and the goodness of fit is better, so the spectrum can be used as the fingerprint of the Chinese gall medicinal materials.

TABLE 67 evaluation results of similarity of bulk drugs (angle times)

Attribution of 6 common peaks and determination of characteristic peaks

Collecting high-resolution mass spectrum data by using a liquid chromatography-mass spectrometry technology according to the liquid chromatography condition of the fingerprint spectrum of the gallnut medicinal material (Ganbtimes), conjointly analyzing and conjecturing the molecular formula of the compound in the gallnut medicinal material by combining with the same digit, and comparing with a reference document and a related reference substance to attribute the common peak of the gallnut medicinal material. Wherein the retention time and ultraviolet spectrum of peak 2, peak 3 and peak 14 are respectively identical to those of gallic acid, gallic acid methyl ester and pentagallic acid acyl glucose in reference substance, so that peak 2, peak 3 and peak 14 in the fingerprint of the test sample are respectively identified as gallic acid, gallic acid methyl ester and pentagallic acid acyl glucose, as shown in FIG. 8. In addition, 11 common peaks were identified by LC-MS, and the results are shown in Table 7.

Collecting high-resolution mass spectrum data by using a liquid chromatography-mass spectrometry technology according to the liquid chromatography condition of the fingerprint spectrum of the gallnut medicinal material (horn fold), conjointly analyzing and conjecturing the molecular formula of the compound in the gallnut medicinal material by combining with the same-digit analysis, and comparing with a reference document and a related reference substance to attribute the common peak of the gallnut medicinal material. Wherein the retention time and ultraviolet spectrum of peak 2, peak 3 and peak 10 are respectively identical to those of gallic acid, gallic acid methyl ester and pentagallic acid acyl glucose in reference substance, so that peak 2, peak 3 and peak 10 in the fingerprint of the test product are respectively identified as gallic acid, gallic acid methyl ester and pentagallic acid acyl glucose, as shown in FIG. 9. In addition, 7 common peaks were identified by LC-MS, and the results are shown in Table 8.

TABLE 7 other chromatographic peak liquid quality analysis results (Ganbtimes medicinal materials)

TABLE 8 other chromatographic peak liquid quality analysis results (horn times crude drug)

Example methodological validation of fingerprint map construction method

(1) Integrity survey

On the same chromatographic condition, the same gradient is kept, the elution time is prolonged by one time, the chromatogram is recorded, no obvious chromatographic peak flows out after 100 minutes, the chromatographic condition basically meets the principle of maximum information content, and the result is shown in figure 10.

(2) Precision survey

The same sample solution in example 1 is continuously injected for 6 times, similarity evaluation software is used for calculating that the similarity of each chromatographic fingerprint is more than 0.990, and the result is shown in table 8, which indicates that the instrument method is stable and the precision is good.

TABLE 9 precision results

(3) Repeatability survey

Taking the gallnut medicinal material powder of the same batch in the example 1, precisely weighing 6 parts, preparing 6 parts of test solution in parallel according to the test preparation method of the example 1, injecting samples respectively, calculating the similarity of each chromatogram fingerprint by using similarity evaluation software to be more than 0.99, and showing that the construction method has good repeatability as shown in a table 9.

TABLE 10 repeatability results

(4) Stability survey

The same sample solution in example 1 was sampled and analyzed at 0,2,4,6,8,10, and 12h, respectively, and similarity evaluation software was used to calculate that the similarity of each spectral fingerprint was greater than 0.990, and the results are shown in table 10, indicating that the sample solution was stable for at least 12 hours.

TABLE 11 stability results

Note: WDX0H-WDH12H in Table 11 represent stability from 0 hour to 12 hours, respectively

(5) Durability examination

1) Investigation of different chromatography columns

4 columns were compared, respectively: waters ACQUITY BEH Shield RP18 (100X 2.1mm, 1.7 μm) (Ser.No.: 01733805218321), Waters ACQUITY BEH Shield RP18 (100X 2.1mm, 1.7 μm) (Ser.No.: 01733805218379), Waters ACQUITY

BEH C18(Waters,100×2.1mm,1.7μm)、ACQUITY

Influence of four chromatographic columns of CORTECS T3(Waters, 100X 2.1mm,1.6 μm) on durability of Chinese gall characteristic spectrum

The results are shown in FIG. 11, which shows that the peak packing phenomenon is liable to occur with other types of columns, and therefore it is suggested that the present invention employs an ultra high performance liquid chromatography column of the type Waters ACQUITY BEH Shield RP18 (100X 2.1mm, 1.7 μm).

2) Investigation of different temperatures

The separation effect of the sample was examined at column temperatures of 20 ℃,25 ℃ and 30 ℃ using a Waters acquisition beam Shield RP18 (100X 2.1mm, 1.7 μm) column.

The results are shown in FIG. 12, and the sample separation effect is better when the column temperature is 20 ℃,25 ℃ and 30 ℃.

3) Investigation of different flow rates

The separation of the samples at flow rates of 0.28ml/min, 0.30ml/min and 0.32ml/min was examined using a Waters ACQUITY BEH Shield RP18 (100X 2.1mm, 1.7 μm) column.

The results are shown in FIG. 13, and the samples were separated well at flow rates of 0.28ml/min, 0.30ml/min, and 0.32 ml/min.

The above examples are intended only to illustrate several embodiments of the present invention, which are described in more detail and detail, but are not to be construed as imposing any limitation on the scope of the present invention. It should be clear that a person skilled in the art can make several variations and modifications without departing from the inventive concept, which fall within the scope of protection of the present invention.

Claims

1. A method for constructing an ultra-high performance liquid chromatography fingerprint of a gallnut medicinal material comprises the following steps:

1) preparing a plurality of test solutions of the nutgall medicinal material and a reference solution of the index components; more preferably, the marker ingredient is at least one selected from gallic acid, methyl gallate and penta-gallate-based glucose, and more preferably, the marker ingredient is gallic acid;

2) respectively carrying out ultra-high performance liquid chromatography analysis on the test solution and the reference solution prepared in the step 1) to obtain corresponding ultra-high performance liquid chromatograms; and (3) determining a plurality of batches of nutgall medicinal materials, and analyzing and comparing to obtain the ultra-high performance liquid chromatography fingerprint spectrum of the common peak.

2. The construction method according to claim 1, characterized in that:

the method also comprises the step 3) of adopting a liquid chromatography-mass spectrometry technology, collecting mass spectrum data according to the ultra-high performance liquid chromatography conditions of the step 2), and attributing common peaks of the mass spectrum data.

3. The construction method according to claim 1 or 2, characterized in that:

the preparation of the test solution in the step 1) comprises the steps of taking a Chinese gall medicinal material, adding a solvent for extraction, preferably, the solvent is methanol, water, a 30-70% methanol water solution or a 30-70% ethanol water solution in volume concentration, the extraction time is preferably 15-60min, and the preferred extraction mode is ultrasonic treatment, shaking extraction or heating reflux.

4. The construction method according to any one of claims 1 to 3, characterized in that:

the conditions of the ultra performance liquid chromatography in the step 2) are as follows: using a reverse phase chromatography column, preferably a Water acquisition BEH Shield RP18 chromatography column; performing gradient elution with methanol as mobile phase A and 0.05-0.2 vol% phosphoric acid water solution or water as mobile phase B, preferably, the mobile phase B is selected from 0.1 vol% phosphoric acid water solution; the column temperature is 20-30 ℃; the flow rate is 0.25-0.35ml/min, and the preferable flow rate is 0.28-0.32 ml/min; the detection wavelength is 273nm or 280 nm; further preferably the procedure of the gradient elution is as follows:

0min → 15min → 20min → 25min → 35min → 45min → 55min → 56min, mobile phase a: 10% → 30% → 31% → 33% → 38% → 45% → 63% → 10%.

5. The construction method according to any one of claims 1 to 4, wherein:

step 1) preparing a plurality of test solution of Ganbtimes medicinal materials, and the ultra high performance liquid chromatography fingerprint spectrum containing 14 common peaks obtained in step 2) takes peak 2 gallic acid as a reference peak, and the relative retention time is respectively as follows: peak 1:0.67 (+ -5%), Peak 2: 1.00(± 5%), peak 3: 2.42(± 5%), peak 4: 3.54(± 5%), peak 5: 4.37(± 5%), peak 6: 5.17(± 5%), peak 7: 5.52(± 5%), peak 8: 6.84(± 5%), Peak 9: 7.69(± 5%), peak 10: 8.35(± 5%), peak 11: 10.28(± 5%), peak 12: 10.78(± 5%), peak 13: 11.61(± 5%), peak 14: 14.29(± 5%).

6. The construction method according to claim 5, wherein:

of the 14 common peaks, peak 2 is gallic acid, peak 3 is gallic acid methyl ester, and peak 14 is pentagallic acid acyl glucose.

7. The construction method according to any one of claims 1 to 4, wherein:

step 1) preparing a plurality of test solution of the horn-fold medicinal material, and the ultra-high performance liquid chromatography fingerprint spectrum containing 10 common peaks obtained in step 2) takes peak 2 gallic acid as a reference peak, and the relative retention time is respectively as follows: peak 1:0.66 (+ -5%), Peak 2: 1.00(± 5%), peak 3: 2.34(± 5%), peak 4: 3.43(± 5%), peak 5: 5.00(± 5%), Peak 6: 5.29(± 5%), peak 7: 6.55(± 5%), Peak 8: 7.95(± 5%), peak 9: 9.69(± 5%), peak 10: 13.52(± 5%); further, of the 10 common peaks, peak 2 is gallic acid, peak 3 is gallic acid methyl ester, and peak 10 is pentagallic acid acyl glucose.

8. An ultra-high performance liquid chromatography fingerprint of a gallnut medicinal material, which is obtained by the construction method according to any one of claims 1 to 7.

9. The use of the ultra-high performance liquid chromatography fingerprint of a Galla chinensis crude drug according to claim 8 in the detection of Galla chinensis crude drug and preparations containing Galla chinensis crude drug.

10. The use according to claim 9, wherein the similarity is above 0.940 for the detection of 17 batches of Ganba medicinal materials; the similarity of 7 batches of the horny tripe medicinal materials is over 0.920.