CN113866306A - Detection method of HPLC (high Performance liquid chromatography) characteristic spectrum of medicinal preparation - Google Patents

Detection method of HPLC (high Performance liquid chromatography) characteristic spectrum of medicinal preparation Download PDF

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CN113866306A
CN113866306A CN202111142138.1A CN202111142138A CN113866306A CN 113866306 A CN113866306 A CN 113866306A CN 202111142138 A CN202111142138 A CN 202111142138A CN 113866306 A CN113866306 A CN 113866306A
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characteristic
peak
pharmaceutical preparation
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solution
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CN113866306B (en
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高婷婷
谢元彪
王玲
李淞明
詹常森
张正光
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Shanghai Hutchison Pharmaceuticals Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N30/04—Preparation or injection of sample to be analysed
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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Abstract

The invention provides a detection method of an HPLC (high performance liquid chromatography) characteristic spectrum of a medicinal preparation. The invention also provides an application of the detection method of the HPLC characteristic spectrum of the pharmaceutical preparation in quality detection of the pharmaceutical preparation. The invention further provides a construction method of the standard characteristic map of the pharmaceutical preparation. The invention further provides a quality control method of the characteristic spectrum of the medicinal preparation. The HPLC characteristic spectrum detection method for the pharmaceutical preparation provided by the invention can obtain the characteristic spectrum of the pharmaceutical preparation containing 7 characteristic peaks, has good precision, good stability and good reproducibility, can truly reflect the quality difference of the pharmaceutical preparation, and improves the quality control system of the pharmaceutical preparation.

Description

Detection method of HPLC (high Performance liquid chromatography) characteristic spectrum of medicinal preparation
Technical Field
The invention belongs to the technical field of traditional Chinese medicine component analysis, and relates to a detection method of an HPLC (high performance liquid chromatography) characteristic spectrum of a medicinal preparation.
Background
The pharmaceutical preparation is first carried in "Beiji Qianjin Yao (prescription of Qianjin of emergency) | fourteenth (small intestine) of Tang-, Sun-Si far away: the recipe is good for forgetting, polygala root, ginseng (four divisions each), poria cocos (two), calamus (two), the four herbs on the upper side are used for sifting down, and the recipe is cun an ancient type of spoon, ri san. The Chinese medicinal preparation is used for treating emotional diseases such as heart-qi deficiency, uneasiness of mind, anxiety and insomnia, depression and severe palpitation and is a basic prescription for improving intelligence, nourishing heart and tranquilizing mind in Chinese medicaments, similar to depression in western medicaments. The modern clinical practice of the pharmaceutical preparation is usually used for treating depression, anxiety and dementia, and the clinical manifestations are symptoms such as restlessness, absentmindedness, anxiety, amnesia, insomnia, palpitation and severe palpitation.
Because an overall evaluation method aiming at the pharmaceutical preparation is lacked at present, a corresponding HPLC characteristic spectrum method is necessary to be established to control the quality of the pharmaceutical preparation and perfect a quality control system of the pharmaceutical preparation.
Disclosure of Invention
In view of the above disadvantages of the prior art, the present invention aims to provide a method for detecting an HPLC profile of a pharmaceutical preparation, which establishes an HPLC profile of the pharmaceutical preparation by using a pretreatment under optimized conditions and a high performance liquid chromatography, and can analyze 7 chemical components in the pharmaceutical preparation, thereby solving the problem of lack of an overall evaluation method for the pharmaceutical preparation in the prior art and comprehensively perfecting a quality control system of the pharmaceutical preparation.
In order to achieve the above and other related objects, the present invention provides, in a first aspect, a method for detecting an HPLC profile of a pharmaceutical preparation, comprising: dissolving a medicinal preparation sample in a solvent, performing ultrasonic extraction, cooling, shaking, filtering, collecting a sample solution obtained from a subsequent filtrate, detecting by high performance liquid chromatography, and performing attribution positioning on index components in the obtained characteristic spectrum of the sample solution according to retention time to obtain the characteristic spectrum of the medicinal preparation.
Preferably, the ratio of the added mass of the drug preparation sample to the added volume of the solvent is 2: 25-75 g/mL. Preferably, the ratio of the mass of the drug formulation sample added to the volume of solvent added is 2:50, g/mL.
Preferably, the solvent is selected from one of 70% methanol, ethanol or water. Preferably, the solvent is 70% methanol. The 70% methanol is 70% methanol aqueous solution by volume percentage.
Preferably, the time of ultrasonic extraction is 30-60 minutes. Preferably, the time of the ultrasonic extraction is 30 minutes.
Preferably, the cooling is to room temperature. The room temperature is 20-30 ℃.
Preferably, the filtration is to filter the supernatant of the shaken solution with a filter membrane.
Preferably, the filter membrane for filter membrane filtration is a 0.22 μm filter membrane.
Preferably, the secondary filtrate is obtained by abandoning the primary filtrate after the filtration of the filter membrane.
Preferably, the detection is performed by high performance liquid chromatography, and comprises the following steps:
1) preparation of reference solutions: mixing ginsenoside Rg1Ginsenoside Re, radix Polygalae
Figure BDA0003284355360000021
Adding a solvent to a reference substance of ketone III, 3,6' -dibastinyl sucrose, tenuifolin A, beta-asarone and pachymic acid for dissolving and fixing the volume to prepare a reference substance solution;
2) and (3) determination: respectively measuring the test solution and the reference solution in the step 1) by adopting a High Performance Liquid Chromatography (HPLC) method to obtain a characteristic spectrum of the test solution and a characteristic spectrum of the reference solution, comparing the retention time of the characteristic spectrum of the test solution with the characteristic spectrum of the reference solution for qualitative determination, and performing attribution positioning on index components in the characteristic spectrum of the test solution, thereby obtaining the characteristic spectrum of the pharmaceutical preparation.
Preferably, in the step 1), the ginsenoside Rg1The CAS number of is 22427-39-0, the CAS number of ginsenoside Re is 52286-59-6, and radix Polygalae
Figure BDA0003284355360000022
The CAS number of ketone III is 162857-78-5, the CAS number of 3,6' -disinapoyl sucrose is 139891-98-8, the CAS number of senegaside A is 139726-35-5, the CAS number of beta-asarone is 5273-86-9, and the CAS number of pachymic acid is 29070-92-6.
The medicinal preparation is brown yellow powder and is prepared from 4 Chinese medicinal materials including ginseng, polygala root, grassleaf sweelflag rhizome and tuckahoe. The main active components of Ginseng radix are ginsenoside, ginsenoside Re and ginsenoside Rg1Suitably used together as a standard for measuring ginsenoside. Polygala tenuifolia is used as a traditional nootropic drug, and the chemical components of the Polygala tenuifolia mainly comprise triterpenoid saponins, ketones, oligosacchrides, alkaloids and the like. Wherein the 3,6' -disinapoyl sucrose is effective component of the drug-containing serum for treating depression. Root of Polygala
Figure BDA0003284355360000023
Ketone III, 3,6' -dibapinyl sucrose, and tenuifolin A, which respectively represent ketones, oligosaccharide lipids, and triterpene saponins of cortex et radix Polygalae. Poria mainly contains pachyman, triterpenic acids, gum, protein, sterol, fatty acid, etc., and pachyman acid is a representative component in Poria. The rhizoma acori graminei medicinal material has high volatile oil content, and the main component is beta-asarone (0.705-1.53%), which is an important basis for playing pharmacological action. Research shows that the beta-asarone has the functions of treating dementia and resisting depression. Therefore, the invention selects the ginsenoside Rg1Ginsenoside Re, radix Polygalae
Figure BDA0003284355360000024
Ketone III, 3,6' -dibapinyl sucrose, tenuifolin A, pachymic acid and beta-asarone are common peaks of characteristic maps.
Preferably, in step 1), the solvent is selected from one of 70% methanol, ethanol or water. More preferably, the solvent is 70% methanol. The 70% methanol is 70% methanol aqueous solution by volume percentage.
Preferably, in step 1), the reference solution is prepared by stepwise dilution.
Preferably, in the step 1), the ginsenoside Rg in the reference solution1Ginsenoside Re, radix Polygalae
Figure BDA0003284355360000025
The concentrations of ketone III, 3,6' -dibapigenin sucrose, tenuifolin A, beta-asarone and pachymic acid are all 10-100 μ g/mL.
Preferably, in the step 2), the detector used in the High Performance Liquid Chromatography (HPLC) is a Diode Array Detector (DAD).
Preferably, in the step 2), the chromatographic column in the high performance liquid chromatography is C18The filler in the chromatographic column is octadecylsilane chemically bonded silica.
More preferably, the column in the high performance liquid chromatography is selected from ZORBAX SB-C18Chromatography column (4.6X 250mm, 5 μm), ORBAX Eclipse XDB-C18Chromatography column (4.6X 250mm, 5 μm), Diamonsil C18One of the chromatography columns (4.6X 250mm, 5 μm).
Further preferably, the column in the high performance liquid chromatography is ZORBAX SB-C18Column (4.6X 250mm, 5 μm).
Preferably, in the step 2), the detection wavelength in the high performance liquid chromatography is 200-205nm, preferably 203 nm.
Preferably, in step 2), the column temperature in the high performance liquid chromatography is 28-32 ℃. More preferably, the column temperature in the high performance liquid chromatography is 30 ℃.
Preferably, in step 2), the flow rate in the high performance liquid chromatography is 0.9-1.1 mL/min. More preferably, the flow rate in the high performance liquid chromatography is 1.0 mL/min.
Preferably, in the step 2), the sample amount in the high performance liquid chromatography is 5-20 μ L. More preferably, the amount of sample in the high performance liquid chromatography is 10. mu.L.
Preferably, in the step 2), the mobile phase in the high performance liquid chromatography is acetonitrile-0.05-0.15% phosphoric acid water solution, wherein the phase A is acetonitrile, and the phase B is 0.05-0.15% phosphoric acid water solution; the analysis time is 102 min; gradient elution.
More preferably, in the high performance liquid chromatography, the mobile phase is acetonitrile-0.10% phosphoric acid aqueous solution, wherein, the phase A is acetonitrile, and the phase B is 0.10% phosphoric acid aqueous solution; the analysis time is 102 min; gradient elution.
The 0.05-0.15% phosphoric acid aqueous solution is 0.05-0.15% phosphoric acid aqueous solution by volume percentage. The 0.10 phosphoric acid aqueous solution is 0.10 percent by volume of phosphoric acid aqueous solution.
More preferably, the specific procedure of the gradient elution is:
0-35 min, phase A: the volume ratio of the phase B is 10: 90-21: 79;
35-53 min, phase A: the volume ratio of the phase B is 21: 79-23: 77;
53-68 min, phase A: the volume ratio of the phase B is 23: 77-45: 55;
68-78 min, phase A: the volume ratio of the phase B is 45: 55-75: 25;
78-100 min, phase A: the volume ratio of the phase B is 75: 25-95: 5;
100-102 min, phase A: the volume ratio of the phase B is 95: 5-95: 5.
the invention provides an application of a detection method of an HPLC characteristic map of a pharmaceutical preparation in quality detection of the pharmaceutical preparation.
Such pharmaceutical formulations include, without limitation, kaixiong, and the like.
The third aspect of the invention provides a method for constructing a standard characteristic map of a pharmaceutical preparation, which comprises the following steps: the detection method of the HPLC characteristic spectrum of the medicinal preparation is adopted to respectively detect a plurality of medicinal preparation samples, the characteristic spectrums of a plurality of medicinal preparations are obtained to generate a common control characteristic spectrum, chromatographic peaks existing in the spectrums are used as common characteristic peaks, the relative retention time of the common characteristic peaks is determined, and the standard characteristic spectrum of the medicinal preparation is established.
Preferably, the generation method of the characteristic map of the pharmaceutical preparation adopts an average method. The averaging method is one of common mode methods.
Specifically, after a batch of fingerprint samples are obtained, similarity analysis and evaluation need to be performed on the fingerprint samples so as to be used for quality control of traditional Chinese medicines. During the similarity evaluation process, a control fingerprint is required to be generated. The current methods for generating the comparison fingerprint mainly comprise a typical fingerprint selection method and a common mode method. Typical fingerprint selection selects a typical or representative fingerprint as a control fingerprint. However, the control fingerprints thus generated do not contain the overall information of the fingerprint sample, but rather the individual characteristics of the sample, and therefore sometimes do not necessarily represent the overall characteristics of the fingerprint sample well. And the selection of the typical fingerprint spectrum also has certain subjectivity. The common mode method includes both an averaging method and a median method. The control fingerprints generated by the two methods contain sample information of the original fingerprints. If no outlier sample is present in the batch, it is generally recommended to use the averaging method.
Preferably, the standard profile of the pharmaceutical preparation is constructed such that the width of the time window for matching the common characteristic peak is 0.1.
Preferably, the standard characteristic map of the pharmaceutical preparation comprises 7 common characteristic peaks, and the specified values of the relative retention times of the No. 2 peak are sequentially No. 1 peak (0.665), No. 3 peak (1.183), No. 4 peak (1.326), No. 5 peak (1.346), No. 6 peak (2.041) and No. 7 peak (2.458) by taking the No. 2 peak as a reference peak (S1 peak, retention time is 1.000), and the relative deviations of the specified values of the relative retention times of the 6 common characteristic peaks except the No. 2 peak are less than or equal to +/-5%.
Preferably, the standard characteristic spectrum of the pharmaceutical preparation is compared with the characteristic spectrum of a reference substance solution, and the number 1 peak is determined to be polygala tenuifolia through localization
Figure BDA0003284355360000041
The peak No. 2 is the characteristic peak of 3,6' -erucyl sucrose, and the peak No. 3 is the characteristic peak of ketone IIIThe peak is the characteristic peak of senegaside A, and the No. 4 peak is ginsenoside Rg1The peak No. 5 is a characteristic peak of ginsenoside Re, the peak No. 6 is a characteristic peak of beta-asarone, and the peak No. 7 is a characteristic peak of pachymic acid.
The fourth aspect of the invention provides a quality control method of a characteristic spectrum of a pharmaceutical preparation, which comprises the following steps: comparing the similarity of relative retention time of chromatographic peaks of the medicinal preparation and corresponding common characteristic peaks of the characteristic spectrum of the medicinal preparation obtained by the detection method of the characteristic spectrum of the medicinal preparation and the standard characteristic spectrum of the medicinal preparation obtained by the construction method of the standard characteristic spectrum of the medicinal preparation.
According to the Chinese pharmacopoeia analysis and detection technical guideline, the characteristic spectrum does not require the same comprehensive evaluation of the similarity of the spectrum as the fingerprint spectrum, and the method is mainly characterized in that the specific components of the characteristic spectrum of the variety and other varieties are highlighted and are used as an important identification means for controlling the quality of the traditional Chinese medicine.
The fifth aspect of the invention provides a method for screening the characteristic maps of a plurality of medicinal materials in a medicinal preparation, which comprises the following steps:
A) preparation of a default negative sample solution: respectively removing any 1 medicinal material from a medicinal preparation sample containing 4 medicinal materials of tuckahoe, ginseng, polygala root and acorus gramineus, and preparing according to the same steps of preparing a test sample solution in the detection method of the characteristic spectrum of the medicinal preparation to respectively obtain 4 sample-lacking negative solutions: poria negative solution, Ginseng radix negative solution, cortex et radix Polygalae negative solution, and rhizoma Acori Graminei negative solution;
B) and (3) determination: respectively measuring the 4 sample-lacking negative solutions by adopting a High Performance Liquid Chromatography (HPLC) method with the same chromatographic conditions as the detection method of the characteristic spectrum of the medicinal preparation to respectively obtain the characteristic spectrums of the 4 sample-lacking negative solutions;
C) and (3) quality detection: comparing the characteristic spectrums of the 4 sample-lacking negative solutions with the standard characteristic spectrums of the pharmaceutical preparation established by the construction method of the standard characteristic spectrums of the pharmaceutical preparation respectively, and identifying the common characteristic peaks of the corresponding single medicinal materials in the 4 sample-lacking negative solutions in the standard characteristic spectrums of the pharmaceutical preparation through relative retention time, thereby performing attribution positioning on the characteristic peaks in the characteristic spectrums of the corresponding single medicinal materials in the 4 sample-lacking negative solutions.
The Poria is dried sclerotium of Wolf of Poria cocos (Schw.) belonging to Polyporaceae.
The Ginseng radix is root of Panax ginseng C.A. Meyer belonging to Araliaceae family.
The radix Polygalae is root of Polygala tenuifolia Willd
The rhizoma Acori Graminei is rhizome of Acorus tatarinowii Schott of Araceae.
Preferably, in the step C), in the standard characteristic spectrum of the pharmaceutical preparation, the common characteristic peaks of the characteristic spectrum of the corresponding single medicinal material in the 4 sample-lacking negative solutions are respectively located, and the peaks 1, 2 and 3 are derived from polygala tenuifolia; the No. 4 and No. 5 peaks are derived from ginseng, the No. 6 peak is derived from rhizoma acori graminei, and the No. 7 peak is derived from poria cocos. The water used in the invention is pure water.
As mentioned above, the detection method of the HPLC characteristic spectrum of the pharmaceutical preparation provided by the invention adopts the pretreatment of optimized conditions and the instrument detection method to carry out qualitative detection on the pharmaceutical preparation. The method can realize the separation and detection of the characteristic components of the medicinal preparation, comprehensively reflect the component information of the medicinal preparation, and realize the application of controlling the quality of the characteristic map of the medicinal preparation, monitoring the production of the medicinal preparation, identifying the authenticity of the medicinal preparation and the like. The method has the following beneficial effects:
(1) according to the detection method of the HPLC characteristic spectrum of the pharmaceutical preparation, provided by the invention, in the process of establishing the HPLC characteristic spectrum determination method of the pharmaceutical preparation, precision, stability and repeatability test investigation and research are carried out by taking relative retention time as an index, 7 common characteristic peaks are confirmed, and the stability and adaptability of the method are ensured.
(2) According to the detection method of the HPLC characteristic spectrum of the pharmaceutical preparation, provided by the invention, as the chemical components in the pharmaceutical preparation are complex, a gradient elution method is adopted in the process of establishing the characteristic spectrum, so that the problems of difficulty in separating characteristic peaks and interference of impurity peaks are solved.
(3) The HPLC characteristic spectrum detection method of the pharmaceutical preparation provided by the invention treats each effective component characteristic spectrum in the pharmaceutical preparation as a whole, pays attention to the front-back sequence and the mutual relation of each characteristic peak, avoids the one-sidedness of determining the whole quality of the pharmaceutical preparation due to the measurement of a small amount of components, and reduces the possibility of manual treatment for reaching the quality standard.
(4) The detection method of the HPLC characteristic spectrum of the pharmaceutical preparation provided by the invention has the advantages of simple operation, high precision, good stability and reproducibility and the like, can truly reflect the quality difference of the pharmaceutical preparation, ensures the stability of the production process and quality among batches, and can comprehensively and scientifically evaluate the quality of the pharmaceutical preparation, so that the quality and the curative effect of the product are ensured, a reference is provided for the perfection of the quality standard of the product, and the quality control system of the pharmaceutical preparation is comprehensively perfected.
Drawings
FIG. 1 shows the precision result characteristic of the pharmaceutical preparation of the present invention.
FIG. 2 shows a similarity evaluation chart for different chromatographic column durability examinations of the pharmaceutical preparation of the present invention.
FIG. 3 shows a similarity evaluation chart for different flow rate durability examinations of the pharmaceutical preparation of the present invention.
FIG. 4 shows a similarity evaluation chart for different column temperature durability examinations of the pharmaceutical preparation of the present invention.
FIG. 5 shows a similarity evaluation chart for different pH durability examinations of the pharmaceutical preparation of the present invention.
FIG. 6 shows a similarity evaluation chart for different instrument durability examinations of the pharmaceutical preparation of the present invention.
FIG. 7 shows a stability result characteristic map of the pharmaceutical formulation of the present invention.
FIG. 8 shows a characteristic spectrum of the repetitive results of the pharmaceutical preparation of the present invention.
FIG. 9 shows a standard characteristic map of the pharmaceutical preparation of the present invention, wherein 1 is Polygala tenuifolia
Figure BDA0003284355360000061
Ketone III, 2 is 3,6' -di-sinapoyl sucrose, 3 is tenuifolin A, 4 is ginsenoside Rg 15 is ginsenoside Re, 6 is beta-asarone and 7 is pachymic acid.
FIG. 10 shows a chromatogram map of the specificity of each herb in the pharmaceutical preparation of the present invention, wherein A is a sample solution; b is reference solution; c is Poria negative solution; d is a ginseng negative solution; e is polygala negative solution: f is acorus gramineus negative solution.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The reagents and equipment used in the following examples are as follows:
1. reagent
The 15 batches of the drug formulation sample were embodied as kaixuan san (manufactured by shanghai and huang pharmaceuticals ltd.) as shown in table 1 below.
TABLE 1
Numbering Batch number Numbering Batch number
A1 2020063001 A9 2020070201
A2 2020063002 A10 2020071302
A3 2020071401 A11 2020071601
A4 2020063003 A12 2020071602
A5 2020063004 A13 2020070202
A6 2020070101 A14 2020071603
A7 2020071301 A15 2020071402
A8 2020070102
Root of Polygala
Figure BDA0003284355360000071
Ketone iii (Polygalaxanthone iii) control (NATURE STANDARD, ST 06290120); 3,6 '-Disinapoyl sucrose (3,6' -disinapoyl sucrose) control (Chinese food and drug testing institute, 111848-202006 ID: QNVP-D9 TF); senegaside a (tenuifoliside a) control (shanghai joy biotechnology ltd, P16A9F 58918); ginsenoside Rg1(Ginsenoside Rg1) The reference (China institute for food and drug assay, 110703-201933 ID: TWYY-M6 VC); ginsenoside Re (ginsenoside Re) reference substance (Chinese food and drug inspection institute, 110754-202028 ID: ZR2T-SE 40); a beta-asarone (beta-asarone) reference substance (China food and drug inspection institute, 112018-; pachymic acid (Pachymic acid) reference (Woodward scientific development Co., Ltd., batch No.: PRF 9090303).
Acetonitrile (chromatographic purity, manufacturer: Fisher chemical, USA, purchaser: chemical reagents of national drug group, Inc., batch No. 204199); methanol, phosphoric acid (analytical purity, manufacturer: CNW technologies, purchaser: national drug group chemical Co., Ltd., batch No. J5640045); ultrapure water (prepared by a water purifier).
2. Instrument for measuring the position of a moving object
Agilent model 1260 ii high performance liquid chromatograph (Agilent, usa, quaternary pump); waters E2695 model high performance liquid chromatograph (Waters, usa, quaternary pump); BT25S model electronic analytical balance (beijing sydolis, china); BSA124S-CW type electronic analytical balance (Sidedoshi, Beijing, China); KQ-250DB type ultrasonic cleaner (kunshan ultrasonic instruments ltd., china); model ZP15D1 ultrasonic water purifier (shanghai zeugo instruments ltd, china).
ZORBAX SB-C18Chromatography column (4.6X 250mm, 5 μm) (Agilent, USA); ORBAX Eclipse XDB-C18Chromatography column (4.6X 250mm, 5 μm) (Agilent, USA); diamonsil C18Chromatography column (4.6X 250mm, 5 μm) (Dima Tech, China).
The detection method for the HPLC characteristic map of the pharmaceutical preparation comprises the following determination processes.
1. Preparation of test solution
Precisely weighing a medicinal preparation sample, adding a solvent for dissolving, wherein the ratio of the adding mass of the medicinal preparation sample to the adding volume of the solvent is 2: 25-75 g/mL, sealing, carrying out ultrasonic treatment for 30-60 minutes, cooling to room temperature, shaking up, filtering a supernatant with a 0.22 mu m filter membrane, and taking a subsequent filtrate to obtain a sample solution. The solvent is selected from 70% methanol, ethanol or water.
2. Preparation of reference solutions
Mixing ginsenoside Rg1Ginsenoside Re, radix Polygalae
Figure BDA0003284355360000081
Adding a solvent to a reference substance of ketone III, 3,6' -dibapigenin sucrose, tenuifolin A, beta-asarone and pachymic acid, dissolving, and diluting to a constant volume to obtain a reference substance solution. The solvent is selected from 70% methanol, ethanol or water.
3. Measurement of
Respectively measuring the test solution and the reference solution by adopting a High Performance Liquid Chromatography (HPLC) method to obtain a characteristic spectrum of the test solution and a characteristic spectrum of the reference solution, comparing the retention time of the characteristic spectrum of the test solution with the characteristic spectrum of the reference solution for qualitative determination, and performing attribution positioning on index components in the characteristic spectrum of the test solution to obtain the characteristic spectrum of the pharmaceutical preparation.
Wherein, the high performance liquid chromatography comprises the following detection conditions:
the detector is a Diode Array Detector (DAD); the chromatographic column is C18A chromatographic column is arranged on the top of the chromatographic column,the filler in the chromatographic column is octadecylsilane chemically bonded silica, specifically ZORBAX SB-C18Chromatography column (4.6X 250mm, 5 μm), ORBAX Eclipse XDB-C18Chromatography column (4.6X 250mm, 5 μm), Diamonsil C18One of a chromatographic column (4.6X 250mm, 5 μm); the detection wavelength is 200-205 nm; the column temperature is 28-32 ℃; the flow rate is 0.9-1.1 mL/min; the sample amount is 5-20 μ L; the mobile phase is acetonitrile-0.05-0.15% phosphoric acid water solution, wherein, the phase A is acetonitrile, and the phase B is 0.05-0.15% phosphoric acid water solution; the analysis time is 102 min; gradient elution.
The specific procedure for gradient elution was:
0-35 min, phase A: the volume ratio of the phase B is 10: 90-21: 79;
35-53 min, phase A: the volume ratio of the phase B is 21: 79-23: 77;
53-68 min, phase A: the volume ratio of the phase B is 23: 77-45: 55;
68-78 min, phase A: the volume ratio of the phase B is 45: 55-75: 25;
78-100 min, phase A: the volume ratio of the phase B is 75: 25-95: 5;
100-102 min, phase A: the volume ratio of the phase B is 95: 5-95: 5.
the detection method of the HPLC characteristic spectrum of the medicinal preparation is adopted to respectively detect a plurality of medicinal preparation samples, the characteristic spectrums of a plurality of medicinal preparations are obtained to generate a common control characteristic spectrum, chromatographic peaks existing in the spectrums are used as common characteristic peaks, the relative retention time of the common characteristic peaks is determined, and the standard characteristic spectrum of the medicinal preparation is established.
The standard characteristic map of the pharmaceutical preparation comprises 7 common characteristic peaks, and the specified values of the relative retention times of the No. 2 peak as a reference peak (S1 peak, retention time of 1.000), and the other 6 common characteristic peaks are sequentially No. 1 peak (0.665), No. 3 peak (1.183), No. 4 peak (1.326), No. 5 peak (1.346), No. 6 peak (2.041) and No. 7 peak (2.458), and the relative deviations of the specified values of the relative retention times of the 6 common characteristic peaks except for the No. 2 peak are all less than or equal to +/-5%.
Comparing the standard characteristic spectrum of the pharmaceutical preparation with the characteristic spectrum of the reference solution, and determining the No. 1 peak as cortex et radix Polygalae
Figure BDA0003284355360000091
The peak 2 is the characteristic peak of 3,6' -erucyl sucrose, the peak 3 is the characteristic peak of tenuifolin A, and the peak 4 is ginsenoside Rg1The peak No. 5 is a characteristic peak of ginsenoside Re, the peak No. 6 is a characteristic peak of beta-asarone, and the peak No. 7 is a characteristic peak of pachymic acid.
Respectively removing any 1 medicinal material from a medicinal preparation sample containing 4 medicinal materials of tuckahoe, ginseng, polygala root and acorus gramineus, and preparing according to the same steps of preparing a test sample solution in the detection method of the characteristic spectrum of the medicinal preparation to respectively obtain 4 sample-lacking negative solutions: poria negative solution, Ginseng radix negative solution, cortex et radix Polygalae negative solution, and rhizoma Acori Graminei negative solution. And respectively measuring the 4 sample-lacking negative solutions by adopting a High Performance Liquid Chromatography (HPLC) method with the same chromatographic conditions as the detection method of the characteristic spectrum of the medicinal preparation to respectively obtain the characteristic spectrums of the 4 sample-lacking negative solutions. Comparing the characteristic spectrums of the 4 sample-lacking negative solutions with the standard characteristic spectrums of the pharmaceutical preparation established by the construction method of the standard characteristic spectrums of the pharmaceutical preparation respectively, and identifying the common characteristic peaks of the corresponding single medicinal materials in the 4 sample-lacking negative solutions in the standard characteristic spectrums of the pharmaceutical preparation through relative retention time, thereby performing attribution positioning on the characteristic peaks in the characteristic spectrums of the corresponding single medicinal materials in the 4 sample-lacking negative solutions.
As shown in fig. 10, in the standard characteristic maps of the pharmaceutical preparation, the common characteristic peaks of the characteristic maps of the corresponding single medicinal materials in the 4 sample-lacking negative solutions are respectively located, and the peaks 1, 2 and 3 are derived from polygala tenuifolia; the No. 4 and No. 5 peaks are derived from ginseng, the No. 6 peak is derived from rhizoma acori graminei, and the No. 7 peak is derived from poria cocos.
Example 1
1. Preparation of test solution
Precisely weighing 2.0g of the medicinal preparation sample, placing in a conical flask with a plug, precisely adding 50ml of methanol, sealing, ultrasonically treating (350w and 53kHz) for 30 minutes, cooling to room temperature, shaking up, filtering the supernatant with 0.22 μm filter membrane, and collecting the subsequent filtrate to obtain sample solution No. 1.
2. Preparation of reference solutions
Mixing ginsenoside Rg1Ginsenoside Re, radix Polygalae
Figure BDA0003284355360000101
Precisely weighing reference substances of ketone III, 3,6' -erucyl sucrose, tenuifolin A, beta-asarone and pachymic acid, adding methanol to constant volume, and making into mixed reference substance solution 1# with mass concentration of 30, 20, 70, 20, 100 and 10 μ g/mL respectively.
3. Measurement of
Detecting the sample solution 1# and the reference solution 1# respectively by adopting a High Performance Liquid Chromatography (HPLC) to obtain a characteristic map of the sample solution 1# and a characteristic map of the reference solution 1#, comparing the characteristic map of the sample solution 1# with the characteristic map of the reference solution 1# for retention time and determining the nature, and performing attribution positioning on index components in the characteristic map of the sample solution 1# so as to obtain the characteristic map of the pharmaceutical preparation.
Wherein, the high performance liquid chromatography comprises the following detection conditions:
the detector is a Diode Array Detector (DAD); the chromatographic column is ZORBAX SB-C18Chromatography column (4.6X 250mm, 5 μm); the detection wavelength is 203 nm; the column temperature is 30 ℃; the flow rate is 1.0 mL/min; the sample injection amount is 10 mu L; the mobile phase is acetonitrile-0.1% phosphoric acid water solution, wherein, the phase A is acetonitrile, and the phase B is 0.1% phosphoric acid water solution; the analysis time is 102 min; gradient elution.
The specific procedure for gradient elution was:
0-35 min, phase A: the volume ratio of the phase B is 10: 90-21: 79;
35-53 min, phase A: the volume ratio of the phase B is 21: 79-23: 77;
53-68 min, phase A: the volume ratio of the phase B is 23: 77-45: 55;
68-78 min, phase A: the volume ratio of the phase B is 45: 55-75: 25;
78-100 min, phase A: the volume ratio of the phase B is 75: 25-95: 5;
100-102 min, phase A: the volume ratio of the phase B is 95: 5-95: 5.
example 2
According to the method in the embodiment 1, 15 batches of medicinal preparation samples are taken for detection, the characteristic maps of a plurality of medicinal preparation samples are obtained to generate a common control characteristic map, chromatographic peaks existing in the map are used as common characteristic peaks, the relative retention time of the common characteristic peaks is determined, and the standard characteristic map of the medicinal preparation is established.
As shown in fig. 9, the standard characteristic pattern of the pharmaceutical preparation includes 7 common characteristic peaks, and the specified values of the relative retention times of the 6 other common characteristic peaks are, in order from the peak No. 2 as the reference peak (S1 peak, retention time 1.000), the peak No. 1 (0.665), the peak No. 3 (1.183), the peak No. 4 (1.326), the peak No. 5 (1.346), the peak No. 6 (2.041), and the peak No. 7 (2.458).
As shown in FIG. 9, the standard characteristic pattern of the pharmaceutical preparation is compared with the characteristic pattern of the reference solution, and the peak No. 1 is determined to be polygala tenuifolia
Figure BDA0003284355360000102
The peak 2 is the characteristic peak of 3,6' -erucyl sucrose, the peak 3 is the characteristic peak of tenuifolin A, and the peak 4 is ginsenoside Rg1The peak No. 5 is a characteristic peak of ginsenoside Re, the peak No. 6 is a characteristic peak of beta-asarone, and the peak No. 7 is a characteristic peak of pachymic acid.
Example 3
A test solution was prepared using pharmaceutical preparation sample A15 (batch No. 2020071402) in step 1 of example 1. Meanwhile, a reference solution was prepared in step 2 of example 1, wherein polygala tenuifolia
Figure BDA0003284355360000111
The content of ketone III is 21.23 μ g/mL, the content of 3,6' -disinapoyl sucrose is 72.19 μ g/mL, the content of tenuifolin A is 45.71 μ g/mL, and the ginsenoside Rg is ginsenoside Rg1Has a content of 28.09 μ g/mL, ginsenoside Re content of 27.26 μ g/mL, beta-asarone content of 143 μ g/mL, and pachymic acid content of 5.78μg/mL。
Removing any one 1 of the 4 medicinal materials of poria cocos, ginseng, polygala tenuifolia and rhizoma acori graminei from the medicinal preparation sample, and preparing according to the step 1) of the detection method of the characteristic spectrum of the medicinal preparation in the embodiment 1 to obtain 4 sample-lacking negative solutions: poria negative solution, Ginseng radix negative solution, cortex et radix Polygalae negative solution, and rhizoma Acori Graminei negative solution.
The High Performance Liquid Chromatography (HPLC) method under the same chromatographic conditions as in step 3) of the method for detecting a characteristic pattern of a pharmaceutical preparation in example 1 was used to measure the test solution, the reference solution, and the 4 sample-lacking negative solutions, respectively, and the characteristic pattern of the test solution, the characteristic pattern of the reference solution, and the characteristic patterns of the 4 sample-lacking negative solutions were obtained, respectively.
The standard characteristic map of the pharmaceutical preparation was established from the characteristic map of the test solution according to the method in example 2. And comparing the characteristic maps of the 4 sample-lacking negative solutions with the standard characteristic map of the pharmaceutical preparation and the characteristic map of the reference solution respectively, and identifying the common characteristic peaks of the corresponding single medicinal materials in the 4 sample-lacking negative solutions in the standard characteristic map of the pharmaceutical preparation through relative retention time, thereby performing attribution positioning on the characteristic peaks in the characteristic maps of the corresponding single medicinal materials in the 4 sample-lacking negative solutions. The specific results are shown in FIG. 10.
As shown in FIG. 10, the result of the negative control map of cortex et radix Polygalae was compared with the mixed standard and sample to show that cortex et radix Polygalae
Figure BDA0003284355360000112
The three index components of ketone III, 3,6' -erucyl sucrose and tenuifolin A exist in polygala tenuifolia medicinal materials only, and the specificity is good. After the ginseng negative control map is compared with a mixed standard and a sample, the result shows that two index components of the ginsenoside Rg1 and the ginsenoside Re only exist in the ginseng medicinal material, and the specificity is good. After the rhizoma acori graminei negative control map is compared with the mixed standard and the sample, the result shows that the beta-asarone component only exists in the rhizoma acori graminei medicinal material, and the specificity is good. After the poria negative control map is compared with the mixed standard and the sample, the result shows that the pachymic acid component only exists in the poriaAmong the tuckahoe medicinal materials, its specificity is good.
Example 4
The detection method of the HPLC characteristic spectrum of the pharmaceutical preparation is verified by methodology, and the performance index result is as follows.
1. Precision degree
A sample A15 (lot 2020071402) of the same pharmaceutical preparation was sampled and analyzed 6 times on the same day by performing sample injection according to step 1 of example 1 under the chromatographic conditions of step 3 of example 1, and the relative retention time of each peak was calculated using 3,6' -erucyl sucrose as a reference peak, and the results are shown in Table 2. As can be seen from table 2, the RSD of the relative retention time of each characteristic peak was less than 5%.
TABLE 2 results of the precision test
Figure BDA0003284355360000121
The obtained characteristic spectrum is introduced into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), the characteristic spectrum of the 2 nd analysis result S2 of the pharmaceutical preparation sample A15 is set as a reference spectrum, the width of a time window is set as 0.1, multi-point correction is carried out, Mark peak matching is carried out, a control characteristic spectrum R is generated by an average method, and the specific result is shown in figure 1. The similarity was calculated and the results are shown in table 3. As can be seen from fig. 1 and table 3, the similarity of the results of 6 injections was 1, indicating that the precision of the apparatus was good.
TABLE 3 precision similarity results
S1 S2 S3 S4 S5 S6 Reference feature map R
S1
1 1 1 1 1 1 1
S2 1 1 1 1 1 1 1
S3 1 1 1 1 1 1 1
S4 1 1 1 1 1 1 1
S5 1 1 1 1 1 1 1
S6 1 1 1 1 1 1 1
Reference feature map R 1 1 1 1 1 1 1
2. Stability of
A sample A15 (batch No. 2020071402) of the same pharmaceutical preparation is taken, a test solution is prepared according to the step 1 in the example 1, the chromatographic conditions of the step 3 in the example 1 are different from 0h, 2h, 4h, 8h, 12h and 24h, the 3,6' -dibapinyl sucrose is taken as a reference peak, the relative retention time of each characteristic peak is calculated, and the specific stability result is shown in a table 4. The results show that the RSD relative to retention time is less than 5%.
Table 4 stability test results
Figure BDA0003284355360000131
The obtained characteristic spectrum is introduced into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), the characteristic spectrum of the 2h analysis result S2 of the pharmaceutical preparation sample A15 is set as a reference spectrum, the width of a time window is set as 0.1, multi-point correction is carried out, Mark peak matching is carried out, a control characteristic spectrum R is generated by an average method, and the specific result is shown in figure 7. The similarity was calculated and the results are shown in table 5. As can be seen from fig. 7 and table 5, the similarity of the feature maps at 6 time points is 1, which indicates that the sample is stable within 24 hours, indicating that the method has good stability.
Table 5 stability similarity results
S1 S2 S3 S4 S5 S6 Reference feature map R
S1
1 1 1 1 1 1 1
S2 1 1 1 1 1 1 1
S3 1 1 1 1 1 1 1
S4 1 1 1 1 1 1 1
S5 1 1 1 1 1 1 1
S6 1 1 1 1 1 1 1
Reference feature map R 1 1 1 1 1 1 1
3. Repeatability of
6 test solutions were prepared in parallel according to step 1 of example 1 from the same pharmaceutical preparation sample A15 (batch No. 2020071402), and analyzed by sample injection according to the chromatographic conditions of step 3 of example 1, and the relative retention time of each peak was calculated using 3,6' -bis-sinapoyl sucrose as the reference peak, and the specific repeatability results are shown in Table 6. The results show that the RSD of the relative retention time of each characteristic peak is less than 5%.
TABLE 6 results of repeated experiments
Figure BDA0003284355360000141
The obtained characteristic spectrum is led into a traditional Chinese medicine chromatogram fingerprint spectrum similarity evaluation system (2012 edition), the characteristic spectrum of the 2 nd test sample solution analysis result S2 of the pharmaceutical preparation sample A15 is set as a reference spectrum, the width of a time window is set as 0.1, multi-point correction is carried out, Mark peak matching is carried out, a reference characteristic spectrum R is generated by an average method, and the specific result is shown in figure 8. The similarity was calculated and the results are shown in table 7. As can be seen from fig. 8 and table 7, the similarity of the feature maps of the 6 samples is 1, indicating that the method has good repeatability.
TABLE 7 repeatability similarity results
S1 S2 S3 S4 S5 S6 Reference feature map R
S1
1 1 1 1 1 1 1
S2 1 1 1 1 1 1 1
S3 1 1 1 1 1 1 1
S4 1 1 1 1 1 1 1
S5 1 1 1 1 1 1 1
S6 1 1 1 1 1 1 1
Reference feature map R 1 1 1 1 1 1 1
4. Durability
4.1 investigation of different chromatography columns
A sample A15 (lot 2020071402) of the same pharmaceutical preparation was sampled and analyzed under the chromatography conditions of step 3 in example 1, to obtain a sample solution according to step 1 in example 1. Comparing ZORBAX SB-C of chromatographic columns of different manufacturers18(4.6×250mm，5μm，P.N.880975-902)、ZORBAX Eclipse XDB-C18(4.6×250mm，5μm，P.N.990967-902)、Diamonsil C18(4.6X 250mm, 5 μm, Cat. no: 99603, Ser. no: 201061838). The relative retention time of each peak was calculated using 3,6' -dibapinyl sucrose as the reference peak, and the results are shown in Table 8. The results show that the RSD of the relative retention time of each characteristic peak is less than 5%.
TABLE 8 relative retention time tables for different chromatography columns
Figure BDA0003284355360000151
The obtained characteristic spectrum is introduced into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), the characteristic spectrum of an SB-C18 chromatographic column analysis result S1 is set as a reference spectrum, the time window width is set as 0.1, multi-point correction is carried out, Mark peak matching is carried out, a control characteristic spectrum R is generated by an average method, and the specific result is shown in figure 2. The similarity was calculated and the results are shown in table 9. As can be seen from fig. 2 and table 9, different columns have a small influence on the detection results, and the durability is good because the similarity is 1.
TABLE 9 results of similarity of different columns
SB-C18 Diamonsil C18 XDB-C18 Comparison fingerprint spectrum R
SB-C18(S1) 1 1 1 1
Diamonsil C18(S2) 1 1 1 1
XDB-C18(S3) 1 1 1 1
Reference feature map R 1 1 1 1
4.2 investigation of different flow rates
A sample A15 (lot 2020071402) of the same pharmaceutical preparation was sampled and prepared by the procedure of step 1 of example 1 to obtain a test solution, the chromatographic conditions of step 3 of example 1 were measured at flow rates of 0.9ml/min, 1.0ml/min and 1.1ml/min, the changes in chromatographic peaks at different flow rates were compared, and the relative retention times of characteristic peaks were calculated using 3,6' -di-sinapoyl sucrose as a reference peak, and the results are shown in Table 10. The results show that the RSD of the relative retention time of each characteristic peak is less than 5%.
TABLE 10 different flow Rate versus Retention time Table
Figure BDA0003284355360000152
The obtained characteristic spectrum is led into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), the characteristic spectrum of an analysis result S1 with the flow rate of 1.0ml/min is set as a reference spectrum, the time window width is set as 0.1, multi-point correction is carried out, Mark peak matching is carried out, a control characteristic spectrum R is generated by an average method, and the specific result is shown in figure 3. The similarity was calculated and the results are shown in Table 11. As can be seen from FIG. 3 and Table 11, the test was conducted at 0.9 to 1.1ml/min, and the degree of similarity was 1, which resulted in good durability.
TABLE 11 results of different flow rate similarity
1.0ml/min 0.9ml/min 1.1ml/min Reference feature map R
1.0ml/min(S1) 1 1 1 1
0.9ml/min(S2) 1 1 1 1
1.1ml/min(S3) 1 1 1 1
Reference feature map R 1 1 1 1
4.3 investigation of different column temperatures
A sample A15 (lot 2020071402) of the same pharmaceutical preparation was sampled and prepared in accordance with step 1 of example 1 to obtain a sample solution, and the sample solution was subjected to measurement at 28 ℃ and 30 ℃ and 32 ℃ under the same chromatographic conditions as in step 3 of example 1, and the peak changes of the chromatographic samples were compared at different column temperatures. The relative retention time of each peak was calculated using 3,6' -dibapinyl sucrose as the reference peak, and the results are shown in Table 12. The results show that the RSD of the relative retention time of each characteristic peak is less than 5%.
TABLE 12 relative retention time tables for different column temperatures
Figure BDA0003284355360000161
The obtained characteristic spectrum is led into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), the characteristic spectrum of an analysis result S1 with the column temperature of 30 ℃ is set as a reference spectrum, the width of a time window is set as 0.1, multi-point correction is carried out, Mark peak matching is carried out, a comparison characteristic spectrum R is generated by an average method, and the specific result is shown in figure 4. The similarity was calculated and the results are shown in Table 13. As can be seen from FIG. 4 and Table 13, the test was carried out at 28 to 32 ℃ and the degree of similarity was 1, and the durability was good.
TABLE 13 results for similarity of different column temperatures
30℃ 28℃ 32℃ Reference feature map R
30℃(S1) 1 1 1 1
28℃(S2) 1 1 1 1
32℃(S3) 1 1 1 1
Reference feature map R 1 1 1 1
4.4 investigation of different phosphoric acid ratios
A sample A15 (lot No. 2020071402) of the same pharmaceutical preparation was sampled and prepared in accordance with step 1 of example 1 to obtain a sample solution, and the sample solution was measured under the chromatographic conditions of step 3 of example 1 using 0.05% phosphoric acid, 0.1% phosphoric acid and 0.15% phosphoric acid as mobile phases, respectively, to compare the change in chromatographic peaks at different pH values. The relative retention times of the characteristic peaks were calculated using 3,6' -dibapinyl sucrose as the reference peak, and the results are shown in Table 14. The results show that the RSD of the relative retention time of each characteristic peak is less than 5%.
TABLE 14 different pH vs. Retention time tables
Figure BDA0003284355360000171
The obtained characteristic spectrum is introduced into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), the characteristic spectrum of a 0.1% phosphoric acid analysis result S1 is set as a reference spectrum, the width of a time window is set as 0.1, multi-point correction is carried out, Mark peak matching is carried out, a control characteristic spectrum R is generated by an average method, and the specific result is shown in figure 5. The similarity was calculated and the results are shown in table 15. As can be seen from fig. 5 and table 15, the tests were carried out with 0.05% phosphoric acid to 0.15% phosphoric acid, and the degree of similarity was 1, and the durability was good.
TABLE 15 results of different pH similarities
0.1% phosphoric acid 0.15% phosphoric acid 0.05% phosphoric acid Reference feature map R
0.1% phosphoric acid (S1) 1 1 1 1
0.15% phosphoric acid (S2) 1 1 1 1
0.05% phosphoric acid (S3) 1 1 1 1
Reference feature map R 1 1 1 1
4.5 different liquid chromatography instrumental investigations
A sample A15 (lot 2020071402) of the same pharmaceutical preparation was sampled and tested by comparing two Agilent infinity 1260 II from different manufacturers and Waters E2695 using chromatographic conditions of step 3 of example 1, wherein Agilent infinity 1260 II was located in 2 different positions of the liquid phase chamber and the standardization center, respectively, to prepare a test solution according to step 1 of example 1. The relative retention time of each peak was calculated using 3,6' -dibapinyl sucrose as the reference peak, and the results are shown in Table 16. The results show that the RSD of the relative retention time of each characteristic peak is less than 5%.
TABLE 16 relative Retention schedules for different instruments
Figure BDA0003284355360000181
The obtained characteristic spectrum is introduced into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), the characteristic spectrum of an Agilent definition 1260 II analysis result S1 positioned in a liquid phase chamber is set as a reference spectrum, the width of a time window is set as 0.1, multi-point correction is carried out, Mark peak matching is carried out, a comparison characteristic spectrum R is generated by an average method, and the specific result is shown in figure 6. The similarity was calculated and the results are shown in Table 17. As can be seen from FIG. 6 and Table 17, the detection is affected little by instruments of different manufacturers, the similarity is greater than 0.999, and the durability is good.
TABLE 17 results of similarity of different instruments
Agilent1260 liquid phase chamber Waters E2695 Agilent1260 standardization centre Reference feature map R
Agilent1260 liquid phase chamber 1 1 1 1
Waters E2695 1 1 0.999 0.999
Agilent1260 standardization centre 1 0.999 1 1
Reference feature map R 1 0.999 1 1
Example 5
15 batches of pharmaceutical formulation samples A1-A15 were collected, and HPLC profiles were obtained for all samples using the test solution prepared in step 1 of example 1, according to the chromatographic conditions of step 3 of example 1. The relative retention times of the characteristic peaks were calculated using 3,6' -dibapinyl sucrose as the reference peak, and the results are shown in Table 18. As can be seen from Table 18, in the characteristic map of the 15 batches of pharmaceutical preparation samples, the RSD of the relative retention time of each characteristic peak is 0-0.15%. And the relative deviation of the other 6 peaks relative to the specified value of the retention time is less than or equal to +/-5 percent by taking 3,6' -digapinyl sucrose as a reference, and the specified values are as follows: 0.665 (Polygala tenuifolia Willd.)
Figure BDA0003284355360000183
Ketone III), 1.000(3,6' -di-sinapoyl sucrose), 1.183 (senegaside A), 1.326 (ginsenoside Rg)1) 1.346 (ginsenoside Re), 2.041 (beta-asarone) and 2.458 (pachymic acid).
TABLE 1815 measurement results of lots of samples
Figure BDA0003284355360000182
Figure BDA0003284355360000191
The theoretical plate number of the reference peak (3,6' -dibapinyl sucrose) was also recorded for 15 samples and the results are shown in Table 19. As can be seen from table 19, the system applicability was good.
TABLE 1815 theoretical plate number results for batches of samples
Figure BDA0003284355360000192
In conclusion, the detection method of the HPLC characteristic spectrum of the pharmaceutical preparation provided by the invention has the advantages of good precision, reproducibility and stability, accuracy and reliability, can truly reflect the quality difference of the pharmaceutical preparation, and perfects the quality control system of the pharmaceutical preparation. Therefore, the present invention overcomes various disadvantages of the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims (10)

1. A method for detecting an HPLC (high Performance liquid chromatography) characteristic spectrum of a pharmaceutical preparation comprises the following steps: dissolving a medicinal preparation sample in a solvent, performing ultrasonic extraction, cooling, shaking, filtering, collecting a sample solution obtained from a subsequent filtrate, detecting by high performance liquid chromatography, and performing attribution positioning on index components in the obtained characteristic spectrum of the sample solution according to retention time to obtain the characteristic spectrum of the medicinal preparation.
2. The method for detecting the HPLC characteristic pattern of a pharmaceutical preparation according to claim 1, wherein the obtaining of the test solution comprises any one or more of the following conditions:
A1) the ratio of the mass of the added medicinal preparation sample to the volume of the added solvent is 2: 25-75 g/mL;
A2) the solvent is selected from one of 70% methanol, ethanol or water;
A3) the ultrasonic extraction time is 30-60 minutes.
3. The method for detecting the HPLC characteristic pattern of the pharmaceutical preparation according to claim 1, wherein the detection is performed by high performance liquid chromatography, comprising the following steps:
1) preparation of reference solutions: mixing ginsenoside Rg1Ginsenoside Re, radix Polygalae
Figure FDA0003284355350000011
Adding a solvent to a reference substance of ketone III, 3,6' -dibastinyl sucrose, tenuifolin A, beta-asarone and pachymic acid for dissolving and fixing the volume to prepare a reference substance solution;
2) and (3) determination: respectively measuring the test solution and the reference solution in the step 1) by adopting a high performance liquid chromatography to obtain a characteristic spectrum of the test solution and a characteristic spectrum of the reference solution, comparing the retention time of the characteristic spectrum of the test solution with the characteristic spectrum of the reference solution for qualitative determination, and performing attribution positioning on index components in the characteristic spectrum of the test solution, thereby obtaining the characteristic spectrum of the pharmaceutical preparation.
4. The method for detecting the HPLC characteristic pattern of the pharmaceutical preparation according to claim 1, wherein in step 2), the measurement conditions of the high performance liquid chromatography are as follows: the detector is a diode array detector; the chromatographic column is C18A filler in the chromatographic column is octadecylsilane chemically bonded silica; the detection wavelength is 200-205 nm; the mobile phase is acetonitrile-0.05-0.15% phosphoric acid water solution, wherein, the phase A is acetonitrile, and the phase B is 0.05-0.15% phosphoric acid water solution; the analysis time is 102 min; gradient elution.
5. The method for detecting the HPLC characteristic pattern of the pharmaceutical preparation according to claim 4, wherein the specific procedure of the gradient elution is as follows: 0-35 min, phase A: the volume ratio of the phase B is 10: 90-21: 79; 35-53 min, phase A: the volume ratio of the phase B is 21: 79-23: 77; 53-68 min, phase A: the volume ratio of the phase B is 23: 77-45: 55; 68-78 min, phase A: the volume ratio of the phase B is 45: 55-75: 25; 78-100 min, phase A: the volume ratio of the phase B is 75: 25-95: 5; 100-102 min, phase A: the volume ratio of the phase B is 95: 5-95: 5.
6. use of a method for the detection of an HPLC profile of a pharmaceutical preparation according to any one of claims 1 to 5 for the quality control of pharmaceutical preparations.
7. A method for constructing a standard characteristic map of a pharmaceutical preparation, comprising: the method for detecting the HPLC characteristic maps of the pharmaceutical preparations according to any one of claims 1 to 5 is adopted to respectively detect a plurality of pharmaceutical preparation samples, the characteristic maps of a plurality of pharmaceutical preparations are obtained to generate a common control characteristic map, chromatographic peaks existing in the maps are used as common characteristic peaks, the relative retention time of the common characteristic peaks is determined, and the standard characteristic maps of the pharmaceutical preparations are established.
8. The method for constructing the standard feature profile of the pharmaceutical preparation according to claim 7, wherein the standard feature profile of the pharmaceutical preparation meets any one or more of the following conditions:
B1) the standard characteristic map of the pharmaceutical preparation comprises 7 common characteristic peaks, wherein the specified values of the relative retention time of the No. 2 peak serving as a reference peak S1 peak and the retention time of the No. 1 peak are 1.665, the specified values of the relative retention time of the other 6 common characteristic peaks are 1 peak, 3 peak, 4 peak, 5 peak, 6 peak and 7 peak, wherein the retention time of the No. 1 peak is 1.183, the retention time of the No. 4 peak is 1.326, the retention time of the No. 5 peak is 1.346, the retention time of the No. 6 peak is 2.041, and the specified values of the relative retention time of the 6 common characteristic peaks except the No. 2 peak have relative deviation of less than or equal to 5 percent;
B2) comparing the standard characteristic spectrum of the pharmaceutical preparation with the characteristic spectrum of a reference solution, and positioning and determining that the No. 1 peak is polygala tenuifolia
Figure FDA0003284355350000021
The peak 2 is the characteristic peak of 3,6' -erucyl sucrose, the peak 3 is the characteristic peak of tenuifolin A, and the peak 4 is ginsenoside Rg1The peak No. 5 is a characteristic peak of ginsenoside Re, the peak No. 6 is a characteristic peak of beta-asarone, and the peak No. 7 is a characteristic peak of pachymic acid.
9. A quality control method of a characteristic spectrum of a pharmaceutical preparation comprises the following steps: comparing the similarity of the relative retention time of the chromatographic peaks of the pharmaceutical preparation with the corresponding common characteristic peaks of the characteristic profile of the pharmaceutical preparation obtained by the method for detecting the characteristic profile of the pharmaceutical preparation according to any one of claims 1 to 5 with the standard characteristic profile of the pharmaceutical preparation obtained by the method for constructing the standard characteristic profile of the pharmaceutical preparation according to any one of claims 7 to 8.
10. A method for screening the characteristic maps of multiple medicinal materials in a medicinal preparation comprises the following steps:
A) preparation of a default negative sample solution: removing any 1 medicinal material from a medicinal preparation sample containing 4 medicinal materials of tuckahoe, ginseng, polygala root and acorus gramineus and preparing the medicinal preparation sample according to the same step of preparing a sample solution in the detection method of the characteristic spectrum of the medicinal preparation according to any one of claims 1 to 5 to respectively obtain 4 sample-lacking negative solutions: poria negative solution, Ginseng radix negative solution, cortex et radix Polygalae negative solution, and rhizoma Acori Graminei negative solution;
B) and (3) determination: respectively measuring the 4 sample-lacking negative solutions by using a high performance liquid chromatography under the same chromatographic conditions as in the detection method of the characteristic pattern of the pharmaceutical preparation according to any one of claims 1 to 5 to respectively obtain the characteristic patterns of the 4 sample-lacking negative solutions;
C) and (3) quality detection: comparing the characteristic maps of the 4 sample-lacking negative solutions with the standard characteristic maps of the pharmaceutical preparation established by the construction method of the standard characteristic maps of the pharmaceutical preparation according to any one of claims 7 to 8, and identifying the common characteristic peaks of the corresponding single medicinal materials in the 4 sample-lacking negative solutions in the standard characteristic maps of the pharmaceutical preparation through relative retention time, thereby performing attribution positioning on the characteristic peaks in the characteristic maps of the corresponding single medicinal materials in the 4 sample-lacking negative solutions.
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