CN113866306B - HPLC (high Performance liquid chromatography) characteristic spectrum detection method of pharmaceutical preparation - Google Patents

HPLC (high Performance liquid chromatography) characteristic spectrum detection method of pharmaceutical preparation Download PDF

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CN113866306B
CN113866306B CN202111142138.1A CN202111142138A CN113866306B CN 113866306 B CN113866306 B CN 113866306B CN 202111142138 A CN202111142138 A CN 202111142138A CN 113866306 B CN113866306 B CN 113866306B
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characteristic
peak
pharmaceutical preparation
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characteristic spectrum
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CN113866306A (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
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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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    • 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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    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
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Abstract

The invention provides a detection method of HPLC characteristic spectrum of a pharmaceutical preparation. The invention also provides an application of the HPLC characteristic spectrum detection method of the pharmaceutical preparation in the 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 pharmaceutical preparation. The HPLC characteristic spectrum detection method of 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 repeatability, can truly reflect the quality difference of the pharmaceutical preparation, and improves the quality control system of the pharmaceutical preparation.

Description

HPLC (high Performance liquid chromatography) characteristic spectrum detection method of pharmaceutical 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 characteristic spectrum of a pharmaceutical preparation.
Background
The pharmaceutical preparation is first loaded in Tangsun Si far away, qianjin Fang, volume fourteenth (small intestine): 'mainly good forgetting prescription', polygala tenuifolia, ginseng (each four parts), poria cocos (two parts) and calamus (one two parts), the upper four ingredients are treated by sieving, and the dagger and the day three are taken orally. The Chinese medicinal composition is used for treating the emotional diseases of heart qi deficiency, restlessness, anxiety, insomnia, depression, and the like, is similar to the depression of Western medicine, and is a basic prescription for improving intelligence, nourishing heart, soothing nerves and stabilizing mind in Chinese medicine. The pharmaceutical preparation is used for treating depression, anxiety and dementia in modern clinic, and the clinical manifestations of the pharmaceutical preparation are usually mental confusion, absentmindedness, anxiety, amnesia, insomnia, palpitation, severe palpitation and the like.
Because of the lack of a method for evaluating the whole pharmaceutical preparation, a corresponding HPLC characteristic spectrum method is necessary to be established, the pharmaceutical preparation is subjected to quality control, and the quality control system of the pharmaceutical preparation is perfected.
Disclosure of Invention
In view of the above drawbacks 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 adopting pretreatment under optimized conditions and a high performance liquid chromatography method, and can analyze 7 chemical components in the pharmaceutical preparation, so as to solve the problem of lack of an overall evaluation method for the pharmaceutical preparation in the prior art, and comprehensively perfect a quality control system of the pharmaceutical preparation.
To achieve the above and other related objects, a first aspect of the present invention provides a method for detecting HPLC profile of a pharmaceutical preparation, comprising: adding a medicinal preparation sample into a solvent for dissolution, performing ultrasonic extraction, cooling, shaking, filtering, taking a sample solution obtained from the subsequent filtrate, detecting by adopting a high performance liquid chromatography, and performing attribution positioning on index components in the characteristic spectrum of the obtained sample solution according to the retention time, thereby obtaining the characteristic spectrum of the medicinal preparation.
Preferably, the ratio of the mass of the drug preparation sample addition to the volume of the solvent addition is 2:25-75, g/mL. Preferably, the ratio of the mass of the drug formulation sample addition to the volume of solvent addition is 2:50, g/mL.
Preferably, the solvent is selected from 70% methanol, ethanol or water. Preferably, the solvent is 70% methanol. The 70% methanol is an aqueous solution of 70% methanol by volume.
Preferably, the time of the 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 filtering is to take supernatant of the solution after shaking up and filter the supernatant with a filter membrane.
Preferably, the filter membrane is a 0.22 μm filter membrane.
Preferably, the subsequent filtrate is obtained by discarding the primary filtrate after filtration by a filter membrane.
Preferably, the detection by high performance liquid chromatography comprises the following steps:
1) Preparation of reference solution: ginsenoside Rg 1 Ginsenoside Re and polygala tenuifolia
Figure BDA0003284355360000021
The reference substances of ketone III, 3,6' -sinapioyl sucrose, tenuifolin A, beta-asarone and pachymic acid are added with a solvent for dissolution and volume fixation to prepare a reference substance solution;
2) And (3) measuring: and (2) respectively measuring the sample solution and the reference solution in the step (1) by adopting a High Performance Liquid Chromatography (HPLC) method, obtaining a characteristic spectrum of the sample solution and a characteristic spectrum of the reference solution, comparing the retention time of the characteristic spectrum of the sample solution and the characteristic spectrum of the reference solution, and qualitatively locating index components in the characteristic spectrum of the sample solution, thereby obtaining the characteristic spectrum of the pharmaceutical preparation.
Preferably, in step 1), the ginsenoside Rg 1 Is of CAS number 22427-39-0, ginsenoside Re is of CAS number 52286-59-6, and cortex et radix Polygalae
Figure BDA0003284355360000022
The CAS number of ketone III is 162857-78-5, 3,6' -sinapoyl sucrose is 139891-98-8, tenatoxin A is 139726-35-5, beta-asarone is 5273-86-9, and pachymic acid is 29070-92-6.
The medicine preparation is brown yellow powder and consists of 4 traditional Chinese medicines of ginseng, polygala tenuifolia, grassleaved sweetflag rhizome and poria cocos. The main active ingredients in Ginseng radix are ginsenoside Re and ginsenoside Rg 1 Is suitable for being used as a standard for measuring ginsenoside. The chemical components of polygala tenuifolia serving as traditional nootropic agents mainly comprise triterpenoid saponins, ketones, oligosaccharide lipids, alkaloids and the like. Wherein 3,6' -sinapiyl sucrose is an anti-depression effective component in the blood serum of the yippee powder. Radix Polygalae
Figure BDA0003284355360000023
Ketone III, 3,6' -sinapioyl sucrose, tenuifolin A, represent ketones, oligosaccharides lipids, triterpene saponins, respectively, of Polygala tenuifolia. Poria mainly contains pachyman, triterpenic acid, gum, protein, sterol, fatty acid, etc., and pachyman acid is representative component of Poria. The rhizoma acori graminei medicinal material has high volatile oil content, and the main component is beta-asarone (0.705-1.53%), so that the rhizoma acori graminei medicinal material is an important basis for playing pharmacological roles. Studies show that the beta-asarone has the functions of treating dementia and resisting depression. Therefore, the invention selects the ginsenoside Rg 1 Ginsenoside Re, polygala tenuifolia->
Figure BDA0003284355360000024
Ketone III, 3,6' -sinapioyl sucrose, tenuifolin A, pachymic acid, and beta-asarone are common peaks of characteristic patterns.
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 an aqueous solution of 70% methanol by volume.
Preferably, in step 1), the reference solution is prepared by stepwise dilution.
Preferably, in step 1), ginsenoside Rg in the reference solution 1 Ginsenoside Re and polygala tenuifolia
Figure BDA0003284355360000025
The concentration range of ketone III, 3,6' -sinapioyl sucrose, tenuifolin A, beta-asarone and poria acid is 10-100 mug/mL.
Preferably, in step 2), the detector used in the High Performance Liquid Chromatography (HPLC) is a Diode Array Detector (DAD).
Preferably, in step 2), the chromatographic column in the high performance liquid chromatography is C 18 The filler in the chromatographic column is octadecylsilane chemically bonded silica.
More preferably, the chromatographic column in the high performance liquid chromatography is selected from ZORBAX SB-C 18 Chromatographic column(4.6×250mm，5μm)、ORBAX Eclipse XDB-C 18 Chromatographic column (4.6X250 mm,5 μm), diamond C 18 One of the columns (4.6X250 mm,5 μm).
Further preferably, the chromatographic column in the high performance liquid chromatography is ZORBAX SB-C 18 Chromatographic column (4.6X250 mm,5 μm).
Preferably, in step 2), the detection wavelength in the high performance liquid chromatography is 200-205nm, preferably 203nm.
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 the step 2), the flow rate in the high performance liquid chromatography is 0.9-1.1mL/min. More preferably, the flow rate in the high performance liquid chromatography is 1.0mL/min.
Preferably, in step 2), the sample injection amount in the high performance liquid chromatography is 5-20 μl. More preferably, the sample injection amount in the high performance liquid chromatography is 10 μl.
Preferably, in the step 2), in the high performance liquid chromatography, the mobile phase is acetonitrile-0.05-0.15% phosphoric acid aqueous solution, wherein the phase A is acetonitrile and the phase B is 0.05-0.15% phosphoric acid aqueous solution; the analysis time is 102min; gradient elution.
More preferably, in the high performance liquid chromatography, the mobile phase is acetonitrile-0.10% phosphoric acid aqueous solution, wherein the A phase is acetonitrile and the B phase is 0.10% phosphoric acid aqueous solution; the analysis time is 102min; gradient elution.
The 0.05-0.15% phosphoric acid aqueous solution is 0.05-0.15% phosphoric acid aqueous solution by volume percent. The 0.10 phosphoric acid aqueous solution is a phosphoric acid aqueous solution with a volume percentage of 0.10.
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, a step of selecting a specific type of material;
78-100 min, phase A: the volume ratio of the phase B is 75:25-95:5, a step of;
100-102 min, phase A: the volume ratio of the phase B is 95:5-95:5.
the second aspect of the invention provides the use of a method for detecting the HPLC characteristic spectrum of a pharmaceutical preparation in the quality detection of the pharmaceutical preparation.
The pharmaceutical formulation includes, but is not limited to, pistachio powder and the like.
The third aspect of the invention provides a method for constructing a standard characteristic map of a pharmaceutical preparation, comprising: the HPLC characteristic spectrum detection method of the pharmaceutical preparation is adopted to detect a plurality of pharmaceutical preparation samples respectively, a plurality of characteristic spectrums of the pharmaceutical preparation 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 pharmaceutical preparation is established.
Preferably, the profile generation method of the characteristic profile of the pharmaceutical preparation adopts an average method. The average method is one of the common mode methods.
Specifically, when a batch of fingerprint samples are obtained, similarity analysis and evaluation are required for the fingerprint samples, so that the fingerprint samples can be used for quality control of traditional Chinese medicines. In the similarity evaluation process, a control fingerprint needs to be generated. The current methods for generating the comparison fingerprint mainly comprise a typical fingerprint selection method and a common mode method. The typical fingerprint selection method selects a fingerprint with typical meaning or representation as a reference fingerprint. However, the control fingerprint thus generated does not contain the overall information of the fingerprint sample, but is merely an individual feature of the sample, and thus may not necessarily represent the overall feature of the fingerprint sample well. And the selection of the typical fingerprint has a certain subjectivity. The common mode method includes two methods, namely a mean method and a median method. The comparison fingerprint generated by the two methods contains sample information of the original fingerprint. If no unusual samples are present in the batch, it is generally recommended to use the average method.
Preferably, the standard characteristic spectrum of the pharmaceutical preparation is constructed, and the width of the time window for matching the common characteristic peak is 0.1.
Preferably, the standard characteristic pattern of the pharmaceutical preparation comprises 7 common characteristic peaks, the reference peak (S1 peak, retention time 1.000) is the peak 1 (0.665), the peak 3 (1.183), the peak 4 (1.326), the peak 5 (1.346), the peak 6 (2.041) and the peak 7 (2.458) are the relative deviation of the relative retention time of the other 6 common characteristic peaks, and the relative deviation of the relative retention time of the 6 common characteristic peaks except the peak 2 is less than or equal to + -5%.
Preferably, the standard characteristic spectrum of the pharmaceutical preparation is compared with the characteristic spectrum of the reference solution, and the peak 1 is determined to be polygala tenuifolia by positioning
Figure BDA0003284355360000041
A characteristic peak of ketone III, wherein the No. 2 peak is a characteristic peak of 3,6' -sinapiyl sucrose, the No. 3 peak is a characteristic peak of tenuifolin A, and the No. 4 peak is ginsenoside Rg 1 The No. 5 peak is the characteristic peak of ginsenoside Re, the No. 6 peak is the characteristic peak of beta-asarone, and the No. 7 peak is the characteristic peak of pachymic acid.
The fourth aspect of the present invention provides a quality control method for a characteristic spectrum of a pharmaceutical preparation, comprising: the characteristic spectrum of the pharmaceutical preparation obtained by adopting the detection method of the characteristic spectrum of the pharmaceutical preparation is compared with the standard characteristic spectrum of the pharmaceutical preparation obtained by adopting the construction method of the standard characteristic spectrum of the pharmaceutical preparation, and the similarity of the relative retention time of the chromatographic peak of the pharmaceutical preparation and the corresponding common characteristic peak is compared.
According to the technical guidelines of analysis and detection of Chinese pharmacopoeia, the characteristic spectrum does not require the comprehensive evaluation of the similarity of the spectrum as the fingerprint spectrum, and is mainly characterized in that the specific components of the characteristic spectrum of the variety and other varieties are highlighted to be used as an important identification means for controlling the quality of the Chinese medicine.
The fifth aspect of the present invention provides a screening method for characteristic patterns of a plurality of medicinal materials in a pharmaceutical preparation, comprising the steps of:
a) Preparation of sample-deficient negative sample solution: removing any 1 medicinal material from medicinal preparation samples containing 4 medicinal materials including poria cocos, ginseng, polygala tenuifolia and grassleaf sweelflag rhizome respectively, preparing the medicinal preparation samples according to the same steps of preparing sample solutions in a detection method of a characteristic spectrum of the medicinal preparation, and respectively obtaining 4 sample deficiency negative solutions: poria cocos negative solution, ginseng negative solution, polygala tenuifolia negative solution and rhizoma acori graminei negative solution;
b) And (3) measuring: respectively measuring 4 sample-lacking negative solutions by adopting a High Performance Liquid Chromatography (HPLC) method under the same chromatographic condition as that in the detection method of the characteristic spectrum of the pharmaceutical preparation, and respectively obtaining the characteristic spectrum of the 4 sample-lacking negative solutions;
c) And (3) quality detection: and comparing the characteristic patterns of the 4 sample-lacking negative solutions with the standard characteristic patterns of the medicinal preparation established by the construction method of the standard characteristic patterns of the medicinal preparation, and identifying common characteristic peaks of corresponding single medicinal materials in the 4 sample-lacking negative solutions in the standard characteristic patterns of the medicinal preparation through relative retention time, so as to carry out attribution positioning on the characteristic peaks in the characteristic patterns of the corresponding single medicinal materials in the 4 sample-lacking negative solutions.
The Poria is dry sclerotium of Polyporaceae fungus Poria cocos (Schw.) Cocos (Wolf).
The Ginseng radix is root of Panax ginseng C.A. Meyer of Araliaceae.
The radix Polygalae is root of Polygala tenuifolia Polygala tenuifolia Willd of Polygalaceae
The rhizoma Acori Graminei is rhizome of rhizoma Acori Graminei Acorus tatarinowii Schott of Araceae.
Preferably, in the step C), the common characteristic peaks of the standard characteristic spectrum of the pharmaceutical preparation and the characteristic spectrum of the corresponding single medicinal material in the 4 sample-lacking negative solutions are respectively located in a belonging way, 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 grassleaf sweelflag rhizome, and the No. 7 peak is derived from poria cocos. The water used in the invention is pure water.
As described above, the HPLC characteristic spectrum detection method of the pharmaceutical preparation provided by the invention adopts pretreatment with optimized conditions and an 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, realize the quality control of the characteristic spectrum of the medicinal preparation, monitor the production of the medicinal preparation, identify the authenticity of the medicinal preparation and the like. The method has the following advantages:
(1) In the method for detecting the HPLC characteristic spectrum of the pharmaceutical preparation, provided by the invention, in the process of establishing the HPLC characteristic spectrum measuring method of the pharmaceutical preparation, the precision, stability and repeatability test, investigation and research are carried out by taking the 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 method for detecting the HPLC characteristic spectrum of the pharmaceutical preparation, disclosed by the invention, as 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 that characteristic peaks are difficult to separate and impurity peaks are interfered are solved.
(3) The HPLC characteristic spectrum detection method of the pharmaceutical preparation provided by the invention takes the characteristic spectrum of each active ingredient in the pharmaceutical preparation as a whole to be regarded, and focuses on the front-back sequence and the interrelation of each characteristic peak, thereby avoiding the one-sided performance of judging the whole quality of the pharmaceutical preparation by only measuring a small amount of ingredients, and reducing the possibility of artificial treatment for reaching the quality standard.
(4) The HPLC characteristic spectrum detection method of the pharmaceutical preparation 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 production process and the quality stability between batches, can comprehensively and scientifically evaluate the quality of the pharmaceutical preparation, ensures the quality and the curative effect of the product, provides reference for the improvement of the quality standard of the pharmaceutical preparation, and comprehensively improves the quality control system of the pharmaceutical preparation.
Drawings
FIG. 1 shows a characteristic map of the precision results of the pharmaceutical preparation of the present invention.
FIG. 2 shows evaluation patterns of the durability of different chromatographic columns of the pharmaceutical preparation according to the invention.
FIG. 3 shows a plot of similarity scores for different flow rate durability studies for the pharmaceutical formulations of the present invention.
FIG. 4 shows a graph of similarity evaluation for different column temperature durability studies of the pharmaceutical formulation of the present invention.
Fig. 5 shows a plot of similarity evaluation for different pH durability studies for the pharmaceutical formulation of the present invention.
Fig. 6 shows a graph of similarity evaluation for durability studies of different instruments of the pharmaceutical preparation of the present invention.
Fig. 7 shows a characteristic spectrum of the stability results of the pharmaceutical preparation of the present invention.
FIG. 8 shows a characteristic map of the repetitive results of the pharmaceutical formulation of the present invention.
FIG. 9 shows a standard characteristic spectrum of the pharmaceutical preparation of the present invention, wherein 1 is polygala tenuifolia
Figure BDA0003284355360000061
Ketone III, 2 is 3,6' -sinapioyl sucrose, 3 is tenuifolin A,4 is ginsenoside Rg 1 5 is ginsenoside Re,6 is beta-asarone and 7 is pachymic acid.
FIG. 10 shows a specific chromatogram of each drug in the drug formulation of the present invention, wherein A is a sample solution; b is a reference solution; c is Poria cocos negative solution; d is a ginseng negative solution; e is polygala tenuifolia negative solution: f is rhizoma Acori Graminei negative solution.
Detailed Description
The invention is further illustrated below in connection with specific examples, which are to be understood as being illustrative of the invention and not limiting the scope of the invention.
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
The reagents and instrumentation used in the following examples were as follows:
1. reagent(s)
The 15 batches of the pharmaceutical formulation samples were specifically Happy powder (manufactured by Shanghai and Huang pharmaceutical industries, ltd.) as shown in Table 1 below.
TABLE 1
Numbering device Lot number Numbering device Lot 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
Radix Polygalae
Figure BDA0003284355360000071
Ketone III (Polygalaxanthine III) control (NATURE STANDARD, ST 06290120); 3,6 '-sinapiyl sucrose (3, 6' -disinapoyl sucrose) control (China food and drug verification institute, 111848-202006ID: QNVP-D9 TF); tenuifolin A (tenuifoliside A) control (Shanghai achievements Yi Biotechnology Co., ltd., P16A9F 58918); ginsenoside Rg 1 (Ginsenoside Rg 1 ) Reference (national food and drug verification institute, 110703-201933ID: TWYY-M6 VC); ginsenoside Re (Ginsenoside Re) reference (national food and drug administration institute,110754-202028ID: ZR2T-SE 40); beta-asarone (beta-asarone) reference (China food and drug institute, 112018-201802ID: N7Q2-LG 99); poria acid (Pachymic acid) control (Chengdu Pure technology development Co., ltd., lot number PRF 9090303).
Acetonitrile (chromatographic purity, manufacturer: fisher chemical, U.S., purchaser: national pharmaceutical systems chemical, inc., lot number 204199); methanol, phosphoric acid (analytical grade, manufacturer: CNW technologies, purchaser: national pharmaceutical group chemical reagent Co., ltd., lot number: J5640045); ultrapure water (prepared by a pure water meter).
2. Instrument for measuring and controlling the intensity of light
Agilent 1260 ii high performance liquid chromatograph (Agilent, quaternary pump, usa); waters E2695 high performance liquid chromatograph (Waters, usa, quaternary pump); BT25S electronic analytical balance (beijing cerdolis corporation, china); BSA124S-CW electronic analytical balance (Beijing Sidoris Corp., china); KQ-250DB type ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd., china); ZP15D1 type ultrasonic pure water apparatus (Shanghai right instruments, limited, china).
ZORBAX SB-C 18 Chromatographic column (4.6X250 mm,5 μm) (Agilent, USA); ORBAX Eclipse XDB-C 18 Chromatographic column (4.6X250 mm,5 μm) (Agilent, USA); diamond C 18 Chromatographic column (4.6X250 mm,5 μm) (Di Ma technology, china).
The method for detecting the HPLC characteristic spectrum of the pharmaceutical preparation comprises the following determination process.
1. Preparation of test solutions
Taking a medicinal preparation sample, precisely weighing, adding a solvent for dissolution, wherein the ratio of the added mass of the medicinal preparation sample to the added volume of the solvent is 2:25-75, g/mL, sealing, performing ultrasonic treatment for 30-60 minutes, cooling to room temperature, shaking uniformly, taking supernatant fluid, and filtering with a 0.22 mu m filter membrane, and taking subsequent filtrate to obtain the sample solution. The solvent is selected from 70% methanol, ethanol or water.
2. Preparation of reference solutions
Ginsenoside Rg 1 Ginsenoside Re and polygala tenuifolia
Figure BDA0003284355360000081
The reference substances of ketone III, 3,6' -sinapioyl sucrose, tenuifolin A, beta-asarone and pachymic acid are added into a solvent for dissolution and volume fixation to prepare a reference substance solution. The solvent is selected from 70% methanol, ethanol or water.
3. Measurement
And respectively measuring the sample solution and the reference solution by adopting a High Performance Liquid Chromatography (HPLC) method, obtaining a characteristic spectrum of the sample solution and a characteristic spectrum of the reference solution, comparing the retention time of the characteristic spectrum of the sample solution and the characteristic spectrum of the reference solution, performing qualitative determination, and performing attribution positioning on index components in the characteristic spectrum of the sample solution, thereby obtaining 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 C 18 The filler in the chromatographic column is octadecylsilane chemically bonded silica gel, and is specifically selected from ZORBAX SB-C 18 Chromatographic columns (4.6X250 mm,5 μm), ORBAX Eclipse XDB-C 18 Chromatographic column (4.6X250 mm,5 μm), diamond C 18 One of the chromatographic columns (4.6X250 mm,5 μm); the detection wavelength is 200-205nm; the column temperature is 28-32 ℃; the flow rate is 0.9-1.1mL/min; the sample injection amount is 5-20 mu L; the mobile phase is acetonitrile-0.05-0.15% phosphoric acid aqueous solution, wherein the A phase is acetonitrile, and the B phase is 0.05-0.15% phosphoric acid aqueous solution; the analysis time is 102min; gradient elution.
The specific procedure of 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, a step of selecting a specific type of material;
78-100 min, phase A: the volume ratio of the phase B is 75:25-95:5, a step of;
100-102 min, phase A: the volume ratio of the phase B is 95:5-95:5.
the HPLC characteristic spectrum detection method of the pharmaceutical preparation is adopted to detect a plurality of pharmaceutical preparation samples respectively, a plurality of characteristic spectrums of the pharmaceutical preparation 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 pharmaceutical preparation is established.
The standard characteristic pattern of the pharmaceutical preparation comprises 7 common characteristic peaks, wherein the peak No. 2 is taken as a reference peak (S1 peak, retention time is 1.000), the relative retention time of other 6 common characteristic peaks is sequentially defined as peak No. 1 (0.665), peak No. 3 (1.183), peak No. 4 (1.326), peak No. 5 (1.346), peak No. 6 (2.041) and peak No. 7 (2.458), and the relative deviation of the relative retention time of the 6 common characteristic peaks except the peak No. 2 is less than or equal to +/-5%.
Comparing the standard characteristic spectrum of the pharmaceutical preparation with the characteristic spectrum of the reference solution, and locating and determining that the No. 1 peak is polygala tenuifolia
Figure BDA0003284355360000091
A characteristic peak of ketone III, wherein the No. 2 peak is a characteristic peak of 3,6' -sinapiyl sucrose, the No. 3 peak is a characteristic peak of tenuifolin A, and the No. 4 peak is ginsenoside Rg 1 The No. 5 peak is the characteristic peak of ginsenoside Re, the No. 6 peak is the characteristic peak of beta-asarone, and the No. 7 peak is the characteristic peak of pachymic acid.
Removing any 1 medicinal material from medicinal preparation samples containing 4 medicinal materials including poria cocos, ginseng, polygala tenuifolia and grassleaf sweelflag rhizome respectively, preparing the medicinal preparation samples according to the same steps of preparing sample solutions in a detection method of a characteristic spectrum of the medicinal preparation, and respectively obtaining 4 sample deficiency negative solutions: poria cocos negative solution, ginseng negative solution, polygala tenuifolia negative solution and rhizoma acori graminei negative solution. And then adopting a High Performance Liquid Chromatography (HPLC) method with the same chromatographic conditions as the detection method of the characteristic spectrum of the pharmaceutical preparation to respectively detect 4 sample-lacking negative solutions, and respectively obtaining the characteristic spectrum of the 4 sample-lacking negative solutions. And comparing the characteristic patterns of the 4 sample-lacking negative solutions with the standard characteristic patterns of the medicinal preparation established by the construction method of the standard characteristic patterns of the medicinal preparation, and identifying common characteristic peaks of corresponding single medicinal materials in the 4 sample-lacking negative solutions in the standard characteristic patterns of the medicinal preparation through relative retention time, so as to carry out attribution positioning on the characteristic peaks in the characteristic patterns of the corresponding single medicinal materials in the 4 sample-lacking negative solutions.
As shown in fig. 10, in the standard characteristic spectrum of the pharmaceutical preparation, the common characteristic peak attribution positioning of the characteristic spectrum of the corresponding single medicinal material in the 4 sample-lacking negative solutions is that the 1, 2 and 3 peaks are derived from polygala tenuifolia respectively; the No. 4 and No. 5 peaks are derived from ginseng, the No. 6 peak is derived from grassleaf sweelflag rhizome, and the No. 7 peak is derived from poria cocos.
Example 1
1. Preparation of test solutions
Taking 2.0g of a medicine preparation sample, precisely weighing, placing into a conical flask with a plug, precisely adding 50ml of methanol, sealing, performing ultrasonic treatment (350 w and 53 kHz) for 30 minutes, cooling to room temperature, shaking uniformly, taking supernatant, and filtering with a 0.22 mu m filter membrane to obtain subsequent filtrate, namely sample solution No. 1.
2. Preparation of reference solutions
Ginsenoside Rg 1 Ginsenoside Re and polygala tenuifolia
Figure BDA0003284355360000101
The reference substances of ketone III, 3,6' -sinapioyl sucrose, tenuifolin A, beta-asarone and pachymic acid are precisely weighed, methanol is added for constant volume, and the mixed reference substance solution 1# with mass concentration of 30, 20, 70, 20, 100 and 10 mug/mL is prepared.
3. Measurement
And (3) respectively detecting the sample solution 1# and the reference solution 1# by adopting a High Performance Liquid Chromatography (HPLC), obtaining a characteristic spectrum of the sample solution 1# and a characteristic spectrum of the reference solution 1#, comparing the retention time of the characteristic spectrum of the sample solution 1# with that of the reference solution 1# for qualitative determination, and carrying out attribution positioning on index components in the characteristic spectrum of the sample solution 1# so as 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 ZORBAX SB-C 18 Chromatographic column (4.6X250 mm,5 μm); the detection wavelength is 203nm; the column temperature is 30 ℃; the flow rate is 1.0mL/min; the sample injection amount is 10 mu L; the mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, wherein the A phase is acetonitrile, and the B phase is 0.1% phosphoric acid aqueous solution; the analysis time is 102min; gradient elution.
The specific procedure of 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, a step of selecting a specific type of material;
78-100 min, phase A: the volume ratio of the phase B is 75:25-95:5, a step of;
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, a plurality of characteristic patterns of the medicinal preparation samples are obtained to generate a common control characteristic pattern, chromatographic peaks existing in the patterns are used as common characteristic peaks, the relative retention time of the common characteristic peaks is determined, and a standard characteristic pattern 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 reference peak (S1 peak, retention time 1.000) is 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) in this order.
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 1 is determined to be polygala tenuifolia by positioning
Figure BDA0003284355360000102
A characteristic peak of ketone III, wherein the No. 2 peak is a characteristic peak of 3,6' -sinapiyl sucrose, the No. 3 peak is a characteristic peak of tenuifolin A, and the No. 4 peak is ginsenoside Rg 1 The No. 5 peak is the characteristic peak of ginsenoside Re, the No. 6 peak is the characteristic peak of beta-asarone, and the No. 7 peak is the characteristic peak of pachymic acid.
Example 3
Sample A15 (lot 2020071402) was taken and a test solution was prepared in step 1 of example 1. Meanwhile, a reference solution was prepared as in step 2 of example 1, in which polygala tenuifolia
Figure BDA0003284355360000111
The content of ketone III is 21.23 mug/mL, the content of 3,6' -sinapioyl sucrose is 72.19 mug/mL, the content of tenuifolin A is 45.71 mug/mL, and the ginsenoside Rg 1 The content of the ginsenoside Re is 28.09 mug/mL, the content of the ginsenoside Re is 27.26 mug/mL, the content of the beta-asarone is 143 mug/mL, and the content of the pachymic acid is 5.78 mug/mL.
Removing any 1 medicinal material from medicinal preparation samples containing 4 medicinal materials including poria cocos, ginseng, polygala tenuifolia and grassleaf sweelflag rhizome, preparing according to the step 1) of the detection method of the characteristic spectrum of the medicinal preparation in the embodiment 1, and obtaining 4 sample-lack negative solutions respectively: poria cocos negative solution, ginseng negative solution, polygala tenuifolia negative solution and rhizoma acori graminei negative solution.
The sample solution, the reference solution, and the 4 sample-deficient negative solutions were measured by a High Performance Liquid Chromatography (HPLC) method under the same chromatographic conditions as in step 3) of the method for detecting a characteristic spectrum of a pharmaceutical preparation of example 1, respectively, to obtain a characteristic spectrum of the sample solution, a characteristic spectrum of the reference solution, and a characteristic spectrum of the 4 sample-deficient negative solutions.
The characteristic pattern of the test solution was set up as in example 2 to establish a standard characteristic pattern of the pharmaceutical preparation. And comparing the characteristic patterns of the 4 sample-lacking negative solutions with the characteristic patterns of the standard characteristic patterns of the medicinal preparation and the characteristic patterns of the reference solution, and identifying common characteristic peaks of corresponding single medicinal materials in the 4 sample-lacking negative solutions in the standard characteristic patterns of the medicinal preparation through relative retention time, so as to carry out attribution positioning on the characteristic peaks in the characteristic patterns 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 comparison of the radix Polygalae negative control pattern with the mixed label and the sample shows that
Figure BDA0003284355360000112
The three index components of ketone III, 3,6' -sinapioyl sucrose and tenuifolin A only exist in the polygala tenuifolia medicinal material, and the specificity is good. After the negative control spectrum of ginseng is compared with the mixed standard and the sample, the result shows that two index components of ginsenoside Rg1 and ginsenoside Re only exist in the ginseng medicinal material, and the specificity is good. After the negative control spectrum of the grassleaf sweelflag rhizome is compared with the mixed standard and the sample, the result shows that the beta-asarone component only exists in the grassleaf sweelflag rhizome medicinal material, and the specificity is good. After the poria negative control spectrum is compared with the mixed standard and the sample, the result shows that the poria acid component only exists in the poria medicinal material, and the specificity is good.
Example 4
The HPLC characteristic spectrum detection method of the pharmaceutical preparation provided by the invention is subjected to methodological verification, and the performance index results are as follows.
1. Precision of
Sample A15 (lot number 2020071402) of the same pharmaceutical preparation was taken, a sample solution was prepared in accordance with step 1 of example 1, the sample was continuously introduced and analyzed 6 times a day under the chromatographic conditions of step 3 of example 1, the relative retention time of each peak was calculated with 3,6' -disjuncyl sucrose as a reference peak, and the results are shown in Table 2. As can be seen from Table 2, the relative retention time of each characteristic peak was less than 5% RSD.
TABLE 2 results of precision experiments
Figure BDA0003284355360000121
The obtained characteristic spectrum is imported into a traditional Chinese medicine chromatographic 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 time window width is set to be 0.1, multipoint 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 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 6 sample injection results is 1, which indicates that the instrument precision is good.
TABLE 3 precision similarity results
S1 S2 S3 S4 S5 S6 Contrast characteristic spectrum 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
Contrast characteristic spectrum R 1 1 1 1 1 1 1
2. Stability of
Sample A15 (lot number 2020071402) of the same pharmaceutical preparation was taken, a sample solution was prepared according to step 1 in example 1, sample analysis was performed according to the chromatographic conditions of step 3 in example 1, which were different from those of 0h, 2h, 4h, 8h, 12h and 24h, the relative retention time of each characteristic peak was calculated by using 3,6' -sinapiyl sucrose as a reference peak, and the specific stability results were shown in Table 4. The results show that the RSD relative to the retention time is less than 5%.
TABLE 4 stability test results
Figure BDA0003284355360000131
The obtained characteristic spectrum is imported into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), the characteristic spectrum of the analysis result S2 of the pharmaceutical preparation sample A15 in 2h is set as a reference spectrum, the time window width is set to be 0.1, multipoint 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 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 patterns at 6 time points is 1, which indicates that the sample is stable within 24 hours, and the method has good stability.
TABLE 5 stability similarity results
S1 S2 S3 S4 S5 S6 Contrast characteristic spectrum 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
Contrast characteristic spectrum R 1 1 1 1 1 1 1
3. Repeatability of
The same pharmaceutical preparation sample A15 (batch No. 2020071402) was taken, 6 parts of test solutions were prepared in parallel according to step 1 of example 1, the respective sample was taken and analyzed under the chromatographic conditions of step 3 of example 1, the relative retention time of each peak was calculated by taking 3,6' -sinapiyl sucrose as a reference peak, and the specific results of reproducibility are shown in Table 6. The results show that the relative retention time of each characteristic peak has an RSD of less than 5%.
TABLE 6 results of repeatability experiments
Figure BDA0003284355360000141
The obtained characteristic spectrum is imported into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), the characteristic spectrum of the 2 nd sample solution analysis result S2 of the pharmaceutical preparation sample A15 is set as a reference spectrum, the time window width is set as 0.1, multipoint 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 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 characteristic patterns of the 6 samples is 1, which indicates that the method has good repeatability.
TABLE 7 repeatability similarity results
S1 S2 S3 S4 S5 S6 Contrast characteristic spectrum 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
Contrast characteristic spectrum R 1 1 1 1 1 1 1
4. Durability of
4.1 different chromatographic column investigation
Sample A15 (lot 2020071402) of the same pharmaceutical preparation was prepared as in step 1 of example 1 to obtain a sample solution, which was subjected to sample analysis as in step 3 of example 1. Comparing ZORBAX SB-C columns of different manufacturers 18 (4.6×250mm，5μm，P.N.880975-902)、ZORBAX Eclipse XDB-C 18 (4.6×250mm，5μm，P.N.990967-902)、Diamonsil C 18 (4.6X105 mm,5 μm, cat.no. 99603, ser.no. 201061838). The relative retention time of each peak was calculated using 3,6' -sinapiyl sucrose as reference peak and the results are shown in Table 8. The results show that the relative retention time of each characteristic peak has an RSD of less than 5%.
TABLE 8 relative retention time tables for different chromatographic columns
Figure BDA0003284355360000151
Introducing the obtained characteristic spectrum into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), setting the characteristic spectrum of an analysis result S1 of an SB-C18 chromatographic column as a reference spectrum, setting the width of a time window as 0.1, performing multipoint correction, performing Mark peak matching, and generating a reference characteristic spectrum R by an average method, wherein 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, the different chromatographic columns have little influence on the detection result, the similarity is 1, and the durability is good.
TABLE 9 similarity results for different chromatographic columns
SB-C 18 Diamonsil C 18 XDB-C 18 Reference fingerprint R
SB-C 18 (S1) 1 1 1 1
Diamonsil C 18 (S2) 1 1 1 1
XDB-C 18 (S3) 1 1 1 1
Contrast characteristic spectrum R 1 1 1 1
4.2 different flow Rate investigation
Sample A15 (lot number 2020071402) of the same pharmaceutical preparation was taken, a sample solution was prepared in accordance with step 1 of example 1, the measurement was carried out at flow rates of 0.9ml/min, 1.0ml/min and 1.1ml/min under the chromatographic conditions of step 3 of example 1, the changes of the chromatographic peaks at different flow rates were compared, and the relative retention time of each characteristic peak was calculated by using 3,6' -sinapiyl sucrose as a reference peak, and the results are shown in Table 10. The results show that the relative retention time of each characteristic peak has an RSD of less than 5%.
TABLE 10 relative retention time table for different flow rates
Figure BDA0003284355360000152
The obtained characteristic spectrum is imported into a traditional Chinese medicine chromatographic 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, multipoint 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 3. The similarity was calculated and the results are shown in Table 11. As is clear from FIG. 3 and Table 11, the tests were carried out at 0.9 to 1.1ml/min, and the degree of similarity was 1, so that the durability was good.
TABLE 11 different flow rate similarity results
1.0ml/min 0.9ml/min 1.1ml/min Contrast characteristic spectrum 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
Contrast characteristic spectrum R 1 1 1 1
4.3 different column temperature investigation
Sample A15 (lot number 2020071402) of the same pharmaceutical preparation was prepared in the same manner as in step 1 of example 1 to obtain a sample solution, and the changes in the chromatographic peaks at different column temperatures were compared by measuring the same conditions of the chromatography in step 3 of example 1 at 28℃and 30℃and 32 ℃. The relative retention time of each peak was calculated using 3,6' -sinapiyl sucrose as a reference peak and the results are shown in Table 12. The results show that the relative retention time of each characteristic peak has an RSD of less than 5%.
TABLE 12 relative retention time Table for different column temperatures
Figure BDA0003284355360000161
The obtained characteristic spectrum is imported into a traditional Chinese medicine chromatographic 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 time window width is set as 0.1, multipoint 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 is clear from FIG. 4 and Table 13, the tests were carried out at 28 to 32℃and the similarity was 1, so that the durability was good.
TABLE 13 results of similarity between column temperatures
30℃ 28℃ 32℃ Contrast characteristic spectrum R
30℃(S1) 1 1 1 1
28℃(S2) 1 1 1 1
32℃(S3) 1 1 1 1
Contrast characteristic spectrum R 1 1 1 1
4.4 investigation of the different phosphoric acid ratios
Sample A15 (lot 2020071402) of the same pharmaceutical preparation was prepared in the same manner as in step 1 of example 1 to obtain a sample solution, and the chromatographic peak changes at different pH values were compared by measuring the sample solution using 0.05% phosphoric acid, 0.1% phosphoric acid and 0.15% phosphoric acid as mobile phases under the chromatographic conditions of step 3 of example 1. The relative retention time of each characteristic peak was calculated using 3,6' -sinapiyl sucrose as a reference peak and the results are shown in Table 14. The results show that the relative retention time of each characteristic peak has an RSD of less than 5%.
TABLE 14 relative retention time Table for different pH values
Figure BDA0003284355360000171
The obtained characteristic spectrum is imported into a traditional Chinese medicine chromatographic 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 time window width is set as 0.1, multipoint 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 5. The similarity was calculated and the results are shown in Table 15. As is clear 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, so that the durability was good.
TABLE 15 similarity results for different pH' s
0.1% phosphoric acid 0.15% phosphoric acid 0.05% phosphoric acid Contrast characteristic spectrum 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
Contrast characteristic spectrum R 1 1 1 1
4.5 different liquid chromatography instruments
Sample A15 (lot 2020071402) of the same pharmaceutical preparation was taken, a sample solution was prepared according to step 1 of example 1, and two Agilent definition 1260 II devices of different manufacturers were compared for detection according to the chromatographic conditions of step 3 of example 1, wherein Agilent definition 1260 II was located at 2 different sites of the liquid phase chamber and the standardization center, respectively. The relative retention time of each peak was calculated using 3,6' -sinapiyl sucrose as a reference peak and the results are shown in Table 16. The results show that the relative retention time of each characteristic peak has an RSD of less than 5%.
Table 16 relative retention time table for different instruments
Figure BDA0003284355360000181
And (3) introducing the obtained characteristic spectrum into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), setting the characteristic spectrum of an Agilent definition 1260 II analysis result S1 positioned in a liquid phase chamber as a reference spectrum, setting the width of a time window to be 0.1, performing multipoint correction, performing Mark peak matching, and generating a comparison characteristic spectrum R by an average method, wherein 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 instruments of different manufacturers have less influence on detection, the similarity is more than 0.999, and the durability is good.
Table 17 results of similarity of different instruments
Agilent1260 liquid phase chamber Waters E2695 Agilent1260 standardization center Contrast characteristic spectrum R
Agilent1260 liquid phase chamber 1 1 1 1
Waters E2695 1 1 0.999 0.999
Agilent1260 standardization center 1 0.999 1 1
Contrast characteristic spectrum R 1 0.999 1 1
Example 5
15 batches of the pharmaceutical preparation samples A1-A15 were collected, and the sample solution was prepared in step 1 of example 1, and HPLC profiles of all samples were obtained according to the chromatographic conditions of step 3 of example 1. The relative retention time of each characteristic peak was calculated using 3,6' -sinapiyl sucrose as a reference peak and the results are shown in Table 18. As can be seen from Table 18, the relative retention time of each characteristic peak in the characteristic spectrum of 15 batches of the pharmaceutical preparation samples was shown to be RSD0 to 0.15 percent. 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% by taking 3,6' -sinapiyl sucrose as a reference, wherein the specified value is: 0.665 (Polygala tenuifolia)
Figure BDA0003284355360000183
Ketone III), 1.000 (3, 6' -sinapioyl sucrose), 1.183 (tenuifolin A), 1.326 (ginsenoside Rg) 1 ) 1.346 (ginsenoside Re), 2.041 (beta-asarone), 2.458 (pachymic acid). />
TABLE 18 measurement results for 15 samples
Figure BDA0003284355360000182
Figure BDA0003284355360000191
The theoretical plate numbers of 15 batches of sample reference peaks (3, 6' -sinapiyl sucrose) were also recorded and the results are shown in Table 19. As can be seen from table 19, the system applicability was good.
TABLE 18 theoretical plate number results for 15 samples
Figure BDA0003284355360000192
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In conclusion, the HPLC characteristic spectrum detection method 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 improves the quality control system of the pharmaceutical preparation. Therefore, the invention overcomes various defects in the prior art and has high industrial utilization value.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Claims (7)

1. A method of detecting an HPLC profile of a pharmaceutical formulation comprising: adding a medicinal preparation sample into a solvent for dissolution, performing ultrasonic extraction, cooling, shaking, filtering, taking a sample solution obtained from the subsequent filtrate, detecting by adopting a high performance liquid chromatography, and performing attribution positioning on index components in the characteristic spectrum of the obtained sample solution according to the retention time, thereby obtaining the characteristic spectrum of the medicinal preparation; the medicinal preparation is Happy powder;
The detection by adopting the high performance liquid chromatography comprises the following steps:
1) Preparation of reference solution: ginsenoside Rg 1 Reference substances of ginsenoside Re, polygala tenuifolia xanthone III, 3,6' -sinapioyl sucrose, tenuifolin A, beta-asarone and pachymic acid are added into a solvent for dissolution and volume fixation to prepare a reference substance solution;
2) And (3) measuring: respectively measuring the sample solution and the reference solution in the step 1) by adopting a high performance liquid chromatography to obtain a characteristic spectrum of the sample solution and a characteristic spectrum of the reference solution, comparing the retention time of the characteristic spectrum of the sample solution with the characteristic spectrum of the reference solution for qualitative determination, and carrying out attribution positioning on index components in the characteristic spectrum of the sample solution so as to obtain the characteristic spectrum of the pharmaceutical preparation;
in the 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 C 18 The filler in the chromatographic column is octadecylsilane chemically bonded silica gel; the detection wavelength is 200-205nm; the mobile phase is acetonitrile-0.05-0.15% phosphoric acid aqueous solution, wherein the A phase is acetonitrile, and the B phase is 0.05-0.15% phosphoric acid aqueous solution; the analysis time is 102min; gradient elution;
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, a step of selecting a specific type of material; 78-100 min, phase A: the volume ratio of the phase B is 75:25-95:5, a step of; 100-102 min, phase A: the volume ratio of the phase B is 95:5-95:5.
2. the method for detecting HPLC profile 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 added mass of the pharmaceutical preparation sample to the added volume of the solvent is 2:25-75 g/mL;
a2 The solvent is selected from 70% methanol, ethanol or water;
a3 The time of the ultrasonic extraction is 30-60 minutes.
3. Use of a method for the detection of HPLC profile of a pharmaceutical formulation according to any one of claims 1-2 in the quality detection of pharmaceutical formulations; the medicinal preparation is Happy powder.
4. A method of constructing a standard profile of a pharmaceutical formulation comprising: detecting a plurality of pharmaceutical preparation samples by adopting the detection method of the HPLC characteristic spectrum of the pharmaceutical preparation according to any one of claims 1-2, obtaining characteristic spectrums of the pharmaceutical preparations, generating a common control characteristic spectrum, taking chromatographic peaks existing in the spectrums as common characteristic peaks, determining the relative retention time of the common characteristic peaks, and establishing a standard characteristic spectrum of the pharmaceutical preparation; the medicinal preparation is Happy powder.
5. The method of claim 4, wherein the standard profile of the pharmaceutical formulation meets any one or more of the following conditions:
b1 The standard characteristic pattern of the pharmaceutical preparation comprises 7 common characteristic peaks, the peak 2 is taken as a reference peak S1, the retention time is 1.000, the relative retention time of other 6 common characteristic peaks is defined as a peak 1 with the retention time of 0.665, a peak 3 with the retention time of 1.183, a peak 4 with the retention time of 1.326, a peak 5 with the retention time of 1.346, a peak 6 with the retention time of 2.041 and a peak 7 with the retention time of 2.458, and the relative deviation of the relative retention time of the 6 common characteristic peaks except the peak 2 is 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, positioning and determining that the No. 1 peak is the characteristic peak of polygala tenuifolia xanthone III, the No. 2 peak is the characteristic peak of 3,6' -sinapiyl sucrose, the No. 3 peak is the characteristic peak of tenuifolin A, and the No. 4 peak is ginsenoside Rg 1 The No. 5 peak is the characteristic peak of ginsenoside Re, the No. 6 peak is the characteristic peak of beta-asarone, and the No. 7 peak is the characteristic peak of pachymic acid.
6. A quality control method of a characteristic spectrum of a pharmaceutical formulation, comprising: comparing the characteristic pattern of the pharmaceutical preparation obtained by the detection method of the characteristic pattern of the pharmaceutical preparation according to any one of claims 1 to 2 with the standard characteristic pattern of the pharmaceutical preparation obtained by the construction method of the standard characteristic pattern of the pharmaceutical preparation according to any one of claims 4 to 5, and comparing the similarity of the relative retention time of the chromatographic peak of the pharmaceutical preparation with the corresponding common characteristic peak; the medicinal preparation is Happy powder.
7. A screening method of characteristic patterns of a plurality of medicinal materials in a medicinal preparation, wherein the medicinal preparation is yippee powder, and comprises the following steps of:
a) Preparation of sample-deficient negative sample solution: removing any 1 medicinal material from medicinal preparation samples containing 4 medicinal materials of poria cocos, ginseng, polygala tenuifolia and grassleaf sweelflag rhizome respectively, preparing the medicinal preparation samples according to the same steps of preparing sample solutions in the detection method of the characteristic spectrum of the medicinal preparation according to any one of claims 1-2, and obtaining 4 sample deficiency negative solutions respectively: poria cocos negative solution, ginseng negative solution, polygala tenuifolia negative solution and rhizoma acori graminei negative solution;
b) And (3) measuring: respectively measuring 4 sample-missing negative solutions by adopting a high performance liquid chromatography under the same chromatographic conditions as those in the detection method of the characteristic spectrum of the pharmaceutical preparation according to any one of claims 1-2, and respectively obtaining the characteristic spectrums of the 4 sample-missing negative solutions;
C) And (3) quality detection: comparing the characteristic patterns of the 4 sample-lacking negative solutions with the standard characteristic patterns of the pharmaceutical preparation established by the method for constructing the standard characteristic patterns of the pharmaceutical preparation according to any one of claims 4-5, and identifying common characteristic peaks of corresponding single medicinal materials in the 4 sample-lacking negative solutions in the standard characteristic patterns of the pharmaceutical preparation through relative retention time, thereby carrying out attribution positioning on the characteristic peaks in the characteristic patterns of the corresponding single medicinal materials in the 4 sample-lacking negative solutions.
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