CN115032299A - Rhizoma corydalis pain-relieving oral liquid fingerprint detection method and quantitative detection method - Google Patents
Rhizoma corydalis pain-relieving oral liquid fingerprint detection method and quantitative detection method Download PDFInfo
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- CN115032299A CN115032299A CN202210602681.3A CN202210602681A CN115032299A CN 115032299 A CN115032299 A CN 115032299A CN 202210602681 A CN202210602681 A CN 202210602681A CN 115032299 A CN115032299 A CN 115032299A
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- G01N30/28—Control of physical parameters of the fluid carrier
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Abstract
The invention discloses a rhizoma corydalis pain-relieving oral liquid fingerprint detection method and a quantitative detection method, and belongs to the technical field of medicine detection. The method specifically comprises the following steps: preparing different batches of rhizoma corydalis pain-relieving oral liquid into test solution; injecting the sample solution into a high performance liquid chromatograph for detection to obtain the rhizoma corydalis pain-relieving oral liquid fingerprint spectrum with the common peak, wherein the detection conditions of the high performance liquid chromatograph are as follows: adopting an NH2P-504E chromatographic column, and taking water (A) and acetonitrile (B) as mobile phases to carry out gradient elution in the following way: for 0-10 min, 78-74% of B; for 10-28 min, 74-50% of B; 28-30 min, 50% B. The method fills the blank that the sugar component in the rhizoma corydalis pain-relieving oral liquid is not controlled sufficiently by the existing method, and can be used for scientifically evaluating the quality condition of the rhizoma corydalis pain-relieving oral liquid.
Description
Technical Field
The invention belongs to the technical field of medicine detection, and particularly relates to a fingerprint detection method and a quantitative detection method for a rhizoma corydalis pain-relieving oral liquid.
Background
The corydalis tuber pain-relieving oral liquid is prepared by soaking corydalis tuber and dahurian angelica root in vinegar, refluxing, concentrating, centrifuging and other processes, and can be used for treating stomachache, hypochondriac pain, headache and dysmenorrheal caused by qi stagnation and blood stasis. The 2020 version of Chinese pharmacopoeia (one part) uses high performance liquid chromatography to determine the content of tetrahydropalmatine in the rhizoma corydalis analgesic oral liquid, and uses the content as the quality control of the rhizoma corydalis analgesic oral liquid, and adopts two reference medicinal materials of rhizoma corydalis processed with vinegar and radix angelicae to perform qualitative identification. Considering the complexity of a traditional Chinese medicine compound system, a method for quantitatively detecting only tetrahydropalmatine in pharmacopoeia cannot show the quality of the preparation completely, so that a fingerprint method needs to be established for comprehensive overall evaluation.
The fingerprint spectrum is an effective method for reflecting the quality of the Chinese medicinal preparation on the whole, and is adopted in Chinese pharmacopoeia. Compared with quantitative detection of individual components, the fingerprint spectrum can more comprehensively reflect the quality condition of the traditional Chinese medicine, so the traditional Chinese medicine quality control is emphasized in recent years. By means of fingerprint similarity calculation and other methods, it may be determined quantitatively whether the quality of Chinese medicine is identical in different batches.
According to the existing research, corydalis tuber contains various saccharide components such as glucose, lactose, fructose, glucuronic acid and mannose (Haofeng, Zhang Jing, Zhang Mei, corydalis tuber chemical components, pharmacological activity and toxic and side effect research progress [ J ]. Shanghai medicine journal, 2017,51(11):4.), and angelica dahurica contains various saccharide components such as sucrose, glucose, rhamnose, arabinose and xylose (Li, Xiao Yong Geng. angelica dahurica water-soluble part chemical component research [ J ]. Chinese medicine journal, 2002,27(4):279 and 280; Kangjun, Qu Jiang Song, Guozui. analysis of angelica dahurica polysaccharide [ J ]. analytical chemistry, 2006,34(4):533 and 535.), and the saccharide components can generally serve as bioactive substances to maintain or improve the physiological functions of organisms. In addition, the auxiliary materials added in the preparation of the rhizoma corydalis pain-relieving oral liquid also comprise glucide such as beta-cyclodextrin, sucrose, stevioside and the like. Therefore, the saccharide component is used for controlling the quality of the rhizoma corydalis pain-relieving oral liquid and has important significance and value.
Stevioside is a natural sweetening agent, has the characteristics of high sweetness and low calorie, can replace cane sugar to be applied to the fields of food, medicine, daily chemical products and the like, and has wide development prospect. The main component of the tea beverage is stevioside, and various glycoside components such as rebaudioside A, B, C, D, F, dulcoside A, rubusoside, steviolbioside and the like are usually also accompanied. Steviol Glycosides have a number of important bioactive functions, such as antioxidation (CASAS-GRAJALES, RAMOS-TOVAR E, CH a VEZ-ESTARDA E, et al.Antixiadant and immunomodulating activity induced by stevioside In lift damage: In vivo, In vitro and In silico assays [ J ]. Life Sciences,2019,224:187-96.), auxiliary hypoglycaemia and hypotension (GUPTA E, PURWAR S, SUNDARAM S, et al.Stevioside and Rebaudioside A-Predominant Ent-kaureide Glycosides of Therapeutic Potential: a Review [ J ]. Journal of Czochralski, 2016 (4):281-99, 281-99, for example, cancer suppressing action (KHARE N, CHANDRA S. Stevioside-mediated chemotherapy fragments and cytoxicity assay on Breast cancer cells lines MDA-MB-231and SKBR3[ J ]. Saudi journal of biological sciences,2019,26(7): 1596-. Therefore, it is necessary to perform quality detection on the stevioside compounds.
But the quality control method of the corydalis tuber analgesic oral liquid is reported less in the literature at present. Particularly, a quantitative fingerprint method of carbohydrate components in the corydalis tuber analgesic oral liquid is not reported.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a method for detecting a fingerprint of a corydalis tuber analgesic oral liquid, which can make up for the deficiency of the existing analysis method in quality control of main components, thereby more comprehensively evaluating the quality of the corydalis tuber analgesic oral liquid, and having an important effect on controlling the quality and clinical efficacy of the corydalis tuber analgesic oral liquid.
In order to solve the technical problems, the invention adopts the following technical scheme:
a fingerprint detection method of rhizoma corydalis oral liquid for relieving pain comprises the following medicinal materials: the fingerprint detection method comprises the following steps:
step a1, preparation of test solution: preparing different batches of corydalis tuber analgesic oral liquid into test solution;
step a2, high performance liquid chromatography determination: injecting the sample solution in the step a1 into a high performance liquid chromatograph for detection to obtain the rhizoma corydalis pain-relieving oral liquid fingerprint spectrum with the common peak;
wherein the detection conditions of the high performance liquid chromatograph are as follows: adopting an NH2P-504E chromatographic column, and taking water (A) and acetonitrile (B) as mobile phases to carry out gradient elution, wherein the elution mode is as follows:
the volume percentage of acetonitrile in the mobile phase is reduced from 78% to 74%, and the volume percentage of water is increased from 22% to 26% in 0-10 min;
the volume percentage of acetonitrile in the mobile phase is reduced from 74% to 50% and the volume percentage of water is increased from 26% to 50% within 10-28 min;
and 28-30 min, wherein the volume percent of acetonitrile in the mobile phase is 50%, and the volume percent of water is 50%.
Preferably, the preparation of the test solution comprises the following steps: transferring rhizoma corydalis oral liquid for relieving pain, placing in volumetric flask, diluting with ultrapure water to constant volume, shaking, centrifuging, and collecting supernatant.
Preferably, the flow rate of the mobile phase is 0.6ml/min, and the column temperature of the NH2P-504E chromatographic column is 30 ℃.
Preferably, the high performance liquid chromatography is performed by using an electrospray detector, wherein the atomization temperature is 35 ℃, the sampling frequency is 10Hz, the optical filter is 5s, the power function is 1.0, and the sample injection volume is 10 mu L.
Preferably, the saccharide component is one or a mixture of more than two of stevioside, rebaudioside C, rebaudioside A, glucose and sucrose.
On the other hand, the invention also provides a quantitative detection method of the rhizoma corydalis pain-relieving oral liquid, which comprises the following steps: preparing the rhizoma corydalis pain-relieving oral liquid into a solution to be detected, injecting the solution to be detected into a high performance liquid chromatograph for detection to obtain a chromatogram of a saccharide component of the solution to be detected, and calculating the content of the saccharide component according to the peak area of a peak corresponding to the saccharide component in the chromatogram; wherein:
the detection conditions of the high performance liquid chromatograph are as follows: adopting an NH2P-504E chromatographic column, and taking water (A) and acetonitrile (B) as mobile phases to carry out gradient elution, wherein the elution mode is as follows:
the volume percentage of acetonitrile in the mobile phase is reduced from 78% to 74%, and the volume percentage of water is increased from 22% to 26% in 0-10 min;
the volume percentage of acetonitrile in the mobile phase is reduced from 74% to 50% and the volume percentage of water is increased from 26% to 50% within 10-28 min;
28-30 min, wherein the volume percentage of acetonitrile in the mobile phase is 50%, and the volume percentage of water is 50%;
an electrospray detector is adopted, the atomization temperature is 35 ℃, the sampling frequency is 10Hz, the optical filter is 5s, the power function is 1.0, and the sample injection volume is 10 mu L.
In another aspect, the invention further provides a quality evaluation method of the rhizoma corydalis analgesic oral liquid, which comprises the following steps:
preparing the rhizoma corydalis pain-relieving oral liquid into a solution to be detected, detecting the solution to be detected by adopting high performance liquid chromatography to obtain a chromatogram of saccharide components of the solution to be detected, comparing the chromatogram with the fingerprint of the rhizoma corydalis pain-relieving oral liquid obtained by the fingerprint detection method, and calculating the similarity; evaluating the quality of the rhizoma corydalis pain-relieving oral liquid according to the similarity; wherein, the detection conditions of the high performance liquid chromatograph are as follows: adopting an NH2P-504E chromatographic column, and taking water (A) and acetonitrile (B) as mobile phases to carry out gradient elution, wherein the elution mode is as follows:
the volume percentage of acetonitrile in the mobile phase is reduced from 78% to 74%, and the volume percentage of water is increased from 22% to 26% in 0-10 min;
the volume percentage of acetonitrile in the mobile phase is reduced from 74% to 50% and the volume percentage of water is increased from 26% to 50% within 10-28 min;
28-30 min, wherein the volume percentage of acetonitrile in the mobile phase is 50%, and the volume percentage of water is 50%;
an electrospray detector is adopted, the atomization temperature is 35 ℃, the sampling frequency is 10Hz, the optical filter is 5s, the power function is 1.0, and the sample injection volume is 10 mu L.
Compared with the prior art, the invention has the beneficial effects that:
the invention establishes the fingerprint spectrum, the quantitative detection method and the quality evaluation method of the saccharide component in the corydalis tuber analgesic oral liquid for the first time, fills the blank of insufficient control of the saccharide component by the existing method, and can be used for accurately measuring the qualitative measurement of the saccharide component in different batches of corydalis tuber analgesic oral liquid and scientifically evaluating the quality condition of the saccharide component.
The invention proves that the high performance liquid chromatography detection method adopted by the invention has high precision, good repeatability, good sample stability and certain specificity through the methodological verification of the fingerprint.
The methodological verification of the quantitative detection shows that the quantitative detection method has high linear fitting degree, high precision, good repeatability and good solution stability, and the sample recovery rate meets the requirements, and can be used for measuring the content of the saccharide component in the rhizoma corydalis pain-relieving oral liquid.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the description of the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a total ion flow diagram of a part of compounds of saccharide component in an LC/Q-TOF-MS anion mode in a rhizoma corydalis analgesic oral liquid;
FIG. 2 is a fingerprint of a control solution;
FIG. 3 is a fingerprint of a test solution;
FIG. 4 is a comparison of similarity between fingerprints of 3 batches of corydalis tuber analgesic oral liquid (R: comparison, S1-S3: fingerprint of 3 batches of test solutions).
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.
The fingerprint spectrum detection method of the corydalis tuber analgesic oral liquid provided by the embodiment of the invention comprises the following steps of: preparing different batches of rhizoma corydalis pain-relieving oral liquid into test solution; and injecting the sample solution into a high performance liquid chromatograph for detection to obtain the rhizoma corydalis pain-relieving oral liquid fingerprint spectrum with the common peak. The corydalis tuber analgesic oral liquid comprises the following medicinal materials: vinegar corydalis tuber and angelica dahurica, and the detection conditions of a high performance liquid chromatograph are as follows: adopting an NH2P-504E chromatographic column, and carrying out gradient elution by taking water (A) and acetonitrile (B) as mobile phases, wherein the water is the mobile phase A, the acetonitrile is the mobile phase B, and the elution mode is as follows:
reducing the volume percent of acetonitrile from 78% to 74% and increasing the volume percent of water from 22% to 26% for 0-10 min;
reducing the volume percent of acetonitrile from 74% to 50% and increasing the volume percent of water from 26% to 50% for 10-28 min;
and (3) 28-30 min, wherein the volume percentage of acetonitrile is 50%, and the volume percentage of water is 50%.
The invention constructs the fingerprint of the saccharide component of the corydalis tuber analgesic oral liquid, 5 common peaks are calibrated by adopting fingerprint software, and the similarity is more than 0.99, which indicates that the constructed fingerprint can provide a basis for the quality evaluation of the corydalis tuber analgesic oral liquid.
The fingerprint is compared with a reference substance map and a mass spectrometry analysis result, and peaks 2, 3, 4, 5 and 6 are identified to be stevioside, rebaudioside C, rebaudioside A, glucose and sucrose respectively.
In further methodological experimental verification, the method is proved to be simultaneously suitable for measuring the content of the sucrose in the rhizoma corydalis analgesic oral liquid.
Therefore, the invention provides a test basis for the quality research of the rhizoma corydalis analgesic oral liquid saccharide component, provides a comprehensive quality evaluation method for the rhizoma corydalis analgesic oral liquid saccharide component, provides a good basis for the internal quality control and improvement of the rhizoma corydalis analgesic oral liquid saccharide component, and has extremely important significance for ensuring the clinical curative effect.
In some embodiments, the preparation of the test solution comprises the steps of: transferring 50 μ L of rhizoma corydalis oral liquid for relieving pain, placing into 10ml volumetric flask, diluting with ultrapure water to constant volume, shaking, centrifuging, and collecting supernatant.
In some embodiments, the flow rate of the mobile phase is 0.6ml/min and the column temperature of the NH2P-504E column is 30 ℃.
In some embodiments, the HPLC assay uses an electrospray detector, an atomization temperature of 35 deg.C, a sampling frequency of 10Hz, a filter of 5s, a power function of 1.0, and a sample volume of 10 μ L. The method combines High Performance Liquid Chromatography (HPLC) and an electrospray detector (CAD) to detect the saccharide components in the rhizoma corydalis analgesic oral liquid, and provides a key support for the construction of the fingerprint spectrum of the rhizoma corydalis analgesic oral liquid and the realization of quantitative detection.
In some specific embodiments, the carbohydrate component is one or a mixture of two or more of stevioside, rebaudioside C, rebaudioside a, glucose, and sucrose. The saccharide component can be used as a reference substance for verifying the precision, the repeatability and the sample stability of the fingerprint detection method.
The invention provides a quantitative detection method of corydalis tuber analgesic oral liquid, which comprises the following steps: preparing rhizoma corydalis pain-relieving oral liquid into a solution to be tested, detecting the solution to be tested by adopting high performance liquid chromatography to obtain a chromatogram of the solution to be tested, and calculating the content of the saccharide component according to the peak area of the peak corresponding to the saccharide component in the chromatogram, for example, according to a standard curve of the peak area integral value and the concentration.
In some embodiments, the method for quantitatively detecting the corydalis tuber analgesic oral liquid comprises the following steps:
step b1, preparation of control solution: precisely weighing sucrose as reference, respectively dissolving with ultrapure water, fixing the volume, and shaking up to obtain sucrose reference stock solution with certain concentration;
respectively transferring stock solutions with different volumes into volumetric flasks, and performing constant volume by using ultrapure water to obtain a series of sucrose reference substance solutions with concentration gradients;
step b2, high performance liquid chromatography determination: injecting the sucrose reference substance solution in the step b1 into a high performance liquid chromatograph for detection to obtain a chromatogram; wherein, the detection conditions of the high performance liquid chromatograph are as follows: adopting an NH2P-504E chromatographic column, and taking water (A) and acetonitrile (B) as mobile phases to carry out gradient elution, wherein the elution mode is as follows:
reducing the volume percent of acetonitrile from 78% to 74% and increasing the volume percent of water from 22% to 26% for 0-10 min;
reducing the volume percent of acetonitrile from 74% to 50% and increasing the volume percent of water from 26% to 50% for 10-28 min;
28-30 min, wherein the volume percentage of acetonitrile is 50%, and the volume percentage of water is 50%.
An electrospray detector is adopted, the atomization temperature is 35 ℃, the sampling frequency is 10Hz, the optical filter is 5s, the power function is 1.0, and the sample introduction volume is 10 mu L;
drawing a standard curve by taking the concentration of the sucrose reference substance solution as a horizontal coordinate and the peak area as a vertical coordinate to obtain a regression equation;
preparing the rhizoma corydalis pain-relieving oral liquid into a solution to be detected, detecting according to the step b2 to obtain a chromatogram of the saccharide component of the solution to be detected, substituting the peak area of the corresponding peak of the sucrose on the chromatogram into a regression equation, and calculating to obtain the content of the sucrose in the solution to be detected.
The quality evaluation method of the rhizoma corydalis oral liquid for relieving pain provided by the embodiment of the invention comprises the following steps:
preparing the rhizoma corydalis pain-relieving oral liquid into a solution to be detected, detecting the solution to be detected by adopting high performance liquid chromatography to obtain a chromatogram of saccharide components of the solution to be detected, comparing the chromatogram with the fingerprint of the rhizoma corydalis pain-relieving oral liquid obtained by the fingerprint detection method, and calculating the similarity; evaluating the quality of the rhizoma corydalis pain-relieving oral liquid according to the similarity; wherein, the detection conditions of the high performance liquid chromatograph are as follows: adopting an NH2P-504E chromatographic column, and taking water (A) and acetonitrile (B) as mobile phases to carry out gradient elution, wherein the elution mode is as follows:
reducing the volume percent of acetonitrile from 78% to 74% and increasing the volume percent of water from 22% to 26% for 0-10 min;
for 10-28 min, reducing the volume percent of acetonitrile from 74% to 50%, and increasing the volume percent of water from 26% to 50%;
28-30 min, wherein the volume percentage of acetonitrile is 50%, and the volume percentage of water is 50%.
The following are specific examples.
1. Laboratory instruments and materials
1.1 Instrument
Thermo Scientific Ultimate3000 hplc equipped with Thermo Scientific Dionex Corona Veo electrospray detector, online degasser, quaternary pump, autosampler, column oven, and Thermo Scientific Dionex Chromeleon 7 chromatographic data system (version 7.2SR 5); LC/Q-TOF-MS analysis System: an Agilent 6546 quadrupole-time-of-flight mass spectrometer with Agilent MassHunter Workstation Data Acquisition software (version 10.1), Agilent MassHunter Qualitative Analysis Data processing software (version 10.0); the chromatographic column is NH2P-504E chromatographic column (4.6X 250mm, 5 μm); electronic balance (AB204-N, Mettler Toledo). A small high speed centrifuge (AB204-N, Mettler Toledo). The fingerprint similarity evaluation software is a Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition of national pharmacopoeia committee).
1.2 reagents
Acetonitrile (chromatographically pure, Merck, Germany), Mili-Q ultrapure water, sucrose control (HPLC > 99%, Shanghai Po pharmaceutical science and technology development Co., Ltd., batch No.: 210620).
2. Conditions of the experiment
2.1 preparation of control solutions
Precisely weighing sucrose reference substance, dissolving with ultrapure water, diluting to 50ml, and shaking to obtain sucrose reference substance stock solution with concentration of 9.9974 mg/ml. 200, 300, 400, 500 and 600 mul of stock solutions are respectively transferred into a 5ml volumetric flask, and the volume is fixed by ultrapure water to obtain sucrose reference solution with the concentration of 0.39990, 0.59984, 0.79979, 0.99974 and 1.1997 mg/ml.
2.2 preparation of test solutions
Transferring 50 μ L of rhizoma corydalis oral liquid for relieving pain into 10mL volumetric flask, diluting with ultrapure water to constant volume, centrifuging, and collecting supernatant.
2.3 chromatographic conditions
The chromatographic conditions are as follows: a chromatographic column: asahipak NH2P-504E column (4.6X 250mm, 5 μm) was eluted with a gradient of water (A) -acetonitrile (B) as mobile phase in the following manner: for 0-10 min, 78-74% of B; for 10-28 min, 74% -50% of B; 28-30 min, 50% B, flow rate: 0.6 ml/min; column temperature: 30 ℃; the injection volume was 10. mu.L.
2.4 LC/Q-TOF-MS (liquid chromatography-triple quadrupole-time of flight mass Spectrometry) conditions
The above-mentioned liquid chromatography conditions were used as the chromatographic conditions for high-resolution mass spectrometry.
The mass spectrometry conditions were as follows: an ion source: electrospray ion source (ESI); an acquisition mode: adopting an anion mode; scanning mode: MS; scanning range: m/z 100-1500; the temperature of the drying gas is 320 ℃, and the flow rate of the drying gas is 8L/min; atomizer pressure 35 psi; the temperature of the sheath gas is 350 ℃, and the flow of the sheath gas is 11L/min; capillary voltage 3500V; nozzle voltage 1000V; the crushing voltage is 175V; the taper hole voltage is 65V; octupole rod peak-to-peak radio frequency voltage: 750V.
After the analysis method is determined, the total ion flow diagram of the test solution is obtained by detecting the test solution by using high-resolution mass spectrometry, as shown in fig. 1. According to the accurate relative molecular mass obtained by the high resolution mass spectrometry, chemical components of 5 chromatographic peaks in the common peaks (shown in figure 3) of the test solution are estimated and compared by adopting a reference solution (shown in figure 2), and the serial numbers and the inference results are shown in table 1.
TABLE 1 LC/Q-TOF-MS analysis of sugar component part compounds of corydalis tuber analgesic oral liquid samples
3. Methodology validation
3.1 methodological verification of finger prints
3.1.1 Experimental methods
The methodological verification of the fingerprint mainly comprises the investigation of precision, repeatability and sample stability.
And (3) sample injection precision experiment: the same sample solution is continuously injected for 6 times. Selecting a reference peak, and respectively calculating the ratio of the retention time of each common peak to the reference peak to the peak area to obtain the relative retention time of each common peak and the Relative Standard Deviation (RSD) value of the relative peak area.
Method repeatability experiment: taking six test solution prepared in parallel, and carrying out sample injection analysis respectively. Selecting a reference peak, and respectively calculating the ratio of the retention time of each common peak to the reference peak to the peak area to obtain the relative retention time of each common peak and the RSD value of the relative peak area.
Sample stability experiment: and taking the same sample solution, carrying out sample injection analysis for 0, 2, 4, 8, 12 and 24 hours, selecting a reference peak, and respectively calculating the retention time and peak area ratio of each common peak to the reference peak to obtain the relative retention time of each common peak and the RSD value of the relative peak area.
3.1.2 results of the experiment
Different batches of rhizoma corydalis analgesic oral liquids with serial numbers of S1-S3 are measured, 5 common peaks are determined under the condition, the peak area of sucrose is relatively large, and the separation degree of sucrose from adjacent chromatographic peaks is good, the common peaks are determined as reference peaks, and the average value and the RSD value of the relative retention time of each chromatographic peak under the experimental items of sample injection precision, repeatability and sample stability are respectively calculated, and the results are shown in tables 2 and 3.
The result shows that under the conditions of sampling precision, repeatability and stability, the relative retention time of each chromatographic peak and the RSD value of the relative retention peak area are both less than 4 percent, and the requirements of the fingerprint spectrum are met.
TABLE 2 sample introduction precision, repeatability and sample stability experiment peak relative retention time results
TABLE 3 sample introduction precision, repeatability and sample stability experiment peak area results
3.2 assay methodology verification
3.2.1 Experimental methods
The high performance liquid chromatography analysis method is also suitable for measuring the content of the saccharide component in the rhizoma corydalis pain-relieving oral liquid, and the methodological verification comprises the investigation of the linearity, precision, stability, repeatability and sample-adding recovery experiment of each content measurement component.
Linear investigation: a series of sucrose reference solutions with different concentrations were injected into 10. mu.L sample for analysis. And (3) taking the peak area measured by each component as a vertical coordinate and the concentration as a horizontal coordinate to prepare a standard curve, obtaining a linear regression equation and an analysis range, and respectively calculating the detection Limit (LOD) and the quantification Limit (LOQ) of the method, wherein the calculation mode of the LOD is shown in a formula (3.1), and the calculation mode of the LOQ is shown in a formula (3.2).
Where σ is the deviation of the response value and s is the slope of the standard curve.
And (3) sample injection precision experiment: and taking the same sample solution to be tested, carrying out continuous sample introduction for 6 times, and calculating to obtain peak areas of all components and RSD values of retention time.
Intermediate precision experiment: and taking the same sample solution, carrying out sample injection analysis on days 1, 2 and 3, and calculating to obtain peak areas of all components and RSD values of retention time.
And (3) repeatability experiment: taking six parts of test solution prepared in parallel, respectively carrying out sample injection analysis, and calculating to obtain the RSD value of each component.
Solution stability experiments: and taking the same sample solution, carrying out sample injection analysis for 0, 2, 4, 8, 12 and 24 hours, and calculating to obtain the RSD value of each component content.
Sample adding and recovering experiment: 9 parts of sample solution with known content are respectively taken and divided into 3 groups. Setting three concentration levels of low, medium and high, controlling the ratio of the addition amount of the reference substance to the amount of the component to be detected in the test sample to be about 0.8:1.0, 1.0:1.0 and 1.2:1.0 respectively, and preparing 3 parts of test sample solution for determination in parallel for each concentration. Precisely transferring 25 mul of sample and a proper amount of reference substance stock solution, fixing the volume to 10mL by ultrapure water, shaking up, centrifuging, taking supernatant, injecting and analyzing.
3.2.2 results of the experiment
And combining the control product map and the mass spectrum detection result to identify peaks 2, 3, 4, 5 and 6 as stevioside, rebaudioside C, rebaudioside A, glucose and sucrose respectively. The main component sucrose with larger peak area and better peak shape is selected for content determination.
The regression equation, linear range, detection limit and quantitative limit results of the sucrose are shown in table 4, and the linear fitting results are all more than 0.999, which meets the requirements of Chinese pharmacopoeia. The sample injection precision experiment results are shown in tables 5 and 6, the intermediate precision experiment results are shown in tables 7 and 8, the repeatability experiment results are shown in table 9, the solution stability experiment results are shown in table 10, wherein the precision, the repeatability and the stability RSD are all less than 5%, the method meets the requirements of Chinese pharmacopoeia, the method is good in linearity, and the sample solution is stable within 24 h. The sample adding and recycling experimental result is shown in a table 11, the average recycling rate meets the requirements, and the RSD values are less than 5%, so that the method obtained through optimization is accurate and reliable, and can be used for determining the content of the saccharide component in the corydalis tuber analgesic oral liquid.
TABLE 4 regression equation, correlation coefficient and Linear analysis Range for sucrose Components
TABLE 5 sample introduction precision results (peak area)
TABLE 6 sample introduction precision results (retention time)
TABLE 7 intermediate precision results (peak area)
TABLE 8 intermediate precision results (retention time)
TABLE 9 results of repeated experiments
TABLE 10 results of solution stability experiments
TABLE 11 sample recovery test results
3.3 sample content measurement results and quality control example
The results of quantitative component content measurement of 3 test sample solutions are shown in Table 12. The sucrose content of each batch of rhizoma corydalis pain-relieving oral liquid is 130.83-168.90 mg/ml. Pharmaceutical enterprises of the rhizoma corydalis pain-relieving oral liquid can set internal control standards of sucrose content, such as not less than 100mg/ml, and then can judge whether new batches of products meet the internal control standards according to the internal control standards.
TABLE 123 determination of sucrose content in corydalis tuber analgesic oral liquid
3.4 fingerprint similarity evaluation
The map data of the test solution of 3 batches (S1-S3) of rhizoma corydalis pain-relieving oral liquid are led into the software of the traditional Chinese medicine chromatogram fingerprint similarity evaluation system, and a control map is generated, as shown in figure 4. The fingerprint spectrum of each test solution is compared with the comparison spectrogram, and the similarity result is shown in a table 13.
As can be seen from the table, the similarity between the fingerprint spectrum of each sample solution and the comparison spectrogram is over 0.99, and the rhizoma corydalis pain-relieving oral liquid sugar components of each batch have better quality consistency. The corydalis tuber pain-relieving oral liquid obtained in the subsequent industrial production can be compared with a reference spectrogram, and the quality consistency of medicines in different batches can be judged after the similarity is calculated. Or the more representative control map can be obtained by continuously adding the normal batches of the corydalis tuber analgesic oral liquid into a fingerprint map library.
TABLE 133 evaluation results of similarity of finger prints of test solutions of normal batches of rhizoma corydalis analgesic oral liquid
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof.
Claims (10)
1. A fingerprint detection method of rhizoma corydalis oral liquid for relieving pain comprises the following medicinal materials: vinegar corydalis tuber and dahurian angelica root, which is characterized in that: the fingerprint detection method comprises the following steps:
step a1, preparation of test solution: preparing different batches of corydalis tuber analgesic oral liquid into test solution;
step a2, high performance liquid chromatography determination: injecting the sample solution in the step a1 into a high performance liquid chromatograph for detection to obtain the rhizoma corydalis pain-relieving oral liquid fingerprint spectrum with the common peak;
wherein, the detection conditions of the high performance liquid chromatograph are as follows: adopting an NH2P-504E chromatographic column, and taking water (A) and acetonitrile (B) as mobile phases to carry out gradient elution, wherein the elution mode is as follows:
the volume percent of acetonitrile in the mobile phase is reduced from 78% to 74% and the volume percent of water is increased from 22% to 26% within 0-10 min;
the volume percentage of acetonitrile in the mobile phase is reduced from 74% to 50% and the volume percentage of water is increased from 26% to 50% within 10-28 min;
and 28-30 min, wherein the volume percent of acetonitrile in the mobile phase is 50%, and the volume percent of water is 50%.
2. The rhizoma corydalis pain-relieving oral liquid fingerprint spectrum detection method as claimed in claim 1, is characterized in that: the preparation of the test solution comprises the following steps: transferring rhizoma corydalis oral liquid for relieving pain, placing in volumetric flask, diluting with ultrapure water to constant volume, shaking, centrifuging, and collecting supernatant.
3. The fingerprint spectrum detection method of the rhizoma corydalis oral liquid for relieving pain according to claim 2, characterized in that: the flow rate of the mobile phase is 0.6ml/min, and the column temperature of the NH2P-504E chromatographic column is 30 ℃.
4. The rhizoma corydalis pain-relieving oral liquid fingerprint spectrum detection method as claimed in claim 3, is characterized in that: the high performance liquid chromatography adopts an electrospray detector, the atomization temperature is 35 ℃, the sampling frequency is 10Hz, the optical filter is 5s, the power function is 1.0, and the sample injection volume is 10 mu L.
5. The rhizoma corydalis pain-relieving oral liquid fingerprint spectrum detection method as claimed in claim 4, is characterized in that: the saccharide component is one or more of stevioside, rebaudioside C, rebaudioside A, glucose and sucrose.
6. A quantitative detection method of rhizoma corydalis oral liquid for relieving pain is characterized by comprising the following steps: the method comprises the following steps: preparing the rhizoma corydalis pain-relieving oral liquid into a solution to be detected, injecting the solution to be detected into a high performance liquid chromatograph for detection to obtain a chromatogram of a saccharide component of the solution to be detected, and calculating the content of the saccharide component according to the peak area of a peak corresponding to the saccharide component in the chromatogram, wherein the detection conditions of the high performance liquid chromatograph are as follows: adopting an NH2P-504E chromatographic column, and taking water (A) and acetonitrile (B) as mobile phases to carry out gradient elution, wherein the elution mode is as follows:
the volume percent of acetonitrile in the mobile phase is reduced from 78% to 74% and the volume percent of water is increased from 22% to 26% within 0-10 min;
reducing the volume percentage of acetonitrile in the mobile phase from 74% to 50% and increasing the volume percentage of water in the mobile phase from 26% to 50% for 10-28 min;
and 28-30 min, wherein the volume percent of acetonitrile in the mobile phase is 50%, and the volume percent of water is 50%.
7. The quantitative detection method of rhizoma corydalis pain-relieving oral liquid according to claim 6, characterized in that: the high performance liquid chromatography adopts an electrospray detector, the atomization temperature is 35 ℃, the sampling frequency is 10Hz, the optical filter is 5s, the power function is 1.0, and the sample injection volume is 10 mu L.
8. The quantitative detection method of rhizoma corydalis pain-relieving oral liquid as claimed in claim 7, wherein: the saccharide component is sucrose.
9. A quality evaluation method of rhizoma corydalis pain-relieving oral liquid is characterized in that: the method comprises the following steps:
preparing the rhizoma corydalis pain-relieving oral liquid into a solution to be detected, detecting the solution to be detected by adopting high performance liquid chromatography to obtain a chromatogram of saccharide components of the solution to be detected, comparing the chromatogram with the fingerprint of the rhizoma corydalis pain-relieving oral liquid obtained by the fingerprint detection method according to any one of claims 1-5, and calculating the similarity;
evaluating the quality of the rhizoma corydalis pain-relieving oral liquid according to the similarity;
wherein, the detection conditions of the high performance liquid chromatograph are as follows: adopting an NH2P-504E chromatographic column, and taking water (A) and acetonitrile (B) as mobile phases to carry out gradient elution, wherein the elution mode is as follows:
the volume percent of acetonitrile in the mobile phase is reduced from 78% to 74% and the volume percent of water is increased from 22% to 26% within 0-10 min;
the volume percentage of acetonitrile in the mobile phase is reduced from 74% to 50% and the volume percentage of water is increased from 26% to 50% within 10-28 min;
and 28-30 min, wherein the volume percent of acetonitrile in the mobile phase is 50%, and the volume percent of water is 50%.
10. The method for evaluating the quality of the rhizoma corydalis analgesic oral liquid as claimed in claim 9, wherein the method comprises the following steps: the high performance liquid chromatography adopts an electrospray detector, the atomization temperature is 35 ℃, the sampling frequency is 10Hz, the optical filter is 5s, the power function is 1.0, and the sample injection volume is 10 mu L.
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| CN116019856A (en) * | 2023-02-23 | 2023-04-28 | 河南福森药业有限公司 | New use of rhizoma corydalis pain relieving oral liquid |
| CN116660408A (en) * | 2023-05-18 | 2023-08-29 | 嘉亨(珠海横琴)医药科技有限公司 | Method for constructing rhizoma corydalis pain-relieving oral liquid fingerprint spectrum and measuring content and application thereof |
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