CN114544816A - Quantitative fingerprint detection method for carbohydrate components of bupleurum tenue capsules - Google Patents
Quantitative fingerprint detection method for carbohydrate components of bupleurum tenue capsules Download PDFInfo
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Abstract
The invention discloses a quantitative fingerprint detection method for saccharide components of bupleurum tenue capsules, which comprises the following steps: (1) preparing a mixed solution of a test sample solution and a reference substance, wherein the preparation method of the mixed solution of the reference substance comprises the following steps: respectively dissolving ribitol, fructose, sucrose, and stachyose in solvent, and mixing; (2) analyzing the two solutions prepared in the step (1) by adopting a high performance liquid chromatograph, wherein the chromatographic conditions are as follows: the instrument adopts a high performance liquid chromatography system, and the chromatographic column adopts an Asahipak NH2P-504E chromatographic column (4.6 multiplied by 250mm, 5 mu m); gradient elution was carried out with water (a) -acetonitrile (B) as mobile phase, elution mode: for 0-10 min, 78-74% of B; for 10-28 min, 74-50% of B; 28-33 min, 50% B, flow: 0.6 ml/min; column temperature: 30 ℃; (3) determining common peaks and quantitative detection indexes according to the chromatograms of all different batches, calculating the content and similarity of different batches, and providing scientific basis for quality control of the bupleurum tenue capsules.
Description
Technical Field
The invention relates to a detection method of traditional Chinese medicine, in particular to a quantitative fingerprint detection method of a carbohydrate component of a bupleurum tenue capsule.
Background
The capsule is prepared from seven medicinal materials of radix bupleuri, radix scutellariae, liquorice, codonopsis pilosula, Chinese date, ginger and ginger processed pinellia tuber through the processes of decoction, percolation, concentration, drying and the like, and can be used for treating exogenous diseases, pathogenic factors attacking shaoyang syndrome, and the symptoms of alternating cold and heat, fullness in chest and hypochondrium, inappetence, dysphoria with joy and vomiting, bitter taste in mouth, dry throat and the like. The 2020 version of Chinese pharmacopoeia (one part) adopts high performance liquid chromatography to determine baicalin content in XIAOCHUI Capsule for quality control, and adopts bupleuri radix, Glycyrrhrizae radix and radix Codonopsis as reference materials to qualitatively identify the three materials. Considering that the bupleurum tenue capsules have more medicinal taste, the method for quantitatively detecting only one component, namely baicalin in the pharmacopoeia cannot show the quality of the preparation completely, and therefore a fingerprint method needs to be established for comprehensive overall evaluation.
The simultaneous quantitative detection of multiple index components is widely used in 'Chinese pharmacopoeia' (one part) of 2020 edition, and is a feasible method for detecting the quality of traditional Chinese medicines. The fingerprint is another effective method for detecting the quality of the traditional Chinese medicine, and the whole quality condition of the traditional Chinese medicine can be quantitatively reflected by adopting indexes such as similarity and the like to totally reflect the spectrum or chromatographic peak information of the fingerprint, so that the fingerprint technology is greatly developed in recent years.
But the quality control method of the bupleurum tenue capsule is reported less in the current literature. Especially, a quantitative fingerprint method of carbohydrate components in the bupleurum tenue capsules is not reported.
Disclosure of Invention
The invention mainly aims to provide a quantitative fingerprint spectrum of a carbohydrate component of a bupleurum tenue capsule, and the detection method can make up the defect of the existing analysis method in the quality control of the pharmacodynamic components, so that the quality of the bupleurum tenue capsule can be more comprehensively evaluated, and the quantitative fingerprint spectrum has an important effect in controlling the quality and the clinical curative effect of the bupleurum tenue capsule.
In order to achieve the purpose, the invention adopts the following technical scheme:
one of the objects of the present invention: the fingerprint detection method of the carbohydrate component of the bupleurum tenue capsule is provided, and the bupleurum tenue capsule comprises the following medicinal materials: radix bupleuri, radix scutellariae, liquorice, radix codonopsis, Chinese date, ginger and pinellia ternate. The fingerprint detection method comprises the following steps:
(1) preparing a reference substance mixed solution and a test solution:
preparation of control mixed solution: precisely weighing reference substances of ribitol, fructose, sucrose and stachyose, respectively dissolving with 60% acetonitrile solution, fixing the volume to 10ml, shaking up to obtain 4 reference substance solutions, transferring appropriate amount of 4 single labels into the same 50ml volumetric flask, adding 60% acetonitrile solution, precisely fixing the volume, and shaking up to obtain mixed standard stock solution. Diluting the stock solution 5 times to obtain control mixed solution containing 0.6512mg/ml ribitol, 2.189mg/ml fructose, 1.636mg/ml sucrose, and 0.6767mg/ml stachyose.
Preparation of a test solution: taking the content of the bupleurum tenue capsule in a mortar, grinding, precisely weighing, placing in a 25mL volumetric flask, adding 60% acetonitrile solution, heating and ultrasonically dissolving, cooling to room temperature, fixing the volume, shaking up, centrifuging, and taking the supernatant to obtain the bupleurum tenue capsule.
(2) High performance liquid chromatography assay
Injecting the reference substance mixed solution and the test substance solution in the step (1) into a liquid chromatography for analysis and determination, wherein the apparatus is as follows: thermo Scientific Ultimate3000 high performance liquid chromatograph; the chromatographic conditions are as follows: a chromatographic column: NH2P-504E column (4.6X 250mm, 5 μm) with water (A) -acetonitrile (B) as mobile phase, gradient elution, elution mode: for 0-10 min, 78-74% of B; for 10-28 min, 74-50% of B; 28-33 min, 50% B, flow: 0.6 ml/min; column temperature: 30 ℃; the atomization temperature of an electrospray detector (CAD) 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.
(3) Establishing quantitative fingerprint and evaluating quality
Taking 10 bupleurum tenue capsules of different batches to prepare 10 test solution of different batches according to the method in the step (1), carrying out high performance liquid chromatography analysis according to the step (2), and recording a chromatogram; and determining common peaks and quantitative detection indexes by using chromatographic peaks common to different batches according to chromatograms of 10 different batches. Meanwhile, the quality of the bupleurum tenue capsule is evaluated through the established fingerprint similarity and the determined saccharide component.
Preferably, the atomization temperature in the step (2) is 35 ℃, and at the atomization temperature, chromatographic peaks are well separated, baselines are stable, peak areas are large, and responses are good.
Preferably, the injection volume in step (2) is 10 μ L, and the peak area is larger and the response is better at this injection volume.
Preferably, the quantitative detection indexes in the step (3) are as follows: ribitol, fructose, sucrose, stachyose. Under the detection conditions of the present invention, the above four components can be sufficiently separated.
Preferably, the number of common peaks in step (3) is 7.
The second object of the present invention is: provides a method for measuring the content of saccharide components in bupleurum tenue capsules.
The chromatographic conditions were as follows: the instrument comprises the following steps: thermo Scientific Ultimate3000 high performance liquid chromatograph; the chromatographic conditions are as follows: a chromatographic column: NH2P-504E column (4.6X 250mm, 5 μm) with water (A) -acetonitrile (B) as mobile phase, gradient elution, elution mode: for 0-10 min, 78-74% of B; for 10-28 min, 74-50% of B; 28-33 min, 50% B, flow: 0.6 ml/min; column temperature: 30 ℃; the injection volume was 10. mu.L.
Precisely weighing appropriate amount of reference substances of ribitol, fructose, sucrose and stachyose, respectively adding 60% acetonitrile to dilute into solutions with different concentrations, injecting into high performance liquid chromatography for analysis, drawing a standard curve with the concentration of each compound as abscissa and the peak area as ordinate, calculating a regression equation, and calculating the content of index components in different batches of samples.
The third object of the present invention is: provides an application of a quantitative fingerprint of a bupleurum tenue capsule carbohydrate component in the quality control of the bupleurum tenue capsule.
Introducing chromatograms of test solution of bupleurum tenue capsules of 10 batches into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition of the national pharmacopoeia committee), generating a comparison fingerprint, introducing the test solution of bupleurum tenue capsules of different batches into similarity evaluation software, calculating the similarity, and regarding the batches with the similarity lower than 0.9, considering that the quality difference is large, and not recommending the release.
Enterprises can set the content standard of the saccharide components by themselves, and if the content standard does not reach the internal control standard of the enterprises, the quality can be considered to be unqualified, and the release is not recommended.
The invention has the beneficial effects that:
the invention establishes the quantitative fingerprint of the carbohydrate component in the bupleurum tenue capsule for the first time, fills the blank of insufficient control of the carbohydrate component by the prior method, and the quantitative fingerprint can be used for comprehensively evaluating the quality of the bupleurum tenue capsule. Aiming at 7 Chinese medicinal materials in the bupleurum tenue capsule, 4 saccharide components are screened out for determination. Wherein, stachyose is a functional oligomeric tetrasaccharide, can promote the growth of probiotics, plays a role in regulating intestinal flora, and is necessary to detect and control the quality of the stachyose as a medicinal component; ribitol is a reduction product of D-ribose, exists in roots of radix bupleuri in a free state, is a characteristic component of radix bupleuri, and can embody partial quality information of radix bupleuri in the Xiaochaihu capsule by establishing a fingerprint spectrum; fructose and sucrose are contained in bupleurum tenue capsules in a large amount, so that the fructose and sucrose are also required to be detected.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the invention without undue limitation of the invention.
FIG. 1 is a total ion flow diagram of a part of compounds of saccharide components of a bupleurum tenue capsule sample in an LC/Q-TOF-MS negative ion mode.
FIG. 2 shows the chromatogram of the mixed control (A) and sample (B).
FIG. 3 shows HPLC fingerprint of saccharide components of 10 batches of XIAOCHAIHU Capsule.
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. Furthermore, it will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, steps, operations, and/or combinations thereof.
1. Laboratory instruments and materials
1.1 instruments
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 the national pharmacopoeia committee).
1.2 reagents
Acetonitrile (chromatographically pure, Merck, Germany) and water, Mili-Q ultrapure water.
Ribitol control (HPLC > 99%, Shanghai Huo medicine science and technology development Co., Ltd., lot number: 210916), fructose control (HPLC > 99%, Shanghai Huo medicine science and technology development Co., Ltd., lot number: 210519), sucrose control (HPLC > 99%, Shanghai Huo He medicine science and technology development Co., Ltd., lot number: 210620), stachyose control (HPLC > 99%, Shanghai Huo He medicine science and technology development Co., Ltd., lot number: 211026).
2. Conditions of the experiment
2.1 preparation of control mix solution
Precisely weighing reference substances of ribitol, fructose, sucrose and stachyose, respectively dissolving with 60% acetonitrile solution, fixing the volume to 10ml, shaking up to obtain 4 reference substance solutions, transferring appropriate amount of 4 single labels into the same 50ml volumetric flask, adding 60% acetonitrile solution, precisely fixing the volume, and shaking up to obtain mixed standard stock solution. Diluting the stock solution 5 times to obtain control mixed solution containing 0.6512mg/ml ribitol, 2.189mg/ml fructose, 1.636mg/ml sucrose, and 0.6767mg/ml stachyose.
2.2 preparation of test articles
Taking the content of the bupleurum tenue capsule in a mortar, grinding, precisely weighing, placing in a 25mL volumetric flask, adding 60% acetonitrile solution, heating and ultrasonically dissolving, cooling to room temperature, fixing the volume, shaking up, centrifuging, and taking the supernatant to obtain the bupleurum tenue capsule.
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; 28-33 min, 50% B, flow: 0.6 ml/min; column temperature: 30 ℃; the injection volume was 10. mu.L.
2.4 liquid chromatography-triple quadrupole-time of flight mass spectrometry (LC/Q-TOF-MS) 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 of the drying gas is 8 l/min; atomizer pressure 35 psi; the temperature of the sheath gas is 350 ℃, and the flow of the sheath gas is 11 l/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. And (4) after the analysis method is determined, detecting the sample by using a high-resolution mass spectrum to obtain a total ion flow graph of the sample. The chemical compositions of 7 chromatographic peaks are estimated and determined by comparison with a reference substance according to the accurate relative molecular mass obtained by the high-resolution mass spectrum, and the numbering and the deduction results are shown in the table 1.
TABLE 1 LC/Q-TOF-MS analysis of part of the carbohydrate component of Bupleurum tenue Capsule 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 injection 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: six samples of the test solution prepared in parallel are taken and respectively subjected to sample injection analysis. 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 test: and taking the same sample solution, carrying out sample injection analysis for 0, 4, 8, 12, 16 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.
3.2.2 results of the experiment
The bupleurum tenue capsules with the serial numbers of S1-S10 of different batches are measured, 7 common peaks are determined under the condition, the reference peak is determined as the peak area of fructose (peak 3) is relatively large and the separation degree of fructose and adjacent chromatographic peaks is good, 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. Under the conditions of sample injection precision and repeatability, 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. Under the stability experiment condition, the relative retention time of each chromatographic peak and the RSD value of the relative retention peak area are both less than 4%, which indicates that the test solution is stable within 24 h.
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 analysis method is also suitable for measuring the content of the saccharide component in the bupleurum tenue capsule, 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 control substance mixed solutions with different concentrations are taken, and 10 mu L of the control substance mixed solutions are injected respectively 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.
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 (3) taking the same sample solution, carrying out sample injection analysis for 0, 4, 8, 12, 16 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 adding 50mg of sample and a proper amount of mixed reference stock solution, ultrasonically heating and dissolving by using 60% acetonitrile, fixing the volume to 25mL after the temperature is restored to room temperature, shaking up, centrifuging, and taking supernatant for sample injection analysis.
3.2.2 results of the experiment
Combining the reference substance positioning and mass spectrum results, identifying peaks 2, 3, 5, 6, 7, 8 and 9 as ribitol, fructose, glucose, sucrose, maltose, raffinose and stachyose respectively. Selecting 4 components of ribitol, fructose, sucrose and stachyose with large peak area and excellent peak shape as content determination components.
The regression equation, linear range, detection limit and quantitative limit results of ribitol, fructose, sucrose and stachyose are shown in table 4, the linear fitting results of the 4 compounds are all more than 0.999, and the requirements of Chinese pharmacopoeia are met. The sample injection precision experiment results are shown in tables 5 and 6, the repeatability experiment results are shown in table 7, the solution stability experiment results are shown in table 8, wherein the precision, the repeatability and the stability RSD are all less than 3%, the method meets the requirements of Chinese pharmacopoeia, and the method is good in linearity and stable in 24 h. The sample adding and recovering experimental result is shown in table 9, the average recovering rate of each component meets the requirement, and the RSD value is less than 4%, so that the method obtained by optimization is accurate and reliable, and can be used for measuring the content of the saccharide component in the bupleurum tenue capsules.
TABLE 4 regression equation, correlation coefficient and Linear analysis Range of the ingredients
TABLE 5 sample introduction precision results (peak area)
TABLE 6 sample introduction precision results (retention time)
TABLE 7 results of repeated experiments
TABLE 8 results of solution stability experiments
TABLE 9 sample addition recovery test results
3.3 sample content measurement results and quality control example
The results of quantitative component content measurement of 11 test sample solutions are shown in Table 10. The content of ribitol in each batch of bupleurum tenue capsules is 0.8985-2.281%, the content of fructose is 1.815-9.018%, the content of sucrose is 2.735-5.320%, and the content of stachyose is lower than 2.430%, which have great difference. Wherein, the fructose and sucrose content is higher.
If the content of ribitol is not less than 1%, the content of fructose is not less than 2%, the content of sucrose is not less than 3%, and the content of stachyose is not less than 0.5% set by the internal control standard of the enterprise, the batches S4 and S11 are considered not to meet the internal control standard, and the release is not recommended.
Table 1011 batch test article solution quantitative component content determination result
Note: "0" means that the content of the component is less than the limit of quantitation
3.4 fingerprint similarity evaluation and quality control example
Introducing original chromatogram data of the test samples of 10 normal batches (S1-S10) of XIAOCHAIHU Capsule into traditional Chinese medicine chromatogram fingerprint similarity evaluation system software, and generating a control chromatogram, as shown in FIG. 3. The test samples were compared with the control spectra, and the similarity results are shown in Table 11. As can be seen from the table, the similarity between the fingerprint spectrum of each sample and the comparison spectrogram is over 0.90, and the carbohydrate components of the bupleurum tenue capsules of each batch have better quality consistency.
Table 1110 shows the similarity evaluation results of the finger prints of the normal batches of bupleurum tenue capsule test solution
Comparing the sample of another new batch S11 with the reference fingerprint, calculating to obtain similarity of 0.888 and similarity of less than 0.9, and determining that the quality of the batch of bupleuri radix capsule is different from that of the normal batch, and recommending that the quality is not approved.
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 (4)
1. A quantitative fingerprint detection method for saccharide components of bupleurum tenue capsules is characterized by comprising the following steps:
(1) preparation of control mixed solution and test solution
Preparing a reference substance mixed solution: taking four saccharide components including ribitol, fructose, sucrose and stachyose as reference substances, and preparing reference substance mixed solution;
preparing a test solution: weighing a plurality of batches of bupleurum tenue capsule contents as a test sample, respectively carrying out ultrasonic dissolution by using a solvent, shaking up, centrifuging, and obtaining a supernatant which is a test sample solution;
(2) high performance liquid chromatography assay
Injecting the reference substance mixed solution and the test substance solution in the step (1) into a liquid chromatography for analysis and determination, wherein the chromatographic conditions are as follows: a chromatographic column: NH2P-504E column, inner diameter 4.6mm x column length 250mm, particle size 5 μm, using water (A) -acetonitrile (B) as mobile phase for gradient elution, elution mode: for 0-10 min, 78-74% of B; for 10-28 min, 74-50% of B; 28-33 min, 50% B, flow: 0.6 ml/min; column temperature: 30 ℃;
(3) establishing quantitative fingerprint and evaluating quality
Preparing a plurality of different batches of bupleurum tenue capsules into different batches of test solution according to the method in the step (1), carrying out high performance liquid chromatography analysis according to the step (2), and recording a chromatogram; determining common peaks and quantitative detection indexes according to chromatograms of all different batches; meanwhile, the quality of the bupleurum tenue capsule is evaluated through the similarity of the fingerprint and the content of the carbohydrate.
2. The quantitative fingerprint detection method for the saccharide component of the bupleurum tenue capsule as claimed in claim 1, wherein the test solution is prepared by the following method: adding 60% acetonitrile solution into the capsule content, heating and ultrasonic dissolving, standing naturally until room temperature is recovered, diluting to desired volume, shaking, centrifuging, and collecting supernatant.
3. The method for quantitative fingerprint detection of the saccharide component of the bupleurum tenue capsule as claimed in claim 1, wherein the control mixed solution is prepared by the following method: precisely weighing reference substances of ribitol, fructose, sucrose and stachyose, dissolving with 60% acetonitrile solution respectively, fixing volume, shaking to obtain 4 reference substance solutions, mixing the 4 reference substance solutions, and shaking to obtain reference substance mixed solution.
4. The method for detecting the quantitative fingerprint spectrum of the carbohydrate component of the bupleurum tenue capsule as claimed in claim 1, wherein the total number of the peaks in the fingerprint spectrum is 7.
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