CN112924586B - Method for detecting bupleurum tenue particles - Google Patents
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- 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
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- 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
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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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- G—PHYSICS
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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- 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
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- 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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
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- G—PHYSICS
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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
Abstract
The invention relates to a detection method of bupleurum tenue particles. The detection method of the bupleurum tenue particles comprises the following steps: preparation of mixed control solution: collecting baicalin, baicalein, wogonin, wogonoside, ammonium glycyrrhizinate, and saikosaponin b2And saikosaponin b1Diluting the reference substance with solvent to obtain mixed reference substance solution; preparing a reference medicinal material solution; preparation of a test solution: dissolving bupleuri radix granule with extraction solvent, filtering, and collecting filtrate as sample solution; and respectively injecting the mixed reference substance solution, the reference medicinal material solution and the test solution into a high performance liquid chromatograph for determination. The invention can simultaneously carry out qualitative analysis on 15 components of 5 medicinal materials of the bupleurum tenue granules and 2 components of 2 medicinal materialsThe method for quantifying the components has the advantages of simple and convenient operation, high repeatability and good chromatographic peak pattern.
Description
Technical Field
The invention relates to the field of quality control of traditional Chinese medicines, in particular to a detection method of bupleurum tenue particles.
Background
The bupleurum tenue granules are Chinese medicinal granules prepared from Chinese medicinal materials such as bupleurum, scutellaria, liquorice, codonopsis pilosula, ginger processed pinellia and Chinese date, have the effects of relieving exterior syndrome, dissipating heat, soothing liver and harmonizing stomach, and are used for treating exogenous diseases and the syndrome of pathogen attacking shaoyang. The bupleurum tenue particles make important contribution in preventing and treating epidemic diseases such as SARS, influenza A, dengue fever, new coronary pneumonia and the like in recent years, are approved by a plurality of doctors and patients, have good public praise and wide market prospect.
In the current pharmacopoeia load standard, the thin layer identification of bupleurum tenue granules respectively carries out qualitative determination on baicalin, a liquorice control medicinal material and a bupleurum root control medicinal material, the content determination index is baicalin, and the product quality cannot be comprehensively and accurately controlled.
The intensive research on the quality control method of the Chinese medicinal compound preparation is an important problem for ensuring the stability and the clinical use safety of the Chinese medicinal compound preparation; the Chinese patent with publication number CN105486771B discloses a fingerprint detection method for a compound preparation of Xiaochaihu granules, wherein the constructed fingerprint respectively identifies 12 characteristic peaks which are respectively exclusive characteristic peaks of bupleurum, scutellaria, liquorice, codonopsis pilosula and ginger. The above method has obvious disadvantages: (1) detecting the fingerprint and the content by different methods; (2) the fingerprint is measured by adopting a nonlinear gradient, is not easy to reproduce and has relatively poor reproducibility; (3) three solutions of methanol, acetonitrile and 0.2% phosphoric acid are adopted as mobile phases, so that more mobile phase channels are used, more organic reagents are consumed, and the detection method is complicated; (4) the detection time is 105 minutes, the time consumption is long, and the efficiency is not high.
Disclosure of Invention
Based on the above, the invention aims to provide a method for qualitatively determining 15 components of 5 medicinal materials in Xiaochaihu granules and quantitatively determining 2 components of 2 medicinal materials, which is simple and convenient to operate and has high repeatability.
The specific technical scheme is as follows:
a method for constructing a fingerprint of bupleurum tenue particles comprises the following steps:
preparation of mixed control solution: collecting baicalin, baicalein, wogonin, wogonoside, ammonium glycyrrhizinate, and saikosaponin b2And saikosaponin b1Diluting the reference substance with solvent to obtain mixed reference substance solution;
preparation of reference drug solution: dissolving bupleuri radix, Scutellariae radix, Glycyrrhrizae radix, radix Codonopsis, and fructus Jujubae with extraction solvent, filtering, and collecting filtrate as control solution;
preparation of a test solution: dissolving bupleuri radix granule with extraction solvent, filtering, and collecting filtrate as sample solution;
respectively injecting the mixed reference substance solution, the reference medicinal material solution and the test solution into a high performance liquid chromatograph for determination to obtain fingerprint spectra;
the high performance liquid chromatograph adopts the following mobile phases: the organic phase is acetonitrile, the water phase is 0.15 wt% -0.25 wt% phosphoric acid water solution, and linear gradient elution is carried out.
In some of these embodiments, the linear gradient elution procedure is:
0-70 min, 10-20)% → (55-65)%, and 80-90)% → (35-45)%.
In some of these embodiments, the aqueous phase is a 0.18 to 0.22 wt% aqueous phosphoric acid solution;
the procedure for the linear gradient elution was:
0-70 min, 13-17)% → (58-62)%, and 83-87)% → (38-42)%.
In some of these embodiments, the column temperature of the liquid chromatography is 22 to 28 ℃, preferably 24 to 26 ℃.
In some embodiments, the extraction solvent is an aqueous ethanol solution, preferably an aqueous ethanol solution with a volume concentration of 60-80%.
In some of these embodiments, the solvent is methanol.
In some embodiments, the mixed control solution comprises baicalin, baicalein, wogonin, ammonium glycyrrhizinate, and saikosaponin b2And saikosaponin b1The concentration of the reference substance is 48-52 mug/mL.
In some of these embodiments, the chromatographic conditions further comprise: the flow rate is 0.7-0.9 ml/min, the detection wavelength is 250-255 nm, the sample injection amount is 8-12 mu L, and the chromatographic column is C18A chromatographic column.
In some of these embodiments, the method of dissolution is sonication.
The invention also aims to provide a method for measuring the content of bupleurum tenue particles, which comprises the following steps:
preparation of mixed control solution: taking baicalin and ammonium glycyrrhizinate as reference substances, adding extraction solvent to dilute, and using as mixed reference substance solution;
preparation of a test solution: dissolving bupleuri radix granule with extraction solvent, filtering, and collecting filtrate as sample solution;
respectively injecting the mixed reference substance solution and the test solution into a high performance liquid chromatograph for determination, and determining the contents of baicalin and glycyrrhizic acid in the bupleurum tenue particles;
the high performance liquid chromatograph adopts the following mobile phases: the organic phase is acetonitrile, the water phase is 0.15 wt% -0.25 wt% phosphoric acid water solution, and linear gradient elution is carried out.
In some of these embodiments, the linear gradient elution procedure is:
0-70 min, 10-20)% → (55-65)%, and 80-90)% → (35-45)%.
In some of these embodiments, the aqueous phase is a 0.18 to 0.22 wt% aqueous phosphoric acid solution;
the procedure for the linear gradient elution was:
0-70 min, 13-17)% → (58-62)%, and 83-87)% → (38-42)%.
In some of these embodiments, the column temperature of the liquid chromatography is 22 to 28 ℃, preferably 24 to 26 ℃.
In some embodiments, the extraction solvent is an aqueous ethanol solution, preferably an aqueous ethanol solution with a volume concentration of 60-80%.
In some embodiments, the concentration of baicalin in the mixed control solution is 0.25-0.27 mg/mL, and the concentration of glycyrrhizic acid is 38-42 μ g/mL.
In some of these embodiments, the chromatographic conditions further comprise: the flow rate is 0.7-0.9 ml/min, the detection wavelength is 250-255 nm (for glycyrrhizic acid content determination) and 313-317 nm (for baicalin content determination), the sample injection amount is 8-12 mu L, and the chromatographic column is C18A chromatographic column.
In some of these embodiments, the method of dissolving comprises sonication.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a method for simultaneously realizing fingerprint construction of bupleurum tenue particles and content determination of baicalin and glycyrrhizic acid. The inventor finds that when the fingerprint of the bupleurum tenue particles is constructed and the contents of baicalin and glycyrrhizic acid are measured, the concentration of the phosphoric acid aqueous solution has a large influence on the elution procedure of a detection system, and when the mobile phase aqueous phase is the phosphoric acid aqueous solution with the specific concentration of 0.15-0.25 wt%, and the organic phase is acetonitrile, the linear gradient elution procedure with simple operation and good universality can be realized. Moreover, the inventor unexpectedly finds that after a phosphoric acid aqueous solution with the concentration of 0.15-0.25 wt% is combined with a linear gradient elution program, the obtained chromatogram has good peak pattern, can simultaneously detect 15 components, and has high separation degree and repeatability of each chromatogram peak.
The fingerprint spectrum construction method of the bupleurum particles and the baicalin and glycyrrhizic acid content determination method can achieve good precision and stability, are favorable for comprehensively improving the quality control level of the bupleurum tenue particles, and ensure the safety, effectiveness and stable uniformity of products.
In addition, the invention adopts saikosaponin b2With saikosaponin b1Replaces saikosaponin a or saikosaponin d as detection index component, and realizes saikosaponin b in chromatogram2With saikosaponin b1Completely separating from other index components, and preventing saikosaponin a or saikosaponin d from being converted into saikosaponin b2With saikosaponin b1Adverse effect on detection accuracy is facilitated to improve the detection accuracy.
Drawings
FIG. 1 is a high performance liquid chromatogram overlay (A) of 10 batches of Xiaochaihu granules in example 1 of the present invention and a comparison fingerprint (B, in the figure, characteristic peaks 1 to 15 are respectively from left to right);
FIG. 2 is a chromatogram of the granule of Bupleurum tenue and the radix bupleuri (S1: Small Bupleurum tenue granule, S2: radix bupleuri, the number in the figure indicates the peak number of the chromatographic peak attributed to radix bupleuri in the granule of Small Bupleurum tenue);
FIG. 3 is a chromatogram of radix bupleuri granule and Scutellariae radix (S1: radix bupleuri granule, S2: Scutellariae radix, the number in the figure indicates the peak number of the chromatographic peak belonging to Scutellariae radix in the radix bupleuri granule);
FIG. 4 is a chromatogram of radix bupleuri granule and Glycyrrhrizae radix (S1: radix bupleuri granule, S2: Glycyrrhrizae radix, the number in the figure indicates the peak number of chromatographic peak belonging to Glycyrrhrizae radix in radix bupleuri granule);
FIG. 5 is a chromatogram of radix bupleuri granule and radix Codonopsis (in the figure, S1: radix bupleuri granule, S2: radix Codonopsis, numeral in the figure indicates the peak number of chromatographic peak belonging to radix Codonopsis in radix bupleuri granule);
FIG. 6 is a chromatogram of the contrast of XIAOCHAIHU granule and fructus Jujubae (S1: XIAOCHAIHU granule, S2: fructus Jujubae, numeral in the figure indicates the peak number of chromatographic peak belonging to fructus Jujubae in XIAOCHAIHU granule);
FIG. 7 is a schematic diagram showing the similarity between the finished product of small radix bupleuri granules and a standard fingerprint (in the figure, characteristic peaks 1 to 15 are respectively shown from left to right; R is a comparison fingerprint; S1-S10 are fingerprints of K00226, K00227, K00228, K00229, K00230, K00231, K00232, K00233, K00234 and K00235);
FIG. 8 is a chromatogram with the aqueous phase being water;
FIG. 9 is a chromatogram of an aqueous phase of 0.1 wt% phosphoric acid in water;
FIG. 10 is a chromatogram of an aqueous phase of 0.2 wt% phosphoric acid in water;
FIG. 11 shows C of three different manufacturers18Chromatographic peaks of the column (A: Feinigen; B: Welch; C: Phecda);
FIG. 12 shows the chromatographic peaks at different column temperatures (A: 25 ℃ C.; B: 20 ℃ C.; C: 30 ℃ C.).
Detailed Description
Experimental procedures according to the invention, in which no particular conditions are specified in the following examples, are generally carried out under conventional conditions, or under conditions recommended by the manufacturer. The various chemicals used in the examples are commercially available.
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. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to only those steps or modules listed, but may alternatively include other steps not listed or inherent to such process, method, article, or device.
The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.
The existing establishment of the fingerprint of the bupleurum tenue granules has the following technical problems: (1) the material types in the granule are complex, and the conversion between multiple effective components occurs, for example, saikosaponin a and saikosaponin d in bupleurum medicinal material are easily converted into saikosaponin b by heating2With saikosaponin b1In the existing detection method, the saikosaponin a is used as a detection index component, which can cause adverse effect on the accuracy of a detection result. (2) The existing method adopts a nonlinear gradient elution program, which is provided with a multi-step elution gradient, has complex operation and longer time consumption, and often causes poor reproducibility because of the complex elution program. (3) The existing method can not realize fingerprint map construction and content measurement of effective substances at the same time.
Based on the defects of the existing method, the invention finally provides a method for constructing the fingerprint spectrum, which can solve the technical problems, and the method comprises the following steps:
preparation of mixed control solution: collecting baicalin, baicalein, wogonin, wogonoside, ammonium glycyrrhizinate, and saikosaponin b2And saikosaponin b1Diluting the reference substance with solvent to obtain mixed reference substance solution;
preparation of reference drug solution: dissolving bupleuri radix, Scutellariae radix, Glycyrrhrizae radix, radix Codonopsis, and fructus Jujubae with extraction solvent, filtering, and collecting filtrate as control solution;
preparation of a test solution: dissolving bupleuri radix granule with extraction solvent, filtering, and collecting filtrate as sample solution;
respectively injecting the mixed reference substance solution, the reference medicinal material solution and the test solution into a high performance liquid chromatograph for determination to obtain fingerprint spectra;
the high performance liquid chromatograph adopts the following mobile phases: the organic phase is acetonitrile, the water phase is 0.15 wt% -0.25 wt% phosphoric acid water solution, and linear gradient elution is carried out.
The method for controlling the quality of the bupleurum tenue particles adopts the combination of the specific mobile phase and the specific linear gradient, can simultaneously realize the construction of the fingerprint and the content determination of the characteristic substances, has short time consumption and high reproducibility, and adopts the saikosaponin b2With saikosaponin b1Replacing saikosaponin a or saikosaponin d as detection index component, and realizing saikosaponin b in chromatogram2With saikosaponin b1Completely separating from other index components to obtain chromatographic peak with good peak shape and separation degree, and has good precision, stability and repeatability.
In some of these embodiments, the linear elution gradient in the present invention is: 0-70 min, 10-20)% → (55-65)%, and 80-90)% → (35-45)%. Further preferably, the water phase is 0.18-0.22 wt% phosphoric acid water solution; the procedure for the linear gradient elution was: 0-70 min, 13-17)% → (58-62)%, and 83-87)% → (38-42)%. In the research process, the invention discovers that whether the good detection effect of the bupleurum tenue particles can be realized by a linear gradient elution procedure or not, the selection of a mobile phase is very important, the chromatographic peak with good peak shape and separation degree can be obtained only by matching 0.18-0.22 wt% phosphoric acid aqueous solution with acetonitrile, and the separation effect is not ideal by using other aqueous phases such as water or phosphoric acid aqueous solutions with other concentrations.
Further, on the basis of the method, the column temperature of the liquid chromatogram is selected to be 22-28 ℃, and preferably 24-26 ℃. The obtained chromatographic peaks are best in comprehensive.
The present invention will be described in further detail with reference to specific examples.
Example 1
Instrument and reagent
The instrument comprises the following steps: ultra-fast high performance liquid mass spectrometer (LC-20AD-XR binary pump, SIL-20AD-XR autosampler, CTO-20A column oven, SPD-M10A-PDA detector) manufactured by SHIMADZU of Japan; agilent1260 hplc (Agilent, germany, G1311B single quad pump, G1329B autosampler, G1316A column oven, G1315D detector, OPENLAB CDS ChemStation Edition data processing software); dionex Ultimate 3000DGLC high performance liquid chromatograph (DGP-3600SD double triple pump, SRD-3600 degasser, WPS-3000SL autosampler, TCC3000-RS column incubator, DAD detector); a chromatographic column: Phecda-C18 (4.6X 250mm, 5 μm).
Reagent testing: 10 batches of finished product were supplied by Guanghua pharmaceutical GmbH, white cloud mountain, Guangzhou (K00226, K00227, K00228, K00229, K00230, K00231, K00232, K00233, K00234, K00235). In the experiment, acetonitrile and phosphoric acid used as reagents for liquid chromatography are both in chromatographic grade, the rest of the reagents are analytically pure, and water is ultrapure water.
The reference substance and the reference medicinal material are purchased from China food and drug testing research institute and are shown in Table 1:
table 1 reference
Example 1 construction of fingerprint
(1) Preparation of a test solution: grinding bupleuri radix granule, weighing 1.0g, precisely weighing, placing into conical flask with plug, precisely adding 70% ethanol 10ml, sealing, weighing, ultrasonic treating for 30min, cooling, weighing again, supplementing with 70% ethanol, shaking, filtering with 0.45 μm filter membrane, and collecting filtrate.
(2) Preparation of mixed control solution: weighing baicalin, baicalein, wogonin, wogonoside, ammonium glycyrrhizinate, and water extract,Saikosaponin b2And saikosaponin b1An appropriate amount of the control was placed in the same volumetric flask, and methanol was added to prepare a mixed control solution containing 50. mu.g of each of the above controls per ml.
(3) Preparation of reference drug solution: weighing bupleuri radix, Scutellariae radix, Glycyrrhrizae radix, radix Codonopsis, and fructus Jujubae respectively, grinding, taking about 1.0g, precisely weighing, placing into a conical flask with a plug, precisely adding 10ml of 70% ethanol, sealing, weighing, ultrasonically treating for 30min, cooling, weighing again, supplementing lost weight with 70% ethanol, shaking, filtering with 0.45 μm filter membrane, and taking the filtrate.
(4) And (3) analyzing by high performance liquid chromatography: precisely sucking 10 mul of test solution, injecting: chromatographic conditions are as follows: the chromatographic column is Phecda-C18(4.6X 250mm, 5 μm); acetonitrile is taken as a mobile phase A, 0.2 wt% phosphoric acid solution is taken as a mobile phase B, and the following gradient elution mode is adopted; the detection wavelength is 252 nm; flow rate: 0.8 ml/min; column temperature: obtaining the standard fingerprint of the high performance liquid chromatography of the bupleurum tenue particles at 25 ℃, as shown in figure 1, and figure 1 is the comparison fingerprint of the bupleurum tenue particles.
Gradient elution mode
| Time (minutes) | Mobile phase A (%) | Mobile phase B (%) |
| 0～70 | 15→60 | 85→40 |
(5) Determination of common peaks:
respectively taking batch numbers: preparing test solution according to the method for preparing and measuring small radix bupleuri particles of K00226, K00227, K00228, K00229, K00230, K00231, K00232, K00233, K00234 and K00235 to obtain a high performance liquid chromatogram superposition map of 10 batches of small radix bupleuri particles, specifically as shown in figure 1(A), comparing the high performance liquid chromatogram fingerprints of the 10 batches of small radix bupleuri particles by a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), determining 15 common characteristic peaks and obtaining a common mode (comparison fingerprint); FIG. 1(B) is a fingerprint of the granule of Bupleurum tenue of the present invention, wherein the characteristic peaks 1 to 15 are shown from left to right in FIG. 1.
By comparing with the control solution, the peak attribution is as follows:
the characteristic peaks belonging to the bupleurum are No. 2, No. 13 and No. 14 peaks; FIG. 2 is a chromatogram of control of XIAOCHAIHU granule and bupleuri radix.
Characteristic peaks belonging to scutellaria baicalensis are No. 3, 4, 6, 7, 8, 9, 10, 11 and 15 peaks; FIG. 3 is a chromatogram of control of bupleuri radix granule and Scutellariae radix.
The characteristic peak belonging to licorice is peak number 12; FIG. 4 is a chromatogram of control of bupleuri radix granule and Glycyrrhrizae radix.
The characteristic peak belonging to the codonopsis pilosula is the No. 1 peak; FIG. 5 is a chromatogram of control of bupleuri radix granule and radix Codonopsis.
The characteristic peak belonging to the Chinese date is No. 5 peak; FIG. 6 is a chromatogram of control of bupleuri radix granule and fructus Jujubae.
FIG. 7 is a schematic view of the similarity between the finished product of Xiaochaihu granules and the standard fingerprint spectrum.
Performing chromatographic peak attribution and peak purity detection on the main peak by using a DAD detector and UFLC-TRIPLE TOF-DAD-MS/MS technical means; mass spectrum working parameters: ESI electrospray ion source, ion spray voltage positive mode 5500V, negative mode-4500V; auxiliary gas 1: 60 PSI; auxiliary gas 2: 50 PSI; ion source temperature: 500 ℃; air curtain air 40 PSI; scanning ranges of m/z 50-1000, and detecting by adopting a positive ion mode and a negative ion mode respectively; high performance liquid chromatography conditions: c18 (2.1X 100mm, 1.8 μm) as column, column temperature: and (3) performing gradient elution by using acetonitrile as a mobile phase A and 0.1% formic acid solution as a mobile phase B at 40 ℃, wherein the gradient elution is performed in a time period of 0min → 15 min: 5% → 30% a, 15min → 30 min: 30% → 95% a, 30min → 32 min: 95% A, 32min → 32.1 min: 95% → 5% a, 32.1min → 35 min: 5% → 0% a, flow rate: 0.25 ml/min; sample introduction amount: 1 μ l.
And (3) identifying molecular ion peaks and cracking fragment information of the combined mass spectrum by comparing UV absorption curves: the 1 st peak is codonopsitis alkali, the 2 nd peak is kaempferol-glucosyl-rhamnoside, the 3 rd peak is scutellarin, the 4 th peak is 6-arabinosyl-8-glucosyl-5, 7-dihydroxyflavone, the 5 th peak is quercetin, the 6 th peak is baicalin, the 7 th peak is dihydrooroxin A, the 8 th peak is baicalin isomer, the 9 th peak is luteolin, the 10 th peak is wogonoside, the 11 th peak is baicalin, the 12 th peak is glycyrrhizic acid, the 13 th peak is saikosaponin b2, the 14 th peak is saikosaponin b1, and the 15 th peak is wogonin.
Taking baicalin 6 # peak as a reference peak, the list of relative retention values of common peaks of the fingerprint spectrum of the bupleurum tenue granules is as follows:
(6) methodology study
6.1 precision test: precisely weighing the same bupleurum tenue particle sample solution, continuously injecting the sample solution for 6 times on a high performance liquid chromatograph, detecting a fingerprint, and evaluating by adopting a traditional Chinese medicine chromatography fingerprint similarity evaluation system (2012 edition), wherein the similarity of the sample fingerprint and a comparison fingerprint is 1.00, and the result shows that the precision of the instrument is good and is shown in a table 2.
TABLE 2 precision test similarity results of Xiaochaihu granules
6.2 stability test: precisely weighing the same small radix bupleuri particle test sample solution, injecting samples in 0, 2, 4, 6, 12, 24 and 48 hours respectively, detecting a fingerprint, evaluating by a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), wherein the similarity of the test sample fingerprint and a comparison fingerprint is 1.00, the similarity of the test sample fingerprint and the comparison fingerprint is 1.00, and the result shows that the test sample solution is stable in 48 hours and is shown in a table 3.
TABLE 3 stability test similarity results for Xiaochaihu granules
6.3 repeatability tests: precisely weighing 6 parts of the same batch of bupleurum tenue particles, operating according to the method under the preparation item of the test solution, respectively injecting samples, detecting fingerprint spectrums, and evaluating by adopting a traditional Chinese medicine chromatogram fingerprint spectrum similarity evaluation system (2012 edition), wherein the similarity of the test sample fingerprint spectrum and the comparison fingerprint spectrum is 1.00, and the result shows that the repeatability of the method is good, and the result is shown in table 4.
TABLE 4 results of similarity of the Bupleurum tenue granule repeatability tests
| S1 | S2 | S3 | S4 | S5 | S6 | Comparison fingerprint | |
| S1 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S2 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S3 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.999 | 1.000 |
| S4 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S5 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S6 | 1.000 | 1.000 | 0.999 | 1.000 | 1.000 | 1.000 | 1.000 |
| Comparison fingerprint | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
6.4 intermediate precision: precisely weighing the same batch of small radix bupleuri particles, determining according to the method under the condition of variable factors such as different dates, different analysts, different instruments and the like, detecting the fingerprint, and evaluating by adopting a traditional Chinese medicine chromatography fingerprint similarity evaluation system (2012 edition). The result shows that the method has good intermediate precision, the HPLC fingerprint quality control method of the bupleurum tenue granules is feasible, and the result is 5-7.
(1) Different analysis times: taking the same batch of bupleurum tenue particles, operating according to the method under the item of preparation of test solution on different dates, and adopting the traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition) to evaluate, wherein the similarity of the fingerprint of the test sample and the comparison fingerprint is 1.00.
TABLE 5 similarity results of different analysis time tests of Xiaochaihu granules
| |
|
Comparison | |
| Analysis time | |||
| 1 | 1.000 | 1.000 | 1.000 |
| |
1.000 | 1.000 | 1.000 |
| Comparison fingerprint | 1.000 | 1.000 | 1.000 |
(2) Different analysts: taking the same batch of bupleurum tenue particles, and operating different personnel according to the method under the item of preparation of test solution, and adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition) to evaluate, wherein the similarity of the fingerprint of the test sample and the comparison fingerprint is 1.00.
TABLE 6 similarity results of different analyst tests of Xiaochaihu granules
| |
|
| |
| Analysts | |||
| 1 | 1.000 | 1.000 | 1.000 |
| |
1.000 | 1.000 | 1.000 |
| Comparison fingerprint | 1.000 | 1.000 | 1.000 |
(3) Different instruments: taking the same batch of bupleurum tenue particles, operating according to the method under the item of preparation of test solution, respectively determining in different instruments according to the method, and adopting the similarity evaluation system of traditional Chinese medicine chromatogram fingerprint (2012 edition) to evaluate, wherein the similarity of the test sample fingerprint and the comparison fingerprint is 1.00.
TABLE 7 similarity results of different analyzers for small Bupleurum particles
| |
|
Comparison fingerprint | |
| |
1.000 | 1.000 | 1.000 |
| |
1.000 | 1.000 | 1.000 |
| Comparison fingerprint | 1.000 | 1.000 | 1.000 |
In conclusion, through methodology research, the fingerprint detection method provided by the invention is good in repeatability, stability and precision, can detect 15 components of 5 medicinal materials, and can control the quality of the bupleurum tenue particles more comprehensively.
Example 2 measurement of index component content of Xiaochaihu granule
1. Preparation of a test solution: grinding bupleuri radix granule, weighing 1.0g, precisely weighing, placing into conical flask with plug, precisely adding 70% ethanol 10ml, sealing, weighing, ultrasonic treating for 30min, cooling, weighing again, supplementing with 70% ethanol, shaking, filtering with 0.45 μm filter membrane, and collecting filtrate.
2. Preparation of mixed control solution: weighing appropriate amount of baicalin and ammonium glycyrrhizinate, placing in the same volumetric flask, adding 70% ethanol to obtain mixed reference solution containing baicalin 0.26mg and glycyrrhizic acid 40 μ g per ml. (glycyrrhizic acid-ammonium glycyrrhizinate/1.0207).
3. High performance liquid chromatography conditions: precisely sucking 10 mu l of sample, injecting: chromatographic conditions are as follows: the column was Phecda-C18 (4.6X 250mm, 5 μm); acetonitrile is used as a mobile phase A, 0.2 wt% phosphoric acid solution is used as a mobile phase B, and the following gradient elution mode is adopted:
| time (minutes) | Mobile phase A (%) | Mobile phase B (%) |
| 0～70 | 15→60 | 85→40 |
Detection wavelength: 252nm (for glycyrrhizic acid content determination) and 315nm (for baicalin content determination); flow rate: 0.8 ml/min; column temperature: at 25 ℃.
4. Methodology study
4.11 Linear relationship examination: taking the concentration of the baicalin reference substance respectively: 0.0001mg/ml, 0.0256mg/ml, 0.0512mg/ml, 0.1024mg/ml, 0.2049mg/ml and 0.4098mg/ml, injecting samples according to the chromatographic conditions, and drawing a standard curve by taking the concentration of a reference substance as an abscissa (x) and the peak area as an ordinate (y), wherein the regression equation is as follows: y-342.34 x-0.3355, r-1, results show that in the concentration range: the linear relation between the baicalin and the peak area in 0.0001-0.4098 mg/ml is good.
Taking the concentration of ammonium glycyrrhizinate as a reference substance respectively: 0.0004mg/ml, 0.0036mg/ml, 0.0073mg/ml, 0.0146mg/ml, 0.0291mg/ml, 0.0582mg/ml, were injected under the above chromatographic conditions, and a standard curve was plotted with the concentration of the control as abscissa (x) and the peak area as ordinate (y), the regression equation: y-123.79 x-0.0139, r-1, results show that in the concentration range: the linear relation between the peak area and the peak area is good within 0.0004-0.0582 mg/ml.
4.12 lower limit of quantitation:
taking appropriate amount of baicalin control solution (concentration: 0.0001mg/ml), injecting 10 μ l, and measuring according to the above method. When the signal to noise ratio is about 10:1, continuous sample injection is carried out for 5 times, and the peak area RSD is 5.38% (see the table below), so the lower limit of the quantification of the content determination method is 0.0001 mg/ml.
Lower limit of baicalin quantification
Taking a proper amount of glycyrrhizic acid reference substance solution (concentration: 0.0004mg/ml), injecting 10 mul of sample, and measuring by the method. When the signal to noise ratio is about 10:1, sample introduction is carried out for 5 times continuously, and the peak area RSD is 3.13% (see the table below), so the lower limit of the quantification of the content determination method is 0.0004 mg/ml.
Lower limit of glycyrrhizic acid quantification
4.2 precision test: taking baicalin and glycyrrhizic acid reference substance solution, carrying out continuous sample injection for 6 times according to the method, recording a chromatogram, and calculating RSD of peak area precision of the baicalin and glycyrrhizic acid to be 0.10% and 0.11% respectively, which shows that the instrument of the invention has good precision.
4.3 repeatability tests: taking 6 parts of the same batch of bupleurum particles, each part of which is about 1.0g, precisely weighing, preparing according to a preparation method of a test solution, carrying out sample injection measurement according to the chromatographic conditions, and calculating to obtain the RSD of the baicalin and glycyrrhizic acid content of 0.57 percent and 0.65 percent respectively, which shows that the method has good repeatability, and is shown in the following table.
Baicalin content determination repeatability test of small radix bupleuri particles
Glycyrrhetic acid content determination repeatability test of bupleurum tenue granules
4.4 stability test: taking about 1.0g of bupleurum tenue particles, precisely weighing, preparing according to a preparation method of a test solution, respectively carrying out sample injection measurement for 0, 2, 4, 6, 12, 24 and 48 hours according to the chromatographic conditions, and respectively carrying out sample injection measurement on the baicalin and glycyrrhizic acid peak areas with RSD of 0.08 percent and 0.48 percent by calculation, which shows that the test solution has good stability within 48 hours.
4.5 sample recovery test: taking 9 parts of bupleurum tenue particles, each part of which is about 0.5g, precisely weighing, respectively adding a proper amount of each reference substance, preparing according to a preparation method of a test solution, carrying out sample injection measurement according to the chromatographic conditions, and calculating to obtain the average sample injection recovery rates of baicalin and glycyrrhizic acid which are respectively 98.88 percent and 98.55 percent, and RSD which are respectively 1.13 percent and 1.34 percent, which shows that the method has good accuracy.
4.6 content determination results and content limitation of Xiaochaihu granules
Taking 10 batches of bupleuri radix granules, preparing according to the preparation method of test solution, performing sample injection measurement according to the chromatographic conditions, and calculating the contents of baicalin and glycyrrhizic acid in the sample; regulating the content of baicalin in the obtained granule of Bupleurum tenue by 20% according to the average value to obtain granule of Bupleurum tenue with baicalin content limit of 25.0 mg/bag and glycyrrhizic acid content limit of 2.5 mg/bag.
TABLE 810 measurement of the contents of two components in a batch of Xiaochaihu granules (mg/bag)
Example 3
This example differs from example 1 in that the aqueous phase of the mobile phase is selected from one of the following groups (1) to (3):
(1) ultrapure water;
(2)0.1 wt% phosphoric acid;
(3)0.2 wt% phosphoric acid (example 1).
The chromatograms are shown in fig. 8-10, and the data such as the separation degree of the chromatograms and the symmetry degree of the peak patterns show that the good chromatographic peak shown in fig. 10 can be realized only by matching the water phase of the mobile phase selected as phosphoric acid aqueous solution with the concentration of 0.2 wt% in example 1 with the linear elution gradient of the invention, and the separation degree and the symmetry degree of each peak are good, and meanwhile, the chromatogram has the advantages of simple operation and high repeatability. When the aqueous phase using pure water or an aqueous solution of phosphoric acid having a concentration of 0.1 wt% as a mobile phase was combined with the linear elution gradient, it was found in fig. 8 and 9 that the overall peak shape was poor and the detection effect was poor.
Example 4
This example differs from example 1 in that the column is selected from the following group (1) to (3):
(1)Phecda C18(4.6×250mm，5μm)；
(2)Feinigen RedClassical II C18(4.6×250mm，5μm)；
(3)Welch Ultimate XB-C18(4.6×250mm，5μm)；
the results are shown in FIG. 11 (A: Feinigen; B: Welch; C: Phecda), C from three different manufacturers18The chromatographic column has good chromatographic peak separation degree and peak type, and the invention is suitable for different C18The chromatographic column has good durability.
Example 5
This example is different from example 1 in that column temperatures of 20 ℃, 25 ℃ and 30 ℃ were selected, respectively.
As a result, as shown in FIG. 12 (A: 25 ℃ C.; B: 20 ℃ C.; C: 30 ℃ C.), the chromatographic peak patterns at 25 ℃ C. were the best because the separation degrees of peaks No. 8 and 9 from peaks No. 11 and 12 at 20 ℃ C. were poor and the separation degree of peak No. 15 at 30 ℃ C. was poor.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. The method for constructing the fingerprint of the bupleurum tenue particles is characterized by comprising the following steps:
preparation of mixed control solution: collecting baicalin, baicalein, wogonin, wogonoside, ammonium glycyrrhizinate, and saikosaponin b2And saikosaponin b1Diluting the reference substance with solvent to obtain mixed reference substance solution;
preparation of reference drug solution: dissolving bupleuri radix, Scutellariae radix, Glycyrrhrizae radix, radix Codonopsis, and fructus Jujubae with extraction solvent, filtering, and collecting filtrate as control solution;
preparation of a test solution: dissolving bupleuri radix granule with extraction solvent, filtering, and collecting filtrate as sample solution;
respectively injecting the mixed reference substance solution, the reference medicinal material solution and the test solution into a high performance liquid chromatograph for determination to obtain fingerprint spectra;
the high performance liquid chromatograph adopts the following mobile phases: carrying out linear gradient elution on an organic phase which is acetonitrile and a water phase which is 0.18-0.22 wt% of phosphoric acid aqueous solution;
the procedure for the linear gradient elution was:
0-70 min, 13-17)% → (58-62)%, and 83-87)% → (38-42)%.
2. The method of claim 1, wherein the aqueous phase is a 0.2 wt% phosphoric acid aqueous solution;
the procedure for the linear gradient elution was:
0-70 min, 15% → 60% of organic phase and 85% → 40% of aqueous phase.
3. The construction method according to claim 1, wherein the column temperature of the liquid chromatography is 22 to 28 ℃.
4. The method according to claim 3, wherein the column temperature of the liquid chromatography is 24 to 26 ℃.
5. The method according to any one of claims 1 to 4, wherein the extraction solvent is an aqueous ethanol solution;
and/or, the solvent is methanol;
and/or, the chromatographic conditions further comprise: the flow rate is 0.7-0.9 ml/min, the detection wavelength is 250-255 nm, the sample injection amount is 8-12 mu L, and the chromatographic column is C18A chromatographic column;
and/or the method of dissolution comprises sonication.
6. A method for measuring the content of bupleurum tenue particles is characterized by comprising the following steps:
preparation of mixed control solution: taking baicalin and ammonium glycyrrhizinate as reference substances, adding extraction solvent to dilute, and using as mixed reference substance solution;
preparation of a test solution: dissolving bupleuri radix granule with extraction solvent, filtering, and collecting filtrate as sample solution;
respectively injecting the mixed reference substance solution and the test solution into a high performance liquid chromatograph for determination, and determining the contents of baicalin and glycyrrhizic acid in the bupleurum tenue particles;
the high performance liquid chromatograph adopts the following mobile phases: carrying out linear gradient elution on an organic phase which is acetonitrile and a water phase which is 0.18-0.22 wt% of phosphoric acid aqueous solution;
the procedure for the linear gradient elution was:
0-70 min, 13-17)% → (58-62)%, and 83-87)% → (38-42)%.
7. The method according to claim 6, wherein the aqueous phase is a 0.2 wt% phosphoric acid aqueous solution;
the procedure for the linear gradient elution was:
0-70 min, 15% → 60% of organic phase and 85% → 40% of aqueous phase.
8. The method according to claim 6, wherein the column temperature of the liquid chromatography is 22 to 28 ℃.
9. The method according to claim 8, wherein the column temperature of the liquid chromatography is 24 to 26 ℃.
10. The content measurement method according to any one of claims 6 to 9, wherein the chromatographic conditions of the liquid chromatography further include:
the extraction solvent is ethanol water solution;
and/or, the chromatographic conditions further comprise: the flow rate is 0.7-0.9 ml/min, the detection wavelength is 250-255 nm and the dual wavelength of 313-317 nm, the sample injection amount is 8-12 mu L, and the chromatographic column is C18A chromatographic column;
and/or the method of dissolution comprises sonication.
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| CN114113436A (en) * | 2021-12-02 | 2022-03-01 | 广州白云山光华制药股份有限公司 | Method for measuring total flavone content of scutellaria baicalensis based on fingerprint spectrum and application |
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| CN114324666B (en) * | 2021-12-31 | 2023-11-24 | 成都中医药大学 | HPLC fingerprint detection method for Tibetan radix codonopsis |
| CN114563496B (en) * | 2022-03-04 | 2023-03-21 | 浙江赞生药业有限公司 | Quantitative fingerprint analysis method for components in ginger, ginger and pinellia tuber percolate |
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