CN110568108B - Multi-component content determination method of Ganfule preparation - Google Patents
Multi-component content determination method of Ganfule preparation Download PDFInfo
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Abstract
The invention relates to a method for measuring the content of medicinal components, in particular to a method for measuring the content of multiple components of a Ganfule preparation, which comprises the following steps: s1, preparing a reference solution; s2, preparing a test solution; s3, content determination: respectively obtaining a reference chromatogram of the reference solution and a test chromatogram of the test solution by adopting an HPLC method; and analyzing the content of the corresponding component in the test solution according to the predetermined concentration of the standard component, the peak area in the chromatogram of the reference product and the peak area in the chromatogram of the corresponding component corresponding to the standard component. Compared with the prior art, the invention has the beneficial effects that: the determination method provided by the invention has the advantages of high sensitivity, strong anti-interference, simple and convenient operation and the like, and can quickly and efficiently determine the content of the components in the sample.
Description
Technical Field
The invention relates to a method for measuring the content of a medicinal component, in particular to a method for measuring the content of multiple components of a Ganfule preparation.
Background
The Chinese herbal compound 'Ganfule' is the first three new Chinese herbal medicines for treating liver diseases in national level, has received the first-grade prize of scientific and technological progress of the State administration of traditional Chinese medicine in 1992, and is listed as an anti-tumor medicine in the national basic insurance medical medicine catalogue in 2004.
The existing Ganfule clinical medicine is mainly in the dosage forms of decoction, tablets, capsules and the like, the prescription is composed of 21 traditional Chinese medicines such as barbed skullcap herb, largehead atractylodes rhizome, astragalus, dried orange peel, rhubarb and the like, and is used for treating primary liver cancer mainly caused by liver stasis and spleen deficiency, and the symptoms of primary liver cancer include abdominal mass, hypochondrium pain, mental fatigue and hypodynamia, anorexia, abdominal fullness, vexation and irritability, bitter taste and dry throat and the like. The prescription is divided into three groups according to the efficacy: spleen invigorating and qi regulating group (radix Codonopsis, parched Atractylodis rhizoma, radix astragali, Poria, Coicis semen, rhizoma Cyperi preparata, pericarpium Citri Tangerinae, bupleuri radix, lignum Aquilariae Resinatum, and caulis Clematidis Armandii); blood stasis removing and hard mass softening group (carapax Trionycis processed with vinegar, Eupolyphaga Seu Steleophaga, semen Persicae, lignum sappan, Concha Ostreae, radix Curcumae, and radix et rhizoma Rhei); group for clearing away heat and toxic materials (rhizoma paridis, herba Patriniae, herba Artemisiae Scopariae, and herba Scutellariae Barbatae).
Accordingly, the main characteristic components detected by the invention are as follows: atractylenolide I, calycosin glycoside, hesperidin and narirutin are respectively main effective and characteristic components in atractylis ovata, astragalus and dried orange peel in the group for strengthening spleen and regulating qi; aloe-emodin is the main effective and characteristic component of radix et rhizoma Rhei in the group of removing blood stasis and softening hard masses; scutellarin and baicalin are main effective and characteristic components of the sculellaria barbata for clearing away heat and toxic materials.
The fingerprint study on Ganfule capsules is reported in Linxinwen, Chenna, Chenzhen, Ganfule capsules, 2019,17(7):42-44, but only 22 common peaks in the capsules are determined and the common peaks are subjected to medicinal material attribution, and characteristic components representing dose-effect relationship in the prescription are not quantitatively analyzed. Ganfule capsules implement the 'national food and drug administration Standard (YBZ 12132006)', Ganfule tablets implement the 'national food and drug administration Standard (WS3-108(Z-018) -2004 (Z))', and only hesperidin is quantified in the 2 preparation Standard, but the single index quantification cannot really and comprehensively control the inherent quality of the preparation.
Therefore, in view of the quality control problem of the existing ganfule preparation, the invention of atractylenolide I, calycosin glycoside, hesperidin and narirutin which can respectively play a role in strengthening spleen and regulating qi needs to be urgently invented; aloe-emodin for removing blood stasis and softening hard masses; the medicinal effect characteristic components of the heat-clearing and detoxifying group, such as scutellarin, baicalin and the like, are used as index components to carry out multi-component content determination in the Ganfule preparation, and the quality of the Ganfule preparation can be more comprehensively controlled.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a method for measuring the contents of multiple components of a Ganfule preparation, simultaneously measure the contents of multiple main components in the Ganfule preparation and more comprehensively control the quality of the Ganfule preparation.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for measuring the contents of multiple components of Ganfule preparation comprises the following steps:
s1, preparation of control solution: weighing a standard component preparation, dissolving the standard component preparation in a first predetermined solvent to prepare a reference substance solution with the standard component at a predetermined concentration; the standard component preparation comprises a plurality of standard components;
s2, preparation of a test solution: weighing a Ganfule preparation sample with a predetermined mass, adding a second predetermined solvent, and performing extraction treatment to obtain a test sample solution;
s3, content determination: respectively obtaining a reference chromatogram of the reference solution and a test chromatogram of the test solution under the same chromatographic conditions by adopting an HPLC method;
the chromatographic conditions include:
mobile phase: the phase A is acetonitrile, the phase B is 0.1 percent formic acid water, and gradient elution is carried out by a preset gradient elution process;
detection wavelength: 285nm under the condition of time of 0-30 min and 35-50 min; 260nm under the conditions of 30-35 min and 50-80 min;
and S4, respectively analyzing the content of the corresponding component according to the preset concentration of the standard component, the peak area in the chromatogram of the reference product and the peak area in the chromatogram of the corresponding component corresponding to the standard component in the test solution.
Preferably, the method for measuring the multi-component content of the ganfule preparation comprises the following steps: hesperidin, scutellarin, baicalin, atractylenolide I, calycosin glycoside, narirutin, and aloe-emodin.
Preferably, the chromatographic conditions further include:
flow rate: 0.8-1 mL/min;
specification of chromatographic column: 250mm × 4.6mm, 5 μm;
column temperature: 20 to 30 ℃.
Preferably, the method for measuring the multi-component content of the ganfule preparation comprises the following steps:
0-5 min: 10% mobile phase a, 90% mobile phase B;
5-45 min: 10% → 25% mobile a phase, 90% → 75% mobile B phase;
45-65 min: 25% → 40% mobile a phase, 75% → 60% mobile B phase;
65-75 min: 40% → 80% mobile a phase, 60% → 20% mobile B phase;
75-80 min: 80% mobile phase A, 20% mobile phase B.
Preferably, the method for measuring the content of multiple components in the Ganfule preparation comprises the steps of firstly, measuring the content of the multiple components in the Ganfule preparation, and then, measuring the content of the multiple components in the Ganfule preparation, wherein the first preset solvent is a 25-100% methanol solvent or a 25-100% ethanol solvent;
the second predetermined solvent is a 25-100% methanol or 25-100% ethanol solvent.
Preferably, in the method for measuring the content of multiple components of the ganfule preparation, in step S2, the extraction is ultrasonic extraction;
the extraction conditions include:
ultrasonic frequency: 40-60 KHz;
extraction time: and (4) 1 h.
Preferably, in the method for measuring the content of multiple components in the ganfule preparation, step S2 includes:
grinding a predetermined mass of Ganfule preparation sample, adding a first predetermined volume of the second predetermined solvent, weighing an initial weight, carrying out ultrasonic extraction treatment, adding the second predetermined solvent, complementing the weight loss to the initial weight, and shaking up;
filtering with microporous membrane to obtain secondary filtrate, diluting the secondary filtrate with water by predetermined times, and filtering with C18Solid phase micro extraction column, eluting, and collecting methanol elution part;
and (3) after the methanol solvent is evaporated in a water bath, adding the second predetermined solvent to dissolve and fixing the volume to a second predetermined volume to obtain the sample solution.
Preferably, the microporous filter membrane is a 0.22 μm microporous filter membrane.
Preferably, the method for measuring the content of the multiple components of the Ganfule preparation comprises decoction, tablets and capsules.
Compared with the prior art, the invention has the beneficial effects that:
1. the determination method provided by the invention has the advantages of high sensitivity, strong anti-interference, simple and convenient operation and the like, and can quickly and efficiently determine the content of components in a sample;
2. according to the multi-component content determination method provided by the invention, the standard component preparation has corresponding components in the Ganfule preparation, the HPLC method is adopted for simultaneous detection, and a plurality of index components are simultaneously detected in the same chromatogram, so that the contradiction that the components cannot be detected under a single wavelength due to large difference of absorption wavelengths is solved, the interference of impurities is reduced while each component is ensured to have large absorption, the specificity and the detection accuracy of each component chromatographic peak are improved, the detection method is simple, convenient and feasible, the detection time of different index components is shortened, and the quality of the Ganfule preparation is effectively controlled;
3. the determination method provided by the invention is simple, convenient, stable, high in precision, good in reproducibility and easy to master.
Drawings
FIGS. 1a-1b are sample solution chromatograms of a mobile phase system of example 1, which is provided by the present invention, of methanol-formic acid aqueous solution and acetonitrile-formic acid aqueous solution, respectively;
FIGS. 2a-2c are sample solution chromatograms in Table 1, Table 2, and Table 3, respectively, showing elution procedures in example 1 provided by the present invention;
FIGS. 3a to 3c are chromatograms of the sample solution in example 1 provided by the present invention, in which the wavelengths are respectively 260nm and 285nm, and the wavelengths are converted in different time periods;
FIGS. 4a-4b are chromatograms of the test solution at flow rates of 0.8mL/min and 1.0mL/min, respectively, in example 1 provided by the present invention;
FIGS. 5a to 5c are chromatograms of a test solution at column temperatures of 20 deg.C, 25 deg.C and 30 deg.C, respectively, in example 1 provided by the present invention;
FIGS. 6a to 6c are chromatograms of sample solutions of the present invention in example 1, wherein the predetermined solvents are 25% methanol, 50% methanol, and 100% methanol, respectively; (ii) a
FIG. 7 is a chromatogram of a test solution of Ganfule capsule of lot 20180505 according to example 2 of the present invention;
FIG. 8 shows HPLC finger prints of 10 Ganfule capsules in example 4.
RSD (relative standard deviation)
DAD (photo-diode-array detector),
HPLC (High Performance Liquid Chromatography)
Detailed Description
In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
1. Instruments and reagents
The instrument equipment comprises: a DAD detector; one-ten-thousandth electronic analytical balance; the chromatographic column is 250mm multiplied by 4.6mm and 5 mu m; a 12-bit solid phase chromatography extraction instrument; numerical control ultrasonic cleaner.
Control, i.e. standard ingredient formulation: atractylenolide I with a purity of 98%, baicalin with a purity of 98%, narirutin with a purity of 98%, hesperidin with a purity of 98%, aloe-emodin with a purity of 98%, scutellarin with a purity of 98% and calycosin glucoside with a purity of 97.6%.
Reagent materials: chromatographic acetonitrile, chromatographic methanol; the water is ultrapure water; the purity of the formic acid is analytically pure; ion chromatography using C18And (4) molding a pretreatment column.
2. Sources of drugs
Ganfule capsule, batch number: 20180219, 20180301, 20180302, 20180505, 20180506, 20171206, 20171205, 20171113, 20171114, 20171003.
Ganfule tablets, batch number: 20180408, 20180520, 20171101, 20171208, 20170409.
Example 1
The first step,Preparation of a reference solution: accurately weighing a proper amount of each reference substance respectively, adding a proper amount of 50% methanol, dissolving by using ultrasonic waves, fixing the volume, and preparing a reference substance solution, wherein the content of each characteristic component in the reference substance solution is as follows: atractylodes macrocephala lactone I60 mug. mL-120.4. mu.g/mL of calycosin glucoside-1108. mu.g/mL scutellarin-1Naringin, Rutaceae, 800 ug/mL-1Hesperidin 400. mu.g.mL-1Baicalin 110. mu.g/mL-1Aloe-emodin 20 microgram/mL-1。
Step two, preparing a test solution: weighing 1.0g of pulverized GANFULE Capsule (batch No. 20180505), precisely weighing, placing in a conical flask with a plug, extracting with 25mL of 50% methanol under ultrasonic wave (40kHz) for 1h, cooling to room temperature, adding 50% methanol to balance weight, centrifuging at 15000r/min for 10min, precisely weighing supernatant 1.0mL, diluting with water to 5mL, and passing through C18And (3) performing solid phase extraction on the type pretreatment column (activating by using chromatographic methanol and ultrapure water respectively before use), sequentially eluting by using 5mL of 10% methanol and 5mL of methanol, collecting methanol eluent, evaporating to dryness in a water bath at 60 ℃, dissolving by using 50% of methanol, fixing the volume to 1.0mL, and filtering by using a 0.22 mu m microporous membrane to obtain the product. Specifically, in the process of preparing the test solution, the ganfule capsules are selected from one or more of ten batch numbers of the ganfule capsules in a medicine source, and multiple batch numbers are selected, wherein each batch number is used for preparing the test solution.
Step three, respectively carrying out content detection on the reference substance solution and the test solution by adopting an HPLC method;
chromatographic conditions during detection:
a chromatographic device: a high performance liquid chromatograph, the high performance liquid chromatograph being a DAD detector;
a chromatographic column: 250 mm. times.4.6 mm, 5 μm.
Specifically, the selection of the mobile phase elution system:
the results of the deep comparison of the application effects of the methanol-formic acid aqueous solution and the acetonitrile-formic acid aqueous solution system show that the methanol-formic acid aqueous solution system has poor separation degree, small response of each component, and high baseline noise, and the acetonitrile-formic acid aqueous solution has the best effect as the elution system of A, B mobile phase in the experiment, so the acetonitrile-formic acid aqueous solution is selected as the mobile phase elution system, wherein the effect of 0.1% formic acid aqueous solution is better, and particularly refer to fig. 1a-1 b. Mobile phase: acetonitrile is used as a mobile phase A, and 0.1% formic acid aqueous solution is used as a mobile phase B for gradient elution.
Examination of elution gradient:
octadecylsilane chemically bonded silica is used as a filler, and a chromatographic column is 250mm multiplied by 4.6mm and 5 mu m; acetonitrile is taken as a mobile phase A, 0.1% formic acid aqueous solution is taken as a mobile phase B, a gradient program is optimized, elution gradient conditions are shown in tables 1-3, and the flow rate is 0.8 mL/min; the detection wavelength is 285 nm; the column temperature was 25 ℃.
According to the test results: preferably, elution procedure III is the elution conditions of the present invention.
Table 1 solvent elution procedure I
TABLE 2 solvent elution procedure II
TABLE 3 solvent elution procedure III
The results of the tests are shown in detail in FIGS. 2a-2c, and according to the results of the tests, it is preferred that elution procedure III be the elution conditions according to the invention, i.e.the morphology shown in FIG. 2 c.
Selection of detection wavelength:
performing full-wavelength scanning on the reference solution at 200-400 nm by using a DAD detector, analyzing an equivalent absorption chart, extracting chromatograms with more absorption peaks at 260nm and 285nm and converting the wavelengths in different time periods for comparison, and displaying that the segmented conversion wavelengths can comprehensively reflect chemical components in the Ganfule sample, and each chromatogram peak has good separation and stable base line, so that the segmented conversion wavelengths are selected as the acquisition wavelengths of the Ganfule preparation, and particularly refer to fig. 3a-3 c. The wavelength is converted to 285nm in different time periods under the conditions of 0-30 min and 35-50 min; the particle size is 260nm under the conditions of 30-35 min and 50-80 min.
The method provided by the invention has the advantages that different detection wavelengths are required to be used in different time periods, chromatographic data of two frequency bands can be complemented, and the detection effect can be embodied in an optimal mode.
Selection of flow rate:
the flow rates are respectively tested at 0.8mL/min and 1.0mL/min to obtain corresponding maps, specifically referring to FIGS. 4a-4b, the result is that the separation degree and peak shape of each component at three flow rates are all satisfactory, but when the flow rate is 0.8mL/min, the separation degree and peak shape of each component in the map shown in FIG. 4a are better, therefore, the flow rate is selected to be 0.8 mL/min.
Column temperature experiment:
the experimental column temperature is 20 ℃, 25 ℃ and 30 ℃ respectively, and corresponding spectra are obtained, specifically referring to fig. 5a-5c, as a result, the separation degree of each component and the symmetry of a chromatographic peak are better when the column temperature is 20 ℃, 25 ℃ and 30 ℃, the chromatogram difference is not large, and the column temperature is selected in consideration of the temperature control capability of a laboratory and an instrument: at 25 ℃.
The first/second predetermined solvent is selected as extraction solvent:
ganfule capsule (batch No. 20180505), removing capsule shell, grinding, precisely weighing 3 parts, each part is about 1g, precisely adding 25% methanol, 50% methanol, 100% methanol each 25mL respectively, ultrasonic extracting (120W, 40KHz) for 1h, cooling, filtering, collecting the subsequent filtrate, filtering with microporous membrane, precisely measuring the subsequent filtrate 1mL, diluting with water to 5mL volumetric flask, diluting to constant volume, shaking, and filtering with C18The solid phase extraction column was eluted sequentially with 5mL 10% methanol and 5mL methanolCollecting methanol eluent, evaporating to dryness in water bath at 60 deg.C, dissolving with 50% methanol, diluting to constant volume of 1.0mL, and injecting into high performance liquid chromatograph under the same chromatographic condition for detection. As shown in FIGS. 6a-6c, the peak pattern, number and peak area of the chromatographic peak of the 50% methanol extract sample are all better, so 50% methanol was selected as the extraction solvent. The microporous filter membrane is a 0.22 mu m microporous filter membrane. Specifically, the first/second predetermined solution may be ethanol solution with a concentration of 50-100%, and the properties thereof are similar to those of methanol with the same concentration, which is not described herein again.
In summary, the preferable preparation conditions of the test solution are as follows: weighing 1.0g of the ground Ganfule sample, precisely weighing, placing in a conical flask with a stopper, precisely adding 25mL of 50% methanol, performing ultrasonic extraction (40kHz) for 1h, cooling to room temperature, adding 50% methanol to make up the weight, centrifuging at 15000r/min for 10min, precisely weighing 1.0mL of supernatant, adding water to dilute to 5mL, and passing through C18And (3) performing solid phase extraction on the type pretreatment column (activating by using chromatographic methanol and ultrapure water respectively before use), sequentially eluting by using 5mL of 10% methanol and 5mL of methanol, collecting methanol eluent, evaporating to dryness in a water bath at 60 ℃, dissolving by using 50% of methanol, fixing the volume to 1.0mL, and filtering by using a 0.22 mu m microporous membrane to obtain the product.
Preferred chromatographic conditions after this are:
a chromatographic device: a DAD detector;
a chromatographic column: YMC-Pack ODS-A C18, 250 mm. times.4.6 mm, 5 μm;
mobile phase: performing gradient elution by using acetonitrile as a mobile phase A and 0.1% formic acid water as a mobile phase B;
flow rate: 0.8 mL/min;
detection wavelength: 285nm (0-30 min, 35-50 min), 260nm (30-35 min, 50-80 min);
column temperature: 25 ℃;
procedure of gradient elution:
respectively and precisely sucking 10 mu L of reference solution and test solution, injecting the reference solution and the test solution into the DAD detector, recording a chromatogram map for 80 minutes, then carrying out corresponding analysis to obtain the content of corresponding special-effect components, and judging whether the special-effect components reach the standard or not.
Referring to fig. 7-8 again, the chromatogram obtained by the method of the present invention has a total of 29 common peaks, wherein the component content of 7 peaks is determined as follows: 31.59 + -0.01 min (atractylenolide I is No. 10 peak), 34.006 + -0.03 min (calycosin glucoside is No. 11 peak), 36.95 + -0.04 min (scutellarin is No. 12 peak), 40.19 + -0.01 min (narirutin is No. 14 peak), 43.37 + -0.03 min (hesperidin is No. 16 peak), 50.47 + -0.04 min (baicalin is No. 19 peak), 73.7 + -0.01 min (aloe-emodin is No. 26 peak).
Example 2 data analysis and calculation of the results of example 1
Step one, preparing a reference substance solution: the procedure is as in example 1 above.
Step two, preparation of a test solution: weighing 1.0g of the ground Ganfule sample, precisely weighing, placing in a conical flask with a stopper, precisely adding 25mL of 50% methanol, performing ultrasonic extraction (40kHz) for 1h, cooling to room temperature, adding 50% methanol to make up the weight, centrifuging at 15000r/min for 10min, precisely weighing 1.0mL of supernatant, adding water to dilute to 5mL, and passing through C18And (3) performing solid phase extraction on the type pretreatment column (activating by using chromatographic methanol and ultrapure water respectively before use), sequentially eluting by using 5mL of 10% methanol and 5mL of methanol, collecting methanol eluent, evaporating to dryness in a water bath at 60 ℃, dissolving by using 50% of methanol, fixing the volume to 1.0mL, and filtering by using a 0.22 mu m microporous membrane to obtain the product. And respectively preparing test solution for ten batches of Ganfule capsules and five batches of Ganfule tablets.
Step three, chromatographic conditions:
a chromatographic device: a DAD detector;
a chromatographic column: YMC-Pack ODS-A C18, 250 mm. times.4.6 mm, 5 μm;
mobile phase: performing gradient elution by using acetonitrile as a mobile phase A and 0.1% formic acid water as a mobile phase B;
flow rate: 0.8 mL/min;
detection wavelength: 285nm (0-30 min, 35-50 min), 260nm (30-35 min, 50-80 min);
column temperature: 25 ℃;
procedure of gradient elution:
and precisely sucking 10 mu L of each of the reference solution and fifteen test solutions, injecting the solutions into the DAD detector, and recording the chromatogram map for 80 minutes.
Please refer to fig. 7, which is a chromatogram of the sample solution of ganfule capsule of lot 20180505 in this embodiment.
Calculating the content of 7 components in the test solution: calculating the content of 7 components including hesperidin, scutellarin, baicalin, atractylenolide I, calycosin glycoside, narirutin and aloe-emodin in the test solution by a standard curve method.
The results are shown in Table 4, where n is the number of repeated measurements per batch of samples.
Table 415 measurement of 7 ingredients in Ganfule preparations (n ═ 3)
The standard curve method is as follows: when the component content in the sample is determined, a chromatogram is drawn under the same chromatographic conditions as the standard curve, the chromatographic peak area or peak height is measured, and then the concentration of the component in the sample injected into the chromatographic column is directly checked out on the standard curve according to the peak area and the peak height. In this embodiment, by obtaining the predetermined concentration of the standard component, the peak area in the chromatogram of the reference product, and the peak area in the chromatogram of the corresponding component corresponding to the standard component in the sample solution, the content of the response of 7 corresponding components in fifteen batches of the sample solution can be determined.
Example 3
Experiment of linear relationship
In order to more accurately determine the mass concentration of the corresponding component and the linear relationship between the peak areas corresponding to the components on the spectrum, 0.5, 1.25, 2.5, 5, 7.5 and 10mL of reference substance solution are respectively precisely measured, 50% methanol is used for fixing the volume to 10mL, 6 parts of mixed reference substance solutions with different mass concentrations are prepared, 10 mu L of sample injection is sequentially carried out according to the chromatographic conditions in example 1, and the peak areas of the chromatogram map are recorded.
After the chromatographic analysis of the 6 reference solutions, a linear regression equation of each component was obtained, with the peak area Y of the corresponding component as the ordinate and the mass concentration X (μ g. mL)-1) In abscissa, see table 5 for details.
TABLE 57 Linear regression equation for corresponding components
Stability test of corresponding component in test solution
The test solution was prepared according to the preparation protocol and the preferred chromatographic conditions in example 1 using the ganfule capsule of lot number 20180505, and according to the chromatographic conditions in example 1, samples were injected at 0, 2, 4, 8, 16, and 24h time points, respectively, and the peak area RSD value on the chromatogram of each corresponding component at each time point was calculated, and the results showed: the change ranges of hesperidin, scutellarin, baicalin, atractylenolide I, calycosin glycoside, narirutin and aloe-emodin RSD are all less than 1.5%, which indicates that the test solution is stable within 24 h; normally, the RSD range of the corresponding counterpart is stable to within 2%, and therefore, this assay is satisfactory within 24 hours.
Repeatability test
6 parts of the same test solution is prepared in parallel by using the Ganfule capsule with the batch number of 20180505 according to the preparation scheme in case 1, sample injection is carried out according to the same chromatographic conditions, the change ranges of hesperidin, scutellarin, baicalin, atractylenolide I, calycosin glucoside, narirutin and aloe-emodin RSD are all less than 1.5 percent, and the result shows that the method has good repeatability; normally, the corresponding RSD ranges of the corresponding components are stable within 2%, and thus, the method has good repeatability.
Sample application recovery test
Precisely weighing 1.0g of 6 parts of Ganfule capsule (lot number 20180505), placing in a conical flask with a plug, and adding 0.312 mg/mL of atractylenolide I-10.066 mg/mL calycosin glucoside-1Scutellarin 0.578 mg/mL-12.618 mg/mL of rutin-12.144 mg/mL hesperidin-1Baicalin 0.232 mg/mL-1Aloe-emodin 0.042 mg/mL-1The sample solution is prepared by the method of the second step in example 1, the peak area of each corresponding component is respectively measured, recorded and analyzed by the chromatographic conditions of example 1, the sample recovery rate of each component is calculated, and the result is shown in table 6, wherein n is the repeated measurement times of each batch of samples, and as shown in table 6, the RSD value of each corresponding component is less than or equal to 2.0%, which indicates that the content measurement method meets the measurement requirement.
Table 6 sample application recovery test of 7 ingredients of ganfule capsule (n ═ 6)
Example 4 Ganfule fingerprint test
Detection of 10 batches of Ganfule capsules by fingerprint spectrum
Selecting the batch numbers as follows: 20171114, 20180301, 20171003, 20180302, 20171206, 20180219, 20171205, 20180505, 20171113 and 20180506, the preparation and detection of the reference solution and the test solution are carried out according to the basic steps of example 2, and the fingerprint of Ganfule capsules of different batches is shown in figure 8.
Determination of common peak and acquisition of contrast fingerprint
Fingerprint spectra of 10 batches of Ganfule capsules are exported and imported into a traditional Chinese medicine chromatogram fingerprint spectrum similarity evaluation system, chromatographic peaks existing in 10 batches of Ganfule capsules are selected as common peaks, and the similarity is calculated by taking the fingerprint spectra of a reference substance solution as reference, so as to obtain a table 7. Table 7 shows the fingerprint similarity results for 10 groups of ganfule.
TABLE 710 fingerprint similarity results for Ganfule capsules
The above experimental results show that the similarity of the fingerprints of 10 batches of Ganfule is more than 0.95, so that the fingerprint standard of Ganfule is determined as follows: the sample fingerprint should be consistent with the reference fingerprint, and the similarity of the sample fingerprint and the reference fingerprint is not less than 0.90 by similarity calculation according to the traditional Chinese medicine chromatogram fingerprint similarity evaluation system.
It should be understood that equivalents and modifications of the technical solution and inventive concept thereof may occur to those skilled in the art, and all such modifications and alterations should fall within the scope of the appended claims.
Claims (5)
1. A method for measuring the multi-component content of a Ganfule preparation is characterized by comprising the following steps:
s1, preparation of control solution: weighing a standard component preparation, dissolving the standard component preparation in a first predetermined solvent to prepare a reference substance solution with the standard component at a predetermined concentration; the standard component preparation comprises a plurality of standard components; the standard component preparation comprises: hesperidin, scutellarin, baicalin, atractylenolide I, calycosin glycoside, narirutin, and aloe-emodin;
s2, preparation of a test solution: weighing a Ganfule preparation sample with a predetermined mass, adding a second predetermined solvent, and performing extraction treatment to obtain a test sample solution;
the method comprises the following specific steps: grinding a predetermined mass of Ganfule preparation sample, adding a first predetermined volume of the second predetermined solvent, weighing an initial weight, carrying out ultrasonic extraction treatment, adding the second predetermined solvent, complementing the weight loss to the initial weight, and shaking up;
filtering with microporous membrane to obtain secondary filtrate, diluting the secondary filtrate with water by predetermined times, and filtering with C18Solid phase micro extraction column, eluting, and collecting methanol elution part;
after the methanol solvent is evaporated to dryness in a water bath, adding the second predetermined solvent to dissolve and fixing the volume to a second predetermined volume to obtain the sample solution;
the first predetermined solvent is a 25-100% methanol or 25-100% ethanol solvent;
the second predetermined solvent is a 25-100% methanol or 25-100% ethanol solvent;
s3, content determination: respectively obtaining a reference chromatogram of the reference solution and a test chromatogram of the test solution under the same chromatographic conditions by adopting an HPLC method;
the chromatographic conditions include:
octadecylsilane chemically bonded silica is used as a filling agent;
mobile phase: the phase A is acetonitrile, the phase B is 0.1 percent formic acid water, and gradient elution is carried out by a preset gradient elution process;
detection wavelength: 285nm under the condition of time of 0-30 min and 35-50 min; 260nm under the conditions of 30-35 min and 50-80 min;
the predetermined gradient elution protocol was as follows:
0-5 min: 10% mobile phase a, 90% mobile phase B;
5-45 min: 10% → 25% mobile a phase, 90% → 75% mobile B phase;
45-65 min: 25% → 40% mobile a phase, 75% → 60% mobile B phase;
65-75 min: 40% → 80% mobile a phase, 60% → 20% mobile B phase;
75-80 min: 80% mobile phase a, 20% mobile phase B;
and S4, respectively analyzing the content of the corresponding component according to the preset concentration of the standard component, the peak area in the chromatogram of the reference product and the peak area in the chromatogram of the corresponding component corresponding to the standard component in the test solution.
2. The method for determining the multi-component content of ganfule preparation according to claim 1, wherein the chromatographic conditions further comprise:
flow rate: 0.8-1 mL/min;
specification of chromatographic column: 250mm × 4.6mm, 5 μm;
column temperature: 20 to 30 ℃.
3. The method for measuring the multicomponent content of Ganfule preparation according to claim 1, wherein in step S2, the extraction is ultrasonic extraction;
the extraction conditions include:
ultrasonic frequency: 40-60 KHz;
extraction time: and (4) 1 h.
4. The method for measuring the multicomponent content of Ganfule preparation according to claim 1, wherein the microfiltration membrane is a 0.22 μm microfiltration membrane.
5. The method for measuring the contents of multiple components in Ganfule preparation according to claim 1, wherein the Ganfule preparation comprises decoction, tablet and capsule.
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