CN103592384B - The content assaying method of Calycosin-7-O-BETA-D-glucoside in a kind of Zhenqi Fuzheng prepn - Google Patents
The content assaying method of Calycosin-7-O-BETA-D-glucoside in a kind of Zhenqi Fuzheng prepn Download PDFInfo
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Abstract
The invention discloses the content assaying method of Calycosin-7-O-BETA-D-glucoside in a kind of Zhenqi Fuzheng prepn, it is with Calycosin-7-O-BETA-D-glucoside reference substance for contrast, with methyl alcohol: water/0.01% ~ 5% phosphate aqueous solution/0.01% ~ 5% glacial acetic acid/0.01% ~ 0.5% aqueous formic acid=30 ~ 70:70 ~ 30 or acetonitrile: water/0.01% ~ 5% phosphate aqueous solution/0.01% ~ 5% glacial acetic acid/0.01% ~ 0.5% aqueous formic acid=20 ~ 50:80 ~ 50 are the high performance liquid chromatography of mobile phase.The specificity of described method is strong, precision is high, reproducible, the recovery is high, stability is high, measurement result is accurate, reaches the object effectively controlling drug quality, ensure that the safe, effective of the stable of product quality and clinical application.
Description
Technical field
The present invention relates to the content assaying method of Calycosin-7-O-BETA-D-glucoside in a kind of Zhenqi Fuzheng prepn, belong to traditional Chinese medicine quality detection technique field.
Background technology
Effect that loyal stilbene preparation has boosting qi and nourishing yin, strengthens the body resistance to consolidate the constitution.Modern pharmacology experiment proof can improve immune function of human body, protection marrow and adrenal cortex function, promotes that interferon produces; Coordinate tumor patient Radiotherapy chemotherapy, alleviate the toxic and side effect during patient's Radiotherapy chemotherapy, promote the effect of the recovery of normal function.Its prescription is made up of the Radix Astragali, the fruit of glossy privet.At present, loyal stilbene preparation has tablet, capsule, granule and pill etc. for the formulation of selection of clinical.
And the principle active component in the Radix Astragali is saponin(e and flavone compound, the flavones ingredient that research confirms in the Radix Astragali have multiple pharmacologically active, can NO concentration in scavenging activated oxygen, anti-lipid peroxidation and maintenance blood, protection ischemical reperfusion injury.Therefore, Radix Astragali flavone constituents is one of key agents effective constituent of Zhenqi Fuzheng prepn, and Calycosin-7-O-BETA-D-glucoside is one important in Radix Astragali flavone constituents, measures also seem particularly important to the content of Calycosin-7-O-BETA-D-glucoside.
But, the quality determining method of existing Zhenqi Fuzheng prepn does not measure the content of Calycosin-7-O-BETA-D-glucoside composition, for improving the quality inspection standard of Zhenqi Fuzheng prepn further, to guarantee its drug quality, the assay project setting up Calycosin-7-O-BETA-D-glucoside in Zhenqi Fuzheng prepn is very necessary.
Summary of the invention
The object of the invention is to, the content assaying method of Calycosin-7-O-BETA-D-glucoside in a kind of Zhenqi Fuzheng prepn is provided, the method specificity is strong, precision is high, reproducible, the recovery is high, stability is high, measurement result is accurate, reach the object effectively controlling drug quality, ensure that the safe, effective of the stable of product quality and clinical application.
Zhenqi Fuzheng prepn of the present invention comprises any Zhenqi Fuzheng prepn product (particle, capsule, tablet etc.) of the prior art, such as, according to the preparation that the method described in patent of invention 200310122216.7,200610051146.4 prepares.
For solving the problems of the technologies described above, the present invention adopts following technical scheme:
The content assaying method of Calycosin-7-O-BETA-D-glucoside in a kind of Zhenqi Fuzheng prepn, it is with Calycosin-7-O-BETA-D-glucoside reference substance for contrast, with methyl alcohol: water/0.01% ~ 5% phosphate aqueous solution/0.01% ~ 5% glacial acetic acid/0.01% ~ 0.5% aqueous formic acid=30 ~ 70:70 ~ 30 or acetonitrile: water/0.01% ~ 5% phosphate aqueous solution/0.01% ~ 5% glacial acetic acid/0.01% ~ 0.5% aqueous formic acid=20 ~ 50:80 ~ 50 are the high performance liquid chromatography of mobile phase.
Concrete content assaying method is: according to high performance liquid chromatography, measure in the steps below:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With methyl alcohol: water/0.01% ~ 5% phosphate aqueous solution/0.01% ~ 5% glacial acetic acid/0.01% ~ 0.5% aqueous formic acid=30 ~ 70:70 ~ 30 or acetonitrile: water/0.01% ~ 5% phosphate aqueous solution/0.01% ~ 5% glacial acetic acid/0.01% ~ 0.5% aqueous formic acid=20 ~ 50:80 ~ 50 are that (acetonitrile is more obvious compared with the advantage of methyl alcohol, as few in produced ghost peak for mobile phase; Pressure is little); Column temperature is 20 ~ 40 DEG C; Determined wavelength is 215 ~ 275nm; Flow velocity is 0.5 ~ 1.5mLmin
-1; Number of theoretical plate calculates should be not less than 3000 by Calycosin-7-O-BETA-D-glucoside peak;
(2) preparation of reference substance solution: it is appropriate that precision takes Calycosin-7-O-BETA-D-glucoside reference substance, adds the solution that methyl alcohol makes 0.1mg/mL, for subsequent use;
(3) preparation of need testing solution: adopt following method 1.-5. in any one:
1. precision takes Zhenqi Fuzheng prepn or its content 2.0g, and precision adds methyl alcohol 100mL, weighed weight, and ultrasonic or refluxing extraction 30 ~ 60min, after cooling, supplies weight with methyl alcohol; Filter, get subsequent filtrate, to obtain final product;
2. precision takes Zhenqi Fuzheng prepn or its content 2.0g, and with appropriate sherwood oil surname extraction 30min, after residue volatilizes, precision adds methyl alcohol 100mL, weighed weight, and ultrasonic or refluxing extraction 30 ~ 60min, after cooling, supplies weight with methyl alcohol; Filter, get subsequent filtrate, to obtain final product;
3. precision takes Zhenqi Fuzheng prepn or its content 2.0g, and precision adds the ethanol 100mL of 70% ~ 80%, weighed weight, and ultrasonic extraction lh, supplies weight with ethanol, filters; Precision measures subsequent filtrate 50mL, be evaporated to dry, residue adds suitable quantity of water makes dissolving, upper large pore resin absorption column, standing adsorption 30min, use water, 40% ethanol, 70% ethanol elution successively, collect 70% ethanol eluate, be concentrated into dry, dissolve with methyl alcohol and be transferred in the measuring bottle of 50mL, add methyl alcohol to scale, shake up, to obtain final product;
4. precision takes Zhenqi Fuzheng prepn or its content 2.0g, and precision adds methyl alcohol 100mL, weighed weight, and ultrasonic or refluxing extraction 1h, supplies weight with methyl alcohol, filters; Precision measures subsequent filtrate 50mL, is concentrated into dry, and residue adds watery hydrochloric acid and is adjusted to pH5 ~ 6 after dissolving with 0.3% sodium hydroxide solution, then with ethyl acetate or water saturated extracting n-butyl alcohol four times, organic layer is merged, evaporated under reduced pressure, residue methanol constant volume, to 50mL, shakes up, and to obtain final product;
5. precision takes Zhenqi Fuzheng prepn or its content 2.0g, and precision adds methyl alcohol 100mL, weighed weight, and ultrasonic or refluxing extraction 1h, supplies weight with methyl alcohol, filters; Precision measures subsequent filtrate 50mL, is concentrated into dry, and residue adds appropriate warm water and dissolves, extract 4 times with water saturated normal butyl alcohol, after fully extracting, merge n-butanol extracting liquid, 3 times are washed with ammonia solution, discard ammonia solution, normal butyl alcohol liquid evaporate to dryness, the residue 10mL that adds water makes dissolving, pass through large pore resin absorption column, with water 50mL wash-out, discard water liquid, then use 40% ethanol elution, discard eluent, continue and use 70% ethanol elution, collect eluent, evaporate to dryness, residue methyl alcohol dissolves and is transferred in 50mL measuring bottle, add methyl alcohol to scale, shake up, to obtain final product;
(4) determination method: accurate absorption reference substance solution and each 10 μ L of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
In aforesaid Zhenqi Fuzheng prepn Calycosin-7-O-BETA-D-glucoside content assaying method in, described mobile phase is acetonitrile: 0.2% aqueous formic acid=20:80.
In aforesaid Zhenqi Fuzheng prepn Calycosin-7-O-BETA-D-glucoside content assaying method in, the determined wavelength in step (1) is 260nm, and flow velocity is 0.8 ~ 1.0mL/min, and column temperature is 35 DEG C.
For guaranteeing content assaying method science of the present invention, reasonable, feasible, applicant carried out series of experimental research and investigation.
One, instrument and reagent (see table 1)
Table 1
Two, method and result
1, the selection of chromatographic condition
1.1 column temperatures are selected
Adopt different column temperature to test, find optimum column temperature.The results are shown in Table 2.
Table 2
| Column temperature (DEG C) | 25 | 35 | 50 |
| Observations | Degree of separation is poor | Degree of separation is good | Solvent viscosity is large |
As shown in Table 2,35 DEG C should be selected as column temperature, this easy control of temperature, and under low pressure be conducive to the viscosity reducing solvent, thus reduce post pressure.
1.2 flow velocitys are selected
Adopt different in flow rate to test, find optimum flow rate.The results are shown in Table 3.
Table 3
As shown in Table 3, arranging flow velocity is that 0.8 ~ 1.0mL/min is best.
1.3 mobile phases are selected
Adopt different mobile phase to carry out UV detection, find optimal flow phase.The results are shown in Table 4.
Table 4
| Mobile phase | Chromatographic peak |
| Acetonitrile: 0.2% aqueous formic acid=20:80 | Generation ghost peak is few, and peak shape is good, and degree of separation is high |
| Methyl alcohol: water=30:70 | Peak shape is slightly poor, and degree of separation is higher |
| Methyl alcohol: 0.05% glacial acetic acid=20:80 | Peak shape is poor, and degree of separation is low |
| Methyl alcohol: 0.05% phosphate aqueous solution=40:60 | Peak shape is slightly poor, and degree of separation is higher |
| Acetonitrile: water=60:40 | Peak shape is poor, and degree of separation is low |
| Acetonitrile: 0.05% glacial acetic acid=30:70 | Peak shape is better, and degree of separation is high |
As seen from the above table, select acetonitrile: 0.2% aqueous formic acid=20:80 is as mobile phase, and the chromatographic peak effect that it obtains is best.
2, the preparation of reference substance solution
It is appropriate that precision takes Calycosin-7-O-BETA-D-glucoside reference substance, adds the solution that methyl alcohol makes 0.1mg/mL, for subsequent use.
3, the preparation of need testing solution
Can adopt following method 1.-5. in any one:
1. precision takes Zhenqi Fuzheng prepn or its content 2.0g, and precision adds methyl alcohol 100mL, weighed weight, and ultrasonic or refluxing extraction 30 ~ 60min, after cooling, supplies weight with methyl alcohol; Filter, get subsequent filtrate, to obtain final product;
2. precision takes Zhenqi Fuzheng prepn or its content 2.0g, and with appropriate sherwood oil surname extraction 30min, after residue volatilizes, precision adds methyl alcohol 100mL, weighed weight, and ultrasonic or refluxing extraction 30 ~ 60min, after cooling, supplies weight with methyl alcohol; Filter, get subsequent filtrate, to obtain final product;
3. precision takes Zhenqi Fuzheng prepn or its content 2.0g, and precision adds the ethanol 100mL of 70% ~ 80%, weighed weight, and ultrasonic extraction lh, supplies weight with ethanol, filters; Precision measures subsequent filtrate 50mL, be evaporated to dry, residue adds suitable quantity of water makes dissolving, upper large pore resin absorption column, standing adsorption 30min, use water, 40% ethanol, 70% ethanol elution successively, collect 70% ethanol eluate, be concentrated into dry, dissolve with methyl alcohol and be transferred in the measuring bottle of 50mL, add methyl alcohol to scale, shake up, to obtain final product;
4. precision takes Zhenqi Fuzheng prepn or its content 2.0g, and precision adds methyl alcohol 100mL, weighed weight, and ultrasonic or refluxing extraction 1h, supplies weight with methyl alcohol, filters; Precision measures subsequent filtrate 50mL, is concentrated into dry, and residue adds watery hydrochloric acid and is adjusted to pH5 ~ 6 after dissolving with 0.3% sodium hydroxide solution, then with ethyl acetate or water saturated extracting n-butyl alcohol four times, organic layer is merged, evaporated under reduced pressure, residue methanol constant volume, to 50mL, shakes up, and to obtain final product;
5. precision takes Zhenqi Fuzheng prepn or its content 2.0g, and precision adds methyl alcohol 100mL, weighed weight, and ultrasonic or refluxing extraction 1h, supplies weight with methyl alcohol, filters; Precision measures subsequent filtrate 50mL, is concentrated into dry, and residue adds appropriate warm water and dissolves, extract 4 times with water saturated normal butyl alcohol, after fully extracting, merge n-butanol extracting liquid, 3 times are washed with ammonia solution, discard ammonia solution, normal butyl alcohol liquid evaporate to dryness, the residue 10mL that adds water makes dissolving, pass through large pore resin absorption column, with water 50mL wash-out, discard water liquid, then use 40% ethanol elution, discard eluent, continue and use 70% ethanol elution, collect eluent, evaporate to dryness, residue methyl alcohol dissolves and is transferred in 50mL measuring bottle, add methyl alcohol to scale, shake up, to obtain final product.
Applicant compares above-mentioned 5 kinds of test sample preparation methods, and experimental result is in table 5.
Table 5
As seen from the above table, although first two method is easy and simple to handle, Calycosin-7-O-BETA-D-glucoside extraction ratio is high, and Calycosin-7-O-BETA-D-glucoside purity is very low.Though rear three kinds of method steps are slightly aobvious loaded down with trivial details, the slightly low purity of Calycosin-7-O-BETA-D-glucoside extraction ratio is high, eliminates the impact of impurity on testing result to a great extent.
4, the selection of determined wavelength
Carry out spectral scan at 215 ~ 275nm to Calycosin-7-O-BETA-D-glucoside reference substance solution, its maximum absorption wavelength is 260nm, therefore selects 260nm to be determined wavelength.
5, the preparation of typical curve
Get reference substance solution 0.25mL, 0.5mL, 1mL, 2mL, 4mL in 10mL volumetric flask, add methanol dilution, constant volume, each sample introduction 10 μ L, with the corresponding peak area of Calycosin-7-O-BETA-D-glucoside reference substance solution of variable concentrations to concentration drawing standard curve, calculating regression equation is Y=2.3 × 10
5x-1.86 × 10
5, r=0.9995, wherein, Y is peak area, and X is Calycosin-7-O-BETA-D-glucoside reference substance solution concentration, and Calycosin-7-O-BETA-D-glucoside is good linear relation at 2.5 ~ 40 μ g/mL.
6, Precision Experiment
Obtain reference substance solution according to legal system below the preparation of reference substance solution, get reference substance solution 10 μ L, repeat sample introduction 6 times, calculating Calycosin-7-O-BETA-D-glucoside peak area RSD is 1.25%, shows that the method precision is good.
7, stability experiment
Need testing solution is obtained according to legal system below the preparation of need testing solution, the same need testing solution of accurate absorption, under room temperature respectively at 0,2,4,8,12,24h respectively inhales 10 μ L, sample introduction 6 times altogether, the RSD calculating Calycosin-7-O-BETA-D-glucoside peak area is 0.98%, at room temperature 24h is interior stable to show the solution after processing, and has good stability.
8, repeated experiment
Obtain need testing solution according to legal system below the preparation of need testing solution, get same sample solution 6 parts, each sample introduction 10 μ L, the RSD calculating peak area is 1.35%, shows that repeatability is good.
9, average recovery experiment
Precision takes 6 parts, the Zhenqi Fuzheng prepn powder of known content, precision adds Calycosin-7-O-BETA-D-glucoside reference substance respectively, prepare by the preparation method of need testing solution, and sample introduction measures, measurement result is in table 6, and to calculate its average recovery rate be 98.40%, RSD is 0.67%, the recovery is high, shows that this method is feasible.
Table 6 average recovery experimental result
10, sample size measures
Get 5 batches of Zhenqi Fuzheng prepns appropriate, accurately weighed, be prepared by method under the preparation of the preparation of reference substance solution, need testing solution respectively, assay (need testing solution, reference substance solution be sample introduction 10 μ L respectively) under the investigation item of linearly relation, calculate the content of Calycosin-7-O-BETA-D-glucoside, the results are shown in Table 7.
Table 7 sample size measurement result (n=5)
| Sample lot number | Average content (mg/g) |
| 20120801 | 0.834 |
| 20120802 | 0.826 |
| 20120803 | 0.852 |
| 20120804 | 0.850 |
| 20120805 | 0.843 |
Compared with prior art, the present invention is measured the content of Calycosin-7-O-BETA-D-glucoside composition in Zhenqi Fuzheng prepn by high performance liquid chromatography, the specificity of described method is strong, precision is high, reproducible, the recovery is high, stability is high, measurement result is accurate, reach the object effectively controlling drug quality, ensure that the safe, effective of the stable of product quality and clinical application.
Embodiment
The embodiment of the present invention 1: the content assaying method of Calycosin-7-O-BETA-D-glucoside in a kind of Zhenqi Fuzheng prepn, measures in the steps below:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With acetonitrile: 0.2% aqueous formic acid=20:80 is mobile phase; Determined wavelength is 260nm; Flow velocity is 1.0mL/min; Column temperature is 35 DEG C; Number of theoretical plate calculates should be not less than 3000 by Calycosin-7-O-BETA-D-glucoside peak;
(2) preparation of reference substance solution: it is appropriate that precision takes Calycosin-7-O-BETA-D-glucoside reference substance, adds the solution that methyl alcohol makes 0.1mg/mL, for subsequent use;
(3) preparation of need testing solution: precision takes Zhenqi Fuzheng prepn or its content 2.0g, and precision adds the ethanol 100mL of 70% ~ 75%, weighed weight, and ultrasonic extraction lh, supplies weight with ethanol, filters; Precision measures subsequent filtrate 50mL, be evaporated to dry, residue adds suitable quantity of water makes dissolving, upper large pore resin absorption column, standing adsorption 30min, use water, 40% ethanol, 70% ethanol elution successively, collect 70% ethanol eluate, be concentrated into dry, dissolve with methyl alcohol and be transferred in the measuring bottle of 50mL, add methyl alcohol to scale, shake up, to obtain final product;
(4) determination method: accurate absorption reference substance solution and each 10 μ L of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
The embodiment of the present invention 2: the content assaying method of Calycosin-7-O-BETA-D-glucoside in a kind of Zhenqi Fuzheng prepn, measures in the steps below:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With acetonitrile: 0.05% glacial acetic acid=30:70 is mobile phase; Determined wavelength is 260nm; Flow velocity is 0.8mL/min; Column temperature is 35 DEG C; Number of theoretical plate calculates should be not less than 3000 by Calycosin-7-O-BETA-D-glucoside peak;
(2) preparation of reference substance solution: it is appropriate that precision takes Calycosin-7-O-BETA-D-glucoside reference substance, adds the solution that methyl alcohol makes 0.1mg/mL, for subsequent use;
(3) preparation of need testing solution: precision takes Zhenqi Fuzheng prepn or its content 2.0g, and precision adds methyl alcohol 100mL, weighed weight, and ultrasonic or refluxing extraction 1h, supplies weight with methyl alcohol, filters; Precision measures subsequent filtrate 50mL, is concentrated into dry, and residue adds watery hydrochloric acid and is adjusted to pH5 ~ 6 after dissolving with 0.3% sodium hydroxide solution, then with ethyl acetate or water saturated extracting n-butyl alcohol four times, organic layer is merged, evaporated under reduced pressure, residue methanol constant volume, to 50mL, shakes up, and to obtain final product;
(4) determination method: accurate absorption reference substance solution and each 10 μ L of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
The embodiment of the present invention 3: the content assaying method of Calycosin-7-O-BETA-D-glucoside in a kind of Zhenqi Fuzheng prepn, measures in the steps below:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With methyl alcohol: 0.05% phosphate aqueous solution=40:60 is mobile phase; Column temperature is 35 DEG C; Determined wavelength is 275nm; Flow velocity is 0.9mLmin
-1; Number of theoretical plate calculates should be not less than 3000 by Calycosin-7-O-BETA-D-glucoside peak;
(2) preparation of reference substance solution: it is appropriate that precision takes Calycosin-7-O-BETA-D-glucoside reference substance, adds the solution that methyl alcohol makes 0.1mg/mL, for subsequent use;
(3) preparation of need testing solution: precision takes Zhenqi Fuzheng prepn or its content 2.0g, and precision adds methyl alcohol 100mL, weighed weight, and ultrasonic or refluxing extraction 1h, supplies weight with methyl alcohol, filters; Precision measures subsequent filtrate 50mL, is concentrated into dry, and residue adds appropriate warm water and dissolves, extract 4 times with water saturated normal butyl alcohol, after fully extracting, merge n-butanol extracting liquid, 3 times are washed with ammonia solution, discard ammonia solution, normal butyl alcohol liquid evaporate to dryness, the residue 10mL that adds water makes dissolving, pass through large pore resin absorption column, with water 50mL wash-out, discard water liquid, then use 40% ethanol elution, discard eluent, continue and use 70% ethanol elution, collect eluent, evaporate to dryness, residue methyl alcohol dissolves and is transferred in 50mL measuring bottle, add methyl alcohol to scale, shake up, to obtain final product;
(4) determination method: accurate absorption reference substance solution and each 10 μ L of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
The embodiment of the present invention 4: the content assaying method of Calycosin-7-O-BETA-D-glucoside in a kind of Zhenqi Fuzheng prepn, measures in the steps below:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With methyl alcohol: water=30:70 is mobile phase; Determined wavelength is 215nm, and flow velocity is 1.5mL/min, and column temperature is 40 DEG C; Number of theoretical plate calculates should be not less than 3000 by Calycosin-7-O-BETA-D-glucoside peak;
(2) preparation of reference substance solution: it is appropriate that precision takes Calycosin-7-O-BETA-D-glucoside reference substance, adds the solution that methyl alcohol makes 0.1mg/mL, for subsequent use;
(3) preparation of need testing solution: precision takes Zhenqi Fuzheng prepn or its content 2.0g, and precision adds methyl alcohol 100mL, weighed weight, and ultrasonic or refluxing extraction 30 ~ 60min, after cooling, supplies weight with methyl alcohol; Filter, get subsequent filtrate, to obtain final product;
(4) determination method: accurate absorption reference substance solution and each 10 μ L of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
The embodiment of the present invention 5: the content assaying method of Calycosin-7-O-BETA-D-glucoside in a kind of Zhenqi Fuzheng prepn, measures in the steps below:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With acetonitrile: water=40:60 is mobile phase; Column temperature is 20 DEG C; Determined wavelength is 240nm; Flow velocity is 0.8mLmin
-1; Number of theoretical plate calculates should be not less than 3000 by Calycosin-7-O-BETA-D-glucoside peak;
(2) preparation of reference substance solution: it is appropriate that precision takes Calycosin-7-O-BETA-D-glucoside reference substance, adds the solution that methyl alcohol makes 0.1mg/mL, for subsequent use;
(3) preparation of need testing solution: precision takes Zhenqi Fuzheng prepn or its content 2.0g, with appropriate sherwood oil surname extraction 30min, after residue volatilizes, precision adds methyl alcohol 100mL, weighed weight, ultrasonic or refluxing extraction 30 ~ 60min, after cooling, weight is supplied with methyl alcohol; Filter, get subsequent filtrate, to obtain final product;
(4) determination method: accurate absorption reference substance solution and each 10 μ L of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
The embodiment of the present invention 6: the content assaying method of Calycosin-7-O-BETA-D-glucoside in a kind of Zhenqi Fuzheng prepn, measures in the steps below:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With methyl alcohol: 0.5% glacial acetic acid=70:30 is mobile phase; Column temperature is 30 DEG C; Determined wavelength is 250nm; Flow velocity is 0.5mLmin
-1; Number of theoretical plate calculates should be not less than 3000 by Calycosin-7-O-BETA-D-glucoside peak;
(2) preparation of reference substance solution: it is appropriate that precision takes Calycosin-7-O-BETA-D-glucoside reference substance, adds the solution that methyl alcohol makes 0.1mg/mL, for subsequent use;
(3) preparation of need testing solution: precision takes Zhenqi Fuzheng prepn or its content 2.0g, and precision adds the ethanol 100mL of 75% ~ 80%, weighed weight, and ultrasonic extraction lh, supplies weight with ethanol, filters; Precision measures subsequent filtrate 50mL, be evaporated to dry, residue adds suitable quantity of water makes dissolving, upper large pore resin absorption column, standing adsorption 30min, use water, 40% ethanol, 70% ethanol elution successively, collect 70% ethanol eluate, be concentrated into dry, dissolve with methyl alcohol and be transferred in the measuring bottle of 50mL, add methyl alcohol to scale, shake up, to obtain final product;
(4) determination method: accurate absorption reference substance solution and each 10 μ L of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
The embodiment of the present invention 7: the content assaying method of Calycosin-7-O-BETA-D-glucoside in a kind of Zhenqi Fuzheng prepn, measures in the steps below:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With methyl alcohol: 0.5% aqueous formic acid=50:50 is mobile phase; Column temperature is 25 DEG C; Determined wavelength is 260nm; Flow velocity is 1.2mLmin
-1; Number of theoretical plate calculates should be not less than 3000 by Calycosin-7-O-BETA-D-glucoside peak;
(2) preparation of reference substance solution: it is appropriate that precision takes Calycosin-7-O-BETA-D-glucoside reference substance, adds the solution that methyl alcohol makes 0.1mg/mL, for subsequent use;
(3) preparation of need testing solution: precision takes Zhenqi Fuzheng prepn or its content 2.0g, and precision adds methyl alcohol 100mL, weighed weight, and ultrasonic or refluxing extraction 1h, supplies weight with methyl alcohol, filters; Precision measures subsequent filtrate 50mL, is concentrated into dry, and residue adds watery hydrochloric acid and is adjusted to pH5 ~ 6 after dissolving with 0.3% sodium hydroxide solution, then with ethyl acetate or water saturated extracting n-butyl alcohol four times, organic layer is merged, evaporated under reduced pressure, residue methanol constant volume, to 50mL, shakes up, and to obtain final product;
(4) determination method: accurate absorption reference substance solution and each 10 μ L of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
The embodiment of the present invention 8: the content assaying method of Calycosin-7-O-BETA-D-glucoside in a kind of Zhenqi Fuzheng prepn, measures in the steps below:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With acetonitrile: 0.5% aqueous formic acid=50:50 is mobile phase; Column temperature is 30 DEG C; Determined wavelength is 230nm; Flow velocity is 1.0mLmin
-1; Number of theoretical plate calculates should be not less than 3000 by Calycosin-7-O-BETA-D-glucoside peak;
(2) preparation of reference substance solution: it is appropriate that precision takes Calycosin-7-O-BETA-D-glucoside reference substance, adds the solution that methyl alcohol makes 0.1mg/mL, for subsequent use;
(3) preparation of need testing solution: precision takes Zhenqi Fuzheng prepn or its content 2.0g, and precision adds methyl alcohol 100mL, weighed weight, and ultrasonic or refluxing extraction 1h, supplies weight with methyl alcohol, filters; Precision measures subsequent filtrate 50mL, is concentrated into dry, and residue adds appropriate warm water and dissolves, extract 4 times with water saturated normal butyl alcohol, after fully extracting, merge n-butanol extracting liquid, 3 times are washed with ammonia solution, discard ammonia solution, normal butyl alcohol liquid evaporate to dryness, the residue 10mL that adds water makes dissolving, pass through large pore resin absorption column, with water 50mL wash-out, discard water liquid, then use 40% ethanol elution, discard eluent, continue and use 70% ethanol elution, collect eluent, evaporate to dryness, residue methyl alcohol dissolves and is transferred in 50mL measuring bottle, add methyl alcohol to scale, shake up, to obtain final product;
(4) determination method: accurate absorption reference substance solution and each 10 μ L of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
Zhenqi Fuzheng prepn described in above-described embodiment can be any Zhenqi Fuzheng prepn product (particle, capsule, tablet etc.) of the prior art, such as, according to the preparation that the method described in patent of invention 200310122216.7,200610051146.4 prepares.
The combination in any of all conditions parameter in above-described embodiment, all can obtain consistent assay result.
Claims (2)
1. the content assaying method of Calycosin-7-O-BETA-D-glucoside in a Zhenqi Fuzheng prepn, it is characterized in that: it is with Calycosin-7-O-BETA-D-glucoside reference substance for contrast, with methyl alcohol: water/0.01% ~ 5% phosphate aqueous solution/0.01% ~ 5% glacial acetic acid/0.01% ~ 0.5% aqueous formic acid=30 ~ 70:70 ~ 30 or acetonitrile: water/0.01% ~ 5% phosphate aqueous solution/0.01% ~ 5% glacial acetic acid/0.01% ~ 0.5% aqueous formic acid=20 ~ 50:80 ~ 50 are the high performance liquid chromatography of mobile phase;
According to high performance liquid chromatography, measure in the steps below:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With methyl alcohol: water/0.01% ~ 5% phosphate aqueous solution/0.01% ~ 5% glacial acetic acid/0.01% ~ 0.5% aqueous formic acid=30 ~ 70:70 ~ 30 or acetonitrile: water/0.01% ~ 5% phosphate aqueous solution/0.01% ~ 5% glacial acetic acid/0.01% ~ 0.5% aqueous formic acid=20 ~ 50:80 ~ 50 are mobile phase; Determined wavelength is 260nm; Flow velocity is 0.8 ~ 1.0mL/min; Column temperature is 35 DEG C; Number of theoretical plate calculates should be not less than 3000 by Calycosin-7-O-BETA-D-glucoside peak;
(2) preparation of reference substance solution: it is appropriate that precision takes Calycosin-7-O-BETA-D-glucoside reference substance, adds the solution that methyl alcohol makes 0.1mg/mL, for subsequent use;
(3) preparation of need testing solution: adopt following method 1.-5. in any one:
1. precision takes Zhenqi Fuzheng prepn or its content 2.0g, and precision adds methyl alcohol 100mL, weighed weight, and ultrasonic or refluxing extraction 30 ~ 60min, after cooling, supplies weight with methyl alcohol; Filter, get subsequent filtrate, to obtain final product;
2. precision takes Zhenqi Fuzheng prepn or its content 2.0g, and with appropriate sherwood oil surname extraction 30min, after residue volatilizes, precision adds methyl alcohol 100mL, weighed weight, and ultrasonic or refluxing extraction 30 ~ 60min, after cooling, supplies weight with methyl alcohol; Filter, get subsequent filtrate, to obtain final product;
3. precision takes Zhenqi Fuzheng prepn or its content 2.0g, and precision adds the ethanol 100mL of 70% ~ 80%, weighed weight, and ultrasonic extraction lh, supplies weight with ethanol, filters; Precision measures subsequent filtrate 50mL, be evaporated to dry, residue adds suitable quantity of water makes dissolving, upper large pore resin absorption column, standing adsorption 30min, use water, 40% ethanol, 70% ethanol elution successively, collect 70% ethanol eluate, be concentrated into dry, dissolve with methyl alcohol and be transferred in the measuring bottle of 50mL, add methyl alcohol to scale, shake up, to obtain final product;
4. precision takes Zhenqi Fuzheng prepn or its content 2.0g, and precision adds methyl alcohol 100mL, weighed weight, and ultrasonic or refluxing extraction 1h, supplies weight with methyl alcohol, filters; Precision measures subsequent filtrate 50mL, is concentrated into dry, and residue adds watery hydrochloric acid and is adjusted to pH5 ~ 6 after dissolving with 0.3% sodium hydroxide solution, then with ethyl acetate or water saturated extracting n-butyl alcohol four times, organic layer is merged, evaporated under reduced pressure, residue methanol constant volume, to 50mL, shakes up, and to obtain final product;
5. precision takes Zhenqi Fuzheng prepn or its content 2.0g, and precision adds methyl alcohol 100mL, weighed weight, and ultrasonic or refluxing extraction 1h, supplies weight with methyl alcohol, filters; Precision measures subsequent filtrate 50mL, is concentrated into dry, and residue adds appropriate warm water and dissolves, extract 4 times with water saturated normal butyl alcohol, after fully extracting, merge n-butanol extracting liquid, 3 times are washed with ammonia solution, discard ammonia solution, normal butyl alcohol liquid evaporate to dryness, the residue 10mL that adds water makes dissolving, pass through large pore resin absorption column, with water 50mL wash-out, discard water liquid, then use 40% ethanol elution, discard eluent, continue and use 70% ethanol elution, collect eluent, evaporate to dryness, residue methyl alcohol dissolves and is transferred in 50mL measuring bottle, add methyl alcohol to scale, shake up, to obtain final product;
(4) determination method: accurate absorption reference substance solution and each 10 μ L of need testing solution respectively, injection liquid chromatography, measures, to obtain final product.
2. the content assaying method of Calycosin-7-O-BETA-D-glucoside in Zhenqi Fuzheng prepn according to claim 1, is characterized in that: described mobile phase is acetonitrile: 0.2% aqueous formic acid=20:80.
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