CN103592391B - The content assaying method of Specnuezhenide in Zhenqi Fuzheng prepn - Google Patents
The content assaying method of Specnuezhenide in Zhenqi Fuzheng prepn Download PDFInfo
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Abstract
The invention discloses the content assaying method of Specnuezhenide in a kind of Zhenqi Fuzheng prepn, it is with Specnuezhenide 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=20 ~ 50:80 ~ 50 or acetonitrile: water/0.01% ~ 5% phosphate aqueous solution/0.01% ~ 5% glacial acetic acid/0.01% ~ 0.5% aqueous formic acid=5 ~ 35:95 ~ 65 are the high performance liquid chromatography of mobile phase.Compared with prior art, the present invention is measured the content of Specnuezhenide 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.
Description
Technical field
The present invention relates to the content assaying method of Specnuezhenide 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 common formulations of selection of clinical.
Wherein, the iridoid glycoside constituents Specnuezhenide in the fruit of glossy privet has immunological regulation and antioxidation, is the principle active component in Zhenqi Fuzheng prepn, therefore carries out accurate assay to it and is very important.Although, the concrete content assaying method of Specnuezhenide in the fruit of glossy privet is described in " Chinese Pharmacopoeia ", but, also without any the record of the content assaying method for the Specnuezhenide composition in existing Zhenqi Fuzheng prepn, therefore, the quality inspection standard of existing Zhenqi Fuzheng prepn is imperfect, inaccurate, needs badly and provides a kind of specially for the content assaying method of Specnuezhenide composition in Zhenqi Fuzheng prepn.
Summary of the invention
The object of the invention is to, the content assaying method of Specnuezhenide in a kind of Zhenqi Fuzheng prepn is provided, the mensuration of quick, accurate, high reappearance, high-recovery can be carried out to the content of Specnuezhenide composition in Zhenqi Fuzheng prepn.
Zhenqi Fuzheng prepn of the present invention: take Milkvetch Root 2084g, Fructus Ligustri Lucidi 1042g and mix, add 10 times amount soak by water 2 hours, filter, collect decocting liquid, doublely in the dregs of a decoction, add 8 times amount soak by water 1 hour, filter, collecting decoction, ultrafiltration, concentrated, dry, pulverize, add appropriate amount of auxiliary materials and make various preparation.
For solving the problems of the technologies described above, the present invention adopts following technical scheme:
The content assaying method of Specnuezhenide in a kind of Zhenqi Fuzheng prepn, it is with Specnuezhenide 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=20 ~ 50:80 ~ 50 or acetonitrile: water/0.01% ~ 5% phosphate aqueous solution/0.01% ~ 5% glacial acetic acid/0.01% ~ 0.5% aqueous formic acid=5 ~ 35:95 ~ 65 are the high performance liquid chromatography of mobile phase.
In aforesaid Zhenqi Fuzheng prepn, the content assaying method of Specnuezhenide shines high performance liquid chromatography, measures 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=20 ~ 50:80 ~ 50 or acetonitrile: water/0.01% ~ 5% phosphate aqueous solution/0.01% ~ 5% glacial acetic acid/0.01% ~ 0.5% aqueous formic acid=5 ~ 35:95 ~ 65 are mobile phase; Column temperature is 20 ~ 40 DEG C; Determined wavelength is 210 ~ 240nm; Flow velocity is 0.5 ~ 1.5mL/min; Number of theoretical plate calculates should be not less than 4000 by Specnuezhenide peak;
(2) preparation of reference substance solution: get Specnuezhenide reference substance appropriate, accurately weighed, add methyl alcohol and make the solution that concentration is 0.2mg/mL, to obtain final product;
(3) preparation of need testing solution: employing method 1. ~ one of be 3. prepared:
1. precision takes Zhenqi Fuzheng prepn or its content 3g, and precision adds methyl alcohol 100mL, weighed weight, and ultrasonic or refluxing extraction 30 ~ 60min, or microwave radiation exaraction 2 ~ 10min, supply weight with methyl alcohol, filters, gets subsequent filtrate and get final product;
2. precision takes Zhenqi Fuzheng prepn or its content 3g, and precision adds methyl alcohol 100mL, weighed weight, ultrasonic or heating and refluxing extraction 30 ~ 60min, or microwave radiation exaraction 2 ~ 10min, supply weight with methyl alcohol, filter, precision measures subsequent filtrate 50mL, decompression and solvent recovery, residue thin up, upper macroreticular resin, is washed to colourless, again with 50% ~ 95% ethanol elution, eluent is concentrated into dry, and residue methyl alcohol dissolves and is transferred in 50mL volumetric flask, add methyl alcohol to scale, shake up, to obtain final product;
3. precision takes Zhenqi Fuzheng prepn or its content 3g, and precision adds methyl alcohol 100mL, weighed weight, ultrasonic or heating and refluxing extraction 30 ~ 60min, or microwave radiation exaraction 2 ~ 10min, supply weight with methyl alcohol, filter, precision measures subsequent filtrate 50mL, decompression and solvent recovery, residue is dissolved in water, and cross silicagel column with chloroform: methyl alcohol: water=5 ~ 45:3:1 wash-out, eluent is concentrated into dry, residue methyl alcohol dissolves and is transferred in 50mL volumetric flask, 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, injects high performance liquid chromatograph, measures, to obtain final product.
In aforesaid Zhenqi Fuzheng prepn Specnuezhenide content assaying method in, described mobile phase is methyl alcohol: 0.05% phosphate aqueous solution=30:70.(methyl alcohol: Specnuezhenide can well be separated by water=30:70; Wherein water phosphoric acid aqueous solution, glacial acetic acid or formic acid replace because acid adding in water, add alkali, salt adding can improve peak shape, improve degree of separation).
In aforesaid Zhenqi Fuzheng prepn Specnuezhenide content assaying method in, described determined wavelength is 224nm; Flow velocity is 0.8 ~ 1.0mL/min; Column temperature is 35 DEG C.
In aforesaid Zhenqi Fuzheng prepn Specnuezhenide content assaying method in, the preparation method of need testing solution 3. in cross silicagel column with chloroform: methyl alcohol: water=20:3:1 wash-out.
In aforesaid Zhenqi Fuzheng prepn Specnuezhenide content assaying method in, described macroreticular resin is nonpolar macroporous adsorption resin D101, nonpolar macroporous adsorption resin HPD100, low pole macroporous absorbent resin AB-8 or intermediate-polarity macroporous adsorption resin DM301.
In aforesaid Zhenqi Fuzheng prepn Specnuezhenide content assaying method in, preferred macroreticular resin is low pole macroporous absorbent resin AB-8.
Adopt the content assaying method of Specnuezhenide in Zhenqi Fuzheng prepn of the present invention, Zhenqi Fuzheng prepn is containing Specnuezhenide C
31h
42o
17must not 1.0% be less than.
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
For finding optimum column temperature, applicant adopts different column temperature to test, and observations, 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
For finding optimum flow rate, applicant adopts different in flow rate to test, and record chromatographic peak, 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
For finding optimal flow phase, applicant adopts different mobile phase to carry out UV detection, and record chromatographic peak, the results are shown in Table 4.
Table 4
| Mobile phase | Chromatographic peak |
| Methyl alcohol: 0.05% phosphate aqueous solution=30:70 | Generation ghost peak is few, and peak shape is good, and degree of separation is high |
| Methyl alcohol: water=60:40 | Peak shape is poor, and degree of separation is low |
| Acetonitrile: 0.05% glacial acetic acid=30:70 | Peak shape is slightly poor, and degree of separation is higher |
| 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=20:80 | Peak shape is good, and degree of separation is high |
As seen from the above table, select methyl alcohol: 0.05% phosphate aqueous solution=30:70 is as mobile phase, and the chromatographic peak effect that it obtains is best.
2, the preparation of reference substance solution
Get Specnuezhenide reference substance appropriate, accurately weighed, add methyl alcohol and make 0.2mg/ml, to obtain final product.
3, the preparation of need testing solution: employing method 1. ~ one of be 3. prepared:
1. precision takes Zhenqi Fuzheng prepn or its content 3g, and precision adds methyl alcohol 100mL, weighed weight, and ultrasonic or refluxing extraction 30 ~ 60min, or microwave radiation exaraction 2 ~ 10min, supply weight with methyl alcohol, filters, gets subsequent filtrate and get final product;
2. precision takes Zhenqi Fuzheng prepn or its content 3g, and precision adds methyl alcohol 100mL, weighed weight, ultrasonic or heating and refluxing extraction 30 ~ 60min, or microwave radiation exaraction 2 ~ 10min, supply weight with methyl alcohol, filter, precision measures subsequent filtrate 50mL, decompression and solvent recovery, residue thin up, upper macroreticular resin, is washed to colourless, again with 50% ~ 95% ethanol elution, eluent is concentrated into dry, and residue methyl alcohol dissolves and is transferred in 50mL volumetric flask, add methyl alcohol to scale, shake up, to obtain final product;
3. precision takes Zhenqi Fuzheng prepn or its content 3g, and precision adds methyl alcohol 100mL, weighed weight, ultrasonic or heating and refluxing extraction 30 ~ 60min, or microwave radiation exaraction 2 ~ 10min, supply weight with methyl alcohol, filter, precision measures subsequent filtrate 50mL, decompression and solvent recovery, residue is dissolved in water, and cross silicagel column with chloroform: methyl alcohol: water=5 ~ 45:3:1 wash-out, eluent is concentrated into dry, residue methyl alcohol dissolves and is transferred in 50mL volumetric flask, add methyl alcohol to scale, shake up, to obtain final product.
The comparison of 3.1 extracting method
Precision takes Zhenqi Fuzheng prepn or its content 3g, and precision adds methyl alcohol 100mL, weighed weight, adopts backflow, ultrasonic or microwave extract method extraction respectively, supplies weight, filter, get subsequent filtrate, obtain three parts of need testing solutions with methyl alcohol; Investigate the extraction ratio of Specnuezhenide, result shows: under the same conditions, microwave method and ultrasonic method extraction ratio more close, all high than the extraction ratio of circumfluence method, and microwave―assisted extraction has extraction time short, the advantage such as extraction efficiency is high, method is easy.
The comparison of 3.2 need testing solution preparation methods
Applicant compares the effect that above-mentioned 3 kinds of methods (method 1. ~ 3.) are extracted Specnuezhenide in Zhenqi Fuzheng prepn, and experimental result is in table 5.
Table 5
As seen from the above table, although method is 1. easy and simple to handle, Specnuezhenide extraction ratio is high, and Specnuezhenide purity is very low.Although 2., 3. step is slightly aobvious loaded down with trivial details for method, Specnuezhenide extraction ratio is slightly low, and Specnuezhenide purity is high, eliminates the impact of impurity on testing result to a great extent.
The selection of 3.3 pairs of silicagel column elution agent
Because the polarity of Specnuezhenide is bigger than normal, therefore the organic solvent that polarity is large can be used, as chloroform, ethyl acetate, methyl alcohol, normal butyl alcohol and water make eluant, eluent.Inventor press need testing solution preparation method 3. in method, following 5 groups of parallel experiments are carried out to same batch of Zhenqi Fuzheng prepn and extracts Specnuezhenide, to compare the effect that in this method, different eluant, eluent extracts Specnuezhenide in Zhenqi Fuzheng prepn, experimental result is in table 6.
Table 6
As shown in Table 6: eluant, eluent is with chloroform: methyl alcohol: the collocation of water is best.
The selection of 3.4 pairs of silicagel column elution agent ratios
Method during 3. inventor adopts need testing solution preparation method, following 4 groups of parallel experiments are carried out to same batch of Zhenqi Fuzheng prepn and extracts Specnuezhenide, to compare the effect that in this method, different eluant, eluent ratio is extracted Specnuezhenide in Zhenqi Fuzheng prepn, experimental result is in table 7.
Table 7
As shown in Table 7, chloroform: methyl alcohol: water=20:3:1 is best eluant, eluent.
4, the selection of macroporous absorbent resin
Dissimilar macroporous absorbent resin to the Static Adsorption of fruit of glossy privet saponins, terpene, flavonoids and Astrageloside, terpene, flavones ingredient and desorption experiment result as shown in table 8.
Table 8
As shown in Table 8, by Static and dynamic adsorption experiment, find that the adsorption and desorption ability of dissimilar macroreticular resin to the effective constituent of the fruit of glossy privet and the Radix Astragali is variant.Comprehensively think adopt D101, HPD-100, DM301 or AB-8 type macroreticular resin time, to the saturated adsorption capacity of terpene, flavonoids and saponins and desorption efficiency all higher, reach about 600mg/g and about 60% respectively, when especially adopting macroreticular resin AB-8, to the saturated adsorption capacity of terpene, flavonoids and saponins and desorption efficiency the highest; Therefore, the present invention, when extracting the effective constituent such as the fruit of glossy privet and Radix Astragali terpene, flavonoids, saponins, adopts D101, HPD-100, DM301 or AB-8 type macroreticular resin, preferred AB-8 macroreticular resin.
5, the selection of determined wavelength
Carry out spectral scan at 200 ~ 400nm to Specnuezhenide reference substance solution, its maximum absorption wavelength is 224nm, therefore selects 224nm to be determined wavelength.
6, 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 Specnuezhenide reference substance solution of variable concentrations to concentration drawing standard curve, calculating regression equation is Y=1.55 × 10
6x+7.31 × 10
3, r=0.9998, wherein, Y is peak area, and X is Specnuezhenide reference substance solution concentration, and Specnuezhenide is good linear relation at 0.005 ~ 0.08g/L.
7, 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 Specnuezhenide peak area RSD is 1.05%, shows that the method precision is good.
8, 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 Specnuezhenide peak area is 0.93%, at room temperature 24h is interior stable to show the solution after processing, and has good stability.
9, 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 of record peak area is 0.82%, shows that repeatability is good.
10, average recovery experiment
Precision takes the Zhenqi Fuzheng prepn 6 parts of known content, and precision adds Specnuezhenide reference substance respectively, prepares by the preparation method of need testing solution, and sample introduction measures, measurement result is in table 9, and calculating its average recovery rate is 98.00%, RSD is 1.47%, and the recovery is high, shows that this method is feasible.
Table 9 average recovery experimental result
10, sample size measures
Get 5 batches of zhenqi fuzheng granuleses 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 Specnuezhenide, the results are shown in Table 10.
Table 10 sample size measurement result (n=5)
| Sample lot number | Average content (mg/g) |
| 20120801 | 14.63 |
| 20120802 | 14.43 |
| 20120803 | 14.58 |
| 20120804 | 14.29 |
| 20120805 | 14.37 |
Compared with prior art, the present invention is measured the content of Specnuezhenide 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: in ZHENQI FUZHENG JIAONANG, the content of Specnuezhenide carries out method for measuring and comprises the following steps:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With methyl alcohol: 0.05% phosphate aqueous solution=30:70 is mobile phase; Column temperature is 35 DEG C; Determined wavelength is 224nm; Flow velocity is 0.9mL/min; Number of theoretical plate calculates should be not less than 4000 by Specnuezhenide peak;
(2) preparation of reference substance solution: get Specnuezhenide reference substance appropriate, accurately weighed, add methyl alcohol and make the solution that concentration is 0.2mg/mL, to obtain final product;
(3) preparation of need testing solution: precision takes Zhenqi Fuzheng prepn or its content 3g, precision adds methyl alcohol 100mL, weighed weight, ultrasonic or heating and refluxing extraction 45min, or microwave radiation exaraction 6min, supply weight with methyl alcohol, filter, precision measures subsequent filtrate 50mL, decompression and solvent recovery, residue is dissolved in water, and cross silicagel column with chloroform: methyl alcohol: water=20:3:1 wash-out, eluent is concentrated into dry, residue methyl alcohol dissolves and is transferred in 50mL volumetric flask, 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, injects high performance liquid chromatograph, measures, to obtain final product.
The embodiment of the present invention 2: in zhenqi fuzheng granules, the content of Specnuezhenide carries out method for measuring and comprises the following steps:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With acetonitrile: 0.05% glacial acetic acid=20:80 is mobile phase; Column temperature is 35 DEG C; Determined wavelength is 224nm; Flow velocity is 0.8mL/min; Number of theoretical plate calculates should be not less than 4000 by Specnuezhenide peak;
(2) preparation of reference substance solution: get Specnuezhenide reference substance appropriate, accurately weighed, add methyl alcohol and make the solution that concentration is 0.2mg/mL, to obtain final product;
(3) preparation of need testing solution: precision takes Zhenqi Fuzheng prepn or its content 3g, precision adds methyl alcohol 100mL, weighed weight, ultrasonic or heating and refluxing extraction 45min, or microwave radiation exaraction 6min, supply weight with methyl alcohol, filter, precision measures subsequent filtrate 50mL, decompression and solvent recovery, residue thin up, upper low pole macroporous absorbent resin AB-8, be washed to colourless, then with 70% ethanol elution, eluent is concentrated into dry, residue methyl alcohol dissolves and is transferred in 50mL volumetric flask, 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, injects high performance liquid chromatograph, measures, to obtain final product.
The embodiment of the present invention 3: in zhenqi fuzheng granules, the content of Specnuezhenide carries out method for measuring and comprises the following steps:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With methyl alcohol: 0.05% glacial acetic acid=35:65 is mobile phase; Column temperature is 40 DEG C; Determined wavelength is 240nm; Flow velocity is 1.5mL/min; Number of theoretical plate calculates should be not less than 4000 by Specnuezhenide peak;
(2) preparation of reference substance solution: get Specnuezhenide reference substance appropriate, accurately weighed, add methyl alcohol and make the solution that concentration is 0.2mg/mL, to obtain final product;
(3) preparation of need testing solution: precision takes Zhenqi Fuzheng prepn or its content 3g, and precision adds methyl alcohol 100mL, weighed weight, ultrasonic or refluxing extraction 45min, or microwave radiation exaraction 6min, supplies weight with methyl alcohol, filters, gets subsequent filtrate and get final product;
(4) determination method: accurate absorption reference substance solution and each 10 μ L of need testing solution respectively, injects high performance liquid chromatograph, measures, to obtain final product.
The embodiment of the present invention 4: in qizhenfuzheng tablet, the content of Specnuezhenide carries out method for measuring and comprises the following steps:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With acetonitrile: water=5:95 mobile phase; Column temperature is 20 DEG C; Determined wavelength is 210nm; Flow velocity is 0.5mL/min; Number of theoretical plate calculates should be not less than 4000 by Specnuezhenide peak;
(2) preparation of reference substance solution: get Specnuezhenide reference substance appropriate, accurately weighed, add methyl alcohol and make the solution that concentration is 0.2mg/mL, to obtain final product;
(3) preparation of need testing solution: precision takes Zhenqi Fuzheng prepn or its content 3g, and precision adds methyl alcohol 100mL, weighed weight, ultrasonic or refluxing extraction 30min, or microwave radiation exaraction 2min, supplies weight with methyl alcohol, filters, gets subsequent filtrate and get final product;
(4) determination method: accurate absorption reference substance solution and each 10 μ L of need testing solution respectively, injects high performance liquid chromatograph, measures, to obtain final product.
The embodiment of the present invention 5: in Zhenqi dripping pill for strengthening resistance, the content assaying method of Specnuezhenide comprises the following steps:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With methyl alcohol: 0.05% aqueous formic acid=50:50 is mobile phase; Column temperature is 30 DEG C; Determined wavelength is 220nm; Flow velocity is 0.8mL/min; Number of theoretical plate calculates should be not less than 4000 by Specnuezhenide peak;
(2) preparation of reference substance solution: get Specnuezhenide reference substance appropriate, accurately weighed, add methyl alcohol and make the solution that concentration is 0.2mg/mL, to obtain final product;
(3) preparation of need testing solution: precision takes Zhenqi Fuzheng prepn or its content 3g, precision adds methyl alcohol 100mL, weighed weight, ultrasonic or heating and refluxing extraction 30min, or microwave radiation exaraction 2min, supply weight with methyl alcohol, filter, precision measures subsequent filtrate 50mL, decompression and solvent recovery, residue thin up, upper intermediate-polarity macroporous adsorption resin DM301, be washed to colourless, then with 50% ethanol elution, eluent is concentrated into dry, residue methyl alcohol dissolves and is transferred in 50mL volumetric flask, 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, injects high performance liquid chromatograph, measures, to obtain final product.
The embodiment of the present invention 6: in ZHENQI FUZHENG JIAONANG, the content assaying method of Specnuezhenide comprises the following steps:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With methyl alcohol: water=20:80 is mobile phase; Column temperature is 25 DEG C; Determined wavelength is 230nm; Flow velocity is 1.3mL/min; Number of theoretical plate calculates should be not less than 4000 by Specnuezhenide peak;
(2) preparation of reference substance solution: get Specnuezhenide reference substance appropriate, accurately weighed, add methyl alcohol and make the solution that concentration is 0.2mg/mL, to obtain final product;
(3) preparation of need testing solution: precision takes Zhenqi Fuzheng prepn or its content 3g, precision adds methyl alcohol 100mL, weighed weight, ultrasonic or heating and refluxing extraction 30min, or microwave radiation exaraction 2min, supply weight with methyl alcohol, filter, precision measures subsequent filtrate 50mL, decompression and solvent recovery, residue is dissolved in water, and cross silicagel column with chloroform: methyl alcohol: water=5:3:1 wash-out, eluent is concentrated into dry, residue methyl alcohol dissolves and is transferred in 50mL volumetric flask, 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, injects high performance liquid chromatograph, measures, to obtain final product.
The embodiment of the present invention 7: in Zhenqi dripping pill for strengthening resistance, the content assaying method of Specnuezhenide comprises the following steps:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With methyl alcohol: 0.01% phosphate aqueous solution=40:60 is mobile phase; Column temperature is 35 DEG C; Determined wavelength is 224nm; Flow velocity is 1.0mL/min; Number of theoretical plate calculates should be not less than 4000 by Specnuezhenide peak;
(2) preparation of reference substance solution: get Specnuezhenide reference substance appropriate, accurately weighed, add methyl alcohol and make the solution that concentration is 0.2mg/mL, to obtain final product;
(3) preparation of need testing solution: precision takes Zhenqi Fuzheng prepn or its content 3g, and precision adds methyl alcohol 100mL, weighed weight, ultrasonic or refluxing extraction 60min, or microwave radiation exaraction 10min, supplies weight with methyl alcohol, filters, gets subsequent filtrate and get final product;
(4) determination method: accurate absorption reference substance solution and each 10 μ L of need testing solution respectively, injects high performance liquid chromatograph, measures, to obtain final product.
The embodiment of the present invention 8: in zhenqi fuzheng granules, the content assaying method of Specnuezhenide comprises the following steps:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With acetonitrile: 0.05% phosphate aqueous solution=35:65 is mobile phase; Column temperature is 30 DEG C; Determined wavelength is 224nm; Flow velocity is 0.6mL/min; Number of theoretical plate calculates should be not less than 4000 by Specnuezhenide peak;
(2) preparation of reference substance solution: get Specnuezhenide reference substance appropriate, accurately weighed, add methyl alcohol and make the solution that concentration is 0.2mg/mL, to obtain final product;
(3) preparation of need testing solution: precision takes Zhenqi Fuzheng prepn or its content 3g, precision adds methyl alcohol 100mL, weighed weight, ultrasonic or heating and refluxing extraction 60min, or microwave radiation exaraction 10min, weight is supplied with methyl alcohol, filter, precision measures subsequent filtrate 50mL, decompression and solvent recovery, residue thin up, upper nonpolar macroporous adsorption resin D101 or nonpolar macroporous adsorption resin HPD100, be washed to colourless, again with 95% ethanol elution, eluent is concentrated into dry, residue methyl alcohol dissolves and is transferred in 50mL volumetric flask, add methyl alcohol to scale, shake up, obtain,
(4) determination method: accurate absorption reference substance solution and each 10 μ L of need testing solution respectively, injects high performance liquid chromatograph, measures, to obtain final product.
The embodiment of the present invention 9: in zhenqi fuzheng granules, the content assaying method of Specnuezhenide comprises the following steps:
(1) chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With acetonitrile: 0.05% aqueous formic acid=30:70 is mobile phase; Column temperature is 35 DEG C; Determined wavelength is 235nm; Flow velocity is 0.9mL/min; Number of theoretical plate calculates should be not less than 4000 by Specnuezhenide peak;
(2) preparation of reference substance solution: get Specnuezhenide reference substance appropriate, accurately weighed, add methyl alcohol and make the solution that concentration is 0.2mg/mL, to obtain final product;
(3) preparation of need testing solution: precision takes Zhenqi Fuzheng prepn or its content 3g, precision adds methyl alcohol 100mL, weighed weight, ultrasonic or heating and refluxing extraction 60min, or microwave radiation exaraction 10min, supply weight with methyl alcohol, filter, precision measures subsequent filtrate 50mL, decompression and solvent recovery, residue is dissolved in water, and cross silicagel column with chloroform: methyl alcohol: water=45:3:1 wash-out, eluent is concentrated into dry, residue methyl alcohol dissolves and is transferred in 50mL volumetric flask, 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, injects high performance liquid chromatograph, measures, to obtain final product.
The combination in any of all conditions parameter in above-described embodiment, all can obtain consistent assay result; In above-described embodiment, Specnuezhenide C in Zhenqi Fuzheng prepn
31h
42o
17content be no less than 1.0%.
Claims (3)
1. the content assaying method of Specnuezhenide in Zhenqi Fuzheng prepn, is characterized in that: it is with Specnuezhenide reference substance for contrast, with methyl alcohol: 0.05% phosphate aqueous solution=30:70 is 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: 0.05% phosphate aqueous solution=30:70 is mobile phase; Column temperature is 20 ~ 40 DEG C; Determined wavelength is 210 ~ 240nm; Flow velocity is 0.5 ~ 1.5mL/min; Number of theoretical plate calculates should be not less than 4000 by Specnuezhenide peak;
(2) preparation of reference substance solution: get Specnuezhenide reference substance appropriate, accurately weighed, add methyl alcohol and make the solution that concentration is 0.2mg/mL, to obtain final product;
(3) preparation of need testing solution: employing method 1. ~ one of be 3. prepared:
1. precision takes Zhenqi Fuzheng prepn or its content 3g, and precision adds methyl alcohol 100mL, weighed weight, and ultrasonic extraction 30 ~ 60min, or microwave radiation exaraction 2 ~ 10min, supply weight with methyl alcohol, filters, gets subsequent filtrate and get final product;
2. precision takes Zhenqi Fuzheng prepn or its content 3g, precision adds methyl alcohol 100mL, weighed weight, ultrasonic extraction 30 ~ 60min, or microwave radiation exaraction 2 ~ 10min, supply weight with methyl alcohol, filter, precision measures subsequent filtrate 50mL, decompression and solvent recovery, residue thin up, upper low pole macroporous absorbent resin AB-8, be washed to colourless, then with 50% ~ 95% ethanol elution, eluent is concentrated into dry, residue methyl alcohol dissolves and is transferred in 50mL volumetric flask, add methyl alcohol to scale, shake up, to obtain final product;
3. precision takes Zhenqi Fuzheng prepn or its content 3g, and precision adds methyl alcohol 100mL, weighed weight, ultrasonic extraction 30 ~ 60min, or microwave radiation exaraction 2 ~ 10min, supply weight with methyl alcohol, filter, precision measures subsequent filtrate 50mL, decompression and solvent recovery, residue is dissolved in water, and cross silicagel column with chloroform: methyl alcohol: water=5 ~ 45:3:1 wash-out, eluent is concentrated into dry, residue methyl alcohol dissolves and is transferred in 50mL volumetric flask, 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, injects high performance liquid chromatograph, measures, to obtain final product.
2. the content assaying method of Specnuezhenide in Zhenqi Fuzheng prepn according to claim 1, is characterized in that: described determined wavelength is 224nm; Flow velocity is 0.8 ~ 1.0mL/min; Column temperature is 35 DEG C.
3. the content assaying method of Specnuezhenide in Zhenqi Fuzheng prepn according to claim 1, is characterized in that: the preparation method of need testing solution 3. in, cross silicagel column with chloroform: methyl alcohol: water=20:3:1 wash-out.
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| CN106918674B (en) * | 2015-12-28 | 2018-12-28 | 广州白云山陈李济药厂有限公司 | It is a kind of treat soreness of waist and knee joint, sciatica Chinese medicine composition detection method |
| CN107238671B (en) * | 2017-08-09 | 2020-07-31 | 吉林省现代中药工程研究中心有限公司 | Method for measuring contents of multiple components in glossy privet fruit and astragalus root body resistance strengthening preparation |
| CN108593819A (en) * | 2018-05-09 | 2018-09-28 | 国珍健康科技（北京）有限公司 | A method of Specnuezhenide content is measured by high performance liquid chromatography |
| CN111487360A (en) * | 2020-04-20 | 2020-08-04 | 山东省药学科学院 | Method for quantitatively detecting content of specnuezhenide in kidney-tonifying and pulse-invigorating prescription by liquid chromatography-mass spectrometry |
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