CN103592392B - Determination method for tanshinol content in Naozhengning preparation - Google Patents
Determination method for tanshinol content in Naozhengning preparation Download PDFInfo
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- CN103592392B CN103592392B CN201310599038.0A CN201310599038A CN103592392B CN 103592392 B CN103592392 B CN 103592392B CN 201310599038 A CN201310599038 A CN 201310599038A CN 103592392 B CN103592392 B CN 103592392B
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Abstract
The invention provides a determination method for tanshinol content in a Naozhengning preparation. Salvianic acid A sodium in the preparation is extracted by using a methanol-glacial acetic acid or methanol-trifluoroacetic acid solution to prepare a solution for test products, octadecylsilane chemically bonded silica chromatographic column is adopted, the methanol to 0.01-0.2 percent of trifluoroacetic acid solution, which equals to (5:95) to (20:80), is used as a mobile phase for carrying out high performance liquid chromatographic analysis, the chromatographic peak of the salvianic acid A sodium is determined the in the position of 280 nm wave length, and the tanshinol content is calculated through an external standard method. The determination method for the tanshinol content, established in the invention, has strong specificity, the linear relation is good, the accuracy is high, the repeatability is good, the determining time is short, the tanshinol content is used as a quality control index in the Naozhengning preparation, the product quality of the Naozhengning preparation can be effectively controlled, and the transfer rate of the salvianic acid A sodium can be appraised more scientifically.
Description
Technical field
The present invention relates to the content assaying method of danshensu, particularly relating to a kind of method for measuring content of Danshensu in Naozhenning preparation.
Background technology
Naozhenning preparation is the various suitable formulation such as granule, capsule, tablet, soft capsule, oral liquid made according to prescription disclosed in ZL93100741, for prevention and therapy postcerebrotraumatic syndrome.
Naozhenning particle is included in " Drug Standard of Ministry of Public Health of the Peoples Republic of China Traditional Chinese medicine historical preparation (the 17) " the 230th page of (standard No.: WS
3-B-3312-98), its prescription is: Radix Angelicae Sinensis 200g, glutinous rehmannia 200g, moutan bark 150g, Ligusticum wallichii 150g, earthworm 150g, red sage root 375g, Poria cocos 250g, dried orange peel 250g, caulis bambusae in taenian 250g, spina date seed (stir-fry) 250g, seed of Oriental arborvitae 250g; Method for making is: above ten simply, Radix Angelicae Sinensis, moutan bark, Ligusticum wallichii, dried orange peel distillating extracting oil, and the another device of the aqueous solution after distillation is collected; The seven taste boiling secondaries such as the dregs of a decoction and all the other reds sage root, each 1.5 hours, collecting decoction, considered, filtrate and above-mentioned aqueous solution merge, be concentrated into the clear cream of relative density 1.35 ~ 1.38 (55 DEG C), add suitable amount of sucrose and dextrin, granulate, dry, add above-mentioned volatile oil, mixing, makes 1000g.Function cooling and activating blood, disperse blood stasis and dredge collateral, beneficial blood and tranquilizing mind, the peaceful heart determines intelligence, relieves restlessness and prevents vomiting.Be used for the treatment of the headache that brain trauma causes, dizzy, irritated insomnia, forgetful palpitation with fear, n and V.
According to the Naozhenning sugar-free particle that above-mentioned Naozhenning particle improves, standard YBZ27112005, its prescription is: Radix Angelicae Sinensis 333.4g, glutinous rehmannia 333.4g, moutan bark 250g, Ligusticum wallichii 250g, earthworm 250g, red sage root 625g, Poria cocos 416.7g, dried orange peel 416.7g, caulis bambusae in taenian 416.7g, spina date seed (stir-fry) 416.7g, seed of Oriental arborvitae 416.7g; Function, to cure mainly and WS
3-B-3312-98 is identical, and just method for making is slightly distinguished, and is specially: above ten simply, Radix Angelicae Sinensis, moutan bark, Ligusticum wallichii, dried orange peel distillating extracting oil, and the another device of the aqueous solution after distillation is collected; The seven taste boiling secondaries such as the dregs of a decoction and all the other reds sage root, each 1.5 hours, collecting decoction, considered, filtrate and above-mentioned aqueous solution merge, and are concentrated into the clear cream of relative density 1.35 ~ 1.38 (55 DEG C), take dextrin as auxiliary material, granulate, drying, adds above-mentioned volatile oil, mixing, make 1000g, to obtain final product.
At present, comprise in the standard of Naozhenning particle and sugar-free particle and all only have Qualitive test to test, do not relate to the quantitative measurement of any active constituent content.The red sage root is one of main flavour of a drug of Naozhenning preparation, danshensu is one of principal ingredient of the red sage root, and stable in properties, take danshensu as index components, a quality control index using content of Danshensu as Naozhenning preparation, controlling the content of the main flavour of a drug red sage root in Naozhenning preparation, is a kind of effective quantitative control methodin.
Summary of the invention
The object of this invention is to provide a kind of easy, sensitive, content of Danshensu assay method fast and accurately in Naozhenning preparation, effectively to control the red sage root content in Naozhenning preparation.
Content of Danshensu assay method in Naozhenning preparation of the present invention comprises the following steps:
1) preparation of reference substance solution: get Sodium Danshensu reference substance, accurately weighed, make the reference substance solution of every 1mL containing 2 ~ 50 μ g Sodium Danshensus with solvent A;
2) preparation of need testing solution: get Naozhenning formulation samples, accurately weighed, add solvent A, weighed weight, add hot reflux or ultrasonic process, let cool, more weighed weight, the weight of less loss is supplied with solvent A, shake up, filter, get subsequent filtrate, be mixed with the need testing solution adapted with reference substance solution Sodium Danshensu concentration;
3) adopt high performance liquid chromatograph to measure, chromatographic condition is: chromatographic column: octadecylsilane chemically bonded silica post; Mobile phase: methyl alcohol: 0.01 ~ 0.2% trifluoroacetic acid aqueous solution=5: 95 ~ 20: 80; Column temperature: 5 ~ 45 DEG C; Determined wavelength: 280nm;
4) precision draws need testing solution and reference substance solution respectively, injection liquid chromatography, measures the peak area of Sodium Danshensu chromatographic peak, calculates the content of Danshensu in Naozhenning formulation samples with external standard method;
Further, the content of Danshensu assay method in Naozhenning preparation comprises the following steps:
1) preparation of reference substance solution: get Sodium Danshensu reference substance, accurately weighed, make the reference substance solution of every 1mL containing 5 ~ 20 μ g Sodium Danshensus with solvent A;
2) preparation of need testing solution: get Naozhenning formulation samples, accurately weighed, add solvent A, weighed weight, with the ultrasound wave process 1 ~ 5 time of power 50 ~ 500W, frequency 25 ~ 80kHz, each 10 ~ 90 minutes, let cool, weighed weight again, supply the weight of less loss with solvent A, shake up, filter, get subsequent filtrate, be mixed with the need testing solution adapted with reference substance solution Sodium Danshensu concentration;
3) adopt high performance liquid chromatography to measure, chromatographic condition is: chromatographic column: octadecylsilane chemically bonded silica post; Mobile phase: methyl alcohol: 0.03 ~ 0.13% trifluoroacetic acid aqueous solution=5: 95 ~ 20: 80; Column temperature: 10 ~ 40 DEG C; Determined wavelength: 280nm; Flow velocity: 0.5 ~ 1.5mL/ minute;
4) precision draws need testing solution and each 2 ~ 25 μ L of reference substance solution respectively, injection liquid chromatography, measures the peak area of Sodium Danshensu chromatographic peak, calculates the content of Danshensu in Naozhenning formulation samples with external standard method.
In said determination method of the present invention, described solvent A can be methanol acetic acid solution or methyl alcohol trifluoroacetic acid solution.Wherein, described methanol acetic acid solution be methyl alcohol and 0.1 ~ 1% glacial acetic acid aqueous solution according to 5: 95 ~ 20: 80 the methanol acetic acid solution that is mixed with of volume ratio, preferably, be methyl alcohol and 0.5% glacial acetic acid aqueous solution according to 10: 90 the methanol acetic acid solution that is mixed with of volume ratio.Described methyl alcohol trifluoroacetic acid solution be methyl alcohol and 0.01 ~ 0.15% trifluoroacetic acid aqueous solution according to 5: 95 ~ 20: 80 the methyl alcohol trifluoroacetic acid solution that is mixed with of volume ratio, preferably, be methyl alcohol and 0.1% trifluoroacetic acid aqueous solution according to 10: 90 the methyl alcohol trifluoroacetic acid solution that is mixed with of volume ratio.
Further, chromatographic condition of the present invention is: chromatographic column: octadecylsilane chemically bonded silica post; Mobile phase: methyl alcohol: 0.1% trifluoroacetic acid aqueous solution=10: 90; Column temperature: 20 ~ 30 DEG C; Determined wavelength: 280nm; Flow velocity: 1mL/ minute.
Usually, liquid chromatography sample size is 10 μ L or 20 μ L.
Concentration described in the present invention, unless otherwise indicated, is concentration expressed in percentage by volume.
Naozhenning preparation described in the present invention refers to the formulations described in any pharmacy such as Naozhenning particle, Naozhenning sugar-free particle, Naozhenning sheet, Naozhenning dispersing tablet, Naozhenning capsule, Naozhenning soft capsule, Naozhenning oral liquid, Naozhenning syrup, Naozhenning ball.
Chinese medicine standard modern is the important component part of TCM Modernization, and selecting to have significant chemical composition in the main flavour of a drug of Chinese patent drug is index components, and measuring its content, is the Main Means of chemical determination Chinese patent drug content.
The present invention selects Sodium Danshensu contained by the main flavour of a drug red sage root in Naozhenning preparation to be index components, by the quality control index adopting the content of Danshensu in high effective liquid chromatography for measuring Naozhenning preparation to be used as Naozhenning preparation, the assay method specificity set up is strong, linear relationship is good, accuracy is high, reproducible, minute is short.Advantage is easy, quick, accurate, can evaluate the rate of transform of Sodium Danshensu more scientificly, effectively improve the quality of product.
Accompanying drawing explanation
Fig. 1 is the Sodium Danshensu high-efficient liquid phase chromatogram in reference substance solution.
Fig. 2 is the Sodium Danshensu high-efficient liquid phase chromatogram in Naozhenning preparation.
Fig. 3 is the high-efficient liquid phase chromatogram of negative controls solution.
Fig. 4 is the linear graph of Sodium Danshensu.
Embodiment
Embodiment 1
Naozhenning particle is (according to the Sanitation Ministry medicine standard WS
3the granule that-B-3312-98 makes) in content of Danshensu measure.
Prescription: Radix Angelicae Sinensis 200g, glutinous rehmannia 200g, moutan bark 150g, Ligusticum wallichii 150g, earthworm 150g, red sage root 375g, Poria cocos 250g, dried orange peel 250g, caulis bambusae in taenian 250g, spina date seed (stir-fry) 250g, seed of Oriental arborvitae 250g.
Method for making: above ten simply, Radix Angelicae Sinensis, moutan bark, Ligusticum wallichii, dried orange peel distillating extracting oil, the another device of the aqueous solution after distillation is collected; The seven taste boiling secondaries such as the dregs of a decoction and all the other reds sage root, each 1.5 hours, collecting decoction, considered, filtrate and above-mentioned aqueous solution merge, be concentrated into the clear cream of relative density 1.35 ~ 1.38 (55 DEG C), add suitable amount of sucrose and dextrin, granulate, dry, add above-mentioned volatile oil, mixing, makes 1000g.
1) chromatographic condition and system suitability:
Chromatographic column: octadecylsilane chemically bonded silica post (5 μm, 4.6mm × 200mm); Mobile phase: methyl alcohol-0.1% trifluoroacetic acid aqueous solution (10: 90); Flow velocity: 1.0mL/min; Wavelength: 280nm; Column temperature: 25 DEG C; Theoretical cam curve is pressed Sodium Danshensu peak and is calculated, and should be not less than 1500.
2) preparation of reference substance solution: be taken at the Sodium Danshensu reference substance 12.5mg that 110 DEG C are dried to constant weight, accurately weighed, put in 50mL measuring bottle, add methyl alcohol-0.5% glacial acetic acid aqueous solution (10: 90) to scale, shake up, precision measures 1mL, to in 25mL measuring bottle, add methyl alcohol-0.5% glacial acetic acid aqueous solution (10: 90) and be diluted to scale, shake up, obtain (1mg Sodium Danshensu is equivalent to 0.89mg) danshensu.
3) preparation of need testing solution: get Naozhenning particle, porphyrize, mixing, get 0.6g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol-0.5% glacial acetic acid aqueous solution (10: 90) 25mL, weighed weight, ultrasonic process (power 500W, frequency 40kHz) 30 minutes, let cool, more weighed weight, the weight of less loss is supplied by methyl alcohol-0.5% glacial acetic acid aqueous solution (10: 90), filter with 0.45 μm of miillpore filter, get subsequent filtrate, to obtain final product.
4) accurate absorption need testing solution and each 10 μ L of reference substance solution, injection liquid chromatography, measures the peak area of Sodium Danshensu chromatographic peak.
5) content of Danshensu in Naozhenning particulate samples is calculated with external standard method.
Measure 3 batches of Naozhenning particulate samples respectively, in every bag, be respectively 3.56 containing danshensu, 4.32,4.10mg.
Embodiment 2
Content of Danshensu in Naozhenning sugar-free particle (the sugar-free particle made according to the Sanitation Ministry medicine standard YBZ27112005) measures.
Prescription: Radix Angelicae Sinensis 333.4g, glutinous rehmannia 333.4g, moutan bark 250g, Ligusticum wallichii 250g, earthworm 250g, red sage root 625g, Poria cocos 416.7g, dried orange peel 416.7g, caulis bambusae in taenian 416.7g, spina date seed (stir-fry) 416.7g, seed of Oriental arborvitae 416.7g.
Method for making: above ten simply, Radix Angelicae Sinensis, moutan bark, Ligusticum wallichii, dried orange peel distillating extracting oil, the another device of the aqueous solution after distillation is collected; The seven taste boiling secondaries such as the dregs of a decoction and all the other reds sage root, each 1.5 hours, collecting decoction, considered, filtrate and above-mentioned aqueous solution merge, and are concentrated into the clear cream of relative density 1.35 ~ 1.38 (55 DEG C), take dextrin as auxiliary material, granulate, drying, adds above-mentioned volatile oil, mixing, make 1000g, to obtain final product.
1) chromatographic condition and system suitability:
Chromatographic column: octadecylsilane chemically bonded silica post (5 μm, 4.6mm × 250mm); Mobile phase: methyl alcohol-0.1% trifluoroacetic acid aqueous solution (10: 90); Flow velocity: 0.5mL/min; Wavelength: 280nm; Column temperature: 30 DEG C; Theoretical cam curve is pressed Sodium Danshensu peak and is calculated, and should be not less than 2000.
2) preparation of reference substance solution: be taken at the Sodium Danshensu reference substance 12.5mg that 110 DEG C are dried to constant weight, accurately weighed, put in 100mL measuring bottle, add methyl alcohol-0.1% trifluoroacetic acid aqueous solution (10: 90) to scale, shake up, precision measures 1mL, to in 25mL measuring bottle, add methyl alcohol-0.1% trifluoroacetic acid aqueous solution (10: 90) and be diluted to scale, shake up, obtain (1mg Sodium Danshensu is equivalent to 0.89mg danshensu).
3) preparation of need testing solution: get Naozhenning sugar-free particle, porphyrize, mixing, get 0.6g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol-0.1% trifluoroacetic acid aqueous solution (10: 90) 60mL, weighed weight, ultrasonic process (power 250W, frequency 40kHz) 45 minutes, let cool, more weighed weight, the weight of less loss is supplied with methyl alcohol-0.1% trifluoroacetic acid aqueous solution (10: 90), filter with 0.45 μm of miillpore filter, get subsequent filtrate, to obtain final product.
4) accurate absorption need testing solution and each 20 μ L of reference substance solution, injection liquid chromatography, measures the peak area of Sodium Danshensu chromatographic peak.
5) content of Danshensu in Naozhenning sugar-free particulate samples is calculated with external standard method.
Measure 3 batches of Naozhenning sugar-free particulate samples respectively, in every bag, be respectively 3.26 containing danshensu, 3.32,3.10mg.
Embodiment 3
Naozhenning sheet is (according to ZL93100741 and the Sanitation Ministry medicine standard WS
3the tablet that-B-3312-98 makes) in content of Danshensu measure.
Prescription: Radix Angelicae Sinensis 1000g, glutinous rehmannia 1000g, moutan bark 750g, Ligusticum wallichii 750g, earthworm 750g, red sage root 1875g, Poria cocos 1250g, dried orange peel 1250g, caulis bambusae in taenian 1250g, spina date seed (stir-fry) 1250g, seed of Oriental arborvitae 1250g.
Method for making: above ten simply, Radix Angelicae Sinensis, moutan bark, Ligusticum wallichii, dried orange peel distillating extracting oil, the another device of the aqueous solution after distillation is collected; The seven taste boiling secondaries such as the dregs of a decoction and all the other reds sage root, each 1.5 hours, collecting decoction, considered, filtrate and above-mentioned aqueous solution merge, and are concentrated into appropriate clear cream, add ethanol and make alcohol content be 60%, mixing, leaves standstill and makes precipitation, filters, filtrate recycling ethanol is also condensed into thick paste, adds right amount of auxiliary materials, mixing, particle processed, dry, add above-mentioned volatile oil, mixing, is pressed into 1000, obtains final product.
1) chromatographic condition and system suitability:
Chromatographic column: octadecylsilane chemically bonded silica post (10 μm, 4.6mm × 200mm); Mobile phase: methyl alcohol-0.05% trifluoroacetic acid aqueous solution (5: 95); Flow velocity: 1.5mL/min; Wavelength: 280nm; Column temperature: 35 DEG C; Theoretical cam curve is pressed Sodium Danshensu peak and is calculated, and should be not less than 2000.
2) preparation of reference substance solution: be taken at the Sodium Danshensu reference substance 12.5mg that 110 DEG C are dried to constant weight, accurately weighed, put in 25mL measuring bottle, add methyl alcohol-0.1% glacial acetic acid aqueous solution (5: 95) to scale, shake up, precision measures 1mL, to in 25mL measuring bottle, add methyl alcohol-0.1% glacial acetic acid aqueous solution (5: 95) and be diluted to scale, shake up, obtain (1mg Sodium Danshensu is equivalent to 0.89mg danshensu).
3) preparation of need testing solution: get Naozhenning sheet, porphyrize, mixing, get 1g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol-0.1% glacial acetic acid aqueous solution (5: 95) 10mL, weighed weight, ultrasonic process (power 50W, frequency 80kHz) 45 minutes, let cool, more weighed weight, the weight of less loss is supplied by methyl alcohol-0.1% glacial acetic acid aqueous solution (5: 95), filter with 0.45 μm of miillpore filter, get subsequent filtrate, to obtain final product.
4) accurate absorption need testing solution and each 5 μ L injection liquid chromatographies of reference substance solution, measure the peak area of Sodium Danshensu chromatographic peak.
5) content of Danshensu in Naozhenning sheet sample is calculated with external standard method.
Measure 3 batches of Naozhenning sheet samples respectively, in every sheet, be respectively 1.10 containing danshensu, 1.04,1.12mg.
Embodiment 4
Content of Danshensu in Naozhenning capsule (capsule made according to ZL93100741 and the Sanitation Ministry medicine standard YBZ27112005) measures.
Prescription: Radix Angelicae Sinensis 1000g, glutinous rehmannia 1000g, moutan bark 750g, Ligusticum wallichii 750g, earthworm 750g, red sage root 1875g, Poria cocos 1250g, dried orange peel 1250g, caulis bambusae in taenian 1250g, spina date seed (stir-fry) 1250g, seed of Oriental arborvitae 1250g.
Method for making: above ten simply, Radix Angelicae Sinensis, moutan bark, Ligusticum wallichii, dried orange peel distillating extracting oil, the another device of the aqueous solution after distillation is collected; The seven taste boiling secondaries such as the dregs of a decoction and all the other reds sage root, each 1.5 hours, collecting decoction, considered, filtrate and above-mentioned aqueous solution merge, and are concentrated into appropriate clear cream, add ethanol and make alcohol content be 60%, mixing, leave standstill and make precipitation, filter, filtrate recycling ethanol is also condensed into thick paste, add right amount of auxiliary materials, mixing, particle processed, drying, adds above-mentioned volatile oil, mixing, incapsulate, make 1000, to obtain final product.
1) chromatographic condition and system suitability:
Chromatographic column: octadecylsilane chemically bonded silica post (5 μm, 4.6mm × 250mm); Mobile phase: methyl alcohol: 0.15% trifluoroacetic acid aqueous solution=15: 85; Flow velocity: 0.6mL/min; Wavelength: 280nm; Column temperature: 40 DEG C; Theoretical cam curve is pressed Sodium Danshensu peak and is calculated, and should be not less than 2000.
2) preparation of reference substance solution: be taken at the Sodium Danshensu reference substance 12.5mg that 110 DEG C are dried to constant weight, accurately weighed, put in 50mL measuring bottle, add methyl alcohol-1% glacial acetic acid aqueous solution (20: 80) to scale, shake up, precision measures 1mL, to in 25mL measuring bottle, add methyl alcohol-1% glacial acetic acid aqueous solution (20: 80) and be diluted to scale, shake up, obtain (1mg Sodium Danshensu is equivalent to 0.89mg danshensu).
3) preparation of need testing solution: get Naozhenning capsule 's content, porphyrize, mixing, get 1.5g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol-1% glacial acetic acid aqueous solution (20: 80) 25mL, weighed weight, ultrasonic process (power 50W, frequency 25kHz) 3 times, each 10min, lets cool, weighed weight again, supplies the weight of less loss by methyl alcohol-1% glacial acetic acid aqueous solution (20: 80), filter with 0.45 μm of miillpore filter, get subsequent filtrate, to obtain final product.
4) accurate absorption need testing solution and each 10 μ L injection liquid chromatographies of reference substance solution, measure the peak area of Sodium Danshensu chromatographic peak.
5) content of Danshensu in Naozhenning capsule sample is calculated with external standard method.
Measure 3 batches of Naozhenning capsule samples respectively, in every, be respectively 0.82 containing danshensu, 0.83,0.86mg.
Embodiment 5
Content of Danshensu in Naozhenning soft capsule (soft capsule made according to ZL93100741 and the Sanitation Ministry medicine standard YBZ27112005) measures.
Prescription: Radix Angelicae Sinensis 1000g, glutinous rehmannia 1000g, moutan bark 750g, Ligusticum wallichii 750g, earthworm 750g, red sage root 1875g, Poria cocos 1250g, dried orange peel 1250g, caulis bambusae in taenian 1250g, spina date seed (stir-fry) 1250g, seed of Oriental arborvitae 1250g.
Method for making: above ten simply, Radix Angelicae Sinensis, moutan bark, Ligusticum wallichii, dried orange peel distillating extracting oil, the another device of the aqueous solution after distillation is collected; The seven taste boiling secondaries such as the dregs of a decoction and all the other reds sage root, each 1.5 hours, collecting decoction, considered, filtrate and above-mentioned aqueous solution merge, and are concentrated into appropriate clear cream, adding ethanol makes alcohol content be 60%, mixing, leaves standstill and makes precipitation, filter, filtrate recycling ethanol is also condensed into thick paste, drying under reduced pressure, be ground into fine powder, add above-mentioned volatile oil and refined soybean oil, mixing, make soft capsule 1000, to obtain final product.
1) chromatographic condition and system suitability:
Chromatographic column: octadecylsilane chemically bonded silica post (5 μm, 4.6mm × 250mm); Mobile phase: methyl alcohol: 0.1% trifluoroacetic acid aqueous solution=10: 90; Flow velocity: 1mL/min; Wavelength: 280nm; Column temperature: 45 DEG C; Theoretical cam curve is pressed Sodium Danshensu peak and is calculated, and should be not less than 2000.
2) preparation of reference substance solution: be taken at the Sodium Danshensu reference substance 12.5mg that 110 DEG C are dried to constant weight, accurately weighed, put in 50mL measuring bottle, add methyl alcohol-0.05% trifluoroacetic acid aqueous solution (10: 90) to scale, precision measures 1mL, in 25mL measuring bottle, adds methyl alcohol-0.05% trifluoroacetic acid aqueous solution (10: 90) and is diluted to scale, shake up, obtain (1mg Sodium Danshensu is equivalent to 0.89mg danshensu).
3) preparation of need testing solution: get Naozhenning soft capsule content 2g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol-0.05% trifluoroacetic acid aqueous solution (10: 90) 25mL, weighed weight, ultrasonic process (power 50W, frequency 25kHz) 80 minutes, let cool, weighed weight again, supplies the weight of less loss with methyl alcohol-0.05% trifluoroacetic acid aqueous solution (10: 90), filter with 0.45 μm of miillpore filter, get subsequent filtrate, to obtain final product.
4) accurate absorption need testing solution and each 10 μ L injection liquid chromatographies of reference substance solution, measure the peak area of Sodium Danshensu chromatographic peak.
5) content of Danshensu in Naozhenning soft capsule sample is calculated with external standard method.
Measure 3 batches of Naozhenning soft capsule samples respectively, in every, be respectively 0.98 containing danshensu, 0.96,1.01mg.
Embodiment 6
Naozhenning particle is (according to the Sanitation Ministry medicine standard WS
3the granule that-B-3312-98 makes) in content of Danshensu measure.
Prescription and method for making are with embodiment 1.
1) chromatographic condition and system suitability:
Chromatographic column: octadecylsilane chemically bonded silica post (5 μm, 4.6mm × 200mm); Mobile phase: methyl alcohol: 0.1% trifluoroacetic acid aqueous solution=10: 90; Flow velocity: 1.0mL/min; Wavelength: 280nm; Column temperature: 20 DEG C; Theoretical cam curve is pressed Sodium Danshensu peak and is calculated, and should be not less than 2000.
2) preparation of reference substance solution: be taken at the Sodium Danshensu reference substance 12.5mg that 110 DEG C are dried to constant weight, accurately weighed, put in 25mL measuring bottle, add methyl alcohol-0.15% trifluoroacetic acid aqueous solution (10: 90) to scale, shake up, precision measures 1mL, to in 25mL measuring bottle, add methyl alcohol-0.15% trifluoroacetic acid aqueous solution (10: 90) and be diluted to scale, shake up, obtain (1mg Sodium Danshensu is equivalent to 0.89mg danshensu).
3) preparation of need testing solution: get Naozhenning particle, porphyrize, mixing, get 2g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol-0.15% trifluoroacetic acid aqueous solution (10: 90) 25mL, weighed weight, ultrasonic process (power 250W, frequency 40kHz) 45 minutes, let cool, more weighed weight, the weight of less loss is supplied with methyl alcohol-0.15% trifluoroacetic acid aqueous solution (10: 90), filter with 0.45 μm of miillpore filter, get subsequent filtrate, to obtain final product.
4) accurate absorption need testing solution and each 20 μ L of reference substance solution, injection liquid chromatography, measures the peak area of Sodium Danshensu chromatographic peak.
5) content of Danshensu in Naozhenning particulate samples is calculated with external standard method.
Measure 3 batches of Naozhenning particulate samples respectively, in every bag, be respectively 4.06 containing danshensu, 4.30,4.21mg.
Embodiment 7
Naozhenning sheet is (according to ZL93100741 and the Sanitation Ministry medicine standard WS
3the tablet that-B-3312-98 makes) in content of Danshensu measure.
Prescription and method for making are with embodiment 3.
1) chromatographic condition and system suitability:
Chromatographic column: octadecylsilane chemically bonded silica post (5 μm, 4.6mm × 200mm); Mobile phase: methyl alcohol: 0.1% trifluoroacetic acid aqueous solution=10: 90; Flow velocity: 1.0mL/min; Wavelength: 280nm; Column temperature: 25 DEG C; Theoretical cam curve is pressed Sodium Danshensu peak and is calculated, and should be not less than 2000.
2) preparation of reference substance solution: be taken at the Sodium Danshensu reference substance 12.5mg that 110 DEG C are dried to constant weight, accurately weighed, put in 25mL measuring bottle, add methyl alcohol-0.5% glacial acetic acid aqueous solution (10: 90) to scale, shake up, precision measures 1mL, to in 25mL measuring bottle, add methyl alcohol-0.5% glacial acetic acid aqueous solution (10: 90) and be diluted to scale, shake up, obtain (1mg Sodium Danshensu is equivalent to 0.89mg danshensu).
3) preparation of need testing solution: get Naozhenning sheet, porphyrize, mixing, get 2g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol-0.5% glacial acetic acid aqueous solution (10: 90) 25mL, weighed weight, ultrasonic process (power 150W, frequency 50kHz) 1 hour, let cool, more weighed weight, the weight of less loss is supplied by methyl alcohol-0.5% glacial acetic acid aqueous solution (10: 90), filter with 0.45 μm of miillpore filter, get subsequent filtrate, to obtain final product.
4) accurate absorption need testing solution and each 10 μ L injection liquid chromatographies of reference substance solution, measure the peak area of Sodium Danshensu chromatographic peak.
5) content of Danshensu in Naozhenning sheet sample is calculated with external standard method.
Measure 3 batches of Naozhenning sheet samples respectively, in every sheet, be respectively 1.16 containing danshensu, 1.06,1.02mg.
Embodiment 8
Naozhenning sheet is (according to ZL93100741 and the Sanitation Ministry medicine standard WS
3the tablet that-B-3312-98 makes) in content of Danshensu measure.
Prescription and method for making are with embodiment 3.
1) chromatographic condition and system suitability:
Chromatographic column: octadecylsilane chemically bonded silica post (5 μm, 4.6mm × 250mm); Mobile phase: methyl alcohol: 0.1% trifluoroacetic acid aqueous solution=10: 90; Flow velocity: 0.8mL/min; Wavelength: 280nm; Column temperature: 40 DEG C; Theoretical cam curve is pressed Sodium Danshensu peak and is calculated, and should be not less than 2000.
2) preparation of reference substance solution: be taken at the Sodium Danshensu reference substance 12.5mg that 110 DEG C are dried to constant weight, accurately weighed, put in 100mL measuring bottle, add methyl alcohol-0.5% glacial acetic acid aqueous solution (10: 90) to scale, shake up, precision measures 1mL, to in 25mL measuring bottle, add methyl alcohol-0.5% glacial acetic acid aqueous solution (10: 90) and be diluted to scale, shake up, obtain (1mg Sodium Danshensu is equivalent to 0.89mg danshensu).
3) preparation of need testing solution: get Naozhenning sheet, porphyrize, mixing, get 2.5g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol-0.5% glacial acetic acid aqueous solution (10: 90) 35mL, weighed weight, ultrasonic process (power 150W, frequency 33kHz) 60 minutes, let cool, more weighed weight, the weight of less loss is supplied by methyl alcohol-0.5% glacial acetic acid aqueous solution (10: 90), filter with 0.45 μm of miillpore filter, get subsequent filtrate, to obtain final product.
4) accurate absorption need testing solution and each 10 μ L injection liquid chromatographies of reference substance solution, measure the peak area of Sodium Danshensu chromatographic peak.
5) content of Danshensu in Naozhenning sheet is calculated with external standard method.
Measure 3 batches of Naozhenning sheet samples respectively, in every sheet, be respectively 1.04 containing danshensu, 1.12,1.08mg.
Embodiment 9
Content of Danshensu in Naozhenning sugar-free particle (the sugar-free particle made according to the Sanitation Ministry medicine standard YBZ27112005) measures.
Prescription and method for making are with embodiment 2.
1) chromatographic condition and system suitability:
Chromatographic column: octadecylsilane chemically bonded silica post (5 μm, 4.6mm × 200mm); Mobile phase: methyl alcohol: 0.1% trifluoroacetic acid aqueous solution=10: 90; Flow velocity: 1mL/min; Wavelength: 280nm; Column temperature: 30 DEG C; Theoretical cam curve is pressed Sodium Danshensu peak and is calculated, and should be not less than 2000.
2) preparation of reference substance solution: be taken at the Sodium Danshensu reference substance 12.5mg that 110 DEG C are dried to constant weight, accurately weighed, put in 100mL measuring bottle, add methyl alcohol-0.5% glacial acetic acid aqueous solution (10: 90) to scale, shake up, precision measures 1mL, to in 25mL measuring bottle, add methyl alcohol-0.5% glacial acetic acid aqueous solution (10: 90) and be diluted to scale, shake up, obtain (1mg Sodium Danshensu is equivalent to 0.89mg danshensu).
3) preparation of need testing solution: get Naozhenning sugar-free particle, porphyrize, mixing, get 2g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol-0.5% glacial acetic acid aqueous solution (10: 90) 50mL, weighed weight, ultrasonic process (power 50W, frequency 25kHz) 90 minutes, let cool, more weighed weight, the weight of less loss is supplied by methyl alcohol-0.5% glacial acetic acid aqueous solution (10: 90), filter with 0.45 μm of miillpore filter, get subsequent filtrate, to obtain final product.
4) accurate absorption need testing solution and each 20 μ L of reference substance solution, injection liquid chromatography, measures the peak area of Sodium Danshensu chromatographic peak.
5) content of Danshensu in Naozhenning sugar-free particulate samples is calculated with external standard method.
Measure 3 batches of Naozhenning sugar-free particulate samples respectively, in every bag, be respectively 3.36 containing danshensu, 3.22,3.16mg.
Embodiment 10
Specificity is investigated according to the prescription ratio of Naozhenning preparation and technique, preparation containing the negative sample of red rooted salvia, then according to preparation method's preparation of need testing solution containing the negative control solution of the red sage root.Draw reference substance solution, need testing solution, each 10 μ L injection liquid chromatographies of negative control solution respectively, obtain the liquid chromatogram of Fig. 1 ~ Fig. 3.
As can be seen from Fig. 1 and Fig. 2, in the high-efficient liquid phase chromatogram of need testing solution, there iing an identical Sodium Danshensu chromatographic peak with the corresponding position of reference substance solution high-efficient liquid phase chromatogram; And as can be seen from Figure 3, in the high-efficient liquid phase chromatogram of negative control solution, noiseless chromatographic peak on this relevant position.Illustrate that assay method provided by the invention has specificity.
Embodiment 11
Linear relationship is investigated precision and is taken Sodium Danshensu reference substance, makes the solution of 0.216mg/mL.Respectively precision measure reference substance solution 0.5,1.0,2.0,3.0,4.0,5.0,6.0,7.0mL puts in 25ml volumetric flask, be diluted to scale by methyl alcohol-0.5% glacial acetic acid aqueous solution (10: 90), shake up, liquid chromatogram measuring peak area.With Sodium Danshensu peak area for ordinate, sample size (μ g) is horizontal ordinate drawing standard curve (Fig. 4), and calculating Sodium Danshensu regression equation is Y=734.03X-7.1915, r=0.9998.Show that Sodium Danshensu is within the scope of 0.0864 ~ 1.2096 μ g, peak area and sample size linear relationship good.
Embodiment 12
Precision test precision draws above-mentioned 4th part of Sodium Danshensu reference substance solution, continuous sample introduction 5 times, measures peak area, calculates RSD, the results are shown in Table 1.Reference substance solution RSD is 0.740%, and result shows that method precision is good.
Embodiment 13
Replica test gets same batch sample 6 parts, measures its Sodium Danshensu content respectively, and Sodium Danshensu average content is 0.4069mg/g, RSD is 1.52%, shows that the assay method repeatability that the present invention sets up is good, the results are shown in Table 2.
Embodiment 14
Same reference substance solution is got in stability test, respectively at preparation after 0,1,3,6,9,12h sample introduction, the RSD of Sodium Danshensu chromatographic peak peak area is 1.15%, shows that reference substance solution is good at 12h internal stability.The results are shown in Table 3.
Embodiment 15
Recovery test adopts application of sample absorption method, gets 6 parts, the sample of known Sodium Danshensu content, adds Sodium Danshensu reference substance respectively, sample preparation measures peak area, calculates average recovery rate and RSD, the results are shown in Table 4, average recovery rate 100.27%, RSD1.45%, the method recovery is better.
Claims (5)
1. the content of Danshensu assay method in Naozhenning preparation, comprises the following steps:
1) preparation of reference substance solution: get Sodium Danshensu reference substance, accurately weighed, make the reference substance solution of every 1mL containing 2 ~ 50 μ g Sodium Danshensus with methyl alcohol trifluoroacetic acid solution;
2) preparation of need testing solution: get Naozhenning formulation samples, accurately weighed, add methyl alcohol trifluoroacetic acid solution, weighed weight, add hot reflux or ultrasonic process, let cool, more weighed weight, the weight of less loss is supplied with methyl alcohol trifluoroacetic acid solution, shake up, filter, get subsequent filtrate, be mixed with the need testing solution adapted with reference substance solution Sodium Danshensu concentration;
3) adopt high performance liquid chromatograph to measure, chromatographic condition is: chromatographic column: octadecylsilane chemically bonded silica post; Mobile phase: methyl alcohol: 0.01 ~ 0.2% trifluoroacetic acid aqueous solution=5: 95 ~ 20: 80; Column temperature: 5 ~ 45 DEG C; Determined wavelength: 280nm;
4) precision draws need testing solution and reference substance solution respectively, injection liquid chromatography, measures the peak area of Sodium Danshensu chromatographic peak, calculates the content of Danshensu in Naozhenning formulation samples with external standard method.
2. assay method according to claim 1, is characterized in that comprising the following steps:
1) preparation of reference substance solution: get Sodium Danshensu reference substance, accurately weighed, make the reference substance solution of every 1mL containing 5 ~ 20 μ g Sodium Danshensus with methyl alcohol trifluoroacetic acid solution;
2) preparation of need testing solution: get Naozhenning formulation samples, accurately weighed, add methyl alcohol trifluoroacetic acid solution, weighed weight, with the ultrasound wave process 1 ~ 5 time of power 50 ~ 500W, frequency 25 ~ 80kHz, each 10 ~ 90 minutes, let cool, weighed weight again, supply the weight of less loss with methyl alcohol trifluoroacetic acid solution, shake up, filter, get subsequent filtrate, be mixed with the need testing solution adapted with reference substance solution Sodium Danshensu concentration;
3) adopt high performance liquid chromatography to measure, chromatographic condition is: chromatographic column: octadecylsilane chemically bonded silica post; Mobile phase: methyl alcohol: 0.03 ~ 0.13% trifluoroacetic acid aqueous solution=5: 95 ~ 20: 80; Column temperature: 10 ~ 40 DEG C; Determined wavelength: 280nm; Flow velocity: 0.5 ~ 1.5mL/ minute;
4) precision draws need testing solution and each 2 ~ 25 μ L of reference substance solution respectively, injection liquid chromatography, measures the peak area of Sodium Danshensu chromatographic peak, calculates the content of Danshensu in Naozhenning formulation samples with external standard method.
3. assay method according to claim 1 and 2, it is characterized in that described methyl alcohol trifluoroacetic acid solution be methyl alcohol and 0.01 ~ 0.15% trifluoroacetic acid aqueous solution according to 5: 95 ~ 20: 80 the methyl alcohol trifluoroacetic acid solution that is mixed with of volume ratio.
4. assay method according to claim 3, it is characterized in that described methyl alcohol trifluoroacetic acid solution be methyl alcohol and 0.1% trifluoroacetic acid aqueous solution according to 10: 90 the methyl alcohol trifluoroacetic acid solution that is mixed with of volume ratio.
5. the assay method according to claim 1 or 2, is characterized in that described chromatographic condition is: chromatographic column: octadecylsilane chemically bonded silica post; Mobile phase: methyl alcohol: 0.1% trifluoroacetic acid aqueous solution=10: 90; Column temperature: 20 ~ 30 DEG C; Determined wavelength: 280nm; Flow velocity: 1mL/ minute.
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