CN103207255A - Content detection method for Naoxintong capsule - Google Patents
Content detection method for Naoxintong capsule Download PDFInfo
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- CN103207255A CN103207255A CN201310077615XA CN201310077615A CN103207255A CN 103207255 A CN103207255 A CN 103207255A CN 201310077615X A CN201310077615X A CN 201310077615XA CN 201310077615 A CN201310077615 A CN 201310077615A CN 103207255 A CN103207255 A CN 103207255A
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Abstract
The invention relates to a content detection method for the Naoxintong capsule. The Naoxintong capsule comprises 66 parts of astragalus root, 27 parts of red peony root, 27 parts of root of red-rooted salvia, 27 parts of Chinese angelica, 27 parts of Szechuan lovage rhizome, 27 parts of peach kernel, 13 parts of safflower, 13 parts of prepared Frankincense, 13 parts of prepared myrrh, 20 parts of suberect spatholobus stem, 27 parts of twotooth achyranthes root, 20 parts of cassia twig, 27 parts of mulberry twig, 27 parts of earthworm, 13 parts of scorpion, and 27 parts of leech. The main chemical components, namely hydroxy safflower yellow A, paeoniflorin, ferulic acid, salvianolic acid B and ligustilide, in the Naoxintong capsule are simultaneously measured by the UPLC (ultra-performance liquid chromatography) technique. Single chromatographic analysis can be completed in 24 minutes. Chromatographic peaks of the main components are well separate, RSD (relative standard deviation) in both precision and receptiveness is smaller than 2.0%, and the quality of Naoxintong capsule can be controlled comprehensively.
Description
Technical field
The present invention relates to a kind of detection method of content of brain heart open capsule, belong to the pharmaceutical preparations technology field.
Background technology
Brain heart open capsule (commercially available prod, quality standard is disclosed in National Drug Administration in 2002 compilation " national standard for traditional Chinese medicines compilation---Chinese patent drug provincial standard rising national standard part " " internal medicine---brain system " fascicle, standard No.: WS-10001(ZD-0001)-2002) be by the Radix Astragali, the radix paeoniae rubrathe, the red sage root, Radix Angelicae Sinensis, Ligusticum wallichii, safflower, peach kernel, frankincense (system), myrrh (system), reticulate millettia, the root of bidentate achyranthes, cassia twig, ramulus mori, earthworm, scorpio, the clinical conventional Chinese medicine compound preparation that leech 16 flavor medicinal materials are processed into, has qi and activate blood circulation, the effect of disperse blood stasis and dredge collateral, be used for blood stagnancy due to deficiency of QI, apoplexy apoplex involving the channels and collaterals and chest impediment and cardialgia due to the venation block, uncomfortable in chest, palpitaition, breathe hard cerebral infarction, the treatment of coronary disease and angina pectoris.Under the assay item of existing brain heart open capsule quality standard, only detect the content of the single index composition of Paeoniflorin, the literature research result of prior art shows simultaneously, the assay of said preparation adopts the HPLC method more, often only detect one or more index components in single certain flavor medicine wherein, multi-flavor medicine, multiple component content do not appear in the newspapers as yet in the brain heart open capsule and measure.In addition, the large usage quantity of the Radix Astragali, the radix paeoniae rubrathe, the red sage root, Radix Angelicae Sinensis, Ligusticum wallichii, safflower in the prescription, once there was bibliographical information to cross main active such as hydroxyl radical carthamin yellow carthamus A in forulic acid in Paeoniflorin, the tanshin polyphenolic acid B in the red sage root, Radix Angelicae Sinensis and the Ligusticum wallichii in the radix paeoniae rubrathe and Ligustilide, the safflower, under different chromatographic conditions, carry out assay respectively, but the achievement in research of measuring simultaneously these compositions under the same testing process of a preparation and condition is not on the books as yet, is worth researchist's further investigation.Along with the development of Ultra Performance Liquid Chromatography (UPLC) technology, the multicomponent imagination of fast detecting is achieved, and can monitor the quality standard of pharmaceutical production better by the multicomponent detection of same preparation.The present invention adopts the UPLC technology, set up the method for measuring hydroxyl radical carthamin yellow carthamus A in the brain heart open capsule, Paeoniflorin, forulic acid, tanshin polyphenolic acid B, 5 chemical composition contents of Ligustilide simultaneously, can finish a stratographic analysis at short notice, this analytical approach is quick, accurate, good reproducibility provides foundation for comprehensively, objectively controlling brain heart open capsule quality.
Summary of the invention
The object of the invention be to provide a kind of quick, accurately, the detection method of content of the brain heart open capsule of stable, good reproducibility.
Technical solution of the present invention is made up of following steps: after (1) is got brain heart open capsule content precise powder and weighed, to tool plug ground conical flask, the a certain amount of organic solvent of accurate adding, after weighing again, extract with suitable way, place room temperature, supply bodies lost weight with organic solvent, shake up, with filtering with microporous membrane, subsequent filtrate namely gets need testing solution; (2) it is an amount of that precision takes by weighing the reference substance of hydroxyl radical carthamin yellow carthamus A, Paeoniflorin, forulic acid, tanshin polyphenolic acid B, Ligustilide, makes the mixed solution of variable concentrations respectively with organic solvent, namely gets the mixing reference substance solution; (3) with octadecylsilane chemically bonded silica as fixing phase chromatography column; Be mixed into mobile phase by a certain percentage with organic solvent-water; Gradient elution; Flow velocity is 0.1-1.0 mLmin
-1Detect wavelength 200-400nm; Column temperature 20-40 ℃; The sample managing actuator temperature is 2-8 ℃; (4) the accurate absorption mixed reference substance solution 1-10 μ L respectively, and need testing solution 1-10 μ L injects liquid chromatograph, measures, namely;
Concrete experimental procedure consists of: (1) is got brain heart open capsule content precise powder and is weighed behind 0.5 g, to 150 mL tool plug ground conical flasks, the accurate 60-80% methyl alcohol 25-75mL that adds, after weighing again, extract 15-45 min with ultrasound wave, place room temperature, supply bodies lost weight with 60-80% methyl alcohol, shake up, with filtering with microporous membrane, subsequent filtrate namely gets need testing solution; (2) it is an amount of that precision takes by weighing the reference substance of hydroxyl radical carthamin yellow carthamus A, Paeoniflorin, forulic acid, tanshin polyphenolic acid B, Ligustilide, makes the mixed solution of variable concentrations respectively with methyl alcohol, namely gets the mixing reference substance solution; (3) with octadecylsilane chemically bonded silica as fixing phase chromatography column; Be the phase that flows with acetonitrile (A)-0.5% aqueous formic acid (B); Gradient elution; Flow velocity is 0.1-1.0 mLmin
-1Detect wavelength 200-400nm; Column temperature 20-40 ℃; The sample managing actuator temperature is 2-8 ℃; (4) the accurate absorption mixed reference substance solution 2 μ L respectively, and need testing solution 2 μ L inject liquid chromatograph, measure, namely.
Wherein preferred experimental procedure is: (1) is got brain heart open capsule content precise powder and is weighed behind 0.5 g, to 150 mL tool plug ground conical flasks, the accurate 75% methyl alcohol 50mL that adds, after weighing again, extract 30 min with ultrasound wave, place room temperature, supply bodies lost weight with 75% methyl alcohol, shake up, with 0.2 μ m filtering with microporous membrane, subsequent filtrate namely gets need testing solution; (2) it is an amount of that precision takes by weighing the reference substance of hydroxyl radical carthamin yellow carthamus A, Paeoniflorin, forulic acid, tanshin polyphenolic acid B, Ligustilide, makes 3.880,13.50,1.345,8.600,5.840 μ gmL respectively with methyl alcohol
-1Mixed solution, namely get the mixing reference substance solution; (3) with WATERS ACQUITY UPLC BEH C
18(2.1 mm * 100 mm, 1.7 μ m) are chromatographic column; Be the phase that flows with acetonitrile (A)-0.5% aqueous formic acid (B); With 0 ~ 1 min, 8%A; 1 ~ 3 min, 8%A → 32%A; 3 ~ 15 min, 32%A → 57%A; 21 ~ 23 min, the rule gradient elution of 57%A → 100%A; Flow velocity is 0.3mLmin
-1Detect wavelength: hydroxyl radical carthamin yellow carthamus A is that 400 nm, Paeoniflorin are that 235 nm, tanshin polyphenolic acid B are that 280 nm, forulic acid and Ligustilide are 324 nm; 28 ℃ of column temperatures; The sample managing actuator temperature is 4 ℃; (4) the accurate absorption mixed reference substance solution 2 μ L respectively, and need testing solution 2 μ L inject liquid chromatograph, measure, namely.
What technical solution of the present invention detected is a medicine multicomponent content that has 16 flavor medicinal materials to form, chemical constitution is very complicated, each compositional polarity differs greatly, in the middle of research in the past, only there is single component to detect the report of index, therefore cause drug quality monitoring shortage science, comprehensive, objective appraisal, influenced the stability that clinical drug is used.If want to overcome the defective of single detection index, need multicomponent to detect simultaneously, but adopt traditional negative sample method, complex operation not only, and be difficult to reflect the specificity of method.Contain same chemical constitution as multiple medicinal material in the prescription, then be prone to false positive and disturb the result, as all containing forulic acid, Ligustilide in Ligusticum wallichii, the Radix Angelicae Sinensis.The three-dimensional information that this research and utilization PDA detecting device is gathered, the ultraviolet absorpting spectrum of the one-tenth swarming to be measured of each in the comparative sample and corresponding standard items chromatographic peak has been verified the specificity of detection method.
For this reason, the realization of technical solution of the present invention can reach following beneficial effect:
(1) in advanced Ultra Performance Liquid Chromatography (UPLC) technology is in 24min, can finish a stratographic analysis, reach the degree of quick monitoring, be conducive to the application of the big production of scale; (2) mode of employing gradient elution can obviously improve the separating effect of each composition, and testing result is more accurate; (3) mode of employing 200-400nm full wavelength scanner is sought the optimum absorb wavelength of each composition, and finally determined the means that multiband detects, and it is better to reach response signal, and other compositions disturb less, specificity is stronger, detects the effect of each component content substantially; (4) ultrasonic extracting mode is adopted in the processing that detects brain heart open capsule; not only improve the dissolution rate of drug ingedient; and the extraction of thermally labile components such as hydroxyl radical carthamin yellow carthamus A, tanshin polyphenolic acid B played a protective role, at utmost avoided the composition loss that higher temperature, longer extraction time cause.Therefore, combination through above-mentioned every technology, making has good degree of separation between each principal ingredient chromatographic peak of medicine of the present invention, and hydroxyl radical carthamin yellow carthamus A, Paeoniflorin, forulic acid, tanshin polyphenolic acid B, 5 composition quality concentration of Ligustilide are respectively at 0.970 ~ 9.70 μ gmL-1(r=0.9993), 3.38 ~ 33.8 μ gmL-1(r=0.9995), 0.336 ~ 3.36 μ gmL-1(r=0.9996), 2.15 ~ 21.5 μ gmL
-1(r=0.9999), 1.46 ~ 14.6 μ gmL
-1(r=0.9998) be good linear relationship with peak area in the scope, the average recovery rate of method (n=6) is respectively 99.47%, 101.3%, 98.45%, 98.54%, 99.02%, and the RSD of precision, repeatability is all less than 2.0%.The multicomponent of having realized complex combination thing assay is monitoring mode simultaneously, for the further research of brain heart open capsule provides technical support.
Specific embodiment
Below be the specific embodiment of content of the present invention, be used for setting forth the technical scheme that present specification is wanted the technical solution problem, help those skilled in the art to understand content of the present invention, but the realization of technical solution of the present invention is not limited to these embodiment.
(1) with Radix Astragali 66g, radix paeoniae rubrathe 27g, red sage root 27g, Radix Angelicae Sinensis 27g, Ligusticum wallichii 27g, peach kernel 27g, safflower 13g, frankincense 13g, myrrh 13g, reticulate millettia 20g, root of bidentate achyranthes 27g, cassia twig 20g, ramulus mori 27g, earthworm 27g, scorpio 13g, leech 27g ten Six-element medicinal material, get earthworm, scorpio, be ground into fine powder; 14 flavors such as all the other Radixs Astragali are ground into fine powder, with earthworm, scorpion facing-up, sieve, and mixing incapsulates, and makes 1000, namely gets capsule.(2) get 20 of capsules, precision is weighed, the content that shifts capsule fully to the measuring cup of dry constant weight, the precision hungry area softgel shell of weighing again, it is heavy to calculate the capsule average particle.About 0.5 g of precision weighing capsule 's content, to 150 mL tool plug ground conical flasks, accurate 75% methyl alcohol, 50 mL that add weigh ultrasound wave (power 280 W, frequency 40 kHz) extract 30 min, place room temperature, supply bodies lost weight with 75% methyl alcohol, shake up, with 0.2 μ m filtering with microporous membrane, subsequent filtrate namely gets need testing solution; It is an amount of that precision takes by weighing the reference substance of hydroxyl radical carthamin yellow carthamus A, Paeoniflorin, forulic acid, tanshin polyphenolic acid B, Ligustilide, makes 3.880,13.50,1.345,8.600,5.840 μ gmL with methyl alcohol
-1Mixed solution, namely get the mixing reference substance solution.(3) chromatographic condition: chromatographic column is WATERS ACQUITY UPLC BEH C
18(2.1 mm * 100 mm, 1.7 μ m); Be the phase that flows with acetonitrile (A)-0.5% aqueous formic acid (B), gradient elution (0 ~ 1 min, 8%A; 1 ~ 3 min, 8%A → 32%A; 3 ~ 15 min, 32%A → 57%A; 21 ~ 23 min, 57%A → 100%A); Flow velocity is 0.3 mLmin
-1Detect wavelength: hydroxyl radical carthamin yellow carthamus A is 400 nm, and Paeoniflorin is 235 nm, and tanshin polyphenolic acid B is 280 nm, and forulic acid and Ligustilide are 324 nm; Column temperature: 28 ℃; The sample managing actuator temperature is 4 ℃; Theoretical cam curve is respectively 58954 and 65956 in hydroxyl radical carthamin yellow carthamus A and tanshin polyphenolic acid B.(4) the accurate absorption mixed reference substance solution 2 μ L respectively, and need testing solution 2 μ L inject liquid chromatograph, measure, namely.Collection of illustrative plates is seen the Figure of description part in detail.
Lot of experiments result through us shows that the key parameter in the above-mentioned experimental procedure can be replaced with following data, and effect also reaches the pharmaceutical production standard-required.
1. the organic phase of chromatogram flow phase can be selected methyl alcohol, normal hexane, ethyl acetate, chloroform of different proportion etc.; 2. the water of chromatogram flow phase can be selected pure water, formic acid, phosphoric acid, phosphate buffer solution of different proportion etc.; 3. detecting wavelength can select in 200 ~ 400 nm scopes; 4. the chemical constitution extracting mode of brain heart open capsule can be selected ultrasound wave extraction, heating and refluxing extraction, percolation extraction, infusion method extraction etc.; 5. the chemical constitution of brain heart open capsule is extracted solvent and can be selected organic solvents such as the ethanol of different proportion, methyl alcohol, ethyl acetate, normal butyl alcohol; 6. chromatographic column can be selected the C of different model
18, C
8, C
4, phenyl post etc.; 7. the gradient elution parameter can be at flow velocity 0.1-1.0 mLmin
-1, column temperature 20-40 ℃; The sample managing actuator temperature is 2-8 ℃; Select in the scopes such as sample size 1-10 μ L.
Description of drawings
1. when Fig. 1 was 235nm for detecting wavelength, a was the capsule sample group, and b is the reference substance group, and the chromatographic peak 2 of both correspondences is the collection of illustrative plates of Paeoniflorin;
2. when Fig. 2 was 280nm for detecting wavelength, a was the capsule sample group, and b is the reference substance group, and the chromatographic peak 4 of both correspondences is the collection of illustrative plates of tanshin polyphenolic acid B;
3. when Fig. 3 was 324nm for detecting wavelength, a was the capsule sample group, and b is the reference substance group, and the chromatographic peak 1,3,4,5 of both correspondences is respectively the collection of illustrative plates of hydroxyl radical carthamin yellow carthamus A, forulic acid, tanshin polyphenolic acid B, Ligustilide;
4. when Fig. 4 was 400nm for detecting wavelength, a was the capsule sample group, and b is the reference substance group, and the chromatographic peak 1 of both correspondences is the collection of illustrative plates of hydroxyl radical carthamin yellow carthamus A.
Claims (3)
1. the detection method of content of a brain heart open capsule, it is characterized in that being formed by following steps: after (1) is got brain heart open capsule content precise powder and weighed, to tool plug ground conical flask, a certain amount of organic solvent of accurate adding is after weighing again, extract with suitable way, place room temperature, supply bodies lost weight with organic solvent, shake up, with filtering with microporous membrane, subsequent filtrate namely gets need testing solution; (2) it is an amount of that precision takes by weighing the reference substance of hydroxyl radical carthamin yellow carthamus A, Paeoniflorin, forulic acid, tanshin polyphenolic acid B, Ligustilide, makes the mixed solution of variable concentrations respectively with organic solvent, namely gets the mixing reference substance solution; (3) with the alkyl silane bonded silica gel as fixing phase chromatography column; Be mixed into mobile phase by a certain percentage with organic solvent-water; Gradient elution; Flow velocity is 0.1-1.0 mLmin
-1Detect wavelength 200-400nm; Column temperature 20-40 ℃; The sample managing actuator temperature is 2-8 ℃; (4) the accurate absorption mixed reference substance solution 1-10 μ L respectively, and need testing solution 1-10 μ L injects liquid chromatograph, measures, namely.
2. the detection method of content of brain heart open capsule as claimed in claim 1, it is characterized in that being made up of following steps: (1) is got brain heart open capsule content precise powder and is weighed behind 0.5 g, to 150 mL tool plug ground conical flasks, the accurate 60-80% methyl alcohol 25-75mL that adds is after weighing again, extract 15-45 min with ultrasound wave, place room temperature, supply bodies lost weight with 60-80% methyl alcohol, shake up, with filtering with microporous membrane, subsequent filtrate namely gets need testing solution; (2) it is an amount of that precision takes by weighing the reference substance of hydroxyl radical carthamin yellow carthamus A, Paeoniflorin, forulic acid, tanshin polyphenolic acid B, Ligustilide, makes the mixed solution of variable concentrations respectively with methyl alcohol, namely gets the mixing reference substance solution; (3) with octadecylsilane chemically bonded silica as fixing phase chromatography column; Be the phase that flows with acetonitrile (A)-0.5% aqueous formic acid (B); Gradient elution; Flow velocity is 0.1-1.0 mLmin
-1Detect wavelength 200-400nm; Column temperature 20-40 ℃; The sample managing actuator temperature is 2-8 ℃; (4) the accurate absorption mixed reference substance solution 2 μ L respectively, and need testing solution 2 μ L inject liquid chromatograph, measure, namely.
3. the detection method of content of brain heart open capsule as claimed in claim 2, it is characterized in that being made up of following steps: (1) is got brain heart open capsule content precise powder and is weighed behind 0.5 g, to 150 mL tool plug ground conical flasks, the accurate 75% methyl alcohol 50mL that adds is after weighing again, extract 30 min with ultrasound wave, place room temperature, supply bodies lost weight with 75% methyl alcohol, shake up, with 0.2 μ m filtering with microporous membrane, subsequent filtrate namely gets need testing solution; (2) it is an amount of that precision takes by weighing the reference substance of hydroxyl radical carthamin yellow carthamus A, Paeoniflorin, forulic acid, tanshin polyphenolic acid B, Ligustilide, makes 3.880,13.50,1.345,8.600,5.840 μ gmL respectively with methyl alcohol
-1Mixed solution, namely get the mixing reference substance solution; (3) with WATERS ACQUITY UPLC BEH C
18(2.1 mm * 100 mm, 1.7 μ m) are chromatographic column; Be the phase that flows with acetonitrile (A)-0.5% aqueous formic acid (B); With 0-1 min, 8%A; 1-3 min, 8%A → 32%A; 3-15 min, 32%A → 57%A; 21-23 min, the rule gradient elution of 57%A → 100%A; Flow velocity is 0.3mLmin
-1Detect wavelength: hydroxyl radical carthamin yellow carthamus A is that 400 nm, Paeoniflorin are that 235 nm, tanshin polyphenolic acid B are that 280 nm, forulic acid and Ligustilide are 324 nm; 28 ℃ of column temperatures; The sample managing actuator temperature is 4 ℃; (4) the accurate absorption mixed reference substance solution 2 μ L respectively, and need testing solution 2 μ L inject liquid chromatograph, measure, namely.
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| CN103411893A (en) * | 2013-07-29 | 2013-11-27 | 陕西步长制药有限公司 | Detection method for near infrared spectrum of Naoxintong capsule |
| CN104127532A (en) * | 2014-07-22 | 2014-11-05 | 陕西步长制药有限公司 | Application of Naoxintong capsule in preparation of tumor disease treatment medicines |
| CN104897831A (en) * | 2015-05-13 | 2015-09-09 | 天津中医药大学 | Construction method of Naoxintong fingerprint |
| CN105044223A (en) * | 2015-04-24 | 2015-11-11 | 贵州景峰注射剂有限公司 | Chemical component identification and active component screening method of Shenxiong glucose injection |
| CN105241980A (en) * | 2015-11-12 | 2016-01-13 | 陕西步长制药有限公司 | Rapid separation liquid chromatography detection method for naoxintong capsules |
| CN105548379A (en) * | 2015-12-08 | 2016-05-04 | 段占娥 | Method for measuring contents of paeoniflorin and ferulic acid in radix codonopsis and radix angelicae sinensis apoplexia rehabilitating particles at the same time |
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| CN108593794A (en) * | 2018-04-25 | 2018-09-28 | 陕西步长制药有限公司 | Active constituent content method in a kind of multi-target ingredient UPLC detection safflower |
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| CN103411893A (en) * | 2013-07-29 | 2013-11-27 | 陕西步长制药有限公司 | Detection method for near infrared spectrum of Naoxintong capsule |
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| CN104897831A (en) * | 2015-05-13 | 2015-09-09 | 天津中医药大学 | Construction method of Naoxintong fingerprint |
| CN105241980A (en) * | 2015-11-12 | 2016-01-13 | 陕西步长制药有限公司 | Rapid separation liquid chromatography detection method for naoxintong capsules |
| CN105241980B (en) * | 2015-11-12 | 2017-05-24 | 陕西步长制药有限公司 | Rapid separation liquid chromatography detection method for naoxintong capsules |
| CN105548379A (en) * | 2015-12-08 | 2016-05-04 | 段占娥 | Method for measuring contents of paeoniflorin and ferulic acid in radix codonopsis and radix angelicae sinensis apoplexia rehabilitating particles at the same time |
| CN106728119A (en) * | 2017-01-19 | 2017-05-31 | 中山大学 | Application of the cerebral ischemic Chinese medicine composition in treatment cerebral hemorrhage medicine is prepared |
| CN108181389A (en) * | 2017-12-20 | 2018-06-19 | 正大青春宝药业有限公司 | It is a kind of while measure the method for tanshin polyphenolic acid B and ferulaic acid content in perhexiline piece |
| CN108593794A (en) * | 2018-04-25 | 2018-09-28 | 陕西步长制药有限公司 | Active constituent content method in a kind of multi-target ingredient UPLC detection safflower |
| CN111999395A (en) * | 2020-06-15 | 2020-11-27 | 陕西步长制药有限公司 | Fingerprint detection method of medicinal preparation |
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