CN103207255B - A kind of detection method of content of NAOXINTONG JIAONANG - Google Patents
A kind of detection method of content of NAOXINTONG JIAONANG Download PDFInfo
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Abstract
The present invention relates to the detection method of content of a kind of NAOXINTONG JIAONANG, by the Radix Astragali 66 parts, Radix Paeoniae Rubra 27 parts, Radix Salviae Miltiorrhizae 27 parts, Radix Angelicae Sinensis 27 parts, Rhizoma Chuanxiong 27 parts, 27 parts of Semen Persicae, 13 parts of Flos Carthami, Olibanum (processed) 13 parts, Myrrha (processed) 13 parts, Caulis Spatholobi 20 parts, Radix Achyranthis Bidentatae 27 parts, Ramulus Cinnamomi 20 parts, Ramulus Mori 27 parts, Pheretima 27 parts, Scorpio 13 parts, Hirudo 27 parts composition, use Ultra Performance Liquid Chromatography (UPLC) technology to measure S-A Hydroxysafflor yellow A in NAOXINTONG JIAONANG simultaneously, peoniflorin, ferulic acid, salvianolic acid B, 5 main chemical compositions of ligustilide, a chromatography can be completed in 24min, good separating degree is had between each main component chromatographic peak, precision, the RSD of repeatability is respectively less than 2.0%, can more fully control the quality of NAOXINTONG JIAONANG.
Description
Technical field
The present invention relates to the detection method of content of a kind of NAOXINTONG JIAONANG, belong to pharmaceutical preparations technology field.
Background technology
NAOXINTONG JIAONANG (commercially available prod, quality standard is disclosed in " internal medicine brain system " fascicle of " country's standard for traditional Chinese medicines compilation Chinese patent medicine provincial standard rises national standard part " of National Drug Administration in 2002 compilation, standard No.: WS-10001(ZD-0001)-2002) it is by the Radix Astragali, Radix Paeoniae Rubra, Radix Salviae Miltiorrhizae, Radix Angelicae Sinensis, Rhizoma Chuanxiong, Flos Carthami, Semen Persicae, Olibanum (is made), Myrrha (processed), Caulis Spatholobi, Radix Achyranthis Bidentatae, Ramulus Cinnamomi, Ramulus Mori, Pheretima, Scorpio, the clinical conventional Chinese medicine compound preparation that Hirudo 16 taste processing of crude drugs is made, there is benefiting QI for activating blood circulation, effect of disperse blood stasis and dredge collateral, for blood stagnancy due to deficiency of QI, apoplexy apoplex involving the channels and collaterals caused by venation block and obstruction of qi in the chest and cardialgia, breast is vexed, cardiopalmus, breathe hard, cerebral infarction, the treatment of angina pectoris.Under the assay item of existing NAOXINTONG JIAONANG quality standard, only detect the content of peoniflorin single index composition, the literature research result of prior art shows simultaneously, assay many employings HPLC method of said preparation, the most only detect one or more index components in certain taste medicine the most single, and measure multi-flavor medicine in NAOXINTONG JIAONANG, there is not been reported for Multiple components content.Additionally, the large usage quantity of the Radix Astragali, Radix Paeoniae Rubra, Radix Salviae Miltiorrhizae, Radix Angelicae Sinensis, Rhizoma Chuanxiong, Flos Carthami in prescription, once there is the main active such as the ferulic acid in salvianolic acid B, Radix Angelicae Sinensis and the Rhizoma Chuanxiong in peoniflorin that document reported in Radix Paeoniae Rubra, Radix Salviae Miltiorrhizae and the S-A Hydroxysafflor yellow A in ligustilide, Flos Carthami, assay is carried out respectively under different chromatographic conditions, but simultaneously a preparation same one-time detection process and under the conditions of to measure the achievement in research of these compositions the most on the books, be worth research worker further investigation.Along with the development of Ultra Performance Liquid Chromatography (UPLC) technology, quickly detect multicomponent imagination and be achieved, can preferably be monitored the quality standard of pharmaceutical production by the multicomponent detection of same preparation.The present invention uses UPLC technology, establish S-A Hydroxysafflor yellow A, peoniflorin, ferulic acid, salvianolic acid B, the method for 5 chemical composition contents of ligustilide in NAOXINTONG JIAONANG that simultaneously measure, a chromatography can be completed at short notice, it is quick, accurately that this analyzes method, reproducible, provide foundation for control NAOXINTONG JIAONANG quality comprehensive, objective.
Summary of the invention
Present invention aim at providing the detection method of content of a kind of quick, accurate, stable, reproducible NAOXINTONG JIAONANG.
Technical solution of the present invention comprises the steps of: (1) takes after NAOXINTONG JIAONANG content precise powder weighs, to tool plug ground conical flask, the a certain amount of organic solvent of accurate addition, after being re-weighed, extract with suitable way, place room temperature, bodies lost weight is supplied with organic solvent, shaking up, with filtering with microporous membrane, subsequent filtrate i.e. obtains need testing solution；(2) precision weigh S-A Hydroxysafflor yellow A, peoniflorin, ferulic acid, salvianolic acid B, ligustilide reference substance appropriate, be respectively prepared the mixed solution of variable concentrations with organic solvent, obtain mixing reference substance solution；(3) using octadecylsilane chemically bonded silica as fixing phase chromatography column；Flowing phase it is mixed into by a certain percentage with organic solvent-water；Gradient elution；Flow velocity is 0.1-1.0mL min-1；Detection wavelength 200-400nm；Column temperature 20-40 DEG C；Sample manager temperature is 2-8 DEG C；(4) precision draws mixing reference substance solution 1-10 μ L, need testing solution 1-10 μ L respectively, injects chromatograph of liquid, measures, to obtain final product；
Concrete experimental procedure consists of: (1) takes NAOXINTONG JIAONANG content precise powder and weighs after 0.5g, to 150mL tool plug ground conical flask, accurate addition 60-80% methanol 25-75mL, after being re-weighed, use ultrasonic extraction 15-45min, place room temperature, bodies lost weight is supplied with 60-80% methanol, shaking up, with filtering with microporous membrane, subsequent filtrate i.e. obtains need testing solution；(2) precision weigh S-A Hydroxysafflor yellow A, peoniflorin, ferulic acid, salvianolic acid B, ligustilide reference substance appropriate, be respectively prepared the mixed solution of variable concentrations with methanol, obtain mixing reference substance solution；(3) using octadecylsilane chemically bonded silica as fixing phase chromatography column；With acetonitrile (A)-0.5% aqueous formic acid (B) for flowing phase；Gradient elution；Flow velocity is 0.1-1.0mL min-1；Detection wavelength 200-400nm；Column temperature 20-40 DEG C；Sample manager temperature is 2-8 DEG C；(4) precision draws mixing reference substance solution 2 μ L, need testing solution 2 μ L respectively, injects chromatograph of liquid, measures, to obtain final product.
Wherein preferably experimental procedure is: (1) takes NAOXINTONG JIAONANG content precise powder and weighs after 0.5g, to 150mL tool plug ground conical flask, accurate addition 75% methanol 50mL, after being re-weighed, use ultrasonic extraction 30min, place room temperature, bodies lost weight is supplied with 75% methanol, shaking up, with 0.2 μm filtering with microporous membrane, subsequent filtrate i.e. obtains need testing solution；(2) precision weigh S-A Hydroxysafflor yellow A, peoniflorin, ferulic acid, salvianolic acid B, ligustilide reference substance appropriate, be respectively prepared 3.880,13.50,1.345,8.600,5.840 μ g mL with methanol-1Mixed solution, obtain mixing reference substance solution；(3) with WATERSACQUITYUPLCBEHC18(2.1mm × 100mm, 1.7 μm) are chromatographic column；With acetonitrile (A)-0.5% aqueous formic acid (B) for flowing phase；With 0 ~ 1min, 8%A；1 ~ 3min, 8%A → 32%A；3 ~ 15min, 32%A → 57%A；The rule gradient elution of 21 ~ 23min, 57%A → 100%A；Flow velocity is 0.3mL min-1；Detection wavelength: S-A Hydroxysafflor yellow A is 400nm, peoniflorin is 235nm, salvianolic acid B be 280nm, ferulic acid and ligustilide be 324nm；Column temperature 28 DEG C；Sample manager temperature is 4 DEG C；(4) precision draws mixing reference substance solution 2 μ L, need testing solution 2 μ L respectively, injects chromatograph of liquid, measures, to obtain final product.
Technical solution of the present invention detection is the medicine multicomponent content having 16 taste medical material compositions, chemical composition is extremely complex, each compositional polarity differs greatly, in the middle of conventional research, only have the report of single component Testing index, thus result in drug quality monitoring shortage science, comprehensive, objective appraisal, have impact on the stability of clinical drug application.If it is desired to overcome the defect of single Testing index, need multicomponent to detect simultaneously, but use traditional negative sample method, not only complex operation, and be difficult to the specificity of reflection method.If medical material multiple in prescription is containing same chemical composition, the most easily occur that false positive disturbs result, all contain ferulic acid, ligustilide as in Rhizoma Chuanxiong, Radix Angelicae Sinensis.The three-dimensional information that this research and utilization PDA detector is gathered, compares the one-tenth swarming each to be measured in sample and the ultraviolet absorpting spectrum of corresponding standard substance chromatographic peak, demonstrates the specificity of detection method.
To this end, the realization of technical solution of the present invention can reach following beneficial effect:
(1) by advanced Ultra Performance Liquid Chromatography (UPLC) technology in 24min in can complete a chromatography, reach the application of the big production of the degree of Fast Monitoring, beneficially scale；(2) using the mode of gradient elution can significantly improve the separating effect of each composition, testing result is the most accurate；(3) mode of 200-400nm full wavelength scanner is used to find the optimum absorb wavelength of each composition, and finally determine the means of multiband detection, it is possible to reaching to respond signal preferable, the interference of other compositions is less, specificity is relatively strong, detects the effect of each component content substantially；(4) process of detection NAOXINTONG JIAONANG uses supersound extraction mode; not only improve the dissolution rate of ingredient; and the extraction to the thermally labile component such as S-A Hydroxysafflor yellow A, salvianolic acid B plays a protective role, at utmost avoid higher temperature, component damages that longer extraction time causes.Therefore, combination through above-mentioned every technology, making to have between each main component chromatographic peak of medicine of the present invention good separating degree, S-A Hydroxysafflor yellow A, peoniflorin, ferulic acid, salvianolic acid B, 5 composition quality concentration of ligustilide are respectively at 0.970 ~ 9.70 μ g mL-1(r=0.9993), 3.38 ~ 33.8 μ g mL-1(r=0.9995), 0.336 ~ 3.36 μ g mL-1(r=0.9996), 2.15 ~ 21.5 μ g mL-1(r=0.9999), 1.46 ~ 14.6 μ g mL-1(r=0.9998) being good linear relationship with peak area in the range of, the average recovery rate (n=6) of method is respectively 99.47%, 101.3%, 98.45%, 98.54%, 99.02%, and precision, the RSD of repeatability are respectively less than 2.0%.Achieving the multicomponent monitoring mode simultaneously of complex combination thing assay, the research further for NAOXINTONG JIAONANG provides technical support.
Specific embodiment
The following is the specific embodiment of present invention, for illustrating the technical scheme of technical problem to be solved in present specification, contribute to skilled artisan understands that present invention, but the realization of technical solution of the present invention is not limited to these embodiments.
Embodiment 1
(1) by Radix Astragali 66g, Radix Paeoniae Rubra 27g, Radix Salviae Miltiorrhizae 27g, Radix Angelicae Sinensis 27g, Rhizoma Chuanxiong 27g, Semen Persicae 27g, Flos Carthami 13g, Olibanum (processed) 13g, Myrrha (processed) 13g, Caulis Spatholobi 20g, Radix Achyranthis Bidentatae 27g, Ramulus Cinnamomi 20g, Ramulus Mori 27g, Pheretima 27g, Scorpio 13g, Hirudo 27g ten Six-element medical material, take Pheretima, Scorpio, be ground into fine powder；14 tastes such as remaining Radix Astragali are ground into fine powder, with Pheretima, scorpion facing-up, sieve, mixing, load capsule, make 1000, obtain capsule.(2) taking capsule 20, precise weighing, the content shifting capsule completely is extremely dried in the weighing botle of constant weight, then precise weighing hungry area softgel shell, calculates capsule average particle weight.Precision weighing capsule 's content about 0.5g, to 150mL tool plug ground conical flask, accurate addition 75% methanol 50mL, weighs, ultrasound wave (power 280W, frequency 40kHz) extract 30min, place room temperature, supply bodies lost weight with 75% methanol, shake up, with 0.2 μm filtering with microporous membrane, subsequent filtrate i.e. obtains need testing solution；Precision weigh S-A Hydroxysafflor yellow A, peoniflorin, ferulic acid, salvianolic acid B, ligustilide reference substance appropriate, make 3.880,13.50,1.345,8.600,5.840 μ g mL with methanol-1Mixed solution, obtain mixing reference substance solution.(3) chromatographic condition: chromatographic column is WATERSACQUITYUPLCBEHC18(2.1mm × 100mm, 1.7 μm)；With acetonitrile (A)-0.5% aqueous formic acid (B) for flowing phase, gradient elution (0 ~ 1min, 8%A；1 ~ 3min, 8%A → 32%A；3 ~ 15min, 32%A → 57%A；21 ~ 23min, 57%A → 100%A)；Flow velocity is 0.3mL min-1；Detection wavelength: S-A Hydroxysafflor yellow A is 400nm, and peoniflorin is 235nm, salvianolic acid B be 280nm, ferulic acid and ligustilide be 324nm；Column temperature: 28 DEG C；Sample manager temperature is 4 DEG C；Theoretical cam curve is respectively 58954 and 65956 in terms of S-A Hydroxysafflor yellow A and salvianolic acid B.(4) precision draws mixing reference substance solution 2 μ L, need testing solution 2 μ L respectively, injects chromatograph of liquid, measures, to obtain final product.Figure of description part is shown in by collection of illustrative plates in detail.
Lot of experiments result through us shows, the key parameter in above-mentioned experimental procedure can be replaced by data below, and effect also reaches pharmaceutical production standard-required.
1. the organic facies of chromatogram flow phase can select the methanol of different proportion, normal hexane, ethyl acetate, chloroform etc.；2. the aqueous phase of chromatogram flow phase can select the pure water of different proportion, formic acid, phosphoric acid, phosphate buffer solution etc.；3. detection wavelength can select in the range of 200 ~ 400nm；4. the chemical composition extracting mode of NAOXINTONG JIAONANG can select the extraction of ultrasonic extraction, heating and refluxing extraction, percolation, immersing extraction etc.；5. the chemical composition of NAOXINTONG JIAONANG is extracted solvent and can be selected the organic solvents such as the ethanol of different proportion, methanol, ethyl acetate, n-butyl alcohol；6. chromatographic column can select the C of different model18、C8、C4, phenyl post etc.；7. Parameters of gradient elution can be: flow velocity 0.1-1.0mL min-1, column temperature 20-40 DEG C；Sample manager temperature is 2-8 DEG C；Select in the range of sample size 1-10 μ L etc..
Accompanying drawing explanation
1., when Fig. 1 is 235nm for detection wavelength, a is capsule sample group, and b is reference substance group, and the chromatographic peak 2 of both correspondences is the collection of illustrative plates of peoniflorin；
2., when Fig. 2 is 280nm for detection wavelength, a is capsule sample group, and b is reference substance group, and the chromatographic peak 4 of both correspondences is the collection of illustrative plates of salvianolic acid B；
3. when Fig. 3 is 324nm for detection wavelength, a is capsule sample group, and b is reference substance group, and the chromatographic peak 1,3,4,5 of both correspondences is respectively S-A Hydroxysafflor yellow A, ferulic acid, salvianolic acid B, the collection of illustrative plates of ligustilide；
4., when Fig. 4 is 400nm for detection wavelength, a is capsule sample group, and b is reference substance group, and the chromatographic peak 1 of both correspondences is the collection of illustrative plates of S-A Hydroxysafflor yellow A.
Claims (1)
1. the detection method of content of a NAOXINTONG JIAONANG, it is characterized in that comprising the steps of: (1) takes NAOXINTONG JIAONANG content precise powder and weighs after 0.5g, to 150mL tool plug ground conical flask, accurate addition 75% methanol 50mL, after being re-weighed, use ultrasonic extraction 30min, place room temperature, supply bodies lost weight with 75% methanol, shake up, with 0.2 μm filtering with microporous membrane, subsequent filtrate i.e. obtains need testing solution；(2) precision weigh S-A Hydroxysafflor yellow A, peoniflorin, ferulic acid, salvianolic acid B, ligustilide reference substance appropriate, make concentration with methanol and be respectively 3.880,13.50,1.345,8.600,5.840 μ g mL-1Mixed solution, obtain mixing reference substance solution；(3) with WATERSACQUITYUPLCBEHC18,2.1mm × 100mm, 1.7 μm are chromatographic column；With acetonitrile A-0.5% aqueous formic acid B for flowing phase；With 0-1min, 8%A；1-3min, 8%A → 32%A；3-15min, 32%A → 57%A；The rule gradient elution of 21-23min, 57%A → 100%A；Flow velocity is 0.3mL min-1；Detection wavelength: S-A Hydroxysafflor yellow A is 400nm, peoniflorin is 235nm, salvianolic acid B be 280nm, ferulic acid and ligustilide be 324nm；Column temperature 28 DEG C；Sample manager temperature is 4 DEG C；(4) precision draws mixing reference substance solution 2 μ L, need testing solution 2 μ L respectively, injects chromatograph of liquid, measures, to obtain final product.
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