CN107966505A - The method for measuring Multiple components in Fufang Danshen Pian at the same time - Google Patents
The method for measuring Multiple components in Fufang Danshen Pian at the same time Download PDFInfo
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Abstract
The invention discloses method that is a kind of while measuring Multiple components in Fufang Danshen Pian, this method uses the detection method of liquid chromatogram gradient elution, change detection wavelength and UV detector cascade evaporation light scattering detector, determine 6 kinds of active ingredient tanshin polyphenolic acid Bs, tanshinone IIA, notoginsenoside R, ginsenoside Rg1, ginsenoside Re and the content of ginsenoside Rb1 in Fufang Danshen Pian at the same time, it not only simplify checkout procedure, saved analysis time, the also quality control for Fufang Danshen Pian provides accurate reference.
Description
Technical field
The present invention relates to traditional Chinese medicine detection technique field, is specifically the side for measuring Multiple components in Fufang Danshen Pian at the same time
Method.
Background technology
The prescription of Fufang Danshen Pian is Radix Salviae Miltiorrhizae 450g, pseudo-ginseng 141g, borneol 8g.It is small that Radix Salviae Miltiorrhizae adds ethanol to be heated to reflux 1.5
When, extracting solution filtration, filtrate recycling ethanol is simultaneously concentrated into right amount, spare；The dregs of a decoction add 50% ethanol be heated to reflux 1.5 it is small when, carry
Liquid is taken to filter, filtrate recycling ethanol is simultaneously concentrated into right amount, spare；The dregs of a decoction add water to cook 2 it is small when, decocting liquid filtration, filtrate is concentrated into
In right amount.Pseudo-ginseng is ground into fine powder, and particle is made with above-mentioned concentrate and suitable auxiliary material, dry.Borneol is finely ground, with above-mentioned particle
Mix, be pressed into 333, film coating；Or 1000 are pressed into, sugar coating or film-coating, to obtain the final product.Danshen Tablets can promoting blood circulation
The stasis of blood, regulating qi-flowing for relieving pain, for the obstruction of qi in the chest of caused by energy stagnation and blood stasis, symptoms include uncomfortable in chest, pareordia shouting pain.Clinically it is used to treat cardiovascular and cerebrovascular
The various diseases such as disease, coronary heart diseases and angina pectoris, myocardial ischemia, its therapeutic effect have obtained the verification of clinic.But current compound
The quality standard of Danshen Tablets only chooses the one of Radix Salviae Miltiorrhizae, two active ingredient or index components carry out assay, and with its content
It is how many to judge quality, there is certain one-sidedness, imperfection, shortage comprehensively and effectively method of quality control.
The content of the invention
Based on this, Multiple components in Fufang Danshen Pian are measured at the same time using HPLC-UV-ELSD methods the present invention provides one kind
Method, simplify checkout procedure, saved analysis time.
To realize above-mentioned technical purpose, particular content is as follows：
1st, instrument, reagent and sample：
（1）Instrument：2695 high performance liquid chromatographs of Waters；2487 UV detector-ALLtech 2000ES evaporative lights dissipate
Penetrate detector detection；MILLI-PROA pure water processors.
（2）Reagent：Tanshin polyphenolic acid B reference substance（National Institute for Food and Drugs Control provides, lot number 111562-201212, supplies
Assay use, content is in terms of 95.4%), tanshinone IIA reference substance（National Institute for Food and Drugs Control provides, lot number
110766-200315, for assay), notoginsenoside R reference substance（National Institute for Food and Drugs Control provides, lot number
110745-200617, for assay), ginsenoside Rg1's reference substance（National Institute for Food and Drugs Control provides, lot number
110703-201128, for assay, content is in terms of 93.4%), ginsenoside Re's reference substance（Chinese food drug assay is ground
Study carefully institute offer, lot number 110754-200822, for assay, content is in terms of 88.8%), ginsenoside Rb1's reference substance（China
Food and medicine calibrating research institute provides, and lot number 110704-201122, for assay, content is in terms of 92.9%).Acetonitrile is color
Compose pure, water is high purity water, and other reagents are that analysis is pure.
（3）Sample：Fufang Danshen Pian is produced by Guangzhou Baiyunshan Heji Huangpu Chinese Medicine Co., Ltd., lot number：E3A030.
2nd, detection method is as follows：
（1）Chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase
A, using 0.1% trifluoroacetic acid solution as Mobile phase B, gradient elution is carried out by the regulation of table 1；The regulation of Detection wavelength according to the form below is set
Fixed, saponin(e is detected with evaporative light scattering detector；
1 gradient elution table of table
（2）The preparation of reference substance solution：Take tanshin polyphenolic acid B reference substance, tanshinone IIA reference substance appropriate, it is accurately weighed, add methanol molten
Solution, is made every 1mL containing 600 μ g tanshin polyphenolic acid Bs, the mixed solution of 50 μ g tanshinone IIAs, as mixed reference substance solution A；Three are taken again
Seven saponin(e R1 reference substances, ginsenoside Rg1's reference substance, ginsenoside Re's reference substance, ginsenoside Rb1's reference substance are appropriate, accurate
It is weighed, add 70% ethanol dissolve, be made every 1mL containing 45 μ g notoginsenoside Rs, 450 μ g ginsenoside Rg1s, 40 μ g ginsenoside Res,
The mixed solution of 200 μ g ginsenoside Rb1s, as mixed reference substance solution B；
（3）The preparation of test solution：10, sample is taken, removes coating, it is accurately weighed, it is finely ground, about 0.9g is taken, it is accurately weighed,
Put in conical flask with cover, precision adds 70% ethanol 25mL, and close plug, weighed weight, is ultrasonically treated（Power 400W, frequency 40KHz）
30 minutes, let cool, then weighed weight, the weight of less loss is supplied with 70% ethanol, is shaken up, filters, takes subsequent filtrate, to obtain the final product；
（4）The durability of the stability of detected solution, chromatographic column and instrument is investigated, and carry out precision, repeatability, specially
Attribute, accuracy（Sample recovery rate）Experiment；
（5）Measure：It is accurate respectively to draw 10 μ L mixed reference substance solutions A, 5 μ L and 15 μ L mixed reference substance solutions B and 10 μ L confessions
Test sample solution, injects liquid chromatograph, measure, the content of tanshin polyphenolic acid B and tanshinone IIA is calculated with one point external standard method, uses external standard
Two-point method logarithmic equation calculates the content of notoginsenoside.
The beneficial effects of the invention are as follows：
The present invention is using liquid chromatogram gradient elution, change detection wavelength and UV detector cascade evaporation light scattering detector
Detection method, while determine 6 kinds of active ingredient tanshin polyphenolic acid Bs, tanshinone IIA, notoginsenoside R, ginseng soap in Fufang Danshen Pian
Glycosides Rg1, ginsenoside Re and the content of ginsenoside Rb1, not only simplify checkout procedure, have saved analysis time, also be multiple
The quality control of square Danshen Tablets provides accurate reference.
Under ultraviolet wavelength, water soluble ingredient tanshin polyphenolic acid B has absorption maximum, liposoluble constituent near 280nm wavelength
Tanshinone IIA has absorption maximum near 270 nm wavelength, and the present invention takes ultraviolet wavelength switching method measure tanshin polyphenolic acid B and pellet
Join II A of ketone, it is ensured that each component has the response of maximum；Because Saponins from Panax notoginseng only has end absorption in ultra-violet (UV) band, this
Invention measures the component of pseudo-ginseng using evaporative light scattering detector, and evaporative light scattering detector has the saponin component in pseudo-ginseng
Preferable response, and baseline noise it is small, from gradients affect.The present invention is used as stream using the trifluoroacetic acid solution of acetonitrile -0.1% system
Dynamic phase, when detection, can obtain preferable chromatography effect.
Brief description of the drawings
Fig. 1 shows scarce Radix Salviae Miltiorrhizae negative control UV chromatograms；
Fig. 2 represents to lack pseudo-ginseng negative control ELSD chromatograms.
Embodiment
The present invention is introduced in order to more detailed, with reference to embodiment, the present invention will be further described.
Embodiment, content are as follows：
1st, instrument, reagent and sample：
（1）Instrument：2695 high performance liquid chromatographs of Waters；2487 UV detector-ALLtech 2000ES evaporative lights dissipate
Penetrate detector detection；MILLI-PROA pure water processors.
（2）Reagent：Tanshin polyphenolic acid B reference substance（National Institute for Food and Drugs Control provides, lot number 111562-201212, supplies
Assay use, content is in terms of 95.4%), tanshinone IIA reference substance（National Institute for Food and Drugs Control provides, lot number
110766-200315, for assay), notoginsenoside R reference substance（National Institute for Food and Drugs Control provides, lot number
110745-200617, for assay), ginsenoside Rg1's reference substance（National Institute for Food and Drugs Control provides, lot number
110703-201128, for assay, content is in terms of 93.4%), ginsenoside Re's reference substance（Chinese food drug assay is ground
Study carefully institute offer, lot number 110754-200822, for assay, content is in terms of 88.8%), ginsenoside Rb1's reference substance（China
Food and medicine calibrating research institute provides, and lot number 110704-201122, for assay, content is in terms of 92.9%).Acetonitrile is color
Compose pure, water is high purity water, and other reagents are that analysis is pure.
（3）Sample：It has collected totally 20 batches of Fufang Danshen Pians of 11 enterprise's production altogether.
2nd, detection method is as follows：
（1）Chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase
A, using 0.1% trifluoroacetic acid solution as Mobile phase B, gradient elution is carried out by the regulation of table 1；The regulation of Detection wavelength according to the form below is set
Fixed, saponin(e is detected with evaporative light scattering detector.Number of theoretical plate is calculated by tanshin polyphenolic acid B peak should be not less than 30000, by Tanshinone II
A peaks, which calculate, should be not less than 1000000, and 50000 should be not less than by being calculated by ginsenoside Rg1 peak；
1 gradient elution table of table
（2）The preparation of reference substance solution：Take tanshin polyphenolic acid B reference substance, tanshinone IIA reference substance appropriate, it is accurately weighed, add methanol molten
Solution, is made every 1mL containing 600 μ g tanshin polyphenolic acid Bs, the mixed solution of 50 μ g tanshinone IIAs, as mixed reference substance solution A；Three are taken again
Seven saponin(e R1 reference substances, ginsenoside Rg1's reference substance, ginsenoside Re's reference substance, ginsenoside Rb1's reference substance are appropriate, accurate
It is weighed, add 70% ethanol dissolve, be made every 1mL containing 45 μ g notoginsenoside Rs, 450 μ g ginsenoside Rg1s, 40 μ g ginsenoside Res,
The mixed solution of 200 μ g ginsenoside Rb1s, as mixed reference substance solution B；
（3）The preparation of test solution：10, sample is taken, removes coating, it is accurately weighed, it is finely ground, about 0.9g is taken, it is accurately weighed,
Put in conical flask with cover, precision adds 70% ethanol 25mL, and close plug, weighed weight, is ultrasonically treated（Power 400W, frequency 40KHz）
30 minutes, let cool, then weighed weight, the weight of less loss is supplied with 70% ethanol, is shaken up, filters, takes subsequent filtrate, to obtain the final product；
（4）The study on the stability of detected solution：To same test solution, measure once, measure 5 times altogether at regular intervals,
Refer to table 2.Experiment shows, interior when 24 is small, and each measured object is stablized；
2 study on the stability result of the test of table
（5）Precision test：To same test solution, by above-mentioned chromatographic condition, METHOD FOR CONTINUOUS DETERMINATION 6 times.As a result 6 measure are each
The RSD of component meets the requirements, and refers to table 3.Experiment shows that the precision of this law is preferable；
3 instrument precision measurement result of table
（6）Repetitive test：Fufang Danshen Pian 20 is taken, removes coating, it is accurately weighed, it is finely ground, about 0.9g is taken, it is accurately weighed,
10 parts of parallel determination, calculates, and result of the test shows, the repeatability of this law preferably refers to table 4
4 repetitive test result of table
（7）Specificity is tested：Scarce Radix Salviae Miltiorrhizae and each 0.9g of the negative sample of pseudo-ginseng are taken respectively, by above-mentioned test solution preparation method
Scarce Radix Salviae Miltiorrhizae and the negative control solution of pseudo-ginseng, sample introduction measure are made in accordance with the law.As a result in ultraviolet chromatogram with tanshinone IIA and pellet
Phenolic acid B compare chromatographic peak relevant position without chromatographic peak occur, in Optical Chromatography is evaporated with pseudo-ginseng 4 compare chromatographic peak relevant position
No chromatographic peak occurs, and sees Fig. 1 and Fig. 2, illustrates that negative control is noiseless；
（8）Accuracy（Sample recovery rate）Measure：
Precision weighs known content（The content of tanshin polyphenolic acid B is 22.7773mg/g, and the content of tanshinone IIA is 1.3859mg/g,
The content of notoginsenoside R is 2.7665mg/g, and the content of ginsenoside Rg1 is 16.7331mg/g, the content of ginsenoside Re
For 1.5459mg/g, the content of ginsenoside Rb1 is 10.7429mg/g,）Totally 6 parts of compound Danshen Root agreement that contracts a film or TV play to an actor or actress 0.45g, respectively essence
Close addition tanshin polyphenolic acid B reference substance about 10mg, then (precision weighs notoginsenoside R control to accurate addition mixed reference substance solution 25mL
Product（Purity 94.0%）12.38mg, ginsenoside Rg1's reference substance（Purity 93.4%）62.40mg, ginsenoside Rb1's reference substance（It is pure
Degree 95.9%）39.97mg is put in 200mL measuring bottles, adds 70% ethanol solution to make dissolving；Precision weighs tanshinone IIA reference substance again
10.03 mg are put in 50mL measuring bottles, are added methanol to make dissolving and are diluted to scale, shake up, and precision measures 25mL and puts above-mentioned 200mL amounts
In bottle；Precision weighs ginsenoside Re's reference substance again（Purity 92.7%）13.01 mg are put in 20mL measuring bottles, add 70% ethanol solution
Make dissolving and be diluted to scale, shake up, precision measures 10mL and puts in above-mentioned 200mL measuring bottles, adds 70% ethanol solution to be shaken to scale
It is even.) by above-mentioned method extraction, measure, calculate sample recovery rate, as a result tanshin polyphenolic acid B average recovery rate for 99.8%, RSD=
1.1%（n=6）, tanshinone IIA average recovery rate is 101.2%, RSD=1.7%（n=6）, notoginsenoside R average recovery rate is
99.6%, RSD=2.7%（n=6）, ginsenoside Rg1's average recovery rate is 100.2%, RSD=2.7%（n=6）, ginsenoside Re puts down
The equal rate of recovery is 98.5%, RSD=0.75%（n=6）, ginsenoside Rb1's average recovery rate is 101.5%, RSD=1.3%（n=6）, symbol
Close the requirement of quantitative analysis；
（9）Durability is investigated：
1）The investigation of different chromatographic columns：In same Waters high performance liquid chromatograph, 3 kinds of chromatographic columns are investigated：Column I
.TechMate C18-ST (5 μm, 4.6 × 250mm)；II .CAPCELLPAK C18 of column (5 μm, 4.6 × 250mm)；Column III
.KromasiL C18 (5 μm, 4.6 × 250mm)；IV .InertsiL ODS-3 of column (5 μm, 4.6 × 250mm), as a result respectively into
Swarming can reach good separating effect, the content no significant difference of each component；
2）The investigation of different instruments：With same pillar [chromatographic column II：CAPCELLPAK C18 (5 μm, 4.6 × 250mm)] respectively
Investigated on 2 kinds of different brands instruments, I, high performance liquid chromatographs Waters 2695, evaporative light dissipates color instrument .ALLtech ELSD
2000ES, II, high performance liquid chromatographs AgiLent 1260, evaporative light dissipate color instrument AgiLent 380-ELSD, as a result each component
Peak can reach good separating effect, and the content no significant difference of each component, meets the requirements；
（10）Measure：It is accurate respectively to draw 10 μ L mixed reference substance solutions A, 5 μ L and 15 μ L mixed reference substance solutions B and 10 μ L
Test solution, injects liquid chromatograph, measure, and the content of tanshin polyphenolic acid B and tanshinone IIA is calculated with one point external standard method, with outer
Mark the content that two-point method logarithmic equation calculates notoginsenoside.The Fufang Danshen Pian that 11 enterprises produce totally 20 batches is determined respectively,
As a result content of danshinolic acid B in 6 batches of samples is not up to standard requirement, remaining meets standard requirement, is shown in Table 5.
5 20 batches of Fufang Danshen Pian assay result tables of table
Claims (1)
1. method that is a kind of while measuring Multiple components in Fufang Danshen Pian, it is characterised in that detection method is as follows：
（1）Chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase
A, using 0.1% trifluoroacetic acid solution as Mobile phase B, carries out gradient elution, elution time 95 minutes；0~71 minute detection ripple of setting
A length of 280nm, Detection wavelength is 270nm within 71~96 minutes, and saponin(e is detected with evaporative light scattering detector；
（2）The preparation of reference substance solution：Take tanshin polyphenolic acid B reference substance, tanshinone IIA reference substance appropriate, it is accurately weighed, add methanol molten
Solution, is made every 1mL containing 600 μ g tanshin polyphenolic acid Bs, the mixed solution of 50 μ g tanshinone IIAs, as mixed reference substance solution A；Three are taken again
Seven saponin(e R1 reference substances, ginsenoside Rg1's reference substance, ginsenoside Re's reference substance, ginsenoside Rb1's reference substance are appropriate, accurate
It is weighed, add 70% ethanol dissolve, be made every 1mL containing 45 μ g notoginsenoside Rs, 450 μ g ginsenoside Rg1s, 40 μ g ginsenoside Res,
The mixed solution of 200 μ g ginsenoside Rb1s, as mixed reference substance solution B；
（3）The preparation of test solution：10, sample is taken, removes coating, it is accurately weighed, it is finely ground, about 0.9g is taken, it is accurately weighed,
Put in conical flask with cover, precision adds 70% ethanol 25mL, and close plug, weighed weight, is ultrasonically treated 30 minutes, lets cool, then weighed heavy
Amount, the weight of less loss is supplied with 70% ethanol, is shaken up, and is filtered, is taken subsequent filtrate, to obtain the final product；
（4）The durability of the stability of detected solution, chromatographic column and instrument is investigated, and carry out precision, repeatability, specially
Attribute, accuracy（Sample recovery rate）Experiment；
（5）Measure：It is accurate respectively to draw 10 μ L mixed reference substance solutions A, 5 μ L and 15 μ L mixed reference substance solutions B and 10 μ L confessions
Test sample solution, injects liquid chromatograph, measure, the content of tanshin polyphenolic acid B and tanshinone IIA is calculated with one point external standard method, uses external standard
Two-point method logarithmic equation calculates the content of notoginsenoside.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109900826A (en) * | 2019-03-26 | 2019-06-18 | 广州莱泰制药有限公司 | A kind of Fufang Danshen Pian Radix Notoginseng Content Test method |
| CN110082460A (en) * | 2019-06-03 | 2019-08-02 | 国药集团精方（安徽）药业股份有限公司 | A kind of quality determining method of Jingshu Granule |
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| CN109900826A (en) * | 2019-03-26 | 2019-06-18 | 广州莱泰制药有限公司 | A kind of Fufang Danshen Pian Radix Notoginseng Content Test method |
| CN110082460A (en) * | 2019-06-03 | 2019-08-02 | 国药集团精方（安徽）药业股份有限公司 | A kind of quality determining method of Jingshu Granule |
| CN110082460B (en) * | 2019-06-03 | 2022-05-20 | 国药集团精方（安徽）药业股份有限公司 | Quality detection method of Jingshu granules |
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Application publication date: 20180427 |