CN108037193A - Using the method for HPLC-UV-ELSD methods detection Fufang Danshen Pian - Google Patents
Using the method for HPLC-UV-ELSD methods detection Fufang Danshen Pian Download PDFInfo
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Abstract
The invention discloses a kind of method using HPLC UV ELSD methods detection Fufang Danshen Pian, using the detection method of liquid chromatogram gradient elution, change detection wavelength and UV detector cascade evaporation light scattering detector, the characteristic spectrum of Radix Salviae Miltiorrhizae in Fufang Danshen Pian and pseudo-ginseng is studied.The present invention can carry out Fufang Danshen Pian the control of full information, simplify checkout procedure, saved analysis time, available for Fufang Danshen Pian quality control and overall merit.
Description
Technical field
The present invention relates to traditional Chinese medicine detection technique field, specifically using HPLC-UV-ELSD methods detection Fufang Danshen Pian
Method.
Background technology
Fufang Danshen Pian is made of Radix Salviae Miltiorrhizae, pseudo-ginseng and borneol, is had the effect of promoting blood circulation and removing blood stasis, regulating qi-flowing for relieving pain, is that clinic is controlled
Treat coronary heart disease, conventional Chinese medicine prescription uncomfortable in chest and anginal.Research shows that the active ingredient of Radix Salviae Miltiorrhizae is mainly Diterpene quinone class liposoluble
Property and salvianolic acid water soluble ingredient, there is anti-oxidant, antiatherosclerosis, reduce the Cardiovascular such as myocardial oxygen consumption
And antitumor action；Notoginsenoside have expansion blood vessel, reduce myocardial oxygen consumption, suppress platelet aggregation, extend the clotting time,
Reducing blood lipid, remove the pharmacological action such as free radical, anti-inflammatory, anti-oxidant.Its current standard is《Chinese Pharmacopoeia》Version one in 2015, is received
The microscopical characters of pseudo-ginseng are carried；Thin layer differentiates that item is respectively with Tanshinone IIA, Panax Notoginseng saponin R1, ginsenoside Rb1, ginsenoside
Rg1It is reference substance with ginsenoside Re, and using borneol and pseudo-ginseng as control medicinal material, differentiates Radix Salviae Miltiorrhizae, pseudo-ginseng and borneol three in prescription
Taste medicinal material；Assay part is then to the liposoluble constituent Tanshinone II, water soluble ingredient tanshin polyphenolic acid B and notoginsenoside of Radix Salviae Miltiorrhizae
Constituents are measured.Not only checkout procedure repetition is cumbersome for the quality standard, but also cannot reflect this product in preparation process comprehensively
The change of middle component.Diterpenoid tanshinone class liposoluble constituent such as dihydrotanshinone Ⅰ, Cryptotanshinone and Radix Salviae Miltiorrhizae known to research report
Ketone I, phenolic acid class water soluble ingredient such as danshensu, protocatechualdehyde, Rosmarinic acid, tanshin polyphenolic acid B etc., notoginsenoside constituents such as ginseng
Saponin(e Rd, fails to be evaluated.
The content of the invention
Based on this, the present invention provides a kind of method using HPLC-UV-ELSD methods measure Fufang Danshen Pian, using liquid
The detection method of phase chromatography gradient elution, change detection wavelength and UV detector cascade evaporation light scattering detector, at the same it is right
Radix Salviae Miltiorrhizae and the characteristic spectrum of pseudo-ginseng are studied in Fufang Danshen Pian.The present invention can carry out Fufang Danshen Pian the control of full information
System, simplifies checkout procedure, has 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：Sodium Danshensu reference substance（National Institute for Food and Drugs Control provide, lot number 110855-200508, for containing
Measure fixed use), protocatechualdehyde reference substance（National Institute for Food and Drugs Control provides, lot number 110810-200506, for content
Measure use), Rosmarinic acid reference substance（National Institute for Food and Drugs Control provides, lot number 111871-201203, for containing measurement
Fixed to use, content is in terms of 98.8%), tanshin polyphenolic acid B reference substance（National Institute for Food and Drugs Control provides, lot number 111562-
201212, for assay, content is in terms of 95.4%), dihydrotanshinone Ⅰ reference substance（National Institute for Food and Drugs Control carries
For, lot number 0868-200103, differentiate and use), Cryptotanshinone reference substance（National Institute for Food and Drugs Control provides, lot number
110852-200808, for assay, content is in terms of 98.7%), salvia miltiorrhiza bge I reference substance（Chinese food drug assay is studied
Institute provide, lot number 110867-200406, differentiate use), Tanshinone IIAReference substance（National Institute for Food and Drugs Control provides,
Lot number 110766-200315, for assay), Panax Notoginseng saponin R1Reference substance（National Institute for Food and Drugs Control provides,
Lot number 110745-200617, for assay), ginsenoside Rg1Reference 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 medicine is examined
Determine research institute offer, lot number 110754-200822, for assay, content is in terms of 88.8%), ginsenoside Rb1Reference substance
（National Institute for Food and Drugs Control provide, lot number 110704-201122, for assay, content is in terms of 92.9%), people
Join saponin(e Rd（National Institute for Food and Drugs Control provides, lot number 110704-201122, for assay, content with
92.9% meter).Acetonitrile is chromatographically pure, and 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；Detection wavelength is set by the regulation of table 1
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 Sodium Danshensu reference substance appropriate, it is accurately weighed, add water that every 1mL is made and contain 100 μ g's
Solution, separately takes protocatechualdehyde reference substance, Rosmarinic acid reference substance, tanshin polyphenolic acid B reference substance, dihydrotanshinone Ⅰ reference substance, hidden Radix Salviae Miltiorrhizae
Ketone reference substance, salvia miltiorrhiza bge I reference substance, tanshinone IIA reference substance, notoginsenoside R reference substance, ginsenoside Rg1's reference substance, people
It is appropriate to join saponin(e Re reference substances, ginsenoside Rb1's reference substance, ginsenoside Rd's reference substance, adds methanol that every 1mL is made and contains former catechu
Each 20 μ g of aldehyde, Rosmarinic acid, dihydrotanshinone Ⅰ, Cryptotanshinone, salvia miltiorrhiza bge I, tanshinone IIA, tanshin polyphenolic acid B, notoginsenoside R,
Ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, the mixed solution of each 50 μ g of ginsenoside Rd；
（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）Durability is investigated：
1）The investigation of different chromatographic columns：In same Waters high performance liquid chromatograph, carried out respectively using different chromatographic columns
Detection；
2）The investigation of different instruments：It is detected respectively on different brands instrument with same pillar；
（5）Measure：It is accurate respectively to draw above-mentioned reference substance solution and each 10 μ L of test solution, inject liquid chromatograph, measure；
（6）Carry out methodology validation：Stability, precision, repeatability and specificity experiment are carried out respectively.
The beneficial effects of the invention are as follows：
The present invention differentiates the thin layer in current standard to be combined with assay, is examined using liquid chromatogram gradient elution, switching
The detection method of wavelength and UV detector cascade evaporation light scattering detector is surveyed, to the spy of Radix Salviae Miltiorrhizae in Fufang Danshen Pian and pseudo-ginseng
Sign collection of illustrative plates is studied.The present invention can carry out Fufang Danshen Pian the control of full information, reduced inspection process, when saving analysis
Between, available for Fufang Danshen Pian quality control and overall merit.
Diode array detector detection is taken to be measured each component of Radix Salviae Miltiorrhizae, the results showed that：Water soluble ingredient Radix Salviae Miltiorrhizae
Plain sodium, protocatechualdehyde, Rosmarinic acid, tanshin polyphenolic acid B have absorption maximum near 280 nm wavelength, are eluted before 50min
Out；Liposoluble constituent dihydrotanshinone Ⅰ, Cryptotanshinone, salvia miltiorrhiza bge I, tanshinone IIA have maximum near 270 nm wavelength
Absorb, and be just eluted out after 70min.The present invention take ultraviolet wavelength switching method measure salvia-soluble and it is fat-soluble into
Point, Multiple components can be measured at the same time and can ensure that each component has the response of maximum.And Saponins from Panax notoginseng is ultraviolet
Area only has end absorption, and the present invention measures the component of pseudo-ginseng in Fufang Danshen Pian using evaporative light scattering detector, and evaporative light dissipates
It is small to penetrate detector baseline noise, from gradients affect, has preferable response to the saponin component in pseudo-ginseng.The present invention is with second
For the trifluoroacetic acid solution of nitrile -0.1% system as mobile phase, when detection, can obtain preferable chromatography effect.The present invention is carried using ultrasound
Active ingredient in method extraction Fufang Danshen Pian is taken, tanshin polyphenolic acid B can be overcome to be heated adverse effect caused by labile property.
Brief description of the drawings
Fig. 1 is to use column I：TechMate C18-ST (5 μm, 4.6 × 250mm), instrument：2695 efficient liquid of Waters
Chromatography, 2487 UV detector-ALLtech 2000ES evaporative light scattering detector detection Fufang Danshen Pian（Guangzhou is white
Chinese medicine Co., Ltd of Yunshan Mountain Hutchison China Trade Holdings, lot number：E3A030）UV characteristic spectrums；
Fig. 2 is to use column I：TechMate C18-ST (5 μm, 4.6 × 250mm), instrument：2695 high-efficient liquid phase colors of Waters
Spectrometer, 2487 UV detector-ALLtech 2000ES evaporative light scattering detector detection Fufang Danshen Pian（Community in Baiyunshan, Guangzhou
Chinese medicine Co., Ltd of Hutchison China Trade Holdings, lot number：E3A030）ELSD characteristic spectrums；
Fig. 3 is to use column II：CAPCELLPAK C18 (5 μm, 4.6 × 250mm), instrument：2695 high-efficient liquid phase colors of Waters
Spectrometer, 2487 UV detector-ALLtech 2000ES evaporative light scattering detector detection Fufang Danshen Pian（Community in Baiyunshan, Guangzhou
Chinese medicine Co., Ltd of Hutchison China Trade Holdings, lot number：E3A030）UV characteristic spectrums；
Fig. 4 is to use column II：CAPCELLPAK C18 (5 μm, 4.6 × 250mm), instrument：2695 high-efficient liquid phase colors of Waters
Spectrometer, 2487 UV detector-ALLtech 2000ES evaporative light scattering detector detection Fufang Danshen Pian（Community in Baiyunshan, Guangzhou
Chinese medicine Co., Ltd of Hutchison China Trade Holdings, lot number：E3A030）ELSD characteristic spectrums；
Fig. 5 is to use column III：Kromasil C18 (5 μm, 4.6 × 250mm), instrument：2695 high performance liquid chromatography of Waters
Instrument, 2487 UV detector-ALLtech 2000ES evaporative light scattering detector detection Fufang Danshen Pian（Community in Baiyunshan, Guangzhou and
Remember Huangpu Chinese medicine Co., Ltd, lot number：E3A030）UV characteristic spectrums；
Fig. 6 is to use column III：Kromasil C18 (5 μm, 4.6 × 250mm), instrument：2695 high performance liquid chromatography of Waters
Instrument, 2487 UV detector-ALLtech 2000ES evaporative light scattering detector detection Fufang Danshen Pian（Community in Baiyunshan, Guangzhou and
Remember Huangpu Chinese medicine Co., Ltd, lot number：E3A030）ELSD characteristic spectrums；
Fig. 7 is to use column IV：Inertsil ODS-3 (5 μm, 4.6 × 250mm), instrument：2695 high-efficient liquid phase colors of Waters
Spectrometer, 2487 UV detector-ALLtech 2000ES evaporative light scattering detector detection Fufang Danshen Pian（Community in Baiyunshan, Guangzhou
Chinese medicine Co., Ltd of Hutchison China Trade Holdings, lot number：E3A030）UV characteristic spectrums；
Fig. 8 is to use column IV：Inertsil ODS-3 (5 μm, 4.6 × 250mm), instrument：2695 high-efficient liquid phase colors of Waters
Spectrometer, 2487 UV detector-ALLtech 2000ES evaporative light scattering detector detection Fufang Danshen Pian（Community in Baiyunshan, Guangzhou
Chinese medicine Co., Ltd of Hutchison China Trade Holdings, lot number：E3A030）ELSD characteristic spectrums；
Fig. 9 is to use chromatographic column II：CAPCELLPAK C18 (5 μm, 4.6 × 250mm), instrument II：High performance liquid chromatograph
Agilent 1260, evaporative light dissipate color instrument Agilent 380-ELSD evaporative light scattering detector detection Fufang Danshen Pian（Guangzhou
Chinese medicine Co., Ltd of White Cloud Mountain Hutchison China Trade Holdings, lot number：E3A030）UV characteristic spectrums；
Figure 10 is to use chromatographic column II：CAPCELLPAK C18 (5 μm, 4.6 × 250mm), instrument II：High performance liquid chromatograph
Agilent 1260, evaporative light dissipate color instrument Agilent 380-ELSD evaporative light scattering detector detection Fufang Danshen Pian（Guangzhou
Chinese medicine Co., Ltd of White Cloud Mountain Hutchison China Trade Holdings, lot number：E3A030）ELSD characteristic spectrums；
Figure 11 is Fufang Danshen Pian（Huqingyutang Pharmaceutical Co., Ltd., Hangzhou City, lot number：100928）UV characteristic spectrums；Peak in figure
1：Sodium Danshensu, peak 2：Protocatechualdehyde, peak 3：Rosmarinic acid, peak 4：Tanshin polyphenolic acid B, peak 5：Dihydrotanshinone Ⅰ peak 6：Hidden Radix Salviae Miltiorrhizae
Ketone, peak 7：Salvia miltiorrhiza bge I, peak 8：Tanshinone IIA；
Figure 12 is Fufang Danshen Pian（Huqingyutang Pharmaceutical Co., Ltd., Hangzhou City, lot number：100928）ELSD characteristic spectrums；In figure
Peak 9：Notoginsenoside R, peak 10：Ginsenoside Rg1, peak 11：Ginsenoside Re, peak 12：Ginsenoside Rb1, peak 13：Ginseng soap
Glycosides Rd；
Figure 13 is Fufang Danshen Pian（Beijing Tongrentang Technology Development Co.ltd. Pharmaceutical Factory, lot number：1125531）UV it is special
Levy collection of illustrative plates；Peak 1 in figure：Sodium Danshensu, peak 2：Protocatechualdehyde, peak 3：Rosmarinic acid, peak 4：Tanshin polyphenolic acid B, peak 5：Dihydrotanshinone Ⅰ
Peak 6：Cryptotanshinone, peak 7：Salvia miltiorrhiza bge I, peak 8：Tanshinone IIA；
Figure 14 is Fufang Danshen Pian（Beijing Tongrentang Technology Development Co.ltd. Pharmaceutical Factory, lot number：1125531）ELSD
Characteristic spectrum；Peak 9 in figure：Notoginsenoside R, peak 10：Ginsenoside Rg1, peak 11：Ginsenoside Re, peak 12：Ginsenoside
Rb1, peak 13：Ginsenoside Rd；
Figure 15 is Fufang Danshen Pian（Shanghai Leiyun Pharmaceutical Industry Co., Ltd., lot number：100523）UV characteristic spectrums；Peak 1 in figure：
Sodium Danshensu, peak 2：Protocatechualdehyde, peak 3：Rosmarinic acid, peak 4：Tanshin polyphenolic acid B, peak 5：Dihydrotanshinone Ⅰ peak 6：Cryptotanshinone,
Peak 7：Salvia miltiorrhiza bge I, peak 8：Tanshinone IIA；
Figure 16 is Fufang Danshen Pian（Shanghai Leiyun Pharmaceutical Industry Co., Ltd., lot number：100523）ELSD characteristic spectrums；Peak in figure
9：Notoginsenoside R, peak 10：Ginsenoside Rg1, peak 11：Ginsenoside Re, peak 12：Ginsenoside Rb1, peak 13：Ginsenoside
Rd；
Figure 17 is to lack Radix Salviae Miltiorrhizae negative control UV chromatograms；
Figure 18 is 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：Sodium Danshensu reference substance（National Institute for Food and Drugs Control provide, lot number 110855-200508, for containing
Measure fixed use), protocatechualdehyde reference substance（National Institute for Food and Drugs Control provides, lot number 110810-200506, for content
Measure use), Rosmarinic acid reference substance（National Institute for Food and Drugs Control provides, lot number 111871-201203, for containing measurement
Fixed to use, content is in terms of 98.8%), tanshin polyphenolic acid B reference substance（National Institute for Food and Drugs Control provides, lot number 111562-
201212, for assay, content is in terms of 95.4%), dihydrotanshinone Ⅰ reference substance（National Institute for Food and Drugs Control carries
For, lot number 0868-200103, differentiate and use), Cryptotanshinone reference substance（National Institute for Food and Drugs Control provides, lot number
110852-200808, for assay, content is in terms of 98.7%), salvia miltiorrhiza bge I reference substance（Chinese food drug assay is studied
Institute provide, lot number 110867-200406, differentiate use), Tanshinone IIAReference substance（National Institute for Food and Drugs Control provides,
Lot number 110766-200315, for assay), Panax Notoginseng saponin R1Reference substance（National Institute for Food and Drugs Control provides,
Lot number 110745-200617, for assay), ginsenoside Rg1Reference 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 medicine is examined
Determine research institute offer, lot number 110754-200822, for assay, content is in terms of 88.8%), ginsenoside Rb1Reference substance
（National Institute for Food and Drugs Control provide, lot number 110704-201122, for assay, content is in terms of 92.9%), people
Join saponin(e Rd（National Institute for Food and Drugs Control provides, lot number 110704-201122, for assay, content with
92.9% meter).Acetonitrile is chromatographically pure, and water is high purity water, and other reagents are that analysis is pure；
（3）Sample：It has collected totally 18 batches of Fufang Danshen Pians of 10 enterprise's production.
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, the regulation according to the form below carries out gradient elution；Detection wavelength by table 1 regulation
Setting, saponin(e are 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 Sodium Danshensu reference substance appropriate, it is accurately weighed, add water that every 1mL is made and contain 100 μ g's
Solution, separately takes protocatechualdehyde reference substance, Rosmarinic acid reference substance, tanshin polyphenolic acid B reference substance, dihydrotanshinone Ⅰ reference substance, hidden Radix Salviae Miltiorrhizae
Ketone reference substance, salvia miltiorrhiza bge I reference substance, tanshinone IIA reference substance, notoginsenoside R reference substance, ginsenoside Rg1's reference substance, people
It is appropriate to join saponin(e Re reference substances, ginsenoside Rb1's reference substance, ginsenoside Rd's reference substance, adds methanol that every 1mL is made and contains former catechu
Each 20 μ g of aldehyde, Rosmarinic acid, dihydrotanshinone Ⅰ, Cryptotanshinone, salvia miltiorrhiza bge I, tanshinone IIA, tanshin polyphenolic acid B, notoginsenoside R,
Ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, the mixed solution of each 50 μ g of ginsenoside Rd.
（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, precision claims
It is fixed, 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）Durability is investigated：
1）The investigation of different chromatographic columns：In same Waters high performance liquid chromatograph, 4 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 equal energy
13 chromatographic peaks are detected, and respectively can reach good separating effect into swarming.It is shown in Table 2,3, Figure 11~18.
The different chromatographic columns of table 2 investigate retention time list（UV characteristic spectrums）
The different chromatographic columns of table 3 investigate retention time list（ELSD characteristic spectrums）
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 can examine
Go out 13 chromatographic peaks, be shown in Table 4~5, Fig. 3,4,9,10.
The different instruments of table 4 investigate retention time list（UV characteristic spectrums）
The different instruments of table 5 investigate retention time list（ELSD characteristic spectrums）
（5）Measure：It is accurate respectively to draw above-mentioned reference substance solution and each 10 μ L of test solution, inject liquid chromatograph, measure.
Ten three chromatographic peaks identical with reference substance solution chromatographic peak retention time should be presented in test sample chromatography.10 are determined respectively
18 batches of Fufang Danshen Pians of enterprise's production, as a result detect 13 characteristic peaks, meet standard requirement, are shown in Table 6,7.
6 18 batches of Coupon Testing Results of table（UV characteristic spectrum retention times）
7 18 batches of Coupon Testing Results of table（ELSD characteristic spectrum retention times）
（6）Methodology validation and result：
1）Stability test：Take and once, investigate the stabilization of sample solution with a test solution, certain interval of time sample introduction
Property.The result shows that test solution is placed at room temperature, the interior stabilization when 24 is small, is shown in Table 8,9.
8 study on the stability result of the test of table（UV characteristic spectrum retention times）
9 study on the stability result of the test of table（ELSD characteristic spectrum retention times）
2）Precision test：Take the precision for a test solution, continuous sample introduction 6 times, investigating instrument.The result shows that this law
Instrument precision it is good, be shown in Table 10,11.
10 Precision test result of table（UV characteristic spectrum retention times）
11 Precision test result of table（ELSD characteristic spectrum retention times）
3）Repetitive test：To same batch of sample, by 10 parts of the method parallel determination of above-mentioned detection, the results showed that, this method weight
Renaturation is good, and 10 samples, 13 characteristic peak separation are good, and each peak relative retention time is less than 1.0%.Result of the test shows, this
The repeatability of method preferably, meets the requirements, is shown in Table 12,13.
12 repetitive test result of table（UV characteristic spectrum retention times）
13 repetitive test result of table（ELSD characteristic spectrum retention times）
4）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 8 contrast colors of Radix Salviae Miltiorrhizae
Spectral peak relevant position occurs without chromatographic peak, and compare chromatographic peak relevant position with pseudo-ginseng 5 in Optical Chromatography is evaporated goes out without chromatographic peak
It is existing, see Figure 17 and Figure 18, illustrate that negative control is noiseless.
Claims (1)
1. using the method for HPLC-UV-ELSD methods detection Fufang Danshen Pian, it is characterised in that the described method includes herein below：
（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 Sodium Danshensu reference substance appropriate, it is accurately weighed, add water that every 1mL is made and contain 100 μ g's
Solution, separately takes protocatechualdehyde reference substance, Rosmarinic acid reference substance, tanshin polyphenolic acid B reference substance, dihydrotanshinone Ⅰ reference substance, hidden Radix Salviae Miltiorrhizae
Ketone reference substance, salvia miltiorrhiza bge I reference substance, tanshinone IIA reference substance, notoginsenoside R reference substance, ginsenoside Rg1's reference substance, people
It is appropriate to join saponin(e Re reference substances, ginsenoside Rb1's reference substance, ginsenoside Rd's reference substance, adds methanol that every 1mL is made and contains former catechu
Each 20 μ g of aldehyde, Rosmarinic acid, dihydrotanshinone Ⅰ, Cryptotanshinone, salvia miltiorrhiza bge I, tanshinone IIA, tanshin polyphenolic acid B, notoginsenoside R,
Ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, the mixed solution of each 50 μ g of ginsenoside Rd；
（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）Durability is investigated：
1）The investigation of different chromatographic columns：In same Waters high performance liquid chromatograph, carried out respectively using different chromatographic columns
Detection；
2）The investigation of different instruments：It is detected respectively on different brands instrument with same pillar；
（5）Detection：It is accurate respectively to draw above-mentioned reference substance solution and each 10 μ L of test solution, inject liquid chromatograph, measure；
（6）Methodology validation：Stability, precision, repeatability and specificity experiment are carried out respectively.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711057070.0A CN108037193B (en) | 2017-11-01 | 2017-11-01 | Method for detecting compound salvia miltiorrhiza tablets by adopting HP L C-UV-E L SD method |
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| CN112763637A (en) * | 2020-12-25 | 2021-05-07 | 上海仪电分析仪器有限公司 | Variable wavelength data acquisition and processing method of liquid chromatograph ultraviolet detector |
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