CN103969346A - Panax notoginseng saponin component analysis method - Google Patents
Panax notoginseng saponin component analysis method Download PDFInfo
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Abstract
The present invention provides a panax notoginseng saponin component analysis method, which comprises content determination and finger print determination of panax notoginseng saponin, and specifically comprises sample solution preparation, reference substance solution preparation, and determination of content and finger print of the panax notoginseng saponin component in compound danshen dripping pills by using ultra performance liquid chromatography. According to the present invention, the same chromatography conditions are adopted to concurrently determine the content and the finger print of the panax notoginseng saponin component, characteristics of good linearity, good repeatability, good stability and good recovery rate are provided, and the quality of the compound danshen dripping pills is easily and completely controlled.
Description
Technical field
The invention belongs to the field of Chinese medicines, be specifically related to the detection method of a kind of notoginsenoside assay and fingerprint map analyzing.
Background technology
Compound danshen dripping pills is the pill of being made up of the red sage root, pseudo-ginseng and borneol, is widely used in clinically prevention, treatment, the first aid of coronary heart diseases and angina pectoris, has now become one of leading brand on domestic cardiovascular market.The principal ingredient of compound danshen dripping pills is danshensu (danshensu, Da), protocatechualdehyde (protocatechuic aldehyde, Pra) and tanshin polyphenolic acid B (salvianolic acid B, Sal B).
In pharmacopeia, the content assaying method of compound danshen dripping pills is at present: taking Sodium Danshensu as reference substance, adopt the content of high effective liquid chromatography for measuring danshensu, concrete grammar is: the preparation of (1) reference substance solution: get Sodium Danshensu reference substance appropriate, accurately weighed, adding methyl alcohol makes every 1ml and is equivalent to danshensu 0.14mg containing 0.16mg() solution, to obtain final product; (2) preparation of need testing solution: get compound danshen dripping pills 5 balls, put in 10ml measuring bottle, add methyl alcohol appropriate, ultrasonic processing 2 hours, lets cool, and adds methyl alcohol to scale, shakes up, and with miillpore filter filtration, gets filtrate and get final product; (3) precision is drawn reference substance solution 5 μ l and need testing solution 5-10 μ l respectively, and injection liquid chromatography is measured, and to obtain final product.
Traditional Chinese medicine fingerprint is can be a kind of method of the thoroughly evaluating traditional Chinese medicine quality pattern accepted extensively both at home and abroad at present.U.S. FDA allows declarer that the chromatographic fingerprinting data of product is provided in autonomic drug goods governing principle, German medicinal plant association, British Herbal Pharmacopoeia, India herbal medicine allusion quotation and Canada is medicinal and also one of content using finger-print as quality control standard all of fragrant plant association.Domestic scholars thank training mountain also think, traditional Chinese medicine quality needs comprehensive evaluation, fingerprint map analyzing is that Chinese medicine and preparation are carried out to one of feasible means of comprehensive macroanalysis.But have about the research of the finger-print aspect of the notoginsenoside constituents in compound danshen dripping pills and there is not yet report at home and abroad.
Compound danshen dripping pills listing was progressively set up the perfect processing quality hierarchy of control in more than ten years, carried out FDA with the form of medicine at present and declared research.But Composite Salvia Dropping Pill group divides middle assay component still lower, improving product processing quality level of control, need to further investigate for more chemical composition of Chinese materia medica wherein, thereby hold more exactly product quality, reduce drug risk, simultaneously for international registration provides support.
In compound danshen dripping pills, the main active of ministerial drug pseudo-ginseng is notoginsenoside and ginsenoside, is different from the peculiar active component of ginseng and notoginsenoside is pseudo-ginseng.But also there is no at present the analytical approach of effective specificity for the notoginsenoside constituents in compound danshen dripping pills.
In order to control better the quality of compound danshen dripping pills, need a kind of analytical approach of notoginsenoside constituents, comprise assay and the finger print measuring method of notoginsenoside constituents, especially to Panax Notoginseng saponin R
1, ginsenoside Rg
1, ginsenoside Re, ginsenoside Rb
1fingerprint discrimination method and quantivative approach.
Summary of the invention
The object of the present invention is to provide a kind of analytical approach of notoginsenoside constituents, comprise assay and the determining fingerprint pattern of notoginsenoside.
According to the analytical approach of notoginsenoside constituents of the present invention, comprise the following steps:
(1) preparation of need testing solution: get compound danshen dripping pills, accurately weighed, add ammonia spirit, ultrasonic; Cross ODS post, water, methanol-eluted fractions successively, collects meoh eluate, filters, and to obtain final product;
(2) preparation of reference substance solution: precision takes Panax Notoginseng saponin R respectively
1reference substance, ginsenoside Rg
1reference substance, ginsenoside Re's reference substance, ginsenoside Rb
1reference substance is appropriate, dissolves and dilutes with methyl alcohol, to obtain final product;
(3) assay of notoginsenoside: get described need testing solution and reference substance solution, adopt Ultra Performance Liquid Chromatography to analyze, measure the content of notoginsenoside;
(4) determining fingerprint pattern of notoginsenoside: get described need testing solution, adopt Ultra Performance Liquid Chromatography to analyze, obtain finger-print.
One of according to the embodiment of the present invention, being prepared as of described need testing solution: get compound danshen dripping pills 10-30 ball, accurately weighed being placed in 15ml graduated tube, adds the ammonia spirit 1-10ml of 0.5%-10%, ultrasonic to dissolving; Cross the ODS pillar of 1.0cm internal diameter, with after 50ml washing by 100% methanol-eluted fractions to 10ml volumetric flask constant volume, shook up the organic filter membrane of 0.22 μ m, to obtain final product.
According to another embodiment of the present invention, being prepared as of described reference substance solution: precision takes Panax Notoginseng saponin R respectively
1reference substance, ginsenoside Rg
1reference substance, ginsenoside Re's reference substance, ginsenoside Rb
1reference substance is appropriate, dissolves and dilutes with methyl alcohol, makes containing Panax Notoginseng saponin R
1concentration is 0.01-0.1mg/ml, ginsenoside Rg
1concentration is that 0.1-0.5mg/ml, ginsenoside Re's concentration are 0.01-0.06mg/ml, ginsenoside Rb
1concentration is the mixing reference substance solution of 0.1-0.5mg/ml.
According to an embodiment more of the present invention, the chromatographic condition of described Ultra Performance Liquid Chromatography is: chromatographic column is C18 chromatographic column, and mobile phase A is acetonitrile or methyl alcohol mutually, and Mobile phase B is water mutually, gradient elution; UV-detector, detection wavelength is 200-210nm.
Preferably, described mobile phase A is acetonitrile mutually, and the gradient of described gradient elution is:
For reducing the impact of solvent on sample analysis, the preferred acetonitrile-water of the present invention is flow phase system.
Investigate the gradient condition of multiple gradient elution, under the gradient condition of gradient elution of the present invention, 4 notoginsenoside constituents have all reached baseline separation, and each chromatographic peak is effectively separated entirely, peak shape is all comparatively sharp-pointed upright, also can meet the requirement of finger-print.
Preferably, the detection wavelength of described Ultra Performance Liquid Chromatography is 203nm.
The conventional detection method of notoginsenoside constituents has ultraviolet detection, evaporative light-scattering to detect.Uv detection method is easy, mature and stable, the range of linearity is wide, instrument is universal, therefore preferred uv detection method.Notoginsenoside constituents all has uv absorption within the scope of ultraviolet end 200-210nm, and its maximum absorption wavelength is 203nm, and therefore the preferred 203nm of the present invention is the ultraviolet detection wavelength of notoginsenoside constituents.
Another object of the present invention is to provide a kind of finger-print of notoginsenoside constituents.
One of according to the embodiment of the present invention, described finger-print is made up of 8 total fingerprint peakses.
With No. 2 peak ginsenoside Rgs
1for with reference to peak, the equal < 0.1% of the relative retention time RSD at 7 total peaks, relative peak area RSD except No. 8 peaks all 3.0%, No. 8 peak-to-peak area of < affect because of separation case, RSD more greatly 8.74%.
According to another embodiment of the present invention, described finger-print is made up of 5 total fingerprint peakses.
Preferably, described 5 total peaks are respectively with respect to relative retention time and the relative peak area at reference peak:
No. 1 peak, relative retention time is 0.77min, relative peak area is 0.19;
S peak, relative retention time is 1.00min, relative peak area 1;
No. 2 peaks, relative retention time is 1.04min, relative peak area 0.11;
No. 3 peaks, relative retention time is 1.93min, relative peak area 0.64;
No. 4 peaks, relative retention time is 2.46min, relative peak area 0.13;
Compared with S peak, the relative retention time at all the other each peaks and relative peak area all should setting ± 5% in.
With the ginsenoside Rg of peak area maximum
1for with reference to peak, get rid of peak area and be less than the total peak with reference to peak-to-peak area 5%, and under different instruments, different chromatographic column condition, relatively go out peak position and change chromatographic peak greatly, the finger-print of notoginsenoside constituents in the compound danshen dripping pills that obtains being formed by 5 total peaks.This finger-print is with ginsenoside Rg
1for with reference to peak, the equal < 0.1% of the relative retention time RSD at all the other 4 total peaks, the equal < 3.0% of relative peak area RSD.
According to the present invention, sample and described finger-print are through Chinese chromatographic fingerprinting similarity evaluation system-computed, and similarity is between 0.8-1.0.
Sample of the present invention is compound danshen dripping pills, adopts the method for the invention to measure the content of the notoginsenoside constituents in testing sample, then through the similarity of Chinese chromatographic fingerprinting similarity evaluation system-computed and finger-print of the present invention.Chinese chromatographic fingerprinting similarity evaluation system of the present invention is promulgated by the Chinese Pharmacopoeia council.
Preferably, sample and described finger-print are through Chinese chromatographic fingerprinting similarity evaluation system-computed, and similarity is not less than 0.9.
The present invention adopts identical chromatographic condition, can measure content and the finger-print of notoginsenoside constituents in compound danshen dripping pills simultaneously, has good linearity, repeatability, stability and the recovery, contributes to control the quality of compound danshen dripping pills more comprehensively.
Brief description of the drawings:
The separation case of the different chromatographic columns of Fig. 1 is investigated test findings one, and chromatographic column is Aqucity UPLC 1.
tMbEH C
18(2.1 × 100mm, 1.7 μ m).
The separation case of the different chromatographic columns of Fig. 2 is investigated test findings two, and chromatographic column is Waters Aqucity UPLC 2.
tM(2.1 × 100mm, 1.8 μ m) for HSS T3.
The separation case of the different chromatographic columns of Fig. 3 is investigated test findings three, and chromatographic column is Aqucity UPLC 3.
tMbEH SHIELD RPC
18(2.1 × 100mm, 1.7 μ m).
The typical color spectrogram of the investigation test of Fig. 4 eluent gradient ratio, chromatographic column is Aqucity UPLC 3.
tMbEH SHIELD RPC
18(2.1 × 100mm, m), corresponding gradient elution program is in table 2 for 1.7 μ.
The typical color spectrogram of the investigation test of Fig. 5 eluent gradient ratio, chromatographic column is Aqucity UPLC 1.
tMbEH C
18(2.1 × 100mm, m), corresponding gradient elution program is in table 3 for 1.7 μ.
Fig. 6 heterogeneity reference substance comparison diagram.
Fig. 7 pseudo-ginseng, compound Danshen Root medicinal extract, compound danshen dripping pills, negative sample, blank solvent comparison diagram, chromatographic column is AqucityUPLC 1.
tMbEH C
18(2.1 × 100mm, m), corresponding gradient elution program is in table 3 for 1.7 μ.
The finger-print of the definite compound danshen dripping pills notoginsenoside constituents of Fig. 8 embodiment mono-.
Tri-kinds of different manufacturers instrument compound danshen dripping pills pseudo-ginseng reference fingerprints of Fig. 9, chromatographic column is Aqucity UPLCTM BEHC
18(2.1 × 100mm, 1.7 μ m).
The compound danshen dripping pills pseudo-ginseng reference fingerprint (lot number is 120509) that tri-kinds of different brands chromatographic columns of Figure 10 record.
The compound danshen dripping pills pseudo-ginseng reference fingerprint (lot number is 120502) that three groups of two different experiments chambers of Figure 11 experimenter records.
The notoginsenoside constituents finger-print that Figure 12 embodiment bis-is definite.
Figure 13 heterogeneity reference substance comparison diagram.
Embodiment:
Further illustrate by the following examples the present invention, but not as limitation of the present invention.
The assay of test example one notoginsenoside and the optimization of finger print measuring method test condition
1, instrument, reagent
Instrument: Waters Ultra Performance Liquid Chromatography instrument, UPLC-PDA Detector; Agilent1290 Ultra Performance Liquid Chromatography instrument, UHPLC-DAD Detector; SHIMADZU Nexera LC-30A Ultra Performance Liquid Chromatography instrument, UHPLC-DADDetector.
Reference substance: Panax Notoginseng saponin R
1(lot number 110745-200415, for assay), ginsenoside Rb
1(lot number 110704-200318, for assay), ginsenoside Rg
1(lot number 110703-200424, for assay), ginsenoside Re's (lot number 110754-200421, for assay), all purchased from Nat'l Pharmaceutical & Biological Products Control Institute, put dry more than 12 hours use in phosphorus pentoxide vacuum drying apparatus.
Similarity evaluation software: Chinese chromatographic fingerprinting similarity evaluation system 2004A version, 2004B version (the Chinese Pharmacopoeia council).
Chromatographic column: Aqucity UPLC
tMbEH C
18(2.1 × 100mm, 1.7 μ are m); Waters Aqucity UPLC
tMhSST3 (2.1 × 100mm, 1.8 μ are m); Aqucity UPLC
tMbEH SHIELD RP C
18(2.1 × 100mm, 1.7 μ are m); ThermoHypersil GOLD Aq (2.1 × 100mm, 1.9 μ are m); Agilent ZORBAX SB-C18 (2.1 × 100mm, the 1.8 μ different model chromatographic column such as m).
Acetonitrile: chromatographically pure (Merck company);
Methyl alcohol: analyze pure, chromatographically pure (Tianjin Concord Technology Co., Ltd.);
Ammoniacal liquor: analyze pure (Tianjin Fengchuan Chemical Reagent Science & Technology Co., Ltd.);
Water: ultrapure water.
2, the selection of test sample disposal route
Amount to sample weighting amount in recipe quantity ratio, preferably test sample disposal route is as follows: compound danshen dripping pills 20 balls (about 0.5g), accurately weighed being placed in 15ml graduated tube, adds 4% ammonia spirit 5ml, ultrasonic to dissolving.Cross the ODS pillar (80-100 order 1.0g, methyl alcohol 20ml, water 20ml activation) of 1.0cm internal diameter, with after 50ml washing by 100% methanol-eluted fractions to 10ml volumetric flask constant volume, shook up the organic filter membrane of 0.22 μ m, to obtain final product.
3, the selection of chromatographic condition
3.1, detecting wavelength selects
The conventional detection method of notoginsenoside constituents has ultraviolet detection, evaporative light-scattering to detect.Uv detection method is easy, mature and stable, the range of linearity is wide, instrument is universal, therefore preferred uv detection method.Notoginsenoside constituents all has uv absorption within the scope of ultraviolet end 200-210nm, and its maximum absorption wavelength is 203nm, and therefore the preferred 203nm of the present invention is the ultraviolet detection wavelength of notoginsenoside constituents.
3.2, mobile phase solution is selected:
In the conventional mobile phase of high performance liquid chromatography, organic phase is mostly chromatogram methyl alcohol or chromatogram acetonitrile, and notoginsenoside constituents detection wavelength is 203nm, under this wavelength condition, methyl alcohol, acetonitrile all exist end to absorb, test shows, the absorbance (approximately 0.15) of acetonitrile is far below methyl alcohol (approximately 1.0).For reducing the impact of solvent on sample analysis, the preferred acetonitrile-water of the present invention is flow phase system.
3.3, the investigation of chromatographic column:
Adopt respectively 1. Aqucity UPLC
tMbEH C
18(2.1 × 100mm, 1.7 μ are m); 2. Waters Aqucity UPLC
tMhSS T3 (2.1 × 100mm, 1.8 μ are m); 3. Aqucity UPLC
tMbEH SHIELD RP C
18(2.1 × 100mm, 1.7 μ are different chromatographic columns m); Detect wavelength: 203nm; Taking acetonitrile as mobile phase A; Taking aqueous solution as Mobile phase B, gradient elution ratio, with reference to enterprises data initial setting, in table 1, is carried out the investigation of separation case to 4 compositions of notoginsenoside class.Different chromatographic columns are investigated chromatogram and are seen Fig. 1～3.
As seen from the figure, 1. ginsenoside Rg in collection of illustrative plates of chromatographic column
1, Re do not reach completely and to separate, but have separately trend; The chromatographic peak number that 2. chromatographic column retains under the same terms is less; Chromatographic column is 3. better to the overall separation situation of sample, but ginsenoside Rg1 place is seemingly wrapped with little assorted peak, therefore preliminary project adopts chromatographic column 3. to carry out the investigation of eluent gradient ratio.
The investigation of 3.4 eluent gradient ratios
3.4.1 adopt 3. Aqucity UPLC of chromatographic column
tMbEH SHIELD RP C
18(2.1 × 100mm, 1.7 μ m), carry out the investigation of eluent gradient ratio.
First press gradient elution program list 1, use UPLC-PDA Detector to investigate the peak purity of four quantitative composition chromatographic peaks, No. 2 peak-to-peak purity can not meet the demands as a result, for ensureing that four quantitative composition chromatographic peaks all reach baseline separation, 27 kinds of different eluent gradient ratios are investigated altogether, typical case investigates chromatogram and sees Fig. 4, and corresponding gradient elution program is in table 2.As shown in Figure 4, in spectrogram the first half section time elongate, make each chromatographic peak peak shape short and stout, not sharp-pointed, and if the second half section launch completely, the holistic approach time will extend to about 50 minutes.
Table 1 gradient elution program 1
Table 2 gradient elution program 2
3.4.2 in view of above problem, use 1. Aqucity UPLC of chromatographic column instead
tMbEH C
18(2.1 × 100mm, 1.7 μ m) re-start the investigation of eluent gradient,
First, the gradient elution program in employing table 2 is investigated equally, and result peak shape makes moderate progress.On this basis, carry out further optimization, adjust altogether 8 kinds of different eluent gradient ratio conditions, result has tentatively been determined the condition of gradient elution showing in table 3, and under this condition, 4 notoginsenoside constituents have all reached baseline separation, and each chromatographic peak is effectively separated entirely, peak shape is all comparatively sharp-pointed upright, also can meet the requirement of finger-print, sees Fig. 5.
Table 3 gradient elution program 3
The methodology checking of the assay of test example two notoginsenosides
1, linearity
Precision takes Panax Notoginseng saponin R respectively
1reference substance, ginsenoside Rg
zreference substance, ginsenoside Re's reference substance, ginsenoside Rb
1reference substance is appropriate, dissolves and dilutes with methyl alcohol, makes containing Panax Notoginseng saponin R
1concentration is 0.04836mg/ml, ginsenoside Rg
1concentration is that 0.2404mg/ml, ginsenoside Re's concentration are the mixing reference substance solution that 0.0328mg/ml, ginsenoside Rb1's concentration are 0.2361mg/ml.Get each reference substance appropriate, accurately weighed after, add methyl alcohol and make every 1ml respectively containing Panax Notoginseng saponin R
1, ginsenoside Rg
1, ginsenoside Re, ginsenoside Rb
1each 0.2034,0.9192,0.12168, the solution of 0.9072mg, as storing solution.Above-mentioned storing solution is diluted to 1 times, 2 times, 4 times, 8 times, 16 times, 32 times successively, as each concentration standard curve solution, the accurate each 1 μ l of above-mentioned solution that draws, injection liquid chromatography, taking sample size, (μ is g) as horizontal ordinate, and peak area is ordinate, drawing standard curve.Result shows, Panax Notoginseng saponin R
1, ginsenoside Rg
1, ginsenoside Re, ginsenoside Rb
1good in 6.35625～203.4,28.725～919.2,3.8025～121.68,28.35～907.2 μ g scope internal linear relations respectively.In Table 4-7.
Table 4 Panax Notoginseng saponin R
1typical curve
Table 5 ginsenoside Rg
1typical curve
Table 6 ginsenoside Re typical curve
Table 7 ginsenoside Rb
1typical curve
2, precision test
Get same lot number (lot number: 111206) sample, get 1 part, precision takes 20 balls, analyzes continuous sample introduction 6 times, Panax Notoginseng saponin R in working sample by method described in embodiment mono-
1, ginsenoside Rg
1, ginsenoside Re, ginsenoside Rb
1peak area, recording peak area mean value is Panax Notoginseng saponin R
143003, the RSD of peak area is 0.98%; Ginsenoside Rg
1221149, the RSD of peak area is 0.57%; Ginsenoside Re 24461, and the RSD of peak area is 1.37%; Ginsenoside Rb
1137625, the RSD of peak area is 0.61%, meets the requirements, and the results are shown in Table 8.
Table 8 precision test
3, replica test
Get same lot number (lot number: 111206) sample, totally 6 parts, analyze by method described in embodiment mono-, the accurate need testing solution 1 μ l that draws, injection liquid chromatography, measures the each index components content of every duplicate samples, the results are shown in Table 9.
Table 9 replica test
4, stability test
Get same lot number (lot number: 111206) sample, get 1 part, precision takes 20 balls, analyze according to method described in embodiment mono-, respectively at 0,0.5,2,4,6.5,10,14,20 and 22 hour, Panax Notoginseng saponin R in working sample
1, ginsenoside Rg
1, ginsenoside Re, ginsenoside Rb
1peak area separately, it is 1.83%, 0.51%, 0.88%, 1.72% that the RSD that records peak area value is respectively, result shows that need testing solution measured stablely in 22 hours, the results are shown in Table 10.
Table 10 stability test
5, accuracy test
Get same lot number (lot number: 111206) sample, totally 6 parts, every part of 10 balls, accurately weighed, precision adds every 1ml containing Panax Notoginseng saponin R
10.05408mg, ginsenoside Rg
10.1645mg, ginsenoside Re 0.02524mg, ginsenoside Rb
1the solution 5ml of 0.2102mg, analyzes according to method described in embodiment mono-, measures calculate recovery rate.Result Panax Notoginseng saponin R
1average recovery rate is that 98.55%, RSD is 1.45%, ginsenoside Rg1's average recovery rate is that 95.95%, RSD is 0.71%, ginsenoside Re's average recovery rate is that 102.6%, RSD is 1.83%, ginsenoside Rb
1average recovery rate is that 96.81%, RSD is 1.10%.The results are shown in Table 11-14.
Table 11 Panax Notoginseng saponin R
1recovery test
Table 12 Panax Notoginseng saponin R g
1recovery test
Table 13 Panax Notoginseng saponin R e recovery test
Table 14 Panax Notoginseng saponin R b
1recovery test
6, serviceability test
6.1 different personnel test investigation
Getting lot number is 111208 batch samples, and by 3, laboratory, different tests personnel analyze by method described in embodiment mono-respectively, Panax Notoginseng saponin R in working sample
1, ginsenoside Rg
1, ginsenoside Re, ginsenoside Rb
1peak area separately, calculates, and the results are shown in Table 15.
The different personnel of table 15 measure notoginsenoside class result (mg/ ball)
6.2, different instrument tests are investigated
Use Aqucity UPLC
tMbEH C
18(2.1 × 100mm, 1.7 μ m), Lot No.0202320241 chromatographic column, adopt respectively Waters UPLC, Agilent1290UHPLC, tri-different instruments of SHIMADZU LC-30A UHPLC to compare method, be that 120104,120105,120,109 3 batch samples are analyzed to lot number respectively, the results are shown in Table 16, basic indifference between the different instruments of notoginsenoside constituents content.
The different instrument comparisons of table 16 content of the total saponins in radix notoginseng (mg/ ball)
6.3 different chromatographic column tests are investigated
Use respectively Aqucity UPLC
tMbEH C
18(2.1 × 100mm, 1.7 μ are m); Thermo Hypersil GOLD Aq (2.1 × 100mm, 1.9 μ are m); (2.1 × 100mm, 1.8 μ m) three different brands chromatographic columns analyze 120509,120510 batch samples Agilent ZORBAX SB-C18.The results are shown in Table 17, the certain difference existing because of separation case difference between the different chromatographic columns of notoginsenoside constituents content, the RSD of three different brands chromatographic columns of two batch samples is respectively 6.39%, 5.79%.
The different chromatographic column comparisons of table 17 content of the total saponins in radix notoginseng (mg/ ball)
Embodiment mono-
1, instrument, reagent
Instrument: Waters Ultra Performance Liquid Chromatography instrument, UPLC-PDA Detector; Agilent1290 Ultra Performance Liquid Chromatography instrument, UHPLC-DAD Detector; SHIMADZU Nexera LC-30A Ultra Performance Liquid Chromatography instrument, UHPLC-DADDetector.Totally three chromatographs.
Reference substance: Panax Notoginseng saponin R
1(lot number 110745-200415, for assay), ginsenoside Rb
1(lot number 110704-200318, for assay), ginsenoside Rg
1(lot number 110703-200424, for assay), ginsenoside Re's (lot number 110754-200421, for assay), all purchased from Nat'l Pharmaceutical & Biological Products Control Institute, put dry more than 12 hours use in phosphorus pentoxide vacuum drying apparatus.
Similarity evaluation software: Chinese chromatographic fingerprinting similarity evaluation system 2004A version, 2004B version (the Chinese Pharmacopoeia council)
Chromatographic column: Aqucity UPLC
tMbEH C
18(2.1 × 100mm, 1.7 μ m).
Acetonitrile: chromatographically pure (Merck company)
Methyl alcohol: analyze pure, chromatographically pure (Tianjin Concord Technology Co., Ltd.)
Ammoniacal liquor: analyze pure (Tianjin Fengchuan Chemical Reagent Science & Technology Co., Ltd.)
Water: ultrapure water
2, chromatographic condition
Detect wavelength: 203nm; Taking acetonitrile as mobile phase A; Taking aqueous solution as Mobile phase B, gradient elution, gradient is as follows:
3, the preparation of need testing solution: compound danshen dripping pills 20 balls (about 0.5g), accurately weighed being placed in 15ml graduated tube, adds 4% ammonia spirit 5ml, ultrasonic to dissolving.Cross the ODS pillar (80-100 order 1.0g, methyl alcohol 20ml, water 20ml activation) of 1.0cm internal diameter, with after 50ml washing by 100% methanol-eluted fractions to 10ml volumetric flask constant volume, shook up the organic filter membrane of 0.22 μ m, to obtain final product.
4, the preparation of reference substance solution: precision takes Panax Notoginseng saponin R respectively
1reference substance, ginsenoside Rg
1reference substance, ginsenoside Re's reference substance, ginsenoside Rb
1reference substance is appropriate, dissolves and dilutes with methyl alcohol, makes containing Panax Notoginseng saponin R
1concentration is 0.04836mg/ml, ginsenoside Rg
1concentration is that 0.2404mg/ml, ginsenoside Re's concentration are 0.0328mg/ml, ginsenoside Rb
1concentration is the mixing reference substance solution of 0.2361mg/ml.
5, the assay of notoginsenoside constituents: the accurate each 1 μ l of above-mentioned two kinds of solution that draws, adopt Ultra Performance Liquid Chromatography to analyze, measure the content of notoginsenoside constituents.
Get 3 batch samples and measure, Panax Notoginseng saponin R in every batch sample
1, ginsenoside Rg
1, ginsenoside Re, ginsenoside Rb
1content in table 18.
Table 18 sample size measurement result
6, the determining fingerprint pattern of notoginsenoside:
6.1, the preliminary of the total peak of finger-print determined
For the preliminary number of determining total peak, randomly draw compound Danshen Root medicinal extract and pseudo-ginseng each a collection of, test investigation.
6.1.1 be 0.1g by preparation prescription amount conversion compound Danshen Root medicinal extract sample weighting amount, operate by the preparation method of need testing solution described in embodiment mono-, obtain medicinal extract need testing solution.
6.1.2 get medicinal powder 2.5g in Backflow bottle, add distilled water 50ml, backflow 1.5h, filters, and adds 50ml distilled water in filter residue, backflow 1h, and merging filtrate, water is settled in 100ml volumetric flask, shakes up and obtain extract mother liquor.Precision measures in 2ml to 5ml volumetric flask, adds methanol constant volume to groove, shakes up, and crosses the organic filter membrane of 0.45 μ m, obtains pseudo-ginseng need testing solution.
6.1.3 the preparation of negative sample solution
According to prescription proportioning, get other two tastes medicinal materials except pseudo-ginseng, make negative sample according to technique, then operate according to the preparation method of need testing solution described in embodiment mono-, make negative sample solution.Accurate 1 μ l, the injection liquid chromatography drawn.
Above-mentioned three kinds of need testing solutions are pressed to chromatographic condition analysis described in embodiment mono-, record test sample chromatogram, and with compound danshen dripping pills and blank solvent chromatogram comparison (Fig. 6).Can be found out by comparison diagram, between chromatographic peak medicinal material-medicinal extract-dripping pill three of retention time before 20min, correspondence is all better, and the chromatographic peak of retention time after 20min can be divided into three groups, in I group medicinal material, all content is lower in medicinal extract and dripping pill for 2 chromatographic peaks; 2 chromatographic peaks content in dripping pill corresponding in II group medicinal material and medicinal extract is extremely low; III group is solvent peak.According to above analysis, in finger-print, terminate in that retention time is before 20min analysis time, and the collection of illustrative plates of measuring during according to the condition of investigation is tentatively determined 8 total peaks, as shown in Figure 7.
6.2, the preparation of reference substance solution
To have No. 2 peak ginsenoside Rgs of coneincone area maximum
1for with reference to peak.Precision takes ginsenoside Rg
1reference substance is appropriate, dissolves and dilutes with methyl alcohol, makes containing ginsenoside Rg
1concentration is the reference substance solution of 0.2404mg/ml.
6.3, determining of finger-print
Get described need testing solution, adopt Ultra Performance Liquid Chromatography to analyze, adopt Chinese chromatographic fingerprinting similarity evaluation system 2004A version to carry out matching to the finger-print of 34 batch samples, obtain reference fingerprint, see Fig. 8.
7, finger print measuring method is learned checking
7.1, precision test
Get same lot number (lot number: 111206) sample, get 1 part, precision takes 20 balls, analyzes continuous sample introduction 6 times by method described in embodiment mono-.In 6 duplicate samples, the total relative retention time at peak and the RSD of relative peak area ratio the results are shown in Table 19,20, taking No. 2 peaks as reference peak, the equal < 0.1% of relative retention time RSD at all the other 7 total peaks, relative peak area RSD is equal < 3.0% except No. 8 peaks, No. 8 peak-to-peak areas affect because of separation case, RSD more greatly 8.74%.
The total peak of table 19 relative retention time ratio precision test
The total peak of table 20 relative peak area ratio precision test
7.2, replica test
Get same lot number (lot number: 111206) sample, totally 6 parts, by " described in embodiment mono-, method is analyzed, the accurate need testing solution 1 μ l that draws, injection liquid chromatography, measures.In 6 duplicate samples, the total relative retention time at peak and the RSD of relative peak area ratio the results are shown in Table 21,22, taking No. 2 peaks as reference peak, the equal < 0.3% of relative retention time RSD at all the other 7 total peaks, relative peak area RSD is equal < 3.0% except 7, No. 8 peaks, 7, No. 8 peak-to-peak areas affect because of separation case, and RSD is respectively more greatly 8.08,10.12%.
The replica test of the total peak of table 21 relative retention time ratio
The total peak of table 22 relative peak area ratio replica test
7.3 stability test
Get same lot number (lot number: 111206) sample, get 1 part, precision takes 20 balls, analyze according to method described in embodiment mono-, respectively at 0,0.5,2,4,6.5,10,14,20 and 22 hour, measure.The total relative retention time at peak of different time points and the RSD% of relative peak area ratio the results are shown in Table 22,23, taking No. 2 peaks as reference peak, the equal < 0.3% of relative retention time RSD at all the other 7 total peaks, relative peak area RSD is equal < 4.0% except No. 8 peaks, No. 8 peak-to-peak areas affect because of separation case, and RSD is respectively more greatly 8.31%.
The stability test of the total peak of table 23 relative retention time ratio
Table 24 has peak relative peak area ratio stability test
Determining of 7.4 total peak compositions
For determining the composition at 8 total peaks in pseudo-ginseng reference fingerprint, adopt reference substance Comparison Method to carry out the chromatographic peak in sample qualitative, see Fig. 7, result determines that No. 6 peaks are ginsenoside Rd.
Embodiment bis-
1, instrument, reagent
Instrument: Waters Ultra Performance Liquid Chromatography instrument, UPLC-PDA Detector; Agilent1290 Ultra Performance Liquid Chromatography instrument, UHPLC-DAD Detector; SHIMADZU Nexera LC-30A Ultra Performance Liquid Chromatography instrument, UHPLC-DADDetector.Totally three chromatographs.
Reference substance: Panax Notoginseng saponin R
1(lot number 110745-200415, for assay), ginsenoside Rb
1(lot number 110704-200318, for assay), ginsenoside Rg
1(lot number 110703-200424, for assay), ginsenoside Re's (lot number 110754-200421, for assay), all purchased from Nat'l Pharmaceutical & Biological Products Control Institute, put dry more than 12 hours use in phosphorus pentoxide vacuum drying apparatus.
Similarity evaluation software: Chinese chromatographic fingerprinting similarity evaluation system 2004A version, 2004B version (the Chinese Pharmacopoeia council)
Chromatographic column: Aqucity UPLC
tMbEH C
18(2.1 × 100mm, 1.7 μ are m); Waters Aqucity UPLC
tMhSST3 (2.1 × 100mm, 1.8 μ are m); Aqucity UPLC
tMbEH SHIELD RP C
18(2.1 × 100mm, 1.7 μ are m); ThermoHypersil GOLD Aq (2.1 × 100mm, 1.9 μ are m); Agilent ZORBAX SB-C18 (2.1 × 100mm, m) totally 5 different model chromatographic columns of 1.8 μ.
Acetonitrile: chromatographically pure (Merck company)
Methyl alcohol: analyze pure, chromatographically pure (Tianjin Concord Technology Co., Ltd.)
Ammoniacal liquor: analyze pure (Tianjin Fengchuan Chemical Reagent Science & Technology Co., Ltd.)
Water: ultrapure water
2, chromatographic condition
Detect wavelength: 203nm; Taking acetonitrile as mobile phase A; Taking aqueous solution as Mobile phase B, gradient elution, gradient is as follows:
3, the preparation of need testing solution: compound danshen dripping pills 20 balls (about 0.5g), accurately weighed being placed in 15ml graduated tube, adds 4% ammonia spirit 5ml, ultrasonic to dissolving.Cross the ODS pillar (80-100 order 1.0g, methyl alcohol 20ml, water 20ml activation) of 1.0cm internal diameter, with after 50ml washing by 100% methanol-eluted fractions to 10ml volumetric flask constant volume, shook up the organic filter membrane of 0.22 μ m, to obtain final product.
4, the preparation of reference substance solution: precision takes Panax Notoginseng saponin R respectively
1reference substance, ginsenoside Rg
1reference substance, ginsenoside Re's reference substance, ginsenoside Rb
1reference substance is appropriate, dissolves and dilutes with methyl alcohol, makes containing Panax Notoginseng saponin R
1concentration is that 0.04836mg/ml, ginsenoside Rg1's concentration are that 0.2404mg/ml, ginsenoside Re's concentration are 0.0328mg/ml, ginsenoside Rb
1concentration is the mixing reference substance solution of 0.2361mg/ml.
5, the assay of notoginsenoside: the accurate each 1 μ l of above-mentioned two kinds of solution that draws, adopt Ultra Performance Liquid Chromatography to analyze, measure the content of notoginsenoside constituents.
6, the determining fingerprint pattern of notoginsenoside: get described need testing solution, adopt Ultra Performance Liquid Chromatography to analyze, adopt Chinese chromatographic fingerprinting similarity evaluation system 2004A version to carry out matching to the finger-print of 36 batch samples, in collection of illustrative plates processing procedure, find that lot number is that in 20080710,090509 2 batch samples, No. 8 peaks are too little, can not meet by No. 2 peak ginsenoside Rgs
1peak area 5% is the integral condition of smallest peaks area.
6.1 different instrument tests are investigated
Use Aqucity UPLC
tMbEH C
18(2.1 × 100mm, 1.7 μ m), Lot No.0202320241 chromatographic column, adopting respectively Waters UPLC, Agilent 1290 UHPLC, tri-different instruments of SHIMADZU LC-30A UHPLC to compare method, is that 120104,120105,120,109 3 batch samples are analyzed to lot number respectively.Table 25 shows, the basic indifference of similarity of finger-print, but there is notable difference (see figure 9) in No. 4 chromatographic peak peak shapes and separation case.
The different instrument comparisons of table 25 pseudo-ginseng fingerprint similarity
6.2 different chromatographic column tests are investigated
Use respectively Aqucity UPLC
tMbEH C
18(2.1 × 100mm, 1.7 μ are m); Thermo Hypersil GOLD Aq (2.1 × 100mm, 1.9 μ are m); (2.1 × 100mm, 1.8 μ m) three different brands chromatographic columns analyze 120509,120510 batch samples Agilent ZORBAX SB-C18.Table 26 shows, the basic indifference of similarity of finger-print exists certain deviation but AgilentZORBAX SB-C18 chromatogram column analysis obtains the position at No. 4 peaks in chromatogram compared with other two chromatographic columns, sees Figure 10.
The different chromatographic column comparisons of table 26 pseudo-ginseng fingerprint similarity
6.3, different experiments chamber is investigated
Three groups of 2 different experiments chambers experimenter, 120413,120501,120,502 3 batch samples are analyzed respectively, be the results are shown in Table 27.The similarity RSD that table 27 shows notoginsenoside constituents finger-print in three batch samples is all in 3%, but the position at No. 4 peaks has necessarily and departs from.Figure 11 is shown in by two three groups of different experiments chambers experimenter's fingerprint analysis collection of illustrative plates.
The different chromatographic column comparisons of table 27 pseudo-ginseng fingerprint similarity
6.4, determining of pseudo-ginseng reference fingerprint
Comprehensive above different instruments, different chromatographic columns etc. are investigated result, in 8 total peaks finding tentatively to draft, 4,7, No. 8 peaks are not all single components, and No. 4 peak changes greatly because condition difference goes out peak position relatively, 7, No. 8 peak-to-peak areas are less, and in 36 batch samples, find that 2 batches of existence lose peak phenomenon, determine that total peak number is 5 peaks therefore finally give up 4,7, No. 8 peaks, liquid phase writing time is 20min, adopt Chinese chromatographic fingerprinting similarity evaluation system 2004 A versions to carry out matching to the finger-print of 36 batch samples, obtain reference fingerprint, see Figure 12.
7, finger print measuring method is learned checking
7.1, precision test
Get same lot number (lot number: 111206) sample, get 1 part, precision takes 20 balls, analyzes continuous sample introduction 6 times by method described in embodiment mono-.In 6 duplicate samples, the total relative retention time at peak and the RSD of relative peak area ratio the results are shown in Table 28,29, with the ginsenoside Rg of peak area maximum
1for reference peak (S peak in Figure 12), the equal < 0.1% of relative retention time RSD at all the other 4 total peaks, the equal < 3.0% of relative peak area RSD4.
The total peak of table 28 relative retention time ratio precision test
The total peak of table 29 relative peak area ratio precision test
7.2, replica test
Get same lot number (lot number: 111206) sample, totally 6 parts, by " described in embodiment mono-, method is analyzed, the accurate need testing solution 1 μ l that draws, injection liquid chromatography, measures.In 6 duplicate samples, the total relative retention time at peak and the RSD of relative peak area ratio the results are shown in Table 30,31, with the ginsenoside Rg of peak area maximum
1for reference peak (S peak in Figure 12), the equal < 0.3% of relative retention time RSD at all the other 4 total peaks, the equal < 3.0% of relative peak area RSD.
The replica test of the total peak of table 30 relative retention time ratio
The total peak of table 31 relative peak area ratio replica test
7.3 stability test
Get same lot number (lot number: 111206) sample, get 1 part, precision takes 20 balls, analyze according to method described in embodiment mono-, respectively at 0,0.5,2,4,6.5,10,14,20 and 22 hour, measure.The total relative retention time at peak of different time points and the RSD% of relative peak area ratio the results are shown in Table 32,33, with the ginsenoside Rg of peak area maximum
1for reference peak (S peak in Figure 12), the equal < 0.3% of relative retention time RSD at all the other 4 total peaks, the equal < 4.0% of relative peak area RSD.
The stability test of the total peak of table 32 relative retention time ratio
Table 33 has peak relative peak area ratio stability test
Determining of 7.4 total peak compositions
For determining the composition at 5 total peaks in pseudo-ginseng reference fingerprint, adopt reference substance Comparison Method to carry out the chromatographic peak in sample qualitative, see Figure 13, result determines that No. 4 peaks are ginsenoside Rd.
Claims (11)
1. an analytical approach for notoginsenoside constituents, comprises and it is characterized in that assay and the determining fingerprint pattern of notoginsenoside, comprises the following steps:
(1) preparation of need testing solution: get compound danshen dripping pills, accurately weighed, add ammonia spirit, ultrasonic; Cross ODS post, water, methanol-eluted fractions successively, collects meoh eluate, filters, and to obtain final product;
(2) preparation of reference substance solution: precision takes Panax Notoginseng saponin R respectively
1reference substance, ginsenoside Rg
1reference substance, ginsenoside Re's reference substance, ginsenoside Rb
1reference substance is appropriate, dissolves and dilutes with methyl alcohol, to obtain final product;
(3) assay of notoginsenoside: get described need testing solution and reference substance solution, adopt Ultra Performance Liquid Chromatography to analyze, measure the content of notoginsenoside;
(4) determining fingerprint pattern of notoginsenoside: get described need testing solution, adopt Ultra Performance Liquid Chromatography to analyze, obtain finger-print.
2. method according to claim 1, is characterized in that, being prepared as of described need testing solution: get compound danshen dripping pills 10-30 ball, accurately weighed being placed in 15ml graduated tube, adds the ammonia spirit 1-10ml of 0.5%-10%, ultrasonic to dissolving; Cross the ODS pillar of 1.0cm internal diameter, with after 50ml washing by 100% methanol-eluted fractions to 10ml volumetric flask constant volume, shook up the organic filter membrane of 0.22 μ m, to obtain final product.
3. method according to claim 1, is characterized in that, being prepared as of described reference substance solution: precision takes Panax Notoginseng saponin R respectively
1reference substance, ginsenoside Rg
1reference substance, ginsenoside Re's reference substance, ginsenoside Rb
1reference substance is appropriate, dissolves and dilutes with methyl alcohol, makes containing Panax Notoginseng saponin R
1concentration is 0.01-0.1mg/ml, ginsenoside Rg
1concentration is that 0.1-0.5mg/ml, ginsenoside Re's concentration are 0.01-0.06mg/ml, ginsenoside Rb
1concentration is the mixing reference substance solution of 0.1-0.5mg/ml.
4. method according to claim 1, is characterized in that, the chromatographic condition of described Ultra Performance Liquid Chromatography is: chromatographic column is C18 chromatographic column, and mobile phase A is acetonitrile or methyl alcohol mutually, and Mobile phase B is water mutually, gradient elution; UV-detector, detection wavelength is 200-210nm.
5. method according to claim 4, is characterized in that, described mobile phase A is acetonitrile mutually, and the gradient of described gradient elution is:
6. method according to claim 4, is characterized in that, described detection wavelength is 203nm.
7. the finger-print of the notoginsenoside obtaining by claim 1-6 any one.
8. finger-print according to claim 7, is characterized in that, described finger-print is made up of 8 total fingerprint peakses.
9. finger-print according to claim 7, is characterized in that, described finger-print is made up of 5 total fingerprint peakses.
10. finger-print according to claim 9, is characterized in that, described 5 total peaks are respectively with respect to relative retention time and the relative peak area at reference peak:
No. 1 peak, relative retention time is 0.77min, relative peak area is 0.19;
S peak, relative retention time is 1.00min, relative peak area 1;
No. 2 peaks, relative retention time is 1.04min, relative peak area 0.11;
No. 3 peaks, relative retention time is 1.93min, relative peak area 0.64;
No. 4 peaks, relative retention time is 2.46min, relative peak area 0.13;
Compared with S peak, the relative retention time at all the other each peaks and relative peak area all should setting ± 5% in.
11. according to the finger-print described in claim 7-10 any one, it is characterized in that, sample and described finger-print are through Chinese chromatographic fingerprinting similarity evaluation system-computed, and similarity is between 0.8-1.0.
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| CN107764926A (en) * | 2017-09-27 | 2018-03-06 | 哈尔滨珍宝制药有限公司 | The finger-print and its method for building up of a kind of extract of panax notoginseng saponins |
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| CN110274978A (en) * | 2018-03-13 | 2019-09-24 | 天士力医药集团股份有限公司 | The more saponin constituent content assaying methods of Radix Notoginseng in a kind of QISHEN YIQI DIWAN |
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| CN112285239A (en) * | 2020-10-24 | 2021-01-29 | 云南农业大学 | HPLC method for detecting notoginsen leaf saponin |
| CN113960218A (en) * | 2021-11-23 | 2022-01-21 | 天津师范大学 | Method for rapidly and quantitatively detecting notoginsenoside |
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| CN110274978A (en) * | 2018-03-13 | 2019-09-24 | 天士力医药集团股份有限公司 | The more saponin constituent content assaying methods of Radix Notoginseng in a kind of QISHEN YIQI DIWAN |
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| CN112213404A (en) * | 2019-07-12 | 2021-01-12 | 云南希陶绿色药业股份有限公司 | Fingerprint construction and content determination method of pseudo-ginseng medicinal liquor |
| CN112285239A (en) * | 2020-10-24 | 2021-01-29 | 云南农业大学 | HPLC method for detecting notoginsen leaf saponin |
| CN113960218A (en) * | 2021-11-23 | 2022-01-21 | 天津师范大学 | Method for rapidly and quantitatively detecting notoginsenoside |
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Address after: 300410 Tianjin city Beichen District Huaihe road and road intersection Dingjiang tianzhijiao Park forensic Center for Intellectual Property Department Patentee after: Tasly Pharmaceutical Group Limited by Share Ltd Address before: 300410 Tianjin city Beichen District Huaihe road and road intersection Dingjiang tianzhijiao Park forensic Center for Intellectual Property Department Patentee before: Tasly Pharmaceutical Group Co., Ltd. |