CN102914598B - Quality testing method for Pien Tze Huang and medicinal materials thereof - Google Patents
Quality testing method for Pien Tze Huang and medicinal materials thereof Download PDFInfo
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Abstract
The invention provides a quality testing method for Panax notoginseng in Pien Tze Huang and further provides a quality testing method for Pien Tze Huang. Extraction of Panax notoginseng is confirmed, chromatographic conditions are optimized, and a liquid chromatography reference fingerprint for Panax notoginseng and Pien Tze Huang is established. The quality of Pien Tze Huang can be more effectively monitored and evaluated by means of the established reference fingerprint and mass spectrometry, a Pien Tze Huang quality control system is more complete, and stability and controllability in internal quality of Pien Tze Huang can be reflected overall.
Description
Technical field
The present invention relates to the quality determining method of Chinese crude drug, particularly the quality determining method of Pien Tze Huang crude drug and Pien Tze Huang.
Background technology
The quality control standard of Pien Tze Huang is recorded in the 18 WS of Ministry of Public Health's standard Traditional Chinese medicine historical preparation
3-B-3381-98, only records the thin-layer chromatography of two reactions and pseudo-ginseng and differentiates project.Pien Tze Huang is by the refining Chinese traditional compound medicine forming of the multiple rare traditional Chinese medicines such as pseudo-ginseng, cow-bezoar, snake gall, Moschus, and manufacturing condition is national maintaining secrecy.Chen Haibin etc. differentiate and improve the thin layer of pseudo-ginseng in Pien Tze Huang, increased the thin-layer chromatography of cow-bezoar and snake gall is differentiated, utilize the content of gas chromatography determination muskone simultaneously; The employing Gradient Elution methods such as Lai Yanjin have been measured Panax Notoginseng saponin R simultaneously
1, ginsenoside Rg
1, ginsenoside Rb
1and the content of four kinds of compositions of natrium taurocholicum; In addition, also having some correlative studys, is mainly to measure certain one-component in Pien Tze Huang, as respectively with HPLC and determined by ultraviolet spectrophotometry cholerythrin, utilize gas Chromatographic Determination muskone etc.And Pien Tze Huang is refined the Chinese traditional compound medicine forming by multiple rare traditional Chinese medicines such as pseudo-ginseng, cow-bezoar, snake gall, Moschus, its composition is quite complicated, comprises a hundred or so kind of chemical composition, and its drug effect is the result of each composition overall coordination.Therefore, only use limited chemical reference substance, measure a few chemical composition or the large chemical composition of concentration in medicine, effective chemical composition in authentic representative medicine not necessarily, and the little chemical composition of other chemical composition and concentration also likely has a certain impact to curative effect.Qualitative or the quantitative target of single or a small amount of chemical composition, be difficult to guarantee the steady quality of preparation, need to further develop control method and the means of Pien Tze Huang quality, set up science more, perfect quality standard, meet international drug quality and control requirement, guarantee Pien Tze Huang steady quality, make product have more international market and unexpectedly strive power.
Traditional Chinese medicine fingerprint refers to that take the chemical constitution study of system and pharmaceutical research is basis, certain obtaining through chemical assay or identified for genes or certain place of production Chinese medicine and preparation thereof common, there is specific component colony (certain class or number constituents) characteristic spectrum or image.The base attribute of traditional Chinese medicine fingerprint is globality and ambiguity, requires exclusive, stable, practically, has the features such as systematicness, characteristic and stability.Finger-print is tool qualitative (by characteristic peak group, peak position) function both, quantitative (peak height, peak area) function of tool again, can control drug quality to a certain extent more comprehensively, simultaneously by the finger-print of crude drug and finished product is controlled, the consistency problem between can substantially solving batch.
Summary of the invention
The object of the invention is to provide the quality determining method of pseudo-ginseng in a kind of Pien Tze Huang; The object of the invention is also to provide a kind of quality determining method of Pien Tze Huang.
The present invention seeks to be achieved through the following technical solutions.
1, the quality determining method of pseudo-ginseng in Pien Tze Huang of the present invention, comprises following fingerprint atlas detection method:
According to high performance liquid chromatography: chromatographic condition and system suitability: select Welch XB-C18 chromatographic column (250mm * 4.6mm, 5 μ m), take octadecylsilane chemically bonded silica as filling agent, take acetonitrile and water carries out gradient elution as mobile phase, gradient elution program is as follows: 0～40 minute 22% acetonitrile, flow velocity 0.5mL/min, 40～50 minutes 22～30% acetonitriles, flow velocity 0.5mL/min, 50～80 minutes 30～55% acetonitriles, flow velocity 0.5mL/min, 80～95 minutes 55% acetonitriles, flow velocity 0.5mL/min, 95～115 minutes 55～90% acetonitriles, flow velocity 0.8mL/min, 115～135 minutes 90% acetonitriles, flow velocity 0.8mL/min, 135～140 minutes 90～22% acetonitriles, flow velocity 0.5mL/min, column temperature is 25 ℃, detection wavelength is 203nm,
Need testing solution preparation: get pseudo-ginseng powder 1～10 weight portion, accurately weighed, in tool plug conical flask, accurately add 50～200 parts by volume 70% ethanolic solution (V ethanol: V water=7: 3), weigh, placement is spent the night; Putting 80 ℃～90 ℃ water-baths refluxes 1～3 hour, room temperature to be chilled to, weigh again, with 70% ethanolic solution, supply the weight of less loss, shake up, filter, accurately pipette 25～100 parts by volume filtrates and be placed in beaker, 100 ℃ of water-baths top is concentrated into dry, uses 70% dissolve with ethanol solution, be transferred in 10 parts by volume volumetric flasks constant volume; With 0.45 μ m pin type membrane filtration to 2 parts by volume sample bottle, obtain pseudo-ginseng need testing solution, standby.
Reference substance solution preparation: accurately take respectively reference substance Panax Notoginseng saponin R
10.00569g, ginsenoside Rg
10.00896g, Rb
10.00809g, Re0.00103g, Rg
20.00605g, Rh
10.00409g, Rd0.00787g, Rf0.01180g, Rc0.00619g, add methyl alcohol and dissolve constant volume 25mL, makes reference substance mixed solution.
Determination method: draw respectively need testing solution and reference substance mixed solution 0.005 parts by volume, in injection liquid chromatography, record 170 minutes chromatograms, maximum, the more stable total peak Rg1 of the peak area of usining is as internal standard compound is as object of reference, using its retention time and peak area respectively as 1, calculate each total peak relative retention time; Test sample finger-print and standard finger-print (seeing accompanying drawing 1) similarity is 1;
Gained finger-print should have 23 total peaks, be respectively 1-12,14-19,23,26,28,29, No. 32 peaks, the relative retention time at total peak is respectively: 27.125,37.341,39.333,57.772,63.659,64.751,65.441,66.391,67.619,68.662,69.171,69.773,71.53,72.633,73.478,81.212,83.71,85.151,91.732,104.717,109.83,110.29,113.46.
Relative peak area ratio is respectively: 0.199,1.000,0.118,0.006,0.020,0.021,0.006,0.670,0.038,0.004,0.026,0.047,0.175,0.007,0.029,0.006,0.006,0.013,0.012,0.004,0.0019,0.004,0.009.
In Pien Tze Huang of the present invention, the quality determining method of pseudo-ginseng, also comprises the steps:
According to liquid phase chromatogram condition as mentioned above, carry out LC-MS (LC-MS/TOF) analysis, according to the position in the corresponding numbering in each peak, chromatogram and corresponding mass spectrogram, infer each peak chemical formula, mass spectrum condition is: atomizer pressure :-35psi, dry gas flow velocity: 9.0L/min, baking temperature: 325 ℃;
The chemical formula that each total peak is corresponding is respectively: No. 1-3, No. 8,11-12 peak and be respectively for No. 14 Panax Notoginseng saponin R
1, ginsenoside Rg
1, Rb
1, Re, Rg
2, Rh
1, Rd; No. 4 peaks are C
41h
70o
13; No. 5 peaks are C
59h
100o
27; No. 6 peaks are C
59h
100o
27; No. 7 peaks are C
42h
72o
14and C
54h
90o
23; No. 9 peaks are C
41h
70o
13; No. 10 peaks are C
53h
90o
22; No. 15 peaks are C
36h
62o
9; No. 16 peaks are C
48h
82o
18; No. 17 peaks are C
42h
70o
13; No. 18 peaks are C
36h
60o
8; No. 19 peaks are C
36h
60o
8; No. 23 peaks are C
25h
8o
18and C
21h
8o
17; No. 26 peaks are C
18h
32o
3; No. 28 peaks are C
29h
12o
22; No. 29 peaks are C
15h
21o
2; No. 32 peaks are C
29h
14o
25.
2, the quality determining method of Pien Tze Huang of the present invention, comprises that following finger-print detects:
According to high performance liquid chromatography: chromatographic condition and system suitability: select Welch XB-C18 chromatographic column (250mm * 4.6mm, 5 μ m), take octadecylsilane chemically bonded silica as filling agent, take acetonitrile and water carries out gradient elution as mobile phase, gradient elution program is as follows: 0～40 minute 22% acetonitrile, flow velocity 0.5mL/min, 40～50 minutes 22～30% acetonitriles, flow velocity 0.5mL/min, 50～80 minutes 30～55% acetonitriles, flow velocity 0.5mL/min, 80～95 minutes 55% acetonitriles, flow velocity 0.5mL/min, 95～115 minutes 55～90% acetonitriles, flow velocity 0.8mL/min, 115～135 minutes 90% acetonitriles, flow velocity 0.8mL/min, 135～140 minutes 90～22% acetonitriles, flow velocity 0.5mL/min, column temperature is 25 ℃, detection wavelength is 203nm,
Need testing solution preparation: get commercially available Pien Tze Huang (lozenge) 1～10 weight portion, through mortar porphyrize, sieve No. 5 or 80 orders, accurately weighed, be placed in tool plug conical flask, add 50～200 parts by volume 70% ethanolic solution (V
ethanol: V
water=7: 3), weigh, placement is spent the night; Putting 80 ℃～90 ℃ water-baths refluxes 1～3 hour, room temperature to be chilled to, weigh again, with 70% ethanolic solution, supply the weight of less loss, shake up, filter, accurately pipette 25～100 parts by volume filtrates and be placed in beaker, 100 ℃ of water-baths top is concentrated into dry, uses 70% dissolve with ethanol solution, be transferred in 10 parts by volume volumetric flasks constant volume; With 0.45 μ m pin type membrane filtration to 2 parts by volume sample bottle, standby.
Reference substance solution preparation: accurately take respectively reference substance Panax Notoginseng saponin R
10.00569g, ginsenoside Rg
10.00896g, Rb
10.00809g, Re0.00103g, Rg
20.00605g, Rh
10.00409g, Rd0.00787g, Rf0.01180g, Rc0.00619g, add methyl alcohol and dissolve constant volume 25mL, makes reference substance mixed solution.
Determination method: draw respectively need testing solution and reference substance mixed solution 0.005 parts by volume, in injection liquid chromatography, record 170 minutes chromatograms, maximum, the more stable total peak Rg1 of the peak area of usining is as internal standard compound is as object of reference, using its retention time and peak area respectively as 1, calculate each total peak relative retention time; Test sample finger-print and standard finger-print (seeing accompanying drawing 3) similarity is greater than 0.998.
Gained finger-print should have 16 total peaks, total peak is respectively 1-9,11,12,14-16,19 and No. 27 peaks, and the relative retention time at total peak is respectively: 27.557,37.956,39.968,57.919,63.809,64.917,65.613,66.549,67.785,69.34,69.948,71.733,72.821,73.697,85.395,105.602.
Relative peak area ratio is respectively: 0.204,1.000,0.116,0.006,0.020,0.021,0.006,0.677,0.040,0.026,0.050,0.183,0.015,0.033,0.012,0.013.
The quality determining method of Pien Tze Huang finished product of the present invention, also comprises the steps:
According to described liquid phase chromatogram condition, carry out LC-MS (LC-MS/TOF) analysis, according to the position in the corresponding numbering in each peak, chromatogram and corresponding mass spectrogram, infer each peak chemical formula, mass spectrum condition is: atomizer pressure (Nebulizer)-35psi, dry gas flow velocity (Dry Gas)-9.0L/min ,-325 ℃ of baking temperatures (Dry Temp).
The chemical formula that each total peak is corresponding is respectively: No. 1-3, No. 8,11-12 peak and No. 14 peaks are respectively Panax Notoginseng saponin Rs
1, ginsenoside Rg
1, Rb
1, Re, Rg
2, Rh
1, R
d; No. 4 peaks are C
41h
70o
13; No. 5 peaks are C
59h
100o
27; No. 6 peaks are C
59h
100o
27; No. 7 peaks are C
42h
72o
14or C
54h
90o
23; No. 9 peaks are C
41h
70o
13; No. 15 peaks are C
36h
62o
9; No. 16 peaks are C
48h
82o
18; No. 19 peaks are C
36h
60o
8; No. 27 peaks are C
18h
32o
3.
Pien Tze Huang of the present invention can be commercially available, by Zhongzhou Pianziguang Pharmaceutical Industry Co., Ltd., is manufactured and is sold.
The unit corresponding relation of weight portion of the present invention and parts by volume is g/ml.
Quality determining method of the present invention has been confirmed the extracting method of pseudo-ginseng, optimized chromatographic condition, set up the liquid chromatography reference fingerprint of the middle product of pseudo-ginseng and Pien Tze Huang and Pien Tze Huang finished product, determined that the more stable total peak of relative retention time and chromatographic peak is respectively 23,18 and 16 peaks, similarity is respectively 1, all > 0.981 and equal > 0.998, and the relative standard deviation of the total area at total peak is respectively 1.24%, 3.26% and 1.64%.Rely on the reference fingerprint connexus analysis of spectrum of setting up can more effectively monitor and evaluate the quality of Pien Tze Huang, make Pien Tze Huang system of quality control more complete, can reflect the stable, controlled of Pien Tze Huang inherent quality from integral body.
Accompanying drawing explanation
Fig. 1 Pien Tze Huang pseudo-ginseng standard finger-print
Fig. 2 Pien Tze Huang finished product standard finger-print
Following experimental example and embodiment are used for further illustrating but are not limited to the present invention.
The selection of experimental example 1 pseudo-ginseng extracting method
1, extract tentatively choosing of solvent
Get 0.6g Radix Notoginseng powder (crossing sieve No. 4,65 orders, particle diameter 250 ± 9.9 μ m), accurately weighed, first attempt first with 30mL ether, dividing 3 ultrasonic extractions, Filter paper filtering, residue volatilizes after ether solvent, then divides 3 ultrasonic extractions, Filter paper filtering with 60mL methyl alcohol or 70% methyl alcohol, filtrate decompression is concentrated into dry, with coordinative solvent, dissolves, and constant volume is in 25mL volumetric flask, filter (0.45 μ m pin type filter membrane) to 2mL sample bottle, accurately draw 5 μ L, injection liquid chromatography, detects.Chromatographic condition, with reference to Chinese Pharmacopoeia, slightly makes an amendment, and column temperature is 25 ℃, and flow velocity is 1.0mL/min, and detection wavelength is 203nm, and acetonitrile and water are mobile phase, and chromatographic condition I program is as follows:
Testing result is in Table 1.
Table 1 step is extracted the comparison (ultrasonic extraction-reduced pressure concentration) of extracting with two steps
As can be seen from Table 1, directly with methyl alcohol, extract and first by extracted by ether, extract with methyl alcohol again, the peak area at 6 total peaks is more or less the same; Directly with 70% methyl alcohol, extract and first by extracted by ether, extract with 70% methyl alcohol again, it is also little that the peak area at 6 total peaks differs.Therefore in the leaching process of pseudo-ginseng, can save this step of extracted by ether.
Use respectively 60mL methyl alcohol, methanol-water (70%), water, alcohol-water (70%) minute three ultrasonic extraction Radix Notoginseng powder (0.6000g), Filter paper filtering, filtrate decompression is concentrated into dry, with coordinative solvent, dissolve, constant volume 25mL, filters (0.45 μ m pin type filter membrane) and, to sample bottle, carries out liquid-phase chromatographic analysis, chromatographic condition is the same, and testing result is in Table 2.
Table 2 extracts the initial option (ultrasonic extraction-reduced pressure concentration) of solvent
As can be seen from Table 2, the total area at 6 peaks that methyl alcohol extracts is minimum, show that its extraction ratio is minimum, and the total area that water extraction is got is maximum, illustrates that its extraction ratio is maximum, and this is consistent with the result of table 3.The extract of methyl alcohol, 70% methyl alcohol and 70% ethanol has 6 total peaks, and water extract only has 5 total peaks, has lacked unknown peak No. 6, but many No. 0 unknown peaks.In addition, by table 2, can obviously be found out, in water extract, Re and Rd relative content are higher, and Rb
1relative content lower.According to data analysis, the extracted amount of 70% methyl alcohol and 70% ethanol, totally the former is bigger for each peak area, between them the RSD of peak area except the RSD of Re be 2.73%, remaining RSD is all less than 2%.Illustrate, the extraction effect of 70% methyl alcohol and 70% ethanol is suitable.Because the toxicity of methyl alcohol is larger than ethanol, therefore select 70% ethanolic solution as extraction agent.
Table 3 extracts the relation between solvent and extracted amount
2, the selection of extracting method
Main ultrasonic extraction and two kinds of methods of refluxing extraction investigated.Get 0.6g Radix Notoginseng powder, accurately weighed, add 70% ethanolic solution, soaked overnight, refluxing extraction (85 ℃) or ultrasonic extraction 2 hours, Filter paper filtering is to 100mL beaker, filtrate is concentrated into dry in water-bath (60 ℃), uses 70% dissolve with ethanol solution, constant volume 25mL.Filter (0.45 μ m pin type filter membrane) and, to sample bottle, carry out liquid-phase chromatographic analysis, chromatographic condition is with reference to Chinese Pharmacopoeia, slightly make an amendment, column temperature is 25 ℃, and detection wavelength is 203nm, flow velocity is 1.2mL/min, and acetonitrile and water are mobile phase, and chromatographic condition II program is as follows:
Test result is in Table 4.
The selection of table 4 extracting method
As can be seen from Table 4, the difference of refluxing extraction and ultrasonic extraction is not very large, what but generally select due to ultrasonic extraction is ultrasonic cleaning machine, the frequency of different ultrasonic cleaning machines has difference, and frequency can not regulate again, and ultrasonic meeting raises water temperature, temperature is wayward, for batch quantity analysis, inconvenient operation, therefore extracting method is selected refluxing extraction.
3, the selection of method for concentration
Main investigation water-bath is concentrated extracts two kinds of methods of filtrate with reduced pressure concentration.Accurately take 0.6g Radix Notoginseng powder, add 70% ethanolic solution, soaked overnight, refluxing extraction (85 ℃) is extracted 2 hours, and Filter paper filtering, concentrates in water-bath (60 ℃) or reduced pressure concentration (50 ℃) is extremely done, and uses 70% dissolve with ethanol solution, constant volume 25mL.Filter by (0.45 μ m pin type filter membrane), carry out liquid-phase chromatographic analysis, chromatographic condition: column temperature is 25 ℃, detection wavelength is 203nm, and flow velocity is 1.2mL/min, and acetonitrile and water are mobile phase, adopts chromatographic condition II program wash-out.Measurement result is in Table 5.
The selection of table 5 method for concentration
As can be seen from Table 5, the concentrated difference of reduced pressure concentration and water-bath is not very large, considers that water-bath is concentrated to be suitable for batch quantity analysis, therefore select water-bath concentrated.
4, the selection of reflux temperature
Extraction effect while investigating respectively reflux temperature and be 80 ℃ and 90 ℃.Get respectively 1.0g Radix Notoginseng powder, accurately weighed, in 250mL tool plug conical flask, accurately add 50.00mL70% ethanolic solution (V
ethanol: V
water=7: 3), weigh, placement is spent the night.Putting 80 ℃ or 90 ℃ of water-baths refluxes 2 hours, room temperature to be chilled to, weigh again, with 70% ethanolic solution, supply the weight of less loss, shake up, filter, accurately pipette 25.00mL filtrate and be placed in 50mL beaker, 100 ℃ of water-baths top is concentrated into dry, uses 70% dissolve with ethanol solution, be transferred in 10mL volumetric flask constant volume.With 0.45 μ m pin type membrane filtration, to 2mL sample bottle, carry out liquid-phase chromatographic analysis, chromatographic condition: column temperature is 25 ℃, detection wavelength is 203nm, and acetonitrile and water are mobile phase, and chromatographic condition III program is as follows:
Test result is in Table 6.
The selection of table 6 reflux temperature (refluxing 2 hours)
As can be seen from Table 6,90 ℃ of backflows are compared with 80 ℃ of backflows, and difference is not very large, considers that temperature low power consuming is few, therefore selected 80 ℃ is refluxing extraction temperature.
5, the selection of return time
Investigate respectively return time and be the extraction effect of 1 hour, 2 hours and 3 hours.Chromatographic condition: column temperature is 25 ℃, detection wavelength is 203nm, acetonitrile and water are mobile phase, adopt chromatographic condition III program wash-out.Test result is in Table 7.
The selection of table 7 return time (reflux temperature is 80 ℃)
As can be seen from Table 7, along with the prolongation of return time, every milligram of peak area and the total peak area of 13 main peaks increase gradually, illustrate that return time has certain influence to extraction efficiency.When extraction time is increased to 2h by 1h, the total area of its extract has increased by 570, then while being increased to 3h, the total area has only increased by 214, and the return time that shows to increase again 1h is not remarkable on the impact of extraction efficiency, and therefore, return time is tentatively defined as 2 hours.
According to above-mentioned experimental result and discussion, the extracting method of pseudo-ginseng is tentatively defined as: alcohol-water (70%) is extraction solvent, soaked overnight, and refluxing extraction (80 ℃ of micro-boiling) 2 hours, water-bath is concentrated.
Determining of experimental example 2 pseudo-ginseng fingerprint analysis methods
1. instrument
Agilent 1200 high performance liquid chromatographs comprise: vacuum degassing machine, quaternary gradient pump, automatic sampler, UV-detector.
LC-MS instrument: Agilent 1200 high performance liquid chromatographs (diode array detector); 6320 ion trap mass spectrometers; ESI electric spray ion source.For unknown material or without the Components identification of tester, and definite chromatographic resolution degree.
LC-MS instrument: Agilent 1200 high performance liquid chromatographs; 6420 time of-flight mass spectrometers; ESI electric spray ion source.For unknown material or without the Components identification of tester, and definite chromatographic resolution degree.
Chromatographic column: Welch XB-C18 chromatographic column (250mm * 4.6mm, 5 μ m).
2. the preparation of need testing solution
The preparation of 2.1 pseudo-ginseng need testing solutions
(1) get 0.6g Radix Notoginseng powder, accurately weighed, add 70% ethanolic solution, soaked overnight, ultrasonic extraction 2 hours, Filter paper filtering is to 100mL beaker, and filtrate decompression concentrated (50 ℃), to dry, is used 70% dissolve with ethanol solution, constant volume 25mL, with 0.45 μ m pin type membrane filtration, to 2mL sample bottle, obtain pseudo-ginseng need testing solution (I), standby.
(2) get 1.0g Radix Notoginseng powder, accurately weighed, in 250mL tool plug conical flask, accurately add 50.00mL70 % ethanolic solution (V
ethanol: V
water=7: 3), weigh, placement is spent the night.Put 80 ℃ of water-baths and reflux 2 hours, room temperature to be chilled to, then weigh, with 70% ethanolic solution, supply the weight of less loss, shake up, filter, accurately pipette 25.00mL filtrate and be placed in 50mL beaker, 100 ℃ of water-bath tops are concentrated into dry, use 70% dissolve with ethanol solution, be transferred in 10mL volumetric flask constant volume.With 0.45 μ m pin type membrane filtration, to 2mL sample bottle, obtain pseudo-ginseng need testing solution (II), standby.
The preparation of 2.2 Pien Tze Huang need testing solutions
Get 1.0g Pien Tze Huang powder (through mortar porphyrize, sieving No. 5 or 80 orders), accurately weighed, be placed in 250mL tool plug conical flask, accurately add 50.00mL 70% ethanolic solution (V
ethanol: V
water=7: 3), weigh, placement is spent the night.Put 80 ℃ of water-baths and reflux 2 hours, room temperature to be chilled to, then weigh, with 70% ethanolic solution, supply the weight of less loss, shake up, filter, accurately pipette 25.00mL filtrate and be placed in 50mL beaker, 100 ℃ of water-bath tops are concentrated into dry, use 70% dissolve with ethanol solution, be transferred in 10mL volumetric flask constant volume.To 2mL sample bottle, standby with 0.45 μ m pin type membrane filtration.
3. the preparation of object of reference solution
According to the feature of pseudo-ginseng and Pien Tze Huang, and the saponin(e that can buy from commercial channel mainly contains notoginsenoside R, ginsenoside Rg1 and Rb1, Re, Rg2, Rh1, Rd, Rf, Rc.
Accurately take respectively reference substance notoginsenoside R (0.01019g), ginsenoside Rg1 (0.01067g, content 97.7%), Rb1 (0.00984g), Rg2 (0.01021g), Rh1 (0.01292g), Rd (0.00947g), Rf (0.01115g), Rc (0.00959g), add respectively methyl alcohol and dissolve constant volume in 10mL volumetric flask; Accurately take Re (0.01643g, content 88.8%), add methyl alcohol and dissolve constant volume in 25mL volumetric flask.
Accurately pipette respectively the Rg of the above-mentioned reference substance solution of 5mL
1and Rb
1in 50mL beaker, room temperature volatilizes solvent, dissolves and moves in 10mL volumetric flask, more accurately pipette respectively the above-mentioned reference substance solution R of 1mL with methyl alcohol
1, Re, Rd, the Rh of 0.6mL
1rg with 0.5mL
2in same volumetric flask, constant volume, must contain R
1, ginsenoside Rg
1, Rb
1, Re, Rg
2, Rh
1, Rd reference substance mixed solution (I).
Accurately take respectively reference substance Panax Notoginseng saponin R
1(0.00569g), ginsenoside Rg
1(0.00896g, content 97.7%), Rb
1(0.00809g), Re (0.00103g, content 88.8%), Rg
2(0.00605g), Rh
1(0.00409g), Rd (0.00787g), Rf (0.01180g), Rc (0.00619g), add methyl alcohol and dissolve constant volume 25mL, make reference substance mixed solution (II).
Accurately take respectively reference substance Panax Notoginseng saponin R
1(0.00402g), ginsenoside Rg
1(0.01515g, content 97.7%), Rb
1(0.01540g), Re (0.00244g, content 88.8%), Rg
2(0.00064g), Rh
1(0.00112g), Rd (0.00356g), add methyl alcohol and dissolve constant volume 10mL, make reference substance mixed solution (III).
4. system suitability
Precision is drawn Panax Notoginseng saponin R respectively
1, ginsenoside Rg
1, Rb
1, Re, Rg
2, Rh
1, Rd, Rf, Rc and reference substance mixed solution (II) and and each 5 μ L of pseudo-ginseng need testing solution (I), according to chromatographic condition III sample detection respectively, record chromatogram.By comparison Panax Notoginseng saponin R
1, ginsenoside Rg
1, Re, Rf, Rb
1, Rc, Rg
2, Rh
1with the chromatogram of Rd reference substance solution and the chromatogram of reference substance mixed solution (II), can determine that, under chromatographic condition III, the peak sequence of these nine kinds of saponin(es is: Panax Notoginseng saponin R
1, ginsenoside Rg
1, Re, Rf, Rb
1, Rc, Rg
2, Rh
1and Rd.By comparing retention time and the peak sequence of the chromatogram of reference substance mixed solution (II) and pseudo-ginseng need testing solution (I), the ownership that can infer each peak in pseudo-ginseng need testing solution chromatogram, contains Panax Notoginseng saponin R in pseudo-ginseng need testing solution
1, ginsenoside Rg
1, Re, Rb
1, Rg
2, Rh
1with Rd component, the Rf and the Rc component that do not contain or contain minute quantity.
In addition, draw respectively each 5 μ L of reference substance mixed solution (II) and pseudo-ginseng need testing solution (II), inject liquid chromatograph-mass spectrometer (LC-MS/Ion Trap), according to chromatographic condition III and mass spectrum condition (Nebulizer-40psi, Dry Gas-12.0L/min, Dry Temp-350 ℃, Target Mass-950m/z, Negative, Scan-300-2000m/z) analyze, according to mass-to-charge ratio, further confirm synergy.Can substantially infer, the mass-to-charge ratio at 1-3 peak is respectively 931.7,799.6 and 945.7, respectively with notoginsenoside [R
1-H]
-, ginsenoside [Rg
1-H]
-[Re-H]
-corresponding, the mass-to-charge ratio at No. 6 peaks is 553.5 and 1107.8, respectively with ginsenoside [Rb
1-2H]
2-[Rb
1-H]
-corresponding, the mass-to-charge ratio at 8-10 peak is respectively 783.6,637.5 and 946.0, respectively with ginsenoside [Rg
2-H]
-, [Rh
1-H]
-[Rd-H]
-corresponding, the mass-to-charge ratio at No. 7 peaks and No. 11 peaks be respectively 769.6 and the mass-to-charge ratio at 945.8,4-5 peak be 619.5[M-2H]
2-and 1239.7[M-H]
-, may be isomers, do not find the peak corresponding with ginsenoside Rf and Rc, show in pseudo-ginseng need testing solution (II) containing or contain minute quantity Rf and Rc composition.
5. linear relationship is investigated
Accurate reference substance mixed solution (III) 1.0,2.0,3.0,4.0,5.0,7.0, the 10.0 μ L that draw, carry out stratographic analysis, measure the peak area of each component.Take sample size (μ g) as horizontal ordinate (X), and peak area (A) is ordinate (Y), drawing standard curve.Obtain regression equation as follows respectively:
Panax Notoginseng saponin R
1: y=379.27x-6.8114, r=0.9998; The range of linearity is 0.402～4.02 μ g/mL;
Ginsenoside Rg
1: y=391.75x-21.555, r=0.9999; The range of linearity is 1.480～14.801 μ g/mL;
Ginsenoside Re: y=357.28x-5.3594, r=0.9998; The range of linearity is 0.217～2.167 μ g/mL;
Ginsenoside Rb
1: y=306.82x+9.6694, r=0.9998; The range of linearity is 1.426～14.260 μ g/mL;
Ginsenoside Rg
2: y=539.04x-2.0947, r=0.9998; The range of linearity is 0.064～0.64 μ g/mL;
Ginsenoside Rh
1: y=422.61x-2.4438, r=0.9998; The range of linearity is 0.112～1.12 μ g/mL;
Ginsenoside Rd: y=348.69x-5.0984, r=0.9998; The range of linearity is 0.356～3.56 μ g/mL.
6. stability test
Get the same pseudo-ginseng need testing solution (II) of preparing according to 2.1 methods (2), at 0,3,6,9,12,23,26,29 hour sample introduction 5 μ L, with chromatographic condition, III detected respectively.The peak area of usining is maximum, more stable total peak Rg1 is the finger-print of internal standard compound as object of reference, using its retention time and peak area respectively as 1, calculate other each relative retention time of total fingerprint peaks and the ratio of peak area, its relative standard deviation relatively, in Table 8.Result shows, the RSD of the relative retention time of 13 chromatographic peaks is all less than 0.271%, and the RSD of peak area ratio is all less than 1.63%, illustrates within 32 hours stable.
Table 8 stability test (pseudo-ginseng)
Get the same Pien Tze Huang need testing solution of preparing according to 2.3 methods, at 0,3,6,129,132,135,138,141 hour sample introduction 5 μ L, with chromatographic condition III, detect (in Table 9) respectively.Result shows, the RSD of the relative retention time of 12 chromatographic peaks is all less than 1%, and the RSD of peak area ratio is all less than 3%, illustrates that need testing solution is basicly stable within 6 days.
Table 9 stability test (Pien Tze Huang)
7. precision test
Get the same pseudo-ginseng need testing solution (II) of preparing according to 2.1 methods (2), continuous sample introduction 5 times (each 5 μ L), detects (in Table 10) with chromatographic condition III.Result shows, the RSD of the retention time of 13 chromatographic peaks is all less than 0.242%, and the RSD of peak area is all less than 3%, shows that instrument precision is good.
Table 10 precision test
8. reappearance test
Get 5 parts of the pseudo-ginseng test samples of same lot number, according to 2.1 methods (2), prepare need testing solution (II), every part of sample introduction 3 times (each 5 μ L) respectively, detects (in Table 11) with chromatographic condition III.Result shows, the RSD of the relative retention time of 13 total chromatographic peaks is all less than 1%, and the RSD of peak area ratio is all less than 3%, shows that the reappearance of the method is better, meets the requirement of finger-print.
The test of table 11 reappearance
The foundation of the finger-print of experimental example 3 pseudo-ginsengs
Get the pseudo-ginseng powder of ten lot numbers, according to 2.1 methods (2) of experimental example 2, prepare same pseudo-ginseng need testing solution (II), every part of difference sample introduction 2 times (each 5 μ L), with chromatographic condition, III detects, and notes down 170 minutes chromatograms.Utilize " traditional Chinese medicine fingerprint area of computer aided similarity software for calculation ", through data importing, Supplements and Data Matching, set up reference fingerprint with average method.According to peak match result, take peak area as parameter, calculate finger-print to be measured and the overall similarity that contrasts collection of illustrative plates.
According to the testing result of 10 batches of pseudo-ginsengs, demarcate and have 23 relatively stable fingerprint peakses.Adopt relative retention time to demarcate fingerprint peaks, because analysis time is longer, therefore select No. 2 (Rg
1) chromatographic peak and No. 8 (Rb
1) chromatographic peak is as internal reference peak, utilization " traditional Chinese medicine fingerprint area of computer aided similarity software for calculation " is proofreaied and correct coupling to spectrogram, obtains 23 total peaks of coupling completely.
It has peak data matching result table 1210 batch pseudo-ginseng test sample
The total peak data matching result of 10 batches of pseudo-ginsengs is in Table 12, and its similarity is 1, and the relative standard deviation of the total area at total peak is 1.24%, shows this 10 batches of pseudo-ginseng stable material quality.
With No. 2 maximum, more stable total peak (Rg of peak area
1) be that internal standard compound is as the finger-print of object of reference, using its retention time and peak area respectively as 1, calculate other each relative retention time of total fingerprint peaks and the ratio of peak area, compare its relative standard deviation, result shows, the relative standard deviation of the relative retention time at 23 total peaks is all less than 0.34%, illustrates that the relative retention time at total peak is more fixing.In 23 total peaks, the RSD of relative peak area is all less than 30%, is wherein less than 3% 14 peaks that have, and between 3 peaks that have of 3-5%, all the other 6 peaks, between 5-10.23%, illustrate that the area ratio of each total fingerprint peaks is relatively fixing.With reference to " technical requirement of traditional Chinese medicine finger-print research ", can determine the total peak that these 23 peaks are pseudo-ginseng, they are respectively 1-12,14-19,23,26,28,29, No. 32 peaks.Determine that principle is: every batch sample all contains this composition; Degree of separation R >=0.8, peak; RSD≤5% of each peak relative retention time; RSD≤30% of each peak-to-peak area.
According to above-mentioned experimental result, in the chromatogram of 10 batches of pseudo-ginsengs, the more stable peak of its relative retention time and chromatographic peak has 23, and what peak area was relatively stable has 14, and similarity is 1 to show that pseudo-ginseng quality of materials is relatively stable.Meanwhile, also reflected that the method for separating and analyzing of setting up can be used for the finger-print foundation of pseudo-ginseng and analyzes.
In addition, also to wherein a collection of (S10 in table 12) batch medicinal material according to chromatographic condition III and mass spectrum condition (Nebulizer-35psi, Dry Gas-9.0L/min, Dry Temp-325 ℃) carried out LC-MS (LC-MS/TOF) and analyzed, according to the position in the corresponding numbering in each peak, chromatogram and corresponding mass spectrogram, inferred each peak chemical formula: No. 1-3, No. 8,11-12 peak and No. 14 corresponding with reference substance (notoginsenoside R, ginsenoside Rg1 and Rb1, Re, Rg2, Rh1, Rd) respectively; In the mass spectrogram at No. 4 peaks, base peak be [M-H]-, its mass-to-charge ratio M/Z is 769.4748, corresponding formula weight is that 770.482, TOF software infers that its chemical formula is C
41h
70o
13(theoretical chemistry formula weight is 770.4816); In the mass spectrogram at No. 5 peaks, base peak be [M-H]-, its mass-to-charge ratio M/Z is that 1239.6334, the second peaks are [M-2H] 2-, its mass-to-charge ratio M/Z is 619.3155, corresponding formula weight is that 1240.6455, TOF software infers that its chemical formula is C
59h
100o
27(theoretical chemistry formula weight is 1240.6452); In the mass spectrogram at No. 6 peaks, base peak is [M-2H] 2-, and its mass-to-charge ratio M/Z is 619.3168, corresponding formula weight be 1240.6467, the second peaks be [M-H]-, its mass-to-charge ratio M/Z is that 1239.6355, TOF software infers that its chemical formula is C
59h
100o
27(theoretical chemistry formula weight is 1240.6452); The base peak of the mass spectrogram at No. 7 peaks be [M-H]-, its mass-to-charge ratio M/Z is 799.4851, corresponding formula weight is that 800.4924, TOF software infers that its chemical formula is C
42h
72o
14(theoretical chemistry formula weight is 800.4922), second and third peak be respectively [M-2H] 2-, [M-H]-, mass-to-charge ratio is respectively 552.2891,1105.5743, corresponding formula weight is respectively 1106.5900,1106.5837, the chemical formula that TOF software is inferred is C
54h
90o
23(theoretical chemistry formula weight is 1106.5873); The base peak of the mass spectrogram at No. 9 peaks be [M-H]-, its mass-to-charge ratio M/Z is 769.4752, corresponding formula weight is that the chemical formula that 770.4824, TOF software is inferred is C
41h
70o
13(theoretical chemistry formula weight is 770.4816); The base peak of the mass spectrogram at No. 10 peaks is [M-2H] 2-, and its mass-to-charge ratio M/Z is 538.2908, and corresponding formula weight is 1078.5945, the second peak be [M-H]-, its mass-to-charge ratio M/Z is 1077.5854, and corresponding formula weight is that the chemical formula that 1078.5928, TOF software is inferred is C
53h
90o
22(theoretical chemistry formula weight is 1078.5924); The base peak of the mass spectrogram at No. 15 peaks be [M-H]-, its mass-to-charge ratio M/Z is 637.4327, corresponding formula weight is that the chemical formula that 638.44, TOF software is inferred is C
36h
62o
9(theoretical chemistry formula weight is 638.4394); The base peak of the mass spectrogram at No. 16 peaks be [M-H]-, its mass-to-charge ratio M/Z is 945.5434, corresponding formula weight is 946.5506, third high peak is [M-2H] 2-, its mass-to-charge ratio M/Z is 472.2698, and corresponding formula weight is that the chemical formula that 946.5525, TOF software is inferred is C
48h
82o
18(theoretical chemistry formula weight is 946.5501); The base peak of the mass spectrogram at No. 17 peaks be [M-H]-, its mass-to-charge ratio M/Z is 783.4904, corresponding formula weight is 784.4977, third high peak is [M-2H] 2-, its mass-to-charge ratio M/Z is 391.2442, and corresponding formula weight is that the chemical formula that 784.5001, TOF software is inferred is C
42h
70o
13(theoretical chemistry formula weight is 784.4973); The base peak of the mass spectrogram at No. 18 peaks be [M-H]-, its mass-to-charge ratio M/Z is 619.4227, corresponding formula weight is that the chemical formula that 620.4300, TOF software is inferred is C
36h
60o
8(theoretical chemistry formula weight is 620.4288); The base peak of the mass spectrogram at No. 19 peaks be [M-H]-, its mass-to-charge ratio M/Z is 619.4223, corresponding formula weight is that the chemical formula that 620.4295, TOF software is inferred is C
36h
60o
8(theoretical chemistry formula weight is 620.4288); The base peak of the mass spectrogram at No. 23 peaks be [M-H]-, its mass-to-charge ratio M/Z is 594.9642, corresponding formula weight is that the chemical formula that 595.9715, TOF software is inferred is C
25h
8o
18(theoretical chemistry formula weight is 595.9711), the peak suitable with base peak intensity be [M-H]-, its mass-to-charge ratio M/Z is 530.9682, corresponding formula weight is that the chemical formula that 531.9754, TOF software is inferred is C
21h
8o
17(theoretical chemistry formula weight is 531.9761); The base peak of the mass spectrogram at No. 26 peaks be [M-H]-, its mass-to-charge ratio M/Z is 295.2288, corresponding formula weight is that the chemical formula that 296.2360, TOF software is inferred is C
18h
32o
3(theoretical chemistry formula weight is 296.2351); The base peak of the mass spectrogram at No. 28 peaks be [M-H]-, its mass-to-charge ratio M/Z is 710.9722, corresponding formula weight is that the chemical formula that 711.9795, TOF software is inferred is C
29h
12o
22(theoretical chemistry formula weight is 711.9782); The base peak of the mass spectrogram at No. 29 peaks be [M-H]-, its mass-to-charge ratio M/Z is 233.1554, corresponding formula weight is that the chemical formula that 234.1627, TOF software is inferred is C
15h
21o
2(theoretical chemistry formula weight is 234.1620); The base peak of the mass spectrogram at No. 32 peaks be [M-H]-, its mass-to-charge ratio M/Z is 760.9695, corresponding formula weight is that the chemical formula that 761.9768, TOF software is inferred is C
29h
14o
25(theoretical chemistry formula weight is 761.9824)
The foundation of experimental example 4 Pien Tze Huang finished product finger-prints
Get the Pien Tze Huang of ten lot numbers corresponding with experimental example 3 pseudo-ginseng used, according to 2.3 methods of experimental example 2, prepare need testing solution, every part of difference sample introduction 2 times (each 5 μ L), with chromatographic condition, III detects, and notes down 170 minutes chromatograms.Utilize " traditional Chinese medicine fingerprint area of computer aided similarity software for calculation ", through data importing, Supplements and Data Matching, set up reference fingerprint with average method.According to peak match result, take peak area as parameter, calculate finger-print to be measured and the overall similarity that contrasts collection of illustrative plates.
According to the testing result of 10 batches of Pien Tze Huang test samples, demarcate and have 19 relatively stable fingerprint peakses.Adopt relative retention time to demarcate fingerprint peaks, and select (Rg No. 2
1) chromatographic peak and No. 8 (Rb
1) chromatographic peak is as internal reference peak, utilization " traditional Chinese medicine fingerprint area of computer aided similarity software for calculation " is proofreaied and correct coupling to spectrogram, obtains 19 total peaks of coupling completely.The total peak data matching result of 10 batches of preparations is in Table 14, the equal > 0.998 of its similarity, and the relative standard deviation of the total area at total peak is 1.64%, shows that these 10 batches of qualities of the pharmaceutical preparations are stable.Meanwhile, also reflected that the method for separating and analyzing of setting up can be used for the finger-print foundation of preparation and analyzes.
Table 1410 batch Pien Tze Huang test sample has peak data matching result
With maximum, the more stable total peak Rg of peak area
1for internal standard compound is as object of reference, using respectively its retention time and peak area as 1, calculate other each relative retention time of total fingerprint peaks and the ratio of peak area, its relative standard deviation relatively, in Table 15.
As can be seen from Table 15, the relative standard deviation of the relative retention time at 19 total peaks is all less than 0.83%, illustrates that the relative retention time at total peak is more fixing, can using this foundation as finger-print.In 19 total peaks, wherein the RSD of the relative peak area at 17,21 and No. 23 peaks is greater than 30%, can not be as the total peak of preparation; All the other 16 peaks can be used as the total peak of preparation, and they are respectively 1-9,11,12,14-16,19 and No. 27 peaks.
Relative retention time and the peak area ratio at the total peak of table 1510 batch Pien Tze Huang test sample
In addition, also to product in the middle of wherein a collection of (S10 in table 15) according to chromatographic condition III and mass spectrum condition (Nebulizer-35psi, Dry Gas-9.0L/min, Dry Temp-325 ℃) carried out LC-MS (LC-MS/TOF) and analyzed, according to the position in the corresponding numbering in each peak, chromatogram and corresponding mass spectrogram, inferred the chemical formula at each peak: No. 1-3, No. 8,11-12 peak and No. 14 respectively with reference substance (Panax Notoginseng saponin R
1, ginsenoside Rg
1, Rb
1, Re, Rg
2, Rh
1, Rd) corresponding; In the mass spectrogram at No. 4 peaks, base peak is [M-H]
-, its mass-to-charge ratio M/Z is 769.4746, corresponding formula weight is that 770.4820, TOF software infers that its chemical formula is C
41h
70o
13(theoretical chemistry formula weight is 770.4816); In the mass spectrogram at No. 5 peaks, base peak is [M-H]
-, its mass-to-charge ratio M/Z is that 1239.6373, the second peaks are [M-2H]
2-, its mass-to-charge ratio M/Z is 619.3156, corresponding formula weight is that 1240.6459, TOF software infers that its chemical formula is C
59h
100o
27(theoretical chemistry formula weight is 1240.6452); In the mass spectrogram at No. 6 peaks, base peak is [M-H]
-, its mass-to-charge ratio M/Z is that 1239.6368, the second peaks are [M-2H]
2-, its mass-to-charge ratio M/Z is 619.3168, corresponding formula weight is that 1240.6467, TOF software infers that its chemical formula is C
59h
100o
27(theoretical chemistry formula weight is 1240.6452); The base peak of the mass spectrogram at No. 7 peaks is [M-H]
-, its mass-to-charge ratio M/Z is [M-H]
-, mass-to-charge ratio is 799.4848, the chemical formula that corresponding formula weight is respectively the supposition of 800.4922, TOF software is C
42h
72o
14(theoretical chemistry formula weight is 800.4922), the second peak is [M-H]
-, mass-to-charge ratio is respectively 1105.5786, and the chemical formula that corresponding formula weight is respectively the supposition of 1106.5861, TOF software is C
54h
90o
23(theoretical chemistry formula weight is 1106.5873); The base peak of the mass spectrogram at No. 9 peaks is [M-H]
-, its mass-to-charge ratio M/Z is 769.4751, corresponding formula weight is that the chemical formula that 770.4823, TOF software is inferred is C
41h
70o
13(theoretical chemistry formula weight is 770.4816); The base peak of the mass spectrogram at No. 15 peaks is [M-H]
-, its mass-to-charge ratio M/Z is 637.4331, corresponding formula weight is that the chemical formula that 638.4403, TOF software is inferred is C
36h
62o
9(theoretical chemistry formula weight is 638.4394); The base peak of the mass spectrogram at No. 16 peaks is [M-H]
-, its mass-to-charge ratio M/Z is 945.5407, corresponding formula weight is that the chemical formula that 946.5484, TOF software is inferred is C
48h
82o
18(theoretical chemistry formula weight is 946.5501); The base peak of the mass spectrogram at No. 17 peaks is [M-H]
-, its mass-to-charge ratio M/Z is 783.4898, corresponding formula weight is that the chemical formula that 784.4972, TOF software is inferred is C
42h
70o
13(theoretical chemistry formula weight is 784.4973); The base peak of the mass spectrogram at No. 18 peaks is [M-H]
-, its mass-to-charge ratio M/Z is 619.4233, corresponding formula weight is that the chemical formula that 620.4305, TOF software is inferred is C
36h
60o
8(theoretical chemistry formula weight is 620.4288); The base peak of the mass spectrogram at No. 19 peaks is [M-H]
-, its mass-to-charge ratio M/Z is 619.4218, corresponding formula weight is that the chemical formula that 620.4291, TOF software is inferred is C
36h
60o
8(theoretical chemistry formula weight is 620.4288); The base peak of the mass spectrogram at No. 20 peaks is [M-H]
-, its mass-to-charge ratio M/Z is 437.2918, corresponding formula weight is that the chemical formula that 438.2991, TOF software is inferred is C
25h
42o
6(theoretical chemistry formula weight is 438.2981); The base peak of the mass spectrogram at No. 21 peaks is [M-H]
-, its mass-to-charge ratio M/Z is 387.2552, corresponding formula weight is that the chemical formula that 388.2625, TOF software is inferred is C
24h
36o
4(theoretical chemistry formula weight is 388.2614), its second peak is [M-H]
-, its mass-to-charge ratio M/Z is 811.5734, corresponding formula weight is that the chemical formula that 812.5807, TOF software is inferred is C
49h
80o
9(theoretical chemistry formula weight is 812.5801); The base peak of the mass spectrogram at No. 23 peaks is [M-H]
-, its mass-to-charge ratio M/Z is 391.2871, corresponding formula weight is that the chemical formula that 392.2943, TOF software is inferred is C
24h
40o
4(theoretical chemistry formula weight is 392.2927), its second peak is [M-H]
-, its mass-to-charge ratio M/Z is 783.5789, corresponding formula weight is that the chemical formula that 784.5862, TOF software is inferred is C
48h
80o
8(theoretical chemistry formula weight is 784.5853); The base peak of the mass spectrogram at No. 27 peaks is [M-H]
-, its mass-to-charge ratio M/Z is 295.2286, corresponding formula weight is that the chemical formula that 296.2359, TOF software is inferred is C
18h
32o
3(theoretical chemistry formula weight is 296.2351); The base peak of the mass spectrogram at No. 28 peaks is [M-H]
-, its mass-to-charge ratio M/Z is 596.9769, corresponding formula weight is that the chemical formula that 597.9841, TOF software is inferred is C
25h
10o
18(theoretical chemistry formula weight is 597.9867); The base peak of the mass spectrogram at No. 29 peaks is [M-H]
-, its mass-to-charge ratio M/Z is 233.1548, corresponding formula weight is that the chemical formula that 234.1621, TOF software is inferred is C
15h
22o
2(theoretical chemistry formula weight is 234.162); The base peak of the mass spectrogram at No. 32 peaks is [M-H]
-, its mass-to-charge ratio M/Z is 425.3043, corresponding formula weight is that the chemical formula that 426.3116, TOF software is inferred is C
28h
42o
3(theoretical chemistry formula weight is 426.3134).
The correlativity of experimental example 5 preparations and pseudo-ginseng HPLC chromatographic fingerprinting
Utilize " traditional Chinese medicine fingerprint area of computer aided similarity software for calculation ", the experimental example 3 of take 10 batches of pseudo-ginsengs used and 10 batches of corresponding preparations are foundation, by produced contrast collection of illustrative plates, through data importing, Supplements and Data Matching, with average method, set up reference fingerprint, take retention time as parameter, calculate finger-print to be measured and the overall similarity that contrasts collection of illustrative plates.With maximum, the more stable total peak Rg of peak area
1for internal standard compound is as object of reference, using its peak area as 1, calculate the ratio of the peak area of other each total fingerprint peaks, its relative standard deviation relatively, in Table 16.
As can be seen from Table 16, the peak of the principal ingredient of pseudo-ginseng all embodies to some extent in preparation, and total peak has 19.
In order to reflect more realistically the associated situation between medicinal material, preparation, the data of 10 batches of medicinal materials and 10 batches of preparations are imported simultaneously to " traditional Chinese medicine fingerprint area of computer aided similarity software for calculation " system, time window elects 0.5 as, mates.Relatively its peak area ratio, the results are shown in Table 17.
As can be seen from Table 17, in 20 batch samples, the peak of coupling has 16 completely, wherein the relative standard deviation of the peak area ratio at 1-9,11-12,14,16, No. 19 peaks is less than 30%, and these 14 peaks can be used as the total peak of association of finger-print between medicinal material and preparation; The relative standard deviation of the peak area ratio at another two 15 and No. 17 peaks is respectively 45.3% and 64.1%, can be used as reference.
Table 1710 batch pseudo-ginseng and corresponding preparation have the RSD of peak retention time and peak area ratio
In sum, the total peak that pseudo-ginseng has the finger-print of continuity in preparation is 14, they are respectively 1-9,11,12,14,16 and No. 19 peaks, and corresponding retention time is respectively 27.24,37.48,39.48,57.81,63.71,64.81,65.50,66.44,67.67,69.22,69.82,71.58,73.54 and 85.21min left and right.
Embodiment
Following embodiment all can realize effect described in above-mentioned experimental example.
Pseudo-ginseng finger print quality detecting method in embodiment 1 Pien Tze Huang
According to high performance liquid chromatography: chromatographic condition and system suitability: select Welch XB-C18 chromatographic column (250mm * 4.6mm, 5 μ m), take octadecylsilane chemically bonded silica as filling agent, take acetonitrile and water carries out gradient elution as mobile phase, gradient elution program is as follows: 0～40 minute 22% acetonitrile, flow velocity 0.5mL/min, 40～50 minutes 22～30% acetonitriles, flow velocity 0.5mL/min, 50～80 minutes 30～55% acetonitriles, flow velocity 0.5mL/min, 80～95 minutes 55% acetonitriles, flow velocity 0.5mL/min, 95～115 minutes 55～90% acetonitriles, flow velocity 0.8mL/min, 115～135 minutes 90% acetonitriles, flow velocity 0.8mL/min, 135～140 minutes 90～22% acetonitriles, flow velocity 0.5mL/min, column temperature is 25 ℃, detection wavelength is 203nm,
Need testing solution preparation:
Get pseudo-ginseng powder 1.0g, accurately weighed, in 250mL tool plug conical flask, accurately add 50.00ml 70% ethanolic solution (V ethanol: V water=7: 3), weigh, placement is spent the night; Put 80 ℃ of water-baths and reflux 2 hours, room temperature to be chilled to, then weigh, with 70% ethanolic solution, supply the weight of less loss, shake up, filter, accurately pipette 25.00ml filtrate and be placed in 50mL beaker, 100 ℃ of water-bath tops are concentrated into dry, use 70% dissolve with ethanol solution, be transferred in 10mL volumetric flask constant volume; With 0.45 μ m pin type membrane filtration, to 2mL sample bottle, obtain pseudo-ginseng need testing solution, standby.
The preparation of internal standard substance solution: take reference substance ginsenoside Rg
10.01067g, adds methyl alcohol and dissolves constant volume in 10mL volumetric flask, standby.
Determination method: draw respectively need testing solution and internal standard substance solution 5uL, in injection liquid chromatography, note down 170 minutes chromatograms.Maximum, the more stable total peak Rg1 of the peak area of usining, as internal standard compound is as object of reference, is usingd respectively its retention time and peak area as 1, calculates each total peak relative retention time.Test sample finger-print and standard finger-print similarity are 1; Pseudo-ginseng finger-print has 23 total peaks, be respectively 1-12,14-19,23,26,28,29, No. 32 peaks, the relative retention time at total peak is respectively: 27.125,37.341,39.333,57.772,63.659,64.751,65.441,66.391,67.619,68.662,69.171,69.773,71.53,72.633,73.478,81.212,83.71,85.151,91.732,104.717,109.83,110.29,113.46.
Relative peak area ratio is respectively: 0.199,1.000,0.118,0.006,0.020,0.021,0.006,0.670,0.038,0.004,0.026,0.047,0.175,0.007,0.029,0.006,0.006,0.013,0.012,0.004,0.0019,0.004,0.009.
The finger-print of embodiment 2 Pien Tze Huang HPLC
According to high performance liquid chromatography: chromatographic condition and system suitability: select Welch XB-C18 chromatographic column (250mm * 4.6mm, 5 μ m), take octadecylsilane chemically bonded silica as filling agent, take acetonitrile and water carries out gradient elution as mobile phase, gradient elution program is as follows: 0～40 minute 22% acetonitrile, flow velocity 0.5mL/min, 40～50 minutes 22～30% acetonitriles, flow velocity 0.5mL/min, 50～80 minutes 30～55% acetonitriles, flow velocity 0.5mL/min, 80～95 minutes 55% acetonitriles, flow velocity 0.5mL/min, 95～115 minutes 55～90% acetonitriles, flow velocity 0.8mL/min, 115～135 minutes 90% acetonitriles, flow velocity 0.8mL/min, 135～140 minutes 90～22% acetonitriles, flow velocity 0.5mL/min, column temperature is 25 ℃, detection wavelength is 203nm,
Need testing solution preparation: get the Pien Tze Huang (through mortar porphyrize, sieving No. 5 or 80 orders) of ten lot numbers corresponding with pseudo-ginseng, every crowd of 1.0g, accurately weighed, be placed in 250mL tool plug conical flask, accurately add 50.00ml70% ethanolic solution (V
ethanol: V
water=7: 3), weigh, placement is spent the night; Put 80 ℃ of water-baths and reflux 2 hours, room temperature to be chilled to, then weigh, with 70% ethanolic solution, supply the weight of less loss, shake up, filter, accurately pipette 25.00ml filtrate and be placed in 50mL beaker, 100 ℃ of water-bath tops are concentrated into dry, use 70% dissolve with ethanol solution, be transferred in 10mL volumetric flask constant volume; To 2mL sample bottle, standby with 0.45 μ m pin type membrane filtration.
Reference substance solution preparation: accurately take respectively reference substance Panax Notoginseng saponin R
1(0.00569g), ginsenoside Rg
1(0.00896g, content 97.7%), Rb
1(0.00809g), Re (0.00103g, content 88.8%), Rg
2(0.00605g), Rh
1(0.00409g), Rd (0.00787g), Rf (0.01180g), Rc (0.00619g), add methyl alcohol and dissolve constant volume 25mL, make reference substance mixed solution.
Determination method: draw respectively reference substance mixed solution and need testing solution 5ul, in injection liquid chromatography, record 170 minutes chromatograms.Maximum, the more stable total peak Rg1 of the peak area of usining, as internal standard compound is as object of reference, is usingd respectively its retention time and peak area as 1, calculates each total peak relative retention time.Test sample finger-print and standard finger-print similarity are 0.998.
Pien Tze Huang finger-print has 16 total peaks, total peak is respectively 1-9,11,12,14-16,19 and No. 27 peaks, and the relative retention time at total peak is respectively: ask 27.557,37.956,39.968,57.919,63.809,64.917,65.613,66.549,67.785,69.34,69.948,71.733,72.821,73.697,85.395,105.602.
Relative peak area ratio is respectively: 0.204,1.000,0.116,0.006,0.020,0.021,0.006,0.677,0.040,0.026,0.050,0.183,0.015,0.033,0.012,0.013.
Claims (6)
1. a detection method for pseudo-ginseng in Pien Tze Huang, is characterized in that the method comprises following fingerprint atlas detection method:
According to high performance liquid chromatography: chromatographic condition and system suitability: select Welch XB-C18 chromatographic column, take octadecylsilane chemically bonded silica as filling agent, take acetonitrile and water carries out gradient elution as mobile phase, gradient elution program is as follows: 0～40 minute 22% acetonitrile, flow velocity 0.5mL/min, 40～50 minutes 22～30% acetonitriles, flow velocity 0.5mL/min, 50～80 minutes 30～55% acetonitriles, flow velocity 0.5mL/min, 80～95 minutes 55% acetonitriles, flow velocity 0.5mL/min, 95～115 minutes 55～90% acetonitriles, flow velocity 0.8mL/min, 115～135 minutes 90% acetonitriles, flow velocity 0.8mL/min, 135～140 minutes 90～22% acetonitriles, flow velocity 0.5mL/min, column temperature is 25 ℃, detection wavelength is 203nm,
Need testing solution preparation: get pseudo-ginseng powder 1～10 weight portion, accurately weighed, in tool plug conical flask, accurately add 50～200 parts by volume 70% ethanolic solutions, to weigh, placement is spent the night; Putting 80 ℃～90 ℃ water-baths refluxes 1～3 hour, room temperature to be chilled to, weigh again, with 70% ethanolic solution, supply the weight of less loss, shake up, filter, accurately pipette 25～100 parts by volume filtrates and be placed in beaker, 100 ℃ of water-baths top is concentrated into dry, uses 70% dissolve with ethanol solution, be transferred in 10 parts by volume volumetric flasks constant volume; With 0.45 μ m pin type membrane filtration to 2 parts by volume sample bottle, obtain pseudo-ginseng need testing solution, standby;
Reference substance solution preparation: accurately take respectively reference substance Panax Notoginseng saponin R
10.00569g, ginsenoside Rg
10.00896g, Rb
10.00809g, Re0.00103g, Rg
20.00605g, Rh
10.00409g, Rd0.00787g, Rf0.01180g, Rc0.00619g, add methyl alcohol and dissolve constant volume 25mL, makes reference substance mixed solution;
Determination method: draw respectively need testing solution and reference substance mixed solution 0.005 parts by volume, in injection liquid chromatography, record 170 minutes chromatograms, maximum, the more stable total peak Rg1 of the peak area of usining is as internal standard compound is as object of reference, using its retention time and peak area respectively as 1, calculate each total peak relative retention time; Test sample finger-print and standard finger-print similarity are 1;
Gained finger-print should have 23 total peaks, be respectively 1-12,14-19,23,26,28,29, No. 32 peaks, the relative retention time at total peak is respectively: 27.125,37.341,39.333,57.772,63.659,64.751,65.441,66.391,67.619,68.662,69.171,69.773,71.53,72.633,73.478,81.212,83.71,85.151,91.732,104.717,109.83,110.29,113.46;
Relative peak area ratio is respectively: 0.199,1.000,0.118,0.006,0.020,0.021,0.006,0.670,0.038,0.004,0.026,0.047,0.175,0.007,0.029,0.006,0.006,0.013,0.012,0.004,0.0019,0.004,0.009.
2. the detection method of pseudo-ginseng in Pien Tze Huang as claimed in claim 1, is characterized in that the method also comprises the steps:
According to liquid phase chromatogram condition described in claim 1, carry out LC-MS analysis, according to the position in the corresponding numbering in each peak, chromatogram and corresponding mass spectrogram, infer each peak chemical formula, mass spectrum condition is: atomizer pressure :-35psi, dry gas flow velocity: 9.0L/min, baking temperature: 325 ℃;
The chemical formula that each total peak is corresponding is respectively: No. 1-3, No. 8,11-12 peak and be respectively for No. 14 Panax Notoginseng saponin R
1, ginsenoside Rg
1, Rb
1, Re, Rg
2, Rh
1, Rd; No. 4 peaks are C
41h
70o
13; No. 5 peaks are C
59h
100o
27; No. 6 peaks are C
59h
100o
27; No. 7 peaks are C
42h
72o
14and C
54h
90o
23; No. 9 peaks are C
41h
70o
13; No. 10 peaks are C
53h
90o
22; No. 15 peaks are C
36h
62o
9; No. 16 peaks are C
48h
82o
18; No. 17 peaks are C
42h
70o
13; No. 18 peaks are C
36h
60o
8; No. 19 peaks are C
36h
60o
8; No. 23 peaks are C
25h
8o
18and C
21h
8o
17; No. 26 peaks are C
18h
32o
3; No. 28 peaks are C
29h
12o
22; No. 29 peaks are C
15h
21o
2; No. 32 peaks are C
29h
14o
25.
3. the detection method of pseudo-ginseng in Pien Tze Huang as claimed in claim 1 or 2, is characterized in that the need testing solution in the method is prepared as:
Get pseudo-ginseng powder 1.0g, accurately weighed, in 250mL tool plug conical flask, accurately add 50.00ml70% ethanolic solution, to weigh, placement is spent the night; Put 80 ℃ of water-baths and reflux 2 hours, room temperature to be chilled to, then weigh, with 70% ethanolic solution, supply the weight of less loss, shake up, filter, accurately pipette 25.00ml filtrate and be placed in 50mL beaker, 100 ℃ of water-bath tops are concentrated into dry, use 70% dissolve with ethanol solution, be transferred in 10mL volumetric flask constant volume; With 0.45 μ m pin type membrane filtration, to 2mL sample bottle, obtain pseudo-ginseng need testing solution, standby.
4. a detection method for Pien Tze Huang, is characterized in that the method comprises that following finger-print detects:
According to high performance liquid chromatography: chromatographic condition and system suitability: selecting Welch XB-C18 chromatographic column specification is 250mm * 4.6mm, 5 μ m, take octadecylsilane chemically bonded silica as filling agent, take acetonitrile and water carries out gradient elution as mobile phase, gradient elution program is as follows: 0～40 minute 22% acetonitrile, flow velocity 0.5mL/min, 40～50 minutes 22～30% acetonitriles, flow velocity 0.5mL/min, 50～80 minutes 30～55% acetonitriles, flow velocity 0.5mL/min, 80～95 minutes 55% acetonitriles, flow velocity 0.5mL/min, 95～115 minutes 55～90% acetonitriles, flow velocity 0.8mL/min, 115～135 minutes 90% acetonitriles, flow velocity 0.8mL/min, 135～140 minutes 90～22% acetonitriles, flow velocity 0.5mL/min, column temperature is 25 ℃, detection wavelength is 203nm,
Need testing solution preparation: getting commercially available formulation is Pien Tze Huang 1～10 weight portion of lozenge, through mortar porphyrize, sieves No. 5 or 80 orders, accurately weighed, is placed in tool plug conical flask, adds 50～200 parts by volume 70% ethanolic solutions, and described ethanolic solution is V
ethanol: V
water=7:3, weighs, and placement is spent the night; Putting 80 ℃～90 ℃ water-baths refluxes 1～3 hour, room temperature to be chilled to, weigh again, with 70% ethanolic solution, supply the weight of less loss, shake up, filter, accurately pipette 25～100 parts by volume filtrates and be placed in beaker, 100 ℃ of water-baths top is concentrated into dry, uses 70% dissolve with ethanol solution, be transferred in 10 parts by volume volumetric flasks constant volume; With 0.45 μ m pin type membrane filtration to 2 parts by volume sample bottle, standby;
Reference substance solution preparation: accurately take respectively reference substance Panax Notoginseng saponin R
10.00569g, ginsenoside Rg
10.00896g, Rb
10.00809g, Re0.00103g, Rg
20.00605g, Rh
10.00409g, Rd0.00787g, Rf0.01180g, Rc0.00619g, add methyl alcohol and dissolve constant volume 25mL, makes reference substance mixed solution;
Determination method: draw respectively need testing solution and reference substance mixed solution 0.005 parts by volume, in injection liquid chromatography, record 170 minutes chromatograms, maximum, the more stable total peak Rg1 of the peak area of usining is as internal standard compound is as object of reference, using its retention time and peak area respectively as 1, calculate each total peak relative retention time; Test sample finger-print and standard finger-print similarity are greater than 0.998;
Gained finger-print should have 16 total peaks, total peak is respectively 1-9,11,12,14-16,19 and No. 27 peaks, and the relative retention time at total peak is respectively: 27.557,37.956,39.968,57.919,63.809,64.917,65.613,66.549,67.785,69.34,69.948,71.733,72.821,73.697,85.395,105.602;
Relative peak area ratio is respectively: 0.204,1.000,0.116,0.006,0.020,0.021,0.006,0.677,0.040,0.026,0.050,0.183,0.015,0.033,0.012,0.013.
5. the detection method of Pien Tze Huang as claimed in claim 4, is characterized in that the method also comprises the steps:
According to described liquid phase chromatogram condition, carry out LC-MS analysis, according to the position in the corresponding numbering in each peak, chromatogram and corresponding mass spectrogram, infer each peak chemical formula, mass spectrum condition is: atomizer pressure-35psi, dry gas flow velocity-9.0L/min, baking temperature-325 ℃;
The chemical formula that each total peak is corresponding is respectively: No. 1-3, No. 8,11-12 peak and No. 14 peaks are respectively Panax Notoginseng saponin Rs
1, ginsenoside Rg
1, Rb
1, Re, Rg
2, Rh
1, R
d; No. 4 peaks are C
41h
70o
13; No. 5 peaks are C
59h
100o
27; No. 6 peaks are C
59h
100o
27; No. 7 peaks are C
42h
72o
14or C
54h
90o
23; No. 9 peaks are C
41h
70o
13; No. 15 peaks are C
36h
62o
9; No. 16 peaks are C
48h
82o
18; No. 19 peaks are C
36h
60o
8; No. 27 peaks are C
18h
32o.
6. the detection method of the Pien Tze Huang as described in claim 4 or 5, is characterized in that the need testing solution in the method is prepared as:
Need testing solution preparation: get the Pien Tze Huang of ten lot numbers corresponding with pseudo-ginseng, every crowd of 1.0g, accurately weighed, be placed in 250mL tool plug conical flask, accurately add 50.00ml70% ethanolic solution, to weigh, placement is spent the night; Put 80 ℃ of water-baths and reflux 2 hours, room temperature to be chilled to, then weigh, with 70% ethanolic solution, supply the weight of less loss, shake up, filter, accurately pipette 25.00ml filtrate and be placed in 50mL beaker, 100 ℃ of water-bath tops are concentrated into dry, use 70% dissolve with ethanol solution, be transferred in 10mL volumetric flask constant volume; To 2mL sample bottle, standby with 0.45 μ m pin type membrane filtration.
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