CN104764828A - Construction method of fingerprint of Ilex rotunda thunb medicinal material and detection method of Ilex rotunda thunb medicinal material - Google Patents

Construction method of fingerprint of Ilex rotunda thunb medicinal material and detection method of Ilex rotunda thunb medicinal material Download PDF

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CN104764828A
CN104764828A CN201510188936.6A CN201510188936A CN104764828A CN 104764828 A CN104764828 A CN 104764828A CN 201510188936 A CN201510188936 A CN 201510188936A CN 104764828 A CN104764828 A CN 104764828A
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mobile phase
holly bark
iron holly
medicinal materials
bark medicinal
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CN104764828B (en
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蒋莉娟
姚江雄
许招懂
方铁铮
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GUANGZHOU XINGQUN PHARMACEUTICAL CO Ltd
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GUANGZHOU XINGQUN PHARMACEUTICAL CO Ltd
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Abstract

The invention discloses a construction method of a fingerprint of Ilex rotunda thunb medicinal material and a detection method of the Ilex rotunda thunb medicinal material. The construction method comprises the following steps: (1) preparation of a reference solution; (2) preparation of a test solution; and (3) determination, namely respectively precisely sucking up the test solution and the reference solution, and injecting the sucked solutions into a high performance liquid chromatograph for determination, thereby obtaining the fingerprint of Ilex rotunda thunb medicinal material composed of 13 common characteristic peaks. For the construction method and the detection method, through a lot of experiments and research by the inventor, the method for preparing the test solution and the reference solution is determined, optimal parameters of chromatographic conditions of the high performance liquid chromatograph are also determined, and therefore, the fingerprint of the Ilex rotunda thunb medicinal material is constructed. With the fingerprint constructed by the method disclosed by the invention and the detection method, the Ilex rotunda thunb medicinal material can be identified accurately, precision is high, and a reagent is saved.

Description

The construction method of iron holly bark medicinal materials fingerprint and detection method thereof
Technical field
The present invention relates to technical field of analysis and detection, particularly relate to a kind of construction method and detection method thereof of iron holly bark medicinal materials fingerprint.
Background technology
Iron holly bark is the dry bark of holly plant ilexrotunda, and its property is bitter, cold.The micro-perfume (or spice) of gas, bitter, micro-puckery.There is effect that is clearing heat and detoxicating, dampness removing pain relieving, be applicable to summer-heat and damp heating, throat swells and ache, damp-heat dysentery, abdominal distention, arthralgia due to wind-dampness, eczema, sore furuncle, traumatic injury etc.Modern pharmacological research shows, iron holly bark also has anti-arrhythmia and function of resisting myocardial ischemia, step-down and reducing heart rate effect, antitumor action, anastalsis etc.
Whether iron holly bark is dry bark, the confusion varieties that current Chinese crude drug has various appearances similar, and be that the method for iron holly bark is comparatively complicated for detecting in prior art, and resultant error is larger.Therefore, be necessary to set up one comprehensively, intuitively, detection method fast.
Summary of the invention
Based on this, the invention provides a kind of construction method of iron holly bark medicinal materials fingerprint.
Concrete technical scheme is as follows:
A construction method for iron holly bark medicinal materials fingerprint, comprises the steps:
(1) preparation of reference substance solution:
Precision takes Syringin reference substance, adds methanol dilution, obtains reference substance solution;
(2) preparation of need testing solution:
Precision takes iron holly bark medicinal powder, adds the ethanol that volumn concentration is 45-55%, ultrasonic extraction or refluxing extraction 50-70min, filters, obtains described need testing solution;
(3) measure
Accurate absorption need testing solution and reference substance solution respectively, injects high performance liquid chromatograph and measures, obtain the iron holly bark medicinal materials fingerprint be made up of 13 common characteristic peaks.
Wherein in an embodiment, in step (3), the condition of high performance liquid chromatograph is: chromatographic column is C18 chromatographic column; Mobile phase: methyl alcohol is mobile phase A, 0.5% (volumetric concentration) glacial acetic acid aqueous solution is Mobile phase B, adopts gradient elution mode; Flow velocity is 0.8-1.2ml/min; Determined wavelength is 288-292nm; Column temperature 23-27 DEG C.
Wherein in an embodiment, described gradient elution mode is: 0-5min, and the percent by volume of mobile phase A is changed to 25% by 20%; 6-15min, the percent by volume of mobile phase A is changed to 35% by 25%; 15-45min, the percent by volume of mobile phase A is changed to 45% by 35%; 45-50min, the percent by volume of mobile phase A is changed to 60% by 45%.
Wherein in an embodiment, determined wavelength is 290nm, and column temperature is 25 DEG C.
Wherein in an embodiment, in step (2), the volumn concentration of ethanol is 50%, and extraction time is 60min.
Another object of the present invention is to provide a kind of detection method of precise Identification iron holly bark medicinal material.
Concrete technical scheme is as follows:
A detection method for iron holly bark medicinal materials fingerprint, comprises the steps:
(1) preparation of reference substance solution:
Precision takes Syringin reference substance, adds methanol dilution, obtains reference substance solution;
(2) preparation of need testing solution:
Precision takes iron holly bark medicinal powder, adds the ethanol that volumn concentration is 45-55%, ultrasonic extraction or refluxing extraction 50-70min, filters, obtains described need testing solution;
(3) measure
Accurate absorption need testing solution and reference substance solution respectively, injects high performance liquid chromatograph and measures, to obtain final product.
Wherein in an embodiment, in step (3), the condition of high performance liquid chromatograph is: chromatographic column is C18 chromatographic column; Mobile phase: methyl alcohol is mobile phase A, 0.5% glacial acetic acid aqueous solution is Mobile phase B, adopts gradient elution mode; Flow velocity is 0.8-1.2ml/min; Determined wavelength is 288-292nm; Column temperature 23-27 DEG C.
Wherein in an embodiment, described gradient elution mode is: 0-5min, and the percent by volume of mobile phase A is changed to 25% by 20%; 6-15min, the percent by volume of mobile phase A is changed to 35% by 25%; 15-45min, the percent by volume of mobile phase A is changed to 45% by 35%; 45-50min, the percent by volume of mobile phase A is changed to 60% by 45%.
Wherein in an embodiment, in step (2), the volumn concentration of ethanol is 50%, and extraction time is 60min; In step (3), the condition of high performance liquid chromatograph is: chromatographic column is C18 chromatographic column; Mobile phase: methyl alcohol is mobile phase A, 0.5% glacial acetic acid aqueous solution is Mobile phase B, adopts gradient elution mode; Flow velocity is 1.0ml/min; Determined wavelength is 290nm; Column temperature 25 DEG C.
Beneficial effect of the present invention is as follows:
The present invention, through the great many of experiments of inventor and research, determines the method preparing need testing solution and reference substance solution, and the optimized parameter of the chromatographic condition of high performance liquid chromatograph, constructs the finger-print of iron holly bark medicinal material thus.The finger-print adopting the application's method to build and detection method can identify iron holly bark medicinal material exactly, and precision is high, and save reagent.
Accompanying drawing explanation
Fig. 1 a is solvent peak comparison diagram;
Fig. 1 b is that standard items Syringin HPLC schemes;
Fig. 2 is that different mobile phase HPLC schemes;
Fig. 3 is the different start gradient HPLC figure of mobile phase;
Fig. 4 is the different gradient HPLC figure of mobile phase;
Fig. 5 is different in flow rate HPLC figure;
Fig. 6 is that different column temperature HPLC schemes;
Fig. 7 is that different wave length HPLC schemes;
Fig. 8 is that different chromatographic column HPLC schemes;
Fig. 9 is that six kinds of Extraction solvent HPLC scheme;
Figure 10 is that the different volume HPLC that extracts schemes;
Figure 11 is that extracting method HPLC not of the same race schemes;
Figure 12 is that different extraction time HPLC schemes;
Figure 13 is that iron holly bark all samples derives HPLC collection of illustrative plates;
Figure 14 is the contrast spectrogram of iron holly bark medicinal materials fingerprint;
Figure 15 is the HPLC collection of illustrative plates of embodiment 2 medicinal material to be measured.
Embodiment
By the following examples the application is further elaborated.
The construction method of embodiment 1 iron holly bark medicinal materials fingerprint
One, instrument, reagent and material
1, instrument
KQ-100B type ultrasonic cleaner (Kunshan ultrasonic instrument company limited); T-214 type electronic analytical balance (Denver company); Waterse2695 highly effective liquid phase chromatographic system, Empower workstation, containing quaternary gradient pump, automatic sampler, UV-detector (Waters company).
2, reagent
Glacial acetic acid (chromatographically pure, Tianjin City Chemical Agent Research Institute); Methyl alcohol, acetonitrile (chromatographically pure, Honeywell company); Water is ultrapure water;
Reference substance is Syringin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number 111574-200603).
3, material
(Guangzhou Baiyunshan Xingqun (Pharmaceutical) Co., Ltd. provides iron holly bark medicinal material, totally 12 batches, lot number or the place of production are respectively XG1404-01, XG1307-01, XG1105-01, XG1110-02,20110401,20111201,2014-0501,2014-0502,2014-0503,2014-0504, Fujian, Guangxi)
Two, method and result
1, chromatographic condition
Chromatographic column: 100RP-18endcapped (5 μm), rT250-4.6 with methyl alcohol (A)-0.5% glacial acetic acid aqueous solution (B) for flow phase system gradient elution (0-5min, 20% → 25%A; 6-15min, 26% → 35%A; 15-45min, 35% → 45%A; 45-50min, 45% → 60%A), flow velocity is 1.0mLmin -1, determined wavelength 290nm; Column temperature 25 DEG C, sample size 10 μ L.
2, the preparation of reference substance solution
Precision takes Syringin reference substance and is about 12mg, adds methyl alcohol appropriate, is mixed with the solution of every 1ml methyl alcohol containing 1.22mg Syringin, product solution in contrast.
3, the preparation of need testing solution
Get iron holly bark medicinal powder (crossing 60 mesh sieves) about 1g, accurately weighed, put in 100ml tool plug conical flask, precision adds 50% ethanol 25ml, and close plug, weighs, ultrasonic process (power 160W, frequency 59kHz) 60 minutes, lets cool, weighed weight again, supply the weight of less loss with 50% ethanol, shake up, 0.22 μm of miillpore filter filters, get subsequent filtrate, to obtain final product.
4, methodological study
The investigation of 4.1 solvent peaks
Get iron holly bark medicinal powder (crossing 60 mesh sieves) about 1g, accurately weighed, put in 100ml tool plug conical flask, precision adds 50% ethanol 25ml, and close plug, weighs, ultrasonic process (power 160W, frequency 59kHz) 60 minutes, lets cool, weighed weight again, supplies the weight of less loss, shakes up with 50% ethanol, 0.22 μm of miillpore filter filters, get subsequent filtrate, HPLC analyzes iron holly bark medicinal material sample and 50% alcohol solvent is blank, obtains Fig. 1.
From Fig. 1 a, by comparing 50% ethanol and iron holly bark medicinal material collection of illustrative plates, can find, solvent is on main chromatographic peak without too large impact, and main chromatographic peak is elected at the obvious peak of uv absorption 1,2,3,4,5,6,7,8,9,10,11,12,13 as.
Fig. 1 b is the standard HPLC spectrogram of reference substance Syringin.
The selection of 4.2 mobile phase compositions
Get 1 part, about 1g iron holly bark medicinal powder sample, accurately weighed, put in 100ml tool plug conical flask, precision adds methyl alcohol 20ml, and close plug, weighs, ultrasonic process (power 160W, frequency 59kHz) 30 minutes, lets cool, weighed weight again, supplies the weight of less loss, shakes up with methyl alcohol, 0.22 μm of miillpore filter filters, get subsequent filtrate HPLC to analyze, mobile phase composition and gradient, in table 1, obtain Fig. 2.
Table 1 four kinds of mobile phase composition information
Numbering Mobile phase forms Time Gradient
JBY1 Acetonitrile (A)-water (B) 0-60min 5％→95％A
JBY2 Acetonitrile (A)-0.5% glacial acetic acid water (B) 0-60min 5％→95％A
JBY3 Methyl alcohol (A)-water (B) 0-60min 5％→95％A
JBY4 Methyl alcohol (A)-0.5% glacial acetic acid water (B) 0-60min 5％→95％A
As shown in Figure 2, methyl alcohol-glacial acetic acid aqueous solution system peak shape is better, goes out peak many, and retention time is applicable to, and selects methanolic acid water system.
The selection of 4.3 mobile phase start gradients
Table 2 three kinds of start gradient information
As shown in Figure 3, when the start gradient of mobile phase methanol-0.5% glacial acetic acid water is 20:80, in figure, the retention time of main peaks is comparatively suitable, and baseline is comparatively steady, therefore selects start gradient 20% methyl alcohol: 80% acetic acid aqueous solution.
4.4 the determination of gradient
The different gradient information of table 3
As shown in Figure 4, retention time is selected to be suitable for, the good gradient of degree of separation and JBY13.
4.5 the determination of flow velocity
As shown in Figure 5, when flow velocity is 1.0ml/min, in figure, the retention time of main peaks is comparatively suitable and baseline steady, therefore selects flow velocity to be 1.0ml/min.
4.6 the determination of column temperature
As shown in Figure 6, along with the rising of column temperature, peak shape is deteriorated, and degree of separation is deteriorated, and considers the retention time of characteristic peak and the protection to chromatographic column, selects column temperature to be 25 DEG C.
4.7 the investigation of determined wavelength
Under above-mentioned chromatographic condition, compare the characteristic peak under 245,254,265,290 and 320nm tetra-wavelength, under 290nm wavelength, go out the more and characteristic peak in peak obvious, therefore select 290nm as optimum absorb wavelength.The results are shown in Figure 7.
The investigation of 4.8 chromatographic columns
The different chromatographic column information of table 4
As shown in Figure 8, the characteristic peak peak shape of four kinds of chromatogram column analysis gained collection of illustrative plates is all better, and the HPLC that all can be used for iron holly bark medicinal material analyzes, this experimental selection LiChrospher chromatographic column.
4.9 Extraction solvent compare
Get 6 parts, about 1g iron holly bark medicinal powder sample, accurately weighed, put in 100ml tool plug conical flask, add methyl alcohol, ethanol, 50% methyl alcohol, 50% ethanol, 75% methyl alcohol, 75% ethanol 20ml respectively, close plug, weighs, ultrasonic process (power 160W, frequency 59kHz) 30 minutes, lets cool, weighed weight again, supply the weight of less loss with methyl alcohol, shake up, 0.22 μm of miillpore filter filters, get subsequent filtrate HPLC and analyze main chromatographic peak peak area in table 5, the results are shown in Figure 9.
The main chromatographic peak peak area of table 5 six kinds of Extraction solvent
Peak number Methyl alcohol Ethanol 50% methyl alcohol 50% ethanol 75% methyl alcohol 75% ethanol Retention time
1 355414 43287 1126145 612666 665064 427967 6.198
2 753862 348529 1202101 1208814 1019759 1064223 7.245
3 7852180 2402752 9562149 11734748 9889989 11219206 9.077
4 6675474 5339880 7459819 8231534 7883031 2276640 10.791
5 2078796 2116441 1935412 2587377 2345356 7894752 12.325
6 4248298 3606709 4807587 5828795 5054246 2409729 23.128
7 14909553 13614380 14743204 16624503 16671205 5552001 24.962
8 892737 899382 973659 1234650 968381 17085179 27.69
9 4286287 3888864 4202010 4470733 4319736 1117468 30.11
10 3690457 3127449 3588076 3850652 3697020 4321404 30.946
11 14211286 11359612 15620417 18132619 17630133 3704611 33.515
12 816490 709192 758246 832788 853132 827163 39.895
13 1923303 1541635 1487506 2164681 2280596 17860407 51.55
The relatively extraction effect of six kinds of solvents, find that the peak area of 50% alcohol extract is maximum, extraction effect is best, so select 50% ethanol to be Extraction solvent.
4.10 extract volume ratio comparatively
Get 4 parts, about 1g iron holly bark medicinal powder sample, accurately weighed, put in 100ml tool plug conical flask, add 50% ethanol 10ml respectively, 20ml, 25ml, 30ml, close plug, weighs, ultrasonic process (power 160W, frequency 59kHz) 30 minutes, let cool, more weighed weight, supply the weight of less loss with 50% ethanol, shake up, 0.22 μm of miillpore filter filters, get subsequent filtrate HPLC to analyze, main chromatographic peak peak area, in table 6, the results are shown in Figure 10.
Four kinds, table 6 extracts the main chromatographic peak peak area of volume
Peak number 10ml 20ml 25ml 30ml
1 550745 382698 225014 182273
2 795399 311685 328838 280487
3 10751015 5431845 4923235 3763573
4 9543766 4814070 3894628 811879
5 2738545 1386119 1114375 3326749
6 5636997 2798505 2270353 957544
7 19302851 9789492 7915744 1918172
8 1084801 527263 429392 6687581
9 5133661 2519510 2036385 367101
10 4366384 2172675 1741729 1714553
11 21334680 10704375 8418868 1430309
12 822726 545299 309345 260308
13 3034318 313810 1072752 7156968
More several extraction volume peak area, the extraction effect of 25ml and 30ml is better than 10ml and 20ml effect, consider save solvent, select 25ml for extract volume.
4.11 DNA extration
Get 2 parts, about 1g iron holly bark medicinal powder sample, accurately weighed, put in 100ml tool plug conical flask, add 50% ethanol 25ml, close plug, weighs, the ultrasonic 30min of close plug (power 160W, frequency 59kHz) and 100 DEG C of water-bath backflow 30min respectively, after letting cool, 50% ethanol is mended heavy, shake up, 0.22 μm of miillpore filter filters, and gets subsequent filtrate HPLC and analyzes, main chromatographic peak peak area, in table 7, the results are shown in Figure 11.
The main chromatographic peak peak area of table 7 two kinds of extracting method
In conjunction with Figure 11 and table 7, ultrasonic suitable with the extraction effect of backflow, consideration method convenient, selects ultrasonic extracting mode.
The comparison of 4.12 ultrasonic extraction times
Get 4 parts, about 1g iron holly bark medicinal powder sample, accurately weighed, put in 100ml tool plug conical flask, precision adds 50% ethanol 25ml, and close plug, weighs, ultrasonic process (power 160W, frequency 59kHz) 15,30,45 and 60 minutes, lets cool respectively, weighed weight again, supplies the weight of less loss, shakes up with 50% ethanol, 0.22 μm of miillpore filter filters, get subsequent filtrate HPLC to analyze, main chromatographic peak peak area, in table 8, the results are shown in Figure 12.
Four kinds, table 8 extraction time main chromatographic peak peak area
Peak number 15min 30min 45min 60min
1 206536 209613 210454 216481
2 339823 353675 363272 337255
3 4291818 4389423 4449125 4474635
4 2062215 2097434 2137429 2180412
5 715504 738274 756388 769509
6 1650413 1541333 1694002 1718805
7 5352594 5204680 5298542 5487156
8 305974 308096 307083 305670
9 1489215 1497402 1503256 1496711
10 1261974 1260612 1259837 1274872
11 5789286 5906794 5949940 6045942
12 313921 319080 321176 327706
13 595234 607105 876232 980172
The peak area of main chromatographic peak, during ultrasonic extraction 60min, extraction effect is best, therefore the selective extraction time is 60min.
5, methodology
Chromatographic condition: chromatographic column is C18 chromatographic column; Mobile phase: methyl alcohol is mobile phase A, 0.5% glacial acetic acid aqueous solution is Mobile phase B, adopts gradient elution mode; Flow velocity is 1.0ml/min; Determined wavelength is 290nm; Column temperature 25 DEG C.
Gradient elution mode is: 0-5min, and the percent by volume of mobile phase A is changed to 25% by 20%; 6-15min, the percent by volume of mobile phase A is changed to 35% by 25%; 15-45min, the percent by volume of mobile phase A is changed to 45% by 35%; 45-50min, the percent by volume of mobile phase A is changed to 60% by 45%.
5.1 precision are investigated
Get the iron holly bark medicinal material need testing solution that lot number is 20111201, under above-mentioned chromatographic condition, continuous sample introduction 5 times, record chromatographic fingerprinting, result shows, each relative retention time at total peak and the RSD of relative peak area are all less than 2%, meet the requirements.Concrete data are in table 9, table 10.
Table 9 relative retention time precision
Peak number 1 2 3 4 5 RSD％
1 0.267095 0.266995 0.267292 0.266914 0.267076 0.05
2 0.312683 0.312719 0.313043 0.312711 0.312943 0.05
3 0.391081 0.391158 0.391411 0.391126 0.391449 0.06
4 0.466284 0.466572 0.466799 0.466647 0.467059 0.04
5 0.533069 0.533342 0.53346 0.53331 0.533676 0.04
6 1 1 1 1 1 0
7 1.078829 1.079174 1.079578 1.079927 1.080019 0.05
8 1.1988 1.199404 1.199291 1.199516 1.199983 0.04
9 1.304179 1.304508 1.304618 1.304675 1.304989 0.02
10 1.339924 1.340507 1.340562 1.340491 1.340654 0.02
11 1.447332 1.448291 1.448525 1.448198 1.448426 0.03
12 1.719824 1.720688 1.721087 1.72047 1.72082 0.03
13 2.224486 2.22663 2.22612 2.225957 2.22752 0.05
Table 10 relative peak area precision
Peak number 1 2 3 4 5 RSD％
1 0.131753 0.131685 0.131815 0.132281 0.133781 0.67
2 0.192835 0.193003 0.192044 0.19164 0.193077 0.33
3 2.647182 2.673209 2.645649 2.649438 2.643687 0.46
4 1.228096 1.233376 1.222277 1.218588 1.216263 0.57
5 0.436703 0.440103 0.433092 0.436298 0.433922 0.63
6 1 1 1 1 1 0
7 3.155146 3.179746 3.149672 3.150997 3.144388 0.44
8 0.175927 0.177852 0.175339 0.177087 0.175512 0.61
9 0.849321 0.850986 0.841967 0.845215 0.840952 0.52
10 0.705366 0.713068 0.700637 0.700755 0.700099 0.78
11 3.535997 3.515968 3.48736 3.494529 3.440437 1.03
12 0.194209 0.18953 0.187805 0.18635 0.188006 1.60
13 0.614107 0.617204 0.608314 0.6328 0.634347 1.87
5.2 repeatability are investigated
Get the iron holly bark medicinal material test sample 6 parts that lot number is 20111201, accurately weighed, need testing solution is prepared by under " need testing solution preparation method " item, sample introduction analysis under above-mentioned chromatographic condition, with the value of relative retention time and relative peak area for index, calculate its RSD, its RSD is all less than 2.0%, meets the requirements.Concrete data are in table 11, table 12.
Table 11 relative retention time repeatability
Peak number 1 2 3 4 5 6 RSD％
1 0.267492 0.267703 0.267746 0.267616 0.267997 0.267767 0.06
2 0.313444 0.313656 0.313625 0.313539 0.314012 0.313369 0.07
3 0.391761 0.392091 0.39188 0.39175 0.392321 0.393097 0.13
4 0.46731 0.467842 0.467581 0.467538 0.468032 0.468191 0.07
5 0.533945 0.534324 0.53415 0.533977 0.534479 0.534537 0.05
6 1 1 1 1 1 1 0
7 1.080135 1.080471 1.080549 1.080809 1.081209 1.081374 0.04
8 1.200294 1.200615 1.200744 1.200831 1.20116 1.201161 0.03
9 1.305352 1.306033 1.305964 1.305834 1.306264 1.305963 0.02
10 1.341353 1.342024 1.341759 1.341716 1.34215 1.341692 0.02
11 1.44931 1.450041 1.449879 1.449749 1.450284 1.450002 0.02
12 1.721951 1.723158 1.723165 1.723122 1.723259 1.722966 0.03
13 2.229328 2.231106 2.229744 2.22983 2.230769 2.231129 0.03
Table 12 relative peak area repeatability
Peak number 1 2 3 4 5 6 RSD％
1 0.133211 0.13127 0.135826 0.134222 0.130898 0.130535 1.59
2 0.195433 0.192876 0.192096 0.192142 0.188766 0.188926 1.32
3 2.652451 2.647589 2.642564 2.6977 2.615283 2.609127 1.20
4 1.221193 1.184481 1.210676 1.20396 1.17561 1.208106 1.43
5 0.436519 0.429028 0.432835 0.427659 0.422544 0.426969 1.13
6 1 1 1 1 1 1 0
7 3.139474 3.148943 3.135862 3.165643 3.135045 3.133823 0.39
8 0.175117 0.17525 0.172635 0.173291 0.172633 0.17121 0.91
9 0.837758 0.835354 0.826243 0.824007 0.826659 0.822924 0.75
10 0.703122 0.699284 0.698885 0.705663 0.6969 0.695484 0.55
11 3.504016 3.505718 3.483028 3.501747 3.45838 3.496906 0.52
12 0.181122 0.185691 0.181703 0.182372 0.181716 0.183711 0.93
13 0.419946 0.402179 0.424806 0.414647 0.40952 0.412618 1.92
5.3 study on the stability
Get with a need testing solution, put in ambient temperatare, respectively 0,4,8,12,15,24h sample introduction analyzes, calculate relative retention time and the relative peak area of each chromatographic peak, its RSD is all less than 2.0%, and experimental result shows, need testing solution is good at 24h internal stability.Concrete data are in table 13, table 14.
Table 13 relative retention time stability
Peak number 1 2 3 4 5 6 RSD％
1 0.267492 0.267368 0.267718 0.267865 0.268022 0.268543 0.16
2 0.313444 0.313336 0.313699 0.313816 0.314058 0.314527 0.14
3 0.391761 0.391681 0.392221 0.392594 0.392735 0.393289 0.16
4 0.46731 0.467558 0.467848 0.468125 0.468421 0.468889 0.12
5 0.533945 0.533957 0.534183 0.534604 0.534874 0.535311 0.10
6 1 1 1 1 1 1 0
7 1.080135 1.080855 1.081115 1.08181 1.082015 1.082052 0.07
8 1.200294 1.201489 1.201037 1.201343 1.201396 1.200909 0.04
9 1.305352 1.306237 1.305791 1.30628 1.306168 1.305651 0.03
10 1.341353 1.342163 1.341616 1.342139 1.342061 1.341459 0.03
11 1.44931 1.450331 1.449784 1.451278 1.451385 1.450357 0.06
12 1.721951 1.72298 1.721953 1.723127 1.723395 1.722407 0.04
13 2.229328 2.22971 2.226966 2.231269 2.23278 2.231262 0.09
Table 14 relative peak area stability
Peak number 1 2 3 4 5 6 RSD％
1 0.138813 0.132942 0.135711 0.139004 0.134821 0.13906 1.91
2 0.195433 0.191587 0.19259 0.194203 0.197755 0.19433 1.12
3 2.652451 2.638877 2.612331 2.634957 2.620315 2.612812 0.61
4 1.221193 1.207863 1.224737 1.227889 1.225567 1.20658 0.77
5 0.436519 0.4325 0.431123 0.433084 0.431592 0.428844 0.59
6 1 1 1 1 1 1 0
7 3.139474 3.118189 3.098322 3.101288 3.092839 3.102113 0.56
8 0.175117 0.17367 0.173267 0.172861 0.172163 0.172834 0.58
9 0.837758 0.829948 0.825173 0.827874 0.822607 0.822159 0.70
10 0.703122 0.69394 0.687885 0.690568 0.691131 0.689601 0.79
11 3.616057 3.561395 3.537886 3.546473 3.550033 3.553876 0.79
12 0.181122 0.185233 0.180531 0.184996 0.178451 0.181668 1.46
13 0.408742 0.39996 0.403322 0.400054 0.401984 0.401775 0.81
2, iron holly bark medicinal materials fingerprint result of study
2.1 all samples
The iron holly bark medicinal material of different batches selected by sample, totally 12 batches, in table 15.
Table 15 all samples information
2.2 iron holly bark medicinal material collection of illustrative plates
Use similarity evaluation (2.0 editions), after importing sample, reference collection of illustrative plates is S1, contrast collection of illustrative plates generation method adopts average method, time window width is located between 0.1-0.5 respectively, and similarity result is stablized, so select time window width is 0.1, Auto-matching generates contrast spectrogram, and overall output sees Figure 13.Use similarity evaluation 2.0 editions, import contrast collection of illustrative plates, output image, obtains Figure 14.
By Figure 13 more each batch of chromatogram, all have 13 stable peaks, and degree of separation is good.Therefore determine that these 13 peaks are for total peak.
2.2 calculate similarity
The similarity calculating all 12 batches of medicinal materials is followed successively by 0.987,0.995,0.973,0.981,0.945,0.991,0.993,0.946,0.939,0.942,0.969,0.942, is all greater than 0.9, illustrates that iron holly bark medicinal material sample has good similarity.
From above data, the iron holly bark medicinal material of the different lot numbers that 1-12 sample provides for same manufacturing enterprise, what wherein two samples were indicated is the place of production is not lot number, 12 batches of iron holly bark medicinal materials all can detect 13 total peaks, quality of medicinal material situation between the difference having peak-to-peak peak area in finger-print reflects to a certain extent and criticizes.Medicinal material sample has good similarity, and similarity is all more than 0.9.
The detection method of embodiment 2 iron holly bark medicinal materials fingerprint
The detection method of a kind of iron holly bark medicinal materials fingerprint of the present embodiment, comprises the steps:
(1) preparation of reference substance solution:
Precision takes Syringin reference substance and is about 12mg, adds methyl alcohol appropriate, is mixed with the solution of every 1ml methyl alcohol containing 1.22mg Syringin, product solution in contrast.
(2) preparation of need testing solution:
Get medicinal powder to be measured (lot number is: 20140801, crosses 60 mesh sieves) about 1g, accurately weighed, put in 100ml tool plug conical flask, precision adds 50% ethanol 25ml, close plug, weigh, ultrasonic process (power 160W, frequency 59kHz) 60 minutes, let cool, more weighed weight, the weight of less loss is supplied with 50% ethanol, shake up, 0.22 μm of miillpore filter filters, and gets subsequent filtrate, to obtain final product.
(3) measure
Accurate absorption need testing solution and reference substance solution respectively, injects high performance liquid chromatograph and measures, to obtain final product.
Spectrogram as shown in figure 15.
By comparing with Figure 14, known, the finger-print of this medicinal material sample is 0.964 with the similarity contrasting spectrogram, can think that this medicinal material sample is iron holly bark medicinal material.
Each technical characteristic of the above embodiment can combine arbitrarily, for making description succinct, the all possible combination of each technical characteristic in above-described embodiment is not all described, but, as long as the combination of these technical characteristics does not exist contradiction, be all considered to be the scope that this instructions is recorded.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be construed as limiting the scope of the patent.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.

Claims (9)

1. a construction method for iron holly bark medicinal materials fingerprint, is characterized in that, comprises the steps:
(1) preparation of reference substance solution:
Precision takes Syringin reference substance, adds methanol dilution, obtains reference substance solution;
(2) preparation of need testing solution:
Precision takes iron holly bark medicinal powder, adds the ethanol that volumn concentration is 45-55%, ultrasonic extraction or refluxing extraction 50-70min, filters, obtains described need testing solution;
(3) measure
Accurate absorption need testing solution and reference substance solution respectively, injects high performance liquid chromatograph and measures, obtain the iron holly bark medicinal materials fingerprint be made up of 13 common characteristic peaks.
2. the construction method of iron holly bark medicinal materials fingerprint according to claim 1, is characterized in that, in step (3), the condition of high performance liquid chromatograph is: chromatographic column is C18 chromatographic column; Mobile phase: methyl alcohol is mobile phase A, 0.5% glacial acetic acid aqueous solution is Mobile phase B, adopts gradient elution mode; Flow velocity is 0.8-1.2ml/min; Determined wavelength is 288-292nm; Column temperature 23-27 DEG C.
3. the construction method of iron holly bark medicinal materials fingerprint according to claim 2, is characterized in that, described gradient elution mode is: 0-5min, and the percent by volume of mobile phase A is changed to 25% by 20%; 6-15min, the percent by volume of mobile phase A is changed to 35% by 25%; 15-45min, the percent by volume of mobile phase A is changed to 45% by 35%; 45-50min, the percent by volume of mobile phase A is changed to 60% by 45%.
4. the construction method of iron holly bark medicinal materials fingerprint according to claim 2, is characterized in that, determined wavelength is 290nm, and column temperature is 25 DEG C.
5. the construction method of the iron holly bark medicinal materials fingerprint according to any one of claim 1-4, is characterized in that, in step (2), the volumn concentration of ethanol is 50%, and extraction time is 60min.
6. a detection method for iron holly bark medicinal materials fingerprint, is characterized in that, comprises the steps:
(1) preparation of reference substance solution:
Precision takes Syringin reference substance, adds methanol dilution, obtains reference substance solution;
(2) preparation of need testing solution:
Precision takes iron holly bark medicinal powder, adds the ethanol that volumn concentration is 45-55%, ultrasonic extraction or refluxing extraction 50-70min, filters, obtains described need testing solution;
(3) measure
Accurate absorption need testing solution and reference substance solution respectively, injects high performance liquid chromatograph and measures, to obtain final product.
7. the detection method of iron holly bark medicinal materials fingerprint according to claim 6, is characterized in that, in step (3), the condition of high performance liquid chromatograph is: chromatographic column is C18 chromatographic column; Mobile phase: methyl alcohol is mobile phase A, 0.5% glacial acetic acid aqueous solution is Mobile phase B, adopts gradient elution mode; Flow velocity is 0.8-1.2ml/min; Determined wavelength is 288-292nm; Column temperature 23-27 DEG C.
8. the detection method of iron holly bark medicinal materials fingerprint according to claim 7, is characterized in that, described gradient elution mode is: 0-5min, and the percent by volume of mobile phase A is changed to 25% by 20%; 6-15min, the percent by volume of mobile phase A is changed to 35% by 25%; 15-45min, the percent by volume of mobile phase A is changed to 45% by 35%; 45-50min, the percent by volume of mobile phase A is changed to 60% by 45%.
9. the detection method of iron holly bark medicinal materials fingerprint according to claim 6, is characterized in that, in step (2), the volumn concentration of ethanol is 50%, and extraction time is 60min; In step (3), the condition of high performance liquid chromatograph is: chromatographic column is C18 chromatographic column; Mobile phase: methyl alcohol is mobile phase A, 0.5% glacial acetic acid aqueous solution is Mobile phase B, adopts gradient elution mode; Flow velocity is 1.0ml/min; Determined wavelength is 290nm; Column temperature 25 DEG C.
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CN111948316A (en) * 2020-08-13 2020-11-17 广西中医药大学 Establishment method and detection method of Zhuang medicine Jinmu granule fingerprint spectrum
CN113219095A (en) * 2021-05-07 2021-08-06 广东在田药业股份有限公司 Construction method of fingerprint of Fukean tablet and fingerprint thereof
CN114644674A (en) * 2020-12-21 2022-06-21 广西大学 Method for separating eclipta saponin I from ovate leaf holly bark

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Publication number Priority date Publication date Assignee Title
CN111948316A (en) * 2020-08-13 2020-11-17 广西中医药大学 Establishment method and detection method of Zhuang medicine Jinmu granule fingerprint spectrum
CN114644674A (en) * 2020-12-21 2022-06-21 广西大学 Method for separating eclipta saponin I from ovate leaf holly bark
CN114644674B (en) * 2020-12-21 2023-05-12 广西大学 Method for separating eclipta saponin I from holly bark
CN113219095A (en) * 2021-05-07 2021-08-06 广东在田药业股份有限公司 Construction method of fingerprint of Fukean tablet and fingerprint thereof

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