CN106525989A - Detection method of fructus aurantii medicine material fingerprint and standard fingerprint - Google Patents
Detection method of fructus aurantii medicine material fingerprint and standard fingerprint Download PDFInfo
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- G—PHYSICS
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Abstract
The invention discloses a detection method of a fructus aurantii medicine material fingerprint and a standard fingerprint, and application of the fructus aurantii medicine material fingerprint or the standard fingerprint in identifying a fructus aurantii medicine material. The method includes the steps that firstly, the fructus aurantii medicine material is obtained and smashed into coarse powder and precisely weighed, methyl alcohol is added, extraction is conducted, a subsequent filtrate obtained, and a test sample solution is obtained; secondly, a reference substance solution is prepared; thirdly, the test sample solution and the reference substance solution are precisely sucked and injected into a high performance liquid chromatograph respectively to be measured, and the fructus aurantii fingerprint is obtained. The measurement method is easy and convenient to operate, characteristic components are maintained completely, repeatability and stability are good, precision is high, specificity is high, and the method plays a positive role in guiding production charging and guaranteeing quality reliability. The fructus aurantii medicine material fingerprint built through the method can be effectively used for monitoring the quality of the fructus aurantii medicine material and identifying whether the fructus aurantii medicine material is true or fake.
Description
Technical field
The invention belongs to pharmaceutical analysiss technical field, more particularly to a kind of Fructus Aurantii medicinal materials fingerprint and its method for building up.In particular it relates to a kind of high performance liquid chromatography of Fructus Aurantii medical material（HPLC）Changing Wave finger printing and its method for building up, and the standard finger-print of Fructus Aurantii.The invention further relates to a kind of good and bad discrimination method of Fructus Aurantii quality of medicinal material.
Background technology
Fructus Aurantii is that Rutaceae citrus plant Citrus aurantium Linn. Citrus aurantium L and its variety are dried immature fruit.《Chinese Pharmacopoeia》The variety that version in 2015 is recorded is citrus aurantium "Huangpi" (Citrus
Aurantium ' Huangpi '), the pool orange（Citms aurantium ' Tangcheng '), Citrus aurantium L. Var decumana Bonar (Citrus aurantium ' Chuluan '), CitrusaurantiumL.Var.amara Engl. (Citrus aurantium ' Daidai ') be dried immature fruit, Fructus Aurantii is to harvest when July peel is still green, is parts of generic medicinal plants.It is Fructus Aurantii bitter in the mouth, pungent, sour, temperature, returns spleen, stomach, with promoting the circulation of QI to alleviate the stagnation in middle-JIAO, help digestion, the function such as resolving sputum, for chest distress distending pain, accumulation of food in the stomach and intes tine due to indigestion, phlegm retention, gastroptosis, proctoptosis, uterine prolapse.Fructus Aurantii crude drug source is in Citrus aurantium Linn. and Fructus Citri sinensiss, due to the growth conditionss such as the geographical environment in each place of production, weather, soil are different and harvesting after processing and concocting method it is different, make Fructus Aurantii Immaturus widely different on chemical composition composition and content, thus directly influence quality of medicinal material.
2015 editions States Pharmacopoeia specifications, using naringin, neohesperidin as Fructus Aurantii quality control index（Chinese Pharmacopoeia Commission compiles, Pharmacopoeia of People's Republic of China version in 2015, Beijing：Chinese Medicine industry publishing house, 2015, one：246）, the method for carrying out Chinese medicine quality control using two indices composition can not reflect the type and quantity of Chinese medicine intrinsic chemical composition comprehensively, there is certain limitation, it is impossible to control quality of medicinal material comprehensively.
Main active in Fructus Aurantii is volatile oil, flavone compound and a small amount of alkaloid compound, and its pharmacological action should be the result of Multiple components comprehensive function.Therefore it is incomplete only with single component or several principle active components come the quality for controlling and evaluating Fructus Aurantii.And finger printing is used as a kind of comprehensive, quantifiable detection meanss for evaluating Chinese medicine quality, just suitable for the multicomponent quality evaluation of Chinese medicine.
Research for Fructus Aurantii relates generally to cultivation technique and extraction and separation technology, and quality determining method discloses less, such as Chinese patent 201410837547.7（Publication number CN104587014A）The quality determining method being related to mainly carries out total flavones detection to Fructus Aurantii extract, is not that Fructus Aurantii medical material is detected, also quality is not controlled comprehensively, only determine the total constituents of total flavones.《Strait Pharmaceutical Journal》6th phase in 2003《The research of green clothing Fructus Aurantii finger printing》Describe one kind application gas chromatogram（GC）The method for determining Naphtha GC fingerprint in green clothing Fructus Aurantii；《Chinese Pharmaceutical Journal》O. 11th in 2005《Chinese medicine Fructus Aurantii HPLC fingerprint map analyzings and Chemical Pattern Recognition sort research》Describe and apply efficient liquid phase（HPLC）The fingerprint spectrum method of Fructus Aurantii is determined, but only 8 total peaks and only points out three index components such as naringin, Hesperidin, neohesperidin；《Chinese herbal medicine》3rd phase in 2009《Different cultivar Fructus Aurantii HPLC finger printing researchs》The HPLC fingerprint spectrum methods for determining Fructus Aurantii are described, but only with single wavelength, are pointed out without index components being carried out to total peak；《CHINA JOURNAL OF CHINESE MATERIA MEDICA》20th phase in 2008《The capillary electrophoresis fingerprint research of different sources and kind Fructus Aurantii medical material》Describe the fingerprint spectrum method using Capillary Electrophoretic Determination Fructus Aurantii；《Contemporary Chinese Chinese medicine》12nd phase in 2009《The research of parched with bran Fructus Aurantii HPLC finger printing》The fingerprint spectrum method using high-performance liquid chromatogram determination Fructus Aurantii processed product is described, but is determined only with single wavelength, only identify naringin, neohesperidin, 3 characteristic peaks of Hesperidin；《Chinese Journal of Modern Applied Pharmacy》4th phase in 2015《The quality research of commercially available Fructus Aurantii Praeparata is studied with finger printing》The fingerprint spectrum method using high-performance liquid chromatogram determination Fructus Aurantii Praeparata is described, is only had 5 total peaks using single wavelength and is only pointed out four index components such as Folium Symplocoris Caudatae naringin, naringin, Hesperidin, neohesperidin.In prior art, the index components of identification are few, it is impossible to reflect the quality good or not of medical material comprehensively, it is ensured that the curative effect of medication, therefore, the fingerprint atlas detection method and standard finger-print method for setting up Fructus Aurantii medical material has very important significance.
The content of the invention
The technical problem to be solved is to provide a kind of assay method of Fructus Aurantii finger printing, quality of the method in control Fructus Aurantii, used in effectively instructing clinical and production, ensures the effective of medication, safety and reliable has positive role.The method of foundation and Fructus Aurantii medical material HPLC standard finger-prints can be used in identifying or differentiating that the Fructus Aurantii quality of medicinal material true and false is good and bad, with higher credibility and high sensitivity.Thus provide following inventions：
A kind of detection method of Fructus Aurantii medicinal materials fingerprint of the present invention, comprises the following steps：
（1）The preparation of need testing solution：Fructus Aurantii medicinal material coarse powder is taken, it is accurately weighed, add methanol, ultrasound or reflux, extract, extracting solution to take subsequent filtrate with micro-pore-film filtration, obtain final product need testing solution；
（2）The preparation of reference substance solution：Folium Symplocoris Caudatae naringin reference substance, naringin reference substance, Hesperidin reference substance, neohesperidin reference substance, limonin reference substance, Nobiletin reference substance, Nomilin reference substance, Exocarpium Citri Rubrum element reference substance is taken respectively, it is accurately weighed, it is separately added into methanol and makes reference substance solution；
（3）High performance liquid chromatography detection：Accurate respectively to draw need testing solution and reference substance solution, injection high performance liquid chromatograph is determined, and obtains final product Fructus Aurantii finger printing；
Wherein, chromatographic condition is：Adopt octadecylsilane chemically bonded silica for filler chromatographic column, 4.6 × 250mm, 5 μm；Column temperature：20~40℃ ；Detection wavelength is 280nm, 215nm, and switching time is 0min, 24.01min；Sample size is 10 μ l；Flow velocity：1.0ml/min；Analysis time is 70min；It is acetonitrile that mobile phase is mobile phase A, and Mobile phase B is 0.1% phosphoric acid solution；Using gradient elution mode：0min → 25min → 25.01min → 35min → 60min → 70min, acetonitrile 20% → 24% → 30% → 40% → 95% → 100%.
The step（1）Fructus Aurantii coarse powder and methanol ratio be 0.2g:50ml.
The step（1）Ultrasound or reflux extracting time be 1 hour.
The step（2）In reference substance solution be specially：Per the 1ml respectively 70ng of naringin containing Folium Symplocoris Caudatae, naringin 80ng, Hesperidin 40ng, neohesperidin 80ng, limonin 70ng, Nobiletin 80ng, Nomilin 70ng, the mixed solution of Exocarpium Citri Rubrum element 60ng.
In Fructus Aurantii finger printing, 12 chromatographic peaks are confirmed as total peak, by the comparison with reference substance retention time, determine that peak 1 is Folium Symplocoris Caudatae naringin, peak 2 is naringin, and peak 3 is Hesperidin, peak 4 is neohesperidin, peak 8 is limonin, and peak 9 is Nobiletin, and peak 10 is Nomilin, peak 11 is Exocarpium Citri Rubrum element, and the relative retention time ranges of 12 total fingerprint peakses of the finger printing and relative standard deviation are as follows：
No. 1 peak：0.680～
0.692, RSD is 0.68%；
No. 2 peaks：0.789～
0.804, RSD is 0.92%；
No. 3 peaks：0.862～
0.877, RSD is 0.75%；
No. 4 peaks：1, RSD is 0%；
No. 5 peaks：1.277～
1.296, RSD is 0.51%；
No. 6 peaks：1.893～
1.924, RSD is 0.74%；
No. 7 peaks：2.322～
2.345, RSD is 0.65%；
No. 8 peaks：2.530～
2.547, RSD is 1.02%；
No. 9 peaks：2.670～
2.696, RSD is 0.84%；
No. 10 peaks：2.765～
2.828, RSD is 0.34%；
No. 11 peaks：2.834～
2.854, RSD is 0.59%；
No. 12 peaks：3.560～
3.571, RSD is 0.64%.
The present invention compared with prior art, has the advantages that：
（1）The wavelength switching finger printing that the present invention sets up can more fully react the material information of Chinese medicine compared with Single wavelength finger printing, overcome the effective ingredient detected by Fructus Aurantii quality determining method single, it is impossible to the defect of reaction Fructus Aurantii quality comprehensively.By the conversion of wavelength, the visualization for enhancing fingerprint peakses is compared, and has pointed out 8 index components, has more effectively characterized the quality information of Fructus Aurantii, is more beneficial for strengthening the quality monitoring to medical material；
（2）The standard finger-print that the present invention is obtained compares, and can effectively monitor the quality of Fructus Aurantii medical material, the perfect quality evaluation system of Fructus Aurantii medical material, and for comprehensive, the effective control of Fructus Aurantii quality of medicinal material, to provide theory and practice basic；
（3）The inventive method is applied to the discriminating of the Fructus Aurantii medical material true and false and quality, is conducive to guidance to feed intake, specification production operation, really guarantee clinical application safely, effectively, it is controllable；
（4）The inventive method is easy to operate, stable, reproducible, and conclusion is objective, accurate.
Description of the drawings
Fig. 1 is Fructus Aurantii medical material standard finger-print, and wherein peak 1 is Folium Symplocoris Caudatae naringin, and peak 2 is naringin, and peak 3 is Hesperidin, and peak 4 is neohesperidin, and peak 8 is limonin, and peak 9 is Nobiletin, and peak 10 is Nomilin, and peak 11 is Exocarpium Citri Rubrum element.
Fig. 2 is 10 batch Fructus Aurantii medicinal materials fingerprints.
Fig. 3 is 8 reference substance mixed solution collection of illustrative plates.
Fig. 4 is the problematic medical material of two qualities of lot that detects and standard finger-print comparison diagram in embodiment 2.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limit the scope of the present invention.In addition, it is to be understood that after the content for having read instruction of the present invention, people in the art
Member can be made various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Scope.
Medical material of the present invention, reference substance source are as follows：
Neohesperidin reference substance（National Institute for Food and Drugs Control, 111857-201102）；Hesperidin reference substance（National Institute for Food and Drugs Control, 110738-201337）；Naringin reference substance（National Institute for Food and Drugs Control, 110721-201316）；Limonin reference substance（National Institute for Food and Drugs Control, 110800-201400）；Nomilin reference substance（Hubei Tuo Chukangyuan medication chemistries company limited, 1163770-201137）；Nobiletin reference substance（Shanghai Chun You bio tech ltd, 487013-201393）；Exocarpium Citri Rubrum element reference substance（Yan Yu chemical reagent company limited, 481538-2015422）；Folium Symplocoris Caudatae naringin is compareed（Nanjing Jing Zhu bio tech ltd）.
10 batches pick up from the Fructus Aurantii medical material of different producing area includes Hunan Yuanjiang, Hunan Hanshou, Sichuan Suining, Jinhua, Zhejiang, Zhejiang Lanxi, Jiangxi Xingan, high, Jiangxi Xinyu on Jiangxi；The essential information such as tables 1 such as each Fructus Aurantii medical material test sample source.
| Sample | Source |
| Sample 1 | Yuanjiang Yangmeishan |
| Sample 2 | Yuanjiang Chi Shan |
| Sample 3 | Three pools of Yuanjiang |
| Sample 4 | Hanshou egression bridge |
| Sample 5 | Sichuan Suining |
| Sample 6 | Jinhua, Zhejiang |
| Sample 7 | Zhejiang Lanxi |
| Sample 8 | Jiangxi Xingan |
| Sample 9 | It is high on Jiangxi |
| Sample 10 | Jiangxi Xinyu |
Embodiment 1：The foundation and checking of Fructus Aurantii wavelength switching finger printing.
1.1 instruments and reagent：
1260 high performance liquid chromatograph of Agilent, is contained in line vacuum degassing machine（G-1311C）, binary pump（G-1311C）, standard automatic sampler（G-1329B）, intelligent column oven（G-1316A）, DAD detectors（G-1314B）、Agilent1260
Infinity chromatographic work stations（Anjelen Sci. ＆ Tech. Inc of the U.S.）；SB-5200D ultrasonic washing instruments（NingBo XinZhi Biology Science Co., Ltd）；FAZ004B analytical balances（Section is helped in Shanghai）；BT125D electronic balances（Sai Duolisi scientific instrument（Beijing）Company limited）.
The selection of 1.2 chromatographic conditions.
（1）The selection of mobile phase
Select -0.01% phosphoric acid solution of acetonitrile, -0.1% phosphoric acid solution of acetonitrile, -4% phosphoric acid solution of acetonitrile, -0.01% formic acid solution of acetonitrile, -0. 1% formic acid solution of acetonitrile, , -4% formic acid solution of acetonitrile, -0.01% glacial acetic acid solution of acetonitrile, -0. 1% glacial acetic acid solution of acetonitrile, , -4% glacial acetic acid solution of acetonitrile, Deng being tested for mobile phase, as a result show, when above-mentioned each solution is mobile phase, the peak shape of chromatographic peak is more satisfactory, each peak separates more complete, when wherein -0.1% phosphoric acid solution of acetonitrile is mobile phase, effect is best, therefore from -0.1% phosphoric acid solution of acetonitrile as optimal flow phase.
Due to complicated component in sample, under constant current conditions, each composition separating degree in sample is poor, it is more difficult to embody the overall permanence of finger printing；Mobile phase is run with such as Gradient, and in sample, each component has preferably separation, and baseline is steady, therefore is optimal gradient from the gradient, is shown in Table 2.
2 gradient table of table.
| Time（min） | Acetonitrile（%） | 0.1% phosphoric acid solution（%） |
| 0～25 | 20～24 | 80～76 |
| 25.01～35 | 30～40 | 70～60 |
| 35～60 | 40～95 | 60～5 |
| 60～70 | 95～100 | 5～0 |
（2）The selection of Detection wavelength
Compared by carrying out all-wave length measure to test sample, when the program of program wavelength switching is that 0 ~ 24min selects 300nm wavelength, when 24.01 ~ 70min selects 215nm wavelength, greatly enhances the visualization of fingerprint peakses, make sample characteristic peak most abundant.
（3）The selection of chromatographic column
By comparing Agilent ZORBAX XDB-C18（5 μm, 4.6 × 250mm）, Thermo Syncronis-C18（5 μm, 4.6 × 250mm）, Unitary-C18,（5 m, 4.6mm × 250mm）, enlightening horse Diamonsil-C18（5 μm, 4.6 × 250mm）The chromatographic column of 4 kinds of different model specifications, as a result shows, the chromatographic column of 4 kinds of brands of the above can reach that retention time is moderate, and peak number mesh is more, the good comparatively ideal chromatograph effect of separating degree.
（4）The selection of column temperature
Investigated 20 DEG C of different column temperatures, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C when separating effect to same sample.As a result show, separation of the different temperatures on chromatographic peak affects less, all more satisfactory with separating effect when 20 DEG C ~ 40 DEG C.
（5）Selection different in flow rate
In the case of other chromatographic condition identicals, investigation flow velocity is 0.8 ml/min, 1.0 ml/min, 1.2ml/min on the detached impact of same sample chromatographic peak respectively.As a result show, when flow velocity is 1.0ml/min, chromatographic peak retention time is moderate, preferably, repeatability is best for separating degree.
The preparation of 1.3 reference substance solution
Precision weighs Folium Symplocoris Caudatae naringin reference substance, naringin reference substance, Hesperidin reference substance, neohesperidin reference substance, limonin reference substance, Nobiletin reference substance, Nomilin reference substance, Exocarpium Citri Rubrum element reference substance respectively, it is placed in volumetric flask, plus methanol dissolves and dilutes and makes mixed solutions of every 1ml containing 40ug, 70ug, 40ug, 80ug, 40ug, 40ug, 40ug, 80ug, obtains final product reference substance solution.
The preparation of 1.4 need testing solutions
Fructus Aurantii medicinal material coarse powder 0.2g is taken respectively, it is accurately weighed, put in conical flask with cover, accurate methanol 50ml, close plug, weighed weight, reflux, extract, 1h let cool, then weighed weight, supply the weight of less loss with methanol, shake up, filtration, take subsequent filtrate, as need testing solution.
1.5 algoscopy
It is accurate respectively to draw 10 μ l of need testing solution and reference substance solution, high performance liquid chromatograph is injected, is determined.
The determination of 1.6 analysis times
Under above-mentioned chromatographic condition, sample introduction is investigated 120 minutes, is as a result shown, in 70 minutes, in sample, all peaks can be eluted completely, therefore be defined as 70 minutes analysis time.
1.7 Fructus Aurantii methodological studies.
（1）Stability test
The same need testing solution of Fructus Aurantii being taken respectively, under above-mentioned liquid phase chromatogram condition, being detected at 0,2,4,6,12,24 hours respectively, 10 μ l of each sample introduction investigate the concordance of the relative retention time and relative peak area of main chromatographic peak.As a result RSD ＜ 3% of the relative retention time and relative peak area of the main chromatographic peak of Fructus Aurantii, show more stable in need testing solution 24 hours.
（2）Precision test
Take with portion Fructus Aurantii need testing solution, under above-mentioned liquid phase chromatogram condition, repeat sample introduction 6 times, 10 μ l of each sample introduction investigate the concordance of the relative retention time and relative peak area of main chromatographic peak.As a result the RSD ＜ 3% of the relative retention time and relative peak area of the main chromatographic peak of Fructus Aurantii, show that instrument precision is good.
（3）Replica test
Same batch of sample of Fructus Aurantii being taken, 6 parts of need testing solutions being prepared by test sample preparation method, under above-mentioned liquid phase chromatogram condition, the concordance of the relative retention time and relative peak area of main chromatographic peak is investigated in sample introduction analysis.As a result the RSD ＜ 3% of the relative retention time and relative peak area of the main chromatographic peak of Fructus Aurantii, show that the method is reproducible.
The formulation of the quasi- finger printing of 1.8 Fructus Aurantiis
10 batch Fructus Aurantii medical material test samples are prepared by the method drafted, is determined in accordance with the law, record chromatogram；Standard finger-print is obtained with similarity evaluation based on ten batches of finished product finger printing.See Fig. 1,2.
By Software match, determine 12 total peaks, compare with reference substance solution, wherein No. 1 peak is Folium Symplocoris Caudatae naringin, and No. 2 peaks are naringin, No. 3 peaks are Hesperidin, it is limonin that No. 4 peaks are neohesperidin, No. 8 peaks, No. 9 peaks be Nobiletin, No. 10 peaks be Nomilin, No. 11 peaks be Exocarpium Citri Rubrum element, contain known multiple compounds in Fructus Aurantii Immaturus in the standard finger-print of the present invention, reliability with height, is shown in accompanying drawing 3.
1.9 Fructus Aurantii medical material similarity evaluations
With the Fructus Aurantii standard finger-print of chromatographic fingerprints of Chinese materia medica similarity evaluation Software Create as reference, similarity is calculated, the similarity of 10 batches of Fructus Aurantii medical materials is more than 0.90, and similarity is preferable, it is thus determined that being to compare with standard finger-print, the limit standard of Fructus Aurantii medical material is set to similarity and should be greater than 0.90.The results are shown in Table 3.
3 10 batches of Fructus Aurantii HPLC-FPS similarity evaluation result tables of table.
| S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | S9 | S10 | Control collection of illustrative plates | |
| S1 | 1.000 | 0.919 | 0.959 | 0.915 | 0.904 | 0.961 | 0.988 | 0.958 | 0.965 | 0.906 | 0.997 |
| S2 | 0.919 | 1.000 | 0.905 | 0.983 | 0.909 | 0.973 | 0.901 | 0.922 | 0.941 | 0.962 | 0.939 |
| S3 | 0.959 | 0.905 | 1.000 | 0.900 | 0.934 | 0.904 | 0.937 | 0.903 | 0.909 | 0.915 | 0.956 |
| S4 | 0.915 | 0.983 | 0.900 | 1.000 | 0.923 | 0.973 | 0.906 | 0.956 | 0.905 | 0.953 | 0.932 |
| S5 | 0.904 | 0.909 | 0.934 | 0.923 | 1.000 | 0.976 | 0.979 | 0.934 | 0.952 | 0.920 | 0.905 |
| S6 | 0.961 | 0.973 | 0.904 | 0.973 | 0.976 | 1.000 | 0.915 | 0.960 | 0.977 | 0.951 | 0.972 |
| S7 | 0.988 | 0.901 | 0.937 | 0.906 | 0.979 | 0.915 | 1.000 | 0.990 | 0.980 | 0.947 | 0.979 |
| S8 | 0.958 | 0.922 | 0.903 | 0.956 | 0.934 | 0.960 | 0.990 | 1.000 | 0.967 | 0.911 | 0.945 |
| S9 | 0.965 | 0.941 | 0.909 | 0.905 | 0.952 | 0.977 | 0.980 | 0.967 | 1.000 | 0.908 | 0.948 |
| S10 | 0.906 | 0.962 | 0.915 | 0.953 | 0.920 | 0.951 | 0.947 | 0.911 | 0.908 | 1.000 | 0.927 |
| Control collection of illustrative plates | 0.997 | 0.939 | 0.956 | 0.932 | 0.905 | 0.972 | 0.979 | 0.945 | 0.948 | 0.927 | 1.000 |
Embodiment 2：Purposes of the standard finger-print in Fructus Aurantii medical material is differentiated.
1.1 instruments and reagent：
1260 high performance liquid chromatograph of Agilent, is contained in line vacuum degassing machine（G-1311C）, binary pump（G-1311C）, standard automatic sampler（G-1329B）, intelligent column oven（G-1316A）, DAD detectors（G-1314B）、Agilent1260
Infinity chromatographic work stations（Anjelen Sci. ＆ Tech. Inc of the U.S.）；SB-5200D ultrasonic washing instruments（NingBo XinZhi Biology Science Co., Ltd）；FAZ004B analytical balances（Section is helped in Shanghai）；BT125D electronic balances（Sai Duolisi scientific instrument（Beijing）Company limited）.
The measure of 1.2 Fructus Aurantii HPLC-FPSs.
（1）The preparation of need testing solution
Fructus Aurantii medicinal material coarse powder 0.2g is taken respectively, it is accurately weighed, put in conical flask with cover, accurate methanol 50ml, close plug, weighed weight, supersound extraction 1h let cool, then weighed weight, supply the weight of less loss with methanol, shake up, filtration, take subsequent filtrate, as need testing solution.
（2）Chromatographic condition is analyzed：
Chromatographic column is Unitary-C18,（5 m, 4.6mm × 250mm）；Column temperature：30℃ ；Detection wavelength is 280nm, 215nm, and switching time is 0min, 24.01min；Sample size is 10 μ l；Flow velocity：1.0ml/min；Analysis time is 70min；It is acetonitrile that mobile phase is mobile phase A, and Mobile phase B is 0.1% phosphoric acid solution；Using gradient elution mode：0min → 25min → 25.01min → 35min → 60min → 70min, acetonitrile 20% → 24% → 30% → 40% → 95% → 100%.
（3）Measurement result is analyzed
The Fructus Aurantii medical material of many batches of stochastic buyings is determined, and is detected wherein 2 batches and significantly total peak disappearance has been compared with Fructus Aurantii standard finger-print（Such as accompanying drawing 4, wherein No. 3 peaks, No. 4 peaks, No. 5 peaks, No. 9 peaks, No. 11 peak disappearances）.In addition, the similarity evaluation of the fingerprint similarity evaluation software promulgated using national Bureau of Drugs Supervision.As a result show this two batches medical material with the similarity of standard finger-print below 0.2.Therefore, it is possible to judge that the batch Fructus Aurantii quality of medicinal material is unqualified.According to the perk purity identification result compared according to embodiment 1 and reference substance solution, wherein No. 3 peaks, No. 4 peaks, No. 9 peaks, No. 11 peaks are respectively Hesperidin, neohesperidin, Nobiletin, Exocarpium Citri Rubrum element, these index components are very important active component in Fructus Aurantii, and this is correct from the conclusion that other side demonstrates above-mentioned " the batch Fructus Aurantii quality of medicinal material is unqualified ".Therefore, the quality and the purpose discerned the false from the genuine of monitoring Fructus Aurantii medical material can by building HPLC finger printing and analysis, with the quality condition of effective evaluation Fructus Aurantii medical material, be reached.
Claims (5)
1. a kind of detection method of Fructus Aurantii medicinal materials fingerprint, it is characterised in that comprise the following steps：
（1）The preparation of need testing solution：Fructus Aurantii medicinal material coarse powder is taken, it is accurately weighed, add methanol, ultrasound or reflux, extract, extracting solution to take subsequent filtrate with micro-pore-film filtration, obtain final product need testing solution；
（2）The preparation of reference substance solution：Folium Symplocoris Caudatae naringin reference substance, naringin reference substance, Hesperidin reference substance, neohesperidin reference substance, limonin reference substance, Nobiletin reference substance, Nomilin reference substance, Exocarpium Citri Rubrum element reference substance is taken respectively, it is accurately weighed, it is separately added into methanol and makes reference substance solution；
（3）High performance liquid chromatography detection：Accurate respectively to draw need testing solution and reference substance solution, injection high performance liquid chromatograph is determined, and obtains final product Fructus Aurantii finger printing；
Wherein, chromatographic condition is：Adopt octadecylsilane chemically bonded silica for filler chromatographic column, 4.6 × 250mm, 5 μm；Column temperature：20~40℃ ；Detection wavelength is 280nm, 215nm, and switching time is 0min, 24.01min；Sample size is 10 μ l；Flow velocity：1.0ml/min；Analysis time is 70min；It is acetonitrile that mobile phase is mobile phase A, and Mobile phase B is 0.1% phosphoric acid solution；Using gradient elution mode：0min → 25min → 25.01min → 35min → 60min → 70min, acetonitrile 20% → 24% → 30% → 40% → 95% → 100%.
2. the detection method of a kind of Fructus Aurantii medicinal materials fingerprint according to claim 1, it is characterised in that：The step（1）Fructus Aurantii coarse powder and methanol ratio be 0.2g:50ml.
3. the detection method of a kind of Fructus Aurantii medicinal materials fingerprint according to claim 1, it is characterised in that：The step（1）Ultrasound or reflux extracting time be 1 hour.
4. the detection method of a kind of Fructus Aurantii medicinal materials fingerprint according to claim 1, it is characterised in that：The step（2）In reference substance solution be specially：Per the 1ml respectively 70ng of naringin containing Folium Symplocoris Caudatae, naringin 80ng, Hesperidin 40ng, neohesperidin 80ng, limonin 70ng, Nobiletin 80ng, Nomilin 70ng, the mixed solution of Exocarpium Citri Rubrum element 60ng.
5. the finger printing of the Fructus Aurantii medical material set up by a kind of detection method of the Fructus Aurantii finger printing as described in claim 1, it is characterised in that：In Fructus Aurantii finger printing, 12 chromatographic peaks are confirmed as total peak, by the comparison with reference substance retention time, determine that peak 1 is Folium Symplocoris Caudatae naringin, peak 2 is naringin, and peak 3 is Hesperidin, peak 4 is neohesperidin, peak 8 is limonin, and peak 9 is Nobiletin, and peak 10 is Nomilin, peak 11 is Exocarpium Citri Rubrum element, and the relative retention time ranges of 12 total fingerprint peakses of the finger printing and relative standard deviation are as follows：
No. 1 peak：0.680～0.692, RSD are 0.68%；
No. 2 peaks：0.789～0.804, RSD are 0.92%；
No. 3 peaks：0.862～0.877, RSD are 0.75%；
No. 4 peaks：1, RSD is 0%；
No. 5 peaks：1.277～1.296, RSD are 0.51%；
No. 6 peaks：1.893～1.924, RSD are 0.74%；
No. 7 peaks：2.322～2.345, RSD are 0.65%；
No. 8 peaks：2.530～2.547, RSD are 1.02%；
No. 9 peaks：2.670～2.696, RSD are 0.84%；
No. 10 peaks：2.765～2.828, RSD are 0.34%；
No. 11 peaks：2.834～2.854, RSD are 0.59%；
No. 12 peaks：3.560～3.571, RSD are 0.64%.
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