CN106525988A - Detection method for fructus aurantii immaturus medicine finger-print, and standard finger-print - Google Patents
Detection method for fructus aurantii immaturus medicine finger-print, and standard finger-print Download PDFInfo
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Abstract
The invention discloses a detection method for a fructus aurantii immaturus medicine finger-print, a standard finger-print thereof, and application of the fructus aurantii immaturus medicine finger-print or the standard finger-print in identifying fructus aurantii immaturus medicines. The method comprises the following steps: (1) taking the fructus aurantii immaturus medicines, smashing into medium-granularity powder, precisely weighting, adding methyl alcohol, carrying out extraction, and taking subsequent filtrate to obtain a solution of a sample for testing; (2) preparing reference substance solution; (3) respectively precisely absorbing the solution of the sample for testing and the reference substance solution, and injecting into a high performance liquid chromatograph to measure, so as to obtain the fructus aurantii immaturus finger-print. The measurement method disclosed by the invention has the advantages of convenience in operation, good repeatability and stability, high accuracy and high specificity, and has an active function on guiding production feeding and guaranteeing quality reliability, and characteristic ingredients are integrally kept. The fructus aurantii immaturus medicine finger-print established by the method can be used for effectively monitoring the quality of the fructus aurantii immaturus medicines and identifying fakes from originals.
Description
Technical field
The invention belongs to pharmaceutical analysiss technical field, more particularly to a kind of Fructus Aurantii Immaturus finger printing and its method for building up.In particular it relates to a kind of high performance liquid chromatography of Fructus Aurantii Immaturus（HPLC）Changing Wave finger printing and its method for building up, and the standard finger-print of Fructus Aurantii Immaturus.The invention further relates to a kind of discrimination method of Fructus Aurantii Immaturus quality good or not.
Background technology
Fructus Aurantii Immaturus is rutaceae Citrus aurantium Linn. Citrus aurantium L. and its variety Fructus Citri sinensiss C. Sinensis（L）Osbeck's is dried young fruit, and main product is in Jiangxi, Sichuan, Fujian, Jiangsu and other places.Version in 2015《Chinese Pharmacopoeia》Record Fructus Aurantii Immaturus and there is dispelling the stagnated QI except painful abdominal mass, transforming phlegm and dispersing accumulations, modern age is clinically used for treating the visceroptosiss diseases such as flatulence, gastroptosis, uterine prolapse, proctoptosis, is tcm clinical practice common drug.Fructus Aurantii Immaturus derive from 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 chemical composition constitute and content on it is poor
It is different very big, thus directly influence quality of medicinal material.
Main active in Fructus Aurantii Immaturus 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 Immaturus.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.
2015 editions States Pharmacopoeia specifications, using Neosynephrine as the index of Fructus Aurantii Immaturus quality control（Chinese Pharmacopoeia Commission compiles, Pharmacopoeia of People's Republic of China version in 2015, Beijing：Chinese Medicine industry publishing house, 2015, one：247）, the method for carrying out Chinese medicine quality control using single component 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.
Research for Fructus Aurantii Immaturus such as relates generally to extract, processes at the research, and quality determining method discloses less, such as Chinese patent 200410077903.6（Publication number CN100486586C）The detection of the total constituents of total flavones in Furctus Aurantii Immaturus extract is referred to, is not that Fructus Aurantii Immaturus are detected, also quality is not controlled comprehensively；Chinese patent 200710120306.0（Publication number CN101366849B）Refer to quality control be carried out using infrared spectrum to Fructus Aurantii Immaturus, only detect three index components, have certain limitation；Chinese patent 201410106425.0（Publication number CN 103837621B）Fructus Aurantii Immaturus HPLC finger printing is referred to, is detected only with single wavelength, Fructus Aurantii Immaturus test sample preparation method is complicated.《China Dispensary》39th phase in 2011《The 2 kinds of Fructus Aurantii Immaturus HPLC finger printing researchs of Citrus aurantium Linn. and Fructus Citri sinensiss and its naringin and Neosynephrine content analysis》Describe one kind application efficient liquid phase（HPLC）The fingerprint spectrum method of Fructus Aurantii Immaturus is determined, but only points out four index components such as naringin, Hesperidin, neohesperidin, Neosynephrine；《Strait Pharmaceutical Journal》2nd phase in 2004《The research of Fingerprint of Fructus Ponciri Trifoliatae Immaturi》Describe one kind application gas chromatogram（GC）The method for determining Naphtha GC fingerprint in Fructus Ponciri Trifoliatae；《R＆D of modern TCM and practice》Supplementary issue of volume 18 in 2004《Chemical Pattern Recognition accumulates the application in real the classification in Chinese medicine》Describe one kind application efficient liquid phase（HPLC）The method for determining chromatogram fingerprint, but using single wavelength detection, only 8 total peaks and only point out three index components such as 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 Immaturus has very important significance.
The content of the invention
The technical problem to be solved is to provide a kind of assay method of chromatogram fingerprint, quality of the method in control Fructus Aurantii Immaturus, 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 Immaturus HPLC standard finger-prints can be used in identifying or differentiating that the Fructus Aurantii Immaturus quality 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 Immaturus finger printing of the present invention, comprises the following steps：
（1）The preparation of need testing solution：Powder in Fructus Aurantii Immaturus 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, Nobiletin 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 chromatogram fingerprint；
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%.
A kind of detection method of Fructus Aurantii Immaturus finger printing according to claim 1, it is characterised in that：The step（1）Fructus Aurantii Immaturus in powder and methanol ratio be 0.2g:50ml.
A kind of detection method of Fructus Aurantii Immaturus finger printing according to claim 1, it is characterised in that：The step（1）Ultrasound or reflux extracting time be 1 hour.
A kind of detection method of Fructus Aurantii Immaturus finger printing 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, Nobiletin 80ng, the mixed solution of Exocarpium Citri Rubrum element 60ng.
A kind of finger printing of the Fructus Aurantii Immaturus set up by detection method of the chromatogram fingerprint as described in claim 1, it is characterised in that：In chromatogram fingerprint, 17 chromatographic peaks are confirmed as total peak, by the comparison with reference substance retention time, determine that peak 3 is Folium Symplocoris Caudatae naringin, peak 4 is naringin, peak 5 is Hesperidin, and peak 6 is neohesperidin, and peak 14 is Nobiletin, peak 15 is Exocarpium Citri Rubrum element, and the relative retention time ranges of 17 total fingerprint peakses of the finger printing and relative standard deviation are as follows：
No. 1 peak：0.266～
0.279, RSD is 0.95%；
No. 2 peaks：0.637～
0.648, RSD is 0.45%；
No. 3 peaks：0.862～
0.889, RSD is 0.61%；
No. 4 peaks：1, RSD is 0%；
No. 5 peaks：1.095～
1.115, RSD is 0.39%；
No. 6 peaks：1.268～
1.284, RSD is 0.85%；
No. 7 peaks：1.617～
1.635, RSD is 0.53%；
No. 8 peaks：2.278～
2.301, RSD is 1.03%；
No. 9 peaks：2.386～
2.406, RSD is 0.81%；
No. 10 peaks：2.697～
2.714, RSD is 0.96%；
No. 11 peaks：2.824～
2.835, RSD is 0.72%；
No. 12 peaks：2.923～
2.944, RSD is 0.88%；
No. 13 peaks：3.229～
3.245, RSD is 0.47%；
No. 14 peaks：3.357～
3.379, RSD is 0.55%；
No. 15 peaks：3.562～
3.602, RSD is 0.83%；
No. 16 peaks：4.475～
4.496, RSD is 1.08%；
No. 17 peaks：4.545～4.569, RSD are 0.61%.
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 Immaturus quality determining method single, it is impossible to the defect of reaction Fructus Aurantii Immaturus quality comprehensively.By the conversion of wavelength, the visualization for enhancing fingerprint peakses is compared, and has pointed out 6 index components, and total 17, the peak of matching more effectively characterizes the quality information of Fructus Aurantii Immaturus, 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 Immaturus, the perfect quality evaluation system of Fructus Aurantii Immaturus, and for comprehensive, the effective control of Fructus Aurantii Immaturus quality, to provide theory and practice basic；
（3）The inventive method is applied to the discriminating of the Fructus Aurantii Immaturus 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 Immaturus standard finger-print, and wherein peak 3 is Folium Symplocoris Caudatae naringin, and peak 4 is naringin, and peak 5 is Hesperidin, and peak 6 is neohesperidin, and peak 14 is Nobiletin, and peak 15 is Exocarpium Citri Rubrum element.
Fig. 2 is 10 batch Fructus Aurantii Immaturus finger printing.
Fig. 3 is 6 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）；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 Immaturus 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 Immaturus test sample source：
Embodiment 1：The foundation and checking of Fructus Aurantii Immaturus wavelength switching finger printing.
1.1 instrument 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）.
1.2 the selection of chromatographic condition.
（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
（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.
1.3 the preparation of reference substance solution
Precision weighs Folium Symplocoris Caudatae naringin reference substance, naringin reference substance, Hesperidin reference substance, neohesperidin reference substance, Nobiletin 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, 80ug, obtains final product reference substance solution.
1.4 the preparation of need testing solution
Powder 0.2g in Fructus Aurantii Immaturus 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 Immaturus methodological studies.
（1）Stability test
The same need testing solution of Fructus Aurantii Immaturus 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 Immaturus, show more stable in need testing solution 24 hours.
（2）Precision test
Take with portion Fructus Aurantii Immaturus 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 Immaturus, show that instrument precision is good.
（3）Replica test
Same batch of sample of Fructus Aurantii Immaturus 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 Immaturus, show that the method is reproducible.
The formulation of the quasi- finger printing of 1.8 Fructus Aurantii Immaturuss
10 batch Fructus Aurantii Immaturus 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 17 total peaks, compare with reference substance solution, wherein No. 3 peaks are Folium Symplocoris Caudatae naringin, No. 4 peaks are naringin, No. 5 peaks are Hesperidin, and No. 6 peaks are neohesperidin, and No. 14 peaks are Nobiletin, No. 15 peaks are Exocarpium Citri Rubrum element, known multiple compounds in Fructus Aurantii Immaturus are contained in the standard finger-print of the present invention, and the reliability with height is shown in accompanying drawing 3.
1.9 Fructus Aurantii Immaturus similarity evaluations
With the Fructus Aurantii Immaturus 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 Immaturus 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 Immaturus is set to similarity and should be greater than 0.9.The results are shown in Table 3.
3 10 batches of Fructus Aurantii Immaturus HPLC fingerprint similarity evaluation result tables of table
| S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | S9 | S10 | Control collection of illustrative plates | |
| S1 | 1.000 | 0.909 | 0.942 | 0.923 | 0.945 | 0.917 | 0.935 | 0.951 | 0.951 | 0.902 | 0.946 |
| S2 | 0.909 | 1.000 | 0.916 | 0.992 | 0.984 | 0.904 | 0.994 | 0.975 | 0.975 | 0.936 | 0.978 |
| S3 | 0.942 | 0.916 | 1.000 | 0.924 | 0.946 | 0.945 | 0.926 | 0.908 | 0.916 | 0.913 | 0.975 |
| S4 | 0.923 | 0.992 | 0.924 | 1.000 | 0.969 | 0.916 | 0.918 | 0.920 | 0.980 | 0.958 | 0.951 |
| S5 | 0.945 | 0.984 | 0.946 | 0.969 | 1.000 | 0.907 | 0.972 | 0.953 | 0.953 | 0.962 | 0.961 |
| S6 | 0.917 | 0.904 | 0.945 | 0.916 | 0.907 | 1.000 | 0.918 | 0.946 | 0.958 | 0.907 | 0.958 |
| S7 | 0.935 | 0.994 | 0.926 | 0.918 | 0.972 | 0.918 | 1.000 | 0.912 | 0.927 | 0.911 | 0.982 |
| S8 | 0.951 | 0.975 | 0.908 | 0.920 | 0.953 | 0.946 | 0.912 | 1.000 | 0.902 | 0.915 | 0.939 |
| S9 | 0.951 | 0.975 | 0.916 | 0.980 | 0.953 | 0.958 | 0.927 | 0.902 | 1.000 | 0.906 | 0.943 |
| S10 | 0.902 | 0.936 | 0.913 | 0.958 | 0.962 | 0.907 | 0.911 | 0.915 | 0.906 | 1.000 | 0.917 |
| Control collection of illustrative plates | 0.946 | 0.978 | 0.975 | 0.951 | 0.961 | 0.958 | 0.982 | 0.939 | 0.943 | 0.917 | 1.000 |
Embodiment 2：Purposes of the standard finger-print in Fructus Aurantii Immaturus are differentiated.
1.1 instrument 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 Immaturus HPLC finger printing.
（1）The preparation of need testing solution
Powder 0.2g in Fructus Aurantii Immaturus 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 Immaturus of many batches of stochastic buyings are determined, and are detected wherein 2 batches and significantly total peak disappearance have been compared with Fructus Aurantii Immaturus standard finger-print（Such as accompanying drawing 4, wherein No. 2 peaks, No. 4 peaks, No. 6 peaks, No. 7 peaks, No. 8 peaks, No. 10 peaks, No. 11 peaks, No. 12 peaks, No. 13 peaks, No. 16 peaks, No. 17 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 Immaturus are off quality.According to the perk purity identification result compared according to embodiment 1 and reference substance solution, wherein No. 4 peaks are naringin, No. 6 peaks are neohesperidin, these index components are very important active component in Fructus Aurantii Immaturus, and this is correct from the conclusion that other side demonstrates above-mentioned " the batch Fructus Aurantii Immaturus are off quality ".Therefore, the quality and the purpose discerned the false from the genuine of monitoring Fructus Aurantii Immaturus can by building HPLC finger printing and analysis, with the quality condition of effective evaluation Fructus Aurantii Immaturus, be reached.
Claims (5)
1. a kind of detection method of Fructus Aurantii Immaturus finger printing, it is characterised in that comprise the following steps：
（1）The preparation of need testing solution：Powder in Fructus Aurantii Immaturus 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, Nobiletin 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 chromatogram fingerprint；
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 Immaturus finger printing according to claim 1, it is characterised in that：The step（1）Fructus Aurantii Immaturus in powder and methanol ratio be 0.2g:50ml.
3. the detection method of a kind of Fructus Aurantii Immaturus finger printing 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 Immaturus finger printing 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, Nobiletin 80ng, the mixed solution of Exocarpium Citri Rubrum element 60ng.
5. the finger printing of the Fructus Aurantii Immaturus set up by a kind of detection method of the chromatogram fingerprint as described in claim 1, it is characterised in that：In chromatogram fingerprint, 17 chromatographic peaks are confirmed as total peak, by the comparison with reference substance retention time, determine that peak 3 is Folium Symplocoris Caudatae naringin, peak 4 is naringin, peak 5 is Hesperidin, and peak 6 is neohesperidin, and peak 14 is Nobiletin, peak 15 is Exocarpium Citri Rubrum element, and the relative retention time ranges of 17 total fingerprint peakses of the finger printing and relative standard deviation are as follows：
No. 1 peak：0.266～0.279, RSD are 0.95%；
No. 2 peaks：0.637～0.648, RSD are 0.45%；
No. 3 peaks：0.862～0.889, RSD are 0.61%；
No. 4 peaks：1, RSD is 0%；
No. 5 peaks：1.095～1.115, RSD are 0.39%；
No. 6 peaks：1.268～1.284, RSD are 0.85%；
No. 7 peaks：1.617～1.635, RSD are 0.53%；
No. 8 peaks：2.278～2.301, RSD are 1.03%；
No. 9 peaks：2.386～2.406, RSD are 0.81%；
No. 10 peaks：2.697～2.714, RSD are 0.96%；
No. 11 peaks：2.824～2.835, RSD are 0.72%；
No. 12 peaks：2.923～2.944, RSD are 0.88%；
No. 13 peaks：3.229～3.245, RSD are 0.47%；
No. 14 peaks：3.357～3.379, RSD are 0.55%；
No. 15 peaks：3.562～3.602, RSD are 0.83%；
No. 16 peaks：4.475～4.496, RSD are 1.08%；
No. 17 peaks：4.545～4.569, RSD are 0.61%.
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