CN105136936A - Rapid detection method for psoralen and isopsoralen - Google Patents
Rapid detection method for psoralen and isopsoralen Download PDFInfo
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- CN105136936A CN105136936A CN201510598922.1A CN201510598922A CN105136936A CN 105136936 A CN105136936 A CN 105136936A CN 201510598922 A CN201510598922 A CN 201510598922A CN 105136936 A CN105136936 A CN 105136936A
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Abstract
The invention provides a rapid detection method for psoralen and isopsoralen. The method comprises steps as follows: 1) preparation of samples; 2) PY-GC/MS (pyrolysis-gas chromatography/mass spectrum) detection; 3) data analysis as well as qualitative and quantitative analysis. A PY-GC/MS technology is selected for detecting the content of the psoralen and the isopsoralen, according to the problem of different quality due to differences in origins, seasons, preparation methods and the like of Chinese angelica in actual production and life, the rapid and trace detection method for the psoralen and the isopsoralen is provided, and then the quality of fructus psoraleae is preliminarily judged; the method has the characteristics of simplicity in operation, low detection limit, good experimental reproducibility, direct and reliable experimental data and the like, is particularly applicable to trace sample detection, can be used for preliminarily comparing quality differences of different varieties and different batches of fructus psoraleae samples from different origins, is wide in application range and provides a preliminary judgment basis for the quality standard and the modernization application of the fructus psoraleae.
Description
Technical field
The present invention relates to a kind of method detecting psoralen and isopsorapen content based on fast pyrogenation-Gas chromatographyMass spectrometry (PY-GC/MS method) fast.
Background technology
Psoralea corylifolia is the dry mature fruit of legumes psoraleae, has warming kidney and enhancing yang, receives effect of gas, antidiarrheal.Its principal ingredient is Coumarins and flavone compound, and wherein psoralen and isopsorapen is its important effective constituent.As traditional Chinese medicine, psoralen and isopsorapen is usually used in prevention and therapy osteoporosis, and modern pharmacology experiment shows, psoralen and isopsorapen is a kind of effective Ca
+channel antagonist, it has obvious facilitation to the osteoblastic increment of rat and differentiation.
Nearest research shows that psoralen and isopsorapen can be used for treatment T cell lymph cancer, human breast cancer cell and some autoimmune diseases.The conbined usage of psoralen and isopsorapen, to the cancer cell of multidrug resistance, as the strain of human breast cancer cell Adriamycin resistant, has obvious inhibiting effect.Therefore, after the failure of other drug treatment leukaemia, psoralen and isopsorapen likely becomes one of leukemic active drug for the treatment of.Fast, the psoralen and isopsorapen in efficient detection Chinese medicine or its compound preparation is very important to druggability and modernization of Chinese medicine application.
The existing analysis to psoralen and isopsorapen detects the method that will adopt efficient liquid phase.Often need to carry out comparatively complicated sample pre-treatments to sample, carried by heat, coldly to carry or psoralen and isopsorapen is separated by the method such as ultrasonic extraction from medicinal material or its compound preparation, adopt the liquid-phase chromatography method analyses such as HPLC/UPLC/SFCLC subsequently.The sample handling processes of this analytical approach is complicated, the aggregate analysis time is long.
Fast pyrogenation-gas chromatography/mass spectrometry technology (PY-GC/MS) is a kind of technology of efficient detection effumability composition, and development in recent years is rapid.Pyrolytic process under low temperature is a kind of very effective thermal release means, can be realized preliminary solution-air or the vapor solid separation of lower boiling complex mixture by the temperature and time controlling pyrolysis; For gas chromatography provide a kind of online with real-time sample pre-treatments and initial gross separation ability.Pyrolysis time in analyte detection process, temperature, heating rate, flow rate of carrier gas, split ratio, ionization temperature etc. are very large on the impact of analysis result.Therefore, based on the feature of PY-GC/MS technology, need badly at present and propose a kind of quick, Sparklet testing method for psoralen and isopsorapen.
Summary of the invention
The object of the present invention is to provide a kind of method for quick of psoralen and isopsorapen, and based on this, tentatively judge the height of Psoralea corylifolia and the quality of the pharmaceutical preparations thereof.
For achieving the above object, present invention employs following technical scheme:
1) accurately take Psoralea corylifolia or its compound preparation 0.01 ~ 10.00mg obtains sample, sample is added in pyrolysis cup;
2) fast pyrogenation-gas chromatograph-mass spectrometer (GCMS) is adopted to measure the sample added in pyrolysis cup;
3) through step 2) after, according to the original chromatographic data measured and mass spectrometric data, the chromatographic peak of psoralen and isopsorapen in qualitative sample, and obtain the relative content of psoralen and isopsorapen in sample.
Described sample is block or pulverulent solids.
The condition of described pyrolysis is: pyrolysis temperature is 200 ~ 400 DEG C, and pyrolysis time is 0.1 ~ 2min.
The condition of work of described gas chromatography is: carrier gas is the helium of flow velocity 0.1 ~ 4.0mL/min, split ratio is 0 ~ 500:1, and heating schedule is: initial temperature is 40 ~ 60 DEG C, and keeps 0 ~ 4min, then rise to 150 DEG C with≤35 DEG C/min, then rise to 300 DEG C with≤15 DEG C/min.
Described mass spectrographic condition of work is: adopt EI source, positive ion detection, electron energy is 40 ~ 80eV, and ion source temperature is 210 ~ 240 DEG C, single level Four bar temperature is 140 ~ 180 DEG C, scan mode is full scan mode, and electron-multiplier voltage is 1070 ~ 3000V, and solvent delay is 0 ~ 3min.
Described relative content adopts area normalization method to calculate.
The qualitative employing NIST08 software Auto-matching of the chromatographic peak of described psoralen and isopsorapen calculates.
Beneficial effect of the present invention is embodied in:
1) thermal release and chromatographic resolution combine, and realize quick separating.Existing method be by Psoralea corylifolia or its preparation by ultrasonic extraction or solvent extraction, after filtration treatment, utilize liquid-phase chromatographic analysis, or thin-layer chromatography-spectrum coupling technique (TLC-SP) etc. detects its effective constituent; The general analysis time of the method is 4 ~ 24 hours.Directly initial gross separation is carried out to the low-boiling point material in Psoralea corylifolia or Compound Buguzhi by thermal release method in the present invention, gas chromatography is adopted to be separated psoralen and isopsorapen further subsequently, greatly save analysis time, the conventional analysis time is 0.5 ~ 1 hour, improves analysis efficiency.
2) required sample size is few, and sensitivity is good.Existing method obtains crude product solution because needs are first separated, and the sample size therefore needed is comparatively large, and normally several grams to tens grams not etc.; The analysis specimen in use amount of the inventive method is generally 0.01 ~ 1mg, improves sensitivity for analysis.
3) present invention optimizes the testing conditions such as pyrolysis, chromatogram, mass spectrum, can chromatographic peak needed for fast qualitative, achieve and detect fast and accurately.
Embodiment
Below in conjunction with embodiment, the invention will be further described.
The present invention is directed to psoralen and isopsorapen and melt the relatively low and volatile feature of boiling point, adopt fast pyrogenation-gas chromatography/mass spectrometry technology (PY-GC/MS), fully thermal release and chromatographic resolution are combined, realize the quick separating of psoralen and isopsorapen, adopt mass spectrometry subsequently and quantitatively detect, reaching object that is quick, trace detection.
Embodiment
Detect a method for psoralen and isopsorapen based on PY-GC/MS, for Psoralea corylifolia preparation, but protection scope of the present invention is not limited to this embodiment, and concrete steps are as follows:
One, preparation of samples
The Psoralea corylifolia capsule obtained (Shaanxi Si Nuote Bioisystech Co., Ltd, Xi'an) is accurately taken the pyrolysis cuvette that 1.00mg (± 0.02mg) puts into 50 μ L;
Two, pyrolysis:
Pyrocrack furnace adopts the PY2020is of Frontiers;
1. pyrolysis temperature: 300 DEG C;
2. pyrolysis time: 0.20min.
Three, be separated, detect
Chromatogram:
Gas chromatography adopts Agilent 7890A:
1. analytical column is HP-5MS fused-silica capillary column (30m*0.25id*0.25 μm);
2. carrier gas: high-purity helium, flow velocity is 1.0mL/min;
3. input mode is split sampling, and split ratio is 50:1;
4. injector temperature is 280 DEG C, and transmission line temperature is 290 DEG C;
5. temperature programme: initial temperature is 60 DEG C, keeps 1min, then rises to 150 DEG C with 30 DEG C/min, then rise to 300 DEG C ° with 10 DEG C/min.
Mass spectrum (adopting Agilent 5975C):
1. EI source, positive ion detects, and electron energy is 70eV;
2. ion source temperature is 230 DEG C, and single level Four bar temperature is 150 DEG C;
3. scan mode is full scan mode;
4. electron-multiplier voltage is 2312V, and solvent delay is 2.75min.
Four, data analysis and process
Retention time and the integral area of chromatogram are completed automatically by workstation, utilize NIST08 to compose the qualitative chromatographic peak in storehouse.The relative content of each volatile ingredient is obtained by area normalization method.
In this sample Psoralea corylifolia preparation pyrolyzed components, identifiable peak is 26, and composition and retention time thereof list in table 1.Retention time and peak area Agilent Chemstation calculate, and the method for relative content area normalization calculates.
The composition of volatile ingredient and content (t in table 1. Psoralea corylifolia
r=10.161: psoralen, t
r=10.789: Isopsoralen)
Five, precision, reappearance and stability experiment
1. Precision Experiment: to take from 5 parts, the sample of same Psoralea corylifolia capsule, continuous sample introduction 5 times according to the method described above, 26 chromatographic peaks relative peak area being greater than to 0.5% are analyzed, the RSD (relative standard deviation) of its relative retention time and relative peak area, respectively between 0.45 ~ 1.78% and 1.2 ~ 4.97%, illustrates that the inventive method precision is good.
2. reappearance experiment: 5 parts, the sample getting same batch of Psoralea corylifolia capsule, according to the method described above continuous sample introduction 5 times.The relative retention time of 26 chromatographic peaks and the RSD of relative peak area are respectively between 0.23 ~ 5.7% and 1.31 ~ 4.63% as a result, illustrate that the inventive method reappearance is good.
3. stability experiment: get 1 part, the sample prepared, respectively 0,2,4,8,12,24h sample introduction measures, the relative retention time of 26 chromatographic peaks and the RSD of relative peak area are all less than 4.445% as a result, and it is stable for illustrating that the inventive method measures in 24h.
Main active basically in Psoralea corylifolia or its preparation is psoralen and isopsorapen, and it can be used as one of main criterion of Psoralea corylifolia or its preparation quality.Chinese crude drug is because of the difference of the place of production, season, process of preparing Chinese medicine mode, and its water cut, density etc. have trickle difference, and the content then in its compound preparation is also different.Therefore, pyrolysis, the separation of needs are all not identical with analysis condition, draw the condition and range in the present invention accordingly.
The present invention selects PY-GC/MS technology to detect psoralen and isopsorapen content, for the Psoralea corylifolia problem that quality is different because the place of production, season, the process of preparing Chinese medicine etc. are different in actual production, life, a kind of quick, micro-detection method of psoralen and isopsorapen is provided, and then tentatively judges the quality of Psoralea corylifolia.The present invention has simple to operate, detectability is low, experiment favorable reproducibility, the features such as experimental data is intuitive and reliable, be specially adapted to micro-example and detect (sample size can be low to moderate 10 μ g), and can be used for carrying out quantitative comparison to the difference in quality between different Psoralea corylifolia or its formulation samples, application is wide, for the quality standard of Psoralea corylifolia and modernization application provide one can the thinking and countermeasure of reference.
Claims (7)
1. a method for quick for psoralen and isopsorapen, is characterized in that: comprise the following steps:
1) accurately take Psoralea corylifolia or its compound preparation 0.01 ~ 10.00mg obtains sample, sample is added in pyrolysis cup;
2) fast pyrogenation-gas chromatograph-mass spectrometer (GCMS) is adopted to measure the sample added in pyrolysis cup;
3) through step 2) after, according to the original chromatographic data measured and mass spectrometric data, the chromatographic peak of psoralen and isopsorapen in qualitative sample, and obtain the relative content of psoralen and isopsorapen in sample.
2. the method for quick of a kind of psoralen and isopsorapen according to claim 1, is characterized in that: described sample is block or pulverulent solids.
3. the method for quick of a kind of psoralen and isopsorapen according to claim 1, it is characterized in that: the condition of described pyrolysis is: pyrolysis temperature is 200 ~ 400 DEG C, pyrolysis time is 0.1 ~ 2min.
4. the method for quick of a kind of psoralen and isopsorapen according to claim 1, it is characterized in that: the condition of work of described gas chromatography is: carrier gas is the helium of flow velocity 0.1 ~ 4.0mL/min, split ratio is 0 ~ 500:1, heating schedule is: initial temperature is 40 ~ 60 DEG C, and keep 0 ~ 4min, then rise to 150 DEG C with≤35 DEG C/min, then rise to 300 DEG C with≤15 DEG C/min.
5. the method for quick of a kind of psoralen and isopsorapen according to claim 1, it is characterized in that: described mass spectrographic condition of work is: adopt EI source, positive ion detection, electron energy is 40 ~ 80eV, ion source temperature is 210 ~ 240 DEG C, single level Four bar temperature is 140 ~ 180 DEG C, scan mode is full scan mode, and electron-multiplier voltage is 1070 ~ 3000V, and solvent delay is 0 ~ 3min.
6. the method for quick of a kind of psoralen and isopsorapen according to claim 1, is characterized in that: described relative content adopts area normalization method to calculate.
7. the method for quick of a kind of psoralen and isopsorapen according to claim 1, is characterized in that: the qualitative employing NIST08 software Auto-matching of the chromatographic peak of described psoralen and isopsorapen calculates.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107436331A (en) * | 2017-07-04 | 2017-12-05 | 浙江工业大学 | The method for flashing index composition in gas chromatography measure Sishen wan |
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| CN101963599A (en) * | 2010-05-25 | 2011-02-02 | 江西中烟工业有限责任公司 | Method for measuring main effective ingredient content of psoralen in cigarettes and main-flow flue gas particle-phase matter |
| CN104251890A (en) * | 2013-06-27 | 2014-12-31 | 天津中医药大学 | Method for detecting metabolite relative to psoralea corylifolia hepatotoxicity and application thereof |
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2015
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Patent Citations (2)
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| CN101963599A (en) * | 2010-05-25 | 2011-02-02 | 江西中烟工业有限责任公司 | Method for measuring main effective ingredient content of psoralen in cigarettes and main-flow flue gas particle-phase matter |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107436331A (en) * | 2017-07-04 | 2017-12-05 | 浙江工业大学 | The method for flashing index composition in gas chromatography measure Sishen wan |
| CN107436331B (en) * | 2017-07-04 | 2019-11-29 | 浙江工业大学 | The method for flashing index ingredient in gas chromatography measurement Sishen wan |
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Effective date of registration: 20200331 Address after: 116033 floor 3, No. 300-8, jinlongsi Road, Ganjingzi District, Dalian City, Liaoning Province Patentee after: Dalian Runsheng Kangtai Medical Laboratory Co.,Ltd. Address before: 710054 No. 58, Yanta Road, Shaanxi, Xi'an Patentee before: XI'AN UNIVERSITY OF SCIENCE AND TECHNOLOGY |