CN105136936B - A kind of rapid detection method of psoralen and isopsorapen - Google Patents
A kind of rapid detection method of psoralen and isopsorapen Download PDFInfo
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- CN105136936B CN105136936B CN201510598922.1A CN201510598922A CN105136936B CN 105136936 B CN105136936 B CN 105136936B CN 201510598922 A CN201510598922 A CN 201510598922A CN 105136936 B CN105136936 B CN 105136936B
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Abstract
The present invention provides a kind of rapid detection method of psoralen and isopsorapen: 1) preparation of sample；2) PY-GC/MS is detected；3) data analysis and qualitative and quantitative analysis.The present invention selects PY-GC/MS technology to detect psoralen and isopsorapen content, it is for actual production, when the attribution place of production in life, season, the different and quality different problems such as process, it is quick to provide a kind of psoralen and isopsorapen, micro detection method, and then tentatively judge the quality of psoralea corylifolia, the present invention has easy to operate, detection limit is low, test favorable reproducibility, the features such as experimental data is intuitive and reliable, it is detected especially suitable for micro-example, it can be used for different sources, kind, the difference in quality of the psoralea corylifolia sample room of batch carries out preliminary comparison, application is wide, a kind of preliminary interpretation foundation is provided for the quality standard and modernization application of psoralea corylifolia.
Description
Technical field
The present invention relates to one kind quickly to be detected based on fast pyrogenation-Gas chromatographyMass spectrometry (PY-GC/MS method)
The method of psoralen and isopsorapen content.
Background technique
Psoralea corylifolia is the dry mature fruit of legumes psoraleae, has effects that warming kidney and enhancing yang, receives gas, antidiarrheal.Its
Main component is Coumarins and flavone compound, and wherein psoralen and isopsorapen is its important effective component.Make
For traditional Chinese medicine, psoralen and isopsorapen is usually used in preventing and treating osteoporosis, and modern pharmacology experiment shows
Psoralen and isopsorapen is a kind of effective Ca+Channel antagonist, increment and differentiation to the osteoblast of rat
With apparent facilitation.
Recent studies have shown that psoralen and isopsorapen can be used for treating T cell lymph cancer, human breast cancer cell
With some autoimmune diseases.The cancer cell of psoralen and isopsorapen being used in combination to multidrug resistance, such as people
Breast cancer cell Adriamycin resistant strain, there is apparent inhibiting effect.Therefore, after other drugs treatment leukaemia failure, Psoralen
Rouge element and Isopsoralen are likely to become one of the active drug for the treatment of leukaemia.Quickly, efficient detection Chinese medicine or its compound
Psoralen and isopsorapen in preparation is very important druggability and modernization of Chinese medicine application.
The existing method that efficient liquid phase is used to the analysis detection of psoralen and isopsorapen.It generally requires to sample
Product carry out more complicated sample pre-treatments, are mentioned, cold are mentioned or the methods of ultrasonic extraction is by psoralen and different psoralea corylifolia by heat
Element is separated from medicinal material or its compound preparation, then using the analysis of the liquid-phase chromatography methods such as HPLC/UPLC/SFCLC.The analysis side
The sample handling processes of method are complicated, the aggregate analysis time is long.
Fast pyrogenation-gas chromatography/mass spectrometry technology (PY-GC/MS) is a kind of efficient detection effumability ingredient
Technology, quickly grow in recent years.Pyrolytic process under low temperature is a kind of very effective thermal release means, is pyrolyzed by control
Temperature and time may be implemented low-boiling complex mixture preliminary solution-air or vapor solid separation；It is mentioned for gas-chromatography
For a kind of online and real-time sample pre-treatments and initial gross separation ability.Pyrolysis time, temperature in analyte detection process, liter
The influence to analysis result such as warm rate, flow rate of carrier gas, split ratio, ionization temperature is very big.Therefore, it is based on PY-GC/MS
The characteristics of technology, needs to propose a kind of quick, Sparklet testing method for psoralen and isopsorapen at present.
Summary of the invention
The purpose of the present invention is to provide a kind of rapid detection methods of psoralen and isopsorapen, and are based on this,
Tentatively judge the height of psoralea corylifolia and its quality of the pharmaceutical preparations.
In order to achieve the above objectives, the invention adopts the following technical scheme:
1) it accurately weighs psoralea corylifolia or 0.01~10.00mg of its compound preparation obtains sample, sample is added in pyrolysis cup；
2) sample being added in pyrolysis cup is measured using fast pyrogenation-gas chromatograph-mass spectrometer (GC-MS)；
3) after step 2), according to the original chromatographic data and mass spectrometric data of measurement, in qualitative sample psoralen and
The chromatographic peak of Isopsoralen, and obtain the relative amount of psoralen and isopsorapen in sample.
The sample is blocky or pulverulent solids.
The condition of the pyrolysis are as follows: pyrolysis temperature is 200~400 DEG C, and pyrolysis time is 0.1~2min.
The operating condition of the gas-chromatography are as follows: carrier gas be 0.1~4.0mL/min of flow velocity helium, split ratio be 0~
500:1, temperature program are as follows: initial temperature is 40~60 DEG C, and keeps 0~4min, then rises to 150 DEG C with≤35 DEG C/min,
Then 300 DEG C are risen to≤15 DEG C/min.
The mass spectrographic operating condition are as follows: use the source EI, positive ion detection, electron energy is 40~80eV, ion source temperature
Degree is 210~240 DEG C, and single level four bars temperature is 140~180 DEG C, and scanning mode is full scan mode, and electron multiplier voltage is
1070~3000V, solvent delay are 0~3min.
The relative amount is calculated using area normalization method.
The qualitative of the chromatographic peak of the psoralen and isopsorapen is calculated using NIST08 software Auto-matching.
The beneficial effects of the present invention are embodied in:
1) thermal release and chromatographic isolation combine, and realize quick separating.Existing method is to pass through psoralea corylifolia or its preparation
Ultrasonic extraction or solvent extraction after filtration treatment, utilize liquid-phase chromatographic analysis or thin-layer chromatography-spectrum joint technology (TLC-
) etc. SP its effective component is detected；The general analysis time of this method is 4~24 hours.It is straight by thermal release method in the present invention
It connects and initial gross separation is carried out to the low-boiling point material in psoralea corylifolia or Compound Buguzhi, benefit is further then separated using gas-chromatography
Analysis time is greatly saved in bone fat element and Isopsoralen, and the conventional analysis time is 0.5~1 hour, improves analysis effect
Rate.
2) sample size needed for is few, and sensitivity is good.Existing method is because needing first isolated crude product solution, it is therefore desirable to sample
Product amount is larger, and usually several grams to tens grams are differed；The analysis sample used amount of the method for the present invention is generally 0.01~1mg, mentions
High sensitivity for analysis.
3) present invention optimizes the testing conditions such as pyrolysis, chromatography, mass spectrum, can be realized with the chromatographic peak needed for fast qualitative
Quickly and accurate detection.
Specific embodiment
Below with reference to embodiment, the invention will be further described.
The present invention melts the relatively low and volatile feature of boiling point for psoralen and isopsorapen, using fast speed heat
Solution-gas chromatography/mass spectrometry technology (PY-GC/MS), sufficiently combines thermal release and chromatographic isolation, realizes psoralen
With the quick separating of Isopsoralen, mass spectrometry and quantitative detection are then used, achievees the purpose that quick, trace detection.
Embodiment
A method of psoralen and isopsorapen is detected based on PY-GC/MS, by taking psoralea corylifolia preparation as an example, but this
The protection scope of invention is not limited to the embodiment, the specific steps are as follows:
One, preparation of samples
By the psoralea corylifolia capsule (Shaanxi Si Nuote Bioisystech Co., Ltd, Xi'an) of acquirement accurately weigh 1.00mg (±
It 0.02mg) is put into the pyrolysis cuvette of 50 μ L；
Two, it is pyrolyzed:
Pyrocrack furnace uses the PY2020is of Frontiers；
1. pyrolysis temperature: 300 DEG C；
2. pyrolysis time: 0.20min.
Three, it separates, detect
Chromatography:
Gas-chromatography uses 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 1.0mL/min；
3. input mode is split sampling, split ratio 50:1；
4. injector temperature is 280 DEG C, transmission line temperature is 290 DEG C；
5. temperature programming: initial temperature is 60 DEG C, keeps 1min, then 150 DEG C is risen to 30 DEG C/min, then with 10
DEG C/min rises to 300 DEG C °.
Mass spectrum (uses Agilent 5975C):
1. the source EI, positive ion detection, electron energy 70eV；
2. ion source temperature is 230 DEG C, single level four bars temperature is 150 DEG C；
3. scanning mode is full scan mode；
4. electron multiplier voltage is 2312V, solvent delay 2.75min.
Four, data analysis and processing
The retention time and integral area of chromatogram are automatically performed by work station, compose the qualitative chromatographic peak in library using NIST08.
The relative amount of each volatile component is obtained by area normalization method.
Identifiable peak is 26 in this sample psoralea corylifolia preparation pyrolyzed components, and ingredient and its retention time are listed in table 1.It protects
Time and peak area Agilent Chemstation is stayed to calculate, the method for relative amount area normalization calculates.
The composition and content (t of volatile component in 1. psoralea corylifolia of tabler=10.161: psoralen, tr=10.789: different benefit
Bone fat element)
Five, precision, reproducibility and stability experiment
1. Precision Experiment: to be derived from 5 parts of sample of same psoralea corylifolia capsule, continuous sample introduction 5 times, right according to the method described above
26 chromatographic peaks of the relative peak area greater than 0.5% are analyzed, and the RSD of relative retention time and relative peak area is (opposite
Standard deviation) respectively between 0.45~1.78% and 1.2~4.97%, illustrate that the method for the present invention precision is good.
2. reproducibility is tested: taking 5 parts of sample of same batch psoralea corylifolia capsule, according to the method described above continuous sample introduction 5 times.Knot
The RSD of the relative retention time of 26 chromatographic peaks of fruit and relative peak area respectively 0.23~5.7% and 1.31~4.63% it
Between, illustrate that the method for the present invention reproducibility is good.
3. stability experiment: take 1 part of sample prepared, respectively 0,2,4,8,12, sample introduction measures for 24 hours, as a result 26
The relative retention time of chromatographic peak and the RSD of relative peak area are respectively less than 4.445%, illustrate the method for the present invention in interior measurement for 24 hours
It is stable.
Basically the main active in psoralea corylifolia or its preparation is psoralen and isopsorapen, can be made
For one of psoralea corylifolia or the main judgment criteria of its preparation quality.Chinese medicine contains because of the place of production, season, the difference for processing mode
Water, density etc. have subtle difference, and then the content in its compound preparation is also different.Therefore, it is necessary to pyrolysis, point
Analysis of variance condition is all different, and obtains the condition and range in the present invention accordingly.
The present invention selects PY-GC/MS technology to detect psoralen and isopsorapen content, is for practical raw
It produces, psoralea corylifolia quality different problems due to the difference such as the place of production, season, processing in life, a kind of psoralen and different benefit is provided
Quick, the micro detection method of bone fat element, and then tentatively judge the quality of psoralea corylifolia.The present invention has easy to operate, detection
It limits low, tests favorable reproducibility, the features such as experimental data is intuitive and reliable, (sample size can be down to especially suitable for micro-example detection
10 μ g), and can be used for carrying out the difference in quality different psoralea corylifolias or its formulation samples quantitative comparison, application is wide, is
The quality standard of psoralea corylifolia and modernization application provide the idea and method that can be referred to.
Claims (4)
1. a kind of rapid detection method of psoralen and isopsorapen, it is characterised in that: the following steps are included:
1) it accurately weighs psoralea corylifolia or 0.01~1mg of its compound preparation obtains sample, sample is added in pyrolysis cup；
2) sample being added in pyrolysis cup is measured using fast pyrogenation-gas chromatograph-mass spectrometer (GC-MS)；
3) after step 2), according to the original chromatographic data and mass spectrometric data of measurement, psoralen and different benefit in qualitative sample
The chromatographic peak of bone fat element, and obtain the relative amount of psoralen and isopsorapen in sample；
The condition of the pyrolysis are as follows: pyrolysis temperature is 200~400 DEG C, and pyrolysis time is 0.1~2min；
The operating condition of the gas-chromatography are as follows: carrier gas is the helium of 0.1~4.0mL/min of flow velocity, and split ratio is 0~500:1,
Temperature program are as follows: initial temperature be 40-60 DEG C, and keep 0~1min, then rise to 150 DEG C with 30-35 DEG C/min, then with
10-15 DEG C/min rises to 300 DEG C；
The mass spectrographic operating condition are as follows: use the source EI, positive ion detection, electron energy is 40~80eV, and ion source temperature is
210~240 DEG C, single level four bars temperature is 140~180 DEG C, and scanning mode is full scan mode, and electron multiplier voltage is 1070
~3000V, solvent delay are 0~3min.
2. a kind of rapid detection method of psoralen and isopsorapen according to claim 1, it is characterised in that: described
Sample is blocky or pulverulent solids.
3. a kind of rapid detection method of psoralen and isopsorapen according to claim 1, it is characterised in that: described
Relative amount is calculated using area normalization method.
4. a kind of rapid detection method of psoralen and isopsorapen according to claim 1, it is characterised in that: described
The qualitative of the chromatographic peak of psoralen and isopsorapen is calculated using NIST08 software Auto-matching.
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| CN104251890B (en) * | 2013-06-27 | 2016-05-25 | 天津中医药大学 | A kind of method that detects the metabolite relevant with Psoralea corylifolia hepatotoxicity wind agitation and uses thereof |
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| SPME/ GC/ MS法分析补骨脂挥发性成分;廖堃 等;《中药材》;20031031;第26卷(第10期);文章摘要、第1-2节 |
| 用裂解气相色谱-质谱法分析丁香油中的成分;丁毅等;《中国生化药物杂志》;20031231;第24卷(第1期);文章第2-3节 |
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