CN110487942A - A kind of fingerprint atlas detection method of antidepressant - Google Patents
A kind of fingerprint atlas detection method of antidepressant Download PDFInfo
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- CN110487942A CN110487942A CN201910943147.7A CN201910943147A CN110487942A CN 110487942 A CN110487942 A CN 110487942A CN 201910943147 A CN201910943147 A CN 201910943147A CN 110487942 A CN110487942 A CN 110487942A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
Abstract
The invention discloses a kind of fingerprint atlas detection methods of antidepressant, using liquid chromatography, the characterization of the reason active constituent of antidepressant for being formed with hypericum perforatum, Radix Paeoniae Alba, cape jasmine, Flos Albiziae, cortex moutan, it provides one kind and method is effectively detected, realize the quality control of Chinese medicine composition, fingerprint atlas detection method provided by the invention has the advantages that repeatability and stability are good.
Description
Technical field
The present invention relates to Analysis of Chinese Traditional Medicine field, the finger-print detection side of more particularly to a kind of antidepressant
Method.
Background technique
After traditional Chinese medicine fingerprint refers to that certain Chinese medicines or Chinese patent drug are appropriately processed, using present information acquisition technique and
The image that can show Chinese medicine or pcm chemical feature, figure, spectrum spectrogram and its data that quality analysis means obtain
A kind of map.A kind of mode that traditional Chinese medicine fingerprint is controlled as quality, is gradually valued by the people, and chromatographic fingerprinting
Most basic attribute be integrality and ambiguity, it is a kind of synthesis, and quantifiable identification of means, it is built upon Chinese medicine
On the basis of chemical component system research, it is mainly used for evaluating the authenticity of Chinese medicine and Chinese materia medica preparation semi-manufactured goods quality, excellent
Benign and stability.Finger-print should have fingerprint, it may be assumed that (1) characteristic and specificity refer to that specified line map is this
Chinese medicine is exclusive, distinguishes with other Chinese medicines, such as the difference of kind, the place of production or collecting season, and map is showed
The chemical information come has the selectivity of height；(2) stability, reproducibility, the finger-print of Chinese medicine should be from certain Chinese medicine
The general character summarized in multiple batches of, under defined method and condition, shared peak or characteristic peak in map should be relatively stable；
(3) validity, integrality, specified finger-print should be able to reproduce fingerprint characteristic (such as shared peak number mesh, greatly under prescribed conditions
Small, position etc.), error should be in allowed limits.
Depressive emotion is common mood disorder venereal disease disease, low for primary symptom with mental state, and has corresponding thinking and behavior to change
A kind of disturbance of emotion disease become, clinical manifestation are low mental state, retardation of thinking, loss of appetite, sleep disturbance etc., serious danger
Victimize physical and mental health.Nowadays rhythm of life is accelerated, and employment competition aggravation, the number for depressive emotion occur increases year by year, sternly
The depressive emotion of weight can develop as depression, or even the moods such as occur committing suiside.
Fingerprint spectrum method for drug test have certain limitation for example: the difference in quality of Chinese medicine itself compared with
Greatly, also there is large effect in different batches, place of production etc. to the detection of finger-print；The detection of finger-print is by environment and instrument
Be affected, the standard of formulation is not able to satisfy the requirement of each period or different experiments room；In addition medicinal material extract separates
Standardization degree to analysis detection is inadequate, also limits the detection of finger-print.
Therefore, in conjunction with the above problem, a kind of fingerprint atlas detection method of antidepressant is provided, is those skilled in the art
The problem of member's urgent need to resolve.
Summary of the invention
In view of this, this method is using efficiently the present invention provides a kind of fingerprint atlas detection method of antidepressant
Liquid chromatography has carried out effective table for the middle pharmacological component of hypericum perforatum, Radix Paeoniae Alba, cape jasmine, Flos Albiziae, cortex moutan
Sign, can effectively realize the quality control to detected Chinese medicine composition, which has repeatability, stability
Good advantage.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of fingerprint atlas detection method of antidepressant, using liquid chromatography, chromatographic condition are as follows:
Chromatographic column uses octadecylsilane chemically bonded silica for filler；
Mobile phase A is acetonitrile, and Mobile phase B is phosphate aqueous solution, gradient elution；
Detection wavelength is 230-300nm；
Flow velocity is 1ml/min；
Sample volume is 10 μ l；
Theoretical cam curve is in terms of standard items Hyperoside peak, and >=3000；
Standard solution preparation: taking Hyperoside, accurately weighed, adds 50% methanol aqueous solution that every 1ml is made containing the molten of 1mg
Liquid；
Condition of gradient elution are as follows:
Preferably, the antidepressant is composed of the following raw materials in parts by weight: 3 parts of hypericum perforatum, 12 parts of Radix Paeoniae Alba, Cape jasmine
Sub 6 parts, 10 parts of cortex moutan, 10 parts of Flos Albiziae.
Preferably, specific step is as follows for antidepressant preparation:
S1 weighs hypericum perforatum, Radix Paeoniae Alba, cape jasmine, Flos Albiziae, cortex moutan in proportion, stock；
S2, hypericum perforatum add 10 times of 60% ethyl alcohol of amount, and heating and refluxing extraction 2 times, 1.5 hours every time, combined extract was dense
Contracting, is dried under reduced pressure；Radix Paeoniae Alba adds 10 times of 70% ethyl alcohol of amount, and heating and refluxing extraction 2 times, 1.5 hours every time, combined extract was concentrated,
It is dried under reduced pressure；Cape jasmine adds 10 times of 80% ethyl alcohol of amount, and heating and refluxing extraction 2 times, 1.5 hours every time, combined extract, concentration subtracted
It press dry dry；Flos Albiziae adds 10 times of amount pure water, and heating and refluxing extraction 2 times, 1.5 hours every time, combined extract, concentration was depressurized dry
It is dry；Cortex moutan adds 10 times of amount pure water, obtains extract through steam distillation.
S3, above-mentioned extract obtained A-E is uniform by weight 15:14:7:5:0.6 ground and mixed, 80 mesh pharmacopeia sieve is crossed,
Obtain antidepressant compound formulation.
Preferably, the finger-print of the antidepressant are as follows:
Finger-print includes 15 shared peaks: the relative retention time at each peak is respectively as follows:
(1)9.153、(2)11.695、(3)15.111、(4)16.559、(5)19.563、(6)20.383、(7)32.511、
(8)44.247、(9S)44.856、(10)45.764、(11)49.397、(12)51.643、(13)53.324、(14)88.658、
(15)96.463。
Preferably, the shared peak of the finger-print are as follows:
No. 1 peak, retention time 9.153, RSD 0.08%, peak area 1336.402, accounting for total peak area percentage is
4.432%；
No. 2 peaks, retention time 11.695, RSD 0.04%, peak area 521.328, accounting for total peak area percentage is
1.729%；
No. 3 peaks, retention time 15.111, RSD 0.04%, peak area 305.260, accounting for total peak area percentage is
1.012%；
No. 4 peaks, retention time 16.559, RSD 0.04%, side, peak area 437.930 account for total peak area percentage
It is 1.452%；
No. 5 peaks, retention time 19.563, RSD 0.06%, peak area 406.334, accounting for total peak area percentage is
1.348%；
No. 6 peaks, retention time 20.383, RSD 0.04%, peak area 1151.954, accounting for total peak area percentage is
3.820%；
No. 7 peaks, retention time 32.511, RSD 0.04%, peak area 820.504, accounting for total peak area percentage is
2.721%；
No. 8 peaks, retention time 44.247, RSD 0.09%, peak area 1617.883, accounting for total peak area percentage is
5.365%；
The peak 9S, retention time 44.856, RSD 0.07%, peak area 1299.504, accounting for total peak area percentage is
4.309%；
No. 10 peaks, retention time 45.764, RSD 0.09%, peak area 1336.627, accounting for total peak area percentage is
4.433%；
No. 11 peaks, retention time 49.397, RSD 0.09%, peak area 2051.489, accounting for total peak area percentage is
6.803%；
No. 12 peaks, retention time 51.643, RSD 0.05%, peak area 563.488, accounting for total peak area percentage is
1.869%；
No. 13 peaks, retention time 53.324, RSD 0.03%, peak area 895.859, accounting for total peak area percentage is
2.971%；
No. 14 peaks, retention time 88.658, RSD 0.02%, peak area 646.792, accounting for total peak area percentage is
2.145%；
No. 15 peaks, retention time 96.463, RSD 0.04%, peak area 9089.165, accounting for total peak area percentage is
30.142%；
Preferably, the pH of mobile phase A described in the method is 2.0-7.0.
Preferably, the pH of mobile phase A described in the method is 2.5.
Preferably, the reference substance solution of liquid chromatography for measuring, preparation method are as follows: take Hypericum Chinense are used in the method
Glycosides is placed in volumetric flask, methanol aqueous solution is added, ultrasonic dissolution, constant volume, filtering, obtaining filtrate is reference substance solution.
Preferably, the test solution of liquid chromatography for measuring, preparation method are used in the method are as follows: take described anti-
In depressed dry powder drug merging volumetric flask, methanol aqueous solution is added, ultrasonic dissolution, constant volume, filtering, obtaining filtrate is that test sample is molten
Liquid.
Preferably, the methanol aqueous solution is the methanol solution of 40%-60%.
As can be seen from the above technical solutions, compared with prior art, beneficial effects of the present invention are as follows:
1, the present invention establish using a kind of Chinese medicine composition of high performance liquid chromatography fingerprint atlas detection method and
Its finger-print has carried out effective table for pharmacological component in its hypericum perforatum, Radix Paeoniae Alba, cape jasmine, Flos Albiziae, cortex moutan
Sign, provides one kind and method is effectively detected, and realizes the quality control of certain Chinese medicine compositions；
2, its repeatability of fingerprint atlas detection method provided by the invention and stability are preferable.
3, by fingerprint atlas detection method can effective monitoring different batches, the medicinal material of different sources, be medicinal material into factory examine
Offer reference frame is provided
4, the Chinese prescription in the present invention is helped to alleviate depressive emotion, be had antidepressant by animal effect experiment
Effect, the present invention carries out the method for quality control of Anti-depressive traditional Chinese medicinal composition on the basis of being guiding with drug effect, only not bright
It really illustrates that the relationship of its fingerprint characteristic and drug effect provides foundation, the matter of the active constituent of Chinese medicine composition can be efficiently controlled
Amount, provides theoretical foundation for the expanding production and clinical rational drug use of Chinese medicine composition.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
The embodiment of invention for those of ordinary skill in the art without creative efforts, can also basis
The attached drawing of offer obtains other attached drawings.
Fig. 1 is the finger-print of 1 Hypericum Perforatum P.E of the embodiment of the present invention；
Fig. 2 is the finger-print of 2 radix paeoniae alba extraction of the embodiment of the present invention；
Fig. 3 is the finger-print of 3 capejasmine extract of the embodiment of the present invention；
Fig. 4 is the finger-print of 4 Cortex Moutan extract of the embodiment of the present invention；
Fig. 5 is the finger-print of 5 Flos Albiziae extract of the embodiment of the present invention.
Fig. 6 is the finger-print of 6 Chinese medicine composition of the embodiment of the present invention；
Specific embodiment
The following is a clear and complete description of the technical scheme in the embodiments of the invention, it is clear that described embodiment
Only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
Art personnel every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1:
The embodiment of the present invention 1 discloses hypericum perforatum determining fingerprint pattern, the technical solution adopted is as follows:
1, preparation method
Hypericum perforatum adds 10 times of 60% ethyl alcohol of amount, and heating and refluxing extraction 2 times, 1.5 hours every time, combined extract was concentrated,
It is dried under reduced pressure；0.71g is weighed, as 50% methanol aqueous solution in 25ml volumetric flask, is added, ultrasonic 20min dissolution is added 50%
Methanol-water be settled to scale, filter to get Hypericum Perforatum P.E sample solution.
2, key instrument
Chromatographic system: 1260 Infinity II of Agilent Technologies (1260 Quat Pump of G7111B,
G7129A 1260 Vialsampler,G7116A 1260 MCT,G7117B 1290 DAD)；
Ultrasonic washing instrument: KQ-600DE, Kunshan Ultrasonic Instruments Co., Ltd.；
Rotary Evaporators: RE-52A, Shanghai Yarong Biochemical Instrument Plant；
Circulating water type vacuum pump: SHZ-D (III), Yu Hua Instrument Ltd., Gongyi City.
3, reagent
Methanol: 20180606, purity >=99.9%, Chinese medicines group chemical reagent；
Acetonitrile: 20180731, purity >=99.9%, Chinese medicines group chemical reagent；
Phosphoric acid: purity >=85%, Tianjin Kermel Chemical Reagent Co., Ltd.；
Pure water: 20180927, Watson.
4, chromatographic condition
Chromatographic column is using octadecylsilane chemically bonded silica as filler, and using gradient elution, mobile phase A is acetonitrile, Mobile phase B
For phosphoric acid water, Detection wavelength 300nm, flow velocity 1ml/min, 30 DEG C of column temperature, sampling volume 10ul；Gradient elution program
Are as follows:
5, it measures
Above-mentioned Hypericum Perforatum P.E sample solution injection liquid chromatograph is drawn, according to high effective liquid chromatography for measuring,
Hypericum Perforatum P.E finger-print is obtained, wherein fingerprint peaks have:
No. 1 peak, retention time RT are 14.972, RSD 0.8%, and peak area 755.783, accounting for peak area ratio is
2.603%；
No. 2 peaks, retention time RT are 16.424, RSD 0.4%, and peak area 1195.265, accounting for peak area ratio is
4.116%；
No. 3 peaks, retention time RT are 19.465, RSD 0.3%, and peak area 1032.728, accounting for peak area ratio is
3.556%；
No. 4 peaks, retention time RT are 20.287, RSD 0.4%, and peak area 2684.446, accounting for peak area ratio is
9.244%；
No. 5 peaks, retention time RT are 21.278, RSD 0.5%, and peak area 968.582, accounting for peak area ratio is
3.335%；
No. 6 peaks, retention time RT are 26.564, RSD 0.2%, and peak area 523.280, accounting for peak area ratio is
1.802%；
No. 7 peaks, retention time RT are 44.227, RSD 0.3%, and peak area 4200.473, accounting for peak area ratio is
14.464%；
No. 8 peaks, retention time RT are 44.847, RSD 0.3%, and peak area 3439.290, accounting for peak area ratio is
11.843%；
No. 9 peaks, retention time RT are 45.769, RSD 0.5%, and peak area 3686.598, accounting for peak area ratio is
12.695%；
No. 10 peaks, retention time RT are 51.697, RSD 0.4%, and peak area 1622.576, accounting for peak area ratio is
5.587%；
No. 11 peaks, retention time RT are 53.377, RSD 0.3%, and peak area 737.842, accounting for peak area ratio is
2.541%；
Embodiment 2:
The embodiment of the present invention 2 discloses radix paeoniae alba extraction determining fingerprint pattern, the technical solution adopted is as follows:
1, preparation method
Radix Paeoniae Alba adds 10 times of 70% ethyl alcohol of amount, and heating and refluxing extraction 2 times, 1.5 hours every time, combined extract was concentrated, decompression
It is dry, 0.71g is weighed, as 50% methanol aqueous solution in 25ml volumetric flask, is added, 50% first is added in ultrasonic 20min dissolution
Alcohol water is settled to scale, filters to get radix paeoniae alba extraction sample solution.
2, key instrument
Chromatographic system: 1260 Infinity II of Agilent Technologies (1260 Quat Pump of G7111B,
G7129A 1260 Vialsampler,G7116A 1260 MCT,G7117B 1290 DAD)；
Ultrasonic washing instrument: KQ-600DE, Kunshan Ultrasonic Instruments Co., Ltd.；
Rotary Evaporators: RE-52A, Shanghai Yarong Biochemical Instrument Plant
Circulating water type vacuum pump: SHZ-D (III), Yu Hua Instrument Ltd., Gongyi City
3, reagent
Methanol: 20180606, purity >=99.9%, Chinese medicines group chemical reagent；
Acetonitrile: 20180731, purity >=99.9%, Chinese medicines group chemical reagent；
Phosphoric acid: purity >=85%, Tianjin Kermel Chemical Reagent Co., Ltd.；
Pure water: 20180927, Watson.
4, chromatographic condition
Chromatographic column is using octadecylsilane chemically bonded silica as filler, and using gradient elution, mobile phase A is acetonitrile, Mobile phase B
For phosphoric acid water, Detection wavelength 230nm, flow velocity 1ml/min, 30 DEG C of column temperature, sampling volume 10ul；Gradient elution program
Are as follows:
5, it measures
Above-mentioned radix paeoniae alba extraction sample solution injection liquid chromatograph is drawn to obtain according to high effective liquid chromatography for measuring
Radix paeoniae alba extraction finger-print, wherein fingerprint peaks have:
No. 1 peak, retention time RT are 9.374, RSD 0.5%, and peak area 9410.198, accounting for peak area ratio is
5.785%；
No. 2 peaks, retention time RT are 11.937, RSD 0.9%, and peak area 2016.463, accounting for peak area ratio is
1.240%；
No. 3 peaks, retention time RT are 27.011, RSD 0.5%, and peak area 22562.261, accounting for peak area ratio is
13.871%；
No. 4 peaks, retention time RT are 30.912, RSD 0.6%, and peak area 68138.645, accounting for peak area ratio is
41.892%；
No. 5 peaks, retention time RT are 44.947, RSD 0.7%, and peak area 18202.291, accounting for peak area ratio is
11.191%；
No. 6 peaks, retention time RT are 50.365, RSD 0.6%, and peak area 4852.267, accounting for peak area ratio is
2.983%；
No. 7 peaks, retention time RT are 56.724, RSD 0.5%, and peak area 2196.713, accounting for peak area ratio is
1.351%；
No. 8 peaks, retention time RT are 99.365, RSD 0.2%, and peak area 1948.910, accounting for peak area ratio is
1.198%.
Embodiment 3:
The embodiment of the present invention 3 discloses capejasmine extract determining fingerprint pattern, the technical solution adopted is as follows:
1, preparation method
Cape jasmine adds 10 times of 80% ethyl alcohol of amount, and heating and refluxing extraction 2 times, 1.5 hours every time, combined extract was concentrated, decompression
It is dry；0.71g is weighed, as 50% methanol aqueous solution in 25ml volumetric flask, is added, 50% first is added in ultrasonic 20min dissolution
Alcohol water is settled to scale, filters to get capejasmine extract sample solution.
2, key instrument
Chromatographic system: 1260 Infinity II of Agilent Technologies (1260 Quat Pump of G7111B,
G7129A 1260 Vialsampler,G7116A 1260 MCT,G7117B 1290 DAD)；
Ultrasonic washing instrument: KQ-600DE, Kunshan Ultrasonic Instruments Co., Ltd.；
Rotary Evaporators: RE-52A, Shanghai Yarong Biochemical Instrument Plant
Circulating water type vacuum pump: SHZ-D (III), Yu Hua Instrument Ltd., Gongyi City.
3, reagent
Methanol: 20180606, purity >=99.9%, Chinese medicines group chemical reagent；
Acetonitrile: 20180731, purity >=99.9%, Chinese medicines group chemical reagent；
Phosphoric acid: purity >=85%, Tianjin Kermel Chemical Reagent Co., Ltd.；
Pure water: 20180927, Watson.
4, chromatographic condition
Chromatographic column is using octadecylsilane chemically bonded silica as filler, and using gradient elution, mobile phase A is acetonitrile, Mobile phase B
For phosphoric acid water, Detection wavelength 300nm, flow velocity 1ml/min, 30 DEG C of column temperature, sampling volume 10ul；Gradient elution program
Are as follows:
5, it measures
Above-mentioned capejasmine extract sample solution injection liquid chromatograph is drawn to obtain according to high effective liquid chromatography for measuring
Capejasmine extract finger-print, wherein fingerprint peaks have:
No. 1 peak, retention time RT are 14.912, RSD 0.4%, and peak area 1933.598, accounting for peak area ratio is
4.781%；
No. 2 peaks, retention time RT are 15.761, RSD 0.4%, and peak area 819.484, accounting for peak area ratio is
2.026%；
No. 3 peaks, retention time RT are 16.615, RSD 0.2%, and peak area 608.844, accounting for peak area ratio is
1.505%；
No. 4 peaks, retention time RT are 18.178, RSD 0.5%, and peak area 907.286, accounting for peak area ratio is
2.243%；
No. 5 peaks, retention time RT are 19.971, RSD 0.8%, and peak area 2888.059, accounting for peak area ratio is
7.141%；
No. 6 peaks, retention time RT are 32.594, RSD 0.8%, and peak area 27584.143, accounting for peak area ratio is
68.205%；
Embodiment 4:
The embodiment of the present invention 4 discloses Cortex Moutan extract determining fingerprint pattern, the technical solution adopted is as follows:
1, preparation method
Cortex moutan adds 10 times of amount pure water, obtains extract through steam distillation.
2, key instrument
Chromatographic system: 1260 Infinity II of Agilent Technologies (1260 Quat Pump of G7111B,
G7129A 1260 Vialsampler,G7116A 1260 MCT,G7117B 1290 DAD)；
Ultrasonic washing instrument: KQ-600DE, Kunshan Ultrasonic Instruments Co., Ltd.；
Rotary Evaporators: RE-52A, Shanghai Yarong Biochemical Instrument Plant
Circulating water type vacuum pump: SHZ-D (III), Yu Hua Instrument Ltd., Gongyi City.
3, reagent
Methanol: 20180606, purity >=99.9%, Chinese medicines group chemical reagent；
Acetonitrile: 20180731, purity >=99.9%, Chinese medicines group chemical reagent；
Phosphoric acid: purity >=85%, Tianjin Kermel Chemical Reagent Co., Ltd.；
Pure water: 20180927, Watson.
4, chromatographic condition
Chromatographic column is using octadecylsilane chemically bonded silica as filler, and using gradient elution, mobile phase A is acetonitrile, Mobile phase B
For phosphoric acid water, Detection wavelength 274nm, flow velocity 1ml/min, 30 DEG C of column temperature, sampling volume 10ul；Gradient elution program
Are as follows:
5, it measures
Above-mentioned Cortex Moutan extract sample solution injection liquid chromatograph is drawn to obtain according to high effective liquid chromatography for measuring
To Cortex Moutan extract finger-print, wherein fingerprint peaks have:
No. 1 peak, retention time RT are 97.110, RSD 0.09%, and peak area 3076.943, accounting for peak area ratio is
99.650%
Embodiment 5:
The embodiment of the present invention 5 discloses Flos Albiziae determining fingerprint pattern, the technical solution adopted is as follows:
1, preparation method
Flos Albiziae adds 10 times of amount pure water, and heating and refluxing extraction 2 times, 1.5 hours every time, combined extract, concentration was depressurized dry
It is dry；0.71g is weighed, as 50% methanol aqueous solution in 25ml volumetric flask, is added, 50% methanol is added in ultrasonic 20min dissolution
Water is settled to scale, filters to get Flos Albiziae extract sample solution.
2, key instrument
Chromatographic system: 1260 Infinity II of Agilent Technologies (1260 Quat Pump of G7111B,
G7129A 1260 Vialsampler,G7116A 1260 MCT,G7117B 1290 DAD)；
Ultrasonic washing instrument: KQ-600DE, Kunshan Ultrasonic Instruments Co., Ltd.；
Rotary Evaporators: RE-52A, Shanghai Yarong Biochemical Instrument Plant
Circulating water type vacuum pump: SHZ-D (III), Yu Hua Instrument Ltd., Gongyi City.
3, reagent
Methanol: 20180606, purity >=99.9%, Chinese medicines group chemical reagent；
Acetonitrile: 20180731, purity >=99.9%, Chinese medicines group chemical reagent；
Phosphoric acid: purity >=85%, Tianjin Kermel Chemical Reagent Co., Ltd.；
Pure water: 20180927, Watson.
4, chromatographic condition
Chromatographic column is using octadecylsilane chemically bonded silica as filler, and using gradient elution, mobile phase A is acetonitrile, Mobile phase B
For phosphoric acid water, Detection wavelength 256nm, flow velocity 1ml/min, 30 DEG C of column temperature, sampling volume 10ul；Gradient elution program
Are as follows:
5, it measures
Above-mentioned Flos Albiziae extract sample solution injection liquid chromatograph is drawn to obtain according to high effective liquid chromatography for measuring
To Flos Albiziae extract finger-print, wherein fingerprint peaks have:
No. 1 peak, retention time RT are 3.133, RSD 0.5%, and peak area 3036.946, accounting for peak area ratio is
10.294%；
No. 2 peaks, retention time RT are 3.956, RSD 0.3%, and peak area 5068.172, accounting for peak area ratio is
17.179%；
No. 3 peaks, retention time RT are 7.329, RSD 0.2%, and peak area 536.413, accounting for peak area ratio is
1.818%；
No. 4 peaks, retention time RT are 15.243, RSD 0.2%, and peak area 2024.062, accounting for peak area ratio is
6.861%；
No. 5 peaks, retention time RT are 20.146, RSD 0.5%, and peak area 396.857, accounting for peak area ratio is
1.345%；
No. 6 peaks, retention time RT are 45.385, RSD 0.6%, and peak area 434.784, accounting for peak area ratio is
1.474%；
No. 7 peaks, retention time RT are 54.011, RSD 0.8%, and peak area 7220.747, accounting for peak area ratio is
24.475%；
Embodiment 6:
The embodiment of the present invention 6 discloses the finger-print detection assay of antidepressant Chinese medicine composition, the technology of use
Scheme is as follows:
Raw material: hypericum perforatum 3g, Radix Paeoniae Alba 12g, cape jasmine 6g, Flos Albiziae 10g, cortex moutan 10g.
1, preparation method
Hypericum perforatum adds 10 times of 60% ethyl alcohol of amount, and heating and refluxing extraction 2 times, 1.5 hours every time, combined extract was concentrated,
It is dried under reduced pressure；Radix Paeoniae Alba adds 10 times of 70% ethyl alcohol of amount, and heating and refluxing extraction 2 times, 1.5 hours every time, combined extract, concentration subtracted
It press dry dry；Cape jasmine adds 10 times of 80% ethyl alcohol of amount, and heating and refluxing extraction 2 times, 1.5 hours every time, combined extract was concentrated, decompression
It is dry；Flos Albiziae adds 10 times of amount pure water, and heating and refluxing extraction 2 times, 1.5 hours every time, combined extract, concentration was dried under reduced pressure；
Cortex moutan adds 10 times of amount pure water, obtains extract through steam distillation.
2, key instrument
Chromatographic system: 1260 Infinity II of Agilent Technologies (1260 Quat Pump of G7111B,
G7129A 1260 Vialsampler,G7116A 1260 MCT,G7117B 1290 DAD)；
Ultrasonic washing instrument: KQ-600DE, Kunshan Ultrasonic Instruments Co., Ltd.；
Rotary Evaporators: RE-52A, Shanghai Yarong Biochemical Instrument Plant
Circulating water type vacuum pump: SHZ-D (III), Yu Hua Instrument Ltd., Gongyi City.
3, reagent
Methanol: 20180606, purity >=99.9%, Chinese medicines group chemical reagent；
Acetonitrile: 20180731, purity >=99.9%, Chinese medicines group chemical reagent；
Phosphoric acid: purity >=85%, Tianjin Kermel Chemical Reagent Co., Ltd.；
Pure water: 20180927, Watson.
4, chromatographic condition
Chromatographic column is using octadecylsilane chemically bonded silica as filler, and using gradient elution, mobile phase A is acetonitrile, Mobile phase B
For phosphoric acid water, Detection wavelength 230nm, 238nm, 256nm, 274nm, 300nm, flow velocity 1ml/min, 30 DEG C of column temperature, sample introduction
Volume is 10ul；Gradient elution program are as follows:
5, it measures
Above-mentioned Chinese medicine composition sample solution injection liquid chromatograph is drawn to obtain according to high effective liquid chromatography for measuring
Chinese medicine composition finger-print, wherein fingerprint peaks have:
No. 1 peak, retention time 9.153, RSD 0.08%, peak area 1336.402, accounting for total peak area percentage is
4.432%；
No. 2 peaks, retention time 11.695, RSD 0.04%, peak area 521.328, accounting for total peak area percentage is
1.729%；
No. 3 peaks, retention time 15.111, RSD 0.04%, peak area 305.260, accounting for total peak area percentage is
1.012%；
No. 4 peaks, retention time 16.559, RSD 0.04%, side, peak area 437.930 account for total peak area percentage
It is 1.452%；
No. 5 peaks, retention time 19.563, RSD 0.06%, peak area 406.334, accounting for total peak area percentage is
1.348%；
No. 6 peaks, retention time 20.383, RSD 0.04%, peak area 1151.954, accounting for total peak area percentage is
3.820%；
No. 7 peaks, retention time 32.511, RSD 0.04%, peak area 820.504, accounting for total peak area percentage is
2.721%；
No. 8 peaks, retention time 44.247, RSD 0.09%, peak area 1617.883, accounting for total peak area percentage is
5.365%；
No. 9 peaks, retention time 44.856, RSD 0.07%, peak area 1299.504, accounting for total peak area percentage is
4.309%；
No. 10 peaks, retention time 45.764, RSD 0.09%, peak area 1336.627, accounting for total peak area percentage is
4.433%；
No. 11 peaks, retention time 49.397, RSD 0.09%, peak area 2051.489, accounting for total peak area percentage is
6.803%；
No. 12 peaks, retention time 51.643, RSD 0.05%, peak area 563.488, accounting for total peak area percentage is
1.869%；
No. 13 peaks, retention time 53.324, RSD 0.03%, peak area 895.859, accounting for total peak area percentage is
2.971%；
No. 14 peaks, retention time 88.658, RSD 0.02%, peak area 646.792, accounting for total peak area percentage is
2.145%；
No. 15 peaks, retention time 96.463, RSD 0.04%, peak area 9089.165, accounting for total peak area percentage is
30.142%；
Embodiment 7:
The embodiment of the present invention 7 is methodology validation, the technical solution adopted is as follows:
1, the preparation of reference substance solution
Take appropriate Hyperoside, add 50% methanol, be configured to concentration 1mg/ml, 0.5mg/ml, 0.25mg/ml,
The reference substance solution of 0.125mg/ml, 0.0625mg/ml, 0.03125mg/ml.
2, standard curve preparation and the range of linearity determine
Take appropriate Hyperoside standard solution through 0.45 μm of filtering with microporous membrane, by the chromatographic condition of setting, respectively into
10 μ L of sample, using concentration as abscissa, draws standard curve using chromatographic peak area as ordinate, obtains the linear of Hyperoside and returns
Return equation and the range of linearity.
Hyperoside regression equation is Y=19800.1399X-148.0636, R2=0.99975；Hyperoside is linear
It is good with peak area linear relationship in range.
3, Precision Experiment
Precision draws above-mentioned Hyperoside standard solution, is measured with HPLC, continuous sample introduction 5 times, every time 10 μ L, surveys
Fixed its peak area RSD value, detects the precision of its high performance liquid chromatograph.
Hyperoside retention time RSD is 0.06%；Peak area RSD is 1.07%
4, stability experiment
Chinese medicine composition sample is taken, above-mentioned chromatographic condition is pressed in 3h, 6h, 9h, 12h, 15h respectively, is measured with HPLC,
10 μ L of sample volume.Its peak area is integrated, the relative standard deviation of calculating integral value detects its stability.
Hyperoside retention time RSD is 0.04%；Peak area RSD is 5.05%.
5, reproducibility is tested
Same batch of sample is taken, by sample solution preparation method, prepares part test solution, by above-mentioned chromatographic condition, is used
HPLC is measured, 10 μ L of sample volume.Its area is integrated, relative standard deviation is calculated, detects its reproducibility.
Hyperoside retention time RSD is 0.09%；Peak area RSD is 3.05%
6, sample recovery rate is tested
Known content Chinese medicine composition sample is weighed, totally 5 parts, is separately added into a certain amount of Hyperoside reference substance, by confession
Test solution preparation method prepares 5 parts of sample, and every part of sample continuous sample introduction time 10 μ L of each sample introduction measures content with calibration curve method,
Calculate the mean sample recovery rate of reference substance.
Determination of Hyperoside RSD is 0.12%；Peak area RSD is 2.19%.
Each embodiment in this specification is described in a progressive manner, the highlights of each of the examples are with other
The difference of embodiment, the same or similar parts in each embodiment may refer to each other.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest scope of cause.
Claims (7)
1. a kind of fingerprint atlas detection method of antidepressant, which is characterized in that use liquid chromatography, chromatographic condition are as follows:
Chromatographic column uses octadecylsilane chemically bonded silica for filler；
Mobile phase A is acetonitrile, and Mobile phase B is phosphate aqueous solution, gradient elution；
Detection wavelength is 230-300nm；
Flow velocity is 1ml/min；
Sample volume is 10 μ l；
Theoretical cam curve is in terms of standard items Hyperoside peak, and >=3000；
Standard solution preparation: taking Hyperoside, accurately weighed, adds 50% methanol aqueous solution that solution of every 1ml containing 1mg is made；
Condition of gradient elution are as follows:
2. a kind of fingerprint atlas detection method of antidepressant according to claim 1, which is characterized in that described is anti-
Depressed drug is composed of the following raw materials in parts by weight: 3 parts of hypericum perforatum, 12 parts of Radix Paeoniae Alba, 6 parts of cape jasmine, 10 parts of cortex moutan, Flos Albiziae
10 parts.
3. a kind of fingerprint atlas detection method of antidepressant according to claim 1, which is characterized in that the anti-suppression
The finger-print of strongly fragrant drug are as follows:
Finger-print includes 15 shared peaks: the relative retention time at each peak is respectively as follows:
(1)9.153、(2)11.695、(3)15.111、(4)16.559、(5)19.563、(6)20.383、(7)32.511、(8)
44.247、(9S)44.856、(10)45.764、(11)49.397、(12)51.643、(13)53.324、(14)88.658、(15)
96.463。
4. a kind of fingerprint atlas detection method of antidepressant according to claim 1, which is characterized in that the method
Described in mobile phase A pH be 2.0-7.0.
5. a kind of fingerprint atlas detection method of antidepressant according to claim 1, which is characterized in that the method
In be used for liquid chromatography for measuring reference substance solution, preparation method are as follows: take Hyperoside merging volumetric flask in, be added methanol
Aqueous solution, ultrasonic dissolution, constant volume, filtering, obtaining filtrate is reference substance solution.
6. a kind of fingerprint atlas detection method of antidepressant according to claim 1, which is characterized in that the method
In be used for the test solution of liquid chromatography for measuring, preparation method are as follows: take antidepressant dry powder merging volumetric flask
In, methanol aqueous solution is added, ultrasonic dissolution, constant volume, filtering, obtaining filtrate is test solution.
7. the preparation method of test solution according to claim 6, which is characterized in that the methanol aqueous solution is
The methanol solution of 40%-60%.
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