CN109239250A - The measuring method and its standard finger-print of sharp brain lamination finger-print - Google Patents
The measuring method and its standard finger-print of sharp brain lamination finger-print Download PDFInfo
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- CN109239250A CN109239250A CN201811133459.3A CN201811133459A CN109239250A CN 109239250 A CN109239250 A CN 109239250A CN 201811133459 A CN201811133459 A CN 201811133459A CN 109239250 A CN109239250 A CN 109239250A
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
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Abstract
The present invention discloses the measuring method and its standard finger-print of a kind of sharp brain lamination HPLC finger-print, the wherein measuring method, comprising the following steps: (1) preparation of reference substance solution: Sodium Danshensu, protocatechualdehyde, Puerarin, Paeoniflorin, liquiritin, fleece-flower root glycosides, tanshin polyphenolic acid B reference substance solution are prepared respectively；(2) preparation of test solution: sharp brain lamination is taken, is extracted, extracting solution filtering with microporous membrane is to get test solution；(3) measuring method: being measured using high performance liquid chromatography, wherein chromatographic condition are as follows: chromatographic column is using octadecylsilane chemically bonded silica as filler；Using gradient elution, mobile phase A is 0.01~3% phosphate aqueous solution, and B is acetonitrile；0.3~1.5ml/min of flow velocity；20~45 DEG C of column temperature；Detection wavelength 230nm.
Description
Technical field
The present invention relates to traditional Chinese medicine fingerprint analysis methods, and in particular to sharp brain lamination HPLC finger print measuring method,
And its obtained sharp brain lamination preparation finger.
Technical background
Cardiovascular and cerebrovascular disease is the common disease for seriously endangering human health in recent years.Due to mentioning for human life quality
Height, cardiovascular and cerebrovascular disease are in rising trend.Cardiovascular and cerebrovascular disease is treated, although Chinese medicine list target spot action intensity is weaker than Western medicine,
The small advantage of multipath, multiple target point, dynamic wholistic therapy, toxic side effect then outclass Western medicine, it is especially curative for effect at
The combined therapy effect of medicine is better than Western medicine.
Sharp brain lamination is by Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong, pueraria lobata, pheretima, radix paeoniae rubra, safflower, Radix Curcumae, prepared fleece flower root, rhizoma alismatis, fructus lycii, acid
Jujube kernel (stir-fry), Radix Polygalae, acorus graminens soland, radix achyranthis bidentatae, 15 taste Chinese medicinal composition of Radix Glycyrrhizae, the medical instrument have promoting blood circulation, promoting the circulation of qi resolving sputum, dredging collateral to stop
The effects of pain.Wherein, Radix Salviae Miltiorrhizae is the monarch drug in a prescription of this preparation, and principle active component has danshensu, tanshin polyphenolic acid B etc., has treatment chest
Rib hypochondriac pain, abdominal mass caking, sore swell and ache curative, falls the effects of falling forward the pain of injury, irregular menstruation, amenorrhea and algomenorrhea, postpartum stasis caused pain at rheumatic arthralgia；
The Paeoniflorin contained in radix paeoniae rubra can be used for treating coronary heart disease, build up health with immune function, anti-inflammatory cough-relieving, eliminating phlegm and relieving asthma etc., especially
It is that can be used as ancillary drug in the treatment of senile chronic respiratory disease；The ferulic acid (sodium ferulate) contained in Rhizoma Chuanxiong has
Platelet aggregation-against, analgesia, alleviates the effects of vasopasm at enhancing prostaglandin activity；Puerarin in pueraria lobata, which has, to be improved
It is immune, enhancing myocardial contractive power, protection cardiac muscle cell, reduce blood pressure, platelet aggregation-against the effects of.
Sharp brain lamination contains 15 taste Chinese medicines, and the assay of existing quality standard only detects a kind of ingredient of danshensu.Furthermore
According to the literature, current content assaying method at most measures 6 kinds of effective components in preparation, including danshensu, former youngster simultaneously
Tea aldehyde, tanshin polyphenolic acid B, Paeoniflorin, ferulic acid, Puerarin.
Existing detection method is as follows:
In State Food and Drug Administration's national drug standards (YBZ04282005-2011Z), surveyed using HPLC method
Determine the content of danshensu.Using methanol as Mobile phase B, 0.5% glacial acetic acid solution is mobile phase A, and gradient elution program is as follows: 0min
→ 15min → 20min → 25min → 30min, mobile phase A 95% → 95% → 70% → 70% → 70% → 95%, flow velocity are
1ml/min, Detection wavelength 280nm.The preparation method of test solution is: this product 10 taken, coating is removed, it is finely ground, and it weighs
2g, it is accurately weighed, it sets in stuffed conical flask, hydrochloric acid solution (1ml → 100ml) 50ml, close plug is added in precision, and weighed weight surpasses
Sonication (power 100W, frequency 50KHz) 30 minutes, lets cool, then weighed weight, is supplied and subtracted with hydrochloric acid solution (1ml → 100ml)
The weight of mistake, shakes up, and filtration, precision measures subsequent filtrate 10ml, and sodium chloride 1g dissolution is added, and is extracted 4 times with ethyl acetate shaking,
Each 15ml, combined ethyl acetate liquid, is evaporated, and residue flowing phased soln is simultaneously transferred in 5ml measuring bottle, adds mobile phase to quarter
Degree, shake up, filter, take subsequent filtrate to get.
Jiang Hui, Wang Shaozhi et al. measure the content of 6 kinds of ingredients in brain-invigorating and heart-soothing capsule, including Radix Salviae Miltiorrhizae using HPLC method simultaneously
Element, protocatechualdehyde, tanshin polyphenolic acid B, Paeoniflorin, ferulic acid and Puerarin.Mobile phase is -0.5% phosphate aqueous solution of acetonitrile-methanol
(gradient elution), gradient elution program is as follows: 0min → 7min → 10min → 35min → 45min, mobile phase A 95% → 95%
→ 70% → 70% → 70% → 95%, flow velocity 1.0ml/min, Detection wavelength 280nm, 230nm, 250nm, 320nm, column
Temperature is 30 DEG C.The preparation method of test solution is: brain-invigorating and heart-soothing capsule about 0.5g is taken, it is accurately weighed, and it is placed in 10ml measuring bottle, adds
Ultrapure water dissolution, is ultrasonically treated (power: 80W, frequency: 20kHz) 30min, is cooled to room temperature, and ultrapure water is added and supplies capacity,
To be centrifuged radius 5cm, 1 500r/min centrifugation 30min, with 0.45 μm of filtering with microporous membrane, take subsequent filtrate to get.
It has been found that being also difficult to efficiently control its total quality by measuring mentioned component, the peace of medication not can guarantee
Quan Xing, validity.For control benefit brain lamination quality on the whole, need to conduct further research it.
Summary of the invention
It is an object of the invention to: the finger print measuring method and its characteristic fingerprint pattern of a kind of sharp brain lamination are provided.
The present invention provides a kind of new method by the research to sharp brain lamination liquid-phase fingerprint, for the quality control of sharp brain lamination,
The disadvantage for compensating for existing Quality Control Technology deficiency keeps sharp brain lamination quality of the pharmaceutical preparations control technology more perfect, also more section
It learns.
For this purpose, the present invention provides a kind of foundation of sharp brain lamination liquid chromatogram standard finger-print and uses the fingerprint image
Compose the method being measured to the sharp brain lamination in production and selling.
A kind of benefit brain lamination HPLC detection method, the described method comprises the following steps:
(1) preparation of test solution: taking sharp brain lamination, extract, and extracting solution filtering with microporous membrane is to get for examination
Product solution；
(2) measuring method: taking test solution, injects high performance liquid chromatograph, obtains chromatic graph spectrum, wherein chromatographic condition
Are as follows:
Chromatographic column is using octadecylsilane chemically bonded silica as filler；
Gradient elution, mobile phase A are 0.01~3% phosphate aqueous solution, and B is acetonitrile；0.3~1.5ml/min of flow velocity；Column temperature
20~45 DEG C；Elution program: 0min → 7min → 10min → 30min → 50min → 65min, acetonitrile 1% → 10% → 15%
→ 38% → 60% → 60%；Detection wavelength 230nm；
(3) gained chromatogram and standard finger-print are compareed, it is qualified product that similarity, which is greater than 0.75, and similarity evaluation is adopted
It is evaluated with " similarity evaluation 2012 editions ".
Wherein, the concentration of reference substance solution described in step (1) is 20~500 μ g/mL.
Wherein, benefit brain lamination described in step (2) weighs as 0.1~10g.
Extraction step described in step (2) is using methanol aqueous solution ultrasonic extraction or to use distilled water ultrasonic extraction, is mentioned
Taking the time is 10~80min.
Miillpore filter aperture described in step (2) is 0.2 μm~0.8 μm.
Wherein, the detection program of the detection of program HPLC described in step (3) are as follows: Detection wavelength 230nm.
In step (3) described measurement, 0.5~20 μ l of high performance liquid chromatograph autosampler sample introduction, according to high-efficient liquid phase color
Spectrometry measurement, obtains finger-print.
A kind of method for building up of standard finger-print, it is characterised in that: steps are as follows:
(1) preparation of reference control solution:
A. the preparation of Sodium Danshensu reference substance solution: it is appropriate that precision weighs Sodium Danshensu reference substance, is prepared with 50% methanol
At the solution of every 1ml 20~500 μ g containing danshensu；
B. the preparation of protocatechualdehyde reference substance solution: it is appropriate that precision weighs protocatechualdehyde reference substance, is prepared with 50% methanol
At the solution of every 1ml 20~500 μ g containing protocatechualdehyde；
C. the preparation of Puerarin reference substance solution: it is appropriate that precision weighs Puerarin reference substance, is configured to often with 50% methanol
The solution of 1ml 20~500 μ g containing Puerarin；
D. the preparation of Paeoniflorin reference substance solution: it is appropriate that precision weighs Paeoniflorin reference substance, is configured to often with 50% methanol
The solution of 1ml 20~500 μ g containing Paeoniflorin；
E. the preparation of liquiritin reference substance solution: it is appropriate that precision weighs liquiritin reference substance, is configured to often with 50% methanol
The solution of 1ml 20~500 μ g containing liquiritin；
F. the preparation of fleece-flower root glycosides reference substance solution: it is appropriate that precision weighs fleece-flower root glycosides reference substance, is prepared with 50% methanol
At the solution of every 20~500 μ g of 1ml glycosides containing the fleece-flower root；
G. the preparation of tanshin polyphenolic acid B reference substance solution: it is appropriate that precision weighs tanshin polyphenolic acid B reference substance, is configured to 50% methanol
The solution of every 1ml 20~500 μ g containing tanshin polyphenolic acid B；
(2) preparation of test solution: weighing the sharp brain lamination of multiple batches of qualification, removes coating, finely ground, weigh 0.1~
0%~100% methanol aqueous solution, ultrasonic extraction is added in 10g, and extraction time is 10~80min, and extraction time is preferably
30min, extracting solution miillpore filter (aperture is 0.2 μm~0.8 μm, preferably 0.45 μm) filtering is to get test solution；
(3) reference control solution and test solution are taken, high performance liquid chromatograph is injected, 0.5~20 μ l of sample volume is obtained
To the sharp brain lamination chromatogram of multiple batches of qualification；
Wherein chromatographic condition are as follows: chromatographic column is using octadecylsilane chemically bonded silica as filler；Using gradient elution, mobile phase
A is 0.01~3% phosphate aqueous solution, and Mobile phase B is acetonitrile；The gradient elution time is 20~180min, and gradient elution program is such as
Under: 0min → 7min → 10min → 30min → 50min → 65min, acetonitrile 1% → 10% → 15% → 38% → 60% →
60%, 0.3~1.5ml/min of flow velocity；20~45 DEG C of column temperature；Detection wavelength 230nm.
(4) the sharp brain lamination chromatogram of the multiple batches of qualification of gained is fitted to by a standard control fingerprint image using computer
Spectrum.
Wherein (1) reference control solution the preparation method is as follows: take each standard items, set in stuffed conical flask, precision plus
Enter 50% methanol 20ml, close plug, weighed weight is ultrasonically treated 30min, lets cool, then weighed weight, supply less loss with Extraction solvent
Weight, with 0.45 μm of filtering with microporous membrane, subsequent filtrate is reference control solution.
Wherein (2) test solution the preparation method is as follows: take sharp brain lamination, remove coating, it is finely ground, weigh 1g, set tool
It filling in conical flask, 50% methanol 20ml, close plug is added in precision, and weighed weight is ultrasonically treated 30min, lets cool, then weighed weight,
The weight that less loss is supplied with Extraction solvent, with 0.45 μm of filtering with microporous membrane, subsequent filtrate is test solution.
Preferably, the model Eclipse XDB-C18 (4.6 × 15cm, 3.5um) of liquid phase capillary column used.Institute
State multiple batches of, at least 10 batches, preferably 20 batches, 30 batches, 50 batches.
The technical scheme is that obtained by screening, screening process is as follows:
1. instrument and reagent
1.1 instrument
Ten a ten thousandth electronic balances (METTLER TOLEDO), Waters H-Class liquid chromatograph, Agilent
Eclipse XDB-C18 (4.6 × 15cm, 3.5um) chromatographic column.
1.2 reagent
Sodium Danshensu reference substance, protocatechualdehyde reference substance, Puerarin reference substance, Paeoniflorin reference substance, the control of fleece-flower root glycosides
Product, liquiritin reference substance, tanshin polyphenolic acid B reference substance, methanol, acetonitrile
2. liquid chromatographic system condition:
Using gradient elution, mobile phase A is 0.5% phosphate aqueous solution, and Mobile phase B is acetonitrile, elution time 65min,
Flow velocity 1.0ml/min；35 DEG C of column temperature；Ultraviolet detection wavelength: 230nm；
Gradient elution program: 0min → 7min → 10min → 30min → 50min → 65min, acetonitrile 1% → 10% →
15% → 38% → 60% (percent by volume).
3. prepared by test solution
Sharp brain lamination is taken, coating is removed, it is finely ground, 1g is weighed, is set in stuffed conical flask, 50% methanol 20ml is added in precision,
Close plug, weighed weight are ultrasonically treated 30min, let cool, then weighed weight, the weight of less loss is supplied with Extraction solvent, with 0.45 μm
Filtering with microporous membrane, subsequent filtrate are test solution.
4. prepared by reference substance solution
A. the preparation of Sodium Danshensu reference substance solution: it is appropriate that precision weighs Sodium Danshensu reference substance, is prepared with 50% methanol
At the solution of every 1ml 55.8 μ g containing danshensu；
B. the preparation of protocatechualdehyde reference substance solution: it is appropriate that precision weighs protocatechualdehyde reference substance, is prepared with 50% methanol
At the solution of every 1ml 52.6 μ g containing protocatechualdehyde；
C. the preparation of Puerarin reference substance solution: it is appropriate that precision weighs Puerarin reference substance, is configured to often with 50% methanol
The solution of 1ml 55.3 μ g containing Puerarin；
D. the preparation of Paeoniflorin reference substance solution: it is appropriate that precision weighs Paeoniflorin reference substance, is configured to often with 50% methanol
The solution of 1ml 27.6 μ g containing Paeoniflorin；
E. the preparation of fleece-flower root glycosides reference substance solution: it is appropriate that precision weighs fleece-flower root glycosides reference substance, is prepared with 50% methanol
At the solution of every 25.5 μ g of 1ml glycosides containing the fleece-flower root；
F. the preparation of liquiritin reference substance solution: it is appropriate that precision weighs liquiritin reference substance, is configured to often with 50% methanol
The solution of 1ml 51.8 μ g containing liquiritin；
G. the preparation of tanshin polyphenolic acid B reference substance solution: it is appropriate that precision weighs liquiritin reference substance, is configured to often with 50% methanol
The solution of 1ml 86.3 μ g containing tanshin polyphenolic acid B；
5. methodology validation
5.1 extraction times were investigated
4 parts of this product are weighed in parallel, each 1g is accurately weighed, sets in stuffed conical flask, and 50% methanol 20ml is added in precision, close
Plug, weighed weight are ultrasonically treated 10min, 20min, 30min and 40min, let cool, then weighed weight, supplied and subtracted with Extraction solvent
The weight of mistake, with 0.45 μm of filtering with microporous membrane, subsequent filtrate to obtain the final product.
It is preferable by comparing experimental result discovery ultrasound 30min and 40min effect.Therefore, select ultrasound 30 minutes as
Extraction time.
5.2 precision test
It takes with a test solution, continuously repeats 6 needle of sample introduction, investigate precision.Retain the result shows that shared peak is opposite
Time RSD is respectively less than 0.08%, and no more than 2.00%, the similarity repeated between 6 needle of sample introduction is relative peak area RSD
1.000 precision is good.Table 1, table 2 are that precision investigates result.
1 Precision test result of table (relative retention time)
2 Precision test result of table (relative peak area)
5.3 repeated
It weighs with a collection of 6 parts of test sample, according to sample solution preparation method, operation repetitive investigates repeatability.As a result table
Bright, shared peak relative retention time RSD is no more than 0.10%, no more than 3.65%, 6 part sample room of relative peak area RSD
Similarity be 1.000, repeatability is good.Table 3, table 4 are that repeatability investigates result.
3 repetitive test result (relative retention time) of table
4 repetitive test result (relative peak area) of table
5.4 study on the stability
Same test solution is taken, respectively at 0h, 2h, 4h, 6h, 8h, 10h injects liquid chromatograph.The result shows that shared
Peak relative retention time RSD is no more than 0.06%, and relative peak area RSD is no more than 3.05%, similarity 1.000, supplies
Test sample solution is stablized in 10h.Table 5, table 6 are that repeatability investigates result.
5 stability test result (relative retention time) of table
6 stability test result (relative peak area) of table
The measurement of 5.5 samples
10 batches of sharp brain lamination are extracted by test sample extracting method, and are measured by the finger print measuring method, are remembered
Record each chromatogram.
6. data are analyzed
It is calculated, is obtained by the Chinese Pharmacopoeia committee " chromatographic fingerprints of Chinese materia medica similarity evaluation software " version in 2012
Each batch similarity value.Sample similarity is 0.75~0.99.
Compared with prior art, improvement of the invention is:
1. fingerprint atlas detection method of the invention can identify at least seven kinds of effective components in sharp brain lamination simultaneously.
2. standard finger-print of the invention possesses 19 fingerprint characteristic peaks, it can reflect the total quality of sharp brain lamination, have
Effect characterizes sharp brain lamination Contents of Main Components.
The method of the present invention has the advantages that following significant and purposes:
1, the first public foundation using HPLC detection for sharp brain lamination finger-print of the present invention, shares peak and summarizes 19
It is a, it can effectively characterize the quality of sharp brain lamination main component；
2, finger-print focuses on each correlation for constituting fingerprint characteristic peak, focuses on whole facial feature, both avoided
Total quality one-sidedness caused by due to measuring individual chemical ingredient, and reduce the possibility artificially handled for requisite quality
Property；
3, test sample is simple for production, and chromatographic condition is easy to accomplish；
4, the peak of the got profit brain lamination finger-print of the method for the present invention is more, and peak shape is good, is easy to identify, and similitude is high, accurately
Reliably；
5, the Efficient Characterization quality of sharp brain lamination, is conducive to product quality monitoring；It is easy with method, stable, accurate
Degree height, favorable reproducibility；The true and false superiority and inferiority of product can quickly and accurately be identified.
Below with reference to embodiment and attached drawing, the present invention is described further.
Detailed description of the invention
Fig. 1 benefit brain lamination standard finger-print
Fig. 2 benefit brain lamination reference substance map
Fig. 3 benefit brain lamination extraction comparison map
Fig. 4 benefit brain 10 batches of finger-prints of lamination
Specific embodiment
The present invention is further described with reference to embodiments, to make more detailed understanding to the present invention.
Embodiment 1
Step (1) sample solution preparation method are as follows: sharp brain lamination is taken, coating is removed, it is finely ground, 1g is weighed, tool plug cone is set
In shape bottle, 50% methanol 20ml, close plug is added in precision, and weighed weight is ultrasonically treated 30min, lets cool, then weighed weight, with mentioning
Solvent is taken to supply the weight of less loss, with 0.45 μm of filtering with microporous membrane, subsequent filtrate is test solution.
It is 55.8 μ g/ml, former youngster that step (2) reference substance solution preparation concentration, which is respectively as follows: the danshensu reference substance concentration,
Tea aldehyde reference substance concentration is 52.6 μ g/ml, and Puerarin reference substance concentration is 55.3 μ g/ml, and Paeoniflorin reference substance concentration is 27.6 μ
G/ml, fleece-flower root glycosides reference substance concentration are 25.5 μ g/ml, and liquiritin reference substance concentration is 51.8 μ g/ml, tanshin polyphenolic acid B reference substance
Concentration is 86.3 μ g/ml.
Test solution, reference substance solution injecting chromatograph are obtained chromatogram by step (3).
Liquid chromatographic system condition:
Using gradient elution, using gradient elution, mobile phase A is 0.5% phosphate aqueous solution, and Mobile phase B is acetonitrile, elution
Time is 65min, flow velocity 1.0ml/min；35 DEG C of column temperature；Ultraviolet detection wavelength: 230nm；Chromatographic column is Agilent Eclipse
XDB-C18 (4.6 × 15cm, 3.5um), 10 μ l of sample volume.
Gradient elution program: 0min → 7min → 10min → 30min → 50min → 65min, acetonitrile 1% → 10% →
15% → 38% → 60% → 60% (percent by volume).
It is calculated, is compared by the Chinese Pharmacopoeia committee " chromatographic fingerprints of Chinese materia medica similarity evaluation software " version in 2012
The similarity of the finger-print of standard finger-print and said determination, similarity are qualification 0.75~0.99, are lower than 0.75
It is unqualified.
Embodiment 2
Determining fingerprint pattern is carried out to sharp 171202 batch of brain lamination, the specific steps are as follows:
Step (1) sample solution preparation method are as follows: sharp brain lamination 171202 is taken, coating is removed, it is finely ground, 1g is weighed, is set
In stuffed conical flask, distilled water 20ml, close plug is added in precision, and weighed weight is ultrasonically treated 30min, lets cool, then weighed weight,
The weight that less loss is supplied with Extraction solvent, with 0.45 μm of filtering with microporous membrane, subsequent filtrate is test solution.
It is 55.8 μ g/ml, former youngster that step (2) reference substance solution preparation concentration, which is respectively as follows: the danshensu reference substance concentration,
Tea aldehyde reference substance concentration is 52.6 μ g/ml, and Puerarin reference substance concentration is 55.3 μ g/ml, and Paeoniflorin reference substance concentration is 27.6 μ
G/ml, fleece-flower root glycosides reference substance concentration are 25.5 μ g/ml, and liquiritin reference substance concentration is 51.8 μ g/ml, tanshin polyphenolic acid B reference substance
Concentration is 86.3 μ g/ml.
Test solution, reference substance solution injecting chromatograph are obtained chromatogram by step (3).
Liquid chromatographic system condition:
Using gradient elution, using gradient elution, mobile phase A is 0.5% phosphate aqueous solution, and Mobile phase B is acetonitrile, elution
Time is 65min, flow velocity 1.0ml/min；35 DEG C of column temperature；Ultraviolet detection wavelength: 230nm；Chromatographic column is Agilent Eclipse
XDB-C18 (4.6 × 15cm, 3.5um), 10 μ l of sample volume.
Gradient elution program: 0min → 7min → 10min → 30min → 50min → 65min, acetonitrile 1% → 10% →
15% → 38% → 60% → 60% (percent by volume).
It is calculated, is compared by the Chinese Pharmacopoeia committee " chromatographic fingerprints of Chinese materia medica similarity evaluation software " version in 2012
The similarity of the finger-print of standard finger-print and said determination.
Test result: compared with standard diagram, similarity 0.88 meets not less than 0.75 171202 batch benefit brain lamination
Requirement.
Embodiment 3
Determining fingerprint pattern is carried out to sharp 180303 batch of brain lamination, the specific steps are as follows:
Step (1) sample solution preparation method are as follows: sharp brain lamination 180303 is taken, coating is removed, it is finely ground, 1g is weighed, is set
In stuffed conical flask, distilled water 20ml, close plug is added in precision, and weighed weight is ultrasonically treated 30min, lets cool, then weighed weight,
The weight that less loss is supplied with Extraction solvent, with 0.45 μm of filtering with microporous membrane, subsequent filtrate is test solution.
It is 55.8 μ g/ml, former youngster that step (2) reference substance solution preparation concentration, which is respectively as follows: the danshensu reference substance concentration,
Tea aldehyde reference substance concentration is 52.6 μ g/ml, and Puerarin reference substance concentration is 55.3 μ g/ml；Paeoniflorin reference substance concentration is 27.6 μ
g/ml；Fleece-flower root glycosides reference substance concentration is 25.5 μ g/ml；Liquiritin reference substance concentration is 51.8 μ g/ml, tanshin polyphenolic acid B reference substance
Concentration is 86.3 μ g/ml.
Test solution, reference substance solution injecting chromatograph are obtained chromatogram by step (3).
Liquid chromatographic system condition:
Using gradient elution, using gradient elution, mobile phase A is 0.5% phosphate aqueous solution, and Mobile phase B is acetonitrile, elution
Time is 65min, flow velocity 1.0ml/min；35 DEG C of column temperature；Ultraviolet detection wavelength: 230nm；Chromatographic column is Agilent Eclipse
XDB-C18 (4.6 × 15cm, 3.5um), 10 μ l of sample volume.
Gradient elution program: 0min → 7min → 10min → 30min → 50min → 65min, acetonitrile 1% → 10% →
15% → 38% → 60% → 60% (percent by volume).
It is calculated, is compared by the Chinese Pharmacopoeia committee " chromatographic fingerprints of Chinese materia medica similarity evaluation software " version in 2012
The similarity of the finger-print of standard finger-print and said determination.
Test result: compared with standard diagram, similarity 0.85 meets not less than 0.75 180303 batch benefit brain lamination
Requirement.
Embodiment 4
Determining fingerprint pattern is carried out to sharp 180701 batch of brain lamination, the specific steps are as follows:
Step (1) sample solution preparation method are as follows: sharp brain lamination 180701 is taken, coating is removed, it is finely ground, 1g is weighed, is set
In stuffed conical flask, distilled water 20ml, close plug is added in precision, and weighed weight is ultrasonically treated 30min, lets cool, then weighed weight,
The weight that less loss is supplied with Extraction solvent, with 0.45 μm of filtering with microporous membrane, subsequent filtrate is test solution.
It is 55.8 μ g/ml, former youngster that step (2) reference substance solution preparation concentration, which is respectively as follows: the danshensu reference substance concentration,
Tea aldehyde reference substance concentration is 52.6 μ g/ml, and Puerarin reference substance concentration is 55.3 μ g/ml, and Paeoniflorin reference substance concentration is 27.6 μ
G/ml, fleece-flower root glycosides reference substance concentration are 25.5 μ g/ml, and liquiritin reference substance concentration is 51.8 μ g/ml, tanshin polyphenolic acid B reference substance
Concentration is 86.3 μ g/ml.
Test solution, reference substance solution injecting chromatograph are obtained chromatogram by step (3).
Liquid chromatographic system condition:
Using gradient elution, using gradient elution, mobile phase A is 0.5% phosphate aqueous solution, and Mobile phase B is acetonitrile, elution
Time is 65min, flow velocity 1.0ml/min；35 DEG C of column temperature；Ultraviolet detection wavelength: 230nm；Chromatographic column is Agilent Eclipse
XDB-C18 (4.6 × 15cm, 3.5um), 10 μ l of sample volume.
Gradient elution program: 0min → 7min → 10min → 30min → 50min → 65min, acetonitrile 1% → 10% →
15% → 38% → 60% → 60% (percent by volume).
It is calculated, is compared by the Chinese Pharmacopoeia committee " chromatographic fingerprints of Chinese materia medica similarity evaluation software " version in 2012
The similarity of the finger-print of standard finger-print and said determination.
Test result: compared with standard diagram, similarity 0.90 meets not less than 0.75 180701 batch benefit brain lamination
Requirement.
Claims (10)
1. a kind of benefit brain lamination HPLC detection method, the described method comprises the following steps:
(1) preparation of test solution: sharp brain lamination is taken, is extracted, extracting solution filtering with microporous membrane is molten to get test sample
Liquid；
(2) measuring method: taking test solution, injects high performance liquid chromatograph, obtains chromatic graph spectrum, wherein chromatographic condition are as follows:
Chromatographic column is using octadecylsilane chemically bonded silica as filler；
Gradient elution, mobile phase A are 0.01~3% phosphate aqueous solution, and B is acetonitrile；0.3~1.5ml/min of flow velocity；Column temperature 20~
45℃；Elution program: 0min → 7min → 10min → 30min → 50min → 65min, acetonitrile 1% → 10% → 15% →
38% → 60% → 60%；Detection wavelength 230nm；
(3) gained chromatogram and standard finger-print are compareed, it is qualified product that similarity, which is greater than 0.75, and similarity evaluation uses
" similarity evaluation 2004A editions " is evaluated.
2. according to the method described in claim 1, it is characterized by: the concentration of reference substance solution described in step (1) is 20
~500 μ g/mL.
3. according to the method described in claim 1, it is characterized by: the weighing as 0.1 of benefit brain lamination described in step (2)~
10g。
4. according to the method described in claim 1, it is characterized by: extraction step described in step (2) is water-soluble using methanol
Liquid ultrasonic extraction uses distilled water ultrasonic extraction, and extraction time is 10~80min.
5. according to the method described in claim 1, it is characterized by: miillpore filter aperture described in step (2) be 0.2 μm~
0.8μm。
6. according to the method described in claim 1, it is characterized by: the detection program of the detection of program HPLC described in step (3)
Are as follows: Detection wavelength 230nm.
7. according to the method described in claim 1, it is characterized by: high performance liquid chromatograph is automatic in step (3) described measurement
0.5~20 μ l of feeder obtains finger-print according to high effective liquid chromatography for measuring.
8. the method for building up of standard finger-print described in claim 1, it is characterised in that: steps are as follows:
(1) preparation of reference control solution:
A. the preparation of Sodium Danshensu reference substance solution: it is appropriate that precision weighs Sodium Danshensu reference substance, is configured to often with 50% methanol
The solution of 1ml 20~500 μ g containing danshensu；
B. the preparation of protocatechualdehyde reference substance solution: it is appropriate that precision weighs protocatechualdehyde reference substance, is configured to often with 50% methanol
The solution of 1ml 20~500 μ g containing protocatechualdehyde；
C. the preparation of Puerarin reference substance solution: it is appropriate that precision weighs Puerarin reference substance, is configured to every 1ml with 50% methanol and contains
The solution of 20~500 μ g of Puerarin；
D. the preparation of Paeoniflorin reference substance solution: it is appropriate that precision weighs Paeoniflorin reference substance, is configured to every 1ml with 50% methanol and contains
The solution of 20~500 μ g of Paeoniflorin；
E. the preparation of liquiritin reference substance solution: it is appropriate that precision weighs liquiritin reference substance, is configured to every 1ml with 50% methanol and contains
The solution of 20~500 μ g of liquiritin；
F. the preparation of fleece-flower root glycosides reference substance solution: it is appropriate that precision weighs fleece-flower root glycosides reference substance, is configured to often with 50% methanol
The solution of 20~500 μ g of 1ml glycosides containing the fleece-flower root；
G. the preparation of tanshin polyphenolic acid B reference substance solution: it is appropriate that precision weighs tanshin polyphenolic acid B reference substance, is configured to every 1ml with 50% methanol
The solution of 20~500 μ g containing tanshin polyphenolic acid B；
(2) preparation of test solution: weighing the sharp brain lamination of multiple batches of qualification, removes coating, finely ground, weighs 0.1~10g,
0%~100% methanol aqueous solution, ultrasonic extraction is added, extraction time is 10~80min, and extraction time is preferably 30min, is mentioned
Take liquid miillpore filter (aperture is 0.2 μm~0.8 μm, preferably 0.45 μm) filtering to get test solution；
(3) reference control solution and test solution are taken, high performance liquid chromatograph is injected, 0.5~20 μ l of sample volume is obtained more
The sharp brain lamination chromatogram of batch qualification；
Wherein chromatographic condition are as follows: chromatographic column is using octadecylsilane chemically bonded silica as filler；Using gradient elution, mobile phase A is
0.01~3% phosphate aqueous solution, Mobile phase B are acetonitrile；The gradient elution time is 20~180min, and gradient elution program is as follows:
0min → 7min → 10min → 30min → 50min → 65min, acetonitrile 1% → 10% → 15% → 38% → 60% → 60%,
0.3~1.5ml/min of flow velocity；20~45 DEG C of column temperature；Detection wavelength 230nm.
(4) the sharp brain lamination chromatogram of the multiple batches of qualification of gained is fitted to by a standard control finger-print using computer.
9. method for building up according to claim 8, it is characterised in that: the wherein preparation method of (1) reference control solution
It is as follows: to take each standard items, set in stuffed conical flask, 50% methanol 20ml, close plug, weighed weight, ultrasonic treatment is added in precision
30min is let cool, then weighed weight, and the weight of less loss is supplied with Extraction solvent, and with 0.45 μm of filtering with microporous membrane, subsequent filtrate is
For reference control solution.
10. method for building up according to claim 8, it is characterised in that: wherein the preparation method of (2) test solution is such as
Under: sharp brain lamination is taken, coating is removed, it is finely ground, 1g is weighed, is set in stuffed conical flask, precision 50% methanol 20ml of addition, close plug,
Weighed weight is ultrasonically treated 30min, lets cool, then weighed weight, the weight of less loss is supplied with Extraction solvent, with 0.45 μm of micropore
Membrane filtration, subsequent filtrate are test solution.
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