CN106501434B - A kind of HPLC finger print measuring methods of Double Harmonizing Decoction standard soup - Google Patents
A kind of HPLC finger print measuring methods of Double Harmonizing Decoction standard soup Download PDFInfo
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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
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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
- G01N30/86—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 present invention provides a kind of HPLC finger print measuring methods of Double Harmonizing Decoction standard soup, belong to technical field of traditional Chinese medicines, the preparation of (1) test sample solution；(2) preparation of mixed reference substance solution；(3) preparation of single medicinal material negative controls solution；(4) accurate test sample solution, mixed reference substance solution and the single medicinal material negative controls solution injection high performance liquid chromatograph drawn is measured respectively, respectively obtains the liquid chromatogram of test sample solution, mixed reference substance solution and single medicinal material negative controls solution；(5) the similarity evaluation software formulated using Chinese Pharmacopoeia Commission, Data Matching is carried out to get standard finger-print to the liquid chromatogram of test sample solution, mixed reference substance solution and single medicinal material negative controls solution.The present invention is effectively controlled the quality of Double Harmonizing Decoction standard particle agent, the effect of ensure that drug, and classics recipe is made to have obtained more regular quality control.
Description
Technical field
It is specifically a kind of HPLC determining fingerprint pattern sides of Double Harmonizing Decoction standard soup the present invention relates to technical field of traditional Chinese medicines is belonged to
Method.
Background technology
Double Harmonizing Decoction is first recorded in Southern Song Dynasty's period《The formulary of peaceful benevolent dispensary》In volume five, all void is controlled, is Baoqing side of increasing newly.It is former
Side is recorded as RADIX PAEONIAE ALBA (seven two halves), Radix Angelicae Sinensis (washing, steeping in wine), Radix Astragali (honey toast), Rhizoma Chuanxiong, prepared rhizome of rehmannia (washing, wine steam, each three
Two), Radix Glycyrrhizae (toast), Chinese cassia tree (peeling loses fire, each 2 two 2 money half).Upper is smalls, often takes two money, water one and half, ginger three
Piece, one piece of jujube are decocted and go six points, hollow, clothes before eating.Man, married woman's exhaustion or lesion of the five internal organs, sextupole, seven kinds of impairments are controlled, heart kidney is all empty, and essence and blood gas is few, then
Into consumptive disease.Bones of the body collectively is withered overworked, four limbs burnout, fevers and chills alternate, and weary, yellow complexion is breathed heavily in dry throat of coughing, action, is slightly touched, easily into his disease.
Or wound in cold, then place food does not disappear, spleen pain abdominal pain, rush down dysentery is spat inverse；Or wound is in heat, then dizzy to feel dizzy, sputum shortness of breath is dysphoria in chestpalms-soles；
Or because of the full action of famine, happiness anger is terrified, disease with and arrive or empty swollen without thinking food or more foods without myogenic meat, vexed then abnormal sweating is stolen
Sweat, all consumptive diseases dare not take dry medicine person, and preferably take it.It fosters the spirit of nobility in informal dress tune, beneficial blood educates god and stomach feed, qi-restoratives damage.Avoid life
The objects such as cold, fruit.
Song dynasty's period《The augmentation formulary of peaceful benevolent dispensary》It rolls up in five Baoqing sides of increasing newly, qi-restoratives damage.Composition is same《It is peaceful
The Bureau of People's Welfare Pharmacies side》.
Period in the Yuan Dynasty, Wei Yilin《The physicians from a family for generations obtain efficacious prescriptions》It rolls up in the 80th side's arteries and veins specialty of miscellaneous diseases, it is deficient.Composition is same《It is peaceful
The Bureau of People's Welfare Pharmacies side》.
Ming Dynasty's period, Dong Su's《Special effect good recipe》In 21 all empty logical sides of controlling of volume, man married woman, the exhaustion or lesion of the five internal organs seven are controlled
Wound, insufficiency of vital energy and blood, sallow complexion, four limbs are tired weary, will be the card of consumptive disease, and informal dress is fostered the spirit of nobility beneficial blood.Father's nozzle suitable for reading, often takes three money, water
One, ginger three pieces, one piece of jujube is decocted, hollow clothes.Composition is same《The formulary of peaceful benevolent dispensary》.
Qing Dynasty's period, Wu Qian's《Yizong Jinjian》Roll up 26 Ce Buming hospital opinion (one), miscellaneous diseases heart method gist is rolled up
40 consumptive disease therapies, gynaecology's heart method gist are rolled up 44 menstruation regulatings card and are controlled and gynaecology's heart method gist 44 menstruation regulatings of volume
Double and drink in Men Huifang, after controlling serious disease, consumptive disease gas is weary.Enriching the blood and tonifying qi, it is not hot not cold, temperature and adjust it.Two money of Radix Paeoniae Alba, Radix Astragali
A money half is processed, liquorice seven is divided, and middle osmanthus seven is divided, and when normalizing money, one money of prepared rhizome of rehmannia, Rhizoma Chuanxiong seven divides, ginger three pieces, two pieces of jujube,
Two, water is decocted one, is warmly taken.Consumptive disease, if in not anxious, abdomen do not have the person that is card bitterly, when with warm qi and blood invigorating.Fuke Tiaojing is demonstrate,proved
It controls, flat to fill blood, i.e., Shiquan Dabu Tang subtracts ginseng, Poria cocos, Rhizoma Atractylodis Macrocephalae.Wu Qian's《Name hospital opinion》Also double and drink is recorded.
Modern Wang Shimin《It does not cut out office side》Chapter 6, Double Harmonizing Decoction is recorded in tonifying recipes, " warm blood " (1992 editions).It thanks
Hai Zhou's《Traditional Chinese medical science successive dynasties good recipe pandect》In, Double Harmonizing Decoction is《The formulary of peaceful benevolent dispensary》Square also known as double and drink.Radix Paeoniae Alba, cultivated land,
Each 10 grams of Radix Astragali, Radix Angelicae Sinensis, each 4.5 grams of Rhizoma Chuanxiong, honey-fried licorice root, Chinese cassia tree.It is decocted in water for oral dose.Function tonifying yin blood, gas of establishing the yang function.The serious disease later stage is controlled,
Cloudy blood yang-energy is not multiple, and shortness of breath and fatigue, dizzy, palpitaition, tongue nature is light, and arteries and veins moistens weak.It is empty to be usually used in fatigue, prolonged illness constitution on modern clinic
The symptom that weak, energy lacks.The clinical application research of Double Harmonizing Decoction has been reported in recent years：The graduate Ma Zhenlie of Korea Spro hospital of South Korea
Et al. be prepared into two kinds of health foods using the lactobacillus fermented product of Double Harmonizing Decoction or Double Harmonizing Decoction, caused by one kind is applied to alcohol
It is still drank after a night；Another kind is used to treat osteoporosis.
Main dosage form of the Chinese medicine classics recipe decoction as traditional tcm clinical practice medication, it is true with reasonable recipe, curative effect
It cuts, is easy to absorb, work the advantages that very fast, it is deep to be trusted by many patients.But because allocate, carry, decoct temporarily, be long placed in easily
Generation is mould to be lost rotten, soup bitter and measures the shortcomings of big and standard is difficult to unification, seriously affects its clinical efficacy, it is impossible to suitable
Answer the life requirement of modern.In order to keep the advantage of decoction, a variety of deficiencies of decoction are overcome, it is " great new in the Ministry of Science and Technology
Medicine is formulated " scientific and technological key special subjects project support energetically lower and academician, industry specialists discussion repeatedly after, with classics recipe benchmark
The qualitative attribute of soup is evaluation index, produces the standard particle agent being consistent substantially in quality and drug effect with it.
" classics recipe standard particle " means the Key Quality attribute using benchmark decoction as evaluation index, to particle preparation mistake
The critical processes such as medicinal material extract, concentration, the drying of journey are studied, and the key with benchmark decoction is prepared using modern production technology
The basically identical particle of qualitative attribute.
According to《The classics recipe standard particle researcher common recognition-definition of benchmark decoction, preparation, qualitative attribute and application》
Related requirement carries out the related of Double Harmonizing Decoction benchmark soup and investigates and study.
Compared with the mass analysis method that index components content measures, finger-print can be than more fully reflecting Chinese medicine
The type and quantity studied point, it can be achieved that in Chinese medicine under the present situation not yet illustrated completely in compound Chinese medicinal preparation active ingredient
It is the effective means of current Chinese medicine and its quality of the pharmaceutical preparations control in the overall merit of quality and effective control to its whole substance
One of.The detection method of traditional Chinese medicine fingerprint has chromatography, spectroscopic methodology, spectral method etc..Chromatography mainly includes thin-layer chromatography, height
Effect liquid phase chromatogram, gas-chromatography, Capillary Electrophoresis etc., wherein high performance liquid chromatography have efficient, quick, sensitive, reappearance
The characteristics of good, with UV detector (UV), diformazan pipe array detector (DAD), evaporative light scattering detector (ELSD) and mass spectrum
A variety of detector combinations such as detector (MS), available for the analysis detection of Various Complex ingredient in Chinese medicine, refer to as Chinese medicine
The main stream approach of line collection of illustrative plates research.
The content of the invention
In view of this, the present invention is intended to provide a kind of HPLC finger print measuring methods of Double Harmonizing Decoction standard soup, effectively
The effect of controlling the quality of Double Harmonizing Decoction standard particle agent, ensure that drug makes classics recipe obtain more regular quality
Control.
In order to achieve the above objectives, the technical proposal of the invention is realized in this way：A kind of HPLC of Double Harmonizing Decoction standard soup refers to
Line collection of illustrative plates assay method, includes the following steps：
(1) preparation of test sample solution：Precision measures the standard soup of the Double Harmonizing Decoction of different batches, loads on and has activated
SPE columns, first eluted with the pure water of 30 volume parts, discard eluent, then eluted with the methanol of 10 parts by weight, collect elution
Eluent low temperature is concentrated to dryness by liquid, is finally dissolved with the methanol of 2 volume parts, with filtering with microporous membrane, is taken subsequent filtrate, obtain
Test sample solution, it is spare；
(2) preparation of mixed reference substance solution：Precision weighs 5-HMF, Paeoniflorin, Calycosin-7-O-BETA-D-glucoside, liquiritin, asafoetide
Acid, cinnaldehydrum, glycyrrhizic acid, 6-gingerol reference substance are appropriate, and methanol is added to be configured to every 1 volume parts containing 5-HMF, Paeoniflorin, Mao Rui
Isoflavone aglycone, liquiritin, forulic acid, cinnaldehydrum, glycyrrhizic acid, 6-gingerol are respectively 0.01,0.12,0.03,0.01,0.01,
0.04th, the mixed reference substance solution of 0.05 and 0.03 parts by weight, shakes up, filtration, spare；
(3) preparation of single medicinal material negative controls solution：Each single medicinal powder is weighed according to 3 times of recipe quantities, is crushed,
Sieve to obtain coarse powder, takes 3 times of recipe quantity coarse powder into full-automatic decocting medicine pot, and precision measures pure water, impregnates, and decocts, is stirred during decoction
It mixes, filters while hot, filtrate is uniformly mixed, and takes and is settled in 250mL volumetric flasks as single medicinal material negative control with pure water in right amount
Product solution, it is spare；
(4) it is accurate respectively to draw test sample solution, mixed reference substance solution and single medicinal material negative controls solution note
Enter high performance liquid chromatograph to be measured, it is right to respectively obtain test sample solution, mixed reference substance solution and single medicinal material feminine gender
According to the liquid chromatogram of product solution；
(5) the similarity evaluation software formulated using Chinese Pharmacopoeia Commission, to for trying
The liquid chromatogram of sample solution, mixed reference substance solution and single medicinal material negative controls solution carries out Data Matching to get mark
Quasi- finger-print；
The relation of the parts by weight and the volume parts is g/mL.
Further, the chromatographic condition of the step (4) is as follows：
Chromatographic column：Accurasil C18 chromatographic columns, 4.6mm × 250mm, 5 μm；
Mobile phase：- 0.1% phosphate aqueous solution of acetonitrile；
Flow velocity：1.0mL/min；
Wavelength：280nm；
Sample size：10μL；
Column temperature：30℃；
Theoretical cam curve is calculated by Paeoniflorin peak not less than 7000；
Gradient elution program is shown in Table 1.
1 finger-print gradient elution program of table
Further, the standard finger-print includes 16 shared peaks：Peak 2,3,4 comes from Radix Paeoniae Alba, and peak 5 is yellow from toast
Stilbene, peak 6,11,14 come from honey-fried licorice root, and peak 7,8,9,16 comes from prepared RADIX ANGELICAE SINENSIS with yellow rice wine and Rhizoma Chuanxiong, and peak 10,12,13 comes from Chinese cassia tree, and peak 15 comes from
Ginger, peak 1 is from wine cultivated land, Rhizoma Chuanxiong, using No. 4 peaks as with reference to peak.
Further, No. 1 peak is 5-HMF in the standard finger-print, and No. 4 peaks are Paeoniflorin, and No. 5 peaks are yellow for Mao Ruiyi
Ketoside, No. 6 peaks are liquiritin, and No. 7 peaks are forulic acid, and No. 13 are cinnaldehydrum, and No. 14 peaks are glycyrrhizic acid, and No. 15 are 6-gingerol.
Further, with 0.22 μm of filtering with microporous membrane in the step (1).
Further, the preparation method of the single medicinal material negative controls solution is as follows：It is weighed respectively according to 3 times of recipe quantities
Single medicinal powder crushes, and crosses 10-40 mesh sieves and obtains coarse powder；3 times of recipe quantity coarse powder are taken into full-automatic decocting medicine pot, precision measures
900mL pure water pours into, and impregnates 30min, decocts 75min, is stirred during decoction three times, while hot with 300 mesh filter-cloth filterings, filtrate body
Product is about 500mL, and filtrate is uniformly mixed, and it is that single medicinal material is negative to take 1/3rd to be settled to pure water in 250mL volumetric flasks
Reference substance solution, it is spare.
Further, the preparation method of the standard soup of the Double Harmonizing Decoction is as follows：Nine taste medicinal materials, powder are weighed according to prescription ratio
It is broken, it crosses 10-40 mesh sieves and obtains coarse powder；Three times recipe quantity coarse powder is taken into full-automatic decocting medicine pot, precision measures 900mL pure water and pours into,
30min is impregnated, 75min is decocted, is stirred during decoction three times, while hot with 300 mesh filter-cloth filterings, filtrate volume is about 500mL, filter
Liquid is uniformly mixed, and it is standard Tangyuan County liquid to take 1/3rd to be settled to pure water in 250mL volumetric flasks, spare.
The present invention also provides application of the above method in the quality testing and quality control of the pharmaceutical preparation of Double Harmonizing Decoction.
The present invention also provides a kind of method of quality control of Double Harmonizing Decoction standard particle, comprise the following steps：
(1) standard finger-print of Double Harmonizing Decoction standard soup is established according to said determination method；
(2) Double Harmonizing Decoction standard particle to be checked is taken, finger-print is obtained according to said determination method；
(3) standard finger-print that the finger-print that step (2) obtains is obtained with step (1) is compared, met i.e.
For qualified products, do not meet as substandard product.
The preparation of Double Harmonizing Decoction standard particle test sample solution：The Double Harmonizing Decoction standard particle under content uniformity item is taken, it is finely ground,
About 5g is taken, it is accurately weighed, it puts in 100mL measuring bottles, adds appropriate amount of water, supersound process (power 250W, frequency 50kHz) 10min makes molten
Solution, lets cool, is diluted with water to scale, shake up, and precision measures 4ml suspensions into 10mL volumetric flasks, adds methanol constant volume to scale,
(power 250W, frequency 50kHz) 10min is ultrasonically treated, is then centrifuged for (rotating speed is 8000 turns per minute) 15min, precision pipettes
Supernatant, filtration, take subsequent filtrate to get.
Compared with the prior art, the present invention has the advantage that：
(1) present invention is during the finger-print of Double Harmonizing Decoction standard particle is established, it is thus identified that 16 common characteristic peaks, and
Its relative retention time and relative peak area are studied, ensure that the chemical composition stability of preparation and safe to use
Property.
(2) complex chemical composition in Double Harmonizing Decoction standard particle will realize that the separating difficulty at its characteristic fingerprint peak is big, the present invention
During finger-print is set up, the method that employs gradient elution solves fingerprint characteristic peak and is difficult to separate and impurity
The interference problem at peak.
(3) finger-print of Double Harmonizing Decoction standard particle is established, single component assay is overcome and is difficult to reflect whole contain
The effect of the defects of amount, can on the whole, macroscopically control the inherent quality of Double Harmonizing Decoction standard particle agent, ensure that drug,
Classics recipe is made to have obtained more regular quality control.
(4) each active ingredient fingerprint graph is treated as an entirety using in Double Harmonizing Decoction standard particle, focuses on each feature
The tandem and correlation at peak had both avoided and have judged that Double Harmonizing Decoction standard particle is whole when only measuring one, two chemical composition
The one-sidedness of weight, and reduce the possibility artificially handled for requisite quality.For complete, accurate evaluation Double Harmonizing Decoction standard
The quality of particle provides new ways and means.
(5) the method for the present invention stability is good, precision is high, favorable reproducibility, convenient and be easy to grasp.
Description of the drawings
Fig. 1 compares collection of illustrative plates for different manufacturers chromatographic column HPLC.
Fig. 2 compares collection of illustrative plates for different column temperature HPLC.
Fig. 3 is 200-400nm full wavelength scanner figures.
Fig. 4 compares collection of illustrative plates for different wave length HPLC.
Fig. 5 compares collection of illustrative plates for different gradient HPLC.
Fig. 6 compares collection of illustrative plates for different pre-treatments HPLC.
Fig. 7 compares collection of illustrative plates for 10 crowdes of standard soup HPLC.
Fig. 8 is 10 batches of standard soup finger-prints (after matching).
Fig. 9 is 10 batches of standard soup finger-prints (common pattern collection of illustrative plates).
Figure 10 compares collection of illustrative plates for mixing reference substance and full side HPLC.
Figure 11 is full side and single medicinal material negative control finger-print Comparative map.
Specific embodiment
The HPLC finger print measuring methods of one Double Harmonizing Decoction standard soup of embodiment
1.1 instruments and reagent
Instrument：
Reagent：
1.2 sample preparation：
1.2.1 the preparation of sample and test sample solution
(1) preparation of sample：Nine taste medicinal materials are weighed according to prescription ratio, are crushed, 10-40 mesh sieves is crossed and obtains coarse powder；Take three times
Into full-automatic decocting medicine pot, precision measures 900mL pure water and pours into recipe quantity coarse powder, impregnates 30min, 75min is decocted, during decoction
Three times, while hot with 300 mesh filter-cloth filterings, filtrate volume is about 500mL, and filtrate is uniformly mixed, and takes 1/3rd to use pure water for stirring
It is standard Tangyuan County liquid to be settled in 250mL volumetric flasks, spare, and the standard soup of 10 batches of variant batches is prepared according to the method.
(2) preparation of test sample solution：It is accurate respectively from 10 batches of standard soup to measure 4mL, load on what is activated
SPE columns are first eluted with 30mL pure water, discard eluent, then are eluted with 10mL methanol, collect eluent, and eluent low temperature is dense
Be reduced to it is dry, finally with 2mL methanol dissolve, with 0.22 μm of filtering with microporous membrane, take subsequent filtrate, obtain test solution, it is spare.
1.2.2 the preparation of mixed reference substance solution：Precision weigh 5-HMF, Paeoniflorin, Calycosin-7-O-BETA-D-glucoside, liquiritin, Ah
Wei's acid, cinnaldehydrum, glycyrrhizic acid, 6-gingerol reference substance are appropriate, methanol are added to be configured to every 1mL yellow containing 5-HMF, Paeoniflorin, Mao Ruiyi
Ketoside, liquiritin, forulic acid, cinnaldehydrum, glycyrrhizic acid, 6-gingerol are respectively 0.01,0.12,0.03,0.01,0.01,0.04,
The mixed reference substance solution of 0.05 and 0.03mg, shakes up, filtration, spare.
Paeoniflorin (lot number：110736-201539 purity >=96.4%), forulic acid (lot number：110773-201313 purity
>=99.6%), liquiritin (lot number：11610-201106 purity >=93.7%), glycyrrhizic acid (lot number：110731-201418 purity
>=93.1%), cinnaldehydrum (lot number：110710-201408 purity >=99.4%) it is purchased from National Institute for Food and Drugs Control；
5-HMF (lot numbers：H40807 purity >=99.0%), Calycosin-7-O-BETA-D-glucoside (lot number：AB019C purity >=98.0%), 6-gingerol
(lot number：AW025G01 purity >=99.0%) it is purchased from one side Science and Technology Ltd. of Tianjin.
1.2.3 the preparation of single medicinal material negative controls solution：Each single medicinal powder, powder are weighed according to 3 times of recipe quantities
It is broken, it crosses 10-40 mesh sieves and obtains coarse powder；3 times of recipe quantity coarse powder are taken into full-automatic decocting medicine pot, precision measures 900mL pure water and pours into, and soaks
30min is steeped, 75min is decocted, is stirred during decoction three times, while hot with 300 mesh filter-cloth filterings, filtrate volume is about 500mL, filtrate
It is uniformly mixed, it is single medicinal material negative controls solution to take 1/3rd to be settled to pure water in 250mL volumetric flasks, spare.
1.3 chromatographic condition
Chromatographic column：Accurasil C18 chromatographic columns, 4.6mm × 250mm, 5 μm；
Mobile phase：- 0.1% phosphate aqueous solution of acetonitrile；
Flow velocity：1.0mL/min；
Wavelength：280nm；
Sample size：10μL；
Column temperature：30℃；
Theoretical cam curve is calculated by Paeoniflorin peak not less than 7000；
Gradient elution program is shown in Table 1：
1 finger-print gradient elution program of table
1.3.1 the selection of chromatographic column
The chromatographic column of tri- producers of Shiseido C18, Cosmosil C18, Accurasil C18 is respectively compared, the result is shown in figures
1.(A：PAK C18 chromatographic columns B：Cosmosil C18 chromatographic columns C：Accurasil C18 chromatographic columns)
As can be seen from Figure 1：The separating degree of sample each chromatographic peak under AccurasilC18 chromatographic columns is good, characteristic peak it is apparent and
Peak shape is preferable.Therefore chromatographic column of the final choice AccurasilC18 chromatographic columns as HPLC finger-prints.
1.3.2 the selection of column temperature
20 DEG C, 30 DEG C and 40 DEG C of column temperature condition is screened and optimized respectively, the result is shown in Fig. 2.(A：20℃B：40℃
C：30℃)
As can be seen from Figure 2：Chromatographic peak separating degree is good under the conditions of 30 DEG C of column temperature, and the factor that is disturbed influences small.Therefore final choosing
Select 30 DEG C of optimum column temperature conditions as HPLC finger-prints of column temperature.
1.3.3 the selection of Detection wavelength
Full wavelength scanner in the range of 200-400nm is carried out to sample using PDA detectors, to 230,254,280nm wavelength
Under chromatogram be compared, the result is shown in Fig. 3, Fig. 4.(A：Wavelength 230nm B：Wavelength 254nm C：Wavelength 280nm)
From the graph 3, Fig. 4 is understood：Number, absorption intensity, the separating degree of chromatographic peak have differences under three wavelength, and synthesis is examined
Consider, when wavelength is in 280nm, chromatographic peak number is most, and each chromatographic peak absorption intensity is average, and separating degree is good, therefore
Final choice 280nm wavelength is best detection wavelength.
1.3.4 the selection of mobile phase
First to flow phase system water-acetonitrile, 0.1% phosphate aqueous solution-acetonitrile, 1% acetic acid-acetonitrile, water-methanol,
0.1% phosphate aqueous solution-methanol, 1% aqueous acetic acid-methanol are screened.As a result 0.1% phosphate aqueous solution-acetonitrile separation
Degree is good, and characteristic peak is apparent, and finally definite 0.1% phosphate aqueous solution-acetonitrile is flow phase system.Secondly optimize again not on year-on-year basis
Example gradient, gradient 1,2,3 such as following table 2-1, table 2-2, table 2-3, the result is shown in Fig. 5.(A：Gradient 1B：Gradient 2C：Gradient
3)
Table 2-1 Double Harmonizing Decoction HPLC finger-prints gradient 1 elutes table
Table 2-2 Double Harmonizing Decoction HPLC finger-prints gradient 2 elutes table
Table 2-3 Double Harmonizing Decoction HPLC finger-prints gradient 3 elutes table
As can be seen from Figure 5：Chromatographic peak separating degree is good under the conditions of gradient 3, and retention time is suitable, therefore final choice ladder
Degree 3 is used as optimal gradient.
1.3.5 the selection of sample pre-treatments
Sample stoste, 60% methanol alcohol deposition method and SPE solid phase extractions are screened, the result is shown in Fig. 6.(A：SPE consolidates
Mutually extraction B：60% first alcohol deposition method 2C：Sample stoste)
As can be seen from Figure 6：It is more that SPE Solid Phase Extraction follows the example of enriched sample chromatographic peak number under chromatographic condition, and each chromatography
Peak UV absorption and separating degree are good, therefore final choice SPE solid phase extractions concentration method is optimal pretreatment process.
1.4 methodological study
1.4.1 instrument precision is tested
1.2.2 lower section methods is taken to prepare mixed reference substance solutions, by the chromatographic condition under 1.3, continuous sample introduction 5 times, often
Secondary 10 μ L of sample introduction using the retention time of Paeoniflorin chromatographic peak (No. 4 peaks) and peak area as reference, calculate the opposite of each shared peak
The RSD values of retention time and relative peak area, RSD are respectively 0.4% and 2.2%, are respectively less than 3.0%, show this instrument precision
Degree is good.
1.4.2 stability test
Take legal system available test agent solution below 1.2.1 (2) item, by the chromatographic condition under 1.3, respectively 0,2,4,8,
12nd, sample introduction 1 time for 24 hours, each 10 μ L of sample introduction using the retention time of Paeoniflorin chromatographic peak and chromatographic peak as reference, are calculated each common
There are the relative retention time at peak and the RSD values of relative peak area, RSD is respectively 0.7% and 2.8%, is respectively less than 3.0%, shows
The sample is good in internal stability for 24 hours.
1.4.3 repetitive test
It is formed by Double Harmonizing Decoction original prescription, it is parallel to weigh each 5 parts of taste medicine, it is prepared by method below 1.2.1 (1) item, and by 1.2.1
(2) 5 parts of legal system available test agent solution below item, by the chromatographic condition under 1.3, every part of 10 μ L of test solution sample introduction, with
The retention time of Paeoniflorin chromatographic peak and peak area calculate each shared peak relative retention time and relative peak area as reference
Relative standard deviation RSD values, RSD are respectively 1.3% and 2.5%, are respectively less than 3.0%, show that this method repeatability is good.
The foundation of 1.5HPLC finger-prints and technical parameter analysis
1.5.1 collection of illustrative plates gathers
10 batches of standard soup that below 1.2.1 (1) item prepared by method are taken, it is molten by legal system available test agent below 1.2.1 (1) item
Liquid is measured by the chromatographic condition under 1.3,10 μ L of sample size, records HPLC finger-prints, the result is shown in Fig. 7.
1.5.2 with reference to the selection at peak
Paeoniflorin is the main active of monarch drug in a prescription Radix Paeoniae Alba first, secondly it was found from test solution finger-print, Chinese herbaceous peony
Glycosides is known chemical composition, and chemical property is stablized, and chromatographic peak is in each chromatographic peak, and separating degree is preferably and peak area is larger
, therefore choose reference peak of No. 4 peaks (Paeoniflorin) as finger-print.
1.5.3 the calibration at peak and the calculating of the relative retention time and relative peak area at shared peak are shared
The similarity evaluation software (2012 editions) formulated using Chinese Pharmacopoeia Commission, it is right
10 batches of decoction collection of illustrative plates carry out Data Matching, and the result is shown in Fig. 8.As seen from Figure 8, wherein common to 10 batches of decoctions, therefore 16 peaks are
It is shared chromatographic peak to determine 16 peaks, common pattern is determined with median method, the result is shown in Fig. 9, with the guarantor at Paeoniflorin peak (No. 4 peaks)
It is the relative retention time and relative peak area that shared peak is calculated with reference to peak to stay time and chromatographic peak area, the results are shown in Table 3, table
4。
3 Double Harmonizing Decoction standard decoction of table shares peak relative retention time
4 Double Harmonizing Decoction standard decoction of table shares peak relative peak area
From table 3, table 4：Relative retention time RSD≤0.82% at the shared peak of 10 batches of Double Harmonizing Decoction standard decoctions, phase
To peak area RSD≤0.82%, both less than 3%, therefore this method can be used for the HPLC determining fingerprint patterns of Double Harmonizing Decoction.
1.5.4 characteristic peak is pointed out
Characteristic peak is carried out using mixing reference substance comparison method first to point out, it is accurate respectively to draw test solution, mixing pair
According to each 10 μ L of product solution, high performance liquid chromatograph is injected, records chromatogram, structure is shown in Figure 10.A(1：5-HMF 4：Paeoniflorin 5：
Calycosin-7-O-BETA-D-glucoside 6：Liquiritin 7：Forulic acid 13：Cinnaldehydrum 14：Glycyrrhizic acid 15：6-gingerol)
As seen from Figure 10, No. 1 peak is determined as 5-HMF, and No. 4 peaks are Paeoniflorin, and No. 5 peaks are Calycosin-7-O-BETA-D-glucoside, and No. 6 peaks are
Liquiritin, No. 7 peaks are forulic acid, and No. 13 are cinnaldehydrum, and No. 14 peaks are glycyrrhizic acid, and No. 15 are 6-gingerol, it is thus determined that this 8 peaks
For the characteristic peak of Double Harmonizing Decoction HPLC finger-prints.
1.5.5 medicinal material attribution analysis
Take No. 1 decoction test sample solution under 1.2.1 (2) item and the single medicinal material feminine gender of 1.2.3 lower section methods preparations
Each 10 μ L of reference substance solution, are measured according to the chromatographic condition under 1.3, and it is right to obtain each single medicinal material feminine gender in full side and side
According to the HPLC collection of illustrative plates of liquid, the result is shown in Figure 11.(A：Full side B：Radix Paeoniae Alba negative control C：Radix Angelicae Sinensis negative control D：Rhizoma Chuanxiong negative control E：
Cultivated land negative control F：Radix Astragali negative control G：Radix Glycyrrhizae negative control H：Chinese cassia tree negative control I：Ginger negative control J：Jujube is cloudy
Property control)
From Figure 11：By comparing relative retention time, the source that peak is shared in Double Harmonizing Decoction standard decoction is chased after
It traces back, obtains appearance 2,3,4 from monarch drug in a prescription Radix Paeoniae Alba, peak 5 comes from astragalus root, and peak 6,11,14 comes from honey-fried licorice root, and peak 7,8,9,16 comes from wine
Radix Angelicae Sinensis and Rhizoma Chuanxiong, peak 10,12,13 come from Chinese cassia tree, and peak 15 comes from ginger, and peak 1 is from wine cultivated land, Rhizoma Chuanxiong.
3.7.6 10 batches of Double Harmonizing Decoction standard soup sample similarity evaluations
The similarity evaluation software (2012 editions) formulated using Chinese Pharmacopoeia Commission, with
The chromatographic peak of control spectrum chart display carries out Supplements, and result of calculation similarity, the results are shown in Table 5 as match point.
5 10 batches of Double Harmonizing Decoction standard decoction similarity calculation results of table
As seen from Table 5：Between 0.938-0.983, average value is the similarity of 10 batches of Double Harmonizing Decoction standard soup finger-prints
0.978, the results showed that：10 batches of Double Harmonizing Decoction standard decoction quality conformances are higher.
The method of quality control of two Double Harmonizing Decoction standard particle of embodiment
A kind of method of quality control of Double Harmonizing Decoction standard particle, comprises the following steps：
(1) Double Harmonizing Decoction standard soup is prepared according to the method in 1.2.1 (1) in embodiment one, using the survey in embodiment one
The method of determining establishes the standard finger-print of Double Harmonizing Decoction standard soup；
(2) Double Harmonizing Decoction standard particle to be checked is taken, finger-print is obtained according to the assay method in embodiment one；
(3) standard finger-print that the finger-print that step (2) obtains is obtained with step (1) is compared, met i.e.
For qualified products, do not meet as substandard product.
The preparation of Double Harmonizing Decoction standard particle test sample solution：The Double Harmonizing Decoction standard particle under content uniformity item is taken, it is finely ground,
About 5g is taken, it is accurately weighed, it puts in 100mL measuring bottles, adds appropriate amount of water, supersound process (power 250W, frequency 50kHz) 10min makes molten
Solution, lets cool, is diluted with water to scale, shake up, and precision measures 4ml suspensions into 10mL volumetric flasks, adds methanol constant volume to scale,
(power 250W, frequency 50kHz) 10min is ultrasonically treated, is then centrifuged for (rotating speed is 8000 turns per minute) 15min, precision pipettes
Supernatant, filtration, take subsequent filtrate to get.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
With within principle, any modifications, equivalent replacements and improvements are made should all be included in the protection scope of the present invention god.
Claims (8)
1. a kind of HPLC finger print measuring methods of Double Harmonizing Decoction standard soup, it is characterised in that：Include the following steps：
(1) preparation of test sample solution：Precision measures the standard soup of the Double Harmonizing Decoction of different batches, loads on what is activated
SPE columns are first eluted with the pure water of 30 volume parts, discard eluent, then are eluted with the methanol of 10 parts by weight, collect elution
Eluent low temperature is concentrated to dryness by liquid, is finally dissolved with the methanol of 2 volume parts, with filtering with microporous membrane, is taken subsequent filtrate, obtain
Test sample solution, it is spare；
(2) preparation of mixed reference substance solution：Precision weigh 5-HMF, Paeoniflorin, Calycosin-7-O-BETA-D-glucoside, liquiritin, forulic acid,
Cinnaldehydrum, glycyrrhizic acid, 6-gingerol reference substance are appropriate, methanol are added to be configured to every 1 volume parts yellow containing 5-HMF, Paeoniflorin, Mao Ruiyi
Ketoside, liquiritin, forulic acid, cinnaldehydrum, glycyrrhizic acid, 6-gingerol are respectively 0.01,0.12,0.03,0.01,0.01,0.04,
The mixed reference substance solution of 0.05 and 0.03 parts by weight, shakes up, filtration, spare；
(3) preparation of single medicinal material negative controls solution：Each single medicinal powder is weighed according to 3 times of recipe quantities, is crushed, sieving
Coarse powder is obtained, takes 3 times of recipe quantity coarse powder into full-automatic decocting medicine pot, precision measures pure water, impregnates, and decocts, stirs, take advantage of during decoction
Heat filtering, filtrate are uniformly mixed, take in right amount with pure water be settled in 250mL volumetric flasks be single medicinal material negative controls it is molten
Liquid, it is spare；
(4) it is accurate respectively to draw test sample solution, mixed reference substance solution and single medicinal material negative controls solution injection height
Effect liquid phase chromatogram instrument is measured, and respectively obtains test sample solution, mixed reference substance solution and single medicinal material negative controls
The liquid chromatogram of solution；
(5) the similarity evaluation software formulated using Chinese Pharmacopoeia Commission, to test sample
The liquid chromatogram of solution, mixed reference substance solution and single medicinal material negative controls solution carries out Data Matching and refers to get standard
Line collection of illustrative plates；
The relation of the parts by weight and the volume parts is g/mL；
The chromatographic condition of the step (4) is as follows：
Chromatographic column：Accurasil C18 chromatographic columns, 4.6mm × 250mm, 5 μm；
Mobile phase：- 0.1% phosphate aqueous solution of acetonitrile；
Flow velocity：1.0mL/min；
Wavelength：280nm；
Sample size：10μL；
Column temperature：30℃；
Theoretical cam curve is calculated by Paeoniflorin peak not less than 7000；
Gradient elution program is shown in Table 1.
1 finger-print gradient elution program of table
2. the HPLC finger print measuring methods of Double Harmonizing Decoction standard soup according to claim 1, it is characterised in that：The mark
Quasi- finger-print includes 16 shared peaks：Peak 2,3,4 comes from Radix Paeoniae Alba, and peak 5 comes from astragalus root, and peak 6,11,14 comes from honey-fried licorice root, peak
7th, 8,9,16 prepared RADIX ANGELICAE SINENSIS with yellow rice wine and Rhizoma Chuanxiong being come from, peak 10,12,13 comes from Chinese cassia tree, and peak 15 comes from ginger, and peak 1 comes from wine cultivated land, Rhizoma Chuanxiong,
Using No. 4 peaks as with reference to peak.
3. the HPLC finger print measuring methods of Double Harmonizing Decoction standard soup according to claim 1, it is characterised in that：The mark
No. 1 peak is 5-HMF in quasi- finger-print, and No. 4 peaks are Paeoniflorin, and No. 5 peaks are Calycosin-7-O-BETA-D-glucoside, and No. 6 peaks are liquiritin, No. 7
Peak is forulic acid, and No. 13 are cinnaldehydrum, and No. 14 peaks are glycyrrhizic acid, and No. 15 are 6-gingerol.
4. the HPLC finger print measuring methods of Double Harmonizing Decoction standard soup according to claim 1, it is characterised in that：The step
Suddenly with 0.22 μm of filtering with microporous membrane in (1).
5. the HPLC finger print measuring methods of Double Harmonizing Decoction standard soup according to claim 1, it is characterised in that：The list
The preparation method of taste medicinal material negative controls solution is as follows：Each single medicinal powder is weighed according to 3 times of recipe quantities, is crushed, crosses 10-
40 mesh sieves obtain coarse powder；3 times of recipe quantity coarse powder are taken into full-automatic decocting medicine pot, precision measures 900mL pure water and pours into, and impregnates 30min,
75min is decocted, is stirred during decoction three times, while hot with 300 mesh filter-cloth filterings, filtrate volume is about 500mL, and filtrate mixing is equal
Even, it is single medicinal material negative controls solution to take 1/3rd to be settled to pure water in 250mL volumetric flasks, spare.
6. the HPLC finger print measuring methods of Double Harmonizing Decoction standard soup according to claim 1, it is characterised in that：It is described double
It is as follows with the preparation method of the standard soup of soup：Nine taste medicinal materials are weighed according to prescription ratio, are crushed, 10-40 mesh sieves is crossed and obtains coarse powder；It takes
Into full-automatic decocting medicine pot, precision measures 900mL pure water and pours into three times recipe quantity coarse powder, impregnates 30min, decocts 75min, decocts
Period stirs three times, and while hot with 300 mesh filter-cloth filterings, filtrate volume is about 500mL, and filtrate is uniformly mixed, and takes 1/3rd use
It is standard Tangyuan County liquid that pure water, which is settled in 250mL volumetric flasks, spare.
7. claim 1-6 any one of them method answering in the quality testing and quality control of the pharmaceutical preparation of Double Harmonizing Decoction
With.
8. a kind of method of quality control of Double Harmonizing Decoction standard particle, it is characterised in that：Comprise the following steps：
(1) standard finger-print of Double Harmonizing Decoction standard soup is established according to claim 1-6 any one of them assay methods；
(2) Double Harmonizing Decoction standard particle to be checked is taken, fingerprint image is obtained according to claim 1-6 any one of them assay methods
Spectrum；
(3) standard finger-print that the finger-print that step (2) obtains is obtained with step (1) is compared, met as closing
Lattice product, does not meet as substandard product.
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