CN107167544B - The measuring method and fingerprint of effective component in a kind of invigorating the spleen middle benefit gas, the pharmaceutical composition of cold dispelling antidiarrheal - Google Patents
The measuring method and fingerprint of effective component in a kind of invigorating the spleen middle benefit gas, the pharmaceutical composition of cold dispelling antidiarrheal Download PDFInfo
- Publication number
- CN107167544B CN107167544B CN201610128775.6A CN201610128775A CN107167544B CN 107167544 B CN107167544 B CN 107167544B CN 201610128775 A CN201610128775 A CN 201610128775A CN 107167544 B CN107167544 B CN 107167544B
- Authority
- CN
- China
- Prior art keywords
- methanol
- mobile phase
- reference substance
- measuring method
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
-
- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
-
- 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
Abstract
The present invention provides the measuring methods and fingerprint of effective component in a kind of invigorating the spleen middle benefit gas, the pharmaceutical composition of cold dispelling antidiarrheal, wherein the measuring method includes the preparation of reference substance solution, the step of the preparation of test solution, measurement.Wherein, the chromatographic condition of the high performance liquid chromatograph are as follows: chromatographic column is using octadecylsilane chemically bonded silica as filler, mobile phase A is methanol, Mobile phase B is selected from formic acid, phosphoric acid or acetic acid, carry out gradient elution, and the flow velocity of mobile phase is 0.8-1.2ml/min, 25-40 DEG C of column temperature, Detection wavelength: 244~264nm.Further, the present invention also provides the methods for establishing corresponding finger-print with this method.
Description
Technical field
The present invention relates to drug detection fields, more particularly, to a kind of high performance liquid chromatography detection of Chinese patent drug.
Background technique
By cloves, cortex cinnamomi, three taste Chinese medicinal composition of fructus piperis longi pharmaceutical composition DINGHUIER QITIE, have warming middle-JIAO and strengthening the spleen, cold dispelling
The effect of antidiarrheal.The quality inspection standard of the kind is not recorded in " Chinese Pharmacopoeia ".Shortcomings in existing standard are as follows: thin
Cloves, cortex cinnamomi and fructus piperis longi method are different in layer identification, and organic solvent consumption is big, pollute environment, and detection time is long, and
It cannot quantify completely, it is poor to the quality controllability of product.
The method that Chinese patent 201210035570.5 improves its quality testing, but this method is laid particular emphasis on to this drug
The Qualitive test and quantitative detection of composition main component lack the control to drug total quality.
Accordingly, it is desirable to provide the pharmaceutical composition of a kind of new, quick, accurate and suitable industrialized production demand
The method of quality control of object.
Summary of the invention
In order to solve at least one above-mentioned technical problem, the present invention provides the drugs of a kind of invigorating the spleen middle benefit gas, cold dispelling antidiarrheal
The measuring method of effective component in composition, and the method for establishing corresponding finger-print with this method.
According to some embodiments of the present invention, a kind of invigorating the spleen middle benefit gas, effective component in the pharmaceutical composition of cold dispelling antidiarrheal
Measuring method, which comprises the following steps:
(1) preparation of reference substance solution: weighing cinnaldehydrum reference substance, adds methanol that reference substance solution is made.(2) test sample is molten
The preparation of liquid: taking described pharmaceutical composition, crushes, and methanol solution is added in sieving, extracts 1~2 hour, puts to room temperature, first is added
Alcohol supplies the weight of less loss, shakes up, and filtration takes filtrate to get test solution.(3) reference substance solution and confession measuring method: are drawn
Test sample solution injects high performance liquid chromatograph, obtains chromatogram, wherein
The chromatographic condition of high performance liquid chromatograph is as follows: chromatographic column is using octadecylsilane chemically bonded silica as filler: stream
Dynamic phase A is methanol, and Mobile phase B is selected from formic acid, phosphoric acid or acetic acid, carries out gradient elution, Gradient program such as following table
The flow velocity of mobile phase be 0.8-1.2ml/min, 25-40 DEG C of column temperature, Detection wavelength: 244~264nm.
The selected testing conditions of the present invention are compared and verify repeatedly by experiment, compared with the prior art compared with having
The characteristics of accuracy is high, and semiquantitative determination may be implemented.
One of embodiment according to the present invention, wherein in step (1) in reference substance solution cinnaldehydrum concentration 0.1mg/
ml。
Another embodiment according to the present invention, wherein the concentration of methanol is 70~100% in step (1) and (2).It is excellent
Selection of land, the concentration of methanol is respectively 100% and 75% in step (1) and (2).
Another embodiment according to the present invention, wherein pharmaceutical composition described in step (2) adds after being crushed, being sieved
Enter methanol, so that contained medicinal powder is 1g~5g in every 100ml Extraction solvent.
Another embodiment according to the present invention, extracting method is heating and refluxing extraction in step (2).
According to the present invention, the concentration of Mobile phase B is 0.2% in step (3).Further, Mobile phase B can be 0.2%
Formic acid.
One of embodiment according to the present invention, most preferred gradient elution program can be with are as follows:
One of embodiment according to the present invention, chromatographic column are Agilent ZORBAX SB-C18Column.
According to the preferred embodiment of the present invention, preferably 30 DEG C of the column temperature of chromatographic column.
According to the preferred embodiment of the present invention, the preferred 1ml/min of the flow velocity of mobile phase.
According to the preferred embodiment of the present invention, Detection wavelength is preferably 254nm.
According to another aspect of the present invention, the pharmaceutical composition efficient liquid phase fingerprint image of a kind of invigorating the spleen middle benefit gas, cold dispelling antidiarrheal
The method for building up of spectrum, comprising the following steps:
(1) preparation of reference substance solution: weighing cinnaldehydrum reference substance, and obtained pair of methanol that concentration is 70~100% is added
According to product solution.(2) preparation of test solution: taking described pharmaceutical composition, crushes, sieving, and it is 70~100% that concentration, which is added,
Methanol solution heating and refluxing extraction 1-2 hours, is put to room temperature so that contained medicinal powder is 1g~5g in every 100ml Extraction solvent,
The weight that the methanol that concentration is 70~100% supplies less loss is added, shakes up, filters, takes filtrate to get test solution.(3) it obtains
It obtains chromatogram: using reference substance solution as object of reference, test solution and control solution being injected into high performance liquid chromatograph, are obtained
To chromatogram, wherein
The chromatographic condition of the high performance liquid chromatograph is as follows: chromatographic column is filling with octadecylsilane chemically bonded silica
Agent, mobile phase A are methanol, and Mobile phase B is selected from formic acid, phosphoric acid or acetic acid, carry out gradient elution, Gradient program such as following table
The flow velocity of mobile phase is 0.8-1.2ml/min, and 25-40 DEG C of column temperature, Detection wavelength is 244~264nm.
(4) it generates reference fingerprint: more batches of qualified described pharmaceutical composition chromatograms is selected, using cinnaldehydrum as reference
Peak obtains the map at 12 shared peaks, calculates by median method and generates standard control finger-print, wherein finger-print is opposite
Retention time are as follows:
Inventors have found that cinnaldehydrum chromatographic peak peak area proportion in test article fingerprint is relatively large, and pass through
Multiple sample actual measurements, it is relatively stable as the result is shown, therefore selected ginseng of No. 6 peak cinnaldehydrum as DINGHUIER QITIE finger-print
According to object.Gained standard finger-print of the invention is referring to attached drawing 23.
The present invention takes HPLC finger-print to detect the kind for the first time, by mobile phase and gradient elution item
The methods of part optimizes, so that the main peak separation of map is good, while the peak shape for compareing peak also obviously improves, and can control comprehensively
The total quality of this product processed, while meeting the requirement of industrialized production.The kind that the method provided through the invention is established
Standard finger-print can provide good quality standard for the internationalization of this product kind.
The additional aspect of the present invention and advantage will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Detailed description of the invention
Above-mentioned and/or additional aspect of the invention and advantage will become from the following description of the accompanying drawings of embodiments
Obviously and it is readily appreciated that, in which:
Fig. 1 reference substance finger-print obtained by providing method according to the present invention；
Fig. 2 gained test article fingerprint according to embodiments of the present invention；
Fig. 3 210~350nm sample to be tested chromatogram according to embodiments of the present invention；
Fig. 4 sample to be tested chromatogram under different wave length according to the present invention；
Sample chromatogram figure obtained by Fig. 5 various concentration and the Extraction solvent of type；
Sample chromatogram figure obtained by Fig. 6 difference extracting mode；
Sample chromatogram figure obtained by Fig. 7 difference extraction time；
Sample chromatogram figure obtained by Fig. 8 different brands chromatographic column；
Sample chromatogram figure obtained by Fig. 9 difference column temperature；
Figure 10 gained sample chromatogram figure different in flow rate；
Figure 11 determines sample chromatogram figure obtained by Chromatographic information acquisition time；
Figure 12 is using sample chromatogram figure obtained by acetonitrile-water mobile phase；
Figure 13 is using sample chromatogram figure obtained by methanol-water mobile phase；
Figure 14 is using sample chromatogram figure obtained by -0.2% formic acid mobile phase of methanol；
Figure 15 is using sample chromatogram figure obtained by -0.2% phosphoric acid mobile phase of methanol；
Figure 16 is using sample chromatogram figure obtained by -0.2% acetic acid mobile phase of methanol；
Figure 17 system suitability finger-print；
Figure 18 repetitive test finger-print；
Figure 19 Intermediate precision tests finger-print；
Figure 20 stability test finger-print；
Figure 21 instrument serviceability test finger-print；
Figure 22 chromatographic column serviceability test finger-print；
Figure 23 according to the present invention provided by standard finger-print.
Specific embodiment
The embodiment of the present invention is described below in detail.The embodiments described below with reference to the accompanying drawings are exemplary, only
It is used to explain the present invention, and is not construed as limiting the claims.
1 finger-print of embodiment is established
1.1 instrument
1260 high performance liquid chromatograph of Agilent, Shimadzu LC-20AT high performance liquid chromatograph, Agilent1200 are efficient
Liquid chromatograph, chromatographic column (Agilent ZORBAX SB-C18,4.6 × 250mm, 5 μm, S/N:USCL 060354), chromatography
Column (Agilent ZORBAX SB-C18,4.6 × 250mm, 5 μm, S/N:USCL 058060), chromatographic column (Agilent
ZORBAX SB-C18,4.6 × 250mm, 5 μm, S/N:USCL 036549), chromatographic column (Agilent ZORBAX SB-C18,
4.6 × 250mm, 5 μm, S/N:USCL 059648), chromatographic column (Phenomenex Gemiri C18,4.6 × 250mm, 5 μm,
S/N:695685-14), chromatographic column (Thermo ODS-2HYPERSIL, 4.6 × 250mm, 5 μm, S/N:0905415N4), chromatography
Column (Inertsil ODS-3,4.6 × 250mm, 5 μm, S/N:1A7143425), chromatographic column (Unitary C18,4.6 ×
250mm, 5 μm, S/N:Z2013112901).
1.2 reagent
Reagent: formic acid (AR, Sinopharm Chemical Reagent Co., Ltd.), (AR, Chinese medicines group chemical reagent are limited for glacial acetic acid
Company), phosphoric acid (AR, Beijing Jing Qiu chemical industry Co., Ltd), pure water (Hangzhou Wahaha Group Co., Ltd), methanol (AR,
Sinopharm Chemical Reagent Co., Ltd.), methanol (HPLC, Honeywell).
Reference substance: eugenol reference substance (110725-200711, purity 99.3%, middle inspection institute), cinnaldehydrum reference substance
(110710-201016, purity 98.6%, middle inspection institute), piperine reference substance (0775-200203, purity 100%, middle inspection institute).
1.3 test sample
DINGHUIER QITIE (lot number: 151012,151013,151014,151015,151016,151020,151065,
151066,151067,151069), Shanxi Yabao Pharmaceutical Group Corp. provides.
1.4 test method
It is measured according to high performance liquid chromatography (four general rules 0512 of Chinese Pharmacopoeia version in 2015).
The preparation of reference substance solution takes each reference substance appropriate, accurately weighed, adds methanol that solution of every 1ml containing 0.1mg is made,
To obtain the final product.
The preparation of test solution takes DINGHUIER QITIE, shreds, and mixes.1g is taken, it is accurately weighed, it sets in round-bottomed flask, essence
75% methanol 50ml of close addition, weighed weight are heated to reflux 1 hour, are taken out, are put to room temperature, then weighed weight, with 75% methanol
The weight for supplying less loss, shakes up, filtration, take subsequent filtrate to get.
Chromatographic condition and system suitability are using octadecylsilane chemically bonded silica as filler (chromatographic column: Agilent
ZORBAX SB-C18 250mm × 4.6mm, 5 μm)；Using methanol as mobile phase A, 0.2% formic acid solution is Mobile phase B, according to the form below
Carry out gradient elution:
Detection wavelength 254nm；30 DEG C of column temperature；Flow velocity 1.0ml/min；Number of theoretical plate should must not be lower than by the calculating of cinnaldehydrum peak
10000。
Measuring method is accurate respectively to draw reference solution and each 10 μ l of test solution, injects liquid chromatograph, measures, note
The chromatogram of record 90 minutes.The result is shown in Figure 1,2.
Attached drawing 1 is reference substance finger-print, according to chemical component contained by each flavour of a drug of DINGHUIER QITIE, selects eugenol, osmanthus
Skin aldehyde, piperine carry out identification as object of reference.In the visible sample chromatogram figure of attached drawing 2, present and cinnaldehydrum reference substance
The consistent chromatographic peak of chromatographic peak retention time, and 12 characteristic peaks corresponding with reference fingerprint should be presented.By test sample
It is found that No. 6 peaks are cinnaldehydrum (retention time are as follows: 37.7min), No. 7 peaks are that eugenol (is protected for finger-print and control trace analysis
Stay the time are as follows: 47.2min), No. 8 peaks are piperine (retention time are as follows: 56.0min).Wherein cinnaldehydrum chromatographic peak is in test sample
Peak area proportion is relatively large in finger-print, and surveys through multiple samples, it is relatively stable as the result is shown, therefore selected
Object of reference (S) of No. 6 peak cinnaldehydrum as DINGHUIER QITIE finger-print.
2 optimum chromatogram condition of embodiment is investigated
Instrument and reagent are the same as embodiment 1.
The selection of 2.1 Detection wavelengths
Test sample is detected using PDA detector, investigates sample chromatogram peak information, selective mechanisms wavelength.
The preparation of test solution: taking this product, shreds, and mixes.1g is taken, it is accurately weighed, it sets in stuffed conical flask, precision adds
Enter 50% methanol 50ml, close plug, weighed weight is ultrasonically treated (500W, 40KHZ) 30 minutes, takes out, puts to room temperature, with 50%
Methanol supplies the weight of less loss, shakes up, filtration, take subsequent filtrate to get.
Using Agilent ZORBAX SB-C18 chromatographic column, 30 DEG C of column temperature, flow velocity 0.8ml/min, methanol is mobile phase A,
Aqueous solution is Mobile phase B, and according to the form below carries out gradient elution.Sample introduction 10ul, acquisition time 150min.
Comparative sample chromatogram in 210nm-350nm, chromatographic absorption is relatively abundanter in 230nm-270nm wave band and baseline
Steadily, see Fig. 3.Comparative sample chromatographic peak situation at 234nm (A), 244nm (B), 254nm (C), 264nm (D), is shown in Fig. 4.
The finger-print information content of 254nm is relatively more, can more fully embody the chemical component of this product, and baseline drift is little.
The prescription of DINGHUIER QITIE has cloves, cortex cinnamomi, three taste of fructus piperis longi, and quality control composition is respectively eugenol, cinnaldehydrum and pepper
Alkali, these three ingredient absorption maximums are between 280nm~343nm.Above-mentioned three kinds of ingredients are comprehensively compared absorption at 254nm
And figure is preferable, therefore it is preferred that Detection wavelength is 254nm.
The preparation of 2.2 test solutions is investigated
The preparation of reference substance solution and measuring method are the same as embodiment 1.
(1) Extraction solvent concentration is investigated
Drug paste (lot number 151012) is taken, is shredded, is mixed.Take 6 parts, each 1g, it is accurately weighed, it sets in stuffed conical flask, respectively
Precision be added dehydrated alcohol, methanol, 75% methanol, 50% methanol, 25% methanol, water 50ml, close plug, weighed weight, ultrasound at
It manages (500W, 40KHZ) 1 hour, takes out, put to room temperature, then weighed weight, supply the weight of less loss with coordinative solvent respectively, shake
It is even, filtration, take subsequent filtrate to get.
Using octadecylsilane chemically bonded silica as filler, (Agilent ZORBAX SB-C18 chromatographic column, column length are
250mm, internal diameter 4.6mm, S/N:USCL060354), using methanol as mobile phase A, 0.2% formic acid be Mobile phase B, according to the form below into
Row gradient elution；Flow velocity 1.0ml/min；Detection wavelength is 254nm；Column temperature is 30 DEG C.
Precision draws each 10 μ l of test solution, injects liquid chromatograph, measurement.It the results are shown in Table 1, Fig. 5.
Chromatographic peak area obtained by 1 various concentration of table and the Extraction solvent of type
As seen from the above table, 75% methanol be solvent extraction when, acquired finger-print information content is more, peak response value compared with
Greatly, therefore most preferably 75% methanol is Extraction solvent.
(2) extracting mode is investigated
This product (lot number 151012) is taken, is shredded, is mixed.Take 2 parts, each 1g, it is accurately weighed, stuffed conical flask and circle are set respectively
In the flask of bottom, accurate respectively that 75% methanol 50ml is added, close plug, weighed weight, it is small that portion is ultrasonically treated (500W, 40KHZ) 1
When, another heating water bath flows back 1 hour, and it takes out, puts to room temperature, then weighed weight, the weight of less loss is supplied with 75% methanol,
Shake up, filter, take subsequent filtrate to get.
Measuring method is the same as (1).It the results are shown in Table 2, Fig. 6.
Chromatographic peak area obtained by the different extracting modes of table 2
When as seen from the above table, using refluxing extraction, acquired finger-print information content is more, and peak response value is larger, therefore excellent
Select refluxing extraction.
(3) extraction time is investigated
This product (lot number 151012) is taken, is shredded, is mixed.Take 3 parts, each 1g, it is accurately weighed, it sets in round-bottomed flask respectively, point
Accurate to be added 75% methanol 50ml, weighed weight, heating water bath flows back 0.5 hour, 1 hour, 2 hours respectively, takes out, put to
Room temperature, then weighed weight, the weight of less loss is supplied with 75% methanol, is shaken up, filtration, take subsequent filtrate to get.
Measuring method is the same as (1).It the results are shown in Table 3, Fig. 7.
Chromatographic peak area obtained by the different extraction times of table 3
As seen from the above table, flowing back 1 hour can be obtained most information content, and peak response value is also larger, therefore it is preferred that reflux 1 is small
When.
Chromatographic peak main in the obtained chromatogram of method made above is integrated, the peak under each retention time is compared
Area selects chromatographic peak abundant information, and extraction efficiency is high, the biggish method of total peak area.
In conclusion the best preparation method of test article finally determined are as follows: take this product, shred, mix.1g is taken, precision claims
It is fixed, it sets in round-bottomed flask, 75% methanol 50ml is added in precision, and weighed weight is heated to reflux 1 hour, and it takes out, puts to room temperature, then
Weighed weight is supplied the weight of less loss with 75% methanol, is shaken up, filtration, take subsequent filtrate to get.
The selection of 2.3 chromatographic columns
The filler and type of feed of chromatographic column may have an impact drug chemistry ingredient separating effect.Inventor couple
Agilent ZORBAX SB-C18、Phenomenex Gemiri C18、Thermo ODS-2、Inertsil ODS-3、
The C18 chromatographic column (5 μm, 250 × 4.6mm) of five different brands of Unitary C18 is investigated.Other chromatographic conditions are the same as implementation
Example 1.The separating effect and peak type of Agilent ZORBAX SB-C18 column are superior to remaining four kinds, therefore preferentially select Agilent
ZORBAX SB-C18 chromatographic column.As a result see Fig. 8.
The selection of 2.4 column temperatures
In chromatogram finger print measuring, column temperature often will affect separating effect, and inventor is respectively in 30 DEG C, 35 DEG C, 40
Under the conditions of DEG C three column temperatures, the finger-print of DINGHUIER QITIE test solution is analyzed.The result shows that each at 30 DEG C
The separating effect of a chromatographic peak is relatively good, therefore 30 DEG C of optimal selection are column temperature when detecting.As a result see Fig. 9.
2.5 flow velocitys are investigated
In chromatogram finger print measuring, flow velocity will affect separating effect to a certain extent, this test exists respectively
Under tetra- flow conditions of 0.6ml/min, 0.8ml/min, 1.0ml/min, 1.2ml/min, to DINGHUIER QITIE test solution
Finger-print analyzed, the results showed that in 1.0ml/min, the separating effect of each chromatographic peak is relatively good, thus select
1.0ml/min is most suitable flow velocity.The result is shown in Figure 10.
2.6 Chromatographic information acquisition times determine
To investigate Chromatographic information acquisition time, this product is detected, chromatographic condition is as follows: chromatographic column Agilent
ZORBAX SB-C18 chromatographic column (250mm × 4.6mm, 5 μm)；Detection wavelength 254nm；30 DEG C of column temperature；Flow velocity 1.0ml/min；Into
10 μ l of sample amount；Mobile phase: using methanol as mobile phase A, 0.2% formic acid solution is Mobile phase B, and according to the form below carries out gradient elution.It adopts
Collect the Chromatographic information in 120min, gained chromatogram is shown in attached drawing 11.
As seen from the figure, without chromatographic peak after 90min, therefore the Chromatographic information in 90min is only acquired.
3 flow phase system comparative studies of embodiment
According to preliminary result, different flow visualizings is investigated, screening chromatographic peak information is most abundant, and separating degree is best
Chromatographic condition.
The preparation of test solution: taking this product, shreds, and mixes.1g is taken, it is accurately weighed, it sets in stuffed conical flask, precision adds
Enter 50% methanol 50ml, close plug, weighed weight is ultrasonically treated (500W, 40KHZ) 30 minutes, takes out, puts to room temperature, with 50%
Methanol supplies the weight of less loss, shakes up, filtration, take subsequent filtrate to get.
Using Agilent ZORBAX SB-C18 chromatographic column, 30 DEG C of column temperature, Detection wavelength 254nm, flow velocity 1.0ml/min,
Investigate acetonitrile-aqueous solution, methanol-water solution, -0.2% formic acid solution of methanol, -0.2% phosphoric acid solution of methanol, methanol -0.2%
5 systems of acetic acid solution.
(1) system 1: acetonitrile is mobile phase A, and aqueous solution is Mobile phase B, acquisition time 65min.Gradient see the table below,
Gained chromatogram is referring to attached drawing 12.
(2) system 2: methanol is mobile phase A, and aqueous solution is Mobile phase B, acquisition time 75min.Gradient see the table below,
Gained chromatogram is referring to attached drawing 13.
(3) system 3: methanol is mobile phase A, and 0.2% formic acid solution is Mobile phase B, acquisition time 70min.Gradient
It see the table below, gained chromatogram is referring to attached drawing 14.
(4) system 4: methanol is mobile phase A, and 0.2% phosphoric acid solution is Mobile phase B, acquisition time 70min.Gradient
It see the table below, gained chromatogram is referring to attached drawing 15.
(5) system 5: methanol is mobile phase A, and 0.2% acetic acid solution is Mobile phase B, acquisition time 70min.Gradient
It see the table below, gained chromatogram is referring to attached drawing 16.
As a result determine -0.2% formic acid solution system (system 3) of methanol to the test solution chromatographic peak point of DINGHUIER QITIE
It is preferable from effect.
The verifying of 4 fingerprint spectrum method of embodiment
4.1 blank test
75% methanol is taken, by 1 chromatographic condition direct injected of embodiment, 90 minutes chromatograms of record, the results showed that system
There is no residuals and interference.
4.2 system suitability
This product (lot number 151012) is taken, is operated according to 1 method of embodiment, is prepared into test solution, direct injected 6 times,
It is to calculate the relative retention time and relative peak area of each main chromatographic peak referring to peak with No. 6 peaks.12 shared peaks as the result is shown
Relative retention time stablize, RSD is respectively less than 1%, and relative peak area is relative constant, and RSD is respectively less than 3%；Show method is
Applicability of uniting is good.It the results are shown in Table 4, table 5, Figure 17.
4 system suitability relative retention time of table
5 system suitability relative peak area of table
3.3 repetitive test
Testing crew A takes same batch DINGHUIER QITIE (lot number 151012), operates according to 1 method of embodiment, preparation 6
Part test solution, detection are to calculate the relative retention time and relative peak area of each main chromatographic peak referring to peak with No. 6 peaks.
The relative retention time at 12 shared peaks is stablized as the result is shown, and RSD is respectively less than 1%, and relative peak area is relative constant, and RSD is small
In 3%；Show that this method repeatability is good.It the results are shown in Table 6, table 7, Figure 18.
6 repetitive test relative retention time of table
7 repetitive test relative peak area of table
The test of 3.4 Intermediate precisions
Testing crew B takes same batch DINGHUIER QITIE (lot number 151012), operates according to 1 method of embodiment, preparation 6
Part test solution, with the high performance liquid chromatograph different from experimenter A, different chromatography post detections, using No. 6 peaks as reference
Peak calculates the relative retention time and relative peak area of each main chromatographic peak.As the result is shown when the opposite reservation at 12 shared peaks
Between stablize, RSD is respectively less than 1%, and relative peak area is relative constant, and RSD is respectively less than 3%；Show that this method repeatability is good.As a result
It is shown in Table 8, table 9, Figure 19.
8 Intermediate precision of table tests relative retention time
9 Intermediate precision of table tests relative peak area
3.5 stability test
Take this product (lot number 151012), according to 1 method of embodiment operate, respectively at test sample prepare 0,2,4,8,12,18,
24,32,40,48 hours sample detections are that the relative retention time of each main chromatographic peak and opposite is calculated referring to peak with No. 6 peaks
Peak area.The relative retention time at 12 shared peaks is stablized as the result is shown, and RSD is respectively less than 1%, and relative peak area is relative constant,
RSD is respectively less than 3%；Show that the test solution of this product is stable within 48 hours.It the results are shown in Table 10, table 11, Figure 20.
10 stability test relative retention time of table
11 stability test relative peak area of table
3.6 serviceability test
3.6.1 different instrument durabilities
This product (lot number 151012) is taken, operates according to 1 method of embodiment, is detected using the instrument of different model, with
1260 instrument map of Agilent is reference, to calculate the similarity of map between different instruments referring to map, the results showed that no
Roughly the same with measurement result between instrument, fingerprint similarity is all larger than 0.98, illustrates the model pair of high performance liquid chromatograph
This fingerprint spectrum method influences little.It the results are shown in Table 12, Figure 21.
12 instrument serviceability test result of table
3.6.2 different batches chromatographic column durability
This product (lot number 151012) is taken, is operated according to 1 method of embodiment, is carried out using same model different batches chromatographic column
Detection calculates similarity, the results showed that no using one of batch-wise chromatography column (sequence number USCL 060354) map as reference
Roughly the same with batch-wise chromatography intercolumniation measurement result, fingerprint similarity is all larger than 0.99, illustrates the batch of chromatographic column to this
Method influences little.It the results are shown in Table 13, Figure 22.
13 chromatographic column serviceability test result of table
3.7 relative retention time
With No. 6 peaks (cinnaldehydrum) be the relative retention time of each shared chromatographic peak is calculated referring to peak should be shown in the table 14
In range.It is shown in Table 14.
14 relative retention time of table
5 reference fingerprint of embodiment is established
To Shanxi Yabao Pharmaceutical Group Corp. production lot number be 151012,151013,151014,151015,
151016,151020,151065,151066,151067,151069 DINGHUIER QITIE amounts to 10 batch samples, respectively by real
The finger print measuring method for applying the foundation of example 1 is detected, using Chinese Pharmacopoeia Commission's " chromatographic fingerprints of Chinese materia medica similarity
Evaluation system " (2012.0 version) fitting generation reference fingerprint, see Figure 23.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (12)
1. the measuring method of a kind of invigorating the spleen middle benefit gas, the medicine composition fingerprint of cold dispelling antidiarrheal, which is characterized in that including following
Step:
(1) preparation of reference substance solution
Cinnaldehydrum reference substance is weighed, the methanol that concentration is 100% is added, reference substance solution is made；
(2) preparation of test solution
Described pharmaceutical composition is taken, is crushed, the methanol solution that concentration is 75% is added in sieving, extracts 1~2 hour, puts to room
Temperature is added the weight that the methanol that concentration is 75% supplies less loss, shakes up, filters, take filtrate to get test solution；
(3) measuring method:
The reference substance solution and the test solution are drawn, high performance liquid chromatograph is injected, obtains chromatogram,
Wherein, the chromatographic condition of the high performance liquid chromatograph is as follows:
Chromatographic column using octadecylsilane chemically bonded silica as filler,
Mobile phase A is methanol, and Mobile phase B is 0.2% formic acid, carries out gradient elution, Gradient program such as following table
The flow velocity of mobile phase be 0.8-1.2ml/min, 25-40 DEG C of column temperature, Detection wavelength: 244~264nm.
2. measuring method according to claim 1, wherein the concentration of cinnaldehydrum in reference substance solution described in step (1)
0.1mg/ml。
3. measuring method according to claim 1, which is characterized in that pharmaceutical composition described in step (2) is through crushing, mistake
Methanol is added after sieve, so that contained medicinal powder is 1g~5g in every 100ml Extraction solvent.
4. measuring method according to claim 1, which is characterized in that extracting method is to be heated to reflux in the step (2)
It extracts.
5. measuring method according to claim 1, which is characterized in that chromatographic column is Agilent ZORBAX SB-C18Column.
6. measuring method according to claim 1, which is characterized in that preferably 30 DEG C of column temperature.
7. measuring method according to claim 1, which is characterized in that the preferred 1ml/min of the flow velocity of mobile phase.
8. measuring method according to claim 1, which is characterized in that Detection wavelength is preferably 254nm.
9. a kind of method for building up of the pharmaceutical composition high-efficiency liquid-phase fingerprint of invigorating the spleen middle benefit gas, cold dispelling antidiarrheal, it is characterised in that
The following steps are included:
(1) preparation of reference substance solution
Cinnaldehydrum reference substance is weighed, the methanol that concentration is 100% is added, reference substance solution is made；
(2) preparation of test solution
Described pharmaceutical composition is taken, is crushed, the methanol solution that concentration is 75% is added, so that in every 100ml Extraction solvent in sieving
Contained medicinal powder is 1g~5g, heating and refluxing extraction 1-2 hours, puts to room temperature, the methanol that concentration is 75% is added and supplies less loss
Weight shakes up, and filtration takes filtrate to get test solution；
(3) chromatogram is obtained
Using the reference substance solution as object of reference, the test solution and the reference substance solution are injected into high-efficient liquid phase color
Spectrometer obtains chromatogram,
Wherein, the chromatographic condition of the high performance liquid chromatograph is as follows:
Chromatographic column using octadecylsilane chemically bonded silica as filler,
Mobile phase A is methanol, and Mobile phase B is 0.2% formic acid, carries out gradient elution, Gradient program such as following table
The flow velocity of mobile phase be 0.8-1.2ml/min, 25-40 DEG C of column temperature, Detection wavelength: 244~264nm；
(4) reference fingerprint is generated:
More batches of qualified described pharmaceutical composition chromatograms of selection obtain the map at 12 shared peaks using cinnaldehydrum as reference peak,
It is calculated by median method and generates standard control finger-print, wherein finger-print relative retention time are as follows:
10. according to the method described in claim 9, it is characterized in that, chromatographic column is Agilent ZORBAX SB-C18Column.
11. according to the method described in claim 9, it is characterized in that, in reference substance solution cinnaldehydrum concentration 0.1mg/ml.
12. according to the method described in claim 9, it is characterized in that, Detection wavelength is 254nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610128775.6A CN107167544B (en) | 2016-03-07 | 2016-03-07 | The measuring method and fingerprint of effective component in a kind of invigorating the spleen middle benefit gas, the pharmaceutical composition of cold dispelling antidiarrheal |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610128775.6A CN107167544B (en) | 2016-03-07 | 2016-03-07 | The measuring method and fingerprint of effective component in a kind of invigorating the spleen middle benefit gas, the pharmaceutical composition of cold dispelling antidiarrheal |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107167544A CN107167544A (en) | 2017-09-15 |
| CN107167544B true CN107167544B (en) | 2019-02-12 |
Family
ID=59848669
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610128775.6A Active CN107167544B (en) | 2016-03-07 | 2016-03-07 | The measuring method and fingerprint of effective component in a kind of invigorating the spleen middle benefit gas, the pharmaceutical composition of cold dispelling antidiarrheal |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107167544B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1991358A (en) * | 2005-12-28 | 2007-07-04 | 劲牌有限公司 | Method for checking Cinnamomum cassia medicinal material by using traditional medicine fingerprint pattern technology |
| CN102621268B (en) * | 2012-02-17 | 2015-02-18 | 亚宝药业集团股份有限公司 | Quality detection method for spleen-invigorating, cold-dispersing and antidiarrheal medicine composition |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2000272667A1 (en) * | 2000-09-13 | 2002-04-29 | Jiangsu Kanion Pharmaceutical Co. | Pharmaceutical composition treating gynecological blood stasis diseases, cardio and cerebral vascular diseases, respiratory diseases and the like |
-
2016
- 2016-03-07 CN CN201610128775.6A patent/CN107167544B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1991358A (en) * | 2005-12-28 | 2007-07-04 | 劲牌有限公司 | Method for checking Cinnamomum cassia medicinal material by using traditional medicine fingerprint pattern technology |
| CN102621268B (en) * | 2012-02-17 | 2015-02-18 | 亚宝药业集团股份有限公司 | Quality detection method for spleen-invigorating, cold-dispersing and antidiarrheal medicine composition |
Non-Patent Citations (5)
| Title |
|---|
| 不同产地肉桂HPLC 指纹图谱研究及桂皮醛含量分析;刘月 等;《海峡药学》;20121231;第24卷(第4期);第59-61页 |
| 五积散酒的多成分HPLC指纹图谱;刘春海 等;《中国医药工业杂志》;20101231;第41卷(第8期);第607-609页 |
| 香桂化浊胶囊的HPLC-PDA指纹图谱研究;张辰辰 等;《医药导报》;20140131;第33卷(第1期);第86-89页 |
| 高效液相色谱法测定丁桂儿脐贴中丁香酚和桂皮醛含量;王也 等;《中国药业》;20111231;第20卷(第10期);第43-44页 |
| 高效液相色谱法测定丁桂儿脐贴中桂皮醛和胡椒碱含量;杨晓宁 等;《环球中医药》;20140131;第7卷(第1期);第17-20页 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107167544A (en) | 2017-09-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104713957B (en) | A kind of assay method of root of fangji Caulis Sinomenii extract finger-print | |
| CN109633001B (en) | Fingerprint detection method of wine glossy privet fruit | |
| CN110907546B (en) | HPLC fingerprint detection method of traditional Chinese medicine for tonifying kidney and strengthening bone | |
| CN107356691A (en) | Build the detection method of bent finger-print | |
| CN110031570A (en) | The fingerprint atlas detection method of Kangganmao Granule | |
| CN109406645A (en) | A kind of Antisathmatic oral liquid for child epheday intermedia, the detection method for frying semen armeniacae amarae, Radix Glycyrrhizae, radix scutellariae | |
| CN108896681B (en) | Nerve-soothing brain-tonifying liquid multi-index quantitative fingerprint establishment method and application thereof | |
| CN104198619A (en) | Quality detection method of cough syrup for children | |
| CN104316613B (en) | A kind of method for building up of dispelling wind detoxicating capsule finger printing | |
| CN102890124B (en) | Fingerprint constructing method of total flavonoid components and total alkaloids components in loranthus parasiticus-kudzuvine root preparation and quality detecting method | |
| CN107576739B (en) | HPLC fingerprint detection method of Longmu Zhuanggu granules | |
| CN109406682A (en) | The UPLC characteristic spectrum construction method and detection method of ginger medicinal material | |
| CN102133333A (en) | Quality control method for shenmai injection mass spectrum finger prints | |
| CN106918673B (en) | A kind of method for building up of the finger-print of Chinese medicine composition | |
| CN110441413A (en) | The construction method and detection method of qianbai biyan tablets HPLC finger-print | |
| CN107167544B (en) | The measuring method and fingerprint of effective component in a kind of invigorating the spleen middle benefit gas, the pharmaceutical composition of cold dispelling antidiarrheal | |
| CN104833754B (en) | A kind of attached sweet drug detection method | |
| CN108693289B (en) | Method for determining content of magnoflorine in herringbone fruit medicinal material | |
| CN114216980B (en) | Method for establishing HPLC-ELSD fingerprint of starwort root | |
| AU2021106279A4 (en) | Method for establishing hplc-elsd fingerprints of shenlingbaizhu pills and standard fingerprints thereof | |
| CN110031564A (en) | The quality determining method of natural plants anticoccidial feed addictive based on HPLC finger-print | |
| CN109738391A (en) | A kind of rhizoma zingiberis evaluation of medical materials' quality method based on near-infrared spectrum technique | |
| CN102441058B (en) | Gas chromatography fingerprint detection method for blood-nourishing brain-refreshing grain | |
| CN107782798B (en) | Method for detecting qi-tonifying astragalus-ginseng dropping pills by using dual-wavelength UPLC | |
| CN108445115A (en) | A kind of method that high performance liquid chromatography detects neoline and/or songorine and/or Fuziline |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |