CN105510488A - Fingerprint spectrum of pain-relieving patch and quality detection method thereof - Google Patents
Fingerprint spectrum of pain-relieving patch and quality detection method thereof Download PDFInfo
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- CN105510488A CN105510488A CN201610049485.2A CN201610049485A CN105510488A CN 105510488 A CN105510488 A CN 105510488A CN 201610049485 A CN201610049485 A CN 201610049485A CN 105510488 A CN105510488 A CN 105510488A
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
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- 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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- G01N2030/027—Liquid chromatography
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- G—PHYSICS
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- 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 invention provides a measuring method for effective ingredients of a pain-relieving patch and a building method for a fingerprint spectrum. The method comprises the steps of preparation of a comparison solution, preparation of a test solution, measuring and the like. Octadecylsilane chemically bonded silica is used as a filling agent for a chromatographic column of a high performance liquid chromatograph, the mobile phase is acetonitrile-0.2% formic acid, gradient elution is carried out according to a specific program, the moving speed of the mobile phase is 0.8-1.2 ml/min, the column temperature is 30-40 DEG C, and the detection wavelength is 290+/-3 nm. The selected mobile phase and gradient elution are verified through experiments, and compared with the prior art, fingerprint spectrum detection and content measurement of two kinds of flavonoid effective ingredients in the pain-relieving path can be achieved at the same time.
Description
Technical field
The present invention relates to medicine detection field, the high performance liquid chromatography especially relating to a kind of Chinese patent drug detects.
Background technology
It is be the external use plaster of primary raw material medicine with local medicinal material sand sagebrush (Artemisia filifolia) that swelling and pain relieving pastes, through zoopery and for many years Clinical practice verify, determined curative effect, has extraordinary anti-inflammatory analgetic effect.Clinical for furuncle sore from the beginning of, the supplemental treatment of painful swelling of joints.Since the listing of this kind, through constantly technology upgrading, be obtained in product quality and production technology and improved significantly.
But for the quality control aspect of this product, lack systematically study and unify, be applicable to industrial standard.The people such as Zhang Yuanyuan once studied the finger-print condition (Zhang Yuanyuan, Qiao Yanjiang, HPLC method measures the content of two kinds of flavanones in the sub-seed of sand sagebrush (Artemisia filifolia), Beijing University of Chinese Medicine's journal, 2008,31 (1): 58 ~ 60.) of sand sagebrush (Artemisia filifolia).But during effective constituent sub-according to the sand sagebrush (Artemisia filifolia) in the subsides of its condition detection swelling and pain relieving, find that its peak type is very poor, degree of separation is bad, does not reach Chinese Pharmacopoeia requirement, is unfavorable for that the large production of enterprise is suitable for.Li Shasha etc. have studied the assay of effective constituent in sand sagebrush (Artemisia filifolia), but it adopts is isocratic elution, can not meet the requirement that finger-print detects.(Li Shasha, history Singapore dollar, Zhan Xiao etc., the assay of general flavone and eriodictyol-7-methylether, isosakuranetin during sand sagebrush (Artemisia filifolia) is sub-, Chinese traditional Chinese medicine magazine, 2009,24 (9): 1223 ~ 1225.)
Therefore, need to provide a kind of new detection method, assay and the finger-print that can meet plurality of active ingredients in the sub-seed of sand sagebrush (Artemisia filifolia) detect simultaneously.
Summary of the invention
In order to solve at least one technical matters above-mentioned, the invention provides a kind of assay method of swelling and pain relieving patch effective constituent.
Assay method provided by the invention, comprises the steps:
(1) preparation of reference substance solution: take eriodictyol-7-methylether, isosakuranetin reference substance, adds ethanol obtained mixing reference substance solution.
(2) preparation of need testing solution: the medicine cancelling swollen pain relieving plaster pastes, and pulverizes, sieves, add ethanolic solution, extracts 20-50min, put to room temperature, add the weight that less loss supplied by ethanol, shake up, filter, get filtrate, evaporate to dryness, residue adds ethanol and dissolves, and shakes up and obtains need testing solution.
(3) determination method: draw mixing reference substance solution and need testing solution, inject high performance liquid chromatograph, obtain chromatogram, wherein, the chromatographic condition of high performance liquid chromatograph is as follows:
Chromatographic column take octadecylsilane chemically bonded silica as filling agent,
Mobile phase: acetonitrile-0.2% formic acid is mobile phase, and carry out gradient elution, Gradient program is as following table
The flow velocity of mobile phase is 0.8-1.2ml/min, column temperature 30-40 DEG C,
Determined wavelength: 290 ± 3nm.
Mobile phase selected by the present invention and gradient are through experimental verification, and prior art compares, and can meet the assay of finger-print detection and two kinds of flavonoids effective constituents in swelling and pain relieving patch simultaneously.
According to one of embodiment of the present invention, in step (1), in mixing reference substance solution, the concentration of eriodictyol-7-methyl ether and isosakuranetin is respectively 40 μ g/ml and 20 μ g/ml.
According to one of embodiment of the present invention, gradient program can be: 0 ~ 10min, acetonitrile 12%; 10 ~ 15min, acetonitrile 12% ~ 16%; 15 ~ 30min, acetonitrile 16%; 30 ~ 65min, acetonitrile 16% ~ 30%; 65 ~ 80min, acetonitrile 30% ~ 35%; 80 ~ 110min, acetonitrile 35%.Namely adopt gradient as shown in the table.
According to one of embodiment of the present invention, the concentration of ethanol is 55 ~ 85%.
According to one of embodiment of the present invention, the concentration of ethanol is preferably 80%.
According to one of embodiment of the present invention, medicine described in step (2) pastes and add ethanol after pulverizing, sieving, and makes contained medicinal powder in every 100ml Extraction solvent be 1g ~ 5g.
According to one of embodiment of the present invention, in step (2), extracting method is ultrasonic extraction or heating and refluxing extraction.
According to one of embodiment of the present invention, in step (2), extracting method is ultrasonic extraction 25 ~ 35min.Further, ultrasonic power can be 500W, and frequency is 40kHz.
According to one of embodiment of the present invention, chromatographic column can be AgilentZORBAXSB-C
18post.
According to one of embodiment of the present invention, column temperature preferably 35 DEG C.
According to one of embodiment of the present invention, the preferred 1ml/min of flow velocity of mobile phase.
According to one of embodiment of the present invention, determined wavelength is 290nm.
Present invention also offers a kind of method for building up of swelling and pain relieving patch high-efficiency liquid-phase fingerprint, it is characterized in that comprising the following steps:
(1) preparation of reference substance solution: take eriodictyol-7-methylether, isosakuranetin reference substance, adds the ethanol obtained mixing reference substance solution that concentration is 55 ~ 85%.
(2) preparation of need testing solution: the medicine cancelling swollen pain relieving plaster pastes, and pulverizes, sieves, add the ethanolic solution that concentration is 55 ~ 85%, make contained medicinal powder in every 100ml Extraction solvent be 1g ~ 5g, ultrasonic extraction or heating and refluxing extraction 20-50min, put to room temperature, add concentration be 55 ~ 85% ethanol supply the weight of less loss, shake up, filter, get filtrate, evaporate to dryness, residue adds the ethanol dissolving that concentration is 55 ~ 85%, shakes up and obtains need testing solution.
(3) obtain chromatogram: using reference substance solution as object of reference, need testing solution and reference substance solution are injected high performance liquid chromatograph, and obtain chromatogram, wherein, the chromatographic condition of described high performance liquid chromatograph is as follows:
Chromatographic column take octadecylsilane chemically bonded silica as filling agent,
Mobile phase: acetonitrile-0.2% formic acid is mobile phase, and carry out gradient elution, Gradient program is as follows:
The flow velocity of mobile phase is 0.8-1.2ml/min, column temperature 30-40 DEG C,
Determined wavelength: 290 ± 3nm.
(4) reference fingerprint is generated: the finger-print selecting many batches of qualified swelling and pain relieving patches, be reference peak with eriodictyol-7-methylether, obtain the collection of illustrative plates at 11 total peaks, calculate by median method and generate standard control finger-print, wherein, finger-print relative retention time is:
Figure is shown in accompanying drawing 2.
One of according to the embodiment of the present invention, the concentration of ethanol is 80%.
One of according to the embodiment of the present invention, chromatographic column can be AgilentZORBAXSB-C
18post.
One of according to the embodiment of the present invention, in step (1), in mixing reference substance solution, the concentration of eriodictyol-7-methyl ether and isosakuranetin is respectively 40 μ g/ml and 20 μ g/ml.
One of according to the embodiment of the present invention, determined wavelength is 290nm.
One of according to the embodiment of the present invention, in step (2), extracting method is ultrasonic extraction 25 ~ 35min.
It should be noted that, it is be the external use plaster of primary raw material medicine with local medicinal material sand sagebrush (Artemisia filifolia) that the swelling and pain relieving that adopts in the present invention pastes, such as, the lot number that Shanxi Yabao Pharmaceutical Group Corp. produces is the swelling and pain relieving subsides of 1504288,1505048,1505201,1505202,1505203,1505204,1505205,1505206,1505207,1505208,1505209,1505210,1505211,1505212,1505213,1505214,1505215,1505216,1505219,1505221,1505223,1505224.
The present invention is optimized HPLC fingerprint spectrum method of the prior art, through improving the method such as mobile phase and condition of gradient elution, the main peak degree of separation of collection of illustrative plates is obviously improved, reach pharmacopoeial requirements, the peak shape simultaneously contrasting peak also obviously improves, and can meet the needs of industrialized production.
The aspect that the present invention adds and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage will become obvious and easy understand from the following description of the accompanying drawings of embodiments, wherein:
Fig. 1 is according to the invention provides method gained reference substance finger-print;
Fig. 2 is according to embodiment of the present invention gained test sample chromatogram;
Fig. 3 eriodictyol-7-methylether standard items uv absorption figure;
Fig. 4 isosakuranetin standard items uv absorption figure;
Fig. 5 swelling and pain relieving patch uv absorption figure;
Fig. 6 variable concentrations Extraction solvent gained test sample chromatogram;
Fig. 7 different solvents consumption gained test sample chromatogram;
Fig. 8 different extracting mode gained test sample chromatogram;
Fig. 9 gained test sample chromatogram of different extraction time;
Figure 10 different ultrasound condition gained test sample chromatogram;
Figure 11 different brands chromatographic column gained test sample chromatogram;
Figure 12 different column temperature gained test sample chromatogram;
Figure 13 different in flow rate gained test sample chromatogram;
Figure 14 precision test finger-print;
Figure 15 replica test finger-print;
Figure 16 Intermediate precision test finger-print;
Figure 17 stability test finger-print;
Figure 18 different instrument serviceability test finger-print;
Figure 19 chromatographic column serviceability test finger-print;
Figure 20 swelling and pain relieving patch standard finger-print;
Figure 21 eriodictyol-7-methylether reference substance typical curve;
Figure 22 isosakuranetin reference substance typical curve;
Figure 23 literature method gradient elution gained reference substance chromatogram;
Figure 24 literature method gradient elution gained sample chromatogram figure;
Figure 25 optimization method gradient elution of the present invention gained reference substance chromatogram;
Figure 26 optimization method gradient elution of the present invention gained sample chromatogram figure;
Figure 27 mobile phase is acetonitrile-0.1% formic acid gained sample chromatogram figure;
Figure 28 mobile phase is acetonitrile-0.2% formic acid gained sample chromatogram figure.
Embodiment
Embodiments of the invention are described below in detail.Being exemplary below by the embodiment be described with reference to the drawings, only for explaining the present invention, and can not limitation of the present invention being interpreted as.
Embodiment 1 finger-print is set up
1.1 instrument
Agilent1260 high performance liquid chromatograph, Shimadzu LC-20AT high performance liquid chromatograph, chromatographic column (AgilentZORBAXSB-C18, 4.6 × 250mm, 5 μm, S/N:USCL060354), chromatographic column (AgilentZORBAXSB-C18, 4.6 × 250mm, 5 μm, S/N:USCL058060), chromatographic column (AgilentZORBAXSB-C18, 4.6 × 250mm, 5 μm, S/N:USCL059648), chromatographic column (PhenomenexGemiriC18, 4.6 × 250mm, 5 μm, S/N:696685-14), chromatographic column (InertsilODS-3C18, 4.6 × 250mm, 5 μm, S/N:1A7143425).Analytical balance AB204-L (METTLERTOLEDO), analytical balance XP-6 (METTLERTOLEDO), LQ-500 ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.).
1.2 reagent
Formic acid (AR, Chemical Reagent Co., Ltd., Sinopharm Group), ethanol (AR, Beijing Chemical Plant), acetonitrile (HPLC, Honeywell), methyl alcohol (HPLC, Honeywell).
Reference substance: eriodictyol-7-methylether reference substance, purity 90.0%, self-control; Isosakuranetin reference substance, purity 93.5%, ChromaDex.
1.3 detect sample
Swelling and pain relieving pastes, and lot number 1505048, is provided by Shanxi Yabao Pharmaceutical Group Corp..
1.4 method of testing
Measure according to high performance liquid chromatography (Chinese Pharmacopoeia version in 2015 four general rules 0512).
Cancel swollen anodyne to paste, test according to following methods.
Eriodictyol-7-methylether is got in the preparation of reference substance solution, isosakuranetin reference substance is appropriate, accurately weighed, adds 80% ethanol and makes the solution of every 1ml containing eriodictyol-7-methylether 40 μ g, isosakuranetin 20 μ g, shake up, to obtain final product.
The preparation of need testing solution is got it filled subsides, pulverizes, crosses No. three pharmacopeia sieves.Get powder and be about 2g, accurately weighed, put in tool plug conical flask, precision adds 80% ethanol 100ml, close plug, weighed weight, ultrasonic process (power 500W, frequency 40kHz) 30 minutes, take out, put to room temperature, supply the weight of less loss with 80% ethanol, shake up, filter, precision measures subsequent filtrate 50ml, evaporate to dryness, residue adds 80% ethanol and dissolves, and is transferred in 10ml measuring bottle, and be diluted to scale, shake up, to obtain final product.
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent (AgilentZORBAXSB-C18 chromatographic column, column length is 250mm, internal diameter is 4.6mm, S/N:USCL059648), take acetonitrile as mobile phase A, 0.2% formic acid is Mobile phase B, and according to the form below carries out gradient elution:
Flow velocity 1.0ml/min; Determined wavelength is 290nm; Column temperature is 35 DEG C.Theoretical cam curve should be not less than 50000 in eriodictyol-7-methylether.
Determination method is accurate respectively draws reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, records the chromatographic peak in 110 minutes, to obtain final product.
Accompanying drawing 1 is reference substance finger-print, and wherein peak 8 is eriodictyol-7-methylether, and peak 11 is isosakuranetin.In the visible test sample chromatogram of accompanying drawing 2, present the chromatographic peak consistent with eriodictyol-7-methylether reference substance chromatographic peak retention time, and 11 characteristic peaks corresponding with reference fingerprint should be presented.
Embodiment 2 optimum chromatogram condition is investigated
Instrument and reagent are with embodiment 1.
The selection of 2.1 determined wavelength
Get eriodictyol-7-methylether reference substance solution, in the interscan of 200 ~ 600nm scope, result has absorption maximum at 288nm place, the results are shown in Figure 3.
Get isosakuranetin reference substance solution, in the interscan of 200 ~ 600nm scope, result has absorption maximum at 292nm place, the results are shown in Figure 4.
Cancel swollen pain relieving patch need testing solution, in the interscan of 200 ~ 600nm scope, result has absorption maximum at 293nm and 329nm place, the results are shown in Figure 5.
Because two assay composition eriodictyol-7-methylethers and isosakuranetin all have absorption maximum near 290nm, and 290nm place chromatographic peak abundant information, therefore select 290nm as best detection wavelength.
The preparation of 2.2 need testing solutions is investigated
The preparation of reference substance solution, chromatographic condition and determination method are with embodiment 1.
(1) Extraction solvent concentration is investigated
Detect medicine and paste lot number 1505205, pulverize, cross No. three pharmacopeia sieves.Get 12 parts, powder, every part of about 2g, accurately weighed, put in tool plug conical flask, be divided into six groups, precision adds methyl alcohol, 95% ethanol, 80% ethanol, 60% ethanol, 30% ethanol, each 150ml of water respectively, close plug, weighed weight, ultrasonic process (power 500W, frequency 40kHz) 30 minutes, take out, put to room temperature, supply the weight of less loss respectively with coordinative solvent, shake up, filter, precision measures subsequent filtrate 75ml, evaporate to dryness, and residue adds 80% ethanol and dissolves, be transferred in 10ml measuring bottle, and be diluted to scale, shake up, to obtain final product.
Measurement result in table 1, table 2, Fig. 6.
Table 1 variable concentrations Extraction solvent gained chromatographic peak area
Table 2 variable concentrations Extraction solvent extraction ratio
Above result shows, when 80% ethanol is solvent extraction, to obtain finger-print quantity of information more, peak response value is comparatively large, and extraction ratio is the highest, therefore preferably 80% ethanol is Extraction solvent.
(2) solvent load is investigated
Get and detect medicine subsides (lot number 1505205), pulverize, cross No. three pharmacopeia sieves.Get 8 parts, powder, every part of about 2g, accurately weighed, put in tool plug conical flask, be divided into four groups, precision adds 80% ethanol 50ml respectively, 100ml, 150ml, 200ml, close plug, weighed weight, ultrasonic process (power 500W respectively, frequency 40kHz) 30 minutes, take out, put to room temperature, the weight of less loss is supplied respectively with 80% ethanol, shake up, filter, precision measures subsequent filtrate 25ml respectively, 50ml, 75ml, 100ml, evaporate to dryness, residue adds 80% ethanol and dissolves, be transferred in 10ml measuring bottle, and be diluted to scale, shake up, filter, accurate absorption subsequent filtrate 10 μ l, injection liquid chromatography, measure, calculate, obtain.The results are shown in Table 3, table 4, Fig. 7.
Table 3 different solvents consumption gained chromatographic peak area
Table 4 different solvents consumption extraction ratio
Above result shows, what 80% ethanol 100ml extraction obtained contains much information, and peak response value is high, and extraction ratio is the highest, therefore determines that preferred Extraction solvent consumption is 100ml.
(3) extracting mode is investigated
Get and detect medicine subsides (lot number 1505205), pulverize, cross No. three pharmacopeia sieves.Get 4 parts, powder, every part of about 2g, accurately weighed, put in tool plug conical flask, precision adds 80% ethanol 100ml respectively, close plug, and weighed weight is divided into two groups, and first group adds hot reflux 30 minutes; Second group is extracted with ultrasonic process (power 500W, frequency 40kHz), takes out, put to room temperature for 30 minutes, supply the weight of less loss respectively with 80% ethanol, shake up, filter, precision measures subsequent filtrate 50ml, evaporate to dryness, and residue adds 80% ethanol and dissolves, be transferred in 10ml measuring bottle, and be diluted to scale, shake up, filter, accurate absorption subsequent filtrate 10 μ l, injection liquid chromatography, measure, calculate, to obtain final product.The results are shown in Table 5, table 6, Fig. 8.
Table 5 different extracting mode gained chromatographic peak area
The different extracting mode extraction ratio of table 6
Above result shows, ultrasonic extraction and refluxing extraction result basically identical, little on extraction efficiency impact.
(4) extraction time is investigated
Get and detect medicine subsides (lot number 1505205), pulverize, cross No. three pharmacopeia sieves.Get 6 parts, powder, every part of about 2g, accurately weighed, put in tool plug conical flask, be divided into three groups, precision adds 80% ethanol 100ml respectively, close plug, weighed weight, ultrasonic process (power 500W respectively, frequency 40kHz) first group 10 minutes, second group 30 minutes, 3rd group 50 minutes, take out, put to room temperature, the weight of less loss is supplied respectively with 80% ethanol, shake up, filter, precision measures subsequent filtrate 50ml, evaporate to dryness, residue adds 80% ethanol and dissolves, be transferred in 10ml measuring bottle, and be diluted to scale, shake up, filter, accurate absorption subsequent filtrate 10 μ l, injection liquid chromatography, measure, calculate, obtain.The results are shown in Table 7, table 8, Fig. 9.
Table 7 gained chromatographic peak area of different extraction time
Table 8 extraction ratio of different extraction time
Above result shows, ultrasonic extraction can obtain maximum quantity of information in 30 minutes, and peak response value is also comparatively large, and extracts the most complete, therefore preferred ultrasonic time 30 minutes.
(5) ultrasonic power, frequency are investigated
Get and detect medicine subsides (lot number 1505205), pulverize, cross No. three pharmacopeia sieves.Get 6 parts, powder, every part of about 2g, accurately weighed, put in tool plug conical flask, be divided into three groups, precision adds 80% ethanol 100ml respectively, close plug, weighed weight, ultrasonic extraction 30 minutes, first group of ultrasonic power 500W/ frequency 40kHz, second group of ultrasonic power 250W/ frequency 40kHz, 3rd group of ultrasonic power 50W/ frequency 53kHz, take out, put to room temperature, the weight of less loss is supplied respectively with 80% ethanol, shake up, filter, precision measures subsequent filtrate 50ml respectively, evaporate to dryness, residue adds 80% ethanol and dissolves, be transferred in 10ml measuring bottle, and be diluted to scale, shake up, filter, accurate absorption subsequent filtrate 10 μ l, injection liquid chromatography, measure, calculate, obtain.The results are shown in Table 9, table 10, Figure 10.
Table 9 different ultrasound condition gained chromatographic peak area
The different ultrasound condition extraction ratio of table 10
Above result shows, ultrasonic power and frequency affect less on this product extraction ratio, but comparatively speaking, selects when ultrasonic power 500W, frequency 40kHz and can obtain best Chromatographic information.
In the chromatogram obtain above preparation method, main chromatographic peak carries out integration, contrasts the peak area under each retention time, and select chromatographic peak abundant information, extraction efficiency is high, the method that total peak area is larger.
Comprehensive above-mentioned experimental result, finally can determine that preferred test sample preparation method provided by the invention is: subsides of getting it filled, and pulverizes, and crosses No. three pharmacopeia sieves.Get powder and be about 2g, accurately weighed, put in tool plug conical flask, precision adds 80% ethanol 100ml, close plug, weighed weight, ultrasonic process (power 500W, frequency 40kHz) 30 minutes, take out, put to room temperature, supply the weight of less loss with 80% ethanol, shake up, filter, precision measures subsequent filtrate 50ml, evaporate to dryness, residue adds 80% ethanol and dissolves, and is transferred in 10ml measuring bottle, and be diluted to scale, shake up, to obtain final product.
The selection of 2.3 chromatographic columns
The filler of chromatographic column and type of feed may have an impact to drug chemistry component separating effect.The C18 chromatographic column (5 μm, 250 × 4.6mm) of inventor to Agilent, Shimadzu and Phenomenex tri-different brands is investigated, and other condition is with embodiment 1.Result as shown in Figure 11.The separating effect of AgilentZORBAXSB-C18 post and peak type are all better than all the other two kinds, therefore select AgilentZORBAXSB-C18 chromatographic column.
The selection of 2.4 column temperatures
In chromatogram finger print measuring, column temperature often affects separating effect, and inventor is respectively under 30 DEG C, 35 DEG C, 40 DEG C three column temperature conditions, and analyze the finger-print of detumescence pain relieving plaster need testing solution, other condition is with embodiment 1.The results are shown in accompanying drawing 11.Show that the separating effect of each chromatographic peak 35 DEG C time is relatively good, therefore selection 35 DEG C is suitable column temperature.
2.5 flow velocitys are investigated
In chromatogram finger print measuring, flow velocity can affect separating effect to a certain extent, this test is respectively under 0.8ml/min, 1.0ml/min, 1.2ml/min tri-flow conditions, and analyze the finger-print of detumescence pain relieving plaster need testing solution, other condition is with embodiment 1.The results are shown in accompanying drawing 13.Show that the separating effect of each chromatographic peak when 1.0ml/min is relatively good, therefore select 1.0ml/min to be suitable flow velocity.
Embodiment 3 fingerprint spectrum method is verified
3.1 blank test
Get 80% ethanol, by embodiment 1 chromatographic condition direct injected, record the chromatogram of 110 minutes, result shows that system does not exist residual and interference.
3.2 precision test
Get and detect medicine subsides (lot number 1505205), need testing solution is prepared into according to embodiment 1 method, according to embodiment 1 chromatographic condition direct injected 6 times, with No. 8 peak eriodictyol-7-methylethers for object of reference, calculate relative retention time and the relative peak area of each main chromatographic peak.The relative retention time that result shows 11 total peaks is stablized, and RSD% is all less than 1%, relative peak area relative constancy, and RSD% is all less than 2%; Show that the precision of this instrument is good.The results are shown in Table 11, table 12, Figure 14.
Table 11 precision test relative retention time
Table 12 precision test relative peak area
3.3 replica test
Get same batch of swelling and pain relieving patch, be prepared into six parts of need testing solutions according to embodiment 1 method, detect according to embodiment 1 chromatographic condition, with No. 8 peak eriodictyol-7-methylethers for object of reference, calculate relative retention time and the relative peak area of each main chromatographic peak.The relative retention time that result shows 11 total peaks is stablized, and RSD% is all less than 1%, relative peak area relative constancy, and RSD% is all less than 3%; Show that this method repeatability is good.The results are shown in Table 13, table 14, Figure 15.
Table 13 replica test relative retention time
Table 14 replica test relative peak area
3.4 Intermediate precision
Get same batch of swelling and pain relieving patch, be prepared into six parts of need testing solutions according to embodiment 1 method, detect according to embodiment 1 chromatographic condition, with No. 8 peak eriodictyol-7-methylethers for object of reference, calculate relative retention time and the relative peak area of each main chromatographic peak.The relative retention time that result shows 11 total peaks is stablized, and RSD% is all less than 1%, relative peak area relative constancy, and RSD% is all less than 1%; Show that this method repeatability is good.The results are shown in Table 15, table 16, Figure 16.
Table 15 Intermediate precision test relative retention time
Table 16 Intermediate precision test relative peak area
3.5 stability test
Get medicine to be measured to paste, lot number 1505205, need testing solution is prepared into according to embodiment 1 method, according to embodiment 1 chromatographic condition respectively at 0,4,8,12,18,24,32,40,48,60,72 hour sample detection, with No. 8 peak eriodictyol-7-methylethers for object of reference, calculate relative retention time and the relative peak area of each main chromatographic peak.The relative retention time that result shows 11 total peaks is stablized, and RSD is all less than 1%, relative peak area relative constancy, and RSD is all less than 4%; Show that the need testing solution of this product is at least stable in 72 hours.The results are shown in Table 17, table 18, Figure 17.
Table 17 stability test relative retention time
Table 18 stability test relative peak area
3.6 durability researchs
3.6.1 different model HPLC durability
By embodiment 1 finger print measuring method, use the instrument of different model to paste (lot number 1505205) to same batch of swelling and pain relieving to detect, with Agilent1260 instrument collection of illustrative plates for reference, to calculate the similarity of collection of illustrative plates between different instrument with reference to collection of illustrative plates.Result shows, between different instrument, measurement result is roughly the same, and fingerprint similarity is all greater than 0.99, illustrates that the model of high performance liquid chromatograph is little on the impact of this fingerprint spectrum method.The results are shown in Table 19, Figure 18.
Table 19 different instrument serviceability test result
3.6.2 different batches chromatographic column durability
By embodiment 1 finger print measuring method, use different batches chromatographic column to the same batch of capable detection of swelling and pain relieving exchange premium, with one of them batch-wise chromatography post (sequence number USCL059648) collection of illustrative plates for reference, calculate similarity.Result shows, between different batches chromatographic column, measurement result is roughly the same, and fingerprint similarity is all greater than 0.99, and the batch little on this method impact of chromatographic column is described.The results are shown in Table 20, Figure 19.
Table 20 chromatographic column serviceability test result
The foundation of embodiment 4 standard finger-print
The lot number produced Shanxi Yabao Pharmaceutical Group Corp. is 1504288, 1505048, 1505201, 1505202, 1505203, 1505204, 1505205, 1505206, 1505207, 1505208, 1505209, 1505210, 1505211, 1505212, 1505213, 1505214, 1505215, 1505216, 1505219, 1505221, 1505223, the swelling and pain relieving of 1505224 pastes, amount to 22 batch sample, detect by the finger print measuring method of embodiment 1 respectively, adopt Chinese Pharmacopoeia Commission " similarity evaluation " (2012.0 version) matching to generate swelling and pain relieving and paste standard finger-print, see Figure 20.
Embodiment 5 swelling and pain relieving patch active constituent content measuring
According to the method for embodiment 1, measure the content of eriodictyol-7-methyl ether and isosakuranetin in 22 batches of swelling and pain relievings subsides, the results are shown in Table 21.
Table 21 sample size measurement result
Result: during above 22 batches of swelling and pain relievings paste, eriodictyol-7-methyl ether average content is 0.386mg/g (being equivalent to 0.1544mg/ paste), isosakuranetin average content is 0.173mg/g (being equivalent to 0.0692mg/ paste).
Embodiment 6 Method validation
6.1 specificity tests
Only have sand sagebrush (Artemisia filifolia) medicinal material simply because swelling and pain relieving pastes the subsides of spongy medicine, and there is no auxiliary material, therefore there is not the interference of other compositions; Get the sub-medicinal material of sand sagebrush (Artemisia filifolia), make medicinal material test liquid by test sample preparation method; Get reference substance solution, need testing solution, medicinal material test liquid and solvent (that is: 80% ethanol), respectively sample introduction 10 μ l, chromatographic condition, with embodiment 1, records chromatogram.Result shows, need testing solution and sand sagebrush (Artemisia filifolia) medicinal material test liquid present chromatographic peak on the position corresponding to reference substance solution chromatogram; And there is not chromatographic peak in solvent chromatogram on relevant position.Show that the method is noiseless, measure eriodictyol-7-methyl ether and the content with isosakuranetin in this product with this law and there is specificity.
6.2 Peak homogeneity
By embodiment 1 method, prepare need testing solution and reference substance solution, adopt PDA detecting device to carry out full wavelength detecting, calculate peak purity, result is that impurity peaks does not detect; The peak purity index of eriodictyol-7-methylether and isosakuranetin, all more than 0.999, proves that the peak purity of eriodictyol-7-methylether and isosakuranetin chromatographic peak under this chromatographic condition all meets the requirements.
6.3 system suitability
By embodiment 1 method, prepare need testing solution and reference substance solution, respectively sample introduction 6 times, the peak area of record eriodictyol-7-methylether and isosakuranetin, calculate relative standard deviation, the results are shown in Table 22, table 23.
Table 22 eriodictyol-7-methylether system suitability result
Table 23 isosakuranetin system suitability result
As a result, under above-mentioned chromatographic condition, in eriodictyol-7-methylether and isosakuranetin chromatographic peak and sample, adjacent chromatographic peak all can reach baseline separation, and degree of separation is greater than 1.5; Number of theoretical plate reaches more than 50000 in eriodictyol-7-methylether peak; Tailing factor is all between 0.95 ~ 1.15; Peak area RSD% is all less than 1.0%.Above result shows, eriodictyol-7-methylether and isosakuranetin are under above-mentioned chromatographic condition, and well, system suitability meets the requirements for the precision of instrument and repeatability.
6.4 linear relationships are investigated
(1) eriodictyol-7-methylether linear test
Get eriodictyol-7-methylether reference substance 6.947mg (purity 90.0%), accurately weighed, put in 25ml measuring bottle, add 80% ethanol to scale, shake up, obtain the storing solution of 0.2501mg/ml.
Accurate draw above-mentioned solution 0.5,1.0,1.0,2.0,4.0ml, be placed in 10ml, 10ml, 5ml, 5ml, 5ml measuring bottle respectively, scale is diluted to 80% ethanol, shake up, be mixed with the reference substance solution of 12.5046,25.0092,50.0184,100.0368 and 200.0736 μ g/ml, shake up.The each 10 μ l of the above-mentioned solution of accurate absorption respectively, injection liquid chromatography, measures by embodiment 1 chromatographic condition.With sample size (μ g) for horizontal ordinate, peak area is ordinate mapping, drawing standard curve.In table 24, Figure 21.
Table 24 eriodictyol-7-methylether reference substance linear test result
Result: regression equation y=3844.11x+16.60, coefficient R 2=1.00, shows that eriodictyol-7-methylether sample size is good in 0.1250 μ g ~ 2.0007 μ g scope internal linear relation.
(2) isosakuranetin linear test
Get isosakuranetin reference substance 3.417mg (purity 93.5%), accurately weighed, put in 25ml measuring bottle, add 80% ethanol to scale, shake up, obtain the storing solution of 0.1278mg/ml.
Accurate draw above-mentioned solution 0.5,1.0,1.0,2.0,4.0ml, be placed in 10ml, 10ml, 5ml, 5ml, 5ml, measuring bottle respectively, scale is diluted to 80% ethanol, shake up, be mixed with the reference substance solution of 6.3898,12.7796,25.5592,51.1183 and 102.2366 μ g/ml, shake up.The each 10 μ l of the above-mentioned solution of accurate absorption respectively, injection liquid chromatography, measures by embodiment 1 chromatographic condition.With sample size (μ g) for horizontal ordinate, peak area is ordinate mapping, drawing standard curve.In table 25, Figure 22.
Table 25 isosakuranetin reference substance linear test result
Result: regression equation y=3524.12x-2.31, coefficient R 2=1.00, shows that isosakuranetin sample size is good in 0.0639 μ g ~ 1.0224 μ g scope internal linear relation.
6.5 solution stability testing
Get medicine to be measured and paste (lot number 1505048), by the operation of embodiment 1 method, respectively at 0,4,8,12,18,24,32,40,48,60,72 hour sample detection, the peak area of record eriodictyol-7-methylether and isosakuranetin.Measurement result is in table 26.
Table 26 stability test result
Result shows, RSD%<1.0%, in need testing solution eriodictyol-7-methyl ether and isosakuranetin good at 72 hours internal stabilities.
6.6 precision test
6.6.1 replica test
Get medicine to be measured and paste (lot number 1505048), operated by embodiment 1 determination method by experimenter A, prepare six parts of need testing solutions, sample detection.The results are shown in Table 27.
Table 27 replica test result
Result: the average content of this product eriodictyol-7-methylether is that 0.331mg/g, RSD% are less than 2.0%; The average content of isosakuranetin is that 0.136mg/g, RSD% are less than 2.0%.Result shows, the repeatability of this method is good.
6.6.2 Intermediate precision
Select the time of different mensuration, different high performance liquid chromatographs, different experimenter (B); operate by embodiment 1 determination method; measure the content of eriodictyol-7-methyl ether and isosakuranetin in same batch sample, the measurement result of comprehensive A and B, in table 28, table 29.
Table 28 eriodictyol-7-methylether Intermediate precision result
Table 29 isosakuranetin Intermediate precision result
As a result, in the sample that measures of A and B, eriodictyol-7-methyl ether average content is 0.326mg/g, the average content of isosakuranetin is that 0.138mg/g, RSD% are all less than 3.0%.Result shows, this method Intermediate precision is good.
6.7 accuracy test
Adopt application of sample absorption method accuracy of measurement.Precision takes eriodictyol-7-methylether reference substance 3.893mg, puts in 10ml measuring bottle, adds 80% appropriate amount of ethanol and makes dissolving and be diluted to scale, shake up, be mixed with the reference substance solution of 0.3504mg/ml; Precision takes isosakuranetin reference substance 1.512mg, puts in 10ml measuring bottle, adds 80% appropriate amount of ethanol and makes dissolving and be diluted to scale, shake up, be mixed with the reference substance solution of 0.1414mg/ml.
Get medicine to be measured and paste (lot number 1505048), pulverize, cross No. three pharmacopeia sieves, get 9 parts, powder, each about 1g, accurately weighed, split in tool plug conical flask, be divided into 3 groups, often organize 3 parts, each group respectively precision add above-mentioned two reference substance solution each 0.8, 1.0, 1.2ml, solvent evaporated, precision adds 80% ethanol 100ml respectively, weighed weight, ultrasonic process (power 500W, frequency 40kHz) 30 minutes, take out, put to room temperature, the weight of less loss is supplied with 80% ethanol, shake up, filter, precision measures subsequent filtrate 50ml, evaporate to dryness, residue adds 80% ethanol and dissolves, be transferred in 10ml measuring bottle, and be diluted to scale, shake up, filter with miillpore filter (0.45 μm), get subsequent filtrate, obtain.
Respectively accurately draw each 10 μ l of above-mentioned solution, injection liquid chromatography, measure by embodiment 1 chromatographic condition, the results are shown in Table 30, table 31.
During this batch of swelling and pain relieving pastes, the content of eriodictyol-7-methyl ether is 0.331mg/g, isosakuranetin content 0.136mg/g.
Note: the recovery (%)=(recording the amount in total amount-sample)/contrast addition × 100%.
Table 30 eriodictyol-7-methylether application of sample recovery test result
Table 31 isosakuranetin application of sample recovery test result
Result: the recovery of eriodictyol-7-methylether and isosakuranetin is all between 95% ~ 105%.Result shows, this method average recovery is good.
6.8 scopes are investigated
(1) high limit adopts application of sample absorption method, and precision takes isosakuranetin reference substance 7.042mg, puts in 50ml measuring bottle, adds 80% appropriate amount of ethanol and makes dissolving and be diluted to scale, shake up, and is mixed with the isosakuranetin reference substance solution that concentration is 0.131685mg/ml.Get medicine to be measured and paste (lot number 1505048), pulverize, cross No. three pharmacopeia sieves, get 6 parts, powder, respectively about 5g, accurately weighed, puts in tool plug conical flask respectively; Respectively precision take and add eriodictyol-7-methylether reference substance 1.982,1.986,1.909,1.897,1.994,1.861mg; Precision measures and adds above-mentioned isosakuranetin reference substance solution 5ml respectively; Solvent evaporated, precision adds 80% ethanol 100ml respectively, weighed weight, ultrasonic process (power 500W, frequency 40kHz) 30 minutes, take out, put to room temperature, supply the weight of less loss with 80% ethanol, shake up, filter, precision measures subsequent filtrate 50ml, evaporate to dryness, and residue adds 80% ethanol and dissolves, be transferred in 10ml measuring bottle, and be diluted to scale, shake up, filter with miillpore filter (0.45 μm), get subsequent filtrate, to obtain final product.
Respectively accurately draw each 10 μ l of above-mentioned solution, injection liquid chromatography, measure by embodiment 1 chromatographic condition, the results are shown in Table 32, table 33.
During this batch of swelling and pain relieving pastes, the content of eriodictyol-7-methyl ether is 0.331mg/g, isosakuranetin content 0.136mg/g.
Note: the recovery (%)=(recording the amount in total amount-sample)/contrast addition × 100%.
Table 32 eriodictyol-7-methylether high limit investigates test findings
Table 33 isosakuranetin high limit investigates test findings
Result: the recovery of eriodictyol-7-methylether and isosakuranetin is all between 95% ~ 105%.
(2) lower bound adopts application of sample absorption method, precision takes eriodictyol-7-methylether reference substance 1.858mg, puts in 10ml measuring bottle, adds 80% appropriate amount of ethanol and makes dissolving and be diluted to scale, shake up, be mixed with the eriodictyol-7-methylether reference substance solution that concentration is 0.1672mg/ml; Precision takes isosakuranetin reference substance 1.756mg, puts in 50ml measuring bottle, adds 80% appropriate amount of ethanol and makes dissolving and be diluted to scale, shake up, and is mixed with the isosakuranetin reference substance solution that concentration is 0.0328mg/ml.Get medicine to be measured and paste (lot number 1505048), pulverize, cross No. three pharmacopeia sieves, get 6 parts, powder, respectively about 0.5g, accurately weighed, puts in tool plug conical flask respectively; Precision measures and adds above-mentioned eriodictyol-7-methylether reference substance solution 1ml, isosakuranetin reference substance solution 2ml respectively; Solvent evaporated, precision adds 80% ethanol 100ml respectively, weighed weight, ultrasonic process (power 500W, frequency 40kHz) 30 minutes, take out, put to room temperature, supply the weight of less loss with 80% ethanol, shake up, filter, precision measures subsequent filtrate 50ml, evaporate to dryness, and residue adds 80% ethanol and dissolves, be transferred in 10ml measuring bottle, and be diluted to scale, shake up, filter with miillpore filter (0.45 μm), get subsequent filtrate, to obtain final product.
Respectively accurately draw each 10 μ l of above-mentioned solution, injection liquid chromatography, measure by embodiment 1 chromatographic condition, the results are shown in Table 34, table 35.
During this batch of swelling and pain relieving pastes, the content of eriodictyol-7-methyl ether is 0.331mg/g, isosakuranetin content 0.136mg/g.
Note: the recovery (%)=(recording the amount in total amount-sample)/contrast addition × 100%.
Table 34 eriodictyol-7-methylether lower bound investigates test findings
Table 35 isosakuranetin lower bound investigates test findings
Result: the recovery of eriodictyol-7-methylether and isosakuranetin is all between 95% ~ 105%.
Scope is investigated result and is shown; during swelling and pain relieving pastes, the content of eriodictyol-7-methyl ether is at the content of 0.16mg/g ~ 1.77mg/g (being equivalent to 0.064mg/ subsides ~ 0.708mg/ paste), isosakuranetin in 0.07mg/g ~ 0.69mg/g (being equivalent to 0.028mg/ subsides ~ 0.276mg/ paste) scope, and accuracy of measurement is good.
6.9 serviceability test
Investigate different column temperature, flow velocity, wavelength, acid concentration and different manufacturers chromatographic column to the durability of this chromatographic condition.Get medicine to be measured and paste (lot number 1505048), pulverize, cross No. three pharmacopeia sieves, get powder and be about 2g, accurately weighed, operate by embodiment 1 method.The results are shown in Table 36, table 37.
Table 36 eriodictyol-7-methylether serviceability test result
Table 37 isosakuranetin serviceability test result
Result: under fixed chromatographic condition; record medicine to be measured and paste (lot number: 1505048), eriodictyol-7-methyl ether content is 0.331mg/g, isosakuranetin content is 0.136mg/g; change column temperature, flow velocity, wavelength, acid concentration and different manufacturers chromatographic column, the RSD% of the content measured under each investigation item is all less than 4.0%.Result shows, the good tolerance of this chromatographic condition.
Embodiment 7 and literature method comparative studies
The method that document " saltliving wormwood leaf seed antiasthmatic activity composition selection and quality research thereof " (Zhang Yuanyuan, Master degree candidate's academic dissertation, 2006) method and the embodiment of the present invention 1 provide is compared research.
7.1 mobile phase compositions and gradient list are compared:
Finger-print condition in list of references, detects the agent of detumescence pain relieving plaster.
Chromatographic column ZORBAXEclipseXDB-C18 (4.6 × 250mm, 5 μm); Determined wavelength: 288nm; Column temperature: 30 DEG C; Sample size: 10 μ L.Mobile phase: acetonitrile-0.1% aqueous formic acid; Flow velocity 1mLmin-1; Mobile phase according to the form below carries out gradient elution:
The preparation of reference substance solution: get eriodictyol-7-methylether, isosakuranetin reference substance is appropriate, accurately weighed, add 80% ethanol and make the solution of every 1ml containing eriodictyol-7-methylether 40 μ g, isosakuranetin 20 μ g, shake up, to obtain final product.
The preparation of need testing solution: get the spongy medicine of this product and paste, pulverize, crosses No. three pharmacopeia sieves.Get powder and be about 2g, accurately weighed, put in tool plug conical flask, precision adds 80% ethanol 100ml, close plug, weighed weight, ultrasonic process (power 500W, frequency 40kHz) 30 minutes, take out, put to room temperature, more weighed weight, the weight of less loss is supplied with 80% ethanol, shake up, filter, precision measures subsequent filtrate 50ml, evaporate to dryness, residue adds 80% ethanol and dissolves, and is transferred in 10ml measuring bottle, and be diluted to scale, shake up, to obtain final product.
Determination method is accurate respectively draws reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product.The results are shown in accompanying drawing 23,24.
Result shows, eriodictyol-7-methylether peak symmetrical factor 0.74; Isosakuranetin peak symmetrical factor 0.76, degree of separation 1.47, does not still reach the requirement of " degree of separation between component to be measured and adjacent concurrent should be greater than 1.5 " required by pharmacopeia.(D of the Pharmacopoeia of the People's Republic of China (version in a 2010) annex VI).Because these two compositions will be listed in containing surveying index, so need to be optimized chromatographic condition.The chromatographic peak of 15.183min is comparatively large in addition, and account for 57.76% of total peak area, but peak type is very poor, degree of separation is bad, needs to optimize chromatographic condition to improve peak type and degree of separation.
According to the chromatographic condition after optimization provided by the present invention, the agent of detumescence pain relieving plaster is detected again.
Take octadecylsilane chemically bonded silica as filling agent (chromatographic column AgilentZORBAXSB-C18250mm × 4.6mm, 5 μm), take acetonitrile as mobile phase A, with 0.2% formic acid solution for Mobile phase B, the regulation according to the form below carries out gradient elution; Flow velocity 1.0ml/min; Determined wavelength is 290nm; Column temperature is 35 DEG C.Theoretical cam curve should be not less than 50000 in eriodictyol-7-methylether.
Eriodictyol-7-methylether is got in the preparation of reference substance solution, isosakuranetin reference substance is appropriate, accurately weighed, adds 80% ethanol and makes the solution of every 1ml containing eriodictyol-7-methylether 40 μ g, isosakuranetin 20 μ g, shake up, to obtain final product.
The preparation of need testing solution is got the spongy medicine of this product and is pasted, and pulverizes, and crosses No. three pharmacopeia sieves.Get powder and be about 2g, accurately weighed, put in tool plug conical flask, precision adds 80% ethanol 100ml, close plug, weighed weight, ultrasonic process (power 500W, frequency 40kHz) 30 minutes, take out, put to room temperature, more weighed weight, the weight of less loss is supplied with 80% ethanol, shake up, filter, precision measures subsequent filtrate 50ml, evaporate to dryness, residue adds 80% ethanol and dissolves, and is transferred in 10ml measuring bottle, and be diluted to scale, shake up, to obtain final product.
Determination method is accurate respectively draws reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product.See accompanying drawing 25,26.
Result shows, eriodictyol-7-methylether symmetrical factor 0.84; Isosakuranetin symmetrical factor 0.84, degree of separation 1.59, higher than standards of pharmacopoeia.Compared with the collection of illustrative plates of literature method, the peak type of these two compositions all improves significantly, and degree of separation also can reach assay requirement.In the collection of illustrative plates of literature method in addition, the chromatographic peak retention time in the figure of 15.183min is 33.013min, all reach separating effect, and peak type also improves significantly with the chromatographic peak of front and back.Chromatographic peak whole separation effect is better than literature method, and baseline is also more steady.
7.2 mobile phase acid concentrations compare:
In list of references, mobile phase composition, compares the difference of mobile phase acetonitrile-0.1% formic acid and acetonitrile-0.2% formic acid, the results are shown in accompanying drawing 27,28.
Result shows, and in the chromatogram of 0.1% formic acid, retention time is the chromatographic peak of 32.602min, and there is an acromion, peak type is bad, and in the chromatogram of 0.2% formic acid, this chromatogram peak-to-peak type has clear improvement, and symmetrical factor becomes 0.93 from 1.18; The symmetrical factor at the peak of 37.741min also becomes 1.07 from 1.30 in addition, and peak type improves.Therefore select acetonitrile-0.2% formic acid to be more conducive to detecting as mobile phase.
7.3 chromatographic column models compare:
Due to ZORBAXEclipseXDB-C18 (4.6 × 250mm, 5 μm) compare, ZORBAXSB-C18 is separated under lower pH condition, there is longer chromatographic column life-span and the reappearance of Geng Jia, more meet the mobile phase environment finally determined, therefore can be preferably ZORBAXSB-C18 (4.6 × 250mm, 5 μm) chromatographic column.
Although illustrate and describe embodiments of the invention, for the ordinary skill in the art, be appreciated that and can carry out multiple change, amendment, replacement and modification to these embodiments without departing from the principles and spirit of the present invention, scope of the present invention is by claims and equivalents thereof.
Claims (18)
1. an assay method for swelling and pain relieving patch effective constituent, is characterized in that, comprises the following steps:
(1) preparation of reference substance solution
Take eriodictyol-7-methylether, isosakuranetin reference substance, add ethanol obtained mixing reference substance solution;
(2) preparation of need testing solution
The medicine cancelling swollen pain relieving plaster pastes, and pulverizes, sieves, add ethanolic solution, extracts 20-50min, puts to room temperature, add the weight that less loss supplied by ethanol, shake up, and filters, gets filtrate, evaporate to dryness, and residue adds ethanol and dissolves, and shakes up and obtains need testing solution;
(3) determination method
Draw mixing reference substance solution and need testing solution, inject high performance liquid chromatograph, obtain chromatogram,
Wherein, the chromatographic condition of described high performance liquid chromatograph is as follows:
Chromatographic column take octadecylsilane chemically bonded silica as filling agent,
Mobile phase: acetonitrile-0.2% formic acid is mobile phase, and carry out gradient elution, Gradient program is as following table
The flow velocity of mobile phase is 0.8-1.2ml/min, column temperature 30-40 DEG C,
Determined wavelength: 290 ± 3nm.
2. assay method according to claim 1, is characterized in that, wherein, in step (1), in mixing reference substance solution, the concentration of eriodictyol-7-methyl ether and isosakuranetin is respectively 40 μ g/ml and 20 μ g/ml.
3. assay method according to claim 1, is characterized in that, the concentration of ethanol is 55 ~ 85%.
4. assay method according to claim 3, is characterized in that, the concentration of ethanol is 80%.
5. assay method according to claim 1, is characterized in that, medicine described in step (2) pastes and add ethanol after pulverizing, sieving, and makes contained medicinal powder in every 100ml Extraction solvent be 1g ~ 5g.
6. assay method according to claim 1, is characterized in that, in described step (2), extracting method is ultrasonic extraction or heating and refluxing extraction.
7. assay method according to claim 6, is characterized in that, in described step (2), extracting method is ultrasonic extraction 25 ~ 35min.
8. assay method according to claim 7, is characterized in that, ultrasonic power is 500W, and frequency is 40kHz.
9. assay method according to claim 1, is characterized in that, chromatographic column is AgilentZORBAXSB-C
18post.
10. assay method according to claim 1, is characterized in that, column temperature preferably 35 DEG C.
11. assay methods according to claim 1, is characterized in that, the preferred 1ml/min of flow velocity of mobile phase.
12. assay methods according to claim 1, is characterized in that, determined wavelength is 290nm.
The method for building up of 13. 1 kinds of swelling and pain relieving patch high-efficiency liquid-phase fingerprints, is characterized in that comprising the following steps:
(1) preparation of reference substance solution
Take eriodictyol-7-methylether, isosakuranetin reference substance, add the ethanol obtained mixing reference substance solution that concentration is 55 ~ 85%;
(2) preparation of need testing solution
The medicine cancelling swollen pain relieving plaster pastes, and pulverizes, sieves, add the ethanolic solution that concentration is 55 ~ 85%, make contained medicinal powder in every 100ml Extraction solvent be 1g ~ 5g, ultrasonic extraction or heating and refluxing extraction 20-50min, put to room temperature, add concentration be 55 ~ 85% ethanol supply the weight of less loss, shake up, filter, get filtrate, evaporate to dryness, residue adds the ethanol dissolving that concentration is 55 ~ 85%, shakes up and obtains need testing solution;
(3) chromatogram is obtained
Using reference substance solution as object of reference, need testing solution and reference substance solution are injected high performance liquid chromatograph, obtain chromatogram,
Wherein, the chromatographic condition of described high performance liquid chromatograph is as follows:
Chromatographic column take octadecylsilane chemically bonded silica as filling agent,
Mobile phase: acetonitrile-0.2% formic acid is mobile phase, and carry out gradient elution, Gradient program is as follows:
The flow velocity of mobile phase is 0.8-1.2ml/min, column temperature 30-40 DEG C,
Determined wavelength: 290 ± 3nm;
(4) reference fingerprint is generated:
Selecting the finger-print of many batches of qualified swelling and pain relieving patches, take eriodictyol-7-methylether as reference peak, obtains the collection of illustrative plates at 11 total peaks, and calculate by median method and generate standard control finger-print, wherein, finger-print relative retention time is:
。
14. methods according to claim 13, is characterized in that, the concentration of ethanol is 80%.
15. methods according to claim 13, is characterized in that, chromatographic column is AgilentZORBAXSB-C
18post.
16. methods according to claim 13, is characterized in that, in step (1), in mixing reference substance solution, the concentration of eriodictyol-7-methyl ether and isosakuranetin is respectively 40 μ g/ml and 20 μ g/ml.
17. methods according to claim 13, is characterized in that, determined wavelength is 290nm.
18. methods according to claim 13, is characterized in that, in described step (2), extracting method is ultrasonic extraction 25 ~ 35min.
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| 李沙沙等: "沙漠嘎子中总黄酮及圣草酚-7-甲醚、异樱花素的含量测定", 《中华中医药杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108956835A (en) * | 2017-05-19 | 2018-12-07 | 亚宝药业集团股份有限公司 | A kind of fingerprint atlas detection method of the antipyretic oral drugs of clearing |
| CN111474276A (en) * | 2020-05-08 | 2020-07-31 | 亚宝药业集团股份有限公司 | Quality control method of yang invigorating tablet preparation |
| CN114813983A (en) * | 2021-01-29 | 2022-07-29 | 武汉康乐药业股份有限公司 | Detection method of compound Chinese patent medicine containing red ginseng |
| CN113075319A (en) * | 2021-03-26 | 2021-07-06 | 北京斯利安药业有限公司 | HPLC detection method of Gentongping traditional Chinese medicine preparation |
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