CN106442846A - Detecting method of fuyankang tablet - Google Patents
Detecting method of fuyankang tablet Download PDFInfo
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- CN106442846A CN106442846A CN201610898459.7A CN201610898459A CN106442846A CN 106442846 A CN106442846 A CN 106442846A CN 201610898459 A CN201610898459 A CN 201610898459A CN 106442846 A CN106442846 A CN 106442846A
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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
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention relates to a detecting method of a fuyankang tablet, and belongs to the field of Chinese pharmaceutical manufacturing detecting. The detecting method comprises the steps of conducting thin-layer chromatographic identification of root of common peony and curcuma zedoary and measuring the content of tanshinol in a finished product through high performance liquid chromatography. The detecting method of a fuyankang tablet has the advantages of starting from a drug which has a major function of invigorating blood circulation and eliminating stasis, aiming at properties of materials containing effective constituents, taking the factor that effective constituent paeoniflorin is unstable in Chinese herbaceous peony into consideration so as to increase an identification item; taking the factor that the property of effective constituent tanshinol in the root of red-rooted salvia is stable into consideration so as to increase a content determination; adding into the identification items an identification item of curcuma zedoary which is a drug of promoting qi circulation to relieve pain, in this way, one content determination and two identifications are added to a 13-odor traditional Chinese medicine formula, and through the combination of the added one content determination and two identifications with the original one content determination and three identifications, the quality of the product is controlled effectively; being capable of meeting the requirement of producing high quality products so as to ensure that the products have the best therapeutic effects.
Description
Technical field
The invention belongs to herbal pharmaceutical detection field, and in particular to the detection method of FUYANKANG PIAN.
Background technology
FUYANKANG PIAN be with Radix Paeoniae Rubra 60g, Rhizoma Smilacis Glabrae 100g, stir-baked RHIZOMA SPARGANII with vinegar 60g, Fructus Toosendan (parched) 60g, stir-baked RHIIZOMA CURCUMAE with vinegar 60g, vinegar Rhizoma Corydalis
Rope 60g, Semen Euryales (parched) 100g, Radix Angelicae Sinensis 100g, Radix Sophorae Flavescentiss 60g, Rhizoma Cyperi (processed with vinegar) 40g, Cortex Phellodendri 60g, Radix Salviae Miltiorrhizae 100g, Rhizoma Dioscoreae 120g composition.
Above 13 taste, stir-baked RHIIZOMA CURCUMAE with vinegar, Rhizoma Dioscoreae are ground into fine powder, sieve, and remaining stir-baked RHIZOMA SPARGANII with vinegar etc. ten simply, adds water to cook three
Secondary, 2 hours for the first time, second and third time was each 1 hour, and decocting liquid is filtered, merging filtrate, is concentrated in right amount, mixes with above-mentioned powder,
Fine powder is dry, pulverize into, with sucrose, starch and Magnesium Stearate proper quantity, is made pellet, dry, be pressed into 1000 (small pieces), bag sugar
Clothing or film-coat, the weight 0.25g per piece；Or be pressed into 500 (sheets), film coating, per piece, weight 0.52g, obtains final product.
This product is coated tablet or Film coated tablets, shows yellowish-brown to sepia after removing coating；Feeble QI, mildly bitter flavor.
Radix Salviae Miltiorrhizae, Radix Paeoniae Rubra, Radix Angelicae Sinensis blood circulation promoting and blood stasis dispelling in Chinese medicine compound, Rhizoma Corydalis, Fructus Toosendan, Rhizoma Cyperi share promoting QI circulation for relieving depression pain relieving, plus
Rhizoma Sparganii, Rhizoma Curcumae strengthen promoting the circulation of QI to relieve pain and hard masses softening and resolving, Rhizoma Dioscoreae, Semen Euryaless nourishing kidney leukorrhagia stopping, Radix Sophorae Flavescentiss, Cortex Phellodendri heat clearing and damp drying, Rhizoma Smilacis Glabrae
Detoxification and promoting urination, all medicine compatibilities can reach blood circulation promoting and blood stasis dispelling, and heat-clearing and toxic substances removing, hard masses softening and resolving, the purpose of pain relieving dampness removing can diminish inflammation,
Patient's waist abdomen pain is released, reduces leucorrhea, have the curative effect for curing chronic pelvic inflammation.
3 are had to differentiate and 1 assay, which is respectively in existing FUYANKANG PIAN detection method：
Differentiate that 1, Rhizoma Corydalis control medicinal material and tetrahydropalmatine reference substance differentiate Rhizoma Corydalis in product；
Differentiate that 2, Cortex Phellodendri control medicinal material and berberine reference substance differentiate Cortex Phellodendri in product；
Differentiate that 3, Radix Salviae Miltiorrhizae control medicinal material and protocatechualdehyde reference substance differentiate Radix Salviae Miltiorrhizae in product.
Assay：Containing Radix Sophorae Flavescentiss to determine matrine content according to high performance liquid chromatography (general rule) in product.
FUYANKANG PIAN is the product that applicant researches and develops the eighties, so far the history of more than 30 year, good effect, deep by wide
The favorable comment of big patient, it has also become the gynopathic good medicine for the treatment of.But due to being limited to production technology, detection meanss etc. at that time during research and development
Technical merit, the detection method level of formulation is low, although done certain raising the end of the nineties, but on product inspection method,
The active constituent content measurings such as drug for invigorating blood circulation and eliminating stasis Radix Salviae Miltiorrhizae, Radix Paeoniae Rubra, discriminating etc. could not be brought in quality standard.But with Radix Sophorae Flavescentiss
In Radix Sophorae Flavescentiss alkali composition as assay, in Rhizoma Corydalis, Cortex Phellodendri, alkaloid is used as discriminating, and Radix Salviae Miltiorrhizae control medicinal material is reflected as Radix Salviae Miltiorrhizae
Not.Reason is in Radix Salviae Miltiorrhizae that danshensu and other compositions will be completely separated needs unique treatment technology, and in Radix Paeoniae Rubra, peoniflorin is not
Stable, make to differentiate to be also required to special method with thin layer chromatography.Additionally, original detection method is concentrated mainly on heat clearing and damp drying
The detection of medicine, does not detect the carrying out of blood circulation promoting and blood stasis dispelling pharmaceutically active ingredient, and takes into account the detection of other effective ingredient so that product
Effectiveness, safety preferably can not embody so that cannot ensure the due clinical efficacy of product.Accordingly, it would be desirable to I
On the basis of original detection method, by study blood circulation promoting and blood stasis dispelling pharmaceutically active ingredient, formulate its assay and differentiate item
Mesh, the quality of General Promotion product, it is ensured that clinical efficacy, clinical application safety.
Content of the invention
The present invention provides a kind of detection method of FUYANKANG PIAN, to solve present in current detection method not promoting blood circulation
The carrying out of blood stasis dispelling pharmaceutically active ingredient is detected, and takes into account the detection of other effective ingredient so that the effectiveness of product, safety can not
Preferably embody, so that cannot ensure the problem of the due clinical efficacy of product.
The present invention is adopted the technical scheme that：Including following discrimination method and content assaying method：
Differentiate (1), 6 small pieces of this product or 3 large stretch of, coated tablet removing sugar-coats are taken, finely ground, plus methanol 30ml, supersound process 30
Minute, filtration, filtrate is evaporated, and the residue 20ml that adds water makes dissolving, with ether extraction 2 times, each 20ml, merges ether solution, standby；
Aqueous solution is extracted 2 times with water-saturated n-butanol again, each 20ml, merges n-butyl alcohol liquid, is steamed to nearly 2ml, is added 100～200 mesh
Neutral alumina 2g, mixes thoroughly in water-bath, dries, is added on neutral alumina column, 100～200 mesh, 2g, internal diameter 0.9cm, according to
Secondary with ethyl acetate：Methanol=2：1st, ethyl acetate：Methanol=1：1 and 80% each 10ml eluting of methanol, collect 80% methanol and wash
De- liquid, is evaporated, and residue adds methanol 1ml to make dissolving, used as need testing solution；Peoniflorin reference substance is separately taken, plus methanol makes every 1ml
Solution containing 1mg, as reference substance solution, tests according to thin layer chromatography, general rule 0502, draws each 4 μ l of above two solution, point
Other point on same silica gel g thin-layer plate, with chloroform：Acetic acid ethyl ester：Methanol：Water=15：40：22：10th, 10 DEG C arranged below
Lower floor's solution be developing solvent, launch, take out, dry, spray with 5% vanillin-sulfuric acid ethanol solution (1 → 10), at 105 DEG C
It is heated to spot development clear, in test sample chromatograph, on position corresponding with reference substance chromatograph, the speckle of aobvious same color；
Differentiate (2), the diethyl ether solution for taking under discriminating (1) item, volatilize, residue adds methanol 1ml that dissolving is made, molten as test sample
Liquid, separately takes Rhizoma Curcumae control medicinal material 0.5g, plus methanol 20ml, supersound process 30 minutes, filtration, and filtrate is evaporated, and residue adds methanol 2ml
Dissolving is made, as control medicinal material solution；Test according to thin layer chromatography, general rule 0502, each 4 μ l of above two solution is drawn, respectively
Put on same silica gel g thin-layer plate, with toluene：Ethyl acetate=93：7 is developing solvent, launches, takes out, dry, sprays with 5%
Vanillin-sulfuric acid ethanol solution (1 → 10), is heated to spot development at 105 DEG C clear；In test sample chromatograph, with control medicinal material
On the corresponding position of chromatograph, the speckle of aobvious same color；
Assay (3), danshensu shine high performance liquid chromatography, 0512 determination step of general rule：
Chromatographic condition and system suitability：With octadecylsilane chemically bonded silica as filler；With acetonitrile：Methanol：
1% acetic acid=2.25：2.25：95.5 it is mobile phase；Detection wavelength is 283nm；Number of theoretical plate presses the calculating of danshensu peak should not be low
In 8000；
The preparation of reference substance solution：Take danshensu sodium reference substance appropriate, accurately weighed, plus methanol makes every 1ml containing 40 μ g
Solution, 36 μ g of suitable danshensu, obtain final product；
The preparation of need testing solution：This product 10 is taken, coated tablet removes sugar-coat, finely ground, and 0.5g is taken, accurately weighed, puts tool
In plug conical flask, 5% oxalic acid solution 5ml of accurate addition, close plug, supersound process 5 minutes, power 25W, frequency 33kHz, add
100～200 mesh neutral alumina 1.5g, stir, 5% oxalic acid 20ml solution of accurate addition, close plug, 30 points of supersound process
Clock, lets cool, and supplies the weight of less loss with 5% oxalic acid solution, shakes up, and 5000 revs/min are centrifuged 5 minutes, take supernatant, and filtration takes
Subsequent filtrate, obtains final product；
Algoscopy：Accurate absorption reference substance solution 10 μ l each with need testing solution, injects chromatograph of liquid, determines respectively,
Obtain final product；
This product is per piece containing Radix Salviae Miltiorrhizae with danshensu C9H10O5Meter, piece weight 0.25g must not be less than 0.44mg, and piece weight 0.52g must not
Less than 0.88mg.
The invention has the beneficial effects as follows：From indication, drug for invigorating blood circulation and eliminating stasis is started with, and for effective ingredient property is contained, examines
Consider effective ingredient peoniflorin unstable factor in Radix Paeoniae, increase discriminating project；Effective component in red sage danshensu is stable in properties, increases
Plus assay；The discriminating project of promoting the circulation of QI to relieve pain medicine Rhizoma Curcumae is increased in item is differentiated, which increases 13 taste Chinese prescriptions
In 1 assay, 2 discriminatings, in conjunction with original 1 containing measuring and 3 discriminatings make product quality be effectively controlled；Can be full
Foot produces the needs of quality product, makes product can ensure that optimal therapeutic effect.
Description of the drawings
Fig. 1 is that Radix Paeoniae Rubra thin layer differentiates chromatogram；
Fig. 2 is that Radix Paeoniae Rubra differentiates toleration thin layer Fig. 1；
Fig. 3 is that Radix Paeoniae Rubra differentiates toleration thin layer Fig. 2；
Fig. 4 is that Radix Paeoniae Rubra differentiates toleration thin layer Fig. 3；
Fig. 5 is that Radix Paeoniae Rubra differentiates toleration thin layer Fig. 4；
Fig. 6 is Rhizoma Curcumae thin-layer chromatogram；
Fig. 7 is Rhizoma Curcumae toleration thin layer Fig. 1；
Fig. 8 is Rhizoma Curcumae toleration thin layer Fig. 2；
Fig. 9 is Rhizoma Curcumae toleration thin layer Fig. 3；
Figure 10 is Rhizoma Curcumae toleration thin layer Fig. 4；
Figure 11 is danshensu sodium mark chromatogram；
Figure 12 is danshensu sodium blank reagent chromatogram；
Figure 13 is danshensu sodium uv absorption spectra；
Figure 14 is danshensu sodium canonical plotting；
Figure 15 is FUYANKANG PIAN HPLC chromatogram.
Specific embodiment
Including following discrimination method and content assaying method：
Differentiate (1), 6 small pieces of this product or 3 large stretch of, coated tablet removing sugar-coats are taken, finely ground, plus methanol 30ml, supersound process 30
Minute, filtration, filtrate is evaporated, and the residue 20ml that adds water makes dissolving, with ether extraction 2 times, each 20ml, merges ether solution, standby；
Aqueous solution is extracted 2 times with water-saturated n-butanol again, each 20ml, merges n-butyl alcohol liquid, is steamed to nearly 2ml, is added 100～200 mesh
Neutral alumina 2g, mixes thoroughly in water-bath, dries, is added on neutral alumina column, 100～200 mesh, 2g, internal diameter 0.9cm, according to
Secondary with ethyl acetate：Methanol=2：1st, ethyl acetate：Methanol=1：1 and 80% each 10ml eluting of methanol, collect 80% methanol and wash
De- liquid, is evaporated, and residue adds methanol 1ml to make dissolving, used as need testing solution；Peoniflorin reference substance is separately taken, plus methanol makes every 1ml
Solution containing 1mg, as reference substance solution, tests according to thin layer chromatography, general rule 0502, draws each 4 μ l of above two solution, point
Other point on same silica gel g thin-layer plate, with chloroform：Acetic acid ethyl ester：Methanol：Water=15：40：22：10th, 10 DEG C arranged below
Lower floor's solution be developing solvent, launch, take out, dry, spray with 5% vanillin-sulfuric acid ethanol solution (1 → 10), at 105 DEG C
It is heated to spot development clear, in test sample chromatograph, on position corresponding with reference substance chromatograph, the speckle of aobvious same color；
Differentiate (2), the diethyl ether solution for taking under discriminating (1) item, volatilize, residue adds methanol 1ml that dissolving is made, molten as test sample
Liquid, separately takes Rhizoma Curcumae control medicinal material 0.5g, plus methanol 20ml, supersound process 30 minutes, filtration, and filtrate is evaporated, and residue adds methanol 2ml
Dissolving is made, as control medicinal material solution；Test according to thin layer chromatography, general rule 0502, each 4 μ l of above two solution is drawn, respectively
Put on same silica gel g thin-layer plate, with toluene：Ethyl acetate=93：7 is developing solvent, launches, takes out, dry, sprays with 5%
Vanillin-sulfuric acid ethanol solution (1 → 10), is heated to spot development at 105 DEG C clear；In test sample chromatograph, with control medicinal material
On the corresponding position of chromatograph, the speckle of aobvious same color；
Assay (3), danshensu shine high performance liquid chromatography, 0512 determination step of general rule：
Chromatographic condition and system suitability：With octadecylsilane chemically bonded silica as filler；With acetonitrile：Methanol：
1% acetic acid=2.25：2.25：95.5 it is mobile phase；Detection wavelength is 283nm；Number of theoretical plate presses the calculating of danshensu peak should not be low
In 8000；
The preparation of reference substance solution：Take danshensu sodium reference substance appropriate, accurately weighed, plus methanol makes every 1ml containing 40 μ g
Solution, 36 μ g of suitable danshensu, obtain final product；
The preparation of need testing solution：This product 10 is taken, coated tablet removes sugar-coat, finely ground, and 0.5g is taken, accurately weighed, puts tool
In plug conical flask, 5% oxalic acid solution 5ml of accurate addition, close plug, supersound process 5 minutes, power 25W, frequency 33kHz, add
100～200 mesh neutral alumina 1.5g, stir, 5% oxalic acid 20ml solution of accurate addition, close plug, 30 points of supersound process
Clock, lets cool, and supplies the weight of less loss with 5% oxalic acid solution, shakes up, and 5000 revs/min are centrifuged 5 minutes, take supernatant, and filtration takes
Subsequent filtrate, obtains final product；
Algoscopy：Accurate absorption reference substance solution 10 μ l each with need testing solution, injects chromatograph of liquid, determines respectively,
Obtain final product；
This product is per piece containing Radix Salviae Miltiorrhizae with danshensu C9H10O5Meter, piece weight 0.25g must not be less than 0.44mg, and piece weight 0.52g must not
Less than 0.88mg.
The following effect for illustrating to further illustrate the present invention by the research of detection method.
In order to effectively improve product inspection method, on the basis of former detection method, through experimental study, increased Radix Paeoniae Rubra and
Rhizoma Curcumae indentification by TLC；Increase using content of Danshensu in high effective liquid chromatography for measuring finished product, the Radix Paeoniae Rubra being now increased by and
The thin layer discrimination test of Rhizoma Curcumae is described as follows：
First, differentiate that the thin layer under (1) item for Radix Paeoniae Rubra differentiates
With peoniflorin reference substance as control, the experimental observation through three batch samples and negative control, as a result show that this method is reappeared
Property good, negative noiseless, therefore this method is listed in detection method text.Thin-layer chromatogram is shown in Fig. 1.While We conducted thin layer color
Spectrum differentiates tolerance test, i.e., carry out thin layer discriminating at different conditions, selects four conditions to be tested：
(1) room temperature environment, makes silica gel g thin-layer plate by oneself.
(2) room temperature environment, the silica gel G precoated plate of Haiyang Chemical Plant, Qingdao's production.
(3) artificial environment：10 DEG C of temperature, relative humidity 40%, handss spread silica gel g thin-layer plate.
(4) artificial environment：35 DEG C of temperature, relative humidity 75%, handss spread silica gel g thin-layer plate.
As a result show that the performance of Radix Paeoniae Rubra indentification by TLC tolerance test is good, as a result see Fig. 2～5；
Radix Paeoniae Rubra thin layer discrimination test can be operated using self-control silica gel g thin-layer plate.
2nd, with Rhizoma Curcumae control medicinal material as control, the experimental observation through three batch samples and negative control, as a result show this method weight
Existing property is good, negative noiseless, therefore this method is listed in detection method text.Thin-layer chromatogram is shown in Fig. 6.While We conducted thin layer
Chromatographic identification tolerance test, i.e., carry out thin layer discriminating at different conditions, selects four conditions to be tested：
(1) room temperature environment, makes silica gel g thin-layer plate by oneself.
(2) room temperature environment, the silica gel G precoated plate of Haiyang Chemical Plant, Qingdao's production.
(3) artificial environment：10 DEG C of temperature, relative humidity 40%, handss spread silica gel g thin-layer plate.
(4) artificial environment：35 DEG C of temperature, relative humidity 75%, handss spread silica gel g thin-layer plate.
As a result show that the performance of Rhizoma Curcumae indentification by TLC tolerance test is good, as a result see Fig. 7～10, Rhizoma Curcumae thin layer differentiates
Test can be operated using self-control silica gel g thin-layer plate.
3rd, in other side, in Radix Salviae Miltiorrhizae, the content of contained effective ingredient danshensu is measured, and method is as follows：
1 instrument and reagent：High performance liquid chromatograph：Japanese Shimadzu LC-10AT high performance liquid chromatograph；Detector：SPD-
10A UV-detector；KQ-250 type processor for ultrasonic wave, danshensu sodium reference substance.
2 chromatographic conditions：Chromatographic column is Agilent chromatographic column (5 μm, 4.6mm × 250mm)；With -1% acetic acid of acetonitrile-methanol
(2.25：2.25：95.5) it is mobile phase；The a length of 283nm of detection filter；Number of theoretical plate is calculated by danshensu peak and should be not less than 8000.
The selection of 3 different mobile phases we in an experiment, investigated using multiple mobile phases, once adopted acetonitrile -1%
Acetic acid (2：98), methanol-(water-dimethylformamide-glacial acetic acid 93：2：1)10：90th, -0.05% phosphoric acid (93 of methanol：7) do not have
Promising result is obtained, by groping using -1% acetic acid (2.25 of acetonitrile-methanol：2.25：95.5) preferable separating effect is obtained, row
Enter in quality standard.
The preparation of 4 need testing solutions
4.1 extracting method Selection experiment
For select suitable prepare need testing solution method, prepare need testing solution from four kinds of distinct methods, and to carrying
Take effect to be investigated, concrete operations are as follows：
4.1.1 this product finely ground powder about 0.6g is taken, precision is weighed, and is put in conical flask with cover, 50% methanol of accurate addition
50ml, close plug, weighed weight, supersound process (power 25W, frequency 33kHz) 30 minutes, let cool, then weighed weight, use 50% first
The weight of less loss supplied by alcohol, shakes up, filtration, accurate absorption filtrate 25ml, is evaporated, and residue methanol makes dissolving in right amount, and shifts
To 5ml measuring bottle, plus methanol is to scale, filters, takes subsequent filtrate, as need testing solution (one).
4.1.2 this product finely ground powder about 0.5g is taken, accurately weighed, put in conical flask with cover, 5% oxalic acid solution of accurate addition
5ml, close plug, supersound process (power 25W, frequency 33kHz) 5 minutes, neutral alumina 1.5g is added, is mixed thoroughly, then accurate addition
5% oxalic acid solution 20ml, close plug, supersound process (power 25W, frequency 33kHz) 30 minutes, let cool, be transferred in centrifuge tube, from
The heart (5000 revs/min) 5 minutes, takes supernatant, filtration, takes subsequent filtrate, as need testing solution (two).
4.1.3 this product finely ground powder about 0.6g is taken, accurately weighed, put in conical flask with cover, accurate addition methanol 50ml, close
Plug, weighed weight, supersound process (power 25W, frequency 33kHz) 30 minutes, let cool, then weighed weight, less loss is supplied with methanol
Weight, shake up, filtration, accurate draw filtrate 25ml, be evaporated, residue methanol makes dissolving in right amount, and is transferred to 5ml measuring bottle
In, plus methanol is to scale, filters, takes subsequent filtrate, as need testing solution (three).
4.1.4 this product finely ground powder about 1.2g is taken, accurately weighed, put in conical flask with cover, 2% hydrochloric acid solution of accurate addition
50ml, supersound process (power 25W, frequency 33kHz) 30 minutes, add Sodium Chloride 5g, dissolving, filtration, absorption filtrate 25ml, use
Ethyl acetate is extracted 4 times, each 20ml, combined ethyl acetate liquid, is vaporized solvent and is done near, and residue methanol makes dissolving in right amount,
And be transferred in 10ml measuring bottle, plus methanol is to scale, filters, takes subsequent filtrate, as need testing solution (four).
4.1.5 each 10 μ l of need testing solution of accurate absorption, is operated by method under assay item, is determined in need testing solution
Content of Danshensu, measurement result is shown in Table 1.
Table 1, Different Extraction Method result of the test
As known from Table 1, using method two：The impurity that can remove in need testing solution is extracted with 5% oxalic acid, can be made again point
The content of danshensu not being affected more preferably and from effect, therefore prepares need testing solution from 5% oxalic acid for Extraction solvent.
5 extraction times investigated test
In order to select the suitable supersound extraction time, we are examined the extraction effect to the different supersound extraction times
Examine, operate as follows：This product finely ground powder about 0.5g is taken, three parts, accurately weighed, operate by method in quality standard, respectively ultrasound
Process (power 25W, frequency 33kHz) 20,30,40 minutes, let cool, centrifugation (5000 revs/min) 5 minutes, supernatant is taken, filtration,
Subsequent filtrate is taken, the content of danshensu is determined, the results are shown in Table 2.
Table 2, the comparison of different supersound extraction times
2 result of table shows, supersound extraction more than 30 minutes, extracts can danshensu complete, in order to shorten detection time, therefore
Determine the supersound extraction time for 30 minutes.
6 reference substances source and purity
Danshensu sodium reference substance (110855-201513) is purchased from National Institute for Food and Drugs Control, for assay.
Danshensu sodium purity testing test operation is as follows：Take danshensu sodium reference substance appropriate, accurately weighed, plus 50% methanol make per
Solution of the 1ml containing 1mg, accurate absorption 20 μ l of this solution, chromatograph of liquid is injected, selected liquid phase chromatogram condition is separated, and is pressed
Normalization method is calculated, and danshensu sodium content is 100%, to can be used for assay.The high performance liquid chromatography of danshensu sodium reference substance
Figure is shown in Figure 11～12.
7 methodological studies
(1) selection of danshensu sodium Detection wavelength
It is danshensu sodium reference substance solution of every 1ml containing 42.9 μ g to take concentration, determines in 190～400nm wave-length coverage
Trap, draws curve, determines Detection wavelength for 283nm, and danshensu sodium uv absorption spectra is shown in Figure 13.
(2) investigation of linear relationship
Precision weighs danshensu sodium reference substance in right amount, plus methanol makes solution of every 1ml containing 0.0429mg, and precision draws this
Each 2,4,8,12,16, the 20 μ l of solution, is injected separately into chromatograph of liquid, determines integrating peak areas value, and measurement result is shown in Table 3.To enter
Sample amount is abscissa, is mapped with peak area value as vertical coordinate, draws standard curve, and obtaining regression equation is：Y=343564.588x+
1706.595, r=0.9999, show danshensu sodium sample size in 0.0858～0.858 μ g range, sample size is accumulated with peak area
Score value linear relationship is good, sees Figure 14.
Measurement result linearly investigated by table 3, danshensu sodium reference substance
(3) blank assay
The full prescription flavour of a drug of Radix Salviae Miltiorrhizae are removed, is operated according under preparation method item, prepare negative sample.Negative sample 0.5g is taken, by containing
Measure and determine the operation of " preparation of need testing solution " method, prepared negative controls under item.Draw danshensu sodium reference substance solution, woman
Scorching health piece need testing solution and each 10 μ l of Radix Salviae Miltiorrhizae negative control solution, inject hplc determination.In negative HPLC collection of illustrative plates,
Do not absorb in retention time corresponding with danshensu sodium, show that feminine gender is noiseless and see Figure 15.
(4) investigation of precision
This product finely ground powder about 0.5g is taken, accurately weighed, need testing solution is prepared, accurate absorption 10 μ l of this solution, repeats
Sample introduction 6 times, determines the peak area of danshensu sodium, calculates RSD value, the results are shown in Table 4.
The investigation result of table 4, precision
4 result of the test of table shows, precision is good.
(5) need testing solution stability test
FUYANKANG PIAN need testing solution is taken, in placing 0 hour, 2 hours, 4 hours, 6 hours, 8 hours and 24 hours, respectively
10 μ l of sample introduction, determines the integrating peak areas value of danshensu sodium, the results are shown in Table 5.
Table 5, need testing solution stability test result
5 result of table shows, need testing solution is at least stable in 24 hours.
(6) repeatability experiment
FUYANKANG PIAN 0.5g is taken, 6 parts, accurately weighed, press the method operation under assay Radix Salviae Miltiorrhizae prime implicant respectively, determine red
Ginseng cellulose content, measurement result is shown in Table 6.
6 reproducible test results of table
(7) average recovery experiment
Accurate absorption danshensu sodium reference substance solution (0.195mg/ml) 2.5ml, volatilizes, adds and predicted content of Danshensu
Sample about 0.25g (content of Danshensu：2.1190mg/g), accurately weighed, method operation under item, system is determined by content of Danshensu
Available test sample solution, 10 μ l of sample introduction, peak area is determined, the response rate is calculated, measurement result is shown in Table 7.
Table 7, danshensu recovery test measurement result
The assay of danshensu in 8 FUYANKANG PIAN
Operate by method below FUYANKANG PIAN assay Radix Salviae Miltiorrhizae prime implicant, need testing solution is prepared, determine ten batches of FUYANKANG PIAN
Middle content of Danshensu content, measurement result is shown in Table 8.
Danshensu measurement result in 8, ten batches of FUYANKANG PIAN of table
As a result showing, in FUYANKANG PIAN, content of Danshensu is basicly stable, FUYANKANG PIAN is limited per piece containing Radix Salviae Miltiorrhizae with danshensu
(C9H10O5) meter, piece weight 0.25g must not be less than 0.44mg, and piece weight 0.52g must not be less than 0.88mg.
The assay of danshensu in 9 red rooted salvias
In experimentation, we adopt and preparation identical liquid phase chromatogram condition, to content of Danshensu in red rooted salvia
It is determined, method is as follows：
Three batches of red rooted salvias are taken, coarse powder is ground into, about 0.8g is taken, accurately weighed, put in round-bottomed flask, 80ml water is added,
Heating and refluxing extraction 4 hours, takes out, lets cool, filtration, and filtrate is transferred in 100ml measuring bottle, is diluted with water to scale, is shaken up, filter
Cross, subsequent filtrate, as need testing solution.Draw reference substance solution 10 μ l each with need testing solution respectively, liquid phase is injected, is surveyed
Fixed, the results are shown in Table 9.
Content of Danshensu measurement result in 9, three batches of red rooted salvias of table
Shown by 9 result of table：In three batches of medical materials, content of Danshensu is stable, temporarily limits Radix Salviae Miltiorrhizae and presses dry product calculating, containing Radix Salviae Miltiorrhizae
Element (C9H10O5) 0.70% must not be less than.
Claims (1)
1. a kind of detection method of FUYANKANG PIAN, it is characterised in that including following discrimination method and content assaying method：
Differentiate (1), 6 small pieces of this product or 3 large stretch of, coated tablet removing sugar-coats are taken, finely ground, plus methanol 30ml, supersound process 30 minutes,
Filtration, filtrate is evaporated, and the residue 20ml that adds water makes dissolving, with ether extraction 2 times, each 20ml, merges ether solution, standby；Water-soluble
Liquid is extracted 2 times with water-saturated n-butanol again, each 20ml, merges n-butyl alcohol liquid, is steamed to nearly 2ml, adds 100～200 mesh neutrality
Aluminium oxide 2g, mixes thoroughly in water-bath, dry, be added on neutral alumina column, 100～200 mesh, 2g, internal diameter 0.9cm, successively with
Ethyl acetate：Methanol=2：1st, ethyl acetate：Methanol=1：1 and 80% each 10ml eluting of methanol, 80% meoh eluate is collected,
It is evaporated, residue adds methanol 1ml to make dissolving, used as need testing solution；Peoniflorin reference substance is separately taken, plus methanol makes every 1ml containing 1mg
Solution, as reference substance solution, test according to thin layer chromatography, general rule 0502, draw each 4 μ l of above two solution, respectively point
On same silica gel g thin-layer plate, with chloroform：Acetic acid ethyl ester：Methanol：Water=15：40：22：10th, 10 DEG C arranged below under
Layer solution is developing solvent, to launch, and takes out, dries, and sprays with 5% vanillin-sulfuric acid ethanol solution (1 → 10), heats at 105 DEG C
Clear to spot development, in test sample chromatograph, on position corresponding with reference substance chromatograph, the speckle of aobvious same color；
Differentiate (2), the diethyl ether solution for taking under discriminating (1) item, volatilize, residue adds methanol 1ml that dissolving is made, as need testing solution,
Rhizoma Curcumae control medicinal material 0.5g, plus methanol 20ml separately taken, supersound process 30 minutes, filtration, filtrate is evaporated, and residue adds methanol 2ml to make
Dissolving, used as control medicinal material solution；Test according to thin layer chromatography, general rule 0502, each 4 μ l of above two solution is drawn, respectively point
On same silica gel g thin-layer plate, with toluene：Ethyl acetate=93：7 is developing solvent, launches, takes out, dry, sprays with 5% perfume (or spice)
Oxalaldehyde ethanol solution of sulfuric acid (1 → 10), is heated to spot development at 105 DEG C clear；In test sample chromatograph, with comparison medicine wood color
Compose on corresponding position, the speckle of aobvious same color；
Assay (3), danshensu shine high performance liquid chromatography, 0512 determination step of general rule：
Chromatographic condition and system suitability：With octadecylsilane chemically bonded silica as filler；With acetonitrile：Methanol：1% second
Acid=2.25：2.25：95.5 it is mobile phase；Detection wavelength is 283nm；Number of theoretical plate is calculated by danshensu peak and should be not less than
8000；
The preparation of reference substance solution：Take danshensu sodium reference substance appropriate, accurately weighed, plus methanol makes every 1ml containing the molten of 40 μ g
Liquid, 36 μ g of suitable danshensu, obtain final product；
The preparation of need testing solution：This product 10 is taken, coated tablet removes sugar-coat, finely ground, and 0.5g is taken, accurately weighed, put tool plug cone
In shape bottle, accurate add 5% oxalic acid solution 5ml, close plug, supersound process 5 minutes, power 25W, frequency 33kHz, add 100～
200 mesh neutral alumina 1.5g, stir, 5% oxalic acid 20ml solution of accurate addition, close plug, and supersound process 30 minutes is put
Cold, the weight of less loss is supplied with 5% oxalic acid solution, is shaken up, 5000 revs/min are centrifuged 5 minutes, take supernatant, and filtration takes continuous filter
Liquid, obtains final product；
Algoscopy：Accurate absorption reference substance solution 10 μ l each with need testing solution, injects chromatograph of liquid, determines, obtain final product respectively；
This product is per piece containing Radix Salviae Miltiorrhizae with danshensu C9H10O5Meter, piece weight 0.25g must not be less than 0.44mg, piece weight 0.52g and must not be less than
0.88mg.
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