CN106442846B - A kind of detection method of 'Fuyankang ' tablet - Google Patents
A kind of detection method of 'Fuyankang ' tablet Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—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
- G01N30/02—Column chromatography
Abstract
The present invention relates to a kind of detection method of 'Fuyankang ' tablet, belong to herbal pharmaceutical detection field.Include the content of danshensu in the radix paeoniae rubrathe and curcuma zedoary indentification by TLC, and high effective liquid chromatography for measuring finished product, the beneficial effects of the invention are as follows：Drug for invigorating blood circulation and eliminating stasis is started with from major function, for containing active ingredient property, it is contemplated that active ingredient Paeoniflorin destabilizing factor in Chinese herbaceous peony, increases discriminating project；Effective component in red sage danshensu property is stable, increases assay；The discriminating project of promoting qi circulation and relieving pain medicine curcuma zedoary is added in item is differentiated, 1 assay in 13 taste Chinese prescriptions, 2 discriminatings is so added, product quality is effectively controlled containing measurement and 3 discriminatings with reference to original 1；The needs for producing quality product can be met, product is can ensure that optimal therapeutic effect.
Description
Technical field
The invention belongs to herbal pharmaceutical detection field, and in particular to the detection method of 'Fuyankang ' tablet.
Background technology
'Fuyankang ' tablet is prolonged recklessly with radix paeoniae rubrathe 60g, smilax 100g, stir-baked RHIZOMA SPARGANII with vinegar 60g, stir-baked FRUCTUS TOOSENDAN 60g, stir-baked RHIIZOMA CURCUMAE with vinegar 60g, vinegar
Rope 60g, stir-baked SEMEN EURYALES 100g, Radix Angelicae Sinensis 100g, kuh-seng 60g, prepared RHIZOMA CYPERI with vinegar 40g, golden cypress 60g, red sage root 100g, Chinese yam 120g compositions.
The taste of the above 13, stir-baked RHIIZOMA CURCUMAE with vinegar, Chinese yam are ground into fine powder, sieving, 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, decocting liquid filtration, merging filtrate, is concentrated into right amount, is mixed with above-mentioned powder,
Fine powder is dry, pulverize into, adds sucrose, starch and Magnesium Stearate proper quantity, is made pellet, dries, is pressed into 1000 (small pieces), bag sugar
Clothing or film-coating, every weight 0.25g；Or 500 (sheets) are pressed into, film coating, every weight 0.52g, produce.
This product is sugar coated tablet or Film coated tablets, shows yellowish-brown to sepia after removing coating；Gas is micro-, mildly bitter flavor.
The red sage root, the radix paeoniae rubrathe, Radix Angelicae Sinensis are promoting blood circulation and removing blood stasis in Chinese medicine compound prescription, and corydalis tuber, Fructus meliae toosendan, rhizoma cyperi share promoting qi circulation and removing obstruction in the collateral analgesic, add
Rhizoma Sparganii, curcuma zedoary enhancing promoting qi circulation and relieving pain and softening and resolving hard mass, Chinese yam, Gorgon fruit nourshing kidney stop-band, kuh-seng, golden cypress heat-clearing and damp-drying drug, smilax
Detoxification and promoting urination, all medicine compatibilities can reach it is promoting blood circulation and removing blood stasis, clearing heat and detoxicating, softening and resolving hard mass, relieve pain dampness removing purpose, can diminish inflammation,
Patient's waist abdomen pain is released, reduces leukorrhea, there is the effect of curing chronic pelvic inflammation.
There are 3 discriminatings and 1 assay in existing 'Fuyankang ' tablet detection method, it is respectively：
Differentiate that 1, corydalis tuber control medicinal material and tetrahydropalmatine reference substance differentiate corydalis tuber in product；
Differentiate that 2, golden cypress control medicinal material and jamaicin reference substance differentiate golden cypress in product；
Differentiate that 3, red sage root control medicinal material and protocatechualdehyde reference substance differentiate the red sage root in product.
Assay：Containing kuh-seng with according to high performance liquid chromatography (general rule) measure matrine content in product.
'Fuyankang ' tablet is the product that applicant researches and develops the eighties, and the history of more than 30 years, good effect are deep by wide so far
The favorable comment of big patient, it has also become treat the good medicine of gynaecological disease.But due to being limited to production technology, detection means etc. at that time during research and development
Technical merit, the detection method level of formulation is low, although having done certain raising the end of the nineties, on product inspection method,
The active constituent content measurings such as the drug for invigorating blood circulation and eliminating stasis red sage root, the radix paeoniae rubrathe, discriminating etc. could not be brought into quality standard.But with kuh-seng
In kuh-seng alkali composition as assay, alkaloid is as differentiating in corydalis tuber, golden cypress, and red sage root control medicinal material is as red sage root mirror
Not.Reason in the red sage root danshensu and other compositions to be completely separated and need unique treatment technology, Paeoniflorin is not in the radix paeoniae rubrathe
Stable, work, which differentiates, is also required to special method with TLC.In addition, original detection method is concentrated mainly on heat-clearing and damp-drying drug
The detection of medicine, pharmaceutically active ingredient promoting blood circulation and removing blood stasis detect, and take into account the detection of other active ingredients so that product
Validity, security can not preferably embody so that cannot ensure the due clinical efficacy of product.Therefore, it is necessary to I
On the basis of original detection method, by studying pharmaceutically active ingredient promoting blood circulation and removing blood stasis, formulate its assay and differentiate item
Mesh, the quality of General Promotion product, it is ensured that clinical efficacy, clinical application security.
The content of the invention
The present invention provides a kind of detection method of 'Fuyankang ' tablet, to solve present in current detection method not promoting blood circulation
Stagnation resolvation pharmaceutically active ingredient detect, and takes into account the detection of other active ingredients so that validity, the security of product can not
Preferably embody, so that the problem of cannot ensure product due clinical efficacy.
The present invention adopts the technical scheme that：Including following discrimination method and content assaying method：
Differentiate (1), take the small pieces of this product 6 or 3 large stretch of, sugar coated tablet removing sugar-coats, it is finely ground, add methanol 30ml, be ultrasonically treated 30
Minute, filtration, filtrate is evaporated, and residue adds water 20ml to make dissolving, with extracted by ether 2 times, each 20ml, merges ether solution, standby；
The aqueous solution is extracted 2 times, each 20ml with water-saturated n-butanol again, merges n-butanol liquid, is steamed to nearly 2ml, is added 100~200 mesh
Neutral alumina 2g, is mixed thoroughly in water-bath, is dried, is added on neutral alumina column, 100~200 mesh, 2g, internal diameter 0.9cm, according to
It is secondary with ethyl acetate：Methanol=2：1st, ethyl acetate：Methanol=1：1 and 80% each 10ml of methanol elution, collect 80% methanol and wash
De- liquid, is evaporated, residue adds methanol 1ml to make dissolving, as need testing solution；Paeoniflorin reference substance separately is taken, adds methanol that every 1ml is made
Solution containing 1mg, as reference substance solution, tested according to thin-layered chromatography, general rule 0502, draw each 4 μ l of above two solution, point
Other point is on same silica gel g thin-layer plate, with chloroform：Acetic acid ethyl ester：Methanol：Water=15：40：22：10th, 10 DEG C it is arranged below
Lower floor's solution be solvent, deploy, take out, dry, the vanillin-sulfuric acid ethanol solution (1 → 10) with 5% sprayed, at 105 DEG C
It is clear to be heated to spot development, in test sample chromatogram, on position corresponding with reference substance chromatogram, shows the spot of same color；
Differentiate (2), take diethyl ether solution under discriminating (1) item, volatilize, residue adds methanol 1ml to make dissolving, molten as test sample
Liquid, curcuma zedoary control medicinal material 0.5g is separately taken, add methanol 20ml, be ultrasonically treated 30 minutes, filtration, filtrate is evaporated, and residue adds methanol 2ml
Make dissolving, as control medicinal material solution；Tested according to thin-layered chromatography, general rule 0502, draw each 4 μ l of above two solution, respectively
Point is on same silica gel g thin-layer plate, with toluene：Ethyl acetate=93：7 be solvent, is deployed, and takes out, dries, spray with 5%
Vanillin-sulfuric acid ethanol solution (1 → 10), it is clear to be heated to spot development at 105 DEG C；In test sample chromatogram, with control medicinal material
On the corresponding position of chromatogram, show the spot of same color；
Assay (3), danshensu are according to high performance liquid chromatography, the determination step of general rule 0512：
Chromatographic condition and system suitability：Using 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 is calculated by danshensu peak should not be low
In 8000；
The preparation of reference substance solution：Take Sodium Danshensu reference substance appropriate, it is accurately weighed, add methanol that every 1ml is made and contain 40 μ g
Solution, the suitable μ g of danshensu 36, produce；
The preparation of need testing solution：This product 10 is taken, sugar coated tablet removes sugar-coat, finely ground, takes 0.5g, accurately weighed, puts tool
Fill in conical flask, precision adds 5% oxalic acid solution 5ml, close plug, is ultrasonically treated 5 minutes, power 25W, frequency 33kHz, adds
100~200 mesh neutral alumina 1.5g, stir, and precision adds 5% oxalic acid 20ml solution, close plug, is ultrasonically treated 30 points
Clock, let cool, the weight of less loss is supplied with 5% oxalic acid solution, is shaken up, 5000 revs/min centrifuge 5 minutes, take supernatant, filter, take
Subsequent filtrate, produce；
Determination method：Accurate absorption reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatograph, measure,
Produce；
This product every is containing the red sage root 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 beneficial effects of the invention are as follows：Drug for invigorating blood circulation and eliminating stasis is started with from major function, for containing active ingredient property, examining
Consider active ingredient Paeoniflorin destabilizing factor in Chinese herbaceous peony, increase discriminating project；Effective component in red sage danshensu property is stable, increases
Add assay；The discriminating project of promoting qi circulation and relieving pain medicine curcuma zedoary is added in item is differentiated, so adds 13 taste Chinese prescriptions
In 1 assay, 2 discriminating, with reference to original 1 containing measure and 3 discriminatings product quality is effectively controlled；Can be full
Foot produces the needs of quality product, product is can ensure that optimal therapeutic effect.
Brief description of the drawings
Fig. 1 is that radix paeoniae rubrathe thin layer differentiates chromatogram；
Fig. 2 is that the radix paeoniae rubrathe differentiates tolerance thin layer Fig. 1；
Fig. 3 is that the radix paeoniae rubrathe differentiates tolerance thin layer Fig. 2；
Fig. 4 is that the radix paeoniae rubrathe differentiates tolerance thin layer Fig. 3；
Fig. 5 is that the radix paeoniae rubrathe differentiates tolerance thin layer Fig. 4；
Fig. 6 is curcuma zedoary thin-layer chromatogram；
Fig. 7 is curcuma zedoary tolerance thin layer Fig. 1；
Fig. 8 is curcuma zedoary tolerance thin layer Fig. 2；
Fig. 9 is curcuma zedoary tolerance thin layer Fig. 3；
Figure 10 is curcuma zedoary tolerance thin layer Fig. 4；
Figure 11 is Sodium Danshensu mark chromatogram；
Figure 12 is Sodium Danshensu blank reagent chromatogram；
Figure 13 is Sodium Danshensu uv absorption spectra；
Figure 14 is Sodium Danshensu canonical plotting；
Figure 15 is 'Fuyankang ' tablet HPLC chromatogram.
Embodiment
Including following discrimination method and content assaying method：
Differentiate (1), take the small pieces of this product 6 or 3 large stretch of, sugar coated tablet removing sugar-coats, it is finely ground, add methanol 30ml, be ultrasonically treated 30
Minute, filtration, filtrate is evaporated, and residue adds water 20ml to make dissolving, with extracted by ether 2 times, each 20ml, merges ether solution, standby；
The aqueous solution is extracted 2 times, each 20ml with water-saturated n-butanol again, merges n-butanol liquid, is steamed to nearly 2ml, is added 100~200 mesh
Neutral alumina 2g, is mixed thoroughly in water-bath, is dried, is added on neutral alumina column, 100~200 mesh, 2g, internal diameter 0.9cm, according to
It is secondary with ethyl acetate：Methanol=2：1st, ethyl acetate：Methanol=1：1 and 80% each 10ml of methanol elution, collect 80% methanol and wash
De- liquid, is evaporated, residue adds methanol 1ml to make dissolving, as need testing solution；Paeoniflorin reference substance separately is taken, adds methanol that every 1ml is made
Solution containing 1mg, as reference substance solution, tested according to thin-layered chromatography, general rule 0502, draw each 4 μ l of above two solution, point
Other point is on same silica gel g thin-layer plate, with chloroform：Acetic acid ethyl ester：Methanol：Water=15：40：22：10th, 10 DEG C it is arranged below
Lower floor's solution be solvent, deploy, take out, dry, the vanillin-sulfuric acid ethanol solution (1 → 10) with 5% sprayed, at 105 DEG C
It is clear to be heated to spot development, in test sample chromatogram, on position corresponding with reference substance chromatogram, shows the spot of same color；
Differentiate (2), take diethyl ether solution under discriminating (1) item, volatilize, residue adds methanol 1ml to make dissolving, molten as test sample
Liquid, curcuma zedoary control medicinal material 0.5g is separately taken, add methanol 20ml, be ultrasonically treated 30 minutes, filtration, filtrate is evaporated, and residue adds methanol 2ml
Make dissolving, as control medicinal material solution；Tested according to thin-layered chromatography, general rule 0502, draw each 4 μ l of above two solution, respectively
Point is on same silica gel g thin-layer plate, with toluene：Ethyl acetate=93：7 be solvent, is deployed, and takes out, dries, spray with 5%
Vanillin-sulfuric acid ethanol solution (1 → 10), it is clear to be heated to spot development at 105 DEG C；In test sample chromatogram, with control medicinal material
On the corresponding position of chromatogram, show the spot of same color；
Assay (3), danshensu are according to high performance liquid chromatography, the determination step of general rule 0512：
Chromatographic condition and system suitability：Using 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 is calculated by danshensu peak should not be low
In 8000；
The preparation of reference substance solution：Take Sodium Danshensu reference substance appropriate, it is accurately weighed, add methanol that every 1ml is made and contain 40 μ g
Solution, the suitable μ g of danshensu 36, produce；
The preparation of need testing solution：This product 10 is taken, sugar coated tablet removes sugar-coat, finely ground, takes 0.5g, accurately weighed, puts tool
Fill in conical flask, precision adds 5% oxalic acid solution 5ml, close plug, is ultrasonically treated 5 minutes, power 25W, frequency 33kHz, adds
100~200 mesh neutral alumina 1.5g, stir, and precision adds 5% oxalic acid 20ml solution, close plug, is ultrasonically treated 30 points
Clock, let cool, the weight of less loss is supplied with 5% oxalic acid solution, is shaken up, 5000 revs/min centrifuge 5 minutes, take supernatant, filter, take
Subsequent filtrate, produce；
Determination method：Accurate absorption reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatograph, measure,
Produce；
This product every is containing the red sage root 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 effect of the present invention is further illustrated by the research explanation of detection method below.
In order to effectively improve product inspection method, on the basis of former detection method, through experimental study, add the radix paeoniae rubrathe and
Curcuma zedoary indentification by TLC；Increase use high effective liquid chromatography for measuring finished product in content of Danshensu, the radix paeoniae rubrathe being now increased by and
The thin layer discrimination test of curcuma zedoary is described as follows：
First, differentiate and differentiate under (1) item for the thin layer of the radix paeoniae rubrathe
Using Paeoniflorin reference substance as control, the experimental observation through three batches of samples and negative control, the results showed that this method is reappeared
Property it is good, it is negative noiseless, therefore this method is included in detection method text.Thin-layer chromatogram is shown in Fig. 1.We conducted thin layer color simultaneously
Spectrum differentiates tolerance test, i.e., carries out thin layer discriminating at different conditions, selects four conditions to be tested：
(1) room temperature environment, silica gel g thin-layer plate is made by oneself.
(2) room temperature environment, the silica G prefabricated board of Haiyang Chemical Plant, Qingdao's production.
(3) artificial environment：10 DEG C of temperature, relative humidity 40%, hand paving silica gel g thin-layer plate.
(4) artificial environment：35 DEG C of temperature, relative humidity 75%, hand paving silica gel g thin-layer plate.
As a result show that the performance of radix paeoniae rubrathe indentification by TLC tolerance test is good, as a result see Fig. 2~5；
Radix paeoniae rubrathe thin layer discrimination test can use self-control silica gel g thin-layer plate to be operated.
2nd, using curcuma zedoary control medicinal material as control, the experimental observation through three batches of samples and negative control, the results showed that this method weight
Existing property is good, negative noiseless, therefore this method is included in into detection method text.Thin-layer chromatogram is shown in Fig. 6.We conducted thin layer simultaneously
Chromatographic identification tolerance test, i.e., thin layer discriminating is carried out at different conditions, select four conditions to be tested：
(1) room temperature environment, silica gel g thin-layer plate is made by oneself.
(2) room temperature environment, the silica G prefabricated board of Haiyang Chemical Plant, Qingdao's production.
(3) artificial environment：10 DEG C of temperature, relative humidity 40%, hand paving silica gel g thin-layer plate.
(4) artificial environment：35 DEG C of temperature, relative humidity 75%, hand paving silica gel g thin-layer plate.
As a result show that the performance of curcuma zedoary indentification by TLC tolerance test is good, as a result see Fig. 7~10, curcuma zedoary thin layer differentiates
Experiment can use self-control silica gel g thin-layer plate to be operated.
3rd, the content of contained active ingredient danshensu is measured in the red sage root in other side, and method is as follows：
1 instrument and reagent：High performance liquid chromatograph：Japanese Shimadzu LC-10AT high performance liquid chromatographs；Detector：SPD-
10A UV-detectors；KQ-250 type processor for ultrasonic wave, Sodium Danshensu reference substance.
2 chromatographic conditions：Chromatographic column is Agilent chromatographic column (5 μm, 4.6mm × 250mm)；With the acetic acid of acetonitrile-methanol -1%
(2.25：2.25：95.5) it is mobile phase；A length of 283nm is filtered in detection；Number of theoretical plate is calculated by danshensu peak should be not less than 8000.
The selection of 3 different mobile phases we in an experiment, investigated using a variety of mobile phases, once using acetonitrile -1%
Acetic acid (2：98), methanol-(water-dimethylformamide-glacial acetic acid 93：2：1)10：90th, the phosphoric acid of methanol -0.05% (93：7) do not have
Promising result is obtained, by groping to use the acetic acid of acetonitrile-methanol -1% (2.25：2.25：95.5) preferable separating effect is obtained, is arranged
Enter in quality standard.
The preparation of 4 need testing solutions
4.1 extracting method Selection experiments
For select it is suitable prepare need testing solution method, prepare need testing solution from four kinds of distinct methods, and to carrying
Effect is taken to be investigated, concrete operations are as follows：
4.1.1 this product finely ground powder about 0.6g is taken, precision is weighed, put in conical flask with cover, and precision adds 50% methanol
50ml, close plug, weighed weight is ultrasonically treated (power 25W, frequency 33kHz) 30 minutes, let cool, then weighed weight, with 50% first
Alcohol supplies the weight of less loss, shakes up, and filtration, precision draws filtrate 25ml, is evaporated, residue methanol makes dissolving in right amount, and shifts
Into 5ml measuring bottles, methanol is added filtration, subsequent filtrate to be taken, as need testing solution (one) to scale.
4.1.2 the finely ground powder of this product about 0.5g is taken, it is accurately weighed, put in conical flask with cover, precision adds 5% oxalic acid solution
5ml, close plug are ultrasonically treated (power 25W, frequency 33kHz) 5 minutes, add neutral alumina 1.5g, mix thoroughly, then accurate addition
5% oxalic acid solution 20ml, close plug are ultrasonically treated (power 25W, frequency 33kHz) 30 minutes, let cool, be transferred in centrifuge tube, from
The heart (5000 revs/min) 5 minutes, takes supernatant, filtration, subsequent filtrate is taken, as need testing solution (two).
4.1.3 the finely ground powder of this product about 0.6g is taken, it is accurately weighed, put in conical flask with cover, precision adds methanol 50ml, close
Plug, weighed weight is ultrasonically treated (power 25W, frequency 33kHz) 30 minutes, let cool, then weighed weight, and less loss is supplied with methanol
Weight, shake up, filter, precision draw filtrate 25ml, be evaporated, residue methanol makes dissolving in right amount, and is transferred to 5ml measuring bottles
In, add methanol filtration, subsequent filtrate to be taken, as need testing solution (three) to scale.
4.1.4 the finely ground powder of this product about 1.2g is taken, it is accurately weighed, put in conical flask with cover, precision adds 2% hydrochloric acid solution
50ml, is ultrasonically treated (power 25W, frequency 33kHz) 30 minutes, adds sodium chloride 5g, dissolves, filtration, draw filtrate 25ml, uses
Ethyl acetate extracts 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 bottles, add methanol filtration, subsequent filtrate to be taken, as need testing solution (four) to scale.
4.1.5 it is accurate to draw each μ l of need testing solution 10, operate, determined in need testing solution by method under assay item
Content of Danshensu, measurement result are shown in Table 1.
Table 1, Different Extraction Method result of the test
As known from Table 1, using method two：Impurity in need testing solution can be removed with the extraction of 5% oxalic acid, and can makes point
It is more preferable from effect and do not influence the content of danshensu, therefore it is that Extraction solvent prepares need testing solution to select 5% oxalic acid.
5 extraction times investigated experiment
In order to select the suitable ultrasonic extraction time, we are examined the extraction effect to the different ultrasonic extraction times
Examine, operation is as follows：The finely ground powder of this product about 0.5g is taken, it is three parts, accurately weighed, operated by method in quality standard, respectively ultrasound
Handle (power 25W, frequency 33kHz) 20,30,40 minutes, let cool, centrifugation (5000 revs/min) 5 minutes, take supernatant, filter,
Subsequent filtrate is taken, the content of danshensu is determined, the results are shown in Table 2.
Table 2, the comparison of different ultrasonic extraction times
The result of table 2 shows, ultrasonic extraction more than 30 minutes, danshensu extraction can be made complete, in order to shorten detection time, therefore
It is 30 minutes to determine the ultrasonic extraction time.
6 reference substance sources and purity
Sodium Danshensu reference substance (110855-201513) is purchased from National Institute for Food and Drugs Control, for assay.
Sodium Danshensu purity testing test operation is as follows：Take Sodium Danshensu reference substance appropriate, it is accurately weighed, add 50% methanol to be made often
Solution of the 1ml containing 1mg, precision draw this solution 20 μ l, inject liquid chromatograph, the separation of selected liquid phase chromatogram condition, press
Normalization method calculates, and Sodium Danshensu content is 100%, can be used for assay.The high performance liquid chromatography of Sodium Danshensu reference substance
Figure is shown in Figure 11~12.
7 methodological studies
(1) selection of Sodium Danshensu Detection wavelength
It is to contain 42.9 μ g Sodium Danshensu reference substance solution per 1ml to take concentration, is determined in 190~400nm wave-length coverages
Trap, curve is drawn, it is 283nm to determine Detection wavelength, and Sodium Danshensu uv absorption spectra is shown in Figure 13.
(2) investigation of linear relationship
Precision weighs that Sodium Danshensu reference substance is appropriate, adds methanol that solution of every 1ml containing 0.0429mg is made, and precision draws this
Each 2,4,8,12,16,20 μ l of solution, liquid chromatograph is injected separately into, determines integrating peak areas value, measurement result is shown in Table 3.To enter
Sample amount is abscissa, is mapped by ordinate of peak area value, draws standard curve, obtaining regression equation is：Y=343564.588x+
1706.595, r=0.9999, show Sodium Danshensu sample size in 0.0858~0.858 μ g ranges, sample size accumulates with peak area
Score value linear relationship is good, sees Figure 14.
Table 3, Sodium Danshensu reference substance linearly investigate measurement result
(3) blank test
The full prescription flavour of a drug of the red sage root are removed, according to being operated under preparation method item, prepare negative sample.Take negative sample 0.5g, by containing
The operation of " preparation of need testing solution " method is determined under item in measurement, and negative controls are made.Draw Sodium Danshensu reference substance solution, woman
Scorching each 10 μ l of health piece need testing solution and red sage root negative control solution, inject hplc determination.In negative HPLC collection of illustrative plates,
Do not absorbed in retention time corresponding with Sodium Danshensu, show that feminine gender is noiseless and see Figure 15.
(4) investigation of precision
The finely ground powder of this product about 0.5g is taken, it is accurately weighed, need testing solution is prepared, precision draws this solution 10 μ l, repeats
Sample introduction 6 times, the peak area of Sodium Danshensu is determined, calculate RSD values, the results are shown in Table 4.
The investigation result of table 4, precision
The result of the test of table 4 shows that precision is good.
(5) need testing solution stability test
'Fuyankang ' tablet need testing solution is taken, in placing 0 hour, 2 hours, 4 hours, 6 hours, 8 hours and 24 hours, respectively
The μ l of sample introduction 10, determine the integrating peak areas value of Sodium Danshensu, the results are shown in Table 5.
Table 5, need testing solution stability test result
The result of table 5 shows that need testing solution is stable at least in 24 hours.
(6) reappearance is tested
'Fuyankang ' tablet 0.5g is taken, it is 6 parts, accurately weighed, operated respectively by the method under assay red sage root prime implicant, measure is red
Join cellulose content, measurement result is shown in Table 6.
The reproducible test results of table 6
(7) average recovery is tested
Precision draws Sodium Danshensu reference substance solution (0.195mg/ml) 2.5ml, volatilizes, and addition has predicted content of Danshensu
Sample about 0.25g (content of Danshensu：2.1190mg/g), it is accurately weighed, determine method under item by content of Danshensu and operate, system
Available test sample solution, the μ l of sample introduction 10, peak area is determined, calculate the rate of recovery, measurement result is shown in Table 7.
Table 7, danshensu recovery test measurement result
The assay of danshensu in 8 'Fuyankang ' tablets
By method operation below 'Fuyankang ' tablet assay red sage root prime implicant, need testing solution is prepared, determines ten batches of 'Fuyankang ' tablets
Middle content of Danshensu content, measurement result are shown in Table 8.
Danshensu measurement result in 8, ten batches of 'Fuyankang ' tablets of table
As a result show, content of Danshensu is basicly stable in 'Fuyankang ' tablet, limits 'Fuyankang ' tablet every containing the red sage root with danshensu
(C9H10O5) count, 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 experimental study, we using 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, are ground into coarse powder, take about 0.8g, it is accurately weighed, put in round-bottomed flask, add 80ml water,
Heating and refluxing extraction 4 hours, take out, let cool, filter, filtrate is transferred in 100ml measuring bottles, is diluted with water to scale, shakes up, and filters
Cross, subsequent filtrate, as need testing solution.Reference substance solution and each 10 μ l of need testing solution are drawn respectively, are injected liquid phase, are surveyed
It is fixed, it the results are shown in Table 9.
Content of Danshensu measurement result in 9, three batches of red rooted salvias of table
Shown by the result of table 9：Content of Danshensu is stable in three batches of medicinal materials, temporarily limits the red sage root and is calculated by dry product, containing the red sage root
Element (C9H10O5) 0.70% must not be less than.
Claims (1)
1. a kind of detection method of 'Fuyankang ' tablet, it is characterised in that including following discrimination method and content assaying method：
Differentiate (1), take the small pieces of this product 6 or 3 large stretch of, sugar coated tablet removing sugar-coats, it is finely ground, add methanol 30ml, be ultrasonically treated 30 minutes,
Filtration, filtrate is evaporated, and residue adds water 20ml to make dissolving, with extracted by ether 2 times, each 20ml, merges ether solution, standby；It is water-soluble
Liquid is extracted 2 times, each 20ml with water-saturated n-butanol again, merges n-butanol liquid, steams to nearly 2ml, it is neutral to add 100~200 mesh
Aluminum oxide 2g, is mixed 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 of methanol elution, 80% meoh eluate is collected,
It is evaporated, residue adds methanol 1ml to make dissolving, as need testing solution；Paeoniflorin reference substance separately is taken, adds methanol that every 1ml is made and contains 1mg
Solution, as reference substance solution, tested according to thin-layered chromatography, general rule 0502, draw each 4 μ l of above two solution, respectively point
In on same silica gel g thin-layer plate, with chloroform：Ethyl acetate：Methanol：Water=15：40：22：10th, 10 DEG C it is arranged below under
Layer solution is solvent, is deployed, and takes out, dries, spray the vanillin-sulfuric acid ethanol solution with 5%, being heated to spot at 105 DEG C shows
Color is clear, in test sample chromatogram, on position corresponding with reference substance chromatogram, shows the spot of same color；
Differentiating (2), take diethyl ether solution under discriminating (1) item, volatilize, residue adds methanol 1ml to make dissolving, as need testing solution,
Curcuma zedoary control medicinal material 0.5g separately is taken, adds methanol 20ml, is ultrasonically treated 30 minutes, filtration, filtrate is evaporated, and residue adds methanol 2ml to make
Dissolving, as control medicinal material solution；Tested according to thin-layered chromatography, general rule 0502, draw each 4 μ l of above two solution, respectively point
In on same silica gel g thin-layer plate, with toluene：Ethyl acetate=93：7 be solvent, is deployed, and takes out, dries, spray the perfume (or spice) with 5%
Oxalaldehyde ethanol solution of sulfuric acid, it is clear to be heated to spot development at 105 DEG C；In test sample chromatogram, corresponding to control medicinal material chromatogram
Position on, show same color spot；
Assay (3), danshensu are according to high performance liquid chromatography, the determination step of general rule 0512：
Chromatographic condition and system suitability：Using 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 to be not less than
8000；
The preparation of reference substance solution：Take Sodium Danshensu reference substance appropriate, it is accurately weighed, add methanol that every 1ml is made molten containing 40 μ g
Liquid, the suitable μ g of danshensu 36, is produced；
The preparation of need testing solution：This product 10 is taken, sugar coated tablet removes sugar-coat, finely ground, takes 0.5g, accurately weighed, puts tool plug cone
In shape bottle, precision adds 5% oxalic acid solution 5ml, close plug, is ultrasonically treated 5 minutes, power 25W, frequency 33kHz, and addition 100~
200 mesh neutral alumina 1.5g, stir, and precision adds 5% oxalic acid 20ml solution, close plug, is ultrasonically treated 30 minutes, puts
It is cold, the weight of less loss is supplied with 5% oxalic acid solution, is shaken up, 5000 revs/min centrifuge 5 minutes, take supernatant, filter, take continuous filter
Liquid, produce；
Determination method：It is accurate respectively to draw reference substance solution and each 10 μ l of need testing solution, liquid chromatograph is injected, measure, is produced；
The 'Fuyankang ' tablet every is containing the red sage root with danshensu C9H10O5Meter, piece weight 0.25g are no less than 0.44mg, and piece weight 0.52g is many
In 0.88mg.
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