CN103983735B - A kind of detection method preparing medical capsule for treating pelvic inflammatory disease - Google Patents
A kind of detection method preparing medical capsule for treating pelvic inflammatory disease Download PDFInfo
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Abstract
The present invention relates to a kind of detection method preparing medical capsule for treating pelvic inflammatory disease, the method harvested with relating to, Radix zanthoxyli, radix fici simplicissimae indentification by TLC, the high performance liquid chromatography assay of forulic acid acid and limit thereof.
Description
Technical field
The present invention relates to a kind of detection method preparing medical capsule for treating pelvic inflammatory disease.
Background technology
Pelvic infecton (Pelvicinflammatorydiseade, PID) inflammation of genital tract and surrounding tissue thereof in women is referred to, mainly comprise endometritis (endometritis), salpingitis (salpingitis), tubo-ovarian abscess (tubo-ovarianabscess, TOA), pelvic peritonitis (peritonitis).Inflammation can be confined to a position, also can involve several position simultaneously, and modal is salpingitis, salpingo-oothecitis.Pelvic infecton mostly occurs property active period, the women having menstruation, and before first menses, post menopausal or unmarried seldom pelvic infecton occur, if there is the diffusion of pelvic infecton also adjacent organs inflammation often.Acute and chronic two kinds can be divided into by its pathogenic process, clinical manifestation.
The acute inflammation that acute pelvitis of pelvic cavity refers to female internal genital organs and surrounding connective tissue thereof, pelvic peritoneum occurs, can be confined to a position, also can fall ill at several position simultaneously.Common pathogen is staphylococcus, streptococcus, Escherichia coli, anaerobion and the pathogen that spreads through sex intercourse, as gonococcus, mycoplasma, Chlamydia etc.Cause to pelvic cavity through lymph, blood or direct spreading.Common acute endometritis, myometritis, salpingitis, pyosalpinx, tubo-ovarian abscess, connective tissue inflammation of pelvic cavity, pelvic peritonitis, severe patient can cause septicemia and septicopyemia.As controlled not in time, can occur that infectious shock is even dead.
Chronic pelvic inflammatory disease refers to female internal genital organs and surrounding connective tissue, pelvic peritoneal chronic inflammation.Its main clinical manifestation is paramenia, leucorrhea increasing, waist abdomen pain and infertile etc., as formed chronic appendages inflammation, then can touch lump.
The medicine of the acute and chronic pelvic infecton of current treatment mostly is GONGYANPING PIAN agent.Due to the easy moisture absorption of tablet, easily oxidized, stability is not high, and drug effect is poor.
Medical capsule for treating pelvic inflammatory disease changes formulation by GONGYANPING PIAN, clinically for the treatment of acute and chronic pelvic infecton, has good effect.The preparation method relating to medical capsule for treating pelvic inflammatory disease has been had to wait patented claim in currently available technology, such as 2005101214259,2005100032342,2005100352266,2008100028798, and 2012101521990 etc. in patented claim, all disclose the preparation method or method of quality control that relate to medical capsule for treating pelvic inflammatory disease.
But disclosed in above-mentioned patented claim still there is the major defect of two aspects in correlation technique:
On the one hand, still there is the defect causing difficulty to discriminating lacking specificity and cause, or disturb excessive defect because TLC Identification detects the not obvious impurity caused, and lack assay and detect the defect that well can not be controlled to quality caused.
And on the other hand, in order to realize being controlled to quality, steady, Radix zanthoxyli, Radix Angelicae Sinensis and radix fici simplicissimae etc. all carry out medicinal material discriminating over the ground to adopt thin-layered chromatography for the whole composition of medical capsule for treating pelvic inflammatory disease, significantly increase production cost again, although energy Mass Control, can not meet the requirement of actual production.Therefore, in currently available technology, the preparation method of Related product cannot meet the needs of actual production.
Summary of the invention
The present invention, just from prior art, provides a kind of detection method preparing medical capsule for treating pelvic inflammatory disease, can realize accurately being controlled to quality, again can production control cost, is suitable in widespread use actual production process.
The invention provides a kind of detection method preparing medical capsule for treating pelvic inflammatory disease, wherein said medical capsule for treating pelvic inflammatory disease is harvested by ground, Radix zanthoxyli, Radix Angelicae Sinensis, radix fici simplicissimae, the wooden one-tenth of three-bristle cudrania, harvest describedly, Radix zanthoxyli, Radix Angelicae Sinensis, radix fici simplicissimae, three-bristle cudrania wood weight ratio be 450: 170: 140: 100: 140; Described medical capsule for treating pelvic inflammatory disease adopts following preparation method to obtain:
Harvest 450g Radix zanthoxyli 170g Radix Angelicae Sinensis 140g
Radix fici simplicissimae 100g three-bristle cudrania wood 140g
The above five tastes, boiling secondary, add water 12 times amount at every turn, decocts 2 hours, filter, merging filtrate, is concentrated into relative density and is determined as 1.25 at 55 ~ 60 DEG C, add ethanol and reach 50% to alcohol content, leave standstill 24 hours, filter, filtrate recycling ethanol, is concentrated into relative density is determined as 1.35 ~ 1.40 thick paste at 60 DEG C, dry, be ground into fine powder, add appropriate amount of auxiliary materials, mixing, granulate, incapsulate, make this product 1000;
The detection method preparing described medical capsule for treating pelvic inflammatory disease is:
(1) get this product 10, incline and content, porphyrize, add methyl alcohol 30ml, ultrasonic process 30 minutes, filter, filtrate evaporate to dryness, the residue 10ml that adds water makes dissolving, adds hydrochloric acid 2ml, add hot reflux 30 minutes, cool immediately, extract 2 times with ethyl acetate jolting, each 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Harvest control medicinal material 1g with separately getting, add methyl alcohol 10ml, be made in the same way of control medicinal material solution, get gallic acid reference substance again, add methyl alcohol and make the solution of every 1ml containing 0.5mg, compare product solution, test according to thin-layered chromatography, draw above-mentioned three kinds of each 10ul of solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride-acetic ether-methanoic acid (9: 3: 1) for developping agent, launch, take out, dry, spray with 1% ferric trichloride ethanolic solution, in test sample chromatogram, with in control medicinal material chromatogram and reference substance chromatogram relevant position, show the spot of same color;
(2) get this product content 2g, porphyrize, add water 30ml, grinds 2 minutes, and filter, filtrate extracts 2 times with methenyl choloride jolting, each 20ml, and merge methenyl choloride liquid, evaporate to dryness, residue adds methenyl choloride 1ml makes dissolving, as need testing solution; Separately get Radix zanthoxyli control medicinal material 1g, boiling 20 minutes, filter, filtrate makes control medicinal material solution with need testing solution preparation method, test according to thin-layered chromatography, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same with sodium carboxymethyl cellulose be binder silica gel g thin-layer plate on, with methenyl choloride-acetone-formic acid (18: 2: 1) for developping agent, launch, take out, dry, inspect under putting 365nm length ultraviolet light modulation, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the fluorescence principal spot of aobvious same color;
(3) get this product content 3g, add methenyl choloride 30ml, add hot reflux 2 hours, let cool, filter, filtrate is concentrated into 0.5ml, as need testing solution, separately get psoralen reference substance to add methenyl choloride and make every 1ml containing the solution of 1mg, product solution in contrast, test according to thin-layered chromatography, draw above-mentioned need testing solution 20 μ l, reference substance solution 5 μ 1, put respectively in same with sodium carboxymethyl cellulose be binder silica gel g thin-layer plate on, with n-hexane-ethyl acetate (9: 1) for developping agent, launch, take out, dry, spray with 10% potassium hydroxide solution colour developing, inspect under putting 365nm length ultraviolet light modulation, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color,
(4) assay
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filling agent, and acetonitrile-1% glacial acetic acid (15: 85) is mobile phase, and determined wavelength is 323nm, and number of theoretical plate calculates by forulic acid peak and is not less than 1500,
The preparation of reference substance solution learns from else's experience the phosphorus pentoxide drying under reduced pressure forulic acid reference substance of 24 hours in right amount, accurately weighed, adds methyl alcohol and makes the solution of every 1ml containing 6.0 μ g,
This product content is got in the preparation of need testing solution, porphyrize, precision takes about 5g, be placed in 100ml tool plug conical flask, add 50ml water, ultrasonic process 40 minutes, let cool, filter, the accurate subsequent filtrate 25ml that draws is in separating funnel, with salt acid for adjusting pH value to 2, 5 times are extracted with ether jolting, each 15ml, merge ether solution, 5 times are extracted with 5% sodium bicarbonate solution jolting, each 30ml, merge sodium bicarbonate solution, with salt acid for adjusting pH value to 2, 5 times are extracted with ether jolting, each 15ml, merge ether solution, volatilize, residue methyl alcohol dissolves and moves in the brown measuring bottle of 5ml, add methanol constant volume to scale, shake up, filter with miillpore filter, get subsequent filtrate and get final product,
Determination method is accurate respectively draws reference substance solution and each 20 μ 1 of need testing solution, injection liquid chromatography, measures, to obtain final product.
Preferably, wherein every containing Radix Angelicae Sinensis with forulic acid (C
10h
10o
4) meter, be no less than 2.5 μ g.
Preferably, the power 90W of wherein ultrasonic process, frequency 59KHz.
Adopt detection method of the present invention, harvest with relating generally in method, Radix zanthoxyli, radix fici simplicissimae indentification by TLC, the high performance liquid chromatography assay of forulic acid acid and limit thereof, on the one hand, long-standing owing to lacking the defect causing difficulty to discriminating that specificity causes before solving, on the other hand, also arranged except disturbing excessive defect and detecting owing to lacking assay the defect that well can not be controlled to quality caused.Meanwhile, while Mass Control, also reduce testing cost, the requirement of actual production can be met completely.
Accompanying drawing explanation
The thin-layer chromatogram harvested Fig. 1
The employment and suitability test (E & ST) of Fig. 2 thin layer plate
The employment and suitability test (E & ST) of Fig. 3 temperature
Fig. 4 medical capsule for treating pelvic inflammatory disease Radix zanthoxyli medicinal material TLC collection of illustrative plates
Fig. 5 medical capsule for treating pelvic inflammatory disease radix fici simplicissimae medicinal material TLC collection of illustrative plates
Embodiment
Below, the specific embodiment that the present invention prepares the detection method of medical capsule for treating pelvic inflammatory disease is provided.
Embodiment 1
Harvest 450g Radix zanthoxyli 170g Radix Angelicae Sinensis 140g
Radix fici simplicissimae 100g three-bristle cudrania wood 140g
Method for making method:
The above five tastes, boiling secondary, add water 12 times amount at every turn, decocts 2 hours, filter, merging filtrate, being concentrated into relative density is 1.25 (55 ~ 60 DEG C), adds ethanol and reaches 50% to alcohol content, leave standstill 24 hours, filter, filtrate recycling ethanol, be concentrated into the thick paste that relative density is 1.35 ~ 1.40 (60 DEG C), dry, pulverize into fine powder, add appropriate amount of auxiliary materials, mixing, granulate, incapsulate, make 1000, to obtain final product.
Detection method:
(1) get this product 10, incline and content, porphyrize, add methyl alcohol 30ml, ultrasonic process 30 minutes, filter, filtrate evaporate to dryness, the residue 10ml that adds water makes dissolving, adds hydrochloric acid 2ml, add hot reflux 30 minutes, cool immediately, extract 2 times with ethyl acetate jolting, each 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Harvest control medicinal material 1g with separately getting, add methyl alcohol 10ml, be made in the same way of control medicinal material solution.Get gallic acid reference substance again, add methyl alcohol and make the solution of every 1ml containing 0.5mg, compare product solution.Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VIB), draw above-mentioned three kinds of each 10ul of solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride-acetic ether-methanoic acid (9: 3: 1) for developping agent, launch, take out, dry, spray with 1% ferric trichloride ethanolic solution.In test sample chromatogram, with in control medicinal material chromatogram and reference substance chromatogram relevant position, show the spot of same color.
(2) get this product content 2g, porphyrize, add water 30ml, grinds 2 minutes, and filter, filtrate extracts 2 times with methenyl choloride jolting, each 20ml, and merge methenyl choloride liquid, evaporate to dryness, residue adds methenyl choloride 1ml makes dissolving, as need testing solution.Separately get Radix zanthoxyli control medicinal material 1g, boiling 20 minutes, filter, filtrate makes control medicinal material solution with need testing solution preparation method.Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VIB), draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same with sodium carboxymethyl cellulose be binder silica gel g thin-layer plate on, with methenyl choloride-acetone-formic acid (18: 2: 1) for developping agent, launch, take out, dry, inspect under putting ultraviolet lamp (365nm), in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the fluorescence principal spot of aobvious same color.
(3) get this product content 3g, add methenyl choloride 30ml, add hot reflux 2 hours, let cool, filter, filtrate is concentrated into 0.5ml, as need testing solution.Separately get psoralen reference substance to add methenyl choloride and make every 1ml containing the solution of 1mg, product solution in contrast.Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VIB), draw above-mentioned need testing solution 20 μ l, reference substance solution 5 μ l, put respectively in same with sodium carboxymethyl cellulose be binder silica gel g thin-layer plate on, with n-hexane-ethyl acetate (9: 1) for developping agent, launch, take out, dry, spray with 10% potassium hydroxide solution colour developing, inspect under putting ultraviolet lamp (365nm).In test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color.
(4) assay
According to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 annex VID).
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filling agent, and acetonitrile-1% glacial acetic acid (15: 85) is mobile phase, and determined wavelength is 323nm.Number of theoretical plate calculates should be not less than 1500 by forulic acid peak.
The preparation of reference substance solution learns from else's experience the phosphorus pentoxide drying under reduced pressure forulic acid reference substance of 24 hours in right amount, accurately weighed, adds methyl alcohol and makes the solution of every 1ml containing 6.0 μ g.
This product content is got in the preparation of need testing solution, porphyrize, get, precision takes about 5g, be placed in 100ml tool plug conical flask, add 50ml water, ultrasonic process (power 90W, frequency 59KHz) 40 minutes, let cool, filter, the accurate subsequent filtrate 25ml that draws is in separating funnel, with salt acid for adjusting pH value to 2, 5 times are extracted with ether jolting, each 15ml, merge ether solution, 5 times are extracted with 5% sodium bicarbonate solution jolting, each 30ml, merge sodium bicarbonate solution, with salt acid for adjusting pH value to 2, 5 times are extracted with ether jolting, each 15ml, merge ether solution, volatilize, residue methyl alcohol dissolves and moves in the brown measuring bottle of 5ml, add methanol constant volume to scale, shake up, filter with miillpore filter (0.45 μM), get subsequent filtrate and get final product.
Determination method is accurate respectively draws reference substance solution and each 20 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product.
This product every contains Radix Angelicae Sinensis with forulic acid (C
10h
10o
4) meter, 2.5 μ g must not be less than.
Experimental example 1: TLC distinguish is harvested on ground
The preparation of need testing solution: get this product 10, incline and content, porphyrize, add methyl alcohol 30ml, ultrasonic process 30 minutes, filter, filtrate evaporate to dryness, the residue 10ml that adds water makes dissolving, adds hydrochloric acid 2ml, add hot reflux 30 minutes, cool immediately, extract 2 times with ethyl acetate jolting, each 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.
The preparation of control medicinal material solution: harvest control medicinal material 1g with getting, adds methyl alcohol 10ml, is made in the same way of control medicinal material solution.
The preparation of reference substance solution: get gallic acid reference substance, adds methyl alcohol and makes the solution of every 1ml containing 0.5mg, product solution in contrast.
The preparation of negative control solution: get the negative sample harvested, makes with need testing solution preparation method and scarce harvests negative control solution scarcely.
Thin-layer chromatography: according to thin-layered chromatography (annex VI B) test, draw each 10 μ l of above-mentioned four kinds of solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride-acetic ether-methanoic acid (9: 3: 1) for developping agent, launch, take out, dry, spray with 1% ferric trichloride ethanolic solution.In test sample chromatogram, on the position corresponding with reference substance chromatogram to control medicinal material, the spot of aobvious same color.By test of many times, reproducible, negative control is noiseless, therefore lists text in.The results are shown in Figure 1, wherein, 1 for negative control 2.3.4. be test sample, lot number: 130606130607130608; 5 for harvest control medicinal material; 6 is gallic acid reference substance, T=25 DEG C, RH=58%.
Durability is investigated
The comparison of different thin layer plate
Get same ground and harvest control medicinal material solution, gallic acid reference substance solution, 3 batch sample solution (the method preparation worked out by text), put respectively on homemade silica gel g thin-layer plate and common prefabricated silica gel g thin-layer plate (production of east, Shanghai medicine Scientific and Technical Industry Co., Ltd), the silica gel g thin-layer plate of result two kinds of different brands all can reach good separating effect, result is basically identical, the results are shown in Figure 2.Wherein, 1.2.3. is test sample, lot number: 130606130607130608; 4 for harvest control medicinal material; 5 is gallic acid reference substance, T=25 DEG C, RH=58%.
The comparison of different temperatures
Get same ground and harvest control medicinal material solution, gallic acid reference substance solution, 3 batch sample solution (the method preparation worked out by text), point is on homemade silica gel g thin-layer plate, investigate ambient temperature and moisture (A:T=25 DEG C respectively, and low temperature often wet (B:T=8 DEG C RH:=58%), RH=58%) separation case under two kinds of conditions, result shows all can reach good separating effect under above temperature conditions, and temperature is little on separation impact, sees Fig. 3.Wherein, 1.2.3. is test sample, lot number: 130606130607130608; 4 for harvest control medicinal material; 5 is gallic acid reference substance, T=8 DEG C, RH=58%.
The comparison of different humidity
Get same ground and harvest control medicinal material solution, gallic acid reference substance solution, 3 batch sample solution, point is on homemade silica gel g thin-layer plate, investigate normal temperature high humidity (B:T=25 DEG C respectively, and normal temperature low humidity (C:T=25 DEG C RH=88%), RH=32%) separation case under three kinds of conditions, result shows all can reach good separating effect under above damp condition, humidity is little on separation impact, see Fig. 3, in Fig. 3,1.2.3. be test sample, lot number: 130606130607130608; 4 for harvest control medicinal material; 5 is gallic acid reference substance; T=25 DEG C, RH=32%.
Experimental example 2: the indentification by TLC of Radix zanthoxyli
Ministerial drug in the Radix zanthoxyli side of being, the discriminating item of this medicinal material has been set up in former method, but find that control medicinal material chromatogram is corresponding with test sample chromatogram spot neat not through test of many times, control medicinal material occurs four spots and test sample can only correspondence wherein two spots, and therefore this method is revised on the basis of former method.The ethanol water bath that test sample extracts and adopts the grinding that adds water, chloroform extraction replaces former method adds hot reflux, and extraction efficiency is higher.Control medicinal material uses chloroform extraction after then adopting soak by water, because adopt chloroform better as solvent spot mouldability.Developping agent changes to chloroform-acetone-formic acid (18: 2: 1), and other inspection methods remain unchanged.Separately get not containing other flavour of a drug of Radix zanthoxyli by prescription, make negative sample by preparation method, carry out negative control experiments, result feminine gender is noiseless, and control medicinal material is corresponding with test sample spot neatly, rounding, clear, shows that this method is feasible.Through many batch samples Germicidal efficacy, in test sample chromatogram, on the position corresponding to contrast medicine chromatogram, the fluorescence spot of aobvious same color, all conforms with the regulations.TLC collection of illustrative plates Fig. 4 of Annex III batch sample, Radix zanthoxyli control medicinal material is provided by Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 121014-200302.In figure, 1, Radix zanthoxyli control medicinal material 2,3,4 three batch sample 5, negative sample.
Experimental example 3: the characteristic component psoralen indentification by TLC of radix fici simplicissimae
With reference to the development system in related documents, expand into the best with normal hexane-ethyl acetate (7: 3), inspect under having ultraviolet lamp (365nm).All the other a few taste medicinal materials separately got except radix fici simplicissimae medicinal material make negative sample by method for making, carry out negative test, test findings shows that negative sample does not disturb principal spot, through several batch sample Germicidal efficacy, control medicinal material neat, rounding corresponding to test sample spot, clear, the method that is indicated as is easy, feasible.The TLC collection of illustrative plates 5 of Annex III batch sample, psoralen reference substance is provided by Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 110739-200209.In figure, 1, Psoralen refers to plain reference substance 2,3,4 three batch sample 5, negative sample.
Effective constituent ferulaic acid content in experimental example 4 Radix Angelicae Sinensis medicinal material measures
Medical capsule for treating pelvic inflammatory disease is harvested by ground, Radix zanthoxyli, Radix Angelicae Sinensis, radix fici simplicissimae, cudrania root five kinds of Chinese medicine form, and now drafts assay item to control this quality further.Harvest as monarch drug in a prescription in side, due to ground, to harvest medicinal material fundamental research weak, and effective constituent is unclear, is difficult to accurate quantitative analysis.And Radix zanthoxyli has promoting qi circulation and relieving pain, promoting blood circulation and removing blood stasis, effect of dispelling wind and removing obstruction in the meridians, but adopt HPLC method to carry out assay to its effective constituent Nitidine Chloride, result is extremely low because of the Nitidine Chloride content in this preparation test product, is difficult to accurate quantitative analysis, therefore transfer to content assaying method research is carried out to the effective constituent forulic acid in Radix Angelicae Sinensis medicinal material, the features such as this method of result has accurately, favorable reproducibility, specificity strong, fast and simple, can be used for the inherent quality controlling this product.
One, Radix Angelicae Sinensis medicinal material ferulaic acid content measures
Table 10 batches of Radix Angelicae Sinensis medicinal material ferulaic acid content measurement results
According to ten batches of Radix Angelicae Sinensis medicinal material forulic acid measurement results, the settled medicinal material internal control quality standard ferulaic acid content of returning of my Inc. must not be less than 0.01%.
Two, the assay of forulic acid in sample
(1) instrument and reagent
AgilentG1311AQualPump, G1314SVWD, G1379ADEGASSER, AT-330 column oven, Thermo-C18 (250mm × 4.6mm, 5um) chromatographic column, Sai Duolisi full-automatic electronic balance, SG2200H ultrasonic cleaner.
Forulic acid reference substance is provided (assay is used) by Nat'l Pharmaceutical & Biological Products Control Institute, lot number 0773-9910.
Acetonitrile is chromatographically pure, and water is distilled water, and it is pure that other reagent is analysis.
(2) chromatographic condition
Chromatographic column: octadecyl silane post (ODS) 4.6 × 250mm; Mobile phase: acetonitrile-1% glacial acetic acid (15: 85); Determined wavelength: 323nm; Room temperature; Flow velocity 1.0ml/min.Number of theoretical plate is all greater than 1500.
Condition analysis like this, main peak and other component of the forulic acid in preparation all can reach baseline separation, and wherein the retention time of forulic acid is 17.8 points.
(3) determination of determined wavelength
Get forulic acid reference substance solution to detect under above-mentioned chromatographic condition, carry out termination of pumping UV scanning, obtain absorption curve when main peak concentration is the highest, its maximum absorption band is 323nm, consistent with bibliographical information, therefore selects 323nm wavelength as determined wavelength.
(4) preparation of need testing solution
Forulic acid dissolves in methyl alcohol, ethanol, is soluble in ether, once adopts the direct ultrasonic extraction of methyl alcohol, because in this product, the content of forulic acid is lower, the amount of accurate quantitative analysis test sample to need to strengthen, the color of such need testing solution is very dark, direct injected chromatographic column is easily damaged, therefore need process further.Then adopt the ultrasonic extraction of water herein, then pH value to 2 is regulated with hydrochloric acid, make forulic acid fully free, with ether, it is extracted from water, then extract with 5% sodium bicarbonate solution, hydrochloric acid adjusts pH value to 2, extracted with diethyl ether, sample light proposed like this, impurity is few, and respond high, be easy to accurate quantitative analysis.
(5) blank test
Remove the negative sample of Radix Angelicae Sinensis medicinal material by prescription, measure according to content assaying method in text, compare need testing solution chromatogram and forulic acid reference substance solution chromatogram, in result negative sample, other compositions are noiseless to forulic acid chromatographic peak.
(6) investigation of linear relationship
Precision takes through the phosphorus pentoxide drying under reduced pressure asafoetide reference substance 11.6mg of 24 hours, put in the brown measuring bottle of 50ml, add methyl alcohol to dissolve, and be diluted to scale, shake up (232 μ g/ml), precision draws 0.5ml respectively again, 1ml, 2ml, 3ml, 6ml is placed in the brown measuring bottle of 50ml respectively, with methanol dilution to scale.Be mixed with the reference substance solution of variable concentrations: 1.55 μ g/ml, 3.10 μ g/ml, 6.20 μ g/ml, 9.28 μ g/ml, 18.56 μ g/ml.The each 20 μ l of the above-mentioned variable concentrations reference substance solution of accurate absorption, measure according to method in text, the results are shown in Table 2 respectively.
Table 2 linear relationship investigates result
With integrating peak areas value for ordinate, forulic acid amount is horizontal ordinate drawing standard curve, calculates regression equation: Y=5419.1X-3.72, r=1.0000.Show that forulic acid amount has good linear relation within the scope of 0.031 μ g ~ 0.3712 μ g.
(7) stability test
Get the reference substance solution (6.20 μ g/ml) under above-mentioned typical curve item and each 20 μ l of need testing solution, injection liquid chromatography, sample introduction measures once at regular intervals, and place in 8 hours in lucifuge, peak area is basically identical.Show that asafoetide reference substance solution and need testing solution are placed in 8 hours stable in lucifuge.(see table 3,4)
Table 3 reference substance solution stability test result
Table 4 sample solution stability test result
(8) precision test
Accurate absorption reference substance solution (9.28 μ g/ml), repeat sample introduction 5 times, the relative standard deviation of forulic acid area integral value is less than 2%, shows that this method precision is good.(see table 5)
Table 5 Precision test result
(9) reappearance test
Get same batch sample 5 parts respectively, by method test under assay item in text, measure the content of forulic acid, the results are shown in Table 6, the relative standard deviation of ferulaic acid content is less than 2%, shows that this method reappearance is good.
Table 6 reproducible test results
(10) recovery test actual measurement forulic acid amount-sample suitable forulic acid amount
Add forulic acid reference substance amount
Adopt application of sample absorption method.Get the medical capsule for treating pelvic inflammatory disease (content 14.27 μ g/g) with a collection of known content, precision takes porphyrize sample and is about 5g, totally 5 parts, each precision adds forulic acid reference substance solution (78 μ g/ml) 1ml, then method under assay item of pressing in text measures ferulaic acid content.The results are shown in Table 7, average recovery rate is 9542%.
Table 7 recovery test result
(11) sample size measures and limit formulation
Get this product 10 batches, measure content by text method, the results are shown in Table 8
Table 8
According to said determination result, tentative this product every must not lower than 2.5 μ g containing forulic acid.This product every contains Radix Angelicae Sinensis with forulic acid (C
10h
l0o
4) meter, 2.5 μ g must not be less than.
Claims (2)
1. prepare the detection method of medical capsule for treating pelvic inflammatory disease for one kind, wherein said medical capsule for treating pelvic inflammatory disease is harvested by ground, Radix zanthoxyli, Radix Angelicae Sinensis, radix fici simplicissimae, the wooden one-tenth of three-bristle cudrania, harvest describedly, Radix zanthoxyli, Radix Angelicae Sinensis, radix fici simplicissimae, three-bristle cudrania wood weight ratio be 450: 170: 140: 100: 140; Described medical capsule for treating pelvic inflammatory disease adopts following preparation method to obtain:
Harvest 450g Radix zanthoxyli 170g Radix Angelicae Sinensis 140g
Radix fici simplicissimae 100g three-bristle cudrania wood 140g
The above five tastes, boiling secondary, add water 12 times amount at every turn, decocts 2 hours, filter, merging filtrate, is concentrated into relative density and is determined as 1.25 at 55 ~ 60 DEG C, add ethanol and reach 50% to alcohol content, leave standstill 24 hours, filter, filtrate recycling ethanol, is concentrated into relative density is determined as 1.35 ~ 1.40 thick paste at 60 DEG C, dry, be ground into fine powder, add appropriate amount of auxiliary materials, mixing, granulate, incapsulate, make this product 1000;
The detection method preparing described medical capsule for treating pelvic inflammatory disease is:
(1) get this product 10, incline and content, porphyrize, add methyl alcohol 30ml, ultrasonic process 30 minutes, filter, filtrate evaporate to dryness, the residue 10ml that adds water makes dissolving, adds hydrochloric acid 2ml, add hot reflux 30 minutes, cool immediately, extract 2 times with ethyl acetate jolting, each 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Harvest control medicinal material 1g with separately getting, add methyl alcohol 10ml, be made in the same way of control medicinal material solution; Get gallic acid reference substance again, add methyl alcohol and make the solution of every 1ml containing 0.5mg, compare product solution, according to thin-layered chromatography test, draw each 10 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, methenyl choloride-acetic ether-methanoic acid with 9: 3: 1 is for developping agent, launch, take out, dry, spray is with 1% ferric trichloride ethanolic solution, in test sample chromatogram, with in control medicinal material chromatogram and reference substance chromatogram relevant position, show the spot of same color;
(2) get this product content 2g, porphyrize, add water 30ml, grinds 2 minutes, and filter, filtrate extracts 2 times with methenyl choloride jolting, each 20ml, and merge methenyl choloride liquid, evaporate to dryness, residue adds methenyl choloride 1ml makes dissolving, as need testing solution; Separately get Radix zanthoxyli control medicinal material 1g, boiling 20 minutes, filter, filtrate makes control medicinal material solution with need testing solution preparation method, test according to thin-layered chromatography, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same with sodium carboxymethyl cellulose be binder silica gel g thin-layer plate on, the methenyl choloride-acetone-formic acid with 18: 2: 1, for developping agent, launches, take out, dry, inspect under putting 365nm length ultraviolet light modulation, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the fluorescence principal spot of aobvious same color;
(3) get this product content 3g, add methenyl choloride 30ml, add hot reflux 2 hours, let cool, filter, filtrate is concentrated into 0.5ml, as need testing solution; Separately get psoralen reference substance to add methenyl choloride and make every 1ml containing the solution of 1mg, product solution in contrast, test according to thin-layered chromatography, draw above-mentioned need testing solution 20 μ l, reference substance solution 5 μ l, put respectively in same with sodium carboxymethyl cellulose be binder silica gel g thin-layer plate on, n-hexane-ethyl acetate with 9: 1 is developping agent, launches, and takes out, dry, spray with 10% potassium hydroxide solution colour developing, inspect under putting 365nm length ultraviolet light modulation, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color;
(4) assay
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filling agent, and acetonitrile-1% glacial acetic acid with 15: 85 is mobile phase, and determined wavelength is 323nm, and number of theoretical plate calculates by forulic acid peak and is not less than 1500,
The preparation of reference substance solution learns from else's experience the phosphorus pentoxide drying under reduced pressure forulic acid reference substance of 24 hours in right amount, accurately weighed, adds methyl alcohol and makes the solution of every 1ml containing 6.0 μ g,
This product content is got in the preparation of need testing solution, porphyrize, precision takes about 5g, be placed in 100ml tool plug conical flask, add 50ml water, ultrasonic process 40 minutes, let cool, filter, the accurate subsequent filtrate 25ml that draws is in separating funnel, with salt acid for adjusting pH value to 2, 5 times are extracted with ether jolting, each 15ml, merge ether solution, 5 times are extracted with 5% sodium bicarbonate solution jolting, each 30ml, merge sodium bicarbonate solution, with salt acid for adjusting pH value to 2, 5 times are extracted with ether jolting, each 15ml, merge ether solution, volatilize, residue methyl alcohol dissolves and moves in the brown measuring bottle of 5ml, add methanol constant volume to scale, shake up, filter with miillpore filter, get subsequent filtrate and get final product,
Determination method is accurate respectively draws reference substance solution and each 20 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product,
Wherein every contains Radix Angelicae Sinensis with forulic acid (C
10h
10o
4) meter, be no less than 2.5 μ g.
2. detection method as claimed in claim 1, the power 90W of wherein ultrasonic process in step (4), frequency 59KHz.
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| CN115327001A (en) * | 2022-09-27 | 2022-11-11 | 广州科曼生物科技有限公司 | Ficus simplicissima lour formula particle contrast extract and preparation method thereof |
| CN117437980A (en) * | 2023-12-20 | 2024-01-23 | 广东罗浮山国药股份有限公司 | Screening method of core genes acting on chronic pelvic inflammatory disease by using gongyan-Ping |
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