CN106841499A - A kind of method of quality control of old iron stop dysentery soluble powder preparation - Google Patents
A kind of method of quality control of old iron stop dysentery soluble powder preparation Download PDFInfo
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- CN106841499A CN106841499A CN201611226259.3A CN201611226259A CN106841499A CN 106841499 A CN106841499 A CN 106841499A CN 201611226259 A CN201611226259 A CN 201611226259A CN 106841499 A CN106841499 A CN 106841499A
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Abstract
The invention provides a kind of method of quality control of old iron stop dysentery soluble powder preparation, belong to drug tests, the first step, preparation of preparation；Second step, with Berberine hydrochloride, palmatin hydrochloride as reference substance, whether thin-layered chromatography contains Chinese mahonia composition in differentiating old iron stop dysentery soluble powder；3rd step, with iron holly bark as control medicinal material, Syringin as reference substance, whether thin-layered chromatography contains iron holly bark composition in differentiating old iron stop dysentery soluble powder；4th step, with matrine, Sophoridine as reference substance, whether thin-layered chromatography contains kuh-seng composition in differentiating old iron stop dysentery soluble powder；5th step, with Syringin as reference substance, the iron holly bark content in the old iron stop dysentery soluble powder prescription of Syrups by HPLC.Beneficial effects of the present invention are：The quality standard control method of formulation, with the degree of accuracy is high, favorable reproducibility the features such as.The controllability of the quality standard of preparation is can ensure that simultaneously, it is ensured that product inherent quality and curative effect, have to clinical application and know meaning.
Description
Technical field
The present invention relates to the analysis technical field of pharmaceutical preparation, more particularly to a kind of matter of old iron stop dysentery soluble powder preparation
Amount control method.
Background technology
The white scour of piglet is the common enteric infectious disease of piglet caused by ETEC, and this disease often betides 10-30 age in days piglets, faces
It is characterized with discharging milky or the sticky stench excrement of canescence on bed.The disease is popular wide, propagates fast, and the incidence of disease is high, though it is dead
Rate is relatively low, but such as treats not in time or malpractice, the lighter's influence Piglet Development, and serious meeting causes cad pig, to pig
Industry brings about great losses.
The piglet of pullorum disease is suffered from treatment, needs clearing heat and detoxicating, anti-inflammation, convergence, stop dysentery.Old iron stop dysentery soluble powder is main
For treating the white scour of piglet.We use Chinese mahonia, relieving cough and reducing sputum with heat-clearing qi-restoratives.Control tuberculosis hemoptysis, osteopyrexia and fever, head
Dizzy tinnitus, waist soreness is vexed, hot eyes；Iron holly bark cures mainly laryngalgia, cold, fever, stomachache, stomachache, anxious slow hepatitis, diarrhoea intestines
Inflammation, heatstroke stomachache, rheumathritis；Geranium wilfordii have antibacterial action, antivirus action, anti-diarrhea effect, hemoglutination, eliminating the phlegm,
Anthelmintic action；Acalypha copperleaf cures mainly clearing heat and detoxicating, dampness removing, astringing to arrest bleeding, for enteritis, dysentery, haematemesis, bleeding from five sense organs or subcutaneous tissue, have blood in stool, hematuria,
Uterine bleeding；Control carbuncle furuncle sore, dermatitis and eczema outward；Kuh-seng is used for hot dysentery, has blood in stool, jaundice renal shutdown, leukorrhea with reddish discharge, swelling of vulva pruritus vulvae, eczema,
Wet sore, pruitus, mange leprosy.Complete square compatibility 5 rationally, is treated both principal and secondary aspect of disease, and can play clearing heat and detoxicating, convergence, the mesh of stop dysentery
's.
Its curative effect of this prescription is indubitable, still has larger gap relative to the requirement of current tcm product quality control,
Fully effective the quality of old iron stop dysentery soluble powder preparation can not be controlled, so as to the production and sales of product and quality can be influenceed to protect
Card.
The content of the invention
It is an object of the invention to provide a kind of method of quality control of old iron stop dysentery soluble powder preparation.
For achieving the above object, the present invention is realized by following measure：A kind of old iron stop dysentery soluble powder system
The method of quality control of agent, comprises the following steps：
The first step, preparation of preparation：By Chinese mahonia, iron holly bark, geranium wilfordii, acalypha copperleaf, kuh-seng gomi herbs according to 3:2:
3:3:3 quality parts ratio is extracted, concentrated, spray drying, and mixed accessories obtain old iron stop dysentery soluble powder preparation；
Second step, with Berberine hydrochloride, palmatin hydrochloride as reference substance, thin-layered chromatography differentiates old iron stop dysentery soluble powder
In whether contain Chinese mahonia composition；
3rd step, with iron holly bark as control medicinal material, Syringin as reference substance, thin-layered chromatography differentiates that old iron stop dysentery is solvable
Property powder in whether contain iron holly bark composition；
4th step, with matrine, Sophoridine as reference substance, whether thin-layered chromatography contains in differentiating old iron stop dysentery soluble powder
There is kuh-seng composition；
5th step, with Syringin as reference substance, rescuing in the old iron stop dysentery soluble powder prescription of Syrups by HPLC must
Answer content.
Wherein, the detailed process of the first step is：
By Chinese mahonia, iron holly bark, geranium wilfordii, acalypha copperleaf, kuh-seng gomi herbs according to 3:2:3:3:3 ratio is mixed
Close, adding the water of 2 times of amounts carries out 2 extractions, and relative density is 1.2 when carrying out being concentrated under reduced pressure into 60 DEG C by extract solution after extraction
Lotion, carefully carries out lotion spray drying and obtains pre-composition in 45 DEG C of condition, mixes the starch and 1% carvacrol of pre-composition 10%
Obtain old iron stop dysentery soluble powder preparation.
Wherein, the detailed process of second step is：
1. the preparation of need testing solution：Old iron stop dysentery soluble powder sample 5g is taken, methyl alcohol 25ml, ultrasonic 30min, mistake is added
Filter, filtrate is evaporated, plus methyl alcohol 3ml makes dissolving, used as need testing solution；
2. the preparation of reference substance solution：Berberine hydrochloride, palmatin hydrochloride reference substance are taken, plus methyl alcohol is made every 1ml and contains
The mixed solution of 0.2mg, as reference substance solution；
3. differentiate：Need testing solution and each 5 μ l of two kinds of solution of reference substance solution are drawn, is put respectively in same silica G thin layer
On plate, with benzene-ethyl acetate-methanol-isopropanol-strong ammonia solution according to 6:3:1.5:1.5:0.5 quality parts ratio is mixed into
Solvent, puts in the chromatography cylinder of ammonia saturated with vapor, launches, and takes out, and dries, and puts and inspect under 365nm ultraviolet lamps, test sample chromatogram
In, on position corresponding with reference substance chromatogram, show two identical yellow fluorescence spots.
Wherein, the detailed process of the 3rd step is：
1. the preparation of need testing solution：Take old iron stop dysentery soluble powder sample 1.0g, plus ethanol 25ml, it is ultrasonically treated
20min, filtration, filtrate is evaporated, and the residue 20ml that adds water makes dissolving, is shaken with water saturated n-butanol and extracted 2 times, each 25ml,
Merge n-butanol liquid, wash with ammonia solution 20ml, discard ammoniacal liquor, take n-butanol liquid, be evaporated, residue adds the methyl alcohol 1ml to make dissolving, work
It is need testing solution；
2. the preparation of control medicinal material solution and reference substance solution：Iron holly bark control medicinal material 0.5g, plus ethanol 25ml separately are taken, is surpassed
Sonication 20min, filtration, filtrate is evaporated, and the residue 20ml that adds water makes dissolving, is shaken with water saturated n-butanol and extracts 2 times, every time
25ml, merges n-butanol liquid, is washed with ammonia solution 20ml, discards ammoniacal liquor, takes n-butanol liquid, is evaporated, and it is molten that residue adds methyl alcohol 1ml to make
Solution, is made control medicinal material solution；Syringin reference substance is taken again, plus methyl alcohol is made solution of every 1ml containing 1mg, as reference substance
Solution；
3. differentiate：Need testing solution, control medicinal material solution and each 2 μ l of three kinds of solution of reference substance solution are taken, is put in same respectively
On one silica gel g thin-layer plate, with chloroform-acetate-methanol-water-formic acid according to 8:24:5:2:2 quality parts ratio is mixed
Solvent is combined into, is launched, taken out, dried, sprayed with 10% ethanol solution of sulfuric acid, colour developing is heated at 105 DEG C clearly, day is put respectively
Inspected under light and ultraviolet lamp (365nm), in test sample chromatogram, on position corresponding with reference substance chromatogram, show same color
Spot.
Wherein, the detailed process of the 4th step is：
1. the preparation of need testing solution：Take old iron stop dysentery soluble powder sample 1g, enriching ammonia solution 0.3ml, three filter methane
25ml, stands overnight, filtration, and filtrate is evaporated, and residue adds the three filter methane 0.5ml to make dissolving, used as need testing solution；
2. the preparation of reference substance solution：Matrine reference substance, Sophoridine reference substance are taken, plus ethanol is made every 1ml and contains
The mixed solution of 0.2mg, as reference substance solution；
3. differentiate：Need testing solution and each 4 μ l of two kinds of solution of reference substance solution are drawn, is put respectively in the same silicon being made
On glue G lamellaes, with toluene-acetone-methyl alcohol according to 8：3：0.5 quality parts ratio is mixed into solvent, ammonia steam presaturation
Expansion cylinder in launch, exhibition is taken out away from 8cm, is dried, then with toluene-ethyl acetate-methanol-water according to 2：4：2：1 mass parts
Number is solvent than mixing and in 10 DEG C of upper solutions arranged below, after presaturation 30min, is launched, and is taken out, and is dried, successively
Spray with bismuth potassium iodide test solution and natrium nitrosum ethanol solution, inspect, in test sample chromatogram, with control medicinal material and reference substance solution
On corresponding position, show the spot of same color.
Wherein, the detailed process of the 5th step is：
1. chromatographic condition：With octadecylsilane key as filler, with acetonitrile as mobile phase A, with water as Mobile phase B, press
Regulation in following table carries out gradient elution；Detection wavelength is 210nm, and number of theoretical plate is calculated according to Syringin peak and should be not less than
3000；
2. the preparation of reference substance solution：Accurately weighed Syringin, plus 50% methyl alcohol is made purple fourths of every 1ml containing 120 μ g
Fragrant glycosides solution, obtains final product reference substance solution；
3. the preparation of need testing solution：Old iron stop dysentery soluble powder 1g is taken, it is accurately weighed, put in 25ml measuring bottles, plus 50%
Methyl alcohol dissolved dilution to scale, shake up, take subsequent filtrate, obtain final product need testing solution；
4. differentiate：It is accurate respectively to draw reference substance solution and each 10 μ l of need testing solution, liquid chromatograph is injected, determine,
Per 1g samples containing iron holly bark with Syringin (C17H24O9) count no less than 2.50mg.
Beneficial effects of the present invention are：Invention increases the thin layer discriminating of Chinese mahonia, the thin layer of iron holly bark is optimized
Condition, is improved during kuh-seng differentiates to adding the requirement of 2% NaOH in silica gel g thin-layer plate by creating alkaline environment；It is excellent
Change effect in the discriminating of iron holly bark medicinal material, optimize thin layer condition；Silica gel g thin-layer plate is set to be applied to the thin of kuh-seng by setting
Layer differentiates；The quality standard control method of formulation, with the degree of accuracy is high, favorable reproducibility the features such as.The matter of preparation is can ensure that simultaneously
The controllability of amount standard, it is ensured that product inherent quality and curative effect, has to clinical application and knows meaning.
Brief description of the drawings
The thin layer of the old iron stop dysentery soluble powder Chinese mahonias of Fig. 1 differentiates.In figure, 1. Berberine hydrochloride, palmatin hydrochloride pair
According to the test sample 1 (lot number 20160601) of product 2. 3. test sample 2 (lot number 20160602) 4. test sample 3 (lot number 20160603) is 5. cloudy
Property control sample.
The thin layer of the old iron stop dysentery soluble powder iron holly barks of Fig. 2 differentiates (solvent：Chloroform-methyl alcohol-anhydrous formic acid
(16:4:1)).In figure, 1. the test sample 1 (lot number 20160601) of 2. iron holly bark control medicinal material of Syringin reference substance 3. is 4. for examination
Product 2 (lot number 20160602) 5. test sample 3 (lot number 20160603) 6. negative control sample.
The thin layer of the old iron stop dysentery soluble powder iron holly barks of Fig. 3 differentiates (solvent chloroform-acetate-methanol-water-
Formic acid (8:24:5:2:2)).In figure, the 1. (lot number of 2. iron holly bark control medicinal material of Syringin reference substance, 3. test sample 1
20160601) 4. test samples 2 (lot number 20160602) 5. test sample 3 (lot number 20160603) 6. negative control sample.
The thin layer of the old iron stop dysentery soluble powder kuh-sengs of Fig. 4 differentiates.In figure, 1. matrine, the test sample of Sophoridine reference substance 2.
1 (lot number 20160601) 3. test sample 2 (lot number 20160602) 4. test sample 3 (lot number 20160603) 5. negative control sample.
Fig. 5 Syringin reference substance liquid chromatograms.
The old iron stop dysentery soluble powder liquid chromatograms of Fig. 6.
Fig. 7 negative control solution liquid chromatograms.
Fig. 8 Syringins peak area-concentration standard curve.
Specific embodiment
For the technical characterstic for illustrating this programme can be understood, below by specific embodiment, this programme is illustrated.
A kind of method of quality control of old iron stop dysentery soluble powder preparation, comprises the following steps：
The first step, preparation of preparation：By Chinese mahonia, iron holly bark, geranium wilfordii, acalypha copperleaf, kuh-seng gomi herbs according to 3:2:
3:3:3 quality parts ratio is extracted, concentrated, spray drying, and mixed accessories obtain old iron stop dysentery soluble powder preparation；
Second step, with Berberine hydrochloride, palmatin hydrochloride as reference substance, thin-layered chromatography differentiates old iron stop dysentery soluble powder
In whether contain Chinese mahonia composition；
3rd step, with iron holly bark as control medicinal material, Syringin as reference substance, thin-layered chromatography differentiates that old iron stop dysentery is solvable
Property powder in whether contain iron holly bark composition；
4th step, with matrine, Sophoridine as reference substance, whether thin-layered chromatography contains in differentiating old iron stop dysentery soluble powder
There is kuh-seng composition；
5th step, with Syringin as reference substance, rescuing in the old iron stop dysentery soluble powder prescription of Syrups by HPLC must
Answer content.
Wherein, the detailed process of the first step is：
By Chinese mahonia, iron holly bark, geranium wilfordii, acalypha copperleaf, kuh-seng gomi herbs according to 3:2:3:3:3 ratio is mixed
Close, adding the water of 2 times of amounts carries out 2 extractions, and relative density is 1.2 when carrying out being concentrated under reduced pressure into 60 DEG C by extract solution after extraction
Lotion, carefully carries out lotion spray drying and obtains pre-composition in 45 DEG C of condition, mixes the starch and 1% carvacrol of pre-composition 10%
Obtain old iron stop dysentery soluble powder preparation.
Wherein, the detailed process of second step is：
1. the preparation of need testing solution：Old iron stop dysentery soluble powder sample 5g is taken, methyl alcohol 25ml, ultrasonic 30min, mistake is added
Filter, filtrate is evaporated, plus methyl alcohol 3ml makes dissolving, used as need testing solution；
2. the preparation of reference substance solution：Berberine hydrochloride, palmatin hydrochloride reference substance are taken, plus methyl alcohol is made every 1ml and contains
The mixed solution of 0.2mg, as reference substance solution；
3. differentiate：Need testing solution and each 5 μ l of two kinds of solution of reference substance solution are drawn, is put respectively in same silica G thin layer
On plate, with benzene-ethyl acetate-methanol-isopropanol-strong ammonia solution according to 6:3:1.5:1.5:0.5 quality parts ratio is mixed into
Solvent, puts in the chromatography cylinder of ammonia saturated with vapor, launches, and takes out, and dries, and puts and inspect under 365nm ultraviolet lamps, test sample chromatogram
In, on position corresponding with reference substance chromatogram, show two identical yellow fluorescence spots.
Wherein, the detailed process of the 3rd step is：
1. the preparation of need testing solution：Take old iron stop dysentery soluble powder sample 1.0g, plus ethanol 25ml, it is ultrasonically treated
20min, filtration, filtrate is evaporated, and the residue 20ml that adds water makes dissolving, is shaken with water saturated n-butanol and extracted 2 times, each 25ml,
Merge n-butanol liquid, wash with ammonia solution 20ml, discard ammoniacal liquor, take n-butanol liquid, be evaporated, residue adds the methyl alcohol 1ml to make dissolving, work
It is need testing solution；
2. the preparation of control medicinal material solution and reference substance solution：Iron holly bark control medicinal material 0.5g, plus ethanol 25ml separately are taken, is surpassed
Sonication 20min, filtration, filtrate is evaporated, and the residue 20ml that adds water makes dissolving, is shaken with water saturated n-butanol and extracts 2 times, every time
25ml, merges n-butanol liquid, is washed with ammonia solution 20ml, discards ammoniacal liquor, takes n-butanol liquid, is evaporated, and it is molten that residue adds methyl alcohol 1ml to make
Solution, is made control medicinal material solution；Syringin reference substance is taken again, plus methyl alcohol is made solution of every 1ml containing 1mg, as reference substance
Solution；
3. differentiate：Need testing solution, control medicinal material solution and each 2 μ l of three kinds of solution of reference substance solution are taken, is put in same respectively
On one silica gel g thin-layer plate, with chloroform-acetate-methanol-water-formic acid according to 8:24:5:2:2 quality parts ratio is mixed
Solvent is combined into, is launched, taken out, dried, sprayed with 10% ethanol solution of sulfuric acid, colour developing is heated at 105 DEG C clearly, day is put respectively
Inspected under light and ultraviolet lamp (365nm), in test sample chromatogram, on position corresponding with reference substance chromatogram, show same color
Spot.
Wherein, the detailed process of the 4th step is：
1. the preparation of need testing solution：Take old iron stop dysentery soluble powder sample 1g, enriching ammonia solution 0.3ml, three filter methane
25ml, stands overnight, filtration, and filtrate is evaporated, and residue adds the three filter methane 0.5ml to make dissolving, used as need testing solution；
2. the preparation of reference substance solution：Matrine reference substance, Sophoridine reference substance are taken, plus ethanol is made every 1ml and contains
The mixed solution of 0.2mg, as reference substance solution；
3. differentiate：Need testing solution and each 4 μ l of two kinds of solution of reference substance solution are drawn, is put respectively in the same silicon being made
On glue G lamellaes, with toluene-acetone-methyl alcohol according to 8：3：0.5 quality parts ratio is mixed into solvent, ammonia steam presaturation
Expansion cylinder in launch, exhibition is taken out away from 8cm, is dried, then with toluene-ethyl acetate-methanol-water according to 2：4：2：1 mass parts
Number is solvent than mixing and in 10 DEG C of upper solutions arranged below, after presaturation 30min, is launched, and is taken out, and is dried, successively
Spray with bismuth potassium iodide test solution and natrium nitrosum ethanol solution, inspect, in test sample chromatogram, with control medicinal material and reference substance solution
On corresponding position, show the spot of same color.
Wherein, the detailed process of the 5th step is：
1. chromatographic condition：With octadecylsilane key as filler, with acetonitrile as mobile phase A, with water as Mobile phase B, press
Regulation in following table carries out gradient elution；Detection wavelength is 210nm, and number of theoretical plate is calculated according to Syringin peak and should be not less than
3000；
| Time/minute | Mobile phase A/% | Mobile phase B/% |
| 0~10 | 10 | 90 |
| 10~20 | 10→40 | 90→60 |
| 20~30 | 40 | 60 |
2. the preparation of reference substance solution：Accurately weighed Syringin, plus 50% methyl alcohol is made purple fourths of every 1ml containing 120 μ g
Fragrant glycosides solution, obtains final product reference substance solution；
3. the preparation of need testing solution：Old iron stop dysentery soluble powder 1g is taken, it is accurately weighed, put in 25ml measuring bottles, plus 50%
Methyl alcohol dissolved dilution to scale, shake up, take subsequent filtrate, obtain final product need testing solution；
4. differentiate：It is accurate respectively to draw reference substance solution and each 10 μ l of need testing solution, liquid chromatograph is injected, determine,
Per 1g samples containing iron holly bark with Syringin (C17H24O9) count no less than 2.50mg.
Embodiment 1 is tested
The step of a kind of method of quality control of old iron stop dysentery soluble powder preparation, its method is：
First, project experiment is differentiated：
The thin layer discrimination test of 1 Chinese mahonia:
Old iron stop dysentery soluble powder quality standard differentiates and the thin layer of Chinese mahonia is differentiated, tests as follows:
1.1 experiment materials:
Berberine hydrochloride reference substance:(110713-201212 supplies assay to National Institute for Food and Drugs Control's offer
With)；
Palmatin hydrochloride reference substance：(110732-201510 supplies assay to National Institute for Food and Drugs Control's offer
With)；
Silica gel g thin-layer plate:Qingdao Sheng Hai fine silica gels Chemical Co., Ltd., 5 × 10cm.
The preparation of 1.2 need testing solutions：
Old iron stop dysentery soluble powder sample 5g is taken, methyl alcohol 25ml, ultrasonic 30min is added, filtered, filtrate is evaporated, plus methyl alcohol
3ml makes dissolving, used as need testing solution.Test sample 1 (lot number 20160601), test sample 2 (lot number 20160602), test sample 3
(lot number 20160603).
The preparation of 1.3 reference substance solutions：
Berberine hydrochloride, palmatin hydrochloride reference substance are taken, plus methyl alcohol is made every 1ml respectively mixed solutions containing 0.2mg, as
Reference substance solution.
The preparation of 1.4 negative control solutions：
In prescription ratio, the group's medicine without Chinese mahonia in prescription is taken, extracted by this product preparation technology, prepared, obtain ten
Big contribution negative control sample.Chinese mahonia negative control sample 5g is taken, by Chinese mahonia " preparation of need testing solution " method
It is made negative control solution.
1.5 experimental conditions
Solvent:Benzene-ethyl acetate-methanol-isopropanol-strong ammonia solution (6:3:1.5:1.5:0.5), ammonia steam is pre- full
With；
Lamellae:Silica gel g thin-layer plate；
Point sample amount:Each 5 μ l of reference substance solution, need testing solution；
Inspection method:Put and inspect under ultraviolet lamp (365nm).
1.6 experimental results:
In test sample chromatogram, on position corresponding with reference substance chromatogram, show the fluorescence spot of same color.Negative control
Solution is noiseless.This differentiates clear spot, easy to operate, stablizes feasible, with characteristic differentiation meaning.See Fig. 1.
The thin layer discrimination test of 2 iron holly barks:
Old iron stop dysentery soluble powder quality standard differentiates and the thin layer of iron holly bark is differentiated, tests as follows:
2.1 experiment materials:
Iron holly bark control medicinal material:For National Institute for Food and Drugs Control provides, (121076-201303 supplies drug inspection
With)；
Syringin reference substance：National Institute for Food and Drugs Control provides (111574-201504 is for assay).
Silica gel g thin-layer plate:Qingdao Sheng Hai fine silica gels Chemical Co., Ltd., 5 × 10cm.
The preparation of 2.2 need testing solutions：
Old iron stop dysentery soluble powder sample 1.0g, plus ethanol 25ml, ultrasonically treated 20min are taken, is filtered, filtrate is evaporated, residual
The slag 20ml that adds water makes dissolving, is shaken with water saturated n-butanol and extracted 2 times, and each 25ml merges n-butanol liquid, uses ammonia solution
20ml is washed, and discards ammoniacal liquor, takes n-butanol liquid, is evaporated, and residue adds the methyl alcohol 1ml to make dissolving, used as need testing solution.Test sample 1
(lot number 20160601), test sample 2 (lot number 20160602), test sample 3 (lot number 20160603).
The preparation of 2.3 reference substance solutions：Iron holly bark control medicinal material 0.5g separately is taken, control medicinal material solution is made in the same way of, then take
Syringin reference substance, plus methyl alcohol is made solution of every 1ml containing 1mg, used as reference substance solution.
The preparation of 2.4 negative control solutions：In prescription ratio, the group's medicine without iron holly bark in prescription is taken, prepared by this product
Technique is extracted, prepared, and obtains iron holly bark negative control sample.Iron holly bark negative control sample 1.0g is taken, by iron holly bark " test sample
The preparation of solution " method is made negative control solution.
2.5 experimental conditions
2.5.1 the investigation of beast official method
Solvent:Chloroform-methyl alcohol-anhydrous formic acid (16:4:1)；
Lamellae:Silica gel g thin-layer plate；
Point sample amount:Each 2 μ 1 of control medicinal material solution, reference substance solution, need testing solution；
Inspection method:Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to colour developing at 105 DEG C, and daylight and ultraviolet is put respectively
Inspected under light lamp (365nm).
2.5.2 the optimization of solvent
Solvent:Chloroform-acetate-methanol-water-formic acid (8:24:5:2:2)；
Lamellae:Silica gel g thin-layer plate；
Point sample amount:Each 2 μ 1 of control medicinal material solution, reference substance solution, need testing solution；
Inspection method:Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to colour developing at 105 DEG C, and daylight and ultraviolet is put respectively
Inspected under light lamp (365nm).
2.6 experimental results:
2.6.1 the investigation of beast official method
This differentiates that spot is not clear enough, and the Rf value of reference substance is also larger, sees Fig. 2.Condition also needs optimization.
2.6.2 the optimization of thin layer condition
In test sample chromatogram, on position corresponding with control medicinal material, reference substance chromatogram, show the spot of same color and glimmering
Light spot.Negative control solution is noiseless.This differentiates clear spot, easy to operate, stablizes feasible, with characteristic differentiation meaning
Justice.See Fig. 3.
The thin layer discrimination test of 3 kuh-sengs:
Old iron stop dysentery soluble powder quality standard differentiates and the thin layer of Chinese mahonia is differentiated, tests as follows:
3.1 experiment materials:
Matrine reference substance:National Institute for Food and Drugs Control provides (110805-200508 is for assay)；Chinese scholartree
Determine alkali reference substance：National Institute for Food and Drugs Control provides (110784-201405 is for assay).Silica gel g thin-layer plate:
Qingdao Sheng Hai fine silica gels Chemical Co., Ltd., 5 × 10cm.
The preparation of 3.2 need testing solutions：Soluble powder powder 1.0g, enriching ammonia solution 0.3ml, three filter methane 25ml are taken,
Stand overnight, filter, filtrate is evaporated, residue adds the three filter methane 0.5ml to make dissolving, used as need testing solution.(the lot number of test sample 1
20160601), test sample 2 (lot number 20160602), test sample 3 (lot number 20160603).
The preparation of 3.3 reference substance solutions：Matrine reference substance, Sophoridine reference substance are taken, plus ethanol is made every 1ml and contains
The mixed solution of 0.2mg, as reference substance solution.
The preparation of 3.4 negative control solutions：In prescription ratio, the group's medicine without kuh-seng in prescription is taken, work is prepared by this product
Skill is extracted, prepared, and obtains kuh-seng negative control sample.Kuh-seng negative control sample 1.0g is taken, by the kuh-seng " system of need testing solution
It is standby " method is made negative control solution.
3.5 experimental conditions
Solvent:1. toluene-acetone-methyl alcohol (8：3：0.5), ammonia steam presaturation, opens up away from 8cm；
2. toluene-ethyl acetate-methanol-water (2：4：2：1) 10 DEG C of upper solutions arranged below；
Lamellae:Silica gel g thin-layer plate；
Point sample amount:Each 4 μ 1 of reference substance solution, need testing solution；
Inspection method:Put observation under ultraviolet lamp (365nm).
3.6 experimental results:
In test sample chromatogram, on position corresponding with reference substance chromatogram, show the spot of same color.Negative control solution
It is noiseless.This differentiates clear spot, easy to operate, stablizes feasible, with characteristic differentiation meaning.See Fig. 4.
4 other
All contain gallic acid in geranium wilfordii and acalypha copperleaf, do not have specificity in discriminating, do not list method of quality control in.
2nd, content detection project experiment：
Syringin content detection is tested, and step is as follows：
1.1 instruments and reagent
Instrument:Agilent high performance liquid chromatograph (HPLC-1260), electronic balance AB204-S.
Reagent:Acetonitrile is chromatographically pure, and water is redistilled water；It is pure that other reagents are analysis.
Reference substance:For National Institute for Food and Drugs Control provides, (111574-201504 supplies content to Syringin reference substance
Determine and use).
Sample:Old iron stop dysentery soluble powder (Qingdao Agricultural University's offer).Test sample 1 (lot number 20160601), test sample 2
(lot number 20160602), test sample 3 (lot number 20160603).
1.2 chromatographic conditions and system suitability
Chromatographic column：Agilent ZORBAX Eclipse Plus(5μm,250mm×4.6mm)；Octadecyl silane
It is filler；Mobile phase:Acetonitrile-water, gradient elution (see the table below)；Flow velocity:1ml/min；Column temperature:30℃；Wavelength:210nm.
| Time/minute | Mobile phase A/% | Mobile phase B/% |
| 0-10 | 10 | 90 |
| 10-20 | 10→40 | 90→60 |
| 20-30 | 40 | 60 |
The preparation of need testing solution：This product 1g is taken, it is accurately weighed, in putting 25ml measuring bottles, the methyl alcohol dissolved dilution for plus 50%
To scale, shake up, take subsequent filtrate, obtain final product.
Reference substance solution prepares accurately weighed Syringin, plus 50% methyl alcohol is made lilacs of every 1ml containing 120 μ g
Glycosides solution, obtains final product.
The preparation of negative sample solution：In prescription ratio, the group's medicine without iron holly bark in prescription is taken, by this product preparation technology
Extract, prepare, obtain iron holly bark negative control sample.Take iron holly bark negative control sample 1g, by iron holly bark " need testing solution
Preparation " method is made negative control solution.
Determination method is accurate respectively to draw reference substance solution, need testing solution, each 10 μ l of negative control solution, injects liquid phase
Chromatograph, determines, and obtains final product.Liquid chromatogram is shown in Fig. 5, Fig. 6, Fig. 7 respectively.
The investigation of 1.3 linear relationships
Precision weighs Syringin reference substance 16.15mg, in putting the volumetric flask of 25ml, plus the dissolving of 50% methyl alcohol, and dilute
To scale, shake up, be 646 μ g/ml reference substance stock solutions as concentration.Respectively precision pipette stock solution 0.25ml, 0.5ml,
1.0ml, 2.0ml, 4.0ml are put in 10ml volumetric flasks, plus 50% methanol dilution is to scale, obtain 16.15,32.3,64.6,129.2,
The Syringin standard solution of 258.4 μ g/ml series concentrations.
Determination method is accurate respectively to draw the μ l of series concentration reference substance solution 10, injects liquid chromatograph, determines, and obtains final product.Knot
Fruit is shown in Table 1.
The Syringin reference substance measurement result of table 1
Peak area-concentration standard curve is shown in Fig. 8：
Regression equation y=34.52x-27.45 (R2=1).Result shows concentration in 16.15-258.4 μ g/ml scopes interior lines
Sexual intercourse is good.
1.4 precision tests
Reference substance solution (129.2 μ g/ml) is drawn, sample introduction is repeated 6 times, each 10 μ 1, the relative standard for calculating peak area is inclined
Difference, the results are shown in Table 2.
The Precision test result of table 2
Result of the test shows that the precision of instrument is good.
1.5 stability
Take this product and prepare need testing solution by need testing solution compound method, respectively at 0h, 2h, 4h, 6h, 8h, 12h sample introduction
Determine.The results are shown in Table 3.
The stability test result of table 3
Result shows that need testing solution is good in 12 hours internal stabilities.
1.6 reappearances are tested
6 parts of the sample of the old iron stop dysentery soluble powder (20150601) of same lot number is taken, is prepared by method of working out respectively, surveyed
Fixed, as a result Syringin content is shown in Table 4.
The reproducible test results of table 4
The μ g/ml of reference substance concentration 129.2, reference substance peak area 4395.2.
Result shows that the reappearance of test method is good.
1.7 recovery tests
Take the old iron stop dysentery soluble powder (lot number of known content:20150601 content 3.12mg/g), precision is weighed and taken
0.5g, puts in 25ml measuring bottles, Syringin reference substance solution (646 μ g/ml) 2.5ml is added, by " preparation of need testing solution "
Prepared by method, sample introduction, calculates the rate of recovery, the results are shown in Table 5.
The recovery test result of table 5
The μ g/ml of reference substance concentration 129.2, reference substance peak area 4395.2.
Result shows:The average recovery rate of this experiment is 0.84% for 106.75%, RSD, and sample-adding reclaims good.
1.8 sample determinations:Three batches of samples are taken, content is determined by the method in quality standard, the results are shown in Table 6.
Syringin content is calculated with external standard peak area method.
Syringin assay result in the sample of table 6
Note:The μ g/ml of reference substance concentration 129.2, reference substance peak area 4395.2.
Result shows that the content assaying method of Syringin is feasible in old iron stop dysentery soluble powder, and this product is per 1g containing rescue must
1.03mg should must not be less than in terms of Syringin (C17H24O9), 3 batches of sample sizes meet standard requirement.
It is of the invention to be realized by or using prior art without the technical characteristic for describing, will not be repeated here, certainly,
Described above is not limitation of the present invention, and the present invention is also not limited to the example above, the ordinary skill of the art
Change, remodeling, addition or replacement that personnel are made in essential scope of the invention, should also belong to protection model of the invention
Enclose.
Claims (6)
1. a kind of method of quality control of old iron stop dysentery soluble powder preparation, it is characterised in that comprise the following steps：
The first step, preparation of preparation：By Chinese mahonia, iron holly bark, geranium wilfordii, acalypha copperleaf, kuh-seng gomi herbs according to 3:2:3:3:3
Quality parts ratio extract, concentration, spray drying, mixed accessories obtain old iron stop dysentery soluble powder preparation；
Second step, with Berberine hydrochloride, palmatin hydrochloride as reference substance, thin-layered chromatography differentiate old iron stop dysentery soluble powder in be
It is no to contain Chinese mahonia composition；
3rd step, with iron holly bark as control medicinal material, Syringin as reference substance, thin-layered chromatography differentiates old iron stop dysentery soluble powder
In whether contain iron holly bark composition；
4th step, with matrine, Sophoridine as reference substance, whether thin-layered chromatography contains hardship in differentiating old iron stop dysentery soluble powder
Ginseng composition；
5th step, with Syringin as reference substance, the iron holly bark in the old iron stop dysentery soluble powder prescription of Syrups by HPLC contains
Amount.
2. the method for quality control of old iron stop dysentery soluble powder preparation according to claim 1, it is characterised in that the first step
Detailed process be：
By Chinese mahonia, iron holly bark, geranium wilfordii, acalypha copperleaf, kuh-seng gomi herbs according to 3:2:3:3:3 ratio is mixed,
Adding the water of 2 times of amounts carries out 2 extractions, and relative density is 1.2 cream when carrying out being concentrated under reduced pressure into 60 DEG C by extract solution after extraction
Body, carefully carries out lotion spray drying and obtains pre-composition in 45 DEG C of condition, adds starch and the mixing of 1% carvacrol of pre-composition 10%
Obtain old iron stop dysentery soluble powder preparation.
3. the method for quality control of old iron stop dysentery soluble powder preparation according to claim 1, it is characterised in that second step
Detailed process be：
1. the preparation of need testing solution：Old iron stop dysentery soluble powder sample 5g is taken, methyl alcohol 25ml, ultrasonic 30min is added, filtered,
Filtrate is evaporated, plus methyl alcohol 3ml makes dissolving, used as need testing solution；
2. the preparation of reference substance solution：Berberine hydrochloride, palmatin hydrochloride reference substance are taken, plus methyl alcohol is made every 1ml and respectively contains 0.2mg
Mixed solution, as reference substance solution；
3. differentiate：Need testing solution and each 5 μ l of two kinds of solution of reference substance solution are drawn, is put respectively in same silica gel g thin-layer plate
On, with benzene-ethyl acetate-methanol-isopropanol-strong ammonia solution according to 6:3:1.5:1.5:0.5 quality parts ratio is mixed into exhibition
Agent is opened, is put in the chromatography cylinder of ammonia saturated with vapor, launched, taken out, dried, put and inspect under 365nm ultraviolet lamps, test sample chromatogram
In, on position corresponding with reference substance chromatogram, show two identical yellow fluorescence spots.
4. the method for quality control of old iron stop dysentery soluble powder preparation according to claim 1, it is characterised in that the 3rd step
Detailed process be：
1. the preparation of need testing solution：Old iron stop dysentery soluble powder sample 1.0g, plus ethanol 25ml, ultrasonically treated 20min are taken, is filtered
Cross, filtrate is evaporated, the residue 20ml that adds water makes dissolving, shaken with water saturated n-butanol and extracted 2 times, each 25ml merges positive fourth
Alcohol liquid, is washed with ammonia solution 20ml, discards ammoniacal liquor, takes n-butanol liquid, is evaporated, and residue adds the methyl alcohol 1ml to make dissolving, used as test sample
Solution；
2. the preparation of control medicinal material solution and reference substance solution：It is another to take iron holly bark control medicinal material 0.5g, plus ethanol 25ml, at ultrasound
Reason 20min, filtration, filtrate is evaporated, and the residue 20ml that adds water makes dissolving, is shaken with water saturated n-butanol and extracts 2 times, every time
25ml, merges n-butanol liquid, is washed with ammonia solution 20ml, discards ammoniacal liquor, takes n-butanol liquid, is evaporated, and it is molten that residue adds methyl alcohol 1ml to make
Solution, is made control medicinal material solution；Syringin reference substance is taken again, plus methyl alcohol is made solution of every 1ml containing 1mg, as reference substance
Solution；
3. differentiate：Need testing solution, control medicinal material solution and each 2 μ l of three kinds of solution of reference substance solution are taken, is put respectively in same silicon
On glue G lamellaes, with chloroform-acetate-methanol-water-formic acid according to 8:24:5:2:2 quality parts ratio is mixed into
Solvent, launches, and takes out, and dries, and sprays with 10% ethanol solution of sulfuric acid, and it is clear to be heated to colour developing at 105 DEG C, put respectively daylight and
Ultraviolet lamp（365nm）Under inspect, in test sample chromatogram, on position corresponding with reference substance chromatogram, show same color spot
Point.
5. the method for quality control of old iron stop dysentery soluble powder preparation according to claim 1, it is characterised in that the 4th step
Detailed process be：
1. the preparation of need testing solution：Old iron stop dysentery soluble powder sample 1g, enriching ammonia solution 0.3ml, three filter methane 25ml are taken,
Stand overnight, filter, filtrate is evaporated, residue adds the three filter methane 0.5ml to make dissolving, used as need testing solution；
2. the preparation of reference substance solution：Matrine reference substance, Sophoridine reference substance are taken, plus ethanol is made every 1ml and respectively contains 0.2mg's
Mixed solution, as reference substance solution；
3. differentiate：Need testing solution and each 4 μ l of two kinds of solution of reference substance solution are drawn, is put respectively in the same silica G being made
On lamellae, with toluene-acetone-methyl alcohol according to 8：3：0.5 quality parts ratio is mixed into solvent, the pre-saturated exhibition of ammonia steam
Open and launch in cylinder, exhibition is taken out away from 8cm, is dried, then with toluene-ethyl acetate-methanol-water according to 2：4：2：1 quality parts ratio
Mix and 10 DEG C of upper solutions arranged below be solvent, after presaturation 30min, launch, take out, dry, spray successively with
Bismuth potassium iodide test solution and natrium nitrosum ethanol solution, inspect, in test sample chromatogram, corresponding to control medicinal material and reference substance solution
Position on, show same color spot.
6. the method for quality control of old iron stop dysentery soluble powder preparation according to claim 1, it is characterised in that the 5th step
Detailed process be：
1. chromatographic condition：With octadecylsilane key as filler, with acetonitrile as mobile phase A, with water as Mobile phase B, according to the form below
In regulation carry out gradient elution；Detection wavelength is 210nm, and number of theoretical plate is calculated according to Syringin peak and should be not less than 3000；
2. the preparation of reference substance solution：Accurately weighed Syringin, plus 50% methyl alcohol be made every 1ml containing 120 μ g Syringin it is molten
Liquid, obtains final product reference substance solution；
3. the preparation of need testing solution：Old iron stop dysentery soluble powder 1g is taken, it is accurately weighed, in putting 25ml measuring bottles, the methyl alcohol for plus 50%
Dissolved dilution shakes up to scale, takes subsequent filtrate, obtains final product need testing solution；
4. differentiate：It is accurate respectively to draw reference substance solution and each 10 μ l of need testing solution, liquid chromatograph is injected, determine, per 1g
Sample is containing iron holly bark with Syringin（C17H24O9）Meter is no less than 2.50mg.
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| CN112180024A (en) * | 2020-09-30 | 2021-01-05 | 西安雨田农业科技股份有限公司 | Quality control method of traditional Chinese medicine compound preparation for treating piglet diarrhea |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1125123A (en) * | 1995-08-21 | 1996-06-26 | 广西中兽药厂 | Anti-white-flux traditional Chinese medicine and Western medicine compound preparation for domestic animal and fowl |
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| 代蕾 等: "救必应及其常用同属药用植物高效薄层指纹图谱研究", 《广东药学院学报》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112180024A (en) * | 2020-09-30 | 2021-01-05 | 西安雨田农业科技股份有限公司 | Quality control method of traditional Chinese medicine compound preparation for treating piglet diarrhea |
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