CN109709256A - Scutelloside and chlorogenic acid thin layer identify detection method in a kind of Compound Jinyinhua Granules - Google Patents
Scutelloside and chlorogenic acid thin layer identify detection method in a kind of Compound Jinyinhua Granules Download PDFInfo
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- CN109709256A CN109709256A CN201811556742.7A CN201811556742A CN109709256A CN 109709256 A CN109709256 A CN 109709256A CN 201811556742 A CN201811556742 A CN 201811556742A CN 109709256 A CN109709256 A CN 109709256A
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Abstract
The present invention provides scutelloside and chlorogenic acid thin layer in a kind of Compound Jinyinhua Granules and identifies detection method, comprising the following steps: step 1, prepares test solution, negative controls solution, scutelloside reference substance solution and chlorogenic acid reference substance solution；Prepare solvent and color developing agent；Solvent includes ethyl acetate, methanol and acetic acid, and the volume ratio of ethyl acetate, methanol and acetic acid is (1~7): (2~5): (7~15)；Step 2, thin-layer chromatography: using polyamide film as lamellae, by on test solution, negative controls solution, scutelloside reference substance solution and the equal point sample to lamellae of chlorogenic acid reference substance solution, the lamellae for having put sample is put into the expansion cylinder of solvent saturation and is unfolded；Then lamellae is taken out, is dried；Step 3, it inspects, inspects chlorogenic acid, scutelloside under ultraviolet and daylight respectively.This method can carry out the identification of specificity thin layer to scutelloside in Compound Jinyinhua Granules and chlorogenic acid.
Description
Technical field
The invention belongs to medical science analysis fields, and in particular to scutelloside and chlorogenic acid in a kind of Compound Jinyinhua Granules
Thin layer identifies detection method.
Background technique
Compound Jinyinhua Granules are to extract its effective component through the modern times by honeysuckle, Fructus Forsythiae and 3 taste Chinese medicine of radix scutellariae, are added
The refined Chinese materia medica preparation of suitable sucrose.Its major function be clearing heat and detoxicating and cool blood detumescence, be mainly used for anemopyretic cold,
Pharyngitis, tonsillitis, mesh pain, toothache and carbuncle swells boil.It is now the common medicine of clinical treatment anemopyretic cold, curative effect does not accommodate
It doubts, but the quality of the product is controlled, the scutelloside and chlorogenic acid thin-layer identification method specificity of existing national standards are poor, spot
It loses count of clear, effective control for product quality is unable to, to influence the production and quality assurance of product.
Summary of the invention
In order to overcome the defects of above-mentioned standard, the purpose of the present invention is to provide radix scutellariaes in a kind of Compound Jinyinhua Granules
Glycosides and chlorogenic acid thin layer identify detection method, and this method can be realized to scutelloside in Compound Jinyinhua Granules and chlorogenic acid progress
Specificity thin layer identifies, and excludes the interference of other substances and separating degree is good, favorable reproducibility.
The present invention is to be achieved through the following technical solutions:
Scutelloside and chlorogenic acid thin layer identify detection method in a kind of Compound Jinyinhua Granules, comprising the following steps:
Step 1, it is molten that test solution, negative controls solution, scutelloside reference substance solution and chlorogenic acid reference substance are prepared
Liquid；Prepare solvent and color developing agent；Solvent includes ethyl acetate, methanol and acetic acid, the volume of ethyl acetate, methanol and acetic acid
Than for (1~7): (2~5): (7~15)；
Step 2, thin-layer chromatography: using polyamide film as lamellae, by test solution, negative controls solution, Huang
In a kind of reed mentioned in ancient books glycosides reference substance solution and the equal point sample to lamellae of chlorogenic acid reference substance solution, the lamellae for having put sample is put into solvent
It is unfolded in the expansion cylinder of saturation；Then lamellae is taken out, is dried；
Step 3, it inspects, inspects chlorogenic acid, scutelloside under ultraviolet and daylight respectively.
Preferably, in step 1, the preparation method of test solution are as follows: Compound Jinyinhua Granules are finely ground, methanol is added,
Ultrasound, filtering, takes filtrate to be evaporated, residue adds methanol to dissolve, and constant volume obtains test solution.
Preferably, in step 1, the preparation method of scutelloside reference substance solution and chlorogenic acid reference substance solution are as follows: take radix scutellariae
Glycosides and chlorogenic acid reference substance, are separately added into methanol, ultrasonic dissolution, and constant volume respectively obtains scutelloside reference substance solution and chlorogenic acid
Reference substance solution.
Further, ultrasonic time is 10~30 minutes.
Preferably, in step 1, color developing agent be 1%~2% ferric trichloride ethanol solution.
Preferably, in step 2, test solution point sample amount is 1~2 μ L.
Preferably, it in step 3, inspects specifically: chlorogenic acid is inspected under 365nm ultraviolet light, the lamellae dried is uniform
Color developing agent is sprayed, is placed, inspects scutelloside under daylight.
Compared with prior art, the invention has the following beneficial technical effects:
The main component caffeoylquinic acid that three taste medicinal material honeysuckles, radix scutellariae, Fructus Forsythiae contain in Compound Jinyinhua Granules
(chlorogenic acid, neochlorogenic acid, isochlorogenic acid etc.), flavonoids (scutelloside, wogonoside, baicalein etc.), forsythol, forsythin etc.
Contain hydroxyl, hydrogen bond can be formed with the carbonyl of amido bond in polyamide film, because forming bonding ability difference adsorption capacity not
It separates together.In chromatography process, solvent can be with the separated object matter competitively carbonyl with amido bond in polyamide film
Hydrogen bond is formed, to select solvent that separated object matter distribution coefficient between solvent and polyamide surface is made to have larger difference, is passed through
The exhibition layer process of adsorption and desorption is crossed, different order separation is formed.So the present invention by using the lesser ethyl acetate of polarity,
The solvent of the biggish methanol of polarity and acetic acid according to certain volume than preparation, and using polyamide film as lamellae, it can
It realizes and the identification of specificity thin layer is carried out to scutelloside in Compound Jinyinhua Granules and chlorogenic acid, specificity is strong, can exclude negative dry
It disturbs, clear spot, separating degree are high, favorable reproducibility.The solvent that the present invention uses directly is prepared and is used, and takes upper layer without standing
Solution, simplifies the preparation steps of solvent, while having saved the time, improves efficiency.
Further, sample is prepared using ultrasonic method, simplifies experimental procedure, shortens the sample preparation time.It is finally fixed
Amount dissolution, accurately controls point sample amount, point sample amount becomes smaller, easily operated.
Further, identify with polyamide film thin layer, scutelloside is in ultraviolet lower color and lamellae color similarity
It is high, it is not easy to it is clear to differentiate, and the present invention inspects scutelloside under daylight after spraying color developing agent and clearly easily distinguishes.
Detailed description of the invention
Fig. 1 using volume ratio for the ethyl acetate of 8:1:1, methanol and formic acid mixture as solvent.
Fig. 2 using volume ratio for the toluene of 10:3:1:2, ethyl acetate, methanol and formic acid mixture as solvent.
Fig. 3 using volume ratio for the ethyl acetate of 4:3:10, methanol and acetic acid mixture as solvent.
Fig. 4 is different point sample volume thin-layer chromatography effects.
Fig. 5 is thin-layer chromatography effect under optimum condition.
Fig. 6 is that specificity investigates thin-layer chromatography effect.
Fig. 7 lamellae is the thin-layer chromatography effect of Shanghai Jin Sui Biotechnology Co., Ltd.
Fig. 8 lamellae is City of Taizhou road and bridge tetramethyl biochemistry plastic molding and processing plant thin-layer chromatography effect.
Fig. 9 is 5 DEG C of expansion results.
Figure 10 is 30 DEG C of expansion results.
Figure 11 be humidity 32% under the conditions of result is unfolded.
Figure 12 be humidity 72% under the conditions of result is unfolded.
Figure 13 is that result is unfolded in embodiment 1.
Figure 14 is that result is unfolded in embodiment 2.
Point sample sequence is as follows in Fig. 1,2,3: 1,2,3 be test solution, and 4 be negative controls solution, and 5 be scutelloside
Reference substance solution, 6 be chlorogenic acid reference substance solution；
In Fig. 4 point sample sequence it is as follows: 1,2,3,4 be point sample volume be respectively 0.5 μ L, 1 μ L, 2 μ L and 3 μ L test sample it is molten
Liquid, 5 be 1 μ L negative controls solution, and 6 be 1 μ L scutelloside reference substance solution, and 7 be 1 μ L chlorogenic acid reference substance solution；
In Fig. 5,6,7,8,9,10,11,12,13,14 point sample sequence it is as follows: 1,2,3 be 1 μ L test solution, 4 are
1 μ L negative controls solution, 5 be 1 μ L scutelloside reference substance solution, and 6 be 1 μ L chlorogenic acid reference substance solution.
Specific embodiment
Below with reference to specific embodiment, the present invention is described in further detail, it is described be explanation of the invention and
It is not to limit.
Scutelloside and chlorogenic acid thin layer identify detection method, including following step in Compound Jinyinhua Granules of the present invention
It is rapid:
1, it prepares Compound Jinyinhua Granules scutelloside and chlorogenic acid thin layer identifies test solution
The method being ultrasonically treated using methanol, obtains Compound Jinyinhua Granules scutelloside and chlorogenic acid thin layer identifies measurement and supplies
Test sample solution.
2, it prepares Compound Jinyinhua Granules scutelloside and chlorogenic acid thin layer identifies negative controls solution
Radix scutellariae and honeysuckle negative sample are prepared according to Compound Jinyinhua Granules prescription, is prepared according still further to the method for step 1
Obtain negative controls solution.
3, it prepares Compound Jinyinhua Granules scutelloside and chlorogenic acid thin layer identifies reference substance solution
The method being ultrasonically treated using methanol obtains scutelloside reference substance solution and chlorogenic acid reference substance solution.
4, lamellae
Three kinds of lamellaes, silica gel g thin-layer plate, 4% sodium acetate silica G plate and polyamide film (thin-layer chromatography).
5, the preparation of solvent
Respectively prepare 3 kinds of solvents, solvent 1 include toluene, ethyl acetate, methanol and formic acid, toluene, ethyl acetate,
The volume ratio of methanol and formic acid is 10:3:1:2；Solvent 2 includes ethyl acetate, methanol and formic acid, ethyl acetate, methanol and first
The volume ratio of acid is 8:1:1；Solvent 3 includes ethyl acetate, methanol and acetic acid, the volume ratio of ethyl acetate, methanol and acetic acid
For 4:3:10；And 3 kinds of development systems are compared.
6, the preparation of color developing agent
Ferric trichloride 2g is weighed in 100mL volumetric flask, after adding 95% ethyl alcohol to dissolve on a small quantity, continues to be settled to 100mL,
It is spare.It is determined by embodiment, ferric trichloride concentration can be 1-2%, can reach desired effects.
7, the thin layer of scutelloside and chlorogenic acid identifies detection in Compound Jinyinhua Granules
Measure steps 1 and 2 respectively with quantitative capillary tube, compound honeysuckle test solution, negative controls made from 3
Solution, scutelloside reference substance solution and chlorogenic acid reference substance solution point are in the expansion cylinder on lamellae, being put into solvent saturation
More than being expanded at lamellae 2/3, takes out, dry, be respectively placed under rated condition and inspect.
When point sample, spot is round point shape, away from 10~15mm of lamellae bottom edge, away from two left and right edges 15mm, round point shape diameter 2
~3mm, 1~2 μ L of point sample amount.
When expansion, solvent is first saturated 15~30 minutes, before lamellae immerses solvent depth requirements solvent initially
Along line-spacing origin 5mm.
When inspecting, chlorogenic acid is inspected under ultraviolet 365, the lamellae of naturally dry is uniformly sprayed to color developing agent, after placement,
It is inspected under daylight.
8, discrimination condition optimizes
1) test article treating method selects
Extraction solvent methanol, 75% methanol, ethyl alcohol and 60% ethyl alcohol have been investigated respectively, the results showed that methanol extraction effect
It is good, it has investigated different extracting methods and has been ultrasonically treated 30 minutes, be heated to reflux 30 minutes, it is high with extraction efficiency, method simplicity
Principle, selection ultrasonic treatment.It is determined by embodiment, ultrasonic time is that can reach desired effects in 10-30 minutes.
2) selection of lamellae
It is respectively that sample is (thin in silica gel g thin-layer plate, 4% sodium acetate silica G plate, polyamide film with quantitative capillary tube point
Analyse layer by layer) on, expansion is inspected, comparison expansion effect, final choice polyamide film (thin-layer chromatography).
3) selection of solvent
Solvent 1: toluene, ethyl acetate, methanol and formic acid ratio are 10:3:1:2；Solvent 2: ethyl acetate, methanol
It is 8:1:1 with formic acid ratio；Solvent 3: ethyl acetate, methanol and proportion of acetic acid are 4:3:10；Sample spot is thin in polyamide
Film lamellae is respectively adopted solvent 1, solvent 2 and solvent 3 and is unfolded, solvent 1, solvent 2 and solvent 3
It is as shown in Figure 1, 2, 3 that effect difference is unfolded, 1,2,3 be test solution in figure, and 4 be negative controls solution, and 5 be scutelloside
Reference substance solution, 6 be chlorogenic acid reference substance solution, and effect is unfolded to it respectively and carries out evaluation selection, optimization, determines best expansion
Agent is solvent 3.It is determined by embodiment, the volume ratio of ethyl acetate, methanol and acetic acid is (1~7) in solvent: (2~
5): (7~15).
4) selection of point sample volume
Take 0.5 μ L of test solution, 1 μ L, 2 μ L and 3 μ L and scutelloside reference substance solution, chlorogenic acid reference substance molten respectively
Liquid and each 1 μ L point of negative controls solution are on polyamide film lamellae, such as Fig. 4.Compare chromatographic effect, in figure, 1,2,3 and
4 be the test solution that point sample volume is respectively 0.5 μ L, 1 μ L, 2 μ L and 3 μ L, and 5 be 1 μ L of negative controls solution, and 6 be radix scutellariae
1 μ L of glycosides reference substance solution, 7 be 1 μ L of chlorogenic acid reference substance solution, determines that best point sample volume is 1~2 μ L.
5) best thin-layer chromatography condition is determined
According to the optimum results of above each factor, determine that the best thin layer discrimination condition of scutelloside and chlorogenic acid is test sample
Methanol is added to be ultrasonically treated 30 minutes, lamellae selects polyamide film, and solvent selects solvent 3: ethyl acetate, methanol and second
Sour ratio is 4:3:10, and point sample volume is 1~2 μ L.Such as Fig. 5.
9, methodology validation
Compound Jinyinhua Granules scutelloside of the present invention, chlorogenic acid Qualitive test, using thin-layered chromatography, according to yellow in prescription
The case where a kind of reed mentioned in ancient books, honeysuckle, main component contained by three taste medicinal material of Fructus Forsythiae, establishes detection chromatographic condition；To the chromatographic condition of foundation into
The methodology validation of row system is studied, and guarantees the specificity and durability of established chromatographic condition.
1) specificity is investigated:
Compound Jinyinhua Granules lot number 180623,180623-1,180624 samples are taken to prepare confession respectively by the method for the present invention
Test sample solution, scutelloside reference substance solution, chlorogenic acid reference substance solution and negative control sample solution, and point sample is in same polyamides
Amine film (thin-layer chromatography), is as a result shown in Fig. 6, wherein 1,2 and 3 be respectively Compound Jinyinhua Granules lot number 180623,180623-1
It is 1 μ L of negative controls solution with each 1 μ L of 180624 test solutions, 4,5 be 1 μ L of scutelloside reference substance solution, and 6 be chlorogenic acid
1 μ L of reference substance solution；It sets and is inspected under ultraviolet lamp (365nm), in sample chromatogram, aobvious with chlorogenic acid reference substance corresponding position
The fluorescence spot of same color.Sprayed again with 2% ferric trichloride ethanol solution, set and inspected under daylight, in sample chromatogram, with Huang
The spot of same color and negative interference are shown on the corresponding position of a kind of reed mentioned in ancient books glycosides reference substance chromatography.
2) durability is investigated
1. lamellae: the polyamide film (thin-layer chromatography) produced using 2 different manufacturers, respectively Shanghai gold fringe biology
Science and Technology Ltd., City of Taizhou road and bridge tetramethyl biochemistry plastic molding and processing plant.Fig. 7 is the life of Shanghai Jin Sui Biotechnology Co., Ltd
The polyamide film of production, the polyamide film that Fig. 8 is the production of City of Taizhou road and bridge tetramethyl biochemistry plastic molding and processing plant.
2. temperature investigate: by the lamellae after point sample set respectively 5 DEG C (refrigerators) and 30 DEG C saturation 30 minutes after, be unfolded, examine
The separating degree of principal spot is examined, Fig. 9 is 5 DEG C of expansion as a result, Figure 10 is 30 DEG C of expansion results.
3. humidity is investigated: being separately added into 68% and 27.5% sulfuric acid solution control relative humidity difference in expansion cylinder side
32% and 72%, the lamellae after point sample is put into another side channel, it is 15-30 minutes closed, immediately by lamellae dislocation in Sheng
Have in the expansion cylinder of solvent, is unfolded, investigates the separating degree of principal spot.Expansion the result is shown in Figure 11 under 32% damp condition, 72%
The result is shown in Figure 12 (22 DEG C of operation room temperature) is unfolded under damp condition.
Durability is investigated the results show that the polyamide film of different manufacturers production does not have significant shadow to identification testing result
It rings, temperature and humidity also has no significant effect identification testing result, and it is resistance to well to illustrate that present invention identification detection method has
The property used.
Specific embodiment is as follows.
Compound Jinyinhua Granules used in the embodiment of the present invention are " golden flower Compound Jinyinhua Granules ", national drug standard
Z20053982。
Embodiment 1:
It includes following steps that Compound Jinyinhua Granules scutelloside disclosed by the invention, chlorogenic acid thin layer, which identify detection method measurement,
It is rapid:
1, the preparation of test solution:
It takes sample finely ground, weighs 2.5g, methanol 50mL is added, be ultrasonically treated 10 minutes, filtering takes filtrate to be evaporated, residue
Methanol 2mL dissolution is added, that is, completes the preparation of test solution.
2, prepared by negative controls solution
Step a: according to 5000g prescription, Fructus Forsythiae (prescription without honeysuckle, radix scutellariae) medicinal material 750g is weighed, is added 12 times
The water of amount extracts 2 times, and 1 hour every time, combined extract, when being concentrated into relative density 1.26 (60~70 DEG C), weighing was added 3
The ethyl alcohol of amount again, it is stirring while adding, 24 hours are stood, ethyl alcohol is recycled, is concentrated into 1.26 (60~70 DEG C) pastes, sucrose is added
Powder mixes, particle is made, is drying to obtain；
Step b: taking in step a that particle obtained is finely ground, weighs 2.5g, and methanol 50mL is added, and is ultrasonically treated 10 minutes, mistake
Filter, takes filtrate to be evaporated, and methanol 2mL dissolution is added in residue, that is, completes the preparation of negative controls solution.
3, prepared by reference substance solution
Precision weighs scutelloside reference substance (National Institute for Food and Drugs Control) 10.88mg, chlorogenic acid reference substance respectively
A small amount of methanol ultrasonic dissolution is added in 25mL volumetric flask in (National Institute for Food and Drugs Control) 10.09mg, then is settled to
25mL is configured to the solution of the 0.4mg/mL containing reference substance.
The preparation of 4 solvents
The ratio for being 4:3:10 according to ethyl acetate, methanol and acetic acid volume ratio measures ethyl acetate 4mL, methanol respectively
3mL, acetic acid 10mL shake up in expansion cylinder, are saturated 15 minutes.
5, color developing agent is prepared
Ferric trichloride 1g is weighed in 100mL volumetric flask, after adding 95% ethyl alcohol to dissolve on a small quantity, continues to be settled to 100mL,
It is configured to 1% ferric trichloride ethanol solution.
6, thin-layer chromatography
1) point sample
Polyamide film (thin-layer chromatography) 10*20cm is selected, with quantitative capillary tube point sample, point sample 2~3mm of diameter, control
Product point sample amount is 1 μ L, and test sample point sample amount is 1 μ L.
2) it is unfolded
The lamellae for having put sample is put into solvent 15 minutes expansion cylinders of saturation, lamellae immerses solvent depth
It is required that being the initial frontal line of solvent away from origin 5mm or so.Exhibition is away from the 2/3 of lamellae or so, taking-up, naturally dry.
Chlorogenic acid is inspected under ultraviolet 365.
3) it develops the color
The lamellae dried is uniformly sprayed to 1% ferric trichloride ethanol solution, is placed, inspects scutelloside under daylight.
4) condition is inspected
Chlorogenic acid is inspected under ultraviolet 365, scutelloside is inspected under daylight, as a result such as Figure 13.
5) result judgement
In sample chromatogram, in the fluorescence spot for showing same color with chlorogenic acid reference substance position, compareed with scutelloside
Grade sets the spot of aobvious same color.
Embodiment 2
It includes following steps that Compound Jinyinhua Granules scutelloside disclosed by the invention, chlorogenic acid thin layer, which identify detection method measurement,
It is rapid:
1, the preparation of test solution:
It takes sample finely ground, weighs 2.5g, methanol 50mL is added, be ultrasonically treated 30 minutes, filtering takes filtrate to be evaporated, residue
Methanol 2mL dissolution is added, that is, completes the preparation of test solution.
2, prepared by negative controls solution
Step a: according to 5000g prescription, Fructus Forsythiae (prescription without honeysuckle, radix scutellariae) medicinal material 750g is weighed, is added 12 times
The water of amount extracts 2 times, and 1 hour every time, combined extract, when being concentrated into relative density 1.26 (60~70 DEG C), weighing was added 3
The ethyl alcohol of amount again, it is stirring while adding, 24 hours are stood, ethyl alcohol is recycled, is concentrated into 1.26 (60~70 DEG C) pastes, sucrose is added
Powder mixes, particle is made, is drying to obtain；
Step b: taking in step a that particle obtained is finely ground, weighs 2.5g, and methanol 50mL is added, and is ultrasonically treated 30 minutes, mistake
Filter, takes filtrate to be evaporated, and methanol 2mL dissolution is added in residue, that is, completes the preparation of negative controls solution.
3, prepared by reference substance solution
Precision weighs scutelloside reference substance 10.88mg, chlorogenic acid reference substance 10.09mg in 25mL volumetric flask respectively, adds
Enter a small amount of methanol ultrasonic dissolution, then be settled to 25mL, is configured to the solution of the 0.4mg/mL containing reference substance.
The preparation of 4 solvents
The ratio for being 7:2:7 according to ethyl acetate, methanol and acetic acid volume ratio measures ethyl acetate 7mL, methanol respectively
2mL, acetic acid 7mL shake up in expansion cylinder, are saturated 15 minutes.
5, color developing agent is prepared
Ferric trichloride 2g is weighed in 100mL volumetric flask, after adding 95% ethyl alcohol to dissolve on a small quantity, continues to be settled to 100mL,
It is configured to 2% ferric trichloride ethanol solution.
6, thin-layer chromatography
1) point sample
Polyamide film (thin-layer chromatography) 10*20cm is selected, with quantitative capillary tube point sample, point sample 2~3mm of diameter, control
Product point sample amount is 1 μ L, and test sample point sample amount is 2 μ L.
2) it is unfolded
The lamellae for having put sample is put into solvent 15 minutes expansion cylinders of saturation, lamellae immerses solvent depth
It is required that being the initial frontal line of solvent away from origin 5mm or so.Exhibition is away from the 2/3 of lamellae or so, taking-up, naturally dry.
Chlorogenic acid is inspected under ultraviolet 365.
3) it develops the color
The lamellae dried is uniformly sprayed to 2% ferric trichloride ethanol solution, is placed, inspects scutelloside under daylight.
4) condition is inspected
Chlorogenic acid is inspected under ultraviolet 365, scutelloside is inspected under daylight, as a result such as Figure 14.
5) result judgement
In sample chromatogram, in the fluorescence spot for showing same color with chlorogenic acid reference substance position, compareed with scutelloside
Grade sets the spot of aobvious same color.
Embodiment 3:
It includes following steps that Compound Jinyinhua Granules scutelloside disclosed by the invention, chlorogenic acid thin layer, which identify detection method measurement,
It is rapid:
1, the preparation of test solution:
It takes sample finely ground, weighs 2.5g, methanol 50mL is added, be ultrasonically treated 20 minutes, filtering takes filtrate to be evaporated, residue
Methanol 2mL dissolution is added, that is, completes the preparation of test solution.
2, prepared by negative controls solution
Step a: according to 5000g prescription, Fructus Forsythiae (prescription without honeysuckle, radix scutellariae) medicinal material 750g is weighed, is added 12 times
The water of amount extracts 2 times, and 1 hour every time, combined extract, when being concentrated into relative density 1.26 (60~70 DEG C), weighing was added 3
The ethyl alcohol of amount again, it is stirring while adding, 24 hours are stood, ethyl alcohol is recycled, is concentrated into 1.26 (60~70 DEG C) pastes, sucrose is added
Powder mixes, particle is made, is drying to obtain；
Step b: taking in step a that particle obtained is finely ground, weighs 2.5g, and methanol 50mL is added, and is ultrasonically treated 20 minutes, mistake
Filter, takes filtrate to be evaporated, and methanol 2mL dissolution is added in residue, that is, completes the preparation of negative controls solution.
3, prepared by reference substance solution
Precision weighs scutelloside reference substance (National Institute for Food and Drugs Control) 10.88mg, chlorogenic acid reference substance respectively
A small amount of methanol ultrasonic dissolution is added in 25mL volumetric flask in (National Institute for Food and Drugs Control) 10.09mg, then is settled to
25mL is configured to the solution of the 0.4mg/mL containing reference substance.
The preparation of 4 solvents
The ratio for being 7:5:15 according to ethyl acetate, methanol and acetic acid volume ratio measures ethyl acetate 7mL, methanol respectively
5mL, acetic acid 15mL shake up in expansion cylinder, are saturated 15 minutes.
5, color developing agent is prepared
Ferric trichloride 1.5g is weighed in 100mL volumetric flask, after adding 95% ethyl alcohol to dissolve on a small quantity, continues to be settled to
100mL is configured to 1.5% ferric trichloride ethanol solution.
6, thin-layer chromatography
1) point sample
Polyamide film (thin-layer chromatography) 10*20cm is selected, with quantitative capillary tube point sample, point sample 2~3mm of diameter, control
Product point sample amount is 1 μ L, and test sample point sample amount is 1.5 μ L.
2) it is unfolded
The lamellae for having put sample is put into solvent 15 minutes expansion cylinders of saturation, lamellae immerses solvent depth
It is required that being the initial frontal line of solvent away from origin 5mm or so.Exhibition is away from the 2/3 of lamellae or so, taking-up, naturally dry.
Chlorogenic acid is inspected under ultraviolet 365.
3) it develops the color
The lamellae dried is uniformly sprayed to 1.5% ferric trichloride ethanol solution, is placed, inspects scutelloside under daylight.
4) condition is inspected
Chlorogenic acid is inspected under ultraviolet 365, inspects scutelloside under daylight.
5) result judgement
In sample chromatogram, in the fluorescence spot for showing same color with chlorogenic acid reference substance position, compareed with scutelloside
Grade sets the spot of aobvious same color.
The present invention passes through the measures such as screening solvent composition, solvent ratio, point sample amount, it is determined that optimal detection condition；
It is detected using the condition, thin layer identifies in map, scutelloside, chlorogenic acid clear spot, and separating degree is high, and it is accurate to improve detection
Degree.It has been determined that the specificity for drafting testing conditions, durability are (lamellaes of different brands, warm and humid by the checking research of system
Degree), solve the problems, such as that spot is unintelligible in primary standard, separating degree is poor.The present invention is suitable for Compound Jinyinhua Granules scutelloside
The detection identified with chlorogenic acid thin layer, more effectively control product quality, guarantee clinical efficacy.
Claims (7)
1. scutelloside and chlorogenic acid thin layer identify detection method in a kind of Compound Jinyinhua Granules, which is characterized in that including following
Step:
Step 1, test solution, negative controls solution, scutelloside reference substance solution and chlorogenic acid reference substance solution are prepared；Match
Solvent and color developing agent processed；Solvent includes ethyl acetate, methanol and acetic acid, and the volume ratio of ethyl acetate, methanol and acetic acid is
(1~7): (2~5): (7~15)；
Step 2, thin-layer chromatography: using polyamide film as lamellae, by test solution, negative controls solution, scutelloside
In reference substance solution and the equal point sample to lamellae of chlorogenic acid reference substance solution, the lamellae for having put sample is put into solvent saturation
Expansion cylinder in be unfolded；Then lamellae is taken out, is dried；
Step 3, it inspects, inspects chlorogenic acid, scutelloside under ultraviolet and daylight respectively.
2. scutelloside and chlorogenic acid thin layer identify detection method in Compound Jinyinhua Granules according to claim 1, special
Sign is, in step 1, the preparation method of test solution are as follows: and Compound Jinyinhua Granules are finely ground, methanol, ultrasound, mistake is added
Filter, takes filtrate to be evaporated, residue adds methanol to dissolve, and constant volume obtains test solution.
3. scutelloside and chlorogenic acid thin layer identify detection method in Compound Jinyinhua Granules according to claim 1, special
Sign is, in step 1, the preparation method of scutelloside reference substance solution and chlorogenic acid reference substance solution are as follows: take scutelloside and green original
Sour reference substance is separately added into methanol, and ultrasonic dissolution, constant volume, respectively obtains scutelloside reference substance solution and chlorogenic acid reference substance is molten
Liquid.
4. scutelloside and chlorogenic acid thin layer identify detection method in Compound Jinyinhua Granules according to claim 2 or 3,
It is characterized in that, ultrasonic time is 10~30 minutes.
5. scutelloside and chlorogenic acid thin layer identify detection method in Compound Jinyinhua Granules according to claim 1, special
Sign is, in step 1, color developing agent be 1%~2% ferric trichloride ethanol solution.
6. scutelloside and chlorogenic acid thin layer identify detection method in Compound Jinyinhua Granules according to claim 1, special
Sign is, in step 2, test solution point sample amount is 1~2 μ L.
7. scutelloside and chlorogenic acid thin layer identify detection method in Compound Jinyinhua Granules according to claim 1, special
Sign is, in step 3, inspects specifically: inspects chlorogenic acid under 365nm ultraviolet light, the lamellae dried is uniformly sprayed colour developing
Agent is placed, and inspects scutelloside under daylight.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811556742.7A CN109709256A (en) | 2018-12-19 | 2018-12-19 | Scutelloside and chlorogenic acid thin layer identify detection method in a kind of Compound Jinyinhua Granules |
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Cited By (1)
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| CN112326864A (en) * | 2020-11-30 | 2021-02-05 | 天津中新药业集团股份有限公司隆顺榕制药厂 | Thin-layer chromatography method for identifying medicinal component of sculellaria barbata in zilongjin tablets and application |
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| CN112326864A (en) * | 2020-11-30 | 2021-02-05 | 天津中新药业集团股份有限公司隆顺榕制药厂 | Thin-layer chromatography method for identifying medicinal component of sculellaria barbata in zilongjin tablets and application |
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