CN109596768A - A kind of detection method of Children stasis relieving particle - Google Patents
A kind of detection method of Children stasis relieving particle Download PDFInfo
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Abstract
The invention discloses a kind of method of quality control of Children stasis relieving particle, carry out Qualitive test to charred FRUCTUS CRATAEGI, kaladana in Children stasis relieving particle using thin-layered chromatography；Quantitative control is carried out using the content of the arecaline activating component in high effective liquid chromatography for measuring Children stasis relieving particle, using the organic acid content in potentiometric determination Children stasis relieving particle.The method of the present invention is simple, separating degree is good, specificity is strong, favorable reproducibility, has effectively ensured the quality and curative effect of Children stasis relieving particle, has very strong practicability.
Description
Technical field
The invention belongs to medicine quality detection technique fields, and in particular to the detection method of Children stasis relieving particle.
Background technique
The prescription of Children stasis relieving particle is led a cow by charred FRUCTUS CRATAEGI, coloured malt, Medicated Leaven (bran stir-fry), Semen Arecae (parched), Endothelium Corneum Gigeriae Galli (parched), stir-fry
Sub- Six-element medicinal material composition, is not included in 2015 editions Chinese Pharmacopoeias at present.
Chinese invention application documents CN101229329A discloses a kind of Chinese patent drug indigestion tablet and its production technology, to change
The scattered prescription of product: 166.67g charred FRUCTUS CRATAEGI, 166.67g coloured malt, 166.67g Medicated Leaven (bran stir-fry), 166.67g Semen Arecae (parched),
166.67g Endothelium Corneum Gigeriae Galli (parched), 166.67g stir-baked SEMEN PHARBITIDIS are mainly used for treating the spleen and stomach in children discord, cream of stopping eating, accumulation lump in the abdomen, tripe
The diseases such as abdominal distention, four limbs burnout, sallow complexion, do not feel like eating.
But the detection method of Children stasis relieving related preparations is not recorded in 2015 editions pharmacopeia one, children's helping digestion is related
The discrimination method of preparation is on the books in 2015 editions Chinese Pharmacopoeia one, wherein the indentification by TLC side of kaladana and hawthorn
Method specifically:
(1) kaladana identifies in children's helping digestion oral solution: taking this product 25ml, adds salt acid for adjusting pH value extremelyAdd acetic acid
Ethyl ester shaking is extracted 2 times, each 25ml, and combined ethyl acetate liquid is evaporated, and residue adds methanol 0.5m l to make to dissolve, as examination
Product solution.Kaladana control medicinal material 0.5g separately is taken, adds water to cook 1 hour, lets cool, is filtered, filtrate adds salt acid for adjusting pH value extremelyIt is made in the same way of control medicinal material solution.It is tested according to thin-layered chromatography (general rule 0502), draws each 10 μ l of above two solution,
It is put respectively on same silica gel g thin-layer plate, with ether-chloroform-formic acid (10:50:1) for solvent, is unfolded, takes out,
It is dry, it is inspected at ultraviolet light (365nm).In sample chromatogram, on position corresponding with reference medicine chromatography, same color is shown
Fluorescence spot.
(2) hawthorn identifies in children's helping digestion ball: taking this product 3g, shreds, add osmanthus diatomaceous earth 1g, grind well, add diethyl ether 20ml, ultrasound
Processing 15 minutes, filtration, filtrate volatilize, and residue adds dehydrated alcohol lm l to make to dissolve, as test solution.Separately hawthorn is taken to compare
Medicinal material 1g, add diethyl ether 15ml, is made in the same way of control medicinal material solution.Ursolic acid reference substance is taken again, is added dehydrated alcohol that every 1ml is made and is contained
The solution of 1mg, as reference substance solution.It is tested according to thin-layered chromatography (general rule 0502), draws 10 μ l of test solution, comparison medicine
Material solution and each 2 μ l of reference substance solution are put respectively on same silica gel g thin-layer plate, with hexamethylene-chloroform-ethyl acetate
(20:5:8) is solvent, is unfolded, and takes out, dries, and spray is heated 10 minutes with 10% ethanol solution of sulfuric acid at 110 DEG C.Test sample
In chromatography, on position corresponding with reference medicine chromatography and reference substance chromatography, identical aubergine spot is shown.
But the TLC Identification of above-mentioned kaladana and hawthorn is not particularly suited for Children stasis relieving particle, this be by
It is different dosage forms in Children stasis relieving particle and children's helping digestion oral solution, children's helping digestion ball, has on prescription and preparation method certain
Difference, it is therefore desirable to be detected according to distinct methods.
Children stasis relieving granule have take, carry, storing, convenient transportation, the larger advantage of drugloading rate is worth further
Quality standard is improved, improves product quality controllability, therefore, the present invention improves existing detection method, Ke Yipai
Except interference, the result made is accurate.
Summary of the invention
The object of the present invention is to provide one kind simply, specificity is strong and the detection of reproducible Children stasis relieving particle
Method, to ensure that the reliable in quality of Children stasis relieving particle provides effective way.
A kind of detection method of Children stasis relieving particle, the Children stasis relieving particle are made by following prescription: charred FRUCTUS CRATAEGI fries wheat
Bud, Medicated Leaven, Semen Arecae (parched), Endothelium Corneum Gigeriae Galli (parched), stir-baked SEMEN PHARBITIDIS, include the following steps:
(1) Qualitive test is carried out to hawthorn in Children stasis relieving particle and kaladana using thin-layered chromatography；
(2) quantitative identification is carried out to the active constituent arecaline in Children stasis relieving particle using high performance liquid chromatography；
(3) quantitative identification is carried out to active constituent citric acid in Children stasis relieving particle using potentiometric titration.
Children stasis relieving particle of the invention is by charred FRUCTUS CRATAEGI, Semen Arecae (parched), coloured malt, stir-baked SEMEN PHARBITIDIS, Endothelium Corneum Gigeriae Galli (parched), Medicated Leaven
(bran stir-fry) 6 taste medicinal materials composition, the function of row consumer product dredge stasis, wherein hawthorn is conventional Chinese medicine, there is relieving dyspepsia, the function of scattered stasis
The effective component of effect, treatment for relieving indigestion and constipation effect is mainly organic acid.
Modern pharmacological research thinks that organic acid has the function of digesting and appetizing, but organic acid content is higher in hawthorn health product,
It is big to gastrointestinal irritation between 5%~9%, therefore hawthorn after processing is used, reducing in hawthorn to a certain extent after processing always has
The content of machine acid, has mitigated irritation；According to the selected requirement of new Chinese medicine quality standard research assay, selection and this product
The assay index components of major function and the relevant principle active component of indication as this product.
Quantitative item is organic acid in Children stasis relieving particle, and organic acid all exists in a variety of medicinal materials, there is certain defect, because
This increases the quantitative control of arecaline on this basis.Therefore, detection method of the invention establishes TLC chromatography to Jiaoshan Hill
The identification item of short, bristly hair or beard, stir-baked SEMEN PHARBITIDIS, chromatography clear spot in photochrome is negative noiseless；Each result meets rule in check item
It is fixed；The method that content determination item establishes HPLC method measurement betel nut alkali content and potentiometric determination organic acid content, method
Result of study is learned to meet the requirements.The method established is easy, accurate, reproducible, has operability.
In step (1), the discrimination method of the hawthorn includes the following steps:
(1) the Children stasis relieving particle is taken, EtOH Sonicate is added to handle, is dissolved in water after boiling off ethyl alcohol, then pass through polyamide
Column is first eluted with water, then with ethanol elution, collects ethanol eluate, and residue is dissolved in water, is extracted with ethyl acetate 3 times, merges
Acetic acid ethyl fluid, residue add methanol to dissolve, as test solution；
(2) hawthorn control medicinal material is taken, water is added to be heated to reflux, filtrate is evaporated, and residue adds EtOH Sonicate to handle, and is made in the same way of pair
According to medicinal material solution；
(3) it is tested according to thin-layered chromatography, draws test solution and control medicinal material solution point respectively in same silica G thin layer
On plate, the cyclohexane-acetone for being 2~15: 0.5~10 using volume ratio takes out after expansion and dries as solvent, spray colour developing
Agent, heating, which is placed under ultraviolet lamp, inspects.
In the hawthorn discrimination method of Children stasis relieving particle of the present invention, experiment shows that it is 2~15 that solvent is changed to volume ratio:
When 0.5~10 cyclohexane-acetone is identified, have good repeatability and specificity, testing result more acurrate.And it uses
Hawthorn differential method identifies Children stasis relieving particle in existing children's helping digestion ball, by investigate sample chromatogram in, with it is right
According on medicinal material chromatography corresponding position, the fluorescence spot of different colours is shown；And in negative control chromatography with reference medicine chromatography phase
It answers on position, there is the fluorescence spot of same color, this method does not have specificity.
Preferably, in step (1), the discrimination method of the hawthorn includes the following steps:
(1) the Children stasis relieving particle 10g is taken, 30~80% 20~80ml of ethyl alcohol are added, is ultrasonically treated 20~60min, is steamed
Ethyl alcohol is removed, water is added to make to dissolve, by polyamide column, is eluted with water, discards, then is eluted with 30~80% 20~80ml of ethyl alcohol, is received
Collect ethanol eluate, residue adds water to make to dissolve, and is extracted 3 times, each 15ml with ethyl acetate shaking, combined ethyl acetate liquid is residual
Slag adds methanol to make to dissolve, as test solution；
(2) hawthorn control medicinal material 2g is taken, water 30ml is added, is heated to reflux 1h, filtrate is evaporated, and residue adds 30~80% ethyl alcohol 20
~80ml, is made in the same way of control medicinal material solution；
(3) it is tested according to thin-layered chromatography, draws test solution and each 10 μ l point of control medicinal material solution respectively in same silicon
On glue G lamellae, for the cyclohexane-acetone for being 2~15: 0.5~10 using volume ratio as solvent, solvent first sets chromatography cylinder
Interior 10~50min of presaturation takes out after expansion and dries, and sprays 10% sulfuric acid ethanol, and 105 DEG C of heating are placed under ultraviolet lamp and examine
Depending on.
In above-mentioned preferred hawthorn discrimination method, each step of identification is specifically defined, experiment shows in above-mentioned specific item
Identified under part, not only control medicinal material usage amount is lower, moreover it is possible to keep final thin-layer chromatography spot separating degree preferable, apparent.
In step (1), the discrimination method of the kaladana includes the following steps:
(1) take the Children stasis relieving particle that methanol is added to be ultrasonically treated, filtrate is evaporated, and residue is dissolved in water, the dilute salt of filtrate
Acid for adjusting pH value, then shaken and extracted with ethyl acetate, combined ethyl acetate liquid is evaporated, and residue adds methanol to dissolve, as test sample
Solution；
(2) kaladana's control medicinal material is taken, water is added to be heated to reflux, filtrate is adjusted with dilute hydrochloric acid, and it is molten to be made in the same way of control medicinal material
Liquid；
(3) it is tested according to thin-layered chromatography, draws test solution and control medicinal material solution point respectively in same silica G thin layer
On plate, it is solvent using butyl acetate-formic acid-water upper solution that volume ratio is 2~15: 1~10: 1~10, is taken after expansion
It dries out, sprays 5% ferric trichloride ethanol solution as color developing agent, heating is inspected.
In kaladana's discrimination method of Children stasis relieving particle of the present invention, experiment shows its reproducible and temperature and humidity pair
Effect is unfolded without influence, finally obtained thin-layer chromatography spot is obvious, clear, in addition, the sample solution preparation method is easy,
Control medicinal material usage amount is lower, and practicability is stronger, is suitble to large-scale application.
And kaladana's discrimination method in existing children's helping digestion oral solution is used to identify Children stasis relieving particle, discovery is led
The characteristic spots of ox control medicinal material are unintelligible, and in sample chromatogram, on position corresponding with reference medicine chromatography, phase
Clear spot degree with color is also unobvious, and effect, poor repeatability is unfolded vulnerable to the influence of environment temperature.
Preferably, in step (1), the discrimination method of the kaladana includes the following steps:
(1) the Children stasis relieving particle 10g is taken, methanol 50ml is added, is ultrasonically treated 40min, filtrate is evaporated, and residue adds water
20ml makes to dissolve, and adjusts pH value to 1~2 with dilute hydrochloric acid, then extracted 3 times, each 20ml with ethyl acetate shaking, merges acetic acid second
Ester liquid, is evaporated, and residue adds methanol 1ml to make to dissolve, as test solution；
(2) kaladana control medicinal material 1g is taken, water 30ml is added, is heated to reflux 40min, filtrate adjusts pH value to 1 with dilute hydrochloric acid
~2, it is made in the same way of control medicinal material solution；
(3) it is tested according to thin-layered chromatography, draws 10~15 μ l of test solution, 8 μ l point of control medicinal material solution in same silicon
It is solvent using butyl acetate-formic acid-water upper solution that volume ratio is 2~15: 1~10: 1~10 on glue G lamellae,
It takes out and dries after expansion, spray 5% ferric trichloride ethanol solution as color developing agent, heating is inspected.
In above-mentioned preferred kaladana's discrimination method, experiment shows to be identified under the above specified conditions, not only compares
Medicinal material usage amount is lower, moreover it is possible to keep final thin-layer chromatography spot separating degree preferable, apparent.
In step (2), the high-efficient liquid phase chromatogram condition of the arecaline are as follows: use with octadecylsilane chemically bonded silica
For the chromatographic column of filler, using volume ratio be 5~25: 75~95 acetonitrile-triethylamine solution as mobile phase, Detection wavelength is
215nm, number of theoretical plate is calculated by arecaline peak is not less than 6000.
In step (2), the high performance liquid chromatography discrimination method of the arecaline are as follows:
(1) Arecoline hydrobromide reference substance is weighed, the solution for adding mobile phase that every 1ml 30 μ g containing Arecoline hydrobromide is made is made
For reference substance solution；
(2) Children stasis relieving particle 5g is taken to add diethyl ether 50ml, then plus carbonate buffer solution 3ml, place 30 minutes, be heated to reflux
30 minutes, divide and take ether solution, residue adds diethyl ether heating and refluxing extraction 2 times again, merges ether solution three times, and phosphoric acid solution l is added
Ml, recycle ether, residue add 50% acetonitrile dissolve, be transferred in 25ml volumetric flask, and be diluted to scale, take subsequent filtrate to get
Test solution；
(3) above-mentioned reference substance solution and test solution are drawn, injection liquid chromatograph is measured.
Semen Arecae (parched) promoting the circulation of qi dissipate-swelling, lower gas stop stagnant push away such as abdomen turgor, inhibited defecation caused by the stomach of place to cream food and swing
Promoting the circulation of qi effect, arecaline are the principle active component of betel nut medicinal material, and arecaline is m receptor agonist, have stronger rush stomach and intestine to transport
Movement is used, and is promoted stomach and intestine glandular secretion, is significant to enhance gastric emptying, therefore select arecaline as this product quality detecting index
Ingredient.
Arecaline in measurement Children stasis relieving particle is matched with discrimination condition using above-mentioned high performance liquid chromatography discrimination method
Content, it is good separating effect, sensitive, accurate, ensure that the quality stable uniformity and curative effect of this product.
In step (3), the potentiometric titration are as follows: take the Children stasis relieving particle that water is added to shake up, after ultrasonic treatment, use
The sodium hydroxide titration liquid constant-current titration pH value of 0.05mol/L is to 7.0~9.5.
Compared with the prior art, the present invention has the following beneficial effects:
(1) present invention carries out qualitative mirror to hawthorn, kaladana in Children stasis relieving particle using the thin-layered chromatography improved
Not, the indentification by TLC clear spot obtained, it is negative noiseless, as a result more satisfied and repeatability, have good stability；
(2) present invention utilizes the content of arecaline in high effective liquid chromatography for measuring Children stasis relieving particle, and this method has
Good separating effect, it is sensitive, accurate the advantages that, ensure that the quality stable uniformity and curative effect of this product；
(3) content of the present invention using organic acid in potentiometric determination Children stasis relieving particle, this method simplicity, standard
Really, reproducible, there is operability；
(4) method of quality control of the invention is simple, specificity is strong, favorable reproducibility, has effectively ensured Children stasis relieving
The quality and curative effect of grain have very strong practicability.
Detailed description of the invention
Fig. 1 is the thin-layer chromatogram of charred FRUCTUS CRATAEGI in hawthorn of the present invention identification, wherein 1 being control medicinal material, 2~4 being the small suffixation of a nonsyllabic "r"
Product particulate samples, 5 be indigestion tablet, 6 be negative control sample；
Fig. 2 is the thin-layer chromatogram of kaladana in kaladana of the present invention identification, wherein 1 being control medicinal material, 2~4 being children
Indigestion eliminating granules sample, 5 be indigestion tablet, 6 be negative control sample；
Fig. 3 is the high-efficient liquid phase chromatogram of arecaline reference substance in arecaline assay of the present invention；
Fig. 4 is the high-efficient liquid phase chromatogram of arecaline feminine gender test sample in arecaline assay of the present invention；
Fig. 5 is the high-efficient liquid phase chromatogram of arecaline test sample in arecaline assay of the present invention.
Specific embodiment
It is described in further detail below in conjunction with detection method of the specific embodiment to Children stasis relieving particle of the invention.This
Children stasis relieving particle used by inventing is produced by Hainan cucurbit baby's medicine company Group Co., Ltd, Children stasis relieving granule packaging specification
It is filled for one bag of 3g.
Embodiment
(1) it checks
The granularity of Children stasis relieving particle, moisture, melting, content uniformity are measured, then to heavy metal and arsenic
It is checked, establishes microbial limit tests.
Microbial limit shines " Chinese Pharmacopoeia " 2015 editions four non-sterile product limit test of microbe: microorganism count method
(general rule 1105), Control bacteria examination method (general rule 1106), non-sterile drug limitation standard in microbe (general rule 1107) check.
This product 10g is taken, adds pH7.0 sodium chloride peptone buffer agent to be diluted to 100ml, 1: 10 test liquid is made.Take 1:
10 test liquid 1ml carries out aerobic bacteria sum, yeast and mold sum inspection in accordance with the law.1:10 test liquid 10ml is taken, is set
In the pancreas junket soya peptone fluid nutrient medium of 100ml, escherichia coli inspection is carried out in accordance with the law；This product 10g is taken, the pancreas junket of 100ml is set
In soya peptone fluid nutrient medium, salmonella examination is carried out in accordance with the law.
In the every 1g test sample of this product, aerobic bacteria sum must not cross 1000cfu, and yeast and mold sum must not mistake
100cfu, must not detect escherichia coli, in every 10g test sample, must not detect detection of Salmonella.
Other should meet related every regulation (four general rules 0104 of " Chinese Pharmacopoeia " version in 2015) under granule item
(2) hawthorn identifies
(1) hawthorn control medicinal material 2g is weighed, water 30ml is added, is heated to reflux 1 hour, is filtered, filtrate is evaporated, and residue adds 60%
Ethyl alcohol 30ml, is made in the same way of control medicinal material solution；
(2) this product 10g is weighed, it is finely ground, add 65% ethyl alcohol 50ml, is ultrasonically treated 20 minutes, filtration, filtrate boils off ethyl alcohol,
Add water 20ml to make to dissolve, by processed polyamide column, eluted with water 70ml, discard water lotion, then with 75% ethyl alcohol
70ml elution, collects ethanol eluate, is evaporated, and residue adds water 20ml to make to dissolve, with ethyl acetate shaking extraction 3 times, every time
15ml, combined ethyl acetate liquid, is evaporated, and residue adds methanol 1ml to make to dissolve, as test solution；
(3) 2g charred FRUCTUS CRATAEGI negative extraction fine powder is weighed, is operated according to step (2) the method, it is molten as negative control
Liquid；
(4) it is tested according to thin-layered chromatography (four general rules 0502 of Chinese Pharmacopoeia version in 2015), draws above two solution
Each 10 μ l puts respectively on same silica gel g thin-layer plate, with cyclohexane-acetone (10: 3) for solvent, sets presaturation in chromatography cylinder
20 minutes, it is unfolded, takes out, dry, spray is heated several minutes at 105 DEG C with 10% sulfuric acid ethanol, set ultraviolet lamp (365nm)
Under inspect.
Identification result on reference medicine chromatography corresponding position, shows phase as shown in Figure 1, in sample chromatogram
With the spot of color；In negative control chromatography and on reference medicine chromatography corresponding position, the spot of no same color.
(3) kaladana identifies
(1) 1g kaladana's control medicinal material is weighed, water 30ml is added, is heated to reflux 40 minutes, is filtered, filtrate is with dilute hydrochloric acid tune PH
Value 1~2, then extracted 3 times, each 20ml with ethyl acetate shaking, combined ethyl acetate liquid is evaporated, and it is molten that residue adds methanol 1ml to make
Solution, as control medicinal material solution；
(2) it weighs and takes this product 10g, it is finely ground, add methanol 50ml, be ultrasonically treated 40 minutes, filtration, filtrate is evaporated, and residue adds
Water 20ml makes to dissolve, and with dilute hydrochloric acid tune pH value 1~2, is made in the same way of test solution；
(3) 1.2g stir-baked SEMEN PHARBITIDIS negative extraction fine powder is weighed, is operated according to step (2) the method, as negative right
According to solution；
(4) according to thin-layered chromatography (four general rules 0502 of Chinese Pharmacopoeia version in 2015) test, draw test solution and
Negative each 10~15 μ l of test solution, 8 μ l of control medicinal material solution are put respectively on same silica gel g thin-layer plate, with acetic acid fourth
Ester-formic acid-water (10: 2: 1.5) upper solution is solvent, is unfolded, and takes out, dries, and is sprayed with 5% ferric trichloride ethanol solution,
It is clear to be heated to spot development.
Identification result shows the spot of same color as shown in Fig. 2, in sample chromatogram and on reference medicine chromatography corresponding position
Point；In negative control chromatography and on reference medicine chromatography corresponding position, the spot of no same color.
(4) arecaline assay
(1) it is appropriate to weigh Arecoline hydrobromide reference substance, it is accurately weighed, add flowing phased soln concentration is made to be
The Arecoline hydrobromide reference substance stock solution (arecaline weight=Arecoline hydrobromide weight/1.5214) of 0.3604mg/ml,
Arecaline reference substance Stock concentrations are 0.2369mg/ml.Precision draws Arecoline hydrobromide reference substance stock solution 1.0ml,
It sets in 10ml measuring bottle, is diluted to scale with mobile phase, shakes up to get reference substance solution；
(2) take this product, it is finely ground, take about 5g, it is accurately weighed, set in stuffed conical flask, add diethyl ether 50ml, then plus carbonate it is slow
Fliud flushing (take sodium carbonate 1.91g and sodium bicarbonate 0.56g, add water make to be dissolved into 100ml to get) 3ml, place 30 minutes, constantly
Shaking；It is heated to reflux 30 minutes, divides and take ether solution, residue adds diethyl ether heating and refluxing extraction 2 times (50ml, 50ml), every time 15 points
Clock merges ether solution three times, and phosphoric acid solution (5 → 1000) l ml is added, and mixes, and recycles ether, and residue adds 50% acetonitrile solution
Dissolution, is transferred in 25ml volumetric flask, adds 50% acetonitrile to scale；It shakes up, filters, take subsequent filtrate to get test solution；
(3) this product prescription preparation method is pressed, the negative granules of scarce Semen Arecae (parched) are made, yin is prepared by step (2) the method
Property test solution；
(4) accurate respectively to draw Arecoline hydrobromide reference substance solution, test solution, each 10 μ of negative test solution
L, injection liquid chromatograph are measured, wherein the condition of high performance liquid chromatography are as follows: use Dikma Diamonsil-C18 chromatography
Column (4.6mm × 250mm, 5 μm)；(2 → 1000, phosphoric acid regulating ph value to 7.4) (24: 76) is flowing to acetonitrile-triethylamine solution
Phase；Detection wavelength is 215nm；Volume flow 1.0ml/min；30 DEG C of column temperature；
Arecoline hydrobromide reference substance solution, test solution, negative test solution chromatogram are respectively such as Fig. 3~5, knot
Fruit shows under the chromatographic condition that the chromatographic peak peak shape of Arecoline hydrobromide is good in reference substance solution, hydrogen bromine in test solution
Chromatographic peak peak shape at the corresponding retention time of sour arecaline is good, and the separating degree of Arecoline hydrobromide chromatographic peak and impurity peaks is greater than
1.5, occur at the corresponding retention time of Arecoline hydrobromide chromatographic peak without chromatographic peak in negative test solution, the chromatographic condition
Specificity is good.
(5) citric acid quantitative determines
This product is taken, it is finely ground, about 2g is taken, it is accurately weighed, it sets in stuffed conical flask, water 50ml is added in precision, it shakes up, close plug,
Weighed weight, ultrasonic treatment (power 120W, frequency 40KHz) 30 minutes are taken out, then weighed weight, and the weight of less loss is supplied with water
Amount, shakes up, and filters, and precision measures subsequent filtrate 25ml, according to potentiometric titration, with sodium hydroxide titration liquid (0.05mol/L) current potential
Be titrated to PH8.0 to get.
Every 1ml sodium hydroxide titration liquid (0.05mol/L) is equivalent to the citric acid of 3.202mg.Every bag of this product contains organic acid
With citric acid (C6H8O7) meter, 15.0mg must not be less than.
This product prescription preparation method is pressed in the preparation of negative test solution, blank auxiliary particle is made, by the preparation side of test sample
Method is prepared to get negative test solution.
Measuring method: shine potentiometric titration, with 0.05mol/L sodium hydroxide titration liquid constant-current titration to PH8.0 to get.Often
1ml sodium hydroxide titration liquid (0.05mol/L) is equivalent to the citric acid of 3.202mg.Measurement result are as follows: 25.98mg/ bags.
The result shows that the volume of the sodium hydroxide titration liquid of negative test solution consumption is atomic in titration, it is therefore, negative
Test solution is noiseless.
In above-mentioned steps (four), methodological study is carried out to HPLC, is made a concrete analysis of as follows:
(1) specificity is investigated
Under the chromatographic condition, the chromatographic peak peak shape of Arecoline hydrobromide is good in reference substance solution, hydrogen in test solution
Chromatographic peak peak shape at the corresponding retention time of bromic acid arecaline is good, and the separating degree of Arecoline hydrobromide chromatographic peak and impurity peaks is big
Occur at the corresponding retention time of Arecoline hydrobromide chromatographic peak without chromatographic peak in 1.5, negative test solution, the chromatostrip
Part specificity is good.
(2) linear test is investigated
Accurate respectively to draw above-mentioned Arecoline hydrobromide reference substance stock solution, concentration is 0.3604mg/ml (i.e. arecaline
Reference substance Stock concentrations be 0.2369mg/ml), 0.2,0.5,1.0,2.0,3.0,4.0,5.0ml set in 10ml measuring bottle, with stream
Dynamic phase dilution is shaken up, is measured by above-mentioned chromatographic condition, respectively 10 μ l of sample introduction to scale, records chromatogram, reading peak area, with
Arecaline sample volume is abscissa, and peak area is ordinate mapping, obtains standard curve.The result shows that being 0.04738 in sample volume
Between~1.1845 μ g, arecaline is in good linear relationship.Regression equation is: y=3424.352x -2.5918, phase relation
Number: r=1.0000.
(3) precision test
This product is taken, by legal system available test sample solution, continuous sample introduction 6 times, is measured in accordance with the law, Arecoline hydrobromide chromatographic peak is recorded
As a result area see the table below 1.
1 precision test data of table
| Number | 1 | 2 | 3 | 4 | 5 | 6 | Average value |
| Test sample peak area | 785.886 | 785.498 | 786.005 | 767.332 | 785.367 | 768.631 | 779.786 |
The result shows that it is respectively 1.17% that Arecoline hydrobromide average peak area, which is respectively 779.8, RSD, in test sample, essence
Density is good.
(4) stability test
Take this product, by legal system available test sample solution and Arecoline hydrobromide reference substance solution respectively at be placed at room temperature for 0,2,4,
8,12, distinguish sample introduction afterwards for 24 hours, measure in accordance with the law, record Arecoline hydrobromide chromatographic peak area, the results are shown in Table 2.
2 stability test data of table
The result shows that test solution is 0.47% being placed at room temperature for stabilization in for 24 hours, peak area RSD, reference substance solution exists
It is placed at room temperature in for 24 hours and stablizes, peak area RSD is respectively 0.35%.
(5) repetitive test
This product about 5g is taken, it is accurately weighed, it is prepared by sample solution preparation method, prepares 6 parts, measure in accordance with the law, by external standard
Method calculates the content of Arecoline hydrobromide in this product, the results are shown in Table 3.
3 repetitive test result of table
| Sample number | 1 | 2 | 3 | 4 | 5 | 6 |
| Betel nut alkali content (mg/g) | 0.2055 | 0.2064 | 0.2058 | 0.2056 | 0.2068 | 0.2050 |
The result shows that betel nut alkali content average value is respectively 0.2059mg/g, RSD 0.32%, repeatability is good.
(6) sample recovery rate is tested
Take 9 parts of this product, every part of about 2.5g, it is accurately weighed, it parallel 9 parts, is respectively placed in 9 conical flasks, respectively takes 3 parts of difference
Precision be added Arecoline hydrobromide reference substance stock solution, i.e., arecaline reference substance Stock concentrations be 0.6ml, 1.2ml,
1.8ml is added ether 50ml, adds carbonate buffer solution 3ml again respectively, according to content determination processing, operates according to methods, prepares 9 parts
Test solution, sample introduction, measurement calculate the rate of recovery,
As the result is shown: in 80%~120% concentration range, the rate of recovery is that 92.71%~94.36%, RSD is
0.43%~2.47%, it meets the requirements.
Claims (8)
1. a kind of detection method of Children stasis relieving particle, the Children stasis relieving particle are made by following prescription: charred FRUCTUS CRATAEGI fries wheat
Bud, Medicated Leaven, Semen Arecae (parched), Endothelium Corneum Gigeriae Galli (parched), stir-baked SEMEN PHARBITIDIS, which comprises the steps of:
(1) Qualitive test is carried out to hawthorn in Children stasis relieving particle and kaladana using thin-layered chromatography；
(2) quantitative identification is carried out to the active constituent arecaline in Children stasis relieving particle using high performance liquid chromatography；
(3) quantitative identification is carried out to active constituent citric acid in Children stasis relieving particle using potentiometric titration.
2. the detection method of Children stasis relieving particle according to claim 1, which is characterized in that in step (1), the mountain
The discrimination method of short, bristly hair or beard includes the following steps:
(1) the Children stasis relieving particle is taken, EtOH Sonicate is added to handle, is dissolved in water after boiling off ethyl alcohol, then by polyamide column, first
It is eluted with water, then with ethanol elution, collects ethanol eluate, residue is dissolved in water, is extracted with ethyl acetate 3 times, merges acetic acid
Ethyl ester liquid, residue add methanol to dissolve, as test solution；
(2) hawthorn control medicinal material is taken, water is added to be heated to reflux, filtrate is evaporated, and residue adds EtOH Sonicate to handle, and is made in the same way of comparison medicine
Material solution；
(3) it is tested according to thin-layered chromatography, draws test solution and control medicinal material solution point respectively in same silica gel g thin-layer plate
On, the cyclohexane-acetone for being 2~15: 0.5~10 using volume ratio takes out after expansion and dries as solvent, color developing agent is sprayed,
Heating, which is placed under ultraviolet lamp, inspects.
3. the detection method of Children stasis relieving particle according to claim 1 or 2, which is characterized in that described in step (1)
The discrimination method of hawthorn includes the following steps:
(1) the Children stasis relieving particle 10g is taken, 30~80% 20~80ml of ethyl alcohol are added, 20~60min is ultrasonically treated, boils off second
Alcohol adds water to make to dissolve, and by polyamide column, is eluted with water, discards, then is eluted with 30~80% 20~80ml of ethyl alcohol, collects second
Alcohol eluen, residue add water to make to dissolve, and are extracted 3 times, each 15ml with ethyl acetate shaking, combined ethyl acetate liquid, residue adds
Methanol makes to dissolve, as test solution；
(2) take hawthorn control medicinal material 2g, add water 30ml, be heated to reflux 1h, filtrate is evaporated, residue add 30~80% ethyl alcohol 20~
80ml is made in the same way of control medicinal material solution；
(3) it is tested according to thin-layered chromatography, draws test solution respectively and each 10 μ l point of control medicinal material solution is thin in same silica G
On laminate, the cyclohexane-acetone for being 2~15: 0.5~10 using volume ratio as solvent, first set pre- in chromatography cylinder by solvent
It is saturated 10~50min, takes out and dries after expansion, sprays 10% sulfuric acid ethanol, 105 DEG C of heating are placed under ultraviolet lamp and inspect.
4. the detection method of Children stasis relieving particle according to claim 1, which is characterized in that described to lead in step (1)
The discrimination method of Niu Zi includes the following steps:
(1) take the Children stasis relieving particle that methanol is added to be ultrasonically treated, filtrate is evaporated, and residue is dissolved in water, filtrate dilute hydrochloric acid tune
PH value is saved, then is shaken and is extracted with ethyl acetate, combined ethyl acetate liquid is evaporated, and residue adds methanol to dissolve, molten as test sample
Liquid；
(2) kaladana's control medicinal material is taken, water is added to be heated to reflux, filtrate is adjusted with dilute hydrochloric acid, is made in the same way of control medicinal material solution；
(3) it is tested according to thin-layered chromatography, draws test solution and control medicinal material solution point respectively in same silica gel g thin-layer plate
On, it is solvent using butyl acetate-formic acid-water upper solution that volume ratio is 2~15: 1~10: 1~10, is taken out after expansion
It dries, sprays 5% ferric trichloride ethanol solution as color developing agent, heating is inspected.
5. the detection method of Children stasis relieving particle according to claim 1, which is characterized in that described to lead in step (1)
The discrimination method of Niu Zi includes the following steps:
(1) the Children stasis relieving particle 10g is taken, methanol 50ml is added, is ultrasonically treated 40min, filtrate is evaporated, and residue adds water 20ml to make
Dissolution adjusts pH value to 1~2 with dilute hydrochloric acid, then is extracted 3 times, each 20ml with ethyl acetate shaking, combined ethyl acetate liquid,
It is evaporated, residue adds methanol 1ml to make to dissolve, as test solution；
(2) kaladana control medicinal material 1g is taken, water 30ml is added, is heated to reflux 40min, filtrate adjusts pH value to 1~2 with dilute hydrochloric acid,
It is made in the same way of control medicinal material solution；
(3) it is tested according to thin-layered chromatography, it is thin in same silica G to draw 10~15 μ l of test solution, 8 μ l point of control medicinal material solution
It is solvent using butyl acetate-formic acid-water upper solution that volume ratio is 2~15: 1~10: 1~10, after expansion on laminate
Taking-up is dried, and sprays 5% ferric trichloride ethanol solution as color developing agent, heating is inspected.
6. the detection method of Children stasis relieving particle according to claim 1, which is characterized in that in step (2), the Bin
The high-efficient liquid phase chromatogram condition of bulky alkali are as follows: the chromatographic column using octadecylsilane chemically bonded silica as filler is used, with volume ratio
Acetonitrile-triethylamine solution for 5~25: 75~95 is mobile phase, and Detection wavelength 215nm, number of theoretical plate is based on arecaline peak
It calculates and is not less than 6000.
7. the detection method of Children stasis relieving particle according to claim 1 or 6, which is characterized in that described in step (2)
The high performance liquid chromatography discrimination method of arecaline are as follows:
(1) Arecoline hydrobromide reference substance is weighed, adds mobile phase that the solution conduct pair of every 1ml 30 μ g containing Arecoline hydrobromide is made
According to product solution；
(2) Children stasis relieving particle 5g is taken to add diethyl ether 50ml, then plus carbonate buffer solution 3ml, place 30 minutes, be heated to reflux 30 points
Clock divides and takes ether solution, and residue adds diethyl ether heating and refluxing extraction 2 times again, merges ether solution three times, and phosphoric acid solution l ml is added, and returns
Ether is received, residue adds 50% acetonitrile to dissolve, is transferred in 25ml volumetric flask, and be diluted to scale, takes subsequent filtrate to get test sample
Solution；
(3) above-mentioned reference substance solution and test solution are drawn, injection liquid chromatograph is measured.
8. the detection method of Children stasis relieving particle according to claim 1, which is characterized in that in step (3), the electricity
Position titration are as follows: take the Children stasis relieving particle that water is added to shake up, after ultrasonic treatment, with the sodium hydroxide titration liquid of 0.05mol/L
Constant-current titration pH value is to 7.0~9.5.
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| WO2020140670A1 (en) * | 2018-12-31 | 2020-07-09 | 海南葫芦娃药业集团股份有限公司 | Method for detecting infantile accumulation-eliminating granules |
| CN115144251A (en) * | 2021-03-30 | 2022-10-04 | 山东新时代药业有限公司 | Method for processing biological sample |
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