CN109164200B - Quality detection method of cough and asthma relieving granules of polietilenii - Google Patents
Quality detection method of cough and asthma relieving granules of polietilenii Download PDFInfo
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Abstract
The invention discloses a quality control method of granules for relieving cough and asthma of a pediatric Malong, which adopts a thin-layer chromatography to qualitatively identify earthworm, loquat leaf and liquorice in the granules for relieving cough and asthma of the pediatric Malong; the content of three active ingredients, namely amygdalin, ephedrine and forsythiaside A in the cough and asthma relieving granule of the pediatric ephedra herb, the ephedra herb and the forsythiaside A, is measured by adopting a high performance liquid chromatography for quantitative control. The method is simple, has good separation degree, strong specificity and good reproducibility, effectively ensures the quality and the curative effect of the granules for relieving cough and asthma of the children, and has strong practicability.
Description
Technical Field
The invention belongs to the technical field of medicine quality detection, and particularly relates to a quality control method of granules for treating cough and asthma of infant ma long-noded pit viper.
Background
The cough and asthma relieving granule of the infant ma long is prepared from the following medicinal components: 1-3 parts of fried bitter almond, 1-3 parts of fried peach kernel, 2-4 parts of rhizoma pinelliae preparata, 1-2 parts of liquorice, 4-9 parts of gypsum, 1-2 parts of ephedra, 1-3 parts of earthworm, 1-3 parts of fructus forsythiae, 1-3 parts of cortex mori radicis, 1-3 parts of honey radix stemonae, 1-3 parts of radix peucedani and 2-4 parts of loquat leaves, and has the effects of clearing lung, eliminating phlegm, dispelling wind and cold, and depressing qi to relieve cough.
The ephedra and the earthworm are combined, the heat clearing and lung ventilating, the wind and cold dispelling, the spasmolysis and cough relieving are carried out together, the ephedra and the earthworm are monarch drugs, the bitter apricot seed, the sessile stemona root, the loquat leaf, the gypsum and the rhizoma pinelliae preparata are ministerial drugs, and the weeping forsythia capsule, the whiteflower hogfennel root and the white mulberry root-bark are adjuvant drugs.
The method for identifying the medicinal materials of earthworm, loquat leaf and licorice is recorded in the first part of the Chinese pharmacopoeia of 2015 edition.
Earthworm medicinal material identification: taking the powder lg, adding 20ml of trichloromethane, carrying out ultrasonic treatment for 20 minutes, filtering, evaporating filtrate to dryness, and adding trichloromethane lm l to residues for dissolving to obtain a test solution; preparing control material lg of Lumbricus, and making into control material solution by the same method. Performing thin layer chromatography, sucking the above two solutions 5 μ l each, dropping on the same silica gel G thin layer plate, developing with toluene-acetone (9: 1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp. In the chromatogram of the test solution, fluorescent spots of the same color appear at corresponding positions of the chromatogram of the reference solution.
Loquat leaf identification: collecting powder lg, adding methanol 20ml, ultrasonic treating for 20 min, filtering, evaporating filtrate, and dissolving residue with methanol 5ml to obtain test solution. Preparing another loquat leaf reference medicinal material lg, and preparing a reference medicinal material solution by the same method; adding methanol into ursolic acid control to obtain lmg solution per lm as control solution; performing thin layer chromatography test, sucking the three solutions 1 μ l each, dropping on the same silica gel G thin layer plate, developing with toluene-acetone (5:1) as developing agent, taking out, air drying, spraying 10% sulphuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the control solution and the reference solution.
And (3) identifying liquorice: collecting powder 1g, adding diethyl ether 40ml, heating and refluxing for 1 hr, filtering, removing ether solution, adding methanol 30ml into the residue, heating and refluxing for 1 hr, filtering, evaporating the filtrate to dryness, dissolving the residue with water 40ml, extracting with n-butanol for 3 times (20 ml each time), mixing n-butanol solutions, washing with water for 3 times, removing water solution, evaporating n-butanol solution to dryness, dissolving the residue with methanol 5ml to obtain sample solution; preparing 1g of Glycyrrhrizae radix control material, and making into control solution by the same method; adding methanol to the monoammonium glycyrrhizinate control to obtain a solution containing 2mg per lm l as a control solution. And (3) performing thin-layer chromatography test, sucking 1-2 mu l of each of the three solutions, respectively dropping the three solutions on the same silica gel G thin-layer plate prepared from 1% sodium hydroxide solution, developing by using ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as a developing agent, taking out, drying in the air, spraying 10% sulfuric acid ethanol solution, heating at 105 ℃ until the spots are clear in color, and inspecting under an ultraviolet lamp. In the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution; at the position corresponding to the control chromatogram, the same orange-yellow fluorescent spot appeared.
The earthworm, the loquat leaf and the liquorice are identified by the reference pharmacopoeia identification method, negative interference exists, and the result is unstable, so that the identification method is improved, the interference can be eliminated, and the obtained result is accurate.
Disclosure of Invention
The invention provides a simple quality control method of the children's Malong cough and asthma relieving granules with strong specificity and good repeatability, and provides guarantee for the quality of the children's Malong cough and asthma relieving granules.
A quality control method of XIAOERFULONG granule for relieving cough and asthma comprises the following steps:
(1) qualitatively identifying Lumbricus, folium Eriobotryae and Glycyrrhrizae radix in the infantile MALONG granule with effects of relieving cough and asthma by thin layer chromatography;
(2) quantitatively identifying active ingredients of amygdalin, ephedrine and forsythiaside A in the infantile Malong cough and asthma relieving granule by high performance liquid chromatography;
in the step (1), the chromatographic conditions for earthworm identification are as follows: taking earthworm as a reference medicinal material, taking n-butanol-glacial acetic acid-water with the volume ratio of 3-7: 0.5-1.5: 1 as a developing agent, taking ninhydrin solution as a color developing agent, and developing at 105 ℃;
the chromatographic conditions for loquat leaf identification are as follows: loquat leaves are used as a reference medicinal material, a chloroform-methanol-water lower layer solution with the volume ratio of 3-6: 4-8: 2-5 is used as a developing agent, a 10% phosphomolybdic acid ethanol solution is used as a color developing agent, and color development is carried out at 105 ℃;
the chromatographic conditions for identifying the liquorice are as follows: the liquorice is used as a reference medicinal material, chloroform-methanol-ethyl acetate-formic acid-water with the volume ratio of 15-22: 4-8: 2-4: 0.2-0.6: 0.15 is used as a developing agent, and a 10% sulfuric acid ethanol solution is used as a color developing agent, so that the color is developed at 105 ℃.
In the step (2), the ephedrine is ephedrine hydrochloride and pseudoephedrine hydrochloride.
According to the invention, the traditional Chinese medicinal materials in the prescription are carefully researched, various methods are investigated for extraction, various expansion systems are adopted for expansion, and the honey radix stemonae has good characteristic spots, but multiple tests show that the method is influenced by more interference factors and has poor stability and repeatability; the cortex mori radicis also provided with better characteristic spots, but the interference cannot be eliminated; the gypsum and the rhizoma pinelliae preparata have no obvious characteristic spots; therefore, the experimental research results of the radix stemonae preparata, the cortex mori radicis, the rhizoma pinelliae preparata and the gypsum are not ideal, and the thin-layer identification repeatability is poor or the interference cannot be eliminated. And the thin-layer chromatography of the earthworm, the loquat leaf and the liquorice has clear identification spots, good reproducibility and no interference on negative.
Therefore, in order to effectively control the medicine quality, a scientific quality control method is established, four medicinal materials of ephedra herb, fried bitter apricot seed, fried peach seed and weeping forsythia are quantitatively controlled, and 3 thin-layer identification items are established for the other 8 medicinal materials through experimental research, wherein licorice, earthworm and loquat leaf are respectively used as reference medicinal materials for qualitative identification.
Earthworm is the monarch drug in the granules for relieving cough and asthma, the main components of the earthworm are protein, polypeptide, amino acid, succinic acid of nucleotide, hypoxanthine, xanthine, adenine and the like, and quantitative control indexes can not be established through research, so thin-layer identification research is carried out. The product is identified by referring to the pharmacopoeia identification method, and the negative is interfered; through multiple experiments, the development system of benzene-ethyl acetate and n-butyl alcohol-glacial acetic acid-water is investigated, and the result is that n-butyl alcohol-glacial acetic acid-water (3-7: 0.5-1.5: 1) is used as a developing agent, ninhydrin solution is sprayed, and color development is carried out at 105 ℃, so that a satisfactory result can be obtained.
The loquat leaves mainly contain ursolic acid and oleanolic acid, the method in the pharmacopoeia is to identify the ursolic acid, the test adopts the method in the pharmacopoeia to identify, but negative results have interference. Through experimental research, the control medicinal material and the test solution are alkalized by sodium hydroxide solution and then extracted by ethyl acetate, so that interference can be eliminated; according to investigation, a lower layer solution of trichloromethane-methanol-water (3-6: 4-8: 2-5) is used as a developing agent, a 10% phosphomolybdic acid ethanol solution is sprayed, and color development is carried out at 105 ℃, so that a satisfactory result is obtained.
The liquorice is firstly identified by adopting a method of pharmacopoeia, and the test sample chromatogram has corresponding spots with the reference medicinal material chromatogram, but the spots have deeper background and appear to have interference, so the method is not adopted. The invention takes trichloromethane-methanol-ethyl acetate-formic acid-water (15-22: 4-8: 2-4: 0.2-0.6: 0.15) as a developing agent, sprays 10% sulfuric acid ethanol solution, develops color at 105 ℃, and has satisfactory result and good repeatability and stability.
According to the selected requirement of quality standard research content determination of new Chinese medicine, the main effective components related to the main functional indications and indications of the product are selected as the content determination index components of the product, ephedrine has the effects of resisting allergy and relieving the spasm of bronchial smooth muscle, amygdalin has the remarkable effects of relieving cough and asthma, and forsythoside A has the effects of resisting inflammation and resisting allergy. Therefore, ephedrine hydrochloride serving as a main component of the monarch drug ephedra, amygdalin serving as a main component of the ministerial drug semen armeniacae amarae and forsythiaside A serving as a main component of the left drug fructus forsythiae are selected as content determination indexes for quality control of the product.
The cough and asthma relieving granule of the infantile Malong contains bitter apricot kernel and peach seed medicinal materials, amygdalin is the main effective component of the two medicinal materials, and the amygdalin can be slowly decomposed in vivo to gradually generate trace hydrocyanic acid, so that the cough and asthma relieving granule can play a role in slightly inhibiting the respiratory center, and leads the respiratory system to be quiet and has obvious cough and asthma relieving efficacy. Therefore, amygdalin is selected as the quality control index component of the product, the content of amygdalin is determined by an HPLC method, and a methodology study is carried out.
The ephedra is a monarch drug in the prescription of the children's ephedra stem cough and asthma relieving granule, and the ephedrine is a main effective component of ephedra medicinal materials and has the effect of relieving asthma and cough, so the ephedrine is selected as a quality control index component of the product, a method for simultaneously measuring the content of the ephedrine by an HPLC method is established, and the methodology research is carried out.
Forsythia suspensa is one of the medicinal ingredients consisting of the Malong cough and asthma relieving granules, forsythiaside A is the main effective component of the medicinal material, modern pharmacological research shows that the forsythiaside A has anti-inflammatory and anti-allergic effects, and literature reports that the forsythiaside A is poor in stability, so that the forsythiaside A is selected as one of a plurality of quantitative control index components of the product, a method for determining the content of the forsythiaside A by an HPLC method is established, and methodology research is carried out.
In the step (1), the earthworm identification method comprises the following steps: dissolving 6g of the product in water, adding trichloromethane for ultrasonic treatment for 20-60 minutes, taking the filtrate, evaporating to dryness, and dissolving the residue in 1ml of methanol to obtain a test solution; adding 20-60 ml of methanol into a earthworm control medicinal material, carrying out ultrasonic treatment for 20-60 minutes, evaporating filtrate to dryness, and dissolving residues in methanol to obtain a control medicinal material solution; testing according to thin layer chromatography Chinese pharmacopoeia 2015 edition four parts general rule 0502, sucking test solution and control solution 10 μ l each, respectively dropping on the same silica gel G thin layer plate, developing in developing agent, taking out, air drying, spraying color developing agent, drying at 105 deg.C until spots are clearly developed, and displaying spots with the same color in the test chromatogram and the corresponding position of the control chromatogram.
In the step (1), the identification method of the loquat leaves comprises the following steps: dissolving 6g of the product in water, adjusting the pH value to 8-11 with 1% sodium hydroxide solution, extracting with 15-30 ml of ethyl acetate for 2 times, evaporating to dryness, and dissolving the residue with 0.5ml of methanol to obtain a sample solution; taking a loquat leaf control medicinal material, adding water, carrying out reflux extraction for 30-60 minutes, adjusting the pH value of filtrate to 8-11 by using 1% sodium hydroxide solution, respectively extracting with 15-30 ml of ethyl acetate for 2 times, evaporating to dryness, and dissolving residues by adding methanol to obtain a control medicinal material solution; testing according to thin layer chromatography Chinese pharmacopoeia 2015 edition four parts general rule 0502, sucking test solution and control solution 10 μ l each, respectively dropping on the same silica gel G thin layer plate, developing in developing agent, taking out, air drying, spraying color developing agent, drying at 105 deg.C until spots are clearly developed, and displaying spots with the same color in the test chromatogram and the corresponding position of the control chromatogram.
In the step (1), the identification method of the liquorice comprises the following steps: taking 3g of the product, adding 30ml of methanol, carrying out ultrasonic treatment for 20-60 minutes, evaporating filtrate to dryness, dissolving residues in water, extracting with 10-40 ml of petroleum ether for 3 times respectively, combining water layers, extracting with 20-50 ml of ethyl acetate for 2 times respectively, evaporating ethyl acetate extracting solution to dryness, and dissolving residues in 2ml of methanol to obtain a sample solution; weighing a liquorice control medicinal material, adding methanol for ultrasonic treatment for 20-60 minutes, evaporating filtrate to dryness, dissolving residues in water, extracting with 10-30 ml of ethyl acetate for 2 times respectively, combining ethyl acetate solutions, evaporating to dryness, and dissolving residues in methanol to obtain a control medicinal material solution; testing according to thin layer chromatography Chinese pharmacopoeia 2015 edition four parts general rule 0502, sucking test solution and control solution 10 μ l each, respectively dropping on the same silica gel G thin layer plate, developing in developing agent, taking out, air drying, spraying color developing agent, drying at 105 deg.C until spots are clearly developed, and displaying spots with the same color in the test chromatogram and the corresponding position of the control chromatogram.
In the step (2), the high performance liquid chromatography conditions of the amygdalin are as follows: a chromatographic column using octadecylsilane chemically bonded silica as a filler is adopted, methanol-0.05% phosphoric acid aqueous solution with the volume ratio of 15-25: 75-85 is used as a mobile phase, the detection wavelength is 210nm, and the number of theoretical plates is not less than 6000 calculated according to the amygdalin peak;
the high performance liquid chromatography conditions of the ephedrine are as follows: octadecylsilane chemically bonded silica is used as a filling agent, acetonitrile-0.1% phosphoric acid aqueous solution with the volume ratio of 2-8: 92-98 is used as a mobile phase, the detection wavelength is 210nm, and the number of theoretical plates is not less than 6000 calculated according to the ephedrine hydrochloride peak;
the conditions of the high performance liquid chromatography of the forsythia are as follows: octadecylsilane chemically bonded silica is used as a filling agent, methanol-0.05% phosphoric acid aqueous solution with the volume ratio of 30-38: 62-70 is used as a mobile phase, the detection wavelength is 330nm, and the number of theoretical plates is not less than 5000 calculated according to a forsythoside A peak.
In the step (2), the high performance liquid chromatography identification method of amygdalin comprises the following steps: weighing amygdalin reference substance, precisely weighing, and adding 50% methanol to obtain 0.1mg solution per 1ml to obtain reference substance solution; taking 0.2g of the product, adding 25ml of 50-80% methanol, carrying out ultrasonic treatment for 10-30 minutes, complementing the weight loss by using 50-80% methanol, shaking up, and taking a filtrate to obtain a test solution; sucking amygdalin reference solution and test solution 10 μ l each, and injecting into liquid chromatograph for determination; the cough and asthma relieving granule of the infant ma long-noded pit viper contains bitter apricot kernels and peach kernels, and the content of the bitter apricot kernels and the peach kernels is not less than 3.9mg/g calculated by amygdalin.
In the step (2), the high performance liquid chromatography identification method of ephedrine comprises: weighing ephedrine hydrochloride reference substance and pseudoephedrine hydrochloride reference substance, precisely weighing, and dissolving in 50% methanol to obtain mixed solution containing ephedrine hydrochloride 0.04mg and pseudoephedrine hydrochloride 0.01mg per 1ml to obtain reference solution; weighing the product, precisely adding 25ml of 50-80% methanol containing 1.0-2.0% phosphoric acid, carrying out ultrasonic treatment for 10-40 minutes, complementing the lost weight with 50-80% methanol containing 1.0-2.0% phosphoric acid, and taking a subsequent filtrate to obtain a test solution; precisely sucking 10 μ l of each of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement; the granule for relieving cough and asthma of the infantile Mallotus comprises herba Ephedrae (calculated on ephedrine hydrochloride and pseudoephedrine hydrochloride) with total amount not less than 0.5 mg/g.
In the step (2), the high performance liquid chromatography identification method of forsythiaside A comprises the following steps: weighing forsythoside A reference substance, precisely weighing, and dissolving with 50% methanol to obtain forsythoside A solution containing 0.05mg per 1ml to obtain reference solution; weighing the product, adding 25ml of 50-80% methanol, carrying out ultrasonic treatment for 10-40 minutes, complementing the lost weight with 50-80% methanol, shaking up, and taking a subsequent filtrate to obtain a test solution; precisely sucking 10 μ l of each of the forsythiaside A reference substance and the sample, and injecting into a liquid chromatograph for determination; the cough and asthma relieving granule containing fructus forsythiae in terms of forsythiaside is not less than 0.07 mg/g.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, the improved thin-layer chromatography is utilized to qualitatively identify the earthworm, the loquat leaf and the liquorice in the granules for relieving cough and asthma of the pediatric Malong, and the obtained thin-layer chromatography identification spots are clear, negative is free from interference, the result is satisfactory, and the repeatability and the stability are good;
(2) the method utilizes the high performance liquid chromatography to measure the contents of ephedrine hydrochloride, amygdalin and forsythiaside A in the granules for relieving cough and asthma of the infantile ephedrine hydrochloride, has the advantages of good separation effect, sensitivity, accuracy and the like, and ensures the quality stability, uniformity and curative effect of the product;
(3) the quality control method of the invention is simple, has strong specificity and good reproducibility, effectively ensures the quality and the curative effect of the granules for relieving cough and asthma of the children, and has strong practicability.
Drawings
FIG. 1 is a thin layer chromatogram of Pheretima of example 1, wherein 1 is a negative control, 2 is a control, and 3-5 are samples;
FIG. 2 is a thin layer chromatogram of loquat leaves of example 2, wherein 1 is a reference, 2 is a negative reference, and 3-5 are samples;
FIG. 3 is a thin layer chromatogram of Glycyrrhiza uralensis of example 3, wherein 1 is a reference substance, 2 is a negative reference substance, and 3-5 are test substances;
FIG. 4 is a high performance liquid chromatogram of the amygdalin control of example 4;
FIG. 5 is a high performance liquid chromatogram of the amygdalin test sample of example 4;
FIG. 6 is a high performance liquid chromatogram of an amygdalin negative test sample of example 4;
FIG. 7 is a high performance liquid chromatogram of ephedrine hydrochloride and pseudoephedrine hydrochloride controls of example 5;
FIG. 8 is a high performance liquid chromatogram of the ephedrine test sample of example 5;
FIG. 9 is a high performance liquid chromatogram of the ephedrine negative test sample of example 5;
FIG. 10 is a high performance liquid chromatogram of a forsythoside A control of example 6;
FIG. 11 is a high performance liquid chromatogram of the forsythoside A test sample of example 6;
FIG. 12 is a high performance liquid chromatogram of a forsythoside A negative test sample of example 6.
Detailed Description
The Agilent1100 high performance liquid chromatograph comprises a G1310A unit pump, a G1313A autosampler, a G1314AVWD detector, a CO-201 column temperature box and an Agilent chromatographic workstation; type AB204-N electronic analytical balance (METTLERTOCO).
The methanol is chromatographically pure, other reagents are analytically pure, and the solution is filtered by a 0.45-micron filter membrane; the control drugs Lumbricus, folium Eriobotryae and Glycyrrhrizae radix were purchased from China food and drug testing research institute.
Example 1
(1) Weighing 0.5g Lumbricus control material, adding 20ml methanol, ultrasonic treating for 40 min, cooling, filtering, evaporating filtrate, and dissolving residue with 2ml methanol to obtain control material solution;
(2) weighing 6g of ground granules for relieving cough and asthma of the pediatric Malong, adding 20ml of water for dissolving, using 30ml of trichloromethane for ultrasonic treatment for 50 minutes, cooling, taking the trichloromethane solution separately, evaporating to dryness, and adding 1ml of methanol to dissolve residues to be used as a test solution;
(3) weighing 3g of ground earthworm negative samples, and operating according to the method in the step (2) to serve as a negative control solution;
(4) according to thin layer chromatography (general rule of four parts of Chinese pharmacopoeia 2015 edition 0502), respectively dropping 10 μ l of test solution, control solution and negative test solution on the same silica gel G thin layer plate, developing with n-butanol-glacial acetic acid-water (5: 1.5: 1) as developing agent, taking out, air drying, spraying ninhydrin solution, and drying at 105 deg.C until the spots are clearly developed.
The identification result is shown in fig. 1, and spots with the same color are displayed in the corresponding positions of the chromatogram of the test solution and the chromatogram of the reference solution; there is no spot of the same color in the negative control chromatogram corresponding to the control chromatogram.
Example 2
(1) Weighing 1g of loquat leaf control drug, adding 20ml of water, carrying out reflux extraction for 60 minutes, cooling, filtering, adjusting the pH of the filtrate to 11 with 1% sodium hydroxide solution, extracting with ethyl acetate for 2 times, each time with 30ml, evaporating the ethyl acetate extract to dryness, and dissolving the residue with 0.5ml of methanol to obtain a control drug solution;
(2) weighing 6g of ground granules for relieving cough and asthma of the pediatric Malong, adding 20ml of water for dissolving, adjusting the pH value to 11 by using 1% sodium hydroxide solution, extracting for 2 times by using ethyl acetate, 30ml each time, evaporating the ethyl acetate extracting solution to dryness, and adding 0.5ml of methanol for dissolving residues to obtain a test solution;
(3) weighing 2.5g of a loquat leaf negative sample, grinding, and operating according to the method in the step (2) to serve as a negative control solution;
(4) according to the thin layer chromatography (the four parts general rule of Chinese pharmacopoeia 2015 edition 0502), respectively sucking 10 mul of the test solution, the control solution and the negative test solution, respectively dropping on the same silica gel G thin layer plate, taking out the lower layer solution of trichloromethane-methanol-water (5: 4) as a developing agent, developing, taking out, airing, spraying 10% phosphomolybdic acid ethanol solution, and drying at 105 ℃ until the spots are clearly developed.
The identification result is shown in FIG. 2, and spots of the same color appear in the chromatogram of the test solution at the positions corresponding to those in the chromatogram of the control solution; there is no spot of the same color in the negative control chromatogram corresponding to the control chromatogram.
Example 3
(1) Weighing 0.5g Glycyrrhrizae radix control, adding 30ml methanol, ultrasonic treating for 50 min, cooling, filtering, evaporating filtrate to dryness, dissolving residue with 20ml water, extracting with ethyl acetate for 2 times (30 ml each time), mixing ethyl acetate solutions, evaporating to dryness, dissolving residue with 2ml methanol to obtain control solution;
(2) weighing 3g of ground granules for relieving cough and asthma of the pediatric Malong, adding 30ml of methanol, carrying out ultrasonic treatment for 50 minutes, cooling, filtering, evaporating filtrate to dryness, dissolving residues in 20ml of water, extracting with petroleum ether (60-90 ℃) for 3 times, 40ml each time, combining aqueous solution layers for later use, extracting with ethyl acetate for 2 times, 40ml each time, combining ethyl acetate extracting solutions, evaporating to dryness, and adding 2ml of methanol to the residues for dissolving to obtain a sample solution;
(3) weighing 1.5g of the ground liquorice negative sample, and operating according to the method in the step (2) to serve as a negative control solution;
(4) testing according to thin layer chromatography Chinese pharmacopoeia 2015 edition four parts general rule 0502), sucking test solution, control solution and negative control solution 10 μ l each, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-ethyl acetate-formic acid-water (16: 5: 4: 0.3: 0.15) as developing agent, taking out, air drying, spraying 10% sulphuric acid ethanol solution, and drying at 105 deg.C until spots are clearly developed.
The identification result is shown in FIG. 3, and spots of the same color appear in the chromatogram of the test solution at the positions corresponding to those in the chromatogram of the control solution; there is no spot of the same color in the negative control chromatogram corresponding to the control chromatogram.
Example 4
(1) Weighing amygdalin reference substance (purity 85.8%) 12.49mg, adding 50% methanol, and dissolving to obtain 0.50mg/ml amygdalin reference substance stock solution; then 2.0ml of the amygdalin reference substance stock solution is sucked and placed in a 10ml measuring flask, diluted to the scale with 50 percent methanol and shaken up to obtain a reference substance solution;
(2) taking the product with different filling amount, grinding, taking 0.2g, precisely weighing, placing in a conical flask with a plug, adding 25ml of 60% methanol, sealing, weighing, ultrasonically treating for 30 minutes, cooling, weighing again, supplementing the lost weight with 60% methanol, shaking up, filtering with 0.45 μm microporous membrane, and taking the subsequent filtrate to obtain the sample solution;
(3) preparing negative granules of the bitter almond and the peach kernel with the lack of taste according to the preparation method of the prescription of the product, and preparing a negative test sample solution according to the method in the step (2);
(4) respectively injecting 10 mul of amygdalin reference solution, test solution and negative test solution into a liquid chromatograph for determination, wherein the conditions of the high performance liquid chromatograph are as follows: dikma Diamonsil-C18(4.6 mm. times.250 mm, 5 μm) was used as a column, methanol-0.05% phosphoric acid aqueous solution (15: 85) was used as a mobile phase, the detection wavelength was 210nm, the volume flow rate was 1.0ml/min, and the column temperature was 30 ℃.
The chromatograms of the obtained reference solution, test solution and negative test solution are respectively shown in figures 4-6, and the results show that: under the chromatographic condition, the peak shape of the chromatographic peak of the amygdalin in the reference solution is good; the chromatographic peak shape of the amygdalin in the test solution at the corresponding retention time is good, and the separation degree of the amygdalin chromatographic peak and the impurity peak is more than 1.5; the negative test sample solution has no chromatographic peak at the corresponding retention time of the amygdalin chromatographic peak, and the chromatographic condition has good specificity.
Example 5
(1) Weighing ephedrine hydrochloride reference substance 10.12mg, dissolving in methanol to obtain ephedrine hydrochloride reference substance stock solution with concentration of 0.405mg/ml, weighing pseudoephedrine hydrochloride reference substance 6.25mg, and dissolving in methanol to obtain pseudoephedrine hydrochloride reference substance stock solution with concentration of 0.250 mg/ml; precisely sucking 1.0ml of ephedrine hydrochloride reference substance stock solution and 0.5ml of pseudoephedrine hydrochloride reference substance stock solution, placing in a 10ml measuring flask, diluting with 50% methanol to scale, and shaking to obtain reference substance solution;
(2) weighing the product with different contents, grinding, weighing 1.0g precisely, placing in a conical flask with a plug, adding 25ml of 60% methanol containing 2.0% phosphoric acid precisely, sealing the plug, weighing, ultrasonically treating for 20 min, cooling, weighing again, supplementing the lost weight with 60% methanol containing 2.0% phosphoric acid, shaking, filtering with 0.45 μm microporous membrane, and collecting the subsequent filtrate to obtain the sample solution;
(3) preparing negative granules of the ephedra without taste according to the preparation method of the prescription, and preparing a negative test sample solution according to the method in the step (2);
(4) precisely sucking 10 μ l of each of the control solution, the test solution and the negative test solution, injecting into a liquid chromatograph for determination, wherein the chromatographic conditions adopt Dikma Diamonsil-C18(4.6mm × 250mm, 5 μm) chromatographic column, acetonitrile-0.1% phosphoric acid aqueous solution (0.2 ml triethylamine (8: 92) is added to each 100 ml) as mobile phase, the detection wavelength is 210nm, the volume flow is 1.0ml/min, and the column temperature is 30 ℃.
The chromatograms of the obtained reference solution, test solution and negative test solution are respectively shown in fig. 7-9, and the results show that: under the chromatographic condition, the chromatographic peak shapes of ephedrine hydrochloride and pseudoephedrine hydrochloride in the reference solution are good; the chromatographic peak shapes of the corresponding retention time of the ephedrine hydrochloride and the pseudoephedrine hydrochloride in the test solution are good, and the separation degree of the chromatographic peaks of the ephedrine hydrochloride and the pseudoephedrine hydrochloride and the impurity peak is more than 1.5; the negative sample solution has no chromatographic peak at the corresponding retention time of the chromatographic peaks of ephedrine hydrochloride and pseudoephedrine hydrochloride, and the chromatographic condition has good specificity.
Example 6
(1) Accurately weighing forsythoside A reference substance 12.42mg (purity is 94.1%), and dissolving in methanol to obtain forsythoside A reference substance stock solution with concentration of 0.497 mg/ml. Precisely sucking 1.0ml of forsythoside A reference substance stock solution, placing in a 10ml measuring flask, diluting with 50% methanol to scale, and shaking to obtain reference substance solution;
(2) grinding the product with different contents, weighing about 0.7g, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 80% methanol, sealing, weighing, ultrasonically treating for 30 min, cooling, weighing again, supplementing the weight loss with 70% methanol, shaking, filtering with 0.45 μm microporous membrane, and collecting the filtrate to obtain the sample solution;
(3) preparing negative granules of the fructus forsythiae medicinal material with the deficiency according to the preparation method of the prescription, and preparing a negative test sample solution according to the method in the step (2);
(4) precisely sucking 10 μ l of each of forsythoside A reference substance, test sample and negative reference solution, injecting into liquid chromatograph, and measuring with high performance liquid chromatography using Dikma Diamond-C18 (4.6mm × 250mm, 5 μm) chromatographic column, methanol-0.05% phosphoric acid water solution (38: 62) as mobile phase, detection wavelength of 330nm, volume flow of 1.0ml/min, and column temperature of 30 deg.C.
The chromatograms of the obtained control solution, test solution and negative control solution are respectively shown in fig. 10-12, and the results show that: under the chromatographic condition, the peak shape of the forsythiaside A in the reference solution is good; the chromatographic peak shape of the forsythiaside A in the test solution at the corresponding retention time is good, and the separation degree of the chromatographic peak of the forsythiaside A and the impurity peak is more than 1.5; the negative test sample solution has no chromatographic peak at the corresponding retention time of the forsythoside A chromatographic peak, and the chromatographic condition has good specificity.
Claims (7)
1. A quality detection method of a pediatric 'Maslong' cough and asthma relieving granule comprises the following steps:
(1) qualitatively identifying Lumbricus, folium Eriobotryae and Glycyrrhrizae radix in the infantile MALONG granule with effects of relieving cough and asthma by thin layer chromatography;
(2) quantitatively identifying active ingredients of amygdalin, ephedrine and forsythiaside A in the infantile Malong cough and asthma relieving granule by high performance liquid chromatography;
in the step (1), the chromatographic conditions for earthworm identification are as follows: taking Lumbricus as reference medicinal material, n-butanol-glacial acetic acid-water at volume ratio of 5: 1.5: 1 as developing agent, and ninhydrin solution as developer, and developing at 105 deg.C;
the chromatographic conditions for loquat leaf identification are as follows: folium Eriobotryae as control, chloroform-methanol-water lower layer solution at volume ratio of 5: 4 as developing agent, 10% phosphomolybdic acid ethanol solution as developer, and developing at 105 deg.C;
the chromatographic conditions for identifying the liquorice are as follows: taking Glycyrrhrizae radix as reference material, chloroform-methanol-ethyl acetate-formic acid-water as developing agent at volume ratio of 16: 5: 4: 0.3: 0.15, and 10% sulphuric acid ethanol solution as developer, developing at 105 deg.C;
in the step (2), the high performance liquid chromatography conditions of the amygdalin are as follows: a chromatographic column which takes octadecylsilane chemically bonded silica as a filler is adopted, methanol-0.05 percent of phosphoric acid aqueous solution with the volume ratio of 15: 85 is taken as a mobile phase, the detection wavelength is 210nm, and the number of theoretical plates is not less than 6000 according to the peak of amygdalin;
the high performance liquid chromatography conditions of the ephedrine are as follows: octadecylsilane chemically bonded silica is used as a filler, acetonitrile-0.1% phosphoric acid aqueous solution with the volume ratio of 8: 92 is used as a mobile phase, the detection wavelength is 210nm, and the number of theoretical plates is not less than 6000 according to the peak of ephedrine hydrochloride;
the conditions of the high performance liquid chromatography of the forsythia are as follows: octadecylsilane chemically bonded silica is used as a filling agent, methanol-0.05% phosphoric acid aqueous solution with the volume ratio of 38: 62 is used as a mobile phase, the detection wavelength is 330nm, and the number of theoretical plates is not less than 5000 according to the peak of forsythoside A.
2. The quality detection method for the granules for treating cough and asthma of the children's Malong dragon according to claim 1, wherein in the step (1), the identification method for the earthworm is as follows: dissolving 6g of the product in water, adding trichloromethane for ultrasonic treatment for 20-60 minutes, taking the filtrate, evaporating to dryness, and dissolving the residue in 1ml of methanol to obtain a test solution; adding 20-60 ml of methanol into a earthworm control medicinal material, carrying out ultrasonic treatment for 20-60 minutes, evaporating filtrate to dryness, and dissolving residues in methanol to obtain a control medicinal material solution; testing according to thin layer chromatography Chinese pharmacopoeia 2015 edition four parts general rule 0502, sucking test solution and control solution 10 μ l each, respectively dropping on the same silica gel G thin layer plate, developing in developing agent, taking out, air drying, spraying color developing agent, drying at 105 deg.C until spots are clearly developed, and displaying spots with the same color in the test chromatogram and the corresponding position of the control chromatogram.
3. The quality detection method for the granules for relieving cough and asthma of children's Malong as claimed in claim 1, wherein in the step (1), the identification method for the loquat leaves comprises the following steps: dissolving 6g of the product in water, adjusting the pH value to 8-11 with 1% sodium hydroxide solution, extracting with 15-30 ml of ethyl acetate for 2 times, evaporating to dryness, and dissolving the residue with 0.5ml of methanol to obtain a sample solution; taking a loquat leaf control medicinal material, adding water, carrying out reflux extraction for 30-60 minutes, adjusting the pH value of filtrate to 8-11 by using 1% sodium hydroxide solution, respectively extracting with 15-30 ml of ethyl acetate for 2 times, evaporating to dryness, and dissolving residues by adding methanol to obtain a control medicinal material solution; testing according to thin layer chromatography Chinese pharmacopoeia 2015 edition four parts general rule 0502, sucking test solution and control solution 10 μ l each, respectively dropping on the same silica gel G thin layer plate, developing in developing agent, taking out, air drying, spraying color developing agent, drying at 105 deg.C until spots are clearly developed, and displaying spots with the same color in the test chromatogram and the corresponding position of the control chromatogram.
4. The quality detection method for the granules with the functions of relieving cough and asthma for children according to claim 1, wherein in the step (1), the identification method for the liquorice comprises the following steps: taking 3g of the product, adding 30ml of methanol, carrying out ultrasonic treatment for 20-60 minutes, evaporating filtrate to dryness, dissolving residues in water, extracting with 10-40 ml of petroleum ether for 3 times respectively, combining water layers, extracting with 20-50 ml of ethyl acetate for 2 times respectively, evaporating ethyl acetate extracting solution to dryness, and dissolving residues in 2ml of methanol to obtain a sample solution; weighing a liquorice control medicinal material, adding methanol for ultrasonic treatment for 20-60 minutes, evaporating filtrate to dryness, dissolving residues in water, extracting with 10-30 ml of ethyl acetate for 2 times respectively, combining ethyl acetate solutions, evaporating to dryness, and dissolving residues in methanol to obtain a control medicinal material solution; testing according to thin layer chromatography Chinese pharmacopoeia 2015 edition four parts general rule 0502, sucking test solution and control solution 10 μ l each, respectively dropping on the same silica gel G thin layer plate, developing in developing agent, taking out, air drying, spraying color developing agent, drying at 105 deg.C until spots are clearly developed, and displaying spots with the same color in the test chromatogram and the corresponding position of the control chromatogram.
5. The quality detection method for the children's marathon cough and asthma relieving granules according to claim 1, wherein in the step (2), the high performance liquid chromatography identification method for amygdalin comprises the following steps: weighing amygdalin reference substance, precisely weighing, and adding 50% methanol to obtain 0.1mg solution per 1ml to obtain reference substance solution; taking 0.2g of the product, adding 25ml of 50-80% methanol, carrying out ultrasonic treatment for 10-30 minutes, complementing the weight loss by using 50-80% methanol, shaking up, and taking a filtrate to obtain a test solution; sucking amygdalin reference solution and test solution 10 μ l each, and injecting into liquid chromatograph for determination; the cough and asthma relieving granule of the infant ma long-noded pit viper contains bitter apricot kernels and peach kernels, and the content of the bitter apricot kernels and the peach kernels is not less than 3.9mg/g calculated by amygdalin.
6. The method for detecting the quality of the children's ephedrine hydrochloride, the ampelopsis grossedentata cough and asthma relieving granules according to the claim 1, wherein in the step (2), the high performance liquid chromatography identification method of the ephedrine is as follows: weighing ephedrine hydrochloride reference substance and pseudoephedrine hydrochloride reference substance, precisely weighing, and dissolving in 50% methanol to obtain mixed solution containing ephedrine hydrochloride 0.04mg and pseudoephedrine hydrochloride 0.01mg per 1ml to obtain reference solution; weighing the product, precisely adding 25ml of 50-80% methanol containing 1.0-2.0% phosphoric acid, carrying out ultrasonic treatment for 10-40 minutes, complementing the lost weight with 50-80% methanol containing 1.0-2.0% phosphoric acid, and taking a subsequent filtrate to obtain a test solution; precisely sucking 10 μ l of each of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement; the granule for relieving cough and asthma of the infantile Mallotus comprises herba Ephedrae (calculated on ephedrine hydrochloride and pseudoephedrine hydrochloride) with total amount not less than 0.5 mg/g.
7. The method for detecting the quality of the granules for relieving cough and asthma of the children's Malong according to claim 1, wherein in the step (2), the method for identifying the forsythiaside A by the high performance liquid chromatography comprises the following steps: weighing forsythoside A reference substance, precisely weighing, and dissolving with 50% methanol to obtain forsythoside A solution containing 0.05mg per 1ml to obtain reference solution; weighing the product, adding 25ml of 50-80% methanol, carrying out ultrasonic treatment for 10-40 minutes, complementing the lost weight with 50-80% methanol, shaking up, and taking a subsequent filtrate to obtain a test solution; precisely sucking 10 μ l of each of the forsythiaside A reference substance and the sample, and injecting into a liquid chromatograph for determination; the cough and asthma relieving granule containing fructus forsythiae in terms of forsythiaside A for children is not less than 0.07 mg/g.
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