CN112526057A - Thin-layer chromatography identification method of Fuyanjing capsule - Google Patents
Thin-layer chromatography identification method of Fuyanjing capsule Download PDFInfo
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- 238000004809 thin layer chromatography Methods 0.000 title claims abstract description 69
- 238000000034 method Methods 0.000 title claims abstract description 49
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- 239000003814 drug Substances 0.000 claims abstract description 12
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- 238000004451 qualitative analysis Methods 0.000 claims abstract description 6
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- 238000006073 displacement reaction Methods 0.000 description 3
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- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- NOTFZGFABLVTIG-UHFFFAOYSA-N Cyclohexylethyl acetate Chemical compound CC(=O)OCCC1CCCCC1 NOTFZGFABLVTIG-UHFFFAOYSA-N 0.000 description 1
- 208000005171 Dysmenorrhea Diseases 0.000 description 1
- 206010013935 Dysmenorrhoea Diseases 0.000 description 1
- 244000110343 Elephantopus scaber Species 0.000 description 1
- KIWBPDUYBMNFTB-UHFFFAOYSA-N Ethyl hydrogen sulfate Chemical compound CCOS(O)(=O)=O KIWBPDUYBMNFTB-UHFFFAOYSA-N 0.000 description 1
- 208000019255 Menstrual disease Diseases 0.000 description 1
- 241000203475 Neopanax arboreus Species 0.000 description 1
- 206010049677 Salpingo-oophoritis Diseases 0.000 description 1
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- 238000009825 accumulation Methods 0.000 description 1
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- CAQXGPRORCTUAZ-UHFFFAOYSA-N acetonitrile;n,n-diethylethanamine;hydrate Chemical compound O.CC#N.CCN(CC)CC CAQXGPRORCTUAZ-UHFFFAOYSA-N 0.000 description 1
- OWAQXCQNWNJICI-UHFFFAOYSA-N benzene;chloroform Chemical compound ClC(Cl)Cl.C1=CC=CC=C1 OWAQXCQNWNJICI-UHFFFAOYSA-N 0.000 description 1
- XRIVRYRXJYCJTQ-UHFFFAOYSA-N benzene;ethyl acetate;formic acid Chemical compound OC=O.CCOC(C)=O.C1=CC=CC=C1 XRIVRYRXJYCJTQ-UHFFFAOYSA-N 0.000 description 1
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- 238000004090 dissolution Methods 0.000 description 1
- ROAYSRAUMPWBQX-UHFFFAOYSA-N ethanol;sulfuric acid Chemical compound CCO.OS(O)(=O)=O ROAYSRAUMPWBQX-UHFFFAOYSA-N 0.000 description 1
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- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
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- 239000010414 supernatant solution Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
- G01N30/91—Application of the sample
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
- G01N30/94—Development
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
- G01N30/95—Detectors specially adapted therefor; Signal analysis
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- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a thin-layer chromatography identification method of a Fuyanjing capsule, which is characterized in that a thin-layer chromatography identification method of picria felterrae lour in the Fuyanjing capsule recorded in the first part of 'Chinese pharmacopoeia' 2015 edition is adopted to identify the picria felterrae lour medicinal material in the Fuyanjing capsule, a thin-layer chromatography identification method of a Chinese angelica medicinal material and a radix zanthoxyli medicinal material is added, and the thin-layer chromatography identification method of 3 medicinal materials is combined to perform qualitative analysis on the Fuyanjing capsule so as to ensure the medication safety of the Fuyanjing capsule. The components in the thin-layer chromatography can be effectively separated, the spots are clear and round, and the separation effect is good; the Fuyanjing capsule is qualitatively analyzed by a thin-layer chromatography identification method, so that the sampling amount is small, the cost is low, and the operation is quick and simple.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine identification, in particular to a thin-layer chromatography identification method of Fuyanjing capsules.
Background
Fuyanjing capsule is made up by using Chinese medicinal materials of picria felterrae lour, elephantopus scaber, Chinese angelica root, spatholobus stem, shinyleaf pricklyash root, transversus cusia root and five-finger fig, etc. and possesses the functions of clearing away heat, removing dampness, regulating menstruation and stopping leukorrhagia, and can be used for curing the diseases of leukorrhagia, menoxenia, dysmenorrhea, chronic pelvic inflammation and adnexitis due to accumulation of damp-heat. The specification is divided into two types (1) and each tablet is filled with 0.3g (equivalent to 2.44g of decoction pieces); (2) each capsule is filled with 0.4g (equivalent to 3.25g of decoction pieces). Fuyanjing capsules are unique varieties of Guangxi Wuzhou pharmaceutical (group) GmbH, approved by Ministry of health in 1988, and are loaded into the drug standards of Ministry of health in 1992, the new drug changes the first volume of the standards, and the standard number is as follows: WS3-1-87-92 (Z); the quality standard is recorded in the part of Chinese pharmacopoeia in 1995, and then continuously recorded in the parts of Chinese pharmacopoeia in 2000, 2005 and 2010 versions, the existing quality standard is recorded in the part of Chinese pharmacopoeia in 2015, and only thin-layer identification and content measurement items of picria fel-terrae are recorded in the standard.
Liupeng Han et al established a quality control method for Fuyanjing granules (prescription from < pharmacopoeia of people's republic of China) for collecting variety Fuyanjing capsules). The method comprises the following steps: qualitatively identifying herba Ajugae Bracteosae, nitidine chloride and ferulic acid in FUYANJING granule by thin layer chromatography; the content of nitidine chloride in the medicine is determined by high performance liquid chromatography. High performance liquid chromatography conditions are Vp-ODSC18 column (4.6 mm × 150mm,5 μm), mobile phase: acetonitrile-water-triethylamine (25:75:0.3) at a flow rate of 1 mL/min; detection wavelength: 328 nm; the column temperature was 35 ℃. As a result: the thin-layer chromatography identification spots are clear and easy to identify; the nitidine chloride is in a good linear relation between 2.0 and 20 mu g/mL by high performance liquid chromatography, r = 0.9998, and the average recovery rate is 99.58% (RSD is 0.46%). Thin-layer identification of nitidine chloride: grinding Fuyanjing granule 10g, adding 2% hydrochloric acid 20mL, ultrasonic extracting for 30min, extracting with chloroform for 3 times, each time 30mL, mixing chloroform solutions, evaporating to dryness, adding residue and fermenting 1mL to obtain sample solution, preparing negative control solution by the same method, adding nitidine chloride control solution and methanol to obtain O.5mg solution per L as control solution. Respectively sucking 10mL of each of the 3 solutions, dropping on the same silica gel G plate with sodium carboxymethylcellulose as binder, developing with benzene-ethyl acetate-methanol-isopropanol-concentrated ammonia solution (20: 5:3: l: O.12) as developing agent, taking out, air drying, and viewing under ultraviolet lamp (365nm) to show the same yellow-green fluorescent spot in the chromatogram of the test sample at the position corresponding to the chromatogram of the control sample, and to show no fluorescent spot with the same color in the corresponding position of the negative control sample. Thin-layer identification of ferulic acid: collecting Fuyanjing granule 20g, grinding, adding methanol 50mL, ultrasonic extracting for 30min, filtering, recovering methanol, dissolving the residue in water 50mL, extracting with diethyl ether for 5 times (20, 20, 15, 15, 10mL), mixing the diethyl ether solutions, and adding 2% Na2CO3Extracting for 5 times (20, 20, 15, 15, 10mL), discarding ether solution, washing alkaline solution with ethyl acetate for 3 times (15 mL each), discarding ethyl acetate, adjusting pH of the alkaline solution with dilute hydrochloric acid to 2-3, eluting with benzene for 3 times (15 mL each), discarding benzene solution, extracting with hexyl ether for 5 times (20, 20, 15, 15, 10mL), mixing ether solutions, volatilizing, adding 1mL of methanol into residue to obtain test solution, preparing negative control solution by the same method, taking ferulic acid control, preparing solution containing 0.5mg per 1mL with methanol to obtain control solution. Drawing lO μ L of each of the 3 solutions, placing on the same silica gel G plate, developing with benzene-ethyl acetate-formic acid (4: L: 0.1) as developing agent, taking out, air drying, placing under ultraviolet lamp (365nm) for inspection, displaying the same deep blue fluorescent spot on the chromatogram of the sample at the position corresponding to the chromatogram of the reference substance, and displaying the same spot with the same color on the corresponding position of the negative reference substance (Liupenghan, Zhao Dian Feng, Lu Xiang, Wei Henlong, Pan Song]"Chinese patent medicine" 2006 6 th page 815 Yi 817). However, in the identification method of the article, the extraction method of angelica and radix zanthoxyli is complex, the steps are more, benzene with high toxicity is used, and the method is not beneficial to establishing the quality detection standard of the Fuyanjing granules/capsules and improving the qualitative analysis speed of the Fuyanjing granules/capsules.
Disclosure of Invention
The invention provides a thin-layer chromatography identification method of Fuyanjing capsules, which is used for carrying out qualitative analysis on the Fuyanjing capsules by the thin-layer chromatography identification method, has the advantages of less sampling amount, low cost and quick and simple operation, and in the thin-layer chromatography, each component can be effectively separated, the spots are clear and round, and the separation effect is good.
The technical scheme of the invention is as follows:
a thin-layer chromatography identification method of Fuyanjing capsules is characterized in that the thin-layer chromatography identification method of picria felterrae lour in Fuyanjing capsules recorded in the first part of the 'Chinese pharmacopoeia' 2015 edition is adopted to identify the picria felterrae lour medicinal material in the Fuyanjing capsules, then the thin-layer chromatography identification method of the angelica sinensis medicinal material and the radix zanthoxyli medicinal material is added, and the thin-layer chromatography identification method of 3 medicinal materials is combined to perform qualitative analysis on the Fuyanjing capsules so as to ensure the medication safety of the Fuyanjing capsules.
The thin-layer chromatography identification method of the angelica comprises the following specific steps:
(1) taking 10 granules of Fuyanjing capsule content, adding 50mL of ethanol, performing ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, dissolving the residue in 20mL of water, shaking and extracting with 20mL of ethyl acetate, evaporating the ethyl acetate solution to dryness, and dissolving the residue in 1mL of methanol to obtain a sample solution.
(2) Taking 1g of angelica sinensis reference medicinal material, adding 50mL of ethanol, performing ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, dissolving residue in 20mL of water, shaking and extracting with 20mL of ethyl acetate, evaporating ethyl acetate solution to dryness, and dissolving residue in 1mL of methanol to obtain a reference medicinal material solution.
(3) Performing thin layer chromatography (general rule 0502) test, sucking 5-10 μ L of test solution and 5 μ L of control solution, respectively dropping on the same silica gel G plate, spreading with upper layer solution of toluene-ethyl acetate-formic acid (8: 1: 0.5) as developing agent, taking out, air drying, spraying phosphomolybdic acid test solution, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution.
The thin-layer chromatography identification method of the radix zanthoxyli comprises the following specific steps:
(1) taking 10 granules of Fuyanjing capsule content, adding 50mL of 1% methanol hydrochloride, refluxing for 1 hour, filtering, passing the filtrate through a neutral alumina column, eluting with 50mL of methanol, collecting the eluent, evaporating to dryness, and dissolving the residue with 1mL of methanol to obtain a test solution.
(2) Taking a nitidine chloride reference substance, adding ethanol to prepare a solution containing 1mg of nitidine chloride per 1mL, and taking the solution as a reference substance solution.
(3) Performing thin layer chromatography (general rule 0502) test, sucking 5-10 μ L of sample solution and 3 μ L of reference solution, respectively dropping on the same silica gel G plate, developing with chloroform-methanol (7: 1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp light (365 nm). The test chromatogram shows fluorescent spots of the same color at the positions corresponding to those of the control chromatogram.
The invention has the beneficial effects that:
the thin-layer chromatography identification method of the invention adopts the angelica medicinal material as a reference medicinal material to identify the angelica component in the Fuyanjing capsule, and adopts the nitidine chloride as a reference substance to identify the nitidine component in the Fuyanjing capsule, so that the sample application amount is small, the cost is low, the thin-layer chromatography separation effect is good, the spots are clear, the specific displacement value is moderate, the reproducibility is good, the negative control is free of interference, and the specificity is strong. By adding the thin-layer chromatography identification method of 2 medicinal materials of angelica and radix zanthoxyli and combining the thin-layer chromatography identification method of picria felterrae lour in the existing quality standard of the Fuyanjing capsule, the Fuyanjing capsule is subjected to qualitative analysis, the identification effect is more comprehensive, and the medication safety of the Fuyanjing capsule can be effectively ensured.
Drawings
FIG. 1 is a chromatogram of thin-layer chromatography identification of radix Angelicae sinensis in FUYANJING Capsule, wherein the chromatogram is marked as follows: 1. angelica sinensis reference medicinal material, 2, a fuyanjing capsule sample (batch 190802), 3, a fuyanjing capsule sample (batch 190801), 4, a fuyanjing capsule sample (batch 190701), 5, a fuyanjing capsule sample (batch 190602), 6, a fuyanjing capsule sample (batch 190601), 7, a fuyanjing capsule sample (batch 190301), 8, a fuyanjing capsule sample (batch 190401), 9, a fuyanjing capsule sample (batch 190402), 10, a fuyanjing capsule sample (batch 190403), 11, a fuyanjing capsule sample (batch 190404), 12 and angelica sinensis reference medicinal material; a represents a blue brown spot. Of these, 1 and 12 belong to the repeated spotting, which avoids edge effects and is more favorable for speckle contrast. Chromatographic conditions are as follows: using a thin layer plate produced by Qingdao ocean chemical Co., Ltd, developing agent: toluene-ethyl acetate-formic acid (8: 1: 0.5), spot amount: 5 μ L, temperature: 25 ℃, relative humidity: 75% RH, splay: 9 cm; color development method: spraying phosphomolybdic acid solution, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight.
FIG. 2 is a chromatogram of thin-layer chromatography identification of radix Angelicae sinensis in FUYANJING Capsule, wherein the chromatogram is marked as follows: 1. the Fuyanjing capsule lacks an angelica negative sample, 2, the angelica control medicine, 3, the Fuyanjing capsule sample (batch No. 190802), 4, the Fuyanjing capsule sample (batch No. 190801), 5, the Fuyanjing capsule sample (batch No. 190701), 6, the Fuyanjing capsule sample (batch No. 190601), and 7, the Fuyanjing capsule lacks an angelica negative sample; a represents a blue brown spot. Of these, 1 and 7 belong to the repeated spotting, which avoids edge effects and is more favorable for speckle contrast. Chromatographic conditions are as follows: developing agent: toluene-ethyl acetate-formic acid (8: 1: 0.5), spot amount: 5 μ L, color development mode: spraying phosphomolybdic acid solution, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight.
FIG. 3 is a chromatogram of thin-layer chromatography identification of radix Angelicae sinensis in FUYANJING Capsule, wherein the chromatogram is marked as follows: 1. ferulic acid reference substance, 2, angelica sinensis reference medicinal material, 3, adopting ethanol as an extraction solvent to carry out heating reflux extraction and then evaporating to dryness, adding water to dissolve residues and then extracting with ethyl acetate to obtain a gynecological inflammation removal capsule test solution, 4, adopting ethanol as an extraction solvent to carry out ultrasonic extraction and then evaporating to dryness, adding water to dissolve residues and then extracting with ethyl acetate to obtain a gynecological inflammation removal capsule test solution, 5, adopting methanol as an extraction solvent to carry out ultrasonic extraction to obtain a gynecological inflammation removal capsule test solution, and 6, adopting ethyl acetate as an extraction solvent to carry out ultrasonic extraction to obtain a gynecological inflammation removal capsule test solution; a represents a blue brown spot. Chromatographic conditions are as follows: developing agent: toluene-ethyl acetate-formic acid (8: 1: 0.5), spot amount: 5 μ L, color development mode: spraying phosphomolybdic acid solution, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight.
FIG. 4 is a chromatogram of thin-layer chromatography identification of radix Angelicae sinensis in FUYANJING Capsule, wherein the chromatogram is marked as follows: 1. the method comprises the following steps of carrying out ultrasonic extraction by adopting ether as an extraction solvent to obtain an angelica sinensis reference medicinal material solution, 2, carrying out ultrasonic extraction by adopting ethanol as an extraction solvent, evaporating to dryness, dissolving residues in water, extracting by using ethyl acetate to obtain an angelica sinensis reference medicinal material solution, 3, a ferulic acid reference substance, 4, carrying out ultrasonic extraction by adopting methanol as an extraction solvent to obtain a fuyanjing capsule test solution, 5, carrying out ultrasonic extraction by adopting trichloromethane as an extraction solvent to obtain a fuyanjing capsule test solution, 6, carrying out ultrasonic extraction by adopting ethyl acetate as an extraction solvent to obtain a fuyanjing capsule test solution, and 7, carrying out ultrasonic extraction by adopting petroleum ether (60-90 ℃) as an extraction solvent to obtain a fuyanjing capsule test solution. Chromatographic conditions are as follows: developing agent: n-hexane-ethyl acetate (4: 1), spot amount: 5 μ L, color development mode: viewing under 365nm ultraviolet lamp.
FIG. 5 is a chromatogram of thin-layer chromatography identification of radix Angelicae sinensis in FUYANJING Capsule, wherein the chromatogram is marked as follows: 1. ferulic acid reference substance, 2, angelica sinensis reference medicinal material, 3, adopting ethanol as an extraction solvent to carry out heating reflux extraction and then evaporating to dryness, adding water to dissolve residues and then extracting with ethyl acetate to obtain a gynecological inflammation removal capsule test solution, 4, adopting ethanol as an extraction solvent to carry out ultrasonic extraction and then evaporating to dryness, adding water to dissolve residues and then extracting with ethyl acetate to obtain a gynecological inflammation removal capsule test solution, 5, adopting methanol as an extraction solvent to carry out ultrasonic extraction to obtain a gynecological inflammation removal capsule test solution, and 6, adopting ethyl acetate as an extraction solvent to carry out ultrasonic extraction to obtain a gynecological inflammation removal capsule test solution. Chromatographic conditions are as follows: developing agent: petroleum ether (30-60 ℃) -ethyl ether-glacial acetic acid (10: 10: 0.5), sample size: 5 μ L, color development mode: spraying phosphomolybdic acid solution, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight.
FIG. 6 is a chromatogram of the two-side needle thin layer chromatography identification in Fuyanjing capsule, in which the following are marked: 1. fuyanjing capsule sample (batch 190802), 2 fuyanjing capsule sample (batch 190801), 3 fuyanjing capsule sample (batch 190701), 4 fuyanjing capsule sample (batch 190602), 5 fuyanjing capsule sample (batch 190601), 6, nitidine chloride reference substance, 7, fuyanjing capsule sample (batch 190301), 8, fuyanjing capsule sample (batch 190401), 9 fuyanjing capsule sample (batch 190402), 10, fuyanjing capsule sample (batch 190403), 11 fuyanjing capsule sample (batch 190404); b represents a yellow-green fluorescent spot. Chromatographic conditions are as follows: using a thin layer plate produced by Qingdao ocean chemical Co., Ltd, developing agent: chloroform-methanol (7: 1); sample amount of spotting: 5 μ L (test solution), 3 μ L (nitidine chloride reference solution); temperature: 25 ℃, relative humidity: 75% RH, splay: 18 cm; color development method: inspecting under ultraviolet lamp light (365 nm).
FIG. 7 is a chromatogram of two-side needle thin layer chromatography identification in Fuyanjing capsule, in which the following are marked: 1. radix zanthoxyli contrast medicinal material solution 2, nitidine chloride contrast, 3, Fuyanjing capsule sample (batch No. 190802), 4, Fuyanjing capsule sample (batch No. 190801), 5, Fuyanjing capsule sample (batch No. 190701), 6, Fuyanjing capsule lack two-side needle negative sample; b represents a yellow-green fluorescent spot. Chromatographic conditions are as follows: developing agent: chloroform-methanol (7: 1), spotting amount: 5 μ L (3 μ L of nitidine chloride control solution), color development mode: inspecting under ultraviolet lamp light (365 nm).
FIG. 8 is a chromatogram of the two-side needle thin layer chromatography identification in Fuyanjing capsule, in which the following are marked: 1. the method comprises the following steps of preparing a nitidine reference medicinal material solution 2, a nitidine chloride reference substance 3, a sample extracting solution 5, 4, a Fuyanjing capsule sample extracting solution obtained by adopting methanol as an extracting solvent and carrying out ultrasonic extraction for 30 minutes, a Fuyanjing capsule sample extracting solution obtained by adopting trichloromethane as an extracting solvent and carrying out ultrasonic extraction for 30 minutes, and a nitidine reference medicinal material 2. Chromatographic conditions are as follows: developing agent: chloroform-methanol-concentrated ammonia solution (30: 1: 0.2), sample application amount: 5 μ L (3 μ L of nitidine chloride control solution), color development mode: spraying 10% ethanol sulfate solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under ultraviolet light (365 nm).
FIG. 9 is a chromatogram of the two-side needle thin layer chromatography identification in Fuyanjing capsule, in which the following are marked: 1. the method comprises the following steps of preparing a nitidine reference medicinal material solution 2, a nitidine chloride reference substance 3, a test sample extracting solution 5, 4, a Fuyanjing capsule test sample extracting solution obtained by adopting methanol as an extraction solvent and carrying out ultrasonic extraction for 30 minutes, a Fuyanjing capsule test sample extracting solution obtained by adopting ethyl acetate as an extraction solvent and carrying out ultrasonic extraction for 30 minutes, and a Fuyanjing capsule test sample extracting solution obtained by adopting petroleum ether (60-90 ℃) as an extraction solvent and carrying out ultrasonic extraction for 30 minutes. Chromatographic conditions are as follows: developing agent: toluene-n-hexane-petroleum ether (60-90 ℃) (1: 3: 1), and the sample amount is as follows: 5 μ L (3 μ L of nitidine chloride control solution), color development mode: inspecting under ultraviolet lamp light (365 nm).
FIG. 10 is a chromatogram of two-side needle thin layer chromatography identification in Fuyanjing capsule, in which the following are marked: 1. the method comprises the following steps of (1) preparing a nitidissimilis contrast medicinal material solution 2 (the sample application amount is 10 mu L), 2, a nitidissimilis contrast medicinal material solution 2 (the sample application amount is 5 mu L), 3, a nitiduline chloride contrast product (the sample application amount is 5 mu L), 4, a test product extracting solution 5, a Fuyanjing capsule test product extracting solution obtained by ultrasonic extraction for 30 minutes by using methanol as an extraction solvent, 6, a Fuyanjing capsule test product extracting solution obtained by ultrasonic extraction for 30 minutes by using ethyl acetate as the extraction solvent, and 7, and a Fuyanjing capsule test product extracting solution obtained by ultrasonic extraction for 30 minutes by using petroleum ether (60-90 ℃) as the extraction solvent. Chromatographic conditions are as follows: developing agent: benzene-chloroform (1: 1), spotting amount: 5 μ L (sample extract), color development method: inspecting under ultraviolet lamp light (365 nm).
FIG. 11 is a chromatogram of the two-side needle thin layer chromatography identification in Fuyanjing capsule, in which the following are marked: 1. the method comprises the following steps of preparing a nitidine reference medicinal material solution 2, a nitidine chloride reference substance 3, a sample extracting solution 2, 4, a nitidine chloride reference medicinal material solution 2, 5, a nitidine chloride reference substance 6 and a sample extracting solution 2, and carrying out spotting on the same thin-layer plate twice by using the same reagents. Chromatographic conditions are as follows: developing agent: chloroform-acetone-formic acid (15: 4: 1), saturated development cylinder with ammonia test solution, spotting amount: 5 μ L (3 μ L of nitidine chloride control solution), color development mode: inspecting under ultraviolet lamp light (365 nm).
FIG. 12 is a chromatogram of the two-side needle thin layer chromatography identification in Fuyanjing capsule, in which the following are marked: 1. chlorinated nitidine reference substance (sample amount of 3 muL), 2, nitidine reference medicinal material solution 2 (sample amount of 5 muL), 3, sample extracting solution 2 (sample amount of 1 muL), 4, sample extracting solution 2 (sample amount of 1 muL), 5, sample extracting solution 2 (sample amount of 5 muL), 6, and sample extracting solution 2 (sample amount of 10 muL). Chromatographic conditions are as follows: developing agent: chloroform-methanol (9: 1), color development mode: inspecting under ultraviolet lamp light (365 nm).
FIG. 13 is a chromatogram of the two-side needle thin layer chromatography identification in Fuyanjing capsule, in which the following are marked: 1. sample extracting solution 3 (sample batch No. 190602), sample extracting solution 2, sample extracting solution 3 (sample batch No. 190701), sample extracting solution 3 (sample batch No. 190801), and sample chloridized nitidine reference substance 4; b represents a yellow-green fluorescent spot. Chromatographic conditions are as follows: developing agent: chloroform-methanol (7: 1), spotting amount: 5 μ L (3 μ L of nitidine chloride control solution), color development mode: inspecting under ultraviolet lamp light (365 nm).
FIG. 14 is a chromatogram of two-side needle thin layer chromatography identification in Fuyanjing capsule, in which the following are marked: 1. sample extracting solution 3, 2, radix zanthoxyli reference medicinal material solution 2, 3, nitidine chloride reference substance 4, sample extracting solution 2 (sample batch No. 190602), 5, sample extracting solution 2 (sample batch No. 190701), 6 and sample extracting solution 2 (sample batch No. 190801); 7. and (4) a test sample extracting solution. Chromatographic conditions are as follows: developing agent: toluene-ethyl acetate-methanol-isopropanol-concentrated ammonia solution (20: 5:3:1: 0.1), sample application amount: 5 mu L (the content of the control solution of the radix zanthoxyli to the medicinal material 2 and the content of the chlorinated nitidine to the control solution is 3 mu L), and the color development mode is as follows: inspecting under ultraviolet lamp light (365 nm).
FIG. 15 is a chromatogram of the two-side needle thin layer chromatography identification in Fuyanjing capsule, in which the following are marked: 1. sample extracting solution 3 (spot sample amount 5 muL), sample extracting solution 2, sample extracting solution 3 (spot sample amount 10 muL), 3, nitidine chloride reference sample (spot sample amount 5 muL), 4, radix zanthoxyli reference medicinal material solution 1 (spot sample amount 5 muL), and 5, radix zanthoxyli reference medicinal material solution 2 (spot sample amount 5 muL). Chromatographic conditions are as follows: developing agent: chloroform-methanol-concentrated ammonia test solution (25: 1: 0.2), color development mode: inspecting under ultraviolet lamp light (365 nm).
Detailed Description
In order to describe the present invention in more detail, the present invention will be further described with reference to the following examples.
Examples
Fuyanjing capsule sample sources: 10 samples of lot numbers 190802, 190801, 190701, 190602, 190601, 190301, 190401, 190402, 190403, 190404, manufactured by cantonese pharmaceutical (group) shares, inc.
A thin-layer chromatography identification method of Fuyanjing capsules is characterized in that the thin-layer chromatography identification is respectively carried out on 3 medicinal materials of picria felterrae lour, Chinese angelica and shinyleaf pricklyash root in the Fuyanjing capsules, and the specific identification is as follows:
1. the thin-layer chromatography identification method of picria felterrae lour comprises the following specific steps
(1) Taking 1.2g of Fuyanjing capsule content, adding 10mL of ethanol, heating and refluxing for 5 minutes, and filtering. The filtrate was evaporated to dryness, and 1mL of ethanol was added to dissolve the residue to obtain a sample solution.
(2) Taking 1g of picria felterrae lour reference medicinal material, adding 10mL of ethanol, heating and refluxing for 5 minutes, and filtering. The filtrate was evaporated to dryness, and the residue was dissolved in 1mL of ethanol to give a control solution.
(3) Performing thin layer chromatography (general rule 0502) test, sucking 10 μ L of each of the test solution and the control solution, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol (4: 1) as developing agent, taking out, air drying, spraying 5% vanillin-sulfuric acid solution, and heating at 105 deg.C for 5 min. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution.
2. The thin-layer chromatography identification method of the angelica comprises the following specific steps:
(1) taking 10 granules of Fuyanjing capsule content, adding 50mL of ethanol, performing ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, dissolving the residue in 20mL of water, shaking and extracting with 20mL of ethyl acetate, evaporating the ethyl acetate solution to dryness, and dissolving the residue in 1mL of methanol to obtain a sample solution.
(2) Taking 1g of angelica sinensis reference medicinal material, adding 50mL of ethanol, performing ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, dissolving residue in 20mL of water, shaking and extracting with 20mL of ethyl acetate, evaporating ethyl acetate solution to dryness, and dissolving residue in 1mL of methanol to obtain a reference medicinal material solution.
(3) Performing thin layer chromatography (general rule 0502) test, sucking 5-10 μ L of test solution and 5 μ L of control solution, respectively dropping on the same silica gel G plate, spreading with upper layer solution of toluene-ethyl acetate-formic acid (8: 1: 0.5) as developing agent, taking out, air drying, spraying phosphomolybdic acid test solution, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight.
10 batches of Fuyanjing capsule samples are identified, and the chromatogram is shown in figure 1. The result shows that spots with the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference drugs, the separation effect of the thin-layer chromatography is good, the spots are clear, the specific displacement value is moderate, and the reproducibility is good.
In order to verify the specificity of thin-layer chromatography identification of angelica, the applicant weighs the other medicines except the angelica according to the prescription proportion, prepares an angelica-deficient negative sample according to the preparation method of the Fuyanjing capsule, takes the content of 10 Fuyanjing capsules in the angelica-deficient negative sample, adds 50mL of ethanol, performs ultrasonic treatment for 30 minutes, filters, evaporates the filtrate to dryness, adds 20mL of water to the residue to dissolve the residue, performs shaking extraction with 20mL of ethyl acetate, evaporates the ethyl acetate solution to dryness, adds 1mL of methanol to the residue to dissolve the residue to serve as a negative control solution. Then, performing thin-layer chromatography (general rule 0502) test, sucking 5-10 μ L of negative control solution, dropping on a silica gel G plate, developing with an upper layer solution of toluene-ethyl acetate-formic acid (8: 1: 0.5) as a developing agent, taking out, air drying, spraying phosphomolybdic acid test solution, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight. The chromatogram is shown in figure 2, and the result shows that the negative is free from interference, and the specificity of the thin-layer chromatography identification method of the angelica is good.
In addition, the applicant also examined the extraction conditions and developing agent of angelica, respectively:
firstly, examining the extraction conditions of angelica, respectively examining methanol, diethyl ether, ethyl acetate, chloroform, petroleum ether (60-90 ℃) and the like as extraction solvents, and also examining reflux extraction and ultrasonic extraction modes.
Second, the developing agent was examined and the supernatant solution of n-hexane-ethyl acetate (4: 1), cyclohexane-ethyl acetate (9: 1), toluene-ethyl acetate-formic acid (8: 1: 0.5), and petroleum ether (30-60 ℃) -diethyl ether-glacial acetic acid (10: 10: 0.5) were examined.
Ferulic acid was added as a control during the study: taking ferulic acid control, adding ethanol to make into solution containing 1mg ferulic acid per 1mL, and using as control solution. The amount of spots was 5. mu.L.
Wherein normal hexane-ethyl acetate (4: 1) is used as a developing agent, the inspection is carried out under the ultraviolet lamp of 365nm, other developing agents are inspected under the sunlight, and the chromatogram is shown in figures 3-5.
The result shows that two test solution obtained by refluxing or ultrasonically extracting the Fuyanjing capsule content for 30 minutes by using ethanol have blue brown spots consistent with those of the control medicinal material, but the test solution obtained by ultrasonic extraction has better color development effect. When the upper solution of toluene-ethyl acetate-formic acid (8: 1: 0.5) is used as a developing agent for development, the separation effect of each component in the angelica is better, and the Rf value is more appropriate.
The applicant also respectively uses silica gel G plates produced by different manufacturers to develop under different temperature conditions, so as to investigate the durability of the thin-layer chromatography identification method of the angelica, and the result shows that the durability of the thin-layer chromatography identification method of the angelica is good.
3. The thin-layer chromatography identification method of the radix zanthoxyli comprises the following specific steps:
(1) taking 10 Fuyanjing capsule contents, adding 50mL of 1% methanol hydrochloride, refluxing for 1 hour, filtering, passing the filtrate through a neutral alumina column (100-200 meshes, 20g, inner diameter of 1.5 cm), eluting with 50mL of methanol, collecting the eluent, evaporating to dryness, and dissolving the residue with 1mL of methanol to obtain a sample solution.
(2) Taking a nitidine chloride reference substance, adding ethanol to prepare a solution containing 1mg of nitidine chloride per 1mL, and taking the solution as a reference substance solution.
(3) Performing thin layer chromatography (general rule 0502) test, sucking 5-10 μ L of sample solution and 3 μ L of reference solution, respectively dropping on the same silica gel G plate, developing with chloroform-methanol (7: 1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp light (365 nm). 10 batches of Fuyanjing capsule samples were identified and the chromatogram is shown in FIG. 6. The result shows that in the chromatogram of the test sample, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference substance, the separation effect of the thin-layer chromatogram is good, the spots are clear, the specific displacement value is moderate, and the reproducibility is good.
In order to verify the specificity of thin-layer chromatography identification of the double-faced needles, the applicant weighs the other medicines except the double-faced needles according to the formula proportion, prepares a double-faced needle-free negative sample according to a preparation method of a Fuyanjing capsule, takes the content of 10 Fuyanjing capsule double-faced needle-free negative samples, adds 50mL of 1% methanol hydrochloride, reflows for 1 hour, filters, passes the filtrate through a neutral alumina column (100-200 meshes, 20g, the inner diameter of 1.5 cm), elutes with 50mL of methanol, collects the eluent, evaporates to dryness, and adds 1mL of methanol to the residue for dissolution to serve as a negative control solution. Then testing by thin layer chromatography (general rule 0502), sucking 5-10 μ L of negative control solution on silica gel G plate, developing with chloroform-methanol (7: 1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp light (365 nm). The chromatogram is shown in figure 7, and the result shows that the negative is free from interference, and the thin-layer chromatography identification method of the two-side needle has good specificity.
In addition, the present applicant also examined the conditions for extracting Zanthoxylum nitidum and the developing agent, respectively:
firstly, the extraction conditions of radix zanthoxyli are examined, solvents such as methanol, ethyl acetate, trichloromethane, petroleum ether (60-90 ℃) and the like are respectively examined, and reflux extraction and ultrasonic extraction modes are also examined.
1. Preparing a test solution:
1) taking 10 capsules of Fuyanjing capsules, adding 50mL of methanol, chloroform, ethyl acetate and petroleum ether (60-90 ℃) respectively, and carrying out ultrasonic extraction for 30 minutes to obtain a sample extracting solution.
2) Taking 10 Fuyanjing capsule contents, adding 50mL of methanol, performing ultrasonic treatment for 30 minutes, filtering, concentrating the filtrate to 30mL, adjusting the pH value to 2-3 with 10% hydrochloric acid solution, shaking and extracting with water-saturated n-butanol for 3 times, 30mL each time, combining the n-butanol solution, evaporating to dryness, and dissolving the residue with 2mL of methanol to obtain a sample extracting solution 2.
3) Taking 10 Fuyanjing capsule contents, adding 50mL of 1% methanol hydrochloride, refluxing for 1 hour, filtering, passing the filtrate through a neutral alumina column (100-200 meshes, 20g, inner diameter of 1.5 cm), eluting with 50mL of methanol, collecting the eluent, evaporating to dryness, and dissolving the residue with 1mL of methanol to obtain a sample solution 3.
4) Taking 10 Fuyanjing capsule contents, adding 50mL of 1% methanol hydrochloride, refluxing for 1 hour, filtering, evaporating filtrate to dryness, dissolving residue with 25mL of 5% sodium hydroxide solution, extracting with 30mL of chloroform twice, mixing extractive solutions, evaporating to dryness, and dissolving residue with methanol to obtain sample extractive solution 4.
5) Taking 10 capsules of Fuyanjing, adding 50mL of ethanol, refluxing for 1 hour, filtering, evaporating filtrate to dryness, dissolving residue in 20mL of water, extracting with 20mL of ethyl acetate twice, mixing extractive solutions, evaporating to dryness, and dissolving residue in methanol to obtain sample extractive solution 5.
2. Preparing a reference medicinal material solution:
1) taking 2g of radix zanthoxyli medicinal material, adding ethanol for refluxing for 30 minutes, filtering, evaporating filtrate to dryness, and dissolving residues in methanol to obtain a reference medicinal material solution 1.
2) Taking 2g of the radix zanthoxyli as a reference medicinal material, adding a proper amount of water, decocting for 30 minutes, filtering, concentrating the filtrate to 30mL, adjusting the pH value to 2-3 by using a 10% hydrochloric acid solution, shaking and extracting for 3 times by using water saturated n-butyl alcohol, 30mL each time, combining the n-butyl alcohol solutions, evaporating to dryness, and adding 2mL of methanol to dissolve residues to obtain a reference medicinal material solution 2.
3. Preparation of a reference solution:
taking a nitidine chloride reference substance, adding ethanol to prepare a solution containing 1mg of nitidine chloride per 1mL, and taking the solution as a reference substance solution.
Second, the developing agent was examined, and a chloroform-methanol-concentrated ammonia test solution (30: 1: 0.2), a chloroform-methanol-concentrated ammonia test solution (25: 1: 0.2), a chloroform-acetone-formic acid (15: 4: 1), a toluene-n-hexane-petroleum ether (60-90 ℃) (1: 3: 1), a benzene-chloroform (1: 1), a chloroform-methanol (9: 1), a chloroform-methanol (7: 1), and a toluene-ethyl acetate-methanol-isopropanol-concentrated ammonia test solution (20: 5:3:1: 0.1) were examined.
Performing thin layer chromatography (general rule 0502) test by spotting 5 or 10 μ L of each sample solution, 5 or 10 μ L of each control solution, and 3 μ L of control solution on silica gel G plate, developing with chloroform-methanol-concentrated ammonia test solution (30: 1: 0.2), chloroform-methanol-concentrated ammonia test solution (25: 1: 0.2), chloroform-acetone-formic acid (15: 4: 1), toluene-n-hexane-petroleum ether (60-90 ℃) (1: 3: 1), benzene-trichloromethane (1: 1), trichloromethane-methanol (9: 1), trichloromethane-methanol (7: 1), toluene-ethyl acetate-methanol-isopropanol-concentrated ammonia test solution (20: 5:3:1: 0.1) as developing agents, taking out, airing, and inspecting under ultraviolet light (365 nm). Or spraying 10% sulphuric acid ethanol solution, heating until the color of the spot is clear, and inspecting under ultraviolet light (365 nm). The chromatogram is shown in FIGS. 8-15.
The result shows that when the chloroform-methanol (7: 1) is used as the developing agent for development, the components in the Zanthoxylum nitidum have better separation effect and the Rf value is more appropriate. However, when other developing agents are used for development, even if the extraction method is perfected to extract the Zanthoxylum nitidum, a chromatogram with good separation effect cannot be obtained.
The applicant also respectively uses silica gel G plates produced by different manufacturers to develop under different temperature conditions, so as to investigate the durability of the thin-layer chromatography identification method of the two-sided needle, and the result shows that the thin-layer chromatography identification method of the two-sided needle has good durability.
Claims (1)
1. A thin-layer chromatography identification method of Fuyanjing capsules is characterized in that the identification method is to identify the picria felterrae lour medicinal material in the Fuyanjing capsules according to the thin-layer chromatography identification method of the picria felterrae lour in the Fuyanjing capsules recorded in the first part of the 'Chinese pharmacopoeia' 2015 edition, then to add the thin-layer chromatography identification methods of the angelica medicinal material and the radix zanthoxyli medicinal material, and to perform qualitative analysis on the Fuyanjing capsules by combining the thin-layer chromatography identification methods of 3 medicinal materials so as to ensure the medication safety of the Fuyanjing capsules;
the thin-layer chromatography identification method of the angelica sinensis medicinal material comprises the following specific steps:
(1) taking 10 granules of Fuyanjing capsule content, adding 50mL of ethanol, performing ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, dissolving residue in 20mL of water, shaking and extracting with 20mL of ethyl acetate, evaporating ethyl acetate solution to dryness, dissolving residue in 1mL of methanol to obtain a sample solution;
(2) taking 1g of angelica sinensis reference medicinal material, adding 50mL of ethanol, performing ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, adding 20mL of water into residue for dissolving, shaking and extracting by using 20mL of ethyl acetate, evaporating ethyl acetate solution to dryness, and adding 1mL of methanol into residue for dissolving to obtain a reference medicinal material solution;
(3) testing by adopting a general rule of thin-layer chromatography 0502, sucking 5-10 mu L of a test solution and 5 mu L of a reference medicinal material solution, respectively dropping the solutions on the same silica gel G plate, taking an upper layer solution of toluene-ethyl acetate-formic acid as a developing agent in a volume ratio of 8:1:0.5, developing, taking out, drying, spraying a phosphomolybdic acid test solution, heating at 105 ℃ until spots are clearly developed, and inspecting in sunlight; spots of the same color appear in the chromatogram of the test solution at the positions corresponding to those in the chromatogram of the reference solution;
the thin-layer chromatography identification method of the radix zanthoxyli medicinal material comprises the following specific steps:
(1) taking 10 granules of Fuyanjing capsule content, adding 50mL of 1% methanol hydrochloride, refluxing for 1 hour, filtering, passing the filtrate through a neutral alumina column, eluting with 50mL of methanol, collecting the eluent, evaporating to dryness, and dissolving the residue with 1mL of methanol to obtain a sample solution;
(2) adding ethanol into a nitidine chloride reference substance to prepare a solution containing 1mg of nitidine chloride per 1mL, and taking the solution as a reference substance solution;
(3) testing by adopting a general rule of thin-layer chromatography 0502, sucking 5-10 mu L of a test solution and 3 mu L of a reference solution, respectively dropping the solutions on the same silica gel G plate, developing by taking a trichloromethane-methanol solution with a volume ratio of 7:1 as a developing agent, taking out, drying in the air, and inspecting under 365nm ultraviolet light; the test chromatogram shows fluorescent spots of the same color at the positions corresponding to those of the control chromatogram.
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| CN113156037A (en) * | 2021-04-23 | 2021-07-23 | 广西壮族自治区食品药品检验所 | Method for rapidly identifying duck feet and erythrina indica |
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