CN104165962B - The quality determining method of WEINAIAN sheet - Google Patents
The quality determining method of WEINAIAN sheet Download PDFInfo
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Abstract
The invention discloses a kind of quality determining method of WEINAIAN sheet, described quality determining method adopts following discriminating and one or more in assay project: the TLC distinguish of the Radix Astragali; The TLC distinguish of the Radix Astragali, red ginseng and pseudo-ginseng; The TLC distinguish of calculus bovis factitius; The high performance liquid chromatography qualitative, quantitative discrimination method of WEINAIAN sheet; The high performance liquid chromatography quantitative identification method of Astragaloside IV.The present invention improves and has revised original capsule for relieving gastric trouble quality standard, has revised and enlarged the TLC distinguish of four kinds of flavones ingredients and Astragaloside IV in the Radix Astragali, has revised and enlarged the TLC distinguish of red ginseng simultaneously, and to index components Astragaloside IV, ginseng soap Rg
1, Rb
1and Panax Notoginseng saponin R
1differentiate simultaneously; Adopt HPLC-PDA-ELSD coupling technique to combine assay and the characteristic spectrum thereof of setting up its main active, realize effective control of WEINAIAN tablet quality, and there is efficient, that quantitatively accurate, good stability, precision are high and repeatability is good advantage simultaneously.
Description
Technical field
The present invention relates to a kind of quality determining method of WEINAIAN sheet.
Background technology
Capsule for relieving gastric trouble, primarily of compositions such as the Radix Astragali, pseudo-ginseng, red ginseng, nacreous layer powder, calculus bovis factitiuses, has air making-up and spleen enlivening, the function of promoting blood circulation and stopping pain, for deficiency of spleen-QI and stomach-QI, and the stomachache caused by extravasated blood retardance, disease sees that vague epigastralgia or shouting pain, indigestion and loss of appetite food are few; Chronic gastritis, gastric and duodenal ulcer are shown in above-mentioned disease person, its quality standard is now recorded in " Chinese Pharmacopoeia " version in 2010, China Patent No. ZL200510037077.7, denomination of invention is that the patent of invention of " a kind of Chinese patent drug for the treatment of peptic ulcer and preparation method thereof " discloses its composition and method of making the same.WEINAIAN sheet is on the basis that the former prescription of maintenance capsule for relieving gastric trouble is constant, be instruct with traditional Chinese medical theory, based on clinical efficacy, the novel form that the modern Chinese herbal medicine preparation new technology of application of advanced, new equipment, new technology and new material carry out secondary development to capsule for relieving gastric trouble and develop.Its preparation method is the Radix Astragali, pseudo-ginseng, red ginseng mixing and water adding decoction secondary, and filter, filtrate merges, and filtrate reduced in volume becomes thick paste; Nacreous layer powder ultramicro grinding, dry, pulverize after mixing with thick paste, and bedding-in adds the calculus bovis factitius of auxiliary material and ultramicro grinding; Fluidized bed granulation, drying is pressed into and forms, and film coating to obtain final product, and the preparation method of above-mentioned WEINAIAN sheet has applied for Chinese invention patent (number of patent application: 201210267101.6).
The quality inspection standard relatively backwardness of former capsule for relieving gastric trouble, only carries out quality testing to the amount of activated material of pseudo-ginseng, calculus bovis factitius and the Radix Astragali, comprehensively can not detect its quality; The invention provides a kind of WEINAIAN sheet efficient, accurately differentiate and content assaying method.
Summary of the invention
Based on this, the invention provides a kind of discriminating and content assaying method of WEINAIAN sheet.
The concrete technical scheme solved the problems of the technologies described above is as follows:
An assay method for WEINAIAN sheet, described detection method adopts following discriminating and one or more in assay project:
(1) TLC distinguish of the Radix Astragali:
A prepares need testing solution: by WEINAIAN sheet porphyrize, get 1.8-2.2g, add methyl alcohol 48-52ml, ultrasonic process, filter, evaporate to dryness filtrate, the residue 18-22ml that adds water makes dissolving, extract 3-4 time with ethyl acetate again, each 28-32ml, merges upper strata acetic acid ethyl fluid, evaporate to dryness, residue adds methyl alcohol 0.8-1.2ml makes dissolving, as need testing solution;
B prepares Radix Astragali control medicinal material solution: get Radix Astragali control medicinal material 1.8-2.2g, add methyl alcohol 48-52ml, ultrasonic process, filter, evaporate to dryness filtrate, the residue 18-22ml that adds water makes dissolving, then extracts 3-4 time with ethyl acetate, each 28-32ml, merge upper strata acetic acid ethyl fluid, evaporate to dryness, residue adds methyl alcohol 0.5ml makes dissolving, as Radix Astragali control medicinal material solution;
C prepares reference substance solution: get onocerin reference substance, calycosin reference substance, ononin reference substance and Calycosin-7-O-BETA-D-glucoside reference substance, adds methyl alcohol respectively and makes the methanol solution of every 1ml methyl alcohol containing the above-mentioned reference substance of 0.8-1.2mg, product solution in contrast;
D detects: get reference substance solution 1.8-2.2 μ l described in Radix Astragali control medicinal material solution each 9-10 μ l and step c described in need testing solution described in step a, step b, put respectively on same silica GF254 thin layer plate, with the toluene-ethyl acetate-methyl alcohol of volume ratio 19-21 ︰ 19-21 ︰ 8-10 for developping agent, launch, take out, dry, inspect under putting the ultraviolet lamp of 254nm, in test sample chromatogram, on the position corresponding with reference substance chromatogram to control medicinal material chromatogram, the fluorescence spot of aobvious same color; Spray 1, the 1-diphenyl-2-trinitrophenyl-hydrazine ethanol solution with 0.08-0.12% again, dry, inspect under putting daylight, in test sample chromatogram, on the position corresponding with reference substance chromatogram to control medicinal material chromatogram, the spot of aobvious same color;
(2) TLC distinguish of the Radix Astragali, red ginseng and pseudo-ginseng, step is as follows:
A prepares need testing solution: by WEINAIAN sheet porphyrize, get 1.8-2.2g, add methyl alcohol 48-52ml, ultrasonic process, filter, evaporate to dryness filtrate, the residue 18-22ml that adds water makes dissolving, then extracts 3-4 time with ethyl acetate, each 28-32ml, merge lower layer of water liquid, extract 3-4 time with water-saturated n-butanol jolting, each 28-32ml, merge normal butyl alcohol liquid, with ammonia solution washing 2-3 time, each 28-32ml, discards ammoniacal liquor, by normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 0.45-0.55ml makes dissolving, as need testing solution;
B prepares control medicinal material solution: get Radix Astragali control medicinal material 1.8-2.2g, add methyl alcohol 48-52ml, ultrasonic process, filter, evaporate to dryness filtrate, the residue 18-22ml that adds water makes dissolving, extract 3-4 time with ethyl acetate again, each 28-32ml, merges lower layer of water liquid, extracts 3-4 time with water-saturated n-butanol jolting, each 28-32ml, merge normal butyl alcohol liquid, with ammonia solution washing 2-3 time, each 28-32ml, discard ammoniacal liquor, by normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 0.45-0.55ml makes dissolving, as Radix Astragali control medicinal material solution; Separately get red ginseng control medicinal material 0.45-0.55g and pseudo-ginseng control medicinal material 0.65-0.75g, add water 90-110ml respectively, decoct 55-65min, filter, filtrate is extracted 3-4 time with ethyl acetate, each 28-32ml, discards acetic acid ethyl fluid, then extracts 3-4 time with water-saturated n-butanol jolting, each 28-32ml, merge normal butyl alcohol liquid, rear ammonia solution washing 2-3 time, each 28-32ml, discard ammoniacal liquor, by normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 0.45-0.55ml makes dissolving, respectively obtained red ginseng control medicinal material solution and pseudo-ginseng control medicinal material solution;
C prepares reference substance mixed solution: get Astragaloside IV reference substance, ginsenoside Rg
1reference substance, ginsenoside Rb
1reference substance and Panax Notoginseng saponin R
1reference substance, adds methyl alcohol and makes every 1ml methyl alcohol respectively containing the mixed solution of the above-mentioned reference substance of 0.8-1.2mg, product mixed solution in contrast;
D detects: get reference substance mixed solution each 1.8-2.2 μ l described in need testing solution described in step a and step c, Radix Astragali control medicinal material solution 8-12 μ l described in step b, red ginseng control medicinal material solution 5-7 μ l and pseudo-ginseng control medicinal material solution 0.8-1.2 μ l, put respectively on same silica gel g thin-layer plate, with the upper solution of the normal butyl alcohol-ethyl acetate of volume ratio 3.5-4.5:1:4.5-5.5-rare ammonia for developping agent, be placed in the strong ammonia solution presaturation expansion cylinder of 20 minutes, launch, take out, dry, spray is with 9-11% ethanol solution of sulfuric acid, spot development is heated to clear at 100-110 DEG C, in test sample chromatogram, on the position corresponding with reference substance chromatogram to control medicinal material chromatogram, the spot of aobvious same color, inspect under putting 365nm ultraviolet lamp, the fluorescence spot of aobvious same color,
(3) TLC distinguish of calculus bovis factitius:
A prepares need testing solution: by WEINAIAN sheet porphyrize, get 0.18-0.22g, add methyl alcohol 18-22ml, ultrasonic process, and filter, evaporate to dryness filtrate, residue adds methyl alcohol 0.8-1.2ml makes dissolving, as need testing solution;
B prepares control medicinal material solution: get calculus bovis factitius control medicinal material 8-12mg, add methyl alcohol 1.5-2.5ml, ultrasonic process, centrifugal, gets supernatant medicinal material solution in contrast;
C prepares reference substance solution: get cholic acid reference substance, hyodesoxycholic acid reference substance, adds methyl alcohol respectively and makes the solution containing the above-mentioned reference substance of 0.8-1.2mg in every 1ml methyl alcohol, as cholic acid reference substance solution and hyodesoxycholic acid reference substance solution;
D detects: get cholic acid reference substance solution described in control medicinal material solution described in need testing solution described in step a, step b and step c and hyodesoxycholic acid reference substance solution each 0.8-1.2 μ l, put respectively on same silica gel g thin-layer plate, take volume ratio as the upper solution of the cyclohexane-ethyl acetate-methanol-acetic acid of 18-22:23-27:2.5-3.5:1.5-2.5 be developping agent, launch, take out, dry, spray is with 9-11% ethanol solution of sulfuric acid, be heated to spot development at 105 DEG C clear, inspect under putting 365nm ultraviolet lamp; In test sample chromatogram, on the position corresponding with reference substance chromatogram to control medicinal material chromatogram, the fluorescence spot of aobvious same color;
(4) the high performance liquid chromatography qualitative identification method of WEINAIAN sheet, comprises the steps:
A chromatographic condition: take octadecylsilane chemically bonded silica as filling agent; Acetonitrile is mobile phase A, and 0.1% formic acid is Mobile phase B, gradient elution, PDA/ELSD Series detectors; The determined wavelength of described PDA is 260nm; ELSD parameter is: drift tube temperature: 60 DEG C, flow rate of carrier gas: 35psi, yield value: 200; Number of theoretical plate presses Calycosin-7-O-BETA-D-glucoside peak and ginsenoside Rb
1peak calculates all should be not less than 5000; Described gradient elution is: 0-32min, 5% → 18% acetonitrile; 32-38min, 18% → 22% acetonitrile; 38-58min, 22% acetonitrile; 58-70min, 22% → 32% acetonitrile; 70-84min, 32% → 34% acetonitrile; 84-96min, 34% acetonitrile; 96-102min, 34% → 40% acetonitrile; 102-125min, 40% → 67% acetonitrile;
B prepares object of reference solution: get Calycosin-7-O-BETA-D-glucoside and ginsenoside Rb
1reference substance, adds methyl alcohol respectively and makes the ginsenoside Rb that Calycosin-7-O-BETA-D-glucoside methanol solution that concentration is 38-42 μ g/ml and concentration are 0.13-0.17mg/ml
1methanol solution;
C prepares need testing solution: by WEINAIAN sheet porphyrize, get powder 0.8-1.2g, add methyl alcohol 48-52ml, ultrasonic process, put to room temperature, supply the weight of minimizing with methyl alcohol, shake up, filter, get filtrate 23-27ml, evaporate to dryness, residue adds methyl alcohol and dissolves, and with methyl alcohol constant volume in 5ml volumetric flask, to obtain final product;
D detects: detect under chromatographic condition described in step a;
(5) the high performance liquid chromatography quantitative identification method of WEINAIAN sheet, comprises the steps:
A chromatographic condition: take octadecylsilane chemically bonded silica as filling agent; Acetonitrile is mobile phase A, and 0.1% formic acid is Mobile phase B, and gradient elution uses PDA/ELSD Series detectors; The determined wavelength of described PDA is 260nm; ELSD parameter is: drift tube temperature: 60 DEG C, flow rate of carrier gas: 35psi, yield value: 200; Number of theoretical plate presses Calycosin-7-O-BETA-D-glucoside peak and ginsenoside Rb
1peak calculates all should be not less than 5000; Described gradient elution is: 0 ~ 32min:5% → 18% acetonitrile; 32 ~ 38min:18% → 22% acetonitrile; 38 ~ 58min:22% acetonitrile; 58 ~ 70min:22% → 32% acetonitrile; 70 ~ 84min:32% → 34% acetonitrile; 84 ~ 96min:34% acetonitrile; 96 ~ 102min:34% → 40% acetonitrile; 102 ~ 125min:40% → 67% acetonitrile;
B preparation mixing reference substance solution: get Calycosin-7-O-BETA-D-glucoside, ononin, calycosin, onocerin, Panax Notoginseng saponin R
1, ginsenoside Rg
1, ginsenoside Re, ginsenoside Rb
1, cholic acid and hyodesoxycholic acid reference substance, add methyl alcohol and make every 1ml methyl alcohol containing Calycosin-7-O-BETA-D-glucoside 38-42 μ g, ononin 7-9 μ g, calycosin 18-22 μ g, onocerin 5-7 μ g, Panax Notoginseng saponin R
10.13-0.17mg, ginsenoside Rg
10.28-0.32mg, ginsenoside Re 38-42 μ g, ginsenoside Rb
1the mixing reference substance solution of 0.13-0.17mg, cholic acid 0.45-0.55mg, hyodesoxycholic acid 0.35-0.45mg;
C prepares need testing solution: by WEINAIAN sheet porphyrize, get powder 0.8-1.2g, add methyl alcohol 48-52ml, ultrasonic process, put to room temperature, supply the weight of minimizing with methyl alcohol, shake up, filter, get filtrate 23-27ml, evaporate to dryness, residue adds methyl alcohol and dissolves, and with methyl alcohol constant volume in 5ml volumetric flask, to obtain final product;
D detects: under the chromatographic condition described in step a, draws reference substance solution 5 μ l, 20 μ l, need testing solution 20 μ l, injection liquid chromatography, measures, and wherein PDA detects and calculates with one point external standard method, ELSD detects and calculates with external standard two-point method logarithmic equation, to obtain final product;
(5) the high performance liquid chromatography quantitative identification method of Astragaloside IV:
A chromatographic condition: take octadecylsilane chemically bonded silica as filling agent; Volume ratio is the acetonitrile-water of 30 ︰ 70 is mobile phase; Evaporative light-scattering detector detects; Number of theoretical plate calculates should be not less than 5000 by Astragaloside IV peak;
The preparation of b reference substance solution: get Astragaloside IV reference substance and add methyl alcohol and make the Astragaloside IV reference substance methanol solution that concentration is 0.45-0.55mg/ml;
The preparation of c need testing solution: by WEINAIAN sheet porphyrize, get powder 1.3-1.7g, add methyl alcohol 48-52ml, ultrasonic process, put to room temperature, supply the weight of minimizing with methyl alcohol, shake up, filter, get filtrate 23-27ml, evaporate to dryness, the residue 18-22ml that adds water makes dissolving, extract 3-4 time with water-saturated n-butanol jolting, each 38-42ml, merges normal butyl alcohol liquid, with ammonia solution washing 2-3 time, each 38-42ml, discard ammoniacal liquor, by normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol and dissolves, with methyl alcohol constant volume in 5ml volumetric flask, to obtain final product;
D detects: draw reference substance solution 5 μ l, 15 μ l, need testing solution 20 μ l, injection liquid chromatography, measure, calculate, to obtain final product with external standard two-point method logarithmic equation.
Wherein in some embodiments, in the high performance liquid chromatography qualitative identification method of WEINAIAN sheet and the high performance liquid chromatography quantitative identification method of WEINAIAN sheet, described in step a, the flow velocity of chromatographic condition is 0.8-1.2ml/min; Column temperature is 30 DEG C.
Wherein in some embodiments, the sample size of chromatographic condition described in step a in the high performance liquid chromatography qualitative identification method of WEINAIAN sheet: 20 μ l.
The detection method of a kind of WEINAIAN sheet of the present invention has the following advantages and beneficial effect:
1, in detection method of the present invention one or the multinomial method of use in conjunction is adopted, improve and revised original capsule for relieving gastric trouble quality standard, the effective control to WEINAIAN sheet end product quality can be realized simultaneously, the method has efficiently, quantitatively accurately, high, the repeated good advantage of good stability, precision.
2, the thin-layer identification method in detection method of the present invention, revise and enlarge the TLC distinguish of four kinds of flavones ingredient Calycosin-7-O-BETA-D-glucosides, ononin, calycosin, onocerin and Astragaloside IVs in the Radix Astragali, also revised and enlarged the TLC distinguish of red ginseng simultaneously, and to index components Astragaloside IV, ginsenoside Rg
1, ginsenoside Rb
1and Panax Notoginseng saponin R
1differentiate simultaneously.
3, the present invention carries the efficient liquid phase quantitative identification method in described detection method; adopt HPLC-PDA-ELSD coupling technique; set up characteristic component Calycosin-7-O-BETA-D-glucoside, ononin, calycosin and the onocerin in the main active Radix Astragali of WEINAIAN sheet; characteristic component notoginsenoside R in pseudo-ginseng; characteristic component ginsenoside Rg1 in red ginseng, ginsenoside Re and ginsenoside Rb1, the characteristic component cholic acid in calculus bovis factitius and the assay of hyodesoxycholic acid and characteristic spectrum.
Accompanying drawing explanation
Fig. 1 is the characteristic spectrum that embodiment 4 test sample PDA detects;
Fig. 2 is the characteristic spectrum that embodiment 4 test sample ELSD detects.
Embodiment
The prescription of WEINAIAN sheet involved in the present invention is: the Radix Astragali, pseudo-ginseng, red ginseng, nacreous layer powder, calculus bovis factitius.
The preparation method of described WEINAIAN sheet is: the above-mentioned Radix Astragali, pseudo-ginseng, red ginseng are decocted secondary in prescription ratio mixing and water adding, filters, filtrate merges, and filtrate reduced in volume becomes thick paste; Nacreous layer powder ultramicro grinding, dry, pulverize after mixing with thick paste, and bedding-in adds the calculus bovis factitius of auxiliary material and ultramicro grinding; Fluidized bed granulation, dry tabletted, to obtain final product.
Below with reference to embodiment, the present invention will be further described.
The TLC distinguish of the Radix Astragali in embodiment 1 WEINAIAN sheet
A detection method for WEINAIAN sheet, described detection method adopts TLC Identification to measure the Radix Astragali, comprises the steps:
A prepares need testing solution: by WEINAIAN sheet finished product porphyrize, get 2g, add methyl alcohol 50ml, ultrasonic process, filter, evaporate to dryness filtrate, the residue 20ml that adds water makes dissolving, 3 times are extracted again with ethyl acetate, each 30ml, merges the upper strata acetic acid ethyl fluid of gained, by acetic acid ethyl fluid evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution;
B prepares Radix Astragali control medicinal material solution: get Radix Astragali control medicinal material 2g, add methyl alcohol 50ml, ultrasonic process, filter, evaporate to dryness filtrate, the residue 20ml that adds water makes dissolving, then extracts 3 times with ethyl acetate, each 30ml, merge the upper strata acetic acid ethyl fluid of gained, by upper strata acetic acid ethyl fluid evaporate to dryness, residue adds methyl alcohol 0.5ml makes dissolving, as Radix Astragali control medicinal material solution;
C prepares reference substance solution: get onocerin reference substance, calycosin reference substance, ononin reference substance and Calycosin-7-O-BETA-D-glucoside reference substance, adds methyl alcohol respectively and makes the methanol solution of every 1ml methyl alcohol containing the above-mentioned reference substance of 1mg, product solution in contrast;
D detects: get the reference substance solution 2 μ l described in each 8 μ l and step c of Radix Astragali control medicinal material solution described in need testing solution described in step a, step b, put respectively on same silica GF254 thin layer plate, be that the toluene-ethyl acetate-methyl alcohol of 20 ︰ 20 ︰ 9 is for developping agent with volume ratio, launch, take out, dry, inspect under putting the ultraviolet lamp of 254nm, in test sample chromatogram, on the position corresponding with reference substance chromatogram to control medicinal material chromatogram, the fluorescence spot of aobvious same color; Spray 1, the 1-diphenyl-2-trinitrophenyl-hydrazine ethanol solution with 0.1% again, dry, inspect under putting daylight, in test sample chromatogram, on the position corresponding with reference substance chromatogram to control medicinal material chromatogram, the spot of aobvious same color.
In embodiment 2 WEINAIAN sheet, the Radix Astragali, red ginseng and pseudo-ginseng carry out indentification by TLC simultaneously
A detection method for WEINAIAN sheet, described detection method adopts the Radix Astragali, red ginseng and pseudo-ginseng in TLC Identification Simultaneously test WEINAIAN sheet, comprises the steps:
A prepares need testing solution: by WEINAIAN sheet finished product porphyrize, get 2g, add methyl alcohol 50ml, ultrasonic process, filter, evaporate to dryness filtrate, the residue 20ml that adds water makes dissolving, extract 3 times with ethyl acetate again, each 30ml, merge lower layer of water liquid, extract 3 times with water-saturated n-butanol jolting, each 30ml, merge normal butyl alcohol liquid, wash 2 times with ammonia solution, each 30ml, discards ammoniacal liquor, by normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 0.5ml makes dissolving, as need testing solution;
B prepares control medicinal material solution: get Radix Astragali control medicinal material 2g, add methyl alcohol 50ml, ultrasonic process, filter, evaporate to dryness filtrate, the residue 20ml that adds water makes dissolving, extract 3 times with ethyl acetate again, each 30ml, merge lower layer of water liquid, extract 3 times with water-saturated n-butanol jolting, each 30ml, merge normal butyl alcohol liquid, wash 2 times with ammonia solution, each 30ml, discards ammoniacal liquor, by normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 0.5ml makes dissolving, as Radix Astragali control medicinal material solution; Separately get red ginseng control medicinal material 0.5g and pseudo-ginseng control medicinal material 0.7g, add water 100ml respectively, decocts 60min, filter, filtrate extracts 3 times with ethyl acetate, each 30ml, discard acetic acid ethyl fluid, extract 3 times with water-saturated n-butanol jolting, each 30ml, merge normal butyl alcohol liquid, wash 2 times with ammonia solution, each 30ml, discard ammoniacal liquor, by normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 0.5ml makes dissolving, respectively obtained red ginseng control medicinal material solution and pseudo-ginseng control medicinal material solution;
C prepares reference substance mixed solution: get Astragaloside IV reference substance, ginsenoside Rg
1reference substance, ginsenoside Rb
1reference substance and Panax Notoginseng saponin R
1reference substance, adds methyl alcohol and makes every 1ml methyl alcohol respectively containing the mixed solution of the above-mentioned reference substance of 1mg, product mixed solution in contrast;
D detects: get need testing solution and each 2 μ l of reference substance mixed solution, Radix Astragali control medicinal material solution 10 μ l, red ginseng control medicinal material solution 6 μ l, pseudo-ginseng control medicinal material solution 1 μ l, put respectively on same silica gel g thin-layer plate, be that normal butyl alcohol-ethyl acetate-rare ammonia of 4:1:5 (gets strong ammonia solution 5ml with volume ratio, be diluted with water to 50ml, obtain) upper solution be developping agent, be placed in the strong ammonia solution presaturation expansion cylinder of 20 minutes, launch, take out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot development at 105 DEG C clear; In test sample chromatogram, on the position corresponding with reference substance chromatogram to control medicinal material chromatogram, the spot of aobvious same color; Inspect under putting 365nm ultraviolet lamp, the fluorescence spot of aobvious same color.
The indentification by TLC of calculus bovis factitius in embodiment 3 WEINAIAN sheet
A detection method for WEINAIAN sheet, described detection method adopts TLC Identification to measure calculus bovis factitius, comprises the steps:
A prepares need testing solution: by WEINAIAN sheet finished product porphyrize, get 0.2g, add methyl alcohol 20ml, ultrasonic process, and filter, evaporate to dryness filtrate, residue adds methyl alcohol 1ml makes dissolving, as need testing solution;
B prepares control medicinal material solution: get calculus bovis factitius control medicinal material 10mg, add methyl alcohol 2ml, ultrasonic process, centrifugal, gets supernatant medicinal material solution in contrast;
C prepares reference substance solution: get cholic acid reference substance, hyodesoxycholic acid reference substance, adds methyl alcohol respectively and makes the solution containing the above-mentioned reference substance of 1mg in every 1ml methyl alcohol, as cholic acid reference substance solution and hyodesoxycholic acid reference substance solution;
D detects: get the need testing solution described in step a, the control medicinal material solution described in step b and the cholic acid reference substance solution described in step c and each 1 μ l of hyodesoxycholic acid reference substance solution, put respectively on same silica gel g thin-layer plate, take volume ratio as the upper solution of the cyclohexane-ethyl acetate-methanol-acetic acid of 20:25:3:2 be developping agent, launch, take out, dry, spray with 10% ethanol solution of sulfuric acid, be heated to spot development at 105 DEG C clear, inspect under putting 365nm ultraviolet lamp; In test sample chromatogram, on the position corresponding with reference substance chromatogram to control medicinal material chromatogram, the fluorescence spot of aobvious same color.
The high performance liquid chromatography Qualitive test of embodiment 4 WEINAIAN sheet
A detection method for WEINAIAN sheet, described detection method adopts high performance liquid chromatography Qualitive test WEINAIAN sheet, comprises the steps:
A chromatographic condition: take octadecylsilane chemically bonded silica as filling agent; Acetonitrile is mobile phase A, and 0.1% formic acid is Mobile phase B, gradient elution; Flow velocity is 1.0ml/min; Column temperature is 30 DEG C; Sample size: object of reference solution and each 20 μ l of need testing solution; Adopt PDA/ELSD Series detectors, the determined wavelength of described PDA is 260nm; ELSD parameter is: drift tube temperature: 60 DEG C, flow rate of carrier gas: 35psi, yield value: 200; Number of theoretical plate presses Calycosin-7-O-BETA-D-glucoside peak and ginsenoside Rb
1peak calculates all should be not less than 5000; Described gradient elution is see table 1:
The gradient elution of table 1 high performance liquid chromatography
B prepares object of reference solution: get Calycosin-7-O-BETA-D-glucoside and ginsenoside Rb
1reference substance, adds methyl alcohol respectively and makes the ginsenoside Rb that Calycosin-7-O-BETA-D-glucoside methanol solution that concentration is 40 μ g/ml and concentration are 0.15mg/ml
1methanol solution;
C prepares need testing solution: by WEINAIAN sheet porphyrize, get powder 1.0g, add methyl alcohol 50ml, ultrasonic process, put to room temperature, supply the weight of minimizing with methyl alcohol, shake up, filter, get filtrate 25ml, evaporate to dryness, residue adds methyl alcohol and dissolves, and with methyl alcohol constant volume in 5ml volumetric flask, to obtain final product;
D detects: under the chromatographic condition described in step a, PDA detects in the test sample characteristic spectrum obtained 4 characteristic peaks (see Fig. 1), the peak corresponding with Calycosin-7-O-BETA-D-glucoside reference peak is 1(S1) peak, all the other 3 peaks are peak 2(ononin), peak 3(calycosin), peak 4(onocerin), calculate the relative retention time at these 3 characteristic peaks and S1 peak, its relative retention time should setting ± 5% within, setting is: 1.00(peak S1), 1.49(peak 2), 1.69(peak 3), 2.52(peak 4); ELSD detects in the test sample characteristic spectrum obtained has 19 characteristic peaks (see Fig. 2), with ginsenoside Rb
1corresponding peak, object of reference peak is S2 peak, calculate peak 5(notoginsenoside R), peak 6(ginsenoside Rg1), peak 7(ginsenoside Re), peak 10(ginsenoside Rb1), peak 13(Astragaloside IV), peak 16(cholic acid), peak 17(hyodesoxycholic acid) relative retention time at number characteristic peak and S2 peak, its relative retention time should setting ± 5% within, setting is: 0.58(peak 5), 0.63(peak 6), 0.64(peak 7), 1.00(peak S2), 1.07(peak 13), 1.30(peak 16), 1.36(peak 17).
The high performance liquid chromatography quantitative measurement of embodiment 5 WEINAIAN sheet
A detection method for WEINAIAN sheet, described detection method adopts High Performance Liquid Chromatography/Photodiode Array Detection-evaporative light-scattering detector coupling technique quantitatively to differentiate WEINAIAN sheet, comprise the steps:
A chromatographic condition: chromatographic column is octadecylsilane chemically bonded silica; Mobile phase is acetonitrile-0.1% formic acid, gradient elution; Flow velocity is 1.0ml/min; Column temperature is 30 DEG C; Sample size: reference substance solution 5 μ L, 20 μ L, need testing solution 20 μ L; Adopt PDA/ELSD Series detectors, the determined wavelength of described PDA is 260nm; ELSD parameter is: drift tube temperature: 60 DEG C, flow rate of carrier gas: 35psi, yield value: 200; Number of theoretical plate presses Calycosin-7-O-BETA-D-glucoside peak and ginsenoside Rb
1peak calculates all should be not less than 5000; Described gradient elution is: 0 ~ 32min:5% acetonitrile → 18% acetonitrile; 32 ~ 38min:18% acetonitrile → 22% acetonitrile; 38 ~ 58min:22% acetonitrile; 58 ~ 70min:22% acetonitrile → 32% acetonitrile; 70 ~ 84min:32% acetonitrile → 34% acetonitrile; 84 ~ 96min:34% acetonitrile; 96 ~ 102min:34% acetonitrile → 40% acetonitrile; 102 ~ 125min:40% acetonitrile → 67% acetonitrile;
B preparation mixing reference substance solution: get Calycosin-7-O-BETA-D-glucoside, ononin, calycosin, onocerin, Panax Notoginseng saponin R
1, ginsenoside Rg
1, ginsenoside Re, ginsenoside Rb
1, cholic acid and hyodesoxycholic acid reference substance be appropriate, add methyl alcohol and make every 1ml methyl alcohol containing Calycosin-7-O-BETA-D-glucoside 40 μ g, ononin 8 μ g, calycosin 20 μ g/ml, onocerin 6 μ g, Panax Notoginseng saponin R
10.15mg, ginsenoside Rg
10.3mg, ginsenoside Re 40 μ g, ginsenoside Rb
1the mixing reference substance solution of 0.15mg, cholic acid 0.5mg, hyodesoxycholic acid 0.4mg;
C prepares need testing solution: by WEINAIAN sheet porphyrize, get powder 1.0g, add methyl alcohol 50ml, ultrasonic process, put to room temperature, supply the weight of less loss with methyl alcohol, shake up, filter, get filtrate 25ml, evaporate to dryness, residue adds methyl alcohol and dissolves, and with methyl alcohol constant volume in 5ml volumetric flask, to obtain final product;
D detects: under the chromatographic condition described in step a, draws reference substance solution 5 μ l, 20 μ l, need testing solution 20 μ l, injection liquid chromatography, measures, and wherein PDA detects and calculates with one point external standard method, ELSD detects and calculates with external standard two-point method logarithmic equation.
A: linear relationship is investigated
In mixing reference substance solution 2 μ L, 5 μ L respectively described in accurate aspiration step b, 10 μ L, 15 μ L, 20 μ L, 30 μ L injection liquid chromatographies, measure by the chromatographic condition described in step (1), record each chromatographic peak area, wherein PDA detect Calycosin-7-O-BETA-D-glucoside, ononin, calycosin and onocerin be with each reference substance sample size (X, μ g) be horizontal ordinate, with peak area (Y) for ordinate; The notoginsenoside R that ELSD detects, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, cholic acid and hyodesoxycholic acid are with the logarithm value (X of each reference substance sample size, μ g) be horizontal ordinate, with peak area logarithm value (Y) for ordinate, respectively drawing standard curve go forward side by side line linearity return calculate, obtain each ingredient standard Regression Equations, the range of linearity, related coefficient, detectability and quantitative limit, the results are shown in Table 2:
The typical curve of each component of table 2 and detectability thereof and quantitative limit
B: replica test
(lot number: 20110301) 6 parts, by the parallel preparation of need testing solution preparation method described in step c, measures in accordance with the law, the results are shown in Table 3: result shows that the method repeatability is good to get test sample under same batch of weight differential item.
C: precision test
Get the same bottle need testing solution of replica test under B item, by above-mentioned chromatographic condition continuous sample introduction 6 times, measure peak area and also calculate RSD, the results are shown in Table 3; Show that this instrument precision is good.
D: stability test
Get the same bottle need testing solution of replica test under B item, respectively at 0h, 5h, 10h, 20h, 30h, 72h sample introduction, measure peak area and also calculate RSD, the results are shown in Table 3; Show that need testing solution is basicly stable in 72h.
E: average recovery is tested
Get the test sample (lot number: 20110301) 6 parts of above-mentioned known content, get 0.5g, accurately weighed, put tool plug conical flask kind, precision adds above-mentioned each reference substance appropriate (about suitable with contained amount in test sample) respectively, by the parallel preparation of need testing solution preparation method described in step c, measure in accordance with the law, calculate the recovery and the results are shown in Table 3.
Table 3 Method validation result
F: assay
Adopt the discrimination method described in the present embodiment, measure the content of three batches of WEINAIAN sheets, the results are shown in Table 4:
Table 4 three batches of WEINAIAN sheet assay results
As known from Table 4, the advantage that this experiment high performance liquid chromatography quantitative detecting method has efficiently, quantitatively accurate, good stability, precision are high, repeatability is good.
The high performance liquid chromatography quantitative measurement of embodiment 6 Astragaloside IV
A detection method for WEINAIAN sheet, described detection method adopts the Astragaloside IV in high performance liquid chromatography Qualitive test WEINAIAN sheet, comprises the steps:
A chromatographic condition: take octadecylsilane chemically bonded silica as filling agent; Volume ratio is the acetonitrile-water of 30 ︰ 70 is mobile phase; Detect by evaporative light-scattering detector; Number of theoretical plate calculates should be not less than 5000 by Astragaloside IV peak;
The preparation of b reference substance solution: get Astragaloside IV reference substance and add methyl alcohol and make the Astragaloside IV reference substance methanol solution that concentration is 0.5mg/ml;
The preparation of c need testing solution: by WEINAIAN sheet porphyrize, get powder 1.5g, add methyl alcohol 50ml, ultrasonic process, put to room temperature, supply the weight of minimizing with methyl alcohol, shake up, filter, get filtrate 25ml, evaporate to dryness, residue adds water 20ml, and low-grade fever makes dissolving, extract 3-4 time with water-saturated n-butanol jolting, each 38-42ml, merge normal butyl alcohol liquid, with ammonia solution washing 2-3 time, each 38-42ml, discards ammoniacal liquor, by normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol and dissolves, and with methyl alcohol constant volume in 5ml volumetric flask, to obtain final product;
D detects: draw reference substance solution 5 μ l, 15 μ l, need testing solution 20 μ l, injection liquid chromatography, measure, calculate, to obtain final product with external standard two-point method logarithmic equation.
Adopt Astragaloside IV (C in every sheet WEINAIAN sheet that described in the present embodiment, high performance liquid chromatography records
41h
68o
14) content be 1.35mg.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (4)
1. a quality determining method for WEINAIAN sheet, is characterized in that: described detection method adopts following discriminating and one or more in assay project:
(1) TLC distinguish of the Radix Astragali:
A prepares need testing solution: by WEINAIAN sheet porphyrize, get 1.8-2.2g, add methyl alcohol 48-52ml, ultrasonic process, filter, evaporate to dryness filtrate, the residue 18-22ml that adds water makes dissolving, extract 3-4 time with ethyl acetate again, each 28-32ml, merges upper strata acetic acid ethyl fluid, evaporate to dryness, residue adds methyl alcohol 0.8-1.2ml makes dissolving, as need testing solution;
B prepares Radix Astragali control medicinal material solution: get Radix Astragali control medicinal material 1.8-2.2g, add methyl alcohol 48-52ml, ultrasonic process, filter, evaporate to dryness filtrate, the residue 18-22ml that adds water makes dissolving, then extracts 3-4 time with ethyl acetate, each 28-32ml, merge upper strata acetic acid ethyl fluid, evaporate to dryness, residue adds methyl alcohol 0.45-0.55ml makes dissolving, as Radix Astragali control medicinal material solution;
C prepares reference substance solution: get onocerin reference substance, calycosin reference substance, ononin reference substance and Calycosin-7-O-BETA-D-glucoside reference substance, adds methyl alcohol respectively and makes the methanol solution of every 1ml methyl alcohol containing the above-mentioned reference substance of 0.8-1.2mg, product solution in contrast;
D detects: get reference substance solution 1.8-2.2 μ l described in Radix Astragali control medicinal material solution each 9-10 μ l and step c described in need testing solution described in step a, step b, put respectively on same silica GF254 thin layer plate, with the toluene-ethyl acetate-methyl alcohol of volume ratio 19-21 ︰ 19-21 ︰ 8-10 for developping agent, launch, take out, dry, inspect under putting the ultraviolet lamp of 254nm, in test sample chromatogram, on the position corresponding with reference substance chromatogram to control medicinal material chromatogram, the fluorescence spot of aobvious same color; Spray 1, the 1-diphenyl-2-trinitrophenyl-hydrazine ethanol solution with 0.08-0.12% again, dry, inspect under putting daylight, in test sample chromatogram, on the position corresponding with reference substance chromatogram to control medicinal material chromatogram, the spot of aobvious same color;
(2) TLC distinguish of the Radix Astragali, red ginseng and pseudo-ginseng, step is as follows:
A prepares need testing solution: by WEINAIAN sheet porphyrize, get 1.8-2.2g, add methyl alcohol 48-52ml, ultrasonic process, filter, evaporate to dryness filtrate, the residue 18-22ml that adds water makes dissolving, then extracts 3-4 time with ethyl acetate, each 28-32ml, merge lower layer of water liquid, extract 3-4 time with water-saturated n-butanol jolting, each 28-32ml, merge normal butyl alcohol liquid, with ammonia solution washing 2-3 time, each 28-32ml, discards ammoniacal liquor, by normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 0.45-0.55ml makes dissolving, as need testing solution;
B prepares control medicinal material solution: get Radix Astragali control medicinal material 1.8-2.2g, add methyl alcohol 48-52ml, ultrasonic process, filter, evaporate to dryness filtrate, the residue 18-22ml that adds water makes dissolving, extract 3-4 time with ethyl acetate again, each 28-32ml, merges lower layer of water liquid, extracts 3-4 time with water-saturated n-butanol jolting, each 28-32ml, merge normal butyl alcohol liquid, with ammonia solution washing 2-3 time, each 28-32ml, discard ammoniacal liquor, by normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 0.45-0.55ml makes dissolving, as Radix Astragali control medicinal material solution; Separately get red ginseng control medicinal material 0.45-0.55g and pseudo-ginseng control medicinal material 0.65-0.75g, add water 90-110ml respectively, decoct 55-65min, filter, filtrate is extracted 3-4 time with ethyl acetate, each 28-32ml, discards acetic acid ethyl fluid, then extracts 3-4 time with water-saturated n-butanol jolting, each 28-32ml, merge normal butyl alcohol liquid, rear ammonia solution washing 2-3 time, each 28-32ml, discard ammoniacal liquor, by normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 0.45-0.55ml makes dissolving, respectively obtained red ginseng control medicinal material solution and pseudo-ginseng control medicinal material solution;
C prepares reference substance mixed solution: get Astragaloside IV reference substance, ginsenoside Rg
1reference substance, ginsenoside Rb
1reference substance and Panax Notoginseng saponin R
1reference substance, adds methyl alcohol and makes every 1ml methyl alcohol respectively containing the mixed solution of the above-mentioned reference substance of 0.8-1.2mg, product mixed solution in contrast;
D detects: get reference substance mixed solution each 1.8-2.2 μ l described in need testing solution described in step a and step c, Radix Astragali control medicinal material solution 8-12 μ l described in step b, red ginseng control medicinal material solution 5-7 μ l and pseudo-ginseng control medicinal material solution 0.8-1.2 μ l, put respectively on same silica gel g thin-layer plate, with the upper solution of the normal butyl alcohol-ethyl acetate of volume ratio 3.5-4.5:1:4.5-5.5-rare ammonia for developping agent, be placed in the strong ammonia solution presaturation expansion cylinder of 20 minutes, launch, take out, dry, spray is with 9-11% ethanol solution of sulfuric acid, spot development is heated to clear at 100-110 DEG C, in test sample chromatogram, on the position corresponding with reference substance chromatogram to control medicinal material chromatogram, the spot of aobvious same color, inspect under putting 365nm ultraviolet lamp, the fluorescence spot of aobvious same color,
(3) the high performance liquid chromatography Qualitive test of WEINAIAN sheet, comprises the steps:
A chromatographic condition: take octadecylsilane chemically bonded silica as filling agent; Acetonitrile is mobile phase A, and 0.1% formic acid is Mobile phase B, gradient elution, PDA/ELSD Series detectors; The determined wavelength of described PDA is 260nm; ELSD parameter is: drift tube temperature: 60 DEG C, flow rate of carrier gas: 35psi, yield value: 200; Number of theoretical plate presses Calycosin-7-O-BETA-D-glucoside peak and ginsenoside Rb
1peak calculates all should be not less than 5000; Described gradient elution is: 0-32min, 5% → 18% acetonitrile; 32-38min, 18% → 22% acetonitrile; 38-58min, 22% acetonitrile; 58-70min, 22% → 32% acetonitrile; 70-84min, 32% → 34% acetonitrile; 84-96min, 34% acetonitrile; 96-102min, 34% → 40% acetonitrile; 102-125min, 40% → 67% acetonitrile;
B prepares object of reference solution: get Calycosin-7-O-BETA-D-glucoside and ginsenoside Rb
1reference substance, adds methyl alcohol respectively and makes the ginsenoside Rb that Calycosin-7-O-BETA-D-glucoside methanol solution that concentration is 38-42 μ g/ml and concentration are 0.13-0.17mg/ml
1methanol solution;
C prepares need testing solution: by WEINAIAN sheet porphyrize, get powder 0.8-1.2g, add methyl alcohol 48-52ml, ultrasonic process, put to room temperature, supply the weight of minimizing with methyl alcohol, shake up, filter, get filtrate 23-27ml, evaporate to dryness, residue adds methyl alcohol and dissolves, and with methyl alcohol constant volume in 5ml volumetric flask, to obtain final product;
D detects: detect under chromatographic condition described in step a;
(4) the high performance liquid chromatography quantitative measurement of WEINAIAN sheet, comprises the steps:
A chromatographic condition: take octadecylsilane chemically bonded silica as filling agent; Acetonitrile is mobile phase A, and 0.1% formic acid is Mobile phase B, and gradient elution uses PDA/ELSD Series detectors; The determined wavelength of described PDA is 260nm; ELSD parameter is: drift tube temperature: 60 DEG C, flow rate of carrier gas: 35psi, yield value: 200; Number of theoretical plate presses Calycosin-7-O-BETA-D-glucoside peak and ginsenoside Rb
1peak calculates all should be not less than 5000; Described gradient elution is: 0 ~ 32min:5% → 18% acetonitrile; 32 ~ 38min:18% → 22% acetonitrile; 38 ~ 58min:22% acetonitrile; 58 ~ 70min:22% → 32% acetonitrile; 70 ~ 84min:32% → 34% acetonitrile; 84 ~ 96min:34% acetonitrile; 96 ~ 102min:34% → 40% acetonitrile; 102 ~ 125min:40% → 67% acetonitrile;
B preparation mixing reference substance solution: get Calycosin-7-O-BETA-D-glucoside, ononin, calycosin, onocerin, Panax Notoginseng saponin R
1, ginsenoside Rg
1, ginsenoside Re, ginsenoside Rb
1, cholic acid and hyodesoxycholic acid reference substance, add methyl alcohol and make every 1ml methyl alcohol containing Calycosin-7-O-BETA-D-glucoside 38-42 μ g, ononin 7-9 μ g, calycosin 18-22 μ g, onocerin 5-7 μ g, Panax Notoginseng saponin R
10.13-0.17mg, ginsenoside Rg
10.28-0.32mg, ginsenoside Re 38-42 μ g, ginsenoside Rb
1the mixing reference substance solution of 0.13-0.17mg, cholic acid 0.45-0.55mg, hyodesoxycholic acid 0.35-0.45mg;
C prepares need testing solution: by WEINAIAN sheet porphyrize, get powder 0.8-1.2g, add methyl alcohol 48-52ml, ultrasonic process, put to room temperature, supply the weight of minimizing with methyl alcohol, shake up, filter, get filtrate 23-27ml, evaporate to dryness, residue adds methyl alcohol and dissolves, and with methyl alcohol constant volume in 5ml volumetric flask, to obtain final product;
D detects: under the chromatographic condition described in step a, draws reference substance solution 5 μ l, 20 μ l, need testing solution 20 μ l, injection liquid chromatography, measures, and wherein PDA detects and calculates with one point external standard method, ELSD detects and calculates with external standard two-point method logarithmic equation, to obtain final product.
2. the quality determining method of WEINAIAN sheet according to claim 1, is characterized in that: described detection method also adopts one or two in following discriminating and assay project:
(1) TLC distinguish of calculus bovis factitius:
A prepares need testing solution: by WEINAIAN sheet porphyrize, get 0.18-0.22g, add methyl alcohol 18-22ml, ultrasonic process, and filter, evaporate to dryness filtrate, residue adds methyl alcohol 0.8-1.2ml makes dissolving, as need testing solution;
B prepares control medicinal material solution: get calculus bovis factitius control medicinal material 8-12mg, add methyl alcohol 1.5-2.5ml, ultrasonic process, centrifugal, gets supernatant medicinal material solution in contrast;
C prepares reference substance solution: get cholic acid reference substance, hyodesoxycholic acid reference substance, adds methyl alcohol respectively and makes the solution containing the above-mentioned reference substance of 0.8-1.2mg in every 1ml methyl alcohol, as cholic acid reference substance solution and hyodesoxycholic acid reference substance solution;
D detects: get cholic acid reference substance solution described in control medicinal material solution described in need testing solution described in step a, step b and step c and hyodesoxycholic acid reference substance solution each 0.8-1.2 μ l, put respectively on same silica gel g thin-layer plate, take volume ratio as the upper solution of the cyclohexane-ethyl acetate-methanol-acetic acid of 18-22:23-27:2.5-3.5:1.5-2.5 be developping agent, launch, take out, dry, spray is with 9-11% ethanol solution of sulfuric acid, be heated to spot development at 105 DEG C clear, inspect under putting 365nm ultraviolet lamp; In test sample chromatogram, on the position corresponding with reference substance chromatogram to control medicinal material chromatogram, the fluorescence spot of aobvious same color;
(2) the high performance liquid chromatography quantitative measurement of Astragaloside IV:
A chromatographic condition: take octadecylsilane chemically bonded silica as filling agent; Volume ratio is the acetonitrile-water of 30 ︰ 70 is mobile phase; Evaporative light-scattering detector detects; Number of theoretical plate calculates should be not less than 5000 by Astragaloside IV peak;
The preparation of b reference substance solution: get Astragaloside IV reference substance and add methyl alcohol and make the Astragaloside IV reference substance methanol solution that concentration is 0.45-0.55mg/ml;
The preparation of c need testing solution: by WEINAIAN sheet porphyrize, get powder 1.3-1.7g, add methyl alcohol 48-52ml, ultrasonic process, put to room temperature, supply the weight of minimizing with methyl alcohol, shake up, filter, get filtrate 23-27ml, evaporate to dryness, the residue 18-22ml that adds water makes dissolving, extract 3-4 time with water-saturated n-butanol jolting, each 38-42ml, merges normal butyl alcohol liquid, with ammonia solution washing 2-3 time, each 38-42ml, discard ammoniacal liquor, by normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol and dissolves, with methyl alcohol constant volume in 5ml volumetric flask, to obtain final product;
D detects: draw reference substance solution 5 μ l, 15 μ l, need testing solution 20 μ l, injection liquid chromatography, measure, calculate, to obtain final product with external standard two-point method logarithmic equation.
3. the quality determining method of WEINAIAN sheet according to claim 1 and 2, it is characterized in that, in the high performance liquid chromatography Qualitive test of described WEINAIAN sheet and the high performance liquid chromatography quantitative measurement of WEINAIAN sheet, in chromatographic condition described in step a, flow velocity is 0.8-1.2ml/min; Column temperature is 25-35 DEG C.
4. the quality determining method of WEINAIAN sheet according to claim 1 and 2, is characterized in that, sample size in chromatographic condition described in step a in the high performance liquid chromatography Qualitive test of described WEINAIAN sheet: 20 μ l.
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| CN105403654B (en) * | 2015-12-11 | 2017-04-05 | 陇西保和堂药业有限责任公司 | A kind of method for improving the identification of Radix Astragali thin-layer chromatography |
| CN110057961B (en) * | 2019-05-21 | 2020-05-19 | 国药集团同济堂（贵州）制药有限公司 | Detection method of Xianlinggubao capsule finished product |
| CN110082460B (en) * | 2019-06-03 | 2022-05-20 | 国药集团精方（安徽）药业股份有限公司 | Quality detection method of Jingshu granules |
| CN110286186B (en) * | 2019-06-25 | 2021-06-18 | 厦门中药厂有限公司 | Method for detecting fingerprint of Xinhuang tablets, Xinhuang tablet standard fingerprint and application |
| CN111579686A (en) * | 2020-06-01 | 2020-08-25 | 金华市中心医院 | Method for determining content of astragaloside in ryegrass formula particles by high performance liquid chromatography |
| CN112697949B (en) * | 2020-12-09 | 2022-05-27 | 浙江金城阜通制药有限公司 | Thin-layer identification method for Baoyuan decoction, similar formula extract and preparation thereof |
| CN113567574B (en) * | 2021-07-13 | 2022-06-21 | 数源汇通（北京）医药科技有限公司 | Method for simultaneously determining contents of multiple components in Xiaopi stomach harmonizing capsule |
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