CN102218122B - Detection method for sea dragon and gecko oral liquid - Google Patents
Detection method for sea dragon and gecko oral liquid Download PDFInfo
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- CN102218122B CN102218122B CN2011100920838A CN201110092083A CN102218122B CN 102218122 B CN102218122 B CN 102218122B CN 2011100920838 A CN2011100920838 A CN 2011100920838A CN 201110092083 A CN201110092083 A CN 201110092083A CN 102218122 B CN102218122 B CN 102218122B
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Abstract
The invention discloses a quality control and detection method for a sea dragon and gecko oral liquid. In the quality control and detection method, on the basis of the original detection standard, a thin-layer chromatography detection method for dried tangerine or orange peel and astragalus root in the prescription of the sea dragon and gecko oral liquid is added, a thin-layer chromatography for ginseng and the astragalus root is improved, and a high performance liquid chromatography for measuring the content of epimedim herb of in the prescription is added. The detection project designed by the invention is reasonable; and the detection method provided by the invention has the advantages of advancement and good accuracy and repeatability, is used for carrying out the quality control on the sea dragon and gecko oral liquid, and can carry out the comprehensive quality control on the sea dragon and gecko oral liquid to ensure the stability and uniformity of product quality.
Description
Technical field
The invention discloses a kind of warming kidney to invigorate yang that has, the detection method of the sea otter gekko oral liquid of enrich and benefit essence and blood effect.
Background technology
" sea otter gekko oral liquid " function cures mainly: warming kidney to invigorate yang, enrich and benefit essence and blood.It is aching and limp to be used for the waist foot, and the complexion white light is white, impotence and seminal emission, and the palace is cold infertile, vertigo.
The former examination criteria of " sea otter gekko oral liquid " records in the 13 in Drug Standard of Ministry of Public Health of the Peoples Republic of China Chinese traditional patent formulation preparation.Have only the thin-layer chromatography of the medicinal material genseng and the root of large-flowered skullcap to differentiate that because the test item of primary standard is few, method is single in the primary standard, therefore can not guarantee the quality of this medicine is comprehensively controlled and detected.
Summary of the invention
The invention provides the detection method of sea otter gekko oral liquid; Be used for the quality control of sea otter gekko oral medicine liquid; The test item of the present invention's design is reasonable, and the detection method that provides is advanced, precision and favorable reproducibility; Can comprehensively carry out quality control, guarantee the stable homogeneity of product quality sea otter gekko oral liquid.
The detection method of sea otter gekko oral liquid of the present invention was with sea otter 39.1g, gekko 0.73g, genseng 4.68g, sheep whip 4.68g, ram testes 4.68g, root of large-flowered skullcap 4.68g, prepared rhizome of rehmannia 3.1g, seed of Chinese dodder 3.1g, fleece-flower root 3.1g, glutinous rehmannia 3.1g, dried orange peel 3.1g, Radix Angelicae Sinensis 1.56g, Radix Astragali 4.68g, actinote 1.56g, stamen nelumbini 1.56g, Radix Glycyrrhizae 1.56g, Ligusticum wallichii 1.56g, rhizoma alismatis 1.56g, cynomorium songaricum 1.56g, cardamom 1.56g, agalloch eaglewood 1.56g, pilose antler 1.56g, fruit of Chinese wolfberry 1.56g, saline cistanche 0.78g, sheep oil Herba Epimedii Preparata 1.56g, Chinese cassia tree 1.56g, semen allii tuberosi 1.56g, frutus cnidii 1.56g, Chinese prickly ash 0.31g 29 flavor composition boiling secondaries, each 2 hours; Collecting decoction, being concentrated into relative density at 80 ℃ is 1.04～1.06 clear cream, adds the ethanol qdx; Refrigerate 12 hours, filter, concentrate; Add water, refrigeration filters; Filtrating adds benzoic acid makes dissolving in right amount; Transfer pH value 4.6～4.8 with 8% NaOH, add flavouring xylitol 3%, the adjustment total amount is to 1000ml; Filter; Packing, sterilization promptly gets.The liquid of light brown to rufous; Mildly bitter flavor.
The detection method of sea otter gekko oral liquid of the present invention, 1) get sea otter gekko oral liquid 30ml, the 20ml that adds diethyl ether, jolting is extracted; Place, discard ether solution, water layer extracts 3 times with water saturated normal butyl alcohol, and consumption is followed successively by 20ml, 15ml, 15ml; Merge normal butyl alcohol liquid, add 4% potassium hydroxide solution 30ml washing once, discard washing lotion, use the saturated washing of normal butyl alcohol again to neutral; Discard water liquid, normal butyl alcohol liquid is put evaporate to dryness in the evaporating dish, and residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance, adds methyl alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution; According to the thin-layered chromatography test, draw need testing solution 10 μ l, reference substance solution 5 μ l; Putting respectively on same silica gel g thin-layer plate, is developping agent with 10 ℃ of lower floor's solution with held of methenyl choloride-methanol-water of 65:35:10, launches; Take out; Dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
2) get sea otter gekko oral liquid 40ml, use hcl acidifying, pH value 3～4 adds the ethyl acetate jolting and extracts 3 times, each 20ml, and combined ethyl acetate liquid is put evaporate to dryness in the evaporating dish, and residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the scutelloside reference substance, adds methyl alcohol and processes the solution that every 1ml contains 2mg, as reference substance solution; According to thin-layered chromatography test, draw need testing solution 10 μ l, reference substance solution 5 μ l put respectively on same silica gel g thin-layer plate, are developping agent with butyl acetate-formic acid-water of 1:1:1, launch, and take out, and dry, and spray is with 3% ferric trichloride ethanolic solution.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
3) get sea otter gekko oral liquid 60ml, add the ethyl acetate jolting and extract 3 times, each 40ml, combined ethyl acetate liquid is put evaporate to dryness in the evaporating dish, and residue adds ethyl acetate 0.5ml makes dissolving, as need testing solution; Other gets the aurantiamarin reference substance, adds methyl alcohol and processes the solution that every 1ml contains 0.5mg, as reference substance solution; According to thin-layered chromatography test, draw need testing solution 15 μ l, reference substance solution 10 μ l put respectively on the same silica gel g thin-layer plate that contains 0.5% NaOH; Ethyl acetate-methanol-water with 100:17:13 is a developping agent, launches 5cm, and the upper solution with toluene-ethyl acetate-formic acid-water of 20:10:1:1 is a developping agent again; Launch 13cm, take out, dry; Spray is put under the ultraviolet lamp and is inspected with the aluminium choride test solution, and wavelength is 365nm; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
4) get sea otter gekko oral liquid 60ml, add water saturated normal butyl alcohol jolting and extract 3 times, each 30ml merges normal butyl alcohol liquid; Add 1% sodium hydroxide solution jolting and extract 3 times, each 20ml discards sodium hydroxide solution; N-butanol layer adds the saturated water washing of normal butyl alcohol 3 times, and each 20ml discards water liquid; Normal butyl alcohol liquid is put evaporate to dryness in the evaporating dish, and residue adds methyl alcohol 0.5ml makes dissolving, as need testing solution; Other gets the Astragaloside IV reference substance, adds methyl alcohol and processes the solution that every 1ml contains 0.5mg, as reference substance solution; According to the thin-layered chromatography test, draw need testing solution 10 μ l, reference substance solution 5 μ l; Putting respectively on same silica gel g thin-layer plate, is developping agent with methenyl choloride-ethyl acetate-10 ℃ of lower floor's solution with held of methanol-water of 10:20:11:5, launches; Take out; Dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
5)
Inspection:The pH value is 4.0～5.0, and the relative density Sugarless type should be not less than 1.01; Low-sugar type should be not less than 1.05; Other should meet each item regulation relevant under an appendix I of mixture Chinese Pharmacopoeia version in 2010 the J item;
6) assay: icariin is according to high effective liquid chromatography for measuring
Icariin:According to an appendix VI of Chinese Pharmacopoeia version in 2010 D high effective liquid chromatography for measuring;
Chromatographic condition and system suitability test:Use octadecylsilane chemically bonded silica to be filling agent; Acetonitrile-water with 25:75 is a moving phase; The detection wavelength is 270nm, and column temperature is a room temperature.Number of theoretical plate calculates by the icariin peak should be not less than 1500;
The preparation of reference substance solution: it is an amount of that precision takes by weighing the icariin reference substance, uses dissolve with methanol, and be diluted to scale with methyl alcohol, shakes up, and promptly gets, and the every 1ml of icariin contains 8 μ g;
The preparation of need testing solution: precision is measured sea otter gekko oral liquid 40ml, and the jolting that adds diethyl ether is extracted 3 times, and each 40ml discards ether solution; Water layer adds the water-saturated n-butanol jolting and extracts 4 times, and each 40ml merges normal butyl alcohol liquid; Add the jolting of 0.5mol/L sodium carbonate liquor and extract 3 times, each 40ml discards sodium carbonate liquor; Evaporate to dryness in the normal butyl alcohol stratification evaporating dish, residue add methyl alcohol 25ml makes dissolving, as need testing solution;
Determination method:Accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing inject liquid chromatograph, measure, and calculate, and promptly get.
It is C with the icariin molecular formula that the every 1ml of sea otter gekko oral liquid contains barrenwort
33H
40O
15Meter must not be less than 1.35 μ g.
Good effect of the present invention is: the thin-layered chromatography detection method that on former examination criteria basis, has increased dried orange peel, the Radix Astragali in the sea otter gekko oral liquid prescription; Improve the thin-layered chromatography of the genseng and the root of large-flowered skullcap, increased the high effective liquid chromatography for measuring content of barrenwort in the prescription; The test item of the present invention's design is reasonable; The detection method that provides is advanced, and precision and favorable reproducibility are used for the quality control of sea otter gekko oral medicine liquid; Can comprehensively carry out quality control, guarantee the stable homogeneity of product quality sea otter gekko oral liquid.
The present invention is on the basis of former low sugar oral liquid, and innovation has increased the Sugarless type oral liquid, makes the suitable person of a lot of diabetes can take this oral liquid, has increased the indication crowd.
Description of drawings
Fig. 1: genseng thin-layer chromatogram
1, ginsenoside reference substance Rg1 2,3,4, three lot sample article; 5, negative control
Fig. 2: root of large-flowered skullcap thin-layer chromatogram
1, scutelloside; 2,3,4, three lot sample article; 5, negative control
Fig. 3: dried orange peel thin-layer chromatogram
1, aurantiamarin; 2; 3; 4, three lot sample article; 5, negative control
Fig. 4: Radix Astragali thin-layer chromatogram
1, Astragaloside IV; 2,3,4, three lot sample article; 5, negative control
Fig. 5: icariin reference substance uv absorption figure
Fig. 6: icariin reference substance canonical plotting
Fig. 7: icariin reference substance HPLC chromatogram
Fig. 8: the HPLC chromatogram of need testing solution
Fig. 9: the HPLC chromatogram of negative controls
Embodiment
For the ease of understanding the present invention, special case is lifted following examples.Its effect is understood that it is to explaination of the present invention but not to any type of restriction of the present invention.
Get sea otter 39.1g, gekko 0.73g, genseng 4.68g, sheep whip 4.68g, ram testes 4.68g, root of large-flowered skullcap 4.68g, prepared rhizome of rehmannia 3.1g, seed of Chinese dodder 3.1g, fleece-flower root 3.1g, glutinous rehmannia 3.1g, dried orange peel 3.1g, Radix Angelicae Sinensis 1.56g, Radix Astragali 4.68g, actinote 1.56g, stamen nelumbini 1.56g, Radix Glycyrrhizae 1.56g, Ligusticum wallichii 1.56g, rhizoma alismatis 1.56g, cynomorium songaricum 1.56g, cardamom 1.56g, agalloch eaglewood 1.56g, pilose antler 1.56g, fruit of Chinese wolfberry 1.56g, saline cistanche 0.78g, barrenwort (sheep oil is processed) 1.56g, Chinese cassia tree 1.56g, semen allii tuberosi 1.56g, frutus cnidii 1.56g, Chinese prickly ash 0.31g 29 flavor composition boiling secondaries, each 2 hours, collecting decoction; Being concentrated into relative density at 80 ℃ is 1.04～1.06 clear cream, adds the ethanol qdx, refrigerates 12 hours; Filter, concentrate, add water; Refrigeration filters, and filtrating adds benzoic acid makes dissolving in right amount; Transfer pH value 4.6～4.8 with 8% NaOH, add flavouring xylitol 3%, the adjustment total amount is to 1000ml; Filter; Packing, sterilization promptly gets.Its detection method comprises step:
1) get sea otter gekko oral liquid 30ml, the 20ml that adds diethyl ether, jolting is extracted, and places; Discard ether solution, water layer extracts 3 times with water saturated normal butyl alcohol, and consumption is followed successively by 20ml, 15ml, 15ml, merges normal butyl alcohol liquid; Add 4% potassium hydroxide solution 30ml washing once, discard washing lotion, use the saturated washing of normal butyl alcohol again, discard water liquid to neutral; Normal butyl alcohol liquid is put evaporate to dryness in the evaporating dish, and residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Other gets ginsenoside Rg1's reference substance, adds methyl alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution.According to the thin-layered chromatography test, draw need testing solution 10 μ l, reference substance solution 5 μ l; Putting respectively on same silica gel g thin-layer plate, is developping agent with 10 ℃ of lower floor's solution with held of methenyl choloride-methanol-water of 65:35:10, launches; Take out; Dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 105 ℃.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color, see accompanying drawing 1;
2) get sea otter gekko oral liquid 40ml, use hcl acidifying, pH value 3～4 adds the ethyl acetate jolting and extracts 3 times, each 20ml, and combined ethyl acetate liquid is put evaporate to dryness in the evaporating dish, and residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Other gets the scutelloside reference substance, adds methyl alcohol and processes the solution that every 1ml contains 2mg, as reference substance solution.According to thin-layered chromatography test, draw need testing solution 10 μ l, reference substance solution 5 μ l put respectively on same silica gel g thin-layer plate, are developping agent with butyl acetate-formic acid-water of 1:1:1, launch, and take out, and dry, and spray is with 3% ferric trichloride ethanolic solution.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color, see accompanying drawing 2;
3) get sea otter gekko oral liquid 60ml, add the ethyl acetate jolting and extract 3 times, each 40ml, combined ethyl acetate liquid is put evaporate to dryness in the evaporating dish, and residue adds ethyl acetate 0.5ml makes dissolving, as need testing solution.Other gets the aurantiamarin reference substance, adds methyl alcohol and processes the solution that every 1ml contains 0.5mg, as reference substance solution.According to thin-layered chromatography test, draw need testing solution 15 μ l, reference substance solution 10 μ l put respectively on the same silica gel g thin-layer plate that contains 0.5% NaOH; Ethyl acetate-methanol-water with 100:17:13 is a developping agent, launches 5cm, and the upper solution with toluene-ethyl acetate-formic acid-water of 20:10:1:1 is a developping agent again; Launch 13cm, take out, dry; Spray is put under the ultraviolet lamp and is inspected with the aluminium choride test solution, and wavelength is 365nm.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color, see accompanying drawing 3;
4) get sea otter gekko oral liquid 60ml, add water saturated normal butyl alcohol jolting and extract 3 times, each 30ml merges normal butyl alcohol liquid; Add 1% sodium hydroxide solution jolting and extract 3 times, each 20ml discards sodium hydroxide solution; N-butanol layer adds the saturated water washing of normal butyl alcohol 3 times, and each 20ml discards water liquid; Normal butyl alcohol liquid is put evaporate to dryness in the evaporating dish, and residue adds methyl alcohol 0.5ml makes dissolving, as need testing solution.Other gets the Astragaloside IV reference substance, adds methyl alcohol and processes the solution that every 1ml contains 0.5mg, as reference substance solution.According to the thin-layered chromatography test, draw need testing solution 10 μ l, reference substance solution 5 μ l; Putting respectively on same silica gel g thin-layer plate, is developping agent with methenyl choloride-ethyl acetate-10 ℃ of lower floor's solution with held of methanol-water of 10:20:11:5, launches; Take out; Dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to spot colour developing at 105 ℃.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color, see accompanying drawing 4;
5)
Inspection:The pH value is 4.0～5.0, and the relative density Sugarless type should be not less than 1.01; Low-sugar type should be not less than 1.05; Other should meet each item regulation relevant under an appendix I of mixture Chinese Pharmacopoeia version in 2010 the J item;
6)
Assay: according to high effective liquid chromatography for measuring;
Icariin:According to a record of Chinese Pharmacopoeia version in 2005 VI D high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test: use octadecylsilane chemically bonded silica to be filling agent; Acetonitrile-water with 25:75 is a moving phase; The detection wavelength is 270nm, and column temperature is a room temperature.Number of theoretical plate calculates by the icariin peak should be not less than 1500;
The preparation of reference substance solution:It is an amount of that precision takes by weighing the icariin reference substance, uses dissolve with methanol, and be diluted to scale with methyl alcohol, shakes up, and promptly gets, and the every 1ml of icariin contains 8 μ g;
The preparation of need testing solution:Precision is measured sea otter gekko oral liquid 40ml, and the jolting that adds diethyl ether is extracted 3 times, and each 40ml discards ether solution; Water layer adds the water-saturated n-butanol jolting and extracts 4 times, and each 40ml merges normal butyl alcohol liquid; Add the jolting of 0.5mol/L sodium carbonate liquor and extract 3 times, each 40ml discards sodium carbonate liquor; Evaporate to dryness in the normal butyl alcohol stratification evaporating dish, residue add methyl alcohol 25ml makes dissolving, as need testing solution;
Determination method:Accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing inject liquid chromatograph, measure, and calculate, and promptly get.
The every 1ml of sea otter gekko oral liquid contains barrenwort with icariin (C
33H
40O
15) meter, must not be less than 1.35 μ g.
High-performance liquid chromatogram determination icariin content method standard is confirmed explanation:
One, instrument, reagent and reagent: instrument: high performance liquid chromatograph (TSP, the U.S.); The UV100 UV-detector; Reagent: the icariin reference substance is purchased in Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 110737-200413; Specification: supply assay to use.Reagent: acetonitrile is a chromatographically pure; Water is double distilled water.
Two, the preparation of reference substance solution: it is an amount of that precision takes by weighing the icariin reference substance, uses dissolve with methanol, and be diluted to scale with methyl alcohol, shakes up, and promptly gets, and the every 1ml of icariin contains 8 μ g;
Three, the preparation of need testing solution and mensuration: precision is measured sea otter gekko oral liquid 40ml, and the jolting that adds diethyl ether is extracted 3 times, and each 40ml discards ether solution; Water layer adds the water-saturated n-butanol jolting and extracts 4 times, and each 40ml merges normal butyl alcohol liquid; Add the jolting of 0.5mol/L sodium carbonate liquor and extract 3 times, each 40ml discards sodium carbonate liquor; Evaporate to dryness in the normal butyl alcohol stratification evaporating dish, residue add methyl alcohol 25ml makes dissolving, as need testing solution;
Four, confirming of maximum absorption wavelength: the maximum absorption wavelength that records icariin standard items (methanol constant volume) with 762 type ultraviolet spectrophotometers is 269nm; Close with the wavelength of assay under 2010 editions epimedium herb items of Chinese Pharmacopoeia; Therefore confirm that detecting wavelength is 270nm, sees accompanying drawing 5.
Five, moving phase is selected: acetonitrile-water (25:75) is as this preparation barrenwort assay moving phase.
Six, chromatographic condition: use octadecylsilane chemically bonded silica to be filling agent; With acetonitrile-water (25:75) is moving phase; The detection wavelength is 270nm; 40 ℃ of column temperatures; Flow velocity 1ml/min.Seven, the calculating of number of theoretical plate: measure according to aforementioned chromatographic condition, measure the result and count: N=5.54 with the icariin peak * (46.3/1.4)
2=6046, so confirm to be no less than 1500 with icariin peak theory of computation plate number.
Eight, methodological study
1, the preparation of typical curve
It is an amount of that precision takes by weighing the icariin reference substance, adds methyl alcohol and process the reference substance solution that every 1ml contains 37.00,18.50,9.25,4.63,2.31,1.16 μ g respectively, draws 20 μ l respectively; Injecting liquid chromatograph, measure by above-mentioned chromatographic condition, is ordinate with the peak area score value; Icariin concentration is horizontal ordinate, and the drawing standard curve gets a straight line; Regression equation Y=74988x+22920, r=1.0, the result shows; Icariin has good linear relationship in 0.0232 μ g～0.74 μ g scope, see accompanying drawing 6.
2, blank test
According to prescription ratio and sea otter gekko oral liquid preparation technology, process the blank sample that does not contain barrenwort, the preparation method by test liquid processes blank solution again, measures in accordance with the law, and the result shows that blank is noiseless to sample determination, sees accompanying drawing 7,8,9.
3, precision test
3.1 replica test
To same test sample replication 6 times, calculate relative standard deviation, icariin RSD is 1.73% as a result.Show that this method repeatability is good, the result sees table 1.
3.2 reappearance test
Different experiments personnel, different experiments chamber, different experiments instrument to same test sample replication 6 times, calculate relative standard deviation, and icariin RSD is 2.84% as a result.Show that this method repeatability is good, the result sees table 2.
4, stability test
Get same need testing solution, measure at 0,2,4,6,8,10,12 hour sample introduction respectively, results sample is stable in 12 hours, and icariin relative standard deviation RSD is 2.27%.Show that this method has good stability, the result sees table 3.
5, recovery test
System adopts the application of sample recovery test, gets the sample 20ml that predicts content, and accurate the title decides; The accurate icariin reference substance that adds is an amount of, extracts, measures according to the test sample preparation method, calculates; The icariin average recovery rate is 99.07% as a result, n=6, RSD=2.31%.Show that this law method is accurate, the result sees table 4.
÷ B * 100% A: the contained tested component content B of sample: add pure article amount C: measured value of the recovery=(C-A)
Nine, sample determination
1, five lot sample article assays
5 lot sample article are measured, the result sees table 5 in accordance with the law.According to measuring the result, icariin content is that every ml is not less than 1.35 μ g in the tentative preparation.Limit is formulated foundation: 5 batches of average contents are 2.25 μ g/ml, float downward 40%, i.e. 1.35 μ g/ml.
2, the medicinal material rate of transform
2.1 " 2005 editions epimedium herb assay methods of Chinese pharmacopoeia
Icariin is measured according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D).
Chromatographic condition and system suitability test use octadecylsilane chemically bonded silica to be filling agent; Acetonitrile-water (25:75) is a moving phase; The detection wavelength is 270nm.Number of theoretical plate calculates by the icariin peak should be not less than 1500.
The preparation of reference substance solution: it is an amount of that precision takes by weighing the icariin reference substance, adds methyl alcohol and process the solution that every 1ml contains 45 μ g, promptly gets.
The preparation of need testing solution: get barrenwort blade medicinal powder (crossing sieve No. three) 0.2g, the accurate title, decide, and puts in the tool plug conical flask; The accurate Diluted Alcohol 20ml that adds claims to decide weight, sonicated 1 hour; Claim again to decide weight, supply the weight that subtracts mistake, shake up with Diluted Alcohol; Filter, get subsequent filtrate, promptly get.
Determination method: accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing, inject liquid chromatograph, measure, promptly get.
Sea otter gekko oral liquid blade is pressed dry product and is calculated, and contains icariin (C
33H
40O
15) meter, must not be less than 0.50%, the result sees table 6.
2.2 epimedium herb preparation content determining method
Icariin: measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D).
Chromatographic condition and system suitability test: use octadecylsilane chemically bonded silica to be filling agent; Acetonitrile-water (25:75) is a moving phase; The detection wavelength is 270nm.Number of theoretical plate calculates by the icariin peak should be not less than 1500.
The preparation of reference substance solution: it is an amount of that precision takes by weighing the icariin reference substance, adds methyl alcohol and process the solution that every 1ml contains 0.18mg, promptly gets.
The preparation of need testing solution: get barrenwort blade medicinal powder (cross No. three sieve) 1.0g, accurate claim fixed, according to preparation process with the medicinal material poach after, filter; The filtrating jolting that adds diethyl ether is extracted 3 times, and each 40ml discards ether solution; Water layer adds the water-saturated n-butanol jolting and extracts 4 times, and each 40ml merges normal butyl alcohol liquid; Add the jolting of 0.5mol/L sodium carbonate liquor and extract 3 times, each 40ml discards sodium carbonate liquor; Evaporate to dryness in the normal butyl alcohol stratification evaporating dish, residue add methyl alcohol 25ml makes dissolving, as need testing solution.
Determination method: accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing, inject liquid chromatograph, measure, promptly get, the result sees table 7.
2.3 the medicinal material rate of transform
Because sea otter gekko oral liquid is an oral liquid; Be not suitable for measuring medicinal material content, therefore measure, calculate its rate of transform average out to 55.02% then with the method for preparation with official method; Three batches of lot numbers of three batches of corresponding respectively preparations of medicinal material are 0603001,0603002,0603003, and the result sees table 8.
Claims (1)
1. the detection method of a sea otter gekko oral liquid was with sea otter 39.1g, gekko 0.73g, genseng 4.68g, sheep whip 4.68g, ram testes 4.68g, root of large-flowered skullcap 4.68g, prepared rhizome of rehmannia 3.1g, seed of Chinese dodder 3.1g, fleece-flower root 3.1g, glutinous rehmannia 3.1g, dried orange peel 3.1g, Radix Angelicae Sinensis 1.56g, Radix Astragali 4.68g, actinote 1.56g, stamen nelumbini 1.56g, Radix Glycyrrhizae 1.56g, Ligusticum wallichii 1.56g, rhizoma alismatis 1.56g, cynomorium songaricum 1.56g, cardamom 1.56g, agalloch eaglewood 1.56g, pilose antler 1.56g, fruit of Chinese wolfberry 1.56g, saline cistanche 0.78g, sheep oil Herba Epimedii Preparata 1.56g, Chinese cassia tree 1.56g, semen allii tuberosi 1.56g, frutus cnidii 1.56g, Chinese prickly ash 0.31g boiling secondary, each 2 hours; Collecting decoction, being concentrated into relative density at 80 ℃ is 1.04～1.06 clear cream, adds the ethanol qdx; Refrigerate 12 hours, filter, concentrate; Add water, refrigeration filters; Filtrating adds benzoic acid makes dissolving in right amount, with 8% NaOH adjust pH 4.6～4.8, adds flavouring xylitol 3%; The adjustment total amount is to 1000ml; Filter packing, sterilization; Promptly get, its characteristic may further comprise the steps:
1) get sea otter gekko oral liquid 30ml, the 20ml that adds diethyl ether, jolting is extracted, and places; Discard ether solution, water layer extracts 3 times with water saturated normal butyl alcohol, and consumption is followed successively by 20ml, 15ml, 15ml, merges normal butyl alcohol liquid; Add 4% potassium hydroxide solution 30ml washing once, discard washing lotion, use the saturated washing of normal butyl alcohol again, discard water liquid to neutral; Normal butyl alcohol liquid is put evaporate to dryness in the evaporating dish, and residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance, adds methyl alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution; According to the thin-layered chromatography test, draw need testing solution 10 μ l, reference substance solution 5 μ l; Putting respectively on same silica gel g thin-layer plate, is developping agent with 10 ℃ of lower floor's solution with held of methenyl choloride-methanol-water of 65:35:10, launches; Take out; Dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
2) get sea otter gekko oral liquid 40ml, use hcl acidifying, pH value 3～4 adds the ethyl acetate jolting and extracts 3 times, each 20ml, and combined ethyl acetate liquid is put evaporate to dryness in the evaporating dish, and residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the scutelloside reference substance, adds methyl alcohol and processes the solution that every 1ml contains 2mg, as reference substance solution; According to thin-layered chromatography test, draw need testing solution 10 μ l, reference substance solution 5 μ l put respectively on same silica gel g thin-layer plate, are developping agent with butyl acetate-formic acid-water of 1:1:1, launch, and take out, and dry, and spray is with 3% ferric trichloride ethanolic solution; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
3) get sea otter gekko oral liquid 60ml, add the ethyl acetate jolting and extract 3 times, each 40ml, combined ethyl acetate liquid is put evaporate to dryness in the evaporating dish, and residue adds ethyl acetate 0.5ml makes dissolving, as need testing solution; Other gets the aurantiamarin reference substance, adds methyl alcohol and processes the solution that every 1ml contains 0.5mg, as reference substance solution; According to thin-layered chromatography test, draw need testing solution 15 μ l, reference substance solution 10 μ l put respectively on the same silica gel g thin-layer plate that contains 0.5% NaOH; Ethyl acetate-methanol-water with 100:17:13 is a developping agent, launches 5cm, and the upper solution with toluene-ethyl acetate-formic acid-water of 20:10:1:1 is a developping agent again; Launch 13cm, take out, dry; Spray is put under the ultraviolet lamp and is inspected with the aluminium choride test solution, and wavelength is 365nm; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
4) get sea otter gekko oral liquid 60ml, add water saturated normal butyl alcohol jolting and extract 3 times, each 30ml merges normal butyl alcohol liquid; Add 1% sodium hydroxide solution jolting and extract 3 times, each 20ml discards sodium hydroxide solution; N-butanol layer adds the saturated water washing of normal butyl alcohol 3 times, and each 20ml discards water liquid; Normal butyl alcohol liquid is put evaporate to dryness in the evaporating dish, and residue adds methyl alcohol 0.5ml makes dissolving, as need testing solution; Other gets the Astragaloside IV reference substance, adds methyl alcohol and processes the solution that every 1ml contains 0.5mg, as reference substance solution; According to the thin-layered chromatography test, draw need testing solution 10 μ l, reference substance solution 5 μ l; Putting respectively on same silica gel g thin-layer plate, is developping agent with methenyl choloride-ethyl acetate-10 ℃ of lower floor's solution with held of methanol-water of 10:20:11:5, launches; Take out; Dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
5)
Inspection: the pH value is 4.0～5.0, and the relative density Sugarless type is not less than 1.01, and low-sugar type is not less than 1.05; Other should meet each item regulation relevant under an appendix I of mixture Chinese Pharmacopoeia version in 2010 the J item;
6)
Assay: icariin is according to high effective liquid chromatography for measuring
Icariin:According to an appendix VI of Chinese Pharmacopoeia version in 2010 D high effective liquid chromatography for measuring;
Chromatographic condition and system suitability test: use octadecylsilane chemically bonded silica to be filling agent; Acetonitrile-water with 25:75 is a moving phase; The detection wavelength is 270nm, and column temperature is a room temperature; Number of theoretical plate calculates by the icariin peak should be not less than 1500;
The preparation of reference substance solution: it is an amount of that precision takes by weighing the icariin reference substance, uses dissolve with methanol, and be diluted to scale with methyl alcohol, shakes up, and promptly gets, and the every 1ml of icariin contains 8 μ g;
The preparation of need testing solution:Precision is measured sea otter gekko oral liquid 40ml, and the jolting that adds diethyl ether is extracted 3 times, and each 40ml discards ether solution; Water layer adds the water-saturated n-butanol jolting and extracts 4 times, and each 40ml merges normal butyl alcohol liquid; Add the jolting of 0.5mol/L sodium carbonate liquor and extract 3 times, each 40ml discards sodium carbonate liquor; Evaporate to dryness in the normal butyl alcohol stratification evaporating dish, residue add methyl alcohol 25ml makes dissolving, as need testing solution;
Determination method: accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing, inject liquid chromatograph, measure, calculate, promptly get.
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