CN101879198B - Method for identification of Xindakang preparation - Google Patents
Method for identification of Xindakang preparation Download PDFInfo
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- CN101879198B CN101879198B CN201010145817XA CN201010145817A CN101879198B CN 101879198 B CN101879198 B CN 101879198B CN 201010145817X A CN201010145817X A CN 201010145817XA CN 201010145817 A CN201010145817 A CN 201010145817A CN 101879198 B CN101879198 B CN 101879198B
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Abstract
The invention is the divisional application of Patent Application No. 200610022691.0 and relates to a method for the identification of Xindakang preparation, which comprises the step of thin layer chromatography identification for quercet and isorhamnetin in the preparation. Compared with the prior art, the thin layer chromatography identification for quercet and isorhamnetin is added in the method according to the invention, and the method adopted has the characteristics of strong specificity, high precision, good reproducibility and high recovery rate, and enhances quality standards of original Xindakang preparation so as to ensure clinical therapeutic effect of the preparation.
Description
The original bill application number of this division application is 200610022691.0, and the applying date is December in 2006 29 days, and denomination of invention is " method of quality control of Xindakang preparation ".
Technical field
The present invention relates to a kind of discrimination method of Xindakang preparation, belong to the technical field of medicine being carried out quality control.
Background technology
Xindakang preparation is that the effective site Fructus Hippophae total flavones and the adjuvant that from plant Fructus Hippophae (Hippophae L.), extract are prepared from.Fructus Hippophae total flavones is used to treat cardiovascular and cerebrovascular disease and has obtained universally acknowledgedly, and modern pharmacology research shows with clinical practice: Fructus Hippophae total flavones has coronary artery dilator, increase heart and brain tissues amount of blood supply; Reduce cardiac load; Anticoagulant, microcirculation improvement prevents thrombosis and atherosclerotic generation; Suppress lipid peroxidation, effects such as slow down aging." XINDAKANG PIAN " wherein and " Flavonihippophae capsule " are published respectively on the 14 in Chinese ministry standard Chinese traditional patent formulation preparation and new drug are become a full member 33 of standards; And use for many years clinically, obtaining than satisfactory therapeutic effects aspect the treatment cardiovascular and cerebrovascular disease.But find that after deliberation existing Xindakang preparation exists the irrational shortcoming of method of quality control, like the XINDAKANG PIAN on the present market; Comprise coated tablet and Film coated tablets; Because its method for making, prescription are in fact the same, just coating is different, and (one is WS3-B-2666-97 and carry out two standards; One is WS3 one B one 2666-2002), wasted standard resource; In version Chinese Pharmacopoeia appendix XVG reference substance in 2005, control medicinal material, reference extract, do not recorded in addition in the existing standard still at the standard substance isorhamnetin glucoside that uses, this brings certain degree of difficulty for quality control of product; Existing Xindakang preparation exists quality control standard simple; The uppity shortcoming of product quality; Be embodied in: be the general name of flavone material because of Fructus Hippophae flavone; And its discriminating and content control are all weighed with single isorhamnetin, so can not effectively control the quality of said preparation, thus its clinical efficacy influenced.
Summary of the invention
The object of the invention provides a kind of discrimination method of Xindakang preparation.It is simple to the present invention is directed to original Xindakang preparation quality control standard; The uppity shortcoming of product quality; Method of quality control to this preparation is studied, and has worked out the thin layer chromatography discriminating of Quercetin, isorhamnetin in the preparation, has improved the quality control standard of Xindakang preparation; Control method in this quality standard can be controlled the quality of Xindakang preparation effectively, thereby has guaranteed the clinical efficacy of said preparation.
Xindakang preparation of the present invention is to be prepared from Fructus Hippophae total flavones (Fructus Hippophae flavone) and adjuvant.Its method for making be Fructus Hippophae through extract Fructus Hippophae flavone; Get Fructus Hippophae flavone 50g (containing total flavones) in isorhamnetin 5g; Be ground into fine powder, add appropriate amount of auxiliary materials and process the different preparations that comprise tablet (comprising buccal tablet, dispersible tablet, chewable tablet), capsule, soft capsule, drop pill according to conventional method.
The discrimination method of this Xindakang preparation, said preparation comprises tablet, capsule, soft capsule, drop pill, said discriminating comprises that with Quercetin reference substance, isorhamnetin reference substance be the thin layer chromatography discriminating that flavone aglycone in the preparation is differentiated in contrast.The discrimination method of Quercetin, isorhamnetin is to be contrast with Quercetin reference substance and isorhamnetin reference substance respectively, and with toluene: ethyl acetate: formic acid=2～10: 1～8: 0.1～1.5 solution is the thin layer chromatography of developing solvent.
Said discrimination method comprises following:
Get this preparation or its content, porphyrize adds ethanol and makes dissolving, filters, and filtrating is as need testing solution; Other gets Quercetin and isorhamnetin reference substance, adds ethanol and processes mixed solution, as reference substance solution; According to the test of Chinese Pharmacopoeia thin layer chromatography, draw above-mentioned need testing solution, reference substance solution, put respectively on same silica gel g thin-layer plate; With toluene: ethyl acetate: formic acid=2～10: 1～8: 0.1～1.5 is developing solvent; Launch, take out, dry; Spray is put under the ultra-violet lamp and is inspected with 1～5% aluminum chloride alcoholic solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
Discrimination method comprises following more specifically:
Get this preparation or its content, porphyrize is got fine powder 2～20mg, adds dehydrated alcohol 2～20ml, and supersound process 1～15 minute filters, and filtrating is as need testing solution; Other gets Quercetin and isorhamnetin reference substance, adds dehydrated alcohol and processes the mixed solution that every 1ml contains 1mg～10mg, as reference substance solution; According to an appendix VIB of Chinese Pharmacopoeia thin layer chromatography test, draw each 1～20ul of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose; With toluene: ethyl acetate: formic acid=2～10: 1～8: 0.1～1.5 is developing solvent; Launch, take out, dry; Spray is put under the 365nm ultra-violet lamp and is inspected with 1～5% aluminum chloride alcoholic solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
The assay of this Xindakang preparation comprises that total flavones is measured in the preparation.
The assay of this Xindakang preparation; Also comprise mensuration to Quercetin, isorhamnetin, kaempferol total amount in the preparation; This assay method is to be contrast with isorhamnetin, Quercetin, kaempferol reference substance, and with methanol: 0.4% phosphoric acid=30～60: 30～60 is the HPLC of mobile phase.
The applicant finds after deliberation, adopts the following method of quality control quality of this preparation of control more easily, more helps guaranteeing the clinical efficacy of preparation:
Character:
For tablet: content be pale brown color to brown, mildly bitter flavor;
For capsule: content is fawn to dun granule and a powder; Feeble QI, mildly bitter flavor;
For soft capsule: content is the yellow oily liquid that contains a small amount of suspended solid extractum; Feeble QI, mildly bitter flavor;
For drop pill: these article are brown drop pill; Feeble QI, mildly bitter flavor;
Differentiate:
Get 1 in this preparation or 1, porphyrize adds ethanol 5ml or 10ml, and supersound process 5 minutes is processed the solution of 1mg/ml, filters, and filtrating is as need testing solution; Other gets Quercetin and isorhamnetin reference substance, adds ethanol and processes the mixed solution that every 1ml contains 1mg, as reference substance solution; According to an appendix VIB of Chinese Pharmacopoeia thin layer chromatography test, draw each 1～2ul of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose; With toluene: ethyl acetate: formic acid=5: 4: 0.5 is developing solvent; Launch, take out, dry; Spray is put under the 365nm ultra-violet lamp and is inspected with 1～3% aluminum chloride alcoholic solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
Inspection:
Should meet in appendix of Chinese Pharmacopoeia each pertinent regulations under tablet, capsule, soft capsule or the drop pill item.
Assay
(1) total flavones
The preparation of reference substance solution: precision takes by weighing and adopts P
2O
5Dry isorhamnetin reference substance more than 12 hours adds dehydrated alcohol and processes the solution that every 1ml contains isorhamnetin 20ug, promptly gets;
The preparation of standard curve: precision is measured reference substance solution 1,2,3,4,5,6ml, puts respectively in the 10ml measuring bottle, adds 1～3% aluminum chloride ethanol solution 1.0ml; Be diluted to scale with dehydrated alcohol, shake up, placed 10 minutes; With the reagent corresponding is blank, according to ultraviolet visible spectrophotometry (appendix VA of Chinese Pharmacopoeia), measures absorbance in the wavelength of 430nm; With the absorbance is vertical coordinate, and concentration is abscissa, the drawing standard curve;
Algoscopy: this preparation is got 20 in tablet, is got 10～20 seed lac wafers, drop pill content, and accurate the title decides, and the porphyrize mixing is got about 0.1g, and accurate title is fixed; Put in the tool plug conical flask, it is an amount of to add dehydrated alcohol, and reflux is 15 minutes in 80 ℃ of water-baths, put cold, in the dislocation 100ml measuring bottle; Be diluted to scale with dehydrated alcohol, shake up, filter, discard filtrating just, precision is measured subsequent filtrate 10ml; Put in the 25ml measuring bottle, be diluted to scale with dehydrated alcohol, shake up, precision is measured 3ml, puts in the 10ml measuring bottle; Add 1～% aluminum chloride ethanol solution 1.0ml, be diluted to scale, shake up, as need testing solution with dehydrated alcohol; Other gets the same amount need testing solution that does not add developer and does blank; Prepare the method under the item according to above-mentioned standard curve, from " placing 10 minutes ", according to ultraviolet visible spectrophotometry (appendix VA of Chinese Pharmacopoeia); Wavelength at 430nm is measured absorbance; From standard curve, read the weight (ug) of isorhamnetin in the test sample, calculate, promptly get;
When this preparation contains total flavones (in isorhamnetin) and is 5mg, contain Fructus Hippophae flavone with isorhamnetin (C
16H
12O
7) meter, every in tablet should be labelled amount 90.0～110.0%, capsule and every of soft capsule should be labelled amount 90.0～110.0%, the every ball of drop pill should be 90.0～110.0% of labelled amount.
When this preparation contains total flavones (in isorhamnetin) and is 10mg, contain Fructus Hippophae flavone with isorhamnetin (C
16H
12O
7) meter, every in tablet should be labelled amount 90.0～110.0%, capsule and every of soft capsule should be labelled amount 90.0～110.0%, the every ball of drop pill should be labelled amount 90.0～110.0%,
(2) Quercetin, isorhamnetin, kaempferol adopt high effective liquid chromatography for measuring: chromatographic column is the C18 post; With methanol: 0.4% phosphoric acid=50: 50 is mobile phase; The detection wavelength is 370nm; Theoretical cam curve is calculated by the isorhamnetin peak should be not less than 3000; Precision takes by weighing with P
2O
5For desiccant drying under reduced pressure isorhamnetin more than 12 hours, kaempferol, 4 hours Quercetin reference substance of 120 ℃ of dryings an amount of; Add methanol and process the mixed solution that every 1ml contains isorhamnetin 20ug, kaempferol 2ug, Quercetin 25ug respectively, promptly get reference substance solution; This preparation is got 20 in tablet and is removed coating, gets 10 seed lac wafers, drop pill content, accurate claims surely, and the porphyrize mixing is got and is equivalent to total flavones 10mg, accurate claim fixed; Put in the tool plug conical flask, the accurate methanol 25ml that adds claims to decide weight, supersound process (power 135W, frequency 59kHz) 30 minutes; Put coldly, claim again decide weight, supply the weight that subtracts mistake, shake up filtration with methanol; Discard filtrating just, precision is measured subsequent filtrate 5ml, puts in the tool plug conical flask, adds methanol 15ml, hydrochloric acid (1 → 2) 5ml; Put in the water-bath reflux 30 minutes, cooling is transferred in the 50ml measuring bottle rapidly, is diluted to scale with methanol; Shake up, filter, get subsequent filtrate, promptly get need testing solution with the 0.45um microporous filter membrane; Accurate respectively reference substance solution and each 10ul of need testing solution of drawing injects chromatograph of liquid, measures, and promptly gets;
When this preparation contained total flavones (in isorhamnetin) and is 5mg, each dosage form contained Quercetin (C
15H
10O
7), isorhamnetin (C
16H
12O
7), kaempferol (C
15H
10O
6) total amount: every in tablet should be no less than labelled amount 80%, capsule and every every of soft capsule should be no less than labelled amount 80%, the every ball of drop pill should be no less than 80% of labelled amount.
When this preparation contained total flavones (in isorhamnetin) and is 10mg, each dosage form contained Quercetin (C
15H
10O
7), isorhamnetin (C
16H
12O
7), kaempferol (C
15H
10O
6) total amount: every in tablet should be no less than labelled amount 80%, capsule and every every of soft capsule should be no less than labelled amount 80%, the every ball of drop pill should be no less than 80% of labelled amount.
This method of quality control is the improvement that the method for quality control that has the Xindakang preparation product now is carried out, and it is improved one's methods and comprises: will have two kinds of product standards of XINDAKANG PIAN coated tablet and Film coated tablets now and merge; Being combined the product standard character adjusts; The chemical reaction colour developing of a former total flavones of differentiating is differentiated that the specificity that deletion replaces Quercetin in the Fructus Hippophae flavone, isorhamnetin differentiates; The reference substance isorhamnetin adopts P by the pharmacopeia new demand in the assay item
2O
5Seasoning; Increase with high effective liquid chromatography for measuring Quercetin, isorhamnetin, kaempferol total content.
Because the present invention compared with prior art; The total content of Quercetin, isorhamnetin, kaempferol in the high effective liquid chromatography for measuring preparation is differentiated and adopted to the thin layer chromatography that has increased Quercetin, isorhamnetin; The being specific to property of method that is adopted is strong, precision is high, favorable reproducibility, and the response rate is high; Improve the quality control standard of former Xindakang preparation, thereby guaranteed the quality and the clinical efficacy of said preparation.
In order to ensure method of quality control science of the present invention, reasonable, feasible, the applicant studies discriminating in the method and content assaying method, and concrete testing data is following:
[discriminating] adopts Quercetin and isorhamnetin to compare the discriminating Xindakang preparation through test, and its feminine gender is noiseless, clear spot.The point sample amount adopts 10ul, and speckle is hangover a bit, and adjustment point sample amount is 1～2ul.
Adopt the silica gel g thin-layer plate of different manufacturers, launch, all can obtain satisfied separating resulting with above-mentioned thin layer chromatography condition.
[inspection] should meet Chinese Pharmacopoeia tablet (appendix ID of Chinese Pharmacopoeia), capsule, soft capsule (appendix IL of Chinese Pharmacopoeia) or drop pill (appendix IK of Chinese Pharmacopoeia) item pertinent regulations down.
[assay] total flavones is only studied the drying mode of isorhamnetin reference substance with the primary standard method, and the result sees table 1.
Table 1
(XINDAKANG PIAN sample lot number: 060301, specification: the 5mg/ sheet)
Above result shows: after adopting the dry isorhamnetin reference substance of dual mode, survey the content of total flavone no significant difference, adopt P so the isorhamnetin reference substance changes into
2O
5More than the dry 12h, more to meet the requirement of pharmacopeia to the reference substance drying condition.
The content assaying method of the Quercetin in the employing high effective liquid chromatography for measuring XINDAKANG PIAN that Quercetin, isorhamnetin, kaempferol increase newly, isorhamnetin, kaempferol total amount.Method is carried out assay with reference to the method for the high effective liquid chromatography for measuring isorhamnetin that records under Fructus Hippophae item of Chinese Pharmacopoeia to Quercetin, isorhamnetin, kaempferol in the Xindakang preparation.
1. maximum absorption wavelength is selected
Get Quercetin, isorhamnetin, kaempferol reference substance; Process reference substance solution with methanol respectively; In the interscan of 200-600nm wave-length coverage; Quercetin has maximum absorption wavelength at the 371nm place, and isorhamnetin has maximum absorption wavelength at the 369.5nm place, and kaempferol has maximum absorption wavelength at the 366.5nm place.With reference to the detection wavelength of isorhamnetin under Fructus Hippophae item of Chinese Pharmacopoeia version in 2005,, Quercetin, isorhamnetin, kaempferide be chosen as 370nm so being detected wavelength.
2. the selection of mobile phase
The assay item current downflow of isorhamnetin is methanol mutually under Fructus Hippophae item of Chinese Pharmacopoeia version in 2005: 0.4% phosphoric acid=58: 42; The assay item current downflow of Quercetin, isorhamnetin, kaempferol is methanol mutually in the Folium Ginkgo: 0.4% phosphoric acid=50: 50; These article are the single medicinal material preparation; So, confirm that mobile phase the best is methanol: 0.4% phosphoric acid=50: 50 with reference to Chinese Pharmacopoeia.
3. chromatographic condition and system suitability test
Mobile phase: methanol: 0.4% phosphoric acid=50: 50
Chromatographic column: (5um 250 * 4.6mm) for the DiamonsilC18 of Di Ma company
Flow velocity: 1ml/s, 35 ℃ of column temperatures;
Under this condition in the sample Quercetin, isorhamnetin, kaempferol can reach baseline separation with other component, meet the requirements with adjacent chromatographic peak separating degree.Theoretical cam curve is calculated by the isorhamnetin peak should be not less than 3000.
4. the method for preparing research of need testing solution
The main component of Xindakang preparation is a Fructus Hippophae total flavones; Form by isorhamnetin-3-rutinoside, isorhamnetin-3-rhamnoside, Quercetin-3-glucoside, kaempferol-multiple flavonoid glycoside compounds such as 3-glucoside; Said composition is difficult directly to be measured; And its aglycon is mainly Quercetin, isorhamnetin, kaempferol, so carry out assay after adopting acid hydrolysis to make flavonoid glycoside change into flavone aglycone.
Because Quercetin, isorhamnetin, kaempferol all can be dissolved in methanol,, serve as to extract solvent to extract with methanol 25ml with reference to the method for preparing of need testing solution in the Flavonihippophae capsule quality standard.20 of these article of getting are removed coating, and accurate the title decides, and porphyrize is got an amount of (being equivalent to total flavones 10mg), and precision is claimed fixed; Put in the tool plug conical flask, the accurate methanol 25ml that adds claims to decide weight, and supersound process (power 135W, frequency 59kHz) is put cold; Claim to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, discard filtrating just, precision is measured subsequent filtrate 5ml; Put in the tool plug conical flask, add methanol 15ml, hydrochloric acid is put reflux in the water-bath, and cooling is transferred in the 50ml measuring bottle rapidly; Be diluted to scale with methanol, shake up, filter, get subsequent filtrate, promptly get with microporous filter membrane (0.45um).Adopt above-mentioned chromatographic condition, measure.
4.1 extraction time research 20 (30 or 40) minute.
Add hydrochloric acid (1 → 2) 3 (5 or 7) ml 4.2 add the research of acid amount.
4.3 reflux 20 in the investigation water-bath of hydrolysis time (30 or 40) minute.
Experimental result is seen table 2.
Table 2
Result: through test relatively, confirm that the test liquid method for preparing is: get 20 of these article, remove coating, the accurate title, decided porphyrize; Get an amount of (being equivalent to total flavones 10mg), the accurate title, decide, and puts in the tool plug conical flask, and the accurate methanol 25ml that adds claims to decide weight; Supersound process (power 135W, frequency 59kHz) 30 minutes is put coldly, claims to decide weight again, supplies the weight that subtracts mistake with methanol; Shake up, filter, discard filtrating just, precision is measured subsequent filtrate 5ml, puts in the tool plug conical flask; Add methanol 15ml, hydrochloric acid (1 → 2) 5ml put in the water-bath reflux 30 minutes, and cooling is transferred in the 50ml measuring bottle rapidly; Be diluted to scale with methanol, shake up, filter, get subsequent filtrate, promptly get with microporous filter membrane (0.45um).
5. specificity test
With the XINDAKANG PIAN negative sample that lacks Fructus Hippophae flavone, press the test sample disposal methods after, measure, the result is noiseless on the position at Quercetin, isorhamnetin, kaempferol peak.
6. the preparation of standard curve
Precision takes by weighing Quercetin reference substance 20.69mg, puts in the 50ml measuring bottle, adds dissolve with methanol and is diluted to scale, shakes up, as the reference substance stock solution.Precision takes by weighing kaempferol reference substance 3.23mg, puts in the 10ml measuring bottle, adds dissolve with methanol and is diluted to scale, shakes up, as the reference substance stock solution.Precision takes by weighing isorhamnetin reference substance 8.46mg, puts in the 25ml measuring bottle, adds dissolve with methanol and is diluted to scale, shakes up, as the reference substance stock solution.Precision is measured Quercetin reference substance stock solution 10ml, kaempferol reference substance stock solution 1ml, isorhamnetin reference substance stock solution 10ml in same 50ml measuring bottle respectively, is diluted to scale with methanol, shakes up, as mixing reference substance solution.Precision is measured mixing reference substance solution 1,2,4,6,8,10ml in the 10ml measuring bottle respectively again, is diluted to scale with methanol, shakes up, and draws the 10ul sample introduction.With the sample size is abscissa, and peak area is a vertical coordinate drawing standard curve.Calculate regression equation.
Quercetin regression equation: A=4636757.285C-18655.16986 (r
2=0.999989135)
Kaempferol regression equation: A=4025498.961C-4063.736986 (r
2=0.999377663)
Isorhamnetin regression equation: A=4551361C-22470.2 (r
2=0.999995)
The result shows: under above-mentioned chromatographic condition; Quercetin is between 0.08276-0.8276ug; Sample feeding amount and peak area linear relationship are good, and kaempferol is between 0.00646-0.0646ug, and sample feeding amount and peak area linear relationship are good; Isorhamnetin is between 0.06768-0.6768ug, and sample feeding amount and peak area linear relationship are good.
7. precision test
7.1 accurate Quercetin, kaempferol, each 10ul of isorhamnetin mixing reference substance solution of drawing of instrument precision, the repetition sample introduction is 5 times under above-mentioned chromatographic condition.The result sees table 3.
The test of table 3 instrument precision
| The sample introduction number of times | Quercetin | Kaempferide | Isorhamnetin |
| 1 | 1486544 | 118686 | 767727 |
| 2 | 1451610 | 113661 | 746763 |
| 3 | 1483973 | 120058 | 768565 |
| 4 | 1483038 | 119708 | 762612 |
| 5 | 1485164 | 120285 | 766082 |
| RSD(％) | 1.0 | 2.3 | 1.2 |
7.2 replica test
Parallel getting with 6 parts in batch XINDAKANG PIAN sample by " assay " test liquid method for preparing test down, adopted above-mentioned chromatographic condition, measures Quercetin, kaempferol, isorhamnetin content.The result sees table 4.
Table 4 replica test result
| Test number (TN) | 1 | 2 | 3 | 4 | 5 | 6 | RSD(％) |
| Quercetin, kaempferide, isorhamnetin total content (mg/ sheet) | 4.82 | 4.83 | 4.82 | 4.82 | 4.80 | 4.82 | 1.1 |
Above result shows: Quercetin, and kaempferide, isorhamnetin content has repeatability preferably
8, accuracy (average recovery test)
Precision is measured Quercetin reference substance solution (concentration 0.9348mg/ml) 3ml, nimbecetin reference substance solution (concentration 0.2196mg/ml) 1ml, isorhamnetin reference substance solution (concentration 0.54mg/ml) 3ml; Totally 6 parts; Put respectively in 6 tool plug conical flasks, solvent evaporated in the water-bath, subsequent use.Get the known sample that contains Quercetin 19.07mg/g, kaempferol 1.40mg/g, isorhamnetin 11.60mg/g; The accurate title, decide; Put in the above-mentioned tool plug conical flask, measure Quercetin, kaempferol, the total metering method of isorhamnetin and carry out application of sample and reclaim experiment by drafting, the result sees table 5, table 6, table 7.
Table 5 Quercetin average recovery result of the test
Table 6 kaempferol average recovery result of the test
Table 7 isorhamnetin average recovery result of the test
Above result's test shows: this method has the response rate preferably, and accuracy is good.
9, ruggedness experiment
9.1 stability test is got need testing solution and measured content in the preparation back after 0,1,3,6,12,24 hour, its result sees table 8.
Table 8 stability test result
| Standing time | 0 | 1 | 3 | 6 | 12 | 24 | RSD(％) |
| Quercetin | 1547874 | 1540112 | 1543021 | 1532368 | 1485664 | 1566521 | 1.8 |
| Kaempferol | 124117 | 122975 | 125646 | 125481 | 119476 | 126826 | 2.1 |
| Isorhamnetin | 801384 | 797535 | 800284 | 793973 | 765418 | 816134 | 2.1 |
Above result shows: test sample is better at 24 hours internal stabilities.
9.2 the test that the mobile phase ratio changes
Sample thief prepares need testing solution by the method for preparing of above-mentioned definite need testing solution, and mobile phase is respectively: 1. methanol-0.4% phosphoric acid (45: 55)); 2. methanol-0.4% phosphoric acid (50: 50); 3. methanol-0.4% phosphoric acid (55: 45).Constant at other chromatographic conditions, only change under the situation of mobile phase, investigate measuring the result.Measure by sample introduction under the above-mentioned chromatographic condition, measure the result and see table 9.
The investigation that table 9 mobile phase ratio changes
According to above mensuration result, RSD is 1.3%, and the less variation of mobile phase ratio does not have influence to measuring the result.
9.3 the investigation of different brands chromatographic column
Get with the lot sample article, prepare need testing solution by the method for preparing of above-mentioned definite need testing solution, chromatographic column is respectively: (1) Kromasil.200 * 4.6mmID, 5um; (2) Dickma 250 * 4.mmID 5um; (3) phenomemex 150 * 4.6ID 5um.Constant in mobile phase and other chromatographic conditions, only change employed chromatographic column, investigate measuring the result.Measure by sample introduction under the above-mentioned chromatographic condition, measure the result and see table 10.
The investigation of table 10 different brands chromatographic column
According to above mensuration result, RSD is 1.09%, and the chromatographic column of different brands does not have obvious influence to measuring the result.
Can know that according to above result of the test the ruggedness of this method is better.
10, sample size is measured
By the Xindakang preparation Quercetin of drafting, kaempferide, isorhamnetin content assaying method, former XINDAKANG PIAN that keeps sample for a long time and commercially available prod have been carried out assay, the result sees table 11, table 12, table 13.
Five batches of XINDAKANG PIANs of table 11 (specification: 5mg) assay result
| Lot number | Quercetin (mg) | Nimbecetin (mg) | Isorhamnetin (mg) | Total amount (mg/ sheet) | Account for labelled amount (%) |
| 060103 | 2.88 | 0.20 | 1.67 | 4.75 | 95.0 |
| 060202 | 2.78 | 0.20 | 1.62 | 4.60 | 92.0 |
| 060203 | 2.91 | 0.20 | 1.71 | 4.83 | 96.6 |
| 060301 | 2.96 | 0.21 | 1.73 | 4.90 | 98.0 |
| 060402 | 2.96 | 0.21 | 1.73 | 4.90 | 98.0 |
Three batches of XINDAKANG PIANs of table 12 (specification: 10mg) assay result
| Lot number | Quercetin (mg) | Nimbecetin (mg) | Isorhamnetin (mg) | Total amount (mg/ sheet) | Account for labelled amount (%) |
| 060101 | 6.14 | 0.25 | 3.27 | 9.66 | 96.6 |
| 060201 | 5.76 | 0.34 | 3.27 | 9.37 | 93.7 |
| 060401 | 5.47 | 0.37 | 3.22 | 9.06 | 90.6 |
Three batches of XINDAKANG PIANs of table 13 (specification: 5mg) assay result
| Manufacturer | Lot number | Quercetin (mg) | Nimbecetin (mg) | Isorhamnetin (mg) | Total amount (mg/ sheet) | Account for labelled amount (%) |
| Shanghai Tiancifu Biological Engineering Co., Ltd. | 051102 | 1.12 | 0.37 | 2.59 | 4.08 | 81.6 |
| Chuanda Huaxi Pharmaceutical Industry Co., Ltd. Sichuan Prov. | 050401 | 0.99 | 0.30 | 2.92 | 4.21 | 84.2 |
| The refined Pharmaceutical limited company that reaches in Sichuan | 050610 | 1.01 | 0.22 | 3.02 | 4.25 | 85 |
Can find out that from above result Quercetin, kaempferol, isorhamnetin total content are equivalent to 81.6%～98.0% of its total flavones labelled amount in the employing high effective liquid chromatography for measuring XINDAKANG PIAN.This standard of considering is just carried out assay to existing procucts, therefore is its limit standard with the minimum data that records temporarily.Promptly adopt Quercetin in the high effective liquid chromatography for measuring XINDAKANG PIAN, kaempferol, isorhamnetin total amount, should be not less than 80.0% of labelled amount.
The specific embodiment
Below further specify the present invention through embodiment, but not as limitation of the present invention, method of quality control of the present invention also can be used for the quality control of following other Flavonihippophae dosage form medicine.
Embodiment one: said tablet quality control method comprises
Character: these article are coated tablet or Film coated tablets, remove to show pale brown color behind the coating to brown, mildly bitter flavor.
Differentiate: get 1 of these article, remove coating, porphyrize adds every 5ml of ethanol (5mg) or 10ml (10mg), and supersound process 5 minutes filters, and filtrating is as need testing solution; Other gets Quercetin and isorhamnetin reference substance, adds ethanol and processes the mixed solution that every 1ml contains 1mg, as reference substance solution; According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia) test, draw each 1～2ul of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose; With toluene: ethyl acetate: formic acid=5: 4: 0.5 is developing solvent; Launch, take out, dry; Spray is put under the 365nm ultra-violet lamp and is inspected with 1% aluminum chloride alcoholic solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
Inspection: should meet each relevant under tablet item regulation (appendix ID of Chinese Pharmacopoeia).
Assay
(1) total flavones
The preparation of reference substance solution: precision takes by weighing the isorhamnetin reference substance, adds dehydrated alcohol and processes the solution that every 1ml contains isorhamnetin 20ug, promptly gets.
The preparation of standard curve: precision is measured reference substance solution 1,2,3,4,5,6ml, puts respectively in the 10ml measuring bottle, adds 1% aluminum chloride ethanol solution 1.0ml; Be diluted to scale with dehydrated alcohol, shake up, placed 10 minutes; With the reagent corresponding is blank, according to ultraviolet visible spectrophotometry (appendix VA of Chinese Pharmacopoeia version in 2005), measures absorbance in the wavelength of 430nm; With the absorbance is vertical coordinate, and concentration is abscissa, the drawing standard curve.
Algoscopy: get 20 in this tablet, remove coating, the accurate title, decide, and porphyrize is got 0.1g; The accurate title, decide, and puts in the tool plug conical flask, and it is an amount of to add dehydrated alcohol, and reflux is 15 minutes in 80 ℃ of water-baths, puts cold; In the dislocation 100ml measuring bottle, be diluted to scale, shake up, filter, discard filtrating just with dehydrated alcohol; Precision is measured subsequent filtrate 10ml, puts in the 25ml measuring bottle, is diluted to scale with dehydrated alcohol, shakes up, and precision is measured 3ml; Put in the 10ml measuring bottle, add 1% aluminum chloride ethanol solution 1.0ml, be diluted to scale, shake up, as need testing solution with dehydrated alcohol; Other gets the same amount need testing solution that does not add developer and makes blank, and the sighting target directrix curve prepares the method under the item, from " placing 10 minutes ", measures absorbance in accordance with the law, from standard curve, reads the weight (ug) of isorhamnetin in the test sample, calculates, and promptly gets.
Every in this tablet contains Fructus Hippophae flavone with isorhamnetin (C
16H
12O
7) meter, should be 90.0%～110.0% of labelled amount.
Every in this tablet contains total flavones (in isorhamnetin) 1. 5mg, 2. 10mg.
(2) Quercetin, isorhamnetin, kaempferol are measured according to HPLC (appendix VID of Chinese Pharmacopoeia version in 2005):
Chromatographic column is the C18 post, and flow velocity is 1ml/s, and column temperature is 35 ℃, and with methanol: 0.4% phosphoric acid=50: 50 is mobile phase; The detection wavelength is 370nm; Theoretical cam curve is calculated by the isorhamnetin peak should be not less than 3000.
The preparation of reference substance solution: precision takes by weighing with P
2O
5An amount of for desiccant drying under reduced pressure isorhamnetin more than 12 hours, kaempferol, 4 hours Quercetin reference substance of 120 ℃ of dryings, add methanol and process the mixed solution that every 1ml contains isorhamnetin 20ug, kaempferol 2ug, Quercetin 25ug respectively, promptly get.
The preparation of need testing solution: get 20 of these article, remove coating, the accurate title, decide, and porphyrize is got an amount of (being equivalent to total flavones 10mg), and precision is claimed fixed; Put in the tool plug conical flask, the accurate methanol 25ml that adds claims to decide weight, and supersound process (power 135W, frequency 59kHz) 30 minutes is put cold; Claim to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, discard filtrating just, precision is measured subsequent filtrate 5ml; Put in the tool plug conical flask, add methanol 15ml, hydrochloric acid (1 → 2) 5ml put in the water-bath reflux 30 minutes, and cooling is transferred in the 50ml measuring bottle rapidly; Be diluted to scale with methanol, shake up, filter, get subsequent filtrate, promptly get with microporous filter membrane (0.45um).
Algoscopy: accurate respectively reference substance solution and each 10ul of need testing solution of drawing, inject chromatograph of liquid, measure, promptly get.
Every in this tablet contains Quercetin (C
15H
10O
7), isorhamnetin (C
16H
12O
7), kaempferol (C
15H
10O
6) total amount be no less than 80.0% of total flavones labelled amount;
[function with cure mainly] reinforce functional activities of the heart, blood stasis dispelling is promoted blood circulation, the expectorant activating the spleen.Be used for a little less than the insufficiency of heart-QI, heart arteries and veins stasis of blood resistance, phlegm-damp is stranded nervous, cardiopalmus, pained due to the spleen, and it is uncomfortable in chest to breathe hard, and blood vessels are not smooth, cough the tired disease of waiting.
[usage and consumption] is oral, a 10mg, and 3 times on the one, three months is a course of treatment.
[specification] every contains 1. 2. 10mg of 5mg of total flavones (in isorhamnetin)
Dry place is put in [storage a surname] sealing.
Embodiment two: said capsule method of quality control comprises
Character: content is fawn to dun granule and a powder; Feeble QI, mildly bitter flavor.
Differentiate: get this capsule content 5mg or 10mg, add ethanol 5ml or 10ml, supersound process 5 minutes is processed the solution of 1mg/ml, filters, and filtrating is as need testing solution; Other gets Quercetin and isorhamnetin reference substance, adds ethanol and processes the mixed solution that every 1ml contains 1mg, as reference substance solution; According to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin layer chromatography test, draw each 1～2ul of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose; With toluene: ethyl acetate: formic acid=5: 4: 0.5 is developing solvent; Launch, take out, dry; Spray is put under the 365nm ultra-violet lamp and is inspected with 3% aluminum chloride alcoholic solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
Inspection: should meet pertinent regulations under the capsule item (appendix IL of Chinese Pharmacopoeia version in 2005).。
Assay
(1) total flavones
The preparation of reference substance solution: precision takes by weighing the isorhamnetin reference substance, adds dehydrated alcohol and processes the solution that every 1ml contains isorhamnetin 20ug, promptly gets;
The preparation of standard curve: precision is measured reference substance solution 1,2,3,4,5,6ml, puts respectively in the 10ml measuring bottle, adds 3% aluminum chloride ethanol solution 1.0ml; Be diluted to scale with dehydrated alcohol, shake up, placed 10 minutes; With the reagent corresponding is blank, according to ultraviolet visible spectrophotometry (appendix VA of Chinese Pharmacopoeia version in 2005), measures absorbance in the wavelength of 430nm; With the absorbance is vertical coordinate, and concentration is abscissa, the drawing standard curve.
Algoscopy: it is tolerant to get this capsule 10～20 intragranulars, and accurate the title decides, porphyrize, and mixing is got 0.1g; The accurate title, decide, and puts in the tool plug conical flask, and it is an amount of to add dehydrated alcohol, and reflux is 15 minutes in 80 ℃ of water-baths, puts cold; In the dislocation 100ml measuring bottle, be diluted to scale, shake up, filter, discard filtrating just with dehydrated alcohol; Precision is measured subsequent filtrate 10ml, puts in the 25ml measuring bottle, is diluted to scale with dehydrated alcohol, shakes up, and precision is measured 3ml; Put in the 10ml measuring bottle, add 3% aluminum chloride ethanol solution 1.0ml, be diluted to scale, shake up, as need testing solution with dehydrated alcohol; Other gets the same amount need testing solution that does not add developer and makes blank, and the sighting target directrix curve prepares the method under the item, from " placing 10 minutes ", measures absorbance in accordance with the law, from standard curve, reads the weight (ug) of isorhamnetin in the test sample, calculates, and promptly gets;
This capsule contains Fructus Hippophae flavone with isorhamnetin (C
16H
12O
7) meter, should be 90.0%～110.0% of labelled amount.
Every of this capsule contains flavone (in isorhamnetin) 1. 5mg, 2. 10mg.
(2) Quercetin, isorhamnetin, kaempferol are measured according to HPLC (appendix VID of Chinese Pharmacopoeia version in 2005).
Chromatographic column is the C18 post, and flow velocity is 1ml/s, and column temperature is 35 ℃, and with methanol: 0.4% phosphoric acid=50: 50 is mobile phase; The detection wavelength is 370nm.Theoretical cam curve is calculated by the isorhamnetin peak should be not less than 3000.
The preparation of reference substance solution: precision takes by weighing with P
2O
5An amount of for desiccant drying under reduced pressure isorhamnetin more than 12 hours, kaempferol, 4 hours Quercetin reference substance of 120 ℃ of dryings, add methanol and process the mixed solution that every 1ml contains isorhamnetin 20ug, kaempferol 2ug, Quercetin 25ug respectively, promptly get;
The preparation of need testing solution: get content an amount of (being equivalent to total flavones 10mg) under the determination of total flavonoids item, the accurate title, decide, and puts in the tool plug conical flask, and the accurate methanol 25ml that adds claims to decide weight; Supersound process (power 135W, frequency 59kHz) 30 minutes is put coldly, claims to decide weight again, supplies the weight that subtracts mistake with methanol; Shake up, filter, discard filtrating just, precision is measured subsequent filtrate 5ml, puts in the tool plug conical flask; Add methanol 15ml, hydrochloric acid (1 → 2) 5ml put in the water-bath reflux 30 minutes, and cooling is transferred in the 50ml measuring bottle rapidly; Be diluted to scale with methanol, shake up, filter, get subsequent filtrate, promptly get with microporous filter membrane (0.45um).
Accurate respectively reference substance solution and each 10ul of need testing solution of drawing of algoscopy injects chromatograph of liquid, measures, and promptly gets.
Every of this capsule contains Quercetin (C
15H
10O
7), isorhamnetin (C
16H
12O
7), kaempferol (C
15H
10O
6) total amount be no less than 80.0% of total flavones labelled amount.
Embodiment three: this soft capsule method of quality control comprises
Character: content is the yellow oily liquid that contains a small amount of suspended solid extractum; Feeble QI, mildly bitter flavor.
Differentiate: get this capsule content 5mg or 10mg, add ethanol 5ml or 10ml, supersound process 5 minutes is processed the solution of 1mg/ml, filters, and filtrating is as need testing solution; Other gets Quercetin and isorhamnetin reference substance, adds ethanol and processes the mixed solution that every 1ml contains 1mg, as reference substance solution; According to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin layer chromatography test, draw each 1～2ul of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose; With toluene: ethyl acetate: formic acid=5: 4: 0.5 is developing solvent; Launch, take out, dry; Spray is put under the 365nm ultra-violet lamp and is inspected with 3% aluminum chloride alcoholic solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
Inspection: should meet pertinent regulations under the capsule item (appendix IL of Chinese Pharmacopoeia version in 2005).
Assay
(1) total flavones
The preparation of reference substance solution: precision takes by weighing the isorhamnetin reference substance, adds dehydrated alcohol and processes the solution that every 1ml contains isorhamnetin 20ug, promptly gets;
The preparation of standard curve: precision is measured reference substance solution 1,2,3,4,5,6ml, puts respectively in the 10ml measuring bottle, adds 3% aluminum chloride ethanol solution 1.0ml; Be diluted to scale with dehydrated alcohol, shake up, placed 10 minutes; With the reagent corresponding is blank, according to ultraviolet visible spectrophotometry (appendix VA of Chinese Pharmacopoeia version in 2005), measures absorbance in the wavelength of 430nm; With the absorbance is vertical coordinate, and concentration is abscissa, the drawing standard curve.
Algoscopy: it is tolerant to get this preparation 10～20 intragranulars, and accurate the title decides, and mixing is got 0.1g, and accurate title is fixed; Put in the tool plug conical flask, it is an amount of to add dehydrated alcohol, and reflux is 15 minutes in 80 ℃ of water-baths, put cold, in the dislocation 100ml measuring bottle; Be diluted to scale with dehydrated alcohol, shake up, filter, discard filtrating just, precision is measured subsequent filtrate 10ml; Put in the 25ml measuring bottle, be diluted to scale with dehydrated alcohol, shake up, precision is measured 3ml, puts in the 10ml measuring bottle; Add 3% aluminum chloride ethanol solution 1.0ml, be diluted to scale, shake up, as need testing solution with dehydrated alcohol; Other gets the same amount need testing solution that does not add developer and makes blank, and the sighting target directrix curve prepares the method under the item, from " placing 10 minutes ", measures absorbance in accordance with the law, from standard curve, reads the weight (ug) of isorhamnetin in the test sample, calculates, and promptly gets;
This soft capsule contains Fructus Hippophae flavone with isorhamnetin (C
16H
12O
7) meter, should be 90.0%～110.0% of labelled amount.
Every of this soft capsule contains flavone (in isorhamnetin) 1. 5mg, 2. 10mg.
(2) Quercetin, isorhamnetin, kaempferol are measured according to HPLC (appendix VID of Chinese Pharmacopoeia version in 2005).
Chromatographic column is the C18 post, and flow velocity is 1ml/s, and column temperature is 35 ℃, and with methanol: 0.4% phosphoric acid=50: 50 is mobile phase; The detection wavelength is 370nm; Theoretical cam curve is calculated by the isorhamnetin peak should be not less than 3000.
The preparation of reference substance solution: precision takes by weighing with P
2O
5An amount of for desiccant drying under reduced pressure isorhamnetin more than 12 hours, kaempferol, 4 hours Quercetin reference substance of 120 ℃ of dryings, add methanol and process the mixed solution that every 1ml contains isorhamnetin 20ug, kaempferol 2ug, Quercetin 25ug respectively, promptly get;
The preparation of need testing solution: get total flavones and measure item content an amount of (being equivalent to total flavones 10mg) down, the accurate title, decide, and puts in the tool plug conical flask, and the accurate methanol 25ml that adds claims to decide weight; Supersound process (power 135W, frequency 59kHz) 30 minutes is put coldly, claims to decide weight again, supplies the weight that subtracts mistake with methanol; Shake up, filter, discard filtrating just, precision is measured subsequent filtrate 5ml, puts in the tool plug conical flask; Add methanol 15ml, hydrochloric acid (1 → 2) 5ml put in the water-bath reflux 30 minutes, and cooling is transferred in the 50ml measuring bottle rapidly; Be diluted to scale with methanol, shake up, filter, get subsequent filtrate, promptly get with microporous filter membrane (0.45um).
Algoscopy: accurate respectively reference substance solution and each 10ul of need testing solution of drawing, inject chromatograph of liquid, measure, promptly get.
Every of this soft capsule contains Quercetin (C
15H
10O
7), isorhamnetin (C
16H
12O
7), kaempferol (C
15H
10O
6) total amount be no less than 80.0% of total flavones labelled amount.
Embodiment four: this drop pill method of quality control comprises
Character: content is fawn to dun granule and a powder; Feeble QI, mildly bitter flavor.
Differentiate: get this drop pill content 5mg or 10mg, add ethanol 5ml or 10ml, supersound process 5 minutes is processed the solution of 1mg/ml, filters, and filtrating is as need testing solution; Other gets Quercetin and isorhamnetin reference substance, adds ethanol and processes the mixed solution that every 1ml contains 1mg, as reference substance solution; According to an appendix VIB of Chinese Pharmacopoeia version in 2005 thin layer chromatography test, draw each 1～2ul of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose; With toluene: ethyl acetate: formic acid=5: 4: 0.5 is developing solvent; Launch, take out, dry; Spray is put under the 365nm ultra-violet lamp and is inspected with 1% aluminum chloride alcoholic solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
Inspection: should meet pertinent regulations under the drop pill item (appendix IK of Chinese Pharmacopoeia version in 2005).
Assay
(1) total flavones
The preparation of reference substance solution: precision takes by weighing the isorhamnetin reference substance, adds dehydrated alcohol and processes the solution that every 1ml contains isorhamnetin 20ug, promptly gets;
The preparation of standard curve: precision is measured reference substance solution 1,2,3,4,5,6ml, puts respectively in the 10ml measuring bottle, adds 1% aluminum chloride ethanol solution 1.0ml; Be diluted to scale with dehydrated alcohol, shake up, placed 10 minutes; With the reagent corresponding is blank, according to ultraviolet visible spectrophotometry (appendix VA of Chinese Pharmacopoeia version in 2005), measures absorbance in the wavelength of 430nm; With the absorbance is vertical coordinate, and concentration is abscissa, the drawing standard curve.
Algoscopy: this preparation 10～20 intragranulars are tolerant, and accurate the title decides, and mixing is got 0.1g, and accurate title is fixed; Put in the tool plug conical flask, it is an amount of to add dehydrated alcohol, and reflux is 15 minutes in 80 ℃ of water-baths, put cold, in the dislocation 100ml measuring bottle; Be diluted to scale with dehydrated alcohol, shake up, filter, discard filtrating just, precision is measured subsequent filtrate 10ml; Put in the 25ml measuring bottle, be diluted to scale with dehydrated alcohol, shake up, precision is measured 3ml, puts in the 10ml measuring bottle; Add 1% aluminum chloride ethanol solution 1.0ml, be diluted to scale, shake up, as need testing solution with dehydrated alcohol; Other gets the same amount need testing solution that does not add developer and makes blank, and the sighting target directrix curve prepares the method under the item, from " placing 10 minutes ", measures absorbance in accordance with the law, from standard curve, reads the weight (ug) of isorhamnetin in the test sample, calculates, and promptly gets;
This drop pill contains Fructus Hippophae flavone with isorhamnetin (C
16H
12O
7) meter, should be 90.0%～110.0% of labelled amount.
The every ball of this drop pill contains flavone (in isorhamnetin) 1. 5mg, 2. 10mg.
(2) Quercetin, isorhamnetin, kaempferol are measured according to HPLC (appendix VID of Chinese Pharmacopoeia).
Chromatographic column is the C18 post, and flow velocity is 1ml/s, and column temperature is 35 ℃, and with methanol: 0.4% phosphoric acid=50: 50 is mobile phase; The detection wavelength is 370nm.Theoretical cam curve is calculated by the isorhamnetin peak should be not less than 3000.
The preparation of reference substance solution: precision takes by weighing with P
2O
5An amount of for desiccant drying under reduced pressure isorhamnetin more than 12 hours, kaempferol, 4 hours Quercetin reference substance of 120 ℃ of dryings, add methanol and process the mixed solution that every 1ml contains isorhamnetin 20ug, kaempferol 2ug, Quercetin 25ug respectively, promptly get;
The preparation of need testing solution: get total flavones and measure the content an amount of (being equivalent to total flavones 10mg) under the item, accurate title is fixed, puts in the tool plug conical flask, and precision adds methanol 25ml, claims to decide weight; Supersound process (power 135W, frequency 59kHz) 30 minutes is put coldly, claims to decide weight again, supplies the weight that subtracts mistake with methanol; Shake up, filter, discard filtrating just, precision is measured subsequent filtrate 5ml, puts in the tool plug conical flask; Add methanol 15ml, hydrochloric acid (1 → 2) 5ml put in the water-bath reflux 30 minutes, and cooling is transferred in the 50ml measuring bottle rapidly; Be diluted to scale with methanol, shake up, filter, get subsequent filtrate, promptly get with microporous filter membrane (0.45um).
Accurate respectively reference substance solution and each 10ul of need testing solution of drawing of algoscopy injects chromatograph of liquid, measures, and promptly gets.The every ball of this drop pill contains Quercetin (C
15H
10O
7), isorhamnetin (C
16H
12O
7), kaempferol (C
15H
10O
6) total amount be no less than 80.0% of total flavones labelled amount.
Claims (3)
1. the discrimination method of an Xindakang preparation, said preparation comprises tablet, capsule, drop pill, it is characterized in that,
Said discriminating comprises that with Quercetin reference substance, isorhamnetin reference substance be the thin layer chromatography discriminating that flavone aglycone in the preparation is differentiated in contrast; Said discrimination method comprises following:
Get this preparation or its content, porphyrize adds ethanol and makes dissolving, filters, and filtrating is as need testing solution; Other gets Quercetin and isorhamnetin reference substance, adds ethanol and processes mixed solution, as reference substance solution; According to the test of Chinese Pharmacopoeia thin layer chromatography, draw above-mentioned need testing solution, reference substance solution, put respectively on same silica gel g thin-layer plate; With toluene: ethyl acetate: formic acid=2～10: 1～8: 0.1～1.5 is developing solvent; Launch, take out, dry; Spray is put under the ultra-violet lamp and is inspected with 1～5% aluminum chloride alcoholic solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
2. according to the discrimination method of the said Xindakang preparation of claim 1, it is characterized in that concrete discrimination method comprises following:
Get this preparation or its content, porphyrize is got fine powder 2～20mg, adds dehydrated alcohol 2～20ml, and supersound process 1～15 minute filters, and filtrating is as need testing solution; Other gets Quercetin and isorhamnetin reference substance, adds dehydrated alcohol and processes the mixed solution that every 1ml contains 1mg～10mg, as reference substance solution; According to an appendix VIB of Chinese Pharmacopoeia thin layer chromatography test, draw each 1～20ul of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose; With toluene: ethyl acetate: formic acid=2～10: 1～8: 0.1～1.5 is developing solvent; Launch, take out, dry; Spray is put under the 365nm ultra-violet lamp and is inspected with 1～5% aluminum chloride alcoholic solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
3. according to the discrimination method of the said Xindakang preparation of claim 2, it is characterized in that further concrete discrimination method comprises following:
Get 1 in this preparation or 1, porphyrize adds ethanol 5ml or 10ml, and supersound process 5 minutes is processed the solution of 1mg/ml, filters, and filtrating is as need testing solution; Other gets Quercetin and isorhamnetin reference substance, adds ethanol and processes the mixed solution that every 1ml contains 1mg, as reference substance solution; According to an appendix VIB of Chinese Pharmacopoeia thin layer chromatography test, draw each 1～2ul of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose; With toluene: ethyl acetate: formic acid=5: 4: 0.5 is developing solvent; Launch, take out, dry; Spray is put under the 365nm ultra-violet lamp and is inspected with 1～3% aluminum chloride alcoholic solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
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| CN109444128B (en) * | 2018-12-04 | 2021-08-27 | 中北大学 | Kit for detecting seabuckthorn flavone and method for detecting seabuckthorn flavone |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1552317A (en) * | 2003-05-26 | 2004-12-08 | 四川美大康药业股份有限公司 | Preparation of Xindakang |
| CN1833651A (en) * | 2006-01-11 | 2006-09-20 | 北京因科瑞斯生物制品研究所 | Xindakang drip pill and prepn. thereof |
| CN100593402C (en) * | 2006-01-11 | 2010-03-10 | 北京因科瑞斯生物制品研究所 | 'Xindakang' dispersion tablets and preparation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1552317A (en) * | 2003-05-26 | 2004-12-08 | 四川美大康药业股份有限公司 | Preparation of Xindakang |
| CN1833651A (en) * | 2006-01-11 | 2006-09-20 | 北京因科瑞斯生物制品研究所 | Xindakang drip pill and prepn. thereof |
| CN100593402C (en) * | 2006-01-11 | 2010-03-10 | 北京因科瑞斯生物制品研究所 | 'Xindakang' dispersion tablets and preparation method |
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