CN105467059A - Quality detecting method for traditional Chinese medicine composition for treating hematuresis - Google Patents
Quality detecting method for traditional Chinese medicine composition for treating hematuresis Download PDFInfo
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- CN105467059A CN105467059A CN201511020492.1A CN201511020492A CN105467059A CN 105467059 A CN105467059 A CN 105467059A CN 201511020492 A CN201511020492 A CN 201511020492A CN 105467059 A CN105467059 A CN 105467059A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Abstract
The invention relates to a quality detecting method for a traditional Chinese medicine compound preparation, in particular to a quality detecting method for a traditional Chinese medicine composition for treating hematuresis. According to the technical scheme, the quality detecting method for the traditional Chinese medicine composition for treating hematuresis is prepared from cortex phellodendri, lalang grass rhizome, clerodendranthus spicatus and linarin in herba cepbalanoplosis segeti. The invention further provides a method for detecting the content of berberine hydrochloride in cortex phellodendri and the content of rosmarinic acid in clerodendranthus spicatus. High performance liquid chromatography is adopted. According to the method, lalang grass rhizome, cortex phellodendri, clerodendranthus spicatus and herba cepbalanoplosis segeti in the traditional Chinese medicine composition for treating hematuresis are qualitatively detected through thin-layer chromatography, and the method is simple and stable. The high performance liquid chromatography is used for detecting the content of berberine hydrochloride in cortex phellodendri and the content of rosmarinic acid in clerodendranthus spicatus, the method is easy and convenient and high in repeatability and stability, the result is accurate, the method serves as a method for detecting the content of ingredients of the traditional Chinese medicine compound preparation, and the rationality and reliability of the whole quality control method are enhanced.
Description
Technical field
The present invention relates to a kind of measuring method of compound Chinese medicinal preparation, particularly relate to a kind of quality determining method for the treatment of the Chinese medicine composition of blood urine.
Background technology
The prescription of the Chinese medicine composition for the treatment of blood urine is the secret recipe handed down in the family in the Xishuangbanna Prefecture, Yunnan Province Jinghong City Ming Lao Dai Nationality of Zhong Daiyi hospital doctor's ripple beautiful ripple more than 40 years of application, clinical treatment renal edema, hematuria, blood urine.Repeatedly apply through clinical, determined curative effect, do not find bad reaction and toxic and side effect.Dedicated Lixiang Pharmaceutical Ind. Co. Ltd.'s development research voluntarily in 1996, and agree to produce by drug standards Yunnan Q/WS1333-1998 in Department of Public Health of Nikkei in April 12 Yunnan Province reply in 1999; By the requirement of National Drug Administration's " notice about the management work of strengthening Chinese patent drug national standard ", rise to the national drug standards, obtain the national drug standards (trying) on November 30th, 2002 and promulgate part, the national drug standards (trying) number are WS-10793 (ZD-0793)-2002.In June, 2003, according to the requirement of " drug registration management method ", proposes the application for registration that medicine tentative standard transfers official standard to, in Nikkei State Food and Drug Administration examination March 19 in 2012, agrees to that tentative standard transfers official standard to.Standard No.: WS-10793 (ZD-0793)-2002-2012Z.
Dai Nationality's doctor's traditional medicine has history in several thousand, theoretical for core with " four towers " (wind, fire, water, soil) at theoretical side, carrys out the physiological phenomenon of researching human body, pathological change, instructs clinical treatment based on disease differentiation and medication." four towers ", ancient Dai Nationality language is called " tower all shelves is thin ", now referred to as " four towers " namely: (1) watt medicine tower one refers to " wind ", like the function of the traditional Chinese medical science " gas ".(2) " father Zhuo Tafei " (fire), fire is like the function of the first day after tomorrow " fire " of the traditional Chinese medical science.(3) " A Bota is difficult " (water, blood), the character in human body with " wetting " is then " water " supervisor.(4) be " bar tower tailing column " (soil), like the function of traditional Chinese medical science taste the foundation of acquired constitution " soil ".The Dai nationality's medical science is thought, the generation of disease, due to internal cause or thus to cause outward " four towers " dysfunction in human body not enough or have a surplus; Inclined Sheng or partially decline and cause.So, cure the disease, to adjust " four towers ".For " blood urine " one disease then think that " tower is luxuriant and rich with fragrance " (fire) in body is too much, water deficiency can not fire processed, the rich bright blood of hyperactivity of fire and hemorrhage." tooth glutinous second " i.e. used in side kidney tea bitter cool in nature, merit is to rush down clearly " tower is luxuriant and rich with fragrance " (fire poison), Li Shui, hemostasia and detumescence, for controlling nephritic dropsy, the key medicine of hematuria, blood urine." heartily " namely the flat taste of cogongrass rhizome is sweet, and the medicine that the flat taste of the Dai nationality's medical science property thought is sweet specially enters the disease of water blood and blood of harnessing the river, therefore cogongrass rhizome has the merit that water is grown in hemostasis of relieving inflammation or internal heat.Prodrug is helped to play a greater role." middleman is many " i.e. field thistle micro-hardship cool in nature is micro-sweet, and purging intense heat poison cool in nature, removing heat from blood is stopped blooding, and also helps the power of two medicines." kiss buries and strangles " i.e. golden cypress bitter cool in nature specially enters lower wall (lower wall and kidney, bladder, intestine and small intestine, uterus, lower limb etc.).Blood urine is then that lower wall " father Zhuo Tafei " crosses Sheng, therefore golden cypress specially disappears, lower wall fire poison, jointly plays purging intense heat poison with upper all medicines, obtain urine, the merit of hemostasis and pain-relieving.
But in national official standard, differentiate that item has two, adopt thin-layered chromatography to authenticated golden cypress wherein and cogongrass rhizome.Only use the qualification of golden cypress and cogongrass rhizome as the testing index in this lozenge method of quality control, well can not reflect the quality good or not of the Chinese medicine composition for the treatment of blood urine.
Summary of the invention
Technical scheme provided by the invention is a kind of quality determining method for the treatment of the Chinese medicine composition of blood urine, and Chinese medicine composition comprises linarin in golden cypress, cogongrass rhizome, kidney tea and field thistle, differentiates linarin in golden cypress, cogongrass rhizome, kidney tea and field thistle;
Described golden cypress differentiates that step is as follows:
Get the Chinese medicine composition 4g for the treatment of blood urine, add ammonia solution 2ml, methenyl choloride 10ml, ultrasonic process 15 minutes, filter, filtrate evaporate to dryness, residue adds methyl alcohol 1ml and dissolves, as need testing solution, separately get golden cypress control medicinal material 0.1g, add strong ammonia solution 1ml, methenyl choloride 5ml, ultrasonic process 15 minutes, filter, filtrate evaporate to dryness, residue adds methyl alcohol 1ml and dissolves, make control medicinal material solution, test according to thin-layered chromatography, draw need testing solution and each 5 μ l of control medicinal material solution, put respectively on same silica gel g thin-layer plate, with toluene: ethyl acetate: methyl alcohol: isopropyl alcohol: strong ammonia solution=6:3:1.5:1.5:0.5 is developping agent, put in the expansion cylinder of ammonia saturated with vapor, launch, take out, dry, inspect under putting ultraviolet lamp, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the fluorescence spot of aobvious same color,
Described cogongrass rhizome differentiates that step is as follows:
Get the Chinese medicine composition 2g for the treatment of blood urine, add ethyl acetate 20ml, ultrasonic process 10 minutes, filter, filtrate volatilizes, residue adds ethyl acetate 1ml makes it dissolve, as need testing solution, separately get cogongrass rhizome control medicinal material 0.5g, add ethyl acetate 10ml, ultrasonic process 10 minutes, filter, filtrate volatilizes, residue adds ethyl acetate 0.5ml makes it dissolve, medicinal material solution in contrast, test according to thin-layered chromatography, draw need testing solution and each 8 ~ 10 μ l of control medicinal material solution, put respectively on same silica gel g thin-layer plate, with methylene chloride: ethyl acetate=4:1 is developping agent, launch, take out, dry, spray is with 10% sulfuric acid ethanol, 105 DEG C to be heated to spot development clear, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color,
Described kidney tea differentiates that step is as follows:
Get the Chinese medicine composition 5g for the treatment of blood urine, add water 20ml, ultrasonic process 25 minutes, centrifugal, get the jolting of supernatant ethyl acetate and extract 2 times, each 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds ethyl acetate 1ml makes it dissolve, as need testing solution, separately get kidney tea control medicinal material 2g, add water 40ml, boil 30 minutes, filter, filtrate is concentrated into 20ml, 2 times are extracted with ethyl acetate jolting, each 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds ethyl acetate 1ml makes it dissolve, make control medicinal material solution, test according to thin-layered chromatography, draw need testing solution and each 5 ~ 10 μ l of control medicinal material solution, put respectively on same silica gel g thin-layer plate, with toluene: ethyl acetate: formic acid=20:20:1 is developping agent, launch, take out, dry, inspect under putting ultraviolet lamp, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the fluorescence principal spot of aobvious same color,
In described field thistle, linarin differentiates that step is as follows:
Get the Chinese medicine composition 0.5g for the treatment of blood urine, add methyl alcohol 10ml, ultrasonic process 30 minutes, filter, filtrate evaporate to dryness, residue water 3ml makes dissolving, be added on polyamide column, with water 50ml wash-out, discard water elution liquid, use ethanol 50ml wash-out again, collect eluent, evaporate to dryness, residue adds methyl alcohol 1ml makes it dissolve, as need testing solution, separately get linarin reference substance, add methyl alcohol and make the solution of every lml containing 0.5mg, product solution in contrast, test according to thin-layered chromatography, draw need testing solution and each 1 μ l of control medicinal material solution, put respectively on same polyamide film, with diacetone: butanone: ethanol: water=1:3:3:13 is developping agent, launch, take out, dry, spray is with aluminium choride test solution, dry 1 hour, inspect under putting ultraviolet lamp, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color.
The present invention also provides a kind of method of the Rosmarinic acid in the Berberine hydrochloride in golden cypress and kidney tea being carried out to assay, and content assaying method adopts high performance liquid chromatography, and step is as follows:
The assay of the Berberine hydrochloride in described golden cypress:
High-efficient liquid phase chromatogram condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With acetonitrile: 0.1% phosphoric acid solution=45:55 is mobile phase; 0.1% phosphoric acid solution of every 100ml adds sodium dodecylsulphonate 0.1g, and determined wavelength is 349nm, and number of theoretical plate calculates should be not less than 4000 by Berberine hydrochloride peak;
The preparation of reference substance solution: get Berberine hydrochloride reference substance, accurately weighed, add hydrochloric acid: the mixed solution of methyl alcohol=1:100 makes the solution of 1ml containing 0.1mg, to obtain final product;
The preparation of need testing solution: the Chinese medicine composition getting the treatment blood urine under content uniformity item, mixing, gets 0.3g, accurately weighed, put in tool plug conical flask, precision adds hydrochloric acid: the mixed solution 50ml of methyl alcohol=1:100, weighed weight, ultrasonic process 40 minutes, lets cool, with hydrochloric acid: the mixed solution of methyl alcohol=1:100 supplies the weight of less loss, shake up, filter, get subsequent filtrate, to obtain final product;
Determination method: accurate absorption reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product;
The assay of the Rosmarinic acid in described kidney tea:
High-efficient liquid phase chromatogram condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take acetonitrile as mobile phase A, with 0.1% phosphoric acid solution for Mobile phase B, eluent gradient is 0min, and mobile phase A is 9%, and Mobile phase B is 91%; Eluent gradient is 40min, and mobile phase A is 30%, and Mobile phase B is 70%; Determined wavelength is that 330nm number of theoretical plate calculates should be not less than 4000 by Rosmarinic acid peak;
The preparation of reference substance solution: get Rosmarinic acid reference substance, accurately weighed, add 50% methyl alcohol and make the solution of 1ml containing 20 μ g, to obtain final product;
The preparation of need testing solution: the Chinese medicine composition getting treatment blood urine, mixing, gets 2g, accurately weighed, put in tool plug conical flask, precision adds 50% methyl alcohol 50ml, weighed weight, put in water-bath and add hot reflux 1 hour, let cool, supply the weight of less loss with 50% methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;
Determination method: accurate absorption reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product
Every that treats the Chinese medicine composition of blood urine contains golden cypress in Berberine hydrochloride, must not be less than 2.5mg this product every and contain kidney tea in Rosmarinic acid, must not be less than 0.08mg.
Compared with prior art, beneficial effect of the present invention is:
1. in the present invention, thin-layered chromatography qualitative detection goes out to treat the middle cogongrass rhizome of the Chinese medicine composition of blood urine, golden cypress, kidney tea and field thistle, and the method is simple, stable, as the qualitative checking method of the Chinese medicine composition for the treatment of blood urine.The content of the Rosmarinic acid in the Berberine hydrochloride in high effective liquid chromatography for measuring golden cypress and kidney tea, method is easy, repeatability and good stability, and result accurately and reliably, as the detection method of content of the Chinese medicine composition for the treatment of blood urine, enhance rationality and the reliability of whole method of quality control.
2. the indentification by TLC of pair cogongrass rhizome is optimized, and spot launches effective.
Accompanying drawing explanation
Fig. 1: exhibition distance 10cm, temperature: 25 DEG C, humidity: 38%, developping agent: methylene chloride: methyl alcohol=9:1
1. negative sample lacks cogongrass rhizome; 2. cogongrass rhizome control medicinal material lot number: 121145-200502; 3. treat the lot number of the Chinese medicine composition of blood urine: 20150101; 4. treat the lot number of the Chinese medicine composition of blood urine: 20141015; 5. treat the lot number of the Chinese medicine composition of blood urine: 20150104;
Fig. 2: exhibition distance 10cm, temperature: 26 DEG C, humidity: 36%, developping agent: methylene chloride: ethyl acetate=4:1
1. negative sample lacks cogongrass rhizome; 2. cogongrass rhizome control medicinal material lot number: 121145-200502; 3. treat the lot number of the Chinese medicine composition of blood urine: 20150101; 4. treat the lot number of the Chinese medicine composition of blood urine: 20141015; 5. treat the lot number of the Chinese medicine composition of blood urine: 20150104;
Fig. 3: exhibition is apart from 9cm; Temperature: 15 DEG C, humidity: 66%; Developping agent: methylene chloride: ethyl acetate=4:1
1. cogongrass rhizome control medicinal material lot number: 121145-200502; 2. negative sample lacks the lot number that cogongrass rhizome 3. treats the Chinese medicine composition of blood urine: 20150101; 4. treat the lot number of the Chinese medicine composition of blood urine: 20141015; 5. treat the lot number of the Chinese medicine composition of blood urine: 20150104;
Fig. 4: exhibition is apart from 9cm; Temperature: 8 DEG C, humidity: 50%; Developping agent: methylene chloride: ethyl acetate=4:1
1. cogongrass rhizome control medicinal material lot number: 121145-200502; 2. negative sample lacks cogongrass rhizome; 3. treat the lot number of the Chinese medicine composition of blood urine: 20150101; 4. treat the lot number of the Chinese medicine composition of blood urine: 20141015; 5. treat the lot number of the Chinese medicine composition of blood urine: 20150104;
Fig. 5: 1 ~ 3,5 ~ 11 test sample lot numbers of Chinese medicine composition being followed successively by treatment blood urine: 20150101,20150102,20150103,20141011,20141012,20141013,20141014,20141015,20150104,20140304,4. cogongrass rhizome control medicinal material;
Fig. 6: exhibition is apart from 12cm; Temperature: 18 DEG C, humidity: 44%; Developping agent: toluene: ethyl acetate: formic acid=20:20:1
1. treat the lot number of the Chinese medicine composition of blood urine: 20150101; 2. treat the lot number of the Chinese medicine composition of blood urine: 20141015; 3. treat the lot number of the Chinese medicine composition of blood urine: 20150104; 4. negative sample lacks kidney tea; 5. kidney tea control medicinal material lot number: 121326-200301;
Fig. 7: exhibition is apart from 9cm; Temperature: 15 DEG C, humidity: 68%; Developping agent: toluene: ethyl acetate: formic acid=20:20:1
1. treat the lot number of the Chinese medicine composition of blood urine: 20150101; 2. treat the lot number of the Chinese medicine composition of blood urine: 20141015; 3. kidney tea medicinal material contrast lot number: 121326-200301; 4. negative sample lacks kidney tea; 5. treat the lot number of the Chinese medicine composition of blood urine: 20150104;
Fig. 8: exhibition is apart from 9cm; Temperature: 8 DEG C, humidity: 50%; Developping agent: toluene: ethyl acetate: formic acid=20:20:1
1. treat the lot number of the Chinese medicine composition of blood urine: 20150101; 2. treat the lot number of the Chinese medicine composition of blood urine: 20141015; 3. treat the lot number of the Chinese medicine composition of blood urine: 20150104; 4. kidney tea medicinal material contrast lot number 121326-200301; 5. negative sample lacks kidney tea;
Fig. 9: 1. negative sample lacks kidney tea; 2. kidney tea control medicinal material; The test sample lot number of the Chinese medicine composition of 3 ~ 12. treatment blood urines is followed successively by: 20150101,20150102,20150103,20141011,20141012,20141013,20141014,20141015,20150104,20140304;
Figure 10: exhibition is apart from 9cm; Temperature: 19 DEG C, humidity: 55%; Developping agent: ethyl acetate: formic acid-water=8:1:1
1. treat the lot number of the Chinese medicine composition of blood urine: 20150101; 2. negative sample lacks field thistle; 3. field thistle control medicinal material lot number: 121436-201102; 4. linarin reference substance lot number 111528-201308;
Figure 11: exhibition is apart from 9cm; Temperature: 18 DEG C, humidity: 44%; Developping agent: ethyl acetate: formic acid-water=8:1:1
1. treat the lot number of the Chinese medicine composition of blood urine: 20150101; 2. treat the lot number of the Chinese medicine composition of blood urine: 20141015; 3. treat the lot number of the Chinese medicine composition of blood urine: 20150104; 4. linarin reference substance lot number 111528-201308; 5. negative sample lacks field thistle;
Figure 12: exhibition is apart from 9cm; Temperature: 18 DEG C, humidity: 46%; Developping agent: diacetone: butanone: ethanol: water=1:3:3:13
1. property sample lacks field thistle; 2. linarin reference substance lot number 111528-201308; 3. treat the lot number of the Chinese medicine composition of blood urine: 20150101; 4. treat the lot number of the Chinese medicine composition of blood urine: 20141015; 5. treat the lot number of the Chinese medicine composition of blood urine: 20150104;
Figure 13: exhibition is apart from 9cm; Temperature: 15 DEG C, humidity: 68%; Developping agent: diacetone: butanone: ethanol: water=1:3:3:13
1. treat the lot number of the Chinese medicine composition of blood urine: 20150101; 2. treat the lot number of the Chinese medicine composition of blood urine: 20141015; 3. treat the lot number of the Chinese medicine composition of blood urine: 20150104; 4. negative sample lacks field thistle; 5. linarin reference substance;
Figure 14: exhibition is apart from 9cm; Temperature: 8 DEG C, humidity: 50%; Developping agent: diacetone: butanone: ethanol: water=1:3:3:13
1. treat the lot number of the Chinese medicine composition of blood urine: 20150101; 2. treat the lot number of the Chinese medicine composition of blood urine: 20141015; 3. treat the lot number of the Chinese medicine composition of blood urine: 20150104; 4. negative sample lacks field thistle; 5. linarin reference substance;
Figure 15: 1 ~ 10 test sample lot numbers of Chinese medicine composition being followed successively by treatment blood urine: 20150101,20150102,20150103,20141011,20141012,20141013,20141014,20141015,20150104,20140304; 11. negative samples lack field thistle, 12. linarin reference substances;
Figure 16 Berberine hydrochloride reference substance high-efficient liquid phase chromatogram;
Figure 17 Berberine hydrochloride test sample high-efficient liquid phase chromatogram;
Figure 18 lacks golden cypress blank sample high-efficient liquid phase chromatogram;
Figure 19 Rosmarinic acid reference substance high-efficient liquid phase chromatogram;
Figure 20 Rosmarinic acid test sample high-efficient liquid phase chromatogram;
Figure 21 lacks kidney tea blank sample high-efficient liquid phase chromatogram.
Specific embodiment
Instrument and reagent
Balance: SARTORIUSBS224S electronic balance;
Instrument: UE10SFD Ultrasound Instrument;
Thin layer plate: MERCKTLCSilicagel60;
Cogongrass rhizome control medicinal material, National Institute for Food and Drugs Control, lot number 121145-200502;
Kidney tea control medicinal material, Nat'l Pharmaceutical & Biological Products Control Institute, lot number 121326-200301;
Field thistle control medicinal material, Nat'l Pharmaceutical & Biological Products Control Institute, lot number 121436-201102;
Linarin reference substance, Nat'l Pharmaceutical & Biological Products Control Institute, lot number 111528-201308;
The sample of the Chinese medicine composition for the treatment of blood urine: provide by Lixiang Pharmaceutical Ind. Co. Ltd., lot number is respectively: 20150101,20150102,20150103,20141011,20141012,20141013,20141014,20141015,20150104,20140304,20120102,20120302,20120301,20120106,20120104,20120105,20120303,20120306,20130903,20130904;
Negative sample: use medicinal material to provide by Lixiang Pharmaceutical Ind. Co. Ltd.;
It is pure that reagent is analysis.
Embodiment 1
The indentification by TLC of 1 cogongrass rhizome
The revision of 1.1 cogongrass rhizome TLC Identifications
During to cogongrass rhizome TLC test, with methylene chloride: methyl alcohol=9:1 is developping agent, after launching, spray is with 10% sulfuric acid ethanol, and 105 DEG C to be heated to spot development clear, and result is see Fig. 1.Principal spot position is high, launches effect bad.Use methylene chloride instead: ethyl acetate=4:1 is developping agent, and result is see Fig. 2.Result display thin layer clear spot, and negative control is noiseless, therefore adopts methylene chloride: ethyl acetate=4:1 is that developping agent carries out cogongrass rhizome indentification by TLC.
The cogongrass rhizome TLC Identification finally determined is as follows:
Get the Chinese medicine composition 2g of this product treatment blood urine, place in tool plug conical flask, add ethyl acetate 20ml, ultrasonic process 10 minutes, power 250W, frequency 40kHz, filter, filtrate volatilizes, and residue adds ethyl acetate 1ml makes dissolving, as need testing solution.Separately get cogongrass rhizome control medicinal material 0.5g, add ethyl acetate 10ml, ultrasonic process 10 minutes, power 250W, frequency 40kHz, filter, filtrate volatilizes, and residue adds ethyl acetate 0.5ml makes dissolving, medicinal material solution in contrast.According to thin-layered chromatography test, draw need testing solution and each 8 ~ 10 μ l of control medicinal material solution, put respectively on same silica gel g thin-layer plate, with methylene chloride: ethyl acetate=4:1 is developping agent, launch, take out, dry, spray with 10% sulfuric acid ethanol, 105 DEG C to be heated to spot development clear.In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.
The serviceability test of 1.2 cogongrass rhizome TLC Identifications
Investigate under different temperature and humidity conditions by set up cogongrass rhizome TLC Identification: result shows, Fig. 2, Fig. 3, Fig. 4 is seen under different temperature and humidity conditions, in test sample chromatogram, on the position corresponding to cogongrass rhizome control medicinal material, all can show the spot of same color.
The discriminating of the middle cogongrass rhizome thin-layer chromatography of the Chinese medicine composition of 1.310 batches for the treatment of blood urines
Get in accordance with the law testing of the Chinese medicine composition of 10 batches for the treatment of blood urines, result is see Fig. 5.Result shows in the test sample chromatogram of the Chinese medicine composition of 10 batches for the treatment of blood urines, on the position corresponding to cogongrass rhizome control medicinal material chromatogram, and the spot of all aobvious same color, and good separating effect, clear spot.
By the test of specificity, durability and 10 batch samples, in result display test sample chromatogram, on the position corresponding to cogongrass rhizome control medicinal material chromatogram, the spot of all aobvious same color, and good separating effect, clear spot, negative control is noiseless, the method is easy and simple to handle, and specificity is strong, favorable reproducibility.
Embodiment 2
The foundation of 2 kidney tea TLC Identifications
The 2.1 Chinese medicine composition lot numbers getting treatment blood urine are 20130903,20130904,20131012 each 5g, add water 20ml, ultrasonic process 25 minutes, centrifugal, get the jolting of supernatant ethyl acetate and extract 2 times, each 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds ethyl acetate 1ml and dissolves, as need testing solution.Separately get kidney tea control medicinal material 2g, add water 40ml, boils 30 minutes, and filter, filtrate is concentrated into about 20ml, extracts 2 times with ethyl acetate jolting, each 20ml, combined ethyl acetate liquid, evaporate to dryness, and residue adds ethyl acetate 1ml and dissolves, and makes control medicinal material solution.Thin-layered chromatography is tested, and draws need testing solution and each 5 ~ 10 μ l of control medicinal material solution, puts respectively on same silica gel g thin-layer plate, with toluene: ethyl acetate: formic acid=20:20:1 is developping agent, launch, take out, dry, inspect under putting 365nm ultraviolet lamp.In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the fluorescence principal spot of aobvious same color.Result is see Fig. 6.In result display test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the principal spot of aobvious same color, and good separating effect, clear spot, negative control is noiseless.
The serviceability test of 2.2 kidney tea TLC Identifications
Investigate under different temperature and humidity conditions by set up kidney tea TLC Identification: Fig. 6, Fig. 7, Fig. 8 result shows, under different temperature and humidity environment, in test sample chromatogram, on the position corresponding to kidney tea control medicinal material, all can show the fluorescence principal spot of same color.
The discriminating of the middle kidney tea thin-layer chromatography of the Chinese medicine composition of 2.310 batches for the treatment of blood urines
Get in accordance with the law testing of the Chinese medicine composition of 10 batches for the treatment of blood urines, result is see Fig. 9.Result shows in the test sample chromatogram of the Chinese medicine composition of 10 batches for the treatment of blood urines, and on the position corresponding to kidney tea control medicinal material chromatogram, the fluorescence principal spot of all aobvious same color, and good separating effect, clear spot, negative control is noiseless.
Tested by specificity, durability and 10 batch samples, in result display test sample chromatogram, on the position corresponding to kidney tea control medicinal material chromatogram, the fluorescence principal spot of all aobvious same color, and good separating effect, clear spot, negative control is noiseless, the method is easy and simple to handle, and specificity is strong, favorable reproducibility.
Embodiment 3
The foundation of 3 field thistle TLC Identifications
Linarin in 3.1 field thistles is one of compound that in field thistle, in numerous flavone compound, activity is high, is also the flavones ingredient that in field thistle medicine materical crude slice, content is the highest.Now plan to build the TLC Identification of field thistle and linarin in vertical preparation.
Get the Chinese medicine composition 0.5g of this product treatment blood urine, add methyl alcohol 10ml, ultrasonic process 30 minutes, filter, filtrate evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Separately get field thistle control medicinal material 1g, add methyl alcohol 10ml, ultrasonic process 30 minutes, filter, filtrate evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, makes control medicinal material solution.Get linarin reference substance again, add methyl alcohol and make the solution of every lml containing 0.5mg, product solution in contrast.Test according to thin-layered chromatography, draw need testing solution and each 1 μ l of control medicinal material solution, put respectively on same polyamide film, with ethyl acetate: formic acid: water=8:1:1 is developping agent, launch, take out, dry, spray, with aluminium choride test solution, is dried about 1 hour, is put ultraviolet lamp 365nm and inspect.Result is see Figure 10.
In test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color, lacks in the negative sample of field thistle and also has no interference.On the position corresponding to control medicinal material chromatogram, the only fluorescence spot of aobvious 2 same colors, and negative control has interference.Therefore in this indentification by TLC, only retain linarin reference substance contrast.
Because background color in test sample is dark, now test sample is further processed, by need testing solution evaporate to dryness, residue water 3ml makes dissolving, is added in 14 ~ 30 orders, 2g, internal diameter is on the polyamide column of 1cm, with water 50ml wash-out, discard water elution liquid, then use ethanol 50ml wash-out, collect eluent, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.All the other operations are the same, and result is see Figure 11.In result display test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color, but spot and R f (Rf value) value is high, and be separated unintelligible with adjacent spots, therefore reselect developping agent.Get reference substance solution and need testing solution developping agent diacetone: butanone: ethanol: water=1:3:3:13 tests.Result is see Figure 12.In result display test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color, and good separating effect, clear spot, negative control is noiseless.
Other developping agents are adopted to test: ethyl acetate: butanone: methenyl choloride: formic acid: water=15:15:6:4:1, methylene chloride: ethyl acetate: butanone: formic acid: water=8:15:15:2:1, the spot of result test sample is separated bad, there is interference.
In the field thistle finally determined, linarin TLC Identification is as follows:
Get the Chinese medicine composition 0.5g of this product treatment blood urine, add methyl alcohol 10ml, ultrasonic process 30 minutes, filter, filtrate evaporate to dryness, residue water 3ml makes dissolving, be added in 14 ~ 30 orders, 2g, internal diameter is 1cm is on polyamide column, with water 50ml wash-out, discard water elution liquid, then use ethanol 50ml wash-out, collect eluent, evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Separately get linarin reference substance, add methyl alcohol and make the solution of every lml containing 0.5mg, product solution in contrast.Test according to thin-layered chromatography, draw need testing solution and each 1 μ l of control medicinal material solution, put respectively on same polyamide film, with diacetone: butanone: ethanol: water=1:3:3:13 is developping agent, launch, take out, dry, spray, with aluminium choride test solution, is dried about 1 hour, is inspected under putting 365nm ultraviolet lamp.In test sample chromatogram, on the position corresponding to reference substance chromatogram, see the fluorescence spot of same color.
The serviceability test of 3.2 field thistle TLC Identifications
Investigate under different temperature and humidity conditions by set up field thistle TLC Identification: Figure 12, Figure 13, Figure 14 result shows, under different temperature and humidity environment, in test sample chromatogram, on the position corresponding to linarin reference substance, all can show the fluorescence spot of same color.
The discriminating of the middle field thistle thin-layer chromatography of the Chinese medicine composition of 3.310 batches for the treatment of blood urines
Get in accordance with the law testing of the Chinese medicine composition of 10 batches for the treatment of blood urines, result is see Figure 15.Result shows in the test sample chromatogram of the Chinese medicine composition of 10 batches for the treatment of blood urines, and on the position corresponding to linarin reference substance chromatogram, the fluorescence spot of all aobvious same color, and good separating effect, clear spot, negative control is noiseless.
By the test of specificity, durability and 10 batch samples, in result display test sample chromatogram, on the position corresponding to linarin reference substance chromatogram, the spot of all aobvious same color, and good separating effect, clear spot, negative control is noiseless, the method is easy and simple to handle, and specificity is strong, favorable reproducibility.
Embodiment 4
4.1 instruments and reagent
High performance liquid chromatograph: Agilent1200 type high performance liquid chromatograph
AgilentZORBAXSB-C
18performance liquid chromatographic column
Acetonitrile, methyl alcohol, sodium dodecylsulphonate are chromatographically pure, and water is ultrapure water, and it is pure that other reagent is analysis.
Balance: Sai Duolisi electronic balance, TB-215D electronic analytical balance.
Ultrasound Instrument: AS7240AUltrasonicCleaner
Berberine hydrochloride reference substance: Nat'l Pharmaceutical & Biological Products Control Institute, lot number 110713-200911, for assay, purity 86.8%, without the need to process before using.
The Chinese medicine composition for the treatment of blood urine: provide lot number by Lixiang Pharmaceutical Ind. Co. Ltd.: 20150101,20150102,20150103,20141011,20141012,20141013,20141014,20141015,20150104,20140304,2012010243,2012030299,20120301100,20120106101,20120104102,20120105103,20120303104,20120306113,2013090315,20130904173
Prepare the medicinal material of negative control: provide by by Lixiang Pharmaceutical Ind. Co. Ltd..
The preparation of 4.2 solution
4.2.1 the preparation of reference substance solution
Precision takes Berberine hydrochloride reference substance 0.03770g, puts in 50ml measuring bottle, adds hydrochloric acid: methyl alcohol=1:100 mixed solution dissolves and to scale, shakes up, and obtaining concentration is 0.6545mgml
-1reference substance solution A.Precision measures reference substance solution A5ml and puts in 50ml measuring bottle, adds hydrochloric acid: methyl alcohol=1:100 mixed solution dissolves and to scale, shakes up, and obtaining concentration is 65.45 μ gml
-1reference substance solution B.
4.2.2 the preparation of need testing solution
With hydrochloric acid: methyl alcohol=1:100 mixed solution is Extraction solvent, adopts the method for ultrasonic process as extracting method.
Get the lot number of the Chinese medicine composition for the treatment of blood urine: under 2012010243 content uniformity items, mixing, gets about 0.3g, accurately weighed, precision adds hydrochloric acid: methyl alcohol=1:100 mixed solution 50ml, put in tool plug conical flask, weighed weight, ultrasonic 10,20,30,40,50,60 minutes respectively, power 250W, frequency 40kHz, obtained need testing solution, result is see table 1.
Table 1 measures the investigation of the need testing solution ultrasonic time of Berberine hydrochloride
Seen by measurement result, ultrasonic 40 minutes little with 50 minutes difference, within ultrasonic 60 minutes, can propose the Berberine hydrochloride in preparation completely, but due to overlong time, ultrasonic 40 minutes of golden cypress employing as extraction time.
Compare through above test, the preparation method of need testing solution is defined as: the Chinese medicine composition getting this product treatment blood urine under content uniformity item, mixing, get about 0.3g, accurately weighed, put in tool plug conical flask, precision adds hydrochloric acid: methyl alcohol=1:100 mixed solution 50ml, weighed weight, ultrasonic process 40 minutes, let cool, with hydrochloric acid: methyl alcohol=1:100 mixed solution supplies the weight of less loss, shakes up, filter, get subsequent filtrate, to obtain final product.
4.2.3 the preparation of negative sample solution
Get other medicinal material in prescription except golden cypress, by prescribed dose and method for making and technological requirement preparation containing the blank sample of golden cypress, the preparation method according to above-mentioned 4.2.2 need testing solution makes negative sample solution.
4.3 chromatographic condition
4.3.1 the confirmation of maximum absorption wavelength
Get reference substance solution, scan by ultraviolet spectrophotometry in the wavelength coverage of 190nm-400nm, result is presented at 348.8nm wavelength place and has absorption maximum.Therefore choosing 349nm is determined wavelength.
4.3.2 the selection of mobile phase
The mobile phase adopted: acetonitrile: 0.1% phosphoric acid solution=45:55.0.1% phosphoric acid solution, every 100ml adds sodium dodecylsulphonate 0.1g.Result shows, when adopting above-mentioned mobile phase, survey the degree of separation at composition Berberine hydrochloride peak and satisfied result can be obtained analysis time.
4.3.3 the determination of chromatographic condition
Chromatographic column: AgilentZORBAXSB-C
18(150mm × 4.6mm, 5 μm);
Mobile phase: acetonitrile: 0.1% phosphoric acid solution=45:55; 0.1% phosphoric acid solution of every 100ml adds sodium dodecylsulphonate 0.1g
Flow velocity: 1.0ml/min;
Determined wavelength: 349nm.
4.4 specificity tests
Get reference substance solution, need testing solution and negative control solution, measure by chromatographic condition under 4.3.3 item, result is in the retention time position identical with reference substance Berberine hydrochloride, occur without other Interference Peaks, show other medicinal materials except golden cypress and the mensuration of auxiliary material to Berberine hydrochloride noiseless, result is see Figure 16,17,18.
Accurate absorption reference substance solution A is 0.6545mgml
-1each 2,5,10,15 μ l, reference substance solution B concentration is 65.45 μ gml
-1each 2,5,10,15 μ l, injection liquid chromatography, measures its peak area respectively by chromatographic condition under 4.3.3 item.With reference substance quality X for horizontal ordinate, integrating peak areas value Y is ordinate, drawing standard curve.Calculate, obtain linear regression equation: Y=4070.73X+52.22, correlation coefficient r
2=1.0000.Show that Berberine hydrochloride has good linear relationship with integrating peak areas value within the scope of 0.98 ~ 9.82 μ g.Determination data is in table 2.
Table 2 Berberine hydrochloride linear relationship is investigated
4.5 precision test
By chromatographic condition under 4.3.3 item, accurate absorption concentration is 65.45 μ gml
-1reference substance solution B, continuous sample introduction 6 times, each 5 μ l, carry out efficient liquid phase mensuration in accordance with the law, measure integrating peak areas value and are respectively 1351.80,1352.20,1355.76,1358.87,1359.88,1351.92.Mean value is 1355.07, RSD (relative standard deviation) is 0.27%.
4.6 replica test
Get the lot number of the Chinese medicine composition for the treatment of blood urine: 2012010243, by legal system available test sample solution 6 parts below 4.2.2 item.By chromatographic condition under 4.3.3 item, get Berberine hydrochloride reference substance solution and be measured in the same method, calculate content of berberine hydrochloride respectively, measurement result is see table 3.Reference substance solution concentration is 65.45 μ gml
-1, average peak area is: 1355.07.
Table 3 Berberine hydrochloride replica test result
Result mean value is 9.3987mg/g, RSD is 0.14%, illustrates that this method is reproducible.
4.7 stability test
Get the lot number of the Chinese medicine composition for the treatment of blood urine: 2012010243 need testing solutions, by chromatographic condition under 4.3.3 item respectively 0,5,10,15,20, within 25 hours, sample introduction measures, and result Berberine hydrochloride peak area is respectively 1147.15,1146.53,1147.71,1152.82,1148.52 and 1149.63, average peak area is 1148.73, RSD is 0.20%, shows that need testing solution is stable in 25 hours.
4.8 accuracy test-application of sample recovery test
It is 0.6545mgml that precision measures Berberine hydrochloride reference substance A
-16 parts, every part of 2mL, put respectively in conical flask, low temperature volatilizes, and precision takes the lot number of the Chinese medicine composition of the treatment blood urine of 4.7 lower replica test known content: 2012010243, gets about 0.15g, add in 6 conical flasks respectively, make test solution by the method process under 4.2.2 item, then under pressing 4.3.3 item, chromatographic condition measures content, get reference substance solution concentration is 65.45 μ gml simultaneously
-1, average peak area is: 1355.07 calculate.The average recovery rate obtaining Berberine hydrochloride is 104.6%, and result is see table 4.
Table 4 Berberine hydrochloride application of sample recovery test result
Test findings, average recovery rate is 100.6%; RSD=0.69%.Show that this law is applicable to the assay of the middle Berberine hydrochloride of the Chinese medicine composition for the treatment of blood urine, and obtain good result.
4.9 sample size
Get the Chinese medicine composition of the treatment blood urine of 20 batches of different lot numbers respectively, by chromatographic condition under 4.3.3 item, measure content of berberine hydrochloride.Get reference substance solution concentration is 65.45 μ gml simultaneously
-1, average peak area is: 1355.07, calculates.Test findings is see table 5.
Table 5 sample size measurement result
Test findings, average content is 3.76mg/ grain.In primary standard, the content of Berberine hydrochloride must not be defined as and is less than 2.5mg/ grain, and the content of the Chinese medicine composition of these 20 batches treatment blood urines are all in limit.
Embodiment 5
The assay of Rosmarinic acid
According to the discussion of the Dai nationality's medical science to this prescription, think that kidney tea bitter cool in nature, merit are to rush down clearly tower phenanthrene i.e. fire poison, Li Shui, hemostasia and detumescence, for controlling nephritic dropsy, the key medicine of hematuria, blood urine.Namely kidney tea is the monarch drug in a prescription of said preparation.Therefore increasing the assay item of kidney tea, is the key of the validity ensureing preparation.Rosmarinic acid is the principal ingredient in kidney tea, now uses high performance liquid chromatography to measure the Rosmarinic acid in preparation.
5.1 instruments and reagent
5.1.1 instrument and test condition
High performance liquid chromatograph: Agilent1200 type high performance liquid chromatograph.
AgilentZORBAXSB-C
18performance liquid chromatographic column.
Acetonitrile is chromatographically pure, and water is ultrapure water, and it is pure that other reagent is analysis.
Balance: Sai Duolisi electronic balance, TB-215D electronic analytical balance.
Ultrasound Instrument: AS7240AUltrasonicCleaner.
5.1.2 reagent and sample
Rosmarinic acid reference substance: National Institute for Food and Drugs Control, lot number 111871-201404, for assay, content, in 98.6%, uses front without the need to process.
The Chinese medicine composition for the treatment of blood urine: sample provides by Lixiang Pharmaceutical Ind. Co. Ltd..Lot number: 20150101,2150102,20150103,20141011,20141012,20141013,20141014,20141015,20150104,20140304.
Prepare the medicinal material of negative control: provide by Lixiang Pharmaceutical Ind. Co. Ltd..
5.2 chromatographic condition
5.2.1 the confirmation of maximum absorption wavelength
Get reference substance solution, scan by ultraviolet spectrophotometry in the wavelength coverage of 190nm-400nm, result shows it and has absorption maximum at 330nm wavelength place.Therefore 330nm is selected to be determined wavelength.
5.2.2 the determination of chromatographic condition
Chromatographic column: AgilentZORBAXSB-C
18(150mm × 4.6mm, 5 μm);
Flow velocity: 1.0ml/min;
Determined wavelength: 330nm.
Mobile phase:
5.3 solution preparations
5.3.1 the preparation of reference substance solution
Get Rosmarinic acid reference substance 0.02312g, put in 25ml measuring bottle, add methyl alcohol dissolve and to scale, obtaining concentration is 0.9119mgml
-1reference substance solution A.Separately get reference substance solution A5ml to put in 10ml measuring bottle, add methanol dilution to scale, shake up, obtaining concentration is 0.45595mgml
-1reference substance solution B.Separately get reference substance solution A1ml to put in 50ml measuring bottle, add methanol dilution to scale, shake up, obtaining concentration is 18.238 μ gml
-1reference substance solution C.
5.3.2 the preparation of need testing solution
5.3.2.1 need testing solution extracts the selection of solvent and extracting method
Get the lot number of the Chinese medicine composition for the treatment of blood urine: 20150101, mixing, gets about 1g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol and 50% methanol solution 50ml respectively, weighed weight, put respectively and water-bath adds hot reflux 30 minutes or ultrasonic process 30 minutes, let cool, supply less loss weight with coordinative solvent, shake up, filter with the miillpore filter of 0.45um, get subsequent filtrate, obtained need testing solution.
Table 6 extracts the selection result table of solvent and extracting method
Result shows, and adopts 50% methyl alcohol to be Extraction solvent, and measured by the extracting method adding hot reflux, rosmarinic acid contents is the highest.
5.3.2.2 the investigation of need testing solution extraction time
Get the lot number of the Chinese medicine composition for the treatment of blood urine: the Chinese medicine composition of 20150101 treatment blood urines, mixing, gets about 2g, accurately weighed, put in tool plug conical flask, precision adds 50% methanol solution 50ml, weighed weight, put respectively in water-bath and add hot reflux 30,40,60,90 minutes, let cool, supply less loss weight with 50% methanol solution, shake up, filter with the miillpore filter of 0.45um, get subsequent filtrate, obtained need testing solution.Measure by 5.2 chromatographic conditions, result is see table 7.
The investigation result table of table 7 extraction time
Result shows, and adds the Rosmarinic acid that hot reflux can propose to treat in the Chinese medicine composition of blood urine for 60 minutes completely.
5.3.2.3 the investigation of need testing solution sampling amount
Get the lot number of the Chinese medicine composition for the treatment of blood urine: 20150101, mixing, gets about 1g, 2g, 3g, 4g, accurately weighed, put in tool plug conical flask, precision adds 50% methanol solution 50ml respectively, weighed weight, put respectively in water-bath and add hot reflux 60 minutes, let cool, supply less loss weight with 50% methanol solution, shake up, filter with the miillpore filter of 0.45um, get subsequent filtrate, obtained need testing solution.Measure by 5.2 chromatographic conditions, result is see table 8.
The investigation result table of table 8 extraction time
Measurement result shows, and sampling amount is that the measurement result of 1g and 2g is high, but sampling amount to be the integrating peak areas value of 1g low, error is large, selects 2g to be final sampling amount.
5.3.2.4 the brief summary prepared of need testing solution
Compare through above test, determine that the extracting method of need testing solution is: the Chinese medicine composition getting treatment blood urine, mixing, get about 2g, accurately weighed, place in tool plug conical flask, precision adds 50% methanol solution 50ml, weighed weight, puts in water-bath and adds hot reflux 1 hour, let cool, supply less loss weight with 50% methanol solution, shake up, filter with the miillpore filter of 0.45um, get subsequent filtrate, obtain need testing solution.
5.3.3 the preparation of negative sample solution
Get other medicinal material in prescription except kidney tea, by prescribed dose and method for making and technological requirement preparation containing the blank sample of kidney tea, make negative sample solution according to the preparation method of need testing solution under 5.3.2 item.
5.4 specificity tests
Get reference substance solution, need testing solution and negative control solution, measure by chromatographic condition under 5.2.2 item, result is in the retention time position identical with reference substance Rosmarinic acid, occur without other Interference Peaks, show other medicinal materials except kidney tea and the mensuration of auxiliary material to kidney tea noiseless, result is see Figure 19,20,21
5.5 linear relationships and the range of linearity are investigated
Accurate absorption concentration is 18.238 μ gml
-1each 2,5,10, the 15 μ l of reference substance solution C, concentration is 0.45595mgml
-1each 2,5, the 10 μ l of reference substance solution B, injection liquid chromatography, measures its peak area respectively by chromatographic condition under 5.2.2 item.With sample size X for horizontal ordinate, integrating peak areas value Y is ordinate, drawing standard curve.Calculate, obtain regression equation: Y=2997.8X+1.0688, correlation coefficient r
2=1.0000.Show that Rosmarinic acid has good linear relationship with integrating peak areas value within the scope of 0.036476 ~ 4.5595 μ g.Determination data is in table 9.
Table 9 Rosmarinic acid linear relationship is investigated
5.6 precision test
Under given chromatographic condition, accurate absorption concentration is 18.238 μ gml
-1reference substance solution C, continuous sample introduction 6 times, injection liquid chromatography, measures its peak area respectively by chromatographic condition under 5.2.2 item.Measure integrating peak areas value and be respectively 549.38,549.59,552.40,551.16,550.29,551.32.Mean value is 550.69, RSD is 0.21%.
5.7 replica test
Get the lot number of the Chinese medicine composition for the treatment of blood urine: 20150101, by legal system available test sample solution 6 parts below 5.3.2.4 item, measure in accordance with the law.Calculate rosmarinic acid contents, measurement result is see table 10.
Table 10 Rosmarinic acid replica test result
Result mean value is 0.4084mg/g, RSD is 1.93%, illustrates that this method is reproducible.
5.8 stability test
Get the lot number of the Chinese medicine composition for the treatment of blood urine: 20150101 need testing solutions, by chromatographic condition under 5.2.2 item respectively 0,2,8,16,24, within 30 hours, sample introduction measures, and result Rosmarinic acid peak area is respectively 485.57,479.02,486.90,477.95,474.13 and 475.72, and average peak area is 479.88, RSD is 1.09%, shows that need testing solution is stable in 30 hours.
5.9 accuracy tests-application of sample recovery test
It is 0.45595mgml that precision measures concentration
-1rosmarinic acid reference substance solution B6 part, every part of 1mL, put respectively in conical flask, low temperature volatilizes, precision takes the lot number of the Chinese medicine composition of the treatment blood urine of 5.7 lower replica test known content: 20150101, gets about 1g, adds in 6 conical flasks respectively, make test solution by method process under 5.3.2.4 item, then under pressing 5.2.2 item, chromatographic condition measures content.Result is see table 11.
Table 11 Rosmarinic acid average recovery is tested
Test findings, average recovery rate is 95.61%; RSD=0.48%.
5.10 sample size measures
Get the Chinese medicine composition of 10 batches for the treatment of blood urines, by legal system available test sample solution below 5.3.2.4 item, then press chromatographic condition mensuration peak area under 5.2.2 item, calculate rosmarinic acid contents by external standard method, result is see table 12.
The rosmarinic acid contents measurement result of table 1210 batch sample
Brief summary
In 10 batches of preparations, the average content 0.1055mg/ grain of Rosmarinic acid, counts 0.0844mg/ grain with 80%, in draft standard, therefore specify that its limit is: this product every in Rosmarinic acid, must not be less than 0.08mg containing kidney tea.
Claims (2)
1. treat a quality determining method for the Chinese medicine composition of blood urine, Chinese medicine composition comprises linarin in golden cypress, cogongrass rhizome, kidney tea and field thistle, it is characterized in that, comprises and differentiating linarin in golden cypress, cogongrass rhizome, kidney tea and field thistle;
Described golden cypress differentiates that step is as follows:
Get the Chinese medicine composition 4g for the treatment of blood urine, add ammonia solution 2ml, methenyl choloride 10ml, ultrasonic process 15 minutes, filter, filtrate evaporate to dryness, residue adds methyl alcohol 1ml and dissolves, as need testing solution, separately get golden cypress control medicinal material 0.1g, add strong ammonia solution 1ml, methenyl choloride 5ml, ultrasonic process 15 minutes, filter, filtrate evaporate to dryness, residue adds methyl alcohol 1ml and dissolves, make control medicinal material solution, test according to thin-layered chromatography, draw need testing solution and each 5 μ l of control medicinal material solution, put respectively on same silica gel g thin-layer plate, with toluene: ethyl acetate: methyl alcohol: isopropyl alcohol: strong ammonia solution=6:3:1.5:1.5:0.5 is developping agent, put in the expansion cylinder of ammonia saturated with vapor, launch, take out, dry, inspect under putting ultraviolet lamp, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the fluorescence spot of aobvious same color,
Described cogongrass rhizome differentiates that step is as follows:
Get the Chinese medicine composition 2g for the treatment of blood urine, add ethyl acetate 20ml, ultrasonic process 10 minutes, filter, filtrate volatilizes, residue adds ethyl acetate 1ml makes it dissolve, as need testing solution, separately get cogongrass rhizome control medicinal material 0.5g, add ethyl acetate 10ml, ultrasonic process 10 minutes, filter, filtrate volatilizes, residue adds ethyl acetate 0.5ml makes it dissolve, medicinal material solution in contrast, test according to thin-layered chromatography, draw need testing solution and each 8 ~ 10 μ l of control medicinal material solution, put respectively on same silica gel g thin-layer plate, with methylene chloride: ethyl acetate=4:1 is developping agent, launch, take out, dry, spray is with 10% sulfuric acid ethanol, 105 DEG C to be heated to spot development clear, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color,
Described kidney tea differentiates that step is as follows:
Get the Chinese medicine composition 5g for the treatment of blood urine, add water 20ml, ultrasonic process 25 minutes, centrifugal, get the jolting of supernatant ethyl acetate and extract 2 times, each 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds ethyl acetate 1ml makes it dissolve, as need testing solution, separately get kidney tea control medicinal material 2g, add water 40ml, boil 30 minutes, filter, filtrate is concentrated into 20ml, 2 times are extracted with ethyl acetate jolting, each 20ml, combined ethyl acetate liquid, evaporate to dryness, residue adds ethyl acetate 1ml makes it dissolve, make control medicinal material solution, test according to thin-layered chromatography, draw need testing solution and each 5 ~ 10 μ l of control medicinal material solution, put respectively on same silica gel g thin-layer plate, with toluene: ethyl acetate: formic acid=20:20:1 is developping agent, launch, take out, dry, inspect under putting ultraviolet lamp, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the fluorescence principal spot of aobvious same color,
In described field thistle, linarin differentiates that step is as follows:
Get the Chinese medicine composition 0.5g for the treatment of blood urine, add methyl alcohol 10ml, ultrasonic process 30 minutes, filter, filtrate evaporate to dryness, residue water 3ml makes dissolving, be added on polyamide column, with water 50ml wash-out, discard water elution liquid, use ethanol 50ml wash-out again, collect eluent, evaporate to dryness, residue adds methyl alcohol 1ml makes it dissolve, as need testing solution, separately get linarin reference substance, add methyl alcohol and make the solution of every lml containing 0.5mg, product solution in contrast, test according to thin-layered chromatography, draw need testing solution and each 1 μ l of control medicinal material solution, put respectively on same polyamide film, with diacetone: butanone: ethanol: water=1:3:3:13 is developping agent, launch, take out, dry, spray is with aluminium choride test solution, dry 1 hour, inspect under putting ultraviolet lamp, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the fluorescence spot of aobvious same color.
2. detection method according to claim 1, is characterized in that, also comprises and carries out assay to the Rosmarinic acid in the Berberine hydrochloride in golden cypress and kidney tea, and content assaying method adopts high performance liquid chromatography, and step is as follows:
The assay of the Berberine hydrochloride in described golden cypress:
High-efficient liquid phase chromatogram condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With acetonitrile: 0.1% phosphoric acid solution=45:55 is mobile phase; 0.1% phosphoric acid solution of every 100ml adds sodium dodecylsulphonate 0.1g, and determined wavelength is 349nm, and number of theoretical plate calculates should be not less than 4000 by Berberine hydrochloride peak;
The preparation of reference substance solution: get Berberine hydrochloride reference substance, accurately weighed, add hydrochloric acid: the mixed solution of methyl alcohol=1:100 makes the solution of 1ml containing 0.1mg, to obtain final product;
The preparation of need testing solution: the Chinese medicine composition getting the treatment blood urine under content uniformity item, mixing, gets 0.3g, accurately weighed, put in tool plug conical flask, precision adds hydrochloric acid: the mixed solution 50ml of methyl alcohol=1:100, weighed weight, ultrasonic process 40 minutes, lets cool, with hydrochloric acid: the mixed solution of methyl alcohol=1:100 supplies the weight of less loss, shake up, filter, get subsequent filtrate, to obtain final product;
Determination method: accurate absorption reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product;
The assay of the Rosmarinic acid in described kidney tea:
High-efficient liquid phase chromatogram condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take acetonitrile as mobile phase A, with 0.1% phosphoric acid solution for Mobile phase B, eluent gradient is 0min, and mobile phase A is 9%, and Mobile phase B is 91%; Eluent gradient is 40min, and mobile phase A is 30%, and Mobile phase B is 70%; Determined wavelength is that 330nm number of theoretical plate calculates should be not less than 4000 by Rosmarinic acid peak;
The preparation of reference substance solution: get Rosmarinic acid reference substance, accurately weighed, add 50% methyl alcohol and make the solution of 1ml containing 20 μ g, to obtain final product;
The preparation of need testing solution: the Chinese medicine composition getting treatment blood urine, mixing, gets 2g, accurately weighed, put in tool plug conical flask, precision adds 50% methyl alcohol 50ml, weighed weight, put in water-bath and add hot reflux 1 hour, let cool, supply the weight of less loss with 50% methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;
Determination method: accurate absorption reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, measures, to obtain final product
Every that treats the Chinese medicine composition of blood urine contains golden cypress in Berberine hydrochloride, must not be less than 2.5mg this product every and contain kidney tea in Rosmarinic acid, must not be less than 0.08mg.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN108267537A (en) * | 2016-12-31 | 2018-07-10 | 九芝堂股份有限公司 | A kind of method of Cortex Phellodendri in discriminating shangqing pill |
| CN108535399A (en) * | 2018-04-09 | 2018-09-14 | 吉林省中研药业有限公司 | A kind of detection method of Fuyankang pill |
| CN109991327A (en) * | 2019-04-04 | 2019-07-09 | 西安医学院 | One surveys the methods for commenting method evaluation field thistle quality more |
| CN110632208A (en) * | 2019-10-10 | 2019-12-31 | 神威药业集团有限公司 | Detection method for main components of traditional Chinese medicine composition for clearing lung, eliminating phlegm, relieving cough and asthma |
| CN110927320A (en) * | 2019-02-25 | 2020-03-27 | 广州白云山医药集团股份有限公司白云山何济公制药厂 | Identification method of pseudo-ginseng ointment for treating traumatic injury and rheumatism |
| CN111505156A (en) * | 2020-05-08 | 2020-08-07 | 四川新绿色药业科技发展有限公司 | Fingerprint spectrogram quality determination method for herba Cirsii formulation granules |
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| CN108267537A (en) * | 2016-12-31 | 2018-07-10 | 九芝堂股份有限公司 | A kind of method of Cortex Phellodendri in discriminating shangqing pill |
| CN108267537B (en) * | 2016-12-31 | 2020-08-28 | 九芝堂股份有限公司 | Method for identifying phellodendron amurense in Shangqing pills |
| CN107389810A (en) * | 2017-07-03 | 2017-11-24 | 广西中医药大学 | A kind of content assaying method of strong drug composition |
| CN108535399B (en) * | 2018-04-09 | 2020-05-08 | 吉林省中研药业有限公司 | Detection method of Fuyankang pills |
| CN108535399A (en) * | 2018-04-09 | 2018-09-14 | 吉林省中研药业有限公司 | A kind of detection method of Fuyankang pill |
| CN110927320B (en) * | 2019-02-25 | 2022-03-04 | 广州白云山医药集团股份有限公司白云山何济公制药厂 | Identification method of pseudo-ginseng ointment for treating traumatic injury and rheumatism |
| CN110927320A (en) * | 2019-02-25 | 2020-03-27 | 广州白云山医药集团股份有限公司白云山何济公制药厂 | Identification method of pseudo-ginseng ointment for treating traumatic injury and rheumatism |
| CN109991327A (en) * | 2019-04-04 | 2019-07-09 | 西安医学院 | One surveys the methods for commenting method evaluation field thistle quality more |
| CN110632208A (en) * | 2019-10-10 | 2019-12-31 | 神威药业集团有限公司 | Detection method for main components of traditional Chinese medicine composition for clearing lung, eliminating phlegm, relieving cough and asthma |
| CN113125627A (en) * | 2020-01-16 | 2021-07-16 | 云南雷允上理想药业有限公司 | Detection method for effective components of traditional Chinese medicine composition for treating nephropathy |
| CN111505156A (en) * | 2020-05-08 | 2020-08-07 | 四川新绿色药业科技发展有限公司 | Fingerprint spectrogram quality determination method for herba Cirsii formulation granules |
| CN111505156B (en) * | 2020-05-08 | 2022-03-01 | 四川新绿色药业科技发展有限公司 | Fingerprint spectrogram quality determination method for herba Cirsii formulation granules |
| CN113030365A (en) * | 2021-03-10 | 2021-06-25 | 贵州百灵企业集团制药股份有限公司 | A Chinese medicinal preparation for treating excess heat and toxic fire, and excess heat in triple warmer, and its detection method |
| CN114660194A (en) * | 2022-03-16 | 2022-06-24 | 西双版纳傣族自治州民族医药研究所(西双版纳傣族自治州傣医医院) | HPLC fingerprint construction method and detection method of Yatouniu Hazhangbo prescription |
| CN114660194B (en) * | 2022-03-16 | 2023-10-10 | 西双版纳傣族自治州民族医药研究所(西双版纳傣族自治州傣医医院) | HPLC fingerprint construction method and detection method of Yakui Niu Hazhan wave prescription |
| CN114720622A (en) * | 2022-04-06 | 2022-07-08 | 贵州良济药业有限公司 | Detection method of swelling and pain relieving vinegar paste |
| CN114720622B (en) * | 2022-04-06 | 2023-10-17 | 贵州良济药业有限公司 | Detection method of detumescence pain vinegar paste |
| CN115420816A (en) * | 2022-07-21 | 2022-12-02 | 广东万年青制药股份有限公司 | Content determination method of traditional Chinese medicine composition for nourishing yin and cooling blood |
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