CN107884480A - A kind of detection method of the peaceful preparation of colon - Google Patents
A kind of detection method of the peaceful preparation of colon Download PDFInfo
- Publication number
- CN107884480A CN107884480A CN201610870711.3A CN201610870711A CN107884480A CN 107884480 A CN107884480 A CN 107884480A CN 201610870711 A CN201610870711 A CN 201610870711A CN 107884480 A CN107884480 A CN 107884480A
- Authority
- CN
- China
- Prior art keywords
- preparation
- colon
- peaceful
- reference substance
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000001072 colon Anatomy 0.000 title claims abstract description 41
- 238000002360 preparation method Methods 0.000 title claims abstract description 40
- 238000001514 detection method Methods 0.000 title claims abstract description 24
- 239000013558 reference substance Substances 0.000 claims abstract description 39
- 238000000034 method Methods 0.000 claims abstract description 37
- 238000012360 testing method Methods 0.000 claims abstract description 32
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 102
- GQODBWLKUWYOFX-UHFFFAOYSA-N Isorhamnetin Natural products C1=C(O)C(C)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 GQODBWLKUWYOFX-UHFFFAOYSA-N 0.000 claims description 25
- IZQSVPBOUDKVDZ-UHFFFAOYSA-N isorhamnetin Chemical compound C1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 IZQSVPBOUDKVDZ-UHFFFAOYSA-N 0.000 claims description 25
- 235000008800 isorhamnetin Nutrition 0.000 claims description 25
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 24
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 23
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 20
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 14
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 8
- 239000000706 filtrate Substances 0.000 claims description 8
- 238000010992 reflux Methods 0.000 claims description 7
- 238000002604 ultrasonography Methods 0.000 claims description 7
- 238000004090 dissolution Methods 0.000 claims description 6
- 238000012546 transfer Methods 0.000 claims description 6
- 238000010829 isocratic elution Methods 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 4
- 238000004458 analytical method Methods 0.000 claims description 2
- 238000003810 ethyl acetate extraction Methods 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims 1
- 238000011084 recovery Methods 0.000 abstract description 11
- 239000003814 drug Substances 0.000 abstract description 4
- 229940079593 drug Drugs 0.000 abstract description 3
- 230000002349 favourable effect Effects 0.000 abstract 1
- 238000005259 measurement Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 39
- 239000012071 phase Substances 0.000 description 13
- 241000233948 Typha Species 0.000 description 12
- 238000000605 extraction Methods 0.000 description 12
- 238000003556 assay Methods 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 8
- 239000000203 mixture Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- QHLKSZBFIJJREC-SPSUIZEHSA-N Isorhamnetin-3-O-nehesperidine Chemical compound C1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O[C@H]2[C@@H]([C@H](O)[C@@H](O)[C@H](C)O2)O)=C1 QHLKSZBFIJJREC-SPSUIZEHSA-N 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 241001108764 Ludwigia sphaerocarpa Species 0.000 description 4
- POMAQDQEVHXLGT-QDYYQVSOSA-N Typhaneoside Chemical compound C1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO[C@H]3[C@@H]([C@H](O)[C@@H](O)[C@H](C)O3)O)O2)O[C@H]2[C@@H]([C@H](O)[C@@H](O)[C@H](C)O2)O)=C1 POMAQDQEVHXLGT-QDYYQVSOSA-N 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- POMAQDQEVHXLGT-UHFFFAOYSA-N isorhamnetin 3-(2-rhamnosyl rutinoside) Natural products C1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)OC2C(C(O)C(O)C(COC3C(C(O)C(O)C(C)O3)O)O2)OC2C(C(O)C(O)C(C)O2)O)=C1 POMAQDQEVHXLGT-UHFFFAOYSA-N 0.000 description 4
- 239000012452 mother liquor Substances 0.000 description 4
- FRBRLXJEQSHWAA-UHFFFAOYSA-N typhaneoside Natural products COc1cc(ccc1O)C2=C(OC3OC(COC4OC(C)C(O)C(O)C4OC5OC(C)C(O)C(O)C5O)C(O)C(O)C3O)C(=O)c6c(O)cc(O)cc6O2 FRBRLXJEQSHWAA-UHFFFAOYSA-N 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 206010009887 colitis Diseases 0.000 description 3
- 239000007791 liquid phase Substances 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 230000035508 accumulation Effects 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 238000005903 acid hydrolysis reaction Methods 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 2
- 208000004429 Bacillary Dysentery Diseases 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 241000792859 Enema Species 0.000 description 1
- 206010017915 Gastroenteritis shigella Diseases 0.000 description 1
- 244000000231 Sesamum indicum Species 0.000 description 1
- 235000003434 Sesamum indicum Nutrition 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- FGDQGIKMWOAFIK-UHFFFAOYSA-N acetonitrile;phosphoric acid Chemical compound CC#N.OP(O)(O)=O FGDQGIKMWOAFIK-UHFFFAOYSA-N 0.000 description 1
- 230000001142 anti-diarrhea Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- OAABHEHWRQAHEJ-UHFFFAOYSA-N butan-1-ol;chloroform Chemical compound ClC(Cl)Cl.CCCCO OAABHEHWRQAHEJ-UHFFFAOYSA-N 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000007920 enema Substances 0.000 description 1
- 229940095399 enema Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- ZOOODBUHSVUZEM-UHFFFAOYSA-N ethoxymethanedithioic acid Chemical compound CCOC(S)=S ZOOODBUHSVUZEM-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 229930182486 flavonoid glycoside Natural products 0.000 description 1
- 150000007955 flavonoid glycosides Chemical class 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 229940074391 gallic acid Drugs 0.000 description 1
- 235000004515 gallic acid Nutrition 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- LBDROUOCQSGOFI-UHFFFAOYSA-N methanol;phosphoric acid Chemical compound OC.OP(O)(O)=O LBDROUOCQSGOFI-UHFFFAOYSA-N 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 201000005113 shigellosis Diseases 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000002137 ultrasound extraction Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012991 xanthate Substances 0.000 description 1
Classifications
-
- 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
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a kind of detection method of the peaceful preparation of colon.Method includes：1) preparation of reference substance solution；2) preparation of need testing solution；3) HPLC is detected.The inventive method precision is high, favorable reproducibility, and the rate of recovery is high, and measurement result is accurate, improves colon peaceful quality controllability and stability, it is ensured that the security and validity of people's medication.
Description
Technical field
The invention belongs to drug measurement techniques field, the secondary metabolite of cattail pollen more particularly in the measure peaceful preparation of colon
Content.
Background technology
The peaceful preparation of colon has the effect of promoting blood circulation and removing blood stasis, gut purge antidiarrheal, is treatment chronic colitis, colitis, exedens
The characteristic enema of colitis, bacillary dysentery.Reach a conclusion after weighing a matter since city, its quality standard is being lifted always.The peaceful preparation of colon be by
Cattail pollen and the taste medicine of false loosestrife two composition prescription preparation, at present enterprises standard be mainly to its cattail pollen, false loosestrife it is qualitative
The quantitative discriminating of gallic acid in discriminating and false loosestrife；But in prescription, cattail pollen dosage is the several times of false loosestrife, as materials
Most raw materials, its quantitative approach are always the emphasis problem of the peaceful preparation detection of colon.
The content of the invention
The object of the invention is directed to the missing for the appropriate method that the peaceful preparation of colon controls for the content of cattail pollen at present and developed
Detection method.The method comprises the following steps：
(1) preparation of reference substance solution：
Isorhamnetin reference substance is weighed, is placed in volumetric flask, adds 25%~75% ethanol or methanol, ultrasonic dissolution and constant volume
To scale, shake up, reference substance solution is made；
(2) preparation of need testing solution：
The preparation of the peaceful need testing solution of colon：The peaceful sample of colon is taken to be placed in volumetric flask, it is accurately weighed, add 25%~75%
Ethanol or methanol, 30~90min of ultrasound, are cooled to room temperature, are settled to scale, filtering；Precision measures subsequent filtrate and is evaporated, and adds water-soluble
Solution is transferred in 100ml triangular pyramidal bottles, adds 1ml hydrochloric acid；It is placed in water-bath and is heated to reflux 30~60min, is cooled to room temperature
Afterwards n-butanol or chloroform or ethyl acetate extraction are closed so that water is full；Transfer is settled in volumetric flask after extract is evaporated, and is produced.
(3) HPLC is detected：
Isorhamnetin reference substance solution and the peaceful preparation need testing solution of colon obtained by aspiration step (1) and (2) respectively, point
It Zhu Ru be measured in high performance liquid chromatograph；
Wherein, chromatographic condition is：Chromatographic column is C18Reverse-phase chromatographic column；UV-detector；Column temperature is 30~35 DEG C；Detect ripple
A length of 350~370nm；Mobile phase A is acetonitrile, and Mobile phase B is 0.1~1% phosphoric acid solution, and flow velocity is 0.8~1.2ml/min；
30 minutes analysis times；Isocratic elution, mobile phase A:Volume ratio=45~55 of Mobile phase B:55~45.
The preparation of step (1) need testing solution is preferably that following methods are carried out：
Weigh Isorhamnetin reference substance 5mg to be placed in volumetric flask, add 25~75% ethanol or methanol, ultrasonic dissolution and constant volume
To scale, shake up, reference substance solution is made.
The preparation of step (2) need testing solution is preferably that following methods are carried out：
The peaceful sample of colon about 1~5g is taken to be placed in 50ml volumetric flasks, it is accurately weighed, add 30~70% methanol or ethanol
45mL, 30~90min of ultrasound, is cooled to room temperature, is settled to 50ml, filtering；Precision measures 10~30ml of subsequent filtrate and is evaporated, and adds water
20ml solution transfers add 1ml hydrochloric acid into 100ml triangular pyramidal bottles；It is placed in water-bath and is heated to reflux 30~90min, cools down
Extracting n-butyl alcohol is closed so that water is full 1~5 time, every time 10~30ml after to room temperature；Transfer is settled to 10ml appearances after extract is evaporated
Measuring bottle, produce.
The C that the preferred column length of chromatographic column in the step (3) is 250mm18Reverse-phase chromatographic column.
Detection wavelength is preferably 370nm in the step (3).
Mobile phase B is preferably 1% phosphoric acid solution in the step (3).
Flow velocity is preferably 1ml/min in the step (3).
Ethanol or methanol concentration are preferably 50% in the step (1) and (2).
Mobile phase A in the step (3):The volume ratio of Mobile phase B is preferably 53:47.
Sonication treatment time is preferably 30 minutes in the step (2).
Return time is 60 minutes in the step (2).
In the step (2) extracting n-butyl alcohol is closed so that water is full.
Preferably to show the essence of the present invention, by the methodological study of the present invention, details are as follows：
1 instrument and reagent
Instrument：Waters e2695-2998 type high performance liquid chromatographs, AR220 assay balances, BS2202D balances,
CP225D balances, KQ300DE types numerical control ultrasonic cleaner, Kromasil 100-5 C18 (250mm × 4.6mm, 5 μm),
Inertsil ODS-2 C18(250mm×4.6mm,5μm)、Hypersil ODS2 C18(250mm×4.6mm,5μm)、DZKW
Type electric-heated thermostatic water bath etc..
Reagent：Colon peaceful (5g/ branch, lot number 20150401,20150402,20150403, by the nine limited public affairs of sesame hall share
Department provides)；Isorhamnetin (110860-201109, National Institute for Food and Drugs Control).
Reagent：Methanol (AR/ chromatographically pures), ethanol (AR), glacial acetic acid (AR), phosphoric acid (AR), acetonitrile (chromatographically pure), water is super
Pure water.
2 methods and result
Selection, mobile phase, extracting mode, Extraction solvent, the selection of extraction time, extractant of 2.1 Detection wavelengths.
Full wavelength scanner (see accompanying drawing 1) has been carried out to sample using SPD-M20A detectors, has selected 370nm to detect ripple
It is long.
The elution effect of the flowing relative sample such as methanol-phosphoric acid solution, acetonitrile-phosphoric acid solution of different proportion has been investigated,
Consider the results such as collection of illustrative plates separating degree, post effect, symmetrical factor, it is mobile phase finally to select the phosphoric acid of acetonitrile -1%.Again according to sample
The testing result (see accompanying drawing 2 to Fig. 3) of product, Isorhamnetin chromatographic peak whole appearances within detection 30min are found, so that it is determined that
Detection time is 30min.
The peaceful 3g of colon is weighed, respectively with ultrasound, backflow 30 minutes, is as a result shown, difference is little, but ultrasound is easy
It is easy, so choosing ultrasound is used as extracting method.Secondly, methanol recovery rate is found using methanol, ethanol as Extraction solvent respectively
It is higher, so use methanol；Simultaneously respectively with 30% methanol, 50% methanol, 70% methanol, methanol ultrasonic extraction, as a result show
50% methanol recovery rate highest, 50% methanol is also finally selected as optimum extraction solvent.Subsequent ultrasonic time is to recovery rate
Influenceing, ultrasound 30,60,90 minutes, it is found that its recovery rate at 30 minutes is larger respectively, so it is super to select optimal ultrasonic time
Sound 30 minutes.And then return time is investigated for acid-hydrolyzed influence, is flowed back 30,60,90 minutes respectively, is found it at 60 points
The recovery rate of clock is higher, therefore it is 60 minutes to choose optimal return time.Influence of the extractant to extraction effect is finally investigated,
The full conjunction n-butanol of chloroform, ethyl acetate, water is respectively adopted, as a result shows that the full conjunction n-butanol of water and chloroform recovery efficiency are higher, though
The right chloroform n-butanol that closes fuller than water is higher, but based on the management that malicious reagent is easily made in enterprise, selection water is full to close butanol solution
As optimal extractant.
By being investigated to Extraction solvent, method, volume, time, the extracting method is finally determined.
Each step of the Research on Methods of table 1 result containing survey and relative deviation data
Isorhamnetin symmetrical factor and appearance time under 2 each mobile phase of table
2.2 chromatographic condition：
By investigating detector, flow visualizing, Detection wavelength, chromatographic column, column temperature and flow velocity, by optimizing repeatedly, obtain
It is to optimal chromatographic condition：UV-detector, the phosphoric acid of acetonitrile -1% (57:43) isocratic elution, Hypersil ODS2C-18 colors
Post (4.6 × 250mm, 5 μm) is composed, column temperature is 35 DEG C, flow velocity 1ml/min, and sample size is 10 μ l, Detection wavelength 370nm, is examined
Survey time 30min.
It is prepared by 2.3 test liquids：
By investigating the investigation of extraction time, Extraction solvent, extracting mode, determine that test liquid preparation method is：Take this product
3g, it is accurately weighed, put in 50ml volumetric flasks, add 50% methanol 45ml, be ultrasonically treated (250W, frequency 20KHz) 30 minutes, put
It is cold, add methanol to shake up to scale, filter, precision measures subsequent filtrate 25ml, is evaporated, and residue adds water 20ml to dissolve, and adds hydrochloric acid 1ml,
Backflow 60 minutes, lets cool, is transferred to separatory funnel, with water-saturated n-butanol shaking extraction 3 times, each 20ml, merges water saturation
N-butanol extracting liquid, it is evaporated, residue is dissolved with methanol, is transferred in 10ml measuring bottles, adds methanol to shake up, produce for examination to scale
Product solution.
2.4 specificities are investigated
The negative sample for lacking cattail pollen is prepared with method with the peaceful technique of colon, test sample, reference substance solution prepares and assay method
Ibid, negative sample is noiseless.See accompanying drawing 10.
2.5 linear investigations
Isorhamnetin reference substance 11.81mg is taken, precision is weighed, and dissolving is settled in 100ml volumetric flasks, produces mother liquor.Take
59.05,47.24,23.62,11.81,4.724,2.362 μ g/ml reference substance solution is respectively prepared in mother liquor, accurate respectively to draw
Each 10 μ l injections liquid chromatograph of Isorhamnetin reference substance solution of above-mentioned various concentrations, measure, using peak area as ordinate, enters
Sample amount (μ g) is abscissa, draws standard curve.Its regression equation is：Y=0.0002x+3.4937, R2=0.9994.Thus
Understand, Isorhamnetin content is linear in 23.62~590.5 μ g ranges.See accompanying drawing 11.
The linear relationship result of the test of table 3
2.6 test limits are investigated with quantitative limit
2.6.1 test limit dilutes 4 times, obtained using the linear middle μ g/ml of concentration 2.362 reference substance solution as mother liquor
0.5905 μ g/ml, sample introduction 3 μ l, s/n are about 3 or so, therefore calculate to detect and be limited to 1.7715ng, as a result see accompanying drawing 12.
2.6.2 quantitative limit dilutes 4 times, obtained using the linear middle μ g/ml of concentration 2.362 reference substance solution as mother liquor
0.5905 μ g/ml, sample introduction 7 μ l, s/n are about 10 or so.Therefore calculate to detect and be limited to 4.1335ng.METHOD FOR CONTINUOUS DETERMINATION 6 times, Qi Feng faces
Product integrated value RSD is 2.57%, the results are shown in Table 4, accompanying drawing 13.
The quantitative limit result of table 4
2.7 precision, repeatability and stability experiment
Reference substance solution is taken to calculate Isorhamnetin peak area RSD% in repeating sample introduction continuous sample introduction in same 1d 6 times, investigate
Instrument withinday precision.It the results are shown in Table 5.
It is 20150402 samples to take lot number, prepares 6 parts respectively, is measured, and calculates content and its RSD%, investigates method
Reappearance.It the results are shown in Table 6.
Same need testing solution is measured 0,6,12,18,24h respectively, records peak area, calculates relative reservation
Time and relative peak area RSD%, to investigate the stability of sample.As a result show, Isorhamnetin had stable in 24 hours
Property.It the results are shown in Table 7.
The precision result of table 5
6 repeated data of table
The stability data of table 7
2.8 average recovery
Precision weighs 6 parts of the test sample (Isorhamnetin content is 93.50 μ g/g) of known content, accurate respectively to add necessarily
The Isorhamnetin reference substance of amount, according to recovery test liquid is prepared under 2.3, determine in accordance with the law, calculate the rate of recovery, the results are shown in Table 8.
The method recovery test result (n=6) of table 8
As a result show, colon is rather good with Isorhamnetin weight calculation renaturation.
2.9 durabilities are investigated
Using the method drafted, using different chromatographic columns, using the chromatographic column of 3 models to three batches of samples
(20150402,20150403,20150501) are measured, and the results are shown in Table 9,10.As a result show, this method good tolerance.
The chromatogram column type number of table 9
The Isorhamnetin serviceability test result of table 10
2.10 10 batches of sample assays：
Add up 20 assay data in 10 batches of samples.It the results are shown in Table 11.
11 10 batches of sample assay data accumulations of table
This product (C in terms of Isorhamnetin16H12O7), 69mg/g must not be less than.
Quality control composition of the cattail pollen in Chinese Pharmacopoeia is typhaneoside and Isorhamnetin-3-O-neohespeidoside, the two
Belong to flavonoid glycoside composition；There is water decoction process in the peaceful technique of colon, have certain destruction to the constituents in heating process, with
The method of pharmacopeia detects to it, and as a result the two most content of total content of display is relatively low or is free of, and sees accompanying drawing 6~9.In not shadow
On the premise of ringing the peaceful preparation oeverall quality of colon, establishing a kind of suitable detection method makes detected composition more protrude very
It is necessary.
Beneficial effects of the present invention：To its index composition, (cattail is new different from cattail pollen in pharmacopeia for the inventive method
Glycosides and Isorhamnetin-3-O-neohespeidoside) carry out Content evaluation, and to the acid hydrolysis products of its index composition (Isorhamnetin-
Acid hydrolysis products-Isorhamnetin of 3-O- neohesperidins) quantify so as to carry out it indirect content control.This method ensures
The peaceful preparation of colon controls quality, filled up in the peaceful preparation of colon to Pu well in the case where original effectively technique is constant
Xanthate material carries out the blank of quality control.The detection method of the peaceful middle cattail pollen of colon of the present invention, it both can be used for control colon peaceful
Quality, other prescriptions for being possible to be destroyed to its index composition can be boiled containing cattail pollen and progress decocting for controlling again
And the control of preparation.The easy to operate of the inventive method, the degree of accuracy is high, specificity is good, precision is higher, reappearance is good, has
Beneficial to the steady quality and clinical efficacy for ensureing the peaceful preparation of colon.
Brief description of the drawings
Fig. 1 is Detection wavelength full wavelength scanner figure；
Fig. 2 is the HPLC chromatogram of the Isorhamnetin reference substance of embodiment 1；
Fig. 3 is the peaceful HPLC chromatogram of the colon of embodiment 1；
Fig. 4 is the HPLC chromatogram of the Isorhamnetin reference substance of comparative example 1；
Fig. 5 is the peaceful HPLC chromatogram of the colon of comparative example 1；
Fig. 6 is the typhaneoside reference substance HPLC chromatogram of comparative example 2；
Fig. 7 is the Isorhamnetin-3-O-neohespeidoside reference substance HPLC chromatogram of comparative example 2；
Fig. 8 is the peaceful HPLC chromatogram of the colon of comparative example 2；
Fig. 9 is that the colon of comparative example 2 rather contrasts HPLC chromatogram with reference substance.
Figure 10 is specificity HPLC chromatogram；
Figure 11 is Isorhamnetin reference substance canonical plotting；
Figure 12 is Isorhamnetin test limit；
Figure 13 is Isorhamnetin quantitative limit.
Embodiment
Embodiment 1
1st, the detection method of the peaceful preparation of a kind of colon, comprises the following steps：
(1) preparation of reference substance solution：
Isorhamnetin reference substance 5mg is weighed, is placed in volumetric flask, adds 50% methanol, ultrasonic dissolution is simultaneously settled to scale, shaken
It is even, reference substance solution is made.
(2) preparation of need testing solution：
Colon peaceful sample 3g is taken, it is accurately weighed, put in 50ml volumetric flasks, add 50% methanol 45ml, be ultrasonically treated (250W,
Frequency 20KHz) 30 minutes, let cool, add 50% methanol to shake up, filter, precision measures subsequent filtrate 25ml, is evaporated, residue to scale
Add water 20ml to be transferred in 100ml triangular pyramidal bottles, add 1ml hydrochloric acid.It is placed in water-bath and is heated to reflux 60min, is cooled to room
Extracting n-butyl alcohol 3 times, each 20ml is closed so that water is full after temperature, combining extraction liquid, is shifted after extract is evaporated and is settled to 10ml appearances
Measuring bottle, produce.
(3) HPLC is detected：
Reference substance solution and the peaceful preparation need testing solution of colon obtained by aspiration step (1) and (2) respectively, is injected separately into height
It is measured in effect liquid phase chromatogram instrument；Wherein, chromatographic condition is：
UV-detector, the phosphoric acid of acetonitrile -1% (57:43) isocratic elution, ODS2C-18 chromatographic columns (4.6 × 250mm, 5 μ
M), column temperature is 35 DEG C, flow velocity 1ml/min, and sample size is 10 μ l, Detection wavelength 370nm, detection time 30min.
(4) sample size determines
3 batches of sample assay data.12 are the results are shown in Table, sees accompanying drawing 2,3.
12 3 batches of sample assay data accumulations of table
Comparative example 1
A kind of peaceful detection method of colon, is comprised the following steps：
(1) preparation of reference substance solution：
Isorhamnetin reference substance 5mg is weighed, is placed in volumetric flask, adds methanol ultrasonic dissolution and is settled to scale, shake up, is made
Into reference substance solution.
(2) preparation of need testing solution：
Colon peaceful sample 3g is taken, it is accurately weighed, put in 50ml volumetric flasks, add methanol 45ml, be ultrasonically treated (250W, frequency
20KHz) 30 minutes, let cool, add methanol to shake up, filter, precision measures subsequent filtrate 25ml, is evaporated, and residue adds water 20ml to scale
It is transferred in 100ml triangular pyramidal bottles, adds 1ml hydrochloric acid.It is placed in water-bath and is heated to reflux 30min, is cooled to after room temperature with chlorine
Imitative extraction 3 times, each 20ml, combining extraction liquid, shifted after extract is evaporated and be settled to 10ml volumetric flasks, produced.
(3) HPLC is detected：
Reference substance solution and the peaceful preparation need testing solution of colon obtained by aspiration step (1) and (2) respectively, is injected separately into height
It is measured in effect liquid phase chromatogram instrument；Wherein, chromatographic condition is：
UV-detector, methanol：0.4% phosphoric acid (52:48) isocratic elution, ODS2C-18 chromatographic columns (4.6 × 250mm, 5 μ
M), column temperature is 35 DEG C, flow velocity 1ml/min, and sample size is 10 μ l, Detection wavelength 370nm, detection time 30min.
Under such an approach, Isorhamnetin is not kept completely separate with other impurity peaks in test sample, can not obtain accurate content
As a result, accompanying drawing 4,5 is as a result seen.
Comparative example 2
Colon is rather detected with Chinese Pharmacopoeia cattail pollen content assaying method, comprised the following steps：
(1) preparation of reference substance solution：Take typhaneoside, Isorhamnetin-3-O-neohespeidoside reference substance appropriate, precision claims
It is fixed, add methanol that every 1ml is made containing 50；
(2) preparation of need testing solution：This product about 0.5g is taken, it is accurately weighed, put in conical flask with cover, precision adds methanol
50ml, weighed weight, cold soaking is heated to reflux 1 hour after 12 hours, let cool, then weighed weight, and the weight of less loss is supplied with methanol,
Shake up, filter, take subsequent filtrate, produce.
(3) HPLC is detected：It is accurate respectively to draw above two reference substance solution and each 10 μ 1 of need testing solution, note people's liquid phase
Chromatograph, measure；Testing conditions are：Using octadecylsilane chemically bonded silica as filler；With the phosphoric acid solution of acetonitrile -0.05%
(15:85) it is mobile phase；Detection wavelength is 254nm.
(4) sample size determines：3 batches of sample assay data.Three batches are free of typhaneoside and Isorhamnetin -3-O-
Neohesperidin, as a result see accompanying drawing 6,7,8,9.
Claims (10)
1. a kind of detection method of the peaceful preparation of colon, comprises the following steps：
(1) preparation of reference substance solution：
Isorhamnetin reference substance is weighed, is placed in volumetric flask, adds 25~75% ethanol or methanol, ultrasonic dissolution is simultaneously settled to quarter
Degree, shakes up, reference substance solution is made；
(2) preparation of need testing solution：
The preparation of the peaceful need testing solution of colon：Take the peaceful sample of colon to be placed in volumetric flask, it is accurately weighed, add 25~75% methanol or
Ethanol, 30~90min of ultrasound, is cooled to room temperature, is settled to scale, filtering；Precision measures subsequent filtrate and is evaporated, and be dissolved in water transfer
Into 100ml triangular pyramidal bottles, add 1ml hydrochloric acid；It is placed in water-bath and is heated to reflux 30~60min, is cooled to after room temperature with water
It is full to close n-butanol or chloroform or ethyl acetate extraction；Transfer is settled in volumetric flask after extract is evaporated, and is produced；
(3) HPLC is detected：
Reference substance solution and the peaceful preparation need testing solution of colon obtained by aspiration step (1) and (2) respectively, is injected separately into efficient liquid
It is measured in chromatography；
Wherein, chromatographic condition is：Chromatographic column is C18Reverse-phase chromatographic column；UV-detector；Column temperature is 30~35 DEG C；Detection wavelength is
350~370nm；Mobile phase A is acetonitrile, and Mobile phase B is 0.1~1% phosphoric acid solution, and flow velocity is 0.8~1.2ml/min；Analysis
30 minutes time；Isocratic elution, mobile phase A:Volume ratio=45~55 of Mobile phase B:55~45.
2. according to the method for claim 1, it is characterised in that：The preparation of step (1) reference substance solution is according to following
Method is carried out：
Isorhamnetin reference substance 5mg is weighed, is placed in volumetric flask, adds 25~75% ethanol or methanol, ultrasonic dissolution is simultaneously settled to
Scale, shake up, reference substance solution is made.
3. according to the method for claim 1, it is characterised in that：The preparation of step (2) need testing solution is according to following
Method is carried out：
The preparation of the peaceful need testing solution of colon：
The peaceful sample of colon about 1~5g is taken to be placed in 50ml volumetric flasks, it is accurately weighed, add 30~70% methanol or ethanol 45mL, surpass
30~90min of sound, is cooled to room temperature, is settled to 50ml, filtering；Precision measures 10~30ml of subsequent filtrate and is evaporated, and adds water 20ml molten
Solution is transferred in 100ml triangular pyramidal bottles, adds 1ml hydrochloric acid；It is placed in water-bath and is heated to reflux 30~90min；It is cooled to room temperature
Extracting n-butyl alcohol is closed so that water is full 1~5 time, every time 10~30ml afterwards；Transfer is settled to 10ml volumetric flasks after extract is evaporated, i.e.,
.
4. according to the method described in claim 1-3 any claims, it is characterised in that：Chromatographic column in the step (3)
It is the C that column length is 250mm18Reverse-phase chromatographic column.
5. according to the method described in claim 1-3 any claims, it is characterised in that：Detection wavelength in the step (3)
For 370nm.
6. according to the method described in claim 1-3 any claims, it is characterised in that：Mobile phase B is in the step (3)
1% phosphoric acid solution.
7. according to the method described in claim 1-3 any claims, it is characterised in that：Flow velocity is in the step (3)
1.0ml/min。
8. according to the method described in claim 1-3 any claims, it is characterised in that：Methanol in the step (1) and (2)
Or concentration of alcohol is 50%.
9. according to the method described in claim 1-3 any claims, it is characterised in that：Mobile phase A in the step (3)：
Volume ratio=53 of Mobile phase B:47.
10. according to the method described in claim 1-3 any claims, it is characterised in that：It is ultrasonically treated in the step (2)
Time is 30 minutes, and return time is 60 minutes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610870711.3A CN107884480A (en) | 2016-09-30 | 2016-09-30 | A kind of detection method of the peaceful preparation of colon |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610870711.3A CN107884480A (en) | 2016-09-30 | 2016-09-30 | A kind of detection method of the peaceful preparation of colon |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107884480A true CN107884480A (en) | 2018-04-06 |
Family
ID=61769980
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610870711.3A Pending CN107884480A (en) | 2016-09-30 | 2016-09-30 | A kind of detection method of the peaceful preparation of colon |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107884480A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1392146A (en) * | 2002-06-11 | 2003-01-22 | 张在雪 | Water cress extract and its preparing method and use |
| CN101279055A (en) * | 2008-05-19 | 2008-10-08 | 王世锋 | Novel therapeutic use of ginseng and astragali pollen preparations and quality control method |
| EP2258391A1 (en) * | 2009-06-05 | 2010-12-08 | Stallergenes S.A. | Reduction of in vitro genotoxicity of pollen extracts by removal of flavonoids |
| CN102707008A (en) * | 2012-06-15 | 2012-10-03 | 湖南麓山天然植物制药有限公司 | Detection method of gynostemma total saponin granules |
-
2016
- 2016-09-30 CN CN201610870711.3A patent/CN107884480A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1392146A (en) * | 2002-06-11 | 2003-01-22 | 张在雪 | Water cress extract and its preparing method and use |
| CN101279055A (en) * | 2008-05-19 | 2008-10-08 | 王世锋 | Novel therapeutic use of ginseng and astragali pollen preparations and quality control method |
| EP2258391A1 (en) * | 2009-06-05 | 2010-12-08 | Stallergenes S.A. | Reduction of in vitro genotoxicity of pollen extracts by removal of flavonoids |
| CN102707008A (en) * | 2012-06-15 | 2012-10-03 | 湖南麓山天然植物制药有限公司 | Detection method of gynostemma total saponin granules |
Non-Patent Citations (2)
| Title |
|---|
| 唐光明等: "HPLC法测定蒲黄片中异鼠李素的含量", 《中国药事》 * |
| 文红梅等: "HPLC法测定蒲黄中总黄酮的含量", 《现代中药研究与实践》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108717095B (en) | Detection method, identification method and content determination method of loquat leaves or medicine containing loquat leaf raw material | |
| CN110308213B (en) | Method for constructing fingerprint spectrum of kidney-nourishing and fetus-growing pill and application of fingerprint spectrum in quality detection | |
| CN107356691B (en) | The detection method of building music fingerprint | |
| CN104730158B (en) | A kind of content assaying method of JIAWEI HUOXIANG ZHENGQI RUANJIAONANG | |
| CN104502518B (en) | A kind of detection method for the treatment of the Chinese medicine preparation of baby anorexia | |
| CN104062374B (en) | The detection method of the Chinese medicine composition of invigorating Qi and tonifying kidney | |
| CN110320311A (en) | A kind of quality determining method of Cortex Eucommiae Traditional Chinese medicine bolus for strengthening bones of human body | |
| CN108445125B (en) | HPLC Fingerprint Detection Method for Zhitong Huazheng Capsules | |
| CN111398437A (en) | The Establishment Method of the Fingerprint of Zexie Decoction and Its Fingerprint | |
| CN107356684A (en) | The detection method of cassia seed finger-print | |
| CN106442789A (en) | Establishment and active component quantitative analysis methods of compound Xuezhining extract fingerprint map | |
| CN117054567A (en) | HPLC (high Performance liquid chromatography) characteristic spectrum construction method for stir-fried papermulberry fruit decoction pieces, standard decoction and formula granules | |
| CN104597139B (en) | Method for simultaneously determining three kinds of phenylethanoid glycoside compositions in callicarpa nudiflora preparation through HPLC | |
| CN106053702B (en) | A kind of multicomponent content assaying method of JIAWEI XIAOYAOWAN | |
| CN106290645B (en) | A kind of construction method and its standard finger-print of Lhasa rhubarb finger-print | |
| CN102048906A (en) | Content measurement method of abrus herb capsules | |
| CN105301130B (en) | A high-performance liquid chromatography method for detecting the content of flavonoids in Radix Ophiopogon japonicus | |
| CN109709222B (en) | A kind of component detection method of Ganmaoling and compound Ganmaoling | |
| CN104458954B (en) | A kind of dodder formulation granule finger printing and method for building up thereof | |
| CN107179374B (en) | Method for detection of fingerprint of Jingxue Patch | |
| CN107884480A (en) | A kind of detection method of the peaceful preparation of colon | |
| CN109865014A (en) | A kind of processing procedure of honey Exocarpium Citri Grandis | |
| CN104597168B (en) | Ramulus Mori refines decoction pieces content assaying method | |
| CN104345108A (en) | Qualitative quantitative determination method for liver-heat-clearing tablet | |
| CN115524424A (en) | Capsella bursa-pastoris sample quality control method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180406 |