CN104407072B - A kind of method of 11 kinds of active constituent contents in Reduning injection of mensuration simultaneously - Google Patents
A kind of method of 11 kinds of active constituent contents in Reduning injection of mensuration simultaneously Download PDFInfo
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Abstract
The invention discloses a kind of method of 11 kinds of active constituent contents in Reduning injection of mensuration simultaneously, 11 kinds of effective ingredient are neochlorogenic acid, chlorogenic acid, 4-dicaffeoylquinic acid, 3,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, 4,5-Dicaffeoylquinic acid, Fructus Gardeniae glycosides, Geniposidic acid, genipin gentiobioside with cape jasmine, jasminoidin and disconnected oxidation loganin, the method uses Agilent ZORBAX SB C18 chromatographic column (3.0 × * 100mm, 1.8um), flowing is acetonitrile (A) 0.1% phosphoric acid (B) mutually, gradient elution, flow velocity 0.35 0.45mL min‑1, detect wavelength 324 326nm and 236 238nm, column temperature: 25 35 DEG C.The inventive method accurate quick is sensitive, further increases Reduning injection quality control level.
Description
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to the side of 11 kinds of compositions in a kind of Reduning injection of mensuration simultaneously
Method.
Background technology
Reduning injection prescription is made up of Flos Lonicerae, Herba Artemisiae Annuae, Fructus Gardeniae, has heat clearing away, dispelling wind, detoxicating functions, clinically
For treating upper respiratory tract infection[1].Reduning injection composition Study shows, it mainly contains coffee mesitoyl quinine acid and ring
Alkene ether terpenoid[2].Early-stage Study establishes Reduning injection HPLC finger printing[3]Survey with one comments method to measure pyretic toxicity more
9 kinds of compositions in injection for curing[4], above-mentioned HPLC and surveys and comments the method operation time longer more, is unfavorable for industrialized great production
The quick of product is detected by journey, and only have detected this kind of iridoids composition of jasminoidin, and iridoids material
In prescription medical material Fructus Gardeniae, content is higher[5-6], other compositions such as including Gardenoside, Fructus Gardeniae glycosides, genipin gentiobioside with cape jasmine,
These compositions are respectively provided with a certain degree of therapeutical effect to upper respiratory tract infection, and only detection Determination of Gardenoside can not preferably reflect
The quality of product and curative effect.Therefore, the present invention establishes a kind of new UPLC method, increases Fructus Gardeniae glycosides, capital Buddhist nun in detection Fructus Gardeniae
The multiple pharmacodynamics index compositions such as flat thuja acid and genipin gentiobioside with cape jasmine, it is achieved 6 kinds of Determination of Organic Acids and 5 in Reduning injection
Measure while planting iridoid constituents, reach to shorten the detection time in actual production, increase relevant to product curative effect
Index, promotes the purpose of the quality control standard of Reduning injection further.
Summary of the invention
It is an object of the invention to overcome above-mentioned weak point to provide a kind of UPLC method, Redujing Granules injection can be measured simultaneously
In liquid, 11 kinds of compositions (include 6 kinds of organic phenol acids and Fructus Gardeniae glycosides, Geniposidic acid and genipin gentiobioside with cape jasmine etc. 5 kinds
Iridoids material).The method be use UPLC-DAD method measure simultaneously neochlorogenic acid in Reduning injection, chlorogenic acid,
4-dicaffeoylquinic acid, 3,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, 4,5-Dicaffeoylquinic acid, Fructus Gardeniae glycosides, Geniposidic acid, genipin gentiobioside with cape jasmine, Cape jasmine
Sub-glycosides, the content of disconnected oxidation 11 kinds of effective ingredient of loganin.The method compared with prior art, when can be greatly shortened analysis
Between, improve the sensitivity and resolution analyzed, and add Con trolling index, further increase Reduning injection Quality Control water
Flat.
It is an object of the invention to be accomplished by:
A kind of method of 11 kinds of active constituent contents in Reduning injection of mensuration simultaneously, the method comprises the following steps:
A, the preparation of reference substance solution: precision weighs neochlorogenic acid, chlorogenic acid, 4-dicaffeoylquinic acid, 3,5-Dicaffeoylquinic acid, different respectively
Chlorogenic acid B, 4,5-Dicaffeoylquinic acid, Fructus Gardeniae glycosides, Geniposidic acid, genipin gentiobioside with cape jasmine, jasminoidin and disconnected oxidation loganin pair
Appropriate according to product, add the methanol that concentration is 50%～100% and make concentration and be respectively 0.12-0.25,0.28-0.58,0.12-
0.25,0.01-0.04,0.02-0.06,0.02-0.05,0.01-0.04,0.02-0.05,0.23-0.48,0.40-0.81,
0.05-0.12mg·ml-1Mixing reference substance solution.
B, the preparation of need testing solution
Accurate 1ml Reduning injection of drawing is put in volumetric flask, dissolves with the methanol that concentration is 50%～100% and dilutes
To 100ml, shaking up, centrifugal, supernatant is filtered by microporous filter membrane, to obtain final product.
C, algoscopy: use UPLC-DAD method precision respectively to draw reference substance solution and need testing solution, inject liquid phase color
Spectrometer, measures, to obtain final product；
Chromatographic condition: chromatographic column Agilent ZORBAX SB-C18 (3.0 × 100mm, 1.8um)；With acetonitrile for flowing phase
A, with 0.1% phosphoric acid as Mobile phase B, is carried out according to the following table gradient elution；Detection wavelength: 324-326nm and 236-238nm；Stream
Speed: 0.35-0.45mL.min-1；Column temperature: 25-35 DEG C.
The methanol that the methanol that concentration is 50%-100% both preferably concentration is 50～70% used described in the method.
Above-mentioned measure the method for 11 kinds of active constituent contents in Reduning injection simultaneously, preferably include following steps:
A, the preparation of reference substance solution: precision weighs neochlorogenic acid, chlorogenic acid, 4-dicaffeoylquinic acid, 3,5-Dicaffeoylquinic acid, different respectively
Chlorogenic acid B, 4,5-Dicaffeoylquinic acid, Fructus Gardeniae glycosides, Geniposidic acid, genipin gentiobioside with cape jasmine, jasminoidin and disconnected oxidation loganin pair
Appropriate according to product, add the methanol that concentration is 50% and make concentration and be respectively 0.12-0.15,0.28-0.30,0.12-0.15,0.01-
0.02、0.02-0.04、0.02-0.03、0.01-0.03、0.02-0.03、0.23-0.30、0.40-0.50、0.05-
0.08mg·ml-1Mixing reference substance solution；Most preferably 0.12225,0.28953,0.12357,0.01606,0.02849,
0.02323、0.01670、0.02468、0.23663、0.40770、0.05745mg·ml-1Mixing reference substance solution.I.e. every
1ml reference substance solution contains neochlorogenic acid 0.12225mg, chlorogenic acid 0.28953mg, 4-dicaffeoylquinic acid 0.12357mg, 3,5-Dicaffeoylquinic acid
0.01606mg, 3,4-Dicaffeoylquinic acid 0.02849mg, 4,5-Dicaffeoylquinic acid 0.02323mg, Fructus Gardeniae glycosides 0.01670mg, Geniposidic acid
0.02468mg, genipin gentiobioside with cape jasmine 0.23663mg, jasminoidin 0.40770mg, disconnected oxidation loganin 0.05745mg.
B, the preparation of need testing solution
Accurate 1ml Reduning injection of drawing is put in volumetric flask, is that 50% methanol dissolves and is diluted to 100ml by concentration,
Shaking up, centrifugal, supernatant is crossed 0.22um microporous filter membrane and is filtered, and to obtain final product.
C, algoscopy: use UPLC-DAD method precision respectively to draw reference substance solution and need testing solution, inject liquid phase color
Spectrometer, measures, to obtain final product；
Chromatographic condition: chromatographic column Agilent ZORBAX SB-C18 (3.0 × 100mm, 1.8um)；With acetonitrile for flowing phase
A, with 0.1% phosphoric acid as Mobile phase B, is carried out according to the following table gradient elution, and then rerun 10-12min；Detection wavelength: 324-
326nm and 236-238nm；Flow velocity: 0.4mL.min-1；Column temperature: 30 DEG C.
The concentration of methanol of the present invention is concentration of volume percent.
The present invention has investigated acetonitrile-water, methanol-water, acetonitrile-phosphoric acid, methanol-phosphoric acid, acetonitrile-acetic acid, methanol-acetic acid etc.
Flow visualizing, is found with methanol for flowing phase time, Fructus Gardeniae glycosides, Geniposidic acid appearance time by substantial amounts of creative work
Close, do not reach baseline separation；With water for flowing phase time, chromatographic peak hangover is serious；With acetic acid for flowing phase time, baseline drift is tight
Weight.Therefore preferably acetonitrile-phosphoric acid is flowing phase, chromatographic peak peak shape is preferable, and separating degree is higher.
The present invention compares different gradient condition under acetonitrile-Phosphoric Acid, owing to coffee mesitoyl quinine acid composition is at 324nm
With all have absworption peak under 238nm so that under 238nm 4-dicaffeoylquinic acid peak interference genipin gentiobioside with cape jasmine peak, even both merge
Go out peak.Screen layer by layer through many condition, determine suitable gradient condition, make genipin gentiobioside with cape jasmine appearance time 12.4min,
4-dicaffeoylquinic acid appearance time 12.8min, both of which reaches baseline separation.
In experimentation, inventor finds that when 25 DEG C and 35 DEG C, the separating degree of chromatographic peak is low compared with during column temperature 30 DEG C, and
Consider that liposoluble ingredient is unstable in Reduning injection, it may happen that the degraded of composition under the conditions of 35 DEG C, so, column temperature
It is preferably 30 DEG C.
To mixing reference substance solution at 200～800nm full wavelength scanners, result shows that iridoid constituents is at 238nm
There is absorption maximum at place；Coffee mesitoyl quinine acid composition has 3 absworption peaks in 220,245 and 324nm, wherein the strongest at 324nm, because of
This preferred 324nm and 238nm is as assay wavelength.
Beneficial effects of the present invention compared with the prior art: survey many with existing Reduning injection HPLC finger printing and one
The 9 kinds of compositions, methods commenting method to measure in Reduning injection are compared, and the UPLC that the present invention sets up measures Reduning injection simultaneously
In the method for 11 kinds of component contents, go up the most at runtime and foreshortened to 30min by 60min, and add Redujing Granules prescription
Fructus Gardeniae glycosides, Geniposidic acid and the genipin gentiobioside with cape jasmine these three pharmacodynamics index composition contained in Fructus Gardeniae in medical material, its
Middle genipin gentiobioside with cape jasmine content in Reduning injection is up to 6.2mg ml-1,, can more effectively control medicine
Quality.
Method of the present invention measure neochlorogenic acid in Reduning injection, chlorogenic acid, 4-dicaffeoylquinic acid, 3,5-Dicaffeoylquinic acid,
3,4-Dicaffeoylquinic acid, 4,5-Dicaffeoylquinic acid, Fructus Gardeniae glycosides, Geniposidic acid, genipin gentiobioside with cape jasmine, jasminoidin, disconnected oxidation loganin
11 components all reach baseline separation, have good linear relationship in the range of linear, and correlation coefficient is all higher than 0.999, flat
All response rate (n=6) are respectively 102.89% (RSD=1.25%), 103.25% (RSD=0.39%), 103.79% (RSD
=1.11%), 98.11% (RSD=1.07%), 97.08% (RSD=1.11%), 98.09% (RSD=0.67%),
101.97% (RSD=1.37%), 99.32% (RSD=1.31%), 103.75% (RSD=1.33%), 102.12% (RSD
=0.41%), therefore, the inventive method specificity is strong, accuracy, precision, repeatability are equal for 103.58% (RSD=1.10%)
Preferably, not only it is adapted to the detection of Reduning injection finished product, is also adapted to the detection of Reduning injection intermediate.The method
Can reflect that the main chemical compositions Reduning injection and chemical composition were producing from qualitative and quantitative angle comprehensively
Change situation in journey, is the simple and easy to do method being worthy to be popularized, for improving the quality mark of Reduning injection further
Standard has great importance.
Optimal case of the present invention is below used to carry out system suitability experiment:
1. instrument, material and reagent
Agilent 1290 Ultra Performance Liquid Chromatography instrument (be furnished with DAD detector, quaternary gradient pump, online degasser, from
Dynamic injector, Agilent company of the U.S.)；SB25-12DTD-500 numerical control ultrasonic cleaner (Ningbo new sesame biotechnology share
Company limited)；METTLER TOLEDO XP6 electronic balance (prunus mume (sieb.) sieb.et zucc. Teller-torr benefit instrument Shanghai company limited)；H1650-W platform
Formula high speed centrifuge (Hunan Xiang Yi Laboratory Instruments development corporation, Ltd.)；The Millipore Milli-Q water purification machine (U.S.
Millipore company).
Reference substance chlorogenic acid (lot number: 110753-201314), jasminoidin (lot number: 110749-201115) are purchased from China
Food and medicine calibrating academy；Reference substance neochlorogenic acid (lot number: MUST-13013001), 4-dicaffeoylquinic acid (lot number: MUST-
13013002), Geniposidic acid (lot number: MUST-12121502), 3,4-Dicaffeoylquinic acid (lot number: MUST-14022612), different green former
Acid A (lot number: MUST-13081402), 4,5-Dicaffeoylquinic acid (lot number: MUST-13081401) are purchased from Chengdu Man Site biotechnology
Company limited；Reference substance Fructus Gardeniae glycosides (lot number: BBP01688) is purchased from Yunnan Province Xili Bioisystech Co., Ltd；Reference substance genipin
Gentiobioside with cape jasmine (lot number: 121120) is purchased from Chengdu Puffy moral Bioisystech Co., Ltd；Reference substance disconnected oxidation loganin by
Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov provides.Chlorogenic acid reference substance purity is 96.6%, and other reference substance purity are all higher than
98%.Acetonitrile is chromatographically pure, and phosphoric acid is analytical pure, and water is ultra-pure water.
Reduning injection is provided by Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov.
2. chromatographic condition
Chromatographic column Agilent ZORBAX SB-C18 (3.0mm × 100mm, 1.8um)；Pre-column Agilent UHPLC
Guard ZORBAX SB-C18(3.0mm×5mm)；Flowing phase acetonitrile (A)-0.1% phosphoric acid (B)；Flow velocity: 0.4mL.min-1；
Gradient elution (0～10min, 5%A → 10%A；10～15min, 10%A → 15%A；15～30min, 15%A → 30%A)；
After carrying out gradient elution, rerun 10min；Sample size: 2 μ l；Column temperature: 30 DEG C；Detection wavelength 324nm and 238nm is for content
Measure.
3. the preparation of solution
3.1 mixing reference substance storing solutions
Precision weighs neochlorogenic acid, chlorogenic acid, 4-dicaffeoylquinic acid, 3,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, 4,5-Dicaffeoylquinic acid, mountain respectively
Cape jasmine glycosides, Geniposidic acid, genipin gentiobioside with cape jasmine, jasminoidin and disconnected oxidation loganin reference substance are appropriate, accurately weighed, put
In 100ml volumetric flask, add 50% methanol and dissolve and constant volume, make concentration be respectively 0.12225,0.28953,0.12357,
0.01606、0.02849、0.02323、0.01670、0.02468、0.23663、0.40770、0.05745mg·ml-1Mixing
Reference substance storing solution.
3.2 need testing solution
Precision measures 1ml Reduning injection and puts in 100ml volumetric flask, dissolves with 50% methanol and is diluted to scale, shaking
Even, centrifugal, supernatant is crossed 0.22um microporous filter membrane and is filtered, and to obtain final product.
4. multicomponent assay
4.1 linear relationships, detection limit (LOD) and the investigation of quantitative limit (LOQ)
Respectively precision measure mixing reference substance storing solution 1,2,4,6,8,10ml put in 20ml volumetric flask, molten with 50% methanol
Solve and constant volume, take above-mentioned solution 2 μ l respectively and inject chromatograph of liquid, by chromatographic condition under " 2 " item, measure peak area.With face, peak
Long-pending integrated value y is to concentration x (ug ml-1) carry out regression analysis, obtain equation of linear regression and the range of linearity of each reference substance, knot
Fruit is shown in Table 1.Measure after mixing reference substance solution is carried out stepwise dilution with 50% methanol, obtain the LOD (S/N=3) of each composition
With LOQ (S/N=10), the results are shown in Table 1.
Result investigated by table 1 linear relationship
4.2 precision test
Precision measures reference substance storing solution, by chromatographic condition under " 2 " item, continuous sample introduction 6 times, records peak area, and result is new
Chlorogenic acid, chlorogenic acid, 4-dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, 3,5-Dicaffeoylquinic acid, 4,5-Dicaffeoylquinic acid, Fructus Gardeniae glycosides, Geniposidic acid, genipin
The RSD (n=6) of gentiobioside with cape jasmine, jasminoidin and disconnected oxidation loganin peak area is respectively 0.74%, 0.50%, 0.75%,
0.74%, 0.84%, 0.82%, 0.65%, 0.68%, 0.38%, 0.58%, 0.77%, illustrate that the precision of instrument is good.
4.3 replica test
Take same batch (lot number Z140203) Reduning injection, prepare 6 parts of need testing solutions by method below " 3.2 " item,
Measure by chromatographic condition sample introduction under " 2 " item, result neochlorogenic acid, chlorogenic acid, 4-dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, 3,5-Dicaffeoylquinic acid, different
Chlorogenic acid C, Fructus Gardeniae glycosides, Geniposidic acid, genipin gentiobioside with cape jasmine, jasminoidin and disconnected oxidation loganin average content (n=
6) be respectively 2.24,6.55,2.49,0.40,0.23,0.37,0.27,0.50,6.27,9.83,1.10mg ml-1, RSD is respectively
Be 0.65%, 0.47%, 0.59%, 0.95%, 1.01%, 1.24%, 0.59%, 0.65%, 0.90%, 0.38%,
0.53%, illustrate that the method repeatability is good.
4.4 stability test
Taking the need testing solution of same batch (lot number Z140203) Reduning injection, ambient temperatare is put, respectively in difference
Time point (0,2,4,8,16,24h) sample introduction, measure by chromatographic condition under " 2 " item, record peak area, result neochlorogenic acid, green former
Acid, 4-dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, 3,5-Dicaffeoylquinic acid, 4,5-Dicaffeoylquinic acid, Fructus Gardeniae glycosides, Geniposidic acid, genipin gentiobioside with cape jasmine,
The RSD of jasminoidin and disconnected oxidation loganin peak area is respectively 1.04%, 0.75%, 1.08%, 1.21%, 1.36%,
1.36%, 1.39%, 1.21%, 1.23%, 0.70%, 1.25%, illustrate that in sample solution, each composition is stable in 24h.
4.5 average recovery tests
Take same a collection of (lot number Z140203) Reduning injection 0.5ml of known content, put in 100ml volumetric flask, accurate
Add according to the mixing reference substance storing solution 12.5ml of preparation under " 3.1 " item, add 50% methanol and dissolve and be diluted to scale, shake
Even, filter with 0.22um microporous filter membrane.Preparing 6 parts of need testing solutions with method, by chromatographic condition sample introduction 2 μ l under " 2 " item, calculating adds
The sample response rate, the results are shown in Table 2:
Table 2 recovery test result (n=6)
4.6 serviceability test
4.6.1 column temperature is adjusted to 25,30,35 DEG C to the impact of system by column temperature respectively, takes with a need testing solution, presses
Under " 2 " item, chromatographic condition sample introduction measures, result neochlorogenic acid, chlorogenic acid, 4-dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, 3,5-Dicaffeoylquinic acid, different green
Ortho acid C, Fructus Gardeniae glycosides, Geniposidic acid, genipin gentiobioside with cape jasmine, jasminoidin and the RSD (n=of disconnected oxidation loganin content
3) be respectively 1.16%, 0.84%, 1.05%, 1.17%, 1.38%, 1.26%, 1.57%, 1.64%, 1.25%,
0.92%, 1.45%.When this law column temperature changes between 25～35 DEG C, good tolerance.
4.6.2 flow velocity flow velocity is adjusted to by the impact of system respectively 0.35,0.4,0.45mL min-1, take with a for examination
Product solution, is measured by chromatographic condition sample introduction under " 2 " item, result neochlorogenic acid, chlorogenic acid, 4-dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, different green
Ortho acid A, 4,5-Dicaffeoylquinic acid, Fructus Gardeniae glycosides, Geniposidic acid, genipin gentiobioside with cape jasmine, jasminoidin and disconnected oxidation loganin content
RSD (n=3) be respectively 1.39%, 1.15%, 1.23%, 1.41%, 1.56%, 1.52%, 1.76%, 1.74%,
1.18%, 1.27%, 1.39%.This law flow velocity is at 0.35～0.45mL min-1Between change time, good tolerance.
4.7 sample determination
Take the Reduning injection of 6 lot numbers, according to legal system available test sample solution below " 3.2 " item, by chromatograph under " 2 " item
Condition sample introduction measures, and calculates each component content, the results are shown in Table 3, and UPLC chromatogram is shown in Fig. 1.
Table 3 Reduning injection assay result (mg mL-1, n=6)
Accompanying drawing explanation
Fig. 1 is the HPLC chromatogram of each composition detection of the present invention.
A is mixing reference substance UPLC chromatogram under 324nm, and B is mixing reference substance UPLC chromatogram under 238nm, and C is
Reduning injection UPLC chromatogram under 324nm, D is Reduning injection UPLC chromatogram under 238nm.
In figure, 1. neochlorogenic acid (Neochlorogenic acid) 2. chlorogenic acid (Chlorogenic acid) is the most hidden green
Ortho acid (Cryptochlorogenic acid) 4. 3,4-Dicaffeoylquinic acid (Isochlorogenic acid B) 5. 3,5-Dicaffeoylquinic acid
(Isochlorogenic acid A) 6. 4,5-Dicaffeoylquinic acid (Isochlorogenic acid C) 7. Fructus Gardeniae glycosides
(Shanzhiside) 8. Geniposidic acid (Geniposidic acid) 9. genipin gentiobioside with cape jasmine (Genipin-1-β-D-
Gentiobioside) 10. jasminoidin (Geniposide) 11. is disconnected aoxidizes loganin (Secoxyloganin)
Detailed description of the invention
The present invention be expanded on further in the following manner:
Embodiment 1
A, the preparation of reference substance solution: precision weighs neochlorogenic acid, chlorogenic acid, 4-dicaffeoylquinic acid, 3,5-Dicaffeoylquinic acid, different respectively
Chlorogenic acid B, 4,5-Dicaffeoylquinic acid, Fructus Gardeniae glycosides, Geniposidic acid, genipin gentiobioside with cape jasmine, jasminoidin, disconnected oxidation loganin pair
Appropriate according to product, the methanol adding 50% make concentration be respectively 0.12225,0.28953,0.12357,0.01606,0.02849,
0.02323、0.01670、0.02468、0.23663、0.40770、0.05745mg·ml-1Mixing reference substance solution.
B, the preparation of need testing solution
Accurate 1ml Reduning injection of drawing is put in volumetric flask, dissolves with 50% methanol and is diluted to 100ml, shaking up, from
The heart, supernatant is crossed 0.22um microporous filter membrane and is filtered, to obtain final product.
C, algoscopy: use UPLC-DAD method precision respectively to draw reference substance solution and need testing solution, inject liquid phase color
Spectrometer, measures, to obtain final product；
Chromatographic condition: chromatographic column Agilent ZORBAX SB-C18 (3.0 × 100mm, 1.8um)；With acetonitrile for flowing phase
A, with 0.1% phosphoric acid as Mobile phase B, after being carried out according to the following table gradient elution, rerun 10min；Detection wavelength: 324nm and
238nm；Sample size: 2 μ l；Flow velocity: 0.4mL.min-1；Column temperature: 30 DEG C.
Measure 11 kinds of composition neochlorogenic acids, chlorogenic acid, 4-dicaffeoylquinic acid, 3,5-Dicaffeoylquinic acid, isochlorogenic acid in Reduning injection
B, 4,5-Dicaffeoylquinic acid, Fructus Gardeniae glycosides, Geniposidic acid, genipin gentiobioside with cape jasmine, jasminoidin, disconnected oxidation 11 kinds of components of loganin
All reaching baseline separation, have good linear relationship in the range of linear, correlation coefficient is all higher than 0.999, average recovery rate
(n=6) it is respectively 102.89% (RSD=1.25%), 103.25% (RSD=0.39%), 103.79% (RSD=
1.11%), 98.11% (RSD=1.07%), 97.08% (RSD=1.11%), 98.09% (RSD=0.67%),
101.97% (RSD=1.37%), 99.32% (RSD=1.31%), 103.75% (RSD=1.33%), 102.12% (RSD
=0.41%), 103.58% (RSD=1.10%).Method repeatability is good, and the precision of instrument is good, each in sample solution
Composition is stable in 24h.
Embodiment 2
A, the preparation of reference substance solution: precision weighs neochlorogenic acid, chlorogenic acid, 4-dicaffeoylquinic acid, 3,5-Dicaffeoylquinic acid, different respectively
Chlorogenic acid B, 4,5-Dicaffeoylquinic acid, Fructus Gardeniae glycosides, Geniposidic acid, genipin gentiobioside with cape jasmine, jasminoidin, disconnected oxidation loganin pair
Appropriate according to product, add methanol make concentration be respectively 0.20001,0.38333,0.18445,0.02661,0.0408,0.02865,
0.04000、0.04845、0.36654、0.60076、0.11475mg·ml-1Mixing reference substance solution.
B, the preparation of need testing solution
Accurate 1ml Reduning injection of drawing is put in volumetric flask, dissolves with methanol and is diluted to 100ml, shaking up, centrifugal,
Supernatant is crossed 0.22um microporous filter membrane and is filtered, and to obtain final product.
C, algoscopy: use UPLC-DAD method precision respectively to draw reference substance solution and need testing solution, inject liquid phase color
Spectrometer, measures, to obtain final product；
Chromatographic condition: chromatographic column Agilent ZORBAX SB-C18 (3.0 × 100mm, 1.8um)；With acetonitrile for flowing phase
A, with 0.1% phosphoric acid as Mobile phase B, is carried out according to the following table gradient elution, and then rerun 10min；Detection wavelength: 326nm and
236nm；Sample size: 2 μ l；Flow velocity: 0.45mL.min-1；Column temperature: 25 DEG C.
Measure 11 kinds of composition neochlorogenic acids, chlorogenic acid, 4-dicaffeoylquinic acid, 3,5-Dicaffeoylquinic acid, isochlorogenic acid in Reduning injection
B, 4,5-Dicaffeoylquinic acid, Fructus Gardeniae glycosides, Geniposidic acid, genipin gentiobioside with cape jasmine, jasminoidin, disconnected oxidation 11 kinds of components of loganin
All reaching baseline separation, have good linear relationship in the range of linear, correlation coefficient is all higher than 0.999.Method repeatability
Well, the precision of instrument is good, and in sample solution, each composition is stable in 24h.
List of references
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Claims (3)
1. one kind measures the method for 11 kinds of active constituent contents in Reduning injection simultaneously, it is characterised in that the method include with
Lower step:
A, the preparation of reference substance solution: precision weighs neochlorogenic acid, chlorogenic acid, 4-dicaffeoylquinic acid, 3,5-Dicaffeoylquinic acid, different green former respectively
Acid B, 4,5-Dicaffeoylquinic acid, Fructus Gardeniae glycosides, Geniposidic acid, genipin gentiobioside with cape jasmine, jasminoidin and disconnected oxidation loganin reference substance
In right amount, add the methanol that concentration is 50%-100% and make concentration and be respectively 0.12-0.25,0.28-0.58,0.12-0.25,
0.01-0.04,0.02-0.06,0.02-0.05,0.01-0.04,0.02-0.05,0.23-0.48,0.40-0.81,0.05-
0.12mg·ml-1Mixing reference substance solution；
B, the preparation of need testing solution
Accurate 1ml Reduning injection of drawing is put in volumetric flask, dissolves with the methanol that concentration is 50%-100% and is diluted to
100ml, shakes up, and centrifugal, supernatant is filtered by microporous filter membrane, to obtain final product；
C, algoscopy: use UPLC-DAD method precision respectively to draw reference substance solution and need testing solution, inject chromatograph of liquid,
Measure, to obtain final product；
Chromatographic condition: chromatographic column Agilent ZORBAX SB-C18；With acetonitrile as mobile phase A, with 0.1% phosphoric acid for flowing phase
B, is carried out according to the following table gradient elution；Detection wavelength: 324-326nm and 236-238nm；Flow velocity: 0.35-0.45mL.min-1；Post
Temperature: 25-35 DEG C；
The method of 11 kinds of active constituent contents, its feature in the Reduning injection of mensuration simultaneously the most according to claim 1
It is that described methanol is the methanol that concentration is 50～70%.
The method of 11 kinds of active constituent contents, its feature in the Reduning injection of mensuration simultaneously the most according to claim 1
It is that the method comprises the following steps:
A, the preparation of reference substance solution: precision weighs neochlorogenic acid, chlorogenic acid, 4-dicaffeoylquinic acid, 3,5-Dicaffeoylquinic acid, different green former respectively
Acid B, 4,5-Dicaffeoylquinic acid, Fructus Gardeniae glycosides, Geniposidic acid, genipin gentiobioside with cape jasmine, jasminoidin and disconnected oxidation loganin reference substance
In right amount, add the methanol that concentration is 50% make concentration be respectively 0.12-0.15,0.28-0.30,0.12-0.15,0.01-0.02,
0.02-0.04、0.02-0.03、0.01-0.03、0.02-0.03、0.23-0.30、0.40-0.50、0.05-0.08mg·ml-1
Mixing reference substance solution；
B, the preparation of need testing solution
Accurate 1ml Reduning injection of drawing is put in volumetric flask, is that 50% methanol dissolves and is diluted to 100ml by concentration, shakes up,
Centrifugal, supernatant is crossed 0.22um microporous filter membrane and is filtered, and to obtain final product；
C, algoscopy: use UPLC-DAD method precision respectively to draw reference substance solution and need testing solution, inject chromatograph of liquid,
Measure, to obtain final product；
Chromatographic condition: chromatographic column Agilent ZORBAX SB-C18；With acetonitrile as mobile phase A, with 0.1% phosphoric acid for flowing phase
B, is carried out according to the following table gradient elution, and then rerun 10-12min；Detection wavelength: 324-326nm and 236-238nm；Flow velocity:
0.4mL.min-1；Column temperature: 30 DEG C；
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| CN107677750A (en) * | 2017-11-01 | 2018-02-09 | 广西壮族自治区食品药品检验所 | The multicomponent content assaying method of Mussaenda hirsutula |
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| CN111413429A (en) * | 2020-04-15 | 2020-07-14 | 江苏康缘药业股份有限公司 | Detection method of gardenia intermediate and fingerprint spectrum construction method thereof |
| CN111896637B (en) * | 2020-06-05 | 2021-07-23 | 江苏康缘药业股份有限公司 | Detection method of Jinqing intermediate and fingerprint spectrum construction method thereof |
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