CN104237416B - The HPLC method of 6 kinds of flavones ingredient content in Simultaneously test poacynum hendersonii woodson leaf - Google Patents
The HPLC method of 6 kinds of flavones ingredient content in Simultaneously test poacynum hendersonii woodson leaf Download PDFInfo
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Abstract
The present invention relates to the HPLC method of 6 kinds of flavones ingredient content in a kind of Simultaneously test poacynum hendersonii woodson leaf, the method is using microemulsion as the high performance liquid chromatography of mobile phase, in micro emulsion mobile phase, the ratio of aqueous phase is general all more than 90%, greatly reduce the use of organic solvent compared with mobile phase in current conventional high-performance liquid phase chromatography, greatly reduce the time to the harm of health, the pollution of environment and analysis and cost.By the optimization to micro emulsion chromatographic condition, under isocratic condition, in poacynum hendersonii woodson leaf, 6 kinds of flavones ingredients obtain effective separation simultaneously, establish a kind of environmental protection, accurately and reliably, stable method for quick.Overcome the defect in existing poacynum hendersonii woodson leaf flavones ingredient mensuration.
Description
Technical field
The present invention relates to analytical chemistry field, be specifically related to a kind of method utilizing 6 kinds of flavones ingredients in HPLC Simultaneously test poacynum hendersonii woodson leaf.
Background technology
Aprocynum henderson hook (Poacynum hendersonii (Hook.F.) Woods.) is Apocynaceae abutilon, formal name used at school largeleaf poacynum leaf, medicinal part is leaf, mainly be distributed in Xinjiang of China area, growth saline and alkaline, sandy wasteland is regional and perennial root plant resources on Gobi desert, the growing environment of its uniqueness makes it have very special medicinal health effect.Xinjiang aprocynum henderson hook is used as medicine history in existing thousand, its Pharmacological be it is found that and very early for clinical, record according to Compendium of Material Medica, herbal for Relief of Famines, the effect such as its Folium Apocyni Veneti has reducing fever and causing diuresis, flat liver, calm the nerves, clinically be used for the treatment of the diseases such as hypertension, coronary heart disease, cervical spondylopathy, ephritis edema, neurasthenia, menopausal syndrome, the formal typing Pharmacopoeia of the People's Republic of China in 1977.Modern pharmacology research proves that Flavonoid substances is the topmost effective constituent of poacynum hendersonii woodson leaf, Flavonoid substances mainly 6 kinds of flavones ingredients such as Quercetin-3-O-sophorose, isoquercitrin, Quercetin, Hyperoside, astragalin and trifolin in poacynum hendersonii woodson leaf.Along with poacynum hendersonii woodson leaf widespread use clinically, be necessary very much to carry out assay and analysis to wherein flavones ingredient, this contributes to the quality control of poacynum hendersonii woodson leaf, also for lay the foundation to the further research of aprocynum henderson hook from now on.The domestic and international detection method to poacynum hendersonii woodson leaf flavones ingredient mainly spectrophotometric method and high performance liquid chromatography (HPLC) at present.
Spectrophotometry be the content of aprocynum henderson hook leaf flavonoids, but this can only reflect roughly the total amount containing flavones ingredient in poacynum hendersonii woodson leaf, the situation of each flavones ingredient of poacynum hendersonii woodson leaf can not be reflected, cannot be used for evaluating the authenticity of aprocynum henderson hook leaf quality, Optimality and stability.In recent years, along with the raising of the deep and modern separation technology of material foundation of tcm, pharmacology activity research, traditional Chinese medicine quality level is significantly improved.After recognizing that herbal medicine efficacy is Mutiple Targets, multicomponent synergy feature, multi-target ingredient quantitatively becomes the development trend of Chinese medicine assay.And in the current document about high effective liquid chromatography for measuring poacynum hendersonii woodson leaf flavone component, only have and the individual components in poacynum hendersonii woodson leaf Flavonoid substances is measured, and the multiple flavones ingredients such as Quercetin-3-O-sophorose, isoquercitrin, Quercetin, Hyperoside, astragalin and trifolin are not measured simultaneously, thus cannot comprehensively reflect poacynum hendersonii woodson leaf flavones ingredient, and then also just cannot reflect the quality condition of poacynum hendersonii woodson leaf.Simultaneously also due to the larger difference of physicochemical property between each flavones ingredient, it is little all to there is separation selectivity in existing HPLC separation method, the shortcoming of degree of separation difference and disengaging time length.In addition, in current HPLC separation method, mobile phase contains a high proportion of methyl alcohol or acetonitrile, and this is also extremely harmful to health and environment.Thus, setting up a kind of environmental protection, to analyze the method for multiple flavones ingredient in poacynum hendersonii woodson leaf that measures efficiently significant.
Microemulsion liquid chromatography (microemulsion liquid chromatography, MELC) method take micro emulsion as the high performance liquid chromatography of mobile phase.Micro emulsion mobile phase is normally made up of aqueous phase, oil phase, surfactant and cosurfactant, is a kind of transparent or semitransparent, low viscous, isotropy and thermodynamically stable water oil commingled system, particle diameter is less than 100nm.MELC method compared with conventional H PLC method, have do not use or few with an organic solvent, separation efficiency is high and velocity of separation is fast advantage, can be used for the separation of complex sample, complex sample just can direct injection analysis after simple dilution, without the need to pre-service.Obvious advantage is had in low ultraviolet wavelength (200nm) context of detection.
The present invention establishes a kind of high performance liquid chromatography (i.e. Microemulsion liquid chromatography (MELC)) using microemulsion as mobile phase and carries out assay to 6 kinds of flavones ingredients such as Quercetin-3-O-sophorose, isoquercitrin, Quercetin, Hyperoside, astragalin and trifolins in poacynum hendersonii woodson leaf simultaneously, and apply the method and respectively 6 batch samples are analyzed, and then compare the similarities and differences of different batches poacynum hendersonii woodson leaf medicinal material on chemical composition composition and content, for the application of poacynum hendersonii woodson leaf medicinal material clinically provides better theoretical direction.
Summary of the invention
The object of the invention is, the HPLC method of 6 kinds of flavones ingredient content in a kind of Simultaneously test poacynum hendersonii woodson leaf is provided, the method is using microemulsion as the high performance liquid chromatography of mobile phase, in micro emulsion mobile phase, the ratio of aqueous phase is general all more than 90%, greatly reduce the use of organic solvent compared with mobile phase in current conventional high-performance liquid phase chromatography, greatly reduce the time to the harm of health, the pollution of environment and analysis and cost.By the optimization to micro emulsion chromatographic condition, under isocratic condition, in poacynum hendersonii woodson leaf, 6 kinds of flavones ingredients obtain effective separation simultaneously, establish a kind of environmental protection, accurately and reliably, stable method for quick.Overcome the defect in existing poacynum hendersonii woodson leaf flavones ingredient mensuration.
The HPLC method of 6 kinds of flavones ingredient content in a kind of Simultaneously test poacynum hendersonii woodson leaf of the present invention, follows these steps to carry out:
Prepared by a, sample solution: take the poacynum hendersonii woodson leaf powder 0.5g of vacuum drying to constant weight, accurately weighed, crosses No. 3 sieves, adding 40mL concentration is 70% ethanol, soaks 1h, ultrasonic process 30min, let cool, filter, accurately pipette subsequent filtrate 2mL, put in 10mL volumetric flask, be 70% ethanol constant volume with concentration, after shaking up, with 0.45 μm of membrane filtration, get subsequent filtrate, obtain sample solution;
Prepared by b, reference substance solution: take vacuum drying respectively appropriate to the Quercetin-3-O-sophorose of constant weight, isoquercitrin, Quercetin, Hyperoside, astragalin and trifolin reference substance, accurately weighed, adding concentration is that 70% EtOH Sonicate dissolves, make the mixing reference substance solution of 2mg/ml, as stock solution, sealing, temperature 4 DEG C preservation;
C, micro emulsion mobile phase are prepared: by volume fraction 2.0% surfactant Genapol X-080, volume fraction 3.0% cosurfactant normal butyl alcohol, volume fraction 0.6% oil phase ethyl acetate, volume fraction 0.2% triethylamine and 94.2% aqueous solution, use 20mM H
3pO
4regulate pH=5.0, ultrasonic 30min, through 0.45 μm of membrane filtration, ultrasonic 30min, obtains the microemulsion solution of transparent and stable, hold over night, for subsequent use;
D, chromatographic condition: chromatographic column is Zorbax SB-C
184.6mm × 150mm, 5 μm of posts, flow velocity 0.8mL/min, determined wavelength 360nm, column temperature 30 DEG C, mobile phase is 2.0%Genapol X-080-3.0% normal butyl alcohol-0.6% ethyl acetate-0.2% triethylamine-94.2% aqueous solution, uses 20mM H
3pO
4regulate pH=5.0, sample size 10 μ L, adopt quantified by external standard method, chromatogram record is completed by workstation with process, and theoretical cam curve calculates by isoquercitrin peak and is not less than 8000.
The present invention adopts Microemulsion liquid chromatography method, the content of flavones ingredient in Simultaneously test poacynum hendersonii woodson leaf 6, and these 6 kinds of flavone components of result reach baseline separation in 15min, within the scope of 1-500mg/L, and the linearly dependent coefficient r of 6 flavone components
2>=0.9996, recovery 98.9-100.8%.This method can be applicable to the quality analysis of 6 kinds of Main Flavonoids compositions in poacynum hendersonii woodson leaf.
The HPLC method of 6 kinds of flavones ingredient content in Simultaneously test poacynum hendersonii woodson leaf of the present invention, the advantage of the method is, in Microemulsion liquid chromatography, 6 kinds of Main Flavonoids component separating efficiency high (being separated completely), velocity of separation fast (disengaging time is only 1/3 of routine HPLC methods), this is because solute is at Stationary liquid, distribute between micro emulsion drop and aqueous phase, be conducive to the separating power improving chromatogram, compared with conventional H PLC, the dissolving power of Microemulsion liquid chromatography to compound is stronger, be particularly useful for polarity or the larger complex sample of dissolubility difference is separated, isocratic elution can reach baseline separation, and consumption of organic solvent is few, analysis cost is low, there is the significant advantage of economy and environmental protection aspect.
The HPLC method of 6 kinds of flavones ingredient content in Simultaneously test poacynum hendersonii woodson leaf of the present invention, the innovative point of the method is to employ the high performance liquid chromatography (HPLC) that micro emulsion is mobile phase, compared with conventional H PLC method, have do not use or few with an organic solvent, separation efficiency is high and velocity of separation is fast advantage, can be used for the separation of complex sample, complex sample just can direct injection analysis after simple dilution, without the need to pre-service.Not only environmental protection, analysis time shortens greatly.Number of patent application 201010120856 is conventional HPLC methods, and not only mobile phase employs the toxic organic solvents such as methyl alcohol and acetonitrile in a large number, and reaches more than 70 minutes analysis time.And in mobile phase of the present invention, do not use any toxic organic solvents, wherein the overwhelming majority is water (94.2%), and only has about 10 minutes analysis time.Which greatly enhances analysis efficiency.Simultaneously according to HPLC side's ratio juris, briefly, be exactly sample dissolution is in mobile phase, enter chromatographic column with mobile phase, when Stationary liquid (filler), the absorption of Stationary liquid can be subject to.The absorption that in sample, different component is subject to is different, so the time sequencing flowing out chromatographic column is just different.
Accompanying drawing explanation
Fig. 1 is the reference substance solution high-efficient liquid phase chromatogram that the present invention sets up MELC method;
Fig. 2 is the sample solution high-efficient liquid phase chromatogram that the present invention sets up MELC method;
Fig. 3 is the high-efficient liquid phase chromatogram that the present invention sets up MELC method (A) and compares with conventional H PLC method (B).
Embodiment
One, experimental section
Instrument and reagent:
Agilent 1100 high performance liquid chromatograph, quaternary gradient pump, DAD detecting device, Agilent chromatographic work station (Agilent company); FZ102 plant comminutor (Tianjin Stettlen Instrument Ltd.); DGSY electric-heated thermostatic water bath (Beijing Chang Yuan experimental facilities factory); TGL-16G table model high speed centrifuge (Shanghai medical analytical instrument factory); SK5200LHC ultrasonic processor (Shanghai High Kudos Science Instrument Co., Ltd.); RE-3000C Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant); The accurate pH meter (Shanghai precision instrumentation company limited) of PHS-3C;
Quercetin-3-O-sophorose, isoquercitrin, Quercetin, Hyperoside, astragalin and the equal available from Sigma of trifolin reference substance (purity is greater than 98%); Poacynum hendersonii woodson leaf medicinal material is provided by this thatched cottage prepared slices of Chinese crude drugs company limited of Xinjiang, is accredited as aprocynum henderson hook (Poacynum hedersonni (Hook.F.) Woodson) through Xinjiang Chinese medicine ethnic drug research institute; Ethoxylated dodecyl alcohol (Genapol X-080, analyze pure, Sigma company), ethyl acetate, heptane, toluene, n-octyl alcohol, n-propanol and normal hexane (analyzing pure, Merck company), phosphoric acid, triethylamine (are analyzed pure, Beijing chemical reagents corporation), acetonitrile (chromatographically pure, Fisher company), water is ultrapure water, and it is pure that all the other reagent are analysis;
Liquid phase chromatogram condition:
Chromatographic column: Zorbax SB-C
18(4.6mm × 150mm, 5 μm) post;
Mobile phase: 2.0%Genapol X-080-3.0% normal butyl alcohol-0.6% ethyl acetate-0.2% triethylamine-94.2% aqueous solution (uses 20mM H
3pO
4regulate pH 5.0);
Flow velocity: 0.8mL/min;
Determined wavelength: 360nm;
Column temperature: 30 DEG C;
Sampling volume: 10 μ L;
Chromatographic resolution the results are shown in Figure 1, as shown in Figure 1, and the separation that 6 kinds of flavones ingredient energy are good;
The preparation of reference substance solution:
Take vacuum drying respectively appropriate to the Quercetin-3-O-sophorose of constant weight, isoquercitrin, Quercetin, Hyperoside, astragalin and trifolin reference substance, accurately weighed, adding concentration is that 70% EtOH Sonicate dissolves, make the mixing reference substance solution of 2mg/ml, as stock solution, seal 4 DEG C of preservations;
Prepared by sample solution:
Take poacynum hendersonii woodson leaf powder (the cross No. 3 sieves) 0.5g of vacuum drying to constant weight, accurately weighed, adding 40mL concentration is 70% ethanol, soak 1h, ultrasonic process 30min, lets cool, filter, accurately pipetting subsequent filtrate 2mL, put in 10mL volumetric flask, is 70% ethanol constant volume with concentration, after shaking up, with 0.45 μm of membrane filtration, get subsequent filtrate, to obtain final product.
Two, result and discussion
The optimization of chromatographic condition
The selection of surfactant:
The surfactants such as SDS, Brij35, Genapol X-080, TritonX-114 are investigated to the separating effect of 6 kinds of flavones ingredients in Cistanche tubulosa, result shows, Genapol X-080 is added in mobile phase, the scope (=2 ~ 8) that the retention factors of 6 kinds of compositions can be made to reach suitable, Chromatogram Baseline is steady simultaneously.When adding SDS in mobile phase, because SDS is ionic surfactant, it is poor to the hydrophilic compounds degree of separation that chemical constitution is closely similar, and 6 kinds of flavones ingredients almost divide too busy to get away; And when adding the non-ionics such as Brij35, TritonX-114 in mobile phase, because Brij35, TritonX-114 exist absorption at 210nm place, Chromatogram Baseline is also unstable, can interference measurement.And non-ionics Genapol X-080 absorbs in 210nm place nothing, Chromatogram Baseline is stablized, therefore selects GenapolX-080 as optimum surfactant.Prepare the micro emulsion mobile phase respectively containing 0.5%, 1.0%, 1.5%, 2.0% and 2.5%Genapol X-080 by a certain percentage, investigate the content of Genapol X-080 to the impact of the separation selectivity of 6 kinds of flavones ingredients in poacynum hendersonii woodson leaf.Result shows: along with the increase of Genapol X-080 content, in poacynum hendersonii woodson leaf, the retention factors of 6 kinds of flavones ingredients obviously reduces, when Genapol X-080 content is 2.0%, the retention factors of 6 kinds of flavones ingredients reaches suitable scope, separating effect is best, therefore selects Genapol X-080 content to be 2.0%.
The selection of cosurfactant:
Select normal butyl alcohol, n-amyl alcohol, n-propanol, isopropyl alcohol to be cosurfactant respectively, investigate its impact on chromatographic behavior.Result shows, and when taking normal butyl alcohol as cosurfactant, the retention time of 6 kinds of flavonoids constituents all reduces, and changes the content 1.0-5.0% of normal butyl alcohol, investigates its impact on 6 kinds of flavonoids constituents separating effects.Result shows: levels of n-butanol is before 3.0%, along with the increase of levels of n-butanol, the retention time of 6 kinds of phenylethanoid glycosides is all along with shortening to some extent, when higher than 3.0% time, retention time is corresponding prolongation again, therefore when determining that cosurfactant levels of n-butanol is 3.0%, separating effect is ideal.
The selection of oil phase:
Normal heptane, toluene, ethyl acetate, normal octane are investigated as the impact of oily relative separation.Result shows: oil phase to the eluting power of 6 kinds of flavones ingredients the strongest for ethyl acetate, and ethyl acetate is best as Component seperation degree each during oil phase, and normal octane, normal heptane and toluene can not make tested composition reach baseline separation, therefore ethyl acetate is selected to be oil phase.Change the content of oil phase ethyl acetate, the degree of separation of material can be changed.Investigate the impact of ethyl acetate content when 0.2%-1.0% range on separating effect, find the increase along with ethyl acetate content, the corresponding raising of degree of separation of 6 kinds of flavones ingredients.When the content of ethyl acetate is 0.6%, separating effect is the most desirable, therefore determines that the content of oil phase ethyl acetate is 0.6%.
Adjuvant and pH value are on the impact be separated:
Because flavones ingredient multi-band has hydroxyl, therefore the retention time of triethylamine concentration to flavones ingredient has considerable influence.When investigation triethylamine concentration is 0%, 0.1%, 0.2%, 0.3%, 0.4%, on the impact of 6 kinds of flavones ingredient separating effects.Result shows: when triethylamine concentration is 0.3%, the retention time of 6 kinds of flavones ingredients all shortens, and finally selects triethylamine concentration to be 0.2%.On the basis of optimum micro emulsion mobile phase, change pH value and (use 20mM H
3pO
4regulate), scope, at 3.0-7.0, has impact to the retention time of 6 kinds of flavones ingredients and degree of separation, along with the increase of pH, the retention time of 6 kinds of flavones ingredients shortens to some extent, but as pH=5.0,6 kinds of flavones ingredients are separated best, therefore, select pH=5.0 (to use 20mMH
3pO
4regulate).
Column temperature is on the impact be separated:
Adopt Genapol X-080-normal butyl alcohol-ethyl acetate-triethylamine-aqueous solution micro emulsion mobile phase, under the condition of temperature 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C and 40 DEG C, investigate the impact of column temperature on Microemulsion liquid chromatography separation selectivity respectively.Result shows: along with column temperature raises, and the retention factors of each component reduces, and when column temperature is lower than 30 DEG C, the peak width of each component is comparatively large, and post effect is lower; When column temperature is equal to or higher than 30 DEG C, the peak shape of each component is better, and post effect significantly improves.Consider that sample and chromatographic column are to the tolerance of temperature, final selection column temperature is 30 DEG C.
Method validation:
The typical curve of method, detection limit and quantitative limit:
Get reference substance storing solution, the standard solution of a series of concentration is diluted to micro emulsion mobile phase, measure by the chromatographic condition of " in experimental section 2 ", make line linearity with peak area Y to concentration X (mg/L) to return, show that the equation of linear regression of 6 kinds of flavones ingredients, quantitative limit (LOQ) and detection limit (LOD) are in table 1:
Table 1 MELC method measures typical curve, quantitative limit and the detection limit of 6 kinds of flavones ingredient standard items
| Composition | The range of linearity (mg/L) | Linear equation | Related coefficient (r 2) | Detection limit (mg/L) | Quantitative limit (mg/L) |
| Quercetin-3-O-sophorose | 1-500mg/L | 146.8X+61.29 | 0.9997 | 0.16 | 0.52 |
| Isoquercitrin | 1-500mg/L | 82.18X+46.73 | 0.9998 | 0.20 | 0.64 |
| Quercetin | 1-500mg/L | 110.12X+51.15 | 0.9998 | 0.22 | 0.71 |
| Hyperoside | 1-500mg/L | 70.51X+30.17 | 0.9997 | 0.18 | 0.68 |
| Astragalin | 1-500mg/L | 63.68X+22.02 | 0.9996 | 0.14 | 0.58 |
| Trifolin | 1-500mg/L | 28.63X+13.81 | 0.9997 | 0.13 | 0.53 |
Result (table 1) shows: 6 flavones ingredients in the typical curve range of linearity (1-200mg/L) in good linear (r
2>=0.9996).The amount of lowest detectable limit is signal to noise ratio (S/N ratio) when being 3 sample, when being 10 using signal to noise ratio (S/N ratio), the amount of sample is as quantitative limit.
The precision of method, repeatability and stability test:
The investigation of precision be measure reference substance solution in a few days, day to day precision.Withinday precision is to same reference substance solution METHOD FOR CONTINUOUS DETERMINATION 6 times within the same day, and day to day precision is then measure once every day same reference substance solution, continuous 6 days.
Table 2 MELC method measures the precision of 6 kinds of flavones ingredients, repeatability and stability
Result is as shown in table 2, and the relative standard deviation (RSD) of withinday precision and day to day precision is respectively 0.42-1.24% and 0.81-2.17%, illustrates that the method that this experiment is set up has good reappearance.
Get same poacynum hendersonii woodson leaf medicinal material 6 parts, as stated above operation repetitive, analysis, calculating, evaluate the repeatability of the method with relative standard deviation.Result (table 2) shows: in 6, the relative standard deviation of flavones ingredient is in the scope of 1.14-2.63%, illustrates that the method repeatability set up is good.
Get poacynum hendersonii woodson leaf medicinal material and prepare sample solution according to the method described above, measure at 0h, 2h, 4h, 8h, 16h, 24h, 36h and 48h respectively, evaluate stability with relative standard deviation.Result is as shown in table 2, shows that sample has good stability in 48h.
The recovery test of method:
Recovery test is used for the accuracy of further evaluation method.The test sample that precision takes known 6 kinds of flavone component content is appropriate, adds 6 kinds of flavones reference substances of equivalent, presses set up chromatographic condition and detects, and calculates the recovery by the ratio calculating theoretical value and the actual value measured.
Table 3 MELC method measures the recovery (n=6) of 6 kinds of flavones ingredients
| Composition | Original vol (mg) | Addition (mg) | Measured amount (mg) | The recovery (%) | RSD(％) |
| Quercetin-3-O-sophorose | 125.4 | 126.9 | 251.8 | 99.6 | 0.72 |
| Isoquercitrin | 82.5 | 83.4 | 166.1 | 100.3 | 1.13 |
| Quercetin | 71.1 | 70.9 | 141.2 | 98.9 | 0.94 |
| Hyperoside | 64.2 | 63.4 | 127.1 | 99.3 | 1.26 |
| Astragalin | 58.5 | 59.8 | 118.8 | 100.8 | 1.52 |
| Trifolin | 33.4 | 35.2 | 68.7 | 100.4 | 1.47 |
Table 3 result shows: the recovery of the method is within the scope of 98.9-100.8%, and relative standard deviation, in the scope of 0.72-1.52%, shows that the method has good reliability and accuracy.
Sample analysis:
Utilize method of the present invention, Simultaneously test has been carried out to 6 kinds of flavone components in 6 batches of poacynum hendersonii woodson leafs.The results are shown in Table 4 and Fig. 2, as shown in Figure 2, the separation that in poacynum hendersonii woodson leaf, 6 kinds of flavones ingredient energy are good, result is satisfactory.
The assay (n=6) of 6 kinds of flavone components in table 46 batches of poacynum hendersonii woodson leafs
With comparing of conventional H PLC separation method:
Have studied the routine HPLC methods measuring 6 kinds of flavone components in poacynum hendersonii woodson leaf (mobile phase A is 0.2% phosphate aqueous solution, and B is acetonitrile solution, gradient elution: 0-10min, A-B 70:30 simultaneously; 10-20min, A-B 60:40; 50-50min, A-B 40:60; 40-60min, A-B 30:70.Determined wavelength 360nm, flow velocity 1.0mL/min, sample size 10 μ L), its analysis time is 60min, completes a sample analysis, and the consumption of organic solvent (acetonitrile) is about 32mL; And namely the MELC method that the present invention sets up completes the analysis to 6 kinds of flavone components in 15min, and only have minute quantity organic solvent in mobile phase, the consumption completing 1 sample analysis is about 1.2mL.The results are shown in Figure 3.As can be seen here, adopt MELC method significantly can shorten detection time, a large amount of consumption reducing organic solvent, reduces costs, environmental protection.
Three, conclusion:
The present invention establishes a kind of new clastotype (MELC) and measures 6 kinds of flavone component content in poacynum hendersonii woodson leaf, by the investigation of the factors such as Surfactant kind and concentration, cosurfactant kind and concentration, oil phase kind and concentration, mobile phase acidity, adjuvant and column temperature, obtain best microemulsion system and chromatographic condition, and carried out methodological study, 6 batch samples are analyzed.Result shows, the method that the present invention sets up is quick, accurate, can be used for the quality analysis to 6 kinds of flavone components in poacynum hendersonii woodson leaf and monitoring.
Claims (1)
1. the HPLC method of 6 kinds of flavones ingredient content in Simultaneously test poacynum hendersonii woodson leaf, is characterized in that following these steps to carry out:
Prepared by a, sample solution: take the poacynum hendersonii woodson leaf powder 0.5g of vacuum drying to constant weight, accurately weighed, crosses No. 3 sieves, adding 40 mL concentration is 70% ethanol, soaks 1 h, ultrasonic process 30 min, let cool, filter, accurately pipette subsequent filtrate 2mL, put in 10 mL volumetric flasks, be 70% ethanol constant volume with concentration, after shaking up, with 0.45 μm of membrane filtration, get subsequent filtrate, obtain sample solution;
Prepared by b, reference substance solution: take vacuum drying appropriate to the Quercetin-3-O-sophorose of constant weight, isoquercitrin, Quercetin, Hyperoside, astragalin and trifolin reference substance, adding concentration is respectively after 70% EtOH Sonicate dissolves, be transferred in 25 mL volumetric flasks, constant volume, shake up, product stock solution in contrast, sealing, temperature 4 DEG C preservation;
C, micro emulsion mobile phase are prepared: by volume fraction 2.0 % surfactant Genapol X-080, volume fraction 3.0% cosurfactant normal butyl alcohol, volume fraction 0.6% oil phase ethyl acetate, volume fraction 0.2% triethylamine and 94. 2% aqueous solution, use 20mM H
3pO
4regulate pH=5.0, ultrasonic 30 min, through 0.45 μm of membrane filtration, ultrasonic 30 min, obtain the microemulsion solution of transparent and stable, hold over night, for subsequent use;
D, chromatographic condition: chromatographic column is Zorbax SB-C
184.6mm × 150mm, 5 μm of posts, flow velocity 0.8
ML/min, determined wavelength 360 nm, column temperature 30 DEG C, mobile phase is 2.0% Genapol X-080-3.0% normal butyl alcohol-0.6% ethyl acetate-0.2% triethylamine-94. 2% aqueous solution, uses 20mM H
3pO
4regulate pH=5.0, sample size 10 μ L, adopt quantified by external standard method, chromatogram record is completed by workstation with process, and theoretical cam curve calculates by isoquercitrin peak and is not less than 8000.
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| CN110927311B (en) * | 2019-12-03 | 2022-04-01 | 广东一方制药有限公司 | Construction method of UPLC (ultra performance liquid chromatography) characteristic spectrum of dogbane leaf medicinal material and method for measuring content of flavonoid component of dogbane leaf medicinal material |
| CN112730688B (en) * | 2020-12-29 | 2022-04-29 | 四川新绿色药业科技发展有限公司 | Method for identifying characteristic spectrum of dogbane leaf formula particle |
| CN114354811B (en) * | 2022-01-11 | 2023-07-07 | 吉林化工学院 | Method for measuring content of three flavonoid chemical components in apocynum venetum leaves |
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