CN107014923A - A kind of method that ASE HPLC methods determine content of oleanolic acid in the root of Chinese clematis - Google Patents
A kind of method that ASE HPLC methods determine content of oleanolic acid in the root of Chinese clematis Download PDFInfo
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- CN107014923A CN107014923A CN201710259576.3A CN201710259576A CN107014923A CN 107014923 A CN107014923 A CN 107014923A CN 201710259576 A CN201710259576 A CN 201710259576A CN 107014923 A CN107014923 A CN 107014923A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a kind of method that ASE HPLC methods determine content of oleanolic acid in the root of Chinese clematis, this method extraction time is short, reproducible, and accuracy of detection is high.Belong to field of chemical detection.This method extracts root of Chinese clematis powder using ASE methods, and collects methanol extraction liquid；The content of the oleanolic acid in methanol extraction liquid is determined using HPLC methods；Wherein, the extraction described in step 1 includes using ethyl acetate degreasing at 80 DEG C successively, and ethanol is extracted at 120 DEG C.The present invention can be extracted and assay instead of official method to oleanolic acid in the root of Chinese clematis.
Description
Technical field
The invention belongs to field of chemical detection, especially a kind of ASE-HPLC methods determine content of oleanolic acid in the root of Chinese clematis
Method.
Background technology
Version official method in 2015：This product powder (crossing No. four sieves) about 4g is taken, it is accurately weighed, put in apparatus,Soxhlet's, plus
Appropriate ethyl acetate, is heated to reflux 3 hours, discards acetic acid ethyl fluid, the dregs of a decoction volatilize solvent, and conical flask is transferred to together with filtration paper cylinder
In, precision adds Diluted Alcohol 50ml, and weighed weight is heated to reflux 1 hour, let cool, then weighed weight, and less loss is supplied with Diluted Alcohol
Weight, shake up, filter, precision measures subsequent filtrate 25ml, put and be evaporated in water-bath, it is molten that residue adds 2mol/L hydrochloric acid solutions 30ml to make
Solution, is heated to reflux 2 hours.Cool down, moved into separatory funnel immediately, with water 10ml gradation washing containers, washing lotion is incorporated to a point liquid leakage
In bucket.Plus ethyl acetate shaking is extracted 3 times, each 15ml, combined ethyl acetate liquid, less than 70 DEG C are concentrated into and closely do, plus methanol
Dissolving, is transferred in 10ml measuring bottles, plus methanol is to scale, shakes up, and produces.
This method extraction time is long, and requires higher to the professional standards of technical staff.
The content of the invention
For above-mentioned deficiency, the present invention is intended to provide a kind of ASE-HPLC methods determine the side of content of oleanolic acid in the root of Chinese clematis
Method, this method extraction time is short, reproducible, and accuracy of detection is high.
In order to realize above-mentioned technique effect, the technical scheme that the present invention is provided is such：A kind of ASE-HPLC methods are determined
The method of content of oleanolic acid in the root of Chinese clematis, comprises the steps successively：
Step 1：Root of Chinese clematis powder is extracted using ASE methods, and collects methanol extraction liquid；
Step 2：The content of the oleanolic acid in methanol extraction liquid is determined using HPLC methods；
Wherein, the extraction described in step 1 includes using ethyl acetate degreasing at 80 DEG C successively, and ethanol is extracted at 120 DEG C.
Preferably, described step 1 includes following sub-steps：
Step S1：The root of Chinese clematis is crushed, No. three sieves is crossed, takes 1g to be mixed with 1g quartz sands；
Step S2：Mixture is loaded on and is placed with the diatomaceous ASE abstraction pools of filter membrane, 1g, plus quartz sand is put down to Chi Kou
OK；
Step S3：Ethyl acetate degreasing is used, then is extracted with ethanol；
Step S4：It is evaporated, precipitation is used into 30ml dissolving with hydrochloric acid, 2h is heated to reflux；
Step S5：Cooling, is moved into separatory funnel, plus 45ml ethyl acetate is extracted, concentration；
Step S6：Add methanol to dissolve the concentrate obtained by step S5, and add methanol constant volume to 10ml, centrifugation takes supernatant
Liquid.
Preferably, the degreasing parameter described in step S3 is：Pressure is 1500psi, and temperature is 80 DEG C, and extraction time is
3min, cycle-index is 2 times, and flush volume is 100%, and purge time is 60s.
Preferably, the extracting parameter described in step S3 is：Pressure is 1500psi, and temperature is 120 DEG C, and extraction time is
5min, cycle-index is 3 times, and flush volume is 100%, and purge time is 60s.
Preferably, the concentration of hydrochloric acid described in step S4 is 2mol/L.
Preferably, described step 5 is specially：Cooling, is moved into separatory funnel, is extracted and is added ethyl acetate extraction 3 times, often
Secondary 15ml, collects No. 3 extract solutions, concentration.
Preferably, the detection parameter of the HPLC methods described in step 2 is：Chromatographic column is Thermo Acclaim C30；Column temperature
For 20 DEG C；
Flow velocity is 0.5mL/min；Mobile phase is acetonitrile-water；Detection wavelength is 205nm.
Preferably, the specification of described chromatographic column is 2.1*150mm, 3 μm.
Preferably, described mobile phase is that volume ratio is equal to 62：38 acetonitrile-water.
The present invention is compared with conventional method, with advantages below：
The present invention is once investigated to the solvent that Accelerate solvent extraction is used,《Chinese Pharmacopoeia》Using ethyl acetate removal of impurities,
Oleanolic acid conjugate is extracted using Diluted Alcohol and is hydrolyzed into oleanolic acid again, impurity is less, therefore to using ethyl acetate, n-hexane
As removal of impurities solvent, methanol, Diluted Alcohol, ethanol have carried out solvent investigation as Extraction solvent, as a result show ethyl acetate removal of impurities,
Diluted Alcohol extracts the content after being hydrolyzed after oleanolic acid using 2mol/L hydrochloric acid and version in 2015《Chinese Pharmacopoeia》Content is consistent, and
Impurity peaks are essentially identical, therefore use the Extraction solvent consistent with pharmacopeia.Period our pilot productions ethyl acetate removal of impurities, using 5%
Ammoniacal liquor Diluted Alcohol, 3% phosphoric acid Diluted Alcohol, as extraction is hydrolyzed in the extractor of instrument, are extracted as extraction and hydrating solution
Effect is not good, therefore individually uses and be hydrolyzed outside the miscellaneous instrument of 2mol/L hydrochloric acid solutions of pharmacopeia.
In dedoping step, official method adds ethyl acetate backflow 3h, and the time is relatively long, and we employ multiple extraction
Miscellaneous mode is removed, 80 DEG C are found, 3min is extracted, 2 times are that can reach the most of impurity of removing, then many extraction times and number of times
It has been had no significant effect that, and high temperature relatively can be the reduction of oleanolic acid result, therefore finally determine optimal impurity removal process combination
For 80 DEG C of Extracting temperature, extraction time 3min, extraction time 2 times.120 DEG C are used in extraction process.5min is extracted, is extracted 3 times
It can reach and approached with pharmacopeia content, more extraction times, temperature, number of times have had no significant effect to result.It is final to determine
Optimum extraction process is combined as 120 DEG C of Extracting temperature, extraction time 5min, extraction time 3 times.
Brief description of the drawings
Fig. 1 is the linear regression graph carried out with concentration (μ g/ml)-peak area of oleanolic acid reference substance；
Fig. 2 is the chromatogram of oleanolic acid reference substance solution；
Fig. 3 is the chromatogram using the need testing solution obtained by official method extraction；
Fig. 4 is the chromatogram using the need testing solution obtained by the extraction of ASE methods.
Embodiment
With reference to embodiment, the claim to the present invention is described in further detail, but is not constituted pair
Any limitation of the present invention, any limited number of time modification made in the claims in the present invention protection domain, still the present invention's
In claims.
Embodiment 1
1st, instrument and equipment and reagent
1.1 instrument：
Electronic analytical balance (XA205DU), ASE350 Accelerate solvent extractions instrument (DIONEX companies of the U.S.), Thermo
U3000UHPLC liquid chromatographs
1.2 reagent：
Water：Meet one-level water as defined in GB/T 6682；
Methanol (CH4O)：Chromatographically pure；
Ethanol (C2H60), ethyl acetate (C4H8O2)：Analysis is pure；
Quartz sand：2mm.
2nd, method
The preparation of 2.1 reference substance solutions
Take oleanolic acid reference substance appropriate, it is accurately weighed, plus solution of every 1ml containing 1mg is made in mobile phase, produces.
The preparation of 2.2 need testing solutions
2.2.1 version official method in 2015
This product powder (crossing No. four sieves) about 4g is taken, it is accurately weighed, put in apparatus,Soxhlet's, plus appropriate ethyl acetate, heating
Backflow 3 hours, discards acetic acid ethyl fluid, the dregs of a decoction volatilize solvent, are transferred to together with filtration paper cylinder in conical flask, and precision adds Diluted Alcohol
50ml, weighed weight is heated to reflux 1 hour, let cool, then weighed weight, and the weight of less loss is supplied with Diluted Alcohol, is shaken up, filtration,
Precision measures subsequent filtrate 25ml, puts and is evaporated in water-bath, and residue adds 2mol/L hydrochloric acid solutions 30ml to make dissolving, is heated to reflux 2 hours.
Cool down, move into separatory funnel, with water 10ml gradation washing containers, washing lotion is incorporated in separatory funnel immediately.Plus ethyl acetate shakes
Shake extraction 3 times, each 15ml, combined ethyl acetate liquid, less than 70 DEG C are concentrated near dry, plus methanol dissolves, and is transferred to 10ml amounts
In bottle, plus methanol is to scale, shakes up, and produces.
2.2.2 Accelerate solvent extraction method (ASE) prepares test sample method
The size-reduced machine of sample is crushed, and crosses No. three sieves, and about 1g is accurately weighed, is well mixed with appropriate amount of quartz sand, stand-by,
Put filter membrane well in advance1g diatomite is first added in 10ml abstraction pools, it is rear to add well mixed sample, add
Appropriate amount of quartz sand, gently shaking is allowed at 2mm, tighten and cover on abstraction pool under same horizontal line with Chi Kou.Extraction (including removal of impurities
With extract 2 steps, see ASE conditions methods) terminate after, put and be evaporated in water-bath, residue adds 2mol/L hydrochloric acid solutions 30ml to make dissolving, plus
Heat backflow 2 hours.Cool down, move into separatory funnel, with water 10ml gradation washing containers, washing lotion is incorporated in separatory funnel immediately.
Plus ethyl acetate shaking is extracted 3 times, each 15ml, combined ethyl acetate liquid, less than 70 DEG C are concentrated near dry, plus methanol dissolves,
It is transferred in 10ml measuring bottles, plus methanol is to scale, shakes up, and centrifuges 3min under 15000r/min, takes supernatant, is determined into LC
Produce.
2.2.3 the preparation of reference substance solution
Take root of Chinese clematis reference substance appropriate, it is accurately weighed, plus solution of every 1ml containing 1mg is made in mobile phase, produces.
2.3ASE extraction conditions
Degreasing parameter is：Pressure is 1500psi, and temperature is 80 DEG C, and extraction time is 3min, and cycle-index is 2 times, is rinsed
Volume is 100%, and purge time is 60s.
Extracting parameter is：Pressure is 1500psi, and temperature is 120 DEG C, and extraction time is 5min, and cycle-index is 3 times, punching
It is 100% to wash volume, and purge time is 60s.
2.4 chromatographic conditions and system suitability
A) instrument：The double ternary liquid phases (U-3000) of Thermo；
B) chromatographic column：Thermo Acclaim C30,2.1*150mm, 3 μm
C) column temperature：20℃；
D) flow velocity：0.5mL/min；
E) mobile phase：Acetonitrile-water (62：38)；
F) Detection wavelength：205nm；
Using octadecylsilane chemically bonded silica as filler；Number of theoretical plate is calculated by oleanolic acid peak should be not less than 3000.
2.5 determination method
Determined according to high performance liquid chromatography (general rule 0512)；
It is accurate respectively to draw reference substance solution and each 10 μ l of need testing solution, liquid chromatograph is injected, determines, produces.
The requirement of 2.6 standard limited values
This product is calculated by dry product, and 0.30% must not be less than containing oleanolic acid (C30H48O3).
2.7 calculate (external standard method)
CR-reference substance solution concentration in formula, unit is micro- gram per liter (mg/L)；
The peak area of AX-test sample；
AR-reference substance peak area.
Note：
It should be dismantled and cleaned out using preceding extraction bottom of pond portion, otherwise easily cause pressure instability；
Otherwise the filter paper of abstraction pool bottom should cause seepage in sealing ring；
Abstraction pool fills elastic moderate during sample, and too loose to be easily caused extract solution excessive；
Check whether gas cylinder air pressure reaches 1Mpa before start；
Using being cleaned out after terminating, abstraction pool will in time dry and (get rusty easily).
3rd, result
3.1 linear relationship
Take oleanolic acid reference substance appropriate, it is accurately weighed, put in brown measuring bottle, plus every 1ml is made containing oleanolic acid in methanol
1mg solution, then the accurate solution 0.1 μ l, 0.2 μ l, 0.5 μ l, 0.8 μ l, the 1.0 μ l of drawing are determined into LC respectively, and according to
Determined according to the above method, the results detailed in Table 1.
Linear regression is carried out with concentration (μ g/ml)-peak area, regression equation Y=0.00084x+0.03077, R=is tried to achieve
0.99991.Oleanolic acid is in good linear relationship in the range of 108.4~1084 μ g/ml, refers to Fig. 1.
The Linear Experiment result of table 1
3.2 reappearances are tested
Take the sample (lot number of identical lot number：150410 (Z)) 1.0g, it is totally 6 parts, accurately weighed, extracted by ASE extracting methods
Need testing solution, sample size is 1 μ L, and with above-mentioned chromatographic condition parallel test, the content for measuring oleanolic acid in sample is shown in Table
2, RSD be 2.0%, as a result shows that ASE extracting methods repeatability is good.
Table 2
3.3 precision and stability experiment
Oleanolic acid reference substance solution (1.084mg/ml) is taken, continuous sample introduction 6 times records peak area, and peak area RSD is
0.5%, show that instrument precision is good.
Same need testing solution is taken, is placed at room temperature, is determined in accordance with the law respectively at 0,1,3,6,12h.As a result Astragaloside IV peak
The RSD of area is 1.5%, shows that need testing solution is stable in 12h.
3.4 samples are using official method and SAE method assays result (3 batches)
Table 3 is it can be seen that using between the result and ASE instruments after ASE instruments 1, ASE instruments 2 and official method extraction
Repeatability it is good.
Table 3
4th, discuss：
The selection of 4.1 ASE Extraction solvents：
In experiment, once the solvent that Accelerate solvent extraction is used was investigated,《Chinese Pharmacopoeia》Using ethyl acetate removal of impurities,
Oleanolic acid conjugate is extracted using Diluted Alcohol and is hydrolyzed into oleanolic acid again, impurity is less, therefore to using ethyl acetate, n-hexane
As removal of impurities solvent, methanol, Diluted Alcohol, ethanol have carried out solvent investigation as Extraction solvent, as a result show ethyl acetate removal of impurities,
Diluted Alcohol extracts the content after being hydrolyzed after oleanolic acid using 2mol/L hydrochloric acid and version in 2015《Chinese Pharmacopoeia》Content is consistent, and
Impurity peaks are essentially identical, therefore use the Extraction solvent consistent with pharmacopeia.Period our pilot productions ethyl acetate removal of impurities, using 5%
Ammoniacal liquor Diluted Alcohol, 3% phosphoric acid Diluted Alcohol, as extraction is hydrolyzed in the extractor of instrument, are extracted as extraction and hydrating solution
Effect is not good, therefore individually outer outside instrument is again hydrolyzed using the 2mol/L hydrochloric acid solutions of pharmacopeia.
The optimization of 4.2 ASE extraction conditions
In dedoping step, official method adds ethyl acetate backflow 3h, and the time is relatively long, and we employ multiple extraction
Miscellaneous mode is removed, 80 DEG C are found, 3min is extracted, 2 times are that can reach the most of impurity of removing, then many extraction times and number of times
It has been had no significant effect that, and high temperature relatively can be the reduction of oleanolic acid result, therefore finally determine optimal impurity removal process combination
For 80 DEG C of Extracting temperature, extraction time 3min, extraction time 2 times.120 DEG C are used in extraction process.5min is extracted, is extracted 3 times
It can reach and approached with pharmacopeia content, more extraction times, temperature, number of times have had no significant effect to result.It is final to determine
Optimum extraction process is combined as 120 DEG C of Extracting temperature, extraction time 5min, extraction time 3 times.
Above-described is only presently preferred embodiments of the present invention, all timess done in the range of the spirit and principles in the present invention
What modifications, equivalent substitutions and improvements etc., should be included within the scope of the present invention.
Claims (9)
1. a kind of method that ASE-HPLC methods determine the content of oleanolic acid in the root of Chinese clematis, it is characterised in that successively including following
Step：
Step 1：Root of Chinese clematis powder is extracted using ASE methods, and collects methanol extraction liquid；
Step 2：The content of the oleanolic acid in methanol extraction liquid is determined using HPLC methods；
Wherein, the extraction described in step 1 includes using ethyl acetate degreasing at 80 DEG C successively, and ethanol is extracted at 120 DEG C.
2. the method that a kind of ASE-HPLC methods according to claim 1 determine the content of oleanolic acid in the root of Chinese clematis, it is special
Levy and be, described step 1 includes following sub-steps：
Step S1：The root of Chinese clematis is crushed, No. three sieves is crossed, takes 1g to be mixed with 1g quartz sands；
Step S2：Mixture is loaded on and is placed with the diatomaceous ASE abstraction pools of filter membrane, 1g, plus quartz sand is extremely parallel with pond mouthful；
Step S3：Ethyl acetate degreasing is used, then is extracted with ethanol；
Step S4：It is evaporated, precipitation is used into 30ml dissolving with hydrochloric acid, 2h is heated to reflux；
Step S5：Cooling, is moved into separatory funnel, plus 45ml ethyl acetate is extracted, concentration；
Step S6：Add methanol to dissolve the concentrate obtained by step S5, and add methanol constant volume to 10ml, centrifugation takes supernatant.
3. the method that a kind of ASE-HPLC methods according to claim 2 determine the content of oleanolic acid in the root of Chinese clematis, it is special
Levy and be, the degreasing parameter described in step S3 is：Pressure is 1500psi, and temperature is 80 DEG C, and extraction time is 3min, circulation time
Number is 2 times, and flush volume is 100%, and purge time is 60s.
4. the method that a kind of ASE-HPLC methods according to claim 2 determine the content of oleanolic acid in the root of Chinese clematis, it is special
Levy and be, the extracting parameter described in step S3 is：Pressure is 1500psi, and temperature is 120 DEG C, and extraction time is 5min, circulation time
Number is 3 times, and flush volume is 100%, and purge time is 60s.
5. the method that a kind of ASE-HPLC methods according to claim 2 determine the content of oleanolic acid in the root of Chinese clematis, it is special
Levy and be, the concentration of hydrochloric acid described in step S4 is 2mol/L.
6. the method that a kind of ASE-HPLC methods according to claim 2 determine the content of oleanolic acid in the root of Chinese clematis, it is special
Levy and be, described step 5 is specially：Cooling, is moved into separatory funnel, extracts plus ethyl acetate is extracted 3 times, each 15ml,
Collect No. 3 extract solutions, concentration.
7. the method that a kind of ASE-HPLC methods according to claim 1 determine the content of oleanolic acid in the root of Chinese clematis, it is special
Levy and be, the detection parameter of the HPLC methods described in step 2 is：Chromatographic column is Thermo Acclaim C30；Column temperature is 20 DEG C；
Flow velocity is 0.5mL/min；Mobile phase is acetonitrile-water；Detection wavelength is 205nm.
8. the method that a kind of ASE-HPLC methods according to claim 7 determine the content of oleanolic acid in the root of Chinese clematis, it is special
Levy and be, the specification of described chromatographic column is 2.1*150mm, 3 μm.
9. the method that a kind of ASE-HPLC methods according to claim 7 determine the content of oleanolic acid in the root of Chinese clematis, it is special
Levy and be, described mobile phase is equal to 62 for volume ratio：38 acetonitrile-water.
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Application publication date: 20170804 |