CN107703237A - Method that is a kind of while determining the content of pine camphor and sequoyitol in ginkgo biloba p.e - Google Patents
Method that is a kind of while determining the content of pine camphor and sequoyitol in ginkgo biloba p.e Download PDFInfo
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- G—PHYSICS
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- 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
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Abstract
Present invention relates particularly to method that is a kind of while determining the content of pine camphor and sequoyitol in ginkgo biloba p.e.Present invention research discovery, under the following conditions：Chromatographic column using aminopropyl bonded silica gel as filler is chromatographic column, with acetonitrile：Water volume ratio is 85~95：5~15 mixed solution is mobile phase, column temperature is 20~40 DEG C, Detection wavelength is 205~215nm, flow velocity is 0.5~2.5mL/min, detected with EISD, the content of sequoyitol in ginkgo biloba p.e and pine camphor can be realized and determined simultaneously, be advantageous to the overall quality control of ginkgo biloba p.e.
Description
Technical field
The invention belongs to Pharmaceutical Analysis field, and in particular to a kind of to determine pine camphor and sequoyitol in ginkgo biloba p.e simultaneously
Content method.
Background technology
Ginkgo (Ginkgo biloba L.) is Ginkgoaceae ginkgo platymiscium, and ginkgo leaf is the dried leaf of ginkgo, mild-natured,
It is sweet, bitter, puckery, the thoughts of returning home, lung channel, there is the effect of astringing the lung, relieving asthma, be promoting blood circulation and removing blood stasis, analgesic.Using ginkgo leaf as raw material, by carrying
Extract made of isolating and purifying is taken to turn into ginkgo biloba p.e.Research shows that ginkgo biloba p.e has the function that hypoglycemic.
Sequoyitol also known as sequoyitol (Sequoyitol), be inositol methyl-derivatives in one kind, be distributed widely in
In various plants, especially in taxaceaes such as taxusyunnanensis, U.S. Chinese yew, Taxus x media and cloud south Miho flower China firs
It is widely distributed in plant.Pine camphor is the isomer of sequoyitol.Recent studies indicate that sequoyitol and pine camphor have drop blood
The effect of sugar.There is document report in the prior art, contain sequoyitol and pine camphor in ginkgo, such as：Nanjing University of Traditional Chinese Medicine master learns
Degree thesis whole-length《Separate sources ginkgo leaf resource chemistry is studied》(in June, 2013) has extracted sequoyitol isolated from ginkgo leaf；
Ohm oto etc. are isolated to sequoyitol, pine camphor etc. in gingko pollen particle.However, still not on ginkgo biloba p.e
The relevant report of the content assaying method of middle sequoyitol and pine camphor.
In spite of document report：The content of D- pine camphors in HPLC-ELSD methods measure cuneate lespedeza herb with root, still, due to cuneate lespedeza herb with root and
The difference of chemical composition contained by ginkgo leaf, the separating effect of other chemical compositions of pine camphor with ginkgo leaf is poor, causes the above method
And it is not used to the assay of pine camphor in ginkgo biloba p.e.Although also there is document report, HPLC-ELSD methods measure sequoyitol
The content of sequoyitol in bulk drug, but due to the difference of chemical composition contained by sequoyitol bulk drug and ginkgo biloba p.e,
Cause the above method and be not used to the assay of pine camphor in ginkgo biloba p.e.
Therefore, a kind of enough methods for determining the content of sequoyitol and pine camphor in ginkgo biloba p.e simultaneously are studied, for silver
The overall quality control of apricot leaf extract is significant.
The content of the invention
It is an object of the invention to provide method that is a kind of while determining the content of pine camphor and sequoyitol in ginkgo biloba p.e.
The present invention provides method that is a kind of while determining the content of pine camphor and sequoyitol in ginkgo biloba p.e, including following
Step：
(1) preparation of need testing solution
Ginkgo biloba p.e solution is prepared, as need testing solution；
(2) preparation of reference substance solution
The solution containing pine camphor is prepared, as pine camphor reference substance solution；
The solution containing sequoyitol is prepared, as sequoyitol reference substance solution；
(3) chromatographic condition
Chromatographic column using aminopropyl bonded silica gel as filler is chromatographic column, using the mixed solution of acetonitrile and water as flowing
Phase, detected with EISD；
(4) determine
Need testing solution and reference substance solution are drawn, injects high performance liquid chromatograph, measure.
Preferably, method that is above-mentioned while determining the content of pine camphor and sequoyitol in ginkgo biloba p.e, with
COSMOSIL5NH2-MS is chromatographic column, with acetonitrile:The volume ratio of water is 85~95:5~15 mixed solution is mobile phase.
It is further preferred that method that is above-mentioned while determining the content of pine camphor and sequoyitol in ginkgo biloba p.e, described
In chromatographic condition, flow velocity is 0.5~2.5mL/min, and column temperature is 20~40 DEG C, and drift tube temperature is 100~110 DEG C, yield value
For 0.5~1.5, nebulizer gas pressure is 2.0~4.0Bar.
It is further preferred that method that is above-mentioned while determining the content of pine camphor and sequoyitol in ginkgo biloba p.e, including
Following steps：
(1) preparation of need testing solution
Ginkgo biloba p.e is taken, adds suitable quantity of water, the ginkgo biloba p.e solution that concentration is 1-10mg/mL is made, shakes up,
Filtration, as need testing solution；
(2) preparation of reference substance solution
Take pine camphor appropriate, add suitable quantity of water, the solution for the pine camphor that concentration is 0.5-2.0mg/mL is made, shakes up, filters, makees
For pine camphor reference substance solution；
Take sequoyitol appropriate, add suitable quantity of water, the solution for the sequoyitol that concentration is 0.5-2.0mg/mL is made, shakes up, filters
Cross, as sequoyitol reference substance solution；
(3) chromatographic condition
Chromatographic column using aminopropyl bonded silica gel as filler is chromatographic column, with acetonitrile：Water volume ratio is 85~95：5~
15 mixed solution is mobile phase, and column temperature is 20~40 DEG C, and Detection wavelength is 205~215nm, and flow velocity is 0.5~2.5mL/
Min, detected with EISD, drift tube temperature is 100~110 DEG C, and yield value is 0.5~1.5, and nebulizer gas pressure is
2.0~4.0Bar；
(4) determine
Need testing solution 5-10 μ L, pine camphor reference substance solution 1-10 μ L and sequoyitol reference substance solution 1-10 μ are drawn respectively
L, inject high performance liquid chromatograph, measure.
It is further preferred that method that is above-mentioned while determining the content of pine camphor and sequoyitol in ginkgo biloba p.e, described
In chromatographic condition, drift tube temperature is 105 DEG C, yield value 1, nebulizer gas pressure 3.0Bar.
It is further preferred that method that is above-mentioned while determining the content of pine camphor and sequoyitol in ginkgo biloba p.e, with second
Nitrile：Water volume ratio is 90.5：9.5 mixed solution is mobile phase.
It is further preferred that method that is above-mentioned while determining the content of pine camphor and sequoyitol in ginkgo biloba p.e, column temperature
For 30 DEG C, flow velocity 1.5mL/min.
It is further preferred that method that is above-mentioned while determining the content of pine camphor and sequoyitol in ginkgo biloba p.e, described
The preparation method of ginkgo biloba p.e comprises the following steps：
Alcohol extracting：Ginkgo leaf is taken, in terms of ginkgo leaf weight, adds the second that the 5-15 times of volumetric concentration measured is 70-95% every time
Alcohol solution extracts 0.5-6.0h, and heating and refluxing extraction 1-5 times merges extract solution, filtering, and filtrate is concentrated into ginkgo leaf weight
0.1~0.5 times, stand at low temperature 12-120h, upper strata oil slick is removed, subnatant is taken, obtains solution A；
Water sinks：In terms of ginkgo leaf weight, the 0.5-5.0 times of water measured, stand at low temperature 46-50h, mistake are added into solution A
Filter, filtrate are concentrated into 0.1-0.5 times of ginkgo leaf weight, put to room temperature, obtain solution B；
Alcohol precipitation：The ethanol water that volumetric concentration is 85-95% is added into solution B makes alcohol content to 70- 90%, will
Solution after alcohol precipitation carries out stand at low temperature, filtering and filtrate and concentrates and repeat 1-5 times successively, obtains solution C；
Ion exchange resin adsorbs：The 0.5-5 times of water measured of ginkgo leaf weight is added to solution C, first uses cation exchange
Resin adsorption is handled 1-5 time, then with anion exchange resin adsorption treatment 1-5 time, collection eluent, is concentrated, obtained ginkgo leaf and carry
Take thing.
It is further preferred that method that is above-mentioned while determining the content of pine camphor and sequoyitol in ginkgo biloba p.e,
In the cationic exchange resin adsorption processing step, the cationic ion-exchange resin is handed over selected from 732 type cations
Change resin, the dosage of the resin cation is measured for 0.5-3.5 times of ginkgo leaf weight；
In the anion exchange resin adsorption treatment step, the anion exchange resin is selected from 711 types, it is described it is cloudy from
The dosage of subtree fat is 0.2-1.0 times of ginkgo leaf weight and measured.
It is further preferred that method that is above-mentioned while determining the content of pine camphor and sequoyitol in ginkgo biloba p.e, described
The preparation method of ginkgo biloba p.e comprises the following steps：
Alcohol extracting：Ginkgo leaf is taken, in terms of ginkgo leaf weight, the volumetric concentration for adding 10 times of amounts every time is water-soluble for 85% ethanol
Liquid extracts 3.0h, heating and refluxing extraction 3 times, merges extract solution, filtering, and filtrate is concentrated into 0.3 times of ginkgo leaf weight, and low temperature is quiet
72h is put, upper strata oil slick is removed, takes subnatant, obtain solution A；
Water sinks：In terms of ginkgo leaf weight, the water of 3.0 times of amounts is added into solution A, stand at low temperature 48h is filtered, and filtrate is dense
0.3 times of ginkgo leaf weight is reduced to, puts to room temperature, obtains solution B；
Alcohol precipitation：The ethanol water that volumetric concentration is 90% is added into solution B makes alcohol content to 80%, after alcohol precipitation
Solution carries out stand at low temperature, filtering and filtrate and concentrates and repeat 3 times successively, obtains solution C；
Ion exchange resin adsorbs：The water of 3 times of amounts of ginkgo leaf weight is added to solution C, first with the 2.0 of ginkgo leaf weight
732 type cationic exchange resin adsorptions of amount are handled 3 times again, then 711 type anion exchanges of 0.6 times of amount with ginkgo leaf weight
Resin adsorption is handled 3 times, is collected eluent, concentration, is obtained ginkgo biloba p.e.Compared with prior art, technical side of the invention
Case has the following advantages that：
(1) present invention research discovery, under the following conditions：Chromatographic column using aminopropyl bonded silica gel as filler is chromatogram
Post, using the mixed solution of acetonitrile and water as mobile phase, column temperature is 20~40 DEG C, and flow velocity is 0.5~2.5mL/min, with evaporative light
Scatter detector is detected, you can the content of the sequoyitol in ginkgo biloba p.e and pine camphor is realized and determined simultaneously, is advantageous to silver
The overall quality control of apricot leaf extract.
(2) present invention further study show that, using COSMOSIL5NH2-MS as chromatographic column, using acetonitrile and water volume ratio as
85~95：5~15 mixed solution is mobile phase, not only causes sequoyitol and pine camphor and other chemistry in ginkgo biloba p.e
Composition can be separated preferably, and sequoyitol and pine camphor are preferably separated, therefore, the content of sequoyitol and pine camphor
Measurement result is more accurate.
(3) present invention further study show that, in the chromatographic condition, drift tube temperature be 100~110 DEG C, yield value
For 0.5~1.5, nebulizer gas pressure is 2.0~4.0Bar, can make it that the specificity of this method is relatively strong, accuracy is good, repeatability
Well, the rate of recovery is good, i.e.,：Each side's science of law test meets regulation.
Brief description of the drawings
In order that present disclosure is more likely to be clearly understood, specific embodiment and combination below according to the present invention
Accompanying drawing, the present invention is further detailed explanation, wherein：
Fig. 1 is the HPLC chromatogram of specificity experiment reclaimed water of the present invention；
Fig. 2 is the HPLC chromatogram of pine camphor in specificity experiment of the present invention；
Fig. 3 is the HPLC chromatogram of sequoyitol in specificity experiment of the present invention；
Fig. 4 is the HPLC chromatogram of need testing solution in specificity experiment of the present invention.
Embodiment
In following examples of the present invention and experimental example, pine camphor refers to D- pine camphors.
Embodiment 1
The preparation method of ginkgo biloba p.e comprises the following steps in the present embodiment：
Alcohol extracting：Ginkgo leaf is taken, in terms of ginkgo leaf weight, the volumetric concentration for adding 10 times of amounts every time is 85% ethanol water
Solution extracts 3.0h, and heating and refluxing extraction 3 times merges extract solution, filtering, and filtrate is concentrated into 0.3 times of ginkgo leaf weight, low temperature
72h is stood, upper strata oil slick is removed, takes subnatant, obtain solution A；
Water sinks：In terms of ginkgo leaf weight, the water of 3.0 times of amounts is added into solution A, stand at low temperature 48h is filtered, and filtrate is dense
0.3 times of ginkgo leaf weight is reduced to, puts to room temperature, obtains solution B；
Alcohol precipitation：The ethanol water that volumetric concentration is 90% is added into solution B makes alcohol content to 80%, after alcohol precipitation
Solution carries out stand at low temperature, filtering and filtrate and concentrates and repeat 3 times successively, obtains solution C；
Ion exchange resin adsorbs：The water of 3 times of amounts of ginkgo leaf weight is added to solution C, first with the 2.0 of ginkgo leaf weight
732 type cationic exchange resin adsorptions of amount are handled 3 times again, then 711 type anion exchanges of 0.6 times of amount with ginkgo leaf weight
Resin adsorption is handled 3 times, is collected eluent, concentration, is obtained ginkgo biloba p.e.
Embodiment 2
The preparation method of ginkgo biloba p.e comprises the following steps in the present embodiment：
Alcohol extracting：Ginkgo leaf is taken, in terms of ginkgo leaf weight, the volumetric concentration for adding 5 times of amounts every time is water-soluble for 95% ethanol
Liquid extracts 0.5h, heating and refluxing extraction 5 times, merges extract solution, filtering, and filtrate is concentrated into 0.1 times of ginkgo leaf weight, and low temperature is quiet
120h is put, upper strata oil slick is removed, takes subnatant, obtain solution A；
Water sinks：In terms of ginkgo leaf weight, the water of 0.5 times of amount is added into solution A, stand at low temperature 50h is filtered, and filtrate is dense
0.1 times of ginkgo leaf weight is reduced to, puts to room temperature, obtains solution B；
Alcohol precipitation：The ethanol water that volumetric concentration is 95% is added into solution B makes alcohol content to 90%, after alcohol precipitation
Solution carries out stand at low temperature, filtering and filtrate and concentrates and repeat 5 times successively, obtains solution C；
Ion exchange resin adsorbs：The water of 0.5 times of amount of ginkgo leaf weight is added to solution C, first with ginkgo leaf weight
732 type cationic exchange resin adsorptions of 3.5 times of amounts are handled 1 time, then 711 type anion of 1.0 times of amounts with ginkgo leaf weight
Exchanger resin adsorption treatment 1 time, eluent is collected, concentration, obtains ginkgo biloba p.e.
Embodiment 3
The preparation method of ginkgo biloba p.e comprises the following steps in the present embodiment：
Alcohol extracting：Ginkgo leaf is taken, in terms of ginkgo leaf weight, the volumetric concentration for adding 15 times of amounts every time is 70% ethanol water
Solution extracts 6.0h, and heating and refluxing extraction 1 time merges extract solution, filtering, and filtrate is concentrated into 0.5 times of ginkgo leaf weight, low temperature
12h is stood, upper strata oil slick is removed, takes subnatant, obtain solution A；
Water sinks：In terms of ginkgo leaf weight, the water of 5.0 times of amounts is added into solution A, stand at low temperature 46h is filtered, and filtrate is dense
0.5 times of ginkgo leaf weight is reduced to, puts to room temperature, obtains solution B；
Alcohol precipitation：The ethanol water that volumetric concentration is 85% is added into solution B makes alcohol content to 90%, after alcohol precipitation
Solution carries out stand at low temperature, filtering and filtrate and concentrates and repeat 1 time successively, obtains solution C；
Ion exchange resin adsorbs：The water of 5 times of amounts of ginkgo leaf weight is added to solution C, first with the 0.5 of ginkgo leaf weight
732 type cationic exchange resin adsorptions of amount are handled 5 times again, then 711 type anion exchanges of 0.2 times of amount with ginkgo leaf weight
Resin adsorption is handled 5 times, is collected eluent, concentration, is obtained ginkgo biloba p.e.
Embodiment 4
The preparation method of ginkgo biloba p.e comprises the following steps in the present embodiment：
Alcohol extracting：Ginkgo leaf is taken, in terms of ginkgo leaf weight, the volumetric concentration for adding 8 times of amounts every time is water-soluble for 85% ethanol
Liquid extracts 3.0h, heating and refluxing extraction 3 times, merges extract solution, filtering, and filtrate is concentrated into 0.3 times of ginkgo leaf weight, and low temperature is quiet
60h is put, upper strata oil slick is removed, takes subnatant, obtain solution A；
Water sinks：In terms of ginkgo leaf weight, the water of 3.0 times of amounts is added into solution A, stand at low temperature 48h is filtered, and filtrate is dense
0.3 times of ginkgo leaf weight is reduced to, puts to room temperature, obtains solution B；
Alcohol precipitation：The ethanol water that volumetric concentration is 90% is added into solution B makes alcohol content to 80%, after alcohol precipitation
Solution carries out stand at low temperature, filtering and filtrate and concentrates and repeat 3 times successively, obtains solution C；
Ion exchange resin adsorbs：The water of 3.0 times of amounts of ginkgo leaf weight is added to solution C, first with ginkgo leaf weight
732 type cationic exchange resin adsorptions of 2.5 times of amounts are handled 4 times, then 711 type anion of 0.5 times of amount with ginkgo leaf weight
Exchanger resin adsorption treatment 4 times, eluent is collected, concentration, obtains ginkgo biloba p.e.
Embodiment 5
The method that the present embodiment determines the content of pine camphor and sequoyitol in ginkgo biloba p.e simultaneously, comprises the following steps：
(1) preparation of need testing solution
Ginkgo biloba p.e is taken, adds suitable quantity of water, the ginkgo biloba p.e solution that concentration is 5mg/mL is made, shakes up, filters
Cross, as need testing solution；
(2) preparation of reference substance solution
Take pine camphor appropriate, add suitable quantity of water, the solution for the pine camphor that concentration is 1.5mg/mL is made, shakes up, filters, as pine
Alcohol reference substance solution；
Take sequoyitol appropriate, add suitable quantity of water, the solution for the sequoyitol that concentration is 0.5mg/mL is made, shakes up, filters, makees
For sequoyitol reference substance solution；
(3) chromatographic condition
Using C18 as chromatographic column, using aminopropyl bonded silica gel as filler, with COSMOSIL5NH2- MS (250mm*
It is 4.6mm) chromatographic column, with acetonitrile：Water volume ratio is 90.5：9.5 mixed solution is mobile phase, and column temperature is 30 DEG C, and flow velocity is
1.5mL/min, detected with EISD, drift tube temperature is 105 DEG C, yield value 1, and nebulizer gas pressure is
3.0Bar；
(4) determine
μ L of need testing solution 8, the μ L of pine camphor reference substance solution 3 and the μ L of sequoyitol reference substance solution 10 are drawn respectively, and injection is high
Effect liquid phase chromatogram instrument, measure.
Embodiment 6
The method that the present embodiment determines the content of pine camphor and sequoyitol in ginkgo biloba p.e simultaneously, comprises the following steps：
(1) preparation of need testing solution
Ginkgo biloba p.e is taken, adds suitable quantity of water, the ginkgo biloba p.e solution that concentration is 10mg/mL is made, shakes up, filters
Cross, as need testing solution；
(2) preparation of reference substance solution
Take pine camphor appropriate, add suitable quantity of water, the solution for the pine camphor that concentration is 0.5mg/mL is made, shakes up, filters, as pine
Alcohol reference substance solution；
Take sequoyitol appropriate, add suitable quantity of water, the solution for the sequoyitol that concentration is 2.0mg/mL is made, shakes up, filters, makees
For sequoyitol reference substance solution；
(3) chromatographic condition
Using C18 as chromatographic column, using aminopropyl bonded silica gel as filler, with COSMOSIL5NH2- MS (250mm*
It is 4.6mm) chromatographic column, with acetonitrile：Water volume ratio is 85：15 mixed solution is mobile phase, and column temperature is 40 DEG C, and Detection wavelength is
205nm, flow velocity 2.5mL/min, is detected with EISD, and drift tube temperature is 100 DEG C, yield value 1.5, is carried
Atmospheric pressure is 2.0Bar；
(4) determine
μ L of need testing solution 5, the μ L of pine camphor reference substance solution 10 and the μ L of sequoyitol reference substance solution 1 are drawn respectively, and injection is high
Effect liquid phase chromatogram instrument, measure.
Embodiment 7
The method that the present embodiment determines the content of pine camphor and sequoyitol in ginkgo biloba p.e simultaneously, comprises the following steps：
(1) preparation of need testing solution
Ginkgo biloba p.e is taken, adds suitable quantity of water, the ginkgo biloba p.e solution that concentration is 2mg/mL is made, shakes up, filters
Cross, as need testing solution；
(2) preparation of reference substance solution
Take pine camphor appropriate, add suitable quantity of water, the solution for the pine camphor that concentration is 2.0mg/mL is made, shakes up, filters, as pine
Alcohol reference substance solution；
Take sequoyitol appropriate, add suitable quantity of water, the solution for the sequoyitol that concentration is 0.5mg/mL is made, shakes up, filters, makees
For sequoyitol reference substance solution；
(3) chromatographic condition
Using C18 as chromatographic column, using aminopropyl bonded silica gel as filler, with COSMOSIL5NH2- MS (250mm*
It is 4.6mm) chromatographic column, with acetonitrile：Water volume ratio is 95：5 mixed solution is mobile phase, and column temperature is 20 DEG C, and Detection wavelength is
215nm, flow velocity 0.5mL/min, is detected with EISD, and drift tube temperature is 110 DEG C, yield value 0.5, is carried
Atmospheric pressure is 4.0Bar；
(4) determine
μ L of need testing solution 10, the μ L of pine camphor reference substance solution 1 and the μ L of sequoyitol reference substance solution 10, injection are drawn respectively
High performance liquid chromatograph, measure.
Embodiment 8
The method that the present embodiment determines the content of pine camphor and sequoyitol in ginkgo biloba p.e simultaneously, comprises the following steps：
(1) preparation of need testing solution
Ginkgo biloba p.e is taken, adds suitable quantity of water, the ginkgo biloba p.e solution that concentration is 1mg/mL is made, shakes up, filters
Cross, as need testing solution；
(2) preparation of reference substance solution
Take pine camphor appropriate, add suitable quantity of water, the solution for the pine camphor that concentration is 1.0mg/mL is made, shakes up, filters, as pine
Alcohol reference substance solution；
Take sequoyitol appropriate, add suitable quantity of water, the solution for the sequoyitol that concentration is 1.0mg/mL is made, shakes up, filters, makees
For sequoyitol reference substance solution；
(3) chromatographic condition
Using C18 as chromatographic column, using aminopropyl bonded silica gel as filler, with COSMOSIL5NH2- MS (250mm*
4.6mm) it is chromatographic column, with acetonitrile：Water volume ratio is 90：10 mixed solution is mobile phase, and column temperature is 35 DEG C, and flow velocity is
1.5mL/min, detected with EISD, drift tube temperature is 105 DEG C, yield value 1.0, and nebulizer gas pressure is
3.5Bar；
(4) determine
μ L of need testing solution 5, the μ L of pine camphor reference substance solution 5 and the μ L of sequoyitol reference substance solution 5 are drawn respectively, and injection is high
Effect liquid phase chromatogram instrument, measure.
Experimental example 1Method validation
1st, experiment purpose
Method validation is carried out to the method for embodiment 5.
2nd, experimental method
2.1 specificities are tested
Take pine camphor reference substance, sequoyitol reference substance appropriate respectively, it is accurately weighed, add water to be respectively prepared in every 1mL and contain pine camphor
The solution of the 0.5mg containing sequoyitol, shakes up, filters, as positioning reference substance solution in 1.5mg solution, every 1mL.
Accurate respectively to draw μ L of reference substance solution 5, position and use the μ L of reference substance solution 5, the μ L of need testing solution 5, the μ L of water 5 (are done
For blank) injection liquid chromatograph, measure.
Specific experiment result is as Figure 1-4.From Fig. 1-4, each chromatographic peak in test sample is kept completely separate, system
It is noiseless, show that this law specificity is stronger.
2.2 ranges of linearity investigate experiment
It is accurate respectively to draw μ L of pine camphor reference substance solution 2,3 μ L, 5 μ L, 7 μ L, 10 μ L, 15 μ L sample introductions measure.With pine camphor pair
Logarithm according to the sample introduction quality of product is abscissa, using peak area logarithm as ordinate, draws pine camphor standard curve.Pine camphor standard is bent
Line equation is lgY=1.663lgX+2.475 (R2=0.998), pine camphor sample introduction quality is linear good in 3.02-22.65 μ g ranges
It is good.
It is accurate respectively to draw μ L of sequoyitol reference substance solution 2,3 μ L, 5 μ L, 7 μ L, 10 μ L, 15 μ L sample introductions measure.With Chinese larch
Alcohol reference substance sample introduction quality logarithm is abscissa, using peak area logarithm as ordinate, draws sequoyitol standard curve.Sequoyitol mark
Directrix curve equation is lgY=1.493lgX+2.805 (R2=0.999), sequoyitol sample introduction quality is in 0.788-5.91 μ g ranges
It is interior linear good.
2.3 Precision Experiment
The ginkgo biloba p.e prepared using embodiment 1 distinguishes sample introduction 6 times as test sample, according to the method for embodiment 5, respectively
Pine camphor, the peak area of sequoyitol are recorded, calculates its RSD value respectively, specific experiment result is as shown in table 1-2.
The pine camphor Precision Experiment result of table 1
The sequoyitol Precision Experiment result of table 2
From table 1-2, in Precision Experiment, the RSD of pine camphor is 2.37%, and the RSD of sequoyitol is 1.99%, is shown
The precision of this method is good.
2.4 repeated experiment
The ginkgo biloba p.e prepared using embodiment 1 is determined according to the method for embodiment 5 as test sample, calculates pine camphor, red
China fir alcohol content, and RSD values are calculated, specific experiment result is as shown in table 3-4.
The pine camphor repeated experiment result of table 3
The sequoyitol repeated experiment result of table 4
From table 3-4, in repeated experiment, the RSD of pine camphor is 2.27%, and the RSD of sequoyitol is 1.14%, is shown
This method repeatability is good.
2.5 rate of recovery are tested
Pine camphor reference substance 4mg, China fir alcohol reference substance 1.5mg is taken to put in 5mL measuring bottles respectively, it is accurately weighed, known to precision addition
(content of pine camphor is 1.434mg/mL to the parenteral solution of ginkgo biloba p.e prepared by the embodiment 1 of content, and the content of sequoyitol is
0.562g/mL) 2.5mL, ultrasound, makes dissolving, adds water to scale, shake up, produce, parallel to prepare 6 parts.According to the side of embodiment 5
Method carries out assay, calculates the rate of recovery.Specific experiment result is as illustrated in tables 5-6.
The pine camphor rate of recovery experimental result of table 5
The sequoyitol rate of recovery experimental result of table 6
From table 5-6, in rate of recovery experiment, the RSD of pine camphor is 4.01%, and the RSD of sequoyitol is 4.74%, is shown
This method rate of recovery is good.
3rd, experiment conclusion
Content is investigated by the above method, it is tested as shown by data method provided by the invention and meets testing goal and want
Ask, be enough in the analysis detection of ginkgo biloba p.e and medicine sample during the manufacturing containing pine camphor and sequoyitol.
Obviously, above-described embodiment is only intended to clearly illustrate example, and is not the restriction to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of change or
Change.There is no necessity and possibility to exhaust all the enbodiments.And the obvious change thus extended out or
Among changing still in the protection domain of the invention.
Claims (10)
1. method that is a kind of while determining the content of pine camphor and sequoyitol in ginkgo biloba p.e, it is characterised in that including following
Step：
(1) preparation of need testing solution
Ginkgo biloba p.e solution is prepared, as need testing solution；
(2) preparation of reference substance solution
The solution containing pine camphor is prepared, as pine camphor reference substance solution；
The solution containing sequoyitol is prepared, as sequoyitol reference substance solution；
(3) chromatographic condition
Chromatographic column using aminopropyl bonded silica gel as filler is chromatographic column, using the mixed solution of acetonitrile and water as mobile phase, with
EISD detects；
(4) determine
Need testing solution and reference substance solution are drawn, injects high performance liquid chromatograph, measure.
2. method that is according to claim 1 while determining the content of pine camphor and sequoyitol in ginkgo biloba p.e, it is special
Sign is, using COSMOSIL5NH2-MS as chromatographic column, with acetonitrile:The volume ratio of water is 85~95:5~15 mixed solution is
Mobile phase.
3. method that is according to claim 1 or 2 while determining the content of pine camphor and sequoyitol in ginkgo biloba p.e, its
It is characterised by, in the chromatographic condition, flow velocity is 0.5~2.5mL/min, and column temperature is 20~40 DEG C, drift tube temperature is 100~
110 DEG C, yield value is 0.5~1.5, and nebulizer gas pressure is 2.0~4.0Bar.
4. the side of the content of pine camphor and sequoyitol in ginkgo biloba p.e is determined while described according to any one of claim 1-3
Method, it is characterised in that comprise the following steps：
(1) preparation of need testing solution
Ginkgo biloba p.e is taken, adds suitable quantity of water, the ginkgo biloba p.e solution that concentration is 1-10mg/mL is made, shakes up, filters
Cross, as need testing solution；
(2) preparation of reference substance solution
Take pine camphor appropriate, add suitable quantity of water, the solution for the pine camphor that concentration is 0.5-2.0mg/mL is made, shakes up, filters, as pine
Alcohol reference substance solution；
Take sequoyitol appropriate, add suitable quantity of water, the solution for the sequoyitol that concentration is 0.5-2.0mg/mL is made, shakes up, filters, makees
For sequoyitol reference substance solution；
(3) chromatographic condition
Chromatographic column using aminopropyl bonded silica gel as filler is chromatographic column, with acetonitrile：Water volume ratio is 85~95：5~15
Mixed solution is mobile phase, and column temperature is 20~40 DEG C, and Detection wavelength is 205~215nm, and flow velocity is 0.5~2.5mL/min, with
EISD detect, drift tube temperature be 100~110 DEG C, yield value be 0.5~1.5, nebulizer gas pressure be 2.0~
4.0Bar；
(4) determine
Need testing solution 5-10 μ L, pine camphor reference substance solution 1-10 μ L and sequoyitol reference substance solution 1-10 μ L, note are drawn respectively
Enter high performance liquid chromatograph, determine.
5. the side of the content of pine camphor and sequoyitol in ginkgo biloba p.e is determined while described according to any one of claim 1-4
Method, it is characterised in that in the chromatographic condition, drift tube temperature is 105 DEG C, yield value 1, nebulizer gas pressure 3.0Bar.
6. the side of the content of pine camphor and sequoyitol in ginkgo biloba p.e is determined while described according to any one of claim 1-5
Method, it is characterised in that with acetonitrile：Water volume ratio is 90.5：9.5 mixed solution is mobile phase.
7. the side of the content of pine camphor and sequoyitol in ginkgo biloba p.e is determined while described according to any one of claim 1-6
Method, it is characterised in that column temperature is 30 DEG C, flow velocity 1.5mL/min.
8. the side of the content of pine camphor and sequoyitol in ginkgo biloba p.e is determined while described according to any one of claim 1-7
Method, it is characterised in that the preparation method of the ginkgo biloba p.e comprises the following steps：
Alcohol extracting：Ginkgo leaf is taken, in terms of ginkgo leaf weight, adds the ethanol water that the 5-15 times of volumetric concentration measured is 70-95% every time
Solution extracts 0.5-6.0h, and heating and refluxing extraction 1-5 times merges extract solution, filtering, and filtrate is concentrated into the 0.1 of ginkgo leaf weight
~0.5 times, stand at low temperature 12-120h, upper strata oil slick is removed, subnatant is taken, obtains solution A；
Water sinks：In terms of ginkgo leaf weight, the 0.5-5.0 times of water measured, stand at low temperature 46-50h, filtering, filtrate are added into solution A
0.1-0.5 times of ginkgo leaf weight is concentrated into, puts to room temperature, obtains solution B；
Alcohol precipitation：The ethanol water that volumetric concentration is 85-95% is added into solution B makes alcohol content to 70-90%, after alcohol precipitation
Solution carry out stand at low temperature, filtering and filtrate successively and concentrate and repeat 1-5 times, obtain solution C；
Ion exchange resin adsorbs：The 0.5-5 times of water measured of ginkgo leaf weight is added to solution C, first uses cationic ion-exchange resin
Adsorption treatment 1-5 times, then with anion exchange resin adsorption treatment 1-5 times, collect eluent, concentration, obtain ginkgo biloba p.e.
9. method that is according to claim 8 while determining the content of pine camphor and sequoyitol in ginkgo biloba p.e, it is special
Sign is,
In the cationic exchange resin adsorption processing step, the cationic ion-exchange resin is selected from 732 type cation exchange trees
Fat, the dosage of the resin cation are measured for 0.5-3.5 times of ginkgo leaf weight；
In the anion exchange resin adsorption treatment step, the anion exchange resin is selected from 711 types, the anion tree
The dosage of fat is 0.2-1.0 times of ginkgo leaf weight and measured.
10. the method for the content of pine camphor and sequoyitol in ginkgo biloba p.e is determined while according to claim 8 or claim 9,
Characterized in that, the preparation method of the ginkgo biloba p.e comprises the following steps：
Alcohol extracting：Ginkgo leaf is taken, in terms of ginkgo leaf weight, the volumetric concentration for adding 10 times of amounts every time carries for 85% ethanol water
3.0h is taken, heating and refluxing extraction 3 times merges extract solution, filtering, and filtrate is concentrated into 0.3 times of ginkgo leaf weight, stand at low temperature
72h, upper strata oil slick is removed, subnatant is taken, obtains solution A；
Water sinks：In terms of ginkgo leaf weight, the water of 3.0 times of amounts is added into solution A, stand at low temperature 48h is filtered, and filtrate is concentrated into
0.3 times of ginkgo leaf weight, puts to room temperature, obtains solution B；
Alcohol precipitation：The ethanol water that volumetric concentration is 90% is added into solution B makes alcohol content to 80%, by the solution after alcohol precipitation
Stand at low temperature, filtering and filtrate are carried out successively to concentrate and repeat 3 times, obtain solution C；
Ion exchange resin adsorbs：The water of 3 times of amounts of ginkgo leaf weight is added to solution C, first with 2.0 times of amounts of ginkgo leaf weight
732 type cationic exchange resin adsorptions handle 3 times, then 711 type anion exchange resin with 0.6 times of ginkgo leaf weight amount
Adsorption treatment 3 times, eluent is collected, concentration, obtains ginkgo biloba p.e.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112114063A (en) * | 2020-08-06 | 2020-12-22 | 浙江省农业科学院 | Method for detecting D-pinitol in soybean |
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