CN107703237B - Method for simultaneously determining contents of pinitol and sequoyitol in ginkgo leaf extract - Google Patents
Method for simultaneously determining contents of pinitol and sequoyitol in ginkgo leaf extract Download PDFInfo
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Abstract
The invention particularly relates to a method for simultaneously measuring the contents of pinitol and sequoyitol in a ginkgo leaf extract. The research of the invention finds that under the following conditions: taking a chromatographic column with aminopropyl bonded silica gel as a filler as a chromatographic column, and taking acetonitrile: the volume ratio of water is 85-95: 5-15 of the mixed solution is a mobile phase, the column temperature is 20-40 ℃, the detection wavelength is 205-215 nm, the flow rate is 0.5-2.5 mL/min, and an evaporative light scattering detector is used for detecting, so that the content of sequoyitol and pinitol in the ginkgo leaf extract can be simultaneously measured, and the comprehensive quality control of the ginkgo leaf extract is facilitated.
Description
Technical Field
The invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for simultaneously determining the contents of pinitol and sequoyitol in a ginkgo leaf extract.
Background
Ginkgo biloba (Ginkgo biloba L.) is a plant of Ginkgoaceae and Ginkgo, and Ginkgo biloba is dry leaf of Ginkgo biloba, has mild nature, sweet, bitter, astringent taste, heart-fire and lung channel, and has effects of astringing lung, relieving asthma, promoting blood circulation, removing blood stasis, and relieving pain. The ginkgo leaf extract is prepared by extracting, separating and purifying ginkgo leaves serving as raw materials. Research shows that the ginkgo leaf extract has the function of reducing blood sugar.
Sequoyitol, also known as Sequoyitol, is one of the methyl derivatives of inositol and is widely distributed in various plants, particularly in plants of the family Taxaceae, such as Taxus yunnanensis, Taxus americana, Taxus media, and Taxus sciaefolia. Pinitol is an isomer of sequoyitol. Recent studies show that sequoyitol and pinitol both have the effect of reducing blood sugar. In the prior art, the ginkgo contains sequoyitol and pinitol, such as: sequoyitol is extracted and separated from folium Ginkgo in the university of Nanjing Chinese medicine Master academic paper of different sources Ginkgo Biloba resources chemical research (6 months in 2013); ohmoto et al isolated sequoyitol, pinitol, and the like from ginkgo pollen grains. However, there is no report on a method for measuring the content of sequoyitol and pinitol in a ginkgo biloba leaf extract.
Although reported in the literature: the HPLC-ELSD method is used for measuring the content of D-pinitol in the Lespedeza cuneata, but the separation effect of pinitol from other chemical components of ginkgo leaves is poor due to the difference of the chemical components contained in Lespedeza cuneata and ginkgo leaves, so that the method cannot be used for measuring the content of pinitol in ginkgo leaf extract. Although the HPLC-ELSD method is also reported to determine the content of sequoyitol in sequoyitol bulk drug, the method cannot be used for determining the content of pinitol in ginkgo biloba extract due to the difference of chemical components contained in sequoyitol bulk drug and ginkgo biloba extract.
Therefore, the research on a method for simultaneously measuring the contents of the sequoyitol and the pinitol in the ginkgo biloba extract has important significance for the comprehensive quality control of the ginkgo biloba extract.
Disclosure of Invention
The invention aims to provide a method for simultaneously measuring the contents of pinitol and sequoyitol in a ginkgo leaf extract.
The invention provides a method for simultaneously measuring the contents of pinitol and sequoyitol in a ginkgo leaf extract, which comprises the following steps:
(1) preparation of test solution
Preparing a ginkgo biloba extract solution as a test solution;
(2) preparation of control solutions
Preparing a solution containing pinitol as a pinitol reference solution;
preparing a solution containing sequoyitol as a sequoyitol control solution;
(3) chromatographic conditions
Detecting by an evaporative light scattering detector with a chromatographic column taking aminopropyl bonded silica gel as a filler and a mixed solution of acetonitrile and water as a mobile phase;
(4) measurement of
Sucking the test solution and the reference solution, injecting into high performance liquid chromatograph, and measuring.
Preferably, the method for simultaneously determining the contents of pinitol and sequoyitol in the ginkgo biloba extract uses COSMOSIL5NH2-MS as a chromatographic column and uses a mixed solution of acetonitrile and water in a volume ratio of 85-95: 5-15 as a mobile phase.
Further preferably, in the method for simultaneously measuring the contents of pinitol and sequoyitol in the ginkgo leaf extract, the flow rate is 0.5-2.5 mL/min, the column temperature is 20-40 ℃, the drift tube temperature is 100-110 ℃, the gain value is 0.5-1.5, and the carrier gas pressure is 2.0-4.0 Bar under the chromatographic conditions.
Further preferably, the method for simultaneously determining the content of pinitol and sequoyitol in the ginkgo biloba extract comprises the following steps:
(1) preparation of test solution
Adding appropriate amount of water into folium Ginkgo extract, making into 1-10mg/mL folium Ginkgo extract solution, shaking, and filtering to obtain sample solution;
(2) preparation of control solutions
Taking a proper amount of pinitol, adding a proper amount of water to prepare a pinitol solution with the concentration of 0.5-2.0mg/mL, shaking up, and filtering to obtain a pinitol reference solution;
taking appropriate amount of sequoyitol, adding appropriate amount of water, making into sequoyitol solution with concentration of 0.5-2.0mg/mL, shaking, and filtering to obtain sequoyitol reference solution;
(3) chromatographic conditions
Taking a chromatographic column with aminopropyl bonded silica gel as a filler as a chromatographic column, and taking acetonitrile: the volume ratio of water is 85-95: 5-15 of the mixed solution is a mobile phase, the column temperature is 20-40 ℃, the detection wavelength is 205-215 nm, the flow rate is 0.5-2.5 mL/min, the detection is carried out by an evaporative light scattering detector, the temperature of a drift tube is 100-110 ℃, the gain value is 0.5-1.5, and the pressure of carrier gas is 2.0-4.0 Bar;
(4) measurement of
Respectively sucking 5-10 μ L of test solution, 1-10 μ L of pinitol reference solution and 1-10 μ L of sequoyitol reference solution, injecting into high performance liquid chromatograph, and measuring.
Further preferably, in the method for simultaneously measuring the contents of pinitol and sequoyitol in the ginkgo leaf extract, in the chromatographic conditions, the drift tube temperature is 105 ℃, the gain value is 1, and the carrier gas pressure is 3.0 Bar.
Further preferably, the above method for simultaneously determining the content of pinitol and sequoyitol in ginkgo biloba extract is performed by mixing acetonitrile: the water volume ratio is 90.5: the mixed solution of 9.5 is a mobile phase.
Further preferably, the above method for simultaneously determining the contents of pinitol and sequoyitol in ginkgo biloba extract has a column temperature of 30 ℃ and a flow rate of 1.5 mL/min.
Further preferably, the method for simultaneously determining the content of pinitol and sequoyitol in the ginkgo biloba extract comprises the following steps:
alcohol extraction: taking ginkgo leaves, adding 5-15 times of ethanol water solution with volume concentration of 70-95% into the ginkgo leaves by weight of the ginkgo leaves for extraction for 0.5-6.0h each time, heating and refluxing for 1-5 times, combining extracting solutions, filtering, concentrating the filtrate to be 0.1-0.5 time of the weight of the ginkgo leaves, standing at low temperature for 12-120h, removing upper-layer floating oil, taking lower-layer liquid, and obtaining solution A;
water precipitation: adding 0.5-5.0 times of water into the solution A based on the weight of folium Ginkgo, standing at low temperature for 46-50h, filtering, concentrating the filtrate to 0.1-0.5 times of the weight of folium Ginkgo, and standing at room temperature to obtain solution B;
alcohol precipitation: adding 85-95 vol% ethanol water solution into the solution B to make the ethanol content reach 70-90%, sequentially standing the ethanol-precipitated solution at low temperature, filtering, concentrating the filtrate, and repeating for 1-5 times to obtain solution C;
ion exchange resin adsorption: adding 0.5-5 times of water to the solution C, adsorbing with cation exchange resin for 1-5 times, adsorbing with anion exchange resin for 1-5 times, collecting eluate, and concentrating to obtain folium Ginkgo extract.
Further preferably, the above method for simultaneously determining the contents of pinitol and sequoyitol in a ginkgo biloba leaf extract,
in the step of cation exchange resin adsorption treatment, the cation exchange resin is selected from 732 type cation exchange resin, and the dosage of the cation exchange resin is 0.5-3.5 times of the weight of the ginkgo leaves;
in the step of the adsorption treatment of the anion exchange resin, the anion exchange resin is selected from 711 types, and the dosage of the anion exchange resin is 0.2-1.0 times of the weight of the ginkgo leaves.
Further preferably, the method for simultaneously determining the content of pinitol and sequoyitol in the ginkgo biloba extract comprises the following steps:
alcohol extraction: taking folium Ginkgo, adding 10 times of 85% ethanol water solution by volume concentration for 3.0h each time based on the weight of folium Ginkgo, extracting under heating and refluxing for 3 times, mixing extractive solutions, filtering, concentrating the filtrate to 0.3 times of folium Ginkgo weight, standing at low temperature for 72h, removing upper layer oil slick, and taking lower layer liquid to obtain solution A;
water precipitation: adding 3.0 times of water into the solution A based on the weight of the ginkgo leaves, standing at a low temperature for 48 hours, filtering, concentrating the filtrate to be 0.3 times of the weight of the ginkgo leaves, and standing to room temperature to obtain a solution B;
alcohol precipitation: adding an ethanol water solution with the volume concentration of 90% into the solution B to enable the alcohol content to reach 80%, sequentially carrying out low-temperature standing, filtering and filtrate concentration on the ethanol-precipitated solution, and repeatedly carrying out 3 times to obtain a solution C;
ion exchange resin adsorption: adding water 3 times the weight of folium Ginkgo into solution C, adsorbing with 732 type cation exchange resin 2.0 times the weight of folium Ginkgo for 3 times, adsorbing with 711 type anion exchange resin 0.6 times the weight of folium Ginkgo for 3 times, collecting eluate, and concentrating to obtain folium Ginkgo extract. Compared with the prior art, the technical scheme of the invention has the following advantages:
(1) the research of the invention finds that under the following conditions: the method is characterized in that a chromatographic column taking aminopropyl bonded silica gel as a filler is taken as a chromatographic column, a mixed solution of acetonitrile and water is taken as a mobile phase, the column temperature is 20-40 ℃, the flow rate is 0.5-2.5 mL/min, and an evaporative light scattering detector is used for detecting, so that the content of sequoyitol and pinitol in the ginkgo leaf extract can be simultaneously measured, and the comprehensive quality control of the ginkgo leaf extract is facilitated.
(2) Further research of the invention discovers that the method takes COSMOSIL5NH2-MS as a chromatographic column, and acetonitrile and water are in a volume ratio of 85-95: the 5-15 mixed solution is a mobile phase, so that not only can sequoyitol and pinitol be well separated from other chemical components in the ginkgo leaf extract, but also sequoyitol and pinitol can be well separated, and therefore the content determination results of sequoyitol and pinitol are more accurate.
(3) Further research of the invention finds that under the chromatographic conditions, the temperature of the drift tube is 100-110 ℃, the gain value is 0.5-1.5, and the pressure of the carrier gas is 2.0-4.0 Bar, so that the method has the advantages of strong specificity, good precision, good repeatability and good recovery rate, namely: all the methodological tests conform to the regulations.
Drawings
In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the present disclosure taken in conjunction with the accompanying drawings, in which:
FIG. 1 is an HPLC chromatogram of water in a proprietary experiment of the invention;
FIG. 2 is an HPLC chromatogram of pinitol in a specific experiment of the invention;
FIG. 3 is an HPLC chromatogram of sequoyitol in a proprietary experiment of the invention;
FIG. 4 is an HPLC chromatogram of a test solution in a proprietary experiment of the invention.
Detailed Description
In the following examples and experimental examples of the present invention, pinitol means D-pinitol.
Example 1
The preparation method of ginkgo biloba extract in this embodiment comprises the following steps:
alcohol extraction: taking folium Ginkgo, adding 10 times of 85% ethanol water solution by volume concentration for 3.0h each time based on the weight of folium Ginkgo, extracting under heating and refluxing for 3 times, mixing extractive solutions, filtering, concentrating the filtrate to 0.3 times of folium Ginkgo weight, standing at low temperature for 72h, removing upper layer oil slick, and taking lower layer liquid to obtain solution A;
water precipitation: adding 3.0 times of water into the solution A based on the weight of the ginkgo leaves, standing at a low temperature for 48 hours, filtering, concentrating the filtrate to be 0.3 times of the weight of the ginkgo leaves, and standing to room temperature to obtain a solution B;
alcohol precipitation: adding an ethanol water solution with the volume concentration of 90% into the solution B to enable the alcohol content to reach 80%, sequentially carrying out low-temperature standing, filtering and filtrate concentration on the ethanol-precipitated solution, and repeatedly carrying out 3 times to obtain a solution C;
ion exchange resin adsorption: adding water 3 times the weight of folium Ginkgo into solution C, adsorbing with 732 type cation exchange resin 2.0 times the weight of folium Ginkgo for 3 times, adsorbing with 711 type anion exchange resin 0.6 times the weight of folium Ginkgo for 3 times, collecting eluate, and concentrating to obtain folium Ginkgo extract.
Example 2
The preparation method of ginkgo biloba extract in this embodiment comprises the following steps:
alcohol extraction: taking ginkgo leaves, adding 5 times of ethanol water solution with volume concentration of 95% into the ginkgo leaves by weight for extraction for 0.5h each time, heating and refluxing the mixture for extraction for 5 times, combining the extracting solutions, filtering the extracting solution, concentrating the filtrate to be 0.1 time of the weight of the ginkgo leaves, standing the mixture at a low temperature for 120h, removing upper-layer floating oil, and taking lower-layer liquid to obtain solution A;
water precipitation: adding 0.5 times of water into the solution A based on the weight of the ginkgo leaves, standing at a low temperature for 50 hours, filtering, concentrating the filtrate to be 0.1 times of the weight of the ginkgo leaves, and standing to room temperature to obtain a solution B;
alcohol precipitation: adding an ethanol water solution with the volume concentration of 95% into the solution B to enable the alcohol content to reach 90%, and sequentially carrying out low-temperature standing, filtering and filtrate concentration on the ethanol-precipitated solution for 5 times to obtain a solution C;
ion exchange resin adsorption: adding 0.5 times of water of folium Ginkgo weight into solution C, adsorbing with 732 type cation exchange resin 3.5 times of folium Ginkgo weight for 1 time, adsorbing with 711 type anion exchange resin 1.0 times of folium Ginkgo weight for 1 time, collecting eluate, and concentrating to obtain folium Ginkgo extract.
Example 3
The preparation method of ginkgo biloba extract in this embodiment comprises the following steps:
alcohol extraction: taking folium Ginkgo, adding 15 times of 70% ethanol water solution each time based on the weight of folium Ginkgo, extracting for 6.0h, heating and reflux extracting for 1 time, mixing extractive solutions, filtering, concentrating the filtrate to 0.5 times of folium Ginkgo weight, standing at low temperature for 12h, removing upper layer oil slick, and taking lower layer liquid to obtain solution A;
water precipitation: adding 5.0 times of water into the solution A based on the weight of folium Ginkgo, standing at low temperature for 46h, filtering, concentrating the filtrate to 0.5 times of the weight of folium Ginkgo, and standing at room temperature to obtain solution B;
alcohol precipitation: adding an ethanol water solution with the volume concentration of 85% into the solution B to enable the alcohol content to reach 90%, sequentially carrying out low-temperature standing, filtering and filtrate concentration on the solution subjected to alcohol precipitation, and repeatedly carrying out the steps for 1 time to obtain a solution C;
ion exchange resin adsorption: adding 5 times of water of folium Ginkgo weight into solution C, adsorbing with 732 type cation exchange resin 0.5 times of folium Ginkgo weight for 5 times, adsorbing with 711 type anion exchange resin 0.2 times of folium Ginkgo weight for 5 times, collecting eluate, and concentrating to obtain folium Ginkgo extract.
Example 4
The preparation method of ginkgo biloba extract in this embodiment comprises the following steps:
alcohol extraction: taking folium Ginkgo, adding 8 times of 85% ethanol water solution by volume concentration for 3.0h each time based on folium Ginkgo weight, heating and reflux extracting for 3 times, mixing extractive solutions, filtering, concentrating the filtrate to 0.3 times of folium Ginkgo weight, standing at low temperature for 60h, removing upper layer oil slick, and taking lower layer liquid to obtain solution A;
water precipitation: adding 3.0 times of water into the solution A based on the weight of the ginkgo leaves, standing at a low temperature for 48 hours, filtering, concentrating the filtrate to be 0.3 times of the weight of the ginkgo leaves, and standing to room temperature to obtain a solution B;
alcohol precipitation: adding an ethanol water solution with the volume concentration of 90% into the solution B to enable the alcohol content to reach 80%, sequentially carrying out low-temperature standing, filtering and filtrate concentration on the ethanol-precipitated solution, and repeatedly carrying out 3 times to obtain a solution C;
ion exchange resin adsorption: adding water 3.0 times the weight of folium Ginkgo into solution C, adsorbing with 732 type cation exchange resin 2.5 times the weight of folium Ginkgo for 4 times, adsorbing with 711 type anion exchange resin 0.5 times the weight of folium Ginkgo for 4 times, collecting eluate, and concentrating to obtain folium Ginkgo extract.
Example 5
The method for simultaneously measuring the contents of pinitol and sequoyitol in the ginkgo leaf extract comprises the following steps of:
(1) preparation of test solution
Adding appropriate amount of water into folium Ginkgo extract, making into folium Ginkgo extract solution with concentration of 5mg/mL, shaking, and filtering to obtain test solution;
(2) preparation of control solutions
Taking a proper amount of pinitol, adding a proper amount of water to prepare a pinitol solution with the concentration of 1.5mg/mL, shaking up, and filtering to obtain a pinitol reference solution;
taking a proper amount of sequoyitol, adding a proper amount of water to prepare 0.5mg/mL sequoyitol solution, shaking up, and filtering to obtain sequoyitol reference solution;
(3) chromatographic conditions
Using C18 as a chromatographic column, aminopropyl bonded silica gel as a filler, COSMOSIL5NH2-MS (250 mm. times.4.6 mm) as a chromatographic column, and acetonitrile: the water volume ratio is 90.5: the mixed solution of 9.5 is a mobile phase, the column temperature is 30 ℃, the flow rate is 1.5mL/min, the detection is carried out by an evaporative light scattering detector, the drift tube temperature is 105 ℃, the gain value is 1, and the carrier gas pressure is 3.0 Bar;
(4) measurement of
Respectively sucking 8 μ L of test solution, 3 μ L of pinitol reference solution and 10 μ L of sequoyitol reference solution, injecting into high performance liquid chromatograph, and measuring.
Example 6
The method for simultaneously measuring the contents of pinitol and sequoyitol in the ginkgo leaf extract comprises the following steps of:
(1) preparation of test solution
Adding appropriate amount of water into folium Ginkgo extract, making into folium Ginkgo extract solution with concentration of 10mg/mL, shaking, and filtering to obtain test solution;
(2) preparation of control solutions
Taking a proper amount of pinitol, adding a proper amount of water to prepare a pinitol solution with the concentration of 0.5mg/mL, shaking up, and filtering to obtain a pinitol reference solution;
taking a proper amount of sequoyitol, adding a proper amount of water to prepare sequoyitol solution with the concentration of 2.0mg/mL, shaking up, and filtering to obtain sequoyitol reference solution;
(3) chromatographic conditions
Using C18 as a chromatographic column, aminopropyl bonded silica gel as a filler, COSMOSIL5NH2-MS (250 mm. times.4.6 mm) as a chromatographic column, and acetonitrile: the water volume ratio is 85: 15 is a mobile phase, the column temperature is 40 ℃, the detection wavelength is 205nm, the flow rate is 2.5mL/min, the detection is carried out by an evaporative light scattering detector, the drift tube temperature is 100 ℃, the gain value is 1.5, and the carrier gas pressure is 2.0 Bar;
(4) measurement of
Respectively sucking 5 μ L of test solution, 10 μ L of pinitol reference solution and 1 μ L of sequoyitol reference solution, injecting into high performance liquid chromatograph, and measuring.
Example 7
The method for simultaneously measuring the contents of pinitol and sequoyitol in the ginkgo leaf extract comprises the following steps of:
(1) preparation of test solution
Adding appropriate amount of water into folium Ginkgo extract, making into folium Ginkgo extract solution with concentration of 2mg/mL, shaking, and filtering to obtain sample solution;
(2) preparation of control solutions
Taking a proper amount of pinitol, adding a proper amount of water to prepare a pinitol solution with the concentration of 2.0mg/mL, shaking up, and filtering to obtain a pinitol reference solution;
taking a proper amount of sequoyitol, adding a proper amount of water to prepare 0.5mg/mL sequoyitol solution, shaking up, and filtering to obtain sequoyitol reference solution;
(3) chromatographic conditions
Using C18 as a chromatographic column, aminopropyl bonded silica gel as a filler, COSMOSIL5NH2-MS (250 mm. times.4.6 mm) as a chromatographic column, and acetonitrile: the water volume ratio is 95:5 is a mobile phase, the column temperature is 20 ℃, the detection wavelength is 215nm, the flow rate is 0.5mL/min, the detection is carried out by an evaporative light scattering detector, the drift tube temperature is 110 ℃, the gain value is 0.5, and the carrier gas pressure is 4.0 Bar;
(4) measurement of
Respectively sucking 10 μ L of test solution, 1 μ L of pinitol reference solution and 10 μ L of sequoyitol reference solution, injecting into high performance liquid chromatograph, and measuring.
Example 8
The method for simultaneously measuring the contents of pinitol and sequoyitol in the ginkgo leaf extract comprises the following steps of:
(1) preparation of test solution
Adding appropriate amount of water into folium Ginkgo extract, making into folium Ginkgo extract solution with concentration of 1mg/mL, shaking, and filtering to obtain test solution;
(2) preparation of control solutions
Taking a proper amount of pinitol, adding a proper amount of water to prepare a pinitol solution with the concentration of 1.0mg/mL, shaking up, and filtering to obtain a pinitol reference solution;
taking a proper amount of sequoyitol, adding a proper amount of water to prepare a sequoyitol solution with the concentration of 1.0mg/mL, shaking up, and filtering to obtain a sequoyitol reference solution;
(3) chromatographic conditions
Using C18 as a chromatographic column, aminopropyl bonded silica gel as a filler, COSMOSIL5NH2-MS (250 mm. times.4.6 mm) as a chromatographic column, and acetonitrile: the water volume ratio is 90: 10, the mixed solution is a mobile phase, the column temperature is 35 ℃, the flow rate is 1.5mL/min, the detection is carried out by an evaporative light scattering detector, the drift tube temperature is 105 ℃, the gain value is 1.0, and the carrier gas pressure is 3.5 Bar;
(4) measurement of
Respectively sucking 5 μ L of test solution, 5 μ L of pinitol reference solution and 5 μ L of sequoyitol reference solution, injecting into high performance liquid chromatograph, and measuring.
Experimental example 1Methodology validation
1. Purpose of experiment
The methodology of example 5 was validated.
2. Experimental methods
2.1 specificity experiments
Respectively taking appropriate amount of pinitol reference substance and sequoyitol reference substance, precisely weighing, adding water to obtain solution containing pinitol 1.5mg per 1mL and sequoyitol 0.5mg per 1mL, shaking, filtering, and making into reference substance solution for positioning.
Precisely sucking 5 μ L of control solution, 5 μ L of positioning control solution, 5 μ L of test solution, and 5 μ L of water (as blank), injecting into liquid chromatograph, and measuring.
The specific experimental results are shown in FIGS. 1-4. As can be seen from FIGS. 1-4, the peaks of the samples were completely separated, and the system was not interfered, indicating that the specificity of the method is strong.
2.2 Linear Range investigation experiments
Precisely sucking 2 μ L, 3 μ L, 5 μ L, 7 μ L, 10 μ L and 15 μ L of the pinitol reference solution respectively for sample injection determination. And drawing a pinitol standard curve by taking the logarithm of the sample injection quality of the pinitol reference substance as an abscissa and taking the logarithm of the peak area as an ordinate. The standard curve equation of pinitol is lgY-1.663 lgX +2.475 (R)20.998), the injection quality of pinitol is good linearly in the range of 3.02-22.65 μ g.
Precisely sucking 2 μ L, 3 μ L, 5 μ L, 7 μ L, 10 μ L and 15 μ L of sequoyitol reference substance solution respectively, and measuring. And drawing a sequoyitol standard curve by taking the mass logarithm of the sample injection of the sequoyitol reference substance as a horizontal coordinate and taking the logarithm of the peak area as a vertical coordinate. The standard curve equation of sequoyitol is 1.493lgX +2.805 (R)20.999), the sampling quality of sequoyitol is good within the range of 0.788-5.91 mug.
2.3 precision test
The ginkgo biloba leaf extract prepared in example 1 was used as a test sample, and the sample injection was performed 6 times according to the method of example 5, the peak areas of pinitol and sequoyitol were recorded, and the RSD values thereof were calculated, respectively, and the specific experimental results are shown in tables 1-2.
TABLE 1 pinitol precision test results
TABLE 2 result of precision experiment of sequoyitol
As can be seen from tables 1-2, the RSD of pinitol is 2.37% and that of sequoyitol is 1.99% in the precision experiment, indicating that the precision of the method is good.
2.4 repeatability test
The ginkgo biloba leaf extract prepared in example 1 was used as a test sample, the contents of pinitol and sequoyitol were calculated by the method of example 5, and the RSD value was calculated, and the specific experimental results are shown in tables 3 to 4.
TABLE 3 pinitol repeatability test results
TABLE 4 result of repeatability experiment of sequoyitol
As can be seen from tables 3-4, the RSD of pinitol is 2.27% and that of sequoyitol is 1.14% in the repeatability experiments, indicating that the method has good repeatability.
2.5 recovery test
Respectively taking 4mg of pinitol reference substance and 1.5mg of sequoyitol reference substance, placing in a 5mL measuring flask, precisely weighing, precisely adding 2.5mL of the known injection (pinitol content is 1.434mg/mL, sequoyitol content is 0.562g/mL) of folium Ginkgo extract prepared in example 1, ultrasonically dissolving, adding water to scale, and shaking uniformly to obtain 6 parts in parallel. The content measurement was performed according to the method of example 5, and the recovery rate was calculated. The results of the specific experiments are shown in tables 5-6.
TABLE 5 pinitol recovery test results
TABLE 6 result of recovery of sequoyitol
As is clear from tables 5 to 6, in the recovery rate test, the RSD of pinitol was 4.01% and that of sequoyitol was 4.74%, indicating that the recovery rate was good.
3. Conclusion of the experiment
Through the research content of the methodology, the experimental data show that the method provided by the invention meets the detection purpose and requirement, and can be used for analyzing and detecting samples of the ginkgo leaf extract and the medicine containing pinitol and sequoyitol in the production and manufacturing processes.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (7)
1. A method for simultaneously measuring the contents of pinitol and sequoyitol in a ginkgo leaf extract is characterized by comprising the following steps:
(1) preparation of test solution
The preparation method of the ginkgo leaf extract comprises the following steps:
alcohol extraction: taking ginkgo leaves, adding 5-15 times of ethanol water solution with volume concentration of 70-95% into the ginkgo leaves by weight of the ginkgo leaves for extraction for 0.5-6.0h each time, heating and refluxing for 1-5 times, combining extracting solutions, filtering, concentrating the filtrate to be 0.1-0.5 time of the weight of the ginkgo leaves, standing at low temperature for 12-120h, removing upper-layer floating oil, taking lower-layer liquid, and obtaining solution A;
water precipitation: adding 0.5-5.0 times of water into the solution A based on the weight of folium Ginkgo, standing at low temperature for 46-50h, filtering, concentrating the filtrate to 0.1-0.5 times of the weight of folium Ginkgo, and standing at room temperature to obtain solution B;
alcohol precipitation: adding 85-95 vol% ethanol water solution into the solution B to make the ethanol content reach 70-90%, sequentially standing the ethanol-precipitated solution at low temperature, filtering, concentrating the filtrate, and repeating for 1-5 times to obtain solution C;
ion exchange resin adsorption: adding 0.5-5 times of water to the solution C, adsorbing with cation exchange resin for 1-5 times, adsorbing with anion exchange resin for 1-5 times, collecting eluate, and concentrating to obtain folium Ginkgo extract;
adding appropriate amount of water into folium Ginkgo extract, making into 1-10mg/mL folium Ginkgo extract solution, shaking, and filtering to obtain sample solution;
(2) preparation of control solutions
Taking a proper amount of pinitol, adding a proper amount of water to prepare a pinitol solution with the concentration of 0.5-2.0mg/mL, shaking up, and filtering to obtain a pinitol reference solution;
taking appropriate amount of sequoyitol, adding appropriate amount of water, making into sequoyitol solution with concentration of 0.5-2.0mg/mL, shaking, and filtering to obtain sequoyitol reference solution;
(3) chromatographic conditions
Taking a chromatographic column with aminopropyl bonded silica gel as a filler as a chromatographic column, and taking acetonitrile: the volume ratio of water is 85-95: 5-15 of the mixed solution is a mobile phase, the column temperature is 20-40 ℃, the detection wavelength is 205-215 nm, the flow rate is 0.5-2.5 mL/min, the detection is carried out by an evaporative light scattering detector, the temperature of a drift tube is 100-110 ℃, the gain value is 0.5-1.5, and the pressure of carrier gas is 2.0-4.0 Bar;
(4) and (4) measuring, sucking the test solution and the reference solution, injecting into a high performance liquid chromatograph, and measuring.
2. The method of claim 1, wherein the content of pinitol and sequoyitol in the Ginkgo biloba leaf extract is determined simultaneously,
in the step (4), 5-10 μ L of the test solution, 1-10 μ L of the pinitol reference solution and 1-10 μ L of the sequoyitol reference solution are respectively absorbed and injected into a high performance liquid chromatograph for determination.
3. The method of claim 1 or 2, wherein the temperature of the drift tube is 105 ℃, the gain value is 1, and the pressure of the carrier gas is 3.0Bar under the chromatographic conditions.
4. The method for simultaneously determining the contents of pinitol and sequoyitol in a ginkgo biloba leaf extract according to claim 1 or 2, characterized in that the contents of acetonitrile: the water volume ratio is 90.5: the mixed solution of 9.5 is a mobile phase.
5. The method for simultaneously determining the contents of pinitol and sequoyitol in a ginkgo biloba leaf extract according to claim 1 or 2, characterized in that the column temperature is 30 ℃ and the flow rate is 1.5 mL/min.
6. The method of claim 1, wherein the content of pinitol and sequoyitol in the Ginkgo biloba leaf extract is determined simultaneously,
in the step of cation exchange resin adsorption treatment, the cation exchange resin is selected from 732 type cation exchange resin, and the dosage of the cation exchange resin is 0.5-3.5 times of the weight of the ginkgo leaves;
in the step of the adsorption treatment of the anion exchange resin, the anion exchange resin is selected from 711 types, and the dosage of the anion exchange resin is 0.2-1.0 times of the weight of the ginkgo leaves.
7. The method for simultaneously determining the contents of pinitol and sequoyitol in a ginkgo biloba leaf extract according to claim 1 or 6, wherein the preparation method of the ginkgo biloba leaf extract comprises the following steps:
alcohol extraction: taking folium Ginkgo, adding 10 times of 85% ethanol water solution by volume concentration for 3.0h each time based on the weight of folium Ginkgo, extracting under heating and refluxing for 3 times, mixing extractive solutions, filtering, concentrating the filtrate to 0.3 times of folium Ginkgo weight, standing at low temperature for 72h, removing upper layer oil slick, and taking lower layer liquid to obtain solution A;
water precipitation: adding 3.0 times of water into the solution A based on the weight of the ginkgo leaves, standing at a low temperature for 48 hours, filtering, concentrating the filtrate to be 0.3 times of the weight of the ginkgo leaves, and standing to room temperature to obtain a solution B;
alcohol precipitation: adding an ethanol water solution with the volume concentration of 90% into the solution B to enable the alcohol content to reach 80%, sequentially carrying out low-temperature standing, filtering and filtrate concentration on the ethanol-precipitated solution, and repeatedly carrying out 3 times to obtain a solution C;
ion exchange resin adsorption: adding water 3 times the weight of folium Ginkgo into solution C, adsorbing with 732 type cation exchange resin 2.0 times the weight of folium Ginkgo for 3 times, adsorbing with 711 type anion exchange resin 0.6 times the weight of folium Ginkgo for 3 times, collecting eluate, and concentrating to obtain folium Ginkgo extract.
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