CN110824071B - Method for detecting lignans and flavonol glycosides in ginkgo leaf extract or preparation thereof - Google Patents
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Abstract
The invention relates to the technical field of detection of ginkgo leaf extracts, and particularly discloses a method for detecting lignans and flavonol glycosides in a ginkgo leaf extract or a preparation thereof, wherein the detection method is a UPLC-MS method, and the detection conditions of UPLC are as follows: a chromatographic column: octadecylsilane chemically bonded silica gel column; mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.08-0.12 vt% formic acid aqueous solution, the volume ratio of the mobile phase A to the mobile phase B is 10-35: 90-60; column temperature: 35-40 ℃; flow rate: 0.1-0.5 ml/min; and (3) an elution mode: isocratic elution; the detection mode of the MS is a negative ion mode. The invention can realize the simultaneous quantitative detection of two lignans compounds and two flavonol glycosides compounds in the ginkgo leaf extract or the preparation thereof with complex components, and has high precision and accuracy of detection and good reproducibility and stability.
Description
Technical Field
The invention relates to the technical field of detection of ginkgo biloba extract, in particular to a method for detecting lignans and flavonol glycosides in the ginkgo biloba extract or a preparation thereof.
Background
The folium Ginkgo extract is effective substance extracted from folium Ginkgo, contains total ginkgetin and bilobalide, and has effects of dilating blood vessel, protecting vascular endothelial tissue, regulating blood lipid, protecting low density lipoprotein, inhibiting PAF (platelet activating factor), inhibiting thrombosis, and scavenging free radicals. Wherein the component capable of scavenging free radicals is lignan compound; the main component playing roles of expanding blood vessels, protecting vascular endothelial tissues, regulating blood fat and the like is flavonol glycoside compounds, so that the content of lignan compounds and flavonol glycoside compounds in the ginkgo leaf extract or the preparation thereof directly influences the playing of the drug effect.
However, after two lignans compounds, namely lariciresinol-4, 4-diglucoside and pinoresinol diglucoside in the ginkgo leaf extract, are scanned by ultraviolet full wavelength, the two lignans compounds are found to be maximally absorbed at a UV277nm position, and the flavonol glycosides (the flavonol glycosides are Urolignoside and cosmosiin) have stronger absorption at the wavelength, so that the contents of the lignans compounds and the flavonol glycosides cannot be detected by common high performance liquid chromatography.
Disclosure of Invention
The invention provides a method for detecting lignans and flavonol glycosides in ginkgo leaf extract or a preparation thereof, aiming at the problem that the content of the lignans (lariciresinol-4, 4-diglucoside and pinoresinol diglucoside) and the content of the flavonol glycosides (Urolinoside and cosmosiin) in the ginkgo leaf extract or the preparation thereof cannot be simultaneously and quantitatively detected by the existing high performance liquid chromatography detection method.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
a method for detecting lignans and flavonol glycosides compounds in a ginkgo leaf extract or a preparation thereof comprises the steps of preparing larch 4, 4-diglucoside and pinoresinol diglucoside, and preparing a UPLC-MS method, wherein the detection conditions of the UPLC are as follows:
a chromatographic column: octadecylsilane chemically bonded silica gel column;
mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.08-0.12 vt% formic acid aqueous solution, the volume ratio of the mobile phase A to the mobile phase B is 10-35: 90-60;
column temperature: 35-40 ℃;
flow rate: 0.1-0.5 ml/min;
and (3) an elution mode: isocratic elution;
the detection mode of the MS is an ESI negative ion mode.
Wherein the structural formula of Urolignoside is as follows:
compared with the prior art, the method for detecting lignans and flavonol glycosides in the ginkgo leaf extract or the preparation thereof adopts a UPLC-MS combined method, selects the UPLC chromatographic condition and the MS detection negative ion mode, can realize the simultaneous quantitative detection of two lignanoid compounds of lariciresinol-4, 4-diglucoside and pinoresinol diglucoside and two flavonol glycoside compounds of Urolignoside and cosmosiin with the same ultraviolet absorption wavelength in the ginkgo leaf extract or the preparation thereof (such as the Shuxuening injection) with complex components, simplifies the detection process, saves the analysis time, has high precision and accuracy of detection and good reproducibility and stability, can be used for quality control and comprehensive evaluation of the ginkgo leaf extract or the preparation thereof, and provides reliable guarantee for improving and controlling the quality of the ginkgo leaf extract or the preparation thereof.
Preferably, the mobile phase B is 0.1 vt% aqueous formic acid.
Preferably, the volume ratio of the mobile phase A to the mobile phase B is 10: 90.
The preferable mobile phase proportion can better separate impurities, is beneficial to detecting the lariciresinol-4, 4-diglucoside, Urolinoside, pinoresinol diglucoside and cosmosiin, can effectively improve the peak shape, and enables the accuracy and precision of the detection result to be higher.
Preferably, the column temperature is 40 ℃ and the flow rate is 0.1 ml/min.
Preferably, the injection volume in the detection method is 0.2-1.5 muL.
The preferable chromatographic conditions are favorable for further improving the sensitivity of the detection of the lariciresinol-4, 4-diglucoside, Urolignoside, pinoresinol diglucoside and cosmosiin.
Preferably, the dilution solvent of the control solution and the test solution in the detection method is methanol.
Preferably, the mass concentration of the component to be detected in the control solution in the detection method is 13-20 times of that in the test solution.
Preferably, the concentration of the ginkgo biloba extract in the test solution in the detection method is 0.3-0.4 mg/mL.
Drawings
FIG. 1 is an EIC chromatogram for the investigation of mass spectrometry conditions in example 1 of the present invention; wherein, A is a positive ion mode [ M + H ] +, B is a negative ion mode [ M-H ] -, C is a positive ion mode [ M + Na ] +, and D is a negative ion mode [ M + COOH ] -;
fig. 2 is a chromatogram of the UPLC-MS method for determining shuxuening injection in example 1 of the present invention, wherein. A blank methanol, B and C Shuxuening injection and D are mixed with a reference solution; peak 1: lariciresinol-4, 4-diglucoside, peak 2: pinoresinol diglucoside, peak 3: urolignoside, peak 4: cosmosiin.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The following examples are provided to better illustrate the embodiments of the present invention.
Example 1
Materials and methods
The instrument comprises the following steps: waters Xevo GX-XS quadrupole series-connection flight time mass spectrum
Reagent: acetonitrile, methanol (chromatographically pure), purified water, and other reagents are analytically pure; lariciresidin-4, 4-diglucoside, pinoresinol diglucoside, Urolignoside and cosmosiin reference substance
Sample preparation: ginkgo leaf extract and Shuxuening injection
Preparing a solution:
preparation of control solutions:
accurately weighing 12.16mg of lariciresinol-4, 4-diglucoside in a 50ml volumetric flask, 10.90mg of pinoresinol diglucoside in a 50ml volumetric flask, 9.20mg of Urolinoside in a 50ml volumetric flask, and 10.26mg of cosmosiin in a 50ml volumetric flask, dissolving in methanol and fixing the volume to obtain a reference mother solution. Precisely measuring 2ml of lariciresinol-4, 4-diglucoside, 2ml of pinoresinol diglucoside, 5ml of Urolinoside and 1ml of cosmosiin reference mother liquor into the same 25ml volumetric flask, dissolving with methanol and fixing volume to obtain a mixed reference solution.
Preparation of a test solution:
precisely measuring 180mg of folium Ginkgo extract in a 50mL volumetric flask, dissolving with methanol and fixing the volume, precisely measuring 1mL of the above solution to 10mL volumetric flask, and fixing the volume with methanol to obtain folium Ginkgo extract test solution.
Precisely measuring 1ml Shuxuening injection 10ml volumetric flask, and fixing the volume with methanol to obtain the test solution of the Shuxuening injection.
Investigation of mass spectrometry conditions:
the pinoresinol diglucoside is found to be the lowest relative to other lignans by extracting the precise molecular weight of each component to be detected in the Shuxuening injection, therefore, the pinoresinol diglucoside is taken as an investigation object, mass spectrum conditions are considered, and by comparing pinoresinol diglucoside Extraction Ion Current (EIC) chromatograms in a positive ion mode and a negative ion mode, as shown in figure 1, the pinoresinol diglucoside is good in correspondence in the negative ion mode, the addition product [ M + COOH ] peak (M/z is 727.2449) of parent ions is obviously higher than the [ M-H ] -molecular ion peak (M/z is 681.2395), so that the precise molecular weight M/z of the pinoresinol diglucoside in a sample and a reference product can be extracted simultaneously (error range is 0.02Da), and the peak areas of the respective EIC chromatograms are calculated to achieve the quantitative purpose.
The detection method comprises the following steps:
determining the contents of lariciresinol-4, 4-diglucoside, pinoresinoside, Urolignoside and cosmosiin in the Shuxuening injection by a UPLC-MS method, wherein the detection conditions are as follows:
a chromatographic column: octadecylsilane chemically bonded silica gel column, 2.1mm × 100mm, 1.6 μm; mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.1 vt% formic acid aqueous solution, and the volume ratio of the mobile phase A to the mobile phase B is 10: 90; column temperature: 40 ℃; flow rate: 0.1 ml/min; and (3) an elution mode: isocratic elution; the sample injection volume is 1 mu L; the MS detection mode is an ESI negative ion mode, the obtained chromatogram is shown in figure 2, and the separation degrees between the detection peaks of lariciresin-4, 4-diglucoside, pinoresinol diglucoside, Urolignoside and cosmos glycoside and between the detection peaks and other miscellaneous peaks are good.
Example 2
Methodology investigation and results
The linear relationship is: diluting the mixed reference substance solution by 2 times, 5 times, 10 times, 20 times and 40 times respectively, precisely sucking 1 μ L of each of the mixed reference substance solution and the diluted solution, injecting into a liquid chromatograph, extracting the precise molecular weight of each component to be measured, and recording the peak area.
Wherein, the examination result of the linear relation of the lariciresinol-4, 4-diglucoside is shown in the table 1;
TABLE 1 Larix Gmelini extract-4, 4-diglucoside Linear relationship study
According to the table, taking the sample injection amount (ug) as the abscissa (X) and the peak area as the ordinate (Y), drawing a standard curve to obtain a regression equation of lariciresinol-4, 4-diglucoside as follows:
y is 23574.7465x +35594.1644, R2 is 0.9997(n is 6). The results show that the lariciresinol-4, 4-diglucoside in the control product has good linear relationship in the range of 0.486ug/ml-19.456 ug/ml.
The results of the linear relationship examination of pinoresinol diglucoside are shown in table 2;
TABLE 2 Linearity of pinoresinol diglucoside
According to the above table, taking the sample amount (ug) as the abscissa (X) and the peak area as the ordinate (Y), a standard curve is drawn to obtain a regression equation of pinoresinol diglucoside as: 19588.7904x +1355.5158, R20.9996(n 6). It was shown that pinoresinol diglucoside is in a good linear relationship in the range of 0.436ug-17.440 ug/ml.
The results of the linear relationship examination of Urolignoside are shown in Table 3;
TABLE 3 Urolignoside Linear relationship examination
According to the above table, taking the sample injection amount (ug) as the abscissa (X) and the peak area as the ordinate (Y), a standard curve is drawn, and the regression equation of the uroligoside is obtained as: y 36689.2702x +23247.2047,R20.9996(n 6). Shows that Urolignoside has a good linear relationship in the range of 0.920ug-36.800 ug/ml. The results are shown in Table 3.
The results of the linear relationship examination of cosmosiin are shown in table 4;
TABLE 4 cosmosiin Linear relationship examination
According to the above table, taking the sample volume (ug) as the abscissa (X) and the peak area as the ordinate (Y), a standard curve is drawn to obtain a regression equation of cosmosiin as: 72588.2689x +8307.9403, R20.9998(n 6). Shows that the cosmosiin has a good linear relationship in the range of 0.205ug-8.208 ug/ml.
Precision: the mixed control solution diluted 10 times was continuously injected for 6 times, 1ul each time, the precise molecular weight of each component to be measured was extracted and the peak area was determined, the results are shown in table 5.
TABLE 5 precision investigation
The RSD values of the lariciresinol-4, 4-diglucoside, pinoresinol diglucoside, Urolignoside and cosmos glycoside obtained by analysis are all less than 1.1 percent, which shows that the detection method has higher precision.
Repeatability: taking 6 parts of Shuxuening injection with the batch number of 17080111, preparing 6 samples according to the preparation method of the test solution of the Shuxuening injection, injecting 1ul of sample into each sample, extracting the accurate molecular weight of each component to be measured, measuring the peak area, and measuring the results as shown in tables 6-9.
TABLE 6 lariciresin-4, 4-diglucoside repeatability test results
TABLE 7 pinoresinol diglucoside repeatability test results
TABLE 8 Urolignoside repeatability test results
TABLE 9 cosmosiin repeatability test results
The RSD values of the lariciresinol-4, 4-diglucoside, pinoresinol diglucoside, Urolignoside and cosmos glycoside obtained by analysis are all less than 3%, which shows that the detection method has higher reproducibility.
Stability: the Shuxuening injection (batch No. 17080111) was used to prepare samples according to the preparation method of the test solution, the peak areas of the components to be measured after the samples were left for 0, 1.5, 3, 6, 12 and 24 hours were respectively examined, and the examination results are shown in Table 10.
TABLE 10 stability test results
The RSD values of the lariciresinol-4, 4-diglucoside, pinoresinol diglucoside, Urolignoside and cosmos glycoside obtained by analysis are all less than 3%, which shows that the detection method has higher stability.
And (3) sample recovery rate: according to the results of the repeated experiments, the concentrations of the components to be measured in the shuxuening test sample solution with the batch of 17080111 are respectively as follows: larch 4, 4-diglucoside 10.191ug/ml, pinoresinol diglucoside 10.423ug/ml, Urolignoside27.722ug/ml, cosmosiin 4.203 ug/ml;
therefore, 17080111 Shuxuening injection is precisely measured in 2.5ml to 50ml volumetric flasks in parallel of 9 parts. Wherein 2ml of mixed reference substance mother liquor is respectively added into 3 parts of the solution, and methanol is used for constant volume to be used as a medium-concentration test solution; adding 1ml of mixed reference substance mother liquor into 3 parts of the solution respectively, and fixing the volume by using methanol to obtain a low-concentration test solution; 3 parts of the mixed reference substance mother liquor are respectively added with 3ml of methanol to constant volume to be used as a high-concentration test solution. Injecting sample into each of the 9 test solutions 1ul, extracting the accurate molecular weight of each component to be measured, and measuring the peak area of each component to be measured, wherein the measurement results are shown in tables 11-14.
TABLE 11 sample recovery test results for lariciresin-4, 4-diglucoside
TABLE 12 pinoresinol diglucoside sample recovery test results
TABLE 13 Urolignoside sample recovery test results
TABLE 14 cosmosiin sample recovery test results
The RSD values of the lariciresinol-4, 4-diglucoside, pinoresinol diglucoside, Urolignoside and cosmos glycoside obtained by analysis are all less than 2.55 percent, which shows that the detection method has higher sample adding recovery rate.
Example 3
The contents of lariciresinol-4, 4-diglucoside, pinoresinoside, Urolignoside and cosmosiin in ginkgo biloba leaf extract were determined by the UPLC-MS method in example 1.
Taking 10 batches of ginkgo biloba extract, 2 parts each, preparing a solution to be detected according to the preparation method of the ginkgo biloba extract test solution in the embodiment 1, precisely sucking 1ul of the solution to be detected, injecting the solution into a chromatograph, extracting the precise molecular weight of each component to be detected, recording the peak area, calculating the content of each component to be detected in the ginkgo biloba extract, and obtaining the content detection result shown in tables 15-18.
TABLE 15 determination of the content of lariciresin-4, 4-diglucoside in Ginkgo biloba leaf extract
TABLE 16 measurement results of pinoresinol diglucoside content in Ginkgo biloba leaf extract
TABLE 17 measurement results of Urolignoside content in Ginkgo biloba leaf extract
TABLE 18 determination of cosmosiin content in Ginkgo biloba leaf extract
Example 4
The UPLC-MS method in example 1 is used to detect the contents of lariciresin-4, 4-diglucoside, pinoresinol diglucoside, Urolignoside and cosmosiin in Shuxuening injection.
Taking 12 batches of Shuxuening injection (specification 2 mL: A, B, C; specification 5 mL: D, E, F; specification 10 mL: G, H, I; specification 20 mL: J, K, L;), preparing a to-be-detected sample solution according to the to-be-detected sample solution of the Shuxuening injection in example 1, precisely sucking 1ul of the to-be-detected sample solution, injecting the to-be-detected sample solution into a chromatograph, extracting the precise molecular weight of each to-be-detected component, recording the peak area, calculating the content of each to-be-detected component in the sample, and obtaining the content detection result shown in tables 19-22.
TABLE 19 measurement results of the content of lariciresin-4, 4-diglucoside in Shuxuening injection
TABLE 20 measurement of pinoresinol diglucoside content in Shuxuening injection
TABLE 21 Urolignoside content determination results in Shuxuening injection
TABLE 22 determination of cosmosiin content in Shuxuening injection
Example 5
Preparation of control solutions:
accurately weighing 12.16mg of lariciresinol-4, 4-diglucoside in a 50ml volumetric flask, 10.90mg of pinoresinol diglucoside in a 50ml volumetric flask, 9.20mg of Urolinoside in a 50ml volumetric flask, and 10.26mg of cosmosiin in a 50ml volumetric flask, dissolving in methanol and fixing the volume to obtain a reference mother solution. Precisely measuring 2ml of lariciresinol-4, 4-diglucoside, 2ml of pinoresinol diglucoside, 5ml of Urolinoside and 1ml of cosmosiin reference mother liquor into the same 25ml volumetric flask, dissolving with methanol and fixing volume to obtain a mixed reference solution.
Preparation of a test solution:
precisely measuring 1ml Shuxuening injection 10ml volumetric flask, and fixing the volume with methanol to obtain the test solution of the Shuxuening injection.
The detection method comprises the following steps:
determining the contents of lariciresinol-4, 4-diglucoside, pinoresinoside, Urolignoside and cosmosiin in the Shuxuening injection by a UPLC-MS method, wherein the detection conditions are as follows:
a chromatographic column: octadecylsilane chemically bonded silica gel column, 2.1mm × 100mm, 1.6 μm; mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.08 vt% formic acid aqueous solution, and the volume ratio of the mobile phase A to the mobile phase B is 20: 80; column temperature: 35 ℃; flow rate: 0.2 ml/min; and (3) an elution mode: isocratic elution; the sample injection volume is 0.2 mu L; the MS detection mode is an ESI negative ion mode, and the chromatogram obtained has better separation degrees between detection peaks of lariciresin-4, 4-diglucoside, pinoresinol diglucoside, Urolignoside and cosmos glycoside and between other peaks.
Example 6
Preparation of control solutions:
accurately weighing 12.16mg of lariciresinol-4, 4-diglucoside in a 50ml volumetric flask, 10.90mg of pinoresinol diglucoside in a 50ml volumetric flask, 9.20mg of Urolinoside in a 50ml volumetric flask, and 10.26mg of cosmosiin in a 50ml volumetric flask, dissolving in methanol and fixing the volume to obtain a reference mother solution. Precisely measuring 2ml of lariciresinol-4, 4-diglucoside, 2ml of pinoresinol diglucoside, 5ml of Urolinoside and 1ml of cosmosiin reference mother liquor into the same 25ml volumetric flask, dissolving with methanol and fixing volume to obtain a mixed reference solution.
Preparation of a test solution:
precisely measuring 1ml Shuxuening injection 10ml volumetric flask, and fixing the volume with methanol to obtain the test solution of the Shuxuening injection.
The detection method comprises the following steps:
determining the contents of lariciresinol-4, 4-diglucoside, pinoresinoside, Urolignoside and cosmosiin in the Shuxuening injection by a UPLC-MS method, wherein the detection conditions are as follows:
a chromatographic column: octadecylsilane chemically bonded silica gel column, 2.1mm × 100mm, 1.6 μm; mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.12 vt% formic acid aqueous solution, and the volume ratio of the mobile phase A to the mobile phase B is 35: 60; column temperature: 38 ℃; flow rate: 0.5 ml/min; and (3) an elution mode: isocratic elution; the sample injection volume is 1.5 mu L; the MS detection mode is an ESI negative ion mode, and the chromatogram obtained has better separation degrees between detection peaks of lariciresin-4, 4-diglucoside, pinoresinol diglucoside, Urolignoside and cosmos glycoside and between other peaks.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (8)
1. A method for detecting lignans and flavonol glycosides compounds in a ginkgo leaf extract or a preparation thereof, wherein the lignans compounds are lariciresinol-4, 4-diglucoside and pinoresinol diglucoside, and the flavonol glycosides compounds are Urolignoside and cosmosiin, is characterized in that: the detection method is a UPLC-MS method, wherein the detection conditions of the UPLC are as follows:
a chromatographic column: octadecylsilane chemically bonded silica gel column;
mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.08-0.12v% formic acid water solution, and the volume ratio of the mobile phase A to the mobile phase B is 10-35: 90-60;
column temperature: 35-40 ℃;
flow rate: 0.1-0.5 ml/min;
and (3) an elution mode: isocratic elution;
the MS detection mode is an ESI negative ion mode.
2. The detection method according to claim 1, characterized in that: the mobile phase B was 0.1v% aqueous formic acid.
3. The detection method according to claim 1, characterized in that: the volume ratio of the mobile phase A to the mobile phase B is 10: 90.
4. The detection method according to claim 1, characterized in that: the column temperature was 40 ℃ and the flow rate was 0.1 ml/min.
5. The detection method according to claim 1, characterized in that: the sample injection volume in the detection method is 0.2-1.5 mu L.
6. The detection method according to claim 1, characterized in that: the diluting solvent of the reference substance solution and the test solution in the detection method is methanol.
7. The detection method according to claim 1, characterized in that: the mass concentration of the component to be detected in the reference solution in the detection method is 13-20 times of that in the test solution.
8. The detection method according to claim 1, characterized in that: the concentration of the ginkgo biloba extract in the test solution in the detection method is 0.3-0.4 mg/mL.
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| CN112592377B (en) * | 2020-12-16 | 2022-07-22 | 神威药业集团有限公司 | Method for separating flavonoid chemical components from ginkgo leaf extract |
| CN113899841B (en) * | 2021-11-08 | 2023-05-12 | 黑龙江珍宝岛药业股份有限公司 | Method for detecting pinoresinol diglucoside in ginkgo leaf extraction intermediate or preparation thereof |
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