CN112213410B - Method for detecting ginkgo leaves - Google Patents
Method for detecting ginkgo leaves Download PDFInfo
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- CN112213410B CN112213410B CN202010536921.5A CN202010536921A CN112213410B CN 112213410 B CN112213410 B CN 112213410B CN 202010536921 A CN202010536921 A CN 202010536921A CN 112213410 B CN112213410 B CN 112213410B
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- ginkgolide
- methanol
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- ginkgolic acid
- acid
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/047—Standards external
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Medicines Containing Plant Substances (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to a method for detecting ginkgo leaves, which is characterized by comprising the following steps: taking a ginkgo leaf test sample for detection, wherein the chromatographic conditions of the detection comprise: a chromatographic column: agilent Eclipse Plus C183.0X 50mm, 1.8 μm; mobile phase: 0.1% formic acid-methanol (a): 0.1% formic acid-water (B), the elution gradient detected was 0-3 min: 25% -95% of A; 3-6 min: 95% of A; 6-7 min: 95% -25% of A; 7-8 min: 25% of A. The methodological investigation confirms that the technology of the invention is simple, convenient, rapid and accurate, can finish the detection of various index components in a short time, and is an effective means for determining the content of the multi-index components of the ginkgo leaves.
Description
Technical Field
The invention relates to a detection method, in particular to a method for determining ginkgo leaves by adopting a liquid chromatogram-series triple quadrupole mass spectrometry.
Background
Folium Ginkgo is dried leaf of Ginkgo biloba (Ginko biloba L.) belonging to Ginko family. Sweet, bitter, astringent and neutral in nature, and has the effects of activating blood circulation to dissipate blood stasis, clearing and activating the channels and collaterals, astringing lung to relieve asthma, eliminating turbid pathogen and reducing blood fat, and the like, and the heart and lung meridians are entered. Folium ginkgo is widely applied to various preparation products as a traditional Chinese medicine, and is mainly used for treating coronary heart disease, angina, hyperlipidemia and other diseases.
Bilobalide is one of the main effective components in Ginkgo leaf (Ginkgo leaf), the structure of the bilobalide is mainly diterpene and sesquiterpene structure, and the bilobalide is the only natural substance which is discovered so far and has a tert-butyl functional group. Research shows that the components can antagonize Platelet Activating Factor (PAF) receptor specifically. The common bilobalide in folium Ginkgo includes bilobalide A, bilobalide B, bilobalide C, bilobalide, and bilobalide J. The content of the bilobalide compounds plays a key role in the curative effect and the internal quality of the ginkgo leaf preparation and is an important basis for controlling the quality of the ginkgo leaves and the extracts and the plant medicines thereof.
Ginkgolic Acids (GA) are the main toxic substances in ginkgo leaves and have sensitization, embryotoxicity, cytotoxicity and immunotoxicity. Is also a main substance causing clinical adverse reactions and has sensitization and mutagenic toxicity. The ginkgo leaf has a plurality of ginkgolic acids, and the limit of the ginkgolic acids is one of key indexes for evaluating the quality of the ginkgo leaf preparation.
At present, the detection method of bilobalide components and ginkgolic acid components in ginkgo leaves is available. For ginkgolide components, because ultraviolet absorption is poor, the components are easy to interfere in detection, a high performance liquid chromatography and Evaporative Light Scattering Detector (ELSD) combined method is adopted for detection at present, but the sensitivity of the ELSD is low, and the ELSD is mainly used for detecting ginkgolide components in ginkgo leaf extracts and preparations at present. For ginkgolic acid components, a high performance liquid chromatography and Diode Array Detector (DAD) combined method is mainly adopted for detection at present, ginkgolic acid and total ginkgolic acid are mostly used as reference, and the content of ginkgolic acid in ginkgo leaves and preparations thereof is calculated by the method, so that the actual existence condition of various ginkgolic acid in the ginkgo leaves and the preparations thereof cannot be reflected. Different pre-treatments are needed to be carried out for detecting the two components respectively, and the process is complicated and time-consuming, so that a detection means with high sensitivity is needed to be adopted to establish a quick, simple and convenient method for simultaneously determining the contents of ginkgolides and ginkgolic acids in the ginkgo leaves and the preparation thereof.
Disclosure of Invention
In view of the above, the present invention provides a method for simultaneously determining the content of multiple index components in ginkgo leaves by HPLC-MS/MS, which is directed to the above problems in the prior art.
The purpose of the invention can be realized by the following technical scheme: a method for detecting ginkgo leaves is characterized by comprising the following steps: taking a ginkgo leaf test sample for detection, wherein the chromatographic conditions of the detection comprise:
a chromatographic column: agilent Eclipse Plus C183.0X 50mm, 1.8 μm; mobile phase: 0.1% formic acid-methanol (a): 0.1% formic acid-water (B), the elution gradient detected was 0-3 min: 25% -95% of A; 3-6 min: 95% of A; 6-7 min: 95% -25% of A; 7-8 min: 25% of A.
Further, the chromatographic conditions further comprise: the column temperature is 25-40 ℃, and the flow rate is 0.4-0.6 mL/min.
In particular, the chromatographic conditions comprise: column temperature 40 ℃, flow rate 0.6mL/min, sample size: 1 μ L.
Further, the detection method further comprises detecting the control solution, wherein the chromatographic conditions of the detection comprise:
a chromatographic column: agilent Eclipse Plus C183.0X 50mm, 1.8 μm; mobile phase: 0.1% formic acid-methanol (a): 0.1% formic acid-water (B), the elution gradient detected was 0-3 min: 25% -95% of A; 3-6 min: 95% of A; 6-7 min: 95% -25% of A; 7-8 min: 25% of A;
the control may be one or more of ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, bilobalide, ginkgolic acid C13:0, ginkgolic acid C15:0, ginkgolic acid C15:1, ginkgolic acid C17:1, and ginkgolic acid C17: 2.
According to the detection result of the test sample or the reference sample, the component information of the ginkgo leaf or the component and the content information thereof can be obtained.
Further, an optional internal standard in the detection method is tolbutamide.
Specifically, the ginkgo biloba leaf test sample can be prepared by extracting with an alcoholic solution; the specific method comprises the following steps:
using 75% methanol as extraction solvent, ultrasonic extracting folium Ginkgo and 75% methanol solution at a ratio of 0.2g:25mL, centrifuging, collecting supernatant, diluting with pure methanol solution, and filtering with 0.22 μm filter membrane.
More specifically, the preparation of the test article comprises:
extracting ginkgo leaves and 75-100% methanol extraction solvent according to the proportion of 1: 125-1: 25, performing ultrasonic extraction for 20-40 min, centrifuging, taking supernatant, diluting by 100 times with pure methanol solution, and filtering with a 0.22 mu m filter membrane;
preferably, 0.2g of ginkgo leaf powder is weighed, 25mL of 75% methanol solution is added, after weighing, ultrasonic extraction is carried out for 30min, after cooling to room temperature, the weight loss is complemented by 75% methanol solution, after centrifugation, 1mL of supernatant is taken, the supernatant is diluted by 100 times by pure methanol solution, and the solution is filtered through a 0.22 mu m filter membrane, thus obtaining the ginkgo leaf test solution.
Specifically, the preparation method of the reference solution comprises the following steps:
taking 10mg of a reference substance, placing the reference substance in a 20mL measuring flask, adding methanol to dissolve and dilute the reference substance to the scale, shaking up the reference substance to prepare a reference substance stock solution of 0.5 mg/mL.
The invention also provides a method for rapidly determining the multi-index components in the ginkgo leaves, which adopts an ultra-high performance liquid chromatography-mass spectrometry technology to detect the ginkgo leaves; wherein,
the chromatographic conditions of the ultra-high liquid phase-tandem mass spectrometry comprise:
a chromatographic column: agilent Eclipse Plus C183.0X 50mm, 1.8 μm; mobile phase: 0.1% formic acid-methanol (a): 0.1% formic acid-water (B), the elution gradient detected was 0-3 min: 25% -95% of A; 3-6 min: 95% of A; 6-7 min: 95% -25% of A; 7-8 min: 25% of A;
the mass spectrum in the ultra-high performance liquid chromatography-tandem mass spectrum is a triple quadrupole mass spectrum.
Further, the chromatographic conditions further comprise: the column temperature is 25-40 ℃, and the flow rate is 0.3-0.45 mL/min.
In particular, the chromatographic conditions comprise: column temperature 40 ℃, flow rate 0.4mL/min, sample size: 1 μ L.
Specifically, the mass spectrum conditions of the ultra-high liquid phase-tandem mass spectrum comprise: the negative ion ESI ionization source, the triple quadrupole detector, the ion source temperature 500 ℃, the atomizing gas and the auxiliary gas are nitrogen, and the scanning mode is a multi-reaction monitoring mode (MRM).
Further, an optional internal standard in the method is tolbutamide.
Specifically, the multi-index component may include bilobalide A, bilobalide B, bilobalide C, bilobalide J, bilobalide, and ginkgolic acid.
Specifically, the ginkgoic acid may include ginkgoic acid C13:0, ginkgoic acid C15:0, ginkgoic acid C15:1, ginkgoic acid C17:1, or ginkgoic acid C17: 2.
Further, the mass spectrum parameters of the multi-index component and the internal standard comprise:
methodological considerations in the present invention may include:
and (3) linear relation investigation: preparing a proper amount of reference stock solution according to the method, mixing, precisely diluting with methanol solution in sequence to prepare a series of reference solutions, and adding a certain amount of internal standard solution into each concentration of reference solution to keep the concentration of the internal standard solution at 20 ng/mL; detecting by using the LC-MS/MS detection method (ultra-high performance liquid chromatography-mass spectrometry combined technology); taking the concentration (C, ng/mL) of the object to be detected as a horizontal coordinate, taking the peak area ratio (Y) of the target analyte to the internal standard as a vertical coordinate, and performing regression operation by adopting a least square method to obtain a linear regression equation of the object to be detected;
precision investigation: taking the same mixed reference solution, performing LC-MS/MS analysis, continuously repeating sample injection for 6 times, recording peak areas of target analyte and internal standard, and calculating ratio (A)Analyte/AIS) RSD value of (d);
and (3) stability investigation: preparing test solution according to the method, performing LC-MS/MS analysis for 0h, 2h, 4h, 6h, 8h, 12h and 24h respectively, and recording each target analyte and internal standard peakArea, calculating its ratio (A)Analyte/AIS) RSD value of (d);
and (3) repeatability inspection: preparing a sample solution according to the method, preparing 6 parts in parallel, measuring the peak areas of a target analyte and an internal standard, and calculating the RSD value of the content of the target analyte and the internal standard;
and (3) sample recovery rate investigation: taking about 0.1g of dried folium Ginkgo powder, precisely weighing, randomly dividing into 3 groups, adding one or more (such as 10 of the multi-index components) mixed reference substance solutions (low, medium and high concentrations, each 3 parts) respectively, analyzing the rest according to the above-mentioned ultra performance liquid chromatography-mass spectrometry, and recording AAnalyte/AISThe average recovery rate and RSD value of 10 index components were calculated, respectively.
In the content determination of the index components in the sample of the invention: the ginkgo leaves from different sources can be used for preparing a sample solution according to the corresponding method, and the measurement is carried out under the condition of the combination of the ultra performance liquid chromatography and the mass spectrum. The pre-treatment process of ginkgo leaf may include: drying fresh folium Ginkgo sample with 60 deg.C hot air to constant weight, and pulverizing to obtain dried folium Ginkgo powder.
The detection method established by the invention can be used for qualitatively or quantitatively detecting as many as 10 main index components in ginkgo leaf samples from different sources, thereby being particularly suitable for quality control of medicinal materials and preparations. The invention uses HPLC-MS/MS method to research the content determination of ginkgolide and ginkgolic acid compounds in folium ginkgo for the first time, the total analysis time is 8 minutes, and the rapid analysis of 10 index components in a short time can be realized. Compared with the traditional HPLC-ELSD and HPLC-UV methods, the method has higher sensitivity and selectivity, and can detect the components with the content of ng/mL grade.
In conclusion, the HPLC-MS/MS method is established, and the contents of ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, bilobalide and ginkgolic acid (such as ginkgolic acid C13:0, ginkgolic acid C15:0, ginkgolic acid C15:1, ginkgolic acid C17:1 and ginkgolic acid C17: 2) in the ginkgo leaves are simultaneously measured, so that the method is simple, convenient and rapid, has high sensitivity, and has important scientific significance for development and utilization of ginkgo leaf resources and quality evaluation.
Drawings
FIG. 1 shows MRM chromatograms of mixed standards.
FIG. 2 is the MRM chromatogram of Ginkgo biloba leaves.
FIG. 3 is a second-order mass spectrum of ginkgolide A.
FIG. 4 is a second-order mass spectrum of ginkgolide B.
FIG. 5 is a second-order mass spectrum of ginkgolide C.
FIG. 6 is a second-order mass spectrum of ginkgolide J.
FIG. 7 is a second-order mass spectrum of bilobalide.
Fig. 8 shows ginkgolic acid C13:0, secondary mass spectrum.
Fig. 9 shows ginkgolic acid C15: second order mass spectrum of 0.
Fig. 10 shows ginkgolic acid C15:1, secondary mass spectrum.
Fig. 11 shows ginkgolic acid C17:1, secondary mass spectrum.
Fig. 12 is ginkgolic acid C17:2, secondary mass spectrum.
FIG. 13 is a secondary mass spectrum of tolbutamide.
FIG. 14 is a graph showing the effect of different extraction methods on the extraction efficiency of ginkgolides and ginkgolic acids.
FIG. 15 is a graph showing the effect of five different extraction solvent species on the extraction efficiency of ginkgolides and ginkgolic acids.
FIG. 16 is a graph showing the effect of extraction time on the extraction efficiency of ginkgolides and ginkgolic acids.
FIG. 17 is a graph showing the effect of extraction solvent ratio on the extraction efficiency of ginkgolides and ginkgolic acids.
FIG. 18 is a graph showing the effect of material-to-liquid ratio (g: mL) on the extraction efficiency of ginkgolides and ginkgolic acids.
FIG. 19 is a graph showing the content change of bilobalide in ginkgo leaves at different time periods.
FIG. 20 is a graph showing the content of ginkgolides in different ages of ginkgo leaves.
FIG. 21 is a graph showing the content change of bilobalide in old ginkgo leaves at different time periods.
Detailed Description
The invention discloses a method for detecting multiple index components in ginkgo simultaneously, which can be realized by appropriately improving process parameters by referring to the content of the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention.
In the method for measuring the content of the multi-index components in the ginkgo leaves, methanol is a mass spectrum-grade reagent of Merck company; formic acid was from ROE corporation, usa; the ultrapure water is self-made by a Pacific TII 7 ultrapure water instrument of Saimei Fei company for laboratories; purity of reference substances (ginkgolide C purity 94.0%, ginkgolic acid C17:2 purity 96.85%, and the rest more than 98%), wherein ginkgolic acid compound and ginkgolide J are obtained from Shanghai leaf Biotech limited, and the rest are obtained from China food and drug testing institute.
In the method for measuring the content of the multi-index components in the ginkgo leaves, the used instruments are as follows: ultra high performance liquid chromatography (LC-30AD, Shimadzu, Japan); triple quadrupole mass spectrometer (API 6500)+AB SCIEX corporation); one in ten thousand electronic analytical balance (ME 204E, mettler-toledo shanghai instruments ltd); a one-tenth-ten-thousandth electronic balance (Mettler XS205, mertler-toledo shanghai ltd); DHG-9023A electric heating constant temperature air-blast drying oven (Shanghai essence macro instrument and equipment Co., Ltd.); an ultrasonic cleaner (KH 300DB, kunshan grass ultrasonic instruments ltd).
Except for special points, the medicines, reagents and instruments used in the technical scheme provided by the invention can be purchased from conventional channels or markets.
Method for detecting multi-index components in ginkgo leaves
1 Instrument and reagent
Ultra high performance liquid chromatography (LC-30AD, Shimadzu, Japan); mass spectrometer (API 6500)+AB SCIEX corporation); one in ten thousand electronic analytical balance (ME 204E, mettler-toledo shanghai instruments ltd); a one-tenth-ten-thousandth electronic balance (Mettler XS205, mertler-toledo shanghai ltd); ultrasonic cleaner (KH 300DB, Kun Yin)Mountain standing grain ultrasonic instruments ltd); pacific tii 7 ultrapure water meter (saimmer flyer, usa); eppendorf Centrifuge 5424R Centrifuge (edbend, germany); rotary evaporator (model BUCHI R-3).
A ginkgolide A reference substance (China institute for food and drug assay, batch No. 110862-; a ginkgolide B reference (China institute for testing and testing food and drug; batch No. 110863-one 201611); a ginkgolide C reference substance (China institute for food and drug assay, batch No. 110864-201508); bilobalide J reference substance (Shanghai leaf Biotech Co., Ltd., batch No. D11J9G 63336); a bilobalide reference substance (China institute for food and drug assay, batch No. 110865-201507); ginkgolic acid C13:0 control (China institute for food and drug testing, lot No. 111690-201604); ginkgolic acid C15:0 control (China pharmaceutical biologicals institute, lot number: D27M9C 56540); a ginkgolic acid C15:1 reference substance (China institute for testing and testing food and drug, lot number: 111762) -200601); ginkgolic acid C17:1 control (Shanghai leaf Biotech Co., Ltd., lot number: Y23M9H 62018); ginkgolic acid C17:2 control (Shanghai leaf Biotech Co., Ltd., lot number Y04J9H 64929); methanol (chromatographically pure, Merck, Germany, LOT: 10934235805); formic acid (HPLC, ROE, USA, LOT: 6F 2914); ultrapure water was prepared by Pacific TII 7 ultrapure water purification system (Thermo Fisher Scientific, USA).
The source of the medicine is as follows: the Ginkgo leaf sampling time is 4 months to 9 months in 2018, and dried leaves of Ginkgo biloba Ginkgo biloba L. of Ginko are identified by a practitioner Wuzhou of Jiangsu Kangyuan pharmaceutical industry GmbH in Jiangsu Kangyuan.
2 experimental methods and results
2.1 preparation of the solution
Preparing a reference solution: weighing ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, bilobalide, ginkgolic acid C13:0, ginkgolic acid C15:0, ginkgolic acid C15:1, ginkgolic acid C17:1 and ginkgolic acid C17:2, and accurately weighing about 10mg of a reference substance, placing the reference substance into a 20mL measuring flask, adding methanol to dissolve and dilute the reference substance to a scale, shaking uniformly, preparing a reference substance stock solution of 0.5mg/mL, and storing the reference substance stock solution in a refrigerator at 4 ℃ for later use.
Taking another 10mg of tolbutamide (internal standard IS) reference substance, precisely weighing, placing into a 20mL measuring flask, adding methanol to dissolve and dilute to scale, shaking up, and preparing into stock solution with concentration of 0.5 mg/mL; a100. mu.L to 50mL measuring flask was precisely pipetted to prepare an internal standard solution at a concentration of 1. mu.g/mL.
2.2 LC-MS/MS detection conditions
2.2.1 optimization of Mass Spectrometry conditions
(1) Preparing a mixed reference substance and an internal standard stock solution according to the method under the item 2.2, and diluting the mixed reference substance and the internal standard stock solution by using 50 percent methanol water solution to ensure that the concentration of a compound to be optimized is about 10 ng/mL;
(2) activating MassOnly on an Analyst workstation, and double-clicking to run Manual Tuning;
(3) sucking a mixed reference substance solution to be optimized into a needle pump, manually connecting a mass spectrum to an injection pump, injecting a sample in a constant flow mode, and setting the flow rate to be 10 mu L/min;
(4) determining the mass-to-charge ratio of parent ions of each compound by using Q1 Scan;
(5) determining the mass-to-charge ratio of each compound daughter Ion by using Product Ion Scan;
(6) and (4) establishing an MRM ion pair according to the parent ions and the daughter ions selected in the steps (4) and (5), and optimizing the CE value and the DP value by using the Ramp, wherein the optimization result is shown in a table 1.
(7) The ion source is an ESI source; ion source Temperature (TEM)500 ℃; the atomizing gas (GS1) and the auxiliary gas (GS2) are nitrogen; the scanning mode is multi-reaction monitoring (MRM), and the negative ion mode; the ion pairs for quantitative analysis and detection are shown in table 110 for mass spectrum parameters of the components to be detected and the internal standard compound in table 1.
TABLE 110 Mass Spectrometry parameters for the Components to be tested and for the internal Standard Compound
2.2.2 chromatographic conditions
The type of the chromatographic column: agilent Eclipse Plus C18 chromatography column (3.0 x 50mm, 1.8 μm), column temperature: at 40 ℃. Mobile phase 0.1% formic acid methanol (a) -0.1% formic acid water (B), flow rate: 0.6mL/min, gradient elution: 0-3 min: 25% -95% of A; 3-6 min: 95% of A; 6-7 min: 95% -25% of A; 7-8 min: 25% -25% of A, sample injection amount: 1 μ L.
The results of the above-described method are shown in FIGS. 1 to 13.
2.3 preparation method of test article
2.3.1 examination of extraction methods
(1) Taking 1g of dried ginkgo leaf medium powder, precisely weighing, placing in a round bottom flask, adding 50mL of pure water, heating, refluxing and extracting for 1h, cooling, performing suction filtration, performing shaking extraction on the subsequent filtrate for multiple times, 20mL each time, by using petroleum ether (30-60 ℃) until a discarded petroleum ether layer is colorless, performing shaking extraction on a water layer by using ethyl acetate for 3 times, 30mL each time, combining ethyl acetate solutions, heating and concentrating to dryness, adding 50% methanol into residues for dissolving and fixing the volume to 25mL, shaking uniformly, filtering, diluting, injecting a sample and detecting.
(2) Taking 1g of dried ginkgo leaf medium powder, precisely weighing, placing in a round-bottom flask, adding 50mL of pure water, heating, refluxing and extracting for 1h, cooling, performing suction filtration, performing oscillation extraction on the subsequent filtrate for 3 times by using ethyl acetate, each time extracting for 30mL, combining ethyl acetate solutions, heating and concentrating to dryness, adding 50% methanol into residues to dissolve, and performing constant volume to 25mL, shaking, filtering, diluting, and performing sample injection and detection.
(3) Taking 1g of dry ginkgo leaf medium powder, precisely weighing, placing in a conical flask with a plug, adding 50mL of pure water, ultrasonically extracting for 1h, cooling, performing suction filtration, performing shaking extraction on the subsequent filtrate for multiple times, 20mL each time, by using petroleum ether (30-60 ℃) until the discarded petroleum ether layer is colorless, performing shaking extraction on the water layer for 3 times, 30mL each time, combining ethyl acetate solutions, heating and concentrating to dryness, adding 50% methanol into residues to dissolve and fix the volume to 25mL, shaking uniformly, filtering, diluting, and performing sample injection detection.
(4) Taking 1g of dry ginkgo leaf medium powder, precisely weighing, placing in a conical flask with a plug, adding 50mL of pure water, ultrasonically extracting for 1h, cooling, performing suction filtration, continuously extracting the filtrate for 3 times by using ethyl acetate in a shaking way, each time extracting for 30mL, combining ethyl acetate solutions, heating and concentrating to be dry, adding 50% methanol into residues to dissolve, fixing the volume to 25mL, shaking uniformly, filtering, diluting, and then injecting a sample for detection.
(5) Taking 0.5g of dried ginkgo leaf powder, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of methanol, weighing, ultrasonically treating for 30min, taking out, cooling, supplementing the lost weight with corresponding solvent, shaking, filtering, diluting, and detecting by sample injection.
(6) Taking 0.2g of dried ginkgo leaf powder, precisely weighing, placing in a 50mL conical flask, precisely adding 10mL of hemicellulase solution (80U/mL) dissolved in pure water into the conical flask, placing in a water bath at 50 ℃ for 3h for enzymolysis, then adding 30mL of methanol into the conical flask, carrying out ultrasonic treatment for 30min, taking out, cooling, complementing weight loss reduction amount with corresponding solvent, shaking uniformly, filtering, diluting, and carrying out sample injection detection.
As shown in FIG. 14, the ultrasonic extraction method of method 5 can achieve the highest extraction rate, and the operation is simple and convenient, so that the methanol ultrasonic method is subsequently used as an extraction method for subsequent research.
2.3.2 examination of extraction solvent
Taking 5 parts of dried ginkgo leaf powder, each part of the dried ginkgo leaf powder is about 0.5g, precisely weighing, respectively placing the dried ginkgo leaf powder into 5 conical flasks with the volume of 50mL, precisely adding 25mL of methanol, ethanol, acetone, chloroform and ethyl acetate into the conical flasks, weighing, ultrasonically treating for 1h, taking out, cooling, supplementing the weight loss by using a corresponding solvent, shaking uniformly, filtering, decompressing and evaporating to dryness, redissolving by using methanol, fixing the volume to 25mL, diluting, and then injecting a sample and detecting.
As shown in FIG. 15, the extraction solvent used in the method was chloroform, acetone, ethyl acetate, and methanol, and the best extraction was obtained when methanol was used as the extraction solvent.
2.3.3 examination of extraction time
Taking 4 parts of dried ginkgo leaf powder, each part of which is about 0.2g, precisely weighing, respectively placing in 4 conical flasks with the volume of 50mL, precisely adding 50mL of methanol, weighing, ultrasonically treating for 20min, 30min, 40min and 60min, taking out, cooling, supplementing the weight loss with methanol, shaking, filtering, diluting, and detecting.
The result is shown in fig. 16, a better extraction effect can be obtained by ultrasonic treatment for 20-40 min, wherein the best extraction effect can be obtained by ultrasonic treatment for 30min, so that the subsequent research is carried out by taking 30min as ultrasonic time.
2.3.4 examination of the proportion of extraction solvent
Taking 4 parts of dried ginkgo leaf powder, each part of which is about 0.2g, precisely weighing, respectively placing the dried ginkgo leaf powder into 4 conical flasks with the volume of 50mL, precisely adding 25%, 50%, 75% and 50mL of 100% methanol respectively, weighing, ultrasonically treating for 30min, taking out, cooling, supplementing the weight loss with methanol, shaking uniformly, filtering, diluting, and then carrying out sample detection.
As shown in fig. 17, when 75% and 100% methanol were used, the total extraction rate of ginkgolides and ginkgolic acids was high, 75% methanol had a good extraction effect with respect to ginkgolides, and 75% methanol was preferably used as the extraction solvent for the purpose of taking account of the measurement of the content of minor components, and the subsequent studies were conducted.
2.3.5 investigation of the ratio of liquid to feed
Taking 4 parts of dried ginkgo leaf powder, each part of which is about 0.2g, precisely weighing, respectively placing the dried ginkgo leaf powder into 4 conical flasks with the volume of 50mL, precisely adding 5mL, 10mL, 25mL and 50mL of methanol into the conical flasks, weighing, ultrasonically treating for 30min, taking out, cooling, complementing the weight loss by using methanol, shaking, filtering, respectively diluting, and carrying out sample injection detection.
The results are shown in fig. 18, when the ratio of liquid to feed is 1: 125. 1: 50. 1: 25, obtaining better extraction effect, and when the ratio of material to liquid is 1: the extraction rate is highest at 125 days, so that the extraction ratio is 1: 125(0.2g:25mL) was used as the feed-to-liquid ratio for subsequent studies.
In conclusion, the preparation method of the test solution is determined as follows: taking 0.2g of dried ginkgo leaf powder, accurately weighing, placing in a 50mL conical flask, accurately adding 25mL of 75% methanol, weighing, ultrasonically treating for 30min, taking out, cooling, supplementing the lost weight with corresponding solvent, centrifuging, taking supernatant, diluting with 100 times of methanol, and carrying out sample detection.
2.4 methodological validation
2.4.1 Linear relationship
Taking a proper amount of reference stock solution prepared by the method under '2.1', mixing, diluting with methanol, preparing into 6 series of mixed reference solutions with gradient concentration, and measuring under the condition of a proposed LC-MS/MS. And (3) taking the concentration (C, ng/mL) of the object to be tested as a horizontal coordinate, taking the peak area ratio (Y) of the target analyte to the internal standard as a vertical coordinate, performing regression operation by adopting a least square method to obtain a linear regression equation of the object to be tested, and automatically calculating the standard curve equation and the related coefficient of each object to be tested by AB Sciex Multi Quant 2.1 data processing software. The specific results are shown in Table 2, and it is understood that 10 components are in good linear relationship within the selected range.
TABLE 210 concentrations of the test ingredients in the control solutions
2.4.2 quantitation Limit and detection Limit
Taking the reference substance solution with the lowest concentration of the standard curve, sequentially diluting the reference substance solution with methanol to form a series of standard solutions with concentration, and respectively injecting samples for analysis. And calculating the signal-to-noise ratio (S/N) of the chromatographic peak of the reference substance, wherein the concentration of the reference substance when the S/N is 3 is the lowest detection Limit (LOD), and the concentration of the reference substance when the S/N is 10 is the lowest quantification Limit (LOQ). The results are shown in Table 3.
TABLE 3 Standard Curve Range, quantitation Limit and detection Limit
2.4.3 precision
Taking the same mixed reference solution, performing LC-MS/MS analysis, continuously feeding sample for 6 times, recording peak areas of target analyte and internal standard, and calculating the ratio (A)Analyte/AIS) The results in Table 4 show that the RSD of the components to be measured is less than 4%, which indicates that the precision of the instrument is good.
TABLE 4 results of precision test
2.4.4 stability
Preparing test solution according to the method of the test preparation method, performing LC-MS/MS analysis for 0h, 2h, 4h, 6h and 8h respectively, recording peak areas of each target analyte and internal standard, and calculating the ratio (A)Analyte/AIS) The results in Table 5 show that the RSD of the components to be tested is less than 4%, which indicates that the reference solution and the test solution are stable within 8 h.
TABLE 5 stability test results
2.4.5 repeatability
And preparing a sample solution according to a method under the item of a sample preparation method, paralleling 6 parts, measuring the peak area ratio of the target analyte to the internal standard, and calculating the content and the RSD value. According to the test results, the RSD of the components to be tested is less than 6%, and the RSD meets the related regulation on repeatability investigation of 2015 edition pharmacopoeia of the people's republic of China, which shows that the method has good repeatability.
TABLE 6 results of the repeatability tests
2.4.6 sample recovery
Precisely weighing about 0.1g of dried folium Ginkgo, preparing 9 parts in parallel, randomly dividing into 3 groups, adding 10 mixed reference solutions (low, medium and high concentrations, 3 parts each) respectively, analyzing according to the above detection conditions, and adding AAnalyte/AISThe value of (A) is substituted into a standard curve, the concentration of each substance to be detected is obtained by calculating AB Sciex Multi Quant 2.1 data processing software, the recovery rate and the RSD value of a reference substance are calculated, and the result is shown in the following table. The experimental results show that the high-temperature-resistant material,the average recovery rates of ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, bilobalide, ginkgolic acid C13:0, ginkgolic acid C15:0, ginkgolic acid C15:1, ginkgolic acid C17:1 and ginkgolic acid C17:2 are respectively 99.17%, 95.59%, 97.55%, 93.46%, 94.30%, 97.39%, 92.53%, 106.41%, 98.89% and 101.33%, and the method conforms to the regulation of the sample loading recovery rate limit of the Chinese pharmacopoeia of 2015 edition, and shows that the method has good accuracy.
TABLE 7 sample recovery test results
2.5 application of the detection method
The ginkgo leaf samples collected in the areas of the State of Peruvian seconds are detected and analyzed by the method, and the results are shown in the following table.
TABLE 8 determination of bilobalide component content in test sample
Referring to fig. 19, the results show that the content of ginkgolide in the folium ginkgo in the te area is generally increased and then decreased in one year, and generally reaches the highest value in seven to august and then decreases.
TABLE 9 determination of bilobalide component content in test samples
As shown in table 9 and also referring to fig. 20, the results showed that the content of the lactone-type compound was different among ginkgo leaves of different ages, wherein the content of the bilobalide-type component was the highest among the two years. With the increase of the tree age, the content of the ginkgolide component is reduced.
TABLE 10 determination of ginkgolic acid compound content in test sample
In addition, the invention researches the content change of ginkgolides in different time periods of the large-age ginkgo leaves, as shown in fig. 21, the content change of ginkgolic acid compounds in the large-age ginkgo leaves in one year shows the trend of increasing firstly and then decreasing, and the content of ginkgolic acid compounds reaches the highest value in July.
The method developed by the invention can be used for rapidly detecting the ginkgo leaves and is particularly suitable for quality control of medicinal materials and preparations. By utilizing the method, the content change of ginkgolide components of ginkgo leaves at different time periods, the content of ginkgolide components of ginkgo leaves at different ages and the content change of ginkgolide components of ginkgo leaves at different time periods of old ginkgo leaves are researched, and a high-value ginkgo leaf medicinal material is found, so that a basis is provided for planting and developing the ginkgo leaves.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. A method for detecting ginkgo leaves is characterized in that an ultra-high performance liquid chromatography-mass spectrometry technology is adopted to determine multi-index components in the ginkgo leaves; the method comprises the following steps:
preparing a ginkgo leaf test sample:
extracting ginkgo leaves and 75-100% methanol extraction solvent according to the proportion of 1 g: 125 mL-1 g: performing ultrasonic extraction for 20-40 min at a feed-liquid ratio of 25mL, centrifuging, taking supernatant, diluting by 100 times with pure methanol solution, and filtering with a 0.22-micron filter membrane;
taking a ginkgo leaf sample for detection:
the chromatographic conditions of the assay include:
and (3) chromatographic column: agilent Eclipse Plus C183.0X 50mm, 1.8 μm; mobile phase: 0.1% formic acid-methanol a: 0.1% formic acid-water B, the elution gradient detected was 0-3 min: 25% -95% of A; 3-6 min: 95% of A; 6-7 min: 95% -25% of A; 7-8 min: 25% of A;
the mass spectrometric conditions of the assay include:
negative ion ESI ionization source, triple quadrupole detector;
wherein the multi-index components comprise ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, bilobalide, ginkgolic acid C13:0, ginkgolic acid C15:0, ginkgolic acid C15:1, ginkgolic acid C17:1 and ginkgolic acid C17: 2;
the mass spectrum parameters of the multi-index components comprise:
2. the detection method according to claim 1, wherein the chromatographic conditions further comprise:
the column temperature is 25-40 ℃, and the flow rate is 0.4-0.6 mL/min.
3. The test method of claim 1, further comprising testing the control solution under chromatographic conditions comprising:
a chromatographic column: agilent Eclipse Plus C183.0X 50mm, 1.8 μm; mobile phase: 0.1% formic acid-methanol a: 0.1% formic acid-water B, the elution gradient detected was 0-3 min: 25% -95% of A; 3-6 min: 95% of A; 6-7 min: 95% -25% of A; 7-8 min: 25% of A;
the reference substance is selected from one or more of ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, bilobalide, ginkgolic acid C13:0, ginkgolic acid C15:0, ginkgolic acid C15:1, ginkgolic acid C17:1 and ginkgolic acid C17: 2.
4. The detection method according to claim 1, wherein an internal standard selected in the detection method is tolbutamide.
5. The detection method as claimed in claim 1, wherein the ginkgo biloba test sample is prepared by using 75% methanol as an extraction solvent, subjecting ginkgo biloba to ultrasonic extraction with the 75% methanol solution at a ratio of 0.2g:25mL, centrifuging, collecting the supernatant, diluting the supernatant with a pure methanol solution by 100 times, and filtering the diluted supernatant through a 0.22 μm filter.
6. The detection method according to claim 3, wherein the preparation method of the control solution comprises:
taking 10mg of a reference substance, placing the reference substance in a 20mL measuring flask, adding methanol to dissolve and dilute the reference substance to the scale, shaking up the reference substance to prepare a reference substance stock solution of 0.5 mg/mL.
7. The detection method according to claim 1, wherein the ion source temperature of the mass spectrometry conditions of the ultra performance liquid chromatography-mass spectrometry is 500 ℃, the atomizing gas and the auxiliary gas are nitrogen, and the scanning mode is a multi-reaction monitoring mode.
8. The detection method according to claim 1, wherein the mass spectral parameters of the multi-index components include:
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| CN104407082B (en) * | 2014-11-27 | 2016-03-09 | 中国科学院上海药物研究所 | The detection method of alkylphenol compounds in a kind of ginkgo leaf raw material and preparation |
| CN107315058A (en) * | 2017-05-04 | 2017-11-03 | 黑龙江珍宝岛药业股份有限公司 | A kind of method of total ginkgoic acid in detection ginkgo biloba succi |
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