Ginkgo leaf detection method and the construction and application of multi-component fingerprint

Abstract

The present application provides a method for detecting ginkgo biloba leaves and the construction and application of a multi-component fingerprint spectrum simultaneously, including: taking a ginkgo biloba reference substance and a test substance for detection, and the chromatographic conditions include: a chromatographic column ZORBAX SB-C18; a mobile phase of 0.1 volume % formic acid water (A)-methanol (B). Flow rate 0.9-1.1mL / min; detection wavelength 360nm, column temperature 25-35℃, injection volume 10μL; evaporative light scattering detector parameters: drift tube temperature 110℃, carrier gas flow rate 3.0mL / min, gain value 4. Through methodological investigation, it is confirmed that the detection method of the present application is simple, fast and accurate, and the fingerprint spectrum determination of multiple index components in ginkgo biloba leaves can be completed in a short time, providing a reliable analysis method for the effective control of the quality of ginkgo biloba products.

Description

Technical Field

[0001] The present application relates to the field of analytical chemical detection technology, and specifically to a method for detecting chemical components of ginkgo leaves and constructing a fingerprint spectrum by combining high performance liquid chromatography (HPLC)-evaporative light scattering detection (ELSD) with traditional Chinese medicine fingerprint spectrum similarity calculation software. Background Art

[0002] Ginkgo biloba is the dried leaf of Ginkgo biloba L., a plant of the Ginkgoaceae family. It is sweet, bitter, astringent and neutral in nature, and enters the heart and lung meridians. It has the effects of promoting blood circulation and removing blood stasis, promoting blood circulation and activating collaterals, astringing the lungs and relieving asthma, and removing turbidity and lowering blood lipids. As a traditional Chinese medicine, Ginkgo biloba has been widely used in various traditional Chinese medicine preparations, mainly for the treatment of coronary heart disease, angina pectoris, hyperlipidemia and other diseases.

[0003] Terpene lactones and flavonoids contained in Ginkgo biloba are the main active ingredients of Ginkgo biloba. The main substance causing clinical adverse reactions in Ginkgo biloba is ginkglic acid, which has sensitization, nerve damage and mutagenic toxicity. The sensitization mechanism may be inhibition of enzyme activity, cytotoxicity and allergic reaction. If the content of ginkglic acid cannot be accurately known and controlled, the utilization of Ginkgo biloba will be limited and its medicinal value will be reduced.

[0004] Ginkgo biloba leaves are produced in many places and have complex ingredients. How to effectively reflect their overall information and conduct quality evaluation is a difficult problem that needs to be solved urgently. With the development of modern analytical technology, fingerprint technology is an internationally recognized quality control model for controlling the quality of traditional Chinese medicine. It can provide a full range of quality assurance from the cultivation and introduction of raw medicinal materials and the standardization and optimization of the production process of Chinese patent medicines to the formulation of product quality standards. Traditional Chinese medicine fingerprint is a comprehensive and quantifiable quality control method. Its basic attributes are integrity and fuzziness, which are consistent with the holistic principle of traditional Chinese medicine theory and the fuzziness of the mechanism of action of traditional Chinese medicine. The rise of traditional Chinese medicine fingerprint technology provides a reliable analytical method for the effective control of the quality of ginkgo leaf products. It has the advantages of high separation, integrity and specificity, and can be applied to the evaluation of the overall quality of ginkgo leaf medicinal materials of different origins, grades and different growth years.

[0005] At present, the quality of ginkgo leaves evaluated by fingerprints is only for ginkgo lactones or flavonoids. The industry generally does not pay attention to the content of ginkgolic acid in ginkgo leaves. This makes it impossible to solve the sensitization reactions, nerve damage and gene mutations that may be caused by the toxic side effects of ginkgolic acid; thus, the quality of ginkgo leaf products cannot be guaranteed. In addition, the physical and chemical properties of flavonoids and lactones in ginkgo leaves are very different, and different methods need to be used to detect these two types of substances. Among them, the detection method of ginkgo lactones is particularly complex, time-consuming and laborious. The content of ginkgo lactones and flavonoids and the limit of ginkgo acid are all key indicators for evaluating the quality of ginkgo leaf preparations, but it is still impossible to reflect ginkgo acid, ginkgo lactones and ginkgo flavonoids in one fingerprint at the same time. The overall detection process is complex, with many steps, long time and great difficulty.

[0006] Although the fingerprint of ginkgo terpenoid lactones in ginkgo leaf extract has been established, the fingerprint of ginkgo acid is limited to total ginkgo acid. For the ginkgo lactone components in ginkgo leaf extract and preparations, due to their poor ultraviolet absorption, they are easily interfered during detection, so high performance liquid chromatography combined with evaporative light scattering detector (ELSD) is often used for detection. Ginkgo flavonoids are also the main evaluation index of the quality of ginkgo extract. There are many types of flavonoid compounds, and their components cannot be fully clarified. At the same time, it is difficult to obtain reference substances, making it difficult to directly determine their content. At present, the main method for detecting ginkgo flavonoid components is high performance liquid chromatography combined with diode array detector (DAD). Quercetin 3-O-glucoside is used as the internal standard, and a mixed standard composed of the internal standard and other 12 flavonol glycosides is established to establish a fingerprint detection method for ginkgo flavonoids. This application is based on the current lack of fingerprint detection methods that can simultaneously reflect ginkgolides, ginkgo flavonoids and ginkgo acids. The HPLC-ELSD combination method is used to solve the problem that ultraviolet light has a poor detection effect on ginkgolides with weak ultraviolet absorption, and can achieve the purpose of simultaneous, simple and rapid detection. This method has high repeatability and accuracy, and can comprehensively and simultaneously reflect the overall characteristics of the chemical composition of ginkgo leaf medicinal materials. It is suitable for the authenticity and quality identification of ginkgo leaf medicinal materials, and provides a strong technical basis for improving the quality control level of ginkgo leaves and their preparations and accurately and comprehensively evaluating their intrinsic quality. Summary of the invention

[0007] In view of the above background technology, the present application aims at the problem that the quality of ginkgo leaf products cannot be comprehensively evaluated simply, quickly and sensitively, and provides a method for combining HPLC-ELSD combined method with traditional Chinese medicine fingerprint similarity calculation software to establish a simple, reliable and stable ginkgo leaf fingerprint determination method for simultaneously characterizing the lactone components, flavonoid components and ginkgolic acid components in ginkgo leaves, and further applies this method to analyze the chemical components in ginkgo leaves, so as to obtain a new method for reliable quality evaluation of ginkgo leaves and their products, and provide a better method for comprehensive evaluation and comprehensive control of the intrinsic quality of ginkgo leaf medicinal materials.

[0008] In order to achieve the above purpose, this application provides the following technical solutions:

[0009] The fingerprint detection of Ginkgo biloba leaves includes the following steps: taking the Ginkgo biloba leaves for testing and the reference substance for testing, and the chromatographic conditions are as follows:

[0010] Chromatographic column: ZORBAX SB-C18 (4.6×250 mm, 5 μm); mobile phase A is 0.1 vol% formic acid water, mobile phase B is methanol;

[0011] Gradient elution program: 0-10min: 90%-80% A by volume; 10-25min: 80%-70% A by volume; 25-35min: 70% A by volume; 35-45min: 70%-58% A by volume; 45-70min: 58%-45% A by volume; 70-85min: 45%-30% A by volume; 85-95min: 30%-5% A by volume; 95-115min: 5% A by volume;

[0012] Evaporative light scattering (ELSD) was used for detection, and the specific parameters were as follows: drift tube temperature was 110-115°C, carrier gas flow rate was 3.0 mL / min, and gain value was 4.

[0013] Furthermore, the chromatographic conditions also include: column temperature 25-35°C, flow rate 0.9-1.1 mL / min, and detection wavelength 360 nm.

[0014] Among them, the reference substance is selected from one or more of ginkgolide A, ginkgolide B, ginkgolide C, bilobalide, 3-O-{2-O-[6-O-(p-hydroxy-trans-coumaryl)-glucosyl]-rhamnosyl} kaempferol, ginkgo biloba flavonoids, golden pine flavonoids, ginkgolic acid C15:1, ginkgolic acid C17:1 and rutin.

[0015] The present application further provides the preparation of reference substance and test substance solution

[0016] The preparation method of the test solution is as follows: Ginkgo leaves and 50 volume%-90 volume% methanol are mixed at a solid-liquid ratio of 1g:100ml to 1g:10ml, and then weighed, ultrasonically extracted for 55 to 65 minutes, centrifuged, and the supernatant is taken and filtered through a filter membrane to obtain the test solution.

[0017] Optionally, ginkgo leaves are mixed with the 90 volume % methanol at a ratio of 0.5 g:5 mL, the weight is determined, ultrasonic extraction is performed for 60 min, the mixture is taken out and allowed to cool to room temperature, and the lost weight is supplemented with 90 volume % methanol, the supernatant is taken after centrifugation, and filtered through a 0.45 μm filter membrane to obtain the product.

[0018] The preparation method of the reference solution is as follows:

[0019] Take an appropriate amount of each reference substance and add anhydrous methanol to prepare a mixed reference substance solution containing each component at a concentration of 100 μg / mL.

[0020] This application further provides a method for constructing a fingerprint of ginkgo leaves, as follows:

[0021] First, prepare the test solution: weigh the dried ginkgo leaf powder, place it in a centrifuge tube, add 90% methanol by volume, weigh it, treat it with ultrasound, take it out and let it cool to room temperature, then add 90% methanol by volume to make up the lost weight, take the supernatant after centrifugation, filter it through a filter membrane, and obtain the test solution;

[0022] Prepare the reference solution again: weigh ginkgolide A, ginkgolide B, ginkgolide C, bilobalide, 3-O-{2-O-[6-O-(p-hydroxy-trans-coumaryl)-glucosyl]-rhamnosyl}kaempferol, ginkgo biloba flavonoids, golden pine biloba flavonoids, ginkgolic acid C15:1, ginkgolic acid C17:1 and rutin reference substances respectively, place them in a volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and prepare a 100 μg / mL reference substance stock solution;

[0023] The following chromatographic conditions were used for separation:

[0024] Chromatographic column: ZORBAX SB-C18 (4.6×250mm, 5μm); mobile phase: 0.1 vol% formic acid water (A): methanol (B), gradient elution program: 0-10min: 90 vol%-80 vol% A; 10-25min: 80 vol%-70 vol% A; 25–35min: 70 vol% A; 35–45min: 70 vol%-58A; 45-70min: 58 vol%-45 vol% A; 70-85min: 45 vol%-30 vol% A; 85-95min: 30 vol%-5 vol% A; 95-115min: 5 vol% A; column temperature was 25-35°C, flow rate was 0.9-1.1 mL / min, injection volume: 10 μL, detection wavelength was 360 nm.

[0025] The detection parameters of the evaporative light scattering detector are:

[0026] Drift tube temperature: 110-115°C; carrier gas flow rate: 3.0 L / min, gain value 4;

[0027] According to the detection results of HPLC-ELSD, the fingerprint of Ginkgo biloba leaves was obtained.

[0028] The construction method also includes methodological investigation:

[0029] Precision investigation: Take the same test solution, perform HPLC-ELSD analysis, repeat the injection 6 times, record the relative retention time and relative peak area of ​​each common peak, calculate its RSD (relative standard deviation) value, and use the Chinese medicine chromatographic fingerprint similarity evaluation software for evaluation;

[0030] Stability study: Prepare the test solution according to the above method, and measure it at 0h, 2h, 4h, 8h, 12h, 16h, and 24h, record the fingerprint spectrum and the relative retention time and relative peak area of ​​each common peak, and calculate its RSD value;

[0031] Repeatability investigation: Six samples of the same batch of ginkgo leaves were processed in parallel according to the test solution preparation method, and the samples were injected and measured according to the above method. The fingerprint spectrum and the relative retention time and relative peak area of ​​each common peak were recorded, and the RSD value was calculated. The Chinese medicine chromatographic fingerprint similarity evaluation software (2012 version) was used for evaluation.

[0032] The present application further provides a method for analyzing the chemical components in Ginkgo biloba leaves, using HPLC-tandem ELSD to analyze the Ginkgo biloba leaves;

[0033] Preparation of the test solution: Weigh the dried Ginkgo leaf powder, place it in a centrifuge tube, add 90% methanol by volume, weigh it, treat it with ultrasound, take it out and let it cool to room temperature, then add 90% methanol by volume to make up the lost weight, take the supernatant after centrifugation, filter it through a filter membrane, and obtain the test solution;

[0034] Preparation of reference solution: Weigh ginkgolide A, ginkgolide B, ginkgolide C, bilobalide, 3-O-{2-O-[6-O-(p-hydroxy-trans-coumaryl)-glucosyl]-rhamnosyl}kaempferol, ginkgo biloba, ginkgol, ginkgolic acid C15:1, ginkgolic acid C17:1 and rutin reference substances respectively, place them in a volumetric flask, add anhydrous methanol to dissolve and dilute to the scale, shake well, and prepare a 100 μg / mL reference substance stock solution;

[0035] Determination: Inject 10 μl of each reference solution and test solution, analyze, and record the chromatogram within 115 minutes;

[0036] Among them, the chromatographic conditions and evaporative light scattering parameters are as follows:

[0037] Chromatographic conditions:

[0038] Chromatographic column: ZORBAX SB-C18 (4.6×250 mm, 5 μm); mobile phase A is 0.1 vol% formic acid water, mobile phase B is methanol;

[0039] The elution program is 0-10min: 90% by volume - 80% by volume A; 10-25min: 80% by volume - 70% by volume A; 25-35min: 70% by volume A; 35-45min: 70% by volume - 58% by volume A; 45-70min: 58% by volume - 45% by volume A; 70-85min: 45% by volume - 30% by volume A; 85-95min: 30% by volume - 5% by volume A; 95-115min: 5% by volume A;

[0040] Column temperature 25-35°C, flow rate 0.9-1.1 mL / min, detection wavelength 360 nm;

[0041] ELSD detection parameters: drift tube temperature 110-115°C, carrier gas flow rate 3.0 mL / min, gain value 4;

[0042] Analyze the chemical components of the Ginkgo leaf test sample based on the test results.

[0043] As mentioned above, the present application establishes a detection method for Ginkgo biloba leaves and constructs its fingerprint spectrum by HPLC-ELSD combination, selects 10 representative chromatographic peaks of Ginkgo biloba lactones, Ginkgo biloba acids, and flavonoids as common peaks to effectively separate them, and realizes that under the same conditions, the Ginkgo biloba lactones, Ginkgo biloba acids, and flavonoids in Ginkgo biloba leaves are simultaneously reflected on a chromatogram, solving the problems of cumbersome, low efficiency, and time-consuming current detection methods. This method can comprehensively reflect the overall characteristics of the chemical composition of Ginkgo biloba medicinal materials, can simply and quickly analyze the components of Ginkgo biloba leaves, can be applied to the authenticity and quality identification of Ginkgo biloba medicinal materials, and provides a strong technical basis for improving the quality control level of Ginkgo biloba leaves and their preparations, and accurately and comprehensively evaluating their intrinsic quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Spectra of different extraction solvents.

[0045] Figure 2 These are the spectra obtained with different extraction solid-liquid ratios.

[0046] Figure 3 These are the spectra examined at different extraction times.

[0047] Figure 4 It is the sum of the peak areas of 10 characteristic peaks at different extraction times.

[0048] Figure 5 The chromatograms of the test solution under different detectors (1. bilobalide, 2. ginkgolide C, 3. ginkgolide A, 4. ginkgolide B, 5. rutin, 6. 3-O-{2-O-[6-O-(p-hydroxy-trans-coumaryl)-glucosyl]-rhamnosyl} kaempferol, 7. ginkgo biloba flavonoids, 8. cyperus rotundus flavonoids, 9. ginkgolic acid C15:1, 10. ginkgolic acid C17:1).

[0049] Figure 6 The chromatograms of the test solution at different drift tube temperatures.

[0050] Figure 7 These are the results of the precision test (1. bilobalide, 2. ginkgolide C, 3. ginkgolide A, 4. ginkgolide B, 5. rutin, 6. 3-O-{2-O-[6-O-(p-hydroxy-trans-coumaryl)-glucosyl]-rhamnosyl} kaempferol, 7. ginkgo biloba flavonoids, 8. scutellaria baicalensis flavonoids, 9. ginkgolic acid C15:1, 10. ginkgolic acid C17:1).

[0051] Figure 8 These are the results of the stability test (1. bilobalide, 2. ginkgolide C, 3. ginkgolide A, 4. ginkgolide B, 5. rutin, 6. 3-O-{2-O-[6-O-(p-hydroxy-trans-coumaryl)-glucosyl]-rhamnosyl} kaempferol, 7. ginkgo biloba flavonoids, 8. ginkgolide flavonoids, 9. ginkgolic acid C15:1, 10. ginkgolic acid C17:1).

[0052] Fig. 9 These are the results of a repeatability test (1. bilobalide, 2. ginkgolide C, 3. ginkgolide A, 4. ginkgolide B, 5. rutin, 6. 3-O-{2-O-[6-O-(p-hydroxy-trans-coumaryl)-glucosyl]-rhamnosyl} kaempferol, 7. ginkgo biloba flavonoids, 8. cinnamomea flavonoids, 9. ginkgolic acid C15:1, 10. ginkgolic acid C17:1).

[0053] Fig.10 This is the fingerprint of the reference substance of Ginkgo biloba leaves (1. bilobalide, 2. ginkgolide C, 3. ginkgolide A, 4. ginkgolide B, 5. rutin, 6. 3-O-{2-O-[6-O-(p-hydroxy-trans-coumaryl)-glucosyl]-rhamnosyl} kaempferol, 7. ginkgo biloba flavonoids, 8. cyperus rotundus flavonoids, 9. ginkgolic acid C15:1, 10. ginkgolic acid C17:1).

[0054] Fig.11 These are the fingerprints of 35 batches of ginkgo leaf samples. DETAILED DESCRIPTION

[0055] Unless otherwise specified, the drugs, reagents, and instruments used in the technical solutions provided in this application can be purchased from conventional channels or the market. This application discloses a method for determining the fingerprint spectrum of Ginkgo biloba leaves. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application.

[0056] The present application is further described in detail below in conjunction with specific examples. It should be understood that these examples are used to illustrate the basic principles, main features and advantages of the present application, and the present application is not limited by the scope of the following examples. The implementation conditions adopted in the examples can be further adjusted according to specific requirements, and the unspecified implementation conditions are generally the conditions in routine experiments.

[0057] High performance liquid chromatography (HPLC) is used to analyze organic compounds with high boiling points, low volatility, thermal instability and large molecular weight. The instrument consists of a reservoir, a pump, an injector, a chromatographic column, a detector, a recorder and other parts. The mobile phase in the reservoir is pumped into the system by a high-pressure pump, and the sample solution enters the mobile phase through the injector and is carried into the chromatographic column (stationary phase) by the mobile phase. Since the components in the sample solution have different distribution coefficients in the two phases, when they move relative to each other in the two phases, after repeated adsorption-desorption distribution processes, the components have large differences in movement speed and are separated into individual components that flow out of the column in turn. When passing through the detector, the sample concentration is converted into an electrical signal and transmitted to the recorder, and the data is analyzed by software. HPLC has the advantages of high resolution, high sensitivity, fast speed, reusable chromatographic columns, and easy collection of effluent components.

[0058] Due to the low ultraviolet absorption characteristics of ginkgolide, it is easily interfered by flavonoids. The detection effect of using ultraviolet detector is not good or it must be derivatized before detection. The evaporative light detector ELSD does not rely on the optical properties of the sample. As long as the volatility is less than the substance of the mobile phase, it can produce a response on the ELSD. Secondly, ELSD can support gradient elution well, and ELSD can eliminate the impact of changes in mobile phase ratio on the baseline. Therefore, this application uses HPLC-ELSD to detect Ginkgo biloba.

[0059] This application consists of the following experiments:

[0060] 1. Preparation of reference solution

[0061] 2. Preparation of test solution, including: optimization of extraction solvent, solid-liquid ratio and extraction time 3. Optimization of detection conditions, including: selection of chromatographic conditions and optimization of evaporative light scattering temperature 4. Methodological validation, including: precision test, stability test, repeatability test

[0062] 5. Construction and specific application of fingerprint detection method

[0063] 6. Other instructions

[0064] The following is the specific content of each part of the experiment

[0065] 1. Preparation of reference solution

[0066] (1) Selection of reference substances: Based on the known components in Ginkgo biloba leaves, one or more of ginkgolide A, ginkgolide B, ginkgolide C, bilobalide, 3-O-{2-O-[6-O-(p-hydroxy-trans-coumaryl)-glucosyl]-rhamnosyl}kaempferol, ginkgo biloba flavonoids, golden pine biloba flavonoids, ginkgolic acid C15:1, ginkgolic acid C17:1, and rutin were selected to represent lactones, flavonoids, and phenolic acids, respectively.

[0067] (2) Preparation of reference solution: 2 mg of each of ginkgolide A, ginkgolide B, ginkgolide C, bilobalide, 3-O-{2-O-[6-O-(p-hydroxy-trans-coumaryl)-glucosyl]-rhamnosyl}kaempferol, ginkgo biloba flavonoids, golden pine biloba flavonoids, ginkgolic acid C15:1, ginkgolic acid C17:1 and rutin reference substances were weighed using an electronic balance with an accuracy of 1 part per 100,000 (Mettler XS205, Mettler-Toledo Shanghai Co., Ltd.) and placed in a 20 mL volumetric flask. Anhydrous methanol was added to dissolve the solution and diluted to the mark. The solution was shaken to prepare a 100 μg / mL reference solution and stored in a refrigerator at 4°C for later use.

[0068] Among them, ginkgolide A (batch number: 110862-201612), ginkgolide B (batch number: 110863-201611), ginkgolide C (110864-201508), bilobalide (110865-201507), ginkgolic acid C15:1 (111762-200601) and rutin (100080-201610) were produced by the National Institute for the Control of Pharmaceutical and Biological Products; ginkgolic acid C17:1 (Y23M9H62018) (Y04J9H64929) was produced by Shanghai Yuanye Biotechnology Co., Ltd.; 3-O-{2-O-[6-O-(p-hydroxy-trans-coumaryl)-glucosyl]-rhamnosyl} kaempferol, ginkgo biloba flavonoids and golden pine flavonoids were produced by Shanghai Yuanye Biotechnology Co., Ltd.

[0069] 2. Preparation of test solution

[0070] The sampling time of the ginkgo leaves for testing was from April to September 2018 in Pizhou, Jiangsu Province. Wu Zhou, a practicing pharmacist of Jiangsu Kangyuan Pharmaceutical Co., Ltd., identified them as dried leaves of Ginkgo biloba L., a plant of the Ginkgoaceae family.

[0071] Optimization method of extraction solvent concentration and solid-liquid ratio: accurately weigh the dried ginkgo leaf powder, use different concentrations of methanol as the extraction solvent, fix the solid-liquid ratio, ultrasonically treat for 60 minutes, evaluate the HPLC peak response value and characteristic peak area, and compare the results to obtain the appropriate methanol concentration.

[0072] Then, the same concentration of methanol was used as the extraction solvent and ultrasonic extraction was performed for 60 minutes at different solid-liquid ratios. The HPLC peak response value and the characteristic peak area were evaluated, and the appropriate solid-liquid ratio was obtained by comparing the results.

[0073] Optimization of ultrasonic extraction time: After determining the methanol concentration and the solid-liquid ratio, different ultrasonic times were set, the HPLC peak response value and the characteristic peak area were evaluated, and the appropriate ultrasonic time was obtained by comparing the results.

[0074] According to the above extraction method, the extraction efficiency of 90 volume % methanol, 75 volume % methanol, 60 volume % methanol, and 50 volume % methanol solvents was investigated, among which 90 volume % methanol had the best extraction effect (see Figure 1 ).

[0075] Then, 90 vol% methanol was used as the extraction solvent to investigate the extraction effects at solid-liquid ratios (g:mL) of 1:10, 1:20, 1:50, and 1:100. The results showed that the best extraction effect was obtained when the solid-liquid ratio was 1:10 (see Figure 2 ).

[0076] Furthermore, 90% methanol by volume was used as the extraction solvent, and the solid-liquid ratio (g:mL) was 1:10. The extraction effects at extraction times of 30 min, 45 min, 60 min, and 75 min were investigated. The results showed that the components could be completely extracted after 60 min of extraction (see Figure 3 and Figure 4 ). Therefore, 90 volume % methanol was used as the extraction solvent, the solid-liquid ratio (g:mL) was 1:10, and ultrasonic extraction was performed for 60 min to prepare the test solution.

[0077] According to the above extraction condition optimization results, the ginkgo leaf samples were dried with hot air at 60°C to constant weight in a DHG-9023A electric constant temperature blast drying oven (Shanghai Jinghong Instrument Equipment Co., Ltd.), and then crushed to obtain dry ginkgo leaf powder. Use an electronic analytical balance with a precision of 1 part per thousand (ME 204E, Mettler-Toledo Shanghai Instrument Co., Ltd.) to accurately weigh 0.5 g of dried Ginkgo leaf powder, place it in a 50 mL centrifuge tube, add 5 mL of 90 volume % methanol (prepared by diluting anhydrous methanol with ultrapure water produced by Pacific TⅡ7 ultrapure water instrument (Thermo Fisher Scientific, USA), weigh the weight, and use an ultrasonic cleaner (KH-300DB, Kunshan Hechuang Ultrasonic Instrument Co., Ltd.) to ultrasonically treat for 60 min (power 300 W, temperature 25°C) to fully dissolve the components, take out and cool to room temperature, and then use 90 volume % methanol to make up the lost weight, use an Eppendorf Centrifuge5424R centrifuge (Eppendorf, Germany) to centrifuge to remove the precipitate and collect the supernatant, filter through a 0.45 μm filter membrane, and collect the filtrate to obtain the product.

[0078] 3. Optimization of Chromatographic Conditions

[0079] The maximum absorption wavelength of the terpene lactone components of Ginkgo biloba is 220nm, and the ultraviolet absorption ability is weak, and the sensitivity is low under the UV detector. Therefore, ELSD was used in this study to detect the terpene lactone components (see Figure 5 The chromatographic column used in the experiment was ZORBAXSB-C18, with specifications of 4.6×250 mm, 5 μm, and the mobile phase was 0.1 vol% formic acid water (A)-methanol (B) gradient elution. The elution program is shown in Table 1.

[0080] Table 1 Gradient elution program of fingerprint detection method

[0081]

[0082] The flow rate is 1.0 mL min -1 ; The detection wavelength was 360 nm and the column temperature was 30 °C.

[0083] 4. Optimization of ELSD drift tube temperature

[0084] ELSD drift tube temperature selection:

[0085] (1) The ELSD detector used in the present application can adopt two modes of split injection and splitless injection. Different split modes can adopt different drift tube temperatures to adapt to the boiling point of the solvent.

[0086] (2) When the temperature decreases, the larger the particles of the volatile components, the higher the ELSD response value. However, when the temperature is too low, part of the mobile phase will not evaporate, which will cause the detection noise to increase and reduce the signal-to-noise ratio.

[0087] (3) The workstation was used to simulate the acquisition effect of 110°C, splitless injection and drift tube temperature of 65°C. The results showed that when the ELSD detector was in splitless mode and the drift tube temperature was 110°C, the signal-to-noise ratio of the ginkgolide components was the largest. Therefore, the drift tube temperature of 110°C and splitless injection were selected (see Figure 6 ).

[0088] According to the above test conditions optimization results, the following tests are performed:

[0089] Each sample was prepared in parallel in duplicate and injected and analyzed twice. 10ul of the test and reference solutions were injected into the automatic sampler. When the drift tube temperature was 110°C, the carrier gas flow rate was 3.0mL / min, and the gain value was 4, the detection was carried out to generate a response signal, and the chromatogram within 115 minutes was recorded. The relative retention time and RSD value of the relative peak area of ​​common peaks 1 to 10 were calculated, and the similarity of each chromatographic fingerprint was calculated by the traditional Chinese medicine fingerprint similarity evaluation software.

[0090] The test solution was tested according to the above method, and the component information of the test sample was obtained by comparing the chromatographic peak characteristics with those of the reference sample.

[0091] 5. Methodological Validation

[0092] (1) Precision test

[0093] Take the same test solution and perform HPLC-ELSD analysis. Repeat the injection 6 times in a row. Take the No. 3 peak Ginkgolide A as the reference peak and calculate the relative retention time and relative peak area RSD value of the common peaks 1 to 10 (see Figure 7 ), calculated the RSD (relative standard deviation) value, and evaluated it using the Chinese medicine chromatographic fingerprint similarity evaluation software. The results showed that the RSD of the relative retention time of each common peak was <0.2%, the RSD of the relative peak area was <5.0%, and the chromatogram similarity was >0.99 (see Tables 2-1, 2-2 and 2-3), indicating that the instrument had good precision.

[0094] Table 2-1 Precision test results

[0095]

[0096] Table 2-2 Precision-Relative Retention Time

[0097]

[0098] Table 2-3 Precision-Relative Peak Area

[0099]

[0100]

[0101] (2) Repeatability test

[0102] Six samples of the same batch of ginkgo leaf were processed in parallel according to the preparation method of the test solution, and the samples were injected and measured according to the above method. Peak 3 was used as the reference peak, and the relative retention time and relative peak area of ​​each common peak were calculated (see Figure 8 ), calculated its RSD value, and evaluated it using the Chinese medicine chromatographic fingerprint similarity evaluation software (2012 version). The results showed that the relative retention time RSD of each common peak was <0.2%, the relative peak area RSD was <5.0%, and the chromatogram similarity was >0.99 (see Tables 3-1, 3-2 and 3-3), indicating that the method had good repeatability.

[0103] Table 3-1 Repeatability test results

[0104]

[0105] Table 3-2 Repeatability-Relative Retention Time

[0106]

[0107] Table 3-3 Repeatability-Relative Peak Area

[0108]

[0109] (3) Stability test

[0110] Take the same test solution for HPLC-ELSD analysis, measure at 0, 2, 4, 8, 12, 16, and 24 hours, record the fingerprint spectrum, take peak 3 as the reference peak, calculate the relative retention time and relative peak area of ​​each common peak (see Fig. 9 ), and calculate its RSD value. The results show that the relative retention time RSD of each common peak is <0.4%, the relative peak area RSD is <4.5%, and the chromatogram similarity is >0.99 (see Tables 4-1, 4-2 and 4-3), indicating that the test solution is stable within 24 hours.

[0111] Table 4-1 Stability-similarity evaluation results

[0112]

[0113] Table 4-2 Stability-Relative Retention Time

[0114]

[0115]

[0116] Table 4-3 Stability-Relative Peak Area

[0117]

[0118] 6. Construction of Ginkgo Leaf Fingerprint

[0119] Ginkgo biloba leaves collected in Pizhou in July and August with an age of two to four years were used as samples. The samples were tested according to the above detection method, and the fingerprint of the ginkgo leaves was obtained (see Fig.10 ).

[0120] 7. Application of fingerprint detection method

[0121] The fingerprint of ginkgo leaves obtained by the above method was used as the reference fingerprint to detect the similarity of 35 batches of ginkgo leaf samples. The results showed that the similarity between the fingerprint of the test sample and the reference fingerprint was not less than 0.9 (see Fig.11 ).

[0122] The results showed that the relative retention times of fingerprint peaks in the fingerprints of 35 batches of Ginkgo biloba leaves from different sources were basically consistent, and the similarity between the fingerprints of each sample and the control fingerprint was greater than 0.9, indicating that the composition of the chemical components contained in the samples was basically the same.

[0123] 8. Other instructions

[0124] This application establishes a fingerprint map of ginkgo leaves, which can comprehensively reflect the information of the three main chemical components in ginkgo leaves, fully display the chemical composition information of ginkgo leaf medicinal materials, and use similarity evaluation software to conduct overall quality evaluation of ginkgo leaf medicinal materials, which is conducive to the quality control of multi-component and multi-target medicinal plants.

[0125] The above is only a preferred implementation of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application.

Claims

1. A method for detecting fingerprints of ginkgo leaves, characterized in that: The method comprises the following steps: taking a reference substance and a test substance solution for detection, and the chromatographic conditions for the detection are: Chromatographic column: ZORBAX SB-C18, specification: 4.6×250 mm, 5 μm; mobile phase A is 0.1 wt. % formic acid water, mobile phase B is methanol; Gradient elution program: 0-10min: 90%-80% A; 10-25min: 80%-70% A; 25-35min: 70% A; 35-45min: 70%-58% A; 45-70min: 58%-45% A; 70-85min: 45%-30% A; 85-95min: 30%-5% A; 95-115min: 5% A; According to the test results, obtaining the component information of the ginkgo leaf; Among them, the component information of the ginkgo leaf obtained is: ginkgolide A, ginkgolide B, ginkgolide C, bilobalide, 3-O-{2-O-[6-O-(p-hydroxy-trans-coumaryl)-glucosyl]-rhamnosyl} kaempferol, ginkgo biloba flavonoids, golden pine flavonoids, ginkgolic acid C15:1, ginkgolic acid C17:1 and rutin.

2. The detection method according to claim 1, characterized in that: The column temperature was 25-35°C and the flow rate was 0.9-1.1 mL / min.

3. The detection method according to claim 1, characterized in that: The reference substance is selected from one or more of ginkgolide A, ginkgolide B, ginkgolide C, bilobalide, 3-O-{2-O-[6-O-(p-hydroxy-trans-coumaryl)-glucosyl]-rhamnosyl}kaempferol, ginkgo biloba flavonoids, golden pine flavonoids, ginkgolic acid C15:1, ginkgolic acid C17:1 and rutin.

4. The detection method according to claim 1, characterized in that: The preparation method of the test solution is as follows: The ginkgo leaf is mixed with 50 volume % to 90 volume % methanol at a solid-liquid ratio of 1 g:100 mL to 1 g:10 mL, and then weighed, ultrasonically extracted for 30 to 75 minutes, centrifuged, and the supernatant is collected and filtered through a filter membrane to obtain the product.

5. The detection method according to claim 4, characterized in that: The ginkgo leaf is mixed with 90 volume % methanol at a ratio of 0.5 g:5 mL, weighed, ultrasonically extracted for 60 min, taken out and allowed to cool to room temperature, and then supplemented with 90 volume % methanol to make up for the lost weight, centrifuged and collected the supernatant, and filtered through a 0.45 μm filter membrane to obtain the product.

6. The detection method according to claim 3, characterized in that: The preparation method of the reference solution includes: Take each of the reference substances and add methanol to prepare a mixed reference substance solution containing each component at a concentration of 80-120 μg / mL.

7. The detection method according to claim 1, characterized in that: The detector is an evaporative light scattering detector (ELSD), and the detection parameters are: drift tube temperature is 110-115°C; carrier gas flow rate: 3 mL / min, gain value 4.

8. A method for analyzing the chemical composition of a ginkgo leaf product, characterized in that: Ginkgo biloba leaves were analyzed using HPLC-tandem ELSD; Preparation of the test solution: Weigh the ginkgo leaf powder, place it in a centrifuge tube, add 90% by volume methanol, weigh it, treat it with ultrasound, take it out and let it cool to room temperature, then add 90% by volume methanol to make up the lost weight, take the supernatant after centrifugation, filter it through a filter membrane, and obtain the test solution; Preparation of reference solution: weigh ginkgolide A, ginkgolide B, ginkgolide C, bilobalide, 3-O-{2-O-[6-O-(p-hydroxy-trans-coumaryl)-glucosyl]-rhamnosyl}kaempferol, ginkgo biloba flavonoids, golden pine biloba flavonoids, ginkgolic acid C15:1, ginkgolic acid C17:1 and rutin reference substances respectively, place them in a volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and prepare reference substance stock solution; Determination: Pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, measure and record the chromatogram within 115 minutes; The chromatographic conditions for the determination are: Chromatographic column: ZORBAX SB-C18, specification: 4.6×250 mm, 5 μm; mobile phase A is 0.1 volume % formic acid water, mobile phase B is methanol; Gradient elution program: 0-10min: 90vol%-80vol%A; 10-25min: 80vol%-70vol%A; 25-35min: 70vol%A; 35-45min: 70vol%-58A; 45-70min: 58vol%-45vol%A; 70-85min: 45vol%-30vol%A; 85-95min: 30vol%-5vol%A; 95-115min: 5vol%A; The ELSD mass spectrometry conditions were: drift tube temperature of 110°C, carrier gas flow rate of 3.0 mL / min, and gain value of 4. The chemical components of the Ginkgo biloba sample were analyzed based on the test results; The chemical components are: ginkgolide A, ginkgolide B, ginkgolide C, bilobalide, 3-O-{2-O-[6-O-(p-hydroxy-trans-coumaryl)-glucose]-rhamnosyl} kaempferol, ginkgo biloba flavonoids, golden pine flavonoids, ginkgolic acid C15:1, ginkgolic acid C17:1 and rutin.

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