Ginkgo biloba extract total ginkgolic acid determination method based on ethanol-chloroform synergistic extraction and polarity regulation

Abstract

The invention relates to a method for determining the content of total ginkgolic acid in a ginkgo biloba extract, which comprises the following steps: dissolving an extract sample in 50% ethanol, and extracting with chloroform. Adding a proper amount of ethanol into the water layer, and extracting with chloroform; and combining all extraction liquids, concentrating to be dry, dissolving residues by using methanol, and detecting by using a liquid chromatograph. By investigating a linear range test, a precision test, a stability test, a repeatability test and a standard recovery test of the method, the method can remarkably improve the purity and the detection sensitivity of a target object, reduce the matrix effect and solve the problem of impurity co-extraction of a traditional single solvent, so that the content detection result of ginkgolic acid is more accurate, and the method is suitable for industrial production. The method plays an important role in guaranteeing the safety of the ginkgo biloba extract product.

Description

Technical Field

[0001] The invention belongs to the field of detection technology, and particularly relates to a method for determining total ginkgolic acid in ginkgo leaf extract based on ethanol-chloroform synergistic extraction and polarity regulation. Background Art

[0002] Ginkgo biloba extract is an extract made from the dried leaves of the Ginkgo biloba L. plant of the Ginkgoaceae family. Its preparation has the function of promoting blood circulation, removing blood stasis and unblocking the meridians. It is commonly used to treat chest pain, heart pain, stroke, hemiplegia, stable angina pectoris of coronary heart disease, cerebral infarction and other symptoms caused by blood stasis blocking the meridians. Ginkgolic acid is a class of alkyl acids or alkylphenol compounds, mainly found in ginkgo leaves, fruits and outer seed coats, and is also present in ginkgo biloba extract at a high level. As a toxic component in ginkgo biloba extract, ginkgolic acid has sensitization, mutagenicity and strong cytotoxicity, which can cause allergic reactions, gene mutations, and nerve damage, leading to adverse reactions such as nausea, heartburn, anaphylactic shock, allergic purpura, convulsions, and paralysis. It has potential harm to human health. Countries around the world have set ginkgolic acid content as an important control indicator for ginkgo biloba extract. The United States, the European Union and the Chinese Pharmacopoeia all regulate ginkgolic acid content to no more than 5.0 ppm. Therefore, accurately measuring the content of ginkgolic acid is of great significance to ensuring the safety of ginkgo biloba extract.

[0003] There are many methods for detecting ginkgo bilobalic acid. Thin-layer chromatography (TLC) is easy to operate, requires simple equipment, has a fast separation speed, and is inexpensive. However, this method has large errors, limited precision, and poor reproducibility, making it only suitable for qualitative detection. Liquid chromatography-mass spectrometry is highly efficient and sensitive, but the instrument is relatively expensive, making it difficult to popularize. Currently, the most common method for determining ginkgo bilobalic acid is high-performance liquid chromatography (HPLC), which has high resolution, high sensitivity, and good reproducibility, making it more suitable for detecting low-content, multi-component substances.

[0004] Ginkgo biloba extract is a key quality control indicator. Due to its low content, accurate determination of its content requires efficient pretreatment technology to remove interferences. The current mainstream pretreatment methods have the following problems: (1) Poor selectivity of single solvent extraction: For example, the Chinese Pharmacopoeia, the European Pharmacopoeia, and the United States Pharmacopoeia all use only methanol as a single solvent to dissolve the extract sample. CN201010117078B discloses that methanol as a high-polarity solvent is easy to co-extract polysaccharides and flavonoids impurities, resulting in baseline drift in subsequent chromatographic analysis. (2) Insufficient liquid-liquid extraction efficiency: CN112684087A uses a methanol-petroleum ether system and does not combine polarity control technology. Ginkgo biloba acid remains seriously in the aqueous phase and the recovery rate is low. (3) Matrix interference and sensitivity limitation: Fat-soluble impurities (such as chlorophyll) in ginkgo biloba extract are difficult to remove by conventional filtration, which easily contaminates the chromatographic column and reduces the detection sensitivity. Some methods rely on LC-MS equipment, which improves sensitivity but is expensive and difficult to popularize. Therefore, it is of great significance to establish a more scientific and accurate pretreatment method. Summary of the Invention

[0005] The purpose of the present invention is to propose a more efficient and accurate method for determining the total ginkgolic acid content in ginkgo biloba extract, which effectively improves the purity and detection sensitivity of the target and reduces the matrix effect through ethanol-chloroform synergistic extraction and polarity control.

[0006] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0007] A method for determining total ginkgolic acid in ginkgo biloba extract based on ethanol-chloroform synergistic extraction and polarity control comprises the following steps:

[0008] (1) Preparation of test solution: Accurately weigh 2 g of Ginkgo biloba extract sample, dissolve it in 100 mL of 50% ethanol, extract with 100 mL of chloroform, add 20 mL of ethanol to the aqueous layer, and extract with 100 mL of chloroform. Combine all extracts, concentrate to dryness, dissolve the residue in 10 mL of methanol, filter, and take the filtrate to obtain the product;

[0009] (2) Preparation of reference solution: Take an appropriate amount of ginkgo biloba acid reference substance, accurately weigh it, and add methanol to make a solution containing 1 μg per 1 ml, which is used as the reference solution; take an appropriate amount of total ginkgo biloba acid reference substance, and use methanol to make a solution containing 20 μg per 1 ml, which is used as the positioning reference solution;

[0010] (3) Content determination: Accurately aspirate 50 μl of each of the test solution, reference solution and positioning reference solution, inject them into the liquid chromatograph, calculate the total peak area of ​​the chromatographic peaks corresponding to the total ginkgo acid reference in the test solution, and calculate the total ginkgo acid content using the external standard method of the ginkgo acid reference.

[0011] Preferably, the chromatographic column of the liquid chromatograph in step (3) is filled with octadecylsilane bonded silica gel, has a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; gradient elution is performed using acetonitrile containing 0.1% trifluoroacetic acid as mobile phase A and water containing 0.1% trifluoroacetic acid as mobile phase B; the detection wavelength is 310 nm. The number of theoretical plates calculated based on the ginkgo biloba acid peak should be no less than 4000.

[0012] Preferably, the gradient elution is specifically:

[0013] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～30 75→90 25→10 30～35 90 10 35～36 90→75 10→25 36～45 75 25

[0014] Compared with the prior art, the technical solution of the present invention has at least the following beneficial effects:

[0015] (1) Mixed solvent dissolution and preliminary purification: Compared with the traditional methanol solvent, the use of 50% ethanol as the sample solvent can not only disperse the sample but also inhibit the dissolution of highly polar impurities such as polysaccharides.

[0016] (2) Ethanol-chloroform synergistic extraction and polarity control: For the first time, 50% ethanol dissolution combined with chloroform step-by-step extraction was used. By dynamically adjusting the solvent polarity (adding ethanol to the aqueous phase), the total ginkgo acid distribution coefficient was optimized and the directional separation of impurities was achieved (for example, polysaccharides were retained in the aqueous phase, and chlorophyll was transferred to the chloroform phase and removed in the concentration step).

[0017] (3) Concentration, enrichment and detection adaptation: After concentration to dryness, methanol is used for re-dissolution, which increases the concentration of the target compound by 10 times and is suitable for conventional HPLC detection. At the same time, fat-soluble interfering substances can be removed simultaneously, and the impurity peaks in the chromatogram are reduced by 80%, with low baseline noise. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a sample liquid chromatogram of the present invention;

[0019] Figure 2 This is the sample liquid chromatogram of the existing method. DETAILED DESCRIPTION

[0020] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] Equipment and materials

[0022] (1) Instruments and equipment

[0023] 1200 liquid chromatograph (Agilent Technologies), XS105 electronic balance (Mettler-Toledo), SK8200LHC ultrasonic extractor (Shanghai Kedao Ultrasonic Instrument Co., Ltd.), HH-4 digital display constant temperature water bath (Changzhou Guohua Electric Co., Ltd.).

[0024] (2) Reagents and materials

[0025] Ginkgo biloba extract (production batch number: C0253250108), ginkgolic acid (China Food and Drug Inspection Institute, batch number: 111690-201604, content: 99.3%), total ginkgolic acid A (China Food and Drug Inspection Institute, batch number: 111594-201605, for limit inspection and positioning purposes), total ginkgolic acid B (China Food and Drug Inspection Institute, batch number: 111594-201605, for limit inspection and positioning purposes). Methanol: analytical grade, chromatographic grade; trifluoroacetic acid: chromatographic grade; acetonitrile: chromatographic grade; ultrapure water.

[0026] The following examples adopt the method for determining total ginkgolic acid in ginkgo biloba extract based on ethanol-chloroform synergistic extraction and polarity control of the present invention, comprising the following steps:

[0027] (1) Preparation of test solution: Accurately weigh 2 g of Ginkgo biloba extract sample, dissolve it in 100 mL of 50% ethanol, extract with 100 mL of chloroform, add 20 mL of ethanol to the aqueous layer, and extract with 100 mL of chloroform. Combine all extracts, concentrate to dryness, dissolve the residue in 10 mL of methanol, filter, and take the filtrate to obtain the product;

[0028] (2) Preparation of reference solution: Take an appropriate amount of ginkgo biloba acid reference substance, accurately weigh it, and add methanol to make a solution containing 1 μg per 1 ml, which is used as the reference solution; take an appropriate amount of total ginkgo biloba acid reference substance, and use methanol to make a solution containing 20 μg per 1 ml, which is used as the positioning reference solution;

[0029] (3) Content determination: Accurately pipette 50 μl of each of the test solution, reference solution, and positioning reference solution, inject them into a liquid chromatograph, calculate the total peak area of ​​the chromatographic peaks corresponding to the total ginkgolic acid reference in the test solution, and calculate the total ginkgolic acid content using the external standard method of the ginkgo biloba reference. The chromatographic column of the liquid chromatograph is filled with octadecylsilane bonded silica gel, with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm. Gradient elution is performed using acetonitrile containing 0.1% trifluoroacetic acid as mobile phase A and water containing 0.1% trifluoroacetic acid as mobile phase B. The detection wavelength is 310 nm. The number of theoretical plates calculated based on the ginkgo biloba peak should be no less than 4000.

[0030] The gradient elution is specifically:

[0031] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～30 75→90 25→10 30～35 90 10 35～36 90→75 10→25 36～45 75 25

[0032] Example 1

[0033] Accurately weigh 2.06 mg of the ginkgo biloba acid reference substance into a 50 mL volumetric flask, add methanol and ultrasonically dissolve it and dilute to the scale. Accurately measure an appropriate amount and dilute it 5 times, 25 times, 100 times, 200 times and 500 times respectively. Chromatographic conditions are used to analyze the peak area of ​​the ginkgo biloba acid reference substance at each concentration. The standard curve is established with the concentration C of the ginkgo biloba acid reference substance solution as the horizontal axis and the peak area A of each ginkgo biloba acid reference substance solution as the vertical axis. The concentrations and peak areas of the ginkgo biloba acid standard curve series solutions are as follows:

[0034] Table 1 Linear equation, correlation coefficient and linear range of ginkgo biloba acid

[0035]

[0036] The results show that ginkgo biloba acid has a good linear relationship in the concentration range of 0.0818 to 40.9116 mg / L, its linear equation is Y=6030X-221, and the correlation coefficient R is 1.0000.

[0037] Example 2

[0038] A test solution was prepared according to the test solution preparation method. Six consecutive injections were performed under the above chromatographic conditions. The peak areas for each analysis were obtained, and the relative standard deviation (RSD) values ​​for the peak areas were calculated. The RSD for the precision experiment was calculated to be 0.92%. Relevant data for the precision experiment are shown in Table 2.

[0039] Table 2 Experimental data of total ginkgo acid precision

[0040] name 1 2 3 4 5 6 RSD (%) Peak area 2072 2060 2058 2081 2044 2098 0.92

[0041] Example 3

[0042] Prepare a test solution according to the test solution preparation method. Under the above chromatographic conditions, inject the sample and analyze it at 0, 1, 2, 4, 8, and 12 hours. Obtain the chromatographic peak area for each analysis and calculate the relative standard deviation (RSD) of the peak area. The calculated RSD for the stability study was 0.68%. Relevant data for the stability study are shown in Table 3.

[0043] Table 3 Total Ginkgo Bilobalic Acid Stability Experimental Data

[0044] Time (h) 0 1 2 4 8 12 RSD (%) Peak area 2072 2060 2058 2044 2080 2079 0.68

[0045] It can be seen from the experimental data in the table that the test solution has good stability when placed at room temperature for 12 hours.

[0046] Example 4

[0047] Six test solutions were prepared according to the test solution preparation method. Chromatographic analysis was performed under the above-described chromatographic conditions. The established standard curve was used to calculate the content of ginkgolic acid in the test solutions. The total ginkgolic acid content in the ginkgo biloba extract samples was then calculated, and the RSD value for repeatability was calculated. The calculated average total ginkgolic acid content in the ginkgo biloba extract samples was 1.94 ppm, and the RSD value for repeatability was 1.07%. Relevant data for the repeatability experiment are shown in Table 4.

[0048] Table 4 Total Ginkgo Bilobalic Acid Repeatability Experimental Data

[0049] name 1 2 3 4 5 6 RSD (%) Peak area 2072 2120 2116 2137 2141 2116 Sample weight (g) 2.0003 2.0086 2.0007 2.0010 2.0009 2.0042 Concentration (mg / L) 0.3802 0.3883 0.3876 0.3911 0.3917 0.3876 Content (ppm) 1.90 1.93 1.94 1.95 1.96 1.93 1.07

[0050] Example 5

[0051] Six 1g portions of Ginkgo biloba extract were weighed accurately in stoppered Erlenmeyer flasks. 4.74 μL of a 40.9116 mg / L ginkgolic acid reference solution was added to each sample. Six test solutions were prepared according to the test solution preparation method. Chromatographic analysis was performed under the above-described chromatographic conditions to determine the total ginkgolic acid content in the test solutions. The total ginkgolic acid recovery of the samples was calculated based on the amount of reference solution added and the total ginkgolic acid content measured in the repeatability test. The RSD value was also calculated. Data from the spike recovery experiments are shown in Table 5.

[0052] Table 5 Total Ginkgo acid spike recovery experimental data

[0053]

[0054] The experimental results showed that the method for determining the content of total ginkgolic acid in Ginkgo biloba extract was accurate and reliable, with an average recovery rate of 100.70% and an RSD value of 1.89%.

[0055] Example 6

[0056] According to the test solution preparation method and chromatographic conditions of the present invention (new method) and the "total ginkgolic acid" content determination method under the ginkgo leaf extract in the 2020 edition of the Chinese Pharmacopoeia (current method), the total ginkgolic acid content in the ginkgo leaf extract (production batch number: C0253250108) sample was detected. The results are shown in Table 6 and the liquid chromatogram is shown in Figure 1 and Figure 2 .

[0057] Table 6 Comparison of total ginkgo acid content detection results of two methods

[0058] Sample name Production batch number New method (ppm) Current method (ppm) Ginkgo biloba extract C0253250108 1.94 1.67

[0059] Compared with the current method, the new method is more sensitive. A comparison of the chromatograms shows that the impurity peak area in the current method's chromatogram is larger, resulting in a relatively smaller total ginkgo acid chromatographic peak. Its content calculation often requires strict automatic integration parameters or manual integration. The new method's chromatogram has significantly reduced the impurity peak area, resulting in a more distinct total ginkgo acid chromatographic peak. This indicates that the new method's sample pretreatment makes the test solution cleaner, reduces the interference of impurity peaks on the test results, and makes the test results more accurate.

Claims

1. A method for determining total ginkgolic acid in ginkgo biloba extract based on ethanol-chloroform synergistic extraction and polarity control, characterized by: The following steps are involved: (1) Preparation of test solution: Accurately weigh 2 g of Ginkgo biloba extract sample, dissolve it in 100 mL of 50% ethanol, extract with 100 mL of chloroform, add 20 mL of ethanol to the aqueous layer, and extract with 100 mL of chloroform. Combine all extracts, concentrate to dryness, dissolve the residue in 10 mL of methanol, filter, and take the filtrate to obtain the product; (2) Preparation of reference solution: Take an appropriate amount of ginkgo biloba acid reference substance, accurately weigh it, and add methanol to make a solution containing 1 μg per 1 ml, which is used as the reference solution; take an appropriate amount of total ginkgo biloba acid reference substance, and use methanol to make a solution containing 20 μg per 1 ml, which is used as the positioning reference solution; (3) Content determination: Accurately aspirate 50 μl of each of the test solution, reference solution and positioning reference solution, inject them into the liquid chromatograph, calculate the total peak area of ​​the chromatographic peaks corresponding to the total ginkgo acid reference in the test solution, and calculate the total ginkgo acid content using the external standard method of the ginkgo acid reference.

2. The method according to claim 1, characterized in that The chromatographic column of the liquid chromatograph in step (3) is filled with octadecylsilane bonded silica gel, acetonitrile containing 0.1% trifluoroacetic acid is used as mobile phase A, and water containing 0.1% trifluoroacetic acid is used as mobile phase B for gradient elution; the detection wavelength is 310 nm.

3. The method according to claim 2, characterized in that The chromatographic column has a column length of 250 mm, a column inner diameter of 4.6 mm, and a particle size of 5 μm.

4. The method according to claim 2, characterized in that The theoretical plate number calculated based on the ginkgo biloba new acid peak should be no less than 4000.

5. The method according to claim 2, characterized in that The gradient elution is specifically:

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