CN111487345B - Method for rapidly detecting total ginkgoic acid in sample in production process of ginkgo leaf extract - Google Patents
Method for rapidly detecting total ginkgoic acid in sample in production process of ginkgo leaf extract Download PDFInfo
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- YXHVCZZLWZYHSA-FPLPWBNLSA-N Ginkgoic acid Chemical compound CCCCCC\C=C/CCCCCCCC1=CC=CC(O)=C1C(O)=O YXHVCZZLWZYHSA-FPLPWBNLSA-N 0.000 title claims abstract description 126
- 238000000034 method Methods 0.000 title claims abstract description 29
- 239000009429 Ginkgo biloba extract Substances 0.000 title claims abstract description 28
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 36
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 124
- 239000000243 solution Substances 0.000 claims description 59
- 239000000523 sample Substances 0.000 claims description 44
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 39
- YXHVCZZLWZYHSA-UHFFFAOYSA-N (Z)-6-[8-pentadecenyl]salicylic acid Natural products CCCCCCC=CCCCCCCCC1=CC=CC(O)=C1C(O)=O YXHVCZZLWZYHSA-UHFFFAOYSA-N 0.000 claims description 36
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 24
- 239000000945 filler Substances 0.000 claims description 22
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- 239000002253 acid Substances 0.000 claims description 17
- 238000010828 elution Methods 0.000 claims description 16
- 239000003480 eluent Substances 0.000 claims description 14
- 238000011068 loading method Methods 0.000 claims description 14
- 238000012360 testing method Methods 0.000 claims description 14
- 238000005303 weighing Methods 0.000 claims description 14
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- 238000002156 mixing Methods 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 11
- 239000012528 membrane Substances 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 8
- 239000012071 phase Substances 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- 239000012088 reference solution Substances 0.000 claims description 7
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- 238000007865 diluting Methods 0.000 claims description 5
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- 239000000463 material Substances 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 3
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- 239000000377 silicon dioxide Substances 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 2
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims description 2
- 238000004704 ultra performance liquid chromatography Methods 0.000 claims description 2
- 238000012856 packing Methods 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 19
- 239000007787 solid Substances 0.000 abstract description 15
- 238000012544 monitoring process Methods 0.000 abstract 1
- 235000008100 Ginkgo biloba Nutrition 0.000 description 14
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- 238000001035 drying Methods 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
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- 238000002347 injection Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 150000002596 lactones Chemical class 0.000 description 7
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- 229930003944 flavone Natural products 0.000 description 6
- 150000002212 flavone derivatives Chemical class 0.000 description 6
- 235000011949 flavones Nutrition 0.000 description 6
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 6
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- 244000194101 Ginkgo biloba Species 0.000 description 2
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- PMZXXNPJQYDFJX-UHFFFAOYSA-N acetonitrile;2,2,2-trifluoroacetic acid Chemical compound CC#N.OC(=O)C(F)(F)F PMZXXNPJQYDFJX-UHFFFAOYSA-N 0.000 description 2
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- 229940106580 ginkgo biloba leaf extract Drugs 0.000 description 2
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- XXMFJKNOJSDQBM-UHFFFAOYSA-N 2,2,2-trifluoroacetic acid;hydrate Chemical compound [OH3+].[O-]C(=O)C(F)(F)F XXMFJKNOJSDQBM-UHFFFAOYSA-N 0.000 description 1
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- 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
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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- Pathology (AREA)
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Abstract
The invention discloses a method for quickly detecting total ginkgoic acid in a sample in the production process of a ginkgo leaf extract, which comprises the steps of quick pretreatment and detection, can quickly detect the total ginkgoic acid in a liquid sample and a solid sample in the production process of the ginkgo leaf extract, greatly shortens the detection time of the index, and effectively ensures the product quality and simultaneously realizes high continuity of production by monitoring the sample in the process in real time.
Description
Technical Field
The invention belongs to the technical field of biomass rapid detection, and particularly relates to a method for rapidly detecting total ginkgoic acid in a sample in a production process of a ginkgo leaf extract.
Background
The ginkgo leaf extract is a medicinal extract collected in Chinese pharmacopoeia, and the process flow is summarized as follows: (1) pretreatment of medicinal materials: pulverizing folium Ginkgo; (2) extracting: extracting pulverized folium Ginkgo with diluted ethanol under reflux, filtering, and mixing filtrates to obtain extractive solution; (3) recovering ethanol: concentrating the extractive solution under reduced pressure to recover ethanol, and concentrating to appropriate amount; (4) solid-liquid separation: centrifuging/filtering the concentrated extract to obtain supernatant; (5) resin adsorption and elution: adding the supernatant into a treated macroporous adsorption resin column, eluting with water, 10-20% ethanol, 50-80% ethanol and other ethanol with different concentrations in sequence, and collecting corresponding eluates; (6) deacidifying: passing the collected eluate through deacidification resin to remove ginkgolic acids; (7) recovering ethanol: collecting deacidified resin effluent, recovering ethanol, and concentrating to appropriate amount; and (8) drying: and (4) carrying out spray drying or vacuum drying on the deacidified concentrated solution and then crushing to obtain the deacidified concentrated solution.
The total ginkgolic acid in the ginkgo leaves has strong toxic effects, mainly comprises potential sensitization, mutagenesis and strong cytotoxicity, can cause serious allergy, gene mutation and nerve injury, and can cause various adverse reactions such as nausea, heartburn, anaphylactic shock, digestive tract mucosa allergy, spasm, allergic purpura, exfoliative dermatitis, nerve paralysis and the like. Therefore, the current pharmacopoeias of various countries in the world have strict control standards for the limit of total ginkgoic acid in ginkgo biloba extract, such as: the content of the compound should not exceed 10mg/kg as specified in the Chinese pharmacopoeia, and the content of the compound should not exceed 5mg/kg as specified in the United states pharmacopoeia and European pharmacopoeia.
Therefore, the extract adds a deacidification procedure after the production of the macroporous resin is finished, and because the limit is too low, the risk of leakage still exists after deacidification, thereby having important influence on the quality of products. Therefore, it is necessary to monitor the ginkgolic acid content of the process product before and after elution to ensure the quality of the final product. The content of ginkgolic acid in the liquid product cannot be directly detected due to low solid content. At present, solid powder is used as a test sample in various pharmacopoeias (including Chinese pharmacopoeias) for detecting the content of ginkgolic acid in a product. Therefore, in order to ensure accurate detection results, the process product is detected by referring to a solid powder method and needs to be concentrated into a solid, so that the pretreatment period is prolonged, and the whole detection period is further prolonged.
The detection method of total ginkgolic acid in ginkgo biloba extract in the Chinese pharmacopoeia comprises the following steps: weighing 2g of the powder, precisely weighing, placing into a conical flask with a plug, precisely adding 10ml of methanol, weighing, ultrasonically dissolving, cooling, shaking uniformly with methanol of less than the initial weight, filtering, and removing the filtrate. Taking 50uL sample of the sample filtered by the filter membrane for detection (the mobile phase is 0.1% trifluoroacetic acid water solution and 0.1% trifluoroacetic acid acetonitrile solution). In order to ensure that the total ginkgoic acid in the produced ginkgo biloba extract is qualified, production process samples (mostly solutions in different production procedures) need to be monitored, if a pharmacopeia method is adopted for control, a certain amount of uniform solution needs to be taken for concentration and drying, then the dry powder samples are pretreated and detected for a long time, and in the actual production process, the condition of production discontinuity often occurs in order to wait for a detection result, so that the production period is prolonged, and the production cost is increased.
In the ginkgoic acid detection process, because the content of a large amount of flavone and lactone components in the ginkgo biloba extract is (for example, the content of total flavone is more than or equal to 24% and the content of lactone components is more than or equal to 6% specified in Chinese pharmacopoeia), in order to detect low content of total ginkgoic acid, the sample injection amount is extremely large, so that the flavone and lactone components are seriously overloaded, the service life of a chromatographic column is influenced, and a large amount of acetonitrile waste liquid is generated.
Disclosure of Invention
The invention aims to provide a rapid detection method for total ginkgoic acid, which is relatively environment-friendly, simple to operate, low in cost and short in time consumption, can quickly provide reference for workshop ginkgo leaf extract production, and ensures the continuity of production and the quality of final products.
The technical scheme of the invention is as follows:
a method for rapidly detecting total ginkgoic acid in a sample in the production process of a ginkgo leaf extract comprises the following steps:
(1) Preparation of C18 packed column
Weighing 2g of octadecylsilane chemically bonded silica (C18) filler into a small column for solid-phase extraction with a plug plate, adding 95% ethanol to fully infiltrate the filler, washing and activating the filler by using 20mL of 95% ethanol, and washing and balancing the filler by using 30mL of 60% ethanol for later use;
(2) Preparation of test solution
Weighing 50mL of 60% ethanol eluent containing ginkgo leaf extract, adding 50 mu L of trifluoroacetic acid, mixing uniformly, loading 2g C18 filler at 4-6 mL/min, washing residual liquid medicine with 20mL of 60% ethanol after loading is finished, and discarding; eluting with 95% ethanol at a speed of 1-2 mL/min, collecting 25mL of effluent, and filtering with 0.22 μm filter membrane;
or,
weighing 500mL of deacidification solution containing ginkgo leaf extract, adding 500 mu L of trifluoroacetic acid, mixing uniformly, loading 2g of C18 filler at 4-6 mL/min, washing residual liquid medicine with 20mL of 60% ethanol after loading is finished, discarding, eluting with 95% ethanol at the speed of 1-2 mL/min, collecting 25mL of effluent liquid, mixing uniformly, and filtering with a 0.22 mu m filter membrane to obtain the ginkgo leaf extract-containing deacidification solution;
or,
weighing 5.0g of ginkgo leaf extract, adding 500mL of 60% ethanol for ultrasonic dissolution, adding 500 mu L of trifluoroacetic acid, uniformly mixing, loading 2g of C18 filler at 4-6 mL/min, washing residual liquid medicine by using 20mL of 60% ethanol after loading is finished, discarding, eluting by using 95% ethanol at the speed of 1-2 mL/min, collecting 25mL of effluent liquid, and filtering by using a 0.22 mu m filter membrane to obtain the ginkgo leaf extract.
(3) Preparation of control solutions
Weighing neoacid of semen Ginkgo, ultrasonic dissolving with methanol, diluting to desired volume to obtain control solution containing 1 μ g of neoacid per 1mL for positioning; taking another appropriate amount of total ginkgoic acid reference substance, performing ultrasonic dissolution with methanol, diluting to constant volume, and preparing into reference solution containing 20 μ g per 1mL for positioning.
(4) Determination of content
And (3) respectively taking 5 mu L of the reference substance solution and the test solution, injecting into a liquid chromatograph for determination, and determining the content of the total ginkgoic acid.
Preferably, the 60% ethanol in the C18 filler is filtered to dryness by suction before the 95% ethanol is used for elution in the step (2).
Preferably, the column temperature in the liquid chromatograph in the step (4) is 30 ℃, the injection volume is 5 μ L, and the gradient elution is carried out by taking acetonitrile-trifluoroacetic acid solution as a mobile phase, and the flow rate of the mobile phase is 0.4mL/min.
Preferably, the gradient elution is in particular:
| time/min | Acetonitrile (%) | 0.1% trifluoroacetic acid (%) |
| 0 | 65 | 35 |
| 5.5 | 72 | 28 |
| 10 | 72 | 28 |
| 15 | 100 | 0 |
| 18 | 100 | 0 |
| 18.01 | 65 | 35 |
| 24 | 65 | 35 |
Preferably, in the step (4), a standard curve is established by taking the concentration of the ginkgolic acid reference substance solution as an abscissa and the chromatographic peak area as an ordinate, the total peak area of the chromatographic peak corresponding to the total ginkgolic acid reference substance in the test substance solution is calculated, the total peak area is substituted into the standard curve to obtain the concentration of the total ginkgolic acid in the test substance solution, and then the content of the total ginkgolic acid in the test substance is calculated.
Preferably, the total ginkgolic acid content of the solid after drying the 60% ethanol eluate is calculated as follows:
X 1 -the content of total ginkgolic acid in the solid obtained after drying the 60% ethanol eluate, unit: mg of/kg；
C 1 -concentration of total ginkgolic acid in 60% ethanol eluate, units: mg/L;
M 1 -solid content of 60% ethanol eluent, unit: g/L.
Preferably, the total ginkgolic acid content in the solid after drying the deacidification liquid is calculated according to the following formula:
X 2 -the content of total ginkgolic acid in the solid obtained after drying the deacidification liquid, unit: mg/kg;
C 2 concentration of total ginkgolic acid in the test solution (deacidification solution), unit: mg/L;
M 2 solid content of deacidification liquid, unit: g/L.
Preferably, the total ginkgolic acid content in the ginkgo biloba extract is calculated according to the following formula:
X 3 -the content of total ginkgolic acid in the solid obtained after drying the deacidification liquid, unit: mg/kg;
C 3 -concentration of total ginkgolic acid in test solution, unit: mg/L;
m-weighing amount of ginkgo biloba leaf extract, unit: g.
preferably, the solid contents of the 60% ethanol eluent and the deacidified solution are respectively calculated after the corresponding solutions are dried.
Compared with the prior art, the technical scheme of the invention at least has the following beneficial effects:
1. the environment is relatively friendly: according to the invention, methanol is not needed in the sample pretreatment process, the flow velocity of the flowing phase in the detection process is only 1/2.5 of that in the prior art, and the required time is only 1/2 of that in the prior art, so that the generated acetonitrile waste liquid is only about 1/5 of that in the prior art, the use of an organic solvent is greatly reduced, and the pollution of the organic solvent to the environment is reduced.
2. The time consumption is short: the liquid sample does not need to be dried, can be directly enriched by the C18 filler and then eluted (25 mL of 95% ethanol eluent is collected), can be subjected to sample injection detection, only needs 2-3h from sampling to sample detection completion, and needs 6-8h by using the prior art (pharmacopoeia detection method).
3. The service life of the chromatographic column is prolonged: when the sample solution passes through the C18 filler, the flavone and lactone components in the sample solution are not reserved, only the total ginkgoic acid is adsorbed on the filler, the sample solution is eluted to prepare a test solution, and 5uL of sample injection is carried out, wherein the test solution only contains the total ginkgoic acid components or contains the flavone and lactone components in trace. In the existing method, 2g of extract powder is extracted by 10mL of methanol, 50uL of sample is introduced, flavone and lactone components in a test solution are seriously overloaded, a platform peak is formed in front of total ginkgoic acid, and the performance of a chromatographic column can be reduced due to frequent sample introduction, so that the service life of the chromatographic column is influenced.
Drawings
FIG. 1 is a chromatogram of a control solution for total ginkgolic acid localization in example 3.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Instrumentation and materials
1. Instrument for measuring the position of a moving object
Chromatograph: waters acquisition H-CLASS ultra performance liquid chromatograph (quaternary gradient pump, autosampler, column oven, PDA detector);
and (3) chromatographic column: an ACQUITY UPLC BEH column (2.1X 50mm,1.7 μm);
an electronic balance: the model number of the one in ten thousand electronic balances is Sartorius SQP QUINTIX125D-1CN, and the model number of the one in ten thousand electronic balances is Sartorius SQP QUINTIX224-1CN;
an ultrasonic extractor: SK8200HP shanghai science leader.
1. Material
Experimental samples: ginkgo biloba extract (C0253180301, C0253180901), 60% alcohol eluent (C0253190402-1, C0253190402-2), deacidified liquid (C0253190502-1, C0253190502-2) (note, "C0253190402-1" means the first sub-batch in the production process of C0253190402 ginkgo biloba extract, and the like).
Neoacid ginkgo: chinese food and drug testing research institute (batch No. 111690-201604; content 99.3%) for content determination;
total ginkgoic acid a: china food and drug testing research institute (batch No. 111594-201605) for limited quantity inspection and positioning.
Total ginkgoic acid B: china food and drug testing research institute (batch No. 111594-201605) for quantitative examination and positioning.
Methanol: the chromatogram is pure and the analysis is pure; trifluoroacetic acid: carrying out chromatographic purification; acetonitrile: carrying out chromatographic purification; ultrapure water;
c18 Filler (UniSil 30-120C18, suzhou nano micro-technology GmbH)
Example 1
1 preparation method of reference solution:
reference is made to the summary of the invention for the preparation of the reference solutions.
2, a preparation method of a test solution comprises the following steps:
the preparation method of the test solution is referred to the summary of the invention.
Precisely sucking the reference solution, the test solution and the positioning reference solution by 5 μ l each, injecting into a liquid chromatograph, calculating the total peak area of the corresponding chromatographic peak of the total ginkgoic acid reference in the test solution, and calculating the content of the total ginkgoic acid by using a ginkgolic neo-acid reference external standard method.
1.0g,1.5g and 2.0g of C18 filler are respectively used for filling columns, sample pretreatment is carried out according to 2.1, 60% ethanol cleaning solution tail liquid is respectively collected before elution is carried out by using 95% ethanol, sample injection is directly carried out after filtration by using a 0.22 mu m filter membrane, and as a result, total ginkgoic acid is detected in collection liquid of 1.0g and 1.5g of C18 filler columns, and the content of the C18 filler is determined to be 2.0g because 2.0g of C18 filler columns are not detected.
Example 2
Loading a column by using 2.0g of C18 filler, loading the sample until 20mL of 60% ethanol is used for cleaning residual liquid medicine, preparing three samples, then respectively using 10mL of methanol, 15mL of methanol, 20% of methanol and 25mL of 95% ethanol to elute at the speed of 2mL/min, after the elution is finished, respectively collecting 1mL of elution tail liquid, filtering by using a 0.22-micrometer filter membrane, directly injecting a sample for detection, and detecting the total ginkgoic acid in the elution tail liquid of the three previous samples and not detecting the total ginkgoic acid in the elution tail liquid of 25mL of 95% ethanol, wherein the elution tail liquid of 25mL of 95% ethanol is used as an elution solvent, so that the use amount of 25mL of 95% ethanol is determined, and the pollution of ethanol is smaller.
Example 3
Chromatography column with octadecylsilane chemically bonded silica as filler (inner diameter 2.1mm, particle size 1.7/1.8 μm), uv detector/DAD/PDA, λ =310nm, column temperature T =30 ℃, sample injection volume: 5 mu L, using acetonitrile-0.1% trifluoroacetic acid solution as mobile phase, performing gradient elution according to table 1, wherein the flow rate of the mobile phase is 0.4mL/min, the chromatogram of the positioning control solution is shown in figure 1, and the total ginkgoic acid contains 5 components including ginkgolic acid and 2-5 components.
TABLE 1 gradient elution Table
| Time/min | Acetonitrile (%) | 0.1% trifluoroacetic acid (%) |
| 0 | 65 | 35 |
| 5.5 | 72 | 28 |
| 10 | 72 | 28 |
| 15 | 100 | 0 |
| 18 | 100 | 0 |
| 18.01 | 65 | 35 |
| 24 | 65 | 35 |
Example 4
Precisely weighing 5.95mg to 50mL of ginkgo neo-acid reference substance in a volumetric flask, adding methanol for ultrasonic dissolution, fixing the volume to scale, precisely weighing a proper amount, respectively diluting the solution by 5 times, 10 times, 25 times, 100 times and 200 times, analyzing according to the chromatographic conditions listed in the example 3 to obtain the peak areas of the ginkgo neo-acid reference substance with various concentrations, and establishing a standard curve by respectively taking the concentration C of the ginkgo neo-acid reference substance solution as an abscissa and the peak area A of the ginkgo neo-acid reference substance solution as an ordinate. The concentration and peak area of the ginkgo neo-acid standard curve series solution are as follows:
TABLE 2 Linear equation, correlation coefficient and Linear Range of ginkgolic acids
From the results, it can be seen that: the linear relation of the ginkgolic acid in the concentration range of 0.591-118.167 mg/L is good, the linear equation is Y =6950X-443, and the correlation coefficient R is 1.0000.
Example 5
Diluted ginkgo biloba was used as a control, diluted to two solutions of appropriate concentration, injected under the chromatographic conditions as listed in example 3, so that the signal-to-noise ratio (S/N) of neoacid of ginkgo biloba in the corresponding chromatograms was close to 10 (LOQ) and 3:1 (LOD), respectively, and 20 needles were injected for each solution.
The data relating to LOQ and LOD experiments (retention time average (RT)) are shown in the table below.
TABLE 3 Total ginkgolic acid LOQ and LOD Experimental data
According to the experimental result, under the chromatographic condition, the LOD of the ginkgolic acid is 0.186mg/L, the LOQ is 0.466mg/L, and the lower the solution concentration is, the smaller the peak area is, and the larger the peak area RSD is.
Example 6
Using 60% ethanol eluent (C015390402-1) as an experimental sample, preparing 6 parts of each sample solution as a repeatability experimental sample according to the sample preparation method, performing chromatographic analysis under the chromatographic condition described in example 3, using ginkgoneoic acid as a reference, calculating the concentration of total ginkgoic acid in each sample solution, calculating the content of total ginkgoic acid in solid after drying 60% ethanol eluent, and calculating the RSD value of the repeatability experiment. The data of the repeatability tests are shown in Table 4.
Table 4 total ginkgolic acid (60% ethanol eluent) repeatability experimental data
The deacidification solution (as the total ginkgoic acid content in the normal deacidification solution is lower or not contained, in order to verify the applicability of the method in a sample with low total ginkgoic acid content, 60% ethanol eluent C015390402-1100mL and deacidification solution C0253190502-1 1700mL are mixed and prepared) is used as an experimental sample, 6 test sample solutions are respectively prepared according to the test sample preparation method of the invention and used as repeatability experimental samples, chromatographic analysis is carried out under the chromatographic condition, the concentration of the total ginkgoic acid in each test sample solution is calculated by using the established standard curve, the content of the total ginkgoic acid in the solid after the deacidification solution is dried is calculated, and the RSD value of the repeatability experiment is calculated. The data of the repeatability tests are shown in Table 5.
TABLE 5 Total ginkgolic acid (deacidification solution) repeatability test data
The experimental results show that: the method for detecting the total ginkgoic acid of the 60% ethanol eluent and the deacidified liquid has good repeatability, and the RSD of the method is 0.69% and 1.17% respectively.
Example 7
The sample solution was prepared in the same manner as in example 6 by continuously feeding samples 6 times under the chromatographic conditions described in example 3, and the peak areas and RSD values of 5 total ginkgoic acid components were calculated, and the experimental results are shown in Table 6.
TABLE 6 Total ginkgolic acid (60% ethanol eluate) precision experimental data
The sample solution was prepared in the same manner as in example 6 by continuously feeding samples 6 times under the chromatographic conditions described in example 3, and the peak areas and RSD values of 5 total ginkgoic acid components were calculated, and the experimental results are shown in Table 7.
TABLE 7 Total ginkgolic acid (deacidification liquid) precision experimental data
The experimental results show that: the method for detecting the total ginkgoic acid in the 60% ethanol eluent and the deacidification solution has good precision, and the RSD is 0.96% and 0.40% respectively.
Example 8
The experimental sample was recovered using the 60% ethanol eluate (C015390402-1) from example 6 as a spiked sample. 25mL of the solution was measured, and 9 parts were taken and divided into 3 groups of 3 parts each. Adding 118.167mg/L ginkgoneoic acid reference substance solutions 0.43mL, 0.53mL and 0.64mL into the 3 groups of solutions respectively, adding 25mL of 60% ethanol respectively, mixing, and performing parallel operation on 3 samples in each group. And (3) preparing a sample according to the ratio of 2.2, taking the sample as a standard-added recovery experiment sample, carrying out chromatographic analysis under the chromatographic condition, calculating the content of the total ginkgoic acid in each test sample solution by taking the neoginkgoic acid as a reference, calculating the recovery rate of the total ginkgoic acid in the sample, and calculating the RSD value. The sample recovery data are shown in Table 8.
TABLE 8 Total ginkgolic acid (neoginkgolic acid) recovery data in 60% ethanol eluate
The deacidified liquid in example 6 was used as a standard for recovering experimental samples. 250mL of the solution was measured, and 9 parts were taken and divided into 3 groups of 3 parts each. Adding 118.167mg/L ginkgoneoic acid reference substance solutions 0.24mL, 0.29mL and 0.34mL into the 3 groups of solutions respectively, adding 250mL of 60% ethanol respectively, mixing, and performing parallel operation on 3 samples in each group. According to the invention, the sample preparation is carried out, the sample is used as a standard-adding recovery experiment sample, the chromatographic analysis is carried out under the chromatographic condition, the content of the total ginkgoic acid in each sample solution is calculated by taking the ginkgolic neo-acid as a reference, the recovery rate of the total ginkgoic acid in the sample is calculated, and the RSD value is calculated, and the experimental data are shown in a table 9.
TABLE 9 Total ginkgolic acid (neoginkgolic acid) spiked recovery data in deacidification solution
The experimental results show that: the method for detecting the total ginkgoic acid in the 60% ethanol eluent and the deacidification liquid has good precision, the average recovery rates are 100.89% and 101.78% respectively, and the RSD is 1.40% and 2.43% respectively.
Example 9
The sample of the precision experiment is taken as a stability experiment sample, sample injection analysis is carried out at different time points respectively according to the chromatographic conditions described in the embodiment 3, the chromatogram of the total ginkgoic acid at each time point is obtained, and the peak areas and RSD values of 5 components are calculated. The data relating to the stability experiments are shown in tables 10 and 11.
TABLE 10 Total ginkgolic acid (60% ethanol eluate) stability Experimental data
TABLE 11 Total ginkgolic acid (deacidification solution) stability experimental data
As can be seen from the experimental data, the test solution has good stability after being placed at room temperature for 25 hours.
Example 10
The reference solution for positioning ginkgo biloba leaf extract was sampled and analyzed by using different chromatographic columns under the chromatographic conditions described in example 3, and the retention time and the separation degree of 5 components are shown in table 12.
TABLE 12 durability examination experimental data
Example 11
According to the sample preparation method of the present invention and the chromatographic condition detection (new method) in example 3 and the content determination method of "total ginkgoic acid" under the item of "ginkgo leaf extract" in the first part of "chinese pharmacopoeia" 2015 edition (existing method), the sample ginkgo leaf extract (C0253180301, C0253180901), 60% alcohol eluent (C0253190402-1, C0253190402-2), deacidification solution (C0253190502-1, C0253190502-2) are respectively processed, and sample detection is performed to calculate the total ginkgoic acid content therein, and the results are shown in table 13.
TABLE 13 comparison of the results of two methods for detecting total ginkgolic acid
Compared with the existing method, the newly developed method is more sensitive in detection, and the pretreatment and detection time of the sample is obviously shortened (the average time is about 2-3 h). In addition, flavonoid alcohol glycoside components and lactone components in the ginkgo leaf extracting solution are discarded during sample pretreatment, and only total ginkgolic acid in the ginkgo leaf extracting solution is enriched, so that the damage to a chromatographic column is reduced, and the service life of the chromatographic column can be prolonged.
Claims (2)
1. A method for rapidly detecting total ginkgoic acid in a sample in the production process of a ginkgo leaf extract is characterized by comprising the following steps:
(1) Preparation of C18 packed column
Weighing 2g of octadecylsilane chemically bonded silica (C18) filler into a small column for solid-phase extraction with a plug plate, adding 95% ethanol to fully infiltrate the filler, washing and activating the filler by using 20mL of 95% ethanol, and washing and balancing the filler by using 30mL of 60% ethanol for later use;
(2) Preparation of test solution
Measuring 50mL of 60% ethanol eluent containing the ginkgo leaf extract, adding 50 muL of trifluoroacetic acid, uniformly mixing, loading the C18 packed column at 4-6 mL/min, washing residual liquid with 20mL of 60% ethanol after loading is finished, and discarding; eluting with 95% ethanol at a speed of 1-2 mL/min, collecting 25mL of effluent, and filtering with a 0.22 mu m filter membrane to obtain the product;
or,
measuring 500mL of deacidification solution containing ginkgo leaf extract, adding 500 muL of trifluoroacetic acid, mixing uniformly, loading the sample into a C18 packed column at 4-6 mL/min, after loading is finished, using 20mL of 60% ethanol to clean residual solution, discarding, using 95% ethanol to elute at a speed of 1-2 mL/min, collecting 25mL of effluent liquid, and filtering by using a 0.22 mu m filter membrane after mixing uniformly to obtain the ginkgo leaf extract deacidification solution;
or,
weighing 5.0g of ginkgo leaf extract, adding 500mL of 60% ethanol for ultrasonic dissolution, adding 500 muL of trifluoroacetic acid, uniformly mixing, loading a C18 packed column with 4-6 mL/min, after loading is finished, using 20mL of 60% ethanol for cleaning residual liquid, discarding, using 95% ethanol for elution at the speed of 1-2 mL/min, collecting 25mL of effluent liquid, and filtering by using a 0.22μm filter membrane to obtain the ginkgo leaf extract;
(3) Preparation of control solutions
Weighing neoginkgoic acid, ultrasonically dissolving with methanol, diluting to a constant volume, and preparing a control solution containing 1 microgram per 1mL for positioning; taking a proper amount of total ginkgoic acid reference substances, carrying out ultrasonic dissolution by using methanol, fixing the volume, and diluting to prepare a control solution for positioning containing 20 microgram per 1 mL;
(4) Determination of content
Injecting 5 μ L of each of the reference solution and the sample solution into an ultra high performance liquid chromatograph for determination to determine the content of total ginkgoic acid;
a chromatographic column in the liquid chromatograph is an ACQUITY UPLC BEH chromatographic column, 2.1 x 50mm and 1.7 mu m, the column temperature is 30 ℃, the sample introduction volume is 5 mu L, and gradient elution is carried out by taking acetonitrile-0.1% trifluoroacetic acid solution as a mobile phase, and the flow rate of the mobile phase is 0.4 mL/min;
the gradient elution is specifically as follows:
；
And establishing a standard curve by taking the concentration of the ginkgoneoic acid reference substance solution as an abscissa and the chromatographic peak area as an ordinate, calculating the total peak area of the chromatographic peak corresponding to the total ginkgolic acid reference substance in the test substance solution, substituting the total peak area into the standard curve to obtain the concentration of the total ginkgolic acid in the test substance solution, and calculating the content of the total ginkgolic acid in the test substance.
2. The method of claim 1, wherein the step (2) comprises suction filtering 60% ethanol from the C18 packing material to dryness before eluting with 95% ethanol.
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