CN101366875B - Quality control method and uses of antirheumatic medicament - Google Patents
Quality control method and uses of antirheumatic medicament Download PDFInfo
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- 239000003435 antirheumatic agent Substances 0.000 title claims abstract description 26
- 239000003814 drug Substances 0.000 title claims abstract description 26
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- 238000003908 quality control method Methods 0.000 title abstract description 14
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Abstract
The invention provides a quality control method for an antirheumatic medicine. Besides the characters and inspection, the method also comprises the steps of identification, extract determination and assaying, wherein the identification step is to dissolve the content of the antirheumatic medicine--a rheumatalgia-relieving preparation in alcohol, carry out ultrasonic extraction to prepare a solution to be tested, prepare a comparison solution with the comparison crude drug of lilac daphne strips, and then carry out thin layer chromatographic comparison. The extract determination step is to carry out the alcohol backflow and leaching of the content of the antirheumatic medicine--the rheumatalgia-relieving preparation and determine the dry substance amount of the methanol extract. The assaying step is to carry out methanol backflow and leaching of the content of the antirheumatic medicine--the rheumatalgia-relieving preparation to prepare the solution to be tested, carry out the high pressure liquid chromatography detection together with the reference substance of daphnetin, and determine the content of the daphnetin in the content of the antirheumatic medicine--the rheumatalgia-relieving preparation. The method is suitable for the quality control of the antirheumatic medicine -the Rheumatalgia-relieving preparation including tablets, capsules, granules, pills, and the like.
Description
Technical Field
A quality control method of antirheumatic medicine for relieving pain in collaterals belongs to an analysis method of traditional Chinese medicine preparations, and particularly relates to a quality control method of antirheumatic medicine. Is suitable for quality control of preparations such as tablet, capsule, granule or pill of antirheumatic drug "Xiaoluotong" on the market.
Background
A Chinese patent medicine for treating rheumatism, XIAOLUOTONG, is a product which is sold on the market for many years, has high safety and good curative effect, and is exported from southeast Asia to China, so that the Chinese patent medicine is well received by the wide range of patients with rheumatism.
The antirheumatic drug "Xiaoluotong" is mainly prepared from extracts of lilac daphne flower bud, the quality control method in the prior art only has two items of properties and inspection, and the content of the preparation is controlled to be dark brown; light smell, bitter and numb taste; and compliance with regulations under pharmacopoeia tablet terms. The quality of the medicine can not be scientifically and accurately judged, and is questioned in the international market, so that the quality of the medicine can be comprehensively and accurately controlled, the reputation of the traditional Chinese medicine in the international market is ensured, and the quality control method of the medicine is necessarily researched.
Disclosure of Invention
The invention aims to establish a quality control method of an antirheumatic drug Xiaoluotong which can scientifically and accurately judge the quality of the drug.
The invention of the application is implemented by the following technical scheme:
the quality control method for preparing antirheumatic medicine is characterized by that besides character and inspection, said method also includes the steps of identification, extract determination and content determination: wherein,
the identification step is that the anti-rheumatism medicine pain-relieving preparation content is dissolved by ethanol, ultrasonic extraction is carried out, a test solution is prepared, and thin-layer chromatography comparison is carried out on the test solution and a control solution prepared from the lilac daphne flower bud control medicinal material;
the extract determination step is that the anti-rheumatism medicine pain-relieving preparation content is extracted by ethanol reflux, and the dry product amount of the ethanol extract is determined;
the content determination step comprises the steps of carrying out methanol reflux extraction on the content of the antirheumatic medicine rheumatalgia-relieving preparation to prepare a test solution, carrying out High Performance Liquid Chromatography (HPLC) detection on the test solution and a daphnoretin reference substance, and determining the content of daphnoretin in the content of the antirheumatic medicine rheumatalgia-relieving preparation.
The above-mentioned identification step is:
firstly, taking the contents of the antirheumatic medicine pain-relieving preparation, grinding, adding ethanol for dissolving, ultrasonically extracting, filtering and concentrating to prepare a test solution;
collecting flos Genkwa (branches) contrast medicinal material, pulverizing, dissolving in ethanol, ultrasonic extracting, filtering, and making into contrast medicinal solution;
respectively sucking 5 mul of test solution and control solution, respectively dropping on a same silica gel G thin-layer plate with sodium carboxymethylcellulose as binder, developing with n-hexane, ethyl acetate and formic acid at ratio of 1: 0.01 as developing agent, air drying, and inspecting under 365nm ultraviolet lamp to obtain the same blue fluorescent spot at the position corresponding to the control material chromatogram in the test solution chromatogram.
The extract measuring steps are as follows:
taking the contents of the preparation for relieving pain in collaterals, grinding, precisely weighing, taking 95% ethanol as a solvent, and performing reflux extraction, wherein the dry content of the ethanol extract in the test sample is calculated.
The content determination steps are as follows:
precisely weighing a proper amount of daphnoretin reference substance, placing the reference substance in a brown measuring flask, and adding methanol to prepare a solution containing 10 mu g of reference substance per 1 ml;
secondly, grinding and precisely weighing the contents of the Xiaoluotong preparation, placing the mixture into a conical flask with a plug, precisely adding methanol, weighing, refluxing and extracting, cooling, weighing again, complementing the weight loss amount by methanol, shaking up, filtering, and preparing a test solution containing 20-40 mg of the contents of the Xiaoluotong preparation per 1 ml;
using Octadecylsilane chemically bonded silica (ODS for short) as a filler, and using methanol or acetonitrile and 0.2% phosphoric acid solution (50-55: 50-45) as a mobile phase; the high performance liquid detection wavelength is 346nm, and the theoretical plate number is according to the daphnoretin C19H12O7Not less than 3000;
fourthly, respectively and precisely absorbing 20 mul of reference solution and test solution, injecting into a high performance liquid chromatograph, measuring, and using daphnoretin C in the test solution19H12O7The content of the powder is counted.
An application of the quality control method of antirheumatic medicine is characterized by being applied to the quality control of tablets, capsules, granules or pills of antirheumatic medicine Xiaoluotong.
The preparation for relieving pain comprises:
in the ethanol extract measurement, the ethanol extract content: the content of the tablet is not less than 10.0%;
the content of capsule is not less than 18.0%;
the granule should not be less than 4.0%;
the pill content should not be less than 12.0%
In the content measurement, each gram of the preparation contains daphnetin C19H12O7Counting:
the tablet is not less than 0.25 mg;
the content of capsule should not be less than 0.5 mg;
the granule should not be less than 0.1 mg;
the pill should not be less than 0.30 mg.
The two items of the property and the check are related to the prior art and are not detailed.
In the present application, the term "preparation content" refers to a core layer of a tablet (sugar-coated tablet or film-coated tablet) from which a coating layer has been removed; or removing the capsule from the capsule (in the original capsule); or granule and pill preparation.
The dosage of each preparation form is consistent with the content of common turnip strips in each administration, and the unit size of each preparation can be properly changed according to the amount of auxiliary materials.
The method of the invention has the advantages that:
1. daphnoretin (Daphnoretin C) as one of main effective components of Daphnoretin19H12O7) The molecular structural formula and the physicochemical constant are confirmed through extensive research. The antirheumatic drug "Xiaoluotong" is prepared by taking daphne genkwa strips (or called as daphne genkwa branches) as main raw materials, and can be used as a control method for effectively controlling the quality of the antirheumatic drug "Xiaoluotong" by measuring the content of daphnetin in the Chinese and western medicine.
2. The quality control method of the invention is verified by a plurality of batches of anti-rheumatism pharmaceutical preparations 'Xiaoluotong' products, and has the advantages of simple method, good reproducibility, no interference in negative and strong specificity.
Drawings
FIG. 1 HPLC chromatogram of Xiaoluotong tablet sample;
FIG. 2 HPLC chromatogram of daphnetin control;
FIG. 3 negative control sample HPLC profile;
FIG. 4 HPLC chromatogram of flos Genkwa strip medicinal material;
FIG. 5 is a reference curve for a reference daphnetin.
Detailed Description
The invention is explained below with reference to the figures and examples;
in the drawings, FIGS. 1 to 4 are comparative test charts of quality control study of pain in elimination, wherein the ordinate of the chart is signal intensity of a sample passing through a detector, and the abscissa of the chart is retention time of the sample.
Fig. 5 is a reference curve of daphnoretin reference, wherein the ordinate is the peak area of the reference and the abscissa is the sample size of the reference. Wherein,
FIG. 1 is the chromatographic peak of XIAOLUOTONG tablet under the extraction method and chromatographic conditions, which is evidence that the product contains daphnetin;
FIG. 2 is a chromatogram peak of a daphnoretin reference substance under the chromatographic condition, which is a positive standard control chromatogram of daphnoretin;
FIG. 3 shows that the negative control sample (a sample map without daphne genkwa strips) has no chromatographic peak under the chromatographic condition, and the other substances in the sample do not interfere with the content determination of daphnetin;
FIG. 4 is the chromatographic peak of Daphne genkwa strip in the chromatographic condition, which is the basis for proving that Daphne genkwa strip contains daphnetin.
Attached notes: a commercial antirheumatic medicinal preparation, XIAOLUOTONG, is prepared from flos Genkwa strip and other adjuvants. The sample is a sample with main drug daphne genkwa strips removed, and is a blank control sample in the conventional sense.
Second, content determination test of Chinese and western daphnetin (taking tablet as example)
(1) Selection of extraction conditions
Selection of an extraction solvent: 30 samples of 071002 batches were taken, the coating was removed, the samples were ground, three parts were weighed, 2.0g of each part was precisely weighed, placed in a conical flask with a stopper, 25ml of each of methanol, absolute ethanol and ethyl acetate was added, the weight was weighed, sonicated (power 100W, frequency 25kHz) for 30min, cooled, weighed again, the weight loss was reduced by adding the corresponding solvent, filtered through an organic microporous membrane with a pore size of 0.45 μm, and injected into a liquid chromatograph (a dean high performance liquid chromatograph including a P680 pump, a UVD170 uv detector, chmel data processing software) to measure the content, the results are shown in table-1.
TABLE-1 extraction solvent selection results Table
The results show that: the highest sample content is obtained when methanol is used as the extraction solvent, so the extraction solvent is selected to be methanol.
Selecting an extraction method: 30 samples of 071002 batches were taken, the coating was removed, the fine powder was ground, 2.0g of the fine powder was weighed, triplicate was weighed, precisely weighed, placed in a conical flask with a stopper, 25ml of methanol was added, the weight was weighed, samples were prepared by ultrasonic method (power 100W, frequency 25kHz), reflux method, soxhlet extraction method, extraction for 1 hour, cooling down, weighing again, the weight loss was reduced by methanol supplementation, filtered with an organic microporous membrane with a pore size of 0.45 μm, and injected into a liquid chromatograph (a dean hplc including a P680 pump, a UVD170 uv detector, CHROMELE data processing software, for testing the content), the results are shown in table-2.
TABLE-2 extraction method selection results Table
The results show that: the reflux extraction method is selected as the reflux extraction method because the sample content is highest when the reflux extraction method is used for extraction.
Selecting extraction time: taking a proper amount of 071002 batch samples, removing coatings, grinding, weighing about 2.0g of fine powder in triplicate, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of methanol, weighing, reflux-extracting for 45min, 60min, 75min, cooling, weighing again, complementing weight loss with methanol, filtering with organic microporous membrane with pore diameter of 0.45 μm, using as test solution, injecting into a liquid chromatograph (American dean high performance liquid chromatograph including p680 pump, UVD170 ultraviolet detector, CHROMELE data processing software) to measure the content, and finding in Table-3.
TABLE-3 extraction time selection results Table
The results show that: when the sample is extracted for 60min under reflux, the daphnoretin is completely extracted, so that the extraction time is determined to be 60 min.
Selecting the extracted sample quantity: taking a proper amount of 071002 batch samples, removing coatings, grinding, respectively weighing 2.0g, 1.0g and 0.5g of fine powder, precisely weighing, placing in a conical flask with a plug, respectively and precisely adding 25ml of methanol, respectively, reflux-extracting for 1h, cooling, weighing again, complementing weight loss with methanol, filtering with organic microporous membrane with pore diameter of 0.45 μm, using as test solution, injecting into a liquid chromatograph (American dean high performance liquid chromatograph including P680 pump, UVD170 ultraviolet detector, CHROMELE data processing software) to measure the content, and finding in Table-4.
TABLE-4 extraction time selection results Table
The results show that: the content of the 1.0g sample reaches the highest when the 1.0g sample is extracted by 25ml of methanol, and the content of the 1.0g sample has no obvious change when the 0.5g sample is extracted by the methanol, which indicates that the 1.0g sample of daphnoretin is completely extracted by 25ml of methanol. In order to save reagents and ensure that components can be extracted to the maximum extent, the sample amount is determined to be 1.0g, and the extraction solvent amount is 25 ml.
Through the above experimental studies, the preparation of the test article solution is finally described as: taking 30 tablets of the product, removing sugar coating, grinding, taking about 1.0g, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of methanol, weighing, reflux-extracting for 60min, cooling, weighing again, supplementing weight loss with methanol, shaking, and filtering.
(2) Linear range investigation: taking a proper amount of daphnoretin reference substance, precisely weighing, adding methanol to prepare a solution containing 40 μ g per 1ml, precisely sucking 20 μ l, 15 μ l, 10 μ l, 5 μ l and 2 μ l samples, and measuring the peak area. The results are shown in Table 4.
And (5) carrying out regression on the sample amount (x) by using the peak area (y) to obtain a standard curve equation. y 52.069x +0.3883(r 0.9998). The results show that the daphnoretin peak area value has a good linear relation with the sample injection amount within the range of 0.08-0.80 mu g.
TABLE-5 daphnetin reference assay results
The test results are shown in the attached figure 5 in the specification.
(3) Negative control test: taking a negative trial sample (the sample is a sample without the daphne genkwa strips, and a blank control sample in the conventional meaning) which does not contain the daphne genkwa strips, respectively weighing 1.0g of the pain relieving tablet sample, 1.5g of the daphne genkwa strip medicinal material (powder), respectively adding 25ml of methanol, refluxing and extracting for 1h, cooling, weighing again, complementing the weight loss with methanol, and filtering to respectively obtain a negative control solution, a pain relieving tablet sample solution and a daphne genkwa strip medicinal material solution. Respectively injecting the daphnoretin reference substance and the sample solution into a liquid chromatograph by 20 mu l respectively, and recording the chromatogram.
The results show that: there was no daphnoretin peak in the negative control profile.
The test results are shown in the attached drawings 1-4 in the specification:
(4) and precision test: the sample injection was repeated 6 times by sucking the control solution (19. mu.g/ml) and 071002 batch of the same sample solution (1.0230g25ml), 20. mu.l of each sample injection was performed, and the RSD of the peak areas of the two samples was calculated, respectively, and the results are shown in Table-6.
TABLE-6 precision test results
The results show that: the average peak area of the reference sample is 20.077, the RSD is 0.63%, the average peak area of the test sample is 12.036, the RSD is 0.92%, and the precision is good.
(5) And a reproducibility test: 5 parts of 071002 batches of samples were taken, and the results were prepared and determined according to the test article preparation items, respectively, and the content of daphnoretin is shown in Table-7.
TABLE-7 reproducibility test results
The results show that: the test method has good reproducibility with 5 parts of the test sample having an average content of 0.282mg/g and an RSD of 1.71%.
Relative Standard Deviation (RSD) is often used to indicate precision.
(6) And a stability test: the same sample solution (1.023g, 25ml) of 071002 batch was injected at 0 hour, 2 hours, 4 hours, 6 hours, and 8 hours, respectively. The results are shown in Table-8.
TABLE-8 stability test results
The results show that: the RSD of the test article is 0.74 percent within 8 hours, and the stability of the test article solution is good within 8 hours.
(7) And a recovery rate test: 071002 batches (with known content of 0.28mg/g) of fine powder 0.5g are precisely weighed (by sample-adding recovery method), 5 parts are weighed, the same daphnoretin reference substance is added respectively, the total content is measured, and the recovery rate is calculated. Recovery rate (total amount-amount in sample)/amount of added pure product. The results are shown in Table-9.
TABLE-9 recovery test results
The results show that: the average recovery was 99.31%, and the RSD (Relative Standard development Relative Standard Deviation, i.e., Standard Deviation/average). generally, the smaller the RSD, the better the proximity between the measured values, and the RSD% of the measured data by HPLC was required to be < 2.0). The sample recovery was 1.09%.
In the oral preparation, each gram of XIAOLUOTONG tablet contains daphnetin (C)19H12O7) Calculated, not less than 0.25 mg; one gram of capsule contains daphnetin (C)19H12O7) Calculated, not less than 0.50 mg; one gram of the granule contains daphnetin (C)19H12O7) Calculated, not less than 0.10 mg; one gram of the pill contains daphnetin (C)19H12O7) Calculated, the content of the active ingredient should not be less than 0.30 mg.
The following are examples of the application of the method of the present invention to the detection of pain-relieving tablets, capsules, granules or pills.
Example 1 example of the testing of pain-relieving tablets using the method provided by the invention
1. The identification and determination steps are as follows:
preparing a test solution: taking 6 tablets of the pain-relieving tablet, grinding, taking about 0.1-0.6 g of the sample, adding 5-15 ml of ethanol for dissolving, carrying out ultrasonic extraction (power 100W and frequency 25kHz) for 30min, filtering, and concentrating the filtrate to 1ml to serve as a test solution.
Preparing a reference medicinal material solution: preparing 0.5g of Genkwa flower control medicinal material, and preparing into control medicinal material solution by the same method.
And (3) thin-layer chromatography determination: according to VIB thin-layer chromatography which is an appendix of the 2005 edition of Chinese pharmacopoeia, 5 μ l of each of the two solutions is drawn, and the two solutions are respectively spotted on the same silica gel G thin-layer plate which takes sodium carboxymethylcellulose as an adhesive, and n-hexane-ethyl acetate-formic acid (1: 0.01) is taken as a developing agent, developed, taken out, dried and inspected under an ultraviolet lamp (365 nm). The sample chromatogram should show the same blue fluorescent spot at the position corresponding to the control chromatogram.
2. The examination and determination steps are as follows: the tablet is in accordance with the relevant regulations in Chinese pharmacopoeia tablet.
3. The extract determination procedure was as follows:
20 tablets of the pain-relieving tablet are taken and ground, about 2.0g of the pain-relieving tablet is precisely weighed and is subjected to hot dipping method in an alcohol-soluble extract measuring method in appendix XA of the first edition of Chinese pharmacopoeia (2005 edition), 95% ethanol is used as a solvent, and the content of the alcohol-soluble extract in the tablet is not less than 10.0%.
4. The content determination steps are as follows:
adopting high performance liquid chromatography, and using octadecylsilane chemically bonded silica as filler in a chromatographic column; methanol or acetonitrile and 0.2% phosphoric acid solution (50-55: 50-45) are taken as mobile phases; the detection wavelength was 346 nm. The number of theoretical plates should not be less than 3000 calculated as daphnoretin.
Preparation of a reference solution: accurately weighing appropriate amount of daphnoretin reference substance, placing in brown measuring flask, and adding methanol to obtain solution containing 10 μ g per 1 ml.
Preparing a test solution: precisely weighing about 1-2 g of XIAOLUOTONG tablet, precisely adding 25-50 ml of methanol into a conical flask with a plug, refluxing and extracting for 1h with 15-25 ml or 30-50 ml of methanol, cooling, supplementing the weight loss with methanol, shaking, and filtering.
Precisely sucking 20 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring content.
One gram of XIAOLUOTONG tablet contains daphnetin (C) and flos Genkwa strip19H12O7) Calculated, not less than 0.25 mg.
Example 2 example of testing pain-relieving capsules using the method provided by the invention
During identification and determination, 3 capsules are sampled; the rest of the procedure is the same as in example 1;
when checking and measuring, the capsule accords with various relevant regulations under Chinese pharmacopoeia capsule items;
during the measurement of the extract, 10 capsules are sampled; the content of alcohol soluble extract in capsule should not be less than 18.0%; the rest of the procedure is the same as in example 1;
when the content is measured, about 0.5-1 g of capsule is sampled, and each gram of capsule contains daphnetin (C)19H12O7) Calculated, not less than 0.50 mg; the rest is the same as example 1.
Example 3 example of testing of pain-relieving granules using the method provided by the invention
During identification and measurement, 1-2 bags of granules are sampled; the rest of the procedure is the same as in example 1;
when checking and measuring, the Chinese pharmacopoeia granule should be in accordance with various relevant regulations under the Chinese pharmacopoeia granule item;
when measuring the extract, 10 bags of granules are sampled; the alcohol-soluble extract content of the granule should not be less than 4.0%; the rest of the procedure is the same as in example 1;
when the content is measured, the granules are sampled about 2-6 g, and each gram of the granules contains daphnetin (C)19H12O7) Calculated, not less than 0.10 mg; the rest is the same as example 1.
Example 4 example of testing pain-relieving pills using the method provided by the invention
When identifying and determining, 6 pills are sampled; the rest of the procedure is the same as in example 1;
when checking and measuring, the pill should accord with various related regulations under the Chinese pharmacopoeia pill item;
when measuring the extract, the pill samples 20 granules; the content of alcohol soluble extract in pill is not less than 12.0%; the rest of the procedure is the same as in example 1;
when the content is measured, the pill is sampled about 1-2 g, and each gram of the pill contains daphnetin (C)19H12O7) Calculated, not less than 0.30 mg; the rest is the same as example 1.
Claims (2)
1. A method for detecting antirheumatic medicine is characterized in that besides characters and inspection, the method also comprises the steps of identification, extract determination and content determination: wherein,
(1) the identification steps are as follows:
firstly, taking the contents of the antirheumatic medicine pain-relieving preparation, grinding, adding ethanol for dissolving, ultrasonically extracting, filtering and concentrating to prepare a test solution;
collecting flos Genkwa contrast medicinal material, pulverizing, dissolving in ethanol, ultrasonic extracting, filtering, and making into contrast medicinal solution;
respectively sucking 5 mul of test solution and control solution, respectively dropping on the same silica gel G thin-layer plate with sodium carboxymethylcellulose as binder, developing with n-hexane, ethyl acetate and formic acid at ratio of 1: 0.01 as developing agent, air drying, and inspecting under 365nm ultraviolet lamp to obtain the same blue fluorescent spot at the position corresponding to the control material chromatogram in the test solution chromatogram;
(2) the extract determination step is that the content of the preparation for relieving pain in collaterals is taken, ground and precisely weighed, 95 percent ethanol is taken as a solvent, and reflux extraction is carried out, wherein the ethanol extract in a test sample is calculated by dry matter;
(3) the content determination steps are as follows:
precisely weighing a proper amount of daphnoretin reference substance, placing the reference substance in a brown measuring flask, and adding methanol to prepare a solution containing 10 mu g of reference substance per 1 ml;
secondly, grinding and precisely weighing the contents of the Xiaoluotong preparation, placing the mixture into a conical flask with a plug, precisely adding methanol, weighing, refluxing and extracting, cooling, weighing again, complementing the weight loss amount by methanol, shaking up, filtering, and preparing a test solution containing 20-40 mg of the contents of the Xiaoluotong preparation per 1 ml;
③ using octadecylsilane chemically bonded silica as filler, using methanol or acetonitrile and 0.2% phosphoric acid solution (50-55: 50-45) as mobile phase, detecting wavelength at 346nm with high performance liquid phase, and counting theoretical plate number according to daphnoretin C19H12O7Not less than 3000;
fourthly, respectively and precisely absorbing 20 mul of reference solution and test solution, injecting into a high performance liquid chromatograph, measuring, and using daphnoretin C in the test solution19H12O7The measured content;
the content of the preparation refers to the core layer of the tablet after the coating layer is removed; or removing the capsule from the original capsule; or granule and pill preparation.
2. Use of the method of testing for an antirheumatic drug according to claim 1, characterised in that the method is used for testing tablets, granules or pellets of an antirheumatic drug for pain relief.
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| 卫生部.消络痛胶囊.《卫生部颁药品标准 中药成方制剂第十二册》.1997, * |
| 吕武清等.高效液相色谱法测定了哥王中西瑞香素含量.《中华中医药学会制剂分会学术交流会论文汇编》.2003, * |
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