CN117191976A - Method for detecting deoxyarteannuin content in sweet wormwood - Google Patents
Method for detecting deoxyarteannuin content in sweet wormwood Download PDFInfo
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- 229930186526 Deoxyarteannuin Natural products 0.000 title claims abstract description 55
- 235000001405 Artemisia annua Nutrition 0.000 title claims abstract description 41
- 240000000011 Artemisia annua Species 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 24
- 239000013558 reference substance Substances 0.000 claims abstract description 45
- 238000012360 testing method Methods 0.000 claims abstract description 30
- 239000012488 sample solution Substances 0.000 claims abstract description 24
- ZQGMLVQZBIKKMP-NNWCWBAJSA-N deoxyartemisinin Chemical compound C([C@](O1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2OC(=O)[C@@H]4C ZQGMLVQZBIKKMP-NNWCWBAJSA-N 0.000 claims abstract description 23
- ZQGMLVQZBIKKMP-UHFFFAOYSA-N desoxyartemisinin Natural products O1C(O2)(C)CCC3C(C)CCC4C32C1OC(=O)C4C ZQGMLVQZBIKKMP-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000000523 sample Substances 0.000 claims abstract description 22
- 239000000126 substance Substances 0.000 claims abstract description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 111
- 239000000243 solution Substances 0.000 claims description 82
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 30
- 239000007789 gas Substances 0.000 claims description 25
- 238000001914 filtration Methods 0.000 claims description 19
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- 239000000706 filtrate Substances 0.000 claims description 15
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- 238000010438 heat treatment Methods 0.000 claims description 9
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 239000012159 carrier gas Substances 0.000 claims description 2
- 229910052757 nitrogen Inorganic materials 0.000 claims description 2
- 238000005259 measurement Methods 0.000 abstract description 10
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- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 8
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- BLUAFEHZUWYNDE-NNWCWBAJSA-N artemisinin Chemical compound C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2OC(=O)[C@@H]4C BLUAFEHZUWYNDE-NNWCWBAJSA-N 0.000 description 4
- 229960004191 artemisinin Drugs 0.000 description 4
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- PLQMEXSCSAIXGB-SAXRGWBVSA-N (+)-artemisinic acid Chemical compound C1=C(C)CC[C@H]2[C@H](C)CC[C@@H](C(=C)C(O)=O)[C@H]21 PLQMEXSCSAIXGB-SAXRGWBVSA-N 0.000 description 2
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- 229930101531 artemisinin Natural products 0.000 description 1
- PLQMEXSCSAIXGB-UHFFFAOYSA-N artemisininic acid Natural products C1=C(C)CCC2C(C)CCC(C(=C)C(O)=O)C21 PLQMEXSCSAIXGB-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a method for detecting the content of deoxyarteannuin in sweet wormwood, which comprises the following steps: s1, preparing a reference substance solution: taking deoxyarteannuin as a reference substance to prepare a reference substance solution; s2, preparing a sample solution: taking a sweet wormwood sample to be detected as a test sample to prepare a test sample solution; s3, respectively injecting the reference substance solution and the sample solution into a gas chromatograph to obtain chromatograms; and then according to chromatograms of the reference substance and the test substance, calculating to obtain the deoxyartemisinin content in the test substance solution. The invention provides a method for detecting the content of the deoxyarteannuin in the sweet wormwood by gas chromatography for the first time, which has the advantages of strong specificity, good operability, good accuracy, linearity, precision and durability, can realize the rapid quantitative measurement of the content of the deoxyarteannuin in the sweet wormwood, and has important significance for the production and application of the deoxyarteannuin.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a method for detecting the content of deoxyarteannuin in sweet wormwood herb.
Background
Herba Artemisiae Annuae is a Chinese medicinal material, and is dry aerial part of Artemisia annua L. Has effects of clearing away deficiency heat, cooling blood, removing steaming, relieving summer-heat, and preventing malaria. Is mainly used for treating pathogenic warm injury of yin, night fever, early cool, yin deficiency, fever, fatigue heat, bone steaming, summer heat exogenous infection, fever, thirst, malaria and cold and heat.
Herba Artemisiae Annuae contains various sesquiterpenes such as artemisinin as main active ingredient for resisting malaria, and also arteannuic acid, arteannuin, etc.; in addition, flavonoid compounds are contained. Deoxyarteannuin is sesquiterpene compound of arteannuin, has anti-inflammatory and antiulcer activities, can inhibit the absorption of nutrient substances such as surface membrane, cell membrane and mitochondrial membrane of plasmodium, and can achieve killing effect; meanwhile, the antibacterial effect can be achieved, and a certain inhibition effect can be achieved on staphylococcus aureus, mycobacterium tuberculosis and bacillus dysenteriae. The detection of the content of deoxyartemisinin in artemisia annua is of great importance for the application of deoxyartemisinin, but a reliable scheme is lacking.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for detecting the content of deoxyarteannuin in sweet wormwood aiming at the defects in the prior art.
In order to solve the technical problems, the invention adopts the following technical scheme: a method for detecting the deoxyarteannuin content in sweet wormwood herb comprises the following steps:
s1, preparing a reference substance solution: taking deoxyarteannuin as a reference substance to prepare a reference substance solution;
s2, preparing a sample solution: taking a sweet wormwood sample to be detected as a test sample to prepare a test sample solution;
s3, respectively injecting the reference substance solution and the sample solution into a gas chromatograph to obtain chromatograms; and then according to chromatograms of the reference substance and the test substance, calculating to obtain the deoxyartemisinin content in the test substance solution.
Preferably, the step S1 specifically includes:
the deoxyarteannuin is weighed and added into ethanol to prepare 0.02-0.03 mg/1mL of ethanol solution of the deoxyarteannuin as a reference substance solution.
Preferably, the step S1 specifically includes:
3-5 g of sweet wormwood herb is taken and placed in a container, 55-65 mL of petroleum ether is added, heating reflux is carried out at 60-90 ℃ for 0.5-1.5 hours, cooling is carried out to room temperature, filtering is carried out, filtrate is placed in an evaporation dish, the container and filter residues are washed by petroleum ether, the obtained washing liquid is added into the evaporation dish, the mixed liquid in the evaporation dish is evaporated to 1.5-2.5 mL in a water bath at 60-80 ℃, and finally ethanol is added to 4.5-5.5 mL, thus obtaining the sample solution.
Preferably, in the step S3, 1.8 to 2.2 μl of each of the control solution and the sample solution is precisely sucked, and the samples are injected into a gas chromatograph to obtain a chromatogram.
Preferably, the column length of the chromatographic column is 28-35 m, more preferably 30m; the column diameter is 0.150-0.350 mm, more preferably 0.250mm; the film thickness is 0.15 to 0.35. Mu.m, more preferably 0.25. Mu.m.
Preferably, in the step S3, the temperature of the sample inlet of the gas chromatograph is 255-285 ℃, and more preferably 260 ℃; the detector temperature is 255 to 275 ℃, more preferably 260 ℃.
Preferably, the split ratio of the gas chromatograph is 1:18 to 22, more preferably 1:20.
preferably, the column flow rate of the gas chromatograph is 0.8 to 1.2mL/min, more preferably 1mL/min.
Preferably, the theoretical plate number of the gas chromatograph is not less than 5000 as calculated as the deoxyartemisinin peak.
Preferably, the chromatographic conditions of the gas chromatograph are as follows:
capillary chromatographic column HP-5, column length 30m, column diameter 0.25mm, film thickness 0.25 μm;
the detector is a FID detector, the temperature of the sample inlet is 260 ℃, the temperature of the detector is 260 ℃, the carrier gas is nitrogen, and the flow rate is 1mL per minute;
gradient heating conditions: the initial temperature is 60 ℃, kept for 5min, and is raised to 160 ℃ at 10 ℃/min, and is raised to 260 ℃ at 5 ℃/min, and is kept for 5min;
the sample injection amount is 0.5 mu L, and the split ratio is 1:20.
The beneficial effects of the invention are as follows:
the invention provides a method for detecting the content of the deoxyarteannuin in the sweet wormwood by gas chromatography for the first time, which has the advantages of strong specificity, good operability, good accuracy, linearity, precision and durability, can realize the rapid quantitative measurement of the content of the deoxyarteannuin in the sweet wormwood, and has important significance for the production and application of the deoxyarteannuin.
Drawings
FIG. 1 is a chromatogram of a deoxyartemisinin control solution in example 2;
FIG. 2 is a chromatogram of a test solution of Artemisia annua medicinal material with DZ1-1 in example 2.
Detailed Description
The present invention is described in further detail below with reference to examples to enable those skilled in the art to practice the same by referring to the description.
It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
The test methods used in the following examples are conventional methods unless otherwise specified. The material reagents and the like used in the following examples are commercially available unless otherwise specified. The following examples were conducted under conventional conditions or conditions recommended by the manufacturer, without specifying the specific conditions. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The main instrumentation and reagents used in the following examples were:
gas chromatograph 1: agilent model 7890A; a FID detector; number MY-10070;
an electronic balance: meltrel-tolido instruments (Shanghai), model XS205DU; number MY-10048;
ultrapure water: self-making through an ultrapure water device; ultrapure water machine: sidoris Germany; model: 6110V; number MY-10010;
numerical control ultrasonic cleaner: kunshan, ultrosophy instruments Inc.; model KQ-500DE; number MY-10055;
petroleum ether: national pharmaceutical group chemical agents, inc; lot number 20211216; the content is 99.0 percent;
deoxyartemisinin: zhangjiawang Shengshang pharmaceutical Co., ltd; lot number 20210526; the content was 98.6%.
Sweet wormwood herb: zhangjiawang Kong Wipe medical Co., ltd. Lot 20201201.
Example 1
A method for detecting the deoxyarteannuin content in sweet wormwood herb comprises the following steps:
s1, preparing a reference substance solution:
precisely weighing 10.10mg of deoxyarteannuin reference substance, placing into a 10mL measuring flask, adding ethanol for dissolving and diluting to scale, and shaking; 3mL is precisely measured, placed in a 100mL measuring flask, dissolved and diluted to the scale by adding ethanol, and shaken uniformly to serve as a reference solution.
S2, preparing a sample solution:
precisely weighing 5g of sweet wormwood herb, placing the sweet wormwood herb into a conical flask with a plug, adding 60mL of petroleum ether, heating and refluxing for 1 hour at 80 ℃, cooling to room temperature, filtering, placing filtrate into an evaporation dish, washing the conical flask and filter residues with petroleum ether, adding the obtained washing liquid into the evaporation dish, evaporating the mixed liquid in the evaporation dish to about 2mL in a water bath at 70 ℃, transferring the mixed liquid into a 5mL measuring flask, adding ethanol to a scale, and shaking uniformly to obtain the sample solution.
S3, respectively precisely sucking 2 mu L of each of the reference solution and the sample solution, injecting into a gas chromatograph, and measuring to obtain a chromatogram; then the deoxyarteannuin content C in the sample solution is calculated according to the formula 0 ：
Wherein, A sample: area of deoxyarteannuin in the test solution;
and C pair: concentration of control in mg/mL;
p: the content of the reference substance;
and A pair: main peak area of the control solution;
c sample: test sample concentration in mg/mL.
Example 2 System suitability test
Solution preparation
(1) Control solution: precisely weighing 10.10mg of deoxyarteannuin reference substance, placing into a 10mL measuring flask, adding ethanol for dissolving and diluting to scale, and shaking; precisely measuring 3mL, placing in a 100mL measuring flask, adding ethanol for dissolving and diluting to scale, and shaking uniformly; as a reference solution;
(2) Preparation of test solution: precisely weighing 5g of sweet wormwood herb, placing the sweet wormwood herb into a conical flask with a plug, adding 60mL of petroleum ether, heating and refluxing for 1 hour, cooling, filtering, placing the filtrate into an evaporation dish, washing a container and residues with 15mL of petroleum ether in a partitioning way, filtering the washing liquid into the same evaporation dish, volatilizing the washing liquid to about 2mL at low temperature on a water bath, quantitatively transferring the washing liquid into a 5mL measuring flask, adding ethanol to a scale, and shaking uniformly to obtain a sample solution.
(II) measurement method
Precisely sucking 2 μl of each of the control solution and the sample solution, and injecting into a gas chromatograph for measurement.
(III) results data results are shown in tables 1-2
Table 1 results of System suitability test
| Name of the name | Retention time | Peak area | Theoretical plate number |
| Deoxyarteannuin | 29.440 | 289.69 | 1517525 |
TABLE 2 sample injection precision results
As an example, referring to fig. 1, the chromatogram of the above-mentioned deoxyarteannuin control solution is shown in fig. 2, which is a chromatogram of the sample solution of the arteannuin medicinal material with the sequence number DZ 1-1.
(IV) conclusion
The experimental results show that:
sample injection precision: the control solution was repeatedly injected for 6 needles, and the relative standard deviation of the peak area of deoxyartemisinin was 0.138% (1. Mu.g/g Repeatability (RSD) was not more than 8.0%); meets the requirements of Chinese pharmacopoeia.
Example 3 Linear and Range testing
Solution preparation
(1) Deoxyartemisinin control solution: precisely weighing 10.60mg of deoxyarteannuin reference substance, placing into a 10mL measuring flask, adding ethanol for dissolving and diluting to scale, and shaking; precisely measuring 3mL, placing in a 100mL measuring flask, adding ethanol for dissolving and diluting to scale, and shaking uniformly; as control solution 1.
(2) Deoxyartemisinin control stock solution: precisely weighing 10.80mg of deoxyarteannuin reference substance, placing into a 10mL measuring flask, adding ethanol for dissolving and diluting to scale, and shaking; as a control stock solution.
(3) Deoxyarteannuin linear solution
(1) 10% linear solution: precisely measuring 12mL of reference substance solution, placing in a 25mL measuring flask, adding ethanol for dilution to scale, shaking, filtering, and collecting subsequent filtrate.
(2) 40% linear solution: precisely measuring 13mL of reference substance solution, placing in a 10mL measuring flask, adding ethanol for dilution to scale, shaking, filtering, and collecting subsequent filtrate.
(3) 80% linear solution: precisely measuring 13mL of reference substance solution, placing in a 5mL measuring flask, adding ethanol for dilution to scale, shaking, filtering, and collecting subsequent filtrate.
(4) 100% linear solution: precisely measuring 1mL of reference substance solution, placing in a 20mL measuring flask, adding ethanol for dilution to scale, shaking, filtering, and collecting subsequent filtrate.
(5) 130% linear solution: precisely measuring 1mL of reference stock solution, placing in a 25mL measuring flask, adding ethanol for dilution to scale, shaking, filtering, and collecting subsequent filtrate.
(6) 220% linear solution: precisely measuring 1mL of reference stock solution, placing in a 20mL measuring flask, adding ethanol for dilution to scale, shaking, filtering, and collecting subsequent filtrate.
(II) measurement method
And precisely measuring 2 mu L of each linear solution with each concentration level, and injecting into a gas chromatograph for measurement. And (5) carrying out linear regression by taking the concentration as an abscissa and the peak area as an ordinate, and calculating the linear correlation coefficient of the linear regression.
And (5) carrying out linear regression on the peak area as an ordinate, and calculating a linear equation.
(III) results
The data results are shown in Table 3
TABLE 3 results of Linear and Range experiments
(IV) conclusion
The experimental results show that: in the range of 2.54-54.00 mug/mL, the linear equation of the linear relation of the concentration and the peak area of the deoxyartemisinin is Y=9.5549x+0.9026, and r=0.9999, so that the method has good linearity.
Example 4 quantitative limit test
Solution preparation
(1) Deoxyartemisinin control solution: precisely weighing 10.38mg of deoxyarteannuin reference substance, placing into a 10mL measuring flask, adding ethanol for dissolving and diluting to scale, and shaking; precisely measuring 3mL, placing in a 100mL measuring flask, adding ethanol for dissolving and diluting to scale, and shaking uniformly; as control solution 1.
(2) Deoxyarteannuin quantitative limiting solution
(1) Quantitative limit concentration: precisely measuring 2mL of the solution (reference substance solution 1) in a 25mL measuring flask, diluting with ethanol to scale, shaking, filtering, and collecting the filtrate.
(II) measurement method
Precisely measuring 2 μl of deoxyarteannuin quantitative limiting solution, and injecting into gas chromatograph for measurement.
(III) results
The data results are shown in Table 4
TABLE 4 quantitative limit test results
(IV) conclusion
The quantitative limit concentration of the deoxyarteannuin is 2.4912 mug/mL, and the signal-to-noise ratio is 10:1, a step of; a relative standard deviation of 3.07%) of 1 μg/g and a Repeatability (RSD) of not more than 8.0%; meets the regulations.
Example 5 recovery test
Solution preparation
(1) Deoxyartemisinin control solution: precisely weighing 10.40mg of deoxyarteannuin reference substance, placing into a 10mL measuring flask, adding ethanol for dissolving and diluting to scale, and shaking; precisely measuring 3mL, placing in a 100mL measuring flask, adding chloroform for dissolution, diluting to scale, and shaking uniformly; as control solution 1.
(2) Deoxyartemisinin control solution: precisely weighing 10.45mg of deoxyarteannuin reference substance, placing into a 10mL measuring flask, adding ethanol for dissolving and diluting to scale, and shaking; precisely measuring 3mL, placing in a 100mL measuring flask, adding ethanol for dissolving and diluting to scale, and shaking uniformly; as control solution 2.
(3) Deoxyartemisinin control stock solution: precisely weighing 8.8mg of deoxyarteannuin reference substance, placing into a 10mL measuring flask, adding ethanol for dissolving and diluting to scale, and shaking; precisely measuring 3mL, placing in a 100mL measuring flask, adding ethanol for dissolving and diluting to scale, and shaking uniformly; as a control stock solution.
(4) Preparation of test solution: precisely weighing 5.0g of sweet wormwood herb, placing into a conical flask with a plug, adding 60mL of petroleum ether, heating and refluxing for 1 hour, cooling, filtering, placing the filtrate into an evaporation dish, washing the container and residues with 15mL of petroleum ether in a partitioning way, filtering the washing liquid into the same evaporation dish, volatilizing to about 2mL at low temperature on a water bath, quantitatively transferring to a 5mL measuring flask, adding ethanol to a scale, and shaking uniformly to obtain the sweet wormwood herb.
(5) Preparation of recovery solution
(1) Preparation of 100% solution: precisely weighing 5.0g of sweet wormwood herb, placing the sweet wormwood herb into a conical flask with a plug, adding 3mL of reference substance solution stock solution, adding 60mL of petroleum ether, heating and refluxing for 1 hour, cooling, filtering, placing the filtrate into an evaporation dish, washing a container and residues with 15mL of petroleum ether in a fractional manner, filtering the washing liquid into the same evaporation dish, volatilizing the washing liquid to about 2mL at a low temperature in a water bath, quantitatively transferring the washing liquid into a 5mL measuring flask, adding ethanol to a scale, and shaking uniformly to obtain the sweet wormwood herb. Six parts were prepared in the same manner.
Recovery = (C-se:Sub>A)/bx 100%
Wherein A is the measured component content of the sample
B is the amount of the reference substance
C is the measured value;
(II) measurement method
Precisely sucking 2 μl of each of the control solution, the sample solution, and the recovery rate measurement sample solution, and injecting into a gas chromatograph for measurement. And calculating the recovery rate of the deoxyarteannuin according to an external standard method. The recovery rate is 75-120%, and the Relative Standard Deviation (RSD) is not more than 8.0%.
(III) results
The data results are shown in Table 5.
TABLE 5 recovery test results
(IV) conclusion
The experimental results show that: and calculating the recovery rate of the deoxyarteannuin according to an external standard method. The recovery rate is 89.35%, the RSD is 2.61%, and less than 8.0%, and the verification requirement is met. The content method is proved to have good accuracy.
EXAMPLE 6 precision test
6.1 repeatability
Solution preparation
(1) Deoxyartemisinin control solution: precisely weighing 10.40mg of deoxyarteannuin reference substance, placing into a 10mL measuring flask, adding ethanol for dissolving and diluting to scale, and shaking; precisely measuring 3mL, placing in a 100mL measuring flask, adding ethanol for dissolving and diluting to scale, and shaking uniformly; as control solution 1.
(2) Deoxyartemisinin control solution: precisely weighing 10.45mg of deoxyarteannuin reference substance, placing into a 10mL measuring flask, adding ethanol for dissolving and diluting to scale, and shaking; precisely measuring 3mL, placing in a 100mL measuring flask, adding ethanol for dissolving and diluting to scale, and shaking uniformly; as control solution 2.
(3) Preparation of test solution: precisely weighing 5.0g of sweet wormwood herb, placing the sweet wormwood herb into a conical flask with a plug, adding 60mL of petroleum ether, heating and refluxing for 1 hour, cooling, filtering, placing the filtrate into an evaporation dish, washing a container and residues with 15mL of petroleum ether in a partitioning way, filtering the washing liquid into the same evaporation dish, volatilizing the washing liquid to about 2mL at a low temperature on a water bath, quantitatively transferring the washing liquid into a 5mL measuring flask, adding ethanol to a scale, and shaking uniformly to obtain the sweet wormwood herb; 6 parts were prepared in parallel.
(II) measurement method
Precisely sucking 2 μl of each of the control solution and the sample solution, and measuring with gas chromatograph. The marked content of the product is calculated according to an external standard method. Its Relative Standard Deviation (RSD) is not more than 8.0%.
(III) results
The data results are shown in Table 6
TABLE 6 repeatability test results
(IV) conclusion
The experimental results show that: the average content of the repeated test sample is 51.72mg/g, the RSD is 1.911%, and the RSD is less than 8.0%, thereby conforming to the regulations.
6.2 intermediate precision
(1) Solution preparation
The preparation is carried out according to the method for preparing the solution under the repeated item.
(2) Measurement method
2 testers perform repeated tests at different time, respectively precisely sucking 2 mu L of each of the reference substance solution and the test substance solution, injecting into a gas chromatograph, and measuring. The marked content of the product is calculated according to an external standard method. The marked content and the relative standard deviation thereof of the product are calculated according to an external standard method, and the relative standard deviation of 2 testers and 12 measurement results are calculated to be less than 6.0 percent.
(III) results
The data results are shown in Table 7
TABLE 7 results of intermediate precision experiments
(IV) conclusion
The experimental results show that: the average content of the intermediate precision sample is 51.42mg/g, the RSD% of the intermediate precision is 0.97, and the RSD% of the intermediate precision is 0.86, which meets the regulations.
Example 7 solution stability test
Solution preparation
(1) Preparation of control solution for investigation: taking a reference substance solution under the system applicability, and placing the reference substance solution into a gas-phase small bottle.
(2) Preparing a test solution: sample solution 1 was taken under the reproducibility and placed in a gas phase vial.
(II) measurement method
Taking the reference substance solution and the test substance solution, placing for 0, 2, 4, 6, 8, 10, 12 and 24 hours at room temperature, respectively precisely sucking 2 mu L of each of the reference substance solution and the test substance solution, injecting into a gas chromatograph, and measuring. The peak area of the chromatogram was recorded, and the relative standard deviation of the peak area was not more than 8.0%.
(III) results
The data results are shown in tables 8-9.
TABLE 8 results of stability test of control solutions
TABLE 9 test results of stability of test solutions
(IV) conclusion
The experimental results show that: under the condition of room temperature, the relative standard deviation of the deoxyarteannuin measured by the reference substance solution in 24 hours is 0.123%, the relative standard deviation of the deoxyarteannuin measured by the test substance in 20 hours is 2.57%, and the requirements (rule: RSD is less than or equal to 8.0%) are met, and the result shows that: the control solution is relatively stable when left to stand at room temperature for 24 hours, and the test solution is relatively stable when left to stand at room temperature for 20 hours.
Example 8 durability test
8.1 preparation of solutions
(1) Preparation of control solution 1: precisely weighing 101mg of chloroform reference substance, placing into 200mL measuring flask, adding absolute ethanol for dissolving and diluting to scale, and shaking; precisely measuring 3mL, placing in a 25mL measuring flask, adding absolute ethyl alcohol to dissolve and dilute to scale, and shaking uniformly; as a control solution.
(2) Chloroform control solution 2: precisely weighing 102mg of chloroform reference substance, placing into 200mL measuring flask, adding absolute ethanol for dissolving and diluting to scale, and shaking; precisely measuring 3mL, placing in a 25mL measuring flask, adding absolute ethyl alcohol to dissolve and dilute to scale, and shaking uniformly; as a control solution.
2 parts were prepared in parallel.
(3) Preparation of test solution: precisely weighing 5g of herba Artemisiae Annuae, placing in 25mL volumetric flask, dissolving in absolute ethanol, performing ultrasonic treatment (power 720W, frequency 40 kHz) for 30 min, adding absolute ethanol to desired volume, shaking, filtering, and collecting filtrate; 2 parts were prepared in parallel.
8.2 measurement method
Adjusting chromatographic conditions according to the following table, precisely sucking 1 μl of each of the control solution and the sample solution, injecting into gas chromatograph, and measuring. The peak area of the chromatogram was recorded, and the relative standard deviation of the peak area was not more than 11%.
The marked content and the relative standard deviation of the product are calculated according to an external standard method and compared.
(III) results
The data results are shown in Table 10
TABLE 10 durability test results of deoxyartemisinin content
(IV) conclusion
The experimental results show that: the deoxyartemisinin content measurement and detection conditions are different columns, different chromatographic column sample inlet temperatures, different detector temperatures, the average deoxyartemisinin content is 50.68mg/g, and the RSD value is 1.15 to less than 11%, so that the method meets the requirements.
Example 9 detection of deoxyarteannuin content in sample
Three batches of herba Artemisiae Annuae (Zhangjiawei medicine Co., ltd., batch Nos. 20210312, 20210305, 20210317) were prepared into test solutions according to the method under the test solution preparation item, respectively, and were measured under the above chromatographic conditions. The measurement results are shown in Table 11.
TABLE 11 determination of deoxyartemisinin content
Through the detection of the three batches of samples, the result of the deoxyartemisinin content is parallel and stable.
Although embodiments of the present invention have been disclosed above, it is not limited to the use of the description and embodiments, it is well suited to various fields of use for the invention, and further modifications may be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the particular details without departing from the general concepts defined in the claims and the equivalents thereof.
Claims (10)
1. The method for detecting the deoxyarteannuin content in the sweet wormwood herb is characterized by comprising the following steps of:
s1, preparing a reference substance solution: taking deoxyarteannuin as a reference substance to prepare a reference substance solution;
s2, preparing a sample solution: taking a sweet wormwood sample to be detected as a test sample to prepare a test sample solution;
s3, respectively injecting the reference substance solution and the sample solution into a gas chromatograph to obtain chromatograms; and then according to chromatograms of the reference substance and the test substance, calculating to obtain the deoxyartemisinin content in the test substance solution.
2. The method for detecting the deoxyarteannuin content in sweet wormwood according to claim 1, wherein the step S1 is specifically:
the deoxyarteannuin is weighed and added into ethanol to prepare 0.02-0.03 mg/1mL of ethanol solution of the deoxyarteannuin as a reference substance solution.
3. The method for detecting the deoxyarteannuin content in sweet wormwood according to claim 1, wherein the step S2 is specifically:
3-5 g of sweet wormwood herb is taken and placed in a container, 55-65 mL of petroleum ether is added, heating reflux is carried out at 60-90 ℃ for 0.5-1.5 hours, cooling is carried out to room temperature, filtering is carried out, filtrate is placed in an evaporation dish, the container and filter residues are washed by petroleum ether, the obtained washing liquid is added into the evaporation dish, the mixed liquid in the evaporation dish is evaporated to 1.5-2.5 mL in a water bath at 60-80 ℃, and finally ethanol is added to 4.5-5.5 mL, thus obtaining the sample solution.
4. The method for detecting the content of deoxyarteannuin in sweet wormwood according to claim 1, wherein in the step S3, 1.8-2.2 μl of each of the reference substance solution and the test substance solution is precisely sucked, and is injected into a gas chromatograph to obtain a chromatogram.
5. The method for detecting the content of deoxyarteannuin in sweet wormwood according to claim 1, which is characterized in that the column length of the chromatographic column is 28-35 m, the column diameter is 0.150-0.350 mm, and the film thickness is 0.15-0.35 μm.
6. The method for detecting the deoxyarteannuin content in sweet wormwood according to claim 1, wherein in the step S3, the sample inlet temperature of the gas chromatograph is 255-285 ℃, and the detector temperature is 255-275 ℃.
7. The method for detecting the content of deoxyarteannuin in sweet wormwood according to claim 1, wherein the split ratio of the gas chromatograph is 1:18 to 22.
8. The method for detecting the content of deoxyarteannuin in sweet wormwood according to claim 1, wherein the column flow rate of the gas chromatograph is 0.8-1.2 mL/min.
9. The method for detecting the content of deoxyarteannuin in sweet wormwood according to claim 1, wherein the theoretical plate number of the gas chromatograph is not less than 5000 calculated according to the deoxyarteannuin peak.
10. The method for detecting the content of deoxyarteannuin in herba Artemisiae Annuae according to any one of claims 1-9, wherein the chromatographic conditions of the gas chromatograph are as follows:
capillary chromatographic column HP-5, column length 30m, column diameter 0.25mm, film thickness 0.25 μm;
the detector is a FID detector, the temperature of the sample inlet is 260 ℃, the temperature of the detector is 260 ℃, the carrier gas is nitrogen, and the flow rate is 1mL per minute;
gradient heating conditions: the initial temperature is 60 ℃, kept for 5min, and is raised to 160 ℃ at 10 ℃/min, and is raised to 260 ℃ at 5 ℃/min, and is kept for 5min;
the sample injection amount is 0.5 mu L, and the split ratio is 1:20.
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