Method for detecting deoxyartemisinin content in artemisia annua

Abstract

The application discloses a method for detecting the content of desoxyartemisinin in artemisia, which comprises the following steps: S1, preparing a control solution: taking desoxyartemisinin as a control sample and preparing a control solution; S2, preparing a sample solution: taking an artemisia sample to be detected as a sample and preparing a sample solution; S3, respectively injecting the control solution and the sample solution into a gas chromatograph to obtain chromatograms; and then calculating the content of desoxyartemisinin in the sample solution according to the chromatograms of the control sample and the sample. The application provides a method for detecting the content of desoxyartemisinin in artemisia by gas chromatography for the first time, the method has strong specificity, good operability, and good accuracy, linearity, precision and durability, can realize rapid quantitative detection of the content of desoxyartemisinin in artemisia, and has important significance for the production and application of desoxyartemisinin.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a method for detecting the content of deoxyartemisinin in Artemisia annua. Background Technology

[0002] Artemisia annua L., a plant belonging to the genus Artemisia in the family Asteraceae, is a traditional Chinese medicine. It has the effects of clearing heat from deficiency, cooling the blood and relieving steaming sensations, relieving summer heat, and treating malaria. It is mainly used to treat yin deficiency due to febrile diseases, night fever with morning coolness, yin deficiency fever, consumptive fever with bone steaming sensations, summer heat syndrome, fever with thirst, and malaria with chills and fever.

[0003] Artemisia annua contains various sesquiterpenes, such as artemisinin, the main active ingredient in its antimalarial activity, as well as artemisinic acid and artemisinol; it also contains flavonoids. Deoxyartemisinin, a sesquiterpene compound of the artemisinin family, possesses anti-inflammatory and anti-ulcer activities. It can inhibit the surface membrane, cell membrane, and mitochondrial membrane of Plasmodium, blocking the absorption of nutrients and thus achieving a killing effect. It also has antibacterial effects, inhibiting Staphylococcus aureus, Mycobacterium tuberculosis, and Shigella dysenteriae to some extent. The detection of deoxyartemisinin content in Artemisia annua is of great significance for its application, but a reliable method is currently lacking. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for detecting the content of deoxyartemisinin in Artemisia annua, addressing the shortcomings of the prior art.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for detecting the content of deoxyartemisinin in Artemisia annua, comprising the following steps:

[0006] S1. Preparation of reference solution: Deoxyartemisinin was used as a reference standard and a reference solution was prepared.

[0007] S2. Preparation of test solution: Take the Artemisia annua sample to be tested as the test sample and prepare the test solution;

[0008] S3. Inject the reference solution and the test solution into the gas chromatograph to obtain chromatograms; then, based on the chromatograms of the reference solution and the test solution, calculate the content of deoxyartemisinin in the test solution.

[0009] Preferably, step S1 specifically comprises:

[0010] Weigh out deoxyartemisinin and add it to ethanol to prepare an ethanol solution of 0.02–0.03 mg / mL deoxyartemisinin, which will be used as a reference solution.

[0011] Preferably, step S1 specifically comprises:

[0012] Take 3-5g of Artemisia annua and place it in a container. Add 55-65mL of petroleum ether and heat under reflux at 60-90℃ for 0.5-1.5 hours. Cool to room temperature and filter. Place the filtrate in an evaporating dish and wash the container and filter residue with petroleum ether. Add the washing solution to the evaporating dish and evaporate the mixture in the evaporating dish to dryness in a water bath at 60-80℃ to 1.5-2.5mL. Finally, add ethanol to 4.5-5.5mL to obtain the test solution.

[0013] Preferably, in step S3, 1.8–2.2 μL of the reference solution and the test solution are precisely pipetted and injected into the gas chromatograph to obtain chromatograms.

[0014] Preferably, the column length is 28-35m, more preferably 30m; the column diameter is 0.150-0.350mm, more preferably 0.250mm; and the film thickness is 0.15-0.35μm, more preferably 0.25μm.

[0015] Preferably, in step S3, the injection port temperature of the gas chromatograph is 255–285°C, more preferably 260°C; and the detector temperature is 255–275°C, more preferably 260°C.

[0016] Preferably, the split ratio of the gas chromatograph is 1:18 to 22, more preferably 1:20.

[0017] Preferably, the column flow rate of the gas chromatograph is 0.8 to 1.2 mL / min, more preferably 1 mL / min.

[0018] Preferably, the theoretical plate number of the gas chromatograph, calculated based on the deoxyartemisinin peak, is not less than 5000.

[0019] Preferably, the chromatographic conditions of the gas chromatograph are as follows:

[0020] HP-5 capillary chromatography column, column length 30m * column diameter 0.25mm * film thickness 0.25μm;

[0021] The detector is an FID detector, the injection port temperature is 260℃, the detector temperature is 260℃, the carrier gas is nitrogen, and the flow rate is 1 mL per minute.

[0022] Gradient heating conditions: Initial temperature 60℃, hold for 5 min, increase to 160℃ at 10℃ / min, then increase to 260℃ at 5℃ / min, hold for 5 min;

[0023] The injection volume was 0.5 μL, and the split ratio was 1:20.

[0024] The beneficial effects of this invention are:

[0025] This invention provides for the first time a method for detecting the content of deoxyartemisinin in Artemisia annua by gas chromatography. The method of this invention has high specificity, good operability, and good accuracy, linearity, precision and robustness. It can realize the rapid quantitative determination of the content of deoxyartemisinin in Artemisia annua, which is of great significance to the production and application of deoxyartemisinin. Attached Figure Description

[0026] Figure 1 The chromatogram of the deoxyartemisinin reference solution in Example 2;

[0027] Figure 2 The chromatogram is of the Artemisia annua test sample solution with serial number DZ1-1 in Example 2. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0029] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments whose manufacturers are not specified, they are all commercially available products.

[0031] The main instruments, equipment, and reagents used in the following examples are as follows:

[0032] Gas chromatograph 1: Agilent 7890A; FID detector; serial number MY-10070;

[0033] Electronic balance: Mettler Toledo Instruments (Shanghai) Co., Ltd., Model XS205DU; Serial number MY-10048;

[0034] Ultrapure water: self-made using an ultrapure water purifier; Ultrapure water purifier: Sartorius, Germany; Model: 6110V; Serial number: MY-10010;

[0035] CNC ultrasonic cleaner: Kunshan Ultrasonic Instrument Co., Ltd.; Model KQ-500DE; Serial number MY-10055;

[0036] Petroleum ether: Sinopharm Chemical Reagent Co., Ltd.; Batch No. 20211216; Purity 99.0%;

[0037] Deoxyartemisinin: Zhangjiagang Weisheng Pharmaceutical Co., Ltd.; Batch No. 20210526; Content 98.6%.

[0038] Artemisia annua: Zhangjiagang Weisheng Pharmaceutical Co., Ltd., batch number 20201201.

[0039] Example 1

[0040] A method for detecting the content of deoxyartemisinin in Artemisia annua includes the following steps:

[0041] S1. Preparation of reference solution:

[0042] Accurately weigh 10.10 mg of deoxyartemisinin reference standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with ethanol, and shake well; accurately measure 3 mL, place it in a 100 mL volumetric flask, dissolve and dilute to the mark with ethanol, and shake well to obtain the reference solution.

[0043] S2. Preparation of the test solution:

[0044] Accurately weigh 5g of Artemisia annua and place it in a stoppered conical flask. Add 60mL of petroleum ether and heat under reflux at 80℃ for 1 hour. Cool to room temperature and filter. Place the filtrate in an evaporating dish and wash the conical flask and filter residue with petroleum ether. Add the resulting washings to the evaporating dish. Evaporate the mixture in the evaporating dish to dryness in a water bath at 70℃ to about 2mL. Transfer the mixture to a 5mL volumetric flask, add ethanol to the mark, and shake well to obtain the test solution.

[0045] S3. Accurately pipette 2 μL each of the standard solution and the test solution into the gas chromatograph, and determine the chromatograms. Then, calculate the deoxyartemisinin content C0 in the test solution according to the formula.

[0046]

[0047] Among them, Sample A: the area of ​​deoxyartemisinin in the test solution;

[0048] C: Concentration of reference standard, in mg / mL;

[0049] P: Content of reference standard;

[0050] A: Peak area of ​​the reference solution;

[0051] C sample: concentration of the test sample, in mg / mL.

[0052] Example 2 System Suitability Test

[0053] (I) Solution Preparation

[0054] (1) Reference solution: Accurately weigh 10.10 mg of deoxyartemisinin reference standard, place it in a 10 mL volumetric flask, add ethanol to dissolve and dilute to the mark, and shake well; accurately measure 3 mL, place it in a 100 mL volumetric flask, add ethanol to dissolve and dilute to the mark, and shake well; this is the reference solution.

[0055] (2) Preparation of test solution: Accurately weigh 5g of Artemisia annua and place it in a stoppered conical flask. Add 60mL of petroleum ether, heat under reflux for 1 hour, cool, filter, and place the filtrate in an evaporating dish. Wash the container and residue with 15mL of petroleum ether in several portions. Filter the washings into the same evaporating dish and evaporate at low temperature on a water bath to about 2mL. Quantitatively transfer the solution to a 5mL volumetric flask, add ethanol to the mark, and shake well to obtain the test solution.

[0056] (II) Measurement Method

[0057] Accurately pipette 2 μL each of the reference solution and the test solution into the gas chromatograph and determine their properties.

[0058] (III) Results and Data: The results are shown in Tables 1-2.

[0059] Table 1 System Applicability Test Results

[0060] name Retention time Peak area Theoretical number of plates deoxyartemisinin 29.440 289.69 1517525

[0061] Table 2. Injection precision results

[0062]

[0063] As an example, refer to Figure 1 The chromatogram is shown below for the deoxyartemisinin reference solution. Figure 2 The chromatogram of the Artemisia annua sample solution with serial number DZ1-1.

[0064] (IV) Conclusion

[0065] Experimental results show that:

[0066] Injection precision: The relative standard deviation of the deoxyartemisinin peak area was 0.138% after six repeated injections of the reference solution (the repeatability (RSD) of 1 μg / g shall not exceed 8.0%), which meets the requirements of the Chinese Pharmacopoeia.

[0067] Example 3: Linearity and Range Test

[0068] (I) Solution Preparation

[0069] (1) Deoxyartemisinin reference solution: Accurately weigh 10.60 mg of deoxyartemisinin reference standard, place it in a 10 mL volumetric flask, add ethanol to dissolve and dilute to the mark, and shake well; accurately measure 3 mL, place it in a 100 mL volumetric flask, add ethanol to dissolve and dilute to the mark, and shake well; this is reference solution 1.

[0070] (2) Deoxyartemisinin reference stock solution: Accurately weigh 10.80 mg of deoxyartemisinin reference standard, place it in a 10 mL volumetric flask, add ethanol to dissolve and dilute to the mark, and shake well; this is the reference stock solution.

[0071] (3) Linear solution of deoxyartemisinin

[0072] ①10% linear solution: Accurately measure 12 mL of the reference solution, place it in a 25 mL volumetric flask, dilute with ethanol to the mark, shake well, filter, and take the filtrate.

[0073] ②40% linear solution: Accurately measure 13 mL of the reference solution, place it in a 10 mL volumetric flask, dilute with ethanol to the mark, shake well, filter, and take the filtrate.

[0074] ③80% linear solution: Accurately measure 13 mL of the reference solution, place it in a 5 mL volumetric flask, dilute with ethanol to the mark, shake well, filter, and take the filtrate.

[0075] ④100% linear solution: Accurately measure 115 mL of the reference solution, place it in a 20 mL volumetric flask, dilute to the mark with ethanol, shake well, filter, and take the filtrate.

[0076] ⑤ 130% linear solution: Accurately measure 1 mL of the reference stock solution, place it in a 25 mL volumetric flask, dilute to the mark with ethanol, shake well, filter, and collect the filtrate.

[0077] ⑥220% linear solution: Accurately measure 1 mL of the reference stock solution, place it in a 20 mL volumetric flask, dilute to the mark with ethanol, shake well, filter, and collect the filtrate.

[0078] (II) Measurement Method

[0079] Precisely measure 2 μL of each linear solution at each concentration level, inject it into the gas chromatograph, and measure the concentration. Perform linear regression with concentration on the x-axis and peak area on the y-axis, and calculate the linear correlation coefficient.

[0080] Linear regression is performed using the peak area as the ordinate, and the linear equation is calculated.

[0081] (III) Results

[0082] The data results are shown in Table 3.

[0083] Table 3. Results of Linearity and Range Tests

[0084]

[0085]

[0086] (IV) Conclusion

[0087] Experimental results show that within the range of 2.54 μg / mL to 54.00 μg / mL, the linear relationship between concentration and peak area of ​​deoxyartemisinin is Y = 9.5549x + 0.9026, r = 0.9999, indicating good linearity of the method.

[0088] Example 4 Limit of Quantitation Test

[0089] (I) Solution Preparation

[0090] (1) Deoxyartemisinin reference solution: Accurately weigh 10.38 mg of deoxyartemisinin reference standard, place it in a 10 mL volumetric flask, add ethanol to dissolve and dilute to the mark, and shake well; accurately measure 3 mL, place it in a 100 mL volumetric flask, add ethanol to dissolve and dilute to the mark, and shake well; this is reference solution 1.

[0091] (2) Deoxyartemisinin limit of quantitation solution

[0092] ① Quantitative limit concentration: Accurately measure 2 mL of solution (1) (reference solution 1), place it in a 25 mL volumetric flask, dilute with ethanol to the mark, shake well, filter, and take the filtrate.

[0093] (II) Measurement Method

[0094] Accurately measure 2 μL of deoxyartemisinin quantification limit solution, inject it into the gas chromatograph, and determine the result.

[0095] (III) Results

[0096] The data results are shown in Table 4.

[0097] Table 4 Results of Limit of Quantitation Test

[0098]

[0099]

[0100] (IV) Conclusion

[0101] The limit of quantitation for deoxyartemisinin was 2.4912 μg / mL, the signal-to-noise ratio was 10:1, the relative standard deviation was 3.07%, and the repeatability (RSD) of 1 μg / g was not more than 8.0%; all of which met the requirements.

[0102] Example 5 Recovery Rate Test

[0103] (I) Solution Preparation

[0104] (1) Deoxyartemisinin reference solution: Accurately weigh 10.40 mg of deoxyartemisinin reference standard, place it in a 10 mL volumetric flask, add ethanol to dissolve and dilute to the mark, and shake well; accurately measure 3 mL, place it in a 100 mL volumetric flask, add chloroform to dissolve and dilute to the mark, and shake well; this is reference solution 1.

[0105] (2) Deoxyartemisinin reference solution: Accurately weigh 10.45 mg of deoxyartemisinin reference standard, place it in a 10 mL volumetric flask, add ethanol to dissolve and dilute to the mark, and shake well; accurately measure 3 mL, place it in a 100 mL volumetric flask, add ethanol to dissolve and dilute to the mark, and shake well; this is reference solution 2.

[0106] (3) Deoxyartemisinin reference solution stock solution: Accurately weigh 8.8 mg of deoxyartemisinin reference standard, place it in a 10 mL volumetric flask, add ethanol to dissolve and dilute to the mark, and shake well; accurately measure 3 mL, place it in a 100 mL volumetric flask, add ethanol to dissolve and dilute to the mark, and shake well; this is the reference standard stock solution.

[0107] (4) Preparation of test solution: Accurately weigh 5.0 g of Artemisia annua and place it in a stoppered conical flask. Add 60 mL of petroleum ether, heat under reflux for 1 hour, cool, filter, and place the filtrate in an evaporating dish. Wash the container and residue with 15 mL of petroleum ether in several portions. Filter the washings into the same evaporating dish and evaporate at low temperature in a water bath to about 2 mL. Quantitatively transfer the solution to a 5 mL volumetric flask, add ethanol to the mark, and shake well to obtain the test solution.

[0108] (5) Preparation of recovery solution

[0109] ① Preparation of 100% solution: Accurately weigh 5.0g of Artemisia annua and place it in a stoppered conical flask. Add 3mL of the (3) reference solution stock solution and 60mL of petroleum ether. Heat under reflux for 1 hour, cool, and filter. Place the filtrate in an evaporating dish and wash the container and residue with 15mL of petroleum ether in several portions. Filter the washings into the same evaporating dish and evaporate at low temperature in a water bath to about 2mL. Quantitatively transfer the solution to a 5mL volumetric flask, add ethanol to the mark, and shake well. Prepare six portions in the same way.

[0110] Recovery rate = (CA) / B × 100%

[0111] In the formula, A represents the amount of the analyte contained in the test sample.

[0112] B represents the amount of reference standard added.

[0113] C represents the measured value;

[0114] (II) Measurement Method

[0115] Accurately pipette 2 μL each of the reference solution, the test solution, and the sample solution for recovery determination, and inject them into the gas chromatograph for analysis. Calculate the recovery rate of deoxyartemisinin using the external standard method. The recovery rate should be between 75% and 120%, and its relative standard deviation (RSD) should not exceed 8.0%.

[0116] (III) Results

[0117] The data results are shown in Table 5.

[0118] Table 5 Results of the recovery rate test

[0119]

[0120] (IV) Conclusion

[0121] Experimental results showed that the recovery rate of deoxyartemisinin calculated using the external standard method was 89.35%, with an RSD of 2.61%, which was less than 8.0%, meeting the validation requirements. This indicates that the method for determining the content has good accuracy.

[0122] Example 6 Precision Test

[0123] 6.1 Repeatability

[0124] (I) Solution Preparation

[0125] (1) Deoxyartemisinin reference solution: Accurately weigh 10.40 mg of deoxyartemisinin reference standard, place it in a 10 mL volumetric flask, add ethanol to dissolve and dilute to the mark, and shake well; accurately measure 3 mL, place it in a 100 mL volumetric flask, add ethanol to dissolve and dilute to the mark, and shake well; this is reference solution 1.

[0126] (2) Deoxyartemisinin reference solution: Accurately weigh 10.45 mg of deoxyartemisinin reference standard, place it in a 10 mL volumetric flask, add ethanol to dissolve and dilute to the mark, and shake well; accurately measure 3 mL, place it in a 100 mL volumetric flask, add ethanol to dissolve and dilute to the mark, and shake well; this is reference solution 2.

[0127] (3) Preparation of test solution: Accurately weigh 5.0 g of Artemisia annua and place it in a stoppered conical flask. Add 60 mL of petroleum ether, heat under reflux for 1 hour, cool, filter, and place the filtrate in an evaporating dish. Wash the container and residue with 15 mL of petroleum ether in several portions. Filter the washings into the same evaporating dish and evaporate at low temperature on a water bath to about 2 mL. Quantitatively transfer the solution to a 5 mL volumetric flask, add ethanol to the mark, and shake well. Prepare 6 parallel solutions.

[0128] (II) Measurement Method

[0129] Accurately pipette 2 μl each of the reference solution and the test solution into the gas chromatograph and determine the content. Calculate the labeled content of this product using the external standard method. The relative standard deviation (RSD) should not exceed 8.0%.

[0130] (III) Results

[0131] The data results are shown in Table 6.

[0132] Table 6 Results of Repeatability Tests

[0133]

[0134] (IV) Conclusion

[0135] The experimental results showed that the average content of the repeatable test sample was 51.72 mg / g, the RSD was 1.911%, which was less than 8.0%, and met the requirements.

[0136] 6.2 Intermediate Precision

[0137] (1) Solution preparation

[0138] Prepare the solution according to the solution preparation method under the repeatability section.

[0139] (2) Measurement method

[0140] Two testers performed the repeatability test at different times. They precisely pipetted 2 μL each of the reference solution and the test solution into the gas chromatograph and determined the content. The labeled content of the product was calculated using the external standard method. The labeled content and its relative standard deviation were calculated using the external standard method, and the relative standard deviations of the results from the two testers and 12 determinations were also calculated; all relative standard deviations must not exceed 6.0%.

[0141] (III) Results

[0142] The data results are shown in Table 7.

[0143] Table 7 Results of intermediate precision test

[0144]

[0145] (IV) Conclusion

[0146] The experimental results show that the average content of the test sample with intermediate precision is 51.42 mg / g, the RSD% of intermediate precision is 0.97, and the RSD% of precision is 0.86, which meets the requirements.

[0147] Example 7 Solution Stability Test

[0148] (I) Solution Preparation

[0149] (1) Preparation of reference solution for testing: Take the reference solution under the system suitability section and place it in a gas chromatograph vial.

[0150] (2) Preparation of test solution: Take test solution 1 from the repeatability section and place it in a gas phase vial.

[0151] (II) Measurement Method

[0152] Take the above-mentioned reference solution and test solution, and let them stand at room temperature for 0, 2, 4, 6, 8, 10, 12, and 24 hours. Accurately pipette 2 μL of each solution and inject it into the gas chromatograph for determination. Record the peak area of ​​the chromatogram; the relative standard deviation of the peak area should not exceed 8.0%.

[0153] (III) Results

[0154] The data results are shown in Tables 8 and 9.

[0155] Table 8 Results of stability test of reference solution

[0156]

[0157] Table 9 Results of the stability test of the test sample solution

[0158]

[0159]

[0160] (IV) Conclusion

[0161] The experimental results showed that, under room temperature conditions, the relative standard deviation of deoxyartemisinin measured in the reference solution within 24 hours was 0.123%, and the relative standard deviation of deoxyartemisinin measured in the test sample within 20 hours was 2.57%, which met the requirements (specified: RSD≤8.0%). The results indicated that the reference solution was relatively stable after 24 hours at room temperature, and the test sample solution was relatively stable after 20 hours at room temperature.

[0162] Example 8 Durability Test

[0163] 8.1 Solution Preparation

[0164] (1) Preparation of reference solution 1: Accurately weigh 101 mg of chloroform reference standard, place it in a 200 mL volumetric flask, add anhydrous ethanol to dissolve and dilute to the mark, and shake well; accurately measure 3 mL, place it in a 25 mL volumetric flask, add anhydrous ethanol to dissolve and dilute to the mark, and shake well; this is the reference solution.

[0165] (2) 2 Chloroform reference solution: Accurately weigh 102 mg of chloroform reference standard, place it in a 200 mL volumetric flask, add anhydrous ethanol to dissolve and dilute to the mark, and shake well; accurately measure 3 mL, place it in a 25 mL volumetric flask, add anhydrous ethanol to dissolve and dilute to the mark, and shake well; this is the reference solution.

[0166] Prepare two parallel portions.

[0167] (3) Preparation of test solution: Accurately weigh 5g of Artemisia annua, place it in a 25mL volumetric flask, add anhydrous ethanol to dissolve it, sonicate (power 720W, frequency 40kHz) for 30 minutes, add anhydrous ethanol to make up to volume, shake well, filter, and take the filtrate to obtain the test solution; prepare 2 parallel samples.

[0168] 8.2 Determination Method

[0169] Adjust the chromatographic conditions according to the table below. Accurately pipette 1 μL each of the reference solution and the test solution and inject them into the gas chromatograph. Measure the results. Record the peak areas of the chromatograms. The relative standard deviation of the peak areas should not exceed 11%.

[0170] The labeled content and its relative standard deviation of this product were calculated using the external standard method and compared.

[0171]

[0172]

[0173] (III) Results

[0174] The data results are shown in Table 10.

[0175] Table 10 Results of Deoxyartemisinin Content Durability Test

[0176]

[0177] (IV) Conclusion

[0178] Experimental results show that the average deoxyartemisinin content was 50.68 mg / g under different column conditions, different column injection temperatures, and different detector temperatures, with an RSD value of 1.15, which is less than 11%, meeting the requirements.

[0179] Example 9: Detection of deoxyartemisinin content in samples

[0180] Three batches of Artemisia annua (Zhangjiagang Weisheng Pharmaceutical Co., Ltd., batch numbers 20210312, 20210305, and 20210317) were used to prepare test solutions according to the methods described in the "Preparation of Test Solution" section, and the chromatographic conditions were determined as described above. The test results are shown in Table 11.

[0181] Table 11 Results of Deoxyartemisinin Content Determination

[0182]

[0183]

[0184] The results of the above three batches of sample testing showed that the deoxyartemisinin content was parallel and stable.

[0185] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A method for detecting the content of desoxyartemisinin in artemisia annua, characterized in that, Includes the following steps: S1. Preparation of reference solution: Deoxyartemisinin was used as a reference standard and a reference solution was prepared. S2. Preparation of test solution: Take the Artemisia annua sample to be tested as the test sample and prepare the test solution; S3. Inject the reference solution and the test solution into the gas chromatograph to obtain chromatograms; then, based on the chromatograms of the reference solution and the test solution, calculate the content of deoxyartemisinin in the test solution. Step S2 specifically involves: Take 3-5g of Artemisia annua and place it in a container. Add 55-65mL of petroleum ether and heat under reflux at 60-90℃ for 0.5-1.5 hours. Cool to room temperature and filter. Place the filtrate in an evaporating dish and wash the container and filter residue with petroleum ether. Add the washing liquid to the evaporating dish and evaporate the mixture in the evaporating dish to dryness in a water bath at 60-80℃ to 1.5-2.5mL. Finally, add ethanol to 4.5-5.5mL to obtain the test solution. The chromatographic conditions of the gas chromatograph are as follows: HP-5 capillary chromatography column; The detector is an FID detector, with an injection port temperature of 255–285℃ and a detector temperature of 255–275℃. Gradient heating conditions: Initial temperature 60℃, hold for 5 min, increase to 160℃ at 10℃ / min, then increase to 260℃ at 5℃ / min, hold for 5 min.

2. The method for detecting the content of desoxyartemisinins in artemisia according to claim 1, characterized in that, Step S1 specifically involves: Weigh out deoxyartemisinin and add it to ethanol to prepare an ethanol solution of 0.02–0.03 mg / mL deoxyartemisinin, which will be used as a reference solution.

3. The method for detecting the deoxyartemisinin content in Artemisia annua according to claim 1, characterized in that, In step S3, 1.8–2.2 μL of the reference solution and the test solution are precisely pipetted into the gas chromatograph to obtain chromatograms.

4. The method for detecting the deoxyartemisinin content in Artemisia annua according to claim 1, characterized in that, The chromatographic column has a length of 28–35 m, a diameter of 0.150–0.350 mm, and a film thickness of 0.15–0.35 μm.

5. The method for detecting the deoxyartemisinin content in Artemisia annua according to claim 1, characterized in that, The split ratio of the gas chromatograph is 1:18 to 22.

6. The method for detecting the content of desoxyartemisinine in artemisia according to claim 1, characterized in that, The column flow rate of the gas chromatograph is 0.8–1.2 mL / min.

7. The method for detecting the deoxyartemisinin content in Artemisia annua according to claim 1, characterized in that, The theoretical plate number of the gas chromatograph, calculated based on the deoxyartemisinin peak, shall not be less than 5000.

8. The method according to any one of claims 1 to 7, wherein the deoxyarteannuin content of the artemisia is determined. The chromatographic conditions of the gas chromatograph are as follows: HP-5 capillary chromatography column, column length 30m * column diameter 0.25mm * film thickness 0.25μm; The detector is an FID detector, the injection port temperature is 260℃, the detector temperature is 260℃, the carrier gas is nitrogen, and the flow rate is 1 mL per minute. Gradient heating conditions: Initial temperature 60℃, hold for 5 min, increase to 160℃ at 10℃ / min, then increase to 260℃ at 5℃ / min, hold for 5 min; The injection volume was 0.5 μL, and the split ratio was 1:20.

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