Quality control method of artemisia apiacea or artemisia apiacea extract

Abstract

The invention relates to a quality control method of artemisia apiacea or an artemisia apiacea extract, which comprises the following steps: respectively detecting a test solution and a reference solution by adopting a high performance liquid chromatography, generating a reference fingerprint spectrum by adopting a median method, and calculating the similarity of each common peak; and calculating the content according to the peak areas of artemisinin, artemisinic acid and dihydroartemisinic acid in the chromatograms of the sample to be detected and the reference substance by an external standard method. The mobile phase A is a tetrahydrofuran phosphate aqueous solution, the mobile phase B is acetonitrile, isocratic elution is carried out, and the stationary phase is Agilent Infinity Lab Poroshell 120EC-C18 250mm * 4.6 mm, 2.7 mu m, Caprisil C18 250mm * 4.6 mm, 3.0 mu m or an equivalent column. The method is high in accuracy and good in stability and reproducibility, can be used as a detection method for the fingerprint spectrum of the artemisia apiacea medicinal material, and can be used for simultaneously determining the contents of artemisinin, artemisinic acid and dihydroartemisinic acid.

Description

Technical Field

[0001] The invention belongs to the technical field of substance detection and relates to a quality control method for Artemisia annua or an Artemisia annua extract. Background Art

[0002] Artemisia annua L., the dried aerial part of the Asteraceae plant, is widely distributed in North America, Europe, Africa, and Asia, with approximately 70% concentrated in my country. A traditional Chinese antimalarial, Artemisia annua L. is cold in nature, bitter, and pungent in flavor. It clears heat and detoxifies, and acts as a remedy for malaria. It is used for symptoms such as summer heat fever, yin deficiency fever, night heat and morning chill, bone steaming and fatigue fever, malarial fever, damp-heat jaundice, and topical treatment for insect bites, sores, and burns. In the 1970s, Chinese researchers isolated artemisinin from Artemisia annua L., a discovery that broke the theoretical barrier that antimalarial drugs must contain nitrogen-containing heterocycles. Compared to previous antimalarial drugs, it is more effective, rapid, less toxic, and safer.

[0003] The main chemical components of Artemisia annua include sesquiterpenes, volatile oils, flavonoids, fatty acids, polyphenols, coumarins, and esters. Sesquiterpenes account for approximately 35%, aromatics for 29%, triterpenes for 6%, steroids for 6%, fatty acids for 6%, phenols for 5%, aliphatics for 4%, alkaloids for 3%, heterocyclics for 2%, and other flavonoids and terpenes for 4%. Artemisinin, artemisinic acid, and dihydroartemisinic acid are the main active ingredients in Artemisia annua.

[0004] At present, the quality standard for Artemisia annua medicinal materials in the 2025 edition of the "Chinese Pharmacopoeia" has not yet stipulated the fingerprint inspection method, nor included the content determination method, which is not conducive to the comprehensive quality control of this medicinal material.

[0005] Chen Jing et al. reported the determination of artemisinin and its related sesquiterpenes in Artemisia annua leaves by HPLC-UV-ELSD (Journal of Shenyang Pharmaceutical University, 2008, 25(11)). A Venusil XBP-C18 column (200mm×4.6mm, 5μm) was used, with a mobile phase of acetonitrile-water gradient elution, a detection wavelength of 203nm, a flow rate of 1.0mL / min, and a detector UV-ELSD was used to determine the contents of artemisinin, artemisinin B, and 3α-hydroxy-1-deoxyartemisinin in Artemisia annua leaves. Under the same chromatographic conditions, the mobile phase was changed to acetonitrile-water-formic acid (60:40:0.08), a detection wavelength of 215nm, a flow rate of 1.0mL / min, and a UV detector to determine the content of artemisinic acid in Artemisia annua leaves. This method uses UV-ELSD to detect artemisinin, artemisinin B, and 3α-hydroxy-1-deoxyartemisinin, which have weaker UV absorption. When gradient elution was used to detect artemisinin, artemisinin B, and 3α-hydroxy-1-deoxyartemisinin, due to the complex components, terminal absorption detection, uneven chromatographic baseline, and poor separation, and because no acid was added to the mobile phase to adjust the pH, the chromatographic peak of artemisinin detected by UV showed severe tailing, which ultimately led to the analytical method validation sample recovery results showing that the recoveries of the four components were 98.5%, 96.5%, 97.5%, and 95.4%, respectively. The sample recovery rates were low, and the detection specificity and accuracy were insufficient.

[0006] Wang Mengyue et al. reported the simultaneous determination of the contents of seven components in Artemisia annua by UPLC-QQQ-MS / MS, including amorpha-4,11-diene, artemisinic acid, dihydroartemisinic acid, artemisinin, artemisinin B, artemisinene, and artemisinic aldehyde (Journal of Chinese Materia Medica, 2024, 49(5), Molecules, 2019, 24, 1530). An ultra-high pressure liquid chromatography-triple quadrupole mass spectrometer was used, with an Eclipse-Plus RRHDC18 column (50 mm × 2.1 mm, 1.8 μm). The mobile phase consisted of formic acid (0.1%)-ammonium formate (5 mmol / L) aqueous solution (A) and methanol (B), with a gradient elution (0-8 min, 55%-100% B; 8-11 min, 55%-100% B; equilibrium 3 min). The flow rate was 0.6 mL / min, the column temperature was 40°C, and the injection volume was 5 μL. This method did not effectively separate artemisinic acid and dihydroartemisinic acid, their retention times almost overlapped at 3.9-4.0 min, and their peaks were tailing. In the method validation, the average recoveries of artemisinin B, artemisinene, artemisinin, dihydroartemisinin, artemisinic acid, artemisinic aldehyde and Amorpha-4,11-diene were 89.02%, 98.58%, 107.4%, 109.3%, 112.8%, 103.9% and 104.6%, respectively. The recoveries of artemisinin, dihydroartemisinin and artemisinic acid were seriously high, indicating that the method was not accurate. The use of this method involves the purchase of expensive instruments and is not popular.

[0007] Xu Dinghua et al. reported the analysis of the changes in the content of artemisinic acid in Artemisia annua at different growth stages (Chinese Materia Medica, 2012, 35(12)). The detection method used was HPLC-UV method, chromatographic column Wondas IIC18 (250mm×4.6mm, 5μm), mobile phase acetonitrile: 0.2% phosphoric acid aqueous solution = 73:27, flow rate 1.0mL / min, detection wavelength 220nm, column temperature 40±1℃, injection 2μL. Due to the high proportion of organic solvent in the mobile phase, the baseline drift of the artemisinic acid peak position at around 6.5min in the chromatogram of the test solution was serious, the separation degree did not meet the requirements, and the artemisinic acid peak was not effectively separated from the adjacent peaks. Xu Dinghua et al. reported the preparation of artemisinic acid in Artemisia annua and the study of its biostability (Natural Product Research and Development, 2014, 26(10)). The detection method used was HPLC-UV, with a Wondas IIC18 chromatographic column (250 mm × 4.6 mm, 5 μm), a mobile phase of acetonitrile: 0.1% acetic acid aqueous solution = 70:30, a flow rate of 1.0 mL / min, a detection wavelength of 203 nm, a column temperature of 30±1°C, and a 2 μL injection for artemisininic acid purity detection. The resolution did not meet the requirements, the mobile phase pH was inappropriately selected, and the artemisininic acid chromatographic peak was severely tailing, affecting the accuracy of the method.

[0008] Ding Dandan et al. reported a spatiotemporal dynamic study of the content of four artemisinin-like compounds, artemisinin, artemisinic acid, and dihydroartemisinic acid, in Artemisia annua (Master's thesis, China Academy of Chinese Medical Sciences, 2019). The detection method used was UPLC-ESI-QQQ-MS, using an Eclipse Plus C18 column (50 mm × 2.1 mm, 1.8 μm). In positive ion mode, the mobile phase was water (A)-acetonitrile (B), with a gradient elution program of: 0–3 min, 50%–100% B; 3–5 min, 100% B; post-run for 2 min. In negative ion mode, the mobile phase was water (A)-acetonitrile (B), with a gradient elution program of: 0–3 min, 75%–100% B; 3–5 min, 100% B; post-run for 2 min. The method used a 0.2% phosphoric acid aqueous solution ratio of 73:27, a flow rate of 1.0 mL / min, a detection wavelength of 220 nm, a column temperature of 35 ± 1°C, and a 2 μL injection. This method did not add acid to the mobile phase to adjust the pH, resulting in broad chromatographic peaks and some tailing. During validation, the average recoveries of artemisinin, artemisinin B, artemisinic acid, and dihydroartemisinin were 112.32%, 112.6%, 112.2%, and 111.3%, respectively. These recoveries were significantly higher, indicating poor method accuracy.

[0009] Zhang Xiaorong et al. reported a quantitative analysis of artemisinic acid content in Artemisia annua L. at different growth stages based on the spot area method (Chinese Materia Medica, 2013, 36(11)). The detection method used was HPLC-UV, with a Wondas IIC18 column (250 mm × 4.6 mm, 5 μm), a mobile phase of acetonitrile and 0.2% phosphoric acid aqueous solution, a flow rate of 1.0 mL / min, a detection wavelength of 220 nm, a column temperature of 40 ± 1°C, and a 2 μL injection. The method did not report the specific ratio of acetonitrile and 0.2% phosphoric acid aqueous solution in the mobile phase. From the chromatogram of the test sample, it can be seen that the retention time of artemisinic acid is about 10 min. It is speculated that the proportion of organic solvent in the mobile phase is relatively high. In the chromatogram of the test sample solution, the baseline drift of the artemisinic acid peak position is serious, the separation degree does not meet the requirements, and the artemisinic acid peak is not effectively separated from the adjacent peaks.

[0010] Therefore, providing a method with high separation and high accuracy for detecting the content of artemisinin, artemisinic acid and dihydroartemisinic acid has become an urgent problem to be solved in this field. Summary of the Invention

[0011] In view of the deficiencies in the prior art, the present invention aims to provide a method for controlling the quality of Artemisia annua or Artemisia annua extract.

[0012] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0013] In a first aspect, the present invention provides a method for controlling the quality of Artemisia annua or an Artemisia annua extract, the method comprising:

[0014] (1) The sample to be tested and the reference solution are respectively tested by high performance liquid chromatography to obtain chromatograms;

[0015] (2) Based on the chromatogram of the sample to be tested, the control fingerprint is generated using the median method, and the similarity of each common peak in the sample to be tested is calculated, with a similarity ≥ 0.95 as the quality control standard;

[0016] The contents of artemisinin, artemisinic acid and dihydroartemisinic acid were calculated by external standard method based on the peak areas of artemisinin, artemisinic acid and dihydroartemisinic acid in the chromatograms of the test samples and the reference substance;

[0017] The mobile phase of the high performance liquid chromatography comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a tetrahydrofuran phosphate aqueous solution, and the mobile phase B is acetonitrile.

[0018] The present invention provides a quality control method for Artemisia annua medicinal material and its extract. Based on the obtained chromatogram of the Artemisia annua medicinal material test solution, a control fingerprint of the Artemisia annua medicinal material is generated using the median method, and the similarity of each common peak is calculated. The similarity ≥ 0.95 is used as the inspection quality control standard. Based on the peak area corresponding to the obtained chromatogram of the Artemisia annua or Artemisia annua extract test solution and the peak area and concentration corresponding to the chromatogram of the artemisinin, artemisinic acid and dihydroartemisinic acid reference solution, the content of artemisinin, artemisinic acid and dihydroartemisinic acid in the Artemisia annua or Artemisia annua extract test solution is calculated using the external standard reference method. Through this method, the contents of artemisinin, artemisinic acid and dihydroartemisinic acid in the Artemisia annua medicinal material can be determined while establishing the fingerprint of the Artemisia annua medicinal material, and the quality of the Artemisia annua medicinal material can be comprehensively, objectively and accurately evaluated. This is of great significance to the quality control of Artemisia annua medicinal material. The quality control method has the characteristics of high accuracy and separation, good stability and reproducibility, and low equipment requirements. The choice of mobile phase has a very important influence on the separation results. Not adding tetrahydrofuran or using other mobile phase combinations will affect the chromatographic separation.

[0019] Preferably, the stationary phase of the high performance liquid chromatography comprises Agilent InfinityLab Poroshell 120EC-C18 250 mm×4.6 mm, 2.7 μm, Caprisil C18 250 mm×4.6 mm, 3.0 μm or an equivalent column of the above chromatographic columns.

[0020] The selection of chromatographic columns such as Agilent InfinityLab Poroshell 120EC-C18 (250mm×4.6mm, 2.7μm) and CaprisilC18 (250mm×4.6mm, 3.0μm) can improve the chromatographic separation effect. The particle size of the chromatographic column should not be too large, as too large a particle size will affect the separation effect.

[0021] Preferably, the volume fraction of phosphoric acid in the tetrahydrofuran phosphate aqueous solution is 0.1-0.4%, and the volume fraction of tetrahydrofuran is 3-7%.

[0022] The volume fraction of phosphoric acid can be selected from 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, etc., and the volume fraction of tetrahydrofuran can be selected from 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, etc. Other specific values ​​within the above numerical range can be selected, and they will not be repeated here.

[0023] Preferably, the elution mode of the high performance liquid chromatography is isocratic elution, wherein the volume fraction of mobile phase A is 40-50%, and the volume fraction of mobile phase B is 50-60%.

[0024] The volume fraction of mobile phase A can be selected to be 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, etc., and the volume fraction of mobile phase B can be selected to be 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be repeated here.

[0025] Isocratic elution is better than gradient elution, and the ratio of mobile phase A to mobile phase B in isocratic elution is very important to the results.

[0026] Preferably, the detection wavelength of the high performance liquid chromatography is 200-210 nm, for example, 200 nm, 201 nm, 202 nm, 203 nm, 204 nm, 205 nm, 206 nm, 207 nm, 208 nm, 209 nm, 210 nm, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be repeated here.

[0027] The detection wavelength will also affect the detection results.

[0028] Preferably, the column temperature of the HPLC is 25-40°C, for example, 25°C, 28°C, 30°C, 32°C, 35°C, 38°C, 40°C, etc. Other specific point values ​​within the above numerical range can be selected and will not be described here one by one.

[0029] Preferably, the flow rate of the mobile phase is 0.8-1.2 mL / min, for example, 0.8 mL / min, 0.85 mL / min, 0.9 mL / min, 0.95 mL / min, 1 mL / min, 1.05 mL / min, 1.1 mL / min, 1.15 mL / min, 1.2 mL / min, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be repeated here.

[0030] Preferably, the method for preparing the sample to be tested comprises: mixing Artemisia annua or an Artemisia annua extract with petroleum ether, and performing ultrasonic extraction to obtain the sample.

[0031] Preferably, the ultrasonic treatment time is 10-60 min and the power is 300-500W.

[0032] The time can be selected as 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, 60min, etc., and the power can be selected as 300W, 320W, 340W, 360W, 380W, 400W, 420W, 440W, 460W, 480W, 500W, etc. Other specific point values ​​within the above numerical range can be selected, so I will not go into details here.

[0033] Preferably, the ultrasound time is 25-35 min, for example, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be repeated here.

[0034] Preferably, the solid-liquid ratio of the Artemisia annua or Artemisia annua extract to petroleum ether is 0.2 g: (50-100) mL.

[0035] The specific point values ​​in (50-100) can be selected as 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be listed here one by one.

[0036] Preferably, the boiling range of the petroleum ether is 60-90°C, for example, 60°C, 62°C, 65°C, 68°C, 70°C, 72°C, 75°C, 78°C, 80°C, 82°C, 85°C, 88°C, 90°C, etc. Other specific point values ​​within the above numerical range can be selected and will not be repeated here.

[0037] Preferably, the solvent of the reference solution is petroleum ether.

[0038] Preferably, the concentration of artemisinin in the reference solution is 0.01-3 mg / mL, the concentration of artemisinic acid is 0.01-2 mg / mL, and the concentration of dihydroartemisinic acid is 0.01-2 mg / mL.

[0039] The concentration of artemisinin can be selected from 0.01mg / mL, 0.05mg / mL, 0.1mg / mL, 0.2mg / mL, 0.5mg / mL, 0.8mg / mL, 1mg / mL, 1.2mg / mL, 1.5mg / mL, 1.8mg / mL, 2mg / mL, 2.2mg / mL, 2.5mg / mL, 2.8mg / mL, 3mg / mL, etc. The concentration of artemisinic acid can be selected from 0.01mg / mL, 0.05mg / mL, 0.1mg / mL, 0.2mg / mL, 0.5mg / mL The concentration of dihydroartemisinic acid can be selected from 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.5 mg / mL, 1.8 mg / mL, 2 mg / mL, etc. The concentration of dihydroartemisinic acid can be selected from 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.5 mg / mL, 1.8 mg / mL, 2 mg / mL, etc. Other specific point values ​​within the above numerical range can be selected, which will not be repeated here.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The method described in the present invention can detect the fingerprint of Artemisia annua and simultaneously determine the contents of three index components: artemisinin, artemisinic acid, and dihydroartemisinic acid. This quality control method effectively overcomes the shortcomings of existing technologies, such as poor resolution, poor reproducibility, and the inability to accurately and simultaneously determine the three index components. With high accuracy and resolution, good stability, and excellent reproducibility, it can be used not only for detecting the fingerprint of Artemisia annua, but also for simultaneously determining the contents of artemisinin, artemisinic acid, and dihydroartemisinic acid in Artemisia annua extracts. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is the chromatogram result of the test sample in Example 1.

[0043] Figure 2 It is the chromatogram result of the test sample in Example 2.

[0044] Figure 3 It is the chromatogram result of the test product in Example 3.

[0045] Figure 4 It is the chromatogram result of the test product in Example 4.

[0046] Figure 5 It is the chromatogram result of the test sample in Example 5.

[0047] Figure 6 It is the chromatogram result of the test sample in Example 6.

[0048] Figure 7 It is the chromatogram result of the test sample in Example 7.

[0049] Figure 8 It is the chromatogram result of the test sample in Example 8.

[0050] Figure 9 It is the chromatogram result of the test sample in Comparative Example 1.

[0051] Figure 10 It is the chromatogram result of the test sample in Comparative Example 2.

[0052] Figure 11 This is the chromatogram result of the test sample in Comparative Example 3.

[0053] Figure 12 It is the reference fingerprint of Artemisia annua medicinal material.

[0054] Figure 13 It is a common pattern in fingerprints.

[0055] Figure 14 It is the chromatogram of mixed reference solution. DETAILED DESCRIPTION

[0056] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0057] The sources of the functional ingredients contained in the products involved in the following examples and comparative examples are as follows (only the functional ingredients are reflected, and the necessary auxiliary ingredients contained in other commercially available raw materials are not repeated):

[0058] Agilent 1290 Infinity II high-performance liquid chromatograph: including G7104A pump, G7129B autosampler, G7117A diode array detector, G7116B column oven, OpenLab workstation; Mettler XPR205 1 / 100000 electronic balance;

[0059] KQ-800DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.);

[0060] Artemisinin (China Food and Drug Control Institutes, purity ≥98%, batch number: 100202-202107);

[0061] Artemisinic acid reference substance (Chengdu Lemeitian Pharmaceutical Technology Co., Ltd., purity ≥98%, batch number: DSTDQ004802);

[0062] Dihydroartemisinic acid (Chengdu Lemeitian Pharmaceutical Technology Co., Ltd., purity ≥98%, batch number: DST250124-038).

[0063] Example 1

[0064] This embodiment provides a method for detecting the content of artemisinin, artemisinic acid, and dihydroartemisinic acid in Artemisia annua, the method comprising:

[0065] (1) Preparation of test samples:

[0066] Weigh 1 g of Artemisia annua powder (passed through No. 4 sieve) and place it in a conical flask. Add 50 mL of petroleum ether (boiling range of 60-90°C), weigh it, sonicate it for 30 minutes, let it cool, weigh it again, make up the lost weight with the same solvent, shake it well, filter it with a 0.22 μm microporous filter membrane, and take the filtrate to obtain the Artemisia annua test solution.

[0067] (2) Preparation of mixed reference solution

[0068] Accurately weigh artemisinin, artemisinic acid, and dihydroartemisinic acid, respectively, and place them in the same volumetric flask. Dissolve them in petroleum ether (boiling range of 60-90°C) and dilute them to prepare a solution containing 0.8 mg of artemisinin, 0.02 mg of artemisinic acid, and 0.01 mg of dihydroartemisinic acid per mL, as the reference solution.

[0069] (3) High performance liquid chromatography test

[0070] The test solution and reference solution prepared as described above were injected into the high performance liquid chromatograph respectively.

[0071] Chromatographic conditions:

[0072] Stationary phase: Agilent InfinityLab Poroshell 120EC-C18 (250 mm × 4.6 mm, 2.7 μm)

[0073] Mobile phase A: 0.1vt% phosphoric acid 5vt% tetrahydrofuran in water

[0074] Mobile phase B: acetonitrile

[0075] Flow rate: 1 mL min -1

[0076] Detection wavelength: 205nm

[0077] Column temperature: 35°C

[0078] Injection volume: 10 μL

[0079] Elution program: 0-35min, mobile phase A: 40vt%, mobile phase B: 60vt%

[0080] The chromatogram of Artemisia annua is as follows: Figure 1 The chromatogram of the reference solution is shown in Figure 14 shown.

[0081] Example 2

[0082] This embodiment provides a method for detecting the content of artemisinin, artemisinic acid, and dihydroartemisinic acid in an Artemisia annua extract. The method differs from Example 1 only in that step (1) is "weighing 200 mg of the Artemisia annua extract, placing it in a 100 mL volumetric flask, adding petroleum ether (boiling range of 60-90° C.) to make up the volume, shaking well, filtering through a 0.22 μm microporous filter membrane, and taking the filtrate to obtain the Artemisia annua extract test solution", and the other operations remain unchanged.

[0083] Chromatogram Figure 2 shown.

[0084] Example 3

[0085] This example provides a method for detecting the content of artemisinin, artemisinic acid, and dihydroartemisinic acid in Artemisia annua. The only difference from Example 1 is that in step (3), "stationary phase: Agilent InfinityLab Poroshell120EC-C18 (250 mm × 4.6 mm, 2.7 μm)" is replaced with "stationary phase: Agilent ZORBAX SB-C18 (250 mm × 4.6 mm, 5 μm)", and other operations remain unchanged.

[0086] Chromatogram Figure 3 As shown, from Figure 3 It can be seen that after replacing the stationary phase, the separation effect is poor, indicating that the choice of chromatographic column will affect the experimental results.

[0087] Example 4

[0088] This embodiment provides a method for detecting the content of artemisinin, artemisinic acid and dihydroartemisinic acid in Artemisia annua. The difference between this method and Example 1 is that the detection wavelength in step (3) is 254 nm, and other operations remain unchanged.

[0089] Chromatogram Figure 4 As shown, from Figure 4 It can be seen that when the detection wavelength is not within the scope of this application, the peak response values ​​of artemisinin, artemisinic acid and dihydroartemisinic acid are low.

[0090] Example 5

[0091] This embodiment provides a method for detecting the content of artemisinin, artemisinic acid and dihydroartemisinic acid in Artemisia annua. The difference between this method and Example 1 is that the detection wavelength in step (3) is 230 nm, and other operations remain unchanged.

[0092] Chromatogram Figure 5 As shown, from Figure 5 It can be seen that when the detection wavelength is not within the scope of this application, the peak response values ​​of artemisinin, artemisinic acid and dihydroartemisinic acid are low.

[0093] Example 6

[0094] This example provides a method for detecting the content of artemisinin, artemisinic acid, and dihydroartemisinic acid in Artemisia annua. The difference between this example and Example 1 is that the elution procedure of this example is:

[0095] 0-15 min, mobile phase A: 40 vt%, mobile phase B: 60 vt%;

[0096] 15-25 min, mobile phase A: 90 vt%, mobile phase B: 10 vt%;

[0097] 25-35 min, mobile phase A: 40 vt%, mobile phase B: 60 vt%;

[0098] Other operations remain unchanged.

[0099] Chromatogram Figure 6 As shown, from Figure 6 It can be seen that after replacing isocratic elution with gradient elution, the index components emerge later, the peak width is wider, and the separation is poor, indicating that the elution method will affect the separation effect.

[0100] Example 7

[0101] This example provides a method for detecting the content of artemisinin, artemisinic acid, and dihydroartemisinic acid in Artemisia annua. The only difference from Example 1 is that the elution procedure of this example is "0-35 min, mobile phase A: 30 vt%, mobile phase B: 70 vt%", and other operations remain unchanged.

[0102] Chromatogram Figure 7 As shown, the test sample has complex components, artemisinin peaks quickly, and the separation is poor.

[0103] Example 8

[0104] This example provides a method for detecting the content of artemisinin, artemisinic acid, and dihydroartemisinic acid in Artemisia annua. The only difference from Example 1 is that the elution program is "0-35 min, mobile phase A: 60 vt%, mobile phase B: 40 vt%", and other operations remain unchanged.

[0105] Chromatogram Figure 8 As shown in the figure, the index component peaks too late and the analysis and detection efficiency is low, which shows that the volume ratio of mobile phase A and mobile phase B during elution will affect the analysis and detection effect.

[0106] Comparative Example 1

[0107] This comparative example provides a method for detecting the content of artemisinin, artemisinic acid, and dihydroartemisinic acid in Artemisia annua. The difference between the method and Example 1 is that the mobile phase A is replaced by "0.1 vt% phosphoric acid 5 vt% tetrahydrofuran aqueous solution" and the rest remains unchanged.

[0108] Chromatogram Figure 9 As shown, from Figure 9 It can be seen that the separation of the index components is poor, indicating that the addition of tetrahydrofuran is conducive to better separation of the index components.

[0109] Comparative Example 2

[0110] This comparative example provides a method for detecting the contents of artemisinin, artemisinic acid and dihydroartemisinic acid in Artemisia annua. The difference between the comparative example and Example 1 is that the mobile phase B is replaced by methanol from acetonitrile, and the rest remains unchanged.

[0111] Chromatogram Figure 10 As shown, from Figure 10 It can be seen that the separation is poor and the baseline signal-noise is large, indicating that the choice of mobile phase B is very important for the results.

[0112] Comparative Example 3

[0113] This comparative example provides a method for detecting the content of artemisinin, artemisinic acid and dihydroartemisinic acid in Artemisia annua. The difference between the method and Example 1 is that in step (1) and step (2), "petroleum ether (boiling range of 60-90°C)" is replaced by "methanol" in equal amounts, and the other operations remain unchanged.

[0114] Chromatogram Figure 11 As shown, from Figure 11 It can be seen that the solvent effect is serious and the separation is poor.

[0115] Test Example 1

[0116] A total of 7 batches of Artemisia annua medicinal materials were collected from the Youyang Artemisia annua medicinal material GMP planting base. All of them were the above-ground parts dried in a cool place. The collection information is shown in Table 1.

[0117] Table 1

[0118] Sample number batch number Harvest time S1 Y001-2514-6-1 2024.719 S2 Y001-2514-6-2 2024.7.29 S3 Y001-2514-6-3 2024.7.09 S4 Y001-250107-1 2024.8.19 S5 Y001-250107-2 2024.8.29 S6 Y001-250107-3 2024.7.09 S7 Y001-250108-1 2024.8.19

[0119] With reference to the method of Example 1, the content of different batches of Artemisia annua medicinal materials was determined, and the obtained atlas data was imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software. With the S1 atlas as a reference, the median method was used to establish a control fingerprint (see Figure 12 ), the time window width was set to 0.1min, and multi-point calibration Mark peak matching was used to establish each fingerprint spectrum and generate the common pattern of fingerprint spectrum, and 14 common peaks were marked (see Figure 13 ). The similarity of fingerprint spectra is greater than 0.98. The results are shown in Table 2.

[0120] Table 2

[0121] S1 S2 S3 S4 S5 S6 S7 Similarity 1 0.993 0.993 0.987 0.989 0.991 0.992

[0122] The resulting fingerprint of Artemisia annua contains 14 common peaks. Based on similarity values, this fingerprint is used for quality control of Artemisia annua, with a fingerprint similarity greater than 0.98 as the quality control standard. Peak 4 is artemisinin, with a retention time of 7.0-8.0 minutes; peak 9 is dihydroartemisinic acid, with a retention time of 15-16 minutes; and peak 11 is artemisinic acid, with a retention time of 17-18 minutes.

[0123] The results of the determination of artemisinin, dihydroartemisinin and artemisinic acid in Artemisia annua are shown in Table 3.

[0124] Table 3

[0125]

[0126] Test Example 2

[0127] Specificity and accuracy testing

[0128] 1. Exclusivity

[0129] The chromatogram obtained in Example 1 was analyzed, and the separation between each peak of artemisinin, artemisinic acid, and dihydroartemisinic acid and the adjacent peaks is shown in Table 4.

[0130] Table 4

[0131] Element Retention time Separation artemisinin 7.177 2.37 Dihydroartemisinic acid 15.394 7.96 artemisinic acid 17.506 7.06

[0132] As can be seen from the data in Table 4, in the chromatogram obtained by the method of the present invention, artemisinin, artemisinic acid and dihydroartemisinin have a high degree of separation.

[0133] 2. Accuracy

[0134] Accurately weigh appropriate amounts of artemisinin, artemisinic acid, and dihydroartemisinic acid, respectively, and place them in the same volumetric flask. Dissolve them in petroleum ether (boiling range of 60-90°C) and dilute to the mark. Shake well to prepare a solution containing approximately 0.8 mg of artemisinin, 0.02 mg of artemisinic acid, and 0.01 mg of dihydroartemisinic acid per mL, which serves as the stock solution of the reference substance.

[0135] Weigh 1 g of 6 portions of Artemisia annua powder (passed through No. 4 sieve) respectively, place in a conical flask, accurately add 49 mL of petroleum ether (boiling range of 60-90 ° C), add 1 mL of reference substance mother liquor and weigh, ultrasonicate for 30 min, cool, weigh again, make up the lost weight with the same solvent, shake well, filter with a 0.22 μm microporous filter membrane, and take the filtrate to obtain the Artemisia annua test solution.

[0136] The chromatographic conditions were the same as in Example 1. The spiked recovery and RSD were calculated, and the results are shown in Table 6.

[0137] Table 5

[0138]

[0139] It can be seen from the data in Table 5 that the method of the present invention has high accuracy and small error.

[0140] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate a method for quality control of Artemisia annua or an Artemisia annua extract, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments for implementation. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for the raw materials of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

[0141] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0142] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for controlling the quality of Artemisia annua or Artemisia annua extract, characterized in that: The method comprises: (1) The sample to be tested and the reference solution are respectively tested by high performance liquid chromatography to obtain chromatograms; (2) Based on the chromatogram of the sample to be tested, the control fingerprint is generated using the median method, and the similarity of each common peak in the sample to be tested is calculated, with a similarity ≥ 0.95 as the inspection quality control standard; The contents of artemisinin, artemisinic acid and dihydroartemisinic acid were calculated by external standard method based on the peak areas of artemisinin, artemisinic acid and dihydroartemisinic acid in the chromatograms of the test samples and the reference substance; The mobile phase of the high performance liquid chromatography comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a tetrahydrofuran phosphate aqueous solution, and the mobile phase B is acetonitrile.

2. The method for quality control of Artemisia annua or Artemisia annua extract according to claim 1, characterized in that: The stationary phase of the high performance liquid chromatography comprises an Agilent InfinityLab Poroshell 120EC-C18 250 mm×4.6 mm, 2.7 μm, a Caprisil C18 250 mm×4.6 mm, 3.0 μm or an equivalent column of the above chromatographic columns.

3. The method for quality control of Artemisia annua or Artemisia annua extract according to claim 1, characterized in that: The volume fraction of phosphoric acid in the tetrahydrofuran phosphate aqueous solution is 0.1-0.4%, and the volume fraction of tetrahydrofuran is 3-7%.

4. The method for quality control of Artemisia annua or Artemisia annua extract according to claim 1, characterized in that: The elution mode of the high performance liquid chromatography is isocratic elution, wherein the volume fraction of mobile phase A is 40-50%, and the volume fraction of mobile phase B is 50-60%.

5. The method for quality control of Artemisia annua or Artemisia annua extract according to claim 1, characterized in that: The detection wavelength of the high performance liquid chromatography is 200-210 nm.

6. The method for quality control of Artemisia annua or Artemisia annua extract according to claim 1, characterized in that: The preparation method of the test sample comprises: mixing Artemisia annua or Artemisia annua extract with petroleum ether, and performing ultrasonic extraction to obtain the sample.

7. The method for quality control of Artemisia annua or Artemisia annua extract according to claim 6, characterized in that: The ultrasonic treatment time is 10-60 min, and the power is 300-500 W.

8. The method for quality control of Artemisia annua or Artemisia annua extract according to claim 6, characterized in that: The solid-liquid ratio of the Artemisia annua or Artemisia annua extract to petroleum ether is 0.2g:(50-100)mL.

9. The method for quality control of Artemisia annua or Artemisia annua extract according to claim 6, characterized in that: The boiling range of the petroleum ether is 60-90°C.

10. The method for quality control of Artemisia annua or Artemisia annua extract according to claim 1, characterized in that: The concentration of artemisinin in the reference solution is 0.01-3 mg / mL, the concentration of artemisinic acid is 0.01-2 mg / mL, and the concentration of dihydroartemisinic acid is 0.01-2 mg / mL. The solvent of the reference solution is petroleum ether.

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