A method for constructing characteristic maps of Artemisia annua medicinal materials and Artemisia annua drug preparations and their application

Abstract

The present invention belongs to the technical field of quality detection of traditional Chinese medicine preparations, and specifically relates to a characteristic spectrum of an Artemisia annua medicinal material and an Artemisia annua medicinal preparation, and further discloses a method for constructing the characteristic spectrum, as well as a method for quality detection of Artemisia annua medicinal material and an Artemisia annua medicinal preparation. The method for constructing the characteristic spectrum of the Artemisia annua medicinal material and the Artemisia annua medicinal preparation of the present invention establishes a characteristic spectrum method for the medicinal preparation based on high performance liquid chromatography, calibrates 14 common characteristic peaks using a traditional Chinese medicine chromatographic fingerprint, selects peak 9 scopoletin as a reference peak, and specifies the relative retention time of each characteristic peak and the S peak, and the relative retention time should be within the range of ±10% of the specified value. The method for constructing the characteristic spectrum of the present invention can comprehensively reflect the intrinsic quality and medication safety of Artemisia annua medicinal material and the Artemisia annua medicinal preparation, is conducive to its comprehensive quality detection and overall quality control, and has the advantages of good stability, high precision and good repeatability.

Description

Technical Field

[0001] The present invention belongs to the technical field of quality detection of traditional Chinese medicine preparations, and specifically relates to a characteristic spectrum of an Artemisia annua medicinal material and an Artemisia annua medicinal preparation, and further discloses a method for constructing the characteristic spectrum, as well as a quality detection method for the Artemisia annua medicinal material and an Artemisia annua medicinal preparation. Background Art

[0002] Traditional Chinese medicine has complex ingredients, and the effective part is often not a single ingredient. Using a single ingredient as a quality control indicator is increasingly incompatible with the requirements of quality control of traditional Chinese medicine. Therefore, traditional Chinese medicine fingerprint technology came into being. Traditional Chinese medicine fingerprint technology originated from fingerprint identification. It uses modern information technology and quality analysis methods to more comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine. The characteristic spectrum of traditional Chinese medicine is obtained by identifying the common peaks of the characteristics of various component groups. For traditional Chinese medicines, characteristic spectra can be used to identify authenticity and judge the quality; for Chinese patent medicines, characteristic spectra can identify product authenticity, judge the rationality of the preparation process, and effectively control product quality. At present, most of the effective ingredients of traditional Chinese medicines are still unclear. The integrity and ambiguity of the characteristic spectrum of traditional Chinese medicines just adapt to this feature, and are more scientific and comprehensive than the quality control method of a single ingredient. At present, the characteristic spectrum of traditional Chinese medicine has been recognized internationally as a control mode for the quality of traditional Chinese medicine. High performance liquid chromatography has the advantages of high separation efficiency and fast analysis speed, and has become the main analytical method for characteristic spectra today.

[0003] Artemisia annua L. is the dried aerial part of Artemisia annua L., a plant of the genus Artemisia in the Asteraceae family, and has a long history of use. Artemisia annua L. is bitter, pungent, and cold in nature. It enters the liver and gallbladder meridians and has the effects of clearing away deficiency heat, removing bone steaming, relieving summer heat, stopping malaria, and jaundice. It is used for symptoms such as warm evil damaging yin, night heat and morning coolness, yin deficiency fever, bone steaming and fatigue fever, summer heat fever, malaria cold and heat, damp-heat jaundice, etc. Modern research shows that Artemisia annua also has the effects of immunosuppression and cell immunity promotion. my country is the main producer of Artemisia annua medicinal materials, and Artemisia annua is distributed throughout the country, but the quality of Artemisia annua medicinal materials has significant ecological regionality. The content of artemisinin varies greatly with the place of origin, and is greatly related to soil factors and Artemisia annua types. At present, there are many types of Artemisia annua on the market, and the composition is complex. The thin layer chromatography identification method is the most common simple and fast identification method in the current national traditional Chinese medicine preparations. However, there are problems with unsatisfactory separation effect, clarity and reproducibility.

[0004] It is reported that Artemisia annua mainly contains compounds such as flavonoids, coumarins, terpenes, phenylpropionic acids and volatile oils, and the most commonly used quantitative components for the determination of Artemisia annua content are artemisinin, scopoletin, artemisinic acid and artemisinin. However, given the large differences in the contents of the above components in different dosage forms of Artemisia annua drugs, they cannot well reflect the quality of Artemisia annua medicinal materials and drug preparations. Therefore, establishing a method that can comprehensively and systematically detect Artemisia annua medicinal materials and Artemisia annua drug preparations, especially a method for establishing characteristic spectra of Artemisia annua medicinal materials and Artemisia annua drug preparations, is of great significance for their comprehensive quality inspection and overall quality control. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a method for constructing a characteristic spectrum of Artemisia annua medicinal material and Artemisia annua drug preparation, so as to comprehensively reflect the intrinsic quality and drug safety of Artemisia annua medicinal material and Artemisia annua drug preparation;

[0006] The second technical problem to be solved by the present invention is to provide a method for quality detection of Artemisia annua medicinal materials and Artemisia annua pharmaceutical preparations.

[0007] In order to solve the above technical problems, the method for constructing a characteristic spectrum of an Artemisia annua medicinal material and an Artemisia annua pharmaceutical preparation described in the present invention comprises the steps of performing high performance liquid chromatography on a test solution of the Artemisia annua medicinal material and / or the Artemisia annua pharmaceutical preparation;

[0008] The chromatographic conditions include: using octadecylsilane bonded silica gel as a filler, methanol as a mobile phase A, and 0.1 wt % phosphoric acid aqueous solution as a mobile phase B, and performing gradient elution according to the following procedure:

[0009] 0-14min, A:B is 18%:82%→28%:72%;

[0010] 14-16 min, A:B 28%:72% → 32%:68%;

[0011] 16-30min, A:B is 32%:68%→36%:64%;

[0012] 30-36 min, A:B is 36%:64% → 40%:60%;

[0013] 36-55min, A:B is 40%:60%→62%:38%.

[0014] Specifically, in the HPLC detection step, the chromatographic conditions also include: column temperature of 18-25°C, flow rate of 0.5-1.0 ml / min, and detection wavelength of 340 nm. Preferably, the column temperature is 20°C, and the flow rate is 0.7 ml / min.

[0015] Specifically, the method for constructing the characteristic spectrum of the Artemisia annua medicinal material and the Artemisia annua drug preparation, and the method for preparing the test sample solution include: taking the test sample and accurately adding an organic solvent, sealing it with ultrasound, filtering and collecting the filtrate to obtain.

[0016] Specifically, the method for constructing the characteristic spectrum of the Artemisia annua medicinal material and the Artemisia annua drug preparation also includes the step of preparing a control medicinal material reference solution, which specifically includes: taking the Artemisia annua control medicinal material, adding water to perform heating reflux extraction, collecting the extract and filtering, taking the filtrate and evaporating it to dryness, adding an organic solvent to the residue, plugging it and subjecting it to ultrasonic treatment, filtering and collecting the subsequent filtrate to obtain the required control medicinal material reference solution.

[0017] Specifically, the method for constructing the characteristic spectrum of the Artemisia annua medicinal material and the Artemisia annua pharmaceutical preparation further comprises the steps of preparing a reference substance solution and constructing the characteristic spectrum of the reference substance based on the high performance liquid chromatography method;

[0018] The reference substance includes scopoletin.

[0019] Preferably, the step of preparing the reference substance solution comprises the step of taking a selected reference substance and accurately adding an organic solvent to prepare the reference substance solution of a selected concentration.

[0020] Preferably, each 1 ml of the control solution contains 30-50 μg of scopoletin, preferably 40 μg.

[0021] Preferably, the organic solvent comprises methanol at a volume concentration of 60-80 v / v%, more preferably 70 v / v% methanol solution;

[0022] The power of the ultrasonic step is 200-300W, and the frequency is 50-60kHz, preferably the power is 250W, and the frequency is preferably 53KHz.

[0023] Specifically, the method for constructing the characteristic map of the Artemisia annua medicinal material and Artemisia annua drug preparation, the Artemisia annua drug preparation includes Artemisia annua formula granules, Artemisia annua formula granule intermediate extracts, Artemisia annua decoction pieces or Artemisia annua standard decoction.

[0024] The present invention also discloses a characteristic spectrum and / or a comparative characteristic spectrum of an Artemisia annua medicinal material and an Artemisia annua pharmaceutical preparation. The characteristic spectrum or the comparative characteristic spectrum of the Artemisia annua medicinal material and the Artemisia annua pharmaceutical preparation is constructed by the method.

[0025] The present invention also discloses a method for constructing the characteristic spectrum of the Artemisia annua medicinal material and the Artemisia annua pharmaceutical preparation and / or the application of the characteristic spectrum of the Artemisia annua medicinal material and the Artemisia annua pharmaceutical preparation and / or a control characteristic spectrum in the field of quality detection of Artemisia annua medicinal material and the Artemisia annua pharmaceutical preparation.

[0026] The present invention also discloses a method for quality detection of Artemisia annua medicinal material and Artemisia annua pharmaceutical preparation, comprising the steps of constructing the characteristic spectrum and a reference characteristic spectrum according to the method, and comparing the characteristic spectrum with the reference characteristic spectrum.

[0027] The method for constructing the characteristic spectrum of Artemisia annua medicinal materials and Artemisia annua pharmaceutical preparations of the present invention takes Artemisia annua medicinal materials and Artemisia annua pharmaceutical preparations as detection objects, establishes a characteristic spectrum method for the pharmaceutical preparation based on high performance liquid chromatography, calibrates 14 common characteristic peaks using traditional Chinese medicine chromatographic fingerprint, and conducts in-depth research on the identification of characteristic peaks, which are confirmed by positioning with reference substances: Peak 1: neochlorogenic acid; Peak 4: chlorogenic acid; Peak 5: cryptochlorogenic acid; Peak 6: 1,3-O-dicaffeoylquinic acid; Peak 9: scopoletin; Peak 11: 3,5-O-dicaffeoylquinic acid; Peak 12: 4,5-di-O-caffeoylquinic acid. The peak 9 scopoletin was selected as the reference peak, and the relative retention time of each characteristic peak and the S peak was specified, and the relative retention time should be within the range of ±10% of the specified value, and the specified values ​​were 0.32 (peak 1), 0.47 (peak 2), 0.55 (peak 3), 0.58 (peak 4), 0.66 (peak 5), 0.85 (peak 6), 0.93 (peak 7), 0.97 (peak 8), 1.51 (peak 10), 1.69, (peak 11) 2.05 (peak 12), 2.62 (peak 13), 2.72 (peak 14), and the characteristics of the Artemisia annua medicinal material and the Artemisia annua medicinal preparation were determined. The method for constructing the characteristic spectrum of the Artemisia annua medicinal material and the Artemisia annua medicinal preparation of the present invention can fully reflect the intrinsic quality and drug safety of the Artemisia annua medicinal material and the Artemisia annua medicinal preparation, is conducive to its comprehensive quality detection and overall quality control, and has the advantages of good stability, high precision and good repeatability.

[0028] The method for constructing characteristic maps of Artemisia annua medicinal materials and Artemisia annua pharmaceutical preparations described in the present invention can be applied to the construction of characteristic maps of Artemisia annua medicinal materials, Artemisia annua decoction pieces, Artemisia annua standard decoction freeze-dried powder, Artemisia annua formula granule intermediate extract, and Artemisia annua formula granules, and can realize quality detection and quality control of the above preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0030] Figure 1 The detection spectra under different chromatographic columns in Example 1;

[0031] Figure 2 Detection spectra under different mobile phase systems in Example 1;

[0032] Figure 3The detection spectra under different elution programs of the acetonitrile system mobile phase in Example 1;

[0033] Figure 4 The detection spectra under different elution procedures of the methanol system mobile phase in Example 1;

[0034] Figure 5 The detection spectra under different optimized conditions in Example 1 in order to shorten the peak time;

[0035] Figure 6 The detection spectra under different optimized conditions for separating the peak after 26 min in Example 1;

[0036] Figure 7 The reference characteristic spectrum in Example 1;

[0037] Figure 8 The characteristic spectra of multiple batches of Artemisia annua medicinal materials in Example 1;

[0038] Fig. 9 It is the reference substance positioning map in Example 1;

[0039] Fig.10 The chromatograms and UV absorption spectra of different reference substances;

[0040] Fig.11 The characteristic spectrum of the Artemisia annua medicinal material control and the chromatogram of the Artemisia annua medicinal material test sample in Example 2;

[0041] Fig.12 This is the instrumental precision chromatogram of the Artemisia annua medicinal material in Example 2;

[0042] Fig.13 This is the repetitive chromatogram of the Artemisia annua medicinal material in Example 2;

[0043] Fig.14 This is the specific negative control chromatogram of the Artemisia annua medicinal material in Example 2;

[0044] Fig.15 The chromatograms are shown for different operators in Example 2;

[0045] Fig.16 is the stability chromatogram in Example 2;

[0046] Fig.17 It is the chromatogram at different flow rates in Example 2;

[0047] Fig.18 The chromatograms at different column temperatures in Example 2 are shown below:

[0048] Fig.19 The chromatograms under different chromatographic columns in Example 2 are shown below:

[0049] Fig. 20 The chromatograms of different chromatographic instruments in Example 2 are shown below:

[0050] Fig.21 The chromatograms of 15 batches of Artemisia annua medicinal materials in Example 2;

[0051] Fig. 22 The reference characteristic spectrum of the Artemisia annua slices and the chromatogram of the Artemisia annua slices test sample in Example 3;

[0052] Fig.23 The characteristic spectra of 15 batches of Artemisia annua slices in Example 3;

[0053] Fig.24 The reference characteristic spectrum and the chromatogram of the standard decoction lyophilized powder test sample in Example 4;

[0054] Fig.25 The instrument precision chromatogram of the freeze-dried powder of Artemisia annua standard decoction in Example 4;

[0055] Fig.26 This is the reproducibility chromatogram of the freeze-dried powder of the standard decoction of Artemisia annua in Example 4;

[0056] Fig. 27 This is the specific negative control chromatogram of the freeze-dried powder of the standard decoction of Artemisia annua in Example 4;

[0057] Fig.28 The chromatograms are shown for different operators in Example 4;

[0058] Fig.29 is the stability chromatogram in Example 4;

[0059] Fig.30 It is the chromatogram at different flow rates in Example 4;

[0060] Fig.31 The chromatograms at different column temperatures in Example 4 are shown below:

[0061] Fig.32 The chromatograms under different chromatographic columns in Example 4 are shown below:

[0062] Fig.33 The chromatograms of different chromatographic instruments in Example 4 are shown below:

[0063] Fig.34 The chromatograms of 15 batches of freeze-dried powder of Artemisia annua standard decoction in Example 4;

[0064] Fig.35 The reference characteristic spectrum and the chromatogram of the test sample of the Artemisia annua intermediate in Example 5;

[0065] Fig.36 The instrumental precision chromatogram of the extract of the intermediate of Artemisia annua formula granules in Example 5;

[0066] Fig.37 This is a reproducible chromatogram of the extract of the intermediate of the Artemisia annua formula granule in Example 5;

[0067] Fig.38 This is a specific negative control chromatogram of the intermediate extract of the Artemisia annua formula granule in Example 5;

[0068] Fig.39 The chromatograms are shown for different operators in Example 5;

[0069] Fig.40 is the stability chromatogram in Example 5;

[0070] Fig.41 It is the chromatogram at different flow rates in Example 5;

[0071] Fig.42 The chromatograms at different column temperatures in Example 5 are shown below:

[0072] Fig.43 The chromatograms of different chromatographic instruments in Example 5 are shown below;

[0073] Fig.44 The chromatograms of the extracts of three batches of Qinghao Granule intermediates in Example 5;

[0074] Fig.45 The characteristic spectrum of the control and the chromatogram of the test sample of Artemisia annua formula granules in Example 6;

[0075] Fig.46 This is the instrumental precision chromatogram of the Artemisia annua formula granules in Example 6;

[0076] Fig.47 This is a reproducible chromatogram of the Artemisia annua formula granules in Example 6;

[0077] Fig.48 This is a specific negative control chromatogram of the Artemisia annua formula granules in Example 6;

[0078] Fig.49 The chromatograms are shown for different operators in Example 6;

[0079] Fig.50 is the stability chromatogram in Example 6;

[0080] Fig.51 is the chromatogram at different flow rates in Example 6;

[0081] Fig.52 The chromatograms at different column temperatures in Example 6 are shown below:

[0082] Fig.53 The chromatograms of different batches of chromatographic columns in Example 6 are shown below:

[0083] Fig.54 The chromatograms of different chromatographic instruments in Example 6 are shown below:

[0084] Fig.55 This is the chromatogram of three batches of Artemisia annua formula granules in Example 6. DETAILED DESCRIPTION

[0085] In the following embodiments of the present invention, the instruments and reagents involved include:

[0086] High performance liquid chromatograph 1: Agilent 1260 chromatography system, including G1311B quaternary pump, G1367E automatic sample injector, G1316A PDA diode array detector, G1330B column oven, chromatography workstation;

[0087] High performance liquid chromatograph 2: UltiMate 3000 chromatography system, including LPG-3400A quaternary pump, WPS-3000TSL autosampler, PDA diode array detector, chromatography workstation;

[0088] METTLER TOLEDO (Swiss Mettler) ME36S, XS204, XS205, XSE205 (one hundred thousandth);

[0089] SK5200H Shanghai Kedao Ultrasonic Instrument Co., Ltd.;

[0090] Column: CORTECS T3, 4.6×150 mm 2.7 μm;

[0091] Reagents: Acetonitrile and methanol were of chromatographic grade, and water was ultrapure water; other reagents such as phosphoric acid were of analytical grade;

[0092] The information of the tested drugs is shown in Table 1 below.

[0093] Table 1 Drug information

[0094] CAS No. Reference factory batch number purity 327-97-9 Chlorogenic acid China Inspection and Quarantine Agency 10753-202018 ≥98.0％ 905-99-7 Cryptochlorogenic acid Stendhal 3208 ≥98.0％ 906-33-2 Neochlorogenic Acid Stendhal 3209 ≥98.0％ 2450-53-5 3,5-O-dicaffeoylquinic acid China Inspection and Quarantine Agency 111782-201807 ≥94.3％ 57378-72-0 4,5-Di-O-caffeoylquinic acid China Inspection and Quarantine Agency 111894-202104 ≥95.1％ 92-61-5 Scopolamine China Inspection and Quarantine Agency 110768-202105 Contains test 19870-46-3 1,3-O-dicaffeoylquinic acid China Inspection and Quarantine Agency 111717-201402 ≥98.0％

[0095] Example 1 Characteristic Spectrum Construction Method Development

[0096] 1. Investigation of different chromatographic columns

[0097] This example first investigates the effect of different manufacturers' different filler chromatographic columns on the separation effect of Artemisia annua formula granules, and the constructed characteristic spectrum is shown in the attached Figure 1 As shown in (a)-(f).

[0098] The results showed that the Waters CORTECS T3 column had better separation for the peaks in the first 20 minutes, so Waters CORTECS T3 was selected for further exploration.

[0099] 2. Investigation of different mobile phase systems

[0100] This example investigates the effect of acetonitrile-0.1% phosphoric acid system and methanol-0.1% phosphoric acid system as mobile phases on peak separation. The characteristic spectra constructed are shown in the attached Figure 2 As shown in (a)-(b), (a) is an acetonitrile-0.1% phosphoric acid system, and (b) is a methanol-0.1% phosphoric acid system.

[0101] The results showed that the acetonitrile system had more peaks and a better tailing factor, but the overall peak distribution was uneven; the methanol system had a uniform overall peak distribution, but a larger tailing factor and peak inclusion. The mobile phases of the two systems were further explored in the future.

[0102] 3. Investigation of mobile phase in acetonitrile system

[0103] The optimization was attempted in the acetonitrile (A)-0.1% phosphoric acid (B) system. The different gradient programs are shown in Table 2 below. The constructed characteristic spectrum is shown in the attached Figure 3 As shown in (a)-(c).

[0104] Table 2 Gradient program of mobile phase in acetonitrile system

[0105] time Gradient 1 (A:B) Gradient 2 (A:B) Gradient 3 (A:B) 0 18：82 19：81 19：81 18 34：66 30：70 32：68 26 36：64 34：66 37：63 32 40：60 40：60 40：60 50 62：38 64：36 64：36

[0106] The results showed that the peak separation between 18min and 24min was poor and slightly crowded, and the overall retention time of the peak was relatively long. After trying multiple gradients (data not shown), it was found that it was difficult to achieve separation within 60 minutes under acetonitrile.

[0107] 4. Investigation of mobile phase in methanol system

[0108] The optimization was attempted in the methanol (A)-0.1% phosphoric acid (B) system. The different gradient programs are shown in Table 3 below. The constructed characteristic spectrum is shown in the attached Figure 4 As shown in (a)-(b).

[0109] Table 3 Mobile phase gradient program for methanol system

[0110] time Gradient 1 (A:B) Gradient 2 (A:B) 0 19：81 16：84 16 28：72 28：72 26 34：66 34：66 32 40：60 38：62 50 62：38 62：38

[0111] The results showed that using the methanol system, the peak separation in the first 26 minutes was good and the distribution was uniform, so the methanol system was selected for further exploration, but the overall peak retention time was relatively long, so the analysis time was shortened next.

[0112] 5. Shorten the peak time optimization

[0113] In order to shorten the overall retention time of the peak, we tried to increase the organic phase ratio, flow rate and column temperature. The characteristic spectra constructed under each optimized condition are shown in the attached figure. Figure 5 As shown in (a)-(d), (a) is the spectrum under increasing the organic phase ratio (Table 3, gradient 1, 16 minutes (A: B = 32: 68)), (b) is the spectrum under increasing the flow rate (0.8 ml / min), (c) is the spectrum under increasing the column temperature to 35°C, and (d) is the spectrum under increasing the column temperature to 25°C and changing the organic phase ratio (Table 3, gradient 1, 16 minutes (A: B = 32: 68)).

[0114] The results showed that the peak retention time was significantly advanced to 60 minutes by increasing the organic phase ratio, increasing the flow rate or increasing the column temperature, but peaks 3 and 4 overlapped after the column temperature was increased to 30°C. When the column temperature was increased to 25°C and the organic phase ratio was changed, the peak separation effect in the first 26 minutes was better, but the peak separation effect in the last 26 minutes was worse.

[0115] 6. Optimization of the peak after 26 minutes of separation

[0116] In order to further optimize the peak after 26 minutes of separation, we tried to replace different acid solutions and change the organic phase methanol ratio, flow rate, column temperature and other optimization conditions. The characteristic spectra constructed under each optimization condition are shown in the attached Figure 6 As shown in (a)-(f), (a) is the spectrum under methanol-0.1% phosphoric acid, (b) is the spectrum under methanol-0.1% formic acid, (c) is the spectrum under methanol-0.5% formic acid, (d) is the spectrum under changing flow rate (0.8 ml / min), (e) is the spectrum under changing the proportion of organic phase (Table 4), and (f) is the spectrum under changing column temperature (25°C).

[0117] The results show that after changing different acids, peak 11 did not improve, peak 13 separated a small peak, but peaks 3 and 4 were not separated, so phosphoric acid was still selected; and peak 11 was not separated by changing the organic phase ratio, flow rate and column temperature, and peak 13 was not completely improved. After changing the flow rate, the peak shape of peak 9 was poor, and the peak of 20-21min did not achieve baseline separation; after changing the column temperature, the peak of 20-24min did not achieve baseline separation; after changing the organic phase ratio, although peak 11 was not separated, the overall peak shape was better, so the chromatogram after changing the organic phase ratio was selected.

[0118] 7. Determination of chromatographic conditions

[0119] Based on the above investigation results, the characteristic spectrum determination conditions of Artemisia annua formula granules were established: octadecylsilane bonded silica gel was used as the filler (CORTECS T3; 4.6×150mm, 2.7μm), methanol was used as the mobile phase A, and 0.1% phosphoric acid aqueous solution was used as the mobile phase B, and gradient elution was performed according to the provisions in Table 4; detection wavelength: 340nm; flow rate: 0.7mL per minute; column temperature: 20℃; theoretical plate number calculated based on the scopoletin peak should be no less than 5000.

[0120] Table 4 Mobile phase elution program

[0121] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 18 82 14 28 72 16 32 68 30 36 64 36 40 60 55 62 38

[0122] Preparation of test solution: Take about 0.4 g of Artemisia annua granules, weigh accurately, put in a stoppered conical flask, add 25 ml of 70% methanol accurately, weigh the weight, ultrasonically treat for 15 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate.

[0123] Determination method: Accurately pipette 5 μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, and record the chromatogram as shown in the attached Figure 7 As shown, the spectral data are shown in Table 5 below.

[0124] As attached Figure 7 As can be seen from the spectrum in the figure, peak 1: neochlorogenic acid; peak 4: chlorogenic acid; peak 5: cryptochlorogenic acid; peak 6: 1,3-O-dicaffeoylquinic acid; peak 9: scopoletin; peak 11: 3,5-O-dicaffeoylquinic acid; peak 12: 4,5-di-O-caffeoylquinic acid.

[0125] Table 5 Spectrum data

[0126]

[0127]

[0128] Integration parameters: Baseline Skim Mode: Standard, Baseline Correction: Advanced, Valley Baseline: On, Slope Sensitivity: 1, Peak Width: 0.02, Minimum Peak Area: 1, Minimum Peak Height: 1.

[0129] 8. Identification of characteristic peaks in [Characteristic spectrum]

[0130] By combining the characteristic spectrum method determined above with the results of multiple batches of measurements (such as the attached Figure 8 ) 14 characteristic peaks were tentatively identified, and the identification of characteristic peaks was studied in depth. After consulting the literature and positioning the reference substances (such as the attached Fig. 9) confirmed: Peak 1: neochlorogenic acid; Peak 4: chlorogenic acid; Peak 5: cryptochlorogenic acid; Peak 6: 1,3-O-dicaffeoylquinic acid; Peak 9: scopoletin; Peak 11: 3,5-O-dicaffeoylquinic acid; Peak 12: 4,5-di-O-caffeoylquinic acid.

[0131] Attached Fig.10 The chromatograms and ultraviolet absorption spectra of different reference substances, wherein (a)-(g) are the spectra of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, scopoletin, 3,5-O-dicaffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid, respectively; wherein (1)-(3) represent the chromatogram of the reference substance, the ultraviolet absorption spectrum of the reference substance, and the ultraviolet absorption spectrum of the reference substance in the sample, respectively.

[0132] Example 2 Construction of characteristic spectrum of Artemisia annua medicinal material

[0133] 1. Chromatographic conditions and system suitability

[0134] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel as filler, chromatographic column: CORTECS T3; 4.6×150 mm, 2.7 μm, methanol as mobile phase A, 0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution according to the provisions in the above Table 4; detection wavelength: 340 nm; flow rate: 0.7 mL per minute; column temperature: 20°C. The number of theoretical plates calculated based on the scopoletin peak should be no less than 5000.

[0135] Preparation of reference solution: Take about 4g of Artemisia annua reference medicinal material, place it in a stoppered conical flask, add 100ml of water, heat and reflux for 45 minutes, take it out, cool it, filter it, evaporate the filtrate to dryness, add 25ml of 70% methanol to the residue, ultrasonically treat it (power 250W, frequency 53kHz) for 30 minutes, take it out, cool it, filter it, and take the filtrate as the reference medicinal material reference solution. Take an appropriate amount of scopoletin reference substance, accurately weigh it, place it in a brown volumetric flask, add 70% methanol to make a solution containing 40μg of scopoletin per 1ml, as the reference substance solution.

[0136] Preparation of test solution: Take about 4g of Artemisia annua powder (sieve No. 3), accurately weigh, put it in a stoppered conical flask, accurately add 100ml of water, heat and reflux for 45 minutes, take it out, cool, filter, and concentrate the filtrate to dryness under reduced pressure at 80℃, accurately add 25ml of 70% methanol to the residue, stopper it, weigh it, ultrasonically treat it (power 250W, frequency 53KHz) for 30 minutes, take it out, cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate.

[0137] Determination method: Accurately pipette 5 μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, and record the chromatogram.

[0138] The reference characteristic spectrum and the chromatogram of the test product of Artemisia annua are shown in the attached Fig.11 As shown in (a)-(b), the system adaptability parameters are shown in Table 6.

[0139] Table 6 System adaptability parameters

[0140] Peak name Retention time Peak area Peak height Peak width (50%) Asymmetry Separation Number of plates 1 6.53 2.0354 11.08 0.178 0.95 10.76 7480 2 9.547 8.9883 49.83 0.153 1.53 6.19 21540 3 11.18 1.9585 11.44 0.158 1.11 1.99 27680 4 11.73 8.9472 47.83 0.168 1.18 5.95 27072 5 13.403 2.6155 14.49 0.164 1.15 13.12 36927 6 17.223 2.9849 14.8 0.179 1.12 6.61 51118 7 18.95 13.6281 93.38 0.129 1.27 3.56 119913 8 19.77 3.8969 22.51 0.143 1.42 2.28 106252 9 20.327 24.5547 150.04 0.146 1.24 32.38 107475 10 30.653 5.8928 22.76 0.23 1.15 9.38 98072 11 34.357 9.6132 35.77 0.235 1.34 21.36 118068 12 41.67 13.5791 70.44 0.169 1.36 48.55 337852 13 53.24 3.3327 26.37 0.112 1.33 10.7 1242081 14 55.427 5.648 37.7 0.129 1.37 na 1026031

[0141] It can be seen that there should be 14 characteristic peaks in the chromatogram of the test sample, and they should correspond to the relative retention times of the 14 characteristic peaks in the chromatogram of the reference medicinal material. Peak 9 should correspond to the retention time of the reference peak of the scopoletin reference substance. The peak corresponding to the scopoletin reference substance is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are 0.32 (peak 1), 0.47 (peak 2), 0.55 (peak 3), 0.58 (peak 4), 0.66 (peak 5), 0.85 (peak 6), 0.93 (peak 7), 0.97 (peak 8), 1.51 (peak 10), 1.69, (peak 11) 2.05 (peak 12), 2.62 (peak 13), 2.72 (peak 14). Calculate the relative peak area of ​​the sum of the peak areas of Peak 1, Peak 4, and Peak 5 and the S peak, which must not be less than 0.55.

[0142] 2. Instrument precision test

[0143] Take the same Artemisia annua medicinal material test solution and repeat the injection 6 times according to the above characteristic spectrum method to measure the relative retention time and relative peak area of ​​14 common peaks. The results are shown in Tables 7-8 below and the instrument precision chromatogram is shown in the attached Fig.12 , from top to bottom represent precision 1-6 respectively.

[0144] Table 7 Instrument precision relative retention time results

[0145]

[0146]

[0147] Table 8 Instrument precision relative retention peak area results

[0148]

[0149] The results showed that the RSD values ​​of the relative retention time and relative peak area of ​​each common peak and the reference peak (peak S) were all less than 2.0%, indicating that the instrument had good precision.

[0150] 3. Repeatability test

[0151] Take the same batch of Artemisia annua medicinal material test solution, prepare 6 test solution in parallel according to the method under [Characteristic Spectrum], and measure the relative retention time of 14 common peaks. The results are shown in Tables 9-10 below, and the repeatability chromatogram is shown in the attached Fig.13 From top to bottom they represent repeatability 1-6 respectively.

[0152] Table 9 Method repeatability relative retention time results

[0153]

[0154] Table 10 Method repeatability relative retention peak area results

[0155]

[0156] The results showed that the RSD values ​​of the relative retention times of the common peaks and the reference peak (peak S) were less than 2.0%, indicating that the method had good repeatability.

[0157] 4. Exclusivity

[0158] Take the test sample and use 70% methanol as the extraction solvent. Accurately pipette 5 μL of the test sample solution and negative control solution (70% methanol) and inject them into the high performance liquid chromatograph respectively. Test according to the method of [Characteristic Spectrum]. The chromatogram of the negative control (70% methanol) is attached. Fig.14 The results showed that the negative had no interference.

[0159] 5. Different personnel (intermediate precision)

[0160] For the same batch of Artemisia annua medicinal material test solution, experimenters A, B, and C prepared samples according to the test sample preparation method in [Characteristic spectrum], and used the same equipment to determine the relative retention time of each common peak. The detailed results are shown in Tables 11-12 below, and the chromatograms of different operators A, B, and C are shown in the attached Fig.15 As shown, from bottom to top, they are the operation results of A, B, and C.

[0161] Table 11 Relative retention time results for different personnel

[0162]

[0163] Table 12 Relative retention peak area results of different personnel

[0164]

[0165] The results showed that the relative average deviations of each characteristic peak and the reference S peak were less than 2%, indicating that the intermediate precision of this method was good.

[0166] 6. Stability test

[0167] Take the same Artemisia annua medicinal material test solution and measure it according to the chromatographic conditions under [Characteristic Spectrum] at 0h, 4h, 8h, 12h, 16h and 24h respectively, and calculate the relative retention time of 10 characteristic peaks. See Table 13-14 below for details, and the stability chromatogram is attached Fig.16 As shown, from top to bottom, they represent the results of 0h, 4h, 8h, 12h, 16h, and 24h respectively.

[0168] Table 13 Relative retention time results of stability test

[0169]

[0170] Table 14 Relative retention peak area results of stability test

[0171]

[0172]

[0173] The results showed that the test solution was stable within 24 hours and met the measurement requirements.

[0174] 7. Investigation of different flow rates

[0175] Take the same sample solution of Artemisia annua and measure it according to the [Characteristic Spectrum] method at flow rates of 0.6mL / min, 0.7mL / min, and 0.8mL / min, respectively, and measure the relative retention time and relative peak area of ​​14 common peaks. See Tables 15-16 below for details. See the attached chromatograms at different flow rates. Fig.17 As shown, (a)-(c) represent the chromatograms at 0.6mL / min, 0.7mL / min, and 0.8mL / min, respectively.

[0176] Table 15 Relative retention time results at different flow rates

[0177]

[0178] Table 16 Relative retention peak area results at different flow rates

[0179]

[0180] The results show that different flow rates have a certain influence on the relative retention time of characteristic peaks. When the flow rate changes slightly, the retention time of each characteristic chromatographic peak changes slightly, and the relative average deviation is large, which has a greater impact on the separation degree. Therefore, the flow rate selected for this method is 0.7mL / min.

[0181] 8. Investigation of different column temperatures

[0182] Take the same sample solution of Artemisia annua and measure it at column temperatures of 18°C, 20°C and 22°C according to the [Characteristic Spectrum] determination method to determine the relative retention time of 14 common peaks. The specific results are shown in Tables 17-18 below. The chromatograms at different column temperatures are shown in the attached Fig.18 , where (a)-(c) represent the chromatograms at column temperatures of 18, 20, and 22°C, respectively.

[0183] Table 17 Relative retention time results at different column temperatures

[0184]

[0185] Table 18 Relative retention peak area results at different column temperatures

[0186]

[0187] The results showed that the RSD of the relative retention time of each characteristic peak was less than 2%, the relative peak area deviations of peaks 8 and 10 were large, and the RSD of other relative peak areas were less than 6%, indicating that slight changes in column temperature had little effect on the relative retention time of the characteristic peaks, but had a certain effect on the relative peak area, so the column temperature was selected as 20°C.

[0188] 9. Investigation of different chromatographic columns

[0189] The same inspector took the same sample solution at different times and tested it on Waters CORTECS T3 4.6mmx150mm SN:01353120115928, Waters CORTECS T3 4.6mmx150mmSN:01393132315626 and Waters CORTECS T3 4.6mmx150mm SN:01393132315612 HPLC respectively according to the above conditions. The relative retention time of each common peak was determined according to the above chromatographic conditions. The results are shown in Tables 19-20 below and the chromatograms under different chromatographic columns are shown in the attached table. Fig.19 As shown, from bottom to top are the results of Waters CORTECS T3 4.6mmx150mm SN: 01353120115928, Waters CORTECS T3 4.6mmx150mm SN: 01393132315626, and Waters CORTECS T3 4.6mmx150mm SN: 01393132315612.

[0190] Table 19 Relative retention time results of different chromatographic columns

[0191]

[0192] Table 20 Relative retention peak area results of different chromatographic columns

[0193]

[0194] The results showed that the RSD of relative retention time and relative peak area of ​​each characteristic peak was less than 2%.

[0195] 10. Investigation of different instruments

[0196] The same inspector took the same sample solution at different times and tested it on Agilent and Thermo Fisher high performance liquid chromatographs according to the above conditions. The relative retention time of each common peak was determined according to the above chromatographic conditions. The results are shown in Tables 21-22 below. The chromatograms under different instruments are shown in the attached Fig. 20 As shown, (a)-(b) represent the detection results of Thermo Fisher and Agilent high performance liquid chromatography, respectively.

[0197] Table 21 Relative retention time results of different instruments

[0198]

[0199]

[0200] Table 22 Relative retention peak area results of different instruments

[0201]

[0202] The results showed that the RSD of the relative retention time of each characteristic peak was less than 3%, and the relative peak area was greatly affected, which might be due to the different responses on different instruments.

[0203] It can be seen that from the above methodological investigation results, among the 14 common peaks of the established Artemisia annua medicinal material [characteristic spectrum], each chromatographic peak is the scopoletin reference peak and the corresponding peak is the S peak. The relative retention time is calculated. Each chromatographic peak is affected to a certain extent by different chromatographic columns, different high performance liquid phases, column temperature and flow rate factors. In order to adapt to its durability, it is recommended to control the specified value range within ±10%.

[0204] 11. Characteristic spectrum determination of multiple batches of Artemisia annua medicinal materials

[0205] Take 15 batches of Artemisia annua medicinal materials (batch numbers: 1710001, 1811002, 1811003, 1811004, 1811005, 1811006, 1811007, 1811008, 1811009, 1811010, 1811011, 181112, 1811013, 1812016, 1812017) and operate according to the proposed method for determining the characteristic spectrum of Artemisia annua medicinal materials. The relative retention time of each common peak was determined under the above chromatographic conditions. The results are shown in Tables 23-24 below. The characteristic spectrum of 15 batches of Artemisia annua medicinal materials is shown in the attached Fig.21 shown.

[0206] Table 23 Relative retention time of characteristic spectra of 15 batches of Artemisia annua

[0207]

[0208]

[0209] Table 24 Relative peak areas of characteristic spectra of 15 batches of Artemisia annua

[0210]

[0211]

[0212] The measurement results show that the relative retention times of the characteristic peaks of 15 batches of Artemisia annua medicinal materials correspond to the corresponding characteristic peaks of the reference medicinal materials, and the specified relative retention times are all within ±10% of the specified values ​​of the reference atlas of Artemisia annua medicinal materials.

[0213] Relative retention time of characteristic peaks in characteristic spectra

[0214] According to the results of the characteristic spectrum of 15 batches of Artemisia annua, it is determined that 14 characteristic peaks should be present in the chromatogram of the characteristic spectrum of Artemisia annua, and the relative retention time of the 14 characteristic peaks in the chromatogram of the reference material should correspond to that of the reference material of the reference material, among which Peak 9 should correspond to the retention time of the reference material of scopoletin, and the peak corresponding to the reference material of scopoletin is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are 0.32 (peak 1), 0.47 (peak 2), 0.55 (peak 3), 0.58 (peak 4), 0.66 (peak 5), 0.85 (peak 6), 0.93 (peak 7), 0.97 (peak 8), 1.51 (peak 10), 1.69, (peak 11) 2.05 (peak 12), 2.62 (peak 13), 2.72 (peak 14).

[0215] Relative peak area specification value of characteristic peak of characteristic spectrum

[0216] Artemisia annua mainly contains compounds such as flavonoids, coumarins, terpenes, phenylpropionic acids and volatile oils. According to the literature, the most commonly used quantitative components for the determination of the content of Artemisia annua are artemisinin, scopoletin, artemisinic acid, artemisinin B, chlorogenic acid, etc. Among them, the recognized effective ingredients sesquiterpenes, such as artemisinin, artemisinic acid, artemisinin B, etc., are almost insoluble in water and unstable to heat, so they are difficult to detect in water extracts and related preparations. Scopoletin belongs to the coumarin compound, which has the effects of clearing heat, antibacterial, anti-inflammatory, expectorant and antitussive. Phenylpropionic acid components such as chlorogenic acid are also important active ingredients, with anti-inflammatory, antibacterial, antithrombotic and other effects, which can reflect the pharmacological activity of the traditional efficacy of Artemisia annua. By combining the characteristic spectrum peak identification information, peak 1 is neochlorogenic acid; peak 4 is chlorogenic acid; peak 5 is cryptochlorogenic acid; peak 6 is 1,3-O-dicaffeoylquinic acid; peak 11 is 3,5-O-dicaffeoylquinic acid; peak 12 is 4,5-di-O-caffeoylquinic acid, which belongs to the phenylpropionic acid class, and peak 9 is scopoletin; it belongs to the coumarin class, and they are all the main active substances of Artemisia annua. Therefore, the relative peak area data of peak 1, peak 4, peak 5, peak 6, peak 9, peak 11, and peak 12 are analyzed. Peak 9 is scopoletin. At the same time, as a content determination indicator, it is more suitable as the S peak to calculate the relative peak areas of the remaining characteristic peaks for control. The relative peak area range of analysis peak 1 is 0.08-0.21, the relative peak area range of peak 2 is 0.02-0.30, the relative peak area range of peak 4 is 0.30-0.50, the relative peak area range of peak 5 is 0.11-0.23, the relative peak area range of peak 6 is 0.12-0.61, the relative peak area range of peak 11 is 0.08-0.51, and the relative peak area range of peak 12 is 0.18-0.80. Combined with the analysis of raw material and standard soup data, it was found that peak 1 (neochlorogenic acid), peak 4 (chlorogenic acid) and peak 5 (cryptochlorogenic acid) had good transmission in the whole process. Taking into account the small relative peak area of ​​a single characteristic peak, the sum of the areas of peak 1, peak 4 and peak 5 and the relative peak area of ​​peak 9 (scopoletin) were calculated. The relative peak area of ​​(peak 1 + peak 4 + peak 5) / peak 9 in 15 batches of Artemisia annua medicinal materials ranged from 0.55 to 0.92, with an average of 0.68. The relative peak area of ​​(peak 1 + peak 4 + peak 5) / peak 9 of Artemisia annua medicinal materials was tentatively determined as 0.55 based on the lower limit of multiple batches, that is, the relative peak area of ​​the sum of the peak areas of peak 1, peak 4 and peak 5 and the S peak must not be less than 0.55.

[0217] Example 3 Characteristic Spectrum of Artemisia annua Pieces

[0218] 1. Chromatographic conditions and system suitability

[0219] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel as filler, chromatographic column: CORTECS T3; 4.6×150 mm, 2.7 μm, methanol as mobile phase A, 0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution according to the specified procedure in Table 4 above; detection wavelength: 340 nm; flow rate: 0.7 mL per minute; column temperature: 20°C. The number of theoretical plates calculated based on the scopoletin peak should be no less than 5000.

[0220] Preparation of reference solution: Take about 4g of Artemisia annua reference medicinal material, place it in a stoppered conical flask, add 100ml of water, heat and reflux for 45 minutes, take it out, cool it, filter it, evaporate the filtrate to dryness, add 25ml of 70% methanol to the residue, ultrasonically treat it (power 250W, frequency 53kHz) for 30 minutes, take it out, cool it, filter it, and take the filtrate as the reference medicinal material reference solution. Take an appropriate amount of scopoletin reference substance, accurately weigh it, place it in a brown volumetric flask, add 70% methanol to make a solution containing 40μg of scopoletin per 1ml, as the reference substance solution.

[0221] Preparation of test solution: Take about 4g of Artemisia annua powder (No. 3 sieve), accurately weigh, put it in a stoppered conical flask, accurately add 100ml of water, heat and reflux for 45 minutes, take it out, cool, filter, and concentrate the filtrate to dryness under reduced pressure at 80℃, accurately add 25ml of 70% methanol to the residue, stopper it, weigh it, ultrasonically treat it (power 250W, frequency 53KHz) for 30 minutes, take it out, cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate.

[0222] Determination method: Accurately pipette 5 μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, record chromatogram, and obtain. The reference characteristic spectrum and the chromatogram of the test sample of Artemisia annua slices are respectively shown in the attached Fig. 22 As shown in (a)-(b), the system adaptability parameter results are shown in Table 25.

[0223] It can be seen that there should be 14 characteristic peaks in the chromatogram of the test sample, and they should correspond to the relative retention times of the 14 characteristic peaks in the chromatogram of the reference medicinal material. Peak 9 should correspond to the retention time of the reference peak of the scopoletin reference substance. The peak corresponding to the scopoletin reference substance is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are 0.32 (peak 1), 0.47 (peak 2), 0.55 (peak 3), 0.58 (peak 4), 0.66 (peak 5), 0.85 (peak 6), 0.93 (peak 7), 0.97 (peak 8), 1.51 (peak 10), 1.69, (peak 11) 2.05 (peak 12), 2.62 (peak 13), 2.72 (peak 14). Calculate the relative peak area of ​​the sum of the peak areas of Peak 1, Peak 4, and Peak 5 and the S peak, which must not be less than 0.55.

[0224] Table 25 System adaptability parameters

[0225] Peak name Retention time Peak area Peak height Peak width (50%) Asymmetry Separation Number of plates 1 6.537 1.5134 12.48 0.108 1.24 16.3 20217 2 9.563 5.2555 44.21 0.111 1.1 8.11 41173 3 11.203 1.4394 10.07 0.128 1.27 2.5 42614 4 11.753 6.5383 43.21 0.132 1.32 7.36 43892 5 13.43 1.9132 12.43 0.137 1.27 16.18 53360 6 17.26 2.1627 13.35 0.142 1.36 7.63 81295 7 18.967 9.9117 71.81 0.121 1.33 6.12 135024 8 19.787 2.8605 17.06 na na na na 9 20.347 17.9475 110.21 0.145 1.25 33.07 109477 10 30.697 4.1416 16.87 0.225 1.23 9.4 103530 11 34.363 7.1318 26.26 0.236 1.38 21.31 117908 12 41.677 9.9071 51.4 0.169 1.36 48.27 335513 13 53.253 2.4292 19.11 0.114 1.33 10.73 1216429 14 55.467 4.0845 27.26 0.13 1.38 na 1012376

[0226] 2. Characteristic spectrum determination of 15 batches of Artemisia annua slices

[0227] Take 15 batches of Artemisia annua slices (batch numbers: 1802001Y, 1812002Y, 1812003Y, 1812004Y, 1812005Y, 1812006Y, 1812007Y, 1812008Y, 1812009Y, 1812010Y, 1812011Y, 1812012Y, 1812013Y, 1812016Y, 1812017Y) and operate according to the proposed method for determining the characteristic spectrum of Artemisia annua slices. The relative retention time of each common peak was determined under the above chromatographic conditions. The results are shown in Tables 26-27 below. The characteristic spectrum of 15 batches of Artemisia annua slices is shown in the attached Fig.23 shown.

[0228] Table 26 Relative retention time of characteristic spectra of 15 batches of Artemisia annua slices

[0229]

[0230]

[0231] Table 27 Relative peak areas of characteristic spectra of 15 batches of Artemisia annua slices

[0232]

[0233]

[0234] Characteristic peak value of characteristic spectrum

[0235] According to the results of the characteristic spectrum of 15 batches of Artemisia annua slices, it is determined that 14 characteristic peaks should be present in the chromatogram of Artemisia annua slices, and should correspond to the relative retention time of the 14 characteristic peaks in the chromatogram of the reference medicinal material, among which Peak 9 should correspond to the retention time of the reference peak of scopoletin reference substance, and the peak corresponding to the reference peak of scopoletin is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are 0.32 (peak 1), 0.47 (peak 2), 0.55 (peak 3), 0.58 (peak 4), 0.66 (peak 5), 0.85 (peak 6), 0.93 (peak 7), 0.97 (peak 8), 1.51 (peak 10), 1.69, (peak 11) 2.05 (peak 12), 2.62 (peak 13), 2.72 (peak 14).

[0236] Relative peak area specification value of characteristic peak of characteristic spectrum (quantity transfer)

[0237] According to the 15 batches of Artemisia annua medicinal materials and the corresponding 15 batches of Artemisia annua slices selected for the study, the determination was carried out according to the method under the characteristic spectrum in the main text. From the results, it can be seen that the number of characteristic peaks obtained is consistent, and the relative retention time and relative peak area are basically consistent, indicating that the main chemical components of Artemisia annua medicinal materials have not changed in the process of obtaining Artemisia annua slices after simple cleaning, and the material basis is consistent. From the test results, the relative peak area results of the slices are basically consistent with those of the medicinal materials. The relative peak area of ​​the Artemisia annua slices is tentatively determined to be the same as that of the medicinal materials, that is, the relative peak area of ​​the sum of the peak areas of peaks 1, 4, and 5 and the peak S shall not be less than 0.55.

[0238] Example 4 Characteristic spectrum of freeze-dried powder of standard decoction of Artemisia annua

[0239] 1. Chromatographic conditions and system suitability

[0240] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel as filler, chromatographic column: CORTECS T3; 4.6×150 mm, 2.7 μm, methanol as mobile phase A, 0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution according to the specified procedure in Table 4 above; detection wavelength: 340 nm; flow rate: 0.7 mL per minute; column temperature: 20°C. The number of theoretical plates calculated based on the scopoletin peak should be no less than 5000.

[0241] Preparation of reference solution Take about 4g of Artemisia annua reference medicinal material, place it in a stoppered conical flask, add 100ml of water, heat and reflux for 45 minutes, take it out, cool it, filter it, evaporate the filtrate to dryness, add 25ml of 70% methanol to the residue, ultrasonically treat it (power 250W, frequency 53kHz) for 30 minutes, take it out, cool it, filter it, and take the filtrate as the reference medicinal material reference solution. Take an appropriate amount of scopoletin reference substance, accurately weigh it, place it in a brown volumetric flask, add 70% methanol to make a solution containing 40μg of scopoletin per 1ml, as the reference substance solution.

[0242] Preparation of test solution: Take about 0.4 g of standard decoction lyophilized powder, weigh accurately, put it in a stoppered conical flask, add 25 ml of 70% methanol accurately, weigh the weight, ultrasonically treat for 30 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate.

[0243] Determination method: Accurately pipette 5μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, record chromatogram, and obtain. The reference characteristic spectrum and the chromatogram of test sample of Artemisia annua standard decoction freeze-dried powder are shown in the attached Fig.24 As shown in (a) and (b), the system adaptability parameters are shown in Table 28 below.

[0244] It can be seen that there should be 14 characteristic peaks in the chromatogram of the test sample, and they should correspond to the relative retention times of the 14 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 9 should correspond to the retention time of the reference peak of the scopoletin reference substance, and the peak corresponding to the scopoletin reference substance is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are 0.32 (peak 1), 0.47 (peak 2), 0.55 (peak 3), 0.58 (peak 4), 0.66 (peak 5), 0.85 (peak 6), 0.93 (peak 7), 0.97 (peak 8), 1.51 (peak 10), 1.69, (peak 11) 2.05 (peak 12), 2.62 (peak 13), 2.72 (peak 14). Calculate the relative peak area of ​​the sum of the peak areas of Peak 1, Peak 4, and Peak 5 and the S peak, which must not be less than 0.55.

[0245] Table 28 System adaptability parameters

[0246] Peak name Retention time Peak area Peak height Peak width (50%) Asymmetry Separation Number of plates 1 6.53 2.5336 13.19 0.188 0.91 10.41 6693 2 9.543 11.2214 62.19 0.154 1.55 6.08 21358 3 11.183 2.4196 13.69 0.164 1.09 1.9 25654 4 11.733 11.1871 57 0.177 1.12 5.63 24235 5 13.403 3.2417 17.17 0.173 1.12 12.31 33305 6 17.24 3.7621 17.16 0.195 1.01 6.21 43393 7 18.957 17.0812 115.1 0.131 1.28 3.49 115676 8 19.773 4.8987 27.91 0.145 1.39 2.3 102774 9 20.343 30.7702 185.73 0.148 1.22 32.04 104990 10 30.67 7.0717 27.81 0.232 1.22 9.22 96403 11 34.343 12.2075 44.92 0.238 1.36 21.12 115628 12 41.67 17.1529 87.86 0.172 1.35 47.99 326253 13 53.253 4.1924 33.1 0.113 1.32 10.73 1227738 14 55.463 7.1359 47.38 0.13 1.42 na 1008757

[0247] 2. Instrument precision test

[0248] Take the same Artemisia annua decoction piece standard decoction (lyophilized powder) test solution, repeat the injection 6 times according to the characteristic spectrum method, and determine the relative retention time and relative peak area of ​​14 common peaks. The specific results are shown in Tables 29-30 below, and the instrument precision chromatogram is shown in the attached Fig.25, from top to bottom represent precision 1-6 respectively.

[0249] Table 29 Instrument precision relative retention time results

[0250]

[0251]

[0252] Table 30 Instrument precision relative retention peak area results

[0253]

[0254] The results showed that the RSD values ​​of the relative retention time and relative peak area of ​​each common peak and the reference peak (peak S) were all less than 3.5%, indicating that the instrument had good precision.

[0255] 3. Repeatability test

[0256] Take the same batch of Artemisia annua decoction standard decoction (lyophilized powder) test solution, prepare 6 test solutions in parallel according to the method under [Characteristic spectrum], and measure the relative retention time and relative peak area of ​​14 common peaks. The detailed results are shown in Tables 31-32 below, and the repeatability chromatogram is shown in the attached Fig.26 , from top to bottom they represent repeatability 1-6 respectively.

[0257] Table 31 Method repeatability relative retention time results

[0258]

[0259]

[0260] Table 32 Method repeatability relative retention peak area results

[0261]

[0262] The results showed that the RSD values ​​of the relative retention times of the common peaks and the reference peak (peak S) were less than 3.0%, indicating that the method had good repeatability.

[0263] 4. Exclusivity

[0264] The extraction solvent for the test sample was 70% methanol. 5 μL of the test sample solution and negative control solution were accurately drawn and injected into the HPLC respectively. The test was performed according to the method of [Characteristic Spectrum]. The chromatogram of the negative control (70% methanol) is shown in the attached figure. Fig. 27 , the result is negative and there is no interference.

[0265] 5. Different personnel (intermediate precision)

[0266] For the same batch of test solution of standard decoction of Artemisia annua (lyophilized powder), experimenters A, B, and C prepared samples according to the test sample preparation method in [Characteristic spectrum], and used the same equipment to determine the relative retention time of each common peak. The detailed results are shown in Tables 33-34 below, and the chromatograms of different experimenters A, B, and C are shown in the attached Fig.28 , representing the operation results of A, B, and C from bottom to top.

[0267] Table 33 Relative retention time results of different personnel

[0268]

[0269]

[0270] Table 34 Relative retention peak area results of different personnel

[0271]

[0272] The results showed that the relative average deviations of each characteristic peak and the reference S peak were less than 3%, indicating that the intermediate precision of this method was good.

[0273] 6. Stability test

[0274] Take the test solution of the same Artemisia annua slice standard decoction (lyophilized powder) and measure it according to the chromatographic conditions under [Characteristic Spectrum] at 0h, 4h, 8h, 12h, 16h and 24h respectively, and calculate the relative retention time of 14 characteristic peaks. See Tables 35-36 below for details, and the stability chromatogram is attached. Fig.29 , from top to bottom they represent the results of 0h, 4h, 8h, 12h, 16h, and 24h respectively.

[0275] Table 35 Method stability relative retention time results

[0276]

[0277]

[0278] Table 36 Method stability relative retention peak area results

[0279]

[0280] The results showed that the test solution was stable within 24 hours and met the measurement requirements.

[0281] 7. Investigation of different flow rates

[0282] Take the same sample solution of Artemisia annua decoction standard decoction (lyophilized powder) and measure it according to the [Characteristic Spectrum] method at flow rates of 0.6mL / min, 0.7mL / min, and 0.8mL / min, respectively, and measure the relative retention times of 14 common peaks. See Tables 37-38 below for details. See the attached chromatograms at different flow rates. Fig.30 , from bottom to top, the chromatograms at flow rates of 0.7mL / min, 0.6mL / min, and 0.8mL / min are shown.

[0283] Table 37 Relative retention time results at different flow rates

[0284]

[0285] Table 38 Relative retention peak area results at different flow rates

[0286]

[0287] The results show that different flow rates have a certain influence on the relative retention time of characteristic peaks. When the flow rate changes slightly, the retention time of each characteristic chromatographic peak changes slightly, and the separation of characteristic peaks will be affected. Therefore, the flow rate is fixed at 0.7 mL / min in the method of the present invention.

[0288] 8. Investigation of different column temperatures

[0289] Take the same sample solution of Artemisia annua decoction standard decoction (lyophilized powder) and measure it at column temperatures of 18℃, 20℃ and 22℃ according to the [Characteristic Spectrum] determination method. The specific results are shown in Tables 39-40 below, and the chromatograms under different column temperatures are shown in the attached Fig.31 , from top to bottom, the chromatograms at 20, 22, and 18°C ​​are shown.

[0290] Table 39 Relative retention time results at different column temperatures

[0291]

[0292] Table 40 Relative retention peak area results at different flow rates

[0293]

[0294] The results showed that the RSD of the relative retention time of the 14 common peaks was less than 2%, and the relative peak area had a greater impact. A slight change in column temperature had a smaller effect on the relative retention time and relative peak area of ​​the characteristic peaks, so the column temperature was selected as 20℃.

[0295] 9. Investigation of different chromatographic column batch numbers

[0296] Through the process of chromatographic condition optimization, it can be known that this method has higher requirements for the chromatographic column and needs to fix the chromatographic column WatersCORTECS T3 4.6mmx150mm SN:01353120115928. Therefore, chromatographic columns with different production batches were investigated (column 1: SN:01353120115928; column 2: SN:01393132315626; column 3: SN:01393132315612). The same sample solution was taken and measured according to the above conditions. The relative retention time and relative peak area of ​​each common peak were calculated. The results are shown in Tables 41-42 below. The chromatograms under different chromatographic column batch conditions are shown in the attached Fig.32 , where from bottom to top are the results of Waters CORTECST3 4.6mmx150mm SN:01353120115928, Waters CORTECS T3 4.6mmx150mm SN:01393132315626, and Waters CORTECS T3 4.6mmx150mm SN:01393132315612.

[0297] Table 41 Relative retention time results of different chromatographic column batches

[0298]

[0299] Table 42 Relative retention peak area results of different chromatographic column batches

[0300]

[0301] The results showed that the relative retention time and relative peak area of ​​the S peak were less affected than those of the reference substance.

[0302] 10. Investigation of different instruments

[0303] The same inspector took the same sample solution at different times and tested it on Dionex and Agilent HPLC respectively according to the above conditions. The relative retention time of each common peak was determined according to the above chromatographic conditions. The results are shown in Tables 43-44 below. The chromatograms under different instruments are shown in the attached table. Fig.33 , where (a)-(b) represent the chromatographic results of Dionex and Agilent high performance liquid chromatographs, respectively.

[0304] Table 43 Relative retention time results of different instruments

[0305]

[0306] Table 44 Relative retention peak area results of different instruments

[0307]

[0308] The results showed that the relative retention time and relative peak area of ​​each characteristic peak and the reference S peak had little deviation.

[0309] In summary, based on the above methodological investigation results, among the 14 common peaks of the standard decoction of Artemisia annua (lyophilized powder) [characteristic spectrum] established, the peak corresponding to the scopoletin reference peak is taken as the S peak, and the relative retention time is calculated. Each chromatographic peak is affected to a certain extent by different chromatographic columns, different high performance liquid phases, column temperature and flow rate factors. In order to adapt to its durability, it is recommended to control the specified value range within ±10%.

[0310] 11. Characteristic spectrum determination of multiple batches of standard decoctions (lyophilized powder) of Artemisia annua

[0311] Take 15 batches of standard decoction of Artemisia annua (freeze-dried powder) (batch number: 1802001Y, 1812002Y, 1812003Y, 1812004Y, 1812005Y, 1812006Y, 1812007Y, 1812008Y, 1812009Y, 1812010Y, 1812011Y, 1812012Y, 1812013Y, 1812016Y, 1812017Y) and operate according to the proposed method for determining the characteristic spectrum of standard decoction of Artemisia annua (freeze-dried powder). The relative retention time of each common peak was determined under the above chromatographic conditions. The results are shown in Tables 45-46 below. The characteristic spectrum of 15 batches of standard decoction of Artemisia annua (freeze-dried powder) is shown in the attached Fig.34 , among which S1-S15 are: 1802001Y, 1812002Y, 1812003Y, 1812004Y, 1812005Y, 1812006Y, 1812007Y, 1812008Y, 1812009Y, 1812010Y, 1812011Y, 1812012Y, 1812013Y, 1812016Y, 1812017Y respectively.

[0312] Table 45 Relative retention time of characteristic spectra of 15 batches of Artemisia annua freeze-dried powder

[0313]

[0314]

[0315] Table 46 Relative peak areas of characteristic spectra of 15 batches of freeze-dried powder of standard decoction of Artemisia annua

[0316]

[0317]

[0318] Relative retention time of characteristic peaks in characteristic spectra

[0319] According to the results of characteristic spectra measurement of 15 batches of standard decoctions (lyophilized powders) of Artemisia annua slices, it was determined that 14 characteristic peaks should be present in the chromatogram of the characteristic spectrum of the standard decoctions (lyophilized powders) of Artemisia annua slices, and the relative retention times of the 14 characteristic peaks in the chromatogram of the reference medicinal material reference substance should correspond to each other, among which Peak 9 should correspond to the retention time of the scopoletin reference substance peak, and the peak corresponding to the scopoletin reference substance is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are 0.32 (peak 1), 0.47 (peak 2), 0.55 (peak 3), 0.58 (peak 4), 0.66 (peak 5), 0.85 (peak 6), 0.93 (peak 7), 0.97 (peak 8), 1.51 (peak 10), 1.69, (peak 11) 2.05 (peak 12), 2.62 (peak 13), and 2.72 (peak 14).

[0320] Relative peak area specification value of characteristic peak of characteristic spectrum

[0321] Artemisia annua mainly contains compounds such as flavonoids, coumarins, terpenes, phenylpropionic acids and volatile oils. According to the literature, the most commonly used quantitative components for the determination of the content of Artemisia annua are artemisinin, scopoletin, artemisinic acid, artemisinin B, chlorogenic acid, etc. Among them, the recognized effective ingredients sesquiterpenes, such as artemisinin, artemisinic acid, artemisinin B, etc., are almost insoluble in water and unstable to heat, so they are difficult to detect in standard decoctions. Scopoletin belongs to the coumarin compound, which has the effects of clearing heat, antibacterial, anti-inflammatory, expectorant and cough-relieving. Phenylpropionic acid components such as chlorogenic acid are also important active ingredients, with anti-inflammatory, antibacterial, antithrombotic and other effects. All of them can reflect the pharmacological activity of the traditional efficacy of Artemisia annua. By combining the characteristic spectrum peak identification information, peak 1 is neochlorogenic acid; peak 4 is chlorogenic acid; peak 5 is cryptochlorogenic acid; peak 6 is 1,3-O-dicaffeoylquinic acid; peak 11 is 3,5-O-dicaffeoylquinic acid; peak 12 is 4,5-di-O-caffeoylquinic acid, which belongs to the phenylpropionic acid class, peak 2 is scopoletin; peak 9 is scopoletin; which belongs to the coumarin class, and are all the main active substances of Artemisia annua. Therefore, the relative peak area data of peak 1, peak 2, peak 4, peak 5, peak 6, peak 9, peak 11, and peak 12 are analyzed. Peak 9 is scopoletin. At the same time, as a content determination indicator, it is more suitable as the S peak to calculate the relative peak areas of the remaining characteristic peaks for control. The relative peak area range of peak 1 was 0.08-0.26, the relative peak area range of peak 2 was 0.02-0.36, the relative peak area range of peak 4 was 0.30-0.71, the relative peak area range of peak 5 was 0.10-0.27, the relative peak area range of peak 6 was 0.09-0.62, the relative peak area range of peak 11 was 0.09-0.76, and the relative peak area range of peak 12 was 0.18-0.82. Combining the data analysis of raw materials and standard soup, it was found that peak 1 (neochlorogenic acid), peak 4 (chlorogenic acid), and peak 5 (cryptochlorogenic acid) were in the whole process. The transferability is good. Taking into account the small relative peak area of ​​a single characteristic peak, the relative peak area of ​​the sum of the areas of Peak 1, Peak 4, and Peak 5 and Peak 9 (scopoletin) are calculated. The relative peak area of ​​(Peak 1 + Peak 4 + Peak 5) / Peak 9 in 15 batches of standard decoctions ranges from 0.55 to 1.23, with an average of 0.73. The lower limit of the relative peak area of ​​Artemisia annua formula granules (Peak 1 + Peak 4 + Peak 5) / Peak 9 is tentatively determined to be 0.55 based on the lower limit of multiple batches of standard decoctions, that is, the relative peak area of ​​the sum of the peak areas of Peak 1, Peak 4, and Peak 5 and Peak S shall not be less than 0.55.

[0322] Example 5 Characteristic spectrum of extract of Artemisia annua formula granule intermediate

[0323] Based on the establishment of the characteristic spectrum determination method of the "lyophilized powder" of the standard decoction of Artemisia annua slices in the aforementioned Example 4, in accordance with the "Technical Requirements for Quality Control and Standardization of Chinese Herbal Medicine Formula Granules" and the "Technical Requirements for Research and Development of Chinese Medicine Quality Standards" in the "Chinese Pharmacopoeia", by investigating the methodology of Artemisia annua formula granule extracts (system adaptability and specificity, precision and repeatability, instrument durability, chromatographic column durability, solution stability), a characteristic spectrum determination method for Artemisia annua formula granule intermediate extracts was established.

[0324] Trial drug: Artemisia annua formula granule extract: 5181201Y, 5181202Y, 5181203Y.

[0325] 1. Chromatographic conditions and system suitability

[0326] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel as filler, chromatographic column: CORTECS T3; 4.6×150 mm, 2.7 μm, methanol as mobile phase A, 0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution according to the specified procedure in Table 4 above; detection wavelength: 340 nm; flow rate: 0.7 mL per minute; column temperature: 20°C. The number of theoretical plates calculated based on the scopoletin peak should be no less than 5000.

[0327] Preparation of reference solution: Take about 4g of Artemisia annua reference medicinal material, place it in a stoppered conical flask, add 100ml of water, heat and reflux for 45 minutes, take it out, cool it, filter it, evaporate the filtrate to dryness, add 25ml of 70% methanol to the residue, ultrasonically treat it (power 250W, frequency 53kHz) for 30 minutes, take it out, cool it, filter it, and take the filtrate as the reference medicinal material reference solution. Take an appropriate amount of scopoletin reference substance, accurately weigh it, place it in a brown volumetric flask, add 70% methanol to make a solution containing 40μg of scopoletin per 1ml, as the reference substance solution.

[0328] Preparation of test solution: Take about 0.4 g of Artemisia annua intermediate extract, weigh it accurately, put it in a stoppered conical flask, add 25 ml of 70% methanol accurately, weigh it, treat it with ultrasound for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0329] Determination method: Accurately pipette 5 μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, record chromatogram, and obtain. The reference characteristic spectrum and the chromatogram of the test sample of Artemisia annua intermediate extract are attached. Fig.35 , the system adaptability parameters are shown in Table 47 below.

[0330] There should be 14 characteristic peaks in the chromatogram of the test sample, and the relative retention time should correspond to the 14 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 9 should correspond to the retention time of the scopoletin reference peak, and the peak corresponding to the scopoletin reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are 0.32 (peak 1), 0.47 (peak 2), 0.55 (peak 3), 0.58 (peak 4), 0.66 (peak 5), 0.85 (peak 6), 0.93 (peak 7), 0.97 (peak 8), 1.51 (peak 10), 1.69, (peak 11) 2.05 (peak 12), 2.62 (peak 13), 2.72 (peak 14). Calculate the relative peak area of ​​the sum of the peak areas of Peak 1, Peak 4, and Peak 5 and the S peak, which must not be less than 0.55.

[0331] Table 47 System adaptability parameters

[0332] Peak name Retention time Peak area Peak height Peak width (50%) Asymmetry Separation Number of plates 1 6.51 5.2049 26.7 0.193 0.87 9.94 6334 2 9.527 10.9645 57.48 0.166 1.42 5.76 18333 3 11.157 6.0931 33.52 0.169 1.1 1.87 24277 4 11.71 5.9878 30.33 0.18 1.12 5.56 23503 5 13.383 4.9736 26.08 0.176 1.09 12.19 32156 6 17.203 10.9526 50.64 0.194 1.01 6.24 43529 7 18.933 7.7178 51.22 0.133 1.27 3.53 111819 8 19.753 7.2386 43.2 0.141 1.38 2.24 109334 9 20.297 22.4403 137.05 0.145 1.28 32.15 108329 10 30.617 6.179 23.74 0.234 1.16 9.33 95124 11 34.327 6.1667 23.05 0.236 1.3 21.38 117586 12 41.65 11.4334 58.83 0.169 1.41 48.71 338187 13 53.23 2.5056 19.93 0.112 1.33 10.62 1251891 14 55.403 4.24 28.26 0.13 1.34 na 1013100

[0333] 2. Instrument precision test

[0334] Take the same Artemisia annua intermediate extract test solution, repeat the injection 6 times according to the characteristic spectrum method, and measure the relative retention time and relative peak area of ​​14 common peaks. The specific results are shown in Tables 48-49 below, and the instrument precision chromatogram is shown in the attached Fig.36 , from top to bottom represent precision 1-6 respectively.

[0335] Table 48 Instrument precision relative retention time results

[0336]

[0337] Table 49 Instrument precision relative retention peak area results

[0338]

[0339]

[0340] The results showed that the RSD values ​​of the relative retention time and relative peak area of ​​each common peak and the reference peak (peak S) were all less than 2.0%, indicating that the instrument had good precision.

[0341] 3. Repeatability test

[0342] Take the same batch of Artemisia annua intermediate extract test solution, prepare 6 test solutions in parallel according to the method under [Characteristic Spectrum], and measure the relative retention time of 14 common peaks. The detailed results are shown in Tables 50-51 below, and the repeatability chromatogram is shown in the attached Fig.37 , from top to bottom they represent repeatability 1-6 respectively.

[0343] Table 50 Method repeatability relative retention time results

[0344]

[0345] Table 51 Method repeatability relative retention peak area results

[0346]

[0347]

[0348] The results showed that the RSD values ​​of the relative retention times of the common peaks and the reference peak (peak S) were slightly different, indicating that the method had good repeatability.

[0349] 4. Exclusivity

[0350] The extraction solvent for the test sample is 70% methanol. Accurately pipette 5 μL of the test sample solution and negative control solution (70% methanol) and inject them into the high performance liquid chromatograph respectively. Test according to the method of [Characteristic Spectrum]. The chromatogram of the negative control (70% methanol) is attached. Fig.38 , the result is negative and there is no interference.

[0351] 5. Different personnel (intermediate precision)

[0352] For the same batch of Artemisia annua intermediate extract test solution, experimenters A, B, and C prepared samples according to the test sample preparation method in [Characteristic spectrum], and used the same equipment to determine the relative retention time and relative peak area of ​​each common peak. The detailed results are shown in Tables 52-53 below, and the chromatograms under different operators are shown in the attached Fig.39 , from bottom to top, they are the operation results of A, B, and C.

[0353] Table 52 Relative retention time results for different personnel

[0354]

[0355] Table 53 Relative retention peak area results of different personnel

[0356]

[0357]

[0358] The results showed that the relative average deviations of the characteristic peaks and the reference S peak were small, indicating that the intermediate precision of the method was good.

[0359] 6. Stability test

[0360] Take the same Artemisia annua intermediate extract test solution and measure it according to the chromatographic conditions under [Characteristic Spectrum] at 0h, 4h, 8h, 12h, 16h and 24h respectively, and calculate the relative retention time and relative peak area of ​​10 characteristic peaks. See Tables 54-55 below for details, and the stability chromatogram is attached. Fig.40 , from top to bottom they represent the results of 0h, 4h, 8h, 12h, 16h, and 24h respectively.

[0361] Table 54 Method stability relative retention time results

[0362]

[0363] Table 55 Method stability relative retention peak area results

[0364]

[0365]

[0366] The results showed that the test solution was stable within 24 hours and met the measurement requirements.

[0367] 7. Investigation of different flow rates

[0368] Take the same sample solution of Artemisia annua intermediate extract and measure it according to the [Characteristic Spectrum] method at flow rates of 0.6mL / min, 0.7mL / min, and 0.8mL / min, respectively, to measure the relative retention time of 14 common peaks. See Tables 56-57 below for details. See the attached chromatograms at different flow rates. Fig.41 , from bottom to top, the chromatograms at flow rates of 0.7mL / min, 0.6mL / min, and 0.8mL / min are shown.

[0369] Table 56 Relative retention time results at different flow rates

[0370]

[0371] Table 57 Relative retention peak area results at different flow rates

[0372]

[0373] The results show that different flow rates have a certain influence on the relative retention time of characteristic peaks. When the flow rate changes slightly, the retention time of each characteristic chromatographic peak changes slightly, and the relative peak area difference is large. Therefore, the flow rate of the present invention is fixed at 0.7mL / min.

[0374] 8. Investigation of different column temperatures

[0375] Take the same sample solution of Artemisia annua intermediate extract and measure it at column temperatures of 18°C, 20°C and 22°C according to the [Characteristic Spectrum] determination method to determine the relative retention time and relative peak area of ​​14 common peaks. The specific results are shown in Tables 58-59 below. The chromatograms at different column temperatures are shown in the attached Fig.42 , from top to bottom, the chromatograms at 20, 22, and 18°C ​​are shown.

[0376] Table 58 Relative retention time results at different column temperatures

[0377]

[0378] Table 59 Relative retention peak area results at different column temperatures

[0379]

[0380] The results showed that it had a significant impact on the relative retention time and peak area RSD, so a column temperature of 20°C was recommended.

[0381] 9. Investigation of different instruments

[0382] The same inspector took the same sample solution at different times and tested it on Agilent and Thermo Fisher HPLC respectively according to the above conditions. The relative retention time of each common peak was determined according to the above chromatographic conditions. The results are shown in Tables 60-61 below. The chromatograms under Thermo Fisher and Agilent conditions are shown in the attached Fig.43 As shown in (a)-(b).

[0383] Table 60 Relative retention time results of different instruments

[0384]

[0385]

[0386] Table 61 Relative retention peak area results of different instruments

[0387]

[0388] The results show that the individual peaks have little effect, but the relative peak area has a greater effect, which may be due to the different responses on different instruments.

[0389] 10. Characteristic spectrum determination of extracts from 3 batches of Artemisia annua formula granules intermediates

[0390] Take 3 batches of Artemisia annua formula granule intermediate extracts and operate according to the proposed [Characteristic Spectrum] determination method. The results are shown in Tables 62-63 below. The characteristic spectra of the 3 batches of Artemisia annua formula granule intermediate extracts are shown in the attached Fig.44 .

[0391] Table 62 Relative retention time of characteristic spectra of extracts from three batches of Artemisia annua formula granules intermediates

[0392]

[0393] Table 63 Relative peak areas of characteristic spectra of extracts from three batches of Artemisia annua granule intermediates

[0394]

[0395]

[0396] Example 6 Characteristic Spectrum of Artemisia annua Formula Granules

[0397] Artemisia annua formula granules: 1901001Y, 1901002Y, 1901003Y.

[0398] 1. Feature spectrum detection method

[0399] Since this product is a granule made from the water extract obtained by decocting Artemisia annua slices in water, its properties are similar to those of the standard decoction "lyophilized powder". Therefore, the characteristic spectrum method of Artemisia annua formula granules was established by referring to the chromatographic conditions and methods in the determination method of the characteristic spectrum items of the standard decoction "lyophilized powder" of Artemisia annua slices.

[0400] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel as filler, chromatographic column: CORTECS T3; 4.6×150 mm, 2.7 μm, methanol as mobile phase A, 0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution according to the specified procedure in Table 4 above; detection wavelength: 340 nm; flow rate: 0.7 mL per minute; column temperature: 20°C. The number of theoretical plates calculated based on the scopoletin peak should be no less than 5000.

[0401] Preparation of reference solution: Take about 4g of Artemisia annua reference medicinal material, place it in a stoppered conical flask, add 100ml of water, heat and reflux for 45 minutes, take it out, cool it, filter it, evaporate the filtrate to dryness, add 25ml of 70% methanol to the residue, ultrasonically treat it (power 250W, frequency 53kHz) for 30 minutes, take it out, cool it, filter it, and take the filtrate as the reference solution of the reference medicinal material. Take the reference substance under [Assay] as the reference solution of the reference substance.

[0402] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.4g, weigh it accurately, put it in a stoppered conical flask, accurately add 25ml of 70% methanol, weigh it, treat it with ultrasound for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0403] Determination method: Accurately pipette 5μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, record chromatogram, and obtain. The reference characteristic spectrum and the chromatogram of the test sample of Artemisia annua formula granules are attached. Fig.45 As shown in (a)-(b), the system adaptability parameters are shown in Table 64 below.

[0404] There should be 14 characteristic peaks in the chromatogram of the test sample, and the relative retention time should correspond to the 14 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 9 should correspond to the retention time of the scopoletin reference peak, and the peak corresponding to the scopoletin reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are 0.32 (peak 1), 0.47 (peak 2), 0.55 (peak 3), 0.58 (peak 4), 0.66 (peak 5), 0.85 (peak 6), 0.93 (peak 7), 0.97 (peak 8), 1.51 (peak 10), 1.69, (peak 11) 2.05 (peak 12), 2.62 (peak 13), 2.72 (peak 14). Calculate the relative peak area of ​​the sum of the peak areas of Peak 1, Peak 4, and Peak 5 and the S peak, which must not be less than 0.55.

[0405] Table 64 System adaptability parameters

[0406]

[0407]

[0408] 2. Instrument precision test

[0409] Take the same Artemisia annua formula granule test solution and repeat the injection 6 times according to the characteristic spectrum method to measure the relative retention time and relative peak area of ​​14 common peaks. The specific results are shown in Tables 65-66 below, and the instrument precision chromatogram is shown in the attached Fig.46 , from top to bottom represent precision 1-6 respectively.

[0410] Table 65 Instrument precision relative retention time results

[0411]

[0412] Table 66 Instrument precision relative retention peak area results

[0413]

[0414]

[0415] The results showed that the RSD values ​​of the relative retention time and relative peak area of ​​each common peak and the reference peak (peak S) were all less than 2.0%, indicating that the instrument had good precision.

[0416] 3. Repeatability test

[0417] Take the same batch of Artemisia annua formula granules test solution, prepare 6 test solutions in parallel according to the method under [Characteristic Spectrum], and measure the relative retention time of 14 common peaks. The detailed results are shown in Tables 67-68 below, and the repeatability chromatogram is shown in the attached Fig.47 , from top to bottom they represent repeatability 1-6 respectively.

[0418] Table 67 Method repeatability relative retention time results

[0419]

[0420] Table 68 Method repeatability relative retention peak area results

[0421]

[0422]

[0423] The results showed that the RSD values ​​of the relative retention times of the common peaks and the reference peak (peak S) were slightly different, indicating that the method had good repeatability.

[0424] 4. Exclusivity

[0425] The extraction solvent for the test sample is 70% methanol. Accurately pipette 5 μL of the test sample solution and negative control solution (70% methanol) and inject them into the high performance liquid chromatograph respectively. Test according to the method of [Characteristic Spectrum]. The chromatogram of the negative control (70% methanol) is attached. Fig.48 , the result is negative and there is no interference.

[0426] 5. Different personnel (intermediate precision)

[0427] For the same batch of Artemisia annua formula granules test solution, experimenters A, B, and C prepared samples according to the test sample preparation method in [Characteristic spectrum], and used the same equipment to measure the relative retention time and relative peak area of ​​each common peak. The detailed results are shown in Tables 69-70 below, and the intermediate precision spectrum of different operators is shown in the attached Fig.49 , from top to bottom they represent the operation result diagrams of A, B, and C respectively.

[0428] Table 69 Relative retention time results for different personnel

[0429]

[0430] Table 70 Relative retention peak area results of different personnel

[0431]

[0432] The results showed that the relative average deviations of the characteristic peaks and the reference S peak were small, indicating that the intermediate precision of the method was good.

[0433] 6. Stability test

[0434] Take the same Artemisia annua formula granule test solution and measure it according to the chromatographic conditions under [Characteristic Spectrum] at 0h, 4h, 8h, 12h, 16h and 24h respectively, and calculate the relative retention time and relative peak area of ​​10 characteristic peaks. See Tables 71-72 below for details, and the stability chromatogram is attached. Fig.50 , from top to bottom they represent the results of 0h, 4h, 8h, 12h, 16h, and 24h respectively.

[0435] Table 71 Stability relative retention time results

[0436]

[0437] Table 72 Stability relative retention peak area results

[0438]

[0439] The results showed that the test solution was stable within 24 hours and met the measurement requirements.

[0440] 7. Investigation of different flow rates

[0441] Take the same sample solution of Artemisia annua formula granules and measure it according to the [Characteristic Spectrum] method at flow rates of 0.6mL / min, 0.7mL / min, and 0.8mL / min, respectively, and measure the relative retention times of 14 common peaks. See Tables 73-74 below for details, and the chromatograms of different flow rates are attached. Fig.51 , where from top to bottom are the chromatograms at flow rates of 0.7 mL / min, 0.6 mL / min, and 0.8 mL / min.

[0442] Table 73 Relative retention time results at different flow rates

[0443]

[0444] Table 74 Relative retention peak area results at different flow rates

[0445]

[0446] The results show that different flow rates have a certain influence on the relative retention time of characteristic peaks. When the flow rate changes slightly, the retention time of each characteristic chromatographic peak changes slightly, and the relative peak area varies greatly. Therefore, this method selects a fixed flow rate of 0.7 mL / min.

[0447] 8. Investigation of different column temperatures

[0448] Take the same sample solution of Artemisia annua formula granules and measure it at column temperatures of 18°C, 20°C and 22°C according to the [Characteristic Spectrum] determination method to determine the relative retention time and relative peak area of ​​14 common peaks. The specific results are shown in Tables 75-76 below. The chromatograms under different column temperature conditions are shown in the attached Fig.52 , where the chromatograms at 20°C, 22°C, and 18°C ​​are shown from top to bottom.

[0449] Table 75 Relative retention time results at different column temperatures

[0450]

[0451]

[0452] Table 76 Relative retention peak area results at different flow rates

[0453]

[0454] The results showed that it had a significant impact on the relative retention time and peak area RSD, so a column temperature of 20°C was recommended.

[0455] 9. Investigation of different chromatographic column batch numbers

[0456] Through the process of chromatographic condition optimization, it can be known that this method has higher requirements for the chromatographic column and needs to fix the chromatographic column WatersCORTECS T3 4.6mmx150mm SN:01353120115928. Therefore, different production batches of chromatographic columns were investigated (column 1: SN:01353120115928; column 2: SN:01393132315626; column 3: SN:01393132315612). The same sample solution was taken and measured according to the above conditions. The relative retention time and relative peak area of ​​each common peak were calculated. The results are shown in Tables 77-78 below. The chromatograms under different batches of chromatographic columns are shown in the attached Fig.53 , where from bottom to top they represent the results of Waters CORTECS T3 4.6mmx150mm SN:01353120115928, Waters CORTECS T3 4.6mmx150mm SN:01393132315626, and Waters CORTECS T3 4.6mmx150mm SN:01393132315612 respectively.

[0457] Table 77 Relative retention time results of different chromatographic column batches

[0458]

[0459] Table 78 Relative retention peak area results of different chromatographic column batches

[0460]

[0461] The results showed that the relative retention time and relative peak area of ​​the S peak were less affected than those of the reference substance.

[0462] 10. Investigation of different instruments

[0463] The same inspector took the same sample solution at different times and tested it on Agilent and Thermo Fisher HPLC respectively according to the above conditions. The relative retention time of each common peak was determined according to the above chromatographic conditions. The results are shown in Tables 79-80 below. The chromatograms under different instruments (Thermo Fisher, Agilent) are shown in the attached table. Fig.54 As shown in (a)-(b).

[0464] Table 79 Relative retention time results of different instruments

[0465]

[0466] Table 80 Relative retention peak area results of different instruments

[0467]

[0468] The results show that the individual peaks have little effect, but the relative peak area has a greater effect, which may be due to the different responses on different instruments.

[0469] In summary, based on the above methodological investigation results, among the 14 common peaks of the characteristic spectrum of Artemisia annua formula granules established, each chromatographic peak is affected to a certain extent by different chromatographic columns, different high performance liquid phases, column temperature and flow rate factors. In order to adapt to its durability, it is recommended to control the specified value range within ±10%.

[0470] 12. Characteristic spectrum determination of 3 batches of Artemisia annua formula granules

[0471] Take 3 batches of Artemisia annua formula granules and operate according to the proposed [Characteristic spectrum] determination method. The results are shown in Tables 81-82 below. The characteristic spectra of the 3 batches of Artemisia annua formula granules are shown in the attached Fig.55 .

[0472] Table 81 Relative retention time of characteristic spectra of three batches of Qinghao formula granules

[0473]

[0474] Table 82 Relative peak areas of characteristic spectra of three batches of Qinghao formula granules

[0475]

[0476] It can be seen that the construction method of the present invention meets the requirements.

[0477] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for constructing a characteristic spectrum of Artemisia annua medicinal material and Artemisia annua pharmaceutical preparation, characterized in that: The method comprises the steps of conducting high performance liquid chromatography on a test solution of an Artemisia annua medicinal material and / or an Artemisia annua pharmaceutical preparation; The chromatographic conditions include: using octadecylsilane bonded silica gel as filler, methanol as mobile phase A, and 0.1 wt % phosphoric acid aqueous solution as mobile phase B, and performing gradient elution according to the following procedure: 0-14min, A:B is 18%:82%→28%:72%; 14-16 min, A:B 28%:72% → 32%:68%; 16-30min, A:B is 32%:68%→36%:64%; 30-36 min, A:B is 36%:64% → 40%:60%; 36-55min, A:B is 40%:60%→62%:38%; In the HPLC detection step, the chromatographic conditions also include: column temperature 18-25°C, flow rate 0.5-1.0 ml / min, detection wavelength 340 nm; The preparation method of the test solution comprises: taking the test sample and accurately adding an organic solvent, sealing it with ultrasonic treatment, filtering and collecting the filtrate, and obtaining; The method also includes the step of preparing a control medicinal material reference solution, which specifically includes: taking an Artemisia annua control medicinal material, adding water to perform heating reflux extraction, collecting the extract and filtering it, taking the filtrate and evaporating it to dryness, adding an organic solvent to the residue, sealing it with ultrasound treatment, filtering and collecting the subsequent filtrate to obtain the desired control medicinal material reference solution.

2. The method for constructing the characteristic spectrum of the Artemisia annua medicinal material and the Artemisia annua pharmaceutical preparation according to claim 1, characterized in that: The method further comprises the steps of preparing a reference substance solution and constructing a reference substance characteristic spectrum based on the high performance liquid chromatography; The reference substance includes scopoletin, and each 1 ml of the reference substance solution contains 30-50 μg of scopoletin.

3. The method for constructing the characteristic spectrum of the Artemisia annua medicinal material and the Artemisia annua pharmaceutical preparation according to claim 2, characterized in that: The organic solvent comprises methanol at a volume concentration of 60-80 v / v%; The power of the ultrasonic step is 200-300W and the frequency is 50-60kHz.

4. The method for constructing the characteristic spectrum of the Artemisia annua medicinal material and the Artemisia annua pharmaceutical preparation according to any one of claims 1 to 3, characterized in that: The artemisia annua drug preparation includes artemisia annua formula granules, artemisia annua formula granule intermediate extracts, artemisia annua decoction pieces or artemisia annua standard decoction.

5. A characteristic spectrum and / or a reference characteristic spectrum of an Artemisia annua medicinal material and an Artemisia annua pharmaceutical preparation, characterized in that: The characteristic spectrum or reference characteristic spectrum of the Artemisia annua medicinal material and Artemisia annua pharmaceutical preparation is constructed by the method according to any one of claims 1 to 4.

6. Application of the method for constructing the characteristic spectrum of the Artemisia annua medicinal material and the Artemisia annua pharmaceutical preparation according to any one of claims 1 to 4 and / or the characteristic spectrum and / or the control characteristic spectrum of the Artemisia annua medicinal material and the Artemisia annua pharmaceutical preparation according to claim 5 in the field of quality inspection of Artemisia annua medicinal material and the Artemisia annua pharmaceutical preparation.

7. A method for quality testing of Artemisia annua medicinal materials and Artemisia annua pharmaceutical preparations, characterized in that: The method comprises the steps of constructing the characteristic map and the reference characteristic map according to the method of any one of claims 1 to 4, and comparing the characteristic map with the reference characteristic map.

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