Method for detecting dehydroartemisinin, 9-epiartemisinin and artemisinin in artemether raw material and application

Abstract

The invention discloses a method for detecting dehydroartemisinin, 9-epiartemisinin and artemisinin in an artemether raw material and application thereof.The detection method comprises the step that a reference substance solution and a test solution of the artemether raw material are detected through high performance liquid chromatography, specifically, octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, the mobile phase B is used as a mobile phase C, and the mobile phase B is used as a mobile phase D; and carrying out gradient elution by taking water as a mobile phase B. The method disclosed by the invention is simple to operate, strong in specificity, good in precision and high in sensitivity, can be used for simultaneously detecting dehydroartemisinin, 9-epiartemisinin and artemisinin, and can be completely separated from four known impurities (an artemether impurity I, an artemether impurity II, dihydroartemisinin and alpha-artemether) of artemether.

Description

Technical Field

[0001] The present invention belongs to the field of drug analysis and detection, and specifically relates to a method and application for detecting dehydroartemisinin, 9-epiartemisinin and artemisinin in artemether raw materials. Background Art

[0002] Artemether (ARM) has a molecular formula of C 16 H 26 O5, with a molecular weight of 298.37 and a structural formula shown in Formula (I), is a methyl ether derivative of artemisinin. Artemether appears as white crystals or a crystalline powder. As an important API, artemether can be manufactured into various antimalarial dosage forms, including tablets, pills, capsules, injections, and compound preparations. Artemether is obtained by a series of reactions from artemisinin to obtain crude artemether, which is then refined to obtain finished artemether.

[0003]

[0004] Drug-related impurities are closely linked to drug safety and efficacy stability. Scientific analysis and assessment of impurities is a crucial foundation for establishing reliable impurity testing methods and a prerequisite for comprehensive and effective control of impurities in pharmaceuticals. Impurity analysis is a crucial component of drug R&D, yet also presents a significant challenge in many drug development efforts.

[0005] Referring to the current quality control standards for artemether in Part II of the 2020 edition of the Chinese Pharmacopoeia, the relevant substances include artemether impurity I, artemether impurity II, and α-artemether. The chromatographic conditions for detecting the relevant substances include: octadecylsilane bonded silica gel as the filler; acetonitrile-water (62:38) as the mobile phase; a detection wavelength of 216 nm; and an injection volume of 20 μl. The starting material for artemether is artemisinin, and dehydroartemisinin and 9-epiartemisinin are related impurities of artemether. Therefore, research on the process impurities dehydroartemisinin, 9-epiartemisinin, and artemisinin in artemether raw materials is necessary to further improve the quality control of artemether raw materials. Currently, there are no literature or patent reports on the research of detection methods for dehydroartemisinin, 9-epiartemisinin, and artemisinin in artemether raw materials.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the shortcomings of the existing technology and provide a method for detecting dehydroartemisinin, 9-epiartemisinin and artemisinin in artemether raw materials. The method is simple to operate, has strong specificity, good precision and high sensitivity, can simultaneously detect dehydroartemisinin, 9-epiartemisinin and artemisinin, and can completely separate them from the four known impurities of artemether (artemether impurity I, artemether impurity II, dihydroartemisinin and α-artemether).

[0008] In order to solve the above technical problems, the basic concept of the technical solution adopted by the present invention is:

[0009] The present invention provides a method for detecting dehydroartemisinin, 9-epiartemisinin, and artemisinin in an artemether raw material. A reference solution and a test solution of the artemether raw material are detected by high performance liquid chromatography, comprising: using octadecylsilane bonded silica gel as a filler, acetonitrile as a mobile phase A, and water as a mobile phase B, and eluting according to the following gradient:

[0010]

[0011] According to the current quality control standards for artemether, the relevant substances that can be detected include artemether impurity I, artemether impurity II, dihydroartemisinin, and α-artemether, but the process impurities dehydroartemisinin, 9-epiartemisinin, and artemisinin cannot be effectively separated and detected. The present invention uses high-performance liquid chromatography and optimizes the elution gradient to develop a new method that can simultaneously detect dehydroartemisinin, 9-epiartemisinin, and artemisinin, and can completely separate them from the four known impurities of artemether (artemether impurity I, artemether impurity II, dihydroartemisinin, and α-artemether). This method can be used to detect more relevant impurities in artemether raw materials, which can further improve the quality control of artemether raw materials.

[0012] In a further embodiment, the elution gradient is:

[0013]

[0014]

[0015] The present invention has tried a variety of elution gradients. Experiments have found that under the above elution gradient, the peak elution time of each component is appropriate, the peak shape is good, and the chromatographic peaks of each component can be completely separated, with a separation degree greater than 1.5. Among them, the separation degree of the chromatographic peaks of dehydroartemisinin and dihydroartemisinin-II is greater than 1.5, and the separation degree of artemisinin and artemether impurity I is greater than 1.5. Under this elution gradient, dehydroartemisinin, 9-epiartemisinin, and artemisinin can be detected simultaneously, and can be completely separated from the four known impurities of artemether (artemether impurity I, artemether impurity II, dihydroartemisinin, and α-artemether). This shows that the specificity of this method is strong.

[0016] In a further embodiment, the chromatographic column is an Agilent Zorbax SB C18, 4.6 mm×150 mm, 1.8 μm, or a chromatographic column of equivalent performance.

[0017] The experiment found that when the chromatographic column used was Agilent Zorbax SB C18, all components had peaks, laying the foundation for the complete separation of all components.

[0018] In a further embodiment, the detection wavelength of the high performance liquid chromatography method is 210 nm.

[0019] In a further embodiment, in the high performance liquid chromatography conditions, the column temperature is 28°C to 32°C; preferably, the column temperature is 30°C.

[0020] In a further embodiment, under the conditions of high performance liquid chromatography, the flow rate is 0.9 to 1.1 ml / min; preferably, the flow rate is 1.0 ml / min.

[0021] Further solution, preparation of test solution:

[0022] Take artemether raw material, accurately weigh it, place it in a volumetric flask, add an appropriate amount of solvent, sonicate to dissolve it, dilute it to the scale with solvent, and shake well;

[0023] Preferably, the solvent is acetonitrile-water, wherein the volume ratio of acetonitrile to water is 70:30.

[0024] Further solution, preparation of reference solution:

[0025] Take appropriate amount of artemether reference substance and dehydroartemisinin reference substance, weigh accurately, and add diluent to quantitatively dilute to prepare a mixed solution;

[0026] Preferably, the diluent is acetonitrile-water (70:30);

[0027] Preferably, in the mixed solution, the concentrations of artemether and dehydroartemisinin are 20 μg / ml respectively.

[0028] A further solution is that when conducting the test, the test solution and the reference solution are accurately measured and injected into the liquid chromatograph respectively, and dehydroartemisinin is calculated by the external standard method using the peak area, and 9-epiartemisinin and artemisinin are calculated by the main component external standard method with the correction factor using the corrected peak area.

[0029] Dehydroartemisinin calculation formula:

[0030]

[0031] Where:

[0032] Aimp = peak area of ​​dehydroartemisinin in the test solution;

[0033] Astd = peak area of ​​dehydroartemisinin in reference solution;

[0034] Wstd = weight of dehydroartemisinin reference substance (mg);

[0035] Vstd = dilution volume of reference solution (ml);

[0036] Wimp = weight of test sample (mg);

[0037] Vimp = dilution volume of test solution (ml);

[0038] P = dehydroartemisinin reference substance content;

[0039] 9- Table Artemisinin, Artemisinin calculation formula (calculated by peak area using the principal component external standard method with correction factor added):

[0040]

[0041] Aimp = peak area of ​​9-epiartemisinin or artemisinin in the test solution;

[0042] Astd = peak area of ​​artemether in reference solution;

[0043] Wstd = weight of artemether reference substance (mg);

[0044] Vstd = dilution volume of reference solution (ml);

[0045] Wimp = weight of test sample (mg);

[0046] Vimp = dilution volume of test solution (ml);

[0047] P = content of artemether reference substance;

[0048] F = correction factor (the correction factor for 9-epiartemisinin is 0.79, and the correction factor for artemisinin peak is 0.94).

[0049] The present invention also provides an application of the above-mentioned detection method in quality control of artemether raw materials.

[0050] After adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art.

[0051] 1. The existing USP method for analyzing related substances of artemether can detect the known impurities artemether impurity I, artemether impurity II, dihydroartemisinin, and α-artemether. This method was intended to be used to detect the process impurities dehydroartemisinin, 9-epiartemisinin, and artemisinin. The results (results of Test Example 2) showed that the chromatographic peak of dehydroartemisinin overlapped with the chromatographic peak of dihydroartemisinin-II, indicating that the existing related substance analysis method cannot simultaneously detect dehydroartemisinin and dihydroartemisinin.

[0052] The present invention provides a method for detecting dehydroartemisinin, 9-epiartemisinin and artemisinin in artemether raw materials, optimizes chromatographic detection conditions, and newly develops a method that can simultaneously detect dehydroartemisinin, 9-epiartemisinin and artemisinin, and can completely separate them from four known impurities of artemether (artemether impurity I, artemether impurity II, dihydroartemisinin and α-artemether). The method is used to detect more relevant impurity residues in artemether raw materials, and can further improve the quality control of artemether raw materials.

[0053] 2. The detection method of the present invention is simple to operate, has strong specificity, good precision and high sensitivity (the detection limit concentration of dehydroartemisinin is 2.4372 μg / ml, equivalent to 12% of the limit; the detection limit concentration of 9-epiartemisinin is 2.4432 μg / ml, equivalent to 12% of the limit; the detection limit concentration of artemisinin is 2.5222 μg / ml, equivalent to 13% of the limit; and the detection limit concentration of artemether is 2.5112 μg / ml, equivalent to 13% of the limit).

[0054] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The accompanying drawings are part of the present invention and are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention, but do not constitute an undue limitation of the present invention. Obviously, the drawings described below are only some embodiments. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without inventive effort. In the accompanying drawings:

[0056] Figure 1 This is the chromatogram of Example 1 of the present invention; wherein the black spectrum is the chromatogram of the mixed solution with other impurities, the blue spectrum is the chromatogram of the spiked solution, and the chromatographic peaks 1-4 are dehydroartemisinin, 9-epiartemisinin, artemisinin, and artemether, respectively;

[0057] Figure 2-Figure 5 is a chromatogram obtained by using different chromatographic columns for detection in Experiment 1 of the present invention, wherein: Figure 2 Chromatographic peaks 1-10 are artemether impurity II-1, artemether impurity II-2, dihydroartemisinin-1, dihydroartemisinin-2, dehydroartemisinin, 9-epiartemisinin, artemisinin, artemether impurity I, α-artemether and artemether, respectively. Figure 3 Chromatographic peaks 1-8 are artemether impurity II-1, artemether impurity II-2, dehydroartemisinin, 9-epiartemisinin, artemisinin, artemether impurity I, α-artemether and artemether, respectively. Figure 4 Peaks were not marked due to incomplete peaks. Figure 5Chromatographic peaks 1-9 are artemether impurity II-1, artemether impurity II-2, dihydroartemisinin-1, dihydroartemisinin-2, dehydroartemisinin, 9-epiartemisinin, artemisinin, α-artemether and artemether, respectively;

[0058] Figure 6 This is the chromatogram of Experimental Example 2 of the present invention, wherein the black spectrum is the chromatogram of the mixed solution of other impurities, and the blue spectrum is the chromatogram of the dehydroartemisinin positioning solution;

[0059] Figure 7 This is the chromatogram of Experimental Example 3 of the present invention, wherein the brown spectrum is the chromatogram of the mixed solution of other impurities, and the pink spectrum is the chromatogram of the dehydroartemisinin positioning solution;

[0060] Figure 8 This is a chromatogram of Test Example 4 of the present invention, wherein chromatographic peaks 1-8 are artemether impurity II-1, artemether impurity II-2, dehydroartemisinin, 9-epiartemisinin, artemisinin, artemether impurity I, α-artemether, and artemether, respectively;

[0061] Figure 9 This is a chromatogram of Test Example 5 of the present invention, wherein chromatographic peaks 1-10 are artemether impurity II-1, artemether impurity II-2, dihydroartemisinin-1, dihydroartemisinin-2, dehydroartemisinin, 9-epiartemisinin, artemisinin, artemether impurity I, α-artemether, and artemether, respectively;

[0062] Figure 10 This is the chromatogram of Test Example 6 of the present invention, wherein chromatographic peaks 1-10 are artemether impurity II-1, artemether impurity II-2, dihydroartemisinin-1, dihydroartemisinin-2, dehydroartemisinin, 9-epiartemisinin, artemisinin, artemether impurity I, α-artemether and artemether, respectively.

[0063] It should be noted that these drawings and textual descriptions are not intended to limit the conceptual scope of the present invention in any way, but rather to illustrate the concept of the present invention for those skilled in the art by referring to specific embodiments. DETAILED DESCRIPTION

[0064] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention but are not used to limit the scope of the present invention.

[0065] The information of the instruments and reagents used in the detection method of the present invention is shown in the following table:

[0066]

[0067] Example 1

[0068] This embodiment provides a method for detecting dehydroartemisinin, 9-epiartemisinin, and artemisinin in artemether raw materials.

[0069] Chromatographic column: Agilent Zorbax SB C18, 4.6 mm × 150 mm, 5 μm.

[0070] Mobile phase: Mobile phase A: acetonitrile; Mobile phase B: water

[0071] Elution method: gradient elution

[0072]

[0073]

[0074] Flow rate: 1.0ml / min

[0075] Column temperature: 30°C

[0076] Detection wavelength: 210nm

[0077] Injection volume: 40 μl

[0078] Solvent: Acetonitrile-water (70:30)

[0079] Diluent: acetonitrile-water (70:30)

[0080] The detection process is:

[0081] (1) Preparation of reference solution: Take appropriate amount of artemether reference substance and dehydroartemisinin reference substance, weigh accurately, and add diluent to quantitatively dilute to make a mixed solution containing approximately 20 μg of each per 1 ml.

[0082] (2) Preparation of test solution: Take about 100 mg of artemether raw material sample, accurately weigh it, place it in a 10 ml volumetric flask, add an appropriate amount of solvent, sonicate to dissolve it, dilute it to the scale with solvent, and shake well.

[0083] (3) Preparation of other impurity mixed solutions: ① Artemether Impurity II Stock Solution: Weigh the total weight of the Artemether Impurity II reference substance and the bottle to 6421.40 mg, transfer the reference substance to a 50 ml volumetric flask with acetonitrile, dissolve it with acetonitrile, dilute it to the mark and shake it evenly. After blowing the bottle dry, the weight of the bottle is 6392.70 mg. (The concentration of Artemether Impurity II Stock Solution is: (6421.40-6392.70) / 50=0.574 mg / ml) ② Other impurity mixed solution: Accurately weigh 2.060 mg of Artemether Impurity I Reference Substance, 2.332 mg of α-artemether Reference Substance, and 2.742 mg of Dihydroartemisinin Reference Substance, place them in the same 100 ml volumetric flask, add 4 ml of Artemether Impurity II Stock Solution, add solvent to dissolve it by ultrasonication, dilute it to the mark and shake it evenly.

[0084] (4) Preparation of system suitability solution: Accurately weigh appropriate amounts of artemisinin, 9-epiartemisinin, and artemisinin reference substance, dissolve in solvent, and dilute to produce a mixed solution containing 20 μg of each per 1 ml. This solution is used to locate each impurity.

[0085] (5) Preparation of spiked solution: ① Preparation of reference stock solution: Accurately weigh 20.15 mg of artemether reference, 20.31 mg of artemisinin reference, 20.36 mg of 9-epiartemisinin reference, and 20.32 mg of artemisinin reference, place them in the same 100 ml volumetric flask, add appropriate amount of solvent, ultrasonically dissolve, dilute to the mark and shake well, and obtain. ② Preparation of spiked solution: Accurately pipette 1 ml of reference stock solution (A), place it in a 10 ml volumetric flask, add solvent to dissolve, dilute to the mark and shake well, and obtain. This solution is only used as a linear 100% solution during method validation and is used for the location of impurities and main components in this invention.

[0086] (6) Determination: Accurately measure the test solution and the reference solution and inject them into the liquid chromatograph respectively. Dehydroartemisinin is calculated by the external standard method based on the peak area. 9-epiartemisinin and artemisinin are calculated by the main component external standard method with the addition of a correction factor based on the corrected peak area.

[0087] Dehydroartemisinin calculation formula:

[0088]

[0089] Where:

[0090] Aimp = peak area of ​​dehydroartemisinin in the test solution;

[0091] Astd = peak area of ​​dehydroartemisinin in reference solution;

[0092] Wstd = weight of dehydroartemisinin reference substance (mg);

[0093] Vstd = dilution volume of reference solution (ml);

[0094] Wimp = weight of test sample (mg);

[0095] Vimp = dilution volume of test solution (ml);

[0096] P = dehydroartemisinin reference substance content;

[0097] 9- Table Artemisinin, Artemisinin calculation formula (calculated by peak area using the principal component external standard method with correction factor added):

[0098]

[0099] Aimp = peak area of ​​9-epiartemisinin or artemisinin in the test solution;

[0100] Astd = peak area of ​​artemether in reference solution;

[0101] Wstd = weight of artemether reference substance (mg);

[0102] Vstd = dilution volume of reference solution (ml);

[0103] Wimp = weight of test sample (mg);

[0104] Vimp = dilution volume of test solution (ml);

[0105] P = content of artemether reference substance;

[0106] F = correction factor (the correction factor for 9-epiartemisinin is 0.79, and the correction factor for artemisinin peak is 0.94).

[0107] result:

[0108] Chromatogram Figure 1 shown

[0109] Artemether, dehydroartemisinin, 9-epiartemisinin and artemisinin all have peaks, and the peak elution time of each component is appropriate and the peak shape is good. The chromatographic peaks of each component can be completely separated with a separation degree greater than 1.5.

[0110] Test Example 1 Screening of Chromatographic Columns

[0111] Artemether Impurity II Stock Solution: Weigh the total weight of the Artemether Impurity II reference substance and the bottle to 6421.40 mg. Transfer the reference substance to a 50 ml volumetric flask with acetonitrile. Dissolve it in acetonitrile and dilute to the mark, shaking well. After air-drying the bottle, the bottle weighs 6392.70 mg. (Concentration of Artemether Impurity II Stock Solution: (6421.40 - 6392.70) / 50 = 0.574 mg / ml)

[0112] Impurity mixed solution: Accurately weigh approximately 2 mg each of artemether reference substance, dehydroartemisinin reference substance, 9-epiartemisinin reference substance, artemisinin reference substance, artemether impurity I reference substance, α-artemether reference substance, and dihydroartemisinin reference substance, place them in the same 100 ml volumetric flask, add 4 ml of artemether impurity II stock solution, add acetonitrile-water (70:30) and ultrasonically dissolve, dilute to the scale and shake well to obtain the product.

[0113] Use different chromatographic columns and the chromatographic conditions in the table below to inject the impurity mixed solution and examine the peak elution and separation of each component.

[0114]

[0115] Table 1 Column screening results

[0116]

[0117] Conclusion: Considering the elution conditions of each chromatographic peak, Agilent Zorbax SB C18, 4.6mm×150mm, 5μm was selected to continue to optimize the chromatographic conditions.

[0118] Experimental Example 2 Optimization of Chromatographic Conditions

[0119] An Agilent Zorbax SB C18 (4.6*150mm, 1.8μm) column was used to optimize the chromatographic conditions and investigate the separation of specific impurities from known impurities and main components. The specific method is as follows:

[0120]

[0121] Conclusion: The chromatogram is as follows Figure 6 As shown, the chromatographic peaks of dehydroartemisinin and dihydroartemisinin-II overlap, and the method is not applicable.

[0122] Test Example 3

[0123] The chromatographic column in Example 2 was used to optimize the chromatographic conditions and investigate the separation of specific impurities from known impurities and main components. The specific method is as follows:

[0124]

[0125]

[0126] Conclusion: The chromatogram is as follows Figure 7 As shown, the chromatographic peaks of dehydroartemisinin and dihydroartemisinin-II overlap, and the method is not applicable.

[0127] Test Example 4

[0128] The chromatographic column in Example 2 was used to optimize the chromatographic conditions and investigate the separation of specific impurities from known impurities and main components. The specific method is as follows:

[0129]

[0130] Conclusion: The chromatogram is as follows Figure 8 As shown, the separation degree of artemisinin and artemether impurity I is less than 1.5, and the method is not applicable.

[0131] Test Example 5

[0132] The chromatographic column in Example 2 was used to optimize the chromatographic conditions and investigate the separation of specific impurities from known impurities and main components. The specific method is as follows:

[0133]

[0134]

[0135] Conclusion: The chromatogram is as follows Figure 9 As shown, artemether peaked too late and had a poor peak shape, so the method was not applicable.

[0136] Test Example 6

[0137] The chromatographic column in Example 2 was used to optimize the chromatographic conditions and investigate the separation of specific impurities from known impurities and main components. The specific method is as follows:

[0138]

[0139] Conclusion: The chromatogram is as follows Figure 10 As shown, artemether peaks when the gradient changes, and the peak shape is affected, so the method is not applicable.

[0140] Test Example 7 Durability

[0141] In order to investigate the stability of the measurement results when the measurement conditions fluctuate slightly, the effects of changing the following conditions on the measurement results were investigated while keeping other conditions of the system unchanged.

[0142] Table 1 Validation of relevant substance methods - Durability inspection items

[0143]

[0144] Solvent: Acetonitrile-water (70:30)

[0145] System suitability solution: Accurately weigh 2.251 mg of dehydroartemisinin reference substance, 2.181 mg of 9-epiartemisinin reference substance, and 2.089 mg of artemisinin reference substance, place them in the same 100 ml volumetric flask, add solvent to dissolve, dilute to the scale, and shake well to obtain.

[0146] Reference substance stock solution: Accurately weigh 20.15 mg of artemether reference substance, 20.31 mg of dehydroartemisinin reference substance, 20.36 mg of 9-epiartemisinin reference substance, and 20.32 mg of artemisinin reference substance, place them in the same 100 ml volumetric flask, add appropriate amount of DMSO solvent and ultrasonically dissolve them, dilute with solvent to the scale and shake well to obtain.

[0147] Reference substance solution: Accurately pipette 20 ml of reference substance stock solution (A) into a 200 ml volumetric flask, add solvent to dissolve and dilute to the scale and shake well.

[0148] Spiking solution: Accurately weigh 99.59 mg of artemether raw material into a 10 ml volumetric flask, dissolve it with 9 ml of reference solution, sonicate for 5 minutes, and dilute to the mark with reference solution.

[0149] The experiment changed the column temperature, flow rate, chromatographic column, instrument, and the initial ratio of mobile phase A in the chromatographic conditions, and examined the durability of the method with the detection amount of each impurity as an indicator.

[0150] Acceptability criteria: System suitability meets requirements when there are minor changes in the assay parameters. The range of each impurity detected in the spiked solution should be ≤ 0.04%.

[0151] (1) Column temperature durability

[0152] 40 μl of each of blank solvent, system suitability solution, reference solution, and spiked solution were injected into a liquid chromatograph and the chromatograms recorded. System suitability and the range of each impurity content were examined by injection testing at column temperatures of 28°C, 30°C, and 32°C, with all other chromatographic conditions unchanged (see Example 1).

[0153] Table 2 Durability-test results (different column temperatures)

[0154]

[0155] Note: “*” indicates normal conditions.

[0156] When the column temperature was changed to 28℃ and 32℃, the range of the detection amount of dehydroartemisinin in the spiked solution was 0.00% and 0.01%, respectively, both less than 0.04%, meeting the acceptable standard; the range of the detection amount of 9-epiartemisinin was 0.00% and 0.01%, respectively, both less than 0.04%, meeting the acceptable standard; the range of the detection amount of artemisinin was 0.00%, less than 0.04%, meeting the acceptable standard; the results showed that changing the column temperature within the range of 28℃~32℃ had no effect on the test results.

[0157] (2) Flow rate

[0158] 40 μl of each of the blank solvent, system suitability solution, reference solution, and spiked solution were injected into a liquid chromatograph and the chromatograms recorded. System suitability and the range of each impurity content were examined by injection at flow rates of 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min, respectively, while maintaining all other chromatographic conditions (see Example 1).

[0159] Table 3 Durability-Test Results (Flow Rate)

[0160]

[0161] Note: “*” indicates normal conditions.

[0162] When the flow rate was changed to 0.9ml / min and 1.1ml / min, the range of the detection amount of dehydroartemisinin in the spiked solution was 0.01%, which was less than 0.04%, meeting the acceptable standard; the range of the detection amount of 9-epiartemisinin was 0.01% and 0.00%, respectively, both less than 0.04%, meeting the acceptable standard; the range of the detection amount of artemisinin was 0.01% and 0.00%, respectively, both less than 0.04%, meeting the acceptable standard; the results showed that changing the flow rate within the range of 0.9ml / min to 1.1ml / min had no effect on the test results.

[0163] (3) Durability of chromatographic columns and instruments

[0164] 40 μl of each of the blank solvent, system suitability solution, reference solution, and spiked solution were injected into a liquid chromatograph and the chromatograms were recorded. The system suitability and the range of each impurity content were examined by injecting samples using different chromatographic columns and instruments, while maintaining other chromatographic conditions unchanged (see Example 1).

[0165] Table 4 Durability-Test Results (Different Chromatographic Columns and Instruments)

[0166]

[0167]

[0168] Note: “*” indicates normal conditions.

[0169] When replacing different chromatographic columns and instruments, the range of the detection amount of dehydroartemisinin in the spiked solution was 0.00%, less than 0.04%, which met the acceptance standard; the range of the detection amount of 9-epiartemisinin was 0.01%, less than 0.04%, which met the acceptance standard; the range of the detection amount of artemisinin was 0.00%, less than 0.04%, which met the acceptance standard; the results showed that different chromatographic columns and instruments had no effect on the determination results.

[0170] (4) Durability of different mobile phase A starting ratios

[0171] 40 μl of each blank solvent, system suitability solution, reference solution, and spiked solution were injected into a liquid chromatograph and chromatograms were recorded. System suitability and the range of each impurity content were examined using injections with mobile phase A starting at 34%, 35%, and 36% acetonitrile, respectively, while maintaining other chromatographic conditions (see Example 1).

[0172] Table 5 Durability-Test Results (Different Mobile Phase A Starting Ratios)

[0173]

[0174] Note: “*” indicates normal conditions.

[0175] When the starting proportion of mobile phase A was changed to 34% and 36%, the range of the detection amount of dehydroartemisinin in the spiked solution was 0.00% and 0.01%, respectively, both less than 0.04%, meeting the acceptable standard; the range of the detection amount of 9-epiartemisinin was 0.01% and 0.00%, respectively, both less than 0.04%, meeting the acceptable standard; the range of the detection amount of artemisinin was 0.00%, less than 0.04%, meeting the acceptable standard; the results showed that changing the starting proportion of mobile phase A within the range of 34% to 36% had no effect on the test results.

[0176] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the present invention can make slight changes or modifications to equivalent embodiments using the above-mentioned technical contents without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the solution of the present invention.

Claims

1. A method for detecting dehydroartemisinin, 9-epiartemisinin and artemisinin in artemether raw materials, characterized in that: The reference solution and the test solution of artemether raw material were tested by high performance liquid chromatography, including: using octadecylsilane bonded silica gel as a filler, acetonitrile as mobile phase A, water as mobile phase B, and eluting according to the following gradient:

2. The detection method according to claim 1, wherein The elution gradient is:

3. The detection method according to claim 1 or 2, characterized in that The chromatographic column is Agilent Zorbax SBC18, 4.6 mm × 150 mm, 1.8 μm or a chromatographic column of equivalent performance.

4. The detection method according to any one of claims 1 to 3, characterized in that In the high performance liquid chromatography conditions, the detection wavelength was 210 nm.

5. The detection method according to any one of claims 1 to 4, characterized in that In the high performance liquid chromatography conditions, the column temperature is 28°C to 32°C; preferably, the column temperature is 30°C.

6. The detection method according to any one of claims 1 to 5, characterized in that In the high performance liquid chromatography conditions, the flow rate is 0.9 to 1.1 ml / min; preferably, the flow rate is 1.0 ml / min.

7. The detection method according to any one of claims 1 to 6, characterized in that Preparation of test solution: Take artemether raw material, weigh accurately, place in a volumetric flask, add appropriate amount of solvent, sonicate to dissolve, dilute to scale with solvent, and shake well; Preferably, the solvent is acetonitrile-water, wherein the volume ratio of acetonitrile to water is 70:

30.

8. The detection method according to any one of claims 1 to 7, characterized in that Preparation of reference solution: Take appropriate amount of artemether reference substance and dehydroartemisinin reference substance, weigh accurately, and add diluent to quantitatively dilute to prepare a mixed solution; Preferably, the diluent is acetonitrile-water (70:30); Preferably, in the mixed solution, the concentrations of artemether and dehydroartemisinin are 20 μg / ml respectively.

9. The detection method according to any one of claims 1 to 8, characterized in that During the test, precisely measure the test solution and reference solution and inject them into the liquid chromatograph respectively. Dehydroartemisinin is calculated by the peak area according to the external standard method, and 9-epiartemisinin and artemisinin are calculated by the main component external standard method with the addition of correction factors using the corrected peak area.

10. Use of the detection method according to any one of claims 1 to 9 in quality control of artemether raw materials.

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