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Abstract This study was conducted to establish a high performance liquid chromatography （HPLC） method for the determination of artemether in the artemether injection using Hypersil ODS C18 chromatographic column （5 μm， 4.6 mm×150 mm）. Mobile phase， column temperature， flow rate and detection wavelength were optimized. Acetonitrile-water-tetrahydrofuran （62∶37∶1， V/V） was selected as the mobile phase， and the HPLC was performed with column temperature at 30 ℃ and the flow rate at 1.0 ml/min； and the detection wavelength was set at 216 nm. The HPLC detection system of artemether had good suitability. The linearity was good in 100-800 μg/ml concentration range， and the regression equation was y=302.36x-682.02， R2=0. 999 8. The overall average recovery was 97.58%， and the RSD was 1.58%. Three batches of artemether injection samples were determined by the method， showing RSD of 1.42%. The method could be used for the detection of artemether content in artemether injection.
Key words Artemether injection； HPLC； Content determination
Artemisinin was isolated and extracted from Armisia annua L. in composite family for the first time in 1971. It has a better effect of killing agamont in red blood cell of plasmodium. Soon afterwards， many derivatives were synthesized with artemisinin as a lead compound， and artemether is one of them[1]. On September 11， 1987， the Ministry of Health of china held a press conference about artemisinin and its derivatives， and announced and approved large batch production of artemisinin injection as a specific medicine for treating various serious malaria patients. In the same year， World Health Organization （WHO） strictly verified the efficacy of artemether， and affirmed artemether as the best anti-malarial medicine. Artemether becomes the first preparation medicine registered internationally in China[2]. In human medicine field， artemether is mainly used for treating plasmodium， and could rapidly cure parasitemia[3-4].
At present， artemether has not been applied to veterinary drug field. Lanzhou Institute of Husbandry and Pharmaceutical Sciences， Chinese Academy of Agricultural Sciences developed a new artemether injection as a veterinary clinical medicine， and therefore， it is urgent to establish a method for the determination of artemether content in preparation. With reference to the determination of artemether content in human medicine[5-8]， according to the guiding principles of quality control in "Chinese Pharmacopoeia （second section）" and "Collection of Guiding Principles for Veterinary Drug Research Technology （2006-2011）"[10]， the determination method of artemether content was studied， so as to lay a foundation for the clinical application of new artemether injection. Materials and Methods
Agents and reagent
Artemether standard （content： 99.3%， lot number： 100271-201404）， purchased from national institute for food and drug control； new artemether injection （lot number： 20141225， 20150121， 20150202） ， control injection containing no artemether （lot number： 20141223） and new 80%， 100% and 120% artemether injections （lot number： 20141223， 20141224， 20141225）， all of which were developed by Lanzhou Institute of Husbandry and Pharmaceutical Science of CAAS； Watsons pure water， purchased from Guangzhou Food and Beverage Co.， Ltd.； ethanol， acetonitrile and tetrahydrofuran， all of which were chromatographically pure and produced by Fisher Scientific.
Instruments
Waters 2695 high performance liquid chromatograph， Waters 2489 ultraviolet-visible detector， and Empower 2 chromatographic work station， purchased from Waters； Hypersil ODS C18 chromatographic column （5 μm， 4.6 mm × 150 mm）， purchased from Dalian Elite Analytical Instruments Co.， Ltd.； SartoriusME235S electronic scale， purchased from Beijing Sartorius Balance Co.， Ltd.； KQ-300DE ultrasonic cleaner， purchased from Kunshan Ultrasonic Instruments Co.， Ltd.； solvent filtering system， purchased from Phenomen.
Experimental methods
Chromatographic conditions
Column temperature： 30 ℃； injection volume： 10 μl； detection wavelength： 216 nm； mobile phase： acetonitrile-water-tetrahydrofuran （volume ration at 62∶37∶1）； flow rate： 1 ml/min.
Preparation of test solution
A certain amount of artemether injection with a content of 10% （0.4 ml） was accurately added into a 100 ml volumetric flask and diluted with ethanol （containing 10% tetrahydrofuran） to constant volume， obtaining artemether injection with a mass concentration of 400 μg/ml after shaking uniformly. The solution was filtered with 0.22 μm filter membrane （nylon） and injected according to an injection volume of 10 μl.
System suitability test
At first， 0.050 04 g of artemether standard with a content of 99.3% was accurately weighed into a 50 ml of volumetric flask， and diluted with ethanol （containing 10% tetrahydrofuran） to constant volume， obtaining artemether standard stock solution with a mass concentration of 1 mg/ml. Then， 4.0 ml of the stock solution was accurately added into a 10 ml measuring flask and diluted with ethanol （containing 10% tetrahydrofuran） to constant volume， obtaining artemether standard solution with a mass concentration of 400 μg/ml after shaking uniformly. The standard solution was filtered with 0.22 μm filter membrane （nylon） and injected according to an injection volume of 10 μl. The chromatogram was recorded， and the number of theoretical plates was calculated. Impurity interference test
Test solutions of new artemether injection and control injection containing no artemether were prepared according to "Preparation of test solution"， and detected under the injection volume of 10 μl， so as to analyze whether the control injection containing no artemether disturbs the chromatographic peak of artemether.
Preparation of standard solution
At first， 1.0， 2.0， 3.0， 4.0， 5.0， 6.0 and 8.0 ml of the artemether stock solution prepared according to "System suitability test" were accurately transferred to 10 ml measuring flasks， respectively， and the solution was diluted with ethanol （containing 10% tetrahydrofuran） to constant volume， followed by shaking uniformly， obtaining artemether standard solutions with mass concentrations of 100， 200， 300， 400， 500， 600 and 800 μg/ml， respectively.
Linear relation investigation
Under above chromatographic conditions， peak area of each various artemether standard solution was determined for 3 times. With average peak area as f（x） and mass concentration as x， linear regression was performed.
Precision test
Three samples with mass concentrations of 200， 500 and 800 μg/ml were injected on the same day at different time for 5 times， obtaining within-day precision. And the three samples were also detected continuously in 5 d， at the same time every day， obtaining day-to-day precision.
Recovery test
New artemether injection solutions with contents equivalent to 80%， 100% and 120% of that of new artemether injection were prepared. Each content was prepared with three parts， which were determined for artemether content， and recovery was calculated.
Determination of content in samples
Each batch of artemether injection was determined according to above chromatographic condition and method for 3 times in parallel. Specifically， 0.2 ml of artemether injection was accurately measured and added into a 50 ml volumetric flask and diluted with ethanol （containing 10% tetrahydrofuran） to constant volume， followed by shaking uniformly， obtaining the artemether solution with a mass concentration of 400 μg/ml. The obtained solution was determined， and the peak area was calculated according to external standard method （the standard solution for the external standard method was prepared according to "Preparation of standard solution"） and recorded. Meanwhile， the relative density was determined， and sample content was calculated. Results and Analysis
Results of system suitability and impurity interference tests
It could be seen from Fig. 1 that the retention time of artemether standard was about 8.1 min. It could be seen from Fig. 2 that the control injection containing no artemether had no interference to the HPLC determination of artemether.
Standard curve of artemether
Table 1 shows the mass concentrations and average peak areas of the determined artemether standard solutions. With average peak area as f（x） and mass concentration as x， linear regression was performed， obtaining regression equation y=302.36x-682.02 （Fig. 3）. It could be seen that artemether had a good linear relation in the mass concentration range of 100-800 μg/ml.
Results of precision test
It could be seen from Table 2 and Table 3 that the within-day precision and day-to-day precision of artemether had the RSD values of 1.29% and 0.46%， respectively. Both of the two values were lower than 5%， satisfying the requirement for precision by methodology validation.
Results of recovery test
It could be seen from Table 4 that in the artemether injection solutions with contents of 80%， 100% and 120%， recovery values of artemether were in the range of 95.87%-98.82%， with an overall average recovery of 97.58% and a RSD value of 1.58%. It indicates that the established determination method has high accuracy， and satisfies the requirement that recovery should be in the range of 80%-120%.
Determination of artemether contents in samples
The contents of the three batches of new artemether injection products were determined to be in the range of 97.27%-99.31%， with a RSD value of 1.42% （Table 5）.
Conclusions and Discussion
In this study， the chromatographic conditions were based on those for human artemether injection in "Chinese Pharmacopoeia （second section）" and optimized according to the characteristics of the injection itself and experimental conditions. Because the artemether injection uses medical tea oil for injection as a main ingredient， which could not be completely dissolved with mobile phase， and would form emulsion， thereby affecting the accuracy of content determination. Reports show that the application of anhydrous ethanol combined with ultrasonic treatment could give clear solution[5]. It was found in this study that sole application of anhydrous ethanol combined with ultrasonic treatment only changed emulsion drops to smaller ones， but could not completely dissolve them， and that the oil agent in the injection could be completely dissolved by firstly dissolving the tea oil with tetrahydrofuran and then adding ethanol to constant volume， without the need for ultrasonic dissolution. Due to application of the two solvents， i.e.， tetrahydrofuran and ethanol， partial solvent peaks would appear 2.5 min before the occurrence of chromatographic peak of artemether. "Chinese Pharmacopoeia （second section）" specifies the mobile phase for the determination of artemether by HPLC as acetonitrile-water （62∶38）， and in order to dissolve tea oil in the injection， the solvent contains tetrahydrofuran. Therefore， in this study， 1% of tetrahydrofuran was added into the mobile phase， forming the mobile phase acetonitrile-water-tetrahydrofuran （62∶37∶1）. After the addition of tetrahydrofuran， the artemether peak appeared 0.5 min ahead of time. Previous studies[11-15] also show that properly increasing proportion of organic phase and decreasing proportion of water phase could avoid tailing of artemether peak. In the injection， 90% is medical tea oil for injection， which belongs to non-drying oil， contains triolein mainly and less esters formed from saturated acid， and has a lower content of polyunsaturated fatty acids prone to auto-oxidation （lower iodine value）， resulting in higher stability than other liquid oil and low irritation to skin[16-20]. Tea oil itself also would produce chromatographic peak， but the appearance time of peak is different from that of artemether， so it would not influence later determination of artemether content.
It could be seen from that results of this study that the method for determination of artemether content satisfies the requirement by methodology validation， has the advantages of good reliability， high selectivity， good reproducibility， and simple operation， and could be used for the determination of artemether content in artemether injection. This study lays a foundation for the quality evaluation and quality standard formulation of the injection， and provides a basis for its further research and development.
References
[1] TU YY. Artemisia apiacea and artemisinin-based drug[M]. Beijing： Chemical Industry Press， 2009： 227-235. （in Chinese）
[2] XIANG G， LI NG， WANG CZ， et al. Artemether technological innovation process and its enlightenment to independent innovation of new drugs in China[J]. Scientific Research Management， 1997（4） ： 30-35. （in Chinese）
[3] WILLIAM T， MENON J， RAJAHRAM G， et al. Severe plasmodium knowlesi malaria in a tertiary care hospital， Sabah， Malaysia[J]. Emerging Infectious Diseases， 2011， 17（7）： 1248-1255.
[4] ZHANG YH， WANG LJ， GAN LL， et al. Advances in clinical application of artemether and its preparations[J]. Chongqing Medicine， 2014， 43 （29）： 3967-3970. （in Chinese）
[5] YAO J， CHENG QL， ZHANG QM， et al. Determination of artemether injection by HPLC method[J]. Chinese Journal of Pharmaceutical Analysis， 2008（1）： 91-92. （in Chinese）
[6] WANG XY， WANG YL. Determination of artemether in artemether tablets by HPLC[J]. Pharmaceutical Journal of Chinese Peoples Liberation Army， 1999， 15（4）： 7-9. （in Chinese）
[7] ZHOU L， ZHANG XS. Determination of artemether soft capsule by HPLC[J]. West China Journal of Pharmaceutical Sciences， 2005， 20（ 4） ： 341-343. （in Chinese）
[8] ZHONG F， BAO GM， DANG MS. Preparation and quality study of artemether capsules[J]. Journal of Hainan Normal University： Natural Science ， 2012， 25（4）： 402-406. （in Chinese） [9] Chinese Pharmacopoeia Commission. Chinese pharmacopoeia （second section） [M]. Beijing： China Medical Science Press， 2010： 1451-1452. （in Chinese）
[10] Veterinary drug evaluation center. Collection of guiding principles for veterinary drug research technology （2006-2011）[M]. Beijing： Chemical Industry Press， 2012： 32-37. （in Chinese）
[11] LI B， ZHANG J， ZHOU XZ， et al. Determination and pharmacokinetic studies of artesunate and its metabolite in sheep plasma by liquid chromatography-tandem mass spectrometry[J]. J Chromatogr B Analyt Technol Biomed Life Sci， 2015， 997： 146-153.
[12] LI C， QIU YQ， YANG GZ， et al. Simultaneous determination of artemether and its major metabolites dihydroartimisinin in rat plasma using ultra performance liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Analytical Chemistry， 2015， 43（4）： 588-593. （in Chinese）
[13] YANG HS， WANG NJ， LIANG BW， et al. Preparation of artemether patch and its pharmacodynamics in rats[J]. Chinese Pharmaceutical Journal， 2008， 43（24）： 1878-1881. （in Chinese）
[14] SANDRENAN N， SIOUFI A， GODBILLON J， et al. Determination of artemether and its metabolite， dihydroartemisinin， in plasma by high-performance liquid chromatography and electrochemical detection in the reductive mode[J]. J Chromatogr B Biomed Sci Appl， 1997， 691（1） ： 145-153.
[15] CHENG PP， YANG YJ， LI JY. Content determination of flunixin meglumine in a novel compound florfenicol injection by high performance liquid chromatography[J]. Journal of Henan Agricultural Sciences， 2014， 43（3）： 142-146. （in Chinese）
[16] YANG H， TAN XJ. Research advance in antitumor activities of artemisinin and its derivatives[J]. Acta Academiae Medicinae Sinicae， 2013， 35（4）： 466-471. （in Chinese）
[17] XU CY， LIU XW， YANG YJ， et al. Establishment of determination method of chlorogenic acid content in Yinqiaolanqin oral liquid by high performance liquid chromatography[J]. Journal of Henan Agricultural Sciences， 2016， 45（11）： 126-129. （in Chinese）
[18] EL-BESHBISHI S N， TAMAN A， EL-MALKY M， et al. In vivo effect of single oral dose of artemether against early juvenile stages of Schistosoma mansoni Egyptian strain [J]. Experimental Parasitology， 2013， 135（2） ： 240-245.
[19] ZHONG D， JIANG ML， WANG T. Advances in chemical components， pharmacological action and clinical application of tea oil[J]. Central South Pharmacy， 2012， 10 （4） ：299-303. （in Chinese）
[20] WU XH， ZHOU W， LI CB， et al. Study on oxidation stability of camellia oil[J]. Journal of The Chinese Cereals and Oils Association， 2008， 23（3）： 96-99. （in Chinese）
Key words Artemether injection； HPLC； Content determination
Artemisinin was isolated and extracted from Armisia annua L. in composite family for the first time in 1971. It has a better effect of killing agamont in red blood cell of plasmodium. Soon afterwards， many derivatives were synthesized with artemisinin as a lead compound， and artemether is one of them[1]. On September 11， 1987， the Ministry of Health of china held a press conference about artemisinin and its derivatives， and announced and approved large batch production of artemisinin injection as a specific medicine for treating various serious malaria patients. In the same year， World Health Organization （WHO） strictly verified the efficacy of artemether， and affirmed artemether as the best anti-malarial medicine. Artemether becomes the first preparation medicine registered internationally in China[2]. In human medicine field， artemether is mainly used for treating plasmodium， and could rapidly cure parasitemia[3-4].
At present， artemether has not been applied to veterinary drug field. Lanzhou Institute of Husbandry and Pharmaceutical Sciences， Chinese Academy of Agricultural Sciences developed a new artemether injection as a veterinary clinical medicine， and therefore， it is urgent to establish a method for the determination of artemether content in preparation. With reference to the determination of artemether content in human medicine[5-8]， according to the guiding principles of quality control in "Chinese Pharmacopoeia （second section）" and "Collection of Guiding Principles for Veterinary Drug Research Technology （2006-2011）"[10]， the determination method of artemether content was studied， so as to lay a foundation for the clinical application of new artemether injection. Materials and Methods
Agents and reagent
Artemether standard （content： 99.3%， lot number： 100271-201404）， purchased from national institute for food and drug control； new artemether injection （lot number： 20141225， 20150121， 20150202） ， control injection containing no artemether （lot number： 20141223） and new 80%， 100% and 120% artemether injections （lot number： 20141223， 20141224， 20141225）， all of which were developed by Lanzhou Institute of Husbandry and Pharmaceutical Science of CAAS； Watsons pure water， purchased from Guangzhou Food and Beverage Co.， Ltd.； ethanol， acetonitrile and tetrahydrofuran， all of which were chromatographically pure and produced by Fisher Scientific.
Instruments
Waters 2695 high performance liquid chromatograph， Waters 2489 ultraviolet-visible detector， and Empower 2 chromatographic work station， purchased from Waters； Hypersil ODS C18 chromatographic column （5 μm， 4.6 mm × 150 mm）， purchased from Dalian Elite Analytical Instruments Co.， Ltd.； SartoriusME235S electronic scale， purchased from Beijing Sartorius Balance Co.， Ltd.； KQ-300DE ultrasonic cleaner， purchased from Kunshan Ultrasonic Instruments Co.， Ltd.； solvent filtering system， purchased from Phenomen.
Experimental methods
Chromatographic conditions
Column temperature： 30 ℃； injection volume： 10 μl； detection wavelength： 216 nm； mobile phase： acetonitrile-water-tetrahydrofuran （volume ration at 62∶37∶1）； flow rate： 1 ml/min.
Preparation of test solution
A certain amount of artemether injection with a content of 10% （0.4 ml） was accurately added into a 100 ml volumetric flask and diluted with ethanol （containing 10% tetrahydrofuran） to constant volume， obtaining artemether injection with a mass concentration of 400 μg/ml after shaking uniformly. The solution was filtered with 0.22 μm filter membrane （nylon） and injected according to an injection volume of 10 μl.
System suitability test
At first， 0.050 04 g of artemether standard with a content of 99.3% was accurately weighed into a 50 ml of volumetric flask， and diluted with ethanol （containing 10% tetrahydrofuran） to constant volume， obtaining artemether standard stock solution with a mass concentration of 1 mg/ml. Then， 4.0 ml of the stock solution was accurately added into a 10 ml measuring flask and diluted with ethanol （containing 10% tetrahydrofuran） to constant volume， obtaining artemether standard solution with a mass concentration of 400 μg/ml after shaking uniformly. The standard solution was filtered with 0.22 μm filter membrane （nylon） and injected according to an injection volume of 10 μl. The chromatogram was recorded， and the number of theoretical plates was calculated. Impurity interference test
Test solutions of new artemether injection and control injection containing no artemether were prepared according to "Preparation of test solution"， and detected under the injection volume of 10 μl， so as to analyze whether the control injection containing no artemether disturbs the chromatographic peak of artemether.
Preparation of standard solution
At first， 1.0， 2.0， 3.0， 4.0， 5.0， 6.0 and 8.0 ml of the artemether stock solution prepared according to "System suitability test" were accurately transferred to 10 ml measuring flasks， respectively， and the solution was diluted with ethanol （containing 10% tetrahydrofuran） to constant volume， followed by shaking uniformly， obtaining artemether standard solutions with mass concentrations of 100， 200， 300， 400， 500， 600 and 800 μg/ml， respectively.
Linear relation investigation
Under above chromatographic conditions， peak area of each various artemether standard solution was determined for 3 times. With average peak area as f（x） and mass concentration as x， linear regression was performed.
Precision test
Three samples with mass concentrations of 200， 500 and 800 μg/ml were injected on the same day at different time for 5 times， obtaining within-day precision. And the three samples were also detected continuously in 5 d， at the same time every day， obtaining day-to-day precision.
Recovery test
New artemether injection solutions with contents equivalent to 80%， 100% and 120% of that of new artemether injection were prepared. Each content was prepared with three parts， which were determined for artemether content， and recovery was calculated.
Determination of content in samples
Each batch of artemether injection was determined according to above chromatographic condition and method for 3 times in parallel. Specifically， 0.2 ml of artemether injection was accurately measured and added into a 50 ml volumetric flask and diluted with ethanol （containing 10% tetrahydrofuran） to constant volume， followed by shaking uniformly， obtaining the artemether solution with a mass concentration of 400 μg/ml. The obtained solution was determined， and the peak area was calculated according to external standard method （the standard solution for the external standard method was prepared according to "Preparation of standard solution"） and recorded. Meanwhile， the relative density was determined， and sample content was calculated. Results and Analysis
Results of system suitability and impurity interference tests
It could be seen from Fig. 1 that the retention time of artemether standard was about 8.1 min. It could be seen from Fig. 2 that the control injection containing no artemether had no interference to the HPLC determination of artemether.
Standard curve of artemether
Table 1 shows the mass concentrations and average peak areas of the determined artemether standard solutions. With average peak area as f（x） and mass concentration as x， linear regression was performed， obtaining regression equation y=302.36x-682.02 （Fig. 3）. It could be seen that artemether had a good linear relation in the mass concentration range of 100-800 μg/ml.
Results of precision test
It could be seen from Table 2 and Table 3 that the within-day precision and day-to-day precision of artemether had the RSD values of 1.29% and 0.46%， respectively. Both of the two values were lower than 5%， satisfying the requirement for precision by methodology validation.
Results of recovery test
It could be seen from Table 4 that in the artemether injection solutions with contents of 80%， 100% and 120%， recovery values of artemether were in the range of 95.87%-98.82%， with an overall average recovery of 97.58% and a RSD value of 1.58%. It indicates that the established determination method has high accuracy， and satisfies the requirement that recovery should be in the range of 80%-120%.
Determination of artemether contents in samples
The contents of the three batches of new artemether injection products were determined to be in the range of 97.27%-99.31%， with a RSD value of 1.42% （Table 5）.
Conclusions and Discussion
In this study， the chromatographic conditions were based on those for human artemether injection in "Chinese Pharmacopoeia （second section）" and optimized according to the characteristics of the injection itself and experimental conditions. Because the artemether injection uses medical tea oil for injection as a main ingredient， which could not be completely dissolved with mobile phase， and would form emulsion， thereby affecting the accuracy of content determination. Reports show that the application of anhydrous ethanol combined with ultrasonic treatment could give clear solution[5]. It was found in this study that sole application of anhydrous ethanol combined with ultrasonic treatment only changed emulsion drops to smaller ones， but could not completely dissolve them， and that the oil agent in the injection could be completely dissolved by firstly dissolving the tea oil with tetrahydrofuran and then adding ethanol to constant volume， without the need for ultrasonic dissolution. Due to application of the two solvents， i.e.， tetrahydrofuran and ethanol， partial solvent peaks would appear 2.5 min before the occurrence of chromatographic peak of artemether. "Chinese Pharmacopoeia （second section）" specifies the mobile phase for the determination of artemether by HPLC as acetonitrile-water （62∶38）， and in order to dissolve tea oil in the injection， the solvent contains tetrahydrofuran. Therefore， in this study， 1% of tetrahydrofuran was added into the mobile phase， forming the mobile phase acetonitrile-water-tetrahydrofuran （62∶37∶1）. After the addition of tetrahydrofuran， the artemether peak appeared 0.5 min ahead of time. Previous studies[11-15] also show that properly increasing proportion of organic phase and decreasing proportion of water phase could avoid tailing of artemether peak. In the injection， 90% is medical tea oil for injection， which belongs to non-drying oil， contains triolein mainly and less esters formed from saturated acid， and has a lower content of polyunsaturated fatty acids prone to auto-oxidation （lower iodine value）， resulting in higher stability than other liquid oil and low irritation to skin[16-20]. Tea oil itself also would produce chromatographic peak， but the appearance time of peak is different from that of artemether， so it would not influence later determination of artemether content.
It could be seen from that results of this study that the method for determination of artemether content satisfies the requirement by methodology validation， has the advantages of good reliability， high selectivity， good reproducibility， and simple operation， and could be used for the determination of artemether content in artemether injection. This study lays a foundation for the quality evaluation and quality standard formulation of the injection， and provides a basis for its further research and development.
References
[1] TU YY. Artemisia apiacea and artemisinin-based drug[M]. Beijing： Chemical Industry Press， 2009： 227-235. （in Chinese）
[2] XIANG G， LI NG， WANG CZ， et al. Artemether technological innovation process and its enlightenment to independent innovation of new drugs in China[J]. Scientific Research Management， 1997（4） ： 30-35. （in Chinese）
[3] WILLIAM T， MENON J， RAJAHRAM G， et al. Severe plasmodium knowlesi malaria in a tertiary care hospital， Sabah， Malaysia[J]. Emerging Infectious Diseases， 2011， 17（7）： 1248-1255.
[4] ZHANG YH， WANG LJ， GAN LL， et al. Advances in clinical application of artemether and its preparations[J]. Chongqing Medicine， 2014， 43 （29）： 3967-3970. （in Chinese）
[5] YAO J， CHENG QL， ZHANG QM， et al. Determination of artemether injection by HPLC method[J]. Chinese Journal of Pharmaceutical Analysis， 2008（1）： 91-92. （in Chinese）
[6] WANG XY， WANG YL. Determination of artemether in artemether tablets by HPLC[J]. Pharmaceutical Journal of Chinese Peoples Liberation Army， 1999， 15（4）： 7-9. （in Chinese）
[7] ZHOU L， ZHANG XS. Determination of artemether soft capsule by HPLC[J]. West China Journal of Pharmaceutical Sciences， 2005， 20（ 4） ： 341-343. （in Chinese）
[8] ZHONG F， BAO GM， DANG MS. Preparation and quality study of artemether capsules[J]. Journal of Hainan Normal University： Natural Science ， 2012， 25（4）： 402-406. （in Chinese） [9] Chinese Pharmacopoeia Commission. Chinese pharmacopoeia （second section） [M]. Beijing： China Medical Science Press， 2010： 1451-1452. （in Chinese）
[10] Veterinary drug evaluation center. Collection of guiding principles for veterinary drug research technology （2006-2011）[M]. Beijing： Chemical Industry Press， 2012： 32-37. （in Chinese）
[11] LI B， ZHANG J， ZHOU XZ， et al. Determination and pharmacokinetic studies of artesunate and its metabolite in sheep plasma by liquid chromatography-tandem mass spectrometry[J]. J Chromatogr B Analyt Technol Biomed Life Sci， 2015， 997： 146-153.
[12] LI C， QIU YQ， YANG GZ， et al. Simultaneous determination of artemether and its major metabolites dihydroartimisinin in rat plasma using ultra performance liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Analytical Chemistry， 2015， 43（4）： 588-593. （in Chinese）
[13] YANG HS， WANG NJ， LIANG BW， et al. Preparation of artemether patch and its pharmacodynamics in rats[J]. Chinese Pharmaceutical Journal， 2008， 43（24）： 1878-1881. （in Chinese）
[14] SANDRENAN N， SIOUFI A， GODBILLON J， et al. Determination of artemether and its metabolite， dihydroartemisinin， in plasma by high-performance liquid chromatography and electrochemical detection in the reductive mode[J]. J Chromatogr B Biomed Sci Appl， 1997， 691（1） ： 145-153.
[15] CHENG PP， YANG YJ， LI JY. Content determination of flunixin meglumine in a novel compound florfenicol injection by high performance liquid chromatography[J]. Journal of Henan Agricultural Sciences， 2014， 43（3）： 142-146. （in Chinese）
[16] YANG H， TAN XJ. Research advance in antitumor activities of artemisinin and its derivatives[J]. Acta Academiae Medicinae Sinicae， 2013， 35（4）： 466-471. （in Chinese）
[17] XU CY， LIU XW， YANG YJ， et al. Establishment of determination method of chlorogenic acid content in Yinqiaolanqin oral liquid by high performance liquid chromatography[J]. Journal of Henan Agricultural Sciences， 2016， 45（11）： 126-129. （in Chinese）
[18] EL-BESHBISHI S N， TAMAN A， EL-MALKY M， et al. In vivo effect of single oral dose of artemether against early juvenile stages of Schistosoma mansoni Egyptian strain [J]. Experimental Parasitology， 2013， 135（2） ： 240-245.
[19] ZHONG D， JIANG ML， WANG T. Advances in chemical components， pharmacological action and clinical application of tea oil[J]. Central South Pharmacy， 2012， 10 （4） ：299-303. （in Chinese）
[20] WU XH， ZHOU W， LI CB， et al. Study on oxidation stability of camellia oil[J]. Journal of The Chinese Cereals and Oils Association， 2008， 23（3）： 96-99. （in Chinese）