Accepted Manuscript Title: Determination and pharmacokinetic studies of artesunate and its metabolite in sheep plasma by liquid chromatography-tandem mass spectrometry Author: Bing Li Jie Zhang Xu-Zheng Zhou Jian-Yong Li Ya-Jun Yang Xiao-Juan Wei Jian-Rong Niu Xi-Wang Liu Jin-Shan Li Ji-Yu Zhang PII: DOI: Reference:

S1570-0232(15)00263-9 http://dx.doi.org/doi:10.1016/j.jchromb.2015.05.001 CHROMB 19433

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

19-11-2014 29-4-2015 3-5-2015

Please cite this article as: B. Li, J. Zhang, X.-Z. Zhou, J.-Y. Li, Y.-J. Yang, X.-J. Wei, J.-R. Niu, X.-W. Liu, J.-S. Li, J.-Y. Zhang, Determination and pharmacokinetic studies of artesunate and its metabolite in sheep plasma by liquid chromatography-tandem mass spectrometry, Journal of Chromatography B (2015), http://dx.doi.org/10.1016/j.jchromb.2015.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Highlights

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1. A LC-MS/MS method determining artesunate and its metabolite in sheep plasma.

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2. A MRM method was employed with an ESI source in positive mode. 3. The method is simple, sensitive and accurate.

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4. Successful study on the pharmacokinetics of artesunate nanoemulsion in the sheep.

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5. Artesunate nanoemulsion has an average emulsion droplet particle size of 60 nm.

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Determination and pharmacokinetic studies of artesunate and its

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metabolite in sheep plasma by liquid chromatography-tandem mass

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spectrometry

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Bing Li

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Xiao-Juan Wei a,b,c, Jian-Rong Niu a,b,c, Xi-Wang Liu a,b,c, Jin-Shan Li a,b,c, Ji-Yu Zhang

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a,b,c,*

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, Jie Zhang d, Xu-Zheng Zhou a,b,c, Jian-Yong Li a,b,c, Ya-Jun Yang a,b,c,

Key Laboratory of Veterinary Pharmaceutical Development, Ministry of Agriculture,

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a,b,c

Lanzhou, China b

Key Laboratory of New Animal Drug Project of Gansu Province, Lanzhou, China

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c

Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS,

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d

Fulin Animal Science and Veterinary Medicine Officer, Chongqing 408000, China

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Lanzhou 730050 , China

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Corresponding author: Ji-Yu Zhang, Lanzhou Institute of Husbandry and

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Pharmaceutical Sciences of CAAS, Lanzhou 730050, China

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Phone and E-mail:+86013893612415,[email protected]

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Fax: +86-931-2115191

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Postal address: 335 Jiangouyan,Qilihe,Lanzhou, 730050,Gansu,China

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Abstract

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A rapid and sensitive high-performance liquid chromatography–tandem mass

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spectrometry (LC–MS/MS) method was developed and validated to simultaneous

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quantify artesunate and its metabolite in sheep plasma. The plasma samples were

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prepared by liquid-liquid extraction. Chromatographic separation was achieved on a

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C18 column (250×4.6mm, 5 μm) using methanol: water (60:40, v/v) (the water

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included 1mM ammonium acetate, 0.1% formic acid, and 0.02% acetic acid) as the

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mobile phase. Mass detection was carried out using positive electrospray ionization in

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multiple reaction monitoring mode. The calibration curve was linear from 1 ng/mL to

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400 ng/mL (r2=0.9992 for artesunate, r2=0.9993 for its metabolite). The intra-day and

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inter-day accuracy and precision were within the acceptable limits of ±10% at all

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concentrations for both artesunate and its metabolite. The recoveries ranged from 92

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to 98% at the three concentrations for both. In summary, the LC–MS/MS metho

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described herein was fully successfully applied to pharmacokinetic studies of

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artesunate nanoemulsion after intramuscular delivery to sheep.

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Keywords:

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Antiparasitic; Artesunate nanoemulsion; LC/MS/MS; Pharmacokinetics;

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Sheep plasma

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1.Introduction Theileriosis, caused by various intraerythrocytic protozoan parasites of the genus

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Theileria, is a tick-borne disease of domestic and wild animals. Theileriosis, also

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known as blood cryptosporidiosis, is a blood protozoosis caused by parasites to infect

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leukocytes and erythrocytes [1], is a tick-borne disease of domestic and wild animals

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[2] . This epidemic disease is causing serious damage to the cattle and sheep

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industries. It is strongly seasonal and local, and present in many countries, research

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carried out over many years has shown these to be distributed mainly in Northern

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China but also sometimes in Southern China. [3]. Theileria lestoquardi is a highly

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pathogenic parasite of sheep and goats., which occurred in south-eastern Europe,

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northern Africa, western and central Asia [4], India [5] and in West, Central and North

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China such as Qinghai, Gansu, Hubei, Liaoning and Inner Mongolia [6,7,8].

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Non-pathogenic or mildly pathogenic Theileria spp. of small ruminants include

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Theileria separata, Theileria ovis and Theileria recondite [9]. Clinical symptoms in

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sick sheep include high fever, depression, anemia, conjunctival yellowish

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discoloration, superficial swollen lymph nodes, limb rigidity, difficulty walking, rapid

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weight loss, weakness, and finally failure and death [10]. Treatment of the disease is

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problematic, as there are few effective medicines. Despite the large number of studies

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on this disease, efficient new drugs are still lacking. The drugs presently in use are

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mainly berenil, chloroquine phosphate, artesunate, uranidin, and imidocarb. Berenil is

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the most commonly used, but its popularity has lessened due to the major side effects

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that can accompany its injection.

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Artesunate is a semi-synthetic derivative of artemisinin, extracted from the plant

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species of Artemisia. Artesunate is a highly efficacious anti-malarial [11,12] and also

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has anti-tumor activity [13]. It is also effective for treating some non-malarial

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parasites [14,15], for example, it is an effective therapeutic for Theileria annulata

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infection of cattle and sheep. However, artesunate still presents a few problems,

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including limited bioavailability, a short half-life, and also poor water solubility [16],

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which makes it difficult to deliver effectively to the lesion site and intracellular space.

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Further, at present there is only an oral tablet and bulk drugs for clinical use. Also, the

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poor stability of the drug's sodium salt causes its low bioavailability and necessitates

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frequent patient medication, leading to poor tolerance and efficacy, and greatly

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limiting its clinical application. New preparations of derivatives with better

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pharmacokinetic profiles are needed to overcome these issues.

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In our previous study, we have successfully prepared artesunate nanoemulsion

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for the first time in China as an intramuscular preparation that increases the solubility

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of artesunate and its sodium salt. It has a clear, transparent appearance, an average

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emulsion droplet particle size of 60 nm, a good degree of dispersion, thermodynamic

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stability, storage stability, and long-placing non-hierarchical[17]. Acute toxicity

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studies of oral artesunate nanoemulsion in mice indicate that it is nontoxic [17].

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Artesunate nanoemulsion has significant preventive and treatment effects in Taylor

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piroplasmosis of goats [18].

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In order to define the pharmacokinetic profile of artesunate nanoemulsion, an high

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performance liquid chromatography–tandem mass spectrometry (LC–MS/MS)

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method for the determination of artesunate and its metabolite in sheep plasma needs

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to be established. Because the artemisinin and its analogues contains no conjugated

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groups in the structure (Fig. 1), they have no appropriate chromophores for use in

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characterization techniques their quantitation sometimes requires the use of lengthy

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derivatization techniques, and these derivatizing conditions are not necessarily stable

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for all artemisinin analogs and can cause the parent compound to decompose [19].

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Hence, we chose LC-MS/MS as an analytical method that is accurate and sensitive,

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using a simple liquid-liquid extraction as the organic phase is evaporated and

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reconstructed before analysis approach for artesunate nanoemulsion. This method has

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been successfully applied to characterize the pharmacokinetics of artesunate

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nanoemulsion in sheep following intramuscular administration at 5 mg/kg.

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Fig. 1 Chemical structures of Artesunate, Artemisinin, and

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Dihydroartemisinin.

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2. Experimental

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2.1 Reagents and chemicals

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Artesunate (AR), dihydroartemisinin (DHA), and internal standard (IS)

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artemisinin (AS) were provided by the National Institute for the Control of

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Pharmaceutical and Biological Products (Beijing, China) with the batch numbers of

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100200-200202, 100184-200401, and 100202-200603 respectively, the purity of AR,

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DHA and IS are >99%. Acetonitrile and methanol (HPLC grade) were purchased from

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Fisher Chemical (Waltham, USA). Acetic acid, ethyl acetate, ammonium acetate, and

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formic acid were analytically pure and were purchased from Sinopharm Group

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Chemical Reagent Co., Ltd. (Ningbo, China). Water was purified through a Milli-Q

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Plus water system (Millipore Corporation, Bedford, MA, USA) before use. Artesunate

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nanoemulsion (containing artesunate 5%) was supplied by Lanzhou Institute of

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Animal Science and Veterinary Pharmaceutics.

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2.2. Equipment

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The LC–MS/MS equipment (1200-6410A) consisted of a LC system with a binary

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pump-SL and a triple quadrupole mass spectrometer with electrospray ionization(ESI)

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(Agilent Technologies, Inc., Santa Clara, CA, USA). Data were recorded, and the

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system was controlled using MassHunter software (version B.01.04, Agilent

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Technologies).

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2.3 Chromatographic and mass spectrometer conditions

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The analysis was carried out using a Agilent Zorbox C18 column (250×4.6mm, 5μm); the mobile phase used for the analysis consisted of methanol: water (60:40, v/v)

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(the water included 1mM ammonium acetate, 0.1% formic acid, and 0.02% acetic

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acid). The mobile phase was filtered before being used to prevent entry of bubbles or

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impurities in the system. The mobile phase was delivered at a flow rate of 0.4 mL/min

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and 5 μL was injected at 30 ºC.

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The mass spectrometer was operated in positive ion mode with an ESI interface.

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Quantitation was performed by multiple reaction monitoring (MRM). In the positive

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mode, the MS/MS setting parameters were as follows: capillary voltage 4 kV, cone

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voltage 40 V, source temperature 100°C, and desolvation temperature 350°C with a

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desolvation nitrogen gas flow of 11 L/min and a cone gas flow of 9 L/min. The

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optimized fragmentation voltages for AR、DHA and IS were all 100 V, and the Delta

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electron multiplier voltage (EMV) were all 200 V. Data were collected in multiple

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reaction monitoring (MRM) mode using [M+Na] + ion for all AS、DHA and IS with a

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collision energy of 25 eV、18 eV and 20 eV, respectively.

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2.4 Preparation of standard solutions and quality control samples

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Standard solution of AR and DHA: Precisely 2 mg of AR and of DHA were

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respectively placed in separate 10 mL brown volumetric flasks, then methyl alcohol

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was added to form stock solutions of 200 μg·mL-1. A series of mixture of AR and

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DHA working standard solutions were prepared by dilutions of the stock solution with

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methyl alcohol to obtain the following concentrations:

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400 ng·mL-1.

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0.1, 0.2, 1, 4, 20, 100, 200,

Internal standard solution: Precisely 4 mg of AR was weighed and placed in a 50

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mL brown volumetric flask, to which methyl alcohol was then added, forming a

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solution of 80μg·mL-1. Then, 2.5 mL of that solution was placed in a 50 mL

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volumetric flask, followed by methyl alcohol to make a 4 μg·mL-1 internal standard

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solution.

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All of the solutions were stored at 4 ºC and brought to room temperature before

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use. Plasma calibration standards of 1–400 ng/mL were prepared by spiking 100 μL

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aliquots of blank plasma with 10 µL of each of the standard solutions. Quality control

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(QC) samples were prepared in the same way, with four levels of 4(QC-low), 100

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(QC-med), 400 (QC-high), and 1 ng/mL (QC-LLOQ). Both the calibration standard

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and the QC samples were applied in the method validation and the pharmacokinetic

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study.

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2.5 Sample preparation Plasma aliquots (100 μL) were spiked with 10 µL of methanol and artemisinin (10

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μL of 4 μg/mL solution) as an internal standard in centrifuge tubes (when preparing

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calibration and quality control (QC) samples, the standard solution was added instead

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of methanol), and mixed. The centrifuge tubes were initially primed with 200 μL of

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ethyl acetate, followed by vortex concussion for 1 min and centrifugation for 10 min

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at the speed of 12000 rpm. The organic phase was evaporated by use of a stream of 25

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ºC nitrogen. The residue was reconstituted with 100 μL of methanol and immediate

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vortex concussion for 20s, placed for 10 min, and injected into the LC–MS/MS

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system after filtration through a 0.45 μm Millipore filter.

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2.6 Validation

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This method was validated in terms of linearity, specificity, LLOQ, recovery,

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intra- and inter-day variation, accuracy and precision, and stability of the analyte

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during sample storage and processing procedures.

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2.6.1 Selectivity

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Selectivity was evaluated by comparing the chromatograms of six different

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batches of the blank plasma with the corresponding standard plasma samples spiked

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with AR and DHA and the internal standard [19].

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2.6.2 Linearity and LLOQ

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A calibration curve was constructed from plasma standards at six concentrations

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of AR and DHA, ranging from 1 ng/mL to 400 ng/mL. A calibration curve was

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constructed by plotting the peak area ratio of AR/IS versus the nominal concentration

Page 9 of 33

of AR and the peak area ratio of DHA/IS versus the nominal concentration of DHA.

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The correlation coefficient and linear regression equation were used for the

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determination of the analyte concentration in the samples. A weighted (1/X2) linear

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least-squares regression was used as the mathematical model. The LLOQ was

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determined as the lowest concentration that produced an S/N of 5 [20]. The limit of

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detection (LOD) was determined as the lowest concentration that produced an S/N of

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3 [20].

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2.6.3 Accuracy and precision

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Intra-day accuracy and precision were evaluated by analyzing the QC

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concentrations at four levels (4, 100, 400 ng/mL and 1 ng/mL; Table 1) with six

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determinations per concentration on the same day. The inter-day accuracy and

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precision were evaluated by the analysis of the QC concentrations at four levels (4,

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100, 400 ng/mL, and 1 ng/mL; Table 1) with six determinations per each

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concentration over 3 days. Precision and accuracy were based on the criterion that the

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relative standard

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15%, except for LLOQ (not to be more than 20%) [21].

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2.6.4 Recovery and matrix effect

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deviation (RSD) for each concentration should be not more than

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The recovery was determined in quadruplicate by comparing processed QC

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samples at three levels (low, med, high) with reference solutions in blank plasma

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extract at the same levels.

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The matrix effect was determined by comparing the peak areas of the

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post-extracted spiked sample with those of the standards containing equivalent

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amounts of the AS and DHA prepared in the mobile phase, respectively. The

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experiments were performed at the three levels in six different batches.

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2.6.5 Stability [22] The stabilities of AR and DHA in sheep plasma were assessed by analyzing

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replicates (n=6) of QC samples at concentrations of 4, 100, and 400 ng/mL during the

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sample storage and processing procedures. Freshly prepared stability-QC samples

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were analyzed by using a freshly prepared standard curve for the measurement for all

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stability studies. The stabilities of stock solutions of AR and DHA were analyzed at

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room temperature for 24 h and at 4 ºC after 1 month. The short-term stability was

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assessed after exposure of the plasma samples to ambient temperature for 24 h. The

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long-term stability was assessed after storage of the plasma samples at -20 ºC for 60

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days. The freeze/thaw stability was determined after three freeze/thaw cycles (room

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temperature to -20 ºC). The sample stability in the autosampler tray was evaluated at 4

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ºC for 24 h; this sample stability evaluation imitates the residence time of the samples

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in the autosampler for each analytical run.

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2.6.6 Formulation

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The injection of artesunate nanoemulsion was prepared as follows: The component substances for 6% ethyl oleate, 24% Tween-80, 11.5% n-butanol,

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53.5% ultra-pure water, 5% artesunate were weighed. The ethyl oleate, Tween-80,

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n-butanol, and artesunate were combined and stirred under ambient conditions until

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the drug was dissolved. Ultrapure water was slowly added, dropwise, to the mixture

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with stirring. At a certain concentration of water, the system becomes a clarified,

Page 11 of 33

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translucent, pale yellow O/W type intravenous-formulation artesunate nanoemulsion.

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The intravenous formulation was administered to sheep (n=12) at a dose of 5 mg/kg.

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2.7 Animal studies The assay method described above was used to study the pharmacokinetics of

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artesunate nanoemulsion in sheep plasma after intramuscular administration. All the

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experimental procedures were approved and performed in accordance with the

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Guidelines for the Care and Use of Laboratory Animals of the Lanzhou Institute of

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Animal Science and Veterinary Pharmaceutics. Healthy Small Tail Han sheep

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(35±1.27 kg) were obtained from the Small Tail Han sheep-breeding base (Kangle,

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Gansu, China). These sheep were housed in a standard, environmentally controlled

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animal room (temperature: 25±2 ºC, humidity: 50±20%) with a natural light-dark

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cycle for 1 week before the experiment. The sheep were fasted for 12 h before dosing

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but allowed free movement and access to water during the whole experiment. All

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sheep (n=12) were dosed at a dosage of 5 mg/kg. After a single dose was administered

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by intramuscular administration, blood samples (3 mL) were collected in heparinized

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tubes via the jugular vein at 0.083, 0.167, 0.25, 0.333, 0.5, 1.5, 1, 2, 4, 6, 8, 10, 12, 16,

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20, and 24 h. After all blood samples were centrifuged at 12,000 rpm for 10 min, the

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plasma samples were collected, and then immediately stored in a -20 ºC freezer until

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analysis by LC–MS/MS.

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The pharmacokinetic parameters were calculated by use of WinNonlin

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professional software version 5.2 (Pharsight, Mountain View, CA, USA). A

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compartmental model was utilized for data fitting and parameter estimation.The

Page 12 of 33

essential pharmacokinetic model was confirmed by Akaike Information Criterion

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(AIC)[23] for the best characterization. Plasma AUC, plasma clearance (CL/F), peak

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plasma concentration (Cmax), elimination rate constant(K) and apparent volume of

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distribution (Vss) were all obtained from observed data. Half-life (t1/2) was calculated

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directly according to the pharmacokinetic parameters.

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3. Results and discussion

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3.1 Mass spectrometric detection

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In order to optimize positive ESI mode conditions, AR, DHA, and IS were

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dissolved in methanol, and then infused into the mass spectrometer for scans in

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positive ion mode. When AR, DHA, and IS were injected directly into the mass

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spectrometer along with the mobile phase, the analytes yielded predominantly [M+Na]

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+ ions at m /z 407.2 for AR, m /z 307.2 for DHA, and at m /z 305.2 for IS(Fig.2). Each

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of the precursor ions was subjected to collision-induced dissociation to determine the

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resulting product ions from the product ion mass spectra. The most abundant and

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stable fragment ions were generated at m /z 261.1 for AR, m /z 163.0 for DHA, and m

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/z 151.1 for IS. Thus, the mass transitions chosen for quantitation were m /z 407.2 to

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261.10 for AR, m /z 307.2 to 163.0 for DHA, and m /z 305.2 to 151.1 for IS.

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Fig. 2 Full mass spectra scan for artesunate (A), dihydrortemisinin (B), and internal

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standard artemisinin (C); product ions for artesunate (D), dihydrortemisinin (E), and

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internal standard artemisinin (F)

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3.2 Chromatographic separation

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High-performance liquid chromatography with MS/MS separations was run using

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column packed with a small amount of mobile phase and a shorter analysis time. A

Page 13 of 33

250 mm column subjected to an flow rate of 0.4 mL/min isocratic elution of the

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mobile phase for 8.5 min was used for the chromatographic separation. A mobile

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phase consisting of a mixture of methanol: water (the water included 1 mM

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ammonium acetate, 0.1% formic acid, and 0.02% acetic acid) was found to be suitable

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for the separation and ionization of AR, DHA, and the internal standard. Formic acid

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was found to increase the ionization of all three compounds. Under optimized LC and

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MS conditions, AR, DHA, and IS were separated with retention times of 5.45, 6.21,

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and 8.12 min, respectively, and the endogenous substances in plasma does not

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interfere with target detection material(Fig.3).

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Fig. 3 Chromatograms

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(A: blank plasma, B: chromatograms of artesunate-a, dihydroartemisinin-b, and

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3.3 Sample preparation

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Recoveries of AR and DHA using methanol (14%), acetonitrile (12%), and

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chloroform (46%) were found to be less than that from ethyl acetate (95%). Therefore,

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ethyl acetate was selected as the extraction solvent for plasma due to its high recovery

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and sensitivity.

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3.4 Optimization of the intramuscular preparations

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The traditional tablets of artesunate cannot overcome the first-pass effect of the

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liver. Further, as artesunate has poor solubility in water, it is difficult to deliver it

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effectively to the lesion and intracellular space. In addition, the poor stability of its

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sodium salt, which causes low bioavailability, necessitates frequent patient medication,

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leading to poor tolerance and efficacy and greatly limits its clinical use. Our aim was

Page 14 of 33

to find a new preparation of artesunate that would increase the solubility of artesunate

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and its sodium salt. A composition of 6% ethyl oleate, 24% Tween-80, 11.5%

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n-butanol, 53.5% ultra-pure water, and 5% artesunate was chosen to formulate an

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artesunate nanoemulsion for intramuscular administration, which was proved to be

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safe.

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3.5 Method validation

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3.5.1 Selectivity

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The specificity of the method was evaluated by analyzing individual blank plasma

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samples from six different sources. All samples were found to have no interference

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from endogenous substances affecting the retention times of AR, DHA, and IS. There

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was good base-line separation of AR, DHA, and the IS extracted from the sheep

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plasma. Representative chromatograms of blank plasma, blank plasma spiked with

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AR, DHA, and the IS are shown in Fig. 3.

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3.5.2 Linearity and LLOQ

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of AR and DHA, ranging from 1 ng/mL to 400 ng/mL. The ratio of peak areas of AR

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and DHA to that of the IS was used for quantification. The calibration model was

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selected based on the analysis of the data by linear regression with intercepts and a

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1/X2 weighting factor. A typical equation of the calibration curve for AR was:

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y=0.0130x-0.3212 (r2=0.9992), where y is the peak-area ratio of AR to IS and x is the

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plasma concentration of AR. A typical equation of the calibration curve for DHA was:

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y=0.0073x+0.1246 (r2=0.9993), where y is the peak-area ratio of DHA to IS and x is

Page 15 of 33

the plasma concentration of DHA. The calibration curve is shown in Fig. 4. The lower

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limits of quantitation (LLOQ) for AR and DHA were both established at 1 ng/mL,

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with an accuracy of 98.5%. The LOD was found to be 0.1 ng/mL of AR and 0.2

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ng/mL of DHA. Fig. 4 Calibration curve

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3.5.3 Accuracy and precision

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The intra-day and inter-day precision and accuracy of QC samples (4, 100, 400

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ng/mL and 1 ng/mL) is summarized in Table 1. These data demonstrate that the

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current method has satisfactory accuracy, precision, and reproducibility for the

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quantification of AR and DHA in sheep plasma.

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Table 1.

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Intra and inter day precision and accuracy of AR and DHA (n=6) in sheep plasma

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3.5.4 Recovery and matrix effect

The mean extraction recoveries of AR were (95.0±2.28)%, (94.5±2.77)%, and

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(97.7±2.69)% at the concentrations of 4, 100, and 400 ng/mL, respectively, and the

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mean extraction recoveries of DHA were (92.9±3.11)%, (95.1±2.23)%, and

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(93.4±2.31)% at the three concentrations of 4, 100, and 400 ng/mL, respectively. The

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mean extraction recovery of the IS was 96.3±2.2% at 200 ng/mL. These results

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suggest

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concentration-dependent. Recovery values are listed in Table 2.

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that

the

Table 2.

recovery

of

AR

and

DHA was

consistent

and

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Recovery of AR and DHA (n=6) from sheep plasma

The matrix effects ranged from (93.2±2.5)% to (96.4±1.9)% for AR and

Page 16 of 33

(92.7±2.2)% to (95.6±1.3)% for DHA at the three concentrations of 4, 100, and 400

345

ng/mL, respectively, while the matrix effect of the IS was (93.8±2.7)%. This means

346

that limited matrix effects were observed.

347

3.5.5 Stability

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344

The stability tests on AR and DHA include stability data from freeze/thaw,

349

short-term, autosampler, and long-term stability tests. These data are shown in Tables

350

3 and 4. The results demonstrate that no stability issues were observed in any of the

351

experiments. AR and DHA were stable after being placed in sheep plasma for three

352

cycles when stored at -20 °C and thawed to room temperature, and were stable to

353

repeated exposure to room temperature for 24 h, in the autosampler tray at 4 °C over

354

24 h, and when stored at -20 °C for 60 days. AR and DHA were also stable in stock

355

solutions at room temperature for 24 h and at 4 °C for 1 month. Taking all these

356

points into consideration, we conclude that AR and DHA can be stored and processed

357

under routine laboratory conditions without special attention.

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Table 3.

Stability of AR in sheep plasma samples under various conditions (n=6)

359

Table 4.

Stability of DHA in sheep plasma samples under various conditions (n=6)

360 361

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3.6 Application to pharmacokinetic studies The present method was successfully validated and applied to quantitate AR and

362

DHA in plasma samples after intramuscular administration of artesunate

363

nanoemulsion to Small Tail Han sheep at doses of 5mg/kg. The pharmacokinetics of

364

AR were investigated in cattle, humans, and dogs, as plasma samples from sheep

365

pharmacokinetic studies were not yet available. Fig.5 shows the mean plasma

Page 17 of 33

concentration vs. time profile for AR and DHA in sheep after intramuscular artesunate

367

nanoemulsion. Based on AIC (the fitted values is 53), the plasma concentration-time

368

curves for AR and DHA were adequately fitted with a one compartment model, and

369

the major pharmacokinetic parameters were calculated by use of this model and are

370

listed in Table 5. The PK data analysis showed that AR and DHA have a faster

371

clearance rate and smaller volume of distribution. After intramuscular administration

372

of artesunate nanoemulsion to sheep, AR can be quickly converted to its active

373

metabolite DHA, but the peak concentration (Cmax) of AS was higher than that of

374

DHA (the same as the result reported by [24]), and AR was not all converted to DHA.

375

The short half-life (t1/2) of AR and DHA indicates that the compound is removed

376

rapidly from the blood. No significant differences were found in comparing males and

377

females (data not shown). This study gives us some useful information to serve as a

378

basis for further research on artesunate nanoemulsion. Method development and

379

evaluation of the pharmacokinetic properties of this formulation will aid the

380

preparation of new formulations of similar drugs with improved pharmacokinetic

381

profiles.

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382

Fig.5 Mean plasma concentration–time profile after intramuscular of artesunate

383

nanoemulsion to sheep (n=12) at 5 mg/kg.

384

Table 5.

Pharmacokinetic parameters of AR and DHA after intramuscular injection (n=12)

385 386 387

4.Discussion A few methods for the quantification of AR and DHA have been published. As

Page 18 of 33

388

these compounds are thermally labile and do not contain anultraviolet (UV) visible or

389

fluorescent

390

high-performance liquid chromatography (HPLC) have proven difficult. C.S. Lai et

391

al.[26] described a HPLC method with UV , which get a lower limit of quantifications

392

for AS and DHA at 20 ng/1ml with a long cycle time of 14min.

analysis

by

gas

chromatography

(GC)

and

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chromophore[25],

K.N.Bangchang et al.[27], C.S. Lai et al.[28].and V. Navaratnam et al.[29]

394

described a HPLC method with electrochemical detection (ECD) . The drawbacks of

395

the ECD technique are that it requires rigorously controlled anaerobic conditions and

396

deoxygenation of biologic samples as well as the mobile phase which can be very

397

difficult to establish and maintain. ECD methods are also labor-intensive. The HPLC

398

method with ECD described by Na-Bangchang et al.[27] can detect AR and DHA at

399

concentrations as low as 5 ng/ml and 3 ng/ml respectively, but requires a large sample

400

volume (1ml of plasma).S.S. Mohamed et al.[30] developed a GC–MS–SIM

401

method.The total run time was 20.5 min with a solvent delay time of 7.5 min, and the

402

limits of quantification is 5 ng/ml using 1ml of plasma for both AR and DHA.

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403

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393

A LC-MS method for quantification of AR and DHA in human plasma was able to

404

reach limits of quantification of 1 ng/ml using 0.5ml of plasma with a total run time of

405

21 min, which was a long retention time for assay [31].Recently a comparison

406

between an ECD and a LC-MS/MS method indicated good correlation but superior

407

sensitivity for the LC-MS/MS method reaching lower limits of quantification of 2 and

408

4 ng/ml for DHA and ARS, respectively, using 100 μL plasma[25].

409

The advantages of the method developed in the present study over that previously

Page 19 of 33

reported are the sample extraction procedure using liquid–liquid extraction (using

411

only a single extraction with ethyl acetate) was simple and less laborious when

412

compared with the previously described methods of post column alkali decomposition

413

[32] and solid phase extraction for AR and DHA [26,28,30,31,33]. Furthermore, this

414

extraction method was able to separate AR , DHA and IS for separate assays without

415

a laborious precolumn separation step, whilst maintaining the high sensitivity (the

416

sensitivity is comparable to the LC–MS/MS method described by H. Naik et al.[31]

417

and W. Hanpithakpong et al. [33]).

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410

Furthermore, the LC-MS/MS method described in this study reached lower limits

419

of quantification of 1 ng/ml for both AR and DHA requires only 100 μL of plasma

420

and a short run time of 8.5 min, and has satisfactory accuracy, precision, and

421

reproducibility for the quantification of AR and DHA in sheep plasma. This method

422

was simple, fast, less laborious, and has high sensitivity using a small amount of

423

sample volumes, which is very advantageous in pharmacokinetic studies.

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The present pk properties obtained for AR and DHA in this study is in agreement

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418

425

with the previously published data, where the in vivo reported studies carried out on

426

healthy volunteers following a single oral dose [26,27,28,30,31,34] or on dogs

427

following intravenous administration [25]. The reported range of T1/2, Cmax and

428

AUC for AR were 0.30–0.47 h, 50–387 ng/ml and 121–2463 ng h/ml, respectively,

429

and for DHA were 0.75–1.69 h, 35–1003 ng/ml and 573–3262 ng h/ml, respectively,

430

following a single oral dose of 20–300mg AR. However, the T1/2 for AR (1h) is more

431

than 2 times compare to the published data, which shows that the nanoemulsion could

Page 20 of 33

slow the elimination rate and prolong the action time of AR in sheep. It is clear from

433

the published data that there is wide inter-individual variation in the pharmacokinetic

434

data. This has been attributed to the biotransformation of AR to its active metabolites,

435

which is greatly affected by the differences in metabolic rates between individuals and

436

between different species[35].

437

5. Conclusion

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The pharmacokinetic analysis of artesunate nanoemulsion relies on a highly

439

sensitive assay, which can determine AR and DHA in plasma after intramuscular

440

injection. The limited volumes of plasma and interference from the biological matrix

441

all add to the complexity of the trace analysis of artesunate. In this study, a rapid and

442

sensitive LC–MS/MS method was developed, validated, and successfully applied to

443

evaluate the pharmacokinetic parameters after intramuscular of artesunate

444

nanoemulsion to sheep. The assay uses AS as the internal standard. The sample

445

preparation is simple and relatively quick. The analysis requires only 100 μL of

446

plasma and a short run time of 8.5 min, which is very advantageous in a

447

pharmacokinetic study. The method has excellent sensitivity, linearity, precision, and

448

accuracy. Currently, tablets are the only available preparation of artesunate for

449

clinical use. This LC–MS/MS assay is an excellent technique with which to further

450

evaluate the pharmacokinetic properties and the therapeutic potential of the new

451

nanoemulsion preparation of the antiparasitic agent artesunate.

452

Acknowledgments

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This work was supported by the earmarked fund for the China Agriculture

Page 21 of 33

454

Research System (cars-38), and the Special Fund for Agro-scientific Research in the

455

Public Interest (No. 201303038-4).

456

References [1] D. Duh, V. Punda-Polic, T. Trilar, T. Avsic-Zupanc, Vet Parasitol, 151(2008) 327-331. [2] H. Yin, G. Guan, M. Ma, J. Luo, B. Lu, G. Yuan, Q. Bai, C. Lu, Z. Yuan, P. Preston, Vet Parasitol,

ip t

107(2002) 29-35. [3] J. Luo, W. Lu, Trop Anim Health Prod, 29(1997) 4S-7S.

[4] G. Uilenberg, Current Topics in Veterinary Medicine and Animal Science, 14(1981) 4-37.

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[5] R.S. Sisodia, Livestock Advisor, 6(1981) 15-19.

[6] Q. Liu, Y.Q. Zhou, G.S. He, M.C. Oosthuizen, D.N. Zhou, J.L. Zhao, Trop Anim Health Prod,

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[7] H. Yin, L. Schnittger, J. Luo, U. Seitzer, J.S. Ahmed, Parasitol Res, 101 Suppl 2(2007) S191-S195.

an

[8] L. Yu, S. Zhang, W. Liang, C. Jin, L. Jia, Y. Luo, Y. Li, S. Cao, J. Yamagishi, Y. Nishikawa, S. Kawano, K. Fujisaki, X. Xuan, J Vet Med Sci, 73(2011) 1509-1512. [9] G.R. Razmi, H. Eshrati, M. Iran, Vet Parasitol, 140(2006) 239-243. [10] T. Dehe, Chinese journal of traditional veterinary science, 4(2010).

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[11] E.J. Reddy, P.S. Rao, M.L. Narasu, J. Biotechnol, 1501(2010) S91.

[12] V. Dhingra, R.K. Vishweshwar, N.M. Lakshmi, Life Sci, 66(2000) 279-300. [13] N.P. Singh, H. Lai, Life Sci, 70(2001) 49-56.

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[14] D. Boulanger, Y. Dieng, B. Cisse, F. Remoue, F. Capuano, J.L. Dieme, T. Ndiaye, C. Sokhna, J.F. Trape, B. Greenwood, F. Simondon, Trans R Soc Trop Med Hyg, 101(2007) 113-116. [15] Y.K. Goo, M.A. Terkawi, H. Jia, G.O. Aboge, H. Ooka, B. Nelson, S. Kim, F. Sunaga, K.

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Namikawa, I. Igarashi, Y. Nishikawa, X. Xuan, Parasitol Int, 59(2010) 481-486. [16] M. Gabriels, J. Plaizier-Vercammen, J. Pharm Biomed Anal, 31(2003) 655-667. [17] H.W.Hu, X.Z.Zhou, .Y.Li , J.Y.Zhang, Journal of Agri.Sci, 36 (2008) 13648-13649, 13652. [18] X.Z.Zhou., J.Y.Zhang, J.Y.Li, J.S.Li, X.J.Wei, J.R.Niu, B.Li, H.W.Hu, Chinese Journal of

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[19] D. Pabbisetty, A. Illendula, K.M. Muraleedharan, A.G. Chittiboyina, J.S. Williamson, M.A. Avery, B.A. Avery, J Chromatogr B Analyt Technol Biomed Life Sci, 889-890(2012) 123-129.

[20] D.Pabbisetty, A.Illendula, K.M. Muraleedharan, A.G.Chittiboyina, J.S.Williamsonb, M.A.Avery, B.A.Avery, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci.889– 890 (2012) 125.

[21] H. Fan, R.Li, Y.Gu, D.Si, C.Liu, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci.889– 890 (2012) 105. [22] V.P. Shah, K.K. Midha, J.W. Findlay, H.M. Hill, J.D. Hulse, I. J. McGilveray, G. McKay, K.J. Miller, R.N. Patnaik, M.L. Powell, A. Tonelli, C.T. Viswanathan, A. Yacobi, Pharm Res, 17(2000) 1551-1557. [23] S.I. Chien, J.C.Yen, R. Kakadiya, C.H.Chen, T.C.Lee, T.L.Su , T.H.Tsai, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci.917– 918 (2013) 62-70. [24] R.S. Miller, Q. Li, L.R. Cantilena, K.J. Leary, G.A. Saviolakis, V. Melendez, B. Smith, P.J. Weina. Malar J, 11(2012) 255.

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[25] Y. Gu, Q. Li, V. Melendez, P.Weina. J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci.867

510 511 512 513 514 515 516 517 518

[32] K.T. Batty, T.M. Davis, L.T.Thu , T. Q. Binh, T.K. Anh , K. F. Ilett , J. Chromatogr. B Biomed. Appl. 677(2) (1996) 345.

(2008) 213–218. [26] C.S. Lai, N.K. Nair, S.M. Mansor, P.L. Olliaro, V. Navaratnam, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 857 (2007) 308 [27] K. N.Bangchang, K. Congpuong, , L.N. Hung, P. Molunto, J. Karbwang., J. Chromatogr. B. Biomed. [28] C.S. Lai, N.K. Nair, S.M. Mansor, P.L. Olliaro b, V. Navaratnam. J. Chromatogr. B. Biomed.Sci. Appl. 877 (2009) 558–562.

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[29] V. Navaratnam, M.N. Mordi, S.M.Mansor. J. Chromatogr. B Biomed. Sci. Appl. 692 (1) (1997) 157.

[30] S.S. Mohamed , S.A. Khalid , S.A. Ward , T.S.M. Wan , H.P.O. Tang , M. Zheng ,,R.K. Haynes ,

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G. Edwards. J. Karbwang., J. Chromatogr. B. Biomed.Sci. Appl. 731 (1999) 251–260.

[31] H.Naik, D.J. Murry, L.E. Kirsch, L. Fleckenstein. J. Chromatogr. B: Analyt. Technol. Biomed.

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Life Sci.816 (2005) 233–242.

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[33] W. Hanpithakponga, B. Kamanikoma, A.M. Dondorpa,b, P. Singhasivanona, N.J.Whitea,b, N.P.J. Daya,b, N. Lindegardha,b. J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 876 (2008) 61–68. [34] Y.Liua, X.Zenga,, Y.H.Denga, L.Wangb, Y.Fenga, L. Yanga, D. Zhou. Journal of Chromatography B, 877 (2009) 465–470.

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[35] S. Parikh, J.-B. Ouedraogo, J.A. Goldstein, P.J. Rosenthal, D.L. Kroetz, Clin. Pharmacol. Ther. 82 (2007) 203.

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519 520

Figure legends:

522

Fig.1 Chemical structures of artesunate, artemisinin and dihydroartemisinin.

523

Fig.2 Full mass spectra scan for artesunate (A), dihydrortemisinin (B), and internal

524

standard artemisinin (C); product ions for artesunate (D), dihydrortemisinin (E), and

525

internal standard artemisinin (F).

526

Fig.3 Chromatograms(A: blank plasma, B: chromatograms of artesunate,

527

dihydroartemisinin, and internal standard artemisinin in plasma).

528

Fig.4 Calibration curve.

529

Fig.5 Mean plasma concentration–time profile after intramuscular of

530

artesunate nanoemulsion to sheep (n=12) at 5 mg/kg.

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531

Page 23 of 33

531

Table 1. Intra and inter day precision and accuracy of AR and DHA (n=6) in sheep

532

plasma

533

DHA

534

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535

Inter-day precision and accuracy (n=6) Accuracy (%) ±SD RSD (%) 92.1±2.9 3.1 92.5±2.5 2.7 96.9±2.2 2.3 98.9±2.3 2.3 93.5±2.9 3.1 94.7±2.3 2.4 101. 6±2.6 2.6 98.5±1.3 1.3

cr

AR

1 4 100 400 1 4 100 400

Intra-day precision and accuracy (n=6) Accuracy (%) ±SD RSD (%) 91.6±2.4 2.6 93.5±2.5 2.7 102.3±2.2 2.3 98.3±1.3 1.3 91.2±2.1 2.3 95.6±1.7 1.8 96.4±2.7 2.8 99.1±1.5 1.5

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concentration (ng/mL)

Page 24 of 33

Table 2. Recovery of AR and DHA (n=6) from sheep plasma

535 536

Recovery (%, n=6)a

a

95.0±2.2 94.5±2.7 97.7±2.6 92.9±3.1 95.1±2.2 93.4±2.3

2.3 2.9 2.7 3.3 2.3 2.5

ip t

RSD (%)b

Recovery = ratio of response of spiked standard before extraction to that after extraction.

b

RSD, relative standard deviation.

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537 538 539

Mean ± SD

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Spiked Concentration (ng/mL) 4 AR 100 400 4 DHA 100 400

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Page 25 of 33

540

Table 3. Stability of AR in sheep plasma samples under various conditions (n=6)

541

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542

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544

1.2 2.4 2.7 1.3 2.4 2.0 1.3 3.8 2.7 1.0 2.7 3.3 1.4 2.0 2.0

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543

RSD (%)

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Accuracy(%) ± SD 90.1±1.1 94.3±2.3 95.4±2.6 93.5±1.2 104.3±2.5 97.4±1.9 92.1±1.2 93.3±3.5 94.8±2.6 93.5±0.9 93.6±2.5 94.8±3.1 97.6±1.4 103.4±2.1 96.4±1.9

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Concentration (ng/mL) 4 Three freeze-thaw cycles 100 400 4 At room temperature for 24 h 100 400 4 At 20 °C for 60 days 100 400 At 4 °C in the autosamplerfor 4 24 h 100 400 At 4 °C for 1 month 4 100 400 Storage conditions

Page 26 of 33

544

Table 4. Stability of DHA in sheep plasma samples under various conditions (n=6)

545

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546

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1.6 2.7 2.4 0.9 2.3 3.4 1.7 2.4 3.2 1.6 3.4 2.5 1.1 3.3 2.8

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547

RSD (%)

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Accuracy(%) ± SD 89.6±1.4 93.2±2.5 94.4±2.3 94.9±0.9 96.1±2.2 97.2±3.3 92.8±1.6 93.1±2.2 93.6±3.0 93.6±1.5 93.2±3.2 96.7±2.4 102.3±1.1 96.2±3.3 97.0±2.7

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Concentration (ng/mL) 4 Three freeze-thaw cycles 100 400 4 At room temperature for 24 h 100 400 4 At 20 °C for 60 days 100 400 At 4 °C in the autosamplerfor 4 24 h 100 400 At 4 °C for 1 month 4 100 400 Storage conditions

Page 27 of 33

547

Table 5. Pharmacokinetic parameters of AR and DHA after intramuscular injection

548

(n=12) Mean±SD

Parameter

AR

DHA

195.8±27.8

168.2±20.0

0.69±0.08 158.9±18.3 30±1.0 1.0±0.1 0.10±0

0.63±0.04 137.5±27.4 30±2.0 1.1±0.1 0.1±0

cr

AUC (ng·h/mL) b K (1/h) c Cmax (ng/mL) d CL/F (L/h/kg) e t1/2 (h) f Vss (mL/kg)

ip t

a

a

AUC, area under the concentration–time curve.

550

b

K, elimination rate constant.

551

c

Cmax, peak plasma concentration.

552

d

CL/F, plasma clearance.

553

e

t1/2, elimination half life.

554

f

Vss, apparent volume of distrib.

an

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Figure1

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Figure2

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Figure3

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Figure4

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Figure5

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Determination and pharmacokinetic studies of artesunate and its metabolite in sheep plasma by liquid chromatography-tandem mass spectrometry.

A rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneous qu...
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