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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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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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,
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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
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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
280
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
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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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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. 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
318
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
341
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
not
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
ip t
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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348
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
Ac ce
358
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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366
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
ip t
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
cr
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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cr
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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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424
an
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
cr
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432
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
453
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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.
cr
[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,
us
42(2010) 109-114.
[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).
M
[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.
ed
[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.
pt
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
Ac ce
457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495
Veterinary Medicine. 46(2010) 54-56.
[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.
Page 22 of 33
496 497 498 499 500 501 502 503 504 505 506 507 508 509
[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.
ip t
[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 ,
cr
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.
us
Life Sci.816 (2005) 233–242.
an
[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.
M
[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.
us
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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540
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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pt Ac ce
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
ed
543
RSD (%)
cr
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
us
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
an
M
546
ip t
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
Ac ce
pt
ed
547
RSD (%)
cr
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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555
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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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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.
1
Highlights
2
1. A LC-MS/MS method determining artesunate and its metabolite in sheep plasma.
3
2. A MRM method was employed with an ESI source in positive mode. 3. The method is simple, sensitive and accurate.
5 6
4. Successful study on the pharmacokinetics of artesunate nanoemulsion in the sheep.
7
5. Artesunate nanoemulsion has an average emulsion droplet particle size of 60 nm.
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4
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Page 1 of 33
8
Determination and pharmacokinetic studies of artesunate and its
9
metabolite in sheep plasma by liquid chromatography-tandem mass
10
spectrometry
11
Bing Li
12
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
13
a,b,c,*
15
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cr
a
, 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,
us
14
a,b,c
Lanzhou, China b
Key Laboratory of New Animal Drug Project of Gansu Province, Lanzhou, China
17
c
Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS,
19
d
Fulin Animal Science and Veterinary Medicine Officer, Chongqing 408000, China
23 24
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22
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20 21
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Lanzhou 730050 , China
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18
an
16
25
Corresponding author: Ji-Yu Zhang, Lanzhou Institute of Husbandry and
26
Pharmaceutical Sciences of CAAS, Lanzhou 730050, China
27
Phone and E-mail:+86013893612415,[email protected]
28
Fax: +86-931-2115191
29
Postal address: 335 Jiangouyan,Qilihe,Lanzhou, 730050,Gansu,China
30 31
Page 2 of 33
Abstract
33
A rapid and sensitive high-performance liquid chromatography–tandem mass
34
spectrometry (LC–MS/MS) method was developed and validated to simultaneous
35
quantify artesunate and its metabolite in sheep plasma. The plasma samples were
36
prepared by liquid-liquid extraction. Chromatographic separation was achieved on a
37
C18 column (250×4.6mm, 5 μm) using methanol: water (60:40, v/v) (the water
38
included 1mM ammonium acetate, 0.1% formic acid, and 0.02% acetic acid) as the
39
mobile phase. Mass detection was carried out using positive electrospray ionization in
40
multiple reaction monitoring mode. The calibration curve was linear from 1 ng/mL to
41
400 ng/mL (r2=0.9992 for artesunate, r2=0.9993 for its metabolite). The intra-day and
42
inter-day accuracy and precision were within the acceptable limits of ±10% at all
43
concentrations for both artesunate and its metabolite. The recoveries ranged from 92
44
to 98% at the three concentrations for both. In summary, the LC–MS/MS metho
45
described herein was fully successfully applied to pharmacokinetic studies of
46
artesunate nanoemulsion after intramuscular delivery to sheep.
47
Keywords:
48
Antiparasitic; Artesunate nanoemulsion; LC/MS/MS; Pharmacokinetics;
49
Sheep plasma
51
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55
1．Introduction Theileriosis, caused by various intraerythrocytic protozoan parasites of the genus
57
Theileria, is a tick-borne disease of domestic and wild animals. Theileriosis, also
58
known as blood cryptosporidiosis, is a blood protozoosis caused by parasites to infect
59
leukocytes and erythrocytes [1], is a tick-borne disease of domestic and wild animals
60
[2] . This epidemic disease is causing serious damage to the cattle and sheep
61
industries. It is strongly seasonal and local, and present in many countries, research
62
carried out over many years has shown these to be distributed mainly in Northern
63
China but also sometimes in Southern China. [3]. Theileria lestoquardi is a highly
64
pathogenic parasite of sheep and goats., which occurred in south-eastern Europe,
65
northern Africa, western and central Asia [4], India [5] and in West, Central and North
66
China such as Qinghai, Gansu, Hubei, Liaoning and Inner Mongolia [6,7,8].
67
Non-pathogenic or mildly pathogenic Theileria spp. of small ruminants include
68
Theileria separata, Theileria ovis and Theileria recondite [9]. Clinical symptoms in
69
sick sheep include high fever, depression, anemia, conjunctival yellowish
70
discoloration, superficial swollen lymph nodes, limb rigidity, difficulty walking, rapid
71
weight loss, weakness, and finally failure and death [10]. Treatment of the disease is
72
problematic, as there are few effective medicines. Despite the large number of studies
73
on this disease, efficient new drugs are still lacking. The drugs presently in use are
74
mainly berenil, chloroquine phosphate, artesunate, uranidin, and imidocarb. Berenil is
75
the most commonly used, but its popularity has lessened due to the major side effects
76
that can accompany its injection.
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Page 4 of 33
Artesunate is a semi-synthetic derivative of artemisinin, extracted from the plant
78
species of Artemisia. Artesunate is a highly efficacious anti-malarial [11,12] and also
79
has anti-tumor activity [13]. It is also effective for treating some non-malarial
80
parasites [14,15], for example, it is an effective therapeutic for Theileria annulata
81
infection of cattle and sheep. However, artesunate still presents a few problems,
82
including limited bioavailability, a short half-life, and also poor water solubility [16],
83
which makes it difficult to deliver effectively to the lesion site and intracellular space.
84
Further, at present there is only an oral tablet and bulk drugs for clinical use. Also, the
85
poor stability of the drug's sodium salt causes its low bioavailability and necessitates
86
frequent patient medication, leading to poor tolerance and efficacy, and greatly
87
limiting its clinical application. New preparations of derivatives with better
88
pharmacokinetic profiles are needed to overcome these issues.
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In our previous study, we have successfully prepared artesunate nanoemulsion
90
for the first time in China as an intramuscular preparation that increases the solubility
91
of artesunate and its sodium salt. It has a clear, transparent appearance, an average
92
emulsion droplet particle size of 60 nm, a good degree of dispersion, thermodynamic
93
stability, storage stability, and long-placing non-hierarchical[17]. Acute toxicity
94
studies of oral artesunate nanoemulsion in mice indicate that it is nontoxic [17].
95
Artesunate nanoemulsion has significant preventive and treatment effects in Taylor
96
piroplasmosis of goats [18].
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89
97
In order to define the pharmacokinetic profile of artesunate nanoemulsion, an high
98
performance liquid chromatography–tandem mass spectrometry (LC–MS/MS)
Page 5 of 33
method for the determination of artesunate and its metabolite in sheep plasma needs
100
to be established. Because the artemisinin and its analogues contains no conjugated
101
groups in the structure (Fig. 1), they have no appropriate chromophores for use in
102
characterization techniques their quantitation sometimes requires the use of lengthy
103
derivatization techniques, and these derivatizing conditions are not necessarily stable
104
for all artemisinin analogs and can cause the parent compound to decompose [19].
105
Hence, we chose LC-MS/MS as an analytical method that is accurate and sensitive,
106
using a simple liquid-liquid extraction as the organic phase is evaporated and
107
reconstructed before analysis approach for artesunate nanoemulsion. This method has
108
been successfully applied to characterize the pharmacokinetics of artesunate
109
nanoemulsion in sheep following intramuscular administration at 5 mg/kg.
cr
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Fig. 1 Chemical structures of Artesunate, Artemisinin, and
ed
110
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Dihydroartemisinin.
111
2. Experimental
113
2.1 Reagents and chemicals
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114
pt
112
Artesunate (AR), dihydroartemisinin (DHA), and internal standard (IS)
115
artemisinin (AS) were provided by the National Institute for the Control of
116
Pharmaceutical and Biological Products (Beijing, China) with the batch numbers of
117
100200-200202, 100184-200401, and 100202-200603 respectively, the purity of AR,
118
DHA and IS are >99%. Acetonitrile and methanol (HPLC grade) were purchased from
119
Fisher Chemical (Waltham, USA). Acetic acid, ethyl acetate, ammonium acetate, and
120
formic acid were analytically pure and were purchased from Sinopharm Group
Page 6 of 33
Chemical Reagent Co., Ltd. (Ningbo, China). Water was purified through a Milli-Q
122
Plus water system (Millipore Corporation, Bedford, MA, USA) before use. Artesunate
123
nanoemulsion (containing artesunate 5%) was supplied by Lanzhou Institute of
124
Animal Science and Veterinary Pharmaceutics.
125
2.2. Equipment
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The LC–MS/MS equipment (1200-6410A) consisted of a LC system with a binary
127
pump-SL and a triple quadrupole mass spectrometer with electrospray ionization(ESI)
128
(Agilent Technologies, Inc., Santa Clara, CA, USA). Data were recorded, and the
129
system was controlled using MassHunter software (version B.01.04, Agilent
130
Technologies).
131
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)
134
(the water included 1mM ammonium acetate, 0.1% formic acid, and 0.02% acetic
135
acid). The mobile phase was filtered before being used to prevent entry of bubbles or
136
impurities in the system. The mobile phase was delivered at a flow rate of 0.4 mL/min
137
and 5 μL was injected at 30 ºC.
pt
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138
ed
133
The mass spectrometer was operated in positive ion mode with an ESI interface.
139
Quantitation was performed by multiple reaction monitoring (MRM). In the positive
140
mode, the MS/MS setting parameters were as follows: capillary voltage 4 kV, cone
141
voltage 40 V, source temperature 100°C, and desolvation temperature 350°C with a
142
desolvation nitrogen gas flow of 11 L/min and a cone gas flow of 9 L/min. The
143
optimized fragmentation voltages for AR、DHA and IS were all 100 V, and the Delta
Page 7 of 33
electron multiplier voltage (EMV) were all 200 V. Data were collected in multiple
145
reaction monitoring (MRM) mode using [M+Na] + ion for all AS、DHA and IS with a
146
collision energy of 25 eV、18 eV and 20 eV, respectively.
147
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
149
respectively placed in separate 10 mL brown volumetric flasks, then methyl alcohol
150
was added to form stock solutions of 200 μg·mL-1. A series of mixture of AR and
151
DHA working standard solutions were prepared by dilutions of the stock solution with
152
methyl alcohol to obtain the following concentrations:
153
400 ng·mL-1.
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M
an
0.1, 0.2, 1, 4, 20, 100, 200,
Internal standard solution: Precisely 4 mg of AR was weighed and placed in a 50
155
mL brown volumetric flask, to which methyl alcohol was then added, forming a
156
solution of 80μg·mL-1. Then, 2.5 mL of that solution was placed in a 50 mL
157
volumetric flask, followed by methyl alcohol to make a 4 μg·mL-1 internal standard
158
solution.
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159
ed
154
All of the solutions were stored at 4 ºC and brought to room temperature before
160
use. Plasma calibration standards of 1–400 ng/mL were prepared by spiking 100 μL
161
aliquots of blank plasma with 10 µL of each of the standard solutions. Quality control
162
(QC) samples were prepared in the same way, with four levels of 4(QC-low), 100
163
(QC-med), 400 (QC-high), and 1 ng/mL (QC-LLOQ). Both the calibration standard
164
and the QC samples were applied in the method validation and the pharmacokinetic
165
study.
Page 8 of 33
166
2.5 Sample preparation Plasma aliquots (100 μL) were spiked with 10 µL of methanol and artemisinin (10
168
μL of 4 μg/mL solution) as an internal standard in centrifuge tubes (when preparing
169
calibration and quality control (QC) samples, the standard solution was added instead
170
of methanol), and mixed. The centrifuge tubes were initially primed with 200 μL of
171
ethyl acetate, followed by vortex concussion for 1 min and centrifugation for 10 min
172
at the speed of 12000 rpm. The organic phase was evaporated by use of a stream of 25
173
ºC nitrogen. The residue was reconstituted with 100 μL of methanol and immediate
174
vortex concussion for 20s, placed for 10 min, and injected into the LC–MS/MS
175
system after filtration through a 0.45 μm Millipore filter.
176
2.6 Validation
M
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This method was validated in terms of linearity, specificity, LLOQ, recovery,
178
intra- and inter-day variation, accuracy and precision, and stability of the analyte
179
during sample storage and processing procedures.
180
2.6.1 Selectivity
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181
ed
177
Selectivity was evaluated by comparing the chromatograms of six different
182
batches of the blank plasma with the corresponding standard plasma samples spiked
183
with AR and DHA and the internal standard [19].
184
2.6.2 Linearity and LLOQ
185
A calibration curve was constructed from plasma standards at six concentrations
186
of AR and DHA, ranging from 1 ng/mL to 400 ng/mL. A calibration curve was
187
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.
189
The correlation coefficient and linear regression equation were used for the
190
determination of the analyte concentration in the samples. A weighted (1/X2) linear
191
least-squares regression was used as the mathematical model. The LLOQ was
192
determined as the lowest concentration that produced an S/N of 5 [20]. The limit of
193
detection (LOD) was determined as the lowest concentration that produced an S/N of
194
3 [20].
195
2.6.3 Accuracy and precision
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188
Intra-day accuracy and precision were evaluated by analyzing the QC
197
concentrations at four levels (4, 100, 400 ng/mL and 1 ng/mL; Table 1) with six
198
determinations per concentration on the same day. The inter-day accuracy and
199
precision were evaluated by the analysis of the QC concentrations at four levels (4,
200
100, 400 ng/mL, and 1 ng/mL; Table 1) with six determinations per each
201
concentration over 3 days. Precision and accuracy were based on the criterion that the
202
relative standard
203
15%, except for LLOQ (not to be more than 20%) [21].
204
2.6.4 Recovery and matrix effect
M
ed
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deviation (RSD) for each concentration should be not more than
Ac ce
205
an
196
The recovery was determined in quadruplicate by comparing processed QC
206
samples at three levels (low, med, high) with reference solutions in blank plasma
207
extract at the same levels.
208
The matrix effect was determined by comparing the peak areas of the
209
post-extracted spiked sample with those of the standards containing equivalent
Page 10 of 33
210
amounts of the AS and DHA prepared in the mobile phase, respectively. The
211
experiments were performed at the three levels in six different batches.
212
2.6.5 Stability [22] The stabilities of AR and DHA in sheep plasma were assessed by analyzing
214
replicates (n=6) of QC samples at concentrations of 4, 100, and 400 ng/mL during the
215
sample storage and processing procedures. Freshly prepared stability-QC samples
216
were analyzed by using a freshly prepared standard curve for the measurement for all
217
stability studies. The stabilities of stock solutions of AR and DHA were analyzed at
218
room temperature for 24 h and at 4 ºC after 1 month. The short-term stability was
219
assessed after exposure of the plasma samples to ambient temperature for 24 h. The
220
long-term stability was assessed after storage of the plasma samples at -20 ºC for 60
221
days. The freeze/thaw stability was determined after three freeze/thaw cycles (room
222
temperature to -20 ºC). The sample stability in the autosampler tray was evaluated at 4
223
ºC for 24 h; this sample stability evaluation imitates the residence time of the samples
224
in the autosampler for each analytical run.
225
2.6.6 Formulation
227
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226
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213
The injection of artesunate nanoemulsion was prepared as follows: The component substances for 6% ethyl oleate, 24% Tween-80, 11.5% n-butanol,
228
53.5% ultra-pure water, 5% artesunate were weighed. The ethyl oleate, Tween-80,
229
n-butanol, and artesunate were combined and stirred under ambient conditions until
230
the drug was dissolved. Ultrapure water was slowly added, dropwise, to the mixture
231
with stirring. At a certain concentration of water, the system becomes a clarified,
Page 11 of 33
232
translucent, pale yellow O/W type intravenous-formulation artesunate nanoemulsion.
233
The intravenous formulation was administered to sheep (n=12) at a dose of 5 mg/kg.
234
2.7 Animal studies The assay method described above was used to study the pharmacokinetics of
236
artesunate nanoemulsion in sheep plasma after intramuscular administration. All the
237
experimental procedures were approved and performed in accordance with the
238
Guidelines for the Care and Use of Laboratory Animals of the Lanzhou Institute of
239
Animal Science and Veterinary Pharmaceutics. Healthy Small Tail Han sheep
240
(35±1.27 kg) were obtained from the Small Tail Han sheep-breeding base (Kangle,
241
Gansu, China). These sheep were housed in a standard, environmentally controlled
242
animal room (temperature: 25±2 ºC, humidity: 50±20%) with a natural light-dark
243
cycle for 1 week before the experiment. The sheep were fasted for 12 h before dosing
244
but allowed free movement and access to water during the whole experiment. All
245
sheep (n=12) were dosed at a dosage of 5 mg/kg. After a single dose was administered
246
by intramuscular administration, blood samples (3 mL) were collected in heparinized
247
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,
248
20, and 24 h. After all blood samples were centrifuged at 12,000 rpm for 10 min, the
249
plasma samples were collected, and then immediately stored in a -20 ºC freezer until
250
analysis by LC–MS/MS.
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235
251
The pharmacokinetic parameters were calculated by use of WinNonlin
252
professional software version 5.2 (Pharsight, Mountain View, CA, USA). A
253
compartmental model was utilized for data fitting and parameter estimation.The
Page 12 of 33
essential pharmacokinetic model was confirmed by Akaike Information Criterion
255
(AIC)[23] for the best characterization. Plasma AUC, plasma clearance (CL/F), peak
256
plasma concentration (Cmax), elimination rate constant(K) and apparent volume of
257
distribution (Vss) were all obtained from observed data. Half-life (t1/2) was calculated
258
directly according to the pharmacokinetic parameters.
259
3. Results and discussion
260
3.1 Mass spectrometric detection
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254
In order to optimize positive ESI mode conditions, AR, DHA, and IS were
262
dissolved in methanol, and then infused into the mass spectrometer for scans in
263
positive ion mode. When AR, DHA, and IS were injected directly into the mass
264
spectrometer along with the mobile phase, the analytes yielded predominantly [M+Na]
265
+ 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
266
of the precursor ions was subjected to collision-induced dissociation to determine the
267
resulting product ions from the product ion mass spectra. The most abundant and
268
stable fragment ions were generated at m /z 261.1 for AR, m /z 163.0 for DHA, and m
269
/z 151.1 for IS. Thus, the mass transitions chosen for quantitation were m /z 407.2 to
270
261.10 for AR, m /z 307.2 to 163.0 for DHA, and m /z 305.2 to 151.1 for IS.
271
Fig. 2 Full mass spectra scan for artesunate (A), dihydrortemisinin (B), and internal
272
standard artemisinin (C); product ions for artesunate (D), dihydrortemisinin (E), and
273
internal standard artemisinin (F)
274
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261
3.2 Chromatographic separation
275
High-performance liquid chromatography with MS/MS separations was run using
276
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
278
mobile phase for 8.5 min was used for the chromatographic separation. A mobile
279
phase consisting of a mixture of methanol: water (the water included 1 mM
280
ammonium acetate, 0.1% formic acid, and 0.02% acetic acid) was found to be suitable
281
for the separation and ionization of AR, DHA, and the internal standard. Formic acid
282
was found to increase the ionization of all three compounds. Under optimized LC and
283
MS conditions, AR, DHA, and IS were separated with retention times of 5.45, 6.21,
284
and 8.12 min, respectively, and the endogenous substances in plasma does not
285
interfere with target detection material(Fig.3).
an
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277
Fig. 3 Chromatograms
287
(A: blank plasma, B: chromatograms of artesunate-a, dihydroartemisinin-b, and
M
286
289
3.3 Sample preparation
ed
internal standard artemisinin-c in plasma)
288
Recoveries of AR and DHA using methanol (14%), acetonitrile (12%), and
291
chloroform (46%) were found to be less than that from ethyl acetate (95%). Therefore,
292
ethyl acetate was selected as the extraction solvent for plasma due to its high recovery
293
and sensitivity.
294
3.4 Optimization of the intramuscular preparations
Ac ce
295
pt
290
The traditional tablets of artesunate cannot overcome the first-pass effect of the
296
liver. Further, as artesunate has poor solubility in water, it is difficult to deliver it
297
effectively to the lesion and intracellular space. In addition, the poor stability of its
298
sodium salt, which causes low bioavailability, necessitates frequent patient medication,
299
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
301
and its sodium salt. A composition of 6% ethyl oleate, 24% Tween-80, 11.5%
302
n-butanol, 53.5% ultra-pure water, and 5% artesunate was chosen to formulate an
303
artesunate nanoemulsion for intramuscular administration, which was proved to be
304
safe.
305
3.5 Method validation
306
3.5.1 Selectivity
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300
The specificity of the method was evaluated by analyzing individual blank plasma
308
samples from six different sources. All samples were found to have no interference
309
from endogenous substances affecting the retention times of AR, DHA, and IS. There
310
was good base-line separation of AR, DHA, and the IS extracted from the sheep
311
plasma. Representative chromatograms of blank plasma, blank plasma spiked with
312
AR, DHA, and the IS are shown in Fig. 3.
313
3.5.2 Linearity and LLOQ
M
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A calibration curve was constructed from plasma standards at six concentrations
Ac ce
314
an
307
315
of AR and DHA, ranging from 1 ng/mL to 400 ng/mL. The ratio of peak areas of AR
316
and DHA to that of the IS was used for quantification. The calibration model was
317
selected based on the analysis of the data by linear regression with intercepts and a
318
1/X2 weighting factor. A typical equation of the calibration curve for AR was:
319
y=0.0130x-0.3212 (r2=0.9992), where y is the peak-area ratio of AR to IS and x is the
320
plasma concentration of AR. A typical equation of the calibration curve for DHA was:
321
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
323
limits of quantitation (LLOQ) for AR and DHA were both established at 1 ng/mL,
324
with an accuracy of 98.5%. The LOD was found to be 0.1 ng/mL of AR and 0.2
325
ng/mL of DHA. Fig. 4 Calibration curve
326
3.5.3 Accuracy and precision
cr
327
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322
The intra-day and inter-day precision and accuracy of QC samples (4, 100, 400
329
ng/mL and 1 ng/mL) is summarized in Table 1. These data demonstrate that the
330
current method has satisfactory accuracy, precision, and reproducibility for the
331
quantification of AR and DHA in sheep plasma.
an
Table 1.
M
332
us
328
Intra and inter day precision and accuracy of AR and DHA (n=6) in sheep plasma
334
ed
333
3.5.4 Recovery and matrix effect
The mean extraction recoveries of AR were (95.0±2.28)%, (94.5±2.77)%, and
336
(97.7±2.69)% at the concentrations of 4, 100, and 400 ng/mL, respectively, and the
337
mean extraction recoveries of DHA were (92.9±3.11)%, (95.1±2.23)%, and
338
(93.4±2.31)% at the three concentrations of 4, 100, and 400 ng/mL, respectively. The
339
mean extraction recovery of the IS was 96.3±2.2% at 200 ng/mL. These results
340
suggest
341
concentration-dependent. Recovery values are listed in Table 2.
342 343
Ac ce
pt
335
that
the
Table 2.
recovery
of
AR
and
DHA was
consistent
and
not
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
ip t
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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cr
348
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
Ac ce
358
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.
Ac ce
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366
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
ip t
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.
us
an
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pt
Ac ce
403
cr
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]).
us
cr
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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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ed
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The present pk properties obtained for AR and DHA in this study is in agreement
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an
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
cr
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432
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
453
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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,
us
42(2010) 109-114.
[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).
M
[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.
ed
[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.
pt
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
Ac ce
457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495
Veterinary Medicine. 46(2010) 54-56.
[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.
Page 22 of 33
496 497 498 499 500 501 502 503 504 505 506 507 508 509
[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.
ip t
[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.
us
Life Sci.816 (2005) 233–242.
an
[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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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
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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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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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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
us
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
an
M
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
Ac ce
pt
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547
RSD (%)
cr
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
us
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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