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Molecules 2010, 15(12), 8747-8768;

A Simplified Liquid Chromatography-Mass Spectrometry Assay for Artesunate and Dihydroartemisinin, Its Metabolite, in Human Plasma

Department of Immunology and Medicine, United States Army Medical Component, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok 10400, Thailand
Department of Pharmacology, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA
Author to whom correspondence should be addressed.
Received: 18 October 2010 / Revised: 13 November 2010 / Accepted: 27 November 2010 / Published: 1 December 2010
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Artesunate (AS) is a potent antimalarial that is used worldwide for the treatment of malaria. A simple method with a total run time of 12 min was developed and validated for the quantification of AS and dihydroartemisinin (DHA), its active metabolite, in human (heparinized) plasma based on one-step protein precipitation in acetonitrile using artemisinin (ARN) as an internal standard, followed by liquid chromatography with a single quadrupole mass spectrometry system connected to a C18 column. Peak area ratio responses were fitted to the 2nd-order curve type, polynomial equation with weighting (1/concentration) over a quantification range between 3.20/5.33–3,000/5,000 nM (1.23/1.52–1153/1422 ng/mL) of AS/DHA showing linearity with very good correlation (r2 > 0.999). Single ion recordings of 5 µL injections of plasma extracts allowed for limits of detection of 1.02 nM (0.39 ng/mL) for AS and 0.44 nM (0.13 ng/mL) for DHA. The inter-assay and intra-assay accuracy and precision of the method was very good with an inaccuracy of ±12.4% and coefficients of variation of ≤10.7% at all tested concentrations. The recovery of the analytes from plasma was ≥95%. Other commonly used antimalarials including mefloquine, quinine, and chloroquine, did not interfere with the analysis. Post-preparative tests over 24 h in an autosampler (10 °C) showed that the DHA response was only 2.1% of AS from auto-hydrolysis, and β-DHA was the major, stable epimer that was used for quantification of DHA. In contrast, α-DHA increased steadily up to 600%. Artesunate and DHA in plasma were stable through three freeze/thaw cycles for up to 6 h at room temperature and up to one year at -80 °C.
Keywords: artesunate; dihydroartemisinin; human plasma; method validation; LC-MS artesunate; dihydroartemisinin; human plasma; method validation; LC-MS
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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Teja-Isavadharm, P.; Siriyanonda, D.; Siripokasupkul, R.; Apinan, R.; Chanarat, N.; Lim, A.; Wannaying, S.; Saunders, D.; Fukuda, M.M.; Miller, R.S.; Weina, P.J.; Meléndez, V. A Simplified Liquid Chromatography-Mass Spectrometry Assay for Artesunate and Dihydroartemisinin, Its Metabolite, in Human Plasma. Molecules 2010, 15, 8747-8768.

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