Identification of thein vivo metabolites of the antimalarial arteether by thermospray high-performance liquid chromatography/mass spectrometry

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Abstract
The thermospray mass spectra of arteether and 16 of its potential metabolites all showed strong [M + NH4]+ ions and with only a few exceptions these compounds also showed spectral peaks corresponding to [M + NH4 − HOR]+ and [M + H − HOR]+, where OR represents the alkoxy or hydroxy group at the 12-position. A method for quantifying the metabolites was developed in which the plasma was spiked with an internal standard (the propyl ether analog of arteether), extracted using a C-18 solid-phase cartridge, then subjected to thermospray high-performance liquid chromatographic/mass spectrometric analysis using selected ion monitoring and a C-18 reversed-phase analytical column. Following the intravenous administration of arteether (11.6 mg kg−1), the plasma was found to contain 12 metabolites of arteether in the 10–1000 ng ml−1 range 15 min post-injection, and within 60 min two of these metabolites attained higher concentrations than that of the parent compound, while several other of the metabolites attained concentrations similar to the parent compound. The pseudo-first-order half-life of arteether was found to be 10.0 ± 0.6 min, while the apparent half-lives of most of the metabolites were in the 15–30 min range. Nine of these metabolites were identified by comparison to authentic reference standards and the structures of three remaining metabolites were tentatively assigned from their spectral and chromatographic properties. The metabolic pathways leading to these 12 metabolites was a rather complex, multiple-step process, but most of the metabolites arose from an enzymatic oxidation at one of three sites; 3α, 9α, or the CH2 of the side-chain. Conversion of the endoperoxide group to an cyclic ether was not a major pathway. The in vitro antimalarial activity of reference standards of several of the metabolites was determined and all of those tested were found to be active in the low nanogram per milliliter range.