title: A high-performance liquid chromatography-tandem mass spectrometry method for the determination of artemether and dihydroartemisinin in human plasma.
abstract: A liquid chromatography-tandem mass spectrometry method is described for the quantitative determination of artemether (ART) and its metabolite dihydroartimisinin (DHA) in human plasma samples. Quantitation of ART and DHA in plasma is challenging due to the presence of malaria related hemolytic products in patient plasma causing degradation of the compounds when organic solvents are used during sample processing. Furthermore, both compounds consist of two epimeric forms that can interconvert both in solution and during chromatographic separation, an effect that is dependent on temperature and solvent properties and needs to be taken into account. This method utilizes micro-elution solid-phase extraction as sample preparation technique to minimize the need for organic solvents. Reversed-phase HPLC using a C18 50 × 2.1mm column with 3.5 μm particles and a mobile phase of acetonitrile:water (30:70, v/v), followed by a step gradient at 90% acetonitrile, is applied to separate ART from DHA and matrix interferences within a run time of 4 min. Chromatographic conditions were optimized to allow analyte quantitation independent of the (unknown) ratio of the epimers in the injected sample. A triple quadruple mass spectrometer equipped with an atmospheric pressure chemical ionization interface in positive mode was used for detection in order to detect all epimeric forms. The method proved to be linear over a concentration range of 1.00-1,000 ng/mL using 50 μL of plasma. Accuracy and precision were within 15% for bias and CV (20% at the lower limit of quantification). ART and DHA were stable (bias <15%) in plasma for 211 days after storage at -20°C and -70°C, 17 h on melting ice and 2h at room temperature. Furthermore, both compounds were stable in whole blood after storage for 2h on melting ice and at room temperature and after five freeze/thaw cycles. The method was successfully used for the analysis of pharmacokinetic samples originating from a drug-drug interaction study in which the antimalarial drugs artemether/lumefantrine were coadministrated etravirine or darunavir/ritonavir in healthy human immunodeficiency virus (HIV)-negative subjects.
authors.affiliation: PRA Early Development Services, Westerbrink 3, 9405 BJ Assen, The Netherlands. Electronic address: hilhorstmartijn@praintl.com.
authors.forename: M J
authors.initials: MJ
authors.lastname: Hilhorst
authors.affiliation: PRA Early Development Services, Westerbrink 3, 9405 BJ Assen, The Netherlands.
authors.forename: G
authors.initials: G
authors.lastname: Hendriks
authors.affiliation: Janssen Research and Development, a Division of Janssen Pharmaceutica N.V., Turnhoutseweg 30, B-2340 Beerse, Belgium.
authors.forename: R
authors.initials: R
authors.lastname: de Vries
authors.affiliation: Janssen Research and Development, a Division of Janssen Pharmaceutica N.V., Turnhoutseweg 30, B-2340 Beerse, Belgium.
authors.forename: V
authors.initials: V
authors.lastname: Hillewaert
authors.affiliation: Janssen Research and Development, a Division of Janssen Pharmaceutica N.V., Turnhoutseweg 30, B-2340 Beerse, Belgium.
authors.forename: T
authors.initials: T
authors.lastname: Verhaege
authors.affiliation: PRA Early Development Services, Westerbrink 3, 9405 BJ Assen, The Netherlands; Department of Analytical Biochemistry, Centre for Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.
authors.forename: N C
authors.initials: NC
authors.lastname: van de Merbel
mesh_terms: D000077549:Artemether; D037621:Artemisinins / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D016014:Linear Models; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D037621:Artemisinins; C039060:artenimol; D000077549:Artemether
keywords: Artemether; Degradation by malaria related hemolytic products; Dihydryartemisinin; Epimerization; LC–MS/MS; Validation