Quantitative Analysis of Artemether and its Metabolite Dihydroartemisinin in Human Plasma by LC with Tandem Mass Spectrometry

Abstract

We report the development and validation of a high-performance liquid-chromatographic–tandem mass spectrometric method for determination of artemether (ARM) and its active metabolite dihydroartemisinin (DHA) in human plasma; artemisinin was used as internal standard (IS). Chromatographic separation was performed on a 150 mm × 4.6 mm i.d., 5 μm particle, C₁₈ column coupled with a 4.0 mm × 3.0 mm i.d., 5 μm particle, C₁₈ guard column. The mobile phase was acetonitrile–0.1% formic acid solution, 80:20 (v/v), at a flow-rate of 1 mL min⁻¹. An atmospheric-pressure chemical-ionization (APCI) interface was used to produce sample ions, and positive ions were quantified by using the MS detector in selected-reaction-monitoring mode, using the reaction m/z 221 to 163 for determination of ARM and DHA and the reaction m/z 283 to 219 for determination of the IS. Plasma samples were prepared by extraction with methyl t-butyl ether, evaporation of the extract to dryness, and reconstitution of the residue with mobile phase. Extraction recovery for ARM and DHA ranged from 74.74 to 99.39%. High specificity and a limit of quantification of 5 ng mL⁻¹ were achieved for ARM and DHA. Linearity was confirmed over the concentration range 5–500 ng mL⁻¹; the correlation coefficients (R) were >0.99. The relative standard deviation for intra-day and inter-day assay of both compounds was <9.60% and inaccuracy was within ±10.81%. Stock solutions were stable at 4 °C for at least 720 h. Processed extracts were stable at room temperature for at least 24 h and QC samples were stable during three freeze–thaw cycles. In spiked human plasma under ambient conditions ARM was stable for at least 8 h whereas DHA was stable for 2 h only.