Liquid chromatographic–mass spectrometric method for the determination of α-,β-arteether in rat serum

This study reports the development and validation of a sensitive and selective assay method for the determination of α-,β-arteether in rat serum by liquid chromatography–mass spectrometry. The mobile phase was composed of methanol–0.1 mM sodium acetate (pH 5) (80:20%) at a flow-rate of 1 ml min−1 and chromatographic separations were achieved on a Ultracarb, 5 ODS 20, Phenomenex column (5 μm, 30 mm×4.6 mm I.D.). The total effluent from the column was split so that one-tenth was injected into the electrospray LC–MS interface. ESI-MS analysis was carried out using a Micromass Quattro II Triple Quadrupole Mass Spectrometer equipped with an electrospray source. The MS analysis was carried out at a cone voltage of 52 V with a scan range of 100–400 Da. The analytes were quantified from the [M+Na]+ ion chromatograms of α-,β-arteether at m/z 335 and artemisinin at m/z 305. A simple liquid–liquid extraction with 2×2 ml n-hexane was used to isolate α-,β-arteether from rat serum. The method was validated in terms of recovery, linearity, accuracy and precision (within- and between-assay variation). The recovery from spiked control samples ranged from 88.41 to 96.17% with a maximum CV of 10.8% for α-arteether and 69.83–79.69% with a maximum CV of 17.06% for β-arteether. Linearity in serum was observed over the range 20–320 ng ml−1. Percent bias (accuracy) was well within the acceptable range. Within- and between-assay precision were less than 15%. The assay method described here is being applied to study the pharmacokinetics of CDRI developed intramuscular formulation Emal (α-/β-arteether in the ratio of 30:70) in rats. The method is sensitive enough to monitor α-,β-arteether up to 24 h after a single 30 mg kg−1 i.m. dose.