Liquid chromatography–tandem mass spectrometric assay with a novel method of quantitation for the simultaneous determination of bulaquine and its metabolite, primaquine, in monkey plasma

A sensitive and selective liquid chromatography–tandem mass spectrometry method (LC–MS–MS) for the simultaneous estimation of bulaquine and primaquine has been developed and validated in monkey plasma. The mobile phase consisted of acetonitrile/ammonium acetate buffer (20 mM, pH 6) (50:50 v/v) at a flow-rate of 1 ml/min. The chromatographic separations were achieved on two spheri cyano columns (5 μm, 30×4.6 mm I.D.) connected in series. The quantitation was carried out using a Micromass LC–MS–MS with an electrospray source in the multiple reaction monitoring (MRM) mode. The analytes were quantified from the summed total ion value of their two most intense molecular transitions. This is another novel method leading to increased sensitivity and precision. A simple liquid–liquid extraction with 2×1.0 ml n-hexane/ethyl acetate/dimethyloctyl amine (90:10:0.05, v/v) was utilized. The method was validated in terms of recovery, linearity, accuracy and precision (within- and between-assay variation). The recoveries from spiked control samples were ⩾90 and 50% for bulaquine and primaquine, respectively. Linearity in plasma was observed over a dynamic range of 1.56–400 and 3.91–1000 ng/ml for bulaquine and primaquine, respectively.