Liquid chromatography-tandem mass spectrometric determination of a new antibacterial agent (AVE6971) in human white blood cells

An LC-MS/MS assay for the quantitative determination of a new antibacterial agent (AVE6971) has been developed and validated in human white blood cells (WBC). The assay involved a lysing procedure of white blood cells and ultra centrifugation of the extracts. Chromatography was performed on a Supelcosil ABZ+ C(18) (2.1 mm x 50 mm, 5 microm) column using a mobile phase consisting of methanol/acetonitrile/10mM ammonium formate mixture (10:30:60, v/v/v) at a flow rate of 0.2 ml/min. The linearity was within the range of 10-10000 ng/ml of extracts, corresponding to 0.5-500 ng of AVE6971 in WBC pellets tubes. The validated lower limit of quantification was 10 ng/ml. The inter- and intra-run coefficients of variation (CV) for the assay were <12.9% and the accuracy were from -9.0 to -1.2%. AVE6971 was stable in WBC for at least 1 month at -75 degrees C. This assay proved to be suitable for the determination of AVE6971 in WBC from clinical studies.