Simultaneous determination of AMN107 and Imatinib (Gleevec®, Glivec®, STI571) in cultured tumour cells using an isocratic high-performance liquid chromatography procedure with UV detection

A reversed phase high-performance liquid chromatographic (HPLC) method with UV detection was developed for the simultaneous determination of imatinib (Gleevec®, Glivec®, STI571) and AMN107 in cultured tumour cells, using clozapine as an internal standard. The compounds of interest were extracted by liquid–liquid extraction using TOXI-TUBES® A extraction tubes. Chromatographic separation was performed on a Phenomenex Gemini C18 reversed phase column (150 mm × 2.0 mm, 5 μm particle size), using a mixture of 65% CH3OH (methanol) and 35% NH4Ac (Ammonium acetate) buffer (20 mM, pH 10). Separation was achieved under isocratic conditions at a flow rate of 0.5 ml/min. Imatinib, clozapine and AMN107 are detected by UV detection at 260 nm. Calibration curves were linear from 50 to 7500 ng/ml with correlation coefficients (r2) better than 0.998. The limit of quantitation (LOD) was 50 ng/ml. The method has been successfully applied to a cellular kinetics study.