Abstract
A method was developed for the quantitative analysis of the novel anticancer agent ES‐285 (spisulosine; free base) in human, mouse, rat, and dog plasma using high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry in order to support pre‐clinical and clinical studies with the drug. Sample preparation was carried out by protein precipitation with acetonitrile, containing isotopically labeled (d~3~) ES‐285 as internal standard. Aliquots of 10 µl of the supernatant were injected directly on to an Inertsil ODS‐3 column (50 × 2.0 mm i.d., 5 µm). Elution was carried out using methanol–10 mM ammonium formate (pH 4) in water (80 : 20, v/v) pumped at a flow‐rate of 0.2 ml min^−1^ with a run time of 8 min. Multiple reaction monitoring chromatograms obtained on an API365 triple‐quadrupole mass spectrometer were used for quantification. The lower limit of quantitation (LLOQ) was 10 ng ml^−1^ in human, mouse, rat, and dog plasma and the linear dynamic range extended to 500 ng ml^−1^. A full validation of the method was performed in human plasma, and partial validations were performed in mouse, rat and dog plasma. Accuracies and precisions were <20% at the LLOQ concentration and <15% for all other concentrations in all matrices. ES‐285 was stable during all steps of the assay. Thus far this method has been used successfully to analyze over 500 samples in pre‐clinical trials, and will be implemented in the planned clinical phase I studies. Copyright © 2003 John Wiley & Sons, Ltd.