Bioanalysis of aplidine, a new marine antitumoral depsipeptide, in plasma by high-performance liquid chromatography after derivatization with trans-4-hydrazino-2-stilbazole

A sensitive bio-analytical assay in plasma of the depsipeptide aplidine is reported, based on reversed-phase liquid chromatography and fluorescence detection of the trans-4'-hydrazino-2-stilbazole (4'H2S) derivative of the analyte. At ambient temperature, two conformations of the depsipeptide are observed in solution due to cis-trans isomerism at the proline-pyruvoyl peptide bond. Aplidine is isolated from the matrix by solid-phase extraction on an octadecyl modified silica stationary phase. After evaporation of the acetone eluate, a derivatization with 4'H2S is performed in a water-acetonitrile mixture at pH 4. The reaction mixture is injected directly into the chromatograph and the analyte is quantified by fluorescence detection at 410 and 560 nm for excitation and emission, respectively. The method has been validated in the 2-100 ng/ml-range, 2 ng/ml being the lower limit of quantification. Precision and accuracy both meet the current requirements for a bioanalytical assay. The identity of the 4'H2S reaction products of aplidine have been confirmed by mass spectrometric analysis. Finally, the method has been employed for a pilot pharmacokinetic study of aplidine in mice which demonstrated its usefulness for pharmacological research.