Post-column coulometric generation and cyclic voltammetric identification of the free radical of the antineoplastic agent etoposide (VP 16-213)

It has been suggested that etoposide can be transformed ‘in vivo’ into a radical intermediate, which may be involved in the irreversible binding of etoposide to microsomal proteins and in DNA damage. To investigate some physico-chemical properties, the on-line coulometric production of this free radical and its subsequent cyclic voltammetric detection is described. For the synthesis, a coulometric ESA Coulochem 5100A module, equipped with an ESA 5010 analytical cell, has been used. For its detection the computerized cyclic voltammetric detection system after HPLC controlled by an Apple IIe computer, using home-made software and a home-made glassy-carbon wall-jet detector, has been used. Application of a ramdisk allows storage of 68 cyclic voltammograms. In a methanol-phosphate buffer (pH=8) (40:60 w/w) containing mobile phase, the forward scan of the on-line cyclic voltammogram of etoposide shows two oxidation steps, caused by a double one-electron transfer. Post-column electrolysis at +500 mV versus the H(+)/H2 reference electrode results into total disappearance of the first oxidation step, whereas a reduction wave arises. The limiting current of this wave, and the remaining second oxidation wave, are of equal heights, indicating one-electron processes. Under these conditions, the half-life time of the product, and consequently the free radical, is 100 s, determined by stopped-flow analysis. Decreasing the pH of the buffer in the mobile phase to pH 2.2 results in merging of the two oxidation steps to a single two-electron transfer, disabling radical formation. Under physiological conditions (‘in vivo’) the established half-life time of the radical enables participation of the intermediate in metabolic processes.