Improved LC assay for the determination of mitozantrone in plasma: analytical considerations

Preliminary method development studies on mitozantrone (MTZ) revealed a number of characteristics which were found to be important in the analysis of patient samples for pharmacokinetic studies. MTZ rapidly bound to glass, particularly at low concentrations (< 10 ng ml-1), necessitating the use of silanized glassware or polypropylene tubes for the handling of all solutions containing MTZ. MTZ was also found to react with two commonly-used antioxidants; sodium metabisulphite and EDTA. However, solutions containing MTZ were found to be stabilized by the addition of ascorbic acid (0.5% w/v). In the absence of ascorbic acid, MTZ underwent rapid, biphasic degradation in plasma at 24 and 37 degrees C, with terminal half-lives of approximately 70 h. Ascorbic acid (0.5% 2/v) was found to stabilize plasma samples containing MTZ throughout work-up procedures and during frozen storage. The addition of ascorbic acid to the sample collection vial was also necessary to prevent MTZ degradation in the eluting solvent of the solid-phase extraction system. Another important consideration was the requirement for an equilibration period of > 5 min after the addition of ametantrone (AM) internal standard to plasma samples. This was essential, since the slope of the calibration plot obtained using non-equilibrated plasma was approximately 30% of that obtained for calibration plots using equilibrated plasma, and would result in erroneous determination of MTZ plasma concentrations. The fully developed assay was rapid, precise and sensitive (relative errors at 1 ng ml-1 = 2.3%). MTZ concentrations determined using the LC method described in this report correlated well with an independently developed ELISA technique (r = 0.995, n = 20).