Simultaneous determination of the peptide-mitomycin KW-2149 and its metabolites in plasma by high-performance liquid chromatography

A gradient high-performance liquid chromatographic (HPLC) method is described for the quantification of KW-2149 and its two major metabolites in plasma. The method involves a sample clean-up by solid-phase extraction on C18 columns, separation of the respective compounds by HPLC on a YMC ODS-AQ column (5-μm particle size, 150×6 mm I.D.), using a methanol–water gradient system as an eluent, and measurement by UV absorbance detection at 375 nm. The limits of quantitation were 10 ng/ml for KW-2149 and M-16, and 15 ng/ml for M-18. Recoveries from plasma were higher than 92% on C18 extraction columns. Intra-day precision, expressed as %C.V., was between 1.4 and 6.5%. Intra-day accuracy ranged from 94 to 107%. Precision and accuracy of variability of inter-assays increased somewhat; however, were still within acceptable ranges. The ability of the method to quantify KW-2149 and two major metabolites simultaneously, with precision, accuracy and sensitivity, make it useful in monitoring the fate of this new mitomycin in cancer patients.