High-performance liquid chromatographic determination of components of bleomycin preparations.

A fast and sensitive method was developed for the quantitative determination of at least 10 components of pharmaceutical bleomycin sulfate preparations. The method is based on the reversed-phase high-performance liquid chromatographic (HPLC) separation of the components on a muBondapak C18 column with a mobile phase having a linear gradient of 10--40% methanol in aqueous 0.005 M 1-pentanesulfonic acid at pH 4.3. With this assay, the average standard deviations for components A2 and B2 are 0.92 and 0.87, respectively, for a 7.5--22.5 x 10(-3)-mg sample. Regulatory agencies presently use the official Code of Federal Regulations (CFR) method, which is based on CM-Sephadex column chromatography. It was demonstrated that this CFR method does not separate the bleomycin A2 component from some other minor bleomycin components. After elution from the CM-Sephadex column, the "A2 component" was separated into five components by the HPLC method. Bleomycin A2 is stable under these HPLC conditions.