CAPILLARY ELECTROPHORETIC SEPARATION AND LASER-INDUCED FLUORESCENCE DETECTION OF THE MAJOR DNA-ADDUCTS OF CISPLATIN AND CARBOPLATIN

Micellar electrokinetic capillary chromatographic separation of a dans ylated mixture of normal nucleotides and platinated cross-link adducts of d(pGpG) and d(pApGr) was optimized with baseline resolution. The i ntroduction of a laser-induced fluorescence (LIF) detector overcame th e lack of sensitivity characteristic of capillary electrophoresis (CE) due to the small injection volume and the short optical path length. CE/LIF was able to detect 1 adduct/10(4) normal nucleotides/mg DNA by fluorescence postlabeling assay. The enrichment of the adduct, prior t o dansylation, enhanced the detection limit to 1 adduct/10(7) normal n ucleotides/mg DNA. Calf thymus DNA was reacted in vitro with cisplatin and carboplatin with total input drug/nucleotide ratios of 0.05 and 0 .5, respectively. A2780 human ovarian carcinoma cells were exposed in culture to 25 mM cisplatin for 2 h. The cells were incubated with drug -free medium for 3 h before harvesting. The identification of the cros s-link adducts in modified DNA was confirmed by cochromatography with authentic markers. The same 1,2-intrastrand cross-link adducts were in duced by both cisplatin and its second-generation drug carboplatin. Th is report has demonstrated, for the first time, the utility of CE/LIF as an analytical tool for assaying DNA damage. (C) 1995 Academic Press , Inc.