Speciation studies of cis-platin adducts with DNA nucleotides via elemental specific detection (P and Pt) using liquid chromatography-inductively coupled plasma-mass spectrometry and structural characterization by electrospray mass spectrometry

As the -platinum (-Pt) antitumoral effect in mammals seems to be related to its binding to DNA components, experiments with incubation of the individual DNA nucleotides with -Pt and analysis of the products by electrospray mass spectrometry (ESI-Q-TOF) are described here. The only detectable complex of such binding has been the one formed by a -Pt molecule bound to two adjacent guanines (/ 921), as confirmed by collisional induced dissociation. The separation of the -Pt adducts from the unreacted nucleotides has been conducted by high-performance liquid chromatography coupled on-line with inductively coupled plasma mass spectrometry (HPLC-ICP-MS), monitoring P and Pt. Two different chromatographic columns have been evaluated for this purpose: a RP-amide-C and a narrow-bore C. Best separation characteristics for the four nucleotides of DNA (coming from adenine, thymine, cytosine and guanine nucleobases) and the formed -Pt adduct were obtained for the C column using a mobile phase containing 60 mM ammonium acetate (pH = 5.8) and 7.5% MeOH. This HPLC-ICP-MS method allowed an easy separation and detection of free nucleotides (by monitoring P) from the synthesized adduct (containing P and Pt in the same molecule). Quantitative capabilities of the proposed hybrid method, by monitoring P and Pt, have been compared by analysing the -Pt adduct formed by the oligonucleotide of sequence 5[prime or minute]-TCCGGTCC-3[prime or minute] after incubation with -Pt and enzymatic hydrolysis. Final application of this methodology to commercially available calf thymus DNA samples has been also satisfactorily accomplished.