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

Abstract

As the cis-platinum (cis-Pt) antitumoral effect in mammals seems to be related to its binding to DNA components, experiments with in vitro incubation of the individual DNA nucleotides with cis-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 cis-Pt molecule bound to two adjacent guanines (m/z 921), as confirmed by collisional induced dissociation. The separation of the cis-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 ³¹P and ¹⁹⁵Pt. Two different chromatographic columns have been evaluated for this purpose: a RP-amide-C₁₆ and a narrow-bore C₈. Best separation characteristics for the four nucleotides of DNA (coming from adenine, thymine, cytosine and guanine nucleobases) and the formed cis-Pt adduct were obtained for the C₈ 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 ³¹P and ¹⁹⁵Pt, have been compared by analysing the cis-Pt adduct formed by the oligonucleotide of sequence 5′-TCCGGTCC-3′ after incubation with cis-Pt and enzymatic hydrolysis. Final application of this methodology to commercially available calf thymus DNA samples has been also satisfactorily accomplished.