Study of the 'in vivo' interaction of melphalan with DNA using miniaturized LC-MS

In this work the interaction of melphalan with DNA by column switching miniaturized LC-ES MS/MS was investigated. This was done in in vitro- and in vivo-experiments and in cell cultures. The obtained results gave us a better insight in these processes that occur when cancer is treated with melphalan. The applied methodology can be further used in the domain of DNA adduct analysis. Via the synthesis of an internal standard and its use in MS-experiments we obtained quantitative results about the level of DNA adducts in melphalan treated rats.; Interaction of melphalan with 2'-deoxynucleosides revealed the presence of adducts mainly on dGuo, dAdo and dCyd. If 2 '-oligodeoxynucleotides were treated with melphalan, a clear preference for Gua-moieties was shown. In spite of the bifunctional alkylation character of melphalan no cross-link adducts could be observed in these in vitro-experiments. The possibilities of data dependent acquisitions were explored in another chapter and showed the usefulness of a method that gives a maximum of information from single shot analyses. Next to this, the way DNA samples are processed prior to analysis seem to have a dramatic impact on the analytical results. A comparison between enzymatic, acidic and thermal hydrolysis of nitrogen mustard modified DNA proved that acidic hydrolysis gave rise to cross-linked adducts while the samples produced by thermal or enzymatic hydrolysis did not. Therefore, the general accepted theory that the anti-cancer activity of mustards is due to cross-linking of DNA strands, remains therefore from our point of view questionable. The last two chapters of this thesis clearly show the ability of LC-MS to detect and quantify in vivo formed DNA adducts of melphalan. The results show a higher DNA adducts content in bone marrow compared to kidney (x10) and liver (x6) after 24h melphalan incubation. It was our intention to explore a broad field in this thesis: from in vitro reactions to in vivo samples, from the interaction of melphalan with individual 2 '-deoxynucleosides to the interaction with short oligodeoxynucleotide strands, from qualitative work to quantitative work, from off-line work to automated on-line methodologies.