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The study of defects in DNA caused by xenobiotics, more particularly the study of DNA adducts, is an important field in cancer aetiology. The analysis of low abundance DNA adducts formed in vivo both in animals and humans requires the development and implementation of highly sensitive analytical methods. Since only a minute amount of DNA can be isolated (ca. 100 microg) it is evident that the amount of sample consumed per analysis should be as small as possible in order to gather as much analytical information as possible. In this article an example is given of how this problem can be solved by the implementation of data-dependent acquisitions using capillary liquid chromatography coupled to electrospray tandem mass spectrometry. As a case study the alkylation of DNA by melphalan is presented. Slight modifications of the chromatographic conditions (mobile and stationary phases) can allow the automated analysis of other kinds of DNA adducts.

In a general study of DNA adduct formation with melphalan, rats were intravenously injected with a single high dose (10 mg kg(-1)). Adduct formation was studied at the nucleoside level in the target organs liver, bone marrow, kidney and blood with the use of 2D liquid chromatography/tandem mass spectrometry (LC/MS/MS). Adducts of dGuo and dAdo were detected under selected reaction monitoring in liver and bone marrow 10 h after administration of melphalan. In the DNA hydrolysates from kidney and blood a Gua-melphalan adduct was found, although in very low abundance. These first results of the search for in vivo-formed melphalan adducts in the rat showed that our miniaturized LC/MS technique is useful for investigating this type of compound. More experiments will be performed in this area to gather more information about the pharmacokinetics and the quantity of adducts formed.

Rats were intravenously injected with a single high dose (10 mg/kg) of the alkylating agent melphalan in order to study DNA-adduct formation. Quantitation of a dGuo-melphalan adduct was done by isotope dilution mass spectrometry using capillary liquid chromatography/mass spectrometry (LC/MS) and [15N5]-labeled dGuo-melphalan as internal standard. DNA-adduct levels were studied in bone marrow, liver and kidney. The instrumental detection limit of the method was determined to be 900 fg (S/N 3, pure standard). These first results clearly show a 10 times higher adduct level in bone marrow compared to kidney and a 6 times higher level compared to liver. More experiments will be necessary to gather more information on the pharmacokinetics of melphalan-DNA adducts under in vivo conditions.

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