Analysis of melphalan adducts of 2′-deoxynucleotides in calf thymus DNA hydrolysates by capillary high-performance liquid chromatography–electrospray tandem mass spectrometry

Abstract

Melphalan is a bifunctional alkylating agent that covalently binds with intracellular nucleophilic sites. A methodology using electrospray mass spectrometry was developed to detect and identify DNA adducts. Alkylation sites within a particular nucleotide were examined using electrospray tandem mass spectrometry hyphenated to capillary liquid chromatography in combination with a column switching system. In the reaction mixtures resulting from the interaction of 2′-deoxynucleotides and melphalan several base-aklylated adducts were found. In the case of 2′-deoxyadenosine monophosphate, thymidine monophosphate and 2′-deoxyguanosine phosphate alkylation was observed in the mononucleotide reaction mixtures but not in the DNA-hydrolysates. Calf thymus DNA was reacted in vitro with melphalan. The DNA pellet was isolated and enzymatically hydrolyzed with the aid of Nuclease P 1. In this hydrolysate both mono-alkylated 2′-deoxynucleotides and dinucleotides were found. The most important adduct found was identified as the N-7 alkylated dGMP adduct. The alkylated dinucleotides were identified as a pdApdT/melphalan and pdGpdC/melphalan the latter being the most important.