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

Melphalan is a bifunctional alkylating agent that covalently binds with int racellular nucleophilic sites. A methodology using electrospray mass spectr ometry was developed to detect and identify DNA adducts. Alkylation sites w ithin a particular nucleotide were examined using electrospray tandem mass spectrometry hyphenated to capillary liquid chromatography in combination w ith a column switching system. In the reaction mixtures resulting from the interaction of 2'-deoxynucleotides and melphalan several base-aklylated add ucts were found. In the case of 2'-deoxyadenosine monophosphate, thymidine monophosphate and 2'-deoxyguanosine phosphate alkylation was observed in th e mononucleotide reaction mixtures but not in the DNA-hydrolysates. Calf th ymus DNA was reacted in vitro with melphalan. The DNA pellet was isolated a nd enzymatically hydrolyzed with the aid of Nuclease P-1. In this hydrolysa te both mono-alkylated 2'-deoxynucleotides and dinucleotides were found. Th e most important adduct found was identified as the N-7 alkylated dGMP addu ct. The alkylated dinucleotides were identified as a pdApdT/melphalan and p dGpdC/melphalan the latter being the most important. (C) 1999 Elsevier Scie nce B.V. All rights reserved.