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 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 P1. In this hydrolysate both mono-alkylated 2'-deoxynucleotides and dinucleotides were found. The most important adduct found was identified as the N-7 alklylated dGMP adduct. The alkylated dinucleotides were identified as a pdApdT/melphalan and pdGpdC/melphalan the latter being the most important.
Hoes
I
I
Department of Chemistry, University of Antwerp, Belgium.
Lemière
F
F
Van Dongen
W
W
Vanhoutte
K
K
Esmans
E L
EL
Van Bockstaele
D
D
Berneman
Z
Z
Deforce
D
D
Van den Eeckhout
E G
EG
D000478:Alkylation; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000378:metabolism; D018736:DNA Adducts / Q000032:analysis*; D003838:Deoxyadenine Nucleotides / Q000032:analysis; D003843:Deoxycytidine Monophosphate / Q000032:analysis; D003848:Deoxyguanine Nucleotides / Q000032:analysis; D003854:Deoxyribonucleotides / Q000032:analysis*; D013058:Mass Spectrometry / Q000379:methods*; D008558:Melphalan / Q000378:metabolism* / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013938:Thymidine Monophosphate / Q000032:analysis
D018736:DNA Adducts; D003838:Deoxyadenine Nucleotides; D003848:Deoxyguanine Nucleotides; D003854:Deoxyribonucleotides; D003843:Deoxycytidine Monophosphate; C077233:2'-deoxyguanylic acid; D013938:Thymidine Monophosphate; C012873:2'-deoxy-5'-adenosine monophosphate; D004247:DNA; D008558:Melphalan