Characterization of glutathione conjugates of chlorambucil by fast atom bombardment and thermospray liquid chromatography/mass spectrometry.
Chlorambucil (p-(di-2-chloroethyl)amino-gamma-phenylbutyric acid) is a bifunctional alkylating agent which exhibits acquired drug resistance upon repeated dosing in humans. This compound reacts with glutathione both non-enzymatically and enzymatically in the presence of immobilized microsomal glutathione-S-transferases to produce several glutathione conjugates. These conjugates result from displacement of one or both chlorines by the nucleophilic cysteine sulfhydryl moiety of glutathione. The mono- and diglutathionyl conjugates of chlorambucil were purified by reversed-phase high-performance liquid chromatography and characterized by positive ion fast atom bombardment mass spectrometry. In addition, the mono- and dihydroxy hydrolysis products of chlorambucil were characterized by positive ion thermospray liquid chromatography/mass spectrometry (LC/MS). The glutathione conjugates of chlorambucil did not produce molecular ion species in thermospray LC/MS mode, but gave characteristic ions at m/z 147 corresponding to fragmentation of the glutathione moiety. The formation of glutathione conjugates of this class of alkylating agents may play a role in the development of acquired drug resistance.
Dulik
D M
DM
Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
Colvin
O M
OM
Fenselau
C
C
D000818:Animals; D002699:Chlorambucil / Q000032:analysis / Q000138:chemical synthesis*; D004351:Drug Resistance; D004800:Enzymes, Immobilized; D008401:Gas Chromatography-Mass Spectrometry; D005978:Glutathione / Q000032:analysis / Q000138:chemical synthesis*; D005982:Glutathione Transferase; D066298:In Vitro Techniques; D008252:Macaca fascicularis; D008862:Microsomes, Liver / Q000201:enzymology
D004800:Enzymes, Immobilized; D002699:Chlorambucil; D005982:Glutathione Transferase; D005978:Glutathione