Characterization of glutathione conjugates of chlorambucil by fast atom bombardment and thermospray liquid chromatography/mass spectrometry

Chlorambucil ( p ‐(di‐2‐chloroethyl)amino‐γ‐phenylbutyric acid) is a bifunctional alkylating agent which exhibits acquired drug resistance upon repeated dosing in humans. This compound reacts with glutathione both nonenzymatically 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.