Application of thermospray/high-performance liquid chromatography/mass spectrometry to the identification of glutathione conjugates derived from bioactive epoxides

Abstract

Thermospray high-performance liquid chromatography/mass spectrometry (HPLC/MS) in the positive ion mode has proven to be a useful technique for analysing different synthetic models of potential metabolites of 3,4-epoxyprecocene II (1), the postulated bioactive epoxide responsible for the cytotoxic activity exhibited by precocenes on a variety of insect and mammal tissues. Thus, whereas standards of the glutathione adducts 3a afforded poor responses, the corresponding cysteine (3b) and N-acetylcysteine (3c) derivatives gave defined spectral patterns and good sensitivity levels, particularly when analysed as methyl esters. Likewise, 3,4-dihydrodiols (2) also afforded satisfactory responses. In all cases, the peak at m/z 237, assigned to the stabilized electrophilic oxybenzopyranyl species which results from the loss of the sulphur or oxygen substituent at C(4), was present. Finally, the incubation of racemic epoxide (1) with rat liver cytosolic fraction, followed by enzymatic degradation and further thermospray HPLC/MS analysis of the resulting methylated cysteine derivatives, allowed the identification, for the first time in a biological matrix, of the pairs of cis and trans glutathione conjugates (3a).