Use of thermospray liquid chromatography-mass spectrometry for characterization of reactive metabolites of 3'-hydroxyacetanilide, a non-hepatotoxic regioisomer of acetaminophen

3'-Hydroxyacetanilide (AMAP) is a non-hepatotoxic regioisomer of acetaminophen that nonetheless does form reactive metabolites that are trapped as glutathione thioether adducts. These reactive intermediates are, 4-acetamido-o-benzoquinone, 2-acetamido-p-benzoquinone and N-acetyl-3-methoxy-p-benzoquinone. Thermospray liquid chromatography mass spectrometry (TSP LC-MS) was used to characterize products of reactions of these reactive compounds with cysteine or N-acetyl-cysteine. The TSP spectra of the mono- and bis-thioether adducts showed protonated molecular ions and characteristic fragmentation patterns. The chromatographic resolution together with the MS selectivity allowed for unequivocal identification of these conjugates in the urine of mice treated with AMAP.