MULTIPLE SCAN MODES IN THE HYBRID TANDEM MASS-SPECTROMETRIC SCREENING AND CHARACTERIZATION OF THE GLUTATHIONE CONJUGATE OF 2-FURAMIDE

The glutathione conjugate of 2-furamide has been screened for and structurally characterized by tandem mass spectrometry (MS/MS) by using a hybrid instrument of BEqQ design. Mass spectrometry experiments employed fast atom bombardment (FAB)13 ionization of a crude bile extract from a rat dosed with a 1:1 mixture of unlabeled and ]C-13(1)[2-furamide. Initial screening for glutathione conjugates employed constant neutral loss scanning to detect the loss of 129 u, corresponding to the loss of the gamma-glutamyl moiety of the conjugates. By direct comparison with control bile, [M + H]+ ions of m/z 417 and 418 were readily identified as candidate ions corresponding to the glutathione conjugates of unlabeled and C-13-labeled 2-furamide. Complementary screening information was generated by using a methylated bile extract, with constant neutral loss scanning to detect the loss of the methylated gamma-glutamyul moiety (143 u). An alternative screening procedure employing parent ion scanning to detect the sodium adducts of methylated glutathione conjugates was also developed. Structural information was generated by first-generation product ion scanning of the protonated and sodium cationized forms of the candidate species, both native and derivatized. This provided a body of internally consistent evidence that the conjugate retains the pseudoaromatic furan ring system without ring hydroxylation. The utility of sequential mass spectrometry (MS/MS/MS) capability of the hybrid instrument in the analysis of complex biological mixtures was also demonstrated. Using the bile extract, first-generation product ions that formed in either the first or second field-free region of the double-focusing portion of the instrument were subsequently collisionally activated in the rf-only quadrupole followed by mass analysis of the second-generation product ions. Structural information so provided for the glutathione conjugate of 2-furamide further substantiated its retention of the pseudoaromatic furan ring system and facilitated plausible assignment of structures to ionic species generated through multiple decomposition events.