Studying the reaction between clozapine and glutathione with element-selective detection

The target of these investigations was a study of covalent binding the antipsychotic drug clozapine and the tripeptide glutathione. Other workers, primarily using radioisotopes, have found many adducts of clozapine and glutathione. We wanted to see how well the chlorine atom in clozapine could serve as an alternate to the use of a radiolabel using the Chemical Reaction Interface/Mass Spectrometer technique with HPLC introduction (HPLC/CRIMS). Here, we examine the capabilities of two such schemes that were previously used with GC introduction: Cl detection with SO2 as the reactant gas; and Cl and S detection using NF3 as the reactant gas. Detecting chlorine as HCl with SO2 was accomplished giving linearity over an 80-fold range of sample size. Incubations of the drug and glutathione with a peroxidase/peroxide system system yielded several metabolites characterized as novel conjugates of clozapine by electrospray mass spectrometry. This tentative identification of two conjugates was confirmed by examining the incubation mixture with NF3 as the CRIMS reactant gas. The simultaneous appearance of both Cl and S is consistent with covalent binding of clozapine to glutathione. A nearly doubled ratio of S to Cl in one peak confirmed the presence of a di-glutathione conjugate. These experiments support our proposition that element selective detection of HPLC effluents with CRIMS can supply additional information, not previously available using radioisotopic methods.