Characterization of intact hemoglobin and oxaliplatin interaction by nanoelectrospray ionization tandem mass spectrometry

Background: Mass spectrometric (MS) detection of intact hemoglobin (Hb) adducts presents considerable. analytical challenges because of the noncovalent association of the 4 subunits of Hb, and MS characterization of the interaction of intact Hb-with platinum drugs has not been reported. We developed a technique for detecting intact Hb and its drug adduct and studied the interactions between intact Hb and oxaliplatin. Methods: We incubated a series of mixtures of Hb and oxaliplatin at 37 degrees C for 24 h or 5 days to investigate adduct formation. Blood samples from colorectal cancer patients undergoing oxaliplatin treatment were analyzed for novel adducts of intact Hb-oxaliplatin, which were characterized with nanoelectrospray ionization quadrupole time-of-flight MS. Results: Two intact Hb adducts, one with the whole oxaliplatin molecule and the other with oxaliplatin losing the oxalate ligand,-were identified. Analysis of erythrocytes from the cancer patients provided direct evidence that oxaliplatin accumulated as Flb adducts in erythrocytes. A higher fraction (-70%) of Hb was bound to oxaliplatin in erythrocytes from a patient who could not tolerate oxaliplatin treatment than in erythrocytes from another patient who benefited from this treatment. Conclusions: The nanoelectrospray tandem MS technique enabled determination of the intact Hb tetramer and its association with oxaliplatin. Hb-oxaliplatin ad- ducts in erythrocytes may serve as a clinical biomarker for toxic response and treatment efficacy. (c) 2005 American Association for Clinical Chemistry.