Adducts formed between hemoglobin and reactive chemicals or their metabolites provide a means of monitoring of the long-term exposure to chemicals. However, the methods that are generally used for measuring these adducts are selective for one or at most several chemicals, are difficult to perform, and typically involve gas chromatography with mass spectrometry for adduct analysis. The objective of this study will be to develop methods that can be used to study exposure to a variety of agents, rather than selectively targeted to a single analyte. A widely used method for analysis of adducts formed on reaction with the N-terminal valine residue of hemoglobin is the modified Edman degradation. In this technique, the pentafluorophenylthiohydantoin derivative of the valine adduct is quantitated by negative ion chemical ionization mass spectrometry. Preliminary studies conducted in our laboratory indicate that the analysis of hemoglobin adducts of acrylamide and glycidamide released by a modified Edman degradation can be accomplished by liquid chromatraphy-mass spectrometry (LC-MS) of phenylthiohydantoin derivatives, using multiple reaction monitoring on a triple quadruple mass spectrometer. Solid phase extraction has reduced sample preparation time. Our new method of analysis provides considerable time savings both in sample preparation and analysis time. Objectives of the proposed research will be to adapt the methodology to the quantitation of a number of adducts derived from a variety of different chemicals of concern in the same samples, and to derive methods for analysis of adducts that are present in globin using less selective methods. The detection of adducts with valine using the modified Edman degradation coupled with LC-MS analysis on an ion trap MS will provide the necessary sensitivity for identification of the adducts present. The qualitative evaluation of adduct formation in globin by protein digestion and mass spectrometry has been carried out primarily in vitro, with applications in vivo in a limited number of cases. A further aim of these studies will be to apply protein digestion and tandem mass spectrometry to the evaluation of adduct formation in vivo, and to evaluate the sensitivity of this approach for the qualitative evaluation of exposure. However, measuring adducts is still relatively low throughput methodology that is not amenable to large-scale epidemiological studies involving thousands of samples, although this is faster than what is currently available.
