Development of a GC-MS Method to Quantitate CVAA in Hemoglobin Adducts
Tue 11:00 AM - Track 3: Protein Adduct Methods
Joe V. Wooten
Joe V. Wooten, Centers for Disease Control and Prevention
Lewisite (dichloro (2-chlorovinyl) arsine) is an extremely toxic chemical warfare agent (CWA) that rapidly hydrolyses to 2-chlorovinylarsonous acid (CVAA) upon contact with skin and moist mucus membranes. In buffered urine of male Wistar rats percutaneously exposed to low (2 mg/kg body wt) or high (10 mg/kg body wt) doses of lewisite, detectable levels of CVAA can be measured up to 24 hrs post dose. In the present study, a method to detect CVAA in biological samples, more than 24 hrs after exposure to lewisite is developed. CVAA binds with specific blood proteins and given that the average life span of a red blood cell is approximately 100 to 120 days, whole human blood is an ideal matrix for retrospective analysis of CVAA. This method uses blood as the primary matrix; with the main focus on CVAA-Haemoglobin adducts, which can be used as a biomarker of exposure. Whole blood (250 µL) is spiked with different concentrations of native CVAA in vitro. Samples were then spiked with 13C2, D2-labeled CVAA, which is used as an internal standard. Complexation of the samples is achieved by derivatization with 1, 3-Propane Dithiol for 30 min at 37° C. This results in the formation of CVAA-PDT cyclic dithiols. The CVAA-PDT dithiol is extracted twice from the samples with 500 mL of toluene: After each extraction, the organic layer is removed from the samples, and then concentrated to dryness. After the second extraction and concentration, the samples are reconstituted with 20 µL of toluene. GC-MS analysis is preformed on 1 µL of each reconstituted sample. This research shows that CVAA can be quantitated in whole human blood more than 24 hrs after exposure to lewisite. Moreover, the results of this study could potentially aid medical personal in the treatment of patients exposed to lewisite, as well as provide useful information to law enforcement agencies in the event of a terrorist attack in which lewisite is used.