Ortho-toluidine blood protein adducts: HPLC analysis with fluorescence detection after a single dose in the adult male rat

Abstract

Hemoglobin (Hb) and albumin (Alb) adducts of the suspect human carcinogen ortho-toluidine (OT) were quantified in blood samples collected from rats after a single i.p. injection. Mild alkaline hydrolysis of Hb adducted with [ 14C]OT followed by extraction with ethyl acetate resulted in recovery of 63% of the bound radioactivity. HPLC analysis revealed a single radiolabeled peak which was identified as OT by GC-MS. In subsequent experiments Hb and Alb adduct levels were determined by HPLC analysis of this cleavage product using fluorescence detection. 4-Ethylaniline was used as internal standard. The detection limit for OT was approximately 450 pg/injection or 5 pmol/mg Hb. Mean adduct levels for Hb increased rapidly over the first 4 hr with the highest (ng/mg Hb ± SD) 3.7 ± 0.5 detected 24 hr after OT administration at 50 mg/kg body wt. In contrast, adduct levels for pooled Alb samples increased from 0.7 ng/mg Alb at 2 hr to 2.5 ng/mg Alb at 4 hr, but were not detectable 24 hr after dosing. Hb adducts showed a linear relationship for OT doses of 10, 20, 40, 50, and 100 mg/kg body wt. The Hb adduct t 1 2 (11 days) was determined after a single 100 mg/kg OT dose. Hb adduct levels were quantifiable (1.3 ± 0.2 ng/mg Hb) by HPLC/fluorescence 28 days after 100 mg/kg OT. Although Hb and Alb adducts differ in stability, a ratio of such OT adducts may be useful in long-term industrial biomonitoring for evaluation of OT exposure.