Determination of dichlorobenzidine-hemoglobin adducts by GC/MS-NCI.

Abstract

A method based on gas chromatography/mass spectrometry-negative ion chemical ionization detection (GC/MS-NCI) was developed for the determination of 3,3'-dichlorobenzidine (DCB)-hemoglobin adducts. Adducts were released from hemoglobin by mild alkaline hydrolysis and determined by GC/MS-NCI after extraction and derivatization with heptafluorobutyric anhydride (HFBA). 2,2'-DCB was used as internal standard and the recovery of the diarylamine derivatives in the overall procedure was 65-88%. The limit of detection attained was below 0.1 ng/g hemoglobin for DCB as well as for the metabolite N-acetyl-3,3'-dichlorobenzidine (acDCB). The method was shown to be linear up to 150 ng/g hemoglobin. In the NCI mass spectra of the HFB derivatives the dominant ion is (M-HF)-. Due to the presence of two chlorines in the diarylamines, the characteristic ratio of 1.5 for m/z 624 to 626 (for diHFB-DCB and diHFB-2,2'-DCB) and m/z 470 to 472 (for HFB-acDCB) can be observed and used for identification. The method was applied to the determination of DCB-hemoglobin adducts formed in young female Wistar rats after treatment for 4 weeks with 0.006%, 0.0012% or 0.00024% DCB via the drinking water. Two adducts were detectable by GC/MS-NCI after alkaline hydrolysis of hemoglobin samples, extraction and derivatization. The structure of these adducts could be assigned to DCB and acDCB by co-chromatography with the synthetic standards and by the presence of the characteristic ion (M-HF)-. Assessment of the time dependence of hemoglobin adduct formation during subchronic treatment with DCB revealed an increase in adduct levels during weeks 1-3. After this time adduct levels essentially remained constant. In hemoglobin samples isolated from animals treated for 4 weeks with DCB a dose-proportional increase in the total amount DCB- and acDCB-hemoglobin adducts from 8.1 ng DCB/g hemoglobin at 0.3 mg/kg body weight per day (0.00024% in drinking water) to 159.9 ng DCB/g hemoglobin at 5.8 mg/kg body weight per day (0.006% in drinking water) was observed. The ratio of the DCB adduct to the acDCB adduct was strongly dose dependent. At low DCB doses the acDCB- and DCB adducts were formed at similar levels, whereas at high DCB doses the DCB adduct was predominant.