New sensitive determination method of benzidine–hemoglobin adducts by gas chromatography–electron impact mass spectrometry

A gas chromatographic–mass spectrometric assay was developed for the determination of benzidine (BZ)–hemoglobin adducts. Adducts were released from hemoglobin by alkaline hydrolysis and extraction at pH 8 with ethyl ether. The dried extract was completely derivatized with N-methyl-N-(tert.-butyldimethylsilyl) trifluoroacetamide (MTBDMSTFA)–NH4I (1000:3) under catalysis of dithioerythritol. The recovery of BZ, acetylbenzidine (ABZ) and diacetylbenzidine (DABZ) in the extraction procedure was 76–98%. The detection limits of the assay were 0.1 ng/g for both BZ and ABZ, and 0.5 ng/g for DABZ based upon assayed hemoglobin of 0.1 g. The method was applied to the determination of BZ–hemoglobin adducts formed in young female Sprague–Dawley rats after treatment for 1, 2 and 3 weeks with 0.008% BZ via the drinking water. Two adducts were detected by proposed procedure. The structure of these adducts could be assigned to BZ and ABZ. After 1 week, the total mean amount of adducts determined was 2.8 ng/g hemoglobin. The adduct levels increased up to about 7.5 ng/g after a week and, thereafter, remained essentially constant. The relative contribution of BZ and ABZ to the total hemoglobin adduct level was strongly treatment time-dependent. After 1 week, the BZ and ABZ adducts were formed at similar levels, whereas after 3 weeks the ABZ adducts was predominant. Treatment of rats for 3 weeks in the dose range 12.2–36.8 mg of BZ in drinking water resulted in a dose-proportional increase in the total amount of hemoglobin adducts formed.