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)-NH(4)I (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.
Shin
Ho Sang
HS
Department of Environmental Education, RRC/NMR, Kongju National University, 314-701, Kongju, South Korea. hshin@kongju.ac.kr
Heon Lee
Jin
J
Ahn
Hye Sil
HS
Shin
Ueon Sang
US
D000818:Animals; D001560:Benzidines / Q000737:chemistry*; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006454:Hemoglobins / Q000737:chemistry*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001560:Benzidines; D006454:Hemoglobins; C029876:benzidine