Separation and trace estimation of benzidine and its macromolecular adducts using supercritical fluid chromatography.

A sensitive, rapid, selective and reproducible method has been developed to measure blood plasma levels of benzidine (BZ) and its acetylated metabolite, N-OH-N,N'-diacetylbenzidine (N-OH-DABZ), using supercritical fluid chromatography (SFC) for the first time. Benzidine and N-OH-N,N'-diacetylbenzidine were extracted from the plasma using ether. Separation was done on a Nucleosil (250 mm x 4.6 mm) 10 microm, Nucleosil-RP-C18 column with 7.4% (v/v) methanol-modified supercritical fluid carbon dioxide (2.5 ml min(-1)) as mobile phase. The column temperature was 45 degrees C and the outlet pressure was set at 8.83 MPa. The detection was done using a UV-Vis detector set at 280 nm. The limit of quantification was 0.10 ng ml(-1) (BZ) and 0.14 ng ml(-1) (N-OH-diacetylbenzidine) using 1 ml plasma specimen. The mean extraction recovery of BZ was found to be 98.6%. The SFC method was directly compared to a published HPLC-UV method. With respect to speed, organic solvent usage, sensitivity, specificity and accuracy, SFC was found to be superior. The method has been successfully used to estimate the BZ, N-OH-diacetylbenzidine levels in blood plasma of the animals who were administered 15 microg kg(-1) body weight of benzidine.Further, this method has been also applied for the detection and quantification of benzidine DNA and hemoglobin adducts from the blood and tissue samples of the benzidine dosed animals.