Abstract
A gas chromatography/mass spectrometric assay method has been developed for the simultaneous determination of benzidine (BZ), N-acetyl benzidine (ABZ) and N,N-diacetyl benzidine (DABZ) in rat urine. BZ, ABZ and DABZ were extracted from urine at pH 8 with ethyl ether. Conjugated urinary metabolites were extracted at pH 8 after hydrolysis with 1 M HCl for 30 min at 100 $^{\circ}C.$ The dried extract was dissolved in 100 ${\mu}{\ell}$ of ethylacetate and then injected in gas chromatography-mass spectrometric (GC-MS) system without further purification or modification. BZ, ABZ and DABZ have good chromatographic properties and offer very sensitive response for the EI-MS (SIM) without any derivatization. The recoveries for BZ, ABZ and DABZ were about 98.0, 81.8 and 71.4%, respectively, at pH 8.0 and the concentration of 5.0 ng/mL. The coefficients of variation of BZ and ABZ were less than 9.5% from 0.1 to 100 ng/mL and that of DABZ was less than 13% in the same concentration range. The detection limits of the assay were 0.01 ng/mL for both BZ and ABZ, and 0.05 ng/mL for DABZ in urine or plasma 1.0 mL.
