Urinary monitoring method of 3,3'-dichlorobenzidine (DCB) and its metabolites, N-acetyl-DCB and N,N'-diacetyl-DCB.

3,3'-dichlorobenzidine (DCB) is suspected to be arcinogenic in experimental animal and human. Several studies have investigated excretion of metabolites in urine, hemoglobin adduction and cancer incidence among workers exposed occupationally to DCB. In these researches, metabolites of DCB had a very important role. The purpose of this study was to develop the urinary monitoring method of its metabolites from rats exposed with DCB, by easily synthesizing them in the laboratory. N,N'-diacetyl-DCB was easily synthesized with DCB in pyridine by adding sufficient acetyl chloride or acetic anhydride. N-acetyl-DCB was isolated from the supernatant, which made by adding 21/microl acetyl chloride (more 3 times than DCB in moles) to 32 mg DCB in 2 ml pyridine and 0.3 ml acetic acid. Gas chromatography/mass spectrometry (GC/MS) was used for the identification, gas chromatography nitrogen phosphorous detection (GC-NPD) for the quantification and gas chromatography flame ionization detection (GC-FID) for the determination of purity. The base peak of DCB, N-acetyl-DCB and N,N'-diacetyl-DCB was 252 m/z. The other main peaks were 294 m/z for N-acetyl-DCB, and 294 and 336 m/z for N,N'-diacetyl-DCB. The purities of N-acetyl-DCB and N,N'-diacetyl-DCB were identified as 98.82 and 98.72% by GC-FID, respectively. After treatment orally to rats with 20 mg DCB/kg body weight for 2 weeks, the urinary excretion amount of DCB was nearly constant at range of 0.11-0.18 mg/L for 2 weeks. But excretion of N-acetyl-DCB was continually increased from 1.30 mg/L on 1st day to 4.15 mg/L on 14th day. And level of N,N'-diacetyl-DCB in urine was sharply increased from 2.13 mg/L on 1st day to 11.00 mg/L on 14th day.