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

Area of Science:

• Analytical Chemistry
• Toxicology
• Biochemistry

Background:

• Benzidine (BZ) is a known human carcinogen.
• Monitoring BZ exposure and its metabolites is crucial for risk assessment.
• Existing analytical methods may lack the required sensitivity or speed.

Purpose of the Study:

• To develop and validate a novel supercritical fluid chromatography (SFC) method for quantifying benzidine and its acetylated metabolite in blood plasma.
• To compare the performance of the developed SFC method against a conventional High-Performance Liquid Chromatography (HPLC) method.
• To apply the validated SFC method for detecting benzidine DNA and hemoglobin adducts.

Main Methods:

• Supercritical fluid chromatography (SFC) using methanol-modified CO2 mobile phase.
• Ether extraction of benzidine and N-OH-N,N'-diacetylbenzidine from plasma.
• UV-Vis detection at 280 nm.
• Comparison with an established HPLC-UV method.

Main Results:

• The SFC method demonstrated high sensitivity with limits of quantification of 0.10 ng/mL for BZ and 0.14 ng/mL for N-OH-DABZ.
• Extraction recovery for BZ was high at 98.6%.
• SFC proved superior to HPLC in terms of speed, organic solvent usage, sensitivity, specificity, and accuracy.
• The method successfully quantified BZ and its metabolite in animal plasma and detected DNA/hemoglobin adducts.

Conclusions:

• SFC offers a sensitive, rapid, selective, and reproducible method for analyzing benzidine and its metabolites in biological samples.
• This SFC method is a valuable tool for toxicological studies and biomonitoring of benzidine exposure.
• The technique's ability to detect adducts expands its utility in carcinogenicity research.