National Institute for Occupational Safety and Health

“ Start reagent blank (2 mL 0.1 N HCl, 2 mL pH 5.5 sodium acetate buffer, and 0.7 mL TNBS reagent) .
CALIBRATION AND QUALITY CONTROL: 11.
12. 13.
Prepare a series of working standards in the range 0 to 20 μg benzidine/100 mL urine by adding aliquots of calibration stock solution to 100-mL portions of control urine (urine pool previously shown to have <0.1 μg benzidine/100 mL urine) . Prepare and analyze the working standards (steps 3 through 10 and 14 through 16) . Prepare a calibration graph, absorbance at 400 nm vs. concentration of analyte (μg/110 mL urine) . ”

“ Extract the urine twice more with 10-mL portions of chloroform. Combine the three chloroform fractions. Re-extract the combined chloroform mixture with 2 mL 0.1 N HCl for 30 min on a rotator. Transfer the aqueous phase (ca. 2 mL) into a culture tube (16 × 125 mm) using a Pasteur pipet. Add 2 mL pH 5.5 sodium acetate buffer and 0.7 mL of TNBS reagent, mix well, and let stand for 15 min at room temperature. ”

“ NIOSH Manual of Analytical Methods (NMAM) , Fourth Edition, 8/15/93 BENZIDINE in urine: METHOD 8304, Issue 2, dated 15 August 1993 - Page 2 of 4 REAGENTS:
EQUIPMENT:
1. Methyl alcohol. 2. Benzidine, 99% (CAUTION: CARCINOGEN) . * 3. Benzidine stock solution, 500 μg/mL. Weighed 50 mg benzidine. Dissolve in methyl alcohol to make 100 mL solution. Stable one month at -8°C. 4. Calibration stock solution, 10 μg/mL. * Dilute 1.00 mL benzidine stock solution to 50 mL with methyl alcohol. Prepare fresh daily. 5. Chloroform. 6. Hydrochloric acid. a. 1.0 N. Dilute 83 mL conc. ”