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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. HCl to 100 mL with distilled water. b. 0.1 N. Dilute 10 mL 1 N HCl to 100 mL with distilled water. 7. Sodium hydroxide, 1 N. Dissolve 40 g NaOH in water to make 1 L solution. 8. Sodium Chloride, crystals. 9. Sodium acetate buffer, 2 M, pH 5.5 Titrate 2 M sodium acetate with 6 N HCl. Refrigerate. 10. 2,4,6-Trinitrobenzene sulfonic acid (TNBS), 0.1 g/mL. Dissolve 2.5 g TNBS in 25 mL distilled water. Stable seven days when stored in the dark. 11. Acetone. 12. Formic acid. 13. Nitrogen, compressed. 14. Chloroform:formic acid, 90:10 (v/v). Prepare fresh daily.

1. Polyethylene bottles, 250-mL. 2. Spectrophotometer for measuring absorbance at 400 nm and 1-mL semi-microcuvettes. 3. Centrifuge, 400 rpm. 4. Rotator for mixing 25 × 200 mm test tubes. 5. pH meter. 6. TLC plates precoated with silica gel activated at 110 °C for 30 min., without flourescence indicator (0.5 mm thichness). 7. Chromatographic TLC tank. 8. UV source for reading TLC plates. 9. Glass bottles, 180-mL, with PTFE-lined caps. 10. Pipettes, glass, 2-, 5- and 100-mL, with pipet bulb. 11. Separatory funnels, 125-mL. 12. Volumetric flasks, 25- and 100-mL; 10-mL amber. 13. Culture tubes, glass with PTFE-lined caps (16 × 125 mm and 25 × 200 mm). 14. Micropipettes, 0.01-, 0.1- and 0.7-mL. 15. Pasteur pipettes. 16. Plastic gloves. 17. Dessicator. 18. pH paper (pH 2).

See Special Precautions.

SPECIAL PRECAUTIONS: Benzidine is a known human carcinogen. Appropriate precautions should be utilized to minimize exposures. All wastes, including acetone rinses of dirty glassware should be collected and disposed of by approved methods. SAMPLING: 1. 2.

Take pre- and post-shift urine samples (ca. 150 mL) in 250-mL polyethylene bottles. Ship the samples with dry ice in an insulated container.

SAMPLE PREPARATION: 3. 4. 5. 6.

Defrost sample if frozen. Adjust the urine pH to between 5 and 6 with 1 N HCl or 1 N NaOH. Pipet 100 mL urine into a 180-mL glass bottle. Start a blank control urine sample (100 mL) and two control urine samples (100 mL) spiked with benzidine (0.3 to 1.0 µg) at this point. Add 0.2 g NaCl to the pH-adjusted urine. Extract the urine twice more with 10 mL chloroform for 2 min. If an emulsion forms centrifuge to seperate the two phases. Collect and save the chloroform fraction. NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 8/15/93

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