BENZIDINE in urine: METHOD 8306, Issue 2, dated 15 August 1994 - Page 3 of 4 13. Buffer. Dissolve 136 g KH 2PO 4 in 900 mL H2O. Adjust to pH 6 with 10 N NaOH. Dilute to 1 L with H 20 and extract once with 100 mL benzene. 14. Paraffin oil. 15. Heptafluorobutyric anhydride. 16. Nitrogen gas, compressed, purified.
See SPECIAL PRECAUTIONS.
SPECIAL PRECAUTIONS: Samples of urine collected from humans pose a real health risk to laboratory workers who collect and handle these samples. These risks are primarily due to personal contact with infective biological samples and can have serious health consequences, such as infectious hepatitis, and other diseases. There is also some risk from the chemical content of these samples, but this is much less. Those who handle urine specimens should wear protective gloves, and avoid aerosolization of the samples. Mouth pipetting, of course, must be avoided. Benzidine and benzene are documented human carcinogens and must be handled in compliance with 29 CFR 1910.1005 and 1910.1028.
SAMPLING: 1. 2. 3.
Collect a spot urine sample in a polyethylene bottle. Add 2 drops 12 Freeze the samples and ship in an insulated container with dry ice. Keep the samples frozen until analysis.
N HCl as a preservative.
SAMPLE PREPARATION: 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14. 15. 16. 17. 18.
Perform a creatinine determination on an aliquot of the sample (e.g., [4]). Pipet 10 mL of the urine specimen into a 50-mL culture tube containing 10 mL 10 N NaOH. Seal the tube and incubate 2 h at 80 °C. Cool to room temperature, add 20 mL benzene, seal, and shake 1 h. Remove the benzene layer and percolate it through a 2-cm column of anhydrous sodium sulfate. Collect the eluate in a 25 × 200-mm culture tube. Repeat step 8 twice. Combine the extracts. Add three or four drops of paraffin oil to the extracts and concentrate the extracts to near dryness under a gentle stream of nitrogen. Add 1.5 mL benzene and 0.5 mL trimethylamine working solution. Add 50 µL heptafluorobutyric anhydride. Seal and place in a 50 °C waterbath for 20 min. Cool to room temperature. Wash the derivatized extract. a. Add 2 mL pH 6 buffer solution. Seal and shake 2 min. b. Centrifuge 2 min. Discard the bottom (aqueous) layer. c. Repeat the washing with two additional 2-mL portions of pH 6 buffer. Place a small glass wool plug in a chromatographic column and add 1.6 g Florisil. Top with 2 cm anhydrous sodium sulfate. Wash the column with 50 mL hexane. Add the buffer-washed, derivatized extract to the Florisil column. Rinse the culture tube twice with ca. 2 mL benzene and add the rinsings to the Florisil column. When the top of the extract is in the sodium sulfate layer, add 10 mL 3:2 (v/v) hexane:benzene. Discard the eluate. Elute the N,N'-diheptafluorobutyryl benzidine with 15 mL benzene. Add 0.5 mL derivatized methylenedianiline stock solution to the eluate. Concentrate to 10 mL NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 8/15/94
