Page:NIOSH Manual of Analytical Methods - 8304.pdf/3

BENZIDINE in urine: METHOD 8304, Issue 2, dated 15 August 1993 - Page 3 of 4 7. 8. 9. 10.

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. 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).

MEASUREMENT: 14. 15.

16. 17. 18. 19.

Add 2 mL CHCl 3 to the extract in step 10 and shake for 1 min. Measure the absorbance vs. the reagent blank of the organic phase at 400 nm. Retain the organic phase for the benzidine-TLC confirmation (steps 16 through 19) if the sample contains more than 0.3 µg/100 mL. Concentrate the organic phase containing the TNBS-amine derivative by evaporating with nitrigen to ca. 0.2 mL. Spot 10 µL of the concentrated organic phase on an activated silica gel TLC plate. Develop the plate in 90:10 chloroform:formic acid. Compare the R f of the unknown amine derivative with that of a benzidine-spiked derivative. Benzidine produces a spot with R f = 0.41 which is yellow in visible light and dark under UV (254 nm) light.

CALCULATIONS: 20.

Obtain the concentration of the analyte in the urine sample by comparing its absorbance with the calibration graph. NOTE: Corrections for extraction efficiency are not needed since standards are prepared in urine and both standards and samples are treated the same way.

GUIDES TO INTERPRETATION: In this laboratory, normal ranges of urine specimens from NIOSH employees not exposed to benzidine or aromatic amines where: Number of Urine Specimens

Aromatic Amine Conc. (µg/100 mL)

10

<0.1

2

0.1 to 0.2

1

0.2

1

0.3

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 8/15/93