BENZIDINE in urine: METHOD 8306, Issue 2, dated 15 August 1994 - Page 4 of 4 under a gentle stream of nitrogen. CALIBRATION AND QUALITY CONTROL: 19.
20.
Prepare six working standards containing between 0 and 10 µg benzidine. a. Add aliquots of benzidine stock solution to 1.5 mL benzene and 0.5 mL trimethylamine working solution. b. Perform steps 12 and 13. c. Percolate the benzene layer through 2 cm anhydrous sodium sulfate. d. Add 0.5 mL derivatized methylenedianiline stock solution. e. Dilute to 10.0 mL with benzene. f. Analyze the working standards together with samples and blanks (step 21 and 22). g. Prepare calibration graph (concentration of standards vs. ratio of peak areas of benzidine to methylenedianiline). Analyze a spiked urine for every 10 samples (minimum three per study).
MEASUREMENT: 21. 22.
Set the chromatograph according to manufacturer's recommendations and conditions given on page 8306-1. Inject 3 µL derivatized sample extract (step 18). Measure peak areas of both methylenedianiline and benzidine. Calculate the ratio of peak areas (benzidine to methylenedianiline). The t r of methylenedianiline is 7.6 min while the t r of benzidine is 8.0 min under these conditions.
CALCULATIONS: 23.
Determine benzidine urine concentrations (µg/mL) by comparing the ratio of benzidine methylenedianiline peak areas for the samples to those obtained for standards on the calibration graph. Report the results as µg benzidine/g creatinine.
GUIDES FOR INTERPRETATION: Benzidine is an OSHA-regulated human carcinogen. Although no specific standard has been set, the fact that benzidine is a human carcinogen means it or its metabolites should not be detected in urine or any other physiological fluid. However, a level of 0.010 µg/mL has been suggested as an indicator of excessive exposure [1].
REFERENCES: [1] [2]
[3] [4]
Baselt, R. C. Biological Monitoring Methods for Industrial Chemicals , 43, Biomedical Publications, Davis, CA (1980). Nony, C. R., and M. C. Bowman. Trace Analysis of Potentially Carcinogenic Metabolites of an Azo Dye and Pigment in Hamster and Human Urine as Determined by Two Chromatographic Procedures, J. Chromatographic Sci., 18, 64 (February, 1980). Benzidine-Based Dyes, 46, U.S. Department of Health and Human Services, Publ. (NIOSH) 80-109 (1980). Tietz, N. W. Fundamentals of Clinical Chemistry , 2nd ed., 994-997, W. B. Saunders Co., Philadelphia, PA (1976).
METHOD REVISED BY:
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 8/15/94
