HIPPURIC ACID IN URINE: METHOD 8300, Issue 2, dated 15 August 1994 - Page 3 of 3 13. 14. 15.
Stop the reaction by adding 5.0 mL ethanol, followed by mixing on a vibration mixer. Centrifuge at 1500 to 2000 RPM (full speed) for 5 min to reduce turbidity. Remove supernatant with pipet and place in a 1-cm cuvette. Read absorbance at 410 nm using ethanol to zero the instrument. NOTE: If absorbance is above the calibration range, discard the sample and start a new sample (beginning at step 5) with greater dilution.
CALCULATIONS: 16. 17.
Determine the concentration, C s (g/L), of hippuric acid corresponding to the absorbance of the sample from the calibration graph. Calculate the concentration of hippuric acid/g creatinine in the urine sample, C (g/g creatinine), using the dilution factor, D (usually 5), from step 5 and the creatinine value, C r (g creatinine/L urine), from step 4:
GUIDES TO INTERPRETATION: 1. 2. 3. 4.
Tomukuni, et al. [4] reported a normal range in 20 non-exposed adults as 0.44 ± 0.20 g/L (equivalent to approximately 0.7 g/g creatinine). Lauwreys [2] reported a normal range of 1.5 g/g creatinine and a "tentative maximum permissible value" of 2.5 g/g creatinine. Pagnotto, et al. [5] reported that exposure to 100 ppm toluene would produce end-of-shift urinary hippuric acid levels of 4 g/L (equivalent to about 5 g/g creatinine). Urine from non-exposed workers should be collected, as well as pre-shift specimens from exposed workers because levels of hippuric acid in urine vary widely.
EVALUATION OF METHOD: Precision (s r) for within-run and day-to-day runs over the analytical range was found to average 0.06. No method comparison studies have been conducted.
REFERENCES: [1] [2] [3] [4] [5]
NIOSH Manual of Analytical Methods, 2nd Ed., Vol. 6, Method P&CAM 327, U.S. Department of Health and Human Services, Publ. (NIOSH) 80-125 (1980). Lauwerys, R.R. Industrial Chemical Exposure : Guidelines for Biological Monitoring , Biomedical Publications, Davis, California, 57-65 (1983). Tietz, N.W. Fundamentals of Clinical Chemistry , 2nd ed., pp. 994-999, W.B. Saunders Co., Philadelphia, PA (1976). Tomokuni, K. and M. Ogata. Direct Colorimetric Determination of Hippuric Acid in Urine, Clin. Chem. 18, 349-351 (1972). Pagnotto, L.D. and L.M. Lieberman. Am. Ind. Hyg. Assoc. J., 28, 129-134 (1967).
METHOD REVISED BY: Frederick C. Phipps, NIOSH/DBBS.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 8/15/94
