HIPPURIC ACID IN URINE: METHOD 8300, Issue 2, dated 15 August 1994 - Page 2 of 3 EQUIPMENT:
REAGENTS: 1.
2. 3. 4. 5.
Calibration stock solution, 0.5 g/L. Dissolve 50 mg hippuric acid in 100 mL distilled water. Stable one month at 25 C. Benzenesulfonyl chloride.* Pyridine, reagent.* Thymol, USP. Ethanol, absolute.
See Special Precautions.
1. 2.
3. 4. 5. 6. 7. 8. 9.
Bottles, polyethylene, 125-mL. Spectrophotometer, 1-cm light path, to read at 410 nm, band width < 10 nm, with 1-cm cuvettes. Centrifuge, clinical, table top. Centrifuge tubes, 15-mL, conical. Pipets, serological, 0.5-, 1.0-, and 5.0-mL Pipet bulb. Mixer, vibration. Volumetric flasks, 10- and 100-mL. Graduated cylinder, 10-mL.
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. Work with pyridine and benzenesulfonyl chloride only in a fume hood.
SAMPLING: 1.
2.
Collect a spot urine sample of 50 to 100 mL in a 125-mL polyethylene bottle containing a few crystals of thymol. NOTE: Take the sample at the end of the second day of suspected exposure to toluene. Also take pre-exposure samples and samples from non-exposed workers as controls. Pack the samples in an insulated shipper with bagged refrigerant.
SAMPLE PREPARATION: 3. 4. 5.
Thaw the urine samples, if frozen. Perform a creatinine determination on an aliquot of the urine (e.g., [3]). Dilute 1 volume urine with 4 volumes distilled water.
CALIBRATION AND QUALITY CONTROL: 6. 7. 8. 9.
Prepare working standards over the range of 0.005 to 0.5 g/L by dilution of calibration stock solution. Working standards are stable for one week at room temperature. Analyze the working standards (steps 10 through 15). Include a frozen pooled urine control with each measurement run. Plot the absorbance at 410 nm against hippuric acid concentration (g/L) in the working standard to prepare the calibration graph.
MEASUREMENT: 10. 11. 12.
Mix 0.5 mL diluted urine and 0.5 mL pyridine in a conical centrifuge tube. Add 0.2 mL benzenesulfonyl chloride and mix for about 5 sec on a vibration mixer. Let stand for 30 min at 20 to 30 °C. NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 8/15/94
