HIPPURIC and METHYL HIPPURIC ACIDS in urine: METHOD 8301, Issue 3, dated 15 March 2003 - page 3 of 5 6. Add 4 mL ethyl acetate. Mix for 2 minutes by rotation. 7. Centrifuge at 100 x gravity for 5 minutes.
NOTE:
r max = rotor radius Re lative Centrifu gal Force (R CF ) = gra vity 8. Transfer 200 µL of the organic (upper) layer to an HPLC vial and evaporate to dryness (- 30 minutes) using a heated (-30 oC) water bath and a gentle stream of nitrogen. 9. Re-dissolve the residue in 200 µL of distilled water.
CALIBRATION AND QUALITY CONTRO L: 10. Prepare working sta ndards over the ran ge of 1 0 to 1000 µg/m L by dilution of combined hippuric acids stock solution using synthe tic urine. W orking standards are stable for 1 week at room temperature and stab le for 30 da ys at 4 oC [1]. 11. Include control samples from an unex pos ed, m atch ed p opu lation with each an alytical run. If controls were not sent, use pooled, unexposed human urine. 12. Extract and analyze the working standards together with samples, and controls (steps 4 through 9, 14 and 15). 13. Analyze a dup licate field sample injection at a frequency of one replicate per 10 samples to monitor instrum ent ac curacy.
MEASUREMENT: 14. Set up the HPLC according to the manufacturer’s recomm endations and to conditions on page 8301-1. 15. Inject 10 µL of the standards, samples, and control samples into the HPLC. Determine peak heights.
CALCULATIONS: 16. Ca lculate the concentration of each analyte in the urine sample, C u (µg/m L), us ing the standard curve to correlate the peak heigh t with the con cen tration a m oun t. 17. Ca lculate the grams of analyte per grams of creatinine in the urine sample, C (g/g creatinine), using the concentration of an alyte in the urine sam ple, C u (µg/m L), and the concentratio n of creatin ine in urine dete rm ined in step 3, C R (g creatinine/L urine).
GUIDES TO INTERPRETATION: 1. Typical exc retion of h ippuric acid in non-exposed individuals is 1.0 g/g creatinine [2]. Meth yl hippuric acids are not found in non-exposed humans. 2. Th e T hres hold Lim it Value (TL V® ) [9] for u rine sam ples collected at the en d of a work shift is 1.6 g/g creatinine for hippuric acid a nd 1 .5 g/g crea tinine for m ethyl hipp uric acids.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition
