MBOCA in urine: METHOD 8302, Issue 2, dated 15 August 1994 - Page 3 of 4 SAMPLING: 1. 2. 3.
Collect 50 to 100 mL urine in a 125-mL polyethylene bottle containing 3 mL 30% citric acid. Cap the bottle immediately after sample collection. Swirl gently to mix. Ship the sample in dry ice in an insulated container.
SAMPLE PREPARATION: 4. Thaw urine sample. 5. Perform a creatinine determination on an aliquot of the urine (e.g., [5]). 6. Pipet a 5-mL aliquot of urine into a clean culture tube. 7. Adjust pH to >12 using 10 N NaOH (ca. 0.1 mL). 8. Add 1 mL methanol. Mix well. 9. Add 5 mL 1:1 diethyl ether:hexane. Shake steadily for 2 min to extract the MBOCA. Centrifuge to separate phases. Transfer the organic phase by Pasteur pipet to a culture tube. Repeat with two additional 5-mL portions of 1:1 diethyl ether: hexane, combining the organic phases. NOTE: Emulsions may form; break them by adding methanol or by repeated centrifugation. 10. Concentrate the extract to ca. 1 mL under a gentle stream of nitrogen in a hood. 11. Add 50 µL TEA, 50 µL HFBA, and 25 µL MDA to the concentrated extract. Mix well in a hood. 12. Secure the cap loosely. Heat in a waterbath at 50 °C for 15 min. 13. Remove from waterbath. Add 2 mL hexane and 5 mL KH 2PO 4 buffer. Mix well. 14. Centrifuge to separate phases completely. Transfer the organic layer containing the derivative, without disturbing the aqueous phase, with a Pasteur pipet into a 15-mL centrifuge tube. 15. If low concentration (<5 µg/L urine) is expected, use Florisil cleanup (see APPENDIX). Add 0.2 g Na 2SO 4 and mix.
CALIBRATION AND QUALITY CONTROL: 16. 17. 18.
Prepare working standards over the range 0 (control) to 200 µg/L urine by adding aliquots of calibration stock solution to a control urine. Extract 5 mL of each working standard (steps 6 through 15). Analyze the working standards with the samples (steps 20 and 21). Prepare calibration graph (ratio of peak area of analyte to peak area of MDA vs. µg MBOCA/L urine).
MEASUREMENT: 19. 20. 21.
Set gas chromatograph according to manufacturer's recommendations and to conditions on page 8302-1. Inject 1 µL hexane extract from step 15. Measure the peak areas of the samples and internal standard. Divide the peak area of the sample by the peak area of the internal standard on the same chromatogram.
CALCULATIONS: 22. 23.
Determine the MBOCA concentration in the urine sample, C u (µg/L), from the calibration graph. Calculate the concentration of MBOCA/g creatinine in the urine sample, C (µg/g creatinine), using the creatinine value (C r) obtained in step 5.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 8/15/94
