3-BROMOPROPIONIC ACID in URINE: METHOD 8324, Issue 1, dated 29 August 2014 - Page 3 of 9
11. Repeat the extraction (steps 9 and 10) three more times. Collect and combine all ethyl acetate extracts using a glass pipet into a 16 X 150 mm culture tube. 12. Dry the ethyl acetate extract by adding approximately 100 to 200 mg of anhydrous magnesium sulfate and swirl for about 15 seconds. 13. Transfer the extract solution into a 16 X 150 mm culture tube by means of a glass funnel with silanized glass wool patch to remove the wet magnesium sulfate. The glass wool patch must be packed tightly enough to prevent particles of magnesium sulfate from passing through. 14. Rinse the tube and the funnel with ethyl acetate to ensure complete transfer. 15. Concentrate the combined ethyl acetate extract for each sample to 1 mL using a nitrogen sweep at room temperature and transfer the solution to a 2-mL GC autosampler vial. 16. tert-Butyldimethylsilane derivatization: Add 50 μL of MTBSTFA with 1% TBDMCS silanizing reagent to each autosampler vial and cap immediately. 17. Heat the solution for 1.5 hours at 70 oC in a heating block or oven. CALIBRATION AND QUALITY CONTROL: 18. 3-Bromopropionic acid (3-BPA) standards are prepared in blank, non-exposed urine. The 1 mg/mL stock 3-BPA solution is diluted in deionized water to make 0.4, 1, 2, 4, 8, 20, 80, 200, 400, 600, and 800 μg/mL 3-BPA solutions for spiking. 19. Transfer 2.0 mL of non-exposed urine into a 16 X 100 mm (or larger) screw-capped culture tube. 20. Acidify by adding 40 μL of concentrated hydrochloric acid. 21. Add 0.5 mL of the 20 μg/mL 3-CPA internal standard solution. 22. Add 0.5 mL of the appropriate 3-BPA spiking solution described in step 18 to make urine samples equivalent to 0.1, 0.25, 0.5, 1, 2, 5, 20, 50,100, 150 and 200 μg/mL of 3-BPA in the original 2.0 mL volume of urine. 23. Prepare at least one blank urine without a 3-BPA spike to verify the source of blank urine contains no detectable quantity of 3-BPA. 24. Prepare at least two levels of quality control (QC) standard of 3-BPA fortified urine using a separately weighed and prepared 3-BPA stock solution. One level should be within the lower 25% of the calibration curve and one level within the upper 25% of the calibration curve. More than two QC levels can be used. QC samples should be analyzed with every batch such that they constitute at least 5% of the sample batch. 25. QC values should be within ±20% of the spiked values. If not, the batch is considered out of control, the batch data discarded, and corrective actions should be taken before more samples are analyzed. 26. Ethyl acetate extraction: Prepare the spiked standard urine samples, the blank urine sample(s), and the QC standards the same as described in the preceding Sample Preparation section using Steps 9 through 17. MEASUREMENT: 27. Set the gas chromatograph according to the manufacturer’s recommendations and to the conditions listed on page 8324-1. 28. Set the mass selective detector to selected ion monitoring mode for ions m/z 211 (derivative of 3-BPA) and 165 (derivative of 3-CPA). NOTE: The use/non-use of qualifier ions for this method is discussed in the literature [9]. 29. Inject 0.5 μL of each sample, standard, blank, and QC standard extract from Steps 17 and 26. 30. Measure the peak areas of the tert-butyldimethylsilane derivatives of 3-BPA and 3-CPA in the chromatograms of the standards. 31. Divide the peak area of the derivative of 3-BPA by the peak area from the derivative of 3-CPA in the same chromatogram.
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition
