POLYCHLORINATED BIPHENYLS in serum: METHOD 8004, Issue 2, dated 15 August 1994- Page 4 of 4 SAMPLE PREPARATION: 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
Pipet 5 mL serum into a clean culture tube containing 4 mL methanol. Cap the tube and mix 4 min on a rotary mixer. Add 5 mL hexane-ethyl ether (1:1, v/v). Mix on a rotary mixer for 15 min. Centrifuge at 2000 rpm for 5 min. Transfer the upper solvent layer by pipet into a 25-mL Kuderna-Danish concentrator tube. Repeat steps 7 through 10 twice, combining the extracts in the concentrator tube. Concentrate the extract to 0.5 mL under a gentle stream of dry organic-free nitrogen. Add 2 mL methanolic KOH to the concentrator tube. Add a piece of hollow glass (end of a Pasteur pipet) to prevent bumping. Attach a micro-Synder column and bring the contents to a gentle boil with of a tube heater and reduce the volume to 0.3 mL. NOTE: If a precipitate forms, add a few drops of methanolic KOH solution and warm gently with a tube heater until it dissolves. Cool the solution slightly and add 2 mL 1:1 methanol:water. Add 2 mL n-hexane when the solution reaches room temperature. Stopper and shake the tube vigorously. Rinse the prepared chromatography column (EQUIPMENT, 9.) with 20 mL hexane. As the hexane settles into the sodium sulfate bed, add the sample. Place a 25-mL graduated cylinder below the chromatographic column. Rinse the concentrator tube and the micro-Synder column with 1 mL hexane. As the saponified extract enters the sodium sulfate layer, add the concentrator tube and micro-Synder column rinses to the chromatographic column. As the rinses settle in the sodium sulfate layer, add 25 mL hexane. Collect 25 mL eluate from the column in the graduated cylinder. Rinse the walls of the graduated cylinder with 2 mL hexane. Concentrate to 1 mL under a gentle stream of nitrogen. Add 10 mL DDE solution and analyze (steps 29 and 30).
CALIBRATION AND QUALITY CONTROL: 26. Analyze the bulk sample for PCB content [4]. 27. Calibrate daily with at least five working standards over the range 0.005 to 1 mg/mL PCB. a. Add known amounts of calibration stock solution to n-hexane in 10-mL volumetric flasks and dilute to the mark with n-hexane. b. Analyze the working standards together with samples and blanks (steps 29 and 30). c. Prepare calibration graph (concentration of each PCB standard vs. the sum of PCB peak areas). 28. Analyze at least three pooled or spiked serum samples.
MEASUREMENT: 29. Set gas chromatograph according to manufacturer's instructions and conditions on page 8004-1. Inject 2-µL sample together with 1 mL DDE solution. 30. Measure the PCB peak areas. Calculate the retention time of the analyte peaks relative to that of DDE.
CALCULATIONS: 31. Compare sample retention times (relative to DDE) with those of the working standards and bulk sample to ascertain the valid PCB peaks. NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 8/15/94
