PENTACHLOROPHENOL in urine: METHOD 8303, Issue 2, dated 15 August 1994 - Page 3 of 4 12. 13. 14. 15.
16. 17. 18. 19. 20. 21.
Add 10 mL of diazomethane reagent (use hood). Let stand under hood for 1 h. Concentrate under a hood to 0.6 mL under a gentle stream of nitrogen. Add 4 mL hexane. Evaporate the solution to approximately 0.6 mL. Add 4 g acid-washed alumina to a 7-mm ID x 200 mm chromatography column. Cap with 1.6 g anhydrous sodium sulfate. Rinse the column with 20% benzene in hexane. Air-dry. Place the column in a 130 °C oven overnight. Cool the column to room temperature before use. Wet the column with 5 mL hexane (use fume hood). Add the concentrated derivatized extract when the solvent layer reaches the top of the sodium sulfate layer. Rinse the culture tube three times with 0.5 mL hexane. Add the rinsings to the column. Add 3.5 mL hexane to the column. Discard the hexane eluate. Elute pentachloroanisole, the PCP derivative, with 20 mL 10% benzene in hexane. Concentrate the sample to 2.0 mL in a graduated centrifuge tube.
CALIBRATION AND QUALITY CONTROL: 22. 23. 24. 25.
Prepare at least six working standards in the range 5 to 1000 µg/L PCP by dilution of the calibration stock solution with hexane. Prepare a calibration graph (peak area of analyte vs. µg/L PCP). Maintain standardization by injecting a working standard after each five sample injections. Run a spiked urine every ten samples or if less than ten determinations are required, run at least three spiked urines per run or study. NOTE: Urine used for spikes must be analyzed before use to determine background levels.
MEASUREMENT: 26. 27.
Set the gas chromatograph according to manufacturer's instructions and to conditions on page 8303-1. Inject 5-µL sample extract from step 21. Measure peak area of analyte. NOTE: The retention time for pentachloroanisole is about 4.7 to 5.0 min.
CALCULATIONS: 28. 29.
Use the analyte peak area from the samples and the calibration graph to determine the PCP concentration in the extract analyzed, C e (µg/L PCP). Calculate the urine PCP concentration, C (µg/L PCP), by applying the extract concentration factor 2 (4 mL urine yields 2 mL extract): C = 0.5C e, µg/L PCP.
30.
Express PCP concentration as µg PCP/g creatinine.
GUIDES TO INTERPRETATION: Urine pentachlorophenol levels for unexposed populations are reported to be between 20 and 40 µg/L PCP [6,7,8]. Symptoms have been reported at 200 µg/L PCP and levels in excess of 20 mg/L are fatal [7,9]. Lauwerys suggests an upper limit of 1 mg/g creatine [10]. The ACGIH Biological Exposure Index is 2 mg PCP/g creatinine [11].
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 8/15/94
