Page:NIOSH Manual of Analytical Methods - 8001.pdf/3

 PENTACHLOROPHENOL IN BLOOD: METHOD 8001, Issue 2, dated 15 August 1994 - Page 3 of 4 7. 8. 9. 10. 11. 12.

13. 14. 15. 16. 17. 18.

Extract twice more with 2 mL hexane for 2 min. Combine the extracts. Concentrate the combined extracts to approximately 0.6 mL under a gentle stream of nitrogen. Add 10 mL diazomethane reagent to the culture tube in a hood. Let stand in a hood for 1 h. Concentrate to approximately 0.6 mL under a gentle stream of nitrogen in a hood. Add 4 mL hexane. Evaporate the hexane 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. Let the column air dry and place it in an oven overnight at 130 °C. Cool the column to room temperature before use. Wet the column with 5 mL hexane in a 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: 19. 20. 21. 22.

Prepare at least 6 working standards in the range 0.01 to 1 µg/mL PCP by dilution of the calibration stock solution with hexane. Prepare a calibration graph (peak area vs. µg/mL PCP). Maintain standardization by injecting a working standard after each five sample injections. Run a spiked pooled control blood sample every ten samples, or if less than ten determinations are required, run at least three spiked pooled blood samples per study. NOTE: All blood that will be utilized for spiked pooled control blood samples must be analyzed for PCP before use because PCP is ubiquitous.

MEASUREMENT: 23. 24.

Set up the gas chromatograph according to manufacturer's instructions and to the conditions on page 8001-1. Inject 5 µL sample extract (from step 18). Measure peak area of analyte. NOTE: The retention time for pentachloroanisole is about 4.7 to 5.0 min.

CALCULATIONS: 25.

Determine the concentration, C (µg/mL), of PCP in blood by comparison of peak areas of samples with the calibration graph.

GUIDES TO INTERPRETATION: Blood PCP levels for unexposed populations range between 0.016 to 0.32 µg/mL PCP [4,5,6]. The Biological Exposure Index is 5 mg/L for free PCP in end-of-shift plasma [7].

EVALUATION OF METHOD: The precision for three sets of three samples of spiked whole blood was 2% relative standard deviation at levels of 0.05 to 1 µg/mL PCP whole blood, with 88 to 93% recovery.

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 8/15/94