Page:NIOSH Manual of Analytical Methods - 8322.pdf/2

 TRICHLOROACETIC ACID IN URINE: METHOD 8322, Issue 1, dated 17 April 2015 - Page 2 of 5

REAGENTS:

EQUIPMENT:

1.	Sodium trichloroacetate [CAS #650-51-1], 97% purity or greater* 2.	Boron trifluoride-methanol solution, 14%* 3.	Toluene, ACS reagent grade or better* 4.	Sodium sulfate, anhydrous, granular; reagent grade or better 5.	Acetic acid, glacial; reagent grade or better* 6.	Acetone, reagent grade or better* 7.	Methanol, reagent grade or better* 8.	Helium, purified 9.	Nitrogen, Ultra High Purity or P5 10. Water, ASTM Type II [14]

1.	Centrifuge tubes, polypropylene, ~15-mL, with screw caps, or other suitable container for specimen collection and storage 2.	Gas chromatograph with electron capture detector, autosampler, data collection system and column (page 8322-1) 3.	Microliter syringes, various sizes 4.	Volumetric flasks, glass, various sizes 5.	Adjustable pipettor with disposable plastic tips, 0.1 to 1-mL 6.	Disposable Pasteur transfer pipettes, 15 and 23  cm 7.	Culture tubes, 13 mm x 100 mm (~8 mL), with PTFE-lined caps 8.	Vortex mixer 9.	Glass wool 10.	Vials, autosampler, glass, 2-mL with caps 11.	Oven, capable of maintaining 60 ºC

SOLUTIONS: 1.	1:3 glacial acetic acid:deionized water 2.	1:1 acetone:methanol
 * See SPECIAL PRECAUTIONS

SPECIAL PRECAUTIONS: Standard precautions should always be used when handling bodily fluids and/or extracts of bodily fluids [15]. Handle urine specimens and urine extracts using powder-free latex or nitrile gloves. Acetic acid, acetone, toluene, and methanol are flammable; handle with care and use in a chemical fume hood. Handle all chemicals using the required safety precautions. Reagents with manufacturer expiration dates should be observed. The Environmental Protection Agency (EPA) has designated TCAA as a known mouse carcinogen and a possible human carcinogen [16]. SAMPLING: 1.	Collect at least 10 mL of urine in ~15-mL polypropylene tubes or other suitable container. NOTE:	Because of the relatively lengthy half-life values of TCAA, ACGIH recommends sampling at the end of shift at the end of workweek [1]. 2.	Freeze the urine and ship in dry ice in an insulated container. Reminder: commercial shippers have special labeling requirements for packages containing biological samples and dry ice. SAMPLE PREPARATION: 3.	Thaw urine specimens, bring to room temperature, and mix thoroughly. 4.	Place 200 µL of urine specimen in an 8-mL glass culture tube with a PTFE-lined cap. 5.	Add 0.5 mL 14% boron trifluoride in methanol; cap and mix. 6.	Heat in oven at 60 ºC for a minimum of 1.5 hr (maximum 2.5 hr). 7.	Cool to room temperature and then add 2.0 mL toluene. 8.	Vortex or shake vigorously for 1 min. 9.	After the layers separate, transfer the upper toluene layer to a drying column containing anhydrous sodium sulfate. The drying columns are prepared in 15-cm Pasteur pipettes with a glass wool plug and about 200 to 300 mg anhydrous sodium sulfate, sufficient to form a bed depth of ~1 cm. 10. Collect the eluate in a 2-mL GC vial. Cap vial.

NIOSH Manual of Analytical Methods (NMAM), Fifth Edition