ANILINE and @-TO LUIDIN E in urine: MET HO D 831 7, Issue 1, dated 15 M arch 200 3 - Page 3 o f 6 SAMPLING: 1. Timing of Collection: In humans, aniline is rapidly metabolized and excreted in the urine [1]. Rat data sug ges t that @-toluidine likewise is rapidly metabolized and excreted [2]. Thus, biological monitoring using pre and post shift urine samples is recomm ended in order to capture the metabolites quickly after exposure. 2. Collect urine in one or more 250-mL polypropylene bottles. Measure the total volume and transfer app roximately 50 m L to a 60-m L po lypropylen e bo ttle con taining 5.0 g of citric a cid prese rvative. Label the bottle with the code unique to that specimen. Freeze imm ediately on dry ice. 3. Ship in a packing container that is designed for dry ice storage and transport. Store at -65 oC.
SAMPLE PREPARATION: 4. W ith each batch of 20 field urine samples, also process 2 quality control samples, 2 water blanks, and 2 field samples analyzed in a previous batch, a total of 26 samples per batch. 5. Assem ble 26 conical centrifuge tubes in a racks. 6. Add 1.00 ± 0.05 g of NaOH pellets, cap, and label tubes. 7. Thaw sam ples to room temperature. To m inimize analyte loss, keep caps tightly sealed and minimize time between steps 7-14. NOTE: Frozen urine samples are inhomogeneous. Insure that the samples are completely thawed and well m ixed befo re rem oving aliquot. 8. Ad d 4.0 m L of sam ple to each tube and re cap tightly. 9. Heat for 2 hr ± 5 minutes at 80 oC in a water bath. 10. Cool to room temperature, add 8.0 mL of butyl chloride to tubes and recap. 11. Tum ble tubes for 10 minutes at 50 rpm, and centrifuge for 5 minutes at 3000 rpm. 12. Transfer 5.0 mL of the upper butyl chloride layer to a second set of labeled centrifuge tubes. 13. Add 1.0 mL of 0.1 N HCl to tubes containing butyl chloride solution. 14. Tum ble tubes for 10 minutes at 50 rpm, and centrifuge for 5 minutes at 3000 rpm. 15. Rem ove lower aqueous layer with a Pasteur pipette, and transfer to a 3 cc syringe barrel fitted with a 10-m m 0.2 µm filter. 16. Insert syringe plunger and inject the aqueous extract through the filter into a HPLC auto-injector vial and seal. 17. Order the samples and standard solutions in the auto-injector carrousel in a fixed pattern of two standard solutions bracketing every two sample extracts, e.g. standard, unknown, unknown, standard, unknown ......., standard. W ithin the fixed order randomize the standards and samples separately. 18. Analyze sam ples by HPLC (Steps 29 to 32).
CALIBRATION AND QUALITY CONTRO L: 19. Label ten 50-m L volum etric fla sk s with th e concentratio ns listed in the second colum n of the table below. Using the table below, prepare the standard solutions listed by diluting the indicated volume of stock standard solution to 50 mL with mobile phase.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition
