ANILINE and @-TO LUIDIN E in urine: MET HO D 831 7, Issue 1, dated 15 M arch 200 3 - Page 2 o f 6 EQUIPMENT:
REAGENTS: 1. Aniline and @-toluidine stock solution, 100 mg/L, is prepared by dissolving 100 mg @tolu idine and 139 m g of an iline hydrochloride in 1 L of 0 .1 N Hc l. 2. Aniline and @-toluidine calibration solution, 1000 :g/L. Dilute 1.000 mL of the stock solution with water to 100 mL to produce a 1000 :g/L solution for spiking. 3. Aniline and @-toluidine calibration solution, 100
- g/L. Dilute 1.000 mL of the above 1000 :g/L
calibra tion so lution with wate r to 100 m L to produce a 100 :g/L solution. These stand ard solutions are stable at 4 oC for at least two weeks. 4. Mobile phase: Add 23.0 g of NaH 2PO 4CH 2O and 6 m L of 8.5 % phosph oric acid to a 2-L volum etric flas k. Bring to n ear volum e with wate r, m ix, and adjust pH to 3.3 ± 0.05 with 8.5 % phosphoric acid or 50% sodium hydroxide, then bring to 2L mark. Add 1226 mL of m eth anol and 200 ± 0.5 m g of sodium dodecyl sulfate to a 4-L HPLC resevoir and mix. Blend the aqueous buffer with the methanol solution to prepare the m obile phase. Tota l liquid volume will shrink when liquids are mixed. 5. W ater, highly purified. >10 MS. 6. Methanol, HPLC grade. 7. Sodium dihydrogen phosphate monohydrate, NaH 2PO 4 CH 2O. 8. Sodium dodecyl sulfate. 9. Aniline hydrochloride.* 10. @-toluidine .* 11. Phosp horic acid 8 5% .* 12. Butyl chloride, HP LC grad e.* 13. Sodium hydroxide, reag ent grade pellets.* 14. Hydroch loric ac id 0.1 N , Fisher ce rtified.* 15. Citric acid, anhydrous, reagents grade. 16. Urine from non-occupationally exposed persons, who have no exposure to tobacco sm oke.*
1. Bottles, polypropylen e, sc rew-top, 8-oz, 250-mL. 2. Bottles, polypropylene, screw-top, 2-oz, 60mL. 3. HPLC system consisting of the following components in series; 5-L reservoir, in-line degas ser filter, pum p, pulse dam pener, electroch em ical guard c ell (1.0 V ), auto injector, in-line 0.2 :m in-line filter, a guard column, a 300 mm X 4.6 mm analytical column in an oven set at 30 °C, dual electrode coloumetric electrochemical detector connected to a data acquisition system, and a liquid waste container. The column packing material is highly endcapped 3 :m C 18-RP pa rticles (W aters NovaPak or equivalent). The detector has two e lectrodes , one of wh ich is used to remove potential interferences oxidizing below 40 0 m V an d the other for analyte detection at 600 mV. 4. pH m eter. 5. Vortex m ixer. 6. Rotatory m ixer. 7. Centrifuge tubes, 15-mL with teflon lined caps, disposable. 8. Reagent dispenser, 8-mL. 9. Auto-injector vials. 10. W ater bath at 80 °C. 11. Dispensing pipette, 2-mL and 5-m L, disposable. 12. Micropipette, 1000-µL 13. Pasteur transfer pipettes. 14. Volumetric pipettes *, 1-, 2-, 3-, 4-, 5-, and 10-mL. 15. Syringes, 3-cc plastic disposable. 16. Syringe filters, AnotopTM 10, 0.2 µm pore size. 17. Volumetric flasks *, convenient sizes for preparing standard solutions. 18. Scintillation vials, 10-mL polypropylene.
- See SPECIAL PRECAUTIONS
W ash all perm ane nt glas sware w ith 0.1 N NaO H, 0.1 N HCl, water, and then methanol before use.
SPECIAL PRECAUTIONS: Urine samples may contain a number of bacterial and viral agents, including hepatitis B virus, and should be handled using Biosafety Level 2 practices, containment equ ipm ent, and fa cilities. [CD C & NIH , Biosafety in Microbiological and Biomedical Laboratories, 3rd ed., HHS Publication No. (CDC ) 93-8395 (19 93)]. Methanol and bu tyl chloride are NFP A level 3 fire hazards. Concentrate d phosphoric acid and sodium hydroxide are a N FP A level 3 health and level 2 reac tivity hazards, besides be ing co rrosive. @-Toluidine is a bladder carcinogen, and may cause damage to the blood or kidneys. Aniline may be fatal if swallowed, inhaled, or absorbed through the skin. Aniline causes irritation to skin, eyes, and respiratory tract, and may cause cyanosis.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition
