# Aniline and o-Toludine in Urine (8317)

​ANILINE and @-TOLUIDINE in urine (1) Aniline: C6 H7 N (2) @-Toluidine: C7 H9 N
MW: 93.13 MW: 107.16
METHOD: 8317, Issue 1
CAS: 62-53-3 CAS: 95-53-4
8317 RTECS: BW6650000 RTECS: XU2975000
EVALUATION: PARTIAL
Issue 1: 15 March 2003
BIOLOGICAL INDICATOR OF: exposure to (1) aniline and (2) @-toluidine ACGIH BEI: none SYNONYMS: (1) (2)
aniline: benzeneamine; aminobenzene; phenylamine @-toluidine: 2-aminotoluene SAMPLING
MEASUREMENT
SPECIMEN:
Urine
TECHNIQUE:
HPLC with electrochemical detection
VOLUME:
At least 4 mL of sample
ANALYTE:
Aniline and @-toluidine
TREATMENT:
Base hydrolysis and liquid-liquid extraction
PRESERVATIVE: 5 g citric acid SHIPMENT:
SAMPLE STABILITY: CONTROLS:
Freeze urine; ship on dry ice in an insulated container
Sample stable for over 6 months at -65 o C Collect urine from non-occupationally exposed workers
INJECTION VOL.: 50 :L MOBILE PHASE: 37:53 methanol-phosphate buffer (pH 3.3), containing - 67 mg/L sodium dodecyl sulfate. FLOW RATE:
0.8 mL/min
GUARD CELL:
1000 mV
DETECTOR:
Dual electrode coulometric electrochemical monitored at 600 mV.
COLUMN:
Highly endcapped C1 8 -RP column (Waters NovaPak), 300-mm x 4.6-mm, heated to 30 o C with 0.2 :m inline filter and guard column of same sorbent.
CALIBRATION:
Analyte in mobile phase.
QUALITY CONTROL:
Analyte in urine at 0, 4, 20, 100 ng/mL.
RANGE:
1.4 to 1200 ng/mL
ESTIMATED LOD: Aniline 1.4 ng/mL @-Toluidine 0.6 ng/mL PRECISION ( þ r ):
Aniline Acetanilide @-Toluidine n-Acetyltoluidine
0.17 [4] 0.16 @ 18 ng/mL 0.20 0.17 @ 16 ng/mL; 0.10 @ 200 ng/mL
APPLICABILITY: This method, monitors the parent compounds and their acetyl metabolites to remove the ambiguity from the aminophenol’s origin. INTERFERENCES: No interferences were observed for aniline or @-toluidine OTHER METHODS: El Bayoumy developed a biological monitoring method for aniline and @-toluidine in 1986 [3], in which each liter of sample spiked with internal standard was lyophilized, reconstituted, concentrated, liquid/liquid extracted 3 times, evaporated to dryness, reflux hydrolyzed for 2.5 hrs, liquid/liquid again extracted 3 times, evaporated to dryness, reacted with pentafluoropropionic acid, before being analyzed by GC-ECD.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ​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 ​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 ​ANILINE and @-TO LUIDIN E in urine: MET HO D 831 7, Issue 1, dated 15 M arch 200 3 - Page 4 o f 6 Calibration Solution
Volume of Stock Standard Solution (mL) 100 µg/L
Volume of M ob ile Phase
(µg/L)
100 0 µg /L
(mL)
1.
100
5.0
45.0
2.
80
4.0
46.0
3.
60
3.0
47.0
4.
40
2.0
48.0
5.
20
1.0
49.0
6.
10
5.0
45.0
7.
8
4.0
46.0
8.
6
3.0
47.0
9.
4
2.0
48.0
10.
2
1.0
49.0
The calibration solutions are stable a 4 oC for at least 2 w eeks . It is recom m ended to, prepare half of the calibration solutions fresh with each s et of sam ples. Alternate the use of duplicate primary stoc k stand ard s olutions so that every other se t of ca libration s olutions is m ade from the alternate stock standard solutions. 20. Analyze these 10 calibration solutions with each b atch of sam ples, so that every 2 field sam ples are bracketed with calibration solutions. 21. Because the calibration results are non-linear at low concentrations a quadratic curve should be prepared using data from the calibration solutions. Make the nominal mass of amine in pg injected the independent X variable and its peak height response the dependent variable of the calibration curve.
whe re a, b, and c are regress ion co efficients 22. Also, prepare quality control samples by collecting 1 L of fresh urine from unexposed non-smoking individuals, who are not taking medication. 23. To one liter of this fresh urine add 100 g of citric acid. 24. Add 25, 5, and 1 mL of stock 1000-µg/L standard solution to three 250-mL volumetric flasks, and dilute to the mark with the acidified urine. The nominal concentrations of these samples, i.e. 100, 20, and 4 µg /L, respectively, mus t be corrected for the averag e conce ntration of @-toluidine and aniline in the unfortified urine. 25. After each standard solution is added to urine, the fortified and unfortified urine is aliquoted into 10mL polypropylene scintillation vials and stored at -65 oC. 26. Analyze these quality control samples with each batch and maintain quality control charts. 27. Also, analyze at least one replicate sample from a previous batch with each new batch to assess precision with variable matrices. 28. Blind field splits are also recomm ended as a check on method precision.
MEASUREMENT: 29. Set the chromatograph according to manufacturer’s recomm endations and to the conditions given on page 1. Inject 50 :L of final solution in step 16. NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ​ANILINE and @-TO LUIDIN E in urine: MET HO D 831 7, Issue 1, dated 15 M arch 200 3 - Page 5 o f 6 30. Measure peak heights of aniline and @-toluidine in the calibration solutions a nd in the sa m ples. 31. Peak purity can be confirmed, if necessary, by re-analyzing the extract and three calibration solutions, once with Electrode 2 set at 520 mV and once at 600 mV. The response ratios of the unknown at these two voltages should match the response ratio of the calibration solutions within ± 3 standard deviations. 32. If the sample gives a peak for aniline and @-toluidine with a peak height out of the calibration range, re-analyze the extract using a 5 µL injection. If the 5 µL injection still gives a peak out of the calibration range, reduce the detector gain.
CALCULATIONS: 33. Calculate the concentration, C(pg/:L), of aniline or @-toluidine in the unknow n from the peak heigh ts and the calibration curve for aniline or @-toluidine , resp ective ly (C, :g/L).
a, b, c, and Y are defined in step 21 D = :L of injection volume E = initial volume of urine F = volum e of 0 .1 M HC l back extra ctan t G = initial volume of butyl chloride H = transferred volume of butyl chloride This equation does not account for a change in detector gain.
GUIDES TO INTERPRETATION: This method was applied to 171 urine specimens from a chemical plant worker population with a know n exces s of bladder can cer. The m edian levels of @-toluidine were: expos ed p reshift = 11 :g/L; exposed postshift = 65 :g/L; nonexposed preshift = 0.7 :g/L; and n one xpo sed pos tshift = 2.6 :g/L. The m edian levels of aniline were: exposed preshift = 11 :g/L; expo sed pos tshift = 23 :g/L; nonexposed preshift = 2.0 :g/L; and nonexposed postshift = 3.2 :g/L. [4-8] The Karam El-Bayoumy et al. study [3] reported the total mass of aniline and @-toluidine excreted by urine of 19 occ upa tionally exp ose d su bjec ts, and they found that the work ers exc reted from 0.02 to 8.8 :g of aniline and 0.3 to 12.9 :g of @-toluidine during a 24-hour period.
EVALUATION OF METHOD: The American Conference of Governmental Industrial Hygienists has recom m ended a biological expos ure index (BEI) for aniline of 50 mg of 4-aminophenol per milligram of creatinine, measured in end-of-shift urine specimens. 4-Am inophen ol is the ring hydroxylated m etab olite of aniline [1]. Before a BE I for @-toluidine can be established, a relationship between exposure to o-toluidine and excretion of the metabolite must be established. Rat studies sugg est that @-toluid ine is also substantially metabolized by a ring hydroxylation pathw ay, but no one has dem ons trated the presenc e of th e m etab olite in the u rine of hum ans exp ose d to o-toluidine [2]. Other xenobiotics, I.e.. acetoaminophen, reach the same m eta bolic fate of 4-aminophenol as does aniline. T his m etho d, m onitors the parent compounds and their acetyl metabolites to remove the ambiguity from the aminophenol’s origin. The method was characterized during a field study that included 45 batches of samples. Each batch contained QC sam ples of aniline with no m inal levels of aniline sp iked at 6.9 ng/m L, 18 ng/m L, and 77 ng/m L and with nom inal levels of @-toluidine spiked at 4.2 n g/m L, 20 ng/m L, and 10 2 ng /m L. QCs sam lpes were also made using aceta nilide and N-acetyl-@-toluidine spiked to an equivalent free am ine co nce ntration of 19 ng/m L NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ​ANILINE and @-TO LUIDIN E in urine: MET HO D 831 7, Issue 1, dated 15 M arch 200 3 - Page 6 o f 6 and 16 ng/mL, aniline and @-toluidine. The precision and recovery data reported on page 8317-1 was an average of these levels, a nd re porte d be low in a m ore d etailed resu lts table. In add ition, blind field splits were also use d to chara cterize the precision of the m etho d. RECOVERY [4]
aniline aniline aniline acetanilide @-toluidine @-toluidine @-toluidine N-acetyltoluidine
PRECISION ( þ r) [4] aniline aniline aniline acetanilide @-toluidine @-toluidine @-toluidine N-acetyltoluidine
109 97 93 96 101 93 86 83
0.26 0.14 0.12 0.16 0.32 0.17 0.14 0.17
6.9 ng/m L 18 ng/m L 77 ng/mL 19 ng/m L 4.2 ng/m L 20 ng/m L 102 ng/m L 16 ng/m L
6.9 ng/m L 18 ng/m L 77 ng/mL 19 ng/m L 4.2 ng/m L 20 ng/m L 102 ng/m L 16 ng/m L
Du plicate blind splits Pairs of duplicates 43 Range (ng/mL) 10 - 350 Average Relative Standard Deviation 23 %
REFERENCES: [1]
[2] [3] [4]
[5]
[6]
[7]
[8]
ACG IH [1986]. Am erican Con ference of G overnm ental Industrial Hygienists: Biological Exposu re Indice s, 5 th ed, pp. BE151-BE153. Cincinnati, OH: Am erican Conference of Governmental Industrial Hygienists. Cheever KL, Richards DE , Plo tnik HB [1980]. Meta bolism of o rtho-, m eta -, and para-T oluidine in the A dult M ale R at, Toxicol. Appl. Pharm aco l. 56:361-369. El-Bayoum y K, Donahue JM, H echt SS, H offm ann D [1986]. Identification and Qua ntitative Determination of Aniline and Toluidines in Hum an Urine. Cancer Res 46:6064-6067. Brown K K, Teas s AW , Simon S, W ard EM (1995). A Biological Monitoring Method for @-Toluidine and Aniline in Urine Using High Performance Liquid Chromatography with Electrochemical De tection , App l. Occup . Environ. H yg., 10(6):557-565. Teass AW, DeBord DG, Brown KK, Cheever KL, Stettler LE, Savage RE, W eigel W W , Dankovic D, W ard E [1993]. Biological Monitoring for Occupational Exposures to @-Toluidine and Aniline, Int. Arc h. O ccu p. En viron. H ealth, 65:S115-S118. Stettler LE, Savage RE, Brown KK, Cheever KL, W eigel W W , DeBord DG, Teass AW , Dankovic D, W ard EM [1992]. Biological Monitoring for Occupational Exposures to @-Toluidine and Aniline, Scand J. W ork Environ. H ealth; 18: Supple 2: 78-81. W ard EM, Sabbioni G, DeBord DG, Teass AW, Brown KK, Talaska GG, Roberts DR, Ruder AM, Streicher RP [1996]. Monitoring of Arom atic Am ine Expo sures in W ork ers at a C hem ical Plant W ith a Kn own Bladder C anc er Exce ss, J . Nat. Can cer Inst., 88(15): 1046-1052. Ru der A M, W ard E M, R obe rts D R, T eas s AW , Brow n KK , Fingerhu t MA , Stettler L E [19 92]. Re spo nse of the National Ins titute for O ccu pation al Safety and Health to an O ccu pation al He alth Risk from exposure to @-Toluidine and Aniline, S can d J. W ork Environ. H ealth, 18: Supple 2: 82-84.
METHOD DEVELOPED BY: Kenneth K. Brown, Ph.D., NIOSH/ DART
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition