Study of Foreign Ions. About 120 times larger amounts of uranium(VI), 60 times of thorium(IV), 140 times of sodium and potassium, 35 times of rare earths(”, 20 times tungsten(VI), yttrium(III), ammonium; 18 times of zinc(II), 1 2 times of strontium(II), 10 times of copper(I1) and zirconium(IV), 5 times of lead(II), 3 times of antimony(V), tantalum(\’), iron(III), equal amounts of titanium(1V) and 50 times of nitrate, 40 times of chloride and phosphate, 140 times of sulfate did not show significant interference in the estimation of 37.6 pm of niobium in 25 ml by the recommended procedure. However, in the presence of large amounts (greater than 10 times) of zirconium and (i), in presence of 4 to 5 times of tantalum(V), iron(III), aluminum(III), fluoride ion; (ii) in the presence of comparable amounts of nickel(II), tin(I1) and (IV), beryllium(I1); and (iii) in the presence of small amounts of molybdenum( VI), this method gives errors in the range of 10%. The interference of 9 times of iron(II1) could be suppressed completely using ascorbic acid, but for molybdenum(V1) using citric acid as a masking agents, niobium could be estimated with 3% of error by this method. In the following synthetic mixtures in 25 ml of solution, niobium(V) was estimated successfully from (1) niobium (40 pg) with zirconium(1V) (91 pg) and uranium(V1) 3808 pg); (2) niobium(V) (37.16 pg) with zirconium(1V) (4.56 pg)
and uranium (VI) (190 pg); (3) Nb205 (44.59 pg); TazO5 (8.05 pg); Ti02 (1.62 pg); Si02 (0.22 pg); SnOz (1.48 pg); Fez03 (14.77 pg); CaO (0.94 pg); MgO (0.03 pg); A1203 (0.79 pg); Tho2 (1.21 pg); Crz03 (0.81 pg); YzO3 (13.93 p g ) ; MnO (1.60 p g ) ; PbO (2.04 pg); UOz (6.03 pg); NazO (0.29 pg); K20 (0.21 pg) (relative standard deviation of 7 samples is 2.6%); (4) Niobium(V) (92.9 pg) tantalum(V) (90.8 pg); titanium(1V) (11.0 pg); tin(I1) (50 wg); antimony(II1) (50 wg), and tungsten(V1) (800 pg). Niobium estimation by this method in the presence of other metals as given above will be useful for the analysis of fuel elements or other types of alloys where uranium(V1) concentration is high.
LITERATURE CITED (1) I. D. Ali-zade and 0 . A. Gamid-zade, Zh, Anal. Khim., 29,735-9 (1974). (2)A. K. Babko and V. V. Lukachine, Ukr. Khem. Zh., 27,682-7 (1961). (3)V . P. Madhava Menon, N. Mahadevan, K. Srinivasulu, and Ch. Venkateswarulu, J. Sci. hd. Res. (Hardwar, India), 218, 20-23 (1962). (4)V . Patrovsky, Collect. Trav. Chim. Tchec., 23 1774 (1958). (5) S.V. Elinson, L. T. Pobedina, and A. T. Rezova, Zavod. Lab., 37, 391-4
(1971).
RECEIVEDfor review August 21,1975. Accepted December 1, 1975. Financial assistance from the Council of Scientific and Industrial Research, India, to one of the authors (MSR) as an award of Junior Research Fellowship is gratefully acknowledged.
Colorimetric Assay for Aromatic Amines Esther Rinde and Walter Troll” New York University Medical Center, Department of Environmental Medicine, 550 First Avenue, New York, N. Y. 100 16
Aromatic amines can be detected in the nanomole range with the reagent Fluram, with which they form stable yellow derivatives. Fluram In glacial acetic acid reacts only with aromatic amlnes. The reaction is complete in 10 minutes and can be performed on thin-layer (TLC) chromatograms making possible the specific measurement of aromatic amines. The yellow product can be quantitatively eluted from the TLC plates. Fluram is colorless, and the blanks are zero.
The reagent Fluorescamine (Fluram) was introduced by Udenfriend (1) for the quantitation of aliphatic amines in a sensitive fluorescent assay. Aromatic amines, like the aliphatics, form fluorescent products with Fluram which are unstable, but they also form stable yellow derivatives. A number of aromatic amines have been found t o be carcinogens ( 2 ) ;hence, it is important to have sensitive and specific assays for their detection in the environment and in body fluids. Using selective extraction procedures ( 3 ) , the excretion of aromatic amine in the urine of exposed individuals can be measured, and is a good criterion for determining whether exposure has occured. The stability of the product formed when Fluram reacts with aromatic amines makes it possible to perform the assay on TLC from which it can be quantitatively eluted. Moreover by the use of TLC in conjunction with the assay, it is also possible to differentiate one aromatic amine from another ( 4 ) . One of the colorimetric assays we had previously used was a modi542
ANALYTICAL CHEMISTRY, VOL. 48,
NO. 3,
MARCH 1976
fication of the Satake method ( 5 ) using the reagent trinitrobenzene sulfonic acid (TNBS) which reacts with aromatic amines a t pH 5. With TNBS, it is necessary to extract the product formed into organic solvent; otherwise the yellow color of the reagent interferes. Fluram has the advantage of being colorless (blanks are zero), eliminating the need for extraction, and has a sensitivity in the nanomole range.
EXPERIMENTAL Apparatus and Reagents. All solvents were Reagent Grade. Aniline, purchased from Eastman Organic Chemicals was redistilled 2 X . Purified samples of the other amines tested were supplied by Allied Chemical Company: benzidine, 2-naphthylamine, dichlorobenzidine, 0-tolidine. Fluram was purchased from Fisher Scientific (Catalog No. 43023). Monoacetyl benzidine was synthesized from benzidine (6). All of the aromatic amines were dissolved in acetone to give a solution (dichlorobenzidine, because of its limited solubility had to be prepared by first dissolving it in glacial acetic acid (glac. HAC), then diluting with acetone to this concentration). Fluram was used as a 1 mg/ml solution in glacial acetic acid which is stable for weeks a t room temperature. For the aliquoting of microliter quantities, the “Drummond Dialamatic Microdispenser” (Drummond Sci. Glass P6295) was used. Thin-layer chromatography (TLC) plates were purchased from Scientific Glass (Silica Gel 0.25 mm thick. Art 5762/0001). The solvent system for development of the plates was chloroform (90) glac. HAC (5) methanol (5). Spraying was accomplished with the “Lab Reagent Sprayer” (Analtech Catalogue No. A-100). Procedure. Fluram Reaction in Solution. Aliquots of 2 to 50 ~1 were transferred to tubes and the acetone was evaporated with a stream of nitrogen. Fifty gl of the Fluram solution was then added
v BENZIDINE 0
MONO-ACETYL BENZ I D I N E TOLIDINE
A DICHLORO BENZIDINE A 2-NAPHTHYLAMINE
1.4
FLURAM ASSAY
1.2
0
0
> c Y
A
A
46
50
0.4 A
0.2
0.0
0
2
6
10
16
20 26 NANOMOLES
3 6 4 0
Figure 1. Standard curves for the reactionof Fluram with aromatic amines in solution
followed by a 30-sec vortex mix. After 10 min, the reaction was stopped by addition of 0.5 ml of methanol. The optical density of the yellow product formed with each amine was measured a t the wavelength of maximum absorption, as determined in a Gilford Spectrophotometer (0.5-ml quartz cuvettes with a 1-cm light path were used). Measurement of Benzidine on TLC. Varying amounts of the benzidine stock were applied to TLC plates. As soon as the plates were dry after development, they were sprayed with the Fluram solution, and again allowed to dry. The yellow spots were scraped off the plates and the benzidine-Fluram product was eluted with 0.5 ml methanol, by vortexing for 1 min, then centrifuging for 10 min a t 12 000 RPM in a Serval1 Refrigerated Centrifuge. This allowed for almost complete recovery of the methanol, free from the silica particles. Optical density was measured a t 415 mF.
tained by reaction in solution (Figure 2). Precision data for the 5 amines tested and for the TLC results are given in Table I. The method is applicable to the monitoring of aromatic amines in urine or in water. The quantitative recovery of the yellow product (formed in minutes) from TLC makes possible the specific measurement of aromatic amine. In previously published work ( 3 ) ,we described the finding of monoacetyl benzidine (MAB) in the urine of Rhesus mon-
0.6
RESULTS AND DISCUSSION The assay was linear over a wide range of amine concentration (Figure 1).Benzidine gave the largest yield a t every concentration, monoacetyl benzidine and tolidine gave values of about l/z, and dichlorobenzidine and 2-naphthylamine about l/4 that of benzidine. The advantage of having the Fluram in glacial acetic acid is that it will react only with aromatic amines, the pK of aliphatic amines being near 9. The reagent is stable as is the yellow product formed (with the exception of the dichlorobenzidine product which fades in 5 min). The benzidine and monoacetyl benzidine products have absorbance maxima a t 415 mp; tolidine, 2-napthylamine, and dichlorobenzidine yellows read maximally a t 390 mp. These aromatic amines are easily separated using the solvent system given in Experimental section. In a 10-cm run, we obtained a 2-cm separation between benzidine and 2-naphthylamine and a 1-cm separation between benzidine and tolidine. Assay of benzidine on a TLC plate was also linear in the range measured and the color yield identical to that ob-
0.5
0.4 2. .-c Lc c
8 0.3 3 ._ c
CL
0
0.2
0.1
0.0
-J l
2
l
l
l
!
l
l
4 6 8 Benzidine iflanomoles)
/
l
10
Flgure 2. Data obtained for the reaction of Fluram with benzidine on a TLC plate
ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
543
Table I. Precision Data A r o m a t i c amine, nmol
I. Benzidine 10 16 20 26
No. of test results, N
Mean
3 3 3 3 3
0.50 0.78 0.97 1.21 1.58
16
2
0.24
20
2 2
0.28
36 11. 2-Naphthylamine 26
30 36 111. Mono acetyl benzidine 6 16 26 30 36 IV. Tolidine 20 26 30 36 V. Dichlorobenzidine 10
20 30 VI. Benzidine on TLC 2 4 8
Sum of squares of deviation from mean, D 2
0.0008 0.0041 0.0074 0.0165
0.0045
0.0004 0.0004 0.0082 0.0018 0.0032
2
0.52
2
2 2 2 2
0.155 0.405 0.675 0.77 0.87
0.0004 0.0004
2 2 2 2
0.54 0.585 0.74 0.88
0.0008 0.0060
2 2
0.195 0.33 0.445
0.0220
2
2 a As defined in Anal. Chem., 41, 2139 (1969). 10
b
0.1167 0.2367 0.39 0.55 Benzidine, 6 nmol, tolidine 6,
0.0026
0.0037 0.0083 0.0023 0.0008
2
3 3
0.0004
0.0008 0.0032 0.0002 0.0004 0.0098
0.38 0.445
2
Varianceaj b ( D 2 / N- 1 )
0.0082
0.0018 0.0032
0.0008 0.0008
0.0098 0.0084 0.0008 0.0078 0.0050 0.0002
0.0032 0.0002 0.0004 0.0098
0.0008 0.0060 0.0008 0.0008 0.0220 0.0098 0.0084 0.0004 0.0039 0.0050
0.0002
10, 1 6 nmol: variance was less than
0.0001.
key fed benzidine or benzidine azo dye and the measurement of benzidine MAB excretion using TNBS. With this new procedure, it will be possible to measure benzidine or its MAB metabolite separately.
+
ACKNOWLEDGMENT We thank the Allied Chemical Corporation for supplying materials used in this study. LITERATURE CITED (1) S. Udenfriend, S. Stein, P. Bohlen, W. Dairman, and W. Leimgruber, Science. 178. 171 (1972). (2) W. C. Hueper, "Occupational-And-Environmental Cancers of the Urinary System", Yale University Press, New Haven, Conn., 1969, pp 118-180.
544
ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
(3) E. Rinde and W. Troll, J. Nat. Cancer SOC.,55 (1). 181 (1975). (4) E. Rinde and W. Troll, Roc. Am. Assoc. Cancer Res., 16, 79. San Diego, Calif., May 1975, Abstract No. 314. ( 5 ) K. Satake, T. Okuyama, M. Ohashi, and T. Shimoda, J. Biochem., 47 654 (1960). (6) S. Laham. J. P. Farant. and M. Potvin, Occup. Health Rev. 21, 14 (1970).
RECEIVEDfor review September 29, 1975. Accepted December l , 1975. Supported by the National Bladder Cancer Project, Public Health Service Grant (2.415315 from the National Cancer Institute; Public Health Service Core Grant ES00260 from the National Institute for Environmental Health Sciences; and the Allied Chemical Corporation.
