March, 19663 BELKAS AND SOULIOTIS 199 Simultaneous Determination of Iodine and Bromine in Urine by Neutron-activation Analysis BY E. P. BELKAS AND A. G. SOULIOTIS (Chemistry Department, hTuclear Research Centre “Democritus,” Athens, Greece) Neutron-activation analysis was used for the simultaneous determination of iodine and hromine in urine. The activated iodine and bromine were separated by radiochemical methods. The 0.46 Me\’ and 0.55 MeV peak areas of lZR1 and 8zBr, respectively, were measured by nieans of a multi- channel analyser. The amounts of iodine and bromine were found to be of the order of lo-’ and of urine, respectively, for normal human beings of different ages. g ml IODINE is found in urine both in inorganic (80 per cent.) and organic (20 per cent.) chemical f0rms.l Lsually, iodine is found in the various biological tissues as an organically bound species, but it cannot be determined until after mineralisation. The order of magnitude of the mineral iodine so obtained, purified by distillation, extraction, ion exchange and so on, is determined by a classical titrimetric or colorimetric method, or from its catalytic action upon the rate of reduction of ceric sulphate by arsenous oxide.2 The rate of this reaction, slow in the absence of a catalyst, is increased by the presence of micro amounts of iodine.Although the sensitivity of this catalytic reaction is of the order of 04002 pg for iodine determinati~n,~ the results of this method are not good due to the disadvantage of the im- purity of the analytical reagents, and to the presence of iodine in the laboratory.Thus neutron-activation analysis, which presents high sensitivity and good precision in comparison with other analytical methods, has been used by several investigators for the quantitative determination of iodine and bromine in a variety of biological materials. Iodine was deter- mined as thyroglobulin i ~ d i n e , ~ blood-serum and serum organically-bound i ~ d i n e , ~ ,6 blood iodine,’ stable iodine up-taken by the thyroid8 and as a constituent of different biological material^.^,^^ ,11 9 l 2 Bromine was determined in a series of biological material^.^^^^^^^^^^^ 9 1 7 9 1 8 Although there are many papers on radioactive iodine determination in urine,lg y 2 0 ,21 ,22 923 ,24 rnilkg925 and plasma,26 not much work has been done on the determination of stable iodine in urine, especially by radioactivation analysis.As a contribution to these studies, iodine and bromine were simultaneously determined in urine by radioactivation-analysis techniques. On irradiating any material with neutrons, various nuclear reactions take place, those in Table I being of interest for iodine and bromine d e t e r r n i n a t i ~ n . ~ ~ * ~ ~ TABLE I NUCLEAR REACTIOKS Main radiation A4ctivation encrgics, MeV Abund- cross- 7- ancc, section, Half- fl cnergy, y energy r--+ r-\ lZ7I 1271 (n,y) 1281 100 5.60 24-99 min. 1.67 16 0.46 17 2.12 76 0.55 75 0.78 83 81Br 81Br (n,y) 82Br 49.48 1-60 36.87 hr 0.44 100 1.04 29 1.32 28 Nuclide Reaction % barns life I ~ X . 76 Possible interference from lasXe (n,p) lZ8I with a Xe matrix sZKr (n,p) E2Br with a Kr matrix EXPERIMENTAL IRRADIATION- Transfer an aliquot of about 6 ml of biological liquid, by pipette, into a polythene snap-closure tube of diameter 26 mm and height 50mm, having along its axis another polythene200 BELKAS AND SOULIOTIS : SIMULTANEOUS DETERMINATION OF IODINE [Analyst, Vol.91 snap-closure tube of diameter 15 mm and height 50 mm, that can be stoppered and fused. Add 6 ml of the standard iodine and bromine solution, mentioned below, into the central tube and then stopper and fuse it. A = External polythene tube containing B = Central polythene tube containing iodide and potassium bromide urine standard solution of potassium Fig. 1. Target for irradiation Irradiate the target for 15 minutes at a flux of 10l2n.cm-2 second-l. We sent our prepared target, by means of the pneumatic system, to the core of the swimming pool of the “Democritus” nuclear reactor. REAGENTS- Use analytical-grade reagents. Potassium iodide standard solution-Make an aqueous solution containing 10 mg ml-l of Potassium bromide standard solution-Make an aqueous solution containing 10 mg ml-l Potassium iodide and potassium bromide standard solution-Prepare an aqueous solution mg ml-l of bromine of mixture, taken from the standardised iodine. of bromine. of lod4 mg ml-l of iodine and solution of the carrier. Use triply distilled water. Perhydrol. Sodium nitrite solution, 2.5 N and N sodium nitrite. Sulphuric acid, concentrated. Sodium hydroxide solution, 7-5 per cent. sodium hydroxide.Carbon tetrachloride. Nitric acid, concentrated, 6 N and 0-01 N. Sodium hydrogen sulphite solution, N. Silver nitrate solution, 0.1 N. Ethanol. Potassium permanganate, powder. Potassium permanganate solution, 2 per cent. potassium permanganate. Hydroxylammonium chloride solution, M. APPARATUS- and a Plexi-glass filter-funnel were used. Modified apparatus was used to increase chemical yield. A Pyrex distillation apparatus ISOLATION OF IODINE AND BROMINE BY DISTILLATON- After irradiation transfer by pipette into the distillation apparatus, 5 m l of urine con- taining 1 ml of iodine plus 2 ml of bromine carrier solutions, respectively. Add the following reagents : 2 ml of concentrated sulphuric acid, 2 ml of perhydrol and 1 ml of N sodium nitrite solution. Begin distillation by heating and introducing air.Introduce the distillate and air into 10 ml of 7.5 per cent. sodium hydroxide solution contained in a separating funnel im- mersed in an ice - water mixture. After a few minutes add the same volumes of perhydrol and sodium nitrite and continue the distillation until white sulphur trioxide vapour is produced. The duration of the distillation procedure was 10 minutes.March, 19661 AND BROMINE I N URINE BY NEUTRON-ACTIVATION ANALYSIS 201 A A = Compressed air from cylinder D = Asbestos shield B = IO-ml funnel containing sodium E = Air-cooled tube nitrite, sulphuric acid and a 30% solution of hydrogen peroxide F = 250-ml flask containing sodium C = 50-ml round-bottom flask contain- ing urine, potassium iodide and G = Beaker containing ice-water mix- potassium bromide ture Fig.2. Pyrex distillation apparatus hydroxide A = Screw-cylinder B = Filter disc C = Screw-funnel Fig. 3. Plexi-glass filter funnel ISOLATION OF IODINE BY REDOX AND PRECIPITATION- Pour 10 ml of carbon tetrachloride into the solution contained in the separating funnel. Gently neutralise and slightly acidify this alkaline solution with 6 N nitric acid. Add 1 ml of 2.5 per cent. sodium nitrite drop by drop until the organic phase turns pink and separates. Repeat the same oxidation extraction step to the aqueous phase. Keep the aqueous layer containing bromine so that it can later be subjected to the bromine isolation procedure.202 BELKAS AND SOULIOTIS : SIMCLTAKEOUS DETERMINA4TTON OF IODINE [A?ZdySt, VOl.91 Gather the organic phases containing the iodine and subject them to a reduction step by adding 10 ml of distilled water and a few drops of N sodium hydrogen sulphite. Separate the aqueous and organic phases and repeat the same reduction step with the organic phase, which can then be discarded. Collect the portions of aqueous phase and add 1 ml of 6 IC’ nitric acid. Heat the solution so as to expel the sulphur dioxide and the few globules of carbon tetra- chloride present. Add a 2-ml portion of 0.1 N silver nitrate. Filter the silver iodide precipitate, rinse the precipitate with 0.01 N nitric acid solution, distilled water and finally with ethanol. Mount the filter-paper with the precipitate on an aluminium disc for counting. The chemical yield averaged 90 per cent., and the time required was 10 minutes for the redox steps and 5 minutes for the precipitation - filtration procedure.ISOLATION OF BROMINE BY REDOX AND PRECIPITATION- Add a 10-ml portion of carbon tetrachloride, 2 ml of concentrated nitric acid and powdered potassium permanganate, until the pink colour of permanganate is visible. When the organic phase becomes brown, separate it immediately. I t was found that, for an instance when separation of the two phases was not achieved due to the excess of permanganate added, one or more mechanical decantations of the mixture from one separating funnel into another gave satisfactory separation of the two phases. Subject the aqueous phase to two more identical oxidation and extraction processes by using 1 ml of concentrated nitric acid and 2 ml of 2 per cent.permanganate solution. Collect the organic phases and reduce them by using 10 ml of distilled water and 1 ml of M hydroxyl- ammonium chloride. Separate the aqueous and organic layers and repeat the same reduction step, using 0.5 ml of reducing agent, on the organic phase, which can then be discarded. Gather the aqueous phases and subject them to the redox procedures mentioned above by using N sodium hydrogen sulphite. Add a 1-ml portion of 6 x nitric acid to the collected aqueous solutions, and then heat so as to expel sulphur dioxide and carbon tetrachloride. Add a 4-ml portion of 0.1 N silver nitrate. Treat the silver bromide precipitate in the same way as the iodide and mount for counting. We found that the chemical yield averaged 70 per cent., and the time required was 15 minutes for the redox steps and 5 minutes for the precipitation - filtration procedure.Subject the aqueous solution containing bromine to the following treatment. The standards were treated in an identical manner to the urine sample. DETERMINATION O F RADIOACTIVITY- Count the silver iodide precipitate immediately for 10 minutes on a 3 x 3-inch sodium iodide (Tl) crystal counter, connected with an Intertechnique 400-channel transistorised analyser, adjusted to count energies from 0 to 2 MeV. Mount a 8-mm thick Plexi-glass block on the crystal to cut off Bremsstrahlung. Print the 0.46-MeV photopeak area of the iodine-128 and compare it with that of the standard. Count the silver bromide precipitate next day for 10 minutes under the conditions mentioned above.Print the 0.55-ICIeV photopeak area of the bromine-82 and compare it with that of the standard. GAMMA SPECTROMETRIC EXAMINATION OF THE ISOLATED RADIO-ISOTOPES- Gamma-ray spectrometry confirmed the absence of any y-emitting radionuclides as contaminants in the isolated precipitates of iodine-128 and bromine-82 coming from the aiialysed urine sample. DETERMINATION OF THE HALF-LIFE OF THE ISOLATED RADIO-ELEMENTS- This was accomplished by plotting the decay curves for silver iodide and silver bromide precipitates. The half-lives were obtained from the slope of the straight line calculated by the method of the least squares. A value of 25 minutes with a standard error of +0.013 minutes was found for iodine, and a value of 36-88 hours with a standard error of +0-21 hours for bromine, compared with 24-99 minutes and 36.87 hours, respectively, reported in the literature .27 928 RESULTS Iodine and bromine quantitative results in urine were obtained for normal human One special case is reported of a person individuals (male and female) of different ages.March, 19661 AND BROMINE IN URINE BY NEUTRON-ACTIVATION ANALYSIS 203 whose first urine sample indicated high iodine content in comparison with the content of other samples, and with those from the same person after a few days from the first sampling.This was due to the fact that the person concerned had sustained food poisoning and was being treated with Mexaform Ciba medicine containing iodochlorhydroxyquinoline. The values found were of the order of 10-7 mg ml-l and mg ml-l for iodine and bromine, respec- tively. The results are shown in Table 11.TABLE I1 CONCENTRATION OF IODINE AND BROMINE IN HUMAN URINE Name G. -4. S. A.C.C. A.G.V. G.A.S. P.C.C. A.G.S. A. J.S. S.G.S. S.G.S. C.A.K. ,4ge, in years 1 2 24 38 29 30 45 58 58 so Sex male female female female female male male female female female Iodine, in p.p.m. 0-160 0.085 0.195 0.225 0.305 0.170 0.165 1.980 0.150 0.175 Bromine, in p.p.m. Remarks 5.3s - 5.82 - 2.78 - 4.3 1 - 9.47 - 8.29 - 9-63 in medical cure 2.4 1 - 4.28 - 11.9 in pregnancy DISCUSSION By the technique mentioned above iodine and bromine were simultaneously determined by one irradiation. From the values found it is confirmed that urine is enriched in iodine and bromine by a factor of about 2 with respect to The errors in iodine determination by titrimetric, colorimetric and catalytic classical chemical methods, mainly due to the impurity of the analytical reagents, are eliminated.The isolated precipitates of silver iodide and silver bromide showed remarkable radiochemical purity. The results were reproducible within a relative error of less than t 3 per cent., and the sensitivity reached values of p.p.m. for iodine and bromine, respectively, for a neutron flux of 1012 n. cm-2 second-l and an irradiation time of 15 minutes. This technique could be easily applied for precise investigation of different thyroid diseases due to its high sensitivity and good precision. It could also be applied to the simultaneous determination of these two halogens in almost every biological material.The authors wish to express their thanks to A. P. Grimanis for helpful discussions. They also appreciate the valuable technical assistance of the N.R.C. “Democritus” nuclear-radio- chemical analysis group personnel. p.p.m. and 1. 3. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. REFERENCES Fletcher, K., Biochem. J., 1957, 17, 136. Sandell, E. B., and Iiolthoff, I. RI., il.likvochi?n. Acta, 1937, 1, 9. Lachiver, F., Thesis: Facultk des Sciences de Paris 1954, Masson, Paris, 1966. Brues, ;I, &I., and Robcrtson, 0. H., Kept. ANL, AECD-20094 (1947). Leonhardt, !A’., Ir‘evmnevgie, 1961, 4, 305. Manney, T. K., and Larochc, G., Kept. Calif. ITniv. Uerkclcy, Lawrence Kad. Lab. UCKL-9897, Kellershohn, C., Comar, D., and Le-Poec, C., Int.J . A p p l . Ratliat. Isotopes, 1961, 12, 87. LVagner, H. N., Selp, Vr. E., and nowling, J . H., .I. Cliiz. 1117iesf., 1961, 40, 1984. Bergh, H., Second United Nations International Conference on Peaceful Uses of Atomic Energy, Coinar, D., Rept. CEA-2095, p. 61 (1962). Comar, I),, and I<ellershohn, C’., Comptes Reiidzies des Joiwne‘es d‘Etudes stir l’’4 nalyse par ‘4 ctiuation, Comar, D., Le-Poec, C., Joly, M., and Kellershohn, G., Bull. SOC. Chiun. Fr., 1961, 56. Beyermann, K., 2. analyt. Chem., 1961, 183, 199. Bowen, H. J. M., Biochem. J., 1959, 73, 381. Bomen, H. J . M., and Cawse, P. A., U.K. Atomic Energy Authority Report AERE-R 2925, Harwell, Hall, T. A., Nucleonics, 1954, 12, 34. Samsahl, H., and Siiremark, R., Rept. .4E-61, p. 22 (1961). __ ~ , in “Proceedings of the 1961 International Conference on Modern Trends in Activation Analysis, December 15-16, 1961,” College Station, Texas, Agricultural and Mechanical College, 1961, pp. 27 30. Volume 18, paper 586, p. 508. Grenoblc, 1961., 71. 1959, p. 40. 1962, pp. 149-154.204 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. BELKAS AND SOULIOTIS [Analyst, VOl. 91 Arnott, D C., and Wells-Cole, J., Nature, 1953, 171, 269. Craig, A., and Jackson, M., Ibid., 1951, 167, 80. Jaworowski, 2.. Nucleonika, 1960, 5, 81. Marriott, J. E., Analyst, 1959, 84, 33. Purves, H. D., Nature, 1952, 169, 111. Schrodt, A. G., Rept. WASH-736, 112-30 (1957). Farabee, L. E., Rept. ORNL-3347, p. 149-52 (1962). Ekins, R. P., Physics Med. Biol., 1959, 4, 182. Allen, R. A., Smith, D. B., and Hiscott, J. E., U.K. Atomic Energy Authority Report, AERE-R Koch, R. C., “Activation Analysis Handbook,” Academic Press, New York, 1960, pp, 92 and 128. Bowen, H. J. M., Biochcm. J.. 1959, 73, 381. 2938, Harwell, Second Edition, 1961, pp. 42 and 89. Received Murch 12th, 1965