Table I. Determination of Phosphorus in Synthetic Mixtures Treated as Unknowns PhosPhosphorus phorus Foreign ions present (weight excess over P) taken, pg found, pg” Error, 0.150 0.152 +1.33 Ca(20), Fe(lO), Cu(15) 0.295 0.300 - 1.67 Al( 30),Si(30) - 1.33 Zn(30), Pb(20) 0.300 0.296 f5.00 AsIX1(lO), Cd(20) 0.400 0.420 +4.00 Se(20), Td20) 0.250 0.260 0.0 Bi(15), Cu(20) 0.200 0.200 -4.30 W(20), Sb(25), Ge(25) 0.350 0.335 Si( 15), Ni( 10) 0.079 -0.62 0.080 a Each result is the average of two determinations in good agreement.
complex by removal of the excess reagent, was made. The overall cumulative formation constant, k, defined as [RhBMFCl/[Mm[RhB]*, under these conditions was estimated t o b e l . 6 f 0.3 X 101lliterSmole-a. Conclusions. High sensitivity is obtained for the determination of phosphorus by the spectrofluorimetric method developed. The sensitivity achieved is higher than that for most other methods based on the formation of molybdophosphoric acid, and compares favorably with that of more complex enzymatic methods. High selectivity is achieved with the optimum conditions established. Of the ions investigated, only arsenic(V) interferes seriously, by formation and extraction of the corresponding Rhodamine B molybdoarsenate; arsenic (111), however, may be tolerated when present in moderate excess with respect to phosphorus.
which the Rhodamine B recovered in the final butanolchloroform phase from the complex after its formation from known amounts of phosphorus was determined fluorimetrically. Allowance for depletion of the Rhodamine B concentration in the final chloroform-butanol phase due to the chloroform extractions, which promote the dissociation of the
RECEIVED for review April 9, 1971. Accepted May 14, 1971. We thank the Government of Ceylon for the award of a scholarship to one of us (R.N.), the University of Ceylon for the grant of study leave (R.N.), and the Science Research Council for the award o f a grant for the spectrofluorimeter.
Application of an Iodide-Specific Resin to the Determination of Iodine in Biological Fluids by Activation Analysis Michel Heurtebise and W. J. Ross Seccidn Quimica, Instifuto Venezolano de Inuesfigaciones Cientificas, Apartado 1827, Caracas, Venezuela
A reliable method has been developed for the determination of iodine in biological fluids that incorporates the high sensitivity of neutron activation analysis with a very simple, economical, and rapid radiochemical separation procedure. Through the use of a novel and specific “iodinated” resin, la1 is separated from all other radioactive components of an activated sample. The high degree of isolation achieved permits the determination of this isotope with the maximum sensitivity that can be attained by means of gamma spectrometry. The method has been applied to the routine analysis of urine, blood serum, PBI, and saliva. OF THE MANY TYPES of clinical analyses performed each year, one of the most difficult and expensive is the determination of iodine in biological fluids. The analytical method most commonly used is based on the catalytic effect of trace amounts of iodine on the oxidation of As(II1) by Ce(1V) (1, 2). This method is not only complex for routine application but there remain potential sources of error due to both the loss of iodine and to the interferences of other trace elements and organic constituents on the catalytic reaction (3). Because of the importance of this analysis, especially in the physiopathology of the thyroid, continual efforts have been made to improve the reliability of this colorimetric method and to adapt it to automated application (4). During the past decade, efforts also have been made to avoid the problems (1) E. B. Sandell and I. M. Kolthoff, Mikrochim. Acfu, 1 , 9 (1937). (2) H. Hoch and C . G . Lewallen, Clin. Chem.. 15, 204 (1969). (3) H. Hoch, S. L. Sinett, and T. H. McGavack, ibid., 10, 799
(1964).
(4) E. Cornoy, Rer, Fr. E/iides C/h. Bid., 12, 189 (1967). 1438
inherent in the catalytic method through the use of’ neutron activation analysis (5,6). These latter studies have culminated jn the development of a fully-automated system based on the measurement of 1 2 8 1 after isolation of this isotope from other radioactive components produced during neutron irradiation of biological fluids (7). Even though this nuclear method affords more reliable results, it has not been adopted on a large scale because of the requirements of a nuclear reactor and a sophisticated separation system. Nuclear reactors, however, are becoming increasingly more available to analytical chemists in all parts of the world. Consequently, increased simplification of the separation procedure should lead to greater acceptance of this method. One effort in this direction has been the development of a semi-automated separation system much less elaborate and expensive than previously available (8). This paper describes a new concept in chromatographic separation that is specific for iodide and which offers even greater simplicity and economy for large scale routine application of the nuclear approach. Essentially every known separation technique has been investigated for separating iodine from biological fluids. The (5) C. Kellershohn. D. Cornar, and C. Le Poec. I/?!. J. A m / . Radiut, Isotop., 12, 87 (1961). (6) E. M. Smith, J. M. Mozley, and H. N.Wagner, J. N d . Med., 5, 828 (1964). (7) D. Cornar and C. Le Poec, International Conference on Modern Trends in Activation Analysis, IAEA, College Station, Texas, 1965. (8) M. Heurtebise, J . Radiomu/. Chem., 7, in press (1971). .
I
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
use of anion exchange resins is the most suitable for the nuclear method and the most amenable to automation. A very well-known method for the separation of mineral iodide from the protein-bound-iodine (PBI) is based on the adsorption of the former on anion exchange resin and has been made the basis of a method for the determination of PBI by neutron activation analysis (9). Comar and Le Poec (IO) observed that neutron activation of a biological liquid ruptures the organic iodine ligand so that total retention of iodine can subsequently be achieved by anion exchange chromatography. This phenomemon eliminates the need for ashing the sample to liberate the organic iodine. Consequently, iodide can be isolated from other radioactive species (principally 24Na, 38Cl, 42K, aoBr,82Br)through retention on Dowex-2 and subsequent selective elution with nitrate solutions (11). Since the induced radioactivity of the major components is very large, high decontamination factors (-lo5 for 24Naand W1) are required to permit accurate measurement of IZ8I. Such efficiency can be achieved only by relatively lengthy elution procedures. Sansoni (12) has reported that molecular iodine and its complexes 1,- are adsorbed very strongly on anion resins probably through a molecular adsorption mechanism. The degree of retention is not a function of pH, extraneous ions, type of solvent, or basicity of the resin. In this paper it is demonstrated that an anion exchange resin that is saturated by a concentrated solution of I-/12/In-exhibits specific retention for iodide ion. This characteristic has been made the basis of a method for the selective and rapid separation of 1 2 8 1 from all other radioactive components of irradiated biological fluids. EXPERIMENTAL
Resin Column. Resin columns are prepared individually by placing 150 mg of Dowex 2-X8 (200-400 mesh) in a suitable tube and washing the resin with an iodine reagent until no difference can be observed between the colors of the influent and effluent. Ten milliliters of reagent are usually sufficient. An additional 5 ml of reagent is then passed through the resin column to ensure complete saturation. The resin is held in the tip of the tube by means of a small wad of glass wool so that the resin bed is approximately 1 cm high and 0.5 cm in diameter. The iodine reagent is prepared by dissolving 25 g of iodine in 1 liter of 1M K I to form a solution nearly saturated in 12. When not in use the resin columns are stored in the iodine reagent. While separations are being performed, the resin holders are connected to a source of vacuum by small-diameter tubing. Preparation of Sample. SALIVAAND URINE. Two-milliliter aliquots are combined with 0.1 ml of 0.05% K2S205 solution and sealed in a polyethylene irradiation tube. BLOODSERUM.One-milliliter aliquots are diluted with 2 ml of distilled water and 0.1 ml of 0.05 % K2S205 and then encapsulated. PBI. This fraction is separated by anion exchange (8, 9) and subsequently treated in the same manner as serum. IODINESTANDARD.Solutions that contained 10 to 100 ng of iodine per ml were prepared from potassium iodide or commercial lyophilized serum solutions (Versatol). Two(9) C. W. Fang and R. H. Tomlinson, Nuclear Activation Techniques in the Life Sciences, IAEA Symposium, Amsterdam, May 1967. (IO) D. Comar and C. Le Poec, Symposium on Radiochemical Methods of Analysis, IAEA, Salzburg, Austria, 1964. (11) R. C . De Geiso, W. Rieman, and S. Lindenbaum, ANAL. CHEM., 26, 1840 (1954). (12) B. Sansoni, Angrw. Cliern., 73, 493 (1961).
milliliter aliquots and 0.1 ml of 0.05% KzS205 were normally used. Irradiation of Samples. All irradiations were performed in the RV-1 Reactor at a thermal neutron flux of - 5 X 1012 n cm-2 sec-l. Counting Equipment. Gamma measurements were made with a 3-in. X 3411. NaI(T1) well detector coupled to a 400channel pulse-height analyzer. Procedure. Groups consisting of three samples and one standard are irradiated in a single container for 25 minutes. During this period the resin columns are purged of excess iodine reagent by washing them with 1 ml of water. The irradiated solutions are transferred, with minimum delay, to the resin columns that are connected to water aspirators. The radioactive eluent is collected in a trap or is flushed into a “hot” drain. A flow rate of 1 to 4 ml per minute is maintained by regulation of the aspirator. After the sample has completely drained, the resin is washed twice with 2-ml volumes of water and then twice with 5-ml portions of water or, preferably, 2 % NaCl solution. When the column is dry the tube is disconnected from the vacuum line and placed in the counting facility where it is counted for 2 minutes. The activity of each sample, corrected for decay, is compared with that of the standard of its group, that has been processed in the same manner, to obtain the weight of iodine in the sample. RESULTS AND DISCUSSION
Retention of Components of Urine and Blood. Initial studies of the retention characteristics of the “iodinated” resin were performed by adding, one at a time, radioactive tracers (24Na, a8C1,r2K, 82Br,lS1I)to two milliliters of inactive urine or blood serum and then passing these solutions through a resin column. The resin was subsequently washed with 2-ml portions of water. Flow rates of 1 to 4 ml per minute were employed. A large percentage (-90%) of all of these elements, except iodine, passed directly through the resin. More than 99% of the extraneous ions are eliminated by washing the resin only twice with 2 ml of water. Only 0.1 to 0.2 % of the 311activity was found in the sample eluate and none was detected in seven subsequent wash solutions. It was interesting to observe that chloride and bromide are not retained to any greater degree than sodium or potassium. The anion exchange properties of the original resin (Dowex 2-X8) seem to have disappeared after saturation with iodine and its complexes. Contrariwise, the retention of 1 2 8 1 is essentially quantitative and unaffected by water washes. Since >99% of the 24Naand W l are removed by washing the resin with only 4 ml of water, a satisfactorily high decontamination factor is obtained by washing with an additional 10 ml. N o loss of iodine from the resin has been observed after successive washes of 2 ml of water. Similar tests were conducted with samples of PBI fraction and, once again, the retention of iodine was observed to be 99.6 % after the resin had been washed seven times. It was possible to eliminate essentially all trace of 24Na or 38Cl activity from the resin columns and to achieve an even greater decontamination factor by substituting 2 % NaCl solution for water in the last 5 washes. No loss of iodine occurs during these NaCl elutions. Effect of pH. To estimate the effect of the pH on the retention characteristics of the resin, solutions that contained 3 pg of iodide and known quantities of I3II tracer were prepared at different pH. The retention of iodide was at least 99.8 when the pH of the solution was 5 7. Approximately 1 of the iodide was lost when the pH was 9.2; however the retention dropped sharply t o 8 6 z at pH 10.5 and 84% at pH
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
1439
5x’0211
OM,5 M n
0
too
4X102
1128
.
Figure 1. Spectrum of blood sample after separation (Iodine = 58 ppb) Flux: 5 X 10lan cm-l sec-l Irradiation time: 25 min Decay time: 10 min Counting time: 1 min
200
400
300
Channel 12. It is known that IPis not stable in basic solution but disproportionates slowly to IOH and I-. IOH is subsequently converted rapidly to IO-3 and I-. This transformation begins at pH 9.5 and is total near pH 11 (13). The decrease in retention of iodide when the pH is >9 can probably be explained by deterioration of the resin through alteration of the iodine species. Effect of the Amount of Iodide on the Resin. The effect of the amount of iodide in a sample solution was established with solutions that contained 1311tracer and iodide of different concentration at a pH of 6.5. Complete retention was attained with carrier-free l3II and when as much as 2.5 mg of I- was passed through the column. However, when 25 mg of I- was present in the initial solution, only 73 % was retained during passage at a rate of 2 ml per minute. Effect of Reducing Agent. Iodide has been reported to be oxidized during neutron irradiation (10). This would cause negative errors due to the inability of this “iodinated” resin to retain free iodine. Such oxidation and consequent loss of iodine can readily be prevented by adding a small amount of reducing agent to the sample before irradiation. To establish the effect of a reducing agent on the “iodinated” resin, retention studies were performed with 2-ml solutions of K&Oa of different concentrations (at pH 6.5) to which 3 pg of iodide and 1311 tracer were added. The retention of the iodide was 97.5%, 99.0%, 99.2%, and -100% when the concentration of K2SZ05 was 0.4 %, 0.2 %, 0.1 %, and 0.05 %. As expected, a slight decrease in retention was observed as the concentration of the K2S205 solution was increased because of the reduction of free iodine and its complexes on the resin. However, when 2 ml of 0.05% KzS205 solution is used, no interference on the retention characteristics of the resin is observed. The introduction of only 0.1 ml of 0.05 KZSZOS solution to each sample during preparation is sufficient to counteract any oxidation during irradiation. This precaution probably is unnecessary when body fluid samples are analyzed because identical results have been achieved with and without the reductant. Because of the simplicity of the precautionary measure, this step has been retained in the procedure. Repetitive Use of a Resin Column. Even though a relatively insignificant expense of time or money is involved in the preparation of a resin column, it was considered worthwhile
to establish if such a column can be used repetitively. Consequently, one column was used for the analysis of seven urine and four blood samples which had been “spiked” with 12*I. After each analysis, the resin was washed with -2 ml of iodine reagent and stored in this reagent when not in use. activity in the eluates was measured and always The 1281 found to be negligible (99.8%. in these eleven samples was negligible (-1 pg) in relation to the quantity (2.5 mg) that such resin can completely retain. This resin was subsequently used in the routine analyses of many irradiated samples, and the selectivity was observed to be the same as that of a new column. Accuracy and Precision. A value for the overall precision of this method was established through seventeen repetitive determinations of the iodine content of one sample of urine. The average result of these determinations was 107 ppb iodine with a relative standard deviation of 6.5%. When various aliquots (0.2 to 2 ml) of this urine specimen were taken for analysis, a linear relationship was achieved between the weight of iodine found and the volume of the sample. An estimation of the accuracy was made through comparative analyses of four urine samples by this method and by a semiautomated method of proved accuracy which involves the chromatography of I- on a Dowex 2-X8 column (8). In Table I is shown the comparison of the results. Sensitivity. The sensitivity of all techniques that use neutron activation analysis, radiochemical separation, and gamma spectrometry is a function of the following controllable factors: the neutron flux that is used; the yield of the radiochemical procedure; its rapidity, if the half-life of the measured isotope is short; and the degree of purity with which this radionuclide can be isolated. One of the limitations of gamma spectrometry is the accuracy with which a photopeak
(13) G. Charlot, “L’Analyse Qualitative et Les RCactions en Solution” 334 Masson et Cie, Paris, 1963. 1440
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
Table I. Comparative Analysis of Urine Ppb of Iodine Semi-automated Sample method This method 1
2 3 4
120
164 127 84
109
170
126 83
can be distinguished from the background caused by Comptons of higher energy radionuclides. In Figure 1 is presented a spectrum of iodine, separated from an irradiated blood sample, which demonstrates the high degree of decontamination achieved by this method. Isolation of lZ8Ifrom urine samples is equally evident from spectral data. This fulfills onc prcrrquis:te for R sensitive method. In addition this procedure has a radiochemical yield of >99% and can be performed in less than 5 minutes. Therefore, this technique appears to offer the maximum sensitivity that can be achieved through use of activation analysis and gamma spectrometry for l P 8 I . The spectrum in Figure 1 was obtained during the analysis of a sainple of serum that contained 58 ng of iodine. As little as 4 ng of iodine, irradiated and measured under the same conditions, would have been sufficient to yield lZsI activity equal to that of the background which is essentially due to 38Clcontamination. The high decontamination that is attained by this procedure also permits accurate measurements of iodine at normal concentrations in biological fluids with a single-channel analyzer, thereby reducing equipment cost and facilitating data reduc-
tion. The background, primarily W l Compton, is prohibitively large only when the concentration of iodine is
