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0021-972X/98/$03.00/0 Vol. 83, No.

Journal of Clinical Endocrinology and Metabolism Printed in U.S.A.
Copyright © 1998 by The Endocrine Society

Rapid Urinary Iodide Test

Clinic of Nuclear Medicine (J.R., D.B., C.R.), University of Würzburg, D-97080 Würzburg, Germany;
and Merck KGaA (T.G.), D-64271 Darmstadt, Germany

Assessment of iodine deficiency and monitoring of iodine supple- iodide, by high-performance liquid chromatography, we determined
mentation programs demand rapid, simple, and cost-effective meth- the iodide concentrations of 370 random (untimed) urine samples
ods for the determination of urinary iodide concentrations. We pro- from consecutive patients by both high-performance liquid chroma-
pose a semiquantitative rapid test, based on the iodide-catalyzed tography and the rapid test. The results obtained by both methods are
oxidation of 3,39,5,59-tetramethylbenzidine by peracetic acid/H2O2, to in close agreement, with respect to classification of the samples ac-
yield colored products. The color of the chemical reaction is compared cording to the above three ranges, with a maximum difference of less
with color categories of a pictogram corresponding to three ranges: than 5% for each range. Median (y) values of a given distribution of
,100, 100 –300, and .300 mg/L (,0.79, 0.79 –2.36, and .2.36 mmol/L) urinary iodide concentrations can be calculated from the percent (x)
of iodide concentrations. The test is very easy to perform and does not of samples below 100 mg/L (0.79 mmol/L) using the regression equa-
require any instrumentation or apparatus. Sample preparation is tion: y 5 179.78 - 1.60x. This rapid test, therefore, is suited to epi-
simple and consists of the removal of interfering substances by dis- demiological surveys of iodine deficiency, especially in developing
posable columns, 65 3 10.5 mm, packed with purified activated char- countries. (J Clin Endocrinol Metab 83: 1007–1012, 1998)
coal. For comparison with a reference method for measuring urinary

I ODINE deficiency is a major health problem worldwide,

with about 1600 million people (mostly in developing
countries) being currently at risk of iodine deficiency disor-
removing interfering substances; column support; three test cups (25 3
50 mm); and color scale (Fig. 1 pictogram).

ders (IDD) (1– 4). Effective control of iodine deficiency by
iodine supplementation is still a challenge, especially in de- Included were: buffer solution [citrate-hydrochloric acid (pH 4.0),
Merck catalog no. 1.09435]; peracetic acid/H2O2 (dropping bottle), 1.2%
veloping countries (5). Because most iodide is excreted in the in 30% H2O2; and 3,39,5,59-TMB (dropping bottle), 2.5 mmol/L analytical
urine, urinary iodide excretion is currently the most conve- grade in ethanol.
nient laboratory marker of iodine deficiency (6). The present
recommendation (7) is to evaluate the iodine intake of a given Apparatus
population by the measurements of iodide concentrations in Apparatus included a spectrophotometer (Shimadzu Corp., Kyoto,
a representative number (at least 50 –100) of casual urine Japan); and HPLC equipment (all components from Waters Chroma-
samples collected within the population (8). Automated tography Div., Millipore, Milford, MA (Ref. 12).
equipment [e.g. Technicon Autoanalyzer (9, 10) or high- Procedures
performance liquid chromatography (HPLC) (11–13)] make
the analysis of large numbers of samples quite feasible, but Collection of urine samples. For a period of 4 weeks, we collected 370
random urine samples from 370 consecutive patients who were referred
the Technicon Autoanalyzer is no longer commercially avail- to our clinic for thyroid examination. For dilution experiments, an ad-
able, and the HPLC technique is expensive. Therefore, these ditional 51 random urine specimens were assembled from patients
methods do not optimally meet the demands of epidemio- within 1 week.
logical surveys, especially in developing countries. Performance of the rapid urinary iodide test. As discussed below, each urine
Here, we present a rapid urinary iodide test that is very sample must be tested within 2 h after collection. Further, the test cannot
easy to perform and does not require any technical equip- be performed on frozen specimens.
ment or apparatus. Dilution of the urine sample
Using a pipet, 1 mL of the urine sample was placed into test cup 1,
and 4 mL of the buffer solution was added.
Materials and Methods Column preparation
The rapid urinary iodide test was provided as a kit by Merck KGaA. Columns were initially filled with a weak alcoholic solution. This
The kit consists of materials and reagents listed below. solution was removed by pouring it out. Then the column was mounted
to test cup 2 using the column support. First, the column was equili-
Materials brated with 3 mL of the buffer solution. After the column run dry, 2 mL
Included were: disposable columns, 65 3 10.5 mm, packed with of the diluted urine sample in test cup 1 was applied to the column. The
purified activated charcoal (Merck patent number WO 96/27794) for column was allowed to run dry again. Then, the solution in test cup 2
was discarded.
Separation procedure (removal of interfering substances)
Received March 13, 1997. Revision received August 27, 1997. Re- The column was mounted to test cup 3 again using the column
revision received November 7, 1997. Accepted November 20, 1997. support. Using a pipet, 2 mL of the diluted urine sample in test cup 1
Address all correspondence and requests for reprints to: Dr. Johann was applied to the column, and the eluate in test cup 3 was collected.
Rendl, Clinic of Nuclear Medicine, University of Wuerzburg, Josef- The column was discarded, and the column support was removed. It is
Schneider-Strabe 2, D-97080 Wuerzburg, Germany. E-mail: rendl@ necessary to carry out the following step within 10 min after collection of the eluate to avoid deterioration of the sensitivity of the color reaction.


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1008 RENDL ET AL. JCE & M • 1998
Vol 83 • No 3

FIG. 1. Pictogram, showing five color

categories corresponding to three
ranges of iodide concentrations. The
test result of the proposed rapid urinary
iodide test is obtained by comparing the
color of the chemical reaction (see text)
with this pictogram.

Iodide determination
Using a pipet, 150 mL (or 6 drops) TMB was applied to the solution
in test cup 3. The catalytic reaction was started by adding 25 mL (one
drop) of peracetic acid/H2O2, mixing by gently shaking the cup, waiting
approximately 30 – 60 sec, and then comparing the color of the solution
with the color ranges of the pictogram (Fig. 1).
Spectral characteristics/colorimetry. After addition of peracetic acid/H2O2
to the solution in test cup 3, containing the eluate and TMB (see above),
optical spectra were recorded after precisely 90 sec. In the visible region,
two peaks appeared at 370 and 655 nm (Fig. 2). Because of correspon-
dence with the visual appearance of the solution, the absorbance was
measured at 655 nm, in a 1-cm path cell, against a water blank.
Comparison method (HPLC analysis). Free inorganic iodide in the urine
samples was determined using paired-ion, reversed-phase HPLC, as
recently described (12).
Photometric detection. The catalytic effect of iodide in the redox reaction
between the colorless 3,39,5,59-TMB and the peracetic acid/H2O2, to
yield colored products, is the basis of the photometric method used by
the rapid urinary iodide test for determination of iodide in urine. The
FIG. 2. Absorbance spectrum of the iodide catalyzed oxidation of
first colored product is a blue charge-transfer complex (Fig. 3) of the
TMB. There are three maxima. Because of correspondence with the
parent diamine and the diamine oxidation product (14). This species
visual impression, the absorbance of each sample was measured at
exists in rapid equilibrium with the TMB-radical cation (Fig. 3). Incu-
655 nm.
bations with high iodide concentrations turn blue, pass through a green
stage, and finally become yellow. The green solution (see pictogram, Fig.
1) is simply a mixture of the initial blue product and final yellow
component (14). starting urine sample, because 2 mL of the diluted sample were
applied to the column (see previous Separation procedure).
Dilution of urine samples. Fifty-one urine samples, collected in addition,
when showing a color reaction in the test corresponding to color ranges Interobserver reproducibility was excellent and was tested
2 or 3 of the pictogram (Fig. 1), were diluted 1:1 with buffer solution (500 by having four observers read the color of the reaction of the
mL buffer and 500 mL urine). The test procedure was carried out on these first 50 samples and by having two observers for the re-
diluted urines, and the results were compared with those obtained from mainder of the urine samples. Because of the clear appear-
the undiluted ones.
ance of the color, 30 – 60 sec after starting the reaction, no
differences were found between the different observers.
Distribution of the urine samples Possible disturbing factors and influences
The distribution of urinary iodide in 370 consecutive patients To assess the stability of columns and reagents under
is given in Fig. 4. The median of the distribution is 91 mg/L (0.72 conditions of storage, 50 columns and two sets of reagents
mmol/L). The iodide concentrations varied between 5 and 6600 were stored for 10 months at room temperature (varying
mg/L (0.04 –51.97 mmol/L). If patients on medical treatment for from 18 C in wintertime to approximately 30 C in summer-
goiter or with a history of exposure to large amounts of exog- time). No difference was observed, with respect to the in-
enous iodine caused by radiological procedures were excluded tensity of the color reaction between tests performed with
(n 5 76), the median of the resulting distribution would be 71 these columns and reagents and tests carried out with new
mg/L (0.56 mmol/L). This result is in agreement with corre- kits stored at 7 C in the refrigerator.
sponding data from other regions in Germany (15). To investigate whether environmental temperature, espe-
cially heat, can affect the results of the chemical reaction, five
Analytic performance urine samples with iodide concentrations of 72, 95, 139, 160, and
The mean (6 sd) of the final column eluate, as determined 250 mg/L (0.57, 0.75, 1.09, 1.26, and 1.97 mmol/L) were tested
by weighing 39 eluates, was 2.007 (0.044) mL. The volumes, at room temperature (24 C) and at 43 C incubation temperature
therefore, are uniform and can be extrapolated back to the after heating urines, columns, and reagents up to this temper-

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FIG. 3. Iodide catalyzed oxidation of 3,395,59-TMB by peracetic acid/H2O2, yielding colored products. The blue charge-transfer complex exists
in rapid equilibrium with the TMB-radical cation.

FIG. 4. Distribution of urinary iodide in 370 consecutive patients FIG. 5. Comparison of absorbance, measured spectrophotometrically
from Wuerzburg and its surrounding area being referred for thyroid at 655 nm with HPLC data. There is an almost-linear relationship up
examination. The concentrations were determined by HPLC; 76 sam- to 500 mg/L iodide. The data show a high correlation, but several
ples (.2000 mg/L) from patients with a history of exposure to large points lie outside the 95% confidence bands.
amounts of iodine caused by radiological procedures, are not shown.
According to the classification, as recommended by the WHO, the
population is affected by a mild degree of IDD. The test carried out on aqueous solutions of potassium
iodate did not show any color reaction; the method, there-
ature for 2 h in an incubator. The tests done in the incubator and
fore, does not detect iodine in any other biologically relevant
at room temperature gave the same correct results.
form than iodide.
Possible effects of interfering substances were assessed by
adding known amounts of potassium thiocyanate, l-ascorbic
Comparison between spectrophotometry and HPLC
acid, sodium sulfide, and sodium chloride to three urine
samples with 59, 169, and 287 mg iodide/L (0.46, 1.33, and For comparison, the iodide content of the 370 urine sam-
2.26 mmol/L) to a final concentration of 300 mmol/L for ples was determined by both spectrophotometry and HPLC.
thiocyanate, 20 mmol/L for ascorbic acid, 50 mmol/L for The results, in terms of absorbance vs. HPLC data, are given
sulfide, and 100 mmol/L for chloride. Correct results were in Fig. 5. There is an almost-linear relationship up to 500
obtained, and no differences were observed when testing mg/L (3.94 mmol/L) iodide. At higher iodide concentrations,
these urines, native and spiked with the compounds above. the absorbance shows a nonlinear increase, reaching a pla-

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1010 RENDL ET AL. JCE & M • 1998
Vol 83 • No 3

teau at about 1500 mg/L (11.81 mmol/L). The r value is 0.94, 2 or 3, dilution experiments were carried out on an additional
calculated by nonlinear regression analysis. 51 urines [median urinary iodide, 228 mg/L (1.80 mmol/L);
range: 72– 478 mg/L (0.57–3.76 mmol/L)]. In comparison with
Comparison between rapid urinary iodide test and HPLC the iodide concentrations determined by HPLC, the results
The data obtained, using the rapid urinary iodide test for allow the following classification: if the diluted urine gives
determining the iodide content of the same 370 urine sam- no color reaction, the iodide concentration of the undiluted
ples, are plotted in Fig. 6, with HPLC as a reference method. urine is usually less than 200 mg/L (,1.57 mmol/L), but at
Corresponding to the color ranges of the pictogram (see Fig. most, equal to 250 mg/L (1.97 mmol/L); in the case of a color
1), four panels (A-D) are shown, each representing the clas- reaction despite dilution, the iodide content of the undiluted
sification of urines, with respect to the color of the chemical urine is usually more than 200 mg/L (.1.57 mmol/L) but not
reaction observed in the rapid test. As can easily be seen (Fig. lower than 180 mg/L (1.42 mmol/L).
6, A, C, and D), there is good agreement between the rapid The classification of all samples, by number and percent-
test and the HPLC data for urines with high (Fig. 6, C and age of samples in each category, is given in Table 1. A very
D) and low (Fig. 6A) iodide concentrations. The deviations good agreement among HPLC, spectrophotometry, and the
from the HPLC values (shaded areas in Fig. 6) are relatively rapid urinary iodide test is obtained. The same is true for the
small, amounting to approximately 8% per color range. calculated median values.
Larger discrepancies with the HPLC data are seen only in the
lower range of color scales 213 (Fig. 6B), where 17% false- Extrapolating the median of a distribution of urinary iodine
positive results are found. In these cases, the rapid test shows concentrations from the percentage of samples below 100
a color reaction despite the relatively low urinary iodide mg/L (0.79 mmol/L)
concentrations between 50 and 100 mg/L (0.39 – 0.79 mmol/ Data collected between 1993 and 1994 in a prospective
L). To solve the problem of ambiguous results in color ranges nationwide survey in Germany show that it is possible to

FIG. 6. Comparison between the re-

sults obtained by the rapid urinary io-
dide test and the HPLC method. A-D,
Results of the rapid test for each color
range, as given in the pictogram (Fig. 1)
and the corresponding iodide concen-
trations determined by HPLC. Devia-
tions of the rapid test results from the
HPLC values are depicted as shaded
areas. For a more detailed explanation,
refer to the text.

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TABLE 1. Classification of 370 urine samples by three categories: percentage of samples, we constructed (by random number
number and percentage of samples in each category, as assignment) 50 subgroups, each containing 50 samples from
determined by HPLC, spectrophotometry, and the rapid urinary our original data set (n 5 370) and calculated the corre-
iodide test
sponding percentages of samples below 100 mg/L (0.79
Median mmol/L), as classified by the rapid test. The mean value of
mg/L Iodide ,100 100 –300 .300
(mg/L) the percentages of samples below 100 mg/L (0.79 mmol/L)
Rapid test 191 (51.6%) 134 (36.2%) 45 (12.2%) 97 was 51.96%, with a coefficient of variation (CV) of 11.7%.
HPLC 198 (53.5%) 117 (31.6%) 55 (14.9%) 94
Spectrophotometry 187 (50.5%) 125 (33.7%) 58 (15.7%) 99 Discussion
The median (y) values in the last column were calculated using the Suitability of the rapid urinary iodide test for the
percent (x) of samples below 100 mg/L and the regression equation: y 5 assessment of iodine deficiency in epidemiological surveys
179.78 2 1.60x.
Bourdoux (17) has shown that information about the iodine
status of a population can easily be obtained from a set of
50–100 casual urine samples. Because of the important scatter
of urinary iodide concentrations yielding skewed distributions,
Bourdoux (18) proposed a classification (Table 2) of iodine
status, based on his experience with thousands of samples from
several countries throughout the world, using an arbitrary cat-
egorization of urinary iodide concentrations: #20, #50, and
#100 mg/L (#0.16, #0.39, #0.79 mmol/L). These three ranges
are related to 3 levels of iodine deficiency (mild, moderate, and
severe) suggested by international organizations (2, 7, 19, 20),
who have adopted a simplified classification based only on
single median values (2, 7). Table 2 suggests that these median
values may be correlated with the percentage of samples below
100 mg/L (0.79 mmol/L). We could indeed derive from 37
distributions a corresponding relationship showing a high cor-
relation (r 5 20.99). Therefore, it is possible to extrapolate the
FIG. 7. High correlation between the median values of 37 distribu-
tions of urinary iodine concentrations and the corresponding percent- median of a given distribution from the percentage of samples
ages of samples below 100 mg/L. The data were collected between 1993 below 100 mg/L (0.79 mmol/L) by using Fig. 7. As can easily be
and 1994 in a nationwide survey in Germany; 3 data sets from Wuer- seen, the correlation shown in Fig. 7 is in very close agreement
zburg were included, giving a total number of 7849 random urine with Bourdoux’s classification (Table 2). If, for example, 50–
samples. This figure can be used for extrapolating a median of dis- 80% of all samples are below 100 mg/L (0.79 mmol/L), the
tribution from the percent of samples below 100 mg/L. corresponding median is between 50 and 100 mg/L (0.39–0.79
mmol/L), and the iodine intake is mildly deficient; if more than
determine with sufficient precision the median of a given 80% of all samples are below 100 mg/L, the median lies below
distribution of urinary iodine concentrations solely from the 50 mg/L (0.39 mmol/L), and the iodine intake is to be classified
percent of samples with urinary iodine less than 100 mg/L as moderately deficient. Evaluating the precision of the rapid
(0.79 mmol/L). A total number of 6815 subjects, belonging to test at the cut-off level of 100 mg/L (0.79 mmol/L), yields a CV
various age groups and spread over 34 regions throughout of 11.7%. For comparison, the mean CV of method A in Ref. 6
in Germany, could be enrolled in this study. Random sam- is 11.0%.
ples of urine were collected from 6381 clinically euthyroid The other results obtained in the present study are summa-
persons without history of thyroid disease, and the samples rized in Table 1. The data show a very good agreement among
were measured for iodine concentration using a modification HPLC, spectrophotometry, and rapid test results, with respect
of the colorimetric method of Sandell and Kolthoff. Part of to the classification of all samples by number and percentage of
these data was published in 1996 by Hampel (16). With kind samples in each category. In more than 50% of all samples,
permission by the author (R. Hampel, University of Rostock, iodide concentrations # 100 mg/L (0.79 mmol/L) were found by
FRG) and by the sponsor of the study (Merck KGaA), we HPLC, spectrophotometry, and the rapid test. This is equivalent
could use the original data of the whole study to establish a to a mildly deficient iodine intake, according to Table 2, and
relationship between the median values of 37 distributions of
urinary iodine concentrations encompassing 7849 subjects (3 TABLE 2. Classification of iodine intake according to the
data sets from Wuerzburg collected in 1986, 1990, and 1997, distribution of urinary iodide concentrations, suggested by
with a total number of 1468 persons, were included) and the Bourdoux (18) and according to median values recommended by
the WHO-UNICEF-ICCIDD consultation (7,20)
corresponding percentages of samples below 100 mg/L (0.79
mmol/L). The correlation shown in Fig. 7 is excellent (r 5 Iodine intake
20.99) and statistically highly significant (P , 1026). % Urinary iodide Normal Mildly deficient Moderately Severely
Therefore, it is possible to calculate the median (y) of a deficient deficient
given distribution of urinary iodine concentrations, from the #20 mg/L 20 –30 30 –50 .50
percentage (x) of samples below 100 mg/L (0.79 mmol/L) #50 mg/L #70 .70 .80
using the regression equation: y 5 179.78 - 1.60x (Fig. 7), with #100 mg/L ,50 .50 .80 .90
an se of estimate (SEE) of only 2.6 mg/L (0.02 mmol/L). Median urinary .100 50 –99 20 – 49 ,20
iodine, mg/L
To evaluate the precision of the test in determining the

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1012 RENDL ET AL. JCE & M • 1998
Vol 83 • No 3

corresponds to the median of 91 mg/L (0.72 mmol/L) of the determination from a dilution, however, is required only if the
whole distribution. The median values calculated by using the iodine intake of a single individual is to be estimated, especially
regression equation of Fig. 7 also are given in Table 1; they are if iodine contamination should be excluded. In field studies, on
in good agreement with the exact value. Furthermore, because the other hand, it is not necessary to dilute any sample, because
of the correct classification of samples also in the ranges be- the test yields the correct percentages of samples in each range
tween 100 –300 mg/L (0.79–2.36 mmol/L) and more than 300 (Table 1).
mg/L (.2.36 mmol/L), this test allows the rapid identification In conclusion, comparison with the HPLC method clearly
of the percentage of subjects who, in case of supplementation, demonstrates that this rapid test for determination of urinary
move from one category to another. iodide is well suited to the assessment of iodine deficiency
Compared with other simple methods (6, 21, 22), the rapid and to the monitoring of iodine supplementation in epide-
test is extremely easy to perform and does not require any miological surveys. With respect to the determination of
instrumentation or apparatus. Only columns with purified iodide in a single urine sample, the test allows the detection
activated charcoal are needed. One technician can analyze, in or exclusion of iodine contamination (excess iodide), which
parallel, 8 –10 samples; the total analysis time for one sample is important in iodide-induced hyperthyroidism (23).
is 15 min. About 60 – 80 samples can be tested this way within
2 h, at a cost of 0.5–1.0 $(US) per sample (depending on the References
number of kits ordered), labor not included. This rapid test 1. Dunn JT. 1992 Iodine deficiency - the next target for elimination? N Engl J Med.
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tries, and it makes shipping of samples unnecessary. For that 3. World Health Organization. 1994 Forty-Seventh World Health Assembly,
reason, the test is well suited to epidemiological investiga- Geneva, 2–12 May 1994. Resolutions and decisions; annexes. Geneva, World
Health Organization 47 (unpublished document WHA47/1994/REC/1).
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