Radiol Oncol 2024; 58(3): 357-365. doi: 10.2478/raon-2024-0035
357
research article
Introduction of a spectrophotometric method 
for salivary iodine determination on microplate 
based on Sandell-Kolthoff reaction
Adrijana Oblak1,2, Jernej Imperl3, Mitja Kolar3, Gregor Marolt3, Blaz Krhin1,  
Katja Zaletel1,2, Simona Gaberscek1,2
1 Division of Nuclear Medicine, University Medical Centre Ljubljana, Ljubljana, Slovenia
2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
3 Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia
Radiol Oncol 2024; 58(3): 357-365.
Received 16 April 2024 
Accepted 28 May 2024
Correspondence to: Adrijana Oblak, EuSpLM, Division of Nuclear Medicine, University Medical Centre Ljubljana, Ljubljana, Slovenia. E-mail: 
adrijana.oblak@kclj.si
Disclosure: No potential conflicts of interest were disclosed.
This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/).
Background. Iodine is an essential element for the synthesis of thyroid hormones. Therefore, a reliable marker of 
iodine supply is important. Iodine is predominantly excreted via kidneys, but also via salivary glands. Our aim was to 
introduce a new and simple method for determination of salivary iodine concentration (SLIC).
Materials and methods. Self-prepared chemicals and standards for Sandell-Kolthoff reaction on microplate with am-
monium peroxydisulfate (AP) in the range 0−400 µg/L were used. Suitability of water-based standards (WBS) and artificial 
saliva-based standards (ASS) for standard curve were tested. We followed standards for method validation, defined 
concentration of used AP and compared our results with Inductively Coupled Plasma Mass Spectrometry (ICP-MS).
Results. WBS gave more reliable results than ASS as an underestimation of iodine concentration was found for ASS. 
LoB was 6.5 µg/L, LoD 12.0 µg/L, therefore analytical range was 12−400 µg/L. Intra- and inter-assay imprecisions at 
iodine concentrations, namely 20, 100, 165, and 350 µg/L were 18.4, 5.1, 5.7, and 2.8%, respectively, and 20.7, 6.7, 
5.1, and 4.3%, respectively. Suitable molarity of AP was 1.0 mol/L and showed no difference to 1.5 mol/L (P values for 
samples with concentration 40, 100, and 150 µg/L, were 0.761, 0.085, and 0.275, respectively), whereas there was a 
significant change using 0.5 mol/L (P<0.001). Saliva samples could be diluted up to 1:8. There was no interference of 
thiocyanate and caffeine up to 193.5 mg/L. Our original method was comparable to ICP-MS. Spaerman coefficient 
was 0.989 (95% CI: 0.984−0.993).
Conclusions. The new method for SLIC determination is in excellent agreement with ICP-MS and easy-to-use.
Key words: iodine; salivary iodine concentration; Sandell-Kolthoff reaction; Inductively Coupled Plasma Mass 
Spectrometry
Introduction
Iodine is an essential trace element for the syn-
thesis of thyroid hormones thyroxine and triiodo-
thyronine, which importantly influence human 
development and metabolism. The human body 
contains up to approximately 20 mg of iodine, of 
which approximately 15 mg is stored in the thy-
roid gland.1 Iodine also accumulates in choroid 
plexus, gastric mucosa, mammary glands during 
lactation, and in salivary glands.1,2 With adequate 
iodine supply, more than 90% of iodine is excreted 
via urine.3 Small amounts of iodine are excreted 
via feces, sweat, and saliva. With iodine deficiency, 
less iodine is excreted and more is accumulated in 
the thyroid gland.4 Iodine supply significantly af-
fects the incidence and severity of thyroid diseas-
es.5 Therefore, reliable biomarkers are needed for 
Radiol Oncol 2024; 58(3): 357-365.
Oblak A et al. / Spectrophotometric method for salivary iodine based on S-K reaction358
the assessment of iodine supply. Determination of 
urinary iodine concentration (UIC) is most widely 
used. But it has one major drawback: it requires 
collecting a 24-hour urine sample, as a single spot 
urine sample is subject to diurnal variations in UIC 
and is therefore not directly comparable to 24-hour 
urinary iodine excretion.6 In salivary glands, iodine 
is transported from blood to saliva via the Na+/I- 
symporter.7,8 Thus, salivary iodine could be a bio-
marker of iodine supply. Saliva samples are easier 
to obtain and transport than urine samples.  
Iodine concentration can be determined using 
different methods, where Inductively Coupled 
Plasma with Mass Spectrometry (ICP-MS) is con-
sidered a gold standard method. More accessible 
are spectrophotometric methods with different 
approaches to remove potential interferences and 
organic matter in the samples, using different di-
gestion methods followed by manual or automatic 
Sandell-Kolthoff (S-K) reaction, where catalytic 
effect of iodide is used to quantify iodine in the 
sample. Digestion method using chloric(VII) acid 
has its drawbacks, because it represents a poten-
tial hazard, analyses should be performed in fume 
hoods and HI or I2 are formed, thereby falsely 
leading to underestimation of final results. On the 
other hand, ammonium peroxydisulfate is a non-
hazardous, non-explosive oxidizing reagent, that 
has very good analytical performances, as shown 
by Pino et al.9 
To our knowledge, salivary iodine concentra-
tion (SLIC) was measured for the first time using 
S-K reaction in 2012 by Gulaboglu et al., but the 
samples were digested with chloric acid.10 Later, 
SLIC was only measured by ICP-MS.11
ICP-MS is considered the best method for iodine 
concentration determination, but due to high cost 
of the instrumentation is not accessible to all labora-
tories. Our aim was to introduce a simple and low-
cost method for determination of SLIC using am-
monium peroxydisulfate as an oxidizing reagent 
followed by S-K reaction on microplate. We have 
already successfully validated this method for UIC 
determination.12 Therefore, we wanted to use this 
method on a different sample as well, saliva.
Materials and methods
Subjects
Cross-sectional study was conducted at the 
Division of Nuclear Medicine, University Medical 
Centre Ljubljana, Slovenia between May 2022 and 
February 2023. Adult volunteers were invited from 
local population to participate in the study. Only 
volunteers without known thyroid diseases were 
included in the study. 
The study was granted by the Republic of 
Slovenia National Medical Ethics Committee, 
number 0120-271/2021/3 (valid from 12th of July 
2021). Informed consent was obtained from all in-
dividuals included in this study.
In all participants, salivary iodine concentra-
tion was measured. Saliva was collected into 
Salivettes® (Sarstedt, Germany) before and 30, 
60, and 120 min after any meal during the day. 
Samples were then centrifuged for 10 min at 1800G 
at room temperature and frozen at -80°C prior to 
analysis.
Materials
Chemicals: potassium iodate(V) (KIO3) (Sigma-
Aldrich, Germany), ammonium peroxydisulfate 
((NH4)2S2O8) (Sigma-Aldrich, Germany), deion-
ized water (dH2O) (BRAUN, Aqua B. Braun, 
Sterile, Ecotainer®, Germany), sodium hydrox-
ide (NaOH) (Merck, Germany), arsenic(III) oxide 
(As2O3) (Sigma-Aldrich, Germany), sodium chlo-
ride (NaCl) (Sigma-Aldrich, Germany), concen-
trated sulfuric(VI) acid (H2SO4) (Sigma-Aldrich, 
Germany), ammonium cerium(IV) sulfate dihy-
drate ((NH4)4Ce(SO4)4x2H2O) (Sigma-Aldrich, 
Germany), 65% nitric(V) acid (HNO3) (Honeywell, 
USA).
All laboratory equipment was treated with 65% 
nitric(V) acid before preparation of reagents to 
remove any additional iodine from environment. 
Preparation of reagents as well as digestion pro-
cesses and analyses took place in a ventilating 
fume hood. Reagents were prepared in volumetric 
flasks. Brief summary of preparation: 1 mol/L am-
monium peroxydisulfate solution: dissolution of 
228.2 g of (NH4)2S2O8 in 1 L of dH2O, 0.875 mol/L 
sodium hydroxide solution: dissolution of 8.75 g 
of NaOH in 0.25 L of dH2O, 0.05 mol/L arsorous 
acid (H3AsO3) solution: dissolution of 5 g of As2O3 
in 0.1 L of 0.875 mol/L NaOH on magnetic stirrer. 
Afterwards, the solution was put in an ice-cold 
bath and 16 mL of concentrated H2SO4 was added 
while stirring on magnetic stirrer. After cooling, 
12.5 g of NaCl was added and diluted with dH2O 
up to 0.5 L. Solution was then mixed for 90 min 
at 60°C. Afterwards the solution was filtrated, 1.75 
mol/L sulfuric acid solution: on an ice bath 97 mL 
of concentrated H2SO4 was slowly added to 0.5 L of 
dH2O and filled with dH2O up to 1 L, 0.019 mol/L 
ammonium cerium(IV) sulfate solution: dissolu-
Radiol Oncol 2024; 58(3): 357-365.
Oblak A et al. / Spectrophotometric method for salivary iodine based on S-K reaction 359
tion of 6 g of (NH4)4Ce(SO4)4x2H2O in 0.5 L of 1.75 
mol/L H2SO4.
All reagents were stored in amber bottles in the 
dark at room temperature except for ammonium 
peroxydisulfate solution, which was stored in the 
dark at 2–8°C. 
We followed all Clinical Laboratory Standard 
Institute (CLSI) protocols for method establish-
ment, namely EP06, EP07, EP09, EP15-A3, EP17-A2.
Preparation of standards
1.68 g of KIO3 was dissolved in 1 L of dH2O. The 
solution was then used for preparation of inter-
mediate standard with iodine concentration of 
1000 µg/L, which was used later for preparation of 
standards and control samples (CSs). 
Standards were prepared with two different 
matrices, i.e. as artificial saliva to retain properties 
comparable to real saliva and as a water solution 
of iodine, which is used in UIC determination, in 
order to test appropriateness of matrix for stand-
ard curve. 
Artificial saliva was prepared according to 
commercially available products that are available 
for patients after head and neck cancer treatment 
for moistening mouth and throat. Ingredients used 
for the preparation of a 100 mL of artificial saliva 
were 30.45 mg/mL of sorbitol, 10.15 mg/mL of so-
dium carboxymethylcellulose, 1.218 mg/mL of po-
tassium chloride, 0.856 mg/mL of sodium chloride, 
0.348 mg/mL of potassium hydrogen phosphate, 
0.148 mg/mL of calcium chloride dihydrate, 0.052 
mg/mL of magnesium chloride hexahydrate, and 
water for injections as solution with pH set at 7.0. 
Cellulose was added for adjusting viscosity of ma-
trix retaining properties comparable to real saliva.
Both standards and CSs were prepared and 
stored according to previously published method 
for UIC determination.12 Briefly, 6 working stand-
ards were prepared at concentrations of iodide: 
0, 40, 80, 120, 200, and 400 µg/L. For comparison, 
both standards were measured on six microplates 
with S-K reaction. Each standard was measured 
in 6 replicates on each microplate and the mean 
concentration was calculated, whereas standards 
in artificial saliva were also analysed by ICP-MS. 
CSs were prepared at two different levels: 160 and 
280 µg/L.
Preparation of standards
Six pooled saliva samples from different partici-
pants were used for testing analytical recovery 
by adding 10, 20, 40 and 120 µL of intermediate 
standard with iodine concentration of 1000 µg/L 
to 500 µL of saliva sample before the digestion 
step. Samples were measured 8 times and recovery 
was expressed as the percentage of the measured 
amount of iodine over the expected amount of io-
dine.
Three pooled saliva samples from different par-
ticipants were tested for the potential influence 
of the molarity of ammonium peroxydisulfate to 
the final result. For UIC determination, 1 mL of 1.0 
mol/L of ammonium peroxydisulfate was used, 
therefore we tested the same volume of ammoni-
um peroxydisulfate with different concentrations, 
below and above proposed concentration: 1 mL of 
0.5 mol/L, 1.0 mol/L, and 1.5 mol/L.
Linearity of the method was tested using 4 sa-
liva samples, each measured 8 times, in the range 
up to 165 µg/L. Higher concentrations were not 
available for linearity testing. Distilled water was 
used as diluent in ratios of 1:2, 1:4, 1:8, and 1:16. Test 
was considered as linear, if the recovery was in the 
range of 80–120%. 
The interferences of some potential substances 
such as thiocyanate and caffeine in the introduced 
method were tested by adding 19.6, 38.5, 74.1, and 
193.5 mg/L of both, potassium thiocyanate and 
caffeine, to 6 saliva samples. Samples were meas-
ured 8 times and results are reported as recoveries 
expressed as the percentage of the measured over 
expected amount of iodine. The result was consid-
ered as no influences of interferences, if the recov-
ery was in the range of 80–120%.
Limit of blank (LoB) and limit of detection (LoD) 
of the method were determined. Eighty-eight 
measurements for LoB from 8 samples on two mi-
croplates using two different standard curves were 
analysed. To test LoB, dH2O was used. An F-test 
was used to compare values of measurements 
between both microplates. For LoD 180 measure-
ments from 12 samples were analysed on five mi-
croplates using five different standard curves. LoD 
samples were in the concentration range between 
LoB and 4-times LoB value. 
For intra- and inter-assay precision, two differ-
ent types of samples were prepared. Intra-assay 
precision was assessed on one microplate where 
40 replicates were measured, whereas inter-assay 
precision was measured on 15 microplates, where 
at least 5 replicates on each microplate were meas-
ured. Reproducibility was assessed by using 4 
samples, 3 of which were prepared in water solu-
tion at concentration levels 20, 100, and 350 µg/L to 
cover the range of the assay standard curve, and 
Radiol Oncol 2024; 58(3): 357-365.
Oblak A et al. / Spectrophotometric method for salivary iodine based on S-K reaction360
one was prepared as pooled saliva sample, which 
was pooled from random saliva samples and final 
concentration was 165 µg/L. Precisions were meas-
ured as coefficients of variation (CV (%)).
Procedure of the method
All reagents reached room temperature and were 
gently mixed before use. To 250 µL of standards, 
CSs, and samples, 1 mL of 1 mol/L ammonium 
peroxydisulfate solution was added. Oxidation 
of all samples, including standards and CSs, was 
performed manually in 13 x 100 mm glass tubes. 
Solution was then mixed and incubated for 1 h on 
95°C on dry bath (ScientificTM IsotempTM Digital 
Dry Bath/Block Heater, Fisher Scientific, UK). 
After cooling down to room temperature, 50 µL of 
standards, CSs, and samples were transferred in 
duplicate to microplate (Nunc Multiwell plate – 96 
well solid flat bottom, Merck, Germany). To each 
well 100 µL of arsorous acid solution was added. 
Microplate was sealed and incubated for 60 s on a 
microplate shaker. Afterwards 50 µL of ammoni-
um cerium(IV) sulfate solution was added to each 
well within 40 s with an 8-channel semi-automatic 
pipette. Microplate was sealed and put on the mi-
croplate shaker for exactly 30 min. Immediately 
after shaking, an absorbance measurement at 405 
nm on a spectrophotometer (SunriseTM, Tecan, 
Switzerland) was performed. The results were cal-
culated by plotting the linear optical density (OD) 
data on Y-axis and linear iodide ion concentration 
on X-axis. Concentration was inversely proportion-
al to absorbance. Standard curves were developed 
for each microplate separately. A quadratic poly-
nomial was used to calculate concentration from 
OD.
ICP-MS method
For the validation of the introduced method, SLIC 
was measured at the Faculty of Chemistry and 
Chemical Technology of the University of Ljubljana 
by ICP-MS method. The concentrations of iodine 
were determined by inductively coupled plasma 
quadrupole mass spectrometer (ICP-MS 7900ce, 
Agilent Technologies, USA) with the use of internal 
standards and optimal instrumental parameters, 
ensuring the lowest detection limits. A forward RF 
power of 1.5 kW was used with Ar gas flows: car-
rier 0.85 L/min, makeup 0.28 L/min, plasma 1.0 L/
min, cooling 15 L/min, and sample flow rate 0.2 mL/
min. Isotope 127I was measured in 3 replicates with 
integration/dwell time of 1 s. Saliva samples were 
diluted 40-times with ultrapure water (resistivity 
≥18.2 MΩ · cm, Synergy Water Purification System, 
Merck Millipore, Merck, Germany). Working 
standard solutions were prepared by appropriate 
dilution of iodide stock standard solution (iodide, 
1000 mg/L, CGICI1, Inorganic Ventures, USA) with 
ultrapure water. Calibration curve was based on 7 
working standard solutions in the concentration 
range 0.1–10 µg/L. Intra-assay and inter-assay im-
precisions of saliva samples at concentration levels 
40 µg/L were 4.0%, and 5.4%, and at 100 µg/L 5.4%, 
and 5.7%, respectively.
Method comparison
ICP-MS method, considered as a reference method, 
was used for comparison of the methods. One hun-
dred and ten saliva samples were analysed with 
ICP-MS as well as with the new S-K method.
Statistical analysis
The Kolmogorov-Smirnov test was used to assess 
the normality of distribution of analysed data. For 
comparison of LoB values of measured samples 
on two different microplates the F-test was used. 
T-test was used to compare values of different mo-
larity. A P<0.05 was considered statistically signifi-
cant. Data were expressed as means and standard 
deviations (SD), as means and ranges when report-
ing results of recoveries, and for method compari-
son as medians and ranges. The Passing-Bablok re-
gression analysis and Bland-Altman method were 
used for the method comparison of analysed data, 
and the Spearman coefficient was determined. Bias 
was also assessed. Statistical analysis was done 
using MedCalc Statistical Software version 20.014 
(MedCalc Software Ltd, Belgium).
Results 
To determine the suitability of artificial saliva 
standards (ASSs) or water-based standards (WBSs) 
we have measured both types of samples with S-K 
reaction. Iodine concentrations in ASS were lower 
than expected in comparison to WBS, as their OD 
differed up to 26% at highest concentration level. 
Therefore, ICP-MS was also used for the analysis of 
ASS. Comparison of ASS measured by S-K method 
and ICP-MS is presented in Figure 1. We found a 
significant difference between the methods as, on 
the average, 37% lower values were obtained with 
the use of S-K method in comparison to ICP-MS. 
Radiol Oncol 2024; 58(3): 357-365.
Oblak A et al. / Spectrophotometric method for salivary iodine based on S-K reaction 361
Therefore, ASSs are not appropriate for iodine de-
termination by S-K method. For further SLIC de-
termination by the new S-K method, only WBSs 
were used.
Assessment of analytical recovery of 6 differ-
ent saliva samples spiked with different concen-
trations of iodine showed no differences between 
measured and expected iodine concentrations. 
The mean percentage recovery (range) of iodine 
at each concentration level of addition of 19.6, 38.5, 
74.1, and 193.5 µg/L of stock potassium iodate(V) 
solution to saliva samples with mean iodine con-
centration (SD) 34.7 (3.1), 72.2 (2.7), 73.7 (1.5), 113.2 
(3.5), 136.0 (3.6), and 146.4 µg/L (3.0), respectively, 
were 103.0% (95.3–111.4%), 99.5% (94.5–102.6%), 
99.5% (97.1–102.8%), 95.2% (90.0–96.9%), 95.4% 
(91.2–95.7%), and 95.0% (88.6–97.8%), respectively. 
Three saliva samples at concentration levels of 
40, 100, and 150 µg/L were tested with 3 different 
concentrations of ammonium peroxydisulfate, 
namely 0.5, 1.0, and 1.5 mol/L, respectively. Results 
are presented in Figure 2. Based on the results, 
both, 1.0 mol/L and 1.5 mol/L of ammonium per-
oxydisulfate solution, could be used as oxygeniz-
ing agents before the S-K reaction.
LoD for SLIC was 12.0 µg/L, with less than 5% 
of both false positives (α) and false negatives (β) 
less than 5%; based on 268 determinations, with 88 
blanks and 180 low-level samples; LoB = 6.5 µg/L. 
LoB data were non-Gaussian. F-test for comparison 
of different samples for LoB determination showed 
no statistical difference between the values on dif-
ferent microplates using different standard curves, 
P = 0.283, so all data for LoB calculation were used. 
Finally, analytical range was 12–400 µg/L. 
The calculated imprecisions expressed as CV 
(%) for 3 levels of iodine concentrations, at 20, 100, 
300 µg/L, and for pooled sample, are presented in 
Table 1.
Linearity of the test was assessed on 4 saliva 
samples. Results are presented in Table 2. The re-
sults below the measuring range of the method 
were omitted from the table.  
Recovery tests of 6 different saliva samples 
spiked with different concentrations of thiocy-
anate and caffeine showed no significant influence 
on iodine concentrations. Recoveries are expressed 
as percentages of observed amount of iodine over 
expected amount of iodine after potassium thiocy-
anate or caffeine stock solutions added to samples. 
The mean percentage recovery (range) of iodine at 
each concentration level of addition of 19.6, 38.5, 
74.1, and 193.5 mg/L of stock solutions of potas-
sium thiocyanate and of caffeine to saliva samples 
with different iodine concentrations are presented 
in Table 3. 
Comparison of SLIC determination with 
the S-K method and with the ICP-MS 
method
The 110 samples covered the whole analytical 
range. For SLIC the median value (range) using 
FIGURE 1. Comparison of measurements of artificial saliva standards (ASS) by two 
different methods, inductively coupled plasma mass spectrometry (ICP-MS) and 
Sandell-Kolthoff method (S-K) using ammonium peroxydisulfate on microplate.
FIGURE 2. Comparison of different ammonium peroxydisulfate concentrations 
and their influence on the determined salivary iodine concentration. Comparison 
between 0.5 and 1.5 mol/L, as well as between 0.5 and 1.0 mol/L for all three 
samples showed significantly different values (P<0.001) for all three samples, 
whereas P values for samples 1, 2, and 3, showed no difference between 1.0 and 
1.5 mol/L. The corresponding P values were 0.275, 0.085, and 0.761, respectively.
Radiol Oncol 2024; 58(3): 357-365.
Oblak A et al. / Spectrophotometric method for salivary iodine based on S-K reaction362
the S-K method was 124.1 µg/L (25.8–297.3 µg/L), 
whilst the median value using the ICP-MS method 
was 129.8 µg/L (22.1–353.5 µg/L). SLIC results of 
saliva samples obtained by both methods were not 
normally distributed, Spaerman coefficient was 
0.989 (95% CI: 0.984–0.993), and P<0.001. Passing-
Bablok regression analysis and Bland-Altman 
graph are presented in Figure 3 and Figure 4, re-
spectively. The average bias between the methods 
was 5.9%. We did not observe any bias at concen-
trations up to 100 µg/L. With higher iodine concen-
tration, the bias between the methods was 8.2%.
Discussion
Determination of UIC is a standard method for 
iodine supply estimation on the population level. 
Urine sampling, however, is associated with many 
problems. Because iodine is also excreted in saliva, 
which is relatively easy to obtain, we developed a 
simple, easily implementable and low-cost meth-
od for determination of SLIC. Our method is in 
agreement with the reference ICP-MS method. The 
method is also suitable for measuring SLIC in sa-
liva samples and UIC in urine samples on the same 
microplate using the same standard curve as it was 
already shown in our previous paper.12
There is very little published data on the deter-
mination of SLIC and none of which is based on 
the method we describe in this work. The method 
is based on S-K reaction on microplate using am-
monium peroxydisulfate as an oxidizing and di-
gestion reagent, which shows sustainability and 
good performance characteristics.12 The main dis-
advantages are the ‘in-house’ reagent preparation, 
potential poisonous arsorous acid, and manage-
ment of arsenic waste. The main advantages of 
the method are cost-effectiveness, simplicity, and 
availability of laboratory equipment. 
Water based standards were used for UIC deter-
mination. To test the saliva matrix effect, standards 
using artificial saliva were prepared in the same 
concentration range as WBS. Using ASSs, iodine 
TABLE 1. Coefficients of variation (CV [%]) for intra- and inter-assay for 4 different iodine concentrations for salivary iodine 
concentration (SLIC)
Level Target value, µg/L
Number of 
measurements
Intra-assay 
imprecision, %
Number of 
measurements
Inter-assay 
imprecision, %
1 20 40 18.4 198 20.7
2 100 40 5.1 192 6.7
3 350 40 2.8 121 4.3
Pooled sample 165 40 5.7 80 5.1
TABLE 2. Dilutions of 4 saliva samples with deionised water. Each sample was measured 8 times and results are presented as 
mean measured concentration (SD). Recoveries are expressed as the percentages of mean measured amount of iodine over 
expected amount of iodine after dilution
Dilution Measured (M)µg/L (SD) 
Expected (E)
µg/L
M/E
%
Sample 1 Non-diluted 105.7 (1.3) / /
1:2 56.9 (1.3) 52.9 107.7
1:4 28.8 (2.0) 26.4 109
Sample 2 Non-diluted 35.6 (1.0) / /
1:2 19.2 (1.4) 17.8 108.2
Sample 3 Non-diluted 76.7 (1.3) / /
1:2 40.2 (1.7) 38.4 104.8
1:4 17.3 (1.2) 19.2 90.3
Pooled sample Non-diluted 165.1 (5.4) / /
1:2 65.0 (3.6) 61.9 105.0
1:4 33.8 (1.7) 31.0 109.1
1:8 17.2 (1.6) 15.5 111.1
Radiol Oncol 2024; 58(3): 357-365.
Oblak A et al. / Spectrophotometric method for salivary iodine based on S-K reaction 363
concentration was underestimated as compared 
to using WBSs. The results were also confirmed 
by ICP-MS. We assume that iodine in ASSs binds 
to organic molecules in artificial saliva and since 
only available iodide ions participate in S-K reac-
tion, ASSs are not suitable for the use. To verify 
whether WBSs are appropriate, we have spiked 6 
different saliva samples with different amounts of 
iodine in concentration range 34.7–146.4 µg/L. The 
average recoveries were very good, 95.0–103.0%, 
which was within the desirable range 80–120%. 
For further analysis only WBSs were used. The 
analytical range was 12–400 µg/L and could be 
used for both, UIC and SLIC determination on the 
same microplate, respectively. Measurement range 
is wide enough to cover expected values in saliva, 
and the introduced method is sensitive. Although 
the ICP-MS method has lower limits of quantifica-
tion, our new method provides sufficient informa-
tion for SLIC. The method also shows good intra- 
and inter-assay imprecision at different levels: low, 
medium, and high. Linearity of the test showed 
that saliva samples could be diluted up to 1:8 in 
the concentration range up to 165 µg/L. Higher di-
lutions could not be tested, since no samples were 
available in higher concentrations.
The first step of iodine determination was oxi-
dation of samples to obtain accurate measurements 
with the elimination of interfering substances that 
could be present in saliva. This reagent – ammo-
nium peroxydisulfate – was already presented by 
Pino et al.9 For UIC measurements, 1.0 mol/L of am-
monium peroxydisulfate was used. To confirm the 
suitability of the proposed concentration, we have 
also tested lower and higher molarities. With 0.5 
mol/L of ammonium peroxydisulfate significantly 
higher iodine concentrations were measured than 
with 1.0 or 1.5 mol/L (P<0.001 for both). Therefore, 
0.5 mol/L proved to be inappropriate. However, 
no differences were found using either 1.0 or 1.5 
mol/L of ammonium peroxydisulfate. In order to 
be complementary to UIC determination, we con-
tinued to use 1.0 mol/L. This way also less ammo-
nium peroxydisulfate is needed, compared to 1.5 
mol/L. From the literature it is well known that the 
disadvantages of the S-K method are the interfer-
ences of the substances like iodine, KBr, CuSO4, 
MgSO4, nitrites, and ascorbic acid13-15, which may 
affect the results. However, the results also depend 
on the use of oxidizing agent. Ascorbic acid does 
not affect analysis if ammonium peroxydisulfate 
is used as the oxidizing reagent, however it affects 
the result if chloric(VII) acid is used.9,15 No method 
can guarantee the complete removal of all interfer-
FIGURE 3. Comparison of salivary iodine concentration (SLIC) determined by the 
Sandell-Kolthoff (S-K) method using ammonium peroxydisulfate on microplate 
and by the Inductively Coupled Plasma Mass Spectrometry (ICP-MS) using 
Passing-Bablock regression. The solid line shows the regression line, dashed lines 
show 95% confidence interval (CI) for the regression line, and dotted line shows 
the identity line Y = X. The sample size is N = 110. The regression equation is Y = 
0.89*X+6.2. The slope is 0.89 (95% CI, 0.86−0.92), and the intercept is 6.2 (95% CI, 
3.3−8.6). The Cusum test for linearity showed no significant deviation from linearity 
(P = 0.89).
FIGURE 4. Bland-Altman plot of comparison of salivary iodine concentration 
(SLIC) determined by the Sandell-Kolthoff method (S-K) and by the Inductively 
Coupled Plasma Mass Spectrometry (ICP-MS). Differences between the methods 
are plotted against the ICP-MS method, which is a gold standard method. 
Sample size is N = 110. Horizontal lines represent the mean SLIC by ICP-MS, zero 
difference, and 95% confidence interval limits of agreement, which are defined 
as mean difference plus/minus 1.96 times the standard deviation (SD) of the 
differences. The mean shows a positive bias of 5.9%. 
Radiol Oncol 2024; 58(3): 357-365.
Oblak A et al. / Spectrophotometric method for salivary iodine based on S-K reaction364
ing substances. Our focus was on possible interfer-
ences of thiocyanate, which is found in cigarettes, 
and caffeine, which is regularly consumed in the 
form of caffeinated beverages, and are consequent-
ly present in saliva. The thiocyanate influence was 
already tested9, but we used higher concentrations 
of thiocyanate comparable to those found in sa-
liva of smokers.16 The same protocol was followed 
with caffeine, as higher concentrations than those 
found in saliva17 were tested for possible interfer-
ence with the assay. Neither thiocyanate nor caf-
feine had any effect on the assay at concentrations 
up to 193.5 mg/L. However, possible coloration 
of saliva with thiocyanate or caffeine could af-
fect the end result since the reaction is based on 
the absorbance measurement. During the process 
of digestion (i.e. oxidation), the samples were dis-
coloured, which was observed and verified spec-
trophotometrically and results were negative (not 
shown in the Results).
Our method for SLIC determination was com-
pared with a reference ICP-MS method to confirm 
our results. The comparison showed agreement 
between the evaluated methods as there was no 
discrepancy between the two up to 100 µg/L. At 
higher concentrations we found a slight discrep-
ancy, on average 8.2% underestimation of the in-
troduced method. Saliva samples for ICP-MS anal-
ysis were diluted 40 times using MQ to overcome 
matrix effect. With this in mind, it is plausible to 
assume that the slight discrepancy between the 
methods could be due to different media used for 
SLIC determination. 
It is also important to emphasize that there is no 
External quality assurance assessment scheme for 
measuring SLIC. Therefore, comparison of SLIC 
determinations between the laboratories cannot 
be established yet. We believe the best practice 
for SLIC determination at this moment would be 
to verify self-laboratory prepared solutions used 
for reaction through available external program 
quality assessment for urinary iodine samples. If 
measurements are within the range, solutions are 
acceptable, and are also suitable for the use with 
saliva samples. 
In summary, described spectrophotometric 
method on microplate using S-K reaction with 
ammonium peroxydisulfate digestion for SLIC 
determination shows acceptable performance 
characteristics and agreement with the reference 
method ICP-MS, which represents the gold stand-
ard. Implemented method showed good sensitiv-
ity and is suitable for measuring iodine concentra-
tion in various biological samples such as SLIC in 
saliva samples and UIC in urine samples, where 
microplate and the standard curve can be used. 
Method also enables different dilution factors for 
saliva samples, and more importantly, has no in-
terferences to thiocyanate and caffeine, which are 
quite abundant in saliva samples. It is cost-effec-
tive, requires equipment that is usually already 
available in laboratories and can be therefore per-
formed by almost any laboratory.
Acknowledgments 
We extend our gratitude to Darinka Levstek at 
University Medical Centre Ljubljana, Slovenia, for 
her technical support and to Prof. Marjan Veber 
for his assistance that led to collaboration between 
both working groups. We also appreciate all par-
TABLE 3. Recoveries of 6 different saliva samples spiked with 19.6, 38.5, 74.1, and 193.5 mg/L of stock solutions of potassium 
thiocyanate and of caffeine to saliva samples. Recoveries are expressed as percentages of observed amount of iodine over 
expected amount of iodine after thiocyanate or caffeine stock solutions added to samples. Each sample was measured 8 
times and results are presented as mean salivary iodine concentration (SLIC) (SD) and as the mean percentage recovery 
(range) of iodine at each concentration level
Thiocyanate Caffeine
SLIC, µg/L  
(SD)
Recovery, % 
(range)
SLIC, µg/L
(SD)
Recovery, %
(range)
Sample 1 34.7 (3.0) 97.9 (97.3–100.5) 29.1 (1.0) 93.2 (90.7–96.3)
Sample 2 86.0 (7.4) 101.2 (99.0–102.8) 73.0 (1.6) 99.3 (97.0–100.9)
Sample 3 90.7 (3.7) 102.4 (99.0–105.0) 75.2 (1.9) 96.1 (92.0–100.4)
Sample 4 109.0 (3.9) 109.0 (95.9–104.9) 101.3 (3.8) 96.3 (94.1–99.0)
Sample 5 143.0 (2.2) 106.2 (103.8–108.7) 142.1 (2.5) 98.7 (96.1–105.3)
Sample 6 150.6 (3.3) 102.4 (98.2–107.7) 152.8 (3.0) 99.3 (97.3–101.3)
Radiol Oncol 2024; 58(3): 357-365.
Oblak A et al. / Spectrophotometric method for salivary iodine based on S-K reaction 365
ticipants for all their effort in collecting biological 
samples. Research funding: ARIS grant number 
P1-0153.
References
1. Fisher DA, Oddie TH. Thyroid iodine content and turnover in euthyroid sub-
jects: validity of esti-mation of thyroid iodine accumulation from short-term 
clearance studies. J Clin Endocrinol Metab 1969; 29: 721-7. doi: 10.1210/
jcem-29-5-721
2. Zimmermann MB. Iodine deficiency. Endocr Rev 2009; 30: 376-408. doi: 
10.1210/er.2009-0011
3. Nath SK, Moinier B, Thuillier F, Rongier M, Desjeux JF. Urinary excretion of 
iodide and fluoride from supplemented food grade salt. Int J Vitam Nutr 
Res 1992; 62: 66-72. 
4. DeGroot LJ. Kinetic analysis of iodine metabolism. J Clin Endocrinol Metab 
1966; 26: 149-73. doi: 10.1210/jcem-26-2-149
5. Bülow Pedersen I, Knudsen N, Jørgensen T, Perrild H, Ovesen L, Laurberg 
P. Large differences in incidences of overt hyper- and hypothyroidism as-
sociated with a small difference in iodine intake: A prospective comparative 
register-based population survey. J Clin Endocrinol Metab 2002; 87: 4462-9. 
doi: 10.1210/jc.2002-020750
6. Iacone R, Idelson PI, Formisano P, Russo O, Lo Noce C, Donfrancesco C, 
et al. Iodine intake estimated by 24 h urine collection in the Italian adult 
population: 2008-2012 Survey. Nutrients 2021; 13: 1529. doi: 10.3390/
nu13051529
7. Spitzweg C, Joba W, Eisenmenger W, Heufelder AE. Analysis of human 
sodium iodide symporter gene expression in extrathyroidal tissues and 
cloning of its complementary deoxyribo-nucleic acids from salivary gland, 
mammary gland, and gastric mucosa. J Clin Endocrinol Metab 1998; 83: 
1746-51. doi: 10.1210/jcem.83.5.4839
8. Jhiang SM, Cho JY, Ryu KY, DeYoung BR, Smanik PA, McGaughy VR, et al. An 
im-munohistochemical study of Na+/I- symporter in human thyroid tissues 
and salivary gland tissues. Endocrinology 1998; 139: 4416-9. doi: 10.1210/
endo.139.10.6329
9. Pino S, Fang SL, Braverman LE. Ammonium persulfate: a safe alternative 
oxidizing reagent for measuring urinary iodine. Clin Chem 1996; 42: 239-43.
10. Gulaboglu M, Akgül H, Akgül N, Cetin M. Urine and saliva iodine levels 
in patients with dental caries and normal healthy volunteers. Trace Elem 
Electroly 2012; 29: 28-33. doi: 10.5414/tex01186
11. Guo WX, Pan ZY, Zhang Y, Jin Y, Dong SY, Wu W, et al. Saliva iodine concentra-
tion in children and its association with iodine status and thyroid function. J 
Clin Endocrinol Metab 2020; 105: e3451-e9. doi: 10.1210/clinem/dgaa471
12. Oblak A, Arohonka P, Erlund I, Kuzmanovska S, Zaletel K, Gaberšček S. 
Validation of a spectrophotometric method for urinary iodine determina-
tion on microplate based on Sandell-Kolthoff reaction. Lab Med 2022; 53: 
376-80. doi: 10.1093/labmed/lmab117
13. Miura Y, Kusakari K. Flow injection analysis of nitrite based on spectrophoto-
metric measurements of iodine formed by oxidation of iodide with nitrite. 
Anal Sci 1999; 15: 923-6. doi: org/10.2116/analsci.15.923
14. Štolc V. Interference of certain ions with the catalytic action of iodine in 
the Sandell-Kolthoff reaction. Fresenius Z Anal Chem 1961; 183: 262-7. doi: 
10.1007/bf00474698
15. Ford HC, Johnson LA. Ascorbic acid interferes with an automated urinary 
iodide determi-nation based on the ceric-arsenious acid reaction. Clin Chem 
1991; 37: 759. doi: 10.1093/clinchem/37.5.759
16. Flieger J, Kawka J, Tatarczak-Michalewska M. Levels of the thiocyanate in the 
saliva of tobacco smokers in comparison to e-cigarette smokers and non-
smokers measured by HPLC on a phosphatidylcholine column. Molecules 
2019; 24: 3790. doi: 10.3390/molecules24203790
17. Zylber-Katz E, Granit L, Levy M. Relationship between caffeine concentra-
tions in plasma and saliva. Clin Pharmacol Ther 1984; 36: 133-7. doi: 
10.1038/clpt.1984.151